Elsevier

Neuropharmacology

Volume 57, Issue 2, August 2009, Pages 109-120
Neuropharmacology

Ethanol enhances both action potential-dependent and action potential-independent GABAergic transmission onto cerebellar Purkinje cells

https://doi.org/10.1016/j.neuropharm.2009.04.012Get rights and content

Abstract

Ethanol (EtOH) modulates synaptic efficacy in various brain areas, including the cerebellum, which plays a role in motor coordination. Previous studies have shown that EtOH enhances tonic inhibition of cerebellar granule cells, which is one of the possible reasons for the alcohol-induced motor impairment. However, the effects of EtOH on molecular layer interneurons (MLIs) in the mouse cerebellum have remained unknown. Here we found that MLIs were depolarized by EtOH through enhancement of hyperpolarization-activated cationic currents (Ih). Under physiological conditions, a low EtOH concentration (3–50 mM) caused a small increase in the firing rate of MLIs, whereas, in the presence of blockers for ionotropic glutamate and GABA receptors, EtOH (≥10 mM) robustly enhanced MLI firing, suggesting that synaptic inputs, which seem to serve as the phasic inhibition, could suppress the EtOH-mediated excitation of MLIs and Purkinje cells (PCs). Even in the absence of synaptic blockers, a high EtOH concentration (100 mM) markedly increased the firing rate of MLIs to enhance GABAergic transmission. Furthermore, 100 mM EtOH-facilitated miniature IPSCs via a mechanism that depended on intracellular cyclic AMP, voltage-dependent Ca2+ channels, and intracellular Ca2+ stores, but was independent of Ih or PKA. The two distinct effects of a high EtOH concentration (≥100 mM), however, failed to attenuate the EtOH-induced strong depolarization of MLIs. These results suggest that acute exposure to a low EtOH concentration (≤50 mM) enhanced GABAergic synaptic transmission, which suppressed the EtOH-evoked excitation of MLIs and PCs, thereby maintaining precise synaptic integration of PCs.

Introduction

Inhibitory interneurons play a crucial role in the neuronal network of various brain areas because inhibitory synaptic transmission regulates the excitability of postsynaptic neurons through phasic and tonic synaptic signals (Farrant and Nusser, 2005). Therefore, it is necessary to elucidate the mechanisms underlying the regulation of the interneuron firing pattern and release machinery at inhibitory presynaptic terminals. In the cerebellum, molecular layer interneurons (MLIs), basket and stellate cells make GABAergic synapses onto other MLIs and Purkinje cells (PCs) and are able to directly and strongly suppress the activity of the neurons (Llano and Gerschenfeld, 1993, Häusser and Clark, 1997, Kondo and Marty, 1998a). In addition to the activation of ionotropic receptors, such as AMPA receptors, NMDA receptors (Clark and Cull-Candy, 2002) and GABAA receptors (Häusser and Clark, 1997), the activation of G protein-coupled receptors regulates MLI firing by a number of neurotransmitters and neuromodulators, such as glutamate (Karakossian and Otis, 2004), GABA (Than and Szabo, 2002, Harvey and Stephens, 2004), endocannabinoids (Kreitzer et al., 2002), and monoamines (Saitow et al., 2000, Hirono and Obata, 2006). Furthermore, some types of food and drink will modulate the firing pattern of neurons.

Ethanol (EtOH), a psychoactive drug, can widely influence GABAergic transmission in the brain via the facilitation of presynaptic interneuron firing (Siggins et al., 2005, Weiner and Valenzuela, 2006). Previous studies on the rat cerebellum have demonstrated that Golgi cells, which are GABAergic interneurons in the granular layer, show an increase in the firing rate following EtOH exposure, resulting in the enhancement of GABAergic transmission between Golgi cells and granule cells at the glomerulus (Freund et al., 1993, Carta et al., 2004, Hanchar et al., 2005). However, little is known about the effects of EtOH on the spontaneous activity of MLIs.

One of the targets of EtOH action is adenylyl cyclase (AC) (Saito et al., 1985, Hanoune and Defer, 2001), whose activation increases the intracellular cyclic AMP (cAMP) level. Subsequently, cAMP activates a wide array of components, including cyclic nucleotide-gated channels, cAMP-dependent protein kinase (PKA), and other cAMP binding proteins, such as Epac (an exchange protein directly activated by cAMP) (Matulef and Zagotta, 2003, Bos, 2006). To date, nine isoforms of membrane bound mammalian AC (AC1–AC9) have been cloned (Cooper, 2003): cerebellar MLIs express AC7, which is the most sensitive to EtOH (Yoshimura and Tabakoff, 1995, Mons et al., 1998, Yoshimura et al., 2006). Therefore, it is tempting to speculate that EtOH can modulate MLI activity through the cAMP signaling pathway. A recent study using rat cerebellar slices has shown that EtOH increases the frequency of miniature IPSCs (mIPSCs) dose-dependently and causes both an increase and a decrease in the firing rate of PCs (Ming et al., 2006, Kelm et al., 2007). However, the presynaptic effects of EtOH on GABA release from MLIs have not been fully elucidated yet.

Acute EtOH administration causes behavioral disorders dose-dependently, including impairment of motor coordination (Crabbe et al., 2003, Rustay et al., 2003). Our recent rotarod tests demonstrated that at a low blood EtOH concentration (≤50 mM), mice showed normal performance. By contrast, at a higher blood EtOH concentration (∼100 mM), a significant decline in rotarod performance was observed. Therefore, the aim of this study is to explore the dose-dependent effects of EtOH on the intrinsic firing of MLIs in acute slices of the mouse cerebellum using patch-clamp recordings.

Here, we found that MLIs are excited by EtOH through enhancement of hyperpolarization-activated cationic currents (Ih). EtOH increases the intrinsic firing rate of MLIs, resulting in the facilitation of GABAergic transmission onto PCs. Furthermore, 100 mM EtOH induces the facilitation of mIPSCs recorded from PCs, which requires cAMP synthesis, voltage-dependent Ca2+ channel (VDCC) activation, and Ca2+ release from intracellular Ca2+ stores, but not Ih or PKA activation. Therefore, especially at a high concentration (≥100 mM), EtOH likely enhances GABAergic synaptic transmission through the two distinct actions: one is the Ih-dependent increase in the firing rate of presynaptic interneurons, and the other is the cAMP-dependent and PKA-independent enhancement of GABA release at presynaptic terminals. The EtOH-induced facilitation of inhibitory GABAergic synaptic transmission can reduce the EtOH-evoked excitation of MLIs and PCs at a low concentration (≤50 mM), but not at a high concentration (≥100 mM). When the cerebellum is acutely exposed to EtOH at the low concentration, the EtOH-induced enhancement of GABAergic synaptic transmission from MLIs suppresses the EtOH-induced increase in the firing rate of MLIs and PCs to keep normal motor coordination. An abstract on these data has been presented previously (Hirono et al., 2008).

Section snippets

Slice preparation

Cerebellar slices were prepared from C57BL/6 mice (PND 18−25) of either sex as described previously (Hirono et al., 2001). All experimental procedures described here were approved by the RIKEN Animal Research Committee and carried out in accordance with the National Institutes of Health (USA) guidelines. The mice were deeply anesthetized with halothane, and their brains were rapidly removed. Parasagittal slices (230 μm thick) of the cerebellar vermis were cut using a vibrating microtome

Effects of EtOH on firing rates of MLIs

It is possible that EtOH can increase the firing rate of MLIs, as previously reported in Golgi cells, which are interneurons in the granular layer (Carta et al., 2004), and subsequently reduce the spontaneous activity of PCs, as reported for the rat cerebellum (Ming et al., 2006). Therefore, we investigated the effects of EtOH on the spontaneous firing activity of MLIs in the mouse cerebellum by loose cell-attached recording. In the absence of synaptic blockers, MLIs showed spikes at various

Discussion

In the present study, we have shown that EtOH potentiates the spontaneous activity of MLIs and subsequently facilitates inhibitory transmission onto other MLIs and PCs. Under physiological conditions, the facilitation of MLI firing by a low EtOH concentration (≤50 mM) is suppressed by inhibitory synaptic inputs from other MLIs. In the absence of inhibitory synaptic inputs, EtOH increases the firing rate of MLIs dose-dependently. We found that EtOH depolarizes MLIs through the activation of HCN

Acknowledgements

We thank Drs. T. Chimura, S. Konishi, W. Matsunaga, F. Saitow, and H. Suzuki for their invaluable comments and critical reading of this manuscript and Mses S. Hikosaka and N. Kume for their excellent technical support. This work was partially supported by Special Postdoctoral Researchers Program (M.H.) from RIKEN.

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