Ultrafilter co-culture, a new method for estimating the molecular mass of bioactive substances, indicates a small molecule neurotrophic substance is released from cultured cerebellar granule neurons of the BALB/c mouse.
Cultured rat cerebellar granule neurons (CGNs), which require a depolarizing agent in the medium for long-term survival, are widely used for the analysis of mechanisms underlying the activity-dependent survival of neurons. It was recently found that this is not the case for BALB/c mouse CGNs, which survive without a depolarizing agent. Co-culture experiments indicated that the mouse cells release a neurotrophic substance. However, the substance is apparently short-living in the medium, making its molecular identification difficult. Here a novel co-culture method was devised for estimating the relative molecular masses of biologically active substances, using a commercially available dialysis membrane filter unit to separate substance-donor from substance-recipient cells. By this simultaneous fractionation/bioassay, the molecular mass of the assumed neurotrophic substance was estimated to be <3 kDa. Neurotrophic substances previously reported to be effective in rat CGNs, including neurotrophins, pituitary adenylate cyclase-activating polypeptide, parathyroid hormone-related polypeptide, glutamic acid, gamma-aminobutyric acid, and D-serine, were excluded as candidate molecules. Estrogen, however, remained a candidate. It should be stressed that the requirements for the activity-dependent survival of CGNs are species-dependent. Care should be taken in the analysis of activity-dependent neuronal survival using transgenic animals.
Fujikawa
Naoto
N
Department of Biology, Osaka University Graduate School of Science, Toyonaka, Osaka 560-0043, Japan.
Shimonaga
Tomokazu
T
Tominaga-Yoshino
Keiko
K
Ogura
Akihiko
A
D000818:Animals; D002452:Cell Count; D002470:Cell Survival; D002531:Cerebellum / Q000378:metabolism*; D018920:Coculture Techniques / Q000379:methods*; D004967:Estrogens / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014462:Ultrafiltration; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D004967:Estrogens; D009414:Nerve Growth Factors; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid