Sensory perception arises from the integration of externally and internally driven representations of the world. Disrupted balance of these representations can lead to perceptual deficits and hallucinations. The serotonin-2A receptor (5-HT 2A R) is associated with such perceptual alterations, both in its role in schizophrenia and in the action of hallucinogenic drugs. Despite this powerful influence on perception, relatively little is known about how serotonergic hallucinogens influence sensory processing in the neocortex. Using widefield and two-photon calcium imaging and single-unit electrophysiology in awake mice, we find that administration of the hallucinogenic selective 5-HT 2A R agonist DOI (2,5-dimethoxy-4-iodoamphetamine) leads to a net reduction in visual response amplitude and surround suppression in primary visual cortex, as well as disrupted temporal dynamics. However, basic retinotopic organization, tuning properties, and receptive field structure remain intact. Our results provide support for models of hallucinations in which reduced bottom-up sensory drive is a key factor leading to altered perception.

The selective 5-HTR agonist DOI (2,5-dimethoxy-4-iodoamphetamine) is known to be a powerful hallucinogen in humans () and has been used extensively to study 5-HTR function in animal models, particularly of schizophrenia and psychedelic drug action (for reviews, see). In this study, we assessed the impact of DOI on visual processing at multiple scales, from retinotopic maps to individual neurons, using widefield and two-photon calcium imaging and single-unit electrophysiology in awake, head-fixed mice. Our results demonstrating how a serotonergic hallucinogen disrupts sensory processing should provide a deeper understanding of how cortical circuits generate a representation of the world based on sensory input.

The cognitive and perceptual effects of 5-HTR modulation have been extensively studied, particularly in the context of psychedelic drugs (reviewed in). Recent studies have begun to elucidate the action of serotonergic hallucinogens on large-scale brain activity in humans using neuroimaging methods (). However, the impact on sensory information processing at the level of single neurons and populations of neurons is largely unknown. To our knowledge, measures of visually evoked responses after 5-HTR agonist administration in humans are limited to one study, which showed large reductions in pre-stimulus alpha-band LFP synchronization (). There have been few studies of individual V1 neuron responses to visual stimuli following administration of 5-HTR agonists, yielding varying findings of suppression, facilitation, or bidirectional changes in firing rate (). Furthermore, these studies were conducted in anesthetized animals, did not measure individual neuron-tuning properties, and did not address cell type or layer specificity.

The effects of LSD and some analogues on the responses of single cortical neurons of the cat to optical stimulation.

Changes in global and thalamic brain connectivity in LSD-induced altered states of consciousness are attributable to the 5-HT2A receptor.

Both externally (bottom-up) and internally (top-down) driven representations of the world contribute to sensory perception. Disruption of accurate sensory perception, as occurs during hallucination, is hypothesized to result from increased top-down and/or decreased bottom-up signaling, leading to excessive reliance on prediction at the expense of sensory input (). Abnormal serotonin-2A receptor (5-HTR) activity is implicated in sensory hallucination, defined as the misinterpretation of sensory stimuli in space or time or the perception of an absent external stimulus. In particular, hallucinations and altered perception resulting from both schizophrenia and psychedelic drug administration are prevented by antagonism of 5-HTRs, supporting a central role of this receptor in mediating hallucinations ().

Results

2A R activation on spatial and temporal processing in visual cortex, we measured visual responses in mice head-fixed on a spherical treadmill ( Dombeck et al., 2007 Dombeck D.A.

Khabbaz A.N.

Collman F.

Adelman T.L.

Tank D.W. Imaging large-scale neural activity with cellular resolution in awake, mobile mice. 2A R agonist DOI (10 mg/kg; see 2A R signaling, we performed a subset of these passive viewing experiments with animals previously trained on a visually guided task, in addition to standard non-trained animals. As visual responses and surround suppression are modulated by behavioral state ( Niell and Stryker, 2010 Niell C.M.

Stryker M.P. Modulation of visual responses by behavioral state in mouse visual cortex. Ayaz et al., 2013 Ayaz A.

Saleem A.B.

Schölvinck M.L.

Carandini M. Locomotion controls spatial integration in mouse visual cortex. Figure 1 DOI Reduces Visually Evoked Responses in Visual Cortex Show full caption (A) In all experiments, we measured responses to visual stimuli before and 20 min after drug administration using widefield and two-photon GCaMP6s imaging or silicon probe electrophysiology in awake, head-fixed mice on a spherical treadmill. (B) Group-averaged phase maps from widefield responses to bilateral stimulus presentation moving along the azimuth (left hemispheres) or elevation (right hemispheres) before and after drug administration. (C) Correlation coefficients for pre- versus post-phase maps across groups. Circles represent individual animals, and bars represent mean ± SEM. (D) Widefield responses to grating patches presented to the right eye before and after drug administration during stationary periods. Inset shows cortical schematic with left visual areas in red. (E) Cycle averages (top; gray bars represent stimulus period) and spatial spread of response (bottom) measured from a manually selected point in V1 (white asterisk, inset). (F) Changes to visually evoked responses after drug administration across groups. Open circles represent individual animals, bars are mean ± SEM. A value of 1 represents no change, asterisks indicate significant change (p < 0.05; saline naive: n = 5; saline trained: n = 5; DOI naive: n = 6; DOI trained: n = 5). To measure the effects of 5-HTR activation on spatial and temporal processing in visual cortex, we measured visual responses in mice head-fixed on a spherical treadmill () using widefield imaging and two-photon calcium imaging, and single-unit electrophysiology with silicon probes ( Figure 1 A). Following presentation of a set of visual stimuli, mice received a subcutaneous injection of either saline (control) or the 5-HTR agonist DOI (10 mg/kg; see STAR Methods and Figure S1 for an explanation of dose choice), and after a 15- to 20-min waiting period, the stimulus set was repeated. To explore how previous experience with visual stimuli may influence effects of 5-HTR signaling, we performed a subset of these passive viewing experiments with animals previously trained on a visually guided task, in addition to standard non-trained animals. As visual responses and surround suppression are modulated by behavioral state (), we separated data into stationary or running periods for statistical comparison. Notably, neither pupil size nor fraction of time running was different following drug administration ( Figure S2 ), suggesting that changes observed were not due to differences in behavioral state.

Wekselblatt et al., 2016 Wekselblatt J.B.

Flister E.D.

Piscopo D.M.

Niell C.M. Large-scale imaging of cortical dynamics during sensory perception and behavior. Widefield imaging of cortical excitatory neurons in CaMKIIa-tTA:tetO-GCaMP6s mice (GCaMP6s mice;) revealed no change in the retinotopic map of azimuth and elevation in visual cortex ( Figures 1 B and 1C; p = 0.999, Kruskal-Wallis; see also Movie S1 ) but a dramatic reduction in responses to grating patches in visual areas after DOI, but not saline, administration during stationary periods ( Figures 1 D and 1E). Interestingly, this reduction was larger in animals that had previously received training on a visual task than in animals naive to training ( Figure 1 F; p = 0.012, Kruskal-Wallis; paired t test: DOI trained: p = 0.031, n = 5; DOI naive: p = 0.049, n = 6; saline trained: p = 0.192, n = 5, saline naive: p = 0.917, n = 5). Passive stimuli used here were similar to those used in previous behavioral experiments (circular grating patches) but were different in size and location in visual space (see STAR Methods for further details), arguing against effects of perceptual learning. Furthermore, baseline responses between trained and naive animals were not statistically different ( Figure S3 ).

eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyMmU5Y2RlMTk5NTA1ZmRhODJkNWEyZjg3NjEyM2Y1NiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjAwOTI2NzI1fQ.UAl6tqVSO86cXcWfDdlOYyf-mQ67J8lotn2XIWKNEQik89pTWpzRlzbqc_hcauQCPtCQFjCoS17d4d2VolRyDQeibY0yabgK9pqA944XZuAbrno4Vage0CXKtFOCHwh3kxV4bw9lOvJVDBixt8AE3S_E9SidnLtFtQeJQvQzCvOPA3zf99zBw994tizHLPfqe_7L5M9wsl_CaxBpyWm1sc6nqDeYLn0fnx_enBaySRk659aa00Lpa9elbwXhajL-X0LnfiMGwuWMFYSkxqg6KjmzwE3VULZp8A_dS1H9_DDKJJK4ksUKn7xYybJcWvVK30IprXdCol2PMgn1oOK6fg

Ayaz et al., 2013 Ayaz A.

Saleem A.B.

Schölvinck M.L.

Carandini M. Locomotion controls spatial integration in mouse visual cortex. D ) and suppressive (R S ) fields. Both R D and R S were reduced after administration of DOI, but not saline ( D and R S were significant for both naive and trained animals during stationary periods ( D p = 0.021, R S p = 0.010, Kruskal-Wallis; paired t test: DOI trained: R D p = 0.003, R S p = 0.002 n = 9/215; DOI naive: R D p = 0.020, R S p = 0.012, n = 8/144; saline trained: R D p = 0.201, R S p = 0.730 n = 11/197; saline naive: R D p = 0.159, R S p = 0.317, n = 11/269; where n = animals/cells; alpha = 0.025 corrected for multiple comparisons). DOI also reduced R D during running bouts in trained, but not naive animals (not shown; R D p = 0.023, R S p = 0.032, Kruskal-Wallis; paired t test: DOI trained: R D p = 0.015, R S p = 0.026; DOI naive: R D p = 0.084, R S p = 0.357; saline trained: R D p = 0.773, R S p = 0.031; saline naive: R D p = 0.744, R S p = 0.559; alpha = 0.025 corrected for multiple comparisons). Consistent with these changes in R D and R S , DOI reduced the suppression index in naive (stationary only) and trained (stationary and running) animals ( stat = 0.005, p run = 0.014; DOI naive: p stat = 0.034, p run = 0.814; saline trained: p stat = 0.285, p run = 0.150; saline naive: p stat = 0.261, p run = 0.390). Figure 2 DOI Reduces Surround Suppression in V1 L2/3 Excitatory Neurons Show full caption (A) Two-photon images in V1 showing responses to stimuli of increasing size before (top) and after (bottom) DOI administration in an example animal. Note surround suppression in the neuropil response. White scale bar in the top left image represents 200 μm. Data are from stationary periods only (see text for running data). (B) Cycle averages of extracted (see STAR Methods ) individual L2/3 excitatory neurons to corresponding stimuli shown above (gray bars show stimulus period), averaged within then across animals before (black) and after (red) DOI administration. (C) Size tuning curve from data in (B) showing average responses of individual neurons with increasing stimulus size. Data are presented as points with error bars, and divisive normalization fits are shown as lines with shaded error bars. (D) Driving (R D ) and suppressive (R S ) field coefficients and suppression index (SI) from divisive normalization fits of individual animal size-tuning curves for saline (black) and DOI (blue) before and after drug administration. (E) Changes in driving and suppressive field coefficients and SI within each group before and after drug administration. A value of 1 represents no change, and asterisks indicate a significant change (p < 0.025 for R D , R S ; p < 0.05 for SI; n = animals/cells: saline naive: n = 11/269; saline trained: n = 11/144; DOI naive: n = 8/144; DOI trained: n = 9/215). Given that widefield signals represent the summed activity in cell bodies, dendrites, and axons from many different excitatory cortical neurons, we next used two-photon calcium imaging to study the effect at the level of individual neurons, focusing on spatial integration. A key mechanism by which V1 neurons integrate information across space is through surround suppression, where larger stimuli tend to decrease V1 responses. This phenomenon can be explained by divisive normalization of “driving” classical receptive field (CRF) responses by “suppressive” responses in the extra-CRF (eCRF). We performed two-photon imaging in L2/3 of V1 in GCaMP6s mice while showing grating patches of varying sizes (5°–50°), which revealed clear surround suppression in the neuropil responses ( Figure 2 A; see also Movie S2 ). Consistent with widefield imaging, DOI reduced the magnitude of visual responses at the level of neuropil, as well as the visual responses of individual neurons ( Figure 2 B). We computed size tuning curves from the individual neuron data ( Figure 2 C), fit these with a divisive normalization model (; see STAR Methods ), and measured the coefficients of the driving (R) and suppressive (R) fields. Both Rand Rwere reduced after administration of DOI, but not saline ( Figure 2 D). These DOI-induced changes in Rand Rwere significant for both naive and trained animals during stationary periods ( Figure 2 E; Rp = 0.021, Rp = 0.010, Kruskal-Wallis; paired t test: DOI trained: Rp = 0.003, Rp = 0.002 n = 9/215; DOI naive: Rp = 0.020, Rp = 0.012, n = 8/144; saline trained: Rp = 0.201, Rp = 0.730 n = 11/197; saline naive: Rp = 0.159, Rp = 0.317, n = 11/269; where n = animals/cells; alpha = 0.025 corrected for multiple comparisons). DOI also reduced Rduring running bouts in trained, but not naive animals (not shown; Rp = 0.023, Rp = 0.032, Kruskal-Wallis; paired t test: DOI trained: Rp = 0.015, Rp = 0.026; DOI naive: Rp = 0.084, Rp = 0.357; saline trained: Rp = 0.773, Rp = 0.031; saline naive: Rp = 0.744, Rp = 0.559; alpha = 0.025 corrected for multiple comparisons). Consistent with these changes in Rand R, DOI reduced the suppression index in naive (stationary only) and trained (stationary and running) animals ( Figures 2 D and 2E; suppression index paired t test before versus after: DOI trained: p= 0.005, p= 0.014; DOI naive: p= 0.034, p= 0.814; saline trained: p= 0.285, p= 0.150; saline naive: p= 0.261, p= 0.390).

eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhYWQ1YjA3ZDFlMGVkMjM1MjNjN2VjNTA5ZTFiMDg2NiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjAwOTI2NzI1fQ.BAQ9ACAIQqduHufgnEUgNrv_bWX6vmU19tVSN12kkVPUkKptWT9C5Y1T8sn66PRPN1UAwqioJYOeBKZMSE_iqOxBCdQQPAcmEXzyZ5CqXqkClQ9IZKaQp2dcpytuU-eYFmf2S1OGG-oz3nYwkcYYQUsQcCip-lmdlpcPziXT4ODjIQd2yBnjB8EN0F3QUqy2KzY4d919fGKMG7QkQogt0i3mulp05jx9sOH0HwhhuxVacNdy8W_19mVMvB0CakylIrpru1L8TcfFF9XmFtq1ZOFv8JKa7IgC_ZX44T57Z1rKNlIVKF8gnlNH5Gt5UpmnL-lADyFeM0IAPNY48I1VUw

2A R activation affects temporal dynamics of population activity, we recorded local field potentials (LFPs) using silicon probes and found the average LFP power in all cortical layers was reduced across a wide frequency range following administration of DOI in both spontaneous (not shown) and visually evoked activity ( delta = 0.184, p theta = 0.531, p alpha = 0.254, p beta = 0.065, p gamma = 0.0361, n = 12 animals; stationary DOI: p delta = 0.127, p theta = 0.0015, p alpha = 0.002, p beta = 0.0001, p gamma = 0.0001, n = 12 animals; running saline: p delta = 0.072, p theta = 0.995, p alpha = 0.572, p beta = 0.616, p gamma = 0.287, n = 12 animals; running DOI: p delta = 0.766, p theta = 0.0077, p alpha = 0.02, p beta = 0.0003, p gamma = 0.0005, n = 12 animals; alpha = 0.01). Interestingly, the visual stimulus-evoked increase in gamma power (28–35 Hz) was completely abolished after DOI administration. These results are consistent with findings from studies of hallucinogenic drug effects in humans using electroencephalography (EEG) and magnetoencephalography (MEG) ( Kometer et al., 2013 Kometer M.

Schmidt A.

Jäncke L.

Vollenweider F.X. Activation of serotonin 2A receptors underlies the psilocybin-induced effects on α oscillations, N170 visual-evoked potentials, and visual hallucinations. Carhart-Harris et al., 2016 Carhart-Harris R.L.

Muthukumaraswamy S.

Roseman L.

Kaelen M.

Droog W.

Murphy K.

Tagliazucchi E.

Schenberg E.E.

Nest T.

Orban C.

et al. Neural correlates of the LSD experience revealed by multimodal neuroimaging. Figure 3 DOI Reduces LFP Power and Bidirectionally Modulates Visually Evoked Firing Rate Show full caption (A) Average stationary and running LFP power ± SEM before (black) and after (red) administration of saline or DOI in response to sinusoidal drifting gratings (n saline = 12 sessions, n DOI = 12 sessions). (B) Peak visually evoked firing rate before or after saline or DOI during stationary periods. Blue circles represent excitatory units, and red circles represent inhibitory units. 8% of saline units and 3% of DOI units are not shown. Black and gray crosses represent averages of all units and individual animals, respectively, including those not shown (n saline exc = 155 cells, n saline inh = 26 cells, n saline = 15 animals, n DOI exc = 187 cells, n DOI inh = 17 cells, n DOI = 15 animals). (C) Change in peak firing rate as a function of initial peak firing rate. One saline and one DOI unit are not shown. (D) Modulation indices (MIs) calculated from change in visually evoked peak firing rate between pre- and post-blocks. MI of 1 represents complete facilitation of firing rate after drug injection. (E) MI distributions for spontaneous rates. (F) Mean absolute value of MIs shows layer-specific changes between saline and DOI for the L2/3 evoked rate. In order to determine how 5-HTR activation affects temporal dynamics of population activity, we recorded local field potentials (LFPs) using silicon probes and found the average LFP power in all cortical layers was reduced across a wide frequency range following administration of DOI in both spontaneous (not shown) and visually evoked activity ( Figure 3 A; paired t test, corrected for multiple comparisons: stationary saline: p= 0.184, p= 0.531, p= 0.254, p= 0.065, p= 0.0361, n = 12 animals; stationary DOI: p= 0.127, p= 0.0015, p= 0.002, p= 0.0001, p= 0.0001, n = 12 animals; running saline: p= 0.072, p= 0.995, p= 0.572, p= 0.616, p= 0.287, n = 12 animals; running DOI: p= 0.766, p= 0.0077, p= 0.02, p= 0.0003, p= 0.0005, n = 12 animals; alpha = 0.01). Interestingly, the visual stimulus-evoked increase in gamma power (28–35 Hz) was completely abolished after DOI administration. These results are consistent with findings from studies of hallucinogenic drug effects in humans using electroencephalography (EEG) and magnetoencephalography (MEG) (), which also show an overall reduction in oscillatory synchronization.

2A R activation by analyzing responses of isolated single units to drifting sinusoidal gratings. We focused this analysis on L2/3 and L5 because they display distinct response properties ( Niell and Stryker, 2008 Niell C.M.

Stryker M.P. Highly selective receptive fields in mouse visual cortex. 2A R density in mouse neocortex ( Weber and Andrade, 2010 Weber E.T.

Andrade R. Htr2a gene and 5-HT(2A) receptor expression in the cerebral cortex studied using genetically modified mice. Niell and Stryker, 2008 Niell C.M.

Stryker M.P. Highly selective receptive fields in mouse visual cortex. 2 = 0.74, p = 0.679, n = 155; DOI: r2 = 0.44, p = 0.181, n = 187; paired t test). Interestingly, we observed rate-specific modulation of responses; neurons with initially low firing rates were facilitated, and neurons with initially high firing rates were suppressed ( 2A R activation in anesthetized non-human primate and cat V1 ( Watakabe et al., 2009 Watakabe A.

Komatsu Y.

Sadakane O.

Shimegi S.

Takahata T.

Higo N.

Tochitani S.

Hashikawa T.

Naito T.

Osaki H.

et al. Enriched expression of serotonin 1B and 2A receptor genes in macaque visual cortex and their bidirectional modulatory effects on neuronal responses. Rose and Horn, 1977 Rose D.

Horn G. Effects of LSD on the response of single units in cat visual cortex. 2 = 0.73, p = 0.103, n = 26; DOI: r2 = 0.93, p = 0.812, n = 17; paired t test). The same pattern was observed during locomotive states (not shown; saline excitatory: r2 = 0.55, p = 0.057, inhibitory: r2 = 0.75, p = 0.215; DOI excitatory: r2 = 0.44, p = 0.7.15e-05, inhibitory r2 = 0.93, p = 0.577; paired t test). We next aimed to examine how individual V1 neuron activity is affected by 5-HTR activation by analyzing responses of isolated single units to drifting sinusoidal gratings. We focused this analysis on L2/3 and L5 because they display distinct response properties (), and both excitatory and inhibitory neurons in these layers contain the highest 5-HTR density in mouse neocortex (). Units were classified as putative excitatory or narrow-spiking inhibitory based on spike waveform (). As such, inhibitory neurons in this study are likely fast-spiking parvalbumin (PV) cells and not somatostatin (SOM)-expressing cells. Following DOI administration, the peak visually evoked firing rate of excitatory V1 neurons was bidirectionally modulated ( Figure 3 B; saline: r= 0.74, p = 0.679, n = 155; DOI: r= 0.44, p = 0.181, n = 187; paired t test). Interestingly, we observed rate-specific modulation of responses; neurons with initially low firing rates were facilitated, and neurons with initially high firing rates were suppressed ( Figure 3 C), similar to observations with 5-HTR activation in anesthetized non-human primate and cat V1 (). In contrast to the excitatory neuron population, inhibitory neurons did not change their peak evoked firing rate (saline: r= 0.73, p = 0.103, n = 26; DOI: r= 0.93, p = 0.812, n = 17; paired t test). The same pattern was observed during locomotive states (not shown; saline excitatory: r= 0.55, p = 0.057, inhibitory: r= 0.75, p = 0.215; DOI excitatory: r= 0.44, p = 0.7.15e-05, inhibitory r= 0.93, p = 0.577; paired t test).

To determine how strongly each cortical layer was affected by DOI, we calculated modulation indices of stationary peak firing rate across the neural population, where negative (positive) values represent neurons that reduced (increased) their rate following drug administration ( Figures 3 C and 3D). The distributions were shifted overall toward suppression; however, because these distributions were bidirectional, we calculated the mean absolute value for each layer to determine the strength of modulation independent of sign. This revealed visually evoked responses in L2/3 were more affected by DOI than saline (t test: p = 0.005, corrected for multiple comparisons), whereas spontaneous rate was not affected ( Figure 3 E). Thus, the effects of DOI are specific for layer and cell type and differ for spontaneous versus evoked activity.

stat = 0.0001, n = 37, p run = 0.004, n = 61; L5: p stat = 0.001, n = 13, p run = 0.026, n = 19), consistent with more neurons being suppressed than enhanced, whereas inhibitory units were not affected (inhibitory [inh.]: p stat = 0.878, n = 7, p run = 0.878, n = 10). Figure 4 DOI Disrupts Temporal Dynamics in a Layer-Specific Manner but Maintains Tuning Properties Show full caption (A) Mean peristimulus time histograms ± SEM before (black) and after (red) administration of DOI across L2/3, L5, and inhibitory units during stationary and locomotive periods. Gray bars show stimulus period. (B) Mean firing rate for each cell before or after DOI administration across transient and sustained components from PSTHs shown in (A). The transient component is defined as the first 500 ms after stimulus onset, and the sustained component is defined as the 500 ms preceding the stimulus offset (L2/3: n stat = n = 37, n run = 61; L5: n stat = 13, n run = 19; inh. n stat = 7, n run = 10). (C) Preferred orientation of individual neurons before or after saline or DOI administration (n saline = 37, n DOI = 33). (D) Average orientation selectivity index (OSI; circular variance) across populations of visually responsive cells before or after saline or DOI injection (n saline = 100, n DOI = 91). (E) Proportion of visually responsive cells (>2 Hz) selective for preferred spatial frequencies before or after drug treatment (n saline = 100, n DOI = 93). (F) Histograms of 2D correlation coefficients of raw spike triggered average receptive fields of all cells responsive above 2 Hz. A value of 1 represents STAs that did not change after saline or DOI administration (n saline = 28, n DOI = 41). We next determined how DOI affected the time course of V1 responses based on the peristimulus time histogram (PSTH) of responses to drifting gratings ( Figure 4 A). Following DOI administration, we saw layer-specific changes in the mean PSTH of visually responsive cells (neurons with peak visually evoked rate greater than 2 Hz in either the pre- or post-recording block). The mean response of both L2/3 and L5 was significantly reduced (two-sample Kolmogorov-Smirnov test; L2/3: p= 0.0001, n = 37, p= 0.004, n = 61; L5: p= 0.001, n = 13, p= 0.026, n = 19), consistent with more neurons being suppressed than enhanced, whereas inhibitory units were not affected (inhibitory [inh.]: p= 0.878, n = 7, p= 0.878, n = 10).

stat = 0.0002, p run = 0.0035) and was only affected during the sustained component when animals were running (p = 0.0007; trans p stat = 0.0471; L5 trans p run = 0.864; L5 sus p stat = 0.436; L5 sus p run = 0.727; inh trans p stat = 0.587; inh trans p run = 0.875; inh sus p stat = 0.964; inh sus p run = 0.852). Thus, DOI administration disrupts temporal dynamics of visual responses in L2/3 by strongly reducing the onset transient. The time course of the mean PSTH showed a transient response at stimulus onset followed by a smaller sustained response, which was most pronounced in L2/3 neurons ( Figure 4 A). Notably, the transient (first 500 ms after stimulus onset) and sustained (500 ms preceding stimulus offset) components were differentially affected by DOI. We separated the two temporal components and found that L2/3 was strongly suppressed during the transient component (p= 0.0002, p= 0.0035) and was only affected during the sustained component when animals were running (p = 0.0007; Figure 4 B). L5 and inhibitory units, in contrast, did not show a significant net change in either temporal component (L5= 0.0471; L5= 0.864; L5= 0.436; L5= 0.727; inh= 0.587; inh= 0.875; inh= 0.964; inh= 0.852). Thus, DOI administration disrupts temporal dynamics of visual responses in L2/3 by strongly reducing the onset transient.

2 = 0.92, p = 0.346, n = 37; DOI: r2 = 0.87, p = 0.639, n = 33; not shown; saline running: r2 = 0.87, p = 0.425, n = 27; DOI running: r2 = 0.81, p = 0.873, n = 43; paired t test). The mean orientation selectivity index was also unaffected by DOI and saline administration (2 = 0.95 p = 0.912 n = 33, running: r2 = 0.5 p = 0.929 n = 25; DOI stationary: r2 = 0.57 p = 0.861 n = 56; running: r2 = 0.46 p = 0.486 n = 62; DSI saline stationary: p = 0.95 n = 71; running: p = 0.25 n = 71; DOI stationary: p = 0.33, n = 64; running: p = 0.70, n = 64). We also found no change in the distribution of spatial frequency preference for responsive cells, as the same proportions were selective to either low (0.01–0.02 cycles per degree [cpd]; paired t test mean of running and stationary; saline: p = 0.435; DOI: p = 0.823), medium (0.04–0.08 cpd; saline: p = 0.334; DOI: p = 0.397), or high (0.16–0.32 cpd; saline: p = 0.640; DOI: p = 0.485) spatial frequencies or to full-field flicker (saline: p = 0.267, DOI: p = 0.577) following DOI treatment (2 = 0.012, pref ori moving [mv]: r2 = 0.034, orientation selectivity index [OSI] stat: r2 = 0.005, OSI mv: r2 = 0.063; DOI: pref ori stat: r2 = 0.0001; pref ori mv: r2 = 0.0096; OSI stat: r2 = 0.021; OSI mv: r2 = 0.053). We next determined if DOI affected the encoding of low-level stimulus features and feature selectivity. Across the recorded population of neurons, we found no change for the preferred grating orientation following DOI administration ( Figure 4 C; saline: r= 0.92, p = 0.346, n = 37; DOI: r= 0.87, p = 0.639, n = 33; not shown; saline running: r= 0.87, p = 0.425, n = 27; DOI running: r= 0.81, p = 0.873, n = 43; paired t test). The mean orientation selectivity index was also unaffected by DOI and saline administration ( Figure 4 D; Wilcoxon rank sum test on mean of stationary and running; saline: p = 0.362, n = 100; DOI: p = 0.214, n = 91). Preferred direction of grating motion and mean direction selectivity index (DSI) were also unchanged (not shown; Wilcoxon rank sum test; preferred direction saline stationary: r= 0.95 p = 0.912 n = 33, running: r= 0.5 p = 0.929 n = 25; DOI stationary: r= 0.57 p = 0.861 n = 56; running: r= 0.46 p = 0.486 n = 62; DSI saline stationary: p = 0.95 n = 71; running: p = 0.25 n = 71; DOI stationary: p = 0.33, n = 64; running: p = 0.70, n = 64). We also found no change in the distribution of spatial frequency preference for responsive cells, as the same proportions were selective to either low (0.01–0.02 cycles per degree [cpd]; paired t test mean of running and stationary; saline: p = 0.435; DOI: p = 0.823), medium (0.04–0.08 cpd; saline: p = 0.334; DOI: p = 0.397), or high (0.16–0.32 cpd; saline: p = 0.640; DOI: p = 0.485) spatial frequencies or to full-field flicker (saline: p = 0.267, DOI: p = 0.577) following DOI treatment ( Figure 4 E; saline: n = 100; DOI: n = 93). The observed changes in firing rate did not correlate with tuning properties or selectivity (not shown; saline preferred [pref] orientation [ori] stationary [stat]: r= 0.012, pref ori moving [mv]: r= 0.034, orientation selectivity index [OSI] stat: r= 0.005, OSI mv: r= 0.063; DOI: pref ori stat: r= 0.0001; pref ori mv: r= 0.0096; OSI stat: r= 0.021; OSI mv: r= 0.053).