GABA Receptor Subunit Expression Research Articles

Local synthesis of estradiol in the rostral ventromedial medulla protects against widespread muscle pain in male mice.

Animal studies consistently demonstrate that testosterone is protective against pain in multiple models, including an animal model of activity-induced muscle pain. In this model, females develop widespread muscle hyperalgesia, and reducing testosterone levels in males results in widespread muscle hyperalgesia. Widespread pain is believed to be mediated by changes in the central nervous system, including the rostral ventromedial medulla (RVM). The enzyme that converts testosterone to estradiol, aromatase, is highly expressed in the RVM. Therefore, we hypothesized that testosterone is converted by aromatase to estradiol locally in the RVM to prevent development of widespread muscle hyperalgesia in male mice. This was tested through pharmacological inhibition of estrogen receptors (ER), aromatase, or ER-α in the RVM which resulted in contralateral hyperalgesia in male mice (C57BL/6J). ER inhibition in the RVM had no effect on hyperalgesia in female mice. As prior studies show modulation of estradiol signaling alters GABA receptor and transporter expression, we examined if removal of testosterone in males would decrease mRNA expression of GABA receptor subunits and vesicular GABA transporter (VGAT). However, there were no differences in mRNA expression of GABA receptor subunits of VGAT between gonadectomized and sham control males. Lastly, we used RNAscope to determine expression of ER-α in the RVM and show expression in inhibitory (VGAT+), serotonergic (tryptophan hydroxylase 2+), and μ-opioid receptor expressing (MOR+) cells. In conclusion, testosterone protects males from development of widespread hyperalgesia through aromatization to estradiol and activation of ER-α which is widely expressed in multiple cell types in the RVM.Significance Statement This study's findings reveal, for the first time, that testosterone protects males from development of widespread muscle hyperalgesia through aromatization to estradiol and activation of ER-α within the RVM. The findings also show, for the first time, ER-α expression in the RVM in male mice, and the distribution patterns of this expression among multiple cell types. Thus, together our data suggest a unique endogenous mechanism in the RVM for protection against widespread pain in males only.

Altered Expression of GABAergic Markers in the Forebrain of Young and Adult Engrailed-2 Knockout Mice.

Impaired function of GABAergic interneurons, and the subsequent alteration of excitation/inhibition balance, is thought to contribute to autism spectrum disorders (ASD). Altered numbers of GABAergic interneurons and reduced expression of GABA receptors has been detected in the brain of ASD subjects and mouse models of ASD. We previously showed a reduced expression of GABAergic interneuron markers parvalbumin (PV) and somatostatin (SST) in the forebrain of adult mice lacking the Engrailed2 gene (En2-/- mice). Here, we extended this analysis to postnatal day (P) 30 by using in situ hybridization, immunohistochemistry, and quantitative RT-PCR to study the expression of GABAergic interneuron markers in the hippocampus and somatosensory cortex of En2-/- and wild type (WT) mice. In addition, GABA receptor subunit mRNA expression was investigated by quantitative RT-PCR in the same brain regions of P30 and adult En2-/- and WT mice. As observed in adult animals, PV and SST expression was decreased in En2-/- forebrain of P30 mice. The expression of GABA receptor subunits (including the ASD-relevant Gabrb3) was also altered in young and adult En2-/- forebrain. Our results suggest that GABAergic neurotransmission deficits are already evident at P30, confirming that neurodevelopmental defects of GABAergic interneurons occur in the En2 mouse model of ASD.

Tet1 overexpression leads to anxiety-like behavior and enhanced fear memories via the activation of calcium-dependent cascade through Egr1 expression in mice.

Ten-eleven translocation methylcytosine dioxygenase 1 (Tet1) initiates DNA demethylation by converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) at CpG-rich regions of genes, which have key roles in adult neurogenesis and memory. In addition, the overexpression of Tet1 with 5-hmC alteration in patients with psychosis has also been reported, for instance in schizophrenia and bipolar disorders. The mechanism underlying Tet1 overexpression in the brain; however, is still elusive. In the present study, we found that Tet1-transgenic (Tet1-TG) mice displayed abnormal behaviors involving elevated anxiety and enhanced fear memories. We confirmed that Tet1 overexpression affected adult neurogenesis with oligodendrocyte differentiation in the hippocampal dentate gyrus of Tet1-TG mice. In addition, Tet1 overexpression induced the elevated expression of immediate early genes, such as Egr1, c-fos, Arc, and Bdnf, followed by the activation of intracellular calcium signals (i.e., CamKII, ERK, and CREB) in prefrontal and hippocampal neurons. The expression of GABA receptor subunits (Gabra2 and Gabra4) fluctuated in the prefrontal cortex and hippocampus. We evaluated the effects of Tet1 overexpression on intracellular calcium-dependent cascades by activating the Egr1 promoter in vitro Tet1 enhanced Egr1 expression, which may have led to alterations in Gabra2 and Gabra4 expression in neurons. Taken together, we suggest that the Tet1 overexpression in our Tet1-TG mice can be applied as an effective model for studying various stress-related diseases that show hyperactivation of intracellular calcium-dependent cascades in the brain.-Kwon, W., Kim, H.-S., Jeong, J., Sung, Y., Choi, M., Park, S., Lee, J., Jang, S., Kim, S. H., Lee, S., Kim, M. O., Ryoo, Z. Y. Tet1 overexpression leads to anxiety-like behavior and enhanced fear memories via the activation of calcium-dependent cascade through Egr1 expression in mice.

Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury

Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, β2, β3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, β2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei.This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’.

Expression of GABAA α2-, β1- and ɛ-receptors are altered significantly in the lateral cerebellum of subjects with schizophrenia, major depression and bipolar disorder

There is abundant evidence that dysfunction of the γ-aminobutyric acid (GABA)ergic signaling system is implicated in the pathology of schizophrenia and mood disorders. Less is known about the alterations in protein expression of GABA receptor subunits in brains of subjects with schizophrenia and mood disorders. We have previously demonstrated reduced expression of GABAB receptor subunits 1 and 2 (GABBR1 and GABBR2) in the lateral cerebella of subjects with schizophrenia, bipolar disorder and major depressive disorder. In the current study, we have expanded these studies to examine the mRNA and protein expression of 12 GABAA subunit proteins (α1, α2, α3, α5, α6, β1, β2, β3, δ, ɛ, γ2 and γ3) in the lateral cerebella from the same set of subjects with schizophrenia (N=9–15), bipolar disorder (N=10–15) and major depression (N=12–15) versus healthy controls (N=10–15). We found significant group effects for protein levels of the α2-, β1- and ɛ-subunits across treatment groups. We also found a significant group effect for mRNA levels of the α1-subunit across treatment groups. New avenues for treatment, such as the use of neurosteroids to promote GABA modulation, could potentially ameliorate GABAergic dysfunction in these disorders.

Human embryonic stem cell model of ethanol-mediated early developmental toxicity

BackgroundFetal alcohol syndrome is an important clinical problem. Human embryonic stem cells (hESC) have not been widely used to study developmental alcohol toxicity. Here we document the phenotype of hESC exposed to clinically-relevant, low dose ethanol (20mM). MethodsAll cultures were maintained in 3% O2 to reflect normal physiologic conditions. Undifferentiated hESC were expanded with basic fibroblast growth factor (bFGF), with or without ethanol, then differentiated without ethanol. Proliferation and apoptosis in response to ethanol were assayed, and PCR used to examine expression of GABA receptor subunits. Whole cell patch clamping was used to examine GABAA receptor function in undifferentiated hESC. Immunocytochemistry and western blotting were used to follow differentiation of early neurons, astrocytes, and oligodendrocytes, Principal findingsExposure to 20mM ethanol resulted in larger colonies of undifferentiated hESC despite an increase in apoptosis, because proliferation of the undifferentiated cells (and neuroblasts) was significantly increased. Differentiation of hESC (following a week of ethanol exposure) resulted in decreased expression of GFAP (by western) compared to unexposed cells, suggesting that astrocyte differentiation was reduced, while markers of oligodendrocyte and neuron differentiation were unchanged. At the message level, undifferentiated hESC express all GABAA receptor subunits, but functional receptors were not found by whole cell patch clamping. ConclusionOur results in hESC suggest a complex mix of ethanol-induced phenotypic changes when ethanol exposure occurs very early in development. Not only increased apoptosis, but inappropriate proliferation and loss of trophic astrocytes could result from low-dose ethanol exposure very early in development. More generally, these studies support a role for hESC in developing hypotheses and focusing questions to complement animal studies of developmental toxicities.

Differential Expression of GABA Receptor Subunit Messenger Ribonucleic Acids and Immunoreactivity in the Rat Neostriatum during Postnatal Development

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the neostriatum. Functions of GABA are mediated by GABA_A and GABA_B receptors in the neostriatum. In order to investigate the developmental expression of GABA receptor subunits (GABA_Aα₁, GABA_Aα₃ and GABA_Aα₆; GABA_Aβ₂ and GABA_Aβ₃, and GABA_BR₁ and GABA_BR₂) in the rat neostriatum, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence were performed. Tissues were obtained from rats on postnatal day (PND) 1, PND 7 and PND 14, and from adult rats. RT-PCR indicated that GABA_Aα₁ and GABA_Aα₆ mRNA levels were low, but that GABA_Aβ₂ and GABA_Aβ₃ mRNAs levels were high during the early postnatal period. Immunofluorescence revealed that GABA_Aα₁ immunoreactivity was only observed in striatal interneurons in PND 14 and in adult rats. Immunoreactivity for GABA_Aα₆ was only observed in PND 14 and adult animals. GABA_BR₁ immunoreactivity was found to be expressed by perikarya of striatal neurons at all ages examined. In contrast, GABA_BR₂ immunoreactivity was mainly observed in choline acetyltransferase-positive striatal interneurons in PND 14 and adult rats. The present results indicate that there are differential patterns of developmental expression of GABA receptor subunits in the neostriatum, with important implications for the development of GABA systems in rat basal ganglia.

Ethanol and Gene Expression in Brain

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Izuru Matusmoto and Peter A. Wilce. The presentations were (1) GABA receptor subunit expression in the human alcoholic brain, by Tracey Buckley and Peter Dodd; (2) NMDAR gene expression during ethanol addiction, by Jorg Puzke, Rainer Spanagel, Walther Zieglgansberger, and Gerald Wolf; (3) Differentially expressed gene in the nucleus accumbens from ethanol-administered rat, by Shuangying Leng; (4) Expression of a novel gene in the alcoholic brain, by Peter A. Wilce; and (5) Investigations of haplotypes of the dopamine D2-receptor gene in alcoholics, by Hans Rommelspacher, Ulrich Finckh, and Lutz G. Schmidt.

Ethanol and Gene Expression in Brain

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Izuru Matusmoto and Peter A. Wilce. The presentations were (1) GABA receptor subunit expression in the human alcoholic brain, by Tracey Buckley and Peter Dodd; (2) NMDAR gene expression during ethanol addiction, by Jorg Puzke, Rainer Spanagel, Walther Zieglgansberger, and Gerald Wolf; (3) Differentially expressed gene in the nucleus accumbens from ethanol‐administered rat, by Shuangying Leng; (4) Expression of a novel gene in the alcoholic brain, by Peter A. Wilce; and (5) Investigations of haplotypes of the dopamine D2‐receptor gene in alcoholics, by Hans Rommelspacher, Ulrich Finckh, and Lutz G. Schmidt.

12. Sex and brain development

Consistent with the importance of reproductive behavior for survival of the species, the gonadal steroids are major regulators of neural function and brain development. Investigation of these regulatory effects has revealed both mechanisms and principles that underlie emergent behavioral properties. First, activational effects of gonadal steroids comprise both acute, signal transduction-mediated and classical, intracellular receptor-mediated actions, the latter including modulation of the synthesis of the synthetic and metabolic enzymes for neurotransmitters, neuropeptides, neuroeffector receptors, and second messengers. Second, gonadal steroids create a context that influences both formation of neurocircuitry and acute neural response. These actions are largely inferred from three types of observations: structural and functional sexual dimorphisms; changes in neural response during reproductive endocrine change (e.g. the ovulatory cycle); and modulation of response to hormone manipulation (e.g. castration). In fact, all stages of neuronal development—neurogenesis, growth, migration, differentiation, and synaptic organization—are modulated by gonadal steroids. Third, gonadal steroids organize the brain during critical developmental windows. Androgen blockade, for example, alters 5-HT1A receptor m-RNA but not GABA receptor subunit expression during the last prenatal week, while the opposite is observed during the fourth postnatal week. Fourth, the effects of gonadal steroids are context dependent and differ as a function of environment, prior exposure, age, and genotypic characteristics. Studies of the effects of gonadal steroids on behavior promise to help answer the critical question in human behavioral endocrinology: Why do different individuals respond differently to ostensibly the same stimulus?