Expression of horizontal cell phenotypes in monolayer cultures from immature rabbit retina

Abstract

Using the sandwich culture technique introduced by Brewer and Cotman 16 we have studied the in vitro differentiation of A- and B-type horizontal cells which represent two well characterized cell types of the rabbit retina. Neurons from immature (postnatal day 3) rabbit retinae were dissociated and grown on inverted coverslips for up to 5 weeks in a chemically defined medium. On the basis of morphological criteria and the staining pattern for several immunocytochemical and autoradiographic horizontal cell markers we have examined to what extent expression of a distinct mature neuronal phenotype can take place under the artificial conditions of monolayer cultures. After 14 days in vitro neurons could be identified which had acquired elaborate morphological features closely resembling those of A- and B-type horizontal cells, respectively. Axonless A-like cells had 2–4 stout primary dendrites. In agreement with in situ observations these cells showed immunoreactivity for neurofilament proteins (68 kDa, 200 kDa), calbindin-28 kDa and less strongly for vimentin. B-like neurons reached varying states of development. Ideally, they had dendritic trees with 6–8 primary processes extending radially from the soma and a single axon-like process which branched extensively to form a profuse neuritic arbor strikingly similar to axon terminal systems of B-type cells in the intact retina. B-like cells also stained for vimentin, calbindin-28 kDa and unexpectedly also for neurofilament proteins. Interestingly, however, neurofilaments became redistributed during in vitro development eventually resulting in their restricted localization in the ‘axon terminal system’. This apparently reflects a developmental process which has escaped detection in situ so far. Both cell types were intensely labelled with antibodies to γ-aminobutyric acid (GABA), the presumed horizontal cell transmitter, but high affinity uptake of this transmitter was practically undetectable by [ 3H]-GABA autoradiography. This was in agreement with observations in intact retinae. These results support the notion that once a neuron has reached a certain developmental state further differentiation and maintenance of its particular morphological and functional properties are primarily governed by intrinsic factors, but do not exclude that extrinsic signals have important modulatory functions.