Synaptogenesis of cultured striatal neurons in serum-free medium: a morphological and biochemical study.

TLDR

Striatal neurons were cultured from fetal mouse brain and maintained in serum‑free medium for 14–21 days in vitro. Pretreatment of dishes with a polycation and fetal calf serum promoted rapid neuron attachment and neurite outgrowth, and over 9–14 days in vitro the cultures developed mature synapses (confirmed by electron microscopy and Synapsin I localization), showed a decline in neurotransmitter‑stimulated cAMP production but a rise in inositol phosphate signaling, and exhibited progressively greater K⁺‑evoked GABA release—doubling by 11 DIV and quintupled by 14 DIV, with increasing Ca²⁺ dependence—demonstrating that serum‑free culture supports full striatal neuronal differentiation.

Abstract

Striatal neurons were cultured from the fetal mouse brain and maintained in serum-free medium for 14-21 days in vitro (DIV). Pretreatment of the culture dishes successively with a polycation followed by fetal calf serum resulted in rapid neuron attachment and neurite proliferation. After 9-10 DIV, electron microscope observations revealed the presence of vesicles in axon terminals forming mature synapses with axons and perikarya of adjacent neurons and in varicosities along extended axons. Synapsin I, a synaptic vesicle-specific protein, was present only in neuronal perikarya after 3 DIV, in perikarya and in varicosities along extended axons after 6 DIV, and in varicosities and contact points between axon terminals and adjacent axons or perikarya after 11-14 DIV. Neurotransmitter-stimulated intracellular formation of cAMP decreased markedly during neuronal differentiation. Inositol phosphate formation in response to neurotransmitters, however, increased significantly throughout the period of striatal neuronal development. K+ (56 mM) depolarization resulted in a 2-fold increase in endogenous gamma-aminobutyric acid (GABA) release from striatal neurons, 50% of which was Ca2+-dependent, between 3 and 11 DIV. Between 11 and 14 DIV, subsequent to synapse formation (as revealed by electron microscope observations), GABA release evoked by 56 mM K+ increased up to 5-fold, 75% of which was Ca2+-dependent. It appears that the complete differentiation of striatal neurons in serum-free medium may provide a suitable model for the study of the physiological and regulatory mechanisms involved in nerve cell development.