In this study, we analysed the effect of glial signals on synapse formation and function in single CNS neurones using a new microculture preparation and a combination of electrophysiological recordings of autaptic activity, FM1-43 labelling of release sites and immunocytochemical staining of autapses. We found that glia-derived proteinase K-sensitive factor(s) induced a drastic increase in the level of spontaneous and asynchronous release and enhanced the reliability of evoked autaptic transmission. GCM affected autaptic activity not immediately, but with a delay of 24 h after application. The effects on evoked autaptic transmission and asynchronous release occurred before the changes in the frequency of spontaneous events indicating an early strengthening of existing autapses followed by a later increase in autapse number. FM1-43 labelling of functional presynaptic release sites and double-staining of autapses with pre- and postsynaptic markers indicated that the glia-induced effects on autaptic activity were due to the drastic increase in the number of autapses and in the probability of transmitter release.
Neurones form only few and inefficient synapses in glia-free microcultures
CNS neurones form readily excitatory autaptic connections in microcultures containing serum and/or glial cells (Segal, 1991; Johnson & Yee, 1995). Recently, Gomperts et al. (2000) estimated that hippocampal neurones growing on glial microislands form one synapse per hour. We found that single RGCs growing for up to 2 weeks under defined conditions formed autapses. This is in agreement with the previous study by Pfrieger & Barres (1997), who observed ultrastructurally defined synapses in normal, glia-free cultures of RGCs. However, our immunostaining and FM1-43 labelling indicate that the number of autapses formed by individual neurones was very small. Why did RGCs form so few autapses? We conclude that they lacked glia-derived factors that promote autapse formation. Low survival rates and insufficient neurite outgrowth cannot account for the low number of autapses in our microcultures. RGCs appeared healthy as indicated by their electrical excitability and the presence of ramifying neurites with numerous growth cones. RGCs may have formed few synapses, because they lacked instructive signals from their natural partner neurones. This appears unlikely in our case, since purified rat RGCs growing under defined conditions in mass cultures form ultrastructurally defined synapses and addition of collicular neurones does not increase the level of synaptic activity (Pfrieger & Barres, 1997). Furthermore, it has been shown that RGCs form autapses in vitro (Taschenberger et al. 1999) and establish intraretinal connections in vivo (Peterson & Dacey, 1998).
Autapses formed under glia-free conditions appeared very inefficient. This was indicated by the small size of spontaneous EACs, the low frequency of asynchronous events, the high failure rates and the slow FM1-43 destaining rates. Why were the autapses so inefficient? We believe that their development stalled at an immature level due to the lack of signals that promote their further maturation. The functional properties of autapses in glia-free cultures resembled those of immature synapses described previously in vivo during early stages of development (Broadie & Bate, 1993; Wu et al. 1996; Nguyen et al. 1999) and in vitro immediately after contact establishment (for reviews see Grinnell, 1995; Fitzsimonds & Poo, 1998). The absence of autaptic currents from most RGCs raises the question of whether these neurones formed functionally silent autapses (Malenka & Nicoll, 1997), which have been observed in CNS microcultures (Kimura et al. 1997; Gomperts et al. 1998). The presence of presynaptically silent autapses in our microcultures is unlikely, however, since even application of α-latrotoxin did not induce autaptic currents. Postsynaptically silent autapses, transmitting only by NMDA receptors, were not present either, since RGCs lacked NMDA receptor-mediated autaptic currents despite the presence of functional NMDA receptors. Our observation is supported by a previous study that failed to detect NMDA receptor-mediated synaptic currents in mass cultures of RGCs (Taschenberger et al. 1995). In vivo, NMDA receptors contribute to light-evoked responses in RGCs (Diamond & Copenhagen, 1993). A recent study showed that NMDA receptors reside in a subset of postsynaptic densities in the inner plexiform layer of the rat retina in vivo (Fletcher et al. 2000) suggesting that RGCs target NMDA receptors to specific synapses, which they may fail to form in vitro.
Soluble glia-derived proteins stimulate neurones to form new and more efficient autapses
A key finding of our study is that soluble, proteinase K-sensitive factors produced by glial cells induce the formation of new autapses in RGCs. This is indicated by the fact that GCM increased the number of synapsin I- and GRIP1-positive autaptic structures per neurone by up to 10-fold and by remarkably similar factors the number of release sites estimated by FM1-43 labelling and by the quantal content of evoked responses. These effects occurred in single neurones and were thus independent of possible glial effects on neuronal survival. We believe that the GCM-induced increase in the number of autapses caused the 10-fold increase in the rate of spontaneous events. Ultrastructural data from a parallel study (Ullian et al. 2001) support our observation of a glia-induced increase in synapse number. A strong glial effect on synaptogenesis was not apparent in the previous study of Pfrieger & Barres (1997), but it appears likely that this also caused the very similar increase in the frequency of miniature postsynaptic currents.
How could glial proteins stimulate the formation of new autapses? Functional synapses form within minutes to hours after a growth cone has contacted a postsynaptic target (Ahmari et al. 2000; Friedman et al. 2000; for reviews see Grinnell, 1995; Fitzsimonds & Poo, 1998). Our observation that GCM increased autaptic activity with a delay of 24 h suggests that the glial signals do not prompt immediate autapse formation, but trigger a multi-step differentiation process, which subsequently enables neurones to form new autapses. Alternatively, the signals may upregulate stabilizing components (Benson & Tanaka, 1998) and thus render autapses less susceptible to elimination (Wolff et al. 1995) that may occur under glia-free conditions.
In addition to the induction of new autapses, glia-derived proteins enhanced the efficacy of existing synaptic connections supporting the previous conclusion by Pfrieger & Barres (1997). This study reveals two mechanisms by which glial signals strengthen synaptic transmission, a strong increase in presynaptic release probability and an increase in the charge transfer amplitude of spontaneous autaptic events, which is equivalent to the quantal size. Direct evidence for a presynaptic effect, for example an increase in release probability, comes from the increase in FM1-43 destaining rates, the decrease in failure rates and the amplification of asynchronous release. In principle, autaptic activity could also increase due to the larger number of autapses. Two observations argue against this and indicate a distinct effect on efficacy. First, GCM increased the rate of asynchronous release on average by 200-fold, but the frequency of spontaneous events only by 10-fold. Second, GCM changed the failure rates and the asynchronous release, before it affected the frequency and amplitudes of spontaneous autaptic activity. If the glia-derived proteins would only increase the number of autapses, then the spontaneous and asynchronous release should increase by the same factor and the effects on failure rates, asynchronous and spontaneous release should occur synchronously.
The increase in quantal size suggests that glial signals also strengthen synaptic efficacy postsynaptically. An increase in the size of non-NMDA receptor-mediated miniature events occurs at many developing CNS synapses in vitro (Gottmann et al. 1994; Gomperts et al. 1998) and in vivo (Wu et al. 1996; Gao et al. 1998; Nguyen et al. 1999) and could reflect an increase in the postsynaptic clustering of glutamate receptors. However, we cannot exclude a presynaptic effect, for example an increase in intravesicular transmitter concentration. Interestingly, the glia-induced increase in the size of spontaneous events did not require electrical activity, in contrast to effects observed in other culture preparations (Gomperts et al. 2000; see review by Craig, 1998).
So far, the identity of the glia-derived proteins is unknown. It appears possible that the effects on autapse formation and maturation are mediated by different glial signals. We have tested a large number of growth factors and found no effect on synaptic activity in mass cultures of RGCs (Pfrieger & Barres, 1997; D. H. Mauch & F. W. Pfrieger, unpublished observation). Likewise, conditioned media from primary fibroblasts and from different glial cell lines showed no effects (D. H. Mauch and F. W. Pfrieger, unpublished observation) indicating that the synapse-promoting activity is specifically produced by macroglial cells as suggested earlier (Pfrieger & Barres, 1997). Interestingly, Blondel et al. (2000) showed recently that a soluble astrocyte-derived signal promotes synapse formation and enhances glutamate sensitivity in cultured hippocampal neurones, possibly by enhancing neuronal release of neurotrophins and by stabilizing NMDA receptor subunits.
Most neurones that grew in direct contact with glial cells lacked autaptic activity. This was surprising considering the strong effect observed in mass cocultures (Pfrieger & Barres, 1997). It is possible that this was due to the small number of glial cells plated in microcultures and therefore a low concentration of the autapse-promoting signal. The few neurones showing enhanced autaptic activity were probably in close contact with factor-releasing glial cells. A previous study (Hartley et al. 1999) showed that rat astrocytes promote the formation of synapses in NTN2 cells, which are derived from a human carcinoma cell line. In contrast to the results reported here, this effect was strictly contact dependent. However, it occurred with a long delay of 1 month and probably involved changes in the survival rate and differentiation state of these neurone-like cells.
In summary, using a new microculture preparation of CNS neurones, we could show that soluble, glia-derived signals increased the number of autapses formed by single neurones and triggered a maturation process that increased the efficacy of autaptic transmission. This suggests that signals from glial cells may play a crucial role in synapse formation in vivo and opens up new perspectives on the mechanisms of synaptogenesis during development and in the adult brain.