Distinct Mechanisms for Visual and Motor-Related Astrocyte Responses in Mouse Visual Cortex

Abstract

Astrocytes are a major cell type in the mammalian nervous system, are in close proximity to neurons, and show rich Ca²⁺ activity thought to mediate cellular outputs. Astrocytes show activity linked to sensory [1, 2] and motor [3, 4] events, reflecting local neural activity and brain-wide neuromodulatory inputs. Sensory responses are highly variable [5-10], which may reflect interactions between distinct input types [6, 7, 9]. However, the diversity of inputs generating astrocyte activity, particularly during sensory stimulation and behavior, is not fully understood [11, 12]. Using a combination of Ca²⁺ imaging, a treadmill assay, and visual stimulation, we examined the properties of astrocyte activity in mouse visual cortex associated with motor or sensory events. Consistent with previous work, motor activity activated astrocytes across the cortex with little specificity, reflecting a diffuse neuromodulatory mechanism. In contrast, moving visual stimuli generated specific activity patterns that reflected the stimulus' trajectory within the visual field, precisely as one would predict if astrocytes reported local neural activity. Visual responses depended strongly on behavioral state, with astrocytes showing high amplitude Ca²⁺ transients during locomotion and little activity during stillness. Furthermore, the amplitudes of visual responses were highly correlated with pupil size, suggesting a role of arousal. Interestingly, while depletion of cortical noradrenaline abolished locomotor responses, visual responses were only reduced in amplitude and their spatiotemporal organization remained intact, suggesting two distinct types of inputs underlie visual responses. We conclude that cortical astrocytes integrate local sensory information and behavioral state, suggesting a role in information processing.

Keywords: 2-photon microscopy; astrocytes; calcium imaging; neuromodulator; noradrenaline; retinotopic; sensory; visual cortex; widefield microscopy.

Figure 1

Distinct Ca²⁺ Responses in Astrocytes of the Mouse Visual Cortex during Locomotion and Visual Stimulation (A) Mice were head-fixed under a dual widefield and multiphoton microscope. To stimulate the right visual field, an LCD monitor was placed in front of the right eye. The right eye was monitored for changes in pupil size. The mice were implanted with a cranial window over the left visual cortex. AAVs encoding the calcium indicator GCaMP6 were delivered by stereotaxic injection. GCaMP6 fluorescence was measured using 1-photon (left) or 2-photon (right) imaging. Anterior (A) and medial (M). Scale bars, 1 mm for 1-photon field of view (FOV), 50 μm for 2-photon FOV. (B) Visual stimulation paradigm for studying responses of visual cortical astrocytes. Left, top to bottom: time-lapse of checkerboard moving bar stimulus, pseudo-colored schematic of retinotopic response within V1, visually evoked astrocytic Ca²⁺ responses shown in grayscale or pseudo-color coding for time since stimulus onset. Numbers indicate time in seconds. Right, top to bottom: schematic, response maximum intensity projection, and retinotopic map inferred from time to response peak. (C–E) Voluntary locomotion induces widespread, synchronous activation of astrocytes across the cortex. (C) Individual black and white frames are 1-s time slices of the event-related averages at indicated times after onset of locomotion. The magnitude of fluorescence increase (% dF/F₀) is shown in grayscale. (D) Left: pseudo-colored, pixel-wise time to response peak map shows uniform peak response times across the cortex after locomotion onset. Areas of weak GCaMP6 expression (gray) are excluded from analysis (see STAR Methods). Black square indicates ROI for traces to the right. Right: individual GCaMP6 (top) and locomotion speed time traces (bottom) from the same experiment. Dashed line indicates locomotion onset. (E) Cumulative distribution plot showing peak amplitudes of locomotion-related responses (black, n = 102 events), compared to stationary baseline (gray, n = 105 events). Scale bars, 1 mm. See also Video S1. (F–H) Mouse visual cortex astrocytes show retinotopically organized responses to moving bars. (F) Individual black and white frames are 1-s time slices of the stimulus-related averages at indicated times after onset of stimulation. (G) Left: response maps show time-to-response peak for rightward moving (top) and upward moving (bottom) stimuli. Maps are the average across 10 repeated trials for each direction tested. Right: single-trial GCaMP6 time traces for stimuli moving in each of 4 directions, aligned to the onset of stimuli presentation (dashed lines). Top bars indicate the time at which visual stimulation was present. (H) Cumulative distribution plots showing peak amplitudes of responses to visual stimulation (black line, n = 438 trials), compared to pre-stimulus baseline (gray line, n = 210 events). Only stationary pre-stimulus windows (8 s) were considered for baseline. Scale bars, 1 mm. See also Video S2. (I) Cumulative distribution plots showing the half-width at half-maximum of responses to locomotion (gray line, n = 42 events) or visual stimulation (black line, n = 233 trials). See also Figures S1 and S2.

Figure 2

Individual Astrocytes Respond to Visual Stimulation (A) Left: example 2-photon imaging FOV (white square) at the cortical surface. Dashed line indicates V1. Center and right: 2-photon imaging at 160 μm cortical depth. Black and white frames are 1-s averages obtained at indicated times after visual stimulation onset. Position of the stimulus is indicated above. Scale bars, 1 mm for 1-photon FOV, 50 μm for 2-photon FOV. See also Video S3. (B) 2-photon response maps showing time-to-response peak relative to visual stimulus onset. Maps are the average of 10 trials for each direction of motion, recorded from a single animal. The pseudo-color scale is adjusted to match the range of delays observed in responses to the moving bar stimuli. Pixels below a fluorescence threshold are shown in gray. Scale bar, 50 μm. See also Video S3. (C) Cumulative distribution plot showing the amplitudes of locomotion responses (black line, n = 727 events), as compared to activity during preceding stationary epochs (baseline) (gray line, n = 797 events). 7 animals, 133 cells. (D) Cumulative distribution plots showing the amplitudes of responses to visual stimulation (black line, n = 1,733 trials), as compared to pre-stimulus baseline (gray line, n = 533 events). 7 animals, 133 cells. (E) Cumulative distribution plots showing the half-width at half-maximum of responses to visual stimulation obtained with 2-photon (black line, n = 1,129 trials, 133 cells, 7 animals) or 1-photon imaging (gray line, n = 233 trials, 8 animals). See also Figure S1.

Figure 3

Visual Cortex Astrocytes Integrate Information on Sensory Inputs and Arousal State (A) Single-trial 1-photon GCaMP6 (top) and movement speed (bottom) traces aligned to stimulus onset (dashed line). Black and orange curves show responses during locomotion and stationarity (no movement from 1 s before visual stimulation onset and during the presentation period), respectively. Bar indicates the time during which visual stimulation was present. (B) Cumulative distribution plots show peak fluorescence changes upon visual stimulation when the animal is stationary (orange curves) or running (black curves). Data were recorded in 1-photon (left) or 2-photon mode (right). n = 291/1,350 (1-photon/2-photon) trials during running, n = 147/363 trials during stationarity, and n = 210/533 events for pre-stimulation baseline (gray curves). (C and D) Scatter plots showing the peak fluorescence changes recorded in 1-photon mode during visual stimulation concomitant with voluntary locomotion plotted against the average speed of locomotion (C) or changes in pupil size at onset of stimulation (D). (E) Changes in pupil size as a function of locomotion speed. Red lines show results of robust linear regression. See also Figure S3.

Figure 4

Visually Induced and Locomotion-Related Signals Operate through Distinct Mechanisms (A) Noradrenaline depletion abolishes the Ca²⁺ responses associated with locomotion onset. Example Ca²⁺ transients (top) recorded using 1-photon imaging from an animal during voluntary locomotion (bottom) pre- (left) and post- (right) DSP-4 administration. (B) Example Ca²⁺ transients (top) recorded using 1-photon imaging from a mouse during 10 trials of visual stimulation with voluntary locomotion (bottom) pre- (left) and post- (right) DSP-4 administration. (C) Cumulative distribution plots showing the peak fluorescence during periods of stationarity (no movement for 8 s or more; gray curves) and after locomotion onset (black curves, 8-s window) pre- (left, n = 88 events) and post- (right, n = 114 events) DSP-4 administration. Signal from the strongest responders (90% percentile) after locomotion onset was 11.8% dF/F₀ and 1.3% dF/F₀ above baseline fluorescence before or after DSP-4, respectively. (D) Cumulative distribution plots show the peak fluorescence before (gray curves) and after (black curves, 8-s window) visual stimulation onset, pre- (left, n = 318 trials) and post- (right, n = 311 trials) DSP-4 administration. Signal from the strongest responders (90% percentile) during visual stimulation was 15.9% dF/F₀ and 4.7% dF/F₀ above pre-stimulation levels before or after DSP-4, respectively. Activity before visual stimulus onset was measured during an 8-s window irrespective of whether the animals were stationary or locomoting. The analysis window was slid independently to be centered approximate to the response peak (STAR Methods). (E and F) Black and white maps show average activities for vision-induced increases in GCaMP6 fluorescence across trials and stimulus directions. Pseudo-color response maps show the average time to reach maximum GCaMP6 intensity after stimulus onset for the stimulus directions indicated. Maps are the average of 10 trials per stimulus direction in 1 animal pre- (E) and post- (F) DSP-4 administration. Scale bars, 1 mm. See also Figure S4 and Video S4.