5. Interleukin-3 Promotes Neurite Outgrowth and Elevates Choline Acetyltrans f erase Activity in vitro

Introduction. Central cholinergic neurons are reported to be important in memory acquisition and are known to degenerate in Alzheimer's disease.l) Since nerve growth factor (NGF) was found to increase choline acetyltransf erase (ChAT) activity''),.) and prevent cholinergic neuron death,4)-°~ investigators started to look for other neurotrophic factors for these neurons. So far, fibroblast growth factor (FGF) was shown to promote survival of dissociated hippocampal neurons 7 > and to prevent death of damaged central cholinergic neuronss~ but the effect on ChAT activity was determined with peripheral cholinergic neurons.9~ Recently, interactions of the immune-nervous system have attracted great interest. Several cytokines have been found to have neurotrophic effect or to effect neuronal differentiation. For instance, neuroleukin is a factor produced by T cells, which induces B cell differentiation and shows trophic effect on spinal cord neurons.12~ Interleukin-6 (IL-6) also induces differentiation of B cells to plasma cells as well as differentiation of PC12 cells, rat pheochromocytoma line cells, to neuronal cells.l3~ For these reasons, we looked for cytokines that may have trophic effects on central cholinergic neurons. Since cytokines are well characterized in humans and mice, we established a serum-free culture system of murine dissociated neurons. Material and method. The septal regions including the basal forebrain were taken from BALB/c mice (Sankyo Laboratory Service, Tokyo) on 15 embryonic day, and were dissected in calciumand magnesium-free Hanks' balanced salt solution (HESS), pH 7.4. The wet tissue in HBSS (10 mg/ml) was treated with 0.03% trypsin (pH 6.5) for 3 min at 37°C, pipetted and sieved through a 63 μm nylon mesh. Dissociated cells were washed twice with HBSS (pH 6.5) and once with the serum-free basal medium-1 (see below) and centrifuged at 200 g. Cell viability was checked by 0.16% trypan blue and a certain number of viable cells were resuspended in the basal medium-2 (see below) with or without a factor to be tested. After preincubation for 15 min, floating dead cells were removed, and cells were cultured in a humidified air mixture with 5% C02 at 37°C. In this condition, cultured cells were comprised almost entirely of neurons, which was confirmed by immunocytochemical and histochemical stainings. Basal medium-1 is a 1; l mixture of Dulbecco's modified Eagle's medium containing 4.5 g/1 glucose (Gibco, Grand Island, NY) and Ham's F 12 (Gibco), pH 7.4, supplemented with 15 mM Hepes buffer, 30 nM sodium selenate, and 20 mM penicillin-streptomycin, Gibco). Basal medium-2 is composed of the basal