The quantal component of synaptic transmission from sensory hair cells to the vestibular calyx

Abstract

Spontaneous and stimulus-evoked excitatory postsynaptic currents (EPSCs) were recorded in calyx nerve terminals from the turtle vestibular lagena to quantify key attributes of quantal transmission at this synapse. On average, EPSC events had a magnitude of ∼ 42 pA, a rise time constant of τ(0) ∼ 229 μs, decayed to baseline with a time constant of τ(R) ∼ 690 μs, and carried ∼ 46 fC of charge. Individual EPSCs varied in magnitude and decay time constant. Variability in the EPSC decay time constant was hair cell dependent and due in part to a slow protraction of the EPSC in some cases. Variability in EPSC size was well described by an integer summation of unitary quanta, with each quanta of glutamate gating a unitary postsynaptic current of ∼ 23 pA. The unitary charge was ∼ 26 fC for EPSCs with a simple exponential decay and increased to ∼ 48 fC for EPSCs exhibiting a slow protraction. The EPSC magnitude and the number of simultaneous unitary quanta within each event increased with presynaptic stimulus intensity. During tonic hair cell depolarization, both the EPSC magnitude and event rate exhibited adaptive run down over time. Present data from a reptilian calyx are remarkably similar to noncalyceal vestibular synaptic terminals in diverse species, indicating that the skewed EPSC size distribution and multiquantal release might be an ancestral property of inner ear ribbon synapses.

Keywords: auditory; synapse; unitary quanta; vestibular.

Fig. 1.

Voltage-clamp recordings from vestibular calyx terminals. A: asynchronous and evoked excitatory postsynaptic currents (EPSCs) were recorded using ∼6 MΩ patch electrodes (e) attached to the lateral membrane of calyx terminals (gray). Type 1 hair bundles were identified visually and a fluid jet was used to deflect the bundle in the direction of the kinocilium. B: subset of afferents branched to form synaptic contacts with remote hair cells. C: EPSC events exhibited a wide range of amplitudes (asynchronous example shown). D: individual EPSCs were fit with an exponential rise-fall function to determine the amplitude (pA), decay time constant (μs), and total charge (fC). E: EPSCs evoked by deflection of a single hair bundle decayed with the same time constant and could be described a superposition of simultaneous unitary quantal events, e.g., 1, 2, and 8 simultaneous quantal events (black). F: capacitance measured by the patch pipette correlated with number (N) of hair cells enveloped by the patched calyx (C = 12.4 + 11.5 × N pF ± 3.2; n = 15 calyces). Presence of a collateral calyx did not consistently increase the measured capacitance. G: 2-dimensional (2D) bin histogram showing there was very little correlation between asynchronous mEPSC size and time constant (n = 8 calyces; k = 770 events; black dots show the mean time constant at for all EPSCs within each size bin). SC, space clamp.

Fig. 2.

Spontaneous vs. evoked EPSC kinetics. A and B: probability histograms showing amplitudes (pA) and decay time constants (ms) for spontaneous EPSCs (black) and hair bundle deflection evoked EPSCs (red) recorded from a single calyx under whole cell voltage clamp. Average EPSCs are shown in the insets. C and D: stimulus evoked EPSCs recorded from a second calyx during deflection of 3 different hair bundles. EPSC size distributions in this calyx were relatively uniform regardless of which particular hair bundle was deflected, but decay time constants differed substantially. Bundle 1 stimulated the hair cell enveloped by the calyx at the recording site, while bundles 2 and 3 were located ∼30–40 μm from the recording site and presumably stimulated hair cells contacted via a collateral.

Fig. 3.

Diversity of EPSC kinetics. A and B: probability histograms showing the amplitudes (pA) and decay time constants (ms) of spontaneous mEPSCs (black), EPSCs evoked by deflection of hair bundle 1 (red), and EPSCs evoked by hair bundle 2 (green) recorded in a single calyx. Inset: average EPSCs. In this calyx, there were significant differences in size and speed depending upon the specific hair bundle stimulated. The bimodal distribution arises because the population of events includes EPSCs from hair cells 1 and 2. C and D: raw records comparing evoked EPSCs (C) to spontaneous mEPSCs (D). Red curves overlaying the raw data (black) illustrate a slow protracted tail in spontaneous mEPSCs that was absent (or overwhelmed) in bundle 1 evoked EPSCs. The presence or absence of this tail is the primary difference between EPSCs with slow vs. fast kinetics.

Fig. 4.

EPSC rate adapts during tonic hair bundle displacements. Example recordings from a single calyx. A: hair bundle deflection increased the rate of EPSCs from zero to >1,000 s⁻¹ followed by a period of adaptation back to zero. Dots denote the instantaneous EPSC rate and the solid cityscape line (black) is an estimate of unitary quantal release rate reported in 20-ms bins. B: raw voltage-clamp data reveal adaptation of EPSC rate and amplitude with time. C: EPSC time constants did not change with amplitude, suggesting larger events at the onset of the stimulus arise from a higher probability of simultaneous quanta. D: data were well described by an integer convolution of unitary events combined with a model of transmitter clearance to capture the slow recovery to baseline. E: simulated EPSCs with (solid red) and without buildup (dashed) illustrating the slow tail that hypothetically could arise from glutamate buildup in the cleft. F: cumulative charge (thick red dashed) and cumulative quantal events (black) imply a unitary quantal size of ∼53 pC, including the slow tail. Neglecting the tail underpredicts the offset and charge (thin red dashed, U). Simultaneous release causes the cumulative number of quantal events (black) to exceed the number of EPSC events (blue).

Fig. 5.

Quantal EPSCs and nonquantal (nq)EPSCs can be evoked simultaneously. Same notation as Fig. 4 but with longer time scale and recording from a different calyx. A: adaptation of EPSC instantaneous rate and unitary quantal rate. B: raw current trace showing superposition of EPSCs and nqEPSC. C: expanded section showing a modest EPSC tail and tonic nqEPSC offset. D: cumulative quantal charge (subtracting nqEPSC) provides an estimate of ∼44 fC per unitary event in this cell.