Glutamatergic Synapses Research Articles

Regulation of AMPA receptor trafficking by secreted protein factors

AMPA receptors (AMPARs) mediate the majority of fast excitatory transmission in the brain. Regulation of AMPAR levels at synapses controls synaptic strength and underlies information storage and processing. Many proteins interact with the intracellular domain of AMPARs to regulate their trafficking and synaptic clustering. However, a growing number of extracellular factors important for glutamatergic synapse development, maturation and function have emerged that can also regulate synaptic AMPAR levels. This mini-review highlights extracellular protein factors that regulate AMPAR trafficking to control synapse development and plasticity. Some of these factors regulate AMPAR clustering and mobility by interacting with the extracellular N-terminal domain of AMPARs whereas others regulate AMPAR trafficking indirectly via their respective signaling receptors. While several of these factors are secreted from neurons, others are released from non-neuronal cells such as glia and muscle. Although it is apparent that secreted factors can act locally on neurons near their sites of release to coordinate individual synapses, it is less clear if they can diffuse over longer ranges to coordinate related synapses within a circuit or region of the brain. Given that there are hundreds of factors that can be secreted from neuronal and non-neuronal cells, it will not be surprising if more extracellular factors that modulate AMPARs and glutamatergic synapses are discovered. Many open questions remain including where and when the factors are expressed, what regulates their secretion from different cell types, what controls their diffusion, stability, and range of action, and how their cognate receptors influence intracellular signaling to control AMPAR trafficking.

Astrocyte β-Adrenergic Receptor Activity Regulates NMDA Receptor Signaling of Medial Prefrontal Cortex Pyramidal Neurons.

Glutamate spillover from the synapse is tightly regulated by astrocytes, limiting the activation of extrasynaptically located NMDA receptors (NMDAR). The processes of astrocytes are dynamic and can modulate synaptic physiology. Though norepinephrine (NE) and β-adrenergic receptor (β-AR) activity can modify astrocyte volume, this has yet to be confirmed outside of sensory cortical areas, nor has the effect of noradrenergic signaling on glutamate spillover and neuronal NMDAR activity been explored. We monitored changes to astrocyte process volume in response to noradrenergic agonists in the medial prefrontal cortex of male and female mice. Both NE and the β-AR agonist isoproterenol (ISO) increased process volume by ∼20%, significantly higher than changes seen when astrocytes had G-protein signaling blocked by GDPβS. We measured the effect of β-AR signaling on evoked NMDAR currents. While ISO did not affect single stimulus excitatory currents of Layer 5 pyramidal neurons, ISO reduced NMDAR currents evoked by 10 stimuli at 50 Hz, which elicits glutamate spillover, by 18%. After isolating extrasynaptic NMDARs by blocking synaptic NMDARs with the activity-dependent NMDAR blocker MK-801, ISO similarly reduced extrasynaptic NMDAR currents in response to 10 stimuli by 18%. Finally, blocking β-AR signaling in the astrocyte network by loading them with GDPβS reversed the ISO effect on 10 stimuli-evoked NMDAR currents. These results demonstrate that astrocyte β-AR activity reduces extrasynaptic NMDAR recruitment, suggesting that glutamate spillover is reduced.

Sex steroid hormones, the estrous cycle, and rapid modulation of glutamatergic synapse properties in the striatal brain regions with a focus on 17β-estradiol and the nucleus accumbens

The striatal brain regions encompassing the nucleus accumbens core (NAcc), shell (NAcs) and caudate-putamen (CPu) regulate cognitive functions including motivated behaviors, habit, learning, and sensorimotor action, among others. Sex steroid hormone sensitivity and sex differences have been documented in all of these functions in both normative and pathological contexts, including anxiety, depression and addiction. The neurotransmitter glutamate has been implicated in regulating these behaviors as well as striatal physiology, and there are likewise documented sex differences in glutamate action upon the striatal output neurons, the medium spiny neurons (MSNs). Here we review the available data regarding the role of steroid sex hormones such as 17β-estradiol (estradiol), progesterone, and testosterone in rapidly modulating MSN glutamatergic synapse properties, presented in the context of the estrous cycle as appropriate. Estradiol action upon glutamatergic synapse properties in female NAcc MSNs is most comprehensively discussed. In the female NAcc, MSNs exhibit development period-specific sex differences and estrous cycle variations in glutamatergic synapse properties as shown by multiple analyses, including that of miniature excitatory postsynaptic currents (mEPSCs). Estrous cycle-differences in NAcc MSN mEPSCs can be mimicked by acute exposure to estradiol or an ERα agonist. The available evidence, or lack thereof, is also discussed concerning estrogen action upon MSN glutamatergic synapse in the other striatal regions as well as the underexplored roles of progesterone and testosterone. We conclude that there is strong evidence regarding estradiol action upon glutamatergic synapse function in female NAcs MSNs and call for more research regarding other hormones and striatal regions.

Identification and validation of m6A RNA regulatory network in pulpitis

BackgroundN6-methyladenosine (m6A) RNA modification regulators play an important role in many human diseases, and its abnormal expression can lead to the occurrence and development of diseases. However, their significance in pulpitis remains largely unknown. Here, we sought to identify and validate the m6A RNA regulatory network in pulpitis.MethodsGene expression data for m6A regulators in human pulpitis and normal pulp tissues from public GEO databases were analyzed. Bioinformatics analysis including Gene ontology (GO) functional, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were performed by R package, and Cytoscape software was used to study the role of m6A miRNA-mRNA regulatory network in pulpitis. Quantitative real-time PCR (qRT-PCR) was performed to validate the expression of key m6A regulators in collected human pulpitis specimens.ResultsDifferential genes between pulpitis and normal groups were found from the GEO database, and further analysis found that there were significant differences in the m6A modification-related genes ALKBH5, METTL14, METTL3, METTL16, RBM15B and YTHDF1. And their interaction relationships and hub genes were determined. The hub m6A regulator targets were enriched in immune cells differentiation, glutamatergic synapse, ephrin receptor binding and osteoclast differentiation in pulpitis. Validation by qRT-PCR showed that the expression of methylases METTL14 and METTL3 was decreased, thus these two genes may play a key role in pulpitis.ConclusionOur study identified and validated the m6A RNA regulatory network in pulpitis. These findings will provide valuable resource to guide the mechanistic and therapeutic analysis of the role of key m6A modulators in pulpitis.

Metabolic characterization of neurogenetic disorders involving glutamatergic neurotransmission.

The study of inborn errors of neurotransmission has been mostly focused on monoamine disorders, GABAergic and glycinergic defects. The study of the glutamatergic synapse using the same approach than classic neurotransmitter disorders is challenging due to the lack of biomarkers in the CSF. A metabolomic approach can provide both insight into their molecular basis and outline novel therapeutic alternatives. We have performed a semi-targeted metabolomic analysis on CSF samples from 25 patients with neurogenetic disorders with an important expression in the glutamatergic synapse and 5 controls. Samples from patients diagnosed with MCP2, CDKL5-, GRINpathies and STXBP1-related encephalopathies were included. We have performed univariate (UVA) and multivariate statistical analysis (MVA), using Wilcoxon rank-sum test, principal component analysis (PCA), and OPLS-DA. By using the results of both analyses, we have identified the metabolites that were significantly altered and that were important in clustering the respective groups. On these, we performed pathway- and network-based analyses to define which metabolic pathways were possibly altered in each pathology. We have observed alterations in the tryptophan and branched-chain amino acid metabolism pathways, which interestingly converge on LAT1 transporter-dependency to cross the blood-brain barrier (BBB). Analysis of the expression of LAT1 transporter in brain samples from a mouse model of Rett syndrome (MECP2) revealed a decrease in the transporter expression, that was already noticeable at pre-symptomatic stages. The study of the glutamatergic synapse from this perspective advances the understanding of their pathophysiology, shining light on an understudied feature as is their metabolic signature.

CNSC-14. T-TYPE CALCIUM CHANNELS REGULATE NEURON-GLIOBLASTOMA INTERACTIONS AND PROMOTE TUMOR GROWTH

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor with a dismal median survival of 15 months. Glioblastoma utilizes calcium signaling to regulate communication between tumor cells and the neuronal microenvironment. T-type calcium channels (TTCCs) are voltage gated calcium channels expressed in various types of neurons throughout the brain regulating glutamatergic synaptic transmission. We analyzed spatial transcriptomics of human GBM tumors and found that TTCCs are expressed in infiltrating tumor regions and enriched for neuronal development spatial patterns and exhibit neurodevelopmental signatures. Analysis of single cells transcriptomic of human GBM tumors demonstrated that TTCCs are enriched in neuronal subtypes. To complement publicly available data,we treated Glioblastoma Stem cells (GSCs) with an FDA-approved repurposed TTCC blocker, mibefradil, and found downregulation of genes associated with neuron processes such as synapse organization, postsynaptic density and glutamatergic synapses. Treatment of GSCs with mibefradil abrogated cell proliferation in the presence of glutamate. We examined the effects of neuronal Cav3.2 on GBM growth. Genetic Cav3.2 knockout (Cav3.2 KO) in the microenvironment of mice xenografted with syngeneic cell lines (GL261 and CT2A) exhibited significant tumor volume reduction as well as increased survival compared to WT mice in both cell lines. Cav3.2 KO tumors exhibited a decrease in proliferation (Ki67+) and neuronal connections (VGlut1) measured by immunofluorescence compared to WT mice. Cav3.2KO neuron-GBM co-cultures exhibited a significant decrease in proliferation compared to WT neuron-GBM co-cultures. Cav3.2KO neuron-GBM co-cultures exhibited a decrease in synaptic connections and a decrease in neuronal projections to GBM cells compared to WT neuron-GBM co-cultures. Transcriptomic analysis of tumors from Cav3.2KO tumors revealed deregulation of genes associated synaptic transmission, glutamatergic synapses and postsynaptic specialization. Altogether, our findings indicate a role of Cav3.2 in regulating tumors and neurons and promoting GBM progression.

GSG1L-containing AMPA receptor complexes are defined by their spatiotemporal expression, native interactome and allosteric sites

Transmembrane AMPA receptor regulatory proteins (TARPs) and germ cell-specific gene 1-like protein (GSG1L) are claudin-type AMPA receptor (AMPAR) auxiliary subunits that profoundly regulate glutamatergic synapse strength and plasticity. While AMPAR-TARP complexes have been extensively studied, less is known about GSG1L-containing AMPARs. Here, we show that GSG1L’s spatiotemporal expression, native interactome and allosteric sites are distinct. GSG1L generally expresses late during brain development in a region-specific manner, constituting about 5% of all AMPAR complexes in adulthood. While GSG1L can co-assemble with TARPs or cornichons (CNIHs), it also assembles as the sole auxiliary subunit. Unexpectedly, GSG1L acts through two discrete evolutionarily-conserved sites on the agonist-binding domain with a weak allosteric interaction at the TARP/KGK site to slow desensitization, and a stronger interaction at a different site that slows recovery from desensitization. Together, these distinctions help explain GSG1L’s evolutionary past and how it fulfills a unique signaling role within glutamatergic synapses.

Binding of CaMKII to the NMDA receptor is sufficient for long-term potentiation.

Learning and the underlying long-lasting increases in glutamatergic synapse strength [called long-term potentiation (LTP)] require both Ca2+ influx through NMDA-type glutamate receptors (NMDARs) and the kinase CaMKII. New evidence now suggests that CaMKII can induce LTP purely by binding to the NMDAR subunit GluN2B and does not require the catalytic activity of the kinase.

Multiple N-linked glycosylation sites critically modulate the synaptic abundance of neuroligin isoforms

In recent years, elegant glycomic and glycoproteomic approaches have revealed an intricate glycosylation profile of mammalian brain with enormous spatial and temporal diversities. Nevertheless, at a cellular level, it is unclear how these post-translational modifications affect various proteins to influence crucial neuronal properties. Here, we have investigated the impact of N-linked glycosylation on neuroligins (NLGNs), a class of cell-adhesion molecules that play instructive roles in synapse organization. We found that endogenous NLGN proteins are differentially glycosylated across several regions of murine brain in a sex-independent but isoform-dependent manner. In both rodent primary neurons derived from brain sections and human neurons differentiated from stem cells, all NLGN variants were highly enriched with multiple N-glycan subtypes, which cumulatively ensured their efficient trafficking to the cell surface. Removal of these N-glycosylation residues only had a moderate effect on NLGNs' stability or expression levels but particularly enhanced their retention at the endoplasmic reticulum. As a result, the glycosylation-deficient NLGNs exhibited considerable impairments in their dendritic distribution and postsynaptic accumulation, which in turn, virtually eliminated their ability to recruit presynaptic terminals and significantly reduced NLGN overexpression-induced assemblies of both glutamatergic and GABAergic synapse structures. Therefore, our results highlight an essential mechanistic contribution of N-linked glycosylations in facilitating the appropriate secretory transport of a major synaptic cell-adhesion molecule and promoting its cellular function in neurons.

Alteration of Metabolic Profiles during the Progression of Alzheimer's Disease.

(1) Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder that threatens the population health of older adults. However, the mechanisms of the altered metabolism involved in AD pathology are poorly understood. The aim of the study was to identify the potential biomarkers of AD and discover the metabolomic changes produced during the progression of the disease. (2) Methods: Gas chromatography-mass spectrometry (GC-MS) was used to measure the concentrations of the serum metabolites in a cohort of subjects with AD (n = 88) and a cognitively normal control (CN) group (n = 85). The patients were classified as very mild (n = 25), mild (n = 27), moderate (n = 25), and severe (n = 11). The serum metabolic profiles were analyzed using multivariate and univariate approaches. Least absolute shrinkage and selection operator (LASSO) logistic regression was applied to identify the potential biomarkers of AD. Biofunctional enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes. (3) Results: Our results revealed considerable separation between the AD and CN groups. Six metabolites were identified as potential biomarkers of AD (AUC > 0.85), and the diagnostic model of three metabolites could predict the risk of AD with high accuracy (AUC = 0.984). The metabolic enrichment analysis revealed that carbohydrate metabolism deficiency and the disturbance of amino acid, fatty acid, and lipid metabolism were involved in AD progression. Especially, the pathway analysis highlighted that l-glutamate participated in four crucial nervous system pathways (including the GABAergic synapse, the glutamatergic synapse, retrograde endocannabinoid signaling, and the synaptic vesicle cycle). (4) Conclusions: Carbohydrate metabolism deficiency and amino acid dysregulation, fatty acid, and lipid metabolism disorders were pivotal events in AD progression. Our study may provide novel insights into the role of metabolic disorders in AD pathogenesis and identify new markers for AD diagnosis.

Increased presynaptic excitability in a migraine with aura mutation.

Migraine is a common and disabling neurological disorder. The headache and sensory amplifications of migraine are attributed to hyperexcitable sensory circuits, but a detailed understanding remains elusive. A mutation in casein kinase 1 delta (CK1δ) was identified in non-hemiplegic familial migraine with aura and advanced sleep phase syndrome. Mice carrying the CK1δT44A mutation were more susceptible to spreading depolarization (the phenomenon that underlies migraine aura), but mechanisms underlying this migraine-relevant phenotype were not known. We used a combination of whole-cell electrophysiology and multiphoton imaging, in vivo and in brain slices, to compare CK1δT44A mice (adult males) to their wild-type littermates. We found that despite comparable synaptic activity at rest, CK1δT44A neurons were more excitable upon repetitive stimulation than wild-type, with a reduction in presynaptic adaptation at excitatory but not inhibitory synapses. The mechanism of this adaptation deficit was a calcium-dependent enhancement of the size of the readily releasable pool of synaptic vesicles, and a resultant increase in glutamate release, in CK1δT44A compared to wild-type synapses. Consistent with this mechanism, CK1δT44A neurons showed an increase in the cumulative amplitude of excitatory post-synaptic currents, and a higher excitation-to-inhibition ratio during sustained activity compared to wild-type. At a local circuit level, action potential bursts elicited in CK1δT44A neurons triggered an increase in recurrent excitation compared to wild-type, and at a network level, CK1δT44A mice showed a longer duration of 'up state' activity, which is dependent on recurrent excitation. Finally, we demonstrated that the spreading depolarization susceptibility of CK1δT44A mice could be returned to wild-type levels with the same intervention (reduced extracellular calcium) that normalized presynaptic adaptation. Taken together, these findings show a stimulus-dependent presynaptic gain of function at glutamatergic synapses in a genetic model of migraine, that accounts for the increased spreading depolarization susceptibility and may also explain the sensory amplifications that are associated with the disease.

On the origin of F-wave: involvement of central synaptic mechanisms.

Neurophysiological methods are used widely to gain information about motor neuron excitability and axon conduction in neurodegenerative diseases. The F-wave is a common biomarker used to test motor neuron properties in the diagnosis of neurological diseases. Although the origin of the F-wave is a subject of debate, the most widely accepted mechanism posits that the F-wave is generated by the backfiring of motor neurons stimulated antidromically from the periphery. In this study, we developed an ex vivo mouse sciatic nerve-attached spinal cord preparation with sensory axons severed. In this preparation, stimulation of the whole sciatic nerve or its tibial branch evoked responses with the electrophysiological signatures of F-waves. Manipulations of synaptic transmission by either removal of extracellular calcium or block of post-synaptic glutamate receptors abolished these responses. These results suggest that F-waves are mediated by spinal microcircuits activated by recurrent motor axon collaterals via glutamatergic synapses.

Recovery kinetics of short-term depression of GABAergic and glutamatergic synapses at layer 2/3 pyramidal cells in the mouse barrel cortex.

Short-term synaptic plasticity (STP) is a widespread mechanism underlying activity-dependent modifications of cortical networks. To investigate how STP influences excitatory and inhibitory synapses in layer 2/3 of mouse barrel cortex, we combined whole-cell patch-clamp recordings from visually identified pyramidal neurons (PyrN) and parvalbumin-positive interneurons (PV-IN) of cortical layer 2/3 in acute slices with electrical stimulation of afferent fibers in layer 4 and optogenetic activation of PV-IN. These experiments revealed that electrical burst stimulation (10 pulses at 10 Hz) of layer 4 afferents to layer 2/3 neurons induced comparable short-term depression (STD) of glutamatergic postsynaptic currents (PSCs) in PyrN and in PV-IN, while disynaptic GABAergic PSCs in PyrN showed a stronger depression. Burst-induced depression of glutamatergic PSCs decayed within <4 s, while the decay of GABAergic PSCs required >11 s. Optogenetically-induced GABAergic PSCs in PyrN also demonstrated STD after burst stimulation, with a decay of >11 s. Excitatory postsynaptic potentials (EPSPs) in PyrN were unaffected after electrical burst stimulation, while a selective optogenetic STD of GABAergic synapses caused a transient increase of electrically evoked EPSPs in PyrN. In summary, these results demonstrate substantial short-term plasticity at all synapses investigated and suggest that the prominent STD observed in GABAergic synapses can moderate the functional efficacy of glutamatergic STD after repetitive synaptic stimulations. This mechanism may contribute to a reliable information flow toward the integrative layer 2/3 for complex time-varying sensory stimuli.

Uncovering the Early Events Associated with Oligomeric Aβ-Induced Src Activation.

Soluble Aβ1-42 oligomers (AβO) are formed in the early stages of Alzheimer's disease (AD) and were previously shown to trigger enhanced Ca2+ levels and mitochondrial dysfunction via the activation of N-methyl-D-aspartate receptors (NMDAR). Src kinase is a ubiquitous redox-sensitive non-receptor tyrosine kinase involved in the regulation of several cellular processes, which was demonstrated to have a reciprocal interaction towards NMDAR activation. However, little is known about the early-stage mechanisms associated with AβO-induced neurodysfunction involving Src. Thus, in this work, we analysed the influence of brief exposure to oligomeric Aβ1-42 on Src activation and related mechanisms involving mitochondria and redox changes in mature primary rat hippocampal neurons. Data show that brief exposure to AβO induce H2O2-dependent Src activation involving different cellular events, including NMDAR activation and mediated intracellular Ca2+ rise, enhanced cytosolic and subsequent mitochondrial H2O2 levels, accompanied by mild mitochondrial fragmentation. Interestingly, these effects were prevented by Src inhibition, suggesting a feedforward modulation. The current study supports a relevant role for Src kinase activation in promoting the loss of postsynaptic glutamatergic synapse homeostasis involving cytosolic and mitochondrial ROS generation after brief exposure to AβO. Therefore, restoring Src activity can constitute a protective strategy for mitochondria and related hippocampal glutamatergic synapses.

Nurture outpaces nature: fostering with an attentive mother alters social dominance in a mouse model of stress sensitivity.

Maternal care is critical for epigenetic programming during postnatal brain development. Stress is recognized as a critical factor that may affect maternal behavior, yet owing to high heterogeneity in stress response, its impact varies among individuals. We aimed here to understand the connection between inborn stress vulnerability, maternal care, and early epigenetic programming using mouse populations that exhibit opposite poles of the behavioral spectrum (social dominance [Dom] and submissiveness [Sub]) and differential response to stress. In contrast to stress-resilient Dom dams, stress-vulnerable Sub dams exhibit significantly lower maternal attachment, serum oxytocin, and colonic Lactobacillus reuteri populations. Sub offspring showed a reduced hippocampal expression of key methylation genes at postnatal day (PND) 7 and a lack of developmentally-dependent increase in 5-methylcytosine (5-mC) at PND 21. In addition, Sub pups exhibit significant hypermethylation of gene promoters connected with glutamatergic synapses and behavioral responses. We were able to reverse the submissive endophenotype through cross-fostering Sub pups with Dom dams (Sub/D). Thus, Sub/D pups exhibited elevated hippocampal expression of DNMT3A at PND 7 and increased 5-mC levels at PND 21. Furthermore, adult Sub/D offspring exhibited increased sociability, social dominance, and hippocampal glutamate and monoamine levels resembling the neurochemical profile of Dom mice. We postulate that maternal inborn stress vulnerability governs epigenetic patterning sculpted by maternal care and intestinal microbiome diversity during early developmental stages and shapes the array of gene expression patterns that may dictate neuronal architecture with a long-lasting impact on stress sensitivity and the social behavior of offspring.

Relationship between DNA methylation changes and skeletal muscle mass

BackgroundSarcopenia is a disease diagnosed in the elderly. In patients with sarcopenia, the muscle mass decreases every year. The occurrence of sarcopenia is greatly affected by extrinsic factors such as eating habits, exercise, and lifestyle. The present study aimed to determine the relationship between muscle mass traits and genes affected by epigenetic factors with three different adjustment methods using Korean Genome and Epidemiology Study (KOGES) data.ResultsWe conducted a demographic study and DNA methylation profiling by three studies according to the muscle mass index (MMI) adjustment methods: appendicular skeletal muscle mass divided by body weight (MMI1); appendicular skeletal muscle mass divided by square of height (MMI2); appendicular skeletal muscle mass divided by BMI (MMI3). We analyzed differentially methylated regions (DMRs) for each group. We then restricted our subjects to be top 30% (T30) and bottom 30% (B30) based on each MMI adjustment method. Additionally, we performed enrichment analysis using PathfindR to evaluate the relationship between identified DMRs and sarcopenia. A total of 895 subjects were included in the demographic study. The values of BMI, waist, and hip showed a significant difference in all three groups. Among 446 participants, 44 subjects whose DNA methylation profiles were investigated were included for DNA methylation analysis. The results of enrichment analysis showed differences between groups. In the women group through MMI1 method, only the glutamatergic synapse pathway showed a significant result. In the men group through MMI2 method, the adherens junction pathway was the most significant. Women group through MMI2 method showed similar results, having an enriched Rap1 signaling pathway. In men group through MMI3 method, the Fc epsilon RI signaling pathway was the most enriched. Particularly, the notch signaling pathway was significantly enriched in women group through MMI3 method.ConclusionThis study presents results about which factor should be concerned first in muscle mass index (MMI) adjustment. The present study suggested that GAB2 and JPH3 in MMI1 method, HLA-DQB1 and TBCD in MMI2 method, GAB2, NDUFB4 and ISPD in MMI3 method are potential genes that can have an impact on muscle mass. It could enable future epigenetic studies of genes based on annotation results. The present study is a nationwide study in Korea with the largest size up to date that compares adjustment indices for MMI in epigenetic research.

Hydrogel-based 3D human iPSC-derived neuronal culture for the study of rabies virus infection.

Rabies is a highly fatal infectious disease that poses a significant threat to human health in developing countries. In vitro study-based understanding of pathogenesis and tropism of different strains of rabies virus (RABV) in the central nervous system (CNS) is limited due to the lack of suitable culture models that recapitulate the complex communication pathways among host cells, extracellular matrices, and viruses. Therefore, a three-dimensional (3D) cell culture that mimics cell-matrix interactions, resembling in vivo microenvironment, is necessary to discover relevant underlying mechanisms of RABV infection and host responses. The 3D collagen-Matrigel hydrogel encapsulating hiPSC-derived neurons for RABV infection was developed and characterized based on cell viability, morphology, and gene expression analysis of neuronal markers. The replication kinetics of two different strains of RABV [wild-type Thai (TH) and Challenge Virus Standard (CVS)-11 strains] in both 2D and 3D neuronal cultures were examined. Differential gene expression analysis (DEG) of the neuropathological pathway of RABV-infected 2D and 3D models was also investigated via NanoString analysis. The 3D hiPSC-derived neurons revealed a more physiologically interconnected neuronal network as well as more robust and prolonged maturation and differentiation than the conventional 2D monolayer model. TH and CVS-11 exhibited distinct growth kinetics in 3D neuronal model. Additionally, gene expression analysis of the neuropathological pathway observed during RABV infection demonstrated a vast number of differentially expressed genes (DEGs) in 3D model. Unlike 2D neuronal model, 3D model displayed more pronounced cellular responses upon infection with CVS-11 when compared to the TH-infected group, highlighting the influence of the cell environment on RABV-host interactions. Gene ontology (GO) enrichment of DEGs in the infected 3D neuronal culture showed alterations of genes associated with the inflammatory response, apoptotic signaling pathway, glutamatergic synapse, and trans-synaptic signaling which did not significantly change in 2D culture. We demonstrated the use of a hydrogel-based 3D hiPSC-derived neuronal model, a highly promising technology, to study RABV infection in a more physiological environment, which will broaden our understanding of RABV-host interactions in the CNS.

Gene losses, parallel evolution and heightened expression confer adaptations to dedicated cleaning behaviour

BackgroundCleaning symbioses are captivating interspecific interactions in which a cleaner fish removes ectoparasites from its client, contributing to the health and diversity of natural fish communities and aquaculture systems. However, the genetic signatures underlying this specialized behaviour remain poorly explored. To shed light on this, we generated a high-quality chromosome-scale genome of the bluestreak cleaner wrasse Labroides dimidiatus, a dedicated cleaner with cleaning as primary feeding mechanism throughout its life.ResultsCompared with facultative and non-cleaner wrasses, L. dimidiatus was found with notable contractions in olfactory receptors implying their limited importance in dedicated cleaning. Instead, given its distinct tactile pre-conflict strategies, L. dimidiatus may rely more heavily on touch sensory perception, with heightened gene expression in the brain in anticipation of cleaning. Additionally, a reduction in NLR family CARD domain-containing protein 3 might enhance innate immunity of L. dimidiatus, probably assisting to reduce the impacts from parasite infections. In addition, convergent substitutions for a taste receptor and bone development genes across cleaners (L. dimidiatus and facultative cleaners) may provide them with evolved food discrimination abilities and jaw morphology that differentiate them from non-cleaners. Moreover, L. dimidiatus may exhibit specialized neural signal transductions for cleaning, as evidenced by positive selection in genes related to the glutamatergic synapse pathway. Interestingly, numerous glutamate receptors also demonstrated significantly higher expression in L. dimidiatus not engaged in cleaning, as compared to those involved in cleaning. Besides, apparent contractions in L. dimidiatus for protocadherins, which are responsible for neuronal development, may further promote specialized neural signal transductions in this species.ConclusionsThis study reveals that L. dimidiatus harbours substantial losses in specific gene families, convergent evolutions across cleaners and a large-scale high gene expression in preparation for cleaning, allowing for adaptation to the dedicated cleaning behaviour.

Possible Compensatory Role of ASICs in Glutamatergic Synapses.

Proton-gated channels of the ASIC family are widely distributed in central neurons, suggesting their role in common neurophysiological functions. They are involved in glutamatergic neurotransmission and synaptic plasticity; however, the exact function of these channels remains unclear. One problem is that acidification of the synaptic cleft due to the acidic content of synaptic vesicles has opposite effects on ionotropic glutamate receptors and ASICs. Thus, the pH values required to activate ASICs strongly inhibit AMPA receptors and almost completely inhibit NMDA receptors. This, in turn, suggests that ASICs can provide compensation for post-synaptic responses in the case of significant acidifications. We tested this hypothesis by patch-clamp recordings of rat brain neuron responses to acidifications and glutamate receptor agonists at different pH values. Hippocampal pyramidal neurons have much lower ASICs than glutamate receptor responses, whereas striatal interneurons show the opposite ratio. Cortical pyramidal neurons and hippocampal interneurons show similar amplitudes in their responses to acidification and glutamate. Consequently, the total response to glutamate agonists at different pH levels remains rather stable up to pH 6.2. Besides these pH effects, the relationship between the responses mediated by glutamate receptors and ASICs depends on the presence of Mg2+ and the membrane voltage. Together, these factors create a complex picture that provides a framework for understanding the role of ASICs in synaptic transmission and synaptic plasticity.

The effects of the NMDAR co-agonist d-serine on the structure and function of optic tectal neurons in the developing visual system

The N-methyl-d-aspartate type glutamate receptor (NMDAR) is a molecular coincidence detector which converts correlated patterns of neuronal activity into cues for the structural and functional refinement of developing circuits in the brain. d-serine is an endogenous co-agonist of the NMDAR. We investigated the effects of potent enhancement of NMDAR-mediated currents by chronic administration of saturating levels of d-serine on the developing Xenopus retinotectal circuit. Chronic exposure to the NMDAR co-agonist d-serine resulted in structural and functional changes in the optic tectum. In immature tectal neurons, d-serine administration led to more compact and less dynamic tectal dendritic arbors, and increased synapse density. Calcium imaging to examine retinotopy of tectal neurons revealed that animals raised in d-serine had more compact visual receptive fields. These findings provide insight into how the availability of endogenous NMDAR co-agonists like d-serine at glutamatergic synapses can regulate the refinement of circuits in the developing brain.