title: Studying synaptic efficiency by post-hoc immunolabelling.
abstract: BACKGROUND
  In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals. This dynamic scenario serves as the substrate for processes such as scaling, plasticity and synaptic weight redistribution. Presynaptic strength has been associated with the activity of several types of proteins, including the scaffolding proteins that form the active zone cytomatrix and the proteins involved in presynaptic exocytosis.
  
  
  METHODS
  We have combined fluorescence imaging techniques using the styryl dye FM1-43 in primary cultures of cerebellar granule cells with subsequent post-hoc immunocytochemistry in order to study synaptic efficiency in terms of vesicular release. We describe a protocol to easily quantify these results with minimal user intervention.
  
  
  RESULTS
  In this study we describe a technique that specifically correlates presynaptic activity with the levels of presynaptic markers. This method involves the use of the styryl dye FM1-43 to estimate the release capacity of a synaptic terminal, and the subsequent post-hoc immunolabelling of thousands of individual nerve terminals. We observed a strong correlation between the release capacity of the nerve terminal and the levels of the RIM1α but not the Munc13-1 protein in the active zone.
  
  
  CONCLUSIONS
  Our findings support those of previous studies and point out to RIM1α as a crucial factor in determining synaptic efficiency. These results also demonstrate that this technique is a useful tool to analyse the molecular differences underlying the heterogeneous responses exhibited by neuronal networks.
authors.forename: Jorge
authors.initials: J
authors.lastname: Ramírez-Franco
authors.forename: Beatris
authors.initials: B
authors.lastname: Alonso
authors.forename: David
authors.initials: D
authors.lastname: Bartolomé-Martín
authors.affiliation: Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, Madrid 28040, Spain. jsprieto@vet.ucm.es.
authors.forename: José
authors.initials: J
authors.lastname: Sánchez-Prieto
authors.forename: Magdalena
authors.initials: M
authors.lastname: Torres
mesh_terms: D000818:Animals; D002531:Cerebellum / Q000502:physiology; D005456:Fluorescent Dyes; D007150:Immunohistochemistry / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000502:physiology*; D061848:Optical Imaging / Q000379:methods*; D017729:Presynaptic Terminals / Q000378:metabolism; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D009435:Synaptic Transmission / Q000502:physiology*
chemical_list: C073804:FM1 43; D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds