Autoradiographic imaging of altered synaptic alphabetagamma2 and extrasynaptic alphabeta GABAA receptors in a genetic mouse model of anxiety

To image the possible alterations in brain regional GABAA receptor subtype properties in a genetic animal model of human anxiety, mice heterozygous for the deletion of GABAA receptor gamma2 subunit (gamma2+/-) were studied using ligand autoradiographic assays on brain cryostat sections. The [35S]TBPS binding assay was designed to reveal impaired GABA and channel site coupling shown to be more prominent in recombinant alpha1/6beta3 than in alpha1/2beta3gamma2 or beta2 subunit-containing GABAA receptors expressed in HEK 293 cells. Increased GABA-insensitive [35 S]TBPS binding in the gamma2+/- mouse brains was evident in the cerebral cortex and in subcortical regions, the alterations being regionally similar to the loss of gamma2 subnunit-dependent benzodiazepine (BZ) sites as revealed by [3H]Ro 15-4513 autoradiography. As the gamma2 subunit protein is needed for synaptic clustering of GABAA receptors, these results indicate that the extrasynaptic alphabeta3 receptors can be visualized in vitro as atypical GABA-insensitive [35S]TBPS binding sites. The results suggest that GABAAergic synaptic inhibition is widely decreased in the brains of anxiety-prone gamma2+/- mice, while extrasynaptic GABAA receptors are increased. These autoradiographic imaging findings further demonstrate the need to develop GABAA receptor subtype-selective in vivo ligands to aid in assessing the contributions of various subcellular receptor populations in anxious and other patient groups.     

Tolerance to Diazepam-Induced Motor Impairment: A Study with GABAA Receptor α6 Subunit Knockout Mice

Development of tolerance to motor-impairing effects of repeated administration of moderate diazepam doses (5.0–7.5 mg/kg; three times daily PO 3 weeks) was compared between mice deficient in the cerebellar granule cell–restricted GABA_A receptor 6 subunit and their wild-type controls. The 6–/– mice were more impaired by the initial challenge doses of diazepam (5 or 10 mg/kg) than their controls, but acquired partial tolerance by the second tests with the same doses 4–7 days later. Chronic treatment produced complete tolerance in both mouse lines. Ligand autoradiography revealed a significant reduction in baseline benzodiazepine and chloride channel site-bindings in various regions of the 6–/– brains, but the chronic diazepam treatment did not consistently alter baseline or benzodiazepine site agonist and inverse agonist-modulated binding in the 6–/– and wildtype mice. The results indicate that tolerance to motor-impairing actions of diazepam is independent of the diazepam-insensitive 6 subunit-containing receptors, which rules out the possibility that tolerance emerges as an increase in structurally benzodiazepine-insensitive receptor population.     

Spatio‐temporal scaling of biodiversity and the species–time relationship in a stream fish assemblage

1. The increase of species richness with the area of the habitat sampled, that is the species–area relationship, and its temporal analogue, the species–time relationship (STR), are among the few general laws in ecology with strong conservation implications. However, these two scale‐dependent phenomena have rarely been considered together in biodiversity assessment, especially in freshwater systems. 2. We examined how the spatial scale of sampling influences STRs for a Central‐European stream fish assemblage (second‐order Bernecei stream, Hungary) using field survey data in two simulation‐based experiments. 3. In experiment one, we examined how increasing the number of channel units, such as riffles and pools (13 altogether), and the number of field surveys involved in the analyses (12 sampling occasions during 3 years), influence species richness. Complete nested curves were constructed to quantify how many species one observes in the community on average for a given number of sampling occasions at a given spatial scale. 4. In experiment two, we examined STRs for the Bernecei fish assemblage from a landscape perspective. Here, we evaluated a 10‐year reach level data set (2000–09) for the Bernecei stream and its recipient watercourse (third‐order Kemence stream) to complement results on experiment one and to explore the mechanisms behind the observed patterns in more detail. 5. Experiment one indicated the strong influence of the spatial scale of sampling on the accumulation of species richness, although time clearly had an additional effect. The simulation methodology advocated here helped to estimate the number of species in a diverse combination of spatial and temporal scale and, therefore, to determine how different scale combinations influence sampling sufficiency. 6. Experiment two revealed differences in STRs between the upstream (Bernecei) and downstream (Kemence) sites, with steeper curves for the downstream site. Equations of STR curves were within the range observed in other studies, predominantly from terrestrial systems. Assemblage composition data suggested that extinction–colonisation dynamics of rare, non‐resident (i.e. satellite) species influenced patterns in STRs. 7. Our results highlight that the determination of species richness can benefit from the joint consideration of spatial and temporal scales in biodiversity inventory surveys. Additionally, we reveal how our randomisation‐based methodology may help to quantify the scale dependency of diversity components (α, β, γ) in both space and time, which have critical importance in the applied context.     

Cardiovascular effects of the combination of levosimendan and valsartan in hypertensive Dahl/Rapp rats

Hypertension is the main risk factor for left ventricular hypertrophy and development of diastolic heart failure. There is no yet treatment, which can effectively reduce mortality in patients suffering from heart failure with preserved systolic function. We tested whether the calcium sensitizer levosimendan and the AT1-receptor antagonist valsartan could protect from salt-induced hypertension, cardiovascular mortality and heart failure in Dahl/Rapp salt-sensitive rats fed for 7 weeks with a high salt diet (8% NaCl). Levosimendan (1 mg/kg/day via drinking water) and valsartan (30 mg/kg in the food) monotherapies and their combination prevented mortality in Dahl/Rapp rats. The drug combination evoked an additive effect on blood pressure, cardiac hypertrophy, cardiomyocyte cross-sectional area, target organ damage and myocardial ANP mRNA expression. There was a close correlation between systolic blood pressure and cardiac hypertrophy, cardiac and renal damage. As compared to Dahl/Rapp controls kept on low-salt diet (NaCl 0.3%). The high salt rats exhibited impaired diastolic relaxation as assessed by isovolumic relaxation time. Levosimendan alone and in combination with valsartan, improved diastolic relaxation without significantly improving systolic function. Our findings are evidence for an additive effect between levosimendan and valsartan on blood pressure and a blood pressure-dependent protection against the development of salt-induced target organ damage. The present study also demonstrates that levosimendan, alone or in combination with valsartan, can correct diastolic dysfunction induced by salt-dependent hypertension.     

Kainic acid-induced status epilepticus alters GABA receptor subunit mRNA and protein expression in the developing rat hippocampus

Kainic acid-induced status epilepticus leads to structural and functional changes in inhibitory GABAA receptors in the adult rat hippocampus, but whether similar changes occur in the developing rat is not known. We have used in situ hybridization to study status epilepticus-induced changes in the GABAAalpha1-alpha5, beta1-beta3, gamma1 and gamma2 subunit mRNA expression in the hippocampus of 9-day-old rats during 1 week after the treatment. Immunocytochemistry was applied to detect the alpha1, alpha2 and beta3 subunit proteins in the control and treated rats. In the saline-injected control rats, the alpha1 and alpha4 subunit mRNA expression significantly increased between the postnatal days 9-16, whereas those of alpha2, beta3 and gamma2 subunits decreased. The normal developmental changes in the expression of alpha1, alpha2, beta3 and gamma2 subunit mRNAs were altered after the treatment. The immunostainings with antibodies to alpha1, alpha2 and beta3 subunits confirmed the in situ hybridization findings. No neuronal death was detected in any hippocampal subregion in the treated rats. Our results show that status epilepticus disturbs the normal developmental expression pattern of GABAA receptor subunit in the rat hippocampus during the sensitive postnatal period of brain development. These perturbations could result in altered functional and pharmacological properties of GABAA receptors.     

Regional muscle oxygenation differences in vastus lateralis during different modes of incremental exercise

Background

Near infrared spectroscopy (NIRS) is used to assess muscle oxygenation (MO) within skeletal muscle at rest and during aerobic exercise. Previous investigations have used a single probe placement to measure MO during various forms of exercise. However, regional MO differences have been shown to exist within the same muscle which suggests that different areas of the same muscle may have divergent MO. Thus, the aim of this study was to examine whether regional differences in MO exist within the same muscle during different types of incremental (rest, 25, 50, 75, 100 % of maximum) exercise (1 leg knee extension (KE), 2 leg KE, or cycling).

Methods

Nineteen healthy active males (Mean ± SD: Age 27 ± 4 yrs; VO_2max: 55 ± 11 mL/kg/min) performed incremental exercise to fatigue using each mode of exercise. NIRS probes were placed on the distal and proximal portion of right leg vastus lateralis (VL). Results were analyzed with a 3-way mixed model ANOVA (probe × intensity × mode).

Results

Differences in MO exist within the VL for each mode of exercise, however these differences were not consistent for each level of intensity. Comparison of MO revealed that the distal region of VL was significantly lower throughout KE exercise (1 leg KE proximal MO – distal MO = 9.9 %; 2 leg KE proximal MO – distal MO = 13 %). In contrast, the difference in MO between proximal and distal regions of VL was smaller in cycling and was not significantly different at heavy workloads (75 and 100 % of maximum).

Conclusion

MO is different within the same muscle and the pattern of the difference will change depending on the mode and intensity of exercise. Future investigations should limit conclusions on MO to the area under assessment as well as the type and intensity of exercise employed.

     

Association of FCGR2A and FCGR2A-FCGR3A haplotypes with susceptibility to giant cell arteritis

The Fc gamma receptors have been shown to play important roles in the initiation and regulation of many immunological and inflammatory processes and to amplify and refine the immune response to an infection. We have investigated the hypothesis that polymorphism within the FCGR genetic locus is associated with giant cell arteritis (GCA). Biallelic polymorphisms in FCGR2A, FCGR3A, FCGR3B and FCGR2B were examined for association with biopsy-proven GCA (n = 85) and healthy ethnically matched controls (n = 132) in a well-characterised cohort from Lugo, Spain. Haplotype frequencies and linkage disequilibrium (D') were estimated across the FCGR locus and a model-free analysis performed to determine association with GCA. There was a significant association between FCGR2A-131RR homozygosity (odds ratio (OR) 2.10, 95% confidence interval (CI) 1.12 to 3.77, P = 0.02, compared with all others) and carriage of FCGR3A-158F (OR 3.09, 95% CI 1.10 to 8.64, P = 0.03, compared with non-carriers) with susceptibility to GCA. FCGR haplotypes were examined to refine the extent of the association. The haplotype showing the strongest association with GCA susceptibility was the FCGR2A-FCGR3A 131R-158F haplotype (OR 2.84, P = 0.01 for homozygotes compared with all others). There was evidence of a multiplicative joint effect between homozygosity for FCGR2A-131R and HLA-DRB1*04 positivity, consistent with both of these two genetic factors contributing to the risk of disease. The risk of GCA in HLA-DRB1*04 positive individuals homozygous for the FCGR2A-131R allele is increased almost six-fold compared with those with other FCGR2A genotypes who are HLA-DRB1*04 negative. We have demonstrated that FCGR2A may contribute to the 'susceptibility' of GCA in this Spanish population. The increased association observed with a FCGR2A-FCGR3A haplotype suggests the presence of additional genetic polymorphisms in linkage disequilibrium with this haplotype that may contribute to disease susceptibility. These findings may ultimately provide new insights into disease pathogenesis.