Complexin2 null mutation requires a 'second hit' for induction of phenotypic changes relevant to schizophrenia.

Schizophrenia is a devastating disease that affects approximately 1% of the population across cultures. Its neurobiological underpinnings are still unknown. Accordingly, animal models of schizophrenia often lack construct validity. As concordance rate in monozygotic twins amounts to only 50%, environmental risk factors (e.g. neurotrauma, drug abuse, psychotrauma) likely act as necessary 'second hit' to trigger/drive the disease process in a genetically predisposed individual. Valid animal models would have to consider this genetic-environmental interaction. Based on this concept, we designed an experimental approach for modeling a schizophrenia-like phenotype in mice. As dysfunction in synaptic transmission plays a key role in schizophrenia, and complexin2 (CPLX2) gene expression is reduced in hippocampus of schizophrenic patients, we developed a mouse model with Cplx2 null mutation as genetic risk factor and a mild parietal neurotrauma, applied during puberty, as environmental 'second hit'. Several months after lesion, Cplx2 null mutants showed reduced pre-pulse inhibition, deficit of spatial learning and loss of inhibition after MK-801 challenge. These abnormalities were largely absent in lesioned wild-type mice and non-lesioned Cplx2 null mutants. Forced alternation in T-maze, object recognition, social interaction and elevated plus maze tests were unaltered in all groups. The previously reported mild motor phenotype of Cplx2 null mutants was accentuated upon lesion. MRI volumetrical analysis showed a decrease of hippocampal volume exclusively in lesioned Cplx2 null mutants. These findings provide suggestive evidence for the 'second hit' hypothesis of schizophrenia and may offer new tools for the development of advanced treatment strategies.

Munc13-2 differentially affects hippocampal synaptic transmission and plasticity.

The short-term dynamics of synaptic communication between neurons provides neural networks with specific frequency-filter characteristics for information transfer. The direction of short-term synaptic plasticity, that is, facilitation versus depression, is highly dependent on and inversely correlated to the basal release probability of a synapse. Amongst the processes implicated in shaping the release probability, proteins that regulate the docking and priming of synaptic vesicles at the active zone are of special importance. Here, we found that a member of the Munc13 protein family of priming proteins, namely Munc13-2, is essential for normal release probability at hippocampal mossy fiber synapses. Paired pulse and frequency facilitation were strongly increased, whereas mossy fiber long-term potentiation was unaffected in the absence of Munc13-2. In contrast, transmission at 3 other types of hippocampal synapses, Schaffer-collateral, associational-commissural, as well as inhibitory synapses onto CA3 pyramidal neurons was unaffected by the loss of Munc13-2.

Neuroligin-3-deficient mice: model of a monogenic heritable form of autism with an olfactory deficit.

Autism spectrum disorder (ASD) is a frequent neurodevelopmental disorder characterized by variable clinical severity. Core symptoms are qualitatively impaired communication and social behavior, highly restricted interests and repetitive behaviors. Although recent work on genetic mutations in ASD has shed light on the pathophysiology of the disease, classifying it essentially as a synaptopathy, no treatments are available to date. To develop and test novel ASD treatment approaches, validated and informative animal models are required. Of particular interest, in this context are loss-of-function mutations in the postsynaptic cell adhesion protein neuroligin-4 and point mutations in its homologue neuroligin-3 (NL-3) that were found to cause certain forms of monogenic heritable ASD in humans. Here, we show that NL-3-deficient mice display a behavioral phenotype reminiscent of the lead symptoms of ASD: reduced ultrasound vocalization and a lack of social novelty preference. The latter may be related to an olfactory deficiency observed in the NL-3 mutants. Interestingly, such olfactory phenotype is also present in a subgroup of human ASD patients. Tests for learning and memory showed no gross abnormalities in NL-3 mutants. Also, no alterations were found in time spent in social interaction, prepulse inhibition, seizure propensity and sucrose preference. As often seen in adult ASD patients, total brain volume of NL-3 mutant mice was slightly reduced as assessed by magnetic resonance imaging (MRI). Our findings show that the NL-3 knockout mouse represents a useful animal model for understanding pathophysiological events in monogenic heritable ASD and for developing novel treatment strategies in this devastating human disorder.

Increased anxiety-like behavior in mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2.

Neuroligins (NL) are postsynaptic cell adhesion molecules that are thought to specify synapse properties. Previous studies showed that mutant mice carrying an autism-associated point mutation in NL3 exhibit social interaction deficits, enhanced inhibitory synaptic function and increased staining of inhibitory synaptic puncta without changes in overall inhibitory synapse numbers. In contrast, mutant mice lacking NL2 displayed decreased inhibitory synaptic function. These studies raised two relevant questions. First, does NL2 deletion impair inhibitory synaptic function by altering the number of inhibitory synapses, or by changing their efficacy? Second, does this effect of NL2 deletion on inhibition produce behavioral changes? We now show that although NL2-deficient mice exhibit an apparent decrease in number of inhibitory synaptic puncta, the number of symmetric synapses as determined by electron microscopy is unaltered, suggesting that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers. This decrease in inhibitory synaptic function in NL2-deficient mice correlates with a discrete behavioral phenotype that includes a marked increase in anxiety-like behavior, a decrease in pain sensitivity and a slight decrease in motor co-ordination. This work confirms that NL2 modulates inhibitory synaptic function and is the first demonstration that global deletion of NL2 can lead to a selective behavioral phenotype.

Enhanced anxiety, depressive-like behaviour and impaired recognition memory in mice with reduced expression of the vesicular glutamate transporter 1 (VGLUT1).

Three isoforms of a vesicular glutamate transporter (VGLUT1-3) have been identified. Of these, VGLUT1 is the major isoform in the cerebral cortex and hippocampus where it is selectively located on synaptic vesicles of excitatory glutamatergic terminals. Variations in VGLUT1 expression levels have a major impact on the efficacy of glutamate synaptic transmission. Given evidence linking alterations in glutamate neurotransmission to various neuropsychiatric disorders, we investigated the possible influence of a down-regulation of VGLUT1 transporter on anxiety, depressive-like behaviour and learning. The behavioural phenotype of VGLUT1-heterozygous mice (C57BL/6) was compared to wild-type (WT) littermates. Moreover, VGLUT1-3 expression, hippocampal excitatory terminal ultrastructure and neurochemical phenotype were analysed. VGLUT1-heterozygous mice displayed normal spontaneous locomotor activity, increased anxiety in the light-dark exploration test and depressive-like behaviour in the forced swimming test: no differences were shown in the elevated plus-maze model of anxiety. In the novel object recognition test, VGLUT1(+/-) mice showed normal short-term but impaired long-term memory. Spatial memory in the Morris water maze was unaffected. Western blot analysis confirmed that VGLUT1 heterozygotes expressed half the amount of transporter compared to WT. In addition, a reduction in the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35-45% reduction in GABA in the frontal cortex and the hippocampus were observed in the mutant mice. These observations suggest that a VGLUT1-mediated presynaptic alteration of the glutamatergic synapses, in specific brain regions, leads to a behavioural phenotype resembling certain aspects of psychiatric and cognitive disorders.

An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size.

Quantal neurotransmitter release at excitatory synapses depends on glutamate import into synaptic vesicles by vesicular glutamate transporters (VGLUTs). Of the three known transporters, VGLUT1 and VGLUT2 are expressed prominently in the adult brain, but during the first two weeks of postnatal development, VGLUT2 expression predominates. Targeted deletion of VGLUT1 in mice causes lethality in the third postnatal week. Glutamatergic neurotransmission is drastically reduced in neurons from VGLUT1-deficient mice, with a specific reduction in quantal size. The remaining activity correlates with the expression of VGLUT2. This reduction in glutamatergic neurotransmission can be rescued and enhanced with overexpression of VGLUT1. These results show that the expression level of VGLUTs determines the amount of glutamate that is loaded into vesicles and released and thereby regulates the efficacy of neurotransmission.

Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming.

Synaptic neurotransmitter release is restricted to active zones, where the processes of synaptic vesicle tethering, priming to fusion competence, and Ca2+-triggered fusion are taking place in a highly coordinated manner. We show that the active zone components Munc13-1, an essential vesicle priming protein, and RIM1, a Rab3 effector with a putative role in vesicle tethering, interact functionally. Disruption of this interaction causes a loss of fusion-competent synaptic vesicles, creating a phenocopy of Munc13-1-deficient neurons. RIM1 binding and vesicle priming are mediated by two distinct structural modules of Munc13-1. The Munc13-1/RIM1 interaction may create a functional link between synaptic vesicle tethering and priming, or it may regulate the priming reaction itself, thereby determining the number of fusion-competent vesicles.

The synaptic vesicle priming protein Munc13-1 is absent from tonically active ribbon synapses of the rat retina.

Ribbon synapses, for example of the retina, are specialized synapses that differ from conventional, phasically active synapses in several aspects. Ribbon synapses can tonically and yet very rapidly release neurotransmitter via synaptic vesicle exocytosis. This requires an optimization of the synaptic machinery and is at least partly due to the presence of synaptic ribbons that bind large numbers of synaptic vesicles and which are believed to participate in priming synaptic vesicles for exocytosis. In this paper we analyzed whether ribbon synapses of the retina employ similar priming factors, i.e. Munc13-1, as do conventional, non-ribbon containing phasically active synapses. We found that though present in conventional synapses of the retina Munc13-1 was completely absent from ribbon-containing synapses of the retina, both in the outer as well as in the inner plexiform layer. This indicates that ribbon synapses of the retina employ other, possibly more potent priming factors than phasically active conventional synapses.

Synaptotagmin I functions as a calcium regulator of release probability.

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors--which are the gatekeepers to neurotransmission--remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.

Complexins regulate a late step in Ca2+-dependent neurotransmitter release.

Synaptic vesicle fusion at synapses is triggered by increases in cytosolic Ca2+ levels. However, the identity of the Ca2+ sensor and the transduction mechanism of the Ca2+ trigger are unknown. We show that Complexins, stoichiometric components of the exocytotic core complex, are important regulators of transmitter release at a step immediately preceding vesicle fusion. Neurons lacking Complexins show a dramatically reduced transmitter release efficiency due to decreased Ca2+ sensitivity of the synaptic secretion process. Analyses of mutant neurons demonstrate that Complexins are acting at or following the Ca2+-triggering step of fast synchronous transmitter release by regulating the exocytotic Ca2+ sensor, its interaction with the core complex fusion machinery, or the efficiency of the fusion apparatus itself.

The cerebellum-specific Munc13 isoform Munc13-3 regulates cerebellar synaptic transmission and motor learning in mice.

Munc13 proteins form a family of three, primarily brain-specific phorbol ester receptors (Munc13-1/2/3) in mammals. Munc13-1 is a component of presynaptic active zones in which it acts as an essential synaptic vesicle priming protein. In contrast to Munc13-1, which is present in most neurons throughout the rat and mouse CNS, Munc13-3 is almost exclusively expressed in the cerebellum. Munc13-3 mRNA is present in granule and Purkinje cells but absent from glia cells. Munc13-3 protein is localized to the synaptic neuropil of the cerebellar molecular layer but is not found in Purkinje cell dendrites, suggesting that Munc13-3, like Munc13-1, is a presynaptic protein at parallel fiber-Purkinje cell synapses. To examine the role of Munc13-3 in cerebellar physiology, we generated Munc13-3-deficient mutant mice. Munc13-3 deletion mutants exhibit increased paired-pulse facilitation at parallel fiber-Purkinje cell synapses. In addition, mutant mice display normal spontaneous motor activity but have an impaired ability to learn complex motor tasks. Our data demonstrate that Munc13-3 regulates synaptic transmission at parallel fiber-Purkinje cell synapses. We propose that Munc13-3 acts at a similar step of the synaptic vesicle cycle as does Munc13-1, albeit with less efficiency. In view of the present data and the well established vesicle priming function of Munc13-1, it is likely that Munc13-3-loss leads to a reduction in release probability at parallel fiber-Purkinje cell synapses by interfering with vesicle priming. This, in turn, would lead to increases in paired-pulse facilitation and could contribute to the observed deficit in motor learning.

Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells.

In chromaffin cells the number of large dense-core vesicles (LDCVs) which can be released by brief, intense stimuli represents only a small fraction of the 'morphologically docked' vesicles at the plasma membrane. Recently, it was shown that Munc13-1 is essential for a post-docking step of synaptic vesicle fusion. To investigate the role of Munc13-1 in LDCV exocytosis, we overexpressed Munc13-1 in chromaffin cells and stimulated secretion by flash photolysis of caged calcium. Both components of the exocytotic burst, which represent the fusion of release-competent vesicles, were increased by a factor of three. The sustained component, which represents vesicle maturation and subsequent fusion, was increased by the same factor. The response to a second flash, however, was greatly reduced, indicating a depletion of release-competent vesicles. Since there was no apparent change in the number of docked vesicles, we conclude that Munc13-1 acts as a priming factor by accelerating the rate constant of vesicle transfer from a pool of docked, but unprimed vesicles to a pool of release-competent, primed vesicles.

Definition of Munc13-homology-domains and characterization of a novel ubiquitously expressed Munc13 isoform.

Munc13 proteins constitute a family of three highly homologous molecules (Munc13-1, Munc13-2 and Munc13-3). With the exception of a ubiquitously expressed Munc13-2 splice variant, Munc13 proteins are brain-specific. Munc13-1 has a central priming function in synaptic vesicle exocytosis from glutamatergic synapses. In order to identify Munc13-like proteins that may regulate secretory processes in non-glutamatergic neurons or non-neuronal cells, we developed protein profiles for two Munc13-homology-domains (MHDs). MHDs are present in a wide variety of proteins, some of which have previously been implicated in membrane trafficking reactions. Taking advantage of partial sequences in the human expressed sequence tag (EST) database, we characterized a novel, ubiquitously expressed, rat protein (Munc13-4) that belongs to a subfamily of Munc13-like molecules, in which the typical Munc13-like domain structure is conserved. Munc13-4 is predominantly expressed in lung where it is localized to goblet cells of the bronchial epithelium and to alveolar type II cells, both of which are cell types with secretory function. In the present study we identify a group of novel proteins, some of which may function in a Munc13-like manner to regulate membrane trafficking. The MHD profiles described in the present study are useful tools for the identification of Munc13-like proteins, that would otherwise have remained undetected.

Regulation of transmitter release by Unc-13 and its homologues.

Neurotransmitters are released by Ca(2+)-triggered exocytotic fusion of synaptic vesicles. Before fusion, vesicles dock at a specialised presynaptic plasma membrane region, the active zone, where they are primed to a fusion competent state. The nature of this priming reaction has long been enigmatic. Recent evidence demonstrates that priming is an essential and rate-limiting step in secretion from neurons and neuroendocrine cells. Members of the Unc-13 protein family, which are highly conserved during evolution and act as novel targets of the diacylglycerol second-messenger pathway, have been identified to play an essential role in this process.

Synaptic cell adhesion proteins and synaptogenesis in the mammalian central nervous system.

Synapses are asymmetric cell-cell contacts, typically formed between the presynaptic axon terminal of a "sending" nerve cell and the postsynaptic dendrite, the soma or - in some cases - the axon of a "receiving" one. The presynaptic axon terminal is specialized for the complex membrane trafficking mechanisms that underlie regulated secretion of neurotransmitter, while the postsynapse is uniquely specialized for signal transduction. Synaptogenesis, the formation of functional synapses, is the final step in the development of the central nervous system. In the mammalian brain it results in the establishment of a neural network, connecting some 10(12) nerve cells with up to 10(15) synapses. In principle, synaptogenesis takes place in two consecutive steps that are most likely mediated by cell adhesion molecules. First, an arriving axonal growth cone identifies its appropriate partner cell, creating an initial contact, and, second, specific axonal and dendritic protein components are recruited to this initial contact site, forming a functional synapse. Three cell adhesion systems have recently been shown to be specifically enriched at synaptic contacts: the cadherin/catenin system, the cadherinlike neuronal receptors, and the beta-neurexin/neuroligin system. Components of all three cell adhesion systems have been localized to synaptic contacts using immunogold electron microscopy but are also present outside of synapses. The present short review discusses the possible role of these synaptic cell adhesion molecules in synaptogenesis.

Evidence of presynaptic location and function of the prion protein.

The prion protein (PrP(C)) is a copper-binding protein of unknown function that plays an important role in the etiology of transmissible spongiform encephalopathies. Using morphological techniques and synaptosomal fractionation methods, we show that PrP(C) is predominantly localized to synaptic membranes. Atomic absorption spectroscopy was used to identify PrP(C)-related changes in the synaptosomal copper concentration in transgenic mouse lines. The synaptic transmission in the presence of H(2)O(2), which is known to be decomposed to highly reactive hydroxyl radicals in the presence of iron or copper and to alter synaptic activity, was studied in these animals. The response of synaptic activity to H(2)O(2) was found to correlate with the amount of PrP(C) expression in the presynaptic neuron in cerebellar slice preparations from wild-type, Prnp(0/0), and PrP gene-reconstituted transgenic mice. Thus, our data gives strong evidence for the predominantly synaptic location of PrP(C), its involvement in the regulation of the presynaptic copper concentration, and synaptic activity in defined conditions.

Transient, phorbol ester-induced DOC2-Munc13 interactions in vivo.

Munc13-1 and DOC2 have been implicated in the regulation of exocytosis. Here we demonstrate in vivo that these two proteins undergo a transient phorbol ester-mediated and protein kinase C-independent interaction, resulting in the translocation of DOC2 from a vesicular localization to the plasma membrane. The translocation of DOC2 is dependent upon the DOC2 Munc interacting domain that binds specifically to Munc13-1, whereas the association of DOC2 with intracellular membranes is dependent on its C2 domains. This is the first direct in vivo demonstration of a protein-protein interaction between two presynaptic proteins and may represent a molecular basis for phorbol ester-dependent enhancement of exocytosis.

An efficient method for infection of adrenal chromaffin cells using the Semliki Forest virus gene expression system.

We have expanded the use of the Semliki Forest virus (SFV) by infecting chromaffin cells with synaptic proteins at high efficiency. Using the SFV gene expression system, up to 40% of cultured bovine chromaffin cells express the protein of interest within 12-48 h after infection. In order to learn about the basic physiological properties of infected cells, we performed membrane capacitance measurements using the whole-cell patch-clamp technique and monitored catecholamine release with amperometry. We found that chromaffin cells infected with green fluorescent protein (GFP) were comparable to control cells in intracellular calcium concentrations ([Ca2+]i), leak currents and cell sizes. In response to depolarization, calcium currents were elicited and the cells secreted catecholamine. Comparison of the calcium current amplitude and the size of the readily releasable pool of vesicles revealed a small decrease in these parameters compared to control cells. The refilling kinetics after pool depletion, however, were not altered. Overexpressed munc13-1 translocates to the plasma membrane in response to phorbol esters, an effect that is also observed in fibroblasts transfected with conventional methods. Thus, the use of the SFV gene expression system to infect chromaffin cells represents a major improvement in infection efficiency compared to other methods. It opens up new opportunities to introduce synaptic proteins into chromaffin cells and study their role in secretion.

Direct interaction between the ARF-specific guanine nucleotide exchange factor msec7-1 and presynaptic Munc13-1.

Msec7-1, a mammalian homologue of yeast sec7p, is a specific GDP/GTP exchange factor for small G-proteins of the ARF family. Overexpression of msec7-1 in Xenopus neuromuscular junctions leads to an increase in synaptic transmitter release that is most likely caused by an increase in the pool of readily releasable vesicles. However, the molecular mechanisms by which msec7-1 is targeted to presynaptic compartments and enhances neurotransmitter release are not known. In the present study, we demonstrate that msec7-1 interacts directly with Munc13-1, a phorbol ester-dependent enhancer of neurotransmitter release that is specifically localized to presynaptic transmitter release zones. Given that Munc13-1 and msec7-1 participate in very similar presynaptic processes and because Munc13-1 is specifically targeted to presynaptic active zones, we suggest that the msec7-1/Munc13-1 interaction serves to colocalize the two proteins at the active zone, a subcellular compartment with extremely high membrane turnover.

Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles.

Neurotransmitter release at synapses between nerve cells is mediated by calcium-triggered exocytotic fusion of synaptic vesicles. Before fusion, vesicles dock at the presynaptic release site where they mature to a fusion-competent state. Here we identify Munc13-1, a brain-specific presynaptic phorbol ester receptor, as an essential protein for synaptic vesicle maturation. We show that glutamatergic hippocampal neurons from mice lacking Munc13-1 form ultrastructurally normal synapses whose synaptic-vesicle cycle is arrested at the maturation step. Transmitter release from mutant synapses cannot be triggered by action potentials, calcium-ionophores or hypertonic sucrose solution. In contrast, release evoked by alpha-latrotoxin is indistinguishable from wild-type controls, indicating that the toxin can bypass Munc13-1-mediated vesicle maturation. A small subpopulation of synapses of any given glutamatergic neuron as well as all synapses of GABA (gamma-aminobutyric acid)-containing neurons are unaffected by Munc13-1 loss, demonstrating the existence of multiple and transmitter-specific synaptic vesicle maturation processes in synapses.