InteractiveFly: GeneBrief

bruchpilot: Biological Overview | References

Neurotransmitters are released at presynaptic active zones (AZs). In Drosophila, monoclonal antibody (MAB) nc82 specifically labels AZs. Nc82 was used to identify Bruchpilot protein (Brp), a previously unknown AZ component. BRP shows homology to human AZ protein ELKS/CAST/ERC (Note: CAST stands for cytomatrix at the active zone associated structural protein), which binds RIM1 in a complex with Bassoon and Munc13-1. The C terminus of Brp displays structural similarities to multifunctional cytoskeletal proteins. During development, transcription of bruchpilot coincides with neuronal differentiation. Panneural reduction of Brp expression by RNAi constructs permits a first functional characterization of this large AZ protein: larvae show reduced evoked but normal spontaneous transmission at neuromuscular junctions. In adults, loss of T bars at active zones, absence of synaptic components in electroretinogram, locomotor inactivity, and unstable flight (hence 'bruchpilot' - German for crash pilot), were observed. It is proposed that Brp is critical for intact AZ structure and normal-evoked neurotransmitter release at chemical synapses of Drosophila (Wagh, 2007). Further studies show that in brp mutants Ca2+ channels are reduced in density, evoked vesicle release is depressed, and short-term plasticity is altered. Brp-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel, 2006)

Neurotransmitter release during chemical communication between nerve cells takes place at synaptic active zones, specific sites characterized ultrastructurally by presynaptic membrane thickenings decorated with synaptic vesicles and surrounded by additional synaptic vesicle accumulations. Often, electron-dense projections of various shapes (plaques, pyramids, T-shaped structures, ribbons) extend from the active zone into the presynaptic cytoplasm. Considerable efforts have been undertaken in recent years to identify and functionally characterize the protein components of these projections and the cytoskeletal matrix associated with the active zone (CAZ). This complex meshwork of proteins most likely constitutes an essential part of the molecular machinery mediating neurotransmitter release. The fine regulation of this process is believed to be central to nervous system operation including higher functions such as learning, memory, and cognition (Wagh, 2007)

In vertebrates, several components associated with the presynaptic active zone have been characterized. In addition to the general cytoskeletal proteins actin and spectrin, the large protein Bassoon (420 kDa) (tom Dieck, 1998; Shapira, 2003) is specifically found at the CAZ. This protein has been shown to be required for structural active zone formation and/or maintenance. Piccolo (530 kDa) (Fenster, 2000) contains several putative protein-protein interaction domains and together with Bassoon is assumed to organize components of the active zone, including Rab3-interacting molecule (RIM1), Munc-13, and the CAZ-associated structural protein (ELKS/CAST/ERC) (Wagh, 2007)

Vertebrate ELKS/CAST/ERC proteins were identified as AZ components by purifying synaptic densities from rat brain followed by electrophoresis and mass spectrometry (Ohtsuka, 2002) and, independently, in a yeast-two-hybrid screen of a rat-brain cDNA library as proteins interacting with the RIM1α PDZ domain (Wang, 2002). Several isoforms have been reported to be transcribed from two genes (Wang, 2002; Deguchi-Tawarada, 2004). Two isoforms (CAST1/ERC2 and CAST2α/ERC1b) are brain-specific and contain several coiled-coil domains as well as a C-terminal IWA motif essential for binding the PDZ domain of RIM1 (Ohtsuka, 2002). ELKS/CAST/ERCs form large oligomeric protein complexes with the other known proteins of the CAZ (Munc-13, RIM1, Piccolo, Bassoon) and are believed to be involved in the molecular organization of presynaptic active zones (Ko, 2003) where they regulate the release of neurotransmitter (Takao-Rikitsu, 2004; Wagh, 2007 and references therein).

Although most proteins shown to be relevant for structure and/or function of the vertebrate nervous system are conserved in invertebrates, apparently no Bassoon or Piccolo homologs are encoded in the Drosophila genome. This study identified the Drosophila bruchpilot gene (brp) coding for a protein (Brp) that contains an N-terminal domain with significant sequence homology to vertebrate ELKS/CAST/ERC and a large C-terminal domain rich in coiled-coil structures similar to several cytoskeletal proteins. The structure of this gene was analyzed; the Drosophila Brp protein localizes at the presynaptic active zone. By analyzing various transgenic RNAi lines, the effects were characterized of reduction of Brp expression on synaptic ultrastructure, as well as neurotransmitter release and observe behavioral defects, including unstable flight (hence bruchpilot, crash pilot, named after an old German movie about a pilot who always crashes his planes but survives). It is speculated that Brp might combine functions of ELKS/CAST/ERC and a cytoskeletal structural protein in a single polypeptide that is highly conserved among insects (Wagh, 2007).

Over the last years, some insight into assembly and molecular composition of vertebrate presynaptic active zones has been gained. Cytoskeletal elements like actin and spectrin, as well as large active-zone-specific proteins like Piccolo and Bassoon, seem to form a structural meshwork organizing various components of the active zone. The third coiled-coil domain of Bassoon contains a motif that is highly homologous to the corresponding region of Piccolo and binds in competition to Piccolo the fifth predicted coiled-coil domain of ELKSα/CAST1. This binding of ELKSα/CAST1 to Bassoon seems to be involved in neurotransmitter release (Takao-Rikitsu, 2004). ELKSα/CAST1 itself has been shown to bind RIM1 via the C-terminal PDZ binding motif IWA (Ohtsuka, 2002). RIM1 is a target of the Rab3A small G protein (Wang, 1997) and interacts with Munc13-1. Together with vesicular proteins, this complex might control the recruitment of vesicles and regulate their subsequent fusion with the presynaptic membrane. Recently, ELKS has been shown to function in insulin exocytosis of pancreatic β cells (Ohara-Imaizumi, 2005). Deletion of the elks gene in C. elegans neither relocates RIM nor produces an obvious structural or functional phenotype, although C. elegans ELKS (Deken, 2005) like its mammalian homologs binds to the PDZ domain of RIM (Wagh, 2007)

Although there is a wealth of information on the ultrastructure of insect synapses, the molecular composition of their synaptic active zones is almost completely unknown. This work shows that in Drosophila, a protein with homology to the ELKS/CAST/ERC protein family of vertebrates localizes at the presynaptic active zones of neuronal terminals. The rather uniform distribution of tiny nc82 stained spots in all adult and larval neuropil regions suggests that Brp is present at active zones of most if not all synapses of Drosophila. Thus, the Brp protein provides an entry point to study general active-zone formation and function in this species (Wagh, 2007)

The open-reading frame of the cDNA identified by RT-PCR corresponds in size to the protein recognized by MAB nc82. The fact that the prominent Northern blot signal is about 5.5 kb larger than the known cDNA could indicate that the brp mRNA abundantly expressed in heads likely contains a long 3'UTR. Long 3'UTRs are found in several brain mRNAs (e.g., elav, fne). Possibly, the gene contains alternative polyadenylation sites giving rise to an abundant 11 kb mRNA and a less-abundant mRNA that contains the 3'end of cDNA AT09405 but is not detected in the Northern blot. This hypothesis is supported by two RT-PCR experiments with independent primer pairs downstream of the 3'end of cDNA AT09405. The signal at 2.0 kb cannot be interpreted with present cDNA information. Both signals were identically reproduced in two independent head mRNA blots hybridized with probes specific for either CG12933 and CG30336 or CG30337, or containing the entire cDNA sequence. No difference was observed with these three probes. Transcripts containing ORF CG12932, on the other hand, apparently are not abundantly expressed in adult heads and may or may not belong to the brp transcription unit (Wagh, 2007)

The combined evidence from MS analysis, bacterial cDNA expression, ectopic expression of GFP-labeled Brp, and the RNAi experiments proves that Brp represents the active-zone protein recognized by MAB nc82. Analysis of the amino acid sequence of Drosophila Brp predicts two leucine zipper domains and a glutamine-rich C terminus but no transmembrane domains. High homologies among human ELKSα, C. elegans CAST, and Brp are found in three regions of the proteins. However, no PDZ interaction motif (IWA) for RIM interaction as seen in several mammalian ELKS/CAST isoforms and, interestingly, in the C. elegans homolog seems to be present in insect Brp. No Drosophila protein is detected by BLAST analysis with conserved C-terminal sequences of worm or human. However, for the large C terminus of Brp (1260 aa) significant sequence similarities to cytoskeletal proteins such as plectin, myosin heavy chain, and restin are observed, suggesting a possible cytoskeletal role or interaction of the C terminus of Brp (Wagh, 2007)

The expression of Brp is not restricted to the glutamatergic type I boutons of the NMJ, but Brp is present in active zones of presumably all synapses. Consistently, those layers in the visual neuropil that contain high levels of either choline acetyltransferase or GABA/glutamic acid decarboxylase do not show reduced nc82 staining, and nc82 staining is found in both glutamatergic and nonglutamatergic synaptic boutons of larval NMJs. It was furthermore demonstrated that normal Brp levels are required for normal synaptic transmission at histaminergic synapses (Wagh, 2007)

This study directly address the structural, physiological, and systemic function of Brp, a member of the ELKS/CASTl/ERC family. The ultrastructure of synaptic active zones in terminals of larval motorneurons and adult photoreceptors is impaired when Brp protein levels are severely reduced. The loss of T bars in the lamina is compatible with the hypothesis that Brp may be required for anchoring the T bars to the presynaptic membrane. The fact that similar frequencies of T bars is seen in wild-type photoreceptor terminals (16.8%) and of conspicuous membrane densities in brp-RNAi^C8-expressing terminals (19.5%) supports the proposition that these membrane thickenings may in fact represent presynaptic sites without typical T bars. However, it is unlikely that Brp is restricted to T bars because about 13%-37% of the active zones of type Ib boutons at NMJs of larval muscles 6/7 have no T bar, whereas in light microscopical preparations, only 3% of the active zones identified by their postsynaptic receptor fields contained no Brp label. Unfortunately, in these experiments the epitope recognized by MAB nc82 did not tolerate tissue fixation conditions required for immunoelectron microscopical analysis of T bars (Wagh, 2007)

Regarding a role of Brp in synaptic function, two RNAi lines with panneural expression were tested. Both showed a reduction in Brp protein levels and a decrease in evoked transmitter release as reflected by reduced EJC amplitude at larval NMJs, whereas miniature EJC amplitude and frequency were indistinguishable from wild-type controls (Wagh, 2007)

The genotype brp-RNAi^B12/elav-Gal4 caused embryonic lethality. When Brp was suppressed only in the eye, adult brp-RNAi^B12/gmr-Gal4 flies had a strong effect on eye development ('rough eye') and in some cases, in addition to the lack of ON/OFF transients, showed a much smaller ERG receptor potential than the other three lines. However, because this lethal and rough eye phenotype is observed only for a single RNAi line, it cannot be excluded that unspecific side effects might play a role. The description of the true loss-of-function phenotype will therefore have to await the generation of a genuine null mutant. A special advantage in this context is that Brp presumably is present at all synapses. Therefore, it is possible not only to asses the functionality of transgenic constructs at the electrophysiological level but also to score their effects at the behavioral level by genetically controlled, spatially, and/or temporally selective expression or suppression (Wagh, 2007)

In mammals ELKS/CAST/ERC isoforms apparently have both neuronal and nonneuronal roles. Drosophila Brp seems to correspond to the neuronal CAST isoforms, whereas the nonneuronal functions might be specific to vertebrates. Vesides the protein described here, the only published molecule localized specifically at Drosophila presynaptic active zones is the Ca²⁺ channel encoded by the cacophony gene. This channel seems to be responsible for providing the calcium trigger for evoked neurotransmitter release. The findings indicate, however, that the molecular structure of the presynaptic active zone might be more conserved between vertebrate and insect synapses than thought previously because of the lack of Piccolo and Bassoon homologs in insects. In the future, studying active-zone formation and function in Drosophila will be a valuable addition to similar studies in vertebrates, especially considering the powerful genetic tools available for Drosophila (Wagh, 2007)

Maturation of active zone assembly by Drosophila Bruchpilot

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel-clustering defects. This study used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, BRP was identified as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains (Fouquet, 2009).

This study addressed whether BRP signals T-bar formation (without being a direct component of the T-bar) or whether the protein itself is an essential building block of this electron-dense structure. Evidence is provided that BRP is a direct T-bar component. Immuno-EM identifies the N terminus of BRP throughout the whole cross section of the T-bar, and genetic approaches show that a truncated BRP, lacking the C-terminal 30% of the protein's sequence, forms truncated T-bars. Immuno-EM and light microscopy consistently demonstrate that N- and C-terminal epitopes of BRP are segregated along an axis vertical to the AZ membrane and suggest that BRP is an elongated protein, which directly shapes the T-bar structure (Fouquet, 2009).

In brp^5.45 (predicted as aa 1-866), T-bars were not detected, whereas brp^1.3 (aa 1-1,389) formed T-bar-like structures, although fewer and smaller than normal. Moreover, the BRP^D1-3GFP construct (1-1,226) did not rescue T-bar assembly. Thus, domains between aa 1,226 and 1,390 of BRP may also be important for the formation of T-bars. Clearly, however, the assembly scheme for T-bars is expected to be controlled at several levels (e.g., by phosphorylation) and might involve further protein components. Nonetheless, it is highly likely that the C-terminal half of BRP plays a crucial role (Fouquet, 2009).

Since BRP represents an essential component of the electron-dense T-bar subcompartment at the AZ center, it might link Ca²⁺ channel-dependent release sites to the synaptic vesicle cycle. Interestingly, light and electron microscopic analysis has located CAST at mammalian synapses both with and without ribbons. Overall, this study is one of the first to genetically identify a component of an electron-dense synaptic specialization and thus paves the way for further genetic analyses of this subcellular structure (Fouquet, 2009).

The N terminus of BRP is found significantly closer to the AZ membrane than the C terminus, where it covers a confined area very similar to the area defined by the Cac^GFP epitope. Electron tomography of frog NMJs suggested that the cytoplasmic domains of Ca²⁺ channels, reminiscent of pegs, are concentrated directly beneath a component of an electron-dense AZ matrix resembling ribs. In addition, freeze-fracture EM identified membrane-associated particles at flesh fly AZs, which, as judged by their dimensions, might well be Ca²⁺ channels. Peg-like structures were observed beneath the T-bar pedestal. Similar to fly T-bars, the frog AZ matrix extends up to 75 nm into the presynaptic cytoplasm. Based on the amount of cytoplasmic Ca²⁺ channel protein it has been concluded that Ca²⁺ channels are likely to extend into parts of the ribs. Thus, physical interactions between cytoplasmic domains of Ca²⁺ channels and components of ribs/T-bars might well control the formation of Ca²⁺ channel clusters at the AZ membrane. However, a short N-terminal fragment of BRP (aa 1-320) expressed in the brp-null background was unable to localize to AZs efficiently and consistently failed to restore Cac clustering (unpublished data) (Fouquet, 2009).

The mean Ca²⁺ channel density at AZs is reduced in brp-null alleles. In vitro assays indicate that the N-terminal 20% of BRP can physically interact with the intracellular C terminus of Cacaphony (Cac). Notably, it was found that the GFP epitope at the very C terminus of Cac^GFP was closer to the AZ membrane than the N-terminal epitope of BRP. It is conceivable that parts of the Cac C terminus extend into the pedestal region of the T-bar cytomatrix to locally interact with the BRP N terminus. This interaction might play a role in clustering Ca²⁺ channels beneath the T-bar pedestal (Fouquet, 2009).

Clearly, additional work will be needed to identify the contributions of discrete protein interactions in the potentially complex AZ protein interaction scheme. This study should pave the way for a genetic analysis of spatial relationships and structural linkages within the AZ organization. Moreover, the current findings should integrate in the framework of mechanisms for Ca²⁺ channel trafficking, clustering, and functional modulation (Fouquet, 2009).

The imaging assays allowed a temporally resolved analysis of AZ assembly in vivo. BRP is a late player in AZ assembly, arriving hours after DLiprin-α and also clearly after the postsynaptic accumulation of DGluRIIA. Accumulation of Cac was late as well, although it slightly preceded the arrival of BRP, and impaired Cac clustering at AZs lacking BRP became apparent only from a certain synapse size onwards, form at sites distant from preexisting ones and grow to reach a mature, fixed size. Thus, the late, BRP-dependent formation of the T-bar seems to be required for maintaining high Ca²⁺ channel levels at maturing AZs but not for initializing Ca²⁺ channel clustering at newly forming sites. As the dominant fraction of neuromuscular AZs is mature at a given time point, the overall impression is that of a general clustering defect in brp mutants. In reverse, it will be of interest to further differentiate the molecular mechanisms governing early Ca²⁺ channel clustering. Pre- to postsynaptic communication via neurexin-neuroligin interactions might well contribute to this process. A further candidate involved in early Ca²⁺ channel clustering is the Fuseless protein, which was recently shown to be crucial for proper Cac localization at AZs (Fouquet, 2009).

In summary, during the developmental formation of Drosophila NMJ synapses, the emergence of a presynaptic dense body, which is involved in accumulating Ca²⁺ channels, appears to be a central aspect of synapse maturation. This is likely to confer mature release probability to individual AZs and contribute to matching pre- and postsynaptic assembly by regulating glutamate receptor composition. Whether similar mechanisms operate during synapse formation and maturation in mammals remains an open question (Fouquet, 2009).

This study concentrated on developmental synapse formation and maturation. The question arises whether similar mechanisms to those relevant for AZ maturation might control activity-dependent plasticity as well and whether maturation-dependent changes might be reversible at the level of individual synapses. Notably, experience-dependent, bidirectional changes in the size and number of T-bars (occurring within minutes) were implied at Drosophila photoreceptor synapses by ultrastructural means. Moreover, at the crayfish NMJ, multiple complex AZs with double-dense body architecture were produced after stimulation and were associated with higher release probability. In fact, a recent study has correlated the ribbon size of inner hair cell synapses with Ca²⁺ microdomain amplitudes. Thus, a detailed understanding of the AZ architecture might permit a prediction of functional properties of individual AZs (Fouquet, 2009).

Bruchpilot promotes active zone assembly, Ca²⁺ channel clustering, and vesicle release

The molecular organization of presynaptic active zones during calcium influx-triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) are entirely lost, Ca²⁺ channels are reduced in density, evoked vesicle release is depressed, and short-term plasticity is altered. BRP-like proteins seem to establish proximity between Ca²⁺ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel, 2006).

Synaptic communication is mediated by the fusion of neurotransmitter-filled vesicles with the presynaptic membrane at the active zone, a process triggered by Ca²⁺ influx through clusters of voltage-gated channels. The spacing between Ca²⁺ channels and vesicles at active zones is particularly thought to influence the dynamic properties of synaptic transmission (Kittel, 2006).

The larval Drosophila neuromuscular junction (NMJ) is frequently used as a model of glutamatergic synapses. The monoclonal antibody Nc82 specifically stains individual active zones by recognizing a coiled-coil domain protein of roughly 200 kD named Bruchpilot (Brp). Brp shows homologies to the mammalian active zone components CAST [cytoskeletal matrix associated with the active zone (CAZ)-associated structural protein], also called ERC (ELKS, Rab6-interacting protein 2, and CAST). Whereas confocal microscopy recognized diffraction limited spots, the subdiffraction resolution of stimulated emission depletion (STED) fluorescence microscopy revealed donut-shaped Brp structures at active zones. Viewed perpendicular to the plane of synapses, both single and multiple 'rings' were uncovered, of similar size to freeze-fracture-derived estimates of fly active zones. The donuts were up to 0.16 µm high, as judged by images taken parallel to the synaptic plane (Kittel, 2006).

Brp seems to demark individual active zones associated with clusters of Ca²⁺ channels. Transposon-mediated mutagenesis allowed isolation of a mutant chromosome (brp⁶⁹) in which nearly the entire open reading frame of Brp was deleted. brp mutants [brp⁶⁹/df(2R)BSC29] develop into mature larvae but do not form pupae. The Nc82 label is completely lost from the active zones of brp mutant NMJs but can be restored by re-expressing the brp cDNA in the brp mutant background with use of the neuron-specific driver lines ok6-GAL4. This also rescued larval lethality. Mutants had slightly smaller NMJs and somewhat fewer individual synapses. However, individual receptor fields, identified by the glutamate receptor subunit GluRIID, were enlarged in brp mutants. Thus, principal synapse formation occurred in brp mutants, with individual postsynaptic receptor fields increased in size but moderately decreased in number (Kittel, 2006).

In electron micrographs of brp mutant NMJs, synapses with pre- and postsynaptic membranes in close apposition were present at regular density, and consistent with the enlarged glutamate receptor fields postsynaptic densities appeared larger while otherwise normal. However, intermittent rufflings of the presynaptic membrane were noted, and brp mutants completely lacked presynaptic dense projections (T-bars). Occasionally, very little residual electron-dense material attached to the presynaptic active zone membrane was identified. After re-expressing the Brp protein in the mutant background, T-bar formation could be partially restored, although these structures were occasionally somewhat aberrant in shape. Thus, Brp assists in the ultrastructural assembly of the active zone and is essential for T-bar formation (Kittel, 2006).

In brp mutant larvae a drastic decrease was noted in evoked excitatory junctional current (eEJC) amplitudes by using two-electrode voltage clamp recordings of postsynaptic currents at low stimulation frequencies. This drop in current amplitude could be partially rescued through brp re-expression within the presynaptic motoneurons by using either elav-GAL4 or ok6-GAL4. In contrast, the amplitude of miniature excitatory junctional currents (mEJCs) in response to single, spontaneous vesicle fusion events was increased over control levels. This is consistent with the enlarged individual glutamate receptor fields of brp mutants and excludes a lack of postsynaptic sensitivity as the cause of the reduced eEJC amplitudes (Kittel, 2006).

It follows that the number of vesicles released per presynaptic action potential (AP) (quantal content) was severely compromised at brp mutant NMJs and could not be attributed solely to the moderate decrease in synapse number. The ultrastructural defects of brp mutant synapses may interfere with the proper targeting of vesicles to the active zone membrane and thereby impair exocytosis. The number of vesicles directly docked to active zone membranes was slightly decreased in brp mutants. However, the amplitude distribution and sustained frequency of mEJCs showed that brp mutant synapses did not appear to suffer from extrasynaptic release, as would be caused by a misalignment of vesicle fusion sites with postsynaptic receptors. Consistent with the appropriate deposition of exo- and endocytotic proteins, an apparently normal distribution of Syntaxin, Dap160, and Dynamin was observed at brp mutant synapses (Kittel, 2006).

The exact amplitude and time course of AP-triggered Ca²⁺ influx in the nerve terminal governs the amplitude and time course of vesicle . Nerve-evoked responses of brp mutants were delayed when compared with controls, whereas in contrast mEJC rise times were unchanged. Thus, evoked vesicle fusion events were less synchronized with the invasion of the presynaptic terminal by an AP. Spatiotemporal changes in Ca²⁺ influx have a profound effect on short-term plasticity. Whereas at 10 Hz controls exhibited substantial short-term depression of eEJC amplitudes, brp mutants showed strong initial facilitation before stabilizing at a slightly lower but frequency-dependent steady-state current. As judged by the initial facilitation at 10 Hz, neither a reduction in the number of releasable vesicles nor available release sites could fully account for the low quantal content of brp mutants at moderate stimulation frequencies. Furthermore, the altered short-term plasticity of brp mutant synapses suggested a change in the highly Ca²⁺-dependent vesicle release probability. Paired-pulse protocols were applied to the NMJ. Closely spaced stimuli lead to a buildup of residual Ca²⁺ in the vicinity of presynaptic Ca²⁺ channels, enhancing the probability of a vesicle within this local Ca²⁺ domain to undergo fusion after the next pulse. The absence of marked facilitation at control synapses could be explained by a depletion of release-ready vesicles. At brp mutant NMJs, however, the prominent facilitation at short interpulse intervals showed that the enhancement of release probability strongly outweighed the depletion of releasable vesicles. Thus, initial vesicle release probability was low, and release at brp synapses particularly benefited from the accumulation of intracellular Ca²⁺ (Kittel, 2006).

Vesicle fusion is highly sensitive to the spacing between Ca²⁺ channels and vesicles at release sites. It has been calculated that doubling this distance from 25 to 50 nm decreases the release probability threefold, and the larger this distance, the more effective the slow synthetic Ca²⁺ buffer EGTA should become in suppressing release. Indeed, after bath application of membrane permeable EGTA-AM, the reduction of evoked vesicle release was more pronounced at brp mutant than at control NMJs (Kittel, 2006).

The Ca²⁺-channel subunit Cacophony governs release at Drosophila NMJs. By using a fully functional, GFP (green fluorescent protein)-labeled variant (Cac^GFP), Ca²⁺ channels were visualized in vivo. Consistently, Ca²⁺ channel expression was severely reduced over the entire NMJ and at synapses lacking Brp (Kittel, 2006).

Thus, it is concluded that brp mutants suffer from a diminished vesicle release probability due to a decrease in the density of presynaptic Ca²⁺ channel clusters. It is conceivable that Brp tightly surrounds but is not part of the T-bar structure, contained within the unlabeled center of donuts. Brp may establish a matrix, required for both T-bar assembly as well as the appropriate localization of active zone components including Ca²⁺ channels, possibly by mediating their integration into a restricted number of active zone slots. Related mechanisms might underlie functional impairments of mammalian central synapses lacking active zone components (Altrock, 2003) and natural physiological differences between synapse types. Electron microscopy has identified regular arrangements at active zones of mammalian CNS synapses ('particle web') and frog NMJs ('ribs'), where these structures have also been proposed to organize Ca²⁺ channel clustering. At calyx of Held synapses (an axosomatic synapse in the auditory brainstem), both a fast and a slow component of exocytosis have been described. The fast component recovers slowly and is believed to owe its properties to vesicles attached to a matrix tightly associated with Ca²⁺ channels, whereas the slow component recovers faster and is thought to be important for sustaining vesicle release during tetanic stimulation. In agreement with this concept, the absence or impairment of such a matrix at brp synapses has a profound effect on vesicle release at low stimulation frequencies, but this effect subsides as the frequency increases. The sustained frequency of mEJCs at brp synapses could be explained if spontaneous fusion events arise from the slow release component or a pathway independent of evoked vesicle fusion (Kittel, 2006).

Synapses lacking Brp and T-bars exhibited a defective coupling of Ca²⁺ influx with vesicle fusion, whereas the vesicle availability did not appear rate-limiting under low frequency stimulation. The activity-induced addition of presynaptic dense bodies has been proposed to elevate vesicle release probability. This work supports this hypothesis and suggests an involvement of Brp or related factors in synaptic plasticity by promoting Ca²⁺ channel clustering at the active zone membrane (Kittel, 2006).

Fos and Jun potentiate individual release sites and mobilize the reserve synaptic-vesicle pool at the Drosophila larval motor synapse

In all nervous systems, short-term enhancement of transmitter release is achieved by increasing the weights of unitary synapses; in contrast, long-term enhancement, which requires nuclear gene expression, is generally thought to be mediated by the addition of new synaptic vesicle release sites. In Drosophila motor neurons, induction of AP-1, a heterodimer of Fos and Jun, induces cAMP- and CREB-dependent forms of presynaptic enhancement. Light and electron microscopic studies indicate that this synaptic enhancement is caused by increasing the weight of unitary synapses and not through the insertion of additional release sites. Electrophysiological and optical measurements of vesicle dynamics demonstrate that enhanced neurotransmitter release is accompanied by an increase in the actively cycling synaptic vesicle pool at the expense of the reserve pool. Finally, the observation that AP-1 mediated enhancement eliminates tetanus-induced forms of presynaptic potentiation suggests: (1) that reserve-pool mobilization is required for tetanus-induced short-term synaptic plasticity; and (2) that long-term synaptic plasticity may, in some instances, be accomplished by stable recruitment of mechanisms that normally underlie short-term synaptic change (Kim, 2009).

Drosophila larval motor synapses show increased synaptic strength when AP-1 is overexpressed in motor neurons (Sanyal, 2002). This synaptic enhancement is accompanied by increases in the quantal content of neurotransmitter release, and increases in the number of presynaptic varicosities (Sanyal, 2002). This study asked whether AP-1 mediated synapse enhancement can be explained by increases in synapse number, Ca2+ influx, Ca2+ sensitivity of vesicle fusion or synaptic vesicle number. The observations support a model in which: (1) AP-1 induced synaptic enhancement occurs without an accompanying increase in synapse number; (2) AP-1 increases the size of the cycling synaptic vesicle pool through mobilization of the reserve pool; (3) that AP-1 causes persistent synaptic change by stably recruiting a cellular mechanism transiently used for posttetanic potentiation, a ubiquitous but poorly understood form of short-term synaptic facilitation (Kim, 2009).

Previous studies have shown AP-1 overexpression in Drosophila motor neurons enhances glutamate release from motor terminals in a manner that is accompanied by an increase in bouton number (Sanyal, 2002). These conclusions were confirmed using failure frequency analysis, which, under conditions of very low Ca²⁺, measures frequency of 'failure' to release even a single quantum of neurotransmitter. At 0.3 mM Ca²⁺, frequencies of failure events are reduced in C155/+;UAS Fos/+;UAS Jun/+ (hereafter referred to as 'AP-1') compared with control C155/+ hereafter 'control') synapses. Therefore, this analysis confirmed quantal content (m = ln [number of events/number failures]) is significantly increased in motor synapses from AP-1 animals. Similar results were obtained under nonfailure conditions where quantal content is calculated by m = EJP/mEJP. Because quantal amplitude is not increased by AP-1 (SF1Fig. S1), presynaptic mechanisms completely account for the measured synaptic strengthening. These observations, taken together with previous work (Sanyal, 2002), show that AP-1 increases quantal content of transmitter release at both low and physiological Ca²⁺ concentrations (Kim, 2009).

Although AP-1 overexpression increases the number of presynaptic boutons (Sanyal, 2002), the average bouton size is significantly reduced. For this reason, and because individual boutons contain multiple release sites, bouton number is not necessarily a reliable measure of synapse number. The following strategy was used to assess whether AP-1-terminals have more functional synapses, which is defined as presynaptic release sites apposed to postsynaptic receptor clusters. In wild-type neuromuscular junctions (NMJ), ~95% of GluR clusters are coupled to Bruchpilot (brp/CAST) immunopositive presynaptic puncta (Rasse, 2005). This fraction is not altered by AP-1 expression. Thus, the number of Brp-positive puncta provides a measure of synapse number in AP-1 synapses (Kim, 2009).

Since individual Brp spots are clearly resolved, they could be counted and analyzed with a spot-detection/analysis program. This method yielded values that were in good agreement with those derived from previous serial EM studies of wild-type NMJs. Surprisingly, total Brp positive puncta (per NMJ) decreased by 21% in AP-1 synapses. AP-1 induction did not detectably alter the distribution of T-Bar or synapse size assessed by quantitative fluoresence and electron microscopy respectively. Thus, it is concluded that although AP-1 increases total bouton number, the number of functional synapses is significantly reduced. Because the quantal content of neurotransmitter release is N × p (where N is synapse number and p is the average probability of vesicle release per synapse), these observations point to an increase in p at AP-1 terminals (Kim, 2009).

If AP-1 overexpression leads to changes in the probability of release, it was reasoned that forms of short-term plasticity, which also alter p, might be altered at these motor terminals. To test this idea, two separable forms of short-term plasticity observed at the Drosophila larval NMJ at low Ca²⁺ concentrations were measured. The first form, paired-pulse facilitation (PPF) is short-lived and decays within milliseconds. This is easily distinguished from longer-lived presynaptic plasticity, observed during and after tetanic stimulation, which decays more slowly (10s of seconds to minutes). Although multiple processes (e.g., augmentation and posttetanic potentiation) could contribute to this longer-lived form of plasticity, this phenomenon is referred by a single term, tetanus-induced potentiation (TIP) (Kim, 2009).

At interstimulus intervals (ISI) of 25 ms, 50 ms, 100 ms, and 1,000 ms, the paired pulse ratios exhibited by control and AP-1 motor terminals did not differ significantly. The site of action for residual Ca²⁺ during paired pulse facilitation (PPF) has been demonstrated in previous studies to be located in the Ca²⁺ microdomain immediately surrounding clustered Ca²⁺ channels and vesicle release sites (Blatow, 2003; Zucker, 2002). The observation that PPF is normal in AP-1 synapses suggests that Ca²⁺ dynamics in this microdomain are not significantly altered by AP-1 (Kim, 2009).

In contrast, TIP was strikingly altered by AP-1 expression. In control synapses, transmitter release increases during a 2-min train of 10-Hz stimulation, eventually reaching a plateau. Contributions from both facilitation and TIP processes underlie the potentiated response during delivery of the tetanic stimulus train. Facilitation, however, decays within a few hundred milliseconds. Thus, longer-lived components (TIP), which decay on the order of seconds to minutes, can be isolated in the potentiated response after the tetanic train ends. TIP is greatly reduced in AP-1 terminals compared with the control. The potentiation factor immediately after the tetanus is 2.53 ± 0.13 for control and 1.15 ± 0.10 for AP-1. This early potentiation decays with time but lasts for several minutes as evidenced by the values for PF_2.75 measured 2.75 min after stimulation cessation, which are 1.54 ± 0.14 for control and 0.93 ± 0.11 for AP-1. Thus, in AP-1 appears to affect both PF₀ (Kim, 2009).

The absence of TIP components in AP-1 synapses is consistent with a model where individual release sites are 'prepotentiated' in AP-1 motor terminals. Loss of TIP cannot be explained by postsynaptic receptor saturation, because EJPs of twice this magnitude can easily be detected at this motor synapse. The observation that one form of short-term plasticity (PPF) remains unaltered, whereas longer lived forms (TIP) are dramatically diminished argues that AP-1 acts through a selective and relatively specific mechanism normally used for tetanus-induced presynaptic plasticity (Kim, 2009).

To determine the underlying mechanism of synaptic enhancement by AP-1, three key parameters that influence the efficiency of neurotransmitter release were measured: (1) presynaptic Ca²⁺ entry; (2) sensitivity of the exocytotic machinery to Ca²⁺; and (3) the available pool of synaptic vesicles (Kim, 2009).

A simple mechanism for increasing the probability of exocytosis from an active zone is enhanced Ca²⁺ entry, e.g., because of a decreased presynaptic potassium conductance and/or an increased Ca²⁺ current. The highly comparable paired-pulse ratios in AP-1 and control terminals suggest presynaptic Ca²⁺ entry and, particularly, the molecular target of residual Ca²⁺ during PPF, is unchanged in AP-1 expressing motor neurons (Kim, 2009).

Direct Ca²⁺ imaging to support the above argument is difficult, because small changes in single-action potential induced Ca²⁺ entry potentially can account for the observed increase in quantal content. Using an indirect approach, it was instead asked whether summed Ca²⁺ entry during 40-Hz nerve stimulation was increased in AP-1 expressing animals (Kim, 2009).

In motor terminals expressing the genetically encoded Ca²⁺ indicator, GCaMP 1.6, fluorescence was imaged during sustained 40-Hz stimulation. Values for DF/F at a plateau reached in ~2 seconds were similar in AP-1 and control synapses. Unexpectedly, Ca²⁺ rise times in AP-1 terminals were slightly slower than in the control. This cannot be ascribed to faster Ca²⁺ extrusion as GCaMP signal does not decay any faster in AP-1 synapses after stimulation cessation. Instead, these data indicate that less Ca²⁺ enters AP-1 presynaptic terminals per action potential, at least during high-frequency stimulation. Although GCaMP imaging does not provide absolute measurement of presynaptic Ca²⁺ before and after stimulation, these data argue against increased evoked Ca²⁺ entry as being the primary mechanism for AP-1's effect on transmitter release (Kim, 2009).

Another mechanism to enhance transmitter release is to increase sensitivity of the exocytotic machinery to free Ca²⁺. Measurements, however, show Ca²⁺ cooperativity of transmitter release was not significantly altered by AP-1 expression (Kim, 2009).

The last major parameter that influences and often correlates with quantal content is the size of the active cycling vesicle pool (also referred to as exo-endo cycling pool, ECP) available for release (Murthy, 1999). At Drosophila motor synapses, the ECP contributes to transmitter release at low to moderate rates of nerve stimulation, e.g., 3 Hz. A second 'reserve' pool of vesicles (RP) poorly accessed at 3-Hz stimulation, is efficiently mobilized during high frequency stimulation >10 Hz. Two independent approaches, one electrophysiological and the other, optical allow the sizes of the cycling and total synaptic vesicle pools to be compared at the Drosophila NMJ (Kim, 2009).

ECP sizes were compared as follows. First, AP-1 and control synapses were stimulated continuously at 3 Hz in the presence of 1 μM bafilomycin A1, a drug that pharmacologically blocks the refilling of vesicles with neurotransmitter. Initial rates of synaptic depression under these experimental conditions largely reflect depletion of the cycling pool of vesicles. The later phase in the decay plot, after significant ECP depletion, represents vescles that arise from slow mixing between RP and ECP. The initial phase is extended in AP-1 compared with control, consistent with a larger ECP. To quantitatively estimate ECP size, Y-intercept values were determined by linear regression of the points from the later slow phase of depression in a cumulative plot. These ECP estimates were consistent with substantial enlargement of the ECP in AP-1 motor terminals. Because these estimates derive from fitting the observed curves to a specific (previously suggested) model (Delgado, 2000), a second and completely independent technique was used to estimate the ECP. In this technique, optical measurements of styryl dye uptake into individual varicosities were used. Consistent with predictions from electrophysiological measurements, varicosities at AP-1 synapses were more brightly labeled than control synapses when the ECP was loaded with FM1-43 dye by 3-Hz stimulation for 7 min, indicating a larger ECP (Kim, 2009).

To test whether this increased ECP in AP-1 synapses occurs at the expense of the reserve poolwe measured the total vesicle content in AP-1 and control terminals was measured by stimulating them to depletion at 10-Hz frequency in the presence of Bafilomycin. Total vesicle pool size was estimated by integrating the complete depression curve of quantal content versus stimulus number. This direct electrophysiological estimate showed a slightly smaller total pool size in AP-1 terminals. To independently assess the sizes of the total vesicle pool FM1-43 uptake into presynaptic boutons was measured after 7 min of 30-Hz stimulation, conditions that should label both ECP and RP. Remarkably, both control and AP-1 terminals were labeled to very similar levels under these conditions, with AP-1 showing slightly lower labeling. This indicates that the total number of synaptic vesicles is similar in control and AP-1 synapses. Thus, 2 independent approaches-electrophysiological and optical establish that AP-1 increases the actively cycling vesicle pool by partially mobilizing the reserve pool of synaptic vesicles. EM analyses of synaptic-vesicle density in AP-1 and control nerve terminals are also conistent with this conclusion (Kim, 2009).

Based on these observations, AP-1 synapses show 2 major differences from the wild-type. First, they have a larger fraction of actively cycling vesicles. Second, they exhibit highly reduced TIP. These 2 phenotypes can be linked if one proposes that mobilization from the reserve vesicle pool is required for TIP. In such a model, AP-1 synapses cannot be further potentiated because the RP has already been mobilized. It was therefore asked whether tetanus-induced potentiation requires RP mobilization (Kim, 2009).

Previous work has established that RP mobilization depends on activity of the myosin light chain kinase (MLCK) in Drosophila motor terminals (Verstreken, 2005). Blocking the activity of this enzyme results in failure to recruit vesicles from the inactive pool under high frequency stimulation (Verstreken, 2005). Strikingly, the MLCK inhibitor ML-7 also inhibited tetanus-induced potentiation; PF was not examined at later time points because in relevant control preparations, the small amount of DMSO required to dissolve MLCK increased the rate of decay of TIP]. Taken together, the above experiments indicate that (1) TIP requires synaptic-vesicle mobilization from the reserve pool; and, by inference, (2) AP-1 driven prepotentiation of transmitter release is accompanied by a stable expansion of the cycling pool of vesicles through reserve pool mobilization (Kim, 2009).

One important conclusion from this work is that Fos and Jun enhance synaptic strength, not by increasing synapse number, but rather by increasing the average probability of release from individual active zones. This conclusion is based on the following: (1) increases in synaptic strength in AP-1 motor terminals can be completely accounted for by increased transmitter release; and (2) light microscopic studies show no increases in the number of release sites. Thus, there is an increase in the average probability of synaptic vesicle fusion at release sites of AP-1 motor neurons. There are some caveats to this argument. First, the definition of functional synapses as Brp-positive puncta is based on the assumptions that Brp puncta: (1) mark the large majority of release sites; and (2) are mostly capable of transmitter release postsynaptic stimulation. These assumptions are supported by the tight colocalization of presynaptic Ca²⁺ channels and postsynaptic receptors with Brp puncta (Kim, 2009).

Increased vesicle-release probability from active zones could conceivably be explained by several different mechanisms. In AP-1 synapses, a large increase in the size of the actively cycling synaptic vesicle pool (ECP), which arises at the expense of the reserve pool (RP), was demonstrated. An increased ECP can account for the observed synaptic enhancement in AP-1 motor terminals if it increases the number of synaptic vesicles immediately available for fusion. RP mobilization has been associated with specific instances of short-term plasticity: e.g., cocaine-induced increases in dopamine release from rat striatal neurons. This study shows that this process can be initiated by nuclear gene expression (Kim, 2009).

How widely might RP mobilization be deployed for synaptic change in vivo? Studies of the reserve pool in hippocampal synapses do not easily support the idea that vesicle trafficking from this source controls synaptic vesicle availability within these small axonal terminals. However, RP mobilization can regulate the output from larger synapses such as neuromuscular junctions or the calyx of Held, where inhibition of the myosin light chain kinase required for vesicle mobilization has been shown to reduce the stability of the synaptic firing during repetitive stimulation (Verstreken, 2005). Because the actual mechanism of RP mobilization is poorly understood, more experiments will be required to understand exactly how Fos and Jun regulate this process. In one model, phosphorylation of synapsin, which tethers synaptic vesicles to an actin-based cytoskeleton in the central domain of synaptic boutons, may mobilize the reserve pool by triggering the dissociation of vesicles from the cytoskeleton, and their transport/diffusion to peripheral release sites. In Drosophila NMJs, mitochondria within the presynaptic terminal are required for sustained release of neurotransmitter under high frequency stimulation. One study suggests ATP production from mitochondria is required to fuel MLCK-activated, myosin-propelled transport of reserve vesicles from central to peripheral sites (Verstreken, 2005). Although such processes might be involved, it is also possible that different pathways are used for Fos and Jun mediated RP mobilization. For instance, smaller sized boutons, such as those observed in AP-1 animals, may be less efficient at holding a central pool of reserve vesicles. In such a scenario, bouton geometry, rather than a specific regulatory protein, may prove to be the relevant target of AP-1 activity (Kim, 2009).

The simplest interpretation of these observations is that stable reserve pool mobilization underlies the observed loss of tetanus-induced potentiation in AP-1 synapses. Other work at the Drosophila neuromuscular junction has associated mobilization of the reserve pool with the expression of TIP (Kim, 2009).

Cytosolic Ca²⁺ accumulation and signaling is required for the induction of TIP. However, links between Ca²⁺ signaling and the expression of TIP are poorly defined; indeed, tetanus induced potentiation could include multiple Ca²⁺-dependent processes including augmentation and posttetanic potentiation/PTP. If TIP expression requires RP mobilization, then it would be occluded in 1 of 2 ways. Either: (1) by preexisting mobilization of the reserve pool; or (2) by inhibition of reserve pool mobilization. Drosophila dnc mutants with enhanced cAMP signaling, and rut mutants with reduced cAMP signaling respectively illustrate these 2 different mechanisms of inhibition. Both mutants do not show tetanus-induced potentiation. Although dnc mutants show a greatly increased ECP and already enhanced transmitter release, rut mutants show a large, stable reserve pool that cannot be mobilized by tetanic stimulation. These data indicate that AP-1 synapses behave like dnc mutants in which reserve vesicles have already been mobilized (Kim, 2009).

If reserve pool mobilization is required for TIP, then mutations or drugs that inhibit reserve pool mobilization would also be expected to block TIP. Consistent with this prediction, application of an MLCK inhibitor, which blocks reserve pool mobilization, was shown to dramatically inhibit TIP induction in wild-type motor synapses. Thus, although alternative contributing mechanisms cannot be ruled out, this study shows that tetanus induced presynaptic potentiation is tightly linked to reserve pool mobilization (Kim, 2009).

It is possible that many different direct and indirect targets of AP-1 contribute to various observed AP-1 dependent neuronal phenomena: e.g., increased bouton number, reduced bouton size, increased dendritic growth, elevated evoked transmitter release and increased ECP size. In addition, AP-1 may have effects on some phenomena that are not yet measured, e.g., kinetic and spatial features of synaptic Ca²⁺ dynamics. Nonetheless, this work shows functions of Fos and Jun in neurons, and provides substantial evidence for a model in which transcription-dependent changes in synaptic function occur through stable recruitment of mechanisms used in short-term plasticity. Recent observations that short-term forms of presynaptic plasticity are altered following synaptic enhancements induced by either BDNF or postsynaptic PSD-95 overexpression suggest that this could be a viable strategy for long-term information storage in central synapses (Zakharenko, 2003). If long-term plasticity requires stable recruitment of short-term plasticity mechanisms, then the lability of long-term memory traces, as observed in studies of reconsolidation, may not require the elimination of stable synaptic connections representing the stored memory (Kim, 2009).

Rab3 dynamically controls protein composition at active zones

Synaptic transmission requires the localization of presynaptic release machinery to active zones. Mechanisms regulating the abundance of such synaptic proteins at individual release sites are likely determinants of site-specific synaptic efficacy. A role for the small GTPase Rab3 has been identified in regulating the distribution of presynaptic components to active zones. At Drosophila rab3 mutant NMJs, the presynaptic protein Bruchpilot, calcium channels, and electron-dense T bars are concentrated at a fraction of available active zones, leaving the majority of sites devoid of these key presynaptic release components. Late addition of Rab3 to mutant NMJs rapidly reverses this phenotype by recruiting Brp to sites previously lacking the protein, demonstrating that Rab3 can dynamically control the composition of the presynaptic release machinery. While previous studies of Rab3 have focused on its role in the synaptic vesicle cycle, these findings demonstrate an additional and unexpected function for Rab3 in the localization of presynaptic proteins to active zones (Graf, 2009).

Individual neurons can form thousands of discrete synaptic connections with their postsynaptic partners. Each synapse comprises tightly apposed pre- and post-synaptic membranes, a postsynaptic cluster of neurotransmitter receptors, and a presynaptic complex of proteins that promotes neurotransmitter release. For a synapse to function, the proper complement of proteins must localize to the presynaptic release machinery, and the protein composition at the release site is a likely determinant of its synaptic efficacy. While the general properties of synapses formed by a single axon are similar, the release probability of such synapses can vary dramatically. This presynaptic heterogeneity is likely due to mechanisms that control synapse specific plasticity and may represent one aspect of the molecular basis of learning and memory. Thus, identifying mechanisms that control the protein composition and presynaptic release properties of individual synapses will provide insights into plasticity mechanisms in the brain (Graf, 2009).

The Drosophila neuromuscular junction (NMJ) is an excellent system for identifying mechanisms that regulate the protein composition of individual active zones. A single Drosophila motoneuron and single muscle cell form an NMJ comprising hundreds of individual release sites, or presynaptic active zones, each apposed to a postsynaptic glutamate receptor (GluR) cluster. Each release site is akin to a single mammalian central nervous system synapse, and like CNS synapses, there is heterogeneity in their release properties. Drosophila contains orthologs of all of the major vertebrate presynaptic proteins with the exception of Bassoon and Piccolo. Among these, Bruchpilot (Brp), the Drosophila ortholog of CAST, plays an essential role in organizing the presynaptic release machinery. This role is similar in mammals where CAST acts as a molecular scaffold within the cytomatrix at the active zone, interacting with Piccolo, Bassoon, Rim1α, and α-liprins/SYD-2 and in C. elegans where the Brp homolog ELKS-1 acts with SYD-2/α-liprin to promote the assembly of presynaptic active zone components. In Drosophila, Bruchpilot localizes to every active zone, but its distribution is heterogeneous, and the abundance of Brp at an active zone appears to correlate with the release probability of that site. Brp is not required for active zone formation per se, but is an integral component of T bars, electron-dense active zone specializations that likely promote transmitter release, and is required for the continuous accumulation of Ca²⁺-channels at active zones during synapse maturation. These findings with Brp imply that mechanisms exist to (1) ensure that Brp is present at each release site and (2) regulate the level of Brp at each site. Such mechanisms would likely impact site-specific release probability by controlling the protein composition of the release machinery at each site. To identify such mechanisms, a large-scale genetic screen was performed to identify genes required for the proper localization of Brp to active zones (Graf, 2009).

This study found that the small GTPase Rab3 functions to influence the distribution of Brp and other crucial presynaptic active zone components to release sites. In the absence of Rab3, key constituents of the presynaptic release machinery are concentrated at a fraction of available sites, resulting in the formation of a small number of super sites with enhanced release probability and a larger number of sites devoid of key presynaptic release proteins. Rab3 can rapidly recruit Brp to active zones, demonstrating that the protein composition of the release machinery is under dynamic control and that Rab3 is well positioned to participate in synapse-specific plasticity mechanisms. Whereas previous studies have implicated Rab3 in the cycling and docking of synaptic vesicles, this study reports a role for Rab3 in influencing the protein composition of the presynaptic release apparatus at individual active zones (Graf, 2009).

To identify mechanisms that control the molecular composition of individual release sites, a collection of Drosophila mutants was screened for those with defects that differentially affect presynaptic active zones within an NMJ. An anatomical genetic screen was performed on a collection of ∼1500 lines that carry unique insertions of transposable elements in or near genes on the second chromosome. Third-instar homozygous mutant larvae were dissected from each line and stained for the presynaptic active zone protein Bruchpilot (Brp) and the essential glutamate receptor subunit DGluRIII. The immunostained NMJs were stained with fluorescence microscopy and mutants were identified with altered active zones, including changes in Brp puncta size, number, or intensity, as well as those with defects in the apposition of presynaptic Brp and postsynaptic DGluRIII puncta. Within this group, one line, P{SUPor-P}KG07292, was identified that has an active zone phenotype. In this mutant, there is a dramatic loss of Brp-positive active zones, yet the morphology and number of DGluRIII clusters appears grossly normal. As such, most GluR clusters are unapposed to a Brp-positive active zone. The remaining Brp puncta are apposed to GluR clusters, and these Brp puncta are significantly larger than in wild-type. Due to the large number of unapposed GluR clusters, this mutant was named running-unapposed (rup) (Graf, 2009).

While Brp morphology is altered in rup, the gross morphology of the mutant NMJ is normal, and the synaptic terminal area is not significantly different than in wild-type. Staining with an antibody against the vesicular glutamate transporter (DVGLUT) demonstrates that synaptic vesicles are distributed throughout the NMJ, and co-staining for the postsynaptic scaffolding protein Discs-large (Dlg) reveals that the presynaptic terminal is apposed to the postsynaptic specialization across its length. Hence, the presence of unapposed GluR clusters is not due to synaptic retraction of synaptic boutons or branches; instead, affected synapses distribute in a salt-and-pepper pattern throughout the synaptic terminal, suggesting that the defect occurs at the level of individual synapses. Glutamate receptors colocalize with the serine-threonine kinase Pak at the Drosophila NMJ. Pak distribution appears normal in the rup mutant, and like GluR clusters, most Pak clusters are unapposed to Brp-positive active zones. Hence, in rup postsynaptic morphology is relatively normal and the primary morphological defect is likely presynaptic. Despite their abnormal active zones, rup mutant animals are viable and fertile (Graf, 2009).

To investigate the mechanism underlying the defective active zones in rup, it was necessary to identify the responsible gene. Although rup was found in a collection of insertional mutants, the phenotype does not map to the P{SUPor-P}KG07292 transposable element. Instead, rup is a second-site mutation fortuitously present on the chromosome. rup was roughly mapped by meiotic recombination to position 43-48 on the right arm of the second chromosome and a deficiency chromosome (Df(2R)ED2076) was identified that fails to complement the mutation. This deficiency deletes 26 predicted genes in the region between 47A10 and 47C1. The list of candidates was narrowed to 21 by complementation testing with known null mutants in the region. The coding regions of candidate genes was sequenced, ultimately identifying a five base pair deletion near the 3' end of the rab3 gene. This deletion throws rab3 out of frame and would lead to a deletion of the last 35 amino acids of the protein, including the final CXC motif that in other systems is required for lipid modification, the binding of Rab3 to synaptic vesicles, and proper Rab3 localization (Graf, 2009 and references therein).

A single ortholog of Drosophila rab3 was previously cloned and demonstrated to be highly conserved. It was further shown to be expressed throughout the fly nervous system; however, no functional studies were performed. To investigate the localization of Rab3 protein and the nature of the mutant allele, we generated a polyclonal antibody to a peptide epitope in the unique C-terminal region of Drosophila Rab3. This antibody stains synaptic terminals of wild-type NMJs in a pattern similar to synaptic vesicle markers such as synapsin and DVGLUT. However, unlike synapsin and DVGLUT, Rab3 staining is further concentrated in a punctate pattern at active zones, as visualized by costaining with Brp. This punctate localization of Rab3 at active zones is not observed in brp mutant NMJs (brp⁶⁹/Df(2R)BSC29) even though the synaptic vesicle-like distribution of Rab3 staining remains. In addition, the antibody recognizes a single band of the predicted size on immunoblots from wild-type larvae. Both the synaptic staining at the NMJ and the band on the immunoblot are absent in the rup mutant, demonstrating that the antibody is specific for Rab3 and that the rup mutant does not express wild-type Rab3 protein. Since the mutation in rup is located in the C-terminal region of rab3 just upstream of the epitope, it is possible that a truncated protein could be expressed. While such a mutant protein could have residual function, the active zone phenotype of rup homozygotes and transheterozygotes of rup and Df(2R)ED2076 are similar in terms of the percentage of GluR clusters apposed to Brp and the average area of individual Brp punctum. Therefore, rup behaves as a genetic null or a very strong hypomorph (Graf, 2009).

This study shows that the small GTPase Rab3 controls the protein composition and release probability of individual active zones at the Drosophila neuromuscular junction. In a rab3 mutant, key constituents of the presynaptic release machinery are enriched at a subset of active zones while the remaining release sites are apparently devoid of these proteins. Expression of Rab3 rapidly and reversibly rescues this altered protein distribution. Physiological studies are consistent with these morphological findings, demonstrating an increase in release probability from an apparently decreased number of release sites. Mechanistic studies indicate that Rab3 functions to increase the probability that the essential synaptic organizing molecule Bruchpilot will cluster at an active zone. This Rab3-dependent regulation of active zone protein composition and release probability provides a potential mechanism for the synapse-specific control of synaptic efficacy (Graf, 2009).

The Drosophila NMJ consists of a motoneuron axon terminal arranged as a chain of synaptic boutons closely associated with the postsynaptic muscle membrane. Within each string of boutons are hundreds of individual synapses, discrete sites of neurotransmitter release where a presynaptic active zone is directly apposed to a postsynaptic glutamate receptor cluster. Such a synapse comprises (1) the site where the axon and muscle membranes are in closest proximity, likely tethered by trans-synaptic cell adhesion molecules; (2) the presynaptic release apparatus that influences the Ca²⁺-mediated release of the neurotransmitter-filled vesicles; and (3) the neurotransmitter receptors and scaffolding and signaling proteins of the postsynaptic density. This study demonstrates that disrupting rab3 alters the distribution of proteins that make up the presynaptic release machinery without grossly disturbing the other two components of the synapse (Graf, 2009).

In the absence of Rab3, a subset of synapses contain increased amounts of the active zone protein Bruchpilot, higher levels of the calcium channel Cacophony, and more electron dense T bars at the active zone. Since Brp is a component of T bars and influences Ca²⁺-channel accumulation, the altered distribution of these components is likely a direct consequence of changes in Brp distribution. The creation of additional active zone markers will be necessary to determine the full extent of this altered distribution. However, since all three components examined influence the probability of evoked vesicle release, the active zones where they accumulate likely are sites of enhanced vesicle release. Conversely, the remaining sites that are devoid of these components likely exhibit impaired evoked release. Two lines of evidence support this conclusion. First, glutamate receptors preferentially cluster opposite sites with the highest release probability. In the rab3^rup mutant, GluR clusters are larger at Brp-positive than Brp-negative sites, suggesting that those active zones containing Brp have a higher release probability. Second, facilitation resulting from short stimulus trains is reduced in the mutant, consistent with an increased release probability (p). However, since quantal content and quantal size are unchanged, the increase in p must be balanced by a decrease in the number of sites that are firing. Hence, both the morphological and electrophysiological data are consistent with the model that Rab3 controls the distribution of active zone proteins to influence the efficacy of individual release sites (Graf, 2009).

Other Drosophila mutants have active zone phenotypes, but none have the combination of phenotypes described in this study. Mutations in synaptojanin, neurexin, and spectrin affect the size and spacing of the entire array of active zones. Mutations in the Unc-51 kinase and the protein phosphatase PP2A have differential affects on active zones, resulting in a subset of glutamate receptors unapposed to Bruchpilot puncta as in the rab3^rup mutant. However, in the unc-51 and PP2A mutants, the remaining Brp puncta are not enlarged and there is no increase in the proportion of active zones with multiple T bars. Such phenotypes are consistent with defects in active zone formation, rather than in the distribution of proteins across active zones. Finally, GluR clusters unapposed to Brp puncta occurs following synaptic retraction, but in such mutants the active zone defects are secondary to the loss of the entire presynaptic terminal. Hence, Rab3 participates in a previously undescribed mechanism that differentially regulates active zones within an NMJ (Graf, 2009).

These findings demonstrate that Rab3 plays a central role in the localization of Bruchpilot to individual active zones. In the absence of Rab3, approximately 70% of active zones are devoid of Brp while the other 30% contain an excess of Brp. What is the function of Rab3 such that its loss leads to this altered Brp distribution? It is suggested that Brp is present in two pools: one fraction bound in complexes at active zones and a second mobile fraction in the cytosol. It is further suggested that Brp is dynamic and may alternate between these two pools by associating with or dissociating from the active zone complex. As such, unbound Brp in the cytosol may either nucleate a cluster at an active zone, creating a new Brp punctum, or add to a pre-existing Brp punctum making it larger. Given this scenario, the rab3 phenotype may be explained by two alternative models of Rab3 function: (1) Rab3 limits Brp puncta size, or (2) Rab3 increases the ability of Brp to nucleate new Brp clusters at active zones. If Rab3 functions to limit the addition of Brp to already existing sites, disruption of Rab3 would allow Brp clusters to grow to a maximal size, reducing the availability of cytosolic Brp to create new puncta and consequently constraining the number of puncta formed. In such a model, it would be predicted that Brp puncta size would be large at rab3^rup mutant NMJs regardless of Brp expression levels. Instead, decreasing Brp levels in the rab3^rup mutant decreases the size of Brp puncta. Even more telling, increasing Brp levels in the rab3^rup mutant also reduces the size of Brp puncta. These results are inconsistent with the model that primary function of Rab3 is to limit the size of Brp puncta (Graf, 2009).

Instead, it is suggested that Rab3 functions to increase the probability that Brp will nucleate a new cluster at an active zone. The presence of Brp at some active zones demonstrates that Rab3 is not absolutely required for Brp localization. Why then is Rab3 required for Brp localization to the 70% of active zones that are bereft of Brp? Rather than posit that these two classes of active zones are fundamentally different in the rab3^rup mutant, it is suggested that in the absence of Rab3, Brp is much less likely to nucleate a cluster at an active zone (Graf, 2009).

The data presented in this study are consistent with this model. First, late rescue with rab3 leads to the rapid addition of new, small Brp puncta and, on a slower time scale, a decrease in the size of the large Brp puncta. This demonstrates that Brp is dynamic and can move into and out of active zones. Second, reducing the levels of Brp at wild-type synapses leads to a decrease in both the number of Brp puncta formed as well as their size. An increase in Brp expression at a wild-type synapse cannot increase the number of puncta since essentially 100% of active zones already contain a Brp puncta, but it does lead to an increase in the size of the puncta. Hence, Brp levels affect both the likelihood of forming a Brp puncta at an active zone as well as the ultimate size of the Brp puncta. Third, increased Brp expression enhances the ability of Brp to cluster at active zones, overcoming the absence of Rab3 and leading to the formation of more Brp puncta in the rab3^rup mutant. This demonstrates that these mutant active zones do have the capacity to cluster Brp, but that it requires the stronger driving force provided by the additional Brp to overcome the absence of Rab3. Finally, when Brp is overexpressed in the rab3^rup mutant the Brp puncta are smaller than when Brp is expressed at wild-type levels. This apparent paradox suggests that Brp puncta compete for unbound Brp and that the large increase in the number of Brp puncta provides more sites for unbound Brp and so ultimately results in smaller puncta. Hence, this model explains the variation in the number and size of Brp puncta present in the various genetic backgrounds tested above, and highlights a novel role for Rab3 in controlling the protein composition of active zones (Graf, 2009).

Brp appears to play a prominent role in the mechanism by which Rab3 regulates the distribution of active zone components to release sites. However, there is no evidence that Rab3 interacts directly with Brp, and such a direct interaction between Rab3 and members of the CAST/ERC family, of which Brp is an ortholog, has not previously been reported. Other proteins could mediate the interaction between Rab3 and Brp. In other species, Rab3 is known to interact with proteins involved in the Rab3 GTPase cycle such as Rab3-GEF, Rab3-GAP, and GDI, as well as the putative Rab3 effectors Sec15, Rabphilin, and Rim. Among the Rab3 effectors, Rabphilin is an unlikely candidate because Rabphilin knockout mice and worms have no observable morphological or physiological synaptic defects. Rim is a more plausible candidate because it is a constituent of the presynaptic release apparatus and binds to many other presynaptic active zone proteins including orthologs of Brp. Alternatively, Rab3 may act on a yet unidentified target to regulate the molecular properties of Brp. Understanding Rab3 function at the fly NMJ will require the identification of the protein(s) Rab3 interacts with to distribute active zone components among release sites (Graf, 2009).

Previous studies of rab3 knockouts in other organisms suggest that Rab3 is involved in regulating vesicle cycling, docking, and exocytosis. While Rab3 may play a direct role in vesicle dynamics and release at the Drosophila NMJ, this study also suggesta that Rab3 plays a second, separate role in influencing the distribution of the presynaptic release apparatus. Defects at the active zone in the rab3^rup mutant are unlikely to be secondary to altered synaptic vesicle release because (1) other mutants affecting release do not disrupt the composition of the active zone and (2) neither increasing nor decreasing activity in the rab3^rup mutant exacerbates or ameliorates the active zone phenotype (Graf, 2009).

If Rab3 controls the protein composition of the active zone, then why have genetic analyses of rab3 in mice and C. elegans not identified structural abnormalities at the synapse? In Drosophila, loss of rab3 results in a very specific ultrastructural phenotype. The active zone, visualized as an electron dense thickening of tightly apposed pre- and postsynaptic membranes, is normal in Drosophila rab3^rup mutants as it is in worms and mice. However, some synapses, including Drosophila NMJ synapses, contain prominent electron-dense specializations such as T bars that are thought to promote transmitter release. It is the distribution of these T bars that is altered in the Drosophila rab3^rup mutant, which would not be apparent at, for example, hippocampal synapses, where such dense bodies are not readily observed. While structural defects have not been detected in other organisms, the electrophysiological findings in mice show interesting parallels to the fly phenotype. The quadruple knockout of Rab3A, Rab3B, Rab3C, and Rab3D in mice demonstrates that Rab3 increases the release probability of a subset of vesicles in the readily releasable pool. Two hypotheses were proposed to explain these findings. The first stays within the traditional vesicle-centric framework for Rab3, suggesting that Rab3 docks specific vesicles to sites of high release probability. The second hypothesis posits that Rab3 recruits additional proteins to the release machinery at certain synapses, thereby making Ca²⁺-mediated release more efficient. This second possibility is consistent with the findings in Drosophila that Rab3 regulates the distribution of release apparatus proteins to control the efficacy of individual sites (Graf, 2009).

Many neurons differentially regulate the release properties of individual release sites along their axonal lengths through presynaptic, synapse-specific mechanisms. These include the regulation of Ca²⁺-channel localization and function and the selective accumulation of group III metabotropic glutamate receptors to specific presynaptic active zones. It is suggested that Rab3 is well positioned to participate in such synapse-specific plasticity mechanisms. The finding that late expression of Rab3 can rapidly reverse the apposition phenotype of the mutant and redistribute Brp to active zones that previously lacked the protein indicates that (1) Brp is highly mobile and (2) Rab3 can rapidly modulate its distribution among individual sites. Multiple proteins control Rab3 function via its GTPase cycle, so mechanisms that locally activate or inhibit Rab3 could lead to rapid and local changes in active zone structure and function. Thus, Rab3 is a candidate to participate in plasticity mechanisms that regulate the protein composition and efficacy of individual release sites (Graf, 2009).

Naked dense bodies provoke depression

At presynaptic active zones (AZs), the frequently observed tethering of synaptic vesicles to an electron-dense cytomatrix represents a process of largely unknown functional significance. This study identified a hypomorphic allele, brp^nude, lacking merely the last 1% of the C-terminal amino acids (17 of 1740) of the active zone protein Bruchpilot. In brp^nude, electron-dense bodies were properly shaped, though entirely bare of synaptic vesicles. While basal glutamate release was unchanged, paired-pulse and sustained stimulation provoked depression. Furthermore, rapid recovery following sustained release was slowed. These results causally link, with intramolecular precision, the tethering of vesicles at the AZ cytomatrix to synaptic depression (Hallermann, 2010).

The specific impairment of vesicle tethering reported in this study delivers the first direct demonstration that efficient sustained release relies on the ability of the AZ to tether vesicles. While the overall AZ structure, including the distribution of Ca²⁺ channels, was unaffected, the impairment of vesicle tethering provoked pronounced synaptic depression and a slowed first component of recovery (Hallermann, 2010).

The C-terminal half of BRP consists of ~1000 aa of essentially contiguous coiled-coil sequence, reminiscent of Golgi/ER-resident tethering factors such as, e.g., GM130. These coiled-coils typically form rod-like structures, where 100 aa residues extend ~15 nm when dimerized, and proteins such as Uso1p extend over 150 nm. These rod-like proteins are believed to act before SNARE protein assembly by forming contacts between membranes at a distance, thereby increasing the specificity or efficiency of the initial attachment of vesicles (tethering). This study has provided morphological and functional evidence that BRP filaments tether vesicles, and thus further mechanistic comparisons between AZ and Golgi/ER trafficking, e.g., concerning the role of small GTPases, might well be informative (Hallermann, 2010).

The C-terminal half of BRP is very highly conserved in insects but not elsewhere. Interestingly, the Drosophila genome does not appear to encode homologs of the vertebrate AZ components Piccolo and Bassoon, which are key regulators of the vertebrate cytomatrix. At central vertebrate synapses, CAST and Bassoon immunoreactivities (closer and further from the AZ membrane, respectively) were recently found to be associated with filaments that may connect vesicles to the AZ. It is tempting to speculate that at AZs of central vertebrate synapses, CAST associates with coiled-coil domain proteins, such as bassoon, to perform the dual functions of Ca²⁺ channel clustering and vesicle tethering executed by the N-terminal and the C-terminal domains of BRP, respectively (Hallermann, 2010).

How synapses manage to repetitively release transmitter with high precision is intensely investigated. Vesicles tethered to electron-dense bodies may represent a reservoir of vesicles required for sustained release. Consistent with this hypothesis, synaptic stimulation provokes depletion of vesicles tethered at dense bodies. While the supply of vesicles appears rate limiting during the train and the first component of recovery, the maturation of vesicles closer to Ca²⁺ channels appears rate limiting during the second component of recovery. One may argue that the rapid component of depression observed at brp^nude synapses could be partially attributed to fewer docked vesicles (though not significantl) with a higher initial release probability. However, a functional estimation of the number of readily releasable vesicles using back-extrapolation from the cumulative EPSC amplitudes in the trains revealed similar numbers of readily releasable vesicles in brp^nude and controls. Finally, it is pointed out that the C-term of BRP could be involved in endocytotic mechanisms, which have been shown to be crucial for sustained release. Novel techniques have begun to address the spatial organization of local vesicle reuse within active zones. It will have to be clarified via which routes vesicles move within active zones and in which direction Bruchpilot steers their translocation (Hallermann, 2010).

Bruchpilot, a synaptic active zone protein for anesthesia-resistant memory

In Drosophila, aversive associative memory of an odor consists of heterogeneous components with different stabilities. This study report that Bruchpilot (Brp), a ubiquitous presynaptic active zone protein, is required for olfactory memory. Brp was shown before to facilitate efficient vesicle release, particularly at low stimulation frequencies. Transgenic knockdown in the Kenyon cells of the mushroom body, the second-order olfactory interneurons, revealed that Brp is required for olfactory memory. It was further demonstrated that Brp in the Kenyon cells preferentially functions for anesthesia-resistant memory. Another presynaptic protein, Synapsin, was shown previously to be required selectively for the labile anesthesia-sensitive memory, which is less affected in brp knockdown. Thus, consolidated and labile components of aversive olfactory memory can be dissociated by the function of different presynaptic proteins (Knapek, 2011).

In Drosophila, middle-term olfactory memory after a single training cycle comprises functionally dissociable forms of memory: the labile ASM and the stable ARM. In contrast to ASM, the molecular basis of ARM formation is poorly understood. Only a few molecules have been shown to be important for ARM so far. This study demonstrates that Brp in the Kenyon cells of the mushroom body is preferentially required for ARM. Although there is no apparent developmental defect in the downregulation of Brp, the requirement for ARM in the adult was not specified in previous studies (Knapek, 2011).

The Brp protein is specifically localized to the active zone at the presynaptic terminals, in which it forms electron dense projections. Interestingly, the Radish protein that is also required for ARM is highly enriched in the lobes of the mushroom body. Thus, Brp and Radish might interact at the active zones to regulate neurotransmission underlying ARM (Knapek, 2011).

Several memory mutants have been shown to have a selective phenotype in ASM. Consistent with the parallel memory formation of ASM and ARM, the brp knockdown and a rutabaga mutation caused an additive memory deficit. Interestingly, Synapsin is required for ASM preferentially, and the null mutation caused no augmentation of the memory phenotype in the rutabaga single mutant (Knapek, 2010). Thus, the complementary forms of memory might recruit differential signaling mechanisms that rely on distinct presynaptic machineries (Knapek, 2011).

In a current model of memory dynamics, ARM gradually develops after training, whereas ASM occupies the largest part of early memory and decays more quickly. Although Radish and Brp are selectively required for ARM measured at 3 h after training, flies lacking either of the proteins are impaired also in immediate memory. By applying cold anesthesia for STM, it was found that STM does contain a significant ARM component. The consistent requirement of Brp for short-term and 3 h ARM may contribute to a synaptic mechanism of memory that is stable against amnesic treatment (Knapek, 2011).

If ARM and ASM were formed at the same synapses of Kenyon cells, how could the two synaptic proteins Brp and Synapsin dissociate these different forms of memory? Notably, Brp and Synapsin are meant to be required for distinct components of action potential-evoked vesicle release. In vertebrates, Synapsin has been shown to be particularly important for recruitment of synaptic vesicles from reserve pools at high stimulation frequencies. Consistently, synapsin mutants show normal quantal content (number of synaptic vesicles released per action potential) at moderate action potential frequencies. Similarly, Drosophila Synapsin maintains the reserve pool of vesicles and mediates mobilization of the reserve pool during intense stimulation. The brp null mutant in contrast shows decreased quantal contents particularly in response to the first arrival of an action potential. Vesicle release after subsequently following high-frequency spikes however is less affected, suggesting the importance in vesicle release at low-frequency stimulation. The two different modes of neurotransmission (e.g., different release probabilities during high- vs low-frequency stimulation) could therefore differentiate ASM and ARM, even if the traces of these different forms of memory resided in the same synapses of the Kenyon cells (Knapek, 2011).

Alternatively, the memory traces of ASM and ARM could be spatially separated within the same neurons, i.e., localized at different synapse populations. In the lobes, Kenyon cell axons have multiple compartments that are intersected by transverse extrinsic neurons. This study found that brp knockdown in the α/β neurons affected ARM. This is consistent with a previous report, in which inhibition of the output of the α/β neurons impaired ARM. Interestingly, inhibition of a specific type of dopaminergic neurons that synapse onto another restricted compartment of the β lobe selectively affected ASM (Aso, 2010). Thus, associative plasticity underlying ASM and ARM could be formed by stimulating different synapses of the same neurons. This hypothesis may be tested in future by the identification of extrinsic neurons that are specifically required for ARM and corresponding functional imaging of memory traces (Knapek, 2011).

Search PubMed for articles about Drosophila Bruchpilot

Altrock, W. D., et al. (2003). Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon. Neuron 37(5): 787-800. Medline abstract: 12628169

Aso, Y., et al. (2009) The mushroom body of adult Drosophila characterized by GAL4 drivers. J Neurogenet 23: 156-172. PubMed Citation: 19140035

Blatow M, et al. (2003). Ca2+ buffer saturation underlies paired pulse facilitation in calbindin-D28k-containing terminals. Neuron 38: 79-88. PubMed Citation: 12691666

Deguchi-Tawarada, M., et al. (2004). CAST2: identification and characterization of a protein structurally related to the presynaptic cytomatrix protein CAST. Genes Cells 9(1): 15-23. Medline abstract: 14723704

Deken, S. L., et al. (2005). Redundant localization mechanisms of RIM and ELKS in Caenorhabditis elegans. J. Neurosci. 25(25): 5975-83. Medline abstract: 15976086

Delgado, R, et al. (2000). Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire. Neuron 28: 941-953. PubMed Citation: 11163278

tom Dieck, S., et al. (1998). Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals. J. Cell Biol. 142(2): 499-509. Medline abstract: 9679147

Fenster, S. D. et al. (2000). Piccolo, a presynaptic zinc finger protein structurally related to bassoon. Neuron 25: 203-214. Medline abstract: 10707984

Fouquet, W., et al. (2009). Maturation of active zone assembly by Drosophila Bruchpilot. J. Cell Biol. 186(1): 129-45. PubMed Citation: 19596851

Graf, E. R., et al. (2009). Rab3 dynamically controls protein composition at active zones. Neuron 64(5): 663-77. PubMed Citation: 20005823

Hallermann, S, et al. (2010). Naked dense bodies provoke depression. J. Neurosci. 30(43): 14340-5. PubMed Citation: 20980589

Kim, S. M., et al. (2009). Fos and Jun potentiate individual release sites and mobilize the reserve synaptic-vesicle pool at the Drosophila larval motor synapse. Proc. Natl. Acad. Sci. 106(10): 4000-4005. PubMed Citation: 19228945

Kittel, R. J., et al. (2006). Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312(5776): 1051-4. Medline abstract: 16614170

Knapek, S., Gerber B. and Tanimoto, H. (2010) Synapsin is selectively required for anesthesia-sensitive memory. Learn Mem. 17: 76-79. PubMed Citation: 20154352

Knapek, S., Sigrist, S. and Tanimoto, H. (2011). Bruchpilot, a synaptic active zone protein for anesthesia-resistant memory. J. Neurosci. 31(9): 3453-8. PubMed Citation: 21368057

Ko, J., Na, M., Kim, S., Lee, J. R. and Kim, E. (2003). Interaction of the ERC family of RIM-binding proteins with the liprin-alpha family of multidomain proteins. J. Biol. Chem. 278: 42377-42385. Medline abstract: 12923177

Murthy, V. N. and Stevens, C. F. (1999). Reversal of synaptic vesicle docking at central synapses. Nat Neurosci. 2: 503-507. PubMed Citation: 10448213

Ohara-Imaizumi, M., et al. (2005). ELKS, a protein structurally related to the active zone-associated protein CAST, is expressed in pancreatic {beta} cells and functions in insulin exocytosis: interaction of ELKS with exocytotic machinery analyzed by total internal reflection fluorescence microscopy. Mol. Biol. Cell 16: 3289-3300. Medline abstract: 15888548

Ohtsuka, T. et al. (2002). Cast: a novel protein of the cytomatrix at the active zone of synapses that forms a ternary complex with RIM1 and munc13-1. J. Cell Biol. 158(3): 577-90. Medline abstract: 12163476

Rasse, T. M., et al. (2005) Glutamate receptor dynamics organizing synapse formation in vivo. Nat. Neurosci. 8: 898-905. PubMed Citation: 16136672

Sanyal, S., et al. (2002). AP-1 functions upstream of CREB to control synaptic plasticity in Drosophila. Nature 416: 870-874. PubMed Citation: 11976688

Shapira, M., et al. (2003). Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles. Neuron 38: 237-252. Medline abstract: 12718858

Takao-Rikitsu, E., et al. (2004). Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release. J. Cell Biol. 164(2): 301-11. Medline abstract: 14734538

Verstreken, P., et al. (2005). Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions. Neuron 47: 365-378. PubMed Citation: 16055061

Wagh, D. A., (2006). Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila. Neuron 49(6): 833-44. Medline abstract: 16543132

Wang, Y., et al. (2002). A family of RIM-binding proteins regulated by alternative splicing: implications for the genesis of synaptic active zones. Proc. Natl. Acad. Sci. 99: 14464-14469. Medline abstract: 12391317

Wang, Y., et al. (1997). Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion. Nature 388: 593-598. Medline abstract: 9252191

Zakharenko, S. S., et al. (2003). Presynaptic BDNF required for a presynaptic but not postsynaptic component of LTP at hippocampal CA1-CA3 synapses. Neuron 39: 975-990. PubMed Citation: 12971897

Zucker, R. S. and Regehr, W. G. (2002). Short-term synaptic plasticity. Annu. Rev. Physiol. 64: 355-405. PubMed Citation: 11826273

date revised: 15 December 2011

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