Potency of several type I-benzodiazepine receptor ligands for inhibition of [3H]flunitrazepam binding in different rat brain tissues.

The potency of several different non-selective and type I-benzodiazepine receptor-selective ligands as inhibitors of [3H]flunitrazepam binding was measured in different rat brain tissues. In contrast to the non-selective ligands, which had a similar potency in all brain tissues investigated, the potency of the type I-receptor-selective ligands was highest in cerebellar membranes and varied in other brain tissues.     

Estimating the efficiency of benzodiazepines on GABA(A) receptors comprising gamma1 or gamma2 subunits

Background and purpose:     

Localization of GABA-A receptor alpha subunits on neurochemically distinct cell types in the rat locus coeruleus

The locus coeruleus (LC) provides the major source of noradrenaline to the central nervous system and is modulated by neurochemically diverse afferents. LC function is central to arousal, memory, cognition and the stress response, with dysfunction of the LC-noradrenergic axis implicated in debilitating psychiatric disorders. The precise targeting of neurotransmitter receptors within the LC is essential for processing the information contained in diverse afferents and thus LC output. The inhibitory modulation of LC neurons is thought to be effected mainly through GABA-A receptors (GABA(A)Rs). Diverse GABA(A)Rs are pentameric complexes assembled from a repertoire of subunits resulting in substantial diversity in their molecular, functional and pharmacological properties throughout the brain. The precise location of distinct GABA(A) R subunits in subregions of the LC, and the neurochemical identity of the cells that express them, remains to be determined. Here, we show that the GABA(A)R alpha1 subunit is expressed exclusively in neurochemically and morphologically diverse non-noradrenergic cell types within the LC, which may innervate the principal noradrenergic cells. Thus, the GABA(A)R alpha1 subunit could provide a neurochemical signature for a pool of local circuit interneurons in the LC. In contrast, non-overlapping GABA(A)R alpha2 and alpha3 subunit-immunoreactive puncta were enriched on noradrenergic dendrites and, to a lesser extent, on somata. The study reveals a cell-type- and domain-specific expression pattern of distinct GABA(A)R subunits in the LC. These data will serve as a template for understanding inhibitory modulation of this region and facilitate more directed pharmacological strategies for disorders arising from the impairment of LC function.     

Histaminergic pharmacology of homo-oligomeric β3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology

A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric β3 GABA(A) receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to β3 GABA(A) receptors with micromolar affinity (K(D)<300 μM), thus extending previous evidence that β3 GABA(A) receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with β3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric β3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for β3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether β3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site.     

Benzodiazepine-induced spatial learning deficits in rats are regulated by the degree of modulation of α1 GABAA receptors

Despite significant advances in understanding the role of benzodiazepine (BZ)-sensitive populations of GABA_A receptors, containing the α₁, α₂, α₃ or α₅ subunit, factual substrates of BZ-induced learning and memory deficits are not yet fully elucidated. It was shown that α₁-subunit affinity-selective antagonist β-CCt almost completely abolished spatial learning deficits induced by diazepam (DZP) in the Morris water maze. We examined a novel, highly (105 fold) α₁-subunit selective ligand—WYS8 (0.2, 1 and 10 mg/kg), on its own and in combination with the non-selective agonist DZP (2 mg/kg) or β-CCt (5 mg/kg) in the water maze in rats. The in vitro efficacy study revealed that WYS8 acts as α₁-subtype selective weak partial positive modulator (40% potentiation at 100 nM). Measurement of concentrations of WYS8 and DZP in rat serum and brain tissues suggested that they did not substantially cross-influence the respective disposition. In the water maze, DZP impaired spatial learning (acquisition trials) and memory (probe trial). WYS8 caused no effect per se, did not affect the overall influence of DZP on the water-maze performance and was devoid of any activity in this task when combined with β-CCt. Nonetheless, an additional analysis of the latency to reach the platform and the total distance swam suggested that WYS8 addition attenuated the run-down of the spatial impairment induced by DZP at the end of acquisition trials. These results demonstrate a clear difference in the influence of an α₁ subtype-selective antagonist and a partial agonist on the effects of DZP on the water-maze acquisition.     

Austrian Joint ?GGH-?GIR-?GHO-ASSO position statement on the use of transarterial chemoembolization (TACE) in hepatocellular carcinoma

Transarterial chemoembolization (TACE) of hepatocellular carcinoma (HCC) is one of the most frequently applied standard treatments for this disease. The role for TACE is fairly well defined within the most widely used treatment algorithm for HCC, die Barcelona Clinic Liver Cancer (BCLC) staging system and treatment algorithm. But no general treatment algorithm will go into the technical details of any procedure and several patients will not fit ideally into the patient groups predefined in BCLC or any other treatment algorithm. Furthermore, indications and contraindications sometimes are viewed differently by the various medical specialties involved in taking care of such patients. We present here the joint expert position statement of the Austrian Societies of Gastroenterology and Hepatology (?GGH), Interventional Radiology (?GIR), Hematology and Oncology (?GHO), and Surgical Oncology (ASSO) on the technical aspects, indications, and contraindication for the use of TACE in the management of HCC.     

New insights on the role of gephyrin in regulating both phasic and tonic GABAergic inhibition in rat hippocampal neurons in culture

Gephyrin is a tubulin-binding protein that acts as a scaffold for clustering glycine and GABA_A receptors at postsynaptic sites. In this study, the role of gephyrin on GABA_A receptor function was assessed at the post-translational level, using gephyrin-specific single chain antibody fragments (scFv-gephyrin). When expressed in cultured rat hippocampal neurons as a fusion protein containing a nuclear localization signal, scFv-gephyrin were able to remove endogenous gephyrin from GABA_A receptor clusters. Immunocytochemical experiments revealed a significant reduction in the number of synaptic γ2-subunit containing GABA_A receptors and a significant decrease in the density of the GABAergic presynaptic marker vesicular GABA transporter (VGAT). These effects were associated with a slow down of the onset kinetics, a reduction in the amplitude and in the frequency of miniature inhibitory postsynaptic currents (mIPSCs). The quantitative analysis of current responses to ultrafast application of GABA suggested that changes in onset kinetics resulted from modifications in the microscopic gating of GABA_A receptors and in particular from a reduced entry into the desensitized state. In addition, hampering gephyrin function with scFv-gephyrin induced a significant reduction in GABA_A receptor-mediated tonic conductance. This effect was probably dependent on the decrease in GABAergic innervation and in GABA release from presynaptic nerve terminals. These results indicate that gephyrin is essential not only for maintaining synaptic GABA_A receptor clusters in the right position but also for regulating both phasic and tonic inhibition.