Activation of GABAB receptors in central amygdala attenuates activity of PKCδ + neurons and suppresses punishment-resistant alcohol self-administration in rats
Esi DomiLi XuSanne ToivainenJoost WiskerkeAndrea CoppolaLovisa HolmEric AugierMichele PetrellaMarkus Heilig
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Abstract Alcohol use despite negative consequences is a core phenomenon of alcohol addiction. We recently used alcohol self-administration that is resistant to footshock punishment as a model of this behavior, and found that activity of PKCδ + GABAergic neurons in the central amygdala (CeA) is a determinant of individual susceptibility for punishment resistance. In the present study, we examined whether activation of GABA B receptors in CeA can attenuate the activity of PKCδ + neurons in this region, and whether this will result in suppression of punishment- resistant alcohol self-administration in the minority of rats that show this behavior. Systemic administration of the clinically approved GABA B agonist baclofen (1 and 3 mg/kg) dose- dependently reduced punishment-resistant alcohol self-administration. Bilateral microinjections of baclofen into CeA (64 ng in 0.3 µl/side) reduced the activity of PKCδ + neurons, as measured by Fos expression. This manipulation also selectively suppressed punished alcohol self-administration in punishment-resistant rats. Expression analysis indicated that virtually all CeA PKCδ + neurons express the GABA B receptor. Using in vitro electrophysiology, we found that baclofen induced hyperpolarization of CeA neurons, reducing their firing rate in response to depolarizing current injections. Together, our findings provide a potential mechanism that contributes to the clinical efficacy of baclofen in alcohol addiction. Therapeutic use of baclofen itself is limited by problems of tolerance and need for dose escalation. Our findings support a mechanistic rationale for developing novel, improved alcohol addiction medications that target GABA B receptors, and that lack these limitations, such as e.g., GABA B positive allosteric modulators (PAM:s).Keywords:
GABAB receptor
Punishment (psychology)
Self-administration
Abstract Alcohol is consumed orally by humans, and oral self‐administration has been successfully modeled in laboratory animals. Over the last several years, attempts have been made to develop a procedure for the reliable intravenous (IV) self‐administration of alcohol in rodents. IV self‐administration would provide a better tool for investigating neurobiological mechanisms of alcohol reinforcement and dependence because confounding factors associated with oral self‐administration, such as variations in orosensory sensitivity to alcohol and/or its absorption, are avoided. A review of the literature shows that rats, mice and non‐human primates can initiate and maintain IV self‐administration of alcohol. However, there are 50‐ to 100‐fold interspecies differences in the reported alcohol infusion doses required. Most surprising is that the infusion dose (1–2 mg/kg) that reliably maintains IV alcohol self‐administration in rats results in total alcohol intakes of only 20–25 mg/kg/hour, which are unlikely to have significant pharmacological effects. The evidence to support IV self‐administration of such low doses of alcohol in rats as well as the potential biological mechanisms underlying such self‐administration are discussed. The minute amounts of alcohol shown to reliably maintain IV self‐administration behavior in rats challenge the relationship between their blood alcohol levels and the rewarding and reinforcing effects of alcohol.
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Punishment (psychology)
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GABA B receptors mediate slow synaptic inhibition in the nervous system. In transfected cells, functional GABA B receptors are usually only observed after coexpression of GABA B(1) and GABA B(2) subunits, which established the concept of heteromerization for G-protein-coupled receptors. In the heteromeric receptor, GABA B(1) is responsible for binding of GABA, whereas GABA B(2) is necessary for surface trafficking and G-protein coupling. Consistent with these in vitro observations, the GABA B(1) subunit is also essential for all GABA B signaling in vivo . Mice lacking the GABA B(1) subunit do not exhibit detectable electrophysiological, biochemical, or behavioral responses to GABA B agonists. However, GABA B(1) exhibits a broader cellular expression pattern than GABA B(2) , suggesting that GABA B(1) could be functional in the absence of GABA B(2) . We now generated GABA B(2) -deficient mice to analyze whether GABA B(1) has the potential to signal without GABA B(2) in neurons. We show that GABA B(2) -/- mice suffer from spontaneous seizures, hyperalgesia, hyperlocomotor activity, and severe memory impairment, analogous to GABA B(1) -/- mice. This clearly demonstrates that the lack of heteromeric GABA B(1,2) receptors underlies these phenotypes. To our surprise and in contrast to GABA B(1) -/- mice, we still detect atypical electrophysiological GABA B responses in hippocampal slices of GABA B(2) -/- mice. Furthermore, in the absence of GABA B(2) , the GABA B(1) protein relocates from distal neuronal sites to the soma and proximal dendrites. Our data suggest that association of GABA B(2) with GABA B(1) is essential for receptor localization in distal processes but is not absolutely necessary for signaling. It is therefore possible that functional GABA B receptors exist in neurons that naturally lack GABA B(2) subunits.
GABAB receptor
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Anxiety and depression disorders represent common, serious and growing health problems world-wide. The neurobiological basis of anxiety and depression, however, remains poorly understood. Further, there is a clear need for the development of better treatments for these disorders. Emerging data with genetic and pharmacological tools supports a role for GABAB receptors in both anxiety and depression. GABAB receptors are metabotropic GABA receptors that are comprised of two subunits, GABAB1 and GABAB2, which form heterodimers. The GABAB(1) gene is transcribed into two predominate isoforms, GABAB(1a) and GABAB(1b) which differ in sequence primarily by the inclusion of a pair of sushi domains (or short consensus repeats) in the GABAB(1a) N-terminus. Both isoforms heterodimerize with GABAB2 subunits to form functional receptors. The two GABAB(1) isoforms and the GABAB(2) subunit constitute the majority of the molecular diversity of the GABAB receptor. However, in the absence of any isoform-selective ligands for research, the behavioural function of mammalian GABAB1 receptor isoforms has been inscrutable. Recently mice deficient in GABAB(1a) and GABAB(1b) isoforms were generated. Aspects of anxiety- and depression-related behaviour may be modelled in mice, by using traditional animal models, and by examining specific biological and behavioural components of the human symptomatology, or ‘endophenotypes’. A preliminary aim of this thesis was to determine the utility of GABAB(1) isoform-deficient mice for the dissection of GABAB(1a) and GABAB(1b) isoform-mediated behaviour. The main aim of this thesis was to test the hypothesis, using a combination of traditional and endophenotype murine models, that GABAB(1) receptor isoforms play an important role in the mediation and anxiety and depression-related behaviour.
GABAB(1) Isoforms in GABAB Receptor Function:
Preparatory work in this thesis examined the influence of genetic background on GABAB receptor-mediated responses. Genetic background, in the form of different mouse strains, had a strong, differential effect on the classic responses to GABAB receptor activation; hypothermia and ataxia. This underlined the necessity of including multiple experimental endpoints in the examination of GABAB receptor function in subsequent work with GABAB(1) isoform-deficient mice. Importantly, this study also demonstrated that the BALB/c mouse strain was an appropriate genetic background for carrying the GABAB(1) isoform mutations.
Initial studies with GABAB(1) isoform-deficient mice demonstrated that they were free of gross sensory-motor deficits that may preclude their application in behavioural tasks. Furthermore, GABAB(1a) and GABAB(1b) diverged in their influences on locomotor responses to novelty and circadian activity, although the GABAB receptor agonists baclofen or γ-hydroxybutyrate (GHB) were not specific for either isoform and were unable to discriminate these differences. These findings demonstrated that the GABAB(1) isoforms had differential influences on behaviour. Together these studies demonstrated that the GABAB(1a)-/- and GABAB(1b)-/- mice were applicable for testing the hypothesis that the GABAB(1) isoforms were differentially implicated in anxiety and depression related behaviour.
GABAB(1) Isoforms in Endophenotypes of Anxiety and Depression:
Deletion of GABAB(1a) and GABAB(1b) isoforms had profound, differential impacts on the acquisition (GABAB(1a)) and extinction (GABAB(1b)) of aversive memories, as determined in a conditioned taste aversion paradigm. These effects, however, were not accompanied by differences in innate anxiety, as assessed in a comprehensive test battery of unconditioned anxiety tests, including autonomic (stress-induced hyperthermia), active (marble burying) and passive exploratory avoidance (staircase, light-dark box, elevated plus maze, elevated zero maze) behavioural readouts. There was no evidence for a specific influence of either isoform in these tests. This indicated that the GABAB(1) isoforms themselves did not have a defining role in innate anxiety.
GABAB(1a)-/- and GABAB(1b)-/- mice diverged in their cognitive phenotypes. GABAB(1a)-/- mice were impaired in tasks of working spatial and recognition memory, but not in passive avoidance. GABAB(1b)-/- mice were also impaired, to a lesser degree, in a working spatial memory task, but showed preservation of working recognition memory and passive avoidance. Long term recognition memory, however, was also impaired in these mice.
The GABAB(1a) isoform was specifically implicated in depression-related behaviour, as indicated by reduced immobility in a classic test of antidepressant-like behaviour – the forced swim test. This was most probably mediated via the striking interactions of the GABAB(1a) isoform with the serotonergic system, as illustrated in particular by the profound desensitisation of presynaptic 5-HT1A receptors in GABAB(1a)-/- mice. A lack of effect on 5-HT1A receptor expression in GABAB(1a)-/- mice, as indicated by normal 5-HT1A autoradiography densities, suggested an intracellular mechanism for this desensitisation.
Together these studies demonstrated that the GABAB(1) isoforms are functionally important variants of the GABAB receptor, with specific relevance in depression and to aversive learning and memory processes that underlie cognitive symptoms in anxiety disorders.
GABAB receptor
Baclofen
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GABAB receptor
Baclofen
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Metabotropic GABAB receptors predominantly function as heterodimers of GABAB(1) and GABAB(2) subunits, but GABAB(1) can also form functional receptors in the absence of GABAB(2). Mice lacking the GABAB(1) subunit have altered behavioural responses in tests for anxiety and depression. In these studies, we investigated anxiety and depression in GABAB(2)-deficient mice. We compared the effects directly with that of genetic deletion of the GABAB(1) receptor subunit. Both GABAB(1) and GABAB(2)-deficient mice were found to be more anxious than wild type in the light–dark box paradigm. In contrast, these mice exhibited an antidepressant-like behaviour in the forced swim test. Taken together, these data suggest that heterodimeric GABAB(1,2) receptors are required for the normal regulation of emotional behaviour.
GABAB receptor
Baclofen
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Self-administration
Alcohol Dependence
GABAB receptor
Allosteric modulator
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Preface.- Foreword.- History of the amygdala.- Formation of the amygdala.- Amygdala and Limbic system.- Functions of the amygdala.- Dissection of the amygdala.- Morphology of the amygdala.- Connections of the amygdala.- Projections from and toward amygdala.- The relationships of the amygdala.- The BST (bed nucleus of the stria terminalis).- The extended amygdala.- Vascularization of the amygdala.- Consequences of the ablation.- Conclusions and prospects.- Bibliography.- Index.
Extended amygdala
Limbic system
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GABAB receptor
Baclofen
GABAA-rho receptor
Neurotransmitter receptor
GABA receptor
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