Cell-type-specific plasticity in synaptic, intrinsic, and sound response properties of deep-layer auditory cortical neurons after noise trauma

Abstract

Peripheral trauma, such as noise-induced hearing loss (NIHL), triggers compensatory plasticity in the auditory cortex (ACtx) to maintain auditory function. While cortical plasticity in superficial cortical layers has been relatively well studied, the plasticity mechanisms governing deep-layer excitatory projection neurons remain less understood. Here, we investigated the plasticity of layer (L)5 extratelencephalic (ETs) and L6 corticothalamic neurons (CTs) following NIHL. Using a combination of in vitro slice electrophysiology, optogenetics, and in vivo two-photon imaging in a mouse model of NIHL, we characterized changes in evoked thalamocortical (TC) synaptic input strength, intrinsic excitability, and sound response properties. We found that TC input was initially equivalent between ETs and CTs, then shifted to CT-dominant one day after noise exposure. This shift renormalized to equivalent seven days after noise exposure and was associated with a transient increase in both the quantal size (q) in TC→CT synapses and intrinsic CT suprathreshold excitability. ETs maintained stable intrinsic properties and showed minor changes in their TC input. In vivo imaging revealed that CTs displayed a persistent elevation in sound intensity thresholds, whereas ETs transiently shifted their best frequency representation and reduced their responsiveness to high-frequency tones one day after NIHL, followed by recovery at seven days. Together, our findings highlight cell-type-specific plasticity mechanisms in deep-layer cortical neurons, enhance our understanding of cortical adaptation to peripheral damage, and highlight targets for developing therapeutic strategies to mitigate hearing loss and related disorders such as tinnitus and hyperacusis.

Keywords: auditory cortex; excitatory neurons; in vitro electrophysiology; in vivo imaging; noise-induced hearing loss; plasticity; thalamocortical circuits.

Figure 1.. Transient shift in TC synaptic strength from CT and ET equivalent to CT dominant one day after NE.

(A) Schematic illustration of stereotaxic injections of retrograde microspheres to label L5 extratelencephalic neurons (ETs, green), and viral vectors (AAVs) for expression of ChR2 in thalamocortical (TC) inputs and tdTomato in L6 corticothalamic neurons (CTs, red) in Ntsr1-Cre mice. (B) Schematic illustration of slice electrophysiology experiment involving photostimulation of ChR2 expressing TC afferents and simultaneous (dual) recording from an ET (green) and a CT (red). (C) Images in 4X magnification showing the extent of ACtx area in bright-field (left), green-labeled ETs and TC axons (middle) and red-labeled CTs (D) Average CT/ET EPSC ratio after optogenetically stimulating thalamic L5–6 input 1d and 7d after SE and NE. (1d SE: 18 cells/6 mice; 1d NE: 26 cells/9 mice; 7d SE: 10 cells/6 mice; 7d NE: 10 cells/6 mice). Asterisks indicate significant differences (***p<0.001, two-way ANOVA and Bonferroni correction for multiple comparisons). (E) Representative traces of EPSCs in dual recordings from both CT (solid line) and ET (dotted line) neurons evoked by maximal photostimulation of L5–6 thalamocortical inputs in 1d SE (E₁) and 1d NE (E₂); 7d SE (E₃) and 7d NE (E₄). Different colored traces represent different pairs of simultaneously recorded CTs and ETs. Detailed statistical values are listed in Table 1.

Figure 2.. Transient increase in the q of TC→CT synapses 1 day after NE.

(A, C) Average amplitude of light-evoked quantal EPSCs (Sr²⁺-mEPSCs) in CTs in response to L5–6 thalamocortical maximal photostimulation in 1d (A) and 7d (C), post SE (black) and NE (red) mice (1d SE: 7 cells/3 mice; 1d NE: 6 cells/3 mice; 7d SE: 5 cells/2 mice; 7d NE: 6 cells/2 mice). Asterisks indicate significant differences (*p<0.05, unpaired t-test). (B, D) Representative Sr²⁺-mEPSCs traces (top left) and representative average traces of quantal Sr²⁺-mEPSCs traces (top right). Amplitude histogram of events before (background noise) and after stimulation from the same cell (bottom) in 1d (B) and 7d (D) after SE (black) and NE (red) mice. The arrowhead indicates the onset of light stimulus. The dotted line indicates 400ms time window before stimulus (pre-LED). The solid line represents a 400ms time window, which started 100 ms after the stimulus (post-LED) and was used to analyze the amplitude of Sr²⁺-mEPSCs (1d SE: 7 cells/3 mice; 1d NE: 6 cells/3 mice; 7d SE: 5 cells/2 mice; 7d NE: 6 cells/2 mice). (E, G) Summary graph of the average CT EPSC amplitudes in response to L5–6 thalamocortical maximal photostimulation in 1d (D) and 7d (F) SE (black) and NE (red). (1d SE: 18 cells/6 mice; 1d NE: 26 cells/9 mice; 7d SE: 10 cells/6 mice; 7d NE: 10 cells/6 mice). Asterisks indicate significant differences (**p<0.01, Mann-Whitney test). (F, H) Representative traces of L6 CT EPSCs in response to L5–6 thalamocortical maximal photostimulation in 1d SE (E, black line) and 1d NE (E, red line) and 7d SE (G, black line) and 7d NE (G, red line). Detailed statistical values are listed in Table 1.

Figure 3.. Transient increase in suprathreshold CT intrinsic excitability 1 day after NE.

(A) Average CT input resistance (R_input) 1d and 7d after SE (black) or NE (red) (1d SE: 22 cells/8 mice; 1d NE: 23 cells/5 mice; 7d SE: 23 cells/6 mice; 7d NE: 21 cells/4 mice). (B) Average CT resting membrane potential (V_rest) 1d and 7d after SE (black) or NE (red) (1d SE: 22 cells/8 mice; 1d NE: 23 cells/5 mice; 7d SE: 23 cells/6 mice; 7d NE: 21 cells/4 mice). (C) Average CT AP width 1d and 7d after SE (black) or NE (red) (1d SE: 21 cells/8 mice; 1d NE: 21 cells/5 mice; 7d SE: 23 cells/6 mice; 7d NE: 21 cells/4 mice). (D) Average CT AP threshold of 1d and 7d after SE (black) or NE (red) (1d SE: 21 cells/8 mice; 1d NE: 19 cells/5 mice; 7d SE: 23 cells/6 mice; 7d NE: 21 cells/4 mice). (E₁, E₂, G₁, G₂) Representative traces of CT firing in response to depolarizing current (50, 100, 200 pA current injections), 1d SE (E₁) vs. 1d NE (E₂); and 7d SE (G₁) vs. 7d NE (G₂). (F, H) Average firing frequency as a function of injected current amplitude, 1d SE vs. 1d NE (F), and 7d SE vs. 7d NE (H). Current injections from 25 to 250 pA with an increment of 25 pA (1d SE: 21 cells/8 mice; 1d NE: 21 cells/5 mice; 7d SE: 23 cells/6 mice; 7d NE: 21 cells/4 mice). Asterisks indicate significant differences (**p<0.01, two-way ANOVA and Bonferroni correction for multiple comparisons). Detailed statistical values are listed in Table 1.

Figure 4.. No changes in the q of TC→ET synapses after NE.

(A, C) Average amplitude of Sr²⁺-mEPSCs in ETs in response to L5–6 thalamocortical maximal photostimulation in 1d (A) and 7d (B), post SE (black) and NE (red) (1d SE: 5 cells/4 mice; 1d NE: 7 cells/5 mice; 7d SE: 7 cells/2 mice; 7d NE: 5 cells/2 mice). (B, D) Representative Sr²⁺-mEPSCs traces (top left) and representative average traces of quantal Sr²⁺-mEPSCs traces (top right). Amplitude histogram of events before (background noise) and after stimulation from the same cell (bottom) in 1d (B) and 7d (D) after SE (black) and NE (red) mice. The arrowhead indicates the onset of light stimulus. The dotted line indicates 400ms time window before stimulus (pre-LED). The solid line represents a 400ms time window, which started 100 ms after the stimulus (post-LED) and was used to analyze the amplitude of Sr²⁺-mEPSCs (1d SE: 5 cells/4 mice; 1d NE: 7 cells/5 mice; 7d SE: 7 cells/2 mice; 7d NE: 5 cells/2 mice). (E) Representative traces of L5 ET EPSCs in response to L5–6 thalamocortical maximal photostimulation in in 1d SE (E₁, black) vs. 1d NE (E₁, red), 7d SE (E₂, black) vs. 7d NE (E₂, red) mice. (F) Summary graph of the average L5 ET EPSC amplitudes in response to L1–4 thalamocortical maximal photostimulation in 1d SE vs. 1d NE, or 7d SE vs. 7d NE mice (1d SE: 8 cells/3 mice; 1d NE: 7 cells/4 mice; 7d SE: 9 cells/5 mice; 7d NE: 12 cells/4 mice). (G) Representative traces of L5 ET EPSCs in response to L1–4 thalamocortical maximal photostimulation in 1d SE (G₁, black) vs. 1d NE (G₁, red), 7d SE (G₂, black) vs 7d NE (G₂, red) mice. Detailed statistical values are listed in Table 1.

Figure 5.. No changes in ET intrinsic excitability after NE.

(A) Average ET R_input 1d and 7d after SE (black) or NE (red) (1d SE: 21 cells/6 mice; 1d NE: 21 cells/5 mice; 7d SE: 20 cells/5 mice; 7d NE: 23 cells/6 mice). (B) Average ET V_rest 1d and 7d after SE (black) or NE (red) (1d SE: 21 cells/6 mice; 1d NE: 21 cells/5 mice; 7d SE: 20 cells/5 mice; 7d NE: 23 cells/6 mice). (C) Average ET AP width 1d and 7d after SE (black) or NE (red) (1d SE: 21 cells/6 mice; 1d NE: 21 cells/5 mice; 7d SE: 20 cells/5 mice; 7d NE: 23 cells/6 mice). (D) Average ET AP threshold 1d and 7d after SE (black) or NE (red) (1d SE: 21 cells/6 mice; 1d NE: 21 cells/5 mice; 7d SE: 20 cells/5 mice; 7d NE: 23 cells/6 mice). (E₁, E₂, G₁, G₂) Representative traces of ET firing in response to depolarizing current injection (50, 100, 200 pA), 1d SE (E₁) vs. 1d NE (E₂); and 7d SE (G₁) vs. 7d NE (G₂). (F, H) Average firing frequency as a function of injected current amplitude, 1d SE vs. 1d NE (F), and 7d SE vs. 7d NE (H). Current injections from 25 to 350 pA with an increment of 25 pA. (1d SE: 21 cells/6 mice; 1d NE: 21 cells/5 mice; 7d SE: 20 cells/5 mice; 7d NE: 23 cells/6 mice). Detailed statistical values are listed in Table 1.

Figure 6.. Changes in CT sound response responses persist 7 days after NE.

(A) Example two-photon field of view from CTs imaged before and after exposure. (B) Average responses to pure tones across all CTs in SE mice (−1d: n=507 neurons; 1d: n=460 neurons; 7d: n=432 neurons). Asterisks indicate significant pairwise differences. (C) Same as (A) for NE mice (−1d: n=713 neurons; 1d: n=638 neurons; 7d: n=646 neurons). Asterisks indicate significant pairwise differences. (D) Tuning curves aligned to best frequency (BF) in SE mice. (E) Same as (D) for NE mice. Asterisks indicate significant pairwise differences (*p<0.05, Bonferroni corrected for multiple comparisons). (F) Change in BF of matched cells in SE (black) and NE (red) mice (1d SE: n=40 neurons; 1d NE: n=36 neurons; 7d SE: n=35 neurons; 7d NE: n=40 neurons). (G) Accuracy of a multinomial logistic regression classifier trained to decode the frequency of pure tones at 80 dB SPL. Dashed line represents chance level. Error bars represent standard deviation of decoding iterations. (H) Average responses to white noise bursts at different intensities across all ETs in SE mice (−1d: n=438 neurons; 1d: n=460 neurons; 7d: n=411 neurons). Asterisks indicate significant pairwise differences (Bonferroni corrected for multiple comparisons). (I) Same as (H) in NE mice (−1d: n=636 neurons; 1d: n=559 neurons; 7d: n=587 neurons). Asterisks indicate significant pairwise differences (Bonferroni corrected for multiple comparisons). (J) Change in intensity thresholds across days in SE (black) and NE (red) mice (1d SE: n=27 neurons; 1d NE: n=31 neurons; 7d SE: n=29 neurons; 7d NE: n=25 neurons). Asterisks indicate significant pairwise differences (**p<0.01, ***p<0.001, Bonferroni corrected for multiple comparisons). (K) Accuracy of a multinomial logistic regression trained to decode sound intensity. Dashed line represents chance level. Error bars represent standard deviation of decoding iterations. Detailed statistical values are listed in Table 1.

Figure 7.. Transient shift in ET sound response properties one day after NE.

(A) Example two-photon field of view from ETs imaged before and after exposure. (B) Average responses to pure tones across all ETs in SE mice (−1d: n=385 neurons; 1d: n=414 neurons; 7d: n=400 neurons). (C) Same as in (A) for NE mice (−1d: n=268 neurons; 1d: n=235 neurons; 7d: n=296 neurons). Asterisks indicate significant pairwise differences (Bonferroni corrected for multiple comparisons). (D) Tuning curves aligned to best frequency (BF) in SE mice. (E) Same as in (D) for NE mice. Asterisks indicate significant pairwise differences (*p<0.05, ****p<0.0001, Bonferroni corrected for multiple comparisons). (F) Change in BF of matched cells in SE (black) and NE (red) mice (1d SE: n=68 neurons; 1d NE: n=54 neurons; 7d SE: n=68 neurons; 7d NE: n=71 neurons). Asterisks indicate significant pairwise differences (*p<0.05, ****p<0.0001, Bonferroni corrected for multiple comparisons). (G) Accuracy of a multinomial logistic regression classifier trained to decode the frequency of pure tones at 80 dB SPL. Dashed line represents chance level. Error bars represent standard deviation of decoding iterations. Asterisks indicate significant pairwise differences (*p<0.05, ****p<0.0001, bootstrap test Bonferroni corrected for multiple comparisons). (H) Average responses to white noise bursts at different intensities across all ETs in SE mice (−1d: n=331 neurons; 1d: n=270 neurons; 7d: n=299 neurons). (I) Same as (H) in NE mice (−1d: n=226 neurons; 1d: n=222 neurons; 7d: n=252 neurons). Asterisks indicate significant pairwise differences (Bonferroni corrected for multiple comparisons). (J) Change in intensity thresholds across days in SE (black) and NE (red) mice (1d SE: n=42 neurons; 1d NE: n=49 neurons; 7d SE: n=50 neurons; 7d NE: n=51 neurons). Asterisks indicate significant pairwise differences (*p<0.05, **p<0.01, Bonferroni corrected for multiple comparisons). (K) Accuracy of a multinomial logistic regression trained to decode sound intensity. Dashed line represents chance level. Error bars represent standard deviation of decoding iterations. Asterisks indicate significant pairwise differences (*p<0.05, bootstrap test Bonferroni corrected for multiple comparisons). Detailed statistical values are listed in Table 1.