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Mosaic distribution of neurotensin-like immunoreactivity in the cat striatum
176
Brain Research, 274 (1983) 176-179
Elsevier
Mosaic distribution of neurotensin-Iike immunoreactivity in the cat striatum M. GOEDERT, P. W. MANTYH, S. P. HUNT and P. C. EMSON MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Hills Road Cambridge CB2 2QH (UK)
(Accepted May 3rd, 1983) Key words: neurotensin - - Met-enkephalin - - corpus striatum - - striosomes- - ~mmunohistochemistry
Neurotensin-like immunoreactivity was found in nerve fibers and terminals throughout the corpus striatum of the adult cat. The densest staining was observed in the globus pallidus followed by the caudate nucleus and the putamen. In the caudate nucleus neurotensin-like immunoreactivityhad a patchy distribution which was in register with a similar pattern of enkephalin-like immunoreactivi-
ty. Neurotensin is a 13 amino acid peptide first isolated from bovine hypothalamus2. 3 and subsequently from both human and bovine small intestine4,13. Several studies have established that neurotensin-like immunoreactivity (NTLI) is widely distributed in the central nervous system of various mammalian species, including man 17. In the cat, one of the highest concentrations of NTLI is found in the corpus striatum and the immunoreactive material coelutes with synthetic neurotensin on gel chromatography 7. In recent years, it has become apparent that the adult corpus striatum is divided into distinct histochemical compartments in cat, monkey and man 21. A striking pattern exists in the adult cat caudate nucleus taking the form of patches of low acetylcholinesterase activity called 'striosomes 's. These have been shown to correspond to regions of high enkephalinlike immunoreactivity H and opiate receptor binding ~4 and to relate to mosaic patterns of striatal efferent neurones ~0 and of cortical afferent terminations ~9. In the present communication, we report that NTLI shows a mosaic distribution in the adult cat striatum where it is present in register with the enkephalin-rich striosomes. Two adult male cats were placed under deep anaesthesia with pentobarbitone and perfused intracardially with 0.9% saline containing 800 U/liter of heparin. This was followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.40) containing 3.42 g/liter lysine and 0.55 g/liter sodium peri0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
odate. The brains were removed, post-fixed for 2 h in the same fixative and placed in 0.1 M phosphate buffer containing 30% (w/v) sucrose. Blocks containing the corpus striatum were frozen and sectioned in the transverse plane on a freezing microtome at 30/~m and treated with 1% hydrogen peroxide for 10 min. Serial sections were alternately stained for neurotensin and Met-enkephalin. Antisera to neurc,ensin and Met-enkephalin were produced in rabbits. The neurotensin antiserum was raised by immunization against neurotensins_~3 conjugated to keyhole hemocyanin using glutaraldehyde, and the enkephalin antiserum has been described previously 15. The antibodies were diluted with 0.1 M phosphate buffer containing 0.1% Triton X-100 and 1% sheep serum. Free floating sections were incubated with the primary antiserum (diluted 1:400 for neurotensin and 1:1000 for Met-enkephalin) for 72 h at 4 °C. Sections were washed in 0.1 M phosphate buffer and incubated with sheep anti-rabbit serum (Miles) diluted 1:20 for 1 h. Following this, the sections were washed, incubated with rabbit peroxidase-anti-peroxidase (Miles) at a dilution of 1:100 for 1 h and washed again. The tissue then was incubated for 5-10 min in phosphate buffer containing 0,09% 3,3-diaminobenzidine and 0.005% hydrogen peroxide and subsequently washed in phosphate buffer. In control experiments, the primary antibody was left out or incubated in the presence of 10 /~M neurotensin or Met-enkephalin. In addition, the neurotensin antiserum was incubated with 10 ,uM
Fig. 1. Photomicrographs A and B show the distribution of neurotensin-like immunoreactivity (NT) in the striatum of the cat. Note that in the caudate nucleus (Cd) there is a patchy distribution, whereas the globus pallidus (GP) has a homogenous distribution broken up by myelinated fiber bundles. Scale bars = 450/~m. Photomicrographs C and D show enkephalin (ENK) and neurotensin (NT)-like immunoreactivities in adjacent sections of the caudate nucleus. The arrows indicate patches of immunoreactivity which appear to overlap in the two adjacent 30/~m brain sections. Scale bars = 250/~m. Abbreviations: Cd, caudate nucleus; CI, internal capsule: ENK, enkephalin; GP, globus pallidus; NT, neurotensin; Put, putamen.
178 Met-enkephalin and the Met-enkephalin antiserum with 10 ~M neurotensin. The neurotensin antiserum was further characterized by being incubated in the presence of 10~M substance P, somatostatin tetradecapeptide, cholecystokinin octapeptide, vasoactive intestinal polypeptide, dynorphin heptadecapeptide or bombesin. When the tissue sections were incubated with the neurotensin antiserum a dense staining was observed throughout the corpus striatum. It was densest in the globus pallidus, followed by caudate nucleus and putamen. A striking mosaic pattern was found in the caudate nucleus with local patches of dense reaction product (Fig. la and b). The patches or striosomes8 were irregular in shape and present throughout the whole extension of the caudate nucleus. They consisted of an irregularly shaped core of dense immunoreactivity surrounded by a rim of virtually no staining, the whole being embedded in the medium dense background staining of the caudate. A dense reaction product without an indication of patches was seen in the globus pallidus (Fig. la). The dense patches of NTLI in the caudate nucleus were granular in nature and did not correspond to perikaryal staining, in control experiments staining was completely abolished in the presence of synthetic neurotensin and in the absence of the neurotensin antiserum. Neither Metenkephalin, nor any of the 6 other neuropeptides tested abolished the staining. Sets of consecutive sections were stained with the neurotensin or Met-enkephalin antiserum. The striosomal pattern corresponded in all sections studied, the neurotensin-rich patches being matched by enkephalin-rich regions (Fig. 1). Control experiments showed that the Metenkephalin stain was completely abolished by synthetic Met-enkephalin but not by synthetic neurotensin. The present findings indicate that NTLI is unevenly distributed within the caudate neucleus of the adult cat with areas of dense immunoreactivity corre-
sponding to previously described striosomes defined by their high content of enkephalin-like immunoreactivity and low acetylcholinesterase activity. The origin of the striatal neurotensin fibers is unknown at present. The caudate nucleus is innervated by the amygdala, the ventral tegmental area, the substantia nigra, the dorsal raphe nucleus, the thalamus and by cortical areas 9. in the rat, where an irregular distribution of neurotensin-containing nerve fibers has been described in the caudate nucleus~6,21, neurotensin-positive cell bodies have been found in several regions which project to the striatum including the amygdala, the ventral tegmental area and the dorsal raphe nucleus ~6. Since the striatum has not been reported to contain any neurotensin cell bodies these areas probably provide the caudate nucleus with its neurotensin-containing nerve fibers. There is considerable evidence for an interaction of neurotensin with the dopaminergic nigrostriatal system. Electrophysiological experiments have shown that neurotensin activates dopamine cells in the substantia nigra ~ where neurotensin receptors are found on dopmaninergic cell bodies 1~. in addition, neurotensin has been shown to increase striatat dopamine turnover in viv020.22, to enhance the release of dopamine from striatal tissue in vitro5 and to antagonize some of the behavioral changes induced by amphetamine 6. However, the anatomical relationship between neurotensin- and dopaminecontaining neurons is incompletely known. In view of the unusually high levels of neurotensin it contains 7, the cat striatum may represent a good system to study the relationship between neurotensin and dopamine and their respective receptors.
1 Andrade, R. and Aghajanian, G. K., Neurotensin selectively activates dopaminergic neurons of the substantia nigra, Soc. Neurosci. Abstr., 7 (1981) 573. 2 Carraway, R. and Leeman, S. E., The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalamus, J. biol. Chem., 248 (1973)6854-6861. 3 Carraway, R. and Leeman, S. E., The amino acid sequence of a hypothalamicpeptide, neurotensin, J, biol. Chem., 250 (1975) 1907-191l.
4 Carraway, R., Kitabgi, P. and Leeman, S. E., The amino acid sequence of radioimmunoassayable neurotensin from bovine intestine, J. biol. Chem., 253 (1978) 7996-7998. 5 De Quidt, M. E. and Emson, P. C., Neurotensin facilitates dopamine release in vitro from rat striatal slices, Brain Research, in press. 6 Ervin, G. N., Birkemo, L. S., Nemeroff, C. B. and Prange, A. J., Neurotensin blocks certain amphetamine behaviours, Nature (Lond.), 291 (198t) 73-76.
We thank Dr. V. Clement-Jones for the gift of the Met-enkephalin antiserum and Dr. L. L. Iversen for his critical reading of the manuscript. M.G. is supported by the British Council and P.W.M. is a NIH Fellow.
179 7 Goedert, M. and Emson, P. C., The regional distribution of neurotensin-like immunoreactivity in central and peripheral tissues of the cat, Brain Research, in press. 8 Graybiel, A. M. and Ragsdale, C. W., Histochemically distinct compartments in the striatum of human, monkey and cat demonstrated by acetylthiocholinesterase stain, Proc. nat. Acad. Sci, U.S.A., 75 (1978) 5723-5726. 9 Graybiel, A. M. and Ragsdale, C. W. Fiber connections of the basal ganglia. In M. Cu~nod, G. W. Kreutzberg and F. E. Bloom (Eds.), Development and chemical specificity of neurons, Elsevier Biomedical, Amsterdam, 1979, pp. 239-283. 10 Graybiel, A. M., Ragsdale, C. W. and Moon Edley, S., Compartments in the striatum of the cat observed by retrograde cell labeling, Exp. Brain Res., 34 (1979) 189-195. 11 Graybiel, A. M., Ragsdale, C. W., Yoneoka, E. S. and Elde, R. P., An immunohistochemical study of enkephalins and other neuropeptides in the striatum of the cat with evidence that the opiate peptides are arranged to form mosaic patterns in register with the striosomal compartments visible by acetylcholinesterase staining, Neuroscience, 6 (1981) 377-397. 12 Graybiel, A. M., Correlative studies of histochemistry and fiber connections in the central nervous system. In V. Chan-Palay and S. L. Palay (Eds.), Cytochemical Methods in Neuroanatomy, Alan R. Liss, New York, 1982, pp. 45-67. 13 Hammer, R. A., Leeman, S. E., Carraway, R. and Williams, R. H., Isolation of human intestinal neurotensin, J. biol. Chem., 255 (1980) 2476-2480. 14 Herkenham, M. and Pert, C. B., Mosaic distribution of opiate receptors, parafascicular projections and acetylcho-
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linesterase in rat striatum, Nature (Lond.), 291 (1981) 415-418. Hunt, S. P., Kelly, J. S., Emson, P. C., Kimmel, J. R., Miller, R. J. and Wu, J. I., An immunohistochemical study of neuronal populations containing neuropeptides or y-aminobutyrate within the superficial layers of the rat dorsal horn, Neuroscience, 6 (1981) 1883-1898. Jennes, L., Stumpf, W. E. and Kalivas, P. W., Neurotensin: topographical distribution in rat brain by immunohistochemistry, J. comp. Neurol., 210 (1982) 211-224. Nemeroff, C. F., Luttinger, D. and Prange, A. J., Neurotensin and bombesin. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 17, Plenum Press, New York, 1983, pp. 363-466. Palacios, J. M. and Kuhar, M. J., Neurotensin receptors are located on dopamine-containing neurons in rat midbrain, Nature (Lond.), 294 (1981) 587-589. Ragsdale, C. W. and Graybriel, A. M., The fronto-striatal projection in the cat and monkey and its relationship to inhomogeneities established by acetylcholinesterase histochemistry, Brain Research, 208 (1981) 259-266. Reches, A., Burke, R. E., Jiang, D. H., Wagner, H. R. and Fahn, S., Neurotensin increases dopamine turnover in rat brain, Soc. Neurosci. Abstr., 8 (1982) 581. Uhl, G. R., Kuhar, M. J. and Snyder, S. H., Neurotensin: immunohistochemical localisation in rat central nervous system, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 4059--4063. WiderlOv, E., Kilts, C. D., Mailman, R. G., Nemeroff, C. B., Macown, T. J., Prange, A. J. and Breese, G. R., Increase in dopamine metabolites in rat brain by neurotensin, J. Pharmacol. exp. Ther., 222 (1982) 1-6.
Brain Research, 274 (1983) 176-179
Elsevier
Mosaic distribution of neurotensin-Iike immunoreactivity in the cat striatum M. GOEDERT, P. W. MANTYH, S. P. HUNT and P. C. EMSON MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Hills Road Cambridge CB2 2QH (UK)
(Accepted May 3rd, 1983) Key words: neurotensin - - Met-enkephalin - - corpus striatum - - striosomes- - ~mmunohistochemistry
Neurotensin-like immunoreactivity was found in nerve fibers and terminals throughout the corpus striatum of the adult cat. The densest staining was observed in the globus pallidus followed by the caudate nucleus and the putamen. In the caudate nucleus neurotensin-like immunoreactivityhad a patchy distribution which was in register with a similar pattern of enkephalin-like immunoreactivi-
ty. Neurotensin is a 13 amino acid peptide first isolated from bovine hypothalamus2. 3 and subsequently from both human and bovine small intestine4,13. Several studies have established that neurotensin-like immunoreactivity (NTLI) is widely distributed in the central nervous system of various mammalian species, including man 17. In the cat, one of the highest concentrations of NTLI is found in the corpus striatum and the immunoreactive material coelutes with synthetic neurotensin on gel chromatography 7. In recent years, it has become apparent that the adult corpus striatum is divided into distinct histochemical compartments in cat, monkey and man 21. A striking pattern exists in the adult cat caudate nucleus taking the form of patches of low acetylcholinesterase activity called 'striosomes 's. These have been shown to correspond to regions of high enkephalinlike immunoreactivity H and opiate receptor binding ~4 and to relate to mosaic patterns of striatal efferent neurones ~0 and of cortical afferent terminations ~9. In the present communication, we report that NTLI shows a mosaic distribution in the adult cat striatum where it is present in register with the enkephalin-rich striosomes. Two adult male cats were placed under deep anaesthesia with pentobarbitone and perfused intracardially with 0.9% saline containing 800 U/liter of heparin. This was followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.40) containing 3.42 g/liter lysine and 0.55 g/liter sodium peri0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
odate. The brains were removed, post-fixed for 2 h in the same fixative and placed in 0.1 M phosphate buffer containing 30% (w/v) sucrose. Blocks containing the corpus striatum were frozen and sectioned in the transverse plane on a freezing microtome at 30/~m and treated with 1% hydrogen peroxide for 10 min. Serial sections were alternately stained for neurotensin and Met-enkephalin. Antisera to neurc,ensin and Met-enkephalin were produced in rabbits. The neurotensin antiserum was raised by immunization against neurotensins_~3 conjugated to keyhole hemocyanin using glutaraldehyde, and the enkephalin antiserum has been described previously 15. The antibodies were diluted with 0.1 M phosphate buffer containing 0.1% Triton X-100 and 1% sheep serum. Free floating sections were incubated with the primary antiserum (diluted 1:400 for neurotensin and 1:1000 for Met-enkephalin) for 72 h at 4 °C. Sections were washed in 0.1 M phosphate buffer and incubated with sheep anti-rabbit serum (Miles) diluted 1:20 for 1 h. Following this, the sections were washed, incubated with rabbit peroxidase-anti-peroxidase (Miles) at a dilution of 1:100 for 1 h and washed again. The tissue then was incubated for 5-10 min in phosphate buffer containing 0,09% 3,3-diaminobenzidine and 0.005% hydrogen peroxide and subsequently washed in phosphate buffer. In control experiments, the primary antibody was left out or incubated in the presence of 10 /~M neurotensin or Met-enkephalin. In addition, the neurotensin antiserum was incubated with 10 ,uM
Fig. 1. Photomicrographs A and B show the distribution of neurotensin-like immunoreactivity (NT) in the striatum of the cat. Note that in the caudate nucleus (Cd) there is a patchy distribution, whereas the globus pallidus (GP) has a homogenous distribution broken up by myelinated fiber bundles. Scale bars = 450/~m. Photomicrographs C and D show enkephalin (ENK) and neurotensin (NT)-like immunoreactivities in adjacent sections of the caudate nucleus. The arrows indicate patches of immunoreactivity which appear to overlap in the two adjacent 30/~m brain sections. Scale bars = 250/~m. Abbreviations: Cd, caudate nucleus; CI, internal capsule: ENK, enkephalin; GP, globus pallidus; NT, neurotensin; Put, putamen.
178 Met-enkephalin and the Met-enkephalin antiserum with 10 ~M neurotensin. The neurotensin antiserum was further characterized by being incubated in the presence of 10~M substance P, somatostatin tetradecapeptide, cholecystokinin octapeptide, vasoactive intestinal polypeptide, dynorphin heptadecapeptide or bombesin. When the tissue sections were incubated with the neurotensin antiserum a dense staining was observed throughout the corpus striatum. It was densest in the globus pallidus, followed by caudate nucleus and putamen. A striking mosaic pattern was found in the caudate nucleus with local patches of dense reaction product (Fig. la and b). The patches or striosomes8 were irregular in shape and present throughout the whole extension of the caudate nucleus. They consisted of an irregularly shaped core of dense immunoreactivity surrounded by a rim of virtually no staining, the whole being embedded in the medium dense background staining of the caudate. A dense reaction product without an indication of patches was seen in the globus pallidus (Fig. la). The dense patches of NTLI in the caudate nucleus were granular in nature and did not correspond to perikaryal staining, in control experiments staining was completely abolished in the presence of synthetic neurotensin and in the absence of the neurotensin antiserum. Neither Metenkephalin, nor any of the 6 other neuropeptides tested abolished the staining. Sets of consecutive sections were stained with the neurotensin or Met-enkephalin antiserum. The striosomal pattern corresponded in all sections studied, the neurotensin-rich patches being matched by enkephalin-rich regions (Fig. 1). Control experiments showed that the Metenkephalin stain was completely abolished by synthetic Met-enkephalin but not by synthetic neurotensin. The present findings indicate that NTLI is unevenly distributed within the caudate neucleus of the adult cat with areas of dense immunoreactivity corre-
sponding to previously described striosomes defined by their high content of enkephalin-like immunoreactivity and low acetylcholinesterase activity. The origin of the striatal neurotensin fibers is unknown at present. The caudate nucleus is innervated by the amygdala, the ventral tegmental area, the substantia nigra, the dorsal raphe nucleus, the thalamus and by cortical areas 9. in the rat, where an irregular distribution of neurotensin-containing nerve fibers has been described in the caudate nucleus~6,21, neurotensin-positive cell bodies have been found in several regions which project to the striatum including the amygdala, the ventral tegmental area and the dorsal raphe nucleus ~6. Since the striatum has not been reported to contain any neurotensin cell bodies these areas probably provide the caudate nucleus with its neurotensin-containing nerve fibers. There is considerable evidence for an interaction of neurotensin with the dopaminergic nigrostriatal system. Electrophysiological experiments have shown that neurotensin activates dopamine cells in the substantia nigra ~ where neurotensin receptors are found on dopmaninergic cell bodies 1~. in addition, neurotensin has been shown to increase striatat dopamine turnover in viv020.22, to enhance the release of dopamine from striatal tissue in vitro5 and to antagonize some of the behavioral changes induced by amphetamine 6. However, the anatomical relationship between neurotensin- and dopaminecontaining neurons is incompletely known. In view of the unusually high levels of neurotensin it contains 7, the cat striatum may represent a good system to study the relationship between neurotensin and dopamine and their respective receptors.
1 Andrade, R. and Aghajanian, G. K., Neurotensin selectively activates dopaminergic neurons of the substantia nigra, Soc. Neurosci. Abstr., 7 (1981) 573. 2 Carraway, R. and Leeman, S. E., The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalamus, J. biol. Chem., 248 (1973)6854-6861. 3 Carraway, R. and Leeman, S. E., The amino acid sequence of a hypothalamicpeptide, neurotensin, J, biol. Chem., 250 (1975) 1907-191l.
4 Carraway, R., Kitabgi, P. and Leeman, S. E., The amino acid sequence of radioimmunoassayable neurotensin from bovine intestine, J. biol. Chem., 253 (1978) 7996-7998. 5 De Quidt, M. E. and Emson, P. C., Neurotensin facilitates dopamine release in vitro from rat striatal slices, Brain Research, in press. 6 Ervin, G. N., Birkemo, L. S., Nemeroff, C. B. and Prange, A. J., Neurotensin blocks certain amphetamine behaviours, Nature (Lond.), 291 (198t) 73-76.
We thank Dr. V. Clement-Jones for the gift of the Met-enkephalin antiserum and Dr. L. L. Iversen for his critical reading of the manuscript. M.G. is supported by the British Council and P.W.M. is a NIH Fellow.
179 7 Goedert, M. and Emson, P. C., The regional distribution of neurotensin-like immunoreactivity in central and peripheral tissues of the cat, Brain Research, in press. 8 Graybiel, A. M. and Ragsdale, C. W., Histochemically distinct compartments in the striatum of human, monkey and cat demonstrated by acetylthiocholinesterase stain, Proc. nat. Acad. Sci, U.S.A., 75 (1978) 5723-5726. 9 Graybiel, A. M. and Ragsdale, C. W. Fiber connections of the basal ganglia. In M. Cu~nod, G. W. Kreutzberg and F. E. Bloom (Eds.), Development and chemical specificity of neurons, Elsevier Biomedical, Amsterdam, 1979, pp. 239-283. 10 Graybiel, A. M., Ragsdale, C. W. and Moon Edley, S., Compartments in the striatum of the cat observed by retrograde cell labeling, Exp. Brain Res., 34 (1979) 189-195. 11 Graybiel, A. M., Ragsdale, C. W., Yoneoka, E. S. and Elde, R. P., An immunohistochemical study of enkephalins and other neuropeptides in the striatum of the cat with evidence that the opiate peptides are arranged to form mosaic patterns in register with the striosomal compartments visible by acetylcholinesterase staining, Neuroscience, 6 (1981) 377-397. 12 Graybiel, A. M., Correlative studies of histochemistry and fiber connections in the central nervous system. In V. Chan-Palay and S. L. Palay (Eds.), Cytochemical Methods in Neuroanatomy, Alan R. Liss, New York, 1982, pp. 45-67. 13 Hammer, R. A., Leeman, S. E., Carraway, R. and Williams, R. H., Isolation of human intestinal neurotensin, J. biol. Chem., 255 (1980) 2476-2480. 14 Herkenham, M. and Pert, C. B., Mosaic distribution of opiate receptors, parafascicular projections and acetylcho-
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linesterase in rat striatum, Nature (Lond.), 291 (1981) 415-418. Hunt, S. P., Kelly, J. S., Emson, P. C., Kimmel, J. R., Miller, R. J. and Wu, J. I., An immunohistochemical study of neuronal populations containing neuropeptides or y-aminobutyrate within the superficial layers of the rat dorsal horn, Neuroscience, 6 (1981) 1883-1898. Jennes, L., Stumpf, W. E. and Kalivas, P. W., Neurotensin: topographical distribution in rat brain by immunohistochemistry, J. comp. Neurol., 210 (1982) 211-224. Nemeroff, C. F., Luttinger, D. and Prange, A. J., Neurotensin and bombesin. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 17, Plenum Press, New York, 1983, pp. 363-466. Palacios, J. M. and Kuhar, M. J., Neurotensin receptors are located on dopamine-containing neurons in rat midbrain, Nature (Lond.), 294 (1981) 587-589. Ragsdale, C. W. and Graybriel, A. M., The fronto-striatal projection in the cat and monkey and its relationship to inhomogeneities established by acetylcholinesterase histochemistry, Brain Research, 208 (1981) 259-266. Reches, A., Burke, R. E., Jiang, D. H., Wagner, H. R. and Fahn, S., Neurotensin increases dopamine turnover in rat brain, Soc. Neurosci. Abstr., 8 (1982) 581. Uhl, G. R., Kuhar, M. J. and Snyder, S. H., Neurotensin: immunohistochemical localisation in rat central nervous system, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 4059--4063. WiderlOv, E., Kilts, C. D., Mailman, R. G., Nemeroff, C. B., Macown, T. J., Prange, A. J. and Breese, G. R., Increase in dopamine metabolites in rat brain by neurotensin, J. Pharmacol. exp. Ther., 222 (1982) 1-6.