Brain Research, 536 (1990) 309-314 Elsevier
309
BRES 24431
Biochemical and anatomical consequences of adult infraorbital nerve transection for serotonergic afferents within rat trigeminal subnuclei interpolaris and caudalis Bradley G. Klein and William D. Blaker* Department of Biomedical Sciences, Virginia-MarylandRegional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061 (U.S.A.) (Accepted 28 August 1990)
Key words: Immunoeytochemistry; High-performance liquid chromatography with electrochemical detection; Serotonin; 5-Hydroxyindoleaceticacid; Brainstem; Monoamine; Plasticity; Sprouting
Immunocytochemistry and high-performance liquid chromatography with electrochemical detection (HPLC-ED) were used, more than 76 days after infraorbital nerve (ION) transection, to examine the distribution and density of serotonin-immunoreactive (5-HTIR) axons, as well as serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content, within the infraorbital (IO) regions of subnuclei caudalis (SpVc) and interpolaris (SpVi). In SpVi, increases in 5-HT concentration and in density of 5-HTIR axonal varicosities were observed on the lesioned side. No changes were seen in SpVc, Following peripheral nerve or dorsal root damage, changes in receptive field size, location and effective stimulus modality have been reported at the principal postsynaptic targets of these primary afferents 18. Regarding the mechanisms responsible for functional alterations of these second-order neurons, much experimental attention has focused on the role of damaged and spared peripheral inputs 12A3 or upon morphological changes in the second-order neurons themselves 9,26. Considerably less work has been devoted to the importance of non-peripheral afferent systems which normally project to the spinal cord and trigeminal brainstem nuclear complex (TBNC), such as brainstem monoaminergic inputs. Electrical stimulation of noradrenergic and serotonergic afferents to the spinal cord and TBNC, as well as iontophoretic administration of norepinephrine and 5-HT have been shown to modulate the responses of spinal and TBNC neurons to peripheral stimuli 4'7'23. Central monoaminergic neurons have also demonstrated considerable plasticity following direct damage, or disruption of peripheral sensory input 2'22'28'29. This plasticity, along with the ability of these systems and their contents to modulate the function of spinal and TBNC neurons, suggests that these monoaminergic pathways may play a major role in functional reorganization of spinal and
brainstem regions, following peripheral nerve damage. We have previously noted functional alterations within SpVc and SpVi following transection of the ION in adult rats 14'21. To explore the possibility that chronic anatomical or biochemical changes in central monoaminergic afferents to the TBNC are correlated with these functional alterations, we have used immunocytochemistry along with H P L C - E D to examine the distribution and density of 5-HTIR axons, as well as 5-HT and 5-HIAA content, within SpVc and SpVi of these lesioned rats. For the biochemical measures, 19 adult male SpragueDawley rats of the same age were used. Nine of these animals had the left I O N transected (see ref. 19). Between 76 and 79 days after the lesion, rats were decapitated. After freezing on dry ice, transverse 340 # m brainstem sections were cut at - 7 °C, in a cryostat. The pyramidal decussation was used as a marker for the caudal border of SpVc, The caudal limit of the SpVc/SpVi border zone was identified by previously described criteria (see refs. 10 and 17) using darkfield examination of 34 # m samples. This region was not included in our samples. The appearance of the dorsal cochlear nucleus was used as a marker for the rostral border of SpVi. The middle one-third of the dorsoventral axis of the trigeminal spinal tract was used as a guide for the approximate location of the IO region. A cold scalpel
* Present address: Department of Biology, Furman University, Greenville, SC 29613, U.S.A. Correspondence: B.G. Klein, Department of Biomedical Sciences, College of Veterinary Medicine, Virginia Tech, Blaeksburg, VA 24061, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
310 blade was used to slice 1 mm 2 and 2.25 mm 2 samples, respectively, from the IO vicinity of the SpVc and SpVi sections. These values were chosen to sample the mediolateral extent of the respective subnuclei. To verify the location of removed samples, the remaining portion of each brainstem section was fixed in 10% formalin, sectioned at 51 or 68/~m in the cryostat and stained with Cresyl violet. Brain tissue indoleamines were analyzed by HPLC-ED using the procedure of Mayer and Shoup 16. The frozen tissue samples were homogenized in cold 0.04 N perchloric acid, containing the internal standard dihydroxybenzylamine. The homogenate was centrifuged and the entire supernatant was immediately injected onto the HPLC. The protein content of the pellet was determined by the method of Lowry et al. 15. For immunocytochemistry, 13 adult male SpragueDawley rats were used. All animals had the left ION transected as noted above. Different rats were used for the SpVc and SpVi samples. Seventy-six to 187 days after nerve transection, rats were deeply anesthetized with sodium pentobarbital (200 mg/kg) and perfused transcardially with 0.1 M phosphate buffered saline (PBS, pH 7.4, 4 °C), 4% paraformaldehyde in 0.1 M sodium acetate buffer (pH 6.5, 4 °C) and 4% paraformaldehyde in 0.1 M sodium borate buffer (pH 9.5, 4 °C). Brains were postfixed for 4 h in the high pH fixative and placed in 10% sucrose in PBS at 4 °C, overnight. Transverse 17/~m sections were cut in a cryostat and sequentially incubated on slides, at room temperature, in (1) 10% normal goat serum containing 0.15% Triton X-100, (2) PBS and (3) rabbit antiserum to 5-HT/BSA (Incstar Corp., Stiliwater, MN) in PBS with 0.15% Triton X-100 (1000:1, 1500:1 or 2000:1 dilutions, overnight). Tissue was subsequently processed by avidin-biotin histochemistry, using a Vectastain kit (Vector Labs, Burlingame, CA) in conjunction with 0.05% diaminobenzidine in PBS, containing 0.01% H202. Every third
immunoreacted slide and all non-reacted tissue, was stained with Cresyl violet. Sample sections were used for primary antibody omission or preabsorption controls. Brainstem raphe nuclei were used as a positive control. The outline of laminae I/II and the dense band of 5-HTIR axons within, were each traced at l13x, at 100 /~m intervals along the rostrocaudal axis. The density of 5-HTIR varicosities in the laminae I/II immunoreactive band and in laminae Ill/IV were determined at 1111× using a drawing tube and a 2500/~m 2 grid (comprised of 100 ~tm2 units). In the IO region of SpVi, at 200/~m intervals, counts of 5-HTIR varicosities were made at 250 /~m (lateral sample) and 500/~m (medial sample) medial to the medial border of the spinal trigeminal tract. For dependent variables examined using immunocytochemistry, two-tailed t-tests on the difference scores for matched pairs were used to compare the lesioned and intact sides of the brainstem. The alpha level was 0.05. In SpVi, the only significant change observed was in 5-HT concentration (F2.25 = 7.04, P < 0.01). Duncan's multiple range test revealed a significant 45.7% increase in mean 5-HT concentration on the lesioned side compared to the intact side (Table I). This was a 48.6% increase with respect to the normal tissue group. In SpVc, no significant differences were observed. Fig. 1 illustrates the generally homogeneous distribution of 5-HTIR axons along the dorsoventral and mediolateral axes of SpVi, on the intact and lesioned sides. Quantitative analysis revealed that a morphological change had occurred on the side of the nerve transection (Fig. 2). For the lateral sample field, the mean difference in density of 5-HTIR varicosities between the two sides was 4240 (S.E.M. = 641) varicosities/mm 2. This was a significant 16.7% mean increase following the lesion (td(3) = 6.61, P = 0.007). For the medial sample field, no significant difference was observed between the intact and lesioned sides. 5-HTIR axons in SpVc were primarily localized within
TABLE I Mean concentrations of 5-HT and 5-HIAA m subnuclei interpolaris and caudalis of lesioned and normal rats
Concentrations are expressed as pmol/mgprotein + S.E.M. (n). Intact sample is from side contralateral to the lesion. Means among tissue ~oups were compared by one-way analysis of variance (alpha level = 0.05), followed by Duncan's multiple range test. Asterisks denote a significant difference from the value on the lesioned side (P < 0.01). Subnucleus
Sample
5-HT
5-HIAA
5-HIAA/5-HT
Interpolaris
Lesioned Intact Normal Lesioned Intact Normal
22.0 + 1.5 (9) 15.1 + 1.4 (9)* 14.8 + 1.6 (10)* 19.6 + 3.0 (8) 23.1 + 3.4 (8) 17.3 + 2.6 (10)
8.3 + 1.9 (9) 6.6 + 1.5 (9) 6.5 + 1.1 (10) 11.3 + 2.2 (7) 10.1 + 2.1 (7) 9.2 + 1.3 (9)
0.345 + 0,062 (9) 0.433 ___0.095 (9) 0.499 + 0.115 (10) 0.586 _+0.090(7) 0.402 + 0.051 (7) 0.545 ___0.063 (9)
Caudalis
311
TrV
Fig. 1. Dark-field and higher magnification bright-field photomicrographsof the lesioned (A, A') and intact (B, B') sides of a 17/~m section from SpVi, showing 5-HT immunoreactivity. C and C" are respective low- and high-power micrographs of a 5-HTIR section counterstained with Cresyl violet to verify subnuclear location. Inset in C is corresponding dark-field image. Arrows indicate corresponding points among photos with the same letter.
lamina I and outer lamina II in a dense, longitudinally oriented band (Fig. 3). Representation in the magnocellular layers ( I I I - V ) was comparatively sparse (Figs. 2B and 3). No anatomical changes were qualitatively or quantitatively apparent between the lesioned and intact sides with respect to the density of 5-HTIR varicosities in
the laminae UII or III/IV sample fields, or in the percent area of laminae I/II occupied by 5-HTIR axons. No change in the width of laminae I/II was observed, suggesting that the lack of change in primary focus of 5-HTIR axons in SpVc was not confounded by shrinkage of the laminar substrate.
312
A. t"q
E E 03 LIJ I-O O < > rY k-"r
40,000
MEDIAL, LESIONED
INTACT
LESIONED
80
_z
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100
30,000
I
(/3 I,i F.-o3 O L) iv,
SpVi
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[ZZ3 LATERAL, INTACT LATERAL, LESIONED ES~ MEDIAL, INTACT
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RAT B
RAT C
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RAT D
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i
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,
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RAT 3
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I/II, LESIONED lll/IV,INTACT
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~
8E4.
~.
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z
~ Kx~
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o
nl
nl
nl
RAT 1
RAT 2
RAT 3
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131 RAT 4
RAT 1
RAT 2
RAT 3
RAT 4
Fig. 2. Density of 5 - H T I R axonal varicosities, on the lesioned and intact sides of 4 rats, for the medial and lateral sample fields in SpVi (A), and for laminae I/II and Ill/IV sample fields in SpVc (B). C and D, respectively present corresponding data for SpVc regarding the percent area of laminae I/II occupied by 5 - H T I R axons and the combined width of laminae I/iI. For all graphs, each bar represents m e a n and standard error for values from serial transverse sections within a given animal.
The data presented above indicate that adult ION transection produces a chronic increase in 5-HT content within the IO region of SpVi. This biochemical change is accompanied by a morphological alteration in 5-HTIR afferents to this region, as evidenced by an increase in the density of axonal varicosities. In SpVc, no such changes were apparent, nor was a change in the focus of 5-HTIR axon distribution. Thus, adult 1ON transection has differential consequences for 5-HT inputs to SpVi and SpVc. Tooth pulp deafferentation in cat has also been shown to have differential consequences for different TBNC subnuclei, with respect to functional organization 5.6. Plasticity of monoaminergic afferents has previously been demonstrated in brainstem and spinal cord following disruption of specific sensory primary afferents innervating these targets 22'28. Given the organizational similarities between spinal and medullary dorsal horns 3, it is somewhat surprising that no increase in 5-HT content, distribution or innervation density was observed
in SpVc in the present experiment. However, in the spinal cord studies, dorsal rhizotomy was used, in contrast to peripheral nerve transection and regeneration. Compared with dorsal rhizotomy, peripheral nerve transection may not sustain a severe enough reduction of the normal primary afferent input to induce a chronic change in 5-HT axons within SpVc t'25. This does not preclude a transient alteration of 5-HT axons in SpVc, prior to recovery of primary afferents, following peripheral transection. There are several possible explanations for the differential effects of ION transection upon 5-HT afferents within SpVi and SpVc. One possibility is a differential innervation of these two subnuclei by surviving primary afferents following the lesion. Such a phenomenon has been demonstrated following neonatal 1ON transection 12' t3. Another reason might be the rich variety of nonserotonergic axonal populations impinging upon SpVc neurons. For example, convergence of 5-HT, enkephalin and substance-P immunoreactive axons upon individual
313
Fig. 3. Dark-field photomicrographs illustrating the distribution of 5-HTIR axons in SpVc, ipsilateral to the cut nerve, for two different rats (A, A', and B, B', respectively). Arrows denote corresponding points in the low- and high-power photographs. Distribution and density of 5-HTIR axons on the corresponding intact side was identical.
SpVc projection neurons has been demonstrated in cat 27. Also, convergence of somatosensory inputs from different modalities has been reported for neurons in cat and rat SpVc2°'24. Such intermodality convergence is rare in normal rat SpVi s'9'14. Thus, 5-HT axons in SpVc may face more competition from heterotypic fibers following the lesion, compared with 5-HT axons in SpVi. Following ION transection in adult 14 or neonatal s'9 rats, chronic changes in the response properties of postsynaptic cells have been reported in SpVi. Many of these changes are common to both types of lesioned animal. Some qualitatively similar changes have been observed in trigeminal primary afferents following ION cut n'19. The findings of the present experiment demonstrate non-primary afferent anatomical and biochemical correlates for changes in SpVi functional status, following
trigeminal nerve damage in adult rats. Hu et al. 5 have demonstrated marked functional alterations in subnucleus oralis (SpVo) following tooth pulp deafferentation. This manipulation did not alter the normal inhibitory influence of nucleus raphe magnus stimulation upon SpVo neurons 7. However, these studies were performed in cat. Also, in contrast to ION transection, the tooth pulp deafferentation procedure involved less than 5% of the afferents in the trigeminal root, which were primarily of a single modality (nociceptive)5,6. We would like to thank Laura Morse and Greg Thornwall for their technical assistance. This work was supported in part by Grant R01 DE08966 (B.G.K.) from the NIH and by a New Initiative Award (B.G.K.) from the Virginia-Maryland Regional College of Veterinary Medicine.
314
1 Aldskogius, H., Arvidsson, J. and Grant, G., The reaction of primary sensory neurons to peripheral nerve injury with particular emphasis on transganglionic changes, Brain Res. Rev., 10 (1985) 27-46. 2 Alesci, R., La Noce, A., Bagnoti, P. and Ghelarducci, B., Dark-rearing modifies serotonin and 5-hydroxyindoleacetic acid contents in specific regions of rabbit brain, Behav. Brain Res., 31 (1988) 115-120. 3 Dubner, R. and Bennett, G.J., Spinal and trigeminal mechanisms of nociception, Annu. Rev. Neurosci., 6 (1983) 381-418. 4 Gray, B.G. and Dostrovsky, J.O., Inhibition of feline spinal cord dorsal horn neurons following electrical stimulation of nucleus paragigantocellularis lateralis. A comparison with nucleus raphe magnus, Brain Research, 348 (1985) 261-273. 5 Hu, J.W., Dostrovsky, J.O., Lenz, Y.E., Ball, G.J. and Sessle, B.J., Tooth pulp deafferentation is associated with functional alterations in the properties of neurons in the trigeminal spinal tract nucleus, J. Neurophysiol., 56 (1986) 1650-1668. 6 Hu, J.W. and Sessle, B.J., Effects of tooth pulp deafferentation on nociceptive and nonnociceptive neurons of the feline trigeminal subnucleus caudalis (medullary dorsal horn), J. Neurophysiol., 61 (1989)1197-1206. 7 Hu, J.W., Sessle, B.J., Dostrovsky, J.O. and Lenz, Y., Effects of nucleus raphe magnus stimulation on jaw-opening reflex and trigeminal brain-stem neurone responses in normal and tooth pulp deafferented cats, Pain, 27 (1986) 349-360. 8 Jacquin, M.E, Structure-function relationships in rat brainstem subnucleus interpolaris: V. Functional consequences of neonatal infraorbital nerve section, J. Comp. Neurol., 282 (1989) 63-79, 9 Jacquin, M.E, Chiaia, N.L., Klein, B.G. and Rhoades, R.W., Structure-function relationships in the rat brainstem subnucleus interpolafis: VI. Cervical convergence in cells deafferented at birth and a potential primary afferent substrate, J. Comp. Neurol., 283 (1989) 513-585. 10 Jacquin, M.E, Mooney, R.D. and Rhoades, R.W., Morphology, response properties, and collateral projections of trigeminothatamic neurons in brainstem subnucleus interpolaris of the rat, Exp. Brain Res., 61 (1986) 457-468. 11 Jacquin, M.E, Renehan, W.E., Klein, B.G., Mooney, R.D. and Rhoades, R.W., Functional consequences of neonatal infraorbital nerve section in rat trigeminal ganglion, J. Neurosci., 6 (1986) 3706-3720. 12 Jacquin, M.E and Rhoades, R.W., Central projections of the normal and 'regenerate' infraorbital nerve in adult rats subjected to unilateral infraorbital lesions: a transganglionic horseradish peroxidase study, Brain Research, 269 (1983) 137-144. 13 Jacquin, M.E and Rhoades, R.W., Effects of neonatal infraorbital nerve lesions upon central trigeminal primary afferent projections in rat and hamster, J. Comp. Neurol., 235 (1985) 129-143. 14 Klein, B.G., Morse, L.A., Barcia, M., Rhoades, R.W. and Jacquin, M.E, Chronic effects of adult infraorbital (IO) nerve transection upon response properties of cells in trigeminal (V) subnucleus interpolaris (SpVi), Soc. Neurosci. Abstr., 13 (1987) 1664. 15 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275.
16 Mayer, G.S. and Shoup, R.E., Similtaneous multiple electrode liquid chromatographic-electrochemical assay for catecholamines, indolamines and metabolites in brain tissue, J. Chromatogr., 255 (1983) 533-544. 17 Phelan, K.D. and Falls, W.M., An analysis of the cyto- and myeloarchitectonic organization of trigeminal nucleus interpolaris in the rat, Somatosens. Mot. Res., 6 (t989) 333-366. 18 Pubols, L.M. and Sessle, B. (Eds.), Effects of Injury on Trigeminal and Spinal Somatosensory Systems, Alan R. Liss. New York, 1987. 19 Renehan, W.E., Chiaia, N.L., Klein, B.G., Jacquin, M.E and Rhoades, R.W., Physiological and anatomical consequences of infraorbital nerve transection in the trigeminal ganglion and trigeminal spinal tract of the adult rat, J. Neurosci., 9 (1989) 548-557. 20 Renehan, W.E., Jacquin, M.E, Mooney, R.D. and Rhoades, R.W., Structure-function relationships in rat medullary and cervical dorsal horns. II. Medullary dorsal horn cells, J. Neurophysiol., 55 (1986) 1187-1201. 21 Renehan, W.E., Stansel, S. and Klein, B.G., Effects of lntraorbital Nerve Transection on the Structure and Function of Trigeminal Brainstem Neurons in the Adult Rat, Paper presented at Winter Conference on Brain Res., Timberline, OR, 1988. 22 Rhoades, R.W. and Hess, A., Altered catecholaminergic innervation of the superior colliculus after enucleation in adult and neonatal hamsters, Brain Research, 261 (1983) 353-357. 23 Sasa, M., Fugimoto, S., Igarashi, S., Munekiyo, K. and Takaori, S., Microiontophoretic studies on noradrenergic inhibition from locus coeruleus of spinal trigeminal nucleus neurons, J. Pharmacol. Exp. Ther., 210 (1979) 311-315. 24 Sessle, B.J., Hu, J.W., Amano, N. and Zhong, G., Convergence of cutaneous, tooth pulp, visceral, neck and muscle afferents onto nociceptive and non-nociceptive neurones in trigeminal subnucleus caudalis (medullary dorsal horn) and its implications for referred pain, Pain, 27 (1986) 219-235. 25 Stensaas, L.J., Partlow, L.M., Burgess, P.R. and Horch, K.W., Inhibition of regeneration: the ultrastructure of reactive astrocytes and abortive axon terminals in the transition zone of the dorsal root. In EJ. Seil, E. Herbert and B.M. Carlson (Eds.), Progress in Brain Research, VoI. 71, Elsevier, Amsterdam, 1987, pp. 457-468. 26 Sugimoto, T. and Gobel, S., Dendritic changes in the spinal dorsal horn following transection of a peripheral nerve, Brain Research, 321 (1984) 199-208. 27 Tashiro, T., Ruda, M.A., Satoda, T., Matsushima, R. and Mizuno, N., Convergence of serotonin-, enkephalin- and substance P-like immunoreactive afferent fibers onto cat medullary dorsal horn projection neurons: a triple immunocytochemical staining technique combined with the retrograde HRP-tracing method, Brain Research, 491 (1989) 360-365. 28 Wang, S.D., Eckenrode, T., Goldberger, M.E. and Murray, M., Plasticity of intrinsic and extrinsic systems after rhizotomy of rat spinal cord, Soc. Neurosci. Abstr., 13 (1987) 166. 29 Wilkund, L., Bj6rklund, A. and Nobin, A., Regeneration of serotonin neurons in the rat brain after 5,6-dihydroxytryptamine-induced axotomy, Ann. N.Y. Acad. Sci., 305 (t978) 370-384.
309
BRES 24431
Biochemical and anatomical consequences of adult infraorbital nerve transection for serotonergic afferents within rat trigeminal subnuclei interpolaris and caudalis Bradley G. Klein and William D. Blaker* Department of Biomedical Sciences, Virginia-MarylandRegional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061 (U.S.A.) (Accepted 28 August 1990)
Key words: Immunoeytochemistry; High-performance liquid chromatography with electrochemical detection; Serotonin; 5-Hydroxyindoleaceticacid; Brainstem; Monoamine; Plasticity; Sprouting
Immunocytochemistry and high-performance liquid chromatography with electrochemical detection (HPLC-ED) were used, more than 76 days after infraorbital nerve (ION) transection, to examine the distribution and density of serotonin-immunoreactive (5-HTIR) axons, as well as serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content, within the infraorbital (IO) regions of subnuclei caudalis (SpVc) and interpolaris (SpVi). In SpVi, increases in 5-HT concentration and in density of 5-HTIR axonal varicosities were observed on the lesioned side. No changes were seen in SpVc, Following peripheral nerve or dorsal root damage, changes in receptive field size, location and effective stimulus modality have been reported at the principal postsynaptic targets of these primary afferents 18. Regarding the mechanisms responsible for functional alterations of these second-order neurons, much experimental attention has focused on the role of damaged and spared peripheral inputs 12A3 or upon morphological changes in the second-order neurons themselves 9,26. Considerably less work has been devoted to the importance of non-peripheral afferent systems which normally project to the spinal cord and trigeminal brainstem nuclear complex (TBNC), such as brainstem monoaminergic inputs. Electrical stimulation of noradrenergic and serotonergic afferents to the spinal cord and TBNC, as well as iontophoretic administration of norepinephrine and 5-HT have been shown to modulate the responses of spinal and TBNC neurons to peripheral stimuli 4'7'23. Central monoaminergic neurons have also demonstrated considerable plasticity following direct damage, or disruption of peripheral sensory input 2'22'28'29. This plasticity, along with the ability of these systems and their contents to modulate the function of spinal and TBNC neurons, suggests that these monoaminergic pathways may play a major role in functional reorganization of spinal and
brainstem regions, following peripheral nerve damage. We have previously noted functional alterations within SpVc and SpVi following transection of the ION in adult rats 14'21. To explore the possibility that chronic anatomical or biochemical changes in central monoaminergic afferents to the TBNC are correlated with these functional alterations, we have used immunocytochemistry along with H P L C - E D to examine the distribution and density of 5-HTIR axons, as well as 5-HT and 5-HIAA content, within SpVc and SpVi of these lesioned rats. For the biochemical measures, 19 adult male SpragueDawley rats of the same age were used. Nine of these animals had the left I O N transected (see ref. 19). Between 76 and 79 days after the lesion, rats were decapitated. After freezing on dry ice, transverse 340 # m brainstem sections were cut at - 7 °C, in a cryostat. The pyramidal decussation was used as a marker for the caudal border of SpVc, The caudal limit of the SpVc/SpVi border zone was identified by previously described criteria (see refs. 10 and 17) using darkfield examination of 34 # m samples. This region was not included in our samples. The appearance of the dorsal cochlear nucleus was used as a marker for the rostral border of SpVi. The middle one-third of the dorsoventral axis of the trigeminal spinal tract was used as a guide for the approximate location of the IO region. A cold scalpel
* Present address: Department of Biology, Furman University, Greenville, SC 29613, U.S.A. Correspondence: B.G. Klein, Department of Biomedical Sciences, College of Veterinary Medicine, Virginia Tech, Blaeksburg, VA 24061, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
310 blade was used to slice 1 mm 2 and 2.25 mm 2 samples, respectively, from the IO vicinity of the SpVc and SpVi sections. These values were chosen to sample the mediolateral extent of the respective subnuclei. To verify the location of removed samples, the remaining portion of each brainstem section was fixed in 10% formalin, sectioned at 51 or 68/~m in the cryostat and stained with Cresyl violet. Brain tissue indoleamines were analyzed by HPLC-ED using the procedure of Mayer and Shoup 16. The frozen tissue samples were homogenized in cold 0.04 N perchloric acid, containing the internal standard dihydroxybenzylamine. The homogenate was centrifuged and the entire supernatant was immediately injected onto the HPLC. The protein content of the pellet was determined by the method of Lowry et al. 15. For immunocytochemistry, 13 adult male SpragueDawley rats were used. All animals had the left ION transected as noted above. Different rats were used for the SpVc and SpVi samples. Seventy-six to 187 days after nerve transection, rats were deeply anesthetized with sodium pentobarbital (200 mg/kg) and perfused transcardially with 0.1 M phosphate buffered saline (PBS, pH 7.4, 4 °C), 4% paraformaldehyde in 0.1 M sodium acetate buffer (pH 6.5, 4 °C) and 4% paraformaldehyde in 0.1 M sodium borate buffer (pH 9.5, 4 °C). Brains were postfixed for 4 h in the high pH fixative and placed in 10% sucrose in PBS at 4 °C, overnight. Transverse 17/~m sections were cut in a cryostat and sequentially incubated on slides, at room temperature, in (1) 10% normal goat serum containing 0.15% Triton X-100, (2) PBS and (3) rabbit antiserum to 5-HT/BSA (Incstar Corp., Stiliwater, MN) in PBS with 0.15% Triton X-100 (1000:1, 1500:1 or 2000:1 dilutions, overnight). Tissue was subsequently processed by avidin-biotin histochemistry, using a Vectastain kit (Vector Labs, Burlingame, CA) in conjunction with 0.05% diaminobenzidine in PBS, containing 0.01% H202. Every third
immunoreacted slide and all non-reacted tissue, was stained with Cresyl violet. Sample sections were used for primary antibody omission or preabsorption controls. Brainstem raphe nuclei were used as a positive control. The outline of laminae I/II and the dense band of 5-HTIR axons within, were each traced at l13x, at 100 /~m intervals along the rostrocaudal axis. The density of 5-HTIR varicosities in the laminae I/II immunoreactive band and in laminae Ill/IV were determined at 1111× using a drawing tube and a 2500/~m 2 grid (comprised of 100 ~tm2 units). In the IO region of SpVi, at 200/~m intervals, counts of 5-HTIR varicosities were made at 250 /~m (lateral sample) and 500/~m (medial sample) medial to the medial border of the spinal trigeminal tract. For dependent variables examined using immunocytochemistry, two-tailed t-tests on the difference scores for matched pairs were used to compare the lesioned and intact sides of the brainstem. The alpha level was 0.05. In SpVi, the only significant change observed was in 5-HT concentration (F2.25 = 7.04, P < 0.01). Duncan's multiple range test revealed a significant 45.7% increase in mean 5-HT concentration on the lesioned side compared to the intact side (Table I). This was a 48.6% increase with respect to the normal tissue group. In SpVc, no significant differences were observed. Fig. 1 illustrates the generally homogeneous distribution of 5-HTIR axons along the dorsoventral and mediolateral axes of SpVi, on the intact and lesioned sides. Quantitative analysis revealed that a morphological change had occurred on the side of the nerve transection (Fig. 2). For the lateral sample field, the mean difference in density of 5-HTIR varicosities between the two sides was 4240 (S.E.M. = 641) varicosities/mm 2. This was a significant 16.7% mean increase following the lesion (td(3) = 6.61, P = 0.007). For the medial sample field, no significant difference was observed between the intact and lesioned sides. 5-HTIR axons in SpVc were primarily localized within
TABLE I Mean concentrations of 5-HT and 5-HIAA m subnuclei interpolaris and caudalis of lesioned and normal rats
Concentrations are expressed as pmol/mgprotein + S.E.M. (n). Intact sample is from side contralateral to the lesion. Means among tissue ~oups were compared by one-way analysis of variance (alpha level = 0.05), followed by Duncan's multiple range test. Asterisks denote a significant difference from the value on the lesioned side (P < 0.01). Subnucleus
Sample
5-HT
5-HIAA
5-HIAA/5-HT
Interpolaris
Lesioned Intact Normal Lesioned Intact Normal
22.0 + 1.5 (9) 15.1 + 1.4 (9)* 14.8 + 1.6 (10)* 19.6 + 3.0 (8) 23.1 + 3.4 (8) 17.3 + 2.6 (10)
8.3 + 1.9 (9) 6.6 + 1.5 (9) 6.5 + 1.1 (10) 11.3 + 2.2 (7) 10.1 + 2.1 (7) 9.2 + 1.3 (9)
0.345 + 0,062 (9) 0.433 ___0.095 (9) 0.499 + 0.115 (10) 0.586 _+0.090(7) 0.402 + 0.051 (7) 0.545 ___0.063 (9)
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311
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Fig. 1. Dark-field and higher magnification bright-field photomicrographsof the lesioned (A, A') and intact (B, B') sides of a 17/~m section from SpVi, showing 5-HT immunoreactivity. C and C" are respective low- and high-power micrographs of a 5-HTIR section counterstained with Cresyl violet to verify subnuclear location. Inset in C is corresponding dark-field image. Arrows indicate corresponding points among photos with the same letter.
lamina I and outer lamina II in a dense, longitudinally oriented band (Fig. 3). Representation in the magnocellular layers ( I I I - V ) was comparatively sparse (Figs. 2B and 3). No anatomical changes were qualitatively or quantitatively apparent between the lesioned and intact sides with respect to the density of 5-HTIR varicosities in
the laminae UII or III/IV sample fields, or in the percent area of laminae I/II occupied by 5-HTIR axons. No change in the width of laminae I/II was observed, suggesting that the lack of change in primary focus of 5-HTIR axons in SpVc was not confounded by shrinkage of the laminar substrate.
312
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Fig. 2. Density of 5 - H T I R axonal varicosities, on the lesioned and intact sides of 4 rats, for the medial and lateral sample fields in SpVi (A), and for laminae I/II and Ill/IV sample fields in SpVc (B). C and D, respectively present corresponding data for SpVc regarding the percent area of laminae I/II occupied by 5 - H T I R axons and the combined width of laminae I/iI. For all graphs, each bar represents m e a n and standard error for values from serial transverse sections within a given animal.
The data presented above indicate that adult ION transection produces a chronic increase in 5-HT content within the IO region of SpVi. This biochemical change is accompanied by a morphological alteration in 5-HTIR afferents to this region, as evidenced by an increase in the density of axonal varicosities. In SpVc, no such changes were apparent, nor was a change in the focus of 5-HTIR axon distribution. Thus, adult 1ON transection has differential consequences for 5-HT inputs to SpVi and SpVc. Tooth pulp deafferentation in cat has also been shown to have differential consequences for different TBNC subnuclei, with respect to functional organization 5.6. Plasticity of monoaminergic afferents has previously been demonstrated in brainstem and spinal cord following disruption of specific sensory primary afferents innervating these targets 22'28. Given the organizational similarities between spinal and medullary dorsal horns 3, it is somewhat surprising that no increase in 5-HT content, distribution or innervation density was observed
in SpVc in the present experiment. However, in the spinal cord studies, dorsal rhizotomy was used, in contrast to peripheral nerve transection and regeneration. Compared with dorsal rhizotomy, peripheral nerve transection may not sustain a severe enough reduction of the normal primary afferent input to induce a chronic change in 5-HT axons within SpVc t'25. This does not preclude a transient alteration of 5-HT axons in SpVc, prior to recovery of primary afferents, following peripheral transection. There are several possible explanations for the differential effects of ION transection upon 5-HT afferents within SpVi and SpVc. One possibility is a differential innervation of these two subnuclei by surviving primary afferents following the lesion. Such a phenomenon has been demonstrated following neonatal 1ON transection 12' t3. Another reason might be the rich variety of nonserotonergic axonal populations impinging upon SpVc neurons. For example, convergence of 5-HT, enkephalin and substance-P immunoreactive axons upon individual
313
Fig. 3. Dark-field photomicrographs illustrating the distribution of 5-HTIR axons in SpVc, ipsilateral to the cut nerve, for two different rats (A, A', and B, B', respectively). Arrows denote corresponding points in the low- and high-power photographs. Distribution and density of 5-HTIR axons on the corresponding intact side was identical.
SpVc projection neurons has been demonstrated in cat 27. Also, convergence of somatosensory inputs from different modalities has been reported for neurons in cat and rat SpVc2°'24. Such intermodality convergence is rare in normal rat SpVi s'9'14. Thus, 5-HT axons in SpVc may face more competition from heterotypic fibers following the lesion, compared with 5-HT axons in SpVi. Following ION transection in adult 14 or neonatal s'9 rats, chronic changes in the response properties of postsynaptic cells have been reported in SpVi. Many of these changes are common to both types of lesioned animal. Some qualitatively similar changes have been observed in trigeminal primary afferents following ION cut n'19. The findings of the present experiment demonstrate non-primary afferent anatomical and biochemical correlates for changes in SpVi functional status, following
trigeminal nerve damage in adult rats. Hu et al. 5 have demonstrated marked functional alterations in subnucleus oralis (SpVo) following tooth pulp deafferentation. This manipulation did not alter the normal inhibitory influence of nucleus raphe magnus stimulation upon SpVo neurons 7. However, these studies were performed in cat. Also, in contrast to ION transection, the tooth pulp deafferentation procedure involved less than 5% of the afferents in the trigeminal root, which were primarily of a single modality (nociceptive)5,6. We would like to thank Laura Morse and Greg Thornwall for their technical assistance. This work was supported in part by Grant R01 DE08966 (B.G.K.) from the NIH and by a New Initiative Award (B.G.K.) from the Virginia-Maryland Regional College of Veterinary Medicine.
314
1 Aldskogius, H., Arvidsson, J. and Grant, G., The reaction of primary sensory neurons to peripheral nerve injury with particular emphasis on transganglionic changes, Brain Res. Rev., 10 (1985) 27-46. 2 Alesci, R., La Noce, A., Bagnoti, P. and Ghelarducci, B., Dark-rearing modifies serotonin and 5-hydroxyindoleacetic acid contents in specific regions of rabbit brain, Behav. Brain Res., 31 (1988) 115-120. 3 Dubner, R. and Bennett, G.J., Spinal and trigeminal mechanisms of nociception, Annu. Rev. Neurosci., 6 (1983) 381-418. 4 Gray, B.G. and Dostrovsky, J.O., Inhibition of feline spinal cord dorsal horn neurons following electrical stimulation of nucleus paragigantocellularis lateralis. A comparison with nucleus raphe magnus, Brain Research, 348 (1985) 261-273. 5 Hu, J.W., Dostrovsky, J.O., Lenz, Y.E., Ball, G.J. and Sessle, B.J., Tooth pulp deafferentation is associated with functional alterations in the properties of neurons in the trigeminal spinal tract nucleus, J. Neurophysiol., 56 (1986) 1650-1668. 6 Hu, J.W. and Sessle, B.J., Effects of tooth pulp deafferentation on nociceptive and nonnociceptive neurons of the feline trigeminal subnucleus caudalis (medullary dorsal horn), J. Neurophysiol., 61 (1989)1197-1206. 7 Hu, J.W., Sessle, B.J., Dostrovsky, J.O. and Lenz, Y., Effects of nucleus raphe magnus stimulation on jaw-opening reflex and trigeminal brain-stem neurone responses in normal and tooth pulp deafferented cats, Pain, 27 (1986) 349-360. 8 Jacquin, M.E, Structure-function relationships in rat brainstem subnucleus interpolaris: V. Functional consequences of neonatal infraorbital nerve section, J. Comp. Neurol., 282 (1989) 63-79, 9 Jacquin, M.E, Chiaia, N.L., Klein, B.G. and Rhoades, R.W., Structure-function relationships in the rat brainstem subnucleus interpolafis: VI. Cervical convergence in cells deafferented at birth and a potential primary afferent substrate, J. Comp. Neurol., 283 (1989) 513-585. 10 Jacquin, M.E, Mooney, R.D. and Rhoades, R.W., Morphology, response properties, and collateral projections of trigeminothatamic neurons in brainstem subnucleus interpolaris of the rat, Exp. Brain Res., 61 (1986) 457-468. 11 Jacquin, M.E, Renehan, W.E., Klein, B.G., Mooney, R.D. and Rhoades, R.W., Functional consequences of neonatal infraorbital nerve section in rat trigeminal ganglion, J. Neurosci., 6 (1986) 3706-3720. 12 Jacquin, M.E and Rhoades, R.W., Central projections of the normal and 'regenerate' infraorbital nerve in adult rats subjected to unilateral infraorbital lesions: a transganglionic horseradish peroxidase study, Brain Research, 269 (1983) 137-144. 13 Jacquin, M.E and Rhoades, R.W., Effects of neonatal infraorbital nerve lesions upon central trigeminal primary afferent projections in rat and hamster, J. Comp. Neurol., 235 (1985) 129-143. 14 Klein, B.G., Morse, L.A., Barcia, M., Rhoades, R.W. and Jacquin, M.E, Chronic effects of adult infraorbital (IO) nerve transection upon response properties of cells in trigeminal (V) subnucleus interpolaris (SpVi), Soc. Neurosci. Abstr., 13 (1987) 1664. 15 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275.
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