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The effects of prenatal capsaicin on the distribution of substance P in developing primary afferent neurons
Neuroscience Letters, 35 (1983) 25-29
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Elsevier Scientific Publishers Ireland Ltd.
THE EFFECTS OF PRENATAL CAPSAICIN ON THE DISTRIBUTION OF SUBSTANCE P IN DEVELOPING PRIMARY AFFERENT NEURONS
M A R T I N E. A T K I N S O N and J A G D I S H SINGH C H A G G A R
Department of Anatomy and Cell Biology, University of Sheffield, Sheffield $10 2TN (U.K.) (Received November 1st, 1982; Revised version received and accepted December 2nd, 1982)
Key words: capsaicin - substance P - immunofluorescence - sensory neurons - ontogeny - sensory ganglia
Capsaicin was injected subcutaneously into 15-day pregnant mice and by transuterine injection into foetuses of the same age. Indirect immunofluorescence was used to assess its effects on the distribution of substance P. Capsaicin administered to either the mother or the foetus crossed the placenta and depleted substance P from the primary afferent terminal field in the spinal cord and abolished reactivity in the dorsal root ganglia and peripheral terminals of spinal nerves. Subcutaneous capsaicin administration to the pregnant female is, operationally, the easiest method providing low doses are used; otherwise pregnant females seem highly susceptible to respiratory failure. These results suggest the use of capsaicin to study prenatal ontogeny of sensory nerves.
Capsaicin has various neurotoxic effects including abolition of responses to chemical and thermal noxious stimuli and depletion of neuropeptides from unmyelinated primary afferent neurones. Recent investigations of local application of capsaicin to peripheral nerve trunks in adult animals have shown that it causes depletion of substance P, somatostatin, cholecystokinin and vasoactive intestinal polypeptide from the terminal fields of primary afferent neurones in the spinal cord [7]. Physiological changes also occur, such as a reduction in conduction time in C fibres and a decrease in their ability to excite cells in the spinal cord [25]. These actions may be brought about by inhibition of axonal transport [6], but no degenerative changes are observed in the nerve [1] or postsynaptically [7]. In contrast, administration of capsaicin to neonates results in irreversible degeneration of unmyelinated primary afferent nerves [12, 15, 21] as well as loss of peptides and responses to noxious thermal stimuli [12, 19]. As well as demonstrating non-specificity of capsaicin for any subpopulation of primary afferent nerves, recent evidence suggests that its effects are independent of those on nociception; loss of response to noxious stimuli is almost immediate whereas depletion of neuropeptides takes much longer [16, 17]. Nevertheless, capsaicin can still be used as a pharmacological agent in the study of the development 0304-3940/83/0000-0000/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
26 of primary afferent neurones using neuropeptides as markers of such nerves. Of the various peptides present in primary afferent nerves, the development of substance P is best documented. It appears quite early in the foetus [11, 18, 22, 23] and it may be first detectable in primary afferent neurones [8]. Capsaicin has not been used prenatally and we report here the results of an investigation of administration of capsaicin prenatally and its effects on substance P distribution in primary afferent neurones and their central terminal fields. Capsaicin was administered to foetuses by two routes to investigate whether any affects could be exerted transplacentally. Capsaicin (Sigma Chemical Co.) was dissolved in 10% absolute alcohol, 10°70 Tween 80 and 80% isotonic saline. It was injected subcutaneously into the neck skin of ether-anaesthetized female mice of 15 day duration dated pregnancy at a dosage of 25, 30 or 50 mg/kg; control mice were injected with vehicle alone. Another group of 15-day pregnant mice were anaesthetized with pentobarbitone sodium, the abdomen opened and the uterus exteriorized. Under cold light transillumination the foetuses were gently manipulated to present their dorsal surface. They were then injected through the uterine wall, foetal membranes and dorsal skin with capsaicin at a dosage of 25 mg/kg. The uterus was carefully replaced and the abdominal wall sutured. Mothers from both experimental groups were allowed to give birth and then the neonates were killed one week after birth by intracardiac perfusion of ice-cold 4070 paraformaldehyde under ether anaesthesia. The mothers were likewise killed. The cervical spinal cord and cervical spinal ganglia were removed together with skin from the forepaw and a piece of duodenum. After further fixation, 20/zm frozen sections were cut and processed for visualization of substance P by indirect immunofluorescence [31. Sections were incubated in monoclonal anti-substance P antibody (Seralab U.K.) diluted 1:40 in phosphate-buffered saline containing 0.2°70 Triton X-100 at 37°C for 45 rain, then washed repeatedly and stained in fluorescein-conjugated sheep anti-rat antibody (Wellcome Reagents Ltd.) diluted 1:20 in buffer. Control sections were incubated in buffer alone or in anti-substance P antibody adsorbed with excess substance P. Capsaicin can cause severe respiratory distress [24] and pregnant mice seemed particularly susceptible to this adverse effect in doses tolerated by normal adult and neonatal rodents [5]. By reducing the dose, capsaicin was tolerated by pregnant mice and mortality fell. At a dose of 50 m g / k g only 2 out of 10 treated animals survived; 5/10 survived at a dose of 30 m g / k g but 9 out of 10 at a dose of 25 m g / k g . The least toxic dose (25 mg/kg) was used for the main experiments. When foetuses were injected only 6 in each litter were treated resulting in a very small total dose of capsaicin ( < 6 m g / k g to the mother). Nevertheless, two pregnant females developed respiratory distress and died, although this may have been a cumulative effect of general anaesthesia and capsaicin. In 7-day-old mice injected with 25 m g / k g of capsaicin either directly or indirectly through the maternal circulation at El 5, the effect of capsaicin on the distribution
27
of substance P was the same. There was an extensive depletion of substance P immunoreactive fibres from the dorsal horn of the spinal cord which was particularly obvious in the substantia gelatinosa (Fig. 1). The distribution of substance P in the ventral horn and around the central canal (lamina X) was the same in both injected and control animals. Capsaicin administered either directly or transplacentaUy also depleted substance P from cutaneous nerve endings in the skin of the forepaw and from the substance P-immunoreactive cells of the dorsal root ganglia. No depletion of substance P immunoreactive fibres or cells was observed in the gut. Substance P was depleted from the various parts of primary afferent neurones in mothers injected directly with 20, 30 or 50 m g / k g capsaicin, but the depletion was not so marked in mothers of injected foetuses where the dose was much less. It is apparent that capsaicin crosses the placenta and exerts a toxic effect on the peripheral nerves of the foetuses. It crosses either from mother to foetus or from foetus to mother, as shown by the immunohistochemical results and respiratory reaction of mothers at the time of injection. Purely for operational ease, it is preferable to administer capsaicin systematically through the mother in a suitable dose which will not cause respiratory problems to the pregnant female as the effects on substance P are identical. The observed effects of capsaicin administered to 15-day foetal mice were identical to those described many times for neonatal animals [4, 13] and adult animals [7, 19]. Substance P is first detectable around E l 4 to E l 6 in the rodent spinal cord, although estimates vary by a day or two according to species and methodology [2, 8, 14, 23]. Substance P is present in 3 systems in the spinal cord: primary afferents, interneurones and supraspinal projections [10], and recent observations in the mouse suggest they appear in the order interneurones, primary afferent neurones and finally projection neurones as judged by detection of substance P-like im° munoreactivity [2]. E 15 seems to be the critical point in the development of primary afferent neurones as shown by the effect of the single dose of capsaicin at that age. In the mouse, substance P is not detectable in peripheral terminals or sensory
Fig. 1. A: a photomontage of the dorsal horn of the spinal cord of a normal 7-day-old mouse. B: a photomontage of the dorsal horn of the spinal cord of a 7-day-old mouse which received a direct injection of capsaicin as a 15-day-old foetus. Note the marked depletion of substance P-like immunoreactivity from laminae II and III but preservation in lamina I. D, dorsal aspect; L, lateral aspect; II, lamina If.
28
ganglia until El7 but the action of capsaicin is sufficiently powerful to prevent its appearance in these areas even though it is only present in the site of action for a relatively short time [20]. The mechanism of action of capsaicin is still poorly understood. In adult animals it adversely affects synthesis of substance P [9] and inhibits axonal transport of substance P [6]. Why it should cause irreversible degeneration of neonatal primary afferent neurones is even less well understood. Whether the observed effects in the present study are simply due to depletion of substance P or are due to destruction of the nerve and hence loss of its neuropeptide content, require correlative structural studies. Nevertheless, this initial study opens up the possibilities of using capsaicin to study prenatal ontogeny of substance P-containing and other peptidergic fibre systems. I Ainsworth, A., Hall, P., Wall, P.D., Allt, G., McKenzie, M.L., Gibson, S. and Polak, J.M., Effects of capsaicin applied locally to adult peripheral nerve. 11 Anatomy and enzyme and peptide chemistry of peripheral nerve and spinal cord, Pain, 11 (1981) 379 388. 2 Chaggar, J.S., Atkinson, M.E. and Mohamed, S.S., The ontogeny of substance P-like immunoreactivity in developing mouse spinal nerves and spinal cord, Neuroscience, in press. 3 Cuello, A.C., Galfre, G. and Milstein, C., Detection of substance P in the central nervous system by a monoclonal antibody, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 3532--3536. 4 Cuello. A.('., Gamse, R., Holzer, P. and Lembeck, F., Substance P immunoreactive neurons followmg neonatal administration of capsaiein, Naunyn-Schmeideberg's Arch. Pharmacol., 315 (1981) 185-194. 5 Gamse, R., Holzer, P. and Lembeck, F., Decrease of substance P in primary afferent neurones and impairment of neurogenic plasma extravasation by capsaicin, Brit. J. Pharmacol., 68 (1980) 207-213. 6 Gamse, R., Petsche, U., Lembeck, F. and Jancs6, G., Capsaicin applied to peripheral nerve inhibits axoplasmic transport of substance P and somatostatin, Brain Res., 239 (1982) 447 462. 7 Gibson, S.J., McGregor, G., Bloom, S.R., Polak, J.M. and Wall, P.D., Local application of capsaicin to one sciatic nerve of the adult rat induces a marked depletion in the peptide content of the lumbar dorsal horn, Neuroscience, in press. 8 Gilbert, R.F.T. and Emson, P.C., Substance P in rat CNS and duodenum during development, Brain Res., 171 (1979) 166-170. 9 Harmar, A., Schofield, J.G. and Keen, P., Cycloheximide-sensitive synthesis of substance P by isolated dorsal root ganglion, Nature (Lond.), 284 (1980) 267-269. 10 H6kfelt, T., Johansson, O., Ljungdahl, A., Lundberg, J.M. and Schultzberg, M., Peptidergic neurons, Nature (Lond.), 284 (1980) 515-522. 11 lnagaki, S., Sakanaka, M., Shiosaka, S., Senba, E., Takatsuki, K., Takagi, H., Kawai, H., Minagawa, H. and Tohyama, M., Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis. 1 Forebrain and upper brain stem, Neuroscience, 7 (1982) 251-277. 12 Jancs6, G., Kiraly, E. and Jancs6-Gabor, A., Pharmacologically induced selective degeneration of chemosensitive primary sensory neurons, Nature (Lond.), 270 (1977) 741-742. 13 Jancs6, G., H6kfelt, T., Lundberg, J.M. Kiraly, E., Hal~.sz, N., Nilsson, G., Terenius, L., Rehfeld, J., Steinbusch, H., Verhofstad, A., Elde, R., Said, S. and Brown, M., Immunohistochemieal studies on the effect of capsaicin on spinal and medullary peptides and monoamine neurones using antisera to substance P, gastrin-CCK, somatostatin, VIP, enkephalin, neurotensin, and 5-hydroxytryptamine, J. Neurocytol., 10 (1981) 963-980. 14 Kessler, J.A. and Black, I.B., Nerve growth factor stimulates development of substance P in the embryonic spinal cord, Brain Res., 208 (1981) 135-145.
29 15 Lawson, S.N. and Nickels, S.M., The use of morphometric techniques to analyse the effect of neonatal capsaicin treatment on rat dorsal root ganglia and dorsal roots, J. Physiol. (Lond.), 303 (1980) 12P. 16 Lembeck, F. and Donnerer, J., Time course of capsaicin-induced functional impairments in comparison with changes in neuronal substance P content, Naunyn-Schmeideberg's Arch. Pharmacol., 36 (1981) 240-243. 17 Miller, M.S., Buck, S.H., Sipes, 1.G. and Burks, T.F., Capsaicinoid-induced local and systemic antinociception without substance P depletion, Brain Res., 244 (1982) 193-197. 18 Mohamed, S.S. and Atkinson, M.E., The ontogeny of substance P fibres in the mouse trigeminal ganglion, Develop. Brain Res., 4 (1982) 351-355. 19 Nagy, J.l., Hunt, S.P., Iversen, L.L. and Emson, P.C., Biochemical and anatomical observations on the degeneration of peptide-containing primary afferent neurons after capsaicin, Neuroscience, 6 (1981) 1923-1934. 20 Saria, A., Skofitch, G. and Lembeck, F., Distribution of capsaicin in rat tissues after systemic administration, J. Pharm. Pharmacol., 34 (1982) 273-275. 21 Scadding, J.W., The permanent anatomical effects of neonatal capsaicin on somatosensory nerves, J. Anat. (Lond.), 131 (1980) 473-484. 22 Sakanaga, M., Inagaki, S., Shiosaka, S., Senba, E., Takagi, H., Takatsuki, K., Kawai, Y., Iida, M., Hara, Y. and Tohyama, M., Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis. II Lower brainstem, Neuroscience, 7 (1982) 1097-1126. 23 Senba, E., Shiosaka, S., Hara, Y., Inagaki, S., Sakanaka, M., Takatsuki, K., Kawai, Y. and Tohyama, M., Ontogeny of the peptidergic system in the rat spinal cord: immunohistochemical analysis, J. comp. Neurol., 208 (1982) 54-66. 24 Virus, R.M. and Gebhart, F.G., Pharmacological actions of capsaicin: apparent involvement of substance P and serotonin, Life Sci., 25 (1979) 1273-1284. 25 Wall, P.D. and Fitzgerald, M., Effects of capsaicin applied locally to adult peripheral nerve I. Physiology of peripheral nerve and spinal cord, Pain, 11 (1981) 363-377.
25
Elsevier Scientific Publishers Ireland Ltd.
THE EFFECTS OF PRENATAL CAPSAICIN ON THE DISTRIBUTION OF SUBSTANCE P IN DEVELOPING PRIMARY AFFERENT NEURONS
M A R T I N E. A T K I N S O N and J A G D I S H SINGH C H A G G A R
Department of Anatomy and Cell Biology, University of Sheffield, Sheffield $10 2TN (U.K.) (Received November 1st, 1982; Revised version received and accepted December 2nd, 1982)
Key words: capsaicin - substance P - immunofluorescence - sensory neurons - ontogeny - sensory ganglia
Capsaicin was injected subcutaneously into 15-day pregnant mice and by transuterine injection into foetuses of the same age. Indirect immunofluorescence was used to assess its effects on the distribution of substance P. Capsaicin administered to either the mother or the foetus crossed the placenta and depleted substance P from the primary afferent terminal field in the spinal cord and abolished reactivity in the dorsal root ganglia and peripheral terminals of spinal nerves. Subcutaneous capsaicin administration to the pregnant female is, operationally, the easiest method providing low doses are used; otherwise pregnant females seem highly susceptible to respiratory failure. These results suggest the use of capsaicin to study prenatal ontogeny of sensory nerves.
Capsaicin has various neurotoxic effects including abolition of responses to chemical and thermal noxious stimuli and depletion of neuropeptides from unmyelinated primary afferent neurones. Recent investigations of local application of capsaicin to peripheral nerve trunks in adult animals have shown that it causes depletion of substance P, somatostatin, cholecystokinin and vasoactive intestinal polypeptide from the terminal fields of primary afferent neurones in the spinal cord [7]. Physiological changes also occur, such as a reduction in conduction time in C fibres and a decrease in their ability to excite cells in the spinal cord [25]. These actions may be brought about by inhibition of axonal transport [6], but no degenerative changes are observed in the nerve [1] or postsynaptically [7]. In contrast, administration of capsaicin to neonates results in irreversible degeneration of unmyelinated primary afferent nerves [12, 15, 21] as well as loss of peptides and responses to noxious thermal stimuli [12, 19]. As well as demonstrating non-specificity of capsaicin for any subpopulation of primary afferent nerves, recent evidence suggests that its effects are independent of those on nociception; loss of response to noxious stimuli is almost immediate whereas depletion of neuropeptides takes much longer [16, 17]. Nevertheless, capsaicin can still be used as a pharmacological agent in the study of the development 0304-3940/83/0000-0000/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
26 of primary afferent neurones using neuropeptides as markers of such nerves. Of the various peptides present in primary afferent nerves, the development of substance P is best documented. It appears quite early in the foetus [11, 18, 22, 23] and it may be first detectable in primary afferent neurones [8]. Capsaicin has not been used prenatally and we report here the results of an investigation of administration of capsaicin prenatally and its effects on substance P distribution in primary afferent neurones and their central terminal fields. Capsaicin was administered to foetuses by two routes to investigate whether any affects could be exerted transplacentally. Capsaicin (Sigma Chemical Co.) was dissolved in 10% absolute alcohol, 10°70 Tween 80 and 80% isotonic saline. It was injected subcutaneously into the neck skin of ether-anaesthetized female mice of 15 day duration dated pregnancy at a dosage of 25, 30 or 50 mg/kg; control mice were injected with vehicle alone. Another group of 15-day pregnant mice were anaesthetized with pentobarbitone sodium, the abdomen opened and the uterus exteriorized. Under cold light transillumination the foetuses were gently manipulated to present their dorsal surface. They were then injected through the uterine wall, foetal membranes and dorsal skin with capsaicin at a dosage of 25 mg/kg. The uterus was carefully replaced and the abdominal wall sutured. Mothers from both experimental groups were allowed to give birth and then the neonates were killed one week after birth by intracardiac perfusion of ice-cold 4070 paraformaldehyde under ether anaesthesia. The mothers were likewise killed. The cervical spinal cord and cervical spinal ganglia were removed together with skin from the forepaw and a piece of duodenum. After further fixation, 20/zm frozen sections were cut and processed for visualization of substance P by indirect immunofluorescence [31. Sections were incubated in monoclonal anti-substance P antibody (Seralab U.K.) diluted 1:40 in phosphate-buffered saline containing 0.2°70 Triton X-100 at 37°C for 45 rain, then washed repeatedly and stained in fluorescein-conjugated sheep anti-rat antibody (Wellcome Reagents Ltd.) diluted 1:20 in buffer. Control sections were incubated in buffer alone or in anti-substance P antibody adsorbed with excess substance P. Capsaicin can cause severe respiratory distress [24] and pregnant mice seemed particularly susceptible to this adverse effect in doses tolerated by normal adult and neonatal rodents [5]. By reducing the dose, capsaicin was tolerated by pregnant mice and mortality fell. At a dose of 50 m g / k g only 2 out of 10 treated animals survived; 5/10 survived at a dose of 30 m g / k g but 9 out of 10 at a dose of 25 m g / k g . The least toxic dose (25 mg/kg) was used for the main experiments. When foetuses were injected only 6 in each litter were treated resulting in a very small total dose of capsaicin ( < 6 m g / k g to the mother). Nevertheless, two pregnant females developed respiratory distress and died, although this may have been a cumulative effect of general anaesthesia and capsaicin. In 7-day-old mice injected with 25 m g / k g of capsaicin either directly or indirectly through the maternal circulation at El 5, the effect of capsaicin on the distribution
27
of substance P was the same. There was an extensive depletion of substance P immunoreactive fibres from the dorsal horn of the spinal cord which was particularly obvious in the substantia gelatinosa (Fig. 1). The distribution of substance P in the ventral horn and around the central canal (lamina X) was the same in both injected and control animals. Capsaicin administered either directly or transplacentaUy also depleted substance P from cutaneous nerve endings in the skin of the forepaw and from the substance P-immunoreactive cells of the dorsal root ganglia. No depletion of substance P immunoreactive fibres or cells was observed in the gut. Substance P was depleted from the various parts of primary afferent neurones in mothers injected directly with 20, 30 or 50 m g / k g capsaicin, but the depletion was not so marked in mothers of injected foetuses where the dose was much less. It is apparent that capsaicin crosses the placenta and exerts a toxic effect on the peripheral nerves of the foetuses. It crosses either from mother to foetus or from foetus to mother, as shown by the immunohistochemical results and respiratory reaction of mothers at the time of injection. Purely for operational ease, it is preferable to administer capsaicin systematically through the mother in a suitable dose which will not cause respiratory problems to the pregnant female as the effects on substance P are identical. The observed effects of capsaicin administered to 15-day foetal mice were identical to those described many times for neonatal animals [4, 13] and adult animals [7, 19]. Substance P is first detectable around E l 4 to E l 6 in the rodent spinal cord, although estimates vary by a day or two according to species and methodology [2, 8, 14, 23]. Substance P is present in 3 systems in the spinal cord: primary afferents, interneurones and supraspinal projections [10], and recent observations in the mouse suggest they appear in the order interneurones, primary afferent neurones and finally projection neurones as judged by detection of substance P-like im° munoreactivity [2]. E 15 seems to be the critical point in the development of primary afferent neurones as shown by the effect of the single dose of capsaicin at that age. In the mouse, substance P is not detectable in peripheral terminals or sensory
Fig. 1. A: a photomontage of the dorsal horn of the spinal cord of a normal 7-day-old mouse. B: a photomontage of the dorsal horn of the spinal cord of a 7-day-old mouse which received a direct injection of capsaicin as a 15-day-old foetus. Note the marked depletion of substance P-like immunoreactivity from laminae II and III but preservation in lamina I. D, dorsal aspect; L, lateral aspect; II, lamina If.
28
ganglia until El7 but the action of capsaicin is sufficiently powerful to prevent its appearance in these areas even though it is only present in the site of action for a relatively short time [20]. The mechanism of action of capsaicin is still poorly understood. In adult animals it adversely affects synthesis of substance P [9] and inhibits axonal transport of substance P [6]. Why it should cause irreversible degeneration of neonatal primary afferent neurones is even less well understood. Whether the observed effects in the present study are simply due to depletion of substance P or are due to destruction of the nerve and hence loss of its neuropeptide content, require correlative structural studies. Nevertheless, this initial study opens up the possibilities of using capsaicin to study prenatal ontogeny of substance P-containing and other peptidergic fibre systems. I Ainsworth, A., Hall, P., Wall, P.D., Allt, G., McKenzie, M.L., Gibson, S. and Polak, J.M., Effects of capsaicin applied locally to adult peripheral nerve. 11 Anatomy and enzyme and peptide chemistry of peripheral nerve and spinal cord, Pain, 11 (1981) 379 388. 2 Chaggar, J.S., Atkinson, M.E. and Mohamed, S.S., The ontogeny of substance P-like immunoreactivity in developing mouse spinal nerves and spinal cord, Neuroscience, in press. 3 Cuello, A.C., Galfre, G. and Milstein, C., Detection of substance P in the central nervous system by a monoclonal antibody, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 3532--3536. 4 Cuello. A.('., Gamse, R., Holzer, P. and Lembeck, F., Substance P immunoreactive neurons followmg neonatal administration of capsaiein, Naunyn-Schmeideberg's Arch. Pharmacol., 315 (1981) 185-194. 5 Gamse, R., Holzer, P. and Lembeck, F., Decrease of substance P in primary afferent neurones and impairment of neurogenic plasma extravasation by capsaicin, Brit. J. Pharmacol., 68 (1980) 207-213. 6 Gamse, R., Petsche, U., Lembeck, F. and Jancs6, G., Capsaicin applied to peripheral nerve inhibits axoplasmic transport of substance P and somatostatin, Brain Res., 239 (1982) 447 462. 7 Gibson, S.J., McGregor, G., Bloom, S.R., Polak, J.M. and Wall, P.D., Local application of capsaicin to one sciatic nerve of the adult rat induces a marked depletion in the peptide content of the lumbar dorsal horn, Neuroscience, in press. 8 Gilbert, R.F.T. and Emson, P.C., Substance P in rat CNS and duodenum during development, Brain Res., 171 (1979) 166-170. 9 Harmar, A., Schofield, J.G. and Keen, P., Cycloheximide-sensitive synthesis of substance P by isolated dorsal root ganglion, Nature (Lond.), 284 (1980) 267-269. 10 H6kfelt, T., Johansson, O., Ljungdahl, A., Lundberg, J.M. and Schultzberg, M., Peptidergic neurons, Nature (Lond.), 284 (1980) 515-522. 11 lnagaki, S., Sakanaka, M., Shiosaka, S., Senba, E., Takatsuki, K., Takagi, H., Kawai, H., Minagawa, H. and Tohyama, M., Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis. 1 Forebrain and upper brain stem, Neuroscience, 7 (1982) 251-277. 12 Jancs6, G., Kiraly, E. and Jancs6-Gabor, A., Pharmacologically induced selective degeneration of chemosensitive primary sensory neurons, Nature (Lond.), 270 (1977) 741-742. 13 Jancs6, G., H6kfelt, T., Lundberg, J.M. Kiraly, E., Hal~.sz, N., Nilsson, G., Terenius, L., Rehfeld, J., Steinbusch, H., Verhofstad, A., Elde, R., Said, S. and Brown, M., Immunohistochemieal studies on the effect of capsaicin on spinal and medullary peptides and monoamine neurones using antisera to substance P, gastrin-CCK, somatostatin, VIP, enkephalin, neurotensin, and 5-hydroxytryptamine, J. Neurocytol., 10 (1981) 963-980. 14 Kessler, J.A. and Black, I.B., Nerve growth factor stimulates development of substance P in the embryonic spinal cord, Brain Res., 208 (1981) 135-145.
29 15 Lawson, S.N. and Nickels, S.M., The use of morphometric techniques to analyse the effect of neonatal capsaicin treatment on rat dorsal root ganglia and dorsal roots, J. Physiol. (Lond.), 303 (1980) 12P. 16 Lembeck, F. and Donnerer, J., Time course of capsaicin-induced functional impairments in comparison with changes in neuronal substance P content, Naunyn-Schmeideberg's Arch. Pharmacol., 36 (1981) 240-243. 17 Miller, M.S., Buck, S.H., Sipes, 1.G. and Burks, T.F., Capsaicinoid-induced local and systemic antinociception without substance P depletion, Brain Res., 244 (1982) 193-197. 18 Mohamed, S.S. and Atkinson, M.E., The ontogeny of substance P fibres in the mouse trigeminal ganglion, Develop. Brain Res., 4 (1982) 351-355. 19 Nagy, J.l., Hunt, S.P., Iversen, L.L. and Emson, P.C., Biochemical and anatomical observations on the degeneration of peptide-containing primary afferent neurons after capsaicin, Neuroscience, 6 (1981) 1923-1934. 20 Saria, A., Skofitch, G. and Lembeck, F., Distribution of capsaicin in rat tissues after systemic administration, J. Pharm. Pharmacol., 34 (1982) 273-275. 21 Scadding, J.W., The permanent anatomical effects of neonatal capsaicin on somatosensory nerves, J. Anat. (Lond.), 131 (1980) 473-484. 22 Sakanaga, M., Inagaki, S., Shiosaka, S., Senba, E., Takagi, H., Takatsuki, K., Kawai, Y., Iida, M., Hara, Y. and Tohyama, M., Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis. II Lower brainstem, Neuroscience, 7 (1982) 1097-1126. 23 Senba, E., Shiosaka, S., Hara, Y., Inagaki, S., Sakanaka, M., Takatsuki, K., Kawai, Y. and Tohyama, M., Ontogeny of the peptidergic system in the rat spinal cord: immunohistochemical analysis, J. comp. Neurol., 208 (1982) 54-66. 24 Virus, R.M. and Gebhart, F.G., Pharmacological actions of capsaicin: apparent involvement of substance P and serotonin, Life Sci., 25 (1979) 1273-1284. 25 Wall, P.D. and Fitzgerald, M., Effects of capsaicin applied locally to adult peripheral nerve I. Physiology of peripheral nerve and spinal cord, Pain, 11 (1981) 363-377.