Vibrodissociation of Rat Retinal Ganglion Cells Attached With Inhibitory Synaptic Boutons

Abstract

Purpose: : The vibrodissociation method has been applied to various CNS regions, yet never applied to vertebrate retinas. This study is aimed to examine whether this method is valid to isolate ganglion cells from a mammalian retina.

Methods: : Retinas were isolated from freshly enucleated eyes of Wistar rats (P7–P20), cut into 2–4 pieces each, and stored in a chilled culture solution for 10–20 min. The retinal piece was placed ganglion cell layer up in a plastic culture dish, flattened, and incubated in an EDTA–supplemented low–Ca²⁺ solution for 3–5 min. The tip of a fire–blunted glass pipette was lightly placed on the tissue surface using a micromanipulator, and vibrated horizontally at 100 Hz with 0.1–0.3 mm of amplitude. The glass pipette was then slowly moved to scan the entire retinal surface, so that cells were dissociated from the ganglion cell layer. After the scanning, the remaining retinal tissue was removed from the culture dish and the dissociated cells were allowed to settle down for 15–30 min. The culture dish was then filled with the culture medium (supplemented with BSA) and stored for 4–12 hours at room temperature. Ganglion cells were identified based on the morphological and the electrophysiological characteristics.

Results: : In perforated–patch whole–cell recordings, the isolated ganglion cells showed sustained as well as transient spiking in response to depolarizing constant current injections, and showed voltage–gated Na⁺ current in response to depolarization steps. Under current–clamp, the ganglion cells somata showed spontaneous IPSP–like events of which reversal potential was approximately –80 mV. The IPSP–like events were suppressed by superfusion of low–Ca²⁺ solution, indicating a Ca²⁺–dependent process. The IPSP–like events were little affected by applying TTX. Membrane conductance changes during the events were calculated based on a single compartment model. Both amplitude and the decay time constant of the conductance change did not depend on the membrane potential of the recorded cells. Pharmacological characteristics of the IPSP–like events were also examined.

Conclusions: : The present study suggested that the vibrodissociation method is useful to isolate rat retinal ganglion cells attached with inhibitory synaptic boutons.

Keywords: retina • ganglion cells • synapse