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Immunocytochemical localization of rat substance P receptor in the striatum
Neuroscience Letters, 153 (1993) 157-160 0 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$06.00
157
NSL 09447
Immunocytochemical
localization of rat substance P receptor in the striatum
Ryuichi Shigemoto”, Yoshifumi Nakaya”, Sakashi Nomurab, Reiko Ogawa-Meguro”, Hitoshi Ohishi”, Takeshi Kaneko”, Shigetada Nakanishi” and Noboru Mizunoa “Department of MorphologicalBrain Science and ‘Institute for Immunology, Faculty of Medicine and %ollege of Medical Technology, Kyoto Universiry, Kyoto (Japan)
(Received 7 January 1993; Accepted 22 January 1993) Key words:
Substance P receptor; Tachykinin; Antibody; Fusion protein; Striatum; Rat; Immunocytochemistry;
Electron microscopy
A trp E fusion protein containing a C-terminal portion of the rat substance P receptor (SPR) was expressed in bacteria and used to produce an antibody. The antibody specifically reacted with SPR expressed in a mammalian cell line and rat striatum. Light and electron microscope analyses of the rat striatum revealed intense SPR-like immunoreactivity in neuronal somata and dendrites. These immunoreactive neurons constituted - 3% of the total population of striatal neurons; they were putative interneurons of large and medium-sized aspiny types.
Substance P (SP), one of the best characterized of the neuropeptides, is widely distributed in the central nervous system (CNS) and plays important roles as a neurotransmitterlneuromodulator [ 10, 133.The regional distribution of SP receptor (SPR) in the CNS was studied by autoradiographic localization of SP-binding sites [10, 111, and RNase-protection analysis of SPR mRNA [14]. Cellular localization of SPR mRNA was also investigated by in situ hybridization in the rat striatum [4, 51. For more precise studies on the cellular and synaptic localization of SPR, however, immunocytochemical analysis is required. In the present study, we produced a polyclonal antibody against a recombinant SPR protein overexpressed in E. coli, and then used it for immunocytochemical detection of SPR-like immunoreactivity (SPR-LI) in the rat striatum. The bacterial expression vector pATH3 [8] was used to overexpress the trp E fusion protein that contain rat SPR sequence. A RsaI-SpeI fragment of prTKR2 [16] encoding a C-terminal portion (amino acid residues 349407) of rat SPR was subcloned into pATH3 and in-frame fusion of the rat SPR fragment to the trp E gene was confirmed by DNA sequencing. After transformation of E. coli strain HBlOl with this construct (PATH-SPR), induction and purification of the fusion protein were perCorre$ondence: R. Shigemoto, Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan. Fax: (81) (75) 7534340.
formed as described [8]. The fusion protein of 43 kDa was finally purified by preparative SDS-PAGE. The excised gel band was crushed and homogenized with equal volume of Freund’s adjuvant. Rabbits were S.C.injected with - 0.5 mg of the fusion protein, boosted every 4 weeks and bled l-2 weeks after each boost. Purification of the antibody specific for the SPR portion from the collected antisera was performed as described [3]. First, the IgG fraction purified with a protein A column was absorbed by the Sepharose 4B column coupled with the unfused trp E protein to remove antibodies reactive to non-SPR portions of the immunized fusion protein. Secondly, the antibody specific for the SPR portion was affinity purified with the Sepharose 4B column coupled with the trp E-SPR fusion protein. Immunobiot analysis was performed as described [6]. Crude membrane preparations from Chinese hamster ovary (CHO) cell lines transfected with cDNAs of rat SPR, substance K receptor (SKR) and neuromedin K receptor (NKR) [12] and those from rat striatum [14] were separated on 10% SDSPAGE and transferred to PVDF membranes. The membranes were stained with Coomassie brilliant blue or reacted with the affinity-purified antibody (1 &nl). Alkaline phosphatase-labeled second antibody (Cappel) was used to visualize the reacted bands. For immunohistochemical analysis, adult male Sprague-Dawley rats were anesthetized with pentobarbital (100 mg/kg body weight) and perfused transcardially with a mixture containing 3.5% paraformaldehyde,
158
a
b
Fig. 1. Immunoblots of SPR-, SKR- and NKR-expressing CHO cells and rat striatum. Crude membrane preparations from the cells and striatum were subjected to 10% SDS-PAGE, blotted to PVDF filters and stained with Coomassie brilhant blue (a) or affinity-purified antibody against a C-terminal portion of rat SPR (b). SPR, SKR and NKR; crude membrane preparations from CHO cells transfected with respective cDNAs [12], St; crude membrane preparations from rat striatum. Positions of molecular weight (kDa) markers (BioRad) are indicated on the left.
1% picric acid and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. For light microscope analysis, the brains were placed in 20% sucrose in 0.1 M phosphate buffer overnight at 4°C and cut into 40-pm sections. The sections were incubated with 1 pg/ml affinity-purified antibody in phosphate-buffered saline containing 2% normal goat serum and 0.1% Triton X-100 overnight at 4”C. They were then washed and incubated with biotinylated goat anti-rabbit IgG (Vector), washed again and reacted with the ABC elite kit (Vector). The sections were finally reacted with 0.2% diaminobenzidine and 0.003% hydrogen peroxide. For electron microscope (EM) analysis, 40-pm sections were cut on a vibratome and processed as described above; 0.2% photoflo (Kodak) was used instead of Triton X-100. After osmification and blockstaining with uranyl acetate, the sections were dehydrated and flat embedded in Epon. Ultrathin sections were then prepared and counter-stained with lead citrate. The trp E-SPR fusion protein was highly expressed after induction in E. coli transformed with PATH-SPR and easily identified as a major band on SDS-PAGE. The band was excised and used to immunize rabbits. The reactivity and specificity of the affinity-purified antibody to the rat SPR protein was tested using crude membrane preparations from cDNA-transfected CHO cell lines that express each of rat SPR, SKR and NKR [12] and those from rat striatum (Fig. 1). Intense and broad immunoreactive bands were observed specifically in SPRexpressing CHO cells (Fig. 1b). The molecular weights of the major bands ranged from 45 to 80 kDa. Minor bands
of lower molecular weights may be degraded products of SPR generated during membrane preparation. No immunoreactive bands were detected in SKR- or NKR-expressing CHO cells. In the striatal membrane, multiple but specific bands of 5676 kDa were clearly present (Fig. lb). All these immunoreactive bands completely disappeared by absorption of the antibody with the trp E-SPR fusion protein but not with the unfused trp E protein (data not shown). Molecular weights of these multiple bands were substantially higher than the predicted molecular weight of rat SPR (46,364 Da) [16]. This discrepancy and multiplicity of the molecular weights may be due to a variety of carbohydrate moieties linked to possible N-glycosylation sites of SPR [9,16]. Immunohistochemical analysis revealed intensely labeled neuronal somata and dendrites in the rat striatum (Fig. 2). SPR-LI neurons constituted - 3% of the total population of striatal neurons. About one half of the SPR-LI neurons were of the large aspiny type and the other half were of the medium-sized aspiny (or sparsely spiny) type (Fig. 2a,b). These immunoreactivities completely disappeared by absorption of the antibody with the trp E-SPR fusion protein (Fig 2~). EM examination revealed accumulation of dense immunoreaction products within the perikaryal and dendritic cytoplasm (Fig. 2d,e). No profiles of axon terminals were immunopositive. In the immunopositive cell bodies, the reaction products accumulated closely associating with the cell membrane (Fig. 26). The reaction products frequently accumulated on the postsynaptic specialization (Fig. 2e) although they were widely distributed in extrasynaptic sites. In the rat striatum, SPR mRNA has been detected by in situ hybridization in a small population of large neurons [4] or in cholinergic neurons [5]. In the present study, however, not only large aspiny neurons, which constituted 1 - 2% of the total population of striatal neurons [ 151, but also medium-sized aspiny (or sparsely spiny) neurons were SPR-positive. The former appeared to correspond to cholinergic interneurons and the latter to GABAergic and/or peptidergic interneurons [ 151. Thus, the SPR-LI neurons detected in the present study were interneurons. On the other hand, SP-LI terminals in the striatum are in synaptic contact not only with cholinergic interneurons but also with projection neurons of medium-sized spiny type [1,2]. Many lines of evidence have shown ‘mismatches’ between the localization of transmitters and their receptors in the CNS [7]. Although the possibility of the existence of another SPR subtype cannot be entirely excluded, this is one example of the ‘mismatches’ at the synaptic level. Thus, for elucidation of the sites of actual transmission by SP, identification of both presynaptic SP-containing terminal and postsynap-
Fig ,. 2. SPR-LI in a parasagittal section through the striatum (a,b). The immunoreactivity in the striatum completely disappeared by absorptio mnof the antibody with the trp E-SPR fusion protein (c). EM photographs show SPR-LI in a neuronal cell body (d) and a postsynaptic dendrite (e). Arl nowheads in (d) indicate the nuclear membrane. Arrows in (e) indicate the synaptic sites. D, dendrite; GP, globus pallidus; N, nucleus; St, striai turn. Bars = 300 pm (a), 100 pm (b,c), 2 pm (d) and 0.2 flrn (e).
tic SPR is required. SPR antibody generated in the present study is a useful tool for this purpose and further investigation for roles of neurotransmission/neuromodulation by SP in the CNS. 1 Bolam, J.P., Ingham, C.A., Izzo, PN., Levey, AI., Rye, D.B., Smith, A.D. and Wainer, B.H., Substance P-containing terminals in synaptic contact with cholinergic neurons in the neostriatum and basal forebrain: a double immunocytochemical study in the rat, Brain Res., 397 (1986) 279-289. 2 Bolam, J.P. and Izzo, P.N., The postsynaptic targets of substance P-immunoreactive terminals in the rat neostriatum with particular reference to identified spiny striatonigral neurons, Exp. Brain Res., 70 (1988) 361-377. 3 Carroll, S.B. and Laughon, A., Production and purification of poly-
clonal antibodies to the foreign segment of /7-galactosidase fusion proteins, In D.M. Glover (Ed.), DNA Cloning, Vol. III, IRL Press, Oxford, 1987, pp. 89111. Elde, R., Schalling, M., Ceccatelli, S., Nakanishi, S. and Hiikfelt, T., Localization of neuropeptide receptor mRNA in rat brain: initial observations using probes for neurotensin and substance P receptors, Neurosci. Lett., 120 (1990) 134-138. Gerfen, C.R., Substance P (neurokinin-1) receptor mRNA is selectively expressed in choline@ neurons in the striatum and basal forebrain, Brain Res., 556 (1991) 165-170. Harlow, E. and Lane, D., Antibodies, a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988. Herkenham, M., Mismatches between neurotransmitter and receptor localizations in brain: observations and implications, Neuroscience, 23 (1987) l-38. Koerner, T.J., Hill, J.E., Myers, A.M. and Tzagoloff, A., High-
160
9
10 11
12
expression vectors with multiple cloning sites for construction of ?rpE fusion genes: PATH vectors, Methods Enzymol.,l94 (1991) 411-490. Liu, Y.F. and Quirion, R., Presence of various carbohydrate moieties includinga-galactose and N-acetylglucosamine residues on solubilized porcine brain neurokinin-Vsubstanw P receptors, J. Neurochem., 57 (1991) 1944-1950. Maggio, J.E., Tachykinins, Annu. Rev. Neurosci., 11 (1991) 13-28. Mantyh, P.W., Gates, T., Mantyh, C.R. and Maggio, J.E., Autoradiographic localiiation and characterization of tachykinin receptor binding sites in the rat brain and peripheral tissues, J. Neurosci., 9 (1989) 258279. Nakajima, Y., Tsuchida, K., Negishi, M., Ito, S. and Nakanishi, S., Direct linkage of three tachykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades in trans-
13 14
15
16
fected Chinese hamster ovary cells, J. Biol. Chem., 267 (1992) 24372442. Otsuka, M. and Yoshioka, K., Neurotransmitter functions ofmammalian tachykinins, Physiol. Rev., 73 (1993) l-80. Tsuchida, K., Shigemoto, R., Yokota, Y. and Nakanishi, S., Tissue distribution and quantitation of the mRNAs for three rat tachykinin receptors, Eur. J. B&hem., 193 (1990) 751-757. Wilson, C.J., Basal ganglia, In G.M. Shepherd (Ed.), The Synaptic Organization of the Brain, 3rd edn., Oxford University Press, New York, NY, 1990, pp. 219-316. Yokota, Y., Sasai, Y., Tanaka, K., Fujiwara, T., Tsuchida, K., Shigemoto, R., Kakizuka, A., Ohkubo, H. and Nakanishi, S., Molecular characterization of a functional cDNA for rat substance P receptor, J. Biol. Chem., 264 (1989) 1764917652.
157
NSL 09447
Immunocytochemical
localization of rat substance P receptor in the striatum
Ryuichi Shigemoto”, Yoshifumi Nakaya”, Sakashi Nomurab, Reiko Ogawa-Meguro”, Hitoshi Ohishi”, Takeshi Kaneko”, Shigetada Nakanishi” and Noboru Mizunoa “Department of MorphologicalBrain Science and ‘Institute for Immunology, Faculty of Medicine and %ollege of Medical Technology, Kyoto Universiry, Kyoto (Japan)
(Received 7 January 1993; Accepted 22 January 1993) Key words:
Substance P receptor; Tachykinin; Antibody; Fusion protein; Striatum; Rat; Immunocytochemistry;
Electron microscopy
A trp E fusion protein containing a C-terminal portion of the rat substance P receptor (SPR) was expressed in bacteria and used to produce an antibody. The antibody specifically reacted with SPR expressed in a mammalian cell line and rat striatum. Light and electron microscope analyses of the rat striatum revealed intense SPR-like immunoreactivity in neuronal somata and dendrites. These immunoreactive neurons constituted - 3% of the total population of striatal neurons; they were putative interneurons of large and medium-sized aspiny types.
Substance P (SP), one of the best characterized of the neuropeptides, is widely distributed in the central nervous system (CNS) and plays important roles as a neurotransmitterlneuromodulator [ 10, 133.The regional distribution of SP receptor (SPR) in the CNS was studied by autoradiographic localization of SP-binding sites [10, 111, and RNase-protection analysis of SPR mRNA [14]. Cellular localization of SPR mRNA was also investigated by in situ hybridization in the rat striatum [4, 51. For more precise studies on the cellular and synaptic localization of SPR, however, immunocytochemical analysis is required. In the present study, we produced a polyclonal antibody against a recombinant SPR protein overexpressed in E. coli, and then used it for immunocytochemical detection of SPR-like immunoreactivity (SPR-LI) in the rat striatum. The bacterial expression vector pATH3 [8] was used to overexpress the trp E fusion protein that contain rat SPR sequence. A RsaI-SpeI fragment of prTKR2 [16] encoding a C-terminal portion (amino acid residues 349407) of rat SPR was subcloned into pATH3 and in-frame fusion of the rat SPR fragment to the trp E gene was confirmed by DNA sequencing. After transformation of E. coli strain HBlOl with this construct (PATH-SPR), induction and purification of the fusion protein were perCorre$ondence: R. Shigemoto, Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan. Fax: (81) (75) 7534340.
formed as described [8]. The fusion protein of 43 kDa was finally purified by preparative SDS-PAGE. The excised gel band was crushed and homogenized with equal volume of Freund’s adjuvant. Rabbits were S.C.injected with - 0.5 mg of the fusion protein, boosted every 4 weeks and bled l-2 weeks after each boost. Purification of the antibody specific for the SPR portion from the collected antisera was performed as described [3]. First, the IgG fraction purified with a protein A column was absorbed by the Sepharose 4B column coupled with the unfused trp E protein to remove antibodies reactive to non-SPR portions of the immunized fusion protein. Secondly, the antibody specific for the SPR portion was affinity purified with the Sepharose 4B column coupled with the trp E-SPR fusion protein. Immunobiot analysis was performed as described [6]. Crude membrane preparations from Chinese hamster ovary (CHO) cell lines transfected with cDNAs of rat SPR, substance K receptor (SKR) and neuromedin K receptor (NKR) [12] and those from rat striatum [14] were separated on 10% SDSPAGE and transferred to PVDF membranes. The membranes were stained with Coomassie brilliant blue or reacted with the affinity-purified antibody (1 &nl). Alkaline phosphatase-labeled second antibody (Cappel) was used to visualize the reacted bands. For immunohistochemical analysis, adult male Sprague-Dawley rats were anesthetized with pentobarbital (100 mg/kg body weight) and perfused transcardially with a mixture containing 3.5% paraformaldehyde,
158
a
b
Fig. 1. Immunoblots of SPR-, SKR- and NKR-expressing CHO cells and rat striatum. Crude membrane preparations from the cells and striatum were subjected to 10% SDS-PAGE, blotted to PVDF filters and stained with Coomassie brilhant blue (a) or affinity-purified antibody against a C-terminal portion of rat SPR (b). SPR, SKR and NKR; crude membrane preparations from CHO cells transfected with respective cDNAs [12], St; crude membrane preparations from rat striatum. Positions of molecular weight (kDa) markers (BioRad) are indicated on the left.
1% picric acid and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. For light microscope analysis, the brains were placed in 20% sucrose in 0.1 M phosphate buffer overnight at 4°C and cut into 40-pm sections. The sections were incubated with 1 pg/ml affinity-purified antibody in phosphate-buffered saline containing 2% normal goat serum and 0.1% Triton X-100 overnight at 4”C. They were then washed and incubated with biotinylated goat anti-rabbit IgG (Vector), washed again and reacted with the ABC elite kit (Vector). The sections were finally reacted with 0.2% diaminobenzidine and 0.003% hydrogen peroxide. For electron microscope (EM) analysis, 40-pm sections were cut on a vibratome and processed as described above; 0.2% photoflo (Kodak) was used instead of Triton X-100. After osmification and blockstaining with uranyl acetate, the sections were dehydrated and flat embedded in Epon. Ultrathin sections were then prepared and counter-stained with lead citrate. The trp E-SPR fusion protein was highly expressed after induction in E. coli transformed with PATH-SPR and easily identified as a major band on SDS-PAGE. The band was excised and used to immunize rabbits. The reactivity and specificity of the affinity-purified antibody to the rat SPR protein was tested using crude membrane preparations from cDNA-transfected CHO cell lines that express each of rat SPR, SKR and NKR [12] and those from rat striatum (Fig. 1). Intense and broad immunoreactive bands were observed specifically in SPRexpressing CHO cells (Fig. 1b). The molecular weights of the major bands ranged from 45 to 80 kDa. Minor bands
of lower molecular weights may be degraded products of SPR generated during membrane preparation. No immunoreactive bands were detected in SKR- or NKR-expressing CHO cells. In the striatal membrane, multiple but specific bands of 5676 kDa were clearly present (Fig. lb). All these immunoreactive bands completely disappeared by absorption of the antibody with the trp E-SPR fusion protein but not with the unfused trp E protein (data not shown). Molecular weights of these multiple bands were substantially higher than the predicted molecular weight of rat SPR (46,364 Da) [16]. This discrepancy and multiplicity of the molecular weights may be due to a variety of carbohydrate moieties linked to possible N-glycosylation sites of SPR [9,16]. Immunohistochemical analysis revealed intensely labeled neuronal somata and dendrites in the rat striatum (Fig. 2). SPR-LI neurons constituted - 3% of the total population of striatal neurons. About one half of the SPR-LI neurons were of the large aspiny type and the other half were of the medium-sized aspiny (or sparsely spiny) type (Fig. 2a,b). These immunoreactivities completely disappeared by absorption of the antibody with the trp E-SPR fusion protein (Fig 2~). EM examination revealed accumulation of dense immunoreaction products within the perikaryal and dendritic cytoplasm (Fig. 2d,e). No profiles of axon terminals were immunopositive. In the immunopositive cell bodies, the reaction products accumulated closely associating with the cell membrane (Fig. 26). The reaction products frequently accumulated on the postsynaptic specialization (Fig. 2e) although they were widely distributed in extrasynaptic sites. In the rat striatum, SPR mRNA has been detected by in situ hybridization in a small population of large neurons [4] or in cholinergic neurons [5]. In the present study, however, not only large aspiny neurons, which constituted 1 - 2% of the total population of striatal neurons [ 151, but also medium-sized aspiny (or sparsely spiny) neurons were SPR-positive. The former appeared to correspond to cholinergic interneurons and the latter to GABAergic and/or peptidergic interneurons [ 151. Thus, the SPR-LI neurons detected in the present study were interneurons. On the other hand, SP-LI terminals in the striatum are in synaptic contact not only with cholinergic interneurons but also with projection neurons of medium-sized spiny type [1,2]. Many lines of evidence have shown ‘mismatches’ between the localization of transmitters and their receptors in the CNS [7]. Although the possibility of the existence of another SPR subtype cannot be entirely excluded, this is one example of the ‘mismatches’ at the synaptic level. Thus, for elucidation of the sites of actual transmission by SP, identification of both presynaptic SP-containing terminal and postsynap-
Fig ,. 2. SPR-LI in a parasagittal section through the striatum (a,b). The immunoreactivity in the striatum completely disappeared by absorptio mnof the antibody with the trp E-SPR fusion protein (c). EM photographs show SPR-LI in a neuronal cell body (d) and a postsynaptic dendrite (e). Arl nowheads in (d) indicate the nuclear membrane. Arrows in (e) indicate the synaptic sites. D, dendrite; GP, globus pallidus; N, nucleus; St, striai turn. Bars = 300 pm (a), 100 pm (b,c), 2 pm (d) and 0.2 flrn (e).
tic SPR is required. SPR antibody generated in the present study is a useful tool for this purpose and further investigation for roles of neurotransmission/neuromodulation by SP in the CNS. 1 Bolam, J.P., Ingham, C.A., Izzo, PN., Levey, AI., Rye, D.B., Smith, A.D. and Wainer, B.H., Substance P-containing terminals in synaptic contact with cholinergic neurons in the neostriatum and basal forebrain: a double immunocytochemical study in the rat, Brain Res., 397 (1986) 279-289. 2 Bolam, J.P. and Izzo, P.N., The postsynaptic targets of substance P-immunoreactive terminals in the rat neostriatum with particular reference to identified spiny striatonigral neurons, Exp. Brain Res., 70 (1988) 361-377. 3 Carroll, S.B. and Laughon, A., Production and purification of poly-
clonal antibodies to the foreign segment of /7-galactosidase fusion proteins, In D.M. Glover (Ed.), DNA Cloning, Vol. III, IRL Press, Oxford, 1987, pp. 89111. Elde, R., Schalling, M., Ceccatelli, S., Nakanishi, S. and Hiikfelt, T., Localization of neuropeptide receptor mRNA in rat brain: initial observations using probes for neurotensin and substance P receptors, Neurosci. Lett., 120 (1990) 134-138. Gerfen, C.R., Substance P (neurokinin-1) receptor mRNA is selectively expressed in choline@ neurons in the striatum and basal forebrain, Brain Res., 556 (1991) 165-170. Harlow, E. and Lane, D., Antibodies, a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988. Herkenham, M., Mismatches between neurotransmitter and receptor localizations in brain: observations and implications, Neuroscience, 23 (1987) l-38. Koerner, T.J., Hill, J.E., Myers, A.M. and Tzagoloff, A., High-
160
9
10 11
12
expression vectors with multiple cloning sites for construction of ?rpE fusion genes: PATH vectors, Methods Enzymol.,l94 (1991) 411-490. Liu, Y.F. and Quirion, R., Presence of various carbohydrate moieties includinga-galactose and N-acetylglucosamine residues on solubilized porcine brain neurokinin-Vsubstanw P receptors, J. Neurochem., 57 (1991) 1944-1950. Maggio, J.E., Tachykinins, Annu. Rev. Neurosci., 11 (1991) 13-28. Mantyh, P.W., Gates, T., Mantyh, C.R. and Maggio, J.E., Autoradiographic localiiation and characterization of tachykinin receptor binding sites in the rat brain and peripheral tissues, J. Neurosci., 9 (1989) 258279. Nakajima, Y., Tsuchida, K., Negishi, M., Ito, S. and Nakanishi, S., Direct linkage of three tachykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades in trans-
13 14
15
16
fected Chinese hamster ovary cells, J. Biol. Chem., 267 (1992) 24372442. Otsuka, M. and Yoshioka, K., Neurotransmitter functions ofmammalian tachykinins, Physiol. Rev., 73 (1993) l-80. Tsuchida, K., Shigemoto, R., Yokota, Y. and Nakanishi, S., Tissue distribution and quantitation of the mRNAs for three rat tachykinin receptors, Eur. J. B&hem., 193 (1990) 751-757. Wilson, C.J., Basal ganglia, In G.M. Shepherd (Ed.), The Synaptic Organization of the Brain, 3rd edn., Oxford University Press, New York, NY, 1990, pp. 219-316. Yokota, Y., Sasai, Y., Tanaka, K., Fujiwara, T., Tsuchida, K., Shigemoto, R., Kakizuka, A., Ohkubo, H. and Nakanishi, S., Molecular characterization of a functional cDNA for rat substance P receptor, J. Biol. Chem., 264 (1989) 1764917652.