Glutamate receptor IIA and Glutamate receptor IIB

DEVELOPMENTAL BIOLOGY

Embryonic and Larval

See the embryonic expression pattern of Glu-RIIA at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

The RNA expression pattern of DGluRIIB was established by means of embryonic whole-mount in situ hybridization. DGluRIIB RNA is observed exclusively in muscle. It first appears at late stage 12 and reaches its highest levels at stage 14. In stages 15-17, DGluRIIB expression is lower but is still present in somatic musculature. Low levels are observed in the gut-associated muscle. This expression pattern is similar but not identical to that of DGluRIIA. DGluRIIA is also first observed at stage 12 and is expressed exclusively in muscle, but in contrast to DGluRIIB, it increases gradually until it reaches its highest levels in stages 16 and 17 (Currie, 1995 and Peterson, 1997).

Glutamate is the excitatory transmitter at neuromuscular synapses in Drosophila, and electrophysiological studies indicate that the receptors for glutamate are concentrated in muscle fibers at synaptic sites. Acetylcholine is the excitatory transmitter at vertebrate neuromuscular synapses, and previous studies have shown that accumulation of acetylcholine receptors (AChRs) at synaptic sites is controlled both by transcriptional and post-translational mechanisms. The transcriptional pathway culminates in selective expression of AChR subunit genes in nuclei near the synaptic site, causing AChR mRNA to accumulate in the synaptic region of the muscle fiber. A cDNA encoding a subunit of the Drosophila muscle glutamate receptor (DGluR-IIA) was used to determine the temporal and spatial expression pattern of the DGluR-IIA gene during embryogenesis and in larval muscle. DGluR-IIA mRNA is first expressed at stage 12 of embryogenesis and that expression is detected in developing dorsal, lateral, and ventral somatic muscles within the next 2 hr. By stage 16 DGluR-IIA mRNA is expressed in all somatic muscles and in pharyngeal muscles. In third instar larvae DGluR-IIA mRNA is expressed in all body-wall muscle fibers. DGluR-IIA mRNA, however, is expressed throughout the larval muscle fibers and is not concentrated within muscle fibers at neuromuscular synapses. These results indicate that although the DGluR-IIA gene is expressed in somatic muscle cells it is not selectively expressed in nuclei near the synaptic site (Currie, 1995).

To investigate the subcellular localization of DGluRIIA and DGluRIIB, each receptor was tagged with the myc-epitope. The epitope was incorporated immediately following a heterologous signal sequence that was used to replace the endogenous signal sequences of each gene. As such, the myc-epitope is present at the extracellular N terminus of each receptor. Transgenic flies were generated that express the tagged receptors under the control of the muscle-specific myosin heavy-chain promoter. Both DGluRIIB and DGluRIIA are localized to the neuromuscular junctions (NMJs) of body-wall muscles in third instar larvae (Peterson, 1997).

The NMJs of third instar larvae have been subdivided by morphological and physiological criteria. Type I synapses have larger boutons and contain small, clear, glutamate-filled vesicles, while Type II synapses have small boutons and are primarily peptidergic. While many muscles possess only Type I synapses, a number of muscles are innervated by both Type I and Type II synapses. To assess whether glutamate receptors are differentially localized to a particular class of synapse within a single cell, double staining was performed for Synaptotagmin, a marker of all presynaptic terminals and for glutamate receptor. Confocal microscopy reveals that DGluRIIB is localized to the postsynaptic specialization surrounding presynaptic terminals of Type I boutons, but no staining for receptors is observed at Type II boutons. Staining for DGluRIIA gives the same result. This indicates that at the Drosophila NMJ, as in vertebrate central neurons, glutamate receptors are differentially localized to particular synapses within a single cell (Peterson, 1997).

A hallmark of Type I boutons is the presence of an elaborate postsynaptic specialization, the subsynaptic reticulum (SSR), which consists of numerous layers of invaginated membrane surrounding the presynaptic terminal. Molecules localized to the SSR such as the PDZ-containing protein Discs-Large (Dlg) and the cell adhesion molecule Fasciclin II (FasII) appear to form a halo surrounding the entire presynaptic terminal when analyzed by confocal microscopy. In contrast, both DGluRIIA and DGluRIIB appear as bright spots adjacent to the presynaptic terminal. These hot spots of receptor localization are of the appropriate size and pattern to represent postsynaptic receptor clusters opposite presynaptic release sites. To investigate this possibility immunoelectron microscopy was performed. The EM analysis confirms the patchy distribution of receptors surrounding the bouton. Receptors are localized to particular regions of the synaptic cleft and are nearly undetectable in the underlying invaginations of the SSR. These patches of receptors around synaptic boutons are always observed opposite a presynaptic terminal containing accumulations of synaptic vesicles and tightly apposed, parallel presynaptic and postsynaptic membranes that are characteristic of active zones. Hence, the clusters of receptors visible by confocal microscopy appear to be postsynaptic markers of vesicle release sites. Shaker (Sh) potassium channels and FasII require Dlg for clustering at synapses. It was of interest to see whether DGluRIIA or DGluRIIB also require Dlg for their localization. To address this question, the myc-tagged proteins were stained in a dlg mutant in which Shaker fails to cluster to the NMJ. No change was seen in glutamate receptor localization. Hence, other proteins are likely to function in the localization of these glutamate receptors (Peterson, 1997).

Changes in the distribution and density of transmitter receptors in the postsynaptic cell are required steps for functional synapse formation. Antibodies were raised against Drosophila glutamate receptors (DGluR-IIA) and the distribution of receptors were visualized during neuromuscular junction formation in embryos. In wild-type embryos, embryonic development is complete within 22 hr after egg lying (AEL) and neuromuscular junction (NMJ) formation begins at 13 hr AEL. At the time of initial synapse formation, DGluR-IIAs appears as clusters closely associated with some muscle nuclei. Subsequently, these nonjunctional clusters disperse while DGluR-IIAs accumulate at the junctional region. In a paralytic temperature-sensitive mutant, para(ts1), neural activity decreases drastically at restrictive temperatures. When neural activity is blocked throughout synaptogenesis by rearing embryos at a restrictive temperature prior to the beginning of synaptogenesis, 12 hr AEL, the dispersal of extrajunctional clusters is significantly suppressed and no accumulation of receptors at the junction is observed at 22 hr AEL. However, when neural activity is blocked later, by rearing embryos at a restrictive temperature from 13 hr AEL, DGluR-IIAs does not accumulate at the NMJ, although extrajunctional clusters disperse normally. These findings suggest that the neural activity differentially regulates dissipation of receptor clusters in the nonjunctional region and accumulation of receptors at the junctional region (Saitoe, 1997).

During the formation of neuromuscular junctions in Drosophila embryos, glutamate receptors undergo a drastic change in distribution. To study the underlying mechanism of this developmental process, it is desirable to map the distribution of functional receptors with accurate spatial resolution. Since glutamate receptors desensitize within several milliseconds, the agonist must be applied rapidly. To fulfil these requirements laser stimulation of a caged compound was used to release L-glutamate at a focal spot. Since the glutamate receptor channel is permeable to Ca2+, the change in internal Ca2+ concentration was examined using a Ca2+ indicator, fluo-3. Using this approach, the distribution of functional glutamate receptors were mapped in cultured embryonic Drosophila myotubes and myoblasts. Consistent with previous immunofluorescence studies using an antibody against a glutamate receptor subunit, a large increase of internal Ca2+ concentration is observed when laser stimulation is located close to some nuclei in the myotube. No change is detected when the laser stimulus is applied over any regions of the myoblasts. No increase of the internal Ca2+ concentration in myotubes is observed when the external solution contains either glutamate at a desensitizing concentration (1 mM) or a glutamate receptor channel blocker, argiotoxin (1 microg/ml). These results indicate that a rise in intracellular Ca2+ concentration can be used to show the distribution of the functional receptor on the muscle surface membrane (Saitoe, 1998).

Little is known about the functional significance of spontaneous miniature synaptic potentials, which are the result of vesicular exocytosis at nerve terminals. By using Drosophila mutants with specific defects in presynaptic function it has been found that glutamate receptors cluster normally at neuromuscular junctions of mutants that retain spontaneous transmitter secretion but have lost the ability to release transmitter in response to action potentials. In contrast, receptor clustering is defective in mutants in which both spontaneous and evoked vesicle exocytosis are absent. Thus, spontaneous vesicle exocytosis appears to be tightly linked to the clustering of glutamate receptors during development (Saitoe, 2001).

The existence of miniature end-plate potentials provides a basis for the theory of quantal synaptic transmission. 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. During the development of Drosophila neuromuscular junctions (NMJs), glutamate receptors (GluRs) cluster in the postsynaptic membrane in a manner that depends on nerve-muscle contact. To investigate the role of spontaneous secretory events in receptor clustering, Drosophila mutants with distinctive secretory defects were used. Mutations of neuronal-synaptobrevin (n-syb) or cysteine string protein (csp) selectively prevent nerve-evoked exocytosis whereas spontaneous vesicle exocytosis persists. In contrast, syntaxin-1A (syx) or shibire (shi) mutations eliminate both spontaneous and evoked exocytosis, thereby allowing one to distinguish the role of spontaneous secretory events (Saitoe, 2001).

Neuromuscular transmission in wild-type and mutant Drosophila embryos or larvae were characterized. A typical burst of excitatory synaptic currents (ESCs) often exceeded 600 pA in amplitude in a newly hatched wild-type larva (control). In the presence of tetrodotoxin, the bursting of ESCs is suppressed, and ESCs seldom exceeded 400 pA. A similar suppression of bursting and reduction in the amplitude of ESCs is observed when the ventral nerve cord is removed. Thus, propagated activity in the nervous system triggers multiple vesicle exocytosis and contributes to the ESCs. Concomitantly, the residual events [miniature ESCs (mESCs)] in these wild-type larvae are due to spontaneous vesicle exocytosis (Saitoe, 2001).

An n-syb null mutant was investigated in which nerve-evoked ESCs but not mESCs are lost. Consistent with these findings, ESCs were detected in n-syb embryos but virtually no large-amplitude ESCs characteristic of nerve-evoked ESCs. 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 (Saitoe, 2001).

In syx mutants, both nerve-evoked and mESCs are undetectable. In agreement with this phenotype, neither nerve-evoked nor mESCs were detected during observations exceeding 15 min each in seven cells. Although the possibility of missing very infrequent occurrences could not be completely eliminated, 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 syx mutants, the distribution of postsynaptic GluRs was examined in these embryos (Saitoe, 2001).

Preparations were also stained with antibody against horseradish peroxidase (anti-HRP), which binds to a neuronal surface antigen and reveals the presynaptic terminals. Immunoreactive GluRs formed prominent junctional clusters that closely mirrored the presynaptic elements in wild-type and n-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, it should be noted that the n-syb mutation is a more subtle perturbation of this system. Unlike n-syb, however, syx mutants rarely have discernible junctional GluR clusters, although they invariably form neuromuscular contacts (Saitoe, 2001).

These findings raised the possibility that the absence of detectable mESCs in these mutants 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 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, and the development of both the neuron and muscle in syx embryos is likely to be due to small amounts of maternal Syx. 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, it was determined whether syx mutants responded to applied glutamate and also whether junctional GluR clusters are restored in syx mutants by selectively inducing the presynaptic or postsynaptic expression of a syx transgene (Saitoe, 2001).

The application of L-glutamate by pressure ejection onto the junctional region of syx muscles indicates that some sensitivity is lost concomitantly with the loss of clusters. The pressure ejection of L-glutamate at the junctional region yielded robust inward currents in wild-type and n-syb mutants, but glutamate-evoked currents are much smaller in syx mutants. What is important here is that even with this diminished glutamate sensitivity, mESCs would have been detected in syx mutants if their terminals were spontaneously releasing transmitter. Thus, the complete absence of detectable mESCs could not be attributed to a lack of postsynaptic sensitivity (Saitoe, 2001).

The low sensitivity to glutamate in syx mutants could reflect the diminished trafficking of GluRs to the surface. Thus, a test was performed to see whether the clustering defect is due to a pre- or post-synaptic action of syx by the targeted expression of the syx transgene with either the neuron-specific elav-GAL4 driver or the muscle-specific G14-GAL4 driver. Neuron-specific expression of the syx transgene restores GluR clusters but muscle-specific expression does not. Evoked and mESCs are readily observed in transgenic embryos in which neuron-specific expression of the syx transgene is restored but not in transgenic embryos in which syx transgene is expressed in muscles. Thus, a comparison of the phenotypes of n-syb and syx has led to a hypothesis that spontaneous secretory events at the NMJ are critical to the formation of GluR clusters (Saitoe, 2001).

Two temperature-sensitive (ts) paralytic mutants were used to examine further the correlation between spontaneous vesicle exocytosis and GluR clustering. At elevated temperatures, a defect in dynamin in shi^ts blocks synaptic vesicle recycling and thereby depletes the terminals of synaptic vesicles. In contrast, csp^ts mutations appear to interfere with excitation-secretion coupling in the terminal. Synapses in shi^ts mutants become completely silent at a nonpermissive temperature (32°C), whereas csp^ts mutants lose evoked responses while retaining mESCs. Because of these differences in release properties at the nonpermissive temperature, the distribution of GluRs in these lines was compared. At a permissive temperature (25°C), when release properties are similar among these embryos, GluR clustering is comparable for wild-type, csp^ts, and shi^ts mutant embryos. However, this situation changes when embryos are moved to the nonpermissive temperature at 13 hours after fertilization, which is when nerve-muscle contacts first form. The development of GluR clusters is not perceptibly altered in wild-type and csp^ts mutants at 32°C. However, no detectable GluR clusters are observed in shi^ts mutants, as is the case in syx mutants. These results again suggest a tight link between spontaneous vesicle exocytosis and GluR clustering (Saitoe, 2001).

Further insight into the nature of interaction of presynaptic and postsynaptic elements has come from the injection of argiotoxin into wild-type embryos at concentrations that block all muscle contractile activity. In these embryos, GluRs still cluster postsynaptically. Thus, it was not the activation of postsynaptic GluRs that directs GluR clustering. Similar findings have been reported in vertebrates, where alpha-bungarotoxin does not impede the clustering of acetylcholine receptor (AChR). As in vertebrates, secretion of molecules, such as agrin for AChRs, ephrins for N-methyl-D-aspartate (NMDA)-type GluRs, and neuronal activity-regulated pentraxin for AMPA-type GluRs, may drive receptor clustering by being released with, or in parallel to, the neurotransmitter at Drosophila NMJs (Saitoe, 2001).

A positive correlation has been documented 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 neither n-syb nor csp^ts mutants show any demonstrable nerve-evoked ESCs, GluRs still cluster. mECSs persist in both mutants. However, when spontaneous secretory events are absent (as in shi^ts mutants at the nonpermissive temperature or in syx), junctional GluR clusters are exceedingly infrequent. If GluR clustering is solely contingent on the nerve-muscle contact, GluRs should cluster at the contacts in shi^ts 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 are absent. Together with the observations made in this paper, 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 (Saitoe, 2001).

The absence of clusters in Drosophila in syx 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 are detected in syx mutants although they rarely form detectable clusters at the synapse. The link between spontaneous vesicle exocytosis and receptor clustering must now be clarified (Saitoe, 2001).

Experience-dependent strengthening of Drosophila neuromuscular junctions

The genetic analysis of larval neuromuscular junctions (NMJs) of Drosophila has provided detailed insights into molecular mechanisms that control the morphological and physiological development of these glutamatergic synapses. However, because of the chronic defects caused by mutations, a time-resolved analysis of these mechanisms and their functional relationships has been difficult so far. This study provides a first temporal map of some of the molecular and cellular key processes that are triggered in wild-type animals by natural larval locomotor activity and then mediate experience-dependent strengthening of larval NMJs. Larval locomotor activity 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. Within 2 hr of enhanced locomotor activity, NMJs showed a significant potentiation of signal transmission that was rapidly reversed by an induced paralysis of the temperature-sensitive mutant para^ts1. Enhanced locomotor activity was also associated with a significant increase in the number of large subsynaptic translation aggregates. After 4 hr, postsynaptic DGluR-IIA glutamate receptor subunits started to transiently accumulate in ring-shaped areas around synapses, and they condensed later on, after chronic locomotor stimulation at 29°C, into typical postsynaptic patches. These NMJs showed a reduced perisynaptic expression of the cell adhesion molecule Fasciclin II, an increased number of junctional boutons, and significantly more active zones. Such temporal mapping of experience-dependent adaptations at developing wild-type and mutant NMJs will provide detailed insights into the dynamic control of glutamatergic signal transmission (Sigrist, 2003).

One of the prerequisites for a time-resolved analysis of experience-dependent adaptations at Drosophila NMJs was the tight control of larval locomotor activity. Acute enhancement of larval locomotor activity has been achieved by transferring larvae from food vials onto agar plates, a procedure that has been used extensively before as a locomotor reference in the genetic analysis of larval foraging behavior. In addition, larval locomotor activity is persistently modified at different temperatures (18°C, 25°C, and 29°C) that are well within the natural temperature range of Drosophila development. Both paradigms enable the control of larval locomotor activity and therefore allow a time-resolved analysis of experience-dependent adaptations at developing NMJs of Drosophila (Sigrist, 2003).

Three independent lines of evidence suggest that the morphological and physiological changes at NMJs described in this study are triggered by enhanced larval locomotor activity and not caused by temperature treatment or plate transfer itself: (1) the considerable bouton outgrowth seen in wild-type larvae reared at 29°C was significantly suppressed in 29°C reared dglurIIA-ko mutants, which show defective postsynaptic signal transmission, rapid depression of spike train-evoked junctional signal transmission, reduced locomotor activity, and reduced subsynaptic protein synthesis; (2) exposing wild-type and para^ts1 larvae to permissive 22°C agar plates for 2-3 hr resulted in a significant and similar strengthening of junctional signal transmission in both genotypes -- strengthening was rapidly reversed by induced paralysis in para^ts1 animals; (3) wild-type larvae reared at 25°C and exposed to 25°C agar plates for up to 18 hr showed significantly enhanced locomotor activity, and they developed more boutons than comparable animals that remained in the food slurry. These experiments show that whenever synaptic signal transmission and larval locomotor activity was compromised, such as in dglurIIA or paralyzed para^ts1 mutants, the junctional phenotypes were strongly suppressed. It is therefore concluded that the acute and chronic exposure of Drosophila larvae to elevated temperatures or agar plates leads to an enhanced larval locomotor activity, which results initially in reversible physiological changes and later on in molecular and cellular adaptations that ensure persistently enhanced junctional signal transmission and efficient muscle contraction (Sigrist, 2003).

One of the first obvious consequences of enhanced locomotor activity was the fast enhancement of evoked junctional signal transmission, which was already maximal after 2-4 hr of locomotor stimulation on agar plates and was rapidly reversed by paralysis. The observation that the quantal sizes remained unaltered at the indicated time points of locomotor stimulation, whereas evoked junctional responses increased significantly, strongly suggests that the phases of experience-dependent strengthening of Drosophila NMJs are based primarily on an enhanced release of presynaptic vesicles per NMJ (Sigrist, 2003).

Mechanisms that can result in a fast increase in the number of released vesicles include an enhanced presynaptic Ca²⁺ influx, alterations in the Ca²⁺ sensitivity of the presynaptic release process, activation of presynaptic metabotropic glutamate receptors, or signaling mediated by the presynaptic G-protein-coupled receptor Methuselah. These mechanisms are typically involved in transient short-term enhancements of synaptic signal transmission. It appears likely that these or similar mechanisms are active during early phases of the experience-dependent junctional strengthening described in this study; however, their exact involvement as well as their temporal regulation remain to be investigated (Sigrist, 2003).

The number of released presynaptic vesicles can also increase at NMJs with a larger number of active release sites. Ultrastructural and morphological analysis of NMJs has revealed that animals that experienced persistently enhanced locomotor activity (rearing at 29°C) develop larger NMJs with an increased total number of T-bar-harboring active zones and an unaltered density of active zones per bouton. Because active zones represent synapses with a high probability of vesicle release, this mechanism could account for the observed strengthening of junctional signal transmission at larger NMJs. In fact, such a typical relationship between the number of active zones and the number of junctional boutons is readily apparent in the consistently observed correlation between junctional transmission strength and NMJ size. These data suggest that the fast-developing NMJs of Drosophila larvae consolidate induced functional changes by recruiting active zones and controlling their density by growing additional boutons. Recent experiments have shown that such NMJs not only transmit single stimuli more efficiently than control NMJs, they also show an enhanced faithfulness in the transmission of high-frequency stimuli (Sigrist, 2003).

It is intriguing to note that the scored electrophysiological parameters were indistinguishable among most locomotor-stimulated animals. This included larvae, which experienced 2-6 hr of locomotor stimulation. NMJs of these larvae showed no detectable bouton outgrowth compared with their controls, suggesting that this early phase of junctional strengthening does not rely on large-scale morphological alterations. It has been shown that dglurIIA-ko mutants are unable to greatly enlarge their NMJs by bouton addition. This mutant mediates enhanced presynaptic vesicle release by increasing the number of active zones; however, these additional active zones are squeezed into a smaller number of preexisting boutons compared with wild type. It is therefore tempting to speculate that within 2 hr of locomotor stimulation NMJs start to increase the number of active zones by de novo synaptogenesis and by rapidly recruiting presynaptic T-bars (dense bodies) onto a large reservoir of preexisting and T-bar-free synapses. In fact, such a fast recruitment of presynaptic dense bodies to synapses has been proposed for synapses in the fly visual system. It is therefore possible that experience-dependent strengthening of junctional signal transmission is mediated primarily by the functional recruitment of additional active zones, which are later distributed in newly grown boutons at their typical density. These processes would leave the efficacy of junctional signal transmission unchanged even during the morphological expansion of NMJs. Unfortunately, because of the current lack of probes that could specifically recognize T-bars or active zones in vivo or in light-microscopic preparations, it was not possible to address these potentially highly dynamic processes at larval NMJs (Sigrist, 2003).

The results show that enhanced locomotor activity results within 2 hr in a rapid and reversible enhancement of evoked junctional signal transmission and in a similarly fast stimulation of local subsynaptic protein synthesis. Although it remains to be investigated whether localized subsynaptic protein synthesis could play an instructive role during these early physiological events, it has been found that the mRNA encoding the glutamate receptor subunit DGluR-IIA is stored within the subsynaptic compartment of NMJs. It therefore represents a likely substrate of localized subsynaptic protein synthesis. It was found that DGluR-IIA-specific immunoreactivity increases visibly after 4 hr of locomotor stimulation, first in the form of ring-shaped accumulations at the edge of preexisting synapses and after chronic stimulation within typical postsynaptic patches. Given that several ionotropic neurotransmitter receptors perform lateral diffusion movements into and out of postsynaptic complexes, it appears likely that the ring-shaped DGluR-IIA accumulations described in this study similarly reflect a transient step in the maturation of postsynapses. Thus, experience-induced subsynaptic protein synthesis seems to instruct the DGluR-IIA-mediated functional maturation of postsynapses, which together with added presynaptic dense bodies mediates the observed increase in the total number of active zones. Finally, NMJs grow more boutons to reestablish the typical active zone density to consolidate the earlier induced physiological alterations. On the basis of this first temporal map of processes contributing to experience-dependent plasticity at Drosophila NMJs, future experiments will incorporate the behavioral assays introduced here to uncover the dynamic control of glutamatergic signal transmission (Sigrist, 2003).

Glutamate receptor dynamics organizing synapse formation in vivo

Insight into how glutamatergic synapses form in vivo is important for understanding developmental and experience-triggered changes of excitatory circuits. Postsynaptic densities (PSDs) expressing a functional, GFP-tagged glutamate receptor subunit (GluR-IIA^GFP) were imaged at neuromuscular junctions of Drosophila melanogaster larvae for several days in vivo. New PSDs, associated with functional and structural presynaptic markers, form independently of existing synapses and grow continuously until reaching a stable size within hours. Both in vivo photoactivation and photobleaching experiments show that extrasynaptic receptors derived from diffuse, cell-wide pools preferentially enter growing PSDs. After entering PSDs, receptors are largely immobilized. In comparison, other postsynaptic proteins tested (PSD-95, NCAM and PAK homologs) exchange faster and with no apparent preference for growing synapses. New glutamatergic synapses form de novo and not by partitioning processes from existing synapses, suggesting that the site-specific entry of particular glutamate receptor complexes directly controls the assembly of individual PSDs (Rasse, 2005).

Glutamate receptors localized within the PSD region transmit the excitatory responses in the brain and in many other neuronal systems. Thus, a detailed molecular and cell-biological insight into the formation of glutamatergic synapses is important for understanding of the development of excitatory neuronal circuits and for long-term information storage in the CNS. So far, formation of glutamatergic synapses has been studied mainly in cultivated brain neurons. These studies have been helpful, for example, in delineating a temporal sequence of pre- and postsynaptic assembly and characterizing mechanisms of glutamate receptor trafficking during synapse formation. How glutamatergic synapses assemble in vivo remains to be addressed. It is conceivable that synapse formation is more tightly controlled temporally and spatially in vivo than in vitro, particularly when synapses are added to strengthen already functional circuits. It is thus important to follow 'the entire history' of identified synapses over time in the intact organism while monitoring their molecular dynamics and functional features. Analysis of synapse formation in vivo might profit from the use of synaptic models that are optically and genetically highly accessible. The Drosophila neuromuscular junction (NMJ) is a well established glutamatergic model, widely used for functional genetic descriptions of principle glutamatergic transmission. A mature NMJ comprises a few hundred individual synapses, which are ultrastructurally similar to central mammalian synapses and express glutamate receptor subunits (GluR-IIA, GluR-IIB, GluR-IIC, GluR-IID, GluR-IIE) related to mammalian non-NMDA type glutamate receptors. NMJs comprised of motor neurons and somatic muscles form during late embryonic development in Drosophila. The number of individual synaptic sites per NMJ increases throughout subsequent larval development. Thus, the NMJ should be a suitable model for studying the new formation of glutamatergic synapses within a functional circuit (Rasse, 2005).

Functional, GFP-labeled glutamate receptors (GluR-IIA^GFP) have been imaged during the formation of new NMJ synapses in vivo. New small receptor fields form de novo and not by discrete partitioning events. These new small receptor fields correspond to the PSD region of functional synapses, as they are tightly associated with both independent PSD markers and functional (styryl dye labeling) and molecular (active zone components, calcium channels) markers of presynaptic active sites. Small PSDs grow for many hours before finally stabilizing at a mature size. Fluorescence recovery after photobleaching (FRAP) and photoactivation experiments indicate that the incorporation of GluR-IIA-containing glutamate receptor complexes from extrasynaptic pools is directly instructive for PSD formation and growth and, as a result, synapse formation (Rasse, 2005).

This study investigated in fully native settings how glutamatergic synapses form and how glutamate receptor dynamics are organized during this process. The transparent nature of Drosophila larvae make them an ideal subject in which to examine the glutamatergic synapses forming at the developing larval NMJ. To label glutamate receptors for in vivo imaging, enhanced GFP (EGFP) was inserted into the middle of the intracellular C terminus of GluR-IIA. GluR-IIA^GFP was then expressed from a genomic transgene. In Western blots probed with antibodies to GFP, GluR-IIA^GFP was detected at the predicted 140 kDa in extracts of GluR-IIA^GFP transgenic embryos. Next, the subcellular distribution of GluR-IIA^GFP was evaluated by immunofluorescent staining of Drosophila NMJs. In such stainings, GluR-IIA is known to label individual PSDs. Likewise, GluR-IIA^GFP expression was confined to individual PSDs. GluR-IIA^GFP strictly colocalizes with p21/rac1-activated kinase (PAK), an established PSD marker, and with endogenous GluR-IIA. Furthermore, GluR-IIA^GFP patches are surrounded by the typical perisynaptic expression of the neural cell adhesion molecule (NCAM) homolog fasciclin II (FasII) In the GluRIIA and GluRIIB double-mutant background, the transgenic expression of GluR-IIA mediated by either GluR-IIA^GFP or GluR-IIA is indistinguishable, and it is similar to the level of GluR-IIA expression found at wild-type NMJs. Also, GluR-IIA and GluR-IIA^GFP colocalize with GluR-IIC. The GluR-IIC subunit is essential for NMJ neurotransmission, probably by acting as an obligate binding partner of GluR-IIA for forming functional channels. In short, GluR-IIA^GFP is expressed at physiological levels, and individual receptor fields correspond to individual PSDs. From this point on, receptor fields identified by means of GluR-IIA^GFP will be referred to as PSDs (Rasse, 2005).

Several key findings were made. (1) New small glutamatergic PSDs form separately from existing synapses and then grow to a mature size. Mature PSDs are discrete entities that seem to be stable in size and shape over periods of days. (2) Growing receptor fields invariably associate with a presynaptic active zone during further outgrowth. The coordination between pre- and postsynaptic assembly seems to be very tight. In fact, 99.5% of all GluR-IIA spots older than 10 h were associated with active zone markers, and, vice versa, no active zone accumulations were observed without GluR-IIA accumulation. In contrast, not all younger GluR-IIA spots are associated with a corresponding active zone. It was somewhat surprising, given the studies on cultured mammalian neurons, to find that receptor field/PSD assembly might precede certain aspects of presynaptic active zone assembly and maturation in this system. It will be interesting to address whether glutamatergic synapse types differ in this regard, or whether initial synaptogenesis in culture differs intriniscally from the mode chosen for adding synapses to an already functional circuitry (Rasse, 2005).

(3) New synapses form de novo, whereas split-like redistributions of glutamate receptors from existing synapses into new synapses do not seem to contribute to the formation of new synapses. This is important, because splitting events at the glutamatergic PSDs of mammalian CNS synapses have been postulated but are controversial. (4) Two complementary strategies for in vivo photolabeling of glutamate receptors (FRAP and photoactivation) allow a comparison of glutamate receptor dynamics at growing and stable PSDs. Both approaches show that GluR-IIA preferentially enters growing PSDs from diffuse extrasynaptic pools. This entry is directly correlated with PSD growth. Once glutamatergic PSDs have reached a certain size, they stabilize, and their glutamate receptor population becomes largely immobilized. In contrast, other postsynaptic transmembrane proteins (for example, FasII) and scaffolding proteins such as the SAP-97/PSD-95 homolog Dlg show equally high dynamics over all synapses. Previously, genetic experiments showed that more synapses form per NMJ when the level of GluR-IIA expression is increased. Vice versa, the reduction of the GluR-IIA level by one gene dose inhibits the NMJ from producing additional synapses when genetically or behaviorally challenged. Thus, the entry of glutamate receptors into PSDs (but not the entry of the other postsynaptic proteins investigated) might directly control the growth of the postsynaptic specialization and thereby the growth of synapses. Notably, local translation of GluR-IIA has been suggested to promote activity-dependent synapse formation in this system. It will be interesting to further test the role of GluR-IIA as a potential organizer of postsynaptic assembly by combining in vivo imaging and functional genetics at the Drosophila NMJ. However, it is well established in the mammalian brain that entry of specific AMPA-type glutamate receptor complexes into preformed synapses can control synapse efficacy over shorter time periods. It will be interesting to investigate whether glutamate receptor entry can control the formation of mammalian glutamatergic synapses under certain circumstances (Rasse, 2005).

In addressing the origin of receptors integrated into growing synapses, no evidence was obtained for internal stores of glutamate receptors. It will thus be interesting to determine the local cues and signals at PSDs that specifically control GluR-IIA entry and immobilization during PSD growth. It is suspected that GluR-IIA populations residing in the extrasynaptic muscle plasma membrane support PSD growth. Electrophysiology has, in fact, demonstrated the existence of glutamate receptors in the extrasynaptic membrane of Drosophila muscles. Such receptors might diffuse laterally in the membrane partitioning in and out of synapses, where they likely have a low residence time. This pool of unbound receptors might be fundamentally different from a second pool of receptors, which is 'trapped' in PSDs, as demonstrated recently by tracking individual glutamate receptor complexes in cultured mammalian neurons (Rasse, 2005).

During the few days of larval development, the synaptic current per Drosophila NMJ increases by nearly two orders of magnitude to keep pace with the growing postsynaptic muscle cell. The data imply that GluRIIA-containing glutamate receptors seem to become particularly 'invested' the formation and subsequent outgrowth of new synapses, but less so in the enlargement of preexisting synapses. At average NMJ synapses, it is likely that glutamate receptors are not saturated for glutamate during vesicle exocytosis. It is speculated that at small, newly forming synapses, a higher proportion of glutamate receptors might be activated during glutamate release because of the smaller physical distance from the exact position of the presynaptic release site. To achieve an increase in the overall NMJ current, it therefore might be more efficient to insert new receptors into these small synapses than to insert them into the large receptor fields of mature size synapses, which are unlikely to be saturated upon presynaptic stimulation. Thereby, the 'drive' of the Drosophila NMJ toward increasing overall transmission strength might be the deeper reason behind strongly stabilizing receptors once they are integrated (Rasse, 2005).

Examining the dynamic molecular composition of glutamatergic synapses in more detail by further applying both in vivo imaging and the powerful genetics of Drosophila should help clarify principal rules for assembly and remodeling of glutamatergic synapses. Notably, at the Drosophila NMJ, activity-dependent formation of additional synapses has been described on the basis of experience-dependent and genetically evoked processes (Rasse, 2005).

Non-NMDA-type glutamate receptors are essential for maturation but not for initial assembly of synapses at Drosophila neuromuscular junctions

The assembly of glutamatergic postsynaptic densities (PSDs) seems to involve the gradual recruitment of molecular components from diffuse cellular pools. Whether the glutamate receptors themselves are needed to instruct the structural and molecular assembly of the PSD has hardly been addressed. This study engineered Drosophila neuromuscular junctions (NMJs) to express none or only drastically reduced amounts of their postsynaptic non-NMDA-type glutamate receptors. At such NMJs, principal synapse formation proceeded and presynaptic active zones showed normal composition and ultrastructure as well as proper glutamate release. At the postsynaptic site, initial steps of molecular and structural assembly took place as well. However, growth of the nascent PSDs to mature size was inhibited, and proteins normally excluded from PSD membranes remained at these apparently immature sites. Intriguingly, synaptic transmission as well as glutamate binding to glutamate receptors appeared dispensable for synapse maturation. Thus, these data suggest that incorporation of non-NMDA-type glutamate receptors and likely their protein-protein interactions with additional PSD components triggers a conversion from an initial to a mature stage of PSD assembly (Schmid, 2006).

A detailed molecular and cell biological insight into the formation of glutamatergic synapses is important for understanding the development of excitatory neuronal circuits and also the process of long-term information storage in the CNS. So far, studies on cultivated brain neurons analyzed mechanisms of glutamate receptor trafficking during synapse formation and have suggested a temporal sequence of presynaptic and postsynaptic assembly. However, whether in turn the process of incorporating glutamate receptors is needed for the establishment of synaptic structures has hardly been addressed (Schmid, 2006).

The relationship between neurotransmitter receptor incorporation and synapse assembly was addressed by genetically reducing or eliminating the expression of all neurotransmitter receptors at a certain synapse type. Consequences of eliminating all postsynaptic glutamate receptors expressed at a specific glutamatergic synapse has so far not been described. This study showed that a lack of glutamate receptors provoked a specific block in the molecular and ultrastructural maturation of PSDs (Schmid, 2006).

Notably, loss of transmission concomitant with losing glutamate receptor complexes seemed not involved, based on the fact that neither blocking synaptic transmission nor affecting glutamate binding by site-directed mutagenesis did provoke similar defects. Thus, consistent with studies in other synaptic systems, ionic transmission through the postsynaptic neurotransmitter receptors does not appear essential for principal synapse assembly. Instead, the data clearly imply that a critical level of glutamate receptor protein is needed to allow synapse maturation (Schmid, 2006).

A model for the maturation of individual NMJ synapses in either the presence or absence of postsynaptic glutamate receptors is presented in this study. At glutamate receptor-deprived synapses, synaptic vesicles appeared normally distributed, and their activity-mediated release appeared increased, likely as part of a compensation for reduced postsynaptic sensitivity. Moreover, functional active zones with presynaptic dense bodies still formed. Thus, active zones still assemble when the mature organization of synaptic membranes ('tight planar apposition') is not established. Consistently, previous work had shown that the formation of presynaptic dense bodies persisted even after genetic elimination of postsynaptic muscle cells. In contrast, active zone formation is severely affected in bruchpilot mutants, whereas the presynaptic and postsynaptic membranes remain tightly apposed (Schmid, 2006).

At developing NMJs, newly forming 'nascent' PSDs are characterized by small GluRIIA accumulations strictly colocalized with PAK kinase. Even in the complete absence of glutamate receptors (gluRIIC single or gluRIIA&IIB double mutant), postsynaptic PAK patches, as typical for small nascent synapses, still formed, indicating that principal cues for the definition of postsynaptic membrane patches persisted in this situation. However, these PAK patches consistently failed to reach mature size. PAK, which mediates effects of Rho-GEF dPIX has been implicated in postsynaptic maturation, with PAK mutants showing a partial depletion of GluRIIA, and reduced SSR formation. However, neither pak nor dpix mutants have so far been reported to show defects in synaptic membrane apposition. Thus, postsynaptic differentiation is not completely blocked in the absence of glutamate receptors. Instead, two postsynaptic 'assembly modules' (PAK/dPIX signaling and glutamate receptor localization) appear only partly dependent on each other, with glutamate receptor localization being essential for PSD maturation but not for initial PSD assembly (Schmid, 2006).

At the cholinergic mouse NMJ, genetic deletion of the adult acetylcholine receptor subunit leads to severely reduced AChR density. Notably, a profound reorganization of AChR-associated components of the postsynaptic membrane and cytoskeleton has been observed in this situation (Schmid, 2006).

Synaptic membranes are electron dense and apposed to each other, leaving a cleft of consistent width, likely essential for robust timing and efficacy of neurotransmission. In contrast, perisynaptic membranes are less electron dense and tend to undulate. At NMJs lacking glutamate receptors, FasII/Dlg complexes ectopically remained at synaptic sites and membranes now appeared undulated, indicating perisynaptic type of membrane adhesion. Thus, glutamate receptors seem essential to establish the type of membrane adhesion found at the synapse, whereas usually perisynaptic adhesion molecules as FasII mediate a qualitatively different type of membrane adhesion. Notably, undulation of perisynaptic membranes was impaired at NMJs lacking glutamate receptors leading to a less developed SSR. Moreover, boutons often appeared atypically round, further indicating that membrane–membrane adhesion is fundamentally affected at NMJ terminals lacking glutamate receptors (Schmid, 2006).

Several classes of bona fide cell adhesion molecules (CAMs) have been implicated in mediating membrane adhesion at synapses, particularly transsynaptic neurexin–neuroligin pairs and cadherins. The specific contributions of these synaptic CAMs during initial synapse assembly and maturation are under intense investigation. The data are consistent with the idea that the C-terminal, intracellular domains of glutamate receptors might engage in interactions with other PSD components, which in turn cluster postsynaptic CAM-type membrane proteins. These would then mediate interactions to cluster presynaptic CAMs or bind components of the extracellular matrix to allow synaptic membrane apposition. Alternatively, direct interactions of glutamate receptors with other membrane protein complexes, as recently demonstrated for Stargazins/TARPs (transmembrane AMPA receptor regulatory proteins), might be involved (Schmid, 2006).

No CAM single mutant has so far been reported to provoke a defect in synaptic membrane apposition as severe as the one observed in this study for glutamate receptor mutant situations. Thus, multivalent interactions of the heterotetrameric glutamate receptor complexes as well as the redundant involvement of several CAM species might occur (Schmid, 2006).

PAK labeling suggested that initial steps in defining postsynaptic membranes persisted even in the total absence of glutamate receptors, whereas these initial assemblies could not mature on the ultrastructural level when glutamate receptors were lacking. in vivo imaging has been achieved of photolabeled GluRIIA at the developing NMJ, and it was found that newly forming PSDs in fact grow by a continuous incorporation of glutamate receptors, whereby the accumulation of presynaptic active zone material (BRP) appeared slightly delayed. Thereby, the entry of GluRIIA, likely derived from cell-wide plasma membrane pools via lateral diffusion, directly correlated with PSD growth. Once glutamatergic PSDs reached a certain size, they stabilized and GluRIIA was essentially immobilized. In comparison, other postsynaptic proteins showed high turnover equally over all synapses. This slow turnover of glutamate receptors is consistent with the view that multiple interactions of glutamate receptor set the core of a transsynaptic interaction matrix. Several lines of genetic and experience-dependent manipulations point toward a rate-limiting role of GluRIIA levels in NMJ synapse formation. In summary, the available data suggest that incorporation of glutamate receptors might be a key event to allow additional expansion of initial postsynaptic assemblies, finally leading to mature PSDs. Thereby, the overall level of glutamate receptors available in the muscle membrane might control the total number of synapses forming per NMJ (Schmid, 2006).

Understanding the plasticity processes taking place at glutamatergic synapses has been a focus of attention within cellular neuroscience. Hereby, rapid changes in synaptic receptor number were reported to mediate plastic changes of synaptic transmission, often on the timescale of tens of minutes in mammalian preparations. Notably, however, a recent study indicated that the cycling of synaptic glutamate receptors needed 16 h or more. Similar timing was observed for nicotinic acetylcholine and GABA receptors. Thus, parts of the synaptic glutamate receptor population might be needed to reside stably within the PSD to maintain synapse stability. In fact, only severe receptor deprivation interfered with proper postsynaptic assembly at the NMJ, suggesting that the glutamate receptor level should not fall below a certain critical threshold (Schmid, 2006).

Notably, the extracellular domain of the mammalian AMPA receptor subunit GluR2 has been shown to increase the size and density of spines in hippocampal neurons, and to induce spine formation in GABAergic interneurons normally lacking spines. It will be interesting to see whether these structural roles of glutamate receptors have a common mechanistic denominator (Schmid, 2006).

Different types of synapses differ strongly in the ultrastructural detail of their postsynaptic specializations. Thus, a typical brain neuron, acting as a postsynaptic partner for different types of presynaptic inputs, has to establish and maintain different postsynaptic architectures, suggesting the existence of 'identity molecules' allowing the self-assembly of such architectures, and potentially a match with membrane cues of the presynaptic partner cell. Obvious candidates for such molecules are the postsynaptic neurotransmitter receptors themselves. This study is consistent with such a view (Schmid, 2006).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D

The Interactive Fly resides on the
Society for Developmental Biology's Web server.