Activation of metabotropic glutamate receptors enhances synaptic transmission at the Drosophila neuromuscular junction
Introduction
Metabotropic glutamate receptors (mGluRs) were found to be abundantly expressed in rat hippocampus and cerebellum, suggesting their important roles in brain function (Masu et al., 1991). To date, eight types of mGluRs (mGluR1 through mGluR8) have been reported and pharmacologically classified into three groups. Among a variety of responses resulting from activation of these mGluRs, changes in the cyclic AMP (cAMP) level are implicated when Group II receptors (including mGluR2 and mGluR3) are activated. In the majority of cases an activation of Group II mGluRs leads to a decrease of the cAMP level (Pin and Duvoisin, 1995). In some cases, however, they are positively coupled to adenylate cyclase. For example, in neonatal rat hippocampal neurons an activation of mGluRs leads to an increase of the frequency of spontaneous synaptic currents through a pathway involving cAMP and protein kinase A (Sciancalepore et al., 1995).
Recently Drosophila mGluR (DmGluRA) was cloned and expressed in a cell line, and its basic pharmacological properties have been described (Parmentier et al., 1996). Based on the sequence homology and pharmacological properties DmGluRA was classified as Group II, an activation of which leads to a modulation of synaptic transmission through the pathway involving cAMP rather than through the phospholipase C cascade. The expression of DmGluRA in the CNS was culminated at stage 14–17 which coincides with the period of the formation of the neuromuscular junction (Broadie and Bate, 1993), suggesting that DmGluRA may play a role in early synapses.
In Drosophila mutants defective in learning and memory, synaptic plasticity at the glutamatergic neuromuscular junction is altered (Zhong and Wu, 1991). Namely, in dunce larvae, in which cAMP-specific phosphodiesterase II is lacking, and consequently a high level of cAMP is demonstrated (Livingstone et al., 1984), the amplitudes of nerve-evoked synaptic currents are larger than those in the wild-type, and both synaptic facilitation during a tetanus and post-tetanic potentiation are absent, probably due to a high level of synaptic transmission at the resting state. In another mutant, rutabaga, in which Ca2+/calmodulin dependent adenylate cyclase is defective, both synaptic facilitation and post-tetanic potentiation are impaired. These results suggest that cAMP is an important mediator of synaptic plasticity at this synapse. However, the physiological pathways linking extracellular signals to this cAMP cascade are still unknown. In Aplysia an activation of serotonin receptors leads to an elevation of cAMP which closes K+ channels causing a long lasting depolarization. This depolarization in turn activates voltage-gated Ca2+ channels. This sequence of events is presumed to be the basis for short term synaptic plasticity (Kandel and Schwartz, 1982). In an analogy, mGluRs in Drosophila larvae may play a role in linking physiological signals to the cAMP cascade.
It has been reported that in a few preparations cAMP is working directly on the fusion machinery independent from Ca2+ entry to enhance synaptic transmission (Trudeau et al., 1996, Chen and Regehr, 1997, Chavis et al., 1998). Thus it is desirable to separate a direct effect of cAMP on the vesicle fusion machinery from an indirect effect via changes in Ca2+ influx accompanied by a membrane depolarization and subsequent activation of voltage-gated Ca2+ channels. For this purpose the majority of our experiments were carried out in nominally Ca2+-free external saline. However, to assess the role of mGluRs in more physiological conditions, we also examined the effects of mGluR activation on nerve-evoked synaptic currents in the presence of external Ca2+. The effects could be the compound effects through multiple pathways after activation of mGluRs, since the influx of Ca2+ may facilitate synaptic transmission not only through an effect on vesicle fusion but also through a facilitating effect on adenylate cyclase. Some of these results have been briefly reported elsewhere (Zhang et al., 1999).
Section snippets
Preparation
Newly hatched first instar larvae (approximately 24 h after fertilization) of Drosophila melanogaster, Canton S, were used in the majority of experiments. However, in one experiment larvae of a mutant, rutabaga (Livingstone et al., 1984), at a similar stage were also used. Dissecting and recording procedures were the same as previously described (Kidokoro and Nishikawa, 1994, Nishikawa and Kidokoro, 1995). Abdominal body wall muscles were exposed by removing internal organs and the ventral
Low concentrations of glutamate increased the frequency of miniature synaptic currents (mSCs) in nominally Ca2+-free saline
In nominally Ca2+-free saline spontaneous synaptic currents were rare at the resting state, namely, 1.3±0.9 events/min (n=48, mean±SD, n=number of cells examined. Data are expressed in this format throughout the text), in these recording conditions (Fig. 2A, Fig. 7). These were miniature synaptic currents (mSCs) due to spontaneous fusion of synaptic vesicles to the presynaptic membrane because synaptic currents in similar amplitude and frequency were observed even in the presence of 1 μM
Discussion
A cloned Drosophila mGluR (DmGluRA) has been classified as being in Group II from the sequence homology and its pharmacological properties (Parmentier et al., 1996). Considering previous reports we anticipate that an activation of DmGluRs leads to modulation of cAMP levels rather than the phospholipase C cascade. Although in many preparations an activation of Group II mGluRs results in the inhibition of adenylate cyclase (Prézeau et al., 1992, Tanabe et al., 1992), in a few cases an elevation
Acknowledgements
This research was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan to Y. Kidokoro (#09480237). We thank Dr Joy A. Umbach at UCLA for criticism of the manuscript.
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