In 2001-2002, we have made progress in the following areas. 1. 73 genes regulated by PACAP in PC12 cells have been identified by microarray analysis, grouped according to regulation through distinct protein kinase-mediated pathways, and correlated with the dependence on activities of these kinases of several cell physiological responses to PACAP, including neurite extension, cessation of cell proliferation, and expression of functional neurotransmitter traits (Vaudry et al., J. Neurochem., 2002). 2. Bioinformatics tools (pathFinder; the STKE PC12 cell differentiation Connections MAP) have been collaboratively developed that aid in the design and interpretation of cell signaling experiments in PC12 and bovine chromaffin cells. These tools were used to help characterize a cAMP-dependent/PKA-independent pathway regulating gene transcription by PACAP in neuroendocrine cells (Hamelink et al., J. Neurosci. 22, 5310, 2001; Vaudry et al., Science 296, 1648, 2002; Hamelink et al., Ann. N.Y. Acad. Sci., 2002). 3. A PACAP knock-out mouse model has been used to demonstrate the role of this neuropeptide in preventing 'homeostatic fatigue' during metabolic, thermal and ischemic stress (Hamelink et al., Proc. Natl. Acad. Sci. USA 99, 461, 2002; Y. Chen et al., Soc. Neurosci. Abstr. 2002). 4. Combinatorial signaling by PACAP involving the synergistic actions of calcium and cyclic AMP on VIP gene transcription has been mapped to three discrete domains of the VIP gene, a common proximal element responsive to both calcium and cyclic AMP, and upstream and far upstream elements responsive to cyclic AMP and calcium, respectively (C. Hamelink et al., Soc. Neurosci. Abstr. 2002). 5. A long-term project to determine the function of chromogranin A in the biogenesis of large dense-core vesicles (LDCVs), the organells from which release of neuropeptides and hormones in slow transmission occurs, came to fruition in the identification of chromogranin A as the molecular substrate for LDCV biogenesis. This work was carried out in collaboration with the laboratories of Dr. Peng Loh (NICHD) and Dr. Susan Tao-Cheng (NINDS).

In 2003-2004, the Section on Molecular Neuroscience zeroed in on the role of the neuropeptide PACAP as a emergency response peptide in stress-mediated neuronal plasticity in the adrenal gland, in circadian adaptation to extremes of light and dark, and in neuroprotection following stroke. 1. The novel calcium-initiated signaling pathway propagated through calcineurin and CREB and regulating neuropeptide gene expression in bovinechromaffin cells (S. H. Hahm et al., Mol. Pharmacol., 64:1503-1511, 2003) is now being studied with respect to calcium-dependent regulation of PACAP signaling to the VIP gene and other genes involved in post-synaptic (cellular) plasticity mediated PACAP at the adrenomedullary synapse (C. Hamelink et al., J. Neurochem. 88, 1091, 2004). A novel calcium-response element on the VIP gene has been identified that mediates PACAP- and depolarization-induced up-regulation of this gene (C. Hamelink, A. Albert, S. H. Hahm and L. Eiden, in preparation, 2004). 2. The microarray tool mAdb has been refined for use by NIMH and extramural investigators and is being applied to the identification of a set of PACAP response transcripts (genes), neuroprotection transcripts (genes), and the overlap of these two sets in stroke (Y. Chen et al., in preparation, 2004), stress (see C. Hamelink, E. Weihe and L. E. Eiden, Pituitary Adenylate Cyclase-Activating Polypeptide, ed. by H. Vaudry and A. Arimura, 2003, chapter 10), and circadian function (M. Gillette, and P. Lindberg, U. of Illinois, with SMN, in progress; G. Pickard, Colorado State U., with SMN, in progress). 3. A set of cyclic AMP-dependent transcripts (genes) has been identified in neuroendocrine cells that are regulated independently of protein kinase A and may represent a novel neuropeptidestimulated gene ensemble (Grumolato et al., Endocrinol. 144, 2368, 2003; A. Ravni et al, in preparation, 2004). 4. We have continued to examine the chemical coding of neurotransmission in the mammalian nervous system through the use of specific 'isovalent' antibodies directed against the vesicular transporters for biogenic amines, acetylcholine, GABA and glutamate. Histochemical and biochemical studies carried out in collaboration with the laboratory of E. Weihe, Philipps University Marburg, have enabled us to determine strain-specific effects of Mash-1 deficiency on neural development, and the existence of neurons that underlie noradrenergic sweating in humans and dopaminergic neurotransmission in the human gut.

In summary, the work of the Section on Molecular Neuroscience to identify the chemical neuroanatomy, signaling mechanisms and gene targets for slow transmission by PACAP in the nervous system has advanced through the identification of specific novel calcium- and cyclic AMP-dependent signaling pathways, cis-active elements on PACAP-responsive genes, and paraphysiological events in which PACAP signaling is required, including neuroprotective responses in stroke, glucohomeostatic responses in hypoglycemic shock, and circadian periodicity adjustment to extreme changes in, or disruption of, photoperiod. We are in the process of identifying for the first time autonomic neurons, specific to the primate nervous system, that on histochemical grounds support simultaneous cholinergic and noradrenergic neurotransmission. We expect to consolidate these findings and identify additional components of PACAP-specific neurotransmission underlying its role as an emergency response peptide in the coming year, and to demonstrate the existence of neurons in the human autonomic nervous system capable of generating simultaneous slow (noradrenergice) and fast (cholinergic) neurotransmission underlying autonomic behavior specific to primates.