The number and availability of vesicle release sites at the synaptic active zone (AZ) are critical factors governing neurotransmitter release; yet, these fundamental synaptic parameters have remained undetermined. Moreover, how neural activity regulates the spatiotemporal properties of the release sites within individual central synapses is unknown. Here, we combined a nanoscale imaging approach with advanced image analysis to detect individual vesicle fusion events with ∼27 nm localization precision at single hippocampal synapses under physiological conditions. Our results revealed the presence of multiple distinct release sites within individual hippocampal synapses. Release sites were distributed throughout the AZ and underwent repeated reuse. Furthermore, the spatiotemporal properties of the release sites were activity dependent with a reduction in reuse frequency and a shift in location toward the AZ periphery during high-frequency stimulation. These findings have revealed fundamental spatiotemporal properties of individual release sites in small central synapses and their activity-dependent modulation.

S-foci, the first reported mRNA-silencing foci specific to neurons, may control local mRNA translation in response to NMDA receptor stimulation and synaptic plasticity.

Immunophilins (IMMs) are a family of proteins that bind immunosuppressive drugs and possess cis/trans-peptidylprolyl-isomerase activity. They are classified as FKBPs (FK506-binding protein) when they bind FK506, and cyclophilins when they bind cyclosporine A (CsA) . FKBP51 and FKBP52 (gene names FKBP5 and FKBP4, respectively) are highly homologous tetratricopeptide-domain immunophilins whose sequences share 60% identity and 75% similarity. The best characterized interactions of FKBP52 and FKBP51 are with hsp90 and steroid receptor complexes. It has been shown that FKBP52 is required for dynein/dynactin-dependent retrograde movement of glucocorticoid receptor (GR) Address correspondence and reprint requests to Dr Mario D. Galigniana, Departamento de Química Bioló gica, Pabellón II, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria (C1428EGA), Buenos Aires, Argentina. E-mail: mgali@qb.fcen.uba.arAbbreviations used: AIF, apoptosis-inducing factor; CHX, cycloheximide; CREB, cAMP-response element binding protein; CsA, cyclosporine A; DMEM, Dulbecco's modified Eagle's medium; ERK1/2, extracellular signal-regulated kinases 1 and 2; FKBP, FK506-binding protein; GR, glucocorticoid receptor; IBMX, 3-isobutyl-1-methylxanthine; IMM, immunophilin; MEK, mitogen-activated protein kinase/ ERK kinase; PKA, protein kinase A; RAC3, receptor-associated coactivator 3; RAD, radicicol; TRPC, transient receptor potential channel. Abstract FKBP51 and FKBP52 (FK506-binding protein 51 and 52) are tetratricopeptide repeat-domain immunophilins belonging to the tetratricopeptide-protein•hsp90•hsp70•p23 heterocomplex bound to steroid receptors. Immunophilins are related to receptor folding, subcellular localization, and hormonedependent transcription. Also, they bind the immunosuppressant macrolide FK506, which shows neuroregenerative and neuroprotective actions by a still unknown mechanism. In this study, we demonstrate that in both, undifferentiated neuroblastoma cells and embryonic hippocampal neurons, the FKBP52•hsp90•p23 heterocomplex concentrates in a perinuclear structure. Upon cell stimulation with FK506, this structure disassembles and this perinuclear area becomes transcriptionally active. The acquisition of a neuronal phenotype is accompanied by increased expression of bIII-tubulin, Map-2, Tau-1, but also hsp90, hsp70, p23, and FKBP52. During the early differentiation steps, the perinuclear heterocomplex redistributes along the cytoplasm and nascent neurites, p23 binds to intermediate filaments and microtubules acquired higher filamentary organization. While FKBP52 moves towards neurites and concentrates in arborization bodies and terminal axons, FKBP51, whose expression remains constant, replaces FKBP52 in the perinuclear structure. Importantly, neurite outgrowth is favored by FKBP52 over-expression or FKBP51 knock-down, and is impaired by FKBP52 knock-down or FKBP51 over-expression, indicating that the balance between these FK506-binding proteins plays a key role during the early mechanism of neuronal differentiation.

Synaptic vesicle fusion occurs at specialized release sites at the active zone. How refilling of release sites with new vesicles is regulated in central synapses remains poorly understood. Using nanoscale-resolution detection of individual release events in rat hippocampal synapses we found that inhibition of myosin V, the predominant vesicle-associated motor, strongly reduced refilling of the release sites during repetitive stimulation. Single-vesicle tracking revealed that recycling vesicles continuously shuttle between a plasma membrane pool and an inner pool. Vesicle retention at the membrane pool was regulated by neural activity in a myosin V dependent manner. Ultrastructural measurements of vesicle occupancy at the plasma membrane together with analyses of single-vesicle trajectories during vesicle shuttling between the pools suggest that myosin V acts as a vesicle tether at the plasma membrane, rather than a motor transporting vesicles to the release sites, or directly regulating vesicle exocytosis.