- Email: [email protected]
Intrinsic properties of central amygdala dynorphin neurons
Abstracts / Alcohol 60 (2017) 203e243
240
epidemic, where the rate of heroin-related overdose deaths has quadrupled from 2002-2013. There are currently a large number of opioid drugs that are misused and can lead to addiction, and evidence suggests that 80% of new heroin abusers previously abused prescription drugs. The central noradrenergic system plays an extensive role in modulating behaviors associated with addiction and anxiety. There is significant evidence to suggest that norepinephrine (NE) plays a role in both the rewarding properties and the manifestation of withdrawal from opiate use, however there is not much known regarding changes in synaptic physiology or plasticity on NE neurons in opioid dependence. Here we explore these alterations by using a rapid dependence model. To engender opioid dependence, mice were injected once daily with 10 mg/kg morphine s.c. followed 2 h later by 1 mg/kg naloxone s.c. for a total of 3 days. On the fourth day, when the mice were in protracted withdrawal, we examined the electrophysiological properties of LC neurons and found an increase in spontaneous post synaptic currents (sEPSCS) and a decrease in the decay kinetics. The initial implication from this finding is that morphine dependence induces a shift to GluA2 lacking calcium permeable AMPA receptors (CP-AMPARs). We are actively investigating these alterations in both LC and NTS neurons in male and female mice. Additionally, we will investigate sIPSCS in these neurons as well as E/I ratios to gather a thorough understanding of how morphine dependence alters synaptic tone in NE circuits. These studies will provide critical understanding of enduring neuroadaptations of this highly relevant circuitry.
(Dyn+), the endogenous ligand of the kappa opioid receptor. To genetically identify dynorphinergic (Dyn) neurons, we crossed a Cre-dependent tdTomato reporter mouse to a mouse expressing Cre recombinase under the same promoter as preprodynorphin. In this model, only dynorphinergic cells express tdTomato, allowing complete visualization of dynorphinergic circuitry throughout the brain and visually-guided, targeted whole-cell recordings in amygdala slices. We report distinct patterns of c-fos expression in these neurons following stress, pain, and alcohol exposure. We also document the intrinsic electrophysiological properties of these neurons and the strengths of inputs they receive from the parabrachial nucleus and the basolateral amygdala. Furthermore, the morphology of CeA Dyn+ neurons is defined by filling the cells with Neurobiotin. To determine the long-range connectivity of Dyn+ CeA neurons, we utilized cell-type selective expression of reporter viruses to identify these molecularly-defined projections throughout the brain. Together these data provide a base knowledge for further cell-type selective manipulation and observation in vivo. Understanding the mechanisms by which the dynorphin/kappa opioid system regulates emotional processing in the context of stress, chronic pain, and alcohol abuse will provide valuable insight into potential therapeutic targets for these neurological and neuropsychiatric disorders.
P157 INHIBITION OF N-ACYLETHANOLAMINE ACID AMIDASE REVERSES EFFECTS OF CHRONIC BINGE ETHANOL DRINKING ON VENTRAL TEGMENTAL AREA DOPAMINE NEURON FIRING
Todd B. Nentwig, Colleen E. McGonigle, Diane E. Wilson, Erin M. Rhinehart, Judith E. Grisel. Bucknell University, Lewisburg, PA, USA
Regina Mangieri, Heather Aziz, Daniele Piomelli, Richard Morrisett. The University of Texas at Austin, Austin, TX, USA; Italian Institute of Technology, Genova, Italy Peroxisome proliferator-activated receptor a (PPARa) agonists reduce ethanol consumption in rodent drinking models, and decrease ventral tegmental area (VTA) dopamine (DA) neuron firing rate e suggesting a possible mechanism by which PPARa activation could regulate drinking. Signaling by endogenous PPARa agonists (oleoylethanolamide, palmitoylethanolamide) can be enhanced by blocking their hydrolysis with an inhibitor of N-acylethanolamine acid amidase (NAAA), such as the compound ARN726. The aims of this project were to determine if inhibiting NAAA in vitro reduces the spontaneous firing rate of VTA DA neurons or blocks acute potentiation of firing by ethanol, and if chronic binge ethanol experience (six weeks drinking-in-the-dark, DID) alters either of these phenomena. Brain slices containing the VTA were prepared from male Pitx3-egfp mice (which express green fluorescent protein (GFP) in midbrain DA neurons) twenty-four hours after the last DID session, and voltage discharges of GFP+ neurons were recorded in cell-attached mode. Overall, application of ARN726 decreased firing rate relative to vehicle, but this effect was statistically significant only in the ethanol-DID group and not in water-DID controls. Similarly, pretreatment with ARN726 selectively attenuated ethanol-potentiation of firing for the ethanol-DID group alone. These results suggest that pharmacological inhibition of NAAA may be a useful strategy for normalizing VTA DA neuron activity after chronic ethanol experience. Funded AA013517. P158 INTRINSIC NEURONS
PROPERTIES
OF
CENTRAL
AMYGDALA
DYNORPHIN
Jordan G. McCall, Bryan A. Copits, Vijay K. Samineni, Robert W. Gereau, IV. Washington University in St. Louis, St. Louis, MO, USA; Louis College of Pharmacy, St. Louis, MO, USA The central amygdala (CeA) is a critical anatomical substrate for emotional regulation in response to stress, pain, and alcohol-related behaviors. While many cell-types have been identified in the CeA, much less is understood about the unique properties of these molecularly-defined neurons. We focus on a subset of neurons in the CeA expressing the neuropeptide dynorphin
P159 BETA-ENDORPHIN MODULATES BINGE-LIKE ETHANOL DRINKING IN MICE
Binge alcohol drinking is a widespread problem in the Unites States and has been linked to an increased risk for alcohol-related complications including the development of alcohol use disorders and alcoholism (King et al, 2011). Therefore, understanding the neural antecedents of binge drinking could elucidate underlying mechanisms and facilitate identification of potential therapeutic targets to prevent the transition from binge drinking to alcoholism. Beta-endorphin (B-E), an endogenous opioid peptide, has long been documented as a risk factor for alcoholism due in part to low basal plasma levels and an increased B-E response to alcohol observed in at-risk subjects (Gianoulakis et al, 1996). Our lab has previously demonstrated that transgenic mice lacking B-E (KO) exhibit decreased ethanol (EtOH) consumption relative to C57BL/6J controls (B6), suggesting B-E might modulate EtOH reward. We sought to extend these findings to binge drinking using a modified “drinking in the dark” paradigm (Rhodes et al, 2005). Across 4 consecutive days, 3-hours into the dark cycle, B6 and KO mice were given 2-hour access to 20% EtOH in their homecage. Access was extended to a 4-hour binge test on the 4th day, after which mice were immediately sacrificed to collect trunk blood and brains for assessing blood alcohol content, corticosterone, and gene expression of corticotropin-releasing hormone. Preliminary results indicate sex specific effects of B-E. Males KOs showed decreased binge-like EtOH consumption compared to female KOs and male B6s. These results suggest that a lack of B-E blunts excessive binge-like EtOH consumption in males, but not females.Supported R15 AA022506. P160 RESTORATION OF KV7.2/7.3 CHANNEL SIGNALING REDUCES DEPENDENCE-INDUCED ESCALATION OF ETHANOL CONSUMPTION Jennifer A. Rinker, Patrick J. Mulholland. Medical University of South Carolina, Charleston, SC, USA Alcohol (ethanol) dependence is a chronic relapsing brain disorder characterized by uncontrollable, heavy alcohol consumption. Emerging evidence suggests that the expression and function of several potassium (K+) channels, including Kv7 channels, are dysregulated in the post-dependent state. Kv7 channels regulate intrinsic excitability and dependence-induced changes may reduce their capacity to regulate neuronal firing contributing to hyperexcitability during periods of ethanol withdrawal. Additionally, chronic ethanol consumption alters Kv7.2 channel trafficking, and systemic administration of FDA approved Kv7 channel opener, retigabine, reduces ethanol consumption in nondependent, high-drinking rats and
240
epidemic, where the rate of heroin-related overdose deaths has quadrupled from 2002-2013. There are currently a large number of opioid drugs that are misused and can lead to addiction, and evidence suggests that 80% of new heroin abusers previously abused prescription drugs. The central noradrenergic system plays an extensive role in modulating behaviors associated with addiction and anxiety. There is significant evidence to suggest that norepinephrine (NE) plays a role in both the rewarding properties and the manifestation of withdrawal from opiate use, however there is not much known regarding changes in synaptic physiology or plasticity on NE neurons in opioid dependence. Here we explore these alterations by using a rapid dependence model. To engender opioid dependence, mice were injected once daily with 10 mg/kg morphine s.c. followed 2 h later by 1 mg/kg naloxone s.c. for a total of 3 days. On the fourth day, when the mice were in protracted withdrawal, we examined the electrophysiological properties of LC neurons and found an increase in spontaneous post synaptic currents (sEPSCS) and a decrease in the decay kinetics. The initial implication from this finding is that morphine dependence induces a shift to GluA2 lacking calcium permeable AMPA receptors (CP-AMPARs). We are actively investigating these alterations in both LC and NTS neurons in male and female mice. Additionally, we will investigate sIPSCS in these neurons as well as E/I ratios to gather a thorough understanding of how morphine dependence alters synaptic tone in NE circuits. These studies will provide critical understanding of enduring neuroadaptations of this highly relevant circuitry.
(Dyn+), the endogenous ligand of the kappa opioid receptor. To genetically identify dynorphinergic (Dyn) neurons, we crossed a Cre-dependent tdTomato reporter mouse to a mouse expressing Cre recombinase under the same promoter as preprodynorphin. In this model, only dynorphinergic cells express tdTomato, allowing complete visualization of dynorphinergic circuitry throughout the brain and visually-guided, targeted whole-cell recordings in amygdala slices. We report distinct patterns of c-fos expression in these neurons following stress, pain, and alcohol exposure. We also document the intrinsic electrophysiological properties of these neurons and the strengths of inputs they receive from the parabrachial nucleus and the basolateral amygdala. Furthermore, the morphology of CeA Dyn+ neurons is defined by filling the cells with Neurobiotin. To determine the long-range connectivity of Dyn+ CeA neurons, we utilized cell-type selective expression of reporter viruses to identify these molecularly-defined projections throughout the brain. Together these data provide a base knowledge for further cell-type selective manipulation and observation in vivo. Understanding the mechanisms by which the dynorphin/kappa opioid system regulates emotional processing in the context of stress, chronic pain, and alcohol abuse will provide valuable insight into potential therapeutic targets for these neurological and neuropsychiatric disorders.
P157 INHIBITION OF N-ACYLETHANOLAMINE ACID AMIDASE REVERSES EFFECTS OF CHRONIC BINGE ETHANOL DRINKING ON VENTRAL TEGMENTAL AREA DOPAMINE NEURON FIRING
Todd B. Nentwig, Colleen E. McGonigle, Diane E. Wilson, Erin M. Rhinehart, Judith E. Grisel. Bucknell University, Lewisburg, PA, USA
Regina Mangieri, Heather Aziz, Daniele Piomelli, Richard Morrisett. The University of Texas at Austin, Austin, TX, USA; Italian Institute of Technology, Genova, Italy Peroxisome proliferator-activated receptor a (PPARa) agonists reduce ethanol consumption in rodent drinking models, and decrease ventral tegmental area (VTA) dopamine (DA) neuron firing rate e suggesting a possible mechanism by which PPARa activation could regulate drinking. Signaling by endogenous PPARa agonists (oleoylethanolamide, palmitoylethanolamide) can be enhanced by blocking their hydrolysis with an inhibitor of N-acylethanolamine acid amidase (NAAA), such as the compound ARN726. The aims of this project were to determine if inhibiting NAAA in vitro reduces the spontaneous firing rate of VTA DA neurons or blocks acute potentiation of firing by ethanol, and if chronic binge ethanol experience (six weeks drinking-in-the-dark, DID) alters either of these phenomena. Brain slices containing the VTA were prepared from male Pitx3-egfp mice (which express green fluorescent protein (GFP) in midbrain DA neurons) twenty-four hours after the last DID session, and voltage discharges of GFP+ neurons were recorded in cell-attached mode. Overall, application of ARN726 decreased firing rate relative to vehicle, but this effect was statistically significant only in the ethanol-DID group and not in water-DID controls. Similarly, pretreatment with ARN726 selectively attenuated ethanol-potentiation of firing for the ethanol-DID group alone. These results suggest that pharmacological inhibition of NAAA may be a useful strategy for normalizing VTA DA neuron activity after chronic ethanol experience. Funded AA013517. P158 INTRINSIC NEURONS
PROPERTIES
OF
CENTRAL
AMYGDALA
DYNORPHIN
Jordan G. McCall, Bryan A. Copits, Vijay K. Samineni, Robert W. Gereau, IV. Washington University in St. Louis, St. Louis, MO, USA; Louis College of Pharmacy, St. Louis, MO, USA The central amygdala (CeA) is a critical anatomical substrate for emotional regulation in response to stress, pain, and alcohol-related behaviors. While many cell-types have been identified in the CeA, much less is understood about the unique properties of these molecularly-defined neurons. We focus on a subset of neurons in the CeA expressing the neuropeptide dynorphin
P159 BETA-ENDORPHIN MODULATES BINGE-LIKE ETHANOL DRINKING IN MICE
Binge alcohol drinking is a widespread problem in the Unites States and has been linked to an increased risk for alcohol-related complications including the development of alcohol use disorders and alcoholism (King et al, 2011). Therefore, understanding the neural antecedents of binge drinking could elucidate underlying mechanisms and facilitate identification of potential therapeutic targets to prevent the transition from binge drinking to alcoholism. Beta-endorphin (B-E), an endogenous opioid peptide, has long been documented as a risk factor for alcoholism due in part to low basal plasma levels and an increased B-E response to alcohol observed in at-risk subjects (Gianoulakis et al, 1996). Our lab has previously demonstrated that transgenic mice lacking B-E (KO) exhibit decreased ethanol (EtOH) consumption relative to C57BL/6J controls (B6), suggesting B-E might modulate EtOH reward. We sought to extend these findings to binge drinking using a modified “drinking in the dark” paradigm (Rhodes et al, 2005). Across 4 consecutive days, 3-hours into the dark cycle, B6 and KO mice were given 2-hour access to 20% EtOH in their homecage. Access was extended to a 4-hour binge test on the 4th day, after which mice were immediately sacrificed to collect trunk blood and brains for assessing blood alcohol content, corticosterone, and gene expression of corticotropin-releasing hormone. Preliminary results indicate sex specific effects of B-E. Males KOs showed decreased binge-like EtOH consumption compared to female KOs and male B6s. These results suggest that a lack of B-E blunts excessive binge-like EtOH consumption in males, but not females.Supported R15 AA022506. P160 RESTORATION OF KV7.2/7.3 CHANNEL SIGNALING REDUCES DEPENDENCE-INDUCED ESCALATION OF ETHANOL CONSUMPTION Jennifer A. Rinker, Patrick J. Mulholland. Medical University of South Carolina, Charleston, SC, USA Alcohol (ethanol) dependence is a chronic relapsing brain disorder characterized by uncontrollable, heavy alcohol consumption. Emerging evidence suggests that the expression and function of several potassium (K+) channels, including Kv7 channels, are dysregulated in the post-dependent state. Kv7 channels regulate intrinsic excitability and dependence-induced changes may reduce their capacity to regulate neuronal firing contributing to hyperexcitability during periods of ethanol withdrawal. Additionally, chronic ethanol consumption alters Kv7.2 channel trafficking, and systemic administration of FDA approved Kv7 channel opener, retigabine, reduces ethanol consumption in nondependent, high-drinking rats and