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Absence of Junctional Glutamate Receptor Clusters in Drosophila Mutants Lacking Spontaneous Transmitter Release

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Science  20 Jul 2001:
Vol. 293, Issue 5529, pp. 514-517
DOI: 10.1126/science.1061270

Abstract

Little is known about the functional significance of spontaneous miniature synaptic potentials, which are the result of vesicular exocytosis at nerve terminals. Here, by usingDrosophila mutants with specific defects in presynaptic function, we found that glutamate receptors clustered normally at neuromuscular junctions of mutants that retained spontaneous transmitter secretion but had lost the ability to release transmitter in response to action potentials. In contrast, receptor clustering was defective in mutants in which both spontaneous and evoked vesicle exocytosis were absent. Thus, spontaneous vesicle exocytosis appears to be tightly linked to the clustering of glutamate receptors during development.

In 1952, Fatt and Katz described miniature end-plate potentials, which provided a basis for the theory of quantal synaptic transmission (1). A single miniature end-plate potential arises when a synaptic vesicle fuses spontaneously with the presynaptic membrane and releases a quantum of transmitter (spontaneous vesicle exocytosis). However, little is known about the functional importance of this process. Presynaptic and postsynaptic neurotoxins that allow spontaneous vesicle exocytosis to persist have little effect on synaptic development, including postsynaptic accumulation of receptors (2, 3). During the development ofDrosophila neuromuscular junctions (NMJs), glutamate receptors (GluRs) cluster in the postsynaptic membrane in a manner that depends on nerve-muscle contact (4). To investigate the role of spontaneous secretory events in receptor clustering, we used Drosophila mutants with distinctive secretory defects. Mutations of neuronal-synaptobrevin (n-syb) orcysteine string protein (csp) selectively prevent nerve-evoked exocytosis whereas spontaneous vesicle exocytosis persists (5–7). In contrast, syntaxin-1A(syx) or shibire (shi) mutations eliminate both spontaneous and evoked exocytosis (8,9), thereby allowing one to distinguish the role of spontaneous secretory events.

We first characterized neuromuscular transmission in wild-type and mutant Drosophila embryos or larvae (10–12). A typical record of a burst of excitatory synaptic currents (ESCs), which often exceeded 600 pA in amplitude in a newly hatched wild-type larva (control), is shown inFig. 1A (TTX–). In the presence of tetrodotoxin (TTX), the bursting of ESCs was suppressed, and ESCs seldom exceeded 400 pA (TTX+). A similar suppression of bursting and reduction in the amplitude of ESCs was observed when the ventral nerve cord was removed. Thus, propagated activity in the nervous system triggered multiple vesicle exocytosis and contributed to the ESCs. Concomitantly, the residual events [miniature ESCs (mESCs)] in these wild-type larvae (TTX+) were due to spontaneous vesicle exocytosis.

Figure 1

The correlation between mESCs and GluR clustering. (A) Synaptic currents in each column were from the same muscle of embryos or newly hatched larvae. The addition of 1 μM TTX (TTX+) had no substantial effect on synaptic activity in n-syb and syx embryos. Each trace is representative of recordings from at least six embryos of that genotype; the recordings lasted at least 15 min each. The evoked responses were initiated by microelectrode stimulation of the CNS (evoked). (B) GluR localization at embryonic NMJs. Embryos at 22 hours after fertilization were double-stained with anti-HRP (orange) and anti-DGluRIIA (green) (14). Immunofluorescence images are of muscle 12 and 13. (C) GluR localization at embryonic NMJs stained with antibodies directed against different subunits. DGluRIIB was stained with a transgenic embryo in which the subunit was myc-tagged (17) (upper panel). AS5, which recognizes DGluRIIA, DGluRIIB, and DGluRIIC, was used (18) (lower panel). Scale bar, in (B) is 10 μm for (B) and (C).

We first investigated an n-syb null mutant (13, 14), in which nerve-evoked ESCs but not mESCs are lost (5, 6). Consistent with these findings, we detected ESCs in n-syb embryos but virtually no large-amplitude ESCs characteristic of nerve-evoked ESCs (Fig. 1A). This apparent absence of evoked responses (but persistence of mESCs) was confirmed by the fact that TTX had no effect on the frequency or amplitude of ESCs and that no evoked ESCs were elicited by nerve stimulation (Fig. 1A).

In syx mutants, both nerve-evoked and mESCs were undetectable (8). In agreement with this phenotype (Fig. 1A), neither nerve-evoked nor mESCs were detected during observations exceeding 15 min each in seven cells. Although we could not completely eliminate the possibility of missing very infrequent occurrences, it is clear that the frequency of mESCs in syx embryos is far lower than the frequency of mESCs in n-syb embryos. Given the distinct phenotypes of the n-syb and syxmutants, we examined the distribution of postsynaptic GluRs in these embryos.

Figure 1B shows the distribution of GluRs at embryonic NMJs (14). Concomitantly, we stained these preparations with antibody against horseradish peroxidase (anti-HRP), which binds to a neuronal surface antigen (15) and reveals the presynaptic terminals. Immunoreactive GluRs formed prominent junctional clusters that closely mirrored the presynaptic elements in wild-type andn-syb mutants. Although this finding apparently contrasts with the observation in para (Na+ channel) mutants that neural electrical activity is essential for the clustering of receptors (16), it should be noted that then-syb mutation is a more subtle perturbation of this system. Unlike n-syb, however, syx mutants rarely had discernible junctional GluR clusters, although they invariably formed neuromuscular contacts (14). Because the antibody we used recognizes only the DGluRIIA subunit, other subunits might have substituted to form functional receptor channels. To exclude this possibility, we used a transgenic line in which the DGluRIIB subunit was tagged by myc (17). syx embryos carrying this myc-tagged GluR exhibited no clustering of receptors. However, receptor clusters were clearly stained in control organisms carrying this myc-tagged receptor (Fig. 1C). As another control, we used the antibody AS5, which recognizes DGluRIIA, DGluRIIB, and DGluRIIC subunits (18). The result was indistinguishable from that obtained with the DGluRIIB antibody (Fig. 1C).

These findings raised the possibility that the absence of detectable mESCs in these mutants (Fig. 1A) may have arisen from a deficit of postsynaptic GluRs. Moreover, the lack of receptor clusters could either be a developmental consequence of a lack of vesicle fusions in the nerve terminal, or it could be due to a requirement for syntaxin in the trafficking of GluRs to the postsynaptic membrane. Indeed,syx is required for cell viability in Drosophila(19, 20), and the development of both the neuron and muscle in syx embryos is likely to be due to small amounts of maternal Syx (19). If this residual Syx is not adequate for the maintenance of normal vesicular traffic to the cell surface, GluRs may not be inserted appropriately in the sarcolemma. To address these issues, we determined whether syx mutants responded to applied glutamate and also whether junctional GluR clusters were restored in syx mutants by selectively inducing the presynaptic or postsynaptic expression of a syx transgene.

The application of l-glutamate by pressure ejection (14) onto the junctional region of syxmuscles indicated that some sensitivity was lost concomitantly with the loss of clusters. The pressure ejection ofl-glutamate at the junctional region yielded robust inward currents in wild-type (987 ± 277 pA, mean ± SD,n = 6) and n-syb mutants (1099 ± 289 pA, n = 7), but glutamate-evoked currents were much smaller in syx mutants (116 ± 118 pA,n = 6) (Fig. 2A). The small response in syx mutants contrasts with an earlier report that postsynaptic glutamate responsiveness is at the control level in syx mutants (8). Although we cannot account for this discrepancy, what is important here is that even with this diminished glutamate sensitivity, we would have detected mESCs insyx mutants if their terminals were spontaneously releasing transmitter. We could have recognized mESCs as small as 20 pA under the recording conditions we used (10). Thus, the complete absence of detectable mESCs could not be attributed to a lack of postsynaptic sensitivity.

Figure 2

Functional GluRs are expressed insyx mutants. (A) The postsynaptic response to pressure ejection of l-glutamate (arrows). (B) (Upper panels) Junctional GluR clusters were restored in syxmutants by the targeted expression of the syx +transgene in neurons [GAL4-responsive upstream activity sequence (UAS-syx+; elav-GAL4; syx) but not in muscle (UAS-syx+; G14-GAL4; syx)]. Although they did not hatch, UAS-syx+; elav-GAL4; syx embryos showed muscle movements that imply functional synaptic transmission. Scale bar, 10 μm. (Lower panels) Nerve-evoked synaptic currents were observed after stimulation at the CNS (arrows) inUAS-syx+; elav-GAL4; syx embryos but not inUAS-syx+; G14-GAL4; syx embryos (evoked). mESCs were also observed in the UAS-syx+; elav-GAL4; syxmutants but not in the UAS-syx+; G14-GAL4; syxmutants (mini).

The low sensitivity to glutamate in syxmutants could reflect the diminished trafficking of GluRs to the surface. Thus, we tested whether the clustering defect was due to a pre- or postsynaptic action of syx by the targeted expression of the syx transgene with either the neuron-specific embryonic lethal, abnormal vision (elav)–yeast transcription activator GAL4 driver or the muscle-specific G14-GAL4 driver (14). Neuron-specific expression of the syx transgene restored GluR clusters but muscle-specific expression did not (Fig. 2B). Evoked and mESCs were readily observed in transgenic embryos in which neuron-specific expression of the syx transgene was restored but not in transgenic embryos in which syx transgene was expressed in muscles (Fig. 2B, lower panels). Thus, a comparison of the phenotypes of n-syb and syx led us to hypothesize that spontaneous secretory events at the NMJ are critical to the formation of GluR clusters.

We used two temperature-sensitive (ts) paralytic mutants to examine further the correlation between spontaneous vesicle exocytosis and GluR clustering. At elevated temperatures, a defect in dynamin in shits blocks synaptic vesicle recycling and thereby depletes the terminals of synaptic vesicles (9). In contrast, cspts mutations appear to interfere with excitation-secretion coupling in the terminal (7, 21). Synapses in shits mutants became completely silent at a nonpermissive temperature (32°C), whereas cspts mutants lost evoked responses while retaining mESCs (7) (Fig. 3A). Because of these differences in release properties at the nonpermissive temperature, we compared the distribution of GluRs in these lines. At a permissive temperature (25°C), when release properties were similar among these embryos (Fig. 3A), GluR clustering was comparable for wild-type,cspts, and shits mutant embryos (Fig. 3B). However, this situation changed when embryos were moved to the nonpermissive temperature at 13 hours after fertilization, which is when nerve-muscle contacts first form (22, 23). The development of GluR clusters was not perceptibly altered in wild-type and cspts mutants at 32°C. However, no detectable GluR clusters were observed in shits mutants (Fig. 3B), as was the case in syx mutants. These results again suggest a tight link between spontaneous vesicle exocytosis and GluR clustering.

Figure 3

The correlation between mESCs and GluR clustering. (A) Neuromuscular transmission incspts and shits embryos. In each column, ESCs were recorded from the same muscle at the permissive temperature (25°C) and were recorded again 10 min after the temperature was raised to 32°C. Vigorous ESCs were seen in wild-type embryos at both temperatures. In cspts mutants, episodic bursting of ESCs was observed as it was in wild-type embryos at 25°C, whereas only mESCs were seen at 32°C. Inshits mutants, ESCs occurred at 25°C but not at 32°C. (B) GluR localization at embryonic NMJs. Embryos were reared at 25°C throughout development (25°C) or shifted to 32°C at 13 hours after fertilization (32°C). Scale bar, 10 μm.

Further insight into the nature of interaction of presynaptic and postsynaptic elements came from the injection of argiotoxin (14) into wild-type embryos at concentrations that block all muscle contractile activity. In these embryos, GluRs still clustered postsynaptically (Fig. 4). Thus, it was not the activation of postsynaptic GluRs that directed GluR clustering. Similar findings have been reported in vertebrates, where α-bungarotoxin did not impede the clustering of acetylcholine receptor (AChR) (3). As in vertebrates, secretion of molecules, such as agrin for AChRs (24), ephrins forN-methyl-d-aspartate (NMDA)–type GluRs (25), and neuronal activity-regulated pentraxin for α-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA)–type GluRs (26), may drive receptor clustering by being released with, or in parallel to, the neurotransmitter atDrosophila NMJs.

Figure 4

Argiotoxin (argTX) does not affect receptor clustering. ArgTX was injected into embryos at 12 to 13 hours after fertilization. Embryos were double stained with anti-HRP and anti-DGluR-IIA at 21 to 22 hours after fertilization. Scale bar, 5 μm.

We documented a positive correlation between ongoing spontaneous vesicle exocytosis and the embryonic development of GluR clusters at Drosophila NMJs. Nerve-evoked vesicle exocytosis is not necessary for this process, because although neithern-syb nor cspts mutants showed any demonstrable nerve-evoked ESCs, GluRs still clustered. mECSs persisted in both mutants. However, when spontaneous secretory events were absent (as in shits mutants at the nonpermissive temperature or in syx), junctional GluR clusters were exceedingly infrequent. If GluR clustering were solely contingent on the nerve-muscle contact, GluRs should have clustered at the contacts in shits mutants raised at the nonpermissive temperature and in syx mutants. In a recent study of mice lacking an isoform of munc 18-1, there was no demonstrable change in AChR clustering, although both spontaneous and evoked neurotransmitter release were absent (27). Together with our observations, these data suggest that munc 18-1 is not involved in the secretion of the agent that induces clustering of neurotransmitter receptors, whereas syntaxin is essential for this process.

The absence of clusters in Drosophila insyx and shi mutants implies that spontaneous secretory events are related to GluR clustering and probably to cluster stabilization as well. Moreover, it is the clustering of these receptors, rather than their surface expression, that depends on spontaneous secretion: Functional GluRs were detected in syxmutants although they had rarely formed detectable clusters at the synapse. The link between spontaneous vesicle exocytosis and receptor clustering must now be clarified.

  • * To whom correspondence should be addressed. E-mail: saitoe{at}tmin.ac.jp

  • Present address: Division of Neuroscience, 300 Longwood Avenue, Enders, 208 Children's Hospital, Boston, MA 02115, USA.

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