Recombinant GABA(C) receptors expressed in rat hippocampal neurons after infection with an adenovirus containing the human rho1 subunit

Department of Neurobiology, University of Alabama at Birmingham School of Medicine, 1719 Sixth Avenue South CIRC 410, Birmingham, AL 35294-0021, USA.
The Journal of Physiology (Impact Factor: 4.54). 09/2001; 535(Pt 1):145-53. DOI: 10.1111/j.1469-7793.2001.00145.x
Source: PubMed

ABSTRACT 1. A recombinant adenovirus was generated with the human rho1 GABA(C) receptor subunit (adeno-rho). Patch-clamp and antibody staining were employed to confirm functional expression of recombinant rho1 receptors after infection of human embryonic kidney cells (HEK293 cell line), human embryonic retinal cells (911 cell line), dissociated rat hippocampal neurons and cultured rat hippocampal slices. 2. Standard whole-cell recording and Western blot analysis using rho1 GABA(C) receptor antibodies revealed that recombinant rho1 receptors were expressed in HEK293 and 911 cells after adeno-rho infection and exhibited properties similar to those of rho1 receptors after standard transfection. 3. Cultured rat hippocampal neurons (postnatal day (P)3-P5) did not show a native GABA(C)-like current. After adeno-rho infection, however, a GABA(C)-like current appeared in 70-90 % of the neurons. 4. Five days after infection, expression of GABA(C) receptors in hippocampal neurons significantly decreased native GABA(A) receptor currents from 1200 +/- 300 to 150 +/- 70 pA (n = 10). The native glutamate-activated current was unchanged. 5. Hippocampal slices (P8) did not show a native GABA(C)-like current, although recombinant rho1 receptors could be expressed in cultured hippocampal slices after adeno-rho infection. 6. These data indicate that an adenovirus can be used to express recombinant GABA(C) receptors in hippocampal neurons. This finding could represent an important step towards the gene therapy of CNS receptor-related diseases.

Download full-text


Available from: William J. Tyler, Feb 09, 2015
  • Source
    • "Initially, the expression of GABA receptors in this area was suggested by the transient expression of a bicucullineinsensitive , GABA-gated Cl -channel during early development [144]. Later on, several studies reported the localization and electrophysiological expression of GABA receptors from both neurons in culture and slices of pyramidal and granule cells of hippocampus [99] [101] [106] [145] [146] [147] [148]. Recent reports have suggested that activated GABA receptors help to protect against neurotoxicity in hippocampal cultures [149]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The present review discusses the functional and molecular diversity of GABAρ receptors. These receptors were originally described in the mammalian retina, and their functional role in the visual pathway has been recently elucidated; however new studies on their distribution in the brain and spinal cord have revealed that they are more spread than originally thought, and thus it will be important to determine their physiological contribution to the GABAergic transmission in other areas of the central nervous system. In addition, molecular modeling has revealed peculiar traits of these receptors that have impacted on the interpretations of the latest pharmacolgical and biophysical findings. Finally, sequencing of several vertebrate genomes has permitted a comparative analysis of the organization of the GABAρ genes.
    DNA research: an international journal for rapid publication of reports on genes and genomes 12/2010; 8(4):422-33. DOI:10.2174/157015910793358141 · 2.35 Impact Factor
  • Source
    • "containing mature viral particles was removed and desalted against phosphate-buffered saline in a Vivaspin 20 mL column using the manufacturer's protocol (Vivascience AG, Hanover, Germany). Infection units used for transfections were10 pfu/cell for SH-SY5Y cells, and 50–200 pfu/cell for rat hippocampal neurons and astrocytes (Filippova et al. 2001). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Telomeres are specialized structures at the ends of chromosomes that consist of tandem repeats of the DNA sequence TTAGGG and several proteins that protect the DNA and regulate the plasticity of the telomeres. The telomere-associated protein TRF2 (telomeric repeat binding factor 2) is critical for the control of telomere structure and function; TRF2 dysfunction results in the exposure of the telomere ends and activation of ATM (ataxia telangiectasin mutated)-mediated DNA damage response. Recent findings suggest that telomere attrition can cause senescence or apoptosis of mitotic cells, but the function of telomeres in differentiated neurons is unknown. Here, we examined the impact of telomere dysfunction via TRF2 inhibition in neurons (primary embryonic hippocampal neurons) and mitotic neural cells (astrocytes and neuroblastoma cells). We demonstrate that telomere dysfunction induced by adenovirus-mediated expression of dominant-negative TRF2 (DN-TRF2) triggers a DNA damage response involving the formation of nuclear foci containing phosphorylated histone H2AX and activated ATM in each cell type. In mitotic neural cells DN-TRF2 induced activation of both p53 and p21 and senescence (as indicated by an up-regulation of beta-galactosidase). In contrast, in neurons DN-TRF2 increased p21, but neither p53 nor beta-galactosidase was induced. In addition, TRF2 inhibition enhanced the morphological, molecular and biophysical differentiation of hippocampal neurons. These findings demonstrate divergent molecular and physiological responses to telomere dysfunction in mitotic neural cells and neurons, indicate a role for TRF2 in regulating neuronal differentiation, and suggest a potential therapeutic application of inhibition of TRF2 function in the treatment of neural tumors.
    Journal of Neurochemistry 05/2006; 97(2):567-81. DOI:10.1111/j.1471-4159.2006.03779.x · 4.24 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ionotropic gamma-aminobutyric acid (GABA) receptors are known to mediate excitation in neonatal neurones as a crucial developmental factor. In the present study we employed calcium imaging techniques with the calcium indicator Fura-2-AM to study the pharmacology of GABA-induced calcium responses in cultures prepared from neonatal rat superficial superior colliculus (SC), after immunocytochemical labelling confirmed the presence of GABA(C) rho(1) subunits in 35% of neurones. Rises in neuronal intracellular calcium were obtained in response to GABA and also to the subtype-specific GABA(A) agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. However, the GABA(C) agonist cis-4-aminocrotonic acid induced calcium response only at unspecifically high concentrations (500 microM). Co-application of GABA antagonists revealed that both GABA(A&C) agonists' actions could be blocked by the GABA(A) antagonist bicuculline but not the GABA(C) antagonists 1,2,5,6-tetrahydro-(pyridin-4-yl) methylphosphinic acid. This suggests that activation of GABA(A) but not GABA(C) receptors contributes to excitatory GABA responses and related calcium signals in neonatal SC neurones.
    Neuroscience Letters 12/2002; 334(2):79-82. DOI:10.1016/S0304-3940(02)01059-5 · 2.06 Impact Factor
Show more