A role for alpha-7 nAChRs at cerebellar synapses

Abstract

The cerebellum performs a well-known motor control function but accumulating evidence supports its role in cognitive functions. The nicotinic cholinergic system alters cognition through its actions elsewhere in the brain but its role in cerebellar processing is not understood. However, expression of a subtype of nicotinic acetylcholine receptors is increased in the cerebellum of human autism patients. Here, we explore the expression, localisation and functional contribution of these receptors at cerebellar synapses.

Longitudinal cerebellar sections (30 μm) were prepared from C57BL/6 and Swiss Webster male mice (28-48 days old). Positive immunoreactivity for α7 nAChR (Abcam) and established excitatory synaptic proteins, Vesicular Glutamate Transporter 1 (VGLUT1; Synaptic Systems) and Post Synaptic Density-95 (PSD-95; Abcam) was visualised using secondary fluorescent antibodies Alexa488 and Alexa594 (Invitrogen) and confocal microscopy. Sagittal cerebellar slices (250 μm thick) (C57BL/6 male mice, 21-31 days old) were prepared in artificial cerebrospinal fluid (aCSF) for whole-cell patch clamp recordings from Purkinje neurons (PNs). We recorded excitatory post-synaptic currents (EPSCs) following parallel-fibre (PF) stimulation in aCSF (containing 50 µM picrotoxin to block GABA-A receptors) before, during and after 15 minutes application of 10 nM methyllycaconitine (MLA; Tocris) a potent α7 nAChR antagonist. Series and input resistances varied by < 10% throughout the recordings.

The α7 nAChRs were abundantly expressed throughout the cerebellar cortex where they overlapped with the PF excitatory pre-synaptic marker protein VGLUT1 (10 ± 1%) and more strongly with the excitatory post-synaptic marker protein PSD-95 (54 ± 3%) (p < 0.0001, n = 3 animals, Mann-Whitney U-test). Overlapping expression of α7 nAChRs was not evident at inhibitory synapses. Electrophysiological recordings revealed that 10 nM MLA reduced EPSC amplitude, compared with controls, by 30 ± 10% (p < 0.05, n = 5, ANOVA).

This study shows for the first time that α7 nAChR expression in the cerebellum contributes to excitatory synaptic transmission at the important PF-PN synapse. Our findings have wider implications for how nicotinic cholinergic inputs influence cerebellar processing.