Martina Kvist Reimer

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.

Markedly reduced chronic nociceptive response in mice lacking the PAC1 receptor

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been proposed to have a role in nociception. Here we have used the formalin test, thermal laser stimulation and mechanical von Frey stimulation to investigate possible alteration of PAC1−/− mice nociceptive behaviour. Our finding, that PAC1−/− mice have a substantial, 75% decrease in nociceptive response during the late phase, provides clear evidence that the specific PACAP-receptor PAC1 is involved in the mediation of nociceptive responses during chronic conditions such as inflammation. PAC1−/− mice had small or no changes in the response to mechanical and thermal laser stimulation. This suggests a limited, if any, involvement of PAC1 in nociception after short-lasting stimuli. Injury-induced changes in DRG neuropeptide expression were more pronounced in PAC1−/− mice, implying neuroregulatory functions of PAC1.

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.

Local growth factors are beneficial for the autonomic reinnervation of transplanted islets in rats

Introduction Transplanted islets, being avascular and denervated, receive blood vessels and nerves from the recipient. Reinnervation may account in part for the normalization of islet function in islet transplants. Whether reinnervation is possible to augment is not known. Aims and Methodology To explore whether reinnervation of transplanted islets is augmented by local addition of growth factors to the graft, syngeneic islets were transplanted to the pancreas of streptozotocin-diabetic Lewis rats with or without pellets locally releasing nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), alone or in combination. The pellets released growth factors for 14 days at a rate of 20 ng/day. After 7 weeks, pancreatic tissue was processed for immunofluorescence of insulin and the neural markers neuropeptide Y (NPY) and tyrosine hydroxylase (TH). Results Islets were larger and more numerous after treatment with NGF (p = 0.024) and with NGF in combination with VEGF (p = 0.044). Similarly, immunostaining for TH and the C-terminal flanking peptide of NPY (C-PON) was more pronounced after treatment with NGF in combination with VEGF than in controls (both p < 0.05). Conclusion Local growth factor treatment has a beneficial effect on autonomic reinnervation as well as islet integrity and survival of the graft after islet transplantation in rats.

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.

Peripheral but not central axotomy promotes axonal outgrowth and induces alterations in neuropeptide synthesis in the nodose ganglion of the rat

We investigated the effects of central and peripheral axotomy of the sensory neurons in the nodose ganglion on neurite outgrowth and neuropeptide expression. Axonal outgrowth was studied in ganglia subjected to a conditioning lesion of the vagus nerve 6 days prior to in vitro explantation. In such cultures, a conditioning effect, i.e. a shorter initial delay and faster axonal outgrowth, was observed after peripheral axotomy, while central axotomy had no effect. Neuropeptide expression was measured by immunocytochemistry 3 days after axotomy. Peripheral axotomy induced an increase in the number of neurons expressing the C‐terminal flanking peptide of neuropeptide Y (C‐PON), galanin (GAL) and vasoactive intestinal peptide (VIP). In contrast, central axotomy did not affect neuropeptide expression. These results suggest that both axonal outgrowth and expression of neuropeptides in the sensory neurons of the nodose ganglion could be regulated by the contact of the cells with their peripheral, but not their central targets.

Regeneration of axons from central neurons into microchips at the level of the spinal cord

AXONS from central neurons can regenerate into the tissue matrix formed within a silicone tube capped with two pieces of peripheral nerve, one of which had been sutured to a lesion in the spinal cord. Such axons can grow through a transversely positioned microchip in the tube. These observations suggest that it is feasible to establish functional contact between external electronic equipment and regenerating central nervous axons making it possible to monitor and control their electrical activity. The findings open new perspectives for restoration of motor and sensory functions following spinal cord lesions.