Altered resting-state functional connectivity of default mode network in brachioradial pruritus.

BACKGROUND: Patients with chronic pruritus (CP) have a low quality of life, thus it is important to gain a better understanding of the underlying processes. Previous functional magnetic resonance imaging studies at rest (rsfMRI) have shown that mainly areas associated with the default mode network (DMN), sensorimotor (SMN), frontoparietal (FPN) and salience networks (SN) are involved in the processing of itch in patients with chronic pruritus (CP), as well as the cortico-striatal circuit, which is involved in the motoric preparation of scratching. rsfMRI studies on functional connectivity (FC) patterns of resting-state networks (RSNs) in patients with inflammatory atopic dermatitis (AD) or with neuropathic brachioradial pruritus (BRP) compared with healthy controls (HC) are lacking. OBJECTIVES: The main goals of this study were to investigate whether functional connectivity within networks and areas associated with itch detection and processing are altered in patients with AD and BRP compared with matched healthy controls by rsfMRI, respectively. METHODS: Patients with AD (n = 28) and with BRP (n = 28) were compared with corresponding matched healthy controls by rsfMRI. Group-specific RSNs were identified by independent component analysis (ICA) and between-group differences in the RSNs were analysed by dual regression technique. Seed-based functional connectivity was analysed in several itch-related brain regions belonging to the DMN, SN and FPN, respectively. RESULTS: ICA and seed-based analyses revealed decreased functional connectivity in BRP compared with HC specially within the DMN including the precuneus and cingulate cortex. For AD patients in comparison with HC, as well as when BRP and AD patients were compared directly, no significant FC differences at rest were seen. CONCLUSIONS: Our findings point towards decreased FC particularly in the DMN at rest in patients with BRP. These results seem to indicate that central connectivity patterns at rest differentially encode itch in BRP and AD.

Implementation of a fully digital histology course in the anatomical teaching curriculum during COVID-19 pandemic.

BACKGROUND: During the COVID-19 pandemic, many medical schools are forced to switch courses of the mandatory curriculum to online teaching formats. However, little information about feasibility and effectiveness is available yet about distance teaching in anatomy. The aim of this study was to evaluate the implementation of a histology course previously taught in a classroom setting into an online-only format based on video conference software. METHODS: Our course design included theoretical introductions, an online-adaptation of virtual microscopy used previously in the classroom, and active learning elements such as collaborative learning in breakout rooms, annotation assignments and multiple-choice questions. Two preclinical semester cohorts of around 400 second and third semester students were taught in histology in parallel courses, using the Zoom software platform. We analyzed data about student attendance during the course, summative quantitative and qualitative evaluation of the students and results of a written test required to pass the course. RESULTS: We observed that student attendance was high and stable during the 19 course days for both second and third semester, and only few students reported technical problems. There were no significant differences in examination results of second semester compared to the third semester, an unexpected result as the third semester already participated in the dissection course before. Similarly, no significant gender-related effects on the examination performance could be noted in both semesters. However, the age of students was negatively correlated with test scores in the second and third semester. Importantly, the overall evaluation of the digital version of the histology course was at least as positive as the in-person version over the past years. CONCLUSION: Together, we experienced that the implementation of a curricular histology course in an online-format is technically realizable, effective and well accepted among students. We also observed that availability and prior experience with digitized specimen in virtual microscopy facilitates transition into an online-only setting. Thus, our study supports the positive potential of distance learning for teaching anatomy during and after COVID-19 pandemic but also emphasizes the need for a synchronous learning environment with partially personnel-intensive small group settings to overcome passivity and inequality aspects, and to foster active learning elements.

Synaptic assembly of the brain in the absence of neurotransmitter secretion.

Brain function requires precisely orchestrated connectivity between neurons. Establishment of these connections is believed to require signals secreted from outgrowing axons, followed by synapse formation between selected neurons. Deletion of a single protein, Munc18-1, in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles throughout development. However, this does not prevent normal brain assembly, including formation of layered structures, fiber pathways, and morphologically defined synapses. After assembly is completed, neurons undergo apoptosis, leading to widespread neurodegeneration. Thus, synaptic connectivity does not depend on neurotransmitter secretion, but its maintenance does. Neurotransmitter secretion probably functions to validate already established synaptic connections.

SV2A and SV2B function as redundant Ca2+ regulators in neurotransmitter release.

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor isoform (SV2C) that resemble transporter proteins. We now show that SV2B knockout mice are phenotypically normal while SV2A- and SV2A/SV2B double knockout mice exhibit severe seizures and die postnatally. In electrophysiological recordings from cultured hippocampal neurons, SV2A- or SV2B-deficient cells exhibited no detectable abnormalities. Neurons lacking both SV2 isoforms, however, experienced sustained increases in Ca2+-dependent synaptic transmission when two or more action potentials were triggered in succession. These increases could be reversed by EGTA-AM. Our data suggest that without SV2 proteins, presynaptic Ca2+ accumulation during consecutive action potentials causes abnormal increases in neurotransmitter release that destabilize synaptic circuits and induce epilepsy.

Neurexins: three genes and 1001 products.

The human brain has approximately 10(12) neurons, three orders of magnitude more than there are basepairs in the human genome. Each neuron is connected to other neurons by thousands of synapses, creating a dense network of communicating neurons. Cell-recognition events between neurons at, and outside of synapses, are likely to guide the development and maintenance of the complex network formed by neurons. However, little is known about which proteins are important for neuronal cell recognition. Neurexins, a family of polymorphic cell-surface proteins, might mediate some of these cell recognition events. Thousands of neurexin isoforms are generated from three genes by usage of alternative promoters and alternative splicing. These isoforms are displayed on the neuronal cell surface, with different classes of neurons expressing distinct combinations of isoforms. Neurexins probably have a multitude of ligands, some of which interact only with subsets of neurexin isoforms. This review describes the properties of the neurexin protein family and their potential roles in neuronal cell adhesion and intercellular signaling.

alpha-Neurexins are required for efficient transmitter release and synaptic homeostasis at the mouse neuromuscular junction.

Neurotransmission at chemical synapses of the brain involves alpha-neurexins, neuron-specific cell-surface molecules that are encoded by three genes in mammals. Deletion of alpha-neurexins in mice previously demonstrated an essential function, leading to early postnatal death of many double-knockout mice and all triple mutants. Neurotransmitter release at central synapses of newborn knockouts was severely reduced, a function of alpha-neurexins that requires their extracellular sequences. Here, we investigated the role of alpha-neurexins at neuromuscular junctions, presynaptic terminals that lack a neuronal postsynaptic partner, addressing an important question because the function of neurexins was hypothesized to involve cell-adhesion complexes between neurons. Using systems physiology, morphological analyses and electrophysiological recordings, we show that quantal content, i.e. the number of acetylcholine quanta released per nerve impulse from motor nerve terminals, and frequency of spontaneous miniature endplate potentials at the slow-twitch soleus muscle are reduced in adult alpha-neurexin double-knockouts, consistent with earlier data on central synapses. However, the same parameters at diaphragm muscle neuromuscular junctions showed no difference in basal neurotransmission. To reconcile these observations, we tested the capability of control and alpha-neurexin-deficient diaphragm neuromuscular junctions to compensate for an experimental reduction of postsynaptic acetylcholine receptors by a compensatory increase of presynaptic release: Knockout neuromuscular junctions produced significantly less upregulation of quantal content than synapses from control mice. Our data suggest that alpha-neurexins are required for efficient neurotransmitter release at neuromuscular junctions, and that they may perform a role in the molecular mechanism of synaptic homeostasis at these peripheral synapses.

Pre- and postnatal development of the primary visual cortex of the common marmoset. II. Formation, remodelling, and elimination of synapses as overlapping processes.

During ontogenesis changes in the numerical density of synapses are usually assumed to depend essentially on variations in the formation of synapses. Only the final adjustment to adult synapse densities is thought to include the elimination of synapses in some brain regions of certain species. Here, we focus attention on quantitative aspects of synapse elimination throughout development of area 17 of marmoset monkeys (Callithrix jacchus). Mature synapses, various precursor forms, and indicators of lysosomal degradation of synapses were quantitatively analysed by electron microscopy and morphometric methods. A total number of about 135 x 10(9) synapses was calculated for area 17 in each adult hemisphere corresponding to a volume density of 600 x 10(6) synapses/mm3. At 3 months of age, the respective values were 508 x 10(9)/area and 1,159 x 10(6)/mm3, while at birth these values were 69 x 10(9)/area and 328 x 10(6)/mm3. Consequently, at least three out of four synapses are eliminated between 3 months and adulthood. However, the real number of synapses being eliminated during development is probably much larger if the time course of lysosomal degradation is additionally taken into account. The frequency of lysosomes in presynaptic endings is highest before net-elimination of synapses occurs, i.e., between 1 and 3 months. This suggests that lysosomal degradation is not directly responsible for the majority of synapses removed during ontogenesis but apparently represents a second mechanism for synapse remodelling and elimination. Thus, it appears from this study that remodelling and elimination of synapses are quantitatively as important as their formation, and accompany synaptogenesis from its very onset onwards.

Pre- and postnatal development of the primary visual cortex of the common marmoset. I. A changing space for synaptogenesis.

The primary visual cortex of Callithrix jacchus occupies a large portion of the occipital neocortex and can be safely delineated from fetal stages onwards. In 20 animals ranging in age from fetal to adult age the morphological development of area 17 was evaluated and compared with the growth of whole brain, skull, and head size. Cortical thickness, surface area, and volume of the area were determined in addition to predominant growth directions. The volume of area 17 approximately doubles between birth (241 mm3) and three months of age (506 mm3). This maximum value marks an overshoot in growth (volume: 180%, surface area: 150%, thickness: 122%), which is followed by a considerable reduction before adult values (100%) are reached. Although these values seem to indicate that the overall reduction in size is fairly isometric, growth and regression are locally anisometric. For example, layers II-IVc contribute disproportionately to the overshoot; thickening is less pronounced than tangential growth and follows a slightly different time course. These data suggest that the developing visual cortex represents a highly dynamic distribution space for the developing synaptic junctions which should be taken into account in studies on synaptogenesis. By comparison it is suggested that this growth dynamic is not restricted to area 17 but also occurs in some other parts of the cerebral cortex. In contrast, most subcortical brain regions apparently do not undergo overshoot growth. Structural changes of the skull compensate the overshoot in cortex growth, so that head size increases steadily.

Structural dynamics of synapses and synaptic components.

Learning and memory formation are apparently based on cascades of molecular and cellular processes with increasing time constants (ms to days and weeks), but even the most long-lasting effects are transient. Memory traces may permanently modify the behavior (activity patterns, gene expression) of neurons and neuronal networks. Therefore the question is raised whether our current view on the stability of synapses under normal conditions is tenable. Evidence is reviewed suggesting that as direct or indirect effects of modifications in bioelectrical activity and chemical trophicity, synapses may be remodeled and removed within days and weeks, and possibly within hours. Accordingly, species-specific connectivity patterns are not restricted to the standard architecture of the CNS, but (morpho-)genetics allow for a considerable number of alternative wiring patterns, which appear under unusual conditions during ontogenesis and in adulthood. Our present knowledge suggests that, rather than the formation of synapses, they are a selective process. Until now there is no direct method of measuring either synaptic reorganization or the average life span of synapses. Specific cases, however, allow to estimate synapse turnover during ontogenesis, at its lowest possible level. Such data suggest that each synapse is on average remodeled or replaced several to many times during normal developmental, e.g. in the cerebral cortex of Marmoset monkeys at the very least 5 to 10 times (corresponding to 250 million synapses eliminated per hour in area 17!). It is discussed how the consequences of synapse turnover could be utilized by learning processes. Conclusions are followed by an outlook.

Postnatal development of glial fibrillary acidic protein, vimentin and S100 protein in monkey visual cortex: evidence for a transient reduction of GFAP immunoreactivity.

In the cerebral cortex of some species, the gradual appearance of glial fibrillary acidic protein (GFAP) is often interpreted as reflecting the parallel maturation of neuronal connectivity. We studied the postnatal maturation of astrocytes in the primary visual cortex of Callithrix jacchus using antibodies against GFAP, vimentin and S100 protein as immunohistochemical markers. In the cortical grey matter of this species, the overall GFAP-immunoreactivity (IR) as measured by image analysis is high at birth (130% of the adult value), decreases until about 3 months (80%) and increases again towards adult values (100%). Vimentin-IR was high at birth, and declined towards 3 months and later. In contrast, S100-IR augmented postnatally in neuropil, and showed a laminar shift of maximum IR from layer IV to supragranular layers during ontogenesis. The decrease of GFAP-IR is predominantly due to changes in density of GFAP-positive (+) astrocytes within cortical tissue (newborn: 18,600 GFAP+astrocytes/mm3; 1 month: 11,600/mm3; 3 months: 5,700/mm3; adult: 10,200/mm3), while the overall number of astrocytes remained relatively constant as shown by the number of S100-positive astrocytic cell bodies. At times of low GFAP-IR a reduced area density of intermediate filaments was found in astrocytes by electron microscopy. The period of reduced GFAP-expression coincides with the time of prominent synapse remodeling in the visual cortex of marmosets. These data suggest that GFAP-expression may depend on functional conditions rather than time-dependent maturation.

Synaptic reorganization in developing and adult nervous systems.

Evidence is accumulating that synapse reorganization already starts during development, soon after first synapses appear. Although remodeling continues throughout ontogenesis, there are apparently (critical) periods which are characterized by enhanced synaptic reorganization. In certain parts of the peripheral and central nervous system, synapses may undergo remodeling which leads to changes in their transmission efficiency or complete elimination of the synaptic junctions, even in adulthood. Synaptic reorganization includes progressive and regressive changes on branches of dendritic and/or axonal processes that accompany the formation and elimination of synapses. Three modes of elimination are presently known: Physiological cell death of synaptically connected neurons is involved, especially during certain developmental periods, during hormonally induced metamorphosis and in the olfactory bulb. Synaptic disconnection ("stripping") and lysosomal degradation predominantly of presynaptic elements occur under different conditions. In order to undergo plastic changes, neurons seem to respond to exogenous or intrinsic factors such as lesions (partial deafferentation and axotomy), long-lasting changes in neuronal activity (e.g. drug application and sensory deprivation), hormonal influences (e.g. sexual hormones) or learning conditions.

Imaging of molecular surface dynamics in brain slices using single-particle tracking.

Organization of signalling molecules in biological membranes is crucial for cellular communication. Many receptors, ion channels and cell adhesion molecules are associated with proteins important for their trafficking, surface localization or function. These complexes are embedded in a lipid environment of varying composition. Binding affinities and stoichiometry of such complexes were so far experimentally accessible only in isolated systems or monolayers of cell culture. Visualization of molecular dynamics within signalling complexes and their correlation to specialized membrane compartments demand high temporal and spatial resolution and has been difficult to demonstrate in complex tissue like brain slices. Here we demonstrate the feasibility of single-particle tracking (SPT) in organotypic brain slices to measure molecular dynamics of lipids and transmembrane proteins in correlation to synaptic membrane compartments. This method will provide important information about the dynamics and organization of surface molecules in the complex environment of neuronal networks within brain slices.

Common exonic missense variants in the C2 domain of the human KIBRA protein modify lipid binding and cognitive performance.

The human KIBRA gene has been linked to human cognition through a lead intronic single-nucleotide polymorphism (SNP; rs17070145) that is associated with episodic memory performance and the risk to develop Alzheimer's disease. However, it remains unknown how this relates to the function of the KIBRA protein. Here, we identified two common missense SNPs (rs3822660G/T [M734I], rs3822659T/G [S735A]) in exon 15 of the human KIBRA gene to affect cognitive performance, and to be in almost complete linkage disequilibrium with rs17070145. The identified SNPs encode variants of the KIBRA C2 domain with distinct Ca(2+) dependent binding preferences for monophosphorylated phosphatidylinositols likely due to differences in the dynamics and folding of the lipid-binding pocket. Our results further implicate the KIBRA protein in higher brain function and provide direction to the cellular pathways involved.

Early defects of GABAergic synapses in the brain stem of a MeCP2 mouse model of Rett syndrome.

Rett syndrome is a neurodevelopmental disorder caused by mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2) and represents the leading genetic cause for mental retardation in girls. MeCP2-mutant mice have been generated to study the molecular mechanisms of the disease. It was suggested that an imbalance between excitatory and inhibitory neurotransmission is responsible for the behavioral abnormalities, although it remained largely unclear which synaptic components are affected and how cellular impairments relate to the time course of the disease. Here, we report that MeCP2 KO mice present an imbalance between inhibitory and excitatory synaptic transmission in the ventrolateral medulla already at postnatal day 7. Focusing on the inhibitory synaptic transmission we show that GABAergic, but not glycinergic, synaptic transmission is strongly depressed in MeCP2 KO mice. These alterations are presumably due to both decreased presynaptic gamma-aminobutyric acid (GABA) release with reduced levels of the vesicular inhibitory transmitter transporter and reduced levels of postsynaptic GABA(A)-receptor subunits alpha2 and alpha4. Our data indicate that in the MeCP2 -/y mice specific synaptic molecules and signaling pathways are impaired in the brain stem during early postnatal development. These observations mandate the search for more refined diagnostic tools and may provide a rationale for the timing of future therapeutic interventions in Rett patients.

Synaptic remodelling and elimination as integral processes of synaptogenesis.

It has become increasingly clear that the development and maintenance of synaptic connectivity patterns in the central and peripheral nervous system are not only based on the formation of synapses but include the selective elimination of synaptic subpopulations. Synaptic remodeling and elimination apparently also play a key role in the specification of neuronal connections during ontogenesis of the neocortex in various species. At least three types of synapse elimination have been demonstrated until now, i.e. physiological cell death of synaptically connected neurons, synaptic disconnection and lysosomal degradation, predominantly of presynaptic elements. Occurrence of different elimination types appears to depend on (1) whether the presynaptic or postsynaptic element induces the synaptic reorganization and (2) whether or not the neuron inducing the synapse elimination survives. The same type of synapse elimination may be seen during normal development and under pathological conditions. Factors inducing mental retardation may then either retard synapse formation or interfere with the process of synapse removal. In order to undergo plastic changes in synaptic connections, neurons may respond to exogenous factors such as lack of trophic factors or sensitivity for them (e.g. NGF), long-lasting changes in neuronal activity (e.g. due to drug application or sensory deprivation), hormonal influences (e.g. thyroxin or sexual hormones), learning conditions, or lesions (partial deafferentation and axotomy). The neurons, in turn, may change their responsiveness to exogenous stimuli by inducing synaptic reorganisation.

Neurexophilins form a conserved family of neuropeptide-like glycoproteins.

Neurexophilin was discovered as a neuronal glycoprotein that is copurified with neurexin Ialpha during affinity chromatography on immobilized alpha-latrotoxin (Petrenko et al., 1996). We have now investigated how neurexophilin interacts with neurexins, whether it is post-translationally processed by site-specific cleavage similar to neuropeptides, and whether related neuropeptide-like proteins are expressed in brain. Our data show that mammalian brains contain four genes for neurexophilins the products of which share a common structure composed of five domains: an N-terminal signal peptide, a variable N-terminal domain, a highly conserved central domain that is N-glycosylated, a short linker region, and a conserved C-terminal domain that is cysteine-rich. When expressed in pheochromocytoma (PC12) cells with a replication-deficient adenovirus, neurexophilin 1 was rapidly N-glycosylated and then slowly processed to a smaller mature form, probably by endoproteolytic cleavage. Similar expression experiments in other neuron-like cells and in fibroblastic cells revealed that N-glycosylation of neurexophilin 1 occurred in all cell types tested, whereas proteolytic processing was observed only in neuron-like cells. Finally, only recombinant neurexin Ialpha and IIIalpha but not neurexin Ibeta interacted with neurexophilin 1 and were preferentially bound to the processed mature form of neurexophilin. Together our data demonstrate that neurexophilins form a family of related glycoproteins that are proteolytically processed after synthesis and bind to alpha-neurexins. The structure and characteristics of neurexophilins indicate that they function as neuropeptides that may signal via alpha-neurexins.

Neurexophilin binding to alpha-neurexins. A single LNS domain functions as an independently folding ligand-binding unit.

alpha-Neurexins (Ialpha, IIalpha, and IIIalpha) are receptor-like proteins expressed in hundreds of isoforms on the neuronal cell surface. The extracellular domains of alpha-neurexins are composed of six LNS repeats, named after homologous sequences in the Laminin A G domain, Neurexins, and Sex hormone-binding globulin, with three interspersed epidermal growth factor-like domains. Purification of neurexin Ialpha revealed that it is tightly complexed to a secreted glycoprotein called neurexophilin 1. Neurexophilin 1 is a member of a family of at least four genes and resembles a neuropeptide, suggesting a function as an endogenous ligand for alpha-neurexins. We have now used recombinant proteins and knockout mice to investigate which isoforms and domains of different neurexins and neurexophilins interact with each other. We show that neurexophilins 1 and 3 but not 4 (neurexophilin 2 is not expressed in rodents) bind to a single individual LNS domain, the second overall LNS domain in all three alpha-neurexins. Although this domain is alternatively spliced, all splice variants bind, suggesting that alternative splicing does not regulate binding. Using homologous recombination to disrupt the neurexophilin 1 gene, we generated mutant mice that do not express detectable neurexophilin 1 mRNA. Mice lacking neurexophilin 1 are viable with no obvious morbidity or mortality. However, homozygous mutant mice exhibit male sterility, probably because homologous recombination resulted in the co-insertion into the neurexophilin gene of herpes simplex virus thymidine kinase, which is known to cause male sterility. In the neurexophilin 1 knockout mice, neurexin Ialpha is complexed with neurexophilin 3 but not neurexophilin 4, suggesting that neurexophilin 1 is redundant with neurexophilin 3 and that neurexophilins 1 and 3 but not 4 bind to neurexins. This hypothesis was confirmed using expression experiments. Our data reveal that the six LNS and three epidermal growth factor domains of neurexins are independently folding ligand-binding domains that may interact with distinct targets. The results support the notion that neurexophilins represent a family of extracellular signaling molecules that interact with multiple receptors including all three alpha-neurexins.

The making of neurexins.

Neurexins are neuronal cell-surface proteins with up to thousands of isoforms. These isoforms are generated by alternative splicing of transcripts from six promoters in three genes. The structure of neurexins resembles cell-surface receptors with a modular architecture suggestive of a sequential assembly during evolution. Neurexins probably perform multiple functions in the brain. They participate in intercellular junctions in which beta-neurexins tightly bind to a second class of neuronal cell-surface receptors called neuroligins. Intracellularly, the neurexin/neuroligin junction is bound by CASK on the neurexin side and PSD95 on the neuroligin side. CASK and PSD95 are homologous membrane-associated guanylate kinases that bind to the neurexin/neuroligin junction via PDZ domains, creating an asymmetric junction (neurexin/neuroligin) with similar intracellular binding partners. In addition to a function as cell-adhesion molecules, neurexins may also serve as a signalling receptor, because a class of ligands for alpha-neurexins called neurexophilins is similar to peptide hormones. Finally, at least one neurexin isoform, neurexin Ialpha, represents a high-affinity receptor for alpha-latrotoxin, which is a potent excitatory neurotoxin. Thus, neurexins constitute a large family of neuronal receptors that may be involved in multiple interactive functions between neurons.

Rapid morphological changes in astrocytes are accompanied by redistribution but not by quantitative changes of cytoskeletal proteins.

Astrocytes have the potential to acquire very different morphologies, depending on their regional location in the CNS and on their functional interactions with other cell types. Morphological changes between a flat or a fibroblast-like and a stellate or process-bearing appearance, and vice versa, can occur rapidly, but very little is known as to whether morphological transformations are based on quantitative changes of cytoskeletal proteins in microfilaments, intermediate filaments, and/or microtubules. Using a cell culture of selective type 1 astrocytes, we compared the distribution and protein amounts of a number of cytoskeletal proteins both during primary process growth induced by specific media conditions and after secondary transformations induced by dBcAMP. Our data presented in this report support the idea that astrocytes can undergo dramatic changes in their morphology requiring subcellular redistribution of most cytoskeletal proteins but no quantitative modifications of the amount of the respective proteins. After pharmacological treatment with lysophosphatic acid and genistein we show that astrocytes can acquire intermediate morphologies reminiscent of both fibroblast and stellate-like cells. These experiments demonstrate that the recently described RhoA-mediated signaling cascade between the cell surface and cytoskeletal proteins is only one of several signaling pathways acting on the astrocytic cytoskeleton.

Complexins: cytosolic proteins that regulate SNAP receptor function.

A family of proteins called complexins was discovered that compete with alpha-SNAP, but not synaptotagmin, for SNAP receptor binding. Complexins I and II are highly homologous hydrophilic proteins that are tightly conserved, with 100% identity among mouse, rat, and human complexin II. They are enriched in neurons where they colocalize with syntaxin and SNAP-25; in addition, complexin II is expressed ubiquitously at low levels. Complexins bind weakly to syntaxin alone and not at all to synaptobrevin and SNAP-25, but strongly to the SNAP receptor-core complex composed of these three molecules. They compete with alpha-SNAP for binding to the core complex but not with other interacting molecules, including synaptotagmin I, suggesting that the complexins regulate the sequential interactions of alpha-SNAP and synaptotagmins with the SNAP receptor during exocytosis.