Neurochem. Int. Vol. 20, Suppl., pp. 129S--133S, 1992 Printed in Great Britain. All fights rvsvrvext
0197-0186/92 $5.00+0.00 Copyright © 1992 Pergamon Press plc
THE EFFECT OF CONTINUOUS A N D REPEATED ADMINISTRATION OF D1 DOPAMINE RECEPTOR ANTAGONIST ON MIDBRAIN DOPAMINE NEURONS STEPHENR. WACHTELand FRANCISJ. WHITE NeuropsychopharmacoiogyLaboratory, Department of Psychiatry,Cellular and Clinical NeurobiologyProgram, Wayne State University School of Medicine, Lafayette Clinic, 951 E. Lafayette, Detroit, M148201, U.S.A.
Many synergistic effects of D l and D2 dopamine (DA) agonists result from an enabling interaction between D~ and D2 receptors in which Dl receptor stimulation is required for the functional effects produced by D2 receptor stimulation (see Clark & White, 1987 for review). The enabling phenomenon was first indicated by the finding that the Dt receptor antagonist SCH 23390 could block the locomotion and stereotyped behaviors elicited by both mixed (non-selective) Dr/D2 agonists and selective D 2 agonists (e.g. Arnt, 1985; Iorio et al., 1983). Further evidence for this permissive role of Dt receptor stimulation was provided by the findings that acute depletion of endogenous DA, produced by methyl-p-tyrosine (AMPT) and/or reserpine, prevented the stimulation of locomotor activity and stereotypy produced by the administration of selective D2 agonists, and that these behaviors were reinstated by the D~ agonist SKF 38393 (Braun & Chase, 1986; Jackson & Hashizume, 1986; White et al., 1988). Electrophysiological experiments have demonstrated that the enabling role of D~ receptor activation for D2 receptor functional effects is also evident at the cellular level. In the both the dorsal (caudateputamen) and ventral (nucleus accumbens) striatum, acute DA depletion produced by AMPT greatly attenuated the inhibition of firing normally produced by iontophoretic administration of selective D2 agonists (quinpirole and RU 24213). The D 2 agonistinduced neuronal inhibition was restored by coadministration of SKF 38393 (Wachtel et al., 1989; White, 1987). Similar relationships have been reported for the excitatory effects of D2 agonists, administered systemically, on rat globus pallidus cells (Waiters et a/., 1987). The discovery of the enabling role of Dt receptors for D2 receptor-mediated functions has added to the speculation that selective DI receptor antagonists might be useful in the treatment of schizophrenia, a disorder typically thought to involve hyperactive DA transmission and currently treated
with DA receptor blockers. From the perspective of D~ receptor enabling, it would appear that D~ receptor antagonists might reduce DA transmission through both Dt receptors (directly) and D2 receptors (reduced enabling). Particularly interesting are recent reports suggesting that selective D~ receptor antagonists exert effects on DA systems which closely resemble those of atypical antipsychotic drugs (APDs), i.e. those with a reduced liability for producing extrapyramidal motor disorders (e.g. Chipkin and Latranyi, 1987; Coffin et al., 1989).Among the animal models typically used to screen potential APDs for therapeutic utility and side effect liability, the ability to produce a depolarization block of midbrain DA neurons has received considerable attention primarily because it is observed only following repeated administration (as are many clinical effects of APDs). Over the past decade, a series of studies has demonstrated that classical APDs, such as haloperidol or chlorpromazine, cause a timedependent decrease in the number of spontaneously active DA cells in both the ventral tegmental area (VTA, A~0)and substantia nigra (SN, As) (Bunney & Grace, 1978; White & Wang, 1983a,b), whereas atypical APDs, such as clozapine selectively inactivate Al0 DA cells (Chiodo & Bunney, 1983; White & Wang, 1983b). Previous attempts to determine whether selective D~ receptor antagonists can induce depolarization block of DA neurons have provided highly discrepant results. Skarsfeldt (1988) reported that the D1 antagonist SCH 23390 caused a dosedependent reduction in the number of both As and At0 DA neurons following twice daily s.c. injections (0.020.31 mg/kg) for 21 days. However, this report did not include a demonstration ofhyperpolarization-induced reversal (e.g. with apomorphine) of the reduction in active neurons. Goldstein and Litwin (1988) reported that a 28 day treatment of once daily s e l l 23390 injections (0.05 mg/kg, s.c.) produced a selective decrease in the number of Aio DA neurons, an effect reversed by i.e. apomorphine (0.063 mg/kg). However,
129S
130S
l)opammc 911 2.0! I
--Z~-
o
_.}_
1.0
0.5
I
Saline SCH 23390 SCH 39166 Haloperidol NO 112 Ventral T e g m e n t a l area
Saline SCH 23390 SCH 39166 Haloperidol NO 112
S u b s t a n t i a Nigra
Fig. 1. Effects of continuous (28 day) administration of D~ receptor antagonists on the number of spontaneously active A0 and A~0 DA neurons. Bars represent the mean + SEM. Asterisks represent a significant difference IP < 0.05) from the saline control using a Student's two-tailed t-test. Espositio and Bunney (1989) f o u n d no evidence for depolarization block following 21 days o f once daily S C H 23390 injections (0.05-0.t0 mg/kg, s.c.) or a similar time o f m o r e constant administration via the animals' drinking water (5.0 - 10.0 mg/kg).In the present experiments, we have also examined the effects o f SCH 23390 on m i d b r a i n D A neurons following b o t h repeated injections (0.5 mg/kg, s.c. for 28 days) and continuous administration via osmotic minip u m p s (28 days) administration. In addition, we have tested two new selective Di receptor antagonists, N O - 1 1 2 [( + )-8-chioro-7-hydroxy-3-methyl-5(7-benzofuranyl)-2,3,4,5,tetrahydro- l H-3-benzazepin] and SCH 39166 [ ( - ) - t r a n s - 6 , 7 , 7 a , 8 , 9 , 1 3 b - h e x a h y d r o - 3 chloro-2- hydroxy-N-methyl-SH-benzo[d]naptho-2,lbazepine], as well as haloperidol for comparative purposes. Both N O - 1 1 2 and SCH 39166 possess a high affinity for D~ receptors in b o t h ligand binding a n d DA-stimulated adenylate cyclase assays (Andersen et aL, 1988; Chipkin et al., 1988l.
EXPERIMENTAL PROCEDURES
In one set of experiments NO-112. SCH 39166. SCH 23390, haloperidol and saline were administered continuously via model 2002 mini osmotic pumps lAlza Corporation, Palo Alto, CAl. Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 150200 g were anesthetized with Brevital (50 mg/kg, i.p., Eli Lily, Indianapolis, IN) and the pumps inserted subcutaneously on the dorsal flanks of the rat through a 1 cm incision. At the time of replacement a second pump was inserted on the opposite flank. Both pumps were removed 2 hrs before the cell counting began on the last day of drug treatment. The initial concentration of drug in the pumps was 2 mg/ml, which resulted in an approximate daily dose of 0.13 mg/kg/day. When the second pump was inserted for the last two weeks of
treatment, the concentration ofantagonists was increased to 4 mg/ml to account for increased body weights. In a second set of experiments. SCH 39166 and SCH 23390, were administered s.c.. 0.5 mg/kg, twice daily for 28 days. Haloperidol was injected at the same dose but only once each day. Saline injected rats received an equivalent volume and served as controls. Cell counting began two hours after the last injection. The procedure for counting spontaneously active DA cells has been described previously IWachtel and White. 1988). Briefly, the rats were anesthetized with choral hydrate (400 mg/kg, i.p.), mounted in a stereotaxic appartatus, and a single barrel extracellular electrode lowered 12 times through a stereotaxically consistent grid in both the SN (3.0-3.4 mm anterior. 2.0-2.6 mm lateral to lambda) and VTA (3.0-3.4 mm anterior. 0.41.0ram lateral), and the number of spontaneously active DA cells counted. The electrode was lowered from 6.0 to 8.5 mm below the cortical surface in each track. Individual tracks were separated by 200 m. For half of the rats. A9 was recorded before A~0, and the order reversed for the other rats. If a decrease in the number of cells encountered after continuous antagomst treatment was observed, an additional group of rats was treated with the particular antagonist, and the ability of Figure 1 Apomorphine to reverse the decrease in the number of spontaneously active Ao or A~0 DA cells was examined to determine if the decrease was the result of depolarization block. In these animals, apomorphine was injected (t0 or 50 /zg/kg, i.v.)just before cell counting was begun. The counting was performed as described above: however, ira decrease was observed in both A9 and A~0,only six tracks were utilized in each area after apomorphine administration. RESULTS
C o n t i n u o u s administration o f N O - t 12 and S C H 23390 via osmotic m i n i p u m p s resulted in a selective reduction in the n u m b e r o f spontaneously active A~0 D A neurons as c o m p a r e d to vehicle treated controls. C o n t i n u o u s administration o f haloperidol decreased the n u m b e r o f spontaneously active D A cells in b o t h the SN and V T A (Fig. 1). Surprisingly, S C H 39166
Dopamine 90
131S
2.0
1.5 - -
V
T
0
.m 1.0 (D
o
0.5 0
Saline
SCH 23390 Haloperidol SCH 39166
Saline
Ventral Tegmental area
SCH 23390 Haloperidol SCH 39166
Substantia Nigra
Fig. 2. Effects of repeated (28 day) administration of Dt receptor antagonists on the number of spontaneously active A9 and A~0 DA neurons. Bars represent the mean + SEM. Asterisks represent a significant difference (p < 0.01) from the saline control using a Student's two-tailed t-test. Table 1. Effectof apomorphineafter continuousantagonistadministration A9
Ai0
Drug
Mean
SEM
N
Mean
SEM
Saline NO 112 + Apomorphine 10#g/kg + Apomorphine 50 #g/kg SCH 23390 + Apomorphine lO/~g/kg + Apomorphine 50 pg/kg Haloperidol + Apomorphine 50 ,ug/kg
0.92 0.87
0.05 0.10
N
7
1.49
0.11
7
7
1.06*
0.13
8
--
--
1.28
0.13
6
0.68
0.10
8
0.77* 1.03"
0.14 0.16
4 8
--
--
--
1.29
0.07
4
-0.59
-0.12
--
0.71"
0.05
4
89
1.00"
0.17
8
0.83
0.07
4
1.50"*
0.20
5
--
* Significantlydifferentfrom salinecontrol (p < 0.05). ** Significantlydifferentfrom DA antagonistalone (p < 0.05). failed to alter the n u m b e r of spontaneously active D A cells encountered in either area (Fig. 1). In contrast to the continuous administration regimen, repeated administration of SCH 23390 failed to produce a reduction in the n u m b e r of A9 or A~0 D A cells. Again, SCH 39166 was ineffective. As previously reported, haloperidol produced a nonselective decrease in spontaneously active D A neurons in both areas (Fig. 2). Acute injections of apomorphine (10 or 50 gg/kg) failed to reverse the selective decrease in spontaneously active A~0 D A neurons produced by continuous N O - 1 1 2 or SCH 23390 (Table 1.) Infact, the 50 g g dose of apomorphine further decreased the n u m b e r of spontaneously active A~0 D A cells in subjects continuously treated with either N O 112 or SCH 23390. Apomorphine (50 gg/kg) reversed the effect of haloperidol in both areas, indicating that the decrease in active D A cells resulted from depolarization block.
DISCUSSION In the present study, repeated daily injections (4 weeks) of the selective D1 antagonists SCH 23390 and SCH 39166 failed to alter the n u m b e r ofspontaneously active A9 or A~0D A neurons in the rat midbrain. In this respect, our findings are similar to those of Esposito and Bunney (1989) who reported no effect of SCH 23390 following 21 days of repeated injections, at this and higher doses. It is unclear why other investigators were successful in observing decreases in active cells following similar treatments with SCH 23390 (Goldstein and Litwin, 1988; Skarsfeldt, 1988). In contrast to repeated daily injections, continuous administration of the Dm antagonists SCH 23390 and N O l l 2 , but not SCH 39166, produced small, but statistically significant, decreases in the n u m b e r of spontaneously active A~0 D A neurons. This selective inactivation of Am0 D A cells is similar to, although
132S
Dopamine 90
weaker than, those previously observed iollowing repeated injections of the atypical antipsychotic drugs clozapine and thioridazine (Chiodo and Bunney, 1983; Skarsfeldt, 1988; White and Wang, 1983b) and the new potential antipsychotic drug BMY 14802 (Wachtel and White, 1988). The lack of effect of SCH 39166, as compared to the other D~ antagonists, is surprising and suggests possible contributions by mechanisms other than DL receptor blockade. Interestingly, NO 112 and SCH 23390 both possess fairly high affinities for 5-HT2-receptors (Andersen et al., 1988; Hicks et al., 1984), whereas SCH 39166 does not (Chipkin et al., 1988). A previous study found that the selective 5-HT, antagonist ICI 169,369 produced a selective inactivation of A~0 DA neurons following repeated administration (Goldstein et al., 1989), suggesting possible 5-HT2 involvement in the effects observed during the present study. Similar to BMY 14802 (Wachtel and White, 1988), which is only a weak antagonist of DA receptors, i.v. injections of the DA agonist apomorphine at doses of 10 and 50/tg/kg were unable to reverse the inactivation of A,, DA neurons by NO 112 or SCH 23390. This finding is contrary to that of Goldstein and Litwin (1988) demonstrating a reversal of the SCH 23390 effect by apomorphine (63 /~g/kg, i.v.). It is important to note that apomorphine hyperpolarizes DA cells via actions at impulse-regulating somatodendritic autoreceptors (Grace and Bunney, 1985), which are exclusively of the D2 subtype (Wachtel et al., 1989; White and Wang, 1984). Since NO 1 12 and SCH 23390 do not block the D_, autoreceptor, apomorphine would not have to compete with these drugs for the autoreceptor as it must following administration of most neuroleptics. Therefore, the 50 pg/kg dose of apomorphine was probably high enough to hyperpolarize many DA cells to the point of quiescense, as evidenced by the further reduction of spontaneously active cells. At a lower dose of 10 ~ug/kg, apomorphine partially reversed the effects of NO 112 and SCH 23390, but this effect failed to reach statistical significance. Thus,it is still an issue as to whether the decreases in A~0activity produced by NO 112 and SCH 23390 result from depolarization block. Taken together with previous studies, these results emphasize that selective D~ receptor antagonists produce weak and inconsistent effects on the number of spontaneously active midbrain DA neurons following repeated administration. In our hands, only continuous administration resulted in a decrease in A~0 DA cells. To the extent that depolarization block represents an electrophysiological model of APD action, these
findings appear to question the potential usefulness or I)~ antagonists for the treatment of schizophrenia. However. given the many other animal models indicating effective blockade of DA neurotransmission. the relative ineffectiveness of D, antagonists in the present studies suggests the alternative possibility that DP block may not always be a necessary component of APD action.
REFERENCES
Andersen, P.H., Gronvald, F.C., Hohlweg, R., Hansen, L.B., Guddal, E. and Braestrup, C. (1988). NO-112, NO-756 New dopamine D~ selective antagonists. Soc. Neurosci. Abstr., 14, 935. Arnt, J. (1985), Behavioral stimulation is induced by separatedopamine D-I and D-2 receptor sites in reserpine pretreated but not in normal rats. Eur. J. Pharmacol., 113, 79-88, Braun, A.R. and Chase, T.N. (1986). Obligatory DL-D2 receptor coactivation and the generation of dopamine agonist related behaviors. Eur. L Pharmacol.,131,301306. Bunney, B.S. and Grace, A.A. (1978). Acute and chronic haloperidol treatment: Comparison of effects on nigral dopaminergic cell activity. Life Sci.,23,1715-1728. Chipkin, R.E. and Latranyi, M.B. (1987). Similarity of clozapine and SCH 23390 in reserpinized rats suggests a common mechanism of action. Eur. J. PharmacoL,16, 371--375. Chipkin, R.E., lorio, L.C., Coffin, V.L., McQuade, R~D., Berger, J.G. and Barnett, A. (1988). Pharmacological profile of SCH39166: A dopamine D~ selective benzonaphthazepine with potential antipsychotic activity. J. Pharmacol. Exp. Ther., 247, 1093-1102. Chiodo, L.A. and Bunney, B.S. (1983). Typical and atypical neuroleptics:Differentialeffectsof chronic administration on the activity of A9 and A~0dopaminergic neurons. J. Neurosci.,3, 1607-1619. Clark, D. and White, F.J. (1987). Review: D~ dopamine receptor The search for function: A critical evaluation of the DJD2 dopamine receptor classificationand its functional implications. Synapse, 1,347-388. Coffin,V.L., Latranyi, M.B. and Chipkin, R.E. (1989). Acute extrapyramidal syndrome in cebus monkeys: Development by dopamine D2but not D~ receptors. J.Pharmacol Exp. Ther., 249, 769-774. Esposito, E., and Bunney, B.S. (1989). The effect of acute and chronic treatment with SCH 23390 on the spontaneous activity of midbrain dopamine neurons. Eur. J: Pharmacol., 162, 109-I 13. Goldstein, J.M., and Litwin, L.C. (1988). Spontaneous activity of As and A~odopamine neurons after acute and chronic administration of the selective dopamine Dlantagonist SCH 23390. Eur. J. Pharmacol., 155; 175180. Goldstein, J.M., Litwin, L.C., Sutton, E.B. and Malick, J.B. (1989) Effects of ICI 169,369, a selective serotoninantagonist, in electrophysiological tests predictive of antipsychotic activity.J. Pharmacol. Exp. Ther. 249, 673680.
Dopamineg0 Grace, A.A. and Bunney, B.S. (1985). Low doses of apomorphine elicit two opposing influences on dopamine cell electrophysiology. Brain Res., 333, 285-298. Iorio, L.C., Barnett, A., Leitz, F.H., Houser, U.P. and Korduba, C.A. (1983).SCH 23390, a potential benzazepine antipsychotic with unique interactions on dopaminergic systems. J. Pharmacol. Exp. Ther., 226, 462-468. Jackson, D.M. and Hashizume, M. (1986). Bromocriptine induces marked locomotor stimulation in dopaminedepleted mice when D-I dopamine receptors are stimulated with SKF 38393. Psychopharmacology, 90, 147-149. Skarsfeldt, T. (1988). Effect of chronic treatment with SCH 23390 and haloperidol on spontaneous activity of dopamine neurones in substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) in rats. Eur. J. Pharmacol., 145,239-243 Wachtel, S.R. and White, F.J. (1988) Electrophysiological effects of BMY 14802, a new potential antipsychoti¢ drug, on midbraln dopamine neurons in the rat: Acute and chronic studies. J. Pharmacol. Exp. Ther. 244, 410-416. Wachtel, S.R., Hu X.-T., Galloway, M.P., and White, F.J. (1989). D~ dopamine receptor stimulation enables the postsyaptic, but not autoreceptor, effects of D2 dopamine
133S
agouists in nigrostriatal and mesoaccumbens doparuine systems. Synapse, 4, 327-346. Waiters, J.R., lkrgstrom, D.A., Carlson, J.H., Chase, T.N., and Braun, A.R. (1987). D~ dopamine receptor activation required for postsynaptic expression of D2 agonist effects. Science, 236, 719-722. White, F.J. (1987). D-1 dopamine receptor stimulation enables the inhibition of nucleus accumbens neurons by a D2 receptor agouist. Eur. J. Pharmacol., 135, 101-105. White, F.J., and Wang, R.Y. (1983a). Comparison of the effects of chronic haloperidol treatment on A9 and A10 DA neurons in the rat. Life Sci., 32, 983-993. White, F.J., and Wang, R.Y. (1983b). Differential effects of classical and atypical antipsychotic drugs on A9 and A~0DA neurons. Science, 221, 1054-1057. White, F.J. and Wang, R.Y. (1984). Pharmacological characterization of dopamine autoreceptors in the rat ventral tegmental area: Microiontophoretic studies. J. Pharmacol Exp. Ther. 231,275-280. White, F.J., Bednarz, L.M., Wachtel, S.R., Hjorth, S., and Brooderson R.J. (1988). Is stimulation of both D~ and D 2 receptors necessary for the expression of dopamine-rnediated behaviors? Pharmacol. Biochem. Behav., 30, 189-193.
0197-0186/92 $5.00+0.00 Copyright © 1992 Pergamon Press plc
THE EFFECT OF CONTINUOUS A N D REPEATED ADMINISTRATION OF D1 DOPAMINE RECEPTOR ANTAGONIST ON MIDBRAIN DOPAMINE NEURONS STEPHENR. WACHTELand FRANCISJ. WHITE NeuropsychopharmacoiogyLaboratory, Department of Psychiatry,Cellular and Clinical NeurobiologyProgram, Wayne State University School of Medicine, Lafayette Clinic, 951 E. Lafayette, Detroit, M148201, U.S.A.
Many synergistic effects of D l and D2 dopamine (DA) agonists result from an enabling interaction between D~ and D2 receptors in which Dl receptor stimulation is required for the functional effects produced by D2 receptor stimulation (see Clark & White, 1987 for review). The enabling phenomenon was first indicated by the finding that the Dt receptor antagonist SCH 23390 could block the locomotion and stereotyped behaviors elicited by both mixed (non-selective) Dr/D2 agonists and selective D 2 agonists (e.g. Arnt, 1985; Iorio et al., 1983). Further evidence for this permissive role of Dt receptor stimulation was provided by the findings that acute depletion of endogenous DA, produced by methyl-p-tyrosine (AMPT) and/or reserpine, prevented the stimulation of locomotor activity and stereotypy produced by the administration of selective D2 agonists, and that these behaviors were reinstated by the D~ agonist SKF 38393 (Braun & Chase, 1986; Jackson & Hashizume, 1986; White et al., 1988). Electrophysiological experiments have demonstrated that the enabling role of D~ receptor activation for D2 receptor functional effects is also evident at the cellular level. In the both the dorsal (caudateputamen) and ventral (nucleus accumbens) striatum, acute DA depletion produced by AMPT greatly attenuated the inhibition of firing normally produced by iontophoretic administration of selective D2 agonists (quinpirole and RU 24213). The D 2 agonistinduced neuronal inhibition was restored by coadministration of SKF 38393 (Wachtel et al., 1989; White, 1987). Similar relationships have been reported for the excitatory effects of D2 agonists, administered systemically, on rat globus pallidus cells (Waiters et a/., 1987). The discovery of the enabling role of Dt receptors for D2 receptor-mediated functions has added to the speculation that selective DI receptor antagonists might be useful in the treatment of schizophrenia, a disorder typically thought to involve hyperactive DA transmission and currently treated
with DA receptor blockers. From the perspective of D~ receptor enabling, it would appear that D~ receptor antagonists might reduce DA transmission through both Dt receptors (directly) and D2 receptors (reduced enabling). Particularly interesting are recent reports suggesting that selective D~ receptor antagonists exert effects on DA systems which closely resemble those of atypical antipsychotic drugs (APDs), i.e. those with a reduced liability for producing extrapyramidal motor disorders (e.g. Chipkin and Latranyi, 1987; Coffin et al., 1989).Among the animal models typically used to screen potential APDs for therapeutic utility and side effect liability, the ability to produce a depolarization block of midbrain DA neurons has received considerable attention primarily because it is observed only following repeated administration (as are many clinical effects of APDs). Over the past decade, a series of studies has demonstrated that classical APDs, such as haloperidol or chlorpromazine, cause a timedependent decrease in the number of spontaneously active DA cells in both the ventral tegmental area (VTA, A~0)and substantia nigra (SN, As) (Bunney & Grace, 1978; White & Wang, 1983a,b), whereas atypical APDs, such as clozapine selectively inactivate Al0 DA cells (Chiodo & Bunney, 1983; White & Wang, 1983b). Previous attempts to determine whether selective D~ receptor antagonists can induce depolarization block of DA neurons have provided highly discrepant results. Skarsfeldt (1988) reported that the D1 antagonist SCH 23390 caused a dosedependent reduction in the number of both As and At0 DA neurons following twice daily s.c. injections (0.020.31 mg/kg) for 21 days. However, this report did not include a demonstration ofhyperpolarization-induced reversal (e.g. with apomorphine) of the reduction in active neurons. Goldstein and Litwin (1988) reported that a 28 day treatment of once daily s e l l 23390 injections (0.05 mg/kg, s.c.) produced a selective decrease in the number of Aio DA neurons, an effect reversed by i.e. apomorphine (0.063 mg/kg). However,
129S
130S
l)opammc 911 2.0! I
--Z~-
o
_.}_
1.0
0.5
I
Saline SCH 23390 SCH 39166 Haloperidol NO 112 Ventral T e g m e n t a l area
Saline SCH 23390 SCH 39166 Haloperidol NO 112
S u b s t a n t i a Nigra
Fig. 1. Effects of continuous (28 day) administration of D~ receptor antagonists on the number of spontaneously active A0 and A~0 DA neurons. Bars represent the mean + SEM. Asterisks represent a significant difference IP < 0.05) from the saline control using a Student's two-tailed t-test. Espositio and Bunney (1989) f o u n d no evidence for depolarization block following 21 days o f once daily S C H 23390 injections (0.05-0.t0 mg/kg, s.c.) or a similar time o f m o r e constant administration via the animals' drinking water (5.0 - 10.0 mg/kg).In the present experiments, we have also examined the effects o f SCH 23390 on m i d b r a i n D A neurons following b o t h repeated injections (0.5 mg/kg, s.c. for 28 days) and continuous administration via osmotic minip u m p s (28 days) administration. In addition, we have tested two new selective Di receptor antagonists, N O - 1 1 2 [( + )-8-chioro-7-hydroxy-3-methyl-5(7-benzofuranyl)-2,3,4,5,tetrahydro- l H-3-benzazepin] and SCH 39166 [ ( - ) - t r a n s - 6 , 7 , 7 a , 8 , 9 , 1 3 b - h e x a h y d r o - 3 chloro-2- hydroxy-N-methyl-SH-benzo[d]naptho-2,lbazepine], as well as haloperidol for comparative purposes. Both N O - 1 1 2 and SCH 39166 possess a high affinity for D~ receptors in b o t h ligand binding a n d DA-stimulated adenylate cyclase assays (Andersen et aL, 1988; Chipkin et al., 1988l.
EXPERIMENTAL PROCEDURES
In one set of experiments NO-112. SCH 39166. SCH 23390, haloperidol and saline were administered continuously via model 2002 mini osmotic pumps lAlza Corporation, Palo Alto, CAl. Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 150200 g were anesthetized with Brevital (50 mg/kg, i.p., Eli Lily, Indianapolis, IN) and the pumps inserted subcutaneously on the dorsal flanks of the rat through a 1 cm incision. At the time of replacement a second pump was inserted on the opposite flank. Both pumps were removed 2 hrs before the cell counting began on the last day of drug treatment. The initial concentration of drug in the pumps was 2 mg/ml, which resulted in an approximate daily dose of 0.13 mg/kg/day. When the second pump was inserted for the last two weeks of
treatment, the concentration ofantagonists was increased to 4 mg/ml to account for increased body weights. In a second set of experiments. SCH 39166 and SCH 23390, were administered s.c.. 0.5 mg/kg, twice daily for 28 days. Haloperidol was injected at the same dose but only once each day. Saline injected rats received an equivalent volume and served as controls. Cell counting began two hours after the last injection. The procedure for counting spontaneously active DA cells has been described previously IWachtel and White. 1988). Briefly, the rats were anesthetized with choral hydrate (400 mg/kg, i.p.), mounted in a stereotaxic appartatus, and a single barrel extracellular electrode lowered 12 times through a stereotaxically consistent grid in both the SN (3.0-3.4 mm anterior. 2.0-2.6 mm lateral to lambda) and VTA (3.0-3.4 mm anterior. 0.41.0ram lateral), and the number of spontaneously active DA cells counted. The electrode was lowered from 6.0 to 8.5 mm below the cortical surface in each track. Individual tracks were separated by 200 m. For half of the rats. A9 was recorded before A~0, and the order reversed for the other rats. If a decrease in the number of cells encountered after continuous antagomst treatment was observed, an additional group of rats was treated with the particular antagonist, and the ability of Figure 1 Apomorphine to reverse the decrease in the number of spontaneously active Ao or A~0 DA cells was examined to determine if the decrease was the result of depolarization block. In these animals, apomorphine was injected (t0 or 50 /zg/kg, i.v.)just before cell counting was begun. The counting was performed as described above: however, ira decrease was observed in both A9 and A~0,only six tracks were utilized in each area after apomorphine administration. RESULTS
C o n t i n u o u s administration o f N O - t 12 and S C H 23390 via osmotic m i n i p u m p s resulted in a selective reduction in the n u m b e r o f spontaneously active A~0 D A neurons as c o m p a r e d to vehicle treated controls. C o n t i n u o u s administration o f haloperidol decreased the n u m b e r o f spontaneously active D A cells in b o t h the SN and V T A (Fig. 1). Surprisingly, S C H 39166
Dopamine 90
131S
2.0
1.5 - -
V
T
0
.m 1.0 (D
o
0.5 0
Saline
SCH 23390 Haloperidol SCH 39166
Saline
Ventral Tegmental area
SCH 23390 Haloperidol SCH 39166
Substantia Nigra
Fig. 2. Effects of repeated (28 day) administration of Dt receptor antagonists on the number of spontaneously active A9 and A~0 DA neurons. Bars represent the mean + SEM. Asterisks represent a significant difference (p < 0.01) from the saline control using a Student's two-tailed t-test. Table 1. Effectof apomorphineafter continuousantagonistadministration A9
Ai0
Drug
Mean
SEM
N
Mean
SEM
Saline NO 112 + Apomorphine 10#g/kg + Apomorphine 50 #g/kg SCH 23390 + Apomorphine lO/~g/kg + Apomorphine 50 pg/kg Haloperidol + Apomorphine 50 ,ug/kg
0.92 0.87
0.05 0.10
N
7
1.49
0.11
7
7
1.06*
0.13
8
--
--
1.28
0.13
6
0.68
0.10
8
0.77* 1.03"
0.14 0.16
4 8
--
--
--
1.29
0.07
4
-0.59
-0.12
--
0.71"
0.05
4
89
1.00"
0.17
8
0.83
0.07
4
1.50"*
0.20
5
--
* Significantlydifferentfrom salinecontrol (p < 0.05). ** Significantlydifferentfrom DA antagonistalone (p < 0.05). failed to alter the n u m b e r of spontaneously active D A cells encountered in either area (Fig. 1). In contrast to the continuous administration regimen, repeated administration of SCH 23390 failed to produce a reduction in the n u m b e r of A9 or A~0 D A cells. Again, SCH 39166 was ineffective. As previously reported, haloperidol produced a nonselective decrease in spontaneously active D A neurons in both areas (Fig. 2). Acute injections of apomorphine (10 or 50 gg/kg) failed to reverse the selective decrease in spontaneously active A~0 D A neurons produced by continuous N O - 1 1 2 or SCH 23390 (Table 1.) Infact, the 50 g g dose of apomorphine further decreased the n u m b e r of spontaneously active A~0 D A cells in subjects continuously treated with either N O 112 or SCH 23390. Apomorphine (50 gg/kg) reversed the effect of haloperidol in both areas, indicating that the decrease in active D A cells resulted from depolarization block.
DISCUSSION In the present study, repeated daily injections (4 weeks) of the selective D1 antagonists SCH 23390 and SCH 39166 failed to alter the n u m b e r ofspontaneously active A9 or A~0D A neurons in the rat midbrain. In this respect, our findings are similar to those of Esposito and Bunney (1989) who reported no effect of SCH 23390 following 21 days of repeated injections, at this and higher doses. It is unclear why other investigators were successful in observing decreases in active cells following similar treatments with SCH 23390 (Goldstein and Litwin, 1988; Skarsfeldt, 1988). In contrast to repeated daily injections, continuous administration of the Dm antagonists SCH 23390 and N O l l 2 , but not SCH 39166, produced small, but statistically significant, decreases in the n u m b e r of spontaneously active A~0 D A neurons. This selective inactivation of Am0 D A cells is similar to, although
132S
Dopamine 90
weaker than, those previously observed iollowing repeated injections of the atypical antipsychotic drugs clozapine and thioridazine (Chiodo and Bunney, 1983; Skarsfeldt, 1988; White and Wang, 1983b) and the new potential antipsychotic drug BMY 14802 (Wachtel and White, 1988). The lack of effect of SCH 39166, as compared to the other D~ antagonists, is surprising and suggests possible contributions by mechanisms other than DL receptor blockade. Interestingly, NO 112 and SCH 23390 both possess fairly high affinities for 5-HT2-receptors (Andersen et al., 1988; Hicks et al., 1984), whereas SCH 39166 does not (Chipkin et al., 1988). A previous study found that the selective 5-HT, antagonist ICI 169,369 produced a selective inactivation of A~0 DA neurons following repeated administration (Goldstein et al., 1989), suggesting possible 5-HT2 involvement in the effects observed during the present study. Similar to BMY 14802 (Wachtel and White, 1988), which is only a weak antagonist of DA receptors, i.v. injections of the DA agonist apomorphine at doses of 10 and 50/tg/kg were unable to reverse the inactivation of A,, DA neurons by NO 112 or SCH 23390. This finding is contrary to that of Goldstein and Litwin (1988) demonstrating a reversal of the SCH 23390 effect by apomorphine (63 /~g/kg, i.v.). It is important to note that apomorphine hyperpolarizes DA cells via actions at impulse-regulating somatodendritic autoreceptors (Grace and Bunney, 1985), which are exclusively of the D2 subtype (Wachtel et al., 1989; White and Wang, 1984). Since NO 1 12 and SCH 23390 do not block the D_, autoreceptor, apomorphine would not have to compete with these drugs for the autoreceptor as it must following administration of most neuroleptics. Therefore, the 50 pg/kg dose of apomorphine was probably high enough to hyperpolarize many DA cells to the point of quiescense, as evidenced by the further reduction of spontaneously active cells. At a lower dose of 10 ~ug/kg, apomorphine partially reversed the effects of NO 112 and SCH 23390, but this effect failed to reach statistical significance. Thus,it is still an issue as to whether the decreases in A~0activity produced by NO 112 and SCH 23390 result from depolarization block. Taken together with previous studies, these results emphasize that selective D~ receptor antagonists produce weak and inconsistent effects on the number of spontaneously active midbrain DA neurons following repeated administration. In our hands, only continuous administration resulted in a decrease in A~0 DA cells. To the extent that depolarization block represents an electrophysiological model of APD action, these
findings appear to question the potential usefulness or I)~ antagonists for the treatment of schizophrenia. However. given the many other animal models indicating effective blockade of DA neurotransmission. the relative ineffectiveness of D, antagonists in the present studies suggests the alternative possibility that DP block may not always be a necessary component of APD action.
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