Prepro-VIP and preprotachykinin mRNAs in the rat dorsal root ganglion cells following peripheral axotomy

Molecular Brain Research, 6 (1989) 327-330 Elsevier BRESM 80047

327

Short Communication

Prepro-VlP and preprotachykinin mRNAs in the rat dorsal root ganglion cells following peripheral axotomy Koichi Noguchi, Emiko Senba, Yasuhiro Morita, Makoto Sato and Masaya Tohyama Department of Anatomy II, Osaka University Medical School, Osaka (Japan) (Accepted 11 July 1989) Key words: Dorsal root ganglion; Sciatic nerve; Substance P; Vasoactive intestinal polypeptide; mRNA; In situ hybridization; Dorsal rhizotomy

Using in situ hybridization histochemistry, we examined the expression of prepro-vasoactive intestinal polypeptide (VIP) mRNAs and preprotachykinin (PPT) mRNAs which coded for substance P (SP) in the rat dorsal root ganglion (DRG) following spinal nerve transection. VIP mRNAs increased dramatically in the DRG neurons after transection of the peripheral branch of the spinal nerve (sciatic nerve), whereas PPT mRNAs showed a gradual decrease for a few weeks. Dorsal rhizotomy or axotomy of the central branch of DRG cells had little influence on VIP-mRNAs and no effect on PPT mRNA expression. These results demonstrated an activation of VIP biosynthesis in the DRG neurons due to axotomy of the peripheral branch, which was opposite to the reaction of PPT mRNA to the same treatment.

I m m u n o h i s t o c h e m i c a l studies have d e m o n s t r a t e d the presence of a n u m b e r of n e u r o p e p t i d e s in the p r i m a r y sensory afferents. These n e u r o p e p t i d e s are divided into two groups according to the change in i m m u n o r e a c t i v i t y after p e r i p h e r a l axotomy of D R G neurons. O n e group consists of vasoactive intestinal p o l y p e p t i d e (VIP) and a related peptide with Nterminal histidine and C-terminal isoleucine amide ( P H I ) , whose immunoreactivities have been shown to increase after sciatic nerve section in the terminal a r e a of the sciatic nerve in the l u m b a r spinal cord 1'13'17'18. The other group of n e u r o p e p t i d e s , which includes substance P (SP), have been found to show d e p l e t i o n of their immunoreactivities in the dorsal horn 2"6. A n increase in V I P immunoreactivity has also been shown in dorsal root ganglion ( D R G ) cells 1'17'18, and these neurons are attributed to be the source of increased V I P in the dorsal horn 19. In the present study, we have e x a m i n e d the expression of V I P and SP precursor m R N A s in these D R G cells using in situ hybridization histochemistry and d e m o n s t r a t e d different responses in n e u r o p e p t i d e

biosynthesis of a x o t o m i z e d D R G cells. Eighteen male Wistar rats (150-200 g) were used in this study. U n d e r d e e p anesthesia, the right sciatic nerve was exposed and sectioned in the u p p e r leg. A f t e r p o s t o p e r a t i v e survival times of 3, 7, 21 and 35 days, the animals were anesthetized with sodium p e n t o b a r b i t a l (50 mg/kg, i.p.) and perfused intracardially with 2% p a r a f o r m a l d e h y d e in 0.1 M phosphate buffer ( p H 7.2). The bilateral L 5 dorsal r o o t ganglia were excised, p r o m p t l y frozen with powd e r e d dry ice and sectioned in a cryostat into 15-/~m-thick sections. To investigate the effect of proximal a x o t o m y on D R G neurons, dorsal rhizotomy was p e r f o r m e d (n = 3). F o r control experiments, 3 animals received sham operations. Both these groups of animals were killed 7 days after their operations. Oligonucleotide hybridization p r o b e s were provided by N E N / D u P o n t (Boston, M A , U . S . A . ) . T h e VIP p r o b e was p r e p a r e d by labeling an antisense deoxynucleotide sequence for bases 347-375 of the m R N A 14 with [35S]dATP using terminal transferase,

Correspondence: K. Noguchi, Department of Anatomy II, Osaka University Medical School, Nakanoshima 4, Kitaku, Osaka 530, Japan. 0169-328X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

328 TABLE I Percentages o f VIP mRNA- and SP mRNA-positive neurons in the DRG following axotomy

The numbers are calculated from 6 DRG sections from animals at each point in time and are shown as the mean _+S.D. Time after O P (days)

Sciatic nerve axotomy 0 (control) 3 7 21 35 Dorsal rhizotomy 7

Positive neurons (%) V1P mRNA

SP mRNA

0.0 18.3 + 1.9" 28.3 + 2.7* 11.6 + 3.4* 4.1 + 1.9"

23.2 _+3.2 16.2+ 1.1" 14.9 + 2.1" 11.5 + 1.7" 10.5 + 1.5"

1.9+0.9

20.1 _+2.9

*P< 0.01, t-test. yielding a probe specific activity of 1.3 x 109 dpm//~g. The PPT oligonucleotide probe was an antisense sequence for bases 175-204 of the m R N A 8 which coded for SP. This probe was also labeled with [35S]dATP and yielded to 1.3 x 109 dpm//~g of the specific activity. The hybridization procedure employed has been previously described 15'16. To keep consistent data among the animals at each point in time, the hybridization for all animals was run at the same time with the same labeled probes. The specificity of the hybridization signals was confirmed by a competition experiment, in which sections were prehybridized with excess amounts of unlabeled oligonucleotide probes. No appreciable hybridization signal was detected in this experiment. In another control experiment in which a radiolabeled oligonucleotide probe (preproneurotensin probe, 35 base pairs) was used, no hybridization signal was observed. Furthermore, the fact that the VIP and SP probes detect specific m R N A s had been tested in rats by D u P o n t scientists. In the present study, neurons with grain densities at least 5 times higher than the background densities were considered as positive, and the statistical comparison was made with t-test to determine whether the difference between experimental animals of different survival times was significant or not (Table I). No hybridization signal was observed in the D R G cells of the sham-operated control rats when the VIP oligonucleotide probe was used (Fig. 1A, Table i). After sciatic nerve section, prepro-VIP m R N A s

dramatically increased and maintained a high level for a few weeks. Many D R G neurons exhibited an aggregation of grains on their somata, which indicated the presence of VIP m R N A - o l i g o n u c l e o t i d e hybrids (Fig. tB). The VIP mRNA-positive cells were small or medium-sized, and made up 18.3, 28.3, 11.6 and 4.1% of all the D R G neurons at 3, 7~ 21 and 35 days after axotomy, respectively. The increase in VIP m R N A could be detected as early as 3 days and reached a peak after 7 days (Table I). In contrast, dorsal rhizotomy induced only a few VIP-positive neurons (1.9%) 7 days after operation (Fig. 1D, Table I). Fig. 1E shows the autoradiograms of the control D R G with PPT probes which showed many (23.2%) heavily positive neurons. The positive neurons gradually decreased and accounted for 16.2, 14.9, 11.5 and 10.5% of all the D R G neurons at 3, 7, 21 and 35 days after axotomy, respectively. No change in SP m R N A was observed in the D R G after dorsal rhizotomy (Fig. 1H, Table I). The major findings of the present study are that a dramatic increase in VIP precursor m R N A occurs in the D R G following peripheral axotomy, and that D R G cells show the different responses to transection of their peripheral branch and central branch. Considering the electrophysiological studies, the activity of sensory neurons has a tendency to be dependent mainly on the conditions of their peripheral branches rather than on those of their central branches. There are no or only a few VIP-immunoreactive cell bodies found in the D R G of normal rats 5'7"9, which confirms the negative findings of VIP m R N A in the present study. However, peripheral axotomy induces and prolongs synthesis of VIP m R N A in a large number of D R G neurons for as long as several weeks. The increase in V I P m R N A implies an increase in P H I 13 as well as VIP 1"13A7-19, since VIP precursor m R N A codes the sequences of both VIP and PHI 14. The functional significance of VIP response to peripheral axotomy remains to be clarified. Previous biochemical studies w-12 concerning the function of V1P in intact nervous tissue, have suggested that increased VIP may serve to stimulate glycogenolysis and increase glucose utilization in injured nerves. Recently VIP has been shown to stimulate a population of glia to increase the production of substances

329 41P

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Fig. I. Dark-field combined bright-field autoradiograms showing the effects of axotomy on the expression of VIP-mRNAs ( A - D ) and SP-mRNAs ( E - H ) in the rat DRG (L5). Sections are taken from animals that had undergone a sham operation (A,E), 7 days (B,F) and 35 days (C,G) after sciatic axotomy, and 7 days after dorsal rhizotomy (D,H). Note the dramatic increase in VIP mRNAs 7 days after axotomy and the gradual decrease in SP mRNAs in the DRG neurons. Dorsal rhizotomy resulted in only a few VIP mRNA-positive neurons and had no effect on the SP mRNAs. Bar = 200 pm.

~

330 that are necessary for neuronal survival3'4. A neu-

after axotomy 2"6. The pattern of decrease of SP

rotrophic action of VIP is evident in electrically

immunoreactivity is identical to that of other neuropeptides, such as somatostatin (SOM), cholecys-

blocked spinal cord neurons during development, whereas PHI shows no such effect 3"4. It is thought that increased VIP in axotomized D R G neurons may contribute to neural regeneration processes through its glucose metabolism-stimulating and neurotrophic

tokinin (CCK) and fluoride-resistant acid phosphatase ( F R A P ) in primary sensory n e u r o n s 17'18. Ju et al. 7 have reported that some VIP-positive cells that

actions. In contrast to the dramatic increase in VIP m R N A expression, SP m R N A expression decreases gradually in the D R G following axotomy. The decrease in

a p p e a r after axotomy are also immunoreactive to calcitonin gene-related peptide ( C G R P ) . W h e t h e r VIP-positive cells in injured n e u r o n s originally express other neuropeptide m R N A s or not is open to question. The mechanism by which expressions of

SP m R N A synthetic activity may result in the loss of SP-immunoreactivity in the D R G and dorsal horn

VIP and other peptides are differentially regulated need further investigation.

1 Atkinson, M.E. and Shehab, S.A.S., Peripheral axotomy of the rat mandibular trigeminal nerve leads to an increase in VIP and decrease of other primary afferent neuropeptides in the spinal trigeminal nucleus, Regul. Pept., 16 (1986) 69-82. 2 Barbut, D., Polak, J.M. and Wall, P.D., Substance P in spinal cord dorsal horn decreases following peripheral nerve injury, Brain Res., 205 (1981) 289-298. 3 Brenneman, D.E. and Eiden, L.E., Vasoactive intestinal peptide and electrical activity influence neuronal survival, Proc. Natl. Acad. Sci. U.S,A., 83 (1986) 1159-1162. 4 Brenneman, D.E., Neale, E.A., Foster, G.A., d'Autremont, S.E. and Westbrook, G.L., Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide, J. Cell Biol., 104 (1987) 1603-1610. 5 Fuji, K., Senba, E., Fuji, S., Nomura, I., Wu, J.Y. and Tohyama, M., Distribution, ontogeny and projection of cholecystokinin-8, vasoactive intestinal polypeptide and y-aminobutyrate-containing neuron systems in the rat spinal cord; an immunohistochemical analysis, Neuroscience, 14 (1985) 881-894. 6 Jessell, T., Tsunoo, A., Kanazawa, I. and Otsuka, M., Substance P; depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons, Brain Res., 168 (1979) 247-257. 7 Ju, G., H6kfelt, T., Brodin, E., Fahrenkrug, J., Fischer, J.A., Frey, P., Elde, R.P. and Brown, J.C., Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells, Cell Tissue Res., 247 (1987) 417-431. 8 Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S. and Hershey, A.D., Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 881-885. 9 Lundberg, J.M., H6kfelt, T., Nilsson, G., Terenius, L., Rehfeld, J., Elde, R. and Said, S., Peptide neurons in the vagus, splanchnic and sciatic nerves, Acta Physiol. Scand., 104 (1978) 499-501. 10 Magistretti, P.J., Morrison, J.H., Shoemaker, W.J., Sapin, V. and Bloom, EE., Vasoactive intestinal polypeptide

induces glycogenotysis in mouse cortical slices; a possible regulatory mechanism for the local control of energy metabolism, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 6535-6539. 11 Magistretti, P.J. and Schorderet, M., VIP and noradrenaline act synergistically to increase cyclic AMP in cerebral cortex, Nature (Lond.), 308 (1984) 280-282. 12 McCuUock, J. and Kelly, P.A.J., A functional role for vasoactive intestinal polypeptide in anterior cingulate gyrus, Nature (Lond.), 304 (1983) 438-440. 13 McGregor, G.P., Gibson, S.J., Sabate, I.M., Blank, M.A., Christofides, N.D., Wall, P.D., Polak, L.M. and Bloom, S.R., Effect of peripheral nerve section and nerve crush on spinal cord neuropeptides in the rat; increase VIP and PHI in the dorsal horn, Neuroscience, 13 (1984) 207-216. 14 Nishizawa, M., Hayakawa, Y., Yanaihara, N. and Okamoto, H., Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat, FEBS Lett., 183 (1985) 55-59. 15 Noguchi, K., Morita, Y., Kiyama, H., Ono, K. and Tohyama, M., A noxious stimulus induces the preprotachykinin-A gene expression in the rat dorsal root ganglion: a quantitative study using in situ hybridization histochemistry, Mol. Brain Res., 4 (1988) 31-35. 16 Noguchi, K., Morita, Y., Kiyama, H., Sato, M., Ono, K. and Tohyama, M., Preproenkephalin gene expression in the rat spinal core after noxious stimuli, Mol. Brain Res., 5 (1989) 229-236. 17 Shehab, S.A.S. and Atkinson, M.E., Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons, Brain Res., 372 (1986) 37-44. 18 Shehab, S.A.S. and Atkinson, M.E., Vasoactive intestinal polypeptide increases in areas of the dorsal horn of the spinal cord from which other neuropeptides are depleted following peripheral axotomy, Exp. Brain Res., 62 (1986) 422-430. 19 Shehab, S.A.S., Atkinson, M.E. and Payne, J.N., The origins of the sciatic nerve and changes in neuropeptides after axotomy: a double labeling study using retrograde transport of True blue and vasoactive intestinal polypeptide immunohistochemistry, Brain Res., 376 (1986) 180-185.