SYNAPSE 10:206-216 (1992)

Repeated D1 Dopamine Receptor Agonist Administration Prevents the Development of Both D l and D2 Striatal Receptor Supersensitivity Following Denehation XIU-TI HU AND FRANCIS J. WHITE Department of Psychiatry, Wayne State University School of Medicine, and Neuropsychopharmacology Laboratory, Lafayette Clinic, Detroit, Michigan 48207

KEY WORDS

Caudate-putamen, Nigrostriatal pathway, Dl/D2 synergism

ABSTRACT Following 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopamine (DA) pathway, rat caudate-putamen (CPu) neurons are supersensitive to the inhibitory effects of both D1 and D2 dopamine (DA) receptor selective agonists. In addition, both the necessity of D1 receptor stimulation for D2 agonist-induced inhibition and the synergistic inhibitory effects of D1 and D2 agonists are abolished by denervation. The present study attempted to determine the relative roles of D1 and D2 DA receptors in the development of denervation supersensitivity to DA agonists and the “uncoupling” of functional interactions between the receptors following 6-OHDA lesions of the nigrostriatal DA pathway. Beginning on the day after a n intraventricular 6-OHDA (or vehicle) injection, groups of rats received daily injections of either the selective D1 receptor agonist SKF 38393 (8.0 mgkg, s.c.), the D2 agonist quinpirole (0.5 mgkg, s.c.), or saline for 7 days. On the day following the last agonist injection, rats were anesthetized and prepared for extracellular single cell recording with iontophoretic drug administration. Daily administration of quinpirole selectively prevented the development of D2 receptor supersensitivity, whereas daily administration of SKF 38393 prevented the development of both D1 and D2 receptor supersensitivity. In addition, D1, but not D2, agonist treatment prevented the loss of synergistic inhibitory responses typically produced by 6-OHDA lesions. Behavioral observations revealed similar effects; daily injections of SKF 38393, but not quinpirole, prevented contralateral rotational responses to the mixed D1/D2 agonist apomorphine (1.0 mgkg, s.c.) in rats with unilateral 6-OHDA lesions of the nigrostriatal pathway. After a 4-week withdrawal from repeated D1 agonist treatment, both supersensitive inhibitory responses of CPu neurons and contralateral rotations to apomorphine were evident, indicating that the preventative effects on DA receptor supersensitivity were not permanent. These findings indicate that continued agonist occupation of striatal D1 DA receptors following DA denervation not only prevents the development of D1 DA receptor supersensitivity but also exerts a similar regulation of D2 receptor sensitivity. INTRODUCTION Denervation of the nigrostriatal dopamine (DA) neuronal system, produced by 6-hydroxydopamine (6OHDA)or other neurotoxins, results in supersensitivity of DA receptors and enhances behavioral responses to DA receptor agonists. There is clear evidence that the density of striatal D2 DA receptors is increased by 6-OHDA lesions (Creese and Snyder, 1979; Neve et al., 1984; Savasta et al., 1987b; see also Seeman, 1980, for review) and that this effect is probably due to a significant increase in striatal D2 receptor mRNA (Gerfen et al., 1990; Mansour et al., 1990). In contrast, considerable discrepancy exists within the literature regarding @ 1992 WILEY-LISS,INC.

striatal D1 DA receptor up-regulation following DA denervation. Various investigators have reported that D1 receptors are either increased (Buonamici et al., 1986; Porceddu e t al., 1987), decreased (Ariano, 1988; Marshall et al., 1989), or unchanged (Altar and Marien, 1987; Filloux e t a]., 1987; Graham et al., 1990; Savasta et al., 1987a) in the DA-depleted CPu. Supporting a decrease in D1 DA receptor density is the recent finding of diminished striatal D1 DA receptor mRNA levels following 6-OHDA lesions (Gerfen et al., 1990). Disagreement also exists regarding whether DA-stimuReceived July 2,1991; accepted in revised form July 31,1991.

D1 REGULATION OF D2 RECEPTOR SENSITIVITY

lated cyclic adenosine monophosphate (CAMP)production (via D1 receptors) is increased by denervation (Kreuger et al., 1976; Mishra et al., 1974; Parenti et al., 1982; Rosenfeld et al., 1979; Von Voigtlander et al., 1973). In contrast to biochemical studies, behavioral studies have revealed an apparent sensitization of D1 receptors following 6-OHDA lesions. Setler et al. (1978) first reported that although the selective D1 DA receptor agonist SKF 38393 failed to elicit hyperactivity in normal rats, it produced contralateral turning in rats with unilateral 6-OHDA lesions of the nigrostriatal DA system. Many subsequent investigators have confirmed this observation (see Clark and White, 1987, for review). Both hyperactivity and oral stereotyped behavior are produced by selective D1 receptor agonists in rats with bilateral 6-OHDA lesions (Arnt, 1985b; Breese and Meuller, 1985)or followingrepeated reserpine administration (Arnt, 1985a). Perhaps the most interesting behavioral consequence of DA denervation is the “uncoupling”of typical synergistic interactions between D1 and D2 DA receptor subtypes (see Clark and White, 1987, for review). This phenomenon was first reported by Arnt (1985a,b), who found that the ability of the selective D1 DA receptor antagonist SCH 23390 to block the unconditioned behavioral effects of selective D2 agonists in normal rats (Amt, 1985a; Breese and Meuller, 1985; Longoni et al., 1987; Molloy and Waddington, 1985; Walters et al., 1987) was no longer evident in either 6-OHDA-lesioned or reserpine-pretreated rats. Electrophysiological studies have provided evidence for both D1 and D2 DA receptor supersensitivity following 6-OHDA lesions of the nigrostriatal system. In normal rats, local administration of either D1 or D2 selective agonists produces primarily inhibitory effects on striatal neurons recorded extracellularly (BickfordWimer et al., 1990; Hu and Wang, 1988,1989; Hu et al., 1990; Wachtel et al., 1989; White and Wang, 1986; White, 1987; White et al., 1987). In addition, simultaneous administration of D1 and D2 agonists produces a synergistic inhibition of most striatal neurons (Hu and Wang, 1988; Hu et., 1990; White, 1986; White and Wang, 1986). In fact, stimulation of D1 receptors by endogenous DA is necessary for (enables) D2 agonistinduced inhibition (Hu et al., 1990; Johansen et al., 1991; Wachtel et al., 1989; White, 1987 1. In rats with 6-OHDA lesions (90% DA depletion), the normal inhibitory effects produced by either D1 or D2 agonists on striatal neurons are significantly enhanced (Hu et al., 1990). In addition, these lesions also result in an apparent uncoupling of D1D2 receptor interactions within the striatum (Hu et al., 1990). Thus DA denervation abolishes the quantitative synergistic interaction between D1 and D2 receptors and relieves D2 receptors from the requirement of D1 receptor stimulation (Hu et al., 1990).

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The mechanisms underlying the uncoupling of D1 and D2 DA receptors following DA denervation remain to be determined. The present study was designed to investigate this issue using both electrophysiological and behavioral techniques. The first objective was to determine whether selective sensitization of D1 or D2 DA receptor-mediated neuronal responses could be achieved in 6-OHDA lesioned rats. Therefore, we attempted to prevent denervation supersensitivity of either the D1 or D2 DA receptor by repeatedly administering a selective agonist following 6-OHDA lesions of the nigrostriatal DA system. The second objective was to determine whether the synergistic electrophysiological interactions between D1 and D2 receptors would be abolished if only one receptor was rendered supersensitive. Finally, we determined whether the mixed D1D2 DA agonist apomorphine would produce rotational behavior in unilateral 6-OHDAlesioned rats if only the D1 or D2 receptor was supersensitive. MATERIALS AND METHODS Subjects Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) with 250-350 g initial body weight were used in all experiments. Rats were housed (two per cage), with food and water available ad libitum, in a colony room maintained at a constant temperature (21-23°C) and humidity (40-50%) on a 12:12 hr light/ dark cycle (on 07:00/off 19:OO). Nigrostriatal6-OHDA lesions: Electrophysiological studies For electrophysiological studies, the selective catecholamine neurotoxin 6-OHDA (Ungerstedt, 1968) was used in combination with desipramine HC1 pretreatment (25 mg/kg, i.p., 30 min prior to surgery) to prevent the high-affinity uptake of the toxin into noradrenergic terminals (Breese and Traylor, 1970). In addition, the monoamine oxidase inhibitor pargyline (40mgkg, i.p.; Sigma, St. Louis, MO) was also administered 30 min before the injection of 6-OHDA to potentiate the toxic effect of 6-OHDA (Breese and Traylor, 1970). Rats were anesthetized with methohexital sodium (40mgkg, i.p.) and secured in a stereotaxic frame (Activational Systems, Inc., Warren, MI). An incision was made and a burr hole was drilled in the skull over the lateral ventricle at bregma and 1.5 mm lateral to the sagittal sinus. Rats received a single dose of 6-OHDA (100 pg free base in 5 pl vehicle solution) via a Hamilton microliter syringe into the right ventricle (4.3 mm below the cortical surface), according to the atlas of Paxinos and Watson (1986).The injection rate was 1pl/min and the needle was maintained in position for 5-10 min after injection to permit diffusion of the drug away from the injection site. Incisions were closed with stainless steel autoclips (Clay Adams), and the area was infiltrated with the long-acting local anesthetic carbocaine hydro-

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X.-T. HU AND F.J. WHITE

chloride (2%).Rats in the sham-lesioned control group received a single injection of vehicle solution (0.1% ascorbic acid in sterile 0.9% NaC1). All surgical procedures were performed in strict accordance with the Guiding Principles in the Care and Use of Animals of the Society for Neuroscience. Rats with the initial behavioral effects of 6-OHDA lesions (aphagia, adipsia) were fed a palatable wet mash to enhance recovery. Repeated agonist treatment In an attempt to prevent the development of denervation supersensitivity of either D1 or D2 DA receptors following 6-OHDA lesions, separate groups of rats received either the selective D1 receptor agonist SKF 38393 (8.0 mgkg, s.c.) or the selective D2 receptor agonist quinpirole (0.5 mgkg, s.c.)' once daily for 1 week. Doses were based on previous behavioral results from our laboratory (White et al., 1988, 1990). A separate control group received equivalent volumes of normal saline. Injections began 24 hr after surgery, and rats were prepared for electrophysiological recordings 24-48 hr after the last injection. Separate groups of rats received identical treatments but were tested 4 weeks after the last agonist or saline injection (5 weeks postlesion). Single unit recording and microiontophoresis Procedures for extracellular recordings from single CPu neurons and microiontophoretic administration of glutamate, SKF 38393, and quinpirole have been described in considerable detail in our previous publications (He et al., 1990; Wachtel et al., 1989). Briefly, rats were anesthetized with chloral hydrate (400 mgkg, i.p.1 and mounted in a stereotaxic apparatus. Body temperature was maintained a t 36-37.5"C with a thermostatically controlled heating pad and a lateral tail vein was cannulated for administration of additional anesthetic as required. Five-barrel glass micropipettes were pulled and broken back under a microscope (5-8 p,m tip) to achieve an in vitro impedance of 1-3 MO, measured at 135 Hz. The center barrel was used for recording and was filled with a 2 M NaCl solution saturated with 1%fast green dye. One side barrel was filled with a 2 M NaCl solution for automatic current balancing, whereas a second sidebarrel was always filled with I-glutamatic acid monosodium salt (10 mM, pH 8) for activating quiescent CPu neurons. The remaining side barrels contained SKF' 38393 HCl(10 mM, pH 4)and quinpirole HCl(10 mM, 'During the course of these experiments, a new D1-like DA receptor (D5) and two new D2-like DA receptors (D3 and D4) were identified with molecular cloning techniques(Sokoloff et al., 1990; Sunahara et al., 1991; Van To1 et al., 1991). The D5 receptor exhibits a high affinity for SKJ? 38393, whereas both the D3 and D4 receptors exhibit high affinity for quinpirole. Thus it might be technically inaccurate to refer to these agonists as selective. However, given the lack of information regarding functional roles for any of the new receptors and the likelihood that they represent subtypes of the more firmly established D1 and D2 receptors, we have continued to refer to SKF 38393 and quinpirole as D1 and D2 selective agonists, respectively.

pH 4). Retaining currents of 8-10 nA were applied to drug barrels between ejection periods. The impedance of the side barrels was typically between 15 and 50 MO. Micropipettes were hydraulically advanced through the lateral CPu (8.7-8.9 mm anterior t o lambda, 3.0-3.3 mm lateral, and 3.5-5.5 mm ventral). Most CPu neurons either were quiescent o r fired at extremely slow rates (under one spikehec) and were thus activated to fire at four to seven spikeshec by adjusting the ejecting current for glutamate. A few neurons were spontaneously active and were studied without glutamate activation. As described previously, CPu neurons appeared to be of two discernible types (Niesenbaum et al., 1988; Skirboll and Bunney, 1979; White et al., 1987), one exhibiting an apparent biphasic, negative-positive waveform (type I cell) and the other exhibiting positivenegative waveforms (type I1 cell). Because we have failed to detect significant differences between these cell types with respect to their responsiveness to D1 and D2 agonists (Wachtel et al., 1989; Hu et al., 1990), the data from both cell types were pooled for our comparisons. Electrical signals were passed through a high-impedance amplifier and filter, displayed on an oscilloscope, monitored by an audio amplifier, and led into a window discriminator for detection of individual action potentials. Integrated rate histograms were generated by the window discriminator and plotted on a polygraph recorder. Digital counts of firing rates were also obtained. Determination of drug effects on CPu neurons The responses of CPu neurons to microiontophoretic administration of DA agonists were determined by comparing the total number of spikes occurring before onset of any drug administration (basal spontaneous or glutamate-driven firing rate) to that occurring during the period of drug ejections (for detail, see Hu et al., 1990). For glutamate-driven cells, data are collected only during the glutamate application. To compare the sensitivity of CPu neurons to SKI? 38393 and quinpirole in DA-depleted and sham-lesioned control rats, currentresponse curves (1-64 nA) were determined for each neuron tested in both control and DA-depleted rats. Histology At the end of each electrophysiological experiment, the final recording site was marked by passing a 25 pA cathodal current through the recording barrel for 15 min to deposit fast green dye. Rats were then perfused with 0.9%NaC1, followed by 10%buffered formalin for 15 min. Serial coronal sections were cut at 50 pm intervals and stained with cresyl violet and neutral red. The dye spot was observed under a light microscope and served as a reference point for the location of all cells investigated.

D1 REGULATION OF D2 RECEPTOR SENSITIVITY

Nigrostriatal6-OHDA lesions: Behavioral studies Because intraventricular administration of 6-OHDA not only results in substantial depletion of striatal DA in the injected hemisphere but also induces significant depletion of the contralateral CPu (Hu et al., 1990))this treatment might diminish the extent of positional bias probed by rotational measures. Thus, for behavioral studies, 6-OHDA (8 pg free base in 4 p1) was injected into the median forebrain bundle (MFB; 4.5 mm anterior to lambda, 1.6 mm lateral, and 7.9 mm ventral). All surgical and recovery procedures were as described above. Behavioral observations On the day following surgery, rats were randomly assigned to three treatment groups. As in the electrophysiological studies, each group received seven daily injections with either saline (n = 9), SKF 38393 (n = 13)) or quinpirole (n = 11). On the day following the last injection, each rat received a single injection of the nonselective (mixed) DUD2 receptor agonist apomorphine (1 mgkg, s.c.). Full contralateral body rotations (360”)were measured for 60 min beginning 5 min after the injection. Rotational behavior was measured in circular (18-inch diameter) bowls, with sloped floors to facilitate rotational movements. Following a 4-week drug “wash-out” period (i.e., 5 weeks postlesion), rats were again tested for apomorphine-induced rotation. In two cases, rats tested in behavioral studies were subsequently used for electrophysiological tests 24 hr after the behavioral experiment. Measurements of DA depletion On the day following the behavioral test (9 days postlesion), a subset of rats (four per group) was sacrificed for biochemical determination of DA levels within the lesioned CPu. Both 6-OHDA-lesioned (MFB) animals and sham-lesioned controls were used such that any possible effect of the apomorphine injection was negligible. An additional group of four rats was sacrificed 5 weeks after the lesion for biochemical determination. Levels of DA were determined by high-pressure liquid chromatography with electrochemical detection, as detailed previously (Hu et al., 1990). The lateral ventricle 6-OHDA protocol used in this study has been shown to produce approximately 88% DA depletion 7 days postlesion (Hu et al., 1990). Statistical analysis Comparisons between the current-response curves generated with iontophoretic administration of DA agonists in control and pretreated rats were conducted with a multivariate analysis of variance (MANOVA).Posthoc comparisons of specific means were made with the Newman-Keuls test. All other comparisons were made with Student’s t test.

209

RESULTS Biochemistry Unilateral lesions of the nigrostriatal DA system effected by 6-OHDA administration into the MFB resulted in more than 90% reduction in DA levels in the ipsilateral CPu when measured either 1 or 5 weeks postlesion (Table I). Combination of daily repeated treatment (1week) with either SKF 38393 or quinpirole did not produce significant effects on the 6-OHDAinduced DA depletion as shown by the similar levels of depletion in saline pretreated rats. Electrophysiology: Denervation supersensitivity A total of 67 histologically verified rat CPu neurons (15 spontaneously active and 52 glutamate evoked) were studied in the various groups. Consistent with previous observations (Hu and Wang, 1988; Hu et al., 1990)) the great majority of tested neurons (n = 64) exhibited apparent negative/positive waveforms, characteristic of type I striatal cells, as previously defined (Nisenbaum et al., 1988; Skirboll and Bunney, 1979; White et al., 1987).Because of their similar responses to administration of SKF 38393 and quinpirole (Wachtel et al., 1989; Hu et al., 1990) and the limited number of type I1 neurons in each experimental group, the results obtained from type I and I1 neurons were combined in the present data analysis. As was previously demonstrated (Hu and Wang, 1988; Hu et al., 1990; Wachtel et al., 19891, iontophoretic administration of both SKF 38393 and quinpirole resulted in a biphasic current-response effect on most CPu neurons in sham-lesioned control rats. At low currents (50% basal firing) the activity of most CPu neurons tested (n = 11/13 and n = 10/13 cells, respectively), whether spontaneously active or glutamateevoked (Figs. 1A and 2). The remaining cells still showed an inhibitory response to iontophoretic SKF 38393 and quinpirole, although the degree of inhibition (2040%)was less than 50%. Nine days followingthe 6-OHDAlesion, the responses of CPu neurons to iontophoretic administration of both SKF 38393 and quinpirole were significantly altered. Two major changes were observed. First, the enhanced excitatory responses to glutamate during administration of low currents of SKF 38393 or quinpirole were almost completely eliminated (n = 0/12 and n = 2/12, respectively; see Figs. 1 and 2). Second, the inhibitory effects of both SKF 38393 and quinpirole on CPu neurons were significantly enhanced [F(1,23) = 20.69, P < 0.005 and F(1,23) = 24.22, P < 0.002, respectively] compared with those observed in sham-lesioned controls (Figs. 1,2). Consistent with previous findings (Hu

210

X.-T. HU AND F.J. WHITE TABLE I. Levels of dopamine (ng/mg tissue) in the CPu of rats following 6-OHDA lesions (median forebrain bundle injections)

Group

Number

Dopamine

Percent depletion

4 4 4 4 4

10.0 f 1.9 0.4 f 0.2* 0.2 f 0.02* 0.5 f 0.1* 0.3 f 0.1*

0 96.0 98.0 95.0 97.0

Control (sham lesion) 6-OHDA saline (1 week) 6-OHDA SKF 38393 (1 week) 6-OHDA Quinpirole (1 week) 6-OHDA (4 weeks withdrawal from 1 week saline treatment)

+

+ +

* P < 0.01 t test).

UJ

100

0 W

Y

O

Fig. 1. Representative cumulative rate histograms illustrating the responses of CPu neurons to iontophoretic administration of the selective D1 receptor agonist SKF 38393 and the selective D2 receptor agonist quinpirole in rats that had received either a sham lesion (A) or a 6-OHDA lesion plus daily injections of either saline (B), SKF 38393 (C), or quinpirole (D) for 1 week. Note that, a t low ejection currents, both SKF 38393 and quinpirole facilitated neuronal activity evoked by glutamate in a sham-lesioned rat. At higher currents, both SKF 38393 and quinpirole effectively depressed neuronal activity (A). The inhibitory responses of a CPu neuron to both SKF 38393 and quinpirole were markedly enhanced in a saline-treated, DA-denervated rat (B) com-

pared with those in a sham-lesioned control rat (A).However, 1weekof daily SKF 38393 treatment (8 mgkg, s.c.) prevented the enhanced inhibitory neuronal responses to iontophoresis of SKF 38393 and quinpirole (compare A, B, and C), while daily quinpirole treatment (0.5 mgkg, s.c.) prevented the supersensitive inhibitory response to iontophoretic quinpirole but not to SKF 38393 (compare A, B, and D). E: A marked enhancement of inhibitory neuronal responses to SKF 38393 and quinpirole was also observed in a rat 5 weeks postlesion (compare A, B, and E).Lines and numbers represent the duration of iontophoretic current and the amount of current in nA, respectively,

D1 REGULATION OF D2 RECEPTOR SENSITIVITY 120

211

responses of CPu neurons in rats that had received 6-OHDA into the ventricle or into the MFB.

A

100 0

2

80 60

m 4 a?

0

20

A-A Sham 0-0B-OHDA+SKF

(1

0-0 6-OHM+ouin (

a-0 A-A

6-OHM+Soline 6-OHM ( 5 ~ k )

0 1

10 SKF-38393 CURRENT (nA)

100

120r 100 0

80

G

a

Y

6o

A-A

Sham

0-0 B-OHM+SKF

20

0-0 &OHM+Quin

a-0 A-A

O'....t

6-OHM+Salin 6-OHM (5wk) . . '

1

'

. . . ' ' . I

'

'

...'..A

10 QUlNPlROLE CURRENT (nA)

100

Fig. 2. Current-response curves showing the inhibitory effects produced by iontophoretic administration of the selective D1 receptor agonist SKF 38393 (A) and the selective D2 receptor agonist quinpirole (B) on CPu neurons in sham (vehicle&and 6-OHDA-lesionedrats. CPu neurons recorded in saline-treated, DA-denervated rats were significantly more sensitive to the inhibitory effects of both SKF 38393 and quinpirole (12 cells each) compared with sham-lesioned rats (13 cells each). However, the enhanced inhibitory CPu responses to both SKF 38393 and quinpirole (10 cells each) were prevented following daily administration of SKF-38393 (8.0 mgkd, s.c.). In contrast, daily quinpirole injection (0.5mgkg, s.c.) prevented the enhancement of inhibitory responses to quinpirole but failed to affect the supersensitive response to SKF 38393 (10 cells each) compared with sham-lesioned rats. In addition, there was no significant difference in the enhanced inhibitory CPu responses to either SKF 38393 or quinpirole between neurons in rats 9 days (12 cells in saline group) and 5 weeks (12 cells) after 6-OHDA injection. Each data point represents the mean percentage inhibition ? SEM.

et al., 19901,these supersensitive inhibitory effects were observed on both spontaneously active and glutamateevoked cells. Significantly enhanced inhibitory effects of SKF 38393 [F(1,23) = 18.26, P < 0.0011 and quinpirole [F(1,23) = 29.88, P < 0.0011 were also evident 5 weeks postlesion compared with sham-lesioned controls (Figs. l E , 2). In fact, there were no significant differences between the responses observed at 9 days and 5 weeks (n = 12 cells/agonist) postlesion for either SKF 38393 [F(1,22) = 0.92, P > 0.31 or quinpirole [F(1,22) = 0.79, P > 0.31. Among the 12 CPu neurons recorded 5 weeks after 6-OHDA, four were recorded from two rats that had received MFB 6-OHDA injections and exhibited apomorphine-induced contralateral rotation (below). There was no obvious difference between the

Electrophysiology: Effects of agonist treatments As was expected, the supersensitive responses of CPu neurons to iontophoretic administration of SKF 38393 were prevented by daily injections of this D1 agonist (Fig. 1C).Current-response curves (Fig. 2A) revealed no significant difference in the inhibitory efficacy of SKF 38393 observed in 6-OHDA-lesioned, SKF 38393treated rats (n = 10 cells) compared with the shamlesioned, saline-treated control group [F(1,21) = 3.77, P > 0.051 and a significantly decreased inhibitory response compared with the 6-OHDA-lesioned, salinetreated control groups [F(1,20) = 32.33, P < 0.0011. In addition, CPu neurons recorded from SKF 38393treated rats also exhibited the facilitation of glutamatedriven activity when low currents of SKF 38393 were ejected (n = 6/10),an effect observed in normal or shamlesioned rats but not in 6-OHDA-lesioned rats that had received daily saline treatments (see above). Unexpectedly, daily administration of SKF 38393 to 6-OHDA-lesioned rats also prevented the supersensitive responses of CPu neurons to the D2 agonist quinpirole (Fig. 1C). Current-response curves (Fig. 2B) revealed a significantly decreased inhibitory effect of quinpirole in 6-OHDA-lesioned, SKF 38393-treated rats compared with the 6-OHDA-lesioned, salinetreated control group [F(1,20) = 33.61, P < 0.0011. In fact, while there was no overall significant difference in the quinpirole current-response curve observed in the 6-OHDA-lesioned, SKF 38393-treated rats and the sham-lesioned, saline-treated control group [F(1,21) = 2.84, P > 0.051, the inhibition produced by the highest quinpirole current was significantly reduced (P< 0.05) by the SKI? 38393 treatment. As with D1 agonist challenge, CPu neurons recorded from SKF 38393-treated rats also exhibited the facilitation of glutamate-driven activity when low currents of quinpirole were ejected (n = 9/10). In addition, three neurons exhibited no inhibitory response to high currents of quinpirole; two of them were not affected ( 6 1 0 % inhibition), and one showed an excitatory response to administration of quinpirole. In contrast to the results obtained with daily stimulation of the D1 receptor, daily administration of the D2 selective agonist quinpirole selectively prevented supersensitive responses of CPu neurons (n = 10) to iontophoretic quinpirole; i.e., there was no heterologous regulation of D1 receptor sensitivity (Fig. 1D). Current response curves (Fig. 2A,B) revealed no significant difference between the inhibitory effects of quinpirole observed in 6-OHDA-lesioned,quinpirole-treated rats compared with the sham-lesioned, saline-treated control group [F(1,21) = 0.18, P > 0.051 and a significant decrease in sensitivity to quinpirole compared with the 6-OHDA-lesioned,saline-treated control group [F(1,21)=

212

X.-T. HU AND F.J. WHITE A

6.57, P 0.051, but a significant difference compared with the sham-lesioned control group [F(1,21) = 6.57, P< 0.021.

Electrophysiology: D1/D2 interactions As in our previous study (Hu et al., 1990), 6-OHDAinduced denervation of the CPu relieved D2 receptors from the necessity of D1 receptor activation as evidenced by the significant inhibitory effects of quinpirole (Fig. 2B). In addition, 6-OHDA eliminated the ability of SKF 38393 to potentiate quinpirole-induced inhibition of CPu neurons (Hu et al., 1990). Interestingly, prevention of D1 and D2 receptor supersensitivity by repeated treatment with SKF 38393 also prevented the loss of D1/D2 synergism (n = 10 cells) as evidenced by the ability of low iontophoretic currents of SKF 38393 (4 nA) t o potentiate significantly [F(1,18)= 9.91, P< 0.011 the quinpirole current-response effect (Fig. 3). In contrast, repeated treatment with quinpirole in 6-OHDA-lesioned rats (n = 10 cells) failed to prevent the loss of D1D2 synergism. Current-response curves showed no significant differences between the inhibitory effects of quinpirole with and without simultaneous administration of SKF 38393 [F(1,18) = 0.28, P > 0.05; Fig. 41. Behavior A single dose of the mixed DliD2 receptor agonist apomorphine (1mg/kg, s.c.)induced significant [t(12) = 6.06, P 0.051 in response to apomorphine when quinpirole-treated rats were compared with the saline-treated control group (Fig. 5). Following a 4-week withdrawal period from repeated agonist treatments, all 6-OHDA-lesionedrats showed strong contralateral turning responses to the

120 r

0-0 W I N 20

-

0 - 0 QUlN

+ S K F (4 nA)

WINPIROLE

a 0

5 MN

Fig. 3. Comparison of responses of CPu neurons to iontophoretic administration of quinpirole with and without concurrently applied SKF-38393in SKF 38393-treated (8.0 mgkg, s.c.),DAdenervated rats. A Low (subinhibitory) currents of SKF 38393 (4 nA) significantly potentiated the inhibitory effects of quinpirole on the same CPu neurons. B: A representative cumulative-rate histogram showing an enhanced inhibitory response to combined administration of SKF 38393 (4nA) and quinpirole (1-64 nA) on a glutamate-driven CPu neuron compared with that induced by iontophoretic quinpirole alone in a 6-OHDA-lesioned rat with 1 week of repeated SKF 38393 injections.

second apomorphine challenge (Fig. 41, such that there were no significantdifferencesbetween the saline-treated group and either SKF 38393-treated [t(8)= 0.43, P > 0.051 or quinpirole-treated [t(9) = 0.06, P > 0.051 rats. DISCUSSION We have recently reported that denervation of the nigrostriatal DA system produced by 6-OHDA leads to enhanced inhibitory effects of both selective D1 and D2 DA receptor agonists on single rat CPu neurons (Hu et al., 1990). In addition, such denervation alleviated the necessity of D1 receptor stimulation for D2 agonistinduced inhibition (enabling) and abolished the normal synergistic inhibition produced by coadministration of D1 and D2 agonists. The present studies have replicated and extended the previous findings by demonstrating that D1 receptor supersensitivity is required for the development of D2 receptor supersensitivity and the uncoupling of D1D2 receptor interactions. The first novel finding of the present study is that denervation supersensitivity of neuronal responses within the rat CPu could be prevented by initiating repeated daily treatment (for 7 days) with DA receptor agonists 24 hr after the introduction of a unilateral

D1 REGULATION OF D2 RECEPTOR SENSITIVITY

213

6-OHDA LESION WITH ONLY OUlNPlROLE TREATMENT

A

B

120 r

20

.

m-.

QUlN

+ SKF (4 nA)

W

Y

CURRENT (nA)

Fig. 4. Comparison of responses of CPu neurons to iontophoretic administration of quinpirole with and without concurrently applied SKF 38393 in quinpirole-treated (0.5 m a g , s.c.), DA-denervated rats. Low currents of SKF 38393 (4 nA) failed to potentiate the inhibitory effects of quinpirole on the same CPu neurons tested (n = 10 cells;

P > 0.05) (A), whether they were spontaneously active (B) or gluta-

6-OHDA lesion of the nigrostriatal DA system. Thus CPu neurons in 6-OHDA-lesioned rats that had received daily repeated administration of the selective D1 receptor agonist SKI? 38393 failed to exhibit supersensitive inhibitory responses to this agonist administered iontophoretically. Similar prevention of supersensitive D2 agonist-induced inhibition was observed following repeated daily treatment with the selective D2 receptor agonist quinpirole. When rats were allowed a 4-week withdrawal from the chronic agonist treatment (5 weeks postlesion), supersensitivity was evident, indicating that the prevention of supersensitivity was not a permanent effect. Taken together, these findings suggest that, under certain experimental conditions, exogenous DA agonists, a t appropriate doses, can supply relatively normal levels of activation of the respective receptors in the near absence of endogenous DA. The unexpected finding of the present experiments is that daily repeated treatment with a selective D1 receptor agonist also prevented the ability of DA denervation to induce a functional supersensitivity of D2 DA receptors located on CPu neurons. Thus CPu neurons from 6-OHDA lesioned, SKF 38393-treated rats exhibit normal sensitivity not only to this D1 agonist but also to iontophoretic administration of quinpirole. Moreover, prevention of D1 receptor supersensitivity by repeated SKF 38393 treatment also preserved the normal ability of this D1 agonist to potentiate D2 agonist-induced inhibition of CPu neurons when both agonists were

locally administered by microiontophoresis. No such synergism was apparent in either quinpirole- or salinetreated, DA-denervated rats. Therefore, it appears that D1 DA receptors, in some way, regulate the development of D2 receptor denervation supersensitivity and that D1 receptor supersensitivity is intricately involved in the uncoupling of adjacent synergistic D1D2 interactions. Whether selective D2 receptor supersensitivity might also cause an uncoupling of D1D2 receptor synergism cannot be determined from the present studies, since no such selectivity was afforded by the repeated SKF 38393 injections to 6-OHDA-lesioned rats. The ability of D1 receptors to regulate D2 receptor sensitivity and to uncouple functional D1D2 interactions was not reciprocated, since daily repeated administration of quinpirole failed to prevent the development of functional D1 receptor supersensitivity and D1D2 uncoupling in 6-OHDA-lesionedrats. In this regard, the present results resemble previous findings indicating that D1 receptor stimulation in normal rats is required for certain D2 agonist-induced electrophysiological and behavioral effects (Hu et al., 1990;Johansen et al., 1991; Wachtel et al., 1989; Walters et al., 1987; White, 1987;) but that similar effects of D1 agonist do not require D2 receptor stimulation (Wachtel et al., 1989; White et al., 1988). A behavioral parallel of the electrophysiological results was also observed in the present studies. Rats that received unilateral 6-OHDA lesions of the nigrostriatal

mate-driven (C). In addition, after approximately 15 min of administration of SKF 38393 (4 nA), quinpirole still suppressed CPu activity (C), suggesting that continuing ejection of this D1 agonist did not desensitize the D2 receptor-mediated inhibitory CPu response.

214

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Fig. 5. Daily repeated treatment with the selective D1 agonist SKF 38393 prevented apomorphine-stimulated contralateral turning in rats with unilateral 6-OHDA lesions of the medial forebrain bundle. Apomorphine (1mgkg, s.c.) induced strong contralateral rotation in daily saline-treated rats (n = 9) 9 days after 6-OHDA injection but failed to produce a similar effect in sham-lesioned controls (n = 5, **P < 0.01). Daily administration of the selective D1 agonist SKF 38393 (8 mgkg, s.c.,for 1week) almost completely prevented apomorphine-induced rotation (n = 13) compared with saline-treated, DAdenervated rats (#P < 0.01). In fact, there was no significant difference in apomorphine-induced turning response between shamlesioned controls and 6-OHDA-lesioned rats combined with daily injections of SKF 38393. However, 1-week of daily treatment with the selective D2 agonist quinpirole (0.5mgikg, s.c.) failed to attenuate the apomorphine-induced rotation (n = 11)compared with saline-treated, DA-denervated rats. The supersensitive behavioral responses to apomorphine lasted at least 5 weeks in daily saline- and quinpiroletreated, 6-OHDA-lesioned rats (n = 4 rats). Following a 4-week period of withdrawal from daily agonist injections, the contralateral turning response to apomorphine was similar in all three groups.

DA pathway and subsequent daily treatment with SKF 38393 failed to exhibit contralateral rotation t o the nonselective DUD2 DA agonist apomorphine. In contrast, 6-OHDA-lesionedrats that received daily quinpirole injections still exhibited contralateral rotation to apomorphine. The ability of repeated SKF 38393 treatment to prevent apomorphine-induced rotation was not due solely to prevention of D1 receptor supersensitivity, since rotational responses to apomorphine require stimulation of both D1 and D2 DA receptors (Arnt, 1985a; Arnt and Hyttel, 1985; Herrera-Marschitz and Ungerstedt, 1985). Moreover, ongoing experiments in our laboratory have shown that repeated SKF 38393 treatment can also prevent contralateral rotational responses to quinpirole in 6-OHDA-lesionedrats (Hu and White, unpublished findings). It should also be noted that the lack of rotational responses to apomorphine could have occurred due to the ability of repeated D1 agonist treatment to cause supersensitivity of D1 receptors in the intact hemisphere (White et al., 1990).Thus, D1 receptor supersensitivity might have existed in both the lesioned and unlesioned CPu. However, D1 agonistinduced supersensitivity is not evident immediately following the treatment regimen but requires a withdrawal period (White et al., 1990). Moreover, given the electrophysiological evidence for prevention of D1 su-

persensitivity on the lesioned side by the repeated SKF 38393 treatment, supersensitive D1 receptors in the intact CPu should have caused ipsilateral rotation. Several other laboratories have recently reported on the ability of repeated or continuous treatment with selective D1 and D2 DA receptor agonists to regulate rotational behavior in rats with unilateral lesions of the nigrostriatal pathway. However, unlike the present paradigm, all of these studies initiated repeated agonist treatment subsequent to the development of 6-OHDAinduced supersensitivity. These experiments have provided mixed findings with respect to homologous regulation of D1 and heterologous regulation of D2 receptor function following unilateral 6-OHDA lesions. For example, repeated daily injections of SKF 38393 diminished contralateral circling induced by this D1 agonist but increased D2 agonist-induced turning (Rouillard et al., 1988). Continuous administration of SKF 38393 markedly reduced contralateral rotation induced by the same drug, but not that of D2 agonists, in 6-OHDAlesioned mice (Winkler et al., 1988). Following repeated injections with 1-DOPA,contralateral rotation induced by SKF-38393in supersensitive 6-OHDArats was abolished, whereas rotation elicited by D2 selective or mixed DUD2 agonists was enhanced (Engber et al., 1989). When viewed together with the present results, these previous behavioral studies suggest that, while D1 receptors appear to regulate the development of D2 receptor supersensitivity, once it has occurred, D2 receptors are resistant to attempts to normalize their sensitivity. Given the potential clinical relevance of these restoration studies, it will be important to study functional sensitivity of striatal D1 and D2 receptors with electrophysiological techniques using agonist administration subsequent to the development of denervation supersensitivity. While the present results demonstrate that D1 receptor stimulation can affect the functional sensitivity of D2 receptors, others have shown that D1 receptor tone can influence D2 receptor density. For example, repeated stimulation ofDl receptors with the selective D1 receptor agonist SKF-38393 can suppress 6-OHDAinduced up-regulation of D2 receptors in rats (Rouillard et al., 1988). Similar findings have been reported (Falardeau et al., 1988; Gagnon et al., 1990)in monkeys pretreated with the DA neurotoxin 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP). Repeated injections of SKJ? 38393 have also been reported to speed the rate of recovery of D2 receptors inactivated by alkylation (Cameron and Crocker, 1988). The mechanisms by which D1 receptors might exert heterologous regulation of D2 receptor density and functional supersensitivity are important issues for further investigation. In summary, this series of experiments has demonstrated that daily repeated administration of the selective D1 receptor agonist SKF 38393 prevented the development of both D1 and D2 receptor supersensitiv-

D1 REGULATION OF D2 RECEPTOR SENSITIVITY

ity and the uncoupling of adjacent functional interactions between these two receptor subtypes within the rat CPu. These results indicate that D1 DA receptors play a critical role in the development of denervation supersensitivity. Further investigation into the mechanisms underlying these phenomena will be of considerable importance in the future development of new therapeutic agents and methods for the treatment of Parkinson’s disease.

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treatment ofMPTP monkeys with dopamine D-1 and/or D-2 receptor agonists. Eur. J. Pharmacol., 178:115-120. Gerfen, C.R., Engber, T.M., Mahan, L.C., Susel, Z . , Chase, T.N., Monsma, F.J., Jr., and Sibley, D.R. (1990) D1 and D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons. Science, 250:1429-1432. Graham, W.C., Crossman, A.R., and Woodruff, G.N. (1990)Autoradiographic studies in animal models of hemi-parkinsonism reveal dopamine D2 but not D1 receptor supersensitivity. I. 6-OHDA lesions of ascending mesencephalic dopaminergic pathways in the rat. Brain Res., 514:93-102. Herrara-Marschitz, M., and Ungerstedt, U.(1985) Effect of the dopamine D-1 antagonist SCH 23390 on rotational behavior induced by apomorphine and pergolide in 6-hydroxydopamine denervated rats. ACKNOWLEDGMENTS Eur. J . Pharmacol., 109:349-354. Hu, X.-T., Wachtel, S.R., Galloway, M.P., and White, F.J. 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