Visual Information Present in Infragranular Layers of Mouse Auditory Cortex

Abstract

The cerebral cortex is a major hub for the convergence and integration of signals from across the sensory modalities; sensory cortices, including primary regions, are no exception. Here we show that visual stimuli influence neural firing in the auditory cortex of awake male and female mice, using multisite probes to sample single units across multiple cortical layers. We demonstrate that visual stimuli influence firing in both primary and secondary auditory cortex. We then determine the laminar location of recording sites through electrode track tracing with fluorescent dye and optogenetic identification using layer-specific markers. Spiking responses to visual stimulation occur deep in auditory cortex and are particularly prominent in layer 6. Visual modulation of firing rate occurs more frequently at areas with secondary-like auditory responses than those with primary-like responses. Auditory cortical responses to drifting visual gratings are not orientation-tuned, unlike visual cortex responses. The deepest cortical layers thus appear to be an important locus for cross-modal integration in auditory cortex.SIGNIFICANCE STATEMENT The deepest layers of the auditory cortex are often considered its most enigmatic, possessing a wide range of cell morphologies and atypical sensory responses. Here we show that, in mouse auditory cortex, these layers represent a locus of cross-modal convergence, containing many units responsive to visual stimuli. Our results suggest that this visual signal conveys the presence and timing of a stimulus rather than specifics about that stimulus, such as its orientation. These results shed light on both how and what types of cross-modal information is integrated at the earliest stages of sensory cortical processing.

Keywords: audiovisual; cortical; cross-modal; laminar; multisensory; sensory.

Figure 1.

A, Awake, acute recordings in right mouse ACtx using a 16-channel multisite probe (right), experimental sequence: B–D. B, Tone stimuli used for identification of characteristic auditory cortical responses (left) and flash stimuli to test for visual responsiveness (right). C, Auditory and visual MU responses (mean ± standard error) on the same recording channel. D, Probe location visualized using fluorescent dye Di-I, which was applied to the probe before recording. Images at 1× magnification (left) and 4× (right) used to determine location and laminar depth. Scale bar, 500 μm. E, Probe track marked by Di-I, marked with electrode site locations. White circles represent locations of channels shown in F. F, MU responses on all 16 channels from one recording. Left, Responses to tones of BF. Middle, Frequency response area. Right, Response to visual stimulus. *Significant response at q = 0.001 after Benjamini–Hochberg false discovery rate correction (see Materials and Methods). G, Additional examples of statistically significant MU responses to visual stimuli from different mice. H, Examples of statistically significant SU responses to visual stimuli. Inset, Waveforms. Black represents SU. Gray represents MU from corresponding channel. I, Example SU showing both auditory and visual responses.

Figure 2.

A, Probe track in ACtx visualized with Di-I, electrode sites marked by circles. B, Colored arrows indicate channels. Scale bar, 500 μm. B, MU auditory responses (left), frequency response area (middle), and visual responses (right) at sites from cortical depths indicated by colored arrows in A. *Visually responsive MU is located in L6. C, All visual responses shown as a function of depth. Red represents MUs. Purple represents SUs. n = 73 MUs and 15 SUs from 30 recordings in 15 mice. D, Fractions of visually responsive sites by depth. Numbers at left indicate total number of MU visual responses over total number of sites recorded in each layer. E, Ai32/Ntsr1-Cre mice express eYFP-tagged ChR2 in L6; histology showing eYFP (green) on right. F, Representative example of optogenetic identification of L6 through activation of Ntsr1-Cre-positive cells. MU activity shows a band activated strongly during light-on period (cyan, indicated above). Significantly modulated channels indicated by green band at right. G, Top, Superficial channel shows minimal light-related MU activity. Bottom, Deep channel MU activity shows strong effect of light activation. H, Summary plot of all visually responsive MUs (red dots) from all recordings with an identifiable light-activated L6 band (n = 7 MUs from 5 recordings in 4 mice) plotted by depth relative to the lower border of this band (green).

Figure 3.

A, Left, Example recording classified as primary-like, based on short latency of responses to tones at BF, all 16 channels shown. Middle, Primary-like sites show a high degree of tuning to frequency and attenuation. Right, Response to visual stimulation at same site. *Responses on deepest two channels were significant at q = 0.001 (see Materials and Methods). B, Recording classified at secondary-like, based on longer latency of responses to tones of BF, as in A. Significant MU visual response (q = 0.001) recorded on channel third from bottom. C, Locations of visually responsive channels from anatomically reconstructed sites (n = 13 mice), color-coded blue (n = 22 sites) or cyan (n = 24 sites) for primary or secondary classification, respectively (section drawings from Paxinos and Franklin, 2004). Depth and anteroposterior distance determined from electrode track histology (see Fig. 1D). Distance behind the mouse skull landmark bregma and putative positions of primary ACtx (Au1, black) and dorsal ACtx (AuD, gray) are marked on each section. D, MU auditory response latencies by auditory classification; in box plots, black line indicates median, box edges indicate 25th and 75th percentile and whiskers extend to most extreme data points excluding outliers. Latencies at primary-like regions are shorter both in onset (left; Wilcoxon rank-sum Z = 11.75, p = 6.50e-32) and peak (right; Wilcoxon rank-sum Z = 10.34, p = 4.20e-25). E, Average MU FRA showing tuning from all sites classified as primary (left) or secondary (right), centered on BF. Before averaging, all FRAs were normalized to peak response. F, Auditory BF of all auditory-responsive MUs (top) and visually responsive MUs (bottom). G, Total counts of all recorded auditory- and visual-responsive MUs by layer (top). Fraction of visually responsive MUs (bottom), showing biases toward deeper layers and sites with secondary-like responses.

Figure 4.

A, Mean normalized firing rate of visually responsive SUs (q = 0.001 following Benjamini–Hochberg false discovery rate correction) in primary (dark red represents n = 45 MUs from 19 recordings in 12 mice) or secondary (light red represents n = 28 MUs from 11 recordings in 8 mice) ACtx following a flash stimulus. B, Mean normalized firing rate of visually responsive MU sites in primary (dark red represents n = 45 MUs from 19 recordings from 12 mice) or secondary (light red represents n = 28 MUs from 11 recordings from 8 mice) ACtx following a flash stimulus. C, Example probe track of recording in VCtx. Scale bar, 500 μm. D, Example MU response to flash stimulus in VCtx. E, Comparison of response dynamics between ACtx and VCtx, including all significantly visually responsive MUs from both regions (n = 73 ACtx MU sites from 30 recordings in 15 mice; n = 78 VCtx MU sites from 5 recordings from 3 mice). F, Latencies to onset, peak, and offset recorded from VCtx and ACtx, showing VCtx peak and onset responses occur earlier than those in ACtx, whereas offsets in VCtx occur later (see Results). In box plots, black line indicates median, box edges indicate 25th and 75th percentile, and whiskers extend to most extreme data points excluding outliers.

Figure 5.

A, Example ACtx SU response to drifting gratings of varied orientations (colors). Right, SU waveform. B, All peristimulus time histogram responses from A plotted together, revealing little preference for grating orientation. C, Results in B, visualized as baseline-normalized circular histogram. Colors represent visual response over 300 ms poststimulus window. Gray represents baseline firing rate. D, ACtx unit with highest OSI. E, For comparison, VCtx unit with highest OSI. F, Cumulative distribution plot of all OSIs, showing a higher degree of tuning in VCtx SUs (n = 17 units from 7 recordings in 3 mice) than those in ACtx (n = 7 units from 5 recordings in 3 mice).