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Aims This study aimed to investigate whether electroacupuncture (EA) promotes the survival and synaptic plasticity of hippocampal neurons by activating brain‐derived neurotrophic factor (BDNF)/tyrosine receptor kinase (TrkB)/extracellular signal‐regulated kinase (Erk) signaling, thereby improving spatial memory deficits in rats under SD. Methods In vivo, Morris water maze (MWM) was used to detect the effect of EA on learning and memory, at the same time Western blotting (WB), immunofluorescence (IF), and transmission electron microscopy (TEM) were used to explore the plasticity of hippocampal neurons and synapses, and the expression of BDNF/TrkB/Erk signaling. In vitro, cultured hippocampal neurons were treated with exogenous BDNF and the TrkB inhibitor K252a to confirm the relationship between BDNF/TrkB/Erk signaling and synaptic plasticity. Results Our results showed that EA mitigated the loss of hippocampal neurons and synapses, stimulated hippocampal neurogenesis, and improved learning and memory of rats under SD accompanied by upregulation of BDNF and increased phosphorylation of TrkB and Erk. In cultured hippocampal neurons, exogenous BDNF enhanced the expression of synaptic proteins, the frequency of the postsynaptic currents, and the phosphorylation of TrkB and Erk; these effects were reversed by treatment with K252a. Conclusions Electroacupuncture alleviates SD‐induced spatial memory impairment by promoting hippocampal neurogenesis and synaptic plasticity via activation of BDNF/TrkB/Erk signaling, which provided evidence for EA as a therapeutic strategy for countering the adverse effects of SD on cognition., Electroacupuncture may ameliorate spatial memory impairment during sleep deprivation through improving hippocampal neuron survival, neurogenesis and synaptic plasticity via regulating BDNF/TrkB/ERK signaling

Functional differentiation of the hippocampus along its longitudinal septotemporal axis is beyond doubt, though the factors determining this subregional specificity remain controversial. The spatial specificity gradient of hippocampal place cells decreases along its longitudinal axis from the septal pole to the temporal area. This difference in the scale of spatial representation suggests differences in the involvement of neurons in these subregions in the exploration and mapping of territories of different sizes. Using c-Fos as a marker for hippocampal neuron activation in novel environments, we studied the influences of exploration of arenas of different sizes on neuron activation in the septal and temporal subregions of fields CA1 and CA3 and the dentate fascia (DF) in C57BL/6 mice. The results of these studies showed that expression intensity was influenced by arena size, and a relationship between subregion activation factors and area size was found. Elevated c-Fos expression was seen only in the temporal part of the hippocampus, while significant changes in activation were noted in field CA3 and the DF in mice exploring the largest arena. c-Fos expression in the septal subregions showed no differences in arenas of different sizes. However, repeat testing of animals in an arena of a different size led to additional activation of expression in the temporal part of CA3 and the septal part of the DF. High neuron activity in the temporal subregion of the hippocampus may be due not only to the size of the arena explored, but also to the greater indeterminacy of a large space in terms of the biological significance of its different areas.

Objective This study was to detect the protective effects of Houttuynia cordata extract on the damage induced by propofol in hippocampal neuron of rats. Methods Propofol-induced neuron injury model and H. cordata extract administration were conducted. Immunofluorescence and immunoblot were conducted for the effect of H. cordata extract on neuronal activity and inflammation were detected in this model. Results H. cordata extracts increased neuronal activity, and reduced propofol-induced neuronal inflammation levels. H. cordata extract also reduced propofol-induced neuronal apoptosis. Mechanically, we noticed H. cordata extract activated phosphoinositide 3-kinase/AKT pathway and suppressed Toll-like receptor 4/nuclear factor kappaB pathway, therefore protected propofol-induced injury of rat hippocampal neurons. Conclusion Our findings provide references for anesthetic use in infants and young children.

The protective effects of blueberry anthocyanin extracts against damage induced by extremely low-frequency electromagnetic field (ELF-EMF) were investigated in a rat model. Wistar rats were exposed to ELF-EMF with or without the administration of blueberry anthocyanin extracts (50, 100, and 200 mg/kg per day intragastrically once a day) for 30 days. Blueberry anthocyanin extracts supplementation inhibited the decrease in Nissl substance levels, cell membrane integrity, and mitochondrial membrane potential induced by ELF-EMF; prevented the increase in nitric oxide, malondialdehyde, and Ca2+ concentrations; suppressed superoxide dismutase and glutathione depletion; and enhanced the cognitive ability of the rats exposed to ELF-EMF. The protective effects of blueberry anthocyanin extracts against hippocampal neuron injury caused by ELF-EMF were dose-dependent. These results demonstrated that blueberry anthocyanin extracts suppress hippocampal neuron injury caused by ELF-EMF by inhibiting cell membrane damage and oxidative stress pathways, and suggested that blueberry anthocyanin treatment potentially prevents hippocampal neuron injury.

Calpains are calcium‐dependent proteases and play critical roles in neuronal autophagy induced by inflammation. Propofol has been reported to exert anti‐inflammatory effects in neurons. We aimed to identify whether and how propofol‐modulated calpain activity and neuron autophagy in response to tumour necrosis factor‐α (TNF‐α). Mouse hippocampal neurons were pre‐treated with propofol and exposed to TNF‐α. Autophagy was evaluated by fluorescent autophagy assay and by measuring LC3I and LC3II expression. Intracellular calcium concentration was measured by fluorescent assay. Calpain activation was measured by calpain activity assay. The protein expression of intracellular signalling molecules was detected by Western blot analysis. Compared with untreated control neurons, 40 ng/mL TNF‐α treatment for 2 hours induced neuron autophagy, which was attenuated by 25 μmol/L propofol. TNF‐α induced intracellular calcium accumulation, the phosphorylation of calcium/calmodulin‐dependent protein kinase II (CAMK II) and calpain‐2, calpain activation and lysosomal cathepsin B release as well as tyrosine kinase receptor B (TrkB) truncation. These effects were alleviated by propofol, calcium chelator, CAMK II inhibitor, calpain‐2 inhibitor, calpain‐2 siRNA transfection and N‐Methyl‐d‐aspartic acid (NMDA) receptor antagonist. Propofol, via NMDA receptor, inhibited TNF‐α‐mediated hippocampal neuron autophagy. The mechanism may involve calcium and calcium‐dependent signalling pathway, especially CAMK II and calpain‐2.

Changes in structural and functional neuroplasticity have been implicated in various neurological disorders. Sterol regulatory element-binding protein (SREBP)-1c is a critical regulatory molecule of lipid homeostasis in the brain. Recently, our findings have shown the potential involvement of SREBP-1c deficiency in the alteration of novel modulatory molecules in the hippocampus and occurrence of schizophrenia-like behaviors in mice. However, the possible underlying mechanisms, related to neuronal plasticity in the hippocampus, are yet to be elucidated. In this study, we investigated the hippocampus-dependent memory function and neuronal architecture of hippocampal neurons in SREBP-1c knockout (KO) mice. During the passive avoidance test, SREBP-1c KO mice showed memory impairment. Based on Golgi staining, the dendritic complexity, length, and branch points were significantly decreased in the apical cornu ammonis (CA) 1, CA3, and dentate gyrus (DG) subregions of the hippocampi of SREBP-1c KO mice, compared with those of wild-type (WT) mice. Additionally, significant decreases in the dendritic diameters were detected in the CA3 and DG subregions, and spine density was also significantly decreased in the apical CA3 subregion of the hippocampi of KO mice, compared with that of WT mice. Alterations in the proportions of stubby and thin-shaped dendritic spines were observed in the apical subcompartments of CA1 and CA3 in the hippocampi of KO mice. Furthermore, the corresponding differential decreases in the levels of SREBP-1 expression in the hippocampal subregions (particularly, a significant decrease in the level in the CA3) were detected by immunofluorescence. This study suggests that the contributions of SREBP-1c to the structural plasticity of the mouse hippocampus may have underlain the behavioral alterations. These findings offer insights into the critical role of SREBP-1c in hippocampal functioning in mice.

Exposure to fine particulate matter (PM2.5) is implicated in neurodevelopmental disorders including cognitive decline, attention-deficit/hyperactivity disorder, and autism spectrum disorder. However, the specific molecular mechanisms by which PM2.5 impacts neurodevelopment are poorly understood. Accordingly, in the present study, the role of protein kinase A (PKA)/cAMP response element binding protein (CREB)/brain-derived neurotrophic factor (BDNF) signaling in PM2.5-induced neurodevelopmental damage was investigated using primary cultured hippocampal neurons. When hippocampal neurons cultured for 3 days in vitro (DIV3) were exposed to PM2.5 for 24 h and 96 h, neuronal viability decreased by 18.8% and 32.7% respectively, percentage of TUNEL-positive neurons increased by 78.5% and 64.0% separately, caspase-9 expression increased, lower postsynaptic density and shorter active zones were observed by transmission electron microscopy, expression of synapse-related proteins including postsynaptic density-95 (PSD95), growth associated protein-43 (GAP43), and synaptophysin (SYP) were decreased, and the phosphorylation levels of PKA, CREB, and BDNF expression also decreased. However, the PM2.5-induced neuronal damage could be ameliorated or aggravated to varying degrees by up- or down-regulation of the PKA/CREB/BDNF signaling pathway, respectively. Our results indicate that PM2.5 exposure exerts neurodevelopmental toxicity as indicated by lower viability, apoptosis, and synaptic damage in primary cultured hippocampal neurons, and that the PKA/CREB/BDNF pathways could play a vital role in PM2.5-mediated neurodevelopmental toxicity.

Background: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and toxic environmental pollutant. Gestational exposure to TCDD has been linked to cognitive and motor deficits, and increased incidence of autism spectrum disorder (ASD) traits in children. Most animal studies of these neurodevelopmental effects involve acute TCDD exposure, which does not model typical exposure in humans. Objectives: The aim of the study was to establish a dietary low-dose gestational TCDD exposure protocol and performed an initial characterization of the effects on offspring behavior, neurodevelopmental phenotypes, and gene expression. Methods: Throughout gestation, pregnant C57BL/6J mice were fed a diet containing a low dose of TCDD (9 ng TCDD/kg body weight per day) or a control diet. The offspring were tested in a battery of behavioral tests, and structural brain alterations were investigated by magnetic resonance imaging. The dendritic morphology of pyramidal neurons in the hippocampal Cornu Ammonis (CA)1 area was analyzed. RNA sequencing was performed on hippocampi of postnatal day 14 TCDD-exposed and control offspring. Results: TCDD-exposed females displayed subtle deficits in motor coordination and reversal learning. Volumetric difference between diet groups were observed in regions of the hippocampal formation, mammillary bodies, and cerebellum, alongside higher dendritic arborization of pyramidal neurons in the hippocampal CA1 region of TCDD-exposed females. RNA-seq analysis identified 405 differentially expressed genes in the hippocampus, enriched for genes with functions in regulation of microtubules, axon guidance, extracellular matrix, and genes regulated by SMAD3. Discussion: Exposure to 9 ng TCDD/kg body weight per day throughout gestation was sufficient to cause specific behavioral and structural brain phenotypes in offspring. Our data suggest that alterations in SMAD3-regulated microtubule polymerization in the developing postnatal hippocampus may lead to an abnormal morphology of neuronal dendrites that persists into adulthood. These findings show that environmental low-dose gestational exposure to TCDD can have significant, long-term impacts on brain development and function. https://doi.org/10.1289/EHP7352

Down-regulated in renal cell carcinoma 1 (DRR1), a unique stress-induced protein, is highly expressed in the nervous system. This study investigated the roles of DRR1 in the brain by examining its expression pattern at different developmental stages of a rat brain and in cultured primary hippocampal neurons. High expression of DRR1 was observed in all developmental stages of a rat brain and cultured primary hippocampal neurons. We then focused on the role of DRR1 in promoting neurite outgrowth during the early stage of hippocampal neuron development. Results showed that down-regulation of DRR1 suppressed axon outgrowth. Mass spectrometry analysis revealed that tropomodulin-2 (Tmod2) is a novel binding partner of DRR1. Our results showed that both DRR1 and Tmod2 mediate axon formation during the early stage of hippocampal neuron development. Suppression of TMOD2 expression rescued the abnormal axon outgrowth induced by DRR1 knockdown during the early stage of hippocampal neuron development.

Mitochondria possess a small genome that codes for core subunits of the oxidative phosphorylation system and whose expression is essential for energy production. Information on the regulation and spatial organization of mitochondrial gene expression in the cellular context has been difficult to obtain. Here we devise an imaging approach to analyze mitochondrial translation within the context of single cells, by following the incorporation of clickable non‐canonical amino acids. We apply this method to multiple cell types, including specialized cells such as cardiomyocytes and neurons, and monitor with spatial resolution mitochondrial translation in axons and dendrites. We also show that translation imaging allows to monitor mitochondrial protein expression in patient fibroblasts. Approaching mitochondrial translation with click chemistry opens new avenues to understand how mitochondrial biogenesis is integrated into the cellular context and can be used to assess mitochondrial gene expression in mitochondrial diseases., This study monitors mitochondrial protein synthesis with spatial resolution in single cells of multiple cell types.

Oxidative stress is a critical event in neuronal damage following seizures. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been shown to be promising nanotherapeutic agents in neurological disorders. However, the mechanism underlying MSC-EVs therapeutic efficacy for oxidative stress-induced neuronal damage remains poorly understood. Methods: We investigated the antioxidant and restoration activities of MSC-EVs on hippocampal neurons in response to H2O2 stimulation in vitro and seizures in vivo. We also explored the potential underlying mechanism by injecting adeno-associated virus (AAV)-nuclear factor erythroid-derived 2, like 2 (Nrf2), a key antioxidant mediator, in animal models. Results: MSC-EVs were enriched in antioxidant miRNAs and exhibited remarkable antioxidant activity evident by increased ferric ion-reducing antioxidant ability, catalase, superoxide dismutase, and glutathione peroxidase activities and decreased reactive oxygen species (ROS) generation, DNA/lipid/protein oxidation, and stress-associated molecular patterns in cultured cells and mouse models. Notably, EV administration exerted restorative effects on the hippocampal neuronal structure and associated functional impairments, including dendritic spine alterations, electrophysiological disturbances, calcium transients, mitochondrial changes, and cognitive decline after oxidative stress in vitro or in vivo. Mechanistically, we found that the Nrf2 signaling pathway was involved in the restorative effect of EV therapy against oxidative neuronal damage, while AAV-Nrf2 injection attenuated the antioxidant activity of MSC-EVs on the seizure-induced hippocampal injury. Conclusions: We have shown that MSC-EVs facilitate the reconstruction of hippocampal neurons associated with the Nrf2 defense system in response to oxidative insults. Our study highlights the clinical value of EV-therapy in neurological disorders such as seizures.

Botulinum neurotoxins (BoNTs) are extremely potent naturally occurring poisons that act by silencing neurotransmission. Intriguingly, in addition to preventing presynaptic vesicle fusion, BoNT serotype A (BoNT/A) can also promote axonal regeneration in preclinical models. Here we report that the non-toxic C-terminal region of the receptor-binding domain of heavy chain BoNT/A (HCC/A) activates the small GTPase Rac1 and ERK pathway to potentiate axonal outgrowth, dendritic protrusion formation and synaptic vesicle release in hippocampal neurons. These data are consistent with HCC/A exerting neurotrophic properties, at least in part, independent of any BoNT catalytic activity or toxic effect., Highlights • The non-catalytic, non-toxic C-terminal region of the heavy chain of BoNT/A (HCC/A) activates Rac1 and ERK. • HCC/A promotes axonal outgrowth and dendritic filopodia. • HCC/A enhances synaptic vesicle release.

Objective. We aimed to establish a method to determine whether microRNA-193b (miR-193b) levels in ABCA1-labeled serum exosomes might serve as a marker for the diagnosis of Alzheimer’s disease. Methods. We used immunocapture methods to determine the levels of ABCA1-labeled exosomal miR-193b in cultures of white blood cells (WBCs), red blood cells (RBCs), mouse hippocampal neuron HT-22 cells, and primary mouse neuronal cells. ABCA1-labeled exosomal miR-193b levels were also evaluated in the cerebrospinal fluid (CSF) and serum of APP/PS1 double-transgenic mice, as well as control subjects ( n = 60 ) and study participants with subjective cognitive decline (SCD, n = 89 ), stage and mild cognitive impairment (MCI, n = 92 ), and dementia of the Alzheimer type (DAT, n = 92 ). Results. ABCA1 levels of exosomes harvested from the medium of HT-22 cells and neurons were significantly higher than those of RBCs and WBCs ( P < 0.05 ). Exosomal ABCA1 from the CSF of APP/PS1 mice were transmitted to the serum of wild-type mice after injection, and high miR-193b levels were observed in both the serum and CSF after injection. The ABCA1-labeled exosomal miR-193b levels were higher in the CSF of MCI and DAT patients compared with the CSF of the control group ( P < 0.05 ). The ABCA1-labeled exosomal miR-193b were also slightly higher ( P > 0.05 ) in the serum of SCD patients and significantly higher in the serum of MCI and DAT patients compared with the serum of the control group ( P < 0.05 ). Conclusion. This study provides a method to capture specific exosomes. Detection of serum exosomes labeled with ABCA1 may facilitate the early diagnosis of AD.

Culture of primary mouse hippocampal neurons. This protocol is for a defined media culture, meaning the use of a feeder layer is not required.

Aging has been extensively associated with cognitive decline and synaptic dysfunction. Hippocampus has a key role in learning and memory, and undergoes structural and functional alterations upon aging process, despite the preservation of primary hippocampal neuron numbers across different species. Comprehension of the age-related hippocampal dysfunction is crucial not only to understand the aging process itself, but also to understand age-triggered neurodegeneration.

Systematically organizing the anatomical, molecular, and physiological properties of cortical neurons is important for understanding their computational functions. Hippocampome.org defines 122 neuron types in the rodent hippocampal formation (dentate gyrus, CA3, CA2, CA1, subiculum, and entorhinal cortex) based on their somatic, axonal, and dendritic locations, putative excitatory/inhibitory outputs, molecular marker expression, and biophysical properties such as time constant and input resistance. Here we augment the electrophysiological data of this knowledge base by collecting, quantifying, and analyzing the firing responses to depolarizing current injections for every hippocampal neuron type from available published experiments. We designed and implemented objective protocols to classify firing patterns based on both transient and steady-state activity. Specifically, we identified 5 transients (delay, adapting spiking, rapidly adapting spiking, transient stuttering, and transient slow-wave bursting) and 4 steady states (non-adapting spiking, persistent stuttering, persistent slow-wave bursting, and silence). By characterizing the set of all firing responses reported for hippocampal neurons, this automated classification approach revealed 9 unique families of firing pattern phenotypes while distinguishing potential new neuronal subtypes. Several novel statistical associations also emerged between firing responses and other electrophysiological properties, morphological features, and molecular marker expression. The firing pattern parameters, complete experimental conditions (including solution and stimulus details), digitized spike times, exact reference to the original empirical evidence, and analysis scripts are released open-source through Hippocampome.org for all neuron types, greatly enhancing the existing search and browse capabilities. This information, collated online in human-and machine-accessible form, will help design and interpret both experiments and hippocampal model simulations.Significance StatementComprehensive characterization of nerve cells is essential for understanding signal processing in biological neuronal networks. Firing patterns are important identification characteristics of neurons and play crucial roles in information coding in neural systems. Building upon the comprehensive knowledge base Hippocampome.org, we developed and implemented automated protocols to classify all known firing responses exhibited by each neuron type of the rodent hippocampus based on analysis of transient and steady-state activity. This approach identified the most distinguishing elements of every firing phenotype and revealed previously unnoticed statistical associations of firing responses with other electrophysiological, morphological, and molecular properties. The resulting data, freely released online, constitute a powerful resource for designing and interpreting experiments as well as developing and testing hippocampal models.

Jie Lv, Wei Ou, Xiao-Hua Zou, Yin Yao, Jin-Li Wu Department of Anesthesia, Guizhou Medical University Affiliated Hospital, Guiyang, People’s Republic of China Abstract: The study aimed to explore the effect of dexmedetomidine (DEX) on hippocampal neuron development process and on molecular expression of brain-derived neurotrophic factor (BDNF)-tyrosine receptor kinase B (TrkB) signaling pathway in neonatal rats. The hippocampal neuron cells were isolated from newborn neonatal rats and cultured in vitro. One control group and three treated groups with 1, 10, and 100 µmol/L DEX were used for the study. Cell activity and apoptosis were detected by the MTT and terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate (UTP) nick end labeling assays. The synaptophysin (SYN) and postsynaptic density 95 (PSD95) were detected by quantitative polymerase chain reaction. There was no difference in the viability of neuron cells among the different dose groups of DEX and the control group during days 2-10 (P>0.05). Compared to the control group, there was no significant difference (P>0.05) in the expressions of SYN and PSD95 in the groups treated with 1 and 10 µmol/L DEX, whereas significant difference in the expression was observed in the group treated with 100 µmol/L DEX (P

Neuronal atrophy is a common pathological feature occurred in aging and neurodegenerative diseases. A variety of abnormalities including motor protein malfunction and mitochondrial dysfunction contribute to the loss of neuronal architecture; however, less is known about the intracellular signaling pathways that can protect against or delay this pathogenic process. Here, we show that the DYNC1I1 deficiency, a neuron-specific dynein intermediate chain, causes neuronal atrophy in primary hippocampal neurons. With this cellular model, we are able to find that activation of RAS-RAF-MEK signaling protects against neuronal atrophy induced by DYNC1I1 deficiency, which relies on MEK-dependent autophagy in neuron. Moreover, we further reveal that BRAF also protects against neuronal atrophy induced by mitochondrial impairment. These findings demonstrate protective roles of the RAS-RAF-MEK axis against neuronal atrophy, and imply a new therapeutic target for clinical intervention. Electronic supplementary material The online version of this article (doi:10.1007/s13238-016-0301-6) contains supplementary material, which is available to authorized users.

S-allyl-l-cysteine (SAC) is known to have neuroprotective properties. We synthesized various SAC derivatives and tested their effects on endoplasmic reticulum stress-induced neurotoxicity in cultured hippocampal neurons (HPNs). Among the compounds tested, S-propyl-l-cysteine (SPC) exhibited the strongest neuroprotective activity in HPNs, followed by S-ethyl-l-cysteine (SEC) and S-methyl-l-cysteine (SMC). Unlike SAC and SMC, SPC and SEC did not have inhibitory activity on μ-calpain, suggesting that the mechanism underlying the protective activity of SPC and SEC differs from that of SAC.

S-allylcysteine (SAC), a major thioallyl compound contained in mature garlic extract (MGE), is known to be a neuroactive compound. This study was designed to investigate the effects of SAC on primary cultured hippocampal neurons and cognitively impaired senescence-accelerated mice prone 10 (SAMP10). Treatment of these neurons with MGE or SAC significantly increased the total neurite length and number of dendrites. SAMP10 mice fed MGE or SAC showed a significant improvement in memory dysfunction in pharmacological behavioral analyses. The expression of &alpha, amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, N-methyl-d-aspartate (NMDA) receptor, and phosphorylated &alpha, calcium/calmodulin-dependent protein kinase II (CaMKII) in the hippocampal tissue of SAMP10 mice fed MGE or SAC was significantly suppressed, especially in the MGE-fed group. These findings suggest that SAC positively contributes to learning and memory formation, having a beneficial effect on brain function. In addition, multiple components (aside from SAC) contained in MGE could be useful for improving cognitive function by acting as neurotrophic factors.

CRMP family proteins (CRMPs) are abundantly expressed in the developing nervous system mediating growth cone guidance, neuronal polarity and axon elongation. CRMP‑5 has been indicated to serve a critical role in neurite outgrowth. However, the detailed mechanisms of how CRMP‑5 regulates neurite outgrowth remain unclear. In the current study, co-immunoprecipitation was used to identify the fact that CRMP‑5 interacted with the actin and tubulin cytoskeleton networks in the growth cones of developing hippocampal neurons. CRMP‑5 exhibited increased affinity towards actin when compared with microtubules. Immunocytochemistry was used to identify the fact that CRMP‑5 colocalized with actin predominantly in the C-domain and T-zone in growth cones. In addition, genetic inhibition of CRMP‑5 by siRNA suppressed the expression of actin, growth cone development and neurite outgrowth. Overexpression of CRMP‑5 promoted the interaction with actin, growth cone development and hippocampal neurite outgrowth. Taken together, these data suggest that CRMP‑5 is able to interact with the actin cytoskeleton network in the growth cone and affect growth cone development and neurite outgrowth via this interaction in developing hippocampal neurons.

Following traumatic brain injuries (TBI), insulin-like growth factor (IGF) is cortically widely upregulated. This upregulation has a potential role in the recovery of neuronal tissue, plasticity, and neurotrophic activity, though the molecular mechanisms involved in IGF regulation and the exact role of IGF after TBI remain unclear. Vitronectin (VN), an extracellular matrix (ECM) molecule, has recently been shown to be of importance for IGF-mediated cellular growth and migration. Since VN is downregulated after TBI, we hypothesized that insufficient VN levels after TBI impairs the potential beneficial activity of IGF. To test if vitronectin and IGF-1/IGFBP-2 could contribute to neurite growth, we cultured hippocampal neurons on &plusmn, vitronectin-coated coverslips and them treated with &plusmn, IGF-1/IGF binding protein 2 (IGFBP-2). Under same conditions, cell cultures were also subjected to in vitro trauma to investigate differences in the posttraumatic regenerative capacity with &plusmn, vitronectin-coated coverslips and with &plusmn, IGF-1/IGFBP-2 treatment. In both the control and trauma situations, hippocampal neurons showed a stronger growth pattern on vitronectin than on the control substrate. Surprisingly, the addition of IGF-1/IGFBP-2 showed a decrease in neurite growth. Since neurite growth was measured as the number of neurites per area, we hypothesized that IGF-1/IGFBP-2 contributes to the polarization of neurons and thus induced a less dense neurite network after IGF-1/IGFBP-2 treatment. This hypothesis could not be confirmed and we therefore conclude that vitronectin has a positive effect on neurite growth in vitro both under normal conditions and after trauma, but that addition of IGF-1/IGFBP-2 does not have a positive additive effect.

Dendrite morphogenesis is a complex but well-orchestrated process. Various studies reported the involvement of alteration in dendrite morphology in different brain disorders, including neuropsychiatric disorders. Initially, beta B2-crystallin (gene symbol: Crybb2/CRYBB2) has been described as a structural protein of the ocular lens. Mutations of the corresponding gene, Crybb2, lead to cataract. Recent studies in mice suggested that mutations in Crybb2 cause alterations in hippocampal morphology and function, albeit its function in hippocampal neuron development remained elusive. In the current study, we found that Crybb2 contributes to dendritogenesis in vitro and in vivo. Furthermore, screening of previous data on differential expression-arrays, we found Tmsb4X up-regulated in Crybb2 mutants mouse brain. Additionally, Tmsb4X was co-expressed with Crybb2 at actin-enriched cell ruffles. Over-expression of Tmsb4X in cultured hippocampal neurons inhibited dendritogenesis, which phenocopied Crybb2 knockdown. The current study uncovers a new function of Crybb2 in brain development, especially in dendritogenesis, and the possible interplay partner Tmsb4X involved in this process. (C) 2018 Elsevier Inc. All rights reserved.

Depressive disorder (DD) is one of the typical affective disorders with a high morbidity, high suicide rate and high recurrence rate. Dysfunction of the 5-hydroxytryptamine 1A receptor (5-HT1AR) in the brain may serve an important role in the pathogenesis of DD. Currently, selective serotonin reuptake inhibitors are the first line antidepressants with 60–70% efficacy and severe adverse effects. Previous studies have demonstrated that Chinese herbal medicines, including the Shuyu capsule (SYC), are effective antidepressants with few side effects. However, the mechanism remains unclear. In the present study, the effects of the SYC on the 5-HT1AR level and the activation of adenylyl cyclase-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element-binding (CREB) signaling pathway that 5-HT1AR mediates in the cells of hippocampal neurons were investigated in vitro. The SYC demonstrated an antidepressant effect similar to that of fluoxetine in a rat depression model. Treatment of hippocampal neurons with the serum of depressive rats resulted in a decrease in the 5-HT1AR protein level and the activation of the cAMP-PKA-CREB signaling pathway in hippocampal neurons. Exposure to the serum of rats that received chronic mild stress plus SYC treatment led to no alterations in the 5-HT1AR level or the activation of the cAMP-PKA-CREB signaling pathway compared with those of cells exposed to normal rat serum. This effect is similar to the effects of 5-HT1AR antagonist WAY-100635. In addition, the 5-HT1A agonist 8-hydroxy-(dipropylamino) tetralin did not antagonize the effects of the SYC. Furthermore, the SYC exhibited an increased effect compared with fluoxetine on 5-HT1AR levels and CREB activation. The present study suggested that the SYC functions by increasing 5-HT1AR protein levels and the activation of the 5-HT1AR-mediated cAMP-PKA-CREB signaling pathway in hippocampal neurons.

Current strategies in Central Nervous System (CNS) repair focus on the engineering of artificial scaffolds for guiding and promoting neuronal tissue regrowth. Ideally, one should combine such synthetic structures with stem cell therapies, encapsulating progenitor cells and instructing their differentiation and growth. We used developments in the design, synthesis, and characterization of polysaccharide-based bioactive polymeric materials for testing the ideal composite supporting neuronal network growth, synapse formation and stem cell differentiation into neurons and motor neurons. Moreover, we investigated the feasibility of combining these approaches with engineered mesenchymal stem cells able to release neurotrophic factors. We show here that composite bio-constructs made of Chitlac, a Chitosan derivative, favor hippocampal neuronal growth, synapse formation and the differentiation of progenitors into the proper neuronal lineage, that can be improved by local and continuous delivery of neurotrophins. Statement of Significance In our work, we characterized polysaccharide-based bioactive platforms as biocompatible materials for nerve tissue engineering. We show that Chitlac-thick substrates are able to promote neuronal growth, differentiation, maturation and formation of active synapses. These observations support this new material as a promising candidate for the development of complex bio-constructs promoting central nervous system regeneration. Our novel findings sustain the exploitation of polysaccharide-based scaffolds able to favour neuronal network reconstruction. Our study shows that Chitlac-thick may be an ideal candidate for the design of biomaterial scaffolds enriched with stem cell therapies as an innovative approach for central nervous system repair.

We aim to investigate the effects of sevoflurane on the ATPase activity of the hippocampal neurons in rats with cerebral ischemia-reperfusion injury (IRI) via the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) signaling pathway. Sixty rats were assigned into the normal, model and sevoflurane groups (n = 20, the latter two groups were established as focal cerebral IRI models). The ATPase activity was detected using an ultramicro Na (+)-K (+)-ATP enzyme kit. Immunohistochemical staining was used to detect the positive protein expression of cAMP and PKA. The hippocampal neurons were assigned to the normal, IRI, IRI + sevoflurane, IRI + forskolin, IRI + H89 and IRI + sevoflurane + H89 groups. qRT-PCR and Western blotting were performed for the expressions of cAMP, PKA, cAMP-responsive element-binding protein (CREB) and brain derived neurotrophic factor (BDNF). The normal and sevoflurane groups exhibited a greater positive protein expression of cAMP and PKA than the model group. Compared with the normal group, the expressions of cAMP, PKA, CREB and BDNF all reduced in the IRI, model and IRI + H89 groups. The sevoflurane group showed higher cAMP, PKA, CREB and BDNF expressions than the model group. Compared with the IRI group, ATPase activity and expressions of cAMP, PKA, CREB and BDNF all increased in the normal, IRI + sevoflurane and IRI + forskolin groups but decreased in the IRI + H89 group. It suggests that sevoflurane could enhance ATPase activity in hippocampal neurons of cerebral IRI rats through activating cAMP-PKA signaling pathway.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This 'sandwich culture' allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

In the paper, based on the electrophysiological experimental dada, the neuron model under the pathology condition of Alzheimer Disease (AD) is built. The neuron models dynamic variation characteristics before and after the effect of AD are comparatively studied. Under the currents stimulation, compared with the normal neuron model, the AD neuron models dynamic characteristics have been obviously changed. Its changing range of the membranes interspike intervals (ISIs) obviously narrows, and the discharge patterns change rather single. In addition, its discharge threshold is much higher than the normal neuron models. So AD causes the neuron models excitability to depress markedly. The research reveals the neuron models dynamic characteristics changing under the effect of AD, which shows bioinformatics change of the neuron caused by AD. The research provides some theoretic basis for AD research by the neurodynamics theory.

Summary 1. Differential demands on cognitive ability may be expected to result in the evolution of cognition and associated changes in underlying neural mechanisms. While most comparative studies of cognition have focused on volumetric brain measurements, it remains unclear whether neuron morphology, which appears to be directly linked to cognitive functions, may be responsive to differential selection on cognitive ability. 2. Food-caching birds rely on caches to survive winter and use spatial memory to recover previously stored food. Birds in more harsh winter climates have been hypothesized to be more dependent on cached food, and therefore, their winter survival may be expected to be more memory-dependent relative to their conspecifics from the milder winter climates. Here, we show that neuron soma size in the hippocampus, a brain region involved in memory function, exhibits significant population variation associated with different environmental pressures on spatial memory related to differences in winter climate harshness in two species of foodcaching chickadees. Comparing ten populations of black-capped chickadees and three populations of mountain chickadees along a gradient of winter climate harshness, we found that birds from harsher environments had significantly larger hippocampal neuron soma sizes. 3. Using chickadees from the two most divergent populations reared in a laboratory environment, we showed that these differences appear to be at least partly heritable as significant differences between these populations remained in birds sharing the same laboratory environment. At the same time, laboratory-reared birds had significantly smaller neuron soma size compared with the wild-sampled birds, suggesting that at least some variation in neuron soma size may be due to environment-related plasticity. 4. Our data suggest that environment-related selection on memory may generate differences in neuron morphology, which appear to be controlled by some heritable mechanisms and likely underlie population differences in spatial memory.

Stress-responsive neuronal membrane glycoprotein M6a (Gpm6a) functions in neurite extension, filopodium and spine formation and synaptogenesis. The mechanisms of Gpm6a action in these processes are incompletely understood. Previously, we identified the actin regulator coronin-1a (Coro1a) as a putative Gpm6a interacting partner. Here, we used co-immunoprecipitation assays with the anti-Coro1a antibody to show that Coro1a associates with Gpm6a in rat hippocampal neurons. By immunofluorescence microscopy, we demonstrated that in hippocampal neurons Coro1a localizes in F-actin-enriched regions and some of Coro1a spots co-localize with Gpm6a labeling. Notably, the over-expression of a dominant-negative form of Coro1a as well as its down-regulation by siRNA interfered with Gpm6a-induced filopodium formation. Coro1a is known to regulate the plasma membrane translocation and activation of small GTPase Rac1. We show that Coro1a co-immunoprecipitates with Rac1 together with Gpm6a. Pharmacological inhibition of Rac1 resulted in a significant decrease in filopodium formation by Gpm6a. The same was observed upon the co-expression of Gpm6a with the inactive GDP-bound form of Rac1. In this case, the elevated membrane recruitment of GDP-bound Rac1 was detected as well. Moreover, the kinase activity of the p21-activated kinase 1 (Pak1), a main downstream effector of Rac1 that acts downstream of Coro1a, was required for Gpm6a-induced filopodium formation. Taken together, our results provide evidence that a signaling pathway including Coro1a, Rac1, and Pak1 facilitates Gpm6a-induced filopodium formation. Fil: Alvarez Juliá, Anabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina Fil: Frasch, Alberto Carlos C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina Fil: Fuchsova, Beata. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina

Caspase 2 was initially identified as a neuronally expressed developmentally down-regulated gene (HUGO gene nomenclature CASP2) and has been shown to be required for neuronal death induced by several stimuli, including NGF (nerve growth factor) deprivation and Aβ (β-amyloid). In non-neuronal cells the PIDDosome, composed of caspase 2 and two death adaptor proteins, PIDD (p53-inducible protein with a death domain) and RAIDD {RIP (receptor-interacting protein)-associated ICH-1 [ICE (interleukin-1β-converting enzyme)/CED-3 (cell-death determining 3) homologue 1] protein with a death domain}, has been proposed as the caspase 2 activation complex, although the absolute requirement for the PIDDosome is not clear. To investigate the requirement for the PIDDosome in caspase-2-dependent neuronal death, we have examined the necessity for each component in induction of active caspase 2 and in execution of caspase-2-dependent neuronal death. We find that both NGF deprivation and Aβ treatment of neurons induce active caspase 2 and that induction of this activity depends on expression of RAIDD, but is independent of PIDD expression. We show that treatment of wild-type or PIDD-null neurons with Aβ or NGF deprivation induces formation of a complex of caspase 2 and RAIDD. We also show that caspase-2-dependent execution of neurons requires RAIDD, not PIDD. Caspase 2 activity can be induced in neurons from PIDD-null mice, and NGF deprivation or Aβ use caspase 2 and RAIDD to execute death of these neurons.

Neuroscience In the brain, synaptic connections are formed with exquisite specificity, but the underlying molecular mechanisms remain largely unexplored. Synapse formation is thought to involve bidirectional signaling by proteins that bind to each other across the synaptic cleft. Sando et al. used conditional genetic tools and in vitro assays to investigate the mechanisms of synapse formation. They found that synapse formation in the mouse hippocampus requires latrophilins. Latrophilins are G protein–coupled receptors that bind to cell-surface proteins called teneurins and fibronectin leucine-rich repeat transmembrane proteins (FLRTs). Two different latrophilins mediated formation of distinct synapses on the same hippocampal neuron. This function required binding of both teneurins and FLRTs. Thus, latrophilins may guide synapse formation by coincidence signaling, which could help to explain the specificity of synaptic connections. Science , this issue p. [eaav7969][1] [1]: /lookup/doi/10.1126/science.aav7969

Green tea has been receiving considerable attention as a possible neuroprotective agent against neurodegenerative disease. Epigallocatechin-3-gallate (EGCG) is the major compound of green tea. Calcium signaling has profound effects on almost all aspects of neuronal function. Using digital calcium imaging and patch-clamp technique, we determined the effects of EGCG on Ca2+ signals in hippocampal neurons. The results indicated that EGCG caused a dose-dependent increase in intracellular Ca2+ ([Ca2+]i). This [Ca2+]i increase was blocked by depleting intracellular Ca2+ stores with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin and cyclopiazonic acid. Furthermore, EGCG-stimulated increase in [Ca2+]i was abolished following treatment with a PLC inhibitor. However, EGCG inhibited high-voltage activated Ca2+ currents (IHVA) and NMDA-induced inward currents (INMDA). These data suggest that EGCG triggers a cascade of events: it activates phospholipase C (PLC), mobilizes intracellular Ca2+ stores, raises the cytosolic Ca2+ levels, and inhibits the VGCC and NMDA receptors-mediated Ca2+ influx through a process that remains to be determined.

To image synaptic activity within neural circuits, we have tethered the genetically-encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detects the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: the locations of synapses are identified and the reporter displays a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicate that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrates that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum as well as graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses.

Indoleamine 2, 3-dioxygenase (IDO), which catabolizes L-tryptophan (L-TRP) to L-kynurenine (L-KYN), is an immunoregulatory factor that is up-regulated via an interferon-gamma (IFN-gamma)-dependent and/or -independent mechanism. In this study, we investigated the localization of IDO and whether induction of IDO expression is an IFN-gamma-dependent and/or -independent mechanism in the CNS after cerebral ischemia. The expressions of IDO protein and mRNA were investigated at different time points following cerebral ischemia using immunohistochemistry, immunofluorescence and RT-PCR. Hippocampal neuron IDO mRNA and immunohistochemical staining were significantly up-regulated 72h after transient global ischemia. Although IFN-gamma is a dominant inducer of IDO, hippocampal neuron IDO was clearly up-regulated in IFN-gamma KO mice. In summary, this is the first finding that up-regulation of IDO in hippocampal neurons after transient global ischemia occurs via INF-gamma-independent mechanisms.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) fulfils vital signalling roles in an array of cellular processes, yet until recently it has not been possible selectively to visualize real-time changes in PIP(2) levels within living cells. Green fluorescent protein (GFP)-labelled Tubby protein (GFP-Tubby) enriches to the plasma membrane at rest and translocates to the cytosol following activation of endogenous Galpha(q/11)-coupled muscarinic acetylcholine receptors in both SH-SY5Y human neuroblastoma cells and primary rat hippocampal neurons. GFP-Tubby translocation is independent of changes in cytosolic inositol 1,4,5-trisphosphate and instead reports dynamic changes in levels of plasma membrane PIP(2). In contrast, enhanced GFP (eGFP)-tagged pleckstrin homology domain of phospholipase C (PLCdelta1) (eGFP-PH) translocation reports increases in cytosolic inositol 1,4,5-trisphosphate. Comparison of GFP-Tubby, eGFP-PH and the eGFP-tagged C1(2) domain of protein kinase C-gamma [eGFP-C1(2); to detect diacylglycerol] allowed a selective and comprehensive analysis of PLC-initiated signalling in living cells. Manipulating intracellular Ca(2+) concentrations in the nanomolar range established that GFP-Tubby responses to a muscarinic agonist were sensitive to intracellular Ca(2+) up to 100-200 nM in SH-SY5Y cells, demonstrating the exquisite sensitivity of agonist-mediated PLC activity within the range of physiological resting Ca(2+) concentrations. We have also exploited GFP-Tubby selectively to visualize, for the first time, real-time changes in PIP(2) in hippocampal neurons.

Deep seawater (DSW; deep ocean water) is pure, rich in inorganic materials which have attracted attention for various applications. In this study we investigated the effects of the DSW upwelled from the East Sea, offshore Yang Yang (Korea) on the morphological differentiation of cultured rat hippocampal neurons, which were grown in the minimal essential medium containing 10% (v/v) fetal bo-vine serum and 25% (v/v) DSW with various hardness. DSW had no effect on initial morphological differentiation (17 hr post-plating). When observed on DIV3, 7, 14, and 17, low hardness (0 and 200) DSW reduced dendritic branching. However, dendritic branches within 80 μm diameter from the center of soma nearly doubled in neurons grown in hardness 1,000 DSW-containing media. DSW with hardness 600 was more or less same as control groups. These results indicate that DSW with appropriate hardness ameliorates neuronal health.

Mongolian gerbils of the seizure-sensitive strain exhibit epileptic seizures in relation to changes in the environment, a characteristic which has been increased to about 100% by inbreeding. The seizures vary from animal to animal but are rather stable in the individual animal, which makes it possible to study the neuron densities in the hippocampus of the gerbil in relation to seizure type and seizure intensity. Five groups of gerbils with seizures ranging from minor movements and motor arrest to intense generalized convulsions were investigated with a quantitative method including cell counting by light microscope and estimation of possible brain shrinkage, as well as determination of nucleoli and nuclei diameters. The cell densities were determined in different areas of the pyramidal cells of the hippocampus (H-fields). The study discloses a reduction of cell densities in fields H2 and H3 in relation to intense generalized convulsions. It is suggested that the reduction in cell density in field H2 is a result of seizure activity, whereas the field H3 cell loss can be the result of both the hypoxia and the seizure activity.

Objective To clarify the firing characteristics of the hippocampal pyramidal cells and interneurons in the auditory discrimination cognition. Methods Thirteen guinea pigs were studied by the paired (active cognition group, n = 10) or unpaired (passive cognition group, n = 3) training with 1 kHz (CS+) and 500 Hz tones (CS−) and the air puff (US) applied 250 ms after the CS+ onset. Results In active group, 32 pyramidal cells showed exciting response to the CS+ tone, 16 cells inhibited response and 4 cells revealed no response to the high frequency tone and 18 interneurons almost unchanged. In passive group, the pyramidal cells responded to the tone casually and 10 out of the 13 interneurons remained invariably. Conclusion The result suggests that the pyramidal cells play a major role in coding auditory information by the networks, and the interneuons may modulate it via forward and feedback.

Over the past decade, evidence has accumulated indicating that, during development, the construction of synapses – the sites of communication between neurons – might rely on the utilization of preassembled sets of synaptic proteins, which have already accumulated in the axon and are highly mobile, before getting recruited to the sites of contact with the postsynaptic neuron. In this review, we discuss evidence from most recent publications pointing to the existence of active vesicle traffic and turnover in developing neurons, which lead to the construction of new synaptic sites.

Neuronal recruitment in the black-capped chickadee (Poecile atricapillus) hippocampus occurs at a higher rate in the fall than at other times of the year. As a means of determining whether this increase in recruitment results from greater neuron production, chickadees were caught in the wild between October and March and injected with the cell-birth marker 5-bromo-2'-deoxyuridine. Two weeks later, birds were killed by overdose, and hippocampal neuron production, apoptosis, neuron number, and hippocampal volume were determined. Chickadees collected in October, November, January, February, and March did not differ in neuron production, apoptosis, hippocampal volume, or neuron number. These findings indicate that increases in neuronal recruitment in the chickadee hippocampus in the fall do not result from increased neuron production, but instead, enhanced survival of new neurons.

The relations between neurite development and the subcellular structures of the hippocampal neuron somata have been studied with atomic force microscopy (AFM). The conformation of the neuron was achieved by the synapse-like structures found by AFM scanning along a neurite of the cell. Hippocampal neuron somata were divided into two or three subcellular parts by one or two horizontal grooves. The upper parts increased while the middle and the lower parts decreased with the number and the length of the neurites and the formation of the neurosynapse-like structures. When neurites sufficiently developed, the middle parts were lost and the lower parts became very small. Mitosis inhibitors could prevent the formation of such subcellular structures of hippocampal neuron somata, which was accompanied by the loss of ability to form synapse-like structures. These results suggest that the upper parts are responsible for neuritogenesis while the middle and the lower parts only have indirect effect on it.

Here, using a genetic approach, we dissect the roles of EphB receptor tyrosine kinases in dendritic spine development. Analysis of EphB1, EphB2, and EphB3 double and triple mutant mice lacking these receptors in different combinations indicates that all three, although to varying degrees, are involved in dendritic spine morphogenesis and synapse formation in the hippocampus. Hippocampal neurons lacking EphB expression fail to form dendritic spines in vitro and they develop abnormal spines in vivo. Defective spine formation in the mutants is associated with a drastic reduction in excitatory glutamatergic synapses and the clustering of NMDA and AMPA receptors. We show further that a kinase-defective, truncating mutation in EphB2 also results in abnormal spine development and that ephrin-B2–mediated activation of the EphB receptors accelerates dendritic spine development. These results indicate EphB receptor cell autonomous forward signaling is responsible for dendritic spine formation and synaptic maturation in hippocampal neurons.

Glutamate receptors mediate the majority of excitatory responses in the central nervous system. The establishment and refinement of glutamatergic synaptic connections depend on the concerted actions of a-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA),N-methyl-D-aspartate (NMDA), and kainate (KA) type ionotropic glutamate receptors (iGluRs) and G-protein coupled metabotropic receptors. While a lot remains to be clarified, the most is known about the mechanisms by which the iGluR subtypes are targeted and how this is influenced by synaptic activity on both short and long time scales. Changes in their subunit compositions are also input specific and developmentally regulated. The identification of key molecular components of the postsynaptic density (PSD) and novel proteins that influence receptor targeting and clustering have started to reveal the underlying molecular mechanisms of the trafficking and targeting of iGluRs. Here we discuss the evidence that these basic mechanisms are used during developmental synaptic plasticity.

The plating efficiency of neurons in culture is highly dependent on the concentration of cells used to establish the monolayer. A discontinuous iodixanol gradient permits both the production of a viable concentrated suspension of neurons and purification from other brain tissue elements. The gradient that is described in this Protocol Article is applicable to brain tissue from rat and also from human biopsy specimens.

Kaplan, Suleyman/0000-0003-1477-5002; Canan, Sinan/0000-0002-9864-1767; pakkenberg, bente/0000-0003-3851-6494 WOS: 000174095200003 Seven days after 10 minutes bilateral occlusion of internal carotid arteries, the total neuron number and the volume of the hippocampus were estimated using the optical fractionator method for cell counting and Cavalieri's principle for volume estimation. After comparing with sham-operated animals, no significant difference in total neuron number was observed in hippocampi of ischemic chicks; a statistically significant increase in numerical density of hippocampal neurons in hippocampi of ischemic group (p