Scaling down of balanced excitation and inhibition by active behavioral states in auditory cortex

Abstract

Cortical sensory processing is modulated by behavioral and cognitive states. How this modulation is achieved by changing synaptic circuits remains largely unknown. In awake mouse auditory cortex, we found that sensory-evoked spike responses of layer 2/3 (L2/3) excitatory cells were scaled down with preserved sensory tuning when mice transitioned from quiescence to active behaviors, including locomotion, whereas L4 and thalamic responses were unchanged. Whole-cell voltage-clamp recordings revealed that tone-evoked synaptic excitation and inhibition exhibited a robust functional balance. The change to active states caused scaling down of excitation and inhibition at approximately equal levels in L2/3 cells, but resulted in no synaptic changes in L4 cells. This lamina-specific gain control could be attributed to an enhancement of L1-mediated inhibitory tone, with L2/3 parvalbumin inhibitory neurons also being suppressed. Thus, L2/3 circuits can adjust the salience of output in accordance with momentary behavioral demands while maintaining the sensitivity and quality of sensory processing.

Figure 1

Behavioral state-dependent modulation of spike responses in the mouse A1. (a) Experimental setup. R, recording electrode; P, head-fixation post; S, sound stimulation; C, camera; v, velocity meter. (b) Sample records of plate rotation speed in different behavioral states. “Q”, quiescence; “A – L”, active without locomotion; “L”, locomotion. (c) Distribution of average speeds (within a 1 sec epoch) in randomly sampled 2000 epochs (n = 3 animals). (d) Top, sample records of LFP in the A1. Scale: 250 µV and 0.5 s. Middle, simultaneously recorded plate rotating speed. Arrow indicates speed at 0. Scale: 20 cm s⁻¹ and 0.5 s. Bottom, power spectrum of LFP. (e) Top, percentage change in power of low-frequency (1–10 Hz) and high-frequency (20–80 Hz) components of LFP relative to quiescence, for the recording in (d). Power spectrums were generated for each 3.3 sec segment of LFP records. Bar = s.d. N = 5 segments. From left to right, t-test (**P = 0.0035, t = −5.118), Wilcoxon signed rank test (*P = 0.0313, Z = −1.888), t-test (***P = 0.0006, t = 8.268), t-test (**P = 0.0012, t = 6.865). Bottom, the ratio of power of the low versus high-frequency component. One-way ANOVA (P = 6.68×10⁻⁶, F = 37.73) and post hoc test (**P < 0.01, ***P < 0.001, same for the below). (f) Summary of recordings in 6 animals. Power ratio was normalized by the average value in the “Q” state. Top, t-test (**P = 0.0011, 0.0016, ***P = 0.0003, 0.0006; t = −5.802, −5.302, 7.453, 6.637 respectively). Bottom, one-way ANOVA (P = 1.75×10⁻⁵, F = 24.80) and post hoc test. (g–i) Spontaneous firing in L2/3 neurons in different states. (g) Top, records of spontaneous spikes of a L2/3 excitatory cell. Bottom, simultaneously recorded plate rotation speed. Arrow indicates speed at 0. Right inset, superimposed 500 individual spikes. (h) Average spontaneous spike rates in the same cell. Bar = s.d. One-way ANOVA (P = 1.26×10⁻⁵, F = 12.88, n = 30 5-sec segments.) and post hoc test. (i) Summary of 17 recorded L2/3 excitatory cells. Spike rate was normalized by the average value in the “Q” state. One-way ANOVA (P = 2.66×10⁻⁷, F = 21.10) and post hoc test. (j–l) Spontaneous spikes recorded in L4 excitatory cells. Data are presented similarly as in (g–i). (k) One-way ANOVA (P = 0.9841, F = 0.0160, n = 28 segments) and post hoc test. (l) One-way ANOVA (P = 0.1542, F = 1.955, n = 15) and post hoc test. (m) Peri-stimulus spike time histogram (PSTH, bin size = 1 ms) for the responses of a L2/3 excitatory cell to CF tones (black lines) in different states. Inset, sample record of evoked spikes by the tone. (n) Average evoked spike number per stimulus trial plotted for the same cell. Bar = s.d. One-way ANOVA (P = 1.21×10⁻⁵, F = 12.93, n = 25 trials) and post hoc test. (o) Summary of average evoked spike numbers for 17 similarly recorded L2/3 excitatory cells. One-way ANOVA (P = 1.89×10⁻⁵, F = 13.76) and post hoc test. (p–q) Evoked spike responses of a L4 excitatory cell. N = 25 trials. One-way ANOVA (P = 0.7415, F = 0.3004) and post hoc test. (r) Summary of average evoked spike numbers for 15 recorded L4 cells. One-way ANOVA (P = 0.1708, F = 1.844) and post hoc test.

Figure 2

Gain modulation of auditory responses by behavioral state. (a) TRFs of spike responses of a L2/3 cell in quiescence (Q) and active (A) states. Each element in the array represents the PSTH of evoked spikes (60 ms time window, bin size = 2 ms, 10 repeats) to the corresponding tone stimulus. Color map depicts the average spike number evoked by tones. (b) Evoked spike number by a tone in active state plotted against that by the same stimulus in quiescence, for the cell shown in (a). The best-fit linear regression line is shown. (c) Normalized PSTHs for all the tone responses in two states, for the cell shown in (a). Arrows indicate response onsets. Δt is the onset difference. (d–f) TRFs of a L4 cell. Data are presented in the same way as in (a–c). (g) Distribution of correlation coefficients (r) for all the recorded L2/3 cells. (h) Distribution of slopes of the linear regression (i.e. the gain value). Arrow points to the mean value. (i) Distribution of differences in onset latency (“A” – “Q”) of spike responses. (j) Characteristic frequency (CF) of spike TRF in “A” versus “Q” state. The best-fit linear regression line is shown. (k) Bandwidth at 20 dB above the intensity threshold (BW20) of spike TRF in “A” versus “Q” state. (l) Intensity threshold of spike TRF in “A” versus “Q” state. (m–r) Summary for L4 cells. Data are presented in the same way as in (g–l).

Figure 3

Activity of thalamic neurons and summary of signal-to-noise ratio (SNR) under different behavioral states. (a) Left, sample records of spontaneous spikes in an MGBv neuron in “Q” and “A” states. Right, summary of average spontaneous firing rates (N = 12 cells). Data points from the same cell are connected with a line. Solid symbol represents mean ± s.d. (paired t-test, P = 0.4795, t = 0.7320, n = 12 cells). (b) Left, PSTH for CF-tone evoked spikes in different states in the same MGBv neuron as shown in (a). Right, summary of average evoked spike numbers by CF tones in different states (paired t-test, P = 0.7640, t = − 0.3078, n = 12 cells). (c) SNR in “A” relative to “Q” state. There is a significant increase in L2/3 cells. From left to right, Wilcoxon signed rank test (**P = 0.0017, Z = 3.435), t-test (P = 0.5834, t = 0.5614), t-test (P = 0.6282, t = 0.4982).

Figure 4

Properties of synaptic responses in quiescence. (a) Average traces of excitatory and inhibitory currents evoked by a best-frequency (BF) tone at 40 dB SPL in an example pyramidal neuron. Black line indicates tone duration. Red line marks 50% duration of the current. Scale: 50 (exc) /100 (inh) pA, 100 ms. (b) Durations of BF-tone evoked synaptic currents. Short, 50 ms; long, 200 or 500 ms. Wilcoxon signed-rank test, P = 0.81, 0.73; Z = 0.2439, 0.4024; N = 9, 3 for exc and inh respectively. (c) Comparison of peak amplitudes (paired t-test, P = 0.4, 0.89; t = 0.8876, 0.1520; N = 9, 3 for exc and inh respectively). (d) Differences in excitatory and inhibitory onset latency (Inh – Exc). Arrow points to the mean value. (e) Comparison of peak amplitudes. Wilcoxon signed-rank test, ***P = 0.0001, Z = − 4.464, n = 21. (f) Distribution of E/I ratios of peak conductances. (g) Frequency tuning of excitation and inhibition in an example cell. Left, average excitatory and inhibitory currents to tones at different frequencies (interval, 0.2 octave). Scale: 30 (exc) / 100 (inh) pA, 200 ms. Right, peak amplitude of excitatory versus inhibitory current evoked by the same stimulus. Top inset, superimposed normalized frequency tuning curves for excitation (red) and inhibition (blue). (h) Another example cell plotted similarly as in (g). Scale: 100 (exc) / 500 (inh) pA, 200 ms. (i–k) Comparison of frequency range (i), half-peak bandwidth (j), and BF (k) between excitation and inhibition in the same cell (P = 0.52, 0.56, 0.46, respectively, paired t-test, n = 15 cells). The unity line is shown.

Figure 5

Modulation of synaptic responses by behavioral state. (a) Average evoked excitatory currents to BF tones at different intensities in quiescence and active states in a L2/3 excitatory cell. Scale: 200 pA, 100 ms. (b) Left, peak excitatory amplitudes in active versus quiescence state with the best-fit line shown. The near zero point depicts the responses to 10 dB tones not shown in (a). Middle, ratio of response amplitudes (Q/A) for all testing intensities (zero responses excluded). Solid symbol represents mean ± s.d. Right, difference in onset latency of evoked synaptic currents (A – Q). (c–d) Inhibitory responses in “Q” and “A” states recorded in the same L2/3 neuron. Scale: 500 pA, 100 ms. (e–h) Excitatory and inhibitory responses of a L4 excitatory cell. Scale: 100 pA and 100 ms in (e); 200 pA and 100 ms in (g). (i) Excitatory and inhibitory currents to tones of different frequencies in a L2/3 cell. Scale: 40 (Exc)/ 80 (Inh) pA, 200 ms. Inset, superimposed normalized excitatory (top) and inhibitory (bottom) frequency tuning curves in quiescence (black) and active (red) states. (j) Peak response amplitude in active versus quiescence state for the same cell shown in (i). Top, excitation; bottom, inhibition. (k–l) An example L4 cell plotted in the same manner as in (i–j). Scale: 50 (E)/ 150 (I) pA, 200 ms. (m) Cumulative distribution of correlation coefficients for synaptic responses in “A” versus “Q” state for L2/3 neurons. (n) Distribution of ratios between peak synaptic amplitudes in “A” and “Q” states (A/Q) for all tone stimuli in all recorded cells. Arrows indicate mean values. Left, excitation; right, inhibition. (o) Slopes (scaling factors) of the linear regression for “A” versus “Q” peak synaptic responses in all recorded cells. No significant difference between scaling factors of excitation and inhibition in L2/3 cells (P = 0.5481, t = 0.6136, unpaired t-test, n =11, 7 for excitatory and inhibitory respectively). (p) The scaling factors for excitatory responses plotted against that for inhibitory responses in the same cell. For L2/3, P = 0.6177, t = 0.5402, paired t-test, n = 5.

Figure 6

Modulation of resting membrane potential and resting conductance by behavioral state. (a) Sample current-clamp recording records (top) together with the simultaneously recorded speeds (bottom) for an example L2/3 neuron. Arrow labels the level for −70 mV. (b) Normalized distribution of membrane voltages during quiescent and active states for the cell shown in (a). Arrow points to the average spike threshold of the cell. (c–f) Comparison of mean resting membrane potential (c), s.d. of resting V_m (d), spike threshold (e) and percentage of V_m values near spike threshold (not lower than the threshold by more than 10 mV) (f) under different behavioral states for 7 recorded L2/3 neurons. Bar = s.e.m., paired t-test (***P = 0.0002, **P = 0.0083, P = 0.5496, ***P = 0.0002; t = 8.114, 3.862, 0.6338, 8.139 for (c–f) respectively). (g) Average excitatory currents of an example cell in response to BF tones (70 dB SPL) in different states. Thick black line marks tone duration. (h) Average inhibitory currents of the same cell. (i) Resting conductances right before, during and after an epoch of active behaviors for five L2/3 cells. Data points from the same cell are connected by lines. N = 5, bar = s.d., paired t-test (***P = 0.0004, **P = 0.0015; t = 10.65, − 7.810)

Figure 7

Changes of activity of PV neurons between different behavioral states. (a) Confocal image of a brain section showing ChR2-EYFP expression in the A1 region. (b) Left, sample loose-patch recording trace showing a train of evoked spikes of a PV cell to blue LED illumination (marked by the blue bar). Right, sample spike response of the same cell to CF-tone stimulation. Top inset, schematic drawing of loose-patch of a cell. Bottom inset, superimposed 500 individual spikes (black) and the average spike shape (red) of the cell. (c) Spike waveforms of four more PV and excitatory cells. (d) Plot of peak to trough amplitude ratio versus trough-to-peak interval for average spike waveforms of PV and excitatory cells recorded with loose-patch methods. Optogenetically identified PV cells were labeled by open circles. (e) Summary of spontaneous firing rates of recorded L2/3 PV cells in different states. Bar = s.d.. N = 8 cells. **P = 0.0044, t = 4.140, paired t-test. (f) CF-tone evoked spike numbers for the cells in (e). **P = 0.0047, t = 4.086, paired t-test. (g) Summary of spontaneous firing rates for PV cells recorded in L4. P = 0.5013, t = −0.7243, paired t-test, n = 6 cells. (h) CF-tone evoked spike numbers for the cells in (g). P = 0.7286, t = 0.3671, paired t-test.

Figure 8

Role of L1 in the behavioral state-dependent L2/3-specific gain modulation. (a) Left, spontaneous spikes of a L1 neuron in different states. Right, summary of average spontaneous firing rates for 6 L1 neurons. Solid symbol represents mean ± s.d. **P = 0.0081, t = −4.252, paired t-test. (b) Left, PSTH for CF-tone evoked spikes of the same cell as in (a). Right, summary of evoked spike numbers for 6 L1 neurons. **P = 0.0019, t = −5.085, paired t-test. (c) Time courses of CF-tone evoked multiunit spike responses in different A1 layers after the topical application of TTX (at time zero). Shaded area denotes the analysis time window during which L2/3 responses were increased to a stable level while L4 responses remained unaffected. N = 4 animals for each layer. Bar = s.d. (d) Summary of evoked firing rates of individual L2/3 excitatory cells in different behavioral states before and after TTX application. Spike rates were normalized by “Q” state before TTX application. N = 10 cells. Among these 10 cells, 6 were also recorded in active states after TTX application. **P < 0.01, ***P < 0.001, paired t-test. (e) Summary of relative response levels (A/Q) before and after TTX application. **P = 0.0035, t = −5.193, paired t-test, n = 6. (f) Summary of normalized evoked spike numbers in different states before and after TTX application for L4 neurons. N = 6 cells.