Muscarinic receptor subtypes differentially control synaptic input and excitability of cerebellum-projecting medial vestibular nucleus neurons

Abstract

Neurons in the vestibular nuclei have a vital function in balance maintenance, gaze stabilization, and posture. Although muscarinic acetylcholine receptors (mAChRs) are expressed and involved in regulating vestibular function, it remains unclear how individual mAChR subtypes regulate vestibular neuronal activity. In this study, we determined which specific subtypes of mAChRs control synaptic input and excitability of medial vestibular nucleus (MVN) neurons that project to the cerebellum. Cerebellum-projecting MVN neurons were labeled by a fluorescent retrograde tracer and then identified in rat brainstem slices. Quantitative PCR analysis suggested that M2 and M3 were the possible major mAChR subtypes expressed in the MVN. The mAChR agonist oxotremorine-M significantly reduced the amplitude of glutamatergic excitatory post-synaptic currents evoked by stimulation of vestibular primary afferents, and this effect was abolished by the M2-preferring antagonist AF-DX 116. However, oxotremorine-M had no effect on GABA-mediated spontaneous inhibitory post-synaptic currents of labeled MVN neurons. Furthermore, oxotremorine-M significantly increased the firing activity of labeled MVN neurons, and this effect was blocked by the M3-preferring antagonist J104129 in most neurons tested. In addition, AF-DX 116 reduced the onset latency and prolonged the excitatory effect of oxotremorine-M on the firing activity of labeled MVN neurons. Our findings suggest that M3 is the predominant post-synaptic mAChR involved in muscarinic excitation of cerebellum-projecting MVN neurons. Pre-synaptic M2 mAChR regulates excitatory glutamatergic input from vestibular primary afferents, which in turn influences the excitability of cerebellum-projecting MVN neurons. This new information has important therapeutic implications for treating vestibular disorders with mAChR subtype-selective agents. Medial vestibular nucleus (MVN) neurons projecting to the cerebellum are involved in balance control. We found that activation of pre-synaptic M2 muscarinic receptors inhibit glutamatergic input from vestibular primary afferents, whereas stimulation of post-synaptic M3 muscarinic receptors increases the firing activity of cerebellum-projecting MVN neurons. This new information advances our understanding of the cholinergic mechanism regulating the vestibular system.

Keywords: acetylcholine; brainstem; glutamate; muscarinic receptors; synaptic transmission; vestibular system.

Fig. 1. Distribution of vestibular afferent nerve fibers and cerebellum-projecting neurons in the MVN and identification of retrogradely labeled cerebellum-projecting MVN neurons in brainstem slices

A: Confocal images show the spatial relationship between primary afferent nerve fibers labeled with IB₄ (green) and MVN neurons labeled with NeuN (red). B: Confocal images show the distribution of primary afferent nerve fibers identified by IB₄ labeling (green) in the MVN region and retrograde tracer-labeled cerebellum-projecting MVN neurons (red). 4V, fourth ventricle. m, medial; d, dorsal. All images are single confocal optical sections. C: FluoSphere-labeled MVN neuron in the brain slice viewed with fluorescence illumination, and photomicrograph of the same labeled neuron (*) with an attached recording electrode (^) in the slice.

Fig. 2. Oxotremorine-M inhibits glutamatergic input from vestibular afferent terminals to cerebellum-projecting MVN neurons

A: Representative recordings show evoked monosynaptic EPSCs during control, application of 0.1 and 2 μM oxotremorine-M (Oxo), and 20 μM DNQX. Note that the stimulating artifact was removed for clarity, and arrows indicate the original location of the stimulating artifact. B: Concentration-dependent effects of oxotremorine-M on the amplitude of evoked EPSCs of 7 labeled MVN neurons. C: Summary data showing the effect of 2 μM oxotremorine-M on the amplitude of EPSCs in 6 labeled MVN neurons recorded with GDP-β-S in internal solution and 5 labeled MVN neurons recorded without GDP-β-S in internal solution. Data are presented as means ± SEM. *P < 0.05, compared with baseline.

Fig. 3. Oxotremorine-M has no effect on GABAergic sIPSCs in cerebellum-projecting MVN neurons

A: Representative traces of a labeled MVN neuron show sIPSCs during application of 2 μM strychnine or 10 μM gabazine. B: Summary data show the effect of strychnine and gabazine on the frequency and amplitude of sIPSCs in 7 labeled MVN neurons. C: Representative traces of a labeled MVN neuron show the effect of 2 μM oxotremorine-M (Oxo) on sIPSCs. D: Group data show the lack of an oxotremorine-M effect on the frequency or amplitude of sIPSCs in 8 labeled MVN neurons. Data are presented as means ± SEM. *P < 0.05, compared with baseline.

Fig. 4. Oxotremorine-M increases the firing activity of cerebellum-projecting MVN neurons

A: Representative traces show the effect of 2 μM oxotremorine-M (Oxo) on discharges of a labeled MVN neuron. B: Summary data show the effect of oxotremorine-M on the firing frequency of 13 labeled MVN neurons. C: Group data show that atropine blocked the stimulatory effect of 2 μM oxotremorine-M on the firing frequency of 5 labeled MVN neurons. Data are presented as means ± SEM. *P < 0.05, compared with baseline.

Fig. 5. Expression levels of mAChR subtypes in the MVN

Quantitative PCR data show the relative mRNA levels of the M1, M3, M4 and M5 subtypes compared with the M2 subtype (n = 6 rats). Data are presented as means ± SEM. *P < 0.05, compared with the M1 subtype.

Fig. 6. Oxotremorine-M-induced inhibition of glutamatergic input to cerebellum-projecting MVN neurons is mediated by the M2 mAChR

A: Representative current traces show the effects of oxotremorine-M (Oxo) and AF-DX 116 on evoked monosynaptic EPSCs of a labeled MVN neuron. Note that the stimulating artifact was removed for clarity, and arrows indicate the original location of the stimulating artifact. B: Summary data show that 2 μM AF-DX 116 abolished the inhibitory effect of oxotremorine-M on the amplitude of evoked EPSCs in 6 labeled MVN neurons. C: Original recordings show the effects of oxotremorine-M (Oxo) on evoked monosynaptic EPSCs of a labeled MVN neuron before and during J104129 application. D: Group data show that J104129 (50 nM) did not alter the inhibitory effect of oxotremorine-M on the amplitude of evoked EPSCs in 5 labeled MVN neurons. Data are presented as means ± SEM. *P < 0.05, compared with baseline.

Fig. 7. Oxotremorine-M increases the firing activity of cerebellum-projecting MVN neurons predominantly via M3 subtype, and blocking the M2 mAChR prolongs the stimulatory effect of oxotremorine-M

A: Summary data show the effects of oxotremorine-M (Oxo) on the firing activity of 11 labeled MVN neurons in the presence of pirenzepine alone or pirenzepine plus J104129. B: Representative current traces show the effect of oxotremorine-M on the firing activity of a labeled MVN neuron in the presence of J104129 (50 nM). C: Group data show that oxotremorine-M failed to increase the firing frequency of labeled MVN neurons in the presence of J104129 in 11 of 14 labeled MVN neurons. In another 3 labeled MVN neurons, oxotremorine-M still increased the firing frequency in the presence of J104129 but failed to increase the firing frequency in the presence of J104129 plus pirenzepine. D: Summary data show that oxotremorine-M failed to increase the firing frequency of 6 labeled MVN neurons in the presence of J104129. E: Time course of the excitatory effect of oxotremorine-M on the firing activity of labeled MVN neurons in the presence of 2 μM AF-DX 116 (n = 10), AF-DX 116 plus 20 μM DNQX (n = 9), or vehicle (n = 12). Data are presented as means ± SEM. *P < 0.05, compared with the respective baseline (time 0); #P < 0.05, compared with the corresponding value in the vehicle group at the same time point.

Fig. 8. Schematic drawing illustrates that the cerebellum-projecting MVN neuron receives synaptic input from glutamatergic vestibular primary afferents, cholinergic neurons, and GABAergic interneurons

The presynaptic M2 mAChR controls glutamatergic input from vestibular primary afferents, whereas the postsynaptic M3 mAChR predominantly excites cerebellum-projecting MVN neurons.