Forrest F. Weight

Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron

The effects of ethanol (EtOH) on membrane ion currents activated by N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) were studied under voltage-clamp conditions in isolated sensory neurons within hours of being dissociated from adult rats. The amplitude of the ion current activated by NMDA was decreased in the presence of 2.5-100 mM EtOH (IC50, 10 mM or 0.05% EtOH), a concentration range that produces intoxication. The amplitude of the GABA-activated Cl- current, on the other hand, was not significantly affected by this concentration range of EtOH. The observations suggest that some of the neural and cognitive impairments associated with EtOH intoxication may result from inhibition of the NMDA-activated ion current.

Dopaminergic mechanisms in subthalamic nucleus of rat: analysis using horseradish peroxidase and microiontophoresis

Afferent connections to the subthalamic nucleus (STN) were studied by microiontophoretically injecting horseradish peroxidase (HRP) into the STN and studying its retrograde transport. Remotely labelled neurons were frequently observed in both the globus pallidus and the pars compacta region of substantia nigra. In addition, individually labelled neurons were occasionally found in other brain regions. The sensitivity of neurons in the STN to dopamine (DA) was studied by applying DA to neurons in the STN by microiontophoresis. Three patterns of response to DA were observed. The most frequent response, observed in 46% of the STN neurons studied, was a decrease in the discharge frequency. In 15% of the neurons there was an increased frequency of firing. Eleven percent of the neurons exhibited a mixed response consisting of an initial depression of discharge rate followed by a delayed increase. The responses of STN neurons to DA were not antagonized by iontophoretically applied haloperidol. In neurons whose firing frequency was decreased by DA, the iontophoretic application of apomorphine and norepinephrine also decreased discharge rate. The observations of HRP-labelled neurons in the pars compacta region of substantia nigra following injection of HRP into the STN together with the DA responsiveness of STN neurons suggest the possibility of a dopaminergic nigro-subthalamic pathway.

Effects of ethanol administration on parameters of immunocompetency in rats.

Ethanol administered to rats intragastrically in doses sufficient to cause dependency resulted in a rapid cell loss from the thymus and spleen. Cell loss from the peripheral blood was due primarily to a loss of lymphocytes, but a concomitant granulocytosis resulted in only small changes in the total leukocyte count. Lymphocyte proliferation to both T‐ and B‐cell mitogens was severely compromised by ethanol treatment. The cell loss and functional lymphocyte impairment also occurred at half the ethanol dose required to induce dependency. Although cell numbers recovered relatively quickly after ethanol withdrawal, lymphocyte function, as measured by proliferation, recovered more slowly. Ethanol administration before or during immunization with sheep erythrocytes resulted in an impairment in the ability of animals to respond with a primary immune response to this antigen. These data suggest that ethanol given in quantities sufficient to produce dependence impairs in vitro and in vivo parameters of immunocompetency.

Single-channel and whole-cell analysis of ethanol inhibition of NMDA-activated currents in cultured mouse cortical and hippocampal neurons

The effects of 0.1 to 500 mM ethanol on NMDA-activated currents were studied in primary cultures of mouse cortical and hippocampal neurons. In whole-cell recordings the IC50S for inhibition of NMDA-activated currents by ethanol were 129 mM +/- 20 mM in hippocampal neurons and 126 +/- 18 mM in cortical neurons. In single-channel recordings from excised outside-out patches of cortical neurons, ethanol inhibited total charge per minute with an IC50 of 174 +/- 23 mM, which was not significantly different from the IC50S for inhibition of whole-cell current. The reduction in mean open channel lifetime by ethanol was fit by the logistic equation with an apparent IC50 of 340 +/- 28 mM. Analysis of single-channel data indicated that ethanol inhibition of NMDA currents did not involve substantial changes in fast closed state kinetics, changes in open channel conductance, or block of the open channel. At the whole-cell IC50 of ethanol, mean open channel lifetime would decrease by 28% and frequency of opening would decline by 31% to account for the reduction in current. Single-channel data were consistent with ethanol being an allosteric modulator of gating which reduces agonist efficacy.

Ethanol inhibition of N‐methyl‐D‐aspartate‐activated current in mouse hippocampal neurones: whole‐cell patch‐clamp analysis

1 The action of ethanol on N‐methyl‐D‐aspartate (NMDA)‐activated ion current was studied in mouse hippocampal neurones in culture using whole‐cell patch‐clamp recording. 2 Ethanol inhibited NMDA‐activated current in a voltage‐independent manner, and did not alter the reversal potential of NMDA‐activated current. 3 Concentration–response analysis of NMDA‐ and glycine‐activated current revealed that ethanol decreased the maximal response to both agonists without affecting their EC50 values. 4 The polyamine spermine (1 μM) increased amplitude of NMDA‐activated current but did not alter the percentage inhibition of ethanol. 5 Compared to an extracellular pH of 7.0, pH 6.0 decreased and pH 8.0 increased the amplitude of NMDA‐activated current, but these changes in pH did not significantly alter the percentage inhibition by ethanol. 6 The sulphydryl reducing agent dithiothreitol (2 mM) increased the amplitude of NMDA‐activated current, but did not affect the percentage inhibition by ethanol. 7 Mg2+ (10, 100, 500 μM), Zn2+ (5, 20 μM) or ketamine (2, 10 μM) decreased the amplitude of NMDA‐activated current, but did not affect the percentage inhibition by ethanol. 8 The observations are consistent with ethanol inhibiting the function of NMDA receptors by a non‐competitive mechanism that does not involve several modulatory sites on the NMDA receptor–ionophore complex.

Ethanol inhibition of N-methyl-d-aspartate-activated ion current in rat hippocampal neurons is not competitive with glycine

The interaction of ethanol with glycine at the N-methyl-D-aspartate (NMDA)-activated ion channel was investigated in voltage-clamped rat cultured hippocampal neurons. As shown previously, glycine increased, and ethanol inhibited, the NMDA-activated current in these cells. Concentration-response data for glycine (0.1-100 microM) indicate that the inhibition of NMDA-activated current by ethanol does not involve a competitive interaction with glycine. Thus, ethanol appears to inhibit NMDA-activated current at a locus different from the glycine modulatory site.

Glutamate induces a depolarization of adult rat dorsal root ganglion neurons that is mediated predominantly by NMDA receptors

The effects of glutamate, glutamate receptor agonists and substances that affect glutamate responses were studied, using the whole-cell patch-clamp technique, on neurons isolated from adult rat dorsal root ganglia. In current-clamp, glutamate (100 microM) or the excitatory amino acid receptor agonist N-methyl-D-aspartate (NMDA; 10 or 100 microM) induced membrane depolarization. Under voltage-clamp, these compounds induced an inward current that was voltage-dependent in the presence of Mg2+. The NMDA-induced current was inhibited by the NMDA receptor antagonist D,L-2-amino-5-phosphono-valerate (APV; 100 microM), and potentiated by 100 nM glycine. Few neurons responded to the receptor agonists kainate (100 microM), or quisqualate (1-10 microM). These observations suggest that glutamate-induced depolarization of primary sensory neurons is mediated predominantly by NMDA receptors.

Cellular and Molecular Physiology of Alcohol Actions in the Nervous System

Publisher Summary This chapter reviews the physiological actions of alcohol in the nervous system and discusses the possible cellular and molecular basis of alcohols behavioral actions. The chapter focuses on the acute neuronal actions of alcohol, as detected by neurophysiological techniques. The approach to elucidating the cellular actions of alcohol in the nervous system investigates the effects of ethanol on the spike firing of neurons in different regions of the nervous system. Such investigations have revealed a plethora of results. Ethanol has been observed to increase or decrease, have biphasic effects on, or have no effect on neuronal discharge in different brain regions. In several brain regions, certain general patterns of ethanol effects have emerged, with either inhibition or excitation of neuronal firing predominating. On the other hand, in some brain regions, different effects of ethanol administration is observed on the firing of a given neuronal type dependent upon such factors as the preparation used, the presence or absence of anesthetic, the type of anesthetic used, and the method of ethanol administration. This diversity suggests that a variety of different factors may influence the effect of ethanol on neuronal discharge. Thus, to elucidate the cellular mechanisms of ethanols action, it is necessary to minimize the variables that impinge upon neuronal function.

Ethanol Inhibition of Neuronal Giutamate Receptor Function

Acute ethanol intoxication is associated with changes in the activity of neurons in the central nervous system. However, the cellular and molecular mechanisms underlying these changes are poorly understood. We have examined the acute effects of ethanol on excitatory synaptic mechanisms in neurons from mammalian central nervous system, and observed that intoxicating concentrations of ethanol can inhibit the ion current activated by the glutamate receptor agonist N-methyl-D-aspartate in cultured neurons from mouse hippocampus, cortex and spinal cord. This inhibition is seen under a variety of experimental recording conditions. On the other hand, ethanol is less effective in inhibiting ion current produced by activation of non-N-methyl-D-aspartate glutamate receptors. Intoxicating concentrations of ethanol also inhibit excitatory synaptic transmission mediated by N-methyl-D-aspartate receptors in hippocampal slices from adult rodents. These observations support the hypothesis that the N-methyl-D-aspartate receptor/ionophore complex is a target for the neural actions of ethanol, and that inhibition of N-methyl-D-aspartate receptor-mediated responses might contribute to acute ethanol intoxication. The possibility that other receptor-gated ion channels may also be sensitive to ethanol is discussed.

Alcohol and Anesthetic Actions on Excitatory Amino Acid—Activated Ion Channels

The actions of alcohol and anesthetics have been studied on excitatory amino acid activated ion channels in mammalian neurons. Ethanol inhibits NMDA-activated current over a concentration range that produces intoxication, and the potency of several alcohols for inhibiting the NMDA-activated current is correlated with their intoxicating potency, suggesting that alcohol-induced inhibition of responses to NMDA receptor activation may contribute to the neural and cognitive impairments associated with intoxication. Studies on the mechanism of ethanol inhibition of NMDA-activated current indicate that ethanol does not appear to block the ion channel, alter the ion selectivity of the channel, or interact with previously described binding sites on the NMDA receptor/ionophore complex. The linear relation between the potency of several alcohols for inhibiting the NMDA-activated current and the hydrophobicity of the alcohols suggests that ethanol may inhibit the NMDA-activated ion current by a novel type of interaction with a hydrophobic site associated with the NMDA channel. In addition, different types of general anesthetic agents exhibit different inhibitory actions on NMDA-, kainate-, and quisqualate-activated currents, suggesting that differences in the profile of inhibition of excitatory amino acid neurotransmission in the CNS among different classes of general anesthetics may contribute to the differences in their behavioral and physiological effects.