N motivation and reward-seeking whereas more medial ORX neurons (e.g., DMH and PF) are more involvedEur J Neurosci. Author manuscript; available in PMC 2016 March 08.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptMoorman et al.Pagein arousal and stress (Estabrooke et al., 2001; Harris Aston-Jones, 2006). This topographic dichotomy in ORX neuron function has been demonstrated for multiple drugs of abuse and natural rewards (Harris et al., 2005; Harris et al., 2007), as well as for alcoholic beer seeking (Hamlin et al., 2007). However, the specific association between regional ORX neuron activation and alcohol seeking has not been completely described. A focus on activities of ORX neurons is also warranted by the fact that ORX neurons corelease multiple neurotransmitters and peptides including ORX, dynorphin and glutamate, among others (Chou et al., 2001; Schone Burdakov, 2012; Muschamp et al., 2014). Behaviors that activate ORX neurons such as alcohol seeking (Hamlin et al., 2007; Dayas et al., 2008; Kallupi et al., 2010; Millan et al., 2010), likely simultaneously release these cotransmitters indicating that a focus on ORX release itself (via e.g., pharmacology alone) may only reveal part of the picture. Despite this need, there have been a limited number of studies of ORX neuron activity during alcohol seeking, and these have focused on one behavioral paradigm (context-induced reinstatement or renewal) (Hamlin et al., 2007; Dayas et al., 2008). We used Fos immunohistochemistry to measure activities of ORX neurons across a range of alcohol seeking behaviors, including during context-induced (ABA renewal) or cue-induced reinstatement of extinguished responding for alcohol, and also during a test of alcohol-free home cage alcohol seeking. We found that ORX neurons were activated in all three tests, and that the relationship between ORX neuron activation and alcohol seeking differed based on hypothalamic subregion and type of test. These results reveal that ORX neural activity varies substantially with different reinstatement paradigms, implying subtle topographically specific roles for ORX neurons in different aspects of alcohol seeking.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptSubjects ProceduresMaterials and MethodsMale Sprague-Dawley rats (n = 48; initial weight approximately 300?50 g; Charles River, Wilmington, MA) were single-housed under a reversed 12-h light/dark cycle (lights off at 6 a.m.) and had ad libitum access to food and water. Animals were housed in a temperatureand humidity-controlled BL-8040 custom synthesis animal facility at MUSC (AAALAC-accredited). All procedures were approved by the Medical University of South Carolina’s Institutional Animal Care and Use Committee and conducted according to specifications of the NIH as outlined in the Guide for the Care and Use of Laboratory Animals.Animals were trained to drink 20 EtOH (95 EtOH (AAPER, Shelbyville, KY) and filtered water) using the intermittent access (IA) paradigm (Wise, 1975; Simms et al., 2008; Moorman Aston-Jones, 2009). In this BL-8040 site procedure, rats received either 20 EtOH or water for 24 h on alternating days for 2 weeks in home cages with ad lib access to food and water. After IA access to develop alcohol drinking, animals were tested in one of the three following paradigms (Experiments 1?).Eur J Neurosci. Author manuscript; available in PMC 2016 March 08.Moorman et al.PageContext-induced reinstatement–In Experiment 1, 16 rats.N motivation and reward-seeking whereas more medial ORX neurons (e.g., DMH and PF) are more involvedEur J Neurosci. Author manuscript; available in PMC 2016 March 08.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptMoorman et al.Pagein arousal and stress (Estabrooke et al., 2001; Harris Aston-Jones, 2006). This topographic dichotomy in ORX neuron function has been demonstrated for multiple drugs of abuse and natural rewards (Harris et al., 2005; Harris et al., 2007), as well as for alcoholic beer seeking (Hamlin et al., 2007). However, the specific association between regional ORX neuron activation and alcohol seeking has not been completely described. A focus on activities of ORX neurons is also warranted by the fact that ORX neurons corelease multiple neurotransmitters and peptides including ORX, dynorphin and glutamate, among others (Chou et al., 2001; Schone Burdakov, 2012; Muschamp et al., 2014). Behaviors that activate ORX neurons such as alcohol seeking (Hamlin et al., 2007; Dayas et al., 2008; Kallupi et al., 2010; Millan et al., 2010), likely simultaneously release these cotransmitters indicating that a focus on ORX release itself (via e.g., pharmacology alone) may only reveal part of the picture. Despite this need, there have been a limited number of studies of ORX neuron activity during alcohol seeking, and these have focused on one behavioral paradigm (context-induced reinstatement or renewal) (Hamlin et al., 2007; Dayas et al., 2008). We used Fos immunohistochemistry to measure activities of ORX neurons across a range of alcohol seeking behaviors, including during context-induced (ABA renewal) or cue-induced reinstatement of extinguished responding for alcohol, and also during a test of alcohol-free home cage alcohol seeking. We found that ORX neurons were activated in all three tests, and that the relationship between ORX neuron activation and alcohol seeking differed based on hypothalamic subregion and type of test. These results reveal that ORX neural activity varies substantially with different reinstatement paradigms, implying subtle topographically specific roles for ORX neurons in different aspects of alcohol seeking.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptSubjects ProceduresMaterials and MethodsMale Sprague-Dawley rats (n = 48; initial weight approximately 300?50 g; Charles River, Wilmington, MA) were single-housed under a reversed 12-h light/dark cycle (lights off at 6 a.m.) and had ad libitum access to food and water. Animals were housed in a temperatureand humidity-controlled animal facility at MUSC (AAALAC-accredited). All procedures were approved by the Medical University of South Carolina’s Institutional Animal Care and Use Committee and conducted according to specifications of the NIH as outlined in the Guide for the Care and Use of Laboratory Animals.Animals were trained to drink 20 EtOH (95 EtOH (AAPER, Shelbyville, KY) and filtered water) using the intermittent access (IA) paradigm (Wise, 1975; Simms et al., 2008; Moorman Aston-Jones, 2009). In this procedure, rats received either 20 EtOH or water for 24 h on alternating days for 2 weeks in home cages with ad lib access to food and water. After IA access to develop alcohol drinking, animals were tested in one of the three following paradigms (Experiments 1?).Eur J Neurosci. Author manuscript; available in PMC 2016 March 08.Moorman et al.PageContext-induced reinstatement–In Experiment 1, 16 rats.
