GABA receptors inhibited by benzodiazepines mediate fast inhibitory transmission in the central amygdala

The amygdala is intimately involved in emotional behavior, and its role in the generation of anxiety and conditioned fear is well known. Benzodiazepines, which are commonly used for the relief of anxiety, are thought to act by enhancing the action of the inhibitory transmitter GABA. We have examined the properties of GABA-mediated inhibition in the amygdala. Whole-cell recordings were made from neurons in the lateral division of the central amygdala. Application of GABA evoked a current that reversed at the chloride equilibrium potential. Application of the GABA antagonists bicuculline or SR95531 inhibited the GABA-evoked current in a manner consistent with two binding sites. Stimulation of afferents to neurons in the central amygdala evoked an IPSC that was mediated by the release of GABA. The GABA(A) receptor antagonists bicuculline and picrotoxin failed to completely block the IPSC. The bicuculline-resistant IPSC was chloride-selective and was unaffected by GABA(B)-receptor antagonists. Furthermore, this current was insensitive to modulation by general anesthetics or barbiturates. In contrast to their actions at GABA(A) receptors, diazepam and flurazepam inhibited the bicuculline-resistant IPSC in a concentration-dependent manner. These effects were fully antagonized by the benzodiazepine site antagonist Ro15-1788. We conclude that a new type of ionotropic GABA receptor mediates fast inhibitory transmission in the central amygdala. This receptor may be a potential target for the development of new therapeutic strategies for anxiety disorders.

Public health implications of new guidelines for lead in drinking water: a case study in an area with historically high water lead levels

Concern about the neurotoxicity of lead, particularly in infants and young children, has led to a revision of blood lead levels which are considered to involve an acceptable level of human exposure. Drinking water guidelines have also been reviewed in order to reduce this source of population exposure to lead. In the last 20 years, guidelines have been reduced from 100 to 50 to 10 mu g/litre. Lead in tap water used to be a major public health problem in Glasgow because of the high prevalence of houses with lead service pipes, the low pH of the public water supply and the resulting high levels of lead in water used for public consumption. Following two separate programmes of water treatment, involving the addition of lime and, a decade later, lime supplemented with orthophosphate, it is considered that maximal measures have been taken to reduce lead exposure by chemical treatment of the water supply. Any residual problem of public exposure would require large scale replacement of lead service pipes. In anticipation of the more stringent limits for lead in drinking water, we set out to measure current lead exposure From tap water in the population of Glasgow served by the Loch Katrine water supply. to compare the current situation with 12 years previously and to assess the public health implications of different limits. The study was based on mothers of young children since maternal blood lead concentrations and the domestic water that mothers use to prepare bottle feeds are the principal sources of foetal and infant lead exposure. An estimated 17% of mothers lived in households with tap water lead concentrations of 10 mu g/litre (the WHO guideline) or above in 1993 compared with 49% in 1981. Mean maternal blood lead concentrations fell by 69% in 12 years. For a given water lead concentration, maternal blood lead concentrations were 67% lower. The mean maternal blood lead concentration was 3.7 mu g/litre in the population at large, compared with 3.3 mu g/litre in households with negligible or absent tap water lead. Nevertheless, between 63% and 76% of cases of mothers with blood lead concentrations of 10 mu g/dl or above were attributable to tap water lead. The study found that maternal blood lead concentrations were well within limits currently considered safe for human health. About 15% of infants may be exposed via bottle feeds to tap water lead concentrations that exceed the WHO guideline of 10 mu g/litre. In the context of the health and social problems which affect the well-being and development of infants and children in Glasgow, however, current levels of lend exposure are considered to present a relatively minor health problem. (C) 2000 Elsevier Science Ltd. All rights reserved.

Age-related differences in physical activity levels of young adults

Purpose: To examine age-related differences in the physical activity behaviors of young adults. Methods: We examined rates of participation in vigorous- and moderate-intensity leisure-time activity and walking, as well as an index of physical activity sufficient for health benefits in three Australian cross-sectional samples, for the age ranges of 18-19, 20-24, and 25-29 yr. Data were collected in 1991, 1996, and 1997/8. Results: There was at least a 15% difference in vigorous-intensity leisure-time physical activity from the 18-19 yr to the 25-29 yr age groups, and at least a 10% difference in moderate-intensity leisure-time physical activity. For the index of sufficient activity there was a difference between 9 and 21% across age groups. Differences in rates of walking were less than 8%. For all age groups, males had higher rates of participation for vigorous and moderate-intensity activity than did females, bur females had much higher rates of participation in walking than males. Age-associated differences in activity levels were more apparent for males. Conclusions: Promoting walking and various forms of moderate-intensity physical activities to young adult males, and encouraging young adult females to adopt other forms of moderate-intensity activity to complement walking may help to ameliorate decreases in physical activity over the adult lifespan.

Eutectic nucleation and growth in hypoeutectic Al-SI alloys at different strontium levels

The effects of different levels of strontium on nucleation and growth of the eutectic in a commercial hypoeutectic Al-Si foundry alloy have been investigated by optical microscopy and electron backscattering diffraction (EBSD) mapping by scanning electron microscopy (SEM). The microstructural evolution of each specimen during solidification was studied by a quenching technique at different temperatures and Sr contents. By comparing the orientation of the aluminum in the eutectic to that of the surrounding primary aluminum dendrites by EBSD, the eutectic formation mechanism could be determined. The results of these studies show that the eutectic nucleation mode, and subsequent growth mode, is strongly dependent on Sr level. Three distinctly different eutectic growth modes were found, in isolation or sometimes together, but different for each Sr content. At very low Sr contents, the eutectic nucleated and grew from the primary phase. Increasing the Sr level to between 70 and 110 ppm resulted in nucleation of independent eutectic grains with no relation to the primary dendrites. At a Sr level of 500 ppm, the eutectic again nucleated on and grew from the primary phase while a well-modified eutectic structure was still present. A slight dependency of eutectic growth radially from the mold wall opposite the thermal gradient was observed in all specimens in the early stages of eutectic solidification.

Mutant GABA(A) receptor gamma 2-subunit in childhood absence epilepsy and febrile seizures

Epilepsies affect at least 2% of the population at some time in life, and many forms have genetic determinants(1,2). We have found a mutation in a gene encoding a GABA, receptor subunit in a large family with epilepsy. The two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS), There is a recognized genetic: relationship between FS and CAE, yet the two syndromes have different ages of onset, and the physiology of absences and convulsions is distinct. This suggests the mutation has age-dependent effects on different neuronal networks that influence the expression of these clinically distinct, but genetically related, epilepsy phenotypes. We found that the mutation in GABRG2 (encoding the gamma2-subunit) abolished in vitro sensitivity to diazepam, raising the possibility that endozepines do in fact exist and have a physiological role in preventing seizures.

Truncation of the GABA(A)-receptor gamma 2 subunit in a family with generalized epilepsy with febrile seizures plus

Recent findings from studies of two families have shown that mutations in the GABA(A)-receptor gamma2 subunit are associated with generalized epilepsies and febrile seizures. Here we describe a family that has generalized epilepsy with febrile seizures plus (GEFS(+)), including an individual with severe myoclonic epilepsy of infancy, in whom a third GABA(A)-receptor gamma2-subunit mutation was found. This mutation lies in the intracellular loop between the third and fourth transmembrane domains of the GABA(A)-receptor gamma2 subunit and introduces a premature stop codon at Q351 in the mature protein. GABA sensitivity in Xenopus laevis oocytes expressing the mutant gamma2(Q351X) subunit is completely abolished, and fluorescent-microscopy studies have shown that receptors containing GFP-labeled gamma2(Q351X) protein are retained in the lumen of the endoplasmic reticulum. This finding reinforces the involvement of GABA(A) receptors in epilepsy.

Weight reduction in patients with chronic HCV reduces circulating insulin levels

Steatosis occurs in >50% of patients with chronic HCV. In patients with viral genotype 3, steatosis may be a cytopathic effect of the virus. However in many patients with HCV, the pathogenesis of steatosis appears to be the same as for patients with non-alcoholic fatty liver disease (NAFLD) ie related to increased body mass index (BMI). We studied the effect of a 12 week weight reduction program on metabolic parameters in subjects with chronic HCV genotype 1 (Group 1, n = 16), genotype 3 (Group 2, n = 13) and patients with NAFLD (Group 3, n = 13). A liver biopsy was performed prior to and 3-6 months after the intervention period in 15 patients. The mean (SD) BMI of subjects in groups 1, 2 and 3 was 30.7 (4.0), 29.0 (5.2) and 33.3 (7.7), respectively. There was no significant difference in the amount of weight loss, change in waist circumference, change in ALT or reduction in steatosis between the 3 groups. Mean (SD) weight loss was 5.1 (3.7) kg. In those patients who lost weight, serum insulin (mean (SD) mU/L) changed from 17.8 (7.8) to 11.5 (4.8) (p = 0.003), 12.4 (5.0) to 8.4 (4.3) (p = 0.02), and 16.9 (7.3) to 17.8 (8.1) (p = 0.76) in Groups 1, 2 and 3, respectively. A small amount of weight loss is associated with a reduction in circulating insulin levels in patients with chronic HCV, particularly in genotype 1. In patients with NAFLD, the lack of a significant decrease in circulating insulin with weight reduction may reflect the higher initial BMI or may be due to the pathogenesis of this disorder.

Coaxial flattening at deep levels of orogenic belts: evidence from blueschists and eclogites on Syros and Sifnos (Cyclades, Greece)

This work presents new Structural data from a high-pressure/low-temperature (HP/LT) metamorphic terrane exposed on the islands of Syros and Sifnos (Cyclades, Greece). The structure and the metamorphism of a relatively coherent HP/LT rock section were studied in order to elucidate how strain was accommodated at deep crustal levels during the formation and exhumation of HP/LT rocks. At least three deformation phases associated with eclogite- and blueschist-facies conditions (P = 8-15 kbar; T = 400-550 degreesC) were recognised. The earliest deformation fabric (S1), preserved as inclusion trails within garnet porphyroblasts, is aligned to define a sub-vertical schistosity (at present orientation), which is frequently orthogonal to the flat matrix schistosity (S2), and may indicate that deep crustal thickening involved upright folding. The currently dominant fabric in the HP rock section, S2, is Usually moderately dipping and locally contains NW-trending glaucophane lineations, symmetric pressure-shadows and eclogitic boudins. The symmetric structures associated with this fabric seem to indicate coaxial vertical thinning, although the existence of non-coaxial structures out of the study area cannot be excluded. Glaucophane-bearing shear bands (S3), with top-to-NW sense of shearing, locally crosscut the earlier structures. The latest recognised fabric (D4) is scarce and often absent within the HP rocks. It is associated with top-to-NE kinematic criteria that formed at greenschist-facies conditions (P = 4-7 kbar; T = 400-450 degreesC). Based on these observations, it is suggested that partitioning of strain occurred at different crustal levels and at different times. Deep crustal deformation was governed by thickening via upright folding followed by coaxial vertical thinning, whereas non-coaxial shearing occurred when the rocks were already exhumed to relatively shallow crustal levels. The earliest fabrics (D1 to D3) pertain to Alpine orogenesis and possibly to syn-orogenic extension, whereas the latest correspond to whole-crust back-are extension. (C) 2002 Elsevier Science Ltd. All rights reserved.

Altered kinetics and benzodiazepine sensitivity of a GABA(A) receptor subunit mutation [gamma(2)(R43Q)] found in human epilepsy

The gamma-aminobutyric acid type A (GABA(A)) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABA(A) receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABA(A) gamma(2)-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABA(A) receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from alpha(1)beta(2)gamma(2) or alpha(1)beta(2)gamma(2)(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imiclazopyricline zolpidem. These results provide evidence of impaired GABA(A) receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6') common to both the glycine receptor (GlyR) and gamma-aminobutyric acid, type A receptor (GABAAR) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter, and the main pore blocker binding site. Despite their high amino acid sequence homologies and common role in conducting chloride ions, recent studies have suggested that the GlyRs and GABA(A)Rs have divergent open state pore structures at the 6' position. When both the human alpha1(T6'C) homomeric GlyR and the rat alpha1(T6'C)beta1(T6'C) heteromeric GABA(A)R were expressed in human embryonic kidney 293 cells, their 6' residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidizing agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABA(A)R pore structure at the 6' level may vary between different expression systems.

Larval activity levels and delayed metamorphosis affect post-larval performance in the colonial, ascidian Diplosoma listerianum

It is becoming widely recognized that extending the larval period of marine invertebrates, especially of species with non-feeding larvae, can affect post-larval performance. As these carry-over effects are presumed to be caused by the depletion of larval energy reserves, we predicted that the level of larval activity would also affect post-larval performance. This prediction was tested with the cosmopolitan colonial ascidian Diplosoma listerianum in field experiments in southern Australia. Diplosoma larvae, brooded in the parent colony, are competent to settle immediately after spawning, and they remain competent to metamorphose for > 15 h. Some larvae were induced to metamorphose 0 to 6 h after release, whilst others were induced to swim actively by alternating light and dark periods for up to 3 h prior to metamorphosis. Juvenile colonies were then transplanted to a subtidal field site in Port Phillip Bay and left to grow for up to 3 wk. Extending the larval period and increasing the amount of swimming both produced carry-over effects on post-larval performance. Colonies survived at different rates among experiments, but larval experience did not affect survival rates. Delays in metamorphosis and increased swimming activity did, however, reduce colony growth rates dramatically, resulting in 50% fewer zooids per colony. Moreover, such colonies produced initial zooids with smaller feeding structures, with the width of branchial baskets reduced by 10 to 15%. These differences in branchial basket size persisted and were still apparent in newly budded zooids 3 wk after metamorphosis. Our results suggest that, for D. listerianum, larval maintenance, swimming, and metamorphosis all use energy from a common pool, and increases in the allocation to maintenance or swimming come at the expense of post-larval performance.

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6’) common to both the glycine receptor (GlyR) and GABAA receptor (GABAAR) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter and the main pore blocker binding site. Recent studies have suggested that the GlyRs and GABAARs have divergent open state pore structures at the 6’ position. When both the human a1T6’C homomeric GlyR and the rat a1T6’Cb1T6’C heteromeric GABAAR were expressed in HEK293 cells, their 6’ residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidising agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABAAR pore structure at the 6’ level may vary between different expression systems.