| Activity-dependent changes in the structural or transmission properties of synapses is essential for alterations of brain function that depend on experience. At the Drosophila larval neuromuscular junction (NMJ), changes in locomotor activity, as well as mutations that alter neuronal excitability, can modulate synaptic growth. Despite the importance of such synaptic structural plasticity, relatively few molecular signaling pathways have been identified that regulate synaptic architecture and little is known about how neuronal activity modulates these pathways.;Recently, a number of signaling pathways including Bone Morphogenetic Protein (BMP) signaling have been shown to be essential for the normal morphology and function of Drosophila NMJ synapses. Synaptic structural development at the NMJ requires a muscle-derived BMP ligand to activate presynaptic BMP receptors in motoneuron axonal terminals. We have now found that BMP signaling is also required for activity-dependent growth of NMJ synapses.;In Drosophila, BMP signals are transduced by receptor-mediated phosphorylation of Smad proteins, transcription factors that translocate from the cytosol to the nucleus to regulate BMP responsive genes. Phosphorylated Smads (P-MAD in Drosophila) can be detected by phospho-specific Smad antisera. In our initial analysis of BMP signaling in the Drosophila NMJ, we discovered that P-MAD shows a striking, and unexpected, accumulation in synaptic terminals in addition to its previously characterized localization in motoneuron nuclei. This finding led us to analyze the function of synaptic P-MAD, as well as its relationship to the role of P-MAD in the nucleus.;In our initial experiments, we inhibited BMP signaling acutely at different points during larval development, and confirmed that BMP signaling and Mad activation are required continuously for the normal elaboration of the NMJ. Furthermore, we showed that all of the known effects of BMP signaling on NMJ structure and growth depend on site-specific DNA binding by Mad, arguing that P-MAD acts primarily, if not solely, as a transcription factor to transduce BMP signaling in the NMJ.;Both BMP signaling, as well as neural activity, modulate NMJ growth and structure, posing the question of whether the two inputs act independently, or via a common mechanism. Surprisingly, we observed that pre-synaptic P-MAD accumulation in the NMJ is dramatically increased in mutants with increased neural activity. In addition, using a genetic interaction approach, we discovered that BMP signaling and neural activity act synergistically to promote synaptic growth of the NMJ. Together, these two lines of evidence suggest an active role for BMP signaling in mediating the control of synaptic structure by neural activity.;To resolve how neural activity alters BMP signaling, we designed experiments to identify where and how neural activity can influence the BMP pathway. Using a combination of transgenic tools to block neurotransmission or to activate BMP signaling in either muscle cells or motoneurons, we have obtained evidence that enhanced neural activity modulates NMJ growth by elevating the capacity of motoneurons to transduce the BMP signal. Specifically, we have shown that neural activity acts at two levels to elevate the level of Mad protein: (i) acutely, to stabilize Mad protein, and (ii) over longer time intervals, to increase the abundance of mad transcripts. We posit that activity-dependent increases in total Mad protein levels provide higher levels of substrate for BMP receptor-dependent phosphorylation, thereby potentiating the transcriptional and morphological responses of the motoneuron to muscle derived BMP signal.;Taken together our data support a model in which neural activity in motoneurons modulates the elaboration of the NMJ by building on a pre-existing BMP mechanism for controlling NMJ growth during the development. Further, we have shown that it does so by regulating the level of total Mad protein in motoneurons, thereby controlling both the sensitivity and the strength of the response to BMP signal from the muscle.;This is the first description of a trophic factor in Drosophila that functions to enhance synaptic growth in an activity-dependent manner. |
