Specific Synapses Research Articles

Changes in concentrations of NMDA receptor subunit GluN2B, Arc and syntaxin-1 in dorsal hippocampus Schaffer collateral synapses in a rat learned helplessness model of depression.

Major depressive disorder involves changes in synaptic structure and function, but the molecular underpinnings of these changes are still not established. In an initial pilot experiment, whole-brain synaptosome screening with quantitative western blotting was performed to identify synaptic proteins that may show concentration changes in a congenital rat learned helplessness model of depression. We found that the N-methyl-d-aspartate receptor (NMDAR) subunits GluN2A/GluN2B, activity-regulated cytoskeleton-associated protein (Arc) and syntaxin-1 showed significant concentration differences between congenitally learned helpless (LH) and nonlearned helpless (NLH) rats. Having identified these three proteins, we then performed more elaborate quantitative immunogold electron microscopic analyses of the proteins in a specific synapse type in the dorsal hippocampus: the Schaffer collateral synapse in the CA1 region. We expanded the setup to include also unstressed wild-type (WT) rats. The concentrations of the proteins in the LH and NLH groups were compared to WT animals. In this specific synapse, we found that the concentration of NMDARs was increased in postsynaptic spines in both LH and NLH rats. The concentration of Arc was significantly increased in postsynaptic densities in LH animals as well as in presynaptic cytoplasm of NLH rats. The concentration of syntaxin-1 was significantly increased in both presynaptic terminals and postsynaptic spines in LH animals, while pre- and postsynaptic syntaxin-1 concentrations were significantly decreased in NLH animals. These protein changes suggest pathways by which synaptic plasticity may be increased in dorsal hippocampal Schaffer collateral synapses during depression, corresponding to decreased synaptic stability.

Specific heparan sulfate modifications stabilize the synaptic organizer MADD-4/Punctin at Caenorhabditis elegans neuromuscular junctions.

Heparan sulfate (HS) proteoglycans contribute to the structural organization of various neurochemical synapses. Depending on the system, their role involves either the core protein or the glycosaminoglycan chains. These linear sugar chains are extensively modified by HS modification enzymes, resulting in highly diverse molecules. Specific modifications of glycosaminoglycan chains may thus contribute to a sugar code involved in synapse specificity. Caenorhabditis elegans is particularly useful to address this question because of the low level of genomic redundancy of these enzymes, as opposed to mammals. Here, we systematically mutated the genes encoding HS modification enzymes in C. elegans and analyzed their impact on excitatory and inhibitory neuromuscular junctions (NMJs). Using single chain antibodies that recognize different HS modification patterns, we show in vivo that these two HS epitopes are carried by the SDN-1 core protein, the unique C. elegans syndecan ortholog, at NMJs. Intriguingly, these antibodies differentially bind to excitatory and inhibitory synapses, implying unique HS modification patterns at different NMJs. Moreover, while most enzymes are individually dispensable for proper organization of NMJs, we show that 3-O-sulfation of SDN-1 is required to maintain wild-type levels of the extracellular matrix protein MADD-4/Punctin, a central synaptic organizer that defines the identity of excitatory and inhibitory synaptic domains at the plasma membrane of muscle cells.

Upregulation of eIF4E, but not other translation initiation factors, in dendritic spines during memory formation.

Local translation can provide a rapid, spatially targeted supply of new proteins in distal dendrites to support synaptic changes that underlie learning. Learning and memory are especially sensitive to manipulations of translational control mechanisms, particularly those that target the initiation step, and translation initiation at synapses could be a means of maintaining synapse specificity during plasticity. Initiation predominantly occurs via recruitment of ribosomes to the 5' mRNA cap by complexes of eukaryotic initiation factors (eIFs), and the interaction between eIF4E and eIF4G1 is a particularly important target of translational control pathways. Pharmacological inhibition of eIF4E-eIF4G1 binding impairs formation of memory for aversive Pavlovian conditioning as well as the accompanying increase in polyribosomes in the heads of dendritic spines in the lateral amygdala (LA). This is consistent with a role for initiation at synapses in memory formation, but whether eIFs are even present near synapses is unknown. To determine whether dendritic spines contain eIFs and whether eIF distribution is affected by learning, we combined immunolabeling with serial section transmission electron microscopy (ssTEM) volume reconstructions of LA dendrites after Pavlovian conditioning. Labeling for eIF4E, eIF4G1, and eIF2α-another key target of regulation-occurred in roughly half of dendritic spines, but learning effects were only found for eIF4E, which was upregulated in the heads of dendritic spines. Our results support the possibility of regulated translation initiation as a means of synapse-specific protein targeting during learning and are consistent with the model of eIF4E availability as a central point of control.

Expression analysis and single-nucleotide polymorphisms of SYNDIG1L and UNC13C genes associated with thoracic vertebral numbers in sheep (Ovis aries).

The objective of the current study was to analyze expression levels of synapse differentiation inducing 1-like (SYNDIG1L) and unc-13 homolog C (UNC13C) genes in different tissues, while single-nucleotide polymorphisms (SNPs) of two genes were associated with multiple thoracic vertebrae traits in both Small-tailed Han sheep (STH) and Sunite sheep (SNT). The expression levels of SYNDIG1L and UNC13C were analyzed in the brain, cerebellum, heart, liver, spleen, lung, kidney, adrenal gland, uterine horn, longissimus muscle, and abdominal adipose tissues of two sheep breeds with different thoracic vertebral number (TVN) sheep (T13 groups and T14 groups) by real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the polymorphisms of UNC13C gene g.52919279CT and SYNDIG1L gene g.82573325CA in T14 and T13 were genotyped by the Sequenom MassARRAY® SNP assay, and association analysis was performed with the TVN. The results demonstrated that UNC13C gene was extensively expressed in 11 tissues. The expression of UNC13C gene in longissimus muscle of T14 groups of STH was significantly higher than that of T13 groups (). SYNDIG1L gene was overexpressed in brain and cerebellum tissues, and the expression level of UNC13C gene in the brain and cerebellum of T13 groups in SNT was significantly higher than that of T14 groups (). Association analysis showed that SNPs found in the UNC13C gene had no significant effects on TVN for both two genes. The polymorphism of SYNDIG1L g.82573325CA was significantly correlated with the TVN in both STH () and SNT (). Taken together, the SYNDIG1L gene was related to thoracic vertebral development, and this variation may be potentially used as a molecular marker to select the multiple thoracic vertebrae in sheep.

Development and maintenance of vision’s first synapse

Synapses in the outer retina are the first information relay points in vision. Here, photoreceptors form synapses onto two types of interneurons, bipolar cells and horizontal cells. Because outer retina synapses are particularly large and highly ordered, they have been a useful system for the discovery of mechanisms underlying synapse specificity and maintenance. Understanding these processes is critical to efforts aimed at restoring visual function through repairing or replacing neurons and promoting their connectivity. We review outer retina neuron synapse architecture, neural migration modes, and the cellular and molecular pathways that play key roles in the development and maintenance of these connections. We further discuss how these mechanisms may impact connectivity in the retina.

The microtubule cytoskeleton at the synapse

In neurons, microtubules (MTs) provide routes for transport throughout the cell and structural support for dendrites and axons. Both stable and dynamic MTs are necessary for normal neuronal functions. Research in the last two decades has demonstrated that MTs play additional roles in synaptic structure and function in both pre- and postsynaptic elements. Here, we review current knowledge of the functions that MTs perform in excitatory and inhibitory synapses, as well as in the neuromuscular junction and other specialized synapses, and discuss the implications that this knowledge may have in neurological disease

Extracellular Release of ILEI/FAM3C and Amyloid-β Is Associated with the Activation of Distinct Synapse Subpopulations.

Brain amyloid-β (Aβ) peptide is released into the interstitial fluid (ISF) in a neuronal activity-dependent manner, and Aβ deposition in Alzheimer's disease (AD) is linked to baseline neuronal activity. Although the intrinsic mechanism for Aβ generation remains to be elucidated, interleukin-like epithelial-mesenchymal transition inducer (ILEI) is a candidate for an endogenous Aβ suppressor. This study aimed to access the mechanism underlying ILEI secretion and its effect on Aβ production in the brain. ILEI and Aβ levels in the cerebral cortex were monitored using a newly developed ILEI-specific ELISA and in vivo microdialysis in mutant human Aβ precursor protein-knockin mice. ILEI levels in autopsied brains and cerebrospinal fluid (CSF) were measured using ELISA. Extracellular release of ILEI and Aβ was dependent on neuronal activation and specifically on tetanus toxin-sensitive exocytosis of synaptic vesicles. However, simultaneous monitoring of extracellular ILEI and Aβ revealed that a spontaneous fluctuation of ILEI levels appeared to inversely mirror that of Aβ levels. Selective activation and inhibition of synaptic receptors differentially altered these levels. The evoked activation of AMPA-type receptors resulted in opposing changes to ILEI and Aβ levels. Brain ILEI levels were selectively decreased in AD. CSF ILEI concentration correlated with that of Aβ and were reduced in AD and mild cognitive impairment. ILEI and Aβ are released from distinct subpopulations of synaptic terminals in an activity-dependent manner, and ILEI negatively regulates Aβ production in specific synapse types. CSF ILEI might represent a surrogate marker for the accumulation of brain Aβ.

Developmental mechanisms underlying circuit wiring: Novel insights and challenges ahead.

Synaptic connectivity within neural circuits is characterized by high degrees of cellular and subcellular specificity. This precision arises from the combined action of several classes of molecular cues, transmembrane receptors, secreted cues and extracellular matrix components, coordinating transitions between axon guidance, dendrite patterning, axon branching and synapse specificity. We focus this review on recent insights into some of the molecular and cellular mechanisms controlling these transitions and present the results of large-scale efforts and technological developments aimed at mapping neural connectivity at single cell resolution in the mouse cortex as a mammalian model organism. Finally, we outline some of the technical and conceptual challenges lying ahead as the field is starting to explore one of the most challenging problems in neuroscience: the molecular and cellular logic underlying the emergence of the connectome characterizing specific circuits within the central nervous system of mammals.

The Neuromuscular Junction in Health and Disease: Molecular Mechanisms Governing Synaptic Formation and Homeostasis.

The neuromuscular junction (NMJ) is a highly specialized synapse between a motor neuron nerve terminal and its muscle fiber that are responsible for converting electrical impulses generated by the motor neuron into electrical activity in the muscle fibers. On arrival of the motor nerve action potential, calcium enters the presynaptic terminal, which leads to the release of the neurotransmitter acetylcholine (ACh). ACh crosses the synaptic gap and binds to ACh receptors (AChRs) tightly clustered on the surface of the muscle fiber; this leads to the endplate potential which initiates the muscle action potential that results in muscle contraction. This is a simplified version of the events in neuromuscular transmission that take place within milliseconds, and are dependent on a tiny but highly structured NMJ. Much of this review is devoted to describing in more detail the development, maturation, maintenance and regeneration of the NMJ, but first we describe briefly the most important molecules involved and the conditions that affect their numbers and function. Most important clinically worldwide, are myasthenia gravis (MG), the Lambert-Eaton myasthenic syndrome (LEMS) and congenital myasthenic syndromes (CMS), each of which causes specific molecular defects. In addition, we mention the neurotoxins from bacteria, snakes and many other species that interfere with neuromuscular transmission and cause potentially fatal diseases, but have also provided useful probes for investigating neuromuscular transmission. There are also changes in NMJ structure and function in motor neuron disease, spinal muscle atrophy and sarcopenia that are likely to be secondary but might provide treatment targets. The NMJ is one of the best studied and most disease-prone synapses in the nervous system and it is amenable to in vivo and ex vivo investigation and to systemic therapies that can help restore normal function.

SynaptoPAC, an optogenetic tool for induction of presynaptic plasticity.

Optogenetic manipulations have transformed neuroscience in recent years. While sophisticated tools now exist for controlling the firing patterns of neurons, it remains challenging to optogenetically define the plasticity state of individual synapses. A variety of synapses in the mammalian brain express presynaptic long-term potentiation (LTP) upon elevation of presynaptic cyclic adenosine monophosphate (cAMP), but the molecular expression mechanisms as well as the impact of presynaptic LTP on network activity and behavior are not fully understood. In order to establish optogenetic control of presynaptic cAMP levels and thereby presynaptic potentiation, we developed synaptoPAC, a presynaptically targeted version of the photoactivated adenylyl cyclase bPAC. In cultures of hippocampal granule cells of Wistar rats, activation of synaptoPAC with blue light increased action potential-evoked transmission, an effect not seen in hippocampal cultures of non-granule cells. In acute brain slices of C57BL/6N mice, synaptoPAC activation immediately triggered a strong presynaptic potentiation at mossy fiber synapses in CA3, but not at Schaffer collateral synapses in CA1. Following light-triggered potentiation, mossy fiber transmission decreased within 20min, but remained enhanced still after 30min. The optogenetic potentiation altered the short-term plasticity dynamics of release, reminiscent of presynaptic LTP. Our work establishes synaptoPAC as an optogenetic tool that enables acute light-controlled potentiation of transmitter release at specific synapses in the brain, facilitating studies of the role of presynaptic potentiation in network function and animal behavior in an unprecedented manner. Read the Editorial Highlight for this article on page 270.

Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer

Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function.

GABA-Receptive Microglia Selectively Sculpt Developing Inhibitory Circuits

Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapses during development is mediated by specialized microglia is unknown. Here, using in vivo two-photon imaging, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABAB receptors within microglia impairs this process and leads to stereotyped repetitive behavior and hyperactivity. These findings demonstrate that GABA-receptive microglia differentially engage with specific synapse types during development.

Interneuron-specific plasticity at parvalbumin and somatostatin inhibitory synapses onto CA1 pyramidal neurons shapes hippocampal output

The formation and maintenance of spatial representations within hippocampal cell assemblies is strongly dictated by patterns of inhibition from diverse interneuron populations. Although it is known that inhibitory synaptic strength is malleable, induction of long-term plasticity at distinct inhibitory synapses and its regulation of hippocampal network activity is not well understood. Here, we show that inhibitory synapses from parvalbumin and somatostatin expressing interneurons undergo long-term depression and potentiation respectively (PV-iLTD and SST-iLTP) during physiological activity patterns. Both forms of plasticity rely on T-type calcium channel activation to confer synapse specificity but otherwise employ distinct mechanisms. Since parvalbumin and somatostatin interneurons preferentially target perisomatic and distal dendritic regions respectively of CA1 pyramidal cells, PV-iLTD and SST-iLTP coordinate a reprioritisation of excitatory inputs from entorhinal cortex and CA3. Furthermore, circuit-level modelling reveals that PV-iLTD and SST-iLTP cooperate to stabilise place cells while facilitating representation of multiple unique environments within the hippocampal network.

Systematic Quantification of Synapses in Primary Neuronal Culture.

SummaryMost neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset for in vitro synapse screens.

PTPσ Controls Presynaptic Organization of Neurotransmitter Release Machinery at Excitatory Synapses

SummaryLeukocyte common antigen-related receptor tyrosine phosphatases (LAR-RPTPs) are evolutionarily conserved presynaptic organizers. The synaptic role of vertebrate LAR-RPTPs in vivo, however, remains unclear. In the current study, we analyzed the synaptic role of PTPσ using newly generated, single conditional knockout (cKO) mice targeting PTPσ. We found that the number of synapses was reduced in PTPσ cKO cultured neurons in association with impaired excitatory synaptic transmission, abnormal vesicle localization, and abnormal synaptic ultrastructure. Strikingly, loss of presynaptic PTPσ reduced neurotransmitter release prominently at excitatory synapses, concomitant with drastic reductions in excitatory innervations onto postsynaptic target areas in vivo. Furthermore, loss of presynaptic PTPσ in hippocampal CA1 pyramidal neurons had no impact on postsynaptic glutamate receptor responses in subicular pyramidal neurons. Postsynaptic PTPσ deletion had no effect on excitatory synaptic strength. Taken together, these results demonstrate that PTPσ is a bona fide presynaptic adhesion molecule that controls neurotransmitter release and excitatory inputs.

Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism

The trans-synaptic interaction of the cell-adhesion molecules teneurins (TENs) with latrophilins (LPHNs/ADGRLs) promotes excitatory synapse formation when LPHNs simultaneously interact with FLRTs. Insertion of a short alternatively-spliced region within TENs abolishes the TEN-LPHN interaction and switches TEN function to specify inhibitory synapses. How alternative-splicing regulates TEN-LPHN interaction remains unclear. Here, we report the 2.9 Å resolution cryo-EM structure of the TEN2-LPHN3 complex, and describe the trimeric TEN2-LPHN3-FLRT3 complex. The structure reveals that the N-terminal lectin domain of LPHN3 binds to the TEN2 barrel at a site far away from the alternatively spliced region. Alternative-splicing regulates the TEN2-LPHN3 interaction by hindering access to the LPHN-binding surface rather than altering it. Strikingly, mutagenesis of the LPHN-binding surface of TEN2 abolishes the LPHN3 interaction and impairs excitatory but not inhibitory synapse formation. These results suggest that a multi-level coincident binding mechanism mediated by a cryptic adhesion complex between TENs and LPHNs regulates synapse specificity.

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins.

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Components of Endocannabinoid Signaling System Are Expressed in the Perinatal Mouse Cerebellum and Required for Its Normal Development.

Endocannabinoid (eCB) signaling system (ECS), encompassing cannabinoid receptors and enzymes involved in the synthesis and degradation of the endogenous cannabinoid signaling lipids, is highly expressed in the cerebellar cortex of adult humans and rodents. In addition to their well-established role in neuromodulation, eCBs have been shown to play key roles in aspects of neurodevelopment in the fore- and mid-brain, including neurogenesis, cell migration, and synapse specification. However, little is known about the role of ECS in cerebellar development. In this study, we conducted immunohistochemical characterization of ECS components through key stages of cerebellar development in mice using antibodies for 2-arachidonoylglycerol (2-AG) synthetizing and degrading enzymes and the major brain cannabinoid receptor, cannabinoid receptor 1 (CB1), in combination with cerebellar cell markers. Our results reveal a temporally, spatially, and cytologically dynamic pattern of expression. Production, receptor binding, and degradation of eCBs are tightly controlled, thus localization of eCB receptors and the complementary cannabinoid signaling machinery determines the direction, duration, and ultimately the outcome of eCB signaling. To gain insights into the role of eCB signaling in cerebellar development, we characterized gross anatomy of cerebellar midvermis in CB1 knock-out (CB1 KO) mice, as well as their performance in cerebellar-influenced motor tasks. Our results show persistent and selective anatomic and behavioral alterations in CB1 KOs. Consequently, the insights gained from this study lay down the foundation for investigating specific cellular and molecular mechanisms regulated by eCB signaling during cerebellar development.

Functional mosaic organization of neuroligins in neuronal circuits.

Complex brain circuitry with feedforward and feedback systems regulates neuronal activity, enabling neural networks to process and drive the entire spectrum of cognitive, behavioral, sensory, and motor functions. Simultaneous orchestration of distinct cells and interconnected neural circuits is underpinned by hundreds of synaptic adhesion molecules that span synaptic junctions. Dysfunction of a single molecule or molecular interaction at synapses can lead to disrupted circuit activity and brain disorders. Neuroligins, a family of cell adhesion molecules, were first identified as postsynaptic-binding partners of presynaptic neurexins and are essential for synapse specification and maturation. Here, we review recent advances in our understanding of how this family of adhesion molecules controls neuronal circuit assembly by acting in a synapse-specific manner.

Approaches to Determining the Liver Volume and the Fact of Hepatomegalia

Objective: to increase the accuracy of computed tomography (CT) in the diagnosis of hepatomegaly.Material and methods. The investigation is based on the analysis of the results of 603 abdominal CT examinations, which are available in the radiology information systems of the city of Moscow. Six liver parameters (the transverse, vertical, and anteroposterior dimensions of the right and left lobes) were measured. The volume of the organ, the right and left lobes was determined when building three-dimensional images with an IntelliSpace Portal multimodality station (Philips) and a special Synapse 3D software system (Fuji).Results.It was established that there was the most pronounced relationship to the true liver volume among all sizes when using only one parameter for the anteroposterior size of the right lobe (r = 0.66), the sum of the two ones for the vertical and anteroposterior sizes of the right lobe (r = 0.83), the sum of three sizes for the vertical, anteroposterior, and transverse dimensions of the right lobe (r = 0.86). ROC analysis was used to calculate the threshold values of the sum of two and three proposed parameters (34 and 42 cm, respectively). The sensitivity of the technique in identifying hepatomegaly, which was established on the basis of the sum of the vertical and anteroposterior dimensions of the right lobe, compared with only one-parameter orientation, increased from 26% to 87%; the specificity rose from 53% to 86%; when using the sum of the vertical, anteroposterior, and transverse dimensions of the right lobe, that was as much as 89% and 84%, respectively. Approximating the cubic root of the volume with the least squares method allowed one to create new and convenient formulas for calculating the volume of the liver.Conclusion. Determination of the sum of the vertical and anteroposterior dimensions of the right lobe (threshold value, 34 cm) is an optimal approach to diagnosing hepatomegaly. If there is a need for knowledge of the volume of the liver in the absence of special programs for its segmentation, data can be obtained using the created formula that takes into account two proposed liver sizes.