Expression of synaptic membrane proteins in gerbil pinealocytes in primary culture

Pinealocytes of various mammalian species contain abundant synaptic-like microvesicles (SLMVs) which are considered the endocrine equivalent of neuronal synaptic vesicles. Although the pinealocytes may thus be a suitable cellular model for experimental in vitro studies of SLMVs, nothing is known about the presence of SLMVs in isolated pinealocytes maintained under tissue culture conditions. In the present investigation, we prepared dissociated primary cultures of gerbil pinealocytes to study the expression and distribution of protein components of synaptic vesicles/SLMVs and the presynaptic plasmalemma in pinealocytes kept in vitro. Using immunofluorescence microscopy, we found that cultured pinealocytes readily expressed all synaptic membrane proteins investigated, i.e., synaptophysin, synaptotagmin I, synaptobrevin II, syntaxin I and SNAP-25. Punctuate immunoreactivity for the vesicle-associated proteins could be detected throughout the cell bodies of pinealocytes and was also distributed into all of their processes which began to develop within the first days in culture. Outgrowing processes exhibited growth cone-like structures which were enriched in synaptic vesicle-associated proteins. After 1 week in vitro, pinealocytes had frequently formed an elaborate network of long interwoven processes. Accumulations of synaptic vesicle-associated proteins were observed in varicosities and terminal swellings of the processes. The vesicle-rich process swellings often established synaptic-like process swellings often established synaptic-like contacts with somata and processes of other pinealocytes. Some of the pinealocyte processes possessed additional axon-like properties as demonstrated by their lack of immunoreactivity for the somato-dendritic marker MAP2 and the transferrin receptor. The comparison of the staining patterns for synaptophysin and the endocytotic marker transferrin receptor by confocal laser scanning microscopy revealed a largely differential intracellular distribution of the two proteins. This may indicate that a substantial fraction of pinealocyte SLMVs by-passes the early endosomal-related recycling pathway of SLMVs. Herewith, we have shown that isolated gerbil pinealocytes maintained in primary culture can acquire morphological and neurochemical traits which closely mimick those observed in vivo. In particular, these cultures permit experimental studies of the compartment of pinealocyte SLMVs which seem to make up a major secretory pathway for paracrine intrapineal communication.