Expression of catecholamine-synthesizing enzymes, peptidylglycine alpha-amidating monooxygenase, and neuropeptide Y mRNA in the rat adrenal medulla after acute systemic nicotine.

The expression of catecholamine-synthesizing enzymes in the adrenal medulla is upregulated in parallel by stress and pharmacological treatments. In this study we examined whether a neuropeptide and its processing enzyme are regulated in parallel with catecholamine enzyme genes after drug treatment. Because the main effect of stress on the adrenal medulla is via splanchnic nerve stimulation of nicotinic receptors, we used nicotine to stimulate the medulla and visualized expression of catecholamine enzyme genes, the medullary peptide neuropeptide Y (NPY), and the neuropeptide-processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) by in situ hybridization quantified by image analysis of autoradiographic images. Rats received a single injection of nicotine (0, 1, or 5 mg/kg sc). Six hours later, rats were transcardially perfused. Free-floating adrenal gland sections were hybridized with 35S-labeled cDNA probes for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), PAM, and NPY. Nicotine treatment upregulated the expression of TH, PNMT, and NPY genes in a dose-dependent fashion. Small but nonsignificant increases were observed in DBH and PAM mRNA levels. These results suggest that common transcriptional activation mechanisms may upregulate both catecholamine and neuropeptide synthesis in the adrenal medulla after nicotinic stimulation.

Alternate promoters in the rat aromatic L-amino acid decarboxylase gene for neuronal and nonneuronal expression: an in situ hybridization study.

Aromatic L-amino acid decarboxylase (AADC) is found in both neuronal cells and nonneuronal cells, and a single gene encodes rat AADC in both neuronal and nonneuronal tissues. However, two cDNAs for this enzyme have been identified: one from the liver and the other from pheochromocytoma. Exons 1a and 1b are found in the liver cDNA and the pheochromocytoma cDNA, respectively. In the third exon (exon 2), there are two alternatively utilized splicing acceptors specific to these exons, 1a and 1b. Structural analysis of the rat AADC gene showed that both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. To demonstrate whether alternative promoter usage and splicing are tissue specific and whether the exons 1a and 1b are differentially and specifically transcribed in nonneuronal and neuronal cells, respectively, in situ hybridization histochemistry for the rat brain, adrenal gland, liver, and kidney was carried out using these two exon probes. The exon 1a probe specifically identified AADC mRNA only in nonneuronal cells, including the liver and kidney, and the exon 1b probe localized AADC mRNA to monoaminergic neurons in the CNS and the adrenal medulla. Thus, both alternative promoter usage and differential splicing are in fact operative for the tissue-specific expression of the rat AADC gene.

Increased c-fos expression in nucleus of the solitary tract correlated with conditioned taste aversion to sucrose in rats.

The pattern of neuronal activation in the rat nucleus of the solitary tract (NTS) in response to a standard gustatory stimulus was examined using c-Fos-like immunoreactivity (c-FLI) before and after conditioned taste aversion (CTA) formation. While unconditioned oral infusions of sucrose solution did not induce c-FLI in the NTS, after three pairings of sucrose with lithium chloride injections, sucrose induced c-FLI in the medial intermediate NTS 1 h after oral infusion. Extinction of the CTA by repeated infusions of sucrose alone reversed the induction of c-FLI.

Differential spatial and temporal gene expression in response to axotomy and deafferentation following transection of the medial forebrain bundle.

Alterations in the levels of neurotransmitter biosynthetic enzymes are a concomitant of many neurodegenerative disorders. In order to elucidate potential mechanisms for longterm alterations in biosynthetic enzyme gene products in response to neuronal injury, an acute axotomy/deafferentation model was employed. A unilateral microknife transection of the medial forebrain bundle (MFB) axotomizes and/or deafferents phenotypically identified neuronal populations important in the function of the basal ganglia. Semi-quantitative in situ hybridization and immunohistochemical analysis demonstrated that the products of the immediate-early gene c-fos were induced postaxotomy in the noradrenergic neurons of the locus ceruleus (LC), but not in the dopaminergic neurons of the substantia nigra pars compacta (SNc). Analysis of the levels of mRNA, protein, and activity for tyrosine hydroxylase demonstrated that the LC neurons survive the injury while the SNc neurons degenerate. After MFB transection, Fos protein also was induced in the corpus striatum within 1 hr, first in large, putatively cholinergic neuronal populations followed at 3 hr by the small, putatively GABAergic neurons. The substantia nigra pars reticulata and the subthalamic nucleus neuronal populations, deafferented by the MFB transection, also exhibited Fos induction beginning at 3 hr. The data suggest that expression of Fos in a neuronal population is correlative with respect to cell survival following either axotomy or deafferentation. Whether Fos induction following injury is either a necessary mechanism of cell survival or merely a marker of increased neuronal activity requires further investigation.

A dual role for the cAMP-dependent protein kinase in tyrosine hydroxylase gene expression.

Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine, the first and rate-limiting step in catecholamine biosynthesis. The cAMP-dependent protein kinase (PKA) phosphorylates and activates the TH enzyme and is thought to mediate transcriptional induction of the TH gene. To better understand the functional role of PKA in TH gene regulation, we studied TH gene expression at the transcriptional, translational, and post-translational levels in several PKA-deficient cell lines derived from rat PC12 pheochromocytoma cells. Strikingly, all PKA-deficient cell lines analyzed in this study showed substantial deficits in basal TH expression as measured by TH enzymatic activity, level of TH immunoreactivity, TH protein level, and steady-state mRNA level. Interestingly, the steady-state level of mRNA correlated well with levels of TH activity, immunoreactivity, and protein. In addition, PKA-deficient cell lines lacked transcriptional induction of the TH gene following treatment with dibutyryl cAMP. Cotransfection of PKA-deficient cells with an expression plasmid for the catalytic subunit of PKA fully reversed transcriptional defect, as indicated by robust transcriptional induction of a reporter construct containing 2400 bp of TH upstream sequence in all PC12 cells tested. These data indicate that the PKA system regulates both the basal and the cAMP-inducible expression of the TH gene primarily at the transcriptional level in PC12 cells.

Axotomy-induced differential gene induction in neurons of the locus ceruleus and substantia nigra.

The biochemical and molecular events correlated with neuronal injury and survival are not well understood. Previous studies have reported that following axotomy, neurons in the substantia nigra pars compacta (SNc) and the locus ceruleus (LC) exhibit a significant increase in tyrosine hydroxylase (TH) enzyme activity within 24-72 h (Brain Res., 144 (1978) 325-342; Brain Res., 92 (1975) 57-72). To investigate the potential contribution of TH gene induction to this increase a semi-quantitative immunocytochemical and in situ hybridization time course analysis was undertaken. Following axotomy, TH immunoreactivity increased in neurons of both the SNc and LC. In contrast, an increase in TH mRNA was only evident in neurons of the LC. As a possible mechanism for the observed alterations in TH gene expression, the levels of an immediate early gene, c-fos, were examined. C-fos mRNA and Fos protein were not expressed in either normal or axotomized neurons of the SNc. However, the constitutive expression in control LC neurons increased significantly following axotomy. These data demonstrate the differential response of two central catecholaminergic populations to axotomy and suggests a potential role for the immediate early gene, c-fos, in the post-injury reaction.

Parallel upregulation of catecholamine-synthesizing enzymes in rat brain and adrenal gland: effects of reserpine and correlation with immediate early gene expression.

Changes in the mRNA levels of all catecholamine-synthesizing enzymes were examined 24 h after a single injection of reserpine by in situ hybridization. The responses of the midbrain dopaminergic cells in the ventral tegmental area and substantia nigra compacta, locus ceruleus and adrenal gland were studied in three groups of animals receiving either no injection, vehicle injection or reserpine 10 mg/kg subcutaneously. Increases in enzyme message signal observed by in situ hybridization were corroborated by Northern blot analysis for all four enzyme mRNAs species expressed in the locus ceruleus and adrenal gland were found while no change of enzyme message was detected the midbrain. Two distinct subpopulations of adrenomedullary cells could be distinguished by their baseline levels of enzyme mRNA expression: the majority of medullary cells have moderate adrenomedullary cells could be distinguished by their baseline levels of enzyme mRNA expression: the majority of medullary cells have moderate levels of all four enzyme mRNAs but a minority of cells show very high signal for the first three enzymes of the catecholamine synthesis pathway. To test whether reserpine elicits a selective transcriptional response of the catecholamine enzyme genes or induces other neuronal genes, cDNA probes for the growth-associated protein GAP-43 which is highly expressed and neurofilament L which is weakly expressed in monoaminergic neurons were used as independent cellular markers and showed no change in message levels. Changes in mRNA levels of the proto-oncogenes c-fos and c-jun were examined 1 h after injection of reserpine by in situ hybridization and compared to the pattern observed for the Fos protein immunohistochemically. C-fos and c-jun proto-oncogene activation was observed 1 h after reserpine in the locus ceruleus and adrenal medulla, specifically in those catecholaminergic structures that respond with increased enzyme gene transcription; in contrast, the dopaminergic neurons of the substantia nigra did not exhibit detectable proto-oncogene activation, only a small group of neurons in the ventral tegmental area showed c-fos without concomitant c-jun expression after reserpine.

In situ hybridization analysis of c-fos and c-jun expression in the rat brain following transient forebrain ischemia.

Early induction of the mRNAs encoding the c-Fos and c-Jun nuclear proteins was examined in rat brain by in situ hybridization at various timepoints following global forebrain ischemia by the method of four-vessel occlusion. All animals were subjected to 20 min of transient ischemia. This produced a pattern of proto-oncogene activation that was most intense in the granule cells of the dentate gyrus 30 min after ischemia, while the hilar cells in the dentate and the pyramidal cells of the CA3 region in the hippocampus showed a more delayed but robust expression of these immediate early genes at 1 h. The neurons of the CA1 region exhibited a more moderate hybridization signal at 1-2 h postischemia. Very little hybridization signal for either immediate early gene could be detected in animals perfused with fixative immediately following ischemia, suggesting that cellular energy levels may have to be restored to a certain level before efficient de novo mRNA synthesis can occur. In the cerebellum, a similar temporal pattern was observed: the granule cells exhibited a prompt but patchy expression of c-fos and c-jun that was followed by a delayed signal in the Purkinje cells. Without exception c-fos and c-jun appeared to be expressed in unison, although the time course of c-fos and c-jun mRNA accumulation and decay was different in various brain regions: invariably the cerebellum returned rapidly to its baseline with virtually no remaining signal at 3 h postischemia, while c-fos and c-jun activation in the hippocampus remained high at 3 h and returned to baseline by 6 h. Several other brain regions showed early production of c-fos and c-jun mRNAs, such as the medial habenula, piriform cortex, the amygdala, the centromedian, lateral posterior, paracentral, intermediodorsal and reuniens nuclei of the thalamus and the ventromedial and dorsal nuclei of the hypothalamus; in the brainstem, the trapezoid body and the noradrenergic neurons of the locus ceruleus as well as the adrenergic neurons in the ventrolateral medulla (C1 group) and nucleus tractus solitarius (C2 group) regions displayed slightly less intense hybridization signals. In addition, the ependyma of the lateral ventricles and the third ventricle showed a prompt albeit short-lived production of c-fos and c-jun mRNAs. Sham-operated animals as well as animals that had survived to one week postischemia showed either no or only trace levels of hybridization signal.(ABSTRACT TRUNCATED AT 400 WORDS)