Synaptic Encoding of in Vivo Ethanol Experience in the Nucleus Accumbens

The purpose of this study was to determine the effects of in vivo ethanol exposure on the expression of plasticity and intrinsic properties of D1 and D2 dopamine receptor expressing medium spiny neurons (MSNs) in the nucleus accumbens (NAc) shell. To distinguish between the two subtypes of MSNs in the nucleus accumbens we used DRD1a-TdTomato mice that have been back crossed onto C57BL/6. Mice were treated with chronic intermittent ethanol vapor, a model to induce ethanol dependence and increase ethanol drinking (Becker and Lopez, 2004; Lopez and Becker, 2005; Griffin 3rd et al., 2009a, 2009b). In current clamp experiments we found that CIE induced an increase in excitability in D1+ MSNs with no change in D1MSNs. Action potential analysis revealed an increase in fast after hyperpolarization and a decrease in the action potential half width, both of which were selectively expressed in D1+ MSNs. Our previous work in C57BL/6 has shown that the low frequency stimulation induction protocol used for the expression of LTD results in LTP after CIE exposure (Jeanes et al., 2011). In this study we used whole cell voltage clamp to measure excitatory post synaptic currents (EPSCs). Plasticity was induced by pairing low frequency stimulation with post synaptic depolarization. In ethanol naïve mice, 31 NMDAR-dependent LTD was found to be expressed exclusively in D1+ MSNs and not in D1MSNs. In slices prepared from CIE treated mice, the pairing protocol to induce plasticity resulted in LTP in D1+ MSNs. The expression of LTP in D1+ MSNs was accompanied by an increase in the frequency of spontaneous EPSCs. Interestingly, CIE exposure uncovered the expression of LTD in D1MSNs. The cell type specific alterations in the expression of plasticity and the intrinsic properties of MSNs in the NAc shell may constitute an important neuroadaptation necessary for the expression of ethanol induced behaviors. Introduction The nucleus accumbens (NAc), a critical component of the brain reward system and is composed primarily of medium spiny neurons (MSNs) of which there are two distinct subtypes, D1 and D2 MSNs. Neuroadaptations in the NAc may be important for the expression of drug induced behaviors (Luscher and Malenka, 2011). Alterations in the intrinsic properties of neurons may be a means in which drug experience modifies neural circuits (Zhang and Linden, 2003). Repeated in vivo cocaine exposure has been shown to alter the excitability of MSNs in the NAc (cite pierce wolf review) (Dong et al., 2006; Benavides et al., 2007; Ishikawa et al., 2009; Kourrich and Thomas, 2009). Chronic alcohol exposure has also been shown to modulate the intrinsic properties of MSNs in the NAc (Hopf et al., 2010; Marty and Spigelman, 2012a) however it is unknown if ethanol differentially modulates the intrinsic properties of 32 D1 and D2 MSNs. Alterations in the excitability of MSNs in the NAc may be a means in which further drug induced neuroadaptations may develop (Kourrich et al., 2015). Several reports have shown that drugs of abuse and ethanol can disrupt the expression of NMDAR-dependent plasticity in the NAc (Thomas et al., 2001; Brebner et al., 2005; Martin et al., 2006; Mao et al., 2009; Kasanetz et al., 2010; Jeanes et al., 2011, 2014; Pascoli et al., 2011; Shen and Kalivas, 2012; Abrahao et al., 2013). In previous work from our lab using C57Bl/6 mice, we found that the induction protocol which results in LTD in ethanol naïve mice produces LTP after ethanol vapor exposure (Jeanes et al., 2011). At that time we were unable to determine if ethanol differentially modulated plasticity between the two MSN subtypes. To selectively record from D1 or D2 MSNs we used bacterial artificial chromosome (BAC) transgenic mice in which the expression of enhanced green fluorescent protein (eGFP) is controlled by the D1R promoter in mice on a Swiss Webster background (Matamales et al., 2009; Valjent et al., 2009). Using this approach we found that NMDAR-dependent LTD is expressed only in D1 MSNs. After vapor exposure we found a reversal in the expression of plasticity in which LTD could be induced in D2 MSNs but not in D1 MSNs (Jeanes et al., 2014). Our primary goal is to characterize neuroadaptations in the NAc that may contribute to excessive ethanol consumption. In our hands, Swiss Webster mice will not voluntarily drink ethanol (unpublished) therefore our previous work using DRD1a-eGFP mice on the Swiss Webster background may not reflect the 33 alterations in plasticity that contribute to the escalation of ethanol intake. In this study, we use DRD1-TdTomato mice on a C57Bl/6 background, a strain that has a well-documented drinking phenotype. We aimed to characterize ethanol induced neuroadaptations of neuronal excitability and plasticity that may contribute to the formation of ethanol dependence. Methods Chronic intermittent ethanol exposure Mice were exposed to ethanol vapor using the chronic intermittent ethanol (CIE) exposure model (Becker and Lopez, 2004; Jeanes et al., 2011, 2014). Ethanol was volatilized by bubbling air through a flask containing 95% ethanol at a rate of 0.2 to 0.3 liter/min. The resulting ethanol vapor then combined with a separate air stream to give a total flow rate of approximately 4 liters/min, which was delivered to mice in special mouse chamber units that contain an airtight top, a vapor inlet and an exhaust outlet (Allentown Inc., Allentown, NJ). Each round consisted of 16 hours of ethanol vapor exposure followed by an 8 hour withdrawal, repeated for 4 days. Mice received intraperitoneal injections of a loading dose of ethanol (20% v/v, 1.5 g/kg) and pyrazole (68.1 mg/kg) prior to vapor exposure to achieve a blood ethanol concentration (BEC) of 150-200 mg/dl. Air control mice were handled the same but were injected with a solution of only pyrazole.