Rabbit cone bipolar cells: correlation of their morphologies with whole-cell recordings.

The superfused retinal slice preparation was used to examine the morphology and glutamate-activated whole-cell currents of rabbit bipolar cells. There were six morphologically distinct types of cone bipolar cells and a rod bipolar cell that had axon terminals stratifying in stratum 3 to 5 of sublamina-b. All of these bipolar cell types exhibited an outward current in response to the application of the metabotropic glutamate receptor, mGluR6, agonist AP-4 (APB), and had I/V curves indicative of membrane channel closure. Conversely, there were no currents activated during the application of kainate, the AMPA/kainate receptor agonist. These data demonstrate they were on-bipolar cells. In addition, there were six morphologically distinct cone bipolar cells that stratified in sublamina-a. Every cell with axonal arborizations in stratum 1 and 2 exhibited an inward current when the ionotropic glutamate receptor agonist kainate was applied. This current was blocked by application of the AMPA/kainate receptor antagonist CNQX. These cells also decreased their membrane resistance in response to kainate, a characteristic of the opening of channels within the plasma membrane. Without exception, no cells stratifying in sublamina-a responded to the mGluR6 agonist AP-4, further identifying them as off-bipolar cells.

GABA responses of rod bipolar cells in rabbit retinal slices.

GABAergic responses of rabbit rod bipolar cells were reexamined by using whole-cell recordings in the superfused slice preparation to determine if there is GABA(C) receptor input to their axon terminal and to characterize the contribution that GABA(A) and GABA(C) receptors make to the total GABA current on the axon terminals of these cells. Pharmacological agents specifically blocking GABA(A) and GABA(C) receptor currents demonstrated that 37% of the GABA-activated current was blocked by either the GABA(A) antagonists bicuculline or SR-95531, whereas the remaining 63% of the GABA current was blocked by a mixture of bicuculline and the GABA(C) antagonist TPMPA. This indicated that GABA(C) receptors were present on the axon terminal of the rabbit rod bipolar cell and that they were responsible for mediating the bicuculline insensitive GABA current.

Rabbit retinal Müller cells undergo antigenic changes in response to experimentally induced proliferative vitreoretinopathy.

Experimental proliferative vitreoretinopathy (PVR) was induced in the rabbit eye by injecting mitotically active Müller cells into the vitreal chamber. Two weeks after the initiation of PVR, the retina and the epiretinal membrane that formed were examined to ascertain the antigenic expression of Müller cells in the retina and in the epiretinal membrane. Examination of various regions of the retina from the experimental PVR eye demonstrated that vimentin, glial fibrillary acidic protein (GFAP), cellular retinaldehyde binding protein (CRALBP), and beta-amyloid precursor protein (beta-APP), which were present in the Müller cells of the retina from the control eye, increased their expression, while the antigenicity of glutamine synthetase (GS), did not change; these proteins were also present in the cells contained within the experimentally induced epiretinal membrane. Alpha smooth muscle actin (alpha-SMA), a cytoskeletal protein that is associated with migration and tractional forces in many cell types, was not only present in the cells embedded within the epiretinal membrane, but was also present in the Müller cells underlying the epiretinal membrane. However, Müller cells that were in the inferior portion of the retina, where epiretinal membrane pathology was absent, did not express alpha-SMA. Although this protein is not normally found in Müller cells, they do express it de novo when they are maintained in culture. This suggests that a localized mechanism associated with epiretinal membrane formation induces the expression of alpha-SMA in Müller cells while the increased expression of GFAP, beta-APP, vimentin, and CRALBP are probably regulated via a more general mechanism.

Antigenic changes of rabbit retinal Müller cells in culture.

PURPOSE: To determine whether dissociated and cultured Müller cells from the avascular rabbit retina undergo the same phenotypic changes as Müller cells that are dissociated and cultured from a vascular retina. METHODS: Müller cells were dissociated from adult rabbit retinas by using an enzymatic digestion-mechanical trituration technique and a cell attachment method that provided Müller cell- enriched cell cultures. Indirect immunofluorescence localization of vimentin, glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), beta-amyloid precursor protein (beta-APP), and (alpha-smooth muscle actin (alpha-SMA) was carried out on Müller cells that were freshly dissociated, on those that had been in culture 2 and 6 days, and on confluent primary cultures and late-passage cultures. The specificity of the antibodies and changes in protein expression were examined by western blot analysis. RESULTS: The expression of vimentin, GFAP, GS, and beta-APP was present 2 days after dissociation and was retained through 6 days in culture, at which time alpha-SMA began to be expressed in a small number of cells. The confluent, primary cultures no longer expressed GS, but vimentin and beta-APP were still expressed, and the expression of alpha-SMA was increased. During the late-passage stage, the morphologic appearance of the Müller cell cultures was large and amorphous, with additional changes in antigenicity. Although there was loss of expression of the intermediate filament proteins GFAP and vimentin, the expression of beta-APP was maintained, whereas alpha-SMA was increased and appeared to be a major cytoskeletal protein. CONCLUSIONS: Dissociated Müller cells that were maintained in culture underwent phenotypic changes that included a large, amorphous appearance; the loss of detectable vimentin, GFAP, and GS expression; the persistent presence of beta-APP; and the de novo appearance of alpha-SMA. The phenotypic and antigenic changes that occurred in cultured Müller cells from an avascular retina were similar but not identical to the changes observed in cultured Müller cells from a vascular retina.