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The neurotensin antagonist SR 48692 attenuates haloperidol-induced striatal Fos expression in the rat
Neuroscience Letters 303 (2001) 17±20
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The neurotensin antagonist SR 48692 attenuates haloperidolinduced striatal Fos expression in the rat Jim Fadel a,*, Paul R. Dobner b, Ariel Y. Deutch a a
Departments of Psychiatry and Pharmacology and Center for Molecular Neuroscience, Vanderbilt, University Medical Center, Nashville, TN 37212, USA b Department of Molecular Genetics and Microbiology and Program in Neuroscience, University of Massachusetts Medical Center, Worcester, MA 01655, USA Received 1 February 2001; received in revised form 26 February 2001; accepted 27 February 2001
Abstract Neurotensin interacts with central dopamine systems and has been suggested to exert antipsychotic drug-like actions. Antipsychotic drugs such as haloperidol induce striatal immediate-early gene expression. In order to study neurotensin's role in antipsychotic drug actions, rats were pretreated with the neurotensin antagonist SR 48692 and then injected with haloperidol. SR 48692 dose-dependently decreased haloperidol-elicited immediate-early gene expression in the dorsolateral and central striatum but not other striatal areas. SR 48692 reduced Fos expression in the striatal patch (striosome) and matrix compartments, with a signi®cantly greater effect in the patch. These data suggest that neurotensin may play a role in the actions of haloperidol. In view of proposed functional roles of the striatal patch and matrix, we suggest that neurotensin may be important in the therapeutic rather than side effects of antipsychotic drugs. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antipsychotic drugs; Fos; Dopamine; Neurotensin; Schizophrenia; Striatum; Striosome
Neurotensin (NT) is a tridecapeptide transmitter that is widely distributed in the brain [13]. Neurotensin interacts extensively with and in some cases is colocalized in dopamine (DA) neurons [12,15,16]. Several indirect but converging lines of evidence have suggested that NT may play an important role in the therapeutic effects of antipsychotic drug (APD) treatment. Thus, many of the behavioral effects observed in rodents that are administered APDs are also seen after intraventricular injection of NT or NT agonists [14,16]. Moreover, cerebrospinal ¯uid levels of NT are reduced in schizophrenic patients, particularly those with prominent negative symptoms, and tend to normalize with APD treatment [11] Based on these and other data NT has been advanced as an endogenous APD-like peptide [16]. All clinically effective APDs induce the immediate-early gene c-fos and its protein product Fos in the forebrain of mammals with a characteristic regional pattern of expression [7]. Typical APDs, which have substantial extrapyramidal side effect (EPS) liability, induce Fos in the * Corresponding author. Psychiatric Hospital at Vanderbilt, Suite 313 1601 23rd Ave South Nashville, TN 37212, USA. Tel: 11-615-327-7092; fax: 11-615-3221901. E-mail address: [email protected] (J. Fadel).
dorsolateral striatum and nucleus accumbens, whereas atypical APDs induce Fos in the shell of the nucleus accumbens (NAS) but not the dorsolateral striatum [7,8]. These APDs also induce neurotensin/neuromedin N (NT/NMN) mRNA and protein in a similar regionally-speci®c manner, with Fos being induced in most of these NT-like immunoreactive (-li) neurons [4,18]. In view of the suggestion that NT may have endogenous APD-like actions, we sought to determine if pre-treatment with an antagonist of the high-af®nity neurotensin receptor (NTR1) would reduce haloperidol-induced Fos expression in the striatal complex. We further determined if pre-treatment with the NTR1 antagonist SR 48692 would differentially alter haloperidol-elicited Fos expression in the patch (striosome) and matrix compartments of the striatum. These two striatal compartments appear to subserve different functions [6,19,21]. Neurons of the patch have been related to affective processes as well as expression of complex motor patterns, while the cells in the matrix compartment appear to be involved with simple motor behavior. Male Sprague±Dawley rats weighing 250±300 g (Harlan, Birmingham, AL, USA) were housed on a constant 12:12 h light:dark cycle with food and water available ad libitum.
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 70 8- 6
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J. Fadel et al. / Neuroscience Letters 303 (2001) 17±20
Experiments were conducted in accord with institutional Animal Care and Use Committee-approved protocols. Rats were injected with SR 48692 (Sano® Recerche, 0.5 or 2.5 mg/kg, intraperitoneally (i.p.)) or its vehicle (~ 50 ml Tween 20 in 5.0 ml distilled water), followed 30 min later by injection of haloperidol (Research Biochemicals International, Natick, MA; 1.0 mg/kg, i.p.) or vehicle (distilled water acidi®ed with acetic acid to pH ~6.5). Two hours after the second injection, animals were anesthetized with iso¯urane and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After post-®xation and cryoprotection of the brains, serial 40 mm sections were cut on a freezing microtome through the striatal complex and processed to reveal Fos- and FosB-li nuclei, as described previously [8]. Following the development of the sections in diaminobenzidine containing nickel and cobalt to reveal black Fos-li nuclei [8], the sections were washed extensively, incubated in a m-opioid receptor (MOR) antibody (a gift of Dr Robert Elde, University of Minnesota), processed, and developed in diaminobenzidine to stain MOR-li patches brown. The numbers of Fos-li neurons were counted in the dorsolateral, central, ventrolateral, and medial striatum at the level of the crossing of the anterior commissure. Fos-li neurons were also counted in the shell and core of the nucleus accumbens. The data were expressed as Fos-li neurons/mm 2 and were analyzed by analyses of variance; group comparisons were made when indicated using Bonferroni t-tests. Haloperidol signi®cantly increased the number of Fos-li neurons in all four striatal areas and the two accumbal sectors (see Fig. 1); similarly, FosB was induced by haloperidol in all four striatal sectors. SR 48692 pre-treatment signi®cantly reduced both haloperidol-elicited Fos and FosB induction in the dorsolateral striatum (Fig. 1; Table 1). The high but not low dose of SR 48692 also signi®cantly decreased haloperidol-elicited Fos induction in the central
Fig. 1. The effects of SR 48692 (SR) pre-treatment on haloperidol (HAL)-elicited striatal Fos induction (n 4/group). Haloperidol signi®cantly increased Fos expression in all sectors of the striatum. A dose-dependent suppression of the density of Fos-li neurons in the dorsolateral (DL) and central (C) but not the ventrolateral (VL) or medial (M) striatum was observed. *P # 0:05 relative to vehicle-haloperidol (V-HAL) group.
Table 1 The effects of SR 48692 pre-treatment on the number of FosB-li neurons in the striatum of haloperidol-treated rats a
V-V SR-V V-H SR-H
Dorsolateral
Central
Ventrolateral
Medial
0.5 ^ 0.3 0.3 ^ 0.3 25.3 ^ 5.2* 6.3 ^ 1.9*#
0.3 ^ 0.3 0.8 ^ 0.5 19.8 ^ 1.7* 13.5 ^ 4.1*
0.3 ^ 0.3 0.3 ^ 0.3 14.3 ^ 4.5* 7.8 ^ 1.4*
2.0 ^ 1.2 4.3 ^ 2.1 16.8 ^ 2.2* 15.8 ^ 2.2*
a Numbers (mean ^ SEM) of striatal FosB-li neurons/mm 2 in vehicle-vehicle (V-V), SR 48692-vehicle (SR-V), vehicle-haloperidol (V-H), and SR 48692-haloperidol (SR-H) treated rats (n 4/ group). The NT antagonist SR 48692 (2.5 mg/kg) was administered 30 min prior to haloperidol (1.0 mg/kg) *P , 0:05 vs. V-V #P , 0:05 vs. V-H.
striatum. The NTR1 antagonist did not modify the ability of haloperidol to increase the number of Fos- or FosB-li neurons in the medial or ventrolateral striatum or the NAS shell or core (data not shown). SR 48692 had no effect of its own on the number of Fos- or FosB-li neurons in any area. Pre-treatment with SR 48692 had a differential effect on haloperidol-induced Fos expression in the patch and matrix compartments of the dorsolateral striatum (see Figs. 2 and 3). Although pre-treatment with SR 48692 signi®cantly reduced Fos expression in both striatal compartments, the effect was disproportionate: a preferential effect was seen in the patch compartment, with a resultant signi®cant decrease in the ratio of Fos-li neuron density in patch:matrix (see Figs. 2 and 3). These data reveal that pre-treatment with the NT antagonist SR 48692 attenuates the ability of the typical APD haloperidol to induce Fos in neurons of the dorsolateral and central striatum, and suggest that functional NT transmission is necessary for the full actions of haloperidol. The anatomical speci®city of this effect is consistent with the pattern of striatal NT expression induced by haloperidol or other D2 antagonists [4,18], such that signi®cant changes were observed only in the dorsolateral quadrant of the striatum. Haloperidol and other D2 receptor antagonists induce both Fos and NT expression in the striatum. It appears that D2 receptor antagonists induce NT in two populations of cells. One population is found in the medial striatum; these cells are primarily located in the matrix compartment and rarely express Fos in response to D2 antagonist challenge [4]. A second more prominent population is seen in the dorsolateral striatum. These cells express Fos in response to haloperidol challenge and project to the globus pallidus [4]. It seems likely that SR 48692 primarily reduces Fos expression in this latter population. The attenuation of haloperidol-induced Fos expression by SR 48692 is unlikely to re¯ect a direct effect of the antagonist on neurons of the striatum, since striatal neurons do not express NTR1 mRNA or protein [1,3]. This suggests that release of the peptide from striatal NT neurons may alter the functional activity of striatal afferents on which NTR1 is
J. Fadel et al. / Neuroscience Letters 303 (2001) 17±20
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Fig. 2. Effects of SR 48692 pre-treatment on halopeidol-elicted Fos expression in the dorsolateral striatum. The sections were processed to reveal Fos-li nuclei, which appear as dark spots, and the striatal patch (striosome) compartment, as revealed by m opioid receptor immunoreactivity. Scale bar, 100 mM. (A) The dorsolateral striatum of an animal injected with SR 48692 prior to vehicle administration. There are virtually no Fos-li neurons in this case, or in animals treated with vehicle alone. (B) The dorsolateral striatum of a haloperidoltreated rat, in which a very large number of Fos-li can be observed. The density of Fos- li nuclei in cells in the patch compartment is comparable to that seen in the matrix. (C) SR 48692 pre-treatment markedly reduces Fos expression in the dorsolateral striatum; cell counts revealed that the numbers of Fos-li neurons were reduced in both the patch and matrix, but that the reduction was signi®cantly greater in the patch.
present. In vivo microdialysis studies report low but detectable levels of extracellular NT in the striatum [20]. The most likely source of NT is striatal neurons. NT neurons in the substantia nigra project to the striatum, but these are very few in number and unlikely to contribute signi®cantly to the observed striatal extracellular NT levels. Although the high af®nity neurotensin receptor is not expressed on striatal neurons, NTR1 mRNA and protein are present in super®cial layer V cortical neurons [1,3], which provide a glutamatergic projection to the striatum. Haloperidol-induced striatal Fos expression is attenuated by pre-treatment with NMDA receptor antagonists [2] and activation of corticostriatal projections induces Fos in striatal neurons via an NMDA-dependent mechanism [17]. These observations suggest that activation of striatal neurons by glutamate plays an important role in APDinduced Fos expression. Since NT evokes striatal glutamate release [10], we posit that the ability of SR 48692 to attenuate haloperidol-elicited striatal Fos expression is due to a
Fig. 3. Effects of SR 48692 pre-treatment (2.5 mg/kg, i.p.) on the density of Fos-li neurons in the patch and matrix and the ratio of Fos-li neuron density in patch/matrix (n 4/group). Data are expressed as percent of vehicle-haloperidol (V-HAL) treated rat values. The density of Fos-li neurons in both the patch and matrix was decreased, and there was a signi®cant decrease in the patch:matrix ratio. *P # 0:05 relative to vehicle-haloperidol group (V-HAL).
blunting of haloperidol-evoked glutamate release from corticostriatal neurons. This hypothesis awaits direct testing. The ®nding that suppression of haloperidol-induced Fos by SR 48692 occurs preferentially in the MOR-rich patch compartment of the striatum is interesting in light of the observation that typical APDs (such as haloperidol) induce Fos to equivalent degrees in both compartments [5,8], but atypical APDs (such as clozapine) increase Fos to a greater degree in the patch [5]. The dopamine innervation of the matrix compartment preferentially degenerates in MPTPtreated primates [19], suggesting that the matrix may be associated with direct alterations in motoric phenomena. The patch compartment has been associated with reward and affective processes and has also been associated with expression of motor sequence patterns, including psychostimulant-elicited stereotyped behavior [6,21]. However, changes in stereotypy that are re¯ected in an increased Fos patch:matrix ratio are seen after treatment with chronic but not acute psychostimulant administration [6]; it is not clear if the chronic treatment regimen, which results in a sensitized motor (stereotypy) response, does not also involve sensitization to the rewarding aspects of psychostimulant challenge. In summary, we found that animals pretreated with the NTR1 antagonist SR 48692 prior to haloperidol challenge displayed a substantially reduced immediate-early gene response to the APD. These ®ndings correspond well with our recent observation that the striatal Fos response to haloperidol is signi®cantly blunted in the NT knockout mouse [9]. The blunted Fos response was seen in both the patch and matrix but there was a greater reduction in the patch compartment. These data suggest that the full striatal response to haloperidol requires neurotensin. In view of the association of the patch compartment with affective and learned (cognitive) functions, we suggest that the requirement of NT for the full striatal response to haloperidol preferentially re¯ects cognitive and affective (therapeutic) functions rather than acute motoric phenomena (side effects). This interpretation
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J. Fadel et al. / Neuroscience Letters 303 (2001) 17±20
is consistent with the concept that neurotensin exerts atypical antipsychotic drug-like actions. We appreciate the gift of the MOR1 m-opioid receptor antibody from Bob Elde. This work was supported in part by a NARSAD Young Investigator Award (J.F.), HL-33307 (P.R.D.), MH-57795 and MH-45124 (A.Y.D.), and the National Parkinson Foundation Center of Excellence at Vanderbilt University. [1] Alexander, M.J. and Leeman, S.E., Widespread expression in adult rat forebrain of mRNA encoding high-af®nity neurotensin receptor, J. Comp. Neurol., 402 (1998) 475±500. [2] Boegman, R.J. and Vincent, S.R., Involvement of adenosine and glutamate receptors in the induction of c- fos in the striatum by haloperidol, Synapse, 22 (1996) 70±77. [3] Boudin, H., Pelaprat, D., Rostene, W. and Beaudet, A., Cellular distribution of neurotensin receptors in rat brain: immunohistochemical study using an antipeptide antibody against the cloned high af®nity receptor, J. Comp. Neurol., 373 (1996) 76±89. [4] Brog, J.S. and Zahm, D.S., Morphologically distinct subpopulations of neurotensin-immunoreactive striatal neurons observed in rat following dopamine depletions and D2 receptor blockade project to the globus pallidus, Neuroscience, 74 (1996) 805±812. [5] Bubser, M. and Deutch, A.Y., Differential effects of typical and atypical antipsychotic drugs on Fos protein expression in striatal patch and matrix compartments, Soc. Neurosci. Abstracts, 26 (2000) 1487. [6] Canales, J.J. and Graybiel, A.M., A measure of striatal function predicts motor stereotypy, Nat. Neurosci., 3 (2000) 377±383. [7] Deutch, A.Y., Sites and mechanisms of action of antipsychotic drugs as revealed by immediate-early gene expression, In J.G. Csernansky (Ed.), Handbook of Experimental Pharmacology, 120, Springer, Berlin, 1996, pp. 117±161. [8] Deutch, A.Y., Lewis, D.A., Whitehead, R.E., Elsworth, J.D., Iadarola, M.J., Redmond Jr., D.E. and Roth, R.H., Effects of D2 dopamine receptor antagonists on Fos protein expression in the striatal complex and entorhinal cortex of the nonhuman primate, Synapse, 23 (1996) 182±191. [9] Fadel, J., Scruggs, J.L., Dobner, P.R. and Deutch, A.Y., Attenuation of haloperidol-induced striatal Fos expression in neurotensin knockout mice, Soc. Neurosci. Abstracts, 30 (2000) 555.
[10] Ferraro, L., Tanganelli, S., O'Connor, W.T., Bianchi, C., Ungerstedt, U. and Fuxe, K., Neurotensin increases endogenous glutamate release in the neostriatum of the awake rat, Synapse, 20 (1995) 362±364. [11] Garver, D.L., Bissette, G., Yao, J.K. and Nemeroff, C.B., Relation of CSF neurotensin concentrations to symptoms and drug response of psychotic patients, Am. J. Psychiatry, 148 (1991) 484±488. [12] Hokfelt, T., Everitt, B.J., Theodorsson-Norheim, E. and Goldstein, M., Occurrence of neurotensinlike immunoreactivity in subpopulations of hypothalamic, mesencephalic, and medullary catecholamine neurons, , J. Comp. Neurol., 222 (1984) 543±559. [13] Jennes, L., Stumpf, W.E. and Kalivas, P.W., Neurotensin: topographical distribution in rat brain by immunohistochemistry, J. Comp. Neurol., 210 (1982) 211±224. [14] Jolicoeur, F.B., Gagne, M.A., Rivest, R., Drumheller, A. and St-Pierre, S., Atypical neuroleptic-like behavioral effects of neurotensin, Brain Res. Bull., 32 (1993) 487±491. [15] Kalivas, P.W., Burgess, S.K., Nemeroff, C.B. and Prange Jr., A.J., Behavioral and neurochemical effects of neurotensin microinjection into the ventral tegmental area of the rat, Neuroscience, 8 (1983) 495±505. [16] Kinkead, B., Binder, E.B. and Nemeroff, C.B., Does neurotensin mediate the effects of antipsychotic drugs? Biol. Psychiatry, 46 (1999) 340±351. [17] Liste, I., Rozas, G., Guerra, M.J. and Labandeira-Garcia, J.L., Cortical stimulation induces Fos expression in striatal neurons via NMDA glutamate and dopamine receptors, Brain Res., 700 (1995) 1±12. [18] Merchant, K.M. and Dorsa, D.M., Differential induction of neurotensin and c-fos gene expression by typical versus atypical antipsychotics, Proc. Natl. Acad. Sci. USA, 90 (1993) 3447±3451. [19] Moratalla, R., Quinn, B., DeLanney, L.E., Irwin, I., Langston, J.W. and Graybiel, A.M., Differential vulnerability of primate caudate-putamen and striosome- matrix dopamine systems to the neurotoxic effects of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine, Proc Natl Acad Sci USA, 89 (1992) 3859±3863. [20] Wagstaff, J.D., Gibb, J.W. and Hanson, G.R., Dopamine D2receptors regulate neurotensin release from nucleus accumbens and striatum as measured by in vivo microdialysis, Brain Res., 721 (1996) 196±203. [21] White, N.M. and Hiroi, N., Preferential localization of selfstimulation sites in striosomes/patches in the rat striatum, Proc. Natl. Acad. Sci. USA, 95 (1998) 6486±6491.
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The neurotensin antagonist SR 48692 attenuates haloperidolinduced striatal Fos expression in the rat Jim Fadel a,*, Paul R. Dobner b, Ariel Y. Deutch a a
Departments of Psychiatry and Pharmacology and Center for Molecular Neuroscience, Vanderbilt, University Medical Center, Nashville, TN 37212, USA b Department of Molecular Genetics and Microbiology and Program in Neuroscience, University of Massachusetts Medical Center, Worcester, MA 01655, USA Received 1 February 2001; received in revised form 26 February 2001; accepted 27 February 2001
Abstract Neurotensin interacts with central dopamine systems and has been suggested to exert antipsychotic drug-like actions. Antipsychotic drugs such as haloperidol induce striatal immediate-early gene expression. In order to study neurotensin's role in antipsychotic drug actions, rats were pretreated with the neurotensin antagonist SR 48692 and then injected with haloperidol. SR 48692 dose-dependently decreased haloperidol-elicited immediate-early gene expression in the dorsolateral and central striatum but not other striatal areas. SR 48692 reduced Fos expression in the striatal patch (striosome) and matrix compartments, with a signi®cantly greater effect in the patch. These data suggest that neurotensin may play a role in the actions of haloperidol. In view of proposed functional roles of the striatal patch and matrix, we suggest that neurotensin may be important in the therapeutic rather than side effects of antipsychotic drugs. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antipsychotic drugs; Fos; Dopamine; Neurotensin; Schizophrenia; Striatum; Striosome
Neurotensin (NT) is a tridecapeptide transmitter that is widely distributed in the brain [13]. Neurotensin interacts extensively with and in some cases is colocalized in dopamine (DA) neurons [12,15,16]. Several indirect but converging lines of evidence have suggested that NT may play an important role in the therapeutic effects of antipsychotic drug (APD) treatment. Thus, many of the behavioral effects observed in rodents that are administered APDs are also seen after intraventricular injection of NT or NT agonists [14,16]. Moreover, cerebrospinal ¯uid levels of NT are reduced in schizophrenic patients, particularly those with prominent negative symptoms, and tend to normalize with APD treatment [11] Based on these and other data NT has been advanced as an endogenous APD-like peptide [16]. All clinically effective APDs induce the immediate-early gene c-fos and its protein product Fos in the forebrain of mammals with a characteristic regional pattern of expression [7]. Typical APDs, which have substantial extrapyramidal side effect (EPS) liability, induce Fos in the * Corresponding author. Psychiatric Hospital at Vanderbilt, Suite 313 1601 23rd Ave South Nashville, TN 37212, USA. Tel: 11-615-327-7092; fax: 11-615-3221901. E-mail address: [email protected] (J. Fadel).
dorsolateral striatum and nucleus accumbens, whereas atypical APDs induce Fos in the shell of the nucleus accumbens (NAS) but not the dorsolateral striatum [7,8]. These APDs also induce neurotensin/neuromedin N (NT/NMN) mRNA and protein in a similar regionally-speci®c manner, with Fos being induced in most of these NT-like immunoreactive (-li) neurons [4,18]. In view of the suggestion that NT may have endogenous APD-like actions, we sought to determine if pre-treatment with an antagonist of the high-af®nity neurotensin receptor (NTR1) would reduce haloperidol-induced Fos expression in the striatal complex. We further determined if pre-treatment with the NTR1 antagonist SR 48692 would differentially alter haloperidol-elicited Fos expression in the patch (striosome) and matrix compartments of the striatum. These two striatal compartments appear to subserve different functions [6,19,21]. Neurons of the patch have been related to affective processes as well as expression of complex motor patterns, while the cells in the matrix compartment appear to be involved with simple motor behavior. Male Sprague±Dawley rats weighing 250±300 g (Harlan, Birmingham, AL, USA) were housed on a constant 12:12 h light:dark cycle with food and water available ad libitum.
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 70 8- 6
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J. Fadel et al. / Neuroscience Letters 303 (2001) 17±20
Experiments were conducted in accord with institutional Animal Care and Use Committee-approved protocols. Rats were injected with SR 48692 (Sano® Recerche, 0.5 or 2.5 mg/kg, intraperitoneally (i.p.)) or its vehicle (~ 50 ml Tween 20 in 5.0 ml distilled water), followed 30 min later by injection of haloperidol (Research Biochemicals International, Natick, MA; 1.0 mg/kg, i.p.) or vehicle (distilled water acidi®ed with acetic acid to pH ~6.5). Two hours after the second injection, animals were anesthetized with iso¯urane and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After post-®xation and cryoprotection of the brains, serial 40 mm sections were cut on a freezing microtome through the striatal complex and processed to reveal Fos- and FosB-li nuclei, as described previously [8]. Following the development of the sections in diaminobenzidine containing nickel and cobalt to reveal black Fos-li nuclei [8], the sections were washed extensively, incubated in a m-opioid receptor (MOR) antibody (a gift of Dr Robert Elde, University of Minnesota), processed, and developed in diaminobenzidine to stain MOR-li patches brown. The numbers of Fos-li neurons were counted in the dorsolateral, central, ventrolateral, and medial striatum at the level of the crossing of the anterior commissure. Fos-li neurons were also counted in the shell and core of the nucleus accumbens. The data were expressed as Fos-li neurons/mm 2 and were analyzed by analyses of variance; group comparisons were made when indicated using Bonferroni t-tests. Haloperidol signi®cantly increased the number of Fos-li neurons in all four striatal areas and the two accumbal sectors (see Fig. 1); similarly, FosB was induced by haloperidol in all four striatal sectors. SR 48692 pre-treatment signi®cantly reduced both haloperidol-elicited Fos and FosB induction in the dorsolateral striatum (Fig. 1; Table 1). The high but not low dose of SR 48692 also signi®cantly decreased haloperidol-elicited Fos induction in the central
Fig. 1. The effects of SR 48692 (SR) pre-treatment on haloperidol (HAL)-elicited striatal Fos induction (n 4/group). Haloperidol signi®cantly increased Fos expression in all sectors of the striatum. A dose-dependent suppression of the density of Fos-li neurons in the dorsolateral (DL) and central (C) but not the ventrolateral (VL) or medial (M) striatum was observed. *P # 0:05 relative to vehicle-haloperidol (V-HAL) group.
Table 1 The effects of SR 48692 pre-treatment on the number of FosB-li neurons in the striatum of haloperidol-treated rats a
V-V SR-V V-H SR-H
Dorsolateral
Central
Ventrolateral
Medial
0.5 ^ 0.3 0.3 ^ 0.3 25.3 ^ 5.2* 6.3 ^ 1.9*#
0.3 ^ 0.3 0.8 ^ 0.5 19.8 ^ 1.7* 13.5 ^ 4.1*
0.3 ^ 0.3 0.3 ^ 0.3 14.3 ^ 4.5* 7.8 ^ 1.4*
2.0 ^ 1.2 4.3 ^ 2.1 16.8 ^ 2.2* 15.8 ^ 2.2*
a Numbers (mean ^ SEM) of striatal FosB-li neurons/mm 2 in vehicle-vehicle (V-V), SR 48692-vehicle (SR-V), vehicle-haloperidol (V-H), and SR 48692-haloperidol (SR-H) treated rats (n 4/ group). The NT antagonist SR 48692 (2.5 mg/kg) was administered 30 min prior to haloperidol (1.0 mg/kg) *P , 0:05 vs. V-V #P , 0:05 vs. V-H.
striatum. The NTR1 antagonist did not modify the ability of haloperidol to increase the number of Fos- or FosB-li neurons in the medial or ventrolateral striatum or the NAS shell or core (data not shown). SR 48692 had no effect of its own on the number of Fos- or FosB-li neurons in any area. Pre-treatment with SR 48692 had a differential effect on haloperidol-induced Fos expression in the patch and matrix compartments of the dorsolateral striatum (see Figs. 2 and 3). Although pre-treatment with SR 48692 signi®cantly reduced Fos expression in both striatal compartments, the effect was disproportionate: a preferential effect was seen in the patch compartment, with a resultant signi®cant decrease in the ratio of Fos-li neuron density in patch:matrix (see Figs. 2 and 3). These data reveal that pre-treatment with the NT antagonist SR 48692 attenuates the ability of the typical APD haloperidol to induce Fos in neurons of the dorsolateral and central striatum, and suggest that functional NT transmission is necessary for the full actions of haloperidol. The anatomical speci®city of this effect is consistent with the pattern of striatal NT expression induced by haloperidol or other D2 antagonists [4,18], such that signi®cant changes were observed only in the dorsolateral quadrant of the striatum. Haloperidol and other D2 receptor antagonists induce both Fos and NT expression in the striatum. It appears that D2 receptor antagonists induce NT in two populations of cells. One population is found in the medial striatum; these cells are primarily located in the matrix compartment and rarely express Fos in response to D2 antagonist challenge [4]. A second more prominent population is seen in the dorsolateral striatum. These cells express Fos in response to haloperidol challenge and project to the globus pallidus [4]. It seems likely that SR 48692 primarily reduces Fos expression in this latter population. The attenuation of haloperidol-induced Fos expression by SR 48692 is unlikely to re¯ect a direct effect of the antagonist on neurons of the striatum, since striatal neurons do not express NTR1 mRNA or protein [1,3]. This suggests that release of the peptide from striatal NT neurons may alter the functional activity of striatal afferents on which NTR1 is
J. Fadel et al. / Neuroscience Letters 303 (2001) 17±20
19
Fig. 2. Effects of SR 48692 pre-treatment on halopeidol-elicted Fos expression in the dorsolateral striatum. The sections were processed to reveal Fos-li nuclei, which appear as dark spots, and the striatal patch (striosome) compartment, as revealed by m opioid receptor immunoreactivity. Scale bar, 100 mM. (A) The dorsolateral striatum of an animal injected with SR 48692 prior to vehicle administration. There are virtually no Fos-li neurons in this case, or in animals treated with vehicle alone. (B) The dorsolateral striatum of a haloperidoltreated rat, in which a very large number of Fos-li can be observed. The density of Fos- li nuclei in cells in the patch compartment is comparable to that seen in the matrix. (C) SR 48692 pre-treatment markedly reduces Fos expression in the dorsolateral striatum; cell counts revealed that the numbers of Fos-li neurons were reduced in both the patch and matrix, but that the reduction was signi®cantly greater in the patch.
present. In vivo microdialysis studies report low but detectable levels of extracellular NT in the striatum [20]. The most likely source of NT is striatal neurons. NT neurons in the substantia nigra project to the striatum, but these are very few in number and unlikely to contribute signi®cantly to the observed striatal extracellular NT levels. Although the high af®nity neurotensin receptor is not expressed on striatal neurons, NTR1 mRNA and protein are present in super®cial layer V cortical neurons [1,3], which provide a glutamatergic projection to the striatum. Haloperidol-induced striatal Fos expression is attenuated by pre-treatment with NMDA receptor antagonists [2] and activation of corticostriatal projections induces Fos in striatal neurons via an NMDA-dependent mechanism [17]. These observations suggest that activation of striatal neurons by glutamate plays an important role in APDinduced Fos expression. Since NT evokes striatal glutamate release [10], we posit that the ability of SR 48692 to attenuate haloperidol-elicited striatal Fos expression is due to a
Fig. 3. Effects of SR 48692 pre-treatment (2.5 mg/kg, i.p.) on the density of Fos-li neurons in the patch and matrix and the ratio of Fos-li neuron density in patch/matrix (n 4/group). Data are expressed as percent of vehicle-haloperidol (V-HAL) treated rat values. The density of Fos-li neurons in both the patch and matrix was decreased, and there was a signi®cant decrease in the patch:matrix ratio. *P # 0:05 relative to vehicle-haloperidol group (V-HAL).
blunting of haloperidol-evoked glutamate release from corticostriatal neurons. This hypothesis awaits direct testing. The ®nding that suppression of haloperidol-induced Fos by SR 48692 occurs preferentially in the MOR-rich patch compartment of the striatum is interesting in light of the observation that typical APDs (such as haloperidol) induce Fos to equivalent degrees in both compartments [5,8], but atypical APDs (such as clozapine) increase Fos to a greater degree in the patch [5]. The dopamine innervation of the matrix compartment preferentially degenerates in MPTPtreated primates [19], suggesting that the matrix may be associated with direct alterations in motoric phenomena. The patch compartment has been associated with reward and affective processes and has also been associated with expression of motor sequence patterns, including psychostimulant-elicited stereotyped behavior [6,21]. However, changes in stereotypy that are re¯ected in an increased Fos patch:matrix ratio are seen after treatment with chronic but not acute psychostimulant administration [6]; it is not clear if the chronic treatment regimen, which results in a sensitized motor (stereotypy) response, does not also involve sensitization to the rewarding aspects of psychostimulant challenge. In summary, we found that animals pretreated with the NTR1 antagonist SR 48692 prior to haloperidol challenge displayed a substantially reduced immediate-early gene response to the APD. These ®ndings correspond well with our recent observation that the striatal Fos response to haloperidol is signi®cantly blunted in the NT knockout mouse [9]. The blunted Fos response was seen in both the patch and matrix but there was a greater reduction in the patch compartment. These data suggest that the full striatal response to haloperidol requires neurotensin. In view of the association of the patch compartment with affective and learned (cognitive) functions, we suggest that the requirement of NT for the full striatal response to haloperidol preferentially re¯ects cognitive and affective (therapeutic) functions rather than acute motoric phenomena (side effects). This interpretation
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