Synaptic Ultrastructure Research Articles

An ultrastructural connectomic analysis of a higher-order thalamocortical circuit in the mouse.

Many studies exist of thalamocortical synapses in primary sensory cortex, but much less in known about higher-order thalamocortical projections to higher-order cortical areas. We begin to address this gap using genetic labeling combined with large volume serial electron microscopy (i.e., "connectomics") to study the projection from the thalamic posterior medial nucleus to the secondary somatosensory cortex in a mouse. We injected into this thalamic nucleus a cocktail combining a cre-expressing virus and one expressing cre-dependent ascorbate peroxidase that provides an electron dense cytoplasmic label. This "intersectional" viral approach specifically labeled thalamocortical axons and synapses, free of retrograde labeling, in all layers of cortex. Labeled thalamocortical synapses represented 14% of all synapses in the cortical volume, consistent with previous estimates of first-order thalamocortical inputs. We found that labeled thalamocortical terminals, relative to unlabeled ones: were larger, were more likely to contain a mitochondrion, more frequently targeted spiny dendrites and avoided aspiny dendrites, and often innervated larger spines with spine apparatuses, among other differences. Furthermore, labeled terminals were more prevalent in layers 2/3 and synaptic differences between labeled and unlabeled terminals were greatest in layers 2/3. The laminar differences reported here contrast with reports of first-order thalamocortical connections in primary sensory cortices where, for example, labeled terminals were larger in layer 4 than layers 2/3 (Viaene et al., 2011a). These data offer the first glimpse of higher-order thalamocortical synaptic ultrastructure and point to the need for more analyses, as such connectivity likely represents a majority of thalamocortical circuitry.

Targeted Brain Delivery of RVG29-Modified Rifampicin-Loaded Nanoparticles for Treatment and Diagnosis of Alzheimer Disease

Alzheimer's disease (AD) is a neurodegenerative disease related to age. The main pathological features of AD are β-amyloid protein (Aβ) deposition and tau protein hyperphosphorylation. Currently, there are not an effective drug for the etiological treatment of AD. Rifampicin (RIF) is a semi-synthetic broad-spectrum antibiotic with anti-β-amyloid deposition, anti-inflammatory, anti-apoptosis and neuroprotective effects, but its application in AD treatment has been limited by its strong hydrophobicity, high toxicity, short half-life, low bioavailability and blood-brain barrier (BBB) hindrance. We designed a novel brain-targeted and MRI-characteristic nanomedicine through loading rabies virus protein 29 (RVG29), rifampicin and Gd on poly(L-lactide) nanoparticles (RIF@PLA-PEG-Gd/Mal-RVG29). The cytotoxicity assay demonstrated that RIF@PLA-PEG-Gd/Mal-RVG29 had good biocompatibility and security. The results of cellular uptake and fluorescence in vivo imaging showed that PLA-PEG-Gd/Mal-RVG29 could deliver rifampicin into brain by enhancing cellular uptake and brain targeting performance, so that improving the bioavailability of rifampicin. In vivo study, compared with rifampicin, RIF@PLA-PEG-Gd/Mal-RVG29 improved the spatial learning and memory capability of APP/PS1 mice in the Morris water maze. Immunofluorescence, TEM, immunoblotting and HE test showed that RIF@PLA-PEG-Gd/Mal-RVG29 could reduce Aβ deposition in hippocampal and cortex of APP/PS1 mice, improve the damage of synaptic ultrastructure, increase the expression level of PSD95 and SYP, as well as reduce the necrosis of neurons. These findings suggest that RIF@PLA-PEG-Gd/Mal-RVG29 may be an effective strategy for the treatment and diagnosis of AD.

Maternal Exposure to Di-(2-ethylhexyl) Phthalate Impairs Hippocampal Synaptic Plasticity in Male Offspring: Involvement of Damage to Dendritic Spine Development.

Exposure to di-(2-ethylhexyl) phthalate (DEHP), a widely used kind of plasticizer, can result in neurodevelopment impairments and learning and memory disorders. We studied the effects and possible mechanisms of maternal DEHP treatment on hippocampal synaptic plasticity in offspring. Pregnant Wistar rats were randomly divided into four groups and received 0, 30, 300, 750 (mg/kg)/d DEHP by gavage from gestational day (GD) 0 to postnatal day (PN) 21. Our data showed that DEHP exposure impaired hippocampal synaptic plasticity, damaged synaptic ultrastructure, and decreased synaptic protein levels in male pups. Furthermore, DEHP decreased the density of dendritic spines, affected F-actin polymerization, and downregulated the Rac1/PAK/LIMK1/cofilin signaling pathway in male offspring. However, the alterations in the hippocampi of female offspring were not observed. These results illustrate that maternal DEHP exposure could impair hippocampal synaptic plasticity by affecting synaptic structure and dendritic spine development in male offspring, which may be attributed to altered cytoskeleton construction induced by downregulation of the Rac1/PAK/LIMK1/cofilin signaling pathway.

SynapsEM: Computer-Assisted Synapse Morphometry

The structural features of a synapse help determine its function. Synapses are extremely small and tightly packed with vesicles and other organelles. Visualizing synaptic structure requires imaging by electron microscopy, and the features in micrographs must be quantified, a process called morphometry. Three parameters are typically assessed from each specimen: (1) the sizes of individual vesicles and organelles; (2) the absolute number and densities of organelles; and (3) distances between organelles and key features at synapses, such as active zone membranes and dense projections. For data to be meaningful, the analysis must be repeated from hundreds to thousands of images from several biological replicates, a daunting task. Here we report a custom computer program to analyze key structural features of synapses: SynapsEM. In short, we developed ImageJ/Fiji macros to record x,y-coordinates of segmented structures. The coordinates are then exported as text files. Independent investigators can reload the images and text files to reexamine the segmentation using ImageJ. The Matlab program then calculates and reports key synaptic parameters from the coordinates. Since the values are calculated from coordinates, rather than measured from each micrograph, other parameters such as locations of docked vesicles relative to the center of an active zone can be extracted in Matlab by additional scripting. Thus, this program can accelerate the morphometry of synapses and promote a more comprehensive analysis of synaptic ultrastructure.

Cortical response selectivity derives from strength in numbers of synapses.

Single neocortical neurons are driven by populations of excitatory inputs, forming the basis of neuronal selectivity to features of sensory input. Excitatory connections are thought to mature during development through activity-dependent Hebbian plasticity1, whereby similarity between presynaptic and postsynaptic activity selectively strengthens some synapses and weakens others2. Evidence in support of this process ranges from measurements of synaptic ultrastructure to in vitro and in vivo physiology and imaging studies3,4,5,6,7,8. These corroborating lines of evidence lead to the prediction that a small number of strong synaptic inputs drive neuronal selectivity, while weak synaptic inputs are less correlated with the somatic output and modulate activity overall6,7. Supporting evidence from cortical circuits, however, has been limited to measurements of neighboring, connected cell pairs, raising the question of whether this prediction holds for a broad range of synapses converging onto cortical neurons. Here we measure the strengths of functionally characterized excitatory inputs contacting single pyramidal neurons in ferret primary visual cortex (V1) by combining in vivo two-photon synaptic imaging and post hoc electron microscopy (EM). Using EM reconstruction of individual synapses as a metric of strength, we find no evidence that strong synapses play a predominant role in the selectivity of cortical neuron responses to visual stimuli. Instead, selectivity appears to arise from the total number of synapses activated by different stimuli. Moreover, spatial clustering of co-active inputs appears reserved for weaker synapses, enhancing the contribution of weak synapses to somatic responses. Our results challenge the role of Hebbian mechanisms in shaping neuronal selectivity in cortical circuits, and suggest that selectivity reflects the co-activation of large populations of presynaptic neurons with similar properties and a mixture of strengths.

Targeting Functionally Characterized Synaptic Architecture Using Inherent Fiducials and 3D Correlative Microscopy

Brain circuits are highly interconnected three-dimensional structures fabricated from components ranging vastly in size; from cell bodies to individual synapses. While neuronal activity can be visualized with advanced light microscopy (LM) techniques, the resolution of electron microscopy (EM) is critical for identifying synaptic connections between neurons. Here, we combine these two techniques, affording the advantage of each and allowing for measurements to be made of the same neural features across imaging platforms. We established an EM-label-free workflow utilizing inherent structural features to correlate in vivo two-photon LM and volumetric scanning EM (SEM) in the ferret visual cortex. By optimizing the volume SEM sample preparation protocol, imaging with the OnPoint detector, and utilizing the focal charge compensation device during serial block-face imaging, we achieved sufficient resolution and signal-to-noise ratio to analyze synaptic ultrastructure for hundreds of synapses within sample volumes. Our novel workflow provides a reliable method for quantitatively characterizing synaptic ultrastructure in functionally imaged neurons, providing new insights into neuronal circuit organization.

Metabotropic glutamate receptor 5 regulates synaptic plasticity in a chronic migraine rat model through the PKC/NR2B signal

BackgroundThe mechanism of chronic migraine (CM) is complex, central sensitization is considered as one of the pathological mechanism. Synaptic plasticity is the basis of central sensitization. Metabotropic glutamate receptor 5 (mGluR5) plays a vital role in the synaptic plasticity of the central nervous system. However, whether mGluR5 can promote the central sensitization by regulating synaptic plasticity in CM is unknown.MethodsMale Wistar rats were used to establish a CM rat model, and the expression of mGluR5 mRNA and protein were detected by qRT-PCR and western blot. The allodynia was assessed by mechanical and thermal thresholds, and central sensitization was assessed by expression of the phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) at Serine 133(pCREB-S133) and c-Fos. The synaptic-associated protein postsynaptic density protein 95 (PSD), synaptophysin (Syp), and synaptophysin-1(Syt-1), synaptic ultrastructure, and dendritic spines were detected to explore synaptic plasticity. The expression of PKC, total NR2B(tNR2B), and phosphorylation of NR2B at Tyr1472(pNR2B-Y1472) were detected by western blot.ResultsWe found that the expression of mGluR5 was upregulated in CM rats. Downregulated the mGluR5 with MPEP alleviated the allodynia and reduced the expression of CGRP, pCREB-S133, c-Fos, PSD, Syp and Syt-1 and synaptic transmission. Moreover, the administration of MPEP inhibited the upregulation of PKC and pNR2B-Y1472.ConclusionsThese results indicate that mGluR5 contributes to central sensitization by regulating synaptic plasticity in CM through the PKC/NR2B signal, which suggests that mGluR5 may be a potential therapeutic candidate for CM.

Altered synaptic ultrastructure in the prefrontal cortex of Shank3-deficient rats

BackgroundDeletion or mutations of SHANK3 lead to Phelan–McDermid syndrome and monogenic forms of autism spectrum disorder (ASD). SHANK3 encodes its eponymous scaffolding protein at excitatory glutamatergic synapses. Altered morphology of dendrites and spines in the hippocampus, cerebellum, and striatum have been associated with behavioral impairments in Shank3-deficient animal models. Given the attentional deficit in these animals, our study explored whether deficiency of Shank3 in a rat model alters neuron morphology and synaptic ultrastructure in the medial prefrontal cortex (mPFC).MethodsWe assessed dendrite and spine morphology and spine density in mPFC layer III neurons in Shank3-homozygous knockout (Shank3-KO), heterozygous (Shank3-Het), and wild-type (WT) rats. We used electron microscopy to determine the density of asymmetric synapses in mPFC layer III excitatory neurons in these rats. We measured postsynaptic density (PSD) length, PSD area, and head diameter (HD) of spines at these synapses.ResultsBasal dendritic morphology was similar among the three genotypes. Spine density and morphology were comparable, but more thin and mushroom spines had larger head volumes in Shank3-Het compared to WT and Shank3-KO. All three groups had comparable synapse density and PSD length. Spine HD of total and non-perforated synapses in Shank3-Het rats, but not Shank3-KO rats, was significantly larger than in WT rats. The total and non-perforated PSD area was significantly larger in Shank3-Het rats compared to Shank3-KO rats. These findings represent preliminary evidence for synaptic ultrastructural alterations in the mPFC of rats that lack one copy of Shank3 and mimic the heterozygous loss of SHANK3 in Phelan–McDermid syndrome.LimitationsThe Shank3 deletion in the rat model we used does not affect all isoforms of the protein and would only model the effect of mutations resulting in loss of the N-terminus of the protein. Given the higher prevalence of ASD in males, the ultrastructural study focused only on synaptic structure in male Shank3-deficient rats.ConclusionsWe observed increased HD and PSD area in Shank3-Het rats. These observations suggest the occurrence of altered synaptic ultrastructure in this animal model, further pointing to a key role of defective expression of the Shank3 protein in ASD and Phelan–McDermid syndrome.

Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons

ABSTRACT 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.

ADAP1/Centaurin-α1 Negatively Regulates Dendritic Spine Function and Memory Formation in the Hippocampus.

ADAP1/Centaurin-α1 (CentA1) functions as an Arf6 GTPase-activating protein highly enriched in the brain. Previous studies demonstrated the involvement of CentA1 in brain function as a regulator of dendritic differentiation and a potential mediator of Alzheimer’s disease (AD) pathogenesis. To better understand the neurobiological functions of CentA1 signaling in the brain, we developed Centa1 knock-out (KO) mice. The KO animals showed neither brain development nor synaptic ultrastructure deficits in the hippocampus. However, they exhibited significantly higher density and enhanced structural plasticity of dendritic spines in the CA1 region of the hippocampus compared with non-transgenic (NTG) littermates. Moreover, the deletion of Centa1 improved performance in the object-in-place (OIP) spatial memory task. These results suggest that CentA1 functions as a negative regulator of spine density and plasticity, and of hippocampus-dependent memory formation. Thus, CentA1 and its downstream signaling may serve as a potential therapeutic target to prevent memory decline associated with aging and brain disorders.

Disentangling the Roles of RIM and Munc13 in Synaptic Vesicle Localization and Neurotransmission

Efficient neurotransmitter release at the presynaptic terminal requires docking of synaptic vesicles to the active zone membrane and formation of fusion-competent synaptic vesicles near voltage-gated Ca2+ channels. Rab3-interacting molecule (RIM) is a critical active zone organizer, as it recruits Ca2+ channels and activates synaptic vesicle docking and priming via Munc13-1. However, our knowledge about Munc13-independent contributions of RIM to active zone functions is limited. To identify the functions that are solely mediated by RIM, we used genetic manipulations to control RIM and Munc13-1 activity in cultured hippocampal neurons from mice of either sex and compared synaptic ultrastructure and neurotransmission. We found that RIM modulates synaptic vesicle localization in the proximity of the active zone membrane independent of Munc13-1. In another step, both RIM and Munc13 mediate synaptic vesicle docking and priming. In addition, while the activity of both RIM and Munc13-1 is required for Ca2+-evoked release, RIM uniquely controls neurotransmitter release efficiency. However, activity-dependent augmentation of synaptic vesicle pool size relies exclusively on the action of Munc13s. Based on our results, we extend previous findings and propose a refined model in which RIM and Munc13-1 act in overlapping and independent stages of synaptic vesicle localization and release.SIGNIFICANCE STATEMENT The presynaptic active zone is composed of scaffolding proteins that functionally interact to localize synaptic vesicles to release sites, ensuring neurotransmission. Our current knowledge of the presynaptic active zone function relies on structure-function analysis, which has provided detailed information on the network of interactions and the impact of active zone proteins. Yet, the hierarchical, redundant, or independent cooperation of each active zone protein to synapse functions is not fully understood. Rab3-interacting molecule and Munc13 are the two key functionally interacting active zone proteins. Here, we dissected the distinct actions of Rab3-interacting molecule and Munc13-1 from both ultrastructural and physiological aspects. Our findings provide a more detailed view of how these two presynaptic proteins orchestrate their functions to achieve synaptic transmission.

Altered Conformation of α-Synuclein Drives Malfunction of Synaptic Vesicles in a Model of Parkinson's Disease

Misfolded, aggregated forms of alpha-synuclein (αSyn) are the principal protein component of Lewy bodies, the defining lesions of Parkinson’s disease (PD) and dementia with Lewy bodies. Increased copy number, missense mutations, and specific 5’ and 3’ regulatory elements of αSyn can all cause familial PD. Although αSyn participates in the regulation of synaptic vesicles, fundamental questions about the relationship between its structure and function in the presynaptic terminal are unanswered. Here, we systematically examined brain synaptosomes from non-transgenic (NTG) mice and transgenic mice expressing wild-type (WT) human αSyn or an amplified form of the E46K PD mutation (called 3K) that impairs the normal α-helical tetramerization of WT αSyn and increases its membrane affinity. Synaptosomes from transgenic mice expressing the 3K mutant had reduced Ca2+-dependent vesicle exocytosis, altered synaptic vesicle ultrastructure, impaired SNARE-complex assembly, and abnormal levels of select proteins involved in synaptic activity compared with NTG and WT human αSyn mice. We also developed a method to perform intra-synaptosomal NMR that revealed that WT αSyn participates in heterogeneous interactions with synaptic components. We show that these interactions depend on the presence of endogenous αSyn and that the 3K mutation diminishes the interplay between αSyn and the synapse. We conclude that the microenvironment inside presynaptic terminals is necessary for the proper conformation and interaction network of αSyn, where unfolded monomers of αSyn no longer represent the dominant conformation. We speculate that the inability to populate dynamically different conformational ensembles likely represents the early steps in αSyn misfunction that result in the neurotoxicity observed in PD and related synucleinopathies. With the advance of techniques such as cryo-electron tomography, in-cell single-molecule FRET, and in-cell NMR, in situ approaches like that presented here have the potential to address questions about the physiology of proteins such as αSyn that show diverse and changing conformations in their native, highly complex environments.

\u03b2-glucan attenuates cognitive impairment via the gut-brain axis in diet-induced obese mice

Background“Western” style dietary patterns are characterized by a high proportion of highly processed foods rich in fat and low in fiber. This diet pattern is associated with a myriad of metabolic dysfunctions, including neuroinflammation and cognitive impairment. β-glucan, the major soluble fiber in oat and barley grains, is fermented in the lower gastrointestinal tract, potentially impacting the microbial ecosystem and thus may improve elements of cognition and brain function via the gut-brain axis. The present study aimed to evaluate the effect of β-glucan on the microbiota gut-brain axis and cognitive function in an obese mouse model induced by a high-fat and fiber-deficient diet (HFFD).ResultsAfter long-term supplementation for 15 weeks, β-glucan prevented HFFD-induced cognitive impairment assessed behaviorally by object location, novel object recognition, and nesting building tests. In the hippocampus, β-glucan countered the HFFD-induced microglia activation and its engulfment of synaptic puncta, and upregulation of proinflammatory cytokine (TNF-α, IL-1β, and IL-6) mRNA expression. Also, in the hippocampus, β-glucan significantly promoted PTP1B-IRS-pAKT-pGSK3β-pTau signaling for synaptogenesis, improved the synaptic ultrastructure examined by transmission electron microscopy, and increased both pre- and postsynaptic protein levels compared to the HFFD-treated group. In the colon, β-glucan reversed HFFD-induced gut barrier dysfunction increased the thickness of colonic mucus (Alcian blue and mucin-2 glycoprotein immunofluorescence staining), increased the levels of tight junction proteins occludin and zonula occludens-1, and attenuated bacterial endotoxin translocation. The HFFD resulted in microbiota alteration, effects abrogated by long-term β-glucan supplementation, with the β-glucan effects on Bacteroidetes and its lower taxa particularly striking. Importantly, the study of short-term β-glucan supplementation for 7 days demonstrated pronounced, rapid differentiating microbiota changes before the cognitive improvement, suggesting the possible causality of gut microbiota profile on cognition. In support, broad-spectrum antibiotic intervention abrogated β-glucan’s effects on improving cognition, highlighting the role of gut microbiota to mediate cognitive behavior.ConclusionThis study provides the first evidence that β-glucan improves indices of cognition and brain function with major beneficial effects all along the gut microbiota-brain axis. Our data suggest that elevating consumption of β-glucan-rich foods is an easily implementable nutritional strategy to alleviate detrimental features of gut-brain dysregulation and prevent neurodegenerative diseases associated with Westernized dietary patterns.Ajj4hQzaw2wK1LZwmZvEcmVideo

Bushen-Tiansui Formula Improves Cognitive Functions in an Aβ1–42 Fibril-Infused Rat Model of Alzheimer’s Disease

Bushen-Tiansui Formula (BTF) was empirically updated from a classical prescription named Kong-Sheng-Zhen-Zhong pill. It is based on the traditional Chinese medicine theory of the mutual relationship between the brain and the kidney and is intended to treat neurodegenerative diseases. This formulation has been used for several years to treat patients with Alzheimer's disease- (AD-) like symptoms in our clinical department. However, the medicinal ingredients and the mechanisms by which BTF improves cognition and memory functions have not been characterized. In this study, we used UPLC-MS to generate a chromatographic fingerprinting of BTF and identified five possible active ingredients, including stilbene glycoside; epimedin A1, B, and C; and icariin. We also showed that oral administration of BTF reversed the cognitive defects in an Aβ1–42 fibril-infused rat model of AD, protected synaptic ultrastructure in the CA1 region, and restored the expression of BDNF, synaptotagmin (Syt), and PSD95. These effects likely occurred through the BDNF-activated receptor tyrosine kinase B (TrkB)/Akt/CREB signaling pathway. Furthermore, BTF exhibited no short-term or chronic toxicity in rats. Together, these results provided a scientific support for the clinical use of BTF to improve learning and memory in patients with AD.

CYP46A1-dependent and independent effects of efavirenz treatment.

Cholesterol excess in the brain is mainly disposed via cholesterol 24-hydroxylation catalysed by cytochrome P450 46A1, a CNS-specific enzyme. Cytochrome P450 46A1 is emerging as a promising therapeutic target for various brain diseases with both enzyme activation and inhibition having therapeutic potential. The rate of cholesterol 24-hydroxylation determines the rate of brain cholesterol turnover and the rate of sterol flux through the plasma membranes. The latter was shown to affect membrane properties and thereby membrane proteins and membrane-dependent processes. Previously we found that treatment of 5XFAD mice, an Alzheimer’s disease model, with a small dose of anti-HIV drug efavirenz allosterically activated cytochrome P450 46A1 in the brain and mitigated several disease manifestations. Herein, we generated Cyp46a1−/−5XFAD mice and treated them, along with 5XFAD animals, with efavirenz to ascertain cytochrome P450 46A1-dependent and independent drug effects. Efavirenz-treated versus control Cyp46a1−/−5XFAD and 5XFAD mice were compared for the brain sterol and steroid hormone content, amyloid β burden, protein and mRNA expression as well as synaptic ultrastructure. We found that the cytochrome P450 46A1-dependent efavirenz effects included changes in the levels of brain sterols, steroid hormones, and such proteins as glial fibrillary acidic protein, Iba1, Munc13-1, post-synaptic density-95, gephyrin, synaptophysin and synapsin-1. Changes in the expression of genes involved in neuroprotection, neurogenesis, synaptic function, inflammation, oxidative stress and apoptosis were also cytochrome P450 46A1-dependent. The total amyloid β load was the same in all groups of animals, except lack of cytochrome P450 46A1 decreased the production of the amyloid β40 species independent of treatment. In contrast, altered transcription of genes from cholinergic, monoaminergic, and peptidergic neurotransmission, steroid sulfation and production as well as vitamin D3 activation was the main CYP46A1-independent efavirenz effect. Collectively, the data obtained reveal that CYP46A1 controls cholesterol availability for the production of steroid hormones in the brain and the levels of biologically active neurosteroids. In addition, cytochrome P450 46A1 activity also seems to affect the levels of post-synaptic density-95, the main postsynaptic density protein, possibly by altering the calcium/calmodulin-dependent protein kinase II inhibitor 1 expression and activity of glycogen synthase kinase 3β. Even at a small dose, efavirenz likely acts as a transcriptional regulator, yet this regulation may not necessarily lead to functional effects. This study further confirmed that cytochrome P450 46A1 is a key enzyme for cholesterol homeostasis in the brain and that the therapeutic efavirenz effects on 5XFAD mice are likely realized via cytochrome P450 46A1 activation.

The Role Of Dopaminergic Synapse And D2DR In Movement Control

Exercise fatigue is a common physiological phenomenon in sports. The central nervous system (CNS) has important regulating effect on exercise fatigue. The basal ganglia are involved in motor function regulation through direct and indirect pathways. Striatum is the main input nucleus of basal ganglia and plays an important role in movement control. PURPOSE: Explore the role of dopaminergic synapse and D2 dopamine receptors (D2DR) in movement control by investigating synaptic ultrastructural change of the dorsolateral striatum (DLS) and D2DR antagonist/agonist effect on autonomic activity in exercise-induced fatigue rats. METHODS: Male Wistar rats were randomly divided into either the sham control group (SCG), 1-day fatigue group (1FG), 3-day fatigue group (3FG), 7-day fatigue group (7FG), 24-hour recovery group (24RG) or 48-hour recovery group (48RG). The synaptic ultrastructure was observed by transmission electron microscopy. Further a D2DR antagonist and agonist were used to interfere with the autonomic exercise of rats with Open Field Test. RESULTS: We found that: (1) The proportion of asymmetrical synapse of DLS (SCG: 41±2(%), 7FG: 28±4(%), p<0.05), number of presynaptic vesicles (SCG: 13.35±4.58 (number/μm2), 7FG: 5.92±2.7 (number/μm2), p<0.05), number of synaptic terminal mitochondria (SCG: 0.19±0.18 (number/μm2), 7FG: 0.15±0.1 (number/μm2), p<0.05), and the area of synaptic terminals(SCG: 123925.64±54773.42 (μm2), 7FG: 84447.16±29495.42 (μm2), p<0.05), decreased with the increase of exercise days. (2) The total exercise distance of each group decreased gradually with the increase of exercise intensity (p<0.05). Additional rat’s exhaustion time was also significantly shortened after injection of the D2DR antagonist(before: 147.33±5.63, after: 103±4.14, p<0.01). CONCLUSIONS: The activity of synapses in the dorsolateral striatum of rats decreased with the increase of exercise fatigue. Exercise fatigue reduced the autonomic activity of rats, which was further enhanced by D2DR antagonist intervention. This indicates that the regulatory role of D2DR in motion may be related to synaptic plasticity of dopaminergic in substantia nigra and striatum, suggesting that D2DR can be used as an important target to address exercise fatigue.

Involvement of NMDAR/PSD-95/nNOS–NO–cGMP pathway in embryonic exposure to BPA induced learning and memory dysfunction of rats

Bisphenol A (BPA), can lead to learning and memory impairment, but the underlying mechanism is poorly understood. Researchers have indicated that the N-methyl-D-aspartate receptor (NMDAR)/postsynaptic density protein 95 (PSD-95)/neuronal nitric oxide synthase (nNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway greatly contributes to learning and memory process. Pregnant rats were exposed to 0, 0.05, 0.5, 5 and 50 mg/kg/day BPA via oral gavage from gestational day (GD) 5 to GD 19. Morris water maze, transmission electron microscope, western blot, real time PCR, biochemical analysis and ELISA were used to analyze the changes in behavior, synaptic ultrastructure, protein and gene expression of NMDAR, PSD-95, nNOS, together with nNOS activity, NO (Nitrate reductase method) and cGMP levels of the rat pups at different growth stages. Results of this research displayed that exposure to 0.5 mg/kg/day BPA could damage the spatial learning ability of rats at postnatal day (PND) 56. However, spatial memory ability could be affected by exposure to BPA at doses up to 5 mg/kg/day. Moreover, the thickness of the postsynaptic density decreased after exposure to BPA at doses of 5 and 50 mg/kg/day. Levels of NR1, NR2A, PSD-95 protein and mRNA were downregulated to some extent after exposure to BPA, whereas the expression of NR2B increased at GD 20 but decreased at PND 21 and 56. Contrarily, the nNOS expression along with the enzyme activity were promoted after exposure to BPA. Meanwhile, the NO and cGMP levels were suppressed at GD 20 but promoted at PND 21 and 56. In conclusion, these results demonstrated that NMDAR/PSD-95/nNOS–NO–cGMP pathway could be affected by embryonic exposure to BPA, which may involve in the spatial learning and memory dysfunction of rats in later life.

Maternal Immune Activation Induces Neuroinflammation and Cortical Synaptic Deficits in the Adolescent Rat Offspring.

Maternal immune activation (MIA), induced by infection during pregnancy, is an important risk factor for neuro-developmental disorders, such as autism. Abnormal maternal cytokine signaling may affect fetal brain development and contribute to neurobiological and behavioral changes in the offspring. Here, we examined the effect of lipopolysaccharide-induced MIA on neuro-inflammatory changes, as well as synaptic morphology and key synaptic protein level in cerebral cortex of adolescent male rat offspring. Adolescent MIA offspring showed elevated blood cytokine levels, microglial activation, increased pro-inflammatory cytokines expression and increased oxidative stress in the cerebral cortex. Moreover, pathological changes in synaptic ultrastructure of MIA offspring was detected, along with presynaptic protein deficits and down-regulation of postsynaptic scaffolding proteins. Consequently, ability to unveil MIA-induced long-term alterations in synapses structure and protein level may have consequences on postnatal behavioral changes, associated with, and predisposed to, the development of neuropsychiatric disorders.

Enriched Environment Decreases Cognitive Impairment in Elderly Rats With Prenatal Mobile Phone Exposure.

Mobile phone use has rapidly increased worldwide, and pregnant women are passively or actively exposed to the associated electromagnetic radiation. Maternal cell phone exposure is related to behavioral difficulties in young offspring. However, whether prenatal mobile phone exposure can predispose the elderly offspring to cognitive impairment is unclear. The enriched environment (EE) has shown positive effects on cognition in an immature brain, but its impact on aging offspring after prenatal cell phone exposure is unknown. This study aimed to investigate whether prenatal exposure to mobile phone exerts long-term effects on cognition in elderly rat offspring and whether EE during adulthood can rescue cognitive impairment by altering the synaptic plasticity. Pregnant rats were subjected to prenatal short-term or long-term cell phone exposure and offspring rats were randomly assigned to standard or EE. Spatial learning and memory were investigated using Morris water maze (MWM) in elderly rat offspring. Hippocampal cellular morphology was assessed by hematoxylin-eosin staining and synaptic ultrastructure was evaluated with transmission electron microscopy. Expression of synaptophysin (SYN), postsynaptic density-95 (PSD-95), and brain-derived neurotrophic factor (BDNF) were detected by western blot. The results demonstrated that prenatal long-term but not short-term exposure to mobile phone lead to cognitive impairment, morphological changes in the hippocampal cells, reduced synaptic number, decreased SYN, PSD-95, and BDNF expression in elderly offspring, which were alleviated by postnatal EE housing. These findings suggest that prenatal long-term mobile phone exposure may pose life-long adverse effects on elderly offspring and impair cognition by disrupting the synaptic plasticity, which may be reversed by postnatal EE housing.

PTPσ Controls Presynaptic Organization of Neurotransmitter Release Machinery at Excitatory Synapses

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