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

• Published in: Brain research Vol. 947; no. 2; pp. 243 - 251
• Main Authors: Fujikawa, Naoto, Shimonaga, Tomokazu, Tominaga-Yoshino, Keiko, Ogura, Akihiko
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 30.08.2002; Amsterdam Elsevier; New York, NY
• Subjects: Activity-dependent survival; Animals; Biological and medical sciences; Cell Count; Cell Survival; Cerebellum - metabolism; Coculture Techniques - methods; Dialysis membrane; Estrogen; Estrogens - metabolism; Fundamental and applied biological sciences. Psychology; gamma-Aminobutyric Acid - metabolism; Glutamic Acid - metabolism; Isolated neuron and nerve. Neuroglia; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Weight; Nerve Growth Factors - metabolism; Rats; Rats, Wistar; Species-dependency; Strain-dependency; Ultrafiltration; Vertebrates: nervous system and sense organs; Species-dependency; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; DNQX, 6,7-dinitroquinoxaline-2,3-dione; NMDA, N-methyl- d-aspartic acid; Estrogen; GABA, γ-aminobutylic acid; NGF, nerve growth factor; DIV, day(s) in vitro; BDNF, brain-derived neurotrophic factor; Development and regeneration; Dialysis membrane; PACAP, pituitary adenylyl cyclase activating polypeptide; NT, neurotrophin; MCPG, α-methyl-(4-carboxyphenyl)glycine; PTHrP, parathyroid hormone related polypeptide; Activity-dependent survival; CGN, cerebellar granule neuron; Strain-dependency; APV, 2-amino-5-phosphonovaleric acid; MEM, minimal essential medium; Cerebellum; Neurotrophic factor; Vertebrata; Mammalia; Granule neuron; Mouse; Rodentia; Central nervous system; Technique; In vitro; Release; Brain (vertebrata)
• ISSN: 0006-8993; 1872-6240
• DOI: 10.1016/S0006-8993(02)02931-1

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, γ-aminobutylic 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.