Presynaptic impairment of synaptic transmission in Drosophila embryos lacking Gs(alpha)

Abstract

Gs(alpha) is a subunit of the heterotrimeric G-protein complex, expressed ubiquitously in all types of cells, including neurons. Drosophila larvae, which have mutations in the Gs(alpha) gene, are lethargic, suggesting an impairment of neuronal functions. In this study, we examined synaptic transmission at the neuromuscular synapse in Gs(alpha)-null (dgsR60) embryos shortly before they hatched. At low-frequency nerve stimulation, synaptic transmission in mutant embryos was not very different from that in controls. In contrast, facilitation during tetanic stimulation was minimal in dgsR60, and no post-tetanic potentiation was observed. Miniature synaptic currents (mSCs) were slightly smaller in amplitude and less frequent in dgsR60 embryos in normal-K+ saline. In high-K+ saline, mSCs with distinctly large amplitude occurred frequently in controls at late embryonic stages, whereas those mSCs were rarely observed in dgsR60 embryos, suggesting a developmental defect in the mutant. Using the Gal4-UAS expression system, we found that these phenotypes in dgsR60 were caused predominantly by lack of Gs(alpha) in presynaptic neurons and not in postsynaptic muscles. To test whether Gs(alpha) couples presynaptic modulator receptors to adenylyl cyclase (AC), we examined the responses of two known G-protein-coupled receptors in dgsR60 embryos. Both metabotropic glutamate and octopamine receptor responses were indistinguishable from those of controls, indicating that these receptors are not linked to AC by Gs(alpha). We therefore suggest that synaptic transmission is compromised in dgsR60 embryos because of presynaptic defects in two distinct processes; one is uncoupling between the yet-to-be-known modulator receptor and AC activation, and the other is a defect in synapse formation.

Figure 4.

mSCs in high-K ⁺ saline with 0.1 mm Ca ²⁺. A, Sample current traces. Three samples are shown for each strain. B, The mean amplitude for each strain. The bar at the top of each column is the SEM. Double asterisks indicate a statistical difference at p = 0.01 from Gs27, dgs^R60/+ and rut¹. The number in each column is the number of cells examined. C, The skewness of amplitude histogram. The skewness is defined in Materials and Methods. The bar at the top of each column is the SEM. The number is the number of cells examined. Double asterisks indicate a statistical difference at p = 0.01 from Gs27, dgs^R60/+, and rut¹. D, The frequency of mSCs. The bar at the top of each column is the SEM. The number is the number of cells examined. A single asterisk indicates a statistical difference at p = 0.05; double asterisks indicate a statistical difference at p = 0.01 from Gs27 and dgs^R60/+. E, Amplitude histograms from a cell in each strain.

Figure 1.

Nerve-evoked synaptic currents in dgs^R60, Gs27, and dgs^R60/+. External saline contained 0.5 mm Ca ²⁺. A, Three sample traces are shown for each strain. The amplitude varied in a large range. B, The mean amplitudes are not significantly different among these strains. The bar at the top of each column indicates the SEM,and numbers in columns are the number of cells examined.

Figure 2.

Facilitation during tetanic stimulation and PTP in various strains (tetanic stimulation; A₁–D₁) and asynchronous release of quantal events (asynchronous release; A₂–D₂). At first, the nerve was stimulated 40 times at 0.3 Hz,and the failure rate was determined (left open columns in A₁–D₁). Then stimulation was switched to 10 Hz for 10 sec. The failure rates for the first 50 stimuli (left shaded columns) and those for the last 50 stimuli (right shaded columns) were depicted separately. Finally, the stimulation was switched back to 0.3 Hz (40 stimuli) to assess PTP (right open columns). A, dgs^R60; n = 12, where n = number of cells examined. B, Gs27; n = 7. C, dgs^R60/+; n = 7. D, rut¹; n = 6. Bars at the top of each column in A₁–D₁ and at each data point in A₂–D₂ are the SEM. A single asterisks indicates a statistical difference from the pretetanic failure rate at p = 0.05; double asterisks indicate statistical significance at p = 0.01. NS, Nosignificance. This series of experiments was performed in normal saline with 0.2 mm Ca²⁺.

Figure 3.

Miniature synaptic currents (mSCs) in normal saline with 0.2 mm Ca ²⁺. A, Sample current traces. Three traces are shown for each strain. B, The mean frequency of mSCs in each strain. The number in each column is the number of cells examined. The bar at the top of each column is the SEM. Double asterisks indicate a statistical difference at p = 0.01 from Gs27 and dgs^R60/+. C, The mean amplitude of mSCs in each strain. The number in each column is the number of events that were pooled among cells examined. Double asterisks indicate a statistical difference from Gs27, dgs^R60/+,andrut¹.D, Amplitude histograms for each strain. Events from a number of cells recorded in each strain were pooled. The number of events for each strain is the same as shown in C.

Figure 5.

Glutamate-induced currents and single glutamate receptor channels currents. A, Glutamate-induced currents. Glutamate (1 mm) was puffed for 100 msec to the synaptic area in Ca ²⁺-free saline. The holding potential was -35 mV. B, The mean amplitude of each strain. The bar at the top of each column is the SEM, and the number is the number of cells examined. The holding potential was -35 mV. C, Single glutamate receptor channel currents. Three sample synaptic currents are shown in each strain. The peak of synaptic currents is saturated. Spontaneous synaptic currents were recorded in high-K ⁺ saline with 0.05 mm Ca ²⁺. On the falling phase of synaptic currents, there are distinct steps (arrows) that are most likely because of opening of a single channel in the postsynaptic membrane (Nishikawa and Kidokoro, 1995). The amplitude of those steps was measured in >10 synaptic currents for each cell. D, The average of single-channel currents in each strain. The bar at the top of each column is the SEM, and the number is the number of cells examined.

Figure 6.

mGluR responses inGs27(A–C), in DC0(D),and in dgs^R60 embryos(E). A, Glutamate at 100 μm was puff-applied for 40 sec in high-K⁺ saline with 0.05 mm Ca²⁺, and quantal synaptic events were counted individually every 10 sec. The means of four cells are plotted. Bars attached to each point are the SEM. B, A specific mGluR antagonist, MCCG-I, at 200 μm was puff-applied together with 100 μm glutamate. The response was abolished. C, A specific mGluR agonist, (S)4C3HPG, 100 μm,was puff-applied for 40 sec in high-K⁺ saline with 0.05 mm Ca²⁺. The number of cells examined is seven. D, (S)4C3HPG at 100 μm was applied in DC0 embryos. No response was observed. The number of cells examined is four. E, The mGluR response evoked with 100μm (S)4C3HPG in dgs^R60 embryos. The number of cells examined is nine.

Figure 7.

Octopamine receptor responses in Gs27 (A, B), in DC0 (C), and in dgs^R60 embryos (D). A, Octopamine at 10μm was puff-applied in high-K ⁺ saline with 0.05 mm Ca ²⁺ for 40 sec, and quantal synaptic events were counted every 10 sec. The means of four cells are plotted, and the bars attached to each point are the SEM. B, The dose–response curve for octopamine. Neighboring two data points were connected by a straight line, and apparent K_d was estimated as a octopamine dose that produces the half-maximal response. Bars attached to each point are the SEM, and numbers are the number of cells examined. C, Lack of the octopamine receptor responses in DC0. The means of seven cells are plotted, and the bars attached to each point are the SEM. D, The octopamine receptor response in dgs^R60 embryos. The means of six cells are plotted, and the bars attached to each point are the SEM.

Figure 8.

Expression of a Gsα transgene, GsW24, in neurons rescued the synaptic impairment in dgs^R60, whereas its expression in postsynaptic muscles did not. A wild-type transgene, GsW24, was expressed with a driver, elav-Gal4, in neurons and with another driver, MHC82-Gal4, in muscles in the dgs^R60 background. A, Facilitation during tetanic stimulation and PTP. At first, the nerve was stimulated 40 times at 0.3 Hz, and the failure rate was determined (left open columns in Aa, Ab). Then, the stimulation was switched to 10 Hz for 10 sec. The failure rates for the first 50 stimuli (left shaded columns) and that for the last 50 stimuli (right shaded columns) were depicted separately. Finally, the stimulation was switched back to 0.3 Hz (40 stimuli) to assess PTP (right open columns). When GsW24 was expressed in neurons (Aa), facilitation during tetanus was observed, but when GsW24 was expressed in muscles (Ab), it was not. Bars at the top of each column are the SEM. A single asterisk indicates a statistical difference from the pretetanic failure rate at p = 0.05; double asterisks indicate a statistical difference at p = 0.01. NS, No significance. The number of cells examined is 10 for Aa and 7 for Ab. B, Asynchronous release of quantal events occurred during this series of stimulations, which increased during and after tetanus as shown in Ba, in which GsW24 was expressed in neurons, but not in Bb,in which GsW24 was expressed in muscles. This series of experiments was performed in normal saline with 0.2 mm Ca ²⁺.The number of cells examined is 10 for Ba and 7 for Bb. C, mSCs in high-K ⁺ saline with 0.1 mm Ca ²⁺. Ca, The amplitude. Left shaded columns are for a strain in which GsW24 was expressed in neurons (n = 6), and right open columns are for a transgenic strain in which GsW24 was expressed in muscles (n = 8). Cb, The skewness of amplitude histogram. Cc, The frequency of mSCs. The bar at the top of each column is the SEM. The number is the number of cells examined. A single asterisk indicates a statistical difference at p = 0.05; double asterisks indicate a statistical difference at p = 0.01. Cd, Amplitude histogram from a cell in which GsW24 was expressed in neurons. Similar histograms were observed in six cells in which GsW24 was expressed in neurons. Ce, Amplitude histogram from a cell in which GsW24 was expressed in muscles. Similar histograms were observed in eight cells in which GsW24 was expressed in muscles.