Methods for direct visualization of active synapses
AssigneesInstitut Pasteur, Centre Nationale de la Recherche Scientifique
InventorsRafael Vazquez-Martinez, Philippe Brulet
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Abstract
A method for visualizing an active synapse wherein said method comprises: (a) exposing cells forming the active synapse to a biomarker comprising at least fragment C of tetanus toxin and a reporter protein; and (b) visualizing the biomarker; wherein the accumulation of the biomarker into dendritic spines of the cells allows visualization of an active synapse. Also, a method for screening molecules capable of modulating synapse activity is provided. A kit useful for the early diagnosis of neurodegenerative disease comprises a biomarker comprising at least fragment C of tetanus toxin and a reporter protein.
Claims23
A method of evaluating the state of networking activity of a neuron, wherein the method comprises visualizing, ex vivo or in vitro, a biomarker in dendritic spines of the cells of a synapse of the neuron, wherein the biomarker comprises at least fragment C of tetanus toxin (TTC) tagged with a reporter protein chosen from beta-Galactosidase (β-gal), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), Discosoma sp, red fluorescent protein (DsRED), and Heteractis crispa far-red fluorescent variant (HcRED), and wherein an elevated amount of the biomarker in the dendritic spines relative to the amount of the biomarker in dendritic shaft regions indicates a neuron that is forming synaptic connections.
A method of evaluating the state of networking activity of a neuron, wherein the method comprises
- (A) exposing cells of a synapse having dendritic spines to a biomarker in vitro;
- and
- (B) visualizing, ex vivo or in vitro, the biomarker in dendritic spines of the cells of the synapse of the neuron, wherein the biomarker comprises at least fragment C of tetanus toxin (TTC) tagged with a reporter protein chosen from beta-Galactosidase (β-gal), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), Discosoma sp, red fluorescent protein (DsRED), and Heteractis crispa far-red fluorescent variant (HcRED),
- wherein an elevated amount of the biomarker in the dendritic spines relative to the amount of the biomarker in dendritic shaft regions indicates a neuron that is forming synaptic connections.
A method of evaluating the state of networking activity of a neuron, wherein the method comprises
- (A) exposing cells of a synapse having dendritic spines to a biomarker in vivo;
- and
- (B) visualizing, ex vivo or in vitro, the biomarker in dendritic spines of the cells of the synapse of the neuron, wherein the biomarker comprises at least fragment C of tetanus toxin (TTC) tagged with a reporter protein chosen from beta-Galactosidase (β-gal), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), Discosoma sp, red fluorescent protein (DsRED), and Heteractis crispa far-red fluorescent variant (HcRED),
- wherein an elevated amount of the biomarker in the dendritic spines relative to the amount of the biomarker in dendritic shaft regions indicates a neuron that is forming synaptic connections.
A method of evaluating the state of networking activity of a neuron, wherein the method comprises:
- (A) providing an active synapse comprised of cells having dendritic spines;
- (B) transfecting in vitro the cells having dendritic spines with a vector that expresses a biomarker comprising at least fragment C of tetanus toxin (TTC) fused with a reporter protein chosen from β-gal, GFP, RFP, YFP, CFP, DsRED, and HcRED;
- (C) selecting, in vitro, transfected cells that are positive for expression of the biomarker;
- (D) providing a central nervous system (CNS) or a spinal cord of an animal;
- (E) implanting the transfected cells in the CNS or spinal cord;
- and
- (F) visualizing, ex vivo or in vitro, the biomarker in dendritic spines of an active synapse, wherein an elevated amount of the biomarker in the dendritic spines relative to the amount of the biomarker in dendritic shaft regions indicates a neuron that is forming synaptic connections.
A method of evaluating the state of networking activity of a neuron, wherein the method comprises:
- (A) providing cells of an active synapse, wherein the cells have dendritic spines, and the dendritic spines contain an accumulation of a biomarker that has been transported into the dendritic spines;
- and
- (B) visualizing the biomarker in the dendritic spines in vitro;
- wherein the biomarker is a fusion protein comprising at least fragment C of tetanus toxin (TTC) tagged with a reporter protein chosen from β-gal, GFP, RFP, YFP, CFP, DsRED, and HcRED, wherein an elevated amount of the biomarker in the dendritic spines relative to the amount of the biomarker in dendritic shaft regions indicates a neuron that is forming synaptic connections.
An ex vivo or in vitro method of evaluating the state of networking activity of a neuron in an animal, the method comprising injecting a biomarker into a muscle of the animal wherein the biomarker comprises at least fragment C of tetanus toxin (TTC) fused to a reporter protein chosen from beta-Galactosidase (β-gal), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), Discosoma sp, red fluorescent protein (DsRED), and Heteractis crispa far-red fluorescent variant (HcRED), wherein an elevated amount of the biomarker in the dendritic spines relative to the amount of the biomarker in dendritic shaft regions indicates a neuron that is forming synaptic connections.
Dependencies are inferred from the claim text.