Neuropsychopharmacology (2017) 42, 1800–1812 © 2017 American College of Neuropsychopharmacology. All rights reserved 0893-133X/17 www.neuropsychopharmacology.org Ethanol Dependence Abolishes Monoamine and GIRK (Kir3) Channel Inhibition of Orbitofrontal Cortex Excitability 1 2 1,2 *,1,2 Sudarat Nimitvilai , Marcelo F Lopez , Patrick J Mulholland and John J Woodward 1Department of Neuroscience, Medical University of South Carolina, Charleston, SC, USA; 2Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, SC, USA Alcohol abuse disorders are associated with dysfunction of frontal cortical areas including the orbitofrontal cortex (OFC). The OFC is extensively innervated by monoamines, and drugs that target monoamine receptors have been used to treat a number of neuropsychiatric diseases, including alcoholism. However, little is known regarding how monoamines affect OFC neuron excitability or whether this modulation is altered by chronic exposure to ethanol. In this study, we examined the effect of dopamine, norepinephrine, and serotonin on lateral OFC neuronal excitability in naive mice and in those exposed to chronic intermittent ethanol (CIE) treatment. All three α monoamines decreased current-evoked spike firing of lOFC neurons and this action required Giα-coupled D2, 2-adrenergic, and 5HT1A receptors, respectively. Inhibition of firing by dopamine or the D2 agonist quinpirole, but not norepinephrine or serotonin, was prevented by the GABAA receptor antagonist picrotoxin. GABA-mediated tonic current was enhanced by dopamine or the D1 agonist SKF81297 but not quinpirole, whereas the amplitude of spontaneous IPSCs was increased by quinpirole but not dopamine. Spiking was also inhibited by the direct GIRK channel activator ML297, whereas blocking these channels with barium increased firing and eliminated the inhibitory actions of monoamines. In the presence of ML297 or the G-protein blocker GDP-β-S, DA induced a further decrease in spike firing, suggesting the involvement of a non-GIRK channel mechanism. In neurons from CIE-treated mice, spike frequency was nearly doubled and inhibition of firing by monoamines or ML297 was lost. These effects occurred in the absence of significant changes in expression of Gi/o or GIRK channel proteins. Together, these findings show that monoamines are important modulators of lOFC excitability and suggest that disruption of this process could contribute to various deficits associated with alcohol dependence. Neuropsychopharmacology (2017) 42, 1800–1812; doi:10.1038/npp.2017.22; published online 15 March 2017 INTRODUCTION modalities in order to guide behavior based on optimal choices and outcomes. Thus, regions of the OFC mediate Monoamine neurotransmitters such as norepinephrine (NE), serotonin (5HT), and dopamine (DA) are important aspects of choice behavior (Roesch and Olson, 2007; regulators of neuronal function and are critical for a wide Schoenbaum et al, 1998), reversal learning (Bissonette et al, variety of affective behaviors including reward, attention, 2008; McAlonan and Brown, 2003), and the development of emotion, memory, and learning. Alterations in the levels or anticipation in response to either appetitive or aversive function of monoamines and their receptors, either by stimuli (Tremblay and Schultz, 1999). Dysfunction of the disease or genetic differences, are thought to contribute to OFC is associated with numerous neuropsychiatric diseases symptoms associated with many psychiatric diseases includ- including obsessive–compulsive disorder and symptoms of ing addiction (Kirby et al, 2011; Narendran et al, 2014; OFC deficiency are often observed in individuals suffering Volkow et al, 2001). Indeed, drugs that target monoamine from alcohol and drug abuse disorders (Fortier et al, 2008; systems have long been used to treat affective and attention Verdejo-Garcia et al, 2006). In a previous report, we showed disorders (Rothman et al, 2008; Volkow et al, 2002). that alcohol-dependent mice have impaired performance on The ability of these compounds to partially alleviate a reversal learning task (Badanich et al, 2011) that in both symptoms of psychiatric diseases is thought to largely reflect rodents (Brown et al, 2007) and primates (Jedema et al, actions on neurons and circuits within the frontal cortex. Of 2011) requires the OFC. Withdrawal from repeated cycles of these, the orbitofrontal cortex (OFC) is known to play an chronic intermittent ethanol exposure was also shown to important role in integrating information from sensory enhance the intrinsic excitability of OFC neurons (Nimitvilai et al, 2016), suggesting that dysregulation of processes that *Correspondence: Dr JJ Woodward, Department of Neuroscience, modulate excitability may contribute to dysfunction asso- Medical University of South Carolina, 67 President Street, IOP456N, ciated with alcohol use disorders. Understanding the Charleston, SC 29425, USA, Tel: 843 792 5225, Fax: 843 792 7353, E-mail: [email protected] mechanisms that regulate OFC excitability is thus important Received 10 August 2016; revised 18 January 2017; accepted 21 January for developing therapeutic interventions designed to restore 2017; accepted article preview online 31 January 2017 normal control of neuronal activity. Ethanol and monoamine inhibition of OFC firing S Nimitvilai et al 1801 The OFC has been shown to be extensively innervated by In some experiments, GDP-β-S (500 μM) was included in the monoamine neurotransmitters (Agster et al, 2013; potassium gluconate internal solution to examine a role for Homayoun and Moghaddam, 2008; Robbins and Arnsten, G-protein activation on AP spiking. For voltage-clamp 2009) and dysregulation of monoamine function has been experiments, a cesium chloride internal solution (in mM; suggested to underlie deficits in certain OFC-dependent 120 CsCl, 10 HEPES, 2 MgCl2, 1 EGTA, 2 NaATP, 0.3 behaviors (Clarke et al, 2005; Kheramin et al, 2004; NaGTP, and 1 QX-314, adjusted to 294 mOsm, pH = 7.4) Winstanley et al, 2006). However, virtually nothing is known was used. To measure spontaneous inhibitory postsynaptic regarding the functional effects of monoamines on the currents (sIPSCs), the glutamate NMDA antagonist DL-AP5 excitability of OFC neurons or whether this modulation is (100 μM) and AMPA antagonist CNQX (10 μM) were added affected by prior exposure to ethanol. In this study, we used to the aCSF perfusion solution to isolate GABAA-mediated whole-cell patch-clamp electrophysiology to examine the currents, and the neuron was voltage clamped at − 70 mV. effects of DA, NE, and 5HT on the intrinsic excitability of Spontaneous events were detected using a template matching lateral OFC (lOFC) neurons and determined whether the algorithm and a threshold amplitude of 10 pA (AxographX actions of these modulators are altered in ethanol-dependent software, Axograph, Sydney, Australia). All whole-cell mice. The results demonstrate that monoamine neurotrans- recordings were carried out using an Axon MultiClamp mitters reduce excitability via activation of G protein- 700B amplifier (Molecular Devices, Union City, CA) coupled inwardly rectifying potassium (GIRK; Kir3 family) interfaced with an Instrutech ITC-18 analog-digital converter channels and that this regulation is lost in mice made (HEKA Instruments, Bellmore, NY). Data were acquired dependent on alcohol. with AxographX software running on a Macintosh G4 computer (Apple, Cupertino, CA). Events were filtered at 4 kHz and digitized at a sampling rate of 10 kHz. Full details MATERIALS AND METHODS can be found in Supplementary Materials. Animals Male C57BL/6J mice were obtained from Jackson Labora- Drugs tories (Bar Harbor, ME) (https://www.jax.org/strain/00064) Dopamine hydrochloride was purchased from Alfa Aesar at 9 weeks of age. They were group-housed (4/cage) and (Ward Hill, MA). Serotonin hydrochloride, α-methyl-5HT, allowed to acclimatize to the colony room for at least 1 week SKF81297, quinpirole hydrochloride, sulpiride, SCH23390, in a temperature- and humidity-controlled AAALAC- WAY100635, yohimbine, picrotoxin, naftopidil hydrochlor- approved facility. Animals were maintained on a 12 h light/ ide, nomifensine, DL-AP5, and CNQX were purchased from dark cycle with lights off at 0900 h and had ad libitum access Tocris Biosciences (Minneapolis, MN). ML297 was pur- to food and water. All animals were treated in strict chased from Abcam (Cambridge, MA). GDP-β-S was accordance with the NIH Guide for the Care and Use of purchased from Santa Cruz Biotechnology (Dallas, TX). Laboratory Animals and all experimental methods were Norepinephrine, strychnine, barium, and all reagents used to approved by the Medical University of South Carolina’s prepare aCSF, sucrose-containing and internal pipette institutional animal care and use committee. solution were purchased from Sigma (St Louis, MO). Chronic IntermittentEthanolExposure Western Blotting After acclimatization, mice were exposed to four cycles of OFC tissue punches were collected from control and CIE- chronic intermittent ethanol (CIE group) vapor or air treated mice at 3 days following the last exposure and (control) in inhalation chambers as previously described western blotting was performed according to standard (Badanich et al, 2011; Nimitvilai et al, 2016). Briefly, each immunoblotting procedures described previously (McGuier cycle consisted of daily exposure to ethanol vapor for 16 h et al, 2015). Full details can be found in Supplementary followed by 8 h of abstinence