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Neurotransmitters and their receptors have been involved in both proper brain development and neurodevelopmental disorders. The role that nicotinic receptors play in immature cortical neurons was initially investigated by gene profiling using Affymetrix DNA arrays. Both short (15 min) and prolonged (18 h) treatments with nicotine did not induce modification in gene expression, whereas a significant down-regulation of c-fos protein levels was observed after 18 h treatment. Conversely, a brief treatment with the glutamatergic agonist NMDA triggered up-regulation of immediate early genes and transcription factors, which remained unaffected by pre-treatment for 18 h with nicotine. Calcium imaging studies revealed that NMDA activated a sustained increase in intracellular calcium concentration in the majority of neurons, whereas nicotine evoked only a transient calcium increase in a smaller percentage of neurons, suggesting that the calcium signalling response was correlated with activation of gene expression. Nicotine effects on immature cortical neurons perhaps do not require gene regulation but may be still acting on signalling pathways.

In the glial cell line C6, regulation of actinomycin D (Act-D)-sensitive translatable polysomal mRNAs of three key enzymes--glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) and glutamine synthetase (GS) by glucocorticoids and lactate dehydrogenase (LDH; EC 1.1.1.27) by catecholamines--is described. Though the first two enzymes are hydrocortisone (HC)-inducible, the nature of their response to the hyperacetylating agent sodium butyrate is dramatically different. Furthermore the appearance of GPDH translatable poly(A)+ RNA in HC-induced cells is inhibited by the presence of cycloheximide (CHX), whereas the induction of GS is unaffected by CHX. These observations necessitate further probing into an existing model system to explain the varied mechanisms of induction of these two enzymes by a single inducer. In combination with the third enzyme whose induction by catecholamines is glial specific, we believe that the C6 cell represents the most appropriate cell line for molecular neurobiologists to study the mechanisms of hormone action in glia.

—Following hypophysectomy or adrenalectomy, glycerolphosphate dehydrogenase (GPDH) (EC 1.1.1.8) activity decreased exponentially in the cerebral hemispheres and brain stem of adult male rats. The latter region was more affected than the former. Malate dehydrogenase (EC 1.1.1.40), isocitrate dehydrogenase (EC 1.1.1.42), lactate dehydrogenase (EC 1.1.1.27) and mitochondrial glycerolphosphate dehydrogenase (EC 1.1.95.5) activities remained unchanged. Injection of adrenocorticotrophic hormone or cortisol in hypophysectomized rats or cortisol in adrenalectomized rats restored GPDH activity. Thyroidectomy and gonadectomy had no effect on GPDH activity. Liver GPDH was not decreased by hypophysectomy or adrenalectomy. Muscle GPDH was diminished slightly by adrenalectomy and as much as brain GPDH by hypophysectomy. In young rats GPDH developmental increase in activity was inhibited by hypophysectomy. These results clearly show that brain GPDH activity is specifically regulated by cortisol (and probably closely related corticosteroids).

—Following head X-irradiation (750 r) of 2-day-old rats the aerobic formation of lactate in the presence or absence of chloral was markedly increased in medulla slices but remained unchanged in cerebral cortex and brain stem slices. The ratio of lactate dehydrogenase (EC 1.1.1.27) to glycerolphosphate dehydrogenase (EC 1.1.1.8) was slightly increased in cortex and brain stem but more than doubled in medulla. The relation of the increased aerobic formation of lactate in irradiated medulla to the lactate dehydrogenase:glycerolphosphate dehydrogenase ratio is discussed.

Co-cultivation of confluent rat astrocyte cultures with embryonic chick neurons resulted in induction of glutamine synthetase activity in the astrocytes. This induction of glutamine synthetase in astrocytes by neurons was independent of induction by hydrocortisone and forskolin, but was dependent on the length of co-cultivation and the number of neurons present in the co-culture. Cycloheximide and actinomycin D inhibited the induction of glutamine synthetase in astrocytes by neurons, whereas cytosine arabinoside had no apparent effect. Results suggest that this induction of glutamine synthetase in astrocytes is mediated by cell contact with neurons and may represent a specific neuronal and glial interaction.

Biochemical evidence suggests that neuroglia are responsive to glucocorticoids, yet previous studies of glucocorticoid localization have typically failed to demonstrate significant uptake by neuroglial cells. To further investigate this problem, we measured glycerol-3-phosphate dehydrogenase (GPDH) activity and glucocorticoid receptor binding capacity in normal rat optic nerves and in those undergoing Wallerian (axonal) degeneration. Binding studies were also performed on hippocampus and anterior pituitary for comparison purposes. Normal optic nerve preparations possessed a high level of GPDH activity that was glucocorticoid-inducible and that increased further following axonal degeneration. Antibody inactivation experiments demonstrated the presence of more enzyme molecules in the degenerating nerve preparations. Correlative immunocytochemical studies found GPDH-positive reaction product only in morphologically identified oligodendrocytes, a result that is consistent with the previously reported localization of this enzyme in rat brain. Optic nerve cytosol fractions displayed substantial high-affinity binding of both dexamethasone (DEX) and corticosterone (CORT) that, like GPDH, was elevated approximately twofold in degenerating nerves. Finally, in vivo accumulation of [3H]DEX and [3H]CORT by optic nerve and other myelinated tracts was examined using nuclear isolation and autoradiographic methods. Although neither steroid was found to be heavily concentrated by these tissues in vivo, a small preference for DEX was observed in the nuclear uptake experiments. These results are discussed in terms of the hypothesis that glial cells are targets for glucocorticoid hormones.

Calcium- and calmodulin-dependent protein kinase activity was studied in pure neuronal and glial cultures. The addition of calcium and calmodulin stimulated 32P incorporation into several neuronal proteins including two in the 50- and 60-kilodalton (kD) region which comigrated with purified forebrain calmodulin kinase II sub-units (CaM kinase II). In mature astrocytes, CaM kinase activity was also present, and was inhibited by trifluoroperazine and diazepam. Again in homogenates of these cells, two phosphoproteins of apparent molecular masses of 50 and 60 kD comigrated with purified CaM kinase. CaM kinase activity was absent in immature mixed glia and oligodendrocytes. The presence of CaM kinase in neurons and mature astrocytes was confirmed using monoclonal antibodies specific for the 50-kD subunit of the enzyme. No immunoreactivity was observed in oligodendrocytes. The presence of CaM kinase in astrocytes suggests a more ubiquitous role of this enzyme in regulating cellular processes than was previously recognized.