title: High resolution in situ zymography reveals matrix metalloproteinase activity at glutamatergic synapses.
abstract: 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.
authors.affiliation: Department of Neurophysiology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Gawlak
authors.forename: T
authors.initials: T
authors.lastname: Górkiewicz
authors.forename: A
authors.initials: A
authors.lastname: Gorlewicz
authors.forename: F A
authors.initials: FA
authors.lastname: Konopacki
authors.forename: L
authors.initials: L
authors.lastname: Kaczmarek
authors.forename: G M
authors.initials: GM
authors.lastname: Wilczynski
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D001921:Brain / Q000201:enzymology* / Q000648:ultrastructure; D004827:Epilepsy / Q000201:enzymology / Q000503:physiopathology; D005109:Extracellular Matrix / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods; D018698:Glutamic Acid / Q000378:metabolism*; D008297:Male; D020782:Matrix Metalloproteinases / Q000032:analysis / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D009448:Neurochemistry / Q000379:methods; D040901:Proteomics / Q000379:methods; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D013569:Synapses / Q000201:enzymology* / Q000648:ultrastructure; D016610:Tissue Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods
chemical_list: D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D020782:Matrix Metalloproteinases