A feedforward inhibitory circuit mediates lateral refinement of sensory representation in upper layer 2/3 of mouse primary auditory cortex

Abstract

Sensory information undergoes ordered and coordinated processing across cortical layers. Whereas cortical layer (L) 4 faithfully acquires thalamic information, the superficial layers appear well staged for more refined processing of L4-relayed signals to generate corticocortical outputs. However, the specific role of superficial layer processing and how it is specified by local synaptic circuits remains not well understood. Here, in the mouse primary auditory cortex, we showed that upper L2/3 circuits play a crucial role in refining functional selectivity of excitatory neurons by sharpening auditory tonal receptive fields and enhancing contrast of frequency representation. This refinement is mediated by synaptic inhibition being more broadly recruited than excitation, with the inhibition predominantly originating from interneurons in the same cortical layer. By comparing the onsets of synaptic inputs as well as of spiking responses of different types of neuron, we found that the broadly tuned, fast responding inhibition observed in excitatory cells can be primarily attributed to feedforward inhibition originating from parvalbumin (PV)-positive neurons, whereas somatostatin (SOM)-positive interneurons respond much later compared with the onset of inhibitory inputs to excitatory neurons. We propose that the feedforward circuit-mediated inhibition from PV neurons, which has an analogous function to lateral inhibition, enables upper L2/3 excitatory neurons to rapidly refine auditory representation.

Keywords: excitatory-inhibitory balance; inhibitory neuron; lateral inhibition; surround suppression; tonal receptive field; whole-cell recording.

Figure 1.

Frequency representation is refined in the upper L2/3. A, Reconstructed spike TRF of an example L4 pyramidal neuron, displayed as an array of PSTHs for the responses of the cell to pure tones of varying frequency and intensity. Red dashed line depicts the TRF boundary. Each PSTH trace depicts the spike response evoked by a 100 ms tone, averaged over 10 repeats. Bin size, 10 ms. Scale, 0.5 spike count. Right top inset, PSTH generated from the spike responses to all effective tones. Red bar marks the duration of tone stimuli. Right bottom inset, The reconstructed morphology of the cell stained after breaking in the membrane. Cortical layers are marked. B, Spike TRFs for an example L2/3 excitatory cell. Data are presented in a similar manner as in A. C, More examples of spike TRFs for both L4 and upper L2/3 cells. Color maps depict the average evoked spike rate in the frequency–intensity space. Spontaneous activity was subtracted. The color maps in the first column are for the cells shown in A and B. Scale: L4, 30, 17, 27 and 23 Hz for maximum; L2/3, 15, 12, 16 and 20 Hz for maximum. D, Average tuning bandwidths of spike TRF at 10 and 20 dB above the intensity threshold for L4 (n = 44) and L2/3 (n = 29) excitatory neurons. Error bar indicates SD. **p < 0.01, t test. E, Average intensity thresholds of spike TRF for L4 and L2/3 neurons. **p < 0.01, t test. F, Average onset latencies of evoked spike responses. **p < 0.01, t test. G, Left, Confocal image of a coronal section of the A1 of an Scnn1a–Tg3–Cre (L4-specific) tdTomato mouse. Note that fluorescence-labeled neurons are mainly distributed in L4. Scale bar, 100 μm. Middle, Confocal image showing the distribution of thalamocortical axons, which were labeled by injecting AAV–ChR2–EYFP in the MGBv. WM, White matter. Right, Image showing the MGB injection site. The MGB and A1 are outlined. Scale bar, 1 mm. A, Anterior; P, posterior.

Figure 2.

Broader inhibitory sidebands in the upper L2/3 than L4. A, One-tone and two-tone stimulation experiments in an example L4 neuron. Blue, PSTH for the responses to the one-tone stimulation. Red, PSTH for the responses to the two-tone stimulation. Blue curve labels the boundary of the spike TRF of the cell. Red curve labels the boundary of the suppressive region tested with the two-tone stimulation. The interval between the two curves (marked by the double arrowhead) was defined as the inhibitory sideband. Bottom, Color map on the left depicts the suppressive region, and color map on the right depicts the spike TRF of the cell. Scale, 15 and 20 Hz for maximum. B, Spike TRF and suppressive region of an example L2/3 neuron. Data are presented in a similar manner as in A. Scale, 40 and 50 Hz for maximum. C, Average widths of inhibitory sidebands (quantified at 20 dB above the TRF intensity threshold) of L4 (n = 13) and L2/3 (n = 17) neurons. **p < 0.01, t test.

Figure 3.

Inhibition has a broader frequency range than excitation in upper L2/3 excitatory neurons. A, TRFs of inhibitory (upper; Inh) and excitatory (lower; Exc) responses (average of 3 repeats) of an example L4 neuron. Color map depicts the peak response amplitude. Color scale, 414.4 (Inh)/ 249 (Exc) pA. Inset, enlarged response trace to the BF tone at 60 dB SPL. Red line marks the tone duration (100 ms). Calibration, 120 (Inh)/80 (Exc) pA, 20 ms. B, TRFs of synaptic responses of an example L2/3 cell. Data are presented in a similar manner as in A. Color scale, 715.9 (Inh)/265.2 (Exc) pA. Calibration: 240 (Inh)/90 (Exc) pA, 20 ms. C, Comparison of frequency ranges of inhibitory and excitatory responses at 20 dB above the intensity threshold of the excitatory TRF for another three L4 cells. Calibration (from top to bottom): 93 (Inh)/49 (Exc), 58/34, and 105/54 pA, 200 ms. Right, Superimposed normalized inhibitory (black) and excitatory (red) tuning curves. Green dotted line labels the baseline. D, Another three L2/3 neurons. Data are presented in a similar manner as in C. Calibration: 95 (Inh)/50 (Exc), 170/32, and 120/39 pA, 200 ms.

Figure 4.

Summary of synaptic response properties for upper L2/3 and L4 neurons. A, Average frequency bandwidths of excitation and inhibition at 20 dB above the intensity threshold of the excitatory TRF. n = 19 for upper L2/3 and 11 for L4. Error bar indicates SD. *p < 0.05, t test; **p < 0.01, paired t test. B, Average frequency bandwidths at 60 dB SPL for excitation (n = 19), inhibition (n = 19), as well as spike response (n = 29) plus inhibitory sideband (white column, n = 17) for L2/3 cells. **p < 0.01, paired t test. C, Plot of bandwidth ratio (E/I) as a function of cortical depth. Bandwidth was measured at 60 dB SPL as the total frequency range of excitation or inhibition. One data point represents one cell. D, Average onset latencies of excitation and inhibition. *p < 0.05, **p < 0.01, t test. E, Average peak amplitudes of excitation and inhibition in response to the BF tone at 60 dB SPL. *p < 0.05, **p < 0.01, t test. F, Average E/I ratios measured for responses at the BF tone of the cell at 60 dB SPL. *p < 0.05, t test. G, Average half-peak durations of excitation and inhibition in response to the BF tone at 60 dB SPL. *p < 0.01, **p < 0.05, t test. Inset, Sample recorded synaptic response. Red line marks half-peak duration. Calibration: 50 ms, 0.1 nA.

Figure 5.

Modeling of effects of broader inhibition on frequency tuning. A, Left, Temporal profiles of modeled tone-evoked excitatory (red) and inhibitory (blue) currents. Calibration: 20 ms, 0.17 nA. The amplitude of inhibition is 1.5-fold of excitation, and inhibition is delayed by 2 ms relative to excitation. Right, Frequency tuning curves of excitatory (red) and inhibitory (blue) responses. The top panel shows the cotuned (inhibitory/excitatory bandwidth, 1:1) scenario, and the bottom panel shows the broader inhibition (inhibitory/excitatory bandwidth, 1.5:1) scenario. B, Traces of derived V_m responses (trace duration, 120 ms) across tone frequency in the two scenarios. Red dash line marks the level of resting membrane potential. C, Left, Frequency tuning curve of peak depolarizing V_m responses (red) when inhibition is cotuned with excitation. Right, Frequency tuning curves of peak depolarizing (red) and peak hyperpolarizing (black) V_m responses when inhibition is more broadly tuned than excitation. Dotted red curve labels the V_m response tuning in the cotuned scenario. D, BW50% of V_m response plotted against the bandwidth ratio (inhibition/excitation). The interval between tones used in the model was 0.01 octave. E, Frequency range of hyperpolarizing V_m responses (>2 mV, measured within 50 ms time window after stimulus onset) plotted against the bandwidth ratio (inhibition/excitation). Black and gray represent scenarios when the amplitude ratio (E/I) is 1:1.5 and 1:2, respectively. F, Frequency tuning of derived spike response under cotuned (upper) or broader (lower) inhibition. Note that each solid vertical line depicts a spike. E/I amplitude ratio, 1:1.5. G, Frequency range of spike response as a function of bandwidth ratio.

Figure 6.

Dynamic-clamp experiment. A, V_m responses of an example cell to injected excitatory and inhibitory conductances simulating responses to different tone frequencies. Top and bottom panels represent the cotuned inhibition and broader inhibition scenarios, respectively. Red dash line marks the level of spike threshold. Arrow points to the level of resting membrane potential. Calibration: 10 mV, 100 ms. B, Comparison of frequency range of recorded spike response under the two scenarios. Data points for the same cell are connected with a line. **p < 0.01, paired t test. There is no significant difference between L4 and L2/3 cells for either condition (p > 0.05, t test). C, Comparison of spike threshold (relative to the resting V_m) between L4 (n = 8) and upper L2/3 (n = 14) neurons. Data are presented as mean ± SD. D, Comparison of resting V_m.

Figure 7.

Inhibitory inputs to L2/3 excitatory cells are originated from interneurons in the same layer. A, Left, Confocal image of a brain slice showing that ChR2 was locally expressed in the A1 region. Scale bar, 500 μm. Right, Enlarged confocal images showing that ChR2–EYFP was expressed in tdTomato-labeled PV (top) or SOM (bottom) neurons. Scale bar, 20 μm. B, Spikes evoked by 20 Hz pulses (pulse duration, 1 ms) of blue light illumination in an example PV and SOM neuron expressing ChR2, recorded under whole-cell current-clamp mode. Each blue dot indicates a single pulse. Calibration: 20 mV, 150 ms. C, Schematic graph showing the laminar specific activation of cortical inhibitory neurons. Whole-cell voltage-clamp recording was made from an upper L2/3 excitatory neuron while PV or SOM neurons expressing ChR2 were stimulated by small circular blue light spots (diameter, 60–70 μm). D, IPSCs recorded in an example L2/3 excitatory neuron while activating PV (left) or SOM (right) neurons in different cortical layers. Calibration: 20 pA, 10 ms. E, Average IPSC amplitudes to PV- or SOM-cell activation in different layers for all the recorded L2/3 excitatory neurons (n = 5). Data points for the same cell are connected with lines. Gray triangle and bar depict the mean and SD, respectively. F, Schematic graph showing the local stimulation of inhibitory neurons in L2/3 in a horizontal plane. G, Average ± SD IPSC amplitudes to stimulation of PV or SOM neurons at different horizontal distances from the recorded L2/3 excitatory cell (n = 7). H, Average ± SD IPSC amplitudes to stimulation of PV or SOM neurons in the entire A1 region (n = 7). *p < 0.05, t test.

Figure 8.

PV neurons mediate the broad inhibition via a feedforward circuit. A, Latencies of tone-evoked excitatory (red) and inhibitory (blue) responses at 60 dB SPL across tone frequency (relative to the BF) in an example L2/3 neuron. Black represents the Δlatency (inhibition–excitation). B, Distribution of Δlatencies (to tones at 60 dB SPL) within the recorded L2/3 excitatory neuron population (n = 19 cells). Arrow marks the mean value. C, Left, Two-photon image of a td-Tomato-labeled inhibitory neuron in our targeted loose-patch recording (top) and 50 superimposed individual spikes (black) and their average (red) of an example SOM (middle) and PV (bottom) cell. Scale bar (top), 20 μm. Calibration: 0.6 ms, 0.04 nA (middle); 0.6 ms, 0.1 nA (bottom). Middle, Latencies of spike responses to 60 dB tones across frequency for the SOM (upper) and PV (lower) cell. Right, PSTHs of their spike responses. Red bar marks the tone duration. D, Average latencies of spike responses to 60 dB tones across frequency for the recorded SOM (blue, n = 19) and PV (red, n = 17) cells, as well as average latencies of inhibitory responses in the recorded L2/3 excitatory cell population (black, n = 10 cells). Error bars indicate SD. E, Average V_m responses to 60 dB BF tones of an example PV, SOM, and excitatory cell all preferring 8 kHz. Note that spikes are truncated. Red, blue, and green dashed lines mark the stimulus onset, the onset of depolarizing responses of the PV and excitatory cells, and the onset of depolarizing responses of the SOM neuron, respectively. Calibration: 4 mV, 30 ms. F, Distributions of onset latencies of excitatory inputs to the recorded PV (red, n = 8 cells), SOM (blue, n = 6), and excitatory (black, n = 21) cell populations. Bin size, 1 ms. Latencies were measured for depolarizing V_m responses to all effective tones at 60 dB SPL.