0306-4522/90 $3.00 + 0.00 Pergamon Press plc
Neuroscience Vol. 36, No. 2, pp. 425430, 1990 Printed in Great Britain
IBRO
RETROGRADE A X O N A L TRANSPORT OF N E U R O T E N S I N IN THE DOPAMINERGIC NIGROSTRIATAL PATHWAY IN THE RAT M.-N. CASTEL,C. MALGOURIS,J.-C. BLANCHARD a n d P. M. LADURON Rh6ne-Poulenc Sant6, Centre de Recherches de Vitry-Alfortville, 13 Quai Jules Guesde, 94403 Vitry-sur-Seine Cedex, France Al~traet--Although the existence of receptor transport has been clearly demonstrated in peripheral nerves, there is no clear cut evidence in the brain of such a process for neuropeptide receptors. Because of the localization of neurotensin receptors on dopaminergic terminals, the dopaminergic nigrostriatal pathway appears to be the system of choice for studying the axonal transport of neuropeptide receptors in the brain. When labelled neurotensin was injected into the rat striatum, a delayed accumulation of radioactivity in the ipsilateral substantia nigra was observed about 2 h after injection. An essential requirement to clearly observe this phenomenon was the pretreatment of animals with kelatorphan in order to prevent the labelled neurotensin degradation. The appearance of this labelling was prevented by injection of an excess of unlabelled neurotensin or of neurotensin 8-13, an active neurotensin fragment, but not by neurotensin 1-8, which had no affinity for neurotensin receptors. This process was saturable, microtubule-dependent and occurred only in mesostriatal and nigrostriatal dopaminergic neurons as identified after 6-hydroxydopamine lesion and by autoradiography. These results demonstrate that neurotensin was retrogradely transported by a process involving neurotensin receptors. The retrograde transport of receptor-bound neuropeptide may represent an important dynamic process which conveys information molecules from the synapse towards the cell body.
N e u r o t e n s i n is a tridecapeptide ( H - p G l u - L e u - T y r Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH) which was isolated from bovine h y p o t h a l a m i , ~ sequenced 2 a n d synthetized 3 by C a r r a w a y a n d Leeman. M o r e recently the gene encoding for this peptide was isolated in the r a t ) ° I m m u n o h i s t o c h e m i c a l studies have s h o w n t h a t this n e u r o p e p t i d e is distributed widely a n d heterogeneously in the m a m m a l i a n central nervous system. 8'9'25 N e u r o t e n s i n receptors have been identified in the b r a i n o f n u m e r o u s species ~,2~ a n d have recently been purified. ~8'19In the rat striatum, n e u r o t e n s i n binding sites are associated with dopaminergic terminals. 7'2°'23 M o r e recently, the utilization o f a p o t e n t antihistaminic drug, levocabastine, has allowed the identification o f two types o f n e u r o t e n s i n binding sites in the rat striatum: the n e u r o t e n s i n - a c c e p t o r sites which are sensitive b o t h to neurotensin a n d levocabastine, a n d the n e u r o t e n s i n - r e c e p t o r sites which only recognize neurotensin. 22 M o r e detailed subcellular analysis has revealed t h a t only the neurotensin-receptor sites are presynaptically located on dopaminergic terminals in this structure, whereas the neurotensin-acceptor sites are associated with glial cells. 23 F o r the m o m e n t , there is no evidence t h a t neurotensin a n d d o p a m i n e co-exist in the dopaminergic nigrostriatal pathway. Fast axonal a n t e r o g r a d e t r a n s p o r t is required to supply nerve terminals with neurotransmitters, enzymes and secretory materials. 6 In peripheral nerves, ligature experiments have d e m o n s t r a t e d a fast and bidirectional t r a n s p o r t of muscarinic, 14 opiate ~5'~7and o t h e r presynaptic receptors (see Ref. 16 for review).
Moreover, [3H]lofentanil was used in vivo to d e m o n strate a retrograde t r a n s p o r t o f the r e c e p t o r - b o u n d opiate in ligated vagus nerves. 17 Thus, the aim o f this study was to determine if neurotensin was t r a n s p o r t e d in rat brain. EXPERIMENTAL PROCEDURES
Materials
Labelled neurotensin was purchased from Amersham and unlabelled peptides (neurotensin and the 1-8 neurotensin and 8-13 neurotensin fragments) from Sigma. Colchicine was obtained from Prolabo and 6-hydroxydopamine from Serva. Kelatorphan was a generous gift from Prof. B. P. Roques. Methods
Adult male Sprague-Dawley rats (n = 110, Charles River, France) weighing between 280 and 320 g were injected under pentobarbital anaesthesia with 2 #1 of kelatorphan (30/~g) or with saline in the right striatum at the rate of 0.2 ttl/min (coordinates AP 7.5 mm, L 3 mm, V 0 mm according to K6nig and KlippeP2). Ten minutes later, 0.16pmol of monoido-Tyr3 neurotensin (2000Ci/mmol), dissolved in saline, was infused in a total volume of 3 #1 into three different areas of the right striatum (coordinates AP 9.4 mm, L 2mm and V 0.2 mm; AP 6.6 mm, L 3.5mm and V - 0 . 4 mm; AP 4.6 mm, L 4.4mm and V - 0 . 6 mm). The coordinates were regularly verified by injection of a dye. Four hours later, animals were killed and their brains dissected out. The radioactivity in different brain areas (the ipsi- and contralateral substantia nigra, the cerebellum and the ipsilateral hippocampus) was measured in an LKB gamma counter. For autoradiographic studies, the rat brains were frozen rapidly in isopentane at -20°C. Coronal sections (20 ttm thick) were cut on a cryostat at - 1 5 ° C and mounted on lamellae. Finally, sections were apposed to 3H ultrofilm 425
426
M.-N. CASTELet al.
(LKB, France) for 3 weeks at -80°C inside X-ray cassettes. The films were developed in a Kodak D 19 developer for 5 min at 18°C, fixed, washed for 30 min and dried. For neurotoxic experiments, animals were pretreated either with colchicine or 6-hydroxydopamine. Colchicine (2001tg/4/~l of 0.1% NaC1) was injected into the right lateral ventricle (AP 0.6mm, L 1.3 mm, V - 5 m m with respect to Bregma) 30 h before kelatorphan administration. Nigrostriatal dopaminergic neurons were lesioned by unilaterally injecting 8/~g of 6-hydroxydopamine in 2/~1 of saline containing 0.01% L-ascorbic acid in the medial forebrain bundle at coordinates AP 4.2mm, L 1.4mm and V 2.3 mm according to K6nig and Klippel. 12 Animals were treated with 25mg/kg desmethylimipramine (i.p.) 45min prior to the 6-hydroxydopamine injections. The effectiveness of the lesion was examined by assessment of the contralateral circling response to apomorphine (0.63 mg/kg subcutaneously), 3 weeks later. Animals displaying contraversive rotations were retained for use in experiments. For in vivo displacement experiments, rats were coinjected with a 1000-fold excess (162pmol) of unlabelled neurotensin, or of neurotensin fragments 8-13 and 1-8, and with monoiodo-Tyr3 neurotensin in the right striatum. On the other hand, animals were injected with monoiodo-Tyr3 neurotensin alone (0.16 pmol) of in association with 3-, 10-, 30-, 100- and 1000-fold excesses of unlabelled neurotensin. For kinetic experiments, rats were injected with 0.16 pmol of monoiodo-Tyr3 neurotensin as previously described and were killed immediately or 1, 2, 3, 4 and 6 h after the injection. Radioactivity was measured in the right substantia nigra.
RESULTS
When animals were injected in the right striatum with monoiodo-Tyr3 neurotensin after kelatorphan pretreatment, a substantial accumulation of radioactivity (360 + 25 d.p.m./mg of tissue) could be observed in the ipsi- but not in the contralateral substantia nigra 4 h later (Fig. 1). However, the ipsilateral substantia nigra was not labelled in the absence of kelatorphan (Fig. 1). As shown in Fig. 1, a weak labelling was measured in the cerebellum ( 0 . 6 + 0 . 2 d . p . m . / m g of tissue) and in the right hippocampus ( 1 3 + 3 d . p . m . / m g of tissue). When
animals were pretreated with saline, no difference of labelling could be detected in these two structures (0.4 + 0.12 d.p.m./mg of tissue in the cerebellum and l l . 2 + 2 . 7 d . p . m . / m g of tissue in the ipsilateral hippocampus). A more precise location of the radioactivity in the mesencephalon was obtained by autoradiography (Fig. 2). The labelling was restricted to the ipsilateral substantia nigra pars compacta and the adjacent ventral tegmental area. This accumulation of radioactivity in the ipsilateral substantia nigra was prevented by a 1000-fold excess of unlabelled neurotensin or of the neurotensin fragment 8-13 (Table 1), but not by the neurotensin fragment 1-8 (Table 1). Labelling persisted in the cerebellum and in the right hippocampus (Table 1), whatever the treatment. The inhibition of radioactivity accumulating in the ipsilateral substantia nigra was dose-dependent (Fig. 3), as demonstrated by the co-injection into the right striatum of increasing doses of unlabelled neurotensin and monoiodo-Tyr3 neurotensin. The radioactivity started to accumulate in the substantia nigra after about 2 h (Fig. 4) and the highest levels were observed 3 or 4 h after the injection (Fig. 4). When rats were pretreated either with colchicine (Table 2) or 6-hydroxydopamine (Table 2), there was no labelling in the ipsilateral substantia nigra. On the contrary, the cerebellum and the hippocampus were still marked (Table 2).
DISCUSSION
The foregoing results suggest that labelled neurotensin injected into the right striatum moves retrogradely to the ipsilateral substantia nigra and the adjacent ventral tegmental area. The pretreatment of animals with kelatorphan was necessary to measure
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Fig. 1. Effect of kelatorphan on the labelling observed in different cerebral regions 4 h after monoiodoTyr3 neurotensin injection into the right striatum. Rats were pretreated with kelatorphan (30 #g, n = 10) or with saline (n = 5), injected into the right striatum 10min before the injection of 0.16pmol of monoiodo-Tyr3 neurotensin. Results are expressed in d.p.m./mg of tissue ___S.E.M.
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428
M.-N. CASTELet al. Table 1. Rats were injected in the right striatum with 0.16 pmol of monoiodo-Tyr3 neurotensin alone or in association with an excess (162 pmol) of unlabelled neurotensin or neurotensin fragments (fragments 8-13 and 1-8) Experimental conditions
Ipsilateral substantia nigra Contralateral substantia nigra Cerebellum Ipsilateral hippocampus
[nSI]NT +
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unlabelled neurotensin n =6
neurotensin fragment 8-13 n =5
neurotensin fragment 1-8 n=5
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Labelling was measured in various cerebral structures 4 h after the injections. Results are expressed in d.p.m./mg of tissue + S.E.M. []25I]NT, monoiodo-Tyr neurotensin. a detectable signal in the ipsilateral substantia nigra. Indeed, kelatorphan is known to inhibit the neutral endopeptidase 24 11, 27 a peptidase present in large quantities in the rat caudate-putamen. 28'29 This enzyme induces the cleavage of the P r o l 0 - T y r l l and T y r l l - I l e l 2 bonds 4 and effectively inactivates neurotensin, since the degradation products are devoid of biological activity: This process appears to be receptor-dependent, since an excess of neurotensin and the fragment 8-13, which have a high affinity for neurotensin receptors: prevented the accumulation of radioactivity in the ipsilateral substantia nigra. On the contrary, the neurotensin fragment 1-8, which is inactive in binding studies, 5 was unable to prevent the accumulation of radioactivity in this structure. Moreover, the appearance of labelling in the substantia nigra was totally inhibited by a very low dose of unlabelled neurotensin (162 pmol); such a dosedependent and saturable process is compatible with the Kd values obtained in binding studies on neuro-
tensin receptors. 22 In contrast, uptake of extracellular proteins, especially horseradish peroxidase, and the subsequent axonal transport, is not saturable and not specific for a given type of neuron because these macromolecules are not internalized by nerve terminals through a ligand-receptor process. Labelling persisted in the cerebellum and in the hippocampus even when an excess of unlabelled neurotensin or the fragment 8-13 was injected, indicating that in these structures the accumulation of radioactivity was unrelated to neurotensin receptors. All these data support the idea that monoiodoTyr3 neurotensin was transported from the striatum towards the ipsilateral substantia nigra through transport mechanisms involving neurotensin receptors. This transport occurred in dopaminergic neurons as shown by the complete absence of labelling in the substantia nigra after 6-hydroxydopamine pretreatment. This result was confirmed by autoradiography, since the labelling was restricted to the ipsilateral substantia nigra pars compacta and the 60C
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Fig. 3. Labelling measured in the ipsilateral substantia nigra 4 h after the injection of monoiodo-Tyr3 neurotensin alone (0.16pmol, n =7) or in combination with 3 (n =5), 10 (n=6), 30 (n=5), 100 ( n = 6 ) and 1000 ( n = 6 ) fold excesses of unlabelled neurotensin. The results are expressed in terms of percentage radioactivity observed in the absence of unlabelled neurotensin.
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Fig. 4. Time-course of labelling measured in the substantia nigra after injection of monoiodo-Tyr3 neurotensin into the rat striatum. Rats were injected with 0.16pmol of monoiodo-Tyr3 neurotensin and were killed immediately ( n = 4 ) or 1 (n=7), 1.5 (n=6), 2 (n=6), 3 (n=6), 4 (n = 10) and 6 (n = 6) h later. Results are expressed as mean d.p.m. + S.E.M.
Retrograde transport of neurotensin in rat brain Table 2. Effect of neurotoxic treatment on the labelling observed in the ipsilateral substantia nigra after the injection of 0.16pmol of monoiodo-Tyr3 neurotensin in the right striatum
[125I]NT n = 10 Ipsilateral substantia nigra Contralateral substantia nigra Cerebellum Ipsilateral hippocampus
Experimental conditions [125I]NT [12~I]NT + + 6-OH-dopamine colchicine pretreatment pretreatment n =5 n =5
360 + 25
0
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0 0.6 + 0.2
0 0.2 + 0.02
0 0.8 + 0.2
13 + 3
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15 + 5
One group of animals received 8 pg of 6-hydroxydopamine (6-OH-dopamine) in the right medial forebrain bundle after a pretreatment with desmethylimipramine (25 mg/kg, i.p.). Three weeks later, the well-lesioned animals were injected with monoiodo-Tyr3 neurotensin. Another group of rats was pretreated with colchicine (200,ug, i.c.v.) 30 h before the monoiodo-Tyr3 neurotensin injection..Results are expressed in d.p.m./mg of tissue _+ S.E.M. [125I]NT, monoiodo-Tyr3 neurotensin.
adjacent ventral tegmental area, both regions containing the cell bodies of the ascending dopaminergic projections to the striatum. The delayed appearance of labelling in the substantia nigra is compatible with a fast rate of axonal transport (1-5 mm/h). 1~ This transport was microtubule-dependent, as indicated by the prevention of accumulation of radioactivity in the nigra after the colchicine pretreatment) 3 On the contrary, the early appearance of radioactivity in the cerebellum and in the ipsilateral hippocampus (in less than 1 h) and the persistent labelling measured in these structures even
429
in the presence of colchicine indicates the existence of a diffusion phenomenon in these cerebral areas. It appears that the retrograde axonal transport of neurotensin first involves the binding of the labelled neuropeptide to specific receptors presynaptically located on the dopaminergic terminals in the striatum, and then the internalization of the ligand-receptor complex. This specific endocytosis has been reported in vivo for opiate agonists in peripheral nerves ~7 and more recently in vitro for neurotensin receptors in cultured cortical neurons} 6 As to the fate of receptor-bound neurotensin in the neuron, there are a number of possibilities, as recently suggested: ~6first, the complex could be degraded after fusion with lysosomes in the axon or in the cell body; second, it could be reused or recycled through the Golgi apparatus; third, such a complex may serve either directly or indirectly to induce changes in gene expression. This latter hypothesis has already been reported for growth factors} 4
CONCLUSION The results presented here provide the first evidence for the existence of an axonal retrograde transport of neuropeptide-bound receptors in the brain and could represent a major dynamic mechanism for conveying information molecules from the synapse to the cell body. This phenomenon could account for the long-term effects of neuropeptides and might be involved in neurogenesis, neural plasticity and perhaps in long-term m e m o r y ) 6 Acknowledgements--We are grateful to Prof. Bernard P.
Roques for fruitful discussions and for the gift of kelatorphan. The skilful technical assistance of Marc Daniel was highly appreciated. We wish to thank Jeremy Pratt and Karen Birmingham for their help in preparing the manuscript.
REFERENCES
1. Carraway R. and Leeman S. E. (1973) The isolation of a new hypothalamic peptide, neurotensin, from bovine hypothalami. J. biol. Chem. 248, 6854-6861. 2. Carraway R. and Leeman S. E. (1975) The amino-acid sequence of hypothalamic peptide, neurotensin. J. biol. Chem. 250, 1907-1911. 3. Carraway R. and Leeman S. E. (1975) The synthesis of neurotensin. J. biol. Chem. 250, 1912-1918. 4. Checler F., Barelli H., Kitabgi P. and Vincent J. P. (1985) Neurotensin metabolism in various tissues of central and peripheral origins: ubiquitous involvement of a novel neurotensin degrading metalloendopeptidase. Biochimie 70, 75-82. 5. Goedert M., Pittaway K., Williams B. J. and Emson P. C. (1984) Specific binding of tritiated neurotensin to rat brain membranes: characterization and regional distribution. Brain Res. 304, 71-81. 6. Grafstein B. and Forman D. S. (1980) Intracellular transport in neurons. Physiol. Rev. 60, 1167-1283. 7. Herv6 D., Tassin J. P., Studler J. M., Dana C., Kitabgi P., Vincent J. P., Glowinski J. P. and Rostrne W. (1986) Dopaminergic control of ~:5I-labeled neurotensin binding site density in corticolimbic structures of rat brain. Proc. natn. Acad. Sci. U.S.A. 83, 6203~207. 8. H6kfelt T., Everitt B. J., Theodorsson-Norheim E. and Golstein M. (1984) Occurrence of neurotensin-like immunoreactivity in subpopulations of hypothalamic, mesencephalic and medullary catecholamine neurons. J. comp. Neurol. 222, 543-559. 9. Jennes L., Stumpf W. E. and Kalivas P. W. (1982) Neurotensin: topographical distribution in rat brain by immunohistochemistry. J. comp. Neurol. 210, 211-224. 10. Kislauskis E., Bullock B., McNeil S. and Dobner P. R. (1988) The rat gene encoding neurotensin and neuromedin N. J. biol. Chem. 263, 4963-4968. 11. Kitabgi P., Carraway R., Van Rietschoten J., Granier C., Morgat J. P., Menez A., Leeman S. E. and Freychet P. (1977) Neurotensin: specific binding to synaptic membranes from rat brain. Proc. natn. Acad. Sci. U.S.A. 74, 1846-1850.
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12. K6nig F. R. G. and Klippel R. A. (1967) The Rat Brain. A Stereotaxic Atlas o f the Forebrain and the Lower Parts o f the Brain Stem. Robert E. Krieger, New York. 13. Kreutzberg G. W. (1969) Neuronal dynamics and axonal flow. IV. Blockade of intra-axonal enzyme transport by colchicine. Proc. natn. Acad. Sci. U.S.A. 62, 722-728. 14. Laduron P. M. (1980) Axonal transport of muscarinic receptors. Nature 286, 287-288. 15. Laduron P. M. (1984) Axonal transport of opiate receptors in capsaicin-sensitive neurons. Brain Res. 294, 157-160. 16. Laduron P. M. (1987) Axonal transport of neuroreceptors: possible involvement in long-term memory. Neuroscience 22, 767 779. 17. Laduron P. M. and Janssen P. M. F. (1982) Axoplasmic transport and possible recycling of opiate receptors labelled with 3H-lofentanil. Life Sei. 31, 457-462. 18. Mazella J., Chabry J., Zsurger N. and Vincent J. P. (1989) Purification of the neurotensin receptor from mouse brain by affinity chromatography. J. biol. Chem. 264, 5559-5563. 19. Mills A., Demoliou-Mason C. D. and Barnard E. A. (1988) Characterization of neurotensin binding sites in intact and solubilized bovine brain membranes. J. biol. Chem. 263, 13 16. 20. Quirion R., Chiueh C. C., Everist H. D. and Pert A. (1985) Comparative localization of neurotensin receptors on nigrostriatal and mesolimbic dopaminergic terminals. Brain Res. 327, 385-389. 21. Sarrieau A., Javoy-Agid F., Kitagbi P., Dussaillant M., Vial M., Vincent J. P., Agid Y. and Rost6ne W. (1985) Characterization and autoradiographic distribution of neurotensin binding sites in the human brain. Brain Res. 348, 375-380. 22. Schotte A., Leysen J. E. and Laduron P. M. (1986) Evidence for a displaceable non-specific [3H]neurotensin binding site in rat brain. Naunyn-Schmiedeberg's Arch. Pharmac. 333, 400-405. 23. Schotte A., Rost6ne W. and Laduron P. M. (1988) Different subcellular localization of neurotensin-receptor and neurotensin-acceptor sites in the rat brain dopaminergic system. J. Neurochem. 50, 102(~1031. 24. Thoenen H. and Edgar D. (1985) Neurotrophic factors. Science 229, 239-242. 25. Uhl G. R., Kuhar M. J. and Snyder S. H. (1977) Neurotensin: immunohistochemical localization in rat central nervous system. Proc. natn. Acad. Sci. U.S.A. 74, 4059-4063. 26. Vanisberg M. A., Maloteaux J. M., Octave J. N., Bourguet C. and Laduron P. M. (1990) Ligand-induced internalization of neurotensin receptors in rat cultured neurons. Biochem. Pharmac. (in press). 27. Waksman G., Bouboutou R., Devin J., Bourgoin S., Cesselin F., Hamon M., Fourni6-Zaluski M. C. and Roques B. P. (1985) In vitro and in vivo effects of kelatorphan on enkephalin metabolism in rodent brain. Eur. J. Pharmac. 117, 233-243. 28. Waksman G., Hamel E., Delay-Goyet P. and Roques B. P. (1987) Neutral endopeptidase-24.11, ~ and 6 opioid receptors after selective brain lesions: an autoradiographic study. Brain Res. 436, 205-216. 29. Waksman G., Hamel E., Fourni6-Zaluski M. C. and Roques B. P. (1986) Autoradiographic comparison of the distribution of the neutral endopeptidase "enkephalinase" and of # and 6 opioid receptors in rat brain. Proc. natn. Acad. Sci. U.S.A. 83, 1523-1527. (Accepted 9 January 1990)
Neuroscience Vol. 36, No. 2, pp. 425430, 1990 Printed in Great Britain
IBRO
RETROGRADE A X O N A L TRANSPORT OF N E U R O T E N S I N IN THE DOPAMINERGIC NIGROSTRIATAL PATHWAY IN THE RAT M.-N. CASTEL,C. MALGOURIS,J.-C. BLANCHARD a n d P. M. LADURON Rh6ne-Poulenc Sant6, Centre de Recherches de Vitry-Alfortville, 13 Quai Jules Guesde, 94403 Vitry-sur-Seine Cedex, France Al~traet--Although the existence of receptor transport has been clearly demonstrated in peripheral nerves, there is no clear cut evidence in the brain of such a process for neuropeptide receptors. Because of the localization of neurotensin receptors on dopaminergic terminals, the dopaminergic nigrostriatal pathway appears to be the system of choice for studying the axonal transport of neuropeptide receptors in the brain. When labelled neurotensin was injected into the rat striatum, a delayed accumulation of radioactivity in the ipsilateral substantia nigra was observed about 2 h after injection. An essential requirement to clearly observe this phenomenon was the pretreatment of animals with kelatorphan in order to prevent the labelled neurotensin degradation. The appearance of this labelling was prevented by injection of an excess of unlabelled neurotensin or of neurotensin 8-13, an active neurotensin fragment, but not by neurotensin 1-8, which had no affinity for neurotensin receptors. This process was saturable, microtubule-dependent and occurred only in mesostriatal and nigrostriatal dopaminergic neurons as identified after 6-hydroxydopamine lesion and by autoradiography. These results demonstrate that neurotensin was retrogradely transported by a process involving neurotensin receptors. The retrograde transport of receptor-bound neuropeptide may represent an important dynamic process which conveys information molecules from the synapse towards the cell body.
N e u r o t e n s i n is a tridecapeptide ( H - p G l u - L e u - T y r Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH) which was isolated from bovine h y p o t h a l a m i , ~ sequenced 2 a n d synthetized 3 by C a r r a w a y a n d Leeman. M o r e recently the gene encoding for this peptide was isolated in the r a t ) ° I m m u n o h i s t o c h e m i c a l studies have s h o w n t h a t this n e u r o p e p t i d e is distributed widely a n d heterogeneously in the m a m m a l i a n central nervous system. 8'9'25 N e u r o t e n s i n receptors have been identified in the b r a i n o f n u m e r o u s species ~,2~ a n d have recently been purified. ~8'19In the rat striatum, n e u r o t e n s i n binding sites are associated with dopaminergic terminals. 7'2°'23 M o r e recently, the utilization o f a p o t e n t antihistaminic drug, levocabastine, has allowed the identification o f two types o f n e u r o t e n s i n binding sites in the rat striatum: the n e u r o t e n s i n - a c c e p t o r sites which are sensitive b o t h to neurotensin a n d levocabastine, a n d the n e u r o t e n s i n - r e c e p t o r sites which only recognize neurotensin. 22 M o r e detailed subcellular analysis has revealed t h a t only the neurotensin-receptor sites are presynaptically located on dopaminergic terminals in this structure, whereas the neurotensin-acceptor sites are associated with glial cells. 23 F o r the m o m e n t , there is no evidence t h a t neurotensin a n d d o p a m i n e co-exist in the dopaminergic nigrostriatal pathway. Fast axonal a n t e r o g r a d e t r a n s p o r t is required to supply nerve terminals with neurotransmitters, enzymes and secretory materials. 6 In peripheral nerves, ligature experiments have d e m o n s t r a t e d a fast and bidirectional t r a n s p o r t of muscarinic, 14 opiate ~5'~7and o t h e r presynaptic receptors (see Ref. 16 for review).
Moreover, [3H]lofentanil was used in vivo to d e m o n strate a retrograde t r a n s p o r t o f the r e c e p t o r - b o u n d opiate in ligated vagus nerves. 17 Thus, the aim o f this study was to determine if neurotensin was t r a n s p o r t e d in rat brain. EXPERIMENTAL PROCEDURES
Materials
Labelled neurotensin was purchased from Amersham and unlabelled peptides (neurotensin and the 1-8 neurotensin and 8-13 neurotensin fragments) from Sigma. Colchicine was obtained from Prolabo and 6-hydroxydopamine from Serva. Kelatorphan was a generous gift from Prof. B. P. Roques. Methods
Adult male Sprague-Dawley rats (n = 110, Charles River, France) weighing between 280 and 320 g were injected under pentobarbital anaesthesia with 2 #1 of kelatorphan (30/~g) or with saline in the right striatum at the rate of 0.2 ttl/min (coordinates AP 7.5 mm, L 3 mm, V 0 mm according to K6nig and KlippeP2). Ten minutes later, 0.16pmol of monoido-Tyr3 neurotensin (2000Ci/mmol), dissolved in saline, was infused in a total volume of 3 #1 into three different areas of the right striatum (coordinates AP 9.4 mm, L 2mm and V 0.2 mm; AP 6.6 mm, L 3.5mm and V - 0 . 4 mm; AP 4.6 mm, L 4.4mm and V - 0 . 6 mm). The coordinates were regularly verified by injection of a dye. Four hours later, animals were killed and their brains dissected out. The radioactivity in different brain areas (the ipsi- and contralateral substantia nigra, the cerebellum and the ipsilateral hippocampus) was measured in an LKB gamma counter. For autoradiographic studies, the rat brains were frozen rapidly in isopentane at -20°C. Coronal sections (20 ttm thick) were cut on a cryostat at - 1 5 ° C and mounted on lamellae. Finally, sections were apposed to 3H ultrofilm 425
426
M.-N. CASTELet al.
(LKB, France) for 3 weeks at -80°C inside X-ray cassettes. The films were developed in a Kodak D 19 developer for 5 min at 18°C, fixed, washed for 30 min and dried. For neurotoxic experiments, animals were pretreated either with colchicine or 6-hydroxydopamine. Colchicine (2001tg/4/~l of 0.1% NaC1) was injected into the right lateral ventricle (AP 0.6mm, L 1.3 mm, V - 5 m m with respect to Bregma) 30 h before kelatorphan administration. Nigrostriatal dopaminergic neurons were lesioned by unilaterally injecting 8/~g of 6-hydroxydopamine in 2/~1 of saline containing 0.01% L-ascorbic acid in the medial forebrain bundle at coordinates AP 4.2mm, L 1.4mm and V 2.3 mm according to K6nig and Klippel. 12 Animals were treated with 25mg/kg desmethylimipramine (i.p.) 45min prior to the 6-hydroxydopamine injections. The effectiveness of the lesion was examined by assessment of the contralateral circling response to apomorphine (0.63 mg/kg subcutaneously), 3 weeks later. Animals displaying contraversive rotations were retained for use in experiments. For in vivo displacement experiments, rats were coinjected with a 1000-fold excess (162pmol) of unlabelled neurotensin, or of neurotensin fragments 8-13 and 1-8, and with monoiodo-Tyr3 neurotensin in the right striatum. On the other hand, animals were injected with monoiodo-Tyr3 neurotensin alone (0.16 pmol) of in association with 3-, 10-, 30-, 100- and 1000-fold excesses of unlabelled neurotensin. For kinetic experiments, rats were injected with 0.16 pmol of monoiodo-Tyr3 neurotensin as previously described and were killed immediately or 1, 2, 3, 4 and 6 h after the injection. Radioactivity was measured in the right substantia nigra.
RESULTS
When animals were injected in the right striatum with monoiodo-Tyr3 neurotensin after kelatorphan pretreatment, a substantial accumulation of radioactivity (360 + 25 d.p.m./mg of tissue) could be observed in the ipsi- but not in the contralateral substantia nigra 4 h later (Fig. 1). However, the ipsilateral substantia nigra was not labelled in the absence of kelatorphan (Fig. 1). As shown in Fig. 1, a weak labelling was measured in the cerebellum ( 0 . 6 + 0 . 2 d . p . m . / m g of tissue) and in the right hippocampus ( 1 3 + 3 d . p . m . / m g of tissue). When
animals were pretreated with saline, no difference of labelling could be detected in these two structures (0.4 + 0.12 d.p.m./mg of tissue in the cerebellum and l l . 2 + 2 . 7 d . p . m . / m g of tissue in the ipsilateral hippocampus). A more precise location of the radioactivity in the mesencephalon was obtained by autoradiography (Fig. 2). The labelling was restricted to the ipsilateral substantia nigra pars compacta and the adjacent ventral tegmental area. This accumulation of radioactivity in the ipsilateral substantia nigra was prevented by a 1000-fold excess of unlabelled neurotensin or of the neurotensin fragment 8-13 (Table 1), but not by the neurotensin fragment 1-8 (Table 1). Labelling persisted in the cerebellum and in the right hippocampus (Table 1), whatever the treatment. The inhibition of radioactivity accumulating in the ipsilateral substantia nigra was dose-dependent (Fig. 3), as demonstrated by the co-injection into the right striatum of increasing doses of unlabelled neurotensin and monoiodo-Tyr3 neurotensin. The radioactivity started to accumulate in the substantia nigra after about 2 h (Fig. 4) and the highest levels were observed 3 or 4 h after the injection (Fig. 4). When rats were pretreated either with colchicine (Table 2) or 6-hydroxydopamine (Table 2), there was no labelling in the ipsilateral substantia nigra. On the contrary, the cerebellum and the hippocampus were still marked (Table 2).
DISCUSSION
The foregoing results suggest that labelled neurotensin injected into the right striatum moves retrogradely to the ipsilateral substantia nigra and the adjacent ventral tegmental area. The pretreatment of animals with kelatorphan was necessary to measure
400
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Fig. 1. Effect of kelatorphan on the labelling observed in different cerebral regions 4 h after monoiodoTyr3 neurotensin injection into the right striatum. Rats were pretreated with kelatorphan (30 #g, n = 10) or with saline (n = 5), injected into the right striatum 10min before the injection of 0.16pmol of monoiodo-Tyr3 neurotensin. Results are expressed in d.p.m./mg of tissue ___S.E.M.
Retrograde transport of neurotensin in rat brain
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428
M.-N. CASTELet al. Table 1. Rats were injected in the right striatum with 0.16 pmol of monoiodo-Tyr3 neurotensin alone or in association with an excess (162 pmol) of unlabelled neurotensin or neurotensin fragments (fragments 8-13 and 1-8) Experimental conditions
Ipsilateral substantia nigra Contralateral substantia nigra Cerebellum Ipsilateral hippocampus
[nSI]NT +
[nSI]NT +
[J~Sl]NT +
[t25I]NT n = 10
unlabelled neurotensin n =6
neurotensin fragment 8-13 n =5
neurotensin fragment 1-8 n=5
360 + 25
0
0
0 0.6 +__0.2
0 0.6 __+0.05
0 0.3 ___0.07
13 ___3
15 + 5
357 + 78 0 0.4 ___0.07
16 ___2
14 + 5
Labelling was measured in various cerebral structures 4 h after the injections. Results are expressed in d.p.m./mg of tissue + S.E.M. []25I]NT, monoiodo-Tyr neurotensin. a detectable signal in the ipsilateral substantia nigra. Indeed, kelatorphan is known to inhibit the neutral endopeptidase 24 11, 27 a peptidase present in large quantities in the rat caudate-putamen. 28'29 This enzyme induces the cleavage of the P r o l 0 - T y r l l and T y r l l - I l e l 2 bonds 4 and effectively inactivates neurotensin, since the degradation products are devoid of biological activity: This process appears to be receptor-dependent, since an excess of neurotensin and the fragment 8-13, which have a high affinity for neurotensin receptors: prevented the accumulation of radioactivity in the ipsilateral substantia nigra. On the contrary, the neurotensin fragment 1-8, which is inactive in binding studies, 5 was unable to prevent the accumulation of radioactivity in this structure. Moreover, the appearance of labelling in the substantia nigra was totally inhibited by a very low dose of unlabelled neurotensin (162 pmol); such a dosedependent and saturable process is compatible with the Kd values obtained in binding studies on neuro-
tensin receptors. 22 In contrast, uptake of extracellular proteins, especially horseradish peroxidase, and the subsequent axonal transport, is not saturable and not specific for a given type of neuron because these macromolecules are not internalized by nerve terminals through a ligand-receptor process. Labelling persisted in the cerebellum and in the hippocampus even when an excess of unlabelled neurotensin or the fragment 8-13 was injected, indicating that in these structures the accumulation of radioactivity was unrelated to neurotensin receptors. All these data support the idea that monoiodoTyr3 neurotensin was transported from the striatum towards the ipsilateral substantia nigra through transport mechanisms involving neurotensin receptors. This transport occurred in dopaminergic neurons as shown by the complete absence of labelling in the substantia nigra after 6-hydroxydopamine pretreatment. This result was confirmed by autoradiography, since the labelling was restricted to the ipsilateral substantia nigra pars compacta and the 60C
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Fig. 3. Labelling measured in the ipsilateral substantia nigra 4 h after the injection of monoiodo-Tyr3 neurotensin alone (0.16pmol, n =7) or in combination with 3 (n =5), 10 (n=6), 30 (n=5), 100 ( n = 6 ) and 1000 ( n = 6 ) fold excesses of unlabelled neurotensin. The results are expressed in terms of percentage radioactivity observed in the absence of unlabelled neurotensin.
,__/I 0
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3 4 Time (hr$)
5
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Fig. 4. Time-course of labelling measured in the substantia nigra after injection of monoiodo-Tyr3 neurotensin into the rat striatum. Rats were injected with 0.16pmol of monoiodo-Tyr3 neurotensin and were killed immediately ( n = 4 ) or 1 (n=7), 1.5 (n=6), 2 (n=6), 3 (n=6), 4 (n = 10) and 6 (n = 6) h later. Results are expressed as mean d.p.m. + S.E.M.
Retrograde transport of neurotensin in rat brain Table 2. Effect of neurotoxic treatment on the labelling observed in the ipsilateral substantia nigra after the injection of 0.16pmol of monoiodo-Tyr3 neurotensin in the right striatum
[125I]NT n = 10 Ipsilateral substantia nigra Contralateral substantia nigra Cerebellum Ipsilateral hippocampus
Experimental conditions [125I]NT [12~I]NT + + 6-OH-dopamine colchicine pretreatment pretreatment n =5 n =5
360 + 25
0
0
0 0.6 + 0.2
0 0.2 + 0.02
0 0.8 + 0.2
13 + 3
5+ 2
15 + 5
One group of animals received 8 pg of 6-hydroxydopamine (6-OH-dopamine) in the right medial forebrain bundle after a pretreatment with desmethylimipramine (25 mg/kg, i.p.). Three weeks later, the well-lesioned animals were injected with monoiodo-Tyr3 neurotensin. Another group of rats was pretreated with colchicine (200,ug, i.c.v.) 30 h before the monoiodo-Tyr3 neurotensin injection..Results are expressed in d.p.m./mg of tissue _+ S.E.M. [125I]NT, monoiodo-Tyr3 neurotensin.
adjacent ventral tegmental area, both regions containing the cell bodies of the ascending dopaminergic projections to the striatum. The delayed appearance of labelling in the substantia nigra is compatible with a fast rate of axonal transport (1-5 mm/h). 1~ This transport was microtubule-dependent, as indicated by the prevention of accumulation of radioactivity in the nigra after the colchicine pretreatment) 3 On the contrary, the early appearance of radioactivity in the cerebellum and in the ipsilateral hippocampus (in less than 1 h) and the persistent labelling measured in these structures even
429
in the presence of colchicine indicates the existence of a diffusion phenomenon in these cerebral areas. It appears that the retrograde axonal transport of neurotensin first involves the binding of the labelled neuropeptide to specific receptors presynaptically located on the dopaminergic terminals in the striatum, and then the internalization of the ligand-receptor complex. This specific endocytosis has been reported in vivo for opiate agonists in peripheral nerves ~7 and more recently in vitro for neurotensin receptors in cultured cortical neurons} 6 As to the fate of receptor-bound neurotensin in the neuron, there are a number of possibilities, as recently suggested: ~6first, the complex could be degraded after fusion with lysosomes in the axon or in the cell body; second, it could be reused or recycled through the Golgi apparatus; third, such a complex may serve either directly or indirectly to induce changes in gene expression. This latter hypothesis has already been reported for growth factors} 4
CONCLUSION The results presented here provide the first evidence for the existence of an axonal retrograde transport of neuropeptide-bound receptors in the brain and could represent a major dynamic mechanism for conveying information molecules from the synapse to the cell body. This phenomenon could account for the long-term effects of neuropeptides and might be involved in neurogenesis, neural plasticity and perhaps in long-term m e m o r y ) 6 Acknowledgements--We are grateful to Prof. Bernard P.
Roques for fruitful discussions and for the gift of kelatorphan. The skilful technical assistance of Marc Daniel was highly appreciated. We wish to thank Jeremy Pratt and Karen Birmingham for their help in preparing the manuscript.
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