Brain Resrurch Bufktin, Vol. 26. pp. 519,552. Pergamon Press plc. 1991.Printed

0361-9?30/91$3.00 * .OO

in the U.S.A.

Permanent Haloperidol-Induced Dopamine Receptor Up-Regulation in the Ovariectomized Rat JEREMY 2. FIELDS’

and Department

AND JOHN H. GORDON

Research Service (151), Hines V.A. Hospital, Hines, IL 60141 of Pharmacology, Loyola University, Stritch School of Medicine, Maywood, Received

FIELDS, J. Z. AND J. H. GORDON.

IL 60153

14 March 1989

Permanent ~l~p~~idoi-indu~~d dopamine receptor up~regul~ti~~in the ovariectomized rut.

BRAIN RES BULL 24(4) 549-552, 1991.-One of the confounding problems associated with the study of tardive dyskinesia in rodent models has been the inability to produce a permanent su~rsensitivity to dopamine (DA) following neuroleptic drugs. We recently observed that ovariectomy (OVX) results in a permanent dopamine receptor (DA-R) up-regulation in the striatum of the rat. This permanent up-regulation of striatal D, DA-R required three months to fully develop and lasted for at least 12 months postOVX. In the present study we further characterized this model by examining the development of both apomorphine-induced stereotypy and D, DA-R density in the striatum of OVX and Sham-operated rats following haloperidol (16 days at 1.O mgikgiday, IP). Following the chronic haloperidol treatment, both the OVX and the Sham-operated animals increased both their behavioral responses (stereotypic sniffing) to apomotphine, and the density of striatal D, DA-R. However, by 30 days posthaloperidol, the Sham animals had reverted to normal behavioral responses to apomorphine and normal levels of striatal DA-R, while both the behavioral responses and the density of D, DA-R in the OVX animals treated with haloperidol remained up-regulated. These data indicate that 1) the time required to develop this unique animal model of a permanent DA-R up-regulation in the ovariectomized (OVX) rat can be considerably shortened; 2) there is probably a unique neurochemical change induced by haloperidol in the OVX but not in Sham rats that leads to the DA-R up-regulation becoming permanent. D, dopamine

receptors

Neuroleptics

Stereotypy

H~o~ridol

ONE of the major drawbacks to the use of neuroleptic drugs in the treatment of schizophrenia is the possibility of developing tardive dyskinesia (TD). TD is a neurological syndrome which generally consists of dyskinetic movements of the buccal, lingual and facial musculature although symptoms may also include generalized choreoathetosis of the limbs and trunk (1, 5, 23). In severe cases, the symptoms of TD have been reported to mimic those of Huntington’s chorea, a condition thought to be due, in part, to an overactivity of dopaminergic neurotransmission in the basal ganglia (1.2). The dopamine (DA) “overactivity” hypothesis as part of the underlying pathophysiology of TD is supported by the clinical ph~acology of TD (2, 5, 6, 13, 23) as DA antagonists decrease TD symptoms while DA agonists exacerbate the syndrome. According to the “textbook” hypothesis, TD results from the chronic blockade of D2 DA-R in the basal ganglia by neuroleptic drugs. This widely cited hypothesis further states that the chronic blockade of the DA-R in the basal ganglia results in a disuse or “denervation-like” response of the postsynaptic cells that includes an increase (i.e., up-regulation) in the density of the postsynaptic DA-R (1, 3, 5. 11, 13, 18). Supposedly, the neuroleptic-induced ‘Requests for reprints should be addressed

to Jeremy 2. Fields, Ph.D.,

Tardive dyskinesia

increase in DA-R eventually overshoots the compensatory needs of the system and results in an “overactivity” of the nigrostriatal dopaminergic tract. A considerable quantity of data support the above hypothesis. For example, an increased sensitivity to the biochemical, behavioral and electrophysiologic effects of drugs that directly stimulate postsynaptic DA-R has been observed in animals after only two to three weeks of neuroleptic treatment (3, 11, 14, 16, 23, 24, 27-29). Moreover, this increase in sensitivity to DA agonists is paralleled by an increased number of D? DA-R in the striatum (3,ll). In spite of the apparently broad-based support (i.e., behavioral. biochemical and clinical data) for the hy~rdopaminergic hypothesis of TD, the use of animal models. specifically the rat. to study TD has been criticized for several reasons (9). One major discrepancy between the rodent models and the clinical syndrome is that TD can sometimes, especially in older patients, persist for very long times and may be permanent in some cases, while the reported increase in DA-R density and behavioral sensitivity to DA agonists in rats is transient. In a previous communication we reported on the permanent Research

549

Service (151). Hines V.A. Hospital,

Hines. IL 60141

550

up-regulation of striatal D, DA-R in ov~i~tomized (OVX) rats (18). We originally noted that 3 months were required for this DA-R up-regulation to fully develop in the OVX rat (18,19). In the present study we took advantage of the apparent inability of the OVX rat to decrease or down-regulate its striatal DA-R once these receptors have been up-regulated. By injecting OVX rats with a neuroleptic, haloperidol, we were able to 1) dramatically accelerate the development of the permanent DA-R up-regulation in the OVX rat; 2) create a novel animal model of TD that is. like several other commonly used models, induced by haloperidol. but one in which the DA-R up-regulation is permanent. In the present report we document the time course for the development of behavioral indices ~a~mo~hine-induced stereotypy) and biochemical indices (D, DA-R density) of a DA-R up-~guIatio~ in this new and unique animal model of TD. METHOD

Development of Experimental Groups

Female Sprague-Dawley rats (King Labs, Madison, WI) were housed in group cages @/cage) with free access to food and water in a 12:12 ligh~d~k cycle (lights on at 6 a.m.). Ninety-dayold animals were OVXed under ether anesthesia. All animals were allowed 7 days to recover from the surgery prior to the onset of chronic drug or vehicle administration. Sham-operated and OVX animals were injected with either haloperidol (1.0 rn~~~y) or vehicle daily for 16 days between 8:00 and 10:00 a.m. Thus day 17 of the reagent paradigm is the first day of drug withdrawal. This treatment paradigm resulted in 4 experimental groups: 1) Sham + vehicle, 2) OVX + vehicle, 3) Sham + haloperidol (Tram animals because the neuroleptic induced dopamine receptor hypersensitivity is transient), and 4) OVX f haloperidol (Perm animals because the neuroleptic-induced DA-R hypersensitivity is permanent),

On days 1, 5, 10, and 30 posttreatment the incidence of apomorphine-induced stereotyped sniffing was determined on individual rats from each treatment group (Tram, Penn and respective vehicle controls) following an IP injection of 0.25 mg&g apomorphine (12,171. This comp~atively low dose of apomo~~ne was chosen as we did not want to obtain a maximal response in all animals, nor did we want to alter receptor sensitivity and density, as is possible using higher doses of apomorphine. All animals were observed for 10 separate IO-second intervals between lo-20 min postinjection, and the presence of stereotyped sniffmg was recorded. Stereotyped sniffing was defined as intense 8-9 Hz sniffing (11) directed towards either the sides or floor of the observation cage. T’he incidence of stereotyped sniffing was compared statistically between groups using the chi-square statistic. Receptor Binding Assays

Similar groups of animals were sacrificed by decapi~tion and the brains were rapidly removed and placed on solid Call on days 1, 5, and 30 posttreatment, Frozen brains were then wrapped in aluminum foil and stored frozen ( - 80°C) until dissection (20,21) and assay. The binding of [3H]spiroperidol ([3H]spiro) to striatal membranes from individual animals was performed as described previously (17). Tissues were homogenized in 100 volumes of 100 n&l Na’/K+ phosphate buffer, pH 7.4, and centrifuged at 40,000 X g for 1.5minutes. The resulting pellet was washed twice by resuspending in buffer and centrifugation. Assay conditions were 2 mg (original tissue weight), incubated for 45 minutes at

I 0

I

I

I

I

I

1

5

10

15

20

25

30

Time Post-Haloperidol

(days)

FIG. 1. Time course for the haloperidol-induced increase in behavioral sensitivity to apomorphine. Sham or ovariectomized (OVX) animals were injected with haIoperidoi (1 rn~~~day) or an equivalent volume of vehicIe (control animals), daily for 16 days. Animals were tested on days 1. 5, 10and 30 of withdrawal Tom the chronic haloperidol or vehicle treatment. Tram animals =Sham treated with haloperidoi. Perm animals= OVX treated with haloperidol. A total of 100 observation periods were recorded for each experimental group (IO animals and 10 observations/ animal). Each value represents the number of observation periods (incidence) in which stereotyped sniffing was observed in the experimental groups, 10-20 following a~rno~~e (0.25 mg/kg, IP). The enclosed “control range” area representsthe range of values for the eight separate behavioral tests conducted on the control animals (e.g., vehicle injection during the chronic treatment phase). Statistics: chi-square. All values outside the control range are significantly increased QKO.05) in incidence of stereotyped sniffing relative to the control (vehicle-injected) animals run on the same day.

37°C in a final volume of 2.0 ml of phosphate buffer. Nonspecific binding was defined with ( + )butacl~ol (1 x 10 --6 M). A total of 12 concentrations of E3H]spiro (range 5-500 ph?) were used to bracket the predicted I& value (20 to 60 PM). Binding parameters (B,, and KJ were estimated from the f3H]spiro binding isotherms using a nonlinear least-squares regression analysis program (25) based on the inde~ndent site models and assumptions of Feldman (8). RESULTS

The results of the behavioral tests are shown in Fig, 1, Clearly, by day 5 postneuroleptic, both haloperidol-treated groups were hypersensitive to the behavioral effects of apomorpbine. However, by 30 days ~s~e~leptic the Tram animals had reverted to a normal level of response to the test dose of a~rno~~ne, while the Perm animals remained elevated. Additions behavioral tests on similuly treated OVX animals (data not shown) have shown that once developed, the haloperidol-induced behavioral h~~ensitivity to a~mo~hine remains unchanged at 45 and 60 days ~s~~o~~dol. Sixty days ~s~~o~~dol corresponds to 3 months post-OVX, and OVX animals are hypersensitive to apomorphine from 3 to 12 months post-OVX (18,19). Changes in the D2 DA-R density in the striatum of the neuroleptic-treated rats paralleled the behavioral changes (Table 1). Both the Perm and Trans animals displayed an increased receptor density in parallel with the increased behavioral sensitivity to apomorphine on day 5 postbaloperidoi. Similarly, Tram animals displayed a normal D2 DA-R density on day 30 when the behavioral sensitivity to the test dose of a~mo~hine had returned to the control range. Although the density of the D2 DA-R was slightly increased

PERMANENT

DOPAMINE

RECEPTOR

551

UP-REGULATION

TABLE 1 [~7HISPIROPERIDOLBINDING TO STRIATAL MEMBRANES FROM TRANS (SHAM t HALDOL) AND PERM (OVX + HALDOL) ANIMALS Vehicle Injected

Days Posthaloperidol I

5

30

B,,,, Sham

13.2 i. 0.8

16.5 -t 0.8

17.2 c

1.4”

14.4 i

0.5

ovx

13.1 L1. 1.1

14.7 i

21.8

1.0”

18.8 2

1.2*

1.8

I

KJ Sham

24 i. 3

24 t

3

30 k 6

23 i

ovx

?-’ _+ 3

34 2 h

35 s 2

20 2 h

5

--.-^ *1)‘0.05 in receptor density relative to control (vehicle-injected) animals. Each value represents the mean and S.E. of 6 individual animals, B,%I*values are fmolimg tissue and K, values are in pM. Sham or ovariectomized (OVX) animals were injected with haloperidol (1 m&g/day) or an equivalent volume of vehicle, daily for 16 days. The six animals in the vehicle-injected groups were comprised of two Sham and two OVX animals sacrificed on each withdrawal day (e.g., days 1, 5 and 30 posttreatment). Tram animals = sham treated with hai~~ridol. Perm animals = OVX treated with haloperid~l. Statistics: analysis of variance-Duncan’s multiple range test.

on day I of withdrawal. the maximum increase in receptor density did not occur until day 5 postneuroleptic, suggesting some additional compensatory adjustments in DL DA-R density during the withdrawal period. DISCUSSION

The behavioral data clearly show the development of an increased sensitivity to apomorphine in both Sham and OVX rats following 16 days of neuroleptic administration. The data in Fig. 1 were for animals from which both behavioral and biochemical data were available in the same animal. In other groups of animals (unpublished) we observed a behavioral dopaminergic supersensitivity in Perm animals as long as 3 months after withdrawal of haloperidol. Moreover, this data on other Perm animals and ~havioral tests on OVX animals ](18,19); no halope~dol] indicates that once OVX rats develop a behavioral hypersensitivity to apomorphine, they never return to Sham levels. Our published observations include a 12 month post-OVX time point (18). This has recently been extended to include a 16 month post-OVX time point (incidence of sniffing/gnawing: Sham=44 out of 100 observations and OVX=95 out of 100 observations: p=0.003) between D, DA-R density (Table I) and apomo~hine-induced stereotypy (Fig. I ). Thus. as the receptor density increases during the withdrawal period. the sensitivity to apomorphine also increases in both the Perm and Trans animals. Similarly. as the behavioral sensitivity to apomorphine decreases in the Trans animals, the receptor density also decreases. Because the Perm and Trans animals are not different, on day 5 of withdrawal, in the magnitude of the increased D2 DA-R density in the striatum. it appears that this l~eurochetnical parameter is not unique to the Pet-m animals and therefore can not be directly involved in the sequence of events which results in the DA-R upregulation becoming permanent. Because of their permanent DA-R hypersensitivity, both the long-term OVX animals (18.19) and the OVX animals treated with neuroleptic (Perm animals in the present study) circumvent one of the major criticisms for using the rodent as an animal model of TD; namely, that TD in humans is often thought of as a persistent, if not permanent. syndrome. while the reported increase in DA-R density and in behavioral responses to DA agonists in rats is almost always transient in nature (27). The longterm GVX and Perm rats thus represent an improvement over existing models of TD in that it is now possible to model, characterize and investigate one impo~ant aspect of TD, namely. the permanence of the DA-R up-regulation. It is obviously not a “perfect” model of TD. no model is. and has its limitations. Specifically. DA supersensitivity develops in all or almost all

552

FIELDS AND CORDOh

rats given OVX, or chronic haloperidol + OVX. In humans, however, only IO-15% of all patients develop TD, this number being higher, up to 40%, in hospitalized patients where compliance in taking medication can be monitored. One possible explanation is that all humans actually do get DA-R up-regulation from chronic neuroleptics. However, DA-R up-regulation may be a necessary but not a sufficient condition for the expression of the behavior abnormalities of TD (dyskinesias). Those subjects that do not express the TD syndrome may be the same ones that can adequately decrease synthesis and/or release of the neurotransmitter, dopamine, and thus compensate for the DA-R up-regulation. The same reasoning might explain why the TD syndrome is “tardive,” that is, late-occurring, whereas in the rat models the DA-R up-regulation develops rapidly. It would be useful to find, and determine the time course for development of, spontaneous abnormal behaviors that are induced in rats by chronic neuroleptics. To this end, several groups have attempted to develop behavioral assays for this purpose although none is yet in wide use.

Although there is a slow or “tardive” development of a DA-R up-regulation in long-term OVX animals (without haloperidol). this model is not pertinent since it is the DA-R up-regulation, not the development of abnormal, spontaneous movements, that is slow to develop. Thus, although the Perm model, like most other animal models, has limitations, it does represent an improvement over existing models, by virtue of the long-lasting nature of the DA-R upregulation. Future comparisons of Perm and Trans animals. especially the demonstration of unique neurochemical changes in the Perm animals, should produce vital information regarding possible neurochemical or neurophysiologic mechanisms which culminate in the development of this permanent DA-R hypersensitivity. ACKNOWLEDGEMENTS Supported in part by VA Medical Research, VA Hospital, Hines, IL; The NIH (NS 26449); Tourette’s Syndrome Association and Scottish Rite Schizophrenia Research Program, N.M.J., USA.

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