SYNAPSE 12:304-311 (1992)
Interaction of Amfonelic Acid With Antipsychotic Drugs on Dopaminergic Neurons GARY A. GUDELSKY, E. EWANZANI NWAJEI, KRISTEN DEFIFE, AND J. FRANK NASH Departments of Psychiatry (G.A.G.,E.E.N., J.F.N.),Pharmacology (G.A.G.1, and Neuroscience (K.D., J.F.N.), Case Western Reserve University, Cleweland, Ohio 441 06
KEY WORDS
Dopamine, Uptake inhibitors, Nigrostriatal, Striatum
ABSTRACT The effects of two inhibitors of dopamine (DA) reuptake, amfonelic acid and GBR 12909, on the clozapine- and haloperidol-induced increases in D-4 synthesis, release, and metabolism were investigated in the rat. In the striatum, a s well as in the nucleus accumbens, the haloperidol-induced increase in tissue concentrations of dihydroxyphenylacetic acid (DOPAC) or the accumulation of dihydroxyphenylalanine (DOPA)was potentiated or unaltered, respectively, in rats treated with amfonelic acid. In contrast, amfonelic acid attenuated the stimulatory effects of clozapine on DOPAC concentrations and DOPA accumulation in both brain regions. GBR 12909 also differentially affected the haloperidol- and clozapine-induced increases in DOPAC concentrations. However, the clozapine-induced increase in DOPA accumulation in the median eminence was not significantly altered by treatment with amfonelic acid. The haloperidol-induced increase in the extracellular concentrations of DA and DOPAC in the striatum also was potentiated by amfonelic acid, whereas the increase elicited by clozapine was suppressed. The increase in extracellular DA produced by the administration of morphine or the coadministration of ritanserin, a 5-HTz antagonist, and haloperidol also was potentiated by amfonelic acid. The ability of amfonelic acid to distinguish between the actions of clozapine and haloperidol on nigrostriatal and mesocorticolimbic DA neurons does not appear to be related to differences in the effects of the drugs on DA autoreceptors or 5-HTz receptors. Moreover, the mechanism through which clozapine activates tuberoinfundibular DA neurons may differ from that which is involved in the activation of nigrostriatal or mesocorticolimbic DA neurons. o 1992 Wiley-Liss, Inc. INTRODUCTION The acute administration of typical antipsychotic agents results in a n increase in the synthesis, release, and metabolism of dopamine (DA) within the terminals of nigrostriatal and mesocorticolimbic neurons (Carlsson, 1975; Imperato and Di Chiara, 1985; Roth et al., 1976). These actions of antipsychotic drugs are generally believed to be the consequence of the blockade of D,-type DA receptors and the involvement of neuronal feedback and/or autoreceptor mechanisms. The prototypic atypical antipsychotic agent clozapine also increases the synthesis, release, and metabolism of DA in these forebrain dopaminergic systems (Imperato and Angelucci, 1989; Walters and Roth, 1976; Wilk et al., 1975). However, it is not clear whether this clozapine-induced increase in the activity of dopaminergic neurons also is mediated by blockade of D, receptors. Indeed, Imperator and Angelucci (1989)have suggested that the stimulatory effect of low doses of clozapine on 0 1992 WILEY-LISS, INC.
striatal DA release is mediated by the blockade of D,type DA receptors. McMillen (1981) and Waldmeier et al. (1985) have reported that the stimulatory effect of typical antipsychotic agents (e.g., haloperidol) on striatal concentrations of dihydroxyphenylacetic acid (DOPAC) is enhanced by the nonamphetamine stimulant amfonelic acid, whereas the increase induced by clozapine is attenuated. Recently, Rivest et al. (1991) utilized in vivo voltammetry to obtain results consistent with those of McMillen (1981) and Waldmeier et al. (1985). The differential interaction of amfonelic acid with the haloperidol- and clozapine-induced increases in striatal DOPAC concentrations is suggestive that different mechanisms of action underlie this effect of clozapine and haloperidol. I n addition, the differential interac-
Received September 12,1991;accepted in revised form May 28,1992.
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tion of haloperidol and clozapine with amfonelic acid cumbens were prepared for the analysis of DOPAC as has been proposed as a means of distinguishing poten- just described for the analysis of DOPA. tial atypical antipsychotic agents (McMillen, 1981; In vivo microdialysis Rivest et al., 1991; Waldmeier et al., 1985). The microdialysis studies were performed using In the present study, we sought to establish whether the interaction of amfonelic acid with the clozapine- methods described by Gudelsky and Nash (1992). induced activation of dopaminergic neurons was a) spe- Briefly, under chloral hydrate anesthesia (400 mgkg, cific for this inhibitor of DA reuptake, b) reflected in the intravenously [ivl), a loop-style dialysis probe, which release, as well as the synthesis and metabolism of DA, consisted of 3 mm of exposed saponified, cellulose ester and c) regionally specific with respect t o different DA dialysis fiber (285 pm, O.D., molecular cutoff of 10,000 neuronal systems. Additionally, an attempt was made daltons) affixed to a 23 g guide cannula, was implanted to determine the extent to which the effects of haloperi- in the striatum (A: 1.0, L: 3.5, V: -6.5 mm from do1 and clozapine on DA autoreceptors and 5-HTz re- bregma, incisor bar, -3.3 mm). The probe was flushed ceptors contribute to the differential sensitivity to am- with a modified Ringer’s solution (136 mM NaC1, 1.7 mM KC1, 1.2 mM CaCl,, 6 mM Na2HP04, 1 mM fonelic acid. KH,P04, pH 7.4). Twenty-four hours following surgery, METHODS the dialysis probe was connected to an infusion pump Animals that delivered the modified Ringer’s solution at a rate Adult male rats of the Sprague-Dawley strain (Zivic of 1.8 pllmin. Dialysate samples were collected every 30 Miller, Allison Park, PA) were used in these experi- minutes for at least 2 hours or until a stable concentraments. The animals were maintained on a 12 hour tion of DA was obtained. Haloperidol (1mgkg, ip), clozlighvdark cycle (lights on at 0630 hours) and had free apine (20 mgkg, ip), or the vehicle was injected 60 access to food and water. minutes following the administration of amfonelic acid (2.5 mgkg, sc). Dialysis samples were collected every Biochemical determinations 30 minutes for the next 3 hours. Following the compleThe synthesis of DA in the terminals of nigrostriatal, tion of the dialysis experiment, the location of the dialmesocorticolimbic, and tuberoinfundibular DA neurons ysis probe was verified in each animal. was assessed using a procedure similar to that deThe concentrations of DA and DOPAC were deterscribed by Demarest and Moore (1980). Briefly, the for- mined by HPLC with electrochemical detection (Gudelmation of 3,4-dihydroxyphenylalanine(DOPA) in the sky and Nash, 1992). Dialysate samples were injected striatum, nucleus accumbens, and median eminence onto an ultramex 3 pm C18 column (Phenomenex, Torwas quantified 30 minutes after the administration of rance, CAI connected to an LC-4B amperometric detecm-hydroxybenzylhydrazine (NSD 1015, 100 mgkg, intor (BAS, West Lafayette, IN). The mobile phase contraperitoneally [ipl), an inhibitor of DOPA decarboxy- sisted of 35 niM citric acid, 50 mM sodium acetate, 0.67 lase. The rats were injected with haloperidol (0.5 mg/ mM ethyldiaminetetraacetic acid (EDTA), 0.23 mM kg, ip), clozapine (20 mgkg, ip), or the vehicle 120 1-octanesulfonic acid, 0.1% triethylamine, and 5% minutes before decapitation. Amfonelic acid (2.5 mgkg, methanol (pH 4.25). subcutaneously [scl) or the vehicle was injected 5 minDrugs utes before the administration of the antipsychotic agent. The following drugs were generously provided by the After decapitation of the rats, the striatum, nucleus respective pharmaceutical companies: haloperidol (Mcaccumbens, and median eminence were dissected from Neil Laboratories, Fort Washington, PA), clozapine the brains and homogenized in 0.2 N perchloric acid (Sandoz Pharmaceuticals, East Hanover, NJ), ritancontaining 10 ng/ml of dihydroxybenzylamine as an in- serin (Janssen, Beerse, Belgium), and amfonelic acid ternal standard. The homogenates were subjected to (Sterling-Winthrop Research Inst., Rensselaer, NY). centrifugation at 9,500g for 30 minutes. An aliquot of GBR 12909 and m-hydroxybenzylhydrazine were purthe supernatant was analyzed for DOPA by high perfor- chased from Research Biochemicals Inc. (Natick, MA) mance liquid chromatography (HPLC) with electro- and Sigma Chemical Co. (St. Louis, MO), respectively. chemical detection, as described by Berry and Gudelsky Morphine sulfate was obtained commercially, and the (1990). Protein in the pellet was determined by the dose injected was the sulfate form. method of Lowry et al. (1951). Statistics For the determination of the DA metabolite, DOPAC, The data were evaluated using a 2 x 3 analysis of the rats were killed by decapitation 90 minutes following the injection of haloperidol (0.5mgkg, ip), clozapine variance (ANOVA). Treatment means were compared (20 mgkg, ip), or the solvent vehicle (0.1% tartaric using the Tukey’s Standardized Range procedure and acid). Amfonelic acid (2.5 mgkg, sc), GBR 12909 (20 were considered to be significantly different if P 0.05. mgkg, ip), or the vehicle was injected 5 minutes before Data from experiments utilizing in vivo microdialysis decapitation. Samples of the striatum and nucleus ac- were evaluated after calculating the total response,
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which was assessed as the summation of the DA values expressed as a percent of baseline during a 3 hour collection period. Total response values were compared using an ANOVA and a pair-wise comparison for treatment means.
RESULTS The concentration of DOPAC in the striatum and nucleus accumbens of rats treated with haloperidol (0.5 mgkg, ip) or clozapine (20 mgkg, ip) was 2.5-4 times that in vehicle-treated controls. Treatment of rats with amfonelic acid significantly ( P d 0.05) potentiated the haloperidol-induced increase in DOPAC concentrations in both brain regions (Fig. lA,B). In contrast, the clozapine-induced increase in DOPAC concentrations in both the striatum and nucleus accumbens was attenuated greatly in rats treated with amfonelic acid (Fig. lA,B). Amfonelic acid treatment alone did not significantly alter DOPAC concentrations in either brain region. The interaction of haloperidol and clozapine with another inhibitor of DA reuptake, GBR 12909, also was examined in order to determine whether the differential effect of amfonelic acid on the responses to haloperido1 and clozapine was unique to this uptake inhibitor. Treatment of rats with GBR 12909 (20 mgkg, ip) also enhanced significantly ( P 0.05) the haloperidol-induced increase in striatal DOPAC concentrations. However, the clozapine-inducedincrease in DOPAC concentrations, like that in amfonelic acid-treated rats, was abolished completely in rats treated with GBR 12909 (Fig. 2). Treatment with GBR 12909 alone did not alter significantly striatal DOPAC concentrations. The synthesis of DA, as evaluated from the in vivo accumulation of DOPA, in the striatum and nucleus accumbens of haloperidol-(0.5mgkg, ip) and clozapine(20 mgkg, ip) treated rats was 200-400% of that in vehicle-treated controls. The haloperidol-induced increase in DA synthesis in both brain regions was not affected by treatment with amfonelic acid. In contrast, the clozapine-induced increase in DA synthesis in the striatum, as well as in the nucleus accumbens, was prevented completely in rats treated with amfonelic acid (Fig. 3A, B). It has been shown in previous studies that the acute administration of clozapine but not haloperidol increases the activity of tyrosine hydroxylase in the median eminence (Gudelsky et al., 1987; Gudelsky and Meltzer, 1989). Thus, only the interaction of amfonelic acid with the clozapine-induced increase in DA synthesis within the terminals of tuberoinfundibular DA neurons was determined in the present study. The accumulation of DOPA in the median eminence was increased significantly ( P 0.05) following the acute administration of clozapine (20 mgkg, ip). The clozapine-induced increase in DA synthesis in the median eminence was
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not altered significantly by the administration of amfonelic acid (Fig. 3C). Treatment with amfonelic acid alone did not significantly affect DA synthesis in any of the brain regions. The interaction of amfonelic acid with the haloperidol- and clozapine-induced release of striatal DA also was investigated using in vivo microdialysis. Haloperido1 (1mgkg, ip) and clozapine (20 mgkg, ip) each produced a 3 5 4 5 % increase in the extracellular concentration of DA in the striatum (Figs. 4, 5). The extracellular concentration of DOPAC in the striatum also was increased 110-140% by each of the antipsychotic agents (Figs. 4, 5). Treatment of rats with am-
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Fig. 2. Effect of GBR 12909 on the haloperidol- and clozapine-induced increase in striatal DOPAC concentrations. Rats were treated with GBR 12909 (20 mgkg, ip) or the vehicle 5 minutes prior to the administration of haloperidol (0.5 mgkg, ip), clozapine (20 mgkg, ip), or the vehicle. The animals were sacrificed 90 minutes after the administration of the antipsychotic agent. Column heights and vertical lines represent the mean and SE of six rats. *P 0.05 compared to the value for the vehicle-pretreated rats given haloperidol or clozapine.
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VEH fonelic acid greatly potentiated the haloperidol-induced increase in extracellular concentrations of DA and DOPAC. DA and DOPAC concentrations in rats treated with amfonelic acid and haloperidol reached values that were approximately 400% of the respective basal concentrations (Fig. 4A,B). In contrast, amfonelic acid did not potentiate the clozapine-induced increases in extracellular DA and DOPAC concentrations. In fact, the response of striatal DA and DOPAC to clozapine was significantly suppressed in rats treated with amfonelic acid (Fig. 5A,B). Treatment of rats with amfonelic acid alone resulted in a slight, but nonsignificant, decline in DA and DOPAC concentrations when compared to values for vehicle-treated rats (data not shown). The possibility was considered that the suppression of the stimulatory effect of clozapine on DA synthesis, metabolism, and release in the presence of amfonelic acid was due to the activation of DA autoreceptors, since clozapine is relatively ineffective at blocking DA autoreceptors (Walters and Roth, 1976). Morphine (20 mgkg, sc), which acts presumably through mu recep-
Fig. 3. Effect of amfonelic acid on the haloperidol- and clozapineinduced increase in DA synthesis in the striatum (A), nucleus accumbens (B),and median eminence (C). Rats were injected with amfonelic acid (2.5 mgkg, sc), haloperidol (0.5 mgkg, ip), and clozapine (20 rngkg, ip) according to the regimen described in the Methods section. In addition, all rats were injected with m-hydroxybenzylhydraeine (100 mgkg, ip) 30 minutes before decapitation. Column heights and vertical lines represent the mean and SE of 6-12 rats. *P 0.05 compared to the value for the vehicle-pretreated rats given clozapine.
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TIME (hrl Fig. 4. Effect of amfonelic acid on the haloperidol-induced increase in the extracellular concentration of DA (A) and DOPAC (B) in the striatum. Amfonelic acid (2.5 mgkg, sc) or the vehicle was injected at time 0. Haloperidol (1 mgkg, ip) or the vehicle was injected a t 1 hour. Each value represents the mean and SE of 5-10 rats expressed as a percent of the 0 time value. The basal (0 time) values for DA and DOPAC for all groups were 25 2 2 and 4,120 i 279 pg/30 minutes.
tors rather than through D, receptors, increased the extracellular concentration of DA in the striatum (Table I). The morphine-induced increase in the extracellular concentration of DA, unlike that produced by cloza0.05) in pine, was potentiated significantly ( P amfonelic acid-treated rats. The possibility that the 5-HT2antagonist property of clozapine (Altar et al., 1986; Nash et al., 1988) accounts for its unique interaction with amfonelic acid also was investigated. This issue was addressed by examining the effect of amfonelic acid in rats treated with the combination of haloperidol and ritanserin, a 5-HT, antagonist, in order to achieve significant 5-HT2 and D, receptor blockade. Ritanserin treatment alone did not alter the extracellular concentration of DA (data not shown). Of more importance was the finding that the increase in extracellular DA induced by ritanserin and
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haloperidol was potentiated in amfonelic acid-treated rats (Table I).
DISCUSSION Results from the present study in which the haloperidol- and clozapine-induced increases in striatal DOPAC concentrations were potentiated and suppressed, respectively, by amfonelic acid are in accord with previous studies (Fuller and Snoddy, 1985; McMillen, 1981; Waldmeier et al., 1985). In addition, there appeared to be no difference in the discrimination by amfonelic acid of the actions of haloperidol and clozapine on DA metabolism within nigrostriatal compared to mesocorticolimbic DA neurons, inasmuch as the stimulatory effect of clozapine on DOPAC concentrations in the nucleus accumbens also was attenuated by amfonelic acid, whereas that of haloperidol was potentiated.
ANTIPSYCHOTIC DRUGS TABLE 1. Effect of arnfonelic acid on the increase in extracellular doparnine in the striatum induced by morphine or ritanserin and haloueridol
’
DA response Treatment Experiment 1 Vehicle + morphine Amfonelic acid + morphine Experiment 2 Vehicle + ritanserin + haloperidol Amfonelic acid + ritanserin + haloperidol
(% of basal x 3 hr)
*
1,081 70 1,796 ? 254* 1,032 % 35 2,370 ? 318*
’Rats received amfonelic acid 12.5 mgkg, sc) or the vehicle 1 hour prior to the administration of morphine (20 mgkg, sc). In another experiment, rats received ritanserin (2.5 mgkg, ip) or the vehicle 1 hour before an injection of amfonelic acid 12.5 mgkg, sc) or its vehicle and 2 hours prior to the administration of haloperidol (1 mgkg, ip). Dialysate samples were obtained every 30 minutes for 3 hours after the administration of haloperidol or morphine. The values represent the summation of the DA values (expressed as a % ’ of baseline) for the 3 hour period commencing after the administration of morphine or haloperidol. *P< 0.05 compared to the values for the rats treated with morphine or haloperidol alone.
Potentiation of the effects of haloperidol and attenuation of the effects of clozapine on striatal DA metabolism were not limited to the actions of amfonelic acid; a similar interaction was produced by GBR 12909. Thus, discrimination of the effects of typical and atypical antipsychotics on forebrain DA neurons is not unique to amfonelic acid but appears to be shared by other selective inhibitors of DA reuptake. The increase in DA synthesis elicited by haloperidol and clozapine also was affected differentially by amfonelic acid. Amfonelic acid completely prevented the clozapine-induced increase in DA synthesis, as assessed from the accumulation of DOPA, in both the striatum and nucleus accumbens. In contrast, the haloperidol-induced increase in DOPA accumulation was unaltered by amfonelic acid. An explanation for the finding that the haloperidol-induced increase in DOPA formation, unlike the induced increase in DOPAC concentrations, was not actually potentiated by amfonelic acid is the possibility that DA synthesis was maximally stimulated by the dose of haloperidol tested. The ability of amfonelic acid to potentiate and suppress the stimulatory effects of haloperidol and clozapine, respectively, on striatal DA release, as assessed from the extracellular concentrations of DA, also was consistent with the interactions on DA synthesis and metabolism. The potentiation of the haloperidol-induced release of striatal DA by amfonelic acid is in accord with the results of Westerink et al. (1987). Recently, Rivest et al. (1991), using in vivo voltammetry, reported that the increase in extracellular DOPAC in the striatum elicited by haloperidol also was potentiated by amfonelic acid, whereas that induced by clozapine or thioridazine was attenuated. The mechanism whereby amfonelic acid discriminates between the effects of haloperidol and clozapine is unclear. Amfonelic acid, as well as GBR 12909, is presumably a specific inhibitor of DA reuptake (McMillen,
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1983; Van der Zee et al., 1980). In addition, it has been suggested that amfonelic acid facilitates the transfer of DA from a storage pool to a readily releasable pool (McMiIlen, 1983). Either mechanism could account for the potentiation by amfonelic acid of the effects of haloperidol on DA release and metabolism. However, it would be presumed that DA uptake inhibitors, such as amfonelic acid, should potentiate the effects of any drug-induced increase in DA release, simply by preventing the reuptake of DA. It has been argued that the suppressive effect of amfonelic acid on the clozapine-induced increase in striatal DOPAC concentrations may be due to the ineffectiveness of clozapine to block DA autoreceptors (McMillen, 1981; Walters and Roth, 1976). However, the interaction of amfonelic acid with the morphine-induced release of striatal DA is not supportive of this hypothesis. Morphine, in agreement with previous studies (Di Chiara and Imperator, 19881, increased the extracellular concentration of DA in the striatum, and this effect was potentiated by amfonelic acid. The morphine-induced release of striatal DA is mediated, presumably, by mu receptors rather than D,-type DA receptors. Consequently, the morphine-induced release of DA, like that induced by clozapine, occurs in the absence of any blockade of DA autoreceptors. Yet, the DA response to morphine was potentiated by amfonelic acid, whereas the response to clozapine was suppressed. Thus, it is unlikely that failure to block DA autoreceptors accounts for the unique interaction of amfonelic acid with clozapine. The affinity of clozapine for 5-HT, receptors relative to D, receptors is greater than that for typical antipsychotis (Altar et al., 1986; Meltzer et al., 1989), and clozapine is an effective 5-HT2 antagonist (Nash et al., 1988; Rasmussen and Aghajanian, 1988). Brougham et al. (1991)have reported that the haloperidol-induced increase in striatal DOPAC concentrations is potentiated by amfonelic acid, whereas that produced by the coadministration of ritanserin and haloperidol, like that of clozapine, is suppressed. These investigators concluded that the 5-HT2 antagonist property of clozapine is critical to its unique interaction with amfonelic acid. However, in the present study, the increase in extracellular concentrations of striatal DA elicited by the coadministration of ritanserin and haloperidol was potentiated by amfonelic acid in a manner similar to the potentiation of the increase elicited by haloperidol alone. The coadministration of ritanserin and haloperido1 did not result in a profile similar to that of clozapine. The reasons for this discrepancy are unknown. Although DA release was measured in the present study and post mortem DOPAC concentrations were quantified in the aforementioned study, it is clear from the present findings that parallel changes are observed in these two parameters. It is noteworthy that other agents with no appreciable affinity for the 5-HT, recep-
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tor, viz., the sigma ligand BMY 14802 (Matthews et al., 1986) and neurotensin (Rivest et al., 19911, also exhibit a profile of interaction with amfonelic acid similar to that of clozapine. Unless it is argued that a final common pathway exists for neurotensin, sigma, and 5-HT, receptors in their interaction with DA uptake inhibitors, these findings also are unsupportive of a potential role of 5-HT, receptor blockade in the interaction of amfonelic acid with clozapine. The suppressive effect of inhibitors of DA reuptake, such as nomifensine, and presumably amfonelic acid and GBR 12909, on amphetamine-stimulated DA release (Butcher et al., 1988)has been viewed as evidence that amphetamine-induced DA release is carried mediated. On the basis of the present results, it also could be argued that amfonelic acid and GBR 12909 block the access of clozapine t o a mechanism, viz., the DA carrier, through which clozapine facilitates DA release. However, to date there is no evidence that clozapine interacts with the DA carrier. In addition, the increase in DA release produced by clozapine, like that elicited by haloperidol (Westerink et al., 19871, is sensitive to inhibition by tetrodotoxin (Gudelsky, unpublished observations). Thus, clozapine-induced DA release appears to be dependent on nerve impulse flow rather than a DA transporter. Finally, although amfonelic acid suppressed the clozapine-induced increase in DOPA accumulation in the striatum and nucleus accumbens, amfonelic acid did not alter the clozapine-induced increase in DA synthesis in the terminals of tuberoinfundibular neurons in the median eminence. These results are suggestive that the mechanism through which clozapine increases the synthesis of DA within tuberoinfundibular neurons (Gudelsky et al., 1987; Gudelsky and Meltzer, 1989) is different from that which is operative within nigrostriatal and mesocorticolimbic DA neurons. Although various mechanisms other than D, receptor blockade (e.g., antagonism of D1or 5-HT2receptors) have been implicated in the actions of clozapine on nigrostriatal DA neurons (Imperato and Angelucci, 1989; Altar et al., 1986; Meltzer et al., 1989), the mechanism involved in the clozapine-induced activation of tuberinfundibular DA neurons is unresolved. In summary, the stimulatory effects of clozapine on DA release, as well as synthesis and metabolism, within nigrostriatal and mesocorticolimbic DA neurons can be distinguished from that elicited by haloperidol in rats treated with DA reuptake inhibitors. Although the mechanism that underlies this distinction is unresolved, the present findings are inconsistent with a role for DA autoreceptors or 5-HT2receptors. Moreover, amfonelic acid appears to exert a differential effect on the clozapine-induced activation of nigrostriatal and mesocorticolimbic DA neurons compared t o tuberoinfundibular DA neurons.
ACKNOWLEDGMENTS This work was supported by USPHS MH 42868, MH 41684, and by grants from the Schizophrenia Research Program of the Scottish Rite Foundation, NMJ USA, the National Alliance for Research on Schizophrenia and Depression, and the Biomedical Research Support Grant Program of the NIH (BRSG 2 SO7 RR05410-29). The expert technical assistance of Ms. Linda Liatti is gratefully appreciated. REFERENCES Altar, C.A., Wasley, A.M., Neale, R.F., and Stone, G.A. (1986) Typical and atypical antipsychotic occupancy of D, and S, receptors: an autoradiographic analysis in rat brain. Brain Res. Bull., 16:517525. Berry, S.A., and Gudelsky, G.A. (1990)D, receptors function to inhibit the activation of tuberoinfundibular dopamine neurons. J. Pharmacol. Exp. Ther., 254:677-682. Brougham, L.R., Conway, P.G., and Ellis, D.B. (19911 Effect of ritanserin on the interaction of amfonelic acid and neuroleptic-induced striatal dopamine metabolism. Neuropharmacology 30:1137-1 140. Butcher, S.P., Fairbrother, IS., Kelly, J.S., and Arbuthnott, G.W. (1988)Amphetamine-induced dopamine release in the rat striatum: an in vivo microdialysis study. J. Neurochem., 50:346-355. Carlsson, A. (1975) Receptor mediated control of dopamine metabolism. In: Pre and Postsynaptic Receptors. E. Usdin and W.E. Bunney, eds. Marcel Dekker, New York, pp. 49-63. Demarest, K.T., and Moore, K.E. (1980)Accumulation of L-DOPA in the median eminence: an index of tuberoinfundibular nerve activity. Endocrinology, 106:463468. Di Chiara, G., and Imperato, A. (1988) Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and dorsal caudate of freely moving rats. J . Pharm. Exp. Ther., 244:1067-1079. Fuller, R.W., and Snoddy, H.D. (1985) Flumezapine and zotepine: 5-hydroxytryptamine antagonism not involved in the lack of synergism of these antipsychotic drugs with amfonelic acid in rats. J. Pharm. Pharmacol., 37:755-756. Gudelsky, G.A., Koenig, J.I., Simonovic, M., Koyama, T., Ohmori, T., and Meltzer, H.Y. (1987) Differential effects of clozapine and haloperidol on tuberoinfundibular dopamine neurons and prolactin secretion in the rat. J. Neural Transm., 68:227-240. Gudelsky, G.A., and Meltzer, H.Y. (1989)Activation of tuberoinfundibular dopamine neurons following the acute administration of atypical antipsychotics. Neuropsychopharmacology, 2:45-51, Gudelsky, G.A., and Nash, J.F. (1992)Neuroendocrinological and neurochemical effects of sigma ligands. Neuropharmacology, 31:157162. Imperato, A,, and Angelucci, L. (1989) The effect of clozapine and fluperlapine on the in vivo release and metabolism of dopamine in the striatum and in the frontal cortex of freely moving rats. Psychopharmacol. Bull., 25:383-389. Imperato, A., and Di Chiara, G. (1985)Dopamine release and metabolism in awake rats after systemic neuroleptics a s studied by transstriatal dialysis. J. Neurosci., 5:297-306. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193~265-275. Matthews, R.T., McMillen, B.A., Sallis, R., and Blair, D. (1986) Effects of BMY 14802, a potential antipsychotic drug, on rat brain dopaminergic function. J. Pharmacol. Exp. Ther., 239:124-131. McMillen, B.A. (1981) Comparative effects of classical and atypical antipsychotic drugs in combination with a non-amphetamine stimulant on rat brain dopamine metabolism. J. Pharm. Pharmacol.. 33544-546. McMillen. B.A. (1983) CNS stimulants: two distinct mechanisms of action for amphetamine-like drugs. Trends Pharmacol. Sci., 4:429432. Meltzer, H.Y., Matsubara, S., and Lee, J.-C. (1989) Classification of typical and atypical antipsychotic drugs on the basis of dopamine D-I, D-2 and serotonin, pK, values. J. Pharmacol. Exp. Ther., 25 1:238-246. Nash, J.F., Metlzer, H.Y., and Gudelsky, G.A. (1988) Antagonism of serotonin receptor mediated neuroendocrine and temperature re-
ANTIPSYCHOTIC DRUGS sponses by atypical neuroleptics in the rat. Eur. J . Pharmacol., 151:463469. Rasmussen, K., and Aghajanian, G.K. (1988)Potency of antipsychotics in reversing the effect of hallucinogenic drugs on locus coeruleus neurons correlates with 5-HT2 binding affinity. Neuropsychopharmacology, 1:lOl-107. Rivest, R., Jolicoeur, F.B., and Marsden, C.A. (1991) Use of amfonelic acid to discriminate between classical and atypical neuroleptics and neurotensin: an in vivo voltammetric study. Brain Res., 544:86-93. Roth, R.H., Murrin, L.C., and Walters, J.R. (1976) Central dopaminergic neurons: effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid. Eur. J . Pharmacol., 36:163-172. Van der Zee, P., Koger, H.S., Gootjes, J., and Hepse, W. (1980) Aryl 1,4-dialky(en)ylpiperazinesas selective and very potent inhibitors of dopamine uptake. Eur. J . Med. Chem., 15:363-370.
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Waldmeier, P.C., Huber, H., Heinrich, M., and Stoecklin, K. (1985) Discrimination of neuroleptics by means of their interaction with amfonelic acid: a n attempt to characterize the test. Biochem. Pharmacol., 34:39-44. Walters, J.R., and Roth, R.H. (1976) Dopaminergic neurons: an in vivo system for measuring drug interactions with presynaptic receptors. Naunyn Schmiedebergs Arch. Pharmacol., 2965-14. Westerink, B.H.C., Dasma, G., De Vries, J., and Koning, H. (1987) Dopamine re-uptake inhibitors show inconsistent effects on the in vivo release of dopamine as measured by intracerebral dialysis in the rat. Eur. J. Pharmacol., 135123-128. Wilk, S., Watson, E., and Stanley, M.E. (1975) Differential sensitivity of two dopaminergic structures in rat brain to haloperidol and to clozapine. J . Pharmacol. Exp. Ther., 195:265-270.
Interaction of Amfonelic Acid With Antipsychotic Drugs on Dopaminergic Neurons GARY A. GUDELSKY, E. EWANZANI NWAJEI, KRISTEN DEFIFE, AND J. FRANK NASH Departments of Psychiatry (G.A.G.,E.E.N., J.F.N.),Pharmacology (G.A.G.1, and Neuroscience (K.D., J.F.N.), Case Western Reserve University, Cleweland, Ohio 441 06
KEY WORDS
Dopamine, Uptake inhibitors, Nigrostriatal, Striatum
ABSTRACT The effects of two inhibitors of dopamine (DA) reuptake, amfonelic acid and GBR 12909, on the clozapine- and haloperidol-induced increases in D-4 synthesis, release, and metabolism were investigated in the rat. In the striatum, a s well as in the nucleus accumbens, the haloperidol-induced increase in tissue concentrations of dihydroxyphenylacetic acid (DOPAC) or the accumulation of dihydroxyphenylalanine (DOPA)was potentiated or unaltered, respectively, in rats treated with amfonelic acid. In contrast, amfonelic acid attenuated the stimulatory effects of clozapine on DOPAC concentrations and DOPA accumulation in both brain regions. GBR 12909 also differentially affected the haloperidol- and clozapine-induced increases in DOPAC concentrations. However, the clozapine-induced increase in DOPA accumulation in the median eminence was not significantly altered by treatment with amfonelic acid. The haloperidol-induced increase in the extracellular concentrations of DA and DOPAC in the striatum also was potentiated by amfonelic acid, whereas the increase elicited by clozapine was suppressed. The increase in extracellular DA produced by the administration of morphine or the coadministration of ritanserin, a 5-HTz antagonist, and haloperidol also was potentiated by amfonelic acid. The ability of amfonelic acid to distinguish between the actions of clozapine and haloperidol on nigrostriatal and mesocorticolimbic DA neurons does not appear to be related to differences in the effects of the drugs on DA autoreceptors or 5-HTz receptors. Moreover, the mechanism through which clozapine activates tuberoinfundibular DA neurons may differ from that which is involved in the activation of nigrostriatal or mesocorticolimbic DA neurons. o 1992 Wiley-Liss, Inc. INTRODUCTION The acute administration of typical antipsychotic agents results in a n increase in the synthesis, release, and metabolism of dopamine (DA) within the terminals of nigrostriatal and mesocorticolimbic neurons (Carlsson, 1975; Imperato and Di Chiara, 1985; Roth et al., 1976). These actions of antipsychotic drugs are generally believed to be the consequence of the blockade of D,-type DA receptors and the involvement of neuronal feedback and/or autoreceptor mechanisms. The prototypic atypical antipsychotic agent clozapine also increases the synthesis, release, and metabolism of DA in these forebrain dopaminergic systems (Imperato and Angelucci, 1989; Walters and Roth, 1976; Wilk et al., 1975). However, it is not clear whether this clozapine-induced increase in the activity of dopaminergic neurons also is mediated by blockade of D, receptors. Indeed, Imperator and Angelucci (1989)have suggested that the stimulatory effect of low doses of clozapine on 0 1992 WILEY-LISS, INC.
striatal DA release is mediated by the blockade of D,type DA receptors. McMillen (1981) and Waldmeier et al. (1985) have reported that the stimulatory effect of typical antipsychotic agents (e.g., haloperidol) on striatal concentrations of dihydroxyphenylacetic acid (DOPAC) is enhanced by the nonamphetamine stimulant amfonelic acid, whereas the increase induced by clozapine is attenuated. Recently, Rivest et al. (1991) utilized in vivo voltammetry to obtain results consistent with those of McMillen (1981) and Waldmeier et al. (1985). The differential interaction of amfonelic acid with the haloperidol- and clozapine-induced increases in striatal DOPAC concentrations is suggestive that different mechanisms of action underlie this effect of clozapine and haloperidol. I n addition, the differential interac-
Received September 12,1991;accepted in revised form May 28,1992.
ANTIPSYCHOTIC DRUGS
305
tion of haloperidol and clozapine with amfonelic acid cumbens were prepared for the analysis of DOPAC as has been proposed as a means of distinguishing poten- just described for the analysis of DOPA. tial atypical antipsychotic agents (McMillen, 1981; In vivo microdialysis Rivest et al., 1991; Waldmeier et al., 1985). The microdialysis studies were performed using In the present study, we sought to establish whether the interaction of amfonelic acid with the clozapine- methods described by Gudelsky and Nash (1992). induced activation of dopaminergic neurons was a) spe- Briefly, under chloral hydrate anesthesia (400 mgkg, cific for this inhibitor of DA reuptake, b) reflected in the intravenously [ivl), a loop-style dialysis probe, which release, as well as the synthesis and metabolism of DA, consisted of 3 mm of exposed saponified, cellulose ester and c) regionally specific with respect t o different DA dialysis fiber (285 pm, O.D., molecular cutoff of 10,000 neuronal systems. Additionally, an attempt was made daltons) affixed to a 23 g guide cannula, was implanted to determine the extent to which the effects of haloperi- in the striatum (A: 1.0, L: 3.5, V: -6.5 mm from do1 and clozapine on DA autoreceptors and 5-HTz re- bregma, incisor bar, -3.3 mm). The probe was flushed ceptors contribute to the differential sensitivity to am- with a modified Ringer’s solution (136 mM NaC1, 1.7 mM KC1, 1.2 mM CaCl,, 6 mM Na2HP04, 1 mM fonelic acid. KH,P04, pH 7.4). Twenty-four hours following surgery, METHODS the dialysis probe was connected to an infusion pump Animals that delivered the modified Ringer’s solution at a rate Adult male rats of the Sprague-Dawley strain (Zivic of 1.8 pllmin. Dialysate samples were collected every 30 Miller, Allison Park, PA) were used in these experi- minutes for at least 2 hours or until a stable concentraments. The animals were maintained on a 12 hour tion of DA was obtained. Haloperidol (1mgkg, ip), clozlighvdark cycle (lights on at 0630 hours) and had free apine (20 mgkg, ip), or the vehicle was injected 60 access to food and water. minutes following the administration of amfonelic acid (2.5 mgkg, sc). Dialysis samples were collected every Biochemical determinations 30 minutes for the next 3 hours. Following the compleThe synthesis of DA in the terminals of nigrostriatal, tion of the dialysis experiment, the location of the dialmesocorticolimbic, and tuberoinfundibular DA neurons ysis probe was verified in each animal. was assessed using a procedure similar to that deThe concentrations of DA and DOPAC were deterscribed by Demarest and Moore (1980). Briefly, the for- mined by HPLC with electrochemical detection (Gudelmation of 3,4-dihydroxyphenylalanine(DOPA) in the sky and Nash, 1992). Dialysate samples were injected striatum, nucleus accumbens, and median eminence onto an ultramex 3 pm C18 column (Phenomenex, Torwas quantified 30 minutes after the administration of rance, CAI connected to an LC-4B amperometric detecm-hydroxybenzylhydrazine (NSD 1015, 100 mgkg, intor (BAS, West Lafayette, IN). The mobile phase contraperitoneally [ipl), an inhibitor of DOPA decarboxy- sisted of 35 niM citric acid, 50 mM sodium acetate, 0.67 lase. The rats were injected with haloperidol (0.5 mg/ mM ethyldiaminetetraacetic acid (EDTA), 0.23 mM kg, ip), clozapine (20 mgkg, ip), or the vehicle 120 1-octanesulfonic acid, 0.1% triethylamine, and 5% minutes before decapitation. Amfonelic acid (2.5 mgkg, methanol (pH 4.25). subcutaneously [scl) or the vehicle was injected 5 minDrugs utes before the administration of the antipsychotic agent. The following drugs were generously provided by the After decapitation of the rats, the striatum, nucleus respective pharmaceutical companies: haloperidol (Mcaccumbens, and median eminence were dissected from Neil Laboratories, Fort Washington, PA), clozapine the brains and homogenized in 0.2 N perchloric acid (Sandoz Pharmaceuticals, East Hanover, NJ), ritancontaining 10 ng/ml of dihydroxybenzylamine as an in- serin (Janssen, Beerse, Belgium), and amfonelic acid ternal standard. The homogenates were subjected to (Sterling-Winthrop Research Inst., Rensselaer, NY). centrifugation at 9,500g for 30 minutes. An aliquot of GBR 12909 and m-hydroxybenzylhydrazine were purthe supernatant was analyzed for DOPA by high perfor- chased from Research Biochemicals Inc. (Natick, MA) mance liquid chromatography (HPLC) with electro- and Sigma Chemical Co. (St. Louis, MO), respectively. chemical detection, as described by Berry and Gudelsky Morphine sulfate was obtained commercially, and the (1990). Protein in the pellet was determined by the dose injected was the sulfate form. method of Lowry et al. (1951). Statistics For the determination of the DA metabolite, DOPAC, The data were evaluated using a 2 x 3 analysis of the rats were killed by decapitation 90 minutes following the injection of haloperidol (0.5mgkg, ip), clozapine variance (ANOVA). Treatment means were compared (20 mgkg, ip), or the solvent vehicle (0.1% tartaric using the Tukey’s Standardized Range procedure and acid). Amfonelic acid (2.5 mgkg, sc), GBR 12909 (20 were considered to be significantly different if P 0.05. mgkg, ip), or the vehicle was injected 5 minutes before Data from experiments utilizing in vivo microdialysis decapitation. Samples of the striatum and nucleus ac- were evaluated after calculating the total response,
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A . STAIATUM
which was assessed as the summation of the DA values expressed as a percent of baseline during a 3 hour collection period. Total response values were compared using an ANOVA and a pair-wise comparison for treatment means.
RESULTS The concentration of DOPAC in the striatum and nucleus accumbens of rats treated with haloperidol (0.5 mgkg, ip) or clozapine (20 mgkg, ip) was 2.5-4 times that in vehicle-treated controls. Treatment of rats with amfonelic acid significantly ( P d 0.05) potentiated the haloperidol-induced increase in DOPAC concentrations in both brain regions (Fig. lA,B). In contrast, the clozapine-induced increase in DOPAC concentrations in both the striatum and nucleus accumbens was attenuated greatly in rats treated with amfonelic acid (Fig. lA,B). Amfonelic acid treatment alone did not significantly alter DOPAC concentrations in either brain region. The interaction of haloperidol and clozapine with another inhibitor of DA reuptake, GBR 12909, also was examined in order to determine whether the differential effect of amfonelic acid on the responses to haloperido1 and clozapine was unique to this uptake inhibitor. Treatment of rats with GBR 12909 (20 mgkg, ip) also enhanced significantly ( P 0.05) the haloperidol-induced increase in striatal DOPAC concentrations. However, the clozapine-inducedincrease in DOPAC concentrations, like that in amfonelic acid-treated rats, was abolished completely in rats treated with GBR 12909 (Fig. 2). Treatment with GBR 12909 alone did not alter significantly striatal DOPAC concentrations. The synthesis of DA, as evaluated from the in vivo accumulation of DOPA, in the striatum and nucleus accumbens of haloperidol-(0.5mgkg, ip) and clozapine(20 mgkg, ip) treated rats was 200-400% of that in vehicle-treated controls. The haloperidol-induced increase in DA synthesis in both brain regions was not affected by treatment with amfonelic acid. In contrast, the clozapine-induced increase in DA synthesis in the striatum, as well as in the nucleus accumbens, was prevented completely in rats treated with amfonelic acid (Fig. 3A, B). It has been shown in previous studies that the acute administration of clozapine but not haloperidol increases the activity of tyrosine hydroxylase in the median eminence (Gudelsky et al., 1987; Gudelsky and Meltzer, 1989). Thus, only the interaction of amfonelic acid with the clozapine-induced increase in DA synthesis within the terminals of tuberoinfundibular DA neurons was determined in the present study. The accumulation of DOPA in the median eminence was increased significantly ( P 0.05) following the acute administration of clozapine (20 mgkg, ip). The clozapine-induced increase in DA synthesis in the median eminence was
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not altered significantly by the administration of amfonelic acid (Fig. 3C). Treatment with amfonelic acid alone did not significantly affect DA synthesis in any of the brain regions. The interaction of amfonelic acid with the haloperidol- and clozapine-induced release of striatal DA also was investigated using in vivo microdialysis. Haloperido1 (1mgkg, ip) and clozapine (20 mgkg, ip) each produced a 3 5 4 5 % increase in the extracellular concentration of DA in the striatum (Figs. 4, 5). The extracellular concentration of DOPAC in the striatum also was increased 110-140% by each of the antipsychotic agents (Figs. 4, 5). Treatment of rats with am-
307
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VEH fonelic acid greatly potentiated the haloperidol-induced increase in extracellular concentrations of DA and DOPAC. DA and DOPAC concentrations in rats treated with amfonelic acid and haloperidol reached values that were approximately 400% of the respective basal concentrations (Fig. 4A,B). In contrast, amfonelic acid did not potentiate the clozapine-induced increases in extracellular DA and DOPAC concentrations. In fact, the response of striatal DA and DOPAC to clozapine was significantly suppressed in rats treated with amfonelic acid (Fig. 5A,B). Treatment of rats with amfonelic acid alone resulted in a slight, but nonsignificant, decline in DA and DOPAC concentrations when compared to values for vehicle-treated rats (data not shown). The possibility was considered that the suppression of the stimulatory effect of clozapine on DA synthesis, metabolism, and release in the presence of amfonelic acid was due to the activation of DA autoreceptors, since clozapine is relatively ineffective at blocking DA autoreceptors (Walters and Roth, 1976). Morphine (20 mgkg, sc), which acts presumably through mu recep-
Fig. 3. Effect of amfonelic acid on the haloperidol- and clozapineinduced increase in DA synthesis in the striatum (A), nucleus accumbens (B),and median eminence (C). Rats were injected with amfonelic acid (2.5 mgkg, sc), haloperidol (0.5 mgkg, ip), and clozapine (20 rngkg, ip) according to the regimen described in the Methods section. In addition, all rats were injected with m-hydroxybenzylhydraeine (100 mgkg, ip) 30 minutes before decapitation. Column heights and vertical lines represent the mean and SE of 6-12 rats. *P 0.05 compared to the value for the vehicle-pretreated rats given clozapine.
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TIME (hrl Fig. 4. Effect of amfonelic acid on the haloperidol-induced increase in the extracellular concentration of DA (A) and DOPAC (B) in the striatum. Amfonelic acid (2.5 mgkg, sc) or the vehicle was injected at time 0. Haloperidol (1 mgkg, ip) or the vehicle was injected a t 1 hour. Each value represents the mean and SE of 5-10 rats expressed as a percent of the 0 time value. The basal (0 time) values for DA and DOPAC for all groups were 25 2 2 and 4,120 i 279 pg/30 minutes.
tors rather than through D, receptors, increased the extracellular concentration of DA in the striatum (Table I). The morphine-induced increase in the extracellular concentration of DA, unlike that produced by cloza0.05) in pine, was potentiated significantly ( P amfonelic acid-treated rats. The possibility that the 5-HT2antagonist property of clozapine (Altar et al., 1986; Nash et al., 1988) accounts for its unique interaction with amfonelic acid also was investigated. This issue was addressed by examining the effect of amfonelic acid in rats treated with the combination of haloperidol and ritanserin, a 5-HT, antagonist, in order to achieve significant 5-HT2 and D, receptor blockade. Ritanserin treatment alone did not alter the extracellular concentration of DA (data not shown). Of more importance was the finding that the increase in extracellular DA induced by ritanserin and
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TIME (hr) Fig. 5. Effect of amfonelic acid on the clozapine-induced increase in the extracellular concentration of DA (A) and DOPAC (B) in the striatum. Amfonelic acid (2.5 mgkg, ip) or the vehicle was injected at time 0. Clozapine (20 mgkg, ip) or the vehicle was injected at 1 hour. Each value represents the mean and SE of 6-10 rats expressed a s a percent of the 0 time value.
haloperidol was potentiated in amfonelic acid-treated rats (Table I).
DISCUSSION Results from the present study in which the haloperidol- and clozapine-induced increases in striatal DOPAC concentrations were potentiated and suppressed, respectively, by amfonelic acid are in accord with previous studies (Fuller and Snoddy, 1985; McMillen, 1981; Waldmeier et al., 1985). In addition, there appeared to be no difference in the discrimination by amfonelic acid of the actions of haloperidol and clozapine on DA metabolism within nigrostriatal compared to mesocorticolimbic DA neurons, inasmuch as the stimulatory effect of clozapine on DOPAC concentrations in the nucleus accumbens also was attenuated by amfonelic acid, whereas that of haloperidol was potentiated.
ANTIPSYCHOTIC DRUGS TABLE 1. Effect of arnfonelic acid on the increase in extracellular doparnine in the striatum induced by morphine or ritanserin and haloueridol
’
DA response Treatment Experiment 1 Vehicle + morphine Amfonelic acid + morphine Experiment 2 Vehicle + ritanserin + haloperidol Amfonelic acid + ritanserin + haloperidol
(% of basal x 3 hr)
*
1,081 70 1,796 ? 254* 1,032 % 35 2,370 ? 318*
’Rats received amfonelic acid 12.5 mgkg, sc) or the vehicle 1 hour prior to the administration of morphine (20 mgkg, sc). In another experiment, rats received ritanserin (2.5 mgkg, ip) or the vehicle 1 hour before an injection of amfonelic acid 12.5 mgkg, sc) or its vehicle and 2 hours prior to the administration of haloperidol (1 mgkg, ip). Dialysate samples were obtained every 30 minutes for 3 hours after the administration of haloperidol or morphine. The values represent the summation of the DA values (expressed as a % ’ of baseline) for the 3 hour period commencing after the administration of morphine or haloperidol. *P< 0.05 compared to the values for the rats treated with morphine or haloperidol alone.
Potentiation of the effects of haloperidol and attenuation of the effects of clozapine on striatal DA metabolism were not limited to the actions of amfonelic acid; a similar interaction was produced by GBR 12909. Thus, discrimination of the effects of typical and atypical antipsychotics on forebrain DA neurons is not unique to amfonelic acid but appears to be shared by other selective inhibitors of DA reuptake. The increase in DA synthesis elicited by haloperidol and clozapine also was affected differentially by amfonelic acid. Amfonelic acid completely prevented the clozapine-induced increase in DA synthesis, as assessed from the accumulation of DOPA, in both the striatum and nucleus accumbens. In contrast, the haloperidol-induced increase in DOPA accumulation was unaltered by amfonelic acid. An explanation for the finding that the haloperidol-induced increase in DOPA formation, unlike the induced increase in DOPAC concentrations, was not actually potentiated by amfonelic acid is the possibility that DA synthesis was maximally stimulated by the dose of haloperidol tested. The ability of amfonelic acid to potentiate and suppress the stimulatory effects of haloperidol and clozapine, respectively, on striatal DA release, as assessed from the extracellular concentrations of DA, also was consistent with the interactions on DA synthesis and metabolism. The potentiation of the haloperidol-induced release of striatal DA by amfonelic acid is in accord with the results of Westerink et al. (1987). Recently, Rivest et al. (1991), using in vivo voltammetry, reported that the increase in extracellular DOPAC in the striatum elicited by haloperidol also was potentiated by amfonelic acid, whereas that induced by clozapine or thioridazine was attenuated. The mechanism whereby amfonelic acid discriminates between the effects of haloperidol and clozapine is unclear. Amfonelic acid, as well as GBR 12909, is presumably a specific inhibitor of DA reuptake (McMillen,
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1983; Van der Zee et al., 1980). In addition, it has been suggested that amfonelic acid facilitates the transfer of DA from a storage pool to a readily releasable pool (McMiIlen, 1983). Either mechanism could account for the potentiation by amfonelic acid of the effects of haloperidol on DA release and metabolism. However, it would be presumed that DA uptake inhibitors, such as amfonelic acid, should potentiate the effects of any drug-induced increase in DA release, simply by preventing the reuptake of DA. It has been argued that the suppressive effect of amfonelic acid on the clozapine-induced increase in striatal DOPAC concentrations may be due to the ineffectiveness of clozapine to block DA autoreceptors (McMillen, 1981; Walters and Roth, 1976). However, the interaction of amfonelic acid with the morphine-induced release of striatal DA is not supportive of this hypothesis. Morphine, in agreement with previous studies (Di Chiara and Imperator, 19881, increased the extracellular concentration of DA in the striatum, and this effect was potentiated by amfonelic acid. The morphine-induced release of striatal DA is mediated, presumably, by mu receptors rather than D,-type DA receptors. Consequently, the morphine-induced release of DA, like that induced by clozapine, occurs in the absence of any blockade of DA autoreceptors. Yet, the DA response to morphine was potentiated by amfonelic acid, whereas the response to clozapine was suppressed. Thus, it is unlikely that failure to block DA autoreceptors accounts for the unique interaction of amfonelic acid with clozapine. The affinity of clozapine for 5-HT, receptors relative to D, receptors is greater than that for typical antipsychotis (Altar et al., 1986; Meltzer et al., 1989), and clozapine is an effective 5-HT2 antagonist (Nash et al., 1988; Rasmussen and Aghajanian, 1988). Brougham et al. (1991)have reported that the haloperidol-induced increase in striatal DOPAC concentrations is potentiated by amfonelic acid, whereas that produced by the coadministration of ritanserin and haloperidol, like that of clozapine, is suppressed. These investigators concluded that the 5-HT2 antagonist property of clozapine is critical to its unique interaction with amfonelic acid. However, in the present study, the increase in extracellular concentrations of striatal DA elicited by the coadministration of ritanserin and haloperidol was potentiated by amfonelic acid in a manner similar to the potentiation of the increase elicited by haloperidol alone. The coadministration of ritanserin and haloperido1 did not result in a profile similar to that of clozapine. The reasons for this discrepancy are unknown. Although DA release was measured in the present study and post mortem DOPAC concentrations were quantified in the aforementioned study, it is clear from the present findings that parallel changes are observed in these two parameters. It is noteworthy that other agents with no appreciable affinity for the 5-HT, recep-
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tor, viz., the sigma ligand BMY 14802 (Matthews et al., 1986) and neurotensin (Rivest et al., 19911, also exhibit a profile of interaction with amfonelic acid similar to that of clozapine. Unless it is argued that a final common pathway exists for neurotensin, sigma, and 5-HT, receptors in their interaction with DA uptake inhibitors, these findings also are unsupportive of a potential role of 5-HT, receptor blockade in the interaction of amfonelic acid with clozapine. The suppressive effect of inhibitors of DA reuptake, such as nomifensine, and presumably amfonelic acid and GBR 12909, on amphetamine-stimulated DA release (Butcher et al., 1988)has been viewed as evidence that amphetamine-induced DA release is carried mediated. On the basis of the present results, it also could be argued that amfonelic acid and GBR 12909 block the access of clozapine t o a mechanism, viz., the DA carrier, through which clozapine facilitates DA release. However, to date there is no evidence that clozapine interacts with the DA carrier. In addition, the increase in DA release produced by clozapine, like that elicited by haloperidol (Westerink et al., 19871, is sensitive to inhibition by tetrodotoxin (Gudelsky, unpublished observations). Thus, clozapine-induced DA release appears to be dependent on nerve impulse flow rather than a DA transporter. Finally, although amfonelic acid suppressed the clozapine-induced increase in DOPA accumulation in the striatum and nucleus accumbens, amfonelic acid did not alter the clozapine-induced increase in DA synthesis in the terminals of tuberoinfundibular neurons in the median eminence. These results are suggestive that the mechanism through which clozapine increases the synthesis of DA within tuberoinfundibular neurons (Gudelsky et al., 1987; Gudelsky and Meltzer, 1989) is different from that which is operative within nigrostriatal and mesocorticolimbic DA neurons. Although various mechanisms other than D, receptor blockade (e.g., antagonism of D1or 5-HT2receptors) have been implicated in the actions of clozapine on nigrostriatal DA neurons (Imperato and Angelucci, 1989; Altar et al., 1986; Meltzer et al., 1989), the mechanism involved in the clozapine-induced activation of tuberinfundibular DA neurons is unresolved. In summary, the stimulatory effects of clozapine on DA release, as well as synthesis and metabolism, within nigrostriatal and mesocorticolimbic DA neurons can be distinguished from that elicited by haloperidol in rats treated with DA reuptake inhibitors. Although the mechanism that underlies this distinction is unresolved, the present findings are inconsistent with a role for DA autoreceptors or 5-HT2receptors. Moreover, amfonelic acid appears to exert a differential effect on the clozapine-induced activation of nigrostriatal and mesocorticolimbic DA neurons compared t o tuberoinfundibular DA neurons.
ACKNOWLEDGMENTS This work was supported by USPHS MH 42868, MH 41684, and by grants from the Schizophrenia Research Program of the Scottish Rite Foundation, NMJ USA, the National Alliance for Research on Schizophrenia and Depression, and the Biomedical Research Support Grant Program of the NIH (BRSG 2 SO7 RR05410-29). The expert technical assistance of Ms. Linda Liatti is gratefully appreciated. REFERENCES Altar, C.A., Wasley, A.M., Neale, R.F., and Stone, G.A. (1986) Typical and atypical antipsychotic occupancy of D, and S, receptors: an autoradiographic analysis in rat brain. Brain Res. Bull., 16:517525. Berry, S.A., and Gudelsky, G.A. (1990)D, receptors function to inhibit the activation of tuberoinfundibular dopamine neurons. J. Pharmacol. Exp. Ther., 254:677-682. Brougham, L.R., Conway, P.G., and Ellis, D.B. (19911 Effect of ritanserin on the interaction of amfonelic acid and neuroleptic-induced striatal dopamine metabolism. Neuropharmacology 30:1137-1 140. Butcher, S.P., Fairbrother, IS., Kelly, J.S., and Arbuthnott, G.W. (1988)Amphetamine-induced dopamine release in the rat striatum: an in vivo microdialysis study. J. Neurochem., 50:346-355. Carlsson, A. (1975) Receptor mediated control of dopamine metabolism. In: Pre and Postsynaptic Receptors. E. Usdin and W.E. Bunney, eds. Marcel Dekker, New York, pp. 49-63. Demarest, K.T., and Moore, K.E. (1980)Accumulation of L-DOPA in the median eminence: an index of tuberoinfundibular nerve activity. Endocrinology, 106:463468. Di Chiara, G., and Imperato, A. (1988) Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and dorsal caudate of freely moving rats. J . Pharm. Exp. Ther., 244:1067-1079. Fuller, R.W., and Snoddy, H.D. (1985) Flumezapine and zotepine: 5-hydroxytryptamine antagonism not involved in the lack of synergism of these antipsychotic drugs with amfonelic acid in rats. J. Pharm. Pharmacol., 37:755-756. Gudelsky, G.A., Koenig, J.I., Simonovic, M., Koyama, T., Ohmori, T., and Meltzer, H.Y. (1987) Differential effects of clozapine and haloperidol on tuberoinfundibular dopamine neurons and prolactin secretion in the rat. J. Neural Transm., 68:227-240. Gudelsky, G.A., and Meltzer, H.Y. (1989)Activation of tuberoinfundibular dopamine neurons following the acute administration of atypical antipsychotics. Neuropsychopharmacology, 2:45-51, Gudelsky, G.A., and Nash, J.F. (1992)Neuroendocrinological and neurochemical effects of sigma ligands. Neuropharmacology, 31:157162. Imperato, A,, and Angelucci, L. (1989) The effect of clozapine and fluperlapine on the in vivo release and metabolism of dopamine in the striatum and in the frontal cortex of freely moving rats. Psychopharmacol. Bull., 25:383-389. Imperato, A., and Di Chiara, G. (1985)Dopamine release and metabolism in awake rats after systemic neuroleptics a s studied by transstriatal dialysis. J. Neurosci., 5:297-306. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193~265-275. Matthews, R.T., McMillen, B.A., Sallis, R., and Blair, D. (1986) Effects of BMY 14802, a potential antipsychotic drug, on rat brain dopaminergic function. J. Pharmacol. Exp. Ther., 239:124-131. McMillen, B.A. (1981) Comparative effects of classical and atypical antipsychotic drugs in combination with a non-amphetamine stimulant on rat brain dopamine metabolism. J. Pharm. Pharmacol.. 33544-546. McMillen. B.A. (1983) CNS stimulants: two distinct mechanisms of action for amphetamine-like drugs. Trends Pharmacol. Sci., 4:429432. Meltzer, H.Y., Matsubara, S., and Lee, J.-C. (1989) Classification of typical and atypical antipsychotic drugs on the basis of dopamine D-I, D-2 and serotonin, pK, values. J. Pharmacol. Exp. Ther., 25 1:238-246. Nash, J.F., Metlzer, H.Y., and Gudelsky, G.A. (1988) Antagonism of serotonin receptor mediated neuroendocrine and temperature re-
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