Extrasynaptic glutamate receptors get selectively stabilized in growing PSDs

Returning to the concept of spines/PSDs being the substrate for long-term memory, it needs to be clarified how the transitions from new to mature and from mature to complex spines/PSDs are controlled. It seems likely that the transition from an immature to a mature PSD is mediated by stabilizing synaptic glutamate receptors. Consistently, it had been shown that large spine heads are characterized by having many AMPA receptors (Nusser et al., 1998). The synaptic system seems to support the outgrowth of synapses by providing a diffuse pool of glutamate receptors, which are selectively stabilized in or inserted into growing synapses. In contrast, mature PSDs show very little (if any) receptor insertion or removal. The overall recycling in these synapses is very small, if present at all.

To rule out imaging artifacts, these results were confirmed by two independent methods, photo-activation and fluorescence recovery after photo bleaching. Both measurements lead, within the accuracy of the measurement, to the same results. Mature synapses showed little, if any synaptic turnover of glutamate receptors. It is not possible to use these methods to prove that there is no synaptic turnover at all. Calculations however showed that, if there were any turnover it must be less than 10% within 12h (based on the FRAP experiments). Independent calculations based on the photo-activation experiments showed that the recycling of synaptic glutamate receptors must be less than 20%. This indicates that in terms of their glutamate receptors mature PSDs of the Drosophila NMJ have a remarkably stable molecular identity.

In the neuromuscular system the molecular composition of the postsynaptic density is not static per se. FRAP of GFP-labeled Drosophila p21-activated kinase, another PSD localizing protein is about 20 times faster than FRAP of DGluRIIA. This suggests that DGluRIIA somehow gets stabilized, once integrated into PSDs, while the molecular composition of the PSD is per se highly dynamic (Fig. 32 C). Simultaneous FRAP experiments on the C-terminally tagged DGluRIIA^GFP and the N-terminally tagged DGluRIIC^GFP could further prove that the low synaptic turnover is really an inherent characteristic of all glutamate receptors in the muscle. DGluRIIC is an essential subunit

thought to be present in all glutamate receptors present in the muscle (Marrus et al., 2004). Therefore, the synaptic turnover of all types of glutamate receptors is reflected in the synaptic turnover of DGluRIIC^GFP, which is similar to that of DGluRIIA. Thereby it is important to mention, that DGluRIIC was N-terminally tagged with GFP. The C-term of glutamate receptors is known to be important for targeting and anchorage (Dong et al., 1997; Srivastava et al., 1998; Srivastava and Ziff, 1999). Since DGluRIIC was N- terminally tagged any alterations of receptor turnover caused by effects of the insertion of GFP into the C-term could be excluded (Fig. 33).

Where are the receptors supporting the outgrowth of new synapses derived from? It could be shown - by bleaching the entire muscle - that newly synthesized receptors contribute to PSD growth (Fig. 32a) as previously suggested (Sigrist et al., 2000).

Significantly more unbleached DGluRIIA^mRFP entered within 24h, when bleaching only smaller areas compared to bleaching the whole muscle (Fig. 32 A,B). Moreover, the exact position of PSDs (not shown) within the bleached area has no influence on the recovery of the fluorescent signal. Collectively these data lead to the following conclusions: Stores of glutamate receptors in close proximity do not significantly contribute to PSD growth. This is consistent with the lack of any discernable accumulations of DGluRIIA outside the PSDs, which is in contrast to data describing glutamate transport vesicles in day 3-4 neurons (Washbourne et al., 2002). In older neurons (Bresler et al., 2004) however, no similar transport vesicles could be observed, nor were they detectable for other NMDAR subunits (Guillaud et al., 2003). Therefore, glutamate transport vesicles might be used during early stages of neuronal development, while the establishment or strengthening of specific PSDs within an established circuitry might be supported by diffuse pools of glutamate receptors. Electrophysiology has actually shown the existence of diffuse, extrasynaptic pools of glutamate receptors in the membrane of Drosophila muscles (Broadie and Bate, 1993). Such receptors might be recruited into newly forming PSDs.

Thereby laterally diffusing receptors might become “trapped” in PSDs, as recently demonstrated by tracking individual glutamate receptor complexes in cultured mammalian neurons. Likewise these glutamate receptors float freely in the membrane. This enables them to diffuse in and out of synapses where they only have a low residence time (Borgdorff and Choquet, 2002; Tardin et al., 2003).

Fig. 37 Model explaining how glutamate receptor dynamics could be organized during PSD formation. The model suggests that small PSDs grow by stabilizing receptors which enter from a diffuse pool of glutamate receptors into the PSD. Thereby “synaptic tags” might differentiate “accepting” i.e. growing from “non-accepting” i.e. mature PSDs.

This pool of unbound receptors might be fundamentally different from a second pool of tightly bound receptors showing essentially no turnover. The model suggests that

“synaptic tags” differentiate “accepting” (i.e. growing) from “non-accepting” (i.e. mature) PSDs (Fig. 37). These tags must be accessible to entering receptors and might be identical to the “slots” which finally immobilize receptors. At growing PSDs, the availability of both slots and glutamate receptors could be rate limiting for growth. The latter possibility is supported by the previous finding that a genetically triggered increase in the expression of the glutamate receptor DGluRIIA stimulates synapses formation and provokes an additional functional strengthening of the junction (Sigrist et al., 2002). At the Drosophila neuromuscular junction mature PSDs usually keep their glutamate receptors and thus retain their strength, consistent with the fact that the synaptic system has to continuously increase its overall strength (Davis and Goodman, 1998). The rarely observed shrinkage potentially reflects competition between synapses (not shown).

Focusing future work on such local factors as neighbor relationships and local disparities in presynaptic release will hopefully help to decipher the molecular and cellular rules controlling learning and memory in an even more detailed manner.

5.5 Identification of the Drosophila homolog of the CAST/ERC protein