Chronic ascorbate potentiates the effects of chronic haloperidol on behavioral supersensitivity but not D2 dopamine receptor binding

Neuroscience

Vol. 45, No. 2, pp. 373-378,

0306-4522/91$3.00+ 0.00 Pergamon Press plc 0 1991IBRO

1991

Printed in Great Britain

CHRONIC ASCORBATE POTENTIATES THE EFFECTS OF CHRONIC HALOPERIDOL ON BEHAVIORAL SUPERSENSITIVITY BUT NOT D, DOPAMINE RECEPTOR BINDING R. C. PIERCE,*J. K. ROWLETT,~ M. T. BARDOTand G. V.

REBEC*$

*Program in Neural Science, Department of Psychology, Indiana University, Bloomington, IN 47405, U.S.A. tDepartment of Psychology, University of Kentucky, Lexington, KY 40506, U.S.A. Abstract-Ample behavioral evidence suggests that ascorbate parallels the action of haloperidol, a widely used neuroleptic. To determine the extent to which this parallel extends to chronic treatment, 21 days of exposure to ascorbate (100 or 500 mg/kg) alone or combined with haloperidol (0.5 mg/kg) were assessed on stereotyped behavior and neostriatal D, dopamine receptor binding in rats. Our results indicate that when challenged with the dopamine agonist, apomorphine (0.5 mg/kg), animals chronically treated with haloperidol or high-dose ascorbate alone display a supersensitive sniffing response relative to controls, while animals chronically treated with the combination of haloperidol and high-dose ascorbate display a further potentiation of sniffing relative to the haloperidol groups. In addition, [)H]spiperone saturation studies showed, as expected, an up-regulation of striatal D, dopamine receptors in rats treated with haloperidol as reflected by a change in receptor density (B_) but not affinity (I&). Ascorbate treatment, however, had no effect on D, receptor density or the distribution of [‘H]apomorphine in whole brain. Even though chronic treatment with the haloperidol-high-dosascorbate combination produced an up-regulation of striatal D, dopamine receptors, this treatment did not cause a further up-regulation relative to haloperidol alone nor did it have any effect on [3H]apomorphine distribution. Taken together, these findings indicate that although chronic ascorbate produces behavioral supersen-

sitivity to apomorphine through central mechanisms, they appear to differ from those induced by chronic haloperidol. Chronic treatment with dopamine receptor antagonists, such as haloperidol and related neuroleptics, is known to cause a supersensitive behavioral response in rodents to subsequent administration of dopamine agonists (e.g. Refs 21,37). Such treatment also causes an up-regulation of D, dopamine receptors in the neostriatum (see Ref. 26), the presumed mechanism underlying the development of behavioral supersensitivity.” These results have important clinical implications because behavioral supersensitivity is physiologically similar to tardive dyskinesia, the choreiform movement disorder that typically develops during long-term neuroleptic treatment.37*39 In fact, neuroleptic-induced tardive dyskinesia presumably results, at least in part, from supersensitive dopamine receptors.20 Steadily accumulating evidence suggests that ascorbate, which is found in high concentrations in the mammalian brain,3*2s~36 parallels in many respects the action of certain neuroleptics. For example, ascorbate inhibits the binding of labeled dopamine agonists and antagonists in neostriatal homogenates7~r5 and also inhibits dopamine-induced increases in cyclic-AMP36 (but see also Ref. 35). In addition, systemic injections of neuroleptics30*31 or ascorbate’ increase the firing rate of neostriatal neurons in

$To whom correspondence should be addressed.

anesthetized, immobilized animals. This excitatory effect of ascorbate also occurs with iontophoretic application, suggesting a direct action on neostriatal neuronsI Behavioral parallels also exist between haloperidol and ascorbate. Thus, systemic,38 intraventricular,41 or intraneostriata14* administration of ascorbate attenuates the behavioral response to amphetamine, an indirect dopamine agonist, and potentiates the ability of haloperidol to block amphetamine-induced behaviors.32*42 In this report, we assessed the effects of chronic treatment with ascorbate alone or combined with haloperidol on apomorphine-induced behavioral supersensitivity in rats. We also examined if such treatment produced changes in D2 dopamine receptor binding and brain permeability to [3H]apomorphine. EXPERIMENTAL PROCEDURES

Data were collected from 88 male, Sprague-Dawley rats (Harlan Sprague Dawley, Inc.) (275-300 g at the beginning of chronic treatment). All animals were housed individually under standard laboratory conditions; food and water were available continuously. Haloperidol-HCl (McNeil), apomorphine-HCl (Sigma), and sodium ascorbate (Sigma) were mixed in 0.9% saline. ( - )-Sulpiride-HCl (Research Biochemicals, Inc.) was mixed in 0.1% glacial acetic acid. Haloperidol and apo-

373

R. (‘. PIEKC-E et NI

374

morphine were expressed as the free base, ascorbate as the salt form. I’HlSDioerone (snecific activity. 27. I Ciimmoi) was purchased from DuPont-New England Nuclear. [‘HlApomorphine (specific activity, 0.27 Ci/mmol) was purchased from Research Products International. L

_._

.

Chronic treatment Animals were treated once daily for 21 days with either 0.5 mg/kg haloperidol, 500 mg/kg ascorbate, a combination of these doses of haloperidol and ascorbate, or an equal volume of 0.9% saline. In all cases, haloperidol was injected s.c., whereas ascorbate and saline were administered i.p. Following a five-day withdrawal period, some animals were challenged with 0.5 mg/kg apomorphine (s.c.) for assessment of behavioral supersensitivity, while others were assessed for either D, dopamine receptor binding or for [3H]apomorphine distribution in whole brain. Separate animals in the behavioral assessment condition received chronic treatment with 100 mg/kg ascorbate or a combination of 0.5 mg/kg haloperidol and 100 mg/kg ascorbate. Behavioral assessment During the five-day washout period, animals were placed in individual Plexiglass observation chambers (31 x 29 x 30cm; height, width, and depth, respectively). On the test day, apomorphine-induced behavior was recorded by two trained observers who were unaware of pretreatment (interrater reliability above 95%). Individual items of behavior were recorded for I-min periods at IO-min intervals beginning immediately after drug administration and continuing for the duration of the response (approximately 70min). Behavioral items, including locomotion, sniffing, rearing, repetitive head movements, and grooming, were rated according to intensity (0, not present; 1, mild; 2, moderate; 3, extreme) and duration (1, discontinuous; 2, continuous) during each observation period. These ratings, which were multiplied to yield a single value (e.g. the maximum score at each observation interval is 3 x 2) as described previously,33 are sensitive to apomorphine-induced behavioral effects.34

liquid scintillation spectrometry using a Packard .j 1x0 \tith approximately 28% counting efficiency. Specific binding \*ra\ defined as the difference in [3H]spiperone bound in ihe presence and absence of ( -- )-sulpiride. Data from ?ht’ binding assays were transformed by Scatchard analyst5 to determine [‘Hlspiperone-labeled receptor density (H.,, ,j I and dissociation constant (KJ In vivo distribution of [3H]apomorphinr Sixty minutes after injection with [‘H]apomorphmc (0.5 mg/kg, approximately 0.15 ,uCi/g), each animal was killed bv rapid decapitation, and the brain was removed and frozen at -7O’C. $or assay, whole brain was homogenized in 10 vol 0.1 N HClO, and centrifuged at IO.000 p for 10 min. Following decapitation, blood also was collected m 1 .S ml microcentrifuge tubes, allowed to coagulate on ice for approximately 5 min and then centrifuged at 10,000~ for 5 min. The plasma was removed and stored at - 70 C. For assay, 150 ~1 plasma was added to 2.8 ~1 70°C HCIO, and centrifuged at 10,000 g for 10 min. After centrifugation, the supernatant (650 ~1 brain, 100 ~1 plasma) was added to 8 ml scintillation fluid (Research Products International 3a70B), and the radioactivity was measured by liquid scintillation spectrometry (approximately 28% counting efficiency). Stativtkv non-parametric. Kruskal-Wallis, one-way Separate, analyses of variance (ANOVA) were performed on each behavioral measure averaged across all observation periods. Subsequent pairwise comparisons were performed using the Mann-Whitney (i-test. A 2 (ascorbate or saline) x2 (haloperidol or saline) factorial ANOVA was performed on the B,,,,, and & of [lH]spiperone-labeled binding sites in the neostriatum as well as on the radioactivity measured in both plasma and whole brain. Subsequent pairwise comparisons data on the B,,,,,, Ko, plasma and whole brain radioactivity were performed with the use of Tukey’s HSD.

RESULTS D, dopamine receptor binding Binding assays for [3H]spiperone were carried out on the neostriatum of individual rats in each treatment group. Animals were killed by rapid decapitation, and the brain was removed rapidly and placed on an ice-cold dissecting plate. The brain was then sectioned via coronal cuts through the optic chiasm and 4mm anterior to this point. The neostriatum (and extreme anterior globus pallidus) was removed and frozen at -70°C. For assay, the tissue samples were thawed and homogenized (Brinkman Polytron, setting 6) in approximately 5 ml of 50mmol/l Tris buffer (pH 7.4 at 37°C). The tissue suspension was brought to a volume of approximately 40 ml and was centrifuged for 10 min at 40,000 g. This procedure was repeated twice. After the third wash, the tissue was suspended for a final concentration of 1.5 mg/ml. The tissue was incubated in 50 mmol/l Tris containing 120 mmol/l NaCl, 5 mmol/l KC], 2 mmol/l CaCl, and 1 mmol/l MgSO, (pH 7.4 at 37”C), with [‘Hlspiperone (0.02-0.9 nmol/l, six to seven concentrations) in the presence and absence of 10 pmol/l ( - )-sulpiride. Each sample was run in duplicate and the final volume was 2 ml. The incubation was carried for 30min at 37°C to equilibrium. The incubation was terminated by vacuum filtration over Whatman GF/B glass-fiber filters, which were soaked prior to filtration in 0.1% polyethylenimine (30 min) to reduce radioligand binding to the filters. The filters were washed twice with 5 ml ice-cold Tris (no ions) and placed in 8 ml scintillation fluid (Research Products International 3a70B). Radioactivity retained by the filters was determined by

Behavior

Administration

of 0.5 mg/kg

apomorphine

pro-

duced a broad range of stereotyped behaviors across all treatment groups (as shown in Table 1). Chronic treatment with haloperidol, either alone or with ascorbate, significantly enhanced repetitive head movements relative to saline controls (x2 = 23.73, P < 0.0002). The most striking and consistent behavioral response, however, was repetitve sniffing. The Kruskal-Wallis one-way ANOVA for sniffing revealed a significant main effect of drug pretreatment (x2 = 25.76, P < O.OOl), and subsequent pairwise comparisons showed that the sniffing response of animals pretreated with 0.5 mg/kg haloperidol alone, 500 mg/kg ascorbate alone, the combination of these two treatments, as well as the combination of 0.5 mg/kg haloperidol and IO0 mg/kg ascorbate was significantly greater than that of saline controls (P < 0.05, Mann-Whitney U-test). In addition, the sniffing response of animals pretreated with the combination of 0.5 mg/kg haloperidol and 5OOmg/kg ascorbate was significantly greater than all groups except the high dose of ascorbate (P < 0.05, MannWhitney U-test).

37s

Chronic ascorbate potentiation Table 1. Behavioral response to apomorphine

following withdrawal from chronic treatment

Chronic treatment

n

Locomotion

Sniffing

Sal-Sal Hal-Sal Sal-aa Sal-AA Hal-aa Hal-AA

9 9 6 9 6 9

0.27 (0.069) 0.10 (0.044) 0.45 (0.124) 0.43 (0.098) 0.48 (0.085) 0.29 (0.076)

2.59 (0.288) 3.81 (0.21 I)* 2.93 (0.324) 3.64 (0.294)* 3.52 (0.244)’ 4.33 (0.219)**

Rearing

Repetitive head mov~en~

Grooming

0.54 (0.179) 0.33 (0.118) 0.52 (0.164) 0.78 (0.187) 0.62 (0.160) 0.24 (0.084)

1.59 (0.256) 2.11 (0.209)f 2.17 (0.291) 1.70 (0.195) 3.12 (0.246)* 2.14 (0.188)*

0.27 (0.124) 0.32 (0.145) 0.21 (0.010) 0.24 (0.119) 0.21 (0.116) 0.24 (0.077)

Data are presented as the mean interval score ( f S.E.M.) for individual items of behavior (see text for details) produced by 0.5 mg/kg apomorphine in rats five days after chronic (21”day) treatment with various combinations of saline (Sal), 0.5 mg/kg haloperidol (Hal), 100 mg/kg ascorbate (aa), or 500 mg/kg ascorbate (AA) as indicated. Note that the Sal-AA, Hal-Sal, and Hal-aa pretreatments produced similar supersensitive behavioral responses compared to Sal-Sal. In addition, Hal-AA produced a potentiated supersensitive response to apomorpbine as the sniffing score of this group was significantly greater than that of all other pretreatment groups except Sal-AA. n, Number of animals in each group. *P c 0.05 vs Sal-Sal (Mann-Whitney V-test); **P < 0.05 vs all other groups except Sal-AA (Mann-Whitney U-test).

0.8

D, dopamine binding sites

The mean K, and B,,,,, of [3H]spiperone-labeled binding sites in the neostriatum for all pretreatment groups are summarized in Table 2. Scatchard plots for these groups are presented in Fig. 1. The overall ANOVA revealed no significant main effect or interaction of pretreatment on K,, but there was a significant effect on haloperidol pretreatment on B,, [F( 1,19) = 30.45, P < O.OOl]. Subsequent pairwise comparisons showed that neostriatal D, binding site density in animals pretreated with either 0.5 mg/kg haloperidol or the combination of 0.5 mg/kg haloperidol and 500 mg/kg ascorbate was significantly higher than that in animals pretreated with saline or 500 mg/kg ascorbate alone. There was no significant effect of ascorbate on B,,,, values.

The mean radioactivity measured in both whole brain and plasma for each pretreatment group is depicted in Table 3. The overall ANOVAs revealed no effect of pretreatment on the distribution of radioactivity in either brain or plasma. DISCUSSION

Our results indicate that chronic treatment with haloperidol potentiates apomorphine-induced head bobbing and sniffing. Chronic treatment with highdose ascorbate alone also elicits a su~r~nsitive behavioral response similar to that produced by

l saline-holopwidol

Q 5 0.6

4 oscorblc acid-saline

A oacwbic ocld-halopaidol

gkz

ta 2 j 0.4 3 p L z 0.2 G

20

10 Bound

(fmol/mg

30 tissue)

Fig. 1. Scatchard plots of [3H]spiperone binding to D, dopamine sites in neostriatum for all treatment groups as indicated in the legend. See text for drug doses.

chronic haloperidol administration, Unhke haloperidol, however, neither low- nor high-dose ascorbate treatment increased neostriatal D, receptor density. Moreover, the combined treatment of haloperidol and high-dose ascorbate produced an even greater behavioral response to an apomorphine challenge than haloperidol alone, but again the enhanced behavioral response was not accompanied by a further change in the density of neostriatal D, receptors. Thus, concurrent ascorbate-haloperidol treatment did not elevate Dz receptor density beyond that produced by chronic haloperidol alone. It appears, therefore, that although chronic ascorbate elicits a supersensitive behavioral response to apomorphine,

Table 2. Effects of chronic treatment on [3H]spiperone binding to D, receptors in rat striatum Chronic treatment

?I

(pmol/[email protected].)

Saline-saline Haloperidol-saline Saline-ascorbate Haloperidol-ascorbate

6 5 6 6

40.2f5.1 46.6 f 5.5 48.5 f 6.0 51.8 f 6.6

‘P < 0.05 vs saline-saline and ascorbate-saline

(fmof/mg tiz

-f. S.E.M.)

27.8 f 1.1 36.7 + 1.7* 26.8 & 1.1 38.8 + 3.0*

groups frukey’s HSD).

R. C. PIERCE (‘I

376 Table 3. Effects of

ui.

chronic treatment on the subsequent distribution of [3H]apomorphine

Chronic treatment Saline-saline Haloperidol-saline Saline-ascorbate Haloperidol-ascorbate

Brain S.E.M.) 2.86 + 0.022 2.64 + 0.153 2.86 5 0.124 2.59 + 0.143

(nCi/g +

4

this effect cannot be explained by an up-regulation of D2 dopamine receptors. Our results also suggest, however, that the ascorbate-induced potentiation of the apomorphine behavioral response is not mediated by a simple change in the pharmacokinetics of either apomorphine or haloperidol. Chronic ascorbate, for example, failed to alter either brain or plasma radioactivity measured 60min after an injection of [3H]apomorphine, arguing against an ascorbate-induced change in the distribution of this drug. Although residual ascorbate could conceivably influence the action of apomorphine at the receptor level, it is noteworthy that chronic ascorbate treatment failed to influence D, receptor density in the neostriatum, a major site of apomorphine action. Moreover, ample evidence42 indicates that an increase in neostriatal ascorbate actually causes a decline in the behavioral response to dopamine agonists, making it unlikely that residual ascorbate alone is responsible for apomorphine-induced behavioral supersensitivity in rats withdrawn from chronic ascorbate treatment. In addition, such treatment had no effect on the ability of haloperidol to alter D, receptor density, arguing against an effect of chronic ascorbate on haloperidol pharmacokinetics. At this point, however, we cannot rule out a role for other central mechanisms, including a change in glutamate transmission. Indeed, glutaminergic neurons in the corticoneostriatal pathway represent the primary source of neostriatal ascorbate,2 and increases in extracellular ascorbate have been linked to corresponding increases in synaptic glutamate.28 Furthermore, haloperidol is known to alter glutamateinfluenced behaviors. Thus, chronic treatment with haloperidol decreases the behavioral response to both quisqualic acid and glutamic acid diethyl ester.” Until additional information becomes available, however, we cannot speculate on the mechanism by which ascorbate could influence this interaction. It also is possible that ascorbate and haloperidol are acting by entirely independent mechanisms, in which case the potentiated effect of the ascorbatehaloperidol combination may reflect a simple additive effect of each treatment. Efforts to identify an effect of chronic ascorbate treatment at non-D, sites currently are underway, but because ascorbate is known to influence a variety of neurotransmitter systems at either pre- or postsynaptic sites, including release from cholinergic,23 adrenergic,23 and GABAergic4 terminals, as well as binding of D,,41 cholinergic,**

Plasma + S.E.M.)

(nCi/ml

0.0733 & 0.01 I 0.0671 + 0.002 0.0633 f 0.006 0.0617 _+0.004

opioid,’ and alpha-adrenergic” receptors, it is difficult to speculate on which of these systems may contribute to our behavioral results. The failure of the low dose of ascorbate to mimic the behavioral supersensitivity that occurred in haloperidol-treated rats or to enhance the effect of haloperidol on this behavioral measure is not surprising in view of the relatively high levels of endogenous ascorbate already present in neostriatal and other forebrain areas. In neostriatum alone, extracellular ascorbate ranges between 200 and Luw)PM and under certain behavioral conditions may increase to more than twice this amount,‘*” reaching endogenous levels approximately 1000 times higher thati those of extracellular dopamine. 6,‘4.‘8Systemic injections of 1000 mg/kg ascorbate, which are known to alter the spontaneous firing rate of neostriatal neurons, elevate neostriatal ascorbate by several hundred micromolar, well within the normal physiological range.9 Such a dose also has been shown to potentiate the haloperidol-induced blockade of the amphetamine behavioral response. 32Thus, to the extent that ascorbate exerts a neuroleptic-like action, treatment with 100 mg/kg ascorbate may not alter endogenously high levels of ascorbate sufficiently to produce a haloperidol-like supersensitivity, though it is conceivable that a longer treatment period may do so. CONCLUSION

Our results provide additional behavioral evidence for a neuroleptic-like action of ascorbate. Withdrawal from chronic ascorbate treatment potentiates the behavioral response to an apomorphine challenge, and following treatment with the high dose of ascorbate the supersensitive behavioral response is similar to that seen following chronic haloperidol administration. Our results also indicate that the apomorphine sensitization is enhanced still further in rats receiving combined treatment with both highdose ascorbate and haloperidol. This effect cannot be explained by an ascorbate-induced change in the pharmacokinetics of these drugs. Unlike haloperidol, however, ascorbate fails to alter D2 receptor density with chronic treatment, suggesting that the behavioral supersensitivity during the withdrawal period is not mediated by the same mechanism. Acknowledgements-This research was supported by NSF Grant BNS 91-12055 (GVR) and NIDA Grant DA 05312 (MTB). We also acknowledge the expert editorial assistance of Faye Caylor.

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