title: WGA-Alexa transsynaptic labeling in the phrenic motor system of adult rats: Intrapleural injection versus intradiaphragmatic injection.
abstract: BACKGROUND
  Intrapleural injection of CTB-Alexa 488, a retrograde tracer, provides an alternative labeling technique to the surgically invasive laparotomy required for intradiaphragmatic injection. However, CTB-Alexa 488 is incapable of crossing synapses restricting the tracer to the phrenic nuclei and the intercostal motor nuclei in the spinal cord.
  
  
  METHODS
  Intrapleural injection of WGA-Alexa 488, a transsynaptic tracer, provides a method to label the respiratory motor pathway in both the spinal cord and medulla. Intradiaphragmatic injection of WGA-Alexa 594 and vagal nerve injections of True blue were used to confirm the phrenic nuclei and to differentiate between the rVRG and the NA in the medulla.
  
  
  RESULTS
  Following intrapleural injection, WGA-Alexa 488 was retrogradely transported to the phrenic nuclei and to the intercostal motor nuclei. Subsequently WGA-Alexa 488 was transsynaptically transported from the phrenic motoneurons to the pre-motor neurons in the rVRG that provide the descending drive to the phrenic neurons during inspiration. In addition WGA-Alexa 488 was identified in select cells of the NA confirmed by a dual label of both WGA-Alexa 488 and True blue.
  
  
  CONCLUSIONS
  WGA-Alexa 488 demonstrates retrograde transsynaptic labeling following intrapleural injection whereas the previous method of injecting CTB-Alexa 488 only demonstrates retrograde labeling.
  
  
  CONCLUSIONS
  Intrapleural injection of WGA-Alexa fluor conjugates is an effective method to transsynaptically label the phrenic motor system providing an alternative for the invasive laparotomy required for intradiaphragmatic injections. Furthermore, the study provides the first anatomical evidence of a direct synaptic relationship between rVRG and select NA cells.
authors.affiliation: Department of Anatomy and Cell Biology, Wayne State University, School of Medicine, 540 East Canfield Street, Detroit, MI 48201, USA.
authors.forename: Janelle L
authors.initials: JL
authors.lastname: Buttry
authors.affiliation: Department of Anatomy and Cell Biology, Wayne State University, School of Medicine, 540 East Canfield Street, Detroit, MI 48201, USA. Electronic address: hgoshgar@med.wayne.edu.
authors.forename: Harry G
authors.initials: HG
authors.lastname: Goshgarian
mesh_terms: D000818:Animals; D003964:Diaphragm / Q000737:chemistry* / Q000187:drug effects; D007267:Injections; D008297:Male; D009930:Organic Chemicals / Q000008:administration & dosage / Q000032:analysis; D010791:Phrenic Nerve / Q000737:chemistry* / Q000187:drug effects; D035422:Pleural Cavity / Q000737:chemistry* / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods; D013569:Synapses / Q000737:chemistry* / Q000187:drug effects; D014909:Wheat Germ Agglutinins / Q000008:administration & dosage / Q000032:analysis*
chemical_list: C417664:Alexa594; D009930:Organic Chemicals; D014909:Wheat Germ Agglutinins
keywords: Intradiaphragmatic injection; Intrapleural injection; Phrenic nuclei; Respiratory pathway; Rostral ventral respiratory groups; WGA-Alexa