Widespread cortical representations of innate behaviors in the mouse

Reviewer #1 (Public review):

In their manuscript, Michelson et al use a combination of mesoscopic 1p and single-cell resolution 2p imaging to characterise cortical encoding of grooming behaviour. Despite their subcortical locus of control (and non-reliance on cortex), the authors report that grooming movements are accompanied by widespread activation of dorsal cortex. Different grooming movements elicit distinct spatiotemporal cortical activity patterns. They find that cortical engagement is greater at the beginning of grooming episodes than at their end. They also report greater cortical activation for atypical unilateral grooming movements seen under head-restraint in comparison to cortical activity during bilateral movements typical of unrestrained or spontaneous grooming.

While this is not the first study to report cortical representations of subcortically controlled behaviours, and the authors themselves cite many previous reports of cortical activation during locomotion and even grooming (Sjöbom et al 2020), the value of the present study lies in their use of imaging to reveal the widespread nature of cortical activation during execution of a complex, innate behaviour. I also appreciate the systematic approach used by the authors to break down grooming episodes into their constituent movements and reveal their transition structure.

I do have concerns, however, that some of the authors' claims are insufficiently supported by their results, and more analysis is required to convincingly rule out alternative interpretations.

(1) One possible explanation for the gradual decline in cortical activity is that unilateral movements associated with greater cortical activation dominate early in grooming episodes, whereas bilateral movements that elicit weaker cortical activity dominate later (Figure 3G and 2C). The authors could check whether cortical activity associated with the *same* grooming movement is constant or declines during such episodes. A related point: doesn't the regression analysis shown in Figure 3, Supplement 2, assume that a stationary relationship between movement and spatiotemporal patterns of cortical activity?

(2) From the decline in cortical responses during long grooming episodes, the authors suggest that "mesoscale cortical activity mostly reflects the initiation of subcortically-mediated behaviors, rather than the behavior itself". The authors have taken a lot of trouble to come up with a rich, detailed segmentation and clustering of the grooming behaviour into its constituent movements (Figure 1). Therefore, I am somewhat surprised that they make this claim solely from analysis of averaged cortical activity during nearly minute-long grooming episodes rather than a higher time resolution analysis of transitions between distinct grooming movements (like the prior study by Sjöbom et al and related work in striatal encoding of innate movement sequences by Markowitz et al).

(3) The authors find that unilateral, atypical grooming movements elicit cortical activity that is distinct from the more naturalistic bilateral movements. They interpret this as reflecting the temporal transition structure of the behaviour. However, an alternative explanation is that the differences (or similarities) in evoked activity simply reflect differences (or similarities) in the kinematics of these movements, with bilateral movements appearing more similar to each other than to unilateral movements. A related point: there is little description of the "non-grooming forelimb movements". Were these kinematically similar to the unilateral forelimb movements, which may explain why they cluster together in Figure 4H?

(4) Page 13, last paragraph: the authors suggest that similar encoding of non-grooming forelimb movements and unilateral grooming movements may reflect a shared reliance on the cortex. This is rather speculative. Several studies have demonstrated that voluntary unilateral movements employed for reaching or lever pressing are not generally reliant on the cortex (Whishaw et al, Beh Brain Res, 1991; Kawai et al, 2015). There isn't, in my opinion, a broad consensus for the authors' statement that "reaching for food is a cortex-dependent action". Rather than extrapolating from past studies, could the authors not experimentally assess whether unilateral grooming movements are more sensitive to cortical silencing than bilateral ones, possibly revealing a cortical locus of control?

Reviewer #2 (Public review):

Summary:

In this manuscript, Michelson, Gupta, and Murphy use calcium imaging to map the distribution of neural activity across the cerebral cortex of grooming, head-restrained mice. Animals groomed spontaneously and in response to wetting of the face. Individual movement elements, such as bilateral strokes across the face, resembled those observed in freely-moving animals. Sequencing of movement elements was structured, but did not consist of full "syntactic grooming chains." Widefield imaging across the cortex revealed distinct patterns of activity for distinct movement elements. Individual neurons responded strongly during movement and had largely similar properties across cortical areas.

Strengths:

In my opinion, this is a solid paper that will be of interest to the mouse sensorimotor neuroscience community. The experiments are technically sound, the text is well-written, and the figures are clear. The activity maps are presented in standardized Allen Atlas coordinates, and I expect they will be very useful for future studies of orofacial and limb movement.

Weaknesses:

While the manuscript provides a valuable description of cortical activity during head-restrained grooming, I think it could engage a bit more with contemporary theories and debates in cortical physiology and motor control. The Abstract nicely highlights an apparent paradox: the motor cortex sends strong projections to the spinal cord, and is strongly modulated during behaviors like grooming. Nevertheless, blocking corticospinal traffic by inactivating or lesioning the motor cortex leaves such behaviors intact. There are several potential resolutions to this paradox. First, cortical activity during grooming could be confined to an "output-null" subspace that is responsible for monitoring sensorimotor events and preparing voluntary movements, but does not drive muscle activity (c.f. work in the macaque: Kaufman et al., Nature Neuroscience 2014; Churchland & Shenoy, Nature Reviews Neuroscience 2024). Second, cortical activity during grooming could be transmitted to lower centers, but gated out through inhibition. Third, it is possible that cortical activity in intact animals does contribute to muscle activation during grooming, but following a lesion or inactivation, other descending pathways compensate for the cortical deficit. The authors might wish to discuss their findings in light of these considerations.

In the first paragraph of the Introduction, it could be made clearer which results are specific to mice. The Niell & Stryker finding, for example, holds in mice, but not marmosets (Liska et al., eLife 2024).

The "hotspots" in Figure 3G appear to be more anterior during bilateral elliptical than unilateral elliptical movements. How do the authors interpret this finding?

The distribution of single-neuron responses looks relatively similar across cortical areas, including forelimb, hindlimb, and trunk somatosensory cortex, and primary and secondary forelimb motor cortex. What do the authors make of this?

Reviewer #3 (Public review):

Summary:

The authors use a combination of a head-fixed grooming paradigm, single-photon mesoscale, and wide-field-of-view two-photon calcium imaging to characterize cortical activity patterns during evoked grooming. Previous work has shown that grooming behavior does not require cortex, but that there are neuronal representations of grooming in motor cortex. The authors extend these findings by showing cortex-wide activation patterns at the meso-scale that relate to distinct grooming elements. This activation is strongest at grooming onset, but declines over the course of extended grooming periods. They also find similar activity patterns during licking/drinking behavior. Two-photon imaging further revealed that individual neurons across the cortex are preferentially activated by grooming. While their activity also declines after grooming onset, they remain active throughout grooming periods. This work extends previous findings by revealing that grooming and other subcortically-generated behaviors may be represented not only in motor cortex, but across dorsal cortex, both on the mesoscale and single neuron levels. These findings may lead to further investigation into the role of cortical activity during subcortically generated behaviors.

Strengths:

(1) Detailed characterization of grooming behavior in a head-fixed paradigm.

(2) Combination of single photon mesoscale and two-photon wide field-of-view imaging to characterize grooming (and licking)-related activity across dorsal cortex on multiple levels

Weaknesses:

(1) The behavior observed in the head-fixed grooming paradigm only partially resembles spontaneous grooming, lacking typical elements of the syntactic chain, while additionally evoking non-typical behaviors, resembling unilateral reaches, making the interpretation of the observations and their relevance to natural behaviors difficult. Furthermore, the nature of the non-typical movements (which may be cortex-dependent while typical grooming is not) is not explored.

(2) Two important findings in relation to the neural representations of individual grooming behaviors remain unclear:

a) The authors state that individual grooming behaviors did not have distinct neuronal representations (except unilateral grooming; Figure 4G) - it remains unclear how this fits with the observation of distinct activation maps during the different grooming behaviors. Should this differential activation not also correspond to distinct activation patterns of 'grooming' neurons across the cortex? Or do they mean that the activity in the 'grooming' neurons is not consistent across grooming instances and therefore no distinct representation can be detected?

b) The authors state that the 'typical' grooming behaviors do not have consistent activation patterns across animals (Figure 3 and supplements). It remains, therefore, unclear what the averaged activation maps really represent. Furthermore, this observation leaves several open questions: Are the activation patterns consistent in individual animals? Do differences across animals emerge due to differences in their behavior? And most importantly, can the actual behavior be decoded from the activation patterns?

(3) Multiple statements/conclusions are not supported by quantification of the data, but only by qualitative assessments, e.g.: lines 433-435: "In general, the maximally activated networks involved in licking and unilateral grooming behaviors 'appeared' to be the most consistent across animals compared to the bilateral grooming movements (Figure 3G)."; 436-437: "Averaged cortical activation maps associated with licking and elliptical behaviors were 'qualitatively similar' between evoked and spontaneous sessions, where the water drop was not applied".; 480-482: "The unique explained variance maps for the licking behavior 'differed' in the drinking context compared to the grooming context (Figure 3-figure supplement 3F)." The lack of quantification leaves the significance of these observations unclear.

(4) It remains unclear what the ongoing activity in 'grooming' neurons represents, since there is no detailed analysis of the relationship between activity and the detailed kinematics of the grooming movements.

The authors show that neuronal representations of grooming and other subcortical behaviors can be found across dorsal cortex and that these representations are at least to some degree specific to distinct behavioral elements. While this study does not reveal functional insights into the role of cortical representations of subcortically-generated behaviors, it is a step towards more in-depth studies. In the future, it will be important to determine whether these representations are efference copies or sensory-driven, or whether they affect the behavior, and if so, under which circumstances.