The Presynaptic Release Apparatus Is Functional in the Absence of Dendritic Contact and Highly Mobile within Isolated Axons

Abstract

Whether contact of an axon with a dendrite is a necessary inductive signal for the assembly of functional presynaptic machinery is controversial. Combining FM1-43 imaging with retrospective immunocytochemistry, we observe many functional synaptic vesicle (SV) release sites lacking postsynaptic specializations in cultured hippocampal neurons. These "orphan" release sites share the same exocytic machinery and mechanisms of endocytic recycling as mature synaptic sites. Moreover, quantitative analysis of FM1-43 destaining at these orphan release sites reveals similar kinetics with slightly lower release probabilities. Time-lapse imaging of FM1-43 reveals that orphans are generated by complete or partial mobilization of synaptic release sites that retain their functionality in transit. Orphan clusters fuse with existing synaptic release sites or form novel release sites onto dendrites. Mobilization and stabilization of orphan boutons to new sites of dendritic contact may represent a necessary presynaptic counterpart to postsynaptic changes observed during development and plasticity in the CNS.

Bcl-x L induces Drp1-dependent synapse formation in cultured hippocampal neurons

“…7], confirming mitochondrial localization of Bcl-x L . During embryonic development and during maturation in culture, presynaptic vesicle clusters can first be mobile precursors or ''orphans'' that are not associated with postsynaptic densities (PSDs) in an opposing cell before formation of mature excitatory synapses (4,27). To examine the effects of presynaptic Bcl-x L on formation of either precursor vesicle clusters or synapses, parallel cultures of hippocampal neurons were transfected at DIV5 with GFP-Bcl-x L or Mito-GFP and immunostained (DIV13) for presynaptic proteins.…”

“…Fluorescence was quantified as the number of puncta per unit length of axon and as mean fluorescence intensity (see Materials and Methods and ref. 27). The effects of Bcl-x L on synaptophysin staining were already apparent by 2 days posttransfection (DIV7) but were more obvious at 14 and 21 days in culture after synapses had matured (SI Fig.…”

“…Image analysis was performed by using mIPLab software (Scanalytics) as described in ref. 27. Background fluorescence of each image was subtracted.…”

“…For every punctum, the software determined a region of interest comprising a maximum of 10 pixels (0.29 m 2 ) of the highest fluorescent intensity. The summed fluorescence correlates with area (up to 0.29 m 2 ) and depth of the labeled structure (27). Puncta with Ͻ5 pixels were excluded.…”

A Study of Clustered Data and Approaches to Its Analysis

“…This approach has been used by several studies that performed optical recording for some (Pyle et al, 2000;Mozhayeva et al, 2002;Krueger et al, 2003;FernandezAlfonso and Ryan, 2004) or all (Virmani et al, 2006;Willeumier et al, 2006) aspects of their analyses. The studies examined individual synapses but the data from each experiment were averaged, thereby reducing multiple observations to a single observation for each experiment.…”