Buspirone Requires the Intact Nigrostriatal Pathway to Reduce the Activity of the Subthalamic Nucleus Via 5-HT1A Receptors A

Experimental Neurology 277 (2016) 35–45

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Experimental Neurology

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Research Paper Buspirone requires the intact nigrostriatal pathway to reduce the activity of the subthalamic nucleus via 5-HT1A receptors A. Sagarduy a,1,J.Llorentea,1,C.Migueleza,b,T.Morera-Herrerasa,J.A.Ruiz-Ortegaa,b,L.Ugedoa,⁎ a Department of Pharmacology, Faculty of Medicine and Dentistry, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain b Department of Pharmacology, School of Pharmacy, University of the Basque Country (UPV/EHU), 01006 Vitoria-Gasteiz, Spain article info abstract

Article history: The most effective treatment for Parkinson's disease (PD), L-DOPA, induces dyskinesia after prolonged use. We Received 23 July 2015 have previously shown that in 6-hydroxydopamine (6-OHDA) lesioned rats rendered dyskinetic by prolonged Received in revised form 4 December 2015 L-DOPA administration, lesion of the subthalamic nucleus (STN) reduces not only dyskinesias but also buspirone Accepted 10 December 2015 antidyskinetic effect. This study examined the effect of buspirone on STN neuron activity. Cell-attached recordings Available online 11 December 2015 in parasagittal slices from naïve rats showed that whilst serotonin excited the majority of STN neurons, buspirone

Chemical compounds studied in this article: showed an inhibitory main effect but only in 27% of the studied cells which was prevented by the 5-HT1A receptor Serotonin hydrochloride (PubChem CID: selective antagonist WAY-100635. Conversely, single-unit extracellular recordings were performed in vivo on STN 160436) neurons from four different groups, i.e., control, chronically treated with L-DOPA, 6-OHDA lesioned and lesioned Buspirone hydrochloride (PubChem CID: treated with L-DOPA (dyskinetic) rats. In control animals, systemic-buspirone administration decreased the firing 36431) rate in a dose-dependent manner in every cell studied. This effect, prevented by WAY-100635, was absent in WAY-100635 maleate salt (PubChem CID: 6-OHDA lesioned rats and was not modified by prolonged L-DOPA administration. Altogether, buspirone in vivo 11957721) reduces consistently the firing rate of the STN neurons through 5-HT1A receptors whereas ex vivo buspirone L-DOPA (PubChem CID: 6047) seems to affect only a small population of STN neurons. Furthermore, the lack of effect of buspirone in 6-OHDA 6-Hydroxydopamine (PubChem CID: 4624) lesioned rats, suggests the requirement of not only the activation of 5-HT receptors but also an intact Gabazine (SR95531 hydrobromide, PubChem 1A CID: 107895) nigrostriatal pathway for buspirone to inhibit the STN activity. DNQX (PubChem CID: 3899541) © 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license D-AP5 (PubChem CID: 135342) (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Dyskinesia Parkinson's disease Basal ganglia 6-OHDA lesion L-DOPA Serotonin

1. Introduction 2013). Unfortunately, prescription of amantadine raises some controver- sy about its long-term antidyskinetic efficacy (Thomas et al., 2004; Wolf As a consequence of long-term treatment with L-DOPA, the most et al., 2010). effective drug to alleviate motor symptoms in Parkinson's disease (PD), Serotonin (5-HT)-based therapies have been extensively investigated patients develop L-DOPA-induced dyskinesia (LID). This motor disorder in an attempt to find efficacious antidyskinetic drugs. Indeed, numerous can be completely disabling for the patient, sometimes reducing quality 5-HT agonists have been shown to improve dyskinesia, although not of life even more than PD itself. In spite of a large number of clinical trials always without worsening the antiparkinsonian effect (Bibbiani et al., for finding an efficient therapy to reduce LID, the only currently available 2001; Dupre et al., 2007; Kleedorfer et al., 1991). For example, sarizotan, antidyskinetic drug so far, is the anti-influenza agent amantadine (Fox, a 5-HT1A receptor agonist, improves dyskinesia in macaque (Gregoire et al., 2009)andratmodels(Gerlach et al., 2011a). However, it failed in a III-phase clinical trial on PD patients (Goetz et al., 2007) due to the Abbreviations: 5-HT, serotonin; 6-OHDA, 6-hydroxydopamine; aCSF, artificial fact that sarizotan did not counteract LIDs better than placebo. Also, a cerebrospinal fluid; DRN, dorsal raphe nucleus; LID, L-DOPA-induced dyskinesia; PD, single dose of eltoprazine, a 5-HT1A/B-receptor agonist, has recently Parkinson's disease; STN, subthalamic nucleus; TH, tyrosine hydroxylase. shown antidyskinetic effects in a placebo-controlled phase I/IIa study ⁎ Corresponding author. E-mail address: [email protected] (L. Ugedo). (Svenningsson et al., 2015) though no data are yet available about its 1 Considered joined first authors. long treatment efficacy.

http://dx.doi.org/10.1016/j.expneurol.2015.12.005 0014-4886/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 36 A. Sagarduy et al. / Experimental Neurology 277 (2016) 35–45

In this regard, buspirone, a partial agonist of 5-HT1A receptors severity of the 6-OHDA lesion was evaluated by rotational behavior (Peroutka, 1988) has antidyskinetic properties not only in 6- over 90 min after d-amphetamine (3 mg/kg, i.p.) administration, and hydroxydopamine (6-OHDA) lesioned rats (Aristieta et al., 2012; only severely lesioned animals were selected for this study (more Azkona et al., 2014; Dekundy et al., 2007; Eskow et al., 2007; Gerlach than 5 full-body ipsilateral turns/min). et al., 2011b) but also in open-label trials performed in humans Rats were divided in four different groups depending on the lesion (Bonifati et al., 1994; Kleedorfer et al., 1991; Politis et al., 2014). Inter- and pharmacological treatment followed: control, sham L-DOPA, estingly, buspirone, apart from being a partial agonist for 5-HT1A 6-OHDA lesioned and 6-OHDA lesioned plus L-DOPA (dyskinetic) rats. heteroreceptors, behaves as a full agonist for 5-HT1A-autoreceptors Animals were treated chronically over 3 weeks with saline in the control (Sussman, 1998) in the dorsal raphe nucleus (DRN) inhibiting the and 6-OHDA lesion groups; or with L-DOPA (6 mg/kg + benserazide- serotonergic neuron firing activity (VanderMaelen et al., 1986). Also, it HCl (12 mg/kg s.c.) in sham L-DOPA and the dyskinetic groups. On is important to mention that, although buspirone has affinity mainly day 21, the dyskinetic movements were scored as described by for 5-HT1A receptors, it also acts as an antagonist for D2 and D3 Miguelez et al. (2011). All 6-OHDA lesioned animals chronically treated dopamine receptors (Bergman et al., 2013; Dhavalshankh et al., 2007; with L-DOPA developed severe dyskinetic movements that reached the McMillen et al., 1983). In fact, buspirone reduces graft-induced dyskine- peak between 40 and 80 min after a single injection of L-DOPA (Fig. 1A). sia in a rat model of PD by a mechanism which is independent from Tyrosine hydroxylase (TH)-immunostaining was used to examine the activation of either pre- or post-synaptic 5-HT1A receptor (Shin et al., degree of dopaminergic denervation in the striatum and the substantia 2012). All these peculiarities could help to explain why buspirone is nigra following the methodology described by Miguelez et al. (2011) an efficacious antidyskinetic drug and make it interesting to figure out and in all cases the loss of striatal DA fibers was N90% (Fig. 1B). the target through what buspirone elicits its antidyskinetic effect. The subthalamic nucleus (STN) is the only glutamatergic nucleus of 2.3. Electrophysiological recordings the basal ganglia and exerts the principal excitatory effect on the output nuclei of the basal ganglia, playing a critical role in the motor circuit. 2.3.1. Ex vivo recordings

Local infusion of a stimulatory 5-HT2C-receptor agonist into the STN Male Sprague Dawley rats (50–75 g) were anesthetized with chloral induces orofacial dyskinesia in rat (Eberle-Wang et al., 1996). Moreover, hydrate (400 mg/kg, i.p.) and sacrificed by decapitation. The brain was we reported that when the STN is lesioned the antidyskinetic effect of rapidly dissected out and placed in ice-cold cutting solution containing buspirone is decreased (Aristieta et al., 2012). Therefore, the aim of (in mM): 20 NaCl, 2.5 KCl, 0.5 CaCl2,7MgCl2,1.25NaH2PO4, 85 sucrose, this study was to investigate the effect induced by buspirone on the 25 D-glucose and 60 NaHCO3, saturated with 95% O2/5% CO2.The STN neuronal activity not only in brain slices but also in anaesthetized cerebellum was removed and brain hemispheres were separated along control and 6-OHDA lesioned rats chronically treated with saline or the inter-hemispheres line. Parasagittal brain slices (300–350 μmthick) L-DOPA. containing the STN, identified as a lens-shaped dense patch located medial to the internal capsule, were cut and immediately placed in the artificial cerebrospinal fluid (aCSF) composed of (mM) 126 NaCl, 2.5 2. Materials and methods KCl, 1.2 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 11.1 D-glucose, 21.4 NaHCO3, 0.1 ascorbic acid, and saturated with 95% O2/5% CO2 at 33 °C. After 2.1. Animals at least one hour resting at 33 °C, the slice was submerged in a slice chamber (0.5 ml) mounted on the microscope stage and superfused Female Sprague–Dawley rats weighing 200–280 g for in vivo exper- (2.5–3 ml/min) with the aCSF bathing solution at 31 °C. iments and male Sprague–Dawley rats weighing 50–75 g for ex vivo STN neurons were visually identified using an upright microscope experiments were housed in groups of six with free access to food and with infrared optics (Eclipse E600FN, Nikon®) equipped with a 40× water in environmentally controlled conditions (20 ± 2 °C; 65–70% of water-immersion objective. Patch electrodes were pulled from borosil- relative humidity and a 12 h:12 h light/dark cycle). Every effort was icate glass capillaries (World Precision Instruments) on a two-stage made to minimize animal suffering, to use the minimum number of puller (PC-10, Narishige) to a pipette resistance of 3–5MΩ. Recordings animals per group and experiment and to utilize alternatives to in vivo were made with an Axopatch 200B amplifier and digitized with a techniques if available. Experimental procedures were approved by Digidata 1322A (Axon Instruments®). To accurately examine spontane- the Local Ethical Committee of the University of Basque Country (UPV/ ous firing in STN neurons, pipettes were filled with aCSF and recordings EHU, CEBA/185/2011), following European (2010/63/UE) and Spanish were performed in the minimally invasive cell-attached loose patch (RD 1201/2005) regulations for the care and use of laboratory animals. configuration (20–30 mΩ seal resistance). Data were low-pass filtered at 5 kHz before being digitized at a rate of 10 kHz. Acquisition and all 2.2. 6-OHDA lesion and L-DOPA treatment subsequent analysis were carried out with Clampex 10.4. Firing rate histograms were generated by integrating action potential discharge 6-OHDA lesions were performed according to our established proto- in10sbins(Fig. 1C). 54 cells fired spontaneously and regularly at cols (Aristieta et al., 2012; Miguelez et al., 2011). Rats were pretreated 13 ± 1 Hz. Only 5 neurons fired in burst mode and 4 cells remained 30 min before anesthesia with desipramine (25 mg/kg, i.p.) in order to silent. This proportion of regularly firing neurons strongly resembles preserve the noradrenergic terminals. Then, rats were anesthetized what has been described in the literature (98%) for ex vivo STN cells with isoflurane inhalation and placed in the stereotaxic frame (David (Wilson et al., 2004). Also, an increase in the temperature from 31 to Kopf® Instruments). Lesions were performed by two injections of 35 °C induced a reversible increase in the average frequency of sponta- 6-OHDA of 7.5 μgand6μg, respectively (3.5 μg/μl saline with neous firing rate from 13 ± 4 Hz to 19 ± 7 Hz (n = 6; paired t test, 0.02% ascorbic acid) in the right medial forebrain bundle: 2.5 μlat p b 0.05) (Fig. 1D) as it has been described for this nucleus (Heida anteroposterior (AP) −4.4 mm, mediolateral (ML) +1.2 mm and dor- et al., 2008). All drugs were applied in the perfusion solution in soventral (DV) −7.8 mm, relative to bregma and dura with the toothbar known concentrations. set at −2.4, and 2 μlatAP−4.0 mm, ML +0.8 mm, and DV −8 mm, with toothbar at +3.4 (Paxinos and Watson, 1986). Sham animals 2.3.2. In vivo recordings were similarly treated but instead of 6-OHDA received vehicle. For the All STN neuron electrophysiological recordings in vivo were administration of the neurotoxin a 10 μl-Hamilton syringe was used performed as described by Morera-Herreras et al. (2011), twenty-four and the infusion rate was 1 μl/min. Prior to the administration, the hours after the last saline or L-DOPA injection. Rats were anesthetized toxin was kept protected from light and in ice. Two weeks later, the with urethane (1.2 g/kg, i.p.) and placed in a stereotaxic frame with its A. Sagarduy et al. / Experimental Neurology 277 (2016) 35–45 37

Fig. 1. Examples of behavioral, histological and electrophysiological procedures. (A) Dyskinesia scores evaluated after a single injection of L-DOPA on the last testing session (day 21). (B) Representative tyrosine hydroxylase (TH) immunostaining in the striatum and the substantia nigra showing the loss of TH positive fibers in rats microinfused unilaterally with 6-OHDA into the right nigrostriatal pathway. (C) Typical spontaneous spikes (action potentials, ~13 Hz) in a presumed STN neurons recorded ex vivo in cell-attached mode. (D) Pooled data of the average frequency of spontaneous firing rate recorded ex vivo at 31 °C and later at 35 °C showing a significant increase (n = 6; paired t test, *p b 0.05). (E) Distribution of the in vivo recording sites in the STN. The STN is marked in gray, framed and expanded; and perpendicular lines show laterality and depth. Image modified from Paxinos and Watson (1986), with permission from Elsevier. (F) Samples of spontaneous spiking activity recorded in vivo in the STN of tonic firing pattern, in which the density histogram follows a Gaussian distribution (top) and a bursting firing pattern, in which the density histogram represents a distribution significantly different from Poisson distribution (bottom).

head secured in a horizontal orientation. The recording electrode was (0.25 M) dissolved in Dulbecco's buffered saline solution (NaCl, lowered into the right STN (relative to bregma and dura: AP −3.7 mm, 136.9 mM; KCl, 2.7 mM; NaH2PO4, 8.1 mM; KH2PO4, 1.5 mM; MgCl2, ML −2.5 mm and DV −7/−8.5 mm). The extracellular signal from the 0.5 mM and CaCl2, 0.9 mM, pH 7.4). The volume of each ejection pulse electrode was amplified with a high-input impedance amplifier and was measured by monitoring the meniscus movement in the calibrate was monitored on an oscilloscope and on an audio monitor. All recorded pipette. STN neurons exhibited a biphasic waveform and a spike width of 1.0 to At the end of each electrophysiological experiment, a Pontamine Sky 1.5 ms (Hollerman and Grace, 1992). Neuronal spikes were digitized Blue mark was deposited for posterior verification of the recording site using computer software (CED micro 1401 interface and Spike2 soft- using neutral red staining. Only cells recorded within the STN were ware, Cambridge Electronic Design). The basal firing rate was recorded included in this study (Fig. 1E). The firing parameters of STN neurons for 5 min. were analyzed off-line using Spike2 software. The following parameters Local injection of buspirone close to the STN neuron which was were calculated: firing rate, coefficient of variation and firing pattern. being recorded was performed as described by Ruiz-Ortega and Ugedo The different firing patterns could be analyzed according to the method (1997). Pressure pulses (50–150 ms) were applied to a calibrated described by Kaneoke and Vitek (1996) (Fig. 1F). The analyzed spike- pipette, glued adjacent to a recording micropipette, containing buspirone trains lasted more than 120 s and contained at least 300 spikes. 38 A. Sagarduy et al. / Experimental Neurology 277 (2016) 35–45

2.4. Drugs 41 neurons were studied before and after a bath application of 5-HT. The majority of these cells (n = 39) spontaneously fired regular action Drugs used in this study were: 6-OHDA (3.5 μg/μl) dissolved in potentials at a mean rate of 11 ± 1 Hz. Only two cells did not show MiliQ water containing 0.02% ascorbic acid; desipramine hydrochloride spontaneous firing. As it has been previously described (Stanford (25 mg/kg i.p.), amphetamine sulfate (3 mg/kg i.p.), chloral hydrate, et al., 2005) not all of these 41 neurons responded in the same way to L-DOPA (6 mg/kg s.c.), benserazide-HCL (12 mg/kg s.c.), serotonin the perfusion with 5-HT (30 μM). The main effect induced by 5-HT hydrochloride (5-HT, 30 μM ex vivo), buspirone hydrochloride (4 and was a reversible increase in STN-neuron firing rate in 31 out of 41 neu- 8 mg/kg, i.p. in vivo; 10 and 30 μM ex vivo) and WAY-100635 maleate rons, a percentage also observed by others (Stanford et al., 2005). On av- salt (0.5 and 1.5 mg/kg, i.p. in vivo;1μM ex vivo) from Sigma-Aldrich erage, firing rates increased moderately and in a concentration- disolved in 0.9% saline or Dulbecco for in vivo experiments and dissolved dependent manner in the presence of 5-HT (Fig. 2A). 5-HT (30 μM) in- in aCSF for ex vivo experiments. Fast synaptic transmission blockers creased the firing rate by 253 ± 30% (n = 29) in these cells. 5-HT in- were gabazine (5 μM) and DNQX (20 μM) from Tocris and D-AP5 duced firing activity on two silent cells but the magnitude of the effect (50 μM) from Abcam. Urethane was dissolved in MiliQ water. could not be quantified as the percentage change had to be calculated according to the basal firing value. The excitatory effect was not mediat-

2.5. Statistical analysis of data ed through 5-HT1A-receptor activation since perfusion with the 5-HT1A antagonist, WAY-100635 (1 μM), for at least 10 min had no effect on Experimental data were analyzed using the computer program basal firing rate or on the excitatory responses induced by 5-HT GraphPad Prism (v. 5.01, GraphPad Software, Inc). The electrophysio- (30 μM) (n = 5, paired t test p N 0.05) (Fig. 2B). Only in two cases, the logical data were analyzed by paired t test when two situations were neuron recorded responded to 5-HT (30 μM) with a decrease in the fir- compared and one-way analysis of variance (ANOVA) followed by ing rate and in 3 out of 41 no effect was observed. Interestingly, five cells post-hoc comparisons using the Newman Keuls to compare different responded in a robust and repeatable biphasic manner (Fig. 2C). In these groups. The firing pattern of STN neurons was analyzed using Fisher's cells 5-HT induced firstly a significant 64 ± 16% reduction of the firing exact test. The level of statistical significance was set at p b 0.05. Data rate (paired t test, p b 0.01) immediately followed by an increase of are presented as group means ± standard error of the mean (SEM). the firing rate above the basal frequency (54 ± 12%, p b 0.05, n = 5). Subsequent WAY-100635 (1 μM) perfusion specifically and completely 3. Results abolished the inhibitory component of the biphasic response indicating

that it was mediated by the activation of the 5-HT1A receptor (Fig. 2D). 3.1. Characterization of 5-HT and buspirone-effects on STN neurons in The excitatory component did not change after WAY-100635 control brain slices application. We have previously shown that a STN lesion decreases the anti- First of all, to validate our STN preparations we decided to study the dyskinetic effect of buspirone in vivo (Aristieta et al., 2012). Here, we well known effect induced by 5-HT on STN neurons. For that purpose, considered to investigate the possible direct effect of buspirone on

Fig. 2. 5-HT ex vivo induces an excitatory effect on STN-neurons. (A) Example of a firing-rate recording (spikes/10s) of a STN neuron showing the stimulatory effects induced (in the majority of the cells studied) by different concentrations of 5-HT (30 μM, 1 min; 3 μM, 1.5 min; 1 μM, 2 min). The horizontal bar indicates de moment and the duration of the bath perfusion of the drug. (B) Pulled data from five paired normalized excitatory responses induced by 5-HT (30 μM) before and after the bath perfusion of WAY-100635 (WAY; 1 μM; n = 5). (C) Example of a biphasic effect induced by 5-HT (30 μM) on the basal firing rate. (D) Pulled data from five biphasic responses induced by 5-HT showing that bath perfusion of WAY- 100635 (1 μM) completely eliminated the inhibitory component (n = 5). A. Sagarduy et al. / Experimental Neurology 277 (2016) 35–45 39

STN-neurons in a more isolated model such as brain slices. In our study, To better characterize the buspirone effect, a series of supplementa- buspirone was tested in 51 STN neurons where it exerted a limited and ry experiments were performed in the presence of fast synaptic trans- variable effect depending on the cell recorded. In fact, bath application mission blockers consisting in the GABAA receptor antagonist gabazine of buspirone (10–30 μM) failed to induce any effect in 31 of the studied (5 μM), the selective NMDA receptor antagonist D-AP5 (50 μM) and cells (61%). On the other hand, in 14 out of 51 studied cells, buspirone the AMPA and kainate receptor antagonist DNQX (20 μM). 23 STN neu- induced a significant 45 ± 7% inhibition of the firing rate (n = 14). rons were recorded in the presence of the fast synaptic transmission Different concentrations of buspirone were used, 10 μMand30μM, blockers. These neurons showed a regular firing frequency (11.4 ± but since the magnitude of the inhibitory effect was similar in both 1.5 Hz) which slightly changed to 12.5 ± 1.7 Hz (paired t test, n = 19, sets of experiments (t test, p N 0.05), all data were pooled together. p b 0.05) after 5 min of bath perfusion with the blockers. As shown in The inhibitory effect exerted by buspirone was slow to wash out but the Supplementary Fig. 1, all effects induced by buspirone (inhibition, easily reversed and prevented by a bath perfusion of the slice with stimulation and biphasic response) were almost completely blocked WAY-100635 (4 ± 15% change, paired t test; p b 0.05, n = 6). A second by the synaptic transmission blockers perfusion. application with buspirone failed to alter the basal firing rate in the presence of WAY-100635 (1 μM) (−6 ± 3% change, p N 0.05; n = 4) 3.2. Characterization of buspirone-effect on STN neurons in control (Fig. 3A and B). Buspirone rarely induced an excitatory effect (3 out of anesthetized rats 51 cells), increasing the firing rate by 54 ± 12% (Fig. 3C and E). Interest- ingly, buspirone also exerted an unusual biphasic response in 3 STN- We studied the effect of buspirone on subthalamic neuronal activity neurons characterized by an excitatory response (78 ± 22%) immedi- in anesthetized rats. For that purpose, basal electrophysiological param- ately followed by a 56 ± 21% inhibitory one (Fig. 3D and E). Altogether, eters of STN neurons (n = 23) were recorded over a period of 5 min whilst 5-HT main effect was excitation (31/41), buspirone's key effect before and, at least, 20 min after the administration of buspirone (4 was inhibition only in 14 out of 51 studied neurons (Fig. 3F). and 8 mg/kg i.p.) (Fig. 4). All cells recorded displayed most characteristic

Fig. 3. The inhibitory effect induced by buspirone ex vivo on STN-neurons is mediated by the 5-HT1A receptor. (A) Example of a ﬁring-rate recording (spikes/10s) showing how buspirone (30 μM) induces an inhibitory response reversed and prevented by a perfusion of WAY-100635 (1 μM). (B) Pooled data from four paired inhibitory responses induced by buspirone before and after the bath perfusion of WAY-100635 1 μM (n = 4, paired t test *p b 0.05). (C) Example of a ﬁring-rate recording showing how buspirone (10 μM) induces a stimulatory effect and (D) a biphasic effect. (E) Pooled data from three stimulatory and three biphasic responses induced by buspirone. (F) Proportions of different effects induced by 5-HT and buspirone. 40 A. Sagarduy et al. / Experimental Neurology 277 (2016) 35–45

Fig. 4. The effects induced by buspirone in vivo on STN neurons are mediated by the 5-HT1A receptor. (A) Example of a firing-rate recording (spikes/10s) showing the inhibitory effect of intraperitoneal (i.p.) administration of buspirone (4 mg/kg). (B) Histograms illustrating the mean firing rate of STN neurons before and after the administration of buspirone (4 mg/kg; n = 12) and (8 mg/kg; n = 11) and with the pre (0.1 mg/kg; n = 5) and post administration (0.5 and 1.5 mg/kg; n = 4) of WAY-100635. Data are expressed as mean ± SEM. (paired ttest***pb 0.001; **p b 0.01 vs. corresponding basal value) (C) Example of a firing-rate recording showing the inhibitory effect of the administration of buspirone (8 mg/kg i.p.) plus the reversion induced by WAY-100635 (0.5 and 1.5 mg/kg i.p.) on the STN neurons firing rate. (D) Histograms illustrating the mean coefficient of variation (percent) of STN neurons before and after the administration of buspirone (4 mg/kg; n = 12) and (8 mg/kg; n = 11) and with the pre (0.1 mg/kg; n = 5) and post administration (0.5 and 1.5 mg/kg; n = 4) of WAY-100635. Data are expressed as mean ± SEM. (paired t test ***p b 0.001; **p b 0.01; *p b 0.05 vs. corresponding basal value). (E) Example of a firing-rate recording showing no effect exerted by WAY-100635 (0.1 mg/kg i.p.) but the prevention of buspirone-inhibitory effect. (F) Distributions of firing patterns of STN neurons before and after the administration of buspirone and with the pre and post administration of the antagonist (Chi-squared (χ2) test ***p b 0.001; **p b 0.01 vs. corresponding basal value).

firing properties of STN neurons described in our previous publications (Aristieta et al., 2012; Morera-Herreras et al., 2011)(Table 1). However, Table 1 the proportion of busty cells was high compared to our previous studies Electrophysiological parameters of STN neurons. what may be attributed to gender differences as shown in PD patients Control Sham L-DOPA Lesion Dyskinetic (Mrakic-Sposta et al., 2008). In control rats, buspirone administration (n = 33) (n = 6) (n = 5) (n = 7) fi ⁎ ⁎ decreased the ring rate of every STN neuron tested in a dose related Basal Firing Rate (Hz) 8.1 ± 0.3 7.4 ± 1.2 13.0 ± 1.1 14.7 ± 2.8 ⁎ manner. Thus, buspirone 4 mg/kg reduced the firing rate by 35 ± 7% Coefficient of variation (%) 43 ± 2 52 ± 10 56 ± 15 124 ± 12 (p b 0.01, paired t test; n = 12) (Fig. 4A and B) and 8 mg/kg produced Firing pattern (%) − − −# # an inhibition of 76 ± 5% with respect to the basal values (p b 0.001, Burst/Tonic/Random 70/30/ 67/33/ 50/50/ 64/27/8 paired t test; n = 11) (Fig. 4B and C). This inhibitory effect was slowly Rats were treated daily with saline or L-DOPA 6 mg/kg in combination with benserazide reversed to basal levels after 60 min. Buspirone also modified the 12 mg/kg, s.c. for at least 21 days. All neurons were recorded 24 h after the last injection of saline or L-DOPA. Each value represents the mean ± SEM of (n = number of animals). coefficient of variation of the cells, making them more irregular, (54% Each cell was recorded for 180 s. b for 4 mg/kg and 63% for 8 mg/kg; p 0.01; paired t test) (Fig. 4D). Firing ⁎ p b 0.05; one-way ANOVA, Newman-Keuls post-hoc. pattern of STN neurons was also significantly altered after buspirone # p b 0.05; Fisher exact test. A. Sagarduy et al. / Experimental Neurology 277 (2016) 35–45 41 administration, decreasing the percentage of STN neurons discharging in rats was also different than that found in the control group (p b 0.01, burst (p b 0.001; χ2test=13.33andχ2 test = 51.11, for 4 and 8 mg/kg, χ2test=8.33;pb 0.05, χ2 test = 8.42 respectively). respectively) (Fig. 4F). Also, buspirone 8 mg/kg, but not 4 mg/kg, induced In sham L-DOPA rats, in the same way as in control rats, we observed a random pattern in a 30% of the studied population (Fig. 4F). that buspirone administration (8 mg/kg i.p.) induced a 58 ± 12% We next attempted to reverse the inhibitory effect induced by decrease on the firing rate (paired t test, p b 0.01; n = 6) (Fig. 6). buspirone with a 5-HT1A receptor antagonist. For this purpose, WAY- There was also a decrease in the regularity of these neurons, shown by 100635 (0.5 and 1.5 mg/kg i.p.) was administered 10–15 min after the an increase in the coefficient of variation from 52 ± 10% in basal condi- administration of buspirone (8 mg/kg i.p.). After a dose of 8 mg/kg of tions to 98 ± 12% after buspirone administration (paired t test, p b 0.05; buspirone, 1.5 mg/kg but not 0.5 mg/kg of WAY-100635 (i.p.), fully n = 6) (Fig. 6D). In this group of rats the amount of neurons discharging reversed the inhibitory effect induced by buspirone in the firing fre- in burst did not change after buspirone administration, however, a 16% quency (reversal effect 111 ± 5%; F(3,9) = 14.97, p b 0.0001, one-way of neurons turned to show a random pattern (p b 0.001, χ2test= ANOVA; n = 4) (Fig. 4B and C). Furthermore, the highest dose of 21.89) (Fig. 6F). WAY-100635 also reversed the effect exerted by buspirone on the Interestingly, buspirone (8 mg/kg i.p.) did not modify the firing rate regularity of these neurons, bringing back the coefficient of variation of STN neurons neither in 6-OHDA lesioned rats (before 13.0 ± 1.1 Hz vs to basal values (41 ± 5%; F(3,9) = 7.58, p b 0.01, one-way ANOVA) after buspirone 12.5 ± 1.3 Hz; n = 5) (Fig. 5B and C) nor in dyskinetic (Fig. 4D). The firing pattern of these neurons returned to basal condi- rats (before 14.7 ± 2.8 Hz vs after buspirone 19.4 ± 4.6 Hz; n = 7) tions after the administration of WAY-100635 (p b 0.001, Fisher (Fig. 5B and E). Also, the neuron regularity from both groups remained exact test) (Fig. 4F). Next, we applied lower doses of WAY-100635 unchanged, as it is shown by the absence of change in the coefficient of (0.1 mg/kg, i.p.) prior to buspirone administration (15 min). In this variation (lesioned, 42 ± 7% vs 53 ± 12% and dyskinetic 130 ± 12% vs case, WAY-100635 had no effect on STN firing frequency but completely 139 ± 10%, before vs after buspirone) (Fig. 5D). On the contrary, burst- blocked the inhibitory effect of buspirone (F(2,14) = 1.22, n = 5; ing pattern of STN neurons decreased after buspirone administration in p N 0.05, one-way ANOVA) (Fig. 4C and D). However, WAY-100635 6-OHDA lesioned (before 40% vs after buspirone 20%, p b 0.01, χ2test= did not block the effect of buspirone on the coefficient of variation, 9.52) and dyskinetic rats (before 57% vs after buspirone 29%; p b 0.001, which became more irregular after buspirone administration (70 ± χ2 test = 19.79) (Fig. 5F).

9%) (F(2,8) = 3.47, p b 0.05, one-way ANOVA) (Fig. 4D). Interestingly, the administration of WAY-100635 turned neurons more bursty (60% 4. Discussion before and 100% after the administration of WAY-100635) as previously shown (Aristieta et al., 2014). This modiﬁcation was reverted after We have previously shown that the 5-HT1A receptor agonist buspirone was applied (bursty cells: 40%) (p b 0.01, Fisher exact test) buspirone not only improves dyskinesias in a rat model of LID but also (Fig. 4F). reverses some key molecular changes related to these motor complica- To better characterize buspirone effect, buspirone was injected into tions (Azkona et al., 2014). Interestingly, these important effects were the STN near to the STN neuron that was being recorded. In this case attenuated when the STN was lesioned (Aristieta et al., 2012). Here, buspirone caused inhibition only in 2 out of 7 recorded neurons (Fig. 5). we show that buspirone reduces the ﬁring rate of STN neurons through

the activation of 5-HT1A receptors by a complex mechanism. Buspirone ex vivo only caused inhibition in a small population of neurons while 3.3. Characterization of buspirone-effect on STN neurons in 6-OHDA in vivo, when systemically applied, all recorded neurons from control lesioned and dyskinetic rats rats were clearly inhibited. Furthermore, in 6-OHDA-lesioned rats chronically treated or not with L-DOPA, buspirone completely failed to Next we studied the effect of buspirone in three groups with differ- induce any effect on STN activity suggesting that the dopaminergic ent conditions: sham L-DOPA (n = 6), 6-OHDA lesioned (n = 5) and system is also implicated. 6-OHDA lesioned plus L-DOPA (n = 7) rats. Firstly, the spontaneous The STN, and in general the basal ganglia, is innervated by 5-HT-rich electrical activity of STN neurons from these rats was recorded and, as fibers coming primarily from neurons in the DRN (Di Matteo et al., we and others have previously described, we found that the STN 2008; Liu et al., 2007; Mori et al., 1985). It has been shown that not becomes hyperactive after dopamine depletion (Aristieta et al., 2012; only 5-HT1A,5-HT2C and 5-HT4 receptor-mRNAs but also the correspon- Bergman et al., 1994; Hassani et al., 1996; Morera-Herreras et al., dent receptors are expressed in STN cells (Eberle-Wang et al., 1997; 2011). As described in Table 1, firing rate in STN neurons from both Grossman et al., 1993; Maroteaux et al., 1992; Pazos et al., 1985; Pazos 6-OHDA lesioned and dyskinetic rat groups as well as the coefficient and Palacios, 1985; Pompeiano et al., 1992; Waeber et al., 1996). Also, of variation of dyskinetic rats were significantly higher than in the con- 5-HT1B/D-receptors are located in terminals of the motor cortex and ( ) trol rat group (F 3,47) = 10.86 and F(3,47 = 45 respectively; p b 0.001, the external segment of the globus pallidus neurons that project to one-way ANOVA). Firing pattern in 6-OHDA lesioned and dyskinetic the STN (Hannon and Hoyer, 2008; Hoyer et al., 2002; Sari et al.,

Fig. 5. (A) Example of a ﬁring-rate recording (spikes/10s) showing the inhibitory effect of local administration of buspirone (0.25–1 nmol). (B) Example of a ﬁring-rate recording (spikes/ 10s) showing the lack of effect after local administration of buspirone (0.25–2nmol). 42 A. Sagarduy et al. / Experimental Neurology 277 (2016) 35–45

Fig. 6. Effect of the administration of buspirone (8 mg/kg i.p.) on the electrical activity of STN neurons of control, sham L-DOPA, 6-OHDA lesioned and dyskinetic rats. (A) Example of a firing-rate recording (spikes/10s) showing the inhibitory effect of buspirone administration (8 mg/kg i.p.) in sham L-DOPA rats. (B) Histograms illustrating the mean firing rate of STN neurons before and after the administration of buspirone (control (n = 11); sham L-DOPA (n = 6); 6-OHDA lesioned (n = 5) and dyskinetic (n = 7)); **p b 0.01 vs. corresponding basal value (Student's t test). Data are expressed as mean ± SEM. (C) Example of a firing-rate recording showing the absence of effect of buspirone administration (8 mg/kg i.p.) in 6-OHDA lesioned rats. (D) Mean coefficient of variation (percent) of STN neurons before and after the administration of buspirone (control (n = 11); sham L-DOPA (n = 6); 6-OHDA lesioned (n = 5) and dyskinetic (n = 7)); *p b 0.05 vs. corresponding basal value (Student's t test). Data are expressed as mean ± SEM. (E) Example of a firing-rate recording showing the absence of effect of buspirone administration (8 mg/kg i.p.) in dyskinetic rats. (F) Distributions of firing patterns of STN neurons before and after the administration of buspirone in the different groups; ***p b 0.001; **p b 0.01 vs. corresponding basal value (Chi-squared (χ2) test).

1999). Furthermore, 5-HT depletion, systemic administration of a non- 12% of the recorded neurons responded to 5-HT with a biphasic selective 5-HT receptor antagonist as well as lesioning the DRN increase response as the 8% shown previously by Stanford et al. (2005) and in the firing rate of STN neurons (Aristieta et al., 2014; Liu et al., 2007). agreement with the biphasic current found in a 19% of STN neurons by However, according to the literature, the effects of 5-HT on STN neurons Shen et al. (2007). Furthermore, we have shown here that the inhibitory ex vivo are an increase in the firing rate, without changing the firing phase induced by 5-HT is mediated through the activation of the 5-HT1A pattern, through 5-HT2C and 5-HT4 receptors activation and also a less receptor as it was prevented by the selective antagonist WAY-100635, frequent inhibition through 5-HT1A/B receptor activation (Flores et al., compared to what has been shown in those previously cited works 1995; Stanford et al., 2005; Xiang et al., 2005; Shen et al., 2007). In our where it was blocked using the less selective 5-HT1A/B receptors antag- hands, 5-HT ex vivo induces a reversible increase in STN-neuron firing onist WAY-100135. When we applied buspirone ex vivo it failed to rate in a 76% of the studied neurons, in a comparable proportion to induce any effect in the 61% of the studied neurons and primarily the 82% described by Stanford et al. (2005) and in agreement with the inhibited the firing rate only in a 27% of the studied cells through the inward current described for the 64% of the patch-clamped STN neurons activation of the 5-HT1A receptor. Buspirone also induced an excitatory (Shen et al., 2007). 5-HT induced rarely an inhibitory effect in a 5% of the effect in a 6% and a biphasic effect in another 6% of the recorded cells. recorded cells, in a similar way it has been described before. Finally, a These results agree with the fact that 5-HT also induces inhibition in a A. Sagarduy et al. / Experimental Neurology 277 (2016) 35–45 43

small population through 5-HT1A receptor being the inhibitory effect on STN cells (Bouthenet et al., 1987; Boyson et al., 1986; Flores et al., expressed in a selected and scarce group of STN neurons (Stanford 1999; Johnson et al., 1994). Several ﬁndings indicate that the response et al., 2005). However, 5-HT, a full agonist, inhibits the STN in only of STN neurons to dopaminergic drugs is altered after dopaminergic the 5% of the recorded cells, less than the 27% induced by buspirone. denervation of the basal ganglia. Thus, apomorphine, a non selective

This discrepancy could be explained by the probable 5-HT interference dopamine receptor agonist, and D2 dopamine receptor agonists admin- co-activating other excitatory receptors (5-HT2C-4) as well, which istration induces a decrease in the ﬁring rate of STN neurons in intact would eclipse the 5-HT1A receptor effect, especially when these rats and do not have effect on 6-OHDA lesioned rats (Hassani and inhibitory receptors were expressed in lower proportion. Altogether, Feger, 1999). On the other hand, as it has been consistently shown the STN appears to be a very heterogeneous nucleus composed by a that the 5-HT system is severely affected after degeneration of population of neurons which show different responses depending on nigrostriatal dopaminergic neurons, a modulation of this system may the array of serotonergic receptors expressed, being the inhibitory be also considered. However, although several studies have observed a re- response mediated by 5-HT1A receptors present in a small proportion duction of 5-HT1A receptor binding sites in the DRN of patients with PD of neurons. (Doder et al., 2003) and in MPTP-treated monkeys (Frechilla et al.,

It has been previously shown that the 5-HT1A full agonist 8-OH- 2001) no changes have been reported concerning the density of 5-HT1A DPAT administered systemically after 5-HT depletion, or locally admin- receptors in the striatum (Azkona et al., 2014). According to that, al- istered in the STN in vivo, inhibits STN neuron activity (Aristieta et al., though Wang et al. (2009) reported a severe reduction in 8-OH-DPAT po- 2014). Here, we show that also buspirone dose-dependently inhibits tency in the DRN after a unilateral lesion of the nigrostriatal pathway, we the firing rate on every STN neuron recorded in control rats through did not observe it in our model (Miguelez et al., 2014). the activation of the 5-HT1A receptors. As buspirone has been described as a partial agonist at post-synaptic receptors but as a full agonist at DRN 5. Conclusions 5-HT1A somatodendritic autoreceptors (Sprouse and Aghajanian, 1988; Valdizan et al., 2010), collectively this STN inhibition could be attributed In conclusion, our results show that buspirone exerts its inhibitory to a direct effect mediated by the activation of the postsynaptic 5-HT1A effect through 5-HT1A receptors by a complex mechanism that may receptors located in the STN itself or via an indirect effect perhaps via involve the dopaminergic system. We also found that the antidyskinetic DRN autoreceptors. In this sense, it has been recently shown that either effect of buspirone does not imply a direct modulation of the electrical 5-HT1A-receptor agonists that preferentially activate post-synaptic activity of the STN. Dyskinesia is a debilitating motor complication 5-HT1A-receptor, such as F15599, or 5-HT1A-receptor agonist that pref- that appears in the majority of the patients that receive chronic treat- erentially activate pre-synaptic 5-HT1A-receptor like F13714, all reduce ment with L-DOPA. In spite of numerous efforts to develop a pharmaco- LID in animal models of PD (Huot et al., 2015; Iderberg et al., 2015a). logical treatment, there is not yet any new licensed drug to treat or fi Also, a potent and ef cacious pre- and post-synaptic 5-HT1A receptor prevent LIDs better than amantadine which clinical use is limited agonist, F13640, has been recently demonstrated to be a promising because of its partial efficacy and some important tolerability issues antidyskinetic drug (Iderberg et al., 2015b). Nevertheless, present such as constipation, dizziness and hallucinations (Pahwa et al., 2015). findings indicate that buspirone effect on STN neurons involve indirect It is essential to characterize drug targets to develop new treatments. mechanisms: (1) the application of a supramaximal concentration of In this sense the 6-OHDA lesioned rat model has proved to be a useful buspirone ex vivo, in a preparation that preserves only afferent termi- tool for studying both PD and the side effects resulting from treatment nals to the STN, failed to reduce the firing rate in the majority of the with L-DOPA (Bezard and Carta, 2015; Miller and Beninger, 1991; recorded cells, (2) ex vivo buspirone effects were almost completely Schallert et al., 2000; Ungerstedt, 1971). Our present results show that prevented by the presence of fast synaptic transmission blockers and response to buspirone depends on dopaminergic innervation stressing (3) in vivo local injection of buspirone into the STN affects to a small the importance of animal model to identify new antidyskinetic pharma- number of recorded STN neurons. cological tools. Indeed, an indirect effect mediated by 5-HT1A receptors in the Supplementary data to this article can be found online at http://dx. cortex, globus pallidus, substantia nigra pars compacta, thalamus or doi.org/10.1016/j.expneurol.2015.12.005. DRN cannot be excluded, since the STN receives input from all of these structures that are affected by buspirone (Canteras et al., 1990; Funding DeLong and Wichmann, 2007; Lanciego et al., 2004; Nambu et al., 2002; Parent et al., 2011). Interestingly, it was already described in This work has been supported by grants from the Government of the 1995 that buspirone at 4 mg/kg i.p. not only reduced glucose metabo- Basque Country T747-13, the University of the Basque Country (UFI 11/ lism in the cortex, thalamus, globus pallidus and DRN but also in the 32) and the Spanish Government (PI12/00613) co-financed by FEDER. STN of the rat (Freo et al., 1995). A.S. had a predoctoral fellowship from the University of the Basque Buspirone also reduces 5-HT synthesis rate in various rat brain struc- Country (UPV-EHU). tures including basal ganglia and the DRN (Okazawa et al., 1999) and totally inhibits DRN neurons (VanderMaelen et al., 1986). It is important Conflicts of interest to point out that in anesthetized rats buspirone significantly increases the levels of dopamine in cortex and striatum (Silverstone et al., 2012) The authors declare to have no conflicts of interest. whereas, activation of 5-HT1A-receptors attenuates the increase in extracellular dopamine derived from exogenously administrated L- Acknowledgments DOPA in the striatum of 6-OHDA lesioned rats (Kannari et al., 2001). Moreover, when administered to dyskinetic PD patients, buspirone We are thankful to SGIKER (UPV-EHU) for the technical and human not only reduces L-DOPA-evoked striatal synaptic dopamine increases support. but also attenuates LIDs (Politis et al., 2014). Interestingly, we observed that in 6-OHDA lesioned rats either treated with saline or with L-DOPA (dyskinetic rats) buspirone had no effect on the neuronal activity of References the STN. The fact that nigrostriatal pathway must be intact for buspirone to exert its inhibitory effect in the STN suggests that the effect may be Aristieta, A., Azkona, G., Sagarduy, A., Miguelez, C., Ruiz-Ortega, J.A., Sanchez-Pernaute, R., Ugedo, L., 2012. The role of the subthalamic nucleus in L-DOPA induced dyskinesia in mediated by dopamine. In this sense, immunohistochemical studies 6-hydroxydopamine lesioned rats. PLoS One 7, e42652. http://dx.doi.org/10.1371/ have demonstrated the presence of both D1 and D2 dopamine receptors journal.pone.0042652. 44 A. Sagarduy et al. / Experimental Neurology 277 (2016) 35–45

Aristieta, A., Morera-Herreras, T., Ruiz-Ortega, J.A., Miguelez, C., Vidaurrazaga, I., Arrue, A., and effects of sarizotan in the 6-hydroxydopamine-lesioned rat model of Zumarraga, M., Ugedo, L., 2014. Modulation of the subthalamic nucleus activity by Parkinson's disease. J. Neural Transm. 118, 1733–1742. http://dx.doi.org/10. serotonergic agents and fluoxetine administration. Psychopharmacology 231, 1007/s00702-010-0571-8. 1913–1924. http://dx.doi.org/10.1007/s00213-013-3333-0. Gerlach, M., Beck, J., Riederer, P., van den Buuse, M., 2011b. Flibanserin attenuates Azkona, G., Sagarduy, A., Aristieta, A., Vazquez, N., Zubillaga, V., Ruiz-Ortega, J.A., Perez- L: -DOPA-sensitized contraversive circling in the unilaterally 6-hydroxydopamine- Navarro, E., Ugedo, L., Sanchez-Pernaute, R., 2014. Buspirone anti-dyskinetic effect lesioned rat model of Parkinson's disease. J. Neural Transm. 118, 1727–1732. is correlated with temporal normalization of dysregulated striatal DRD1 signalling http://dx.doi.org/10.1007/s00702-010-0570-9. in L-DOPA-treated rats. Neuropharmacology 79, 726–737. http://dx.doi.org/10. Goetz,C.G.,Damier,P.,Hicking,C.,Laska,E.,Muller,T.,Olanow,C.W.,Rascol,O.,Russ,H.,2007. 1016/j.neuropharm.2013.11.024. Sarizotan as a treatment for dyskinesias in Parkinson's disease: a double-blind placebo- Bergman, J., Roof, R.A., Furman, C.A., Conroy, J.L., Mello, N.K., Sibley, D.R., Skolnick, P., 2013. controlled trial. Mov. Disord. 22, 179–186. http://dx.doi.org/10.1002/mds.21226. Modification of cocaine self-administration by buspirone (buspar(R)): potential Gregoire, L., Samadi, P., Graham, J., Bedard, P.J., Bartoszyk, G.D., Di Paolo, T., 2009. involvement of D3 and D4 dopamine receptors. Int. J. Neuropsychopharmacol. 16, Low doses of sarizotan reduce dyskinesias and maintain antiparkinsonian efficacy 445–458. http://dx.doi.org/10.1017/S1461145712000661. of L-DOPA in parkinsonian monkeys. Parkinsonism Relat. Disord. 15, 445–452. Bergman, H., Wichmann, T., Karmon, B., DeLong, M.R., 1994. The primate subthalamic http://dx.doi.org/10.1016/j.parkreldis.2008.11.001. nucleus. II. Neuronal activity in the MPTP model of parkinsonism. J. Neurophysiol. Grossman, C.J., Kilpatrick, G.J., Bunce, K.T., 1993. Development of a radioligand binding 72, 507–520 (PMID: 7983515). assay for 5-HT4 receptors in guinea-pig and rat brain. Br. J. Pharmacol. 109, Bezard, E., Carta, M., 2015. Could the serotonin theory give rise to a treatment for 618–624. http://dx.doi.org/10.1111/j.1476-5381.1993.tb13617.x. levodopa-induced dyskinesia in Parkinson's disease? Brain 138, 829–830. http://dx. Hannon, J., Hoyer, D., 2008. Molecular biology of 5-HT receptors. Behav. Brain Res. 195, doi.org/10.1093/brain/awu407. 198–213. http://dx.doi.org/10.1016/j.bbr.2008.03.020. Bibbiani, F., Oh, J.D., Chase, T.N., 2001. Serotonin 5-HT1A agonist improves motor compli- Hassani, O.K., Feger, J., 1999. Effects of intrasubthalamic injection of dopamine receptor cations in rodent and primate parkinsonian models. Neurology 57, 1829–1834. agonists on subthalamic neurons in normal and 6-hydroxydopamine-lesioned rats: http://dx.doi.org/10.1212/WNL.57.10.1829. an electrophysiological and c-Fos study. Neuroscience 92, 533–543. http://dx.doi. Bonifati, V., Fabrizio, E., Cipriani, R., Vanacore, N., Meco, G., 1994. Buspirone in levodopa- org/10.1016/S0306-4522(98)00765-9. induced dyskinesias. Clin. Neuropharmacol. 17, 73–82. http://dx.doi.org/10.1097/ Hassani, O.K., Mouroux, M., Feger, J., 1996. Increased subthalamic neuronal activity after 00002826-199402000-00008. nigral dopaminergic lesion independent of disinhibition via the globus pallidus. Bouthenet,M.L.,Martres,M.P.,Sales,N.,Schwartz,J.C.,1987.Adetailedmappingofdopamine Neuroscience 72, 105–115. http://dx.doi.org/10.1016/0306-4522(95)00535-8. D-2 receptors in rat central nervous system by autoradiography with [125I]iodosulpride. Heida,T.,Marani,E.,Usunoff,K.G.,2008.The subthalamic nucleus part II: modelling and Neuroscience 20, 117–155. http://dx.doi.org/10.1016/0306–4522(87)90008-X. simulation of activity. Adv. Anat. Embryol. Cell Biol. 199 (1–85), vii (ISBN: 978–3- Boyson, S.J., McGonigle, P., Molinoff, P.B., 1986. Quantitative autoradiographic localization 540-79461-5). of the D1 and D2 subtypes of dopamine receptors in rat brain. J. Neurosci. 6, Hollerman, J.R., Grace, A.A., 1992. Subthalamic nucleus cell firing in the 6-OHDA-treated 3177–3188 (PMID: 3534157). rat: basal activity and response to haloperidol. Brain Res. 590, 291–299. http://dx. Canteras, N.S., Shammah-Lagnado, S.J., Silva, B.A., Ricardo, J.A., 1990. Afferent connections doi.org/10.1016/0006-8993(92)91108-Q. of the subthalamic nucleus: a combined retrograde and anterograde horseradish Hoyer, D., Hannon, J.P., Martin, G.R., 2002. Molecular, pharmacological and functional peroxidase study in the rat. Brain Res. 513, 43–59. http://dx.doi.org/10.1016/0006- diversity of 5-HT receptors. Pharmacol. Biochem. Behav. 71, 533–554 (DOI: 8993(90)91087-W. S0091305701007468). Dekundy, A., Lundblad, M., Danysz, W., Cenci, M.A., 2007. Modulation of L-DOPA-induced Huot, P., Johnston, T.H., Fox, S.H., Newman-Tancredi, A., Brotchie, J.M., 2015. The highly- abnormal involuntary movements by clinically tested compounds: further validation selective 5-HT1A agonist F15599 reduces L-DOPA-induced dyskinesia without of the rat dyskinesia model. Behav. Brain Res. 179, 76–89. http://dx.doi.org/10.1016/j. compromising anti-parkinsonian benefits in the MPTP-lesioned macaque. Neuro- bbr.2007.01.013. pharmacology 97, 306–311. http://dx.doi.org/10.1016/j.neuropharm.2015.05.033. DeLong, M.R., Wichmann, T., 2007. Circuits and circuit disorders of the basal ganglia. Arch. Iderberg, H., McCreary, A.C., Varney, M.A., Cenci, M.A., Newman-Tancredi, A., 2015a. Neurol. 64, 20–24. http://dx.doi.org/10.1001/archneur.64.1.20. Activity of serotonin 5-HT(1A) receptor ‘biased agonists’ in rat models of Parkinson's Dhavalshankh, A.G., Jadhav, S.A., Gaikwad, R.V., Gaonkar, R.K., Thorat, V.M., Balsara, J.J., disease and L-DOPA-induced dyskinesia. Neuropharmacology 93, 52–67. http://dx. 2007. Effects of buspirone on dopamine dependent behaviours in rats. Indian doi.org/10.1016/j.neuropharm.2015.01.012. J. Physiol. Pharmacol. 51, 375–386 (PMID: 18476392). Iderberg, H., McCreary, A.C., Varney, M.A., Kleven, M.S., Koek, W., Bardin, L., Depoortère, R., Di Matteo, V., Pierucci, M., Esposito, E., Crescimanno, G., Benigno, A., Di Giovanni, G., 2008. Cenci, M.A., Newman-Tancredi, A., 2015b. NLX-112, a novel 5-HT1A receptor agonist for Serotonin modulation of the basal ganglia circuitry: therapeutic implication for the treatment of L-DOPA-induced dyskinesia: behavioral and neurochemical profile in Parkinson's disease and other motor disorders. Prog. Brain Res. 172, 423–463. rat. Exp. Neurol. 271, 335–350. http://dx.doi.org/10.1016/j.expneurol.2015.05.021. http://dx.doi.org/10.1016/S0079-6123(08)00921-7. Johnson, A.E., Coirini, H., Kallstrom, L., Wiesel, F.A., 1994. Characterization of dopamine Doder, M., Rabiner, E.A., Turjanski, N., Lees, A.J., Brooks, D.J., 2003. Tremor in Parkinson's receptor binding sites in the subthalamic nucleus. Neuroreport 5, 1836–1838. disease and serotonergic dysfunction: an 11C-WAY 100635 PET study. Neurology http://dx.doi.org/10.1097/00001756-199409080-00038. 60, 601–605. http://dx.doi.org/10.1212/01.WNL.0000031424.51127.2B. Kaneoke, Y., Vitek, J.L., 1996. Burst and oscillation as disparate neuronal properties. Dupre, K.B., Eskow, K.L., Negron, G., Bishop, C., 2007. The differential effects of 5-HT(1A) J. Neurosci. Methods 68, 211–223. http://dx.doi.org/10.1016/S0165-0270(96)00081-7. receptor stimulation on dopamine receptor-mediated abnormal involuntary move- Kannari, K., Yamato, H., Shen, H., Tomiyama, M., Suda, T., Matsunaga, M., 2001. Activation ments and rotations in the primed hemiparkinsonian rat. Brain Res. 1158, 135–143 of 5-HT(1A) but not 5-HT(1B) receptors attenuates an increase in extracellular (DOI: S0006-8993(07)01064–5). dopamine derived from exogenously administered L-DOPA in the striatum with Eberle-Wang, K., Lucki, I., Chesselet, M.F., 1996. A role for the subthalamic nucleus in 5- nigrostriatal denervation. J. Neurochem. 76, 1346–1353. http://dx.doi.org/10.1046/j. HT2C-induced oral dyskinesia. Neuroscience 72, 117–128. http://dx.doi.org/10. 1471-4159.2001.00184.x. 1016/0306-4522(95)00548-X. Kleedorfer, B., Lees, A.J., Stern, G.M., 1991. Buspirone in the treatment of levodopa induced Eberle-Wang, K., Mikeladze, Z., Uryu, K., Chesselet, M.F., 1997. Pattern of expression of the dyskinesias. J. Neurol. Neurosurg. Psychiatry 54, 376–377 (PMID: 2056334). serotonin2C receptor messenger RNA in the basal ganglia of adult rats. J. Comp. Lanciego, J.L., Gonzalo, N., Castle, M., Sanchez-Escobar, C., Aymerich, M.S., Obeso, J.A., Neurol. 384, 233–247. http://dx.doi.org/10.1002/(SICI)1096-9861(19970728)384: 2004. Thalamic innervation of striatal and subthalamic neurons projecting to the 2b233::AID-CNE5N3.0.CO;2-2. rat entopeduncular nucleus. Eur. J. Neurosci. 19, 1267–1277. http://dx.doi.org/10. Eskow, K.L., Gupta, V., Alam, S., Park, J.Y., Bishop, C., 2007. The partial 5-HT(1A) agonist 1111/j.1460-9568.2004.03244.x. buspirone reduces the expression and development of L-DOPA-induced dyskinesia Liu, J., Chu, Y.X., Zhang, Q.J., Wang, S., Feng, J., Li, Q., 2007. 5,7-Dihydroxytryptamine lesion in rats and improves L-DOPA efficacy.Pharmacol.Biochem.Behav.87,306–314. of the dorsal raphe nucleus alters neuronal activity of the subthalamic Nucleus in http://dx.doi.org/10.1016/j.pbb.2007.05.002. normal and 6-hydroxydopamine-lesioned rats. Brain Res. 1149, 216–222 (DOI: Flores, G., Liang, J.J., Sierra, A., Martinez-Fong, D., Quirion, R., Aceves, J., Srivastava, L.K., S0006-8993(07)00485–4). 1999. Expression of dopamine receptors in the subthalamic nucleus of the rat: Maroteaux, L., Saudou, F., Amlaiky, N., Boschert, U., Plassat, J.L., Hen, R., 1992. Mouse characterization using reverse transcriptase-polymerase chain reaction and 5HT1B serotonin receptor: cloning, functional expression, and localization in motor autoradiography. Neuroscience 91, 549–556. http://dx.doi.org/10.1016/S0306- control centers. Proc. Natl. Acad. Sci. U. S. A. 89, 3020–3024. http://dx.doi.org/10. 4522(98)00633-2. 1073/pnas.89.7.3020. Flores, G., Rosales, M.G., Hernandez, S., Sierra, A., Aceves, J., 1995. 5-Hydroxytryptamine McMillen, B.A., Matthews, R.T., Sanghera, M.K., Shepard, P.D., German, D.C., 1983. Dopamine increases spontaneous activity of subthalamic neurons in the rat. Neurosci. Lett. receptor antagonism by the novel antianxiety drug, buspirone. J. Neurosci. 3, 733–738 192, 17–20 (DOI: 030439409511597P). (PMID: 6131948). Fox, S.H., 2013. Non-dopaminergic treatments for motor control in Parkinson's disease. Miguelez, C., Aristieta, A., Cenci, M.A., Ugedo, L., 2011. The locus coeruleus is directly im- Drugs 73, 1405–1415. http://dx.doi.org/10.1007/s40265-013-0105-4. plicated in L-DOPA-induced dyskinesia in parkinsonian rats: an electrophysiological Frechilla, D., Cobreros, A., Saldise, L., Moratalla, R., Insausti, R., Luquin, M., Del Rio, J., 2001. and behavioural study. PLoS One 6, e24679. http://dx.doi.org/10.1371/journal.pone. Serotonin 5-HT(1A) receptor expression is selectively enhanced in the striosomal 0024679. compartment of chronic parkinsonian monkeys. Synapse 39, 288–296. http://dx. Miguelez, C., Navailles, S., Delaville, C., Marquis, L., Lagire, M., Benazzouz, A., Ugedo, L., De doi.org/10.1002/1098-2396(20010315)39:4b288::AID-SYN1011N3.0.CO;2-V. Deurwaerdère, P., 2014. L-DOPA alters dopamine and serotonin release independent- Freo, U., Pietrini, P., Pizzolato, G., Furey-Kurkjian, M., Merico, A., Ruggero, S., Dam, M., ly from electrophysiological changes in the dorsal raphe nucleus in vivo: relevance to Battistin, L., 1995. Dose-dependent effects of buspirone on behavior and cerebral dyskinesia. Poster Communication. Society for Neuroscience, November 15–19, glucose metabolism in rats. Brain Res. 677 (2), 213–220. http://dx.doi.org/10.1016/ Washington, DC, 2014. 0006-8993(95)00140-L. Miller, R., Beninger, R.J., 1991. On the interpretation of asymmetries of posture and loco- Gerlach,M.,Bartoszyk,G.D.,Riederer,P.,Dean,O.,vandenBuuse,M.,2011a.Roleof motion produced with dopamine agonists in animals with unilateral depletion of dopamine D3 and serotonin 5-HT 1A receptors in L: -DOPA-induced dyskinesias striatal dopamine. Prog. Neurobiol. 36, 229–256 (PMID: 1673254). A. Sagarduy et al. / Experimental Neurology 277 (2016) 35–45 45

Morera-Herreras, T., Ruiz-Ortega, J.A., Linazasoro, G., Ugedo, L., 2011. Nigrostriatal dener- Shen, K.Z., Kozell, L.B., Johnson, S.W., 2007. Multiple conductances are modulated by 5-HT vation changes the effect of cannabinoids on subthalamic neuronal activity in rats. receptor subtypes in rat subthalamic nucleus neurons. Neuroscience 148, 996–1003 Psychopharmacology 214, 379–389. http://dx.doi.org/10.1007/s00213-010-2043-0. (DOI: S0306-4522(07)00887–1). Mori, S., Takino, T., Yamada, H., Sano, Y., 1985. Immunohistochemical demonstration of Shin, E., Garcia, J., Winkler, C., Björklund, A., Carta, M., 2012. Serotonergic and dopaminer- serotonin nerve fibers in the subthalamic nucleus of the rat, cat and monkey. gic mechanisms in graft-induced dyskinesia in a rat model of Parkinson's disease. Neurosci. Lett. 62, 305–309 (DOI: 0304–3940(85)90566-X). Neurobiol. Dis. 47, 393–406. http://dx.doi.org/10.1016/j.nbd.2012.03.038. Mrakic-Sposta, S., Marceglia, S., Egidi, M., Carrabba, G., Rampini, P., Locatelli, M., Foffani, G., Silverstone, P.H., Lalies, M.D., Hudson, A.L., 2012. Quetiapine and buspirone both elevate Accolla, E., Cogiamanian, F., Tamma, F., Barbieri, S., Priori, A., 2008. Extracellular spike cortical levels of noradrenaline and dopamine in vivo, but do not have synergistic ef- microrecordings from the subthalamic area in Parkinson's disease. J. Clin. Neurosci. fects. Front. Psychiatry 3, 82. http://dx.doi.org/10.3389/fpsyt.2012.00082. 15, 559–567. http://dx.doi.org/10.1016/j.jocn.2007.02.091. Sprouse, J.S., Aghajanian, G.K., 1988. Responses of hippocampal pyramidal cells to putative Nambu, A., Tokuno, H., Takada, M., 2002. Functional significance of the cortico- serotonin 5-HT1A and 5-HT1B agonists: a comparative study with dorsal raphe neurons. subthalamo-pallidal ‘hyperdirect’ pathway. Neurosci. Res. 43, 111–117. http://dx. Neuropharmacology 27, 707–715. http://dx.doi.org/10.1016/0028-3908(88)90079-2. doi.org/10.1016/S0168-0102(02)00027-5. Stanford, I.M., Kantaria, M.A., Chahal, H.S., Loucif, K.C., Wilson, C.L., 2005. 5- Okazawa, H., Yamane, F., Blier, P., Diksic, M., 1999. Effects of acute and chronic administra- Hydroxytryptamine induced excitation and inhibition in the subthalamic nucleus: action of the serotonin1A agonist buspirone on serotonin synthesis in the rat brain. tion at 5-HT(2C), 5-HT(4) and 5-HT(1A) receptors. Neuropharmacology 49, J. Neurochem. 72, 2022–2031. http://dx.doi.org/10.1046/j.1471-4159.1999.0722022.x. 1228–1234 (DOI: S0028-3908(05)00327–8). Pahwa, R., Tanner, C.M., Hauser, R.A., Sethi, K., Isaacson, S., Truong, D., Struck, L., Ruby, A.E., Sussman, N., 1998. Anxiolytic antidepressant augmentation. J. Clin. Psychiatry 59 (Suppl. McClure, N.L., Went, G.T., Stempien, M.J., 2015. Amantadine extended release for 5), 42–48 (discussion 49-50. PMID: 9635547). levodopa-induced dyskinesia in Parkinson's disease (EASED study). Mov. Disord. 30 Svenningsson, P., Rosenblad, C., Af Edholm Arvidsson, K., Wictorin, K., Keywood, C., (6), 788–795. http://dx.doi.org/10.1002/mds.26159. Shankar, B., Lowe, D.A., Bjorklund, A., Widner, H., 2015. Eltoprazine counteracts L- Parent, M., Wallman, M.J., Gagnon, D., Parent, A., 2011. Serotonin innervation of basal gan- DOPA-induced dyskinesias in Parkinson's disease: a dose-finding study. Brain 138, glia in monkeys and humans. J. Chem. Neuroanat. 41, 256–265. http://dx.doi.org/10. 963–973. http://dx.doi.org/10.1093/brain/awu409. 1016/j.jchemneu.2011.04.005. Thomas, A., Iacono, D., Luciano, A.L., Armellino, K., Di Iorio, A., Onofrj, M., 2004. Duration of Paxinos, G., Watson, C., 1986. The Rat Brain Stereotaxic Coordinates. second ed. Academic, amantadine benefit on dyskinesia of severe Parkinson's disease. J. Neurol. Neurosurg. Orlando (ISBN 9780125476195). Psychiatry 75, 141–143 (PMID: 14707325). Pazos, A., Palacios, J.M., 1985. Quantitative autoradiographic mapping of serotonin recep- Ungerstedt, U., 1971. Postsynaptic supersensitivity after 6-hydroxy-dopamine induced tors in the rat brain. I. Serotonin-1 receptors. Brain Res. 346, 205–230. http://dx.doi. degeneration of the nigro-striatal dopamine system. Acta Physiol. Scand. Suppl. org/10.1016/0006-8993(85)90856-X. 367, 69–93. http://dx.doi.org/10.1111/j.1365-201X.1971.tb11000.x. Pazos, A., Cortes, R., Palacios, J.M., 1985. Quantitative autoradiographic mapping of seroto- Valdizan, E.M., Castro, E., Pazos, A., 2010. Agonist-dependent modulation of G-protein cou- nin receptors in the rat brain. II. Serotonin-2 receptors. Brain Res. 346, 231–249 (DOI: pling and transduction of 5-HT1A receptors in rat dorsal raphe nucleus. Int. 0006–8993(85)90857–1). J. Neuropsychopharmacol. 13, 835–843. http://dx.doi.org/10.1017/S1461145709990940. Peroutka, S.J., 1988. 5-Hydroxytryptamine receptor subtypes: molecular, biochemical and VanderMaelen, C.P., Matheson, G.K., Wilderman, R.C., Patterson, L.A., 1986. Inhibition of physiological characterization. Trends Neurosci. 11, 496–500. http://dx.doi.org/10. serotonergic dorsal raphe neurons by systemic and iontophoretic administration of 1016/0166-2236(88)90011-2. buspirone, a non-benzodiazepine anxiolytic drug. Eur. J. Pharmacol. 129, 123–130 Politis, M., Wu, K., Loane, C., Brooks, D.J., Kiferle, L., Turkheimer, F.E., Bain, P., Molloy, S., (DOI: 0014–2999(86)90343–2). Piccini, P., 2014. Serotonergic mechanisms responsible for levodopa-induced dyski- Waeber, C., Sebben, M., Bockaert, J., Dumuis, A., 1996. Regional distribution and ontogeny nesias in Parkinson's disease patients. J. Clin. Invest. 124, 1340–1349. http://dx.doi. of 5-HT4 binding sites in rat brain. Behav. Brain Res. 73, 259–262. http://dx.doi.org/ org/10.1172/JCI71640. 10.1016/0166-4328(96)00108-8. Pompeiano, M., Palacios, J.M., Mengod, G., 1992. Distribution and cellular localization of Wang, S., Zhang, Q.J., Liu, J., Wu, Z.H., Wang, T., Gui, Z.H., Chen, L., Wang, Y., 2009. Unilat- mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding. eral lesion of the nigrostriatal pathway induces an increase of neuronal firing of the J. Neurosci. 12, 440–453 (PMID: 1531498). midbrain raphe nuclei 5-HT neurons and a decrease of their response to 5-HT(1A) re- Ruiz-Ortega, J.A., Ugedo, L., 1997. The stimulatory effect of clonidine on locus coeruleus ceptor stimulation in the rat. Neuroscience 159, 850–861. http://dx.doi.org/10.1016/j. neurons of rats with inactivated alpha 2-adrenoceptors: involvement of imidazoline neuroscience.2008.12.051. receptors located in the nucleus paragigantocellularis. Naunyn Schmiedeberg's Arch. Wilson, C.L., Puntis, M., Lacey, M.G., 2004. Overwhelmingly asynchronous firing of rat sub- Pharmacol. 355, 288–294 (PMID: 9050025). thalamic nucleus neurones in brain slices provides little evidence for intrinsic inter- Sari, Y., Miquel, M.C., Brisorgueil, M.J., Ruiz, G., Doucet, E., Hamon, M., Verge, D., 1999. Cel- connectivity. Neuroscience 123, 187–200 (DOI: S030645220300705X). lular and subcellular localization of 5-hydroxytryptamine1B receptors in the rat cen- Wolf, E., Seppi, K., Katzenschlager, R., Hochschorner, G., Ransmayr, G., Schwingenschuh, tral nervous system: immunocytochemical, autoradiographic and lesion studies. P., Ott, E., Kloiber, I., Haubenberger, D., Auff, E., Poewe, W., 2010. Long-term Neuroscience 88, 899–915 (DOI: S0306452298002565). antidyskinetic efficacy of amantadine in Parkinson's disease. Mov. Disord. 25, Schallert, T., Fleming, S.M., Leasure, J.L., Tillerson, J.L., Bland, S.T., 2000. CNS plasticity and 1357–1363. http://dx.doi.org/10.1002/mds.23034. assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cor- Xiang, Z., Wang, L., Kitai, S.T., 2005. Modulation of spontaneous firing in rat subthalamic tical ablation, parkinsonism and spinal cord injury. Neuropharmacology 39, 777–787. neurons by 5-HT receptor subtypes. J. Neurophysiol. 93, 1145–1157. http://dx.doi. http://dx.doi.org/10.1016/S0028-3908(00)00005-8. org/10.1152/jn.00561.2004.