fnins-13-00545 June 1, 2019 Time: 10:30 # 1 ORIGINAL RESEARCH published: 04 June 2019 doi: 10.3389/fnins.2019.00545 Inhibition of Morphine- and Ethanol-Mediated Stimulation of Mesolimbic Dopamine Neurons by Withania somnifera Valentina Bassareo1,2†, Giuseppe Talani3†, Roberto Frau1, Simona Porru4, Michela Rosas4, Sanjay B. Kasture5, Alessandra T. Peana6, Eleonora Loi4, Enrico Sanna2,3,4 and Elio Acquas2,4* 1 Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy, 2 Centre of Excellence on Neurobiology of Addiction, University of Cagliari, Cagliari, Italy, 3 Institute of Neuroscience, National Research Council, Cagliari, Italy, 4 Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy, 5 Pinnacle Biomedical Research Institute, Bhopal, India, 6 Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy Morphine- and ethanol-induced stimulation of neuronal firing of ventral tegmental area (VTA) dopaminergic neurons and of dopamine (DA) transmission in the shell of the nucleus accumbens (AcbSh) represents a crucial electrophysiological and Edited by: A. Leslie Morrow, neurochemical response underlying the ability of these compounds to elicit motivated University of North Carolina at Chapel behaviors and trigger a cascade of plasticity-related biochemical events. Previous Hill, United States studies indicate that the standardized methanolic extract of Withania somnifera roots Reviewed by: Mark S. Brodie, (WSE) prevents morphine- and ethanol-elicited conditioned place preference and oral The University of Illinois at Chicago, ethanol self-administration. Aim of the present research was to investigate whether United States WSE may also interfere with the ability of morphine and ethanol to stimulate VTA Scott C. Steffensen, Brigham Young University, dopaminergic neurons and thus AcbSh DA transmission as assessed in male Sprague- United States Dawley rats by means of patch-clamp recordings in mesencephalic slices and *Correspondence: in vivo brain microdialysis, respectively. Morphine and ethanol significantly stimulated Elio Acquas [email protected] spontaneous firing rate of VTA neurons and DA transmission in the AcbSh. WSE, at †These authors have contributed concentrations (200–400 mg/ml) that significantly reduce spontaneous neuronal firing equally to this work of VTA DA neurons via a GABAA- but not GABAB-mediated mechanism, suppressed the stimulatory actions of both morphine and ethanol. Moreover, in vivo administration Specialty section: This article was submitted to of WSE at a dose (75 mg/kg) that fails to affect basal DA transmission, significantly Neuropharmacology, prevented both morphine- and ethanol-elicited increases of DA in the AcbSh. Overall, a section of the journal these results highlight the ability of WSE to interfere with morphine- and ethanol- Frontiers in Neuroscience mediated central effects and suggest a mechanistic interpretation of the efficacy of this Received: 28 February 2019 Accepted: 13 May 2019 extract to prevent the motivational properties of these compounds. Published: 04 June 2019 Keywords: dopamine, ethanol, morphine, nucleus accumbens shell, standardized Withania somnifera extract, Citation: ventral tegmental area, GABA Bassareo V, Talani G, Frau R, Porru S, Rosas M, Kasture SB, Peana AT, Loi E, Sanna E and INTRODUCTION Acquas E (2019) Inhibition of Morphine- and Ethanol-Mediated Stimulation of Mesolimbic Dopamine Withania somnifera (WS) Dunal (family Solanaceae), also known as Ashwagandha or Indian Neurons by Withania somnifera. Ginseng, is a plant widely used in traditional Ayurvedic medicine in India since antiquity (Kulkarni Front. Neurosci. 13:545. and Dhir, 2008). It is included in the Indian Pharmacopoeia (Singh et al., 2011) as a safe official doi: 10.3389/fnins.2019.00545 medication for the treatment of several ailments (Dar et al., 2016) and recent evidence supports its Frontiers in Neuroscience| www.frontiersin.org 1 June 2019| Volume 13| Article 545 fnins-13-00545 June 1, 2019 Time: 10:30 # 2 Bassareo et al. Withania Prevents Stimulated DA Function anti-inflammatory, immunomodulatory, neuroprotective of morphine- and ethanol-elicited CPP (Ruiu et al., 2013; Spina (Bhatnagar et al., 1975; Kulkarni et al., 2008; Dar et al., 2016; et al., 2015) and acquisition and maintenance of ethanol oral self- Yenisetti et al., 2016) and free-radical scavenging (Bhattacharya administration (Peana et al., 2014) is represented by the ability et al., 2001; Davis and Kuttan, 2001; Prakash et al., 2014; Dar of WSE to affect morphine- and ethanol-elicited increase of the et al., 2015) properties. Moreover, WS has been shown to spontaneous firing rate of VTA DA neurons as well as of phasic modulate GABAergic [(g-amino-butyric acid (GABA)] (Mehta DA transmission in the AcbSh. et al., 1991; Kulkarni and Dhir, 2008) and cholinergic (Schliebs Hence, in order to shed light on the mechanism(s) by et al., 1997) neurotransmission and to affect different properties which WSE prevents DA-mediated morphine- and ethanol- of addictive drugs. In fact, several studies have reported that elicited motivated behaviors (Ruiu et al., 2013; Peana et al., 2014; WS prevents the development of tolerance and dependence Spina et al., 2015), the present study aimed at characterizing to morphine (Kulkarni and Ninan, 1997) as well as morphine further the ability of WSE to affect the psychopharmacological withdrawal-dependent reduction of dendritic spine density in properties of morphine and ethanol by assessing whether it may the shell of the nucleus accumbens (AcbSh) (Diana et al., 2006; also interfere with the ability of these substances to stimulate Kasture et al., 2009). In addition, WS has also been shown to VTA DA neuronal firing activity and prevent morphine- and reduce anxiety associated with ethanol-withdrawal and increase ethanol-elicited increases of DA transmission in the AcbSh. In ethanol-induced anxiolysis (Gupta and Rana, 2008). addition, to further characterize the mechanism by which WSE Drug addiction is defined as the progressive loss of may affect the firing activity of VTA DA neurons by itself, we also control over drug taking and results from a long series of applied WSE in the presence of GABAA and GABAB receptors neuroadaptations taking place in selective neural circuits in antagonists, bicuculline and SCH50911, respectively. To this end, response to the progressive exposure to addictive drugs (Volkow ex vivo standard patch-clamp recordings of VTA DA neurons in and Morales, 2015). Notably, among these drugs both morphine mesencephalic slices and in vivo brain microdialysis in the AcbSh, (Di Chiara and Imperato, 1988) and ethanol (Imperato and from male Sprague-Dawley rats, were performed. Di Chiara, 1986; Bassareo et al., 2003) preferentially increase dopamine (DA) transmission in the AcbSh, a property that has been pinpointed as a neurochemical feature shared by MATERIALS AND METHODS all substances with addictive potential (Di Chiara, 1999; Di Chiara et al., 2004). Moreover, this property represents the Subjects neurophysiological correlate of increased spontaneous firing Male Sprague-Dawley rats (Envigo, Italy) bred in our animal rate of DA neurons in the ventral tegmental area (VTA) by facility (19–30 days of age) and male Sprague-Dawley rats addictive drugs, including morphine (Jalabert et al., 2011; Chen (Envigo, Italy) (45–52 days of age) were used for the et al., 2015) and ethanol (Gessa et al., 1985; Brodie et al., electrophysiological and the microdialysis experiments, 1990; Brodie and Appel, 1998; Xiao and Ye, 2008; Theile respectively. Animals had access to water and standard laboratory et al., 2011) on the one hand, and of morphine- and ethanol- food (Stefano Morini, S. Polo D’Enza, RE, Italy) ad libitum. mediated motivated behaviors (Di Chiara, 1999; Di Chiara et al., Animal care and handling throughout the experimental 2004) on the other. However, in spite of decades of research procedures were in accordance with the guidelines for care aimed at understanding its neurobiological basis, drug addiction and use of experimental animals of the European Community Council (2010/63/UE L 276 20/10/2010) and with Italian law represents a complex pathological condition for which a still ◦ limited list of therapeutic tools, restricted mostly to opioids, (DL 04.03.2014, N 26). In particular, this study was approved by nicotine and alcohol dependence, is available (Lyon, 2017). the Organization for Animal Care of the University of Cagliari Hence, the preclinical search of new therapeutic approaches, (OPBA-UniCA) and performed in accordance with the Ministry including the application and development of strategies based of Health authorization number 1177/2016-pr (December 15, on the use of substances obtained from herbal remedies or 2016). Every effort was made to minimize suffering and reduce from whole herbal extracts (Carai et al., 2000; Lu et al., 2009; the number of animals used. Liu et al., 2011; Zhang et al., 2014), appears nowadays highly desirable. In this regard, the standardized methanolic extract Experimental Procedures for of WS roots (WSE) has recently been shown to affect the Electrophysiological Experiments ability of morphine (Ruiu et al., 2013) and ethanol (Spina Preparation of Rat VTA Slices et al., 2015) to elicit acquisition and expression of conditioned Brain slices were prepared as previously described by Talani et al. place preference (CPP) in mice as well as the acquisition and (2011, 2016). In brief, at post-natal day (PND) 19–30, animals