The molecular mechanisms controlling synaptogenesis in the central nervous system (CNS) are poorly understood. Previous reports showed that a glia-derived factor strongly promotes synapse development in cultures of purified CNS neurons. Here, we identify this factor as cholesterol complexed to apolipoprotein E-containing lipoproteins. CNS neurons produce enough cholesterol to survive and grow, but the formation of numerous mature synapses demands additional amounts that must be provided by glia. Thus, the availability of cholesterol appears to limit synapse development. This may explain the delayed onset of CNS synaptogenesis after glia differentiation and neurobehavioral manifestations of defects in cholesterol or lipoprotein homeostasis.

To study the effects of glial cells on synapse formation, we established microcultures of purified rat retinal ganglion cells (RGCs) and monitored synapse (autapse) development in single neurones using electrophysiological recordings, FM1‐43 labelling and immunocytochemistry. Solitary neurones grew ramifying neurites, but formed only very few and inefficient excitatory autapses, when cultured for up to 2 weeks in defined medium and in the absence of glial cells. Treatment of glia‐free microcultures of RGCs with glia‐conditioned medium (GCM) increased the number of autapses per neurone by up to 10‐fold. This was indicated by a similar increase in the frequency of spontaneous events and the number of FM1‐43‐labelled functional release sites and of puncta, where pre‐ and postsynaptic markers colocalized. In addition, GCM treatment enhanced the efficacy of presynaptic transmitter release as indicated by lower failure rates of stimulation‐induced excitatory autaptic currents, a 200‐fold increase in the frequency of asynchronous release and an accelerated stimulation‐induced FM1‐43 destaining. Furthermore, GCM induced an increase in the quantal size. GCM affected autaptic activity not immediately, but with a delay of 24 h, and the effects on stimulation‐induced autaptic currents occurred before changes in the frequency of spontaneous events indicating an early strengthening of existing autapses followed by a later increase in autapse number. The observed effects were mediated by proteinase K‐sensitive factors in GCM and occurred independently of electrical activity. These results suggest that soluble glia‐derived signals induce synapse formation and maturation in neurones of the central nervous system (CNS).

There is increasing evidence that synapse function depends on interactions with glial cells, namely astrocytes. Studies on specific neurons of the central nervous system (CNS) indicated that glial signals also control synapse development, but it remained unclear whether this is a general principle that applies to other neuronal cell types. To address this question, we developed new methods to immunoisolate neurons from different brain regions of postnatal mice and to culture them in a chemically defined medium. Electrophysiological recordings and immunocytochemical staining revealed vigorous synaptogenesis in hippocampal and cerebellar neurons, but not in retinal ganglion cells (RGCs) in the absence of glial cells. Co-culture with glia promoted synapse formation in RGCs as indicated by a strong increase in the incidence and frequency of action potential-independent miniature synaptic currents, but showed no such effects in hippocampal or cerebellar neurons. On the other hand, glial signals promoted the efficacy of excitatory synapses in all regions as indicated by an increase in the size of spontaneous synaptic events in cerebellar cultures and of miniature synaptic currents in hippocampal neurons and RGCs. Inhibitory synaptic currents remained largely unaffected by glia. Our results indicate that in the mammalian CNS, the way that glial signals promote the development of excitatory synapses depends on the type of neuron.