Brain Research, 574 (1992) 333-337 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
333
BRES 25081
Quantitative autoradiographic localization of [1251]neuropeptide Y receptor binding sites in rat spinal cord and the effects of neonatal capsaicin, dorsal rhizotomy and peripheral axotomy S. Kar and R. Quirion Douglas Hospital Research Center and Department of Psychiatry, Faculty of Medicine, McGill University, Verdun, Que. (Canada)
(Accepted 26 November 1991) Key words: Neuropeptide Y; Receptor autoradiography; Spinal cord; Capsaicin; Dorsal rhizotomy; Axotomy
Using in vitro quantitative receptor autoradiography the present study reports on the distribution and possible changes of [~25I]neuropeptide Y (NPY) binding sites in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. In control spinal cord the highest density of [12SI]NPY binding sites was noticed in the superficial layers of the dorsal horn whereas low-to-moderate densities of [125I]NPY binding sites were detected in the deeper dorsal horn and in the ventral horn. In comparison with control rats, neonatally treated capsaicin rats showed a significant (P < 0.001) bilateral decrease in [125I]NPY binding sites in the superficial layers of the dorsal horn. Unilateral dorsal rhizotomy and unilateral sciatic nerve section also exhibited a significant (P < 0.05) depletion in [125I]NPY labeling in the superficial layers of the dorsal horn ipsilateral to the surgery. These results suggest that a certain proportion of [12SI]NPY receptor sites is located on the primary afferent fibers of the superficial layers of the dorsal horn. This peptide thus could play an important role in the modulation of nociceptive transmission by acting directly on primary afferent terminals. Neuropeptide Y (NPY), a m e m b e r of the pancreatic polypeptide family, is an amidated 36-amino acid peptide derived by the posttranslational processing of a 97amino acid precursor molecule 22'28. In the brain, NPYlike immunoreactivity 1'5 and [125I]NpyI9-21 receptor binding sites are widely distributed and it is considered to be one of the most abundant neuropeptides in this tissue. In accordance with its distribution, N P Y has been shown to play an important role in the regulation of cardiovascular and neuroendocrine functions, sympathetic neurotransmission and m e m o r y processing 4'11'17. In the spinal cord a rich system of NPY-like immunoreactivity, particularly in the superficial layers of the dorsal horn and in the intermediolateral cell column has been described 9'16'27'3°. The origin of endogenous N P Y in the dorsal horn as well as in other parts of the normal spinal cord is considered to be primarily from intrinsic interneurones and from descending bulbospinal projections 9'12'16'27'30. Functionally, N P Y has been shown to be a powerful modulator of neurotransmitter release from cultured dorsal root ganglion cells 3~ and to yield a strong inhibition of spinal nociceptive and visceromotor reflexes following intrathecal injection in the unanaesthetized rat t3. In keeping with the increased significance of N P Y in the spinal cord, we have examined the quantitative autoradiography distribution and possible alteration of
[125I]NPY binding sites in rat spinal cord following pharmacological (capsaicin) and surgical (dorsal rhizotomy and peripheral axotomy) manipulations. Sprague-Dawley rats of either sex, obtained from Charles River, St. Constant, Canada, were employed throughout. All rats undergoing surgery were anaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and fentanyl citrate/fluansone (5 mg/kg, i.m.). Neonatal pups (n = 20) obtained from time-pregnant Sprague-Dawley rats were injected with a capsaicin solution (50 mg/kg, i.p.) (Sigma Chemicals, St. Louis, M O ) on day 2 and 3 of postnatal life 15. Capsaicin (10 mg/ml) was emulsified in a vehicle solution containing 10% (w/v) ethanol, 10% (v/v) Tween 80 and 80% (v/v) isotonic saline. Littermate controls (n = 20) were injected with the vehicle solution alone. Rats were killed at 2 months of age. In a group of 8 adult (3-month-old) rats, the right sciatic nerve was exposed at mid-thigh level, ligated at two locations separated by about 1 cm and cut between ligations. These animals were sacrificed 14 days later. In a second group of 8 rats (3 months old), a dorsal hemilaminectomy was performed on the lumbar2-1umbar 6 vertebrae. The corresponding dorsal roots i.e., LE-L 5 were sectioned extradurally on the right side taking care to avoid injury to the dorsal radicular arteries. The ventral roots were left intact. The area was then gently
Correspondence: R. Quirion, Douglas Hospital Research Center, 6875 La Salle Blvd, Verdun, Que. H4H 1R3, Canada. Fax: (1) (514) 766-2503.
334 packed with gelfoam and sutured in layers. The rats were sacrificed 14 days later. All the animals used in the study were decapitated and their spinal cords from lumbar level (L4) w e r e removed rapidly and snap-frozen in 2-methyl-butane at -40°C. The tissues were then serially cut (20 t~m) on a cryostat, thaw-mounted on gelatin-coated slides and processed as described earlier21. In brief, the sections were pre-incubated in Krebs-Ringer phosphate buffer (pH 7.4) for 60 min and then incubated for 120 min in the same buffer containing 0.1% bovine serum albumin, 0.05% bacitracin and 25 pM [125I]Bolton-Hunter porcine NPY (New England Nuclear, Boston, MA; spec. act. 2200 Ci/mmol). The non-specific binding was determined in the presence of 1/~M porcine NPY. All chemicals used in this study were of analytical grade and purchased from Sigma Chemicals (St. Louis, MO) or Fisher Scientific (Montreal, Que.). Following incubation, slides were rinsed four times (1 min each) in ice-cold buffer, dipped in cold water, rapidly air-dried and then exposed to tritium-sensitive films for 6-7 days along with micro-scale standards (Amersham, Canada). The autoradiograms were quantified densitometrically using a MCID image analysis system (Imaging Research Inc., Ontario, Canada) and the data were statistically analysed employing one-way ANOVA test. The overall distribution of [125I]NPY binding sites in control lumbar spinal cord (L4) as detected by in vitro receptor autoradiography is illustrated in Fig. 1A. The highest density of [12SI]NPY binding sites was noticed in superficial layers (laminae I-II) of the dorsal horn. Lowto-moderate densities of [125I]NPY binding sites were detected in deeper dorsal horn (laminae III-VI), ventral horn (laminae VII-IX) and in areas around the central canal (lamina X). White matter exhibited the lowest density of [125I]NPY binding sites. In neonatally treated capsaicin rats, compared to littermate controls, a significant (P < 0.001) bilateral decrease in [125I]NPY binding sites was noticed in the superficial layers (laminae I and II) of the dorsal horn (Figs. 1A,B, 2A). In the deeper dorsal horn and in the ventral horn only laminae
Fig. 1. Photomicrographs of [12SI]NPY binding sites in lumbar (L4) spinal cord of control (A), capsaicin-treated (B), unilateral dorsal root sectioned (C) and unilateral sciatic nerve sectioned (D) rats. Note the bilateral decrease (arrows) of [12SI]NPY binding sites from the superficial layers of the dorsal horn of capsaicin-treated rats (B) compared to the littermate control rats (A). Also note the ipsilateral decrease (arrows) of [12SI]NPY binding sites in superficial layers of the dorsal horn of surgically manipulated rats (C,D) compared to the respective contralateral sides. E represents [125I]NPY binding sites in control spinal cord in presence of 1/~M NPY. NS, non-specific binding. A - E x 15.
335 II, VI, IX and X showed statistically significant decrease in [125I]NPY binding sites whereas other laminae (IV, V, VII and VIII) did not exhibit any significant change in the density of [125I]NPY binding (see Fig. 2A). Similar to capsaicin treatment, unilateral dorsal rhizotomy (Figs. 1C, 2B) and unilateral sciatic nerve section (Figs. 1D, 2C) also significantly (P < 0.05) decrease [125I]NPY binding sites in the superficial layers (laminae I and II) of the dorsal horn ipsilateral to the surgery. However, the depletion of [125I]NPY binding sites in the ipsilateral dorsal horn of surgically manipulated rats is relatively less when compared to capsaicin-treated rats (Figs. 1B, C,D, 2A,B,C). The other region which exhibited statistically significant (P < 0.05) decrease in [125I]NPY bind-
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Fig. 2. Histograms showing [125I]NPY binding sites in the spinal cord of control and capsaicin-treated (A), unilateral dorsal root sectioned (B) and unilateral sciatic nerve sectioned (C) rats. Each value represents the mean + S.E.M. of 16-20 sections from 5-6 rats. L, lamina; WM, white matter. *P < 0.05, **P < 0.001.
ing sites from the corresponding contralateral side includes only lamina IX of the spinal cord (Fig. 2B,C). The present result demonstrates the distribution of [125I]NPY receptor binding sites in rat spinal cord and its alteration following pharmacological and surgical manipulations. The highest densities of [125I]NPY binding sites are located in laminae I and II of the dorsal horn while low to moderate amounts are present in other areas of the grey matter. The distribution of [125I]NPY binding sites, in general, correlates well with the localization of NPY-containing nerve terminals, which are found primarily in laminae I-II of the dorsal horn and to lesser extent in the ventral portion of the spinal cord 9' 16,27,30
The dorsal horn of the spinal cord, particularly laminae I and II, receives projections from small diameter sensory neurones which give rise to myelinated (A6) or unmyelinated (C) fibers with either nociceptive or nonnociceptive field properties 26. Capsaicin, when given neonatally is known to cause a selective lesion of small diameter neurones of the dorsal root ganglia along with marked depletion of several neuropeptides including calcitonin gene-related peptide, substance P, galanin and vasoactive intestinal polypeptide from superficial layers of the dorsal horn 3'8'15. Transection of a peripheral nerve or the dorsal root also evokes alterations in the level of immunoreactive peptides in the superficial layers of the dorsal horn as well as in the neurones of the severed nerves or roots 29. The selective depletion of [125I]NPY receptor binding sites from laminae I and II of the dorsal horn observed following capsaicin treatment, unilateral dorsal rhizotomy or sciatic nerve section suggests that [125I]NPY binding sites are likely associated with primary afferent fibers of the dorsal horn. However, it could be argued that decrease in [125I]NPY binding sites may be associated with intrinsic NPY-immunoreactive fibers or with second order neurones of the dorsal horn. Since NPY immunoreactivity in the superficial layers of the dorsal horn is of central origin (see Introduction) and remains unaltered following peripheral axotomy9'3° or capsaicin treatment 12 it is very unlikely that depletion in [125I]NPY binding sites corresponds to autoreceptors or to sites located postsynaptically on second order neurones of the dorsal horn. Evidence that NPY receptors are present on the primary afferent terminals is further substantiated by the demonstration of high affinity [125I]NPY binding on primary afferent fibers of cultured dorsal root ganglion cells31. However, a certain proportion of [125I]NPY sites which is still present in superficial dorsal horn following lesions appears to be located postsynaptically. Moreover, in comparison to capsaicin treatment, decreases in [125I]NPY binding sites in the ipsilateral dorsal horn of the surgically manipulated rats
336 are relatively lower. This possibly represents the association of [125I]NPY binding sites on the primary afferent fibers originating from the contralateral side of the same segment or from the ipsilateral side of the adjacent segments which are being spared following unilateral dorsal rhizotomy or sciatic nerve section 1s'23"25. In keeping with superficial layers of the dorsal horn, the loss of [125I]NPY binding sites in other areas of the spinal cord (i.e. laminae III, VI, IX and X in capsaicin-treated rats and lamina X in surgically manipulated rats) possibly reflects either the loss of primary afferent fibers known to be terminating in some of these areas 26 or the structural rearrangement that occurred following capsaicin treatment s or the section of spinal nerves or roots 1s'23. However, this probability should be taken with care because of the very low density of [125I]NPY binding sites in these areas of the spinal cord. Similar to N P Y sites, opiate receptors are associated, at least in part, with primary afferent fibers since capsaicin 24, dorsal rhizotomy 2'1° or peripheral nerve section 7
NPY, it has recently been shown that intrathecal injection of this peptide produces a powerful, d o s e - d e p e n d e n t blockade of noxious, thermal and mechanical stimuli which are not antagonized by an opioid or a 2 adrenergic receptor antagonist 13. M o r e o v e r , there is in vitro evidence that N P Y strongly inhibits depolarisation-evoked release of substance P from cultured dorsal root ganglion neurones 31. These results, together with the present findings lend strong support to the hypothesis that antinociceptive effects of N P Y could possibly be m e d i a t e d , at least in part, via the presynaptic inhibition of the release of substance P and other coexisting classical transmitters from primary afferent terminals. However, like for opiates, there is currently little in vivo evidence for the involvement of intrinsic N P Y in the direct regulation of substance P (or any other neuropeptides/classical transmitters) content and release from the primary afferent fibers. Therefore it would be of interest to examine the effects of N P Y on the release of various sensory n e u r o p e p t i d e s particularly that of substance P and the interaction, if any, between the opiates and N P Y using in vivo p a r a d i g m 6.
results in a substantial reduction in opiate receptors in the u p p e r dorsal horn, especially in laminae I and II. These results, together with findings that primary nociceptive afferents release substance P and that opiates inhibit the release of substance P both in vitro 14 and in vivo 33, have led to the suggestion that opiates may, in part, modulate nociceptive transmission by acting directly on primary afferent terminals 32. In the case of
This research was supported by a grant from the Medical Research Council of Canada (PG-38) and a fellowship from Eli Lilly Corp., Canada. R.Q. is a "Chercheur-Boursier' of the 'Fonds de la Recherche en Sant6 du Qu6bec'.
1 Allen, Y.S., Adrian, T.E., Allen, J.M., Tatemoto, K., Crow, T.J., Bloom, S.R. and Polak, J.M., Neuropeptide Y distribution in the rat brain, Science, 221 (1983) 877-879. 2 Besse, D., Lombard, M.C., Zajac, J.M., Roques, B.P. and Besson, J.M., Pre- and postsynaptic distribution of/~, 6 and ~ opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord, Brain Res., 521 (1990) 15-22. 3 Dalsgaard, C.J., The sensory system. In A. BjOrklund, T. H/~kfelt and C. Owman (Eds.), Handbook of Chemical Neuroanatomy, Vol. 6, The Peripheral Nervous System, Elsevier, Amsterdam, 1988, pp. 599-636. 4 Danger, J.M., Tonon, M.C., Jenks, B.G., St-Pierre, S., Martel, J.C., Fasolo, A., Breton, B., Quirion, R., Pelletier, G. and Vaudry, H., Neuropeptide Y: localization in the central nervous system and neuroendocrine functions, Fundam. Clin. Pharmacol., 4 (1990) 307-340. 5 DeQuidt, M.E. and Emson, P.C., Distribution of neuropeptide Y-like immunoreactivity in the rat: central nervous system II. Immunohistoehemical analysis, Neuroscience, 18 (1986) 545618. 6 Duggan, A.W., Hope, P.J., Jarrott, B., Schalble, H.-G. and Fleetwood-Walker, S.M., Release, spread and persistence of immunoreactive neurokinin A in the dorsal horn of the cat following noxious cutaneous stimulation. Studies with antibody microprobes, Neuroscience, 35 (1990) 195-202. 7 Fields, H.L., Emson, P.C., Leigh, BK., Gilbert, R.ET. and Iversen, L.L., Multiple opioid receptors in primary afferent fibers, Nature, 214 (1980) 351-352. 8 Fitzgerald, M., Capsaicin and sensory neurones a review, Pain, 15 (1983) 109-130. 9 Gibson, S.J., Polak, J.M., Allen, J.M., Adrian, T.E., Kelly,
J.S. and Bloom, S.R., The distribution and origin of a novel brain peptide, neuropeptide Y in the spinal cord of several mammals, J. Comp. Neurol., 227 (1984) 78-91. Gouarderes, C., Beaudet, A., Zajac, J.-M., Cros, J. and Quirion, R., High resolution radioautographic localization of [12SI]FK-33-824-1abeledmu opioid receptors in the spinal cord of normal and deafferented rats, Neuroscience, 43 (1991) 197209. Gray, T.S. and Morley, J.E., Neuropeptide Y: anatomical distribution and possible function in mammalian nervous system, Life Sci., 38 (1986) 389-401. H6kfelt, T., Lundberg, J.M., Terenius, L., Jancso, G. and Kimmel, J., Avian pancreatic polypeptide (APP) immunoreactivity neurones in the spinal cord trigeminal nucleus, Peptides, 2 (1981) 82-87. Hua, X.Y., Boublik, J.H., Spicer, M.A., Rivier, J.E., Brown, M.R. and Yaksh, T.L., The antinociceptive effects of spinally administered neuropeptide Y in the rat: systematic studies on structure-activity relationship, J. Pharmacol. Exp. Ther., 258 (1991) 243-248. Jessel, T.M. and Iversen, L.L., Opiate analgesics inhibit substance P release from rat trigeminal nucleus, Nature, 268 (1977) 549-551. Kar, S., Gibson, S.J. and Polak, J.M., Origins and projections of peptide-immunoreactive nerves in the male rat genitofemoral nerve, Brain Res., 512 (1990) 229-237. Krukoff, T.L., Neuropeptide Y-like immunoreactivity in cat spinal cord with special reference to autonomic areas, Brain Res., 415 (1987) 300-308. Lehmann, J., Neuropeptide Y: an overview, Drug Dev. Res., 19 (1990) 329-351.
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337 18 Liu, C.N. and Chambers, W.W., Intraspinal sprouting of dorsal root axons, Arch. Neurol. Psychol., 79 (1958) 46-61. 19 Lynch, D.R., Walker, M.W., Miller, R.J. and Snyder, S.H., Neuropeptide Y receptor binding sites in rat brain: differential autoradiographic localization with [125I]peptide YY and [1251]neuropeptide Y imply receptor heterogeneity, J. Neurosci., 9 (1989) 2607-2619. 20 Martel, J.C., St-Pierre, S. and Quirion, R., Neuropeptide Y receptors in rat brain: autoradiographic localization, Peptides, 7 (1986) 55-60. 21 Martel, J.C., Fournier, A., St-Pierre, S. and Quirion, R., Quantitative autoradiographic distribution of [12SI]BoltonHunter neuropeptide Y receptor binding sites in rat brain. Comparison with [125I]peptide YY receptor sites, Neuroscience, 36 (1990) 255-283. 22 Minth, C.A., Bloom, S.R., Polak, J.M. and Dixon, J.E., Cloning, characterization and DNA sequence of a human cDNA encoding neuropeptide tyrosine, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 4577-4581. 23 Murray, M. and Goldberger, M.E., Restitution of function and collateral sprouting in the cat spinal cord: the partially hemisected animals, J. Comp. Neurol., 158 (1974) 19-36. 24 Nagy, J.I., Vincent, S.R., Staines, W.M.A., Fibiger, H.C., Reisine, T.D. and Yamamura, H.I., Neurotoxic action of capsaicin on spinal substance P neurones, Brain Res., 186 (1980) 435-444. 25 Rustioni, A. and Weinberg, R.J., The somatosensory system. In A. BjSrklund, T. HSkfelt and L.W. Swanson (Eds.), Handbook of Chemical Neuroanatomy, Vol. 7. Integrated Systems of the CNS, Part 11, Elsevier, Amsterdam, 1989, pp. 219-321.
26 Salt, T.E. and Hill, R.G., Neurotransmitter candidates of somatosensory primary afferent fibres, Neuroscience, 10 (1983) 1083-1103. 27 Sasek, C.A. and Elde, R.P., Distribution of neuropeptide Y-like immunoreactivity and its relationship to FMRF-amide-like immunoreactivity in the sixth lumbar and first sacral spinal cord segments of the rat, J. Neurosci., 5 (1985) 1729-1739. 28 Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y: a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature, 296 (1982) 659-660. 29 Villar, M.J., Cortes, R., Theodorsson, E., Wiesenfeld-Hallin, Z., Schalling, M., Fahrenkrug, J., Emson, P.C. and HSkfelt, T., Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin, Neuroscience, 33 (1989) 587-604. 30 Wakisaka, S., Kajander, K.C. and Bennett, G.J., Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurones following peripheral axotomy, Neurosci. Lett., 124 (1991) 200-203. 31 Walker, M.W., Ewald, D.A., Pemey, T.M. and Miller, R.J., Neuropeptide Y modulates neurotransmitter release and Ca 2+ currents in rat sensory neurones, J. Neurosci., 8 (1988) 2438-2446. 32 Yaksh, T.L., Spinal opiate analgesia: characteristics and principles of action, Pain, 11 (1981) 293-346. 33 Yaksh, T.L., Jessel, T.M., Gamse, R., Mudge, A.W. and Leeman, S.E., Intrathecal morphine inhibits substance P release from mammalian spinal cord in vivo, Nature, 286 (1980) 155-157.
333
BRES 25081
Quantitative autoradiographic localization of [1251]neuropeptide Y receptor binding sites in rat spinal cord and the effects of neonatal capsaicin, dorsal rhizotomy and peripheral axotomy S. Kar and R. Quirion Douglas Hospital Research Center and Department of Psychiatry, Faculty of Medicine, McGill University, Verdun, Que. (Canada)
(Accepted 26 November 1991) Key words: Neuropeptide Y; Receptor autoradiography; Spinal cord; Capsaicin; Dorsal rhizotomy; Axotomy
Using in vitro quantitative receptor autoradiography the present study reports on the distribution and possible changes of [~25I]neuropeptide Y (NPY) binding sites in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. In control spinal cord the highest density of [12SI]NPY binding sites was noticed in the superficial layers of the dorsal horn whereas low-to-moderate densities of [125I]NPY binding sites were detected in the deeper dorsal horn and in the ventral horn. In comparison with control rats, neonatally treated capsaicin rats showed a significant (P < 0.001) bilateral decrease in [125I]NPY binding sites in the superficial layers of the dorsal horn. Unilateral dorsal rhizotomy and unilateral sciatic nerve section also exhibited a significant (P < 0.05) depletion in [125I]NPY labeling in the superficial layers of the dorsal horn ipsilateral to the surgery. These results suggest that a certain proportion of [12SI]NPY receptor sites is located on the primary afferent fibers of the superficial layers of the dorsal horn. This peptide thus could play an important role in the modulation of nociceptive transmission by acting directly on primary afferent terminals. Neuropeptide Y (NPY), a m e m b e r of the pancreatic polypeptide family, is an amidated 36-amino acid peptide derived by the posttranslational processing of a 97amino acid precursor molecule 22'28. In the brain, NPYlike immunoreactivity 1'5 and [125I]NpyI9-21 receptor binding sites are widely distributed and it is considered to be one of the most abundant neuropeptides in this tissue. In accordance with its distribution, N P Y has been shown to play an important role in the regulation of cardiovascular and neuroendocrine functions, sympathetic neurotransmission and m e m o r y processing 4'11'17. In the spinal cord a rich system of NPY-like immunoreactivity, particularly in the superficial layers of the dorsal horn and in the intermediolateral cell column has been described 9'16'27'3°. The origin of endogenous N P Y in the dorsal horn as well as in other parts of the normal spinal cord is considered to be primarily from intrinsic interneurones and from descending bulbospinal projections 9'12'16'27'30. Functionally, N P Y has been shown to be a powerful modulator of neurotransmitter release from cultured dorsal root ganglion cells 3~ and to yield a strong inhibition of spinal nociceptive and visceromotor reflexes following intrathecal injection in the unanaesthetized rat t3. In keeping with the increased significance of N P Y in the spinal cord, we have examined the quantitative autoradiography distribution and possible alteration of
[125I]NPY binding sites in rat spinal cord following pharmacological (capsaicin) and surgical (dorsal rhizotomy and peripheral axotomy) manipulations. Sprague-Dawley rats of either sex, obtained from Charles River, St. Constant, Canada, were employed throughout. All rats undergoing surgery were anaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and fentanyl citrate/fluansone (5 mg/kg, i.m.). Neonatal pups (n = 20) obtained from time-pregnant Sprague-Dawley rats were injected with a capsaicin solution (50 mg/kg, i.p.) (Sigma Chemicals, St. Louis, M O ) on day 2 and 3 of postnatal life 15. Capsaicin (10 mg/ml) was emulsified in a vehicle solution containing 10% (w/v) ethanol, 10% (v/v) Tween 80 and 80% (v/v) isotonic saline. Littermate controls (n = 20) were injected with the vehicle solution alone. Rats were killed at 2 months of age. In a group of 8 adult (3-month-old) rats, the right sciatic nerve was exposed at mid-thigh level, ligated at two locations separated by about 1 cm and cut between ligations. These animals were sacrificed 14 days later. In a second group of 8 rats (3 months old), a dorsal hemilaminectomy was performed on the lumbar2-1umbar 6 vertebrae. The corresponding dorsal roots i.e., LE-L 5 were sectioned extradurally on the right side taking care to avoid injury to the dorsal radicular arteries. The ventral roots were left intact. The area was then gently
Correspondence: R. Quirion, Douglas Hospital Research Center, 6875 La Salle Blvd, Verdun, Que. H4H 1R3, Canada. Fax: (1) (514) 766-2503.
334 packed with gelfoam and sutured in layers. The rats were sacrificed 14 days later. All the animals used in the study were decapitated and their spinal cords from lumbar level (L4) w e r e removed rapidly and snap-frozen in 2-methyl-butane at -40°C. The tissues were then serially cut (20 t~m) on a cryostat, thaw-mounted on gelatin-coated slides and processed as described earlier21. In brief, the sections were pre-incubated in Krebs-Ringer phosphate buffer (pH 7.4) for 60 min and then incubated for 120 min in the same buffer containing 0.1% bovine serum albumin, 0.05% bacitracin and 25 pM [125I]Bolton-Hunter porcine NPY (New England Nuclear, Boston, MA; spec. act. 2200 Ci/mmol). The non-specific binding was determined in the presence of 1/~M porcine NPY. All chemicals used in this study were of analytical grade and purchased from Sigma Chemicals (St. Louis, MO) or Fisher Scientific (Montreal, Que.). Following incubation, slides were rinsed four times (1 min each) in ice-cold buffer, dipped in cold water, rapidly air-dried and then exposed to tritium-sensitive films for 6-7 days along with micro-scale standards (Amersham, Canada). The autoradiograms were quantified densitometrically using a MCID image analysis system (Imaging Research Inc., Ontario, Canada) and the data were statistically analysed employing one-way ANOVA test. The overall distribution of [125I]NPY binding sites in control lumbar spinal cord (L4) as detected by in vitro receptor autoradiography is illustrated in Fig. 1A. The highest density of [12SI]NPY binding sites was noticed in superficial layers (laminae I-II) of the dorsal horn. Lowto-moderate densities of [125I]NPY binding sites were detected in deeper dorsal horn (laminae III-VI), ventral horn (laminae VII-IX) and in areas around the central canal (lamina X). White matter exhibited the lowest density of [125I]NPY binding sites. In neonatally treated capsaicin rats, compared to littermate controls, a significant (P < 0.001) bilateral decrease in [125I]NPY binding sites was noticed in the superficial layers (laminae I and II) of the dorsal horn (Figs. 1A,B, 2A). In the deeper dorsal horn and in the ventral horn only laminae
Fig. 1. Photomicrographs of [12SI]NPY binding sites in lumbar (L4) spinal cord of control (A), capsaicin-treated (B), unilateral dorsal root sectioned (C) and unilateral sciatic nerve sectioned (D) rats. Note the bilateral decrease (arrows) of [12SI]NPY binding sites from the superficial layers of the dorsal horn of capsaicin-treated rats (B) compared to the littermate control rats (A). Also note the ipsilateral decrease (arrows) of [12SI]NPY binding sites in superficial layers of the dorsal horn of surgically manipulated rats (C,D) compared to the respective contralateral sides. E represents [125I]NPY binding sites in control spinal cord in presence of 1/~M NPY. NS, non-specific binding. A - E x 15.
335 II, VI, IX and X showed statistically significant decrease in [125I]NPY binding sites whereas other laminae (IV, V, VII and VIII) did not exhibit any significant change in the density of [125I]NPY binding (see Fig. 2A). Similar to capsaicin treatment, unilateral dorsal rhizotomy (Figs. 1C, 2B) and unilateral sciatic nerve section (Figs. 1D, 2C) also significantly (P < 0.05) decrease [125I]NPY binding sites in the superficial layers (laminae I and II) of the dorsal horn ipsilateral to the surgery. However, the depletion of [125I]NPY binding sites in the ipsilateral dorsal horn of surgically manipulated rats is relatively less when compared to capsaicin-treated rats (Figs. 1B, C,D, 2A,B,C). The other region which exhibited statistically significant (P < 0.05) decrease in [125I]NPY bind-
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WM
Fig. 2. Histograms showing [125I]NPY binding sites in the spinal cord of control and capsaicin-treated (A), unilateral dorsal root sectioned (B) and unilateral sciatic nerve sectioned (C) rats. Each value represents the mean + S.E.M. of 16-20 sections from 5-6 rats. L, lamina; WM, white matter. *P < 0.05, **P < 0.001.
ing sites from the corresponding contralateral side includes only lamina IX of the spinal cord (Fig. 2B,C). The present result demonstrates the distribution of [125I]NPY receptor binding sites in rat spinal cord and its alteration following pharmacological and surgical manipulations. The highest densities of [125I]NPY binding sites are located in laminae I and II of the dorsal horn while low to moderate amounts are present in other areas of the grey matter. The distribution of [125I]NPY binding sites, in general, correlates well with the localization of NPY-containing nerve terminals, which are found primarily in laminae I-II of the dorsal horn and to lesser extent in the ventral portion of the spinal cord 9' 16,27,30
The dorsal horn of the spinal cord, particularly laminae I and II, receives projections from small diameter sensory neurones which give rise to myelinated (A6) or unmyelinated (C) fibers with either nociceptive or nonnociceptive field properties 26. Capsaicin, when given neonatally is known to cause a selective lesion of small diameter neurones of the dorsal root ganglia along with marked depletion of several neuropeptides including calcitonin gene-related peptide, substance P, galanin and vasoactive intestinal polypeptide from superficial layers of the dorsal horn 3'8'15. Transection of a peripheral nerve or the dorsal root also evokes alterations in the level of immunoreactive peptides in the superficial layers of the dorsal horn as well as in the neurones of the severed nerves or roots 29. The selective depletion of [125I]NPY receptor binding sites from laminae I and II of the dorsal horn observed following capsaicin treatment, unilateral dorsal rhizotomy or sciatic nerve section suggests that [125I]NPY binding sites are likely associated with primary afferent fibers of the dorsal horn. However, it could be argued that decrease in [125I]NPY binding sites may be associated with intrinsic NPY-immunoreactive fibers or with second order neurones of the dorsal horn. Since NPY immunoreactivity in the superficial layers of the dorsal horn is of central origin (see Introduction) and remains unaltered following peripheral axotomy9'3° or capsaicin treatment 12 it is very unlikely that depletion in [125I]NPY binding sites corresponds to autoreceptors or to sites located postsynaptically on second order neurones of the dorsal horn. Evidence that NPY receptors are present on the primary afferent terminals is further substantiated by the demonstration of high affinity [125I]NPY binding on primary afferent fibers of cultured dorsal root ganglion cells31. However, a certain proportion of [125I]NPY sites which is still present in superficial dorsal horn following lesions appears to be located postsynaptically. Moreover, in comparison to capsaicin treatment, decreases in [125I]NPY binding sites in the ipsilateral dorsal horn of the surgically manipulated rats
336 are relatively lower. This possibly represents the association of [125I]NPY binding sites on the primary afferent fibers originating from the contralateral side of the same segment or from the ipsilateral side of the adjacent segments which are being spared following unilateral dorsal rhizotomy or sciatic nerve section 1s'23"25. In keeping with superficial layers of the dorsal horn, the loss of [125I]NPY binding sites in other areas of the spinal cord (i.e. laminae III, VI, IX and X in capsaicin-treated rats and lamina X in surgically manipulated rats) possibly reflects either the loss of primary afferent fibers known to be terminating in some of these areas 26 or the structural rearrangement that occurred following capsaicin treatment s or the section of spinal nerves or roots 1s'23. However, this probability should be taken with care because of the very low density of [125I]NPY binding sites in these areas of the spinal cord. Similar to N P Y sites, opiate receptors are associated, at least in part, with primary afferent fibers since capsaicin 24, dorsal rhizotomy 2'1° or peripheral nerve section 7
NPY, it has recently been shown that intrathecal injection of this peptide produces a powerful, d o s e - d e p e n d e n t blockade of noxious, thermal and mechanical stimuli which are not antagonized by an opioid or a 2 adrenergic receptor antagonist 13. M o r e o v e r , there is in vitro evidence that N P Y strongly inhibits depolarisation-evoked release of substance P from cultured dorsal root ganglion neurones 31. These results, together with the present findings lend strong support to the hypothesis that antinociceptive effects of N P Y could possibly be m e d i a t e d , at least in part, via the presynaptic inhibition of the release of substance P and other coexisting classical transmitters from primary afferent terminals. However, like for opiates, there is currently little in vivo evidence for the involvement of intrinsic N P Y in the direct regulation of substance P (or any other neuropeptides/classical transmitters) content and release from the primary afferent fibers. Therefore it would be of interest to examine the effects of N P Y on the release of various sensory n e u r o p e p t i d e s particularly that of substance P and the interaction, if any, between the opiates and N P Y using in vivo p a r a d i g m 6.
results in a substantial reduction in opiate receptors in the u p p e r dorsal horn, especially in laminae I and II. These results, together with findings that primary nociceptive afferents release substance P and that opiates inhibit the release of substance P both in vitro 14 and in vivo 33, have led to the suggestion that opiates may, in part, modulate nociceptive transmission by acting directly on primary afferent terminals 32. In the case of
This research was supported by a grant from the Medical Research Council of Canada (PG-38) and a fellowship from Eli Lilly Corp., Canada. R.Q. is a "Chercheur-Boursier' of the 'Fonds de la Recherche en Sant6 du Qu6bec'.
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