Postnatal Hippocampal Neurons Research Articles

Isoflurane Alters Presynaptic Endoplasmic Reticulum Calcium Dynamics in Wild-Type and Malignant Hyperthermia-Susceptible Rodent Hippocampal Neurons.

Volatile anesthetics reduce excitatory synaptic transmission by both presynaptic and postsynaptic mechanisms which include inhibition of depolarization-evoked increases in presynaptic Ca2+ concentration and blockade of postsynaptic excitatory glutamate receptors. The presynaptic sites of action leading to reduced electrically evoked increases in presynaptic Ca2+ concentration and Ca2+-dependent exocytosis are unknown. Endoplasmic reticulum (ER) of Ca2+ release via ryanodine receptor 1 (RyR1) and uptake by SERCA are essential for regulation intracellular Ca2+ and are potential targets for anesthetic action. Mutations in sarcoplasmic reticulum (SR) release channels mediate volatile anesthetic-induced malignant hyperthermia (MH), a potentially fatal pharmacogenetic condition characterized by unregulated Ca2+ release and muscle hypermetabolism. However, the impact of MH mutations on neuronal function are unknown. We used primary cultures of postnatal hippocampal neurons to analyze volatile anesthetic-induced changes in ER Ca2+ dynamics using a genetically encoded ER-targeted fluorescent Ca2+ sensor in both rat and mouse wild-type (WT) neurons and in mouse mutant neurons harboring the RYR1 T4826I MH-susceptibility mutation. The volatile anesthetic isoflurane reduced both baseline and electrical stimulation-evoked increases in ER Ca2+ concentration in neurons independent of its depression of presynaptic cytoplasmic Ca2+ concentrations. Isoflurane and sevoflurane, but not propofol, depressed depolarization-evoked increases in ER Ca2+ concentration significantly more in mouse RYR1 T4826I mutant neurons than in wild-type neurons. The RYR1 T4826I mutant neurons also showed markedly greater isoflurane-induced reductions in presynaptic cytosolic Ca2+ concentration and synaptic vesicle (SV) exocytosis. These findings implicate RyR1 as a molecular target for the effects of isoflurane on presynaptic Ca2+ handling.

Suppression of DNA Double-Strand Break Formation by DNA Polymerase β in Active DNA Demethylation Is Required for Development of Hippocampal Pyramidal Neurons.

Genome stability is essential for brain development and function, as de novo mutations during neuronal development cause psychiatric disorders. However, the contribution of DNA repair to genome stability in neurons remains elusive. Here, we demonstrate that the base excision repair protein DNA polymerase β (Polβ) is involved in hippocampal pyramidal neuron differentiation via a TET-mediated active DNA demethylation during early postnatal stages using Nex-Cre/Polβ fl/fl mice of either sex, in which forebrain postmitotic excitatory neurons lack Polβ expression. Polβ deficiency induced extensive DNA double-strand breaks (DSBs) in hippocampal pyramidal neurons, but not dentate gyrus granule cells, and to a lesser extent in neocortical neurons, during a period in which decreased levels of 5-methylcytosine and 5-hydroxymethylcytosine were observed in genomic DNA. Inhibition of the hydroxylation of 5-methylcytosine by expression of microRNAs miR-29a/b-1 diminished DSB formation. Conversely, its induction by TET1 catalytic domain overexpression increased DSBs in neocortical neurons. Furthermore, the damaged hippocampal neurons exhibited aberrant neuronal gene expression profiles and dendrite formation, but not apoptosis. Comprehensive behavioral analyses revealed impaired spatial reference memory and contextual fear memory in adulthood. Thus, Polβ maintains genome stability in the active DNA demethylation that occurs during early postnatal neuronal development, thereby contributing to differentiation and subsequent learning and memory.SIGNIFICANCE STATEMENT Increasing evidence suggests that de novo mutations during neuronal development cause psychiatric disorders. However, strikingly little is known about how DNA repair is involved in neuronal differentiation. We found that Polβ, a component of base excision repair, is required for differentiation of hippocampal pyramidal neurons in mice. Polβ deficiency transiently led to increased DNA double-strand breaks, but not apoptosis, in early postnatal hippocampal pyramidal neurons. This aberrant double-strand break formation was attributed to active DNA demethylation as an epigenetic regulation. Furthermore, the damaged neurons exhibited aberrant gene expression profiles and dendrite formation, resulting in impaired learning and memory in adulthood. Thus, these findings provide new insight into the contribution of DNA repair to the neuronal genome in early brain development.

Interfacing Neurons with Nanostructured Electrodes Modulates Synaptic Circuit Features.

Understanding neural physiopathology requires advances in nanotechnology-based interfaces, engineered to monitor the functional state of mammalian nervous cells. Such interfaces typically contain nanometer-size features for stimulation and recording as in cell-non-invasive extracellular microelectrode arrays. In such devices, it turns crucial to understand specific interactions of neural cells with physicochemical features of electrodes, which could be designed to optimize performance. Herein, versatile flexible nanostructured electrodes covered by arrays of metallic nanowires are fabricated and used to investigate the role of chemical composition and nanotopography on rat brain cells in vitro. By using Au and Ni as exemplary materials, nanostructure and chemical composition are demonstrated to play major roles in the interaction of neural cells with electrodes. Nanostructured devices are interfaced to rat embryonic cortical cells and postnatal hippocampal neurons forming synaptic circuits. It is shown that Au-based electrodes behave similarly to controls. Contrarily, Ni-based nanostructured electrodes increase cell survival, boost neuronal differentiation, and reduce glial cells with respect to flat counterparts. Nonetheless, Au-based electrodes perform superiorly compared to Ni-based ones. Under electrical stimulation, Au-based nanostructured substrates evoke intracellular calcium dynamics compatible with neural networks activation. These studies highlight the opportunity for these electrodes to excite a silent neural network by direct neuronal membranes depolarization.

Procedures for Culturing and Genetically Manipulating Murine Hippocampal Postnatal Neurons.

Neuronal hippocampal cultures are simple and valuable models for studying neuronal function. While embryonic cultures are widely used for different applications, mouse postnatal cultures are still challenging, lack reproducibility and/or exhibit inappropriate neuronal activity. Yet, postnatal cultures have major advantages such as allowing genotyping of pups before culture and reducing the number of experimental animals. Herein we describe a simple and fast protocol for culturing and genetically manipulating hippocampal neurons from P0 to P3 mice. This protocol provides reproducible cultures exhibiting a consistent neuronal development, normal excitatory over inhibitory neuronal ratio and a physiological neuronal activity. We also describe simple and efficient procedures for genetic manipulation of neurons using transfection reagent or lentiviral particles. Overall, this method provides a detailed and validated protocol allowing to explore cellular mechanisms and neuronal activity in postnatal hippocampal neurons in culture.

High-Content Screening for Protein-Protein Interaction Modulators Using Proximity Ligation Assay in Primary Neurons.

The proximity ligation assay (PLA) allows the detection and subcellular localization of protein-protein interactions with high specificity. We recently developed a high-content screening model based on primary hippocampal neurons cultured in 384-well plates and screened a library of ∼1100 compounds using a PLA between tau and bridging integrator 1, a genetic risk factor for Alzheimer's disease. We developed image-segmentation and spot-detection algorithms to delineate PLA signals in the axonal network, but not in cell bodies, from confocal images acquired via a high-throughput microscope. To compare data generated from different plates and through different experiments, we developed a computational routine to optimize the image analysis parameters for each plate and devised a range of quality-control measures to ultimately identify compounds that consistently increase or decrease our read-out. We provide the following protocols. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Routine culture of rat postnatal hippocampal neurons in 384-well plates Basic Protocol 2: Compound incubation using the high-content screening platform Support Protocol 1: Preparation of intermediate plates for compound screening Support Protocol 2: Preparation of intermediate plates for hit validation (dose-response curves) Basic Protocol 3: Proximity ligation assay in 384-well plates Basic Protocol 4: Image acquisition and analysis Support Protocol 3: Optimization of analysis parameters Basic Protocol 5: Identification of hits Basic Protocol 6: Validation of hits based on dose-response curves.

Impaired neuronal maturation of hippocampal neural progenitor cells in mice lacking CRAF.

RAF kinases are major constituents of the mitogen activated signaling pathway, regulating cell proliferation, differentiation and cell survival of many cell types, including neurons. In mammals, the family of RAF proteins consists of three members, ARAF, BRAF, and CRAF. Ablation of CRAF kinase in inbred mouse strains causes major developmental defects during fetal growth and embryonic or perinatal lethality. Heterozygous germline mutations in CRAF result in Noonan syndrome, which is characterized by neurocognitive impairment that may involve hippocampal physiology. The role of CRAF signaling during hippocampal development and generation of new postnatal hippocampal granule neurons has not been examined and may provide novel insight into the cause of hippocampal dysfunction in Noonan syndrome. In this study, by crossing CRAF-deficiency to CD-1 outbred mice, a CRAF mouse model was established which enabled us to investigate the interplay of neural progenitor proliferation and postmitotic differentiation during adult neurogenesis in the hippocampus. Albeit the general morphology of the hippocampus was unchanged, CRAF-deficient mice displayed smaller granule cell layer (GCL) volume at postnatal day 30 (P30). In CRAF-deficient mice a substantial number of abnormal, chromophilic, fast dividing cells were found in the subgranular zone (SGZ) and hilus of the dentate gyrus (DG), indicating that CRAF signaling contributes to hippocampal neural progenitor proliferation. CRAF-deficient neural progenitor cells showed an increased cell death rate and reduced neuronal maturation. These results indicate that CRAF function affects postmitotic neural cell differentiation and points to a critical role of CRAF-dependent growth factor signaling pathway in the postmitotic development of adult-born neurons.

Autism-Associated Chromatin Regulator Brg1/SmarcA4 Is Required for Synapse Development and Myocyte Enhancer Factor 2-Mediated Synapse Remodeling.

Synapse development requires normal neuronal activities and the precise expression of synapse-related genes. Dysregulation of synaptic genes results in neurological diseases such as autism spectrum disorders (ASD). Mutations in genes encoding chromatin-remodeling factor Brg1/SmarcA4 and its associated proteins are the genetic causes of several developmental diseases with neurological defects and autistic symptoms. Recent large-scale genomic studies predicted Brg1/SmarcA4 as one of the key nodes of the ASD gene network. We report that Brg1 deletion in early postnatal hippocampal neurons led to reduced dendritic spine density and maturation and impaired synapse activities. In developing mice, neuronal Brg1 deletion caused severe neurological defects. Gene expression analyses indicated that Brg1 regulates a significant number of genes known to be involved in synapse function and implicated in ASD. We found that Brg1 is required for dendritic spine/synapse elimination mediated by the ASD-associated transcription factor myocyte enhancer factor 2 (MEF2) and that Brg1 regulates the activity-induced expression of a specific subset of genes that overlap significantly with the targets of MEF2. Our analyses showed that Brg1 interacts with MEF2 and that MEF2 is required for Brg1 recruitment to target genes in response to neuron activation. Thus, Brg1 plays important roles in both synapse development/maturation and MEF2-mediated synapse remodeling. Our study reveals specific functions of the epigenetic regulator Brg1 in synapse development and provides insights into its role in neurological diseases such as ASD.

No dopamine cell loss or changes in cytoskeleton function in transgenic mice expressing physiological levels of wild type or G2019S mutant LRRK2 and in human fibroblasts.

Mutations within the LRRK2 gene have been identified in Parkinson’s disease (PD) patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.

A novel neuroferritinopathy mouse model (FTL 498InsTC) shows progressive brain iron dysregulation, morphological signs of early neurodegeneration and motor coordination deficits

Neuroferritinopathy is a rare genetic disease with a dominant autosomal transmission caused by mutations of the ferritin light chain gene (FTL). It belongs to Neurodegeneration with Brain Iron Accumulation, a group of disorders where iron dysregulation is tightly associated with neurodegeneration. We studied the 498–499InsTC mutation which causes the substitution of the last 9 amino acids and an elongation of extra 16 amino acids at the C-terminus of L-ferritin peptide. An analysis with cyclic voltammetry on the purified protein showed that this structural modification severely reduces the ability of the protein to store iron. In order to analyze the impact of the mutation in vivo, we generated mouse models for the some pathogenic human FTL gene in FVB and C57BL/6J strains.Transgenic mice in the FVB background showed high accumulation of the mutated ferritin in brain where it correlated with increased iron deposition with age, as scored by magnetic resonance imaging. Notably, the accumulation of iron–ferritin bodies was accompanied by signs of oxidative damage. In the C57BL/6 background, both the expression of the mutant ferritin and the iron levels were lower than in the FVB strain. Nevertheless, also these mice showed oxidative alterations in the brain. Furthermore, post-natal hippocampal neurons obtained from these mice experienced a marked increased cell death in response to chronic iron overload and/or acute oxidative stress, in comparison to wild-type neurons. Ultrastructural analyses revealed an accumulation of lipofuscin granules associated with iron deposits, particularly enriched in the cerebellum and striatum of our transgenic mice. Finally, experimental subjects were tested throughout development and aging at 2-, 8- and 18-months for behavioral phenotype. Rotarod test revealed a progressive impaired motor coordination building up with age, FTL mutant old mice showing a shorter latency to fall from the apparatus, according to higher accumulation of iron aggregates in the striatum. Our data show that our 498–499InsTC mouse models recapitulate early pathological and clinical traits of the human neuroferritinopathy, thus providing a valuable model for the study of the disease. Finally, we propose a mechanistic model of lipofuscine formation that can account for the etiopathogenesis of human neuroferritinopathy.

Presenilins regulate calcium homeostasis and presynaptic function via ryanodine receptors in hippocampal neurons

Presenilin (PS) plays a central role in the pathogenesis of Alzheimer's disease, and loss of PS causes progressive memory impairment and age-related neurodegeneration in the mouse cerebral cortex. In hippocampal neurons, PS is essential for neurotransmitter release, NMDA receptor-mediated responses, and long-term potentiation. PS is also involved in the regulation of calcium homeostasis, although the precise site of its action is less clear. Here we investigate the mechanism by which PS regulates synaptic function and calcium homeostasis using acute hippocampal slices from PS conditional knockout mice and primary cultured postnatal hippocampal neurons, in which PS is inducibly inactivated. Using two different calcium probes, Fura-2 and Mag-Fura-2, we found that inactivation of PS in primary hippocampal neurons does not affect calcium concentration in the endoplasmic reticulum. Rather, in the absence of PS, levels of ryanodine receptor (RyR) are reduced in the hippocampus, measured by Western analysis and radioligand binding assay, although the mRNA expression is unaffected. RyR-mediated function is also impaired, as indicated by reduced RyR agonist-induced calcium release from the ER and RyR-mediated synaptic responses in the absence of PS. Furthermore, knockdown of RyR expression in wild-type hippocampal neurons by two independent shRNAs to levels comparable with the RyR protein reduction in PS-deficient hippocampal neurons mimics the defects exhibited in calcium homeostasis and presynaptic function. Collectively, our findings show that PS regulates calcium homeostasis and synaptic function via RyR and suggest that disruption of intracellular calcium homeostasis may be an early pathogenic event leading to presynaptic dysfunction in Alzheimer's disease.

Neuronal Nogo‐A regulates glutamate receptor subunit expression in hippocampal neurons

Nogo-A and its cognate receptor NogoR1 (NgR1) are both expressed in neurons. To explore the function of these proteins in neurons of the CNS, we carried out a series of studies using postnatal hippocampal neurons in culture. Interfering with the binding of Nogo-A to NgR1 either by adding truncated soluble fragment of NgR1 (NgSR) or by reducing NgR1 protein with a specific siRNA, resulted in a marked reduction in Nogo-A expression. Inhibition of Rho-ROCK or MEK-MAPK signaling resulted in a similar reduction in neuronal Nogo-A mRNA and protein. Reducing Nogo-A protein levels by siRNA resulted in an increase in the post-synaptic scaffolding protein PSD95, as well as increases in GluA1/GluA2 AMPA receptor and GluN1/GluN2A/GluN2B NMDA glutamate receptor subunits. siRNA treatment to reduce Nogo-A resulted in phosphorylation of mTOR; addition of rapamycin to block mTOR signaling prevented the up-regulation in glutamate receptor subunits. siRNA reduction of NgR1 resulted in increased expression of the same glutamate receptor subunits. Taken together the results suggest that transcription and translation of Nogo-A in hippocampal neurons is regulated by a signaling through NgR1, and that interactions between neuronal Nogo-A and NgR1 regulate glutamatergic transmission by altering NMDA and AMPA receptor levels through an rapamycin-sensitive mTOR-dependent translation mechanism.

Excitotoxicity Triggered by Neurobasal Culture Medium

Neurobasal defined culture medium has been optimized for survival of rat embryonic hippocampal neurons and is now widely used for many types of primary neuronal cell culture. Therefore, we were surprised that routine medium exchange with serum- and supplement-free Neurobasal killed as many as 50% of postnatal hippocampal neurons after a 4 h exposure at day in vitro 12–15. Minimal Essential Medium (MEM), in contrast, produced no significant toxicity. Detectable Neurobasal-induced neuronal death occurred with as little as 5 min exposure, measured 24 h later. D-2-Amino-5-phosphonovalerate (D-APV) completely prevented Neurobasal toxicity, implicating direct or indirect N-methyl-D-aspartate (NMDA) receptor-mediated neuronal excitotoxicity. Whole-cell recordings revealed that Neurobasal but not MEM directly activated D-APV-sensitive currents similar in amplitude to those gated by 1 µM glutamate. We hypothesized that L-cysteine likely mediates the excitotoxic effects of Neurobasal incubation. Although the original published formulation of Neurobasal contained only 10 µM L-cysteine, commercial recipes contain 260 µM, a concentration in the range reported to activate NMDA receptors. Consistent with our hypothesis, 260 µM L-cysteine in bicarbonate-buffered saline gated NMDA receptor currents and produced toxicity equivalent to Neurobasal. Although NMDA receptor-mediated depolarization and Ca2+ influx may support survival of young neurons, NMDA receptor agonist effects on development and survival should be considered when employing Neurobasal culture medium.

Carboxymethylchitosan/Poly(amidoamine) Dendrimer Nanoparticles in Central Nervous Systems‐Regenerative Medicine: Effects on Neuron/Glial Cell Viability and Internalization Efficiency

The applicability of CMCht/PAMAM dendrimer nanoparticles for CNS applications was investigated. AFM and TEM observations revealed that the nanoparticles possessed a nanosphere-like shape with a size from 22.0 to 30.7 nm. The nanoparticles could be bound to fluorescent-probe FITC for tracing purposes. Post-natal hippocampal neurons and cortical glial cells were both able to internalize the FITC-labeled CMCht/PAMAM dendrimer nanoparticles with high efficiency. The percentage of positive cells internalizing the nanoparticles varied, reaching a peak after 48 h of incubation. Further experiments for periods up to 7 d revealed that the periodical addition of FITC-labelled CMCht/PAMAM dendrimer nanoparticles was needed to maintain the overall percentage of cells internalizing them. Finally, it was also observed that cell viability was not significantly affected by the incubation of dendrimer nanoparticles.

Role of Human Umbilical Cord Mesenchymal Progenitors Conditioned Media in Neuronal/Glial Cell Densities, Viability, and Proliferation

It has been recently reported that mesenchymal progenitor/stem cells isolated from the Wharton's Jelly (WJ) of umbilical cords (UC) ameliorate the condition of animals suffering from central nervous system (CNS)-related conditions. However, little is known on the mechanisms that regulate these actions. Therefore, the objective of the present work was to determine how the conditioned media (CM) of a population of mesenchymal progenitors present in the UC WJ, known as human umbilical cord perivascular cells (HUCPVCs), regulate processes such as cell viability, survival, and proliferation of postnatal hippocampal neurons and glial cells. For this purpose primary hippocampal and cortical cultures of neurons and glial cells, respectively, were incubated with CM from HUCPVCs. Results revealed that HUCPVCs CM increase glial cell viability and proliferation. Furthermore, it was observed that glial cell cultures exhibited higher numbers of GFAP-positive cells (astrocytes) and O4-positive cells (oligodendrocytes) when incubated with the CM. Additionally, it was also observed that the growth factors presents in the CM did not induce an increase on the microglial cells number. For hippocampal neurons similar results were obtained, as cultures exposed to HUCPVCs CM disclosed higher numbers of MAP-2-positive cells. Moreover it was also observed that the cell viability and proliferation in this primary hippocampal cell culture system was also higher, when compared to control cultures. From these results it was possible to conclude that HUCPVCs release neuroregulatory factors that have a direct impact on the densities, viability, and proliferation of glial cells and hippocampal primary cultures.

Vulnerability of postnatal hippocampal neurons to seizures varies regionally with their maturational stage

The mechanism of status epilepticus-induced neuronal death in the immature brain is not fully understood. In the present study, we examined the contribution of caspases in our lithium–pilocarpine model of status epilepticus in 14 days old rat pups. In CA1, upregulation of caspase-8, but not caspase-9, preceded caspase-3 activation in morphologically necrotic cells. Pretreatment with a pan-caspase inhibitor provided neuroprotection, showing that caspase activation was not an epiphenomenon but contributed to neuronal necrosis. By contrast, upregulation of active caspase-9 and caspase-3, but not caspase-8, was detected in apoptotic dentate gyrus neurons, which were immunoreactive for doublecortin and calbindin-negative, two features of immature neurons. These results suggest that, in cells which are aligned in series as parts of the same excitatory hippocampal circuit, the same seizures induce neuronal death through different mechanisms. The regional level of neuronal maturity may be a determining factor in the execution of a specific death program.

Thyroid Hormone (T3)-Induced Up-Regulation of Voltage-Activated Sodium Current in Cultured Postnatal Hippocampal Neurons Requires Secretion of Soluble Factors from Glial Cells

We have previously shown that treatment with the thyroid hormone T(3) increases the voltage-gated Na(+)current density (Nav-D) in hippocampal neurons from postnatal rats, leading to accelerated action potential upstrokes and increased firing frequencies. Here we show that the Na(+) current regulation depends on the presence of glial cells, which secrete a heat-instable soluble factor upon stimulation with T(3). The effect of conditioned medium from T(3)-treated glial cells was mimicked by basic fibroblast growth factor (bFGF), known to be released from cerebellar glial cells after T(3) treatment. Neutralization assays of astrocyte-conditioned media with anti-bFGF antibody inhibited the regulation of the Nav-D by T(3). This suggests that the up-regulation of the neuronal sodium current density by T(3) is not a direct effect but involves bFGF release and satellite cells. Thus glial cells can modulate neuronal excitability via secretion of paracrinely acting factors.

C‐Jun N‐terminal kinases trigger both degeneration and neurite outgrowth in primary hippocampal and cortical neurons

We provide a comprehensive analysis on c-Jun N-terminal kinase (JNK) actions leading to death or differentiation in postnatal hippocampal and cortical neurons. Stimulation with glutamate or 6-hydroxy-dopamine caused activation of caspase-3 and apoptotic neuronal death which were both attenuated by JNK-inhibition. In cortical neurons, stress-induced nuclear JNK distribution was rather complex. We observed a decrease of activated and total JNK in the nucleus after stimulation, but an increase of the phosphorylated transcription factor c-Jun. Isoform-analysis revealed a nuclear translocation of JNK2, while nuclear protein levels of JNK1 decreased. This activation pattern differed from neurite formation. In hippocampal and cortical neurons, JNK activity continuously increased during neuritogenesis, whereas levels of phosphorylated c-Jun gradually declined. Despite these similarities, JNK inhibition by SP600125 only affected neurite outgrowth in hippocampal cells. Furthermore, experiments in JNK-deficient mice demonstrated that all JNK isoforms contributed to neuritogenesis. Summarizing, JNKs are involved in both neuritogenesis and death of primary neurons with differentially regulated nuclear translocation of specific isoforms after degenerative stress, while neuritogenesis is supported by all JNK isoforms.

GAT‐1 acts to limit a tonic GABAA current in rat CA3 pyramidal neurons at birth

Tonic activation of GABA(A) receptors takes place before the development of functional synapses in cortical structures. We studied whether inefficient GABA uptake might explain the presence of a tonic GABA(A)-mediated current (I(GABA-A)) in early postnatal hippocampal pyramidal neurons. The data show, however, that the tonic I(GABA-A) is enhanced by the specific blocker of GABA transporter-1 (GAT-1), NO-711 (1-[2-[[(Diphenylmethyleneimino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), at birth in rat CA3 pyramidal neurons. NO-711 also prolonged the duration of GABA transients during endogenous hippocampal network events (known as giant depolarizing potentials) at postnatal day 0. The endogenous tonic I(GABA-A) was seen and it was enhanced by NO-711 in the presence of tetrodotoxin, which itself had only a minor effect on the holding current under control conditions. This indicates that the source of interstitial GABA is largely independent of action-potential activity. The tonic I(GABA-A) in neonatal CA3 pyramidal neurons was increased by zolpidem, indicating that at least a proportion of the underlying GABA(A) receptors contain gamma2 and alpha1-alpha3 subunits. The present data point to a significant role for GAT-1 in the control of the excitability of immature hippocampal neurons and networks.

Polycystin‐1 can interact with homer 1/Vesl‐1 in postnatal hippocampal neurons

Polycystin-1 (PC-1) has been identified as critical to development of the nervous system, but the significance of PC-1 expression in neurons remains undefined, and little is known of its roles outside the kidney, where mutation results in autosomal dominant polycystic kidney disease (ADPKD). In kidney, PC-1 interacts with cadherins, catenins, and its cognate calcium channel polycystin-2 (PC-2), which in turn interacts with a number of actin-regulatory proteins. Because some of the proteins that interact with PC-1 in kidney also participate in synaptic remodeling and plasticity in the hippocampus, we decided to test PC-1's potential to interact with a recently discovered type of plasticity-associated protein (homer 1a/Vesl-1S) in postnatal mouse hippocampus. Homer 1a/Vesl-1S is an activity-induced protein believed to participate in synaptic remodeling/plasticity responses to temporal lobe seizure and learning. Here we report the following. 1) PC-1 contains a homer-binding motif (PPxxF), which lies within its purported cytoplasmic domain. 2) Immunoreactivity for PC-1 (PC-1-ir) is highly colocalized with homer 1a immunoreactivity (H1a-ir) in primary cultured hippocampal neurons. 3) PC-1-ir and H1a-ir are present and appear to be colocalized in mouse hippocampus but not cortex on postnatal day 2 (P2), when higher frequencies of spontaneous activity are normal for hippocampus compared with cortex. 4) An endogenous PC-1-ir band with the correct size for the reported C-terminal G-protein-sensitive domain cleavage product of PC-1 (approximately 150 kDa) coimmunoprecipitates with endogenous homer 1/Vesl-1 proteins from mouse brain, suggesting that PC-1 can interact with homer 1/Vesl-1 proteins in postnatal hippocampal neurons.

Microglia Kv1.3 Channels Contribute to Their Ability to Kill Neurons

Many CNS disorders involve an inflammatory response that is orchestrated by cells of the innate immune system: macrophages, neutrophils, and microglia (the endogenous CNS immune cell). Hence, there is considerable interest in anti-inflammatory strategies that target these cells. Microglia express Kv1.3 (KCNA3) channels, which we showed previously are important for their proliferation and the NADPH-mediated respiratory burst. Here, we demonstrate the potential for targeting Kv1.3 channels to control CNS inflammation. Rat microglia express Kv1.2, Kv1.3, and Kv1.5 transcripts and protein, but only a Kv1.3 current was detected. When microglia were activated with lipopolysaccharide or a phorbol ester, only the Kv1.3 transcript (but not protein) expression changed. Using a Transwell cell-culture system that allows separate drug treatment of microglia or neurons, we found that activated microglia killed postnatal hippocampal neurons through a process that requires Kv1.3 channel activity in microglia but not in neurons. A major neurotoxic molecule in this model was peroxynitrite, which is formed from superoxide and nitric oxide; thus, it is significant that Kv1.3 channel blockers reduced the respiratory burst, but not nitric oxide production, by the activated microglia. In addressing the biochemical pathway affected by Kv1.3 channel activity, we found that Kv1.3 acts via a different cellular mechanism from the broad-spectrum drug minocycline, which is often used in animal models of neuroinflammation. That is, the dose-dependent reduction in neuron killing by minocycline corresponded with a reduction in p38 mitogen-activated protein kinase activation in microglia; however, none of the Kv1.3 blockers affected p38 activation.