Close Packing of Cells in Vestibular Epithelia Supports Local Electrical Potentials that Reduce Latency of Action Potential Generation

Abstract

In the vestibular system, upon transduction of head motion, ionic currents from type I sensory hair cells alter [K⁺] and electrical potentials in an extended synaptic cleft formed by a calyx terminal of the associated afferent neuron. During excitatory stimuli, these changes in turn modulate post-synaptic currents across the calyx inner face to depolarize the afferent and initiate action potentials. Within the tightly packed columnar vestibular sensory epithelium, electrical currents from the hair cell and calyx must also traverse non-synaptic extracellular spaces and generate local extracellular potentials before dispersing into the perilymph beneath the basement membrane. Here we show that such dynamic electrical potentials enhance action potential generation by reducing outward K⁺ currents on both the inner and outer faces of the calyx. This effect also influences adjacent calyces and may explain the abundance of calyx terminals in amniotes where there is a need for rapid recognition of changes in head orientation and acceleration.

Introduction.

In amniote vestibular sensory epithelia, up to 74% of hair cells can be type-I (1), and upon transduction of head motion, transmit to a cup-like (calyx) terminal of an afferent neuron. Transmission to the afferent neuron can occur without neurotransmitter release (non-quantal transmission, NQT) (2). During NQT the modulation of potassium ion concentration ‘[K⁺]’ and electrical potential ‘ϕ’ in the narrow (3) synaptic cleft alter ionic currents on the post-synaptic terminal of the afferent neuron (4, 5). This process is facilitated by voltage-activated K⁺ channels present on the type-I hair cell, calyx terminal and afferent fiber (6–10) that typically mediate an efflux of K⁺ ions. K⁺ currents primarily flow out of the hair cell through a low voltage-activated potassium conductance comprised of K_V1.8 subunits (11) and into the cleft through K_V7 channels on the inner face of the calyx (3, 6, 7). Until now, the fast encoding of head motion by vestibular primary afferent neurons has mostly been attributed to processes and morphological features upstream from the afferent: variations in hair bundle and otolith morphology (12), rapid transmission across the synaptic cleft due to large hair cell currents (13), and the effect of synaptic K⁺ accumulation (10) on currents across the calyx inner face (CIF). However, while synaptic transmission can initiate afferent depolarization, AP generation is a function of ion channels on the afferent neuron. Calyx afferents can exhibit spontaneous firing independent of synaptic transmission, and AP firing rates change when K_V1 and K_V7 channels are blocked (8, 9, 14). Efferent activity also mediates slow excitation of the afferent neuron by closing K_V7 channels on the calyx outer face (COF) (15). Cells within intact vestibular epithelia are densely packed with synaptic and non-synaptic extracellular spaces (ECS) that are continuous and of comparable width and K⁺ channels are present on the COF and at hemi-nodes (7, 16). This raises the possibility that dynamic changes in electrical potential and [K⁺] occur not only in the synaptic cleft, but throughout the ECS of the epithelium. We hypothesized that when one or more type I hair cells experience bundle deflection: 1) currents flow into ECS of the epithelium surrounding the COF to modulate local extracellular potentials and are in turn affected by them; 2) such extracellular potentials enhance AP recruitment in afferent calyces.

Results.

Based on our previous work (4), we developed a 3D finite element model to compare the in vitro condition where an isolated vestibular hair cell-calyx and the associated afferent are in a perilymph bath (VHCC, Fig 1 A1) with the semi-intact or in vivo situations where the synaptic cleft is continuous with an extensive network of ECS that lead to perilymph past basement membrane (VHCC-E, Fig 1 A2). The VHCC-E model thus represents a more realistic pathway for ion flow and allows for modulation of channels located on the COF. For a 1 μm excitatory hair bundle deflection, AP latency is reduced when simulated within the epithelium (Fig 1B). To distinguish whether electrical potential or K⁺ accumulation in the ECS were primarily responsible for the latency reduction, we added potassium chloride cotransporter (KCC) activity to the COF and supporting cell membranes - in addition to its location on the CIF in the VHCC model (4). Our epithelial model contained five adjacent hair cell-calyx complexes, as can occur in-vivo (especially in the striolar/central regions) (Fig 2A). When only the hair cell in the center is stimulated (Figs. 1B and 2A), a significant reduction in AP latency remained (Fig 1B) even when extracellular [K⁺] was reduced (Fig 2A, top row). Importantly, the extracellular electrical potential surrounding calyceal terminals was still significant (Fig 2A, bottom row). Extracellular potentials were greater in magnitude along the calyx membrane when within the epithelium (VHCC-E, Fig. 2B). We subsequently investigated simultaneous bundle deflection of all five hair cells with greater KCC activity in the epithelia. The type-I hair cells are labelled based on their distance to the cell in the center (C), as the 1^st (1N) and 2^nd nearest neighbors (2N) (inset, Fig 3A1). Excitatory deflection of the central hair bundle caused an AP to fire in the associated afferent and created small nonquantal EPSPs in neighboring afferents (Fig 3A1, A2). When all (C, 1N, 2N) hair bundles were deflected together, by either 1 μm or 0.4 μm, the AP latency in the “C” afferent was further reduced by 1.2 ms (Fig 3B1) or 5.2 ms (Fig 3B2) respectively.

Fig 1. AP latency is reduced when cells are within the epithelium.

A1 A large perilymph bath ([K⁺] = 5 mM and 0 mV) surrounds the entire hair cell and calyx and the perilymph boundary condition is applied at the lip of the calyx where the synaptic cleft would meet perilymph (pink curve); as in (Govindaraju et al. 2023), perilymph bathes the COF. A2 In the epithelial model (VHCC-E) the perilymph boundary (pink curve) is at the basement membrane (BM) and electro-diffusion is calculated in both synaptic (between hair cell and calyx) and non-synaptic extracellular spaces between supporting cell (SC), the hair cell and the calyx. Within the epithelium, surfaces represent cell membranes and the adjoining extracellular space (ECS); examples are marked with dashed lines. B APs generated under VHCC and VHCC-E conditions are shown. The VHCC AP peak occurred 12.3 ms after stimulus onset (blue curve) and was greater than that seen experimentally (black curve, adapted from Songer and Eatock 2013, Figure 5A, 4.7 ms) in a semi-intact preparation. When dynamic changes in extracellular electrical potential and ion concentration are considered in the VHCC-E model, the AP peak (orange curve) occurred at 8 ms, a latency of 3 ms. When large amounts of a potassium chloride cotransporter (KCC) were situated on supporting cell surfaces (VHCC-E; KCC on SC – yellow curve) to minimize changes in extracellular [K⁺], the AP Peak occurred at 9 ms, a latency of 4 ms.

Fig 2. Extracellular electrical potential rises significantly during APs.

A. The 3D spatial gradients in extracellular potassium ion concentration [K⁺] (top row) and electrical potential ϕ (bottom row) are shown at rest and at the AP peaks (black arrowheads) seen in Fig 1B where only the central hair cell is stimulated for the VHCC-E condition (2^nd column) and with KCC on SC (3^rd column). The outer surface (COF) of calyces in the foreground have been partially removed to show the synaptic cleft. B. The spatial profile of extracellular potential along the calyx from the base of the inner face (CIF) to the basement membrane (BM) is shown when only the central hair cell is stimulated. In the epithelial model (red lines, VHCC-E), the magnitude of the extracellular potential is increased both at rest (thin lines) and during the action potential (thick lines) with respect to a calyx in bath (blue lines, VHCC).

Fig. 3. Collective hair bundle deflection reduces AP latency.

When only the hair bundle of the center hair cell is displaced (A1, A2), the rise in extracellular potential surrounding the associated calyx, over the course of the AP (blue curve), caused small depolarizations in the 1st (1N) and 2nd (2N) nearest neighbors (yellow and orange curves). Inset: Top-down view of the arrangement of hair cells. When the hair bundles of all hair cells are displaced together, APs occur in all calyces (blue, yellow and orange curves) (B1, B2). The maximal reduction in AP latency is experienced by the calyx in the center. The improvement in AP latency due to concomitant depolarization of adjacent calyces is greatest for the smaller, 0.4 μm, displacement (1.2 ms vs 5.2 ms, arrows, B1 vs B2) although the larger displacement results in an AP that peaks at an earlier time (8 ms vs 10 ms, blue curve, B1 vs B2).

Discussion.

In the VHCC-E model, following excitatory bundle deflection, the afferent AP occurred with reduced latency (Fig 1B) and was closer to in-vitro experimental data (2) than when surrounded by a perilymph bath. The reduction in AP latency can be attributed, primarily, to greater changes in extracellular electrical potential during afferent depolarization (Fig 2) which alter the driving forces of currents across the CIF and COF. The voltage and ligand-gated K_V7 channels (17) found on the CIF and COF are non-inactivating, depolarization-activated, and modulated by efferent activity. Increases in extracellular electrical potential reduce K⁺ efflux through these channels and notably reduce AP latency for smaller hair bundle displacements applied concomitantly to multiple adjacent hair bundles (Fig 3) – as would occur in-vivo during head motion. In-vivo extracellular recordings, in response to applied head motion stimuli, show shorter time to spike and encoding of higher frequency stimuli than seen in-vitro in recordings from an individual calyx terminal where only the hair bundle of the associated type-I cell is displaced (2, 13, 18). We posit that transient local potentials in the ECS act in concert with hair cell, calyceal and hemi-nodal currents to enhance transmission at the synapse and reduce the decay of the afferent graded potential. This appears to be a way to lower AP latency without significant disruption to subsequent repolarization and may explain differences observed between in-vivo and in-vitro recordings (in addition to other factors such as temperature, age, species and presence of endolymph). The model also rationalizes observations that calyx bearing afferents were sensitive to applied galvanic stimulation (extracellular current injection) (19). Our results suggest this sensitivity reflects a natural ability of calyceal afferents to create and interact with extracellular potentials, within the intact epithelium, to support faster AP generation during head motion.

Methods.

Simulations were performed in COMSOL 6.3. Governing equations were used as in our previous publication (4) with the electro-diffusion equation now applied to the entire extracellular space as opposed to just the synaptic cleft. Other modifications are detailed in the Supporting Information (SI).