Effects of polyunsaturated fatty acids and hormones on synaptogenesis in serum-free medium cultures of mouse fetal hypothalamic cells.
The effects of soluble factors on synaptogenesis by mouse fetal hypothalamic cells cultured in chemically defined conditions have been examined using transmission electron microscopy. Hypothalami taken on the 16th day of gestation were mechanically dissociated and cells were seeded in a minimum serum-free medium supplemented or not with the following components: triiodothyronine, corticosterone and a mixture of polyunsaturated fatty acids (arachidonic acid plus docosahexaenoic acid bound to defatted bovine serum albumin). In the minimum serum free medium synapses were found after 10 days in culture. However, the development of synaptic vesicles was very limited, whereas that of the presynaptic and postsynaptic densities was apparently normal. Supplementation of the minimum serum-free medium with triiodothyronine, corticosterone and polyunsaturated fatty acids added simultaneously, permitted a full development of synapses as attested to by the increase in number and the regular shape and diameter of synaptic vesicles as well as by the complexity and diversity of synapse configurations. Among those three factors, polyunsaturated fatty acids clearly played a key role. The ability of synapses formed in culture to respond to potassium evoked depolarization was examined on cultures grown for 12 days in the simultaneous presence of the three above mentioned supplements. Exposure for 3 min to 60 mM potassium chloride induced in synaptic boutons vesicular depletion, apposition of vesicle clusters onto the presynaptic grid, appearance of a rich filamentous network and of some coated vesicles. Return to 3mM potassium chloride induced in 3 min a massive restoration of the population of vesicles which slightly differed from synaptic vesicles in control cultures. These results show that: (1) the formation of synaptic vesicles in this system is regulated by soluble factors among which polyunsaturated fatty acids play a major role, and (2) synapses formed de novo in chemically defined conditions of culture display the same ability to respond to and to recover from potassium evoked depolarization as adult axon terminals. Thus, they offer a suitable model for analysis of the mechanisms involved in membrane traffic in central neurons.
D000818:Animals; D002478:Cells, Cultured; D003345:Corticosterone / Q000494:pharmacology*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology*; D007031:Hypothalamus / Q000196:embryology* / Q000648:ultrastructure; D007797:Laminin / Q000494:pharmacology; D051379:Mice; D008854:Microscopy, Electron; D011189:Potassium Chloride / Q000494:pharmacology; D013569:Synapses / Q000187:drug effects*; D014284:Triiodothyronine / Q000494:pharmacology*
D005231:Fatty Acids, Unsaturated; D007797:Laminin; D014284:Triiodothyronine; D011189:Potassium Chloride; D003345:Corticosterone