Characterization of interactions between neurotensin and dopamine systems in the rat brain

To study the functional interactions between neurotensin (NT) and dopamine (DA) systems in the rat brain, the effects of altering dopaminergic activity on the levels of NT-like immunoreactivity (NTLI) in the neostriatum, the nucleus accumbens and the substantia nigra were measured. Dopamine Di receptors were found to help regulate NT systems as methamphetamine (METH)-induced elevations in NTLI contents of these structures were completely blocked by the Di antagonist, SCH 23390. Administration of receptor-selective, direct-acting agonists revealed that Di and D2 receptors have opposite effects on NT systems in both the striatum and the nucleus accumbens. In contrast, stimulation of Di activity by itself did not affect the nigral NT systems, but after activation of D2 receptors a significant increase in the nigral level of NTLI was observed; surprisingly, stimulation of D2 receptors alone reduced the nigral NTLI content. These data suggest that although selective activation of D2 receptors opposes the effect of the Di subtype on nigral NT systems, in combination with Di stimulation, they are facilitatory to nigral Di activity. Elimination of greater than 85% of the central dopaminergic activity completely blocked the elevation in NTLI content caused by METH. For these experiments, depletion of dopamine was achieved by either (i) nigral administration of the dopaminergic neurotoxin, 6-hydroxydopamine, 7 to 10 days before administrations of METH or (ii) multiple doses of reserpine and a-methyl-p-tyrosine. Interestingly, these DA-depleting treatments by themselves induced significant increases in the NTLI content of the striatum and the nucleus accumbens. Although the nigral NTLI content was unaffected by the long-lasting treatments, a single dose of reserpine caused a significant decrease in nigral NTLI content by 18 h after the treatment. These results suggest that NT systems examined are tonically regulated by basal dopaminergic activity, although the nigral response appears to be short-lived. Administration of selective Di or D2 agonists with reserpine showed that tonic dopaminergic regulation of NT systems is mediated primarily by D2 receptors in the striatum and the nucleus accumbens but by Di receptors in the substantia nigra. This was further supported by the observations that interruption of dopaminergic transmission by D2 antagonists (e.g., sulpiride, SULP) increased NTLI levels only in the striatum and the nucleus accumbens whereas the Di antagonist decreased the nigral NTLI content. Finally, another transmitter system(s) appears to be involved in dopaminergic regulation of extrapyramidal NT systems, as destruction of striatal interneurons and/or striatal efferents with the excitotoxin, ibotenic acid, or a knife cut, blocked the METH- and SULP-induced alterations in NT activity of these structures. This linking transmitter system does not appear to be cholinergic since the muscarinic antagonist, atropine, did not attenuate the METH-induced increase in the striatum. However, other studies have shown that glutamatergic projections mediate the effects of METH on NT systems as concurrent blockade of N-methyl-D-aspartate receptors with MK 801 blocked the increases in NTLI contents caused by METH. The SULP-induced alterations, on the other hand, were not blocked by MK 801 suggesting that glutamatergic pathways may not be involved in basal D2 receptor-mediated regulation of NT systems.

CHARACfERIZATION OF INTERACITONS BE1WEEN NEUROTENSIN AND DOPAMINE SYSTEMS IN THE RAT BRAIN by Kalpana Mahesh Merchant A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Phannacology Department of Phannacology and Toxicology University of Utah August 1989 THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUI~~~I~VISOI~Y COl\ll\JI'l"rT'EE API)I~OV AL of a dissertation submitted by Kalpana Mahesh Merchant This dissertation has been read by each member of the followiIlg supervisory cOlllmittee and by mZljority vote has been found to be satisfactory. Chair: Dr. Donald N. Franz Dr. Stephen M. Prescott THE U'iIVERSITY OF UTAH GRADUATE SCHOOL I;INAL I{l~AI)ING APPI{(),lj\L, To the Graduate Council of The Ulli\'Crsity of Utah: I have read the dissertation or ......................., .... Mahesh Merchant its final forlll and have foulld that ( I) it s forlllat. citatiolls. alld hibliog ra ph ic st yle are cOllsistent and acceptable; (2) its illllstrati\'c materials including figures, tables. alld charts are ill place; and (3) the fillal manuscript is satisi'anol-Y to Ihe Supervis­ory Committee alld is ready ror suhmissioll to the Craduate School. Dale Approved lor the 1\1;~jor lkp;\IIIl\('llt Dr. B. Gale Dick I k;1I1 01 I 11l' (;1 ;1(11];111' s( 1.11111 Copyright © Kalpana Mahesh Merchant 1989 All Rights Reserved ABSTRACT To study the functional interactions between neurotensin (NT) and dopamine (DA) systems in the rat brain, the effects of altering dopaminergic activity on the levels of NT­like immunoreactivity (NTLI) in the neostriatum, the nucleus accumbens and the substantia nigra were measured. Dopamine DI receptors were found to help regulate NT systems as methamphetamine (METH)-induced elevations in NTLI contents of these structures were completely blocked by the Dl antagonist, SCH 23390. Administration of receptor-selective, direct-acting agonists revealed that Dl and D2 receptors have opposite effects on NT systems in both the striatum and the nucleus accumbens. In contrast, stimulation ofDl activity by itself did not affect the nigra! NT systems, but after activation of D2 receptors a significant increase in the nigral level of NTLI was observed; surprisingly, stimulation of D2 receptors alone reduced the nigral NTLI content. These data suggest that although selective activation of D2 receptors opposes the effect of the Dl subtype on nigral NT systems, in combination with Dl stimulation, they are facilitatory to nigra! Dl activity. Elimination of greater than 85% of the central dopaminergic activity completely blocked the elevation in NlLI content caused by METH. For these experiments, depletion of dopamine was achieved by either (i) nigral administration of the dopaminergic neurotoxin, 6-hydroxydopamine, 7 to 10 days before administrations of METII or (ii) multiple doses of reserpine and a-methyl-p-tyrosine. Interestingly, these DA-depleting treatments by themselves induced significant increases in the N1LI content of the striatum and the nucleus accumbens. Although the nigra! NTLI content was unaffected by the long­lasting treatments, a single dose of reserpine caused a significant decrease in nigral N1LI content by 18 h after the treatment. These results suggest that NT systems examined are tonically regulated by basal dopaminergic activity, although the nigral response appears to be short-lived. Administration of selective Dl or D2 agonists with reserpine showed that tonic dopaminergic regulation of NT systems is mediated primarily by D2 receptors in the striatum and the nucleus accumbens but by D 1 receptors in the substantia nigra. This was further supported by the observations that interruption of dopaminergic transmission by D2 antagonists (e.g., sulpiride, SULP) increased N1LI levels only in the striatum and the nucleus accumbens whereas the D 1 antagonist decreased the nigral NTLI content. Finally, another transmitter system(s) appears to be involved in dopaminergic regulation of extrapyramidal NT systems, as destruction of striatal intemeurons and/or striatal efferents with the excitotoxin, ibotenic acid, or a knife cut, blocked the METH- and SULP-induced alterations in NT activity of these structures. This linking transmitter system does not appear to be cholinergic since the muscarinic antagonist, atropine, did not attenuate the METH-induced increase in the striatum. However, other studies have shown that glutamatergic projections mediate the effects of MElli on NT systems as concurrent blockade of N-methyl-:D-aspartate receptors with MK 801 blocked the increases in N1LI contents caused by METH. The SULP-induced alterations, on the other hand, were not blocked by MK 801 suggesting that glutamatergic pathways may not be involved in basal D2 receptor-mediated regulation of NT systems. v To Mahesh -for making my dream come true TABLE OF CONTENTS ABSTRACT ........................................................................................ iv LIST OF FIGURES ............................................................................. viii ACKNOWLEDGMENTS ........................................................................ ix Chapter 1. INTRODUCTORY REMARKS ............................................................... 1 Neurotensin as a Central Neurotransmitter ................................................... l Interactions with Central Dopaminergic Systems ........................................... 2 References ....................................................................................... 5 2. IDENTIFICATION OF CENTRAL TRANSMITTER SYSTEMS INVOLVEDIN REGULATION OF NEUROTENSIN PATHWA yS .......................................................... 8 Introduction ...................................................................................... 8 Materials and Methods ........................................................................ 10 Results .......................................................................................... 15 Discussion ...................................................................................... 23 References ...................................................................................... 32 3. ROLE OF Dl RECEPTORS IN REGULATION OF NEUROTENSIN SySTEMS ................................................................ 35 Introduction .................................................................................... 35 Materials and Methods ........................................................................ 37 Results .......................................................................................... 38 Discussion ...................................................................................... 43 References ...................................................................................... 5 0 4. ROLE OF D2 RECEPTORS IN REGULATION OF NEUROTENSIN SySTEMS ................................................................ 52 Introduction .................................................................................... 52 Materials and Methods ........................................................................ 53 Results .......................................................................................... 54 Discussion ...................................................................................... 58 References ...................................................................................... 64 CURRICULUM VITAE ......................................................................... 66 LIST OF FIGURES Figure Page 1.1 Effects of METH and/or SULP on tissue NTLI content. .............................. .4 2.1 Effects of lesioning the nigrostriatal DA pathway on striatal and nigral NTLI content. .......................................................... 17 2.2 Effects of DA depletion induced by reserpine plus aMpT on tissue NTLI content .................................................................... 19 2.3 Effects of lesioning the striatal-nigral projections on NTLI content of the striatum and the substantia nigra ......................................... 21 2.4 Effect of concurrent blockade of muscarinic receptors on METH-induced increase in NTLI content of the striatum ............................. 23 2.5 Effect of concurrent blockade of NMDA receptors on SULP-induced increase in striatal NTLI content. ...................................... 24 3.1 Effect of concurrent blockade of DI receptors on MElli-induced alterations in tissue NTLI content. ............................................. 39 3.2 Effect of concurrent blockade of D 1 receptors on SULP-induced alterations in tissue NTLI content. ............................................. 40 3.3 Effect of selective DA receptor agonists on tissue NTLI content ........................................................................ 42 3.4 Effect of Dl receptor blockade on tissue NTLI content. ............................. .43 3.5 Correlation between striatal DA depletion and alterations in nigral NTLI content following treatment with reserpine ...................................... 44 3.6 Effect of selective activation ofDI receptors on reserpine-induced decrease in NTl .. .! content of the substantia nigra ...................................... .45 4.1 Recovery correlation between DA depletion induced by reserpine and NTLI content of the striatum and the nucleus accumbens ........................ 56 4.2 Effect of selective activation of DA receptors on reserpine-induced alterations in tissue NTLI content. ....................................................... 57 4.3 Effect of DA-Dl receptor blockade on reserpine- or SULP-induced increases in NTLI levels os the striatum and the nucleus accun1bens ................ 59 ACKNOWLEDGMENTS I wish to express my sincere gratitude to Dr. Glen R. Hanson for his confidence in my abilities, constant encouragement and guidance. His invaluable contribution to all aspects of my graduate training will long be rembered. Warmest thanks also to Dr. James W. Gibb for his inspiration and kindness. Special thanks to the other members of my supervisory committee- Dr. Donald N. Franz, Dr. Stephen M. Prescott and Dr. Thomas M. McIntyre for their guidance and critical reading of the manuscript. I also wish to thank Dr. Michel Johnson and Lloyd Bush for their assistance in my experimental work. Finally, I wish to thank Mahesh for his patience, unwavering support, love and encouragement which allowed my graduate education to become a reality. I am also indebted to our parents and friends, especially Lalit and Bobbi, for their understanding and encouragement. Financial support was provided by Fellowships from Osco Skaggs and the University of Utah. Portions of the dissertation are adapted from the following sources with permission from Elsevier Science Publishers: Letter et aI., Brain Research, 422 (1987) 200-203; Merchant et aI., Eur. J. PharmacoI., 153 (1988) 1-9; Merchant et aI., Eur. J. Pharmacol., 160 (1989) 409-412 and Merchant et aI., Brain Res., in press. CHAPTER 1 INTRODUCTORY REMARKS Neurotensin As A Central Neurotransmitter Neurotensin (NT) is an endogenous tridecapeptide that satisfies some of the criteria for a central neurotransmitter. It was first isolated from bovine hypothalamus by Carraway and Leeman (1973) and later characterized by the same investigators (Carraway and Leeman, 1975, 1976). Subsequently, it was shown to have a characteristic heterogenous distribution in the central nervous system of rodents, subhuman primates and humans, by both radioimmunoassay (Kobayashi et aI., 1977; Kataoka, et aI., 1979; Emson et aI., 1982; Manberg et aI., 1982) and immunohistochemistry (Uhl et aI., 1979; Jennes et aI., 1982). Furthermore, it is selectively localized in synaptosomal fractions (Uhl and Snyder, 1976) and degraded by endogenous peptidases (Dupont and Merand, 1978; Checler et al., 1983). During depolarization NT is released from brain slices in a calcium-dependent manner (Iversen et aI., 1978). In addition, NT binding sites are present on synaptic membranes and show high affinity, saturability and specificity for the peptide (Uhl et aI., 1977; Kitabgi et aI., 1977, Young and Kuhar, 1981, Quirion et aI., 1982). Pharmacological application of this peptide in the central nervous system of experimental animals causes a variety of neuronal effects such as hypothermia (Bissette et al., 1976), hypotension (Rioux et aI., 1981), enhancement of the depressant effects of pentobarbital (Nemeroff et eI., 1977), antinociception (Clineschmidt and McGuffin, 1977; Kalivas et aI., 1982) and decreased locomotor activity (Nemeroff et aI., 1977). 2 Because the exact physiological role of this peptide in the C~S is not well defined it is sometimes referred to as a neuromodulator. Interactions With Central Dopaminerric Systems A number of recent studies have demonstrated that NT influences dopamine (DA) activity in various brain structures. For example: (1) NT is colocalized with DA in the catecholaminergic cell bodies of the ventral tegmental area, the substantia nigra and the hypothalamus (Hokfelt et al., 1984), (2) NT receptors are highly concentrated in areas rich in dopaminergic perikarya (Young and Kuhar, 1981), with a large fraction located on DA neurons (Palacios and Kuhar, 1981), (3) DA neurons in the substantia nigra are depolarized by NT (Pinnock, 1985), (4) in the neostriatum as well as nucleus accumbens, both basal and K + - induced release of DA are enhanced by NT (Battaini et al., 1986; Okuma et al., 1983), (5) DA synthesis and turnover rates are increased in several DA terminal areas following i.c.v. injections of NT (Widerlov et al., 1982) and (6) NT and neuroleptics have overlapping pharmacological profiles (Nemeroff, 1980). In view of such anatomical, physiological and biochemical relationships between these two transmitters, it is likely that reciprocal regulation by doparninergic systems also affects the activity of NT systems. This hypothesis is supported by several recent research reports. Thus, NT binding sites are upregulated following administration of neuroleptics to rats and humans (Uhl and Kuhar, 1984; Herve et al., 1986). In addition, destruction of dopaminergic pathways by 6-hydroxydopamine alters NT receptor density in a site-specific manner (Herve et al., 1986). Furthermore, treatment of rats with classical (e.g., haloperidol) or nonclassical (e.g., sulpiride, SULP) neuroleptics selectively elevates the content of neurotensin-like immunoreactivity (NTLI) in the striatum and the nucleus accumbens (Govoni et al., 1980; Frey et al., 1986; Letter et al., 1987a,b; Merchant et aI., 1988). Surprisingly, like the neuroleptics, the indirectly acting DA agonist, methamphetamine (MElli), also causes a several-fold increase in the NTLI 3 content of the striatum and the nucleus accumbens, while combined administration of these antagonistic drugs (Le., METII+SULP or METI-I+haloperidol) result in an additive response by the NT systems in both structures (Letter et aI., 1987a, Merchant et ai., 1988). In contrast, the nigral NT systems do not respond to the neuroleptics but are substantially altered (increases in NTLI content to 200-300%" of control) following METH: this effect is blocked by coadministration of haloperidol but not SULP. Data showing METH- and/or SULP-induced alterations in NTLI concentrations are summarized in Figure 1.1. These findings suggest that NT pathways in the striatum, the nucleus accumbens and the substantia nigra regulate and are regulated by associated dopaminergic systems. It is important to understand the functional interactions between central NT and DA pathways especially in view of the putative roles of DA in the etiology of several neurological and psychiatric diseases such as Parkinson's disease and schizophrenia. This thesis attempts to identify the mechanisms underlying the interactions between NT and DA transmitter systems in the striatum, the nucleus accumbens and the substantia nigra. The manuscript is divided into four chapters: following the Introduction, in Chapter 2 the roles of specific transmitter systems in METH and neuroleptic-induced changes in discrete NT systems are examined and in Chapters 3 and 4 the role of dopaminergic D 1 and D2 receptors, respectively, in mediating the regulation of NT pathways is addressed. --e ...... c e ..... Q ~ /:)I,) S c .~. ~ .-Q. . ~ ~ 600 500 400 300 200 100 0 400 300 200 100 0 400 300 200 100 0 striatum *** 11 nucleus accumbens *** 1 treatment D control II SULP ~ MElli II SULP+METII Figure 1.1: Effects of METII andlor SULP on tissue NTLI content. Rats were given five doses of METII (10 mg/kg/dose) or SULP (80 mg/kg/dose) or a combination thereof and were sacrificed 18 h after the last treatment. Each column represents the mean±S.E.M. for the treatment group expressed as percentage of the respective controls (n=6 to 10). The control NTLI levels expressed as pg/mg protein were: striatum=108±20; nucleus accumbens= 326±45 and substantia nigra=516±25. *P<0.02, **P<O.Ol and ***P<O.OOl compared to corresponding controls. tP<O.Ol, ttP<O.OOl compared to the corresponding SULP-treated group. 4 5 References Battaini F., S. Govoni, S. Di Giovine and M. Trabucchi, 1986, Neurotensin effect on dopamine release and calcium transport in rat striatum: interactions with diphenyl­alkylamine calcium antagonists, Naunyn-Schmiedberg's Arch. Phannacol. 332, 267. Bissette, G., C.B. Nemeroff, P.T. Loosen, A.J. Prange, Jr. and M.A. Lipton, 1976, Hypothermia and intolerance to cold induced by intracistern~ administration of the hypothalamic peptide, neurotensin, Nature (Lond.) 262, 607. Carraway, R. and S.E. Leeman, 1973, The isolation of a new hypothalamic peptide, neurotensin, from bovine hypothalami, 1. BioI. Chern. 248, 6854. Carraway, R. and S.E. Leeman, 1975, The amino acid sequence of a hypothalamic peptide, neurotensin, 1. BioI. Chern. 250, 1904. Carraway, R. and S.E. Leeman, 1976, Characterization of radioimmunoassay neurotensin in the rat Its differential distribution in the central nervous system, small intestine and stomach, J. BioI. Chern. 251, 7045. Checler, F., lP. Vincent, and P. Kitabgi, 1983, Degradation of neurotensin by rat brain synaptic membranes: Involvement of a thennolysin-like metalloendopeptidase (enkephalinase), angiotensin converting enzyme and other unidentified peptidases, J. Neurochem. 41, 375. Cline schmidt, B.V. and lC. Mc Guffin, 1977, Neurotensin administered intracisternally inhibits responsiveness of mice to noxious stimuli, Eur. J. Phannacol. 46, 395. Dupont, A. and Y. Merand, 1978, Enzymatic inactivation of neurotensin by thalamic and brain extracts of the rat, Life Sci. 22, 1623 Emson, P.C., M. Goedert, P. Horsfield, F. Rioux and S. St. Pierre, 1982, The regional distribution and chromatographic characterization ofneurotensin-like immunoreactivity in the rat central nervous system, J. Neurochem. 38, 1777. Frey, P., K. Fuxe, P. Eneroth and L.F. Agnati, 1986, Effects of acute and long-term treatment with neuroleptics on regional telencephalic neurotensin levels in the male rat, Neurochem. Int. 8, 429. Govoni, S., J.S. Hong, H.Y.-T. Yang and E. Costa, 1980,. Increase of neurotensin content elicited by neuroleptics in nucleus accumbens, J. Pharmacol. Exp. Ther. 215, 413. Herve, D., J.P. Tassin, J.M. Studler, C. Dana, J.P. Vincent, J. Glowinski and W. Rostene, 1986, Dopaminergic control of 1251-1abeled neurotensin binding site density in corticolimbic structures of the rat brain, Proc. Nat!. Acad. Sci., USA 83, 6203. Hokfelt, T., BJ. Everitt, E. Theodorsson-Norheim and M. Goldstein, 1984, Occurrence of neurotensinlike immunoreactivity in subpopulations of hypothalamic, mesencephalic and medullary catecholamine neurons, 1. Compo Neurol. 222, 543. 6 Iversen, L.L., S.D. Iversen, F.E. Bloom, C. Douglas, M. Brown, and W.Vale, 1978, Calcium-dependent release of somatostatin and neurotensin in rat brain in vitro, Nature (Lond.) 273, 161. Jennes, L., W. Stumph and P.W. Kalivas, 1982, Neurotensin: Topographical distribution in rat brain by immunohistochemistry, 1. Compo Neurol. 210,213. Kalivas, P.W., B. Gau, C.B. Nemeroff and A.1. Prange, Jr., 1982, Antinociception after microinjection of neurotensin in the central amygdaloid nucleus of the rat, Brain Res., 243,279. Kataoka, K., N. Mizuno and L.A. Frohman, 1979, Regional distribution of neurotensin in monkey brain, Brain Res. Bull. 4, 57. Kitabgi, P., R. Carraway, J.V. Rietschoten, C. Graniter, J.L. Morgat, A. Menez, S. Leeman and P. Freychat, 1977, Neurotensin: Specific binding ·of synaptic membranes from the rat brain, Proc. Nat!. Acad. Sci., USA 74, 1846. Kobayashi, K., M. Brown, and W. Vale, 1977, Regional distribution of neurotensin and somatostatin in the rat brain, Brain Res. 126, 584. Letter, A.A., L.A. Matsuda, K.M. Merchant, 1.W. Gibb and G.R. Hanson, 1987a, Characterization of dopaminergic influence on striato-nigral neurotensin systems, Brain Res. 422, 200. Letter, A.A., K. Merchant, J.W. Gibb and G.R. Hanson, 1987b, Effect of methamphetamine on neurotensin concentrations in rat brain regions, J. Pharmacol. Exp. Ther. 241, 443. Man berg , P.J., W.W. Youngblood, C.B. Nemeroff, M.N. Rossor, L.L. Iversen. A.1. Prange, Jr. and J.S. Kizer, 1982, Regional distribution of neurotensin in human brain, J. Neurochem. 38, 1777. Merchant, K.M., A.A. Letter, J.W. Gibb, and G.R. Hanson, 1988, Changes in the limbic neurotensin systems induced by dopaminergic drugs, Eur. 1. Phannacol. 153, 1. Nemeroff, C.D., 1980, Neurotensin: Perchance an endogenous neuroleptic ?, BioI. Psychiatry 15, 283. . Nemeroff, C.B., G. Bissette, A.J. Prange, Jr., P.T. Loosen, T.S. Barlow and M.A. Lipton, 1977, Neurotensin: Central nervous system effects of a hypothalamic peptide, Brain Res. 128, 485. Okuma, Y., Fukuda, Y. and Osumi, Y., 1983, Neurotensin potentiates the potassium­induced release of endogenous dopamine from rat striatal slices, Eur. J. Pharmacol. 93, 27. Palacios, 1.M. and M.J. Kuhar, 1981, Neurotensin receptors are located on dopamine­containing neurons in rat midbrain, Nature (Lond.) 294, 587. Pinnock, R.D., 1985, Neurotensin depolarizes substantia nigra dopamine neurons, Brain Res. 338, 151. 7 Quirion, R., P. Gaudreau, S. ST. Pierre, F. Rioux and C.B. Pert, 1982, Autoradiographic distribution of [3H]neurotensin receptors in rat brain: Visualization by tritium-sensitive film, Peptides 3, 757. Rioux, F., R. Quirion, S. ST. Pierre, D. Regoli, F. Jolicoeur, F. Belanger and A. Barbeau, 1981, The hypotensive effect of centrally administered neurotensin in rats, Eur. J. Pharmacol. 69, 241. Uhl, G.R., J.P. Bennett Jr. and S.H. Snyder, 1977, Neurotensin, a central nervous system peptide: Apparent receptor binding in brain membranes, Brain Res. 130, 299. Uhl, G.R., R.R. Goodman and S. H. Snyder, 1979, Neurotensin-containing cell bodies, fibers and tenninals in the brain stem of the rat: Immunohistochemical mapping, Brain Res. 167, 77. Uhl, G.R. and MJ. Kuhar, 1984, Chronic neuroleptic treatment enhances neurotensin receptor binding in human and rat substantia nigra, Nature 309, 350. Uhl G.R. and S.H. Snyder, 1976, Regional and subcellular distributions of brain neurotensin, Life Sci. 19, 1827. Widerlov, E., C.D. Kilts, R.B. Mailman, C.D. Nemeroff, TJ .. McCown, AJ. Prange, Jr., and G.R. Breese, 1982, Increase in dopamine metabolites in rat brain by neurotensin, J. Pharmacol. Exp. Ther. 222, 1. Young, W.S. III and M.J. Kuhar, 1981, Neurotensin receptor localization by light microscopic autoradiography in rat brain, Brain Res. 206, 273. CHAPTER 2 IDENTIFICATION OF CENTRAL TRANSMITIER SYSTEMS INVOLVED IN REGULATION OF NEUROTENSIN PA THW A YS Introduction Role of dopaminergic pathways Systemic administration of METH substantially increases dopaminergic transmission by causing massive release of endogenous DA (Schmidt et al., 1987). Because of the close relationship between NT and DA systems, as discussed in Chapter 1, it is likely that the METH-induced changes in NT pathways (Figure 1.1; Letter et al., 1987a; Merchant et al., 1988) are mediated by specific action of the endogenously released DA on its receptor SUbtypes. However, METH is also a potent releaser of serotonin and norepinephrine in the CNS (Schmidt et al., 1987; Daly et al., 1966). Consequently, METH-induced changes in NT systems may also be influenced by activation of these other monoaminergic projections. The neuroleptics, SULP and haloperidol, antagonize dopaminergic transmission by blocking primarily dopaminergic D2 receptors. In addition, by virtue of such an activity, this class of drugs also induces DA release either by blocking the inhibitory autoreceptors on DA terminals or the postsynaptic receptors on regulatory feedback loops (Starke et aI., 1978; Scatton, 1981; Lazareno et al., 1985). Hence, it is possible that like METH, the neuroleptics affect NT systems by altering dopaminergic activity. However, these antagonists do not show absolute specificity for DA receptors: SULP inhibits serotonin 5HT-2 and muscarinic receptors (Seeman, 1981) and haloperidol has some affinity for cholinergic and sigma receptors. Because of the nondopaminergic effects of METH and 9 the neuroleptics, it is important to elucidate the role of DA in the effects of these drugs on NT systems. In order to achieve this objective, central dopaminergic activity was eliminated prior to the administration of METH or SULP. The nigrostriatal DA system is the most likely pathway regulating the activity of extrapyramidal. NT projections; hence, this pathway was selectively lesioned by unilateral intranigral administration of the neurotoxin, 6-hydroxydopamine (6-0HDA), according to the methext of Un gerst edt and Arbuthnott (1970). The effects of these lesions on METH-, SULP-, or haloperidol­induced alterations in striatal and nigral NTLI content were measured. The nucleus accumbens, on the other hand, receives the majority of its dopaminergic projections from the ventral tegmental area and only a minor component from the substantia nigra-zona compacta (Anden et al., 1966, Ungerstedt, 1971 ). Consequently, nigral administration of 6-0HDA is not likely to cause substantial destruction of DA afferents in the nucleus accumbens. In order to examine the role of DA in the regulation of NT activity in the nucleus accumbens and also to confirm the 6-0HDA-induced changes in the striatum and the substantia nigra, DA was .depleted by systemic administration of reserpine (this drug prevents vesicular storage of DA) plus a-methyl-p­tyrosine (aMpT; this drug inhibits tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of DA). It was expected that elimination of central dopaminergic activity would block the METH- and the neuroleptic-induced alterations in the NT systems and thereby implicate the role of DA in these drug-induced changes. The effects of restoring synthesis of DA by administering its precursor, L-dihydroxyphenylalanine (L-DOPA), along with the peripheral DOPA decarboxylase inhibitor (R04-4602), in animals treated with reserpine plus aMpT were also studied in order to determine if such a strategy would restore the NT responses to METH and the neuroleptics. 10 Role of striatal interneLUons and efferents Following confmnation of the role of DA in the regulation of the associated NT systems, it was important to determine if alterations in dopaminergic activity directly affected the NT pathways or whether other transmitter systems were involved in mediating the DA-induced changes in these pepetide systems. For example, striatal cholinergic intemeurons are thought to mediate many of the postsynaptic effects of DA (Bartholini et al., 1973; Consolo et aI., 1974, 1975, 1987). Similarly, the corticostriatal glutamatergic projections modulate not only the release of DA in the striatum but also postsynaptic events of dopaminergic transmission (Cheremy et at, 1986». In addition, several striatal efferents projecting into the substantia nigra regulate the activity of nigraI neurons, including the nigrostriatal DA pathway (Mc Geer and Mc Geer, 1976). Studies were carried out to examine the possibility that striatal intemeurons or efferents contributed to the DA-mediated changes in striatal and nigral NT systems. Four distinct strategies were employed to achieve this objective: (1) the axon-sparing neurotoxin, ibotenic acid, was administered into the striatum to destroy striatal intemeurons and efferents prior to the administration of METH or SULP, (2) a knife cut was made in the striatum to sever all striatonigral projections (including ibotenate-insensitive neurons) prior to treatments with MElli, (3) the muscarinic antagonist, atropine, was administered with METH to test if a cholinergic link existed between DA and NT systems in the striatum and (4) MK 801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA)-type of glutamate receptors, was coadministered with SULP to see if glutamatergic projections contributed to the neuroleptic-induced alteration in NTLI contents. Materials and Methods Drugs (+)-Methamphetamine hydrochloride (National Institute on Drug Abuse, Rockville, MD) and MK 801 (Merck Sharp and Dohme Research Lab, Rahway, NJ) were kindly 11 supplied by the indicated sources. All other drugs used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The vehicles used for dissolving each drug were as follows: (±)sulpiride in 2% lactate+25% propylene glycol-saline; haloperidol in 1 % lactate-saline; reserpine in 1 % citrate+20% PEG 4OO-saline; 6-hydroxydopamine in 0.1 % ascorbate-saline; a-methyl-p-tyrosine, atropine sulfate, L-DOPA plus R04-4602 (seryltrihydroxybenzylhydrazine), ibotenic acid, METH and MK 801 in saline. Animals and treatments For the entire study male Sprague Dawley rats (190-240 g) were maintained in a controlled environment with a 12-h light/dark cycle; food and water were available ad libitum. All drugs were administered intraperitoneally, except METH, which was given subcutaneously, in doses calculated for their free fonns (control "animals for each group received identical treatments with the vehicles only). After at least one week of habituation, the animals were subjected to one of the following treatment protocols: Destruction of the nigrostriatal DA projections. The animals were anesthetized with chloral hydrate (375 mg/kg) and ketamine (35 to 40 mg/animal) and placed in a stereotaxic apparatus. A freshly prepared solution of 6-0HDA (8 ).1g in 4 ).11 of 0.1 % ascorbate-saline) was injected unilaterally into the substantia nigra at the rate of 1).1l/min. The coordinates used were 5.1 mm posterior to bregma, 2 mm lateral from the midskull suture and 6.6 nun ventral from the surface of the brain. The needle was kept in position for 5 min after the injection before withdrawal. The contralateral substantia nigra was treated in an identical manner except only the vehicle was injected. The incision was closed with staples and animals were allowed to recover for 7 to "1 0 days. Following the recovery, the rats were reweighed and divided into four treatment groups. Each group received three doses, at 6-h intervals, of METH (10 mg/kg/dose), SULP (80 mglkg/dose), haloperidol (2 mglkg/dose) or the vehicle alone (1 ml/kg/dose). Four groups of unoperated rats were given identical drug treatments to serve as controls for 12 sham-operated tissues. The animals were sacrificed 18 h following the last dose. The extent of the lesions was determined by comparing the tyrosine hydroxylase activities of the lesioned and the sham-treated striata for each animal. Non-selective depletion of brain DA. Rats were given two doses (12-h interval) of reserpine (5 mg/kg/dose) and 30 min following this treatment they received three doses of a-methyl-p-tyrosine (aMpT, 80 mglkg/dose), METH (10 mglkg/dose), SULP (80 mg/kgldose) or a combination thereof. Rats were sacrificed 18 h after the last treatment Axon-sparing lesions of the striatum. Rats were anesthetized and placed in a stereotaxic apparatus as described above for treatment with 6-0HDA. lbotenic acid (20 J.1g/2 ,.11 saline) was injected (1 J.1Vmin) unilaterally into the striatum using the following coordinates: 1.0 mm anterior to bregma, 2.7 mm lateral from the mid sku II suture and 5.0 mm ventral from the surface of the skull. The needle was kept in position for 5 min after the injection. The contralateral striatum was sham-treated with saline only. Following a recovery period of 7 to 10 days the rats were treated with a single dose of saline, MElli (10 mg/kg) or SULP (80 mglkg) and were sacrificed after i2 h. Three groups of unoperated animals were treated in an identical manner to serve as controls for the sham­treated tissues. The extent of lesions was determined by comparing the concentrations of substance P-like immunoreactivity (SPLI) in the lesioned and sham-treated tissues. Data from rats showing more than 50% lesions of the SP projections were used for computation of results. Effects of knife cut-induced lesions of striatal-nigral projections. Animals were anesthetized and placed on a stereotaxic apparatus as in (a). A cut was made by lowering a sharp stainless steel blade (width=3.2 mm) into the brain at the following coordinates: 0.5 mm from the bregma, 2.2 mm lateral to the midline suture and 7 mm ventral from the surface of the brain. After a week of recovery, the rats were given a single dose of MElli (10 mg/kg) and sacrificed 12 h later. 13 Concurrent blockade of muscarinic receptors with METH treatment. Animals were given three doses of either METH (10 mg/kg/dose), atropine (2 mg/kg/dose) or a combination thereof. At each dosing interval, atropine was administered 30 min prior to MElli. The rats were sacrificed 18 h following the last treatment Blockade of glutamate receptors in combination with SULP treatments. Rats were treated with a single dose of MK 801 (lor 2.5 mg/kg), SULP (80 mg/kg) or a combination thereof; animals were sacrificed 12 h following the treatments. Dissections Animals were sacrificed by decapitation always between 12:00 noon and 3 p.m. in order to minimize variability due to diurnal fluctuations. The brains were rapidly removed and after the dissection of the striata, tissues were frozen immediately on dry ice and stored at - 800 C. The nucleus accumbens and the substantia nigra were bilaterally dissected out from I-mm thick coronal slices of the frozen brains using the atlas of Konig and Klippel (1963) as a guide and stored at - 800 C until assayed for neurotensin-like immunoreactivity (NTLI) content Determinations of NJU and SPU contents of tissues The levels of N1LI and SPLI were measured using a radioimmunoassay technique described in detail by Letter et al. (1987b) and Hanson and Lovenberg (1980), respectively. Briefly, the tissues were homogenized in 0.D1N HCI, heated to inactivate peptidases, centrifuged and lyophilized. An aliquot of the homogenate was used to determine the total protein for each tissue by the method of Bradford (1976). Samples were reconstituted in phosphate-buffered saline (pH 7.4) containing 0.1 % gelatin. Duplicate aliquots were mixed with the antiserum (1/40,{)()() dilution for NT and 1/400,000 dilution for SP) and 125I-Iabeled peptide. Following incubation, the antibody bound and free 1251-labeled peptides were separated using dextran-coated charcoal slurry. Quantities of NlLI or SPLI were determined by comparing bound to free 1251_ 14 labeled peptide for each tube to a standard curve. The results were calculated as NTLI or SPLI content, pg/mg protein. Measurement of the tyrosine hydroxylase activity in the striatum Tyrosine hydroxylase activity was measured by a tritium-release assay (Nagatsu et a1., 1964). Briefly, the tissue was homogenized in 50 mM HEPES (4-(2-hydroxylethyl)­I- piperazoneethane acid) buffer containing 0.2% Triton X-lOO (pH 7.4) at 1: 2 weight to volume ratio. Duplicate aliquots of the centrifuged samples were'used for measuring the enzyme activity using dl-6-methyl-5,6,7,8-tetrahydrobiopterin as a cofactor. Quantitation of striatal dopamine (DA) levels Concentration of tissue DA was determined by high-performance liquid chromatography coupled to electrochemical detection (model LC-4B, Bioanalytical Systems, West Lafayette, IN) according to a modification of the method described by Nielsen and Moore (1982). Briefly, the tissues were homogenized in 0.3-0.5 ml of mobile phase buffer (0.15 M monochloroacetic acid buffer containing 2 mM EDT A, 0.1 mM l-octanesulfonic acid sodium salt and 12.5% methanol, pH 2.9), centrifuged at 4,000 X g for 15 min at 4° C. The supernatants were filtered with a 0.2-J1m Microfilter system (Bioanalytical Systems) and 50 J11 were injected onto a 10-cm Microsorb reverse phase column (Rainin Instrument, Woburn, MA). The eluent was monitored using a glassy carbon electrode with the potential set at +0.73 V (vs AglAgCI reference electrode). Tissue levels were quantitated by comparisons with standards of known concentration. Statistical analysis Unless otherwise noted, results are expressed as percentages of the respective controls in order to facilitate comparisons between groups. Each bar represents the mean ± S.E.M. Differences between means were analyzed using one-way ANOV A. If the F 15 ratio was significant, a Fisher-PLSD post-hoc test was used to compare differences between the means of individual groups. Differences were considered significant when the probability that they were zero was less than 5%. Results Effects of prior selective destruction of the nigrostriatal DA pathway on M ETH - and neuroleptic-induced alterations in NfU content To assess the role of the nigrostriatal DA pathway in MElli- and neuroleptic-induced alterations in striatal and nigral NTLI contents, this DA projection was unilaterally lesioned by intranigral administrations of the neurotoxin, 6-0HDA; the contralateral substantia nigra was sham-treated with vehicle only. Multiple doses of MElli, SULP or haloperidol were administered to the animals following the lesions as described above in Materials and Methods. For comparison, unoperated groups of rats were given identical treatments with these drugs. As previously reported (Letter et al., 1987a, Merchant et al., 1988), treatments with MElli and neuroleptics caused approximately 2- to 3-fold increases in striatal and nigral NlLI contents of the unoperated rats (Figure 2.1): the surgery did not modify the NT response to these drugs. However, NlLI content in the lesioned striata of the control group with greater than 85% destruction of the nigrostriatal DA pathway (as judged by the residual tyrosine hydroxylase activity) was significantly increased to 231 % of its respective sham-control; subsequent treatments with MElli, SULP or haloperidol did not significantly alter the NT levels COfllpared to these lesioned controls. In contrast, the NlLI levels in the striata of control animals with less than 85% lesions were very similar to the corresponding sham-control values and subsequent treatments with MElli or haloperidol caused the usual 2- to 3-fold increase in NTLI content compared to the corresponding controls (none of the tissues from SULP-treated animals showed less than 85% lesions). The nigral response was different as NILI levels .-c-- 1200 QJ Q 1000 s.. c. 800 600 400 200 striatum o ..L...I--L.::I~ unoperated sham- lesioned treated «85 % ) 4000 --c "Qj Q 3000 r.. C. bil ~ 2000 -c-. ** unoperated substantia nigra sham­treated lesioned (>85%) lesioned (>60%) D vehicle ISJ MElli l8J haloperidol II SULP 10 WIDe I LS1 MEllI 16 Figure 2.1: Effects of lesioning the nigrostriatal DA pathway on striatal and nigral NTLI content. Rats were treated with 6-0HDA as described in Materials and Methods. After a recovery period of 7 to 10 days, the animals were given three doses (6-h apart) of METH (10 mg/kgldose), haloperidol (2 mglkg/dose), SULP (80 mg/kg/dose) or their vehicle and sacrificed 18 h after the last treatment Four groups of unoperated rats were given identical treatments simultaneously. The data represent combined results obtained from three separate experiments. The lesioned animals were divided into two groups (Le., greater or less than 85% lesions) based on the success of the lesions as detennined by striatal tyrosine hydroxylase activity. Each bar represents the mean ± S.E.M. for the NILI content of each group expressed as pglmg protein; n ranged from 4 to 22. *p < 0.01, **p< 0.001 versus the corresponding vehicle-treated controls. tP<O.OI, versus the corresponding sham-treated groups. 17 were not affected by the lesions even in rats showing the maximum degree of dopaminergic destruction, although the MElli effect (Le., an increase to 244% of control in the N1LI content of the sham-treated tissues) was totally blocked even when only 60% of the DA pathway was destroyed (Figure 2.1). Effects of depletion of brain DA by reserpine plus aMpT on METH- and SULP-induced increases in NTU content In the striatum, treatment with reserpine plus aMpT caused more than 95% depletion of DA (control=10.3±1.2; reserpine ± aMpT=O.39±.08 J,1g DA /g tissue) and increased the NTLI level by 424% of control (Figure 2.2). Administration of MElli and SULP caused the NILI content to increase to 351 and 271 %, respectively, of the control value. However, when administered to rats with DA depletion, these drugs failed to increase the level of N1LI more than that caused by the depletion alone. Interestingly, restoration of DA synthesis by coadministration L-DOPA and the peripheral decarboxylase inhibitor, R04-4602, with reserpine plus aMpT, did not affect the increase in NTLI content caused by the DA depleting drugs, although the DA level increased to 68% of control (control=10.3±1.2; reserpine + aMpT + L-DOPA=6.98±1.06 J,1g DNg tissue). In addition, subsequent treatment with METH or SULP did Dot further increase the corresponding control NTLI content in these animals. L-DOPA by itself caused a slight but significant decrease in striatal N1LI level. Compared to the striatum, the response of nucleus accumbens to DA depletion was qualitatively similar but quantitatively less (Figure 2.2). Thus, the NTLI level was increased to 218% of control following DA depletion and METH or SULP did not modify this response; however, when given to rats with intact dopaminergic functions these drugs increased the level of NTLI to 180 and 196% of control, respectively. However, unlike the striatum, administration of L-DOPA by itself increased the NTLI content to 180% of control and appeared to attenuate the DA depletion-induced increase in --. 0 .s..... = 0 CJ .... = Q"I .E... "Q"I .s..... Q"I s.. c. • -Q"I .C..J ..c Q"I ;a.. c.- o Q"I Cl) ."... = Q"I CJ s.. Q"I -C. ,".".".'J" ~ Z 700 600 500 400 300 200 100 0 400 300 200 100 0 500 400 300 200 100 0 striatum ** * substantia nigra *** vehicle· pretreatment reserpine+ a MpT +L·DOPA 18 D control ~ME1H tIlISULP ~ L-DOPA Figure 2.2: Effects of DA depletion induced by reserpine plus aMpT on tissue NlLI content. Animals were given two doses (12-h apart) of reserpine (5 mg/kgldose) and 30 min later they were given three doses (6-h apart) of aMpT (80 mglkgldose), METH (10 mg/kgldose), SULP (80 mg/kgldose), L-DOPA (50 mglkgldose) plus R04-4602 (25 mglkgldose) or a combination thereof. Rats were sacrificed 18 h after the fmal treatment. Each bar represents the mean±S.E.M. for each treatment group, expressed as the percentage of its vehicle-pretreatment control; n = 4 to 9. The individual control NlLI levels (pglmg protein) were: striatum=161±16; nucleus accumbens=639± 97 and substantia nigra=380±34. *p< 0.05, **P<0.02, ***P<O.OOI versus the corresponding vehicle-pretreatment control, §P<0.05 compared to the respective control group and tP<O.05 compared to vehicle pretreatment-METII group. 19 NTLI content. In addi tion, in the presence of L-DOPA, treatment with MElli and S ULP significantly increased NTLI content of rats that had received reserpine plus aMpT. In contrast to the striatum and the nucleus accumbens, the nigral NTLI content remained unaffected following DA depletion by reserpine plus aMpT (Figure 2.2). Administration of MElli increased the NTLI content to 280% of control; this response was blocked by prior depletion of DA. Interestingly, when DA synthesis was restored by L-DOPA treatments, a greater increase (340% of control) in nigra! NTLI content was caused by MElli treatment Effects of axon-sparing lesions of the striatum induced by ibotenic acid Like the 6-0HDA effects, a bimodal response of the NT systems in the striatum was seen following lesions with ibotenate (Figure 2.3). Thus if the lesions were between 50 to 74% (as assessed by a reduction in striatal SPLI content), the NTLI level in the lesioned striatum was not reduced to (80% of control; statistically not significant). However, lesions of 75% or greater caused the NTLI level to decrease to 480/0 of control. Interestingly, a single administration of MElli or SULP to sham-treated animals increased the content of striatal NTLI to 150% and 252% of control, respectively; similar increases were also observed in the un operated rats. However, these increases did not occur in the striata that had greater than 75% depletion of SPLI content. In contrast, SULP induced a significant increase (211 % of control) in striatal NTLI content in tissues with lesions between 50-74% (there were no animals in the METH-treated group showing 50 to 74% lesions). Although the magnitude of this SULP-induced change was less than that observed in the corresponding sham-treated animals, the percent of increases with respect to the corresponding saline-treated controls were similar in the sham-treated and lesioned (50-74%) tissues (Le., 252%±31 in sham-SULP group versus 211 %±3 in lesioned[50-74%]-SULP group). 600 .".Q=..-.l SOO e Q. 400 t),I) eE:o 300 -Q-. 200 100 striatum unoperated sham lesioned lesioned treated (50·74%) (>75%) o control ~ MElli 81 SULP 20 2000 sUbstantia nigra·ibotenate substantia nigra-knife cut 2800 .- .5 1600 ~ Q '- Q. 1200 800 400 ** un­operated ** ** * ** sham- lesioned un- sham- lesioned treated (>50 % ) operated treated (>50 % ) Z 2400 -3 t- 2000 ..... .- ." 1600 <tt a 1200 ~ ..", 800 sa. t'ri 400 e: Figure 2.3: Effects of lesioning the striatal-nigral projections on NTLI content of the striatum and the substantia nigra. Lesions were induced with ibotenic acid or a knife cut as described in Materials and Methods. After a recovery period of 7 to 10 days, the animals were given 1 dose of MElli (10 mglkg/dose), or SULP (80 mglkg/dose) or their vehicle and sacrificed 12 h later. Three groups of unoperated rats were given identical treatments simultaneously. The data represent combined results obtained from 2 separate experiments; the lesioned animals were divided into two groups (Le., greater or less than 75% lesions) based on the success of the lesions as detennined by SPLI content of the tissues. Each bar represents the mean ± S.E.M. for the NTLI content of each group expressed as pg/mg protein; n ranged from 3 to 12. *p < 0.01, **p< 0.001 versus the corresponding vehicle-treated controls. tP<O.Ol, versus the corresponding sham-treated groups. 21 Like the striatum, the nigral NTLI content was also significantly reduced following ibotenate lesions. However, unlike the striatum, the nigral response was not bimodal in nature. Thus, a maximal decrease of only about 20% was observed in nigral N1LI content in tissues showing more than 50% reduction in SPLI content (Figure 2.3). Importantly, the MElli-induced increases in the N1LI content (expressed as percent of the respective controls) were similar in the unoperated, sham-treated as well as lesioned nigral tissues. The effects of knife cut-induced lesions on nigral N1LI content were similar to the effects of ibotenate lesions described above. Thus, NTLI content on the lesioned side decreased to approximately 70% as compared to the contralateral tissue (this decrease was statistically not significant because of a small number of surviving animals in this group; n=3). While the increase in NILI content caused by a single dose of MElli was 217% of control in the sham-treated tissues, this response was slightly attenuated following knife cut-induced destruction of striatonigral projections (Figure 2.3). Effects of concurrent blockade of muscarinic receptors on MEIH-induced increases in tissue NrU contents As shown in Figure 2.4, multiple doses of MElli induced approximately 2-fold increases in striatal N1LI content. Atropine by itself appeared to increase striatal N1LI level and when concurrently administered with MElli, a significantly greater increase in the NTLI content was observed. Effects of concurrent blockade of glutamatergic receptors on SULP-induced increase in NFL! content of the striatum A single dose of SULP increased the striatal NT content to 159% of the control level (Figure 2.5). Treatments with Img/kg or 2.5 mg/kg of MK 801 did not affect the NILI level in this structure. However, concurrent administration of MK 801 appeared to add to .-.- 300 0 striatum ...... * t D control c0 III atropine CJ c... * IS1 ME1H 0 200 ~ atrop+METH ~ eJ) .e.c.s c ~ .C.J. ~ 100 Q. --- ~ ~ Eo- Z 0 treatment Figure 2.4: Effect of concurrent blockade of muscarinic receptors on METH­induced increase in NTLI content of the striatum. Rats were treated with three doses (6-h intervals) of atropine (atrop; 2 mg/kgldose), METH (10 mg/kgldose) or a combination thereof. The rats were sacrificed 18 h following the treatment. Each bar represents the mean±S.E.M. for each treatment group expressed as the percentage of vehicle-treated control; (n=4 to 9). The control NlLI content was 120±9 pg/mg protein. *p< 0.0001 compared to the control; tP< 0.025 compared to all other groups. 22 300 striatum t# treatment o control ~ MK(1mg/kg) ~ MK(2.5 mglkg) II SULP ~ MK(1mg)+SULP m MK(2.5mg)+SULP Figure 2.5: Effect of concurrent blockade of NMDA receptors on SULP­induced increase in striatal NILI content. Rats were treated with a single dose of MK 801 (1 or 2.5 mglkg), SULP (80 mg/kg) or a combination thereof and were sacrificed 12 h following the final treatment. Each bar represents the mean±S.E.M. expressed as percentage of the control (n=6 for all groups). The control NTLI level was 403±33 pg/mg protein. *p< 0.01, **p< 0.005 compared to the control group. tP<O.05 compared to SULP-treated group. 23 24 the increase caused by SULP alone, especially at the lower dose of 1 mg/kg (P<O.05 compared to SULP-treated group). Discussion The present results demonstrate that prior depletion of central dopaminergic systems block the effects of subsequent treatments with MElli, SULP or haloperidol on NT systems in the striatum, the nucleus accumbens and the substantia nigra. Interestingly, reduction in dopaminergic activity itself increased the NTLI con~ent of the striatum and nucleus accumbens, but only when the depletion of DA was greater than 85% (Figures 2.1, 2.2). Administration of METH or the neuroleptics following such disruption in DA systems did not further increase the NILI content of the striatum and nucleus accumbens. These data suggest that either (a) the same mechanism underlies the increases in NlLI caused by DA depletion and treatments with MElli or the neuroleptics or (b) MElli and the neuroleptics are unable to exert their effect due to lack of sufficient drug-induced alteration in dopaminergic activity following DA depletion. Because MElli is a potent releaser of DA, it is most likely that the second possibility is responsible for the lack of METH effects on N1LI content following elimination of DA. This is the basis for the effects of METH on other peptides, such as substance P (Ritter et aI., 1984) and dynorphin A(l_7) (Hanson et al., 1988). This possibility is examined further in Chapter 3. The neuroleptics, on the other hand, can either block dopaminergic transmission (by blocking D2 receptors) or increase DA release (by blocking autoreceptors or inhibitory feedback loops). Hence, either possibility could explain the results presented here. Since coadministration of the neuroleptics with METH adds to the effects of the latter (Figure 1.1), it is likely that the neuroleptics regulate NT systems by a mechanism distinct from that of MElli. This hypothesis is tested in the folloVling chapters. 25 The precise mechanism underlying the changes in NlLI levels caused by DA depletion is not clear from these data. However, based on the observation that the dopaminergic systems must be severely compromised (>85%) before the NT systems in the striatum and the nucleus accumbens are altered suggests that removal of the basal dopaminergic tone may be responsible for the changes in NT levels. This possibility is investigated and discussed in Chapter 4. Administration of L-DOPA (with its peripheral DOPA decarboxyalse inhibitor, R04-4602) following reserpine plus aMpT, increased the DA level in the striatum to 68% of control and yet failed to affect the increase caused by DA depletion or restore the METH or SULP effects in the striatum (Figure 2.2). This suggests that (1) METH and the neuroleptics require an intact DA system to exert their effect on striatal NT systems or (2) the effects of reserpine-induced DA depletion on NT activity in the striatum are not readily reversible. In the accumbens, however, treatment with L-DOPA slightly attenuated the increase induced by DA depletion and subsequent treatment with SULP or METH significantly increased the NTLI content of this structure compared to the respective saline-treated control. This suggests that the regulatory mechanism underlying DA depletion-induced alterations in the NlLI content of the nucleus accumbens is distinct from that underlying the changes in the striatum. Another difference between the NT systems in the striatum and the nucleus accumbens was evident when treatment with L-DOPA plus R04-4602 by itself decreased and increased, respectively, the NTLI content of these tissues. The present data are not sufficient to explain these differential effects of L-DOPA in the striatum and the nucleus accumbens, but it might be due to differential distribution of DA receptor subtypes in the nucleus accumbens compared to the striatum as discussed in Chapter 4. Contrary to the striatum and the nucleus accumbens, the nigral NTLI content was not affected by disruption in DA transmission (Figure 2.1, 2.2). However, similar to those structures, the METH-induced increase in the NlLI content of the substantia nigra was completely blocked by prior depletion of DA. Interestingly, in contrast to the striatum, 26 even when only 60% of the nigrostriatal DA projections were destroyed following 6- OHDA, the nigral METH effect was totally prevented. These data suggest that the relationship between DA projections and NT systems in the substantia nigra is distinct from that present in the striatum and the nucleus accumbens. This is further supported by the observations that administration of L-DOPA plus R04-4602 following reserpine plus aMpT, completely restored the METH effect in the substantia nigra (although the striatal DA level was restored to only 68% of control by these drugs) but not in the striatum (Figure 2.2). In fact, METH caused a significantly greater increase in nigral N1LI content of rats treated with reserpine+aMpT followed by L-DOPA than the rats which did not receive the DA depleting drugs. This enhanced response to METH may be due to an upregulation of DA receptors following elimination of dopaminergic activity by treatments with reserpine plus aMpT. This hypothesis is supported by previous reports (porceddu et al., 1985) demonstrating an upregulation of DA receptor subtypes following treatment with drugs (such as 6-0HDA and DA receptor antagonists) that disrupt dopaminergic transmission. It is recognized that reserpine is a nonselective drug that depletes not only DA but also norepinephrine, epinephrine as well as serotonin. However, minimal norepinephrine/epinephrine-containing projections are present in the structures being studied. Hence, these transmitters are not likely to be mediating the changes induced by reserpine. This conclusion is also supported by the recent evidence that administration of DSP-4, a norepinephrine-selective toxin, does not affect the NT systems in the basal ganglia (Michel Johnson, personal communication). In addition, intranigral administration of 6-0HDA does not significantly affect serotonergic afferents in the striatum and the substantia nigra. Since the effects of 6-0HDA-induced lesions and DA depletion caused by reserpine plus aMpt appeared to be identical, it is likely that reserpine plus aMpt or 6-0HDA-induced alterations in NT systems were mediated by changes in dopaminergic transmission. This hypothesis is supported by data from 27 experiments in which selective dopaminergic drugs were employed (see Chapters 3 and 4). The results of this study demonstrate an underlying role of DA in MElH- and the neuroleptic-induced changes in discrete NT systems. In order to examine if other transmitter systems serve as a link between DA and NT systems in the striatum, ibotenic acid was injected into the striatum prior to treatments with METH or SULP. Due to the toxicity of ilx>tenate and an apparent increase in the sensitivity of.the lesioned animals to the toxic effects of METH, only a single dose of METH or SULP was administered to the rats in these studies. Ibotenic acid is an excitotoxin by virtue of its affinity for the N­methyl- D-aspartate (NMDA)-type of glutamate receptors. The exact mechanism of the excitotoxin-induced cell death is not known, but it appears to involve an interaction between the glutamate receptors and some presynaptic transmitter system, most likely, glutamatergic in nature. Direct injections of this excitotoxin into discrete brain areas selectively destroys neurons that have their cell bodies at or near the site of injection; axons and terminals appear to be resistant to the effects of this neurotoxin. When injected into the striatum, ibotenate causes destruction of striatal intemeurons and efferents that possess NMDA receptors. Since a glutamate-sensitive striatonigral substance P pathway has been identified, alterations in striatal and nigral SPLI content were used as indicators of the extent of ibotenate-induced lesions in the extrapyramidal tissues. As shown in Figure 2.3, striatal NTLI content was reduced by only 20-25% (statistically not significant) in animals showing 50 to 74% lesions following administration of ibotenate. However, in animals with greater than 75% lesions, striatal NTLI content was reduced to approximately 50% of control. These data suggest the presence of NMDA-sensitive NT cell bodies in the striatum. Supporting this hypothesis, NT mRNA-containing cells have recently been shown to be present in the ventral striatum using in situ hybridization (Alexander et al., 1989) and a striatal-pallidal NT pathway was identified by Zahm and Heimer (1988). Since the striatal lesions caused a maximal decrease in the NTLI content 28 of only 50% of control, it is likely that the total pool of NlLI content in the striatum is distributed equally between NT neurons which originate in the striatum (sensitive to ibotenate) and NT-containing fibers and terminals which project to the striatum (insensitive to ibotenate). However, it is also possible that a portion of striatal NT systems is associated with cell bodies that do not possess NMDA receptors. A confounding factor to such speculations is that changes in NTI...I content may reflect not only loss of neuronal systems but also compensatory changes by surviving NT neurons.Thus, an increase in NT immunoreactive cells has been shown around the site of injection of another excitotoxin, kainic acid, in the cat striatum (Sugimoto et al., 1987). If a similar compensatory increase in NTLI content occurred in surviving neurons adjacent to the ibotenate-induced lesions, perhaps the 50% estimate of affected NT neurons is an underestimation. Histological studies are required to gain a better understanding of ibotenate-sensitive striatal NT systems. The responses of striatal NT systems to acute treatments with METH and SULP following ibotenate lesions are worth noting. Thus, increases caused by a single dose of MElli or SULP were totally blocked in the tissues that showed more than 75% lesions of the SP pathway, although the lesions reduced the NT content to only 48% in these tissues (Figure 2.3). These data suggest that the activity of ibotenate-insensitive striatal NT systems is not affected by alterations in dopaminergic activity. However, another possibility is that the lesions destroyed a linking transmitter system (between striatal DA projections and the surviving NT pathways) that mediates dopaminergic regulation of these NT systems. When the lesions were of an intermediate degree (50 to 74%), the response of the surviving NT systems to SULP was of the same magnitude as that of the sham-treated tissue. This may be due to sufficient survival of putative linking transmitter systems following the lesser destruction such that normal regulation of NT systems by dopaminergic activity is maintained. Further studies are required to determine which explanation is correct. 29 Like the striatum, the nigral NTLI content was also significantly reduced following striatal lesions induced by ibotenate administration or a knife cut (Figure 2.3). However, the maximal reduction in nigral NTLI content was only about 20% irrespective of the extent of lesions. There are two distinct possibilities that can explain this reduction in nigral NTLI level following striatal ibotenate lesions: frrst, a portion of the ibotenate­sensitive striatal NT cell bodies project to the substantia nigra or second, ibotenate destroys a specific striatal transmitter system projecting to the nigra that may be regulating the basal activity of nigral NT systems. The former possibility is not likely since NT­positive striatal-nigral projections have never been reported. However, histological studies are needed to confrrm the mechanism of reduction in nigra! N1LI level following lesions of striatal interneurons or efferents. The response of the nigral NT systems to METH treatment following ibotenate administration was also distinct from that seen in striatal tissues with greater than 75% lesions (Figure 2.3). Thus, the magnitude of METH effects (expressed as percent of the corresponding saline group) was similar in the nigral tissues of lesioned and nonlesioned animals. Interestingly, the METH response was significantly reduced in lesioned tissues following striatal knife cuts. Such a difference between striatal ibotenate lesions- and knife cut-induced alterations suggests that an ibotenate-insensitive striatal-nigral transmitter system also regulates the METH-induced alterations in nigra! NT activity. A similar finding was made for the effects of METH on nigral SP system (Hanson et al., 1981). Further studies are required to identify this transmitter system. Due to an established functional relationship between DA and cholinergic neurons (Bartholini et al., 1973; Consolo et al., 1987), the possibility of the presence of a cholinergic link between striatal DA and NT pathways was examined by coadministering a muscarinic antagonist, atropine, with METH. However, as shown in Figure 2.4, cholinergic activity does not appear to be involved in MElli-induced increase in striatal NTLI content since concurrent administration of atropine did not block this response of 30 the NT systems to MElli. In fact, atropine by itself appeared to cause a slight increase in striatal NTLI level. Other investigators have also reported an increase in striatal NTLI content following treatments with atropine (Frey et al., 1988). Concurrent treatment of atropine with MElli resulted in a significantly greater increase in the NlLI level of the striatum. This enhanced response of striatal NT systems to atropine plus MElli may be similar to the additive increases caused by coadministration of METH and SULP (figure 1.1) since acetylcholine tonically regulates dopaminergic activity. A cholinergic link also does not appear to be involved in the effects of the neuroleptics in the striatum as other investigators have reported that atropine fails to block the increase in NTLI content induced by these drugs (Levant et al., 1988; Frey et al., 1987). Another possible linking system for the striatal DA and NT pathways may be the glutamatergic afferents. Several studies have shown functional interactions between DA and glutamate pathways in the striatum (Cheremy et al., 1986). In addition, the increases in striatal NlLI content caused by multiple doses of METH have recently been shown to be completely blocked by concurrent treatments with the NMDA receptor antagonist, MK 801 (Singh et aI., 1989). This result clearly indicates that some of dopaminergic regulatory effects on NT systems are mediated by glutamatergic pathways. In order to test if glutamatergic pathways also mediate the effects of acute treatments with SULP, MK 801 was administered to rats 30 min prior to a single dose of SULP. In the striatum, MK 801 by itself did not affect the activity of the NT systems at either of the doses tested (Figure 2.5), A single dose of SULP increased the NTLI content by 159% of control. Interestingly, concomitant administration of MK 801 with SULP did not attenuate the increase in striatal NTLI content caused by SULP. This finding demonstrates that while the MElli-induced changes in NT systems are mediated by a NMDA-related mechanism, the effects of SULP on striatal NlLI content appear to be mediated by a NMDA-unrelated mechanism(s). 31 In summary, the studies in this chapter have detennined that dopamine is the primary transmitter mediating the effects of MElli and SULP on the NT systems in the striatum, nucleus accumbens and the substantia nigra. In addition, ibotenate-sensitive, NT­containing cell bodies appear to be present in the striatum. The results presented here also suggest the existence of another transmitter system(s) which helps to mediate the effects of SULP and MElli on NTLI contents of the striatum and possibly the substantia nigra. For the striatum, this linking system appears to be ibotenate-sensitive whereas for the nigra it appears that an ibotenate-insensitive striatal-nigral system is involved. The striatal linking system is not cholinergic in nature although glutamatergic mechanisms appear to playa modulatory role in the effects of MElli on NT systems. 32 References Alexander, MJ., P.R. Dobner, M.A. Miller, B.P. Bullock, D.M. Dorsa and S.E. Leeman, S.E., 1989, Estrogen increases neurotensin mRNA levels in sexually dimorphic nuclei of the rat preoptic region, Science submitted. Anden, N.E., A. Dahlstrom, K. Fuxe, K. Larsson, L. Olson and U. Ungerstedt, 1966, Ascending monoamine neurons to the telencephalon and diencephalon, Acta Physiol. Scand. 67, 313. Bartholini, G., H. Stadler and K.G. Lloyd, 1973, Cholinergic-<iopaminergic interactions in the extrapyramidal system, Adv. Neurol. 3 , 233. Bradford, M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248. Cheremy. A., R. Romo, G. Godedheu, P. Baruch and J. Glowinski, 1986, In vivo presynaptic content of dopamine release in cat caudate nucleus II. Facilitatory or inhibitory influence of I-glutamate, Neurosci. 19, 1081. Consolo, S., F.W. Chun and F. Rossella, 1988, D-1 receptor-linked mechanism modulates cholinergic neurotransmission in rat striatum, 1. Phannacol. Exp. Ther. 242, 300. Consolo, S., R. Fanelli, S. Garattini, D. Ghezzi, A. Jori, H. Ladinsky, V. Marc and Samanin, 1975, Dopaminergic-cholinergic interactions in the striatum: Studies with peribedil., Adv. Neurol. 9, 257. Consolo, S., H. Ladinsky and S. Garattini, 1974, Effects of several dopaminergic drugs and trihexiphenidyl on cholinergic parameters in the rat striatum, J. Phann. Phannacol. 26, 275. Daly, 1. W., C.R. Creveling and B. Witkop, 1966, The chemorelease of norepinephrine from mouse hearts, Structure activity relationship I: Sympathomimetic and related amines, J. Med. Chern. 9, 273. Frey, P., L. Madalenna and D.M. Coward, 1988, Neurotensin concentrations in rat striatum and nucleus accumbens: further studies of their regulation, Neurochem. Int. 12, 33. Hanson, G.R., L. Alphs, W. Wolf, R. Levine and W. Lovenberg, 1981, Haloperidol­induced reduction of nigral substance P-like immunoreactivity: A probe for the interactions between dopamine and substance P neuronal systems, J. Pharmacol. Exp. Ther. 218, 568. Hanson, G.R. and W. Lovenberg, 1980, Elevation of substance P-like immunoreactivity in rat central nervous system by protease inhibitors, 1. Neurochem. 35, 1370. Hanson, G.R., K.M. Merchant, A.A. Lettter, L. Bush and J.W. Gibb, 1988, Characterization of methamphetamine effects on the striatal-nigral dynorphin system, Eur. 1. Phannacol. 155, 11. 33 Konig, J.F.R. and R.A. Klippel, 1963, The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem (Williams and Wilkins Company, Baltimore). Lazareno, S., D.B. Marriott and S.R. Nahorski, 1985, Differential effects of selective and non-selective neuroleptics on intracellular and extracellular cyclic AMP accumulation in rat striatal slices, Brain Res. 361, 91. Letter, A.A., L.A. Matsuda, K.M. Merchant, J.W. Gibb and G.R. Hanson, 1987, Characterization of dopaminergic influence on striatal-nigra! neurotensin systems, Brain Res. 422, 200. Letter, A.A., K.M. Merchant, J.W. Gibb and G.R. Hanson, 1987, Effect of methamphetamine on neurotensin concentrations in rat brain regions, J. Phannacol. Exp. Ther. 241, 443. Levant, B., G. Bissette, G. and C.B. Nemeroff, 1988, Anticholinergic drugs do not alter neurotensin concentration in the nucleus accumbens and the striatum, Soc. Neurosci. Abst. 14, 114. Mc Geer, P.C. and E.G. Mc Geer, 1976, The GABA system and function in the basal ganglia: Huntington's disease, In GABA in nervous System Function, eds. E. Roberts, T. Chase and D. Tower (Raven Press, New York). Merchant, K.M., A.A. Letter, J.W. Gibb and G.R. Hanson, 1988, Changes in the limbic neurotensin systems induced by the dopaminergic drugs, Eur. J. Pharmacol. 153, 1. Nagatsu, T., M. Levitt and S. Udenfriend, 1964, A rapid and simple radioassay for tyrosine hydroxylase activity, Anal. Biochem. 9, 122. Nielsen, J.A. and K.E. Moore, 1982, Measurement of metabolites of dopamine and 5- hydroxytryptamine in cerebroventricular perfusates of unanesthetized, freely moving rats: selective effects of drugs, Pharmacol. Biochem. Behav. 16, 131. Porceddu M.L., E. Ongini and G. Biggio, 1985, [3H]SCH 23390 binding sites increase after chronic blockade of D-l receptors, Eur. J. Phannacol. 118, 367. Ritter, J.K., C.J. Schmidt, J.W. Gibb and G.R. Hanson, 1984, Increases of substance P-like immunoreactivity within striatal-nigral structures after subacute methamphetamine treatment, J. Pharmacol. Exp. Ther. 229, 487. Scatton, B., 1981, Differential changes in DOPAC levels in the hippocampal fonnation, septum and striatum of the rat induced by acute and repeated neuroleptic treatment, European J. Pharmacol. 71, 499. Schmidt, C.J., J.A. Levin and W. Loven berg , 1987, In vitro and in vivo neurochemical effects of methylenedioxymethamphetamine on striatal monoaminergic systems in the rat brain, Biochem. Pharmacol. 36, 747. Seeman, P., 1981, Dopamine receptors, Pharmacol. Rev. 32,229. Singh, N.A., K.M. Merchant, J.W. Gibb and G.R. Hanson, 1989, Role of glutamate in dopamine-mediated neurotensin changes, Soc. Neurosci. Abst submitted. 34 Starke, K., W. Reimann, A. Zumstein and O. Hertting, 1978, Effect of dopamine receptor agonists and antagonists on release of dopamine in rabbit caudate nucleus in vitro, Naunyn Schmiedeberg's Arch. Pharmacol. 305, 27. Sugimoto T., K. Itoh, Y. Yasui, H. Kamiya, Y. Vemura and N. Mizuno, 1987, Changes in neurotensin-like immunoreactivity in the striatum of the cat after intrastriatal injection of kainic acid, J. Compo Neurol. 263, 607. Vngerstedt, V., 1971, Stereotoxic mapping of the monoamine pathways in the rat brain, Acta Physiol. Scand., Suppl. 367, 1. Vngerstedt, V. and O.W. Arbuthnott, 1970, Quantitative recording of rotational behaviour in rats after 6OH-dopamine lesions of the nigro-striatal dopamine systems, Brain Res. 24, 485. . Zahm, D.S. and L. Heimer, 1988, Ventral striatopallidal parts of the basal ganglia in the rat: I. Neurochemical compartmentation as reflected by the distribution of neurotensin and substance P immunoreactivity, J. Compo Neurol. 272,516. CHAPTER 3 ROLE OF Dl RECEPTORS IN REGULA nON OF NEUROTENSIN SYSTEMS Introduction As previously discussed, DA pathways appear to be the primary transmitter systems mediating the effects of METH and the neuroleptics on NT projections of the striatum, the nucleus accumbens and the substantia nigra. METH increases DA transmission by causing massive release of endogenous DA (Schmidt et aI., 1984). This newly released DA in turn affects associated distal transmitter systems (such as those containing NT) by activating postsynaptic receptors. On the basis of phannacological evidence, brain DA receptors have been divided into two categories: D 1 receptors, which increase cAMP synthesis or D2 receptors, which decrease or have no effect on cAMP synthesis (Kebabian and CaIne, 1979; Stoof and Kebabian, 1981). Thus METH-induced alterations in NT systems may be caused by activation of either or both of the DA receptor subtypes. However, activation of D2 receptors does not seem to be involved in the METH-nlediated effects on NT systems of the striatum and the nucleus accumbens as concurrent blockade of the D2 receptors with SULP does not antagonize the increases induced by METH (Figure 1.1). These data suggest that Dl activation is a more likely mechanism underlying changes in NT systems caused by treatment with METH. This possibility is investigated here by concurrent administration of SCH 23390, a D 1- selective antagonist (Iorio et aI., 1983, Hyttel et aI., 1983), with METH. The neuroleptic, SULP, on the other hand, is a selective blocker ofDA-D2 receptors. Unlike METH, treatment with SULP does not affect the nigral NTLI content but does 36 cause substantial increases in the NlLI levels in the striatum and the nucleus accumbens (figure 1.1). Two distinct mechanisms may underly this neuroleptic-induced increase in the NTI..I contents of the striatum and nucleus accumbens: (1) interruption of basal DA transmission by receptor blockade or (2) enhancing DA transmission by increasing the release of DA due to blockade of the autoreceptors on dopaminergic terminals (D2 subtype, Roth, 1979) or postsynaptic receptors on regulatory feedback loops (Starke et al., 1978; Scatton, 1981; Lazareno et al., 1985). The DA released by the second proposed mechanism could activate D1 receptors and thereby affect the associated NT systems. This possibility was tested by studying the effects of coadministering SCH 23390 with SULP. In order to help distinguish the role ofD1 and D2 receptors in regulating NT systems, the effects of direct activation of these receptors by the selective agonists, SKF 38393 (Setler, et aI., 1978), and LY 171555 (Itoh et aI., 1985), respectively, were invesigated. Finally, it is possible that tonic D1 receptor activation by basally released OA has some regulatory influence on NT systems. The presence of basal dopaminergic control over associated NT systems was evident from the results shown in Chapter 2 (i.e., DA depletion with 6-0HDA or reserpine plus aMPT induced large increases in NTLI content of the striatum and the nucleus accumbens, Figures 2.1, 2.2). A possible role for the 01 receptors in this phenomenon was investigated by two strategies: first, the effect of acute and subchronic blockade of 01 receptors was assessed and second, the effect of stimulating D1 receptors (with a selective agonist) in combination with reserpine-induced DA depletion on tissue NTI..I contents was also examined. Materials and Methods Drugs (+ )Methamphetamine hydrochloride (National Institute on Drug Abuse, Rockville, MD), the D1 selective antagonist, SCH 23390 [(R)-(+)-8-chloro-2,3,4,5-tetrahydro-3- 37 methyl-5-phenyl-lH-3-benzozepine-7 -ol-hemimaleate, Schering Corp., Bloomfield, NJ] and the D2 selective agonist, L Y 171555 [trans-( -)-4a-R-4,4a,5,6,7 ,8,8a,9-octahydro-5- propyl-lH(or 2H)-pyrazolo (3,4-g) quinoline mono-hydrochloride, Lilly Research Laboratories, Indianapolis, IN] were generous gifts of the indicated sources. The D 1 selective agonist, SKF 38393 was purchased from Research Biochemicals, Inc. (Natick, MA). All other drugs used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The vehicles used for dissolving each drug were as follows: sulpiride and SKF 38393 in 2% lactate+25% propyleneglycol-saline; reserpine in 1 % citrate+20% PEG 400-saline; L Y 171555 in 1 % lactate-saline; MElli and SCH 23390 in saline. Animals Male Sprague Dawley rats were used for the entire study as described in Chapter 2. Treatments Effect of concurrent blockade of D 1 receptors on MElli- or SULP-induced changes in tissue NTLI content. Rats were given five doses of MElli (10 mglkg/dose) or three doses of SULP (80 mg/kg/dose) every 6 h. At each dosing interval, SCH 23390 (0.5 mglkg/dose) was administered 30 min prior to METH or SULP. The animals were sacrificed 18 h following the last treatment Effect of direct activation of selective dopamine receptors on tissue NTLI content. Rats were given three doses (6-h interval) of the D 1 agonist SKF 38393 (18 mglkg/dose), the D2 agonist, LY 171555 (5 mg/kg/dose) or a combination thereof; animals were sacrificed 18 h after the last treatment Study of possible basal regulation of NT systems mediated by Dl receptors. The following three sets of experiments were carried out for this study. (1) Effects of Dl receptor blockade: rats were given one, three or five doses of SCH 23390 (0.5 mg/kg/dose) at 6-h intervals and sacrificed 18 h following the last treatment. (2) Effects of depleting DA with reserpine: animals were given a single dose of reserpine and 38 sacrificed after 18,66 or 186 h. The correlation between striatal DA depletion and nigral NTLI content was studied. (3) Effects of D1 receptor activation concurrent with dopamine depletion: rats were treated with a single dose of reserpine (5 mg/kg) 30 min following the first dose of the Dl selective agonist, SKF 38393 (18 mg/kg/dose). The treatment with SKF 38393 was continued for a total of four doses given every 3 h and the animals were sacrificed 7 h after the treatments. Methods for dissections, determination of NTLI and DA contents and statistical analysis are discribed in Chapter 2. Results Effects of concurrent blockade of D 1 receptors on METH­or SULP-induced changes in tissue NTU contents Five administrations of SCH 23390 failed to affect significantly the NTLI contents in all structures examined. Administration of METH increased the level of NTLI in the striatum, nucleus accumbens and the substantia nigra to 227%, 211 % and 313%, respectively, of the corresponding control values. Interestingly, coadministration of SCH 23390 with MElli totally blocked the METH-induced increases in the NTLI content of all three structures (Figure 3.1). As previously reported (Figure 1.1), multiple doses of SULP caused significant increases in the NILI content of the striatum and the nucleus accumbens (Figure 3.2) but did not affect the nigrallevel. Unlike the METH-induced increases, the SULP effects were not antagonized by concurrent blockade of the Dl receptors with SCH 23390. 300 ------......-------..... ------- striatum D control ** 200 100 ~ 308 _---...-0:--------::-------. ..L..... nucleus accumbens =ou CO 200 ~ 1:\1) S =t 100 L. ~ -Q-. ....c * ~ 408 -------~--....., Z substantia nigra *** treatment § SCH23390 ~ME1H ~ SCH+MElli Figure 3.1: Effect of concurrent blockade of Dl receptors on METH-induced alterations in tissue NTLI content. Animals were treated with five doses (6-h apart) of MElli (10 mg/kgldose), SCH 23390 (SCH; 0.5 mg/kgldose) or a combination thereof and were sacrificed 18 h following the final treatment. Each bar represents the mean±S.E.M. expressed as percentage of the respective controls (n=6 to 15). The individual control NTLI values were: striatum=136±16; nucleus accumbens=604±66 and substantia nigra=430±19 pglmg protein. *p< 0.05, **P<0.02 and ***P<O.OOI compared to the respective controls. tP<O.02 compared to the corresponding METH-treated group. 39 300 striatum D control ~ SCH *** II 'SULP 200 ~ SCH+SULP -- 100 0 ..s...... C 0 CJ c.- o Q; CJ) 0 ..c..:.o. c 200 nucleus accumbens Q; CJ s.. Q; -Q-. * * ~ ,.J E-c Z 100 o treatment Figure 3.2: Effect of concurrent blockade of D 1 receptors on SULP-induced alterations in tissue NTLI content. Animals were treated \\ith three doses (6-h apart) of SULP (80 mg/kg/dose) or SCH 23390 (SCH; 0.5 mg/kg/dose) or a combination thereof and were sacrificed 18 h following the final treatment. Each bar represents the mean±S.E.M. expressed as percentage of the respective controls (n=4 to 10). The individual control NTLI values were: striatum=231±30 and nucleus accumbens=896±145 pg/mg protein. *p< 0.05, **P<0.02 and ***P<0.005 compared to the respective controls. 40 41 Effects of selective activation of D 1 and/or D2 receptors on tissue NTU level Administration of three doses (at 6-h intervals) of the D1 selective agonist, SKF 38393, increased the NTLI content of the striatum and the nucleus accumbens to 129% and 154%, respectively, of the corresponding control values (Figure 3.3). In contrast, the D2 selective agonist, L Y 171555, decreased the level of NTLI in both structures by about 25%, although the reduction did not reach statistical significance in the nucleus accumbens (P<O.l, Figure 3.3). When the two drugs were coadministered, the levels of NTLI in neither structure differed from the respective controls. On the other hand, the nigral NTLI content did not change following activation of only D1 receptors but was significantly reduced following D2 receptor activation with LY 171555. Interestingly, combined administration of the two agonists caused the nigral NTLI content to increase to 130% of the control (Figure 3.3). Study of basal D 1 activity in regulation of NT systems Eighteen hours following a single or multiple (Le., three or five) doses of SCH 23390, the NTLI content in the striatum and the nucleus accumbens remained unchanged. In contrast, the nigral level of NTLI was reduced significantly to 83% of its corresponding control value following a single administration of this D1 antagonist, although multiple doses of SCH 23390 did not significantly alter the nigral NTLI level (Figure 3.4). These data suggest that acute interruption of tonic D1 activity affects only the nigral NT systems. In order to confmn this hypothesis, the response of nigral NT systems to reserpine-induced DA depletion and concurrent activation of D 1 receptors was examined. Eighteen hours following a single dose of reserpine, the nigra! NTLI level was reduced to 71 % of control (Figures 3.5, 3.6); this reduction in NT activity coincided with the maximal depletion of striatal DA content (Figure 3.5). Administration of the D1 42 200 striatum 0 control ISl SKF * ~ LY lII1 SKF+LY 100 -- 0 s0. 200 ..... nucleus accumbens =0 * CJ ~ 0 ~ ~ eo ..... 100 = ~ CJ s. ~ -Q-. JIIoooC ....J ~ Z 208 substantia nigra * treatment Figure 3.3: Effect of selective DA receptor agonists on tissue N1LI content. Rats were given three doses (6-h apart) of the Dl-selective agonist, SKF 38393 (SKF; 18 mg/kg, Lp.), the D2 selective agonist, L Y 171555 (L Y; 5 mg/kg, i.p.) or a combination thereof and were sacrificed 18 h after the last dose. Each bar represents the mean ± S.E.M. for each treatment group, expressed as percentage of the respective controls (n = 5 to 7). The average control values for NTLI levels (expressed as pg/mg protein) were: striatum=154±10; nucleus accumbens=728±90 and substantia nigra=734+36. *P < 0.05, **P<O.02 compared to the respective controls. striatum 120 80 40 -e- O~----~---c~~==&a~------~ § 160 ----n-u-c':""le-u-s-a-c-cu-m--=-b-e-n-s ------, CJ c... c:> 120 O~ __________________________ -, - 120- - 80- - 40- - o substantia nigra .,*... ~,­r-:.:.:-:.:. r-:.:-:.:.:. r-:.:.:.:.:. r-:.:-:-:.:. treatment o control El SCH-1 dose § SCH-3 doses B SCH-5 doses Figure 3.4: Effect of Dl receptor blockade on tissue N1LI content. Rats were given one, three or five doses of SCH 23390 (SCH; O.Smglkg/dose); the interval between successive treatments was 6 h. Eighteen hours following the final treatment the animals were sacrificed. Each bar represents the mean±S.E.M. for each group expressed as percentage of the respective control. The control NTLI levels expressed as pg/mg protein were: striatum=192± 28; nucleus accumbens=812±105 and substantia nigra= 620±60. *p< 0.05 compared to the respective control. 43 120 120 --0 100 ~ 100 Z ..t..-.. ~ =o t~- u - e.- 80 -c 0 80 ~ .. ~ - t':> ! -- ~ ::I = .. ..... 60 0,') .0- I!I 60 - (JQ ...... . ~ t~- ..... t .. ·... . ·f 0 ...... - ................... * ..., = . ....... OJ 40 .' * 40 t':> U .. ' 0 = .. ::I 0 ... . ...... . u .. .. 2- -< .. 20 .. - Q 20 . .. .. -..- If * 0 0 0 24 72 120 168 hours following treatment Figure 3.5: Correlation between striatal DA depletion and alterations in nigral NILI content following treatment with reserpine. Animals were given a single dose of reserpine (5mg/kg) and sacrificed at 18, 66 or 186 h following the treatment. The graph shows the correlation between striatal DA depletion and decrease in the NTLI content of the substantia nigra expressed as mean ± S.E.M. The data are expressed as percentage of the respective controls; n = 4 to 10. The control DA content in the striatum was 8.06±O.39 Jlg/g of tissue, the control NTI..I content of the substantia nigra was 879±42. *P<O,005 versus the corresponding controls (values represented by the 0 h time point). 44 160 .- "0 140 .."."..". substantia nigra - t 0 control ~ reserpine =0 y 120 c.- o <U 100 · -~ - -· T * T IS] SKF [I] SKF+reserp b.C) ..=.... · = 80 <U Y - · I "<"U" Q. 60 -- -· ~ ~ E-- 40 - Z 20 - 0 treatment Figure 3.6: Effect of selective activation of Dl receptors on reserpine-induced decrease in NTLI content of the substantia nigra. Rats were given four doses (every 3 h) of the Dl-seiective agonist, SKF 38393 (SKF; 18 mg/kg/dose). Reserpine (reserp; 5 mg/kg) was adn1inistered 30 min following the frrst dose of SKF 38393; rats were sacrificed 7 h following the last treatment. Results are expressed as percentage of the respective controls; each bar represents the mean ± S.E.M. for the individual treatment groups; n = 5 to 10. The control NTLI level was 879±42 pg/mg protein. *P<0.OO5 versus the vehicle-treated control. tP<O.OI versus the reserpine-treated group. 45 46 agonist, SKF 38393, to rats treated with reserpine completely prevented this DA depletion-induced decrease in the level of NlLI (Figure 3.6). Discussion The findings of the present study clearly demonstrate that dopamine Dl receptors modulate the activity of NT pathways in the striatum, the nucleus accumbens as well as the substantia nigra. However, two distinct functional links appear to be involved in this Dl regulation of NT activity. Firstly, stimulation of the Dl receptors causes an increase in the contents of NlLI above control levels in all three structures under study. Secondly, basal activity of endogenous DA on the Dl receptors appears to regulate the basal NT activity in the substantia nigra as its removal decreases the nigral NTLI content in this structure. Thus, enhanced dopamine transmission (induced by METH) raised the NlLI content in the striatum, the nucleus accumbens and the substantia nigra to 227 and 211 and 313% of the corresponding controls, respectively (Figure 3.1). That Dl receptor activation was the mechanism underlying these METH-induced increases was demonstrated as concurrent administration of the D1 antagonist, SCH 23390, totally blocked the increase in NlLI content caused by treatment with METH in all structures examined (Figure 3.1). Additional evidence for D I-mediated regulation of NT systems was the observation that the Dl-selective agonist, SKF 38393, also caused an increase in the content of NlLI in the striatum and the nucleus accumbens (Figure 3.3). Although the effects of SKF 38393 were qualitatively similar to METH in the striatum and the nucleus accumbens, quantitatively this direct agonist induced much smaller increases in the NlLI content of these structures than METH. A possible explanation is that SKF 38393 acts as a partial agonist of dopamine Dl receptors (Setler et al., 1978). In contrast, METH mediates its effects by endogenous DA which is much more effective at activating Dl receptors than SKF 38393 as judged by the efficacy of DA and SKF 38393 to activate adenylate cyclase in striatal slices (Setler et al., 1978). Unlike METH, SKF 38393 by itself did not increase the nigral NlLI content but its concurrent administration with the 47 D2 receptor-selective agonist, L Y 171555 (Itoh et al., 1985), caused the nigral NTLI level to increase to 136% of control; although D2 activation by itself significantly reduced nigral NlLI content (Figure 3.3). These findings suggested that activation of both D 1 and D2 receptors was necessary to obtain a significant rise in nigral NTLI content. This was surprising since the METH-induced increase in nigral N1LI content was completely antagonized by Dl receptor blockade (Figure 3.1) but not by D2 receptor blockade (Figure 1.1). A possible explanation is that although Dl receptors playa major role in regulating nigral NT systems, D2 receptors have a facilitatory function in this regulation. A variety of different interactions between D 1 and D2 receptors has been shown to modulate several behavioral as well as biochemical parameters. Thus, D 1 agonists facilitate D2 agonist-induced stereotyped behavior in rats (Amt et al., 1987). In addition, SKF 38393 enhances the ability of D2 receptor antagonists on striatal DA metabolite concentrations (Saller and Salama, 1985). Furthermore, D1 receptor blockade attenuates the biochemical effects of D2 receptor blockade both in vitro and in vivo (Saller and Salama, 1986). In contrast to the substantia nigra, Dl and D2 receptors in the striatum as well as the nucleus accumbens regulate associated NT systems in an antagonistic manner. Thus, activation of D1 and D2 receptors increased and decreased, respectively, the content of NTLI in both these strucutres and concurrent activation of the two receptor SUbtypes failed to cause any change in NlLI levels of these tissues (Figure 3.3). The antagonism seen in this study between the functional roles of the two DA receptor subtypes has also been reported for the striatal-nigral substance P projections (Sonsalla et al., 1984). Interestingly, the response of this substance P pathway was opposite to that of the NT systems described herein in that D 1 activation decreased whereas D2 activation increased the substan.ce P levels. Another major difference between the NT systems in the substantia nigra and the striatum or the nucleus accurnbens was the response of these systems to acute D 1 48 receptor blockade. Thus, nigral NTLI content decreased to about 80% of control following a single dose of SCH 23390 but the NTI...I levels in the striatum or the nucleus accumbens remained unaffected by such a treatment (Figure 3.4). This suggests that Dl receptors maintain basal regulation of NT systems in the substantia nigra but not in the striatum or the nucleus accumbens. This hypothesis was confIrmed when depletion of DA, following a single dose of reserpine, also reduced the nigral NTLI content to approximately 60% of control (Figure 3.5). That the reserpine-induced DA depletion was the mechanism underlying the reduction in the nigral NTI...I content was suggested by the temporal correlation between striatal DA depletion and alterations in the nigral NT activity (Figure 3.5). Thus, 18 h following a single dose of reserpine the striatal DA concentration was maximally decreased by about 92% of control which coincided with the decrease in nigral NTLI content. Interestingly, by 66 h the NTI...I level had completely recovered to the control value although the DA level had recovered to only 45% of control. This indicates that alterations in nigral NT systems induced by removal of basal DA activity are short-lived. This is supported by the observations that while a single dose of SCH 23390 caused a significant decrease in nigral NTI...I content (Figure 3.4), the effect was no longer present following multiple administrations of this drug (figures 3.1, 3.2, 3.4). Similarly, destruction of the nigrostriatal DA pathway did not affect the basal NTLI content in the substantia nigra (Figure 2.1), presumably because during the 1 week of post-surgery recovery, the nigral NT systems had recovered from the effects of DA depletion. The rapid recovery of nigra! NT systems possibly also explains the lack of an effect of treatments with reserpine plus aMpT on nigral NT activity (Figure 2.2) since the time of recovery in that study was approximately 42 h following the fIrst dose of reserpine compared to 18 h for the reserpine experiment shown in Figures 3.5 and 3.6. The role of D 1 receptors in this basal regulation of nigral NT projections was confinned when coadministration of the Dl-selective agonist, SKF 38393, with resetpine, completely antagonized the reserpine-induced decrease in the NTI...I content in 49 the substantia nigra (Figure 3.6). The observation that SKF 38393, a dopamine-specific drug, reversed the effect of reserpine further confmns the hypothesis that the reserpine­induced alterations in NTLI contents are mediated by specific changes in dopaminergic systems although reserpine also affects the activity of other monoaminergic pathways as discussed in Chapter 2. In summary, the results presented here conclusively demonstrate that DA D 1 receptors regulate the NT systems in the striatum, the nucleus accumbens and the substantia nigra. However, two distinct regulatory mechanisms are linked with the Dl receptor mediated interactions between DA and NT systems. Thus, activation of Dl receptors increased the NTLI contents of all three structures (although in the substantia nigra concurrent activation of D2 receptors is required for the Dl-mediated increases). In contrast, the Dl-mediated basal dopaminergic regulation of NT systems appeared to occur only in the substantia nigra. The significance of the D I-mediated alterations in tissue NTLI contents is not clear from these studies. Further research is required to understand if these changes represent alterations in the synthesis and/or release of the peptide to gain a better understanding of the complex interactions between NT and DA neuronal systems. 50 Re.ferences Amt, J., 1. Hyttel and 1. Perregaard, 1987, Dopamine D-1 receptor agonists combined with the selective D-2 agonist quinpirole facilitates the expression of oral stereotyped behavior in rats, Eur. 1. Pharmacol. 133, 137. Hyttel,1., 1983, SCH 23390-The fust selective D-1 antagonist, Eur. J. Pharmacol., 91, 153. Iorio, L.C., A. Barnett, F.H. Leitz, V.P. Hower and C.A. Korduba, 1983, SCH 23390, a potential benzozepine antipsychotic with unique interactions on dopaminergic systems, J. Pharmcol. Exp. TIler. 226, 462. Itoh, Y, M.E. Glodman and J.W. Kebabian, 1985, TL 333, a benzhydro[G] quinoline stimulates both D-1 and D-2 receptors: Implications for the selectivity of LY 141865 towards the D-2 receptors, Eur. J. Phannacol. 108, 99. Kebabian, J.W. and D.B. CaIne, 1979, Multiple receptors for dopamine, Nature (Lond.) 261, 717. Lazareno, S., D.B. Marriott, and S.R. Nahorski, 1985, Differential effects of selective and non-selective neuroleptics on intracellular and extracellular cyclic AMP accumulation in rat striatal slices, Brain Res. 361,91-98. Letter, A.A., L.A. Matsuda, K.M. Merchant, J.W. Gibb and G.R. Hanson, 1987a, Characterization of dopaminergic influence on striato-nigral neurotensin systems, Brain Res.422, 200. Letter, A.A., K. Merchant, J.W. Gibb and G.R. Hanson, 1987b, Effect of methamphetamine on neurotensin concentrations in rat brain regions, J. Pharmacol. Exp. Ther. 241, 443. Merchant, K.M., A.A. Letter, J.W. Gibb, and G.R. Hanson, 1988, Changes in the limbic neurotensin systems induced by dopaminergic drugs, Eur. J. Pharmacol. 153, 1. Ritter, 1.K., C.J. Schmidt, J.W. Gibb, J.W. and G.R. Hanson, 1984, Increases of substance P-like immunoreactivity within striatal-nigral structures after subacute methamphetamine treatment, J. Pharmacol. Exp. Ther. 229, 487. Roth, R., 1979, Dopamine autoreceptors: pharmacology, function and comparison with post-synaptic dopamine receptors, Commun. Psychophannacol. 3,429. Saller, C. and A. Salama, 1985, Dopamine receptor subtypes: in vivo biochemical evidence for functional interaction, Eur. J. Pharmacol. 109,297. Saller, C. and A. Salama, 1986, D-1 and D-2 dopamine receptor blockade: interactive effects in vitro and in vivo, J. Pharmacol. Exp. Ther. 236, 714. Scatton, B., 1981, Differential changes in DOPAC levels in the hippocampal fonnation, septum and striatum of the rat induced by acute and repeated neuroleptic treatment, European 1. Pharmacol. 71,499. 51 Schmidt, C.J., J.A. Levin, and W. Lovenberg, 1987, In vitro and in vivo neurochemical effects of methylenedioxymethamphetamine on striatal monoaminergic systems in the rat brain, Biochem. Pharmacol. 36, 747. Seder, P.E., H.M. Sarau, H.L. Zirkle and S.L. Saunders, 1978, The central effects of a novel dopamine agonist, Eur. 1. Pharmacol. 50,419. Sonsalla, P.K., 1.W. Gibb and G.R. Hanson, 1984, Opposite responses in the striato­nigra! substance P system to Dl and D2 receptor activation, European J. Pharmacol. 105, 185. Starke, K., W. Reimann, A. Zumstein and G. Hertting, 1978, Effect of dopamine receptor agonists and antagonists on release of dopamine in rabbit caudate nucleus in vitro, Naunyn Schmiedeberg's Arch. Pharmacol. 305, 27. CHAPTER 4 ROLE OF D2 RECEPTORS IN REGULATION OF NEUROTENSIN SYSTEMS Introduction As shown in Chapter 3 (Figure 3.1), the effects of METH on the NT systems of the striatum, the nucleus accumbens and the substantia nigra appear to be mediated by activation of DA D1 receptors likely by the massive release of endogenous DA induced by the drug (Schmidt et al., 1987). Evidence for the role of D 1 receptors in the regulation of NT activity in these structures is that treatment of rats with the Dl receptor-selective agonist, SKF 38393, also induces significant increases in the NTLI levels of these structures (Figure 3.3). Surprisingly, like METH, treatment of animals with the DA D2 receptor-selective antagonist, sulpiride (SULP), or the nonselective neuroleptic, haloperidol, also elevates the NTLI conten t of the striatum and the nucleus accumbens (Figure 1.1, Govoni et aI., 1980; Frey et al., 1986, Letter et al., 1987; Merchant et al., 1988). The finding that both a DA agonist and antagonist cause similar changes in the NT systems of the striatum and the nucleus accumbens suggests that two distinct mechanisms are responsible for these effects. This is supported by the observation that coadministration of SULP adds to the MElli-induced increases in the NTLI contents of the striatum and the nucleus accumbens, (Figure 1.1, Letter et al., 1987, Merchant et al., 1988). Since evidence presented in Chapter 3 supports the hypothesis that the effects of METH are mediated by the activation of D 1 receptors (Figure 3.1) and because D 1 and D2 receptors often have antagonistic actions (Sonsalla et al., 1984), it was hypothesized that the neuroleptic-induced blockade of tonic D2 activity causes elevation of NT content. 53 This is supported by several observations: (1) blockade of the D2 receptors elevates NTLI content of the striatum and the nucleus accumbens whereas blockade of the D 1 receptors has no effect on the NTLI levels of these structures (Figures 1.1, 3.4), (2) eliminaton of more than 85% of central dopaminergic transmission causes a several-fold increase in striatal NTLI content and subsequent treatment \\~th SULP or haloperidol does not add to this effect (Figure 2.1) and (3) concurrent block:~e of the D 1 receptors with SCH 23390 does not antagonize SULP-induced increase in the NTLI content of the striatum and the nucleus accumbens (Figure 3.2). The present study was perfonned to test the possibility that elimination of tonic D2 activity was the mechanism by which neuroleptics alter the NTLI contents of the striatum and the nucleus accumbens. Basal dopaminergic activity was eliminated by treatment with reserpine and the effects of concurrent administration of the D 1 or D2 selective agonists, SKF 38393 or L Y 171555, respectivaly, were studied. Materials and Methods Drugs Chapter 3 lists the sources and the vehicles used for each drug employed in this study. Animals Male Sprague Dawley rats (190-240 g) were used for the entire study as described in Chapter 2. Treatments Recovery correlation between dopamine depletion induced by reserpine and NTLI contents of the striatum and the nucleus accumbens. Following a single dose of reserpine (5 mg/kg), rats were killed at 18, 66 or 186 h and tissue DA and NTLI levels were determined. 54 Administration of a selective D1 or D2 agonist with reserpine. Animals were given four doses of SKF 38393 or L Y 171555 (5 mg/kgldose) with a 3-h interval between each dose. Reserpine was administered as a single dose (5 mg/kg) 30 min following the first dose of the agonists and animals were sacrificed 6-7 h after the fmal administration of the agonist. Treatment with SCH 23390 plus reserpine or SCH 23390 plus SULP. A single dose of SCH 23390 (0.5 mg/kg) was given to rats 30 min prior to a single administration of reserpine (5 mg/kg). SULP (80 mg/kg/dose) was administered in three doses (6-h intervals). At each dosing interval, SCH 23330 (0.5 mg/kgldose) was administered 30 min prior to SULPa Rats were killed 18 h following the final treatment. Methods for dissections, determination of NILI and DA contents and statistical analysis are detailed in Chapter 2. Results Recovery correlation between dopamine depletion induced by reserpine and mu content of the striatum and the nucleus accumbens Eighteen hours following a single dose of reserpine, N1LI levels of the striatum and the nucleus accumbens were maximally increased to 586% and 169% of control, respectively (Figure 4.1). This maximal response by the NT systems in both structures coincided with the maximal depletion in striatal DA content (92~ of control) induced by the drug. By 66 h, the N1LI level in the nucleus accumbens and the striatum recovered and were not significantly different from their respective controls. Interestingly, although the NT systems appeared to have recovered at this time point, the DA level in the striatum was only back to 39% of the control value. --e. c 0 c;,J c... o Q) e.o S c Q.J ~ Q.J c.. "-' c:: .S2 ''E L. C Q.J u C 0 u ~ 0 rn -..-, 120 s--· 700 c:: 3 z 100 I ~ 600 t- ~ "-' .... -. ::s ~ 80 500 r:: r:s a. ~ "'=" ~ -::s 0 400 &1 ~ ..;:.; 60 ~ 0;; ** ~ ., . ~ 0 ~ ..... ....., ...................... .. 300 c , § 40 3 Cf -- 200 ~::s e. -rJ:.I "-' 20 100 ! "-' 0 0 24 72 120 168 hours following treatment Figure 4.1: Recovery correlation between striatal DA depletion induced by reserpine and NTLI content of the striatum and the nucleus accumbens. Animals were given a single dose of reserpine (5mglkg) and sacrificed at 18, 66 or 186 h following the treatment. The graph shows the correlation between striatal DA depletion and increases in the NTLI content of the striatum and the nucleus accumbens expressed as mean ± S.E.M. The data are expressed as percentage of the re,~pective controls; n = 4 to 10. The control DA content in the striatum was 8.06 J,lg/g of tissue, the control NTLI contents expressed as pg/mg protein were: striatum=185±26 and nucleus accumbens=741±111. *P<O.005, **p<O.OO 1 versus the corresponding controls (values represented by the 0 h time point). 55 Effect of concurrent activation of selective DA receptors on alterations in NTLJ levels induced by Mpamine depletion 56 A single dose of reserpine depleted the striatal DA content by approximately 90% (control=8.28 ± 0.45; reserpine=O.86 ± 0.29 flg/g tissue) and induced increases in the NlLI content of the striatum and the nucleus accumbens which were 467% and 163% of control, respectively (Figure 4.2). Administration of multiple doses of the D2-selective agonist, L Y 171555, completely blocked the elevation in NTLI levels caused by reserpine-induced DA depletion in both structures. The concentration of DA in the group of animals treated with LY 171555 plus reserpine were similarly depressed (0.65 ± 0.37 Jlg/g tissue) as those in the group that received reserpine alone. In contrast, administration of multiple doses of the D I-selective agonist, SKF 38393, with reserpine, only attenuated the increase in striatal NlLI content induced by reserpine (from 385% to 277% of control, Figure 4.2). In addition, the increase in NTLI level of the nucleus accumbens following rese.rpine-induced DA depletion was not affected by activation of the Dl receptors. The extent of striatal DA depletion following reserpine treatment was similar to the study with L Y 171555 (control=8.4±0.39, reserpine=O.36±.04 and reserpine+SKF 38393=O.33±O.02 Jlg/g tissue) In contrast to the striatum and the nucleus accuInbens, a single dose of reserpine decreased nigral NTLI content to 70% of control. Multiple administrations of LY 171555 also significantly reduced the NTLI level to 80% of control and its coadministration with reserpine did not cause the nigral NILI level to recover to control value. On the other hand, treatment with SKF 38393 in combination with reserpine completely blocked the reserpine-induced decrease in the NlLI level. The results with SKF 38393 and reserpine have been shown previously in Chapter 3 (Figure 3.6) and are repeated here to facilitate their comparison with the effects of LY 171555 on reserpine-induced decrease in nigral NTLI content. ~-----------------------------------,-------------~ striatum 500 400 300 200 100 04-~--~~~~--__ ~~~~auUL--~ g 300 o= CJ c.­o ~ ~ 200 100 ** nucleus accumbens * ~ 04-~--~~~~----~~~~~UL--~ Z 160------------------- substantia nigra t 120 * 80 40 D-l activation D-2 activation D control ~ reserpine ~ SKF 38393 ~ LY 171555 ~ reserp+ SKF [ll reserp+L Y 57 Figure 4.2: Effect of selective activation of DA receptors on reserpine-induced alteration in tissue NlLI content. Rats were given four doses (every 3 h) of SKF 38393 (SKF, 18 mg/kg/dose) or LY 171555 (L Y; 5 mg/kg/dose). Reserpine (5 mg/kg) was administered 30 min following the frrst dose of the agonists; rats were sacrificed 7 h following the last treatment. Results are expressed as percentage of the respective controls; each bar represents the mean ± S.E.M. for the individual treatment groups; n = 4 to 10. The average control NTLI levels expressed as pg/mg protein were: striatum=232±16 and nucleus accumbens=807±99 and substantia nigra=879±46. *P<0.02, **P<O.OOl versus the vehicle-treated control. tP<0.OO5, ttP<O.OOl versus the corresponding reserpine-treated group. Effect of DA D 1 receptor blockade on reserpine- or SULP -induced increases in tissue NTil levels 58 In order to evaluate the role of the D 1 receptors in the effects of reserpine and SULP on NT systems of the striatum and the nucleus accumbens, treatment with the D1 receptor antagonist, SCH 23390, was begun 30 min prior to the administration of reserpine or SULP. The results with SULP have been shown previously in Chapter 3 (Figure 3.2) and are repeated here to facilitate comparisons between reserpine and SULP. Reserpine treatment induced NT increases that were 586% and 169% of control in the striatum and the nucleus accumbens, respectively; SULP increased NlLI content to 204% and 163% of control in the respective tissues (Figure 4.3). Blockade of the D1 receptors did not significantly m<Xiify the NTLI levels of the striatum or the nucleus accumbens by itself and had no effect on the elevation of NTLI contents seen following reserpine or SULP treatments (Figure 4.3). Discussion The present results confrrm the hypothesis that the basal release of DA exerts tonic regulation of the NT systems in the striatum and the nucleus accumbens. Thus, elimination of dopaminergic influence by depletion of DA in the brain with reserpine alone (Figures 4.1,4.2,4.3), caused substantial increases in NT content of the striatum and the nucleus accumbens. This supports the results shown in Chapter 2 that destruction of the nigrostriatal DA pathway with 6-hydroxydopamine or nonselective depletion of DA with reserpine plus a-methyl-p-tyrosine substantially increased the NTLI content of these structures. The association between DA depletion and increases.in NTLI content in the striatum and the nucleus accumbens was confirmed by correlating the 700 striatum o control 600 121 reserpine 500 II SULP E3 SCH 23390 400 ~ SCH+reserp ..- 300 * -..0r... . 200 * DI1 SCH+SULP = 0 (J 100 '- 0 0 ~ ~ ..=... nucleus accumbens =(~J 200 ** ** r. ~ -Q. * * ...... .J E-c :z 100 o reserpine SULP Figure 4.3: Effect of DA-Dl receptor blockade on reserpine- or SULP-induced increases in NTLI levels of the striatum and the nucleus accumbens. Animals were given a single dose of reserpine (5mg/kg) 30 min following a single dose of SCH 23390 (0.5 mg/kg). SULP was adminstered every 6 h for three administrations and at each dosing interval SCH 23390 (0.5 mg/kg/dose) was administered 30 min prior to SULP. The rats were sacrificed 18 h after the fmal treattnent. Each column represents the mean ± S.E.M. for treatment groups expressed as percentages of the respective controls; n = 4 to 10. The control NTLI levels expressed as pg/mg protein were: striatum=231±30 and nucleus accumbens=896±148. *P<0.02, **P<O.OOl versus the respective vehicle-treated controls. 59 60 recoveries of striatal DA levels and NlLI concentrations following reserpine treatment (Figure 4.2). Maximum increases in NTLI levels occurred at 18 h following drug administration and corresponded with the greatest depletion (92%) of DA in the striatum (and likely the nucleus accumbens as well). By 66 h, striatal DA had returned to 39% of control and the NTLI levels in the striatum and the nucleus accumbens had returned to control. These fmdings suggest that DA pathways operating at fractional capacity are able to maintain normal regulation of associated NT systems. This hypothesis is confmned by the results displayed in Figure 2.1: greater than 85% destruction of the nigrostriatal DA pathway was required before significant changes occurred in the striatal NTLI levels. These results are consistent with other reports that only a fraction of surviving DA neurons are sufficient to exert normal regulation over other associated transmitter systems and functions (Hefti et al., 1985; Sonsalla et aI., 1984). The mechanism responsible for changes in the NlLI levels of the striatum and the nucleus accumbens following DA depleting strategies appear to be similar to those underlying the effects of neuroleptics on NT systems. This is supported by the observation that coadministration of high doses of both haloperidol or SULP did not significantly alter the increase in NILI content of the striatum and the nucleus accumbens which resulted from DA-depleting treatments such as 6-0HDA lesions (Figure 2.1) or reserpine plus aMpT administrations (Figure 2.2). Often, if distinct mechanisms mediate similar effects by two separate drugs, the coadministration of these drugs at the maximum-effective doses will cause an additive effect. Such summation of effects did occur in the response of NT systems to concurrent administration of neuroleptics with METH (Figure 1.1), demonstrating that the actions of these two drug types on NT content are mediated by different mechanisms. The study represented by Figure 4.3 further supports the hypothesis that NT changes induced by depletion of DA and treatment with neuroleptic agents occur by a common mechanism. Thus, blockade of the D 1 receptors by coadministration with SCH 23390 did not alter the NT response to either 61 reserpine or SULP administration. Consequently, unlike the METH-induced increase in tissue NTLI level, D1 receptors do not appear to playa role in the response of NT systems to either neuroleptic treatment or DA depletion. Due to the lack of an effect by SCH 23390 on either reserpine or SULP-induced increases in NT content of the striatum and the nucleus accumbens (Figure 3.4), the basal dopaminergic regUlation of these peptide systems is likely a D2-regulated phenomenon. This was confirmed by the study represented by Figure 4.2. If the alterations in NT activity caused by reserpine or the neuroleptics were due to interruption of tonic D2 activity, the coadministration of a D2-selective agonist, such as LY 171555, should prevent the increase resulting from reserpine-mediated DA depletion. As shown in Figure 4.2, multiple administrations of this D2 agonist totally blocked the responses to reserpine by the NT systems in the striatum and the nucleus accumbens. However, the decrease in nigral NTLI content caused by reserpine was not prevented by concurrent activation of D2 receptors. It was critical to have concurrent maximal stimulation of the D2 receptors to block the striatal effects of reserpine since administration of a D2 agonist 6 h following reserpine did not antagonize the effects of the latter on NILI level in the striatum (data not shown). This is in agreement with the findings in Chapter 2 that restoration of DA synthesis with L-DOPA following treatments with reserpine plus aMpT did not alter the increase in striatal NTLI content caused by depletion of DA (Figure 2.2). Administration of the D I-selective agonist, SKF 38393, on the other hand, only attenuated the striatal response to reserpine and did not affect the increase in the NILI content of the nucleus accumbens caused by reserpine (Figure 4.2). However, the nigral decrease was totally blocked by concurrent administration of SKF 38393. These data suggest that in the striatum, although NT regulation by basal dopaminergic activity is mediated primarily by the D2 receptors, the Dl receptors may contribute to this regulation. As discussed in Chapter 3, such biochemical interactions between DA-Dl and D2 receptors are frequently observed in the CNS. In contrast, the basal dopaminergic regulation of NT systems 62 appears to be mediated by only D2 receptors in the nucleus accumbens and only Dl receptors in the substantia nigra. The significance of such tissue-specific, differential regulatory effects of DA on NT pathways is not very clear from these data. However, these results are in agreement with previous studies of the differential effects of centrally administered NT on dopaminergic activity of discrete brain areas. Thus, administration of NT in the nucleus accumbens antagonizes the stimulatory effects of d-amphetamine (Ervin et al., 1981) but intranigral injection of NT increases the activity of dopaminergic neurons in this brain structure (Pinnock, 1985). It should be mentioned that in the experiment represented by Figure 4.2, administration of LY 171555 or SKF 38393 alone did not have a significant effect on NTLI content of the striatum or the nucleus accumbens. In a previous study shown in Chapter 3 it was observed that activation of the D 1 receptors causes an increase while stimulation of the D2 receptors causes a decrease in the NTLI content of both these structures (Figure 3.3). This apparent discrepancy is likely due to differences in the dosing protocols used for the two experiments. In the previous study, animals were treated with three doses of SKF 38393 or L Y 171555 every 6 h and sacrificed 18 h following the drug treattnent (Le., a total of 30 h after the first drug administration). In the present study, four doses of these drug were given at 3- h intervals and the rats were sacrificed 7 h after the final injection (i.e., a total of 16 h after the first drug dose). It has been reported that the response of other peptide systems (e.g., changes in striatal substance P content) to DA agonists requires long-term treatment protocols (Ritter et al., 1984; Sonsalla et al., 1986); perhaps a similar duration of receptor activation is necessary before changes in N1LI levels occur. It is noteworthy that while the responses of NT systems associated with the striatum and the nucleus acccumbens were qualitatively similar, the percentage increases of NT content in the striatum were always substantially greater than those observed in the nucleus accumbens. These differences in the response of the two structures occurred following both D2 receptor blockade (Figure 4.3) and DA depletion (Figures 4.1, 4.2, 63 4.3). The significance of these quantitative differences are not apparent from this study. They might reflect variability in 02 receptor populations, with the striatum reported to have the higher density (Oehlert and Wamsley, 1985); consequently, interference with 02 receptor functioning might have a greater effect in this structure. Another possible explanation is that there exists a difference between the striatum and the nucleus accumbens in the physiological extent to which neurotensin synthesis/release are under dopaminergic control. A third possibility is that while changes in NTLI levels expressed as percent are much greater in the striatum, the changes expressed in terms of absolute NTLI tissue content are quite similar in both structures. This is due to the fact that the control level of NlLI in the nucleus accumbens is approximately three to four times greater than that in the striatum. In summary, the [mdings in this study clearly demonstrate that basally released OA exerts tonic regulation of NT systems in the striatum and the nucleus accumbens. However, unlike the substantia nigra where 01 receptors mediate basal dopaminergic regulation of the NT systems, primarily 02 receptors appear to be involved in basal regulation of NT systems in the striatum and the nucleus accumbens. Thus, blockade of this D2 dopaminergic tone is the mechanism underlying the neuroleptic-induced increases in the NTLI contents of the striatum and the nucleus accumbens (Figure 1.1). In view of the possible role of NT as an endogenous neuroleptic, the findings of this chapter are important. Since neuroleptics exert their effects primarily by blocking the D2 receptors, it is likely that alterations in the activity of NT pathways are involved in some of the clinical effects of the neuroleptics. In addition, monitoring of the changes in the NT systems may be a convenient means to assess neurochemically the postsynaptic consequences of alterations in the activity of the OA receptors in some brain structures. The results presented here are not sufficient to indicate the mechanism(s) underlying the increases in NTLI contents induced by removal of the 02 dopaminergic tone (whether the changes reflect alterations in synthesis or release of the peptide). Clarifications of such 64 mechanisms would help to elucidate the nature of interactions between NT and DA transmitter systems and likely contribute to an understanding of normal as well as pathological states of eNS dopaminergic systems. 65 References Ervin, G.N., L.S. Birkemo, C.B. Nemeroff and AJ. Prange, Jr., 1981, Neurotensin blocks certain amphetamine-induced behaviours, Nature 291, 73. Frey, P., K. Fuxe, P. Eneroth and L.F. Agnati, 1986, Effects of acute and long-tenn treatment with neuroleptics on regional telencephalic neurotensin levels in the male rat, Neurochem. Int. 8, 429. Frey, P., M. Lis and D.M. Coward, 1988, Neurotensin concentrations in rat striatum and nucleus accumbens: further studies of their regulation, Neurochem. Int. 12, 33. Gehlert, D.R. and lK. Wamsley, 1985, Dopamine receptors in the rat brain: quantitative autoradiographic localization using [3H]sulpiride, Neurochem. Int. 7, 717. Govoni, S., J.S. Hong, H.Y.-T. Yang and E. Costa, 1980, Increase of neurotensin content elicited by neuroleptics in nucleus accumbens, J. Pharmacol. Exp. Ther. 215, 413. Hefti, B., A. Enz and E. Melamed, 1985, Partial lesions of the nigrostriatal pathway in the rat. Acceleration of transmitter synthesis and release of sUlViving neurons by drugs, Neuropharmacol. 24, 19. Lazareno, S., D.B. Marriott and S.R. Nahorski, 1985, Differential effects of selective and non-selective neuroleptics on intracellular and extracellular cyclic AMP accumulation in rat striatal slices, Brain Res. 361, 91. Letter, A.A., K.M. Merchant, J.W. Gibb and G.R. Hanson, 1987, Effect of methamphetamine on neurotensin concentrations in rat brain regions, J. Pharmacol. Exp. Ther. 241, 443. Merchant, K.M., A.A. Letter, J.W. Gibb and G.R. Hanson, 1988, Changes in the limbic neurotensin systems induced by the dopaminergic drugs, Eur. J. Pharmacol. 153, 1. Pinnock, R.D., 1985, Neurotensin depolarizes substantia nigra dopamine neurons, Brain Res. 338, 151. Ritter, lK., C.J. Schmidt, J.W. Gibb and G.R. Hanson, 1984, Increases of substance P-like immunoreactivity within striatal-nigra! structures after subacute methamphetamine treatment, 1 Pharmacol. Exp. Ther. 229, 487. Scatton, B., 1981, Differential changes in DOPAC levels in the hippocampal fonnation, septum and striatum of the rat induced by acute and repeated neuroleptic treatment, European J. Pharmacol. 71, 499. Schmidt, C.l, lA. Levin and W. Lovenberg, 1987, In vitro and in vivo neurochemical effects of methylenedioxymethamphetamine on striatal monoaminergic systems in the rat brain, Biochem. Pharmacol. 36, 747. Sonsalla, P.K., J.W. Gibb and G.R. Hanson, 1986, Nigrostriatal dopamine actions on the D2 receptors mediate methamphetamine actions on the striatonigral substance P system, Neuropharm. 25, 1221. 66 Sonsalla, P.K., J.W. Gibb and G.R. Hanson, 1984, Opposite responses in the striato­nigra! substance P system to Dl and D2 receptor activation, European J. Phannacol. 105, 185. Starke, K., W. Reimann, A. Zumstein and G. Hertting, 1987, Effect of dopamine receptor agonists and antagonists on release of dopamine in rabbit caudate nucleus in vitro, Naunyn Schmiedeberg's Arch. Phannacol. 305, 27. CURRICULUM VITAE Personal Information: Name: Kalpana Mahesh Merchant Date of birth: Nov 12, 1957 Education: June 1973 - June 1979 Sept. 1985 - present Research and/or Professional Experience: Aug. 1986 - present June 1985 - July 1986 March 1985 - May 1985 Place of birth: Bombay, India Bachelor of Pharmacy University of Bombay, Bombay, India. Class placed in: First (equivalent to Grade A) Ph. D. in Pharmacology, Aug. 1989 University of Utah School of Pharmacy, Salt Lake City, Utah 84112 Graduate research under the guidance of Dr. G. R. Hanson, Dept. Pharmacol. and Toxicology, University of Utah School of Pharmacy. The project involves characterization of mechanisms underlying the interactions between the neuroactive peptide, neurotensin, and dopamine pathways in the rat brain. Some of the techniques employed are: HPLC-ECD, radioimmunoassay, radioenzymatic assay, receptor autoradiography and DNAJRNA hybridization. Worked as a research assistant under the guidance of Dr. G.R. Hanson, U niversi ty of Utah, Salt Lake City, Utah. Assisted in the development and characterization of immunoglobulins against several neuroactive peptides. Studied the effects of dopamine-modulating drugs on the activity of peptides such as neurotensin, substance P and substance K in discrete brain areas. Worked as a voluntary technician in the laboratory of Dr. W. Nichols, University of Utah, Salt Lake City, Utah. Dec. 1981 - Dec. 1984 Jan. 1981 - May 1981 Aug. 1979 - Dec. 1980 Honors: 1976-1977: 1985-1986: 1987-1988: 1988-1989: Publications: Manuscripts: Helped set up and maintain primary cultures of epithelial cells. 68 Set up and worked as a partner for Wellchem Laboratories in Bombay, India. Helped in the development and manufacturing of fine chemicals and drugs such as ampicillin. Special Project Development Officer, Wyeth Laboratories, Bombay, India. Developed a pharmaceutical product for exports. Research and Development Officer, Boots Company India Ltd., Bombay, India. Developed several formulaions for topical applications and carried out shelf-life studies on the same. University of Bombay Scholarship NIH Predoctoral Training Grant recipient Osco Skaggs Fellowship University of Utah Graduate Research Fellowship Hanson, G. R., A. A. Letter, K. M. Merchant and J. W. Gibb, 1987, Comparison of responses by striatonigral substance P and neurokinin A systems to methamphetamine treatment, Peptides, 7, 983-987. Letter, A. A., K. M. Merchant, J. W. Gibb and G. R. Hanson, 1987, Effects of methamphetamine on neurotensin concentrations in rat brain, J. Pharmacol. Exp. Ther., 241,443-447. Merchant, K. M., A. A. Letter, M. Johnson, D. M. Stone, J. W. Gibb and G. R. Hanson, 1987, Effects of amphetamine analogues on neurotensin concentrations in rat brain, European J. Pharmacol., 138, 151-154. Letter, A. A., L. A. Matsuda, K. M