Abstract
Purpose:
Structural and functional remodeling following retinal degeneration (RD) leads to aberrant hyperactivity and may exacerbate the visual impairment.The purpose of this study was to evaluate the mechanisms of NMDA receptor activation and distribution of synaptic versus extrasynaptic areas and reveal how these functional and structural changes may affect ganglion cells in RD. This is important since differential distributions of glutamate receptor isoforms play a key role in shaping light responses across visual pathways.
Methods:
Retinal whole mounts were prepared from rd10 mice (P60-250) and age matched controls. Excitatory postsynaptic currents (EPSCs) were recorded with patch clamp in whole cell configuration and responses were isolated at different holding potentials (-35 mV and -60 mV). The NMDAR-mediated component was revealed by treating retina with CNQX/DNQX, specific antagonists of non-NMDARs. D-AP5 was used to block all NMDAR activity, and ifenprodil was used to block extrasynaptic NMDARs. For immunohistochemistry, NMDARs subunits with PSD proteins within the same retina as the recorded cell.
Results:
n healthy controls, application of CNQX blocked spontaneous excitatory activity in almost all RGCs consistent with predominant activation of synaptic AMPARs and perisynaptic localization of NMDARs. In contrast, the majority of rd10 RGCs retained their responses. Remaining EPSCs were completely eliminated by application of D-AP5. To test whether this D-AP5-sensitive component in rd10 RGCs was due to spillover activation of perisynaptic NMDARs, we evaluated the presence and distribution of NR2A and NR2B subunits of the synaptic and perisynaptic isoforms of NMDARs, respectively. Ifenprodil, a selective NR2B antagonist, had a significant and moderate effect on EPSCs. However, its effect did not completely eliminate CNQX-resistant sEPSCs, suggesting that the remaining EPSCs were due to synaptic NMDARs. Indeed, immunohistochemichal analysis revealed the atypical presence of both subunits of NMDARs across all sublaminae of IPL in degenerated retina.
Conclusions:
Our data indicate that hyperactivity emerging during RD leads to enhanced activation of NMDARs by multiple mechanisms, involving both a synaptic component as well as spillover activation of perisynatic NMDARs.
Keywords: 508 electrophysiology: non-clinical •
531 ganglion cells •
695 retinal degenerations: cell biology