Gain control by layer six in cortical circuits of vision

Abstract

After entering the cerebral cortex, sensory information spreads through six different horizontal neuronal layers that are interconnected by vertical axonal projections. It is believed that through these projections layers can influence each other's response to sensory stimuli, but the specific role that each layer has in cortical processing is still poorly understood. Here we show that layer six in the primary visual cortex of the mouse has a crucial role in controlling the gain of visually evoked activity in neurons of the upper layers without changing their tuning to orientation. This gain modulation results from the coordinated action of layer six intracortical projections to superficial layers and deep projections to the thalamus, with a substantial role of the intracortical circuit. This study establishes layer six as a major mediator of cortical gain modulation and suggests that it could be a node through which convergent inputs from several brain areas can regulate the earliest steps of cortical visual processing.

Figure 1. Photo-stimulation of L6 suppresses visual responses in the other layers

a. Schematic of L6 projections. b. Left, coronal section of V1 from NTSR1-Cre, floxed-tdTomato, GAD67-GFP mouse. Inset, L6 projection to dLGN (V1 of NTSR1-Cre mouse was injected with floxed-tdTomato virus). Right, The two types of L6 neurons labeled by the NTSR1-Cre line. Dendrites: black; axons: gray. c. Schematic of setup. d. Cortical visual responses with (blue) and without (black) L6 photo-stimulation. Left, raster plot of multiunit activity grouped by depth. Control and photo-stimulation trials were interleaved but are separated here for clarity. Black bar: visual stimulus (1.5 s); blue bar: LED illumination (0.5 s). Right, normalized PSTH; top: upper layers; bottom: L6. e. Summary (n = 6 experiments). Control in black and increasing LED intensities in darker blues. f. Suppression of multiunit activity with increasing L6 activity. g. Visual response of single L4 unit with (blue) and without (black) L6 photo-stimulation. Scale bar, 20 spikes/s. h. Response of each regular spiking unit with and without strong photo-stimulation of L6. i. Average normalized PSTH (n = 47 units tested with 5 LED intensities). Colors as in (e). j. Suppression of single unit activity.

Figure 2. L6 bidirectionally modulates gain of visual responses without altering tuning

a. Visual responses of L5 neuron (2 of 8 tested directions) with (blue) and without (black) L6 photo-stimulation. Scale bar, 40 spikes/s. b. Tuning curves for neuron in (a). c. Orientation selectivity index (OSI) for each neuron with and without photo-stimulation of L6. d. Population tuning curve with (blue) and without (black) L6 photo-stimulation (n = 55). Black curve: fit using sum of two gaussians. Blue curve is black curve scaled by slope of linear fit in (e). e. Control response plotted against response with L6 photo-stimulation (data from c). Linear fit (blue; r² = 0.98). f. Visual response of L4 neuron with (orange) and without (black) L6 photo-suppression. Scale bar, 50 spikes/s. g. Tuning curves for neuron in (f). h. OSI for each isolated unit with and without photo-suppression of L6. i. Population tuning curves with and without L6 photo-suppression (n = 52). Black curve: fit using sum of two gaussians. Orange curve is black curve scaled by slope of linear fit in (j). j. Control response plotted against response with L6 photo-stimulation (data from i). Linear fit (orange; r² = 0.92).

Figure 3. Photo-stimulation of L6 suppresses cortex faster than it suppresses dLGN

a. Schematic of setup. b. Visual response of dLGN unit with (blue) and without (black) L6 photo-stimulation Scale bar, 20 spikes/s. c. Average response of each dLGN unit with and without L6 photo-stimulation. Inset, monotonic suppression of dLGN. d. Schematic of setup for silencing V1 by photo-stimulation of PV inhibitory neurons. e. Visual response of dLGN unit with and without photo-silencing of V1. Scale bar, 30 spikes/s f. Average response of each dLGN unit with and without cortical silencing. g. Schematic of setup. h. Left, time-course of L6-mediated suppression of dLGN (gray) and V1 (black) (n = 4). Bin size: 3 ms. Right, same data on expanded timescale. The first bin at LED onset was blanked to remove LED-induced artifact. Inset, time to suppression exceeding 2 SDs from baseline activity in dLGN and V1 (y-axis: ms) for four experiments (p = 0.012).

Figure 4. Photo-stimulation of L6 recruits intra-cortical synaptic inhibition

a. Schematic of in vitro setup. b. Average IPSCs (blue) and EPSCs (red) recorded in pyramidal cells during photo-stimulation of L6. Synaptic currents are averages of n = 5–12 cells. Inset, onset of EPSC. c. Histogram of excitatory charge as a percentage of total charge. d. Top traces: perforated patch recording from L5 pyramidal cell in response to depolarizing current injection with (right) and without (left) L6 photo-stimulation. Bottom graphs: spike rate with and without L6 photo-stimulation. e. Average spike rate in control versus spike rate with L6 photo-stimulation for each cell. f. Schematic of setup for focal photo-stimulation. g. Top traces: spiking of L5 pyramidal cell to depolarizing current injection with focal photo-stimulation of L6 at three progressively more distant positions (left to right). Bottom graph: spike rate in control (black) and with focal photo-stimulation of L6 (blue) (n = 4). Delta indicates medial/lateral distance from radial axis through recording site. h. Percentage of spike suppression plotted against horizontal displacement.

Figure 5. L6 suppresses upper layers largely through intra-cortical circuits

a. Schematic of setup. b. Simultaneously recorded multiunit responses to increasing contrasts (light to dark) in V1 (top) and dLGN (bottom). All spikes recorded above L6 (≤650 μm) were included in V1 multiunit activity. Scale bar: V1: 200 spikes/s; LGN: 100 spikes/s. Dotted line: baseline activity. Right, contrast response functions. c. dLGN-V1 transfer function derived by plotting normalized response in V1 versus dLGN (from b). Fit: hyperbolic ratio function. d. Simultaneously recorded multiunit responses to maximal contrasts in V1 (top) and dLGN (bottom) in control (black) or while photo-stimulating L6 with increasing LED intensities (progressively darker blue). Same experiment as in b and c. Scale bars: as in (b). e. V1 versus dLGN response to maximal contrast under control condition (black data point) or during three progressively stronger photo-stimulation of L6 (light, medium and dark blue; data from d). V1 responses are suppressed more than predicted by transfer function (red arrows) even for photo-stimulations that reduce dLGN activity only ~ 10% (light blue). f. Average intra-cortical component of suppression as function of suppression of dLGN (n = 5 experiments). Intra-cortical component (red arrow in e) is quantified as fraction of total V1 suppression (gray arrow + red arrow in e). g. Schematic of main finding.