Embryonic Rat Hippocampal Neurons Research Articles

Steady-state intracellular pH and recovery from NH3/NH4+-induced intracellular acid loads in primary cultures of rat hippocampal neurons and astrocytes

Intracellular pH (pH i ) homeostasis is of vital importance because pH i fluctuations alter the net charge on intracellular weak acids and bases (e.g., amino-acid residues of proteins, metabolic intermediates, solutes like inorganic phosphate), and can result in cellular dysfunction. The rate of pH i change (dpH i /dt) is determined by the balance between acid loading (e.g., metabolic production of H + , influx of H + , efflux of HCO 3 − via transmembrane proteins of the SLC4 family) and acid extrusion (e.g., efflux of H + via Na-H exchangers in the SLC9 family, influx of HCO 3 − by members of the SLC4 family). A powerful approach for studying pH i regulation is to impose an acute intracellular acid load using the NH 4 + /NH 3 prepulse technique, and then assess dpH i /dt during the subsequent pH i recovery. Using two such prepulses and recoveries (twin pulses), one can compare 2 experimental conditions. The goal of the present study is to develop a data-analysis approach that facilitates a meaningful presentation of all twin-pulse experiments in a data set. Using primary cultures (~2 weeks) of embryonic (E18–E20) rat hippocampal neurons and astrocytes on coverslips, we loaded cells with the ratiometric pH-sensitive fluorescent dye BCECF, subjected the cells to twin NH 4 + /NH 3 pulses, and calibrated BCECF at the end of each experiment with nigericin at pH=7.0. A standard 12-point nigericin calibration curve allowed us to compute pH i vs. time. Because we studied cells in 21% O 2 but the nominal absence of CO 2 /HCO 3 − , the pH i recoveries mainly reflect Na-H exchange. We fitted the pH i -recovery time course with a double exponential (DExp) to reduce bias. Nevertheless, in both neurons and astrocytes plots of dpH i /dt vs. pH i appeared rather linear, indicating that the pH i recoveries were “quasi” single exponential (SExp). To exploit the near-linearity, for each pH i recovery, we fitted the plot of dpH i /dt vs pH i with a line, the slope of which is the quasi-single exponential rate constant (kqSExp), and the x-intercept of which is the asymptotic pH i of the recovery. We summarize the twin pH i recoveries from individual cells as miniature plots (“thumbnails”) in which we display, for each recovery, both the dpH i /dt values (from the DExp fit of pH i vs. time) vs. pH i and the line of best fit for these dpH i /dt values vs. pH i . A single figure can replicate such twin-pulse experiments from many dozens of cells, so that a reader, at a glance, can surmise the essentials of each pH i recovery. The present work provides background data for additional work in which we studied pH i recoveries at a reduced [O 2 ], with or without CO 2 /HCO 3 − . R01 NS018400, R01 HL160857, R01 DK128315 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ashwagandha and Its Active Ingredient, Withanolide A, Increase Phosphorylation of TrkB in Cultured Hippocampal Neurons

Aims: To primary rat embryonic hippocampal neurons in culture, ashwagandha or one of its active ingredients, withanolide A were added in the presence or absence of nutrient supplementation and then assayed for activity of the BDNF receptor, TrkB.
 Study Design:  Primary hippocampal neurons were cultured and grown in nutrient-rich or nutrient-poor medium.  Ashwagandha or withanolide A were then be added to both types of media with or without an inhibitor of TrkB or either the PI-3K or MAPK pathway.
 Place and Duration of Study: Department of Biological Sciences, California State University, Los Angeles, CA, USA, between July 2021 and August 2022.
 Methodology: Rat embryos were removed by cesarean section from mother rats at 18 days’ gestation and the hippocampi of the former dissected, plated into culture dishes, and treated with the appropriate drug(s) (see Study Design above).  After 4 days, neurons were harvested for Western blotting.  Optical density of Western blot bands were quantified and statistically analyzed in a 2-way ANOVA, using a level of statistical significance at P < .05.
 Results: Under normal conditions (with N2 supplement), ashwagandha, but not withanolide A, increased phospho-TrkB immunoreactivity when compared to the effects of vehicle (controls, F(11, 24) = 22.48, P < .001), although withanolide A did not quite reach statistical significance (P = .069) when compared to that of the controlled condition.  Likewise, under nutrient-deprived conditions, both ashwagandha and withanolide A also increased phosphorylation of TrkB when compared to that of vehicle-nutrient-deprived conditions (P < .0001). The same results were obtained in the presence of inhibitors of TrkB itself and the PI-3K (ashwagandha, P < .001; withanolide A, P < .001) and MAPK (ashwagandha, P = .027; withanolide A, P = .045) pathways.
 Conclusion: Ashwagandha or withanolide A activates TrkB, in nutrient-deprived hippocampal neurons, underscoring its role in neuronal survival signaling.

Pyramidal neuron morphogenesis requires a septin network that stabilizes filopodia and suppresses lamellipodia during neurite initiation

Pyramidal neurons are a major cell type of the forebrain, consisting of a pyramidally shaped soma with axonal and apicobasal dendritic processes. It is poorly understood how the neuronal soma develops its pyramidal morphology, while generating neurites of the proper shape and orientation. Here, we discovered that the spherical somata of immature neurite-less neurons possess a circumferential wreath-like network of septin filaments, which promotes neuritogenesis by balancing the protrusive activity of lamellipodia and filopodia. In embryonic rat hippocampal and mouse cortical neurons, the septin wreath network consists of curvilinear filaments that contain septins 5, 7, and 11 (Sept5/7/11). The Sept5/7/11 wreath network demarcates a zone of myosin II enrichment and Arp2/3 diminution at the base of filopodial actin bundles. In Sept7-depleted neurons, cell bodies are enlarged with hyperextended lamellae and abnormally shaped neurites that originate from lamellipodia. This phenotype is accompanied by diminished myosin II and filopodia lifetimes and increased Arp2/3 and lamellipodial activity. Inhibition of Arp2/3 rescues soma and neurite phenotypes, indicating that the septin wreath network suppresses the extension of lamellipodia, facilitating the formation of neurites from the filopodia of a consolidated soma. We show that this septin function is critical for developing a pyramidally shaped soma with properly distributed and oriented dendrites in cultured rat hippocampal neurons and invivo in mouse perinatal cortical neurons. Therefore, the somatic septin cytoskeleton provides a key morphogenetic mechanism for neuritogenesis and the development of pyramidal neurons.

Accelerating Neurite Growth and Directing Neuronal Connections Constrained by 3D Porous Microtubes.

Investigation of neural growth and connection is crucial in the field of neural tissue engineering. Here, using a femtosecond laser direct writing (fs-DLW) technique, we propose a directionally aligned porous microtube array as a culture system for accelerating the growth of neurons and directing the connection of neurites. These microtubes exhibited an unprecedented guidance effect toward the outgrowth of primary embryonic rat hippocampal neurons, with a wrap resembling the myelin sheaths of neurons. The speed of neurite growth inside these microtubes was significantly faster than that outside these microtubes. We also achieved selective/directing connection of neural networks inside the magnetic microtubes via precise microtube delivery to a gap between two neural clusters. This work not only proposes a powerful microtube platform for accelerated growth of neurons but also offers a new idea for constructing biological neural circuits by arranging the size, location, and pattern of microtubes.

Semi-Automated Quantitative Evaluation of Neuron Developmental Morphology In Vitro Using the Change-Point Test.

Neuron morphology gives rise to distinct axons and dendrites and plays an essential role in neuronal functionality and circuit dynamics. In rat hippocampal neurons, morphological development occurs over roughly one week in vitro. This development has been qualitatively described as occurring in 5 stages. Still, there is a need to quantify cell growth to monitor cell culture health, understand cell responses to sensory cues, and compare experimental results and computational growth model predictions. To address this need, embryonic rat hippocampal neurons were observed in vitro over six days, and their processes were quantified using both standard morphometrics (degree, number of neurites, total length, and tortuosity) and new metrics (distance between change points, relative turning angle, and the number of change points) based on the Change-Point Test to track changes in path trajectories. Of the standard morphometrics, the total length of neurites per cell and the number of endpoints were significantly different between 0.5, 1.5, and 4 days in vitro, which are typically associated with Stages 2-4. Using the Change-Point Test, the number of change points and the average distance between change points per cell were also significantly different between those key time points. This work highlights key quantitative characteristics, both among common and novel morphometrics, that can describe neuron development in vitro and provides a foundation for analyzing directional changes in neurite growth for future studies.

Chlorinated disinfection byproducts of diazepam perturb cell metabolism and induce behavioral toxicity in zebrafish larvae

Numerous byproducts resulting from chlorinated disinfection are constantly being generated during water treatment processes. The potential risks of these new emerging pollutions remain largely unknown. Here, we determined the risks of chlorinated disinfection byproducts of diazepam (DZP) in the cellular and zebrafish exposure experiments. The cytotoxicity of disinfection byproducts (MACB and MBCC) was greater than DZP in macrophage raw 264.7 cells at 10 mg/L. We further found that the effects of MBCC on the metabolism of glycine, serine, threonine and riboflavin were far greater than DZP by the targeted metabolomics methods. Moreover, MBCC significantly decreased the peak amplitude of neuronal action potential in primary embryonic rat (Spragu-Dawley SD) hippocampal neurons. We finally determined behavioral toxicity of DZP and byproducts in zebrafish larvae. MBCC significantly decreased the maximal swim-activity and peak duration of zebrafish after 72 h exposure. Altogether, these findings indicate the MBCC pose serious pressures on public health.

The selective GSK3 inhibitor, SAR502250, displays neuroprotective activity and attenuates behavioral impairments in models of neuropsychiatric symptoms of Alzheimer\u2019s disease in rodents

Glycogen synthase kinase 3 (GSK3) has been identified as a promising target for the treatment of Alzheimer’s disease (AD), where abnormal activation of this enzyme has been associated with hyperphosphorylation of tau proteins. This study describes the effects of the selective GSK3 inhibitor, SAR502250, in models of neuroprotection and neuropsychiatric symptoms (NPS) associated with AD. In P301L human tau transgenic mice, SAR502250 attenuated tau hyperphosphorylation in the cortex and spinal cord. SAR502250 prevented the increase in neuronal cell death in rat embryonic hippocampal neurons following application of the neurotoxic peptide, Aβ25–35. In behavioral studies, SAR502250 improved the cognitive deficit in aged transgenic APP(SW)/Tau(VLW) mice or in adult mice after infusion of Aβ25–35. It attenuated aggression in the mouse defense test battery and improved depressive-like state of mice in the chronic mild stress procedure after 4 weeks of treatment. Moreover, SAR502250 decreased hyperactivity produced by psychostimulants. In contrast, the drug failed to modify anxiety-related behaviors or sensorimotor gating deficit. This profile confirms the neuroprotective effects of GSK3 inhibitors and suggests an additional potential in the treatment of some NPS associated with AD.

Spectroscopic Identifications, Molecular Docking, Neuronal Growth and Enzyme Inhibitory Activities of Steroidal Nitro Olefin: Quantum Chemical Study

AbstractFT‐IR, NMR and ultraviolet spectroscopy by means of DFT / B3LYP‐6‐311G(d, p), molecular docking and biological activity of 6‐nitrocolest‐5‐en‐3‐beta‐yl acetate (steroid nitro‐olefin) was reported in this article. The optimized molecular geometric structure and its associated parameters including vibrational frequencies, carbon and 1H NMR data of the title compound in ground electronic state was obtained at DFT approach applying B3LYP/6‐311G(d,p). The ultraviolet absorbance data is obtained at TDB3LYP/6‐311G(d,p) basis set. The present computational data are found in reasonable agreement with the experimental results. Simulated electronic and NMR (1H and 13C) spectra have been reported in solution phase. Parameters like thermodynamic quantities, dipole moment, FMO energies, MEP and global reactivity descriptors of steroid nitro olefin were determined at same level of theory. The neuromodulatory effect of the compound on central nervous system development of rat embryonic hippocampal neurons has been studied. Based on the outcome; compound demonstrated suppression activity despite the promotion of neuronal cytoarchitecture leading to a negative effect on hippocampal neurons. The nature of the compound found to be toxic to the cells. Besides, steroid compound is also used to perform inhibitory activity against acetylcholinesterase (AChE) compared to the reference drug i. e. Galanthamine. The molecular docking of the synthesized compound is carried out with the structure of the acetylcholinesterase (PDB: 1DX6) protein to check the mode of interactions.

Varying Effects of the G‐Protein Coupled Estrogen Receptor During Development in Embryonic Rat Hippocampal and Cortical Neurons

Estrogen has been known to play a role in enhancing brain development for over half a century. Most studies of estrogen's effect on the nervous system have focused on the traditional estrogen receptors Estrogen Receptor‐α (ERα) and Estrogen Receptor‐β (ERβ). The recent discovery of the G‐Protein Coupled Estrogen Receptor (GPER) suggests knowledge of estrogen's effects may be incomplete. While evidence has suggested the roles GPER plays in the nervous system include neuroprotection, neuronal recovery from stroke and degeneration, neuron proliferation, as well as many others. A caveat is that most studies have looked only at the effect in mature neurons and not the effect GPER has during early neuronal development. Here we sought to study the role of GPER activity in neurite outgrowth and synapse formation during early development.Embryonic day 18 rat cortical and hippocampal neurons were cultured in estrogen‐ and phenol red‐ free medium in the presence of a nonselective ER agonist (E2), a selective GPER agonist (G‐1), and a selective GPER antagonist (G‐15). Neurite growth was measured at 20, 48, 72, and 96 hours in culture using the ImageJ plugin NeuronJ. Synapses were visualized at 7 and 14 days in culture by using immunofluorescence of the pre‐ and post‐synaptic markers, synaptophysin and PSD‐95, respectively. Synapse formation was measured by measuring the overlap between these markers in ImageJ. Our data revealed activation of GPER promoted neurite growth in hippocampal neurons, but not in cortical neurons, but blocking the receptor inhibited neurite growth in cortical neurons. In addition, low levels of G1 (1μM) inhibited neurite outgrowth in cortical neurons. Cortical neurons showed no significant difference in synapse formation, while in hippocampal neurons the effect varied based on time period and dosage. This provides the first evidence that GPER activation may play different roles in brain regions, as well as having specific dosage based effects in specific regions. Future experiments will investigate the molecular and cellular mechanisms underlying the pharmacological differences in GPER effects between developing cortical and hippocampal neurons.Support or Funding InformationThis project was supported by the Saint Louis University Start‐up Fund for Dr. Xu lab and the Sigma XI Grant‐In‐Aid of Research awarded to Mr. Kyle Pemberton.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Therapeutic Assessment and Neuroprotective Effects of a Novel G Protein‐Coupled Estrogen Receptor (GPR30/GPER) Agonist

The G protein‐coupled estrogen receptor (GPER) has been implicated in the neuroprotective effects of 17β‐estradiol and selective GPER agonist, G‐1. Currently, G‐1 remains the standard for GPER agonists, necessitating the design and identification of novel candidates. Previously, our group synthesized and biologically evaluated a series of GPER antagonists. Modification of an aromatic moiety on the scaffold altered the pharmacological profile from antagonists to agonists. Using a fluorescence‐based binding assay for the classical estrogen receptors, ERa and ERb, we discovered off‐target binding. Due to the similarity in functionalization to estradiol, we hypothesized that the hydrogen bonding abilities of the initial candidate were responsible for the observed off‐target binding. To mitigate off‐target binding, modifications were made to reduce the hydrogen bonding ability. For each resulting candidate, the EC50 value was determined through in vitro biochemical screening. From these studies, we identified a lead compound that exhibited an EC50 value below that of our experimentally determined EC50 value of G‐1. Using E18 rat embryonic hippocampal neurons, the lead compound was shown to increase synaptogenesis. These results suggest that the identified lead compound for GPER may be a potential candidate for neurodegenerative diseases.Support or Funding InformationSaint Louis UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The MAP1B Binding Domain of Nav1.6 Is Required for Stable Expression at the Axon Initial Segment.

Nav1.6 (SCN8A) is a major voltage-gated sodium channel in the mammalian CNS, and is highly concentrated at the axon initial segment (AIS). As previously demonstrated, the microtubule associated protein MAP1B binds the cytoplasmic N terminus of Nav1.6, and this interaction is disrupted by the mutation p.VAVP(77-80)AAAA. We now demonstrate that this mutation results in WT expression levels on the somatic surface but reduced surface expression at the AIS of cultured rat embryonic hippocampal neurons from both sexes. The mutation of the MAP1B binding domain did not impair vesicular trafficking and preferential delivery of Nav1.6 to the AIS; nor was the diffusion of AIS inserted channels altered relative to WT. However, the reduced AIS surface expression of the MAP1B mutant was restored to WT levels by inhibiting endocytosis with Dynasore, indicating that compartment-specific endocytosis was responsible for the lack of AIS accumulation. Interestingly, the lack of AIS targeting resulted in an elevated percentage of persistent current, suggesting that this late current originates predominantly in the soma. No differences in the voltage dependence of activation or inactivation were detected in the MAP1B binding mutant relative to WT channel. We hypothesize that MAP1B binding to the WT Nav1.6 masks an endocytic motif, thus allowing long-term stability on the AIS surface. This work identifies a critical and important new role for MAP1B in the regulation of neuronal excitability and adds to our understanding of AIS maintenance and plasticity, in addition to identifying new target residues for pathogenic mutations of SCN8ASIGNIFICANCE STATEMENT Nav1.6 is a major voltage-gated sodium channel in human brain, where it regulates neuronal activity due to its localization at the axon initial segment (AIS). Nav1.6 mutations cause epilepsy, intellectual disability, and movement disorders. In the present work, we show that loss of interaction with MAP1B within the Nav1.6 N terminus reduces the steady-state abundance of Nav1.6 at the AIS. The effect is due to increased Nav1.6 endocytosis at this neuronal compartment rather than a failure of forward trafficking to the AIS. This work confirms a new biological role of MAP1B in the regulation of sodium channel localization and will contribute to future analysis of patient mutations in the cytoplasmic N terminus of Nav1.6.

Structural and biological evaluation of a platinum complex as a potential anti-neurodegenerative agent

A novel mononuclear platinum complex with N2O2 donor atoms was synthesized from the ligand, [N,N′-bis(3-methoxysalicylidene)-2,2-dimethylpropane-1,3-diamine], H2L. The complex was characterized by microanalysis (C, H, N), spectroscopic studies and single crystal X-ray crystallography. The TGA study revealed that the complex degraded in two distinct steps. The effect of the platinum complex was examined by exposing the synthesized complex to rat embryonic hippocampal neurons. The results exhibited significant neuritogenesis at low doses, suggesting it to be a potential therapeutic candidate for combating the neurodegenerative diseases.

Mechanisms Underlying Neuroprotection by the NSAID Mefenamic Acid in an Experimental Model of Stroke

Stroke is a devastating neurological event with limited treatment opportunities. Recent advances in understanding the underlying pathogenesis of cerebral ischemia support the involvement of multiple biochemical pathways in the development of the ischemic damage. Fenamates are classical non-steroidal anti-inflammatory drugs but they are also highly subunit-selective modulators of GABAA receptors, activators of IKS potassium channels and antagonists of non-selective cation channels and the NLRP3 inflammosome. In the present study we investigated the effect of mefenamic acid (MFA) in a rodent model of ischemic stroke and then addressed the underlying pharmacological mechanisms in vitro for its actions in vivo. The efficacy of MFA in reducing ischemic damage was evaluated in adult male Wistar rats subjected to a 2-h middle cerebral artery occlusion. Intracerebroventricular (ICV) infusion of MFA (0.5 or 1 mg/kg) for 24 h, significantly reduced the infarct volume and the total ischemic brain damage. In vitro, the fenamates, MFA, meclofenamic acid, niflumic acid, and flufenamic acid each reduced glutamate-evoked excitotoxicity in cultured embryonic rat hippocampal neurons supporting the idea that this is a drug class action. In contrast the non-fenamate NSAIDs, ibuprofen and indomethacin did not reduce excitotoxicity in vitro indicating that neuroprotection by MFA was not dependent upon anti-inflammatory actions. Co-application of MFA (100 μM) with either of the GABAA antagonists picrotoxin (100 μM) or bicuculline (10 μM) or the potassium channel blocker tetraethylammonium (30 mM) did not prevent neuroprotection with MFA, suggesting that the actions of MFA also do not depend on GABAA receptor modulation or potassium channel activation. These new findings indicate that fenamates may be valuable in the adjunctive treatment of ischemic stroke.

Assessing seizure liability using multi-electrode arrays (MEA)

The purpose of these studies was to develop ex vivo tissue-based and in vitro cell-based assays using multi-electrode array (MEA) technology to predict seizure liability at the early stage of preclinical studies. Embryonic rat hippocampal neurons and adult rat hippocampal slices were used in these studies. Spontaneous activity in cultured neurons and evoked field potentials in hippocampal brain slices were recorded using MEA technology. Six seizurogenic compounds bicuculline, pentylenetetrazole, picrotoxin, gabazine, 4-Aminopyridine and BMS-A increased field potential area and peak number in brain slices and spontaneous spike activity in hippocampal neurons. Physostigmine, another seizurogenic compound, had no effect on brain slices at lower concentrations (0.1, 1, and 10 μM), and mildly increased field potential area at 100 μM. However, physostigmine induced multiple peaks in evoked field potential starting at 10 μM. Physostigmine showed greater potency in the cultured neuron assay, and increased spike rates in the nanomolar range. Two seizurogenic compounds, BMS-B and BMS-C increased the spontaneous activity in hippocampal neurons, but did not increase area and peak number of field potentials in brain slices. These findings suggest that MEA technology and rat hippocampal brain slices or rat embryonic hippocampal neurons, may be useful as early, predictive in vitro assays for seizure liability.

Abstract TP264: Actin Reorganization is a Rapid and Reversible Adaptive Response by Neurons Exposed to Ischemic and Excitotoxic Injury in vitro and in vivo

In initial stages of ischemic injury, glutamate receptor hyperactivation mediates extensive neuronal cell death. Most of this initial death is due to cytotoxic edema, whereby sodium and chloride entry cause cell swelling. Early subcellular responses to neuronal edema are poorly understood. We observed that actin filaments (F-actin) undergo a dramatic reorganization within the somatodendritic domain of neurons exposed to ischemia and/or sublethal excitotoxicity in vivo and in vitro. Fluorescence imaging was performed on embryonic rat hippocampal neurons cultured for 21-days in vitro. “Ministrokes” were induced in vivo by photothrombosis of a single penetrating vessel in mouse sensorimotor cortex. Animals were fixed 4 or 6 hours after vessel occlusion. Fluorescence images were analyzed in infarct, peri-infarct, and contralateral regions. All experiments were repeated 3-4 times, and quantified in a blinded fashion In control tissue F-actin is concentrated within dendritic spines, with little F-actin in the dendrite shaft. Within 5 min of applying 50μM NMDA, F-actin is lost from spines (inducing spine shrinkage) and polymerizes into long filament bundles within the dendrite shaft and soma. A similar redistribution of actin occurs after oxygen/glucose deprivation in vitro and after stroke in vivo. In vitro these actin changes are reversible within 1-3 hours of transient NMDA exposure. Increased Ca2+ is necessary but not sufficient for iniduction. Conditions that reduce cytotoxic edema prevent actinification; conditions that promote cytotoxic edema induce actinfication. The actin nucleator inverted formin-2 (INF2) was found to mediate dendrite actinification. Pharmacological inhibition of formins but not Arp2/3, completely prevented actinification. Moreover, silencing of endogenous INF2 using shRNA prevented NMDA-induced actinification; overexpression of wildtype INF2 sensitized neurons to actinification. Silencing of INF2 rendered neurons more vulnerable to NMDA-induced cell death. Results indicate that ischemia-induced actin filament accumulation within the somatodendritic compartment is a pro-survival response that protects neurons from death induced by cell swelling.

Three dimensional microenvironments on multi-electrode arrays produce neuronal networks that function like the brain

Event Abstract Back to Event Three dimensional microenvironments on multi-electrode arrays produce neuronal networks that function like the brain Justin L. Bourke1, 2, 3*, Anita Quigley1, 2, 3, 4, Cathal O'Connell2, 3, Jeremy Crook2, 4, Gordon Wallace2, 4, Mark Cook1, 2, 3 and Robert Kapsa1, 2, 3, 4 1 University of Melbourne, Department of Clinical Neurosciences, St Vincent's Hospital, Australia 2 Australian Research Council Centre of Excellence for Electromaterials Science, Australia 3 St Vincent's Hospital (Melbourne), Department of Medicine, Australia 4 University of Wollongong, Intelligent Polymer Research Institute, Australia Introduction: Engineered 3D constructs for cell culture mimic in vivo conditions better than traditional 2D substrates, resulting in controlled cellular growth patterns[1] and improved cell function[2-5]. More critically from a tissue engineering perspective, such 3D substrates may improve intercellular interactions and thus whole-tissue and organoid functionality. Previous studies have generated pseudo-3D neuronal networks using beads to separate neuronal positioning[6], with MEA recordings providing insight into changes in function from such substrate designs[7-8]. Functional network differences exist between these bead-based substrate networks and traditional 2D neuronal networking, although the authors point out the limited inter-layer connectivity, with 7 times more neuronal interconnectivity within layers than between due to neurites extending only on bead surfaces. We have developed a true 3D networking model based on collagen where neurite positioning and connectivity is not limited by the substrate. We used microelectrode arrays to investigate functional differences between neuronal networks formed in 2D and 3D, and determined that 3D cultures produce neuronal network function more akin to networks of the intact brain. Methods: Embryonic rat hippocampal neurons were cultured in 2D and in 3D collagen gels upon microelectrode arrays (Multichannel Systems). Cultures were maintained for 56 days, with electrophysiological recordings taken every 7 days. Neuronal cell function at each electrode and network activity between electrodes were compared between 2D and 3D cultures. Results and Discussion: Neural activation originated from spontaneously active pace-making regions within both 2D and 3D cultures. In 2D, activity flowed directly across the planar cultures resulting in a short repetitive and predictable spatial and temporal bursting pattern, with one burst at each electrode per activation of the pace-making region (Fig 1a). In contrast, the 3D constructs produced regular oscillatory activity throughout the higher order neural networks, resulting in spiking patterns of increased burst duration (Fig 1b), a pattern that more closely mimics network activity within the intact brain. Synchronous activation was observed throughout both 2D and 3D networks, indicating network connectivity throughout the cultures. Previous bead-based materials produce network functionality with both short interval regular spiking and longer duration bursts, a mix of network function observed in 2D and true 3D collagen networks observed within this study. The results observed illustrate the importance of a true 3D neuronal network microenvironment to producing network function akin to the in vivo state. Conclusions: Mimicking the natural microenvironment in cell culture applications is critical to achieving more natural single-cell function. In addition, characterisation of intercellular interactions within such tissue engineered constructs is critical to understanding whole-tissue and organoid level functionality. From a neural construct perspective, we have shown that neural network formation and function in three-dimensions mimics network function of the intact brain more accurately than conventional two-dimensional cultures. Measurement of neural network function at the whole-tissue level in three-dimensional neural cultures will optimise outcomes for network studies and allow for investigation of mechanisms behind neural network formation and neural networking disorders such as epilepsy and schizophrenia. Paired with neurons derived from induced pluripotent stem cells from patients, the findings here will facilitate a personalised medicine approach to drug efficacy studies for individuals suffering epilepsy and schizophrenia. This presentation: This presentation will concentrate on the yet to be published methods to generate such 3D networks, analysis of the networking activity, and work currently underway in applying such networks to a clinical model for drug selection for patients suffering from epilepsy. Figure 1: Typical single electrode recordings in (Ai) 2D and (bi) 3D cultures (adapted from Bourke et al [5]). Synchronicity within raster plots of spike times across multiple electrode in (Aii) 2D and (Bii) 3D cultures indicate fully connected neural networks in both culture conditions. Note the fast spiking patterns in 2D cultures and slower, highly consistent bursting patterns in 3D networks. Figure 1 Acknowledgements The authors wish to acknowledge financial support from the Australian Research Council (ARC) Project CE140100012, the ARC Centre of Excellence for Electromaterials Science, and National Health and Medical Research Council project grant 1062569.

Alleviation of Hippocampal Endoplasmic Reticulum Stress by Allomyrina dichotoma Larvae Extract.

In the brain, endoplasmic reticulum (ER) stress results in synaptic dysfunction and eventually leads to neurodegeneration. Allomyrina dichotoma larvae are a Chinese ethnomedicine and are widely used in East Asia. In the present study, we investigated the ability of ethanol extract of A. dichotoma larvae (ADE) to improve synaptic structure and function by activating unfolded protein response (UPR) under ER stress in animal and neuron culture models. ER stress was induced in obese mice fed a high fat diet (HFD) or by treating dissociated cultures of rat embryonic (E19) hippocampal neurons with tunicamycin (TM). Western blot and real-time or conventional RT-PCR were performed to analyze the expressions of ER stress marker proteins. In dissociated hippocampal cultures, immunocytochemistry was performed for synaptic proteins, and cultures were stained with styryl dye FM1-43 to assess presynaptic activities. In HFD-fed obese mice, ADE efficiently reduced the expressions of ER stress markers, such as, xbp-1, chop, atf4, erdi4, and eIf2a, and those of the ER chaperone/foldases Bip/grp78, Ero-1l, and PDI. Unconventionally spliced xbp-1s mRNA was not detected. In primary rat hippocampal cultures under ER stress, ADE significantly lowered the nuclear expression of CHOP, inhibited the downregulations of postsynaptic proteins, such as, GluN2A, GluN2B, and PSD-95, and maintained the pool size of recycling presynaptic vesicles. The study shows that ADE potently suppressed the induction of ER stress and maintained the structure and function of hippocampal neurons, and suggests that ADE is a potentially valuable food supplement and preventive therapeutic for ER stress-related nervous disorders.

Antagonistic action on NMDA/GluN2B mediated currents of two peptides that were conantokin-G structure-based designed

BackgroundThe GluN2B subunit of the N-methyl-d-aspartate receptor (NMDAr) modulates many physiological processes including learning, memory, and pain. Excessive increase in NMDAr/GluN2B activity has been associated with various disorders such neuropathic pain and neuronal death following hypoxia. Thus there is an interest in identifying NMDAr antagonists that interact specifically with the GluN2B subunit. Recently based on structural analysis between the GluN2B subunit and conantokin-G, a toxin that interacts selectively with the GluN2B subunit, we designed various peptides that are predicted to act as NMDAr antagonists by interacting with the GluN2B subunit. In this study we tested this prediction for two of these peptides EAR16 and EAR18.ResultsThe effects of EAR16 and EAR18 in NMDA-evoked currents were measured in cultured rat embryonic hippocampal neurons and in HEK-293 cells expressing recombinant NMDAr comprised of GluN1a–GluN2A or GluN1a–GluN2B subunits. In hippocampal neurons, EAR16 and EAR18 reduced the NMDA-evoked calcium currents in a dose-dependent and reversible manner with comparable IC50 (half maximal inhibitory concentration) values of 241 and 176 µM, respectively. At 500 µM, EAR16 blocked more strongly the NMDA-evoked currents mediated by the GluN1a–GluN2B (84%) than those mediated by the GluN1a–GluN2A (50%) subunits. At 500 µM, EAR18 blocked to a similar extent the NMDA-evoked currents mediated by the GluN1a–GluN2B (62%) and the GluN1a–GluN2A (55%) subunits.ConclusionsThe newly designed EAR16 and EAR18 peptides were shown to block in reversible manner NMDA-evoked currents, and EAR16 showed a stronger selectivity for GluN2B than for GluN2A.

Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons

This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over time since the neuronal culture medium provides the nutrients and trophic factors required for differentiation. In this protocol we describe the methodology for the preparation of both cortical and hippocampal neuronal cultures.

First and second generation antipsychotics differentially affect structural and functional properties of rat hippocampal neuron synapses

The therapy of patients suffering the psychiatric disorder schizophrenia requires the usage of antipsychotic drugs that are classified into two different groups, the first-generation (FGAs) and the second-generation antipsychotics (SGAs).This study compares the effects of the two FGAs haloperidol and flupentixol with those of the SGA olanzapine on synapse formation and synaptic activity of embryonic rat hippocampal neurons. To this end, the development of perineuronal nets (PNNs), the formation of synapses and the resulting spontaneous network activity under control and treatment conditions were studied using an indirect co-culture system of neurons and astrocytes in completely defined media.The number and extent of PNNs that consist of extracellular matrix superstructures surrounding synapses was not altered in hippocampal neurons by exposure to antipsychotic drugs. In contrast treatment of hippocampal neurons with haloperidol led to a slight decrease whereas olanzapine induced a significant increase of the number of structural synapses after 13days. This differential effect concerning synapse numbers was also reflected in the spontaneous activity of neuronal networks, as monitored on multielectrode arrays (MEAs). In that context, application of haloperidol reduced while olanzapine significantly enhanced network activity. Unexpectedly, flupentixol that is regarded as an FGA caused similar effects than the SGA olanzapine in that it augmented synapse number as well as network activity. Our pilot study provides a proof of concept that the neuron-astrocyte co-culture model can be used to investigate the impact of antipsychotics on pivotal parameters of neuronal cell biology. Thereby, it may support the comparative analysis of antipsychotics applied in the therapy of schizophrenia.