European Neuropsychopharmacology (2014) 24, 1524–1533

www.elsevier.com/locate/euroneuro

Reduced striatal dopamine DA D2 receptor function in dominant-negative GSK-3 transgenic mice Raquel Gomez-Sintesa,c,1, Analia Bortolozzib,d,1, Francesc Artigasb,d,n, José J. Lucasa,c,nn Centro de Biología Molecular “Severo Ochoa” (CBM“SO”), CSIC/UAM, 28049 Madrid, Spain Department of Neurochemistry and Neuropharmacology, IIBB – Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CSIC, Barcelona, Spain c Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain d Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Instituto de Salud Carlos III, Madrid, Spain a

b

Received 7 December 2013; received in revised form 7 June 2014; accepted 11 July 2014

KEYWORDS

Abstract

Basal ganglia; Dopamine receptors; Dorsal striatum; Glycogen synthase kinase-3 (GSK-3); Schizophrenia

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase with constitutive activity involved in cellular architecture, gene expression, cell proliferation, fate decision and apoptosis, among others. GSK-3 expression is particularly high in brain where it may be involved in neurological and psychiatric disorders such as Alzheimer's disease, bipolar disorder and major depression. A link with schizophrenia is suggested by the antipsychotic drug-induced GSK-3 regulation and by the involvement of the Akt/GSK-3 pathway in dopaminergic neurotransmission. Taking advantage of the previous development of dominant negative GSK3 transgenic mice (Tg) showing a selective reduction of GSK-3 activity in forebrain neurons but not in dopaminergic neurons, we explored the relationship between GSK-3 and dopaminergic neurotransmission in vivo. In microdialysis experiments, local quinpirole (DA D2-R agonist) in dorsal striatum reduced dopamine (DA) release significantly less in Tg mice than in wild-type (WT) mice. However, local SKF-81297 (selective DA D1-R agonist) in dorsal striatum reduced DA release equally in both control and Tg mice indicating a comparable function of DA D1-R in the

n Corresponding author at: Department of Neurochemistry and Neuropharmacology, IIBB-Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CSIC, Barcelona, Spain and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Madrid, Spain. Tel.: +34 93 363 8315/+34 93 363 2381; fax: +34 93 363 8301. nn Corresponding author at: Centro de Biología Molecular “Severo Ochoa” CSIC/UAM/CiberNed Campus UAM Cantoblanco, 28049 Madrid, Spain. Tel.: +34 91 196 4552/ +34 91 196 4582; fax: +34 91 196 4420. E-mail addresses: [email protected] (F. Artigas), [email protected] (J.J. Lucas). URL: http://www.cbm.uam.es/lineas/lucasgroup.htm (J.J. Lucas). 1 These authors equally contributed to this work.

http://dx.doi.org/10.1016/j.euroneuro.2014.07.004 0924-977X/& 2014 Elsevier B.V. and ECNP. All rights reserved.

Reduced striatal dopamine DA D2 receptor function in dominant-negative GSK-3 transgenic mice

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direct striato-nigral pathway. Likewise, systemic quinpirole administration – acting preferentially on presynaptic DA D2- autoreceptors to modulate DA release-reduced striatal DA release similarly in both control and Tg mice. Quinpirole reduced locomotor activity and induced c-fos expression in globus pallidus (both striatal DA D2-R-mediated effects) significantly more in WT than in Tg mice. Taking together, the present results show that dominant negative GSK-3 transgenic mice show reduced DA D2-R-mediated function in striatum and further support a link between dopaminergic neurotransmission and GSK-3 activity. & 2014 Elsevier B.V. and ECNP. All rights reserved.

1.

Introduction

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that is present in most tissues and that is particularly abundant in the CNS (Woodgett, 1990). This enzyme has two isoforms (GSK-3α and GSK-3β) and participates in multiple signaling transduction cascades such as the insulin and wnt pathways (Woodgett, 1990; Jope and Johnson, 2004). A distinctive feature of GSK-3 is its ability to be active under resting conditions. Activation of above-mentioned signaling pathways results in GSK-3 inhibition by phosphorylation on a serine residue on its N-terminus (Ser21 and Ser9 in GSK-3α and GSK-3β, respectively) (Grimes and Jope, 2001). The many phosphorylation substrates of GSK-3 include cytoskeletal proteins, transcription factors, and metabolic regulators, highlighting a prominent role for GSK-3 in cellular architecture, gene expression, cell division and fate decision, and apoptosis among others (Grimes and Jope, 2001; Jope and Johnson, 2004). Aberrantly increased GSK-3 activity is believed to play a key role in the pathogenesis of chronic metabolic disorders like Type-II diabetes (Eldar-Finkelman, 2002) as well as under CNS conditions such as bipolar disorder, major depression, schizophrenia and Alzheimer's disease (Jope and Johnson, 2004; Jope and Roh, 2006; Amar et al., 2011; Hernandez et al., 2013). In particular, an abnormally high GSK-3 activity has been suggested to occur in bipolar disorder (Jope and Roh, 2006), schizophrenia (Emamian et al., 2004; Lovestone et al., 2007; Karam et al., 2010; Beaulieu, 2012) and Alzheimer's disease (AD) (Avila et al., 2004; Hooper et al., 2008; Hernandez et al., 2013). Accordingly, potent and specific GSK-3 inhibitors are currently under development (Cohen and Goedert, 2004; Jope and Roh, 2006; Medina and Avila, 2010). In psychiatric disorders, given its inhibitory activity (Klein and Melton, 1996; O’brien, Klein, 2009), lithium was used as mood stabilizer and to augment antidepressant efficacy in treatment resistant depression. Recent evidence also suggests a role of GSK-3 inhibition in antidepressant effects (Li and Jope, 2010; Wilkinson et al., 2011; Liu et al., 2013; Zunszain et al., 2013). A role for GSK-3 in schizophrenia is suggested by two main lines of evidence. On the one hand, dopamine DA D2-R blockade by classical and atypical antipsychotic drugs regulates forebrain GSK-3 levels (Alimohamad et al., 2005; Roh et al., 2007). On the other hand, Akt/GSK-3 signaling pathways are involved in dopaminergic neurotransmission (Beaulieu et al., 2007; Del’guidice and Beaulieu, 2008; Beaulieu et al., 2009; Li and Gao, 2011). Thus,

pharmacological inhibition of GSK-3 has been suggested as a new target in the treatment of psychiatric disorders (Eldar-Finkelman, 2002; Gould et al., 2004; Gould and Manji, 2005; Beaulieu et al., 2009; Li and Gao, 2011). Here we further explore the relationship between striatal DA D2-R signaling and GSK-3 in vivo, taking advantage of the previous development of transgenic mice with a regionallyselective reduction of the expression of GSK-3 in forebrain (dominant-negative GSK-3 transgenic mice, DN-GSK-3β; (Gomez-Sintes et al., 2007). We examined the effects of the selective DA D2-R agonist quinpirole (Bolaños-Jiménez et al., 2011) on DA D2 receptor-mediated functions in wild-type (WT) and dominant-negative GSK-3 transgenic (Tg) mice.

2. 2.1.

Experimental procedures Tet/DN-GSK-3 mice

Mice were bred at Centro de Biología Molecular “Severo Ochoa” animal facility. Mice were housed four per cage with food and water available ad libitum and maintained in a temperature-controlled environment on a 12/12 h lightdark cycle with light onset at 07:00 h. DN-GSK-3β mice (Tg mice) were generated, as previously described in GomezSintes et al., 2007, and maintained in a C57/BL6 background (Harlan). The CamkII-tTa mouse line (Mayford et al., 1996) was also maintained in a C57BL/6 background. Finally Tet/ DN-GSK-3 mice are double transgenic mice that result from crossing CamkII-tTa mice with DN-GSK-3β mice. Animal housing and maintenance protocols followed the guidelines of Council of Europe Convention ETS123, recently revised as indicated in the Directive 86/609/EEC. Animal experiments were performed under protocols (P15/P16/P18/P22) approved by the Centro de Biología Molecular Severo Ochoa Institutional Animal Care and Local Utilization Committee (CEEA-CBM), Madrid, Spain.

2.2.

Drug administration

In microdialysis experiments, quinpirole (Sigma-RBI, Spain) was locally (reverse dialysis) or systemically administered (1 mg/kg i.p. at a volume of 4 ml/kg). Likewise, the selective dopamine DA D1-R agonist SKF81297 (Sigma-RBI) and the classical antipsychotic drug haloperidol (dopamine DA D2-R antagonist) (Sigma-RBI) were also used. The drugs were dissolved in saline or artificial cerebrospinal fluid (aCSF), as required. Concentrated solutions (1 mM; pH

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adjusted to 6.5–7 with NaHCO3 when necessary) were stored at 80 1C and working solutions were prepared daily by dilution in aCSF. In behavioral experiments and histological analysis, Tg mice and corresponding controls (WT littermates) were injected intraperitoneally with quinpirole 1 mg/kg or vehicle (0.9% NaCl). Behavioral testing started performed 30 min after injection. For histological assessments, mice were sacrificed 3 h after the injection.

2.3.

Microdialysis

Microdialysis experiments were performed essentially as previously described. A detailed description of the probe manufacture and microdialysis procedures in mice can be found elsewhere (Diaz-Mataix et al., 2005). Briefly, anesthetized (sodium pentobarbital, 30 mg/kg, i.p.) wild-type (WT) and dominant negative GSK-3 transgenic mice (Tg) were stereotaxically implanted with concentric microdialysis probes equipped with a Cuprophan membrane (1.5-mm long) in the caudate-putamen (dorsal striatum) at the following co-ordinates (in mm): AP þ + 0.5, L 1.7, DV 4.5, according to Franklin and Paxinos (1997). Microdialysis was performed in freely moving mice 420 h after surgery. Probes were perfused with aCSF (125 mM NaCl, 2.5 mM KCl, 1.26 mM CaCl2 and 1.18 mM MgCl2) containing 10 mM of the DA transporter inhibitor nomifensine pumped at 1.5 ml/min. Dialysate fractions were collected each 20 min. After a 100-min stabilization period, four fractions were collected to obtain basal values before local (reverse dialysis) or systemic drug administration. Thereafter, successive 20-min dialysate samples were collected. The concentration of DA in dialysate samples was determined by HPLC, using a modification of a previously described method (Ferre et al., 1994). Brain dialysates were collected on micro vials containing 5 ml of 10 mM perchloric acid and were rapidly injected into the HPLC. DA was amperometrically detected at 5–7.5 min with an absolute limit of detection of detection of 2–3 fmol/sample using an oxidation potential of þ + 0.75 V (Hewlett-Packard 1049 amperometric detector)

2.4.

Locomotor activity

Locomotor activity was measured in clear plexiglas boxes measuring 43.2 cm  43.2 cm, outfitted with photo-beam detectors for monitoring horizontal and vertical activity. Activity levels were recorded with a MED Associates' Activity Monitor (MED Associates, St. Albans, VT). Locomotor activity data were collected via a PC and was analyzed with the MED Associates' Activity Monitor Data Analysis software. Thirty minutes after drug or vehicle injections, mice were placed in a corner of the open-field apparatus and left to move freely. Variables recorded included: ambulatory distance (cm), ambulatory counts (number of beam crosses), ambulatory time (s), stereotypies (number of repetitive beam breaks), stereotypic time (s), resting time (s), rearings (vertical counts) and vertical time (s). Data were individually recorded for each animal during 210 min and presented in 10-min blocks, starting 30 min after the injection of quinpirole or saline.

2.5.

Immunofluorescence

Mice were sacrificed using CO2 and brains immediately removed and dissected on an ice-cold plate. Left hemispheres were left overnight in 4% paraformaldehyde in Sorensen's phosphate buffer (PFA), and then immersed in 30% saccharose in PBS for 72 h. Once cryoprotected, the samples were included in OCT compound (Sakura Finetek Europe), frozen and stored at 80 1C until analysis. 30 mm coronal sections were cut on a CM 1950 Ag Protect freezing microtome (Leica) and stored free floating in glycol containing buffer (30% glycerol, 30% ethylenglycol in 0,02 M phosphate buffer) at 20 1C. For immunofluorescence analysis, 30 mm coronal brain sections were pretreated with 0,1% Triton X-100 for 15 min, 1 M Glycin for 30 min, and blocking solution (1% BSA and 0,1% Triton X-100) for 1 h. Sections were then incubated overnight at 4 1C with primary antibodies in the blocking solution at the following concentrations: β-gal (monoclonal, Promega, 1:1000), β-gal (polyclonal, ICN Biomed.-Cappel, 1:1000), DARPP-32 (monoclonal, Promega, 1:1000) and TH (polyclonal, Pel-Freez, 1:400). On the following day, sections were washed in PBS and incubated with donkey anti-rabbit Alexa 488, goat antimouse Alexa 488, donkey anti-rabbit Alexa 555 and goat anti-mouse Alexa 555 (Invitrogen, 1:1000) secondary antibodies for 1 h. Finally, nuclei were counterstained with DAPI (1:5000, Calbiochem). Sections were mounted on glass slides, coverslipped with Mowiol mounting solution and maintained at 4 1C. Colocalization of markers was identified by taking successive Alexa 555 and Alexa 488 fluorescent images using a Laser Confocal LSM710 camera (Zeiss) coupled to an inverted microscope AxioImager.M2 (Zeiss).

2.6.

Immunohistochemistry

Brain sections were pretreated for 30 min in 1% H2O2/PBS followed by 1 h with 1% BSA, 5% FBS and 0.2% Triton X-100 and incubated overnight at 4 1C with cFos K-25, Santa Cruz Biotechnology (1:1000). Finally, brain sections were incubated in avidin–biotin complex using the Elite Vectastain kit (Vector Laboratories). Chromogen reactions were performed with diaminobenzidine (SIGMAFAST™ DAB, Sigma) for 10 min. Sections were mounted on glass slides and coverslipped with Mowiol mounting solution. For quantification of immuno-stainings, three coronal sections matching the lateral 0.26 mm plane as in Paxinos and Franklin mouse brain atlas (1997) were selected from each animal. For quantification, images were taken with a Vertical Axioskop2 plus (Zeiss) microscope coupled to a Color view CCD camera. Counting of c-fos positive cells was performed with Image J software. All analyses were performed blind and results were presented as number of immunoreactive cells per region in a 30 mm section. Number of animals analyzed: wt saline, n = 7; wt quinpirole, n= 4; Tg saline, n =5; Tg quinpirole, n =4.

2.7.

Data analysis

Data were analyzed by repeated or independent measures ANOVA, with genotype and treatment as main factors (time

Reduced striatal dopamine DA D2 receptor function in dominant-negative GSK-3 transgenic mice as repeated factor in microdialysis and behavioral experiments). Results are given as mean7SEM of the number of mice indicated in the text. Statistical significance has been set at the 95% level.

3.

Results

3.1. Tet/DN-GSK-3 mice mice show transgene expression in striatal neurons but not in dopaminergic neurons The K85R mutation in GSK-3 results in a dead kinase thus having a dominant negative effect (Dominguez et al., 1995). Based on this, we previously generated Tet/DN-GSK-3 mice (Tg mice) that are double transgenic mice carrying the CamKIIα-tTA transgene as well as the bidirectional mycK85R-GSK-3-TetO-β-Gal transgene (Gomez-Sintes et al., 2007). As a result of this combination of transgenes, Tg mice have decreased GSK-3 activity selectively in forebrain neurons. Besides, B-Gal staining can be used to monitor the neuronal populations that express the transgenes and that, accordingly, have diminished GSK-3 acivity. We have previously shown that Tg mice express the transgene in striatal neurons (Gomez-Sintes et al., 2007). This is further shown in Fig. 1 that also demonstrates the absence of transgene expression in dopaminergic neurons. More precisely, to

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confirm transgene expression in striatal neurons and to rule out expression in dopamine neurons, double immunofluorescence of β-gal and specific markers were performed (Fig. 1). Expression of the transgene was found in GABAergic neurons of striatum (DAPP32 positive cells) (Fig. 1A and B) but no transgene expression was found in TH-positive cells of substantia nigra (SN) or ventral tegmental area (VTA) (Fig. 1C and D).

3.2.

Basal values of DA in striatum dialysates

Upon demonstration of transgene expression in striatal but not dopaminergic neurons of Tg mice, we then analyzed basal values of DA in striatum dialysates. Basal extracellular levels of DA in dialysates from striatum of WT and Tg mice were 226.3739.1 and 209.8742.7 fmol/fraction-20 min, respectively. Non-significant differences were found between mice.

3.3. Effect of quinpirole on in vivo DA release in dorsal striatum The local application of quinpirole (10 mM, 40 min) by reverse dialysis in the dorsal striatum of WT mice (n= 8) evoked a maximal reduction of the DA output to 5578% of baseline at the end of the perfusion period, with a

Fig. 1 Expression of the reporter of transgene expression β-gal (red) in GABAergic medium sized spiny neurons of the striatum that are positive for DARPP32 (green) (A, B). Absence of β-gal (red) reporter in TH-positive cells of substantia nigra (SN) or ventral tegmental area (VTA) (green) (C, D). Scale bar in A corresponds to 10 mm.

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subsequent slow recovery of the DA output, which did not regain basal values at the end of experiment (3 h after the end of quinpirole infusion) (Fig. 2A). Local quinpirole application in the dorsal striatum of Tg mice evoked a smaller reduction of the DA output, to a maximal reduction of 7377% of baseline and DA concentration regained baseline values rapidly (Fig. 2A). Two-way repeated measures ANOVA indicated a significant effect of the treatment (F15,225 = 4.81, Po0.0001), genotype (F1,15 =5.66, Po0.04) and treatment x genotype interaction (F15,225 = 1.76, Po0.05). The local application of the selective DA D1-R agonist SKF-81297 (10 mM, 40 min) elicited a maximal reduction of the DA output to 3575% and 3676% in WT and Tg mice, respectively (n= 8 each; Fig. 2B). At the end of the collection period (4 h after beginning of drug infusion), DA values were still reduced in both experimental groups. Twoway repeated measures ANOVA indicated a significant effect of the treatment (F15,210 = 0.76, Po0.00001) but not of the group nor of the group x treatment interaction. To examine whether midbrain DA D2 autoreceptors were affected in Tg mice, we performed two additional microdialysis experiments. In one of them, quinpirole was systemically administered (1 mg/kg i.p.) and DA output was measured in the dorsal striatum, as above, under the Fig. 3 A) Vehicle i.p. administration did not alter extracellular DA in dorsal striatum of WT and Tg mice. B) The systemic administration of quinpirole (1 mg/kg s.c.) reduced DA release to the same extent in Tg and WT mice (Po0.00001, treatment effect, non-significant effects of genotype and treatment x genotype interaction). The number of animals in each group is given in brackets.

Fig. 2 The local application of the selective DA D2-R and DA D1-R agonists quinpirole (QUIN, A) and SKF-81297 (SKF, B) (10 mM each) by reverse dialysis for 40 min in the dorsal striatum evoked a marked reduction of DA release in WT and Tg mice. The effect of quinpirole (A) was significantly less marked in Tg than in WT animals (Po0.0001 treatment effect; Po0.04 genotype effect; Po0.05 treatment x genotype interaction). SKF-81297 (B) had a comparable effect in both groups of mice (Po0.00001, treatment effect, non-significant effects of genotype and treatment x genotype interaction). The number of animals in each group is given in brackets.

hypothesis that systemic quinpirole would preferentially target DA D2 autoreceptors in midbrain, more sensitive to agonist activation than postsynaptic DA D2-R in dorsal striatum. Saline injections did not alter the DA output in WT and Tg mice (Fig. 3A). The systemic administration of quinpirole reduced DA output to 2475% of baseline in WT mice (n= 5) and to 3176% of baseline in Tg mice (n =9), with a slower return to baseline values in Tg mice (Fig. 3B). Two-way ANOVA revealed a significant effect of the treatment (F15,180 = 40.35, Po0.00001) but not of the group nor of the treatment x group interaction. Further, we locally perfused the antipsychotic drug haloperidol (DA D2-R antagonist) by reverse dialysis to block terminal DA D2 autoreceptors in the dorsal striatum and thus enhance DA release (Fig. 4). The local application of haloperidol in striatum at low concentration (1 mM, 100 min) increased the DA output to 169721and 175728% of baseline in WT and Tg mice, respectively (n =8 mice per group). Two-way ANOVA revealed a significant effect of the treatment (F8,112 = 10.14, Po0.0001), but not of the group or the treatment x group interaction.

3.4.

Effect of quinpirole on locomotor activity

Mice were injected with saline or quinpirole (1 mg/kg i.p.) and 30 min later placed in the open-field. Ambulatory activity was recorded from 30 min to 240 min post-injection. Mice

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Fig. 4 Effects of the local application of the antipsychotic drug haloperidol (DA D2-R antagonist) on extracellular DA in the dorsal striatum of WT and Tg mice. In this experiment, the perfusion fluid contained 10 mM of the DA transporter inhibitor nomifensine in order to increase extracellular DA and thus, the endogenous tone on terminal DA D2 autoreceptors. The blockade of DA D2 autoreceptors by haloperidol increased comparably DA release in WT and Tg mice (Po0.0001, treatment effect, non-significant effects of genotype and treatment x genotype interaction). The number of animals in each group is given in brackets.

treated with saline (n=9WT, 8Tg) exhibited a slow and progressive decline in the ambulatory activity following the initial inspection of the open-field. The administration of quinpirole (n=10WT, 8Tg) evoked a rapid a robust suppression of locomotor activity. This period was followed by a slow recovery which reached that in saline-treated mice ca. 150 min post-administration. Three-way repeated measures ANOVA showed a significant effect of the time (F20,260 =24.4; Po0.00001), treatment (F1,31 =36.0; Po000001) and genotype (F1,31 =14.0; Po0.01) as well as significant interactions between treatment and time (F20,620 =44.56, Po0.00001) and between treatment, genotype and time F20,620 =2.00, Po0.01). Tg mice had a locomotor activity significantly higher than that of WT mice when treated with either saline or quinpirole. Thus, further two-way repeated measures ANOVA revealed significant differences between genotypes when mice were treated with saline (F1,15 =5.00, Po0.05, genotype effect; F20,300 =48.57, Po0.00001 time effect) and in those treated with quinpirole (F1,16 =19.09, Po0.0005, genotype effect; F20,320 =7.10, Po0.00001, time effect; F20,320 =2.27, Po0.005, genotype x time interaction) (Fig. 5).

3.5. Induction of c-fos following quinpirole administration It has been previously reported that systemic administration of quinpirole induces pallidal expression of c-Fos upon activation of striatal D2 receptors (Marshall et al., 1993; Wirtshafter and Asin, 1994; Ruskin and Marshall, 1997). In good agreement, we observed that systemic quinpirole (1 mg/kg i.p.) induced the expression of c-fos in globus pallidus and ventral pallidum of wt mice 3 h after administration. In contrast, when quinpirole was injected to Tg mice, no c-fos induction was observed. Two-way ANOVA indicated a significant effect of the treatment (F1,16 = 4.63, Po0.05), genotype (F1,16 = 17.74, Po0.001) and treatment x genotype interaction (F1,16 = 5.32, Po0.04) (Fig. 6).

Fig. 5 Locomotor activity in a novel environment (open field). Ambulatory distance was recorded from 30 min to 240 min postinjection. Performance of WT and Tg animals injected with saline solution is represented in Fig. 5 A. As a consequence of quinpirole injection (Fig. 5B) a decrease in locomotor activity was observed both in wt and Tg mice. Interestingly, Tg mice treated with quinpirole recovered faster than WT mice. The number of animals in each group is given in brackets.

4.

Discussion

GSK-3 is a constitutively active serine/threonine kinase which plays a major role in a number of physiological functions of CNS, being also involved in several psychiatric and neurological conditions (see Introduction). Dominant negative GSK-3 transgenic mice (Tg) show a regionally selective reduction of GSK-3 function restricted to forebrain. Also, Tg mice show impaired motor coordination under baseline conditions and reduced striatal c-fos expression in response to an amphetamine challenge (GomezSintes et al., 2007). These differences are not due to an impaired presynaptic DA function since amphetamine increases DA release to the same extent in WT and Tg mice and suggest the existence of alterations in forebrain elements postsynaptic to DA (Gomez-Sintes et al., 2007). Given the potential usefulness of the present Tg mice to further elucidate the role of GSK-3 in neuropsychiatric disorders, here we explored whether the reduced expression of GSK-3 was associated to changes in postsynaptic DA receptor function using in vivo experimental paradigms. Overall, the present results indicate the existence of a reduced function of striatal DA D2-R (but not of DA D1-R) in Tg mice with a reduced GSK-3 expression, further supporting the view that GSK-3 is involved in DA D2-R signaling, as previously observed in response to an amphetamine challenge or in mice lacking the DA transporter (Beaulieu et al., 2004; Beaulieu et al., 2007).

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Fig. 6 (A–F) Induction of c-fos immunohistochemistry in pallidal neurons (A-C, lateral globus pallidus; D–F, ventral pallidum) of WT SAL (A and D), WT QUIN (B and E) and Tg QUIN (C and F) mice. Scale bar in A corresponds to 100 mm. G) Quantification of c-fos induction (Po0.003 treatment effect; Po0.001 genotype effect; Po0.008 treatment x genotype interaction). **po0.003 WT QUIN vs. the rest of groups (Bonferroni post-hoc following significant two-way ANOVA). The number of animals in each group is given in brackets.

The reduction in DA release induced by local quinpirole application in dorsal striatum is due to two different factors: 1) the activation of presynaptic DA D2 autoreceptors located on nerve terminals of nigrostriatal dopaminergic neurons, and 2) the activation of postsynaptic DA D2-R on spiny striato-pallidal GABAergic neurons, which control the activity of substantia nigra compacta (SNc) neurons via the subthalamic nucleus (indirect pathway). (Fig. 7; for review of basal ganglia circuits see also (Alexander and Crutcher, 1990; Gerfen, 2000; Surmeier et al., 2011). The stimulation of postsynaptic DA D2-R by relative high quinpirole concentrations results in a suppression of the excitatory input onto SNc dopaminergic neurons, with a subsequent reduction of striatal DA release (Ferre et al., 1994). In a similar way,,the stimulation of postsynaptic DA D1-R located on striatal GABAergic neurons of the striato-

nigral direct pathway leads to an increased GABA tone on dopaminergic neurons of the SNc, which than translates into a reduction of DA release in dorsal striatum (Ferre et al., 1994). The comparable reduction of DA release evoked by the selective DA D1-R agonist SKF-81297 in the striatum of WT and Tg mice indicates that postsynaptic DA D1-R function in the direct pathway is unaltered in Tg mice. However, local quinpirole application in dorsal striatum produced a marked reduction of DA release which was significantly less marked in Tg than in WT mice. Given the selectivity of quinpirole for DA D2-R (i.e., DA D2/3), this difference most likely accounted for by a deficient DA D2-R-mediated neurotransmission in dorsal striatum in Tg mice which further supports the involvement of GSK-3 in DA D2-R-mediated responses. The activation of presynaptic DA D2-R by this dose of

Reduced striatal dopamine DA D2 receptor function in dominant-negative GSK-3 transgenic mice

Fig. 7 Schematic diagram of the motor circuit of the basal ganglia in normal conditions. Pyramidal neurons of the motor cortex project to the dorsal striatum (caudate/putamen) where their axons synapse on medium spiny GABAergic neruons containing either substance/dynorphin (SP/Dyn) or enkephaline (Enk). These two striatal subpopulations project, directly (direct pathway) or indirectly (indirect pathway) to the output nuclei of basal ganglia, i.e., substantia nigra pars reticulata (SNr), internal globus pallidus (GPi) (or entopeduncular nucleus, EP, in rodents). Striatal neurons of the direct pathway express DA D1-R and the neuropeptides substance P (SP) and Dynorphin. Striatal GABA neurons from the indirect pathway express DA D2R and Enkephalin (Enk), and project to the external globus pallidus (GPe), which in turn projects to the subthalamic nucleus (STN), whose excitatory glutamatergic neurons activate tonically GABAergic neurons of the output nuclei (GPi and SNr) and project to the cell bodies of DA neurons in the substantia nigra pars compacta (SNc) projecting back to the caudate/ putamen. A reduction in the function of DA D2-R in GSK-3 Tg mice reduces the efficacy of the indirect pathway in response to postsynaptic DA D2-R stimulation, as assessed by the two readouts used in the present study: DA release in striatum and motor activity. In contrast, the activity of the D1-R-dependent direct pathway is preserved in Tg mice. Note that DA neurons in the SNc extend their dendritic arbors into the SNr, which allows a regulation of the activity of SNc DA neurons by the D1 receptor-mediated direct pathway. Modified after (Alexander and Crutcher, 1990;Gerfen, 2000).

quinpirole is warranted given their preferential sensitivity to agonists. Since i) the function of presynaptic DA D2 autoreceptors does not differ between Tg and WT mice, given the forebrain-specific loss of GSK-3 expression/function (Gomez-Sintes et al., 2007; see also below), and d ii) both pre- and postsynaptic DA D2-R contribute to reduce DA

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release, it is likely that the actual difference in postsynaptic DA D2-R function are greater than that observed in the present microdialysis experiments. Quinpirole induced the expression of c-fos in the globus pallidus and ventral pallidus of wt mice, but not in Tg mice. The pallidal induction of c-fos by quinpirole in wt mice is consistent with data in the literature (Ruskin and Marshall, 1997; Billings and Marshall, 2003) and is believed to be due to disinhibition of pallidal neurons after the stimulation of DA D2R in striatal output neurons of the indirect pathway (Fig. 7). The lack of c-fos expression in DN GSK-3 Tg mice after quinpirole administration further indicates loss of function of DA D2-R signaling in striato-pallidal neurons. Differences between WT and Tg mice in presynaptic DA D2 autoreceptors can be reliably ruled out given the similar reduction of DA release by systemic quinpirole in both genotypes. Systemic quinpirole also contributes to reduce DA release through the activation of pre- and postsynaptic receptors. However, somatodendritic (in midbrain) and terminal DA D2 autoreceptors (in striatum) are more sensitive to the action of quinpirole and other agonists than postsynaptic DA D2-R (Werkman et al., 2000;Rice et al., 2011). Thus, although postsynaptic DA D2-R are sensitive to the quinpirole dose used herein, their contribution to reduce DA release is much lower than that of presynaptic DA D2-R. This view is further supported by the microdialysis experiments in which haloperidol increased extracellular DA comparably in WT and Tg mice. The presence of nomifensine in the perfusion fluid of microdialysis probes enhanced extracellular DA by locally blocking DA transporter, leading to an activation of presynaptic DA D2 autoreceptors higher than in normal conditions (e.g., without nomifensine in the perfusion fluid). The occupancy of presynaptic DA D2 autoreceptors by haloperidol removed the DA D2 autoreceptor-mediated negative feed-back on DA release, leading to a comparable increase of DA release in Tg and WT mice. Overall, the similar efficacy of presynaptic DA D2 autoreceptors observed in these microdialysis experiments is in agreement with the regional change of GSK-3 activity in Tg mice. Experiments assessing motor activity in response to an acute quinpirole challenge also support the existence of a reduced function of DA D2-R in Tg mice compared with WT mice. Tg mice showed a smaller spontaneous decline in motor activity in the open-field during the observation period. Quinpirole administration markedly reduced locomotor activity in mice, as previously described (Martinez De Lagran et al., 2007). This effect was significantly smaller in Tg mice, a difference not accounted for by presynaptic factor, given the similar fall in striatal DA release produced by systemic quinpirole administration (see above) and most likely reflects a reduced function of postsynaptic DA D2-R in dorsal striatum. However, different scenarios could account for the observed effect on locomotion as the complexity of the circuit and of the genetic manipulation in Tg mice converge in this integrated behavioral outcome. For instance, given the positive role of D2 receptors on locomotion, it could also indicate that their function is reinforced in the Tg mice. Also, the reduced DA D2-R function in output striatal neurons and the consequent imbalance between excitatory (D1-R-mddiated) and inhibitory (D2-R-mediated)

1532 effects of endogenous dopamine might account for the basal motor hyperactivity observed in Tg mice. Besides, neuronal apoptosis is observed in some brain regions of Tg mice including the striatum (Gomez-Sintes et al., 2007) and, although the number of apoptotic neurons is very low, this might contribute to the deficit in motor coordination previously reported in Tg mice. This additional complexity of the system should also be kept in mind when interpreting the results although, as mentioned, we do not expect great impact on the phenotypes analyzed in the present study as only few neurons in the circuit are dying. Tg mice with a reduced constitutive activity of GSK-3 may be useful to gain further insight on the role of this signaling pathway in a number of neuropsychiatric diseases. Hence, the well-known inhibition of GSK-3 activity by lithium (Stambolic et al., 1996) suggest the utility of Tg mice in studies of bipolar disorder. Also, the different cellular roles of GSK-3 suggest an association with neuroplasticity mechanisms evoked by antidepressant drugs (Duman and Aghajanian, 2012). Likewise, several studies have associated GSK-3 activity with schizophrenia (Lovestone et al., 2007;Freyberg et al., 2010;Karam et al., 2010;Lipina et al., 2011). In particular, a new hypothesis on the relationship between GSK-3 and schizophrenia has been put forward suggesting that GSK-3 dysfunction causes neurodevelopmental alterations, on which, abnormal dopaminergic function produced by persistent GSK-3 dysfunction, would evoke psychotic symptoms and cognitive deficits. (Lovestone et al., 2007). Moreover, both classical and second generation (atypical) antipsychotic drugs used in the treatment of schizophrenia and bipolar disorder (only atypical drugs) regulate GSK-3 expression in forebrain (Alimohamad et al., 2005;Roh et al., 2007). Additionally, and given the restricted deficiency in the DA D2-R-dependent indirect pathway of the basal ganglia circuits, the present mice may useful models to examine the specific contribution of this pathway in basal ganglia motor, affective and cognitive circuits. In summary, the present study adds further evidence to the relationship between GSK-3 and schizophrenia by showing that Tg mice with a reduced constitutive activity of GSK3 show a deficient DA D2-R-medaited neurotransmission in forebrain. Further studies will examine the validity of Tg mice as animal models of the pathophysiology and treatment of schizophrenia.

Conflicts of interest All authors declare that they have no conflicts of interest.

Role of the funding source This work was supported by grants PI10/00290 from Instituto de Salud Carlos III and P91C from Centro de Investigación Biomédica en Red de Salud Mental to AB, by grants from Ministerio de Ciencia (MICINN, MINECO) SAF2012-35183 to FA and by Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CiberNed-Instituto de Salud Carlos III) and by grants from Ministerio de Ciencia (MEC, MICINN) SAF2009-08233 and SAF2012-34177 and Fundación Ramón Areces to JJL. The funding sources had no

R. Gomez-Sintes et al. further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Contributors All authors designed the study, wrote the protocol and managed the literature searches. RGS and AB performed the experiments, analyzed data and undertook the statistical analysis. FA wrote the first draft of the manuscript. JJL revised the manuscript. All authors contributed to and have approved the final manuscript.

Acknowledgments We are grateful to Javier Palacín, Desireé Ruiz, Miriam Lucas and Alicia Tomico for technical assistance. This work was supported by the following grants: PI10/00290 and P91C (Instituto de Salud Carlos II) to A.B., SAF2012-35183 (Ministry of Economy and Competitiveness, co-financed by the European Regional Development Fund (ERDF)), to F.A:, SAF2009-08233 and SAF2012-34177 (Ministry of Science, MEC, MICINN) and by Fundación Ramón Areces to JJL. Support from the Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM, Instituto de Salud Carlos III) to FA and by Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED-Instituto de Salud Carlos III) to JJL is also acknowledged.

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