Comparison of quantal size at synapses expressing one or the other receptor reveals that DGluRIIA-expressing synapses exhibit a significantly larger response to transmitter than DGluRIIB-expressing synapses. In addition to the difference in amplitude, there is also a difference in the kinetics of the synaptic potentials. The time constant of the miniature extrajunctional potential (mEJP) decay is significantly shorter in DGluRIIB expressing larvae than in DGluRIIA-expressing larvae (21.6 ± 0.6 msec and 32.9 ± 1.2 msec) (DiAntonio, 1999).

The data above suggest that the ratio of receptor subunits at the wild-type synapse could regulate quantal size. A larger proportion of DGluRIIA would increase quantal size, whereas more DGluRIIB would decrease quantal size. When DGluRIIA is overexpressed in a wild-type background, there is a significant increase in quantal size (Petersen, 1997). However, this result is equally consistent with quantal size being regulated by receptor subunit composition or receptor density. To distinguish between these two possibilities, DGluRIIB was overexpressed in a wild-type background. A late driver, MHC Gal4, initiates expression in the first larval instar after endogenous receptor expression has begun, and an early driver, 24B Gal4, initiates expression in myoblasts. In both cases, there is a significant decrease in quantal size. Late expression of DGluRIIB leads to a 44% reduction in mEJP amplitude, whereas earlier expression produces a 68% decrease. A similar change in mEJP amplitude is seen when DGluRIIB is directly overexpressed from the myosin heavy chain promoter. Despite the likely increase in receptor density caused by overexpression, quantal size fell because of a change in the relative abundance of receptor subtype (DiAntonio, 1999).

Although receptor subunit composition is a primary determinant of quantal size, the data suggest that receptor density may also regulate postsynaptic sensitivity to single quantum. When the double mutant is rescued with increasing gene dosages of DGluRIIA, there is a significant increase in quantal size. There is an 18% increase from one to two genomic copies of A and a further 20% increase from two genomic copies to gross overexpression of the cDNA. Because no DGluRIIB is expressed in any of these genotypes, these results cannot be explained by a change in subunit composition between these two receptors, although the existence of a third receptor that may function at this synapse cannot be ruled out. Similarly, there is a 24% increase in quantal size when the gene dosage of DGluRIIB is doubled while rescuing the null mutant. However, there is no further increase in mEJP amplitude when the DGluRIIB cDNA is overexpressed. These data are consistent with a model in which receptor density is a determinant of quantal size (DiAntonio, 1999).

Since quantal size is reduced in the absence of DGluRIIA and quantal size is increased when DGluRIIA is overexpressed (Petersen, 1997) it has been suggested that receptor density is a primary determinant of postsynaptic responsiveness. However, these observations are equally consistent with a model in which the relative level of DGluRIIA regulates quantal size, with a higher proportion of DGluRIIA favoring a larger postsynaptic response. This second model is supported by the data. Regardless of the level of expression, synapses lacking DGluRIIB have a large quantal size, and synapses lacking DGluRIIA have a small quantal size. In fact, overexpression of the DGluRIIB subunit at a wild-type synapse leads to a dose-dependent decrease in quantal size. In this case, the receptor density should be increasing, but the quantal size is decreasing. This is most easily explained if the primary determinant of quantal size at this synapse is the relative abundance of each receptor subtype (DiAntonio, 1999).

Although subunit composition is the primary factor controlling postsynaptic responsiveness, the data does suggest that receptor density may also regulate quantal size. In the absence of the DGluRIIB subunit, increasing the gene dosage of DGluRIIA increases the quantal size. However, the alternate explanation, that the subunit composition is changing between DGluRIIA and an unidentified third receptor, cannot be excluded (DiAntonio, 1999).

How might the cell exploit the differences in receptor function to regulate synaptic strength? (1) The two receptors could be differentially expressed. During embryonic development, DGluRIIB is initially expressed at a high level and then declines, whereas DGluRIIA expression slowly rises throughout embryogenesis (Petersen, 1997). Such a mechanism is used at the vertebrate NMJ in the switch from a fetal to adult acetylcholine receptor subunit. (2) The two receptors could be differentially regulated by second messengers. Davis (1998) has demonstrated that activation of PKA decreases the quantal size at the Drosophila NMJ and that this modulation requires the presence of DGluRIIA. Similar subunit-specific modulation has been seen for numerous vertebrate transmitter receptors (DiAntonio, 1999).

Does the cell use these postsynaptic mechanisms to regulate synaptic strength? When a Drosophila muscle is hypoinnervated, it compensates with an increase in quantal size (Davis, 1998a). It is suggested that this increase in postsynaptic sensitivity may reflect an increase in the proportion of DGluRIIA at the synapse or a decrease in the PKA-dependent modulation of DGluRIIA (DiAntonio, 1999).

To investigate the underlying biophysical basis for the observed differences in quantal properties, a single-channel analysis of the two receptor subunits was undertaken. Outside-out patches were isolated from extrajunctional regions of muscle 6 of wild-type third instar, as well as DGluRIIA&B^SP22 mutant larvae rescued with either DGluRIIA or DGluRIIB. The patches were held at minus 60 mV, and 10 mM glutamate was applied with a rapid application system. In response to glutamate, the channels open rapidly, flicker between open and closed states, and desensitize in the continued presence of glutamate. There is no significant difference in single-channel current amplitudes in the three genotypes. Their single-channel conductance is very similar to what has been observed previously for wild-type channels from larvae (Heckmann, 1995) and embryos (Broadie, 1993; Nishikawa, 1995). There is, however, a marked difference in the time course of desensitization. When fit with an exponential function, the time constant of decay is 18 msec for channels from wild-type larvae; 19 msec from larvae expressing DGluRIIA, and 2.0 msec for channels from larvae expressing DGluRIIB (DiAntonio, 1999).

Because channels from wild-type patches that desensitize as quickly as the DGluRIIB channels (Heckmann, 1997 and DiAntonio, 1999) have not been observed, DGluRIIB homomultimers must be quite rare in a wild-type cell. The channels analyzed were extrajunctional; however, there is no evidence for a difference in the time course of patch and quantal currents (Heckmann, 1998). Therefore, this difference in the time course of desensitization seen with single channels may explain some of the differences in quantal amplitude and time course seen at synapses in larvae rescued with either DGluRIIA or DGluRIIB (DiAntonio, 1999).

In DGluRIIA mutants, the amplitude of evoked synaptic events remains normal despite a large decrease in quantal size because of a compensatory increase in quantal content; that is, the number of vesicles released by the nerve (Petersen, 1997). These data have been taken as evidence for a retrograde signal linking postsynaptic activity with presynaptic transmitter release properties. Does a similar form of retrograde signaling occur at synapses mutant for DGluRIIB? At synapses lacking DGluRIIB, the quantal size is near wild-type levels. To assess the relationship between quantal size and quantal content over a wide range of values, the double mutant was rescued with a transgenic UAS DGluRIIA cDNA driven by a Gal4 line (H94) that gives quite variable levels of expression. Recordings of spontaneous miniature junctional potentials and evoked excitatory junctional potentials were made from muscle 6, segment A3 of third instar larvae, and quantal content was estimated by dividing the mean EJP amplitude by the mean mEJP amplitude. There is a significant difference in quantal content when cells were grouped by quantal size; cells with the smallest quantal size tend to have the largest quantal content. This suggests that, at synapses lacking DGluRIIB, changes in postsynaptic activity are compensated for by regulating presynaptic transmitter release. In this genotype, the amplitude of the evoked events is significantly larger than in wild type (25.1 ± 1.4 mV and 15.4 ± 2.0 mV respectively). It is argued in this paper that the compensatory mechanism involves an increase in quantal content, or the number of residues released by the nerve (Di Antonio, 1999).

To assess in a more quantitative manner the relationship between gene dosage of DGluRIIA and quantal content, the synaptic response was compared in 0.3 mM external calcium at the wild-type synapse and in the double mutant rescued with one genomic DGluRIIA transgene, two genomic DGluRIIA transgenes, or by overexpression of the DGluRIIA cDNA. As would be expected from the results above, the single genomic DGluRIIA, with the smallest mean quantal size, gave the largest quantal content. The single DGluRIIA shows a significant increase in quantal content, when compared with wild type (237%); two copies of genomic DGluRIIA have a smaller increase (180%), and overexpression of DGluRIIA has no change in quantal content (114%) (DiAntonio, 1999).

Whereas the inverse relationship between the gene dosage of DGluRIIA and quantal content was expected, the magnitude of the change in quantal content was a surprise. Although the null mutant rescued with a single genomic DGluRIIA transgene does have a slightly smaller quantal size than wild type (0.97 ± 0.06 vs 1.19 ± 0.07 mV), the increase in quantal content more than compensates for this postsynaptic deficit. As a result, the postsynaptic response to nerve stimulation is significantly increased (182%; p < 0.05). As the gene dosage of DGluRIIA (and hence the response to a single vesicle) is increased, the response to nerve stimulation decreases because quantal content is no longer upregulated. This result, in addition to the increase seen in the EJP amplitude in the H94-DGluRIIA rescued larvae described above, suggests that the mechanism monitoring postsynaptic activity and regulating presynaptic transmitter release is not directly sensitive to depolarization of the muscle. Thus when low levels of DGluRIIA are expressed postsynaptically, there is a large increase in presynaptic transmitter release that overcompensates for the decrease in postsynaptic sensitivity to transmitter (DiAntonio, 1999).

Activation of ionotropic glutamate receptors leads to the generation of two types of signals. The postsynaptic cell is depolarized by the influx of cations through the open channel, and second messenger systems can be activated through either the influx of calcium or the interaction of the receptor with other signaling molecules. The overcompensation of quantal content seen in the single genomic DGluRIIA rescue suggests that depolarization is not the determinant being sensed in the postsynaptic cell. In fact, this result could suggest that the retrograde signal is not even sensitive to the activity of the channel but instead is measuring the amount of channel present. To distinguish between the activity and amount of postsynaptic receptor, a dominant negative mutant of DGluRIIA was generated. Using site-directed mutagenesis, a single residue in the channel pore M614 to an R. The analogous mutation in homologous vertebrate channels is thought to coassemble with wild-type receptors and produce nonfunctional channels. Transgenic flies were generated carrying the M/R mutant cloned downstream of the UAS promoter. Expression of two copies of this transgene in a wild-type background driven by the strong mesodermal promoter 24B Gal4 is lethal. Driving expression of a single copy of the mutant with 24B Gal4 produces viable adults with no obvious behavioral abnormalities. Staining of the larval neuromuscular junction shows that this mutant receptor does localize to the synapse. As with overexpression of the wild-type receptor, however, much of the transgenic receptor is present extrasynaptically. Recordings of spontaneous excitatory junctional potentials reveal that expression of this mutant receptor leads to a dramatic decrease in quantal size (1.01 ± 0.05 vs 0.33 ± 0.02 mV). Hence, this pore mutant acts as a dominant negative receptor in vivo. Analysis of evoked synaptic potentials revealed no significant change, indicative of a large increase in quantal content in the mutant. These data do not support the model that a low channel density is the signal controlling the retrograde regulation of presynaptic transmitter release. Normal levels of the endogenous DGluRIIA and DGluRIIB receptors are expressed in addition to the transgenic expression of a DGluRIIA pore mutant, and yet quantal content is upregulated. Although the possibility that the mutant channel could disrupt localization of the endogenous receptors to the synapse cannot be ruled out, the model that it acts as a dominant negative by disrupting the pore of the channel is favored. Therefore, these data imply that the activity of the channel and ion flux through the pore are the initiating events for the measurement of postsynaptic activity and the regulation of presynaptic function. These data lead to the simple model of a homeostatic mechanism in which a muscle-to-motoneuron signal regulates presynaptic release to ensure appropriate depolarization of the muscle. Similar compensation may occur at the vertebrate and crayfish NMJ and at central excitatory and inhibitory synapses. Such a mechanism could function during development to match the release capacity of the nerve to the ever growing requirements of the muscle (DiAntonio, 1999).

In this study a transgenic Ca2+ imaging technique was established in Drosophila that enabled the Ca2+ sensor protein yellow Cameleon-2 to be targeted specifically to larval neurons. This noninvasive method allows the measurement of evoked Ca2+ signals in presynaptic terminals of larval neuromuscular junctions (NMJs). Transgenic Ca2+ imaging was combined with electrophysiological recordings and morphological examinations of larval NMJs to analyze the mechanisms underlying persistently enhanced evoked vesicle release in two independent mutants. Persistent strengthening of junctional vesicle release relies on the recruitment of additional active zones, the spacing of which correlates with the evoked presynaptic Ca2+ dynamics of individual presynaptic terminals. Knock-out mutants of the postsynaptic glutamate receptor (GluR) subunit DGluR-IIA, which showed a reduced quantal size, develop NMJs with a smaller number of presynaptic boutons but a strong compensatory increase in the density of active zones. This results in an increased evoked vesicle release on single action potentials and larger evoked Ca2+ signals within individual boutons; however, the transmission of higher frequency stimuli is strongly depressed. A second mutant, pabpP970/+ in a gene coding for a [poly(A)-binding protein (pabp)], shows genetically elevated subsynaptic protein synthesis, which shows unaltered quantal size but strongly increased eEJCs caused by enhanced evoked vesicle release. pabpP970/+ showed enhanced evoked vesicle release triggered by elevated subsynaptic protein synthesis, developed NMJs with an increased number of presynaptic boutons and active zones; however, the density of active zones is maintained at a value typical for wild-type animals. This resulted in wild-type evoked Ca2+ signals but persistently strengthened junctional signal transmission. These data suggest that the consolidation of strengthened signal transmission relies not only on the recruitment of active zones but also on their equal distribution in newly grown boutons (Reiff, 2002).

This study addresses the question of how NMJs of Drosophila larvae achieve the continuous enhancement of evoked vesicle release seen throughout their development and during activity-dependent strengthening. Using wild-type animals and two independent mutants that genetically represent both phases of junctional strengthening, it was found that Ca2+-dependent presynaptic mechanisms, which are known to result in fast and reversible modifications of presynaptic vesicle release, may provide only a minor or transient contribution to enhanced vesicle release during the development and long-term strengthening of junctional signal transmission. Instead, a persistent enhancement of vesicle release relies primarily on the recruitment of active zones. This conclusion is further supported by yCam2-based Ca2+ imaging results, which together with ultrastructural data reveal that evoked presynaptic Ca2+ signals correlate with the density of active zones. The data therefore suggest that enhanced vesicle release is realized by a differential regulation of active zone density in different genotypes: NMJs of dglurIIA-ko mutants compensate for their postsynaptic defect by packing more active zones into preexisting boutons. This leads to a functional compensation, which approaches homeostasis of evoked junctional signal transmission compared with wild type presumably to ensure muscle contraction and animal survival. In contrast, enhanced junctional signal transmission as seen in elav-Campabp animals is mediated by distributing added active zones into newly grown boutons. This leads to homeostasis of active zone density compared with wild-type controls and therefore may reflect the cellular basis of strengthened junctional signal transmission at Drosophila NMJs (Reiff, 2002).

Previous ultrastructural observations from other Drosophila mutants and larvae of the flesh fly Sarcophaga bullata have already suggested that the density of active zones is tightly regulated, presumably to ensure that individual synapses have sufficient access to reserve pool vesicles, vesicle recycling machinery, efficient Ca2+-buffering systems, or neurotransmitter uptake mechanisms, for example. Data from wild-type and elav-Campabp animals show a similar active zone density and evoked Ca2+ signals per bouton and thus suggest that individual boutons represent functional compartments that are likely to be maintained constant during junctional development and its strengthening. This seems to guarantee uncompromised signal transmission on a single bouton level. From these observations a model emerged that predicts that additional active zones need to be distributed in newly grown boutons. This would explain the increasing number of genotypes that show a strict relationship between bouton number and transmission strength. Intriguingly, in several other systems the recruitment of active synapses has been observed, as well as local morphological alterations of synaptic compartments; both are thought to represent long-lasting changes in the strength of synaptic communication (Reiff, 2002).

On the basis of the above considerations, it appears surprising that DGluR-IIA-ko mutants pack the additional active zones in a smaller number of presynaptic boutons. This results in an increased density of active zones, a larger stimulation-evoked Ca2+ entry per bouton, an enhanced evoked vesicle release, and a wild-type muscle depolarization on single action potentials. These phenotypes show that mutants with impaired postsynaptic glutamate receptor function are capable of efficiently triggering the recruitment of active zones to compensate for the mutationally induced postsynaptic defect. However, this recruitment fails to induce the proportional outgrowth of new boutons that can be observed at wild-type NMJs and several other genotypes. Indeed, a recent analysis of the role of DGluR-IIA subunits in junctional development revealed that the increased expression of DGluR-IIA is sufficient to induce bouton outgrowth. Although it is currently not clear why DGluR-IIA-ko mutants accumulate active zones at such an unusual density, it appears that this mechanism alone is not sufficient to ensure uncompromised repetitive signal transmission. Although the latter may be attributable to increased postsynaptic desensitization in this mutant, presynaptic factors like the depletion of the readily releasable vesicle pool also appear likely to contribute to this observation. It is therefore tempting to speculate that this mutant is trapped in a transient phase of junctional strengthening (Reiff, 2002).

The developing neuromuscular junctions (NMJs) of Drosophila larvae can undergo long-term strengthening of signal transmission, a process that has been shown recently to involve local subsynaptic protein synthesis and that is associated with an elevated synaptic accumulation of the postsynaptic glutamate receptor subunit DGluR-IIA. To analyze the role of altered postsynaptic glutamate receptor expression during this form of genetically induced junctional plasticity, the expression levels of two so far-described postsynaptic receptor subunit genes, dglur-IIA and dglur-IIB, were manipulated in wild-type animals and plasticity mutants. Elevated synaptic expression of DGluR-IIA, which was achieved by direct transgenic overexpression, by genetically increased subsynaptic protein synthesis, or by a reduced dglur-IIB gene copy number, results in an increased recruitment of active zones, a corresponding enhancement in the strength of junctional signal transmission, and a correlated addition of boutons to the NMJ. Ultrastructural evidence demonstrates that active zones appear throughout NMJs at a typical density regardless of genotype, suggesting that the space requirements of active zones are responsible for the homogeneous synapse distribution and that this regulation results in the observed growth of additional boutons at strengthened NMJs. These phenotypes were suppressed by reduced or eliminated DGluR-IIA expression, which resulted from either a reduced dglur-IIA gene copy number or transgenic overexpression of DGluR-IIB. These results demonstrate that persistent alterations of neuronal activity and subsynaptic translation result in an elevated synaptic accumulation of DGluR-IIA, which mediates the observed functional strengthening and morphological growth apparently through the recruitment of additional active zones (Sigrist, 2002).

The focus of this study was the question of how local subsynaptic protein synthesis can regulate the transmission strength and the morphological development of larval neuromuscular junctions of Drosophila. The increased synaptic accumulation of the glutamate receptor subunit DGluR-IIA alone is responsible for the functional recruitment of additional synapses within a given NMJ. This was evident in an apparent increase in the number of DGluR-IIA-expressing postsynapses on transgenic overexpression of DGluR-IIA, an increased total number of T-bar-harboring release sites per NMJ, an increased frequency of spontaneous vesicle fusion events, and significantly larger junctional responses on nerve stimulation (eEJCs) compared with control animals. These physiological changes, which have been evoked solely by manipulating the expression level of DGluR-IIA, were indistinguishable from those seen in animals with genetically increased subsynaptic translation, and they were suppressed in the latter genotypes by reducing the dglur-IIA gene doses. These observations suggest that most if not all of the physiological effects of subsynaptic protein synthesis at NMJs are mediated by increased synaptic expression of the glutamate receptor subunit DGluR-IIA. Because subsynaptically stored mRNAs encoding DGluR-IIA represent a likely substrate of synaptic translation, the local synthesis of DGluR-IIA subunits and their subsequent synaptic delivery could therefore provide the means for a site-specific functional recruitment of synapses and thus for local alterations of glutamatergic signal transmission (Sigrist, 2002).

Interestingly, it was found that the synaptic expression level of DGluR-IIA and its associated physiological phenotypes are inversely related to the expression of the glutamate receptor subunit DGluR-IIB: a reduced dglur-IIB gene copy number results in a significant increase of synaptic DGluR-IIA accumulation, whereas the transgenic overexpression of DGluR-IIB reduces synaptic DGluR-IIA levels. One possibility to explain this inverse relationship of both glutamate receptor expression levels could be a competition of both subunits in the formation of hetero-oligomeric receptor complexes. Another possibility may reside in the opposing roles of DGluR-IIA and DGluR-IIB for synaptic signal transmission: synapses expressing DGluR-IIA resemble wild-type transmission characteristics, whereas DGluR-IIB-expressing synapses exhibit very fast desensitization kinetics, resulting in strongly reduced quantal sizes. Given that NMJs with small quantal sizes are accompanied by suppressed subsynaptic protein synthesis this could result in an inefficient subsynaptic synthesis and a reduced synaptic delivery of DGluR-IIA in DGluR-IIB-overexpressing animals. In turn, synapses with reduced or no DGluR-IIB may efficiently activate the DGluR-IIA synthesis and their synaptic deposition. Although it is currently not possible to differentiate between these and other possibilities, it is important to note that NMJs are apparently equipped with two subunit-specific mechanisms, which because of their opposing effects on synaptic DGluR-IIA accumulation are well suited to tightly control the subunit composition of postsynaptic glutamate receptors (Sigrist, 2002).

On the basis of these data, it appears that a crucial factor for the implementation of persistently strengthened junctional signal transmission is the controlled upregulation of DGluR-IIA, which results in the functional recruitment of additional synapses. These added synapses show postsynaptic responses to released quanta of glutamate that are typical for wild-type NMJs, suggesting that increased DGluR-IIA expression results primarily in a larger number of normally operating postsynapses. Very strong overexpression of DGluR-IIA, which has been achieved using a cDNA-based transgene, appears to further increase the amount of DGluR-IIA at individual postsynapses and has been shown to result in a dose-dependent increase of miniature excitatory junctional potential (mEJP) amplitudes. These findings suggest that not only the number of responsive postsynapses can be changed by DGluR-IIA, but also the postsynaptic sensitivity can be changed. They further support the idea that the number of postsynaptic glutamate receptor complexes per synapse determines the amplitudes of mEJPs, and they are consistent with results from hippocampal synapses, which propose that a neurotransmitter from a single vesicle saturates all postsynaptic glutamate receptors of that synapse (Sigrist, 2002).

Strengthening of glutamatergic synapses and activation of silent postsynapses during long-term potentiation has recently gained much attention. Several lines of evidence have suggested that the targeted trafficking of specific glutamate receptor subunits and their incorporation into preexisting synapses represent a prominent route of synaptic activation and functional modification in hippocampal preparations. The results from the Drosophila NMJ indicate that these glutamatergic synapses use similar postsynaptic mechanisms to functionally recruit additional synapses, indicating that the local synthesis and the targeted trafficking of receptor subunits may represent an evolutionary conserved mode to alter glutamatergic circuits in a site-specific manner (Sigrist, 2002).

There is increasing evidence from this and several other recent studies that the strength of junctional signal transmission is correlated with the number of synapse-harboring boutons. Strikingly, the density of active zones, which represent sites of high-probability vesicle release, is approximately constant within individual boutons in all analyzed genotypes, even in NMJs with strongly enhanced signal transmission. This observation is consistent with a recent report suggesting that the spacing of active zones at NMJs and in the visual system of Drosophila and Sarcophaga is tightly regulated, presumably because each active zone requires a large enough surrounding surface area for proper function. It therefore seems likely that synapse recruitment leads to a transient increase in the density of active zones at larval NMJs of Drosophila, which are induced to grow to provide the now-required additional synaptic surface area. Interestingly, this growth does not involve a simple size increase of preexisting boutons, but it uses in a FasII-dependent manner the rather costly addition of new boutons to NMJs. This suggests that the axonal compartmentalization, which is given in form of type I boutons, generates functional units that, similarly to the spacing of active zones, need to be homeostatically preserved. It therefore appears that the consistent correlation between the strength of junctional signal transmission and the number of junctional boutons reflects the consolidation of induced functional changes, which include the functional recruitment of synapses and their distribution in newly grown boutons (Sigrist, 2002).

On the basis of the prominent role of the glutamate receptor subunit DGluR-IIA during long-term strengthening of signal transmission, the question arises whether NMJs can develop without DGluR-IIA. Surprisingly, NMJs with genetically eliminated DGluR-IIA expression develop to a size that resembles that of wild-type NMJs, despite strong defects in synaptic signal transmission. The same effect of eliminated DGluR-IIA expression was found in animals with increased subsynaptic protein synthesis, that normally develop significantly larger NMJs. Moreover, mutants in the synaptic vesicle protein synaptotagmin that have substantial defects in junctional signal transmission show similar basal development of NMJs. These observations demonstrate that neither DGluR-IIA expression itself nor intact synaptic physiology or subsynaptic translation is required to develop NMJs with a relatively simple morphology. They suggest that larval NMJs can develop according to a program that appears to be independent of neuronal activity and the expression of the glutamate receptor subunit DGluR-IIA. This 'programmed development' appears to establish a minimal innervation that would typically ensure baseline synaptic signal transmission and muscle contraction (Sigrist, 2002).

Superimposed on such programmed development, some of the mechanisms underlying the functional and structural modulation of the initially established synaptic connectivity are described in this study. Because postsynaptic DGluR-IIA expression plays a key role in this form of plasticity, which is likely regulated by neuronal activity and local subsynaptic protein synthesis, it is proposed that the 'activity-dependent' mode of junctional development helps adjust the junctional performance to the prevailing needs of the individual animal. A similar concept of activity-induced modifications of previously established neural circuits has been implicated in the development and functional tuning of various neural networks, for example, during the formation of barrels in the somatosensory cortex, and of ocular dominance columns in the primary visual cortex. On the basis of these similarities, the molecular and genetic analysis of developing NMJs of Drosophila might yield further important insights into the mechanisms underlying the activity-dependent remodeling of synaptic networks (Sigrist, 2002).

Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. This retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, it is shown that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, presynaptic BMP receptor Wishful thinking is required for the retrograde signal to function. These results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila (Haghighi, 2003).

Reducing the function of postsynaptic glutamate receptors at the neuromuscular junction (NMJ) of Drosophila triggers a retrograde signal from the postsynaptic muscle to the presynaptic motor neuron, leading to an increase in the amount of neurotransmitter release. This retrograde signal is regulated by the postsynaptic activity of CaMKII. Reducing postsynaptic CaMKII activity by expressing a CaMKII inhibitory peptide in somatic muscles increases quantal content, mimicking the effect of reducing postsynaptic glutamate receptor activity. Furthermore, in glutamate receptor GluRIIA^-/- mutants, constitutive activation of CaMKII in muscles inhibits the retrograde signal and decreases quantal content. These changes in retrograde signaling and neurotransmitter release are not accompanied by any significant changes in the number of synaptic boutons per muscle surface area or any gross structural or ultrastructural alterations. However, upon inhibition of CaMKII postsynaptically the number of T bars per active zone in presynaptic boutons is significantly increased. Similarly, the number of T bars per active zone is doubled in GluRIIA^-/- mutant larvae. This increase is suppressed by constitutive activation of CaMKII in postsynaptic muscles in GluRIIA^-/- mutants. These results point to CaMKII as a key regulator of the retrograde signal controlling homeostasis of synaptic transmission at the NMJ of Drosophila (Haghighi, 2003).

Postsynaptic inhibition of CaMKII activity is sufficient to increase presynaptic neurotransmitter release in a retrograde fashion. This increase in quantal content can be potentiated by expressing additional doses of the inhibitory transgene and suppressed by expressing a constitutively active CaMKII transgene simultaneously. These results suggest a direct involvement of CaMKII in controlling the retrograde signal that maintains the homeostasis of neurotransmitter release at the Drosophila NMJ (Haghighi, 2003).

While increasing the postsynaptic activity of CaMKII in wild-type larvae has no effect on neurotransmitter release, once the retrograde signal is induced (i.e., in GluRIIA^-/- mutants), activation of CaMKII can inhibit the signal. This is consistent with the observation that while removal of GluRIIA causes a decrease in quantal size and an increase in quantal content, overexpression of GluRIIA, which leads to an increase in quantal size, does not change quantal content. These results suggest that quantal content may be increased only when CaMKII activity is reduced to a critical threshold. As long as CaMKII activity remains above this critical threshold, quantal content is unchanged. This could be a mechanism through which the synapse can compensate for any reduction in muscle activity and ultimately maintain homeostasis (Haghighi, 2003).

In a recent study, Kazama (2003) has provided evidence for the involvement of postsynaptic CaMKII in the retrograde control of neurotransmission at the Drosophila NMJ. Changes in the activity of postsynaptic CaMKII have been shown to affect both neurotransmitter release and synaptic structure in early first instar larvae. Kazama also reports an apparent change in localization of postsynaptic glutamate receptors in response to postsynaptic activation of CaMKII. This is in contrast to the current observations; no changes were found in the Highaghi (2003) study in overall synaptic structure or localization of glutamate receptors in response to either inhibition or activation of postsynaptic CaMKII. The differences in these findings could be partially due to differences in the level and pattern of expression of transgenes (using different Gal4 lines) or due to the fact that the NMJ was examined at very different developmental stages. Haghighi (2003) examined late third instar larvae, while Kazama (2003) examined early first instar larvae. Interestingly, the results are in agreement in that both studies observed no changes in the amplitude or kinetics of spontaneous potentials, indicating that CaMKII does not directly modulate glutamate receptors at the Drosophila NMJ; this is not the case with vertebrates (Haghighi, 2003).

When the activity of postsynaptic glutamate receptors at the Drosophila NMJ is reduced, a retrograde signal from the muscle to the motor neuron is triggered that causes an increase in quantal content. It has been suggested that this retrograde signal could be triggered in response to changes in muscle depolarization or in response to Ca²⁺ conducted by glutamate receptors. The data indicate that postsynaptic activity of CaMKII plays an important role in controlling the signal. Both muscle depolarization and Ca²⁺ flux through glutamate receptors could be involved in changing the levels of intracellular Ca²⁺ and thus that of CaMKII. The idea is favored that calcium influx through glutamate receptors is at least in part responsible for activating CaMKII and triggering the retrograde signal. There are several lines of evidence that support this hypothesis (Haghighi, 2003).

One line of evidence is based on the glutamate receptor ion channel properties and how they are changed in GluRIIA^-/- mutants. Compared to wild-type receptors, glutamate receptors in these mutants have a greatly reduced single-channel mean open time; this also affects the kinetics of EPSPs. Therefore, evoked currents that give rise to similar EPSP peak amplitudes in wild-type and GluRIIA^-/- mutants will lead to less ion influx in the mutants (ion flux is a product of time and current). Considering the high Ca²⁺ permeability of glutamate receptors (P_Ca/P_Na = 9.55), due to this reduced ion influx, Ca²⁺ influx will also be reduced in GluRIIA^-/- mutants both during spontaneous and evoked activities. Therefore, it is conceivable that this change in Ca²⁺ influx, monitored by CaMKII, could act as a trigger for the retrograde signal (Haghighi, 2003).