Source Paper
Distribution of Brain-Derived Neurotrophic Factor (BDNF) Protein and mRNA in the Normal Adult Rat CNS: Evidence for Anterograde Axonal Transport
James M. Conner, Julie C. Lauterborn, Qiao Yan, Christine M. Gall, Silvio Varon
Journal of Neuroscience • 1997
Afferent Elimination Study
Objective: Test whether BDNF in target regions is derived from anterograde axonal transport by eliminating select afferent systems and measuring BDNF immunostaining changes
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Equipment2
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Protocol Steps
Immunohistochemical mapping of BDNF
Generate detailed mapping of BDNF immunoreactivity throughout the adult rat CNS using sensitive immunohistochemical technique with highly specific affinity-purified antibodies to BDNF
Note: Identifies groups of cell bodies containing diffuse BDNF-ir and varying degrees of BDNF-ir outside cell bodies in fibers and terminals
View evidence from paper
“A sensitive immunohistochemical technique was used, along with highly specific affinity-purified antibodies to brain-derived neurotrophic factor (BDNF), to generate a detailed mapping of BDNF immunoreactivity (BDNF-ir) throughout the adult rat CNS”
In situ hybridization analysis of BDNF mRNA
Perform parallel analysis of sites of BDNF synthesis using in situ hybridization techniques with cRNA probe to exon encoding mature rat BDNF protein
Note: Identifies fields of cells containing BDNF mRNA and correlates with BDNF-ir cell bodies
View evidence from paper
“A parallel analysis of sites of BDNF synthesis was performed with in situ hybridization techniques using a cRNA probe to the exon encoding mature rat BDNF protein”
Identify regions with BDNF-ir lacking mRNA
Map regions containing heavy BDNF-immunoreactive fiber/terminal labeling that lack BDNF mRNA, suggesting BDNF derives from anterograde axonal transport
Note: Target regions include medial habenula, central nucleus of amygdala, bed nucleus of stria terminalis, lateral septum, and spinal cord
View evidence from paper
“many regions containing extremely heavy BDNF-immunoreactive fiber/terminal labeling that lacked BDNF mRNA (e.g., medial habenula, central nucleus of the amygdala, bed nucleus of stria terminalis, lateral septum, and spinal cord)”
Eliminate select afferent systems
Perform surgical or chemical elimination of select afferent systems to target regions identified as containing BDNF-ir without BDNF mRNA
Note: Two cases tested; specific elimination methods not detailed in provided text
View evidence from paper
“In the two cases in which this hypothesis was tested by the elimination of select afferents”
Assess BDNF immunostaining after afferent elimination
Perform immunohistochemistry on tissue from animals with eliminated afferents to determine if BDNF immunostaining is reduced or eliminated
Note: Complete elimination of BDNF immunostaining observed in tested cases
View evidence from paper
“BDNF immunostaining was completely eliminated”