Calcitonin gene-related peptide immunoreactivity in neuronal perikarya in dorsal root

324 BRES 24118

Brain Research, 519 (1990) 324-328 Elsevier

Short Communications

Calcitonin gene-related peptide immunoreactivity in neuronal perikarya in dorsal root Srdija Jeftinija and Ksenija Jeftinija Department of Veterinary Anatomy, Iowa State University, Ames, IA 50011 (U.S.A.)

(Accepted 20 February 1990) Key words: Spinal dorsal root; Organotypicculture; Calcitonin gene-related peptide; Immunohistochemistry;Primary afferent; Dorsal root neuron

Calcitonin gene-related peptide (CGRP) has been localized in neuronal cell bodies in dorsal root ganglia (DRG) and in fibers of the dorsal horn (DH), where it may be involved in transmission of a sensory signal. The capacity of neurons from DRG and dorsal roots (DR) to produce CGRP in culture was examined in this investigation. CGRP-Iikeimmunoreactivitywas observed in cultured neuronal cell bodies in the DRG and DR. CGRP-positive cell bodies in the DR explains why extirpation of the DRG fails to eliminate CGRP-positive fibers from the DH. Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide encoded by a messenger RNA that results from alternative processing of the calcitonin gene primary transcript 1"14. CGRP-like immunoreactivity (IR) and CGRP m R N A have been localized to various neuronal groups, including neurons of the dorsal root ganglia (DRG) and ventral horn of the spinal cord 6'13. Although the dorsal horn (DH) contains CGRP-IR, no CGRP-IR cell bodies have been reported in the DH 2'6. Vigorous attempts have been made to determine the source(s) of CGRP-IR in DH. It is generally agreed that most CGRP is from sensory fibers arising from neurons in D R G 2'6'16. However, removal of the D R G does not produce complete disappearance of CGRP-IR fibers in the dorsal horn, which has cast doubt on the assumption that all CGRP positive fibers in the dorsal horn are of primary afferent origin 2'16. The displaced or aberrant D R G cells have been described as neurons with their cell bodies located in the proximal peripheral nerve or distal ventral root 3'15A7. By applying immunocytochemistry to organotypic cultures of dorsal roots (DR) we have demonstrated the presence of CGRP-IR somata in dorsal roots. These cells may be the source of the CGRPpositive fibers that remain in the spinal dorsal horn after extirpation of the D R G 2"16, and provide the missing link in support of the hypothesis that all CGRP in the rat lumbar dorsal horn is of primary afferent origin. Two types of organotypic roller tube or Petri dish cultures from postnatal day 8 to adult rats were established using the modified methods described by Gath-

wiler and Dells et al. 4'5. One was a horizontal spinal cord slice with attached D R with or without attached DRG. The other one was dorsal and ventral roots with or without DRG. Isolated explants were placed on glass coverslips that had been completely covered with chicken plasma, coagulated by adding thrombin solution. The coverslips bearing the explants were either inserted into a 10 ml tissue culture tube with 1 ml of medium and placed in a roller tube assembly rotating at 15 rotations per h, or placed into 35 mm Petri dishes. The cultures in the tubes were fed at weekly intervals, and 0.5 ml of medium was replaced every 48 h in the Petri dishes. The culture medium was 25% horse serum, 25% Earl's balanced salt solution, 50% basal medium eagles with glucose (6.4 mg/ml). All cultures were maintained at a temperature of 36 °C + 0.5 °C, and the cultures in Petri dish were kept in a humid 5% CO 2 atmosphere. Immunocytochemistry (ICC): cultures were fixed in 4% paraformaldehyde for 2-4 h, then washed in 30% sucrose in 50 mM potassium phosphate buffer (KPBS) for 24 h. Immunoreactive CGRP was demonstrated by using a modification of the avidin-biotin-peroxidase complex (ABC) technique s. Cultures were washed 3 times for 10 min each in 50 mM KPBS. Inactivation of endogenous hydrogen peroxidase was achieved by rinsing cultures in 0.3% hydrogen peroxide in 50 mM KPBS for 15 min. Next, the cultures were incubated for 30 min in 3% normal goat serum in 50 mM KPBS, 1% bovine serum albumin (BSA) and 0.4% Triton X-100. Primary antiserum against CGRP was diluted to 1:5000 in 50 mM

Correspondence: S. Jeftinija, College of Veterinary Medicine, Department of Veterinary Anatomy, Iowa State University of Science and Technology, Ames, IA 50011, U.S.A.

0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

325 K P B S , 1% B S A and 0.4% Triton X-100. A f t e r incubation with p r i m a r y antiserum for 24 h at 4 °C, the cultures were washed 10 times for 10 min each in 50 m M KPBS and 0.02% Triton X-100. Then they were incubated for 1 h in anti-rabbit biotinylated antibody in 50 m M KPBS, 1 % B S A and 0.02% Triton X-100, washed 4 times in 50 m M K P B S and 0.02% Triton X-100, and incubated for a n o t h e r h o u r in an avidin-biotin-peroxidase conjugate ( A B C ) . A n t i - r a b b i t a n t i b o d y and A B C were applied at r o o m t e m p e r a t u r e using dilutions r e c o m m e n d e d by the supplier (Vectastain, Vector Lab.). Staining was perf o r m e d adding 250 mg nickel and 4 mg 3,3"-diaminoben-

zidine to every 10 ml of 0.1 M sodium acetate used. Controls were processed by omitting the specific antiserum or by using antisera p r e a b s o r b e d with an excess of the immunogen. N o positive immunostaining was detected on the control tissues. Perikarya of sensory n e u r o n s i m m u n o r e a c t i v e for C G R P were identified in dorsal roots that had been grown in organotypic cultures for 5 - 1 4 days. The presence of positive C G R P neurons was not d e p e n d e n t on the presence of a dorsal r o o t ganglion (Fig. 1). The neurons were r a n d o m l y located in the D R ; some were located m o r e distal (towards D R G ) and some were m o r e

A

C

Fig. 1. A: low-power photomicrograph showing a horizontal spinal cord (SC) slice with associated dorsal roots (DR) and dorsal root ganglion (DRG) on one of the lumbar dorsal roots of an 18-day-old rat. The explant was incubated in a rotating test tube for 1 week. Tissue was

processed to demonstrate immunoreactivity to CGRP, visible as black-stained fibers and cell bodies. B: higher-power photomicrograph of the region indicated by the square in A, showing CGRP-IR somata in both roots. Notice the much larger number of CGRP-IR axons in the root with the DRG attached. C: high-power micrograph of the area marked in B, showing immunopositive neurons in a root without an attached ganglion. Notice the visibility of a nucleus with nucleolus, and the granular appearance of CGRP-positive material at this highest magnification. Scale bar: A, 1.0 mm; B, 200/~m; C, 100/zm.

326 proximal (towards the spinal cord) (Fig. 1). Some neurons were located individually; other lay in small clusters of 2-6 neurons (Figs. 1-3). All of the cells in one cluster were of the same size (Figs. 1 and 2). The diameters of the neurons located in DR and D R G on the same root were similar. On the basis of light microscopic analysis the cells appear to be mainly bipolar. Although in some cases it could not be determined whether the cell was bipolar or pseudounipolar, no more than two neurites were seen growing away from any perikarya (Figs. 1 and 2 E - G ) . The immunoreactive neurites ex-

tended through the length of the section of dorsal root, and CGRP-immunostained varicosities were regularly observed (Fig. 1). To establish if CGRP-positive neurons are present only at certain developmental stages, cultures were prepared from rats aged 8 days to adult. One 4-month-old male rat was sacrificed, and the D R from L3-L6 roots on both sides were dissected out and cultured for 2 weeks. Immunoreactive neurons were found in 4 roots (Fig. 2A-C). On the basis of these observations it appears that CGRP-positive neurons are present within D R throughout life. To investigate the

D

A

E

C I ~" :

Fig. 2. A: low-power photomicrographs of a cluster of neurons that reacted positive to C G R P antiserum in a l u m b a r dorsal root isolated from an adult rat and cultured for 14 days in a test tube. B and C: higher-power micrographs of the same cluster of cells. D: photomicrograph of a cluster of C G R P - I R neuronal cell bodies in a dorsal root isolated from an 18-day-old rat and cultured for 7 days. E: micrographs of two immunopositive bipolar neurons in a dorsal root from a 13-day-old rat cultured for 6 days. F: single bipolar n e u r o n from an 18-day-old rat cultured for 7 days. G: groups of paired C G R P - I R cell bodies of pseudounipolar neurons. Scale bar: A , 1.0 m m ; B,D, 200/~m; C , E - G , 100 /~m.

327 possibility that C G R P - p 0 s i t i v e neurons reach the D R as a result of n e u r o n a l migration induced by culturing or i n d u c e d by the injury to the neurons by extirpation, we e x a m i n e d 12 freshly dissected l u m b a r dorsal roots from 18-day-old rats. Single cells or clusters of C G R P - p o s i t i v e n e u r o n s were found in 5 of the roots (Fig. 3 A - C ) . During the process of tissue culturing all fibers that have been severed from their n e u r o n a l cell body loose their immunoreactivity (Fig. 3D). This decreases the total reactivity in the r o o t and facilitates visualization of positive neurons and fibers. H o w e v e r , in material that had not been cultured, the thickness of the tissue and the large n u m b e r of positive bundles m a d e visualization of positive s o m a t a very difficult. N o C G R P - p o s i t i v e neurons were found in cultured ventral roots (n = 25). Because C G R P - I R fibers

d i s a p p e a r within 1 w e e k if they are not in contact with their neuronal somata, no i m m u n o r e a c t i v e fibers were present in ventral root. C G R P - p o s i t i v e fibers were also absent in V R in cultures w h e r e D R and V R were left in contact with the D R G (Fig. 3B), suggesting that C G R P positive fibers originating from D R G do not e n t e r the VR. The significance of these results is that for the first time the presence of C G R P - I R cell bodies is d e m o n s t r a t e d in the D R . These cells explain the failure, o b s e r v e d in our l a b o r a t o r y and r e p o r t e d by others 2'6 of D R G removal to completely eliminate C G R P - I R from fibers in the D H . These d a t a provide the missing evidence supporting the suggestion that p r i m a r y afferent neurons are the only source of C G R P - p o s i t i v e fibers in the dorsal horn. Many

A

I

C

Fig. 3. A: low-power micrograph of a cluster of CGRP-positive neurons in a freshly dissected DR. Individual axons are difficult to resolve because many axons are labeled at varying planes of focus. B: low-power micrograph of a CGRP-IR neuron in a freshly dissected DR from an 18-day-old rat. C: higher-power micrograph of the neuron shown in B. D: absence of immunopositive cell bodies and fibers in a DR collected from a 30-day-old rat and cultured for 7 days. Notice the accumulation of immunoreactive material on the cut ends. E: immunostaining with CGRP antiserum reveals immunoreactive neuronal perikarya and fibers in the rat DRG and DR, and the absence of any immunopositive structures in the ventral root, although it has remained in contact with the dorsal root ganglion. Scale bar: A, 200/~m; B, 160/~m; C, 80/~m; D, 0.5 mm; E, 1.0 ram.

328 studies claim to demonstrate surviving fibers in proximal stump of dorsal rhizotomies or D R gangliectomies9' 10,12,18. It was assumed that their cell bodies are in spinal

Coggeshal111. The present study has shown that there are neuronal cell bodies in the D R which may be the origin of fibers surviving rhizotomy.

cord and the fibers represent dorsal root efferents. Hinsey's 7 argument that fibers surviving a dorsal rhizotomy were regenerating or sprouting fibers from nearby nerves and that there are no dorsal root efferents in mammals

have

been

confirmed

by

Langford

and

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Thanks to Lisa Takes for assistance in performing the immunocytochemistry, and to Drs. Jeanine Carithers and Virginia Seybold for very helpful suggestions with preparation of the manuscript. This work was supported in part by NIH Grant NS 27751 and USDA Grant PL95-113.

10 Kure, K., Saegusa, G., Kawaguchi, K. and Shiraishi, K., On the parasympathetic (spinal parasympathetic) fibers in the dorsal roots and their ceils of origin in the spinal cord, Q. J. Exp. Physiol., 20 (1930) 51-66. 11 Langford, L.A. and Coggeshall, R.E., Branching of sensory axons in the dorsal root and evidence for the absence of dorsal root efferent fibers, J. Comp. Neurol., 184 (1979) 193-204. 12 Okelberry, A.M., Efferent fibers in the lumbar dorsal roots of the dog, J. Comp. Neurol., 62 (1935) 1-16. 13 Rethelyi, M., Metz, C.B. and Lund, P.K., Distribution of neurons expressing calcitonin gene-related peptide mRNAs in the rat brain stem, spinal cord and dorsal root ganglia of rat and guinea-pig, Neuroscience, 29 (1989) 225-239. 14 Rosenfeld, M.G., Mermod, J.-J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W. and Evans, R.M., Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing, Nature (Lond.), 304 (1983) 129-135. 15 Sherington, C.S., On the anatomical constitution of nerves, J. Physiol. (Lond.), 17 (1894) 211-258. 16 Traub, R.J., Solodkin, A. and Ruda, M.A., Calcitonin generelated peptide immunoreactivity in the cat lumbosacral spinal cord and the effects of multiple dorsal rhizotomies, J. Comp. Neurol., 287 (1989) 225-237. 17 Windle, W.E, Neurones of the sensory type in the ventral roots of man and of other mammals, Arch. Neurol. Psychiatry, 26 (1931) 791-800. 18 Young, J.Z. and Zuckerman, S., The course of fibers in the dorsal nerve roots in Macaca mulatta, the rhesus monkey, J. Anat., 71 (1937)447-457.