Effects of prenatal and early postnatal ethanol exposure on [3H]MK-801 binding in rat cortex and hippocampus.

The effects of prenatal and/or early postnatal exposure to ethanol at high concentrations on N-methyl-D-aspartate (NMDA) receptor number and functioning in the weanling rat were examined. The binge-like exposure protocol was used in an animal model of acute ethanol effects at two critical periods of development. [3H]MK-801 binding parameters for the internal channel phencyclidine site were assessed in the presence of 10 microM glutamate and 10 microM glycine activation. Four treatment groups were included: (1) animals exposed to ethanol both prenatal and postnatal; (2) animals exposed only prenatal; (3) animals exposed early postnatal only; and (4) control animals with no exposure to ethanol. The results of the [3H]MK-801 binding experiments showed that both prenatal and postnatal exposure to ethanol resulted in a significant decrease in the density of NMDA receptors. In addition, data indicated an apparent increase in the percentage of high-affinity state (open channel state) relative to low-affinity state (close channel state) receptors in the ethanol-treated groups. These results show that both prenatal and postnatal ethanol exposure decrease NMDA receptor density in the cortex and hippocampus. The findings are consistent with previous observations by our laboratory and others that NMDA-mediated calcium influx is reduced in these regions, as well as in whole brain by prenatal ethanol exposure. It is suggested that after ethanol exposure, the remaining functional NMDA receptors might have altered sensitivity to coagonist activation with an increased probability of channel opening.

Effects of prenatal ethanol exposure on brain region NMDA-mediated increase in intracellular calcium and the NMDAR1 subunit in forebrain.

The effects of prenatal ethanol exposure on N-methyl-D-aspartate (NMDA)-mediated calcium entry into neonatal dissociated neurons from hippocampus, forebrain, and cerebellum were investigated. Dissociated cells were isolated from less than 1-day-old pups of prenatally exposed, pair-fed control and ad libitum control groups and loaded with fura-2. Prenatal ethanol exposure significantly reduced the NMDA-stimulated increase in intracellular calcium in all three brain regions compared to the two control groups. These findings are very similar to those previously observed in neonatal dissociated whole brain neurons using the same ethanol exposure protocol. Studies were also conducted using forebrain to determine if prenatal ethanol exposure alters NMDAR1 subunit protein expression in this major brain area; however, the results indicated no significant differences between ethanol-exposed and control groups.

Effects of prenatal ethanol exposure on voltage-dependent calcium entry into neonatal whole brain-dissociated neurons.

The effect of prenatal ethanol exposure on voltage-dependent calcium entry into neonatal-dissociated neurons was studied. Dissociated whole brain cells were isolated from neonates of prenatally ethanol-treated (ET), pair-fed (PF) control, and ad libitum (AL) control groups and loaded with fura-2. Prenatal ethanol exposure resulted in a significant reduction of calcium entry into K(+)-depolarized cells, compared with AL and PF control treatments. Initially, in dissociated cells from AL control animals, it was found that nifedipine (1 microM), omega-agatoxin (100 nM), and omega-conotoxin (500 nM), to a much lesser extent, significantly inhibited the 45 mM KCl-stimulated calcium entry. To determine the inhibitory action of prenatal ethanol exposure on N-, P-, and L-type voltage-dependent calcium channels, treatment of neonatal-dissociated neurons with different combinations of omega-conotoxin, omega-agatoxin, and nifedipine, respectively, was compared in the prenatal ethanol and control treatment groups. The inhibition of K(+)-stimulated increase in calcium entry by prenatal ethanol exposure was significantly less in the presence or absence of single antagonist conditions (ET < AL and PF). There was no apparent interaction of ethanol exposure and antagonist condition. However, the reduced calcium entry after prenatal ethanol exposure was superseded by the stronger inhibition in dual and triple antagonist conditions. The magnitude of the calcium response inhibition by the antagonist combinations was similar among the ET, PF, and AL groups. Thus, these results suggest that prenatal ethanol exposure decreases voltage-dependent calcium entry into neonatal-dissociated neurons in a manner that does not seem to involve the selective inhibition of any individual N-, P-, or L-type calcium channel.

N-methyl-D-aspartate-stimulated increases in intracellular calcium exhibit brain regional differences in sensitivity to inhibition by ethanol.

This study compared N-methyl-D-aspartate (NMDA)-stimulated increases in intracellular calcium in fura-2-loaded neurons dissociated from newborn rat brainstem (EC50 in microM; 6.4), cerebellum (9.5), forebrain (6.3), and hippocampus (10.6). Ethanol inhibition of the response to 25 microM NMDA differed among the regions. The NMDA response in hippocampus was inhibited by 20 mM ethanol; cortex and cerebellum responses were inhibited by 80 mM ethanol, and no inhibition was seen in the brainstem. Addition of glycine (15 microM) failed to attenuate ethanol inhibition of the NMDA response. These results demonstrate that ethanol inhibition of NMDA-stimulated responses varies according to brain region. In contrast to previous findings from this laboratory using dissociated neurons from whole brain, the addition of glycine did not reverse the inhibitory effects of ethanol on NMDA-stimulated responses.

D, L-(tetrazol-5-yl)glycine stimulation of NMDA receptors in neonatal dissociated neurons: inhibition by ethanol.

Ethanol inhibition of NMDA receptor stimulation by the high-affinity selective agonist D, L-(tetrazol-5-yl)glycine (T5G) was studied using acutely dissociated neonatal whole-brain neurons loaded with the fluorescent indicator fura-2. T5G induced a concentration-dependent increase in intracellular calcium with a maximal increase above basal of 70nM at 16 microM T5G (EC50 of 0.66 +/- 0.18 microM). T5G agonist specificity was verified using the NMDA antagonists MK-801 (40 nM), APV (100 microM), and Mg2+ (1 mM). The T5G stimulation of calcium entry was both blocked and reversed by these antagonists. Ethanol significantly inhibited the T5G-mediated increase in intracellular calcium only at concentrations > or = 100 mM. In addition, the effect of increasing concentrations of ethanol in the presence of the glycine-site antagonist 5, 7-dichlorokynurenic acid (DCKA, 0.37 microM) on T5G-stimulated calcium entry was examined. A significant inhibition of the T5G-stimulated response in the presence of DCKA was observed at ethanol concentrations as low as 20 mM. These results support previous findings that T5G is a potent agonist of the NMDA receptor and indicate that stimulation of calcium entry by this agonist is less sensitive to ethanol inhibition than stimulation by NMDA.

Alteration of [3H]MK-801 binding associated with the N-methyl-D-Aspartate receptor complex by acute ethanol in rat cortex and hippocampus in vitro.

We investigated the effect of ethanol on specific binding of [3H]MK-801 to the intrachannel phencyclidine (PCP) receptor site, as an index of change in the functional response of the N-methyl-D-Aspartate (NMDA)-associated ion channel. Saturation binding experiments were performed on synaptic membrane homogenates from adult rat cortex and hippocampus. [3H]MK-801 binding assays were conducted under conditions of basal, 10 microM glutamate, or 10 microM glutamate + 30 microM D-serine, with and without 50 or 100 mM ethanol. Association experiments of [3H]MK-801 binding (5 nM) were conducted under conditions of 0 or 10 microM glutamate, with varying concentrations of glycine (0.01, 0.10, and 10 microM) with and without 100 mM ethanol. Ethanol (50 and 100 mM) significantly decreased the percentage of high-affinity (open-channel state) MK-801 receptors with a concomitant increase in percentage of low-affinity receptors, but did not change high- and low-affinity constants of the two binding states. An ethanol-induced increase in the closed-channel receptor density in basal and activated conditions was suggested by the saturation experiments. Association experiments further explained this finding, in that ethanol (100 mM) significantly decreased fast component (open-channel) [3H]MK-801 binding in conditions of glycine (0.01-10 microM) only and activated conditions of glutamate + glycine (0.01-0.10 microM). However, the observed fast and slow kinetic rate constants of [3h]MK-801 binding, as well as total specific binding (fast + slow components), were not altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prenatal ethanol exposure on N-methyl-D-aspartate-mediated calcium entry into dissociated neurons.

The effects of prenatal ethanol exposure on N-methyl-D-aspartate (NMDA)-activated calcium entry into dissociated neurons were studied. Dissociated brain cells were isolated from less than 1-day-old pups from prenatally ethanol-exposed, pair-fed control and ad libitum control groups and loaded with fura-2. Prenatal ethanol exposure significantly decreased the NMDA receptor-mediated calcium entry compared to both pair-fed and ad libitum control groups. To determine the mechanisms of the prenatal ethanol exposure on the NMDA-mediated ion channel decrements, possible modulatory sites of the NMDA receptor were studied. Glycine (0.1, 1, 10 and 100 microM) increased calcium entry to an equal extent in the ethanol and control groups, but did not reverse the effect of prenatal ethanol exposure. Furthermore, low concentrations of MK801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5-10-imine hydrogen maleate] (25 and 50 nM) did not further inhibit calcium entry beyond that observed with the prenatal ethanol exposure, but significantly inhibited control group responses. Mg++ showed a similar result. With increasing concentrations of Mg++ the calcium entry in the three groups tended to converge. Thus, these results suggest that prenatal ethanol exposure inhibits the function of NMDA receptor-mediated ion channels by possibly altering the structural properties of the ion channel itself and/or by interacting with inner ion channel modulatory sites activated by Mg++ or MK801, and not the glycine site.