Chronic cellular imaging of mouse visual cortex during operant behavior and passive viewing

Abstract

Nearby neurons in mammalian neocortex demonstrate a great diversity of cell types and connectivity patterns. The importance of this diversity for computation is not understood. While extracellular recording studies in visual cortex have provided a particularly rich description of behavioral modulation of neural activity, new methods are needed to dissect the contribution of specific circuit elements in guiding visual perception. Here, we describe a method for three-dimensional cellular imaging of neural activity in the awake mouse visual cortex during active discrimination and passive viewing of visual stimuli. Head-fixed mice demonstrated robust discrimination for many hundred trials per day after initial task acquisition. To record from multiple neurons during operant behavior with single-trial resolution and minimal artifacts, we built a sensitive microscope for two-photon calcium imaging, capable of rapid tracking of neurons in three dimensions. We demonstrate stable recordings of cellular calcium activity during discrimination behavior across hours, days, and weeks, using both synthetic and genetically encoded calcium indicators. When combined with molecular and genetic technologies in mice (e.g., cell-type specific transgenic labeling), this approach allows the identification of neuronal classes in vivo. Physiological measurements from distinct classes of neighboring neurons will enrich our understanding of the coordinated roles of diverse elements of cortical microcircuits in guiding sensory perception and perceptual learning. Further, our method provides a high-throughput, chronic in vivo assay of behavioral influences on cellular activity that is applicable to a wide range of mouse models of neurologic disease.

Keywords: awake mouse; discrimination; head-fixed; learning; neuron; perception; two-photon calcium imaging; visual cortex.

Figure 1

Overview and setup for two photon calcium imaging in awake and behaving mice. (A) Schematic timeline for combining cellular imaging with visual behavior. Green and gray paths delineate routes for imaging with genetically encoded calcium indicators or bolus-loaded synthetic calcium indicators, respectively. (B) Head-fixed mice view an LCD monitor during concurrent two-photon calcium imaging and eyetracking. Optimized fluorescence excitation and light collection, combined with fast 3D imaging, allow robust measurements of neural responses on single trials. (C) A titanium headpost secured over visual cortex and an imaging well constructed from rubber O-rings provided excellent stability, wide viewing angle, and light shielding (see Section ‘Materials and Methods’).

Figure 2

Mapping visual responses in identified excitatory and inhibitory neurons in awake mice. (A) Average image of neurons loaded with synthetic calcium indicator, OGB1-AM, in an awake, transgenic mouse expressing GFP in all GABAergic neurons (labeled in green; image depth: 145 μm below pia; 45 mW excitation at 800 nm). (B) Orientation tuning curve (cell 1; average of eight trials) demonstrating calcium responses during presentation of drifting oriented gratings in 16 directions (arrows and gray bars). (C) Examples of single-trial responses to repeated presentations of the same stimulus (yellow bar in B), in one excitatory (gray, cell 1) and one inhibitory (green, cell 2) neuron. Thick lines indicate mean responses. (D) Concurrent eyetracking (left panel) demonstrated relatively stable pupil position (slow drifts <10°, with minor changes in dilation) across hundreds of seconds. Right panel: Time-lapse of cross-section of mouse eye (black: pupil). Pupil location (blue line) was estimated using simple offline tracking of pupil edges. Yellow arrows indicate stimulus presentation. (E) Pupil time course following alignment to stimulus onset [N = 24 traces; mean pupil location in (4 s, 4 s) was subtracted from each trace; black line: mean trace]. For the stimuli used in this manuscript (28°/cycle, drifting at 2 Hz), stimulus-evoked eye movements were not consistently observed, and trials with spontaneous eye movements were rare (see also Figures S1 and S2 in Supplementary Material).

Figure 3

GO–NOGO operant visual orientation discrimination task: Basic paradigm and variants. (A) Upper panel: Mice were rewarded with water for licking following upward drifting oriented gratings, and received negative reinforcement for licking to other non-target orientations, including a 500 ms white noise burst, and, early in training, a 2 s time-out and a mild airpuff stimulus. Lower panel: Following the end of the 1 s stimulus (yellow box), mice had 1 s to lick to indicate a response (purple line). Stimuli were presented 7–8 s apart, which encouraged cessation of licking following the end of the previous trial. Mice could also be partially deterred from spurious licking by aborting trials with lick responses immediately following stimulus onset (blue line). Blocks in which the mouse performed this basic discrimination paradigm could be inter-digitated with passive viewing blocks in which the lickspout was removed. (B) Example behavioral session containing active discrimination and passive viewing trials. Behavioral responses are shown for active discrimination trials only, sorted by stimulus type. These data demonstrate correct decisions (green lines) in the majority of trials – Lick responses to target stimuli (top), and suppression of responses following difficult non-targets (middle; 11°) and easy non-targets (bottom; 90°). The average performance and discriminability (d′) is shown at the right. Note that minimal licking occurred prior to stimulus onset or during the enforced lick-suppression window (blue lines: aborted trials). Behavior did not extinguish following passive viewing blocks (gray arrows at right indicate onset of each active discrimination block, not including five initial target trials). (C) Same session, cumulative incidence of target trials containing lick responses. Mice quickly learned to cease licking following target stimuli when the lick-spout was removed during passive viewing blocks (gray bars).

Figure 4

Task acquisition and long-term stability. (A) Discriminability (d′) of target from non-target stimuli improved over days, as shown in four mice (four different colors). Dashed lines: d′ for non-targets oriented 90° from the target. In subsequent sessions, more difficult non-target stimuli including 45° discrimination (solid lines) were used. (B) Example of psychometric curves across days for one mouse. Increased angular distance of non-targets from target resulted in better discrimination performance, as expected. Later training days (lighter curves) showed general improvement in performance. Frequency of stimulus presentation: (target, difficult non-target,…, easiest non-target) = (35, 7, 7, 31, 14, 6%). (C) Using these daily psychometric curves, orientations thresholds were estimated, for this mouse (blue line) and three other mice, as the angular discrimination achievable at a performance level of d′ = 1. Traces in (A,C) were smoothed with a 3-day boxcar filter for presentation. (D) Example of long-term stability in a mouse presented with three stimuli: Target (40% of stimuli), difficult non-target (∼15° from target; 45% of stimuli) and easy non-target (90°; 15% of stimuli). This mouse demonstrated stable performance in 167 sessions spanning over one year (left panel) and was engaged in the discrimination behavior for several hundred trials a day (right panel, number of trials per day, mean ± SD). Later sessions (gray bars) involved alternating discrimination blocks and passive viewing blocks (the latter blocks, while not included in ordinate estimates, provided a useful control during subsequent imaging). For summary statistics of stability and performance across nine mice, see Table 1. These data suggest that the current behavioral method can be used in studies imaging the same neurons across days and months, during task learning or disease progression/therapeutic intervention.

Figure 5

Three-dimensional imaging during behavior improves recording stability. (A) Average imaging volume corresponding to OGB calcium indicator (top left, green fluorescence) and static astrocytic label SR101 (bottom left, red fluorescence). Individual OGB volumes acquired at 4 Hz were co-registered in 3D, and estimated shifts were then applied to SR101 data. (B) SR101 data from a small square region at each imaging depth (yellow rectangular solid in A) surrounding an astrocyte was collapsed (in X–Y plane) for each 250 ms volume, revealing an apparent ‘sinking’ of the astrocyte to deeper planes during lick/jaw motion (top panel). Following post-processing and 3D rigid and subsequent plane-by-plane co-registration, the cell motion was greatly reduced (bottom panel; only a subset of planes were used after motion correction). (C) For typical studies imaging from one plane, SR101 fluorescence from the edge of this astrocyte should be stable over time, but instead shows severe motion artifacts (top panel). Gray ticks indicate licking, which mostly occurred during engaged blocks but not during passive viewing (black line). Using standard 2D rigid co-registration techniques (middle panel), some artifacts are reduced. However, 3D co-registration was necessary for near-complete elimination of artifacts (bottom panel), particularly those concurrent with onset of lick responses. Histograms on right indicate a narrower bandwidth of dF/F values using 3D correction, likely due to improved robustness to licking and other motion artifacts (e.g., breathing, heart rate).

Figure 6

Simultaneous calcium responses from multiple cortical neurons during visual behavior. (A) Responses from 3 out of 20 neurons recorded simultaneously using OGB1-AM indicator during a 2-h behavior session (black traces; 130–178 μm deep; 13–16 mW excitation at 800 nm). Colored vertical bars indicate presentation of target (green) and non-target (red, blue) stimuli (1 s stimuli; bar widths are 3 s wide to emphasize stimulus selectivity of different neurons). Black line indicates task engagement/passive viewing, gray ticks indicate licking, and blue trace indicates online estimate of angular pupil displacement (downsampled to 50 Hz) after correction for translational motion (using corneal reflections). Small nasally directed eye rotations occurred concurrent with reward presentation. Visual responses were not strongly affected by licking or small changes in eye position. (B) Following cessation of behavioral trials (>900), mapping of orientation tuning in these same cells was performed in 16 orientations, including the three orientations used during behavior (colored lines). (C) Orientation responses in cell 2 for 16 consecutive orientations showed high signal-to-noise ratio in single trials (gray traces) and on average (black trace). (D) Neurons in and surrounding the imaged volume (upper panel) expelled the dye over ∼12–36 h (not shown), but were reloaded and imaged 2 days later (lower panel). (E) Average responses (mean ± SE) during behavior (cell 2, left panels) were robust across large numbers of trials and across both these imaging sessions, as well as in a third session (Day 4) imaging calcium dye loaded a day earlier. Yellow box: stimulus presentation. Polar plots of orientation tuning mapped following behavioral sessions (normalized to peak response) were also stable across multiple days (right panels; different rows indicate different days).

Figure 7

Stable recordings during visual behavior using a genetically encoded calcium indicator. (A) Strong calcium responses (black trace) during visual behavior recorded from a cell with virally induced expression of the ratiometric calcium indicator, YC3.6 (132–180 μm deep; 15 mW excitation at 830 nm). The trace was computed as the ratio of YFP to CFP fluorescence (using 4 Hz volume acquisition). All lines and colors are as in Figure 6A. (B) Top: Color coded stimulus directions used. Middle one plane in the volume collected during imaging in (A). Bottom: Lower magnification image illustrates sparsity of expression. (C) Average responses (mean ± SE) to target stimuli presented during first and second hour of behavior. Response strength did not decline over two consecutive hours of imaging. (D) Another neuron in the same mouse demonstrated visual responses during behavioral sessions that were spaced over a month apart. Top: Timeline and images of the same neuron and its processes across behavioral sessions (and number of behavioral trials per session). Bottom: Responses to target stimuli during a large number of behavioral trials in four different imaging sessions.