- Email: [email protected]
Estrogen increases both spiperone-induced catalepsy and brain levels of [3H]spiperone in the rat
360
Brain Research, 172 (1979) 360-366 © Elsevier/North-HollandBiomedicalPress
Estrogen increases both spiperone-induced catalepsy and brain levels of [3H]spiperone in the rat
LOUIS A. CHIODO, ANTHONY R. CAGGIULAand CHARLESF. SALLER Psychobiology Program, Departments of Psychology, Biological Sciences and Pharmacology, University of Pittsburgb, Pittsburgh, Pa. 15260 (U.S.A.)
(Accepted April 26th, 1979)
Clinical evidence suggests that the sex of an individual can influence the behavioral consequences of drug-induced alterations in brain dopamine (DA) activity. For example, women appear more prone than men to develop parkinsonism (3:1), akathesia (3:2) and tardive dyskinesia (2:1) in response to neuroleptic drugs ~. These disorders are thought to be the consequence of disrupting normal activity in the nigrostriatal DA system. Other evidence suggests that at least some of these sex differences might be attributable to hormonal factors. Stevens 20 reported that the antipsychotic efficacy of neuroleptics may fluctuate over the menstrual cycle, and estrogen has been shown to accelerate the onset and increase the intensity of druginduced parkinsonism in both men and women 13. One recent interpretation of these findings is that circulating steroid hormones, particularly estrogen, act by blocking dopaminergic transmission4. Animal studies also suggest that hormones such as estrogen can influence the behavioral response to DA agonists and antagonists. For example, estrogen has been reported to attenuate apomorphine-induced circling in rats 4 and both fl-phenylethylamine and amphetamine-induced stereotyped behaviors in mice18, although estrogen might actually increase amphetamine-induced stereotypy in rats 6. In a study that most closely parallels the clinical reports reviewed above regarding parkinsonism, intact female rats showed a higher incidence of chlorpromazine-induced catalepsy than intact males 17. This study is particularly interesting from a clinical perspective since neuroleptic-induced catalepsy is thought to be a reasonable model of drug-induced parkinsonism in humans 9. The fact that ovariectomy virtually abolished this sex difference led the authors to suggest that the female gonadal hormones, probably estrogen, were responsible for the increased catalepsy. Given the potential importance of this hypothesis as it relates to the mechanisms by which hormones may influence the efficacy of neuroleptic drugs, we undertook a study to investigate directly the effects of estrogen on the catalepsy induced by the DAreceptor blocker spiperone 6. This drug was selected because it is a more specific and potent DA receptor antagonist than chlorpromazine, although cortical serotonergic blocking properties have been suggested 16. We also sought to provide information
361 bearing on estrogen's site of action. That is, estrogen may alter drug-induced catalepsy by a direct action at the level of the central nervous system, as Mislow and Friedhoff suggested 17. Alternately, estrogen may act peripherally, since gonadal steroids have been shown to drastically alter drug metabolism and distribution 15. Therefore, we examined the effects of estrogen on the uptake into the brain of radioactively labelled spiperone given intraperitoneally. Long-Evans hooded female rats (250-350 g; Blue Spruce Farms) were singly housed on a reversed light-dark cycle (dark period: 08.0(020.00 h) with free access to food and water. Bilateral ovariectomy was performed on all animals under ether anesthesia. They then received weekly subcutaneous injections of either estradiol benzoate (EB, 100 #g/kg) or its vehicle sesame oil (1 ml/kg) for a total of 4 weeks. Catalepsy was measured between 10.00 and 15.00 h by placing the animal's hind quarters on an elevated wood block (7.5 × 10.0 × 4.0 cm) which was mounted in the center of a 42 × 42 cm base. An electronic digital timer was started when the experimenter released the animal and was stopped when both of the animal's hind paws came to rest on the base. The step-down latency was recorded to the nearest 0.1 sec and was termed the catalepsy duration for that test. Catalepsy durations which reached 300 sec were considered maximal responses and that test was terminated. Testing took place 48 h after each hormone administration. On weeks 2 and 3, baseline catalepsy measures (i.e. without drugs) were taken once every 0.5 h for a total of 4 h to allow for adaptation to the testing procedure. On the fourth week animals received a single intraperitoneal dose of spiperone (Janssen Pharmaceuticals) or its vehicle (0.9 700 NaC1 adjusted to pH 2.5-3.0 with glacial acetic acid) and catalepsy was measured as before. All catalepsy durations were transformed to equivalent log values prior to statistical analysis (two-way, repeated measures analysis of variance). All data expressed in both the figure and the text are the retransformed values. We first examined the behavioral effects of spiperone (0.10, 0.15, 0.25 and 0.50 mg/kg, i.p.) in ovariectomized rats treated with EB or oil for 4 weeks. Animals were randomly assigned to one of the 4 dose groups or given the vehicle, and catalepsy was measured every 0.5 h for 4 (0.10 and 0.15 mg/kg) or 7 h (0.25 and 0.50 mg/kg). Spiperone induced significant catalepsy in both EB- and oil-treated animals at 0.15, 0.25 and 0.50 mg/kg (P < 0.01), but not at 0.10 mg/kg (P > 0.05; Fig. 1) when compared to vehicle controls (mean step-down latency for controls ~- 0.72 sec; average of 20 animals over 4 h of testing). In addition, EB potentiated the catalepsy produced by the two highest doses of spiperone, an effect which was statistically significant for 0.50 mg/kg (P < 0.01), but not for 0.25 mg/kg (P > 0.05). Next we examined the distribution of spiperone in blood and brain. Estrogen- or oil-treated rats were tested as described above. On the fourth week they were given 0.50 mg/kg of spiperone containing 60/~Ci/kg of [3H]spiperone (26.4 Ci/mmol, New England Nuclear) and were tested for catalepsy. They were then sacrificed by decapitation at either 3 or 6 h after drug administration. Whole brains were rapidly removed from the skull, frozen on dry-ice and stored at --70 °C until analyzed for [3H]spiperone levels. Blood was collected into 10 ml Vacutainer tubes containing disodium E D T A and stored at 4 °C for two days.
362 DOSE OF SPIPERONE 280'
2,;o
=n
z o
c~..---o Oil
160120S Ee z
~8(19)
80" 40-
131
mg/kg
.--.EB
200'
,J
• 25
• I0 m g / k g
0
280n
i
• 15
=
L
x
. (10) . - - , 1 ~ ' ~ ( 1 0 ) I I I
.50mg/kg
mg/kg
ILl
sE,,~
240-
s~-,&," El
2o0l II
16o-
"~E.--E • %~,~i'q,
i(lO)
120• I
(I0)
sO-
t 0
t
2
3
4 HOURS
AFTER
INJECTION
Fig. 1. Mean q- S.E. step-down latencies (retransformed from log transformations) for ovariectomized female rats given either 100 #g/kg of estradiol benzoate s.c. or oil and tested for 4-7 h beginning 0.5 h after spiperone (i.p.) n given in parentheses. Analysis of variance: EB > oil for 0.50 mg/kg P < 0.01. Brains were homogenized in 3 vols. (w/v) methanol-glacial acetic acid (95:5, v/v). The homogenate was centrifuged at 10,000 × g for 10 min at 4 °C. Four ml of the supernatant was transfered to 10 ml tubes and evaporated in a freeze-dryer. The remaining residue was redissolved in 250 #1 of methanol-glacial acetic acid solution. Twenty-five/zl of 0.1 N acetic acid containing 25 Fg of spiperone was then added to each tube. After vortexing, 50 #1 of this solution was transfered to 7 ml polypropylene scintillation vials. Five ml of Scintiverse scintillation cocktail (Fisher Scientific)was added to each vial and the radioactivity was determined by liquid scintillation spectrometry. In order to differentiate between spiperone and its metabolites, 50 #1 aliquots of the redissolved brain residue were spotted onto the absorbent layers of 19channel, 20 × 20 era, 250 # m silica TLC plates (LK6DF, Whatman) and developed in chloroform-methanol (9:1, v/v) or methanol-benzene-water (15:2:5, v/v). The spots corresponding to [3H]spiperone (R1s ---- 0.94 and 0.89, respectively) were visualized under UV light (254 rim), scraped into 7 ml scintillation vials, and eluted with 0.2 ml of 0.2 N acetic acid. Five ml Scintiverse was added to each vial and the samples were counted the next day. Counting efficiency was determined by internal standardization using [3H]spiperone. Values are not corrected for recovery from the TLC plates. Five-hundred/d whole blood was added to tubes containing 100 #1 1.0 N acetic acid. The samples were vortexed and centrifuged at 5000 × g for 10 min at 4 °C. One hundred F1 of the deproteinized supernatant was then transfered to 7 ml scintillation
363 vials. Two hundred #1 of toluene saturated with benzoyl peroxide was added to each vial to bleach the sample. After approximately 5 h, 5 ml of Scintiverse was added to each vial and the samples were counted. Fifty/zl aliquots of the deproteinized blood supernatant were also spotted and developed by the two TLC systems described above. All procedures were the same as those for the whole brain assay, except that 5 /A 0.1 N acetic acid containing 5/~g of spiperone was spotted in each channel of the TLC plates. Spiperone again induced significant catalepsy in all conditions (P < 0.01) which was once again potentiated by EB in the 3 and 6 h groups relative to oil controls (mean duration in sec + S.E. 116.7 ± 9.9 vs 22.6 ± 8.8 at 3 h; P < 0.01 and 182.5 4- 23.3 vs 102.0 4- 39.3 at 6 h; P < 0.03). These same EB-treated rats which showed prolonged catalepsy also had elevated brain spiperone levels following [3H]spiperone administration (Table I). That is, EB treatment resulted in increased whole brain tritium levels, relative to oil-injected controls, at both 3 (129 ~o) and 6 h (129 ~). Tritium levels in blood were also elevated at these times (125~ and 146~o of control values, respectively) in EB-treated animals. Similar results were obtained after chromatography of brain and blood samples, indicating that most of the radioactivity was accounted for by [3H]spiperone. In the present study we have demonstrated that estrogen significantly prolongs the catalepsy produced by spiperone in the rat. However, we have also shown that rats treated with estrogen had significantly higher whole brain and blood levels of [3H]spiperone than ovariectomized controls. This effect of estrogen on spiperone levels may be due to the fact that prior estrogen treatment decreases the activity of enzymes involved in the metabolism of butyrophenone neurolepticsn,la. To the extent that this effect applies to the metabolism of other neuroleptic classes as well, it is possible that the fluctuations in the behavioral response to neuroleptics produced by differences in sex or hormonal state, which has been reported in both the clinical 3,s, 13,~o and experimental animal literature6, a7 may be, at least in part, the result of estrogen's ability to alter the actual levels of drug reaching the central nervous system. In this regard, we are presently investigating the possibility that estrogen may affect the metabolism or distribution of the DA agonists, apomorphine and amphetamine. Central factors may also contribute to the ability of estrogen treatment to potentiate spiperone-induced catalepsy. Since catalepsy produced by butyrophenones such as spiperone is generally thought to be due to blocking postsynaptic DA receptors in the striatum5, our data are consistent with the view that estrogen can directly or indirectly attenuate striatal DA function4. Support for this hypothesis comes from the findings that estrogen reduces both apomorphine-induced stereotypy in micO a, and decreases the accumulation of striatal acetylcholine in response to DA agonisO 2. However, conflicting evidence exists 6,10. An antidopaminergic role for estrogen also is suggested by the finding that estrogen can antagonize the inhibition, by DA or DA agonists, of prolactin release from anterior pituitary cells in vitro 2. Biochemical studies also indicate that estrogen can affect DA function, although the exact nature of that effect is not always clear. For example, normally cycling female rats show decreased steady-state concentrations of
364
I
0
dddo
0
°i
4.,
~5 C,
,5
v .le
o. Q
v
.~S
.x.
V
365 D A in the s t r i a t u m d u r i n g o r j u s t after p e r i o d s associated with high estrogen levels 7, whereas low estrogen p e r i o d s are a c c o m p a n i e d b y elevated striatal D A c o n c e n t r a tions 14. M o r e o v e r , synthetic steroid c o n t r a c e p t i v e s generally elevate D A t u r n o v e r in the s t r i a t u m 1, a l t h o u g h estrogen m a y n o t affect either low o r high affinity b i n d i n g o f spiperone in the s t r i a t u m ( A n d o r n , p e r s o n a l c o m m u n i c a t i o n ) . Therefore, it becomes evident t h a t b o t h central a n d p e r i p h e r a l effects o f estrogen m u s t be c o n s i d e r e d w h e n e x a m i n i n g its role in b e h a v i o r a l o r n e u r o c h e m i c a l consequences o f d r u g - i n d u c e d alterations in D A function. W e w o u l d like to t h a n k D s . M. J. Z i g m o n d , S. M. A n t e l m a n a n d N. R o w l a n d f o r their c o m m e n t s on t h e m a n u s c r i p t , a n d J. H o f f m a n , M. B. D e e r i n g a n d S. O d a c h o w s k i for their l a b o r a t o r y assistance. This research was s u p p o r t e d b y grants MH16581 ( A . R . C . ) a n d MH20620 (Dr. M. J. Z i g m o n d ) .
1 Algeri, S., Ponzio, F., Dolfini, E. and Jori, A., Biochemical effects of treatment with oral contraceptive steroids on the dopaminergic system of the rat, Neuroendocrinology, 22 (1976) 343-351. 2 Beaulieu, M., Raymond, V. and Labrie, F., Antagonism between estrogens and dopamine at the anterior pituitary. In E. Usdin (Ed.), Catecholamines: Basic and Clinical Frontiers, Pergamon Press, New York, in press. 3 Bedard, P., Langelier, P. and Villeneuve, A., Oestrogens and extrapyramidal system, Lancet, ii (1977) 1367-1368. 4 Bedard, P., Dankova, J., Boucher, R. and Langelier, P., Effect of estrogen on apomorphineinduced circling behavior in the rat. Canad. J. PhysioL PharmacoL, 56 (1978) 538-541. 5 Chase, T. N., Antipsychotic drugs, dopaminergic mechanisms and extrapyramidal function in man. In G. Sedvall, B. Uvnas and Y. Zotterman (Eds.), Antipsychotic Drugs: Pharmacodynamics andPharmacokineties, Pergamon Press, New York, 1976, pp. 321-329. 6 Chiodo, L. A. and Caggiula, A. R., Estrogen modulation of spiroperidol-induced catalepsy and amphetamine stereotypy in the rat, Neurosci. Abstr., 4 (1978) 487. 7 Crowley, W. R., O'Donohue, T. L. and Jacobowitz, D. M., Changes in catecholamine content in discrete brain nuclei during the estrous cycle of the rat, Brain Research, 154 (1978) 345-357. 8 Donlon, P. T. and Stenson, R. L., Neuroleptic induced extrapyramidal symptoms, Dis. herr. Syst., 37 (1976) 629-635. 9 Duvoisin, R. C., Parkinsonism: animal analogues of the human disorder. In M. D. Yahr (Ed.), The Basal Ganglia, Raven Press, New York, 1976, pp. 293-303. 10 Eikenburg, D. C., Ravitz, A. J., Gudelsky, G. A. and Moore, K. E., Effect of estrogen on prolactin and tuberinfundibular dopaminergic neurons, J. neurol. Transm., 40 (1977) 235-244. 11 el DeFrawy el Marsy, S. and Mannering, G. J., Sex-dependent differences in drug metabolism in the rat. It. Qualitative changes produced by castration and the administration of steroid hormones and pentobarbital, Drug Metab. Dis., 2 (1974) 279-284. 12 Euvard, C., Labrie, F. and Boissier, J. R., Antagonism between estrogens and dopamine in the rat striatum. In E. Usdin (Ed.), Catecholamines: Basic and Clinical Frontiers, Pergamon Press, New York, in press. 13 Gratton, L., Neuroletiques, parkinsonisme et schizophrenia, Union Med. Canad., 89 (1960) 681-694. 14 Jori, A., Colturani, F., Dolfini, E. and Rutczynski, M., Modifications of striatal dopamine metabolism during the estrous cycle in mice, Neuroendocrinology, 21 (1976) 262-266. 15 Kato, R., Sex-related differences in drug metabolism, Drug Metab. Rev., 3 (1974) 1-32. 16 Leysen, J. E., Niemegeers, C. J. E., Tollenaere, J. P. and Laduron, P. M., Serotonergic components of neuroleptic receptors, Nature (Lond.), 272 (1978) 168-171. 17 Mislow, J. F. and Friedhoff, A. J., A comparison of chlorpromazine-induced extrapyramidal syndrome in male and female rats. In K. Lissak (Ed.), Hormones and Brain Function, Plenum Press, New York, 1973, pp. 315-326.
366 18 Naik, S. R., Kelkar, M. R. and Sheth, U. K., Attenuation of stereotyped behavior by sex steroids, Psychopharmacologia (Berl.), 57 (1978) 211-214. 19 Nerland, D. E. and Mannering, G. J., Species, sex, and developmental differences in the Oand N-dealkylation of ethylmorphine by hepatic microsomes, Drug Metab. Dis., 6 (1978) 150-153. 20 Stevens, J. R., An anatomy of schizophrenia? Arch. gen. Psychiat., 29 (1973) 177-189.
Brain Research, 172 (1979) 360-366 © Elsevier/North-HollandBiomedicalPress
Estrogen increases both spiperone-induced catalepsy and brain levels of [3H]spiperone in the rat
LOUIS A. CHIODO, ANTHONY R. CAGGIULAand CHARLESF. SALLER Psychobiology Program, Departments of Psychology, Biological Sciences and Pharmacology, University of Pittsburgb, Pittsburgh, Pa. 15260 (U.S.A.)
(Accepted April 26th, 1979)
Clinical evidence suggests that the sex of an individual can influence the behavioral consequences of drug-induced alterations in brain dopamine (DA) activity. For example, women appear more prone than men to develop parkinsonism (3:1), akathesia (3:2) and tardive dyskinesia (2:1) in response to neuroleptic drugs ~. These disorders are thought to be the consequence of disrupting normal activity in the nigrostriatal DA system. Other evidence suggests that at least some of these sex differences might be attributable to hormonal factors. Stevens 20 reported that the antipsychotic efficacy of neuroleptics may fluctuate over the menstrual cycle, and estrogen has been shown to accelerate the onset and increase the intensity of druginduced parkinsonism in both men and women 13. One recent interpretation of these findings is that circulating steroid hormones, particularly estrogen, act by blocking dopaminergic transmission4. Animal studies also suggest that hormones such as estrogen can influence the behavioral response to DA agonists and antagonists. For example, estrogen has been reported to attenuate apomorphine-induced circling in rats 4 and both fl-phenylethylamine and amphetamine-induced stereotyped behaviors in mice18, although estrogen might actually increase amphetamine-induced stereotypy in rats 6. In a study that most closely parallels the clinical reports reviewed above regarding parkinsonism, intact female rats showed a higher incidence of chlorpromazine-induced catalepsy than intact males 17. This study is particularly interesting from a clinical perspective since neuroleptic-induced catalepsy is thought to be a reasonable model of drug-induced parkinsonism in humans 9. The fact that ovariectomy virtually abolished this sex difference led the authors to suggest that the female gonadal hormones, probably estrogen, were responsible for the increased catalepsy. Given the potential importance of this hypothesis as it relates to the mechanisms by which hormones may influence the efficacy of neuroleptic drugs, we undertook a study to investigate directly the effects of estrogen on the catalepsy induced by the DAreceptor blocker spiperone 6. This drug was selected because it is a more specific and potent DA receptor antagonist than chlorpromazine, although cortical serotonergic blocking properties have been suggested 16. We also sought to provide information
361 bearing on estrogen's site of action. That is, estrogen may alter drug-induced catalepsy by a direct action at the level of the central nervous system, as Mislow and Friedhoff suggested 17. Alternately, estrogen may act peripherally, since gonadal steroids have been shown to drastically alter drug metabolism and distribution 15. Therefore, we examined the effects of estrogen on the uptake into the brain of radioactively labelled spiperone given intraperitoneally. Long-Evans hooded female rats (250-350 g; Blue Spruce Farms) were singly housed on a reversed light-dark cycle (dark period: 08.0(020.00 h) with free access to food and water. Bilateral ovariectomy was performed on all animals under ether anesthesia. They then received weekly subcutaneous injections of either estradiol benzoate (EB, 100 #g/kg) or its vehicle sesame oil (1 ml/kg) for a total of 4 weeks. Catalepsy was measured between 10.00 and 15.00 h by placing the animal's hind quarters on an elevated wood block (7.5 × 10.0 × 4.0 cm) which was mounted in the center of a 42 × 42 cm base. An electronic digital timer was started when the experimenter released the animal and was stopped when both of the animal's hind paws came to rest on the base. The step-down latency was recorded to the nearest 0.1 sec and was termed the catalepsy duration for that test. Catalepsy durations which reached 300 sec were considered maximal responses and that test was terminated. Testing took place 48 h after each hormone administration. On weeks 2 and 3, baseline catalepsy measures (i.e. without drugs) were taken once every 0.5 h for a total of 4 h to allow for adaptation to the testing procedure. On the fourth week animals received a single intraperitoneal dose of spiperone (Janssen Pharmaceuticals) or its vehicle (0.9 700 NaC1 adjusted to pH 2.5-3.0 with glacial acetic acid) and catalepsy was measured as before. All catalepsy durations were transformed to equivalent log values prior to statistical analysis (two-way, repeated measures analysis of variance). All data expressed in both the figure and the text are the retransformed values. We first examined the behavioral effects of spiperone (0.10, 0.15, 0.25 and 0.50 mg/kg, i.p.) in ovariectomized rats treated with EB or oil for 4 weeks. Animals were randomly assigned to one of the 4 dose groups or given the vehicle, and catalepsy was measured every 0.5 h for 4 (0.10 and 0.15 mg/kg) or 7 h (0.25 and 0.50 mg/kg). Spiperone induced significant catalepsy in both EB- and oil-treated animals at 0.15, 0.25 and 0.50 mg/kg (P < 0.01), but not at 0.10 mg/kg (P > 0.05; Fig. 1) when compared to vehicle controls (mean step-down latency for controls ~- 0.72 sec; average of 20 animals over 4 h of testing). In addition, EB potentiated the catalepsy produced by the two highest doses of spiperone, an effect which was statistically significant for 0.50 mg/kg (P < 0.01), but not for 0.25 mg/kg (P > 0.05). Next we examined the distribution of spiperone in blood and brain. Estrogen- or oil-treated rats were tested as described above. On the fourth week they were given 0.50 mg/kg of spiperone containing 60/~Ci/kg of [3H]spiperone (26.4 Ci/mmol, New England Nuclear) and were tested for catalepsy. They were then sacrificed by decapitation at either 3 or 6 h after drug administration. Whole brains were rapidly removed from the skull, frozen on dry-ice and stored at --70 °C until analyzed for [3H]spiperone levels. Blood was collected into 10 ml Vacutainer tubes containing disodium E D T A and stored at 4 °C for two days.
362 DOSE OF SPIPERONE 280'
2,;o
=n
z o
c~..---o Oil
160120S Ee z
~8(19)
80" 40-
131
mg/kg
.--.EB
200'
,J
• 25
• I0 m g / k g
0
280n
i
• 15
=
L
x
. (10) . - - , 1 ~ ' ~ ( 1 0 ) I I I
.50mg/kg
mg/kg
ILl
sE,,~
240-
s~-,&," El
2o0l II
16o-
"~E.--E • %~,~i'q,
i(lO)
120• I
(I0)
sO-
t 0
t
2
3
4 HOURS
AFTER
INJECTION
Fig. 1. Mean q- S.E. step-down latencies (retransformed from log transformations) for ovariectomized female rats given either 100 #g/kg of estradiol benzoate s.c. or oil and tested for 4-7 h beginning 0.5 h after spiperone (i.p.) n given in parentheses. Analysis of variance: EB > oil for 0.50 mg/kg P < 0.01. Brains were homogenized in 3 vols. (w/v) methanol-glacial acetic acid (95:5, v/v). The homogenate was centrifuged at 10,000 × g for 10 min at 4 °C. Four ml of the supernatant was transfered to 10 ml tubes and evaporated in a freeze-dryer. The remaining residue was redissolved in 250 #1 of methanol-glacial acetic acid solution. Twenty-five/zl of 0.1 N acetic acid containing 25 Fg of spiperone was then added to each tube. After vortexing, 50 #1 of this solution was transfered to 7 ml polypropylene scintillation vials. Five ml of Scintiverse scintillation cocktail (Fisher Scientific)was added to each vial and the radioactivity was determined by liquid scintillation spectrometry. In order to differentiate between spiperone and its metabolites, 50 #1 aliquots of the redissolved brain residue were spotted onto the absorbent layers of 19channel, 20 × 20 era, 250 # m silica TLC plates (LK6DF, Whatman) and developed in chloroform-methanol (9:1, v/v) or methanol-benzene-water (15:2:5, v/v). The spots corresponding to [3H]spiperone (R1s ---- 0.94 and 0.89, respectively) were visualized under UV light (254 rim), scraped into 7 ml scintillation vials, and eluted with 0.2 ml of 0.2 N acetic acid. Five ml Scintiverse was added to each vial and the samples were counted the next day. Counting efficiency was determined by internal standardization using [3H]spiperone. Values are not corrected for recovery from the TLC plates. Five-hundred/d whole blood was added to tubes containing 100 #1 1.0 N acetic acid. The samples were vortexed and centrifuged at 5000 × g for 10 min at 4 °C. One hundred F1 of the deproteinized supernatant was then transfered to 7 ml scintillation
363 vials. Two hundred #1 of toluene saturated with benzoyl peroxide was added to each vial to bleach the sample. After approximately 5 h, 5 ml of Scintiverse was added to each vial and the samples were counted. Fifty/zl aliquots of the deproteinized blood supernatant were also spotted and developed by the two TLC systems described above. All procedures were the same as those for the whole brain assay, except that 5 /A 0.1 N acetic acid containing 5/~g of spiperone was spotted in each channel of the TLC plates. Spiperone again induced significant catalepsy in all conditions (P < 0.01) which was once again potentiated by EB in the 3 and 6 h groups relative to oil controls (mean duration in sec + S.E. 116.7 ± 9.9 vs 22.6 ± 8.8 at 3 h; P < 0.01 and 182.5 4- 23.3 vs 102.0 4- 39.3 at 6 h; P < 0.03). These same EB-treated rats which showed prolonged catalepsy also had elevated brain spiperone levels following [3H]spiperone administration (Table I). That is, EB treatment resulted in increased whole brain tritium levels, relative to oil-injected controls, at both 3 (129 ~o) and 6 h (129 ~). Tritium levels in blood were also elevated at these times (125~ and 146~o of control values, respectively) in EB-treated animals. Similar results were obtained after chromatography of brain and blood samples, indicating that most of the radioactivity was accounted for by [3H]spiperone. In the present study we have demonstrated that estrogen significantly prolongs the catalepsy produced by spiperone in the rat. However, we have also shown that rats treated with estrogen had significantly higher whole brain and blood levels of [3H]spiperone than ovariectomized controls. This effect of estrogen on spiperone levels may be due to the fact that prior estrogen treatment decreases the activity of enzymes involved in the metabolism of butyrophenone neurolepticsn,la. To the extent that this effect applies to the metabolism of other neuroleptic classes as well, it is possible that the fluctuations in the behavioral response to neuroleptics produced by differences in sex or hormonal state, which has been reported in both the clinical 3,s, 13,~o and experimental animal literature6, a7 may be, at least in part, the result of estrogen's ability to alter the actual levels of drug reaching the central nervous system. In this regard, we are presently investigating the possibility that estrogen may affect the metabolism or distribution of the DA agonists, apomorphine and amphetamine. Central factors may also contribute to the ability of estrogen treatment to potentiate spiperone-induced catalepsy. Since catalepsy produced by butyrophenones such as spiperone is generally thought to be due to blocking postsynaptic DA receptors in the striatum5, our data are consistent with the view that estrogen can directly or indirectly attenuate striatal DA function4. Support for this hypothesis comes from the findings that estrogen reduces both apomorphine-induced stereotypy in micO a, and decreases the accumulation of striatal acetylcholine in response to DA agonisO 2. However, conflicting evidence exists 6,10. An antidopaminergic role for estrogen also is suggested by the finding that estrogen can antagonize the inhibition, by DA or DA agonists, of prolactin release from anterior pituitary cells in vitro 2. Biochemical studies also indicate that estrogen can affect DA function, although the exact nature of that effect is not always clear. For example, normally cycling female rats show decreased steady-state concentrations of
364
I
0
dddo
0
°i
4.,
~5 C,
,5
v .le
o. Q
v
.~S
.x.
V
365 D A in the s t r i a t u m d u r i n g o r j u s t after p e r i o d s associated with high estrogen levels 7, whereas low estrogen p e r i o d s are a c c o m p a n i e d b y elevated striatal D A c o n c e n t r a tions 14. M o r e o v e r , synthetic steroid c o n t r a c e p t i v e s generally elevate D A t u r n o v e r in the s t r i a t u m 1, a l t h o u g h estrogen m a y n o t affect either low o r high affinity b i n d i n g o f spiperone in the s t r i a t u m ( A n d o r n , p e r s o n a l c o m m u n i c a t i o n ) . Therefore, it becomes evident t h a t b o t h central a n d p e r i p h e r a l effects o f estrogen m u s t be c o n s i d e r e d w h e n e x a m i n i n g its role in b e h a v i o r a l o r n e u r o c h e m i c a l consequences o f d r u g - i n d u c e d alterations in D A function. W e w o u l d like to t h a n k D s . M. J. Z i g m o n d , S. M. A n t e l m a n a n d N. R o w l a n d f o r their c o m m e n t s on t h e m a n u s c r i p t , a n d J. H o f f m a n , M. B. D e e r i n g a n d S. O d a c h o w s k i for their l a b o r a t o r y assistance. This research was s u p p o r t e d b y grants MH16581 ( A . R . C . ) a n d MH20620 (Dr. M. J. Z i g m o n d ) .
1 Algeri, S., Ponzio, F., Dolfini, E. and Jori, A., Biochemical effects of treatment with oral contraceptive steroids on the dopaminergic system of the rat, Neuroendocrinology, 22 (1976) 343-351. 2 Beaulieu, M., Raymond, V. and Labrie, F., Antagonism between estrogens and dopamine at the anterior pituitary. In E. Usdin (Ed.), Catecholamines: Basic and Clinical Frontiers, Pergamon Press, New York, in press. 3 Bedard, P., Langelier, P. and Villeneuve, A., Oestrogens and extrapyramidal system, Lancet, ii (1977) 1367-1368. 4 Bedard, P., Dankova, J., Boucher, R. and Langelier, P., Effect of estrogen on apomorphineinduced circling behavior in the rat. Canad. J. PhysioL PharmacoL, 56 (1978) 538-541. 5 Chase, T. N., Antipsychotic drugs, dopaminergic mechanisms and extrapyramidal function in man. In G. Sedvall, B. Uvnas and Y. Zotterman (Eds.), Antipsychotic Drugs: Pharmacodynamics andPharmacokineties, Pergamon Press, New York, 1976, pp. 321-329. 6 Chiodo, L. A. and Caggiula, A. R., Estrogen modulation of spiroperidol-induced catalepsy and amphetamine stereotypy in the rat, Neurosci. Abstr., 4 (1978) 487. 7 Crowley, W. R., O'Donohue, T. L. and Jacobowitz, D. M., Changes in catecholamine content in discrete brain nuclei during the estrous cycle of the rat, Brain Research, 154 (1978) 345-357. 8 Donlon, P. T. and Stenson, R. L., Neuroleptic induced extrapyramidal symptoms, Dis. herr. Syst., 37 (1976) 629-635. 9 Duvoisin, R. C., Parkinsonism: animal analogues of the human disorder. In M. D. Yahr (Ed.), The Basal Ganglia, Raven Press, New York, 1976, pp. 293-303. 10 Eikenburg, D. C., Ravitz, A. J., Gudelsky, G. A. and Moore, K. E., Effect of estrogen on prolactin and tuberinfundibular dopaminergic neurons, J. neurol. Transm., 40 (1977) 235-244. 11 el DeFrawy el Marsy, S. and Mannering, G. J., Sex-dependent differences in drug metabolism in the rat. It. Qualitative changes produced by castration and the administration of steroid hormones and pentobarbital, Drug Metab. Dis., 2 (1974) 279-284. 12 Euvard, C., Labrie, F. and Boissier, J. R., Antagonism between estrogens and dopamine in the rat striatum. In E. Usdin (Ed.), Catecholamines: Basic and Clinical Frontiers, Pergamon Press, New York, in press. 13 Gratton, L., Neuroletiques, parkinsonisme et schizophrenia, Union Med. Canad., 89 (1960) 681-694. 14 Jori, A., Colturani, F., Dolfini, E. and Rutczynski, M., Modifications of striatal dopamine metabolism during the estrous cycle in mice, Neuroendocrinology, 21 (1976) 262-266. 15 Kato, R., Sex-related differences in drug metabolism, Drug Metab. Rev., 3 (1974) 1-32. 16 Leysen, J. E., Niemegeers, C. J. E., Tollenaere, J. P. and Laduron, P. M., Serotonergic components of neuroleptic receptors, Nature (Lond.), 272 (1978) 168-171. 17 Mislow, J. F. and Friedhoff, A. J., A comparison of chlorpromazine-induced extrapyramidal syndrome in male and female rats. In K. Lissak (Ed.), Hormones and Brain Function, Plenum Press, New York, 1973, pp. 315-326.
366 18 Naik, S. R., Kelkar, M. R. and Sheth, U. K., Attenuation of stereotyped behavior by sex steroids, Psychopharmacologia (Berl.), 57 (1978) 211-214. 19 Nerland, D. E. and Mannering, G. J., Species, sex, and developmental differences in the Oand N-dealkylation of ethylmorphine by hepatic microsomes, Drug Metab. Dis., 6 (1978) 150-153. 20 Stevens, J. R., An anatomy of schizophrenia? Arch. gen. Psychiat., 29 (1973) 177-189.