Efferent Vestibular Neurons Show Homogenous Discharge Output But Heterogeneous Synaptic Input Profile In Vitro

Abstract

Despite the importance of our sense of balance we still know remarkably little about the central control of the peripheral balance system. While previous work has shown that activation of the efferent vestibular system results in modulation of afferent vestibular neuron discharge, the intrinsic and synaptic properties of efferent neurons themselves are largely unknown. Here we substantiate the location of the efferent vestibular nucleus (EVN) in the mouse, before characterizing the input and output properties of EVN neurons in vitro. We made transverse serial sections through the brainstem of 4-week-old mice, and performed immunohistochemistry for calcitonin gene-related peptide (CGRP) and choline acetyltransferase (ChAT), both expressed in the EVN of other species. We also injected fluorogold into the posterior canal and retrogradely labelled neurons in the EVN of ChAT:: tdTomato mice expressing tdTomato in all cholinergic neurons. As expected the EVN lies dorsolateral to the genu of the facial nerve (CNVII). We then made whole-cell current-, and voltage-clamp recordings from visually identified EVN neurons. In current-clamp, EVN neurons display a homogeneous discharge pattern. This is characterized by a high frequency burst of action potentials at the onset of a depolarizing stimulus and the offset of a hyperpolarizing stimulus that is mediated by T-type calcium channels. In voltage-clamp, EVN neurons receive either exclusively excitatory or inhibitory inputs, or a combination of both. Despite this heterogeneous mixture of inputs, we show that synaptic inputs onto EVN neurons are predominantly excitatory. Together these findings suggest that the inputs onto EVN neurons, and more specifically the origin of these inputs may underlie EVN neuron function.

Fig 1. Immunohistochemical staining and retrograde tracing of the EVN.

VN: vestibular nucleus; G7n: genu of seventh cranial nerve (facial nerve); 6n: sixth cranial nerve nucleus (abducens nucleus). (A&C) Immunohistochemical staining for CGRP (n = 7) and ChAT (n = 4) respectively in transversely sectioned mouse brainstem. The EVN is located dorsolateral to genu of seventh nerve (box). Neurons in the abducens and vestibular nucleus were also labelled. (B&D) Higher power visualization of vestibular efferent nucleus. Arrowheads indicate CGRP and ChAT immuno-positive cells respectively. (E&F) Fluorogold injection into the posterior canal of ChAT:: tdTomato mouse strain under low and high power respectively (n = 4). Arrowheads indicate co-localization (yellow) of fluorogold (green) and tdTomato (red), confirming the location of vestibular efferent nucleus.

Fig 2. Discharge profiles of spontaneous and non-spontaneous EVN neurons.

(A) Schematic view of transversely sectioned mouse brainstem. Inset shows map of recording sites from a subset of EVN neurons (37/54 recorded neurons). VN: vestibular nucleus; G7n: genu of seventh cranial nerve (facial nerve); 6n: sixth cranial nerve nucleus (abducens nucleus); 4V: fourth ventricle; EVN: efferent vestibular nucleus. (B) EVN neurons are either spontaneous firing (n = 16) (top trace) or non-spontaneously firing (n = 38) (bottom trace) at resting membrane potential and display homogenous discharge profiles in response to depolarizing (C) and hyperpolarizing (E) step currents. EVN neurons respond with a short burst (*) of high frequency action potentials (AP) at the onset of a depolarizing stimulus or the cessation of a hyperpolarizing stimulus. (D) Comparison of instantaneous frequencies as a function of injected depolarizing current from a subset of MVN and EVN neurons from which the slope of linear fit was used to calculate the gain of each neuron. *** p<0.001. (F) EVN neurons displayed an afterdepolarization (ADP) following release from inhibition (arrow in (E)). The ADP was mediated by T-type calcium channels—TTX (1 μM) abolished all APs, and TTA-P2 (1 μM) abolished the remaining response.

Fig 3. Identification and classification of excitatory and inhibitory profiles in EVN neurons.

(A) Schematic view of transversely sectioned mouse brainstem. Inset shows map of recording sites (22/23 recorded neurons). VN: vestibular nucleus; G7n: genu of seventh cranial nerve (facial nerve); 6n: sixth cranial nerve nucleus (abducens nucleus); 4V: fourth ventricle; EVN: efferent vestibular nucleus. (B) Top trace: EPSCs recorded under normal conditions before the addition of drugs. Second trace: addition of CNQX (10 μM) and TTX (1 μM). Third trace: mIPSCs recorded under normal conditions before the addition of drugs. Bottom trace: addition of strychnine (1 μM) and bicuculline (10 μM) abolished all synaptic activity. Some neurons received excitatory inputs in conjunction with: GABA_AR-mediated events (C) Bottom trace: addition of bicuculline to the bath abolished activity remaining after the addition of TTX and CNQX (second trace); GlyR-mediated events (D) Bottom trace: addition of strychnine abolished remaining activity following the addition of TTX and CNQX (second trace). (E) Other neurons received a combination of mIPSCs in addition to EPSCs. In these neurons, the addition of bicuculline reduced the frequency of synaptic activity (third trace) that was abolished by addition of strychnine (bottom trace). Scale bar in (B) is the same for all traces. (F) Frequencies of EPSCs and mIPSCs per cell calculated over a period of 30 seconds under the influence of excitatory and inhibitory synaptic activity blockers.

Fig 4. EPSC and mIPSC properties.

(A) AMPA/kainate type glutamate receptor, GABA_AR and GlyR-mediated EPSCs and mIPSCs. (B) Averaged GABA_AR- and GlyR- mediated mIPSCs, and AMPA/kainate glutamate receptor mediated EPSC, isolated from the recordings shown above. (C) Bar graphs showing GABA_AR-, GlyR-mediated mIPSCs and AMPA/kainate glutamate receptor mediated EPSC amplitude, decay time, rise time, and width. * p<0.05, ** p<0.01. Values within bars indicate the number of cells sampled. Double diagonal lines indicate that EPSC and mIPSCs values are not compared, but are presented on same bar graph for ease of demonstration.