For pharmacological experiments, adult male wild type C57BL/6 mice (Charles River, St-Constant, QC, Canada) and mice lacking dopamine D_2L receptors [D_2L receptor knockouts, D_2L(−/−)] and their littermates (Usiello et al., 2000) were used. All mice weighted 20–25 g and were housed five per cage in a temperature-controlled environment maintained under a 12-h light/dark cycle with ad libitum access to food and water. For experiments involving ibotenic acid lesions, we used male Sprague–Dawley rats (Charles River, St-Constant, QC, USA) weighing 280–320 g. Experimental procedures, including means to minimize discomfort, were reviewed and approved by the institutional Animal Ethics Committee of the Université de Montréal and were done in accordance with the Canadian Council on Animal Care guidelines for use of experimental animals.

The selective dopamine D₂/D₃ receptor antagonist eticlopride, cannabinoid CB₁ receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251), muscarinic antagonist scopolamine, and selective adenosine A_2A antagonist SCH 58261 were purchased from Sigma–Aldrich (St. Louis, MO, USA). The selective mGlu5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), mGlu1/5 agonist (RS)-3,5-Dihydroxyphenylglycine (DHPG), selective mGlu5 agonist (RS)-2-Chloro-5-hydroxyphenylglycine sodium salt (CHPG), selective A_2A agonist 4-[2-[[6-Amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene propanoic acid hydrochloride (CGS 21680), and excitatory amino acid transporters 1-2 (EAAT1-2) blocker (3S)-3-[[3-[[4-(Trifluoromethyl)benzoyl] amino]phenyl]methoxy]-l-aspartic acid (TBOA) were purchased from Tocris Bioscience (Avonmouth, UK). Non-competitive NMDA antagonists phencyclidine (PCP) and MK-801 were obtained from Sigma–Aldrich through a restricted importation permit. PCP analogs N-[1-(2-benzo(β)thiophenyl)cyclohexyl]piperidine (BTCP) and N-[1-(2-thiophenyl)cyclohexyl]piperidine (TCP) were obtained as a gift from Dr. J. M. Kamenka and the National Institute of Mental Health Chemical Synthesis Program (Rouillard et al., 1990).

First, we compared the effect of vehicle (NaCl 0.9%) and eticlopride (1 mg/kg) on striatal Nur77 mRNA expression in wild type [D_2L(+ / +)] and D_2L(−/−) mice in order to assess the contribution of D_2S and D_2L receptor isoforms in the effect of the D₂ antagonist. We used eticlopride, a highly selective D₂/D₃ receptor antagonist, because typical antipsychotic drugs used in clinics display a wider pharmacological profile, which could further complicate data interpretation. Note that eticlopride displays activities similar to typical antipsychotic drugs, but it is not used in clinic because of its poor pharmacokinetic properties (Martelle and Nader, 2008). Secondly, we assessed the effect of glutamatergic drugs on Nur77 gene transcription. We acutely treated (0.25 ml, i.p.) five different groups of wild type mice (N = 5) as follows: (a) vehicle (NaCl 0.9%); (b) MK-801 (non-competitive NMDA receptor antagonist, 0.75 mg/kg); (c) PCP (5 mg/kg); (d) BTCP (16 mg/kg); and (e) TCP (0.75 mg/kg). Then, lower doses of MK-801 were used alone or in combination with eticlopride. Groups of mice were formed as follows: (a) vehicle (NaCl 0.9%; N = 6); (b) eticlopride (1 mg/kg; N = 5); (c) MK-801 (0.3 mg/kg; N = 5); (d) MK-801 (0.03 mg/kg; N = 5); (e) eticlopride (1 mg/kg) + MK-801 (0.3 mg/kg); (f) eticlopride (1 mg/kg) + MK-801 (0.03 mg/kg). MK-801 was injected 15 min before eticlopride. In the third experiment, involvement of mGlu5 and A_2A receptor drugs on eticlopride-induced Nur77 upregulation was investigated. Animals were distributed into eight groups and treated as follows: (a) vehicles (NaCl 0.9 and 3% DMSO, 8% PEG600 in sterile water); (b) MPEP (mGlu5 antagonist, 10 mg/kg); (c) SCH58261 (A_2A antagonist, 5 mg/kg); (d) MPEP + SCH58261; (e) eticlopride (dopamine D₂ antagonist, 1 mg/kg); (f) eticlopride (1 mg/kg) + MPEP (10 mg/kg); (g) eticlopride (1 mg/kg) + SCH58261 (5 mg/kg); (h) eticlopride (1 mg/kg) + MPEP + SCH58261 (N = 5 per group). MPEP and SCH58261 were administered 30 min before the dopamine antagonist. A similar paradigm was used to investigate the effect of the CB₁ antagonist AM251 (5 mg/kg, i.p.) and muscarinic m1-4 antagonist scopolamine (2.5 mg/kg, i.p.) on eticlopride-induced Nur77 expression. All the drug doses used were chosen based on previous studies using similar paradigms; eticlopride (Keefe and Adams, 1998; Pozzi et al., 2003; Bourhis et al., 2008), ionotropic glutamatergic drugs (Rouillard et al., 1990; Keefe and Adams, 1998; Chartoff et al., 1999), MPEP (Choe et al., 2002; Parelkar and Wang, 2004), SCH58261 (Pollack and Fink, 1995; Pinna et al., 1999), AM251 (Xi et al., 2006; Rubino et al., 2007), and scopolamine (Guo et al., 1992; Wang and McGinty, 1996). For all treatments, mice were sacrificed by decapitation 60 min after the last drug injection under CO₂ anesthesia. Brains were rapidly removed, immediately immersed into cold 2-methylbutane (−40°C) for a few seconds and kept frozen at −80°C until used.

Probe preparation and in situ hybridization of Nur77 mRNA on brain slices were performed as previously reported (Beaudry et al., 2000; St-Hilaire et al., 2003; Maheux et al., 2005). Briefly, the Nur77 single-stranded riboprobe was synthesized and labeled using Promega riboprobe kit (Promega, Madison, WI, USA), [³⁵S]UTP (PerkinElmer Life and Analytical Sciences, Woodbridge, ON, Canada), and the RNA polymerase T₃. In situ hybridization of the riboprobe with cryostat coronal brain sections (12 μm) mounted on Snowcoat X-tra slides (Surgipath, Winnipeg, MA, Canada) was done at 58°C overnight in a standard hybridization buffer containing 50% formamide. Brain sections were then apposed against BiomaxMR radioactive sensitive films (Eastman Kodak, New Haven, CT, USA) for 2 days.

Levels of autoradiographic labeling on films were quantified by computerized densitometry as previously described (Beaudry et al., 2000; St-Hilaire et al., 2003; Maheux et al., 2005). Optical density of the autoradiograms was translated in nCi/g of tissue using [¹⁴C]-radioactivity standards (ARC 146A-¹⁴C standards, American Radiolabeled Chemicals Inc., St. Louis, MO, USA). Nur77 mRNA levels were measured in the dorsomedial (StDM), dorsolateral (StDL), ventromedial (StVM), and ventrolateral (StVL) portions of the striatum, and the nucleus accumbens shell (AcSh) and core (AcC). The average level of labeling for each area was calculated from three to four adjacent brain sections of the same animal. Background intensity was subtracted from every measurement.

All data are expressed as group mean ± SEM and statistical analysis were performed using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical comparisons between groups were obtained by using a one-way analysis of variance and a Bartlett’s test for equal variance. When the Bartlett’s test showed significant differences between variance, the log, or square root of data were used in the analysis. One-way analyses of variance were followed by a Tukey’s multiple comparison test as a post hoc test when appropriate.

Brain sections were pre-incubated in a 50 mM Tris-HCl buffer, pH 7.4, containing 120 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5.7 mM ascorbic acid, and 10 μM hydroxyquinoline for 15 min. The slides were then incubated for 1 h a room temperature in the same buffer containing 0.2 nM [¹²⁵I]iodosulpride (Martres et al., 1985). Non-specific binding was evaluated in the presence of 2 μM raclopride. After 2 min rinses in ice-cold buffer, sections were dipped for 10 s in cold water, dried, and apposed to BioMax film. Autoradiograms were generated after an exposure time of 24 h.

Rats were anesthetized with isoflurane (2.5–3.5%, O₂ 0.6 L/min) and mounted on a stereotaxic apparatus. The surface of cranium was exposed and the bone and dura above the left or right sensorimotor cortex was removed. A needle (300 μm in diameter) containing 5 μg/μl of ibotenic acid, or its vehicle, was inserted into the cortex at five anterior-posterior (0.5 mm apart) and 2–4 medial-lateral sites using the following flat skull coordinates: between 0.5 posterior and 1.5 mm anterior to Bregma; 1.4–3.3 mm lateral to the sagittal line, and 1.2–2.2 mm below the surface of the cortex. At each site, the needle was lowered to 2.2 mm below the cortex and a volume of 0.15 μl solution was injected over a minute; two (three for the most lateral and anterior sites) similar injections were made 0.5 and 1.0 mm above. The solution was injected using a microinfusion pump to activate a Hamilton microsyringe connected to the injection needle by polyethylene tubing. Similar saline injections were performed in sham animals. Ten days after surgery, animals were injected with eticlopride (1 mg/kg, i.p.) or saline and returned to their home cage. Sixty minutes after the injection, they were anesthetized with isoflurane (3.5%, O₂ 0.6 L/min) and killed by decapitation. The brains were quickly removed dipped in cold 2-methylbutane and stored at −80°C.

Corticostriatal organotypic slice cultures were prepared from 4–5 days old mice using the methods of Stoppini et al. (1991) and Stahl et al. (2009) with minor modifications (Stoppini et al., 1991; Stahl et al., 2009). Briefly, mouse brains were extracted and immerged in complete Hank’s buffer solution supplemented with glucose (5.6 mM) and sucrose (27.8 mM). Coronal 400 μm slices were cut using a McIlwain tissue chopper (Havard Apparatus, St. Laurent, QC, USA). Slices containing the striatum were transferred on Millicell filter inserts (0.4 μm; Millipore, Fisher scientific, Whitby, ON, USA) placed into a six-well plate filled with 1 ml of neurobasal medium containing 10% FBS, 1× N-2 supplement, 1× glutamine, 1× antibio-antimyc, and 0.6% glucose and maintained in culture for 3 days. Slices were then serum deprived for 14 h before pharmacological treatments. Drugs such as TBOA, DHPG, CHPG, and CGS 21680 were applied for 1 h directly to the culture medium. MPEP or quinpirole were applied 15 min prior subsequent treatments. Striata were removed using a glass Pasteur pipette as a tissue punches.

Striatal tissue samples were expelled directly in Trizol reagent for RNA extraction (Sigma–Aldrich, St. Louis, MO, USA). Reverse transcriptase reactions of 2 μg RNA were performed in a final volume of 20 μl using the High Capacity cDNA Reverse Transcription Kit with random primers (Applied Biosystems, Streetsville, ON, USA). Complementary DNA samples (1.5 μl) were used for SYBR green qPCR amplification of Nur77 (Nr4a1; NM_010444.2) using Fast SYBR Green Master Mix (Applied Biosystems, Streetsville, ON, USA). Gene expression level for endogenous controls was determined using pre-validated Taqman Gene Expression Assays (Applied Biosystems, Streetsville, ON, USA). Four endogenous controls [glyceraldehyde-3-phosphate (GAPDH), hypoxanthine guanine phosphoribosyl transferase (HPRT1), β-actin, ACTB, and TATA binding protein (TBP)] were first assessed to determine which ones had the more stable expression in our experimental conditions. Further analysis of each sample was controlled using both GAPDH (NM_008084.2) and HPRT1 (NM_013556). The ABI PRISM^® 7900HT Sequence Detection System (Applied Biosystems, Streetsville, ON, USA) was used to detect amplification levels. All reactions were run in triplicate and average values of cycle thresholds (CTs) were used for quantification. The relative quantification of target genes was determined using the ∆∆CT method.

We first investigated the contribution of D_2S and D_2L receptors in the modulation of Nur77 mRNA expression in the striatum by comparing basal and eticlopride-induced Nur77 mRNA levels in wild type (+/+) and D_2L knockout (−/−) mice (Usiello et al., 2000; Lindgren et al., 2003). D_2L(−/−) mice displayed a significant reduction in basal Nur77 mRNA levels in the dorsomedial striatum, but basal transcript levels were unchanged in all other striatal subterritories, as compared with their wild type littermates (Figure 1). As expected, administration of eticlopride in wild type mice led to a strong increase of Nur77 transcript levels (Figure 1). Surprisingly, this modulation was still present in D_2L mutant mice (Figure 1), and was even significantly stronger when compared to their wild type littermates in most striatal subterritories (Figure 1). These data indicate that D_2L receptors might not play an important role in the effect of eticlopride-induced Nur77 expression in the striatal tissue.

In order to investigate whether integrity of the corticostriatal pathway, and therefore involvement of presynaptic D₂ heteroreceptors in eticlopride-induced Nur77 mRNA in the striatum, we proceeded to unilateral lesions of corticostriatal fibers using intra cortical injections of ibotenic acid. Ibotenic acid injections led to the lesion of most cortical neurons located in the primary motor cortex (M1) and affected all layers of the cortex. In some individuals, the lesion extended to the primary sensorimotor cortex (Figures 2A–C). Nissl staining (data not shown) and [¹²⁵I] iodosulpride specific binding to dopamine D₂ receptors showed that underlying subcortical regions were left intact (compared Figures 2B,C). As expected, eticlopride-induced a strong increase in Nur77 mRNA levels in control animals bearing a sham lesion (Figure 2D). On the other hand, unilateral cortical lesion almost totally prevented eticlopride-induced upregulation of Nur77 mRNA levels in the ipsilateral dorsal striatum (Figure 2D). Eticlopride-induced striatal Nur77 mRNA levels were comparable to sham-lesioned animals in the contralateral side (Figure 2D). These data clearly indicate that the integrity of corticostriatal inputs is necessary for the upregulation of Nur77 mRNA levels by the D₂ antagonist in these striatal subterritories.

We then investigated if glutamate receptors located on postsynaptic striatal medium spiny neurons might contribute to the effect of the D₂ antagonist. To test this hypothesis, we combined the administration of eticlopride with ionotropic or metabotropic glutamate receptor antagonists. Although the modulation of Nur77 mRNA levels by dopamine D₂ antagonists has been previously described (Beaudry et al., 2000; Werme et al., 2000; Maheux et al., 2005), modulation of this transcription factor by glutamate receptors remains largely unexplored. Thus, we first evaluated the effect of NMDA antagonists on striatal Nur77 mRNA levels in wild type mice. Both MK-801 (0.75 mg/kg) and PCP (5 mg/kg) strongly reduced basal Nur77 mRNA levels in the striatal complex (Figure 3). We also used two PCP analogs to confirm the role of NMDA receptors in the effect of PCP. TCP is a PCP analog, which display a stronger affinity for the PCP site on the NMDA receptor than PCP itself, whereas BTCP is a PCP analog characterized by a higher affinity for the dopamine transporter (Rouillard et al., 1990). A dose equivalent of BTCP produced a strong increase in Nur77 expression (Figure 3). This effect is consistent with the BTCP psychostimulant-like property, since it has been previously shown that psychostimulants increased Nur77 mRNA levels (Bäckman and Morales, 2002; Bhardwaj et al., 2003). A PCP dose equivalent of TCP based on their affinity for the PCP site did not induce Nur77 mRNA levels, but rather tended to reduce it (however, it did not reach significance). Thus, we may conclude from these experiments that basal striatal Nur77 expression is under a tonic control by NMDA receptors.

In the next step, we investigated the role of NMDA receptors into the upregulation of Nur77 mRNA expression induced by the D₂ antagonist eticlopride. Since the initial dose of MK-801 (0.75 mg/kg) induced a strong reduction of basal Nur77 expression (Figure 3), which could be mixed up with the putative effect of NMDA on eticlopride-induced Nur77 expression, we investigated two lower doses of MK-801 (0.03 and 0.3 mg/kg) alone or in combination with eticlopride (Figures 4A–D). At these concentrations, MK-801 was still able to reduce basal Nur77 levels, but to a lesser extent (Figures 4A–D). However, both MK-801 doses did not alter eticlopride-induced Nur77 expression in lateral striatal subterritories (Figures 4B,D) and the lower dose of MK-801 (0.03 mg/kg) also remained without effect in medial portions of the striatum (Figures 4A,C). Note that a similar low dose of MK-801 can modulate c-fos mRNA levels in the brainstem, as well as to reduce reward threshold induced by electrical self-stimulation of the ventral tegmental area (Hattori et al., 2004; Clements and Greenshaw, 2005), indicating that this dose is effective.

Since these results suggested a modest contribution of NMDA receptors in eticlopride-induced Nur77 mRNA levels in the striatum, we investigated the role of the metabotropic glutamate mGlu5 receptor and its partner, the adenosine A_2A receptor (Figures 4E–I). Administration of MPEP (mGlu5 antagonist) or SCH58261 (A_2A antagonist) alone had no effect on Nur77 mRNA expression in the brain areas analyzed (Table 1). When co-administered with eticlopride, SCH58261 had no significant effect on the upregulation of Nur77 mRNA expression (Figures 4E–I). Selective blockade of mGlu5 receptor with MPEP tended to reduce eticlopride-induced Nur77 mRNA levels, but this effect reached significance only in the dorsolateral portion of the striatum (Figure 4G). Interestingly, co-administration of both MPEP and SCH58261 strongly reduced eticlopride-induced Nur77 mRNA levels in all striatal subterritories, restoring Nur77 mRNA levels back to baseline (Figures 4E–I).

To complement the pharmacological characterization of the dopamine D₂ receptor antagonist effect, we also investigated the contribution of cannabinoid and muscarinic drugs in the modulation of striatal Nur77 mRNA levels induced by eticlopride. Systemic injections of AM251, a CB₁ receptor antagonist or scopolamine, a muscarinic m1-4 receptor antagonist, alone did not modulate Nur77 mRNA levels in the ventrolateral portion of the striatum (Figure 5). Co-administration of AM251 or scopolamine with the D₂ antagonist did not reduce eticlopride-induced Nur77 expression in the ventrolateral portion of the striatum (Figure 5). In fact, scopolamine significantly further increased eticlopride-induced Nur77 expression (Figure 5).

To directly demonstrate the contribution of mGlu5 and adenosine A_2A receptor subtypes to the modulation of Nur77 mRNA expression, we tested whether an increase of glutamatergic neurotransmission or mGlu5 and A_2A receptor activation can modulate Nur77 mRNA levels in corticostriatal organotypic slices in culture. To this end, we used TBOA, which is a high affinity blocker of glial excitatory amino acid transporters 1 and 2 (EAAT1-2), which has been shown to increase glutamate concentration in acute slice preparations (Beurrier et al., 2009). TBOA alone produced a nice and strong dose-dependent Nur77 mRNA induction (Figure 6A). Pre-treatment of organotypic cultures with MPEP, a selective mGlu5 receptor antagonist, significantly reduced the effect of the low dose of TBOA on Nur77 mRNA induction, confirming the important role of mGlu5 receptor in the induction of Nur77 gene transcription by glutamate (Figure 6B). We therefore tested direct activation of mGlu5 receptor in organotypic corticostriatal slices. Exposition to DHPG, a mGlu1/5 agonist, significantly increased Nur77 mRNA expression in the striatum by approximately fivefold (Figure 6C). This effect was selective for mGlu5 receptors since co-administration of MPEP led to a complete blockade of DHPG-induced Nur77 mRNA upregulation (Figure 6C). Additionally, activation of mGlu5 receptor with a more specific agonist (CHPG) also led to a strong increase of Nur77 mRNA levels (Figure 6D). As previously observed in vivo, this effect can be potentiated by a concomitant activation of A_2A receptors with CGS21680 (Figure 6D). Direct exposure of corticostriatal organotypic slices to eticlopride or quinpirole (dopamine D₂ agonist) alone or in combination with TBOA did not modulate Nur77 mRNA levels (Figures 6E–G). In addition, both D₂ agonist and antagonist drugs were not able to modulate mGlu5/A_2A agonist-induced striatal Nur77 mRNA levels in organotypic slice preparations (Figure 6H). These results suggest that postsynaptic D₂ receptors do not significantly contribute to striatal transcriptional activity in the present experimental conditions.