Superior colliculus drives stimulus-evoked directionally biased saccades and attempted head movements in head-fixed mice

  1. Sebastian H Zahler
  2. David E Taylor
  3. Joey Y Wong
  4. Julia M Adams
  5. Evan H Feinberg  Is a corresponding author
  1. Department of Anatomy, University of California, San Francisco, United States
  2. Neuroscience Graduate Program, University of California, San Francisco, United States
  3. Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, United States

Abstract

Animals investigate their environments by directing their gaze towards salient stimuli. In the prevailing view, mouse gaze shifts entail head rotations followed by brainstem-mediated eye movements, including saccades to reset the eyes. These ‘recentering’ saccades are attributed to head movement-related vestibular cues. However, microstimulating mouse superior colliculus (SC) elicits directed head and eye movements resembling SC-dependent sensory-guided gaze shifts in other species, suggesting that mouse gaze shifts may be more flexible than has been recognized. We investigated this possibility by tracking eye and attempted head movements in a head-fixed preparation that eliminates head movement-related sensory cues. We found tactile stimuli evoke directionally biased saccades coincident with attempted head rotations. Differences in saccade endpoints across stimuli are associated with distinct stimulus-dependent relationships between initial eye position and saccade direction and amplitude. Optogenetic perturbations revealed SC drives these gaze shifts. Thus, head-fixed mice make sensory-guided, SC-dependent gaze shifts involving coincident, directionally biased saccades and attempted head movements. Our findings uncover flexibility in mouse gaze shifts and provide a foundation for studying head-eye coupling.

Editor's evaluation

Animals investigate their environments by directing their gaze towards salient stimuli. However, whether non-foveal mammals like mice can make directed saccades independent of head movements in response to sensory stimuli remains unclear. Feinberg et al. systematically investigate how tactile, auditory and visual stimuli drive saccade and head movement patterns. Mice make sensory-guided gaze shifts that depend on superior colliculus and involve coincident, directionally biased saccades and attempted head movements, with flexibility in saccade kinematics relative to attempted head movements.

https://doi.org/10.7554/eLife.73081.sa0

Introduction

Natural environments are complex and dynamic, and animals frequently redirect their gaze to scrutinize salient sensory stimuli. Gaze shifts employ head and eye movement coupling strategies that depend on context and can vary between species (Goldring et al., 1996; Land, 2019; Land and Nilsson, 2012; Populin, 2006; Populin and Rajala, 2011; Populin et al., 2004; Ruhland et al., 2013; Tollin et al., 2005). Mice are an increasingly important model organism in vision research, yet the strategies they use to shift their gaze remain incompletely understood. Revealing these strategies is essential to understanding mouse visual ethology and the underlying neural mechanisms.

The prevailing view holds that species whose retinae lack high-acuity specializations (afoveates) such as mice generate gaze shifts driven by head movements and followed by ‘recentering’ saccades (Land and Nilsson, 2012). Indeed, recent studies tracking head and eye movements in freely moving mice found that spontaneous and visually evoked mouse gaze shifts matched this description (Meyer et al., 2018; Meyer et al., 2020; Michaiel et al., 2020; Payne and Raymond, 2017). Specifically, during a gaze shift, slow eye movements stabilize the retinal image by countering the rotation of the head and are punctuated by fast saccadic eye movements to recenter the eyes in the orbits as they approach the end of their range of motion. These recentering saccades—also known as ‘compensatory’ saccades or the quick phase of nystagmus—are centripetal, occur in the direction of the head movement, and are thought to be driven by vestibular or optokinetic signals acting on circuits in brainstem (Curthoys, 2002; Hepp et al., 1993; Kitama et al., 1995; Meyer et al., 2020; Michaiel et al., 2020; Payne and Raymond, 2017). These recent observations have buttressed the view that gaze shifts in mice and other afoveates are led by head movements, with eye movements made only to compensate for the effects of head movements. In contrast, primates and other foveate species are capable of an additional form of gaze shift led by directed saccades, with or without directed head movements, to redirect their gaze toward salient stimuli (Bizzi et al., 1972; Freedman, 2008; Lee, 1999; Zangemeister and Stark, 1982). Directed saccades differ from recentering saccades in that they have endpoints specified by the location of the stimulus (and therefore are often centrifugally directed), typically occur simultaneously with head movements during gaze shifts, and are driven by midbrain circuits, particularly the superior colliculus (SC). To date, there is no behavioral evidence that mice or any afoveate species generate directed saccades or gaze shifts not initiated by head movements.

However, three observations are inconsistent with the model that saccades in mice are exclusively recentering and made to compensate for head movements. First, mouse saccades are not only a product of vestibular or optokinetic cues, because head-fixed mice, in which these signals do not occur, generate saccades, albeit less frequently. Second, neuroanatomical and functional studies suggest that the circuits that underlie directed saccades are conserved in mice (May, 2006; Sparks, 1986; Sparks, 2002). Specifically, microstimulation of the mouse superior colliculus (SC) showed that it contains a topographic map of saccade and head movement direction and amplitude (Masullo et al., 2019; Wang et al., 2015) roughly aligned with maps of visual, auditory, and somatosensory space (Dräger and Hubel, 1975). These SC sensory and motor maps resemble those believed to underlie primates’ and cats’ ability to make gaze shifts led by directed saccades toward stimuli of these modalities (Sparks, 1986; Sparks, 2002). Third, saccade-like eye movements in the absence of head movements have occasionally been observed in freely moving mice, albeit infrequently and usually in close proximity to head movements (Meyer et al., 2018; Meyer et al., 2020; Michaiel et al., 2020).

We therefore hypothesized that mice innately generate gaze shifts that incorporate directionally biased saccades. We predicted that this ability was obscured in previous studies for several reasons. First, in freely moving mice it is difficult to uncouple the contributions of reafferent vestibular and optokinetic inputs from those of exafferent (extrinsic) sensory inputs to saccade generation. Second, previous analyses in mice were mostly confined to spontaneous or visually guided gaze shifts, and there is evidence in humans, non-human primates, and cats that gaze shifts in response to different sensory modalities can involve distinct head-eye coupling strategies (Goldring et al., 1996; Populin, 2006; Populin and Rajala, 2011; Populin et al., 2004; Ruhland et al., 2013; Tollin et al., 2005). Third, in freely moving mice it is difficult to present stimuli in specific craniotopic locations. We therefore reasoned that by using a head-fixed preparation both to eliminate vestibular and optokinetic cues and to present stimuli of different modalities at precise craniotopic locations, we could systematically determine whether mice are capable of gaze shifts not initiated by head movements and involving saccades whose endpoints depend on stimulus location and show different coupling to head movements. We found that tactile stimuli evoke saccades whose endpoints depend on stimulus location, that these saccades are coincident with attempted head rotations, and that these touch-evoked gaze shifts are SC-dependent. Together, these results resolve an apparent discrepancy between mouse neuroanatomy and behavior, demonstrating that head-fixed mice are capable of generating gaze shifts involving directionally biased saccades coincident with attempted head movements.

Results

Stimulus-evoked gaze shifts in head-restrained mice

Previous studies established that SC microstimulation or optogenetic stimulation evoke both head and eye movements and that these movements roughly match the topographic map of sensory spatial receptive fields within SC, with each SC hemisphere driving contraversive movements whose amplitudes are larger for more posterior stimulation sites (Masullo et al., 2019; Wang et al., 2015). However, to our knowledge, a simultaneous measurement of eye and attempted head movements elicited by SC microstimulation had not been conducted. To perform this comparison, we pursued an optogenetic approach. We stereotaxically injected adeno-associated virus (AAV) encoding the light-gated ion channel ChR2 under the control of a pan-neuronal promoter and implanted a fiber optic in right SC (Figure 1B). Several weeks later, we head-fixed mice and used infrared cameras to track both pupils and a strain gauge (also known as a load cell) to measure attempted head rotations (Figure 1A). Consonant with previous studies, SC optogenetic stimulation elicited contraversive eye and attempted head movements (Figure 1C and D). Strikingly, optogenetically evoked saccades and attempted head movements were roughly coincident, similar to what has been described for SC-dependent sensory-guided gaze shifts in other species and unlike the temporal relationship previously documented for mouse spontaneous and visually evoked gaze shifts (Figure 1C and E).

Optogenetic stimulation of the superior colliculus evokes coincident and directionally biased attempted head and eye movements.

(A) Behavioral schematic. Naive mice are head-fixed, both eyes are tracked using cameras, and attempted head rotations are measured using a strain gauge (load cell). In subsequent quantification, eye positions to the right of center (nasal for left eye, temporal for right eye) are positive, and eye positions to the left of center (temporal for left eye, nasal for right eye) are negative, with zero defined as the mean eye position. Likewise, attempted rightward head movements are positive, and leftward head movements are negative. (B) Schematic of right SC optogenetic stimulation using ChR2 and example histology for representative mouse. Scale bar, 0.5 mm. (C) Mean attempted head (blue) and eye (black) movement traces (n = 44 trials, 4 mice) in the 1 s period surrounding optogenetic stimulation. Optogenetic illumination (1 mW) was delivered for 40 ms. (D) Relationship between saccade amplitude and attempted head movement amplitude for individual mice. (E) Relationship between saccade latency and head movement latency for individual mice.

In light of this observation, we hypothesized that mice possess an innate ability to make sensory-evoked gaze shifts that incorporate directionally biased saccades coupled to head movements. To test this hypothesis, we head-fixed naive, wild-type adult animals and tracked eye and attempted head movements. Previous studies in head-fixed mice observed occasional undirected saccades in response to changes in the visual environment (Samonds et al., 2018) and visually guided saccades only after weeks of training and at long (~1 s) latencies (Itokazu et al., 2018). We therefore tested a panel of stimuli of different modalities to determine whether they could evoke saccades. We began by presenting the following stimuli from a constant azimuthal location: (1) a multisensory airpuff that provides tactile input to the ears and generates a loud, broadband sound; (2) an auditory stimulus consisting of the same airpuff moved away from the animal so as not to provide tactile input; (3) a tactile stimulus consisting of a bar that nearly silently taps the ear; and (4) a visual stimulus consisting of a bright LED. Stimuli were presented on either side of the animal every 7–12 s in a pseudorandom sequence (Figure 2A). The probability of horizontal eye movements increased sharply and significantly above the low baseline level (1.3% ± 0.2%, mean ± s.d.) in the 100ms period following delivery of multisensory airpuffs (ear airpuff: 29.0% ± 7.5%, p < 0.001 paired Student’s t-test; whisker airpuff: 12.5% ± 2.3%, p < 0.001), auditory airpuffs (3.5% ± 1.2%, p < 0.05), and tactile stimuli (4.5% ± 0.5%, p < 0.001) and remained slightly elevated for at least 500 ms (Figure 2B–F and G–K; Figure 2—figure supplements 12). In contrast, the probability of saccade generation was not changed by visual stimuli (Figure 2F and K; 1.3% ± 0.2%, p = 0.61). We consider these stimulus-evoked eye movements to be saccades because they reached velocities of several hundred degrees per second (Figure 2—figure supplement 1), displayed a main sequence, that is, peak velocity scaled linearly with amplitude (Figure 2—figure supplement 1), and were bilaterally conjugate (Figure 2—figure supplement 1; Bahill et al., 1975). As in previous studies, saccade size for temporal-to-nasal movements was slightly larger than for nasal-to-temporal movements (Meyer et al., 2018), but this asymmetry was eliminated by averaging the positions of both pupils (before averaging: temporal saccade amplitude = 10.9 ± 0.8° (mean ± s.d.), nasal saccade amplitude = 8.4 ± 1.1°, p = 0.0009; after averaging, leftward amplitude = 10.2 ± 1.0°, rightward amplitude = 9.3 ± 0.4°, p = 0.0986).

Figure 2 with 2 supplements see all
Mice innately make sound- and touch-evoked gaze shifts.

(A) Sample eye and attempted head movement traces. Dashed vertical lines indicate right (green) and left (magenta) ear airpuff delivery. (B–F) Saccade rasters for five representative mice in response to (B) ear airpuffs, (C) whisker airpuffs, (D) ear tactile stimuli, (E) auditory airpuffs, and (F) visual stimuli. Each row corresponds to a trial. Each dot indicates onset of a saccade. Vertical black lines denote time of left or right stimulus delivery. Each gray or white horizontal stripe contains data for a different mouse. n = 1000 randomly selected trials (200 /mouse). (G–K) Peri-stimulus time histograms showing instantaneous saccade probabilities in response to (G) ear airpuffs, (H) whisker airpuffs, (I) ear tactile stimuli, (J) auditory airpuffs, and (K) visual stimuli for mice from (B–F). Each light trace denotes a single animal; black traces denote population mean. Dashed lines denote time of stimulus delivery. Horizontal bar indicates the 100ms response window used in subsequent analyses. (L–P) Heatmaps of attempted head movements in response to (L) ear airpuffs, (M) whisker airpuffs, (N) ear tactile stimuli, (O) auditory airpuffs, and (P) visual stimuli for mice from (B–K). Each row corresponds to an individual trial from B-F. Black and white bars at left indicate blocks of trials corresponding to each of five different mice. Dashed line denotes stimulus delivery time.

We next examined attempted head movements. The baseline frequency of attempted head movements was much higher than that of eye movements (27.8% ± 4.8%, mean ± s.d.). Mirroring results for saccades, auditory, tactile, and audiotactile stimuli evoked attempted head movements but visual stimuli did not (Figure 2L–P; auditory: 53.2% ± 21.9%, p = 0.045; tactile: 67.1% ± 10%, p < 0.01; ear airpuff: 86.6% ± 8.7%, p < 10–5; whisker airpuff: 79.2% ± 9.5%, p < 0.001; visual: 26.9% ± 2.0%, p = 0.0505, paired Student’s t-test). These data demonstrate that both auditory and tactile stimuli are sufficient to evoke gaze shifts in head-fixed mice, and that mice make sensory-evoked saccades in the absence of vestibular and optokinetic inputs.

Tactile stimuli evoke directionally biased eye and attempted head movements

To determine whether sensory-evoked saccades are directionally biased, we asked whether saccade endpoints are dependent on stimulus location. We began by examining the endpoints of saccades evoked by left and right ear airpuffs (Figure 3A, Figure 3—figure supplement 1). We found that left ear airpuffs evoked saccades with endpoints far left of center (with center defined as the mean eye position), whereas right ear airpuffs evoked saccades with endpoints far right of center (left: –5.4 ± 4.5°, right: 5.4 ± 3.4°, mean ± s.d., p < 10–10 Welch’s t-test, n = 2155 trials). To understand how this endpoint segregation arises, we examined the trajectories of individual saccades (Figure 3E). We found that left ear airpuffs elicited nearly exclusively leftward saccades (94.2% ± 3.1%, mean ± s.d., n = 5 mice), whereas right ear airpuffs elicited nearly exclusively rightward saccades (96.0% ± 2.1%)—often from the same eye positions. By definition, from any eye position, one of these directions must lead away from center and is thus centrifugal rather than centripetal. In addition, puff-evoked saccades that began toward the center often overshot to reach endpoints at eccentricities of 5–10 degrees. To further test whether saccade endpoints are specified by stimulus location, we repositioned the airpuff nozzles to stimulate the whiskers and repeated the experiments. We reasoned that saccade endpoints should become less eccentric as stimulus eccentricity decreases. Indeed, airpuffs applied to the whiskers evoked saccades with endpoints central to those evoked by ear airpuff stimulation, such that the ordering of saccade endpoints mirrored that of stimulus locations (Mean endpoints: left ear, –5.4 ± 4.5°; left whiskers, –0.4 ± 3.8°; right whiskers, 0.8 ± 3.9°; right ear, 5.4 ± 3.4°; mean ± s.d., p < 0.05 for all pairwise comparisons, paired two-tailed Student’s t-test) (Figure 3A and B; Figure 3—figure supplements 12). In this cohort, the separation between whisker-evoked saccade endpoints, although significant, was small, but in other cohorts we observed larger separation (as well as higher auditory-evoked saccade probabilities) (Figure 3—figure supplement 3). Taken together, these data suggest that airpuff-evoked saccades are biased toward particular eye positions that are specified by stimulus location.

Figure 3 with 3 supplements see all
Sensory-evoked eye and attempted head movements.

(A–D) Endpoints for ear airpuff-, whisker airpuff-, ear tactile-, and auditory airpuff-evoked saccades. Top, schematics of stimuli. Middle, scatter plots showing endpoints of all saccades for all animals (n = see below, 5 animals) made spontaneously (blue) and in response to left (green) and right (magenta) stimuli. Darker shading indicates areas of higher density. Bottom, histograms of endpoint distributions for spontaneous and evoked saccades. (E–H) Trajectories of individual stimulus-evoked saccades. Each arrow denotes the trajectory of a single saccade. Saccades are sorted according to initial eye positions, which fall on the dashed diagonal line. Saccade endpoints are indicated by arrowheads. Because the probability of evoked gaze shifts differed across stimuli, data for ear and whisker airpuffs are randomly subsampled (15% and 30% of total trials, respectively) to show roughly equal numbers of trials for each condition. (I–L) Ear airpuff-, whisker airpuff-, ear tactile-, and auditory airpuff-evoked attempted head displacements associated with saccades in A-D. Top, scatter plots showing displacements of attempted head movements associated with saccades made spontaneously (blue) and in response to left (green) and right (magenta) stimuli (n = see below, 5 animals). Darker shading indicates areas of higher density. Bottom, histograms of attempted displacement distributions for spontaneous and evoked attempted head movements. Saccade numbers in A-L: ear airpuff sessions, spontaneous = 7146, left ear airpuff-evoked = 942 (141 in E), right ear airpuff-evoked = 1213 (182 in E); whisker airpuff sessions: spontaneous = 7790, left whisker airpuff-evoked = 440 (132 in F), right whisker airpuff-evoked = 606 (181 in F); ear tactile sessions, spontaneous = 6706, left ear tactile-evoked = 133, right ear tactile-evoked = 186; auditory sessions, spontaneous = 10240, left auditory-evoked = 140, right auditory-evoked = 158.

We next examined the endpoints of saccades evoked by tactile and auditory stimuli. Similar to the multisensory airpuff stimuli, tactile stimuli delivered to the left and right ears evoked saccades whose endpoints were significantly different (–3.9 ± 5.3° [left] vs. 1.5 ± 5.0° [right]; mean ± s.d., p < 10–10, Welch’s t-test, n = 452 trials) and whose directions were largely opposite from nearly all eye positions (Figure 3C and G, Figure 3—figure supplement 1) (left stimuli evoked 77.5% ± 23.7% leftward saccades, right stimuli evoked 78.3% ± 11.6% rightward saccades, n = 5 mice). This result suggests that tactile stimuli are sufficient to induce gaze shifts that involve directionally biased saccades. We next examined the endpoints and trajectories of saccades evoked by left and right auditory stimuli (Figure 3D and H, Figure 3—figure supplement 1). Strikingly, the endpoint locations did not differ significantly for saccades elicited by left and right auditory stimuli and were located centrally (0.1 ± 5.1° (left) vs. –0.1 ± 5.1° (right); mean ± s.d., p = 0.72, Welch’s t-test, n = 298 trials). Because we had fewer trials with sound-evoked gaze shifts overall, to confirm that this lack of significant endpoint separation was not a result of lower statistical power, we repeated our analyses on equal numbers of sound- and touch-evoked saccades sampled at random, once again observing that left and right ear airpuff-, whisker airpuff-, and ear tactile-evoked saccade endpoints were significantly different (ear airpuff, p < 10–10; whisker airpuff, p = 0.0039; ear tactile, p < 10–10; auditory airpuff, p = 0.50; Welch’s t-test). The central endpoints of sound-evoked saccades arose because, in contrast to touch-evoked saccades, saccades evoked by both right and left stimuli traveled in the same, centripetal direction from all initial eye positions: rightward from eye positions to the left of center, and leftward from eye positions to the right of center (fraction centripetal: left airpuff for initial eye positions left of center, 0.89 ± 0.08; left airpuff for initial eye positions right of center, 0.90 ± 0.09; right airpuff for initial eye positions left of center, 0.87 ± 0.07; right airpuff with initial eye positions right of center, 0.76 ± 0.12). We compared the mean sound-evoked saccade endpoint to the mean overall eye position and found no significant difference, suggesting that sound-evoked saccades function to center the eye (p = 0.93, one-sample Student’s t-test) (Land and Nilsson, 2012; Meyer et al., 2018; Meyer et al., 2020; Michaiel et al., 2020; Paré and Munoz, 2001; Tatler, 2007). These responses are unlikely to be due to an auditory startle response, which is elicited by stimuli louder than 80 dB, because the airpuffs were <65 dB (Gómez-Nieto et al., 2020). Thus, both the auditory and tactile components of the airpuff stimuli are sufficient to evoke saccades but only tactile stimulation evokes directionally biased saccades whose endpoints are specified by the site of stimulation.

We next analyzed the relationship between stimuli and attempted head movements. The amplitude distributions for attempted head movements to left and right ear airpuff stimuli were well separated (left: –1.56 ± 1.25 Z, right: 1.29 ± 1.16 Z, mean ± s.d., p < 10–10 Welch’s t-test, n = 2155 trials), mirroring the separation of the endpoints of saccades elicited by left and right stimuli (Figure 3I). Similarly, whisker and tactile stimuli elicited attempted head movements whose amplitude distributions were separated (left whiskers: –0.22 ± 1.58 Z, right whiskers: 0.20 ± 1.42 Z, p < 10–4, n = 1046; left tactile: –1.45 ± 1.84 Z, right tactile: 0.55 ± 1.75 Z, p < 10–10, n = 319; mean ± s.d., Welch’s t-test) but less so than those of ear airpuffs (Figure 3J and K). Left and right auditory airpuffs elicited attempted head movements whose distributions overlapped (left: –0.25 ± 2.04 Z, right: 0.07 ± 1.94 Z, mean ± s.d., p = 0.16, Welch’s t-test, n = 298 trials), similar to what was observed for saccades (Figure 3L). Thus, it appeared that the patterns of evoked eye and attempted head movements were similar for a given stimulus but different across stimuli, with tactile but not auditory stimuli able to evoke directionally biased movements.

Stimulus-evoked gaze shifts lack pre-saccadic head movements

Having observed that mice are able to make stimulus-evoked directionally biased gaze shifts, similar to what we had observed in SC optogenetic stimulation, we next compared the relationships between attempted head rotations and saccades during spontaneous and stimulus-evoked gaze shifts. We first analyzed the relative timing of eye and attempted head movements. Whereas our SC optogenetic stimulation evoked roughly coincident saccades and attempted head movements, previous studies found that on average, spontaneous and visually evoked saccades in freely moving mice are preceded by head rotations, and that spontaneous saccades in head-fixed mice are similarly preceded by slow attempted head rotations (Figure 1; Meyer et al., 2018; Meyer et al., 2020; Michaiel et al., 2020; Payne and Raymond, 2017). Consistent with these observations, we found that most spontaneous saccades were preceded by attempted head rotations in the same direction as the ensuing saccade (Figure 4B, D and E). Interestingly, attempted head rotations during spontaneous saccades appeared biphasic, with a slow phase starting 100–200 ms before saccade onset followed by a fast phase beginning roughly simultaneously with saccade onset (Figure 4B, D and E). This biphasic response closely resembles the eye-head coupling pattern reported in freely moving mice (Meyer et al., 2020).

Figure 4 with 5 supplements see all
Head-eye coupling during spontaneous and touch-evoked gaze shifts.

(A) Mean trajectories of all rightward (solid traces) and leftward (dashed traces) saccades during spontaneous (blue, n = 7146) and ear airpuff-evoked (red, n = 1437) gaze shifts. Means + s.e.m. (smaller than line width). Gray bar indicates average saccade duration. (B) Mean attempted head displacement accompanying rightward (solid traces) and leftward (dashed traces) saccades during spontaneous (blue) and ear airpuff-evoked (red) gaze shifts. (C) Mean velocities of all rightward (solid traces) and leftward (dashed traces) saccades during spontaneous (blue) and ear airpuff-evoked (red) gaze shifts. (D) Mean attempted head movement velocities accompanying rightward (solid traces) and leftward (dashed traces) saccades during spontaneous (blue) and ear airpuff-evoked (red) gaze shifts. (E, F) Timing of attempted head movements relative to saccades during all spontaneous (E) and ear airpuff-evoked (F) gaze shifts. Each row corresponds to a single gaze shift. Darker shades indicate larger attempted head displacement. Purple hues denote attempted displacement in the same direction as the saccade (ipsiversive), and orange hues denote displacement in the opposite direction of the saccade (contraversive). Dashed vertical line indicates time of saccade onset. Trials are sorted by latency of attempted head movements. (G) Fraction of trials with ipsiversive attempted head displacements at different timepoints relative to saccade onset for spontaneous (blue) and evoked (red) saccades. (H). Head-eye amplitude coupling of spontaneous (blue) and ear airpuff-evoked saccades (red). Each dot corresponds to a single gaze shift. Attempted head amplitude was measured 150ms after saccade onset. Spontaneous: R2 = 0.58, slope = 0.214, p < 10–10. Evoked: R2 = 0.64, slope = 0.162, p < 10–10. Spontaneous and evoked regression slopes were significantly different (p < 10–5, permutation test). Histograms above and beside scatter plot indicate distributions of saccade and attempted head movement amplitudes. Difference in means significant (p < 10–5 for saccades, p < 10–5 for head, permutation test). (I–J) As in (E–F), but for attempted head movement velocity. (K) As in (G), but for attempted head movement velocity. (L) As in (H), but for attempted head movement velocity. Peak attempted head velocity was measured 60ms after saccade onset. Spontaneous: R2 = 0.40, slope = 2.04, p < 10–10. Evoked: R2 = 0.52, slope = 1.98, p < 10–10. Spontaneous and evoked regression slopes were not significantly different (p = 0.08, permutation test). Histograms above and beside scatter plot indicate distributions of saccade amplitudes and peak attempted head velocities. Difference in means was significant (p < 10–5 for saccades, p < 10–5 for head, permutation test).

We hypothesized that touch-evoked gaze shifts are mediated by SC and therefore that the relative timing of eye and attempted head movements would more closely resemble those observed in other species and in our SC optogenetic stimulation. Consistent with our prediction, ear airpuff-evoked saccades were typically not preceded by slow attempted head rotations but were accompanied by roughly coincident fast attempted head rotations (Figure 4B, D and F). On average, these fast attempted head rotations had similar peak velocities and latencies relative to saccade onset to those observed from SC optogenetic stimulation as well as those made during the fast phase of head movements during spontaneous gaze shifts (Figure 4B and D). This pattern was mirrored in the average eye and attempted head movement traces for whisker airpuff-, auditory airpuff-, and ear tactile-evoked saccades (Figure 4—figure supplement 1A-D).

To better understand how these patterns arose, we examined eye and attempted head movement timing at the single-trial level. For spontaneous gaze shifts, attempted head movement onset fell along a continuum starting well before saccade onset, with attempted head displacement roughly 60% predictive of the direction of the ensuing saccade starting as early as 200 ms before saccade onset and approximately 80% predictive by 100 ms before saccade onset (Figure 4E and G). In contrast, for stimulus-evoked gaze shifts, the vast majority of attempted head movements began roughly coincidently with saccade onset, and attempted head displacement was not predictive of head position prior to saccade onset (Figure 4F and G). Attempted head velocity showed similar trends (Figure 4I–K). Thus, these data suggest that spontaneous gaze shifts are typically preceded by attempted head movements in the direction of the ensuing saccade, whereas stimulus-evoked gaze shifts are not.

We next examined the amplitudes of head and eye movements during spontaneous and stimulus-evoked gaze shifts. During spontaneous gaze shifts, both the slow and fast phases of attempted head rotations were in the same direction as the ensuing saccades and scaled with saccade amplitude (Figure 4A, B, E, H, K and L). These data are consistent with eye-head coupling patterns previously observed in both head-fixed and freely moving conditions (Meyer et al., 2020). Similarly, stimulus-evoked gaze shifts involved attempted head rotations that were made in the same direction as saccades and scaled with saccade amplitude (Figure 4A, B, F, H, J, K and L). Interestingly, stimulus-evoked saccades of a given amplitude were coupled to an average of 24% smaller attempted head movements than were spontaneous saccades (linear regression slopes 0.162 vs 0.214, p < 10–5, permutation test) (Figure 4H). This difference was due to the slow pre-saccadic attempted head movements observed during spontaneous gaze shifts, as the fast phase of both spontaneous and evoked gaze shifts was nearly identical (Figure 4L). To confirm that the differences in coupling we observed were not an artifact of differences in saccade size and starting position between saccade types, we performed an additional analysis using subsets of gaze shifts matched for saccade amplitude and initial eye position, observing the same effects (Figure 4—figure supplement 2; linear regression slopes 0.162 vs 0.221, p < 10–5, permutation test). To determine whether these differences could be attributed to experience (for example, if animals learn that attempted head movements in response to sensory stimuli are futile) we compared evoked and spontaneous gaze shifts across five sessions and within sessions (Figure 4—figure supplement 3). Interestingly, the gain of head-eye coupling did appear significantly lower for evoked but not spontaneous gaze shifts by the fifth session, but at every time point analyzed, evoked gaze shifts involved smaller head movements than did spontaneous gaze shifts (Figure 4—figure supplement 3). These data indicate that mice may subtly and slowly change their strategies with experience but differences between spontaneous and evoked gaze shifts do not reflect learning.

Directionally biased saccades reflect different stimulus-dependent relationships between initial eye position and saccade direction and amplitude

We next sought to understand how different stimuli evoke saccades with distinct endpoints. Given the prevailing view that head movements drive saccades during mouse gaze shifts, one possibility was that directionally biased saccade endpoints are the result of larger or more directionally biased attempted head movements. Indeed, the distributions of attempted head displacements seemed similar to those of saccade endpoints, suggesting that this was possible, although there was not a clear relationship between average attempted head movement amplitude and saccade endpoint eccentricity (Figure 3A-D, I-L, Figure 4—figure supplement 1A-D). Therefore, to directly test whether different distributions of attempted head movement direction and amplitude across stimuli could explain the distinct saccade endpoints (e.g. with stimuli that evoke larger head movements causing saccades that overshoot to more directionally biased endpoints) we compared trials across stimuli with matched attempted head movement direction and amplitude (Figure 3A, B, I, J, Figure 4—figure supplement 4). If differences in attempted head movements underlie directionally biased saccade endpoints, then controlling for attempted head movement amplitude in this manner should cause whisker and ear airpuff-evoked saccade endpoints to be similar. Strikingly, however, endpoints for attempted head movement-matched whisker and ear airpuff trials remained well-separated, indicating that the more directionally biased endpoints of ear airpuff-evoked saccades are not simply due to larger or more directionally biased attempted head movements (Figure 4—figure supplement 4).

Because saccade endpoints are a function of both saccade amplitude and initial eye position, we next examined whether stimulus-dependent differences in the relationship between initial eye position and saccade direction and amplitude could contribute to resulting endpoint differences. Indeed, for all stimuli there was an inverse relationship between initial eye position and saccade amplitude but the slopes and intercepts of the lines of best fit describing these relationships differed depending on stimulus modality and location (Figure 5A–D, Figure 5—figure supplement 1). For example, stimuli with central saccade endpoints had lines of best fit passing through the origin, whereas stimuli with more leftward or rightward endpoints had lines of best fit that were shifted downward or upward, respectively. In other words, saccades of a given amplitude were typically elicited from distinct initial eye positions by different stimuli: for example, 5° leftward saccades were evoked by left ear airpuffs from central initial eye positions but by left auditory airpuffs from initial eye positions roughly 5° right of center (Figure 5A and D, Figure 5—figure supplement 1). In this way, otherwise identical 5° saccades have central endpoints in response to an auditory airpuff and leftward endpoints in response to a left ear airpuff. Thus, endpoint differences between stimuli arise at least in part from distinct stimulus-dependent relationships between initial eye position and saccade direction and amplitude.

Figure 5 with 4 supplements see all
Saccade and head movement direction, amplitude, and probability depend on initial eye position.

(A–D) Relationship between saccade amplitude and eye position for ear airpuffs (A), whisker airpuffs (B), ear tactile (C), and auditory airpuffs (D). Dotted lines in B-D are lines of best fit from A for comparison. (E–H) Relationship between attempted head displacement and eye position for ear airpuffs (E), whisker airpuffs (F), ear tactile (G), and auditory airpuffs (H). Dotted lines in F-H are lines of best fit from E. The trials in A-H are the same as those in Figure 2. (I–L) Relationship between initial eye position and saccade probability for left and right saccades. (M–P) Relationship between initial eye position and attempted head movement probability for left and right attempted head movements. Green and magenta lines in I-P indicate population means for movements evoked by left and right stimuli, respectively. Blue lines indicate spontaneous saccades or head movements. Error bars indicate s.e.m. Total trial numbers for I-P: ear airpuff sessions, spontaneous = 13,384, left ear airpuff = 3506, right ear airpuff = 3497; whisker airpuff sessions, spontaneous = 14,511, left whisker airpuff = 3926, right whisker airpuff = 4026; tactile ear sessions, spontaneous = 13,529, left tactile ear = 3646, right tactile ear; = 3695; auditory airpuff, spontaneous = 13,404, left auditory airpuff = 6362, right auditory airpuff = 6385.

Having observed that initial eye position and stimulus location jointly shape the direction and amplitude of stimulus-evoked saccades, we next examined whether they had any relationship with saccade probability. Indeed, initial eye position strongly influenced saccade probability, and the relationship between initial eye position and saccade probability differed across stimuli, with the lowest probability coinciding with the mean endpoint of saccades evoked by that stimulus (Figure 5I–L, Figure 5—figure supplement 2). For all stimuli, leftward eye movements were more likely from initial eye positions to the right of the mean saccade endpoint for that stimulus, whereas rightward eye movements were more likely from initial eye positions to the left of the mean saccade endpoint for that stimulus. In contrast, saccade probability did not differ between trials with high (dilated pupils) or low (constricted pupils) arousal (Figure 5—figure supplement 3; Reimer et al., 2014) and declined only slightly over successive sessions or within sessions.

Finally, because the preceding analyses found that saccade direction and amplitude depend on initial eye position and stimulus location (Figure 5A-D,I-L), and because head and eye movement direction and amplitude are highly correlated, we asked whether initial eye position and stimulus location shape the direction and amplitude of evoked head movements. We first analyzed whisker airpuff-evoked gaze shifts because these involved a mixture of saccade directions. Strikingly, attempted head movement direction and amplitude were dependent on initial eye position (Figure 5F, Figure 5—figure supplement 1). We then analyzed auditory airpuff-evoked gaze shifts, which also involved a mixture of saccade directions. As for whisker airpuffs, both attempted head movement direction and amplitude were dependent on initial eye position (Figure 5D, Figure 5—figure supplement 1). We next examined ear tactile stimuli, which elicited mostly contraversive movements but also some ipsiversive movements. Indeed, as for both whisker and auditory airpuffs, attempted head movement direction and amplitude depended on eye position, but the slope of this relationship was shallower (Figure 5G, Figure 5—figure supplement 1). We then examined ear airpuffs. These stimuli elicited nearly exclusively contraversive movements, such that no effect of eye position on direction could be observed, and effects on amplitude were subtle, with shallower slopes, and significant for stimuli only on one side (Figure 5E, Figure 5—figure supplement 1). However, eye position strongly influenced the probability of attempted head movements evoked by all stimuli, including ear airpuffs (Figure 5M–P, Figure 5—figure supplement 1). As a control, we examined the relationship between the initial position of the head, as measured by the strain gauge, and saccade and attempted head movement direction and amplitude. For all stimuli, initial eye position was a much stronger predictor of saccade direction and amplitude than was initial position of the head (Figure 5—figure supplement 4). Likewise, for each of the stimuli for which attempted head movement direction and amplitude were well predicted by initial eye position—whisker airpuffs, ear tactile stimuli, and auditory airpuffs—initial head position was a weak predictor of attempted head movements (Figure 5—figure supplement 4). Taken together, these data indicate that starting eye position and stimulus location jointly shape attempted head and eye movement probability, direction, and amplitude.

The superior colliculus mediates airpuff-evoked gaze shifts

We next sought to identify the neural circuitry underlying airpuff-evoked gaze shifts. As discussed previously, in other species, stimulus-evoked gaze shifts involving directed head and eye movements are driven by SC (Freedman, 2008; Freedman et al., 1996; Guitton, 1992; Guitton et al., 1980; Paré et al., 1994). In contrast, it is widely believed that the recentering saccades observed in mice are driven by brainstem circuitry in response to head rotation (Curthoys, 2002; Hepp et al., 1993; Kitama et al., 1995; Meyer et al., 2020; Michaiel et al., 2020; Payne and Raymond, 2017). To determine whether SC is required to generate touch-evoked gaze shifts in mice, we pursued an optogenetic strategy to perturb SC activity in the period surrounding airpuff onset. For inhibition experiments, we stereotaxically injected adeno-associated virus (AAV) encoding the light-gated chloride pump eNpHR3.0 under the control of a pan-neuronal promoter and implanted a fiber optic in right SC (Gradinaru et al., 2010). Consistent with data in foveate species (Hikosaka and Wurtz, 1985; Robinson, 1972; Schiller and Stryker, 1972), optically reducing right SC activity shifted airpuff-evoked saccade endpoints to the right (i.e. ipsilaterally) for both left (–3.7 ± 4.3° [control] vs. –2.3 ± 4.7° [LED on], p < 0.001, Welch’s t-test) and right ear airpuffs (4.5 ± 4.1° [control] vs. 5.4 ± 4.7° [LED on], p = 0.011, Welch’s t-test) (Figure 6A–C). To control for potential mismatches in starting eye position between LED-off and LED-on trials, we performed additional analyses using matched trials and found that the endpoint and amplitude differences persisted (Figure 6—figure supplement 1). For stimulation experiments, we again stereotaxically injected AAV encoding the light-gated ion channel ChR2 under the control of a pan-neuronal promoter and implanted a fiber optic in right SC (Gradinaru et al., 2010). Because strong SC stimulation evokes saccades, we used weak stimulation (50–120 μW) in order to bias SC activity. This manipulation caused the reciprocal effect of right SC inhibition, biasing endpoints leftwards (i.e. contraversively) for both left (–5.8 ± 4.6° [control] vs. –8.0 ± 6.1° [LED on], p = 0.0016, Welch’s t-test) and right (5.6 ± 3.5° [control] vs. 1.9 ± 6.1° [LED on], p < 10–5, Welch’s t-test) airpuffs (Figure 6E–G). Once again, controlling for differences in starting eye position between conditions yielded similar results (Figure 6—figure supplement 1).

Figure 6 with 1 supplement see all
Superior colliculus controls touch-evoked gaze shifts.

(A) Schematic of right SC optogenetic inhibition using eNpHR3.0 and example histology for representative mouse. Scale bar, 0.5 mm. The lack of fluorescence immediately surrounding fiber tip is due to photobleaching by high photostimulation intensity (12 mW, as opposed to 50–120 μW for ChR2 experiments in (E–H)). (B) Trial structure. Optogenetic illumination is provided for a 1 s period centered around airpuff delivery. (C) Effects of SC optogenetic inhibition on saccade endpoints. Top, scatter plots and histograms of endpoints for control (white background, n = 296) and LED on (orange background, n = 235) trials. Middle, endpoint histograms for control (black) and LED on (orange) trials. Bottom, saccade vectors for control (black) and LED on (orange) trials. (D) Head-eye amplitude coupling during ear airpuff-evoked gaze shifts for control (black) and LED on (orange) trials. Each dot represents an individual gaze shift. Control: R2 = 0.56, slope = 0.123, p < 10–10. LED on: R2 = 0.53, slope = 0.127, p < 10–10. Control and LED on regression slopes were significantly different (p = 0.01, permutation test) due to differences in eye positions from which gaze shifts were generated, because controlling for initial eye position eliminated this difference (Figure 6—figure supplement 1). Histograms above and beside scatter plot show distributions of saccade amplitudes and attempted head displacements, respectively. Distribution means were significantly different (p < 10–5, permutation test). (E) Schematic of right SC optogenetic subthreshold stimulation using ChR2 and example histology for representative mouse. Scale bar, 0.5 mm. (F) Trial structure. Optogenetic illumination is provided for a 1 s period centered around airpuff delivery. (G) Effects of weak SC optogenetic stimulation on saccade endpoints. Top, scatter plots and histograms of endpoints for control (white background, n = 547) and LED on (blue background, n = 157) trials. We observed fewer trials in the LED-on condition because SC stimulation increased the probability of spontaneous saccades prior to stimulus onset, and trials with saccades in the 500ms before stimulus delivery were excluded from analysis. Middle, histograms of endpoints for control (black) and LED on (blue) trials. Bottom saccade vectors for control (black) and LED on (blue) trials. (H) Head-eye amplitude coupling during ear airpuff-evoked gaze shifts for control (black) and LED on (blue) trials. Each dot represents an individual gaze shift. Attempted head amplitude was measured 150ms after saccade onset. Control: R2 = 0.69, slope = 0.137, p < 10–10. LED on: R2 = 0.52, slope = 0.164, p < 10–10. Control and LED-on regression slopes were significantly different (p < 10–5, permutation test) due to difference in eye positions from which gaze shifts were generated, because controlling for initial eye position eliminated this difference (Figure 6—figure supplement 1). Histograms above and beside scatter plot show distributions of saccade amplitudes and attempted head displacements, respectively. Distribution means were significantly different (p < 10–5, permutation test).

To understand how SC manipulations affect attempted head movements and head-eye coupling, we examined the distribution of head movements as a function of saccade amplitude. As expected given the role of SC in generating both head and eye movements, attempted head movements were shifted to the right (i.e. ipsiversively) by SC inhibition (Figure 6D) and to the left (i.e. contraversively) by SC excitation (Figure 6H). To examine the effects of SC manipulations on head-eye coupling, we identified trials with identical saccade trajectories in LED-on and LED-off conditions and examined the corresponding head movement amplitudes. Interestingly, SC manipulations had no effect on the relationship between saccade and head movement amplitudes, suggesting that SC manipulations do not change head-eye coupling during gaze shifts (Figure 6—figure supplement 1). Taken together, these bidirectional manipulations indicate that SC serves a conserved necessary and sufficient role in generating ear airpuff-evoked gaze shifts.

Discussion

Here, we investigated whether mouse gaze shifts are more flexible than had previously been appreciated. In the prevailing view, mouse gaze shifts are led by head rotations that trigger compensatory eye movements, including saccades that function to reset the eyes (Land, 2019; Land and Nilsson, 2012; Liversedge et al., 2011; Meyer et al., 2020; Michaiel et al., 2020; Payne and Raymond, 2017). These ‘recentering’ saccades are attributed to head movement-related vestibular and optokinetic cues (Curthoys, 2002; Meyer et al., 2020; Michaiel et al., 2020; Payne and Raymond, 2017). Working in a head-fixed context to eliminate vestibular and optokinetic cues and to present stimuli of different modalities at precise craniotopic locations, we found that mouse gaze shifts are more flexible than previously thought. As discussed below, we identified unexpected flexibility in the endpoints of saccades, saccade timing and amplitude relative to attempted head movements, the eye positions from which gaze shifts are made, and the brain regions that drive them.

Touch-evoked saccades are directionally biased

The first indication that mouse gaze shifts are more flexible than previously appreciated was that an analysis of endpoints revealed that touch-evoked saccades are directionally biased rather than recentering. This conclusion is based on three lines of evidence. First, endpoints of saccades evoked by left and right ear airpuffs are near the left and right edges, respectively, of the range of eye positions observed and overlap minimally, despite trial-to-trial variability. In contrast, endpoints for saccades evoked by left and right auditory stimuli are indistinguishable and centrally located. Second, left and right ear airpuffs evoke saccades traveling in opposite directions from most eye positions; by definition, one of these directions must lead away from center and is thus centrifugal rather than centripetal. In contrast, saccades evoked by both left and right auditory stimuli travel centripetally from all initial eye positions. Third, from many eye positions, touch-evoked saccades that begin toward the center pass through to reach endpoints at eccentricities between 5 and 10 degrees and cannot accurately be termed centripetal. For these reasons, we conclude that touch-evoked saccades are directionally biased and do not serve to recenter the eyes.

Our findings contrast with and complement previous studies contending that rodents, like other afoveates, use saccades to reset their eyes to more central locations (Meyer et al., 2018; Meyer et al., 2020; Michaiel et al., 2020; Wallace et al., 2013). One recent analysis suggested that gaze shifts made during visually guided prey capture involve resetting centripetal saccades that ‘catch up’ with the head (Michaiel et al., 2020). Another found that saccades away from the nose recenter the eye, whereas saccades toward the nose move the eye slightly beyond center (Meyer et al., 2020). Although we observed this as well, it does not contribute to our results because we averaged the positions of the left and right eyes, eliminating this asymmetry. Earlier studies in head-fixed mice observed occasional, undirected saccades in response to changes in the visual environment (Samonds et al., 2018) and found that mice could be trained to produce visually guided saccades only after weeks of training and at extremely long (~1 s) latencies (Itokazu et al., 2018). To our knowledge, ours is the first study demonstrating innate gaze shifts involving directionally biased saccades in mice (or any species lacking a fovea). An important caveat is that the present studies were conducted in head-fixed animals, and mice may behave differently in a freely moving context due to head movement-related feedback mechanisms.

Touch-evoked saccades do not follow head movements

The prevailing view holds that head movements initiate and determine the amplitude of mouse gaze shifts, with eye movements compensatory by-products. In support of this model, one study found that spontaneous saccades in head-fixed mice are preceded by attempted head rotations. A careful comparison with gaze shifts occurring during visually guided object tracking and social interactions in freely moving mice led the authors to suggest that head-eye coupling is not disrupted during head-fixation, and that gaze shifts in both contexts are head-initiated (Meyer et al., 2020). Another study tracked the eyes and head during visually guided cricket hunting and found that gaze shifts are driven by the head, with the eyes following to stabilize and recenter gaze (Michaiel et al., 2020). Together, these findings have bolstered the prevailing view that afoveates such as mice generate gaze shifts driven by head movements, with eye movements compensatory by-products (Land, 2019; Land and Nilsson, 2012; Liversedge et al., 2011). Consistent with published findings in both freely moving and head-fixed mice, we found that spontaneous saccades are preceded by slow attempted head movements in the direction of the ensuing saccade, typically beginning 100–200 ms before saccade onset (Meyer et al., 2020; Michaiel et al., 2020). In contrast, we found that touch-evoked gaze shifts are not preceded by head movements, suggesting that mouse saccades are not always made in response to head movements. Additional support for this idea came from an analysis of head and eye movements as a function of eye position. If gaze shifts were determined solely by the location of the stimulus relative to the head and saccades were a compensatory by-product of this calculation, eye position should have no effect on head movements. However, we found that the amplitudes, directions, and/or probabilities of stimulus-evoked saccades and attempted head movements vary with initial eye position. The influence of eye position on evoked eye and attempted head movements further suggests that saccades are not simply compensatory by-products of head movements. Instead, we contend, touch-evoked head movements and saccades are likely to be specified simultaneously as parts of a coordinated movement whose component movements take into account both stimulus location and initial eye position. Nevertheless, it is possible that the observed relationship between evoked saccades and attempted head movements is due to the lack of head movement-related feedback in head-fixed mice.

The relative timing of head and eye movements during touch-evoked gaze shifts in head-fixed mice resembles that observed during gaze shifts in cats and primates. For example, head-fixed cats and primates generate gaze shifts using directed saccades and then maintain their eyes in the new orbital position, similar to what we have observed in mice (Freedman, 2008; Guitton et al., 1980). In addition, saccades in head-fixed cats and primates are often accompanied by attempted head rotations, similar to those we observe during touch-evoked gaze shifts in head-fixed mice (Bizzi et al., 1971; Guitton et al., 1984; Paré et al., 1994). In primates and cats able to move their heads, gaze shifts are usually led by directed saccades (with some exceptions), likely because the eyes have lower rotational inertia and can move faster (Pelisson and Guillaume, 2009; Ruhland et al., 2013). These saccades tend to be followed by a head movement in the same direction that creates vestibular signals that drive slow, centripetal counterrotation of the eyes to maintain fixation (Bizzi et al., 1972; Freedman, 2008; Freedman and Sparks, 1997; Guitton et al., 1984). In this way, the animal can rapidly shift its gaze with a directed saccade yet subsequently reset the eyes to a more central position. It is tempting to speculate that a similar coordinated sequence of head and eye movements occurs during touch-evoked gaze shifts in freely moving mice, enabling mice to rapidly shift gaze with their eyes while eventually resetting the eyes in a more central orbital position.

Head-eye amplitude coupling differs during spontaneous and evoked saccades

An additional feature that distinguishes spontaneous and touch-evoked gaze shifts is the relative contributions of head and eye movements. We found that spontaneous saccades of a given amplitude are coupled to larger head movements than are touch-evoked saccades. This difference arises largely from the absence of a pre-saccadic slow attempted head movement during touch-evoked gaze shifts, as the fast phases are similar. This differential pairing of head and eye movements is reminiscent of reports in primates and cats that the relative contributions of head and eye movements vary for gaze shifts evoked by different sensory modalities (Goldring et al., 1996; Populin, 2006; Populin and Rajala, 2011; Populin et al., 2004; Ruhland et al., 2013; Tollin et al., 2005). However, in those species, vision typically elicits gaze shifts dominated by saccades while hearing typically evokes gaze shifts entailing larger contributions from head movements. In contrast, we observed that sound- and touch-evoked gaze shifts involve larger contributions from saccades than do spontaneous gaze shifts, whereas visual stimuli did not evoke gaze shifts at all. This indicates that although there is general conservation of the involvement of SC in sensory-driven gaze shifts, modality-specific features are not conserved, which may reflect differences in sensory processing across species. We also observed limited variability across mice in the relative contributions of head and eye movements to gaze shifts, which contrasts with the observation that different human subjects are head ‘movers’ and ‘non-movers’ during gaze shifts (Figure 4—figure supplement 5) Thus, our results reveal that head-fixed mice are capable of using multiple strategies to shift their gaze but with key differences from other species.

Directionally biased saccade endpoints reflect different stimulus-dependent relationships between initial eye position and saccade direction and amplitude

Given the prevailing view that head movements drive saccades during mouse gaze shifts, we predicted that directionally biased saccades were the result of different head movements. Indeed, distributions of attempted head displacements and saccade endpoints appeared similar for the stimuli tested. However, an analysis of trials matched for head movement direction and amplitude across stimuli yielded well-separated endpoints, suggesting that more eccentric saccade endpoints and the associated distributions of attempted head displacements are not simply due to differences in the directions and amplitudes of evoked head movements. Instead, we found that saccade endpoint differences across stimuli were associated with distinct stimulus-dependent relationships between initial eye position and saccade amplitude and direction. For example, 5° leftward saccades were evoked by left ear airpuffs from central initial eye positions but by left auditory airpuffs from initial eye positions roughly 5° right of center. In this way, saccades with identical amplitudes, coupled to identical attempted head movements, yielded central endpoints in response to an auditory airpuff and eccentric endpoints in response to a left ear airpuff. In the future, it will be essential to determine the neural mechanisms that instantiate these strategies and to examine these strategies in freely moving mice.

The role of superior colliculus in sensory-evoked mouse gaze shifts

In other species, SC drives sensory-evoked gaze shifts, and microstimulation and optogenetic stimulation of mouse SC has been shown to elicit gaze shifts (Masullo et al., 2019; Wang et al., 2015). However, to our knowledge, no study had identified a causal involvement of SC in mouse gaze shifts. We found that optogenetic stimulation of SC elicits directionally biased saccades that coincide with attempted head movements and resembled those elicited by touch. We therefore performed bidirectional optogenetic manipulations that revealed that touch-evoked gaze shifts depend on SC, identifying a conserved, necessary and sufficient role for SC in directed gaze shifts. In addition, we found that SC manipulations did not alter head-eye amplitude coupling. This observation suggests that SC specifies the overall gaze shift amplitude rather than the individual eye or head movement components, consistent with observations in other species (Freedman et al., 1996; Paré et al., 1994).

Ethological significance

Prior to the present study, it was believed that species with high-acuity retinal specializations acquired the ability to make directed saccades to scrutinize salient environmental stimuli, because animals lacking such retinal specializations were thought incapable of gaze shifts led by directed saccades (Land, 2019; Land and Nilsson, 2012; Liversedge et al., 2011; Walls, 1962). Our discovery that sensory-guided directionally biased saccades are present in head-fixed mice—albeit withless precise targeting of stimulus location than is seen in foveate species—raises the question of what fovea-independent functions these movements ordinarily serve. Although mice have lateral eyes and a large field of view, saccades that shift gaze in the direction of a stimulus, as seems to occur with both ear and whisker tactile stimuli, may facilitate keeping salient stimuli within the field of view. As natural stimuli are often multimodal, directing non-visual stimuli toward the center of view maximizes the likelihood of detecting the visual component of the stimulus. Alternatively, despite mouse retinae lacking discrete, anatomically defined specializations such as foveae or areas centralis, there are subtler nonuniformities in the distribution and density of photoreceptors and retinal ganglion cell subtypes, and magnification factor, receptive field sizes, and response tuning vary across the visual field in higher visual centers; it may be desirable to center a salient tactile stimulus on a particular retinal region (Ahmadlou and Heimel, 2015; Baden et al., 2013; van Beest et al., 2021; Bleckert et al., 2014; Dräger and Hubel, 1976; Feinberg and Meister, 2015; Li et al., 2020; de Malmazet et al., 2018). Although touch-evoked saccades alone may be too small to center the stimulus location on any particular region of the retina, they may do so in concert with directed head movements. Experiments in freely moving mice will be essential to understanding the behavioral functions of these saccades.

Why tactile stimuli evoked directionally biased saccades in our preparation whereas auditory and visual stimuli did not is unclear. One possibility is the aforementioned speed of saccades relative to head movements may be especially beneficial because tactile stimuli typically derive from proximal objects and as a result may demand rapid responses. Alternatively, the high spatial acuity of the tactile system may enable more precise localization (Allen and Ison, 2010; Diamond et al., 2008). Auditory stimuli, in contrast, may alert animals to the presence of a salient stimulus in their environments whose location is less precisely ascertained, and as a result drive gaze shifts whose goal is to reset the eyes to a central position that maximizes their chances of sensing and responding appropriately. Finally, our set of stimuli was not exhaustive, and it is possible that as yet unidentified visual or auditory stimuli could elicit gaze shifts with directionally biased saccades.

Future directions

In this study, we used a head-fixed preparation to eliminate the confound of head movement-related sensory cues and to present stimuli from defined locations. However, in the future, it will be critical to compare touch-evoked gaze shifts in head-fixed and freely moving animals. For example, as noted previously, whereas head-fixed primates and cats generate gaze shifts using directed saccades and then maintain their eyes in the new orbital position, similar to what we have observed, in freely moving primates and cats, gaze shifts are led by directed saccades but typically followed by head movements during which the eyes counterrotate centripetally in order to maintain gaze in the new direction. It will be interesting to know whether similar differences distinguish saccade-led touch-evoked gaze shifts in head-fixed and freely moving mice. By expanding on methods similar to those recently described by Meyer et al. and Michaiel et al., it may be possible to investigate these and other questions (Meyer et al., 2018; Meyer et al., 2020; Michaiel et al., 2020).

Furthermore, a practical implication of our identification of mouse SC-dependent gaze shifts is that this behavioral paradigm could be applied to the study of several outstanding questions. First, there are many unresolved problems regarding the circuitry and ensemble dynamics underlying target selection (Basso and May, 2017) and saccade generation (Gandhi and Katnani, 2011), and the mouse provides a genetically tractable platform with which to investigate these and other topics. Second, gaze shifts are aberrant in a host of conditions, such as Parkinson’s and autism spectrum disorder (Liversedge et al., 2011). This paradigm could be a powerful tool for the study of mouse models of a variety of neuropsychiatric conditions. Third, directing saccades toward particular orbital positions during these gaze shifts requires an ability to account for the initial positions of the eyes relative to the target, a phenomenon also known as remapping from sensory to motor reference frames. Neural correlates of this process have been observed in primates (Groh and Sparks, 1996; Jay and Sparks, 1984) and cats (Populin et al., 2004), but the underlying circuitry and computations remain obscure. This behavior may facilitate future studies of this problem. Fourth, the different types of gaze shifts that rely on distinct head-eye coupling we have identified may be useful for understanding mechanisms that control movement coordination. Thus, touch-evoked saccade behavior is likely to be a powerful tool for myriad lines of investigation.

Conclusions

We have found that mouse gaze shifts are unexpectedly flexible, with mice able to make both spontaneous gaze shifts led by the head and stimulus-evoked gaze shifts involving directionally biased saccades coincident with head movements. Prior studies in species whose retinae lack high-acuity specializations had never observed gaze shifts with these properties, but our study used a broader range of stimuli than previously tested and a head-fixed preparation that allowed spatially precise delivery. Detailed perturbation experiments determined that the circuit mechanisms of sensory-evoked gaze shifts are conserved from mice to primates, suggesting that this behavior may have arisen in a common, afoveate ancestral species long ago. More broadly, our findings suggest that analyzing eye movements of other afoveate species thought not to make directed saccades—such as rabbits, toads, and goldfish—in response to a diverse range of multimodal stimuli may uncover similar flexibility.

Materials and methods

Mice

All experiments were performed according to Institutional Animal Care and Use Committee standard procedures. C57BL/6J wild-type (Jackson Laboratory, stock 000664) mice between 2 and 6 months of age were used. Mice were housed in a vivarium with a reversed 12:12 h light:dark cycle and tested during the dark phase. No statistical methods were used to predetermine sample size. Behavioral experiments were not performed blinded as the experimental setup and analyses are automated.

Surgical procedures

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Mice were administered carprofen (5 mg/kg) 30 min prior to surgery. Anesthesia was induced with inhalation of 2.5% isoflurane and buprenorphine (1.5 mg/kg) was administered at the onset of the procedure. Isoflurane (0.5-2.5% in oxygen, 1 L/min) was used to maintain anesthesia and adjusted based on the mouse’s breath and reflexes. For all surgical procedures, the skin was removed from the top of the head and a custom titanium headplate was cemented to the leveled skull (Metabond, Parkell) and further secured with dental cement (Ortho-Jet powder, Lang Dental). Craniotomies were made using a 0.5 mm burr and viral vectors were delivered using pulled glass pipettes coupled to a microsyringe pump (Micro4, World Precision Instruments) on a stereotaxic frame (Model 940, Kopf Instruments). Following surgery, mice were allowed to recover in their home cages for at least 1 week.

Viral injections and implants

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Coordinates for SC injections were ML: 1.1 mm, AP: 0.6 mm (relative to lambda), DV: -1.9 and -2.1 mm (100 nL/depth). Coordinates for SC implants were ML: 1.1 mm, AP: 0.6 mm (relative to lambda), DV: -2.0 mm. Fiber optic cannulae were constructed from ceramic ferrules (CFLC440-10, Thorlabs) and optical fiber (400 mm core, 0.39 NA, FT400UMT) using low-autofluorescence epoxy (F112, Eccobond).

Behavioral procedures

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To characterize stimulus-evoked gaze shifts (Figures 25 and related supplements), data were collected from 5 mice over 53 days (maximum of 1 session/mouse/day). Session types were randomly interleaved to yield a total of 6 ear airpuff sessions, 6 ear tactile sessions, 6 whisker airpuff sessions, 10 auditory airpuff sessions, and 5 visual sessions. During experiments, headplated mice were secured in a custom 3D-printed mouse holder. Timing and synchronization of the behavior were controlled by a microcontroller (Arduino MEGA 2560 Rev3, Arduino) receiving serial commands from custom Matlab scripts. All behavioral and data acquisition timing information was recorded by a NI DAQ (USB-6001) for post hoc alignment. All experiments were performed using awake mice. Left and right stimuli were randomly selected and presented at intervals drawn from a 7-12 s uniform distribution. Each session consisted of 350 stimulus presentations and lasted ~55 min. No training or habituation was necessary.

Stimuli

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Airpuff stimuli were generated using custom 3D-printed airpuff nozzles (1.5 mm wide, 10 mm long) connected to compressed air that was gated by a solenoid. 3D-printed nozzles were used to standardize stimulus alignment across experimental setups but similar results were obtained in preliminary experiments using a diverse array of nozzle designs. For whisker airpuffs, the nozzles were spaced 24 mm apart and centered 10 mm beneath the mouse’s left and right whiskers. For ear airpuffs, the nozzles were directed toward the ears while maintaining 10 mm of separation between the nozzles and the mouse. For auditory-only airpuffs, the nozzles were directed away from the mouse while maintaining the same azimuthal position as the ear airpuffs. Whisker, ear, and auditory airpuffs produced a 65dB noise measured at the mouse’s head. For tactile-only stimulation, the ears were deflected using a thin metal bar coated in epoxy to soften its edges (7122A37, McMaster). A stepper motor (Trinamic, QSH2818-32-07-006 and TMC2208) was programmed to sweep the bar downward against the ear before sweeping back up. The stepper motor was sandwiched between rubber pads (8514K61, McMaster) and elevated on rubber pedestals (20125K73, McMaster) to reduce any sound or tactile stimulation due to vibration. For visual stimulation, white LEDs (COM-00531, Sparkfun) were mounted 6 inches from the mouse at the same azimuthal position as the airpuff nozzles.

Eye tracking

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The movements of both left and right eyes was monitored at 100 Hz using two high-speed cameras (BFS-U3-28S5M-C, Flir) coupled to a 110 mm working distance 0.5X telecentric lens (#67- 303, Edmund Optics). A bandpass filter (FB850-40, Thorlabs) was attached to the lens to block visible illumination. Three IR LEDs (475-1200-ND, DigiKey) were used to illuminate the eye and one was aligned to the camera’s vertical axis to generate a corneal reflection. Videos were processed post hoc using DeepLabCut, a machine learning package for tracking pose with user-defined body parts (Mathis et al., 2018). Data in this paper were analyzed using a network trained on 1000 frames of recorded behavior from 8 mice (125 frames per mouse). The network was trained to detect the left and right edges of the pupil and the left and right edges of the corneal reflection. Frames with a DeepLabCut-calculated likelihood of p < 0.90 were discarded from analyses. Angular eye position (E) was determined using a previously described method developed for C57BL/6J mice (Sakatani and Isa, 2004).

Attempted head rotation tracking

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Attempted head rotations were measured using a 3D-printed custom headplate holder coupled to a load cell force sensor (Sparkfun, SEN-14727). Load cell measurements (sampling frequency 80 Hz) were converted to analog signals and recorded using a NI DAQ (sampling frequency 2000 Hz). The data were then low-pass filtered at 80 Hz using a zero-phase second-order Butterworth filter and then upsampled to match the pupil sampling rate.

Optogenetics

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Optogenetic experiments were performed using the ear airpuff nozzles. Fiber optic cables were coupled to implanted fibers and the junction was shielded with black heat shrink. A 470 nm fiber-coupled LED (M470F3, Thorlabs) was used to excite ChR2-expressing neurons, and a 545 nm fiber-coupled LED (UHP-T-SR, Prizmatix) was used to inhibit eNpHR3.0-expressing neurons. Optogenetic excitation was delivered on a random 50% of trials using 1 s of illumination centered around airpuff onset.

SC inhibition

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For optogenetic inactivation of SC neurons, AAV1.hSyn.eNpHR3.0 was injected into the right SC of 5 wild-type mice (0.6 AP, 1.1 ML, -2.1 and -1.9 DV; 100 nl per depth). Experiments were performed 35-40 days post injection. LED power was 12 mW. Mice underwent five sessions each.

SC stimulation

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To examine optogenetically-evoked gaze shifts in Figure 1, AAV1.CaMKIIa.hChR2(H134R)-EYFP was injected into the right SC of four wild-type mice. Experiments were performed 60-65 days post injection. For each mouse, SC was stimulated (1mW) for 40-ms every 7-12s for 350 trials.

For subthreshold optogenetic stimulation of SC neurons, AAV1.CaMKIIa.hChR2(H134R)-EYFP was injected into the right SC of four wild-type mice. Experiments were performed 67-71 days post injection. LED power was individually set to an intensity that did not consistently evoke saccades upon LED onset (50-120uW). Mice underwent five sessions each.

Histology

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For histological confirmation of fiber placement and injection site, mice were perfused with PBS followed by 4% PFA. Brains were removed and post-fixed overnight in 4% PFA, and stored in 20% sucrose solution for at least 1 day. Brains were sectioned at 50 μm thickness using a cryostat (NX70, Cryostar), every third section was mounted, and slides were cover-slipped using DAPI mounting medium (Southern Biotech). Tile scans were acquired using a confocal microscope (LSM700, Zeiss) coupled to a 10X air objective.

Behavioral analysis

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Eye position analyses were performed using the averaged left and right pupil positions, and the mean eye position was subtracted from each session prior to combining data across sessions and mice. Similarly, because mice generate different ranges of raw strain gauge measurements when making attempted head rotations, attempted head rotation data was Z-scored prior to combining data across sessions and mice. Saccades were defined as eye movements that exceeded 100°/s, were at least 3° in amplitude, and were not preceded by a saccade in the previous 100 ms. The initial positions and endpoints of saccades were defined as the first points at which saccade velocity rose above 30°/s and fell below 20°/s, respectively. Analyses focused on horizontal saccades because saccades were strongly confined to the azimuthal axis (Figure 2—figure supplement 1). For all subsequent analyses, saccades were defined as being evoked by a sensory or optogenetic stimulus if they occurred within a 100 ms response window following stimulus delivery, selected due to the sharp increase in saccade probability during this period (Figure 2—figure supplement 2). Spontaneous saccades were a catch-all category defined as any saccades made outside of an experimental stimulus (i.e. no stimulus in the 500 ms periods preceding or following the saccade).

To examine stimulation-evoked gaze shifts (Figure 1), only trials in which the head and eyes were fixated in the 500ms period preceding LED-onset and in which the eyes began in a central orbital position (-2o to 0o) were used for analysis. Head movement amplitude during gaze shifts was defined as the head sensor reading 150ms following saccade onset. Head movement onset was defined as the point relative to LED-onset at which head displacement exceeded 5 standard deviations from baseline (~0.05-0.1Z).

To quantify stimulus-evoked saccade probability (Figure 2), we calculated the fraction of trials in which a saccade occurred in the 100 ms period following stimulus onset (i.e. the response window). To quantify stimulus-evoked attempted head movement probability, we calculated the fraction of trials in which the head sensor reading exceeded 0.25Z at the point 150 ms following stimulus onset. To determine the baseline head movement probability, we calculated the fraction of trials in which the head sensor reading exceeded 0.25Z between -500 ms and -350 ms relative to stimulus onset.

To examine saccade endpoints, we first identified trials in which mice maintained fixation in the 500 ms preceding saccade onset. We then considered stimulus-evoked saccades those occurring within 100 ms of stimulus onset. To examine the amplitudes of attempted head movements accompanying stimulus-evoked saccades, we used the head sensor reading 150 ms following saccade onset.

Heatmaps of single-trial head movements (Figure 4 and accompanying supplements) were sorted according to head movement latency. To calculate attempted head movement latencies, we first identified trials in which the mice maintained head fixation from -1 to -0.5 s prior to saccade onset and used this period as the baseline. Latency was defined as the first frame between -0.5 and 0.5 s relative to saccade onset when the attempted head movement amplitude exceeded 5 standard deviations from that trial’s baseline (~0.05-0.1Z).

To examine the timing of attempted head movements relative to saccade onset (Figure 4), we first baseline subtracted attempted head movement traces 500 ms before saccade onset for each trial. For each time point between -500 ms and 500 ms surrounding saccade onset, we calculated the fraction of trials with instantaneous attempted head movements that were ipsiversive to their accompanying saccades.

To examine head-eye amplitude coupling during spontaneous and stimulus-evoked gaze shifts, we defined attempted head rotation displacement as the load cell value 150 ms following saccade onset (the time point at which average load cell value plateaus during stimulus-evoked gaze shifts (Figure 4)). For certain analyses, we identified saccades matched (without replacement) for initial eye position and/or saccade amplitude using Euclidean distance as a metric and a 3° distance cutoff. For a subset of analyses, we used attempted head movement velocity which was measured 60 ms after saccade onset (the time point when average load cell velocity peaks).

To examine the relationship between initial eye position and stimulus-evoked saccade or head movement probability, we identified trials in which mice did not saccade in the 500 ms preceding stimulus onset. Left and right head movements were defined as those less than -0.25Z or greater than 0.25Z, respectively. Qualitatively similar results were obtained using thresholds ranging from 0.1Z to 2Z. To examine the relationship between initial eye position and spontaneous saccade or head movement probability, we identified 1 s long time periods in which no stimuli were delivered and in which mice did not saccade in the first 500 ms. We then determined the probability of a saccade between 500 and 600 ms, and the probability of a head movement using the head sensor value at 650ms.

Tests for statistical significance are described in the text and figure legends. Data were shuffled 10,000 times to generate a null distribution for permutation tests.

Data availability

Annotated data have been uploaded to a Dryad repository (https://doi.org/10.7272/Q6V69GTV).

The following data sets were generated
    1. Zahler SH
    2. Taylor DE
    3. Adams J
    4. Feinberg EH
    5. Wong J
    (2021) Dryad Digital Repository
    Superior colliculus drives stimulus-evoked directionally biased saccades and attempted head movements in head-fixed mice.
    https://doi.org/10.7272/dryad.Q6V69GTV

References

Decision letter

  1. Martin Vinck
    Reviewing Editor; Ernst Strüngmann Institute (ESI) for Neuroscience in Cooperation with Max Planck Society, Germany
  2. Tirin Moore
    Senior Editor; Stanford University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Mice Make Targeted Saccades" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Comments to the Authors:

We are sorry to say that, after consultation with the reviewers, we have decided that your work will not be considered further for publication by eLife. While the reviewers found your paper provocative and addressing an important question, they found that the experiments did not support and justify the main conclusions.

Reviewer #1:

Zahler, Taylor et al. investigate the role of rapid eye movements (saccades) in mice. Recent studies in head-fixed and freely-moving mice did not find evidence for saccades directed at salient visual stimuli, even during goal-directed behaviors relying on vision. Rather, saccades appear to be part of a "saccade and fixate" gaze pattern: during the "fixate" period the eyes are counter-rotating relative to the head to keep the image of the external world on the retinas stable. The "saccade" period seems to "recenter" the eyes in the orbits via saccadic eye movements (by shifting gaze together with the head). These studies support the idea that mice (and potentially most other afoveate animals) do not make targeted saccades.

To challenge this view, Zahler, Taylor et al. developed a paradigm in passive, head-fixed mice in which the visual stimulus used in previous studies was replaced by a combination of tactile and auditory stimuli. In the first experiment, an airpuff to the left or right whiskers was paired with a loud sound coming from the same side as the airpuff. In a small subset of stimulus presentations, mice made saccadic eye movements in response to the stimulus. The authors termed this behavior "whisker-induced saccade-like reflex" (WISLR).

Using the WISLR task, the authors found that saccade endpoints depended on the type of stimulus (e.g., ear airpuff or whisker deflection). Optogenetic deactivation or activation of the superior colliculus (SC), a brain region that plays a key role in controlling head and eye movements, shifted saccade endpoints in the WISLR task. Finally, the authors use simulations to test the idea that WISLR-related saccades keep stimuli within the visual field.

While I think that the authors are addressing an important and timely question, I have some concerns about their conclusions. In particular, most observations seem to be consistent with the "resetting model" that the authors seek to disprove. However, I would like to emphasize that providing evidence either for or against the resetting model would be extremely valuable to the scientific community. The mouse is becoming an increasingly important species in vision research and a major "model" of human disease. Thus it would be important to know which aspects might translate to human research (and which don't).

Major concerns:

1. The paper is quite inconsistent when it comes to the stimuli evoking saccades. The authors state that the main motivation for developing the WISLR task was the absence of targeted saccades towards salient visual stimuli in previous studies. However, the conclusion based on their final simulation (Figure 4F-H) is that the function of saccades in response to tactile/auditory stimuli is to keep visual stimuli in the animal's visual field of view. How do these two things fit together? How are airpuff-evoked saccades related to a visual stimulus leaving the quite large visual field extending to the front and the sides of the animal as assumed in the modeling work? Currently, the relation between the stimuli in Figure 2 that evoked pronounced saccades (e.g., ear airpuff and whisker bar) and the animal's visual field is not clear. In particular, to reconcile the auditory/tactile observations with the simulations it will be important to know whether the tactile/auditory stimuli were within or outside the visual field.

– There is a large body of literature on gaze shifts towards visual, tactile and auditory stimuli in humans and non-human primates. A common observation in these studies is that gaze shifts towards visual targets show a stronger saccade component than gaze shifts towards auditory targets which rely more strongly on head movements. This seems to be somewhat orthogonal to the idea that tactile/auditory but not visual stimuli should result in targeted saccades, in particular if circuits are conserved across species as stated by the authors. It would be important to discuss the motivation/findings in the context of the existing literature which is largely ignored in the current paper.

– The authors show that average saccade trajectories converge on nearly the same endpoint (close to 0 deg) regardless of initial eye position (Figure 1C-E). Would that not support the idea of resetting saccades that move the eyes back to a more central location? There is a statistically significant, yet very small difference in endpoints (< 5 deg) between the left/right stimulus conditions. This seems to be the only evidence for "targeted saccades". However, studies in head-fixed (Sakatani and Isa, 2007; Itokazu et al., 2018) and freely-moving (Meyer et al., 2020) mice have demonstrated that there is a systematic asymmetry (about 5 deg) in the sizes of nasal vs temporal saccades. It would be important to check that this asymmetry cannot explain the difference in saccade endpoints.

– The authors show in Suppl Figure 4 that mice attempted to reorient their heads during saccades and that the magnitude of attempted head movements is predictive of saccade sizes. This indicates that saccades in the WISLR task were part of combined eye/head gaze shifts as reported in freely moving mice (Michaiel et al., 2020; Meyer et al., 2020). Consequently, attempted head movements need to be taken into account when interpreting the data. Indeed, there seems to be a difference in attempted head movements between the two conditions shown in Suppl Figure 4B,C (spontaneous, WISLR). Based on these data, it is not clear if "targeted saccades" were just a by-product of head-free gaze patterns in head-fixed animals (given the low probability of observing a saccade at all).

– The shift in saccade endpoints during optical activation/deactivation of the superior colliculus (SC) is a novel and interesting finding. However, the SC is also involved in generating head movements in non-human primates (e.g., Freedman et al. 1996) and mice (e.g., Wilson et al. (2018)). In particular, Masullo et al. (2019) show that SC stimulation generates both head and eye movements. Again, it would be important to dissociate the contribution of (attempted) head movements to the measured saccades to support the idea that eye movements, and not the associated head movements, determine saccade endpoints.

– The authors use modeling to test the idea that the role of "targeted saccades" is to keep visual targets in the animal's visual field. Why would the U-shaped curve in Figure 4D not be consistent with a resetting saccade model in which the probability of observing a saccade increases with angular distance from the eye's central position? The small differences in trough locations might be related to the asymmetry of nasal vs temporal eye movements (see comment above).

– The probability of observing a saccade following a non-visual stimulus seems to be quite low (Figure 1B and Suppl Figure 5) and saccade sizes are rather small (~5-15 deg, Suppl Figure 2A) compared to the large visual field of the mouse (>250 deg). Even if mice made targeted saccades: their function in the context of natural behavior is not discussed in the paper.

A major concern is that "targeted saccades" are just a by-product of combined eye/head gaze shifts observed in freely moving mice (Michaiel et al., 2020; Meyer et al., 2020). The authors measured attempted head orienting movements in a subset of experiments, and the data strongly indicate that eye/head coupling is preserved in the WISLR task. To test if differences in saccade end points can be explained by head orienting movements, the authors could measure attempted head movements during the different conditions in Figure 2. This could be combined with a regression model to test if saccade end points can be predicted from attempted head movement amplitude.

In the method section it is mentioned that only saccades occurring within a specific response window following stimulus onset were included in the analysis (p 23). First, it might be useful to explain the choise of the 150 ms response window and the impact of response window length on the results. For example, Itokazu et al. (2018) report a much longer response window for visual stimuli. Second, it might be useful to indicate this response window in Suppl Figure 1A. I would expect that based on the criteria mentioned in the methods section the 2nd saccade in Suppl Figure 1A was excluded from the analysis. Is that correct?

Related to this: Figure 1B shows the probability of observing a saccade in different time bins following an airpuff. Maybe I missed this but what was the overall probablity of observing a saccade within the response time window? To get a sense of the reliability of saccade generation, it might be useful to also show cumulative probability (either in the same figure or in a suppl figure). It is hard to guess from the distribution but I would guess that p(saccade in response window) < 0.2.

Reviewer #2:

This study makes a bold and provocative claim – mice make targeted eye saccades. This is a significant claim, since although many studies have shown that species without fovea perform saccades, these are generally coupled to head movements, and serve to shift gaze by recentering the eyes following slower compensatory eye movements. Indeed, recent studies have shown that eye movements in mice follow this pattern, and in fact even the eye movements seen in head-fixed mice are associated with attempted head movements. This study claims that head-fixed mice do make targeted eye saccades, when triggered by tactile stimulation to the ears/whiskers. If it is true that mice make saccades similar to foveate animals, and this had just been missed because previous researchers had not used tactile stimuli, this would be an exciting finding not only for mouse vision, but for the vision field at large.

However, there are two major issues with the claim that mice are making targeted eye saccades. First, the saccades do not appear to be targeted directly to a stimulus location – rather, they return to center with a slight bias of a couple degrees for saccades "to" stimuli at disparate locations, and in fact there is great variability in saccade endpoints (larger than the difference between target locations). Second, it is very possible that the results can all be explained by attempted head movements, consistent with existing findings on mouse eye movements. Thus the findings as presented do not overturn the current thinking, nor suggest that mice are making targeted eye saccades similar to humans.

1. Targeting of eye movements

– The eye movements in response to a left airpuff (Figure 1C) look exactly like one would expect for re-centering saccades – from all initial locations, the eye returns to center. It is only in comparing this to the right airpuff that a 2deg, on average, shift appears. Thus, there is only a +/-1 deg difference for saccades to stimuli on opposite sides of the body. If these saccades are targeting a stimulus, where is it such that left/right are only 2 deg apart? Even for a puff to the ears (Figure 2G) there is at most a 5 deg displacement of target location from central eye position, for a stimulus that is far lateral to the eyes. A much more straightforward description is that these are recentering movements, with a slight bias toward the stimulus side.

– It seems that the authors are assuming that the stimulus location is at the mean endpoint, but that would imply that they had centered the whisker puff stimulus within 2 deg of the center of the visual field for each animal (unlikely), and that the ear puff stimulus is located within 5 deg of the center of the visual field (certainly not true).

– Furthermore, rather than being targeted to a specific location, wherever that happens to correspond to, the saccade endpoints appear highly variable. Indeed, the variability across mean end position based on starting position is similar to the difference due to stimulus location (Figure 2E). The actual variability is probably much greater, as this is masked by averaging a very large number of saccades in figures such as 2C. This averaging makes it appear that all saccades go to the same location, and makes it hard to estimate variability (except to the extent that the error bars after saccade are much larger than for the 5 deg bins pre-saccade). It is important to show the true variability in endpoint position, for example, as a histogram of end positions for left vs right stim. It would also be valuable to see a number of overlaid raw traces for Figures such as 2C,D, to show the true variability before presenting the means.

– The fact that saccade amplitude is equal and opposite to initial eye displacement (negative linear relationship in 1F) is exactly what one would expect for re-centering saccades as well.

2. Confound of head movements/fixation

Previous studies have shown that eye movements are highly disrupted in head-fixed mice, and in fact most eye movements are coupled to attempted head movements. Critically, a previous study (Meyer et al., 2020), and even supplementary data here, show that in head-fixed mice, attempted head movement results in eye saccades in the same direction and proportional in amplitude of the head movement, as one would see in a free moving animal and consistent with the standard gaze-shifting reset model. If the tactile stimulus is evoking attempted head movements (likely) and these are biased in one direction, then that would explain the offset in resulting eye movements. In that case, the reason this study found tactile evoked eye movements is that tactile stimuli evoke head movements, not because they are saccading to the tactile stimulus.

The ideal solution to this would be to perform these experiments in freely moving animals and show that they move their eyes toward the stimulus independent of the head. However, barring that there are a number of key questions that would need to be answered to resolve this issue, potentially based on load cell measurements.

– What is the pattern of head movements resulting from the stimuli? Do stimuli from opposite sides evoke different directions of head movements on average? If so, the bias in head movements could explain the resulting bias in eye movement.

– Do the different types of stimuli (ear, whisker, auditory) evoke different directions/amplitudes of head movement? It could be that ear puff stimulation causes larger eye shifts than whisker not because it is more peripheral, but because it evokes a larger movement. Likewise, auditory stimulation alone may elicit smaller movements or less directionally-biased movements.

– Do the optogenetic manipulations affect head movement as well? This seems likely, since studies in multiple species have shown head movements evoked by SC stimulation or impaired by SC lesion. If so, the shift in eye movements in Figure 3 could be explained by the corresponding change in head movements.

– Finally, which does a better job of predicting the endpoint – head movement or stimulus side? A figure such as 1C-E, but broken up by head movement direction, rather than stimulus side, would test this. Indeed, it would be interesting to see spontaneous eye saccades broken up in this way, to see if there are "targeted" eye movements in the absence of a target.

Additional point

1. Modeling the consequences of eye movements. The authors note that the probability of a saccade increases as a U-shaped curve relative to center position (Figure 4D,E). Indeed, this is exactly what one would expect for recentering saccades – the further the pupil is from center, the more likely it is to reset. However, the authors perform modeling to suggest that instead this represents the goal of keeping the target in the visual field. It's not clear how the modeling supports their claim as presented. The results of the model show that if a stimulus is near the edge of the visual field, then it is more likely to move out of the visual field based on a random movement. Isn't that almost trivially true? It would provide stronger support for their claim if this was quantitative, using physically meaningful values. Specifically, eye position relative to target in 4E varies by roughly +/-10 deg in the data, out of a field of view of 140deg for the mouse. However, this is drastically different than the cartoon shown in 4F, and it doesn't seem that quantitatively a 10deg displacement is going to greatly increase the probability of moving out of a 140deg field of view to the extent shown in Figures4G,H.

Recommendations for the authors

1. The authors state the endpoint should be "strongly dependent" on the stimulus position (p. 3), but highly variable targeting with mean 2 deg shift based on stimulus position does not seem to meet that criteria. If the saccades are indeed targeting a stimulus location, they should be able to systematically vary the stimulus positioning (for example from -30 to 30 deg in the visual field) and show that the targeting eye movements follow.

2. Unless the authors can demonstrate that there is a specific stimulus location that the saccades are targeting, and can explain why they are so highly variable, then the claim that they are making primate-like saccades is not valid. Something like "directionally biased recentering saccades" seems more accurate. But this does not overturn textbook models as they suggest.

3. Throughout the text, there is a notable lack of numerical data for population summary statistics, which makes it difficult to accurately assess the findings. For example, the mean value for left and right endpoints is never provided in the text or figure legends, only the p-value for the comparison (e.g. p 4, line 6). This is not just a style point – it is essential to convey the magnitude of effects observed, as nowhere in the text do the authors state the size of the targeting difference, which is only on the order of a few degrees. In addition, presenting N for number of datapoints (saccades) rather than just number of animals would be valuable.

4. The impact statement, that a "hallmark of human vision" is conserved in mice, seems overstated. In addition to the fact that these saccades don't resemble human saccades, targeted eye movements are not just a hallmark of human vision but many species across the animal kingdom that have retinal specializations.

Reviewer #3:

The submitted article titled "Mice Make Targeted Saccades" by Zahler et al. presents some interesting eye movement data that suggests that mice reorient their visual field toward an object of interest. I think the data are thought-provoking and the authors have presented a careful, thorough systematic description of the behavior. My enthusiasm though is tempered for a couple reasons:

First, no vision is really involved in this paradigm so it is hard to be convinced this demonstrates the mice are actually reorienting their visual field. Meyer et al. (2020), as well as the authors, demonstrate that saccadic eye movements coincide with attempted head movements. The mice only made saccades when the mice were "touched" on their head (whiskers or ears). It is not surprising that the mice would attempt to move their heads with a potential threat like that. Therefore, the eye movements might be simply a consequence of the attempted head movement whether or not it is related to a shift in gaze. It is still possible that mice were shifting (or attempting to shift) their visual field, but I would be more convinced if this was demonstrated for targets represented across other senses, especially vision. It might be that more consistent shifts require stimuli farther out in the periphery (remember that mice have a very large visual field due their laterally oriented eyes). In fact, the authors do nicely show this with the U-shaped function in Figure 4. It might be that more conditions are required more peripherally to trigger higher probabilities of saccades and from stimuli represented by sound and vision.

Second, Michaiel et al. (2020) have demonstrated that mice shift their gaze towards an object of interest and this was based on a visual stimulus (cricket) so it should be possible to demonstrate targeted saccades systematically based on vision. The separation of head and eye movement shifts in gaze is not that important of a distinction as nearly all animals combine head and eye movements in some manner to shift gaze, and the superior colliculus jointly represents these saccadic movements.

Suggestions for authors:

I am surprised there was no eye movement triggered towards the auditory stimulus as I have seen the eyes shift towards sounds during experiments. I have also seen eye movements towards tactile stimuli farther back on the body so I do think it is possible to explore this beyond the ears.

Figure 1. I think the data are clear, but it is important to present it fully so that readers understand the similarities *and* the differences with primate behavior. The current presentation is misleading to make it appear more similar to primate behavior than it is in reality.

Figure 1. C,D. It would be helpful to see actual individual saccades plotted here rather than averages (not just the supplemental figure). As plotted, I think this is a little misleading about the precision of the targeted saccades for mice.

Figure 1E. The rightward bias should be explained. The bias probably arises because you are mostly monitoring left eye position. Saccades in mice are on average convergent so the left eye would move more rightward (nasal) than leftward (temporal) (Itokazu et al. 2018; Meyer et al. 2020).

Figure 1B. What is the aggregate probability of a saccade if you integrate over a ~400 ms window after the airpuff? The binning is not clear, but the number appears to be pretty small. For a primate, I would assume this to be close to or at 100%. I assume these are represented in Figure 4.

Figure 1F. Again, I think averages are misleading. Why not just plot all of the data points?

Figure 1G, H. Was regression performed on all data points or just the means? It would be more appropriate to use all of the individual data points.

Figure 2. It would be nice to see airpuff position plotted versus saccade endpoint for each mouse to see if the shift in gaze consistently and systematically follows the position of the airpuff.

Figure 4. Again, would stimuli closer to the edge of visual field result in P approaching 1? Demonstrating this U function for auditory and visual stimuli would be much more convincing to the overall conclusion.

Figure 4. It might be nice to see what this U function would look like for a primate. Also, with more positions, could we predict if the mouse were shifting gaze based on the entire visual field or some restricted specialized region? How does the primate function relate to the fovea size? That might provide some insight about the mouse.

Discussion. When talking about a specialized region of the retina, you should include how the representation of wavelength and binocularity vary across the visual field. The data in Wallace et al. (2011) suggest that mice shift gaze to try to keep the central upper part of their visual field in front of them.

Methods. How often were mice excluded for not making enough saccades?

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "A new type of mouse gaze shift is led by directed saccades" for further consideration by eLife. Your revised article has been evaluated by Tirin Moore (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below. As you can see, the reviewers see merit in the study, however remain to have major concerns about several of the analyses and interpretations of the analyses and data. This can be partially fixed by carefully interpreting the data in light of the reviewer's comments (who agree with each other on these points) and by reanalyses and experiments. The manuscript should be thoroughly revised to address these concerns that all reviewers agree on.

1) The claim of a 'new' type of eye movement is fairly strong, particularly given that they share many features with standard reset saccades, as well as the fact that this is being demonstrated under the artifact of head fixation. It'd be better to have a less loaded statement, such as "Touch-evoked eye saccade endpoints are biased toward the direction of head motion in head-fixed mice". In any case, the title should certainly include "in head-fixed mice". An alternative suggestion is "Stimulus evoked rapid eye and attempted head movements in the mouse modulated by the superior colliculus".

2) The authors should clarify that *previous studies* have shown that microstimulation in mouse SC produces head and eye movements that are roughly matched to a visual map. What would resolve the major issue that is raised in the introduction of this submission is a direct measurement of combined head and eye movements in mice following SC microstimulation.

3) Short of that, Figure 3 and 4 is the best evidence to support that there is a change in gaze in mice that is consistent with SC eye-head saccades in non-human primates. A major concern with Figure 3 is that it is dependent on how attempted head movements are measured. If there is any sort of threshold in detectable attempted movements in the force meter, weaker/smaller attempted head movements might show an artificial delayed onset. If spontaneous and evoked saccades are genuinely different, then there should be a detectable difference in latency statistics for *matched magnitudes* (not z-scores) of attempted head movements in these two cases. It is possible that spontaneous and evoked saccades are not different, but eye saccades are still genuinely preceding head movements in some cases. Indeed, 30% of spontaneous saccades look just like the evoked saccades with the eye preceding the head. It could be that there is a continuum of gaze changes where smaller/quicker changes have the eye saccade precede the head movement and for larger changes in gaze, the head precedes a resetting of the eye. In that case, it would be important to see it demonstrated in a head-free scenario and/or how it directly relates to SC microstimulation. Figure 2E and 2I is helpful to the case on its own though that the eye movements are not just resetting and maybe could help resolve some of these questions. If the difference holds up in the matched data though, then it would be pretty convincing theses evoked saccades are unique. In that case, maybe the mouse makes faster changes in gaze for more immediate threats, which would definitely be the case for tactile stimulation.

4) For Figure 4, we recommend to see what happens for optogenetic stimulation during spontaneous saccades as well to see if they are driven by circuitry different than SC. it may be difficult to accomplish this with the same experimental timing structure, but it would still be valuable to have the light on for an extended period for some spontaneous saccades and off for other spontaneous saccades. This might not be that beneficial if any of the suggested Figure 3 analysis suggests there is no practical difference between evoked and spontaneous saccades and only a shift in distributions.

5) It is unclear if the data in the end supports the strong claims of a new type of gaze shift that a) is directed toward the stimulus and b) precedes head movement. In the end, these look much like previously described reset saccades, but with an overshoot that is dependent on head movement amplitude. This by itself could be interesting, as it's possible that this represents what happens in an abrupt head movement from rest – a condition that occurs reliably for airpuff stimuli in a head-fixed mouse, and may be present in freely-moving mice albeit less frequently (and hence not seen in previous studies). This would resemble rapid head movements seen in other species, where the eyes "boost" the saccade by moving more rapidly than the head. However, the fact that these are so clearly coupled to head movement in the new data makes it even more important than before to show that this "new" type of movement is present in freely moving mice, rather than trying to interpret attempted head movements in a head-fixed mouse.

6) Although the authors now acknowledge that these are not independent eye movements, but coupled to head movements, they continue to make the association between stimulus location and eye movement. For example, they plot eye movement vs stimulus position, but do not show the equivalent head movements vs stimulus position. They also do not test whether spontaneous saccades, which have zero mean offset, are actually "directed" if they are broken up according to head movement. This was requested in the previous reviews – "What is the pattern of head movements resulting from the stimuli? Do stimuli from opposite sides evoke different directions of head movement on average?" and "Which does a better job of predicting the endpoint – head movement or stimulus side? A figure such as 1C-E, but broken up by head movement direction, rather than stimulus side, would test this. Indeed, it would be interesting to see spontaneous saccades broken up in this way". However, no data is presented in the manuscript to address this. In the review response, the authors describe a regression model but this data is not presented in the text, and it's not clear what the regression is based on – direction of head movement, head displacement during the 0.5 secs before the saccade?

In order to clarify this, it is essential that they show figures such as 2A-F, but broken up by head movement direction rather than stimulus side. This includes breaking up the spontaneous saccades, based on direction of movement during the saccade. Another direct comparison would be side-by-side scatter plots of saccade endpoint vs stimulus side, and saccade endpoint vs head movement direction (and one could do R2 on these plots, rather than a separate regression model across conditions). it can be expected that these will show that head movement predicts the eye movements better than stimulus side does, particularly in cases such as whisker puff. If so, the eye movements are not truly sensory guided or directed toward the stimulus, but are accompanying head movements evoked by getting airpuffed or whiskers touched. Likewise, breaking up spontaneous saccades based on direction of movement will likely show that they are "directed" even in the absence of sensory input.

7) A major claim is that the evoked saccades precede head movements, whereas for spontaneous the head movements precede saccades. This claim is problematic for several reasons. A) For spontaneous, the authors are including movement over the previous 0.5secs, which are slow movements and therefore likely unrelated to the saccade. Saying that movement 0.5 secs before a saccade is "head movements preceding the saccade" is very misleading. B) For both spontaneous and evoked, it's clear that there is a fast movement that occurs right around the time of the saccade. Indeed, the plots of velocity (Figure 3D) show almost identical peaks for evoked and spontaneous, suggesting that these represent the same eye-head coupling, just that evoked saccades do not have movement previous to the stimulus (not surprising, since mice don't know when the stimulus will occur). C) In the plots of velocity, it is clear that both eye and head velocity both rapidly increase right at stimulus onset. Thus, although the peak of head movement is later than eye (presumably due to physical/motor constraints) the onset of the two is nearly simultaneous. Indeed, Figures3E,F would be much more straightforward if presented in terms of head velocity, rather than displacement. Furthermore, calculating latency relative to a threshold on velocity (rather than position), or time of peak head velocity, would likely reveal much closer coupling. Together, these factors make the argument about eye saccades following vs leading very weak. It also points out the challenge in interpreting head movements in a head-fixed animal.

8) The authors clearly demonstrate that there is a higher probability of eye saccade when the eye is initially offset in the opposite direction (Figure 2I-J). This is exactly what one would expect for a reset saccade resulting from head movement, as the eye is reaching the end of its range in that direction. Likewise, they show that amplitude of the eye movement is equal and opposite to initial eye position (Supp Figure 8), again exactly what one would expect for a reset saccade.

9) The endpoint offset relative to center seems to be directly proportional to the head movement amplitude (Supp Figure 6A-D). Combined with the previous point, this suggests that these are similar to previously described saccades, but with an overshoot that is determined by head movement amplitude (rather than stimulus location). Such a mechanism makes sense, since for large head movements this would allow greater dynamic range for the ensuing compensatory phase. Alternately, it could be that this overshoot occurs in head-fixed mice where other feedback mechanisms are lacking.

10) The authors make the claim that head movements depend on eye movement, which would be quite a remarkable finding. However, the actual data is buried in the supplement, and turns out to be a very weak correlation, driven by a small number of outliers with unusually large head movements. Furthermore, only one stimulus condition is presented for this analysis, suggesting that it was not significant for other conditions. Finally, it is easy to envision situations where a weak correlation could arise without a causal role of eye position. For example, if the mouse simply alternates between left and right eye movements on each trial – after a left head movement, the eye would tend to be on the left, so on the subsequent trial when it makes a head movement to the right it would appear to be driven by eye position, even though it's just due to a simple behavioral strategy. If the authors want to support this claim, they should show that it applies for all conditions together, that it is consistent across animals (this is a nested design, and it appears that they used N as # of saccades, rather than # of animals), and that it does not depend on extreme head movements that may represent aberrant conditions.

11) If these are sensory-guided directed saccades, then why do stimuli at the same location result in different saccade locations (2A vs C) and some saccades are directed away from the stimulus (clearly seen in 2G)? At best, these should be described as "directionally biased" rather than "directed".

12) However, a remaining concern is the claim that the data reveal "A new type of mouse gaze shift is led by directed saccades" as stated in the title. The data are fully consistent with the "saccade and fixate" pattern that is widely shared across species (e.g., Land (2019)) and which has recently been identified in freely-moving mice (Michaiel et al. 2020; Meyer et al. 2020). The present study shows that in mice this pattern appears more flexible than previously thought which is a new and important finding. Nevertheless, the rather small (yet important) variations compared to the freely-moving data might not justify the classification as a new type of gaze shift. In particular, Figure 3 suggests that the relative initial contributions of the head and eyes to gaze shifts lie along a continuum and even spontaneous saccades (the baseline condition) can "lead" attempted head rotations; for airpuff-evoked saccades, the head closely follows those saccades (median latency of 30 ms after saccade onset) which corresponds to about half the typical saccade duration (e.g., Sakatani and Isa (2005)). In other words: the saccade and fixate head-eye coupling is preserved for the airpuff-evoked saccades but the relative contributions of the head and eyes are shifted along the continuum. The worry is that the claim of a "new type of mouse gaze shift" might lead to a misperception of this study that shows stronger flexibility of an existing, but not a new, gaze pattern in mice.

13) Figure 2 could use some improvement. They should probably plot saccade start vs saccade end (this data should help their case, but it is presented awkwardly). They should also include average saccade sizes (with direction as sign) versus starting points. Attempted head movement data needs to be looked at in all these scenarios too. It is unclear how well a single trial attempted head movement can be captured. Individual trials look pretty noisy, but the average dynamics look pretty informative. Results may be similar to the eye movements.

14) Another concern is that head-fixed non-human primates will learn to reduce attempted head movements over a short period, once they learn it will not accomplish anything. It is possible the results in Figure 3 are from a similar mechanism. The directed saccades might have provided more reinforcement for learning the failure to move and that is why you rarely see the attempted head movement before the saccade (and why attempted head movements overall appear to be weaker). In addition, spontaneous saccades may decrease over time as the animal is habituated to the experiments and therefore may include fewer saccades during the post-learning period. We recommend that the data from Figure 3 therefore be plotted over time to see if anything observed is from an effect of learning.

https://doi.org/10.7554/eLife.73081.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

[…] Major concerns:

1. The paper is quite inconsistent when it comes to the stimuli evoking saccades. The authors state that the main motivation for developing the WISLR task was the absence of targeted saccades towards salient visual stimuli in previous studies. However, the conclusion based on their final simulation (Figure 4F-H) is that the function of saccades in response to tactile/auditory stimuli is to keep visual stimuli in the animal's visual field of view. How do these two things fit together? How are airpuff-evoked saccades related to a visual stimulus leaving the quite large visual field extending to the front and the sides of the animal as assumed in the modeling work? Currently, the relation between the stimuli in Figure 2 that evoked pronounced saccades (e.g., ear airpuff and whisker bar) and the animal's visual field is not clear. In particular, to reconcile the auditory/tactile observations with the simulations it will be important to know whether the tactile/auditory stimuli were within or outside the visual field.

We thank the reviewers for noting that there was a gap between the stated motivation for our study and the conclusions we drew from our final simulation. We should have explicated an implicit step in our logic, which is that natural stimuli are often multimodal such that touch- or sound-evoked saccades could benefit the animal by maximizing the likelihood of detecting the visual component of a natural stimulus. This is likely why other species make gaze shifts towards auditory and tactile stimuli, and why freely moving mice have been observed to make orienting head movements towards auditory stimuli (as well as visual stimuli such as crickets), despite their large visual field. As an aside, the stimuli we deliver to the whiskers and ears do fall within the mouse’s visual field. Nonetheless, our revised manuscript adopts a different framework and we have removed the simulation.

– There is a large body of literature on gaze shifts towards visual, tactile and auditory stimuli in humans and non-human primates. A common observation in these studies is that gaze shifts towards visual targets show a stronger saccade component than gaze shifts towards auditory targets which rely more strongly on head movements. This seems to be somewhat orthogonal to the idea that tactile/auditory but not visual stimuli should result in targeted saccades, in particular if circuits are conserved across species as stated by the authors. It would be important to discuss the motivation/findings in the context of the existing literature which is largely ignored in the current paper.

We thank Reviewer 1 for pointing out this oversight. In our revised introduction, we place the motivation for our experiments in the context of existing literature on gaze shifts towards visual, tactile, and auditory stimuli (page 2). This literature is even more relevant in light of our additional load-cell experiments and analyses showing that head-eye coupling varies for spontaneous and touch-evoked gaze shifts. We agree that it is interesting that, despite the conserved requirement for SC in driving sensory-driven gaze shifts in mice, certain features such as modality-specific differences in reliance on head and eye movements are not conserved. Our discussion goes into more detail on the interspecies commonalities and differences in gaze shifts evoked by stimuli of different modalities and potential mechanisms (pages 22-25).

– The authors show that average saccade trajectories converge on nearly the same endpoint (close to 0 deg) regardless of initial eye position (Figure 1C-E). Would that not support the idea of resetting saccades that move the eyes back to a more central location? There is a statistically significant, yet very small difference in endpoints (< 5 deg) between the left/right stimulus conditions. This seems to be the only evidence for "targeted saccades". However, studies in head-fixed (Sakatani and Isa, 2007; Itokazu et al., 2018) and freely-moving (Meyer et al., 2020) mice have demonstrated that there is a systematic asymmetry (about 5 deg) in the sizes of nasal vs temporal saccades. It would be important to check that this asymmetry cannot explain the difference in saccade endpoints.

We agree with Reviewer 1 that for some tactile stimuli, such as whisker airpuffs, saccade endpoints are close to center and narrowly separated. However, as noted above, mean endpoints for more peripheral stimuli (i.e., ear airpuffs) are quite well separated and not near the center (Figure 2A). Moreover, left and right ear airpuffs evoke saccades in opposite directions from most eye positions; by definition, one of these directions leads away from center and is thus centrifugal rather than centripetal (Figure 2E). In contrast, both left and right auditory stimuli evoke saccades in the same, centripetal direction from all initial eye positions (Figure 2H). Finally, many puff-evoked saccades that begin towards the center pass through to reach endpoints at eccentricities of 5 to 10 degrees—often more eccentric than the initial eye position—and cannot fairly be termed centripetal (Figure 2E). These results are incompatible with the idea that touch-evoked gaze shifts involve resetting centripetal saccades that move the eyes back to a more central position. Instead, it appears that saccades evoked by more central tactile stimuli may be directed towards more central orbital positions.

The second concern—whether the difference in mean endpoints could be an averaging artifact due to asymmetries in the amplitudes of nasal and temporal saccades—is addressed by showing more raw, unaveraged data, as the reviewers requested. We note in our revised manuscript that we observe this subtle asymmetry. However, it is clear that these asymmetries do not account for the effects we observe. First, in our revision, all data presented in the main figures are from tracking both eyes and represent the average position of both eyes; because saccades are conjugate, each gaze shift involves a nasal saccade of one eye and a temporal for the eye, such that averaging the two eyes eliminates any contribution of the nasal/temporal asymmetry. Second, Figure 2A-D shows the endpoints of all ear airpuff-evoked, auditory airpuff-evoked, whisker-airpuff evoked, and spontaneous saccades for the entire cohort of mice tested. Strikingly, regardless of initial eye position, virtually all saccades elicited by right ear stimulation are directed to the right (nasal), whereas virtually all saccades elicited by left ear stimulation are directed to the left (temporal) (Figure 2E). In other words, from the same initial eye positions, left and right stimuli elicit saccades in opposite directions. Thus, the differences in mean endpoints for these two stimuli are not averaging artifacts of asymmetries in nasal and temporal saccade amplitudes. Moreover, auditory airpuff-evoked saccades obey the same head-eye coupling as touch-evoked saccades, and display the same asymmetries in nasal and temporal saccade amplitudes, yet their endpoints fit the recentering model (Figure 2D, H, Supplementary Figure 6). For these reasons, asymmetries in amplitudes of nasal and temporal saccades do not create an artifact that could explain the directed touch-evoked saccades. We thank the reviewers for raising this potential issue.

In addition, the reviewers’ comments prompted us to examine the ~2-fold discrepancy between the range of saccade amplitudes observed in Meyer et al., 2020 and those reported in our original manuscript. Whereas Meyer et al., 2020 used the methods described in Sakatani and Isa, 2004 to convert pupil position from pixel values into angular eye position, we used the methods described in Stahl et al., 2000. This led to a substantial reduction in the range of saccade amplitudes we observed compared to those in Meyer et al., 2020. In our revised paper, we re-analyze our data using the methods referenced in Meyer et al., 2020 to facilitate direct comparisons. Using this approach, we find that the mean saccade endpoints for left and right ear airpuffs are separated by ~11 degrees (Figure 2A).

– The authors show in Suppl Figure 4 that mice attempted to reorient their heads during saccades and that the magnitude of attempted head movements is predictive of saccade sizes. This indicates that saccades in the WISLR task were part of combined eye/head gaze shifts as reported in freely moving mice (Michaiel et al., 2020; Meyer et al., 2020). Consequently, attempted head movements need to be taken into account when interpreting the data. Indeed, there seems to be a difference in attempted head movements between the two conditions shown in Suppl Figure 4B,C (spontaneous, WISLR). Based on these data, it is not clear if "targeted saccades" were just a by-product of head-free gaze patterns in head-fixed animals (given the low probability of observing a saccade at all).

We agree with the reviewers that we should have devoted more consideration to attempted head movements when interpreting the saccade data. In our revision, head movements are a focus: whereas the original manuscript included only a supplemental figure of head movements that accompany spontaneous and whisker airpuff-evoked saccades, most main figures of the revision (Figures 1, 3, and 4) present both head and eye movements in response to all the stimuli tested, and additional analyses and visualizations that directly address concerns raised by the reviewers.

These efforts confirmed Reviewer 1’s hypothesis that attempted head movements differ between spontaneous and touch-evoked saccades. In fact, there are multiple differences. First, more in-depth analyses found that touch-evoked saccades are coupled to much smaller head movements than are spontaneous saccades of the same amplitude, as Reviewer 1 noticed in our original Supplemental Figure 4. Second, we found that touch-evoked saccades precede attempted head rotations, an observation that is incompatible with the notion that directed eye movements are head-initiated and a “by-product of head-free gaze patterns.” In contrast, spontaneous saccades are coupled to biphasic head rotations, with a slow pre-saccadic phase followed by a fast post-saccadic phase, just as Meyer et al. documented in both freely moving and head-fixed mice (Meyer et al., Figure 5C, 7D). This distinction largely accounts for the difference in head-eye amplitude coupling between spontaneous and evoked gaze shifts. Thus, touch-evoked gaze shifts are “part of combined eye/head gaze shifts,” as the Reviewer notes, but not as reported in freely moving mice by Michaiel et al. and Meyer et al.

The fact that touch-evoked saccades precede rather than react to head movements and are coupled to smaller head movements indicates they are part of a new type of gaze shift: One that permits mice to more rapidly shift their gaze using their eyes. This contrasts with the previously known type of head-eye coupling, present in spontaneous and visually guided gaze shifts, which is driven by slower head movements followed by compensatory saccades that do not appear directed in either head-free or head-fixed contexts. Moreover, the endpoints of touch-evoked saccades are not a “by-product” of attempted head movements. Ff the goal of touch-evoked gaze shifts were merely to reorient the head relative to the location of a tactile stimulus, then head movement direction and amplitude should be constant for stimuli applied to the head (e.g. the ears or whiskers). However, we present new data and analyses that show that attempted head movement direction and amplitude depend on both stimulus location and initial eye position (Revision Supplementary Figure 8). This indicates that touch-evoked saccades are part of a new type of gaze shift involving different eye-head coupling in which saccades are not a product of head movements but part of a coordinated eye and head movement that takes into account the current position of the eyes and the location of the stimulus. In other words, head movements are (at least partly) determined by the eyes.

Nevertheless, it is worth contemplating these gaze shifts in the freely moving context in which they would ordinarily be made. To this end, a discussion of gaze shifts in head-free versus head-fixed cats and primates may be illuminating. Sensory-guided saccades in head-fixed primates and cats are often accompanied by attempted head rotations, similar to those we observe during touch-evoked gaze shifts in head-fixed mice. In head-unrestrained cats and primates, gaze shifts are initiated by directed saccades closely followed by directed head movements that are paired with smooth counter-rotatory eye movements. In this way, saccades rapidly shift gaze, and ensuing head and eye movements allow animals to return the eyes to more central locations while maintaining the new gaze direction. It is possible that mice use a similar strategy to shift gaze in response to tactile stimuli in the freely moving condition. The discussion in our revision addresses these ideas on pages 22 and 25.

– The shift in saccade endpoints during optical activation/deactivation of the superior colliculus (SC) is a novel and interesting finding. However, the SC is also involved in generating head movements in non-human primates (e.g., Freedman et al. 1996) and mice (e.g., Wilson et al. (2018)). In particular, Masullo et al. (2019) show that SC stimulation generates both head and eye movements. Again, it would be important to dissociate the contribution of (attempted) head movements to the measured saccades to support the idea that eye movements, and not the associated head movements, determine saccade endpoints.

We thank each of the reviewers for raising related questions regarding whether saccades and their endpoints are “determined” or “explained” by or “by-products of” head movements. This is an important point and one we did not address adequately in our original manuscript. As mentioned in the previous response, if the goal of touch-evoked gaze shifts were only to reorient the head relative to the location of a tactile stimulus, then head movement direction and amplitude should be constant for stimuli applied to the head (e.g. the ears or whiskers), because their distance from the rest of the head are fairly fixed. However, our data show that attempted head movement direction and amplitude depend on both stimulus location and initial eye position, similar to directed saccades (Revision Supplementary Figure 8). This indicates that rather than being specified solely by the necessary head movements, with saccades a by-product, touch-evoked gaze shifts involve coordinated eye and head movements that take into account the location of the stimulus and the current position of the eyes.

We agree it is interesting to know how SC manipulations affect both head and eye movements during touch-evoke gazed shifts. In our revision, we repeated the optogenetic experiments from our original manuscript while measuring saccades and attempted head movements. We used two unilateral manipulations: inhibition using the light-gated chloride pump eNpHR3.0 and weak optogenetic activation using ChR2. We found that reducing SC activity unilaterally causes ipsiversive shifts in both saccade and head movement amplitudes. In contrast, sub-threshold SC activation causes contraversive shifts in both saccade and head movement amplitudes (Figure 4, Supplementary Figure 10). These results indicate that head and eye movements are coordinately specified by SC.

– The authors use modeling to test the idea that the role of "targeted saccades" is to keep visual targets in the animal's visual field. Why would the U-shaped curve in Figure 4D not be consistent with a resetting saccade model in which the probability of observing a saccade increases with angular distance from the eye's central position? The small differences in trough locations might be related to the asymmetry of nasal vs temporal eye movements (see comment above).

We agree that the U-shaped function relating initial position with saccade probability would also be consistent with a recentering/resetting saccade model; the key difference is the location of the troughs, which for recentering saccades (such as auditory-evoked) are in the center, whereas the troughs for touch-evoked coincide with the more peripheral saccade endpoints (Figure 2I-L). Nevertheless, in our revised manuscript, we remove this modeling. As addressed in response 3, the differences we observe are not related to the asymmetry in nasal and temporal saccade amplitudes.

– The probability of observing a saccade following a non-visual stimulus seems to be quite low (Figure 1B and Suppl Figure 5) and saccade sizes are rather small (~5-15 deg, Suppl Figure 2A) compared to the large visual field of the mouse (>250 deg). Even if mice made targeted saccades: their function in the context of natural behavior is not discussed in the paper.

We thank the reviewers for raising this important point. In our revised manuscript, we have expanded our discussion of what roles these movements may play in natural behaviors in the section “Ethological significance” on pages 24 and 25.

A major concern is that "targeted saccades" are just a by-product of combined eye/head gaze shifts observed in freely moving mice (Michaiel et al., 2020; Meyer et al., 2020). The authors measured attempted head orienting movements in a subset of experiments, and the data strongly indicate that eye/head coupling is preserved in the WISLR task. To test if differences in saccade end points can be explained by head orienting movements, the authors could measure attempted head movements during the different conditions in Figure 2. This could be combined with a regression model to test if saccade end points can be predicted from attempted head movement amplitude.

We thank the reviewer for raising this question. The first part of their question, whether saccade endpoints are a “by-product” of eye-head gaze shifts, is answered in responses 4 and 5. Briefly, the influence of initial eye position on touch-evoked head movement amplitude (Supplementary Figure 8) indicates that touch-evoked head and eye movement amplitudes are coordinated to take into account both stimulus location on the head and current eye position. Thus, touch-evoked saccade endpoints are not “explained by” or “by-products” of head movements.

To address whether eye/head coupling is preserved in the WISLR task, as suggested, we examined the spatiotemporal profiles of attempted head rotations relative to saccades in different stimulus conditions, as well as during spontaneous gaze shifts (Figure 3, Supplementary Figure 6). We found that all stimulus-evoked gaze shifts obeyed a similar head-eye coupling pattern that differed markedly from patterns observed during spontaneous gaze shifts in head-fixed mice, and those previously characterized in freely moving mice. Strikingly, stimulus-evoked saccades preceded attempted head rotations, whereas the opposite held true for head-fixed spontaneous saccades and those previously characterized in freely moving mice. In addition, stimulus-evoked saccades of a given amplitude were consistently coupled to smaller attempted head rotations than were spontaneous saccades. Together, these data indicate that mice are capable of multiple types of gaze shift and that directed saccades are not a “by-product of combined eye/head gaze shifts observed in freely moving” and head-fixed mice.

Second, as requested, we trained and cross-validated a regression model to test if saccade amplitudes can be predicted from attempted head movement amplitude. We trained the model using data collected during spontaneous gaze shifts and compared its predictive ability to that of stimulus location. We found that this model is a worse predictor than stimulus location (R2 = 0.37 vs. R2 = 0.51, P = 0.031). That said, there is a correlation between head movement amplitude and saccade amplitude for touch-evoked gaze shifts. However, as noted in the first paragraph of this response, our new data indicate that rather than being a by-product of attempted head rotations, touch-evoked saccades are part of a coordinated head-eye movement in which attempted head movement amplitude varies according to initial eye position. In other words, there is a correlation between touch-evoked head movement direction and saccade direction, but this is because these movements are specified coordinately, not because either head movements or saccades are a “by-product” of the other.

Third, because touch-evoked and spontaneous gaze shifts appear to obey different head-eye coupling patterns, we developed separate regression models for each to examine how well head movement amplitude predicts saccade amplitude at different timepoints relative to saccade onset. For touch-evoked gaze shifts, head movements are only predictive of saccade amplitude after saccade onset, and never as strongly predictive as stimulus side (Author response image 1) . In contrast, for spontaneous gaze shifts, head movements are strongly predictive of saccade amplitude well before saccade onset (Author response image 1) . Once again, these data indicate that mice are capable of multiple types of gaze shifts and that differences in saccade trajectories are not simply due to differences in head movement amplitudes.

Author response image 1
Performance of linear regression models for saccade amplitude and head position.

Performance of linear models trained separately for spontaneous (blue) and puff-evoked (red) gaze shifts at different times relative to saccade onset. Each model was trained on distributions of trials matched for initial eye position and amplitude (6 mice, 20 sessions, 1920 matched saccades). Traces denote population mean R2 values +/- s.e.m. Shaded gray area indicates mean saccade duration.

Taken together, these new analyses show that saccade endpoints are not determined by head movement and that directed saccades are not a by-product of previously observed head-eye coupling patterns. Instead, they suggest that touch-evoked directed saccades are part of a novel type of mouse gaze shift involving coordinated movements of the head and eyes. Our revision provides data, analyses, and visualizations that illustrate these points.

In the method section it is mentioned that only saccades occurring within a specific response window following stimulus onset were included in the analysis (p 23). First, it might be useful to explain the choice of the 150 ms response window and the impact of response window length on the results. For example, Itokazu et al. (2018) report a much longer response window for visual stimuli. Second, it might be useful to indicate this response window in Suppl Figure 1A. I would expect that based on the criteria mentioned in the methods section the 2nd saccade in Suppl Figure 1A was excluded from the analysis. Is that correct?

We agree that we need to clarify how we selected this response window. We used this time window because it is the period in which saccade probability is clearly elevated above baseline, although probability sags to a very low but slightly above baseline level for at least 500 ms after stimulus delivery. This is now illustrated in Supplementary Figure 2 in which we show that extending the response window beyond 100 ms does not capture additional evoked saccades and merely dilutes the results with spontaneous saccades, which prompted us shorten our response window to 100 ms. In addition, our response window coincides to the latencies of sensory-guided express saccades in other species, The extremely long latencies (~1 s) observed by Itokazu et al., do not resemble those of sensory-guided saccades in other species, or those we observed in mice, suggesting that those saccades were driven by different mechanisms. Reviewer 1 is also correct that the second saccade in the original Supplementary Figure 1A would not be counted as stimulus-evoked. As Reviewer 1 suggested, we have clarified these points.

Related to this: Figure 1B shows the probability of observing a saccade in different time bins following an airpuff. Maybe I missed this but what was the overall probablity of observing a saccade within the response time window? To get a sense of the reliability of saccade generation, it might be useful to also show cumulative probability (either in the same figure or in a suppl figure). It is hard to guess from the distribution but I would guess that p(saccade in response window) < 0.2.

We agree that these values are useful for the reader and will include them in our revision on page 5.

Reviewer #2:

This study makes a bold and provocative claim – mice make targeted eye saccades. This is a significant claim, since although many studies have shown that species without fovea perform saccades, these are generally coupled to head movements, and serve to shift gaze by recentering the eyes following slower compensatory eye movements. Indeed, recent studies have shown that eye movements in mice follow this pattern, and in fact even the eye movements seen in head-fixed mice are associated with attempted head movements. This study claims that head-fixed mice do make targeted eye saccades, when triggered by tactile stimulation to the ears/whiskers. If it is true that mice make saccades similar to foveate animals, and this had just been missed because previous researchers had not used tactile stimuli, this would be an exciting finding not only for mouse vision, but for the vision field at large.

However, there are two major issues with the claim that mice are making targeted eye saccades. First, the saccades do not appear to be targeted directly to a stimulus location – rather, they return to center with a slight bias of a couple degrees for saccades "to" stimuli at disparate locations, and in fact there is great variability in saccade endpoints (larger than the difference between target locations). Second, it is very possible that the results can all be explained by attempted head movements, consistent with existing findings on mouse eye movements. Thus, the findings as presented do not overturn the current thinking, nor suggest that mice are making targeted eye saccades similar to humans.

1. Targeting of eye movements

– The eye movements in response to a left airpuff (Figure 1C) look exactly like one would expect for re-centering saccades – from all initial locations, the eye returns to center. It is only in comparing this to the right airpuff that a 2deg, on average, shift appears. Thus, there is only a +/-1 deg difference for saccades to stimuli on opposite sides of the body. If these saccades are targeting a stimulus, where is it such that left/right are only 2 deg apart? Even for a puff to the ears (Figure 2G) there is at most a 5 deg displacement of target location from central eye position, for a stimulus that is far lateral to the eyes. A much more straightforward description is that these are recentering movements, with a slight bias toward the stimulus side.

We thank the reviewer for pointing out our imprecise and poorly defined use of the term “targeted”. We agree that these movements do not necessarily center the pupil on the stimulus location, particularly given the laterality of the eyes and the limited range of eye positions in mice, and we regret not having made this clear. Rather, our use of “targeted” was meant to indicate that mice direct their pupils to particular orbital positions specified by the location of the tactile stimulus. Nevertheless, a better descriptor for these saccades is “directed,” which we have adopted throughout our revision.

With regards to the movements themselves, this is an instance in which showing more raw, unaveraged data, as the reviewers requested, is clarifying. Figure 2 and Supplemental Figure 4 of the revised manuscript show the endpoints and trajectories of all ear airpuff-evoked, whisker airpuff-evoked, ear tactile-evoked, auditory airpuff-evoked, and spontaneous saccades made by two different cohorts of mice. Although endpoints of whisker airpuff-evoked saccades are somewhat near the center, as the Reviewer noted, those for saccades evoked by the more peripheral ear airpuffs are near the edges of the pupils’ range, with their means separated by ~11 degrees and the distributions overlapping minimally (Figure 2A, Supplementary Figure 4A). Furthermore, virtually all saccades elicited by right ear stimulation are directed to the right (nasal), whereas virtually all saccades elicited by left ear stimulation are directed to the left (temporal) (Figure 2E, Supplementary Figure 4E); by definition, from any eye position, one of these directions leads away from center and is thus centrifugal rather than centripetal (i.e., not recentering). Moreover, many saccades begin towards the center but pass through to reach endpoints at eccentricities of 5 to 10 degrees and as such cannot be accurately described as recentering (Figure 2E, Supplementary Figure 4E). Thus, it is clear that these ear airpuff saccades are directed, not slightly biased recentering. The less eccentric endpoints of whisker-evoked saccades may simply reflect the fact that the whiskers are located more centrally. Sound-evoked saccade endpoints, in contrast, are well described by the recentering model (Figure 2D, H, Supplementary Figure 4D, H).

In addition, the reviewers’ comments prompted us to examine the ~2-fold discrepancy between the range of saccade amplitudes and eye positions observed in Meyer et al., 2020 and those reported in our study. Whereas Meyer et al., 2020 used the methods described in Sakatani and Isa, 2004 to convert pupil position from pixel values into angular position, we used the methods described in Stahl et al., 2000. This led to a substantial reduction in the range of saccade amplitudes we observed compared to those in Meyer et al., 2020. In our revised paper, we will re-analyze our data using the methods referenced in Meyer et al., 2020 to facilitate direct comparisons.

– It seems that the authors are assuming that the stimulus location is at the mean endpoint, but that would imply that they had centered the whisker puff stimulus within 2 deg of the center of the visual field for each animal (unlikely), and that the ear puff stimulus is located within 5 deg of the center of the visual field (certainly not true).

We again thank the reviewer for pointing out our imprecise and poorly defined use of the term “targeted.” As noted above, our use of this term was meant to imply targeting of a particular orbital location, but we failed to define the term. For a variety of reasons, including their lateral eyes and limited range of eye movements, we agree that mouse saccades could never center both pupils on the stimulus location. We regret not conveying this clearly. As described in response 11, we have adopted the term “directed” in the revised manuscript.

– Furthermore, rather than being targeted to a specific location, wherever that happens to correspond to, the saccade endpoints appear highly variable. Indeed, the variability across mean end position based on starting position is similar to the difference due to stimulus location (Figure 2E). The actual variability is probably much greater, as this is masked by averaging a very large number of saccades in figures such as 2C. This averaging makes it appear that all saccades go to the same location, and makes it hard to estimate variability (except to the extent that the error bars after saccade are much larger than for the 5 deg bins pre-saccade). It is important to show the true variability in endpoint position, for example, as a histogram of end positions for left vs right stim. It would also be valuable to see a number of overlaid raw traces for Figures such as 2C,D, to show the true variability before presenting the means.

We thank the reviewer for noting that averaging can obscure variability. These comments spurred us to develop visualizations that better communicate the overall trends while showing raw, unaveraged data. As suggested, we use scatter plots and histograms to portray the complete distributions of endpoints for saccades evoked by left and right ear airpuffs (Figure 2A-H). These illustrate both the extent of endpoint variability and the fact that this variability is small enough that there is minimal endpoint overlap for ear airpuff-evoked saccades. The reviewer noted that average endpoint location varied as a function of initial eye position, which is also evident in the raw amplitude distributions; however, these raw traces also show that the difference due to stimulus side completely overshadows the variability due to starting eye position (Figure 2E-H). Although comparisons to primates are subjective and we agree with the reviewers that those made in our previous draft were excessive, it is worth mentioning that touch-evoked saccades in primates are also highly variable (~20 degree range of endpoints for a single stimulus, Groh and Sparks 1996i, Figure 3) and much of this variability also depends on initial eye position, possibly due to the inability to make saccade-coupled head movements in head-fixed animals (Groh and Sparks 1996i, Figure 5).

– The fact that saccade amplitude is equal and opposite to initial eye displacement (negative linear relationship in 1F) is exactly what one would expect for re-centering saccades as well.

We agree and should have been clearer in our argument. A negative linear relationship is a necessary but not a sufficient criterion for both targeted and directed saccades. The key difference is whether these linear fits pass through the origin, i.e., saccades of amplitude zero are made when the eyes are in the center. This is true for recentering saccades but not for directed saccades, whose linear fits pass through zero at values statistically insignificantly different from the saccade endpoints for those stimuli (Author response image 2) .

Author response image 2
Relationship between saccade amplitude and initial eye position for ear airpuff- and auditory-evoked saccades.

Each point denotes a single saccade. Brighter areas indicate higher point density. Lines are fits of linear regression model. Dashed horizontal line indicates saccade amplitude of 0°. Dashed vertical line indicates initial eye position at which linear fit intersects with dashed horizontal line, i.e., the eye position at which predicted saccade amplitude is 0°. For left and right ear airpuffs, these intercepts are at -6.35° +/- 0.15 and 6.20° +/- 0.10 (mean +/- s.e.m) and R2 values are 0.352 and 0.506, respectively. For left and right auditory airpuffs, intercepts are at 0.14° (+/- 0.45) and 0.19° (+/- 0.43) and R2 values are 0.648 and 0.615, respectively.

2. Confound of head movements/fixation

Previous studies have shown that eye movements are highly disrupted in head-fixed mice, and in fact most eye movements are coupled to attempted head movements. Critically, a previous study (Meyer et al., 2020), and even supplementary data here, show that in head-fixed mice, attempted head movement results in eye saccades in the same direction and proportional in amplitude of the head movement, as one would see in a free moving animal and consistent with the standard gaze-shifting reset model. If the tactile stimulus is evoking attempted head movements (likely) and these are biased in one direction, then that would explain the offset in resulting eye movements. In that case, the reason this study found tactile evoked eye movements is that tactile stimuli evoke head movements, not because they are saccading to the tactile stimulus.

We thank Reviewer 2 for raising this alternative explanation of our data, which we address at length in our revised manuscript. As Reviewer 2 suggested, these questions can be addressed using strain gauge measurements that enable presentation of stimuli at precise craniotopic locations. Indeed, Meyer et al., 2020 performed careful comparisons of head-eye coupling between freely moving and head-fixed mice and found that head-eye coupling—especially during saccades—is highly preserved across these two contexts. In particular, their data show that spontaneous saccades follow head movements or attempted head movements, consistent with the idea that these saccades are a “result” of the head movements. Our new experiments recapitulate this published relationship between saccades and attempted head movements during spontaneous gaze shifts, which we agree is consistent with observations in freely moving mice and the prevailing view of the “gaze-shifting reset model” (Figure 3B, D, G).

The reviewer posits that touch-evoked saccades are also consistent with the standard gaze-shifting recentering model (i.e., tactile stimuli evoke gaze shifts led by head movements that “result” in compensatory recentering saccades). However, our new data show that airpuff-evoked saccades precede attempted head movements—an observation that is incompatible with the idea that they are a “result” of head movements (Figure 3B, D, G). In addition, there are other differences in head-eye coupling between spontaneous and touch-evoked gaze shifts: (1) spontaneous saccades are coupled to biphasic head rotations, with a slow pre-saccadic phase followed by a fast post-saccadic phase, very similar to what is observed during freely moving spontaneous saccades (Figure 3B, D) (Meyer et al., 2020 Figure 5C); and (2) touch-evoked saccades are coupled to smaller head movements overall because they lack a slow pre-saccadic phase (Figure 3B, H). Thus, in both timing and amplitude, touch-evoked gaze shifts are not consistent with the standard model of gaze shifts. We are grateful to Reviewer 2 for suggesting these helpful additional analyses that demonstrate additional differences between touch-evoked and spontaneous gaze shifts.

The ideal solution to this would be to perform these experiments in freely moving animals and show that they move their eyes toward the stimulus independent of the head. However, barring that there are a number of key questions that would need to be answered to resolve this issue, potentially based on load cell measurements.

– What is the pattern of head movements resulting from the stimuli? Do stimuli from opposite sides evoke different directions of head movements on average? If so, the bias in head movements could explain the resulting bias in eye movement.

– Do the different types of stimuli (ear, whisker, auditory) evoke different directions/amplitudes of head movement? It could be that ear puff stimulation causes larger eye shifts than whisker not because it is more peripheral, but because it evokes a larger movement. Likewise, auditory stimulation alone may elicit smaller movements or less directionally-biased movements.

These are interesting points and we thank Reviewer 2 for raising them. They suggest that the endpoints of directed saccades evoked by tactile stimuli can be “explained” by the direction or amplitudes of head movements elicited by these stimuli. As noted in response 5, each of the reviewers asked a version on this question—whether the saccades are “explained” or “determined” by or are “by-products” of head movements—illustrating that this an important point we did not address adequately in our original manuscript. As the reviewer suggests, it is true that, for example, left ear stimuli elicit primarily leftward saccades that are associated with mostly leftward head movements. However, this correlation does not indicate that head movements cause eye movements. First, as the previous response noted, touch-evoked saccades precede attempted head movements. Second, as noted in response 5, if the goal of touch-evoked gaze shifts were merely to reorient the head relative to the location of a tactile stimulus, with saccades a by-product, then head movement direction and amplitude should be constant across trials for stimuli applied to the head because the distance between the ears or whiskers and the tip of the nose is largely invariant. However, our data show that attempted head movement directions and amplitudes vary with both stimulus location and initial eye position (Revision Supplementary Figure 8). This dependence of head movements on eye position is incompatible with the idea that saccade endpoints are “explained” or “determined” by head movements, as it shows that head movements are also determined by the eyes. Instead, this result indicates that head and eye movement amplitudes are coordinated to take into account both stimulus location on the head and current eye position, a result that is incompatible with the “gaze-shifting reset model”.

– Do the optogenetic manipulations affect head movement as well? This seems likely, since studies in multiple species have shown head movements evoked by SC stimulation or impaired by SC lesion. If so, the shift in eye movements in Figure 3 could be explained by the corresponding change in head movements.

We thank the reviewers for raising this point. As noted in response 5 and the immediately preceding response, the dependence of head movement probability, amplitude, and direction on initial eye position and stimulus location indicates that touch-evoked saccades are not “explained” by head movements. Instead, touch-evoked saccades and head movements are specified as coordinated movements that take into account initial eye position and stimulus location.

Although head movements do not explain touch-evoked saccades, we agree that it is important to know how SC manipulations affect touch-evoke gaze shift generation overall and we provide these data in our revision. These manipulations caused parallel effects on saccades and head movements, i.e., weak SC stimulation causes both saccades and head movements to shift contraversively and vice versa for SC inhibition (Figure 4, Supplementary Figure 10). These data indicate that the change in saccade endpoints does not compensate for the effects of these SC manipulations on head movements. Instead, SC activity appears to specify touch-evoked gaze shift amplitude, as is observed in other species.

– Finally, which does a better job of predicting the endpoint – head movement or stimulus side? A figure such as 1C-E, but broken up by head movement direction, rather than stimulus side, would test this. Indeed, it would be interesting to see spontaneous eye saccades broken up in this way, to see if there are "targeted" eye movements in the absence of a target.

We thank the reviewer for raising this issue. As mentioned previously, if one assumes that head-eye coupling is the same for spontaneous and touch-evoked saccades, a regression model trained on a mixture of both is a worse predictor of saccade amplitude than is stimulus side (R2 = 0.37 vs. R2 = 0.51, p = 0.031). However, we subsequently showed that head-eye coupling differs for spontaneous and touch-evoked saccades (Figure 3, Supplementary Figure 6). We therefore trained separate linear regression models on spontaneous and touch-evoked saccade and attempted head movement data. The model trained specifically on puff-evoked gaze shifts performs better than the spontaneous model and almost as well as stimulus side (R2 = 0.46 vs. R2 = 0.51, p > 0.05). Thus, stimulus side is a better predictor of saccade amplitude even using a model trained solely on this new type of gaze shift.

Because of the distinct head-eye coupling for spontaneous and touch-evoked saccades, regression models trained on each class of gaze shift perform differently during the perisaccadic epoch: Spontaneous saccade amplitude can be predicted before saccade onset, but touch-evoked saccade amplitude cannot be predicted until after the saccade has ended (Author response image 1) . More importantly, touch-evoked gaze shifts are a different type from those previously observed in mice. Finally, we agree that it may be possible to “break up” the wide distribution of observed spontaneous gaze shifts to create populations with some separation in their endpoint distributions--although the separation would be smaller than that observed for ear airpuff-evoked saccades. However, as detailed in responses 15 and 16, this correlation between head movements and saccades does not indicate that head movements cause eye movements. Instead, our data indicate that eye and head movements are jointly specified as part of a coordinated movement that takes into account both stimulus location and current eye position.

Additional point

1. Modeling the consequences of eye movements. The authors note that the probability of a saccade increases as a U-shaped curve relative to center position (Figure 4D,E). Indeed, this is exactly what one would expect for recentering saccades – the further the pupil is from center, the more likely it is to reset. However, the authors perform modeling to suggest that instead this represents the goal of keeping the target in the visual field. It's not clear how the modeling supports their claim as presented. The results of the model show that if a stimulus is near the edge of the visual field, then it is more likely to move out of the visual field based on a random movement. Isn't that almost trivially true? It would provide stronger support for their claim if this was quantitative, using physically meaningful values. Specifically, eye position relative to target in 4E varies by roughly +/-10 deg in the data, out of a field of view of 140deg for the mouse. However, this is drastically different than the cartoon shown in 4F, and it doesn't seem that quantitatively a 10deg displacement is going to greatly increase the probability of moving out of a 140deg field of view to the extent shown in Figures 4G,H.

We agree with the reviewer and have removed the model from our revision.

Recommendations for the authors

1. The authors state the endpoint should be "strongly dependent" on the stimulus position (p. 3), but highly variable targeting with mean 2 deg shift based on stimulus position does not seem to meet that criteria. If the saccades are indeed targeting a stimulus location, they should be able to systematically vary the stimulus positioning (for example from -30 to 30 deg in the visual field) and show that the targeting eye movements follow.

Reviewer 2 correctly notes that there are not objective criteria for “strongly dependent” and we should be clearer in defining this. In response to the concern that endpoint differences are small and variable, we would like to point out that endpoints for left and right ear airpuffs are near the edges of the range of eye positions observed and overlap minimally, despite trial-to-trial variability (Figure 2A). In addition, as elaborated in response 11, we recognized a ~2-fold discrepancy between the range of saccade amplitudes observed by us and Meyer et al., 2020 caused by methodological differences. In our revised paper, we will re-analyze our data using the methods referenced in Meyer et al., 2020 to enable direct comparisons. Using this alternate method, the separation of mean endpoints for the ear airpuff stimuli is ~11 degrees. Although comparisons to primates are subjective and those made in our previous draft were excessive, it is worth noting that touch-evoked primate saccades are also highly variable (~20 degree range of endpoints for single stimulus to the hand, Groh and Sparks, 1996i, Figure 1).

As the reviewer suggested, when we varied the locations of airpuffs from peripheral (the ears) to central (the whiskers), the saccade endpoints followed (Figure 2, Supplemental Figure 3, page 8). That said, as noted previously, we agree that these saccades are not necessarily targeting the location of the stimulus itself, particularly with both eyes, given the laterality of the mouse’s eyes and the limited range of eye positions, and that this use of the word “targeted” may have been misleading. In our revision, we more clearly define our terminology and call these movements “directed” rather than “targeted.”

2. Unless the authors can demonstrate that there is a specific stimulus location that the saccades are targeting, and can explain why they are so highly variable, then the claim that they are making primate-like saccades is not valid. Something like "directionally biased recentering saccades" seems more accurate. But this does not overturn textbook models as they suggest.

We thank the reviewers for pointing out the subjectivity inherent in such interspecies comparisons and the overstatement in our claims. As such, and as detailed in the introduction, our revision focuses on our results solely in the context of mouse gaze shifts, even though the caveats listed (saccades targeting a particular orbital location that does not necessarily correspond to the stimulus location and highly variable endpoints) are also observed in primate touch-evoked saccades (cf. Groh and Sparks, 1996i). The textbook model for gaze shifts, found in volumes such as the “Oxford Handbook of Eye Movements” and espoused by recent papers such as Meyer et al. 2020 and Michaiel et al. 2020, posits that mice and all other afoveates make gaze shifts that involve directed head movements followed by recentering saccades. As Michaiel et al. concluded in eLife last year, “mice do not perform either directed eye saccades or smooth pursuit.” Thus, setting aside any comparison to primates, our findings that mouse make gaze shifts that involve directed rather than recentering saccades and that these saccades do not follow head movements is incompatible with the prevailing view that is also found in textbooks.

3. Throughout the text, there is a notable lack of numerical data for population summary statistics, which makes it difficult to accurately assess the findings. For example, the mean value for left and right endpoints is never provided in the text or figure legends, only the p-value for the comparison (e.g. p 4, line 6). This is not just a style point – it is essential to convey the magnitude of effects observed, as nowhere in the text do the authors state the size of the targeting difference, which is only on the order of a few degrees. In addition, presenting N for number of datapoints (saccades) rather than just number of animals would be valuable.

We agree with the reviewers that we need to include more raw data and statistics. We have changed the visualizations to show exclusively raw data and added precise values to the text and figure legends.

4. The impact statement, that a "hallmark of human vision" is conserved in mice, seems overstated. In addition to the fact that these saccades don't resemble human saccades, targeted eye movements are not just a hallmark of human vision but many species across the animal kingdom that have retinal specializations.

We agree. Because our revision uses a different framework, mouse gaze shifts, and has a new impact statement: “Mice are capable of gaze shifts led by the eyes rather than the head.”

Reviewer #3:

The submitted article titled "Mice Make Targeted Saccades" by Zahler et al. presents some interesting eye movement data that suggests that mice reorient their visual field toward an object of interest. I think the data are thought-provoking and the authors have presented a careful, thorough systematic description of the behavior. My enthusiasm though is tempered for a couple reasons:

We thank the reviewer for their kind words and suggestions.

First, no vision is really involved in this paradigm so it is hard to be convinced this demonstrates the mice are actually reorienting their visual field. Meyer et al. (2020), as well as the authors, demonstrate that saccadic eye movements coincide with attempted head movements. The mice only made saccades when the mice were "touched" on their head (whiskers or ears). It is not surprising that the mice would attempt to move their heads with a potential threat like that. Therefore, the eye movements might be simply a consequence of the attempted head movement whether or not it is related to a shift in gaze. It is still possible that mice were shifting (or attempting to shift) their visual field, but I would be more convinced if this was demonstrated for targets represented across other senses, especially vision. It might be that more consistent shifts require stimuli farther out in the periphery (remember that mice have a very large visual field due their laterally oriented eyes). In fact, the authors do nicely show this with the U-shaped function in Figure 4. It might be that more conditions are required more peripherally to trigger higher probabilities of saccades and from stimuli represented by sound and vision.

We agree with Reviewer 3 that the behavioral function of these saccades was not discussed adequately in the original manuscript. Our goal was not to elicit saccades with visual stimuli, but simply to determine whether mice generated sensory-evoked saccades. Implicit in our reasoning was that many objects provide multisensory input, which is presumably why primates, cats, and other species are well known make directed saccades towards auditory and tactile stimuli. Importantly, although sound- and touch-evoked gaze shifts are not elicited by visual stimuli, the visual field, i.e., the portion of the world projected onto the eyes, necessarily changes every time the animal saccades.

Reviewer 3 suggests that the saccades “might simply be a consequence of the attempted head movement.” As noted in response 5 and several subsequent responses, each of the reviewers asked a version on this question—whether the saccades are “explained” or “determined” by or are “by-products” or “consequences” of head movements—illustrating that this an important point we did not address adequately in our original manuscript. As noted in response 5, if the goal of touch-evoked gaze shifts were merely to reorient the head relative to the location of a tactile stimulus, with saccades a by-product, then head movement direction and amplitude should be constant across trials for stimuli applied to the head, because the airpuffs are at fixed locations relative to the head. However, our data show that attempted head movement directions and amplitudes vary with both stimulus location and initial eye position (Revision Supplementary Figure 8). This dependence of head movements on eye position is incompatible with the idea that saccade endpoints are “explained” or “determined” by head movements, as it shows that head movements are also determined by the eyes. Instead, they suggest that head and eye movement amplitudes are coordinated to take into account both stimulus location on the head and current eye position, a result that is incompatible with the “gaze-shifting reset model”. Likewise, that touch-evoked saccades precede attempted head rotations is incompatible with the notion that directed eye movements are a “consequence” of directed head movements (Figure 3B, D, E-G). In contrast, spontaneous saccades are a coupled to biphasic head rotations, with a slow pre-saccadic phase followed by a fast post-saccadic phase (Figure 3B, D, H). An important outcome of this difference is that touch-evoked saccades are coupled to smaller head movements, further evidence that the saccades we observed are not an expected “consequence” of head movements or of the previously published relationship between head and eye movements (Figure 3B, D, H).

We agree that it would be interesting if visual stimuli also evoked directed saccades. That said, and although absence of evidence is not evidence of absence, many groups (including ours) have unsuccessfully attempted to elicit directed saccades using a variety of visual stimuli. For completeness, we include an example of such negative data in our revision, finding that a very bright LED does not evoke gaze shifts in head-fixed mice (Figure 1G, L). The reviewer suggests it may be the physical proximity of a tactile stimulus that explains this difference, but less proximal auditory stimuli also evoked saccades (albeit at a lower probability, and these were not directed) (Figure 1F, K; Figure 2D, H, L; Supplementary Figure 4D, H, L). Similarly, this difference is not simply a result of stimulus eccentricity, as the visual stimulus was placed at the same peripheral location as the ear airpuffs. Moreover, for tactile and auditory stimuli, the probability of making a gaze shift is not simply a function of how peripheral stimuli are. In fact, we see much larger modulation of saccade probability by eye position than by stimulus location. For example, ear airpuffs were roughly twice as likely as whisker airpuffs to elicit saccades overall, but mice were roughly four times as likely to saccade in response to a central tactile stimulus (right whisker airpuff) when the eyes began to the left than they were to saccade in response to a peripheral tactile stimulus (right ear airpuff) when the eyes began to the right (Figure 2I, J). In contrast, mice did not respond to a peripheral visual stimulus regardless of their initial eye position (Figure 1G). Thus, it is not simply that the visual stimuli were insufficiently proximal or peripheral. It is possible that an as yet unidentified visual stimulus could robustly elicit saccades in head-fixed animals, although it is worth noting that every tactile stimulus we have delivered to any location on the mouse’s body, from the tip of the nose to the trunk, elicited saccades. Therefore, it seems more likely that the effects we and others have observed may simply reflect differences across modalities, something we discuss in greater depth in our revision.

Second, Michaiel et al. (2020) have demonstrated that mice shift their gaze towards an object of interest and this was based on a visual stimulus (cricket) so it should be possible to demonstrate targeted saccades systematically based on vision.

We thank the reviewer for mentioning this highly relevant study. However, it is critical to clarify what Michaiel et al., 2020 claimed. They conclude their abstract by asserting that during cricket hunting “orienting movements are driven by the head, with the eyes following in coordination to sequentially stabilize and recenter the gaze.” In their results, they note that “eye movements are not targeting the cricket more precisely, but simply ‘catching up’ with the head, by re-centering following a period of stabilization.” Moreover, they state that “mice do not perform either directed eye saccades or smooth pursuit.” In other words, throughout their manuscript, they explicitly state that mice do not make targeted (or directed) saccades based on vision (for the stimuli they tested, at least). Instead, they contend that visual stimuli evoke head movements directed towards the stimulus location, followed by compensatory saccades that recenter the eyes. This is in marked contrast to our observation that touch-evoked eye movements are directed, precede attempted head rotations, and shape gaze shift direction, amplitude, and probability. As noted in our previous response, to our knowledge no group has observed an innate ability of mice to make directed saccades based on vision; Itokazu et al. were able to train mice to make saccades in response to visual stimuli, but this required many months and the extremely long latencies (~1 s) relative to those of sensory-guided saccades in other species or, as our data show, in mice suggest that these saccades were not sensory-guided. Thus, ours is the first study of which we are aware to show that mice make directed, sensory-guided saccades. We agree it is interesting that we observe this separation on the basis of sensory modality and address this in our revised discussion.

The separation of head and eye movement shifts in gaze is not that important of a distinction as nearly all animals combine head and eye movements in some manner to shift gaze, and the superior colliculus jointly represents these saccadic movements.

We agree that our previous draft failed to place our study in the appropriate context and as such the significance of this distinction was obscured. As mentioned in our previous response, Michaiel et al. explicitly stated that mice do not make directed saccades. Instead, they bolstered the model found in textbooks such as the Oxford Handbook of Eye Movements that mouse gaze shifts are led by the head, with these head movements triggering compensatory saccades that recenter the eyes. The emphasis of this idea in the penultimate sentence of an eLife abstract from last year and by each of the reviewers of our original manuscript underscore the importance of this longstanding distinction and how surprising our findings are.

Another point the reviewer raises is that SC is known to drive head and eye movements. It is important to clarify that although SC is known to drive head and eye movements in many species, the prevailing view holds that recentering saccades driven by head movements (i.e., the quick phase of nystagmus), such as those in the previously observed in mouse gaze shifts, arise independently of SC and rely on brainstem circuits receiving vestibular input from the semicircular canals (reviewed in Curthoys, 2002). Indeed, Michaiel et al. conclude that “orienting movements are driven by the head, with the eyes following in coordination to sequentially stabilize and recenter the gaze,” and Meyer et al. invoke vestibular mechanisms as a likely explanation for the saccades they observed during spontaneous gaze shifts. Once again, such recentering movements are thought to be generated independent of SC circuits, which could explain the different head-eye coupling we observe for spontaneous and touch-evoked saccades. To our knowledge, no study has ever shown that mice make SC-dependent gaze shifts, although multiple studies have shown SC stimulation can evoke saccades and head movements in mice.

We agree that our observations that mice generate SC-dependent, sensory-evoked, saccade-led gaze shifts feel intuitive, but this is because they reconcile the longstanding logical disconnect between the aforementioned studies of mouse gaze shifts and SC microstimulation results. Indeed, it was such an intuition motivated us to undertake this study and to test a broader range of stimuli than had been previously studied. However, the fact that the last half-century of studies of afoveates, including high-profile papers in the last year such as Michaiel et al. and Meyer et al. unanimously asserted that mouse gaze shifts are (1) led by the head and (2) involve recentering saccades that are believed to be SC-independent shows the importance of this distinction and highlights the novelty of our findings. Our revision discusses this literature in more depth in order to more clearly convey the importance and implications of these findings.

I am surprised there was no eye movement triggered towards the auditory stimulus as I have seen the eyes shift towards sounds during experiments. I have also seen eye movements towards tactile stimuli farther back on the body so I do think it is possible to explore this beyond the ears.

We were also surprised. In fact, while doing these experiments we also had the impression that the auditory-evoked saccades we observed were directed. However, once we analyzed the trajectories and endpoints it became clear that this notion was incorrect. That said, and although our tests were not exhaustive, absence of evidence is not evidence of absence, and other auditory stimuli we did not identify may be capable of eliciting directed saccades.

Figure 1. I think the data are clear, but it is important to present it fully so that readers understand the similarities *and* the differences with primate behavior. The current presentation is misleading to make it appear more similar to primate behavior than it is in reality.

Figure 1. C,D. It would be helpful to see actual individual saccades plotted here rather than averages (not just the supplemental figure). As plotted, I think this is a little misleading about the precision of the targeted saccades for mice.

As noted previously, we thank the reviewers for pointing out the limitations of interspecies comparisons and have reframed our manuscript to focus on mouse gaze shifts. In addition, we present much more raw data (e.g., endpoints and trajectories for all saccades rather than means) as the reviewers suggested.

Figure 1E. The rightward bias should be explained. The bias probably arises because you are mostly monitoring left eye position. Saccades in mice are on average convergent so the left eye would move more rightward (nasal) than leftward (temporal) (Itokazu et al. 2018; Meyer et al. 2020).

This is an interesting point. It is possible that this difference is attributable to the asymmetry in nasal and temporal saccades suggested. On the other hand, it seems likely that the apparent bias is a result of the somewhat arbitrarily defined “center” eye position. For example, if we define the center as the mean endpoint of spontaneous saccades, rather than the median eye position, the apparent rightward bias is greatly reduced. Nevertheless, to eliminate any potential nasal/temporal bias caused by recording from one eye, we recorded both eyes and averaged their positions in the revised manuscript.

Figure 1B. What is the aggregate probability of a saccade if you integrate over a ~400 ms window after the airpuff? The binning is not clear, but the number appears to be pretty small. For a primate, I would assume this to be close to or at 100%. I assume these are represented in Figure 4.

Figure 1F. Again, I think averages are misleading. Why not just plot all of the data points?

Figure 1G, H. Was regression performed on all data points or just the means? It would be more appropriate to use all of the individual data points.

Figure 2. It would be nice to see airpuff position plotted versus saccade endpoint for each mouse to see if the shift in gaze consistently and systematically follows the position of the airpuff.

We thank Reviewer 3 for these suggestions. We provide aggregate probabilities in our revision on page 5. We plot all data points throughout the revision. We show airpuff position vs. endpoint in Revision Supplementary Figure 3.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Essential revisions:

1) The claim of a 'new' type of eye movement is fairly strong, particularly given that they share many features with standard reset saccades, as well as the fact that this is being demonstrated under the artifact of head fixation. It'd be better to have a less loaded statement, such as "Touch-evoked eye saccade endpoints are biased toward the direction of head motion in head-fixed mice". In any case, the title should certainly include "in head-fixed mice". An alternative suggestion is "Stimulus evoked rapid eye and attempted head movements in the mouse modulated by the superior colliculus".

We thank the reviewers for their suggestions. We agree that our claims of a new type of gaze shift is fairly strong and have attempted to modulate the tone. Therefore, we have attempted to combine the above suggestions and retitled our manuscript “Superior colliculus drives stimulus-evoked directionally biased saccades and attempted head movements in head-fixed mice.”

2) The authors should clarify that *previous studies* have shown that microstimulation in mouse SC produces head and eye movements that are roughly matched to a visual map. What would resolve the major issue that is raised in the introduction of this submission is a direct measurement of combined head and eye movements in mice following SC microstimulation.

We thank the reviewers for raising this issue. In order to better motivate our studies, we now provide in Figure 1 our own data on combined head and eye movements evoked by SC optogenetic stimulation. These data show that stimulation-evoked saccades are coincident with attempted head movements and directionally biased. This differs from the temporal relationship reported for mouse spontaneous and visually guided gaze shifts which are thought to recenter the eyes. This finding nicely motivates the subsequent experiments and analyses.

3) Short of that, Figure 3 and 4 is the best evidence to support that there is a change in gaze in mice that is consistent with SC eye-head saccades in non-human primates. A major concern with Figure 3 is that it is dependent on how attempted head movements are measured. If there is any sort of threshold in detectable attempted movements in the force meter, weaker/smaller attempted head movements might show an artificial delayed onset. If spontaneous and evoked saccades are genuinely different, then there should be a detectable difference in latency statistics for *matched magnitudes* (not z-scores) of attempted head movements in these two cases. It is possible that spontaneous and evoked saccades are not different, but eye saccades are still genuinely preceding head movements in some cases. Indeed, 30% of spontaneous saccades look just like the evoked saccades with the eye preceding the head. It could be that there is a continuum of gaze changes where smaller/quicker changes have the eye saccade precede the head movement and for larger changes in gaze, the head precedes a resetting of the eye. In that case, it would be important to see it demonstrated in a head-free scenario and/or how it directly relates to SC microstimulation. Figure 2E and 2I is helpful to the case on its own though that the eye movements are not just resetting and maybe could help resolve some of these questions. If the difference holds up in the matched data though, then it would be pretty convincing theses evoked saccades are unique. In that case, maybe the mouse makes faster changes in gaze for more immediate threats, which would definitely be the case for tactile stimulation.

We thank the reviewers for raising this issue and for suggesting analyses whose results we agree are “pretty convincing.” As suggested, we provide an Author response image in which we compare trials matched for the raw attempted head movement magnitude. Because different mice generate different ranges of raw magnitudes of head movements (which is our reason for Zscoring attempted head movements for population analyses), we provide separate plots for 5 mice showing within-session comparisons of interleaved spontaneous and evoked gaze shifts (Author response image 3) . The results from each mouse are consistent with those in Figure 4, with spontaneous saccades on average preceded by slow attempted head movements whereas touch-evoked saccades are not. Thus, this matched analysis shows that spontaneous and touch-evoked gaze shifts differ and that the differences we observe are not related to the amplitudes of the evoked gaze shifts. We agree with the reviewers’ suggestion that the immediacy of tactile threats may warrant faster gaze shifts and raise this possibility in the discussion.

Author response image 3
Ear airpuff-evoked attempted head movement latencies using matched, raw strain gauge measurements for individual mice.

(A) Left, distributions of raw attempted head movement amplitude-matched trials accompanying spontaneous (blue) and ear airpuff-evoked (red) gaze shifts for a single animal. Trials are matched for raw attempted head movement magnitude at 150 ms after saccade onset as used for amplitude analyses in Figure 4. Middle, mean attempted head movement traces for raw magnitude-matched head movements accompanying spontaneous (blue) and ear airpuff-evoked (red) gaze shifts. Saccade onset is at 0 ms. Right, head movement latencies for magnitude-matched head movements accompanying spontaneous (blue) and ear airpuff-evoked (red) gaze shifts. Dashed vertical line indicates saccade onset. (B-E) As in A for four other animals. Medians indicated by blue and red vertical lines. Spontaneous and evoked medians are significantly different for all animals (p< 10-5, permutation test)

We also agree with the reviewers that there are some spontaneous saccades without preceding head movements. However, it is important to note we, like most in the field, use “spontaneous” as a non-specific, catch-all descriptor for any saccade made when the experimenters were not delivering a sensory stimulus. As such, these “spontaneous” saccades likely include saccades evoked by auditory or tactile stimuli not under experimental control, such as sound or vibration from a door closing down the hall or the elevator ascending in the adjacent shaft, or from stochastic activity in the saccadic circuitry, including SC. Likewise, what we quantify as evoked saccades, i.e., those in the post-stimulus period when saccade probability is elevated, undoubtedly includes spontaneous gaze shifts that began before the stimulus was delivered and that were unrelated to the stimulus itself. Thus, we would not expect either population of gaze shifts to be pure.

4) For Figure 4, we recommend to see what happens for optogenetic stimulation during spontaneous saccades as well to see if they are driven by circuitry different than SC. it may be difficult to accomplish this with the same experimental timing structure, but it would still be valuable to have the light on for an extended period for some spontaneous saccades and off for other spontaneous saccades. This might not be that beneficial if any of the suggested Figure 3 analysis suggests there is no practical difference between evoked and spontaneous saccades and only a shift in distributions.

We thank the reviewers for this suggestion. We include Author response images on the effects of weak and strong SC optogenetic stimulation on spontaneous saccades (Author response image 4; Author response image 5). We observed an increase in saccade probability during weak optogenetic stimulation. Saccades generated during weak optogenetic stimulation appear spontaneous-like in that they tend to be preceded by attempted head movements. In contrast, saccades generated during strong optogenetic stimulation resemble touch-evoked saccades in that they are not preceded by head movements. These data suggest that spontaneous saccades may also be modulated by SC.

Author response image 4
Effect of subthreshold ChR2 stimulation on spontaneous saccades.

(A) Saccade probability before and during (blue shading) a 1 second subthreshold ChR2 stimulus. (B) Mean trajectories of spontaneous saccades occurring during LED off (black) and LED on (blue) periods. (C) Mean attempted head movement amplitudes accompanying spontaneous saccades occurring during LED off (black) and LED on (blue) periods. (D) Mean velocities of spontaneous saccades occurring during LED off (black) and LED on (blue) periods. (E) Mean head movement velocities accompanying spontaneous saccades occurring during LED off (black) and LED on (blue) periods. (F, G) Timing of attempted head movements relative to spontaneous saccades occurring during LED off and LED on periods. Each row corresponds to a single gaze shift. Darker shades indicate larger attempted head displacement. Purple hues denote attempted displacement in the same direction as the saccade (ipsiversive; note that this is contraversive relative to stimulated SC), and orange hues denote displacement in the opposite direction of the saccade (contraversive). Dashed vertical line indicates time of saccade onset. Trials are sorted by latency of attempted head movements. (H, I) As in (F, G) but for attempted head movement velocity. (J) Instantaneous fraction of LED off spontaneous saccades with ipsiversive attempted head displacements. (K) As in (J) but for attempted head velocity.

Author response image 5

Effect of suprathreshold ChR2 stimulation on head-eye coupling. (A) Saccade probability during a 40 ms suprathreshold ChR2 stimulus. (B) Mean trajectories of saccades during spontaneous (black) and opto-evoked (blue) gaze shifts. (C) Mean attempted head movement amplitudes accompanying saccades during spontaneous (black) and opto-evoked (blue) gaze shifts. (D) Mean velocities of saccades during spontaneous (black) and opto-evoked (blue) gaze shifts. (E) Mean head movement velocities accompanying saccades during spontaneous (black) and opto-evoked (blue) gaze shifts. (F, G) Timing of attempted head movements relative to saccades occurring spontaneous (black) and opto-evoked (blue) gaze shifts. Each row corresponds to a single gaze shift. Darker shades indicate larger attempted head displacement. Purple hues denote attempted displacement in the same direction as the saccade (ipsiversive, which is contraversive to stimulated SC), and orange hues denote displacement in the opposite direction of the saccade (contraversive). Dashed vertical line indicates time of saccade onset. Trials are sorted by latency of attempted head movements. (H, I) As in (F, G) but for attempted head movement velocity. (J) Instantaneous fraction of trials with ipsiversive attempted head displacements relative to saccade onset for spontaneous (black) and opto-evoked (blue) gaze shifts. (K) As in (J) but for attempted head velocity.

5) It is unclear if the data in the end supports the strong claims of a new type of gaze shift that a) is directed toward the stimulus and b) precedes head movement. In the end, these look much like previously described reset saccades, but with an overshoot that is dependent on head movement amplitude. This by itself could be interesting, as it's possible that this represents what happens in an abrupt head movement from rest – a condition that occurs reliably for airpuff stimuli in a head-fixed mouse, and may be present in freely-moving mice albeit less frequently (and hence not seen in previous studies). This would resemble rapid head movements seen in other species, where the eyes "boost" the saccade by moving more rapidly than the head. However, the fact that these are so clearly coupled to head movement in the new data makes it even more important than before to show that this "new" type of movement is present in freely moving mice, rather than trying to interpret attempted head movements in a head-fixed mouse.

We agree with the reviewers that our claims of a “new” type of gaze shift may have been too strong. We also agree that it would be ideal to examine this behavior in the context of freely moving mice, but do not currently have the ability to do these experiments and could not realistically develop the necessary preparations quickly enough to submit our revision “in a timely manner” as requested. Therefore, we have changed the title of our manuscript, as suggested, and modulated language throughout the revised manuscript to emphasize that our analyses are confined to head-fixed mice.

6) Although the authors now acknowledge that these are not independent eye movements, but coupled to head movements, they continue to make the association between stimulus location and eye movement. For example, they plot eye movement vs stimulus position, but do not show the equivalent head movements vs stimulus position. They also do not test whether spontaneous saccades, which have zero mean offset, are actually "directed" if they are broken up according to head movement. This was requested in the previous reviews – "What is the pattern of head movements resulting from the stimuli? Do stimuli from opposite sides evoke different directions of head movement on average?" and "Which does a better job of predicting the endpoint – head movement or stimulus side? A figure such as 1C-E, but broken up by head movement direction, rather than stimulus side, would test this. Indeed, it would be interesting to see spontaneous saccades broken up in this way". However, no data is presented in the manuscript to address this. In the review response, the authors describe a regression model but this data is not presented in the text, and it's not clear what the regression is based on – direction of head movement, head displacement during the 0.5 secs before the saccade?

In order to clarify this, it is essential that they show figures such as 2A-F, but broken up by head movement direction rather than stimulus side. This includes breaking up the spontaneous saccades, based on direction of movement during the saccade. Another direct comparison would be side-by-side scatter plots of saccade endpoint vs stimulus side, and saccade endpoint vs head movement direction (and one could do R2 on these plots, rather than a separate regression model across conditions). it can be expected that these will show that head movement predicts the eye movements better than stimulus side does, particularly in cases such as whisker puff. If so, the eye movements are not truly sensory guided or directed toward the stimulus, but are accompanying head movements evoked by getting airpuffed or whiskers touched. Likewise, breaking up spontaneous saccades based on direction of movement will likely show that they are "directed" even in the absence of sensory input.

We thank the reviewers for the analyses suggested and inspired by this comment. Specifically, this comment asks us to illustrate head movements elicited by stimuli, now added to Figure 3, for figures showing eye movements broken up by head movement direction (Author response image 6) , and for R2 values of regression models predicting saccade endpoint from stimulus side versus head movement direction. As expected, because head and eye movements are highly correlated, head movements mirror eye movements and thus predict eye movements better than stimulus side for many individual stimuli. The reviewers suggest that such a result would indicate that “eye movements are not truly sensory guided or directed toward the stimulus, but are accompanying head movements evoked by getting airpuffed or whiskers touched”. We respectfully disagree that this correlation implies causation for the following reasons, which we elaborate on below: (1) whisker and ear airpuff trials matched for head movement direction and amplitude yield well-separated saccade endpoint distributions, indicating that head movements alone do not predict saccade endpoints; (2) head movements are worse at predicting saccade endpoints than are stimulus locations for multiple stimuli; (3) the distribution of saccade endpoints for a stimulus reflects the relationship between starting eye position and evoked saccade amplitude, which differs across stimuli; (4) head movement direction, amplitude, and probability depend on initial eye position, suggesting that head movements do not simply “accompany” saccades.

Author response image 6

Endpoints and trajectories of sensory-evoked saccades organized by head movement direction. (A-D) Endpoints for ear airpuff-, whisker airpuff-, ear tactile-, and auditory airpuff-evoked saccade organized by head movement direction. Top, schematics of stimuli. Middle, scatter plots showing endpoints of all saccades for all animals (n = see below, 5 animals) made spontaneously (blue) and during left (green) and right (magenta) attempted head movements. Darker shading indicates areas of higher density. Bottom, histograms of endpoint distributions for spontaneous and evoked saccades. (E) As in (A-D) but with endpoints for spontaneous saccades organized by head movement direction. (F-J) Trajectories of individual stimulus-evoked saccades. Each arrow denotes the trajectory of a single saccade. Saccades are sorted according to initial eye positions, which fall on the dashed diagonal line. Saccade endpoints are indicated by arrowheads. Because the probability of evoked gaze shifts differed across stimuli, data for ear and whisker airpuffs are randomly subsampled (15% and 30% of total trials, respectively) to show roughly equal numbers of trials for each condition. Saccade numbers in A-J: ear airpuff sessions, spontaneous = 7146, evoked left head movement = 951 (143 in E), evoked right head movement = 1204 (181 in E); whisker airpuff sessions: spontaneous = 7790, evoked left head movement = 486 (146 in F), evoked right head movement = 560 (168 in F); ear tactile sessions, spontaneous = 6706, evoked left head movement = 167 evoked right head movement = 152; auditory sessions, spontaneous = 10240 evoked left head movement = 134, evoked right head movement = 164; spontaneous = 7146, spontaneous left head movement = 3565 (171 in J), spontaneous right head movements = 3581 (168 in J).

To test the hypothesis that head movements explain saccade endpoints, we performed an additional analysis in which we matched trials by attempted head movement direction and amplitude across stimuli. If the distribution of head movements determines the distribution of saccade endpoints, then saccade endpoints for different stimuli should be similar if we compare trials matched for head movement direction and amplitude. Comparisons of matched left whisker/ear airpuff trials and comparisons of matched right whisker/ear airpuff trials yielded well separated endpoints (left ear: -3.93 ± 4.42° vs. left whiskers: -1.56 ± 3.93°, p <10-9, n = 253 matched trials; right ear: 4.47 ± 3.00° vs. right whiskers: 2.05 ± 3.33°, p <10-24, n = 403 matched trials; mean ± s.d., Welch’s t-test) (Figure 4—figure supplement 4). This illustrates that the saccade endpoint differences between stimuli are not simply the result of head movement differences between stimuli and argues there are other differences in the gaze shifts evoked by different stimuli.

Because of the strong correlation between saccades and attempted head movements and the symmetry of responses to a given stimulus on opposite sides, it is not surprising that head movement direction is as good or better than stimulus side at predicting saccade endpoints for left and right delivery of a single stimulus. However, this correlation does not indicate causation. For example, if the only association between stimulus location and saccade endpoint is that saccades accompany head movements, then it should be true that head movements alone are also able to predict saccade endpoints across stimulus locations. To test this, we pooled equal numbers of left and right whisker and ear airpuff-evoked gaze shifts (1046 of each) and asked whether saccade endpoints were better predicted by head movement direction and amplitude or stimulus location. These analyses show that stimulus location is a better predictor of saccade endpoint (R2 = 0.537) than is attempted head movement direction (R2 = 0.416) or even head movement direction and amplitude (R2 = .480).

The preceding results suggest that saccade endpoints are not simply the result of biases in evoked head movements. In our revision, we present data suggesting that saccade endpoint differences between stimulus types and locations are due to differences in the relationship between starting eye position and saccade amplitude (Figure 5). For example, a 5° saccade can be directed towards central or peripheral endpoints depending on the initial position of the eyes from which it is made. It follows that changing the average initial eye position from which a 5° saccade is made will shift the distribution of saccade endpoints accordingly. This is exactly what we observe. This is most clearly illustrated by comparing the relationship between initial eye position and saccade amplitude for left and right ear airpuffs. The lines of best fit for these distributions are well separated, consistent with the resultant endpoint separation. In contrast, the lines of best fit for left and right whisker airpuffs are less separated.

Lastly, we present additional data showing that attempted head movement direction and amplitude depend on initial eye position (Figure 5, Figure 5—figure supplement 4). In contrast, both saccade and attempted head movement direction and amplitude depend weakly if at all on initial attempted head displacement (Figure 5—figure supplement 4). This dependence of attempted head movements on initial eye position argues that eye movements do not simply “accompany” head movements. We contend that a more parsimonious explanation is that evoked head and eye movements are jointly specified to account for stimulus location and starting eye position. This explanation is further supported by our data showing that evoked head and eye movements are coincident and SC-dependent. Nevertheless, it is possible that these relationships are an artifact of head fixation and we acknowledge this in our discussion.

7) A major claim is that the evoked saccades precede head movements, whereas for spontaneous the head movements precede saccades. This claim is problematic for several reasons. A) For spontaneous, the authors are including movement over the previous 0.5secs, which are slow movements and therefore likely unrelated to the saccade. Saying that movement 0.5 secs before a saccade is "head movements preceding the saccade" is very misleading. B) For both spontaneous and evoked, it's clear that there is a fast movement that occurs right around the time of the saccade. Indeed, the plots of velocity (Figure 3D) show almost identical peaks for evoked and spontaneous, suggesting that these represent the same eye-head coupling, just that evoked saccades do not have movement previous to the stimulus (not surprising, since mice don't know when the stimulus will occur). C) In the plots of velocity, it is clear that both eye and head velocity both rapidly increase right at stimulus onset. Thus, although the peak of head movement is later than eye (presumably due to physical/motor constraints) the onset of the two is nearly simultaneous. Indeed, Figures3E,F would be much more straightforward if presented in terms of head velocity, rather than displacement. Furthermore, calculating latency relative to a threshold on velocity (rather than position), or time of peak head velocity, would likely reveal much closer coupling. Together, these factors make the argument about eye saccades following vs leading very weak. It also points out the challenge in interpreting head movements in a head-fixed animal.

We thank the reviewer for pointing out the potential flaws in our methods for measuring head movement latencies during spontaneous and evoked saccades. In particular, as the reviewer notes in (A), we agree that latency statistics based on event detection on continuous signals, such as from the load cells, are potentially problematic, and as such have removed the latency measurements in Figure 3G (now Figure 4G). We also agree there is no predictive power of slow head movements at 500 ms prior to saccade onset—as is customary in the field, we chose to show a time window that starts at a baseline timepoint well before movement onset. However, we respectfully disagree with the statement that slow head movements are unrelated to the saccade. In place of latency statistics, Figure 3G (now 4G) now shows a plot demonstrating that slow head movements are predictive of saccade direction starting roughly 200 milliseconds before onset of spontaneous but not evoked saccades. Likewise, we have amended panels 3E and F (now 3E and F) to illustrate whether head displacements are in the same or opposite direction as the saccade. These data more clearly illustrate that presaccadic head displacements are strongly predictive of spontaneous but not evoked saccade direction. In response to the suggestion that we use head velocity rather than displacement in our analyses, we now present both head displacement and velocity for spontaneous and evoked saccades. Although noisier, the velocity data similarly show that, starting roughly 200 ms before saccade onset, slow head movements tend to precede and predict the direction of the ensuing spontaneous saccades.

In addition, we agree with the reviewers’ suggestion in (B) that the fast attempted head movement occurring right around the time of the saccade is similar for spontaneous and evoked gaze shifts. We have attempted to more clearly explain in our revision that the major difference between spontaneous and evoked gaze shifts is the presence or absence of slow-phase movements preceding the saccade.

We respectfully disagree that it is not surprising that there is no preceding head movement since mice don’t know when an airpuff stimulus would occur. Mice also do not know when a visual stimulus will occur. However, visually evoked gaze shifts (e.g., as shown in Figure 6G of Meyer et al., 2020) start with slow head movements followed 100-200 ms later by saccades coupled to fast head movements, similar to spontaneous gaze shifts. Thus, it is quite surprising that touch-evoked gaze shifts do not begin with slow attempted head movements followed 100200 ms later by saccades. Instead, we found, touch-evoked saccades begin on average 30 ms after stimulus delivery, roughly coincident with a fast attempted head movement.

Finally, we agree with (C) that the onsets of saccades and the fast phase of attempted head movements are very close. As the SC microstimulation data we have added to the revision suggest, this close coupling is likely the result of a shared motor command, analogous to what Bizzi and colleagues first described in primates. We therefore place less emphasis in our revision on estimating the precise temporal ordering and instead emphasize that the main difference between spontaneous and evoked gaze shifts is that the latter lack a slow presaccadic head movement. To this end, we have retitled this figure. In addition, we have amended our discussion accordingly.

8) The authors clearly demonstrate that there is a higher probability of eye saccade when the eye is initially offset in the opposite direction (Figure 2I-J). This is exactly what one would expect for a reset saccade resulting from head movement, as the eye is reaching the end of its range in that direction. Likewise, they show that amplitude of the eye movement is equal and opposite to initial eye position (Supp Figure 8), again exactly what one would expect for a reset saccade.

This is an interesting point and one we now mention in our revised discussion. Absent freely moving data, we agree it is important to entertain alternative explanations, such as whether these saccades are resetting. However, we consider this explanation less plausible for several reasons. First, resetting saccades occur to compensate for the eye nearing the end of its range of travel during the slow (100-200 ms) presaccadic head movement (e.g., see Meyer et al., 2020, Figure 5C). As evoked saccades are made at very short latency and there is typically not a presaccadic slow attempted head movement, it is not clear that there are slow counter-rotatory eye movements for which mice would need to reset the eyes. Second, the probabilities of ear airpuff-evoked eye movements are higher when the eyes begin in the center than when the eyes begin on the side towards which the eyes are moving; it is unclear why a reset saccade would be necessary from an already central eye position. In addition, from central eye positions, left and right ear airpuffs evoke saccades in opposite directions, arguing this cannot be the end of the range of travel in either direction. Moreover, for other stimuli, saccade probability as a function of eye positions differs across stimuli such that the probability is lowest for initial eye positions that correspond to the mean endpoint of saccades elicited by that stimulus (Figure 5— figure supplement 2). Third, Figure 5 shows that different stimuli evoke saccades with different endpoints by using different linear relationships between saccade direction/amplitude and initial eye position. This means that saccade amplitude is equal and opposite to the initial eye position only for stimuli that evoke saccades with fairly central endpoints. Instead, for stimuli that evoke saccades with more eccentric endpoints, the amplitude is not equal and opposite to the distance from the center, which the reviewer notes one would expect for a reset saccade. We thank the reviewers for raising this analysis and spurring us to more clearly illustrate these relationships. Nevertheless, because our data are from head-fixed mice, it is possible that there are differences in freely moving mice and we attempt to more fully acknowledge this possibility in the revision.

9) The endpoint offset relative to center seems to be directly proportional to the head movement amplitude (Supp Figure 6A-D). Combined with the previous point, this suggests that these are similar to previously described saccades, but with an overshoot that is determined by head movement amplitude (rather than stimulus location). Such a mechanism makes sense, since for large head movements this would allow greater dynamic range for the ensuing compensatory phase. Alternately, it could be that this overshoot occurs in head-fixed mice where other feedback mechanisms are lacking.

This is an interesting idea and one that we explored but failed to mention in our previous revision. The endpoint offset relative to the center is not proportional to the head movement amplitude. For example, ear airpuff stimuli elicit saccades directed to average endpoints roughly ninefold more eccentric than those elicited by whisker airpuffs (average eccentricity 5.4° vs. 0.6°) but ear airpuffs elicit attempted head movements that are only roughly twice as large (Figure 3; Figure 4—figure supplement 1A, B, red traces in middle right panels). In addition, ear tactile stimuli and auditory airpuffs elicit roughly equal distributions of attempted head movement amplitudes (Figure 4—figure supplement 1C, D, red traces in middle right panels) even though ear tactile-evoked saccades have eccentric (on average, 2.7°) and well separated endpoints whereas auditory airpuff-evoked saccades have central (on average, 0.1°) and minimally separated central endpoints (Figure 3C, D). Moreover, we have performed an analysis to control for any differences in attempted head movement amplitudes by comparing trials with matched head movement amplitudes across stimulus conditions. In this analysis, whisker and ear airpuffs continue to evoke well-separated saccade endpoint distributions, with ear airpuff-evoked saccade endpoints far more eccentric (Figure 4—figure supplement 4). As noted previously, the differences in these saccade endpoints at least partly reflect differences in the initial eye positions from which these gaze shifts were made. Nevertheless, we agree that there are caveats related to head-fixing, including the lack of feedback mechanisms, and address this possibility in the revised discussion.

10) The authors make the claim that head movements depend on eye movement, which would be quite a remarkable finding. However, the actual data is buried in the supplement, and turns out to be a very weak correlation, driven by a small number of outliers with unusually large head movements. Furthermore, only one stimulus condition is presented for this analysis, suggesting that it was not significant for other conditions. Finally, it is easy to envision situations where a weak correlation could arise without a causal role of eye position. For example, if the mouse simply alternates between left and right eye movements on each trial – after a left head movement, the eye would tend to be on the left, so on the subsequent trial when it makes a head movement to the right it would appear to be driven by eye position, even though it's just due to a simple behavioral strategy. If the authors want to support this claim, they should show that it applies for all conditions together, that it is consistent across animals (this is a nested design, and it appears that they used N as # of saccades, rather than # of animals), and that it does not depend on extreme head movements that may represent aberrant conditions.

We thank the reviewer for this suggestion. As requested, we present data for each condition both for the population and individual animals after removing “outlier” trials with large head movements (larger than 3 standard deviations) (Figure 5, Figure 5—figure supplement 1, Author response image 7). We find that across the population, whisker airpuffs, auditory airpuffs, and ear tactile stimuli show a clear relationship between initial eye position and attempted head movement direction and amplitude. These effects are also consistent across animals, particularly for stimuli with central endpoints, which elicit mixtures of ipsiversive and contraversive saccades (whisker airpuffs and auditory airpuffs) (Author response image 7) . These effects on amplitude and direction are more variable and less evident for ear airpuff-evoked saccades and only significant for one side, perhaps because ear airpuffs evoke nearly exclusively contraversive saccades. Nevertheless, it is clear that initial eye position influences ear airpuff-evoked head movements as well because the probability of evoked head movements varies with eye position and in a manner that mirrors saccade probability (Figure 5). Thus, for every stimulus we tested that elicited gaze shifts, initial eye position influences direction, amplitude, and/or probability of attempted head movements. Interestingly, initial eye position has a similar effect on spontaneous saccades, suggesting that these gaze shifts may also take into account initial eye position. We thank the reviewer for suggesting ways to more convincingly illustrate and for their kind words regarding what we agree is “quite a remarkable” finding.

Author response image 7
Evoked attempted head movement amplitude varies according to initial eye position across individual mice.

(A-D) Attempted head movement amplitude as a function of initial eye position for left and right stimulus-evoked gaze shifts for an example animal. Each dot corresponds to a single gaze shift. (E-F) Summary of the slopes of the lines of best fit for initial eye position vs. attempted head movement data for 5 mice after removing outlier head movements. Asterisks indicate p < 0.05 using Student’s t-test.

Regarding the idea that mice are alternating leftward and rightward saccades, it is worth noting that we deliberately do not present the stimuli in an alternating pattern to prevent mice from using strategies such as this. Instead, stimuli are presented in a pseudorandom sequence (e.g., in sample trace in Figure 1), such that a left stimulus is equally likely to be followed by a left or a right stimulus, and vice versa. Thus, a strategy of alternation could not produce the results we observe. In addition, stimuli are presented infrequently, mice do not saccade in response to every stimulus presentation, and between stimulus presentations mice often make spontaneous saccades. The most parsimonious explanation we can offer is that eye position is the driver of these effects, but we agree that without causal studies of the role of eye position this relationship could be correlative and driven by other unidentified factors.

11) If these are sensory-guided directed saccades, then why do stimuli at the same location result in different saccade locations (2A vs C) and some saccades are directed away from the stimulus (clearly seen in 2G)? At best, these should be described as "directionally biased" rather than "directed".

We thank the reviewer for raising this issue. There are several candidate reasons that the ear airpuff and tapper stimuli in Figure 2A and C (now Figure 3A and C) have slightly different endpoints. First, it is difficult to precisely ascertain the relative locations the airpuff and tapper bar stimulate. It is possible that they stimulate different parts of the ear. They also approach the ear from different directions and motion direction may factor into saccade selection. Second, the airpuff stimulus is multisensory, consisting of a mix of a tactile stimulus and an auditory stimulus, and we do not know how these modalities synergize. Third, it is likely that the intensities of these stimuli differ, which may underlie the difference in saccade probabilities, and it is possible that stimulus location and intensity jointly specify saccade parameters. Fourth, and relevant to the comment regarding Figure 2G (now Figure 3G), the arrow plot shows every saccade that occurred in a short time window following stimulus delivery. As noted in a previous response, since the stimuli do not evoke saccades on every trial, and in some instances a spontaneous gaze shift has begun before stimulus delivery, these plots include some spontaneous saccades, which are on average recentering. Given that spontaneous saccade probability is fairly fixed, we would expect a larger fraction of the saccades made in this peristimulus period to be spontaneous (i.e., net recentering) for stimuli that evoke saccades less frequently, e.g, the air tactile stimulus relative to the ear airpuff stimulus. This appears to be the case. In addition, the inclusion of more spontaneous saccades, which are on average recentering, may partly underlie the central shift in the distribution of endpoints of ear tactile-evoked saccades.

12) However, a remaining concern is the claim that the data reveal "A new type of mouse gaze shift is led by directed saccades" as stated in the title. The data are fully consistent with the "saccade and fixate" pattern that is widely shared across species (e.g., Land (2019)) and which has recently been identified in freely-moving mice (Michaiel et al. 2020; Meyer et al. 2020). The present study shows that in mice this pattern appears more flexible than previously thought which is a new and important finding. Nevertheless, the rather small (yet important) variations compared to the freely-moving data might not justify the classification as a new type of gaze shift. In particular, Figure 3 suggests that the relative initial contributions of the head and eyes to gaze shifts lie along a continuum and even spontaneous saccades (the baseline condition) can "lead" attempted head rotations; for airpuff-evoked saccades, the head closely follows those saccades (median latency of 30 ms after saccade onset) which corresponds to about half the typical saccade duration (e.g., Sakatani and Isa (2005)). In other words: the saccade and fixate head-eye coupling is preserved for the airpuff-evoked saccades but the relative contributions of the head and eyes are shifted along the continuum. The worry is that the claim of a "new type of mouse gaze shift" might lead to a misperception of this study that shows stronger flexibility of an existing, but not a new, gaze pattern in mice.

We agree that our claims were somewhat strong and have modulated our descriptions throughout, including the title.

13) Figure 2 could use some improvement. They should probably plot saccade start vs saccade end (this data should help their case, but it is presented awkwardly). They should also include average saccade sizes (with direction as sign) versus starting points. Attempted head movement data needs to be looked at in all these scenarios too. It is unclear how well a single trial attempted head movement can be captured. Individual trials look pretty noisy, but the average dynamics look pretty informative. Results may be similar to the eye movements.

We thank the reviewers for this suggestion. Although the arrow plots previously shown in Figure 2 (now Figure 3) do indicate saccade start vs. saccade end, we have added scatter plots of initial eye position vs. saccade amplitude to Figure 5, plots of average saccade sizes and endpoints in Figure 5—figure supplement 1, and scatter plots of initial eye position vs. saccade endpoint in Figure 3—figure supplement 1. We include in Figure 5 and Figure 5—figure supplement 1 scatter plots and summary data of attempted head movements as a function of eye position. As the reviewers predicted and as discussed in more depth in previous responses, the results are very similar to those for eye movements.

14) Another concern is that head-fixed non-human primates will learn to reduce attempted head movements over a short period, once they learn it will not accomplish anything. It is possible the results in Figure 3 are from a similar mechanism. The directed saccades might have provided more reinforcement for learning the failure to move and that is why you rarely see the attempted head movement before the saccade (and why attempted head movements overall appear to be weaker). In addition, spontaneous saccades may decrease over time as the animal is habituated to the experiments and therefore may include fewer saccades during the post-learning period. We recommend that the data from Figure 3 therefore be plotted over time to see if anything observed is from an effect of learning.

We thank the reviewers for this suggestion. We include analyses in our revision that examine both within sessions (dividing each into 15-minute segments) and across sessions (Figure 4— figure supplement 3). Interestingly, touch-evoked head movements are significantly smaller on the fifth session than on the previous 4 sessions (p < 0.05) but spontaneous head movements are not changed. However, in every time segment of every session (15/15), evoked saccades are coupled to smaller head movements than are spontaneous saccades. Therefore, although we thank the reviewers for raising this interesting idea and find that mice appear to gradually learn the futility of evoked attempted head movements, the differences in attempted head movement amplitudes for evoked and spontaneous gaze shifts are not attributable to learning.

https://doi.org/10.7554/eLife.73081.sa2

Article and author information

Author details

  1. Sebastian H Zahler

    1. Department of Anatomy, University of California, San Francisco, San Francisco, United States
    2. Neuroscience Graduate Program, University of California, San Francisco, San Francisco, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review and editing
    Contributed equally with
    David E Taylor
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0089-3593
  2. David E Taylor

    1. Department of Anatomy, University of California, San Francisco, San Francisco, United States
    2. Neuroscience Graduate Program, University of California, San Francisco, San Francisco, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review and editing
    Contributed equally with
    Sebastian H Zahler
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0476-0299
  3. Joey Y Wong

    Department of Anatomy, University of California, San Francisco, San Francisco, United States
    Contribution
    Investigation, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3697-8951
  4. Julia M Adams

    Department of Anatomy, University of California, San Francisco, San Francisco, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1402-1040
  5. Evan H Feinberg

    1. Department of Anatomy, University of California, San Francisco, San Francisco, United States
    2. Neuroscience Graduate Program, University of California, San Francisco, San Francisco, United States
    3. Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, San Francisco, United States
    Contribution
    Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review and editing
    For correspondence
    evan.feinberg@ucsf.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7040-0980

Funding

National Institute of Mental Health (DP2MH119426)

  • Evan H Feinberg

National Institute of Neurological Disorders and Stroke (R01NS109060)

  • Evan H Feinberg

Simons Foundation Autism Research Initiative (574347)

  • Evan H Feinberg

Esther A. and Joseph Klingenstein Fund

  • Evan H Feinberg

E. Matilda Ziegler Foundation for the Blind

  • Evan H Feinberg

Whitehall Foundation

  • Evan H Feinberg

Brain and Behavior Research Foundation (25337)

  • Evan H Feinberg

Brain and Behavior Research Foundation (27320)

  • Evan H Feinberg

Sandler Foundation

  • Evan H Feinberg

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank M Brainard, J Horton, A Krishnaswamy, M Scanziani, and members of the Feinberg laboratory for helpful discussions and comments on earlier versions of the manuscript. This work was supported by departmental funds and grants from the E M Ziegler Foundation for the Blind, Sandler Foundation, Klingenstein-Simons Fellowship Award in Neuroscience, Brain and Behavior Research Foundation (NARSAD Young Investigator Awards 25,337 and 27320), Whitehall Foundation, Simons Foundation (SFARI 574347), and US National Institutes of Health (DP2 MH119426 and R01 NS109060) to EHF.

Ethics

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the University of California San Francisco Institutional Animal Care and Use Committee (IACUC) (protocol number AN176625), and were conducted in agreement with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Senior Editor

  1. Tirin Moore, Stanford University, United States

Reviewing Editor

  1. Martin Vinck, Ernst Strüngmann Institute (ESI) for Neuroscience in Cooperation with Max Planck Society, Germany

Publication history

  1. Preprint posted: February 11, 2021 (view preprint)
  2. Received: August 16, 2021
  3. Accepted: December 27, 2021
  4. Accepted Manuscript published: December 31, 2021 (version 1)
  5. Version of Record published: January 10, 2022 (version 2)

Copyright

© 2021, Zahler et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Sebastian H Zahler
  2. David E Taylor
  3. Joey Y Wong
  4. Julia M Adams
  5. Evan H Feinberg
(2021)
Superior colliculus drives stimulus-evoked directionally biased saccades and attempted head movements in head-fixed mice
eLife 10:e73081.
https://doi.org/10.7554/eLife.73081

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