The cerebellum as a model for in vitro and in situ gap junctional coupling studies

THE CEREBELLUM AS A MODEL FOR IN VITRO AND IN SITU GAP JUNCTIONAL COUPLING STUDIES^*

MARIA M. FRÓES¹, ANA H. P. CORREIA¹, JOÃO R. L. MENEZES¹, ANTÔNIO C. CAMPOS DE CARVALHO², ROBERTO LENT¹AND VIVALDO MOURA NETO¹

¹

²Instituto de Biofísica Carlos Chagas Filho

Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, RJ, Brazil.

Here we consider the phenomenon of gap junction-mediated intercellular communication (GJC) in the young cerebellum from three different experimental perspectives. By intracellular iontophoretic application of fluorochromes, we were able to characterize the temporal profile of homo- and heterocellular coupling in primary cocultures of cerebellar neurons onto monolayered astrocytes, dissociated from P0 rats. Dye injections of Lucifer yellow (LY) were performed everyday, between the first and fourth days in vitro, and then at the seventh day. Coupling between neurons and between these and astrocytes displayed inverted profiles, with older cocultures enriched in heterocellular coupling. In contrast, coupling between astrocytes was roughly constant. Such increase in neuron/astrocyte communication was accompanied by a decrease in cell proliferation, which paralleled the neuronal uncoupling and gradual neuronal maturation evidenced by morphological and immunolabeling criteria. These results were crossed with a topographic view of dye coupling in cerebellum slices, revealed by the "transection loading technique'' (Menezes J et al. 2000 Dev Neurosci 22:34). Thick (800mm) sagittal sections of P4 cerebella were immersed in a combination of LY and rhodamine-conjugated dextran (RD, 3kDa), washed and fixed in paraformaldehyde, prior to blocking and cryostat resectioning. Directly loaded cells, LY+ and RD+, were visualized along the cut edges of cerebellar slices. LY+ coupled cells, some of them presenting typical migratory aspects could be identified in the inner granule and the molecular layers and were absent in the proliferative external granule layer, in agreement with previous findings. Using a third experimental model, the explant culture, cell migration could also be approached. Explants from the external granule layer (P4) were kept for up 5 days in culture onto polylisine-treated coverslips or, alternatively, onto brain astrocytic monolayers. Centrifugal migration from explants was examined in presence of the uncoupler agent, carbenoxolone (CARBEN). Blockage of GJC had opposite consequences in polylisine and astrocytic monolayers. Briefly, migration of neural precursors was impaired by CARBEN in explant cultures on coverslips and enhanced in monolayers, reverting the patterns evident in control conditions. Taken together, our results point to a range of functional cell pairs, homo- and heterocellular, at early stages of the cerebellum development involved in different aspects of cell differentiation. — ( June 27, 2000 ).

Publication Dates

• Publication in this collection
  05 Oct 2000
• Date of issue
  Sept 2000