2.3.1 Drosophila neuromuscular synapses as a model system to study synaptic function and development
For various reasons, the Drosophila neuromuscular junction (NMJ) is an attractive model system to study fundamental questions concerning neuronal development and activity-dependent plasticity. Drosophila has short generation time and allows to establish, test and to efficiently apply different transgenic and knock out strategies. In its neuromuscular system, all motoneurons are identified and the exact target muscle they innervate is known. Most molecules involved in synaptic transmission are conserved between flies and vertebrates. The possibility to screen very efficiently for mutants in neuronal outgrowth and target recognition as well as in learning and memory has allowed identifying many genes involved in different aspects of neuronal development. Even subtle alterations in synaptic efficacy can reliably be identified since electrophysiological techniques are established for both the embryonic and the larval NMJ.
2.3.2 Organization and development of Drosophila NMJ synapses
The larval musculature together with its innervations is composed of a segmentally repeated set of 30 muscle cells each innervated by identified motoneurons (Goodman et al., 1986). The neuromuscular junction is organized into a series of boutons, which can be added during development and plasticity.
Fig. 8 Organization of Drosophila neuromuscular junctions. The NMJ in mature Drosophila larvae is composed from several boutons, each of them containing several individual synapses as visualized by nc82 (red, marks cytomatrix of active zone) and Drosophila glutamate receptor subunit DGluRIIC (green). The same structure is shown using electron microscopy. The PSDs are visualized as electron dense material between the arrowheads. The presynaptic bouton is filled with small, clear synaptic vesicles containing glutamate. Scale bars (from left to right) 20 µm, 1 µm and 300 nm (EM micrograph taken from (Sigrist et al., 2002)).
Each bouton (Fig. 8 central panel) contains several synapses identified as pairs of a postsynaptic density with corresponding specializations on the presynaptic site (the active zone is marked by the expression of the nc82 epitope and presynaptic Ca2+ channels (Kawasaki et al., 2004)).
This thesis will focus on type 1 boutons of the NMJ. These boutons contain small, clear synaptic vesicles (Atwood et al., 1993). Adjacent to the PSD, the postsynaptic muscle membrane folds in a typical manner. This structure, referred to as the subsynaptic
reticulum, surrounds the presynaptic terminal with multiple layers of elaborately folded muscle membrane. Many proteins shown to be relevant for the proper function of the neuromuscular junction like the N-CaM homolog Fasciclin II (Davis et al., 1996; Schuster et al., 1996, 1996) and the PSD95 homolog discs large (Budnik et al., 1996), have been shown to localize to the subsynaptic reticulum.
2.3.3 Non-NMDA type glutamate receptors are expressed at Drosophila NMJ synapses
The glutamate receptors expressed at the Drosophila neuromuscular junction are structurally and functionally similar to mammalian AMPA-/Kainate-type receptors. So far three different glutamate receptor subunits have been described at the neuromuscular junction. The Drosophila glutamate receptor subunit IIA (DGluRIIA) (Schuster et al., 1991) and IIB (DGluRIIB) (Petersen et al., 1997) share 44 % overall amino acid identity with each other. Animals double mutant for dglurIIA and the related dglurIIB subunit are embryonic lethal, while they can be rescued to adult vitality by transgenic expression of either DGluRIIA or DGluRIIB (Petersen et al., 1997; DiAntonio et al., 1999). Thus, either a DGluRIIA or a DGluRIIB subunit seems to be required to form functional ion channels at the NMJ. Drosophila glutamate receptor subunit IIC (DGluRIIC) is essential for neurotransmission and for synaptic localization of DGluRIIA or DGluRIIB, likely by acting as obligate binding partner of either DGluRIIA or DGluRIIB (Marrus et al., 2004). Like their vertebrate relatives Drosophila receptors desensitize within milliseconds in the presence of glutamate. The kinetics of glutamate binding and channel gating hereby are similar to those of vertebrate non-NMDA-type receptors (Heckmann and Dudel, 1997).
2.3.4 Activity-dependent plasticity of Drosophila neuromuscular junctions induced by genetic means
Analysis of synaptic plasticity at the Drosophila NMJ has allowed the identification of several mutants which suppress or stimulate outgrowth of the NMJ. The cell adhesion molecule Fascicilin II has been shown to mediate growth and activity dependent changes at the neuromuscular junction (Davis et al., 1996; Schuster et al., 1996, 1996). Genetic reduction of Fasciciln II by 50% yields significantly larger NMJs (Davis et al., 1996;
Schuster et al., 1996, 1996). Thus reduction of cell adhesion seems to be an important prerequisite for additional outgrowth in response to increased presynaptic activity (Davis et al., 1996; Schuster et al., 1996, 1996). Such increase in presynaptic activity can be achived using a double mutant combination of both the ether a go-go (eag) and Shaker (Sh) potassium channel in which the frequency of presynaptic action potentials is strongly enhanced (Zhong et al., 1992). This “increase in activity” in turn provokes an increase of cAMP levels and finally enhanced morphological outgrowth of the junction. Increased morphological outgrowth of eag, Sh mutants is cAMP dependent, and the learning mutant dunce (Dudai et al., 1976) shows a very similar phenotype (Zhong et al., 1992). The dunce mutation affects the cAMP specific phosphodiesterase, which leads to elevated cAMP level (Kauvar, 1982). Junctional outgrowth can be suppressed (Zhong et al., 1992) in double mutants of dunce and rutabaga (Dudai and Zvi, 1984; Livingstone et al., 1984;
Dudai and Zvi, 1985; Livingstone, 1985) with the latter mutation reducing cAMP synthesis.
The outgrowth phenotype of Sh and dunce single mutants was further enhanced in double mutants (Zhong et al., 1992). In addition to these morphological changes mediated by the cAMP cascade, changes in glutamate receptor subunit composition have been shown to be able to provoke long-term changes of synaptic performance (Petersen et al., 1997;
DiAntonio et al., 1999; Sigrist et al., 2002). Ultrastructural reconstruction of NMJ boutons has demonstrated the formation of additional synaptic sites in situations of increased DGluRIIA expression (Sigrist et al., 2002). The genetic analysis of plasticity mutants at the NMJ has thus already provided insights into molecular mechanisms controlling the formation of synapses in this model system. However, due to the chronic defects caused by mutations a time-resolved analysis of these mechanisms and their functional relationships is difficult.
2.3.5 Experience-dependent plasticity of Drosophila neuromuscular junctions
Recently, experience-dependent plasticity independent of genetic manipulation could be demonstrated at the NMJ. To this end, the locomotor activity (and therefore the extent of synaptic transmission) of Drosophila larvae was experimentally controlled in an acute and chronic manner (Sigrist et al., 2003; Zhong and Wu, 2004). When larval locomototion was increased either by chronically rearing a larval culture at 29°C instead of 18°C or 25°C, or by acutely transferring larvae from a culture vial onto agar-plates, a significant potentiation of synaptic transmission was detected within 2 hours (Sigrist et al., 2003). Enhanced locomotor activity was also associated with a significant increase in the number of subsynaptic translation aggregates (Sigrist et al., 2003). DGluRIIA, mRNA of which is present at the neuromuscular junction, has been suggested to be a target of local translation activity (Sigrist et al., 2000). In these experiments, an increased occurrence of subsynaptic translation aggregates was shown to be associated with the significant increase of DGluRIIA synaptic immunoreactivity (Sigrist et al., 2000). After 4 hours, postsynaptic DGluRIIA glutamate receptor subunits started to transiently accumulate in ring-shaped areas around synapses. Upon chronic locomotor stimulation at 29°C they condensed into typical postsynaptic patches (Sigrist et al., 2003). These NMJs showed a reduced perisynaptic expression of the cell adhesion molecule Fasciclin II, an increased number of boutons per NMJ and significantly more synapses (Sigrist et al., 2003). When combined with synapse live imaging, this experience dependent plasticity might be an important tool for in vivo study of activity-driven synapse formation.
2.3.6 Addressing the cellular and molecular basis of synaptic long-term changes at the Drosophila NMJ
As the muscles grow from embryo to mid third instar larvae their surface increases more than 100 fold which leads to a drop of input resistance (Jan and Jan, 1976; Broadie and Bate, 1993). Accordingly the synaptic current collectively mediated by the set of synapses within a junction increases more than two orders of magnitude during larval development (Broadie and Bate, 1993; Sigrist et al., 2003). Here, synapse formation was
studied within this time window. A principal question is, in how far these results can be compared to long-term potentiation (LTP) of existing circuitry. Long-term potentiation of synaptic systems means a long lasting increase of synaptic strength in response to a stimulus. In other words, a synaptic system challenged towards higher transmission strength reacts to that stimulus by structural and/or functional changes of its synaptic circuitry. This is in turn very similar to the increase in synaptic strength observed at the neuromuscular junction during development where the system provides additional synapses in order to maintain sufficient depolarization of a postsynaptic muscle cell which dramatically increases its size. Lessons learned “in development” might thus well be helpful in understanding the cellular basis of long-term potentiation and in turn learning and memory processes. This idea is consistent with a hypothesis of Cajal, saying that growth processes involved in the development of the nervous system persist into the adult where they subserve learning and memory (Cajal S., 1911). While this idea recently gained increased popularity (Kandel and O'Dell, 1992) it still needs to be further assayed in studies testing whether the same process is required for both neuronal development and synaptic plasticity. Due to the existence of alternative mechanisms, which mediate learning, this experiment is not easy to perform. Even when the system is severely disturbed as in the α-CamKII knockout mice (Silva et al., 1992; Silva et al., 1992; Silva et al., 1992; Tonegawa et al., 1995) there is still some learning present. Nonetheless, especially the similarities between learning and development in Aplysia, as described for the role of serotonergic axosomatic contacts, the activation of transcription factors, the necessity of an appropriate postsynaptic target, the role of cAMP as a second messenger and the common role of cell adhesion molecules, support the idea that learning and development share many common processes (Marcus et al., 1994). Results obtained from in vivo imaging of synapse formation and plasticity at the Drosophila NMJ might thus contribute substantially to our understanding of the fundamental cellular mechanisms important for establishing synaptic long-term changes.
2.4 High-throughput screen for the systematic identification and