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Using AAV9 to trace bulbospinal inputs to phrenic motoneurons
Author(s) -
Nair Jayakrishnan,
Mandel Ronald J,
Fuller David D
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1294.2
Subject(s) - spinal cord , brainstem , mcherry , anatomy , medulla oblongata , phrenic nerve , medulla , green fluorescent protein , biology , respiratory system , chemistry , medicine , central nervous system , endocrinology , neuroscience , biochemistry , gene
We used adeno associated virus (AAV) serotype 9 driving expression of green (AAV9‐cba‐GFP) or red (AAV9‐cba‐mCherry) fluorescent protein to examine the distribution of bulbospinal projections to the phrenic motor nucleus in the adult rat. Rats were anesthetized with isoflurane and focal injections of AAV9‐GFP (left medulla; titer: 1.19 × 10e13 vg/ml; volume: 0.25μl) and AAV9‐mCherry (right medulla; titer: 1.66 × 10e13 vg/ml; volume: 0.25μl) were targeted to the immediate region of medullary rostral ventral respiratory group (rVRG) as determined in separate neurophysiological recordings. In spinal intact rats, brainstem and spinal cord tissues were harvested 18‐wks following AAV injection. Separate animals received a cervical spinal cord hemisection injury (C2Hx) 4‐wks prior to the brainstem AAV injections. Phrenic motoneurons were retrogradely and bilaterally labeled 3‐days prior to tissue harvest using intrapleural delivery of cholera toxin‐beta (CT‐β, 0.2%, 5μl). When the rVRG showed robust transgene expression as indicated via fluorescence, a dense cluster of AAV9‐GFP or mCherry expressing terminal projections could also be observed in the phrenic nucleus bilaterally. In a few rats, injections were slightly lateral to the rVRG, and these produced minimal if any labeling in the region of the phrenic nucleus. Preliminary observation of medullary injected C2Hx cord (14‐wks) revealed robust AAV9‐GFP and mCherry expression in the phrenic nucleus contralateral to the lesion, and a dramatically reduced but even distribution of GFP and mCherry positive fibers along the ipsilateral phrenic nucleus. These initial results indicate that injection of AAV9 to the rVRG is an effective technique to label bulbospinal projections to the phrenic motor nucleus. Support or Funding Information 1R01NS080180‐01A1 (DDF)