Neurosteroid effects at α4ßδ GABAA receptors alter spatial learning and synaptic plasticity in CA1 hippocampus across the estrous cycle of the mouse.

Fluctuations in circulating levels of ovarian hormones have been shown to regulate cognition (Sherwin and Grigorova, 2011. Fertil. Steril. 96, 399-403; Shumaker et al., 2004. JAMA. 291, 2947-2958), but increases in estradiol on the day of proestrus yield diverse outcomes: In vivo induction of long-term potentiation (LTP), a model of learning, is reduced in the morning, but optimal in the afternoon (Warren et al., 1995. Brain Res. 703, 26-30). The mechanism underlying this discrepancy is not known. Here, we show that impairments in both CA1 hippocampal LTP and spatial learning observed on the morning of proestrus are due to increased dendritic expression of α4ßδ GABAA receptors (GABARs) on CA1 pyramidal cells, as assessed by electron microscopic (EM) techniques, compared with estrus and diestrus. LTP induction and spatial learning were robust, however, when assessed on the morning of proestrus in α4-/- mice, implicating these receptors in mediating impaired plasticity. Although α4ßδ expression remained elevated on the afternoon of proestrus, increases in 3α-OH-THP (3α-OH-5α-pregnan-20-one) decreased inhibition by reducing outward current through α4ßδ GABARs (Shen et al., 2007. Nat. Neurosci. 10, 469-477), in contrast to the usual effect of this steroid to enhance inhibition. Proestrous levels of 3α-OH-THP reversed the deficits in LTP and spatial learning, an effect prevented by the inactive metabolite 3ß-OH-THP (10 mg/kg, i.p.), which antagonizes actions of 3α-OH-THP. In contrast, administration of 3α-OH-THP (10 mg/kg, i.p.) on the morning of proestrus improved spatial learning scores 150-300%. These findings suggest that cyclic fluctuations in ovarian steroids can induce changes in cognition via α4ßδ GABARs that are dependent upon 3α-OH-THP. This article is part of a Special Issue entitled SI: Brain and Memory.

Characterization of neurosteroid effects on hyperpolarizing current at α4ß2δ GABAA receptors.

RATIONALE: The neurosteroid 3α,5ß-THP (3α-OH-5ß-pregnan-20-one, pregnanolone) is a modulator of the GABAA receptor (GABAR), with α4ß2δ GABARs the most sensitive. However, the effects of 3α,5ß-THP at α4ß2δ are polarity-dependent: 3α,5ß-THP potentiates depolarizing current, as has been widely reported, but decreases hyperpolarizing current by accelerating desensitization. OBJECTIVES: The present study further characterized 3α,5ß-THP inhibition of hyperpolarizing current at this receptor and compared effects of other related steroids at α4ß2δ GABARs. METHODS: α4ß2δ GABARs were expressed in HEK-293 cells, and agonist-gated current recorded with whole cell voltage-clamp techniques using a theta tube to rapidly apply agonist before and after application of neurosteroids. RESULTS: The GABA-modulatory steroids (30 nM) 3α,5α-THP (3α-OH-5α-pregnan-20-one, allopregnanolone) and THDOC (3α,21-dihydroxy-5α-pregnan-20-one) inhibited hyperpolarizing GABA (10 µM)-gated current at α4ß2δ GABARs similar to 3α,5ß-THP, while the inactive 3ß,5ß-THP isomer had no effect. Greater inhibition was seen for current gated by the high efficacy agonist gaboxadol (THIP, 100 µM) than for GABA (0.1-1000 µM), consistent with an effect of 3α,5ß-THP on desensitization. Inhibitory effects of the steroid were not seen under low [Cl(-)] conditions or in the presence of calphostin C (500 nM), an inhibitor of protein kinase C. Chimeras swapping the IL (intracellular loop) of α4 with α1, when expressed with ß2 and δ, produced receptors (α[414]ß2δ) which were not inhibited by 3α,5ß-THP when GABA-gated current was hyperpolarizing, while α[141]ß2δ exhibited steroid-induced polarity-dependent modulation. CONCLUSIONS: These findings suggest that numerous neurosteroids exhibit polarity-dependent effects at α4ß2δ GABARs, which are dependent upon protein kinase C and the IL of α4.

One-stage correction of aortic coarctation and severe mitral regurgitation via left posteriolateral thoracotomy in a 2-year-old child.

We successfully treated a case of a 2-year-old male with aortic coarctation coexisting with severe mitral regurgitation via left posteriolateral thoracotomy at one stage. After a mitral valve replacement under perfused ventricular fibrillation with moderate hypothermia, we repaired the aortic coarctation with coarctation resection and end-to-end anastamosis with the aid of deep hypothermic circulatory arrest and selective low-flow cerebral perfusion. The patient had an uneventful hospital course and remains well.

Two novel mutations of the LDL receptor gene associated with familial hypercholesterolemia in a Chinese family.

BACKGROUND: Familial hypercholesterolemia (FH) is a type of dominant autosomal disease that causes high levels of plasma low-density lipoprotein cholesterol (LDL-C). In the past years, molecular data related to FH were limited in China. Now, to gain more information about FH, we analyzed one proband with a severe FH phenotype as well as his relatives. METHODS: After the entire coding sequence and the intron-exon junctions of the low-density lipoprotein receptor (LDLR) gene were amplified using PCR, we sequenced the LDLR gene of a Chinese FH family. RT-PCR was used to detect changes in the mRNA. RESULTS: Two novel mutations were identified in the LDLR gene of this family. One, W165X, was a G > A substitution at the third nucleotide of codon 165. The other, IVS5-1G > A, was also a G > A substitution at the acceptor splice site of intron 5. The most striking discovery is that the proband was heterozygous for W165X but homozygous for IVS5-1G > A. The cDNA sequencing showed that the IVS5-1G > A mutation caused the insertion of 10 nucleotides, namely GCTCTCACAA, between exon 5 and exon 6. CONCLUSIONS: The two nucleotide variations are thought to be the FH-causing mutations because the co-segregation of the mutant allele with the phenotype of FH has been shown in this Chinese family. These data show an increase in the mutational spectrum of FH in China and verify a scarce mutational form in the LDLR gene.

Ethanol effects on GABA-gated current in a model of increased alpha4betadelta GABAA receptor expression depend on time course and preexposure to low concentrations of the drug.

Several recent studies have suggested that alphabetadelta subtypes of gamma-aminobutyric acid type A (GABAA) receptors (delta-GABAR) are a target for low dose ethanol (<30 mM). However, there are also conflicting reports suggesting that only high doses of the drug (100 mM) modulate these receptors. In addition, the studies which have demonstrated a clear effect of low dose ethanol on delta-GABAR find different effective concentrations for this effect. Here, we test the hypothesis that the apparent disparity in effective concentration is due to time-course effects when low (1-3 mM) dose ethanol is preapplied. To this end, we tested ethanol effects on native GABAR in CA1 hippocampus in a model of increased alpha4betadelta GABAR expression following 48h administration of the GABA-modulatory steroid THP (3alpha-OH-5beta-pregnan-20-one) to adult, female rats. GABA(EC20)-gated current was recorded with whole-cell patch clamp procedures from acutely isolated pyramidal cells. We assessed ethanol's effect on GABA-gated current using either (1) 2-5 min application of ethanol in increasing concentrations (0.1-30 mM) or (2) coadministration of ethanol with GABA. Two minute application of 1-3 mM ethanol produced optimal potentiation of GABA-gated current following steroid treatment, with higher concentrations less effective. In contrast, 30 mM ethanol produced optimal effects when ethanol was not preapplied. However, following preapplication of 1mM ethanol, 30 mM ethanol decreased the peak GABA-gated current. These findings suggest that ethanol may act at multiple interacting sites to affect GABAR efficacy and desensitization. These data also suggest that ethanol effects on GABA-gated current are affected by the time course of exposure and previous exposure to low concentrations of the drug.

[Multifactor analysis of postoperative mechanical ventilation supporting time in infants with congenital heart diseases].

OBJECTIVE: To analyze the multiple factors affecting the postoperative mechanical ventilation supporting time in infants less than 10 kg with simple congenital heart diseases and to seize time by the forelock of extube and improve the outcome of surgical treatment. METHODS: Data of 231 infants less than 10 kg with atrial septal defect(ASD),ventricular septal defect, and combining patent ductus arteriosus were retrospectively analyzed. The multivaricate stepwise logistic regression statistics were done for the predisposing factors affecting the ventilative supporting time. RESULTS: The ventilative supporting time was 3~375 (average 23.5 h) h. The multivaricate stepwise logistic regression analysis indicated that severe pulmonary hyperpressure, cross-cramp aortic time, cardiopulmonary bypass time, preoperational pulmonary infection, membrane oxygenator, modified ultrafiltration, weight, and postoperative complications were significantly correlated to the ventilative supporting time. CONCLUSION: Severe pulmonary hyperpressure, preoperational pulmonary infection, long cross-cramp aortic time, long cardiopulmonary bypass time, postoperative complications all prolong the ventilation supporting time; the use of membrane oxygenator and modified ultrafiltration during the operation and big weight can diminish the pulmonary complications and shorten the ventilation supporting time.

Neurosteroid regulation of GABA(A) receptors: Focus on the alpha4 and delta subunits.

Neurosteroids, such as the progesterone metabolite 3alpha-OH-5alpha[beta]-pregnan-20-one (THP or [allo]pregnanolone), function as potent positive modulators of the GABA(A) receptor (GABAR) when acutely administered. However, fluctuations in the circulating levels of this steroid at puberty, across endogenous ovarian cycles, during pregnancy or following chronic stress produce periods of prolonged exposure and withdrawal, where changes in GABAR subunit composition may occur as compensatory responses to sustained levels of inhibition. A number of laboratories have demonstrated that both chronic administration of THP as well as its withdrawal transiently increase expression of the alpha4 subunit of the GABAR in several areas of the central nervous system (CNS) as well as in in vitro neuronal systems. Receptors containing this subunit are insensitive to benzodiazepine (BDZ) modulation and display faster deactivation kinetics, which studies suggest underlie hyperexcitability states. Similar increases in alpha4 expression are triggered by withdrawal from other GABA-modulatory compounds, such as ethanol and BDZ, suggesting a common mechanism. Other studies have reported puberty or estrous cycle-associated increases in delta-GABAR, the most sensitive target of these steroids which underlies a tonic inhibitory current. In the studies reported here, the effect of steroids on inhibition, which influence anxiety state and seizure susceptibility, depend not only on the subunit composition of the receptor but also on the direction of Cl(-) current generated by these target receptors. The effect of neurosteroids on GABAR function thus results in behavioral outcomes relevant for pubertal mood swings, premenstrual dysphoric disorder and catamenial epilepsy, which are due to fluctuations in endogenous steroids.

Reversal of neurosteroid effects at alpha4beta2delta GABAA receptors triggers anxiety at puberty.

Puberty is characterized by mood swings and anxiety, which are often produced by stress. Here we show that THP (allopregnanolone), a steroid that is released as a result of stress, increases anxiety in pubertal female mice, in contrast to its anxiety-reducing effect in adults. Anxiety is regulated by GABAergic inhibition in limbic circuits. Although this inhibition is increased by THP administration before puberty and in adults, during puberty THP reduces the tonic inhibition of pyramidal cells in hippocampal region CA1, leading to increased excitability. This paradoxical effect of THP results from inhibition of alpha4betadelta GABAA receptors. These receptors are normally expressed at very low levels, but at puberty, their expression is increased in hippocampal area CA1, where they generate outward currents. THP also decreases the outward current at recombinant alpha4beta2delta receptors, and this effect depends on arginine 353 in the alpha4 subunit, a putative site for modulation by Cl-. Therefore, inhibition of alpha4beta2delta GABAA receptors by THP provides a mechanism for the generation of anxiety at puberty.

[Surgical treatment of partial atrioventricular septal defect].

OBJECTIVE: To summarize the experience of surgical treatments of partial atrioventricular septal defect in 60 patients. METHODS: From April 1999 to April 2004, 60 patients of partial atrioventricular septal defect were operated. Fifty-eight patients were performed with suture of the cleft of mitral valve and the other 2 were given mitral valve replacement; For closure of primum ASD, 53 patients with pericardial patches and 7 with Dacron patches. Coronary sinus was baffled to left atrium with kirklin procedure in 35 cases and baffled to right atrium with McGoon procedure in other 25 cases. Correct the accompanying cardiac deformity at the same time. RESULTS: The hospital mortality was 3.3% (2/60) due to low cardic output syndrome. The incidence rate of complete atrioventricular block was 8.00% (2/ 35) in the group with Kirklin procedure and 6.06% (2/25) in the group with McGoon procedure. There was no statistical significance between the 2 groups (P > 0.05). The follow-up was from 1 month to 5 years, and there was no late death. All cardiac function were improved except middle mitral regurgitation in 1 patient. CONCLUSION: Reasonable operative design, refined procedures, avoiding damage to conducting bundles and proper perioperative management are the key points in improving theraeutic effect.

Short-term steroid treatment increases delta GABAA receptor subunit expression in rat CA1 hippocampus: pharmacological and behavioral effects.

In this study, 48 h administration of 3alpha-OH-5beta-pregnan-20-one (3alpha,5beta-THP) or 17beta-estradiol (E2)+progesterone (P) to female rats increased expression of the delta subunit of the GABA(A) receptor (GABAR) in CA1 hippocampus. Coexpression of alpha4 and delta subunits was suggested by an increased response of isolated pyramidal cells to the GABA agonist 4,5,6,7- tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), following 48 h steroid treatment, and nearly complete blockade by 300 microM lanthanum (La3+). Because alpha4betadelta GABAR are extrasynaptic, we also recorded pharmacologically isolated GABAergic holding current from CA1 hippocampal pyramidal cells in the slice. The La3+-sensitive THIP current, representative of current gated by alpha4betadelta GABAR, was measurable only following 48 h steroid treatment. In contrast, the bicuculline-sensitive current was not altered by steroid treatment, assessed with or without 200 nM gabazine to block synaptic current. However, 48 h steroid treatment resulted in a tonic current insensitive to the benzodiazepine agonists lorazepam (10 microM) and zolpidem (100 nM). These results suggest that 48 h steroid treatment increases expression of alpha4betadelta GABAR which replace the ambient receptor population. Increased anxiolytic effects of THIP were also observed following 48 h steroid treatment. The findings from the present study may be relevant for alterations in mood and benzodiazepine sensitivity reported across the menstrual cycle.

[Diagnosis and surgical treatment of 102 cases of ventricular septal defect with patent ductus arteriosus].

OBJECTIVE: To summarize the experience of diagnosis and surgical treatment of ventricular septal defect with patent ductus arteriosus. METHODS: We retrospectively analyzed the clinical data of 102 cases of ventricular septal defect combined with patent ductus arteriosus who underwent surgical treatment. Preoperative ultrasonic cardiogram (UCG) showed ventricular septal defect combined with patent ductus arteriosus in 82 cases and ventricular septal defect in 20 cases. RESULTS: The hospital mortality was 4.9% (5/102). The reasons for death included low cardiac output syndrome (1 case), pulmonary hypertension crisis (2 cases) and respiratory failure (2 cases). In the remaining patients,the perioperative complications included lung infection (7 cases), pulmonary atelectasis (5 cases), hydrothorax (1 case), and pulmonary hypertension crisis (2 cases); and all the 15 patients recovered lastly. The pulmonary hypertension of all living patients decreased to some degree. The therapeutical effectiveness was satisfactory. CONCLUSION: Ventricular septal defect with patent ductus arteriosus is easy to be confused with ventricular septal defect clinically. At the same time,it is diffcult to form a correct diagnosis in some patients by UCG preoperatively. To prevent the occurrence of perfusive lung, it is important to reinforce preoperative diagnosis and exploration during operation. Because pulmonary hypertension in patients with ventricular septal defect with patent ductus arteriosus emerges early and develops quickly, it tends to result in organic pulmonary hypertension which can make patients lose operation chances and influence the long-term therapeutical effect. Surgical operation should be performed as soon as possible. Optimal operative timing and proper perioperative management play important roles in surgical results.

Neurosteroid administration and withdrawal alter GABAA receptor kinetics in CA1 hippocampus of female rats.

Withdrawal from the GABA-modulatory steroid 3alpha-OH-5alpha-pregnan-20-one (3alpha,5alpha-THP) following exposure of female rats to the parent compound progesterone (P) produces a syndrome characterized by behavioural excitability in association with up-regulation of the alpha4 subunit of the GABA(A) receptor (GABAR) in the hippocampus. Similar changes are seen after 48 h exposure to its stereoisomer, 3alpha,5beta-THP. Here, we further characterize the effects of P withdrawal on GABAR kinetics, using brief (1 ms) application of 5-10 mm GABA to outside-out patches from acutely isolated CA1 hippocampal pyramidal cells. Under control conditions, GABA-gated current deactivated biexponentially, with tau(fast) = 12-19 ms (45-60% of the current), and tau(slow) = 80-140 ms. P withdrawal resulted in marked acceleration of deactivation (tau(fast) = 3-7 ms and tau(slow) = 30-100 ms), as did 48 h exposure to 3alpha,5beta-THP (tau(fast) = 5-8 ms; tau(slow) = 40-120 ms). When recombinant receptors were tested in HEK-293 cells, a similar acceleration in tau(fast) was observed for alpha4beta2delta and alpha4beta2gamma2 GABARs, compared to alpha1beta2gamma2 and alpha5beta2gamma2 receptors. In addition, tau(slow) was also accelerated for alpha4beta2delta receptors, which are increased following steroid withdrawal. As predicted by the Jones-Westbrook model, this change was accompanied by reduced receptor desensitization as well as an acceleration of the rate of recovery from rapid desensitization. A theoretical analysis of the data suggested that steroid treatment leads to receptors with a greater stability of the bound, activatable state. This was achieved by altering multiple parameters, including desensitization and gating rates, within the model. These results suggest that fluctuations in endogenous steroids result in altered GABAR kinetics which may regulate neuronal excitability.

Ethanol administration rapidly reverses alpha4 GABAA receptor subunit upregulation following steroid exposure.

Both short-term (48 h) exposure to the neuroactive steroid 3alpha,5alpha[beta]-THP and its withdrawal increase expression of the benzodiazepine (BDZ)-insensitive GABAA receptor (GABAR) alpha4 subunit in hippocampus. This increase in alpha4 subunit expression was associated with a relative insensitivity of CA1 hippocampal pyramidal cells to modulation of GABA-gated current by the BDZ lorazepam (LZM), assessed using whole cell patch clamp techniques. Chronic ethanol is also known to regulate expression of the alpha4 subunit. Thus, in the present study we investigated the capacity of ethanol, administered in low doses across a 2 h period (0.5 g/kg, i.p., 3x), to suppress alpha4 expression produced by 48 h exposure to 3alpha,5 beta-THP in adult female rats. We show here that 2 h ethanol administration reverses the increase in alpha4 expression normally observed following 48 h steroid treatment. This effect was correlated with a recovery of responses recorded from CA1 hippocampal pyramidal cells to the GABA-modulatory effects of LZM. Similar effects of ethanol in suppressing alpha4 expression and restoring LZM responsiveness were seen following steroid withdrawal when alpha4 expression is normally increased. These results suggest that increases in expression of the alpha4 subunit produced by steroid exposure or withdrawal are altered by other GABA-modulatory drugs, such as ethanol.

Hormonally regulated alpha(4)beta(2)delta GABA(A) receptors are a target for alcohol.

Here we report that low concentrations of alcohol (1-3 mM) increased Cl(-) currents gated by a recombinant GABA(A) receptor, alpha(4)beta(2)delta, by 40-50% in Xenopus laevis oocytes. We also found greater hippocampal expression of receptors containing alpha(4) and delta subunits, using a rat model of premenstrual syndrome (PMS) in which 1-3 mM alcohol preferentially enhanced GABA-gated currents, and low doses of alcohol attenuated anxiety and behavioral reactivity. The alcohol sensitivity of delta-containing receptors may underlie the reinforcing effects of alcohol during PMS, when eye saccade responses to low doses of alcohol are increased.