Innervation and activity dependent dynamics of postsynaptic oxidative metabolism.

Despite extensive investigations into the mechanisms of aerobic respiration in mitochondria, the spontaneous metabolic activity of individual cells within a whole animal has not been observed in real time. Consequently, little is known about whether and how the level of mitochondrial energy metabolism is regulated in a cell during development of intact systems. Here we studied the dynamics of postsynaptic oxidative metabolism by monitoring the redox state of mitochondrial flavoproteins, an established indicator of energy metabolism, at the developing Drosophila neuromuscular junction. We detected transient and spatially synchronized flavoprotein autofluorescence signals in postsynaptic muscle cells. These signals were dependent on the energy substrates and coupled to changes in mitochondrial membrane potential and Ca2+ concentration. Notably, the rate of autofluorescence signals increased during synapse formation through contact with the motoneuronal axon. This rate was also influenced by the magnitude of synaptic inputs. Thus, presynaptic cells tightly regulate postsynaptic energy metabolism presumably to maintain an energetic balance during neuromuscular synaptogenesis. Our results suggest that flavoprotein autofluorescence imaging should allow us to begin assessing the progress of synapse formation from a metabolic perspective.

Synaptic components necessary for retrograde signaling triggered by calcium/calmodulin-dependent protein kinase II during synaptogenesis.

The development and function of presynaptic terminals are tightly controlled by retrograde factors presented from postsynaptic cells. However, it remains elusive whether major constituents of synapses themselves are necessary for retrograde modulation during synaptogenesis. Here we show that the homophilic cell adhesion molecule Fasciclin II (FasII) as well as the scaffolding protein Discs large (DLG) is indispensable for retrograde signaling initiated by calcium/calmodulin-dependent protein kinase II (CaMKII) at developing Drosophila neuromuscular junctions. Postsynaptic activation of CaMKII increased the area of nerve terminals, the number of active zones, and the frequency of miniature excitatory synaptic currents in wild-type animals. However, all of these retrograde effects were abolished in the fasII or dlg mutant background. On the other hand, the retrograde effects remained in null mutants of the glutamate receptor subunit GluRIIA. Furthermore, we show that CaMKII-induced modulation was independent of the bone morphogenetic protein signaling that is important for retrograde control at mature larvae. These results highlight a novel function of FasII as well as DLG, and more broadly, illustrate that prime synaptic components are necessary for transferring target-derived signals to presynaptic cells at a certain developing synapse.

Developmental stage-dependent modulation of synapses by postsynaptic expression of activated calcium/calmodulin-dependent protein kinase II.

In Drosophila neuromuscular junctions, there is a unique system which consists of two neighboring muscles (M6 and M7) innervated by the same neurons and a gene of interest can be expressed in only M6 or in both muscles by GAL4-upstream activating sequence expression system. By using this system, we previously demonstrated that expression of activated calcium/calmodulin-dependent protein kinase II (CaMKII) in the muscle cell promotes coordinated maturation of pre- and postsynaptic sites of larvae just after hatching (JAH larvae) in a synapse-specific manner. Here we show that the promotive effects are no longer seen in the older larvae, 8-10 h after hatching (8 h AH larvae). Morphological studies indicate that CaMKII activation in fact reduces postsynaptic sites at 8 h AH. This is opposite to the effect observed in JAH larvae. These results suggest that the mode of CaMKII function switches during development, and that regulation of postsynaptic CaMKII activity is necessary for normal synaptic development. Finally, we report that in 8 h AH but not in JAH larvae, synapses on M7, in which CaMKII activity is not manipulated, are affected by the expression of activated CaMKII in M6. This suggests the interesting possibility that at certain developmental stages only, modification of synapses on one target cell can influence the synapses on another target cell innervated by the same neurons.

Postsynaptic activation of calcium/calmodulin-dependent protein kinase II promotes coordinated pre- and postsynaptic maturation of Drosophila neuromuscular junctions.

The interaction between a neuron and its target cell(s) is essential for the development of synapses. To elucidate the role of target cells in synaptogenesis, the activity of postsynaptic calcium/calmodulin-dependent protein kinase II (CaMKII) was manipulated in a mosaic manner and its specific effect was examined at the developing Drosophila neuromuscular junction. We found that postsynaptic expression of constitutively active CaMKII augmented the amplitude of excitatory synaptic currents (ESCs) and the frequency of miniature ESCs. It also promoted morphological maturation of presynaptic as well as postsynaptic specializations, which presumably underlie the enhancement of synaptic activities. Expression of an inhibitory peptide of CaMKII in the postsynaptic cell partially affected the synaptic maturation. These results suggest two significant functions of postsynaptic CaMKII in synaptogenesis-retrograde modulation of presynaptic properties and coordinated regulation of pre- and postsynaptic maturation.