- Email: [email protected]
Dopamine D2 receptors in the striatum and frontal cortex following chronic administration of haloperidol
Neuropharmocology Vol. 26, No. 5, pp. 481483, 1987 Printed in Great Britain. All rights reserved
Ol28-3908/87 $3.00 + 0.00 Copyright 0
1987 PergamonJournals Ltd
DOPAMINE D, RECEPTORS IN THE STRIATUM AND FRONTAL CORTEX FOLLOWING CHRONIC ADMINISTRATION OF HALOPERIDOL D. R. LISKOWSKY’and L. T. POTTERY ‘Behavioral Medicine Building, Department ofPsychology, University of Miami, Coral Gables, FL 33124, U.S.A. and *Department of Pharmacology, P.O. Box 016189, University of Miami School of Medicine, Miami, FL 33101, U.S.A. (Accepted
25 May 1986)
Summary-The effects of chronic treatment with a neuroleptic on D, dopamine receptors in the striatum and frontal cortex were studied. Exposure to haloperidol for 21 days caused an upregulation in the striatum but not in the cortex of D, receptors. These results indicate that dopamine-regulating mechanisms in the cortex may differ from those in the striatum and suggest that the anti-psychotic action of neuroleptics may be due in part to blockade of receptors in the cortex. Key words: D, receptors, frontal cortex, striatum, haloperidol.
The mesocortical dopamine system appears to respond differently to neuroleptics than does the striatal dopamine system, in terms of altered rates of turnover of dopamine and production of metabolites (Laduron, DeBie and Leyson, 1979; Bowers, 1984). Tolerance to the increased metabolism of dopamine, caused by these drugs, develops in the striatum but not in the frontal cortex. Within the striatum, the chronic administration of neuroleptics causes an increase in dopamine D, receptors (Rupniak, Jenner and Marsden, 1983; Fleminger, Rupniak, Hall, Jenner and Marsden, 1983) but it is not known whether there are also compensatory receptor changes in the frontal cortex. Until recently it has been difficult to measure D, receptors in the frontal cortex, primarily because spiroperidol, the ligand of choice for labelling D2 receptors, also binds to serotonin receptors. As much as 80% of the binding of spiroperidol in the cortex is to receptors for serotonin (Howlett and Nahorski, 1980). The present authors have shown that it is possible to measure D2 receptors in the frontal cortex with [‘Hlspiroperidol, by including an antagonist to block serotonin receptors (Liskowsky and Potter, 1985). Using this assay procedure it was possible to demonstrate that the binding of [3H]spiroperidol to the frontal cortex has a biphasic rate of dissociation, with the high affinity component having the same rate of dissociation as that of D, receptors in the striatum. In the presence of 3OOnM cinanserin only the high affinity component remains and it is displaced with high affinity by dopaminergic compounds. Also, this component of the binding is increased after lesioning of the median forebrain bundle. This same assay
procedure was used in the present study to compare the responses of D, dopamine receptors in the cortex and striatum to the chronic administration of haloperidol. METHODS Male Sprague-Dawley rats (150-I 7.5g) were given haloperidol (0.5 mg/kg) through the sub-cutaneous implantation of pellets (Innovative Research, Inc.). These pellets are designed to deliver a constant dose of the drug over a 21-day period. Augmentation of the dose to allow for gain in weight of the animals was accomplished by implantation of a supplemental pellet, containing l/10 the original dose, halfway through the 21-day period of drug administration. Control animals recieved placebo pellets of methyl cellulose. Tissue preparation and determination of numbers of D, receptor, were carried out using procedures described previously (Liskowsky and Potter, 1985). Briefly, after 21 days of administration of drug, the animals were sacrificed and the striata and frontal cortex immediately dissected. Tissue was homogenized in sodium phosphate-EDTA buffer, and left at 4°C for 30 min. The homogenate was centrifuged and the pellet resuspended in Tris-HCI buffer. The assay of D2 receptors, was carried out in 1 ml of Tris-HCl buffer, containing 1 mM MnCl,, 10 PM pargyline, 300 nM cinanserin, membranes from 5 mg of tissue and a saturating concentration of [3H]spiroperidol (1.25 nM). Incubation was for 30 min at 37°C. Nonspecific binding was determined in the presence of 1 ,uM d-butaclamol. Radioactivity was determined by liquid scintillation spectroscopy.
481
D. R. LISKOWSKY and L. T. POTTER
482 Table
I. Effect of administration for 21-days of haloperidol on D, dopamine receptors in the striatum and frontal cortex Specific binding of [‘H]spiroperidol (pm&/g tissue _+SD) Control (N = 14)
Striatum Frontal cortex
17.5 k 2.5 3.1 + 0.5
Halorxridol
Percentage increase
(N = 15)
27.8 + 3.4’ 3.4 + 1.0
58% II%
Membranes from the striatum and frontal, cortex from haloperidol-treated and control rats were incubated with a saturating concentration (1.25 nM) of (‘H]spiroperidol in 1 ml of Tris-HCI buffer containing 1 mM MnCI,, 10 PM pargyline and 300 nM cinanserin for 30min at 37°C. Membranes were then filtered and washed as described in Methods. *Significantly different (P < 0.001) from specific binding of [‘H]spiropetidol in control group (Student’s f-test).
RESULTS
Table 1 shows that the chronic administration of haloperidol caused a significant increase (+.58%, P < 0.001) in the binding of spiroperidol in the striatum, with no significant increase (+ 1l%, P > 0.20) in the frontal cortex. To determine the nature of the change in the binding in the striatum, saturation experiments with [3H]spiroperidol (0.0154.0 nM) and dopamine/[gH]sprioperidol competition experiments were carried out. Scatchard analysis of the binding to striatal receptors (data not shown) indicated that the increase in binding was due to an increase in B,, with no change in antagonist affinity (Kd). Computer analysis, using an iterative least squares method, of the data from the competition experiments (Fig. l), indicated that the ability of dopamine to inhibit the binding of spiroperidol was identical in control and haloperidol-treated tissue. Both curves fitted best to the same two-site model, the two sites representing the presence of high and low agonist-affinity binding states of the D, receptors. These results demonstrated that exposure to the drug had not affected the agonist affinity of the receptor.
but is in keeping with the development of tardive dyskinesias (Rupniak et al., 1983). In contrast, an increase in the metabolism of dopamine is maintained in the cortex, with no suggestion of tolerance occurring (Bacapoulos, Spokes, Bird and Roth 1979; Bacapoulos, Redmond, Baulu and Roth 1980). These results are in keeping with the lack of development of receptor changes in the frontal cortex found in the present study and with the response to the chronic administration of fluphenazine (Bacopoulus, 1981); they also coincide with the concept that dopamineregulating mechanisms may differ between the striatum and cortex. This study suggests that the antipsychotic action of neuroleptics may be due to undiminished blockade of dopamine receptors in the cortex.
I
. control 0
hdc+&l
DISCUSSION
In the present study, exposure for 21 days to haloperidol induced a significant upregulation of D, dopamine receptors in the striatum, but not in the frontal cortex. The increase in the receptors in the striatum was an increase in receptor number, with no change in either antagonist or agonist affinity, in agreement with previous studies (Fleminger et al., 1983; MacKensie and Zigmond, 1984). These results support the view that the response of the mesocortical dopamine system to neuroleptics is different from the response of the striatal system. In the striatum, tolerance to the receptor blocking effects of neuroleptics develops after chronic exposure (Rupniak et nf., 1983). An initial increase in the turnover of dopamine abates and a concomittant increase in the number of dopamine receptors occurs. The development of receptor supersensitivity in the striatum diminishes the possibility that some of the anti-psychotic therapeutic effects of these drugs are due to their actions on mechanisms in the striatum,
ok2 -8
(
I
I
I
1
1
-7
-6
-5
-4
-3
-2
dopamino ( log M I Fig. 1.Competition between [3H]spiroperidol and dopamine in control and haloperidol-treated striata. Membranes, prepared as described in Methods, from halopetidol-treated and control striata, were incubated with 1.25 nM [3H]spiroperidol and various concentrations of dopamine, in I ml of Tris-HCI buffer containing 1 mM MnCl,, 10 PM pargyline and 300 nM cinanserin, for 30 min at 37°C. The membranes were then filtered and washed as described in Methods. Computer analysis, using an iterative least squares method, demonstrated that both sets of data fitted best to the same two site curve, indicating that treatment with haloperidol did not alter the agonist affinity of the receptor. The dashed line is the computer-derived curve, comprised of a high (38%; IC 50 = l.OpM) and low affinity (62%; IC 50 = 100 PM) component. These components represent the high and low agonist-affinity binding states of the D, receptor. Data points are the mean of three experiments for each condition.
D, receptors after haloperidol exposure
Bacopoulos N. (198 1) Biochemical mechanism of tolerance to neuroleptic drugs, regional differences in rat brain. Eur. J. Pharmac. 70: 585-588.
Bacopoulos N., Redmond D., Baulu J. and Roth R. (1980) Chronic haloperidol or fluphenazine effects on dopamine metabolism in brain, cerebrospinal fluid and plasma of Cereopithecus aethiops (Vervet monkey). J. Pharm. exp. Ther. 212: 1-5. Bacopoulos N., Spokes E., Bird E. and Roth R. (1979) Antipsychotic drug action in schizophrenic patients: Effect on cortical dopamine metabolism after long term treatment. Science 205: 1405-1407. Bowers M. (1984) Homovanillic acid in caudate and prefrontal cortex following neuroleptics. Eur. f. ~~urmuc. 99: 103-105. Fleminger S., Rupniak N., Hall M., Jenner P. and Marsden C. (1983) Changes in apomorphine-induced stereotypy as
N.P. 26/5-F
483
a result of subacute neuroleptic treatment correlates with increased D-2 receptors, but not with increase in D-l receptors. Biochem. Pharmac. 32: 2921-2927. Howlett D. and Nahorski S. (1980) Quantitative assessment of heterogeneous ‘H-spiperone binding to rat neostriatum and frontal cortex. Lilp Sci. 26: 511-517. Laduron P., DeBie K. and Leyson J. (1979) Specific effect of haloperidol on dopamine turnover in frontal cortex, Naunyndchmeiakbergs
Arch. Pharmac. 2%: 183-185.
Liskowsky D. and Potter L. (1985) Dz dopamine receptors in the frontai cortex of rat and human. A& Sci. 36: 1551-1559. MacKenzie R. and Zigmond M. (1984) High and low affinity states of striatal D2 receptors are not affected by 6-hydroxydopamine or chronic haloperidol treatment. .l. ~e~roc~m. 43: 131@-1315. Rupniak N., Jenner P. and Marsden C. (1983) The effect of chronic neuroleptic administration on cerebral dopamine receptor function. Life Sci. 32: 2289-2311.
Ol28-3908/87 $3.00 + 0.00 Copyright 0
1987 PergamonJournals Ltd
DOPAMINE D, RECEPTORS IN THE STRIATUM AND FRONTAL CORTEX FOLLOWING CHRONIC ADMINISTRATION OF HALOPERIDOL D. R. LISKOWSKY’and L. T. POTTERY ‘Behavioral Medicine Building, Department ofPsychology, University of Miami, Coral Gables, FL 33124, U.S.A. and *Department of Pharmacology, P.O. Box 016189, University of Miami School of Medicine, Miami, FL 33101, U.S.A. (Accepted
25 May 1986)
Summary-The effects of chronic treatment with a neuroleptic on D, dopamine receptors in the striatum and frontal cortex were studied. Exposure to haloperidol for 21 days caused an upregulation in the striatum but not in the cortex of D, receptors. These results indicate that dopamine-regulating mechanisms in the cortex may differ from those in the striatum and suggest that the anti-psychotic action of neuroleptics may be due in part to blockade of receptors in the cortex. Key words: D, receptors, frontal cortex, striatum, haloperidol.
The mesocortical dopamine system appears to respond differently to neuroleptics than does the striatal dopamine system, in terms of altered rates of turnover of dopamine and production of metabolites (Laduron, DeBie and Leyson, 1979; Bowers, 1984). Tolerance to the increased metabolism of dopamine, caused by these drugs, develops in the striatum but not in the frontal cortex. Within the striatum, the chronic administration of neuroleptics causes an increase in dopamine D, receptors (Rupniak, Jenner and Marsden, 1983; Fleminger, Rupniak, Hall, Jenner and Marsden, 1983) but it is not known whether there are also compensatory receptor changes in the frontal cortex. Until recently it has been difficult to measure D, receptors in the frontal cortex, primarily because spiroperidol, the ligand of choice for labelling D2 receptors, also binds to serotonin receptors. As much as 80% of the binding of spiroperidol in the cortex is to receptors for serotonin (Howlett and Nahorski, 1980). The present authors have shown that it is possible to measure D2 receptors in the frontal cortex with [‘Hlspiroperidol, by including an antagonist to block serotonin receptors (Liskowsky and Potter, 1985). Using this assay procedure it was possible to demonstrate that the binding of [3H]spiroperidol to the frontal cortex has a biphasic rate of dissociation, with the high affinity component having the same rate of dissociation as that of D, receptors in the striatum. In the presence of 3OOnM cinanserin only the high affinity component remains and it is displaced with high affinity by dopaminergic compounds. Also, this component of the binding is increased after lesioning of the median forebrain bundle. This same assay
procedure was used in the present study to compare the responses of D, dopamine receptors in the cortex and striatum to the chronic administration of haloperidol. METHODS Male Sprague-Dawley rats (150-I 7.5g) were given haloperidol (0.5 mg/kg) through the sub-cutaneous implantation of pellets (Innovative Research, Inc.). These pellets are designed to deliver a constant dose of the drug over a 21-day period. Augmentation of the dose to allow for gain in weight of the animals was accomplished by implantation of a supplemental pellet, containing l/10 the original dose, halfway through the 21-day period of drug administration. Control animals recieved placebo pellets of methyl cellulose. Tissue preparation and determination of numbers of D, receptor, were carried out using procedures described previously (Liskowsky and Potter, 1985). Briefly, after 21 days of administration of drug, the animals were sacrificed and the striata and frontal cortex immediately dissected. Tissue was homogenized in sodium phosphate-EDTA buffer, and left at 4°C for 30 min. The homogenate was centrifuged and the pellet resuspended in Tris-HCI buffer. The assay of D2 receptors, was carried out in 1 ml of Tris-HCl buffer, containing 1 mM MnCl,, 10 PM pargyline, 300 nM cinanserin, membranes from 5 mg of tissue and a saturating concentration of [3H]spiroperidol (1.25 nM). Incubation was for 30 min at 37°C. Nonspecific binding was determined in the presence of 1 ,uM d-butaclamol. Radioactivity was determined by liquid scintillation spectroscopy.
481
D. R. LISKOWSKY and L. T. POTTER
482 Table
I. Effect of administration for 21-days of haloperidol on D, dopamine receptors in the striatum and frontal cortex Specific binding of [‘H]spiroperidol (pm&/g tissue _+SD) Control (N = 14)
Striatum Frontal cortex
17.5 k 2.5 3.1 + 0.5
Halorxridol
Percentage increase
(N = 15)
27.8 + 3.4’ 3.4 + 1.0
58% II%
Membranes from the striatum and frontal, cortex from haloperidol-treated and control rats were incubated with a saturating concentration (1.25 nM) of (‘H]spiroperidol in 1 ml of Tris-HCI buffer containing 1 mM MnCI,, 10 PM pargyline and 300 nM cinanserin for 30min at 37°C. Membranes were then filtered and washed as described in Methods. *Significantly different (P < 0.001) from specific binding of [‘H]spiropetidol in control group (Student’s f-test).
RESULTS
Table 1 shows that the chronic administration of haloperidol caused a significant increase (+.58%, P < 0.001) in the binding of spiroperidol in the striatum, with no significant increase (+ 1l%, P > 0.20) in the frontal cortex. To determine the nature of the change in the binding in the striatum, saturation experiments with [3H]spiroperidol (0.0154.0 nM) and dopamine/[gH]sprioperidol competition experiments were carried out. Scatchard analysis of the binding to striatal receptors (data not shown) indicated that the increase in binding was due to an increase in B,, with no change in antagonist affinity (Kd). Computer analysis, using an iterative least squares method, of the data from the competition experiments (Fig. l), indicated that the ability of dopamine to inhibit the binding of spiroperidol was identical in control and haloperidol-treated tissue. Both curves fitted best to the same two-site model, the two sites representing the presence of high and low agonist-affinity binding states of the D, receptors. These results demonstrated that exposure to the drug had not affected the agonist affinity of the receptor.
but is in keeping with the development of tardive dyskinesias (Rupniak et al., 1983). In contrast, an increase in the metabolism of dopamine is maintained in the cortex, with no suggestion of tolerance occurring (Bacapoulos, Spokes, Bird and Roth 1979; Bacapoulos, Redmond, Baulu and Roth 1980). These results are in keeping with the lack of development of receptor changes in the frontal cortex found in the present study and with the response to the chronic administration of fluphenazine (Bacopoulus, 1981); they also coincide with the concept that dopamineregulating mechanisms may differ between the striatum and cortex. This study suggests that the antipsychotic action of neuroleptics may be due to undiminished blockade of dopamine receptors in the cortex.
I
. control 0
hdc+&l
DISCUSSION
In the present study, exposure for 21 days to haloperidol induced a significant upregulation of D, dopamine receptors in the striatum, but not in the frontal cortex. The increase in the receptors in the striatum was an increase in receptor number, with no change in either antagonist or agonist affinity, in agreement with previous studies (Fleminger et al., 1983; MacKensie and Zigmond, 1984). These results support the view that the response of the mesocortical dopamine system to neuroleptics is different from the response of the striatal system. In the striatum, tolerance to the receptor blocking effects of neuroleptics develops after chronic exposure (Rupniak et nf., 1983). An initial increase in the turnover of dopamine abates and a concomittant increase in the number of dopamine receptors occurs. The development of receptor supersensitivity in the striatum diminishes the possibility that some of the anti-psychotic therapeutic effects of these drugs are due to their actions on mechanisms in the striatum,
ok2 -8
(
I
I
I
1
1
-7
-6
-5
-4
-3
-2
dopamino ( log M I Fig. 1.Competition between [3H]spiroperidol and dopamine in control and haloperidol-treated striata. Membranes, prepared as described in Methods, from halopetidol-treated and control striata, were incubated with 1.25 nM [3H]spiroperidol and various concentrations of dopamine, in I ml of Tris-HCI buffer containing 1 mM MnCl,, 10 PM pargyline and 300 nM cinanserin, for 30 min at 37°C. The membranes were then filtered and washed as described in Methods. Computer analysis, using an iterative least squares method, demonstrated that both sets of data fitted best to the same two site curve, indicating that treatment with haloperidol did not alter the agonist affinity of the receptor. The dashed line is the computer-derived curve, comprised of a high (38%; IC 50 = l.OpM) and low affinity (62%; IC 50 = 100 PM) component. These components represent the high and low agonist-affinity binding states of the D, receptor. Data points are the mean of three experiments for each condition.
D, receptors after haloperidol exposure
Bacopoulos N. (198 1) Biochemical mechanism of tolerance to neuroleptic drugs, regional differences in rat brain. Eur. J. Pharmac. 70: 585-588.
Bacopoulos N., Redmond D., Baulu J. and Roth R. (1980) Chronic haloperidol or fluphenazine effects on dopamine metabolism in brain, cerebrospinal fluid and plasma of Cereopithecus aethiops (Vervet monkey). J. Pharm. exp. Ther. 212: 1-5. Bacopoulos N., Spokes E., Bird E. and Roth R. (1979) Antipsychotic drug action in schizophrenic patients: Effect on cortical dopamine metabolism after long term treatment. Science 205: 1405-1407. Bowers M. (1984) Homovanillic acid in caudate and prefrontal cortex following neuroleptics. Eur. f. ~~urmuc. 99: 103-105. Fleminger S., Rupniak N., Hall M., Jenner P. and Marsden C. (1983) Changes in apomorphine-induced stereotypy as
N.P. 26/5-F
483
a result of subacute neuroleptic treatment correlates with increased D-2 receptors, but not with increase in D-l receptors. Biochem. Pharmac. 32: 2921-2927. Howlett D. and Nahorski S. (1980) Quantitative assessment of heterogeneous ‘H-spiperone binding to rat neostriatum and frontal cortex. Lilp Sci. 26: 511-517. Laduron P., DeBie K. and Leyson J. (1979) Specific effect of haloperidol on dopamine turnover in frontal cortex, Naunyndchmeiakbergs
Arch. Pharmac. 2%: 183-185.
Liskowsky D. and Potter L. (1985) Dz dopamine receptors in the frontai cortex of rat and human. A& Sci. 36: 1551-1559. MacKenzie R. and Zigmond M. (1984) High and low affinity states of striatal D2 receptors are not affected by 6-hydroxydopamine or chronic haloperidol treatment. .l. ~e~roc~m. 43: 131@-1315. Rupniak N., Jenner P. and Marsden C. (1983) The effect of chronic neuroleptic administration on cerebral dopamine receptor function. Life Sci. 32: 2289-2311.