Ruth Anne Eatock

Vestibular Hair Cells and Afferents: Two Channels for Head Motion Signals

Vestibular epithelia of the inner ear detect head motions over a wide range of amplitudes and frequencies. In mammals, afferent nerve fibers from central and peripheral zones of vestibular epithelia form distinct populations with different response dynamics and spike timing. Central-zone afferents are large, fast conduits for phasic signals encoded in irregular spike trains. The finer afferents from peripheral zones conduct more slowly and encode more tonic, linear signals in highly regular spike trains. The hair cells are also of two types, I and II, but the two types do not correspond directly to the two afferent populations. Zonal differences in afferent response dynamics may arise at multiple stages, including mechanoelectrical transduction, voltage-gated channels in hair cells and afferents, afferent transmission at calyceal and bouton synapses, and spike generation in regular and irregular afferents. In contrast, zonal differences in spike timing may depend more simply on the selective expression of low-voltage-activated ion channels by irregular afferents.

M-Like K+ Currents in Type I Hair Cells and Calyx Afferent Endings of the Developing Rat Utricle

Type I vestibular hair cells have large K+ currents that, like neuronal M currents, activate negative to resting potential and are modulatable. In rodents, these currents are acquired postnatally. In perforated-patch recordings from rat utricular hair cells, immature hair cells [younger than postnatal day 7 (P7)] had a steady-state K+ conductance (g−30) with a half-activation voltage (V1/2) of −30 mV. The size and activation range did not change in maturing type II cells, but, by P16, type I cells had added a K conductance that was on average fourfold larger and activated much more negatively. This conductance may comprise two components: g−60 (V1/2 of −60 mV) and g−80 (V1/2 of −80 mV). g−80 washed out during ruptured patch recordings and was blocked by a protein kinase inhibitor. M currents can include contributions from KCNQ and ether-a-go-go-related (erg) channels. KCNQ and erg channel blockers both affected the K+ currents of type I cells, with KCNQ blockers being more potent at younger than P7 and erg blockers more potent at older than P16. Single-cell reverse transcription-PCR and immunocytochemistry showed expression of KCNQ and erg subunits. We propose that KCNQ channels contribute to g−30 and g−60 and erg subunits contribute to g−80. Type I hair cells are contacted by calyceal afferent endings. Recordings from dissociated calyces and afferent endings revealed large K+ conductances, including a KCNQ conductance. Calyx endings were strongly labeled by KCNQ4 and erg1 antisera. Thus, both hair cells and calyx endings have large M-like K+ conductances with the potential to control the gain of transmission.

Molecular Microdomains in a Sensory Terminal, the Vestibular Calyx Ending

Many primary vestibular afferents form large cup-shaped postsynaptic terminals (calyces) that envelope the basolateral surfaces of type I hair cells. The calyceal terminals both respond to glutamate released from ribbon synapses in the type I cells and initiate spikes that propagate to the afferents central terminals in the brainstem. The combination of synaptic and spike initiation functions in these unique sensory endings distinguishes them from the axonal nodes of central neurons and peripheral nerves, such as the sciatic nerve, which have provided most of our information about nodal specializations. We show that rat vestibular calyces express an unusual mix of voltage-gated Na and K channels and scaffolding, cell adhesion, and extracellular matrix proteins, which may hold the ion channels in place. Protein expression patterns form several microdomains within the calyx membrane: a synaptic domain facing the hair cell, the heminode abutting the first myelinated internode, and one or two intermediate domains. Differences in the expression and localization of proteins between afferent types and zones may contribute to known variations in afferent physiology.

Functional Development of Hair Cells

Abstract Embryonic hair cells in chicks and mammals have functional transduction channels and voltage-gated outwardly rectifying potassium (K + ) channels, fast inwardly rectifying channels, and voltage-gated sodium (Na + ) and calcium (Ca 2+ ) channels. Together these channels may participate in spiking by the immature hair cells, which may drive rhythmic or bursting activity of neurons at higher levels of the auditory pathway. The electrical activity of immature hair cells may influence afferent synaptogenesis and differentiation indirectly by promoting neurotrophin release or more directly by glutamatergic transmission. With maturation, a number of changes tend to reduce hair cell spiking: Na + , Ca 2+ , and fast inwardly rectifying channels may become less numerous, whereas outwardly rectifying K + channels become more numerous and diverse. These changes signal the transformation from a developing epithelium with active formation of synaptic contacts to a sensing epithelium where receptor potentials represent the mechanical input in a graded fashion. The composition of the late-arriving outwardly rectifying K + channels is specific to the hair cells type and location in the sensory epithelium and confers specialized properties on the receptor potentials. Fast, Ca 2+ -gated channels serve high-quality electrical tuning in certain tall hair cells of the chick cochlea. In rodent cochlear hair cells and type I hair cells of chick and rodent vestibular organs, large outwardly rectifying conductances lower the input resistance, enhancing the speed and linearity of voltage responses.

Tuning and Timing in Mammalian Type I Hair Cells and Calyceal Synapses

Afferent nerve fibers in the central zones of vestibular epithelia form calyceal endings around type I hair cells and have phasic response properties that emphasize fast head motions. We investigated how stages from hair-cell transduction to calyceal spiking contribute tuning and timing to central (striolar)-zone afferents of the rat saccular epithelium. In an excised preparation, we deflected individual hair bundles with rigid probes driven with steps and sinusoids (0.5–500 Hz) and recorded whole-cell responses from hair cells and calyces at room temperature and body temperature. In immature hair cells and calyces (postnatal days (P)1–P4), tuning sharpened at each stage. Transducer adaptation and membrane-charging time produced bandpass filtering of the receptor potential with best frequencies of 10–30 Hz and phase leads below 10 Hz. For small stimuli, electrical resonances sharply tuned the hair-cell membrane in the frequency range of 5–40 Hz. The synaptic delay of quantal transmission added a phase lag at frequencies above 10 Hz. The influence of spike thresholds at the calyceal spike initiation stage sharpened tuning and advanced response phase. Two additional mechanisms strongly advanced response phase above 10 Hz when present: (1) maturing (P7–P9) type I hair cells acquired low-voltage-activated channels that shortened the rise time of the receptor potential and (2) some calyces had nonquantal transmission with little synaptic delay. By reducing response time, the identified inner-ear mechanisms (transducer adaptation, low-voltage-activated channels, nonquantal transmission, and spike triggering) may compensate for transmission delays in vestibular reflex pathways and help stabilize posture and gaze during rapid head motions.

Hair cells in mammalian utricles

Two morphological classes of mechanosensory cells have been described in the vestibular organs of mammals, birds, and reptiles: type I and type II hair cells. Type II hair cells resemble hair cells in other organs in that they receive bouton terminals from primary afferent neurons. In contrast, type I hair cells are enveloped by large cuplike afferent terminals called calyces. Type I and II cells differ in other morphological respects: cell shape, hair bundle properties, and more subtle ultrastructural features. Understanding the functional significance of these strikingly different morphological features has proved to be a challenge. Experiments that correlated the response properties of primary vestibular afferents with the morphologies of their afferent terminals suggested that the synapse between the type I hair cell and calyx ending is lower gain than that between a type II hair cell and a bouton ending. Recently, patch-clamp experiments on isolated hair cells have revealed that type I hair cells from diverse species have a large potassium conductance that is activated at the resting potential. As a consequence, the voltage responses generated by the type I hair cells in response to injected currents are smaller than those generated by type II hair cells. This may contribute to the lower gain of type I inputs to primary afferent neurons. Studies of neonatal mouse utricles show that the type I-specific potassium conductance is not present at birth but emerges during the first postnatal week, a period of morphological differentiation of type I and type II hair cells.

Mechanoelectrical and voltage-gated ion channels in mammalian vestibular hair cells

Mammalian vestibular afferents respond robustly to head movements at low frequencies and provide input to reflexes that control eye, head and body position. Vestibular organs have distinctive regions and hair cells: Type II cells receive bouton afferent endings and type I cells receive large calyx afferent endings. In the rodent utricle, type II cells are broadly tuned to frequencies between 10 and 30 Hz. Other recent data suggest that otolith organs function in this frequency range, which is higher than previously imagined. Some of the tuning derives from adaptation of the transducer current, which is best fitted with a double exponential decay with time constants of ∼4 and 40 ms. Further tuning is provided by basolateral conductances, principally outwardly rectifying, voltage-gated K+ conductances. The kinetics of the K+ currents tend to vary with location in the sensory epithelium and therefore may contribute to regional variation in afferent physiology. Type I hair cells have a large, negatively activating K+ conductance, gK,L, that confers a very low input resistance and therefore attenuates the receptor potential. This may reduce nonlinearity in the receptor potential, a possibly useful feature for the motor reflexes served by the vestibular system. On the other hand, the small receptor potentials together with unusually negative resting potentials are hard to reconcile with calcium-mediated quantal transmission. This problem may be overcome by factors that inhibit gK,L’s activation at resting potential. Also, the calyx may support nonquantal transmission.

Dopaminergic signaling in the cochlea: receptor expression patterns and deletion phenotypes.

Pharmacological studies suggest that dopamine release from lateral olivocochlear efferent neurons suppresses spontaneous and sound-evoked activity in cochlear nerve fibers and helps control noise-induced excitotoxicity; however, the literature on cochlear expression and localization of dopamine receptors is contradictory. To better characterize cochlear dopaminergic signaling, we studied receptor localization using immunohistochemistry or reverse transcriptase PCR and assessed histopathology, cochlear responses and olivocochlear function in mice with targeted deletion of each of the five receptor subtypes. In normal ears, D1, D2, and D5 receptors were detected in microdissected immature (postnatal days 10–13) spiral ganglion cells and outer hair cells but not inner hair cells. D4 was detected in spiral ganglion cells only. In whole cochlea samples from adults, transcripts for D1, D2, D4, and D5 were present, whereas D3 mRNA was never detected. D1 and D2 immunolabeling was localized to cochlear nerve fibers, near the first nodes of Ranvier (D2) and in the inner spiral bundle region (D1 and D2) where presynaptic olivocochlear terminals are found. No other receptor labeling was consistent. Cochlear function was normal in D3, D4, and D5 knock-outs. D1 and D2 knock-outs showed slight, but significant enhancement and suppression, respectively, of cochlear responses, both in the neural output [auditory brainstem response (ABR) wave 1] and in outer hair cell function [distortion product otoacoustic emissions (DPOAEs)]. Vulnerability to acoustic injury was significantly increased in D2, D4 and D5 lines: D1 could not be tested, and no differences were seen in D3 mutants, consistent with a lack of receptor expression. The increased vulnerability in D2 knock-outs was seen in DPOAEs, suggesting a role for dopamine in the outer hair cell area. In D4 and D5 knock-outs, the increased noise vulnerability was seen only in ABRs, consistent with a role for dopaminergic signaling in minimizing neural damage.

Stimulus Processing by Type II Hair Cells in the Mouse Utricle

Abstract: In type II and neonatal hair cells in the mouse utricle, the receptor potentials evoked by low‐frequency sinusoidal deflections of the hair bundle are attenuated by adaptation of the mechanoelectrical transduction current and the voltage‐dependent activation of a large potassium (K)‐selective outwardly rectifying conductance, gDR. These processes may contribute to high‐pass filtering of the responses of some utricular afferents to sinusoidal linear accelerations below 2 Hz. Depolarizing receptor potentials are more attenuated by gDR than are hyperpolarizing receptor potentials. It may therefore reduce nonlinear distortion introduced by mechanoelectrical transduction, which generates larger depolarizing currents than hyperpolarizing currents.

Differences Between the Negatively Activating Potassium Conductances of Mammalian Cochlear and Vestibular Hair Cells

Cochlear and type I vestibular hair cells of mammals express negatively activating potassium (K+) conductances, called gK,n and gK,L respectively, which are important in setting the hair cells’ resting potentials and input conductances. It has been suggested that the channels underlying both conductances include KCNQ4 subunits from the KCNQ family of K+ channels. In whole-cell recordings from rat hair cells, we found substantial differences between gK,n and gK,L in voltage dependence, kinetics, ionic permeability, and stability during whole-cell recording. Relative to gK,L, gK,n had a significantly broader and more negative voltage range of activation and activated with less delay and faster principal time constants over the negative part of the activation range. Deactivation of gK,n had an unusual sigmoidal time course, while gK,L deactivated with a double-exponential decay. gK,L, but not gK,n, had appreciable permeability to Cs+. Unlike gK,L, gK,n’s properties did not change (“wash out”) during the replacement of cytoplasmic solution with pipette solution during ruptured-patch recordings. These differences in the functional expression of gK,n and gK,L channels suggest that there are substantial differences in their molecular structure as well.