Phenotypic Characterization of Neuroleptic- Sensitive Neurons in the Forebrain: Contrasting Targets of Haloperidol and Clozapine Ningning Guo, Ph.D., Steven R. Vincent, Ph.D., and H. Christian Fibiger, Ph.D. The prototypical neuroleptic haloperidol and the atypical (DYN) indicated that clozapine increases c-fos expression antipsychotic clozapine induce distinctly different patterns in both ENK and DYN containing neurons in the NAc of c-fos expression in the forebrain. While haloperidol (ENK 40%, DYN 53%) and LSN (ENK 32%, DYN 59%). appears to increase c-fos expression via its D2 dopamine Haloperidol also increases c-fos expression in ENK and receptor antagonist properties, the receptor mechanisms by DYN containing neurons, albeit in a different pattern which clozapine produces its unique pattern of c-fos (striatum: ENK 93%, DYN 20%; nucleus accumbens: expression are not known. The present experiments sought ENK 46%, DYN 36%; lateral septum: ENK 29%, DYN to address this question by determining the phenotypes of 18%). The present results demonstrate that haloperidol and neurons in which clozapine increases Fos-like clozapine target different populations of neurons even in immunoreactivity (FLI). Fos immunostaining combined regions such as the NAc and LSN, where they both increase with in situ hybridization histochemistry using a cDNA c-fos expression. In addition, the fact that the majority of oligonucleotide probe for D3 receptor mRNA indicated that clozapine-sensitive neurons in NAc, LSN, and ICjM the great majority (95%) of clozapine-induced FLI neurons express D3 receptors suggests that activity at these in the major island of Calleja (ICjM) express D3 receptors. receptors may contribute to the unique clinical profile of Similarly, in the nucleus accumbens (NAc) and lateral this antipsychotic agent. These data indicate that D3 septal nucleus (LSN), the majority of clozapine-induced FLI receptors may represent novel targets in the neurons express D3 receptor mRNA (NAc 69%; LS 73%). pharmacotherapy of schizophrenia. In marked contrast, haloperidol-induced FLI neurons failed [Neuropsychopharmacology 19:133–145, 1998] to express D3 receptors in any brain region. Studies with © 1998 American College of Neuropsychopharmacology. oligonucleotide probes for enkephalin (ENK) and dynorphin Published by Elsevier Science Inc. KEY WORDS: Clozapine; Haloperidol; D3 dopamine Clozapine has been termed an “atypical” antipsychotic receptors; c-fos; Nucleus accumbens; Striatum; Enkephalin; agent in part because at therapeutic doses it fails to pro- Dynorphin duce the extrapyramidal, Parkinsonian-like side effects that are associated with the use of “typical” neurolep- tics such as haloperidol and chlorpromazine. While the From the Divison of Neurological Sciences, Department of Psy- chiatry, University of British Columbia, Vancouver, B.C., Canada. mechanisms by which clozapine generates its unique Address correspondence to: H.C. Fibiger, Division of Neurologi- clinical profile are not established, recent studies from cal Sciences, Department of Psychiatry, University of British Colum- several laboratories have demonstrated that, compared bia, 2255 Wesbrook Mall, Vancouver, B.C. V6T 1Z3 Canada. Received May 9, 1997; revised October 1, 1997; accepted October to typical neuroleptics, clozapine produces a rather 3, 1997. unique regional pattern of Fos-like immunoreactive NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 2 © 1998 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(97)00202-9 134 N. Guo et al. NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 2 (FLI) neurons in the forebrain (Deutch et al. 1992; mechanism (Robertson et al. 1992), at present, the pepti- Nguyen et al. 1992; Robertson and Fibiger 1992; MacGib- dergic phenotype of neurons in the NAc that are tar- bon et al. 1994), Fos being the protein product of the im- geted by haloperidol is not known. Similarly, the extent mediate early gene c-fos which is thought to be an ac- to which clozapine increases FLI in enkephalinergic vs. tivity marker in some neurons (Sagar et al. 1988; dynorphinergic neurons in the NAc has not been inves- Dragunow and Faull 1989). Thus, while haloperidol tigated. To determine the peptidergic nature of neurons and clozapine both increase c-fos expression in the nu- in the NAc which increase c-fos expression in response cleus accumbens (NAc) and lateral septal nucleus to clozapine or haloperidol, Fos immunohistochemistry (LSN), haloperidol also potently targets the dorsal stria- was combined with in situ hybridization histochemis- tum, where clozapine is essentially without effect, try using cDNA oligonucleotide probes for enkephalin whereas clozapine also targets the islands of Calleja and or dynorphin. A similar analysis was applied to the lat- the medial prefrontal cortex (PFC), where haloperidol is eral septal nucleus, major island of Calleja, medial pre- inactive. In the striatum, haloperidol is thought to in- frontal cortex and dorsal striatum. crease c-fos expression by blocking D2 dopamine recep- tors on enkephalin-containing medium spiny neurons that project to the globus pallidus (Robertson et al. MATERIALS AND METHODS 1992). Dopaminergic mechanisms have also been impli- cated in the NAc by virtue of the fact that extensive le- Adult male Wistar rats (280–320 gm) were maintained sions of the mesotelencephalic dopaminergic system on a 12-hr light/12-hr dark cycle, with free access to block the effects of both haloperidol and clozapine on food and water. The rats were handled periodically for c-fos expression in this structure (Robertson and Fibiger 4–5 days prior to the experiment. 1992). Despite this, there are reasons to believe that ha- loperidol and clozapine may increase Fos-like immu- noreactivity in different populations of accumbal neu- Drug Administration and Brain Section Preparation rons. For example, the distribution of FLI neurons within the NAc is not the same after the two drugs Clozapine (H. Lundbeck, Kobenhavn-Valby) and halo- (Robertson and Fibiger 1992), clozapine being relatively peridol (McNeil Pharmaceutical, Stouffville, Canada) m more active in the so-called shell region, and haloperi- were dissolved in 40 l of 20% acetic acid and brought dol being approximately equally active in the core and to final volume (1 ml) with 0.9% saline. Rats were in- shell (Deutch et al. 1992). jected subcutaneously with clozapine (20 mg/kg) or ha- In a recent study, Guo et al. (1995) investigated the loperidol (1 mg/kg). Two hours after the injection, the receptor mechanisms by which clozapine produces its rats were deeply anesthetized with pentobarbital and unique pattern of c-fos expression in the forebrain and perfused with 0.9% saline (200 ml) followed by 150 ml 4% paraformaldehyde (PFA) in 0.1 M phosphate-buff- concluded that while its serotonergic (5-HT2) and norad- renergic (a1) antagonist properties do not contribute, ered saline (PBS). The brains were removed and placed actions at dopamine D3 receptors may be responsible in fresh fixative (4% PFA) for at least 12 hours. Twenty m for this pattern of effects. The present study, therefore, or 25 m sections were cut from each brain using a Vi- combined in situ hybridization histochemistry for D3 bratome. The sections were collected in 0.02 M PBS for receptor mRNA with Fos immunohistochemistry to de- immunostaining and in situ hybridization. termine if D3 receptors are expressed in neurons in which clozapine increases FLI. For comparative pur- Fos-Like Immunohistochemistry poses, identical experiments were conducted with halo- peridol. All solutions were prepared with pyrogen-free, ultra- Medium spiny projection neurons, which represent pure water and autoclaved before using. Brain sections 90–95% of all neurons in the dorsal striatum, can be were processed for Fos immunostaining (Guo et al. subdivided into two major populations: 1) enkephalin- 1992b) using the avidin-biotin-complex (ABC) method ergic, D2-expressing cells with projections to the exter- (Guo et al. 1992a) under RNAase-free conditions. nal division of the globus pallidus; and 2) D1-express- Briefly, the free-floating sections were preincubated ing dynorphin/substance P-containing neurons which with 0.5% H2O2 for 15 min to remove endogenous per- innervate entopeduncular nucleus and the substantia oxidase activity, and then incubated with the Fos pri- nigra pars reticulata (Beckstead and Kersey 1985; Ger- mary antibody (Cambridge Research Biochemicals, CRB fen and Young 1988; Albin et al. 1989; Alexander and OA-11-824; 1:3000 dilution), a biotinylated secondary Crutcher 1990; Anderson and Reiner 1990; Gerfen et al. antibody (Vector Laboratories, Burlingame, CA; 1:200), 1990; Le Moine et al. 1990, 1991; Gerfen 1992). While ha- and avidin-biotinylated horseradish peroxidase com- loperidol selectivity increases c-fos expression in stri- plex (Vector Laboratories; 1:200). The sections were ato-pallidal, enkephalinergic neurons via a D2 receptor stained with 0.1% 3,39-diaminobenzidine (DAB) in NEUROPSYCHOPHARMACOLOGY 1998–VOL. 19, NO. 2 Neuronal Targets of Antipsychotic Drugs 135 35 0.002% H2O2, and then mounted on double chrome- For S-labeled sections, washing solutions contained 14 alum-coated, diethyl pyrocarbonate-treated slides. mM b-mercaptoethanol. The sections were air dried and examined autoradiographically by apposition to X-ray film (X-Omat, Kodak). The exposures were 3–5 Oligonucleotide Probes days for 33P- and 10–14 days