Ro 15-4513 Antagonizes Alcohol-Induced Sedation in Mice Through aß¿2-type GABA(A) Receptors

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Increased Sensitivity of Mice Lacking Extrasynaptic delta-Containing GABA(A) Receptors to Histamine Receptor 3 Antagonists

Histamine/gamma-aminobutyric acid (GABA) neurons of posterior hypothalamus send wide projections to many brain areas and participate in stabilizing the wake state. Recent research has suggested that GABA released from the histamine/GABA neurons acts on extrasynaptic GABA(A) receptors and balances the excitatory effect of histamine. In the current study, we show the presence of vesicular GABA transporter mRNA in a majority of quantified hypothalamic histaminergic neurons, which suggest vesicular release of GABA. As histamine/GABA neurons form conventional synapses infrequently, it is possible that GABA released from these neurons diffuses to target areas by volume transmission and acts on extrasynaptic GABA receptors. To investigate this hypothesis, mice lacking extrasynaptic GABA(A) receptor delta subunit (Gabrd KO) were used. A pharmacological approach was employed to activate histamine/GABA neurons and induce histamine and presumably, GABA, release. Control and Gabrd KO mice were treated with histamine receptor 3 (Hrh3) inverse agonists ciproxifan and pitolisant, which block Hrh3 autoreceptors on histamine/GABA neurons and histamine-dependently promote wakefulness. Low doses of ciproxifan (1 mg/kg) and pitolisant (5 mg/kg) reduced locomotion in Gabrd KO, but not in WT mice. EEG recording showed that Gabrd KO mice were also more sensitive to the wake-promoting effect of ciproxifan (3 mg/kg) than control mice. Low frequency delta waves, associated with NREM sleep, were significantly suppressed in Gabrd KO mice compared with the WT group. Ciproxifan-induced wakefulness was blocked by histamine synthesis inhibitor alpha-fluoromethylhistidine (alpha FMH). The findings indicate that both histamine and GABA, released from histamine/GABA neurons, are involved in regulation of brain arousal states and delta-containing subunit GABA(A) receptors are involved in mediating GABA response.Peer reviewe

Effects of acute lysergic acid diethylamide on intermittent ethanol and sucrose drinking and intracranial self-stimulation in C57BL/6 mice

Background: Psychedelics, like lysergic acid diethylamide (LSD), are again being studied as potential therapies for many neuropsychiatric disorders, including addictions. At the same time, the acute effects of psychedelics on rewarding behaviours have been scarcely studied. Aims: The current study aimed to clarify if LSD decreases binge-like ethanol drinking in mice, and whether the observed acute effects on ethanol consumption are generalizable to a natural reinforcer, sucrose, and if the effects resulted from aversive or reward-attenuating effects caused by LSD. Methods: The effects of acute LSD were examined using 2-bottle choice intermittent ethanol (20%) and sucrose drinking (10%), discrete-trial current-intensity threshold method of intracranial self-stimulation and short-term feeding behaviour assay in C57BL/6 male mice. Results: The results showed that acute 0.1 mg/kg, but not 0.05 mg/kg, dose (i.p.) of LSD reduced 2-h intermittent ethanol drinking transiently without any prolonged effects. No effects were seen in intermittent 2-h sucrose drinking. The tested LSD doses had neither effect on the intracranial self-stimulation current-intensity thresholds, nor did LSD affect the threshold-lowering, or rewarding, effects of simultaneous amphetamine treatment. Furthermore, LSD had small, acute diminishing effects on 2-h food and water intake. Conclusions: Based on these results, LSD decreases binge-like ethanol drinking in mice, but only acutely. This effect is not likely to stem from reward-attenuating effects but could be in part due to reduced consummatory behaviour.Peer reviewe

Early Maternal Alcohol Consumption Alters Hippocampal DNA Methylation, Gene Expression and Volume in a Mouse Model

The adverse effects of alcohol consumption during pregnancy are known, but the molecular events that lead to the phenotypic characteristics are unclear. To unravel the molecular mechanisms, we have used a mouse model of gestational ethanol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first 8 days of gestation (GD 0.5-8.5). Early neurulation takes place by the end of this period, which is equivalent to the developmental stage early in the fourth week post-fertilization in human. During this exposure period, dynamic epigenetic reprogramming takes place and the embryo is vulnerable to the effects of environmental factors. Thus, we hypothesize that early ethanol exposure disrupts the epigenetic reprogramming of the embryo, which leads to alterations in gene regulation and life-long changes in brain structure and function. Genome-wide analysis of gene expression in the mouse hippocampus revealed altered expression of 23 genes and three miRNAs in ethanol-exposed, adolescent offspring at postnatal day (P) 28. We confirmed this result by using two other tissues, where three candidate genes are known to express actively. Interestingly, we found a similar trend of upregulated gene expression in bone marrow and main olfactory epithelium. In addition, we observed altered DNA methylation in the CpG islands upstream of the candidate genes in the hippocampus. Our MRI study revealed asymmetry of brain structures in ethanol-exposed adult offspring (P60): we detected ethanol-induced enlargement of the left hippocampus and decreased volume of the left olfactory bulb. Our study indicates that ethanol exposure in early gestation can cause changes in DNA methylation, gene expression, and brain structure of offspring. Furthermore, the results support our hypothesis of early epigenetic origin of alcohol-induced disorders: changes in gene regulation may have already taken place in embryonic stem cells and therefore can be seen in different tissue types later in life.Peer reviewe

GABA(B) receptor positive allosteric modulators with different efficacies affect neuroadaptation to and self-administration of alcohol and cocaine

Drugs of abuse induce widespread synaptic adaptations in the mesolimbic dopamine (DA) neurons. Such drug-induced neuroadaptations may constitute an initial cellular mechanism eventually leading to compulsive drug-seeking behavior. To evaluate the impact of GABA(B) receptors on addiction-related persistent neuroplasticity, we tested the ability of orthosteric agonist baclofen and two positive allosteric modulators (PAMs) of GABA(B) receptors to suppress neuroadaptations in the ventral tegmental area (VTA) and reward-related behaviors induced by ethanol and cocaine. A novel compound (S)-1-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-4-methyl-6,7,8,9-tetrahydro-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (ORM-27669) was found to be a GABA(B) PAM of low efficacy as agonist, whereas the reference compound (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) had a different allosteric profile being a more potent PAM in the calcium-based assay and an agonist, coupled with potent PAM activity, in the [S-35] GTP gamma S binding assay in rat and human recombinant receptors. Using autoradiography, the high-efficacy rac-BHFF and the low-efficacy ORM-27669 potentiated the effects of baclofen on [S-35] GTP gamma S binding with identical brain regional distribution. Treatment of mice with baclofen, rac-BHFF, or ORM-27669 failed to induce glutamate receptor neuroplasticity in the VTA DA neurons. Pretreatment with rac-BHFF at non-sedative doses effectively reversed both ethanol- and cocaine-induced plasticity and attenuated cocaine i.v. self-administration and ethanol drinking. Pretreatment with ORM-27669 only reversed ethanol-induced neuroplasticity and attenuated ethanol drinking but had no effects on cocaine-induced neuroplasticity or self-administration. These findings encourage further investigation of GABA(B) receptor PAMs with different efficacies in addiction models to develop novel treatment strategies for drug addiction.Peer reviewe

Tankyrase inhibition ameliorates lipid disorder via suppression of PGC-1 alpha PARylation in db/db mice

Objective Human TNKS, encoding tankyrase 1 (TNKS1), localizes to a susceptibility locus for obesity and type 2 diabetes mellitus (T2DM). Here, we addressed the therapeutic potential of G007-LK, a TNKS-specific inhibitor, for obesity and T2DM. Methods We administered G007-LK to diabetic db/db mice and measured the impact on body weight, abdominal adiposity, and serum metabolites. Muscle, liver, and white adipose tissues were analyzed by quantitative RT-PCR and western blotting to determine TNKS inhibition, lipolysis, beiging, adiponectin level, mitochondrial oxidative metabolism and mass, and gluconeogenesis. Protein interaction and PARylation analyses were carried out by immunoprecipitation, pull-down and in situ proximity ligation assays. Results TNKS inhibition reduced body weight gain, abdominal fat content, serum cholesterol levels, steatosis, and proteins associated with lipolysis in diabetic db/db mice. We discovered that TNKS associates with PGC-1 alpha and that TNKS inhibition attenuates PARylation of PGC-1 alpha, contributing to increased PGC-1 alpha level in WAT and muscle in db/db mice. PGC-1 alpha upregulation apparently modulated transcriptional reprogramming to increase mitochondrial mass and fatty acid oxidative metabolism in muscle, beiging of WAT, and raised circulating adiponectin level in db/db mice. This was in sharp contrast to the liver, where TNKS inhibition in db/db mice had no effect on PGC-1 alpha expression, lipid metabolism, or gluconeogenesis. Conclusion Our study unravels a novel molecular mechanism whereby pharmacological inhibition of TNKS in obesity and diabetes enhances oxidative metabolism and ameliorates lipid disorder. This happens via tissue-specific PGC-1 alpha-driven transcriptional reprogramming in muscle and WAT, without affecting liver. This highlights inhibition of TNKS as a potential pharmacotherapy for obesity and T2DM.Peer reviewe

Heterogeneous somatostatin-expressing neuron population in mouse ventral tegmental area

Publisher Copyright: © Nagaeva et al.The cellular architecture of the ventral tegmental area (VTA), the main hub of the brain reward system, remains only partially characterized. To extend the characterization to inhibitory neurons, we have identified three distinct subtypes of somatostatin (Sst)-expressing neurons in the mouse VTA. These neurons differ in their electrophysiological and morphological properties, anatomical localization, as well as mRNA expression profiles. Importantly, similar to cortical Sst-containing interneurons, most VTA Sst neurons express GABAergic inhibitory markers, but some of them also express glutamatergic excitatory markers and a subpopulation even express dopaminergic markers. Furthermore, only some of the proposed marker genes for cortical Sst neurons were expressed in the VTA Sst neurons. Physiologically, one of the VTA Sst neuron subtypes locally inhibited neighboring dopamine neurons. Overall, our results demonstrate the remarkable complexity and heterogeneity of VTA Sst neurons and suggest that these cells are multifunctional players in the midbrain reward circuitry.Peer reviewe

Increased Motor-Impairing Effects of the Neuroactive Steroid Pregnanolone in Mice with Targeted Inactivation of the GABA(A) Receptor gamma 2 Subunit in the Cerebellum

Endogenous neurosteroids and neuroactive steroids have potent and widespread actions on the brain via inhibitory GABA(A) receptors. In recombinant receptors and genetic mouse models their actions depend on the alpha, beta, and delta subunits of the receptor, especially on those that form extrasynaptic GABA(A) receptors responsible for non-synaptic (tonic) inhibition, but they also act on synaptically enriched gamma 2 subunit containing receptors and even on alpha beta binary receptors. Here we tested whether behavioral sensitivity to the neuroactive steroid agonist 5 beta-pregnan-3 alpha-ol-20-one is altered in genetically engineered mouse models that have deficient GABA(A) receptor mediated synaptic inhibition in selected neuronal populations. Mouse lines with the GABA(A) receptor gamma 2 subunit gene selectively deleted either in parvalbumin-containing cells (including cerebellar Purkinje cells), cerebellar granule cells, or just in cerebellar Purkinje cells were trained on the accelerated rotating rod and then tested for motor impairment after cumulative intraperitoneal dosing of 5 beta-pregnan-3 alpha-ol-20-one. Motor impairing effects of 5 beta-pregnan-3 alpha-ol-20-one were strongly increased in all three mouse models in which gamma 2 subunit-dependent synaptic GABA(A) responses in cerebellar neurons were genetically abolished. Furthermore, rescue of postsynaptic GABA(A) receptors in Purkinje cells normalized the effect of the steroid. Anxiolytic/explorative effects of the steroid in elevated plus maze and light:dark exploration tests in mice with Purkinje cell gamma 2 subunit inactivation were similar to those in control mice. The results suggest that, when the deletion of gamma 2 subunit has removed synaptic GABA(A) receptors from the specific cerebellar neuronal populations, the effects of neuroactive steroids solely on extrasynaptic alpha beta or alpha beta delta receptors lead to enhanced changes in the cerebellum-generated behavior.Peer reviewe

Does ethanol act preferentially via selected brain GABAA receptor subtypes? the current evidence is ambiguous

In rodent models, γ-aminobutyric acid A (GABAA) receptors with the α6 and δ subunits, expressed in the cerebellar and cochlear nucleus granule cells, have been linked to ethanol sensitivity and voluntary ethanol drinking. Here, we review the findings. When considering both in vivo contributions and data on cloned receptors, the evidence for direct participation of the α6-containing receptors to increased ethanol sensitivity is poor. The α6 subunit-knockout mouse lines do not have any changed sensitivity to ethanol, although these mice do display increased benzodiazepine sensitivity. However, in general the compensations occurring in knockout mice (regardless of which particular gene is knocked out) tend to fog interpretations of drug actions at the systems level. For example, the α6 knockout mice have increased TASK-1 channel expression in their cerebellar granule cells, which could influence sensitivity to ethanol in the opposite direction to that obtained with the α6 knockouts. Indeed, TASK-1 knockout mice are more impaired than wild types in motor skills when given ethanol; this might explain why GABAA receptor α6 knockout mice have unchanged ethanol sensitivities. As an alternative to studying knockout mice, we examined the claimed δ subunit-dependent/γ2 subunit-independent ethanol/[3H]Ro 15-4513 binding sites on GABAA receptors. We looked at [3H]Ro 15-4513 binding in HEK 293 cell membrane homogenates containing rat recombinant α6/4β3δ receptors and in mouse brain sections. Specific high-affinity [3H]Ro 15-4513 binding could not be detected under any conditions to the recombinant receptors or to the cerebellar sections of γ2(F77I) knockin mice, nor was this binding to brain sections of wild-type C57BL/6 inhibited by 1–100 mM ethanol. Since ethanol may act on many receptor and channel protein targets in neuronal membranes, we consider the α6 (and α4) subunit-containing GABAA receptors unlikely to be directly responsible for any major part of ethanol's actions. Therefore, we finish the review by discussing more generally alcohol and GABAA receptors and by suggesting potential future directions for this research.This study and review was supported by the Finnish Foundation for Alcohol Studies, the Academy of Finland and the Sigrid Juselius Foundation.Peer reviewe

Removal of GABA(A) Receptor gamma 2 Subunits from Parvalbumin Neurons Causes Wide-Ranging Behavioral Alterations

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