Subthalamic nucleus high-frequency stimulation modulates neuronal reactivity to cocaine within the reward circuit

Neurobiology of Disease 80 (2015) 54–62

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Subthalamic nucleus high-frequency stimulation modulates neuronal reactivity to cocaine within the reward circuit Sabira Hachem-Delaunay a,b,1, Marie-Line Fournier a,b,1, Candie Cohen c,d, Nicolas Bonneau a,b, Martine Cador a,b, Christelle Baunez c,d, Catherine Le Moine a,b,⁎ a

Université Bordeaux, INCIA, UMR 5287, Bordeaux F-33000, France CNRS, INCIA, UMR 5287, Bordeaux F-33000, France CNRS, INT, UMR 7289, Marseille F-13385, France d Aix Marseille Université, INT, UMR 7289, Marseille F-13385, France b c

a r t i c l e

i n f o

Article history: Received 19 January 2015 Revised 4 May 2015 Accepted 9 May 2015 Available online 14 May 2015 Keywords: Addiction Parkinson Basal ganglia Arc c-Fos catFISH Nucleus accumbens Striatum Deep brain stimulation

a b s t r a c t The subthalamic nucleus (STN) is a critical component of a complex network controlling motor, associative and limbic functions. High-frequency stimulation (HFS) of the STN is an effective therapy for motor symptoms in Parkinsonian patients and can also reduce their treatment-induced addictive behaviors. Preclinical studies have shown that STN HFS decreases motivation for cocaine while increasing that for food, highlighting its influence on rewarding and motivational circuits. However, the cellular substrates of these effects remain unknown. Our objectives were to characterize the cellular consequences of STN HFS with a special focus on limbic structures and to elucidate how STN HFS may interfere with acute cocaine effects in these brain areas. Male Long–Evans rats were subjected to STN HFS (130Hz, 60 μs, 50–150 μA) for 30 min before an acute cocaine injection (15 mg/kg) and sacrificed 10 min following the injection. Neuronal reactivity was analyzed through the expression of two immediate early genes (Arc and c-Fos) to decipher cellular responses to STN HFS and cocaine. STN HFS only activated c-Fos in the globus pallidus and the basolateral amygdala, highlighting a possible role on emotional processes via the amygdala, with a limited effect by itself in other structures. Interestingly, and despite some differential effects on Arc and c-Fos expression, STN HFS diminished the c-Fos response induced by acute cocaine in the striatum. By preventing the cellular effect of cocaine in the striatum, STN HFS might thus decrease the reinforcing properties of the drug, which is in line with the inhibitory effect of STN HFS on the rewarding and reinforcing properties of cocaine © 2015 Elsevier Inc. All rights reserved.

Introduction The subthalamic nucleus (STN) belongs to the basal ganglia and is classically associated with motor functions. High-frequency stimulation (HFS) of the STN is the most common surgical therapy applied to Parkinsonian patients (PD) and is very effective in alleviating the

Abbreviations: PD, Parkinson’s disease; OCD, obsessive–compulsive disorders; STN, subthalamic nucleus; HFS, high-frequency stimulation; DBS, deep brain stimulation; FISH, fluorescent in situ hybridization; catFISH, cellular compartment analysis of temporal activity by FISH; IEG, immediate early gene; DST, dorsal striatum; NAC, nucleus accumbens; PFC, prefrontal cortex; OFC, orbitofrontal cortex; GP, globus pallidus; VP, ventral pallidum; BLA, basolateral amygdala; CEA, central amygdala; SNC, substantia nigra pars compacta; SNR, substantia nigra reticulata; VTA, ventral tegmental area. ⁎ Corresponding author at: Université de Bordeaux, INCIA “Institut de Neurosciences Cognitives et Intégratives d’Aquitaine”; CNRS UMR 5287, Equipe “Neuropsychopharmacologie de l’addiction”, BP31; Université Bordeaux Segalen; 146 rue Léo Saignat; 33076 Bordeaux Cedex. Fax: + 33 5 56 90 02 78. E-mail address: [email protected] (C. Le Moine). 1 The two authors equally contributed to the work. Available online on ScienceDirect (www.sciencedirect.com).

http://dx.doi.org/10.1016/j.nbd.2015.05.007 0969-9961/© 2015 Elsevier Inc. All rights reserved.

motor disorders associated with this pathology: tremor, rigidity and hypokinesia (Benabid, 2003; Limousin et al., 1995). STN is also a critical component of a network controlling non-motor, associative and limbic functions involving the prefrontal cortex, nucleus accumbens and ventral pallidum (for a review, see Baunez and Lardeux, 2011). In line with these non-motor functions, STN HFS is also applied for the treatment of obsessive–compulsive disorders (OCD) (Mallet et al., 2008) and is proposed to be also useful to treat addiction. Indeed if STN HFS has not yet been tested in addicts, some clinical observations in PD after STN HFS have reported craving for sweet food in some cases, or decreased addictive behavior towards dopaminergic treatment in other cases (for a review, see Pelloux and Baunez, 2013). A recent study has also shown that STN stimulation may significantly reduce the compulsive use of dopaminergic drugs in PD patients without increasing the risk of inducing the disorder in previous non-misusers. In addition, most of the addictive behaviors improve after STN deep brain stimulation (DBS) partly as a result of the lower dosage of dopaminergic medication but also possibly through a specific effect of STN DBS in the limbic circuit of motivation and reward (Eusebio et al., 2013).

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In preclinical models, we have previously shown that STN lesions or STN HFS decrease motivation for cocaine (Baunez et al., 2005; Rouaud et al., 2010). On the other hand, cocaine administration activates c-Fos expression in the STN in parallel with behavioral sensitization (Uslaner et al., 2001, 2003). The effects of STN HFS by itself at the cellular level have been assessed so far mainly on motor structures showing activation of striatal markers (like the cytochrome oxidase and GAD) as well as increased GAD or Fos activity in GP, EP and SNR (Bacci et al., 2004; Oueslati et al., 2007; Salin et al., 2002; Shehab et al., 2014; Vlamings et al., 2009). However, there are no data on the consequences of STN HFS on the neuronal reactivity (activity or plasticity) of the reward circuit. Our hypothesis is that STN HFS alters the acute cellular effects of cocaine on the reward circuit, which could explain an inhibition of the rewarding properties of cocaine in procedures in which a very limited number of injections were taken (Rouaud et al., 2010). The objectives of the present study were thus to determine (1) the cellular consequences of bilateral STN HFS by itself on motor and limbic structures and (2) whether the cellular reactivity to acute cocaine in these brain areas was affected by STN HFS. For this, c-Fos and Arc mRNA expression were analyzed by in situ hybridization following cocaine injection associated or not with STN HFS. These two immediate early genes (IEG) have different dynamics of induction and functions in relation to synaptic activity (for reviews, see Guzowski, 2002; Lanahan and Worley, 1998) so that they can both reflect the acute response to a given stimulus or subsequent plasticity-related processes. Moreover, one advantage of using Arc as a marker of neuronal activity was the possibility to use the fluorescent method of catFISH (cellular compartmental analysis of temporal activity by Fluorescent In Situ Hybridization) that allows to discriminate cellular responses to two types of temporally separated stimuli (for a review, see Guzowski et al., 2005), in the present case STN HFS and cocaine, by dissociating cytoplasmic from nuclear labeling. Thus, to decipher both STN HFS and cocaine effect in the present study, we have combined classical in situ hybridization and catFISH. We have focused our analysis on a network of interconnected limbic structures (nucleus accumbens, amygdala nuclei, infra-limbic and pre-limbic prefrontal cortex, orbitofrontal cortex) known to be involved in rewarding and motivational processes. Materials and methods Animals Adult male Long–Evans rats (n = 24, Janvier Labs, Le Genest-SaintIsle, France) were housed in pairs and maintained on a 12 hr light/ dark cycle (lights on at 07:00 am). The animals had no food restriction. Water was provided ad libitum, except during experimental sessions. All procedures were conducted during the light phase and followed the regulations in accordance with the European Community’s Council Directive (EU Directive 2010⁄6⁄EU86) and our national French Agriculture and Forestry Ministry decree 87-849.

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Electrodes implantation All rats were anesthetized with ketamine (Imalgene® 100 mg/kg i.m.) and medetomidine (Domitor® 30 mg/kg i.m.). They were then secured in a Kopf stereotaxic apparatus, and the bilateral electrodes were implanted at the following coordinates: anteroposterior − 3.8 mm (from bregma); lateral ± 2.4 mm; dorsoventral − 8.35 mm (from skull, according to (Paxinos and Watson, 1997)). After surgery, all rats were awaken up with the medetomidine antagonist (antisédan 15 mg/kg i.m.) Rats had then seven to ten days of recovery. HFS stimulation procedure The day before the experiment, each individual was tested for adjustment of the stimulation parameters. The frequency remained fixed at 130 Hz, as well as the pulse width at 60 μs. The intensity was adjusted for each individual, so that it was just below the threshold of induction of hyperkinetic movements of the contralateral paw as described previously (Rouaud et al., 2010). Starting from 0 to a maximum of 150 μA, the intensity was increased progressively, and the rat was observed in a cylinder made of perspex to observe its circling behavior and the hyperkinetic movements of the front paws. Once the parameters determined for each rat, the animal was placed back to his home cage. On the day of the experiment, each rat was placed in a locomotor activity cage made of perspex panels with a grid floor and equipped with photobeam cells, allowing measurement of the locomotor activity via a computer and interface (Imetronic, France). Stimulation was applied according to the parameters determined the day before. At the end of the 30 min period in the activity cages, the rats received either an acute i.p. injection of cocaine (15 mg/kg) or saline (NaCl 0.9%), and the activity was measured for 9 min. Four experimental groups (n = 6/group) were used: STN HFS OFF-Saline (Sal-OFF), STN HFS OFF-Cocaine (Coc-OFF), STN HFS ON-Saline (Sal-ON) and STN HFS ON-Cocaine (Coc-ON). Rats were sacrificed by decapitation at a 10 min time-point following cocaine injection. Tissue processing and histological control of the electrode’s location Brains were quickly dissected out, frozen and kept at − 80 °C until use. All the in situ hybridization experiments were performed on cryostat sections (12 μm thick), which were collected on gelatin-coated slides and stored at − 80 °C until processing for in situ hybridization. Sections were collected to encompass the following brain regions: the prefrontal and orbitofrontal cortices (PFC and OFC), the dorsal striatum (DST), the nucleus accumbens core and shell (NAC Core and NAC Shell), the globus pallidus (GP), the ventral pallidum (VP), the basolateral and central amygdala nuclei (BLA and CEA), the substantia nigra pars compacta and reticulata (SNC and SNR) and the ventral tegmental area (VTA). For checking the electrode’s location, 30 μm thick sections encompassing the entire STN were collected on gelatin-coated slides in order to be stained with Cresyl violet (Figs. 1A, B). Probe labeling and in situ hybridization

HFS electrodes As described previously (Darbaky et al., 2003), the electrodes were made out of two Platinum-Iridium wires coated with Teflon (75 μm diameter, Phymep, Paris, France). The coating was removed over 0.5 mm at the tips (the average distance between the two tips was of 100 μm), and the wires were then paired and inserted into needles to form the electrodes. Wires were then soldered on an electric connector, allowing the interface with both recording and stimulation devices. Using custom mold, the needles were separated by a distance of 4.8 mm in line with the laterality of the STN and bilateral implantation and deeply bound to the connector with dental cement.

The Arc (Lyford et al., 1995) and c-Fos (Curran et al., 1987) probes were labeled by in-vitro transcription with digoxigenin-11-UTP (Roche Diagnostics, Meylan, France), using appropriate T7 or SP6 RNA polymerases (Promega, Charbonnières, France) and purified as described in (Lucas et al., 2008). Cryostat sections were post-fixed in ice-cold PFA 4% for 5 min, and washed twice in 4× saline-sodium citrate (SSC). After 5 min in 0.1 M triethanolamine/4× SSC, pH 8.0, in which we added 0.25% acetic anhydride for another 5 min, the sections were dehydrated in increasing concentrations of ethanol. For catFISH, an additional step was performed incubation in chloroform for 5 min, and then in ethanol 100% and 95% (1 min each) before drying. Sections were hybridized overnight at 55 °C with 10–20 ng of digoxigenin-

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Fig. 1. Histological control of electrodes locations and schematic representation of analyzed brain areas. (A, B) Example of the electrode location in the STN at −4.16 mm from bregma. Dotted line delineates the STN and black arrows point to the electrode tips. (B) Summary diagram of HFS sites in the cocaine (Coc-ON, dark boxes, left) and saline (Sal-ON, grey boxes, right) groups. (C) Representation of the brain regions which have been quantified.

labeled probes in 50 μl hybridization buffer (20 mM Tris–HCl, 1 mM EDTA, 300 mM NaCl, 50% deionized formamide, 10% dextran sulfate, 1% Denhardt, 250 μg/mL yeast tRNA, 100 μg/mL salmon sperm DNA, 0.1% SDS, 0.1% sodium-thiosulfate). Sections were then placed in 4 × SSC, incubated in RNAse A (37 °C for 15 min) and washed in increasing conditions of stringency, up to 0.1 × SSC for 30 min at 65 °C (twice), before a final wash was in 0.1× SSC at room temperature (RT). For colorimetric staining, slides were then rinsed twice for 5 min in buffer A (1 M NaCl/0.1 M Tris/2 mM MgCl2, pH 7.5), and then for 30 min in buffer A containing 3%normal goat serum and 0.3% Triton X-100. After 5 h incubation at room temperature with alkaline phosphatase-conjugated anti-digoxigenin antiserum (Roche Diagnostic, Meylan, France; 1:1000 in buffer A/3% normal goat serum/0.3% Triton X-100), the sections were rinsed in buffer A (5 min twice), then 10 min twice in STM buffer (1 M NaCl/0.1 M Tris/5 mM MgCl2, pH 9.5), and 10 min twice in 0.1 M STM buffer pH 9.5 (0.1 M NaCl/ 0.1 M Tris/5 mM MgCl2, pH 9.5). The sections were then incubated overnight in the dark at room temperature in 0.1 M STM buffer pH 9.5 containing 0.34 mg/ml nitroblue-tetrazolium and 0.18 mg/ml bromochloro-indolylphosphate (BCIP/NBT, Promega, Charbonnières, France). They were then rinsed in 0.1 M STM buffer pH 9.5 and in distilled water before being mounted in Eukitt without counterstaining. Labeling for c-Fos and Arc are illustrated in Fig. 1. For fluorescent hybridization processing (catFISH), the sections were processed as described by (Ramirez-Amaya et al., 2013) with minor modifications. Briefly, the sections were placed in 2% hydrogen peroxide (H2O2) in 1× SSC for 15 min to inactivate endogenous peroxidases and rinsed several times in TNT buffer (0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.3% Triton X-100). Sections were blocked with TNB (0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.5% blocking reagent from the TSA Amplification kit, Perkin Elmer Life Sciences, Courtaboeuf, France) for 30 min and then incubated with horseradish peroxidaseconjugated digoxigenin antibody (Roche Diagnostic, Meylan, France)

1:1000 in TNB for 2 h under coverslips. After rinsing with TNT, the slides were incubated in Cy3-Plus-Tyramide (1:1000 in TSA amplification diluent, Perkin Elmer Life Sciences, Courtaboeuf, France) for 30 min under coverslips. After rinsing 3 times (5 min each) in TNT then in 2× SSC, the slides were incubated in SYTOX Green (1:80000 in 2× SSC 10 min at RT, Fisher Scientific, Illkirch, France), rinsed 3 times (5 min each) in 2× SSC and mounted with Vectashield hard set mounting medium (Biovalley, Marne-la-Vallée, France). Colorimetric quantification C-fos and arc mRNA expressions were quantified at the cellular level using an image analyzer system for cartography (Mercator, Explora Nova, La Rochelle, France) as described by Lucas et al. (2008) with minor modifications. Briefly regions of interest (illustrated Fig. 1C) were delineated by an experimenter blind to the experimental groups and labeled neurons were automatically counted given that they were above a threshold determined from 4 different random sections. Analyses in cortical regions have been performed without taking into account the different layers. All the sections were then processed for quantification with identical threshold parameters. Positive neurons were counted on each side of the brain and the data obtained in both sides of each section were pooled. Data are expressed as mean ± SEM of the numbers of c-Fos or Arc-positive nuclei per mm2. Confocal microscopy and analysis Analyses were performed according to Ramirez-Amaya et al. (2013) with minor modifications. Confocal images were acquired using a Leica SP5 confocal microscope (Leica, Nanterre, France) with a 40×/1.3 NA oil immersion objective, using a 543 helium/neon laser to excite the CY3 signal, and a 488 argon laser to excite the SYTOX Green signal. Confocal parameters (i.e. offset and detector gain) were established

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from 4 different random sections, and those settings were kept constant for imaging the rest of the sections. For the dorsal striatum, 10 Z-stacks with sections of 1 μm optical thickness were collected for each side (right and left). For the nucleus accumbens, 4 optical Z-sections were collected from each side for both core and shell subregions. Confocal image analysis was performed offline using the Image J software (NIH, USA) by an experimenter blind to the experimental groups. SYTOX Nuclear staining (green) revealed two distinct morphologies (Fig. 2A). Cells with large and diffusely stained nuclei were considered as neurons. The remaining cells had smaller nuclei and were bright and uniformly stained thus corresponding to glia. Thus, only neuron-like cells present in the median planes of each Z-stack were counted. Once neurons were identified, they were classified according to their cytoplasmic and nuclear Arc staining, detected with CY3 signal (Fig. 2A). Neurons with one or two intense CY3 nuclear foci visible across 3 or more Z-section plains were classified as Arc nuclear-positive cells (N, Fig. 2B). Neuronal nuclei surrounded more than 60% with the CY3 signal in 3 or more Z-section plains were classified as Arc cytoplasmic positive (C, Fig. 2C). Finally, neurons with both criteria were classified as double activated cells (N + C, Fig. 2D). The total number of neurons counted in each stacks did not differ between sections or groups.

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were performed to check for electrode’s location. Cresyl violet staining at the STN level showed that four rats were implanted outside the STN and were therefore excluded from the data analysis. The detailed location of the electrodes for the two final ON groups (Saline versus Cocaine) are illustrated in Fig. 1B. The mean HFS intensities were 96.40 μA ± 13.56 SEM for the Sal-ON group and 94.20 μA ± 15.17 SEM for the Coc-ON group and were not significantly different between groups. Effects of bilateral STN HFS on c-Fos expression in motor and limbic areas The global quantification of c-Fos and Arc expression was used here mainly to assess the sole global effect of STN HFS on motor and limbic areas. Our results showed that STN HFS by itself has little effect on these regions in our experimental conditions, except in the GP and the BLA (Fig. 3). Indeed, c-Fos mRNA-positive neurons were increased following 40 min of STN HFS in the GP (p b 0.05, unpaired t test) and the BLA (p b 0.05, unpaired t test). No significant effect was observed in all the other areas analyzed, i.e., PFC, OFC, DST, NAC shell and core, VP, CEA, SNR and VTA. In contrast to these effects on c-Fos mRNA, Arc mRNA expression was not modified by STN HFS in any brain area in saline treated rats (as illustrated for DST and NAC in Figs. 5A’-C’). Effect of STN HFS on the c-Fos and Arc cellular global responses to an acute injection of cocaine

Statistical analysis Cellular data were analyzed using either unpaired t tests or two-way ANOVA, followed by post hoc Fisher’s LSD test. All the statistical analyses were performed using Prism v.4.03 (GraphPad, San Diego, CA, USA). In all cases, p b 0.05 was considered significant. Results Four experimental groups (n = 6/group) were used in the present study: STN HFS OFF-Saline (Sal-OFF), STN HFS OFF-Cocaine (Coc-OFF), STN HFS ON-Saline (Sal-ON), STN HFS ON-Cocaine (Coc-ON). Before starting the in situ hybridization experiments, histological controls

A

As above, the quantification of c-Fos and Arc mRNA using classical colorimetric in situ hybridization allowed assessing the global effect of STN HFS on the response to an acute injection of cocaine within motor and limbic areas. Cocaine (15 mg/kg) injected 10 min before sacrifice strongly activates both c-Fos and Arc expression in the DST and NAC shell and core (Sal-OFF/Coc-OFF, Figs. 4 and 5). No activation following cocaine was observed in all the other areas analyzed, i.e., PFC, OFC, GP, VP, BLA, CEA, SNR, SNC and VTA (data not shown). STN HFS significantly inhibits c-Fos mRNA activation induced by cocaine in the dorsal striatum (DST, Coc-OFF/Coc-ON, Fig. 5A). Two-way ANOVA indicated a significant cocaine × STN HFS interaction

B

C

D

Fig. 2. Examples of Arc expression after catFISH and Z-stacks confocal analysis. (A) Orthogonal views (×40 objective) of Arc mRNA (red) visualization using fluorescent in situ hybridization (FISH) and confocal image acquisition. Nuclei are labeled with SYTOX Green (green) revealing two distinct morphologies. Cells with large and diffusely stained nuclei were considered neurons (arrows). The remaining cells had smaller nuclei and were bright and uniformly stained thus corresponding to glia (arrowheads). Scale bar scale: 15 μm. (B, C) Examples of nuclear labeling with two intra nuclear foci (B) and of cytoplasmic labeling around the nucleus (C). (D) Example of a neuron displaying both nuclear and cytoplasmic labeling.

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Density of c-fos positive neurons (per mm2)

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PFC

*

Arc

*

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GP

BLA

Fig. 3. Cellular effects of STN HFS on c-Fos expression. Histograms showing the density of c-Fos-positive neurons per mm2 (mean ± SEM) in PFC, DST, NAC shell, NAC core, GP and BLA in sham-operated rats (OFF, white bars) and in rats with STN HFS (ON, hatched bars). HFS induced an activation of c-Fos only in GP (p = 0.0333) and in BLA (p = 0.0420). Unpaired t test; *p b 0.05.

(F(1,16) = 7.161, p b 0.05). Post hoc Fisher’s LSD showed significant c-Fos activation by cocaine in the OFF groups (p = 0.01) that is abolished in the ON group (p N 0.05). In the NAC core and shell, the two-way ANOVA showed a significant effect of cocaine injection (F(1,16) = 12.29, p b 0.01 for the core, F(1,16) = 7.945, p = 0.01 for the shell) but no interaction with STN HFS (F(1,16) = 1.247, p N 0.05 for the core, F(1,16) = 1.626, p N 0.05 for the shell), although the cocaine effect is only significant in the OFF group in both shell and core (Figs. 5B, C). The cocaine-induced Arc activation in DST, NAC core and shell was not significantly modified by STN HFS stimulation (Figs. 4 and 5A’, B’, C’). Indeed, two-way ANOVA indicated a significant cocaine effect in all three regions (F(1,15) = 32.06, p b 0.0001 for the DST; F(1,15) = 20.02, p b 0.01 for the core; F(1,15) = 48.00, p b 0.0001 for the shell), with no significant effect of STN HFS nor interaction.

Dissociation of STN HFS and cocaine cellular responses through Arc expression using catFISH Since the cellular effects of cocaine were found only in the DST and NAC core and shell, the catFISH analysis was performed only on these structures. Neuronal reactivity related to STN HFS (after 40 min) was hypothesized to display mainly cytoplasmic labeling. On the other hand, the response to cocaine, initiated 2–12 min before sacrifice (Guzowski et al., 1999; Vazdarjanova et al., 2002), was likely to be visualized by a nuclear labeling. Our data confirmed that STN HFS by itself (in the control groups Sal-ON vs Sal-OFF) had no significant effect on Arc mRNA levels and did not induce any significant labeling changes in all three regions.

Cocaine

Fig. 4. Examples of c-Fos and Arc activation by cocaine in the dorsal striatum (DST). Photomicrographs of c-Fos (A, B) and Arc (C, D) expression following colorimetric in situ hybridization after saline (A, C) or cocaine (B, D) in the DST. Cocaine injection (15 μg/kg) increased both c-Fos and Arc mRNA expression, despite an overall higher level of Arc in control conditions (saline). Scale bar: 100 μm.

Cocaine increased Arc nuclear labeling (N alone) both in the DST, the NAC shell and the core (Figs. 6A–C, left panels). Two-way ANOVA indicated a significant cocaine effect in all three regions (F(1,16) = 16.12, p = 0.001 for the DST; F(1,16) = 45.06, p b 0.0001 for the NAC shell, and F(1,15) = 18.94, p = 0.0006 for the NAC core), with no significant effect of STN HFS by itself. Cocaine as well as STN HFS had however no significant effect nor interaction on the amount of cytoplasmic labeling (C alone, Figs. 6A″–C″, right panels). Interestingly, we observed a significant proportion of neurons exhibiting both type of labeling (N + C neurons) in all three structures following cocaine (Figs. 6A′–C′, middle panels). Two-way ANOVA indicated a significant cocaine effect in all three regions (F(1,15) = 17.14, p = 0.0009 for the DST; F(1,16) = 14.26, p = 0.0017 for the NAC shell; and F(1,15) = 10.62, p = 0.0053 for the NAC core), with no significant effect of STN HFS by itself. A tendency for an increased cocaine effect following STN HFS was observed, but a significant interaction was found only in the NAC shell (F(1,16) = 6.510, p = 0.0213), showing that the cocaine effect on N + C labeling was enhanced under STN HFS (Fig. 6B’, #p b 0.05). Discussion The present work shows that STN HFS applied bilaterally induces increased neuronal activity as measured by c-Fos expression in the GP and the BLA and also alters cocaine-induced activity in the striatum and nucleus accumbens in a complex manner depending of the marker analyzed, c-Fos or Arc. These two IEGs are classically used to map functionally relevant circuits in different experimental situations (for reviews, see Bramham et al., 2008; Kovacs, 2008). However, they have both different dynamics of induction and functions in relation to

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Density of c-fos positive neurons (per mm2)

Sal OFF

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Density of arc positive neurons (per mm2)

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Fig. 5. Effect of STN HFS on the c-Fos and Arc cellular responses to an acute injection of cocaine. Histograms showing the density of c-Fos (A, B, C) and Arc (A′, B′, C′)-positive neurons per mm2 (mean ± SEM) in the DST (A, A′), NAC shell (B, B′) and NAC core (C, C′). Only post hoc Fisher’s LSD were reported (* versus Saline; # vs OFF). Detailed two-way ANOVA analyses are mentioned in the text. Cocaine (15 mg/kg) injected 10 min before sacrifice strongly increases the number of c-Fos and Arc mRNA-positive neurons in the DST and NAC shell and core (Sal-OFF/Coc-OFF, white and grey bars). STN HFS significantly inhibits c-Fos mRNA activation induced by cocaine only in the dorsal striatum (A, DST, Coc-OFF/Coc-ON) despite a tendency in the NAC for which the cocaine effect is only significant in the OFF group in both shell (B) and core (C). The cocaine-induced Arc mRNA activation in DST, NAC core and shell was not significantly modified by STN HFS stimulation (hatched white and grey bars).

synaptic activity and plasticity (for reviews, (Bramham et al., 2008; Guzowski, 2002; Lanahan and Worley, 1998). Effects of STN HFS in the reward circuit The first objective of our study was to analyze the cellular consequences of bilateral STN HFS on the reward circuit along with motor and limbic structures. In our experimental conditions, and without any cocaine stimulation, c-Fos expression was expected to reflect mainly responses induced by the STN HFS. Our data showed that STN HFS alone induced increased c-Fos gene expression only in one motor structure: the GP and one limbic structure: the BLA. The increased c-Fos expression into the GP is not in line with the hypothesis that HFS inactivates STN neurons, which should reduce the level of activity in the GP (for a review, see Gubellini et al., 2009). However, this effect could be the result of stimulation of the fibers despite an inhibition of the STN core structure. Indeed, it has been shown that brief unilateral STN HFS increased cytochrome oxidase and Fos activity in the GP (Benazzouz et al., 2004; Shehab et al., 2014). A microdialysis study has also reported an increase of glutamate level in the GP following

STN HFS (Windels et al., 2003). If STN HFS increases the level of glutamate into the GP, this could lead to an over-activity in the GP, evidenced by increased c-Fos activity, leading to a stronger inhibitory effect on STN neurons. However, it is clear that it may be difficult to reconcile all the cellular data obtained mainly on motor structures since they all rely on different experimental conditions (unilateral versus bilateral stimulation, various durations of the stimulation), which may account for these discrepancies (Bacci et al., 2004; Oueslati et al., 2007; Salin et al., 2002; Vlamings et al., 2009). Despite the behavioral effects of STN HFS on motivational functions and the fact STN HFS significantly affects neurotransmission in the limbic system (Baunez et al., 2007; Rouaud et al., 2010; Winter et al., 2008), the cellular responses induced by STN HFS in limbic and motivational structures are still poorly known. The increased c-Fos expression in the basolateral amygdala (BLA) following STN HFS is interesting given the fact that the connection between STN and BLA was only recently documented functionally in human (Lambert et al., 2012). BLA is classically associated with emotional processes, and STN DBS was reported recently to affect recognition of emotions as well as affective responses in PD patients (Peron et al., 2013). Those effects seem to be related to an

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Fig. 6. Analysis of Arc mRNA expression using catFISH differentiates STN HFS and cocaine cellular responses. Each histogram represents means ± SEM of the density of Arc-positive neurons per mm2 according to the type of labeling: nuclear (N) labeling alone (A, B, C left panels), cytoplasmic (C) labeling alone (A″, B″, C″ right panels) and N + C labeling (A’, B’, C’ middle panels). Empty bars are for the OFF groups, and hatched bars for the ON groups. Saline groups are in white bars, and cocaine groups are in gray. Cocaine injection induced a strong increase in the density of Arc-positive neurons with a high proportion (82–95%) of neurons showing nuclear labeling (both N and N + C) in DTS, NAC core and shell. (A–C) Cocaine strongly activated Arc mRNA N labeling which was not significantly altered by STN HFS. (A′–C′) Cocaine increased the density of neurons displaying N + C labeling, and this effect was more pronounced following STN HFS but significant only in the NAC shell (#p = 0.0107). (A″–C″) No effect of cocaine nor STN HFS was observed for C labeling alone. Only post hoc Fisher’s LSD were reported (* versus Saline; # vs OFF group). Detailed two-way ANOVA analyses are mentioned in the text.

inactivation of STN itself since STN lesions have been shown recently to abolish affective responses in the rat (Pelloux et al., 2014). Since STN inactivation reduces affective responses, as BLA lesions do (for reviews, (Cardinal et al., 2002; LeDoux, 2000), one might either suggest that STN HFS induces a stimulation of the STN-BLA direct glutamatergic projection or inhibits STN neurons affecting the BLA via an inhibitory link that remains to be identified. Interestingly, BLA drives synchronized firing in STN during amygdala kindling (Shi et al., 2007), suggesting that the two structures may interact during STN HFS. No significant effect was observed on Arc activation in any brain areas, suggesting that reactivity processes involving Arc mRNA regulation were poorly involved in the effects of STN HFS on indirect or direct target structures. Further studies will help to understand this lack of response. In summary, the effects of STN HFS by itself represent complex cellular processes that cannot only be restricted to an inactivation of the structure, as suggested by our previous behavioral studies on motivation (Baunez et al., 2005; Rouaud et al., 2010) but are in line with the complex effects observed on motor behavior (Baunez et al., 2007; Darbaky et al., 2003). However, if there is a stimulation of fibers, one might expect changes in the prefrontal cortex (PFC) via a retrograde stimulation of the hyper-direct pathway, which is not the case in our conditions in the cortical territories studied. One can thus hypothesize

little involvement of the hyper-direct pathway originating in the PFC and OFC in the effects of STN HFS on motivation for cocaine. This will also need to be investigated further. STN HFS modulation of acute cocaine-induced cellular responses Since STN HFS decreases motivation for cocaine (Rouaud et al., 2010) and that cocaine induces Fos activity in the STN (Uslaner et al., 2001), we hypothesized that STN HFS may modify the acute effects of cocaine on the reward circuit. As previously stated, in our experimental protocol, c-Fos expression could mainly reflect cellular reactivity to STN HFS, while Arc expression could reflect both cellular reactivity to cocaine and plasticity-related processes involved together with those induced by STN HFS. As expected (e.g., Besnard et al., 2011; Fosnaugh et al., 1995; Fumagalli et al., 2006; Samaha et al., 2004), cocaine induced a strong Arc activation in the DST, as well as in the NAC shell and core in the OFF group. In addition, no cocaine response was found in any other motor or limbic areas examined. At the same time, and most surprisingly, c-Fos was also found to be strongly activated in these striatal areas as early as 10 min following cocaine injection. These data suggest that cocaine induced both Arc and c-Fos gene expression with a very fast dynamic.

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This peculiarity could be specific of the strain of rats used here (Long–Evans). Accordingly, we found in another strain of rats (Wistar) that c-Fos activation was not detectable 10 min after cocaine while Arc activation was (data not shown), suggesting that the peaks of IEGs activation may be different between various strains. This hypothesis corroborates with some studies in mice showing different dynamic of activation between c-Fos and Arc mRNA expression and between C57BL/6 J and DBA/2 J mice (Ziolkowska et al., 2012, 2015). Our data showed that STN HFS prevents the c-Fos activation induced by cocaine in the dorsal striatum (and reduces it in the nucleus accumbens), which might explain how STN HFS may alter (1) the rewarding efficacy of cocaine previously observed in the CPP paradigm and (2) the decreased motivation for cocaine in the progressive ratio schedule of reinforcement (Rouaud et al., 2010). However, cocaine-induced Arc mRNA was not modified by STN HFS, underscoring different mechanisms of expression between the two IEGs. Therefore, to try to more precisely dissociate the cellular responses to cocaine from STN HFS, we took advantage of the catFISH method which allows to differentiate activation of Arc following temporally distinct epochs (Guzowski et al., 2005). The failure of STN HFS alone to activate Arc expression (whatever nuclear or cytoplasmic) was confirmed here, further supporting the idea that plasticity processes involving such activity regulating cytoskeleton proteins are not directly engaged in our experimental conditions. The fact that STN HFS may induce a kind of axonal failure (Zheng et al., 2011) may account for this possibility of an incorrect Arc processing due to an altered capacity of axons to transmit signal to terminals where Arc protein is acting. Our data showed that Arc activation was mainly due to cocaine injection with a high proportion of neurons (80% to 86% according to structures) showing nuclear labeling (N alone or N + C), which was expected from our experimental design). We showed that cocaine-induced nuclear labeling (N alone) was not significantly altered by the STN HFS in any area which is in line with the global Arc activation measured previously. However, we had to point out that cocaine induced a significant proportion of neurons displaying both nuclear and cytoplasmic labeling (N + C), further supporting the idea that the Arc dynamic of response (as for c-Fos) was fast in Long–Evans rats. Interestingly, STN HFS seems to potentiate the cocaine-induced N + C activation, which was significant only in the NAC shell. At a first look, these data are difficult to reconcile with the opposite effects observed for the c-Fos responses. However, their differential roles and dynamic of expression may account for these discrepancies. Indeed if they are similarly activated following some acute events (e.g., Besnard et al., 2011, 2014; de Bartolomeis et al., 2015; Guzowski et al., 2001; Hearing et al., 2008; Serres et al., 2006), we have also previously shown that in more complex situations, opposite responses can be observed (Lucas et al., 2008). Moreover, if Arc expression is not modified by STN HFS alone, its activation by cocaine may depend upon other activity regulated genes or IEGs expressed in the neuron at a given time which could have been indirectly modulated by the HFS (Bramham et al., 2008). Thus, through more global changes in transcriptional activity generated by cocaine administration, STN HFS may indirectly activate plasticity processes (Brody and Korngreen, 2013; Dvorzhak et al., 2013; Shen et al., 2003; Zheng et al., 2011). The mechanisms by which these responses occur remain unknown, but the catFISH analysis has highlighted the complex effects of cocaine molecular responses in combination to STN HFS. Conclusion The present data showed that STN HFS alone has a selective effect on c-Fos gene expression, and in a restricted number of motor or limbic regions, limited to the GP and the BLA. Interestingly, the later effect confirm the functional link between STN and BLA, highlighting the possible consequences of STN manipulation on emotional processes, in line with clinical observations of emotional changes in PD patients subjected to STN DBS.

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By comparing Arc and c-Fos, we clearly show that STN HFS modulates neuronal reactivity to cocaine within the reward circuit in a complex manner, and at least through differential effects on Arc and c-Fos expression. However, the decreased c-Fos response to cocaine induced by STN HFS in the striatum is in line with the inhibitory effect of STN HFS on the rewarding and reinforcing properties of cocaine (Rouaud et al., 2010), even if it cannot be explained only in terms of acute cellular response to a single cocaine injection. Based on these results, further studies will be required to clearly decipher the cellular mechanism by which STN HFS exerts its action on the rewarding and motivational properties of cocaine. Acknowledgments The authors thank Dr Victor Ramirez-Amaya for providing the Arc probe and Gilles Courtand for his help with the confocal microscopy. 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