Maeng-Hee Kang-Park

Presynaptic δ Opioid Receptors Regulate Ethanol Actions in Central Amygdala

Endogenous opioid systems are implicated in the reinforcing effects of ethanol consumption. For example, δ opioid receptor (DOR) knockout (KO) mice show greater ethanol consumption than wild-type (WT) mice (Roberts et al., 2001). To explore the neurobiological correlates underlying these behaviors, we examined effects of acute ethanol application in brain slices from DOR KO mice using whole-cell patch recording techniques. We examined the central nucleus of amygdala (CeA) because the CeA is implicated in alcohol reinforcement (Koob et al., 1998). We found that the acute ethanol effects on GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were greater in DOR KO mice than in WT mice. Ethanol increased the frequency of miniature IPSCs (mIPSCs) significantly more in DOR KO mice than in WT mice. In CeA of WT mice, application of ICI 174864 [[allyl]2-Tyr-α-amino-isobutyric acid (Aib)-Aib-Phe-Leu-OH], a DOR inverse agonist, augmented ethanol actions on mIPSC frequency comparable with ethanol effects seen in DOR KO mice. Superfusion of the selective DOR agonist d-Pen2,d-Pen5-enkephalin decreased the mean frequency of mIPSCs; this effect was reversed by the DOR antagonist naltrindole. These findings suggest that endogenous opioids may reduce ethanol actions on IPSCs of CeA neurons in WT mice through DOR-mediated inhibition of GABA release and that the increased ethanol effect on IPSCs in CeA of DOR KO mice could be, at least in part, due to absence of DOR-mediated inhibition of GABA release. This result supports the hypothesis that endogenous opioid peptides modulate the ethanol-induced augmentation of GABAA receptor-dependent circuitry in CeA (Roberto et al., 2003).

κ-Opioid Receptors in the Central Amygdala Regulate Ethanol Actions at Presynaptic GABAergic Sites

Human and animal studies indicate that κ-opioid receptors (KORs) are involved in ethanol drinking and dependence (Xuei et al., 2006; Walker and Koob, 2008; Walker et al., 2011). Using in vitro single-cell recording techniques in mouse brain slices, we examined the physiologic effects of KOR activation in the central amygdala (CeA) on GABAergic neurotransmission and its interaction with acute ethanol. A selective KOR agonist (U69593, 1 μM) diminished evoked GABAergic inhibitory postsynaptic currents (IPSCs) by 18% (n = 10), whereas blockade of KORs with a selective antagonist (nor-binaltorphimine, 1 μM) augmented the baseline evoked GABAergic IPSCs by 14% (P < 0.01; n = 34), suggesting that the KOR system contributes to tonic inhibition of GABAergic neurotransmission in the CeA. In addition, the enhancement by acute ethanol of GABAergic IPSC amplitudes was further augmented by pharmacologic blockade of KORs, from 14% (n = 36) to 27% (n = 26; P < 0.01), or by genetic deletion of KORs, from 14% in wild-type mice (n = 19) to 34% in KOR knockout mice (n = 13; P < 0.01). Subsequent experiments using tetrodotoxin to block activity-dependent neurotransmission suggest that KORs regulate GABA release at presynaptic sites. Our data support the idea that KORs modulate GABAergic synaptic responses and ethanol effects as one of multiple opioid system-dependent actions of ethanol in the CeA, possibly in a circuit-specific manner.

Differential actions of diazepam and zolpidem in basolateral and central amygdala nuclei

Benzodiazepines are among the most widely prescribed therapeutic agents, having anxiolytic, anticonvulsant, sedative/hypnotic, and amnestic properties (Mehta and Ticku, Brain Res. Rev. 29 (1999) 196). Recent research indicates that these disparate actions are dissociable (Nature 401 (1999) 796; Science 290 (2000) 131; Kralic et al., Neuropharmacology 43 (2002) 685). Behavioral studies indicate that the amygdala plays a critical role in the anxiolytic effect of benzodiazepines (Nagy et al., Neuropharmacology 18 (1979) 573; The amygdala: anxiety and benzodiazepines. The Amygdala: a Functional Analysis. p. 195). However, the neuronal substrates of this anxiolytic effect remain unclear. Our study characterizes the physiological response to acute application of the benzodiazepine diazepam and the non-benzodiazepine sedative zolpidem using whole cell patch recording in two discrete amygdala subnuclei. We found that acute application of diazepam enhances GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) with equal potency in the basolateral (BL) and central (Ce) amygdala subnuclei. However, zolpidem enhanced IPSCs with similar potency only in the BL, and was effective in the Ce only at high concentrations. This finding is in agreement with histochemical data regarding the localization of GABA(A) receptor isoforms in the amygdala (J. Comp. Neurol. 359 (1995) 154; Brain Res. 964 (2003) 91) and suggests that anxiolytic effects of allosteric modulators of the GABA(A) receptor may be further dissociated from their hypnotic/sedative effects.

μ-Opioid Receptors Selectively Regulate Basal Inhibitory Transmission in the Central Amygdala: Lack of Ethanol Interactions

Endogenous opioid systems are implicated in the actions of ethanol. For example, μ-opioid receptor (MOR) knockout (KO) mice self-administer less alcohol than the genetically intact counterpart wild-type (WT) mice (Roberts et al., 2000). MOR KO mice also exhibit less anxiety-like behavior than WT mice (Filliol et al., 2000). To investigate the neurobiological mechanisms underlying these behaviors, we examined the effect of ethanol in brain slices from MOR KO and WT mice using sharp-electrode and whole-cell patch recording techniques. We focused our study in the central nucleus of the amygdala (CeA) because it is implicated in alcohol drinking behavior and stress behavior. We found that the amplitudes of evoked inhibitory postsynaptic currents (IPSCs) or inhibitory postsynaptic potentials (IPSPs) were significantly greater in MOR KO mice than WT mice. In addition, the baseline frequencies of spontaneous and miniature GABAA receptor-mediated inhibitory postsynaptic currents were significantly greater in CeA neurons from MOR KO than WT mice. However, ethanol enhancements of evoked IPSP and IPSC amplitudes and the frequency of miniature IPSCs were comparable between WT and MOR KO mice. Baseline spontaneous and miniature excitatory postsynaptic currents (EPSCs) and ethanol effects on EPSCs were not significantly different between MOR KO and WT mice. Based on knowledge of CeA circuitry and projections, we hypothesize that the role of MOR- and GABA receptor-mediated mechanisms in CeA underlying reinforcing effects of ethanol operate independently, possibly through pathway-specific responses within CeA.

Interaction of CRF and kappa opioid systems on GABAergic neurotransmission in the mouse central amygdala.

The corticotropin-releasing factor (CRF) and kappa-opioid receptor (KOR) systems are both implicated in stress-related behaviors and drug dependence. Although previous studies suggest that antagonism of each system blocks aspects of experimental models of drug dependence, the possible interaction between these systems at the neuronal level has not been completely examined. We used an in vitro brain slice preparation to investigate the interaction of these two peptide systems on inhibitory neurotransmission in the central nucleus of the amygdala (CeA). Application of exogenous CRF increased the mean frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSC) by 20.2%, suggesting an increase in presynaptic GABA release. Although the pharmacological blockade of KORs by norBNI alone did not significantly affect mIPSC frequency, it significantly enhanced the effect of CRF (by 43.9%, P = 0.02). Similarly, the CRF effects in slices from KOR knockout (KO) mice (84.0% increase) were significantly greater than in wild-type (WT) mice (24.6%, P = 0.01), although there was no significant difference in baseline mIPSC frequency between slices from KOR KO and WT mice. The increase in CRF action in the presence of norBNI was abolished by a CRF-1 receptor antagonist but was unaffected by a CRF-2 receptor antagonist. We hypothesize that CRF facilitates the release of an endogenous ligand for KORs and that subsequent activation of KOR receptors modulates presynaptic effects of CRF in CeA. These results suggest that potential pharmacotherapies aimed at neurobehavioral and addictive disorders may need to involve both the KOR/dynorphin and the CRF systems in CeA.

Dopamine attenuates evoked inhibitory synaptic currents in central amygdala neurons

The central nucleus of the amygdala (CeA) plays a critical role in regulating the behavioral, autonomic and endocrine response to stress. Dopamine (DA) participates in mediating the stress response and DA release is enhanced in the CeA during stressful events. However, the electrophysiological effects of DA on CeA neurons have not yet been characterized. Therefore, the purpose of this study was to identify and characterize the effect of DA application on electrophysiological responses of CeA neurons in coronal brain sections of male Sprague–Dawley rats. We used whole‐cell patch‐clamp electrophysiological techniques to record evoked synaptic responses and to determine basic membrane properties of CeA neurons both before and after DA superfusion. DA (20–250 μm) did not significantly alter membrane conductance over the voltage range tested. However, DA significantly reduced the peak amplitude of evoked inhibitory synaptic currents in CeA neurons. Pretreatment with the D2 receptor antagonist eticlopride failed to significantly block the inhibitory effects of DA. In contrast, pretreatment with the D1 receptor antagonist SCH‐23390 significantly reduced the effects of DA on evoked inhibitory neurotransmission in these neurons. Moreover, bath superfusion of the specific D1 receptor agonist SKF‐39393, but not the D2 receptor agonist quinpirole, significantly reduced peak amplitude of evoked inhibitory synaptic events. DA reduced the frequency of miniature IPSCs without altering the amplitude, while having no effect on the amplitude of IPSCs elicited by pressure application of GABA. These results suggest that DA may modulate inhibitory synaptic transmission in CeA through D1 receptor activation primarily by a presynaptic mechanism.