Optogenetic LC stimulation and stress trigger similar responses in astrocytes but not in excitatory or inhibitory neurons.

A, Schematic representation of the viral strategy and experimental approach using fiber photometry recordings in hippocampus and optogenetic LC stimulation during behavior. B, Schematic and histology example of LC targeting using DBH-iCre transgenic mice together with injection of Cre-dependent AAV with ChrimsonR-tdTomato (red). Scale bar, 100 μm. C, Schematic and histology example of HC targeting using injection of AAV with, in this case, GFAP-GCaMP6s (green) to target astrocytes, visualized together with nuclear stain (blue, DAPI). Scale bar, 100 μm. D, Schematic representation of the experiments with corresponding signals below. E-I, NA release in response to (E) a 10-s tail lift (n = 10), (F) a 3-s foot shock (n = 10), (G) optogenetic 10-s 5-Hz LC stimulation (n = 5), and (H) 10-s 20-Hz stimulation (n = 5). Comparison of the peak ΔF/F in (I). J-N, Astrocytic Ca2+ in response to (J) a 10-s tail lift (n = 6), (K) a 3-s foot shock (n = 6), (L) optogenetic 10-s 5-Hz stimulation (n = 6), and (M) 10-s 20-Hz stimulation (n = 6). Comparison of the peak ΔF/F in (N). O-S, Pyramidal cell Ca2+ in response to (O) a 10-s tail lift (n = 7), (P) a 3-s foot shock (n = 7), (Q) 10-s 5-Hz stimulation (n = 6), and (R) 10-s 20-Hz stimulation (n = 6). Comparison of the peak ΔF/F in (S). T-X, Interneuron Ca2+ in response to (T) a 10-s tail lift (n = 6), (U) 3-s foot shock (n = 6), (V) 10-s 5-Hz stimulation (n = 5), and (W) 10-s 20-Hz stimulation (n = 5). Comparison of the peak ΔF/F in (X).

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Chronic two-photon imaging during optogenetic LC stimulation and spontaneous behavior across neurons and astrocytes

A, Schematic representation of the two-photon recording setup. B, Viral strategy to stimulate the LC and record Ca2+ signals from either astrocytes (orange), excitatory neurons (purple) or inhibitory neurons (turquoise). C, Recording paradigms across the imaging days. D, Top: Representative FOV of triple-plane imaging in astrocytes. Bottom: Example cell tracked across recording days. E, Example recording from astrocytes. From top to bottom: sorted time traces of all cells identified in (D) (n = 310 cells; the vertical scale bar, here and in panels (I) and (M), represents 50 cells); average ΔF/F across cells; body movement and pupil diameter extracted from behavioral tracking video. LC stimulations are indicated with a red shaded bar (10 s stimulation at 3 Hz, 5 Hz or 20 Hz). F, Correlation function of body movement and astrocytic Ca2+ signals, averaged across all animals and sessions in dark color, individual sessions (n = 12) indicated in lighter color. The delay τ measures the average delay (median ± S.D. across sessions) of the calcium signal compared to body movement. G, Mean astrocyte response to 20 Hz LC stimulation, averaged across all stimulations (n = 32 stimulations) and animals (N = 4). H, Top: Representative FOV of single-plane imaging in excitatory neurons. Bottom: Example cell tracked across recording days. I, Example recording from pyramidal cells. From top to bottom: rastermap sorting of time traces from selected cells (n = 170 out of 513 detected cells); spike rate estimate averaged across all detected cells; body movement and pupil diameter extracted from behavioral tracking video. LC stimulations are indicated with a red shaded bar (10 s stimulation at 5 Hz or 20 Hz). J, Correlation function of body movement and pyramidal cell Ca2+ signals, averaged across all animals and sessions (n = 16) in dark color, individual sessions indicated in lighter color. K, Mean pyramidal cell response to 20 Hz LC stimulation, averaged across all stimulations (n = 36 stimulations) and animals (N = 5). L, Top: Representative images of the four-plane imaging in interneurons. Bottom: Example cell tracked across recording days. M, Example recording from interneurons. From top to bottom: sorted time traces of all cells identified in (L) (n = 124 cells); average ΔF/F across cells; body movement and pupil diameter extracted from behavioral tracking video. LC stimulations are indicated with a red shaded bar (10 s stimulation at 5 Hz or 20 Hz). N, Correlation function of body movement and interneuronal Ca2+ signals, averaged across all animals and sessions (n = 14) in dark color, individual sessions indicated in lighter color. O, Mean interneuron response to 20 Hz LC stimulation, averaged across all stimulations (n = 39 stimulations) and animals (N = 4).

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Astrocyte subpopulations exhibit stable, cell-specific responses to LC stimulation that diverge from activity during natural arousal.

A, All astrocytic responses to ≥20 Hz LC stimulation, sorted by the ΔF/F change during the 10 s stimulation window compared to the 20 s window before stimulation, with subselection of the 250 most and least responsive cells. Data pooled from 12 sessions across 4 mice. B, Time-averaged response to a strong stimulation as shown in panel (A) as a function of pre-stimulus ΔF/F (average across 20 s before stimulation). C, Left: Response to two strong LC stimulations within a single session (stimulation 1: x-axis, stimulation 2: y-axis) to investigate within-session stability. Right: Marginal distribution of LC stimulation responses, colorcoded as in panel (D). D, Analysis of stability of astrocytic responses within sessions. Left: Cellular responses to stimulation #1, sorted by most to least responsive cells and plotted as the quantiles shown in panel (C, right). Right: Responses of the same cells to stimulation #2 within the same session, with the sorting maintained from stimulation #1. E, Left: Analysis of stability of astrocytic responses towards LC stimulation for an individual session pair (day N vs. day N+1). Here and in panels (H,I,J), only a single session pair is shown as scatter plot due to large differences between mean average response magnitudes across sessions and animals. Right: Correlation of responses across days for each animal and each session pair. Here and in the following panels, the colored dots correspond to the astrocytes shown in panel (G). F, Left: Analysis of stability of astrocytic activity locked to body movement across a session pair (day N vs. day N+1). Right: Correlation of responses across days for each animal and each session pair. G, Examples of distinct astrocytic response types. Episodes of natural arousal encompassed by body movement are highlighted with gray shading (manual annotation). Episodes of optogenetic stimulation of LC are highlighted with red shading. Simultaneously recorded body movement as a proxy for natural arousal is shown below. The same astrocytes are shown for day 1 (left) and day 2 (right). H, Sigmoid fit of activation in response to increasing LC stimulation frequency for the three selected astrocytes displayed in (G). Activation functions are shown normalized to the saturating response of each astrocyte. I, Response sensitivity of astrocytes to LC stimulation, defined as the ratio of responses to 5 Hz vs. 20 Hz stimulations of LC. Left: Example stability of response sensitivity across two imaging sessions. Right: Pearson correlation coefficient for each session pair for all animals. J, Left: Correlation of responses to LC stimulation and movement modulation. Right: Pearson correlation coefficient for all four animals. K, Example activity traces from one mouse, ΔF/F sorted by LC stimulation response (same segment as for Day 2 in F.). Black arrows indicate bouts of body movement. L, Left: Example of population vector correlations for one recording session comparing movement episodes with instances of 5 Hz and 20 Hz LC stimulation. Right: Average Pearson correlation coefficient for each comparison. Each data point indicates an imaging session.

Pyramidal cells exhibit distinct and stable response patterns during natural arousal but respond uniformly to LC stimulation.

A, All cellular responses to ≥20 Hz LC stimulation, shown as the spike rate estimated from ΔF/F traces and sorted by the spike rate change during the 10 s stimulation window compared to the 20 s before stimulation, with subselection of the 250 most and least responsive cells. Data pooled from 15 sessions across 5 mice. B, Same as in (A) but sorted by the highest activity in 10-s time bins for 60 s before and 90 s after LC stimulation. Neurons with maximum activity outside the displayed time window are not shown. The visualization illustrates the sequential activity patterns of pyramidal cells. C, Quantification of number of neurons in each time bin in panel (B). The number of neurons activated in the 10-s bins following LC stimulation is lower than for any other time bins. D, Change of spike rate from a 10-s pre-stimulus window (x-axis) to the 10-s window of LC stimulation (y-axis). Spike rate changes are shown both for random time points (spontaneous, gray) and 20-Hz LC stimulation time points (red). Compared to spontaneous spike rate changes, LC stimulation induces a reduction of spike rates. E, Left: Response to two strong LC stimulations within a single session (stimulation 1: x-axis, stimulation 2: y-axis) to investigate within-session stability. Right: Marginal distribution of LC stimulation responses, color-coded as in panel (C). F, Analysis of stability of pyramidal cell responses within sessions. Left: Cellular responses to stimulation #1, sorted by most to least responsive cells and plotted as the quantiles shown in panel (C, right). Right: The responses of the same cells to stimulation #2 within the same session, with the sorting maintained from stimulation #1. No evidence for response stability can be observed. G, Example recording of negatively-movement modulated (top) and positively-movement modulated pyramidal cell (below), together with behavioral proxies for arousal (pupil diameter, body movement; bottom). Raw ΔF/F traces (light color) as well as estimated spike rates (dark color) are shown, matching the color-code in panels (J,K). Episodes of natural arousal encompassed by body movement are highlighted with gray shading (manual annotation). Episodes of optogenetic stimulation of LC are highlighted with red shading.The same pyramidal cells are tracked for day 1 (left) and day 2 (right) H, Left: Analysis of stability of pyramidal cells responses towards LC stimulation across sessions (day N vs. day N+1), pooled across all sessions and animals. Right: Correlation of responses across days for each animal and each session pair. I, Left: Analysis of stability of pyramidal cell activity locked to body movement across sessions (day N vs. day N+1), pooled across all sessions and animals. Right: Correlation of responses across days for each animal and session pair. J, Comparison of the cellular responses to natural arousal (body movement) vs artificial arousal (LC stimulation), not exhibiting any visible correlation. Cells modulated positively vs. negatively by body movement are color-coded. The two neurons shown in (G) are highlighted with large dark symbols. K, LC response magnitude of positively-modulated (−0.5 ± 1.9 Hz; median response ± S.D. across neurons). vs. negatively-movement modulated cells (−0.3 ± 1.1 Hz). Color-code as in (J).

Distinct interneuron populations across laminar position within deep CA1 in response to movement and LC stimulation.

A, All interneuron responses to ≥20 Hz LC stimulation, sorted by the response during the 10-s stimulation window, with subselection of the 250 most and least responsive cells. Data pooled from 9 sessions across 3 mice. One mouse was excluded due to prominent LC stimulation-related movement (shown separately in Supplementary Figure S5-1). B, Left: Response to two strong LC stimulations within a single session (stimulation 1: x-axis, stimulation 2: y-axis) to investigate within-session stability. Right: Marginal distribution of LC stimulation responses, colorcoded as in panel (C). C, Analysis of stability of interneuron responses within sessions. Left: Cellular responses to stimulation #1, sorted by most to least responsive cells and plotted as the quantiles shown in panel (C, right). Right: The responses of the same cells to stimulation #2 within the same session, with the sorting maintained from stimulation #1. D, Change of spike rate from a 10-s pre-stimulus window (x-axis) to the 10-s window of LC stimulation (y-axis) as in Figure 4D. Spike rate changes are shown both for random time points (spontaneous, gray) and 20-Hz LC stimulation time points (red). Compared to spontaneous spike rate changes, LC stimulation tends to activate silent and suppresses active interneurons. E, Left: Analysis of stability of interneuron responses towards LC stimulation across sessions (day N vs. day N+1), pooled across all sessions and animals. Right: Correlation of responses across days for each animal and each session pair. The right-most bar plot, here and in the next panel, corresponds to the animal described in Supplementary Figure S5-1). F, Left: Analysis of stability of interneuron activity locked to body movement across sessions (day N vs. day N+1), pooled across all sessions and animals. Right: Correlation of responses across days for each animal and session pair. G, Examples of distinct interneuron response types, 2 exemplary neurons for each group described in panel (H). Episodes of natural arousal encompassed by body movement are highlighted with gray shading (manual annotation). Episodes of optogenetic stimulation of LC are highlighted with red shading. Simultaneously recorded body movement and pupil diameter as proxies for natural arousal are shown below. The same interneurons are shown for day 1 (left) and day 2 (right). H, Relationship of natural arousal (x-axis, quantified by body movement modulation of each cell) and response to LC stimulation (y-axis). Upon discarding non-reliably responsive cells (gray), three primary groups remain: Group 1, which responds positively to both LC stimulation and movement (black); Group 2, which responds positively to movement but is primarily inhibited by LC stimulation (blue); and Group 3, which is inhibited both by LC stimulation and negatively modulated by body movement (green). I, Laminar distribution within deep CA1 of the three cell groups shown in panel (H). “Zero” indicates the position of the pyramidal cell layer, with positive distance going deeper into the stratum oriens. Neurons responsive to LC stimulation (black, Group 1) are located closer to the pyramidal cell layer. Neurons negatively modulated by movement and LC stimulation (green, Group 3) are primarily located in deep layers close to the corpus callosum. Neurons from Group 2 (blue) are distributed across all recorded laminae. The overall distribution of recorded interneurons is shown in the background as a gray dashed line. J, Neurons responding positively to movement (Groups 1 and 2) increase their response strength to LC stimulation when located more superficially, i.e., closer to the pyramidal cell layer.

– Cell-type-specific response profiles to LC stimulation across intensities.

A, Mean astrocytic population responses to optogenetic LC stimulation at increasing frequencies (1-40 Hz) for astrocytes. Shaded region indicates stimulation period. Responses are offset on the y-axis to improve readability. B, Astrocytic response properties during the early response window (10-s window during stimulation) for astrocytes. Left: grand-average response magnitude. Right: population similarity across stimulation intensities computed with Pearson’s correlation of the population activity vector. C, Same as in (B) but for the late time window (10 s after stimulation). D-F, Same as in (A-C) but for pyramidal neurons. G-I, Same as in (A-C) but for interneurons. In (G-I), the subpopulation of interneurons that were activated by stimulation intensities ≥20 Hz are shown separately (lighter color) together with the entire population (darker color). The similarity measurements are based on the entire interneuron population. For all panels, values are averaged for astrocytes across 495 cells, with 6 stimulations from 3 sessions and 3 animals for 1 Hz, 495/6/3/3 for 3 Hz, 2207/34/12/4 for 5 Hz, 495/7/3/3 for 10 Hz, 2207/32/12/4 for 20 Hz, and 495/6/3/3 for 40 Hz. For interneurons, 368/7/3/3 for 1 Hz, 368/6/3/3 for 3 Hz, 1710/41/14/4 for 5 Hz, 368/6/3/3 for 10 Hz, 1710/39/14/4 for 20 Hz, and 751/12/6/3 for 40 Hz. For pyramidal cells, 672/4/2/2 for 1 Hz, 672/4/2/2 for 3 Hz, 4425/37/14/6 for 5 Hz, 672/4/2/2 for 10 Hz, 4425/36/14/6 for 20 Hz, and 3457/17/9/3 for 40 Hz.

Overview of functionally defined response characteristics of the investigated cell types.

Subgroups are defined based on different response polarities to arousal or LC stimulation (third and fourth column). Accordingly, there are no subgroups for astrocytes, although astrocytes exhibit a spectrum of sensitivities to LC stimulation. Laminar location is defined as “deep” (more dorsal in dorsal CA1) and “superficial” (more ventral in dorsal CA1). “Aligned with population average” indicates whether the overall response directionality (activation vs. inhibition) was the same between responses to arousal and responses to LC stimulation. “Aligned on cellular level” indicates whether the single-cell response profiles were aligned across arousal vs. LC stimulation. “Response stability” indicates whether single-cell response patterns were stable within and across days.

GFP control experiments related to

Figure 1. A, Viral targeting strategy for control (GFP) or regular (GRAB-NE2m) experiments, each performed in DBH-iCre transgenic mice together with injection of Cre-dependent ChrimsonR-tdTomato. B, Upon optogenetic stimulation of LC, GFP control animals (green) show a pupil response similar to GRAB-NE2m-expressing animals (gray), indicating successful targeting of LC with the optogenetic sensors (ChrimsonR) under both conditions. C-E, No discernible signal change in response to (C) tail lift, (D) foot shock, and (E) LC stimulation in GFP control animals. F, Comparison of peak ΔF/F of GFP control vs. GRAB-NE2m animals (GRAB-NE2m values taken from Figure 1E-G).

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Forced swim stress experiment with photometry of NA.

Mice expressing the NA sensor GRAB-NE2m were subject to a forced swim in 18°C cold water for 3 min minutes. Fiber photometry recordings were performed several minutes before, during, and after forced swim stress. A, Time course of fiber photometry of NA sensor-expressing mice (gray, n = 4) and control mice expressing GFP (green, n = 4). The measured NA release increases briefly even after the end of the acute stressor, and decays slowly back to baseline on a timescale of 10s of minutes. B, Comparison of NA release (peak ΔF/F) during forced swim stress (values from panel A) and strong optogenetic LC stimulation (values from Figure 1H). The values are of a similar magnitude, illustrating that the 20-Hz LC stimulation used in our study results in NA release similar to a strong stressor. Due to tightening regulations on animal welfare, the swim stress paradigm was not included for subsequent experiments with astrocytic and neuronal fiber photometry.

Optogenetic inhibition of LC prevents NA release during stress.

A, Viral targeting strategy to inhibit LC activity (bilateral injection of Cre-dependent Jaws in DBH-iCre mice; bilateral fiber implants) while monitoring NA levels (NA sensors GRAB-NE1m or nLightG) in HC. Inhibition of LC with Jaws decreases NA levels (B) at baseline, (C) during tail lift, and (D) during foot shock (without LC inhibition in green; with LC inhibition in purple). E, Example fiber photometry recording with responses to tail lift with (gray+red shading) and without inhibition (gray shading only).

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NA release in HC upon optogenetic LC stimulation.

A, Viral targeting strategy to optogenetically activate LC (injection of Cre-dependent ChrimsonR in DBH-iCre mice) while monitoring NA levels (NA sensors GRAB-NE1m or nLightG) in HC. B, NA release in response to increasing LC stimulation intensities (10 s of 1 Hz, 3 Hz, 5 Hz, 10 Hz, 15 Hz, 20 Hz, 40 Hz and continuous stimulation, 10-ms pulses, 10 mW laser power, for n = 4 mice).

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Tracking of astrocytes, pyramidal cells and interneurons across days.

A, An astrocytic FOV (central plane of three imaging planes) with manually selected somatic ROIs (colored overlays) for two separate days. The coloring of ROIs does not reflect cell identity across days. B, Example ROIs covering the same astrocytes tracked across days. ROI locations are highlighted with yellow squares in (A). Across 4 mice, 960 cells were tracked in ≥2 sessions, 646 in ≥3 sessions, and 406 in ≥4 sessions. C, A pyramidal cell FOV with sources of neuronal activity, extracted with Suite2p (colored overlays), for two separate days. D, Example ROIs covering the same neurons tracked across days. ROI locations are highlighted with yellow squares in (C). Across 3 mice, 897 pyramidal cells were tracked in ≥2 sessions, 619 in ≥3 sessions, and 324 in ≥4 sessions. E, An interneuron FOV (second plane out of four planes, counted from stratum radiatum to stratum oriens) with manually selected somatic ROIs (colored overlays) for two separate days. F, Example ROIs covering the same cells tracked across days. ROI locations are highlighted with yellow squares in (E). Across 4 mice, 553 interneurons were tracked in ≥2 sessions, 481 in ≥3 sessions, and 385 in ≥4 sessions.

Body movement as a proxy for endogenous arousal.

Visually, pupil dilation appeared as a lowpass-filtered version of body movement. Importantly, as described previously (Rupprecht et al., 2024), body movement reflected not only locomotion but also locomotion-independent body movement that was reflected by pupil dilation. To demonstrate that body movement is predictive of pupil dilation (and therefore arousal), we used dilated linear regression (see Methods) to predict pupil dilation from a temporally extended window of body movement (left). We found that the predicted pupil dilation correlated highly with the experimentally measured pupil diameter (median correlation ± S.D., 0.57 ± 0.13 across 42 imaging sessions from 13 animals). Lower values within this distribution are likely due to imprecisions of pupil diameter measurement because of unstable lighting conditions (e.g., we used an UV LED pointed at the mouse’s eye to preconstrict the pupil; eye movements of the mouse towards or away from the UV light therefore result in pupil dilations or constrictions) and subsequent pupil segmentation errors, as well as from pupil dilations not stemming from natural arousal but from optogenetic LC stimulation (indicated in red here).

Behavioral responses upon LC stimulation for two-photon microscopy experiments.

A, Pupil diameter changes upon stimulation with 10-s 20-Hz protocols were recorded and averaged across stimulations for each imaging session and then averaged across sessions for each animal. For each animal (n = 13), the pupil diameter change is shown with a different color. While the magnitude of evoked pupil diameter changes is variable across animals, each of them shows a clear pupil diameter increase upon stimulation, indicating successful optogenetic stimulation of LC. B, Body movement upon stimulation with 10-s 20-Hz protocols as in (A) (n = 13 animals). On average across animals (blue trace), animals exhibit a reduction of body movement, consistent with previous reports of behavioral arrest upon strong and long-lasting LC stimulation. One animal consistently exhibited brief motion bouts upon LC stimulation (medium blue trace; the neuronal activity extracted from this mouse was therefore analyzed separately from all other mice in Fig. S5-1).

Single-cell correlation functions and pairwise correlations for all cell types.

A, Correlation function of cellular activity with body movement as a proxy for arousal as shown in Figure 2F,J,N, but for individual cells. The cells are sorted according to the value of the movement modulation index of each cell (see Methods). B, Pairwise correlation between the activity patterns of all pairs of simultaneously recorded cells. C, Pairwise correlation as in panel (B) but plotted as distance-dependent fit to the data (fit value with 95% confidence interval of the linear fit as shading). For the condition “within laminae”, cell pairs within an imaging plane are considered, presumably within very close laminar distance. For the condition “across laminae”, cell pairs from non-identical imaging planes are considered only. The result demonstrates that interneuron activity is relatively similar within but more different across laminae. D, Same as in panel (C) but for astrocytes. The result demonstrates that astrocyte activity is similarly synchronized within as well as across laminae.

Cell-type specific responses to LC stimulation during anesthesia with two-photon microscopy.

Average responses (average across all cells, animals and sessions) to LC stimulation for astrocytes (A), pyramidal cells (B) and interneurons (C) during wakefulness (left) and anesthesia (right). Under both conditions, similar overall responses can be observed (activation for astrocytes, inhibition for pyramidal cells and interneurons). For interneurons, a slow activation not observed during wakefulness can be seen riding on top of the inhibition. D, Responses on the cellular level (responses to LC stimulation computed as in Figures 3 to 5) are correlated across states for astrocytes and interneurons but not for pyramidal cells, consistent with results observed for response stability across sessions during wakefulness (Figures 3 to 5).

Cell-type specific modulation during freely moving vs. head-fixed behavior.

To compare movement bout-evoked cellular activity between fiber photometry experiments in freely moving mice (Figure 1) and two-photon imaging experiments in head-fixed mice (Figure 2), we applied the same criteria to detect contiguous movement bouts (Methods). For each behavioral paradigm, the cellular response to each bout was temporally normalized by resampling to the median bout duration (3.1 s for freely moving behavior; 6.7 s for head-fixed behavior). Averages were performed across bouts within a session (gray traces) and then averaged across sessions to obtain the grand average (colored traces) (freely moving: 6 sessions from 6 mice for astrocytes, 5 sessions from 5 mice for interneurons, 5 sessions from 5 mice for pyramidal cells; head-fixed: 12 sessions from 4 mice for astrocytes, 14 sessions from 4 mice for interneurons, 12 sessions from 5 mice for pyramidal cells). A, Movement-evoked activity during freely moving behavior. B, Movement-evoked activity during head-fixed behavior. The longer median bout durations and the stronger astrocytic response observed during head-fixed behavior may reflect the higher effort necessary to initiate locomotion and the presumably more stressful mismatch between intended and actual movement under head-fixed conditions. These observations suggest that movement during head-fixed behavior adds a stressful component that is less prominent during freely moving behavior in an open field.

Correlation between sensitivity to LC responses and response to movement.

Left: Correlation of responses to LC stimulation sensitivity as described in Figure 3I and movement modulation for an example session. Individual dots refer to individual neurons. Right: All Pearson correlation coefficients for 12 sessions from 4 animals.

LC-induced change of spike rates, related to

Figure 4D. Change of spike rate from a 10-s pre-stimulus window (x-axis) to the 10-s window of LC stimulation (y-axis) as in Figure 4D. A, Same as in Figure 4D but only for spontaneous transitions (no LC stimulation), shown as median of the distribution together with the inter-quartile range as shaded corridor. B, Same as in Figure 4D but only for transitions induced by LC stimulation, shown as median of the distribution (red) together with the inter-quartile range as a shaded corridor. For reference, the median of the distribution for spontaneous transitions is shown in gray. C, Same as in panel (B) but for 5-Hz instead of 20-Hz LC stimulation. As in Figure 4D and panels (A,B), an - albeit smaller - reduction of the spike rate upon LC stimulation can be observed across all pre-stimulation spike rates..

Additional analyses related to

Figure 5 (outlier mouse). Additional analyses for the outlier mouse, which was excluded from the analyses in Figure 5 due to LC stimulation-triggered movement bouts. Despite this potential confound, all analyses performed for this mouse only reflected the effects observed for the other mice in Figure 5. A, Interneuron responses to 20 Hz LC stimulation, sorted by the response during the 10-s stimulation window. Data pooled from 3 sessions. This mouse exhibits higher apparent responses to LC stimulation compared to the data shown in Figure 5A, but this difference may be explained by movement systematically triggered by LC stimulation in this specific mouse. B, Relationship of natural arousal (x-axis, quantified by body movement modulation of each cell) and response to LC stimulation (y-axis), analysis performed as for Figure 5G, resulting in three primary groups: Group 1, which responds positively to both LC stimulation and movement (black); Group 2, which responds positively to movement but is primarily inhibited by LC stimulation (blue); and Group 3, which is inhibited both by LC stimulation and negatively modulated by body movement (red). C, Top: Laminar distribution of the three cell groups shown in panel (B), analogous to Figure 5H. “Zero” on the x-axis indicates the position of the pyramidal cell layer, with positive distance going deeper into the stratum oriens. Neurons responsive to LC stimulation (black, Group 1) are located closer to the pyramidal cell layer. Neurons negatively modulated by movement and LC stimulation (red, Group 3) are primarily located in deep layers close to the corpus callosum. Neurons from Group 2 are distributed across all recorded laminae. The overall distribution of recorded interneurons is shown in the background as a gray dashed line. Bottom: Neurons responding positively to movement (Groups 1 and 2) increase their response strength to LC stimulation when located more superficially, i.e., closer to the pyramidal cell layer, as shown for the other mice in Figure 5I.

Stability of interneuron responses to moderate (5 Hz) vs. strong (≥20 Hz) LC stimulation.

A, Interneuron responses to ≥20-Hz LC stimulation (left) and 5-Hz LC stimulation (right), sorted by the response during the 10-s stimulation window for 5-Hz stimulation. The sorted suggests the neurons that are inhibited/activated by strong stimulations of LC are also inhibited/activated by moderate stimulations of LC. Data pooled from all animals (N = 4) across all recorded cells (n = 1710 neurons). B, Correlation of responses to strong vs. moderate stimulation strength, measured as the response magnitude during the 10-s stimulation window, with the statistical outcomes of the computed correlation across all neurons.

LC-induced change of ΔF/F, related to

Figure 5D. Change of ΔF/F from a 10-s pre-stimulus window (x-axis) to the 10-s window of LC stimulation (y-axis) as in Figure 5D. A, Same as in Figure 5D but only for spontaneous transitions (no LC stimulation), shown as median of the distribution together with the inter-quartile range as shaded corridor. B, Same as in Figure 5D but only for transitions induced by LC stimulation, shown as median of the distribution (red) together with the inter-quartile range as a shaded corridor. For reference, the median of the distribution for spontaneous transitions is shown in gray. In addition, we permuted stimulation trials across repetitions for individual neurons and performed the same analysis (black), resulting in no obvious difference compared to the experimental data (red). This control analysis indicates that interneurons do not show a specific sensitivity to LC stimulation based on their activity prior to stimulation, but rather a specific sensitivity to LC stimulation that is coupled with a specific baseline activity level: interneurons with lower baseline activity are more likely to be activated by LC stimulation, while interneurons with higher baseline activity are more likely to be inactivated by LC stimulation. C, Same as in panel (B) but for 5-Hz instead of 20-Hz LC stimulation. For this lower stimulation intensity, the inactivation of highly active interneurons can still be observed, while the activation of inactive neurons as observed for 20-Hz stimulation is not present.

Multi-timepoint modeling to explain interneuron activity with movement.

The modeled activity patterns were subtracted from experimentally recorded data, resulting in a residual pattern that is not or less contaminated by the influence of body movement, thereby providing a cleaner readout of responses to LC stimulation. A, A model based on dilated linear regression (see Methods) was trained in a cross-validated manner for each neuron to explain its activity (ΔF/F) as a linear function of current and past body movement. Movement (top) is used as the regressor to explain single-cell activity (bottom, blue) with a linear model (bottom, red). B, Example of raw imaging data (ΔF/F), excerpt from a single recording session. C, Single-neuron model of the activity patterns seen in (B), based on body movement as regressor. D, Residual activity patterns, obtained by subtracting the modeled activity pattern (C) from the original activity pattern (D). It is clear that the effect of body movement (compare with panel A) is reduced, although not completely eliminated.

Example responses of individual interneurons to LC stimulation (≥20 Hz).

Only neurons that exhibited reliable responses (see Methods) are shown. They are sorted according to the response magnitude during the 10-s stimulation window in a columnar sequence (neurons shown in the left-most column show strongest activating response; neurons in top row of this column show the strongest activating response within this column). Individual stimulation instances are shown with coloring, average across responses in black. Traces of body movement as a proxy for natural arousal and potential confound are shown below, with the colors matching between corresponding trials. Subset of neurons with reliable responses (every 8th neuron) for mice 1-3, upon which most analyses in Figure 5 are based.

Movement modulations as a function of laminar location within deep CA1.

Neuronal responses to movement of Group 1 and 2 interneurons are not well explained by laminar depth (r = −0.13). This is in contrast to responses to LC stimulation, which are well explained by laminar depth for the same set of interneurons (Figure 5J).

Schematic overview of cell-type-specific effects of natural arousal and LC stimulation.

Individual pyramidal cells (purple triangles) are consistently activated (upward-pointing arrows) or inhibited (downward-pointing arrows) during arousal, whereas LC stimulation induces an non-specific, slow inhibitory response. Similarly, individual interneurons (turquoise circles) are consistently activated or inhibited during arousal, but also exhibit consistent responses to LC stimulation, with a transient activation of interneurons located near stratum pyramidale on top of a broader slow inhibitory response. In contrast, astrocytes (orange symbols) are activated during arousal and LC stimulation. At the level of individual cells, responses to LC stimulation differ from arousal responses for all three cell types (red outlines of arrows). At the population level, pyramidal cells and interneurons differ markedly between arousal and LC stimulation (red outlines of cell symbols), while astrocyte population responses are aligned across conditions.

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