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The Journal of Neuroscience, July 2, 2003, 23(13):5897-5905
Previous Article | Next Article
Presynaptic Impairment of Synaptic Transmission in Drosophila Embryos Lacking Gs
Dongmei Hou,1 Kazuhiro Suzuki,1 William J. Wolfgang,2 Catherine Clay,2 Michael Forte,2 andYoshiaki Kidokoro1 1Gunma University School of Medicine, Maebashi 371-8511, Japan, and 2Vollum Institute, Oregon Health & Science University, Portland, Oregon 97201
Abstract
Top
Abstract
Introduction
Gsis 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
Key words: Gs
Introduction
In a variety of cells, extracellular signals induce cellular responses through a family of receptors with seven transmembrane domains (7TMR). Essential components of this cascade are intermediary heterotrimeric G-proteins composed of, and
subunits. These G-proteins couple the receptors to appropriate intracellular effectors (Morris and Malbon, 1999
). This signaling pathway plays a key role in triggering physiological responses to a wide variety of hormones, neurotransmitters, and sensory stimuli. In this scheme, the
The well studied example of this signal transduction pathway involves 7TMR coupling to G-protein complexes containing Gs
At neuromuscular synapses of newly hatched Drosophila larvae, activation of metabotropic glutamate receptors (mGluRs) facilitates synaptic transmission, which is mediated by the cAMP–PKA cascade. Both a membrane-permeant cAMP analog and forskolin, an activator of AC, mimic the mGluR response, and the response is inhibited by a blocker of AC and greatly reduced in rut1. It appears that presynaptic mGluRs are positively coupled to AC, possibly through Gs (Zhang et al., 1999
dgs also appears to be involved in synapse formation. Boutons at neuromuscular synapses in Drosophila third instars are more numerous in a hyperexcitable double mutant, ether-á-go-go, Shaker, than in wild-type (Budnik et al., 1990
We studied neuromuscular synaptic transmission in Gs
Materials and Methods
Fly stocks. Primarily, embryos (19–21 hr after egg laying, AEL) of the strain dgs R60c/CyO-GFP were used for the experiment. Homozygotes, which are embryonic lethal, are called dgs R60 in the text. Heterozygotes are called dgs R60/+ and were used as a control. As an additional control, we used a rescued strain of dgs R60 with a Gs27 transgene, designated Gs27. This rescue construct contains the entire dgs gene, which is located on the X chromosome (Gs27; dgs R60c/dgs R60c) (Wolfgang et al., 2001Electrophysiology. Electrophysiological procedures for voltage-clamping embryonic muscles have been published previously (Nishikawa and Kidokoro, 1995
Nerve stimulation. A tip of micropipette filled with 4 M potassium acetate and having a resistance of
10 M
was placed in the ventral nerve cord at the site from which motor nerves emerged. Rectangular pulses of
Recording of miniature synaptic currents in high-K+ saline. In normal saline with 0.2 mM Ca 2+, the frequency of miniature synaptic currents was low, and it was difficult to determine the mean amplitude accurately. To increase the miniature synaptic current (mSC) frequency, high-K + saline was used (see below for its ionic composition). The frequency was higher in this solution:
The amplitude histograms of mSCs were not normally distributed. Some of them were skewed to larger amplitudes. To quantify the extent of skewness, we calculated the following statistical parameters for each histogram:
where xi is the amplitude of the ith mSC,
is the mean, and n is the total number of mSCs. Here, m3 and m2 are the third and second moments about the mean. Using these parameters, the skewness is defined as follows:
The value of skewness is zero when the amplitude histogram is normally distributed, positive when the distribution is skewed toward larger values (as in Fig. 4 Ea), and negative when it is skewed toward smaller values.
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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 rut 1. 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 rut 1. 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.
Application of glutamate to activate ionotropic glutamate receptors. To estimate the total amount of glutamate receptors in the postsynaptic membrane, glutamate-induced currents were measured at muscle 6 in Ca 2+-free saline. Glutamate was dissolved in a Ca 2+-free bath solution at 1mM and included in a glass pipette that had a tip diameter of
It should be noted that this method for glutamate application, although aimed at the synaptic area, also activates extrasynaptic receptors. The precise contribution of extrasynaptic glutamate receptor channels to the glutamate-induced currents is not known but is probably small, because receptors are highly localized at the postsynaptic area (Saitoe et al., 2001
Application of agonists to activate metabotropic glutamate receptors or octopamine receptors. Glutamate (100 µM) or an agonist of metabotropic glutamate receptor, (S)4C3HPG (100 µM) or octopamine (10 µM), was puff-applied for 40 sec in high-K + saline containing 0.05 mM Ca 2+ and 3 µM tetrodotoxin (TTX). The frequency of mSCs was counted every 10 sec. The starting point of the 10-sec period was aligned at the onset of the puff pulse for comparison among records from different cells.
Solutions. Ca 2+-free saline had the following ionic composition (in mM): 140 NaCl, 2 KCl, 6 MgCl2, and 5 HEPES-NaOH, at a pH of 7.1. To evoke synaptic currents by nerve stimulation, 0.2 or 0.5 mM CaCl2 was added, and the equivalent amount of MgCl2 was reduced. The ionic composition of high-K + saline with 0.1 mM Ca 2+ was as follows (in mM): 78 NaCl, 62 KCl, 5.9 MgCl2, 0.1 CaCl2, and 5 HEPES-NaOH, at a pH of 7.1. Activation of metabotropic glutamate receptors and octopamine receptors was performed in the following high-K + saline (in mM): 78 NaCl, 62 KCl, 5.95 MgCl2, 0.05 CaCl2, and 5 HEPES-NaOH, at a pH of 7.1.
Biochemicals. (S)4C3HPG and MCCG-I were purchased from Tocris (Essex, UK), and TTX, octopamine, and glutamate were purchased from Sigma (St Louis, MO).
Statistics. First, parameters were tested for the normal distribution using the Kolmogorov–Smirnov test at a value of p = 0.05. When they were found to be normally distributed, we used Student's t test to compare two groups, and to compare more than two groups, we used ANOVA with Scheffé's criteria. When they were not normally distributed, we used the Mann–Whitney test.
Results
Synaptic transmission is subtly impaired in dgs-null mutant embryosAlthough the dgs-null mutations are lethal, morphologically the neuromuscular synapse forms normally in embryos (Wolfgang et al., 2001
Synaptic transmission in external saline containing 0.5 mM Ca2+
In the external solution containing 0.5 mM Ca2+, nerve stimulation at 0.3 Hz evoked robust synaptic currents in dgsR60 embryos (dgsR60/dgsR60). The amplitude of synaptic currents varied widely within one postsynaptic muscle cell and among different cells, and nerve stimulation rarely failed to evoke synaptic currents (Fig. 1A, left three traces). As a control, we used embryos of a strain in which a transgene, Gs27, was introduced into the background of dgsR60 (Gs27; dgsR60c/dgsR60c) (Wolfgang et al., 2001
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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.
Synaptic transmission in external saline containing 0.2 mM Ca2+
The large variation of evoked synaptic current amplitudes within a cell and among different cells may prevent detection of subtle impairment of synaptic transmission in dgsR60 embryos. A part of the variation of synaptic current amplitudes in embryos originates from a large variation of quantal synaptic current amplitudes in embryos (mSCs) (Kidokoro and Nishikawa, 1994
In the external solution containing 0.2 mM Ca2+, nerve stimulation at 0.3 Hz often failed to evoke synaptic currents in dgsR60 embryos (failure rate, 0.88 ± 0.11, mean ± SD, n = 16) (Fig. 2A1, left open column). This failure rate was not different from that in Gs27 embryos (0.92 ± 0.05, n = 8) (Fig. 2B1, left open column) but was significantly higher than that in heterozygous embryos, dgsR60/+ (0.76 ± 0.05, n = 8; p < 0.05) (Fig. 2C1, left open column), suggesting that synaptic transmission is slightly impaired in dgsR60 and Gs27 embryos.
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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, rut 1; 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+.
We further examined the synaptic facilitation during tetanus and PTP. When the nerve was stimulated at 10 Hz for 10 sec, facilitation during tetanus in dgsR60 embryos was not as prominent as in Gs27 and far less than in dgsR60/+ (Fig. 2A1–C1, middle two shaded columns). When the failure rate during the last 50 stimuli (right shaded column) was compared with that before tetanus (left open column), it was slightly lower in dgsR60 embryos, whereas it was much lower in Gs27 or in dgsR60/+ embryos (Fig. 2A1–C1). After tetanus, synaptic transmission was potentiated, and consequently the failure rate was reduced for a prolonged period of time in dgsR60/+ (Fig. 2C1, right open column) but not in dgsR60 embryos or in Gs27 embryos (Fig. 2A1,B1, right open columns). Thus, synaptic transmission in dgsR60 and Gs27 embryos appears to be mildly impaired presynaptically. Because the Gs27 transgene includes the entire Gs
In a mutant, rut1, in which AC is defective (Livingstone et al., 1984
During and after tetanus, asynchronous transmitter release was clearly enhanced in the controls (in Gs27 and dgsR60/+) (Fig. 2B2,C2), whereas that in dgsR60 embryos was minimal (Fig. 2A2). In rut1, an enhancement of asynchronous release was clearly observed during tetanus but was much less after tetanus (Fig. 2D2) compared with that in dgsR60/+ (Fig. 2C2). Thus, facilitation of nerve-evoked synaptic transmission and asynchronous release during and after tetanus changed in parallel among different strains, suggesting that the same mechanism, such as an elevation of [Ca2+]i and/or cAMP in the terminal, underlies both of these phenomena.
Miniature synaptic currents were infrequent and smaller in Gs
In saline with a normal Ca2+ concentration (1.5 mM), muscles occasionally contracted and stretched presynaptic nerves, which increased the frequency of mSCs. Thus, it was difficult to accurately estimate the resting mSC frequency. To avoid this problem, mSCs were examined in 0.2 mM Ca2+ saline with 3 µM TTX during a 10 min recording period (Fig. 3A). The frequency was significantly lower in dgsR60 than in Gs27 or in dgsR60/+ embryos (Fig. 3B). This result again suggests that synaptic transmission in dgsR60 embryos is presynaptically compromised. In rut1, the mean frequency was not different in controls (Fig. 3B).
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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/+,andrut 1.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.
We also noticed that amplitudes of mSCs in dgsR60 embryos were somewhat smaller compared with those in Gs27 or in dgsR60/+ embryos (Fig. 3C). Because the amplitude varied widely in all strains and there were not enough events in each cell during the 10-min observation period, all synaptic events were pooled, and the mean amplitudes were compared. The mean amplitude in dgsR60 was significantly smaller than that in Gs27 or in dgsR60/+ embryos (Fig. 3C). This result suggests that the quantal size of synaptic currents is smaller in dgsR60 embryos than that in Gs27 or in dgsR60/+. However, because the numbers of events were small and the amplitudes varied widely (Fig. 3D), this conclusion should be considered tentative. In rut1, the mean amplitude was not different in dgsR60/+ or in Gs27.
To confirm the smaller amplitude of mSCs in dgsR60 embryos, we next measured mSCs in 62 mM K+ saline with 0.1 mM Ca2+, in which the mSC frequency was higher and sufficient numbers of events (>300) were collected within 2 min in each cell (Fig. 4). The amplitudes of mSCs were larger in high-K+ saline in all strains compared with those in normal-K+ saline (Fig. 4A,B). This is, at least in part, a result of the higher conductivity of K+ compared with Na+ through the Drosophila glutamate receptor channel: at the -65 mV membrane potential, K+ ions carry
The frequency of mSCs in high-K+ saline was lower in dgsR60 and rut1 embryos than in controls (Fig. 4D). It should be noted that the mSC frequency in normal saline was not low in rut1 (Fig. 3B). Because the frequency of mSCs was high in high-K+ saline, the lower mSC frequency in rut1 is probably reflecting the smaller size of the exo/endo cycling pool (readily releasable pool) (Kuromi and Kidokoro, 2000
Properties of postsynaptic glutamate receptor channels are not different in dgsR60 embryos from those in controls
The smaller amplitude of mSCs in dgsR60 embryos could be a result of either presynaptic factors, such as smaller amount of glutamate in vesicles, or postsynaptic factors, such as lower densities of glutamate receptors or smaller conductance of synaptic glutamate receptor channels. To distinguish these possibilities, we next examined the properties of postsynaptic glutamate receptor channels.
Glutamate-induced currents
To assess the total number of glutamate receptors in the postsynaptic membrane, 1 mM glutamate was puff-applied at the neuromuscular synapse in abdominal longitudinal muscle 6. The mean amplitude of glutamate-induced inward currents was not different in dgsR60 embryos from that in controls (Fig. 5A,B), suggesting that the total number of postsynaptic receptors is not the cause for smaller amplitudes of mSCs in dgsR60 embryos.
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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
Single-channel current amplitudes
In the falling phase, the synaptic current often changes in steps, revealing underlying single-channel currents. The smallest step amplitude probably corresponds to unitary channel current amplitude of the synaptic glutamate receptor channel (Nishikawa and Kidokoro, 1995
Metabotropic glutamate receptor responses in dgsR60 were indistinguishable from those in controls
Activation of metabotropic glutamate receptors (mGluRs) in the presynaptic terminal clearly increases the frequency of mSCs in Ca2+-free saline and enhances synaptic transmission at the neuromuscular synapse of first instar larvae (Zhang et al., 1999
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Figure 6. mGluR responses inGs27(A–C), in DC0(D),and in dgsR60 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 dgsR60 embryos. The number of cells examined is nine.
In dgsR60 embryos, puff application of 100 µM (S)4C3HPG clearly increased the mSC frequency (Fig. 6E). The mGluR response in dgsR60 embryos was not different from that in Gs27 embryos (Fig. 6C), indicating that the mGluR response at the embryonic neuromuscular synapse is not mediated by Gs
Octopamine receptor responses in dgsR60 were indistinguishable from those in controls
The effect of octopamine on synaptic transmission was also examined in high-K+ saline with low Ca2+ (0.05 mM). Puff application of 10 µM octopamine for 40 sec increased the mSC frequency in G27 embryos (Fig. 7A). The dose–response curve is shown in Figure 7B, indicating that an apparent Kd is
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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 Kd 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.
In dgsR60 embryos, responses similar to those in Gs27 were observed (Fig. 7D), indicating that the octopamine response is not mediated by Gs
Expression of a Gs
In wild-type embryos, Gs
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Figure 8. Expression of a Gs
In contrast, when the transgene, GsW24, was expressed in muscles in the dgsR60 background, none of above-mentioned phenotypes were rescued (Fig. 8Ab,Bb,Ca–Cc,Ce).
Together, we conclude that the synaptic impairment in dgsR60 embryos is the result of lack of Gs
Discussion
Presynaptic defects of synaptic transmission in dgsR60 embryosTwo distinct sets of phenotypes in synaptic transmission at the neuromuscular synapse in dgsR60 embryos were revealed, as follows. (1) Slightly smaller quantal content: The failure rate of stimuli to evoke synaptic currents in dgsR60 embryos was slightly greater in saline containing 0.2 mM Ca2+ compared with heterozygotes (Fig. 2), suggesting smaller quantal contents of evoked synaptic currents. This subtle impairment in nerve-evoked synaptic transmission probably correlates with lower frequencies of mSCs (Figs. 3B, 4D). Synaptic impairment was more clearly demonstrated on stimulation at 10 Hz. In dgsR60 embryos, we found minimal synaptic facilitation during tetanus and no PTP. Furthermore, asynchronous release of quanta during and after tetanus was much less than in controls. The lack of PTP found in dgsR60 embryos was similar to that in rut1 (Fig. 2D1). At the light-microscopic level, the morphology of neuromuscular synapses in dgsR60 embryos is not different from that of controls (Wolfgang et al., 2001
These two distinct sets of phenotypes in synaptic transmission in dgsR60 embryos are both a result of presynaptic defects.
Some phenotypes in dgsR60 embryos are similar to those in rut1, but others are distinctly different
In rut1, Ca2+–calmodulin-responsive AC is defective (Livingstone et al., 1984
During tetanic stimulation, synaptic transmission was slightly facilitated in rut1 embryos and in dgsR60, but PTP was absent in both mutants (Fig. 2). In third instars of a dgs hypomorph, dgsB19, both facilitation during tetanus and PTP were absent (Wolfgang, Clay, Parker, Delgado, Kidokoro, Labarca, and Forte, unpublished observation), and in third instars of rut1, there was slight facilitation during tetanus but no PTP (Zhong and Wu, 1991
Neither mGluRs nor octopamine receptors are coupled to a G-protein containing Gs
Both mGluR and octopamine receptor responses in dgsR60 embryos were indistinguishable from those in Gs27 (Figs. 6, 7), indicating that Gs
Because there are many G-protein-coupled receptors, our negative findings with two synaptic modulator receptors are not surprising. We found that of two distinct sets of phenotypes in dgsR60 embryos, one set, slightly smaller quantal content, is similar to that in rut1. Therefore, it is still possible that an unknown modulator receptor in the presynaptic terminal is coupled to AC through a G-protein containing Gs
Frequent occurrence of large mSCs in high-K+ saline in control embryos
Unexpectedly, we observed in high-K+ saline many distinctly large mSCs in Gs27 and dgsR60/+ embryos at the late embryonic stage. The mean amplitude was
Two peaks in the mSC amplitude distribution have been reported in Xenopus nerve–muscle cultures. In younger cultures, the mean amplitude of mSCs is smaller and the amplitude distribution is skewed toward larger amplitudes. The second symmetrical peak in the large-amplitude range appears in older cultures. This change in the amplitude distribution is considered to be a developmental process (Kidokoro, 1984
Why do large mSCs occur frequently in high-K+ saline? Among other possibilities, we favor the following scenario. In rapidly developing embryos, some release sites may be more mature than others. In high-K+ saline with Ca2+, in which Ca2+ levels in the presynaptic nerve terminal are elevated, fusion of vesicles may occur more frequently at those mature release sites than at immature sites. These mature release sites probably face a postsynaptic membrane with a higher receptor density. In dgsR60 embryos, however, fewer release sites may be mature and face a postsynaptic membrane with a high receptor density. In addition, those release sites may not be responding to an elevated Ca2+ in high-K+ saline to produce large mSCs, resulting in the smaller mean amplitude with a skewed distribution. In normal saline, these mature release sites may be regulated not to initiate excessive vesicle fusion. Because the mean amplitude of glutamate-induced currents reflecting the total number of receptors was not different between dgsR60 embryos and controls (Fig. 5A,B), these mature release sites with high receptor densities could not be more numerous in controls but must be releasing vesicles more frequently in high-K+ saline.
In this study, we found the skewed amplitude distributions of mSCs in dgsR60 embryos in high-K+ saline, whereas in controls, amplitude distributions were broader. Because a transition from a skewed amplitude distribution to a broader one has been observed during synapse formation (Kidokoro, 1984
The effects of the null-mutation in dgs on synaptic transmission were observed in two aspects. One could be because of uncoupling between the as-yet-unknown modulator receptor and AC activation. This phenotype is similar to that in rut1. The other is probably a defect in synapse formation. Mature release sites with high receptor densities may not be well developed in dgsR60 embryos. To pinpoint the process in which Gs
Footnotes
Received Jan. 8, 2003; revised Mar. 7, 2003; accepted Apr. 9, 2003.This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan to Y.K. and by National Institutes of Health Grant R01-NS42841 to M.F. D.H. is supported by a scholarship from the Japanese Government.
Correspondence should be addressed to Dr. Yoshi Kidokoro, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan. E-mail: kidokoro{at}med.gunma-u.ac.jp.
Copyright © 2003 Society for Neuroscience 0270-6474/03/235897-09$15.00/0
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