P2X7 receptor activation modulates light-evoked excitatory synaptic responses and microglial morphology in the mouse retina

S Chavda1, PJ Luthert2,3, TE Salt1. 1Department of Visual Neuroscience, Institute of Ophthalmology, University College London, London, UK, 2Ocular Biology and Therapeutics, Institute of Ophthalmology, University College London, London, UK, 3NIHR Biomedical Research Centre in Ophthalmology, London, UK

ATP-gated P2X7 receptors (P2X7Rs) are expressed by various retinal cell types, including retinal ganglion cells (RGCs) and microglia, and are known to modulate glutamatergic neurotransmission in the central nervous system (CNS) [1, 2]. P2X7Rs are also associated with the induction of microglia-derived cytokine release and neuronal injury, phenomena which are common to many neuroinflammatory conditions in the retina and CNS [3]. However, the role of P2X7Rs in the modulation of retinal synaptic function remains elusive. Using a dark-adapted, ex vivo mouse retinal wholemount preparation, the present study aimed to characterise the effect of P2X7R activation on light-evoked NMDA receptor (NMDAR)-mediated RGC synaptic responses, on bipolar cell output, and on microglial morphology.

ON and OFF field excitatory post-synaptic potentials (fEPSPs) were recorded from the ganglion cell layer of acutely isolated retinal wholemounts (adult, male C57BL/6 mice) in response to a light stimulus (1s-duration peak wavelength 562nm flash, repeated every 3s). The NMDA receptor-mediated component of the responses was pharmacologically isolated with a Mg2+-free Krebs’ medium containing NBQX, picrotoxin, strychnine and tetrodotoxin. In separate experiments, electroretinogram (ERG) responses were recorded from the surface of retinal wholemounts, using a normal Krebs medium. Additional compounds were applied to the bathing medium. Retinae were stained for microglia (α-IBA-1/IgG-Alexa488) and visualised with confocal microscopy. Statistical analyses: Wilcoxon’s matched pairs test and Student’s two-tailed unpaired t-test; statistical significance: P<0.05.

The P2X receptor agonist 2’(3’)-O-(4-benzoylbenzoyl)adenosine 5’-triphosphate (BzATP) induced a concentration-related depression of the ON (100µM, 89±2%, n=6; 300µM, 78±2% of control, n=21; both P<0.05), but not the OFF RGC fEPSP peak amplitude. The effect of BzATP (300µM) on the ON RGC fEPSP was reduced by the selective P2X7R antagonist 3-[[5-(2,3-Dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine (A438079; 10µM, 31±2% reduction; P<0.05, n=6). Additionally, adenosine (300µM) potentiated the ON response (110±2% of control, P<0.05, n=8), ruling out a possible effect of Bz-adenosine (a catabolic product of BzATP) on P1 receptors. Under similar conditions, BzATP-treated retinae showed a significant increase in IBA-1-positive microglial arbour area (4609.1±142.2µm2, n=144 cells, 6 retinae), compared to control (3854.4±134.0µm2, n=100 cells, 5 retinae; P<0.05). BzATP also induced a depression of the ERG b-wave (80±3% of control; P<0.05, n=9), reflecting the modulation of ON-bipolar cell responses.

In summary, P2X7R activation induces a depression of the ON-retinal ganglion and ON-bipolar cell responses, and causes an increase in microglial arbour area, consistent with the early stages of microglial activation. Since P2X7Rs are well known for mediating cytokine release from microglia, this could also have implications for neural-immune interactions in the retina and CNS.

1. Zhang X et al, Inv Ophthal Vis Sci 46:2183, 2005

2. Vessey KA & Fletcher EL, PloS One 7:e29990, 2012

3. Ferrari D et al, J Immunol 176:3877–83, 2006

This work was supported by Lundbeck.