Neurobiology of Disease 78 (2015) 146–161

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Rhes regulates dopamine D2 receptor transmission in striatal cholinergic interneurons Giuseppe Sciamanna a,b,1, Francesco Napolitano c,d,1, Barbara Pelosi e,1, Paola Bonsi b, Daniela Vitucci d,f, Tommaso Nuzzo d, Daniela Punzo d, Veronica Ghiglieri g, Giulia Ponterio a,b, Massimo Pasqualetti e,h, Antonio Pisani a,b,⁎, Alessandro Usiello d,i,⁎⁎ a

Department of Systems Medicine, University of Rome “Tor Vergata”, Via Oxford 1, 00133 Rome, Italy IRCCS Fondazione Santa Lucia, Laboratory of Neurophysiology and Synaptic Plasticity, Via del Fosso di Fiorano 64, 00133 Rome, Italy c Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Via Pansini 5, 80131 Naples, Italy d CEINGE Biotecnologie Avanzate, Via G. Salvatore 486, 80145 Naples, Italy e Department of Biology, University of Pisa, Via L. Ghini 13, 56126 Pisa, Italy f Department of Health and Motor Sciences, DiSMeB, University of Naples “Parthenope”, Via Medina 40, 80143, Naples, Italy g Department of Philosophy and Human Sciences, University of Perugia, Piazza Ermini 1, 06123 Perugia (PG), Italy h Istituto Italiano di Tecnologia, Center for Neuroscience and Cognitive Systems, Corso Bettini 31, 38068 Rovereto (TN), Italy i Department of Environmental Sciences, Second University of Naples (SUN), Via Vivaldi 43, 81100 Caserta, Italy b

a r t i c l e

i n f o

Article history: Received 28 January 2015 Revised 12 March 2015 Accepted 17 March 2015 Available online 26 March 2015 Keywords: Rhes Striatum Cholinergic interneurons D2 dopamine receptors Calcium Dystonia

a b s t r a c t Ras homolog enriched in striatum (Rhes) is highly expressed in striatal medium spiny neurons (MSNs) of rodents. In the present study, we characterized the expression of Rhes mRNA across species, as well as its functional role in other striatal neuron subtypes. Double in situ hybridization analysis showed that Rhes transcript is selectively localized in striatal cholinergic interneurons (ChIs), but not in GABAergic parvalbumin- or in neuropeptide Y-positive cell populations. Rhes is closely linked to dopamine-dependent signaling. Therefore, we recorded ChIs activity in basal condition and following dopamine receptor activation. Surprisingly, instead of an expected dopamine D2 receptor (D2R)-mediated inhibition, we observed an aberrant excitatory response in ChIs from Rhes knockout mice. Conversely, the effect of D1R agonist on ChIs was less robust in Rhes mutants than in controls. Although Rhes deletion in mutants occurs throughout the striatum, we demonstrate that the D2R response is altered specifically in ChIs, since it was recorded in pharmacological isolation, and prevented either by intrapipette BAPTA or by GDP-β-S. Moreover, we show that blockade of Cav2.2 calcium channels prevented the abnormal D2R response. Finally, we found that the abnormal D2R activation in ChIs was rescued by selective PI3K inhibition thus suggesting that Rhes functionally modulates PI3K/Akt signaling pathway in these neurons. Our findings reveal that, besides its expression in MSNs, Rhes is localized also in striatal ChIs and, most importantly, lack of this G-protein, significantly alters D2R modulation of striatal cholinergic excitability. © 2015 Elsevier Inc. All rights reserved.

Introduction Ras homolog enriched in striatum (Rhes) is a small monomeric GTPbinding protein, discovered by a subtractive hybridization procedure, predominantly localized throughout dopaminoceptive neurons of dorsal striatum and nucleus accumbens (Errico et al., 2008; Vargiu et al., 2001,

⁎ Correspondence to: A. Pisani, Department of Systems Medicine, University of Rome Tor Vergata, V. Montpellier 1, 00133 Rome, Italy. Fax: +39 0620903118. ⁎⁎ Correspondence to: A. Usiello, Ceinge Biotecnologie Avanzate, Via G. Salvatore, 486, 80145 Naples, Italy. Fax: +39 0813737808. E-mail addresses: [email protected] (A. Pisani), [email protected] (A. Usiello). 1 These authors equally contributed to this study.. Available online on ScienceDirect (www.sciencedirect.com).

http://dx.doi.org/10.1016/j.nbd.2015.03.021 0969-9961/© 2015 Elsevier Inc. All rights reserved.

2004). Rhes expression is developmentally regulated by thyroid hormone in rodents (Falk et al., 1999; Vallortigara et al., 2008, 2009), and by dopamine (DA) innervation in adult rats (Harrison and LaHoste, 2006; Harrison et al., 2008). Studies in cell lines (Agretti et al., 2007; Harrison and LaHoste, 2006; Harrison et al., 2008; Vargiu et al., 2004), have indicated that Rhes, most likely through its binding to Gαi, reduces G-protein coupled receptor (GPCR)-mediated cAMP accumulation (Harrison and He, 2011). Accordingly, we have shown that lack of Rhes increases striatal cAMP/PKA activity in mutant mice (Errico et al., 2008). In addition, earlier in vitro evidence has revealed that Rhes influences N-type/Cav2.2 calcium channels activity (Thapliyal et al., 2008). In particular, the authors found that Rhes, by modulating Gαi-dependent signaling, reduces basal calcium current density and triggers a voltagedependent inhibition of Cav2.2 channels in HEK293-trasfected cell culture. Aside from its influence on cAMP accumulation and regulation of

G. Sciamanna et al. / Neurobiology of Disease 78 (2015) 146–161

Cav2.2 channels activity, it has been found that Rhes acts as a selective striatal E3 ligase of mutant Huntingtin (mHtt) (Mealer et al., 2013, 2014; Subramaniam et al., 2009, 2010). On the other hand, it has been shown that Rhes also activates mammalian target of rapamycin complex1 (mTORC1), a critical signaling pathway associated, among other processes (Wullschleger et al., 2006), with L-DOPA-induced dyskinesia (LID) in hemiparkinsonian animal models (Santini et al., 2010; Subramaniam et al., 2012). Finally, different in vitro settings have shown that Rhes modulates PI3K/Akt signaling pathway (Bang et al., 2012; Vargiu et al., 2004). Moreover, recent in vivo findings have revealed that Rhes is also able to interact with β-arrestins (Harrison et al., 2013), a scaffolding protein involved in the striatal modulation of DA D2Rdependent Akt/GSK3-β signaling (Beaulieu et al., 2005). Despite the growing interest for the involvement of Rhes in striatal medium spiny neurons (MSNs) dysfunction, many basic issues still need to be addressed, including specific localization and activity of Rhes in other distinct striatal neuronal subtypes. Therefore, in the present work we explored the striatal cell-type expression of Rhes and RASD2 mRNAs in mouse and human brain, respectively. Interestingly, we found a selective expression of Rhes in the striatal cholinergic interneurons (ChIs), but not in those expressing GABAergic parvalbumin (Parv) or neuropeptide Y (NPY). Considering the pivotal role of acetylcholine (ACh) in the modulation of striatal circuit function (Pisani et al., 2007), here we also investigated the functional role of Rhes in modulating dopaminergic transmission in ChIs, by using knockout animals with targeted deletion of Rhes gene. Notably, our data clearly show that lack of Rhes affects the dopamine-dependent ChIs activity, thus highlighting a regulatory role of this small G-protein on dopaminergic responses in such a striatal neuronal subpopulation.

Material and methods Animals In order to prevent the possibility that the presence of the PGK-neo cassette may interfere with normal transcriptional regulation within the Rhes locus (Dhar et al., 1990; Pasqualetti et al., 2002; Pham et al., 1996; Tuan et al., 1985), we crossed Rhes+/loxP-neo founders (Spano et al., 2004) with a CMV-Cre deleter strain in the B6D2 genetic background (Johansson et al., 2013). The validation of Cre-mediated loxPflanked neo cassette excision was assessed by PCR analysis using the following primers: Rhes-EGFP_forward: 5′-CATGGTCCTGCTGGAGTTCG TGA-3′, and Rhes-Ex2_reverse: 5′-ACCACCATGCGGTAGGAGTTCT-3′. The amplification product was ~400 bp. Mice obtained were then genotyped by PCR using the following primers: Rhes-forward 5′-TTTAGGAA TTTCACCTGTGT-3′ and Rhes-Ex2_reverse 5′-ACCACCATGCGGTAGGAG TTCT-3′, for Rhes+/+ allele (~420 bp); Rhes-EGFP_forward 5′-CATGGT CCTGCTGGAGTTCGTGA-3′ and Rhes Ex2_reverse′ACCACCATGCGGTA GGAGTTCT-3′ for Rhes−/− allele (~370 bp). Rhes−/− mice were viable, fertile and did not show alterations in body weight and basal motor activity (data not shown). Male and female Rhes +/+ and Rhes−/− mice, derived from mating of heterozygous animals (Rhes+/−), back-crossed to F11 generation to C57BL/6 strain, were used in this study. For a set of biochemical experiments two-months old male knock-in Tor1a+/Δgag mice heterozygous for ΔE-torsinA and their controls (Tor1a+/+) (Goodchild et al., 2005) were used. Breeding colonies were established at our animal house. Mice were housed in groups in standard cages (29 × 17.5 × 12.5 cm) at constant temperature (22 °C ± 1 °C) and maintained on a 12/12 h light/dark cycle, with food and water ad libitum. Experiments were performed in conformity with protocols approved by the veterinary department of the Italian Ministry of Health and in accordance to the ethical and safety rules and guidelines for the use of animals in biomedical research provided by the relevant Italian laws and European Union's directives (n. 86/609/EC). All efforts were made to minimize the animal's suffering.

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Drugs All drugs used were obtained from Tocris Bioscience, dissolved in distilled water and added to the Krebs' solution at the final concentration. In situ hybridization (ISH) ISH analyses were performed according to protocols previously described (Migliarini et al., 2013). Digoxigenin (DIG)- (Rhes, 0.5 kb; RASD2, 2.8 kb), fluorescein- (Chat, 2.3 kb; Parv, 0.9; Npy, 0.3 kb), or 35 S-labeled (hCHAT, 2 kb) antisense riboprobes were used. For double ISH on adult mouse tissue, sections were hybridized simultaneously with DIG- and fluorescein-labeled riboprobes. A two-step chromogenic reaction using NBT/BCIP and HNPP/Fast Red Fluorescent Detection Set (Roche) was performed to visualize the DIG- and fluorescein-labeled riboprobes, respectively. Specimens were counterstained with DAPI. Double-ISH on human tissue was performed combining DIG-labeled and a 35S-radiolabeled riboprobes. After chromogenic reaction using NBT/BCIP for the detection of the DIG-labeled riboprobe, slides were dipped in Kodak NTB emulsion for autoradiography as previously described (Pasqualetti et al., 1999). Sections were examined using brightfield and darkfield light microscopy. Cell counts on double in situ hybridization from mouse sections were performed as described previously (Pelosi et al., 2014). Briefly, six 14 μm hemisections for each probe combination were examined at the level of caudate nucleus and putamen, and nucleus accumbens. Dorsal striatum was divided in four subdivisions, namely the dorso-medial (DM), dorso-lateral (DL), ventro-medial (VM) and ventro-lateral (VL; Harrison and LaHoste, 2006). In order to avoid counting cells twice, serial sections 130 μm distant one from another were used. For each subregion, images were captured in both brightfield and TRITC channel using a 20× objective in order to visualize presence of NBT/BCIP staining indicating Rhes expression colocalizing with Fast Red-derived fluorescence present in the interneuron populations, respectively. We let the staining with HNPP/Fast Red substrate proceed up to the point fluorescence appeared and then alkaline-phosphatase activity was intentionally stopped in order to prevent masking the blue signal developed in Rhes positive cells by reddish color appearing in brightfield. Images were converted to 8-bit grayscale and a threshold function was manually applied to remove sub-threshold signal using ImageJ software. For each image, only cells showing labeling clearly above the background level were counted. Rhes+/Chat+ vs Chat+, Rhes+/Parv+ vs Parv+ and Rhes+/NPY+ vs NPY+ interneurons were counted and the obtained values were expressed as total number of cells per sub-region. Human samples Human tissue was obtained from the Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore. Samples for cryosectioning were dissected in the region of the putamen nucleus of brains from one male (16 years old, 16 h post-mortem) and one female (35 years old, 6 h post mortem) who had no history of primary neurological or psychiatric disorders and processed as previously described (Pasqualetti et al., 1999). Electrophysiology and calcium imaging Preparation of corticostriatal coronal slices for ChIs recordings was carried out as previously described (Sciamanna et al., 2011). Slices were cut from male and female mice (4–6 weeks old) brains (200 μm thick) using a vibratome. After 30 min recovery, a slice was transferred in the recording chamber (0.5–1 ml volume), on the stage of an upright microscope (BX51WI, Olympus, Milan, Italy), and submerged by oxygenated (95% O2/5% CO2) Krebs' solution flowing at 2.5–3 ml/min, 32–33 °C. The composition of the solution was (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose and 25 NaHCO3. The

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microscope was equipped with a 20×, 0.95 n.a. water immersion objective (XLUMPlan Fl, Olympus). A monochrome CCD camera (C6790, Hamamatsu, Japan) was used to visualize ChIs on a PC monitor. Differential interference contrast optics (DIC, Nomarski) combined to infrared (IR) light were used to identify and impale under visual guidance ChIs up to 150 μm beneath the slice surface. Whole-cell and cell-attached patch-clamp recordings were made with a Multiclamp 700b and AxoPatch 200 amplifier (Axon Instruments), using borosilicate glass pipettes (1.5 mm outer diameter, 0.86 inner diameter) pulled on a P-97 Puller (Sutter Instruments) were filled with (in mM): 125 K+-gluconate, 10 NaCl, 1.0 CaCl2, 2 MgCl2, 0.5 1,2-bis-(2-aminophenoxy)-ethaneN,N,N,N-tetraacetic acid (BAPTA), 19 HEPES, 0.3 GTP, 1.0 Mg-ATP, adjusted to pH 7.3 with KOH. Pipette resistances ranged from 2.5 to 5 MΩ. Current-clamp recordings were also made in the perforated-patch configuration. Gramicidin was used as the pore-forming agent (Sciamanna et al., 2011) and was added to the pipette solution at an approximate concentration of 20 μg/ml. The tip of the pipette was filled with gramicidin-free intracellular solution. The perforation process was considered complete when the amplitude of the action potentials was steady and N 60 mV, and electrode resistance was steady and b50 MΩ. Currents recorded were normalized to cell capacitance to get current density measurements. For experiments with intrapipette BAPTA, current-clamp experiments were performed in the whole-cell configuration. For whole-cell patch-clamp experiments requiring a pharmacological isolation, picrotoxin (50 μM), MK801 (30 μM) and CNQX (10 μM) were added to the perfusion Krebs' solution during actual recording immediately after establishing the whole-cell configuration. Slices were incubated with drugs for at least 10 min and throughout the duration of the experiment. Data were analyzed using pClampfit 9.2 (Molecular Device), Origin 8.0 (Microcal) and Prism 5.0 (GraphPad). In data presented as box plots in the figures, the central line represents the mean, the edges of the box represent the interquartiles, and the “whisker lines” show the extent of the overall distribution. Numerical data are presented as means ± SEM. The evaluation of statistical difference was performed using paired and unpaired parametric statistical tests (Student's t-test) and two-way ANOVA followed by Bonferroni post-tests. The significance level was set at p b 0.05. For calcium imaging experiments we used bis-fura-2 (100– 200 mM) performed with UV flash light delivered by means of a Polychrome IV monochromator (TILL Photonics-FEI Munich GmbH, Germany) (Sciamanna et al., 2014). Calcium transient decay time was fitted with a linear equation: y = a + bx. After establishing the whole-cell configuration, we waited N5 min to allow equilibration of the indicator in the cell. Image sequences were obtained using an ImagEM-CCD digital camera (Hamamatsu, Japan). For sequence acquisition, images were binned into 4 pixel × 4 pixel bins, to achieve frame rates from 30 to 100 Hz. The mean fluorescence within each region of interest constituted the raw fluorescence signal. Fluorophores were excited at 380 ± 15 nm. Fluorescence signals recorded while injecting small hyperpolarizing currents into the soma (to prevent spontaneous spiking) provided a baseline sequence that was subtracted to compensate for bleaching of indicator during long sequences. Baseline subtracted data were expressed as the percentage change from initial fluorescence following shutter opening, normalized to the change in absolute fluorescence during shutter opening (ΔF/F). Signal was corrected by subtracting from the fluorescence of neighboring region. Drop in fluorescence signal correspond to an increase of calcium level. Western blotting Western blotting was performed as previously described (Napolitano et al., 2010). Mice were killed by decapitation and the heads were immediately immersed in liquid nitrogen for 5–6 s, the brains were removed, the striata dissected out within 20 s on an ice-

cold surface, sonicated in 1% SDS and boiled for 10 min. 1% SDS was diluted in distilled water. Aliquots (2 μl) of the homogenate were used for the protein determination by Bio-Rad Protein Assay kit (Bio-Rad). Equal amounts of total proteins for each sample were loaded onto 12% polyacrylamide gels. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto PVDF (polyvinylidene difluoride) membrane (GE Healthcare). Membrane was immunoblotted overnight using a selective antibody against Rhes (1:500, Merck Millipore©, Merck KGaA, Darmstadt, Germany). Blot was then incubated in horseradish peroxidase-conjugated secondary antibody and target protein was visualized by ECL detection (Pierce) followed by quantification using Quantity One software (Bio-Rad). Optical density values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000 Santa Cruz Biotechnology, Inc., Dallas, Texas 75220, U.S.A.) for variation in loading and transfer. Normalized values were then averaged and used as dependent variable. Results were analyzed by unpaired Student's t-test. Results Rhes mRNA is expressed in mouse and human striatal ChIs Differently from the well-established striatal localization of Rhes mRNA in both D1R- and D2R-expressing MSNs (Errico et al., 2008), a more detailed characterization of its transcript within specific subpopulations of striatal interneurons is still lacking. Therefore, we evaluated by double ISH a potential Rhes mRNA expression in three major striatal neuronal subtypes, such as GABAergic parvalbumin (Parv), neuropeptide Y (Npy), and choline acetyltransferase (ChAT)-positive interneurons (ChIs) (Figs. 1A–I). We quantified the expression level of Rhes mRNA in Parv-, NPY- or ChAT-positive interneurons and counted them in the dorso-medial (DM), dorso-lateral (DL), ventro-medial (VM) and ventro-lateral (VL) subdivisions of the dorsal striatum, and in the nucleus accumbens (Table 1). Results showed that Rhes mRNA was detectable neither in Parv- nor in NPY-positive interneurons (Figs. 1A–C and D–F, respectively). Conversely, it was found expressed in virtually all striatal ChAT-positive interneurons (Figs. 1G–I). Furthermore, in order to assess whether the ortholog RASD2 expression in striatal ChIs is conserved also in humans, we extended our morphological analysis from rodent to human putamen. As assessed at high magnification by the presence of autoradiographic silver grain deposition in purple cells and consistently to what observed in mouse striatum, double ISH analysis revealed that the co-expression of CHAT and RASD2 mRNAs similarly occurred in the human putamen nucleus (Figs. 1J–L; Supplemental Fig. 1). Role of Rhes in modulating basic membrane properties of ChIs To decipher the potential physiological role of Rhes in ChIs activity, we investigated whether lack of this gene may affect the basal electrophysiological properties in such a striatal cell population. ChIs were recorded from dorso-lateral striatum and identified on the basis of their somatic morphology observed using IR-DIC imaging (Figs. 2A, A1). Collectively, our data showed no main difference in the intrinsic membrane properties between ChIs recorded from Rhes−/− and control mice, in both genders (Figs. 2B–D and B1–D1, Table 2, n = 81). The recorded neurons had a depolarized resting membrane potential (RMP), a long-lasting afterhyperpolarization (AHP), and a prominent Ih current elicited by hyperpolarizing current steps (Figs. 2B, B1). No significant difference in the intrinsic membrane properties, such as RMP, input resistance or Ih current, was recorded between Rhes−/− and Rhes+/+ mice, for each analyzed gender (males/females RMP: Rhes+/+ vs Rhes−/− respectively p = 0.37/p = 0.54; males and females IR: Rhes+/+ vs Rhes−/− respectively p = 0.61/p = 0.41; males/females Ih: Rhes+/+ vs Rhes−/− respectively p = 0.51/p = 0.55, unpaired Student's t test between genotypes of each gender) (Table 2, Figs. 2B, B1) In the current-clamp configuration,

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Fig. 1. Rhes is expressed in mouse and human striatal ChAT-positive interneurons. (A–I) Double in situ hybridization (ISH) on coronal sections of mouse caudate/putamen using specific antisense riboprobes for Rhes/Parv (A–C), Rhes/NPY (D–F) and Rhes/ChAT (G–I). Boxed regions in (A, D, G) are shown at high magnification in both brightfield (B, E, H) and fluorescence (C, F, I), respectively. Parv- (light reddish, B; red, C) and NPY- (light reddish, E; red, F) positive interneurons are indicated by arrows. Cells indicated by the dashed arrows in B, C, E, F, are shown in the insets at higher magnification in both brightfield (B, E) and fluorescence (C, E) and clearly appear devoid of Rhes mRNA-expression (purple staining in B and E). ChAT- (light reddish, H; red, I) positive interneurons shown by arrows or the dashed arrow present co-expression of Rhes mRNA (purple staining) as clearly demonstrated at higher magnification in the inset (H). (J–L) Double ISH analysis on sections from post mortem human brain using specific antisense riboprobes for RASD2 and CHAT (K, L). (J) Nissl stained cryosection highlighting the neuroanatomical region of the putamen nucleus where RASD2-expressing cells are localized. (K, L) Brightfield and darkfield photomicrographs of a coronal section of putamen showing the distribution of RASD2 mRNA (purple, K) in CHAT-positive (silver grains, L) interneurons. In the inset in K, higher magnification allow to highlight presence of autoradiographic silver grain deposition cluster in a purple cell demonstrating co-expression of RASD2 and CHAT. Abbreviations: i, insula; cl, claustrum; ec, external capsule; pu, putamen nucleus. Scale bar: 750 μm (A, D, G); 75 μm (B, C, E, F, H, I, K, L); 3 mm (J); 15 μm (insets in B, C, E, F, H, I); 40 μm (inset in K).

spontaneous spiking activity was observed in nearly all of the recorded ChIs (~90% cells). Overall, the average firing rate was not significantly different between genotypes and genders (Rhes+/+, males: 2.2 Hz ± 0.28; Rhes−/−, males: 2.6 Hz ± 0.36 p = 0.45; Rhes+/+, females: 2.2 Hz ± 0.4; Rhes−/−, females: 2.7 Hz ± 0.38, n = 61, p = 0.21, unpaired Student's t test) (Figs. 2C, C1, D, D1). Role of Rhes in neurotransmitter-dependent modulation of spontaneous firing activity of ChIs In order to verify the potential influence of Rhes deletion on transmitter-dependent modulation of ChIs excitability, we investigated the effects of muscarinic and GABAergic receptor activation in mutant striata. We first measured the response to muscarinic M2/M4 autoreceptor activation, which produces a membrane hyperpolarization coupled to a complete, although transient, interruption of firing activity

(Calabresi et al., 1998). Regardless of the genotype and gender, we report that bath-application of the selective M2/M4 agonist oxotremorine (300 nM, 3 min) caused a complete and reversible neuronal firing reduction (Figs. 3A–D). Then, we explored the effect of baclofen, a GABAB receptor agonist, which inhibits spontaneous cell firing (Sciamanna et al., 2012a). Bath application of baclofen (10 μM, 2 min) caused a reversible inhibition of spontaneous firing activity in both genotypes and genders (Figs. 3A1–D1). Collectively, these experiments indicated that lack of Rhes in ChIs affects neither intrinsic membrane properties nor their responsiveness to muscarinic and GABAergic receptor activation. Rhes affects electrophysiological responses to dopamine receptor activation in ChIs ChIs excitability is significantly modulated by both dopamine D1and D2-like receptors, whose activation results, respectively, in

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Table 1 Quantification of Rhes expression in striatal interneuron populations. Total cell count for each schematically represented subregion is expressed as mean values on six different hemisections ± s.e.m. DM: dorso-medial; DL: dorso-lateral; VM: ventro-medial; VL: ventro-lateral; NAc: nucleus accumbens.

DM striatum

DL striatum

VM striatum

VL striatum

Accumbens

Rhes– / Parv+

3.7 ± 0.3

6.2 ± 0.5

3.7 ± 0.4

6.8 ± 0.8

3.0 ± 0.7

Rhes+ / Parv+

0

0

0

0

0

Rhes– / NPY+

6.5 ± 0.5

6.3 ± 0.6

6.5 ± 0.6

6.3 ± 0.6

24.3 ± 0.9

Rhes+ / NPY+

0

0

0

0

0

Rhes– / Chat+

0

0

0

0

0

Rhes+ / Chat+

18.7 ± 0.7

17.2 ± 0.9

12.7 ± 0.6

12.0 ± 0.6

8.3 ± 1.1

excitatory and inhibitory effects (Aosaki et al., 1998; Maurice et al., 2004; Pisani et al., 2000). Remarkably, the prevailing effect of dopaminergic inputs onto ChIs via D2R is inhibitory, resulting in a net decrease of ACh release (DeBoer et al., 1996). To evaluate the influence of Rhes in controlling dopaminergic responses of ChIs, we first tested in striatal mutant slices the efficacy of D1R activation in modulating spontaneous

firing activity of these neurons. Brief bath-applications of the D1R agonist, SKF 38398 (10 μM, 2 min), caused in control ChIs an overall significant increase of spontaneous firing activity (firing increase: Rhes+/+, males: 30.6% ± 2.4, females: 31.1% ± 2.3, n = 11 for both genders, p = 0.005, paired Student's t test) (Figs. 4A and B). Conversely, in ChIs recorded from either male or female Rhes knockouts, activation of D1R

Fig. 2. Basic membrane properties of ChIs from Rhes−/− mice. (A) Infrared image of corticostriatal brain slice used for electrophysiological recording. White box (a) shows the field of recording. (A1) Infrared image of ChI during whole-cell recording. (B–B1) Representative traces showing voltage responses to current steps (100 pA, 600 ms) in both depolarizing and hyperpolarizing direction in ChIs of Rhes+/+ and Rhes−/− from both male and female animals. Note the prominent Ih current evoked by hyperpolarizing step (white arrow) and the robust AHP current following the step ending (black arrow). (C, C1) Representative traces of spontaneous firing activity recorded in perforated-patch configuration in ChIs of Rhes+/+ and Rhes−/− from both male and female animals. As summarized in the plot (D, D1), no significant change is observed in basal firing rate between genotypes.

G. Sciamanna et al. / Neurobiology of Disease 78 (2015) 146–161 Table 2 Electrophysiological properties of striatal ChIs recorded from both genders of Rhes+/+ and Rhes−/− mice. Males

RMP (mV) IR (MΩ) Ih (mV)

Females

Rhes+/+

Rhes−/−

Rhes+/+

Rhes−/−

−61 ± 5 45 ± 12 85 ± 17

−58 ± 4 53 ± 11 79 ± 12

−56 ± 7 49 ± 6 91 ± 16

−60 ± 12 43 ± 18 84 ± 13

caused an attenuated increase of firing rate activity (firing increase: Rhes−/−, males: 15.7% ± 3.3; females: 23.8% ± 1.8; n = 13 for both genders; Rhes+/+ vs Rhes−/−, males: p = 0.04; females: p = 0.04, unpaired Student's t test) (Figs. 4A1, B1 and C, D). However, no significant gender dependency was observed (two-way ANOVA: F(9,20) = 0.61, p = 0.77). Next, we explored the influence of Rhes on D2R-related modulation of firing activity in ChIs. Bath application of quinpirole, a selective D2R agonist (10 μM, 2 min), induced a weak decrease in firing frequency, and had no main effect on membrane potential or input resistance in ChIs, recorded from control mice of both genders (n = 8, pre- vs postdrug application, p = 0.55, paired Student's t test) (Figs. 5A, C). On the contrary, quinpirole administration significantly increased the rate of action potential discharge in Rhes mutants (males: from 1.2 ± 0.25 to 2.9 ± 0.44 Hz, n = 14, p = 0.004; females: from 1.4 ± 0.37 to 3.12 ± 0.25 Hz, n = 12, p = 0.004, paired Student's t test) (Figs. 5B, D). Accordingly, interspike interval (ISI) exhibited a clear left-shift of the histogram, confirming the rise in firing rate in Rhes mutants of both genders. No gender dependency was found (two-way ANOVA, F(11,33) = 1.19, p = 0.32). Pre-treatment with the D2R antagonist, sulpiride (3 μM, 10 min), fully prevented the aberrant response to quinpirole (data not shown). Overall, these data highlight a modulatory role of Rhes in maintaining a physiological D2R-mediated response in the striatal ChIs. To rule out the possibility that a ceiling effect is involved in D2R mediated effect in Rhes−/−, a statistical correlation analysis between firing frequency in basal condition and under quinpirole was performed. Basal firing was not significantly correlated with the effect of quinpirole (basal frequency vs quinpirole frequency, males: r2 = 0.02 p = 0.54, n = 18, females: r2 = 0.02 p = 0.49, n = 19, data not shown). Cellular specificity of the abnormal response to D2R activation in ChIs The ubiquitous expression of Rhes in striatal neurons prompted us to address the cellular specificity of the D2R-related alterations seen in mutant ChIs (Fig. 6). In a first set of experiments, whole-cell recordings were made in ChIs from Rhes−/− mice, in the presence of the GABA-A receptor blocker picrotoxin (50 μM) and a combination of ionotropic NMDA and AMPA glutamate receptor blockers, MK801 and CNQX (30 μM, 10 μM), in order to achieve an experimental condition of pharmacological isolation. All drugs were bath-applied for at least 10 min immediately after establishing the whole-cell configuration. In this condition, bath-application of quinpirole (10 μM) still caused a significant increase in the firing rate of the recorded ChIs in both males and females (Figs. 6A, A1). A further demonstration that the altered D2R response is cellspecific was obtained by loading cells with the calcium chelator BAPTA (10 mM) during current-clamp recordings. Intracellular BAPTA did not cause per se significant changes of firing rate either before or after application of quinpirole in ChIs recorded from Rhes+/+ (n = 3, for both genders, males: p = 0.50, females: p = 0.71, paired Student's t test) (Supplemental Figs. 2A, B). Conversely, in the presence of intracellular BAPTA, quinpirole failed to induce the abnormal increase of

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firing frequency in ChIs recorded from Rhes−/− mice, in both genders (n = 6, for both genders, males: p = 0.37, females: p = 0.47, paired Student's t test) (Figs. 6B, B1, B2). Next, to investigate whether the D2R-dependent signaling mechanisms were altered in ChIs from Rhes−/− mice, recording pipettes were loaded with GDP-β-S (500 nM). The non hydrolysable GDP-β-S competes with endogenous GTP for the nucleotide binding on G-proteins, locking G-proteins in an inactive state. In Rhes+/+ mice, the presence of GDP-β-S prevented changes in firing rate after quinpirole application (n = 3, for both genders, males: p = 0.48, females: p = 0.64, paired Student's t test) (Supplemental Figs. 2C, D). In whole-cell current-clamp experiments from Rhes−/− mice, intracellular replacement with GDP-β-S fully prevented the response to quinpirole, in both genders (Figs. 6C, C1, C2). Influence of Rhes in modulating Ca2+ entry in ChIs In addition to the set of experiments performed with intrapipette BAPTA, we measured voltage step-induced intracellular calcium (Ca2+) transients and simultaneously monitored membrane currents before and during quinpirole application. These experiments were performed in the presence of TTX 1 μM to avoid contamination from sodium currents or transmitter-activated conductances. In ChIs obtained from Rhes−/− mice, a transient elevation in intracellular Ca2+ was measured following a voltage step (from −60 mV to 0 mV, 600 ms). No significant difference was measured in the peak Ca2+ transient between Rhes−/− and Rhes+/+ ChIs, as well as between male and female knockouts (ΔF: Rhes+/+; males: 0.96 ± 0.10; females: 0.89 ± 0.13, n = 10 for both genders; ΔF: Rhes−/−; males: 0.98 ± 0.08; females: 0.96 ± 0.07, n = 7 for both genders) (Figs. 7A–E). However, in the presence of quinpirole, the decay of Ca2+ transients to baseline was significantly delayed in ChIs from Rhes−/− mice, as compared to Rhes+/+ mice (males: p = 0.006, n = 10; females: p = 0.02, n = 8; Figs. 7A, E, F, F1) suggesting either a defective clearance or a further mobilization of Ca2+ from internal stores. D2R activation inhibits Cav2.2 channels in striatal ChIs (Yan et al., 1997). Of note, in ChIs from Rhes−/− mice the delayed decay time of Ca2 + transients were completely blocked by the specific Cav2.2 channel blocker, ω-conotoxin GVIA (1 μM) (males: p = 0.67, n = 5; females: p = 1.12, n = 4; Figs. 7D–F1). Moreover, previous data reported that Rhes modulates Cav2.2 Ca2+ channels by interfering with Gαi-dependent signaling (Thapliyal et al., 2008). Altogether, these data prompted us to test ω-conotoxin GVIA on the D2R effect on firing rate in Rhes ChIs mutants (Fig. 8). Whole-cell recordings of ChIs were performed in pharmacological isolation as previously described. After slice preincubation with ω-conotoxin GVIA (1 μM, 10–15 min), quinpirole did not induce significant firing changes in Rhes+/+ mice after treatment with ω-conotoxin GVIA (n = 3, for both genders, males: p = 0.71, females: p = 0.49, paired Student's t test, Supplemental Figs. 2E, F). In Rhes−/− mice the presence of ω-conotoxin GVIA prevented the aberrant excitatory response triggered by quinpirole (n = 4, for both genders, males: p = 0.52; females: p = 0.41, paired Student's t test, Figs. 8A–A2). Next, we performed a set of voltage-clamp experiments by means of a series of voltage steps (10 mV step from − 60 to 0 mV). Whole-cell currents were studied in the presence of TTX to eliminate inward currents at suprathreshold voltages that are generated by sodium channels. Current densities normalized to cell capacitance were measured (Figs. 8B–D1). No significant differences were found between Rhes−/− and Rhes+/+ mice in both males and females (n = 13, for both genders, males: Rhes−/− vs Rhes+/+ p = 0.14, females: Rhes−/− vs Rhes+/+ p = 0.19, unpaired Student's t test, Fig. 8D, D1). Our recordings showed that bath-application of quinpirole (10 μM, 2 min) slightly reduced the

Fig. 3. Pharmacological membrane properties of ChIs from Rhes+/+ and Rhes−/− mice. (A–D1) Traces show firing activity recorded in cell-attached configuration from ChIs of male and female Rhes+/+ and Rhes−/− mice. M2/M4 receptor activation by oxotremorine (300 μM, 3 min) induces a transient firing cessation in Rhes+/+ (A, B) as well as in Rhes−/− (C, D). Similarly, application of baclofen, a GABAB receptor agonist (10 μM, 2 min) reversibly blocks firing activity in Rhes+/+ (A1, B1) and in Rhes−/− mice (C1, D1).

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(Rhes−/−: pre vs post drug application, males: p = 0.02, n = 12, females: p = 0.02, n = 12, paired Student's t test, Figs. 8B1, C1, D1). Subtraction of currents evoked in quinpirole from currents evoked in control condition confirmed the existence of a larger quinpirolesensitive currents in Rhes−/− with respect of Rhes+/+ mice (Fig. 8E). In addition, we tested whether the effect of quinpirole occurs via Cav2.2 Ca2 + channels. In Rhes−/− slices, pre-incubation with ωconotoxin-GVIA (1 μM) prevented the effect of quinpirole on stepevoked whole-cell currents (Rhes−/− pre vs post drug application, males: p = 0.82, n = 6; females: p = 0.55, n = 7, paired Student's t test) (Figs. 8B1, C1, D1). ω-conotoxin-GVIA was also able to prevent the slight reduction in voltage-activated current recorded in Rhes+/+ (Figs. 8B, C, D). The effects of Cav2.2 blockade on quinpirole-related responses in mutant ChIs showed no substantial difference between genders. Altered D2R/Akt signaling in striatal mutant ChIs The canonical signaling pathway activated by D2R stimulation involves G-protein-dependent inhibition of adenylate cyclase (Missale et al., 1998). However, recent evidence has shown that D2R agonists can also modulate PI3K/Akt pathway, in a G-protein independent manner (Beaulieu et al., 2005). Interestingly, recent data have demonstrated that Rhes is able to regulate PI3K/Akt signaling in the striatum (Bang et al., 2012; Harrison et al., 2013; Vargiu et al., 2004). To test the possibility that PI3K/Akt signaling may be involved in the abnormal D2Rmediated response found in Rhes−/− mutant ChIs, striatal slices were pre-treated with a selective PI3K inhibitor, LY294002. First, we tested if PI3K inhibitor could, per se, interfere with the effect of quinpirole in Rhes+/+ mice. The presence of LY294002 (50 μM, 15 min), did not significantly modify the firing frequency increase induced by quinpirole (n = 4 for both genders, males: p = 0.9; females: p = 0.73, paired Student's t test) (Figs. 9A1, B1, C, C1). Interestingly, LY294002 was able to prevent the quinpirole-induced increase of firing rate in mutant ChIs in both females, causing no changes in firing frequency before and after drug application (males: p = 0.21, n = 6; females: p = 0.81, n = 6; paired Student's t test; Figs. 9A2, B2, C, C1), thus suggesting that Rhes is physiologically implicated in the modulation of D2R/Akt signaling pathway in striatal ChIs. Unaltered striatal Rhes protein levels in DYT1 mouse model of dystonia Similarly to Rhes mutant ChIs, previous studies showed a paradoxical excitatory effect, caused by D2R activation, in distinct DYT1 dystonia mouse models (Pisani et al., 2006; Sciamanna et al., 2012a,b). Based on this evidence, we evaluated the putative influence of Rhes expression on the electrophysiological alteration found in DYT1 mutant mice. To this aim, we analyzed the protein levels of Rhes in DYT1 GAG heterozygous knock-in mice. Western blotting analysis, performed in striatal lysates (n = 7/genotype), revealed no significant difference in Rhes expression levels in mutants (p N 0.05, unpaired Student's t test), compared to control animals (Fig. 10). Discussion Fig. 4. Moderate decreased responsiveness to D1R stimulation in ChIs from Rhes−/− mice. (A, B1) Representative traces of ChIs recorded in the perforated-patch configuration and collected before (left column) and during (right column) bath application of SKF38398 (10 μM, 2 min). (A, B) The D1R agonist causes a firing increase in Rhes+/+ animals of both male and female genders. (A1, B1) Otherwise only a slight increase was observed in Rhes−/− mice in both males and females. (C, D) Graph plots summarize the effect of SKF38398 in both Rhes+/+ and Rhes−/− mice.

transient currents at depolarizing holding potentials in both genotypes (Rhes+/+: pre vs post drug application, males: p = 0.54, n = 11; females: p = 0.46, n = 10; Student's t test, Figs. 8B, C, D). However, in Rhes−/− the current density recorded after quinpirole application was significant smaller than the current recorded in control conditions

Rhes is involved in several signaling pathways, including those modulated by the activity of cAMP (Harrison and He, 2011; Vargiu et al., 2004), PI3K (Bang et al., 2012; Harrison et al., 2013; Vargiu et al., 2004), and mTOR (Subramaniam and Snyder, 2011). In addition, recent findings have indicated that striatal interaction between Rhes and mutant Huntingtin protein (mHtt) is responsible for the selective vulnerability of MSNs in Huntington's disease (HD) (Mealer et al., 2014; Subramaniam et al., 2009). Here, we provide evidence that Rhes mRNA expression, so far thought to be localized only in striatal Darpp32-positive MSNs (Errico et al., 2008; Vargiu et al., 2001), also occurs in striatal ChIs. On the other hand, it was not found in Parv- and

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Fig. 5. Abnormal responsiveness to D2R stimulation in ChIs from Rhes−/− mice. (A–D) Representative traces showing the spontaneous firing activity of a ChI recorded in the perforated-patch configuration and collected before (left column) and during (right column) bath application of quinpirole (10 μM, 2 min). In both male and female Rhes+/+ mice (A, C), quinpirole (10 μM, 2 min) causes a slight firing rate reduction. ISI plot (right) confirms the small effect of D2R activation. In Rhes−/− animals (B, D) quinpirole application causes a significant increase in the rate of action potentials. Left-shift of the ISI plot (right) confirms the increased firing frequency induced by quinpirole for both male and female animals. Graph plots (inset) of normalized data summarize the abnormal effect of quinpirole.

Npy-positive striatal populations. Interestingly, in keeping with the observations obtained in mouse striatum, we also showed that RASD2 transcript is co-expressed with human ChAT mRNA-positive

interneurons. These findings extend to ChIs the potential relevance of Rhes in regulating striatal circuit function, thus suggesting a previously undetected role both in physiological and pathological conditions.

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Fig. 7. Rhes modulates cytoplasmic Ca2+ in ChIs. (A–D) Representative traces of fluorometric measurements of Ca2+ level induced by suprathreshold voltage steps (from −60 to 0 mV). (Inset a,b) Representative infrared (left) and fluorescence image (right, 380 nm excitation) of a ChI loaded with bis-fura-2 during whole-cell recording. Red circle mark the region considered for fluorometric measurements. Both ChIs of Rhes+/+ (A, B) and Rhes−/− (D, E) were recorded in presence of TTX (1 μM). During quinpirole application (red traces) the decay of calcium transients to baseline is significantly delayed in ChIs from Rhes−/− mice, as compared to Rhes+/+ mice. Bath application of ω-conotoxin GVIA (Ctx, gray traces) fully prevents the effect of quinpirole. (C, C1, F, F1) Box plots summarize these effects. No genders dependence was observed.

Based on their autonomous pacemaking activity, ChIs physiologically provide a continuous release of ACh in dorsal striatum in the absence of synaptic activity, and represent key modulators of striatal synaptic plasticity and pathophysiology of movement disorders (Pisani et al., 2007). The basal activity of ChIs is mainly modulated by the expression of striatal acetylcholinesterase, which is responsible for ACh degradation in the extracellular space (Zhou et al., 2002), and by M2/M4 muscarinic autoreceptors, able to mediate a negative control on ACh release (Calabresi et al., 1998). However, ACh release is also mediated by dopaminergic inputs from substantia nigra and ventral tegmental area (VTA) (Pan et al., 2010), through the activation of both dopamine D1 and D2 receptors. In particular, it has been demonstrated that D2R activation reduces ChIs firing rate, thus exerting a negative control over ACh release (DeBoer et al., 1996), while D1R activation enhances interneuronal excitability (Aosaki et al., 1998; Maurice et al., 2004; Pisani et al., 2000). While the functional role of RASD2 in human ChIs remains to be established, here, by using Rhes mutant mice, we report that this Gprotein exerts a substantial control on dopaminergic responses in striatal ChIs. Conversely, the responses to muscarinic M2/M4 and GABA-B receptor agonists were unchanged. Indeed, we demonstrate that, in Rhes−/−

striata, application of the selective D2R agonist, quinpirole, significantly alters ChIs excitability, resulting in a paradoxical increase in firing frequency, compared to wild-type controls. In principle, changes in ChIs firing frequency could be a consequence of Rhes being inactivated in MSNs that also express this protein. However, several lines of experimental evidence suggest that the abnormal response to D2R activation is a direct consequence of Rhes deletion in ChIs. First, the aberrant quinpirole effect was measured in whole-cell recordings in a condition of pharmacological isolation, ruling out possible contaminations from both GABA and ionotropic glutamate receptors. Additionally, chelation of intracellular Ca2+ by intrapipette BAPTA fully prevented the D2R-dependent effect. Likewise, when intracellular pipettes were loaded with the nonhydrolysable GDP-β-S, which locks G-proteins in an inactive state, quinpirole failed to cause any change in firing frequency. Both BAPTA and GDP-β-S do not permeate the cell membrane, therefore their antagonistic effects on D2R responses are indeed cell-specific. Finally, both fluorometric measurements of Ca2+ transients, as well as voltage-clamp experiments were performed in the presence of TTX, thereby excluding both sodium and transmitter-dependent conductances. Of interest, these experiments show that in Rhes−/− mice D2R activation is responsible for a prolonged cytosolic Ca2 + elevation, as

Fig. 6. Cellular specificity of the abnormal response to D2R activation in ChIs. (A, A1) Sample traces of a whole-cell recording from males and females Rhes−/− mice. Slices were preincubated with CNQX (10 μM), MK801 (30 μM) and picrotoxin (50 μM). In such pharmacological isolation bath application of quinpirole still induces in ChIs a paradoxical excitation. (B–C1) Representative traces of ChIs recorded in whole-cell current clamp experiments in ChIs of male and female Rhes−/− mice. Cells were loaded with BAPTA [10 mM; (B, B1)] or with GDP-β-S [500 nM, (C, C1)]. Both drugs fully prevent the abnormal response triggered by quinpirole. (B2, C2) Plots summarize the efficacy of BAPTA and GDP-β-S.

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Fig. 8. Ca2+ channels involvement in the abnormal D2-mediated response in ChIs from Rhes−/− mice. (A, A1) Representative traces of ChIs recorded in whole-cell current-clamp experiments in male and female Rhes−/− mice. Slice preincubation with ω-conotoxin GVIA (Ctx, 1 μM) fully prevents the excitation induced by quinpirole. (A2) Box plot summarizes the effect of Ctx on firing frequency. (B–C1) Representative traces from ChIs of voltage-dependent currents evoked by a series of depolarizing steps from an holding potential of −60 to 0 mV (inset). Data are collected from both males and females Rhes+/+ and Rhes−/− mice. Recordings were made in TTX (1 μM). In Rhes+/+ mice, application of the D2R agonist quinpirole (10 μM) slightly reduces the current evoked by depolarizing steps (B, C; left column). Conversely, in Rhes−/− mice D2R activation causes a current inhibition in both genders (B1, C1, left column). Blockade of Cav2.2 Ca2+ channels by ω-conotoxin GVIA (Ctx, 1 μM) prevents the quinpirole effect in both Rhes+/+ and Rhes−/− mice (B–C1; right column). (D, D1) Graphs report the current density induced by single voltage suprathreshold step (black arrows) normalized to cell capacitance under control conditions, in the presence of quinpirole and in quinpirole plus Ctx. (E) Graphs show the quinpirole-sensitive transient currents plotted against voltage and normalized to cell capacitance in both genders. Application of Ctx fully abolishes the quinpirole-sensitive current.

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Fig. 9. Blockade of Akt signaling counteracts the abnormal D2R-mediated response in ChIs from Rhes−/− mice. (A–B) Sample traces of a perforated-patch recording from males and females of Rhes−/− animals showing that in ChIs bath application of quinpirole induces a paradoxical excitation. (A1, B1) In Rhes+/+ slice incubation with PI3K inhibitor LY294002 (50 μM, LY) does not substantially change the slight inhibitory effect of quinpirole. Conversely in Rhes−/− (A2, B2) mice, pretreatment with LY294002 fully prevents the excitatory response to quinpirole. (C, C1) Graph plots summarize the efficacy of LY294002 in both males and females in Rhes+/+ and Rhes−/− animals.

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Fig. 10. Rhes protein levels determined by western blotting in the dorsal striatum of Tor1a+/+ and Tor1a+/Δgag knock-in mice. The top panels show representative blots comparing the different genotypes. Graphs summarize the results collected. All values are expressed as the mean ± SEM.

compared to their littermates, further supporting the primary role of Ca2 + in sustaining the increased firing frequency triggered by quinpirole. Dopamine D2R activation is coupled to inhibition of Cav2.2 channels in a membrane-delimited PKC-insensitive manner (Yan et al., 1997), and previous work has shown that Rhes modulates Cav2.2 channel activity by influencing Gαi-coupled GPCR signaling (Thapliyal et al., 2008). Accordingly, we found that the blockade of Cav2.2 channels was able to counteract the effect of quinpirole on cytoplasmic Ca2 + elevation. Moreover, the selective Cav2.2 blocker, ω conotoxin GVIA, prevented the quinpirole effect on action potentials rate in ChIs from Rhes−/− mice, providing a more direct link between Cav2.2 channel function and changes in firing frequency. Dopamine receptors exert some of their biological effects through G-protein dependent modulation of cAMP/PKA signaling cascade (Missale et al., 1998). On the other hand, they can also affect Akt pathway in a cAMP-independent manner (Beaulieu et al., 2005). In this respect, D2R activation induces changes in Akt/GSK3β signaling cascade through a PI3K-dependent mechanism (Chen et al., 2012) which, via phosphatidyl-dependent kinases 1/2 (PDK1/2), phosphorylates and, in turn, activates Akt (Alessi et al., 1997). Previous in vitro studies showed that Rhes binds to and activates PI3K (Vargiu et al., 2004). In keeping with that, Bang and colleagues demonstrated that growth factor stimulation promotes Rhes interaction with PI3K and Akt, highlighting the role of Rhes as a bridge between PI3K and Akt signaling pathway (Bang et al., 2012). Remarkably, recent in vivo findings have revealed that Rhes interacts with β-Arrestins (Harrison et al., 2013), and participates in the multi-protein complex

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involved in the striatal modulation of DA D2R-dependent Akt/GSK3β signaling (Beaulieu et al., 2005). In particular, the authors have found that lack of Rhes causes an increased striatal phosphorylation of Akt and GSK3β in mutant mice, thus suggesting an alternative route of action for this small GTP-binding protein in modulating the striatal PI3K/ Akt/GSK3β pathway (Harrison et al., 2013). Moreover, PI3K promotes the membrane insertion of non-selective cation channels and calcium-dependent potassium channels (Lhuillier and Dryer, 2002; Martin et al., 2002) and, indeed, PI3K has been reported to modulate changes in excitability by increasing the insertion or prolonging the surface expression Ca2+ channels (Viard et al., 2004). Accordingly, we found that the selective PI3K inhibitor, LY294002, was able to normalize the aberrant D2R responses recorded in striatal mutant ChIs. Therefore, in light of previous evidence, but also of our data, we hypothesize that Rhes deficiency interferes with D2R-dependent activity in ChIs, through an abnormal Ca2 +-dependent modulation of PI3K/Akt signaling. Previous reports have demonstrated that Rhes negatively modulates D1R-dependent activity by interfering with cAMP/PKA signaling in MSNs (Errico et al., 2008; Harrison and He, 2011). On the other hand, differently from MSNs, striatal ChIs preferentially express dopamine D5R subtype (Bergson et al., 1995; Rivera et al., 2002; Yan and Surmeier, 1997), thus suggesting an involvement of signaling pathways other than those described for D1R. This evidence related to different dopamine receptor subtypes selectively expressed in distinct striatal subpopulations may explain the reduced responsivity found in mutant ChIs after SKF38393 administration. Further work is mandatory to shed light on the specific role of Rhes in modulating the activity of dopamine receptor isoforms in peculiar striatal neuronal populations. A growing interest points to Rhes as a crucial factor in the pathogenesis of basal ganglia disorders, such as HD or L-DOPA-induced dyskinesia in Parkinson's disease (Baiamonte et al., 2013; Subramaniam et al., 2009, 2012; Subramaniam and Snyder, 2011). It has been demonstrated that, in the early stages of the disease, both dystonia and parkinsonism commonly accompany not only the juvenile form of HD, but also the adult onset HD cases (Louis et al., 1999). Notably, we have previously demonstrated the existence of the paradoxical D2R-mediated excitation in ChIs in distinct mouse models of DYT1 dystonia (Pisani et al., 2006; Sciamanna et al., 2012a,b). These findings are reminiscent of the D2Rmediated abnormal electrophysiological response observed in ChIs Rhes mutants. However, western blotting analysis of Rhes expression in a knock-in DYT1 dystonia model did not reveal any gross difference in the protein Rhes levels. This observation suggests that convergent electrophysiological findings may arise from distinct elements, which ultimately converge on common D2R-dependent effectors, like Cav2.2. channels and PI3K/Akt pathway, that we found to be altered in both Rhes and DYT1 models (Sciamanna et al., 2011, 2014). Additional focused studies are required to corroborate this crucial point. Overall, our findings enlarge the complexity of the specific role of Rhes in controlling striatal circuitry and, most importantly, strongly suggest that this striatal molecule could represent an interesting novel target of investigation for basal ganglia disorders. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.nbd.2015.03.021. Funding This study was partially supported by PRIN 2010–2011 to AP and AU, by FIRB 2013 (RBFR13S4LE — Italian Minister of Research, MIUR) to GS, FN and VG, and by the Foundation for Dystonia Research (FDR) to AP. Acknowledgments We thank Drs A. Di Maio and V. Lucignano for their excellent technical support. We wish to thank Rose Goodchild for helpful discussion and comments.

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Rhes regulates dopamine D2 receptor transmission in striatal cholinergic interneurons.

Ras homolog enriched in striatum (Rhes) is highly expressed in striatal medium spiny neurons (MSNs) of rodents. In the present study, we characterized...
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