High resolution in situ zymography reveals matrix metalloproteinase activity at glutamatergic synapses.
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.
Gawlak
M
M
Department of Neurophysiology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland.
Górkiewicz
T
T
Gorlewicz
A
A
Konopacki
F A
FA
Kaczmarek
L
L
Wilczynski
G M
GM
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
D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D020782:Matrix Metalloproteinases