title: AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations.
abstract: As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression of the enzyme. A similar vector for expression of green fluorescent protein (GFP) was injected into the same location. For each vector, two versions with minor differences were used, giving similar results. After 4 weeks, the brains were stained to show GFP and products of chondroitinase digestion. Chondroitinase was widely expressed, and the AAV-ChABC and AAV-GFP vectors gave similar expression patterns in many respects, consistent with the known projections from the directly transduced neurons in vibrissal motor cortex and adjacent cingulate cortex. In addition, diffusion of vector to deeper neuronal populations led to labelling of remote projection fields which was much more extensive with AAV-ChABC than with AAV-GFP. The most notable of these populations are inferred to be neurons of cortical layer 6, projecting widely in the thalamus, and neurons of the anterior pole of the hippocampus, projecting through most of the hippocampus. We conclude that, whereas GFP does not label the thinnest axonal branches of some neuronal types, chondroitinase is efficiently secreted from these arborisations and enables their extent to be sensitively visualised. After 12 weeks, chondroitinase expression was undiminished.
authors.affiliation: John van Geest Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK; Department of Physiology Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
authors.forename: João Nuno
authors.initials: JN
authors.lastname: Alves
authors.affiliation: Department of Physiology Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Muir
authors.affiliation: John van Geest Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.
authors.forename: Melissa R
authors.initials: MR
authors.lastname: Andrews
authors.affiliation: Department of Physiology Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
authors.forename: Anneliese
authors.initials: A
authors.lastname: Ward
authors.affiliation: Department of Physiology Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Michelmore
authors.affiliation: Department of Physiology Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
authors.forename: Debayan
authors.initials: D
authors.lastname: Dasgupta
authors.affiliation: Laboratory for Neuroregeneration, Netherlands Institute for Neuroscience, Meibergdreef 47, 1105BA Amsterdam, Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: Verhaagen
authors.affiliation: Laboratory for Neuroregeneration, Netherlands Institute for Neuroscience, Meibergdreef 47, 1105BA Amsterdam, Netherlands.
authors.forename: Elizabeth B
authors.initials: EB
authors.lastname: Moloney
authors.affiliation: Department of Physiology Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Keynes
authors.affiliation: John van Geest Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.
authors.forename: James W
authors.initials: JW
authors.lastname: Fawcett
authors.affiliation: Department of Physiology Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK. Electronic address: jhr11@cam.ac.uk.
authors.forename: John H
authors.initials: JH
authors.lastname: Rogers
mesh_terms: D000818:Animals; D000941:Antigens / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D001369:Axons / Q000378:metabolism / Q000502:physiology*; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D019765:Chondroitin ABC Lyase / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics; D005822:Genetic Vectors / Q000502:physiology*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology*; D037121:Plant Lectins / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats; D034781:Receptors, N-Acetylglucosamine / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods; D000099216:Chondroitin Sulfate Proteoglycan 4
chemical_list: D000941:Antigens; D015703:Antigens, CD; D002135:Calcium-Binding Proteins; D019765:Chondroitin ABC Lyase; D005904:Glial Fibrillary Acidic Protein; D049452:Green Fluorescent Proteins; D008840:Microfilament Proteins; D037121:Plant Lectins; D011509:Proteoglycans; D034781:Receptors, N-Acetylglucosamine; D000099216:Chondroitin Sulfate Proteoglycan 4; C100745:Aif1 protein, rat; C040129:wisteria lectin
keywords: AAV; Chondroitinase; Corticospinal tract; Green fluorescent protein; Pathway tracing; Thalamus; Vibrissal motor cortex; Whisker movements