Somatostatin Interneurons Control a Key Component of Mismatch Negativity in Mouse Visual Cortex.

A detailed description of experimental procedures and rationale are provided in Supplemental Experimental Procedures . Animals, Surgery, and Training All experimental procedures were approved by and carried out in accordance with Columbia University institutional animal care guidelines. Experiments were performed on adult parvalbumin-cre (n=10) or somatostatin-cre transgenic mice (n=21; Jackson Laboratory, C57BL/6 background, 22-32g; n-31 total), at the age of postnatal day (P) P60-P90. Virus injection, titanium head plate fixation (see supplement ), and skull-thinning/craniotomy were carried out in that order over the course of 4 weeks. A glass capillary pulled to a sharp micropipette was advanced with the stereotaxic instrument (coordinates from lambda: X=−2500, Y=200 μm), and 75ul solution of 1:1 diluted AAV1/Syn:GCaMP6s/f (obtained from the University of Pennsylvania Vector Core; n=20) or a 3:7 mixture of AAV1/Syn:GCaMP6s/f and AAV5hsynDIOhM4D(Gi)mCherry (UNC vector core; n=11 SOM-cre mice) was injected into putative layer 2/3 over a 5 min period at a depth of 200-300 μm from the pial surface using a UMP3 microsyringe pump (World Precision Instruments). On the day of the experiment, the mouse was anesthetized again with isoflurane. For local field potential recordings, a small region (approximately 0.5 mm in diameter) was removed left V1 centered just anterior to the injection site. For calcium imaging experiments, a small circle (approximately 1 mm in diameter) was thinned with a dental over the left V1 centered just anterior to the injection site. The skull was thinned until the bone, moistened with saline, was transparent enough so that the underlying vasculature was visible to the naked eye (usually 10 minutes of drilling). The mouse was then allowed to wake up and was transferred to the wheel for recordings.

Multielectrode recordings

Extracellular electrophysiological data are reported on 14 mice (8 female, 22-28g, 5 PV-cre, 8 SOM-cre). Sixteen-channel linear silicon probes (spaced at 50 Um intervals; model a1x16-3mm50-177, Neuronexus Technologies, Ann Arbor MI) were inserted perpendicular to and with the top electrode aligned just at the pial surface (visually confirmed with an adjustable miniature digital microscope (adafruit)). Recordings were referenced to the skull above prefrontal cortex and grounded to the headplate. Continuous data were acquired with a Plexon MiniDigi amplifier and software (Plexon Inc, Dallas, TX). Local field potential (LFP) signals were filtered from 0.5 to 300Hz and sampled at 1kHz. Single electrodes with the peak negative deflection 50-200ms post-stim near putative granular cortex were reserved for LFP analysis, or the whole array was analysed as current source density (see below). Locomotion was recorded as stripes in the running wheel crossed between an infared LED/photodarlington pair (see supplement ).

Two-Photon Calcium Imaging

The activity of cortical neurons was recorded by imaging fluorescence changes under a two-photon microscope (Bruker; Billerica, MA) excited with a Ti:Sapphire laser (Chameleon Ultra II; Coherent) tuned at 940 nm and scanned with resonant galvometers through a 20× (0.95 N.A.; Olympus) water immersion objective. To ensure stability of the imaging meniscus for long duration imaging sessions, a small volume (approx. 1ml) of Aquasonic ultrasound gel (Parker Laboratories Inc.) was centrifuged and dolloped onto a moistened, thinned skull in lieu of water. Scanning and image acquisition were controlled by Prairie View software (≈30 frames per second for 256 × 256 pixels, 200-225 um beneath the pial surface). On imaging days (before and after treatment) mice were allowed 1 hour on the wheel before imaging began. Imaging consisted of a visual stimulation condition (15 minutes), followed by 20-40 minutes of awake rest in a dark room with the monitor off. Data are reported on 18 mice (11 female, 7 PV-cre, 11 SOM-cre). Both 2P-Ca2+ and LFP recording sessions occurred between 11am and 3pm. Mice were monitored by the experimenter to ensure they were awake during data collection. Locomotion was detected as voltage deflections in the photodarlington readout. While previous work has suggested that locomotion enhances visual processing in V1 in mice, most of our mice did not exhibit enough locomotion to enable thorough examination of this effect in our paradigm (< 10% of frames/trials in LFP). Therefore, when detected, frames or trials during locomotion periods were excluded along with the previous and subsequent 60 frames (2-seconds). Large eye movements are uncommon in mice except during periods of locomotion, and, since we also near full field visual stimulation, the input for the recorded V1 retinotopic subfields was likely unaltered across trials and conditions.

Visual Stimulation

Visual stimuli were generated using the MATLAB (MathWorks) Psychophysics Toolbox and displayed on a liquid crystal display monitor (19-inch diameter, 60-Hz refresh rate) positioned 15 cm from the right eye, roughly at 45° to the long axis of the animal ( Fig. 1a ). Stimuli were static full-field square-wave gratings (100% contrast, 0.04 cycles per degree) oriented in 2 separate orientations for the oddball paradigm (45deg and 135deg) or in 8 orientations for the many-standards control (30, 45, 60, 90, 120, 135, 150, 180 deg). Stimuli were presented for 500 ms followed by an interstimulus interval of 1000-1500 ms of mean luminescence gray screen. In the oddball sessions, the “standard” stimulus was presented at a minimum of 3 sequential trials, followed by a linearly increasing probability of the “target” stimulus on each successive trial to yield an overall 12.5% probability of targets. These sessions lasted 10 minutes, and were repeated with the “standard” and “target” stimuli reversed. In the many-standards sessions, stimuli of 6 separate orientations each occurred at random with a 12.5% probability in a session of 10 minutes.

Current source density analysis

LFP data were manually prescreened for excessive artifact (e.g. signal greater than 8 standard deviations) and aberrant trials were removed and noisy channels were interpolated if present (never more than 2, and never 2 adjacent channels). Data were then digitally filtered from 0.1 to 300Hz (bandpass least squares FIR) and with a 60Hz notch filter. Average current source density (CSD) was computed from either the average LFP (for Fig 1d ) or on single trials and averaging across trials (number equalized across conditions; between 32 and 68) by taking the discrete second derivative across the electrode sites and interpolated to produce a smooth CSD map ( Buzsáki et al., 2012 ; Niell and Stryker, 2008 ). Putative laminar subregions (3 adjacent channels) were defined based on CSD demarcations previously published and histologically verified in mouse V1 for each mouse separately based on average CSD plots (see supplement ). Importantly, the presence of MMN-like responses and their D.R.E.A.D.D modulation did not appear to differ dramatically as function of layer, so statistical analyses and subsequent conclusions focus more generally on local (but not layer specific) processing in a V1 column. That is, we calculated a rectified CSD for each “layer” domain and averaged across domains within each mouse for statistical comparisons ( Fig. 1 , 2 ). Average CSD waveforms were then averaged over stimulus orientations.

Image Analysis

Imaging datasets were scored similarly to previous reports (see supplement ). ROIs were selected using a semi-manual PCA-assisted algorithm with halo subtraction (see supplement ). The discrete first derivative on lowess-smoothed tracese was scored as delta-f (within cell/single cell comparisons). A 2-10 second baseline window was manually selected for each which contained no apparent calcium transients. The mean and standard deviation was calculated on the delta-f values in this window for each cell for the whole experiment which was used to i) compute a z-scored delta-f for visualizing and combining activity across cells and ii) determine activation thresholds (see supplement ).

Single cell analyses

Condition averages of normalized delta-f values for redundant (4 th in sequence), deviant, and control stimuli were calculated separately for each stimulus type for each neuron. All analyses focused on the first 10-trials to equate across conditions, cells, and mice with varying numbers of available trials. Initial analyses focused on neurons showing, during control or deviant conditions, an average post-stimulus (0-1second) response of 1.67 stdevs above prestimulus baseline (equating to a 1-tailed p-value of .05; n=160, or 18.9% of all imaged cells; “Responsive” cells). Only responses to one stimulus orientation were considered for each cell (i.e. the orientation with greater magnitude). A minority of mice expressed GCaMP6f (5; 3 SOM-cre) while the rest expressed GCaMP6s (see supplement ). Pharmacogenetic suppression of interneurons On the day of recording, mice viewed visual stimuli with intermittent “rest” periods as described above. Then hM4D-SOM mice and an equivalent number of control mice each received a subcutaneous injection of Clozapine N-Oxide (CNO; 12 mg/kg; within the range of previously reported doses ( Roth, 2016 ; see supplement )), followed by a repeat of visual stimuli and “rest” periods during imaging or LFP recording 30 minutes later. In the presence of CNO, hM4D(Gi) activation functionally silences host cells primarily through the suppression of synaptic current amplitude and release ( Roth, 2016 ).

Statistical procedures

All significance values for t-tests are two-tailed. For establishing the presence of MMN-like potentials: the cross trial average LFP response from 40 to 240ms post-stim was averaged within mice and compared between redundant and deviant stimuli with a paried-samples t-test. SSA and deviance detection were confirmed with CSD and 2P-Ca2+. CSD: Paired t-tests were computed on mouse-wise averages. For SSA we compared responses between control and redundant stimuli (using the 4 th redundant in order to normalize for trial counts and for relative time during the run) in the early time-range (40-80ms). For deviance processing, we compared responses between deviant and control stimuli in the late time-range (120-240ms) ( Chen et al., 2015a ). 2P-Ca2+: Responses were quantified as described above (in Single cell analyses). Initial demonstration of SSA and deviance detection were established with paired t-tests on cell-wise averages of post stimulus activity (0-1 sec) from “responsive” cells. For SSA we compared responses between redundant and control stimuli; for deviance processing, we compared responses between deviant and control stimuli. Trial numbers were equated between stimulus conditions. All standard error bars in all figures reflect within-subjects/within-cells standard error. For determining the effect of SOM-suppression, a slightly different statistical approach was employed. LFP/CSD: For LFP estimates of MMN (deviant minus redundant responses, 120-240 ms time range), CSD estimates of SSA (redundant minus control; 40-80ms), and CSD estimates of deviance detection (deviant minus control; 120-240ms), difference values for each mouse were subjected to a 2-by-2 mixed ANOVA with GROUP (CNO-control; SOM-hM4D) as the between subject variable and TREATMENT (pre/post) as the within subject variable were computed. One-way repeated measures ANOVAs within groups were used to describe interaction effects. As an exploratory step we next focused on stimulus evoked oscillatory power in the LFP of the SOM-hM4D group, and for SSA and deviance detection separately and for each frequency band, we carried a 2-by-2 repeated measures ANOVA on evoked power within mice with STIM (redundant or deviants; control) and TREATMENT (pre/post) as within subject variables. Follow-up paired t-tests for interactions were computed within treatment conditions. 2P-Ca2+: two complimentary statistical approaches tested for single cell effects of SOM suppression. First, focusing on “responsive cells” during the main SSA time window (0-500ms post-stim, control only; number of cells: CNO-controlpre/post=60/63; SOM-hM4Dpre/post=60/60), we computed the proportion of cells showing a redundant response of less than X stdevs below the control response average (for X=.25 to 2.5). Then, focusing on “responsive” cells during the main deviance detection time window (150-750ms post-stim, control or deviant; number of cells: CNO-controlpre/post=82/90; SOM-hM4Dpre/post=100/103), we computed the proportion of cells showing a deviant-stimulus response of greater than X stdevs above the control reponse average for X=.25 to 2.5 in 0.1 stdev steps. Thus each of the 4 conditions (pre/post; control/SOM) had one SSA curve and one deviance detection curve. We then computed a log-rank test on these curves for SSA and deviance detection separately, and for CNO-control and SOM-hM4D separately. Second, we focused on the cells with the top 10% magnitude average responses to the control stimulus (for SSA) or to the deviant stimulus (for deviance detection) in the pre-treatment run. Responses to the redundant stimulus and responses to the deviant stimulus were divided by the average response to the control stimulus for each of the 4 conditions. Then, separately for SSA and DEV, a 2-by-2 mixed ANOVA on individual cells with GROUP (CNO-control; SOM-hM4D) as the between subject variable and TREATMENT (pre/post) as the within subject variable was computed. One-way repeated measures ANOVAs within groups were used to describe interaction effects. Pre-normalized values were also reported in figure S4 , confirming the effects did not depend on changes in baseline response magnitudes.

Supplementary Material 1 2