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The extracellular matrix (ECM) is a complex molecular network distributed throughout the extracellular space of different tissues as well as the neuronal system. Previous studies have identified various ECM components that play important roles in neuronal maturation and signal transduction. ECM components are reported to be involved in neurogenesis, neuronal migration, and axonal growth by interacting or binding to specific receptors. In addition, the ECM is found to regulate synapse formation, the stability of the synaptic structure, and synaptic plasticity. Here, we mainly reviewed the effects of various ECM components on synapse formation and briefly described the related diseases caused by the abnormality of several ECM components.

Accurate and efficient methods for identifying and tracking each animal in a group are needed to study complex behaviors and social interactions. Traditional tracking methods (e.g., marking each animal with dye or surgically implanting microchips) can be invasive and may have an impact on the social behavior being measured. To overcome these shortcomings, video-based methods for tracking unmarked animals, such as fruit flies and zebrafish, have been developed. However, tracking individual mice in a group remains a challenging problem because of their flexible body and complicated interaction patterns. In this study, we report the development of a multi-object tracker for mice that uses the Faster region-based convolutional neural network (R-CNN) deep learning algorithm with geometric transformations in combination with multi-camera/multi-image fusion technology. The system successfully tracked every individual in groups of unmarked mice and was applied to investigate chasing behavior. The proposed system constitutes a step forward in the noninvasive tracking of individual mice engaged in social behavior.

Synapses are the connections between neurons in the central nervous system (CNS) or between neurons and other excitable cells in the peripheral nervous system (PNS), where electrical or chemical signals rapidly travel through one cell to another with high spatial precision. Synaptic analysis, based on synapse numbers and fine morphology, is the basis for understanding neurological functions and diseases. Manual analysis of synaptic structures in electron microscopy (EM) images is often limited by low efficiency and subjective bias.We developed a multifunctional synaptic analysis system based on several advanced deep learning (DL) models. The system achieved synapse counting in low-magnification EM images and synaptic ultrastructure analysis in high-magnification EM images.The synapse counting system based on ResNet18 and a Faster R-CNN model had a mean average precision (mAP) of 92.55%. For synaptic ultrastructure analysis, the Faster R-CNN model based on ResNet50 achieved a mAP of 91.60%, the DeepLab v3 + model based on ResNet50 enabled high performance in presynaptic and postsynaptic membrane segmentation with a global accuracy of 0.9811, and the Faster R-CNN model based on ResNet18 achieved a mAP of 91.41% for synaptic vesicle detection.The proposed multifunctional synaptic analysis system may help to overcome the experimental bias inherent in manual analysis, thereby facilitating EM image-based synaptic function studies.

Synapse is the fundamental structure for neurons to transmit information between cells. The proper synapse formation is crucial for developing neural circuits and cognitive functions of the brain. The aberrant synapse formation has been proved to cause many neurological disorders, including autism spectrum disorders and intellectual disability. Synaptic cell adhesion molecules (CAMs) are thought to play a major role in achieving mechanistic cell-cell recognition and initiating synapse formation via trans-synaptic interactions. Although several synaptic CAMs, such as neurexins, neuroligins, SynCAMs, and LRRTMs, have been identified as synaptogenic molecules, these molecules so far as we know cannot fully explain the mechanism of synapse formation. There should be other synaptogenic adhesion molecules that remain undiscovered. Artificial synapse formation (ASF) assays, the commonly used method for screening synaptogenesis molecules, is time-consuming and labor-intensive due to the long-lasting immunostaining step. To skip this step, we generated synaptotagmin 1-tdTomato (Syt1-TDT) transgenic mice by inserting the tdTomato-fused synaptotagmin 1 coding sequence into the genome of C57BL/6J mice. In the brain of Syt1-TDT transgenic mice, the Syt1-TDT signals were widely observed in different areas. In the cultured hippocampal neurons, the Syt1-TDT signals merged with several synaptic markers, suggesting the well synaptic localization of Syt1-TDT. Compared to the wild-type (WT) mouse neurons, cultured hippocampal neurons from Syt1-TDT transgenic mice presented normal synaptic neurotransmission. In ASF assays, neurons from Syt1-TDT transgenic mice could form synaptic connections with HEK293T cells expressing neuroligin2, LRRTM2, and Slitrk2 without immunostaining. Therefore, our work suggested that the Syt1-TDT transgenic mice with the ability to label synapses by tdTomato will be a convenient tool for screening synaptogenic molecules.

Macroautophagy/autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggest that autophagy is impaired during cerebral ischemia, contributing to neuronal dysfunction and neurodegeneration. However, the outcomes after transient modification in autophagy machinery are not fully understood. This study investigated the effects of ischemic stress on autophagy and synaptic structures using a rat model of oxygen-glucose deprivation (OGD) in hippocampal neurons and a mouse model of middle cerebral artery occlusion (MCAO). Upon acute ischemia, an initial autophagy modification occurred in an upregulation manner. Following, the number of lysosomes increased, as well as lysosomal volume, indicating dysfunctional lysosomal storage. These changes were prevented by inhibiting autophagy via 3-methyladenine (3-MA) treatment or ATG7 (autophagy related 7) knockdown, or were mimicked by rapamycin (RAPA), a known activator of autophagy. This suggests that dysfunctional lysosomal storage is associated with the early burst of autophagy. Dysfunctional lysosomal storage contributed to autophagy dysfunction because the basal level of MTOR-dependent lysosomal biogenesis in the reperfusion was not sufficient to clear undegraded cargoes after transient autophagy upregulation. Further investigation revealed that impairment of synaptic ultra-structures, accompanied by dysfunctional lysosomal storage, may result from a failure in dynamic turnover of synaptic proteins. This indicates a vital role of autophagy-lysosomal machinery in the maintenance of synaptic structures. This study supports previous evidence that dysfunctional lysosomal storage may occur following the upregulation of autophagy in neurons. Appropriate autophagosome-lysosomal functioning is vital for maintenance of neuronal synaptic function and impacts more than the few known synaptic proteins. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALR: autophagic lysosome reformation; ATG7: autophagy related 7; CTSB: cathepsin B; CTSD: cathepsin D; DAPI: 4ʹ,6-diamidino-2-phenylindole; DEGs: differentially expressed genes; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide; GO: Gene Ontology; HBSS: Hanks’ balanced salt solution; HPCA: hippocalcin; i.c.v: intracerebroventricular; KEGG: kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; LSDs: lysosomal storage disorders; MAP2: microtubule-associated protein 2; MCAO: middle cerebral artery occlusion; mCTSB: mature CTSB; mCTSD: mature CTSD; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; OGD/R: oxygen-glucose deprivation/reoxygenation; PBS: phosphate-buffered saline; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; proCTSD: pro-cathepsin D; RAPA: rapamycin; RNA-seq: RNA sequencing; RPS6KB/p70S6K: ribosomal protein S6 kinase; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIM: Structured Illumination Microscopy; SNAP25: synaptosomal-associated protein 25; SQSTM1/p62: sequestosome 1; SYN1: synapsin I; SYT1: synaptotagmin I; TBST: tris-buffered saline Tween-20; TEM: transmission electron microscopy; TFEB: transcription factor EB; tMCAO: transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyltetrazolium chloride; TUBB3: tubulin, beta 3 class III.

Long-term potentiation (LTP) has long been considered as an important cellular mechanism for learning and memory. LTP expression involves NMDA receptor-dependent synaptic insertion of AMPA receptors (AMPARs). However, how AMPARs are recruited and anchored at the postsynaptic membrane during LTP remains largely unknown. In this study, using CRISPR/Cas9 to delete the endogenous AMPARs and replace them with the mutant forms in single neurons, we have found that the amino-terminal domain (ATD) of GluA1 is required for LTP maintenance. Moreover, we show that GluA1 ATD directly interacts with the cell adhesion molecule neuroplastin-65 (Np65). Neurons lacking Np65 exhibit severely impaired LTP maintenance, and Np65 deletion prevents GluA1 from rescuing LTP in AMPARs-deleted neurons. Thus, our study reveals an essential role for GluA1/Np65 binding in anchoring AMPARs at the postsynaptic membrane during LTP.

Parylene-C is a popular polymer material in biomedical applications, with excellent physicochemical properties and microfabrication capability. Like many aromatic polymers, parylene-C also has autofluorescence, which was usually taken as a negative background noise in biomedical detection studies. However, the fluorescence intensity of thin-film (

Three neuronal pentraxins are expressed in brain, the membrane-bound “neuronal pentraxin receptor” (NPR) and the secreted proteins NP1 and NARP (i.e., NP2). Neuronal pentraxins bind to AMPARs at excitatory synapses and play important, well-documented roles in the activity-dependent regulation of neural circuits via this binding activity. However, it is unknown whether neuronal pentraxins perform roles in synapses beyond modulating postsynaptic AMPAR-dependent plasticity, and whether they may even act in inhibitory synapses. Here, we show that NPR expressed in non-neuronal cells potently induces formation of both excitatory and inhibitory postsynaptic specializations in cocultured hippocampal neurons. Knockdown of NPR in hippocampal neurons, conversely, dramatically decreased assembly and function of both excitatory and inhibitory postsynaptic specializations. Overexpression of NPR rescued the NPR knockdown phenotype but did not in itself change synapse numbers or properties. However, the NPR knockdown decreased the levels of NARP, whereas NPR overexpression produced a dramatic increase in the levels of NP1 and NARP, suggesting that NPR recruits and stabilizes NP1 and NARP on the presynaptic plasma membrane. Mechanistically, NPR acted in excitatory synapse assembly by binding to the N-terminal domain of AMPARs; antagonists of AMPA and GABA receptors selectively inhibited NPR-induced heterologous excitatory and inhibitory synapse assembly, respectively, but did not affect neurexin-1β-induced synapse assembly as a control. Our data suggest that neuronal pentraxins act as signaling complexes that function as general trans-synaptic organizers of both excitatory and inhibitory synapses by a mechanism that depends, at least in part, on the activity of the neurotransmitter receptors at these synapses.SIGNIFICANCE STATEMENTNeuronal pentraxins comprise three neuronal proteins, neuronal pentraxin receptor (NPR) which is a type-II transmembrane protein on the neuronal surface, and secreted neuronal pentraxin-1 and NARP. The general functions of neuronal pentraxins at synapses have not been explored, except for their basic AMPAR binding properties. Here, we examined the functional role of NPR at synapses because it is the only neuronal pentraxin that is anchored to the neuronal cell-surface membrane. We find that NPR is a potent inducer of both excitatory and inhibitory heterologous synapses, and that knockdown of NPR in cultured neurons decreases the density of both excitatory and inhibitory synapses. Our data suggest that NPR performs a general, previously unrecognized function as a universal organizer of synapses.

Recent genome-wide association studies (GWAS) have identified multiple risk loci that show strong associations with schizophrenia. However, pinpointing the potential causal genes at the reported loci remains a major challenge. Here we identify candidate causal genes for schizophrenia using an integrative genomic approach. Sherlock integrative analysis shows that ALMS1, GLT8D1, and CSNK2B are schizophrenia risk genes, which are validated using independent brain expression quantitative trait loci (eQTL) data and integrative analysis method (SMR). Consistently, gene expression analysis in schizophrenia cases and controls further supports the potential role of these three genes in the pathogenesis of schizophrenia. Finally, we show that GLT8D1 and CSNK2B knockdown promote the proliferation and inhibit the differentiation abilities of neural stem cells, and alter morphology and synaptic transmission of neurons. These convergent lines of evidence suggest that the ALMS1, CSNK2B, and GLT8D1 genes may be involved in pathophysiology of schizophrenia., More than 100 risk loci for schizophrenia have been identified by genome-wide association studies. Here, the authors apply an integrative genomic approach to prioritize risk genes and validate GLT8D1 and CSNK2B as candidate causal genes by in vitro studies in neural stem cells.

The proper formation of synapses—specialized unitary structures formed between two neurons—is critical to mediating information flow in the brain. Synaptic cell adhesion molecules (CAMs) are thought to participate in the initiation of the synapse formation process. However,in vivofunctional analysis demonstrates that most well known synaptic CAMs regulate synaptic maturation and plasticity rather than synapse formation, suggesting that either CAMs work synergistically in the process of forming synapses or more CAMs remain to be found. By screening for unknown CAMs using a co-culture system, we revealed that protein tyrosine phosphatase receptor type O (PTPRO) is a potent CAM that induces the formation of artificial synapse clusters in co-cultures of human embryonic kidney 293 cells and hippocampal neurons cultured from newborn mice regardless of gender. PTPRO was enriched in the mouse brain and localized to postsynaptic sites at excitatory synapses. The overexpression of PTPRO in cultured hippocampal neurons increased the number of synapses and the frequency of miniature EPSCs (mEPSCs). The knock-down (KD) of PTPRO expression in cultured neurons by short hairpin RNA (shRNA) reduced the number of synapses and the frequencies of the mEPSCs. The effects of shRNA KD were rescued by expressing either full-length PTPRO or a truncated PTPRO lacking the cytoplasmic domain. Consistent with these results, the N-terminal extracellular domain of PTPRO was required for its synaptogenic activity in the co-culture assay. Our data show that PTPRO is a synaptic CAM that serves as a potent initiator of the formation of excitatory synapses.SIGNIFICANCE STATEMENTThe formation of synapses is critical for the brain to execute its function and synaptic cell adhesion molecules (CAMs) play essential roles in initiating the formation of synapses. By screening for unknown CAMs using a co-culture system, we revealed that protein tyrosine phosphatase receptor type O (PTPRO) is a potent CAM that induces the formation of artificial synapse clusters. Using loss-of-function and gain-of-function approaches, we show that PTPRO promotes the formation of excitatory synapses. The N-terminal extracellular domain of PTPRO was required for its synaptogenic activity in cultured hippocampal neurons and the co-culture assay. Together, our data show that PTPRO is a synaptic CAM that serves as a potent initiator of synapse formation.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors are major excitatory receptors that mediate fast neurotransmission in the mammalian brain. The surface expression of functional AMPA receptors (AMPARs) is crucial for synaptic transmission and plasticity. AMPAR auxiliary subunits control the biosynthesis, membrane trafficking, and synaptic targeting of AMPARs. Our previous report showed that α/β-hydrolase domain-containing 6 (ABHD6), an auxiliary subunit for AMPARs, suppresses the membrane delivery and function of GluA1-containing receptors in both heterologous cells and neurons. However, it remained unclear whether ABHD6 affects the membrane trafficking of Glutamate receptor subunits, GluA2 and GluA3. Here, we examine the effects of ABHD6 overexpression in HEK293T cells expressing GluA1, GluA2, GluA3, and stargazin, either alone or in combination. The results show that ABHD6 suppresses the glutamate-induced currents and the membrane expression of AMPARs when expressing GluA2 or GluA3 in the HEK293T cells. We generated a series of GluA2 and GluA3 C-terminal deletion constructs and confirm that the C-terminus of GluAs is required for ABHD6’s inhibitory effects on glutamate-induced currents and surface expression of GluAs. Meanwhile, our pull-down experiments reveal that ABHD6 binds to GluA1–3, and deletion of the C-terminal domain of GluAs abolishes this binding. These findings demonstrate that ABHD6 inhibits the AMPAR-mediated currents and its surface expression, independent of the type of AMPAR subunits, and this inhibitor’s effects are mediated through the binding with the GluAs C-terminal regions.

In the brain, AMPA-type glutamate receptors are major postsynaptic receptors at excitatory synapses that mediate fast neurotransmission and synaptic plasticity. α/β-Hydrolase domain-containing 6 (ABHD6), a monoacylglycerol lipase, was previously found to be a component of AMPA receptor macromolecular complexes, but its physiological significance in the function of AMPA receptors (AMPARs) has remained unclear. The present study shows that overexpression of ABHD6 in neurons drastically reduced excitatory neurotransmission mediated by AMPA but not by NMDA receptors at excitatory synapses. Inactivation of ABHD6 expression in neurons by either CRISPR/Cas9 or shRNA knockdown methods significantly increased excitatory neurotransmission at excitatory synapses. Interestingly, overexpression of ABHD6 reduced glutamate-induced currents and the surface expression of GluA1 in HEK293T cells expressing GluA1 and stargazin, suggesting a direct functional interaction between these two proteins. The C-terminal tail of GluA1 was required for the binding between of ABHD6 and GluA1. Mutagenesis analysis revealed a GFCLIPQ sequence in the GluA1 C terminus that was essential for the inhibitory effect of ABHD6. The hydrolase activity of ABHD6 was not required for the effects of ABHD6 on AMPAR function in either neurons or transfected HEK293T cells. Thus, these findings reveal a novel and unexpected mechanism governing AMPAR trafficking at synapses through ABHD6.

In the original version of this Article, the affiliation details for Qiushuo Shen incorrectly omitted ‘Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, 650204, China’. This has now been corrected in both the PDF and HTML versions of the Article.

Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells. Currently, most protocols for generating high-titer lentivirus require ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory. In this study, the effect of relative centrifugal force (RCF) on the concentration efficiency of the lentivirus was systematically explored and it was found that sucrose gradient centrifugation with a relatively low speed (≤10,000 g) robustly produces a high-titer virus (up to 2 × 108 TU/ml). The optimal sucrose concentration is 10% and the recovery rate of the functional virus is greater than 80%. The infection efficiency of both concentrated and un-concentrated lentivirus decreases rapidly when the viruses are stored at 4 °C (τ ≈ 1.3 days) or subjected to multiple freeze-thaw cycles (τ = 1.1 rounds). In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.

Autism spectrum disorders (ASD) are a pervasive neurodevelopmental disease characterized by deficits in social interaction and nonverbal communication, as well as restricted interests and stereotypical behavior. Genetic changes/heritability is one of the major contributing factors, and hundreds to thousands of causative and susceptible genes, copy number variants (CNVs), linkage regions, and microRNAs have been associated with ASD which clearly indicates that ASD is a complex genetic disorder. Here, we will briefly summarize some of the high-confidence genetic changes in ASD and their possible roles in their pathogenesis.

Dendritic spines are actin-rich membrane protrusions that are the major sites of excitatory synaptic input in the mammalian brain, and their morphological plasticity provides structural basis for learning and memory. Here we report that endophilin A1, with a well-established role in clathrin-mediated synaptic vesicle endocytosis at the presynaptic terminal, also localizes to dendritic spines and is required for spine morphogenesis, synapse formation and synaptic function. We identify p140Cap, a regulator of cytoskeleton reorganization, as a downstream effector of endophilin A1 and demonstrate that disruption of their interaction impairs spine formation and maturation. Moreover, we demonstrate that knockdown of endophilin A1 or p140Cap impairs spine stabilization and synaptic function. We further show that endophilin A1 regulates the distribution of p140Cap and its downstream effector, the F-actin-binding protein cortactin as well as F-actin enrichment in dendritic spines. Together, these results reveal a novel function of postsynaptic endophilin A1 in spine morphogenesis, stabilization and synaptic function through the regulation of p140Cap.