Induction of neuropeptide gene expression and blockade of retrograde transport in facial motor neurons following local peripheral nerve inflammation in severe combined immunodeficiency and BALB/C mice.

Peripheral nerve inflammation is a common clinical problem that accompanies nerve injury and several diseases including Guillain-Barre syndrome and acute and chronic inflammatory demyelinating polyneuropathy. To determine if neuropeptides are induced in motor neurons after inflammation and to study the mechanisms involved, a nerve cuff soaked in complete Freund's adjuvant (CFA) was applied locally to the facial nerve of Balb/C mice. This procedure resulted in an influx of lymphocytes and macrophages to the affected area and a blockade of retrograde axonal transport distal, but not proximal, to the site of application. The same treatment resulted in a strong ipsilateral induction of pituitary adenylyl cyclase activating peptide (PACAP) gene expression in motor neurons in the facial motor nucleus. Because the changes could have occurred due to the loss of target-derived factors or to the production of new factors by immune cells, we studied the effect of the inflammatory stimulus on PACAP mRNA in mice with severe combined immunodeficiency (SCID). As expected, SCID mice showed a severely reduced influx of T-lymphocytes but not macrophages to the peripheral nerve. Moreover, although retrograde transport distal to the inflammation site was blocked similarly in control and SCID mice, the number of motor neurons expressing PACAP mRNA after CFA application was significantly reduced in SCID mice. The data indicate that the induction of PACAP mRNA during nerve inflammation requires the involvement of lymphocytes. However, because the induction of PACAP gene expression was only partially blocked in SCID mice, macrophages, loss of target-derived factors, or other mechanisms may also contribute to the upregulation of PACAP gene expression in motor neurons after nerve inflammation.

Embryonic expression of pituitary adenylyl cyclase-activating polypeptide and its selective type I receptor gene in the frog Xenopus laevis neural tube.

The genes encoding pituitary adenylyl cyclase-activating peptide (PACAP) and its selective type I receptor (PAC1) are expressed in the embryonic mouse neural tube, where they may be involved in neurogenesis and neural tube development. We examined here the early expression and potential actions of PACAP and PAC1 in the vertebrate developmental model Xenopus laevis. PACAP and PAC1 mRNAs were first detected by RT-PCR in stage 16-18 embryos (18 hours after fertilization). Two distinct PACAP precursor mRNAs were identified. One encoded both growth hormone-releasing hormone and PACAP, whereas the other encoded only full-length PACAP. Unlike that in the adult, the latter represented the predominant embryonic PACAP mRNA species. In situ hybridization revealed that PACAP and PAC1 mRNAs were restricted to neural cells. PAC1 gene expression was observed mainly in the ventricular zone in the ventral parts of the prosencephalon, mensencephalon, rhombencephalon, and anterior spinal cord. In contrast, PACAP mRNA was localized exclusively in postmitotic cells in the dorsolateral parts of the rhombencephalon and entire spinal cord. Most PACAP mRNA-containing cells were characterized as Rohon-Beard neurons. Exposure of early embryos to UV irradiation, which ventralizes embryos and inhibits neural induction, reduced the expression of PACAP and PAC1 genes. These results suggest that PACAP may be involved in the early development of the embryonic Xenopus neural tube.

Expression of arginase isozymes in mouse brain.

The two forms of arginase (AI and AII) in man, identical in enzymatic function, are encoded in separate genes and are expressed differentially in various tissues. AI is expressed predominantly in the liver cytosol and is thought to function primarily to detoxify ammonia as part of the urea cycle. AII, in contrast, is predominantly mitochondrial, is more widely expressed, and is thought to function primarily to produce ornithine. Ornithine is a precursor in the synthesis of proline, glutamate, and polyamines. This study was undertaken to explore the cellular and regional distribution of AI and AII expression in brain using in situ hybridization and immunohistochemistry. AI and AII were detected only in neurons and not in glial cells. AI presented stronger expression than AII, but AII was generally coexpressed with AI in most cells studied. Expression was particularly high in the cerebral cortex, cerebellum, pons, medulla, and spinal cord neurons. Glutamic acid decarboxylase 65 and glutamic acid decarboxylase 67, postulated to be related to the risk of glutamate excitotoxic and/or gamma-aminobutyric acid inhibitoxic injury, were similarly ubiquitous in their expression and generally paralleled arginase expression patterns, especially in cerebral cortex, hippocampus, cerebellum, pons, medulla, and spinal cord. This study showed that AI is expressed in the mouse brain, and more strongly than AII, and sheds light on the anatomic basis for the arginine-->ornithine-->glutamate-->GABA pathway.

Axotomy-induced changes in pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP receptor gene expression in the adult rat facial motor nucleus.

It has been demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) promotes the survival of neurons in culture and can inhibit neuronal cell death after experimental injury. Furthermore, peripheral axotomy results in increased PACAP gene expression in sensory and sympathetic neurons, suggesting that PACAP might be a mediator in the injury response in certain parts of the nervous system. However, changes in PACAP expression have not been reported in injured motor neurons, despite the significant problem of motor neuron degeneration in injury and in several neurological diseases. We examined here changes in gene expression of PACAP and two high-affinity PACAP receptors, PAC(1) and VPAC(2), in adult rat motor neurons after facial nerve axotomy by in situ hybridization. PACAP gene expression was very low in facial motor neurons of normal rats. However, a robust time-dependent increase in PACAP mRNA was observed in the facial motor nucleus in most or all axotomized motor neurons. This induction was detectable 6 hr after axotomy, and peaked at 48 hr, when expression on the injured side averaged more than 20-fold higher than that on the contralateral side. Thereafter, PACAP mRNA levels decreased slightly, but remained more than 10-fold elevated for as long as 30 days after axotomy. In contrast to PACAP, gene expression for both the PAC(1) and VPAC(2) receptor was high in facial motor neurons of normal rats. No significant change was observed for VPAC(2) receptor gene expression in facial motor neurons after axotomy, whereas gene expression for the PAC(1) receptor became significantly decreased. The results indicate that the PACAP ligand receptor system is tightly regulated in the facial motor nucleus after axotomy, providing evidence that PACAP may be involved in motor injury responses.

Neural tube expression of pituitary adenylate cyclase-activating peptide (PACAP) and receptor: potential role in patterning and neurogenesis.

Neural tube patterning in vertebrates is controlled in part by locally secreted factors that act in a paracrine manner on nearby cells to regulate proliferation and gene expression. We show here by in situ hybridization that genes for the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and one of its high-affinity receptors (PAC1) are widely expressed in the mouse neural tube on embryonic day (E) 10.5. Transcripts for the ligand are present in differentiating neurons in much of the neural tube, whereas the receptor gene is expressed in the underlying ventricular zone, most prominently in the alar region and floor plate. PACAP potently increased cAMP levels more than 20-fold in cultured E10.5 hindbrain neuroepithelial cells, suggesting that PACAP activates protein kinase A (PKA) in the neural tube and might act in the process of patterning. Consistent with this possibility, PACAP down-regulated expression of the sonic hedgehog- and PKA-dependent target gene gli-1 in cultured neuroepithelial cells, concomitant with a decrease in DNA synthesis. PACAP is thus an early inducer of cAMP levels in the embryo and may act in the neural tube during patterning to control cell proliferation and gene expression.