Control of interneuron firing by subthreshold synaptic potentials in principal cells of the dorsal cochlear nucleus

Abstract

Voltage-gated ion channels amplify, compartmentalize, and normalize synaptic signals received by neurons. We show that voltage-gated channels activated during subthreshold glutamatergic synaptic potentials in a principal cell generate an excitatory→inhibitory synaptic sequence that excites electrically coupled interneurons. In fusiform cells of the dorsal cochlear nucleus, excitatory synapses activate a TTX-sensitive Na(+) conductance and deactivate a resting Ih conductance, leading to a striking reshaping of the synaptic potential. Subthreshold voltage changes resulting from activation/deactivation of these channels subsequently propagate through gap junctions, causing slow excitation followed by inhibition in GABAergic stellate interneurons. Gap-junction-mediated transmission of voltage-gated signals accounts for the majority of glutamatergic signaling to interneurons, such that subthreshold synaptic events from a single principal cell are sufficient to drive spikes in coupled interneurons. Thus, the interaction between a principal cell's synaptic and voltage-gated channels may determine the spike activity of networks without firing a single action potential.

Figure 1

Parallel fiber transmission generates an E/I sequence mediated by AMPA receptors. A) DCN circuit and experimental set-up. Projection neurons (fusiform cells; FC) integrate excitatory synapses from auditory nerve and granule cell parallel fibers. The latter also contact the stellate interneurons that are electrically coupled to fusiform cells. A bipolar stimulating electrode is placed in the molecular layer to activate parallel fibers. B) A single parallel fiber shock generates an E/I sequence in stellate cells that is blocked by the AMPA receptor antagonist NBQX (10 μM; red trace). Black trace is an average of events that had an initial rapidly rising component, whereas the gray trace is an average of events lacking the fast component. The right panel shows the two types of events on a faster time base to highlight the drastic difference in rise times. C) Parallel fiber EPSCs in cx36 −/− mice lack the slow inward and outward components. Black and gray traces represent averages of trials in which the fast component either succeeded or failed, respectively. D) Quantification of EPSCs from wild-type and cx36 −/− mice. Left panel: The total inward charge transfer of parallel fiber EPSCs in wild-type mice was on average −734±164 pA*ms (n=11), whereas it was only −62±6 pA*ms in the KO mice (n=12). Middle and right panels: The inward charge transfer and peak amplitude of the fast rising AMPA EPSC from parallel fiber synapses directly on stellate cells was not significantly different between wild-type and KO mice (WT charge and peak amplitude: −65±8 pA*ms, −29±3 pA. KO charge and peak amplitude: −60±6 pA*ms, −32±3 pA. p=0.67 and 0.39, respectively; unpaired t-test). The fast rising AMPA EPSC was isolated by subtracting the average of slowly-rising events from the average of fast rising events (e.g., black trace minus gray trace).

Figure 2

Subthreshold activation of Na⁺ channels in fusiform cells shapes excitatory transmission in electrically-coupled stellate cells. A) Example traces from a fusiform cell in current-clamp. An EPSC waveform is injected at two different amplitudes (100 and 500 pA) before and after bath application of TTX (black and red traces, respectively). The differential kinetics of the black traces highlights the role of Na⁺ channels in shaping the aEPSP half-width. TTX application reduced the aEPSP half-width and peak amplitude, showing that subthreshold depolarizations activate Na⁺ channels. Note the AHP following the aEPSP evoked with a 500 pA waveform. Resting voltages for 100 pA events: −70 mV (control), −71 mV (TTX); for 500 pA events: −72 mV (control), −71 mV (TTX). B) Example input/output curves for the example cell shown in panel (A). Left panel: aEPSP half-widths as a function of EPSC waveform amplitude before and after TTX (black and red points, respectively). In n=7 cells, aEPSP half-widths evoked with the largest EPSC waveform amplitude (mean: 471±58 pA, range: 250–700 pA) were on average 2.4±0.4 times greater than aEPSP half-widths evoked with the smallest amplitude waveform (mean: 114±24 pA, range: 50–200 pA). This ratio was reduced to 1.0±0.1 in TTX (p=0.007, paired t-test), showing that Na⁺ channels powerfully control the kinetics of subthreshold events. Right panel: aEPSP peak amplitudes plotted as a function of EPSC command waveform in baseline and after TTX. Color scheme is the same as left panel. The dotted lines are linear fits to the data points to calculate the slope of the input/output function. On average, TTX reduced the slope of the aEPSP amplitude function to 77±3% of baseline (0.027±0.004 mV/pA in baseline to 0.020±0.003 mV/pA in TTX, n=7 cells, p=0.007, paired t-test). C) Left: diagram of paired recording experiment. Na⁺ channels are blocked specifically in the stellate cell by the addition of the Na⁺ channel blocker QX314 to the stellate cell pipette internal solution. Right: Average traces showing a subthreshold aEPSP in a fusiform cell (lower panel) and the resulting inward current in the stellate cell (upper panel) before and after TTX (black and red traces, respectively). TTX reduced the amplitude and half-width of aEPSPs in the fusiform cell. TTX similarly reduced the amplitude and charge transfer of the postjunctional inward current in the stellate cell, showing that Na⁺ channels in fusiform cells are a significant source of excitation. Resting voltages for fusiform cell pre-TTX, −75 mV; post-TTX: −74 mV. D) Summary data from 6 pairs similar to C. Left: TTX reduced the amplitude and half-width of fusiform cell aEPSPs to 73±3% and 32±5% of baseline (p=0.0002 and p<0.0001, respectively, one sample t-test). Right: TTX also significantly reduced the amplitude and inward charge transfer of postjunctional currents in stellate cells to 75±6% and 29±8% of baseline (p=0.0097 and p=0.0003, respectively, one sample t-test). Gray points are mean±SEM.

Figure 3

Fusiform cell AHPs and stellate cell off-responses are mediated by deactivation of HCN channels. A) An EPSC waveform (500 pA, lower trace) is injected in a fusiform cell in current clamp. CsCl (red trace) hyperpolarized the cell (see main text), and significantly shortened the half-width and reduced the peak amplitude of aEPSPs. These effects were largely reversed by returning the membrane potential to near baseline values with positive current (gray trace), showing that HCN channels control EPSP kinetics by setting the resting membrane potential near the activation range for subthreshold Na⁺ currents (n=7 cells. Peak amplitude baseline: 7.8±0.6 mV; CsCl 6.0±0.5 mV; CsCl + bias current: 8.8±0.6 mV. p=0.0007 and p=0.13 comparing baseline to CsCl and CsCl + Bias conditions, respectively. Repeated measures ANOVA + Dunnett's test. Half-width baseline: 47±6 ms; CsCl: 15±1 ms; CsCl + bias: 36±3 ms; p=0.002 and p=0.076 comparing baseline to CsCl and CsCl+Bias, respectively. Repeated measures ANOVA + Dunnett's test). Blocking HCN channels reduced the AHP following the depolarizing phase of the aEPSP, but adding bias current in the presence of CsCl failed to recover the AHP following the aEPSP (baseline: −0.39±0.08 mV; CsCl: −0.05 ±0.02 mV; CsCl + Bias current −0.03±0.01 mV, p= 0.014 and p=0.001 comparing baseline to CsCl and CsCl + Bias, respectively. Repeated measures ANOVA + Dunnett's test). Resting voltage of fusiform cell pre CsCl, −69 mV; in CsCl, −75 mV; in CsCl + bias current, −71 mV. B) A stellate cell is recorded in voltage clamp. Parallel fibers are stimulated with a single shock, generating the biphasic E/I sequence shown in Figure 1B. CsCl significantly reduced the slow outward current to 1±2% of baseline (n=8. p<0.0001, one-sample t-test). Additionally CsCl hastened the timecourse of the inward component, quantified as a reduction of the EPSC inward charge transfer (n=8. 26±3% of baseline remaining, p=0.01, one sample t-test). C) A fusiform cell is driven to spike with positive current injection. CsCl (3 mM, middle red panel) significantly reduced the amplitude of the AHP to 54±5% of baseline (n=6. p=0.0002, one sample t-test). Resting voltage of fusiform cell control, −78 mV; in CsCl: −78 mV; wash, −78 mV. Single sweeps are shown. D) Example from a voltage-clamped stellate cell in a VGluT2-ChR2 mouse. Prejunctional fusiform cells are activated with blue light flashes through the microscope objective (blue bar), generating a biphasic postjunctional response. The outward component, which represents the fusiform cell membrane potential returning towards baseline during the AHP, was reversibly reduced by CsCl to 40±12% of baseline (n=7, p<0.0001. One sample t-test).

Figure 4

Subthreshold events in fusiform cells drive spikes in stellate cells. A) Example recording (20 single trials) from a coupled fusiform-stellate cell pair (upper and lower panels, respectively). A subthreshold aEPSP is produced by injecting an EPSC-like waveform into the fusiform cell at ΔT=1 s (peak amplitude: 350 pA). Of note is the clustering of spikes in the stellate cell recording specifically during the fusiform cell aEPSP. B) Raster plot showing 123 trials from the pair in A. Subthreshold events in the fusiform cell cause time-locked spikes in the stellate cell. C) Histogram (50 ms bins) from the same pair as panels A and B. D) Summary histogram (50 ms bins) from 7 pairs as in A–C. The data are normalized to the number of spikes/bin during the 1 s prior to EPSC injection in the fusiform cell. Subthreshold events in fusiform cells caused a 21±12 fold increase in the number of stellate cell spikes/bin at ΔT=1.05–1.1 s, followed by a non-significant reduction in spike count at ΔT=1.2–1.4 s (baseline: 7.4±3.9 spikes/bin; ΔT=1.05–1.1 s: 34.9±9.0 spikes/bin; ΔT=1.2–1.4 s: 6.1±4.4 spikes/bin, p=0.01 and 0.2 compared to baseline, respectively. Repeated measures ANOVA + Dunnett's test). Of note is that the y-axis is plotted on a logarithmic scale.