Regionally selective effects of GABA on hypothalamic GABAA receptor mRNA in vitro

Biochemical Pharmacology

et al. 2007

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CYP2C11, the most commonly expressed isoform of cytochrome P450 in male rat liver, was measured in spleen, thymus and bone marrow by quantitative real-time PCR and enhanced Western blotting. CYP2C11 concentrations in the lymphoid tissues were a fraction of that observed in liver, but like the liver, were sexually dimorphic (M>F) with mRNA and protein levels in agreement. Although the response to hypophysectomy varied according to tissue and sex, expression levels of CYP2C11 in all measured tissues remained greater in males. Further differences in CYP2C11 expression between liver and lymphoid tissue were observed following restoration of the circulating masculine growth hormone profile in hypophysectomized rats. In contrast to the liver where the renaturalized growth hormone profile elevated CYP2C11 expression in both sexes, the response was opposite in spleen and thymus with isoform concentrations declining in both sexes. Lastly, the divergent response of CYP2C11 between the liver and immune system was examined in cultured splenocytes exposed to different mitogens. In contrast to the dramatic depletion of CYP2C11 reported in proliferating hepatocytes, mitogen-stimulation resulted in a significant elevation in splenocyte CYP2C11 expression. In summary, we report for the first time that thymus, spleen and bone marrow express, albeit nominal, sex-dependent levels of CYP2C11 (M>F) whose regulation appears to be under some hormonal control, but very different from that of the hepatic isoform.

Section: Sexually Dimorphic Expression Of Tubulinsupporting

confidence: 93%

Sex-dependent expression of CYP2C11 in spleen, thymus and bone marrow regulated by growth hormone

Thangavel

Dhir

Biochemical Pharmacology

et al. 2007

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“…The reverse transcription-polymerase chain reaction (RT-PCR) procedures used for mRNA quantification were described previously [44] , [48] . Total RNA was extracted from each punch using the RNeasy-mini kit (Qiagen, Valencia, CA), re-dissolved in 50 µl of RNase-free water and quantified by densitometry (BioPhotometer, Eppendorf, Hamburg, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…This, in turn, would enhance the strength of GABAergic inhibition at this site and translate into a stronger sleep-promoting signal, increased subjective sleepiness and deeper sleep. We previously tested selected aspects of this hypothesis by superfusing posterior hypothalamic slices in vitro with either GABAergic agonists (to mimic a period of increased GABA release during sleep), or a GABA A R antagonist (to mimic a state of reduced inhibition) [44] . The results from this reduced model supported the concept that a few hours of increased cellular activity lead to a significant and regionally selective upregulation of mRNA for selected subunits of GABA A Rs in the PF region.…”

Section: Introductionmentioning

confidence: 99%

Time- and Behavioral State-Dependent Changes in Posterior Hypothalamic GABAA Receptors Contribute to the Regulation of Sleep

Stettner

et al. 2014

PLoS ONE

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Sleep-wake behavior is regulated by a circadian rhythm, homeostatically and by additional mechanisms that determine the timing of slow-wave sleep and rapid eye movement sleep (REMS) episodes. The posterior hypothalamus coordinates the neural and humoral signals with the rest-activity cycle. It contains wake-active neurons, and is a site where stimulation of inhibitory GABAA receptors promotes sleep, whereas their antagonism enhances wakefulness. We explored whether GABAergic mechanisms present in the posterior hypothalamus contribute to the homeostatic and other aspects of sleep-wake regulation. Using micropunches of tissue extracted from either the perifornical (PF) or dorsomedial (DM) regions of the posterior hypothalamus of rats, we determined that mRNA levels for selected subunits of GABAA receptors (β1, β3 and ε) were higher at the end of the active period or following sleep deprivation, when the need for sleep is high, than after several hours of sleep, when sleep need is partially fulfilled. Such a pattern was present in the PF region only, and was consistent with changes in β1 subunit and GABA synthesizing enzyme (GAD) protein levels. In contrast, in the DM region, the levels of GABAA receptor subunit mRNAs and proteins (α1, α2, β1) and GAD varied with circadian time, but were not responsive to sleep deprivation. Separate experiments with sleep-wake monitoring and local perfusion of the PF region with the GABAA receptor antagonist bicuculline revealed that the antagonist had a weaker sleep-reducing effect when sleep need was enhanced by sleep deprivation and that the increased amount of REMS characteristic of the late sleep period was dependent on endogenous GABAergic inhibition. These results support the concept that a varying magnitude of GABAergic inhibition exerted within the PF region contributes to the homeostatic regulation of sleep and shapes its temporal pattern, whereas GABAergic mechanisms in the DM region contribute to circadian regulation.

“…1B) and the other from the VLPO region of the anterior hypothalamic slice. One of the samples from each pair was subjected to RNA extraction and quantitative reverse transcription-polymerase chain reaction (RT-PCR) using the protocol described in our earlier study [38]. We quantified mRNA levels for eleven subunits of GABA A R (α1-5, β1-3, γ2 (long and short splicing variants; γ2 L/S ), δ, and ε) that are expressed in the hypothalamus [24].…”

Section: Methodsmentioning

confidence: 99%

“…The images of membranes were digitized and immunostaining density was quantified using ImageJ software (National Institutes of Health, http://rsb.info.nih.gov). Quality controls included histological verification of punch locations and assessment of RNA and PCR quality, as described previously [38].…”

Section: Methodsmentioning

confidence: 99%

Perinatal alcohol exposure leads to prolonged upregulation of hypothalamic GABAA receptors and increases behavioral sensitivity to gaboxadol

2008

Neuroscience Letters

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Prenatal alcohol exposure (AE) is associated with lasting abnormalities of sleep and motor development, but the underlying mechanisms are unknown. We hypothesized that AE alters development of GABAergic signaling in the hypothalamic regions important for the control of sleep and motor activity. Alcohol (5.25 g/(kg day)) was administered intragastrically to male rats on postnatal days (PD) 4-9, a period of brain development equivalent to the human third trimester (AE group). Control pups were sham-intubated (S group). Motor activity was monitored on PD27 and 28. On PD29 and 30, GABA A receptor subunit mRNA levels and alpha4 and delta subunit proteins were quantified by RT-PCR and immunoblotting, respectively, in the wake- and motor activity-promoting perifornical (PF) region of the posterior hypothalamus and the sleep-promoting ventrolateral preoptic (VLPO) region of the anterior hypothalamus. Then, in 47-52-day-old rats, motor activity was quantified following administration of GABA A receptor agonist, gaboxadol (5 mg/kg s.c.). In the PF region, mRNA and protein levels for the alpha4 and delta subunits were significantly higher and beta3 and gamma2 subunit mRNAs were also increased in the AE group. In the VLPO region, only the delta subunit mRNA was increased. Spontaneous motor activity was lower and suppressed more by gaboxadol in the AE than S group, and the latency to a transient total loss of activity after gaboxadol was shorter in the AE group. Thus, perinatal AE leads to GABA A receptor overexpression in the vigilance- and motor activity-promoting hypothalamic PF region, with the neurochemical and functional outcomes lasting long beyond the period of the insult.