Activation of GABAB receptors results in excitatory modulation of calyx terminals in rat semicircular canal cristae.

Previous studies have found GABA in vestibular end organs. However, existence of GABA receptors or possible GABAergic effects on vestibular nerve afferents has not been investigated. The current study was conducted to determine whether activation of GABAB receptors affects calyx afferent terminals in the central region of the cristae of semicircular canals. We used patch-clamp recording in postnatal day 13-18 (P13-P18) Sprague-Dawley rats of either sex. Application of GABAB receptor agonist baclofen inhibited voltage-sensitive potassium currents. This effect was blocked by selective GABAB receptor antagonist CGP 35348. Application of antagonists of small (SK)- and large-conductance potassium (BK) channels almost completely blocked the effects of baclofen. The remaining baclofen effect was blocked by cadmium chloride, suggesting that it could be due to inhibition of voltage-gated calcium channels. Furthermore, baclofen had no effect in the absence of calcium in the extracellular fluid. Inhibition of potassium currents by GABAB activation resulted in an excitatory effect on calyx terminal action potential firing. While in the control condition calyces could only fire a single action potential during step depolarizations, in the presence of baclofen they fired continuously during steps and a few even showed repetitive discharge. We also found a decrease in threshold for action potential generation and a decrease in first-spike latency during step depolarization. These results provide the first evidence for the presence of GABAB receptors on calyx terminals, showing that their activation results in an excitatory effect and that GABA inputs could be used to modulate calyx response properties.NEW & NOTEWORTHY Using in vitro whole cell patch-clamp recordings from calyx terminals in the vestibular end organs, we show that activation of GABAB receptors result in an excitatory effect, with decreased spike-frequency adaptation and shortened first-spike latencies. Our results suggest that these effects are mediated through inhibition of calcium-sensitive potassium channels.

Cholinergic Modulation of Membrane Properties of Calyx Terminals in the Vestibular Periphery.

Vestibular nerve afferents are divided into regular and irregular groups based on the variability of interspike intervals in their resting discharge. Most afferents receive inputs from bouton terminals that contact type II hair cells as well as from calyx terminals that cover the basolateral walls of type I hair cells. Calyces have an abundance of different subtypes of KCNQ (Kv7) potassium channels and muscarinic acetylcholine receptors (mAChRs) and receive cholinergic efferent inputs from neurons in the brainstem. We investigated whether mAChRs affected membrane properties and firing patterns of calyx terminals through modulation of KCNQ channel activity. Patch clamp recordings were performed from calyx terminals in central regions of the cristae of the horizontal and anterior canals in 13-26 day old Sprague-Dawley rats. KCNQ mediated currents were observed as voltage sensitive currents with slow kinetics (activation and deactivation), resulting in spike frequency adaptation so that calyces at best fired a single action potential at the beginning of a depolarizing step. Activation of mAChRs by application of oxotremorine methiodide or inhibition of KCNQ channels by linopirdine dihydrochloride decreased voltage activated currents by ∼30%, decreased first spike latencies by ∼40%, resulted in action potential generation in response to smaller current injections and at lower (i.e., more hyperpolarized) membrane potentials, and increased the number of spikes fired during depolarizing steps. Interestingly, some of the calyces showed spontaneous discharge in the presence of these drugs. Together, these findings suggest that cholinergic efferents can modulate the response properties and encoding of head movements by afferents.

A novel intracochlear injection method for rapid drug delivery to vestibular end organs.

BACKGROUND: Injection into the inner ear through the round window (RW) or a cochleostomy is a reliable method for delivering drugs or viruses to the cochlea. This method has been less effective for fast deliveries to vestibular end organs. NEW METHOD: We describe a novel approach for rapid delivery of drugs to the vestibular end organ via the oval window (OW) and scala vestibuli in 1-3 month old C57BL/6 mice. The OW was directly accessed through the external ear canal after ablating the tympanic membrane and middle ear ossicles. A canalostomy in the superior canal provided a low pressure point for faster transit of injected solution from the OW to the vestibular neuroepithelia, allowing for higher rates of injection. RESULTS: The efficacy of this technique was shown by fast transit times of a colored artificial perilymph from the OW to the utricle and the ampullae of the horizontal and superior canals in ∼2 min. Following injection, the response of the vestibular nerve was preserved, as measured by the vestibular sensory evoked potentials (VsEP). COMPARISON WITH EXISTING METHODS: Previous studies have used posterior semicircular canals or the RW with canalostomy to gain access to vestibular end organs in mice. The OW with canalostomy, provides the means for high injection rates and fast and reliable delivery of drugs to vestibular hair cells and afferent terminals. CONCLUSIONS: The presented method for injections through the OW provides rapid delivery of solutions to vestibular end organs without adversely affecting vestibular nerve responses measured by VsEP.