Depolarization regulates expression of a synaptic vesicle protein in rat superior cervical ganglia in vitro

Abstract

The role of membrane depolarization in the regulation of expression of a neuron specific protein was evaluated by culturing superior cervical ganglia from neonatal rats in defined medium and manipulating neuronal activity by depolarizing agents. P65 is an integral membrane protein of synaptic vesicles and can be used as a marker for general neuronal maturation. P65 antigen levels were quantified by indirect radioimmunoassay, using monoclonal antibodies. The expression of p65 in ganglion explants increased by 40-100% when the cultures were treated with the depolarizing agents, veratridine or high potassium. The veratridine effect could be blocked by simultaneous treatment with the sodium channel blocker, tetrodotoxin (TTX). The rise in p65 was not evident until 36 h after depolarizing treatment had begun and reached peak levels after 48 h, with no further increases observed with sustained treatment. After removal of the depolarizing treatment, p65 levels returned to control values after 24 h. P65 joins a growing number of molecules whose expression is regulated by membrane depolarization.

3?,5?-Cyclic adenosine monophosphate regulates expression of synaptotagmin in neonatal sympathetic gangliain vitro

“…Deafferentation of the SCG in vivo blocks normal postnatal increases in syt (Greif and Trenchard, 1988). Depolarization of ganglia maintained in explant culture using either the Na ϩ channel ionophore, veratridine (Ver), or elevated potassium produces increases in syt levels (Linderman and Greif, 1990). These studies suggest that activitymediated factors may play critical roles in development and maintenance of synaptic components.…”

“…Animals were checked at least twice daily to pinpoint the time of birth of pups. Two-day-old pups (with the date of birth designated P0) were anesthetized on ice prior to decapitation and removal of SCGs; previous work indicated that little change in syt occurred during this period in vivo, while ganglia doubled in protein content (Greif and Reichardt, 1982;Linderman and Greif, 1990). …”

“…Cultures were grown under 1.5% CO 2 at 37°C in a water-jacketed incubator (Kessler et al, 1984;Linderman and Greif, 1990). Cultures were maintained for 2 days in this basal medium to allow stabilization of cultures prior to addition of experimental agents.…”

“…Cultures were then maintained an additional 48 h to assess the effects of treatments on syt levels (cf. Linderman and Greif, 1990). For assay of cyclic AMP, cultures were harvested at different time points after initiation of treatment (see below).…”

“…Veratridine (CAS 71-62-5), a Naϩ channel ionophore, was used at a concentration of 1 M. The cyclic AMP analogs, di-Bu-cyclic AMP (CAS 16980-89-5) and 8-Br-cyclic AMP (CAS 76939-46-3), were used at 1 M, and the adenylyl cyclase activators, cholera toxin and forskolin (CAS 66575-39-9) at 20 M and 100 -300 M,respectively (Sigma-Aldrich)(cf. Akagi and Kudo, 1985;Black et al, 1985;Linderman and Greif, 1990). The adenylyl cyclase inhibitors 2Ј,5Ј dideoxyadenosine (DDA, CAS 6698-26-6) and MDL 12,330 (MDL, were used at concentrations of 10 M and 50 M, respectively (Calbiochem) (Gan et al, 1995;Graness et al, 1997;Wu and Dun, 1997).…”