There is an inhibitory GABAergic input to cerebellar Purkinje neurons (Satake et al., 2004
) and previous work has demonstrated that ethanol can enhance the release of GABA from presynaptic terminals (Ariwodola and Weiner, 2004
; Crowder et al., 2002
; Roberto et al., 2003
; Ziskind-Conhaim et al., 2003
). Because GABA release could be contributing to the inhibitory action of ethanol on firing rate for some, but not all, neurons (Freund et al., 1993
; Yang et al., 2000
), we examined the action of ethanol on GABA release on this cell type. Ethanol produced a concentration-dependent increase in the frequency of bicuculline-sensitive mIPSCs. These data indicate that the ethanol was increasing the presynaptic release of GABA. Ethanol had no effect on the amplitude or decay time of the mIPSCs, indicating that the sensitivity of the postsynaptic receptor to GABA was not changed. This is consistent with the majority of recent reports (see reviews by Breese et al., 2006
; Criswell and Breese, 2005
; and Siggins et al., 2005
). However, Sanna et al. (2004)
reported increased decay time as well as amplitude of mIPSCs from hippocampal neurons following ethanol. Altered intracellular Cl−
levels or other variables altered following dialysis of intracellular contents by the recording pipettes could have interfered with the effect of ethanol on postsynaptic GABAA
receptors. For example, the Cl−
transporters are likely overwhelmed. However, when these interfering variables were removed by the use of extracellular recording, there was still no consistent enhancement of the effect of GABA on rate by ethanol. While the present study did not find a direct action of ethanol on the postsynaptic GABAA
receptor, other studies have suggested that ethanol might act at a specific site on the GABAA
receptor to enhance the effectiveness of GABA (Ueno et al., 2000
), but these actions may require higher concentrations of ethanol. For example Peoples and Weight (1999)
indicated an EC50
concentration of 1.2 M for a direct action of ethanol on GABAA
receptors—a concentration well above the lethal dose and above the concentrations used in the present study. In previous work, we have been unable to see a direct effect of pharmacologically relevant concentrations of ethanol on GABA-gated currents from enzymatically isolated Purkinje neurons (Criswell et al., 2003
). Other studies have indicated that indirect actions on the postsynaptic GABAA
receptor, including phosphorylation of the receptor (Freund and Palmer, 1997
; Harris et al., 1995
; Weiner et al., 1997
) and release of neuroactive steroids (Sanna et al., 2004
) by ethanol could, increase the receptor sensitivity to GABA. The relatively brief application of ethanol in the present study might not have allowed sufficient time for occurrence of these processes. Under these recording conditions, glutamatergic input was completely absent in the vast majority of cells (16 of 18) and minimal in the remaining 2. Based upon these observations, an increased release of GABA with no change in glutamate release would predict an overall decrease in the firing rate of Purkinje neurons following ethanol.
To test the hypothesis that the increased GABA release would evoke a decrease in the firing rate of Purkinje neurons, the effect of the most effective concentration of ethanol to enhance GABA release was examined using extracellular recording. In agreement with results from earlier work (Bloom and Siggins, 1987
; Franklin and Gruol, 1987
; Rogers et al., 1980
; Siggins et al., 1987
), intoxicating concentrations of ethanol did not produce a net decrease in the firing rate of cerebellar Purkinje neurons. However, rather than forming a normal distribution around zero, ethanol caused an increase in firing rate for some neurons but a decrease in rate for others. These results suggest that ethanol has multiple actions on Purkinje neurons to produce this variable pattern of firing. For this reason, the effect of ethanol on the intrinsic pacemaker activity of Purkinje neurons was investigated to account for the variable effects of ethanol.
Previous work has indicated that the bursting pattern of cerebellar Purkinje neuron activity results from an intrinsic pacemaker activity (Edgerton and Reinhart, 2003
). Because cerebellar Purkinje neurons are spontaneously active in the absence of synaptic inputs, we were able to examine the effect of ethanol on this intrinsic pacemaker function when the excitatory input was removed by the glutamate antagonists CNQX and AP-5 and the inhibitory input from GABA and glycine were eliminated by picrotoxin. Under these conditions, ethanol reliably increased the firing rate of the Purkinje neurons. When synaptic inputs are removed, cerebellar Purkinje neurons continue to fire in a bursting pattern. There was a strong correlation between overall change in rate and change in firing pattern following ethanol application such that increases in firing rate were associated with an increase in burst duration and a decrease in burst rate. There was no correlation between the within-burst interspike interval and change in rate. These data are consistent with an effect of ethanol that alters rate by a mechanism that controls the pattern of Purkinje neuronal activity but does not affect within-burst interspike interval. The present correlation between rate and firing pattern by ethanol further suggests that ethanol is having a direct action to alter intrinsic properties of the Purkinje neuron. The bursting pattern of activity in Purkinje neurons can be initiated by climbing fiber stimulation (Eccles et al., 1966
) but occurs even in the absence of input from other neurons (Swensen and Bean, 2003
; Womack and Khodakhah, 2004
). The onset and offset of a burst is under the coordinated control of a number of voltage- and ligand-dependent Na+
, and K+
channels (Swensen and Bean, 2005
). While bursting occurs in acutely dissociated Purkinje neurons that lack their dendritic tree (Swensen and Bean, 2005
), the mechanism may be somewhat different than bursting in a slice preparation, where termination of a burst appears to depend on dendritic calcium spikes (Womack and Khodakhah, 2004
). The effects of ethanol on any one of these ion channels could alter burst characteristics.
The correlation between the sensitivity of cerebellar Purkinje neurons to GABA inhibition of rate and the effect of ethanol on rate would be consistent with varying strengths of the intrinsic drive between neurons such that it is more difficult to perturb the rate and pattern of activity of some neurons than of others by either GABA or ethanol. However, there are other possible mechanisms for the correlation between the effects of ethanol and GABA on rate. For example, neurons less sensitive to the inhibitory action of GABA would be less sensitive to the effect of increased GABA release by ethanol. The present methods do not allow a test of these alternatives.
An important question raised by these data is how an interaction of 2 variables with a normal distribution of either excitatory or inhibitory effects can result in an effect on neuronal activity that is not normally distributed. The most parsimonious explanation is that the excitatory effect of ethanol on the intrinsic pacemaker activity of the Purkinje neuron and the inhibitory effect of increased GABA release by ethanol are moderated by a homeostatic mechanism. Only a net excitation or a net inhibition sufficient to overcome homeostatic control will produce a change in activity of the Purkinje neuron. A second mechanism to produce a change in rate that is not normally distributed is a nonlinear interaction between the excitatory and inhibitory mechanisms. Either of these possibilities could explain the present results but they do not represent the only possible mechanisms.
Taken together, these data are consistent with 2 conflicting actions of ethanol on the cerebellar Purkinje neuron. An increase in inhibition due to a presynaptic action of ethanol to increase GABA release and a postsynaptic action of ethanol to increase the intrinsic excitatory drive of the Purkinje neuron. Interplay of these 2 actions may underlie a part of the complex effect of ethanol on the rate of firing of cerebellar Purkinje neurons.