While imaging Ca 2+ dynamics, we recorded local field potential (LFP) activity from cortical L2/3 and observed spontaneous alternations in brain state every 5.4 ± 0.37 min ( Fig. 1I ; n = 7 animals, 11 30-min trials), closely resembling the switch between REM and SWS ( 41 ⇓ ⇓ – 44 ). This spontaneous switch from a high to low frequency-dominated state, characterized by the clear presence of UP and DOWN states of the slow oscillation and a decrease in the power of frequencies above 2 Hz, has been well-described previously in both awake animals and under various types of anesthesia ( 7 , 41 ⇓ ⇓ – 44 ) ( Fig. 1 I and J and Fig. S1 I–K ), indicating that this is a generalized, essential feature of cortical circuits.

In vivo Ca 2+ imaging of cortical astrocytes with GCaMP6s and LFP recordings under various conditions. (A) Twenty representative Ca 2+ signals from ROIs in the experiment in C. Note the heterogeneity of the amplitude and duration of transients. (B) df/f Ca 2+ traces from all active ROIs in Fig. 1B , with average Ca 2+ traces in dark gray overlaid (Top) and expanded in y axis (Bottom). (C–H) Immunostained cortical slices for GFAP and various proteins in virally injected animals, including sham injections. (Scale bar, 100 μm.) (I) Example of a state transition under urethane anesthesia from a 100-s sample (red line) in Fig. 1I , with raw data (Top), resulting spectrogram (Middle), and traces quantifying the power in low- and high-frequency bands (Bottom). Automatically detected increases in the low-frequency band above threshold (red dot) and decreases in the high-frequency band below threshold (blue dot) are plotted to emphasize the state transition to a slow-oscillation state. Same data as Fig. 1J . (J and K) Similar state transitions are evident in LFP recordings in isoflurane-anesthetized (J) and awake animals (K), as in recordings from the urethane-anesthetized animals in A. The LFP recording in J is courtesy of J. Jackson, Columbia University, New York.

Simultaneous in vivo Ca 2+ imaging of cortical astrocytes with GCaMP6s and LFP recording of the local cortical state. (A) Experimental setup. Injection of AAV1-CAG.FLEX-GCaMP6s into GFAP-Cre mice results in GCaMP6s expression in a subset of cortical astrocytes and allows two-photon imaging and LFP recording. (B, Left) Expression of GCaMP6s in L2/3 of V1. Note the low baseline fluorescence, as is typical for GCaMP6s ( 31 ). (B, Right) Active ROIs from a 5-min movie of an imaging field ( Movie S1 ). (C) Coexpression of GCaMP6s and tdTomato in a single astrocyte demonstrates expression of both fluorescent proteins throughout the astrocyte ( Movie S2 ). [Scale bars, 50 μm (B and C).] (D) df/f Ca 2+ traces from 20 representative ROIs in B. Note the heterogeneity of the amplitude and duration of transients. (E–H) Statistics for Ca 2+ imaging experiments in Fig. 2 (n = 22 5-min trials). (E) Distribution of subcellular localization of active ROIs per 5-min trial. Distribution of the number of Ca 2+ events per experiment for all active ROIs (F) and amplitude (G) and duration (H) of all Ca 2+ transients. ROIs from astrocyte somata are shown in gray and from processes are shown in green. (I) Raw LFP data of a 30-min recording in L2/3 of the cortex (Top) and resulting spectrograms calculated from raw LFP, separated into low (0.25–10 Hz) and high (10–80 Hz) graphs for clarity. Note the periodic state switching from the high frequency-dominated to the low frequency-dominated state. The red box corresponds to 100 s of LFP, marked by a red line in the raw trace. In spectrograms here and throughout paper, color bars are in units of 10*log(mV 2 ). (J) Example of a state transition from a 100-s sample (red line) in I, with raw data (Top), spectrogram (Middle), and traces quantifying the power in low- and high-frequency bands (Bottom). Automatically detected increases in the low-frequency band above threshold (red dots) and decreases in the high-frequency band below threshold (blue dots) are plotted to emphasize the state transition to a slow-oscillation state.

Desynchronized astrocytic Ca 2+ activity, measured with cytosolic and membrane-bound GCaMP6, is also temporally correlated with slow oscillation. Average astrocytic GCaMP6s (A–E) and Lck-GCaMP6f (F–J) activity closely precedes the shift to slow oscillation. (A and F) GCaMP6 expression and active ROIs. (Scale bars, 50 μm.) (B and G) Raw 5-min LFP trace (Top) and resulting spectrogram (0.25–10 Hz; Bottom). (C and H) All active astrocytic ROIs. Compare these desynchronized Ca 2+ signals with the synchronized transients in the example in Fig. 2D . (D and I) Average astrocyte Ca 2+ trace (gray line) and Ca 2+ event (gray dots) plotted with mean of the low-frequency band (0.5–2 Hz; red line) and automatically detected low-frequency increase events (red dots). Note the closely timed gray and red dots. (E and J) Average astrocyte Ca 2+ trace (gray line) and Ca 2+ event (gray dots) plotted with the mean of the high-frequency band (3–10 Hz; blue line) and automatically detected high-frequency decrease events (blue dots). Dashed lines indicate the respective thresholds.

Temporal relationship between astrocytic Ca 2+ and brain state switches. (A) LFP and Ca 2+ data from all 22 trials shown in Fig. 1 and quantified in Fig. 1I . Gray dots represent automatically detected average Ca 2+ events, and red and blue dots show LFP low-frequency increases and high-frequency decreases, respectively. Gray horizontal lines separate individual 5-min trials. (B) Data from A replotted as a histogram to highlight that the majority of low-frequency increase events start in the 20 s following the Ca 2+ event, whereas the high-frequency decrease events are more evenly distributed around the Ca 2+ event. (C) Same data from A and B and Fig. 1 plotted, with all increase (light blue) and decrease (blue) high-frequency events shown. (D) Histogram of all data from C. These two datasets do not have statistically significantly different distributions (two-sample F test). (E) Same data as in A–C replotted with Ca 2+ events (black) triggered from low-frequency increases (red; event time 0). (F) Same analysis as in E, but with Ca 2+ event time (gray) from each low-frequency decrease (red). (G) Frequency histogram of all data from E and F. (H) Ca 2+ data from the examples in Fig. 2 A–F plotted against the ratio of a set of standard frequency bands (0.5–2, 3–8, 8–14, 15–30, and 30–80 Hz) to each other. A gray box shades the four graphs with the lowest-frequency band in the numerator of the ratio, highlighting the dominance of this frequency band’s correspondence with the average astrocyte Ca 2+ activity.

Astrocyte Ca 2+ events precede the switch to the slow-oscillation state. (A) Astrocyte-specific expression of GCaMP6s (Left) and active ROIs (Right). (Scale bars, 50 μm.) (B) Raw 5-min LFP recording at the site shown in A. The expanded area in the red box (Bottom) shows an example of a shift to a slow-oscillation–dominated regime. (C) Power spectrogram of 0.25- to 10-Hz frequencies in the LFP shown in B. Note the transition to the slow-oscillation state (0.5–2 Hz; increase in red). (D) Ca 2+ traces from 22 active ROIs in A (light gray) and the average of all traces in dark gray. (E) Overlay of astrocyte Ca 2+ and the mean of frequency change to bands to 80 Hz. (F) Automatically detected mean Ca 2+ (gray), low-frequency (0.5–2 Hz) increase (red) and decrease (pink) events, and high-frequency (3–10 Hz; blue) decrease events (dots) above thresholds (dashed lines). (G) All low-frequency LFP events from Ca 2+ event time. Only the nearest LFP events to the Ca 2+ event are shown for clarity. Gray dots represent automatically detected average Ca 2+ events, and red and pink dots show LFP low-frequency increase and decrease events, respectively. Gray horizontal lines separate each 5-min trial. Darker gray lines indicate trials from different animals. (H) Distribution of events shown in G. (I) Distribution of all low-frequency increase events plotted with all high-frequency decrease events from all trials.

Using Optogenetics to Drive Ca2+ Increases in Astrocyte Processes.

To test whether there is a causal relationship between astrocyte activity and brain state, we manipulated astrocyte Ca2+ activity and observed its effects on the neural circuit by using a previously unidentified method for specific optogenetic activation of astrocytes with Arch, a tool normally used to inhibit neurons by pumping H+ out of the cell (45). Viral injection of CAG.FLEX-Arch-GFP into a GFAP-Cre mouse resulted in astrocyte-specific expression of Arch-GFP, based on astrocytes’ highly and finely branched morphology that distinguishes them from neuronal and other glial cell types (Fig. 3 A and B). To confirm the morphological observation of expression, we also pressure-injected rhod-2 AM, a Ca2+ indicator that specifically loads astrocytes (46), into the cortex and observed that the somata of the Arch-GFP+ cells were clearly labeled with rhod-2 and that the Arch-GFP+ processes wrapped around vasculature, as is typical for these cells (Fig. 3 A and B). In slice experiments, whole-cell patch clamping of Arch-GFP+ cells and subsequent presentation of yellow light flashes confirmed Arch expression due to the resultant hyperpolarization (−1.37 ± 0.04-mV shift; n = 28; Fig. 3C). To test whether Arch activation increases intracellular Ca2+ concentration in astrocytic branches in a physiological manner in vivo, we coexpressed Arch-tdTomato and GCaMP6s in astrocytes. Double viral injection of Arch-tdTomato and GCaMP6s results in a patchwork of astrocytic expression in the cortex in vivo, with astrocytes labeled with either one of these proteins or with both (mean 3.75 ± 1.9 Arch+, 1.75 ± 0.9 Arch+/GCaMP+ cells per field; Fig. 3 D and E). In cells expressing both proteins, we could image Ca2+ dynamics before and after Arch activation, and found that light stimulation of Arch+ astrocytes consistently evoked Ca2+ transients in branches with durations of the same length, and amplitudes slightly decreased, compared with ongoing spontaneous activity in interleaved no-stim control trials (Fig. 3 F–H and Fig. S4 A–D; n = 4 animals, 16 trials, 571 Ca2+ events; peak Ca2+ response 18.2 ± 2.6 s after stimulus; mean duration 13.9 ± 0.49 s and amplitude 0.46 ± 0.01 df/f; P > 0.05 and P < 0.05, respectively, t test), whereas Arch−/GCaMP+ neighboring cells did not have a Ca2+ response to the light activation, either by mean fluorescence levels (Fig. 3H, Top) or automatically detected Ca2+ events, as in Fig. 1 (Fig. 3H, Bottom). To further confirm that activation of astrocytes was limited to Arch+ astrocytes and was not due to viral coexpression of two proteins in astrocytes or to the light stimulation itself, we also coexpressed GCaMP6s and tdTomato (without Arch) and performed the same experiments. In these experiments, light activation did not evoke Ca2+ transients in the astrocyte branches or processes of tdTomato+/GCaMP+ astrocytes, nor was Ca2+ activity significantly different from neighboring tdTomato−/GCaMP+ controls (Fig. S4 E and F; n = 4 animals, 28 trials; P > 0.1, t test, two-sample F test).

Fig. 3. Optogenetic activation of Archaerhodopsin (Arch) in cortical astrocytes drives Ca2+ increases. (A and B) Astrocyte-specific expression of Arch-GFP (green) throughout the astrocyte processes with stereotyped astrocyte morphology. Astrocyte somata are specifically labeled with rhod-2 (red). (C) Astrocyte in cortical slice expressing Arch-GFP and whole-cell patch-clamped with Alexa Fluor 594 in the pipette (Top). Yellow light stimulation causes the astrocyte to hyperpolarize (Bottom). (D) Cartoon of coexpressed astrocytic GCaMP6s and Arch-tdTomato. (E) Coexpression of GCaMP6s and Arch-tdTomato in vivo (Left), zoomed-in to show a single coexpressing cell (Middle), with automatically detected ROIs used for analysis and active ROIs shaded in green (Right). (F) Three successive stimulation (5-s) trials of the GCaMP+/Arch+ astrocyte from E. Yellow highlights the frame before stimulus onset and white arrows point to GCaMP fluorescence increases in astrocyte processes poststimulus. (G) Mean astrocyte Ca2+ dynamics in each of the three trials in F. Yellow bars denote stimulus throughout the figure. (H, Top) Average Ca2+ across animals in all stimulation (Left) and no-stimulation (control; Right) trials in GCaMP+/Arch+ astrocytes (green) and GCaMP+/Arch− astrocytes (gray) ± SEM. (H, Bottom) Time from light stimulus to first Ca2+ event, as detected in Fig. 1 in stimulation (Left) and control (Right) trials. [Scale bars, 50 μm (A, C, and E) and 25 μm (B).]

Fig. S4. Arch stimulation causes physiological astrocytic Ca2+ increases, whereas light activation alone does not increase astrocytic Ca2+. (A and B) Histogram of Ca2+ transient duration (A) and amplitude (B) following light stimulation of Arch+/GCaMP6+ astrocytes. (C and D) Arch-stimulated Ca2+ responses compared with spontaneous astrocyte Ca2+ activity. (E) Average Ca2+ dynamics ± SEM in the astrocyte soma and processes coexpressing tdTomato and GCaMP6s following 5-s light stimulation, as used in the optogenetic experiments. There is no significant change due to the light stimulus in either tdTomato+/GCaMP+ astrocytes (green) or tdTomato−/GCaMP+ controls (gray). (F) Distribution of automatically detected Ca2+ events from the time of stimulation in tdTomato+/GCaMP+ (green) or tdTomato−/GCaMP+ (gray) cells indicates no stimulus-dependent difference between cell types (two-sample F test). (G) Intracellular alkalinization upon application of NH 4 Cl in the bath causes a decrease in SNARF-1 fluorescence in all loaded cells in the cortical slice.

Interestingly, each 5-s optogenetic activation with Arch (followed by 5 min of imaging and interleaved with a 6-min control trial) evoked Ca2+ signals in different branches of each Arch+ astrocyte (Fig. 3F, arrows), similar to the spatiotemporal branch specificity of Ca2+ changes observed under baseline conditions (33, 34, 40, 47) (Fig. 1 and Movie S2). Astrocytic somata largely did not respond to Arch stimulation, even with visible protein expression there (mean 35.1 ± 9.4 active ROIs in all Arch+/GCaMP+ astrocytes, 33.2 ± 8.9 active ROIs in processes). Other groups have previously tested optogenetic and nonoptogenetic methods to activate astrocytes by measuring Ca2+ increases only in the soma (48⇓⇓–51), even though somatic activity is much more rare under baseline conditions (Fig. 1 E–H).

We next carried out experiments to investigate the mechanisms by which Arch causes Ca2+ increases in astrocyte processes. Because Arch activation causes hyperpolarization of cells, we whole-cell patch-clamped GCaMP6s-expressing astrocytes in slices and imaged the patched cells for hyperpolarization-induced changes in [Ca2+] i (Fig. 4A). Five-second hyperpolarizations (500-ms pulses × 10, mean −15.1 ± 1.2-mV change) were induced to the patched cell, interleaved with control trials. Ca2+ levels significantly increased throughout the cell, including processes, following the hyperpolarization compared with control trials (Fig. 4B). The voltage changes that caused these Ca2+ increases were much larger than those recorded from astrocytic soma during light activation carried out over the entire cell (Fig. 3C). However, this is not surprising; due to the low input resistance of astrocytes (20, 52), the voltage change required at the soma to elicit a similar hyperpolarization at the distant processes, where we observe the Ca2+ changes following light activation, would need to be higher than we record during light stimulation. Because we can observe hyperpolarization-induced Ca2+ increases, our data indicate that hyperpolarization of astrocytes—and its subsequent effects on voltage-dependent channels, receptors, or transporters—may be the mechanism by which Arch stimulation increases [Ca2+] i . While carrying out these experiments, we also found that by using the stereotyped astrocytic electrophysiological marker of network UP states (Fig. 4C, Top) (20, 43) to monitor population activity in the slice, it was evident that hyperpolarization of single astrocytes not only increased astrocytic Ca2+ but also increased the number of UP states in the network in the 100 s following the stimulation compared with control trials (Fig. 4C). This result is consistent with our previous work demonstrating that stimulation of a single astrocyte increases the Ca2+ activity in the local astrocytic network and leads to increased cortical UP states, which constitute the slow oscillation (20).

Fig. 4. Mechanisms of Arch activation of astrocytes. (A) GCaMP6s-expressing astrocyte (green) under whole-cell patch clamp using a pipette filled with Alexa Fluor 594 (red) to confirm the correct cell. Single two-photon image; the pipette outline is shown with dashed lines because the pipette is out of the optical plane. (Scale bar, 50 μm.) (B) GCaMP Ca2+ dynamics in patched astrocytes in hyperpolarization (red) and control (gray) trials. Example of astrocyte electrophysiological recording, including hyperpolarization period, shown below Ca2+ data (*P < 0.05, t test; n = 10 cells, 34 110-s paired trials). (C, Top) Stereotypical astrocytic UP state (20) used to quantify network synchronization following hyperpolarization. (C, Bottom) Increased UP states following single-astrocyte hyperpolarization from same experiment as in B. (D and E) Two-photon single-plane images of SNARF-1 loading of astrocytes and neurons in slice (red) and specific Arch-GFP expression (green) in either astrocytes (D) or neurons (E). White arrowheads indicate double-labeled somata of the respective cell types. (Scale bars, 50 μm.) (F) SNARF-1 fluorescence dynamics before and after Arch stimulation (yellow bar; 5 s) of astrocytes (Top) and neurons (Bottom) in Arch+ cells (green) and surrounding Arch− cells (gray) (astrocytes: n = 15 Arch+ cells, 264 Arch− cells, 8 110-s trials each; neurons: n = 46 Arch+ cells, 313 Arch− cells, 8 110-s trials each). Error bars are ± SEM.