hr. 1. Dwl. Neuroscience. Vol. 6. No. 2. pp. 137-147. IY88. Printed in Great Britain. 0736-57wRR $03.00+0.tx, Pergamon Press pk. Q 1988 ISDN SYNAPSE FORMATION AND DEVELOPMENT OF NEUROTRANSMITTER FUNCTIONS IN NEURONAL CELLS FROM CHICK BRAIN CULTURED IN A SERUM-FREE, DEFINED MEDIUM D. LANGUI,* S. SARHAN,t G. DEVILLIERS,* B. PETTMANN,* J. P. DELAUNOY,* N. SEILER~ and M. SENSENBRENNER~ ‘Centre de Neurochimie du CNRS and INSERM U44, 5, rue Blaise Pascal; and tMerrell Dow Research Institute, Strasbourg Center, 16, rue d’Ankara, 67084 Strasbourg Cedex, France (Received 29 December 1986; in revised form 21 November 1987; accepted 3 December 1987) AbstractXells dissociated from cerebral hemispheres of 8-day-old chick embryos were seeded on poly-L-lysine coated Petri dishes in serum-containing medium. After 24 hr the culture medium was switched to a serum-free, chemically defined medium. These cultures contain mainly neuronal cells until day 14, characterized by the presence of acetylcholinesterase activity and neurotilament proteins. After 2 weeks glial cells progressively contaminated the neuronal culture. Cultures were maintained for a period of 4 weeks. From day 6 on numerous synapses with clear vesicles were observed. The activity of choline acetyltransferase remained low throughout the culture period, while GABA levels increased in parallel with synaptogenesis. Our observations indicate that chick cerebral hemisphere neuronal cultures grown in serum-free, chemically defined medium contain GABAergic neurons that undergo maturation. Key words: Culture. Neurons. Chick, Synapses, GABA In a previous work we have described primary cultures consisting mainly of neuronal cells from chick embryo brains.” These cultured cells, if maintained in a serum-containing medium, undergo morphological and biochemical maturation.“.*” Electron microscopy revealed the presence of synapses containing predominantly dense-core vesicles.” This apparently homogenous population of neuronal cells was predominantly dopaminergic*” and GABAergic.‘“.2”.J’ Recently, biologically active peptides have been shown to be present in these neurons. ” The activity of choline acetyltransferase (ChAcT: EC 2.3.1.6) remained low during the morphological maturation of the chick cerebral hemisphere neurons.” In the present study we describe the maturation of chick neuronal cells in a serum-free defined medium, and the development of neuron-specific functions in relation to synapse formation. EXPERIMENTAL PROCEDURES zyxwvutsrqponmlkjihgfedcbaZYXW Primary neuronal cell culture Cerebral hemispheres from 8-day-old chick embryos were dissected, cleaned of their meningeal membranes, passed through a sterile nylon sieve (48 pm pore size), and the dis- sociated cells were collected in nutrient medium. The nutrient medium was Dulbecco’s modified Eagle’s medium (DMEM: Gibco) plus 20% fetal calf serum (Gibco), 50 U/ml penicillin and 50 Fg/ml streptomycin. The cells (approximately 2 x 10h cells/dish) were plated on poly+-lysine P 2636 (Sigma) coated Falcon Petri dishes (diameter 60 mm), as was previously described.*” Cultures were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO*. After 24 hr, the cultures were washed three times with DMEM and fed with the chemically defined, slightly modified Nl-medium,’ and consisting of a 1: 1 mixture of DMEM and Ham’s F12 medium (Gibco), or DMEM alone plus 5 pg/ml bovine insulin (Sigma), 5, 10, 50 or 100 &ml human transferrin (Sigma), 20 nM progesterone (Sigma), 100 PM putrescine (Sigma), 30 nM sodium selenite (Merck), 50 U/ml penicillin and 50 &ml streptomycin. The medium was changed twice a week. Electron microscopy Cultures were fixed in situ in a 5% solution of glutaraldehyde (in phosphate buffer 0.1 M, pH 7.2) for 30 min at room temperature, rinsed with the buffer and post-fixed in 1% 0~0, for 137