Distinct pattern of c-fos mRNA expression after systemic and intra-accumbens amphetamine and MK-801

PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 4 1 5 - 3

Neuroscience Vol. 115, No. 1, pp. 67^78, 2002 ? 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00

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DISTINCT PATTERN OF c-FOS mRNA EXPRESSION AFTER SYSTEMIC AND INTRA-ACCUMBENS AMPHETAMINE AND MK-801 E. DE LEONIBUS,a A. MELE,a
A. OLIVERIOa and A. PERTb a

Dipartimento di Genetica e Biologia Molecolare, Universita' di Roma ‘La Sapienza’, P. le Aldo Moro, 5, 00185 Roma, Italy b

BPB, National Institute of Mental Health, N.I.H., Bethesda, MD 20892-1272, USA

Abstract9Pharmacological manipulation of both dopamine and glutamate systems a¡ects motor responses in laboratory animals. The two systems, however, seem to act in opposite ways, since direct or indirect activation of dopamine receptors induces similar stimulatory e¡ects to those seen following blockade of N-methyl-D-aspartate receptors. In the present study we compared the pattern of c-fos activation induced by systemic and intra-accumbens administration of the non-competitive N-methyl-D-aspartate antagonist MK-801 and the indirect dopamine agonist amphetamine. Systemic MK-801 induced c-fos mRNA expression in the motor cortex and preferentially in the motor thalamus, i.e. ventrolateral nucleus. Systemic amphetamine, on the other hand, enhanced c-fos mRNA expression in the shell of the accumbens and in limbic thalamic nuclei such as the anteroventral and anterodorsal nuclei. The main e¡ect observed after intra-accumbens administrations of either drug was enhanced c-fos expression in the thalamus, somewhat similar to what seen following systemic administration. In fact also in this case there was a preferential activation of the limbic thalamus by amphetamine and the motor thalamus by MK-801. The present results con¢rm that di¡erent neural substrates underlie behavioral e¡ects induced by systemic administrations of N-methyl-D-aspartate receptor antagonists and dopamine agonists. Further they suggest that intra-accumbens manipulation of the two neural systems could a¡ect di¡erent e¡erent pathways from this structure activating di¡erent thalamic targets. ? 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: dopamine, glutamate, thalamus, locomotor activity, nucleus accumbens.

The DA system, on the other hand, is somewhat more circumscribed with terminal ¢elds and receptors located primarily in the striatal complex ^ caudate putamen and N. accumbens. There is considerable evidence that the striatum has a critical role in mediating a variety of DA induced behavioral responses. In particular, the N. accumbens has been demonstrated to be a key structure in modulating locomotor activity induced by DA activation. For example, it has been demonstrated that 6-hydroxydopamine (6-OHDA) lesions to the N. accumbens completely block locomotor stimulation induced by systemic amphetamine, thus suggesting that DA activation within this structure is a prime determinant of DA agonist induced motor activity (Kelly et al., 1975; Koob et al., 1981). This hypothesis is con¢rmed further by the observation that lesions of the ventral pallidum (VP), the major recipient of N. accumbens e¡erent projections, block motor activity induced by systemic amphetamine (Mele et al., 1998) or apomorphine enhanced motor response in 6-OHDA lesioned rats (Swerdlow et al., 1984). Systemic administration of the glutamate antagonist dizocilpine hydrogen maleate (MK-801), that acts as an N-methyl-D-aspartate (NMDA) channel blocker, and is thus considered a non-competitive antagonist at these receptor sites, also stimulates locomotion (Carlsson and Carlsson, 1989; Ford et al., 1989; Ouagazzal et al., 1994). Early studies suggested that systemic NMDA

Pharmacological manipulations of both glutamate and dopamine (DA) systems induce a variety of behavioral e¡ects that range from alteration in locomotor activity (Kim and Vezina, 1998; Mele et al., 1998; Jacobs et al., 2000; Kretschmer, 2000) to changes in learning and memory functions (Smith-Roe et al., 1999; Adriani et al., 2000; Smith-Roe and Kelley, 2000; Di Ciano and Everitt, 2001) as well as response to novel stimuli (Roullet et al., 1996; Beaufour et al., 2001). Particular attention has been devoted to the study of neural mechanisms that mediate motor responses induced by manipulation of these two systems. Glutamate receptors are quite ubiquitous in brain with highest concentrations in cortical regions (Ottersen and Storm-Mathisen, 1984).

*Corresponding author. Tel.: +39-6-4991-2244; fax: +39-6-4440812. E-mail address: [email protected] (A. Mele). Abbreviations : ADT, anterodorsal thalamus; ANOVA, analysis of variance ; AVT, anteroventral thalamus; DA, dopamine ; DEPC, diethylpyrocarbonate; 2DG, 2-deoxyglucose ; MDT, mediodorsal thalamus; MK-801, dizocilpine hydrogen maleate; NMDA, Nmethyl-D-aspartate; 6-OHDA, 6-hydroxydopamine; PBS, phosphate bu¡ered saline; SSC, salt sodium citrate; VLT, ventrolateral thalamus; VMT, ventromedial thalamus; VP, ventral pallidum ; VTA, ventral tegmental area. 67

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antagonist induced motor activity might be determined by NMDA receptor blockade within the accumbens, possibly mediated by enhanced DA activity. This hypothesis was based on evidence demonstrating that focal administrations of the NMDA antagonist, MK-801, into the N. accumbens enhanced motor activity (Ouagazzal and Amalric, 1995; Mele et al., 1998; Frantz and Van Hartesveldt, 1999; De Leonibus et al., 2001) as well as DA over£ow (Imperato et al., 1990). Contrary to the notion that N. accumbens DA is involved in mediating such increased locomotor activity, however, is the observation that 6-OHDA lesions of this structure do not block systemic MK-801 induced locomotion (Ouagazzal et al., 1994; Mele et al., 1998). Furthermore, while VP lesions have been found to block DA agonist induced increases in locomotor activity, the same lesions do not a¡ect motor activity induced by systemic MK-801 (Mele et al., 1998; Kretschmer, 2000). Such ¢ndings indicate that while NMDA receptor blockade within the accumbens is able to induce motor stimulation, this structure, as well as its output to the VP, are not entirely essential in mediating systemic NMDA antagonist induced motor activity. The nucleus accumbens is part of a cortico^thalamic^ cortical loop (Swerdlow and Koob, 1987; Kalivas et al., 1999). Based on neuroanatomical evidence, it has been suggested that its functional output could be mediated by two distinct e¡erent pathways: the ¢rst one projecting from the lateral component of the VP to the basal ganglia circuit, i.e. the entopeduncular nucleus and the substantia nigra, the second one from the medial component of the VP directly to the thalamus (Zahm, 1989). It has been implied that these two pathways could a¡ect di¡erent cortical areas (Zahm and Brog, 1992) possibly through the modulation of distinct thalamic nuclei (Kalivas et al., 1999). This suggestion is intriguing since the two di¡erent pathways could underlie distinct function of this structure. Interestingly recent evidence seems to provide some support for the view that locomotor activity induced by intra-accumbens DA and NMDA receptor manipulation could be mediated by di¡erent pathways. For example, locomotor activity induced by intra-accumbens amphetamine and MK-801 is a¡ected respectively by lesions of mediodorsal thalamus (MDT) (Swerdlow and Koob, 1987) and ventrolateral thalamus (VLT) (De Leonibus et al., 2001). C-fos is an immediate early gene that is thought to in£uence transcription of other genes in many neural systems (Morgan and Curran, 1989), and has been demonstrated to be a very useful tool to measure relative alterations in neuronal activity in response to pharmacological manipulations or external stimuli (Sagar et al., 1988; Dragunow and Faull, 1989; Wirtshafter et al., 1994). In order to further investigate the circuits mediating amphetamine and MK-801 induced motor e¡ects, in the present study we undertook two di¡erent experiments. In the ¢rst study we compared the e¡ects of systemic MK-801 and amphetamine administrations on c-fos induction in the rat brain, while in the second we evaluated changes in c-fos expression after intra-accumbens focal MK-801 and amphetamine.

EXPERIMENTAL PROCEDURES

Subjects and surgical procedure Male Sprague^Dawley rats (Taconic Farms) weighing 300^ 350 g were group housed (10 per cage) and maintained for 1 week on a light/dark cycle (light 07.00^19.00), with food and water available ad libitum. The animals were adapted to vivarium conditions and handled once a day for 1 week before starting the experiments. Immediately prior to surgery, the animals were injected intraperitoneally (i.p.) with chloral hydrate (400 mg/kg) and placed in a stereotaxic frame (David Kopf, Tujunga, CA, USA). Using standard stereotaxic procedures, they were implanted with 16 mm bilateral stainless steel guide cannulae (23 gauge) aimed at an area 2 mm dorsal to the nucleus accumbens (AP = +10.9, L = T 1.5, DV = +4.0 mm). The coordinates reported are relative to the interaural zero, with the incisor bar set at 33.5 mm, according to Paxinos and Watson (1982). Every possible e¡ort was made to minimize animal su¡ering, and all procedures were in strict accordance with European community laws and regulations on the use of animals in research and National Institutes of Health guidelines on animal care. Behavioral recording Locomotor activity was recorded with Digiscan photocell activity monitors (Omnitech Electronics, Columbus, OH, USA), the chambers being constructed from clear Plexigas (30.5 cm highU42 cm wideU42 cm long). The activity monitor was enclosed in sound-attenuating compartments equipped with 15 W £uorescent light, a ventilation fan that also provided masking noise and one-way mirror (21U21 cm2 ) mounted in the door, to allow visual observation of the animals during testing. A series of equally spaced infrared photocell detectors were located along two adjacent walls of the chamber 4 cm from the £oor surface. Interruptions of the infrared light sources by the animal were recorded and stored by an IBM AT computer. Systemic administration study A week following their arrival, the rats were habituated to the activity chamber for 40 min a day, for seven consecutive days. After this habituation period, the animals were divided into three groups and injected i.p. with either saline (n = 9), 1 mg/ kg of amphetamine (n = 8) or 0.3 mg/kg of MK-801 (n = 6). Following injections, the animals were placed in the activity chambers and their horizontal locomotor activity was monitored for 40 min. Immediately after the end of the behavioral test the animals were decapitated, the brain removed and rapidly frozen. Focal administration study One week after surgery, animals implanted with bilateral cannulae aimed at the accumbens were habituated to the activity chamber 40 min a day for seven consecutive days. On the eighth day the animals were divided into three groups. Animals in the ¢rst group were injected bilaterally in the nucleus accumbens with 0.5 Wl physiological saline (n = 8) through 30 gauge injectors that were 2 mm longer than the guide cannulae. Animals in the second group were injected bilaterally in the accumbens with 5 nmol of amphetamine (n = 9) in 0.5 Wl of saline, while the animals in the third group were focally injected with 25 nmol of MK-801 (n = 6) in 0.5 Wl of saline. The injections were made with a Harvard microinfusion pump, at a £ow rate of 2 Wl/min. The injections needle was removed 30 s following an injection to allow the di¡usion of the drug from the injection site. After an injection, the animals were placed in the activity chamber where horizontal locomotor activity was recorded for 40 min. Immediately after the end of the behavioral test the animals were decapitated, the brains removed, placed in 345‡C isopentane and then stored at 370‡C. The animals

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C-fos activation after MK-801 and amphetamine

were killed 40 min after the injection on the basis of previous studies (Wang et al., 1995) which have demonstrated that c-fos peak expression is between 30 and 60 min after pharmacological manipulations. In situ hybridization and histology Coronal sections (15 Wm) were cut through each frozen brain on a cryostat. The sections were thawed onto gelatinized microscope slides, air dried and stored at 370‡C. Placement of cannulae was veri¢ed by staining with Cresyl Violet solution (0.1% Cresyl Violet, 0.1% glacial acetic acid diluted with deionized water), dehydrating the sections with increasing concentrations of ethanol (70, 90, 95 and 100%), and rinsing in two washes of xylenes. The slides were coverslipped using a solution of 2/3 Permount and 1/3 xylenes, and allowed to dry. An antisense c-fos RNA probe was generated from an 860 bp sense cDNA with Sac1 from full length 3005 bp transcript via SP65 plasmid (gift of Dr. Tom Curran of Roche Institute, NJ, USA). The riboprobe was synthesized by in vitro transcription of the linearized plasmid with Sp6 polymerase. Frozen sections were allowed to thaw for 10^15 min and ¢xed in 4% paraformaldehyde 1Uphosphate bu¡ered saline (PBS) for 5 min, then rinsed with 1UPBS. The sections were treated with 0.25% acetic anhydride, 0.1 M triethanolamine and 0.4% HCl for 10 min at room temperature. The tissue was dehydrated with increasing concentrations of ethanol (70, 90, 95 and 100%). Following defatting of the tissue with chloroform for 5 min, the tissue was rinsed in 95 and 100% ethanol, 1 min each, and allowed to air dry. All the prehybridization solutions were prepared with autoclaved 0.1% diethylpyrocarbonate (DEPC) water (Flucka, Switzerland). The antisense c-fos RNA probe was labeled with [35 S]UTP (NEN Dupont, MA, USA) utilizing Ambion Maxiscript kit (Austin, TX, USA) and ribonucleic acid mix [yeast tRNA 250 Wg/ml (Gibco, NY, USA); yeast total RNA type XI 250 Wg/ml (Segind) DEPC water]; 1U106 disintegrations per minute (d.p.m.) of denatured riboprobe was mixed with ribonucleic acid mix and denatured at 65‡C for 5 min, put on ice for 2 min, then added to the hybridization bu¡er [20 mM Tris^HCl, pH 7.4 (Quality Biologicals, Gaithersburg, MD, USA); 50% formamine; 300 mM NaCl (Flucka); EDTA, pH 8.0 1 mM (Digene Diagnostics, Beltville, MD, USA); 10% dextran sulfate (Oncar, Gaithersburg, MD, USA)]. Dithiothreitol (DTT) (5 M, 3P to 5P), 10% sodium thiosulfate (Sigma) and 10% sodium dodecyl sulfate (SDS) (Oncar) were added to the hybridization bu¡er^ribonucleic acid mix solution, approximately 100 Wl of hybridization bu¡er were added to each slide, which was covered with a glass coverslip and incubated in a Petri dish with ¢lter paper dampened with 4Usalt sodium citrate (SSC) 50% at room temperature. The slides were washed four times with 4USSC, for 5 min each at room temperature while rotating on a platform. The slides were incubated at room temperature in 1USSC with RNase A (¢nal concentration 20 mg/l) (Boehringer Mannheim, Indianapolis, IN, USA). Subsequent washes of decreasing concentrations of SSC occurred for 5 min (1U; 0.5U; 0.1U) at room temperature. Sections were incubated at 65‡C in 0.1USSC for 30 min each, followed by 5 min wash with 0.1USSC at room temperature. The tissue was dehydrated with increasing concentrations of ethanol ammonium acetate (¢nal concentration 300 nM) at room temperature (50, 70, 90 and 95%). Sections were dipped quickly in 100% ethanol and allowed to air dry. Slides were apposed to Biomax ¢lm (Kodak, Rochester, NY, USA) for 2 days. The autoradiographs obtained from the in situ hybridization studies were digitized and analyzed using the National Institute of Health Image software. The program analyzed the distribution of optical density (OD) values (arbitrary units) of pixels. To allow a comparison between the e¡ects of di¡erent drug treatments on c-fos mRNA expression in the same brain regions, the optical density measured in each brain region was divided by the optical density measured in brain areas in the same plane which do not express c-fos mRNA (white matter). After this correction, the e¡ects of either amphetamine or MK-801 treatments

69

on c-fos expression in di¡erent brain regions were compared with the e¡ects of saline treatment on c-fos expression in similar regions. In the systemic administration study, the brain regions analyzed were neocortical and allocortical regions such as the secondary motor cortex (AP = +11.20), the primary motor cortex (AP = +11.20), the cingulate cortex, area1 (AP = +11.20), the prelimbic cortex (AP = +11.20), nucleus accumbens (AP = +11.20), nucleus accumbens core (AP = +11.20), nucleus accumbens shell (AP = +11.20) with the anteroposterior coordinate (AP) relative to interaural zero, according to Paxinos and Watson (1982). Also analyzed were the anterior cingulate cortex (AP = +8.7), and the basolateral amygdala (AP = +7.2), some regions of the basal ganglia such as the substantia nigra reticulata (AP = +4.2), substantia nigra compacta (AP = +4.2), ventral tegmental area (VTA) (AP = +4.2), VP (AP = +8.7), globus pallidus (AP = +8.7) and caudate putamen (AP = +8.7). The e¡ects of systemic administration of MK-801 and amphetamine on c-fos mRNA expression were also analyzed in most of thalamic nuclei, e.g. anteroventral (AP = +7.2), anterodorsal (AP = +7.2), posteroventrolateral (AP = +6.2), mediodorsolateral (AP = +6.2), laterodorsal (AP = +6.2), reticular (AP = +7.2). For the mediodorsal, ventrolateral, ventromedial thalamic nuclei two independent measures were taken at two di¡erent anteroposterior levels, with the intent to reveal possible induction gradients, i.e. mediodorsal anterior (AP = +7.2), mediodorsal posterior (AP = +6.2), ventrolateral anterior (AP = +7.2), ventrolateral posterior (AP = +6.2), ventromedial anterior (AP = +7.2), ventromedial posterior (AP = +6.2) thalamic nuclei. The brain regions analyzed in the focal administration study were the same basal ganglia regions and thalamic nuclei analyzed in the systemic administration study, excluding the nucleus accumbens and its subregions. Densitometric analyses primarily emphasized the basal ganglia structures (including the ventral striatum) and associated thalamic regions. Additional cortical structures were also included for analyses in studies utilizing systemic drug injections. All the areas were analyzed using a template made from coronal plates of the rat brain atlas (Paxinos and Watson, 1982). Statistical analysis The data obtained from behavioral recording during the habituation period were analyzed using one-way analysis of variance (ANOVA), with repeated measures on the day factor (seven levels: day 1^day 7); a two-way ANOVA with one between factor treatment (three levels: saline, amphetamine, MK-801) and one within factor day (seven levels: day 1^day 7) was performed to be sure that the animals assigned to one of the three treatment groups did not di¡er signi¢cantly in their habituation response to the test chamber. The e¡ects of drug administration on horizontal activity were analyzed using a oneway ANOVA, with a between factor treatment (three levels: saline, amphetamine and MK-801). The simple e¡ects were analyzed with the post hoc Newman^Keuls comparison test. A twoway ANOVA for repeated measures was performed to analyze a possible drug (three levels: saline, amphetamine, MK-801)^time (four levels: 10, 20, 30, 40 min interval) interaction. The e¡ect of amphetamine and MK-801 on c-fos mRNA expression in di¡erent brain regions was analyzed using a oneway ANOVA with the between factor treatment (two levels: saline, drug). The level of signi¢cance chosen for all experiments was P 6 0.05.

RESULTS

E¡ects of systemic administration of MK-801 and amphetamine on horizontal activity Table 1 illustrates locomotor activity measured during the habituation period (seven days). As revealed by the

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E. De Leonibus et al. Table 1. Mean activity count ( T S.E.M.) for 7 days habituation period and summary of the statistical analysis for the systemic and focal studies

Treatment Habituation period

Systemic Focal

ANOVA

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

F

P

7601 T 472 7059 T 410

5889 T 238 5260 T 292

7008 T 429 3746 T 261

7278 T 503 4057 T 219

7431 T 195 4549 T 366

5979 T 286 4526 T 406

5422 T 294 4683 T 909

F(6,22) = 7.717 F(6,24) = 6.885

P = 0.0001 P = 0.0001

one-way ANOVA for repeated measures, animals showed a signi¢cant reduction of locomotor activity across the habituation days (F(6,22) = 7.717, P = 0.0001). Figure 1A illustrates the mean locomotor activity induced by systemic administration of saline,

amphetamine and MK-801 on day 8. The one-way ANOVA revealed a signi¢cant e¡ect of treatment (F(2,20) = 16.895, P = 0.0001). Analysis of the simple e¡ects with a post hoc comparison revealed that both amphetamine (P = 0.002) and MK-801 (P = 0.0004)

Fig. 1. Horizontal locomotor activity response to systemic and intra-accumbens administrations of saline, MK-801 and amphetamine. The arrow shows the time of the injection. (A) E¡ect of systemic administrations of saline (n = 9), MK-801 0.3 mg/kg (n = 6), and amphetamine 1 mg/kg (n = 8). (B) E¡ect of intra-accumbens administrations of saline (n = 8), MK-801 25 nmol (n = 6), and amphetamine 5 nmol (n = 9). The inserts represent total activity counts during 40 min testing. *P 6 0.05 saline vs. drug treatment.

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C-fos activation after MK-801 and amphetamine

induced a signi¢cant increase in the locomotor activity when compared with saline, but did not di¡er from each other (P = 0.9134). The two-way ANOVA revealed a signi¢cant e¡ect of time (F(3,22) = 7.815, P = 0.0002) and a signi¢cant e¡ect of treatment^time interaction (F(2,3) = 24.964, P = 0.0001) for amphetamine and MK-801 induced locomotor activity over the test session. E¡ects of systemic administration of MK-801 and amphetamine on c-fos mRNA expression Table 2 shows the e¡ect of systemic administration of amphetamine and MK-801 on c-fos mRNA expression in di¡erent brain regions. Both treatments seemed to a¡ect the c-fos expression in several brain regions, even though the pattern of c-fos expression induced by MK801 and amphetamine is di¡erent. In general, MK-801 increased c-fos expression in the neocortical regions and in several of the thalamic nuclei, while amphetamine seemed to a¡ect c-fos expression selectively in the shell subregion of the accumbens and in the anterior thalamic nuclei. MK-801 increased c-fos expression in the primary motor cortex (P = 0.012) and in the infralimbic cortex (P = 0.04). Amphetamine, on the contrary, had no significant e¡ects on any of the neocortical areas measured, even if the increase in c-fos expression observed in the

71

anterior cingulate cortex approached statistical signi¢cance (P = 0.057). MK-801 did not a¡ect c-fos expression in any of the basal ganglia structures analyzed, while amphetamine produced an increase in c-fos expression in the nucleus accumbens (P = 0.053). Interestingly, when the two nucleus accumbens subregions were separately analyzed, a signi¢cant increase was found only in the shell subregion (P = 0.037), while the c-fos expression in the core subregion was not a¡ected by systemic administration of amphetamine. Compacting the two subregions of the accumbens failed to reveal an e¡ect of systemic MK-801 (Fig. 2). Both drugs a¡ected c-fos expression in thalamic nuclei, although MK-801 and amphetamine induced di¡erent patterns of c-fos expression. MK-801 increased c-fos expression in the mediodorsal nucleus (P = 0.008) (Fig. 3) without any particular di¡erences between its anterior (P = 0.01) and posterior (P = 0.005) components. MK-801 also increased the c-fos expression in the VLT (P = 0.029) (Fig. 3) that showed a clear dorsocaudal gradient of activation, with the posterior component being more active compared to the anterior, in which no signi¢cant increase was observed. In addition, systemic administration of MK-801 signi¢cantly increased c-fos expression in the AV thalamic nucleus (P = 0.048) (Fig. 4), but did not a¡ect c-fos expression in anterodor-

Table 2. E¡ects of systemic administration of MK-801 and amphetamine on c-fos mRNA expression Brain region

Treatment Saline

Neocortex Secondary motor cortex Primary motor cortex Cingulate cortex, area 1 Prelimbic cortex Infralimbic cortex Ant. cing. cortex Basolateral amygdala Basal ganglia Substantia nigra reticulata Substantia nigra compacta VTA Nucleus accumbens Core Shell VP Globus pallidus Caudate putamen Thalamic nuclei Mediodorsal Ventrolateral Ventromedial Anteroventral Anterodorsal Posteroventrolateral Mediodorsolateral Laterodorsal Reticular Paraventricular Reuniens

MK-801

Amphetamine

1.358 1.211 1.453 1.398 1.297 1.304 1.267

(0.096) (0.045) (0.087) (0.085) (0.101) (0.076) (0.066)

1.592 1.421 1.684 1.627 1.661 1.421 1.461

(0.093) (0.051)** (0.077) (0.084) (0.114)* (0.167) (0.157)

1.562 1.328 1.619 1.497 1.370 1.668 1.282

(0.070) (0.038) (0.065) (0.092) (0.078) (0.138) (0.049)

0.910 0.948 1.090 1.091 1.072 1.142 0.962 0.938 0.970

(0.045) (0.038) (0.071) (0.021) (0.021) (0.026) (0.012) (0.017) (0.013)

0.949 1.035 1.165 1.161 1.147 1.182 1.006 0.951 0.972

(0.016) (0.015) (0.051) (0.050) (0.048 (0.056) (0.039) (0.038) (0.030)

0.936 0.986 1.100 1.206 1.149 1.296 1.065 0.969 1.096

(0.032) (0.040) (0.044) (0.042) (0.043) (0.051)* (0.064) (0.056) (0.067)

1.116 1.054 1.104 1.127 1.118 1.017 1.169 1.081 1.029 1.086 1.107

(0.034) (0.016) (0.022) (0.068) (0.059) (0.009) (0.047) (0.049) (0.015) (0.071) (0.040)

1.475 1.143 1.223 1.352 1.196 1.024 1.389 1.225 1.032 1.649 1.327

(0.108)** (0.032)* (0.070) (0.076)* (0.081) (0.028) (0.089) (0.043) (0.021) (0.175)** (0.090)*

1.301 1.151 1.184 1.481 1.405 1.128 1.344 1.225 1.052 1.552 1.234

(0.063)* (0.044) (0.050) (0.101)** (0.070) (0.065) (0.099) (0.054) (0.021) (0.077)*** (0.042)*

Data are presented as (mean T S.E.M.) optical density standardized measures. *P 6 0.05, **P 6 0.01, ***P 6 0.001.

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sal, posteroventrolateral, ventromedial and reticular thalamic nuclei. The increased c-fos expression induced by MK-801 in the laterodorsal (P = 0.051) and in the mediodorsolateral (P = 0.054) thalamic nuclei almost reached statistical signi¢cance, while a signi¢cant increase was observed in the paraventricular (P = 0.0091) and the reuniens (P = 0.038) thalamic nuclei (Fig. 4). Systemic amphetamine administration increased c-fos in the AV (P = 0.014) and AD (P = 0.008) thalamic nuclei (Fig. 4). A similar increase was also observed in the MDT (P = 0.03) (Fig. 4), mainly due to its anterior component (P = 0.004). On the contrary, no signi¢cant changes were observed in the posterior part of MDT. Overall, the increase in c-fos expression observed in the VLT after systemic administration of amphetamine was not signi¢cant. It should be noted, however, that an anteroposterior gradient could be observed with the most anterior component being more active than the posterior. Systemic administration of amphetamine did not a¡ect c-fos mRNA expression in the posteroventrolateral, mediodorsolateral, dorsolateral and in the reticular thalamic nuclei. Interestingly, however, such as for MK-801, increased c-fos expression was observed in the paraventricular (P = 0.0007) and in the reuniens (P = 0.05) thalamic nuclei (Fig. 4).

Fig. 3. Autoradiograms of coronal sections showing the e¡ects of systemic administration of saline (A), MK-801 0.3 mg/kg (B) and amphetamine 1.0 mg/kg (C) on c-fos mRNA expression. MDTh, mediodorsal thalamus; VLTh, ventrolateral thalamus; VMTh, ventromedial thalamus.

E¡ects of nucleus accumbens focal administration of MK-801 and amphetamine on horizontal activity

Fig. 2. Autoradiograms of coronal sections showing the e¡ects of systemic administration of saline (A), MK-801 0.3 mg/kg (B) and amphetamine 1.0 mg/kg (C) on c-fos mRNA expression. Core, nucleus accumbens core ; shell, nucleus accumbens shell.

Table 1 illustrates the locomotor activity measures during the habituation period (seven days). As revealed by a one-way ANOVA for repeated measures, animals showed a signi¢cant reduction of locomotor activity across the habituation days (F(6,24) = 6.885, P = 0.0001). The insert in Fig. 1B shows the mean of locomotor activity induced by nucleus accumbens focal administration of saline, amphetamine and MK-801 on day 8. The one-way ANOVA revealed a signi¢cant e¡ect of treatment (F(2,24) = 10.339, P = 0.0006). Analysis of the simple e¡ects with a post hoc comparison revealed that both amphetamine (P = 0.002) and MK-801 (P = 0.04) induced a signi¢cant increase in locomotor activity when compared with saline, but did not di¡er from each other (P = 0.085). Figure 1B illustrates the time course (10 min intervals) of increased locomotor activity induced by amphetamine and MK-801. The two-way ANOVA revealed a signi¢cant e¡ect of time (F(3,24) = 3.223, P = 0.02) and a signi¢cant treatment^ time interaction (F(2,3) = 4.403, P = 0.0008).

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C-fos activation after MK-801 and amphetamine

73

E¡ects of nucleus accumbens focal administration of MK-801 and amphetamine on c-fos mRNA expression

Fig. 4. Autoradiograms of coronal sections showing the e¡ects of systemic administration of saline (A), MK-801 0.3 mg/kg (B) and amphetamine 1.0 mg/kg (C) on c-fos mRNA expression. ADTh, anterodorsal thalamus; AVTh, anteroventral thalamus.

Table 3 shows the e¡ect of focal administrations of MK-801 and amphetamine on c-fos mRNA expression in di¡erent brain regions. It should be noted that in the focal administration study there was a potent increase in c-fos expression generalized to the whole cortex in all treatment groups, included the saline treated animals, possibly as a result of the cannulae implantation. Compared to saline injected rats the e¡ects of intra-accumbens focal administrations of MK-801 and amphetamine on c-fos expression seem to be circumscribed to the thalamic nuclei, while no signi¢cant change in c-fos expression was observed in basal ganglia regions. MK-801 signi¢cantly increased c-fos expression in MD (P = 0.005), and in VLT (P = 0.007), the increase observed in the other motor nucleus of the thalamus, the ventromedial thalamus (VMT), also approached statistical signi¢cance (P = 0.055) (Fig. 5). Furthermore, increased c-fos expression was also found in the nucleus reuniens of the thalamus (P = 0.054). On the contrary, intra-accumbens MK-801 focal administrations did not a¡ect c-fos expression in any of the anterior thalamic nuclei. Focal administration of amphetamine, like MK-801, increased c-fos expression in MD (P = 0.005) and in the VLT (P = 0.002), but di¡erently from the NMDA antagonist, not in the VMT (Fig. 5). It also should be noted that focal administrations of amphetamine into the N. accumbens increased c-fos expression in the anterodorsal thalamus (ADT) (P = 0.01). Interestingly, when a rostrocaudal gradient within the di¡erent nuclei of the thalamus was taken into account, both amphetamine and MK-801 injections into the

Table 3. E¡ects of nucleus accumbens focal administration of MK-801 and amphetamine on c-fos mRNA expression Brain region

Treatment Saline

Basal ganglia Substantia nigra reticulata Substantia nigra compacta VTA VP Globus pallidus Caudate putamen Thalamic nuclei Mediodorsal Ventrolateral Ventromedial Anteroventral Anterodorsal Posteroventrolateral Mediodorsolateral Laterodorsal Reticular Paraventricular Reuniens

MK-801

Amphetamine

0.994 0.994 1.108 1.005 0.972 1.257

(0.010) (0.008) (0.023) (0.045) (0.044) (0.056)

0.963 0.992 1.040 0.970 0.963 1.182

(0.020) (0.028) (0.039) (0.024) (0.025) (0.042)

0.984 1.005 1.049 1.002 0.959 1.229

(0.016) (0.019) (0.025) (0.018) (0.017) (0.048)

1.108 1.052 1.077 1.160 1.080 1.045 1.096 1.052 1.046 1.258 1.123

(0.020) (0.009) (0.071) (0.018) (0.016) (0.014) (0.012) (0.022) (0.010) (0.036) (0.019)

1.239 1.138 1.170 1.231 1.165 1.096 1.310 1.195 1.092 1.492 1.249

(0.029)** (0.021)** (0.034) (0.036) (0.055) (0.017)* (0.049)** (0.036)** (0.024) (0.113) (0.050)

1.195 1.105 1.112 1.203 1.152 1.119 1.250 1.132 1.066 1.402 1.237

(0.016)** (0.001)** (0.018) (0.022) (0.016)** (0.011)*** (0.022)*** (0.022)* (0.014) (0.073) (0.057)

Data are presented as (mean T S.E.M.) optical density standard measures. *P 6 0.05, **P 6 0.01, ***P 6 0.001.

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Fig. 5. Autoradiograms of coronal sections showing the e¡ects of intra-accumbens administration of saline (A), MK-801 25 nmol (B) and amphetamine 5 nmol (C) on c-fos mRNA expression. MDTh, mediodorsal thalamus; VLTh, ventrolateral thalamus; VMTh, ventromedial thalamus.

nucleus accumbens generally increased c-fos expression in the most anterior components, with the exception of the VLT and MDT nuclei. The posterior component of the VLT nucleus showed increased c-fos expression with both drugs while the posterior component of the MDT was preferentially activated only by amphetamine. Finally, no e¡ect was found after either MK-801 or amphetamine in the mediodorsolateral, laterodorsal, ventroposterolateral and the paraventricular thalamic nuclei.

DISCUSSION

In the present study we report on the e¡ects of systemic and intra-accumbens focal administrations of the NMDA non-competitive antagonist, MK-801, and the indirect DA agonist, amphetamine, on c-fos mRNA expression. C-fos is an immediate early gene that is thought to in£uence transcription of other genes in many neural systems. Systemic as well as focal administrations of both drugs increased c-fos activity with di¡erent pattern in several brain regions. In the present study we did not observe any signi¢cant decreases in c-fos

expression in any areas analyzed. Expression of c-fos is very sensitive to extrinsic signals. It has been demonstrated, for example, that c-fos is activated by single saline injections as well as handling. In order to minimize the e¡ects of handling or novelty on c-fos mRNA induction and possible interaction of these factors with drug e¡ects, we habituated the animals to the motor cages for 1 week before testing them. It is therefore possible that basal levels of c-fos mRNA expression under these conditions were too low to allow the detection of decreases. It should be noted that sustained NMDA receptor activation induces immediate early genes by way of different mechanisms (Platenik et al., 2000). MK-801 is a potent and selective antagonist of NMDA receptor/channel complex and acts by binding to the so called PCP site located within the channel (Reynolds and Miller, 1988), blocking Ca2þ in£ux. It is unlikely that MK-801 induced increase in c-fos expression after systemic and focal MK801 administrations could be ascribed to a direct action of the NMDA antagonists on transcription processes. It seems more feasible to attribute the e¡ect observed with MK-801 to blockade of NMDA receptors on inhibitory neurons which might then lead to enhanced neural activity in cells expressing c-fos. Amphetamine, on the other hand, is a psychostimulant that acts by blocking the uptake and enhancing the release of catecholamines at the terminal regions. Complete and partial blockade of amphetamine induced increases in c-fos expression in the striatum have been demonstrated by D1 and D2 DA receptor antagonists respectively (Graybiel et al., 1990). Therefore in the striatum activation of DA receptors seems the main determinant in amphetamine induced c-fos expression. It should be noted however that in other brain regions alternative mechanisms, such as stimulation of release of other amines, can not be completely excluded. Since the purpose of this study was to determine the possible pathways involved in motor e¡ects induced by amphetamine and MK-801 we used doses that have been demonstrated to have behavioral relevance (Mele et al., 1998; De Leonibus et al., 2001; Kretschmer, 2000) and to induce similar e¡ects. Overall, systemic and focal administrations of the two drugs increased locomotion to comparable levels. It should be mentioned, however, that the time course di¡ered, consistent with results reported in previous experiments (Mele et al., 1998). In interpreting results from the focal administration studies, it is important to consider that at the end of the 40 min post-injection period, the activity levels between MK-801 and amphetamine injected rats were the same. In the ¢rst experiment we analyzed the pattern of c-fos activation in response to systemic administrations of MK-801 and amphetamine. Among the cortical regions analyzed, systemic MK-801 induced a signi¢cant increase in c-fos expression in the primary motor cortex and in the infralimbic cortex (part of the prefrontal cortex), while amphetamine was void of e¡ects on the motor cortex but enhanced c-fos (without reaching a statistical signi¢cance) in the anterior cingulate cortex. These results are consistent with previous reports demonstrating an activation of the medial prefrontal cortex with

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C-fos activation after MK-801 and amphetamine

amphetamine, as assessed with c-fos (Engber et al., 1998). Increases in 2-deoxyglucose (2DG) uptake have also been observed in the prefrontal cortex following systemic MK-801 administrations (Duncan et al., 1999). The prefrontal cortex, like most cortical regions, has a high density of NMDA receptors, and it is also the target of DA projections from the VTA. In light of such anatomical considerations, the e¡ect induced by MK-801 administration is not surprising, while it is more di⁄cult to account for the modest e¡ect induced by amphetamine. It is worth noting, however, that similar di¡erences in the e¡ects of systemic administrations of MK801 and amphetamine have been observed on 2DG uptake in the prefrontal cortex (Duncan et al., 1999). This is of interest since both MK-801 and amphetamine have been suggested to be valid pharmacological models of psychosis (Sams-Dodd, 1995; Zhang et al., 1996). Systemic amphetamine increased c-fos expression also in the nucleus accumbens. This e¡ect was mainly due to increased activity in the shell rather than in core subregion. On the contrary no changes in c-fos expression were observed in the N. accumbens or in the other basal ganglia structures analyzed after MK-801 administrations. Changes in c-fos expression in the striatal complex after amphetamine are consistent with previous reports (Graybiel et al., 1990). However, in those studies systemic amphetamine and cocaine increased activity within both ventral and dorsal striatum (Graybiel et al., 1990; Wang et al., 1995). It should be noted that higher doses were used in those studies compared to the one used in the present one. Further, in light of the functional complexity of the dorsal striatum and considering our limited rostrocaudal analyses, regional changes within this structure can not be completely excluded. The selective e¡ect induced by amphetamine in the shell of the accumbens supports the view, based upon behavioral data (Pontieri et al., 1994; Swanson et al., 1997), that psychostimulant induced behavioral e¡ects might be preferentially mediated through this subregion. The most interesting changes observed in this study after systemic administrations, however, were those in the thalamus. Increased c-fos was observed in several thalamic nuclei after systemic administrations of both drugs. Of special interest is the di¡erence in activity patterns induced by the two drugs in the distinct component of the thalamus (Table 4). It is noteworthy, in this regard, that the nuclei which showed enhanced activity after systemic MK-801 are the VL, MD and AV nuclei of the thalamus. On the contrary, amphetamine was void of e¡ects on the ventromedial part of the thalamus, i.e. VLT and VMT, while it increased c-fos expression in the

75

most anterior and limbic component, i.e. anteroventral thalamus (AVT), ADT and MDT nuclei. The thalamus receives glutamatergic a¡erents from several cortical and allocortical regions (Eaton and Salt, 1996; Gonzalo-Ruiz et al., 1997; Reardon and Mitrofanis, 2000) and evidence from several studies demonstrates the presence of NMDA receptors within these nuclei (Jones et al., 1998; Ibrahim et al., 2000). In particular the major input to the VL and VM nuclei is derived from the basal ganglia, by way of the substantia nigra and the entopeduncular nucleus, and the cerebellum. On the other hand, the major source of input to the AV and AD thalamic nuclei is derived from the hippocampal formation and the hypothalamus (Swanson and Cowan, 1977; Irle and Markowitsch, 1982; Seki and Zyo, 1984), the ¢rst one being glutamatergic (Mengual et al., 2001). Finally the MDT, which is classi¢ed as an associative thalamic nucleus, receives indirect GABAergic a¡erent from the N. accumbens, trough the VP, and further ^ glutamatergic ^ projection from allocortical regions such as the amygdala (O’Donnell et al., 1997; Groenewegen et al., 1999; Reardon and Mitrofanis, 2000). The e¡ect observed after systemic MK-801 administrations therefore could be due to two possible actions not mutually exclusive. First of all, MK-801 could act directly in cortical, allocortical and cerebellar regions that have been demonstrated to possess a high density of NMDA receptors and in this way indirectly modulate c-fos expression within the thalamus. Alternatively, a direct action on NMDA receptors located in this region could be responsible for thalamic activation. It should be noted however that in this case the presence of inhibitory inter-neurons needs to be hypothesized and morphological evidence does not always support this view (Price, 1995). Furthermore, ligand binding studies demonstrate that [3 H]MK-801 binding is higher in the limbic rather than in the motor thalamus (Ibrahim et al., 2000), thus if the e¡ect was due to a direct action on the NMDA receptor in these structures an opposite e¡ect would be expected. In light of the e¡ect observed in the shell of the accumbens after systemic amphetamine the increased activity within the MDT (which is the main recipient of N. accumbens e¡erent projections by way of the VP) is not surprising (O’Donnell et al., 1997; Groenewegen et al., 1999; Reardon and Mitrofanis, 2000). AVT and ADT activation, on the contrary, could be due to enhanced activity in the hippocampus. In the present study we did not ¢nd any change in c-fos expression within the hippocampal region (data not shown) and

Table 4. Summary table of the e¡ects observed on c-fos mRNA expression in the di¡erent thalamic nuclei after systemic and intra-accumbens administration of the non-competitive NMDA antagonist MK-801 and the indirect DA agonist amphetamine

Ventrolateral Ventromedial Mediodorsal Anteroventral Anterodorsal

MK-801 systemic

Amph systemic

MK-801 focal

Amph focal

0 C 0 0 C

C C 0 0 0

0 0 0 C C

0 C 0 C 0

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similar results were obtained on 2DG uptake after systemic administrations of behaviorally relevant doses of amphetamine (Duncan et al., 1999). It should be noted that dense projections to the anterior component of the thalamus originate from the hypothalamus (Seki and Zyo, 1984) and neuroanatomical evidence demonstrates that e¡erent projections from the shell but not the core of the accumbens innervate this structure (Groenewegen and Russchen, 1984; Heimer et al., 1991; Stratford and Kelley, 1999). Therefore it is possible that systemic amphetamine acts within the shell of the accumbens to activate the MDT by way of the VP and anterior thalamus through its e¡erent projections to the hypothalamus. The main e¡ect observed after N. accumbens manipulation of both NMDA and DA receptors was enhanced c-fos expression within the thalamus (Table 3). On the contrary no changes in c-fos activity were observed in other nuclei receiving direct or indirect e¡erent projections form the N. accumbens. MK-801 was found to induce changes in the VLT and MDT while amphetamine a¡ected c-fos in ADT and MDT. The increase induced in the ADT, after focal amphetamine, supports the suggestion that the e¡ect observed after systemic amphetamine could be due to an action of amphetamine within the accumbens. There were two main di¡erences observed between focally and systemically administered MK-801 and amphetamine. C-fos expression was increased in the VMT following focal but not systemic MK-801 and amphetamine increased c-fos expression in the VLT following focal but not systemic administrations (Table 4). It should be noted, however, that intra-accumbens amphetamine administrations, at the doses used in the present study, induced slightly higher levels of locomotor activity, compared to MK-801. Even if this di¡erence did not reach statistical signi¢cance it could partially explain the discrepancies found in c-fos expression in the motor thalamus after systemic and focal administrations of the two drugs. Despite these di¡erences c-fos expression in the limbic thalamus was more evident after amphetamine than after MK-801. Based on the present data it is proposed that the thalamus is activated in di¡erent ways by manipulation of NMDA and DA systems in the accumbens. In particular blockade of NMDA receptors in the N. accumbens enhances neural activity in the motor thalamus while amphetamine acting in this structure activates limbic thalamic targets. Finally it should be noted that the MDT which receives projections from pathways respectively through the entopeduncular nucleus and the VP is activated by systemic and focal administrations of both drugs. These data support the behavioral observation that locomotor activity induced by intra-accumbens adminis-

trations of MK-801 is blocked by selective lesions of the ventrolateral/ventromedial nuclei of the thalamus (De Leonibus et al., 2001). Further it has been proposed that increased DA activity within the N. accumbens, induced by focal and systemic administrations of direct and indirect DA agonists, could be translated into increased motor activity through the MD nucleus. This proposition is based on the partial impairment of amphetamine induced increases in motor activity observed after MD thalamic lesions (Swerdlow and Koob, 1987). Such an hypothesis seems to be partially supported by the present observations. The increased c-fos expression, extended to several limbic nuclei of the thalamus, described in the present study however suggests that activation of the MDT may not be the only determinant of psychostimulant induced activity. This could also possibly explain the partial blockade observed after mediodorsal thalamic lesions.

CONCLUSION

The present data con¢rm previous observations that MK-801 induced e¡ects are mediated by structures di¡erent from those responsible for amphetamine induced activity (Mele et al., 1998; Kretschmer, 2000; De Leonibus et al., 2001). This makes it further unlikely that the motor e¡ect exerted by NMDA antagonists depends on increased DA activity (Imperato et al., 1990), otherwise a somewhat similar pattern of c-fos induction should have been observed. Further it con¢rms the behavioral observation (Mele et al., 1998; De Leonibus et al., 2001) that MK-801 and amphetamine induced activity could be mediated by di¡erent outputs of the accumbens. In particular NMDA blockade within the accumbens seems to exert its e¡ects through the motor component of the thalamus while amphetamine seems to modulate the limbic component. This working hypothesis based on the present as well as previous studies (Swerdlow and Koob, 1987; De Leonibus et al., 2001) of course would need further experimental support from neuropharmacological as well as lesion studies. Finally, it might also be of interest to speculate on the di¡erent roles that the two components of the thalamus may have in modulating complex behavioral responses and the di¡erential roles of NMDA and DA receptors within the N. accumbens in regulating such responses. Acknowledgements.The authors would like to thank dr. Andrea Felici and Mr. Tim Sullivan for their valuable help in running the experiments. This study has been partially supported by a C.N.R. grant to A.M. and a MURST grant ‘Neurofarmacologia dell’apprendimento e della memoria’ to A.O.

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