D1 agonist SKF 38393 antagonizes pentobarbital-induced narcosis and depression of hippocampal and cortical cholinergic activity in rats

Life Sciences, Vol. 49, pp. 595-601 Printed in the U.S.A.

Pergamon Press

D1 AGONIST SKF 38393 ANTAGONIZES PENTOBARBITAL-INDUCED NARCOSIS AND DEPRESSION OF HIPPOCAMPAL AND CORTICAL CHOLINERGIC ACTIVITY IN RATS A. Horita, M.A. Carino and Y. Nishimura Departments of Pharmacology and Psychiatry, University of Washington School of Medicine, Seattle, WA 98195 (Received in final form June 21, 1991)

SUMMARY SKF 38393 (5 mg/kg), but not quinpirole, shortened the duration of loss of righting reflex produced in pentobarbital-narcotized rats. This effect was blocked by atropine (2 mg/kg), but not by atropine methylbromide, suggesting involvement of central cholinergic mechanisms. The analeptic effect was also blocked by SCH 23390 (0.2 mg/kg) or raclopride (2 mg/kg). SKF 38393 also increased sodium dependent high affinity choline uptake (HACU) in cortical and hippocampal synaptosomes that had been depressed by pentobarbital. SCH 23390 or raclopride prevented the SKF 38393 reversal of the depressed HACU, indicating that both D1 and I)2 mechanisms were involved mediating the analeptic effect. These results provide neurochemical evidence that cortical and hippocampal D1mediated cholinergic activation results in a behavioral arousal (analeptic) response. They also suggest that DA mechanisms may be involved in regulation of cortical and hiplx)campal cholinergic neurons. The discovery of D1 and D2 dopamine (DA) receptor agonists and antagonists has prompted investigations to uncover the physiological role of these receptor subtypes. Whereas the D2 subtype represents the binding sites for the antipsychotic drugs (1,2), little is known as to the significance of the D1 receptor. Several behaviors are thought to be associated with the D1 system, but more often this receptor subtype has been described as mediating a "potentiating" or "enabling" effect for full expression of D2 receptor-mediated responses (3-6). We recently demonstrated that the administration of D1 agonist (SKF 38393), but not a D2 agonist (quinpirole), produced in rabbits a shortening of the duration of pentobarbital-induced narcosis. This arousal (analeptic) response was blocked by atropine as well as by the D1 and D2 antagonists, SCH 23390 and raclopride, respectively (7). These results suggested that a dopaminergic mechanism was involved in activating central cholinergic pathways that ultimately led to the arousal response. The present study was undertaken to confirm that the arousal effect of SKF 38393 that was seen previously in rabbits could also be reproduced in rats, and upon so doing, conduct neurochemical experiments to demonstrate that this arousal effect was associated with increased hippocampal and cortical cholinergic activity. 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc

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METHODS Male Sprague-Dawley rats, 300-350 gm (Tyler Labs, WA) were used in these studies. All experiments were conducted in an ambient temperature of 22.0 + 1.0 ° C. ~ . The following drugs, the doses of which are expressed as the base, were dissolved in 0.9% saline solution: R(+)-SKF 38393 (RBI); (-)-quinpirole HC1 (RBI); R(+)-SCH 23390 tartrate (Schering-Plough); raclopride tartrate (Astra); pentobarbital Na; atropine sulfate and atropine methylbromide. All injections were made by the intraperitoneal (IP) route. Analeotic activity. The arousal property of SKF 38393 was measured as a shortening of the duration (recovery of the fighting reflex) of narcosis produced by pentobarbital (40 mg/kg). Integrity of the fighting reflex was determined by placing the animal on its back and observing if it would resume and maintain the upright position. Injections and observations of animals were made by different individuals; i.e., the observer was not aware of the treatments given to each animal. High affinity choline uptake (I-IACU). The method employed was based on the procedure described by Yamamura and Snyder (8) and modified by Zucker et al (9). Synaptosomes of hippocampus or frontal cortex were prepared and incubated with 0.5 uM choline containing 0.4 uCi of 3H-choline chloride (80 Ci/mmol), New England Nuclear. The incubation was started by transfer of the samples from an ice bath to water incubator at 38 ° C. After 4 rain. of incubation, the uptake reaction was terminated by transfer back to the ice bath. The particulate in each sample was collected by centrifugation at 8000 x g for 20 min. The remaining medium was decanted and the pellet surface washed with 1 ml of ice-cold 0.9% saline. After removal of the saline, the pellet was dissolved overnight with 0.7 ml Protosol (NEN). All uptake experiments were performed in triplicate and radioactivity determined by liquid scintillation technique. Protein concentration was measured by the method of Lowry et al (10) with bovine serum albumin as external standard. Sodium-dependent HACU was expressed as pmol/mg protein/4 min. Data analysis. The data were analyzed by one way analysis of variance, and the differences between treatment groups by the Newman-Keuls method. A difference of p<0.05 was considered statistically significant. RESULTS The analeptic effect of SKF 38393 in rats narcotized with pentobarbital is presented in Fig. 1A. One-way analysis of variance showed a significant difference among treatment groups (F[3,43]=16.2, P<0.005). Newman-Keuls test showed that the duration of narcosis in the 5 mg/kg and 10 mg/kg SKF 38393-treated animals were significantly shorter than saline-treated animals (p < 0.01). Quinpirole, in doses of 2 mg/kg and 5 mg/kg, was without analeptic activity (Fig. 1B) (F[2,23]=2.9, P>0.05, n.s.). The effect of atropine and atropine methylbromide on the SKF 38393 analeptic effect is shown in Fig. 2. Analysis of variance showed significant differences among treated and control groups (F[4,38] = 10.4, P < 0.005). Atropine, but not atropine methylbromide, completely blocked the analeptic effect of SKF 38393, indicating that the effect was mediated via central cholinergic mechanisms.

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A

D-I Mediated Cholinergic Arousal

B

Saline or SKF 38393

597

Saline or Quinpirole I I

Saline

~--

]

:Salinei I

SKF (2)

Quin (2)

SKF(5)

Quin (5)

SKF (tO)

I

I' 20

I i i 40 60 80

I

I

I00

'i 20

I

40

l

I

60 80

I

I00

Fig. 1 Effect of varying doses of (A) SKF 38393 and (B) quinpirole on the duration of pentobarbital-induced narcosis in rats. Each bar represents the mean duration of narcosis (min + SEM, N=8-16) after the designated drug treatments. Saline or agonist was administered 20 min after pentobarbital. Figures in parentheses indicate dose (mg/kg IP) administered. One way analysis of variance showed differences among the SKF 38393 groups (F[3,43] = 16.2, P<0.005). *P<0.01 when compared with saline controls, as determined by Newman-Keuls test. One way analysis of variance showed differences among the quinpirole groups (F[3,31] = 4.67, P < 0.025). However, none of the quinpirole animals showed significant differences when compared to saline controls, as determined by Newman-Keuls test. Table 1. Analeptic effect of SKF 38393 (5 mg/kg IP) in pentobarbital-narcotized rats and its reversal by D-1 and D-2 antagonists. Drug 2 was administered 10 min, and drug 3, 20 min after drug 1.

Drug Treatment N

1 A. B. C. D. E. F.

PB PB PB PB PB PB

2 Saline Saline SCH 23390 SCH 23390 Raclop Raclop

3 Saline SKF 38393 SKF 38393 Saline SKF 38393 Saline

13 12 7 6 9 6

Mean Duration of Narcosis (min+SEM) 93 _ 66 _ 103 + 106 + 99 _ 107 _

10 8* 11" 8 13 20

PB=Pentobarbital, 40 mg/kg IP; SKF 38393, 5 mg/kg IP; SCH 23390, 0.2 mg/kg IP; Raclopridc, 2 mg/kg IP. One way analysis of variance indicated significant differences among groups, (F[5,47]=16, P<0.005). *P<0.01 when group B was compared with A, B with C, and B with E, according to Newman-Keuls test.

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SALINE OR

SKF 38393

SALINE

SKF ATR + SKF ATR -ISALINE ME-ATR + SKF

! I

I

I

30

60

I

90

I 120

Fig. 2 Effect of atropine (ATR, 2 mg/kg) or atropine methylbromide (ME ATR, 2 mg/kg) on the analeptic effect of SKF 38393 (SKF, 5 mg/kg). Each bar represents mean duration of narcosis (min _ SEM, N=5-1 i) after the designated treatments. Anticholinergic or saline was administered 10 rain, and saline or SKF 38393 20 min, after pentobarbital. One way analysis of variance showed differences among groups, (F[4,38] = 10.4, P<0.005). *P<0.01 when compared with saline controls, as determined by Newman-Keuls test. Table 1 shows the antagonism of the analeptic effect of SKF 38393 (5 mg/kg) by SCH 23390 (0-.2 mg/kg) or raclopride (2 mg/kg). These doses of antagonists were selected because other investigators had used them for selective blockade of behaviors mediated by agonists of the respective receptor subtypes (11-13). High affinity choline uptake (HACU) activities of cortical and hippocampal synaptosomes were measured under the different conditions to determine whether a neurochemical correlate of cholinergic activity could be found with the SKF 38393induced analeptic response. The effect of pentobarbital and pentobarbital + SKF 38393 is shown in Fig. 3. Pentobarbital lowered HACU in cortex and hippocampi of saline-treated rats by 33%. SKF 38393 (2 or 5 mg/kg) restored cortical and hiplx~.ampal HACU activities to control levels. We also investigated the effects of D1 and D2 antagonists on the HACU reversal effect of SKF 38393 in pentobarbital-narcotized rats. As shown in Table 2 administration of either SCH 23390 (0.2 mg/kg) or raclopride (2 mg/kg) produced total blockade of the SKF 38393-induced reversal of HACU activity that had been depressed by pentobarbital in cortical and hippocampal synaptosomes. Neither SCH 23390 nor raclopride by themselves affected HACU activity of pentobarbital-treated animals.

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F. CORTEX

50

20

I0

I

ii

C Pb Pb-I- Pb+ SKF SKF 2 5

599

HIPPOCAMPUS

I O

C

Pb Pb+ Pb+ SKF SKF 2 5

Fig. 3 Effect of SKF 38393 on the pentobarbital-induced decrease in high affinity choline uptake (HACU) activity in cortical and hippocampal synaptosomes of the rat. Saline or SKF 38393 (SKF, 2 or 5 mg/kg) was administered 20 min after pentobarbital (Pb). Animals were sacrificed 20 rain thereafter, the brains removed and synaptosomes prepared. Each bar represent mean uptake of ~H choline/4 min/mg protein + SEM, N=7-9. One way analysis of variance showed significant differences among treatment groups of cortical and hippocampal synaptosomes (F[3,27] = 9.6, P<0.001; F[3,27] = 7.1, P<0.001, respectively). *P <0.01 when compared with saline controls (C), as determined by Newman-Keuls test. DISCUSSION The present results confirm and extend our earlier findings that SKF 38393 exerted its arousal effect by activating hippocampal and/or cortical cholinergic systems (7). They are also consistent with our earlier studies demonstrating that cholinergic mechanisms were involved in the analeptie actions of peptides, opiates and locomotor stimulants (14-17). Since SKF 38393, but not quinpirole, exerted an analeptic effect, it appeared that a D1 mechanism was involved in cholinergic activation. However, the fact that raclopride also blocked the analeptic effect suggested that a D2 component might also play a role in this response. Such a view is not inconsistent with current ideas of the functions of D1 and D2 receptor subtypes. In a number of behavioral effects of the D1 or D2 agonists, one or the other receptor subtype appears to modulate the other by exerting an "enabling" effect (3-6). For example, Starr and Starr (4) demonstrated that in mice depleted of dopamine stores, neither SKF

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Table 2. Effect of SKF 38393 on the pentobarbital-induced decrease in HACU activity in rat hippocampal and cortical synaptosomes and its blockade by SCH 23390 and raclopride. Saline or SKF 38393. Drugs 2 and 3 were administered IP 10 and 20 min, respectively, after drug I. The animals were sacrificed 20 min after drug 3, the brains removed and synaptosomes prepared.

Drug Treatment 1

A. B. C. D. E. F.

PB PB PB PB PB PB

2

Saline Saline SCH SCH Raclop Raclop

N

HACU (pmol/mg protein/4 min+SEM) Cortex Hippocampus

8 9 6 9 7 6

20 30 23 20 22 21

3

Saline SKF Saline SKF Saline SKF

+ 3 -I- 6** -I- 3 _ 4** _+ 4 + 5*

16 22 19 17 18 17

+ 4 _ 3** -t- 2 _+ 2** _ 4 _ 3*

SKF = SKF 38393 (5 mg/kg IP); PB=Pentobarbital (40 mg/kg IP); SCH = SCH 23390 (0.2 mg/kg IP); Raclop = Raclopride (2 mg/kg IP). One way analysis of variance showed significant differences in synaptosomal HACU activities of cortex (F[5,39] = 5.4, P<0.005), and of hippocampus (F[5,39] = 3.38, P < 0.025). **P<0.01 when treatment A is compared to treatment B, or B is compared to D *P<0.05 when treatment B is compared to treatment F

38393 nor quinpirole produced locomotor activation. However, if quinpirole were given prior to SKF 38393, the latter exhibited a dose-related increase in locomotor activity. In the same way SKF 38393 may require a background of D2 "tone" for its expression of the Dl-mediated arousal effect. The blockade of the SKF 38393-mediated analepsis by SCH 23390 or raclopride is not because of an anticholinergic action. Earlier studies showed that while atropine completely blocked the behavioral effects of physostigmine and oxotremorine, neither of the DA antagonists in the doses used in this study exerted any antagonism of cholinergic activity (7). Instead, the finding that SCH 23390 and raclopride prevented the SKF 38393-induced reversal of HACU that was depressed by pentobarbital, suggested that they exerted the antianaleptie effect by blocking ACh release in cortex and/or hippocampus. The results of this study are also consistent with the EEG findings of Ongini et al (11,18) who reported that SKF 38393 and quinpirole produced arousal EEG in hippocampus and cortex of conscious rats and rabbits. However, these authors did not investigate the possibility of a cholinergic mechanism in these arousal responses. Their impression was that whereas SKF 38393 produced direct EEG arousal, quinpirole might have produced its EEG responses indirectly because it exerted considerable behavioral excitation and stereotypic behaviors. In our studies the latter possibility was obviated because all of the animals were in the anesthetized state.

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We believe that this study presents the first evidence that a measurable Dl-mediated behavior (analeptie activity) is associated with increased cortical and/or h i p ~ m p a l cholinergie function as determined by HACU. Other studies showed that dopamine agonists inhibited s e p t o h i ~ p a l cholinergic neurons via activation of GABA mechanisms (19). Other workers have reported that increased ACh release was produced by administration of D2 agonists, such as bromocriptine (20) and quinpirole (21), but not by SKF 38393. These and other references are indicative of the growing interest in understanding DA's role in regulating cortical and hippocampal ACh release. Such understanding will eventually explain the mechanisms and importance of the DA-cholinergic relationship described for the arousal actions of dopamine agonists and the psychomotor stimulants (22). ACKNOWLEDGEMENTS This research was supported by grants from the USPHS (DA4907) and the Rehabilitation and Research Training Grant, H133B80081. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22.

I. CREESE. Psych0pharmacology: The Third Generation of Progress, H.Y. Meltzer, ed., pp. 257-264, Raven Press, New York (1987). P. SEEMAN. Synapse 1 133-152 (1987). C. CLARK;F.J. WHITE. Synapse 1 347-388 (1987). M.S. STARR, B.S. STARR. Pharmacol. Biochem. Behav. 33 41-44 (1989) J.R. WAITERS, D.A. BERGSTROM,J.H. CARLSON,T.N. CHASE, A.R. BRAUN. Science 236 719-722 (1987) G.S. ROBERTSONand H.A. ROBERTSON. Trends Pharmacol. Sci. 8 295-299 (1987) A. HOR1TAand M.A. CARINO. Pharmacol. Biochem. Behav. (In press, 1991) H . I . YAMAMURA,S.H. SNYDER. J. Neurochem. 21 1355-1374 (1973) J.R. ZUCKER, H. LAI and A. HORITA. J. Pharmacol. Exp. Ther. 235 398-401 (1985) O.H. LOWRY. J. Biol. Chem. 193 265-275 (1951) E. ONGINI, V.G. LONGO. Int. Rev. Neurobiol. 31 239-255 (1989) S.A. PARASHOS, C. MARIN, T.N. CHASE. Neurosci. Lett. 105 169-173 (1989) J. POLLOCK,C. KOP.NETSKY. Neurosci. Lett. 102 291-296 (1989) P.W. KALIVAS, A. HORITA. E. Griffith; G. Bennett, eds. Thyrotropin releasing hormone. New York: Raven; 283-290 (1983) A. HORITA, M.A. CAR1NO. J. Pharmacol. Exp. Ther. 247 863-867 (1988) A. HORITA, M.A. CARINO, C. CHINN. Neuropharmacol. 28 481-486 (1989) M. YABASE, M.A. CARINO, A. HoRrrA. Pharmacol. Biochem. Behav. 37 375-377 (1990) E. ONGINI, M.G. CAPORALI,M. MASSOTTI. G. Biggio, P. F. Spano, G. Toffano, G.L. Gessa, eds. Modulation of Central and Peripheral Transmitter Function. Padova: Liviana Press; 37-46 (1986). E. COSTA, P. PANULA, H.K. THOMPSON, D.L. CHENEY. Life Sci. 32 165-179 (1983) G. PEPEU, P. MANTOVANI,F. PEDATA. D.J. Jensen, ed. Cholinergic Mechanisms and P~ychopharmacology. New York: Plenum; 605-614 (1978) F. CASAMENTI,C. CosI, G. PEPEU. M. J. Dowdall, J.N. Hawthorne, eds. Cellular and Molecular Basis of Cholinergic Function. Weinheim: VCH/Horwood; 245-249. K. YOSrnDA, M. YABASE, M.A. CARtNO, A. HoRrrA. XVII CINP Abst. 2 373 (1990)