Synaptic Spine Research Articles

Novel De Novo Intronic Variant of SYNGAP1 Associated With the Neurodevelopmental Disorders.

SYNGAP1 encodes a Ras/Rap GTPase-activating protein that is predominantly expressed in the brain with the functional roles in regulating synaptic plasticity, spine morphogenesis, and cognition function. Pathogenic variants in SYNGAP1 have been associated with a spectrum of neurodevelopmental disorders characterized by developmental delays, intellectual disabilities, epilepsy, hypotonia, and the features of autism spectrum disorder. The aim of this study was to identify a novel SYNGAP1 gene variant linked to neurodevelopmental disorders and to evaluate the pathogenicity of the detected variant. A novel de novo intronic variant in SYNGAP1 was identified by Whole exome sequencing (WES) and confirmed by Sanger sequencing. Minigene assays were conducted to assess whether the intronic variant in SYNGAP1 influenced the normal splicing of mRNA. A novel de novo intronic variant in SYNGAP1 (c.3582+2T>G) was indentified with clinical features suggestive of neurodevelopmental related disorders. Minigene splicing analysis demonstrated that this noncanonical splice site variant led to the activation of a cryptic acceptor splice site. Consequently, 101 base pairs of intron 16 were aberrantly retained in the mRNA, leading to a frameshift. This frameshift resulted in the introduction of a premature stop codon (TGA) in the coding sequence and the production of a truncated SYNGAP1 protein, potentially leding to loss of function and subsequent disruption of its biological roles. Our findings highlight the significance of de novo pathogenic SYNGAP1 variants at the intron 16/exon 17 junction in the SYNGAP1-related neurodevelopmental disorders, providing novel insights into the genetic basis and diagnosis of these disabilities.

Microglial-Biomimetic Memantine-Loaded Polydopamine Nanomedicines for Alleviating Depression.

Depression is a common psychiatric disorder, and monoamine-based antidepressants as first-line therapy remain ineffective in some patients. The synergistic modulation of neuroinflammation and neuroplasticity could be a major strategy for treating depression. In this study, an inflammation-targeted microglial biomimetic system, PDA-Mem@M, is reported for treating depression. Microglial membrane-coated nanoparticles penetrate the blood-brain barrier and facilitate microglial targeting. Subsequently, owing to the excellent free radical-scavenging capacity, PDA-Mem@M attenuatethe brain inflammatory microenvironment. After on-demand release from the nanoparticles, memantine increasesthe expression of brain-derived neurotrophic factors and reversesthe loss of synaptic dendritic spines. Further, in vivo studies demonstrate that PDA-Mem@M effectively alleviatedepression-like behaviors to a greater extent than memantine or polydopamine nanoparticles (PDA) monotherapy. This synergistic strategy, with satisfactory biosafety and strong anti-inflammatory and synaptic plasticity restoration effects, is conducive to advances in depression therapy.

Exercise therapy facilitates neural remodeling and functional recovery post-spinal cord injury via PKA/CREB signaling pathway modulation in rats.

Neuronal structure is disrupted after spinal cord injury (SCI), causing functional impairment. The effectiveness of exercise therapy (ET) in clinical settings for nerve remodeling post-SCI and its underlying mechanisms remain unclear. This study aims to explore the effects and related mechanisms of ET on nerve remodeling in SCI rats. We randomly assigned rats to various groups: sham-operated group, sham-operated + ET, SCI alone, SCI + H89, SCI + ET, and SCI + ET + H89. Techniques including motor-evoked potential (MEP), video capture and analysis, the Basso-Beattie-Bresnahan (BBB) scale, western blotting, transmission electron microscopy, hematoxylin and eosin staining, Nissl staining, glycine silver staining, immunofluorescence, and Golgi staining were utilized to assess signal conduction capabilities, neurological deficits, hindlimb performance, protein expression levels, neuron ultrastructure, and tissue morphology. H89-an inhibitor that targets the protein kinase A (PKA)/cAMP response element-binding (CREB) signaling pathway-was employed to investigate molecular mechanisms. This study found that ET can reduce neuronal damage in rats with SCI, protect residual tissue, promote the remodeling of motor neurons, neurofilaments, dendrites/axons, synapses, and myelin sheaths, reorganize neural circuits, and promote motor function recovery. In terms of mechanism, ET mainly works by mediating the PKA/CREB signaling pathway in neurons. Our findings indicated that: (1) ET counteracted the H89-induced suppression of the PKA/CREB signaling pathway following SCI; (2) ET significantly alleviated neuronal injury and improved motor dysfunction; (3) ET facilitated neuronal regeneration by mediating the PKA/CREB signaling pathway; (4) ET enhanced synaptic and dendritic spine plasticity, as well as myelin sheath remodeling, post-SCI through the PKA/CREB signaling pathway.

A spatial model of autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) predicts that the lifetime of phospho-CaMKII after induction of synaptic plasticity is greatly prolonged by CaM-trapping.

Long-term potentiation (LTP) is a biochemical process that underlies learning in excitatory glutamatergic synapses in the Central Nervous System (CNS). The critical early driver of LTP is autophosphorylation of the abundant postsynaptic enzyme, Ca2+/calmodulin-dependent protein kinase II (CaMKII). Autophosphorylation is initiated by Ca2+ flowing through NMDA receptors activated by strong synaptic activity. Its lifetime is ultimately determined by the balance of the rates of autophosphorylation and of dephosphorylation by protein phosphatase 1 (PP1). Here we have modeled the autophosphorylation and dephosphorylation of CaMKII during synaptic activity in a spine synapse using MCell4, an open source computer program for creating particle-based stochastic, and spatially realistic models of cellular microchemistry. The model integrates four earlier detailed models of separate aspects of regulation of spine Ca2+ and CaMKII activity, each of which incorporate experimentally measured biochemical parameters and have been validated against experimental data. We validate the composite model by showing that it accurately predicts previous experimental measurements of effects of NMDA receptor activation, including high sensitivity of induction of LTP to phosphatase activity in vivo, and persistence of autophosphorylation for a period of minutes after the end of synaptic stimulation. We then use the model to probe aspects of the mechanism of regulation of autophosphorylation of CaMKII that are difficult to measure in vivo. We examine the effects of "CaM-trapping," a process in which the affinity for Ca2+/CaM increases several hundred-fold after autophosphorylation. We find that CaM-trapping does not increase the proportion of autophosphorylated subunits in holoenzymes after a complex stimulus, as previously hypothesized. Instead, CaM-trapping may dramatically prolong the lifetime of autophosphorylated CaMKII through steric hindrance of dephosphorylation by protein phosphatase 1. The results provide motivation for experimental measurement of the extent of suppression of dephosphorylation of CaMKII by bound Ca2+/CaM. The composite MCell4 model of biochemical effects of complex stimuli in synaptic spines is a powerful new tool for realistic, detailed dissection of mechanisms of synaptic plasticity.

Identification and characterization of human KALRN mRNA and Kalirin protein isoforms.

Kalirin is a multidomain protein with important roles in neurite outgrowth, and synaptic spine formation and remodeling. Genetic and pathophysiological links with various neuropsychiatric disorders associated with synaptic dysfunction and cognitive impairment have sparked interest in its potential as a pharmacological target. Multiple Kalirin proteoforms are detected in the adult human brain, yet we know little about the diversity of the transcripts that encode them or their tissue profiles. Here, we characterized full-length KALRN transcripts expressed in the adult human frontal lobe and hippocampus using rapid amplification of complementary DNA (cDNA) ends and nanopore long-read sequencing. For comparison with non-neural tissue, we also analyzed KALRN transcripts in the aorta. Multiple novel isoforms were identified and were largely similar between the two brain regions analyzed. Alternative splicing in the brain results in preferential inclusion of exon 37, which encodes 32 amino acids upstream of the second guanine nucleotide exchange factor (GEF) domain. Structural modeling predicts that a subset of these amino acids forms a conserved alpha helix. Although deletion of these amino acids had little effect on GEF activity, it did alter Kalirin-induced neurite outgrowth suggesting that this brain-enriched splicing event may be important for neural function. These data indicate that alternative splicing is potentially important for regulating Kalirin actions in the human brain.

Peptidomimetic inhibitors targeting TrkB/PSD-95 signaling improves cognition and seizure outcomes in an Angelman Syndrome mouse model.

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder with profoundly debilitating symptoms with no FDA-approved cure or therapeutic. Brain-derived neurotrophic factor (BDNF), and its receptor tropomyosin receptor kinase B (TrkB), have a well-established role as regulators of synaptic plasticity, dendritic outgrowth and spine formation. Previously, we reported that the association of postsynaptic density protein 95 (PSD-95) with TrkB is critical for intact BDNF signaling in the AS mouse model, as illustrated by attenuated PLCγ and PI3K signaling and intact MAPK pathway signaling. These data suggest that drugs tailored to enhance the TrkB-PSD-95 interaction may provide a novel approach for the treatment of AS and a variety of neurodevelopmental disorders (NDDs). To evaluate this critical interaction, we synthesized a class of high-affinity PSD-95 ligands that bind specifically to the PDZ3 domain of PSD-95, denoted as Syn3 peptidomimetic ligands. We evaluated Syn3 and its analog D-Syn3 (engineered using dextrorotary (D)-amino acids) in vivo using the Ube3a exon 2 deletion mouse model of AS. Following systemic administration of Syn3 and D-Syn3, we demonstrate improvement in the seizure domain of AS. Learning and memory using the novel object recognition assay also illustrated improved cognition following Syn3 and D-Syn3, along with restored long-term potentiation. A pharmacokinetic analysis of D-Syn3 demonstrates that it crosses the blood-brain barrier (BBB), and the brain influx rate is in the range of CNS therapeutics. Finally, D-Syn3 treated mice showed a partial rescue in motor learning. Neither Syn3 nor D-Syn3 improved gross exploratory locomotion deficits, nor gait impairments that have been well documented in the AS rodent models. These findings highlight the need for further investigation of this compound class as a potential therapeutic for AS and other genetic NDDs.

Mitochondrial modulation treating postoperative cognitive dysfunction neuroprotection via DRP1 inhibition by Mdivi1

This study investigated the role of mitochondrial dynamics in postoperative cognitive dysfunction (POCD) and assessed the therapeutic potential of mitochondrial modulation, particularly through the inhibition of dynamin-related protein 1 (DRP1) with Mdivi-1. Our findings indicated that DRP1 inhibition substantially mitigated neuroinflammation mediated by microglial cells, contributing to improved cognitive function in POCD models. The administration of Mdivi-1 led to a notable decrease in mitochondrial fission, reduced reactive oxygen species (ROS) production, and stabilization of mitochondrial membrane potential, all of which correlate with diminished neuroinflammation, as evidenced by lower NOD-like receptor family pyrin domain containing 3 (NLRP3)/ interleukin-1β (IL-1β) expression in microglial cells. Importantly, Mdivi-1 treatment was also found to enhance synaptic plasticity, increasing synaptic spine density in the hippocampal region of POCD mice. This improvement in mitochondrial health and synaptic integrity was paralleled by enhanced cognitive performance, as demonstrated in Y-maze tests. These results underscored the critical role of mitochondrial dynamics in the pathophysiology of POCD and suggested that targeting mitochondrial dysfunction, specifically through DRP1 inhibition, could be an effective approach for POCD treatment.

Ultrastructural characterization of hippocampal inhibitory synapses under resting and stimulated conditions.

The present study uses electron microscopy to document ultrastructural characteristics of hippocampal GABAergic inhibitory synapses under resting and stimulated conditions in three experimental systems. Synaptic profiles were sampled from stratum pyramidale and radiatum of the CA1 region from (1) perfusion fixed mouse brains, (2) immersion fixed rat organotypic slice cultures, and from (3) rat dissociated hippocampal cultures of mixed cell types. Synapses were stimulated in the brain by a 5min delay in perfusion fixation to trigger an ischemia-like excitatory condition, and by treating the two culture systems with 90 mM high K+ for 2-3min to depolarize the neurons. Upon such stimulation conditions, the presynaptic terminals of the inhibitory synapses exhibited similar structural changes to those seen in glutamatergic excitatory synapses, with depletion of synaptic vesicles, increase of clathrin-coated vesicles and appearance of synaptic spinules. However, in contrast to excitatory synapses, no structural differences were detected in the postsynaptic compartment of the inhibitory synapses upon stimulation. There were no changes in the appearance of material associated with the postsynaptic membrane or the length and curvature of the membrane. Also no change was detected in the labeling density of gephyrin, a GABAergic synaptic marker, lining the postsynaptic membrane. Furthermore, virtually all inhibitory synaptic clefts remained rigidly apposed, unlike in the case of excitatory synapses where ~ 20-30% of cleft edges were open upon stimulation, presumably to facilitate the clearance of neurotransmitters from the cleft. The fact that no open clefts were induced in inhibitory synapses upon stimulation suggests that inhibitory input may not need to be toned down under these conditions. On the other hand, similar to excitatory synapse, EGTA (a calcium chelator) induced open clefts in ~ 18% of inhibitory synaptic cleft edges, presumably disrupting similar calcium-dependent trans-synaptic bridges in both types of synapses.

Manipulation of radixin phosphorylation in the nucleus accumbens core modulates risky choice behavior

Ezrin-Radixin-Moesin (ERM) proteins are actin-binding proteins that contribute to morphological changes in dendritic spines. Despite their significant role in regulating spine structure, the role of ERM proteins in the nucleus accumbnes (NAcc) is not well known, especially in in the context of risk-reward decision-making. Here, we measured the relationship between synaptic excitation and inhibition (E/I ratio) from medium spiny neurons in the NAcc core obtained in the rat after a rat gambling task (rGT). Then, after surgery of a phosphomimetic pseudo-active mutant form of radixin (Rdx-T564D) in the NAcc core, we examined its role in synaptic plasticity and the accompanying risk-choice behavior in rGT. We found that basal E/I ratio in the NAcc core was higher in risk-averse rats than risk-seeking rats. However, it was significantly reduced in risk-averse rats similar to that in risk-seeking rats in the presence of Rdx-T564D in the NAcc core. Furthermore, the head sizes of spines were shifted in risk-averse rats expressing Rdx-T564D in the NAcc core, similar to those observed in risk-seeking rats. The effects of Rdx-T564D in risk-averse rats were again manifested as behavioral changes, with reduced selection of optimal choices and increased selection of disadvantageous ones. In this study, we demonstrated that manipulation of radixin phosphorylation status in the NAcc core can alter glutamatergic synaptic transmission and spine structure at this site, as well as risk choice behaviors in the rGT. These novel findings illustrate that radixin in the NAcc core plays a significant role in determining risk preference during the rGT.

Cognitive synaptopathy: synaptic and dendritic spine dysfunction in age-related cognitive disorders.

Cognitive impairment is a leading component of several neurodegenerative and neurodevelopmental diseases, profoundly impacting on the individual, the family, and society at large. Cognitive pathologies are driven by a multiplicity of factors, from genetic mutations and genetic risk factors, neurotransmitter-associated dysfunction, abnormal connectomics at the level of local neuronal circuits and broader brain networks, to environmental influences able to modulate some of the endogenous factors. Otherwise healthy older adults can be expected to experience some degree of mild cognitive impairment, some of which fall into the category of subjective cognitive deficits in clinical practice, while many neurodevelopmental and neurodegenerative diseases course with more profound alterations of cognition, particularly within the spectrum of the dementias. Our knowledge of the underlying neuropathological mechanisms at the root of this ample palette of clinical entities is far from complete. This review looks at current knowledge on synaptic modifications in the context of cognitive function along healthy ageing and cognitive dysfunction in disease, providing insight into differential diagnostic elements in the wide range of synapse alterations, from those associated with the mild cognitive changes of physiological senescence to the more profound abnormalities occurring at advanced clinical stages of dementia. I propose the term "cognitive synaptopathy" to encompass the wide spectrum of synaptic pathologies associated with higher brain function disorders.

TRPC3/6 Channels Mediate Mechanical Pain Hypersensitivity via Enhancement of Nociceptor Excitability and of Spinal Synaptic Transmission.

Patients with tissue inflammation or injury often experience aberrant mechanical pain hypersensitivity, one of leading symptoms in clinic. Despite this, the molecular mechanisms underlying mechanical distortion are poorly understood. Canonical transient receptor potential (TRPC) channels confer sensitivity to mechanical stimulation. TRPC3 and TRPC6 proteins, coassembling as heterotetrameric channels, are highly expressed in sensory neurons. However, how these channels mediate mechanical pain hypersensitivity has remained elusive. It is shown that in mice and human, TRPC3 and TRPC6 are upregulated in DRG and spinal dorsal horn under pathological states. Double knockout of TRPC3/6 blunts mechanical pain hypersensitivity, largely by decreasing nociceptor hyperexcitability and spinal synaptic potentiation via presynaptic mechanism. In corroboration with this, nociceptor-specific ablation of TRPC3/6 produces comparable pain relief. Mechanistic analysis reveals that upon peripheral inflammation, TRPC3/6 in primary sensory neurons get recruited via released bradykinin acting on B1/B2 receptors, facilitating BDNF secretion from spinal nociceptor terminals, which in turn potentiates synaptic transmission through TRPC3/6 and eventually results in mechanical pain hypersensitivity. Antagonizing TRPC3/6 in DRG relieves mechanical pain hypersensitivity in mice and nociceptor hyperexcitability in human. Thus, TRPC3/6 in nociceptors is crucially involved in pain plasticity and constitutes a promising therapeutic target against mechanical pain hypersensitivity with minor side effects.

Amyloid-β Causes NMDA Receptor Dysfunction and Dendritic Spine Loss through mGluR1 and AKAP150-Anchored Calcineurin Signaling.

Neuronal excitatory synapses are primarily located on small dendritic protrusions called spines. During synaptic plasticity underlying learning and memory, Ca2+ influx through postsynaptic NMDA-type glutamate receptors (NMDARs) initiates signaling pathways that coordinate changes in dendritic spine structure and synaptic function. During long-term potentiation (LTP), high levels of NMDAR Ca2+ influx promote increases in both synaptic strength and dendritic spine size through activation of Ca2+-dependent protein kinases. In contrast, during long-term depression (LTD), low levels of NMDAR Ca2+ influx promote decreased synaptic strength and spine shrinkage and elimination through activation of the Ca2+-dependent protein phosphatase calcineurin (CaN), which is anchored at synapses via the scaffold protein A-kinase anchoring protein (AKAP)150. In Alzheimer's disease (AD), the pathological agent amyloid-β (Aβ) may impair learning and memory through biasing NMDAR Ca2+ signaling pathways toward LTD and spine elimination. By employing AKAP150 knock-in mice of both sexes with a mutation that disrupts CaN anchoring to AKAP150, we revealed that local, postsynaptic AKAP-CaN-LTD signaling was required for Aβ-mediated impairment of NMDAR synaptic Ca2+ influx, inhibition of LTP, and dendritic spine loss. Additionally, we found that Aβ acutely engages AKAP-CaN signaling through activation of G-protein-coupled metabotropic glutamate receptor 1 (mGluR1) leading to dephosphorylation of NMDAR GluN2B subunits, which decreases Ca2+ influx to favor LTD over LTP, and cofilin, which promotes F-actin severing to destabilize dendritic spines. These findings reveal a novel interplay between NMDAR and mGluR1 signaling that converges on AKAP-anchored CaN to coordinate dephosphorylation of postsynaptic substrates linked to multiple aspects of Aβ-mediated synaptic dysfunction.

Treadmill exercise prevents recognition memory impairment in VD rat model and enhancement of hippocampal structural synaptic plasticity.

In vascular dementia (VD), memory impairment caused by the damage of synaptic plasticity is the most prominent feature that afflicts patients and their families. Treadmill exercise has proven beneficial for memory by enhancing synaptic plasticity in animal models including stroke, dementia, and mental disorders. The aim of this study was to examine the effects of treadmill exercise on recognition memory and structural synaptic plasticity in VD rat model. Male Sprague-Dawley rats were randomly assigned into four groups: control group (C group, n=6), vascular dementia group (VD group, n=6), treadmill exercise and vascular dementia group (Exe-VD group, n=6), and treadmill exercise group (Exe group, n=6). Four-week treadmill exercise was performed in the Exe-VD and Exe groups. Then, the common carotid arteries of rats in the VD and Exe-VD groups were identified to establish the VD model. Behavior tests (open-field test and novel recognition memory test) were adopted to evaluate anxiety-like behavior and recognition memory. Transmission electron microscopy and Golgi staining were performed to observe synaptic ultrastructure and spine density in the hippocampus. Our study demonstrated that VD rat exhibited significantly anxiety-like behavior and recognition impairment (p <.01), while treadmill exercise significantly alleviated anxiety-like behavior and improved recognition memory in VD rat (p <.01). Transmission electron microscopy revealed that hippocampal synapse numbers were significantly decreased in the VD group compared to the control group (p <.05). These alterations were reversed by treadmill exercise, and the rats exhibited healthier synaptic ultrastructure, including significantly increased synapse (p <.05). Meanwhile, golgi staining revealed that the spine numbers of the hippocampus were significantly decreased in the VD group compared to the control group (p <.05). When compared with the VD group, hippocampal spine numbers were significantly increased in the Exe-VD group (p <.05). The improvement of VD-associated recognition memory by treadmill exercises is associated with enhanced structural synaptic plasticity in VD rat model.

GlyT1 Inhibition by NFPS Promotes Neuroprotection in Amyloid-β-Induced Alzheimer's Disease Animal Model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β, leading to N-methyl-D-aspartate (NMDA) receptor-dependent synaptic depression, spine elimination, and memory deficits. Glycine transporter type 1 (GlyT1) modulates glutamatergic neurotransmission via NMDA receptors (NMDAR), presenting a potential alternative therapeutic approach for AD. This study investigates the neuroprotective potential of GlyT1 inhibition in an amyloid-β-induced AD mouse model. C57BL/6 mice were treated with N-[3-([1,1-Biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine (NFPS), a GlyT1 inhibitor, 24h prior to intrahippocampal injection of amyloid-β. NFPS pretreatment prevented amyloid-β-induced cognitive deficits in short-term and long-term memory, evidenced by novel object recognition and spatial memory tasks. Moreover, NFPS pretreatment curbed microglial activation, astrocytic reactivity, and subsequent neuronal damage from amyloid-β injection. An extensive label-free quantitative UPLC-MSE proteomic analysis was performed on the hippocampi of mice treated with NFPS. In proteomics, KEGG enrichment analysis revealed increased in dopaminergic synapse, purine-containing compound biosynthetic process and long-term potentiation, and a reduction in Glucose catabolic process and glycolytic process pathways. The western blot analysis confirmed that NFPS treatment elevated BDNF levels, correlating with enhanced TRKB phosphorylation and mTOR activation. Moreover, NFPS treatment reduced the GluN2B expression after 6h, which was associated with an increase on CaMKIV and CREB phosphorylation. Collectively, these findings demonstrate that GlyT1 inhibition by NFPS activates diverse neuroprotective pathways, enhancing long-term potentiation signaling and countering amyloid-β-induced hippocampal damage.

APP fragment controls both ionotropic and non-ionotropic signaling of NMDA receptors

NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating synaptic long-term depression (LTD) and spine shrinkage. Here, we identify AETA (Aη), an amyloid-β precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes. AETA inhibits ionotropic NMDAR activity by competing with the co-agonist and induces an intracellular conformational modification of GluN1 subunits. This favors non-ionotropic NMDAR signaling leading to enhanced LTD and favors spine shrinkage. Endogenously, AETA production is increased by invivo chemogenetically induced neuronal activity. Genetic deletion of AETA production alters NMDAR transmission and prevents LTD, phenotypes rescued by acute exogenous AETA application. This genetic deletion also impairs contextual fear memory. Our findings demonstrate AETA-dependent NMDAR activation (ADNA), characterizing AETA as a unique type of endogenous NMDAR modulator that exerts bidirectional control over NMDAR signaling and associated information processing.

Synaptic correlates of the corticocortical circuit in motor learning.

Rodents actively learn new motor skills for survival in reaction to changing environments. Despite the classic view of the primary motor cortex (M1) as a simple muscle relay region, it is now known to play a significant role in motor skill acquisition. The secondary motor cortex (M2) is reported to be a crucial region for motor learning as well as for its role in motor execution and planning. Although these two regions are known for the part they play in motor learning, the role of direct connection and synaptic correlates between these two regions remains elusive. Here, we confirm M2 to M1 connectivity with a series of tracing experiments. We also show that the accelerating rotarod task successfully induces motor skill acquisition in mice. For mice that underwent rotarod training, learner mice showed increased synaptic density and spine head size for synapses between activated cell populations of M2 and M1. Non-learner mice did not show these synaptic changes. Collectively, these data suggest the potential importance of synaptic plasticity between activated cell populations as a potential mechanism of motor learning. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Coenzyme Q10 alleviates depression-like behaviors in mice with chronic restraint stress by down-regulating the pyroptosis signaling pathway

To explore the neuroprotective effect of coenzyme Q10 and its possible mechanism in mice with chronic restraint stress (CRS). Mouse models of CRS were treated with intraperitoneal injections of coenzyme Q10 at low, moderate and high doses (50, 100 and 200 mg/kg, respectively, n=8), VX765 (a caspase-1 specific inhibitor, 50 mg/kg, n=8), or fluoxetine (10 mg/kg, n=8) on a daily basis for 4 weeks, and the changes in depression-like behaviors of the mice were assessed by sugar water preference test, forced swimming test and tail suspension test. The expression of glial fibrillary acidic protein (GFAP) in the hippocampus of the mice was detected using immunohistochemistry, and the number of synaptic spines was determined with Golgi staining. Western blotting was performed to detect the changes in the expressions of GFAP and pyroptosis-related proteins in the hippocampus, and the colocalization of neurons and caspase-1 p10 was examined with immunofluorescence assay. Compared with the normal control mice, the mouse models of CRS showed significantly reduced sugar water preference and increased immobility time in forced swimming and tail suspension tests (P < 0.05), and these depression-like behaviors were obviously improved by treatment with coenzyme Q10, VX765 or FLX. The mouse models showed a significantly decreased positive rate of GFAP and lowered GFAP protein expression in the hippocampus with obviously decreased synaptic spines, enhanced expressions of GSDMD-N, caspase-1 and IL-1β, and increased colocalization of neurons and caspase-1 p10 (all P < 0.05). All these changes were significantly ameliorated in the mouse models after treatment with Q10. Coenzyme Q10 can alleviate depression-like behaviors in mice with CRS by down-regulating the pyroptosis signaling pathway.

PolyI:C Maternal Immune Activation on E9.5 Causes the Deregulation of Microglia and the Complement System in Mice, Leading to Decreased Synaptic Spine Density.

Maternal immune activation (MIA) is a risk factor for multiple neurodevelopmental disorders; however, animal models developed to explore MIA mechanisms are sensitive to experimental factors, which has led to complexity in previous reports of the MIA phenotype. We sought to characterize an MIA protocol throughout development to understand how prenatal immune insult alters the trajectory of important neurodevelopmental processes, including the microglial regulation of synaptic spines and complement signaling. We used polyinosinic:polycytidylic acid (polyI:C) to induce MIA on gestational day 9.5 in CD-1 mice, and measured their synaptic spine density, microglial synaptic pruning, and complement protein expression. We found reduced dendritic spine density in the somatosensory cortex starting at 3-weeks-of-age with requisite increases in microglial synaptic pruning and phagocytosis, suggesting spine density loss was caused by increased microglial synaptic pruning. Additionally, we showed dysregulation in complement protein expression persisting into adulthood. Our findings highlight disruptions in the prenatal environment leading to alterations in multiple dynamic processes through to postnatal development. This could potentially suggest developmental time points during which synaptic processes could be measured as risk factors or targeted with therapeutics for neurodevelopmental disorders.

Autism risk gene Cul3 alters neuronal morphology via caspase-3 activity in mouse hippocampal neurons.

Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders (NDDs) in which children display differences in social interaction/communication and repetitive stereotyped behaviors along with variable associated features. Cul3, a gene linked to ASD, encodes CUL3 (CULLIN-3), a protein that serves as a key component of a ubiquitin ligase complex with unclear function in neurons. Cul3 homozygous deletion in mice is embryonic lethal; thus, we examine the role of Cul3 deletion in early synapse development and neuronal morphology in hippocampal primary neuronal cultures. Homozygous deletion of Cul3 significantly decreased dendritic complexity and dendritic length, as well as axon formation. Synaptic spine density significantly increased, mainly in thin and stubby spines along with decreased average spine volume in Cul3 knockouts. Both heterozygous and homozygous knockout of Cul3 caused significant reductions in the density and colocalization of gephyrin/vGAT puncta, providing evidence of decreased inhibitory synapse number, while excitatory synaptic puncta vGulT1/PSD95 density remained unchanged. Based on previous studies implicating elevated caspase-3 after Cul3 deletion, we demonstrated increased caspase-3 in our neuronal cultures and decreased neuronal cell viability. We then examined the efficacy of the caspase-3 inhibitor Z-DEVD-FMK to rescue the decrease in neuronal cell viability, demonstrating reversal of the cell viability phenotype with caspase-3 inhibition. Studies have also implicated caspase-3 in neuronal morphological changes. We found that caspase-3 inhibition largely reversed the dendrite, axon, and spine morphological changes along with the inhibitory synaptic puncta changes. Overall, these data provide additional evidence that Cul3 regulates the formation or maintenance of cell morphology, GABAergic synaptic puncta, and neuronal viability in developing hippocampal neurons in culture.

Mechanism of Chaijin Jieyu Anshen Tablets in regulating abnormal ACC-vHPC glutaminergic neural circuit to alleviate synaptic remodeling of ventral hippocampal neurons in depressed rats

This study aims to reveal the molecular mechanism of Chaijin Jieyu Anshen Tablets(CJJYAS) in regulating the abnormal anterior cingulate cortex(ACC)-ventral hippocampus(vHPC) glutaminergic neural circuit to alleviate synaptic remodeling of ventral hippocampal neurons in depressed rats. Firstly, the study used chemogenetics to localize glutaminergic adeno-associated virus(AAV) into the ACC brain region of rats. The model of depressed rats was established by chronic unpredictable mild stress(CUMS) combined with independent feeding. The rats were randomly divided into control group, model group, AAV empty group, AAV group, AAV+ glucocorticoid receptors(GR) blocker group, AAV+chemokine receptor 1(CX3CR1) blocker group, and AAV+CJJYAS group. Depressive-like behaviors of rats were evaluated by open-field, forced-swimming, and Morris water maze tests, combined with an animal behavior analysis system. The morphological and structural changes of ACC and vHPC neurons in rats were observed by hematoxylin-eosin(HE) staining. Immunofluorescence and nuclear phosphoprotein(c-Fos) were used to detect glutaminergic neural circuit activation of ACC-vHPC in rats. The changes in dendrites, synaptic spines, and synaptic submicrostructure of vHPC neurons were observed by Golgi staining and transmission electron microscopy, respectively. The expressions of synaptic remodeling-related proteins N-methyl-D-asprtate receptor 2A(GRIN2A), N-methyl-D-asprtate receptor 2B(GRIN2B), Ca~(2+)/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ), mitogen-activated protein kinase-activated protein kinase 2(MK2), and a ubiquitous actin-binding protein(cofilin) in vHPC glutaminergic neurons of rats were detected by immunofluorescence and Western blot, respectively. The results indicated that the activated glutaminergic AAV aggravated the depressive-like behaviors phenotype of rats in the model group and deteriorated the damage of morphology and structure of ACC and vHPC neurons and synaptic ultrastructure. However, both GR and CX3CR1 bloc-kers could reverse the abnormal changes to varying degrees, suggesting that the abnormal activation of ACC-vHPC glutaminergic neural circuit mediated by GR/CX3CR1 signals in gliocytes in the ACC brain region may be closely related to the occurrence and development of depression. Interestingly, CJJYAS significantly inhibited the activation of the ACC-vHPC glutaminergic neural circuit induced by AAV and the elevated Glu level. Furthermore, CJJYAS could also effectively reverse the aggravation of depressive-like behaviors and synaptic remodeling of vHPC neurons of rats in the model group induced by the activated AAV. Additionally, the findings suggested that the molecular mechanism of CJJYAS in improving synaptic damage of vHPC neurons might be related to the regulation of synaptic remodeling-related signals such as NR/CaMKⅡ and MK2/cofilin. In conclusion, this research confirms that CJJYAS effectively regulates the abnormal ACC-vHPC glutaminergic neural circuit and alleviates the synaptic remodeling of vHPC glutaminergic neurons in depressed rats, and the molecular mechanism might be associated with the regulation of synapse-related NR/CaMKⅡ and MK2/cofilin signaling pathways, which may be the crucial mechanism of its antidepressant effect.