High resolution in situ zymography reveals matrix metalloproteinase activity at glutamatergic synapses

Neuroscience. 2009 Jan 12;158(1):167-76. doi: 10.1016/j.neuroscience.2008.05.045. Epub 2008 Jun 7.


Synaptic plasticity involves remodeling of extracellular matrix. This is mediated, in part, by enzymes of the matrix metalloproteinase (MMP) family, in particular by gelatinase MMP-9. Accordingly, there is a need of developing methods to visualize gelatinolytic activity at the level of individual synapses, especially in the context of neurotransmitters receptors. Here we present a high-resolution fluorescent in situ zymography (ISZ), performed in thin sections of the alcohol-fixed and polyester wax-embedded brain tissue of the rat (Rattus norvegicus), which is superior to the current ISZ protocols. The method allows visualization of structural details up to the resolution-limit of light microscopy, in conjunction with immunofluorescent labeling. We used this technique to visualize and quantify gelatinolytic activity at the synapses in control and seizure-affected rat brain. In particular, we demonstrated, for the first time, frequent colocalization of gelatinase(s) with synaptic N-methyl-D-aspartic acid (NMDA)- and AMPA-type glutamate receptors. We believe that our method represents a valuable tool to study extracellular proteolytic processes at the synapses, it could be used, as well, to investigate proteinase involvement in a range of physiological and pathological phenomena in the nervous system.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Assay / methods
  • Brain / enzymology*
  • Brain / ultrastructure
  • Epilepsy / enzymology
  • Epilepsy / physiopathology
  • Extracellular Matrix / metabolism
  • Fluorescent Antibody Technique / methods
  • Glutamic Acid / metabolism*
  • Male
  • Matrix Metalloproteinases / analysis
  • Matrix Metalloproteinases / metabolism*
  • Microscopy, Fluorescence / methods
  • Neurochemistry / methods
  • Proteomics / methods
  • Rats
  • Rats, Wistar
  • Receptors, AMPA / metabolism
  • Receptors, N-Methyl-D-Aspartate / metabolism
  • Synapses / enzymology*
  • Synapses / ultrastructure
  • Tissue Embedding / methods
  • Tissue Fixation / methods


  • Receptors, AMPA
  • Receptors, N-Methyl-D-Aspartate
  • Glutamic Acid
  • Matrix Metalloproteinases