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

Pergamon

PII:

Neuroscience Vol. 88, No. 1, pp. 213–222, 1999 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00240-1

INCREASED EXPRESSION, AXONAL TRANSPORT AND RELEASE OF PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE IN THE CULTURED RAT VAGUS NERVE M. REIMER,*§ K. MOLLER,† F. SUNDLER,† J. HANNIBAL,‡ J. FAHRENKRUG‡ and M. KANJE* *Department of Animal Physiology, University of Lund, Helgonav. 3 B, 223 62 Lund, Sweden †Department of Physiology and Neuroscience, Section for Neuroendocrine Cell Biology, Lund University, Biskopsg. 5, S-223 62 Lund, Sweden ‡University Department of Clinical Biochemistry, Bispebjerg Hospital, DK 2400 Copenhagen, Denmark Abstract––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.  1998 IBRO. Published by Elsevier Science Ltd. Key words: pituitary adenylate cyclase-activating polypeptide (PACAP), nerve regeneration, rat nodose ganglion, vagus nerve, axonal transport, forskolin.

Pituitary adenylate cyclase activating peptide (PACAP) belongs to the vasoactive intestinal peptide (VIP)/secretin/glucagon family of neuropeptides and exists in two biologically active forms, PACAP-27 and PACAP-38. It is normally present in preganglionic fibres of sympathetic neurons and expressed in small- to medium-sized neurons of the dorsal root ganglia (DRG).1,4,19 We and others have found that PACAP is up-regulated in response to axonal injury in a number of neuronal systems including sensory, sympathetic, parasympathetic and enteric ones.12,16,24,27 The up-regulation is rapid and occurs within 24 h following nerve lesions in vivo but is

§To whom correspondence should be addressed. Abbreviations: DRG, dorsal root ganglion; FITC, fluorescein isothiocyanate; GAP-43, growth associated protein43; IR, immunoreactivity/immunoreactive; PACAP, pituitary adenylate cyclase-activating polypeptide; PBS, phosphate-buffered saline; SCG, superior cervical ganglion; TCA, trichloroacetic acid; VIP, vasoactive intestinal peptide.

also evident in cultured ganglia from adult animals.17 The functional significance of this up-regulation is, however, unknown. We have speculated that PACAP, like VIP, may attain a function as a growth promoter in the adult animal during regeneration, since PACAP has been found to stimulate neurite outgrowth from cultured PC12 cells and rat cerebellar neuroblasts.3,6,8 The transient expression of PACAP receptors during synaptogenesis in the developing CNS of the mouse and rat is also consistent with such a role for PACAP.15,20,21 In the present study we investigated the regulation of PACAP expression in a sensory nerve preparation, the vagus nerve of the rat. The aim was to delineate the expression and distribution of this neuropeptide during regeneration. Since conditions which favour non-neuronal cell proliferation have been shown to promote regeneration,22,23 we also studied the effects of PACAP-27 and -38 on proliferation of non-neuronal cells.

213

214

M. Reimer et al. EXPERIMENTAL PROCEDURES

Animals Experiments were carried out according to the European Communities Council Directive regarding care and use of animals for experimental procedures. Adult female Sprague–Dawley rats (Møllegaard Breeding Centre, Denmark) weighing around 200 g were used. The animals were housed in group cages with food and water ad libitum and maintained under a 12 h light/dark cycle. The animals were anaesthetized by an i.p. injection of a 1:2:1 mixture of pentobarbital (Mebumal, 60 mg/ml), diazepam (5 mg/ml) and 0.9% NaCl and killed by heart puncture. Both vagus nerves with their attached nodose ganglion were removed by dissection as previously described.11 Organ culture Dependent on the experiment, the vagus nerves with attached nodose ganglia were cultured as follows: for immunocytochemistry and also for [3H]thymidine incorporation, nerves were mounted on strips of nitrocellulose paper in 35 mm plastic Petri dishes. In some of these preparations a crush lesion was made on the vagus nerve approximately 10 mm distal to the nodose ganglion using a pair of watchmakers’ forceps. For studying axonal outgrowth, vagus nerves with attached nodose ganglia were mounted in 5 ml Matrigel; here, neutralizing polyclonal rabbit antiPACAP antibodies7 (16 µg/ml) were added to some cultures. For in situ hybridization, nodose ganglia were mounted in Matrigel. Freshly dissected ganglia and nerves served as controls and were directly processed for immunocytochemistry or in situ hybridization. In some experiments, the vagus nerves were cut into 3 mm segments and six such segments were cultured together in one culture dish. Forskolin dissolved in dimethylsulphoxide or PACAP was added to some of these preparations and the nerve segments were then processed for [3H]thymidine incorporation (see below). The preparations were maintained at 37C in a humidified atmosphere of 95% O2 and 5% CO2 for various periods of time. Axonal outgrowth Axonal outgrowth from nodose ganglia cultured for 48 h was determined by computer-based image analyses. Pictures of the cultures were taken with a digital camera (Kodak) attached to a phase-contrast microscope. After transfer of the images to a Power Macintosh computer, the analysis was performed using the public domain NIH-image programme written by Wayne Rasband, National Institutes of Health, Bethesda, MD, U.S.A. The areas containing axons emanating from the distal end of the cut vagus nerve were determined and an axonal area index was calculated, which was the quotient between the area of outgrowth from the ipsilateral nerve treated with a polyclonal rabbit antibody against PACAP (ab C523, for reference see Ref. 7) and the untreated contralateral vagus nerve. Immunocytochemistry After the culture period, the vagus nerves with their attached ganglia were removed from the nitrocellulose paper and fixed in Stefanini’s fixative (2% formaldehyde and 0.1% picric acid in phosphate buffer pH 7.2) for at least 4 h. The nerves were then rinsed in phosphate-buffered saline (PBS) and immersed in 20% sucrose in PBS overnight. The preparations were frozen in Tissue-Tek and serially sectioned at 10 µm thickness on a cryostat. The sections were mounted on poly--lysine-treated glass-slides and allowed to air-dry. A mouse monoclonal antibody against PACAP-38 (Mab JHH1), which has been described elsewhere,7 was used at a 1:10 dilution in PBS containing 3% bovine serum albumin

and 0.25% Triton-X. The sections were washed with PBS and incubated overnight with the primary antibody in a humid chamber at 4C. The preparations were washed three times in PBS for 5 min and then incubated with a 1:80 diluted fluorescein isothiocyanate (FITC)-conjugated secondary antibody (goat anti-mouse IgG-FITC; Jackson ImmunoResearch Laboratory Inc., West Grove, PA, U.S.A.) for 45 min at room temperature. After three washes in PBS, the sections were mounted in PBS-glycerol (1:1) and coverslipped. The number of PACAP-immunoreactive (IR) cells was determined in an Olympus BX 60 microscope equipped with proper filter settings for epi-fluorescence. Sections, which were incubated with the secondary antibody only, or where the antibody had been preabsorbed with 10 µM PACAP-38 prior to incubation, served as negative controls and showed no fluorescence. For the determination of axonal regeneration in crushlesioned nerves, a mouse monoclonal antibody against growth-associated protein-43, (GAP-43; Boehringer Mannheim, Germany) was used as primary antibody (diluted 1:50) in combination with the FITC-labelled secondary antibody described above. The site of the crush lesion was determined by examining the sections with phase-contrast microscopy. The site of the crush lesion is indicated by swelling and degeneration of axons distally from the lesion. The nitrocellulose papers, on which the vagus nerve had been cultured were fixed in paraformaldehyde vapours at 75C for 120–160 min. The fixed paper strips were washed and then processed according to a modification of the immunohistochemical procedure (dot blot) described previously.7 After incubation with the PACAP antibody (diluted 1:5) for 24 h at 4C, the nitrocellulose papers were washed in PBS+0.1% Triton X-100 and then incubated with a biotinylated rabbit anti-mouse antiserum (E464, Dako, Copenhagen, Denmark, diluted 1:800) for 1 h. After washing and incubating for 30 min at room temperature in ABC–streptavidin horseradish peroxidase complex diluted 1:125 (Dako, Denmark), the filters were washed and then incubated in biotinylated tyramide using a TSA-kit (Tyramide System Amplification; DuPont NEN., Boston, MA, U.S.A.) diluted 1:100. After another wash in PBS+0.1% Triton X-100 and a new incubation in ABC–streptavidin horseradish peroxidase complex as before, the papers were incubated in a solution of diaminobenzidine (DAB, Sigma, St Louis, MO, U.S.A.) for 15 min. The reaction was terminated by washing the filters with tap water. Control experiments, in which the primary antibody step was omitted, showed no staining of the nitrocellulose papers. In situ hybridization Snap-frozen preparations were sectioned (10 µm), airdried and fixed in 4% paraformaldehyde for 15 min followed by rinsing in PBS 25 min. In situ hybridization was performed as described previously.18 In brief, sections were acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min, then dehydrated and air-dried. A synthetic oligodeoxyribonucleotide, complementary to positions 1038–1068 of the rat PACAP cDNA, was used as a probe. The oligoprobe was labelled with [35]S-dATP with terminal deoxynucleotidinyl transferase, yielding an activity of approximately 2109 c.p.m./mg. Hybridization was performed for 16 h at 37C. After several washes under high stringency conditions and dehydration in a graded ethanol series, slides were air-dried. For autoradiography, slides were exposed to Ilford’s K5 photo emulsion; developed with Kodak D-19 after three weeks, counterstained with Ehrlich’s Haematoxylin and finally mounted and coverslipped. For control purposes, hybridization was performed in the presence of a 100-fold excess of unlabelled probe.

PACAP in the cultured rat vagus nerve Autoradiography After culturing, crush-lesioned nerves were rinsed with Ringer solution and then incubated for 2 h with 50 µCi [methyl-3H]thymidine/ml in Ringer solution at 37C. After extensive rinsing with Ringer solution, the nerves were whole-mounted between two glass slides, one of which was chrome-alum coated. Unincorporated thymidine was extracted with 10% trichloroacetic acid (TCA) overnight. The preparations were briefly rinsed with distilled water and covered with Amplify (Amersham, U.K.) for 1 h. Then nerve preparations were dried and exposed to enhanced chemiluminescence (ECL)-film (Amersham, U.K.) for three to five days before development in Kodak D19 developer. Proliferation of non-neuronal cells as measured by [3H]thymidine incorporation After 48 h in culture, nerve segments were incubated with [3H]thymidine as described above. After extraction of unincorporated thymidine in 10% TCA, nerves were solubilized with 200 ml Soluene (Packard, U.S.A.) overnight. Liquid scintillation cocktail was added and the radioactivity in the samples was measured in a scintillation counter. Some of the nerves were, after incubation in thymidine, cut into 3 mm long segments proximal and distal from the crush and radioactivity in each segment was measured as described above. Statistics The number of PACAP-IR cells in sections of the nodose ganglion was determined and a one-way ANOVA, followed by Dunnett’s multiple range test was used for assessment of statistical differences. All thymidine incorporation experiments were performed with paired controls. First, an experiment/control ratio was calculated from the data, then Student’s one group, two-tailed t-test was used to test if this quotient differed significantly from 1.0. For both comparisons, results were considered to be significant at P<0.05. RESULTS

PACAP-immunoreactivity Figure 1 shows the nodose ganglion immunostained for PACAP. In freshly dissected ganglia, very few cells (Fig. 1a, see also Table 1) exhibited PACAPIR. After 24 h in culture, the number of PACAP-IR cells had increased significantly and 10 times more cells were immunopositive for PACAP compared to freshly dissected ganglia (Fig. 1b). At 48 h the number of PACAP-IR cells had increased further (Fig. 1c). The PACAP-IR in the ganglionic cells had a Golgi-like appearance. After both 24 and 48 h in vitro, PACAP-IR fibres, originating from ganglionic cells, could be observed. An accumulation of PACAP-IR was visible at both the central and the peripheral cut end of the vagus nerve, and the amount of accumulated PACAP-IR increased with time (not shown). PACAP messenger RNA expression In freshly dissected ganglia, a few nerve cells displayed weak PACAP mRNA expression (Fig. 2a). In cultured preparations, more cells were positively labelled for PACAP mRNA and the labelling inten-

215

sity per cell was higher than in the control. The number of cells, which were labelled for PACAP mRNA, appeared somewhat higher at 24 h compared to 48 h in culture (Fig. 2b, c). Axonal transport of PACAP in cultured and ligated nerves In freshly dissected nerves, axons did not display PACAP-IR (not shown). In contrast, in nerves cultured for 48 h, strong PACAP-IR could be demonstrated in axons at the distal end of the vagus nerve (Fig. 3a). PACAP was also found to accumulate proximal to a crush lesion of the vagus nerve after 24–48 h in culture (Fig. 3b). Interestingly, PACAP-IR fibres were found to regenerate through the site of the lesion (Fig. 3b, arrowhead). The accumulation of PACAP at the site of a ligature could be blocked by 0.1 mM vinblastine, a potent inhibitor of axonal transport by virtue of its ability to disrupt microtubules (Fig. 3c). Release of PACAP To study if PACAP was released from the nerves, the nerves with their attached ganglia were cultured on nitrocellulose papers. The assumption was that released PACAP would become attached to the nitrocellulose paper because of the papers’ protein binding capacity. Figure 4 shows such a preparation with the nitrocellulose paper immunostained for PACAP. Staining was observed around the ganglia, but also around the site of the nerve lesion and at the distal cut end of the nerve i.e. sites, where the immunohistochemical experiments had revealed an elevated PACAP content. 3

[H]thymidine incorporation

Autoradiography of a vagus nerve preparations subjected to pulse labelling with [3H]thymidine following 48 h in culture revealed increased labelling proximal to the crush lesion and also at the most distal point of the nerve, i.e. where PACAP was found to be accumulated and released (Fig. 5). The radioactivity in segments proximal and distal to the site of a lesion in crush-lesioned nerves incubated with [3H]thymidine was determined. The amount of radioactivity was significantly higher (P<0.05) in segments proximal to the lesion, as compared to the juxtapositioned segment. In nerves cultured in the presence of 100 nM PACAP-38 the distribution of radioactivity was similar (not shown). One additional set of experiments was performed to test if PACAP affected proliferation of nonneuronal cells. For these experiments, cultured segments of the nerve were used, since in this situation the potential influence of PACAP contained within the ganglionic cells is removed. The addition of PACAP-27 or -38 at concentrations of 50 or 100 nM to such cultured segments

216

M. Reimer et al.

Fig. 1. PACAP-IR in the rat nodose ganglion during culture. Few PACAP-IR cells were found in freshly dissected ganglia (a), whilst there was an increase in the number of PACAP-IR neurons and fibres (arrowhead) after 24 and 48 h in culture (b, c). Note the Golgi-like distribution of the PACAP-IR. Scale bar=100 µm.

PACAP in the cultured rat vagus nerve Table 1. PACAP-immunoreactive neurons in the nodose ganglion Time (h) 0 24 48

PACAP-IR neurons (%)

n

2.60.9 34.26.2* 58.95.8**

4 4 6

The number of labelled cells is expressed as the percentage of the total number of neuronal profiles. Only cells with a visible nucleus were counted. Values are expressed as meanS.D. *P<0.005;**P<0.001.

did not change the incorporation of [3H]thymidine (Table 2). Addition of forskolin, however, significantly (P<0.005) reduced the amount of incorporated [3H]thymidine (Table 2). Axonal outgrowth The outgrowth of axons in cultured preparations subjected to a crush lesion and exposed to 100 nM PACAP-38 during 48 h was determined by staining for GAP-43 in sections of the vagus nerve. The length of GAP-43-IR axons from the site of a crush lesion to the distal end of the axon was measured and compared to control preparations. There was no significant difference between the length of regenerating fibres between PACAP-treated nerves and controls (Table 3). The outgrowth of axons from the vagus nerve preparation cultured in the presence of neutralizing PACAP-antibodies was also studied in Matrigel. After 72 h in culture, the area around the severed end of the vagus nerve, which contained axons, was measured in cultures, where antibodies against PACAP were added and compared to control cultures without antibodies. An axonal area index was calculated, which is the quotient between paired nerves cultured with and without PACAP antibody. The addition of PACAPneutralizing antibodies to the culture did not have any significant effect on the area containing axons (axonal area index=1.451.0 S.D.). DISCUSSION

The present study shows that both PACAP and PACAP mRNA levels are up-regulated in response to axonal injury in the cultured rat vagus nerve, and that PACAP is axonally transported and released at the site of a nerve injury. In addition, we could demonstrate that proliferation of non-neuronal cells, measured as the incorporation of [3H]thymidine, was highest at the site of peptide release. However, addition of PACAP to the cultured vagus preparation did not affect [3H]thymidine incorporation, while forskolin, another potent stimulator of adenylate cyclase (besides PACAP), was shown to markedly decrease the incorporation of [3H]thymidine. Furthermore, initial axonal out-

217

growth from the cultured preparation was unaffected by PACAP-38 or a polyclonal antibody against PACAP. We found that the number of PACAP-IR cell bodies in the nodose ganglion increased during the culture period. This represents an increased expression, since also the mRNA-levels for the peptide were upregulated in neurons of the cultured preparation. In this respect, the nodose ganglion, a sensory ganglion shows the same response as the DRG and also the superior cervical ganglion (SCG).17 The functional significance of the up-regulation of PACAP following axonal injury is unknown. For sensory neurons it has been suggested that the increased synthesis of certain neuropeptides represents a response mechanism to injury aimed at the modulation of nociceptive transmission at the level of the spinal cord.19,25,27 This hypothesis can not apply for the efferent sympathetic neurons of the SCG, as these are motor neurons, suggesting that PACAP may also exert other functions than those of a neurotransmitter/modulator during nerve regeneration. We have speculated that PACAP could be involved in the regeneration process by promoting outgrowth of neurites through direct or indirect mechanisms.24 However, we failed to observe any effect on axonal outgrowth of PACAP and PACAP antibodies, when added to regenerating nerve preparations in vitro. These results imply that PACAP does not possess growth promoting activity, during the early phases of the regeneration process. However, the interpretation of these results warrants some caution. We cannot entirely rule out the possibility that PACAP affects axonal outgrowth, since we measured outgrowth only after a relatively short period. Furthermore, the possibility that the amount of endogenously produced PACAP is sufficient for outgrowth of axons and that not all of it could be neutralized by the added antibody, must be considered. We found that PACAP was axonally transported and that it was released at the site of an injury. Released PACAP may exert effects on cells surrounding the regenerating nerve fibres, e.g., Schwann cells and macrophages. Schwann cells play an important role during regeneration, because they provide the injured neurons with trophic factors and the growing axons with extracellular matrix proteins, like nerve growth factor and laminin, respectively.23 Indeed, Q.-L. Zhang et al.26 showed that Schwann cells increase laminin synthesis in response to VIP, mediated by the action of a low-affinity VIP receptor,26 which probably is the PACAP type 1 receptor. We tested if PACAP affected proliferation of Schwann cells. PACAP failed to affect proliferation in either the whole vagus nerve or cultured nerve segments. In the latter system there should be no endogenous PACAP contribution from neurons. In contrast, forskolin, another potent adenylate cyclase activator, inhibited proliferation of non-neuronal cells as measured by incorporation

218

M. Reimer et al.

Fig. 2. PACAP mRNA labelling in the rat nodose ganglion during culture. A low expression of PACAP mRNA was found in a few neurons in freshly dissected ganglia (a). There was a marked increase both in the number of silver grains per cell and in the number of labelled cells after 24 h (b). PACAP mRNA expression was still elevated after 48 h in culture (c). Scale bar=50 µm.

PACAP in the cultured rat vagus nerve

Fig. 3. Axonal transport of PACAP in the rat vagus nerve. PACAP-IR is shown at the distal end of a vagus nerve cultured with the attached nodose ganglion for 48 h (a). Accumulation of PACAP-IR at a crush lesion (arrow) after 48 h in culture (b). The arrowhead indicates PACAP-IR fibres, which had regenerated through the lesion. Accumulation of PACAP-IR proximal to a crush lesion was blocked in nerves exposed to 0.1 mM vinblastine (c). Scale bar=200 µm.

219

220

M. Reimer et al.

Fig. 4. PACAP immunostaining of nitrocellulose paper, on which a vagus nerve had been cultured for 48 h. The shown result is a representative out of eight experiments. IR for PACAP can be seen. Note the increased PACAP-IR around the ganglion, the distal part of the nerve, and around the site of the crush lesion (arrow). The picture is a photomontage, created by superimposing a photograph of the cultured nerve before removal of the paper and a photograph of the immunostained nitrocellulose paper.

Fig. 5. Whole-mount autoradiography of [3H]thymidine incorporation in the cultured rat vagus nerve. Increased labelling was seen around the ganglion and the segment proximal to the site of the crush lesion (arrowhead). Note also increased amount of incorporated [3H]thymidine at the distal end of the nerve (arrowhead). The arrow indicates the site of the crush lesion. Table 2. Effects of PACAP and forskolin on [3H]thymidine incorporation of non-neuronal cells of the adult rat vagus nerve

Table 3. Effect of 100 nM PACAP-38 on outgrowth of nerve fibres Treatment

Treatment

Concentration (µM)

[3H]Thymidine incorporation ratio

n

PACAP-27 PACAP-27 PACAP-38 PACAP-38 Forskolin

0.05 0.1 0.05 0.1 10

1.21.2 0.80.5 1.51.5 1.30.4 0.30.1*

4 4 4 7 4

Segments of rat vagus nerves were cultured in the presence of the indicated drugs for 48 h. The results are ratios between incorporation in the ipsilateral drug-exposed ganglia and that of the contralateral control, expressed as meansS.D. *P<0.005 Only forskolin affected [3H]thymidine incorporation significantly.

of radioactive thymidine. The latter finding is in agreement with previous results of ours, showing that drugs which elevate cAMP inhibit proliferation of Schwann cells.5

PACAP Control

Axon length (mm)

n

2.80.5 2.60.6

8 6

Rat vagus nerves were crushed and cultured in the presence of PACAP as described in Experimental Procedures. PACAP immunohistochemistry was performed and the length of GAP-43-IR fibres regenerating through a crush lesion was used to measure the effect of PACAP on neurite outgrowth. Values are expressed as meanS.D.

Taken together, these experiments suggest that Schwann cells do not possess the PACAP type I receptor or not sufficient receptors to elicit a cAMP-response, which would inhibit proliferation. Preliminary experiments using [125]I-PACAP-27 for receptor binding on sections of the vagus nerve supported this notion. Interestingly, Delgado et al.2 recently showed that the migratory behaviour of macrophages is markedly

PACAP in the cultured rat vagus nerve

influenced by PACAP; it promotes mobility in these cells and acts as a chemoattractant.2 Macrophages have been shown to have a supporting effect on peripheral nerve regeneration and also CNS regeneration.9,10,13 It is possible that PACAP affects regeneration through attraction of macrophages. Such a mechanism of action would go undetected in our culture system, since the cultured nerve is essentially devoid of macrophages at the time of explantation.14 CONCLUSION

Our present results show that the peptide and mRNA expression of PACAP is up-regulated in

221

the cultured vagus nerve and that PACAP is transported to the site of an injury, where it is released. The functional significance of this upregulation remains to be determined, but could involve stimulation of cells surrounding the regenerating nerve fibres including Schwann cells and macrophages.

Acknowledgements—The present study was supported by grants from the Swedish Medical and Natural Science Research Councils and the Danish Biotechnology Center for Cellular Communication. Thanks are due to Marie Adler Maihofer and Doris Persson for expert technical assistance.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Chiba T., Tanaka K., Tatsuoka H., Dun S. L. and Dun N. J. (1996) The synaptic structure of PACAP immunoreactive axons in the intermediolateral nucleus of the rat. Neurosci. Lett. 214, 65–68. Delgado M., Garrido E., De-La-Fuente M. and Gomariz R. P. (1996) Pituitary adenylate cyclase-activating polypeptide (PACAP-38) stimulates rat peritoneal macrophage functions. Peptides 17, 1097–1105. Deutsch P. J. and Sun Y. (1992) The 38-amino acid form of pituitary adenylate cyclase activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth. J. biol. Chem. 267, 5108–5113. Dun E. C., Huang R. L., Dun S. L. and Dun N. J. (1996) Pituitary adenylate cyclase activating polypeptideimmunoreactivity in human spinal cord and dorsal root ganglia. Brain Res. 721, 233–237. Fex Svenningsen A r . and Kanje M. (1997) Age and sex related effects of forskolin, estrogen and progesterone on Schwann cell proliferation in vitro. J. Neurochem. 69, S226C. Gonzalez B. J., Basille M., Vaudry D., Fournier A. and Vaudry H. (1997) Pituitary adenylate cyclase-activating polypeptide promotes cell survival and neurite outgrowth in rat cerebellar neuroblasts. Neuroscience 78, 419–430. Hannibal J., Mikkelsen J. D., Clausen H., Holst J. J., Wulff B. S. and Fahrenkrug J. (1995) Gene expression of pituitary adenylate cyclase-activating polypeptide (PACAP) in the rat hypothalamus. Regul. Pept. 55, 133–148. Hernandez A., Kimball K., Romanchuk G. and Mulholland M. W. (1995) Pituitary adenylate cyclase-activating polypeptide stimulates neurite growth in PC 12 cells. Peptides 16, 927–932. Hikawa N., Horie H. and Takenaka T. (1993) Macrophage-enhanced neurite regeneration of adult dorsal root ganglia neurones in culture. NeuroReport 5, 41–44. Hirschberg D. L. and Schwartz M. (1995) Macrophage recruitment to acutely injured central nervous system is inhibited by a resident factor: a basis of an immune–brain barrier. J. Neuroimmunol. 61, 89–96. Kanje M. (1991) Survival of the adult rat vagus nerve in culture. Brain Res. 550, 340–342. Larsen J.-O., Hannibal J., Knudsen S. M. and Fahrenkrug J. (1997) Expression of pituitary adenylate cyclaseactivating polypeptide (PACAP) in the mesencephalic trigeminal nucleus of the rat after transsection of the masseteric nerve. Molec. Brain Res. 46, 109–117. Lazarov-Spiegler O., Soloman A. S., Zeev-Brann A. B., Hirschberg D. L., Lavie V. and Schwartz M. (1996) Transplantation of activated macrophages overcomes central nervous system regrowth failure. Fedn Am. Socs exp. Biol. J. 10, 1296–1302. Magnusson S. and Kanje M. (1996) Invading and resident macrophages in the regenerating rat vagus nerve in vivo and in vitro. Restor. Neurol. Neurosci. 9, 161–166. Masuo Y., Tokito F., Matsumoto Y., Shimamoto N. and Fujino M. (1994) Ontogeny of pituitary adenylate cyclase-activating polypeptide (PACAP) and its binding sites in the rat brain. Neurosci. Lett. 170, 43–46. Moller K., Reimer M., Ekblad E., Kanje M. and Sundler F. (1997) Pituitary adenylate cyclase activating polypeptide (PACAP): an important neuropeptide in the peripheral nervous system. In Recent Advances in Microscopy of Cells, Tissues and Organs (ed Motta P. M.), pp. 171–176. Antonio Delfino Editore, Rome. Moller K., Reimer M., Hannibal J., Fahrenkrug J., Sundler F. and Kanje M. (1997) Pituitary adenylate cyclaseactivating polypeptide (PACAP) and PACAP receptor expression in regenerating adult mouse and rat superior cervical ganglia in vitro. Brain Res. 775, 156–165. Moller K. and Sundler F. (1996) Expression of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type-1 receptors in the rat adrenal medulla. Regul. Pept. 63, 129–139. Moller K., Zhang Y.-Z., Ha˚kansson R., Luts A., Sjo¨lund B., Uddman R. and Sundler F. (1993) Pituitary adenylate cyclase activating polypeptide is a sensory neuropeptide: immunocytochemical and immunochemical evidence. Neuroscience 57, 725–732. Sheward W. J., Lutz E. M. and Harmar A. J. (1996) Expression of pituitary adenylate cyclase activating polypeptide receptors in the early mouse embryo as assessed by reverse transcription polymerase chain reaction and in situ hybridization. Neurosci. Lett. 216, 45–48. Shuto Y., Uchida D., Onda H. and Arimura K. (1996) Ontogeny of pituitary adenylate cyclase-activating polypeptide and its receptor mRNA in the mouse brain. Regul. Pept. 67, 79–83. Sjo¨berg J. and Kanje M. (1988) Influence of non-neuronal cells on regeneration of the rat sciatic nerve. Brain Res. 453, 221–226. Sjo¨berg J. and Kanje M. (1990) The initial period of peripheral nerve regeneration and the importance of the local environment for the conditioning lesion effect. Brain Res. 529, 79–84.

222

M. Reimer et al.

24.

Sundler F., Ekblad E., Hannibal J., Moller K., Zhang Y.-Z., Mulder H., Elsa˚s T., Grunditz T., Danielsen N., Fahrenkrug J. and Uddman R. (1996) Pituitary adenylate cyclase-activating peptide in sensory and autonomic ganglia: localization and regulation. Ann. N. Y. Acad Sci. 805, 410–426. 25. Yamamoto T. and Tatsuno I. (1995) Antinociceptive effect of intrathecally administered pituitary adenylate cyclase-activating polypeptide (PACAP) on the rat formalin test. Neurosci. Lett. 184, 32–35. 26. Zhang Q.-L., Lin P.-X., Shi D., Xian H. and Webster de H. F. (1996) Vasoactive intestinal peptide: mediator of laminin synthesis in cultured schwann cells. J. Neurosci. Res. 43, 496–502. 27. Zhang Y.-Z., Hannibal J., Zhao Q., Moller K., Danielsen N., Fahrenkrug J. and Sundler F. (1996) Pituitary adenylate cyclase activating polypeptide expression in the rat dorsal root ganglia: up-regulation after peripheral nerve injury. Neuroscience 74, 1099–1110. (Accepted 20 April 1998)