AMPA receptor-mediated rapid EPSCs in vestibular calyx afferents

Abstract

In the vestibular periphery neurotransmission between hair cells and primary afferent nerves occurs via specialized ribbon synapses. Type I vestibular hair cells (HCIs) make synaptic contacts with calyx terminals, which enclose most of the HCI basolateral surface. To probe synaptic transmission, whole cell patch-clamp recordings were made from calyx afferent terminals isolated together with their mature HCIs from gerbil crista. Neurotransmitter release was measured as excitatory postsynaptic currents (EPSCs) in voltage clamp. Spontaneous EPSCs were classified as simple or complex. Simple events exhibited a rapid rise time and a fast monoexponential decay (time constant < 1 ms). The remaining events, constituting ~40% of EPSCs, showed more complex characteristics. Extracellular Sr²⁺ greatly increased EPSC frequency, and EPSCs were blocked by the AMPA receptor blocker NBQX. The role of presynaptic Ca²⁺ channels was assessed by application of the L-type Ca²⁺ channel blocker nifedipine (20 µM), which reduced EPSC frequency. In contrast, the L-type Ca²⁺ channel opener BAY K 8644 increased EPSC frequency. Cyclothiazide increased the decay time constant of averaged simple EPSCs by approximately twofold. The low-affinity AMPA receptor antagonist γ-d-glutamylglycine (2 mM) reduced the proportion of simple EPSCs relative to complex events, indicating glutamate accumulation in the restricted cleft between HCI and calyx. In crista slices EPSC frequency was greater in central compared with peripheral calyces, which may be due to greater numbers of presynaptic ribbons in central hair cells. Our data support a role for L-type Ca²⁺ channels in spontaneous release and demonstrate regional variations in AMPA-mediated quantal transmission at the calyx synapse.NEW & NOTEWORTHY In vestibular calyx terminals of mature cristae we find that the majority of excitatory postsynaptic currents (EPSCs) are rapid monophasic events mediated by AMPA receptors. Spontaneous EPSCs are reduced by an L-type Ca²⁺ channel blocker and notably enhanced in extracellular Sr²⁺ EPSC frequency is greater in central areas of the crista compared with peripheral areas and may be associated with more numerous presynaptic ribbons in central hair cells.

Keywords: AMPA receptor; crista; cyclothiazide; glutamate; type I hair cell.

Fig. 1.

The isolated calyx preparation. A: schematic of an isolated type I hair cell (HCI) and calyx terminal. The calyx envelops the basolateral regions of the HCI, where several presynaptic ribbons are present. Glutamate receptors are located on the inner face of the postsynaptic calyx in opposition to the presynaptic ribbons. A patch electrode is shown on the outer face of the calyx membrane. Inset: a calyx filled with Alexa 488 introduced via the patch electrode. Scale bar, 4 µm. B: typical whole cell currents from an isolated calyx terminal in the presence of ion channel blockers. In voltage clamp, cells were held at −70 mV and 10-mV steps were applied in increments from −80 to 30 mV after a −120-mV prepulse (voltage protocol shown at bottom). A small, slowly developing inward current preceded small residual outward currents. QX-314 and Cs²⁺ were present in the electrode solution to block Na⁺ and K⁺ channels, respectively.

Fig. 2.

Characterization of EPSC event types. A: spontaneous recording of EPSCs showing a wide range of amplitudes from an isolated calyx. Cell was held at −70 mV in voltage clamp, and events (downward deflections) were identified as simple (black arrowheads) or complex (gray arrowheads). B: example of a simple/monophasic event type with a simple rise time and single exponential decay. C: examples of complex events demonstrating a high degree of variability with staggered rise times, overlapping events, and multiphasic decay. D: averaged monophasic events from a control cell (n = 119, simple events). The sum of 2 single exponential fits to the rise and decay is shown in gray, giving an activation time constant of 0.04 ms and a decay time constant of 0.3 ms. E: decay times for simple events as determined by Mini Analysis. I, current. F: addition of cyclothiazide (CTZ, 40 µM) produced an increase in amplitude and slowing of averaged monophasic events consistent with AMPA receptor involvement. For the calyx shown, averages were constructed from 71 (control, black trace) and 131 (CTZ, gray trace) events. Exponential fits to the rise and decay of averages for control and CTZ are indicated by smooth lines. Decay tau was 0.42 ms in control and 0.75 ms in CTZ.

Fig. 5.

Effect of γ-DGG on EPSCs. A and B: occurrence of simple (A) and complex (B) EPSCs in control cells and after exposure to γ-DGG, a low-affinity AMPA-type glutamate receptor antagonist (n = 8, 6 single and 2 double calyces). There was an overall decrease in simple events and increase in complex events in 2 mM γ-DGG, but the difference was not statistically significant. C: the fraction changed from a majority of simple events to preferentially complex events. Open symbols represent simple events and filled symbols represent complex events for 8 individual cells. D: γ-DGG increased the average amplitude of EPSCs and shifted the cumulative amplitude histogram rightward, changing the median from 27 pA (control shown in black) to 54 pA (γ-DGG shown in gray; n = 8 cells, Kolmogorov-Smirnov test P = 0.001).

Fig. 6.

A comparison of EPSCs in central and peripheral calyces from intact crista slices. A: average of monophasic events from central zone (CZ) calyces (n = 4 cells, 468 events, black trace) and peripheral zone (PZ) calyces (n = 5 cells, 545 events, gray trace) recorded from crista slice preparations. B: EPSC frequency averaged 1.77 ± 0.15 Hz in CZ cells (n = 4) and 0.81 ± 0.15 Hz in PZ cells (n = 5); the difference between zones was statistically significant (*P = 0.003, t-test). C: the decay time for each cell EPSC average was fit with a single exponential, and mean values of 0.40 ± 0.11 for CZ calyces (n = 4) and 0.67 ± 0.16 for PZ calyces (n = 5) were obtained. There was no significant difference in decay tau between zones (P = 0.237, t-test).

Fig. 3.

Effect of Sr²⁺ on EPSCs. A and B: EPSCs in control (A) and in the presence of 8 mM Sr²⁺ (B), where the rate of spontaneous events increased markedly. C: EPSCs in Sr²⁺ were blocked by NBQX (40 µM), confirming that events were mediated by AMPA receptors. D: averages of monophasic events in control (n = 117 events) and Sr²⁺ (n = 603 events) are shown for a single calyx. Exponential fits to the rise and decay phases are shown for control (blue trace, decay tau 0.41 ms) and Sr²⁺ (red trace, decay tau 0.29 ms). E: the increase in frequency of spontaneous events in Sr²⁺ was statistically significant (n = 9, P < 0.001, Mann-Whitney test). F: amplitude histograms for events in control and 8 mM Sr²⁺ for 1 calyx. EPSCs were separated into monophasic (simple) and complex for both groups. The area under the curves (fit with Weibull distribution functions) demonstrates an increase in both frequency and amplitude of EPSCs in Sr²⁺. Complex events (yellow bars, median 231.2 pA) in Sr²⁺ were typically larger than monophasic events (blue bars, median 151.2 pA), and the difference was significant (Kolmogorov-Smirnov test, D = 0.3229, P < 0.001).

Fig. 4.

Effects of calcium channel modulators on EPSCs. A and B: EPSCs in the presence of 8 mM Sr²⁺ (A) followed by the application of nifedipine (20 µM) and 8 mM Sr²⁺ (B), which resulted in a reduction in EPSC frequency. C: frequency of events increased after nifedipine washout. D: frequency of EPSCs in cells bathed in 8 mM Sr²⁺ (control) followed by a combination of Sr²⁺ and 20 µM nifedipine decreased from 3.81 ± 0.20 Hz to 1.09 ± 0.25 Hz (n = 6 cells, t-test, *P < 0.005). E: treatment of cells (control) with the L-type Ca²⁺ channel activator BAY K 8644 (25 µM) resulted in an increase in mean frequency from 1.10 ± 0.84 Hz to 3.16 ± 1.00 Hz (n = 4 cells, t-test, *P < 0.05).