- Email: [email protected]
Comparison of [125I]-Bolton–Hunter substance P binding in young and aged rat spinal cord
Brain Research 786 Ž1998. 263–266
Short communication
Comparison of w 125 Ix-Bolton–Hunter substance P binding in young and aged rat spinal cord Christine M. Maguire, Dominic P. Geraghty
)
Department of Biomedical Science, UniÕersity of Tasmania at Launceston, P.O. Box 1214, Launceston, Tasmania 7250, Australia Accepted 25 November 1997
Abstract Binding of w 125 Ix-labeled Bolton–Hunter substance P Žw 125 Ix-BHSP. to NK1 receptors was investigated in the spinal cord of young Ž3–4 month. and aged Ž14–16 month. rats. In homogenates of whole spinal cord, the affinity Žequilibrium dissociation constant, ; 210 pM. and maximum density of w 125 Ix-BHSP binding sites Ž; 0.25 fmolrmg wet weight. were similar for young and aged rats. Autoradiographic studies revealed a similar distribution of w 125 Ix-BHSP sites in both young and old rats at all spinal levels. Intense binding was observed in the superficial dorsal horn Žlaminae I–III., grey commissure Žlamina X. and thoracic intermediolateral cell column ŽIML. with lower levels of binding in the deeper dorsal horn Žlaminae IV–VI. and ventral horn Žlaminae VII–IX.. However, the density of w 125 Ix-BHSP sites was significantly Ž P - 0.05. lower in lamina X of lumbar sections of aged rats compared with young controls. These studies suggest that ageing is associated with a selective loss of NK1 receptors in lamina X of the lumbar spinal cord, although the affinity of NK1 receptors in aged rats is unchanged. q 1998 Elsevier Science B.V. Keywords: Substance P; NK1 receptor; Ageing; Spinal cord; Autoradiography
The tachykinin, substance P ŽSP., is released from the central terminals of primary afferent nociceptive C-fibres following stimulation of peripheral nerves and has been implicated in the processing of nociceptive information at the spinal cord level w5,15x. NK1 ŽSP-preferring. receptors have been demonstrated in the rat spinal cord using homogenate radioligand binding, autoradiographic, immunocytochemical and functional studies w4,9,12,14,15,17,18x. SP binding sites ŽNK1 receptors. are evenly distributed throughout the spinal grey matter at birth but become highly localized to the dorsal horn Žlaminae I–III. and grey commissure Žlamina X. by the third postnatal week w4,9x. Changes in the synthesis andror release of SP have been demonstrated in peripheral nerves and sensory ganglia of aged rats Ž) 14 months. w1,7,10x. However, the effects of ageing on spinal cord SP receptors is unknown. Thus, the present study compared the localization, density and affinity profile of SP ŽNK1. receptors in the spinal cord of young and aged rats.
) Corresponding author. [email protected]
Fax:
q 61-3-6324-3658;
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 4 6 3 - 7
Young Ž3–4 months. and aged Ž14–16 months. male Hooded–Wistar rats were housed 6–8 per cage Žyoung. and 3–4 per cage Žold. under a 12-h light–dark cycle. Animals were killed by carbon dioxide narcosis and the spinal cord removed using the method of deSousa and Horrocks w19x. Spinal cords were frozen in liquid N2-cooled isopentane and stored at y708C until required. Membranes for w 125 Ix-Bolton–Hunter substance P Žw 125 Ix-BHSP, 2,200 Cirmmol, New England Nuclear. binding were prepared from whole rat spinal cord as previously described for other tissues w11x and suspended in 50 mM Tris incubation buffer ŽpH 7.4 at 258C. containing 0.02% bovine serum albumin, 3 mM MnCl 2 and the peptidase inhibitors ŽSigma., bacitracin Ž40 m grml., chymostatin Ž4 m grml., leupeptin Ž4 m grml., bestatin Ž10 m M. and phosphoramidon Ž5 m M.. For hot saturation binding experiments Žyoung rats only., membranes Ž3% based on original wet weight of tissue. were incubated at 258C for 70 min with 10 pM—0.8 nM w 125 Ix-BHSP in a total volume of 0.25 ml incubation buffer. For cold saturation studies, membranes were incubated with 50 pM w 125 Ix-BHSP and increasing concentrations of SP ŽAuspep, Australia.. Nonspecific binding was defined in all assays
C.M. Maguire, D.P. Geraghtyr Brain Research 786 (1998) 263–266
264
using 1 m M unlabeled SP. Incubations were terminated by rapid filtration over Whatman GFrB filters Žpresoaked in 0.1% polyethyleneimine. and washing Ž4 = 3 ml. with ice-cold 50 mM Tris buffer ŽpH 7.4. containing 3 mM MnCl 2 . Membrane bound w 125 Ix-BHSP was counted in an LKB Multigamma counter at approximately 80% efficiency. Homogenate radioligand binding data were analysed using the nonlinear curve fitting program LIGAND w16x. Data are expressed as the geometric mean and 95% confidence limits, derived from approximate standard errors as described by Munson and Rodbard w16x. For autoradiography, frozen sections Ž15 m M. of spinal cord were thaw-mounted onto gelatine coated slides, desiccated overnight at 48C and stored at y708C. Sections were preincubated Ž2 = 5 min. in 170 mM Tris ŽpH 7.4, 258C. containing BSA Ž0.02%. followed by 10 min in 170 mM Tris ŽpH 7.4, 258C. containing BSA Ž0.02%., 3 mM MnCl 2 , 40 m grml bacitracin, 4 m grml chymostatin, 4 m grml leupeptin, 10 m M bestatin and 5 m M phosphoramidon. This was followed by incubation for 45 min at 258C in the same buffer containing 60 pM w 125 Ix-BHSP in the absence and presence Žnonspecific binding. of 1 m M SP. Sections were washed in ice-cold 170 mM Tris ŽpH 7.4, 48C. followed by distilled water at 48C, dried under a stream of warm air and apposed to 3 H-Hyperfilm ŽAmersham. together with 125 I-microscales ŽAmersham. for 5–10 days at 48C. Hyperfilms were developed, fixed and used as negatives to generate photographic prints. For quantitative autoradiography, the density of w 125 Ix-BHSP binding sites in laminae I–III, IV–VI, VII–IX and X was measured using an MCID image analysis system ŽImaging Research, St. Catherine’s, Ontario.. Measurements from four sections of each spinal segment from three young and three aged rats were obtained. The density of binding sites was expressed as the mean d.p.m.rmm 2 " S.E.M. Data were analysed using analysis of variance ŽANOVA. followed by Fisher’s Least Significant Difference ŽLSD. test Ž P - 0.05.. Specific binding of w 125 Ix-BHSP to membranes prepared from whole spinal cord of young rats was saturable Ž; 0.6 nM. and LIGAND analysis of hot and cold saturation experiments revealed a single class of high affinity sites ŽTable 1.. At or near the K D , specific binding accounted for approximately 70% of total binding. Cold saturation Table 1 Saturation binding characteristics for w 125 Ix-BHSP to spinal cord membranes from young and aged rats Age group
n
Assay method
K D ŽpM.
Bmax Žfmolrmg wet weight.
Young Young Aged
3 6 6
hot cold cold
210 Ž166–265. 219 Ž98–487. 206 Ž113–375.
0.37 Ž0.30–0.45. 0.25 Ž0.22–0.30. 0.26 Ž0.23–0.29.
Values for the equilibrium dissociation constant Ž K D . and maximum density of binding sites Ž Bma x . were generated by LIGAND. Confidence limits Ž95%. are shown in parentheses.
Fig. 1. Photomicrographs showing binding of w 125 Ix-BHSP to sections of rat cervical ŽA,E., thoracic ŽB,F., lumbar ŽC,G. and sacral ŽD,H. spinal cord of young ŽA–D. and aged ŽE–H. rats. Nonspecific binding Žnot shown. to sections was negligible. Bar s1 mm.
data for both young and aged spinal cord yielded similar K D and Bmax values ŽTable 1.. Fig. 1 shows photomicrographs of w 125 Ix-BHSP binding to sections of cervical, thoracic, lumbar and sacral spinal cord from young and aged rats. Under the conditions used, nonspecific binding was extremely low and all quantifiable binding detected was specific. Intense binding was observed in the superficial dorsal horn Žlaminae I–III. of cervical, lumbar and sacral spinal cord sections. Lower levels of binding were found in the deeper dorsal horn Žlaminae IV–VI. and the ventral horn Žlaminae VII–IX. of these regions. Binding was also pronounced in lamina X, particularly in lumbar and thoracic sections. In thoracic sections, highly localized binding occurred in the intermediolateral cell column ŽIML. while binding in the superficial dorsal horn was less pronounced. This distribution of w 125 Ix-BHSP binding sites was in accordance with previous autoradiographic studies and coincided with the immuno-
C.M. Maguire, D.P. Geraghtyr Brain Research 786 (1998) 263–266
histochemical localization of SP in spinal cord sections w9,17x. The highest densities of w 125 Ix-BHSP binding sites ŽNK1 receptors. were generally found in the grey commissure Žlamina X. and superficial dorsal horn Žlaminae I–III. where the majority of SP-containing primary afferent nociceptive C-fibres terminate. The density of w 125 Ix-BHSP binding sites in the cervical, thoracic and sacral spinal cord was similar for young and aged rats. However, in the lumbar cord of aged rats, the density of binding sites in lamina X was significantly lower than in young controls ŽFigs. 1 and 2.. The affinity Ž K D , ; 200 pM. and overall density of w 125 Ix-BHSP binding sites Ž Bmax , ; 0.25
Fig. 2. Specific w 125 Ix-BHSP binding to sections of cervical ŽA. and lumbar ŽB. spinal cord from young and aged rats. The density of binding sites Žd.p.m.rmm 2 . was measured in the superficial dorsal horn Žlaminae I–III., deeper dorsal horn Žlaminae V–VI., ventral horn ŽVII–IX., grey commissure Žlamina X. and total grey area ŽTOTAL.. Values represent the mean"S.E.M. of three rats. ) , P - 0.05, significantly different from young rats.
265
fmolrmg wet weight tissue. was almost identical in homogenates from young and old rats. The apparent loss of NK1 receptors in the grey commissure of the lumbar spinal cord may simply reflect normal age-related neurodegeneration. However, one would expect a uniform decrease in NK1 receptor numbers throughout the entire spinal cord. The plasticity of spinal NK1 receptors in mature rats is well established. Mechanical Žpinching. or chemical Žcapsaicin. stimulation of peripheral nerves releases SP from the central terminals of primary afferent nociceptive C-fibres and leads to rapid but reversible internalization of NK1 receptors in laminae I and II w14x. In contrast, dorsal rhizotomy or prolonged stimulation of peripheral nerves by adjuvant-induced hindpaw inflammation or constriction increases the number of spinal NK1 receptors w20,21x. Perhaps the decrease in NK1 receptor density in lamina X of aged rat spinal cord reflects increased release of SP which chronically downregulates NK1 receptors. For several days to weeks following chemical or surgical manipulation of peripheral nerves, the density of SP binding sites ŽNK1 receptors. in the dorsal horn appears to be inversely related to spinal SP content w6,20x. However, data on SP levels in aged animals is limited and conflicting. The ability of explanted sympathetic ganglia from aged Ž2 years. rats to synthesize SP is reduced compared with ganglia from adult Ž6 months. rats w1x. Buck et al. w3x found no change in SP levels with age in several CNS regions. As the decrease in NK1 receptor number in response to ageing was limited to the lumbar grey commissure, the mechanisms involved appear to be more specific than gross alterations in spinal SP levels. Littlewood et al. w12x have shown that in lamina X, many NK1 receptors are located on GABAergic neurons. This is not the case in the dorsal horn or other spinal cord regions with high NK1 receptor densities. Morphological studies show that neurons in the grey commissure which express the NK1 receptor are distinct from those in the dorsal horn w2x. It may be that ageing is accompanied by a loss of GABAergic neurons and the NK1 receptors they express. The GABAergic system in the spinal cord is activated to counteract chronic pain. Blockade of GABA receptors in monoarthritic rats causes increased release of SP from primary afferent fibres coupled with intense hyperalgesia w13x. Hypersensitivity to both chemical and mechanical stimuli and neuronal sensitivity to SP are characteristic of ageing w7,8x. Loss of GABA inhibitory neurons in the spinal cord could lead to hyperexcitability of dorsal horn and hence to the hyperalgesia observed in aged animals. This would also lead to the reduction in the density of NK1 receptors in lamina X of aged animals observed in this study. In conclusion, this study demonstrates a selective loss of NK1 receptors Žwith no change in affinity. in the lumbar spinal cord of aged rats. This may reflect a loss of GABAergic neurons and thus be partially responsible for the hyperalgesia associated with ageing.
266
C.M. Maguire, D.P. Geraghtyr Brain Research 786 (1998) 263–266
Acknowledgements These studies were supported by a grant to D.P.G. from the National Health and Medical Research Council of Australia. Thanks are due to Dr. A. Lawrence, Department of Pharmacology, Monash University, Victoria, for the use of the MCID system.
w10x
w11x
w12x
References w1x J.E. Adler, I.B. Black, Plasticity of substance P in mature and aged sympathetic neurons in culture, Science 225 Ž1984. 1499–1500. w2x J.L. Brown, H. Liu, J.E. Maggio, S.R. Vigna, P.W. Mantyh, A.I. Basbaum, Morphological examination of substance P receptor-immunoreactive neurons in the rat spinal cord and trigeminal nucleus caudalis, J. Comp. Neurol 356 Ž1995. 327–344. w3x S.H. Buck, P.P. Deshmukh, T.F. Burks, H.I. Yamamura, A survey of substance P, somatostatin, and neurotensin levels in ageing in the rat and human central nervous system, Neurobiol. Aging 2 Ž1981. 257–264. w4x C.G. Charlton, C.J. Helke, Ontogeny of substance P receptors in the rat spinal cord: quantitative changes in receptor number and differential expression in specific loci, Dev. Brain Res. 29 Ž1986. 81–91. w5x R. Dubner, M.A. Ruda, Activity dependent neuronal plasticity following tissue injury and inflammation, Trends Neurosci. 15 Ž1992. 96–103. w6x M.T. Galeazza, M.G. Garry, H.J. Yost, K.A. Strait, K.M. Hargreaves, V.S. Seybold, Plasticity in the synthesis and storage of substance P and calcitonin gene-related peptide in primary afferent neurons during peripheral inflammation, Neuroscience 66 Ž1995. 443–458. w7x Z.-G. Jiang, R.A. Smith, Regulation by nerve growth factor of neuropeptide phenotypes in primary cultured sensory neurons prepared from aged as well as adult mice, Dev. Brain Res. 90 Ž1995. 190–193. w8x B.S.G. Jones, H.R. Olpe, Multiple changes in the sensitivity of cingulate cortical neurones to putative neurotransmitters in ageing rats: substance P, acetylcholine and noradrenaline, Neurosci. Lett. 50 Ž1984. 31–36. w9x S. Kar, R. Quirion, Neuropeptide receptors in developing and adult spinal cord: an autoradiographic study of calcitonin gene-related
w13x
w14x
w15x
w16x
w17x
w18x
w19x
w20x
w21x
peptide, neurokinins, m-opioid, galanin, somatostatin, neurotensin and vasoactive intestinal polypeptide receptors, J. Comp. Neurol. 354 Ž1995. 253–281. Z. Khalil, V. Ralevic, G.J. Dusting, R.D. Helme, Effects of ageing on sensory nerve function in rat skin, Brain Res. 641 Ž1994. 265–272. R. Lew, D.P. Geraghty, G. Drapeau, D. Regoli, E. Burcher, Binding characteristics of w 125 IxBolton–Hunter wSar 9 ,MetŽO 2 .11 xsubstance P, a new selective radioligand for the NK1 receptor, Eur. J. Pharmacol. 184 Ž1990. 97–108. N.K. Littlewood, A.J. Todd, R.C. Spike, C. Watt, S.A.S. Shehab, The types of neurons in spinal dorsal horn which possess neurokinin-1 receptors, Neuroscience 66 Ž1995. 597–608. M. Malcangio, N.G. Bowery, Spinal cord SP release and hyperalgesia in monoarthritic rats: involvement of GABA B receptor system, Br. J. Pharmacol. 113 Ž1994. 1561–1566. P.W. Mantyh, E. DeMaster, A. Malhotra, J.R. Ghilardi, S.D. Rogers, C.R. Mantyh, H. Liu, A.I. Basbaum, S.R. Vigna, J.E. Maggio, D.A. Simone, Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation, Science 268 Ž1995. 1629– 1632. G.E. Marshall, S.A.S. Shehab, R.C. Spike, A.J. Todd, Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat, Neuroscience 72 Ž1996. 255–263. P.J. Munson, D. Rodbard, LIGAND: a versatile computerized approach for the characterization of ligand-binding systems, Anal. Biochem. 107 Ž1980. 220–239. Y. Nakaya, T. Kaneko, R. Shigemoto, S. Nakanishi, N. Mizuno, Immunohistochemical localisation of the substance P receptor in the central nervous system of the adult rat, J. Comp. Neurol. 347 Ž1994. 249–274. V. Neugebauer, H.G. Schaible, F. Weiretter, U. Freudenberger, The involvement of substance P and neurokinin-1 receptors in the responses of rat dorsal horn neurons to noxious but not innocuous mechanical stimuli applied to the knee joint, Brain Res. 666 Ž1994. 207–215. B.N. deSousa, L.A. Horrocks, Development of rat spinal cord: I. Weight and length with a method for rapid removal, Dev. Neurosci. 2 Ž1979. 115–121. C.L. Stucky, M.T. Galeazza, V.S. Seybold, Time-dependent changes in Bolton–Hunter-labelled 125 I-substance P binding in rat spinal cord following unilateral adjuvant-induced peripheral inflammation, Neuroscience 57 Ž1993. 397–409. K. Yashpal, T.-V. Dam, R. Quirion, Effects of dorsal rhizotomy on neurokinin receptor sub-types in the rat spinal cord: a quantitative autoradiographic study, Brain Res. 552 Ž1991. 240–247.
Short communication
Comparison of w 125 Ix-Bolton–Hunter substance P binding in young and aged rat spinal cord Christine M. Maguire, Dominic P. Geraghty
)
Department of Biomedical Science, UniÕersity of Tasmania at Launceston, P.O. Box 1214, Launceston, Tasmania 7250, Australia Accepted 25 November 1997
Abstract Binding of w 125 Ix-labeled Bolton–Hunter substance P Žw 125 Ix-BHSP. to NK1 receptors was investigated in the spinal cord of young Ž3–4 month. and aged Ž14–16 month. rats. In homogenates of whole spinal cord, the affinity Žequilibrium dissociation constant, ; 210 pM. and maximum density of w 125 Ix-BHSP binding sites Ž; 0.25 fmolrmg wet weight. were similar for young and aged rats. Autoradiographic studies revealed a similar distribution of w 125 Ix-BHSP sites in both young and old rats at all spinal levels. Intense binding was observed in the superficial dorsal horn Žlaminae I–III., grey commissure Žlamina X. and thoracic intermediolateral cell column ŽIML. with lower levels of binding in the deeper dorsal horn Žlaminae IV–VI. and ventral horn Žlaminae VII–IX.. However, the density of w 125 Ix-BHSP sites was significantly Ž P - 0.05. lower in lamina X of lumbar sections of aged rats compared with young controls. These studies suggest that ageing is associated with a selective loss of NK1 receptors in lamina X of the lumbar spinal cord, although the affinity of NK1 receptors in aged rats is unchanged. q 1998 Elsevier Science B.V. Keywords: Substance P; NK1 receptor; Ageing; Spinal cord; Autoradiography
The tachykinin, substance P ŽSP., is released from the central terminals of primary afferent nociceptive C-fibres following stimulation of peripheral nerves and has been implicated in the processing of nociceptive information at the spinal cord level w5,15x. NK1 ŽSP-preferring. receptors have been demonstrated in the rat spinal cord using homogenate radioligand binding, autoradiographic, immunocytochemical and functional studies w4,9,12,14,15,17,18x. SP binding sites ŽNK1 receptors. are evenly distributed throughout the spinal grey matter at birth but become highly localized to the dorsal horn Žlaminae I–III. and grey commissure Žlamina X. by the third postnatal week w4,9x. Changes in the synthesis andror release of SP have been demonstrated in peripheral nerves and sensory ganglia of aged rats Ž) 14 months. w1,7,10x. However, the effects of ageing on spinal cord SP receptors is unknown. Thus, the present study compared the localization, density and affinity profile of SP ŽNK1. receptors in the spinal cord of young and aged rats.
) Corresponding author. [email protected]
Fax:
q 61-3-6324-3658;
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 4 6 3 - 7
Young Ž3–4 months. and aged Ž14–16 months. male Hooded–Wistar rats were housed 6–8 per cage Žyoung. and 3–4 per cage Žold. under a 12-h light–dark cycle. Animals were killed by carbon dioxide narcosis and the spinal cord removed using the method of deSousa and Horrocks w19x. Spinal cords were frozen in liquid N2-cooled isopentane and stored at y708C until required. Membranes for w 125 Ix-Bolton–Hunter substance P Žw 125 Ix-BHSP, 2,200 Cirmmol, New England Nuclear. binding were prepared from whole rat spinal cord as previously described for other tissues w11x and suspended in 50 mM Tris incubation buffer ŽpH 7.4 at 258C. containing 0.02% bovine serum albumin, 3 mM MnCl 2 and the peptidase inhibitors ŽSigma., bacitracin Ž40 m grml., chymostatin Ž4 m grml., leupeptin Ž4 m grml., bestatin Ž10 m M. and phosphoramidon Ž5 m M.. For hot saturation binding experiments Žyoung rats only., membranes Ž3% based on original wet weight of tissue. were incubated at 258C for 70 min with 10 pM—0.8 nM w 125 Ix-BHSP in a total volume of 0.25 ml incubation buffer. For cold saturation studies, membranes were incubated with 50 pM w 125 Ix-BHSP and increasing concentrations of SP ŽAuspep, Australia.. Nonspecific binding was defined in all assays
C.M. Maguire, D.P. Geraghtyr Brain Research 786 (1998) 263–266
264
using 1 m M unlabeled SP. Incubations were terminated by rapid filtration over Whatman GFrB filters Žpresoaked in 0.1% polyethyleneimine. and washing Ž4 = 3 ml. with ice-cold 50 mM Tris buffer ŽpH 7.4. containing 3 mM MnCl 2 . Membrane bound w 125 Ix-BHSP was counted in an LKB Multigamma counter at approximately 80% efficiency. Homogenate radioligand binding data were analysed using the nonlinear curve fitting program LIGAND w16x. Data are expressed as the geometric mean and 95% confidence limits, derived from approximate standard errors as described by Munson and Rodbard w16x. For autoradiography, frozen sections Ž15 m M. of spinal cord were thaw-mounted onto gelatine coated slides, desiccated overnight at 48C and stored at y708C. Sections were preincubated Ž2 = 5 min. in 170 mM Tris ŽpH 7.4, 258C. containing BSA Ž0.02%. followed by 10 min in 170 mM Tris ŽpH 7.4, 258C. containing BSA Ž0.02%., 3 mM MnCl 2 , 40 m grml bacitracin, 4 m grml chymostatin, 4 m grml leupeptin, 10 m M bestatin and 5 m M phosphoramidon. This was followed by incubation for 45 min at 258C in the same buffer containing 60 pM w 125 Ix-BHSP in the absence and presence Žnonspecific binding. of 1 m M SP. Sections were washed in ice-cold 170 mM Tris ŽpH 7.4, 48C. followed by distilled water at 48C, dried under a stream of warm air and apposed to 3 H-Hyperfilm ŽAmersham. together with 125 I-microscales ŽAmersham. for 5–10 days at 48C. Hyperfilms were developed, fixed and used as negatives to generate photographic prints. For quantitative autoradiography, the density of w 125 Ix-BHSP binding sites in laminae I–III, IV–VI, VII–IX and X was measured using an MCID image analysis system ŽImaging Research, St. Catherine’s, Ontario.. Measurements from four sections of each spinal segment from three young and three aged rats were obtained. The density of binding sites was expressed as the mean d.p.m.rmm 2 " S.E.M. Data were analysed using analysis of variance ŽANOVA. followed by Fisher’s Least Significant Difference ŽLSD. test Ž P - 0.05.. Specific binding of w 125 Ix-BHSP to membranes prepared from whole spinal cord of young rats was saturable Ž; 0.6 nM. and LIGAND analysis of hot and cold saturation experiments revealed a single class of high affinity sites ŽTable 1.. At or near the K D , specific binding accounted for approximately 70% of total binding. Cold saturation Table 1 Saturation binding characteristics for w 125 Ix-BHSP to spinal cord membranes from young and aged rats Age group
n
Assay method
K D ŽpM.
Bmax Žfmolrmg wet weight.
Young Young Aged
3 6 6
hot cold cold
210 Ž166–265. 219 Ž98–487. 206 Ž113–375.
0.37 Ž0.30–0.45. 0.25 Ž0.22–0.30. 0.26 Ž0.23–0.29.
Values for the equilibrium dissociation constant Ž K D . and maximum density of binding sites Ž Bma x . were generated by LIGAND. Confidence limits Ž95%. are shown in parentheses.
Fig. 1. Photomicrographs showing binding of w 125 Ix-BHSP to sections of rat cervical ŽA,E., thoracic ŽB,F., lumbar ŽC,G. and sacral ŽD,H. spinal cord of young ŽA–D. and aged ŽE–H. rats. Nonspecific binding Žnot shown. to sections was negligible. Bar s1 mm.
data for both young and aged spinal cord yielded similar K D and Bmax values ŽTable 1.. Fig. 1 shows photomicrographs of w 125 Ix-BHSP binding to sections of cervical, thoracic, lumbar and sacral spinal cord from young and aged rats. Under the conditions used, nonspecific binding was extremely low and all quantifiable binding detected was specific. Intense binding was observed in the superficial dorsal horn Žlaminae I–III. of cervical, lumbar and sacral spinal cord sections. Lower levels of binding were found in the deeper dorsal horn Žlaminae IV–VI. and the ventral horn Žlaminae VII–IX. of these regions. Binding was also pronounced in lamina X, particularly in lumbar and thoracic sections. In thoracic sections, highly localized binding occurred in the intermediolateral cell column ŽIML. while binding in the superficial dorsal horn was less pronounced. This distribution of w 125 Ix-BHSP binding sites was in accordance with previous autoradiographic studies and coincided with the immuno-
C.M. Maguire, D.P. Geraghtyr Brain Research 786 (1998) 263–266
histochemical localization of SP in spinal cord sections w9,17x. The highest densities of w 125 Ix-BHSP binding sites ŽNK1 receptors. were generally found in the grey commissure Žlamina X. and superficial dorsal horn Žlaminae I–III. where the majority of SP-containing primary afferent nociceptive C-fibres terminate. The density of w 125 Ix-BHSP binding sites in the cervical, thoracic and sacral spinal cord was similar for young and aged rats. However, in the lumbar cord of aged rats, the density of binding sites in lamina X was significantly lower than in young controls ŽFigs. 1 and 2.. The affinity Ž K D , ; 200 pM. and overall density of w 125 Ix-BHSP binding sites Ž Bmax , ; 0.25
Fig. 2. Specific w 125 Ix-BHSP binding to sections of cervical ŽA. and lumbar ŽB. spinal cord from young and aged rats. The density of binding sites Žd.p.m.rmm 2 . was measured in the superficial dorsal horn Žlaminae I–III., deeper dorsal horn Žlaminae V–VI., ventral horn ŽVII–IX., grey commissure Žlamina X. and total grey area ŽTOTAL.. Values represent the mean"S.E.M. of three rats. ) , P - 0.05, significantly different from young rats.
265
fmolrmg wet weight tissue. was almost identical in homogenates from young and old rats. The apparent loss of NK1 receptors in the grey commissure of the lumbar spinal cord may simply reflect normal age-related neurodegeneration. However, one would expect a uniform decrease in NK1 receptor numbers throughout the entire spinal cord. The plasticity of spinal NK1 receptors in mature rats is well established. Mechanical Žpinching. or chemical Žcapsaicin. stimulation of peripheral nerves releases SP from the central terminals of primary afferent nociceptive C-fibres and leads to rapid but reversible internalization of NK1 receptors in laminae I and II w14x. In contrast, dorsal rhizotomy or prolonged stimulation of peripheral nerves by adjuvant-induced hindpaw inflammation or constriction increases the number of spinal NK1 receptors w20,21x. Perhaps the decrease in NK1 receptor density in lamina X of aged rat spinal cord reflects increased release of SP which chronically downregulates NK1 receptors. For several days to weeks following chemical or surgical manipulation of peripheral nerves, the density of SP binding sites ŽNK1 receptors. in the dorsal horn appears to be inversely related to spinal SP content w6,20x. However, data on SP levels in aged animals is limited and conflicting. The ability of explanted sympathetic ganglia from aged Ž2 years. rats to synthesize SP is reduced compared with ganglia from adult Ž6 months. rats w1x. Buck et al. w3x found no change in SP levels with age in several CNS regions. As the decrease in NK1 receptor number in response to ageing was limited to the lumbar grey commissure, the mechanisms involved appear to be more specific than gross alterations in spinal SP levels. Littlewood et al. w12x have shown that in lamina X, many NK1 receptors are located on GABAergic neurons. This is not the case in the dorsal horn or other spinal cord regions with high NK1 receptor densities. Morphological studies show that neurons in the grey commissure which express the NK1 receptor are distinct from those in the dorsal horn w2x. It may be that ageing is accompanied by a loss of GABAergic neurons and the NK1 receptors they express. The GABAergic system in the spinal cord is activated to counteract chronic pain. Blockade of GABA receptors in monoarthritic rats causes increased release of SP from primary afferent fibres coupled with intense hyperalgesia w13x. Hypersensitivity to both chemical and mechanical stimuli and neuronal sensitivity to SP are characteristic of ageing w7,8x. Loss of GABA inhibitory neurons in the spinal cord could lead to hyperexcitability of dorsal horn and hence to the hyperalgesia observed in aged animals. This would also lead to the reduction in the density of NK1 receptors in lamina X of aged animals observed in this study. In conclusion, this study demonstrates a selective loss of NK1 receptors Žwith no change in affinity. in the lumbar spinal cord of aged rats. This may reflect a loss of GABAergic neurons and thus be partially responsible for the hyperalgesia associated with ageing.
266
C.M. Maguire, D.P. Geraghtyr Brain Research 786 (1998) 263–266
Acknowledgements These studies were supported by a grant to D.P.G. from the National Health and Medical Research Council of Australia. Thanks are due to Dr. A. Lawrence, Department of Pharmacology, Monash University, Victoria, for the use of the MCID system.
w10x
w11x
w12x
References w1x J.E. Adler, I.B. Black, Plasticity of substance P in mature and aged sympathetic neurons in culture, Science 225 Ž1984. 1499–1500. w2x J.L. Brown, H. Liu, J.E. Maggio, S.R. Vigna, P.W. Mantyh, A.I. Basbaum, Morphological examination of substance P receptor-immunoreactive neurons in the rat spinal cord and trigeminal nucleus caudalis, J. Comp. Neurol 356 Ž1995. 327–344. w3x S.H. Buck, P.P. Deshmukh, T.F. Burks, H.I. Yamamura, A survey of substance P, somatostatin, and neurotensin levels in ageing in the rat and human central nervous system, Neurobiol. Aging 2 Ž1981. 257–264. w4x C.G. Charlton, C.J. Helke, Ontogeny of substance P receptors in the rat spinal cord: quantitative changes in receptor number and differential expression in specific loci, Dev. Brain Res. 29 Ž1986. 81–91. w5x R. Dubner, M.A. Ruda, Activity dependent neuronal plasticity following tissue injury and inflammation, Trends Neurosci. 15 Ž1992. 96–103. w6x M.T. Galeazza, M.G. Garry, H.J. Yost, K.A. Strait, K.M. Hargreaves, V.S. Seybold, Plasticity in the synthesis and storage of substance P and calcitonin gene-related peptide in primary afferent neurons during peripheral inflammation, Neuroscience 66 Ž1995. 443–458. w7x Z.-G. Jiang, R.A. Smith, Regulation by nerve growth factor of neuropeptide phenotypes in primary cultured sensory neurons prepared from aged as well as adult mice, Dev. Brain Res. 90 Ž1995. 190–193. w8x B.S.G. Jones, H.R. Olpe, Multiple changes in the sensitivity of cingulate cortical neurones to putative neurotransmitters in ageing rats: substance P, acetylcholine and noradrenaline, Neurosci. Lett. 50 Ž1984. 31–36. w9x S. Kar, R. Quirion, Neuropeptide receptors in developing and adult spinal cord: an autoradiographic study of calcitonin gene-related
w13x
w14x
w15x
w16x
w17x
w18x
w19x
w20x
w21x
peptide, neurokinins, m-opioid, galanin, somatostatin, neurotensin and vasoactive intestinal polypeptide receptors, J. Comp. Neurol. 354 Ž1995. 253–281. Z. Khalil, V. Ralevic, G.J. Dusting, R.D. Helme, Effects of ageing on sensory nerve function in rat skin, Brain Res. 641 Ž1994. 265–272. R. Lew, D.P. Geraghty, G. Drapeau, D. Regoli, E. Burcher, Binding characteristics of w 125 IxBolton–Hunter wSar 9 ,MetŽO 2 .11 xsubstance P, a new selective radioligand for the NK1 receptor, Eur. J. Pharmacol. 184 Ž1990. 97–108. N.K. Littlewood, A.J. Todd, R.C. Spike, C. Watt, S.A.S. Shehab, The types of neurons in spinal dorsal horn which possess neurokinin-1 receptors, Neuroscience 66 Ž1995. 597–608. M. Malcangio, N.G. Bowery, Spinal cord SP release and hyperalgesia in monoarthritic rats: involvement of GABA B receptor system, Br. J. Pharmacol. 113 Ž1994. 1561–1566. P.W. Mantyh, E. DeMaster, A. Malhotra, J.R. Ghilardi, S.D. Rogers, C.R. Mantyh, H. Liu, A.I. Basbaum, S.R. Vigna, J.E. Maggio, D.A. Simone, Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation, Science 268 Ž1995. 1629– 1632. G.E. Marshall, S.A.S. Shehab, R.C. Spike, A.J. Todd, Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat, Neuroscience 72 Ž1996. 255–263. P.J. Munson, D. Rodbard, LIGAND: a versatile computerized approach for the characterization of ligand-binding systems, Anal. Biochem. 107 Ž1980. 220–239. Y. Nakaya, T. Kaneko, R. Shigemoto, S. Nakanishi, N. Mizuno, Immunohistochemical localisation of the substance P receptor in the central nervous system of the adult rat, J. Comp. Neurol. 347 Ž1994. 249–274. V. Neugebauer, H.G. Schaible, F. Weiretter, U. Freudenberger, The involvement of substance P and neurokinin-1 receptors in the responses of rat dorsal horn neurons to noxious but not innocuous mechanical stimuli applied to the knee joint, Brain Res. 666 Ž1994. 207–215. B.N. deSousa, L.A. Horrocks, Development of rat spinal cord: I. Weight and length with a method for rapid removal, Dev. Neurosci. 2 Ž1979. 115–121. C.L. Stucky, M.T. Galeazza, V.S. Seybold, Time-dependent changes in Bolton–Hunter-labelled 125 I-substance P binding in rat spinal cord following unilateral adjuvant-induced peripheral inflammation, Neuroscience 57 Ž1993. 397–409. K. Yashpal, T.-V. Dam, R. Quirion, Effects of dorsal rhizotomy on neurokinin receptor sub-types in the rat spinal cord: a quantitative autoradiographic study, Brain Res. 552 Ž1991. 240–247.