Abstract
Abstract: :
Purpose: Mixed–rod cone bipolar (Mb) cells of goldfish retina have large synaptic terminals (10 µm in diameter) that make 50–70 ribbon synapses mostly onto amacrine cells, rarely to ganglion cells and receive 300–400 synapses from GABAergic amacrine cells. Electrophysiological evidence indicates substantial direct input from Mb cells to ganglion cells in the scotopic state. But, anatomical studies show minimal synaptic contact between Mb cell terminals and ganglion cells and thus there appears to be a mismatch between the anatomical and electrophysiological findings. In this study a process invaginating the Mb terminals and immunoreactive for a pan–specific Na channel (PanNaCh) antibody was studied by confocal and electron microscopy. Methods:Goldfish retinas were fixed in 4% paraformaldehyde and incubated in a rabbit PanNaCh antibody (Upstate) against the conserved intracellular III–IV loop, and mouse mAb–PKC (Chemicon). LM immunohistofluorescence and confocal microscopy were used to localize immunoreactivity (IR). Tissue was also viewed by electron microscopy (EM). Homogenized goldfish retina and brain, and rat brain were electroblotted and incubated in PanNaCh antibody and HRP conjugated anti–rabbit IgG and visualized using enhanced chemiluminescence. Results: In immunoblots, the PanNaCh antibody recognized one ∼220 kDa band in rat brain and two bands at ∼220 kDa and ∼110 kDa in goldfish retina and brain. At the light level, PanNaCh–IR appeared in processes that penetrated deeply into the Mb terminal. The frequency of these processes appeared light–dependent, with a higher frequency and deeper invagination in the dark–adapted state at daytime and midnight, compared to light–adapted daytime. Confocal analysis clearly showed the processes extending from the IPL into the Mb terminal, like "worms in an apple". EM of Mb terminals showed double membrane structures well within the confines of the terminal. There was no indication of membrane specializations on these invaginating processes. The EM supports the impression obtained by the confocal microscopy, indicating that dendritic–like processes penetrate the Mb terminal. Conclusions: The PanNaCh–IR invaginating processes may belong to dendrites of ganglion cells that comprise sites of ephaptic transmission between Mb bipolar cells and ganglion cells. The light–dependent plasticity of these processes could provide the mechanism for transmission of bipolar cell signals to ganglion cells in the scotopic state, and either by retraction of the processes or internalization of the sodium channel become inactive at photopic levels of illumination.
Keywords: ion channels • retina: proximal (bipolar, amacrine, and ganglion cells) • immunohistochemistry