The population of the dorsal root ganglion cells which have central processes in ventral root and their immunoreactivity

Brain Research, 402 (1987) 393-308 Elsevier

393

BRE 22008

The population of the dorsal root ganglion cells which have central processes in ventral root and their immunoreactivity Xiu-bin Fang* Department of Anatomy, UCLA Center for the Health Sciences, Los Angeles, CA 90024 (U.S.A.)

(Accepted 16 September 1986) Key words: Dorsal root ganglion; Ventral root; Afferent fiber: Bifurcation projection; Calcitonin gene-related peptide; Rat

Axonal transport of fluorescent dyes applied to the cut distal ends of rat L~ dorsal and ventral spinal roots was studied in order to characterize the population of dorsal root ganglion (DRG) neurons emitting axons entering the ventral root. Ca. 9% of DRG neurons, principally of small or medium size, can be labeled from the ventral root, and 55% of these display immunoreactivity for the most ubiquitous DRG neuropeptide, calcitonin gene-related peptide (CGRP). Attempts to simultaneously label cut dorsal and ventral roots revealed that double labeling was exceedingly rare and that dorsal root labeling was markedly reduced. The results are consistent with previous reports of small DRG cells emitting axons which loop into the ventral root before entering the spinal cord via the dorsal root. The few cells labeled simultaneously from cut dorsal and ventral roots indicate that axonal bifurcation distal to the DRG is very rare.

Many anatomic and physiologic studies have demonstrated that a large n u m b e r of fibers in mammalian ventral roots are derived from sensory ganglion cells3.4,7.11,15,2o,21,27 many of which loop backwards to

dorsal and ventral roots were soaked separately with FB and Diamidino yellow-dihydrochloride (DY) in

enter the spinal cord through the dorsal root 2"23"25, in-

the same animal, and the dorsal root ganglia were examined. The relation of D R G cells with axons projecting into the ventral root to peptidergic axons was

dicating that the doctrine of functional separation of

examined for colocalization with the most widely dis-

the spinal roots is still valid s. In the L 7 ventral root, the proportion of u n m y e l i n a t e d axon profiles is sub-

tributed thin-fiber neuropeptide, calcitonin gene-related peptide (CGRP).

stantially less in kittens than in adult cats, which means that the proportion increases with age 24 and

325 g, were anesthetized i.p, with Equithesin and an

most of them arise postnatally. Some investigations suggest that only a few afferent fibers in the ventral root enter the spinal cord, course from ventral horn to dorsal horn t72° or innervate the pia mater 5'"~22. The function of these fibers is still u n k n o w n and it seemed important to determine whether many cells of the dorsal root ganglion ( D R G ) give rise to central processes in ventral roots and to explore their immunohistochemical characterization. To this end we initiated a study ill which the distal cut ends of ventral roots were soaked with the fluorescent dyes True blue (TB) or Fast blue (FB), or the distal cut ends of

Eighteen adult rats, ranging in weight from 200 to L3-L 4 laminectomy was performed. The experiments were divided into 3 groups. (1) With the aid of a dissecting microscope, L 4 dorsal roots were cut unilaterally at 0 . 7 - 1 . 0 cm proximal to the D R G . The cut ends of the distal stumps of dorsal roots were soaked with 5% FB in distilled water (7 animals, 2 - 3 days survival) in a plastic tube the open end of which was sealed with heat before surgery and the other with vaseline jelly after placing the distal stump into the tube. In one animal we used propidium iodide (PI), but found that the pattern of labeling was patchy. The ventral roots remained intact. (2) Using the

* Present address: Department of Anatomy, China Medical University, Shenyang, People's Republic of China. Correspondence: L. Kruger. Dcpartment of Anatomy, UCLA Center for the Health Sciences, Los Angeles, CA 90024, U.S.A.

394 same procedures as above, the cut distal stumps of La ventral roots were soaked with 5% TB (3 animals. cases l - 3 , 5 - 7 days survival l or 2% FB (1 animal. case 4, 5 days survival) in distilled water. (3) For double-labeling, the cut ends of the distal stump of La ventral roots were soaked with 2% DY and the cut ends of the distal stump of La dorsal roots were soaked with FB (6 animals. 3 - 4 days survival) ipsilaterally. The animals were anesthetized with an overdose of Equithesin and perfused intracardially with 0.9% saline followed by freshly prepared 4% paraformaldehyde (PFA) in phosphate buffer (PB, p H 7.4). Both L 4 D R G (the right for control) were removed immediately after perfusion and refixed in 10% sucrose in P F A for 1 h and then stored overnight by immersion in 20% sucrose in PB in the refrigerator. The ganglia were embedded in O C T embedding medium and frozen in dry ice. Frozen sections were cut at 20 um on a cryostat, collected onto gelatinized glass slides and stored at - 2 0 °C. After choosing every fifth consecutive section and 5 sections for each ganglion in group 2, pictures were taken under the fluorescent microscope and montaged. Labeled cells were then counted and measured. The right L4 D R G of every animal was cut and processed for control. No labeled cells were seen. except for some background low-level fluorescence. Immunoperoxidase staining, using rabbit antibodies raised to rat C G R P (a gift from Dr. Catia Sternini), was performed in the following manner. Sections, pictures of which were perviously taken in group 2 (ventral root labeled), were rinsed in phosphate-buffered saline (PBS) for 4 x 5 rain. incubated in 2% normal goat antiserum in 0.3% Triton X-100 in PBS for about 30 rain and, without rinsing, directly incubated overnight at room temperature using antirabbit C G R P diluted 1:3000 in PBS. The sections were washed 4 x (10 min for each) with PBS. then incubated for 1 h in biotinylated goat anti-rabbit secondary antibody in PBS. After 4 10-rain washes in PBS, the sections were incubated with avidin-biotin peroxidase complex ( A B C reagent) in PBS for 1 h and then followed by the same wash procedure. To ensure method specificity, some sections were incubated with normal rabbit serum and then put through all t~,e procedures described above. No cells were labeled. The specificity of the antiserum to C G R P

was verified by the lack of staining in the sections incubated with antiserum preabsorbed with I(F 3 M synthetic C G R P (Bachem). The secuons were reacted with a solution of (t. lC2 3.3'-diaminobenzidine tetrahydrochloride ( D A B I in Tris-HCl buffer pH 7.2. for 5 min and hydrogen peroxide (().06%j was added for another 5 min. The~ were then rinsed 3 x 10 min in PBS. counterstained with Toluidine blue. dehydrated and coverslipped with 1tistoclad. Photomicrographs of CGRP-positive cells were montaged and compared with the pictures ot TB labeled cells from the same sections in order to determine the number of both TB- and CGRP-doublelabeled cells. In the colocalization studies, ont~ cells with visible nuclei were counted and sized by measuring two perpendicular diameters with the aid of an eyepiece graticule. The mean of these two measurements was taken for each cell diameter and compared with the total number and size distribution of L~ D R G neurons in selected sections of 4 animals. The counted cells were arbitrarily divided into 3 size groups: large 1>511 t~m~ 21)<~'~.medium 135-5~ mn~ 22%. and small (<35 ~ml 58C~. The total number of cells in selected sections l in each case. out of 25 sections, every fifth) were counted and measured in order to determine the percentage of labeled cells. Virtually all D R G cells were distinctly labeled when the distal cut ends of the dorsal root were soaked with FB and the ventral root was intact (Fig, I I The entire size range displayed unambiguous fluorescence of the transported dye. but small cells were most intenselv labeled with FB as well as with other transported dyes (TB and Pll. By contrast, only ca. 9 . l % (n = 579) of D R G cells were labeled by soaking the distal cut ends of ventral roots with FB (Fig. 2) or TB (Fig. 3), of which 6.8% were small. 1.7% medium and 0.6% large. Clearly, small cells predominate and the proportion of labeled cells could be determined readily by identifying labeled cells in relation to the total population seen in a Nissl stain of the same section (Fig. 2a). In 3 animals, the sections labeled with TB were photographed and subsequently studied for C G R P like immunoreactivity (IR) using the A B C method for demonstrating reaction product. The results (Table I) indicate that slightly more than half (55%) of the sparsely distributed cells labeled from the ven-

Fig. ! Photomicrograph section of rat L, DRG. FB application to the distal stump of dorsal roots, with ventral root intact, re-

sults in Labeling of almost all the cells and most intensely in small-sized cells. x 116.

Fig. 2. L, DRG after the distal stump of ventral roots had beeo soaked with FB. Dorsal roots were intact. A: a few small and medium-sized cells labeled with FB. B: the same section Nisslstained. The proportion of labeled cells was determined by identifying labeled cells in relation to the total population seen in Nissl stain (arrows). X 116.

tral root with TB also displayed CGRP-IR and the vast majority are small neurons (Fig. 3) which constiTABLE I

Them&r andpercentage tI S.E.M.) of ~~~~~~~~~~~~~ {,-I

cells in TB-hheled C& n = 4%. total

Fig. 3. L, DRG. A: fluorescent labeled cells after the distal stump of VentraL ruots was soaked with TB with the dorsal roots intact. B: CGRP-’lmmunoreactive neurons from the same settion as A. Comparing A and B, only some of the sparselv distributed ~dls labeled from the ventral root with TB also di&ayed CGRP-IR (arrows). x 15-7. Fig. 4. A M&stained section in which a neuron (arrow) is dou_ ble-labeled (inset) by FB applied to cut distal dorsal root and DY applied to cut distal ventral root, the latter appearing as a yellow nuclear ring. x456.

number of TB-labeled cells in selected section in

3 cases. - -_-_

SmaN - - - - - n 175 PC%‘) 39.9 -+ 3.7

Medium 58 13.2 5 4.4

-- _. Toolal Large - - - - - - - - - ~241 8 1.8 _e 4.9 55.0 i 3.2

toted 324% of rhe total DRG population in these 3 animls (Table JJ). Large and intermediate size cells were less numerous and often more lightly stained (Fig. 3a).

396 TABLE II The number and percentage (+_ S. E. M. ) o f CGRP-positive ~P) cells in L 4 D R G

n = 5078, total number of cells in selected sections in 3 cases. Small

n P(%)

t 201 23.6+1,2

Medium

276 5.4_+1.3

Large

167 3.3_+ 1.3

Total

t 644 32.4~_1.2

A n u m b e r of attempts were m a d e to double-label D R G cells from both roots simultaneously by applying FB to the cut distal ends of dorsal roots and, s~milarly, D Y to corresponding ventral roots. The result was largely disappointing in that clearly doublelabeled D R G cells were rarely detected m a given section. A few small cells were clearly l a b e l e d with a light yellow ring a r o u n d the nucleus (Fig. 4), indicating D Y uptake. These constitute only a small fraction of 1% of total D R G neurons, but the exact n u m b e r may be a low estimate because the mtense labeling with FB often obscured the light yellow of D Y . rendering exact counts unreliable. Labeling from the dorsal root with FB after cutting the ventral root substantially r e d u c e d the n u m b e r of labeled neurons c o m p a r e d with the virtually complete labeling from dorsal root with the ventral root intact. This was variable in different animals and is p r o b a b l y a consequence of cutting a p r o p o r t i o n of the ventral root afferent fibers that enter the ventral root and the loop back into the dorsal root. The distance from the D R G where the ventral root was cut. p r e s u m a b l y accounted for the p r o p o r t i o n of the looping fibers that were interrupted. The rarity of double labeling also may be indicative of the possibility that few D R G cells actually emit bifurcating axons projecting indep e n d e n t l y via dorsal and ventral roots. The observation that almost all cells in the rat La D R G were labeled when the distal cut ends of dorsal roots were s o a k e d with FB seems to indicate that. as expected, almost all the cells in this ganglion give rise to dorsal root afferent fibers. This 1s consistent with early studies indicating that spinal sensory inputs enter the spinal cord via the dorsal roots, and m o t o r axons leave the spinal cord through the ventral roots and constitutes the law of separation of function of the spinal roots 8. F r o m an anatomic point of view. this implies that afferent fibers are located exclusive-

ly in dorsal roots and efferent fibers exclusively m ventral roots. H o w e v e r . it has been d e m o n s t r a t e d by numerous studies that a significant n u m b e r of sensory fibers are found in m a m m a l i a n ventral roots 347A1 15.2021.27 Thus it becomes i m p o r t a n t to determine whether many D R G cells emit fibers in the ventral root and identify their functional nature The main result of the present study is that 9.1% of D R G neurons are labeled when the distal cut ends of the ventral roots were soaked with fluorescent dyes. and thal most of these are small D R G neurons (6.8% of the total, or 74% of those projecting via the ventral rootl. This merely confirms previous observations that some D R G cells have central processes in ventral roots. The o b s e r v a n o n that only a very small percentage of large cells (0.6%) and m e d i u m (1.7%) cells were labeled, is consistent with findings that the majority of ventral root afferent fibers are unmyelinated because the fibers emitted bv small cells are mostly unmyelinated or small m y e l i n a t e d fibers ..... 18.23.25

Although it is clear that there are many afferent fibers in ventral roots according to previous studies as well as the present results, it is important to understand the a r r a n g e m e n t of these afferent fibers. The results of some studies suggesl that: (1) fibers enter the ventral root but then turn back to the spinal cord through dorsal root 2'2325. or (2) fibers enter the spinal cord directly through the ventral root tv'2°, o1 (3) fibers in the ventral root innervate the spinal pm mater5.11).22. The presence of fibers e n t e r m g the spinal cord via the ventral root are most controversial. Some investigators believe that the central processes of only a few ganglion cells project to the spinal cord through the ventral root 17'2°. including the afferent fibers to p i a mater 5'1°'22. Thus. horseradish peroxidase ( H R P ) injected into the spinal cord l a b e l e d some D R G cells 2° or only a few a b e r r a n t cells in the ventral root 2s after the dorsal roots were cut. bul it ts difficult to determine whether the injected H R P was a b s o r b e d by the axon terminals innervating the pia m a t e r o r by those in the spinal cord. The only obvious axonal pathway for retrograde transport was the fine afferent fibers labeled by H R P entering the spinal cord through the ventral root and ending in the dorsal hornY. In functional studies, there are still physiological reflex effects when p e r i p h e r a l nerves or muscles are snmu-

397 lated after dorsal roots a r e c u t 19'26 and there is some pain sensation produced by stimulation of ventral roots in man 12. Neither dorsal rhizotomy nor ganglionectomy invariably relieves chronic intractable pain 14, and it is possible that persistence of pain can be explained by afferent fibers in the ventral root that innervate the pia mater. Most studies seem to agree that many afferent fibers in the ventral roots loop back to the spinal cord through the dorsal r o o t s 2'23'25 and that some innervate the pia mater 5't°'22. The attempts to achieve double-labeling by simultaneous soaking of cut distal stumps of dorsal and ventral roots proved somewhat disappointing in that examples of double-labeling were extraordinarily rare (Fig. 4), suggesting that few D R G cells have independent processes in both ventral and dorsal roots 4. The double-labeling experiments also revealed numerous clusters of neurons (mostly small) that could not be labeled from the dorsal root, in contrast to the almost complete labeling from the dorsal root when the ventral root was intact. This is presumably explained by observations that most sensory fibers in the ventral roots loop back to enter the spinal cord via the dorsal r o o t s 2'23'25. The labeling of D R G cells from the ventral root was more variable than dorsal root labeling and led to the suspicion that D Y labeling from the ventral root might be ineffective for unknown technical reasons. However, the same batch of DY was used successfully in parallel experiments in the central nervous system and it therefore seems more likely that the variability of ventral root dye uptake and transport is partly related to the distance of root interruption in relation to the D R G . The site of looping back in the ventral root and minimal uptake by uncut axons might be among the several relevant variables. The intense blue label transported from dorsal roots might obscure the light yellow of DY transported by ventral roots, but the failure to find almost 10% even singly labeled with DY

transported from the ventral root indicates that the reliability of dye uptake constitutes a significant factor of uncertainty. This is clearly the case for DY in thin ventral root axons. Despite the uncertainties of quantitative precision, it seems likely that remarkably few D R G cells emit independent branches to dorsal and ventral roots. The variable proportion of single labeling from dorsal root transport in the double-labeling experiments is most probably a function of the proportion of looping ventral root afferent fibers cut. Similarly, variability in single-labeling from the ventral root may be a function of the proportion of cut ventral root afferents exposed to the dye and capable of uptake, and this may, in turn, be reduced with increasing distance from the D R G . The choice of C G R P antibodies for a second label was predicated on evidence that C G R P is the most widely distributed peptide found in small sensory fibers, although the proportion of CGRP-Iike immunoreactivity (32.4%) was slightly less than reported by others 13'16. Evidence that substance P-containing neurons are largely one subclass of C G R P neurons and that C G R P is an exceptionally powerful vasoactive agent I provided further impetus for determining peptidergic overlap with thin sensory neurons whose central axons course via the ventral roots. The finding that slightly more than half, mostly small, D R G cells that labeled from cut ventral roots also display CGRP-Iike IR indicates an unlikelihood for correlating ventral root afferents with a peptidergic class, at least for those peptides currently known for sensory fibers.

1 Brain, S.D., Williams, T.J., Tippins, J,R., Morris, H.R. and MacIntyre, I., Calcitonin gene-related peptide is a potent vasodilator, Brain, 313 (1985) 54-56. 2 Bostock, U., U-turns in rat ventral roots, J.Physiol. (LondonL 312 (1981) 49P-50P. 3 Chung, J.M., Lee, K.H. and Coggeshall, R.E., Nociceprive role of the ventral root afferents, Pain, Suppl. 2 (1984) $242. 4 Chung, J.M., Lee, K.H., Kim, J. and Coggeshall, R.E.,

Activation of dorsal horn cells by ventral root stimulation in the cat, J. Neurophysiol., 54 (1985) 261-272. 5 Clark, S.L., Innervation of the pia mater of the spinal cord and medulla, J. Comp. Neurol., 53 ( 1931) 129-145. 6 Clifton, G.L., Vance, W,H., Applebaum, M.L., Coggeshall, R.E. and Willis, W.D., Responses of unmyelinated afferents in the mammalian ventral root, Brain Research, 82 (1974) 162-t67. 7 Coggeshall, R.E., Afferent fibers in the ventral root, Neu-

I am grateful to Dr. Lawrence Kruger for the hospitality of his laboratory and his personal help and guidance at each stage of this research. I would also like to thank Sharon Sampogna and Anita Roff for their technical and secretarial assistance.

398 rosurgery, 4 (1979) 443-448. 8 Coggeshall, R.E.. Law of separation of function of the spinal roots. Physiol. Rev., 60 (1980) 716-755. 9 Coggeshall, R.E.. Applebaum. M.L.. Frazen. M.. Stubbs. T.B. and Sykes, M.T., Unmyelinated axons in human ventral roots, a possible explanation for the failure of dorsal rhizotomy to relieve pain, Brain. 98 (1975) I57-166. 10 Dalsgaard, C,J., Risling, M. and Cuello. C., Immunohistochemical localization of substance P in the lumbosacral spinal pia mater and ventral roots of the cat. Brain Research 246 (1982) 168-171. 11 Dimsdale, J.A. and Kemp, J.M., Afferent fibers in the ventral nerve roots in the rat. J. Physiol. (London), 187 f1966) 25-26. 12 Frykholm. R,. Hyde, J., Norlen. G. and Skoglund. C.R.. On pain sensations produced by stimulation of ventral roots in man. Acta Physiol. Scand., Suppl., 106 (1953) 455-469. 13 Gibson. S.J., Polak. J.M., Bloom, S.R.. Sabate, I.M., Mulderry, P.M.. Ghatei. M.A.. McGregor, G.P.. Morrison. J.F.B . Kelley, J.S.. Evans, R.M. and Rosenfeld. M.G,. Calcitonin gene related peptide immunoreactivity in the spinal cord of man and of eight other species. J. Neurosci.. a (1984) 3101-3111. 14 Hosobuchi. Y.. The majority of unmyetinated afferent axons in human ventral roots probably conduct pain, Pain. (1980) 167-180. 15 Kato. M. and Tanji. J.. Physiological properties of sensory fibers in the spinal ventral roots in the cat. Jap. J. Physiol. 21 (1971) 71-77. 16 Lee. Y., Takami. K.. Kawai. Y.. Girgis, S.. Hillyard. C.J.. Maclntyre, I.. Emson. P.C. and Tohyama. M.. Distribution of caicitonin gene-related peptide in the rat peripheral nervous system with reference to its coexistence with substance P. Neuroscience. 15 (1985) 1227-1238. 17 Light, A.R and Metz. C.B.. The morphology of spinal cord efferent and afferent neurons contributing to the ventral roots of the cat. J. Comp. Neurol.. 179 f1978) 501-516 18 Loeb, G.E., Ventral projections of myelinated dorsal root ganglion cells in the cal. Brain Research. 106 (1976) 159-165

t9 Longhurst. J.C., Mitchell. J.H. and Moore. M.B . The spinal cord ventral root: an afferent pathway of the hindlimb pressor reflex in cats. J. Physiol. ¢LondonL 301 (19801 467-476. 20 Maynard. C.W.. Leonard. R.B . Coulter. J.D, and Coggeshall. R.E.. Central connections of ventral root afferents as demonstrated by the HRP method. J. Comp. NeuroL. 172 (1977) 601-608 21 O'Donnell. J.E. and Windle. W.F., An experimental study of the sensory neurons in the ventral spinal nerve root~ of the cat. Anat. Rec. 55 (t933) 117-127 22 Risling, M., Dalsgaard. C.J. and Cuello. A.C.. Invasion of lumbosacral ventral roots and spinal pia mater by substance P-immunoreactive axons after scmtic nerve lesion in kittens, Brain Research. 307 (1984) 351--354. 23 Risting, M., Dalsgaard. C.J.. Cukierman. A. and Cuelto, A.C.. Electron microscopic and immunohistochemical evidence that unmyelinated ventral root axons make U-turns or enter the spinal pia mater, L Comp Neurol.. 225 (1984) 53-63. 24 Risling, M.. Hildebrand C. and Aldskogms, H., Developmental changes in the occurrence of feline unmyelinated L7 ventral root axons. Neurosci, Lelt. Suppl. 5 (1980) 309. 25 Risling, M. and Hildebrand, C.. Occurrence of unmyetinated axon profiles at distal, middle and proximal levels in the ventral root L7 of cats and kitten ~. J. Neurol. Sc~.. 56 / 1982 219-231. 26 Voorhoeve. P.E. and Nauta. J.. 1)o nociceptive ventral root afferents exert central somatic effects? In J.J. Bonica. U. Lindblom and A. Iggo (Eds.), Advance~ in Pain Research and Therapy, Vot. 5. Raven, New York. 1983, pp. 105-t10. 27 Willis. W.D., Grace R.R. and Skinner, R.D., Ventral root afferent fibres and the recurrent inhibitory pathway Nature (London J, 216 (1967) 1010-1011 28 Yamamoto. T.. Takahashi. K.. Satomi. 1t and Ise. H.. Origins of primary afferent fibers in the spinal ventral roots m the cat as demonstrated by the horseradish peroxidase method. Brain Research. 126 1977~ 350-354