Hearing loss raises excitability in the auditory cortex

Abstract

Developmental hearing impairments compromise sound discrimination, speech acquisition, and cognitive function; however, the adjustments of functional properties in the primary auditory cortex (A1) remain unknown. We induced sensorineural hearing loss (SNHL) in developing gerbils and then reared the animals for several days. The intrinsic membrane and synaptic properties of layer 2/3 pyramidal neurons were subsequently examined in a thalamocortical brain slice preparation with whole-cell recordings and electron microscopic immunocytochemistry. SNHL neurons displayed a depolarized resting membrane potential, an increased input resistance, and a higher incidence of sustained firing. They also exhibited significantly larger thalamocortically and intracortically evoked excitatory synaptic responses, including a greater susceptibility to the NMDA receptor antagonist AP-5 and the NR2B subunit antagonist ifenprodil. This correlated with an increase in NR2B labeling of asymmetric synapses, as visualized ultrastructurally. Furthermore, decreased frequency and increased amplitude of miniature EPSCs (mEPSCs) in SNHL neurons suggest that a decline in presynaptic release properties is compensated by an increased excitatory response. To verify that the increased thalamocortical excitation was elicited by putative monosynaptic connections, minimum amplitude ventral medial geniculate nucleus-evoked EPSCs were recorded. These minimum-evoked responses were of larger amplitude, and the NMDAergic currents were also larger and longer in SNHL neurons. These findings were supported by significantly longer AP-5-sensitive durations and larger amplitudes of mEPSCs. Last, the amplitudes of intracortically evoked monosynaptic and polysynaptic GABAergic inhibitory synaptic responses were significantly smaller in SNHL neurons. These alterations in cellular properties after deafness reflect an attempt by A1 to sustain an operative level of cortical excitability that may involve homeostatic mechanisms.

Figure 6.

NMDA receptor-mediated minimum-evoked thalamocortical excitatory currents are stronger in SNHL neurons. A, Minimum MGv-evoked excitatory currents were recorded in voltage clamp at V_HOLD of -70 mV in the presence of bicuculline and glycine. They were isolated by stimulating MGv (arrow, stimulus artifact) at 0.2-0.5 Hz in incremental intensities, resulting in larger EPSCs at each stimulus step (grayt races); the intensity at which minimum EPSC was recorded was then chosen for successive recordings (dark EPSC). This stimulation intensity produced a failure rate of ≥50% (dark trace at baseline is a failure). B, Left, In a normal neuron, a minimum MGv-evoked EPSC is shown (first trace). Holding the neuron at -20 mV revealed the initial fast AMPAergic as well as a delayed NMDAergic component (second trace). Addition of DNQX eliminated the AMPAergic component and revealed the NMDAergic EPSC (third trace). This was then blocked by AP-5. B, Right, Note the comparatively larger minimum MGv-evoked EPSC in an SNHL neuron (top). Holding the cell at -20 mV shows the initial AMPAergic component and a much longer (than normal) NMDAergic EPSC component (second trace). Addition of DNQX eliminated the AMPAergic component, revealing the long NMDAergic EPSC (third trace). Addition of AP-5 eliminates the NMDA receptor-mediated EPSC (bottom trace). C-E, Bar graphs of minimum MGv-evoked EPSCs recorded from six normal and six SNHL neurons show the following in the SNHL neurons: C, that the amplitude of mean minimum evoked EPSCs at V_HOLD of -70 mV is significantly larger; D, that the amplitude of pure NMDAergic currents are significantly larger; and E, that the duration of pure NMDAergic currents are significantly longer (asterisks indicate a significant difference; for p values, see Results). Each neuron was obtained from a different animal.

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, each open symbol represents the measurement from one neuron (n values shown on the x-axis). Filled symbols are mean ± SEM. C, Examples of neurons that respond to suprathreshold current injection with an onset (top), sustained (middle trace), and adapting (bottom trace) response. Five suprathreshold pulses (10 pA, 1500 ms) were injected, and firing pattern was established if the fifth pulse did not alter the firing pattern. D, Bar graphs of firing patterns in normal and SNHL neurons show percentile distribution. After SNHL, onset-type neurons were not observed, and there was a significant rise in the sustained and a decrease in adapting neuron patterns, suggesting greater excitability. E, Linear fit of number of spikes by depolarizing current injection. Five 10 pA, 1500 ms depolarizing current steps were injected, and the resultant spikes were counted. The fit shows no significant difference between the stimulus-response characteristics of normal and SNHL adapting neurons; the SNHL sustained neuron fits had a greater correlation coefficient, similar to the correlation in sustained neuron fits of normal neurons (see Results). Therefore, the characteristics of firing patterns did not change; rather, the incidence of sustained firing increased and adapting neurons decreased in SNHL neurons, as shown in D.

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 apical spines from the neuron shown in B. E, An SNHL sustained neuron. F, SNHL adapting (left) and sustained (right) neurons recorded in the same brain slice. G, Normal adapting (left) and onset (right) neurons recorded in the same slice. H, An SNHL adapting neuron. Scale bar: A-C, E-H, 100 μm; D, 10 μm.

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 (V_m). The approximate distance the afferents travel from the MGv around the lateral geniculate (LGN) and hippocampus, radiating to the recording site in layer 2/3, is ∼5.5 mm. B, Maximum EPSP evoked by stimulating MGv (arrowhead). Note the significant duration (in milliseconds) reduction by the NMDA receptor antagonist AP-5 and a greater reduction in an SNHL neuron. Resting membrane potentials (in millivolts) indicated at the left of the traces. Inset shows MGv-evoked EPSP and EPSP-elicited spike (clipped); in all cases, the subthreshold maximum EPSPs were analyzed. C, Scatter plot of the magnitude of reduction in EPSP duration by AP-5 between normal and SNHL neurons shows a significantly greater AP-5-sensitive NMDA receptor-mediated component among SNHL cases. D, EPSCs evoked by stimulating at MGv (arrowhead). Note the reduction in amplitude by the NR2B subunit-specific antagonist ifenprodil and a greater reduction in an SNHL neuron. We do not rule out an effect of ifenprodil on presynaptic NMDA receptors. E, Scatter plot of the magnitude of reduction in EPSC amplitude by ifenprodil between normal and SNHL neurons shows a significantly greater ifenprodil-sensitive NR2B subunit component in SNHL neurons.

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 and presynaptic axon terminal, respectively. Black dots are 10 nm immunogold particles labeling for NR2Bs. The categories of labeling are as follows: Pre, on presynaptic membrane; At, at PSD; Near, near PSD; Ex, on extrasynaptic membrane; S/D, within postsynaptic spine or dendrite away from the synapse; Tml, within axon terminal away from the synapse. Scale bar, 200 nm.

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 recording was acquired ∼2 min apart. Note that amplitudes of mEPSCs in an SNHL neuron (right) appear to be larger, whereas the frequency of mEPSCs have decreased. At the bottom panel of A, expanded mEPSCs from a normal (left) and an SNHL neuron are shown, both before and after AP-5 treatment. The traces indicate that the amplitude, duration, and AP-5 sensitivity in the SNHL neuron are greater. B-D, Bar graphs summarizing the mean amplitude and duration of mEPSCs recorded from five normal and three SNHL neurons. B, The amplitude of mean mEPSCs was significantly larger in SNHL neurons. C, The mean frequency of mEPSCs declined significantly in SNHL neurons. D, The total mEPSC duration was significantly greater in SNHL neurons. Asterisks (and bar in D) indicate that the differences are significant (for p values, see Results). Each neuron was obtained from a different animal.

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 2/3 is shown for a normal cell (top) and an SNHL cell (bottom). Note that the SNHL IPSP amplitude appears to be smaller. These recordings were obtained in the presence of blockers of the ionotropic glutamate receptors DNQX and AP-5. C, The plot of monosynaptic IPSP amplitudes from all recorded neurons shows a significant reduction for SNHL neurons.