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Topographical distribution of substance P in the cat substantia nigra
Neuroscience Letters, 12 (1979) 127--131 © Elsevier/North-Holland Scientific Publishers Ltd.
127
TOPOGRAPHICAL DISTRIBUTION OF SUBSTANCE P IN THE CAT SUBSTANTIA NIGRA
C. G A U C H Y , J.C. B E A U J O U A N , M.J. BESSON, B. K E R D E L H U E , J. G L O W I N S K I and R. MICHELOT"
Groupe NB, INSERM U. 114, College de France, 11, place Marcelin Berthelot, 75231 Paris Cedex 05 and (B.K. ) Laboratoire des Hormones Polypeptidiques, CNRS, 91190 Gif-surYvette (France)
(Received October 26th, 1978) (Revised version received January 2nd, 1979) (Accepted January 3rd, 1979)
SUMMARY
The substance P (SP) content in cat substantia nigra (SN) was estimated by radioimmunoassay in microdiscs punched on successive frontal sections. Only slightdifferences were seen in the regional distribution of SP within the SN. The highest content was found in the pars reticulata(20.7 pmol/mg protein) which was two times higher than in the pars compacta and pars lateralis. Identical resultswere found in the right and left part of the SN. Out of this area, as pedunculus cerebri and zona incerta, very low concentrations were detected.
The largest concentration of substance P (SP) in the central nervous system (CNS) is found in the substantia nigra (SN) [2,15]. After subceUular fractionation this undecepeptide is recovered in a fraction containing particles of nerve endings [8]. In fact, a dense network of SP nerve terminals has been visualized in this area by immunohistochemistry [6,10,13]. According to studies combining lesions and biochemical techniques, SP nerve terminals originate from neurons located in the head of the striatum and possibly in the globus pallidus [3,9,13]. Chronic treatment with neuroleptics produces a large decrease in the level of SP in the SN but not in other structures, such as the striatum and the hypothalamus [11]. In vitro studies performed on slices of rat SN have indicated that SP can be released by potassium through a calcium-dependent process [12]. Furthermore, the spontaneous release of SP was increased by veratridine and this effect was abolished by tetrodotoxine [12]. GABA, which is also found in large amounts in the SN, inhibited the potassium-evoked release of SP and this effect was antagonized by picrotoxin [12]. Recently, due to the availability of [3H] SP of a high specific activity, a saturable, high
128
affinity specific binding of [3H]SP was found in crude synaptic membranes of the rabbit brain. The regional distribution of this high affinity specific binding was heterogenous, a relatively high level of binding being found in the mesencephalon and the striatum [19]. The microiontophoretic application of SP has been shown to activate neurons both in the pars compacta and the pars reticulata of the rat SN [ 7 ]. Recent in vivo release studies made in our laboratory suggest that the SP neurons projecting to the cat SN exert a tonic excitatory influence on the nigro striatal dopaminergic neurons. Indeed, the nigral application of SP (10 -s M) stimulated the release of dopamine in the ipsilateral caudate nucleus [5] whereas the immunoneutralization of nigral SP reduced the release of dopamine [4]. SP nerve terminals could directly interact with dopaminergic cell bodies, however, these effects could be mediated by changes in the dendritic release of dopamine. In fact SP decreased the nigral release of dopamine whereas an increase in the dendritic release of the transmitter was seen during the local immunoneutralization of SP transmission [4]. To further define the role of SP neurons in the control of the activity of nigral efferent pathways and to study the in vivo nigral release of SP, we decided in the present study to first determine the topographical distribution of this peptide in the cat SN. Four cats (male + female) were sacrificed by injecting a lethal dose of Nembutal @ (50 mg/kg i.p.). Animals being fixed in the stereotaxic apparatus, a frontal reference plane was determined by a coronal section anterior to the SN. Their brains were immediately removed and frozen at -15°C on a LeitzKryomat microtome stage. Serial coronal sections (500 ~m thick) were made. After precise localization of the SN according to the atlas of Berman A.L. [1], microdiscs of tissues were punched out with a cool stainless steel tube (inner diameter 1.4 mm) in various parts of the SN and in surrounding structures (as indicated in Fig. 1). The microdiscs were then homogenized by sonication in 250 gl of 0.1 M phosphoric acid. As aliquot (100 ul) of the homogenates was used for protein determination. The remaining homogenates (150 ~1) were heated at 80°C for 20 min to further inactivate peptidases. After adjustment to pH 7.4 with 0.1 N NaOH, the samples were diluted 2-fold with a phosphate saline buffer (in mM: NaH2PO4, 2.8; Na2HPO+, 18.3; NaC1; 150; adjusted to pH 7.4 and containing bovine serum albumin, 0.05%) and centrifuged for 4 min in an Eppendorf centrifuge. SP was then estimated using a double antibody radioimmunological method in aliquots (50 and 100 ~1) of the supernatant. Antisera were generated in rabbits using synthetic SP (2 mg) (Beckman, France) polymerized with guinea pig 7-globulins (2 mg) using glutaraldehyde as a coupling agent. The antigen solution emulsified in complete Freund adjuvant was injected (s.c., into the lymphatic nodule area of each limb) monthly, for 7--10 months. Using 12SI-TyrS-SP as a tracer prepared as for luteinizing hormone--releasing hormone (LH--RH) [ 16], the highest radioimmunological titer (the final dilution required to bind 50% of 2 pg of radioiodinated TyrS-SP) was about 1/300,000. The sensitivity of the method was between 1.5 and 7.5 fmol depending on the 12SI-TyrS-SPbatch used. Various
129 other hypothalamic or pituitary peptides (LRH, TRH, SRIF, Met- or Leuenkephalin, B-endorphin, B-LPH, prolactin, GH and gonadotropins) did n o t cross react in the RIA for concentrations up to 200 ng/ml. Nevertheless, almost 50% cross reactivity was observed with physalaemin, a structural analog o f SP, as previously observed b y other authors [2]. Response curves obtained with aliquots (25--50--100 ~1) of SN samples paralleled that of synthetic SP. Using this procedure of extraction and this radioimmunoassay we have estimated the content o f SP in the rat SN and striatum and found values in the range of those described by other authors [2,3,13,15]. The SP levels estimated in this study correspond to the SP like material reacting with the antibody.
~
A4
Fig. 1. Topographic distribution of SP in microdiscs punched at median (1), intermediate (2) and lateral (3) part of the cat SN on antero-posterior frontal sections~ C, zona compacta; R, zona reticulata; L, pars lateralis; ZI, zona incerta; ST, subthalamic nucleus. Each value represents the mean -*S.E.M. of the combined SP values from left and right SW.
130 As illustrated in Fig. 1, much higher levels of SP were found in the pars compacta and the pars reticulata of the SN than in surrounding structures such as the pedunculus cerebri and the zona incerta. Within the SN, slight differences were seen in the regional distribution of SP, the highest content of the peptide being found in the middle part of the pars reticulata (20.7 -+ 3.4 pmol/mg protein in the microdisc no. 2 of the frontal place A4). The SP levels in microdiscs punched out in the pars compacta and the pars lateralis of the SN were at least two times lower than those found in the middle part of the pars reticulata. Identical results were found in the left and right SN in the four cats examined. The range of SP levels {from 10 to 20 pmol/mg protein) determined within the pars reticulata of the cat SN are comparable to the SP content of pars reticulata of the rat SN. Indeed, the values reported by Mroz et al. [18] and Jessel et al. [13] were respectively 12 and 16 pmol/mg protein. They are also similar to those measured in the pars reticulata of the human SN (up to 10 pmol/mg protein) [14]. In agreement with our results, Mroz et al. [18] found higher levels of SP in the pars reticulata (11.38 pmol/mg protein) than in the pars compacta (2.98 pmol/mg protein) or pars lateralis {2.98 pmol/mg protein) of the rat SN. A slight difference in the extent of SP innervation in the various parts of the rat SN has also been noted b y Ljungdahl et al. [17] in their recent immunohistochemical mapping of SP cell bodies and terminals on the rat brain. However, Jessel et al. [13] did not find significant differences in the SP innervation of the pars compacta and pars reticulata of the rat SN. Although anatomical studies at the electron microscopic level are absolutely required to define the relationships between SP nerve terminals and efferent nigral pathways, the presence of relatively large amounts of SP in the various parts o f the cat SN suggests that various types of nigral neurons besides the dopaminergic neurons may be controlled b y the striatonigral SP projection. In any case, the high content of SP in the middle part of the pars reticulata should facilitate in vivo release studies with push-pull cannulae. ACKNOWLEDGEMENTS This work was supported by grants from DGRST (77.7.0496) and RhSnePoulenc S.A. REFERENCES
I Berman, A.L., The brain stem of the cat. A cytoarchitectonic atlas with stereotaxic coordinates, The University of Wisconsin Press, Madison, 1968. 2 Brownstein, M.J., Mroz, E.A., Kizer, J.S., Palkovitz, M. and Leeman, S.E., Regional distribution of substance P in the brain of the rat, Brain Res. 116 (1976) 299--305. 3 Brownstein, M.J., Mroz, E.A., Tappaz, M.L. and Leernan, S.E., On the origin of substance P and glutarnic acid decarboxylase (GAD) in the substantia nigra, Brain Res., 135 (1977) 315--323. 4 Cheramy, A., Michelot, R., Leviel, V., Nieoullon, A., Glowinski, J. and Kerdelhue, B.,
131
5
6 7 8 9 10
11 12 13 14 15 16
17 18
19
Effect of the immunoneutralization of substance P in the cat substantia nigra on the release of dopamine from dendrites and terminals of dopaminergic neurons, Brain Res., 155 (1978) 404--408. Cheramy, A., Nieoullon, A., Michelot, R. and Glowinski, J., Effects of intranigral applications of dopamine and substance P on the in vivo release of newly synthesized [3H]dopamine in the ipsilateral caudate nucleus of the cat, Neurosci. Lett., 4 (1977) 105--109. Cuello,A.C. and.Kanazawa, I., The distribution of substanceP immunoreactivefibers in the rat central nervoussystem, J. comp. Neurol., 178 (1978) 129---156. Dray,A. and Straughan, D.W., Synaptic mechanismsin the substantia nigra, J. Pharm. Pharmacol., 28 (1976) 400--405. Duffy, M.J., Mulhall, D. and Powell, D., Subcellulardistribution of substance P in bovine hypothalamus and substantia nigra, J. Neurochem., 25 (1975) 305--307. Gale,K., Hong, J.S. and Guidotti, A., Presence of substanceP and GABAin separate striatonigral neurons, Brain Res., 136 (1977) 371--375. Hbkfelt, T., Kellerth, J.O., Nilsson, G. and Pernow, B., Substance P: localization in the central nervoussystem and in some primary sensory neurons, Science, 190 (1975) 889--890. Hong, J.S., Yang, H.Y.T. and Costa, E., Substance P content of substantia nigra after chronic treatment with antischizophrenic drugs, Neuropharmacology, 17 (1978) 83--85. Jessell, T.M., Substance P release from the rat substantia nigra, Brain Res., 151 (1978) 469--478. Jessell, T.M., Emson, P.C., Paxinos, G. and Cuello, A.C., Topographic projections of substance P and GABA pathways in the striato- and pallido-nigral system: a biochemical an immunohistochemical study, Brain Res., 152 (1978) 487--498. Kanazawa, I., Bird, E., O'Connell, R. and Powell, D., Evidence for a decrease in substance P content of substantia nigra in Huntington's chorea, Brain Res., 120 (1977) 387--392. Kanazawa, I. and Jessell, T., Post mortem changes and regional distribution of substance P in the rat and mouse nervous system, Brain Res., 117 (1976) 362--367. Kerdelhue, B., Jutisz, M., Gillessen, D. and Studer, R.O., Obtention of antisera against a hypothalamic decapeptide (luteinizing hormone/follicule stimulating hormone releasing hormone) which stimulates the release of pituitary gonadotropin and development of its radioimmunoassay, Biochim. Biophys. Acta, 297 (1973) 540--548. Ljungdahl, A., Hokfelt, T. and Nilson, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861--943. Mroz, A.E., Brownstein, M.J. and Leeman, S.E., Distribution of immunoassayable substance P in the rat brain: Evidence for the existence of substance P-containing tracts. In U.S. Von Euler and B. Pernow (Eds.), Substance P Raven Press, New York, 1977. Nakata, Y., Kusaka, Y., Segawa, T., Yajima, H. and Kitagawa, K., Substance P: regional distribution and specific binding to synaptic membranes in rabbit central nervous system, Life Sci., 22 (1978) 259--268.
127
TOPOGRAPHICAL DISTRIBUTION OF SUBSTANCE P IN THE CAT SUBSTANTIA NIGRA
C. G A U C H Y , J.C. B E A U J O U A N , M.J. BESSON, B. K E R D E L H U E , J. G L O W I N S K I and R. MICHELOT"
Groupe NB, INSERM U. 114, College de France, 11, place Marcelin Berthelot, 75231 Paris Cedex 05 and (B.K. ) Laboratoire des Hormones Polypeptidiques, CNRS, 91190 Gif-surYvette (France)
(Received October 26th, 1978) (Revised version received January 2nd, 1979) (Accepted January 3rd, 1979)
SUMMARY
The substance P (SP) content in cat substantia nigra (SN) was estimated by radioimmunoassay in microdiscs punched on successive frontal sections. Only slightdifferences were seen in the regional distribution of SP within the SN. The highest content was found in the pars reticulata(20.7 pmol/mg protein) which was two times higher than in the pars compacta and pars lateralis. Identical resultswere found in the right and left part of the SN. Out of this area, as pedunculus cerebri and zona incerta, very low concentrations were detected.
The largest concentration of substance P (SP) in the central nervous system (CNS) is found in the substantia nigra (SN) [2,15]. After subceUular fractionation this undecepeptide is recovered in a fraction containing particles of nerve endings [8]. In fact, a dense network of SP nerve terminals has been visualized in this area by immunohistochemistry [6,10,13]. According to studies combining lesions and biochemical techniques, SP nerve terminals originate from neurons located in the head of the striatum and possibly in the globus pallidus [3,9,13]. Chronic treatment with neuroleptics produces a large decrease in the level of SP in the SN but not in other structures, such as the striatum and the hypothalamus [11]. In vitro studies performed on slices of rat SN have indicated that SP can be released by potassium through a calcium-dependent process [12]. Furthermore, the spontaneous release of SP was increased by veratridine and this effect was abolished by tetrodotoxine [12]. GABA, which is also found in large amounts in the SN, inhibited the potassium-evoked release of SP and this effect was antagonized by picrotoxin [12]. Recently, due to the availability of [3H] SP of a high specific activity, a saturable, high
128
affinity specific binding of [3H]SP was found in crude synaptic membranes of the rabbit brain. The regional distribution of this high affinity specific binding was heterogenous, a relatively high level of binding being found in the mesencephalon and the striatum [19]. The microiontophoretic application of SP has been shown to activate neurons both in the pars compacta and the pars reticulata of the rat SN [ 7 ]. Recent in vivo release studies made in our laboratory suggest that the SP neurons projecting to the cat SN exert a tonic excitatory influence on the nigro striatal dopaminergic neurons. Indeed, the nigral application of SP (10 -s M) stimulated the release of dopamine in the ipsilateral caudate nucleus [5] whereas the immunoneutralization of nigral SP reduced the release of dopamine [4]. SP nerve terminals could directly interact with dopaminergic cell bodies, however, these effects could be mediated by changes in the dendritic release of dopamine. In fact SP decreased the nigral release of dopamine whereas an increase in the dendritic release of the transmitter was seen during the local immunoneutralization of SP transmission [4]. To further define the role of SP neurons in the control of the activity of nigral efferent pathways and to study the in vivo nigral release of SP, we decided in the present study to first determine the topographical distribution of this peptide in the cat SN. Four cats (male + female) were sacrificed by injecting a lethal dose of Nembutal @ (50 mg/kg i.p.). Animals being fixed in the stereotaxic apparatus, a frontal reference plane was determined by a coronal section anterior to the SN. Their brains were immediately removed and frozen at -15°C on a LeitzKryomat microtome stage. Serial coronal sections (500 ~m thick) were made. After precise localization of the SN according to the atlas of Berman A.L. [1], microdiscs of tissues were punched out with a cool stainless steel tube (inner diameter 1.4 mm) in various parts of the SN and in surrounding structures (as indicated in Fig. 1). The microdiscs were then homogenized by sonication in 250 gl of 0.1 M phosphoric acid. As aliquot (100 ul) of the homogenates was used for protein determination. The remaining homogenates (150 ~1) were heated at 80°C for 20 min to further inactivate peptidases. After adjustment to pH 7.4 with 0.1 N NaOH, the samples were diluted 2-fold with a phosphate saline buffer (in mM: NaH2PO4, 2.8; Na2HPO+, 18.3; NaC1; 150; adjusted to pH 7.4 and containing bovine serum albumin, 0.05%) and centrifuged for 4 min in an Eppendorf centrifuge. SP was then estimated using a double antibody radioimmunological method in aliquots (50 and 100 ~1) of the supernatant. Antisera were generated in rabbits using synthetic SP (2 mg) (Beckman, France) polymerized with guinea pig 7-globulins (2 mg) using glutaraldehyde as a coupling agent. The antigen solution emulsified in complete Freund adjuvant was injected (s.c., into the lymphatic nodule area of each limb) monthly, for 7--10 months. Using 12SI-TyrS-SP as a tracer prepared as for luteinizing hormone--releasing hormone (LH--RH) [ 16], the highest radioimmunological titer (the final dilution required to bind 50% of 2 pg of radioiodinated TyrS-SP) was about 1/300,000. The sensitivity of the method was between 1.5 and 7.5 fmol depending on the 12SI-TyrS-SPbatch used. Various
129 other hypothalamic or pituitary peptides (LRH, TRH, SRIF, Met- or Leuenkephalin, B-endorphin, B-LPH, prolactin, GH and gonadotropins) did n o t cross react in the RIA for concentrations up to 200 ng/ml. Nevertheless, almost 50% cross reactivity was observed with physalaemin, a structural analog o f SP, as previously observed b y other authors [2]. Response curves obtained with aliquots (25--50--100 ~1) of SN samples paralleled that of synthetic SP. Using this procedure of extraction and this radioimmunoassay we have estimated the content o f SP in the rat SN and striatum and found values in the range of those described by other authors [2,3,13,15]. The SP levels estimated in this study correspond to the SP like material reacting with the antibody.
~
A4
Fig. 1. Topographic distribution of SP in microdiscs punched at median (1), intermediate (2) and lateral (3) part of the cat SN on antero-posterior frontal sections~ C, zona compacta; R, zona reticulata; L, pars lateralis; ZI, zona incerta; ST, subthalamic nucleus. Each value represents the mean -*S.E.M. of the combined SP values from left and right SW.
130 As illustrated in Fig. 1, much higher levels of SP were found in the pars compacta and the pars reticulata of the SN than in surrounding structures such as the pedunculus cerebri and the zona incerta. Within the SN, slight differences were seen in the regional distribution of SP, the highest content of the peptide being found in the middle part of the pars reticulata (20.7 -+ 3.4 pmol/mg protein in the microdisc no. 2 of the frontal place A4). The SP levels in microdiscs punched out in the pars compacta and the pars lateralis of the SN were at least two times lower than those found in the middle part of the pars reticulata. Identical results were found in the left and right SN in the four cats examined. The range of SP levels {from 10 to 20 pmol/mg protein) determined within the pars reticulata of the cat SN are comparable to the SP content of pars reticulata of the rat SN. Indeed, the values reported by Mroz et al. [18] and Jessel et al. [13] were respectively 12 and 16 pmol/mg protein. They are also similar to those measured in the pars reticulata of the human SN (up to 10 pmol/mg protein) [14]. In agreement with our results, Mroz et al. [18] found higher levels of SP in the pars reticulata (11.38 pmol/mg protein) than in the pars compacta (2.98 pmol/mg protein) or pars lateralis {2.98 pmol/mg protein) of the rat SN. A slight difference in the extent of SP innervation in the various parts of the rat SN has also been noted b y Ljungdahl et al. [17] in their recent immunohistochemical mapping of SP cell bodies and terminals on the rat brain. However, Jessel et al. [13] did not find significant differences in the SP innervation of the pars compacta and pars reticulata of the rat SN. Although anatomical studies at the electron microscopic level are absolutely required to define the relationships between SP nerve terminals and efferent nigral pathways, the presence of relatively large amounts of SP in the various parts o f the cat SN suggests that various types of nigral neurons besides the dopaminergic neurons may be controlled b y the striatonigral SP projection. In any case, the high content of SP in the middle part of the pars reticulata should facilitate in vivo release studies with push-pull cannulae. ACKNOWLEDGEMENTS This work was supported by grants from DGRST (77.7.0496) and RhSnePoulenc S.A. REFERENCES
I Berman, A.L., The brain stem of the cat. A cytoarchitectonic atlas with stereotaxic coordinates, The University of Wisconsin Press, Madison, 1968. 2 Brownstein, M.J., Mroz, E.A., Kizer, J.S., Palkovitz, M. and Leeman, S.E., Regional distribution of substance P in the brain of the rat, Brain Res. 116 (1976) 299--305. 3 Brownstein, M.J., Mroz, E.A., Tappaz, M.L. and Leernan, S.E., On the origin of substance P and glutarnic acid decarboxylase (GAD) in the substantia nigra, Brain Res., 135 (1977) 315--323. 4 Cheramy, A., Michelot, R., Leviel, V., Nieoullon, A., Glowinski, J. and Kerdelhue, B.,
131
5
6 7 8 9 10
11 12 13 14 15 16
17 18
19
Effect of the immunoneutralization of substance P in the cat substantia nigra on the release of dopamine from dendrites and terminals of dopaminergic neurons, Brain Res., 155 (1978) 404--408. Cheramy, A., Nieoullon, A., Michelot, R. and Glowinski, J., Effects of intranigral applications of dopamine and substance P on the in vivo release of newly synthesized [3H]dopamine in the ipsilateral caudate nucleus of the cat, Neurosci. Lett., 4 (1977) 105--109. Cuello,A.C. and.Kanazawa, I., The distribution of substanceP immunoreactivefibers in the rat central nervoussystem, J. comp. Neurol., 178 (1978) 129---156. Dray,A. and Straughan, D.W., Synaptic mechanismsin the substantia nigra, J. Pharm. Pharmacol., 28 (1976) 400--405. Duffy, M.J., Mulhall, D. and Powell, D., Subcellulardistribution of substance P in bovine hypothalamus and substantia nigra, J. Neurochem., 25 (1975) 305--307. Gale,K., Hong, J.S. and Guidotti, A., Presence of substanceP and GABAin separate striatonigral neurons, Brain Res., 136 (1977) 371--375. Hbkfelt, T., Kellerth, J.O., Nilsson, G. and Pernow, B., Substance P: localization in the central nervoussystem and in some primary sensory neurons, Science, 190 (1975) 889--890. Hong, J.S., Yang, H.Y.T. and Costa, E., Substance P content of substantia nigra after chronic treatment with antischizophrenic drugs, Neuropharmacology, 17 (1978) 83--85. Jessell, T.M., Substance P release from the rat substantia nigra, Brain Res., 151 (1978) 469--478. Jessell, T.M., Emson, P.C., Paxinos, G. and Cuello, A.C., Topographic projections of substance P and GABA pathways in the striato- and pallido-nigral system: a biochemical an immunohistochemical study, Brain Res., 152 (1978) 487--498. Kanazawa, I., Bird, E., O'Connell, R. and Powell, D., Evidence for a decrease in substance P content of substantia nigra in Huntington's chorea, Brain Res., 120 (1977) 387--392. Kanazawa, I. and Jessell, T., Post mortem changes and regional distribution of substance P in the rat and mouse nervous system, Brain Res., 117 (1976) 362--367. Kerdelhue, B., Jutisz, M., Gillessen, D. and Studer, R.O., Obtention of antisera against a hypothalamic decapeptide (luteinizing hormone/follicule stimulating hormone releasing hormone) which stimulates the release of pituitary gonadotropin and development of its radioimmunoassay, Biochim. Biophys. Acta, 297 (1973) 540--548. Ljungdahl, A., Hokfelt, T. and Nilson, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861--943. Mroz, A.E., Brownstein, M.J. and Leeman, S.E., Distribution of immunoassayable substance P in the rat brain: Evidence for the existence of substance P-containing tracts. In U.S. Von Euler and B. Pernow (Eds.), Substance P Raven Press, New York, 1977. Nakata, Y., Kusaka, Y., Segawa, T., Yajima, H. and Kitagawa, K., Substance P: regional distribution and specific binding to synaptic membranes in rabbit central nervous system, Life Sci., 22 (1978) 259--268.