Enrichment of the fraction of nociceptive neurones in cultures of primary sensory neurones.
A method for enriching the fraction of nociceptive neurones in cultures of primary sensory neurones is described. Neurones from neonatal rat dorsal root ganglia were isolated, layered on 40% Ficoll and centrifuged, separating the neurones into a low density fraction (LDF) and a high-density fraction (HDF). The LDF had a smaller mean diameter (19.7 microm) than the HDF (27.3 microm) and uncentrifuged cells (23.6 microm). The proportion of cells immunoreactive for antibodies to substance P and calcitonin gene-related peptide, both of which are found in nociceptive neurones, was significantly greater in the LDF than in HDF. A substantial enrichment of the proportion of neurones responding to the algogenic substances capsaicin and bradykinin with an increase in intracellular calcium was observed in the LDF. The proportion of capsaicin and bradykinin-responsive neurones was also found to be increased by culturing the neurones, with a particularly pronounced enhancement in the proportion of bradykinin-responsive cells. We conclude that separation on the basis of density followed by culture is a useful way of enriching the proportion of nociceptive neurones for the purpose of electrophysiological, biochemical or other studies.
Gilabert
R
R
Department of Physiology, King's College London, Strand, UK.
McNaughton
P
P
D000818:Animals; D000831:Animals, Newborn; D000918:Antibody Specificity; D001920:Bradykinin / Q000494:pharmacology; D015740:Calcitonin Gene-Related Peptide / Q000032:analysis / Q000276:immunology; D002118:Calcium / Q000032:analysis; D002211:Capsaicin / Q000494:pharmacology; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000166:cytology; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009475:Neurons, Afferent / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D009619:Nociceptors / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D013373:Substance P / Q000032:analysis / Q000276:immunology
D013373:Substance P; D015740:Calcitonin Gene-Related Peptide; D002211:Capsaicin; D001920:Bradykinin; D002118:Calcium