Ethanol Regulation of Synaptic GABAA 4 Receptors Is Prevented by Protein Kinase A Activation

Ethanol alters GABAA receptor trafficking and function through activation of protein kinases, and these changes may underlie ethanol dependence and withdrawal. In this study, we used subsynaptic fraction techniques and patch-clamp electrophysiology to investigate the biochemical and functional effects of protein kinase A (PKA) and protein kinase C (PKC) activation by ethanol on synaptic GABAAα4 receptors, a key target of ethanol-induced changes. Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or kinase modulators for 4 hours, a paradigm that recapitulates GABAergic changes found after chronic ethanol exposure in vivo. PKA activation by forskolin or rolipram during ethanol exposure prevented increases in P2 fraction α4 subunit abundance, whereas inhibiting PKA had no effect. Similarly, in the synaptic fraction, activation of PKA by rolipram in the presence of ethanol prevented the increase in synaptic α4 subunit abundance, whereas inhibiting PKA in the presence of ethanol was ineffective. Conversely, PKC inhibition in the presence of ethanol prevented the ethanol-induced increases in synaptic α4 subunit abundance. Finally, we found that either activating PKA or inhibiting PKC in the presence of ethanol prevented the ethanol-induced decrease in GABA miniature inhibitory postsynaptic current decay τ1, whereas inhibiting PKA had no effect. We conclude that PKA and PKC have opposing effects in the regulation of synaptic α4 receptors, with PKA activation negatively modulating, and PKC activation positively modulating, synaptic α4 subunit abundance and function. These results suggest potential targets for restoring normal GABAergic functioning in the treatment of alcohol use disorders

Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons

The GABAA α4 subunit exists in two distinct populations of GABAA receptors. Synaptic GABAA α4 receptors are localized at the synapse and mediate phasic inhibitory neurotransmission, while extrasynaptic GABAA receptors are located outside of the synapse and mediate tonic inhibitory transmission. These receptors have distinct pharmacological and biophysical properties that contribute to interest in how these different subtypes are regulated under physiological and pathological states. We utilized subcellular fractionation procedures to separate these populations of receptors in order to investigate their regulation by protein kinases in cortical cultured neurons. Protein kinase A (PKA) activation decreases synaptic α4 expression while protein kinase C (PKC) activation increases α4 subunit expression, and these effects are associated with increased β3 S408/409 or γ2 S327 phosphorylation respectively. In contrast, PKA activation increases extrasynaptic α4 and δ subunit expression, while PKC activation has no effect. Our findings suggest synaptic and extrasynaptic GABAA α4 subunit expression can be modulated by PKA to inform the development of more specific therapeutics for neurological diseases that involve deficits in GABAergic transmission

Perinatal neurosteroid levels influence GABAergic interneuron localization in adult rat prefrontal cortex

Neurosteroids are a class of steroids synthesized de novo in the brain, several of which are potent modulators of GABAA receptor function. In developing brain GABAA receptor, stimulation plays a trophic role. Cortical levels of the GABAergic neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) vary dramatically across development; during the second week of life, elevated levels of 3α,5α-THP are associated with decreased GABAA receptor function. To determine whether alteration of endogenous 3α,5α-THP levels during development alters GABAergic interneurons in prefrontal cortex (PFC) at maturity, rat pups were exposed to 3α,5α-THP (10 mg/kg) on postnatal day 1 (P1), P2, and P5. On P80, frontal cortex tissue was assayed for GABAergic cell localization (parvalbumin and calbindin immunoreactivity), agonist-dependent [3H] dizocilpine (MK-801) binding to NMDA receptors in cortical homogenates, muscimol-mediated 36Cl- influx into synaptoneurosomes, and 3α,5α-THP levels. The localization of parvalbumin-labeled cells was markedly altered; the ratio of cell number in the deep layers (V-VI) versus superficial layers (I-III) of adult PFC increased twofold in animals exposed to 3α,5α-THP on P1 or P5. Relative microtubule-associated protein-2 and calbindin immunoreactivity were not altered by perinatal 3α,5α-THP administration. Agonist-dependent [3H]MK-801 binding was decreased in PFC but not parietal cortex homogenates, whereas muscimolmediated 36Cl- influx and 3α,5α-THP levels were unchanged in frontal cortex of adult males exposed to 3α,5α-THP on P5. These data are consistent with a change in the distribution of a subset of interneurons in response to neurosteroid exposure and suggest that GABAergic neurosteroids are critical for normal development of GABAergic systems in the PFC

Divergent neuroactive steroid responses to stress and ethanol in rat and mouse strains: relevance for human studies

Neuroactive steroids are endogenous or synthetic steroids that rapidly alter neuronal excitability via membrane receptors, primarily GABAA receptors. Neuroactive steroids regulate many physiological processes including hypothalamic-pituitary-adrenal (HPA) axis function, ovarian cycle, pregnancy, aging, and reward. Moreover, alterations in neuroactive steroid synthesis are implicated in several neuropsychiatric disorders

COX-2 and PPARγ expression are potential markers of recurrence risk in mammary duct carcinoma in-situ

<p>Abstract</p> <p>Background</p> <p>In women with duct carcinoma in-situ (DCIS) receiving breast conservation therapy (BCT), in-breast recurrences are seen in approximately 10%, but cannot be accurately predicted using clinical and histological criteria. We performed a case-control study to identify protein markers of local recurrence risk in DCIS.</p> <p>Methods</p> <p>Women treated for DCIS with BCT, who later developed in-breast recurrence (cases) were matched by age and year of treatment to women who remained free of recurrence (controls).</p> <p>Results</p> <p>A total of 69 women were included in the study, 31 cases and 38 controls. Immunohistochemical evaluation of DCIS tissue arrays was performed for estrogen receptor, progesterone receptor, HER-2/neu, cyclin D1, p53, p21, cycloxygenase-2 (COX-2) and peroxisome proliferator activated receptor γ (PPARγ). Two markers were significantly different between cases and controls on univariate analysis: strong COX-2 expression was associated with increased risk of recurrence, with 67% vs. 24% positivity in cases and controls p = 0.006; and nuclear expression of PPARγ was associated with protection from recurrence with 4% vs. 27% positivity in cases and controls, p = 0.024. In a multivariate model which included size, grade, COX-2 and PPARγ positivity, we found COX-2 positivity to be a strong independent risk factor for recurrence (OR 7.90, 95% CI 1.72–36.23)., whereas size and grade were of borderline significance. PPARγ expression continued to demonstrate a protective trend, (OR 0.14, 95% CI 0.06–1.84).</p> <p>Conclusion</p> <p>Our findings suggest that COX-2 and PPARγ should be investigated further as biologic markers to predict DCIS recurrence, particularly since they are also potential therapeutic targets.</p

Role of Acetaldehyde in Ethanol-Induced Elevation of the Neuroactive Steroid 3α-Hydroxy-5α-Pregnan-20-One in Rats

Systemic ethanol administration increases neuroactive steroid levels that increase ethanol sensitivity. Acetaldehyde is a biologically active compound that may contribute to behavioral and rewarding effects of ethanol. We investigated the role of acetaldehyde in ethanol–induced elevations of 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) levels in cerebral cortex

GABA A Receptors Mediate Trophic Effects of GABA on Embryonic Brainstem Monoamine Neurons In Vitro

The inhibitory neurotransmitter GABA may act as a trophic signal for developing monoamine neurons in embryonic rat brain, because GABA neurons and their receptors appear in brainstem during generation of monoamine neurons. To test this hypothesis, we used dissociated cell cultures from embryonic day 14 rat brainstem, which contains developing serotonin (5-HT), noradrenaline (tyrosine hydroxylase; TH), and GABA neurons. Immunocytochemistry and reverse transcription-PCR (RT-PCR) revealed the presence of multiple alpha, beta, gamma, and delta subunits in these cultures. Competitive RT-PCR demonstrated high levels of beta3 subunit transcripts. Expression of functional GABAA receptors was demonstrated using 36Cl- flux assays. To investigate GABAergic regulation of neuronal survival and growth, cultures were treated for 1-3 d in vitro with 10 microM GABA and/or GABAA antagonist (bicuculline or the pesticide dieldrin). The effects of treatments were quantified by analysis of immunoreactive 5-HT, TH, and GABA neurons. GABAA receptor ligands differentially regulated neuronal survival and growth depending on neurotransmitter phenotype. GABA exerted positive effects on monoamine neurons, which were countered by bicuculline (and dieldrin, 5-HT neurons only). By itself, bicuculline produced inhibitory effects on both 5-HT and TH neurons, whereas dieldrin potently inhibited 5-HT neurons only. GABA neurons responded positively to both antagonists, but more strongly to bicuculline. Taken together, these results demonstrate that the activation/inhibition of GABAA receptors produces opposite effects on the development of embryonic monoamine and GABA neurons. This suggests that these neurotransmitter phenotypes may express GABAA receptors that differ in fundamental ways, and these differences determine the developmental responses of these cells to GABAergic stimuli

The Effects of Acute and Chronic Ethanol Exposure on Presynaptic and Postsynaptic GABAA Receptor Function in Cultured Cortical and Hippocampal Neurons

Decades after ethanol was first described as a GABA mimetic, the precise mechanisms that produce the acute effects of ethanol and the physiological adaptations that underlie ethanol tolerance and dependence remain unclear. While a substantial body of evidence suggests that ethanol acts on GABAergic neurotransmission to enhance inhibition in the CNS, the precise mechanisms underlying the physiological effects of both acute and chronic ethanol exposure are still under investigation. We have used in vitro ethanol exposure followed by recording of miniature inhibitory postsynaptic currents (mIPSCs) to determine whether acute or chronic ethanol exposure directly alters synaptic GABAA receptor function or GABA release in cultured cortical and hippocampal neurons. Acute ethanol exposure slightly increased the duration of mIPSCs in hippocampal neurons but did not alter mIPSC kinetics in cortical neurons. Acute ethanol exposure did not change mIPSC frequency in either hippocampal or cortical neurons. One day of chronic ethanol exposure produced a transient decrease in mIPSC duration in cortical neurons but did not alter mIPSC kinetics in hippocampal neurons. Chronic ethanol exposure did not change mIPSC frequency in either hippocampal or cortical neurons. Chronic ethanol exposure also did not produce substantial cross-tolerance to a benzodiazepine in either hippocampal or cortical neurons. The results suggest that ethanol exposure in vitro has limited effects on synaptic GABAAR function and action-potential independent GABA release in cultured neurons and suggests that ethanol exposure in cultured cortical and hippocampal neurons may not reproduce all of the effects that occur in vivo and in acute brain slices

Blunted neuroactive steroid and HPA axis responses to stress are associated with reduced sleep quality and negative affect in pregnancy: a pilot study

Anxiety during pregnancy has been linked to adverse maternal health outcomes, including postpartum depression (PPD). However, there has been limited study of biological mechanisms underlying behavioral predictors of PPD during pregnancy