NMDA receptor stimulation selectively initiates GABA(A) receptor delta subunit mRNA expression in cultured rat cerebellar granule neurons.

Findings in vivo and in culture suggest that neuronal activity selectively regulates GABA(A) receptor delta subunit mRNA expression in cerebellar granule neurons. For example, the onset of delta subunit mRNA expression during postnatal maturation coincides with innervation. Furthermore, depolarizing conditions (25 mM KCl) in culture initiate and maintain increases in the delta subunit transcript level. We have now examined whether similar changes in delta subunit mRNA expression occur in cultured neurons after activation of glutamate receptors of the NMDA subtype, an event that mimics granule neuron depolarization by mossy fiber innervation in vivo. Our studies demonstrate that addition of 50 microM NMDA to cultured rat granule neurons maintained in defined, serum-free medium specifically initiates delta subunit transcript expression. Whereas the level of the delta subunit mRNA is increased fourfold by this treatment, levels of other GABA(A) receptor subunit transcripts are not significantly changed. The level of the delta subunit transcript is further increased when NMDA receptor activation is enhanced by maintaining neurons in a Mg2+-free medium to alleviate Mg2+ blockade of the receptor channel. The NMDA-induced elevation in delta subunit transcript expression involves activation of a Ca2+/calmodulin-dependent protein kinase pathway. These findings suggest that activation of an excitatory pathway may regulate the expression of an inhibitory receptor phenotype in cerebellar granule neurons in vivo.

Expression of the GABAA receptor delta subunit is selectively modulated by depolarization in cultured rat cerebellar granule neurons.

The levels of several GABAA receptor subunit mRNAs increase as cerebellar granule neurons migrate to their adult positions and receive excitatory mossy fiber input. Despite the temporal similarity of these increases in transcript expression in vivo, studies in cultured granule neurons demonstrated that the subunit mRNAs are differentially regulated. To address the possibility that neuronal activity regulates transcript expression, GABAA receptor subunit mRNA levels were assessed in cultured granule neurons grown in chemically defined, serum-free medium containing either nondepolarizing (5 mM) or depolarizing (25 mM) KCl concentrations. Whereas the delta subunit mRNA was almost undetectable in cultures maintained in nondepolarizing medium, an eightfold increase occurred between days 2 and 4 in cultures grown in depolarizing medium. Furthermore, delta subunit transcript expression was reduced by 76 +/- 6% when neurons in depolarizing medium were switched into nondepolarizing medium. The importance of depolarization in the initiation and maintenance of subunit transcript expression in granule neurons was selective for the GABAA receptor delta subunit. These changes in transcript expression involved calcium entry through L-type calcium channels. Nifedipine treatment (1 microM) both reduced intracellular calcium and decreased delta subunit mRNA expression by 79 +/- 4%. Furthermore, inhibition of Ca2+/calmodulin-dependent protein kinases (CaM kinases) by KN-62 (1 microM) also reduced delta subunit transcript expression. These studies demonstrate that KCl-induced depolarization, a condition that mimics the effects of neuronal activity, selectively modulates GABAA receptor delta subunit mRNA expression through a pathway involving calcium entry and activation of a CaM kinase.

Differential regulation of GABA A receptor subunit mRNAs in rat cerebellar granule neurons: importance of environmental cues.

Levels of the GABA A receptor alpha1-, alpha6-, beta2-, beta3-, gamma2-, and delta-subunit mRNAs in cerebellar granule neurons rise concurrently during the second week of postnatal ontogeny. Previous studies in culture have suggested that extrinsic signals control these increases, but little is known about the nature of the regulatory cues. To determine when granule neurons become competent to express these six subunit mRNAs in mature patterns and to gain insight into their regulation, reverse transcriptase-PCR was used to examine transcript expression in cultured granule neurons prepared at 2-day intervals from postnatal days 2 through 10. Although only one pattern of expression was observed in vivo, three patterns were detected in culture. First, the levels of the alpha1- and alpha6-subunit mRNAs were constant in cultures prepared at all ages. Second, the levels of the beta2-, beta3-, and gamma2-subunit mRNAs were constant in cultures prepared at postnatal days 2-6 but increased in those prepared at days 8-10. Third, the delta-subunit mRNA level increased over time in culture regardless of cerebellar age at plating. Moreover, only delta-subunit transcript expression was modulated by cell density. These findings indicate that the subunit transcripts are differentially regulated by multiple environmental cues.

Changes in energy metabolites, cGMP and intracellular pH during cortical spreading depression.

The spatial and temporal distribution of cerebral metabolites and pHi were examined in the cortex during spreading depression. Acidification and marked depression in the energy status of the tissue was evident at the wavefront of spreading depression. In its aftermath, the residual activation of glycolysis and accumulation of cGMP persisted for minutes after a relatively rapid restoration of high-energy phosphates and pHi.

Adrenalectomy attenuates stress-induced elevations in extracellular glutamate concentrations in the hippocampus.

Glucocorticoids and stress have deleterious effects on hippocampal cell morphology and survival. It has been hypothesized that these effects are mediated via an excitatory amino acid mechanism. The present study was designed to evaluate the effects of acute stress on the extracellular levels of glutamate in the hippocampus and to determine if adrenalectomy modifies this response. Rats were adrenalectomized or sham-adrenalectomized and implanted with microdialysis probes in the CA3 region of the hippocampus. Three days later rats were subjected to an acute 1-h period of immobilization stress. Stress significantly increased extracellular glutamate levels in the sham-operated rats, which peaked at 20 min following the initiation of stress. Extracellular glutamate levels also increased immediately following the termination of stress. In the adrenalectomized rats there was a 30% decrease in basal extracellular concentrations of glutamate and a marked attenuation (-70%) of the stress-induced increase in extracellular glutamate levels. Extracellular concentrations of taurine were not modified by adrenalectomy and did not change in response to stress. These results suggest that glucocorticoid-induced elevations in extracellular glutamate concentrations may contribute to the deleterious effects of stress on hippocampal neurons.