Brain Research 871 (2000) 156–159www.elsevier.com/ locate /bres

Short communication

Effects of MK-801 on D1 dopamine receptor-mediated immediateearly gene expression in the dopamine-depleted striatum

*Anindita Ganguly, Kristen A. KeefeDepartment of Pharmacology and Toxicology, University of Utah, 30 South, 2000 East [201, Salt Lake City, UT 84112-5820, USA

Accepted 18 April 2000

Abstract

Previous work indicates that intrastriatal administration of MK-801 does not completely block D1 agonist-induced gene expression indopamine-depleted rats. The present study examined the effects of systemic MK-801 on such gene expression. A low dose of MK-801 didnot affect induction of c-fos or zif268. A high dose completely blocked induction of c-fos, but only slightly suppressed zif268. The datasuggest that NMDA receptor activity may not always be necessary for D1-induced gene expression. 2000 Elsevier Science B.V. Allrights reserved.

Theme: Motor systems and sensorimotor integration

Topic: Basal ganglia

Keywords: NMDA; Basal ganglia; zif268; c-fos; Parkinson; Glutamate

The basal ganglia are subcortical structures, important completely [16,27]. Our data also indicate that induction offor motor and cognitive functions. The striatum is the main c-fos and zif268 in the intact striatum by the full D1input nucleus of the basal ganglia, receiving dopamine receptor agonist SKF 82958 is blocked by antagonists of(DA) input from substantia nigra pars compacta and NR2A subunit-containing NMDA receptors [10]. Together,glutamate input from the cortex and thalamus [4,7,12– these data indicate that D1-mediated IEG expression is14,19,24]. These inputs synapse onto spiny efferent neu- fully dependent on ongoing NMDA receptor activity in therons, and are anatomically positioned to interact with each intact striatum.other. Striatal efferent neurons regulate the output of the Data from studies conducted on DA-depleted animals,basal ganglia via striatonigral and striatopallidal pathways, however, suggest that D1 receptor-mediated IEG expres-which express the D1 and D2 subtypes of DA receptors, sion is not as dependent on NMDA receptors. Intrastriatalrespectively [5,6,17,18]. infusion of NMDA receptor antagonists has no effect on

Numerous findings provide support for interactions induction of zif268 in the DA-depleted striatum by thebetween DA and N-methyl-D-aspartate (NMDA) receptor- partial D1 agonist SKF38393 and only partially (20–40%)mediated processes in the regulation of striatal neuron attenuates c-fos expression [11]. Dopamine depletionfunction. For example, amphetamine-induced immediate therefore seems to alter the involvement of NMDA re-early gene (IEG) expression in the striata of intact animals ceptors in D1 receptor-mediated regulation of striatonigralrequires activation of D1 receptors [2,20,26]. This D1 neurons. However, in the studies on intact rats reviewedreceptor-mediated IEG expression is also dependent on above, the NMDA receptor antagonists were administeredglutamate input via NMDA receptors, as MK-801 blocks it systemically, whereas in the study on DA-depleted rats, the

antagonists were administered intrastriatally. Furthermoredifferent types of DA agonists were used in the studies*Corresponding author. Tel.: 11-801-585-7989; fax: 11-801-585-described above. Thus, the route of administration or the5111.

E-mail address: k.keefe@m.cc.utah.edu (K.A. Keefe) type of drug used could account for the discrepant observa-

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02435-5

A. Ganguly, K.A. Keefe / Brain Research 871 (2000) 156 –159 157

tions obtained from intact versus lesioned rats. Therefore, facility at the University of Utah. The purified probe was35to evaluate the hypothesis that D1 DA receptor-mediated end-labeled with S-dATP and terminal deoxynucleotidyl

IEG expression is independent of NMDA receptors in transferase (Boehringer Mannheim, Indianapolis, IN,DA-depleted animals, we examined the effects of systemic USA).NMDA receptor blockade on the induction of IEGs in All data were analyzed using the image analysis pro-6-hydroxydopamine (6-OHDA)-treated rats by systemic gram Image (NIH) as previously described [10]. Images ofadministration of the full D1 agonist SKF82958. sections from all groups in the experiment were captured

Male Sprague–Dawley rats (Charles River, Wilmington, and measured under constant lighting and camera con-MA, USA) weighing 225–250 g were used in all experi- ditions. Measurements were made over medial and lateralments. Rats were housed in groups of four in hanging thirds of the DA-depleted striatum. Data from the densito-wire-mesh cages in a temperature-controlled room on a metric analysis of film autoradiograms were analyzed with12:12 light:dark cycle. Rats had free access to food and a one-way analysis of variance for both medial and lateralwater. All animal care and experimental manipulations striatum. Post-hoc analysis was performed with thewere approved by the Institutional Animal Care and Use Tukey–Kramer test. Paired Student’s t-tests were used toCommittee of the University of Utah and were in accord- compare the effects of DA-depletion on PPE and THance with the NIH Guide for the Care and Use of mRNA in the ipsilateral versus contralateral striata andLaboratory Animals. substantia nigra, respectively, of the 6-OHDA-treated

All drugs were obtained from Research Biochemicals animals. Statistical significance was set at P,0.05.International (Natick, MA, USA). The doses of SKF82958 All animals chosen for analysis had a greater than 90%and MK-801 were calculated as the salt, whereas the doses loss of DA, as reflected by a significant reduction (.90%,of desipramine HCl and 6-OHDA were calculated as the P,0.0001) in TH mRNA in the ipsilateral substantia nigrafree base. Rats were anesthetized with sodium pentobarbi- and a significant increase (31%, P,0.0001) in PPE mRNAtal (50 mg/kg, i.p.) and then pre-treated with desipramine in the lesioned striatum compared to the contralateral side(25 mg/kg, i.p.) 30 min prior to infusion of 6-OHDA. A [21].29-gauge needle connected to a 25-ml Hamilton syringe on Systemic administration of SKF82958 (1 mg/kg, i.p.)a syringe pump was lowered into the right medial forebrain increased the expression of both c-fos and zif268 in thebundle at the following coordinates relative to bregma and medial and lateral striatum (Fig. 1). Administration of thethe skull (mm): AP 24.0, ML 11.5, DV 28.5. An aliquot low dose of MK-801 (0.01 mg/kg, i.p.) had no effect onof 2 ml of 6-OHDA (8 mg) in 0.02% ascorbic acid–0.9% the D1-induced increases (Fig. 1a and b). Administrationsaline was infused over 5 min. The canula was left in place of the high dose of MK-801 (1.0 mg/kg, i.p.) completelyfor 5 min after the infusion. blocked the D1-induced increase in c-fos expression (Fig.

Experiments were performed 3 weeks post lesion. On 1a). However, this high dose only slightly suppressed thethe day of the experiment, the rats were rehoused in plastic D1 agonist-induced increase in zif268 expression (Fig. 1b).tub cages (4–5 per cage) and transferred to the laboratory. We have shown that systemic NMDA receptor blockadeThe rats were weighed and then injected with either MK- does not fully block D1 agonist-induced IEG expression in801 (0.01 or 1 mg/kg, i.p.) or the vehicle (deionized the DA-depleted striatum. A low dose of MK-801 had nowater). Fifteen min later, each rat received an injection of effect on either D1 agonist-induced c-fos or zif268 expres-SKF82958 (1 mg/kg, i.p.) or the vehicle (deionized water). sion. A high dose of MK-801, on the other hand, markedlyControl rats received two vehicle injections. reduced c-fos expression, but only very slightly attenuated

One hour after the second injection, the rats were zif268 expression. These data stand in marked contrast toeuthanized by exposure to CO for 1 min and then our previous work in intact rats [10], as well as other2

decapitated. The brains were rapidly removed and frozen reports in the literature [16,27], demonstrating that thesein isopentane chilled on dry ice. The brains were stored at doses of MK-801 block D1 receptor-induced increases in2208C until they were cut in 12-mm sections in a cryostat both c-fos and zif268 expression. Taken together, these(Cryocut 1800, Cambridge Instruments, Germany). Sub- data indicate that the involvement of NMDA receptors insequently, slides from all animals were postfixed and D1 agonist-induced gene expression is altered after DA-processed for in situ hybridization for c-fos and zif268 as depleting brain lesions, especially in the case of zif268previously described [10]. The extent of DA depletion was expression.determined by measuring preproenkephalin (PPE) mRNA Increased levels of activated protein kinase A (PKA)in striatum and tyrosine hydroxylase (TH) mRNA in may be responsible for the lack of effect of NMDAsubstantia nigra. In the case of PPE, an antisense ribonu- receptor blockade on D1-mediated IEG expression in thecleotide probe complementary to bases 51–987 (courtesy DA-depleted striatum. Konradi et al. [22] have postulated

35of Dr. C.R. Gerfen) was synthesized using S-UTP and that under low levels of activation of PKA, increased11SP6 RNA polymerase. For the detection of TH mRNA, a Ca influx through the NMDA receptor is primarily

48-base oligonucleotide probe complementary to bases responsible for mediating gene expression. On the other1441–1488 was synthesized by the DNA/peptide synthesis hand, high levels of activation of PKA are thought to be

158 A. Ganguly, K.A. Keefe / Brain Research 871 (2000) 156 –159

Fig. 1. Effects of systemic administration of MK-801 (‘MK’; 0.01 mg/kg, i.p.; 1.0 mg/kg, i.p.) on c-fos (a) and zif268 (b) expression induced in theDA-depleted striatum by systemic administration of the D1 DA receptor agonist SKF82958 (‘SKF’; 1.0 mg/kg, i.p.). MK-801 was injected 15 min beforethe injection of SKF82958. The control rats were given two injections of the vehicle 15 min apart. The rats treated with MK-801 alone received MK-801followed 15 min later by the vehicle. Rats receiving SKF82958 alone received the vehicle followed 15 min later by an injection of SKF82958. All ratswere sacrificed 1 h after the second injection. The graphs show average gray values (6S.E.M.; arbitrary units) in medial and lateral thirds of themid-striatum obtained from densitometric analysis of film autoradiograms. The numbers in parentheses indicate the number of animals in each group.*P,0.05 vs. control; 1P,0.05 vs. SKF82958.

capable of increasing transcription factor activity indepen- DA depletion. Further experiments to delineate the altera-dent of NMDA receptors. We think that the same phenom- tions occurring in these pathways as a consequence of DAenon occurs in the DA-depleted striatum, since D1 DA depletion may provide a greater insight into the basis ofreceptors are supersensitive under such conditions [3,25]. symptoms associated with DA-depleting disorders such asPresumably, the dose of D1 agonist used produced more Parkinson’s disease.pronounced activation of PKA in the DA-depleted animals, In conclusion, our data indicate that in the DA-depleted

11overshadowing the contribution of Ca influx through striatum, the involvement of NMDA receptors in D1 DANMDA receptors. Other studies from our lab also show receptor-mediated regulation of striatonigral neuron func-differential involvement of NMDA receptors in D2 DA tion is altered. Further studies are warranted to understandreceptor antagonist-induced gene expression depending on the signal transduction pathways involved and the altera-the degree of gene induction [1,9]. Further studies examin- tions occurring in these pathways as a consequence of DAing the second messenger pathways involved in D1-in- depletion.duced IEG expression in the DA-depleted striatum shouldhelp clarify the basis for the differential involvement ofNMDA receptors in D1 receptor-mediated changes in Referencesstriatal gene expression.

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