SNHL alters passive and active membrane properties
The data were collected from 164 neurons recorded in 143 brain slices from experiments performed in an approximately equal number of normal and SNHL neurons. An intact thalamocortical projection to A1 was verified by recording a robust MGv-evoked extracellular response (Cruikshank et al., 2002
). The cortex was first observed at 100× magnification, in which the laminar structure is clear, and the recording electrode position was established to be within layer 2/3. Neurons were visually identified under infrared-differential interference contrast optics at 400× magnification, and whole-cell recordings were then obtained. The search strategy excluded neurons in sparsely populated layer 1 and targeted pyramidal neurons that are densely packed in layer 2/3. A blind analyses of over 53 randomly chosen biocytin-filled pyramidal neurons showed that there was no difference in the soma depth from the pial surface between normal and SNHL neurons (normal, 402 ± 16 μm vs SNHL, 392 ± 18 μm; Wilcoxon’s test; χ2
= 0.14; df = 52; p
= 0.7). Therefore, there was no bias in recording site depth between the two groups. The depth range of these cell bodies varied between 125 and 575 μm from the pial surface.
The mean resting membrane potential of neurons from the SNHL animals displayed a small but significant depolarization when compared with the value from normal animals () (mean ± SEM; normal, −63.4 ± 0.8 mV vs SNHL, −59.9 ± 0.8 mV; t test; t = 3; df = 100; p = 0.002). SNHL also led to a significant increase in the mean input resistance () (mean ± SEM; normal, 277 ± 13.5 MΩ vs SNHL, 409 ± 29 MΩ; t test; t = 4; df = 92; p < 0.001). A linear fit between resting potential and input resistance did not reveal a significant correlation in either normal (R2 = 0.01; p > 0.05) or SNHL (R2 = 0.02; p > 0.05) neurons. Furthermore, we compared certain properties of adapting only neurons after hearing loss. This analysis showed a trend similar to that seen in the entire neuron population (resting potential: normal adapting, 64 ± 0.8 vs SNHL adapting, 59.4 ± 0.9 mV, t test, t = 3.4, df = 73, p < 0.0008; input resistance: normal adapting, 271 ± 14 vs SNHL adapting, 452 ± 29 MΩ, t test, t = 4.5, df = 67, p < 0.0001). Whereas there was no effect of age on VREST, there was a small effect for RINPUT. Therefore, a linear fit was performed for data from normal and SNHL neurons that revealed a very small effect of age on RINPUT of SNHL neurons (R2 = 0.11; p < 0.05) but not normal neurons (R2 = 0.06; p > 0.05).
Figure 1 SNHL increases membrane excitability. The resting membrane potential of SNHL neurons is significantly depolarized (A), and the input resistance is significantly higher (B) compared with neurons recorded in normal animals. In this and subsequent figures, (more ...)
The firing patterns of A1 neurons were characterized in response to suprathreshold depolarizing pulses (1500 ms) and fell into three broad categories: (1) onset (phasic) neurons that fired a single action potential; (2) sustained neurons that fired at a high rate with little or no adaptation; and (3) adapting (regular spiking) neurons that fired at a relatively lower rate exhibited a significant degree of spike frequency adaptation (). Whereas 15% of normal neurons were onset type, this response was not found in SNHL cases. Furthermore, the adapting pattern decreased by 13% (), whereas the incidence of sustained type of firing increased by 30% after SNHL.
presents a comparison of additional measures, including spike threshold and half-spike width. These measures strongly suggest that all included recordings were obtained from pyramidal neurons and that differences in firing patterns were induced by hearing loss. For example, there were no significant differences between the half-spike width ( p = 0.7; df = 15), input resistance ( p = 0.4; df = 15), or resting membrane potential ( p = 0.11; df = 15) between the adapting and sustained SNHL neurons. Likewise, there was no difference between the half-spike widths of normal and SNHL neurons ( p = 0.14; df = 20). In contrast, the spike threshold (i.e., magnitude of voltage deflection from the resting potential before the neurons fired) and the threshold current (i.e., the amount of current required to elicit a threshold response) were significantly lower in SNHL neurons compared with normals. Additional comparisons between the resting potentials and input resistance between all adapting neurons (normal vs SNHL) showed a highly significant difference as seen in the entire population of normal versus SNHL neurons (). To compare the stimulus–response curves, a correlation coefficient was obtained for injected current (five current steps of 1500 ms in 10 pA increments) and number of elicited spikes for each firing pattern in . SNHL sustained neurons exhibited a stronger correlation (R2 = 0.6) compared with adapting neurons (normal, R2 = 0.42; SNHL, R2 = 0.43). The stimulus–response curve for normal sustained firing neurons is not plotted in (R2 = 0.59). The maximum instantaneous firing frequency in response to depolarizing current injections (during the first 100 ms) for adapting neurons was 25 Hz (n = 6), whereas that for sustained neurons was 50 Hz (n = 6). The stimulus–response plots () for normal and SNHL adapting neurons showed a similar increment in firing rate. Therefore, hearing loss led to a net increase in the incidence of the sustained firing pattern and a net decrease in the adapting and onset patterns.
Membrane properties of adapting and sustained firing neurons
To confirm that nonpyramidal cells were not included in our sample, biocytin filled neurons were examined after each experiment under 40–600× magnification to identify the dendritic structure and to confirm the presence of spines. This qualitative examination of normal and SNHL neurons indicated the presence of intact basal and apical dendrites with spines in each recovered neuron included in this study ().
Figure 2 Layer 2/3 spiny pyramidal neurons. A selection of nine biocytin-filled layer 2/3 neurons with different firing patterns reveal cell and dendritic architecture. A, A normal adapting neuron. B, A normal sustained neuron. C, A normal onset neuron. D, Dendritic (more ...)
SNHL enhances synaptic excitation
MGv stimulation was used to evoke EPSPs in layer 2/3 pyramidal neurons (). A comparison of maximum EPSP amplitudes between normal and SNHL neurons showed no difference, both before and after treatment with the NMDA receptor blocker AP-5 (mean ± SEM before AP-5 application: normal, 10.9 ± 1.5 mV vs SNHL, 12.1 ± 1.7 mV, t test, t = 0.54, df = 26, p = 0.5; mean ± SEM after AP-5 application: normal, 5.7 ± 1 mV vs SNHL, 4.2 ± 0.7mV, t test, t = −1.1, df = 26, p = 0.2).
Figure 3 SNHL augments NMDA receptor function. A, A Schematic of the thalamocortical brain slice showing the position of a stimulating electrode in the MGv (square pulse), the pathway (dark line) from MGv to A1, and a recording electrode within A1 (Vm). The approximate (more ...)
In contrast to maximum EPSP amplitude, the EPSP duration was significantly longer in SNHL neurons. illustrates that the reduction in EPSP duration by the NMDA receptor blocker AP-5 was significantly greater after SNHL. The mean EPSP duration (milliseconds ± SEM) of normal and SNHL neurons was significantly different before the application of AP-5 (normal, 238 ± 22 ms vs SNHL, 412 ± 69 ms; Wilcoxon’s test; χ2 = 5.89; p = 0.02). Furthermore, the absolute reduction in EPSP duration by application of AP-5 was significantly greater in SNHL neurons (normal, 110 ± 13 ms vs SNHL, 315 ± 44 ms; Wilcoxon’s test; χ2 = 16.62; p < 0.0001). However, the duration of the remainder EPSP, presumably carried by AMPA receptors, was not different between normal and SNHL neurons (normal, 129 ± 20 ms vs SNHL, 123 ± 31 ms).
A similar trend was found for intracortically evoked EPSPs. SNHL led to longer EPSP durations and greater reduction by AP-5 than in normals (mean ± SEM reduction in duration of intracortically evoked EPSPs after AP-5 treatment: normal, 70 ± 14 ms vs SNHL, 190 ± 39 ms; t test; t = 2.8; df = 17; p = 0.01). There was no significant difference in the maximum amplitude of intracortically evoked EPSPs before or after AP-5 treatment.
To test whether the extended EPSP duration in SNHL neurons was attributable to a change in the subunit composition of NMDA receptors, MGv-evoked maximum EPSCs were recorded after blocking sodium and potassium channels in an Mg+2-free ACSF (VHOLD of −50 mV). The NMDA receptor-mediated component was recorded in the presence of DNQX (20 μM), bicuculline (10 μM), and glycine (10 μM). In these experiments, we chose a stimulus intensity that produced an initial (before ifenprodil) EPSC amplitude of 400–500 pA for both normal and SNHL neurons (mean ± SEM EPSC amplitude: normal, 437 ± 89 pA vs SNHL, 474 ± 80 mV; Wilcoxon’s test; χ2 = 3.84; p = 0.05). We maintained this stimulus intensity throughout the experiments. In the absence of the preceding AMPAergic EPSC component (DNQX in bath), we measured the reduction in EPSC amplitude by the antagonist to the NR2B subunit-containing NMDA receptors, ifenprodil, as a function of change in NR2B subunits.
The reduction of EPSC amplitude by the antagonist specific for NR2B subunit-containing NMDA receptors, ifenprodil, was significantly greater in SNHL neurons () (mean ± SEM reduction after application of ifenprodil: normal, 201 ± 41 pA vs SNHL, 318 ± 38 pA; t test; t = 2.1; df = 18; p = 0.05). The remaining EPSC amplitudes, presumably carried by the NR2A subunit, were not significantly different between normal and SNHL neurons. In two normal and two SNHL neurons recorded from four different slices, these remainder EPSCs were completely and reversibly abolished by AP-5 (data not shown), demonstrating that the analyzed EPSCs in this experiment were exclusively NMDAergic.
To assess whether increased sensitivity of EPSCs to ifenprodil in SNHL neurons was attributable to increased expression of NR2B subunits at synapses in layer 2/3, postembedding immunogold staining was performed to examine the localization of these subunits at synapses using an electron microscope (). Gold particles reflecting immunolabeling for NR2B that resided in the vicinity of asymmetric synapses were counted and grouped into the following mutually exclusive categories: (1) on presynaptic membrane, (2) at PSD, (3) near PSD, (4) on extrasynaptic membrane, and (5) within spine/dendrite or within axon terminal. For each category, the average number of gold particles per synapse was more numerous for the SNHL neurons (). The difference was most pronounced at the PSD and over the total synaptic region (i.e., the average of the sum of gold particles in categories 1–3).
Figure 4 Greater occurrence of immunolabeling of NR2B subunits at synapses from SNHL animals. A, B, Electron micrographs from normal brain. C, D, Micrographs from SNHL brain. Arrowheads indicate PSDs of asymmetric synapses. S and T represent postsynaptic spine (more ...)
Average ± SEM number of NR2B-labeling immunogold particles per synapse in layer 2/3 of A1
If the maximum EPSP amplitude is determined primarily by the AMPAergic component (), there could have been a masking effect attributable to the depolarized resting membrane potential () and decreased firing threshold (). Therefore, a series of voltage-clamp recordings were performed. First, to examine whether such postsynaptic alterations are accompanied by a presynaptic change, mEPSCs were recorded in nominal Mg2+-ACSF and in the presence of blockers of Na+ and K+ channels and GABAA receptors under voltage-clamp conditions (see Materials and Methods). Analyses showed a significant decrease in the frequency and an increase in the peak amplitude of mEPSCs in SNHL neurons () (mEPSC frequency, mean ± SEM: normal, 3.3 ± 0.2 Hz vs SNHL, 1.9 ± 0.4 Hz, t test, t = 2.9, df = 7, p = 0.02; mEPSC amplitude, mean ± SEM: normal, 12.7 ± 1.4 pA vs SNHL, 20 ± 2.6 pA, t test, t = 2.6, df = 7, p = 0.03). Furthermore, the duration of total mEP-SCs, and reduction in duration by AP-5, was greater in SNHL neurons than in normals () (total mEPSC duration, mean ± SEM: normal, 51 ± 4.7 vs SNHL, 100.9 ± 4.4, t test, t = 7.6, p < 0.0001; mEPSC duration after AP-5, mean ± SEM: normal, 34.5 ± 3 ms vs SNHL, 46 ± 7 ms; t test, t = 2.8, p < 0.01). Statistical comparisons confirmed that AP-5 significantly reduced mEPSC duration within both the normal and the SNHL groups (data not shown).
Figure 5 Frequency and amplitude of mEPSCs, respectively, decreases and increases in SNHL neurons. A, The left panel shows two sweeps of mEPSCs recorded for 20 s each in a normal (left) and an SNHL neuron (right) at a holding potential of −70 mV. Each (more ...)
To assess whether putative monosynaptic connections from the MGv to layer 2/3 pyramidal neurons exhibit NMDAergic alterations and whether there was a parallel change in AMPAergic function, minimum MGv-evoked EPSCs (≥50% failure rates) were recorded in voltage clamp with normal ACSF, bicuculline (10 μM), and glycine (10 μM) in the bath (). The results showed that the amplitude of minimum EPSCs at VHOLD of −70 was significantly larger in SNHL neurons (minimum MGv-evoked amplitudes at VHOLD of −70 mV, mean ± SEM: normal, 11 ± 2 pA vs SNHL, 28 ± 5 pA; Wilcoxon’s test; χ2 = 5; p = 0.025). To examine the NMDAergic component, cells were held at −20 mV. Comparison of such EPSC durations at this holding potential showed that SNHL neuron EPSCs were significantly longer (minimum MGv-evoked EPSC duration at VHOLD of −20 mV, mean ± SEM: normal, 22 ± 3 ms vs SNHL, 182 ± 40 ms; Wilcoxon’s test; χ2 = 8.3; p = 0.003). Finally, after blockade of AMPA receptors by DNQX, the pure NMDA receptor-mediated minimum-evoked EPSC currents revealed at VHOLD of −20 mV were significantly larger and longer in SNHL neurons (minimum MGv-evoked amplitude in the presence of DNQX at VHOLD of −20 mV, mean ± SEM: normal, 8 ± 1.8 pA vs SNHL, 49 ± 19 pA, Wilcoxon’s test, χ2 = 7.4, p = 0.006; minimum MGv-evoked duration in the presence of DNQX at VHOLD of −20 mV, mean ± SEM: normal, 17 ± 2 ms vs SNHL, 191 ± 38 ms, Wilcoxon’s test, χ2 = 8.3, p = 0.003) (). In all neurons (n = 6 each normal and SNHL neurons), the EPSCs were abolished by AP-5, demonstrating that they were pure minimum-evoked MGv-evoked NMDAergic currents (). For each recorded neuron, we calculated the maximum difference in response latency as a measure of variability in minimum-evoked EPSCs. We compared this measure of variability between normal and SNHL neurons and found no significant difference (normal, 1.5 ± 0.3 ms vs SNHL, 1.1 ± 0.2 ms; n = 12; p = 0.3).
SNHL reduces synaptic inhibition
Because the resting potential of layer 2/3 pyramidal neurons in normal animals is close to ECl
(data not shown), it was often difficult to observe the evoked IPSPs. Furthermore, cortical stimulation produces a highly variable mixed excitatory–inhibitory synaptic response (Cruikshank et al., 2002
). To circumvent these issues, recorded neurons were held at −55 mV to enhance the driving force for IPSPs, and excitatory ionotropic transmission was blocked partially with AP-5. This procedure revealed polysynaptic IPSPs. In three each normal and SNHL neurons, these IPSPs could then be blocked by the sequential addition of GABAB
receptor antagonists, respectively, demonstrating that they were GABAergic (data not shown). In another set of experiments, the AMPA receptor blocker DNQX was also added with AP-5; this strategy blocked recruitment of intracortical excitatory neurons that would drive inhibitory interneurons. However, because such monosynaptic IPSPs were not recorded consistently, IPSP data were first examined with AP-5 application alone.
is a schematic of the stimulation and recording site for monosynaptic IPSPs. The results showed that the amplitudes of both monosynaptic IPSPs (; DNQX and AP-5 in the bath) and polysynaptic IPSPs (data not shown; AP-5 alone in the bath) were significantly reduced in SNHL neurons when compared with those in normal neurons (maximum monosynaptic IPSP amplitude, mean ± SEM: normal, −9.2 ± 1.5 mV vs SNHL, −4.2 ± 1.0 mV, t test, t = 2.6, df = 16, p = 0.02; maximum polysynaptic IPSP amplitude, mean ± SEM: normal, −7.8 ± 0.7 mV vs SNHL, −4.4 ± 0.5 ms, t test, t = 3.5, df = 25, p = 0.001).
Figure 7 SNHL reduces monosynaptic IPSP amplitude. A, Schematic of the thalamocortical brain slice showing a stimulating electrode on layer 2/3 (square pulse) ~1 mm rostral to the recording electrode. B, The maximum monosynaptic IPSP evoked by stimulating layer (more ...)