Expression of the kcnk3 potassium channel gene lessens the injury from cerebral ischemia, most likely by a general influence on blood pressure.

We examined the possible protective effect of TASK-1 (TWIK-related acid-sensitive potassium channel-1, kcnk3) and -3 potassium channels during stroke. TASK-1 and TASK-3, members of the two pore domain (K2P or kcnk) potassium channel family, form hetero or homodimers and help set the resting membrane potential. We used male TASK-1 and TASK-3 knockout mice in a model of focal cerebral ischemia, permanent middle cerebral artery occlusion (pMCAO). Infarct volume was measured 48 h after pMCAO. The TASK-1 knockout brains had larger infarct volumes (P=0.004), and those in TASK-3 knockouts were unchanged. As the TASK-1 gene is expressed in adrenal gland, heart and possibly blood vessels, the higher infarct volumes in the TASK-1 knockout mice could be due to TASK-1 regulating blood vessel tone and hence blood pressure or influencing blood vessel microarchitecture and blood flow rate. Indeed, we found that male TASK-1 knockout mice had reduced blood pressure, likely explaining the increased brain injury seen after pMCAO. Thus to make precise conclusions about how TASK-1 protects neurons, neural- or organ-specific deletions of the gene will be needed. Nevertheless, a consequence of having TASK-1 channels expressed (in various non-neuronal tissues and organs) is that neuronal damage is lessened when stroke occurs.

Changes in expression of some two-pore domain potassium channel genes (KCNK) in selected brain regions of developing mice.

Two P loop domain potassium (K2P or KCNK) channels produce transmitter-modulated K+ currents that could influence brain development. We mapped by in situ hybridization the expression of the K2P gene family in the developing mouse brain. All the K2P genes had different expression patterns, and it is likely that many neuronal types change their K2P channel subunit composition during development. Fitting with a possible role in the control of cell division, three K2P genes (tandem of P domains in a weak inwardly-rectifying K+ channel-related K+ channel (TREK) -1, TREK-2 and weak inwardly-rectifying K+ channel-related acid-sensitive K+ channel (TASK) -2) had high expression in the embryonic subventricular and ventricular zones, and the tandem of P domains in a weak inwardly-rectifying K+ channel (TWIK) -1, TREK-1, TREK-2 and TASK-3 genes were significantly expressed in the external cerebellar granule cell layer. There were also some clear changes in developmental expression of the K2P genes: for example, TREK-1 goes from high to low expression in post-migratory cerebellar granule cells; TREK-2 has one of the highest expressions in the embryonic and early postnatal brain of any K2P gene, but transcript levels fall strongly in the postnatal periods, except for cerebellar granule cells. TASK-1 and tandem pore domain halothane-inhibited K+ channel (THIK) -2 genes both turn on specifically in post-migratory cerebellar granule cells, whereas the TASK-3 gene, for example, is strongly expressed in pre-migratory cells as well as post-migratory cells. On the other hand, young postnatal dentate granule cells express TWIK-1, TREK-1 and TREK-2 before P7, but TASK-3 expression only begins to become clear in these cells in the second postnatal week. THIK-2 mRNA was up-regulated with TASK-1 and TASK-3 transcripts in cerebella of GABAA receptor alpha6 subunit knockout mice, possibly implying a functional association of THIK-2, TASK-1 and TASK-3.

Abolition of zolpidem sensitivity in mice with a point mutation in the GABAA receptor gamma2 subunit.

Agonists of the allosteric benzodiazepine site of GABAA receptors bind at the interface of the alpha and gamma subunits. Here, we tested the in vivo contribution of the gamma2 subunit to the actions of zolpidem, an alpha1 subunit selective benzodiazepine agonist, by generating mice with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the gamma2 subunit. The gamma2F77I mutation has no major effect on the expression of GABAA receptor subunits in the cerebellum. The potency of zolpidem, but not that of flurazepam, for the inhibition of [3H]flunitrazepam binding to cerebellar membranes is greatly reduced in gamma2I77/I77 mice. Zolpidem (1 microM) increased both the amplitude and decay of miniature inhibitory postsynaptic currents (mIPSCs) in Purkinje cells of control C57BL/6 (34% and 92%, respectively) and gamma2F77/F77 (20% and 84%) mice, but not in those of gamma2F77I mice. Zolpidem tartrate had no effect on exploratory activity (staircase test) or motor performance (rotarod test) in gamma2I77/I77 mice at doses up to 30 mg/kg (i.p.) that strongly sedated or impaired the control mice. Flurazepam was equally effective in enhancing mIPSCs and disrupting performance in the rotarod test in control and gamma2I77/I77 mice. These results show that the effect of zolpidem, but not flurazepam, is selectively eliminated in the brain by the gamma2F77I point mutation.

Cerebellar granule cell Cre recombinase expression.

The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells.

Distribution of the gamma-aminobutyric acid(A) receptor complex alpha 5 subunit in chick brain. An immunocytochemical and autoradiographic study.

This work reports the distribution of the gamma-aminobutyric acid(A) (GABA(A)) receptor complex alpha5 subunit in the chick using an antibody raised against this subunit in the rat, an immunoprecipitation study and a comparative autoradiographic study using [(3)H]flunitrazepam in the presence of 1 microM zolpidem, which is considered to bind only to those areas presenting the alpha5 subunit. The specificity of the antibody for the chick GABA(A) receptor complex alpha5 subunit is supported by the similar bands obtained by Western blotting from rat and chick, the immunoprecipitation study and the general agreement in the distribution and pattern of labelling of this antibody in both species. The immunocytochemical and autoradiographic distributions in both the chick and rat are compared and some areas with disagreement between these distributions are discussed. The general conclusion is that the alpha5 subunit of the GABA(A) complex receptor seems to have been conserved along evolution.

The subcommissural organ of the frog Rana perezi is innervated by nerve fibres containing GABA.

The innervation of the frog subcommissural organ was studied by light-microscopic and ultrastructural immunocytochemistry using antisera against serotonin, noradrenaline, dopamine, gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, different GABA receptor subunits and bovine Reissner's fibre material (AFRU). In the proximity of the organ, serotonin- and noradrenaline-containing fibres were rare whereas dopamine-immunoreactive fibres were more numerous. Many GABA- and glutamic acid decarboxylase-containing nerve fibres were found at the basal portion of the ependymal cells of the subcommissural organ. Under the electron microscope, these GABA-immunolabelled nerve endings appeared to establish axoglandular synapses with secretory ependymal cells of the subcommissural organ. In addition, the secretory ependymal cells expressed high amounts of the beta2-subunit of the GABA(A) receptor. Since GABA-immunoreactive neurons were present in the frog pineal organ proper and apparently contributed axons to the pineal tract, we suggest that at least part of the GABAergic fibres innervating the frog subcommissural organ could originate from the pineal organ.

Effect of surgical stress on benzodiazepine receptors as a consequence of placebo pellet implantation in rat: an autoradiographic study.

This paper reports modifications in benzodiazepine (BZ) receptors induced by minimally invasive surgery, such as pellet implantation, a widely used surgical process for chronic drug administration. The intrinsic stress induced by this manipulation on BZ receptors was analysed in an autoradiographic saturation study determining the affinity (K(D)) and total number (Bmax) of binding sites of a number of brain areas from the mesencephalon, cerebral cortex, hippocampus, and cerebellum. The radioligand used for the study was [3H]flunitrazepam, which permitted overall characterization of BZ binding sites. Use of the specific BZ1 agonist zolpidem as an inhibitor of this radioligand permitted the direct characterization of subtype 2 (BZ2) and the indirect characterization of subtype 1 receptor (BZ1). Significant increases in Bmax and K(D) values were observed in pellet-implanted animals with respect to those not implanted. The results support the notion of an up-regulation of these receptors, mainly in BZ1 receptors, following sustained desensitization as a result of the surgical stress induced by pellet implantation.

Distribution of the GABAA receptor complex beta 2/3 subunits in the brain of the frog Rana pipiens.

This report describes the distribution of labeling of the monoclonal antibody bd-17 against the beta 2/3 subunits of the mammalian GABAA receptor complex throughout the brain of the frog Rana pipiens. The distribution matches quite closely those in homologous brain regions as previously described for this antibody in fishes, birds, and mammals, indicating that this antibody also labels beta 2/3 subunits of frog. A semiquantitative analysis of the distribution of labeling throughout the brain is based upon relative optical densities with respect to the structure showing maximal optical density in each brain, using standard illumination conditions. Comparison with distributions in birds and mammals suggests that these GABAA receptor complex subunits are strongly conserved in vertebrate evolution and play an important role in the visual, auditory, olfactory and motor systems.

Identification of beta-adrenoceptors in rat lymph nodes and spleen: an autoradiographic study.

The anatomical localization of beta 1 and beta 2-adrenoceptors was studied in rat lymphoid tissues by quantitative autoradiography using [125I]cyanopindolol as a ligand. In lymph nodes, a significant density of these receptors was found in the medullary cords and the interfollicular cortex, while only low densities were observed in the paracortex. No detectable binding appeared in the remaining areas. In the spleen, these receptors were mainly localized in the capsule, marginal zone of white pulp and red pulp, while the labelling over the white pulp was extremely low. The subtype beta 2 was predominant in both lymph nodes and spleen. The results suggest that beta-adrenoceptors are present in mature cells in lymphoid tissues and are probably not involved in homing mechanisms.