Sanjiv V. Bhave

Ethanol Promotes Apoptosis in Cerebellar Granule Cells by Inhibiting the Trophic Effect of NMDA

Abstract: When primary cultures of cerebellar granule neurons are grown in a physiological concentration of KCl (5 mM) they undergo apoptosis, which can be prevented by growing the cells in the presence of N‐methyl‐d‐aspartate (NMDA). We now show that ethanol inhibits this trophic effect of NMDA, i.e., promotes apoptosis, and also inhibits the NMDA‐induced increase in intracellular Ca2+ concentration in cells grown in 5 mM KCl. Both effects of ethanol show a similar concentration dependence and are reversed by a high concentration of glycine, the co‐agonist at the NMDA receptor. The data suggest that the effect of ethanol on apoptosis is mediated, at least in part, by inhibition of NMDA receptor function. This effect of ethanol to increase apoptosis could contribute to the previously described in vivo sensitivity of the developing cerebellum to ethanol‐induced damage.

Acute Effects of Ethanol on Pharmacologically Isolated Kainate Receptors in Cerebellar Granule Neurons: Comparison with NMDA and AMPA Receptors

Abstract: Comparisons of acute ethanols effects on individual members of the three major families of ionotropic glutamate receptors (kainate, AMPA, and NMDA) have been performed only with recombinant receptors. However, no study has compared the acute effects of ethanol on individual members of each one of these receptor families in the same neuron. We accomplished this task by using cultured cerebellar granule neurons and LY303070 (GYKI‐53784), a noncompetitive and selective AMPA receptor antagonist. Ethanol concentrations of 25, 50, 75, and 100 mM decreased the amplitude of pharmacologically isolated kainate‐activated currents by 3 ± 1, 9 ± 2, 14 ± 2, and 22 ± 3% (n = 8), respectively. The magnitude of the ethanol‐induced inhibition of nonselective kainate‐activated currents, i.e., in the absence of LY303070, and currents activated by submaximal AMPA concentrations was not significantly different from that obtained with isolated kainate currents. However, the magnitude of the ethanol‐induced inhibition of NMDA receptor‐activated currents was about twofold greater than that of kainate and/or AMPA receptors.

Phosphatidylinositol 3′‐OH kinase and protein kinase A pathways mediate the anti‐apoptotic effect of pituitary adenylyl cyclase‐activating polypeptide in cultured cerebellar granule neurons: modulation by ethanol

Cerebellar granule neurons cultured in the presence of 5u2003mm KCl undergo spontaneous apoptosis, which is reduced by exposure to pituitary adenylyl cyclase‐activating polypeptide (PACAP). Previous work has suggested roles for the cyclic AMP/PKA and MAP kinase signaling pathways in the anti‐apoptotic effect of PACAP. In the present study, the use of specific inhibitors confirmed the role of the cyclic AMP/PKA pathway, and also demonstrated a role for the phosphatidylinositol 3′‐OH kinase (PI 3‐kinase) neuroprotective pathway in the action of PACAP. Ethanol exposure accelerates the anti‐apoptotic effect of PACAP by a mechanism that involves the PKA and PI‐3 kinase pathways. The results demonstrate that ethanol can increase neuroprotection induced by PACAP. As previous work has shown that ethanol can increase apoptosis of cerebellar granule neurons by inhibiting the protective effect of agents such as NMDA or IGF‐1, the overall effect of ethanol on cerebellar neuron apoptosis during development may reflect the balance between inhibition and enhancement of the actions of various endogenous neuroprotective agents.

The 71 kDa glutamate-binding protein is increased in cerebellar granule cells after chronic ethanol treatment

Besides the N-methyl-D-aspartate (NMDA) receptor proteins NR1 and NR2, another complex of proteins which has been shown to contain ligand-binding sites characteristic of NMDA receptors is expressed in cerebellar granule cells. One of the proteins in the latter complex is the 71 kDa glutamate-binding protein (GBP). To determine the role of the GBP in the response to NMDA, primary cultures of cerebellar granule cells were treated with an antisense oligonucleotide complementary to mRNA for this protein. This treatment substantially reduced both mRNA and protein levels of the GBP, as well as the response of the cells to NMDA, measured as an increase in intracellular Ca2+ with fura-2 fluorescence. The antisense oligonucleotide treatment did not alter the Ca2+ responses to KC1 or kainate. Chronic ethanol exposure has previously been shown to increase NMDA receptor function and the density of binding sites for the NMDA receptor channel blocker, dizocilpine, in cerebellar granule cells. Chronic exposure of the cells to 100mM ethanol is now shown to result in significant increases in mRNA and protein levels for the GBP (45% and 100%, respectively). Ethanol treatment did not affect mRNA levels for NR1 or NR2A, caused only a small increase (20%) in protein levels for NR1, and resulted in a decrease (30%) in NR2A protein. Although a role of the NMDA receptor NR1/NR2 subunits cannot be ruled out, these results are compatible with the hypothesis of involvement of the GBP in the chronic ethanol-induced increase in NMDA receptor function in cerebellar granule cells.

Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons.

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the ‘developmental switch’ of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol‐treated cells than in control cells. Cross‐linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell‐surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol‐treated cells, as determined by NMDA/glycine‐induced increases in intracellular Ca2+. The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanols effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.

Chronic Ethanol Exposure Attenuates the Anti-Apoptotic Effect of NMDA in Cerebellar Granule Neurons

Abstract: Ethanol, added to primary cultures of cerebellar granule neurons simultaneously with NMDA, was previously shown to inhibit the anti‐apoptotic effect of NMDA. The in vitro anti‐apoptotic effect of NMDA is believed to mimic in vivo protection against apoptosis afforded by innervation of developing cerebellar granule neurons by glutamatergic mossy fibers. Therefore, the results suggested that the presence of ethanol in the brain at a critical period of development would promote apoptosis. In the present studies, we examined the effect of chronic ethanol exposure on the anti‐apoptotic action of NMDA in cerebellar granule neurons. The neurons were treated with ethanol in vitro for 1‐3 days in the absence of NMDA. Even after ethanol was removed from the culture medium, as ascertained by gas chromatography, the protective effect of added NMDA was significantly attenuated. The decreased anti‐apoptotic effect of NMDA was associated with a change in the properties of the NMDA receptor, as indicated by a decrease in ligand binding, decreased expression of NMDA receptor subunit proteins, and decreased functional responses including stimulation of increases in intracellular Ca2+ and induction of brain‐derived neurotrophic factor expression. The latter effect may directly underlie the attenuated protective effect of NMDA in these neurons. The results suggest that ethanol exposure during development can have long‐lasting effects on neuronal survival. The change in the NMDA receptor caused by chronic ethanol treatment may contribute to the loss of cerebellar granule neurons that is observed in animals and humans exposed to ethanol during gestation.

Ethanol sensitivity of NMDA receptor function in developing cerebellar granule neurons.

The mechanism by which ethanol inhibits the function of the NMDA subtype of glutamate receptor has not been elucidated. One possibility that has been suggested is that NMDA receptor subunit composition influences the sensitivity of the receptor to ethanol. We have taken advantage of developmental changes in subunit composition of the NMDA receptor in cultured neurons to examine possible changes in the effect of ethanol. We found an increase in expression of the NR2A subunit, and a decrease in expression of the NR2B subunit of the NMDA receptor in primary cultures of cerebellar granule neurons over time in culture, with no significant change in NR1 expression. This change in NR2 subunit expression was associated with the expected changes in functional properties of the NMDA receptor (measured as the NMDA-induced increase in intracellular Ca2+), i.e., ifenprodil sensitivity and glycine potency were higher when there was a relatively greater proportion of NR2B in the cultured neurons. However, the potency of ethanol to inhibit NMDA receptor function was lower when there was a greater proportion of NR2B subunits. Previous studies showed that ethanol inhibition of NMDA receptor function in cerebellar granule neurons resulted from an ethanol-induced decrease in potency of the co-agonist, glycine, and that this effect of ethanol was blocked by inhibitors of protein kinase C. Our current results suggest that the lower potency of ethanol to inhibit the response of NMDA receptors when cerebellar granule neurons are expressing a greater proportion of NR2B subunits is a result of the higher affinity of the NMDA receptors for endogenous levels of glycine at this point in time.