Effects of chronic lithium treatment on dopamine receptors in the rat corpus striatum. II. No effect on denervation or neuroleptic-induced supersensitivity

Brain Research, 232 (1982) 401-412

401

Elsevier Biomedical Press

EFFECTS OF C H R O N I C L I T H I U M T R E A T M E N T ON D O P A M I N E RECEPTORS IN T H E RAT CORPUS STRIATUM. II. NO E F F E C T ON D E N E R V A T I O N OR N E U R O L E P T I C - I N D U C E D SUPERSENSITIVITY

DAVID A. STAUNTON, PIERRE J. MAGISTRETTI, WILLIAM J. SHOEMAKER, SCOTT N. DEYO and FLOYD E. BLOOM Arthur It'. Davis Center for Behavioral Neurobiology, The Salk Institute, P.O. Box 85800, San Diego, CA 92138 (U.S.A.)

(Accepted June 1lth, 1981) Key words: lithium - - supersensitivity- - dopamine receptors - - 6-hydroxydopamine -- haloperidol -

-

[3H]spiroperidol

SUMMARY The influence of chronic dietary lithium administration was evaluated on dopamine receptor supersensitivity in the rat corpus striatum. Supersensitivity was induced with either unilateral destruction of dopamine-containing fibers in the nigrostriatal pathway or with 3 weeks of treatment with haloperidol (HAL). Both treatments elevated [aH]spiroperidol binding sites, but in neither case was this increase in ligand binding affected by chronic dietary Li (brain levels 0.8 to 1.2 mEq/1 tissue). Our rats receiving 21 daily injections of H A L did show a behavioral supersensitivity to the dopamine agonist, apomorphine, and this effect was attenuated by concurrent treatment with dietary Li (accompanying paper). However, in contrast to previous data z0, this behavioral attenuation could not be linked to the prevention of increased [3H]spiroperidol binding in the corpus striatum. Furthermore, co-administration of dietary Li to subjects injected with H A L for 3 weeks did not reverse the increased density of [aH]spiroperidol binding sites which developed in the corpus striatum. Neither H A L nor Li treatment altered the affinity of the radioligand for its binding site. In the same animals, neostriatal dopamine-sensitive adenylate cylcase was not affected by either long-term dietary Li or chronic neuroleptic treatment, supporting the view that membrane antagonist and agonist sites differentially adapt to chronic alterations of synaptic input. Taken together, the results are incompatible with the hypothesis that the anti-manic action of Li is related to its ability to prevent dopamine receptor supersensitivity.

0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press

402 INTRODUCTION Although the treatment of manic-depressive illness is often successful, little is known of the etiology of the diseaseT,9,11. Recent clinical investigations have demonstrated that, in certain cases, dopamine receptor antagonists such as pimozide or haloperidol (HAL)l°, 16,21 can acutely suppress manic symptomatology, while dopamine agonists such as piribedil 1° or L-DOPA2,19 can precipitate manic episodes in depressed patients. Such findings have given rise to the hypothesis that dopamine receptor sensitivity changes 3,4 underlie the behavioral signs in bipolar disease: mania arising from an abnormal increase and depression from an abnormal decrease. Several investigators have recently reported a variety of preclinical tests of this hypothesis. Because chronic lithium treatment is a powerful anti-manic therapy, the concept has arisen that Li might prevent the development of dopaminergic supersensitivity normally observed with long-term exposure to anti-psychotic drugs such as HALl,S,20, a3. Behavioral evidence of supersensitive dopamine receptors in animals chronically treated with HAL was abolished when the treatment included prolonged exposure to Lil,20, a3. Furthermore, with chronic dietary administration, Li prevented the usual increase in dopamine receptor density assessed with [aH]spiroperidol binding in the corpus striatum following multiple injections of the neuroleptic 20. At face value, these reports supported the hypothesis that Li could prevent mania by blocking dopaminergic supersensitivity. In the present investigation, we attempted to characterize the molecular and cellular bases of Li action more fully. We first sought to compare the effects of Li treatment on dopamine receptor supersensitivity induced by unilateral dopamine denervation of the nigrostriatal pathway 18 with that reported to be produced by chronic neuroleptics 5 in order to evaluate whether Li action could be generalized to all types of postsynaptic receptor proliferation. Because no such actions of Li were observed, the remaining experiments were used to explore systematically whether chronic Li could prevent increases in dopamine-related binding in the neostriatum following HAL treatment 20. METHODS For this series of experiments, male Sprague-Dawley rats were obtained from Charles-River Labs, housed 1-3/cage (consistent within each experiment) and placed on a 12 h light/dark schedule (lights on at 07.00 h). Animals treated with 6-hydroxydopamine had initial body weights of 150-180 g; in all other experiments the initial body weight was 160-310 g. Except for pair-fed experiments (see below), food was available ad libitum; water was always available ad libitum. In all of the experiments some animals were fed a diet of Li-containing food pellets (Teklad Mills: a nutritionally complete formulation containing 1.696 g (40 mmol) LiC1/kg) for periods of 2.5-5 weeks. This diet led to brain Li levels of 0.87 ± 0.12 mEq/1. The control diet was identical, except that it lacked the LiC1. During the first week of this treatment animals on the Li diet did not gain weight; during the remainder of the chronic dietary treatment, subjects receiving Li gained weight at a rate approximately one-half that of

403 animals fed the control diet. Therefore, in one experiment utilizing chronic dietary administration of Li, pair-fed control groups were included in which the subjects were matched to specific members of the groups receiving Li. The pair-fed animals were fed (once daily) an amount of control diet which resulted in weight gain nearly identical to their pair-mates.

6-Hydroxydopamine treatment The method of Ungerstedt al was used to produce unilateral destruction of the nigrostriatal pathway. Briefly, animals were anesthetized with chloral hydrate (50 mg/kg, i.p.) and sodium pentobarbital (25 mg/kg, i.p.) and placed in a stereotaxic apparatus such that the interaural line was 2.4 mm below the incisor bar. A 32-gauge stainless steel cannula was lowered through a burr hole in the skull to a point with the following coordinates: --4.2 mm (from bregma) AP, 1.0 mm lateral to the midsagittal suture and 7.5 mm ventral to the brain surface. A solution of 8 #g of 6-hydroxydopamine HBr in 4 #1 of 0.9 ~ saline (with 0.02 ~ ascorbic acid) was injected with a Hamilton microsyringe driven with a Harvard Apparatus infusion pump (1/A/min). After the injection, the cannula was left in place for 2-4 rain. Three days following surgery, the animals were placed on Li or control diet. One week after 6-hydroxydopamine injection the animals were observed for rotational behavior (contralateral to the side of the lesion) following a test injection of 0.79-2.4/~mol/kg of apomorphine HC1. Non-rotating animals were excluded from the experiment. Three weeks after 6hydroxydopamine treatment the animals were sacrificed for assay of [3H]spiroperidol binding and endogenous neostriatal dopamine content.

Long-term haloperidol treatment In other experiments, animals were randomly assigned into Li and control diet groups. After 5-8 days, HAL (Haldol Injection, McNeil Labs; 2.2/zmol/kg, s.c.) or HAL vehicle (1.8 mg/ml methylparaben, 0.2 mg/ml propylparaben and lactic acid for pH adjustment to 3.4 -4- 0.2; 1 ml/kg) was administered once each day to subjects fed either diet. The injections were terminated after 21 days and series of animals were decapitated for assays of [3H]spiroperidol binding and dopamine-sensitive adenylate cyclase 1, 3, 5, 7 and 14 days after treatment.

Tissue preparation Immediately after decapitation, corpora striata were dissected free of surrounding structures over ice and were then homogenized separately or as pairs in 30 vol. of ice-cold 10 mM Tris-maleate buffer, pH 7.5, using a Teflon-glass homogenizer. For determination of dopamine content (in animals treated with 6-hydroxydopamine), a 75/zl aliquot of each homogenate was combined with 600/~1 of 0.1 N perchloric acid and stored at 0 °C until assayed by high performance liquid chromatography. The remaining crude homogenate was diluted 1 : 2 with cold buffer and an aliquot used within 10 min for determination of adenylate cyclase activity. The last of the homogenate was centrifuged at 20,000 g for 10 min at 4 °C. The pellet was resuspended (final protein concentration approximately 125 #g/ml) in 0.15 M NaC1-20 mM Tris buffer, pH 7.5, for use in binding assays.

404

Dopamine assay Samples were thawed and the pH adjusted to 7.6 with 2 M Tris, pH 8.6. Catecholamines were extracted with activated A1208 (10 mg/sample) and the alumina washed twice with 0.02 M Tris buffer, pH 8.6. The solid phase was suspended in 500/A of 0.4 M perchloric acid with shaking for 15 rain. Aliquots (100/~1) were injected into a #Bondapak CI 8 column using a mobile phase consisting of 0.05 M phosphate buffer, pH 3.0, sodium octyl sulfate, sodium EDTA and methanol. Electrochemical detection was accomplished with an amperometer (Model LC 2A, Bioanalytical Systems) having the oxidation potential adjusted to +0.72 V. The usual sensitivity for dopamine is 40 pg. An internal standard of dihydroxybenzylamine was included with each sample for calibration of peak heights and determination of recovery (typically 85 ~).

Adenylate eyclase assay Basal and dopamine-stimulated adenylate cyclase activity were measured in crude homogenates as the conversion of [a-a2P]ATP to [82P]cyclicAMP. The details of this method 29 and the use ot sequential Dowex and alumina column chromatography for purification of cyclic AMP from other purine nucleotides24,36 have been described previously. In the present case, the concentrations of exogenous dopamine used to define the stimulated enzyme activity were 3, 10, 30, 100 and 300/zM. Each concentration was assayed in triplicate. The recovery from each Dowex-alumina column pair was corrected by adding [3H]cyclic AMP (50,000 cpm) to each sample prior to purification. The corrected cpm of 32p were converted to curies and then divided by the specific activity of the precursor [a-32P]ATP. The enzyme activities were normalized for protein content a7 and expressed as pmol/mg protein/min.

[zH]Spiroperidol binding The assay is a slightly modified version of a recently reported method zg. In brief, a 900 /~1 aliquot of the resuspended membranes was incubated with 100 pl of [aH]spiroperidol (26 Ci/mmol) in a solution containing 1 mM ascorbic acid and 10 #g/ml bovine serum albumin, neither of which affected the binding of the radioligand. A second set of samples also contained 2 pM (+)-butaclamol to define the nonspecific binding. The radioligand concentration was varied from 33 to 90 pM. Each point on the saturation curve was determined in duplicate. The samples were incubated in polypropylene tubes at 37 °C for 15 rain, a period more than sufficient for the binding to reach equilibium. The reaction was terminated by the addition of 10 ml of ice-cold buffered saline and the samples immediately filtered through glass fiber filters (Schleicher and Schuell, no. 30). The filters were washed with and additional 10 ml of buffered saline (4 °C), dried and suspended in 7 ml of scintillation fluid (Aquasol, NEN). Radioactivity was detelmined by a liquid scintillation spectrometric system in which the efficiency for 3H was 38 ~. The specific binding typically represented 80-90 ~ of the total binding. The protein concentration in each resuspended homogenate was determined 17, and the results expressed as fmol/mg protein.

Materials Radiolabeled compounds ([3H]spiroperidol, [a-3'~P]adenosine triphosphate and

405 [3H]cyclic adenosine-3',5'-monophosphate) were obtained from New England Nuclear. (+)-Butaclamol was generously provided by Ayerst Labs. All other drugs were commercially available.

Statistical analysis Scatchard analysis z5 was used to derive Bmax and KD values in all binding experiments. Linear regression of log-dose-response curves (r _> 0.95) was used to calculate ECs0 values for adenylate cyclase activation. Because the activation of adenylate cyclase was complete in the presence of 100 #M dopamine, the Vra ax was defined as the activity in the presence of 100 ~M dopamine minus the basal activity. All results were tested with 2-way analysis of variance with significance assumed when P < 0.05. RESULTS

Chronic lithium exposure following unilateral 6-hydroxydopamine Subcutaneous injection of low doses of apomorphine HC1 elicited the usual rotatory response directed away from the side of 6-hydroxydopamine treatment 31. There was no difference in the mean rotational rate in animals fed the control diet (ad libitum) vs those that ate the Li diet (control: 7.8 ± 1.2 rotations per min; Li; 8.5 41.1 rotations per min). This test was used only to exclude non-rotating subjects from further analysis; possible differences in the total response to apomorphine were not examined. The loss of neostriatal dopamine content on the side of destruction of the 2000 ~

~xxx

o. I000

0

"":'"

500

600 B (fmoles/rag PROT.)

900

IOOI

Fig. 1. Effect of chronic Li diet on binding of [ZH]spiroperidol to neostriatal membranes following unilateral 6-hydroxydopamine treatment. Scatchard 25 analysis of equilibrium binding of [SH]spiroperidol to resuspended membranes prepared from neostriata on the neurotoxin-treated (dotted lines) or intact (solid lines) side. Animals ingested a Li-containing (solid symbol; n = 11) or control (open symbol; n = 8) diet for 18 days.

406 nigrostriatal pathway was 83 ~ in subjects fed the normal diet and 84~o in those given Li (P > 0.05). Thus, by objective measures, the extent of dopamine loss caused by the neurotoxin was identical in the two dietary groups. As reported beforelS, 29 depletion of neostriatal dopamine led to a marked elevation of [aH]spiroperidol binding sites in resuspended membranes prepared from corpora striata on the treated side (F(1,34) = 40.3, P < 0.001 ; Fig. 1). Unexpectedly, the KD values for [3H]spiroperidol binding were significantly lower on the denervated side (intact side: 75.6 4- 2.9, denervated side: 60.8 4- 2.0; F(1,34) -- 16.8, P < 0.001), indicating that the radioligand had higher affinity for the receptor on this side. However, 18 days of dietary treatment with Li did not affect either the Bmax or the KD on either side (P > 0.05). Thus, 18 days of Li exposure resulting in brain Li levels of 0.87 4- 0.12 mEq/l brain tissue completely failed to influence [aH]spiroperidol binding in animals with unilateral destruction of the nigrostriatal pathway.

Chronic L i exposure with haloperidol Animals fed the Li or control diets were injected with H A L or H A L vehicle for 3 weeks and then sacrificed 3 days after withdrawal from the neuroleptic. Scatchard analysis 25 of [3H]spiroperidol binding to neostriatal membranes revealed increased binding sites in HAL-treated rats (Fig. 2; F(1,20) -----66.3, P < 0.001). As in the case of 6-hydroxydopamine, 24 days of exposure to the Li diet (brain Li levels: 0.99 4- 0.16 mEq/l for vehicle-injected group vs 1.0 4- 0.21 for HAL-injected group) had no effect on the density of binding sites in subjects exposed to H A L or the vehicle (P > 0.05). There was no interaction of the dietary and injected drug treatments (P > 0.05).

15ooI

F"

~,

°

50o

""XX.X. , o

300

~,~-~

-...

600 B (frnoles / mg protein)

900

Fig. 2. Effect of chronic Li diet on binding of [aH]spiroperidol to neostriatal tissue following multiple daily injections of HAL. Scatchard analysis25 of saturation binding curves obtained from animals pretreated with HAL vehicle (©--O), HAL (O---©), HAL vehicle and dietary Li ( 0 - - 0 ) and HAL dietary Li (Q---O). The 6 animals in each group were withdrawn from HAL or HAL vehicle injection for 3 days prior to sacrifice. Calculated values for Bmax appear in Table I.

407 Lastly, analysis of the KD values indicated that there were no direct effects of, or interactions between, the drug treatments (vehicle-injected control diet: 69.5 4- 3.2 pM; vehicle-injected Li diet: 66.1 zk 2.0 p M ; HAL-injected control diet: 72.0 zk 3.7 p M ; HAL-injected Li diet: 67.7 ± 3.2 pM). Therefore, the DA-related binding parameters were totally unaffected by the Li treatment. Other experiments were conducted to examine whether altering the duration of withdrawal from H A L might uncover an effect of Li exposure on the binding of [3H]spiroperidol. The H A L and Li treatments were the same as in the experiment described above. In every case, the results were precisely the same (Table I). Li treatment had no influence on the binding of [3H]spiroperidol to neostriatal tissue at short (1 day), intermediate (3, 5 or 7 days) or long (2 weeks) times after the final H A L injection (see Table I). In all these experiments, there was no direct effect of Li treatment nor any interaction between Li and H A L pretreatments. Although the magnitude of the increase in [SH]spiroperidol binding sites declined as the duration of withdrawal was lengthened (Table I), the analysis of each experiment clearly demonstrated that the recovery to normal receptor density was not altered by Li administration. Although animals fed the Li diet for several weeks always steadily gained weight, at the time of sacrifice they generally weighed approximately 100 g less than the subjects fed the control diet ad libitum. However, when the consumption of the control diet was restricted in pair-fed control rats to keep body weight the same as animals on the Li diet, the binding results were the same. For this series of tests, the experiment was terminated 8 days after the last H A L injection. Long-term treatment with the neuroleptic drug induced a 27 ~o increase in [aH]spiroperidol binding sites in animals pair-fed the control diet and a 29 ~o increase in the density of sites in those given Li (Table I; P > 0.05). TABLE I Effect of chronic dietary lithium on the elevation of [3H]spiroperidol binding following long-term haloperidol administration

Values are means ~ S.E.M.; numbers of animals shown in brackets, AC = ad libitum control diet; PC = pair-fed control diet; L ~ lithium diet; V = haloperidol vehicle; H = haloperidol. Days withdrawn from haloperidol

1 3

Bmax(fmol/mg protein)

Bmax (fmol/ng protein)

ACV LV ACV LV

ACH LH ACH LH ACH LH PCH LH ACH LH

5 8 14

PCV LV ACV LV

-425 723 650 --

* zk 11.8 (8) :k 43.0 (6) zk 17.6 (6) *

--

*

492 478 657 631

zk zk ± zk

22.4 12.5 27.8 35.0

(7) (8) (6) (6)

665 ± 17.6 637 ± 15.0 956 -4- 19.0 916 ± 35.0 1022 zk 15.6 1051 zk 51.7 626 + 22.5 617 ± 28.0 764 ± 43.6 739 ± 23.2

% Increase

(8) (10) (6) (6) (6)

56 50 32 41 --

(6)

--

(7) (7) (6) (6)

27 29 16 17

* Vehicle-injected groups not done. Other data indicates no effect of dietary lithium on the Bmax in vehicle-injected controls.

408 TABLE II

Basal adenylate cyclase activity and ECho values following lithium and haloperidol pretreatments Values are means ± S.E.M. ; number of animals shown in brackets.

Pretreatment

Basal activity

Drug

Diet

(pmol/mg protein~rain)

VEH VEH VEH HAL HAL HAL

Control-Ad lib Control-Pair fed Lithium Control-Ad lib Control-Pair fed Lithium

59.7 52.4 53.3 59.5 54.7 51.7

~ ± ~ ~ ± ±

1.9 2.2 2.0 5.0 5.2 2.1

(6) (8) (8) (6) (8) (8)

ECho (tiM dopamine)

10.2 9.8 12.4 11.9 11.1 10.5

± 1.2 =1- 1.2 ± 1.7 ± 2.1 ± 2.3 ± 1.5

Neostriatal adenylate cyclase activity Basal and dopamine-stimulated adenylate cyclase were assayed in crude homogenates of the corpus striatum prepared from animals chronically treated as described and withdrawn from HAL for 8 days. There was no effect of any pretreatment on basal adenylate cyclase activity (P ~ 0.05; Table I1). Similarly, the EC~0 values (Table 1I) and the maximal dopamine-stimulated activity (Fig. 3) were unaffected by either Li or HAL pretreatments. These parameters were also not altered in the case of comparisons with pair-fed controls (Table 1I, Fig. 3; P > 0.05 for all measures).

4O

VEHICLE

HA',OPERIDOL

E E ®

3C

!

g

~

ili!i!i!;i~i~ililil

181:(81

~ ee

i!iii~i~i!i!iiil

g > ' Ic

!!iiiiiii!! i

ilili!iiiiiiiiii!i! ~

: .........

~i:U:i ......................... iiii!!li

:ili AD LIB

PAIR FED

CONTROLS

Li ÷

AD LIB

PAIR FED

Li +

i CONTROLS

BI05Z5

Fig. 3. Maximal dopamine-stimulated adenylate cyclase activity in neostriatal homogenates from animals with Li and H A L pretreatments. Values are mean pmol [32P]cyclic AMP/mg protein/rain ± S.E.M. in the presence of 100/~M (Vm) dopamine minus the basal activity (no exogenous dopamine). The animals in each group (number in parentheses) were withdrawn from H A L (or vehicle) 8 days orior to sacrifice.

409 DISCUSSION The series of investigations reported here represents a systematic attempt to probe the influence of prolonged Li exposure on dopamine receptor supersensitivity. Li treatment was accomplished with a diet which reliably produced serum and brain Li levels in the therapeutic range (0.8-1.2 mEq/l) without overt signs of toxicity. "[he advantageous properties of our Li diet appeared in an earlier report (see accompanying paper), in which we assessed the ability of chronic lithium to alter spontaneous and apomorphine-induced behaviors. In that investigation, we confirmed a previous report 20 that long-term dietary Li administration reduces, at least partially, dopaminergic behavioral supersensitivity elicited by chronic exposure to the antipsychotic, HAL. It has been suggested that the suppression of this measure of behavioral hyperresponsivity by Li might be based upon an ability of the anti-manic drug to: (1) depress the proliferation of neuroleptic binding sites in the corpus striatum which normally accompanies long-term haloperidol treatmentS, 2°, or (2) abolish the heightened sensitivity of the dopamine-containing neurons to inhibition by dopamine agonists s. Our examination began as an attempt to extend the first hypothesis by determining whether chronic Li treatment could abolish dopamine receptor proliferation which occurs following destruction of dopamine terminals in the corpus striatum 6. For this test, the ascending dopamine-containing axons in the nigrostriatal pathway were unilaterally destroyed with 6-hydroxydopamine, a procedure known to lead to exaggerated behavioral responsivity to dopamine agonists31,az as well as increased neuroleptic binding sites in the denervated corpus striatum6,18. Chronic dietary lithium or control diet was instituted three days after 6-hydroxydopamine treatment, when the elevation of dopamine antagonist binding sites was not yet measurable 1a,29. The results of equilibrium binding analysis using [3H]spiroperidcl conclusively showed that lithium treatment was not able to diminish the marked elevation of the density of binding sites. This finding seemed to suggest that in order for chronic Li to depress DA receptor proliferation the presence of dopamine terminals was required. Therefore, in the remaining experiments, we attempted to verify that chronic dietary Li could prevent the elevation of [aH]spiroperidol binding sites in neostriata prepared from animals injected daily with HAL. A major index of the success of chronic neuroleptic treatment - - the increased behavioral responsiveness to the dopamine agonist, apomorphine12, a0 - - was confirmed in the subjects used for receptor analysis (accompanying paper). Furthermore, a pronounced enhancement of the density of [aH]spiroperidol membrane binding sites was certainly evident in each group of animals exposed to HAL for 3 weeks. Yet, regardless of the duration of withdrawal from HAL, co-treatment with dietary Li was totally unable to attenuate the elevation of binding sites. Our results do not agree with previous research 2°. Moreover, we were also unable to confirm another report from the same group in which chronic dietary Li was found to diminish the density of neostriatal [aH]spiroperidol sites in animals not

410 otherwise treated with drugs 2z. The reasons for these important discrepancies are not readily apparent. In fact, careful comparison of experimental protocols reveals only minor differences in animal treatment. For example, Pert et al. 2° discontinued Li treatment when the HAL injections were stopped, but in our experiments, Li ingestion was maintained until sacrifice. Our protocol was designed to avoid the possibility that an effect of Li on dopamine receptor supersensitivity could disappear following withdrawal from HAL. The use of different buffers for membrane resuspension also does not explain the different results of binding analysis, because we have found that the K o and Bmax values for [3H]spiroperidol binding obtained with the two buffers are not statistically different. It may be that the disparate findings are due to the different data analyses employed in these parallel studies; for example, we routinely used Scatchard 25 analysis of saturation binding curves based on 6 ligand concentrations, but Pert et al. 2° utilized single concentration analysis. We conclude that our data do not support the contention that chronic dietary Li administration can either prevent or reverse the proliferation of neuroleptic binding sites in the corpus striatum following prolonged treatment with HAL. Although we could not confirm an effect of Li on [3H]spiroperidol binding sites in HAL-treated animals, another possible basis for diminished dopaminergic behavioral supersensitivity would be an influence of Li on striatal dopamine-sensitive adenylate cyclase. However, reports of chronic receptor blockade with antipsychotic drugs on the sensitivity of this enzyme system to dopamine do not agree. Some investigators found that long-term treatment with HAL la-15 resulted in greater stimulation of neostriatal adenylate cyclase by dopamine, but others could find no such effecta4. We have demonstrated that dopamine-activated adenylate cyclase is unaltered in rats 8 days after withdrawal from 3 weeks administration of HAL. In the same animals, HAL treatment had resulted in a 30 ~ elevation of the density of [aH]spiroperidol binding sites in neostriatal membranes. Thus, the adaptive responses to HAL treatment at agonist and antagonist sites must be distinct (cf. ref. 5). Because dopamine-sensitive adenylate cyclase was not affected by HAL treatment, no effect of chronic Li on this measure could be evaluated. In summary, our results cannot support the hypothesis that Li impairs behavioral supersensitivity of the dopaminergic systeml,Z0, aa, by suppressing proliferation of dopamine receptors. The possibility that Li could prevent supersensitivity by acting at a dopamine receptor-mediated event subsequent to the activation of adenylate cyclase (e.g. protein phosphorylation aS) is tenable, but has not been tested. On the other hand, the ability of Li to reduce dopaminergic behavioral hyper-responsiveness may instead be a product of its action on presynaptic dopamine receptors located in the substantia nigra. Chronic HAL administration has been shown to reduce the ECs0 for apomorphine-induced inhibition of discharge rate in dopamine-containing neurons in the substantia nigra, an effect which is completely prevented by chronic Li 8. Because apomorphine inhibits these neurons by acting at dopamine autoreceptors located on dopaminergic somata and dendrites, Li could, in fact, prevent the enhanced sensitivity to apomorphine by precluding HAL-induced supersensitivity of these dopamine autoreceptors. The possible mechanisms of action of Li on dopamine autoreceptors are not

411 easily t e s t e d because, in c o n t r a s t to n e o s t r i a t a l d o p a m i n e receptors these receptors have no identified i n t r a c e l l u l a r messengers2Z, 27. Finally, the negative influence o f Li on d o p a m i n e - m e d i a t e d b e h a v i o r s may, in fact, be related to a n a c t i o n o f the a n t i - m a n i c d r u g o n systems o u t s i d e the n i g r o s t r i a t a l axis26, 2s. A differential analysis o f the acute a n d c h r o n i c m e c h a n i s m s o f a c t i o n o f Li in defined n e u r o t r a n s m i t t e r systems may, indeed, b e n e e d e d b e f o r e the basis o f the t h e r a p e u t i c a c t i o n o f this a n t i - m a n i c d r u g is understood. ACKNOWLEDGEMENTS W e are i n d e b t e d t o M r s . Viveca Sapin, M r s . L y n n e R a n d o l p h a n d M r . G r e g Baetge f o r c a p a b l e technical assistance a n d to Mrs. N a n c y C a l l a h a n for m a n u s c r i p t preparation. This investigation was s u p p o r t e d b y the N a t i o n a l Institute o f H e a l t h N a t i o n a l R e s e a r c h Service A w a r d M H 08080 to D . A . S . a n d also b y M H 29466 f r o m the N I H . P . J . M . was s u p p o r t e d b y a Swiss N a t i o n a l Science F o u n d a t i o n Fellowship. S . N . D . was s u p p o r t e d b y N I A A A 07273.

REFERENCES 1 Allikmets, L. H., Stanley, M. and Gershon, S., The effect of lithium on chronic haloperidol enhanced apomorphine aggression in rats, Life Sci., 25 (1979) 165-170. 2 Bunney, W. E., Brodie, K. H., Murphy, D. L. and Goodwin, F. K., Studies of alpha-methyl-paratyrosine, L-dopa, and L-tryptophan in depression and mania, Amer. J. Psychiat., 127 (1971) 872-881. 3 Bunney, W. E., Jr. and Murphy, D. L., Neurobiological considerations on the mode of action of lithium carbonate in the treatment of affective disorders, Pharmakopsychiat. Neuropsyehopharmakol., 9 (1976) 132-147. 4 Bunney, W. E., Jr., Post, R. M., Andersen, A. E. and Kopanda, R. T., A neuronal receptor sensitivity mechanism in affective illness (a review of evidence), Commun. Psychopharmacol. 1 (1977) 393-405. 5 Burt, D. R., Creese, I. and Snyder, S. H., Antischizophrenic drugs: chronic treatment elevates dopamine receptor binding in brain, Science, 196 (1977) 326-328. 6 Creese, 1., Burt, D. R. and Snyder, S. H., Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity, Science, 197 (1977) 596-598. 7 Ehrlich, B. E. and Diamond, J. M., Lithium, membranes, and manic-depressive illness, J. Membrane Biol., 52 (1980) 187-200. 8 Gallagher, D. W., Pert, A. and Bunney, W. E., Jr., Haloperidol-induced presynaptic dopamine supersensitivity is blocked by chronic lithium, Nature (Lond.), 273 (1978) 309-312. 9 Garver, D. L. and Davis, J. M., Minireview: biogenic amine hypothesis of affective disorders, Life Sci., 24 (1979) 383-394. 10 Gerner, R. H., Post, R. M. and Bunney, W. E., Jr., A dopaminergic mechanism in mania, Amer. J. Psychiat., 133 (1976) 1177-1180. 11 Gershon, S., Lithium salts in the management of the manic-depressive syndrome, Ann. Rev. Med., 23 (1972) 439-452. 12 Gianutsos, G., Drawbaugh, R. B., Hynes, M. D. and Lal, H., Behavioral evidence for dopaminergic supersensitivity after chronic haloperidol, Life Sci., 14 (1974) 887-898. 13 Gnegy, M., Uzunov, P. and Costa, E., Participation of an endogenous Ca ++ binding protein activator in the development of drug-induced supersensitivity of striatal dopamine receptors, J. Pharmacol. exp. Ther., 202 (1977) 558-564. 14 Gnegy, M. E., Lucchelli, A. and Costa, E., Correlation between drug-induced supersensitivity of dopamine dependent striatal mechanisms and the increase in striatal content of the Ca ++

412

15 16 17 18

19

20 21

22

23 24 25 26

27 28 29

30 31 32 33 34 35 36

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