Simultaneous Dual Recordings From Vestibular Hair Cells and Their Calyx Afferents Demonstrate Multiple Modes of Transmission at These Specialized Endings

Abstract

In the vestibular periphery, transmission via conventional synaptic boutons is supplemented by post-synaptic calyceal endings surrounding Type I hair cells. This review focusses on the multiple modes of communication between these receptors and their enveloping calyces as revealed by simultaneous dual-electrode recordings. Classic orthodromic transmission is accompanied by two forms of bidirectional communication enabled by the extensive cleft between the Type I hair cell and its calyx. The slowest cellular communication low-pass filters the transduction current with a time constant of 10-100 ms: potassium ions accumulate in the synaptic cleft, depolarizing both the hair cell and afferent to potentials greater than necessary for rapid vesicle fusion in the receptor and potentially triggering action potentials in the afferent. On the millisecond timescale, conventional glutamatergic quantal transmission occurs when hair cells are depolarized to potentials sufficient for calcium influx and vesicle fusion. Depolarization also permits a third form of transmission that occurs over tens of microseconds, resulting from the large voltage- and ion-sensitive cleft-facing conductances in both the hair cell and the calyx that are open at their resting potentials. Current flowing out of either the hair cell or the afferent divides into the fraction flowing across the cleft into its cellular partner, and the remainder flowing out of the cleft and into the surrounding fluid compartment. These findings suggest multiple biophysical bases for the extensive repertoire of response dynamics seen in the population of primary vestibular afferent fibers. The results further suggest that evolutionary pressures drive selection for the calyx afferent.

Keywords: calyx; ephaptic transmission; hair cell; ion accumulation; quantal transmission; resistive coupling; synaptic transmission; vestibular.

Figure 1

Morphophysiology of the semicircular canal (SCC) epithelium. (A) Cross section through the saddle-shaped crista reveals distinct innervation patterns: boutons are found peripherally, with calyx and dimorphic afferents located centrally [adapted from (18)]. (B) Type I HCs are internal to an enveloping calyx that creates an extensive synaptic cleft in which ions accumulate. Type I HCs have synaptic bodies associated with quantal transmission onto the inner leaflet of the calyx. Type II HCs also have synaptic bodies indicative of quantal transmission, and synapse onto bouton endings (right) or onto the outer leaflet of an afferent calyx (middle and left). (C) A single myelinated afferent (center, red) may branch into several calyces, each containing one or more Type I HCs. Additional input from Type II HCs may occur via synapses onto the outer face of the afferent (left), or through bouton endings of fine collateral branches (right). Efferent fibers (yellow) from the brainstem synapse on HCs and on the outer faces of calyces (19). (D) Top: SEM plan view of the turtle posterior SCC (20). The epithelium is divided into two hemicristae with central and peripheral regions that extend bilaterally from the torus to the planum on either side. Middle: Map of the density of bouton afferents in each hemicrista. Bottom: Density map of the distribution of calyx-bearing afferents in the central regions of each hemicrista (21). (E) Afferent signaling dynamics and discharge statistics in the posterior SCC of chinchilla (yellow symbols) (22) and turtle (blue symbols) (18) are correlated and map systematically across the epithelia. CV*, a measure of the irregularity of the interval between successive APs, is a continuous variable with the most regular fibers on the left and the most irregular on the right. Typically, when either animal was examined with low-frequency, 0.3 or 2 Hz, rotations, low gain afferents sensitive to velocity are more regular, and high gain afferents are sensitive to accelerations. For the chinchilla, the low gain bouton (yellow diamond) and dimorphic (yellow square) afferents demonstrate that regular discharge can be achieved with either bouton or calyx endings. Data seen on the right side, including high gain bouton (turtle: blue, open circle) and dimorphic (turtle: blue, filled circle; chinchilla: yellow square) fibers demonstrate that irregular discharge can be achieved by afferents containing either type of ending. The irregular low gain “calyx only” afferents in chinchilla (yellow filled circle) remain a puzzle but may simply be afferents that respond maximally to stimulations at higher frequencies (23) or with vibration (24). CV* for chinchilla data transformed from 15 ms inter-spike interval (ISI) CV* to that at 50 ms ISI using a third-order polynomial fit to the CV* vs. ISI data at 50 ms in Figure 1 of (22), equivalent to the CV* and ISI used in the turtle data (18).

Figure 2

Three forms of synaptic transmission between a Type I HC and calyx afferent. (A) In the absence of mechanical stimulation (upper panel), basolateral potassium currents activated at hyperpolarized potentials elevate the [K⁺]_cleft to concentrations near 8 mM, twice that found in perilymph. This results in a shift in E_K and depolarization of the HC to a resting potential between −60 and −50 mV, where Ca_{1, 3} channels are activated. At rest there is occasional vesicle fusion and the production of a background rate of afferent discharge. Upon mechanical stimulation (lower panel), the inward transduction current flows into the HC and out the basolateral conductances into the cleft as a potassium current. The further increase in [K⁺]_cleft is a low-pass integral of the transduction current and further depolarizes E_K for conductances in the HC and afferent membranes facing the cleft. The HC depolarization gates Ca²⁺ influx, resulting in fusion of synaptic vesicles and quantal transmission of glutamate to AMPARs on the calyx inner leaflet. The slow accumulation of potassium thereby facilitates quantal transmission near the resting potential, and for maintained depolarizations toward 0 mV, elevates [K⁺]_cleft to greater than 28 mM within 25 ms. It is likely that the irregularities of vesicle fusion and large quanta that initiate an AP are driven by neighboring Ca-channel noise (108), and as a consequence the degree of regularity of afferent discharge would be determined by the degree to which the post-synaptic afferent conductances have intrinsic memory and oscillatory behavior similar to those found in HCs of the turtle auditory papilla (90, 109). (B) Large conductances in the HC and calyx near the resting potential also create an open resistive pathway between the two that permits a fraction of the transduction current to be communicated across the cleft and into the calyx without synaptic delay. Outward current from the HC (yellow arrow) divides at point (a) into a large component (gray arrow) going along the cleft into the bath at point (b), and a smaller current flowing into the calyx (green arrow). The measured current for the HC (bluish-green current trace) and calyx (vermillion trace) show transmission within 10 μs. (C) Resistive coupling can also be demonstrated during large HC depolarizations that depolarize the calyx to potentials sufficient to generate an AP that travels into the calyx. Currents due to the invasion of an AP will sum with the primary currents illustrated in (B). With an AP, an additional inward current (orange arrow) flows into the calyx, with an amplitude equal to the sum of the corresponding currents flowing into the HC (black arrow) and down the cleft (white arrow). The coupling between the calyx retrograde AP current (vermillion) and the induced HC current (bluish-green) is 12 μs [modified from (63)].