Functional plasticity of GAT-3 in avian Müller cells is regulated by neurons via a glutamatergic input

Abstract

GABA (γ-amino butyric acid) is the major inhibitory transmitter in the central nervous system and its action is terminated by specific transporters (GAT), found in neurons and glial cells. We have previously described that GAT-3 is responsible for GABA uptake activity in cultured avian Müller cells and that it operates in a Na(+) and Cl(-) dependent manner. Here we show that glutamate decreases [(3)H] GABA uptake in purified cultured glial cells up to 50%, without causing cell death. This effect is mediated by ionotropic glutamatergic receptors. Glutamate inhibition on GABA uptake is not reverted by inhibitors of protein kinase C or modified by agents that modulate cyclic AMP/PKA. Biotinylation experiments demonstrate that this reduction in GABA uptake correlates with a decrease in GAT-3 plasma membrane levels. Interestingly, both GAT-1 and GAT-3 mRNA levels are also decreased by glutamate. Conditioned media (CM) prepared from retinal neurons could also decrease GABA influx, and glutamate receptor antagonists (MK-801 + CNQX) were able to prevent this effect. However, glutamate levels in CM were not different from those found in fresh media, indicating that a glutamatergic co-agonist or modulator could be regulating GABA uptake by Müller cells in this scenario. In the whole avian retina, GAT-3 is present from embryonic day 5 (E5) increasing up to the end of embryonic development and post-hatch period exclusively in neuronal layers. However, this pattern may change in pathological conditions, which drive GAT-3 expression in Müller cells. Our data suggest that in purified cultures and upon extensive neuronal lesion in vivo, shown as a Brn3a reduced neuronal cells and an GFAP increased gliosis, Müller glia may change its capacity to take up GABA due to GAT-3 up regulation and suggests a regulatory interplay mediated by glutamate between neurons and glial cells in this process.

Cell Calcium Imaging as a Reliable Method to Study Neuron–Glial Circuits

“…Müller cells are the main glial element in the retina, which intermingles with most, if not all, neurons in this tissue. These cells are active players ( de Melo Reis et al, 2008 ) that transverse from the inner to the outer limiting membranes, spanning the whole retinal length, and control numerous activities such as osmotic (solute and water composition in the extracellular space), metabolic (glutamate-glutamine cycle), signaling (trophic factors and mediators), synaptic (release of transmitters), regulation of plasticity ( Reichenbach and Bringmann, 2013 ), and response to excitotoxicity ( Schitine et al, 2015 ). Müller cells are categorized by the expression of glutamine synthetase and glial fibrillary acidic protein, among other markers, and are activated by high concentrations of ATP due to the expression of purinergic calcium-permeable P2X7 receptor-channel, among others ( De Melo Reis et al, 2011 ; Faria et al, 2017 ).…”

Fragile X Mental Retardation Protein expression in the retina is regulated by light

“…Phospho-FMRP (or pFMRP) antibody was validated by Western blot and immunohistochemistry in wildtype and Fmr1 knockout mice (Supplementary material, Figures 1S and 2S). Mouse PKCα monoclonal antibody (NB600-201, Novus Biologicals) was used as a bipolar cell marker (Quraishi et al, 2007), and mouse Brn3a monoclonal antibody (MAB1585, Millipore) was used as a ganglion cell marker (Schitine et al, 2015). Specificity of the ab17722 FMRP antibody was further validated by preadsorption of the antibody with a human FMRP peptide (ab19074, Abcam) at a 4:1 molar dilution (peptide: ab17722 primary antibody).…”

Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells

“…Variations of free intracellular calcium levels ([Ca 2+ ]i) were evaluated in single cells obtained from retinal cells in culture adapting the protocol from [ 19 ], essentially described in [ 20 ]. Retinal cells in culture were loaded for 40 min with 5μM Fura-2/AM (Molecular Probes), 0.1% fatty acid-free bovine serum albumin (BSA), and 0.02% pluronic acid F-127 (Molecular Probes) in Krebs solution (132mM NaCl, 4mM KCl, 1.4mM MgCl 2 , 2.5mM CaCl 2 , 6mM glucose, 10mM HEPES, pH 7.4), in an incubator with 5% CO 2 and 95% atmospheric air at 37°C.…”

“…A fluorescence-based viability assay was performed exactly as [ 20 ] (Live-dead assay; Invitrogen, Carlsbad, CA).…”