Anoctamin 2-chloride channels reduce simple spike activity and mediate inhibition at elevated calcium concentration in cerebellar Purkinje cells.

Friederike Auer,Dana Fleischhauer,Stephan Frings,Eliana Franco Taveras,Frank Möhrlen,Uli Klein,Céline Kesenheimer,Steven Barnes

doi:10.1371/journal.pone.0247801

Abstract

Modulation of neuronal excitability is a prominent way of shaping the activity of neuronal networks. Recent studies highlight the role of calcium-activated chloride currents in this context, as they can both increase or decrease excitability. The calcium-activated chloride channel Anoctamin 2 (ANO2 alias TMEM16B) has been described in several regions of the mouse brain, including the olivo-cerebellar system. In inferior olivary neurons, ANO2 was proposed to increase excitability by facilitating the generation of high-threshold calcium spikes. An expression of ANO2 in cerebellar Purkinje cells was suggested, but its role in these neurons remains unclear. In the present study, we confirmed the expression of Ano2 mRNA in Purkinje cells and performed electrophysiological recordings to examine the influence of ANO2-chloride channels on the excitability of Purkinje cells by comparing wildtype mice to mice lacking ANO2. Recordings were performed in acute cerebellar slices of adult mice, which provided the possibility to study the role of ANO2 within the cerebellar cortex. Purkinje cells were uncoupled from climbing fiber input to assess specifically the effect of ANO2 channels on Purkinje cell activity. We identified an attenuating effect of ANO2-mediated chloride currents on the instantaneous simple spike activity both during strong current injections and during current injections close to the simple spike threshold. Moreover, we report a reduction of inhibitory currents from GABAergic interneurons upon depolarization, lasting for several seconds. Together with the role of ANO2-chloride channels in inferior olivary neurons, our data extend the evidence for a role of chloride-dependent modulation in the olivo-cerebellar system that might be important for proper cerebellum-dependent motor coordination and learning.

Highlights

The activity of a neuronal network depends on the excitability of the neurons within this network and on the modulation of this excitability
This raised a controversy about whether Anoctamin 2 (ANO2) is expressed in Purkinje cells and, to examine this question, we performed Quantitative real-time PCR (qPCR) and in situ hybridization experiments to investigate the expression of Ano2-mRNA in cerebellar tissue
The signals for in situ hybridization (Fig 1B, upper) and immunohistochemistry for calbindin, a marker for Purkinje cells (Fig 1B, middle), clearly overlapped (Fig 1B, lower). These data demonstrate that Ano2 is expressed in cerebellar Purkinje cells

Summary

Introduction

The activity of a neuronal network depends on the excitability of the neurons within this network and on the modulation of this excitability. Calcium-activated chloride channels are of special interest in this context, because they can either enhance or attenuate neuronal excitability in various ways (see [1] for recent review). While the enhancement of climbing fiber signals by ANO2 is well established, the effect of ANO2 on Purkinje cell excitability remains controversial. We examine the contribution of ANO2 to the modulation of excitability in Purkinje cells To this end, we analyze the excitability of Purkinje cells in acute tissue slices, uncoupled from climbing fiber input, and compare wildtype to Ano2-/- mice. Combined with the proposed role of ANO2 in climbing fibers [9], we integrate these new findings into the concept of chloride-dependent modulation of network activity in the cerebellar cortex

Materials and methods

Results

Discussion