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

Abstract

Peripheral damage drives auditory cortex (ACtx) plasticity, but the underlying synaptic and cellular mechanisms remain poorly understood. We used a combination of in vitro slice electrophysiology, optogenetics, and in vivo two-photon imaging to investigate layer 5 extratelencephalic (ET) and layer 6 corticothalamic (CT) neuronal plasticity in mice, following noise-induced hearing loss (NIHL). Thalamocortical input was initially balanced between CTs and ETs but shifted to CT-dominant 1 day post-NIHL and then normalized by day 7. This transient shift was accompanied by increased quantal size and suprathreshold excitability in CTs, with minimal changes in ETs. In vivo, CTs exhibited persistent elevation in sound intensity thresholds, while ETs showed a transient shift in frequency tuning and reduced high-frequency responsiveness that recovered within a week. These findings reveal distinct, cell type-specific plasticity mechanisms in deep-layer ACtx neurons following peripheral damage and highlight potential targets for treating hearing loss-related disorders such as tinnitus and hyperacusis.

Fig. 1.. Transient shift in TC synaptic strength from CT and ET equivalent to CT dominant 1 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 4× 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-L6 input 1 day (1d) and 7 days (7d) after sham exposure (SE) and noise exposure (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 corrected for multiple comparisons). (E and F) Representative traces of excitatory postsynaptic currents (EPSCs) in dual recordings from both CT (solid line) and ET (dotted line) neurons evoked by maximal photostimulation of L5-L6 thalamocortical inputs in 1d SE [(E), left] and 1d NE [(E), right]; 7d SE [(F), left] and 7d NE [(F), right]. Different colored traces represent different pairs of simultaneously recorded CTs and ETs. Detailed statistical values are listed in Table 1.

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

(A and C) Average amplitude of light-evoked quantal EPSCs (Sr²⁺-mEPSCs) in CTs in response to L5-L6 thalamocortical maximal photostimulation in 1 day (A) and 7 days (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 and 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 1 day (B) and 7 days (D) after SE (black) and NE (red) mice. The arrowhead indicates the onset of light stimulus. The dotted line indicates 400 ms time window before stimulus (pre-LED). The solid line represents a 400-ms 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 and G) Summary graph of the average CT EPSC amplitudes in response to L5-L6 thalamocortical maximal photostimulation in 1 day (E) and 7 days (G) 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 U test). (F and H) Representative traces of L6 CT EPSCs in response to L5-L6 thalamocortical maximal photostimulation in 1d SE [(F), black line] and 1d NE [(F), red line] and 7d SE [(H), black line] and 7d NE [(H), red line]. Detailed statistical values are listed in Table 1.

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

(A) Average CT input resistance (R_input) 1 and 7 days 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) 1 and 7 days 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 1 and 7 days 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 1 and 7 days 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). (E and G) Representative traces of CT firing in response to depolarizing current (50, 100, and 200 pA current injections), 1d SE (E, left) versus 1d NE [(E), right], and 7d SE [(G), left] versus 7d NE [(G), right]. (F and H) Average firing frequency as a function of injected current amplitude, 1d SE versus 1d NE (F), and 7d SE versus 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 corrected for multiple comparisons). Detailed statistical values are listed in Table 1.

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

(A and C) Average amplitude of Sr²⁺-mEPSCs in ETs in response to L5-L6 thalamocortical maximal photostimulation in 1 day (A) and 7 days (C), 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 and 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 1 day (B) and 7 days (D) after SE (black) and NE (red) mice. The arrowhead indicates the onset of light stimulus. The dotted line indicates a 400-ms time window before stimulus (pre-LED). The solid line represents a 400-ms 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 and G) Summary graph of the average L5 ET EPSC amplitudes in response to L5-L6, and L1-L4 thalamocortical maximal photostimulation in 1d SE versus 1d NE, or 7d SE versus 7d NE mice (L5-L6: 1d SE: 18 cells/6 mice; 1d NE: 26 cells/9 mice; 7d SE: 11 cells/6 mice; 7d NE: 10 cells/6 mice; L1-L4: 1d SE: 8 cells/3 mice; 1d NE: 7 cells/4 mice; 7d SE: 9 cells/5 mice; 7d NE: 12 cells/4 mice). (F and H) Representative traces of L5 ET EPSCs in response to L5-L6, and L1-L4 thalamocortical maximal photostimulation in 1d SE [(F) left, (H) left, black] versus 1d NE [(F) left, (H) left, red], and 7d SE [(F) right, (H) right, black] versus 7d NE [(F) right, (H) right, red] mice. Detailed statistical values are listed in Table 1.

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

(A) Average ET R_input 1 and 7 days 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 1 and 7 days 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 1 and 7 days 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 1 and 7 days 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). (E and G) Representative traces of ET firing in response to depolarizing current injection (50, 100, and 200 pA), 1d SE [(E), left] versus 1d NE [(E), right], and 7d SE [(G), left] versus 7d NE [(G), right]. (F and H) Average firing frequency as a function of injected current amplitude, 1d SE versus 1d NE (F), and 7d SE versus 7d NE (H). Current injections from 25 to 350 pA with an increment of 25 pA (1d SE: 21 cells/6 mice; 1d NE: 20 cells/5 mice; 7d SE: 20 cells/5 mice; 7d NE: 23 cells/6 mice). Detailed statistical values are listed in Table 1.

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

(A) Example two-photon field of view (FoV) from CTs imaged before and after NE. (B) Average pure tone responses across all CTs in SE mice (−1d: n = 507 neurons; 1d: n = 460 neurons; 7d: n = 432 neurons). (C) Same as (A) for NE mice (−1d: n = 713 neurons; 1d: n = 638 neurons; 7d: n = 646 neurons). (D) Tuning curves aligned to BF in SE mice. (E) Same as (D) for NE mice. (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 pure tone frequency at 80 dB SPL. The dashed line represents chance level. Error bars represent SD of decoding iterations. (H) Average white noise responses at different intensities across all ETs in SE mice (−1d: n = 438 neurons; 1d: n = 460 neurons; 7d: n = 411 neurons). (I) Same as (H) in NE mice (−1d: n = 636 neurons; 1d: n = 559 neurons; 7d: n = 587 neurons). (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). (K) Accuracy of a multinomial logistic regression trained to decode sound intensity. The dashed line represents chance. Error bars represent SD of decoding iterations. All asterisks indicate significant pairwise differences (*P < 0.05, **P < 0.01, ***P < 0.001, Bonferroni corrected for multiple comparisons). Detailed statistical values are listed in Table 1.

Fig. 7.. Transient shift in ET sound response properties 1 day after NE.

(A) Example two-photon FoV from ETs imaged before and after NE. (B) Average pure tone responses 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). (D) Tuning curves aligned to best frequency (BF) in SE mice. (E) Same as in (D) for NE mice. (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). (G) Accuracy of a multinomial logistic regression classifier trained to decode pure tone frequency at 80 dB SPL. The dashed line represents chance. Error bars represent SD of decoding iterations. (H) Average white noise responses 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). (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). (K) Accuracy of a multinomial logistic regression trained to decode sound intensity. The dashed line represents chance. Error bars represent SD of decoding iterations. All asterisks indicate significant pairwise differences (*P < 0.05, **P < 0.01, ****P < 0.00001, Bonferroni corrected for multiple comparisons). Detailed statistical values are listed in Table 1.

Fig. 8.. Changes in synaptic, intrinsic, and sound response properties correlate with noise trauma severity.

(A) Correlation between ABR threshold shift and CT/ET EPSC ratio after optogenetically stimulating thalamic input (Fig. 1D) in 1d SE (black) and NE (red) mice (1d SE: 18 cells/6 mice; 1d NE: 26 cells/9 mice). The line depicts a linear fit. (B) Same as (A) for amplitude of light-evoked quantal EPSCs in CTs in response to TC photostimulation (Fig. 2A) in 1d SE (black) and NE (red) mice (1d SE: 7 cells/3 mice; 1d NE: 6 cells/3 mice). (C) Same as (A) for average CT EPSC amplitudes in response to TC photostimulation (Fig. 2E) in 1d SE (black) and NE (red) mice (1d SE: 18 cells/6 mice; 1d NE: 26 cells/9 mice). (D) Same as (A) for ET EPSC amplitudes in response to TC photostimulation (Fig. 4E) in 1d SE (black) and NE (red) mice (1d SE: 18 cells/6 mice; 1d NE: 26 cells/9 mice). (E) Same as (A) for CT resting membrane potential (Fig. 3B) in 1d SE (black) and NE (red) mice (1d SE: 22 cells/8 mice; 1d NE: 23 cells/5 mice). (F) Same as (A) for the maximum firing frequency (Fig. 3F) in 1d SE (black) and NE (red) mice (1d SE: 21 cells/8 mice; 1d NE: 21 cells/5 mice). (G and H) Same as (A) for the change in intensity thresholds (Fig. 6J) in 1d (G) or 7d (H) 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). (I and J) Same as (A) for the change in intensity thresholds (Fig. 7J) in 1d (I) or 7d (J) 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).