Kristian Moller

The effects of axotomy and preganglionic denervation on the expression of pituitary adenylate cyclase activating peptide (PACAP), galanin and PACAP type 1 receptors in the rat superior cervical ganglion

The effects of axotomy, chemical sympathectomy and preganglionic denervation on the expression of the neuropeptides, pituitary adenylate cyclase-activating peptide (PACAP), galanin (GAL), and the PACAP type 1 receptor in the rat superior cervical ganglion (SCG) were investigated by immunocytochemistry, in situ hybridization and receptor autoradiography. An antibody recognizing the rat vesicular acetylcholine transporter (VAChT) was used for the detection of preganglionic cholinergic fibers. In the normal SCG, PACAP-immunoreactivity (-IR) was present in numerous, basket-forming, preganglionic nerve fibers, while very few SCG neurons expressed PACAP. GAL-IR was restricted to occasional neurons, and a few nerve fibers, most of which were, in addition, PACAP-IR. PACAP type 1 receptors were expressed in all nerve cell bodies. Axotomy resulted in a rapid and prominent upregulation of PACAP in a large number of nerve cell bodies. There was a large increase also in GAL expression in many nerve cell bodies. In contrast, there was a marked decline in PACAP type 1 receptor expression. Chemical sympathectomy by administration of the catcholaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), gave rise to similar changes. Preganglionic denervation led to the disappearance of PACAP- and VAChT-IR baskets and to the upregulation of PACAP and GAL expression in neurons located close to the entrance of the sympathetic chain, whereas PACAP type 1 receptor expression was not affected. PACAP and GAL were coexpressed in most neurons after axotomy and chemical sympathectomy. Taken together, these results indicate that disruption of target contact and/or the infliction of an injury to the axons of the sympathetic neurons, rather than the preganglionic output, regulates the expression of PACAP, GAL and the PACAP type 1 receptor.

Expression of pituitary adenylate cyclase activating peptide (PACAP) and PACAP type I receptors in the rat adrenal medulla

Pituitary adenylate cyclase activating polypeptide (PACAP) which belongs to the vasoactive intestinal polypeptide family of regulatory peptides, occurs in two variants, PACAP-27 and PACAP-38, and is thought to be an important messenger in both the central and peripheral nervous system. Three cloned G-protein coupled 7 transmembrane spanning receptors bind PACAP with high affinity, one reacting only weakly with VIP (PACAP type I receptor) the other two binding vasoactive intestinal peptide (VIP) with equally high affinity (VIP type I and II receptors). The PACAP type I receptor displays high affinity for both variants of PACAP. In this study, we have investigated the distribution of PACAP and the PACAP type I receptor in the adrenal medulla of newborn and adult rat. Immunocytochemistry revealed, in the adult rat, a dense network of PACAP-immunoreactive nerve fibers terminating on chromaffin cells. Such fibers were few and weakly immunoreactive in the newborn rat. PACAP-immunoreactive medullary cells could not be detected in the adult rat, whereas in the newborn, occasional cells were seen. By in situ-hybridization we detected PACAP type I receptor mRNA in a majority of the adrenal medullary cells of both newborn and adult rat. Receptor autoradiography using 125I-PACAP-27 as ligand revealed binding-sites with a localization virtually identical to the in situ hybridization signal indicating a functional expression of high-affinity type I PACAP receptors in the adrenal medulla. Additionally, in the adult rat, single or clustered large cells, presumably ganglion cells, contained an even higher abundance of PACAP receptor mRNA as well as binding sites than the surrounding chromaffin cells. Our observations on the distribution of PACAP peptide and PACAP receptors in the adrenal medulla suggest that both chromaffin cells and ganglion cells are PACAP targets. The data thus strengthen earlier observations indicating an important regulatory role of PACAP in catecholamine biosynthesis and release. The presence of both ligand and receptors in newborn rats may indicate a role for PACAP in the development of the adrenal medulla.

Pituitary adenylate cyclase activating polypeptide is expressed in autonomic neurons

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide, which is present in neuronal elements of several peripheral organs, and thus a putative neurotransmitter/modulator. In the present study, the expression of PACAP in two parasympathetic ganglia (otic, sphenopalatine) and one mixed parasympathetic/sensory ganglion (jugular-nodose) in rat was characterized by use of in situ hybridization and immunocytochemistry and compared to that of VIP and calcitonin gene-related peptide (CGRP). PACAP and VIP were expressed in virtually all nerve cell bodies in the otic and sphenopalatine ganglia; PACAP and VIP were also expressed in subpopulations of nerve cell bodies in the jugular-nodose ganglion. CGRP was expressed in numerous nerve cell bodies in the jugular-nodose ganglion and in a few, scattered, nerve cell bodies in the sphenopalatine ganglion. In the otic and sphenopalatine ganglia, PACAP- and VIP-like immunoreactivities were frequently co-localized; in the jugular-nodose ganglion, PACAP-like immunoreactivity was frequently co-localized with CGRP-like immunoreactivity in presumably sensory neurons and to a lesser extent with VIP in parasympathetic neurons. Thus, PACAP is synthesized and stored in autonomic parasympathetic neurons as well as in vagal sensory neurons, which provides an anatomical basis for the diverse effects of PACAP previously described.

Pituitary adenylate cyclase-activating peptide (PACAP) and PACAP type 1 receptor expression in regenerating adult mouse and rat superior cervical ganglia in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP), a regulatory peptide belonging to the vasoactive intestinal peptide (VIP) family, is widely distributed in the central and peripheral nervous system. Recent studies have shown that PACAP expression is upregulated in sensory neurons in response to axonal injury. Here we report that PACAP and PACAP type 1 receptors are located in rat and mouse superior cervical ganglia (SCG). PACAP-immunoreactivity (-IR) was demonstrated in preganglionic fibers, whereas only occasional PACAP-IR cell bodies could be observed. In situ hybridization histochemistry using 35S-labeled deoxyribonucleotide probes confirmed that PACAP mRNA was present only in occasional cell bodies. In contrast, PACAP type 1 receptor mRNA was expressed in virtually all cell bodies within the ganglia. After removal and culturing of the SCG for 24 h, there was a marked increase in PACAP mRNA, whilst PACAP type 1 receptor mRNA expression appeared to be downregulated in most nerve cell bodies except for a few scattered neurons displaying a strong upregulation. The total specific binding of PACAP to isolated SCG membranes as assayed by [125I]PACAP-27 binding showed an increase in SCG cultured for 48 h. PACAP-27 neither affected axonal outgrowth from the cultured SCG nor the survival of cells within the SCG. We conclude that PACAP and PACAP receptors are rapidly upregulated in sympathetic ganglia in response to axonal injury and that PACAP may play a role during nerve regeneration.

Increased expression, axonal transport and release of pituitary adenylate cyclase-activating polypeptide in the cultured rat vagus nerve

The expression and axonal transport of pituitary adenylate cyclase-activating polypeptide (PACAP) was studied in the cultured vagus nerve of the rat by immunocytochemistry and in situ hybridization. The number of neurons immunoreactive for PACAP increased markedly within the nodose ganglion during a 24-48 h culture period, as did the number of cells containing messenger RNA for PACAP. PACAP was found to be axonally transported and accumulated at the site of a crush injury. The peptide was also released at this site. Addition of PACAP to regenerating nerves in culture did not affect axonal outgrowth, neither did antibodies against PACAP. Separate experiments showed that neither PACAP-27 nor PACAP-38 affected proliferation of non-neuronal cells measured as the incorporation of [3H]thymidine. In contrast, forskolin, another potent stimulator of adenylate cyclase besides PACAP, dramatically decreased [3H]thymidine incorporation. The results showed that, during regeneration of peripheral nerves, PACAP expression increases and the peptide is transported into the regenerating nerve, where it is released. The functional significance of this release is unknown, but it does not seem to be directly related to the initiation of proliferation of Schwann cells or initial axonal outgrowth.

Phosphatidylethanol; clinical significance and biochemical basis

As shown in figure 1, ethanol can be metabolized by oxidative and nonoxidative pathways (Lieber, 1995). In the oxidative pathway, ethanol is converted to acetal-dehyde through the action of alcohol dehydrogenases, the microsomal ethanol-oxidizing system, or catalase. Acetaldehyde is then subsequently metabolized to acetate through the action of aldehyde dehydrogenases. In one of the nonoxidative pathways of ethanol metabolism, ethanol undergoes esterification with fatty acids by the action of fatty acid ethyl ester synthase to form fatty acid ethyl esters. The nonoxidative ethanol pathway that is the focus of this review is the pathway leading to the synthesis of phosphatidylethanol. Figure 1 shows how ethanol can be inserted as the head group of a phospholipid to form phosphatidylethanol. The transformation occurs through the activation of phospholipase D, mainly on phosphatidylcholine in the presence of ethanol.

Cutaneous field stimulation with moderate intensity current induces nerve proliferation in rat skin but has no effect on dorsal root ganglia.

Cutaneous field stimulation is used to treat localized itch. The aim of the present study was to determine whether such treatment induces neurochemical changes in the dorsal root ganglia in 30 rats using a pan-neuronal marker protein gene-product 9.5 (PGP 9.5) and calcitonin gene-related peptide (CGRP). Electrical stimulation using the currents of either 0.13 mA or 0.53 mA--was given under general anaesthesia for 30 min per day for 10 days. Punch biopsies from the thoracal skín and the corresponding dorsal root ganglia were collected upon sacrifice. Both stimulation regimens induced proliferation of epidermal and dermal nerve fibers in the skin. The mean number of all cutaneous PGP-immunoreactive (IR) nerve fibers after the electrical stimulation with 0.13 mA was increased by 49% (p<0.001), the mean number of epidermal PGP-IR nerve fibers was increased by 25% (p = 0.001) and the mean number of all CGRP-IR nerve fibers was increased by 65% (p<0.001) compared with controls. The mean number of all PGP-IR nerve fibers after the electrical stimulation with 0.53 mA was increased by 39% (p<0.001), the mean number of PGP-IR epidermal nerve fibers was increased by 30% (p = 0.001) and the mean number of CGRP-IR nerve fibers was increased by 65% (p<0.001) compared with controls. Only stimulation with 0.53 mA induced an up-regulation of sensory neuron markers in the dorsal root ganglia. The ratio of positive/negative PGP-IR cells was increased by 17% (p = 0.002), the ratio of positive/negative CGRP-IR cells was increased by 12% (p = 0.003) and the ratio of positive/negative VR1-IR cells was likewise increased by 10% (p = 0.008) as compared with the control ganglia. We conclude that serial cutaneous electrical stimulation by a moderate current in rat does not induce neurochemical changes in the dorsal root ganglia.