A. CHOCYK*, A. CZYRAK, K. WÊDZONY

DOPAMINE D1-LIKE RECEPTORS AGONIST SKF 38393 INCREASEScFOS EXPRESSION IN THE PARAVENTRICULAR NUCLEUS OF THEHYPOTHALAMUS � IMPACT OF ACUTE AND CHRONIC COCAINE

Laboratory of Pharmacology and Brain Biostructure, Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland

The present study indicates that activation of dopamine D1-like receptors byadministration of SKF 38393 leads to dose-dependent (doses: 5, 10 and 20 mg/kg)increases in the expression of cFos proteins in the rat paraventricular nucleus of thehypothalamus (PVN). This effect was abolished by administration of SCH 23390, adopamine D1-like receptor antagonist (0.5 and 1 mg/kg, given 30 min before SKF38393 - 10 mg/kg), suggesting that the apparent effect is specific for activation ofdopamine D1-like receptors. Expression of cFos after SKF 38393 (10 mg/kg) wasobserved in some, but not all, CRF-immunoreactive neurons, as well as in smallportion of oxytocin- but not vasopressin-immunoreactive neurons (double-immunofluorescence experiments). There were also certain populations of nucleithat showed expression of cFos but did not co-localize with the above markers. Wealso found that both acute and repeated (once daily for 5 consecutive days) exposureto cocaine (25 mg/kg) attenuated the induction of cFos expression triggered by SKF38393 when administered 24 hours after single or the last dose of cocaine (25mg/kg). Attenuation was observed at the same level after single and chronic exposureto cocaine, indicating a rapid functional down-regulation of dopamine D1-likereceptors that are resistant to subsequent doses of cocaine. These data provideevidence for the functional role of dopamine D1-like receptors in the PVN andindicate a functional adaptation of dopamine D1-like receptors following a singledose of cocaine without further progression of adaptation or resistance of D1-likereceptor-mediated genomic function in the course of repeated cocaine intake.

K e y w o r d s : cocaine, cFos, corticotropin-releasing factor, dopamine D1-like receptors,oxytocin, paraventricular nucleus of the hypothalamus, vasopressin

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2008, 59, 3, 425�440

www.jpp.krakow.pl

INTRODUCTION

Exposure, either acute or repeated, to addictive psychostimulants evokesseveral adaptations in brain function, adjusting its activity to the presence ofexogenous substance (1, 2). These adaptations are responsible for developmentand maintenance of addiction (2, 3). The effects are not only limited to the braincircuitry responsible for reward (4, 5), but they also affect other systems, such asthe endocrine system (6 - 8). Alterations in the release of such hormones asACTH, corticotropin-releasing factor (CRF), and corticosterone (cortisol inhumans) have a great impact on the susceptibility and the severity of addictionand its maintenance and relapse to drugs of abuse after cessation of their intake(9 - 11). The major center that controls homeostasis of the organism byintegrating brain function with endocrine and autonomic systems is theparaventricular nucleus of the hypothalamus (PVN) (12, 13). The PVNconstitutes the central part of two neuroendocrine axes, including thehypothalamo-pituitary-adrenal (HPA) axis and the hypothalamo-pituitary-thyroidsystem (14). These systems can be further differentiated by the synthesis andrelease of the two principal hormones, CRF and TRH (thyrotropin-releasinghormone). These neuropeptides are produced in the parvocellular compartment ofthe PVN, and they are engaged in regulation of stress responses (CRF) andmetabolism (TRH) (14). The PVN possesses other neurosecretory neurons aswell, including oxytocin and vasopressin neurons that are localized mainly inmagnocellular compartment of the PVN (14). Oxytocin, a peptide that stimulatessmooth muscle contraction, plays an important role in parturition and lactation(14). It is also responsible for expression of maternal and sexual behaviors.Vasopressin exerts water retentive effects by regulating the water balance of anorganism, and it mediates peripheral vasoconstriction. Beside neurosecretoryprojections, the PVN also has numerous nonendocrine synaptic contacts with thebrain stem, spinal cord, and forebrain regions of the brain (many of them beingreciprocal) (12, 14, 15). Activity of PVN neurons can be regulated by manyneurotransmission systems, such as noradrenergic (16), serotonergic (17, 18),glutamatergic (19, 20) and GABA-ergic systems (21). Growing literatureproviding functional and anatomic data indicate that dopaminergic system canalso modulate the functions of the PVN (22 - 24), although the exact role andsignificance of dopamine action on PVN neurons is poorly understood. Sincepsychotropic drugs, including substances of abuse, act via dopaminergicneurotransmission (3 - 5, 25), it is conceivable that they may have substantialimpact on PVN function and subsequently influence the homeostasis of somephysiological processes (2). Available data point towards the important role fordopamine D1 receptors in modulation of activity of the PVN. It is well knownthat the dopamine D1-like receptor agonist, SKF 38393, given peripherally andcentrally (into the third ventricle, i.e. in proximity to the PVN) activates the HPAaxis, which thereby increases levels of ACTH and corticosterone (22, 24). The

426

above effects are blocked by pretreatment with dopamine D1-like receptorantagonist, SCH 23390 (22). SKF 38393 also potently increases CRF mRNAlevels in PVN neurons (23). Moreover, it is well know that cocaine, apsychostimulant drug that enhances dopamine neurotransmission, stronglyaffects the activity of the HPA axis (26 - 28), and it is also known that dopamineD1 receptors are involved in this phenomenon (26, 29). Acute administration ofcocaine increases levels of ACTH and corticosterone (cortisol) (26 - 28). Chronicexposure to cocaine produces more heterogeneous effects in the HPA axisresponse depending on the schedule of administration (30 - 32), which suggeststhat repeated cocaine treatment leads to adaptive changes in PVN neurons,possibly at the level of dopamine D1 receptors among other sites.

There is a growing interest in the potential role of dopamine D1 receptors inthe mechanism of dependence and its pharmacotherapy (33 - 36). Whiletransmission mediated by dopamine molecules requires activation of both D1-likeand D2-like receptors (1), the former family of receptors have an exceptionalimpact on the discriminative and reinforcing effects of cocaine in rat and primates(35, 36). It has been found that self-administration of cocaine is greatly reducedin dopamine D1 but not D2 receptor knock-out mice compared to wild typeanimals (37 and references given there). These findings indicate the importantand almost exclusive role of dopamine D1 receptors in addiction. Although givenobservation are limited to effect mediated by reward circuitry and not to the othereffects mediated by activation of dopamine D1 receptors, they simply may beextended to the HPA axis. Alterations in the function of the HPA axis evoked bycocaine via activation of dopamine D1 receptors and subsequent alterations inACTH and corticosterone release have an important impact on addiction (26, 29,38). Corticosterone, or stress-induced secretion of corticosterone, enhancesvulnerability to cocaine (11, 39, 40). On the contrary, metyrapone reducescocaine-induced locomotor activity and relapse of cocaine self-administration(41). Surgical adrenalectomy completely abolishes the acquisition of i.v. cocaineself-administration without affecting food maintained responses, similar to theeffects found in the D1 receptor knock-out mice (37, 42). These effects should notbe limited only to the role of dopamine D1-like receptors in the control of ACTHand corticosterone release. CRF also participates in the acquisition as well as themaintenance and relapse of cocaine intake (43, 44). However, there is debate overwhether activation of CRF secretion by the PVN neurons is responsible for abovementioned effects (10, 44).

Available data are not limited only to involvement of dopamine D1-likereceptors in the release of CRF, ACTH and corticosterone; they also show thatsecretion of oxytocin can be regulated by pharmacologically specific activationof dopamine D1-like receptors in lactating rats (45).

These data, along with the potential role of CRF, ACTH and corticosterone invarious aspects of addiction (9, 11, 41, 43, 46) and data showing the presence ofdopamine D1 receptors in the PVN (47 - 49) prompted us to investigate whether

427

the dopamine D1-like receptor agonist, SKF 38393 is capable of activating PVNneurons in D1-like receptor-specific manner. To achieve an appropriate level ofanatomically accurate activation of dopamine D1-like receptors, we measured theinduction of cFos proteins using immunohistochemical methods. Moreover, todetermine whether CRF, oxytocin and vasopressin neurons, primary endocrineneurons of the PVN, are activated by the dopamine D1-like receptor agonist, weexamined the co-localizations of SKF 38393-induced cFos with the above-mentioned neuropeptide markers of PVN neurons. Finally, we studied how acuteand repeated cocaine pretreatment affects the function of dopamine D1-likereceptors, as measured by SKF 38393-induced cFos expression in the PVN. Inother words, we determined whether rapid or sustained enhancement ofdopaminergic neurotransmission could evoke any adaptive changes in dopamineD1-like receptors. To accomplish these tasks, rats were injected with cFoschallenging dose of SKF 38393 2 or 24 hours after acute or repeated cocainetreatment.

MATERIALS AND METHODS

Animals and drug treatments

Experiments were carried out on male Wistar rats weighing 200-250 g (bred locally in thefacilities of the Institute of Pharmacology, Polish Academy of Sciences in Kraków), housed ingroups of six under controlled conditions (12h light/dark cycle, 22 ± 2°C). Rats had free access tostandard laboratory food and water. Rats were habituated to the animal room for 1 week. Tominimize stress, the animals were accustomed to handling and intraperitoneal (i.p.) andsubcutaneous (s.c.) injections (0.9% saline, 2 ml/kg) for 4 days before the experiments. Allexperimental protocols were approved by the Local Bioethics Commission of Institute ofPharmacology, Polish Academy of Sciences in Kraków, and they met the internationally acceptedprinciples for the care and use of laboratory animals.

During acute treatments, rats were injected s.c. with R(±)-SKF 38393 hydrobromide (5, 10 and20 mg/kg; Sigma RBI) or with vehicle (0.1% ascorbic acid in saline; 1 ml/kg).

In experiments with dopamine D1-like receptor antagonism, rats were subjected to an acute s.c.administration of either R(+)-SCH 23390 hydrochloride (0.5 and 1 mg/kg, Sigma-RBI) orrespective vehicle (saline, 1 ml/kg) 30 min before SKF 38393 (10 mg/kg) injection.

In experiments with cocaine pretreatment, rats were injected acutely or repeatedly (once dailyfor 5 consecutive days) with either cocaine hydrochloride (25 mg/kg, i.p., Merck) or vehicle (saline,2 ml/kg). Then, 2 or 24 hours after single or the last dose of cocaine, the challenging dose of SKF38393 (10 mg/kg) was given.

Immunohistochemistry of cFos

Two hours following drug treatments, rats were deeply anesthetized and transcardially perfusedwith saline, followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Brains were removed fromthe skulls, postfixed in 4% paraformaldehyde in PBS for 24 h at 4°C, and sectioned on a vibratome(VT1000S, Leica) into 50 µm-thick coronal sections at the level of the paraventricular nucleus of

428

the hypothalamus. The sections corresponding to the Bregma levels from �1.60 to �2.12 mm (50)were collected.

Free-floating sections were washed in 0.01 M PBS (pH 7.4) and incubated for 30 min in PBScontaining 0.3% H2O2 ,0.1% Triton X-100. Sections were then rinsed and transferred to the blockingbuffer (5% normal goat serum, 0.1% Triton X-100 in PBS) for 1h. After the blocking procedure thesections were incubated (48 h, 4°C) with anti-cFos rabbit polyclonal antibodies (1:20 000;Oncogene Research Products: Ab-5). Antibodies were diluted in 3% normal goat serum, 0.1%Triton X-100 in PBS. After washing in PBS, the sections were further incubated with a biotinylatedgoat anti-rabbit IgG (1:500, 1h; Vector Laboratories), followed by incubation with an avidin-biotin-peroxidase complex (1:200, 1h; Vector Laboratories). An immunochemical reaction was developedusing a glucose oxidase-diaminobenzidine-nickel method (51). Finally, the sections were mountedonto gelatin-coated slides, air-dried, dehydrated and coverslipped using Permount (FisherScientific) as a mounting medium.

The sections were examined and photographed using a Leica DM LB microscope equipped witha Cool Snap FX (Roper Scientific Photometrics) digital camera. Two sections (representing ca.�1.80 and �1.95 mm bregma levels, according to Paxinos (50)) from each rat were analyzed. Theabove anatomical levels of the PVN are known to contain highly representative oxytocin- andvasopressin-immunoreactive magnocellular neurons, as well as CRF-immunoreactive parvocellularneurons (52, 53). cFos-immunoreactive nuclei were counted on the captured images using MetaMorph (Universal Imaging Corp.) software. Calculations were done bilaterally yet separately foreach side of the PVN based on the assumption that each inspected side (from two 50 µm-thicksections) represented a slightly different level of the PVN, due to limitation in the method of brainsectioning. Then, the number of immunoreactive nuclei was averaged for each rat.

Double-labeling immunofluorescence

Two hours following drug treatments, rats were anesthetized and perfused, and the brains weresectioned as above. For the double-labeling of cFos and CRF, free-floating sections were washed in0.01 M PBS and blocked in 3% normal donkey serum and 0.1% Triton in PBS (1 h), and they werethen incubated (48 h, 4°C) in a cocktail of the following primary antibodies: rabbit anti-cFos (1:10000; Oncogene Research Products: Ab-5) and goat anti-CRF (1:200; Santa Cruz Biotechnology: sc-1759). After several washes in 0.01 M PBS, the sections were further incubated for 1 h withbiotinylated donkey anti-goat IgG (1:500; Jackson ImmunoResearch, PA), followed by a 1 hincubation with a mixture of Cy2-conjugated donkey anti-rabbit IgG (1:100, JacksonImmunoResearch, PA) and Texas Red-conjugated streptavidin (2 µl/ml; Jackson ImmunoResearch,PA) to visualize cFos and CRF, respectively. For the double-labeling of cFos with oxytocin andvasopressin, the sections were blocked in 5% normal goat serum. Subsequently, the sections wereincubated (48 h, 4°C) in cocktails of the following primary antibodies: rabbit anti-cFos (OncogeneResearch Products: Ab-5) with mouse anti-oxytocin (1:50, kindly provided by Dr. H. Gainer) andrabbit anti-cFos with mouse anti-vasopressin (1:50, as well kindly provided by Dr. H. Gainer). Afterseveral washes, the sections were further incubated (1 h) with a mixture of Cy2-conjugated goatanti-rabbit and Texas Red-conjugated goat anti-mouse antibodies (1:100, JacksonImmunoResearch) to visualize cFos and oxytocin/vasopressin, respectively. Finally, the sectionswere mounted onto gelatin-coated slides and coverslipped using PBS-buffered glycerol.

The sections were examined and photographed using a Leica DM LB microscope equipped withan epifluorescent attachment, narrow-band filters and a Cool Snap FX (Roper ScientificPhotometrics) digital camera. Six subsequent sections (representing the Bregma levels from �1.60to �1.90 mm) from six control and six SKF 38393-treated rats were analyzed. Co-localizations ofimmunoreactivity for cFos and CRF, as well as between cFos and oxytocin and cFos and

429

vasopressin, were ascertained by overlaying the captured images of each fluorescent Cy2- or TexasRed-labeled markers with MethaMorph software. The number of single- and double-labeledneurons was counted from these overlays (for further details and methodological consideration see:(54, 55)). Over 500 cells from each rat were studied.

Statistical analysis

Data (coming from two independent experiments) were expressed as the mean ± SEM andanalyzed for overall significant differences between treatment groups by an analysis of variance(ANOVA) followed by Duncan�s post-hoc analysis. P < 0.05 was considered statisticallysignificant.

RESULTS

Effects of dopamine D1-like receptor agonist SKF 38393 on cFos expression in the PVN

Treatment with SKF 38393 (5, 10 and 20 mg/kg) dose-dependently increasedcFos immunoreactivity in the PVN (Fig. 1). cFos-immunoreactive nuclei, labeledin the PVN after SKF 38393 treatment, were distributed mainly in the

430

Fig. 1. Effect of SKF 38393treatment on cFos expression inthe paraventricular nucleus of thehypothalamus (PVN). SKF 38393was given in doses of 5, 10 and 20mg/kg s.c., and cFos immuno-reactivity was measured 2 h afterthe drug or vehicle injections. Theobtained values are presented asthe mean ± SEM. *P < 0.05compared to the control (vehicle-treated) group (one-way ANOVA,followed by Duncan�s post-hocanalysis); n=6. For representativephotomicrographs showing SKF38393-induced cFos expressionsee Fig. 2B, 4D.

parvocellular part of the PVN but were also present in the magnocellular divisionof the studied structure (Fig. 2B, 4D). Similar induction of cFos expression wasalso observed after SKF 81297 - another dopamine D1-like receptor agonist ofgreater potency/efficacy than SKF 38393 (56, 57) - when administered at dosesof 0.5 and 1 mg/kg but not 0.1 mg/kg (data not shown).

Effect of dopamine D1-like receptor antagonist SCH 23390 on cFos expressioninduced in the PVN by SKF 38393 treatment

It was shown that treatment with the dopamine D1-like receptor antagonistSCH 23390 at a dose of 0.5 mg/kg alone did not change cFos immunoreactivityin the PVN. However, pretreatment with the abovementioned dose of SCH 23390attenuated the SKF 38393-induced increase in cFos immunoreactivity in the PVN(Fig. 2). SCH 23390 alone at a higher dose of 1 mg/kg slightly elevated cFosexpression in the PVN but given prior to SKF 38393 completely blocked SKF38393-induced increase in cFos immunoreactivity in the PVN (Fig. 2). It was alsofound that pretreatment with SCH 23390 (1 mg/kg) attenuated the SKF 81297-induced increase in cFos immunoreactivity in the PVN (data not shown).

431

Fig. 2. Effect of SCH 23390 pretreatment on SKF 38393-induced cFos expression in theparaventricular nucleus of the hypothalamus (PVN). SCH 23390 was given in doses of 0.5 and 1mg/kg, s.c. 30 min before SKF 38393 (10 mg/kg, s.c.) injection, and cFos immunoreactivity wasmeasured 2 h after SKF treatment. Left panel: the obtained values are presented as the mean ± SEM.*P < 0.05 compared to the control group that received vehicle injections; #P < 0.05 compared tothe group treated with SKF 38393 alone (two-way ANOVA, followed by Duncan�s post-hocanalysis); n=6. Right panel: photomicrographs showing cFos immunoreactivity in the PVN of ratstreated with the vehicle � A, SKF 38393 � B, SCH 23390 (1 mg/kg) � C, and SCH 23390administered 30 min before SKF 38393 injection � D. Scale bar in A � D: 100 µm.

Studies of co-localizations of the cFos and CRF, oxytocin, and vasopressinimmunoreactivities in PVN neurons after SKF 38393 treatment

Treatment with SKF 38393 (10 mg/kg) did not change the number of CRF-,oxytocin- and vasopressin-immunoreactive neurons in the PVN, which weremeasured 2 h after the injection (data not shown). It was also observed that SKF38393-induced cFos immunoreactivity was present in some populations (34 ±5%) of CRF-immunoreactive neurons (Fig. 3A, B, C) and in small populations (8± 3%) of oxytocin-immunoreactive neurons (Fig. 3D, E, F). Furthermore, it was

432

Fig. 3. Photomicrographs showing double-labeling immunofluorescent histochemistry visualizingcFos and CRF � A, B, C, cFos and oxytocin � D, E, F, and cFos and vasopressin � G, H, I,performed on rats treated with SKF 38393 (10 mg/kg, s.c.). The presence of theseimmunoreactivities was studied 2 h after SKF injection. Photomicrographs C, F and I representoverlays of photomicrographs A+B, D+E and G+H, respectively. cFos immunoreactivity isvisualized in green � B, E, H, while CRF � A, oxytocin � D, and vasopressin � G appear as redstaining. Arrows point to the examples of double-labeled neurons, while arrowheads indicatesingle-labeled cFos nuclei, and asterisks point to examples of single-labeled oxytocin � D, F andvasopressin neurons � G, I. Scale bar in A � I: 20 µm.

found that 25 ± 3% of cFos-immunoreactive nuclei present in the parvocellularregion of the PVN co-localized with CRF immunoreactivity, and 26 ± 6% ofcFos-immunoreactive nuclei present in the magnocellular region of the PVN co-localized with oxytocin immunoreactivity. No co-localization between cFos andvasopressin immunoreactivities was observed in PVN neurons after SKF 38393treatment (Fig. 3G, H, I). In vehicle-treated rats, the degree of co-localizationswas insignificant in all studied cases.

The effects of acute an repeated cocaine treatment on SKF 38393-induced cFosexpression in the PVN

It was found that neither acute nor repeated (once daily for 5 consecutive days)cocaine (25 mg/kg) pretreatment changed constitutive expression of cFos in thePVN. Moreover, it was found that neither acute nor repeated cocaine pretreatmentchanged SKF 38393-induced cFos expression in the PVN when SKF 38393challenge dose (10 mg/kg) was administered 2 h after a single or the last cocaineinjection (data not shown). However, when SKF 38393 was given 24 h after asimilar cocaine pretreatment, the attenuation of SKF-induced cFosimmunoreactivity in the PVN was observed. (Fig. 4).

433

Fig. 4. Effects of acute and repeated cocaine treatment on SKF 38393-induced cFos expression inthe paraventricular nucleus of the hypothalamus (PVN). Cocaine (25 mg/kg) was injected acutelyor repeatedly (once daily for 5 consecutive days) and SKF 38393 (10 mg/kg) was given 24 h afterthe abovementioned cocaine administration. cFos expression was measured 2 h after SKFtreatment. Left panel: the obtained values are presented as the mean ± SEM. *P < 0.05 comparedto the control group that received vehicle injections; #P < 0.05 compared to the control grouptreated with SKF 38393 alone (two-way ANOVA, followed by Duncan�s post-hoc analysis); n=6.Right panel: photomicrographs showing cFos immunoreactivity in the PVN of rats treated with thevehicle � A, acute cocaine � B, repeated cocaine � C, SKF 38393 administered 24 h after vehicleinjection � D, and SKF administered 24 h after acute � E as well as 24 h after the last dose ofrepeated cocaine treatment � F. Scale bar in A � F: 100 µm.

DISCUSSION

The major findings of the present study indicate that activation ofdopaminergic D1-like receptors, by administration of SKF 38393, dose-dependently increased cFos protein expression in rat PVN. This effect seems tobe specific for dopamine D1-like receptors since we demonstrated that it wasabolished by SCH 23390, a compound that functions as a dopamine D1-likereceptor antagonist (58). Our pharmacological findings are in line with studiesthat show genetic deletion of dopamine D1 receptors abolishing the ability ofselective dopamine D1-like receptor agonist (59) or indirect agonist(amphetamine) (60) to evoke cFos protein expression. We also found out thatSKF 38393-induced cFos expression occurred in certain populations of CRF- andoxytocin-immunoreactive neurons but not in vasopressin-immunoreactiveneurons. We also found numerous cFos positive nuclei that were not co-localizedwith the above-mentioned markers of PVN neurons. By the size of nuclei andtheir localization, the non-co-localized nuclei should be classified as nuclei ofGABA-ergic interneurons or nuclei of TRH-positive neurons, which were notanalyzed in this study. Finally, we found that acute cocaine evoked an attenuationof cFos expression stimulated by dopamine D1-like receptor agonist (when SKF38393 was given 24 h after cocaine) and that this effect was not altered byrepeated exposure to cocaine. These data indicate that there is neither tolerancenor sensitization to the effect evoked by a single dose of cocaine at the level ofdopamine D-1-like receptor function, as measured by expression of cFos proteins.

The appearance of cFos is regarded as a marker of neuronal activation (61).Thus, in functional terms, our data provide evidence that stimulation of dopamineD1-like receptors, which were demonstrated previously in the PVN by expressionof D1 receptor mRNA (49, 62) and distribution of D1 receptor protein (47, 63),may activate neurons in the PVN. The obtained results may also indicate that SKF38393-induced expression of cFos is probably direct. In other words, cFosexpression resulted from activation of D1-like receptors in the PVN. However, itis not possible to rule out an indirect effect, such as increased cFos expression byaltered activity of neurons innervating the PVN, situated outside of the PVN andcontrolled by dopamine D1-like receptors (64). The appearance of cFos proteinfollowing SKF 38393 administration suggesting activation of PVN neurons is inline with data showing that SKF 38393 is capable of enhancing release of CRF,ACTH, corticosterone (22, 24) and oxytocine (45).

Although our findings provide additional functional evidence for the role ofdopamine D1-like receptors in the control of the PVN activity, we should mentionthat the pattern of cFos activation is slightly different form the pattern ofdopamine D1 receptor distribution in the PVN. SKF 38393-induced cFos-positivenuclei have been observed mainly in the parvocellular part of the PVN, while themagnocellular part of the PVN is mainly occupied by dopamine D1 receptors(47). This discrepancy may result from the fact that available data on the

434

localization of D1 receptors are based on the specific markers (mRNA encodingD1 receptors (49, 62), or specific antibody recognizing an epitope of D1 receptors(47)) characterizing one pool of receptors forming dopamine D1 receptor familyand excluding dopamine D5 receptors. SKF 38393 activates both dopamine D1and D5 receptors (56), so its spectrum of action is wider then activation of D1receptors only. Selective pharmacological tools activating separately dopamineD1 and D5 receptors are not yet available.

The apparent desensitization of dopamine D1-like receptors observedfollowing acute cocaine pretreatment at the level of cFos induction stimulated bySKF 38393 in the PVN is in line with similar effects observed in the nucleusaccumbens and striatum (65). In the nucleus accumbens, a single dose of cocaineevoked rapid tolerance of cFos expression (65). Our data and the above findingsindicate that the function of dopamine D1-like receptors, as measured byexpression of cFos after acute and repeated cocaine in the PVN, is regulated inthe similar fashion as in neuronal circuitry associated with the reward system.Such desensitization of dopamine D1-like receptors, which we and othersobserved at the level of transcription factors contrasts data obtained inelectrophysiological studies analyzing SKF 38393-induced responses ofaccumbal and cortical neurons after repeated exposure to cocaine (66 - 68). In theabove paradigm, supersensitivity of dopamine D1-like receptors (withoutconsistent data about changes in the number of dopamine D1, D5 receptors andmRNA encoding their synthesis) has been demonstrated (67) and correlated withthe appearance of sensitization to the locomotor stimulant effects of cocaine (69).It is worthwhile to note that such contrasting effects are observed on differentfunctional levels. In other words, observed effects likely involve surfacedopamine receptors in electrophysiological studies and alterations in the genetranscription controlled by cFos protein that activates neurons in other studies. Itis conceivable that such down-regulation of transcription activity mayparadoxically lead to the enhancement of the function mediated by activation ofsurface dopamine D1-like receptors.

It is of interest that the functional down-regulation of dopamine D1-likereceptors observed in our study following acute administration of cocaine andmeasured as capability of SKF 38393 to induce cFos expression in the PVN wasrelatively slow since it was not observed 2 hours following acute cocaineadministration but 24 later. Such slow onset of adaptation cannot be based onlyon cocaine�induced alternation in neurotransmission since the duration ofcocaine action on dopamine and serotonin reuptake lasts approximately 90 min(1), suggesting the involvement of factors operating slowly. Such slow factorsinduced by alteration in dopaminergic or serotonergic transmission are not knownas yet. However, another transcription factor, FosB/∆FosB protein, may be ofinterest (70, 71). In our recent study, using immunocytochemistry and westernblot techniques, we found that acute cocaine treatment induced a relatively long-lasting (at least 72 h) expression of FosB/∆FosB in the PVN, whereas repeated

435

cocaine administration caused accumulation of FosB/∆FosB in PVN neurons(among others - in CRF-immunoreactive neurons) (72). Accumulation of ∆FosBduring cocaine administration, and persist appearance of this protein in the brainlong after cessation of drug administration suggest that the above transcriptionfactor is responsible for the appearance of adaptive changes concomitant with theprocess of addiction and responsible for its maintenance (73). There is alsoanother important consequence of the long-lasting appearance of FosB/∆FosBafter acute cocaine in structures involved in reward that may be extended to thePVN. Appearance of FosB/∆FosB may suppress induction of cFos since,depending on experimental paradigm, ∆FosB may operate not only as theactivator but also as suppressor of AP1 function (74, 75). Thus, it is conceivablethat long-lasting induction of ∆FosB by acute cocaine in the PVN may suppressthe effects of SKF 38393 on cFos expression.

To summarize our findings, we have shown an important role of dopamineD1-like receptors in regulation of transcriptional activity of PVN neurons at thelevel of cFos induction. Additionally, we have provided evidence thattranscriptional activity initiated by activation of dopamine D1-like receptors ismodulated by acute but not repeated administration of cocaine. Such resistance ofdopamine D1-like receptors to repeated administration of cocaine at the level ofgene transcription regulated by cFos protein may indicate that activity of the HPAaxis controlled by PVN during the course of cocaine is unchanged, andtranscription controlled by cFos is maintained at a constant, though decreased,level following a single dose of cocaine.

Acknowledgments: We thank Dr. H. Gainer for supplying us with excellent anti-oxytocin andanti-vasopressin monoclonal antibodies. The study was supported by statutory activity of Instituteof Pharmacology, Polish Academy of Sciences, Kraków, Poland.

REFERENCES

1. Clouet D, Asghar K, Brown R. Mechanisms of Cocaine Abuse and Toxicity. In NIDA ResaerchMonograph 88. 1988, pp 1-354.

2. Koob GF, Le Moal M. Drug addiction, dysregulation of reward, and allostasis.Neuropsychopharmacology 2001; 24: 97-129.

3. Kuhar MJ, Ritz MC, Boja JW. The dopamine hypothesis of the reinforcing properties ofcocaine. Trends Neurosci 1991; 14: 299-302.

4. Roberts DC, Koob GF, Klonoff P, Fibiger HC. Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens. PharmacolBiochem Behav 1980; 12: 781-787.

5. Roberts DC, Koob GF. Disruption of cocaine self-administration following 6-hydroxydopaminelesions of the ventral tegmental area in rats. Pharmacol Biochem Behav 1982; 17: 901-904.

6. Beveridge TJ, Smith HR, Nader MA, Porrino LJ. Functional effects of cocaine self-administration in primate brain regions regulating cardiovascular function. Neurosci Lett 2004;370: 201-205.

436

7. Kiritsy-Roy JA, Halter JB, Gordon SM, Smith MJ, Terry LC. Role of the central nervous systemin hemodynamic and sympathoadrenal responses to cocaine in rats. J Pharmacol Exp Ther1990; 255: 154-160.

8. Mello NK, Mendelson JH. Cocaine�s effects on neuroendocrine systems: clinical and preclinicalstudies. Pharmacol Biochem Behav 1997; 57: 571-599.

9. Deroche V, Piazza PV, Casolini P, Maccari S, Le Moal M, Simon H. Stress-induced sensitizationto amphetamine and morphine psychomotor effects depend on stress-induced corticosteronesecretion. Brain Res 1992; 598: 343-348.

10. Erb S, Salmaso N, Rodaros D, Stewart J. A role for the CRF-containing pathway from centralnucleus of the amygdala to bed nucleus of the stria terminalis in the stress-inducedreinstatement of cocaine seeking in rats. Psychopharmacology (Berl) 2001; 158: 360-365.

11. Goeders NE, Guerin GF. Role of corticosterone in intravenous cocaine self-administration inrats. Neuroendocrinology 1996; 64: 337-348.

12. Iversen S, Iversen L, Saper CB. The autonomic nervous system and the hypothalamus. InPrinciple of Neural Science, ER Kandel, JH Schwartz, TM Jessel (eds). New York, McGraw-Hill, 2000, pp. 960-981.

13. Swanson LW, Sawchenko PE. Paraventricular nucleus: a site for the integration ofneuroendocrine and autonomic mechanisms. Neuroendocrinology 1980; 31: 410-417.

14. Armstrong WE. Hypothalamic supraoptic and paraventricular nuclei. In The Rat NervousSystem, G Paxinos (ed). New York, Academic Press, 1995, pp. 377-390.

15. Swanson LW, Kuypers HG. The paraventricular nucleus of the hypothalamus: cytoarchitectonicsubdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinalcord as demonstrated by retrograde fluorescence double-labeling methods. J Comp Neurol1980; 194: 555-570.

16. Day HE, Campeau S, Watson SJ, Jr., Akil H. Expression of alpha(1b) adrenoceptor mRNA incorticotropin-releasing hormone-containing cells of the rat hypothalamus and its regulation bycorticosterone. J Neurosci 1999; 19: 10098-10106.

17. Bagdy G. Role of the hypothalamic paraventricular nucleus in 5-HT1A, 5-HT2A and 5-HT2Creceptor-mediated oxytocin, prolactin and ACTH/corticosterone responses. Behav Brain Res1996; 73: 277-280.

18. Van de Kar LD, Javed A, Zhang Y, Serres F, Raap DK, Gray TS. 5-HT2A receptors stimulateACTH, corticosterone, oxytocin, renin, and prolactin release and activate hypothalamic CRFand oxytocin-expressing cells. J Neurosci 2001; 21: 3572-3579.

19. Bartanusz V, Muller D, Gaillard RC, Streit P, Vutskits L, Kiss JZ. Local gamma-aminobutyricacid and glutamate circuit control of hypophyseotrophic corticotropin-releasing factor neuronactivity in the paraventricular nucleus of the hypothalamus. Eur J Neurosci 2004; 19: 777-782.

20. Boudaba C, Szabo K, Tasker JG. Physiological mapping of local inhibitory inputs to thehypothalamic paraventricular nucleus. J Neurosci 1996; 16: 7151-7160.

21. Boudaba C, Schrader LA, Tasker JG. Physiological evidence for local excitatory synapticcircuits in the rat hypothalamus. J Neurophysiol 1997; 77: 3396-3400.

22. Borowsky B, Kuhn CM. D1 and D2 dopamine receptors stimulate hypothalamo-pituitary-adrenal activity in rats. Neuropharmacology 1992; 31: 671-678.

23. Eaton MJ, Cheung S, Moore KE, Lookingland KJ. Dopamine receptor-mediated regulation ofcorticotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus. BrainRes 1996; 738: 60-66.

24. Jezova D, Jurcovicova J, Vigas M, Murgas K, Labrie F. Increase in plasma ACTH afterdopaminergic stimulation in rats. Psychopharmacology (Berl) 1985; 85: 201-203.

437

25. Ritz MC, Lamb RJ, Goldberg SR, Kuhar MJ. Cocaine self-administration appears to bemediated by dopamine uptake inhibition. Prog Neuropsychopharmacol Biol Psychiatry 1988;12: 233-239.

26. Borowsky B, Kuhn CM. Monoamine mediation of cocaine-induced hypothalamo-pituitary-adrenal activation. J Pharmacol Exp Ther 1991; 256: 204-210.

27. Saphier D, Welch JE, Farrar GE, Goeders NE. Effects of intracerebroventricular andintrahypothalamic cocaine administration on adrenocortical secretion. Neuroendocrinology1993; 57: 54-62.

28. Sarnyai Z, Mello NK, Mendelson JH, Eros-Sarnyai M, Mercer G. Effects of cocaine on pulsatileactivity of hypothalamic-pituitary-adrenal axis in male rhesus monkeys: neuroendocrine andbehavioral correlates. J Pharmacol Exp Ther 1996; 277: 225-234.

29. Zhou Y, Spangler R, Yuferov VP, Schlussmann SD, Ho A, Kreek MJ. Effects of selective D1-or D2-like dopamine receptor antagonists with acute �binge� pattern cocaine on corticotropin-releasing hormone and proopiomelanocortin mRNA levels in the hypothalamus. Brain Res MolBrain Res 2004; 130: 61-67.

30. Borowsky B, Kuhn CM. Chronic cocaine administration sensitizes behavioral but notneuroendocrine responses. Brain Res 1991; 543: 301-306.

31. Schmidt ED, Tilders FJ, Janszen AW, Binnekade R, De Vries TJ, Schoffelmeer AN. Intermittentcocaine exposure causes delayed and long-lasting sensitization of cocaine-induced ACTHsecretion in rats. Eur J Pharmacol 1995; 285: 317-321.

32. Zhou Y, Spangler R, Schlussman SD, Ho A, Kreek MJ. Alterations in hypothalamic-pituitary-adrenal axis activity and in levels of proopiomelanocortin and corticotropin-releasing hormone-receptor 1 mRNAs in the pituitary and hypothalamus of the rat during chronic �binge� cocaineand withdrawal. Brain Res 2003; 964: 187-199.

33. Bergman J, Kamien JB, Spealman RD. Antagonism of cocaine self-administration by selectivedopamine D(1) and D(2) antagonists. Behav Pharmacol 1990; 1: 355-363.

34. Callahan PM, Appel JB, Cunningham KA. Dopamine D1 and D2 mediation of thediscriminative stimulus properties of d-amphetamine and cocaine. Psychopharmacology (Berl)1991; 103: 50-55.

35. Xu M, Hu XT, Cooper DC et al. Elimination of cocaine-induced hyperactivity and dopamine-mediated neurophysiological effects in dopamine D1 receptor mutant mice. Cell 1994; 79:945-955.

36. Xu M, Guo Y, Vorhees CV, Zhang J. Behavioral responses to cocaine and amphetamineadministration in mice lacking the dopamine D1 receptor. Brain Res 2000; 852: 198-207.

37. Caine SB, Thomsen M, Gabriel KI et al. Lack of self-administration of cocaine in dopamine D1receptor knock-out mice. J Neurosci 2007; 27: 13140-13150.

38. Zhou Y, Spangler R, Ho A, Jeanne KM. Hypothalamic CRH mRNA levels are differentiallymodulated by repeated �binge� cocaine with or without D(1) dopamine receptor blockade. BrainRes Mol Brain Res 2001; 94: 112-118.

39. Goeders NE. Stress and cocaine addiction. J Pharmacol Exp Ther 2002; 301: 785-789.40. Przegalinski E, Filip M, Siwanowicz J, Nowak E. Effect of adrenalectomy and corticosterone

on cocaine-induced sensitization in rats. J Physiol Pharmacol 2000; 51: 193-204.41. Piazza PV, Marinelli M, Jodogne C et al. Inhibition of corticosterone synthesis by Metyrapone

decreases cocaine-induced locomotion and relapse of cocaine self-administration. Brain Res1994; 658: 259-264.

42. Goeders NE, Guerin GF. Effects of surgical and pharmacological adrenalectomy on theinitiation and maintenance of intravenous cocaine self-administration in rats. Brain Res 1996;722: 145-152.

438

43. Erb S, Shaham Y, Stewart J. The role of corticotropin-releasing factor and corticosterone instress- and cocaine-induced relapse to cocaine seeking in rats. J Neurosci 1998; 18: 5529-5536.

44. Sarnyai Z, Shaham Y, Heinrichs SC. The role of corticotropin-releasing factor in drug addiction.Pharmacol Rev 2001; 53: 209-243.

45. Parker SL, Crowley WR. Activation of central D-1 dopamine receptors stimulates oxytocinrelease in the lactating rat: evidence for involvement of the hypothalamic paraventricular andsupraoptic nuclei. Neuroendocrinology 1992; 56: 385-392.

46. Piazza PV, Le Moal M. Glucocorticoids as a biological substrate of reward: physiological andpathophysiological implications. Brain Res Brain Res Rev 1997; 25: 359-372.

47. Czyrak A, Chocyk A, Mackowiak M, Fijal K, Wedzony K. Distribution of dopamine D1receptors in the nucleus paraventricularis of the hypothalamus in rats: an immunohistochemicalstudy. Brain Res Mol Brain Res 2000; 85: 209-217.

48. Fetissov SO, Meguid MM, Sato T, Zhang LH. Expression of dopaminergic receptors in thehypothalamus of lean and obese Zucker rats and food intake. Am J Physiol Regul Integr CompPhysiol 2002; 283: R905-R910.

49. Fremeau RT, Jr., Duncan GE, Fornaretto MG et al. Localization of D1 dopamine receptormRNA in brain supports a role in cognitive, affective, and neuroendocrine aspects ofdopaminergic neurotransmission. Proc Natl Acad Sci U S A 1991; 88: 3772-3776.

50. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York, Academic Press,1998.

51. Mackowiak M, Chocyk A, Fijal K, Czyrak A, Wedzony K. c-Fos proteins, induced by theserotonin receptor agonist DOI, are not expressed in 5-HT2A positive cortical neurons. BrainRes Mol Brain Res 1999; 71: 358-363.

52. Alonso G, Szafarczyk A, Assenmacher I. Immunoreactivity of hypothalamo-neurohypophysialneurons which secrete corticotropin-releasing hormone (CRH) and vasopressin (Vp):immunocytochemical evidence for a correlation with their functional state in colchicine-treatedrats. Exp Brain Res 1986; 61: 497-505.

53. Sofroniew MV. Vasopressin, oxytocin and their related neurophysins. In Handbook of ChemicalNeuroanatomy. GABA and Neuropeptides in the CNS, Part I, A Bjorklund, T Hokfelt (eds). NewYork, Elseviere Sciences, 1985, 4: pp. 93-153.

54. Wedzony K , Chocyk A, Mackowiak M. A search for colocalization of serotonin 5-HT2A and5-HT1A receptors in the rat medial prefrontal and entorhinal cortices - immunohistochemicalstudies. J Physiol Pharmacol 2008; 59: 229-238.

55. Wedzony K, Chocyk A, Kolasiewicz W, Mackowiak M. Glutamatergic neurons of rat medialprefrontal cortex innervating the ventral tegmental area are positive for serotonin 5-HT1Areceptor protein. J Physiol Pharmacol 2007; 58: 611-624.

56. Andersen PH, Jansen JA. Dopamine receptor agonists: selectivity and dopamine D1 receptorefficacy. Eur J Pharmacol 1990; 188: 335-347.

57. Reavill C, Bond B, Overend P, Hunter AJ. Pharmacological characterization of thediscriminative stimulus properties of the dopamine D1 agonist, SKF 81297. Behav Pharmacol1993; 4: 135-146.

58. Bourne JA. SCH 23390: the first selective dopamine D1-like receptor antagonist. CNS DrugRev 2001; 7: 399-414.

59. Bender M, Drago J, Rivkees SA. D1 receptors mediate dopamine action in the fetalsuprachiasmatic nuclei: studies of mice with targeted deletion of the D1 dopamine receptorgene. Brain Res Mol Brain Res 1997; 49: 271-277.

60. Moratalla R, Xu M, Tonegawa S, Graybiel AM. Cellular responses to psychomotor stimulantand neuroleptic drugs are abnormal in mice lacking the D1 dopamine receptor. Proc Natl AcadSci U S A 1996; 93: 14928-14933.

439

61. Chaudhuri A. Neural activity mapping with inducible transcription factors. Neuroreport 1997;8: v-ix.

62. Rivkees SA, Lachowicz JE. Functional D1 and D5 dopamine receptors are expressed in thesuprachiasmatic, supraoptic, and paraventricular nuclei of primates. Synapse 1997; 26: 1-10.

63. Huang Q, Zhou D, Chase K, Gusella JF, Aronin N, DiFiglia M. Immunohistochemicallocalization of the D1 dopamine receptor in rat brain reveals its axonal transport, pre- andpostsynaptic localization, and prevalence in the basal ganglia, limbic system, and thalamicreticular nucleus. Proc Natl Acad Sci U S A 1992; 89: 11988-11992.

64. Herman JP, Figueiredo H, Mueller NK et al. Central mechanisms of stress integration:hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. FrontNeuroendocrinol 2003; 24: 151-180.

65. Moratalla R, Elibol B, Vallejo M, Graybiel AM. Network-level changes in expression ofinducible Fos-Jun proteins in the striatum during chronic cocaine treatment and withdrawal.Neuron 1996; 17: 147-156.

66. Dong Y, Nasif FJ, Tsui JJ et al. Cocaine-induced plasticity of intrinsic membrane properties inprefrontal cortex pyramidal neurons: adaptations in potassium currents. J Neurosci 2005; 25:936-940.

67. Henry DJ, White FJ. Repeated cocaine administration causes persistent enhancement of D1dopamine receptor sensitivity within the rat nucleus accumbens. J Pharmacol Exp Ther 1991;258: 882-890.

68. Zhang XF, Cooper DC, White FJ. Repeated cocaine treatment decreases whole-cell calciumcurrent in rat nucleus accumbens neurons. J Pharmacol Exp Ther 2002; 301: 1119-1125.

69. Henry DJ, White FJ. The persistence of behavioral sensitization to cocaine parallels enhancedinhibition of nucleus accumbens neurons. J Neurosci 1995; 15: 6287-6299.

70. Chen J, Kelz MB, Hope BT, Nakabeppu Y, Nestler EJ. Chronic Fos-related antigens: stablevariants of deltaFosB induced in brain by chronic treatments. J Neurosci 1997; 17: 4933-4941.

71. Nye HE, Hope BT, Kelz MB, Iadarola M, Nestler EJ. Pharmacological studies of the regulationof chronic FOS-related antigen induction by cocaine in the striatum and nucleus accumbens. JPharmacol Exp Ther 1995; 275: 1671-1680.

72. Chocyk A, Czyrak A, Wedzony K. Acute and repeated cocaine induces alterations inFosB/DeltaFosB expression in the paraventricular nucleus of the hypothalamus. Brain Res2006; 1090: 58-68.

73. Nestler EJ, Barrot M, Self DW. DeltaFosB: a sustained molecular switch for addiction. ProcNatl Acad Sci U S A 2001; 98: 11042-11046.

74. Herdegen T, Leah JD. Inducible and constitutive transcription factors in the mammaliannervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins.Brain Res Brain Res Rev 1998; 28: 370-490.

75. McClung CA, Nestler EJ. Regulation of gene expression and cocaine reward by CREB andDeltaFosB. Nat Neurosci 2003; 6: 1208-1215.

R e c e i v e d : June 10, 2008A c c e p t e d : July 30, 2008

Author�s address: A. Chocyk, Laboratory of Pharmacology and Brain Biostructure, Institute ofPharmacology, Polish Academy of Sciences; 31-343 Kraków, Smêtna 12, Poland; phone: (4812)6623241 fax (4812) 6374500; e-mail: chocyk@if-pan.krakow.pl

440