Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture

Abstract

Dissociated cultures from many species have been important tools for exploring factors that regulate structure and function of central neuronal synapses. We have previously shown that cells harvested from brains of late stage Drosophila pupae can regenerate their processes in vitro. Electrophysiological recordings demonstrate the formation of functional synaptic connections as early as 3 days in vitro (DIV), but no information about synapse structure is available. Here, we report that antibodies against pre-synaptic proteins Synapsin and Bruchpilot result in punctate staining of regenerating neurites. Puncta density increases as neuritic plexuses develop over the first 4 DIV. Electron microscopy reveals that closely apposed neurites can form chemical synapses with both pre- and postsynaptic specializations characteristic of many inter-neuronal synapses in the adult brain. Chemical synapses in culture are restricted to neuritic processes and some neurite pairs form reciprocal synapses. GABAergic synapses have a significantly higher percentage of clear core versus granular vesicles than non-GABA synapses. Gap junction profiles, some adjacent to chemical synapses, suggest that neurons in culture can form purely electrical as well as mixed synapses, as they do in the brain. However, unlike adult brain, gap junctions in culture form between neuronal somata as well as neurites, suggesting soma ensheathing glia, largely absent in culture, regulate gap junction location in vivo. Thus pupal brain cultures, which support formation of interneuronal synapses with structural features similar to synapses in adult brain, are a useful model system for identifying intrinsic and extrinsic regulators of central synapse structure as well as function.

Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

“…3B-D). These same varicosities have been recently shown to be presynaptic specializations by ultrastructural criteria (Oh et al, 2008). These data show that RBO is expressed in synapses, colocalizing with presynaptic markers in central brain neurons.…”

“…3A). In neurons that were double-labeled with the presynaptic marker anti-Bruchpilot (BRP), an overlapping punctate pattern was observed, very similar to that reported by Oh et al (Oh et al, 2008). RBO expression was clearly observed in varicosities along axonal processes, colocalized with BRP and Cysteine string protein (CSP) in synapses ( Fig.…”

The fragile X mental retardation protein developmentally regulates the strength and fidelity of calcium signaling in Drosophila mushroom body neurons

“…It was therefore possible that these structural defects might contribute to the changes in Ca 2+ transients and therefore compromise interpretation of results. To eliminate this variability from our analysis, and to examine Ca 2+ responses at the single neuron level, we performed the same series of experiments on primary MB neuronal cultures (Jiang et al, 2005; Oh et al, 2008; Sicaeros et al, 2007; Su and O’Dowd, 2003). Cultures were made from animals expressing the gCAMP reporter only in MB Kenyon cells (OK107-GAL4 X UAS-gCAMP).…”

“…Cultures were made from animals expressing the gCAMP reporter only in MB Kenyon cells (OK107-GAL4 X UAS-gCAMP). Neurons were grown for either 1) 3 days in vitro (DIV) (a time prior to detectable synaptic development) or 2) 6 DIV (a mature time point with abundant synaptic connections) (Oh et al, 2008). Neurons were acutely depolarized with 70mM KCl, using the same protocol described above (Fig.…”

“…After 15 minutes of depolarization, the total fluorescence changes were approximately 60% lower than those seen in more mature cultured neurons (6 DIV). It is possible that this difference may be linked to the reduced number of processes and the lack of synaptic connections in these immature neurons at this early developmental stage (Oh et al, 2008). The larger release of calcium at 6 DIV may be due to a large contribution from synaptic mechanisms not present at 3 DIV.…”