Brain Research, 561 (1991) 1-10 Elsevier ADONIS 000689939116985N BRES 16985

Research Reports

Dopamine receptor 'supersensitivity' occurring without receptor up-regulation Beth E. Mileson 1'2, Mark H. L e w i s 1'2'3 and Richard B. M a i l m a n 1'2'3'4 Brain and Development Research Center1, Toxicology C u r r i c l d u m 2 and Departments of 3Psychiatry and 4Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (U.S.A.) (Accepted 30 April 1991)

Key words: 6-Hydroxydopamine; D 1 receptor; D 2 receptor; Cytotoxicity; Supersensitivity; Stereotyped behavior; Cyclic AMP; Dopamine receptor

Unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the substantia nigra have been widely used to study various aspects of dopamine neurobiology, and to screen for antiparkinsonian drugs. This study examined the role of receptor alterations in the pharmacological supersensitivity seen in response to lesioning of central dopamine pathways in rats by intracisternal (IC) administration of 6-OHDA (200 #g), as well as by bilateral (BIL) or unilateral (UNI) infusion of 6-OHDA into the substantia nigra (8 #g/side). Both IC and BIL lesions resulted in permanent decreases in dopamine concentration in the striatum, the major terminal projection from the substantia nigra. When challenged with apomorphine (0.3 mg/kg), IC-lesioned rats exhibited bursts of rapid locomotion interspersed by rearing, whereas BIL-lesioned rats displayed intense grooming or gnawing and nose poking of the cage floor; these behaviors were not seen in respective sham (i.e. vehicle)lesioned rats injected with apomorphine. Scatchard analysis of saturation isotherms of both D 1 ([31"1]SCH23390 binding sites) and D 2 ([3H]spiperone binding sites) dopamine receptors in the striatum revealed no difference in either the maximum number of binding sites (B ~ ) , or the dissociation constant (Ko) of either receptor type when BIL and IC lesioned rats were compared to appropriate controls. Conversely, the UNI lesioned rats had, under identical conditions of analysis, the expected increase in the density of D 2 receptors on the lesioned side. There was no change in dopamine-sensitive adenylate cyelase activity in the striata of supersensitive IC-lesioned rats, but there was a shift to the left in the dose-response curve in striata from rats bilaterally-lesioned in the substantia nigra, similar to what occurs in UNI lesioned rats. Together, these data clearly demonstrate that although increases in receptor density and changes in cAMP systems are seen in the UNI model, neither mechanism is a requirement for functional supersensitivity in response to 6-OHDA lesions. These data suggest that other cellular events (e.g. alterations in receptor interactions) may play a role in the response to insult, and raise questions about the utility of the unilateral model as a screen for antiparkinsonian drugs.

INTRODUCTION D e n e r v a t i o n m o d e l s have b e e n widely used in studying the nervous system, in developing m o d e l s of neurological and psychiatric diseases, and in elucidating mechanisms of drug action. D e n e r v a t i o n of p e r i p h e r a l organs o r pathways is k n o w n to result in e n h a n c e d responsiveness to administration of agonists for the d e n e r v a t e d system 5~. Similar m o d e l s have b e e n widely used to study central n e u r o t r a n s m i t t e r function, and again, p o t e n t i a t e d responses to agonist administration are seen after 'recovery'. T h e mechanism responsible for this p h e n o m e n o n of ' d e n e r v a t i o n supersensitivity' is usually assumed to b e r e c e p t o r 'up-regulation' (increases in the density o f rec e p t o r sites), and concomitant functional changes in seco n d - m e s s e n g e r systems (e.g. c A M P ) linked to these receptors.

Studies of central n o r a d r e n e r g i c tracts were the first in which r e c e p t o r 'up-regulation' was d e m o n s t r a t e d in brain. S p o r n et al. 49 r e p o r t e d that administration of the catecholamine-specific toxicant 6 - h y d r o x y d o p a m i n e (6O H D A ) caused loss of n o r a d r e n e r g i c innervation in the cortex, with a concomitant increase in b o t h fl-adrenoreceptor density and c A M P synthesis in response to agonist administration. Such d a t a s u p p o r t e d the hypothesis that r e c e p t o r 'up-regulation' was a critical mechanism in response to denervation. The most widely studied m o d e l in brain, however, is that first described by U n g e r s t e d t 52. In this model, a unilateral lesion of the nigrostriatal dopaminergic p a t h w a y is p r o d u c e d b y injection of 6 - O H D A into one substantia nigra of the rat. This lesion results in a p e r m a n e n t depletion o f d o p a m i n e in the ipsilateral striatum. A f t e r recovery, behavioral supersensitivity (i.e. contralateral circling) is seen when the rat is

Correspondence: R. Mailman, C B ~ 7250 - Brain and Development Research Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7250, U.S.A. Fax: (1) (919) 966-1844.

2 challenged with a direct-acting dopamine agonist. This increased sensitivity to agonist administration has been correlated with an increase in striatal dopamine D 2 receptor density ipsilateral to the lesion 11'1s'19"35. Pharmacological denervation by chronic blockade of D 1 or D 2 receptors in vivo has also been shown to increase the density of the appropriate class of receptor ~2"22. Until recently, the emphasis of most studies has been on D 2 receptors because of the presumed importance of that class in mediating the behavioral effects of dopaminergic drugs 46. Awareness of the fact that D 1 receptors have psychopharmacological e f f e c t s 7"30'4s, and could modulate D 2 receptor function 6'23"3°'53, has led to an appreciation of the importance of D1-D 2 receptor interactions. Nonetheless, because dopamine D 2 receptor density increases after unilateral and pharmacological denervation, it has been assumed that this increase in receptor density is a critical mechanism in the accommodation to denervation insult. Several reports have suggested that receptor up-regulation may not be a principal or universal mechanism of response to such denervation. For example, in the unilateral 6-OHDA model, Staunton et al.50 reported dissociating rotation behavior from dopamine receptor changes following destruction of the nigrostriatal pathway. Moreover, administration of a selective or non-selective dopamine receptor blocker seems to be associated with a large change in receptor density as measured in radioreceptor assays 17'21'32'42"43 b u t much less dramatic psychopharmacological supersensitivity than that observed in toxicant-induced denervation models. Clearly, such paradigms are influenced by both the dose and time of drug administration s . Finally, it has been reported that intracisternal injection of 6-OHDA can cause profound behavioral supersensitivity to agonist challenge, yet change neither receptor density nor affinity4,29,31,33.

In the present study, we have explored this issue further by examining behavioral supersensitivity, receptor density, and receptor function in two lesion models (intracisternal and bilateral substantia nigra injections or injection of 6-OHDA) which, a priori, might be expected to cause similar receptor and psychopharmacological changes. These results were compared directly to dopamine receptor changes observed in rats with unilateral substantia nigra lesions also induced by 6-OHDA. Although it has been hypothesized that 'denervation supersensitivity' of dopamine systems may occur in the absence of receptor up-regulation 4'z9'31, a direct comparison of several models of 6-OHDA administration has not been done. Thus, the goal of the present study was to determine if receptor up-regulation is an essential mechanism in the response to cytochemical injury.

MATERIALS AND METHODS

Animal housing Sprague-Dawley rats (Charles River, Wilmington, MA) weighing between 175 and 275 g at the beginning of the studies were housed in wire cages in an animal facility maintained at 27 °C on a 07.00:19.00 h light:dark schedule. The rats were allowed free access to food and water throughout the study.

Lesioning methods Unilateral and bilateral lesions. Rats receiving unilateral or bilateral nigral injections of 6-OHDA were pretreated with 25 mg/kg desipramine, anesthetized with sodium pentobarbitai and placed in a stereotaxic apparatus. Thirty gauge needles were lowered into the right, or both aspects of the substantia nigra at the following coordinates: inter-aural zero +3.2 mm; lateral -2.0 mm; and dorsoventral -7.7 mm 3s. Four/~1 of 0.1% ascorbic acid in sterile saline containing 8/zg of 6-OHDA were infused in each side over a period of 20 min using 10/tl Hamilton syringes and a Sage syringe pump. The needles were left in place 2 min after infusion to minimize withdrawal of the 6-OHDA solution with the needles. The small burr holes in the skull were plugged with bone wax, and the wound sutured. Post-operative care of these rats included observation, sweet food supplements, and gastric intubation during the aphagic period following lesion. Bilaterally lesioned rats increased their weight by approximately 10% between surgery and the time of behavioral testing, compared to a 50% increase seen in shamlesioned animals during the same period. Lesioned animals which fell below their preoperative weight at the time of behavioral testing were not used in the present studies. Control rats were treated with desipramine, anesthetized, and injected with vehicle. lntracisternal injections, lntracisternal injection of 6-OHDA was accomplished using a 50/~i glass Hamilton syringe fitted with a 30 gauge needle. Rats were pretreated peritoneally with 25 mg/kg desipramine 30 rain prior to 6-OHDA exposure. Rats were anesthetized with ether and received an injection in the cisterna magna of 200/~g 6-OHDA.HBr (as the free base) in 20/~1 of 0.1% astorbic acid in sterile saline. The procedure was repeated 7 days later. Intracisternal control rats were treated the same as the lesioned group, but received a vehicle injection in the cisterna magna. Postoperative care of aphagic rats included observation and dietary supplements of desirable fruit and sweets. Weight gain in IC lesioned animals at the time of behavioral testing was comparable to that observed in sham-lesioned animals.

Pharmacological challenge and behavioral evaluation For the intracisternal lesions, assays were run 15-19 days after lesion, and 7 days after the behavioral challenge with apomorphine. For the bilateral lesions, assays were run 3-6 weeks after lesion, and 7 days after challenge with apomorphine. Lesioned and control rats were allowed to habituate to plastic observation cages at least 45 min prior to administration of a subcutaneous dose of 0.3 mg/kg apomorphine (as the free base). A computer-assisted observational method 27 was utilized to quantify stereotyped behavior ineluding sniffing, licking, gnawing, grooming, locomotion, and rearing. Prior to initiation of the study, observers achieved inter-rater reliability coefficients (Cohen's ~9) of 0.80 or better for each of the behavioral topographies to be scored. Observers were uninformed as to the treatment condition to which each rat was assigned. Each rat was observed for 1 min every 5 min during a period of 30-45 min after injection of apomorphine. Codes for each behavior were entered into laptop computers (TRS-80 model 100, Tandy Corp.) at 15 s intervals, every 1 min of observation. Data were analyzed as percent of 15 s scoring intervals during which a particular behavior was observed in each rat. Analysis of variance (ANOVA) was used to determine whether differences between lesioned and control groups behavior were statistically significant.

Radioreceptor assays Radioreceptor assays were performed using the following

method. Rats were killed by decapitation, the brains removed, and striata dissected on ice. Tissue was homogenized in 50 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (pH 7.5) at a concentration of 10 mg tissue/ml using a Wheaton Teflon-glass homogenizer. Each striatal homogenate from a single brain was separated into aliquots, and used for both D 1 and D 2 receptor binding assays, or for both D 1 and adenylate cyclase assays. A small aliquot (300 #1) of each homogenate was saved for quantification of dopamine and its metabolites. When dopaminestimulated adenylate cyclase was examined in addition to receptor binding, tissue was homogenized first in HEPES buffer containing EGTA, followed by centrifugation and resuspension in 50 mM HEPES. The homogenates were centrifuged for 10 min at 27,000 x g, and the supernatant discarded. The wash was repeated, and the final pellet resuspended at a concentration of 2 mg tissue/mi buffer. Culture tubes (12 × 75 mm) were used for incubation of each 1 ml sample. The 1 ml sample volume included 300 #1 of buffer, 100 #1 of [3H]SCH23390 (approx. 0.05-1.10 nM) or [3H]spiperone (approx. 0.01-0.30 nM) in buffer, 500 #1 tissue homogenate and 100/~1 of unlabeled competitor or 0.1% tartaric acid vehicle. Non-specific binding of [3H]SCH23390 was defined by addition of a final concentration of 1 #M unlabeled SCH23390, and the same concentration of unlabeled chlorpromazine was included to define non-specific binding of [3H]spiperone. Only D 2 assays were done for the unilaterally-lesioned rats due to the lack of adequate quantities of striatal tissue. Six concentrations of each ligand were assayed in triplicate to produce saturation isotherms suitable for Scatchard analysis. In some cases, both D 1 and D 2 receptor binding experiments were performed using a single striatnm, and because of limited amounts of tissue, only 5 concentrations of ligand were used. Ketanserin (final concentration 40 nM) was added to tubes containing [ 3H]spiperone to mask 5-HT 2 binding sites. A 15 min incubation at 37 °C was terminated by vacuum filtration and the tubes were rinsed with ice cold buffer, using a Skatron cell harvester (Skatron Inc., Sterling, VA). Filters were air dried and radioactivity was quantified by liquid scintillation spectroscopy using an LKB Rackbeta counter (LKB Instruments Inc., Gaithersburg, MD) at an average efficiency of 56%. Radioreceptor data were analyzed using custom Lotus 1,2,3 spreadsheets which give output identical to the program EBDA-LIGAND for single site models. All data from these studies fit a single site model with a very high correlation (r > 0.97).

analyzed using a Lineweaver-Burke plot. The apparent maximum velocity of cAMP production by adenylate cyelase in the presence of dopamine (Vm~), and the pseudo-Michaelis Menten constant (Kin) were determined from this analysis.

Determination of dopaminc and metabolite concentrations Three hundred #1 aliquots of the 10 mg/ml striatai homogenates used for the receptor binding and adenylate cyelase assays were diluted 1:1 in 0.5 M perchloric acid and frozen at -80 °C until use. Samples were prepared and analyzed using a modification of the method described by Kilts et al.2s which uses HPLC with electrochemical detection. The HPLC consisted of a Princeton model 400 EC detector (Princeton Applied Research, Princeton, NJ) equipped with a C-18 3 #m column and a mobile phase of 25% methanol, 0.05 M sodium phosphate dibasic, 0.03 M citrate and sodium octyl sulfate at a pH of approximately 3.4.

RESULTS

Psychopharmacological effects of bilateral nigral and intracisternal lesions Apomorphine

a d m i n i s t r a t i o n to rats r e c o v e r e d f r o m

b i l a t e r a l lesions o f t h e substantia n i g r a (n = 5) i n d u c e d a high d e g r e e o f e i t h e r i n t e n s e s t e r e o t y p e d g r o o m i n g (n = 2) o r g n a w i n g / n o s e p o k i n g d i r e c t e d t o w a r d t h e c a g e floor (n = 3), b e h a v i o r s n o t s e e n in s h a m - l e s i o n e d control rats (n = 6) i n j e c t e d w i t h v e h i c l e (see Fig. 1). I n at

100 6-OHDA []

Vehicle

80

Evaluation of dopamine-stimulated adenylate cyclase Evaluation of dopamine-stimulated adenylate cyclase activity was performed using the method described by Schulz et al. 45. Briefly, striatal tissue was dissected from rat brains on ice, and homogenized using a Wheaton Teflon-glass homogenizer in 100 vol of ice cold HEPES buffer containing 2 mM EGTA (pH 7.5). A 20 #1 aliquot of tissue homogenate containing approximately 0.02 mg of protein was added to 80 #1 of an ice-cold prepared reaction mixture containing 0.5 mM ATP, [32p]-ATP (0.5 #Ci), 1 mM cAMP, 0.5 mM isobutylmethylxanthine (IBMX), 70 mM HEPES buffer, 2 pM GTP, 10 mM phosphoereatine, 5 units of creatine phosphokinase and 0-100/~M dopamine. Incubation tubes for 'blank' (no tissue), basal and dopamine-stimulated adenylate cyclase activity were prepared in triplicate. The reaction was initiated by placement of the samples in a water bath at 30 °C and terminated 10 rain later by addition of 100 #1 of a 3% SDS solution. To precipitate proteins and non-cyclic nudeotides, 300/~1 of 4.5% ZnSO 4 and 300 #1 of 10% Ba(OH)2 (pH 2.3) were added to each incubation tube prior to centrifugation at 10 000 x g (5 rain). Constituents of the resulting supernatant were separated by HPLC, using a Waters Z-Module equipped with a C-18, 10 #m cartridge and a mobile phase composed of 150 mM sodium acetate and 20% methanol, with UV detection. Cyclic AMP-containing fractions were collected via a FOXY fraction collector (ISCO Inc., Omaha, NB) and [32p]-cAMP quantified by liquid scintillation spectroscopy (Rackbeta counter, LKB). The quantity of cAMP produced above basal levels at each concentration of dopamine was

i

6O

O

i 20

OIROOM

ONAW

N O S B POK[~

Behavioral Category Fig. 1. Effects of apomorphine (0.3 mg/kg) administration on the behavior of rats with bilateral 6-OHDA-induced substantia nigra lesions and sham-lesioned controls. Behavioral supersensitivitywas evidenced by potentiated grooming and gnawing/nose poking directed toward the cage floor observed in the bilaterally-lesioned rats. Error bars represent the S.E.M.

120 •

6-OHDA

[]

Vehielo

TABLE I

Evaluation of dopamine receptors in homogenates of rat striatum All values are shown -+ S.E.M. Striatal dopamine receptor binding characteristics were evaluated in homogenate binding assays as described in the Materials and Methods section. [3H]SCH23390 was used to label D 1 receptors, [3H]spiperone was used to label D 2 receptors. No significant difference in D~ receptor number or ligand affinity between lesioned and control rats was seen. A slight decrease in D 2 receptor density was seen, though there was no change in either K d.

100

80

60

Ligand

Treatment

K~ n B~a~ (fmoles/mg (nM) protein)

[3H]SCH23390

Bilat. 6-OHDA Bilat. Vehicle

5 1630 -+ 60 0.18 -+ 0.01 5 1590 _+ 30 0.22 + 0.05

[3H]Spiperone

Bilat. 6-OHDA Bilat. Vehicle

3 3

4O

20

REAR

LOC

540 +- 9 574 -+ 9

0.03 -+ 0.00 0.04 _+ 0.00

LICK

Behavioral Category Fig. 2. Effects of apomorphine (0.3 mg/kg) administration on the behavior of rats with intracisternal 6-OHDA-induced lesions and sham-lesioned controls. Behavioral supersensitivity was evidenced by substantially-increased occurrence of rearing and locomotion observed in the intracisternally-lesioned rats. Error bars represent the S.E.M.

least one case, the intense grooming progressed to bouts of self-injurious behavior, which included paw and body nibbling. The percent occurrence of these behaviors for each 1 min interval was averaged and compared with the occurrence of such behavior in control rats also treated with apomorphine (Fig. 1). Statistical analysis of these data showed a significantly elevated occurrence of gnawing (F1,1 = 10.7, P < 0.01) and nose-poking (F1,1 = 6.43, P < 0.05) in lesioned animals. Because the high frequency of occurrence of grooming was accounted for by only 2 of the 5 lesioned animals, this agonist-induced increase did not achieve statistical significance (P > 0.05). Apomorphine-treated control rats exhibited mild stereotypies including sniffing and licking the cage floor. Intracisternally-lesioned rats responded to low dose apomorphine challenge by exhibiting significantly increased levels of locomotor activity (F1,1 = 92.1, P < 0.0001) and rearing (F1,1 = 32.5, P < 0 . 0 0 ~ 1 ) , behaviors only infrequently observed in control rats (see Fig. 2). The locomotor activity displayed by the lesioned animals occurred in rapid bursts, often interrupted by rearing. Sham-lesioned rats responded to apomorphine challenge by exhibiting a high frequency of stereotyped licking, an oral stereotypy not observed in lesioned animals (F1,1 = 329.9, P < 0.0001). Intracisternally-lesioned rats that did not exhibit significant locomotor and

rearing activity following apomorphine challenge were assumed to be insufficiently lesioned, and were not included in the biochemical analyses.

Effects of bilateral nigral and IC lesion on neurotransmitter content Historically, dopamine depletion of greater than 80% has been required for behavioral supersensitivity following agonist administration to occur 55. Samples of striatal tissue from all lesioned and control rats were analyzed by H P L C with electrochemical detection to determine the percent dopamine depletion in lesioned rats. The range of dopamine depletion in the striata of bilateral nigrally lesioned rats was 85-96%, with a mean of 0.78 --- 0.16 for the lesioned rats vs 9.17 - 0.79/~g/g (S.E.M.) for the vehicle-treated controls. For the behaviorally supersensitive, intracisternally-lesioned rats, the range of depletion was 78-93%, with a mean of 1.29 - 0.02 for lesioned rats vs 9.90 --+ 0.46/zg/g (S.E.M.) for controls. Receptor changes after bilateral nigral and IC lesions: comparison with unilateral lesions As noted above, only rats which demonstrated behavioral supersensitivity to agonist challenge were used in these studies. Scatchard analysis of saturation isotherms following bilateral nigral denervation indicated no increase in D 1 receptor density (Bmax) or affinity (Table I), using the antagonist [3H]SCH23390 to characterize these receptors. In addition, no statistically significant difference in the dissociation constant (Kd) or B,~,x of D 2 (i.e. [3H]spiperone) receptors was found in bilaterally lesioned rats. There was no difference in Bmax or g d of either D 1 or D 2 striatal dopamine receptors in the behaviorally supersensitive, intracisternally-lesioned rats

~.

130

0 ~

100

120 o

~no lo0

i.20 I

I

i

7

14

28

-6.0

Time (days after lesion) Fig. 3. Effects of unilateral 6-OHDA substantia nigra lesions on striatal dopamine D E receptors. The data are plotted as the ratio of [3H]spiperone binding on lesioned vs unlesioned side (as % of unlesioned side). The data represent the mean -+ S.E.M. of 3 animals per time point, with each value being from a Scatchard analysis of 5-6 points. Three vehicle-injected control rats were run at days 14 and 28. No significant difference between left and fight sides were seen in these control rats. No significant change in the Kd (range from 0.21 to 0.37 nM) was seen for any comparison. Error bars represent the S.E.M.

I

I

I

I

-5.5

-5.0

-4.5

-4.0

Log [dopamlne (M)] Fig. 4. Dopamine-stimulated cAMP synthesis in intracisternally-lesioned rats compared to vehicle-lesioned controls, cAMP production in response to dopamine stimulation in striatal tissue from intracisternally-lesioned rats (n = 8) was virtually identical to that in control rats (n = 8). Error bars represent the S.E.M.

Dopamine-sensitive adenylate cyclase in bilateral nigral and I C lesioned rats: comparison to unilateral lesion

compared to control rats (Table II). In extensive preliminary studies, similar characterization of D 2 receptors was done in intracisternally-lesioned rats at time ranging from 3 to 12 weeks after lesion 19. In these studies, as

model Dopamine-sensitive adenylate cyclase was used as the biochemical marker of D 1 dopamine receptor function. In our tissue preparation conditions, D E receptor inhibition of adenylate cyclase activity is negligible. No difference in

with the present data, no increase or decrease in D z receptors was seen in behaviorally supersensitive rats. Consistent with earlier literature, unilateral lesions of

the ability of dopamine to stimulate cAMP formation in striatal tissue from intracisternally-lesioned, supersensitive rats was seen compared to c A M P formation in tis-

the substantia nigra caused an increase in striatal D z receptors that continued to increase through at least 1 m o n t h after lesion (Fig. 3). I n a second study (data not shown), an increase of 40.3% ( m e a n of 3 lesioned animals and 3 controls) was seen at day 14. These data are consistent with earlier literature, and demonstrate that well-documented increases in D z receptors were detectable by the techniques used.

sue from vehicle-injected controls (Fig. 4). The apparent K m and Vm~ of d o p a m i n e in stimulation of adenylate cyclase in tissue homogenates of intracisternally-lesioned and control rats were virtually identical. The K m for tissue from intracisternally-lesioned rats was 4.5 /~M and the control Km was 4.9 k~M. The apparent Vm~ was 95 pmoles/mg/min in lesioned tissue, and 91 pmoles/mg/min

.~

TABLE II

60

Scatchard analysis of D 1 and Dz dopamine receptors in striatal homogenates of 6-OHDA lesioned rats All values are shown -+ S.E.M. Homogenate binding data reveal no alteration in striatal D 1 or D 2 receptor density (Bmax) or dissociation constant (Ka) due to intracisternal lesion. Ligand

Treatment

n

B ,,~ Ka (fmoles/mg (riM) protein)

0

-6.0

I

I

I

I

-5.5

-5.0

-4.5

-4.0

Log [dopamine (M)] [3H]SCH23390

IC 6-OHDA IC Vehicle

12 12

1674 + 56 0.23 -+ 0.01 1742 - 56 0.23 -+ 0.01

[3H]Spiperone

IC 6-OHDA IC Vehicle

6 6

501 - 45 0.07 -+ 0.01 562 - 46 0.08 -+ 0.01

Fig. 5. Dopamine-stimulated cAMP synthesis in bilaterally-lesioned rats compared to vehicle-lesioned controls. The cAMP production was increased at every concentration greater than 1.25/aM in bilaterally-lesioned (n -- 4) compared to vehicle-lesioned controls (n = 4). Error bars represent the S.E.M.

in control samples. Conversely, the ability of dopamine to stimulate striatal adenylate cyclase activity was increased in bilateral nigral lesioned rats. In lesioned rats compared to controls, significantly more cAMP was produced at each concentration of dopamine greater than 1.25/~M (Fig. 5). Lineweaver-Burke analysis of the data revealed an apparent Km of dopamine-sensitive adenylate cyclase activity of 5.1/~M in lesioned rats and 7.3/tM in control striata. The Vm~ of 189 pmoles/mg protein/min in lesioned rats was significantly greater than the 126 pmoles/mg protein/min seen in control rats. It should be noted that because the rats used in the intracisternal and bilateral experimental and control groups were of different ages and different cohorts, it is not surprising that the kinetic parameters varied between the control groups. DISCUSSION Chemical and physical denervation of central dopamine neurons, as well as persistent pharmacological receptor blockade, have been shown to result in an upregulation of dopamine receptors in terminal fields. Denervation models in which this occurs include unilateral lesion of the rat substantia nigra, deafferentation of olfactory tubercles, and chronic treatment with D 1 o r D E receptor antagonists 11"17"28'32'42'43. The concomitant recovery of spontaneous behaviors, onset of pharmacological supersensitivity, and increased receptor density in such denervated rats has provided support for the hypothesis that the compensatory up-regulation of receptor systems mediates, or at least is a factor in, the increase in behavioral sensitivity to challenge with a dopamine agonist. Despite this large body of supporting data, there are also data that suggest that this correlation is not mechanistically linked to the pharmacological supersensitivity. As noted earlier, these include reports suggesting a lack of relationship between drug-induced rotation and dopamine receptor changes in the unilateral model 5°, as well as large changes in receptor density seen after chronic receptor blockade and after unilateral lesion, the former associated only with transient and weak supersensitivity 17'21'32'42"43. More directly, we had provided preliminary reports indicating that behavioral supersensitivity to agonist challenge could be seen after 6 - O H D A lesioning of adult 29 or neonatal rats 31 without a change in D 2 receptors. Since the underlying basis for these pharmacological models rests in large measure on the psychopharmacological supersensitivity, it is important to review briefly the changes that occur. An increase in locomotor activity in adult rats lesioned by intracisternal injection of

6 - O H D A has been reported following agonist challenge 4. Most behavioral studies performed using such lesioned rats have relied solely on automated equipment, and, for example, report locomotion as total photobeam crossings per unit of time following agonist challenge. While such an apparatus works well for quantification of locomotor activity, it cannot distinguish behaviors with important qualitative differences. The behavioral observation method used in this study 27 is not so confounded. In response to apomorphine, the locomotor activity of the intracisternal rats is a rapid traverse from one end of a rectangular cage to the other, punctuated by rearing; control rats tend to remain in a single location and sniff or lick the cage floor. Thus, unlike results observed with bilateral nigral lesions, apomorphine did not induce perioral stereotypies such as licking and gnawing in intracisternal rats. In the bilaterally-lesioned rats, on the other hand, apomorphine caused intense grooming or gnawing/nose poking of the cage floor. Indeed, even selfinjurious behavior was observed. This was unexpected in that such self-injurious behavior has been hypothesized to occur at low doses of apomorphine only after neonatal lesions 3. This self-injurious behavior was not seen in apomorphine-challenged intracisternally-lesioned rats. Thus, while both the intracisternal and bilateral lesion caused a profound depletion of dopamine in the striatum, it is clear that the effects of the intracisternal lesion on other terminal fields (such as the nucleus accumbens) may contribute markedly to the differences in drug response. Intracisternal and unilateral models differ in that intracisternal administration of 6 - O H D A causes widespread damage to dopamine systems throughout the brain, whereas the unilateral model primarily affects only those neurons originating in one substantia nigra. This potential confound might explain why increases in D 2 receptors are seen in the unilateral but not in the intracisternal model. This possibility was addressed by the inclusion of the bilateral model in the present comparison. Neither bilateral nor intracisternal lesions with 6-OHDA caused an increase in D 2 receptors, whereas unilateral 6 - O H D A lesions clearly had this effect. In fact, unilateral injection of 6 - O H D A into the substantia nigra has been reported to result in as much as a 70% increase in D 2 receptors in the ipsilateral vs contralateral striatum 7-10 days after lesion 3s, although more modest (but still large) effects were seen in the present data. In a similar fashion, Morelli et al. 37 have reported that unilateral 6 - O H D A lesions also caused an increase in [3H]sulpiride binding in the striatum, and a decrease in the substantia nigra. This is important to note because sulpiride and spiperone may label different populations of receptors or different states of D 2 o r D 3 receptors 2.

If the D 2 receptor up-regulation were the major mechanism mediating the receptor supersensitivity in the unilateral lesion model, then it would be expected that bilateral injection of 6-OHDA in the substantia nigra (causing lesion of both nigrostriatal tracts) should result in a similar increase in striatal D 2 receptor number compared to control rats. In the present study, such bilateral lesions did cause profound pharmacological supersensitivity, yet no increase in either Dx o r D 2 dopamine receptor density was seen in the striata of rats lesioned BIL. In addition, no increase in D 1 o r D E dopamine receptor density occurred in striata from behaviorally supersensitive rats lesioned intracisternally, consistent with earlier reports 4'29. These results cannot be due to insufficient dopamine depletions, as all the animals tested had 80% or greater depletions and were shown to have potentiated behavioral responses to dopamine agonist challenge. As noted, the lack of effect in these models contrasts with the consistent up-regulation in D E receptors in unilateral nigral lesioned animals, or after chronic treatment with dopamine antagonists (like haloperidol or SCH23390). One recent study which essentially agrees with one aspect of the present work is that of Wolterink et al. 54, also using the bilateral 6-OHDA model. While they found no evidence of up-regulation of high affinity D E receptors, the use of a lower affinity ligand ([3H] haloperidol) and the method of data analysis, make their conclusions less certain. The lack of up-regulation in the bilateral model is relevant to the issue of dopamine receptor alterations in Parkinson's disease. Increased receptor density has been reported in tissue taken post-mortem from Parkinson's patients who did not receive L-DOPA in the last weeks of life5°. Other studies, however, have failed to find an up-regulation in unmedicated patients ~°. The unilateral 6-OHDA lesion has been widely used as a model for Parkinsonism and a screen for antiparkinsonian drugs, at least in part, because of the reliable receptor up-regulation that can be obtained. Such receptor up-regulation need not be a criterion, however. Moreover, the neuropathology seen in Parkinson's patients is not unilateral but bilateral, similar to the 6-OHDA model not associated with a change in receptor density. While the lack of a change in either D 1 o r 0 2 receptor density was unexpected, examination of adenylate cyclase activity provided an additional surprising finding. Although a variety of other mechanisms have been suggested, dopamine-sensitive adenylate cyclase (i.e. the function of D 1 receptor) is the only biochemical mechanism that consistently can be shown to be evoked by occupation of dopamine receptors in terminals of the nigrostriatal system. Unilateral nigral lesion of rats by 6-OHDA injection results in increased dopamine-stimu-

lated cAMP production in the ipsilateral striatum 26'35. This finding has been reproduced in our studies (data not shown). It is unclear whether striatal D1 receptor density increases TM or remains unchanged in unilaterally lesioned rats 24'44. It is clear that unilateral denervation alters the spatial relationship between D 1 receptors and cAMP reactive neurons in the striatum 1. In the absence of changes in D 1 receptor density in either the intracisternal or the bilateral model, one might hypothesize that dopamine-sensitive adenylate cyclase activity would not be altered. We found, however, an increased synthesis of cAMP in response to dopamine in the bilateral but not in the intracisternal lesion model. Although unexpected, this finding is not unprecedented. Missale et al. 36 have reported an increased sensitivity of cAMP synthesis, without a change in D 1 receptor binding characteristics, in behaviorally supersensitive rats subchronically treated with reserpine. Zigmond and Stricker reported an increase in dopamine-stimulated cAMP production in striata of rats following 6-OHDA injection in the lateral ventricle 55. Though this lesion method might be expected to destroy dopaminergic neurons in a pattern similar to the intracisternal injection method used in this study, the 2 methodologies may produce discrete lesions. For example, Herve et aL E° report that an intact dopaminergic innervation of the prefrontal cortex is required to elicit striatal D 1 receptor supersensitivity (as measured by dopamine-stimulated cAMP production) in a unilateral lesion model. The intracisternal administration of 6-OHDA may destroy these neurons more readily than intraventricular injection, thereby accounting for the disparity. While a pharmacological supersensitivity is common to the 2 models used here, the present data make it clear that receptor changes alone are insufficient to explain the increased drug responsiveness, and may, in fact, not be an important covariate. An increase in receptor density or an increase in second messenger activity are the 2 mechanisms generally believed to mediate denervation-induced supersensitivity. The changes detected in these 3 models were disparate, and increase in receptor density in the UNI model, and increase in striatal dopamine-sensitive adenylate cyclase activity in the BIL lesioned rats, and no biochemical change detected in IC model. It may be either that other mechanisms are responsible for the supersensitivity, or that the operative pathway mediating the increased locomotor activity is not the nigrostriatal system. The other major central dopaminergic tract, the mesolimbic pathway, arises from cell bodies located in the ventral tegmental area, having terminal fields principally in the nucleus accumbens, olfactory tubercles and frontal cortex. Locomotor activity is elicited readily by do-

pamine stimulation of the nucleus accumbens or olfactory tubercles 39'4°. Though the mesolimbic tract is not fully responsible for dopamine-mediated locomotor activity, lesion of this pathway following intracisternal administration of 6 - O H D A may be responsible for the locomotor supersensitivity exhibited by these rats. Alterations in receptor density or dopamine-stimulated adenylate cyclase activity may occur in terminal areas of the mesolimbic dopamine tracts rather than in nigrostriatal areas in intracisternally-lesioned rats. Several other neural mechanisms may be involved in mediation of behavioral supersensitivity, and these include both cellular and biochemical considerations. These possibilities are currently under study. There are other anatomical considerations related to the differences in the cellular compensatory mechanisms operative following unilateral vs bilateral lesions. Axons of nigrostriatal neurons are largely ipsilateral tracts, though approximately 3% of these dopaminergic neurons project to the contralateral striatum 14'15. These contralateral projections of the substantia nigra cross at the ventral tegmental decussation, and may be destroyed by unilateral 6 - O H D A infusion 13. The major physical difference between the unilateral and bilateral nigral lesion models is that the intact nigrostriatal tract present in the unilaterally lesioned brain is absent in the bilaterally lesioned model. The ipsilateral increase in dopamine receptors in unilateral, but not bilateral, destruction of nigrostriatal neurons may be a function of the intact nigrostriatal fibers. This influence may be due to intact contralateral dopaminergic fibers crossing to the depleted striatum, or by a feedback mechanism initiated by loss of contralateral innervation in the intact striatum. A slight decrease in D 2 receptors in striata of bilaterally lesioned rat was observed, which may be interpreted as a loss of presynaptic receptors concomitant with dopaminergic terminal destruction. In summary, both the bilateral nigral and the intracisternally lesioned rats exhibited behavioral supersensitivity to apomorphine challenge, without a concurrent increase in striatal dopamine receptor number. The lack of an increase in receptor B m a x following dopaminergic denervation indicates alterations in receptor recognition REFERENCES 1 Ariano, M.A., Long-term changes in striatal D1 dopamine receptor distribution after dopaminergic deafferentation, Neuroscience, 32 (1989) 203-212. 2 Bishop, J.E., Mathis, C.A., Gerdes, J.M., Whitney, J.M., Eaton, A. and Mailman, R.B., Synthesis and in vitro evaluation of 2,3-dimethoxy-5-fluoroalkyl-substitutedbenzamides and salicylamides: high affinity ligands for CNS dopamine D E receptors, J. Med. Chem., 34 (1991) 1612-1624. 3 Breese, G.R., Baumeister, A.A., McCown, T.J., Emerick,

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Acknowledgements. This work was supported by PHS Program Grant ES01104, research Grants MH40537 and MH37404, Training Grant ES07040 (BEM), and Center Grants HD03310 and MH133127.

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