Specific Early and Late Oddball-Evoked Responses in Excitatory and Inhibitory Neurons of Mouse Auditory Cortex

Abstract

A major challenge for sensory processing in the brain is considering stimulus context, such as stimulus probability, which may be relevant for survival. Excitatory neurons in auditory cortex, for example, adapt to repetitive tones in a stimulus-specific manner without fully generalizing to a low-probability deviant tone ("oddball") that breaks the preceding regularity. Whether such stimulus-specific adaptation (SSA) also prevails in inhibitory neurons and how it might relate to deviance detection remains elusive. We obtained whole-cell recordings from excitatory neurons and somatostatin- and parvalbumin-positive GABAergic interneurons in layer 2/3 of mouse auditory cortex and measured tone-evoked membrane potential responses. All cell types displayed SSA of fast ("early") subthreshold and suprathreshold responses with oddball tones of a deviant frequency eliciting enlarged responses compared with adapted standards. SSA was especially strong when oddball frequency matched neuronal preference. In addition, we identified a slower "late" response component (200-400 ms after tone onset), most clearly in excitatory and parvalbumin-positive neurons, which also displayed SSA. For excitatory neurons, this late component reflected genuine deviance detection. Moreover, intracellular blockade of NMDA receptors reduced early and late responses in excitatory but not parvalbumin-positive neurons. The late component in excitatory neurons thus shares time course, deviance detection, and pharmacological features with the deviant-evoked event-related potential known as mismatch negativity (MMN) and provides a potential link between neuronal SSA and MMN. In summary, our results suggest a two-phase cortical activation upon oddball stimulation, with oddball tones first reactivating the adapted auditory cortex circuitry and subsequently triggering delayed reverberating network activity. Significance statement: Understanding how the brain encodes sensory context in addition to stimulus feature has been a main focus in neuroscience. Using in vivo targeted whole-cell recordings from excitatory and inhibitory neurons of mouse primary auditory cortex, we report two temporally distinct components of membrane potential responses encoding oddball tones that break stimulus regularity. Both components display stimulus-specific adaptation upon oddball paradigm stimulation in the three recorded cell types. The late response component, in particular, carries signatures of genuine deviance detection. In excitatory but not parvalbumin-positive inhibitory neurons, both early and late components depend on NMDA receptor-signaling. Our work proposes a potential neuronal substrate of a known deviant-evoked event-related potential, which is of fundamental significance in basic and clinical neuroscience.

Keywords: auditory cortex; interneurons; mismatch negativity; mouse; stimulus-specific adaptation.

Figure 1.

Tone-evoked membrane potential responses of neurons in mouse auditory cortex. A, Schematic of experimental design. Left, Whole-cell recordings were obtained from Exc neurons (black), SST (blue) and PV (red) interneurons in L2/3 of A1. Middle, Two sound frequencies f1 and f2, distributed around the BF, were chosen from the tuning curve. Due to asymmetry of the tuning curve, f1 and f2 typically differed in their evoked response amplitudes, referred to here as preferred and nonpreferred frequencies. Right, In an oddball paradigm repeated standard tones of one frequency (f1 or f2; gray) were interleaved with rare and irregular tones (Oddballs; black) of the other frequency. Use of f1 and f2 as standard or oddball tones was swapped between stimulation blocks. For analysis, oddball-evoked responses (Odd) are compared with responses evoked by pre-oddball standard tones (PreOdd) and those by the first standard tone of each block (First). B, A1 was located using red-light intrinsic optical imaging. Left, Example image of exposed cortical surface. Right, Localized tissue reflectance decreases upon presentation of pure tones (8 kHz). C, Example images of an excitatory pyramidal neuron and tdTomato-expressing SST and PV interneurons (left to right) filled with AlexaFluor-488 (green) via whole-cell recording pipettes. D, Pooled data for average membrane potential (V_m), input resistance (R_in), and AP half-width for the three cell types (n = 21, 20, and 11 for Exc, SST, and PV neurons). E, Example V_m responses in Exc, SST, and PV cells evoked by tones of their respective BFs (Exc: 11.3 kHz; SST: 26.9 kHz; PV: 11.3 kHz). Top row, Individual (gray) and average (colored) traces of the original V_m responses. Middle row, Estimated subthreshold V_m obtained by removing APs with median filtering. Bottom row, Colored lines show the average of the corresponding FR and the colored shades their SD. F, Comparisons of the BF-evoked peak V_m amplitudes (ΔV_m) and maximum FR changes (ΔFR) during 100 ms after tone onsets between Exc (n = 22), SST (n = 17), and PV cells (n = 11). D, F, Open circles indicate responses from individual neurons, and bold lines with error bars represent mean ± SEM. **p < 0.01, *p < 0.05.

Figure 2.

Neuronal adaptation during repeated standard tone presentation. A, Mean of filtered V_m responses for Exc, SST, and PV neurons for repeated stimulation with the same tone (colored average traces with shaded SEM range; responses were averaged from the initial 6 consecutive standard tone presentations during the oddball paradigm with P_Odd = 0.1, n = 20, 20, 12 for Exc, SST, and PV cells). Early and late time windows for analysis are indicated as black bars below voltage traces (0–0.1 s and 0.2–0.4 s after tone onset, respectively). Gray bars indicate tone durations. B, Equivalent mean of FR responses, showing rapid adaptation of Exc and SST neurons and a more gradual response decrease in PV neurons. C, Changes in V_m amplitude (mean ± SEM) as a function of tone number in the series of six tones. Peak and mean amplitudes, relative to baseline before the first tone, were analyzed for early (top) and late (bottom) time window, respectively. Note that baseline V_m for PV neurons was elevated due to spontaneous activity. D, FR changes as a function of tone number. Peak and mean amplitude of FR were quantified in early and late time window, respectively. Asterisks indicate significance of paired t test comparisons between responses evoked by the nth and the first tone. **p < 0.01 and *p < 0.05.

Figure 3.

Oddball tones recover subthreshold and spiking responses of A1 neurons. A, Subthreshold V_m responses (mean ± SD) to pre-oddball standard (PreOdd; left) and oddball (Odd; right) tones for exemplary Exc, SST, and PV neurons with P_Odd = 0.1. B, Difference traces for the three example neurons shown in A. Peak amplitude within the early time window and mean amplitude during the late time window were computed for analysis. C, D, Corresponding mean FR changes (±SD) and FR difference traces for the same cells shown in A. E, Peak V_m response amplitude (ΔV_m) for the first standard tone (First), the pre-oddball standard and the oddball tone for the early (top) and late (bottom) time windows for all Exc, SST, and PV neurons. F, Box plot comparison of the adaptation index (d′_SSA), calculated for V_m depolarization in early (top) and late (bottom) time windows, across cell types and for three different oddball probabilities. G, Mean FR change (ΔFR) for first, pre-oddball, and oddball stimuli in early (top) and late (bottom) time windows for all Exc, SST, and PV neurons. H, Comparison of d′_SSA, calculated for FR change in early (top) and late (bottom) time windows, across cell types and for three different oddball probabilities. For E and G, bars and error bars represent mean ± SEM, with asterisks indicating statistical significance of two-sample comparisons between stimulus types. For F and H, top and bottom edges of boxes represent the first and third quartiles, and the middle line the median. Whiskers indicate 1.5 interquartile range and outliers are shown as red crosses. Top asterisks indicate outcome of one-sample right-tailed t test against the null hypothesis d′_SSA = 0. Bottom asterisks indicate statistical significance of two-sample comparisons between cell types. **p < 0.01, *p < 0.05.

Figure 4.

Difference waveform depends on oddball probability and can be used to estimate response onsets. Traces in the top row show the difference traces (V_Diff) of mean oddball-evoked V_m response minus mean pre-oddball evoked V_m response for P_Odd = 0.1, 0.3, and 0.5 in the three cell types (grand averages from n = 20, 20, and 12 for Exc, SST, and PV cells, respectively). The corresponding first derivatives of the difference traces (dV_Diff/dt; smoothed by a moving average filter of 19 ms span at 1 kHz sampling rate) are shown in the bottom row. The onsets of the early responses can be estimated as the latency of the initial peaks of the first derivatives for P_Odd = 0.1 and 0.3 in all three neuronal types. The second local maxima of the first derivative, which are most prominent for P_Odd = 0.1 in Exc and PV cells, specify the onsets of the late components. Gray bars indicate tone duration and black bars the early and late time windows.

Figure 5.

Cell-type-specific modulation of oddball tone responses by frequency preference. A, Grand average wave forms for oddball minus pre-oddball difference of the V_m changes evoked when oddball frequency matched the neuron's preferred (left) of nonpreferred (right) frequency. Gray bars indicate tone duration. Black bars represent the early and late component time windows. B, Comparisons of SSA index d′_SSA for V_m changes in response to oddballs of preferred (P) and nonpreferred (NP) frequency within early and late time windows. Boxplots were calculated as in Figure 3. C, D, Same as A and B, but for FR changes. E, Discriminability of tone frequency (f1 vs f2) for oddball compared with pre-oddball standard tones. Traces show averaged binned classification performance of a Bayesian classifier on V_m responses. Chance performance is 50%. Stars indicate significance of Odd versus PreOdd classification difference over the respective time windows (two-way repeated-measures ANOVA). **p < 0.01 *p < 0.05.

Figure 6.

Subthreshold late oddball-response component carries signs of deviance detection. A, Schematic illustration of the relation between MS and oddball paradigm. In the MS condition, tones of frequencies f1 and f2 are presented within a pseudo-random sequence of tones of 15 different frequencies. Compared with the oddball condition, no regularity is established. Increased activity to oddballs compared with the same frequency tones in MS condition (MS tones) could indicate genuine deviance detection. B, Grand average V_m amplitudes and FR changes (both relative to baseline activity before each tone) for oddball (Odd; P_Odd = 0.1) and MS tones. Average responses are shown as colored lines and the SEM across each subpopulation as gray shades. Tone duration (0.1 s for both oddball and MS tones) is indicated as gray bars below traces and early and late time windows as black bars. C, D, Comparisons of peak V_m amplitudes (C) and FR changes (D) evoked by oddball and MS tones within early and late time windows. Bar graph indicates the averages and error bars indicate 1 SEM. *p < 0.05.

Figure 7.

Oddball membrane potential responses are sensitive to NMDA receptor signaling in a cell-type-specific manner. A, B, Average V_m (A) and firing rate (B) in response to pre-oddball standard (PreOdd) and oddball (Odd; P_Odd = 0.1) tones in example Exc and PV cells with intracellular infusion of NMDA-receptor antagonist MK-801. Colored lines are shown as the average responses and colored shades as the SD for each neuron. Right panels indicate the grand mean difference responses (V_m, FR; oddball − pre-oddball). Solid lines represent the grand average response with MK-801 blockage (n = 11, 9 for Exc and PV cells), dashed lines those under control condition (n = 20, 12 for Exc and PV cells, respectively); colored shades indicate SEM across the subpopulation of each cell type. Tone duration is shown as gray bars and early and late time windows as black bars below traces. Note that MK-801 application leads to a clearly reduced difference waveform for V_m and spiking activity in pyramidal neurons. C, Comparisons of peak V_m amplitudes evoked by the first, pre-oddball standard and oddball tones within early and late time windows under intracellular MK-801 infusion. D, Comparisons of d′_SSA index computed from V_m between control condition and with MK-801 infusion for early and late responses. Data are shown with box plots as represented in Figure 3F,H. Lower stars indicate the outcome of one-sample right-tailed t test against zero and upper stars represent the results of two-sample comparisons between control and MK-801 treatment. E, F, Same as C and D but for FR changes. **p < 0.01, *p < 0.05.

Figure 8.

Schematic model of early and late oddball-evoked responses in L2/3 neurons of A1. Left: In response to oddball tones of either frequency f1 (preferred) or f2 (nonpreferred), the fast activation of a nonadapted synaptic channel originating from thalamus triggers the early responses in L2/3 Exc cells, as well as in SST and PV neurons. As a result a subset of L2/3 neurons becomes reactivated. Right, The recovered early oddball-evoked activity also triggers a late response component in form of a delayed network reverberation, which possibly involves local recurrent excitation and activation of larger-scale networks, e.g., thalamocortical or corticocortical loops.