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Daniel Llewellyn Rathbun, Ayse Sahaboglu, Blanca Arango-Gonzalez, Eberhart Zrenner, Francois Paquet-Durand; Visual Responses in the Organotypically Cultured Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1889.
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© ARVO (1962-2015); The Authors (2016-present)
We investigated the appropriateness of the organotypic retinal preparation for in vitro visual electrophysiology measurements during early development of the mouse retina in comparison with acutely dissected retinas.
Wild-type (C3H) mouse retinas with attached retinal pigment epithelium were harvested at either postnatal day 5 (P5) or P9 and maintained in culture up to P14. Retinal ganglion cell (RGC) spiking responses to a full-field flashing visual stimulus were recorded either from cultured or from age-matched, acutely-dissected control retinas at P11, P12 and P14 using a multielectrode array. Histological assessments of RGC viability were performed in parallel using the TUNEL assay and BRN3A immunostaining.
Retinas acutely prepared at P26 with the present methods demonstrated ON and OFF response latencies, amplitudes and durations similar to previous reports [Carcieri et al. J. Neurophysiol. 90:1704-13, 2003]. While latency and duration decreased from P11 to P26 in acute retina, amplitude increased over this time. At P11 and P12 visual responses from cultured retina were more robust when retinas were cultured at P9 vs. P5 - reflecting the observation that RGC responsiveness degrades quickly in culture, possibly because RGCs are axotomized in preparation. Responses from retinas cultured at P9 and recorded at P11, P12 and P14 had longer latencies, shorter durations, and - at most ages - lower amplitudes relative to acutely prepared control retina. Response degradation correlated with the histologically observed loss of RGCs, an effect that grew stronger with time in culture. Neither exogenous brain-derived neurotrophic factor (BDNF) nor retention of the optic nerve slowed degradation in cultured responses.
For in vitro visual electrophysiology measurements, the organotypic culture protocol provides only a limited methodological basis for comparison of visual responses in healthy and degenerate retinas. Although the number of RGCs numbers and their visual responses decrease quickly in culture, baseline measurements have been established for further investigation of RGC neuroprotection in the organotypic retinal preparation.
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