Rat Hippocampal Cell Cultures Research Articles

SARS-CoV-2 Viroporin E Induces Ca2+ Release and Neuron Cell Death in Primary Cultures of Rat Hippocampal Cells Aged In Vitro.

The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and neurological dysfunctions. The SARS-CoV-2 envelope protein (E protein) is a structural viroporin able to form ion channels in cell membranes, which is critical for viral replication. However, its effects in primary neurons have not been addressed. Here we used fluorescence microscopy and calcium imaging to study SARS-CoV-2 viroporin E localization and the effects on neuron damage and intracellular Ca2+ homeostasis in a model of rat hippocampal neurons aged in vitro. We found that the E protein quickly enters hippocampal neurons and colocalizes with the endoplasmic reticulum (ER) in both short-term (6-8 days in vitro, DIV) and long-term (20-22 DIV) cultures resembling young and aged neurons, respectively. Strikingly, E protein treatment induces apoptosis in aged neurons but not in young neurons. The E protein induces variable increases in cytosolic Ca2+ concentration in hippocampal neurons. Ca2+ responses to the E protein are due to Ca2+ release from intracellular stores at the ER. Moreover, E protein-induced Ca2+ release is very small in young neurons and increases dramatically in aged neurons, consistent with the enhanced Ca2+ store content in aged neurons. We conclude that the SARS-CoV-2 E protein quickly translocates to ER endomembranes of rat hippocampal neurons where it releases Ca2+, probably acting like a viroporin, thus producing Ca2+ store depletion and neuron apoptosis in aged neurons and likely contributing to neurological damage in COVID-19 patients.

Memantine protects the cultured rat hippocampal neurons treated by NMDA and amyloid β1-42.

Alzheimer's disease (AD) is a devastating neurodegenerative condition with no effective treatments. Recent research highlights the role of NMDA receptors in AD development, as excessive activation of these receptors triggers excitotoxicity. Memantine, an NMDA receptor antagonist, shows promise in curbing excitotoxicity. What sets our study apart is our novel exploration of memantine's potential to protect hippocampal neurons from neurotoxicity induced by NMDA and amyloid β1-42, a hallmark of AD. To achieve this, we conducted a series of experiments using rat hippocampal cell cultures. We employed Hoechst and propidium iodide double staining to assess neuronal viability. Analyzing the viability of neurons in normal conditions compared to their status after 24 h of exposure to the respective agents revealed compelling results. The incubation of hippocampal neurons with NMDA or amyloid β1-42 led to a more than twofold increase in the number of apoptotic and necrotic neurons. However, when memantine was co-administered with NMDA or amyloid β1-42, we witnessed a notable augmentation in the number of viable cells. This unique approach not only suggests that memantine may act as a neuroprotective agent but also emphasizes the relevance of hippocampal neuron cultures as valuable models for investigating excitotoxicity and potential AD treatments.

Of the Mechanisms of Paroxysmal Depolarization Shifts: Generation and Maintenance of Bicuculline-Induced Paroxysmal Activity in Rat Hippocampal Cell Cultures.

Abnormal depolarization of neuronal membranes called paroxysmal depolarization shift (PDS) represents a cellular correlate of interictal spikes. The mechanisms underlying the generation of PDSs or PDS clusters remain obscure. This study aimed to investigate the role of ionotropic glutamate receptors (iGluRs) in the generation of PDS and dependence of the PDS pattern on neuronal membrane potential. We have shown that significant depolarization or hyperpolarization (by more than ±50 mV) of a single neuron does not change the number of individual PDSs in the cluster, indicating the involvement of an external stimulus in PDS induction. Based on this data, we have suggested reliable protocols for stimulating single PDS or PDS clusters. Furthermore, we have found that AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors are necessary for PDS generation since AMPAR antagonist NBQX completely suppresses bicuculline-induced paroxysmal activity. In turn, antagonists of NMDA (N-methyl-D-aspartate) and kainate receptors (D-AP5 and UBP310, respectively) caused a decrease in the amplitude of the first action potential in PDSs and in the amplitude of the oscillations of intracellular Ca2+ concentration occurring alongside the PDS cluster generation. The effects of the NMDAR (NMDA receptor) and KAR (kainate receptor) antagonists indicate that these receptors are involved only in the modulation of paroxysmal activity. We have also shown that agonists of some Gi-coupled receptors, such as A1 adenosine (A1Rs) or cannabinoid receptors (CBRs) (N6-cyclohexyladenosine and WIN 55,212-2, respectively), completely suppressed PDS generation, while the A1R agonist even prevented it. We hypothesized that the dynamics of extracellular glutamate concentration govern paroxysmal activity. Fine-tuning of neuronal activity via action on Gi-coupled receptors or iGluRs paves the way for the development of new approaches for epilepsy pharmacotherapy.

Prolactin-induced neuroprotection against excitotoxicity is mediated via PI3K/AKT and GSK3β/NF-κB in primary cultures of hippocampal neurons

Prolactin (PRL) is a polypeptide hormone that has been reported to play a significant role in neuroprotection against neuronal excitotoxicity produced by glutamate (Glu) or kainic acid (KA) in both, in vitro and in vivo models. However, the molecular mechanisms involved in PRL’s neuroprotective effects in the hippocampus have not been completely elucidated. The aim of the present study was to assess the signaling pathways involved in PRL neuroprotection against excitotoxicity. Primary rat hippocampal neuronal cell cultures were used to assess PRL-induced signaling pathway activation. The effects of PRL on neuronal viability, as well as its effects on activation of key regulatory pathways, phosphoinositide 3-kinases/Protein Kinase B (PI3K/AKT) and glycogen synthase kinase 3β / nuclear factor kappa B (GSK3β/NF-κB), were evaluated under conditions of Glutamate-induced excitotoxicity. Additionally, the effect on downstream regulated genes such as Bcl-2 and Nrf2, was assessed. Here, we show that the PI3K/AKT signaling pathway is activated by PRL treatment during excitotoxicity, promoting neuronal survival through upregulation of active AKT and GSK3β/NF-κB, resulting in induction of Bcl-2 and Nrf2 gene expression. Inhibition of the PI3K/AKT signaling pathway abrogated the protective effect of PRL against Glu-induced neuronal death. Overall, results indicate that the neuroprotective actions of PRL are mediated in part, by the activation of the AKT pathway and survival genes. Our data support the idea that PRL could be useful as a potential neuroprotective agent in different neurological and neurodegenerative diseases.

Issue Information

Front cover Calcium-permeable AMPA receptors (CP-AMPARs) are involved in synaptogenesis and formation of neuronal circuitry. However, these receptors provide additional Ca2+ inflow into neurons, contributing dysregulation of Ca2+ homeostasis and neuronal dysfunction in excitotoxicity associated with numerous CNS disorders. Considering these aspects of Ca2+ activity, we have proposed the method of vital identification of neurons containing CP-AMPARs. Using this method we demonstrate that approximately 80% CP-AMPARs-containing neurons in rat hippocampal cell cultures are GABAergic. As with most GABAergic neurons, GABAergic neurons containing CP-AMPARs demonstrate weaker GABA(A)R-mediated innervation compared to glutamatergic neurons. This peculiarity together with the presence of CP-AMPARs may contribute to vulnerability of these neurons to excitotoxicity. Image content Microphotographs of neurons in rat hippocampal neuron-glial culture stained with antibodies against GABA (red) and a marker of a GABAergic synapse, vesicular GABA transporter (green puncta). The panel shows the distribution of GABAergic synapses on two glutamatergic neurons (left image) and GABAergic neuron with (right image) and without CP-AMPARs (middle image). Read the full article ‘A novel approach for vital visualization and studying of neurons containing Ca2+-permeable AMPA receptors’ by S. G. Gaidin, S. A. Maiorov, D. P. Laryushkin, V. P. Zinchenko and A. M. Kosenkov, (J. Neurochem. 2023, vol.164 (5), pp. 583–587) on doi:10.1111/jnc.15729

Magnetic-field-synchronized wireless modulation of neural activity by magnetoelectric nanoparticles

The in vitro study demonstrates wirelessly controlled modulation of neural activity using magnetoelectric nanoparticles (MENPs), synchronized to magnetic field application with a sub-25-msec temporal response. Herein, MENPs are sub-30-nm CoFe2O4@BaTiO3 core-shell nanostructures. MENPs were added to E18 rat hippocampal cell cultures (0.5 μg of MENPs per 100,000 neurons) tagged with fluorescent Ca2+ sensitive indicator cal520. MENPs were shown to wirelessly induce calcium transients which were synchronized with application of 1200-Oe bipolar 25-msec magnetic pulses at a rate of 20 pulses/sec. The observed calcium transients were similar, in shape and magnitude, to those generated through the control electric field stimulation with a 50-μA current, and they were inhibited by the sodium channel blocker tetrodotoxin. The observed MENP-based magnetic excitation of neural activity is in agreement with the non-linear M − H hysteresis loop of the MENPs, wherein the MENPs’ coercivity value sets the threshold for the externally applied magnetic field.

The cell adhesion protein dystroglycan affects the structural remodeling of dendritic spines

Dystroglycan (DG) is a cell membrane protein that binds to the extracellular matrix in various mammalian tissues. The function of DG has been well defined in embryonic development as well as in the proper migration of differentiated neuroblasts in the central nervous system (CNS). Although DG is known to be a target for matrix metalloproteinase-9 (MMP-9), cleaved in response to enhanced synaptic activity, the role of DG in the structural remodeling of dendritic spines is still unknown. Here, we report for the first time that the deletion of DG in rat hippocampal cell cultures causes pronounced changes in the density and morphology of dendritic spines. Furthermore, we noted a decrease in laminin, one of the major extracellular partners of DG. We have also observed that the lack of DG evokes alterations in the morphological complexity of astrocytes accompanied by a decrease in the level of aquaporin 4 (AQP4), a protein located within astrocyte endfeet surrounding neuronal dendrites and synapses. Regardless of all of these changes, we did not observe any effect of DG silencing on either excitatory or inhibitory synaptic transmission. Likewise, the knockdown of DG had no effect on Psd-95 protein expression. Our results indicate that DG is involved in dendritic spine remodeling that is not functionally reflected. This may suggest the existence of unknown mechanisms that maintain proper synaptic signaling despite impaired structure of dendritic spines. Presumably, astrocytes are involved in these processes.

Epileptic seizures of suspected autoimmune origin: a multicentre retrospective study

ObjectiveTo analyse autoantibody status in a well-defined European multicentre cohort of patients with epilepsy of unknown aetiology and to validate the recently proposed Antibody Prevalence in Epilepsy (APE2) and Response...

Design and synthesis of selective and blood-brain barrier-permeable hydroxamate-based gelatinase inhibitors.

Matrix metalloproteinases (MMPs), a family of zinc-containing endopeptidases involved in the degradation of the extracellular matrix, make a major contribution to the progression of a vast number of diseases, such cancer or epilepsy. Although several MMP inhibitors (MMPi) have been developed to date for the treatment of cancer, they have all failed in clinical trials due to lack of efficacy and, most importantly, the presence of severe side effects. The latter can be explained by their lack of selectivity of these inhibitors. In this regard, MMPs' family members have a high structural homology, which challenge the development of selective inhibitors for a specific MMP. Here, we have used in silico calculations and in vitro data to design MMPi that selectively target gelatinases (MMP-2 and MMP-9) and have the capacity to cross the blood-brain barrier. Following this approach, we obtained compound 40 that shows high proteolytic stability and low cytotoxicity. This compound may be of particular interest for the treatment of central nervous diseases such epilepsy or Alzheimer's disease, where gelatinase activity is increased. Our data show the specificity of compound 40 for recombinant MMP-9 and MMP-2 and endogenous MMP-9 from rat hippocampal cell cultures, and reveals its permeability across the blood-brain barrier in vivo.

Time- and polarity-dependent proteomic changes associated with homeostatic scaling at central synapses.

In homeostatic scaling at central synapses, the depth and breadth of cellular mechanisms that detect the offset from the set-point, detect the duration of the offset and implement a cellular response are not well understood. To understand the time-dependent scaling dynamics we treated cultured rat hippocampal cells with either TTX or bicucculline for 2 hr to induce the process of up- or down-scaling, respectively. During the activity manipulation we metabolically labeled newly synthesized proteins using BONCAT. We identified 168 newly synthesized proteins that exhibited significant changes in expression. To obtain a temporal trajectory of the response, we compared the proteins synthesized within 2 hr or 24 hr of the activity manipulation. Surprisingly, there was little overlap in the significantly regulated newly synthesized proteins identified in the early- and integrated late response datasets. There was, however, overlap in the functional categories that are modulated early and late. These data indicate that within protein function groups, different proteomic choices can be made to effect early and late homeostatic responses that detect the duration and polarity of the activity manipulation.

Trkb-IP3 Pathway Mediating Neuroprotection in Rat Hippocampal Neuronal Cell Culture Following Induction of Kainic Acid.

BackgroundFollowing brain injury, development of hippocampal sclerosis often led to the temporal lobe epilepsy which is sometimes resistant to common anti-epileptic drugs. Cellular and molecular changes underlying epileptogenesis in animal models were studied, however, the underlying mechanisms of kainic acid (KA) mediated neuronal damage in rat hippocampal neuron cell culture alone has not been elucidated yet.MethodsEmbryonic day 18 (E-18) rat hippocampus neurons were cultured with poly-L-lysine coated glass coverslips. Following optimisation, KA (0.5 μM), a chemoconvulsant agent, was administered at three different time-points (30, 60 and 90 min) to induce seizure in rat hippocampal neuronal cell culture. We examined cell viability, neurite outgrowth density and immunoreactivity of the hippocampus neuron culture by measuring brain derived neurotrophic factor (BDNF), γ-amino butyric acid A (GABAA) subunit α-1 (GABRA1), tyrosine receptor kinase B (TrkB), and inositol trisphosphate receptor (IP3R/IP3) levels.ResultsThe results revealed significantly decreased and increased immunoreactivity changes in TrkB (a BDNF receptor) and IP3R, respectively, at 60 min time point.ConclusionThe current findings suggest that TrkB and IP3 could have a neuroprotective role which could be a potential pharmacological target for anti-epilepsy drugs.

Retracted: Anti-ribosomal-phosphoprotein autoantibodies penetrate to neuronal cells via neuronal growth associated protein, affecting neuronal cells in vitro.

OBJECTIVE Anti-ribosomal-phosphoprotein antibodies (anti-Ribos.P Abs) are detected in 10-45% of NPSLE patients. Intracerebroventricular administration of anti-ribosomal-P Abs induces depression-like behaviour in mice. We aimed to discern the mechanism by which anti-Ribos.P Abs induce behavioural changes in mice. METHODS Anti-Ribos.P Abs were exposed to human and rat neuronal cell cultures, as well as to human umbilical vein endothelial cell cultures for a control. The cellular localization of anti-Ribo.P Abs was found by an immunofluorescent technique using a confocal microscope. Identification of the target molecules was undertaken using a cDNA library. Immunohistochemistry and an inhibition assay were carried out to confirm the identity of the target molecules. Neuronal cell proliferation was measured by bromodeoxyuridine, and Akt and Erk expression by immunoblot. RESULTS Human anti-Ribos.P Abs penetrated into human neuronal cells and rat hippocampal cell cultures in vitro, but not to endothelial cells as examined. Screening a high-content human cDNA-library with anti-Ribos.P Abs identified neuronal growth-associated protein (GAP43) as a target for anti-Ribos.P Abs. Ex vivo anti-Ribos.P Abs bind to mouse brain sections of hippocampus, dentate and amygdala. Anti-Ribos.P Abs brain-binding was prevented by GAP43 protein. Interestingly, GAP43 inhibited in a dose-dependent manner the anti-Ribos.P Abs binding to recombinant-ribosomal-P0, indicating mimicry between the ribosomal-P0 protein and GAP43. Furthermore, anti-Ribos.P Abs reduced neuronal cell proliferation activity in vitro (P < 0.001), whereas GAP43 decreased this inhibitory activity by a factor of 7.6. The last was related to Akt and Erk dephosphorylation. CONCLUSION Anti-Ribos.P Abs penetrate neuronal cells in vitro by targeting GAP43. Anti -Ribos.P Abs inhibit neuronal-cell proliferation via inhibition of Akt and Erk. Our data contribute to deciphering the mechanism for anti-Ribos.P Abs' pathogenic activity in NPSLE.

High-resolution bioelectrical imaging of A\u03b2-induced network dysfunction on CMOS-MEAs for neurotoxicity and rescue studies

Neurotoxicity and the accumulation of extracellular amyloid-beta1–42 (Aβ) peptides are associated with the development of Alzheimer’s disease (AD) and correlate with neuronal activity and network dysfunctions, ultimately leading to cellular death. However, research on neurodegenerative diseases is hampered by the paucity of reliable readouts and experimental models to study such functional decline from an early onset and to test rescue strategies within networks at cellular resolution. To overcome this important obstacle, we demonstrate a simple yet powerful in vitro AD model based on a rat hippocampal cell culture system that exploits large-scale neuronal recordings from 4096-electrodes on CMOS-chips for electrophysiological quantifications. This model allows us to monitor network activity changes at the cellular level and to uniquely uncover the early activity-dependent deterioration induced by Aβ-neurotoxicity. We also demonstrate the potential of this in vitro model to test a plausible hypothesis underlying the Aβ-neurotoxicity and to assay potential therapeutic approaches. Specifically, by quantifying N-methyl D-aspartate (NMDA) concentration-dependent effects in comparison with low-concentration allogenic-Aβ, we confirm the role of extrasynaptic-NMDA receptors activation that may contribute to Aβ-neurotoxicity. Finally, we assess the potential rescue of neural stem cells (NSCs) and of two pharmacotherapies, memantine and saffron, for reversing Aβ-neurotoxicity and rescuing network-wide firing.

Roles of the µ-opioid receptor and its related signaling pathways in the pathogenesis of premenstrual syndrome liver-qi stagnation.

The present study aimed to investigate the roles of the µ-opioid receptor (MOR) and its related signaling pathways in the pathogenesis of premenstrual syndrome (PMS) liver-qi stagnation, along with the therapeutic effects of the Shu-Yu capsule in treating the condition. A PMS liver-qi stagnation rat model was established using a chronic restraint stress method. The protein expression level of MOR within rat hippocampal tissue was detected via western blot analysis and cyclic adenosine monophosphate (cAMP) levels within the supernatant of a rat hippocampal cell culture were determined by ELISA. The western blot analysis indicated that the hippocampal expression level of MOR was significantly elevated in the PMS liver-qi stagnation model group. However, subsequent treatment with a Shu-Yu capsule was found to significantly decrease the level of MOR expression. In addition, in vitro experiments were performed, whereby primary hippocampal neurons were treated with model rat serum. It was observed that the level of MOR expression was significantly elevated, while brain-derived neurotrophic factor (BDNF) and cAMP levels in the culture supernatant were significantly decreased. These effects were reversed by treatment with serum from the Shu-Yu capsule-treated rats. Furthermore, when treated with the MOR activator DAMGO, the following were significantly decreased in the primary neurons: Phosphorylation levels of cAMP response element binding protein and extracellular signal-regulated protein kinases (ERK); BDNF expression; and cAMP content in the culture supernatant. These effects were reversed in primary neurons treated with DAMGO and Shu-Yu-containing rat serum. Collectively, the data suggest that increased MOR expression and activation of the cAMP/ERK signaling pathway in the hippocampus may be involved in the pathogenesis of PMS liver-qi stagnation. Furthermore, the efficacy of the Shu-Yu capsule in treating the condition may be via its regulation of MOR receptor signaling.

A modulatory role of ASICs on GABAergic synapses in rat hippocampal cell cultures.

Rapid acidification occurring during synaptic vesicle release can activate acid-sensing ion channels (ASICs) both on pre- and postsynaptic neurons. In the latter case, a fraction of postsynaptic current would be mediated by cation-selective acid-sensing ion channels. Additionally, in both cases, activation of acid-sensing ion channels could modulate synaptic strength by affecting transmitter release and/or sensitivity of postsynaptic receptors. To address potential involvement of acid-sensing ion channels in mediation/modulation of synaptic transmission at hippocampal GABAergic synapses, we studied effects of three structurally different blockers of acid-sensing ion channels on evoked postsynaptic currents using the patch-clamp technique. We found that GABAergic postsynaptic currents, recorded below their reversal potential as inward currents, are suppressed by all the employed blockers of acid-sensing ion channels. These currents were suppressed by ~ 20 % in the presence of a novel blocker 5b (1 μM) and by ~30 % in the presence of either amiloride (25 μM) or diminazene (20 μM). In the same cells the suppression of postsynaptic currents, recorded above their reversal potential as outward currents was statistically insignificant. These results imply that the effects of blockers in our experiments are at least partially postsynaptic. On the other hand, in the case of mediation of a fraction of postsynaptic current by acid-sensing ion channels, an increase of outward currents would be expected under our experimental conditions. Our analysis of a bicuculline-resistant fraction of postsynaptic currents also suggests that effects of the blockers are predominantly modulatory. In this work we present evidence for the first time that acid-sensing ion channels play a functional role at hippocampal GABAergic synapses. The suppressing effect of the blockers of acid-sensing ion channels on GABAergic transmission is due, at least partially, to a postsynaptic but (predominantly) modulatory mechanism. We hypothesize that the modulatory effect is due to functional crosstalk between ASICs and GABAA-receptors recently reported in isolated neurons, however, verification of this hypothesis is necessary.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0269-4) contains supplementary material, which is available to authorized users.

Bothriurus bonariensis scorpion venom activates voltage-dependent sodium channels in insect and mammalian nervous systems

Animal venoms have been widely recognized as a major source of biologically active molecules. Bothriurus bonariensis, popularly known as black scorpion, is the arthropod responsible for the highest number of accidents involving scorpion sting in Southern Brazil. Here we reported the first attempt to investigate the neurobiology of B. bonariensis venom (BBV) in the insect and mammalian nervous system. BBV (32 μg/g) induced a slow neuromuscular blockade in the in vivo cockroach nerve-muscle preparations (70 ± 4%, n = 6, p < 0.001), provoking repetitive twitches and significantly decreasing the frequency of spontaneous leg action potentials (SNCAPs) from 82 ± 3 min−1 to 36 ± 1.3 min−1 (n = 6, p < 0.05), without affecting the amplitude. When tested in primary cultures of rat hippocampal cells, BBV induced a massive increase of Ca2+ influx (250 ± 1% peak increase, n = 3, p < 0.0001). The disturbance of calcium homeostasis induced by BBV on the mammalian central nervous system was not accompanied by cellular death and was prevented by the co-treatment of the hippocampal cells with tetrodotoxin, a selective sodium channel blocker. The results suggest that the biological activity of BBV is mostly related to a modulation of sodium channels function. Our biological activity survey suggests that BBV may have a promising insecticidal and therapeutic potential.

Anti-ribosomal-phosphoprotein autoantibodies penetrate to neuronal cells via neuronal growth associated protein, affecting neuronal cells in vitro.

Anti-ribosomal-phosphoprotein antibodies (anti-Ribos.P Abs) are detected in 10-45% of NPSLE patients. Intracerebroventricular administration of anti-ribosomal-P Abs induces depression-like behaviour in mice. We aimed to discern the mechanism by which anti-Ribos.P Abs induce behavioural changes in mice. Anti-Ribos.P Abs were exposed to human and rat neuronal cell cultures, as well as to human umbilical vein endothelial cell cultures for a control. The cellular localization of anti-Ribo.P Abs was found by an immunofluorescent technique using a confocal microscope. Identification of the target molecules was undertaken using a cDNA library. Immunohistochemistry and an inhibition assay were carried out to confirm the identity of the target molecules. Neuronal cell proliferation was measured by bromodeoxyuridine, and Akt and Erk expression by immunoblot. Human anti-Ribos.P Abs penetrated into human neuronal cells and rat hippocampal cell cultures in vitro, but not to endothelial cells as examined. Screening a high-content human cDNA-library with anti-Ribos.P Abs identified neuronal growth-associated protein (GAP43) as a target for anti-Ribos.P Abs. Ex vivo anti-Ribos.P Abs bind to mouse brain sections of hippocampus, dentate and amygdala. Anti-Ribos.P Abs brain-binding was prevented by GAP43 protein. Interestingly, GAP43 inhibited in a dose-dependent manner the anti-Ribos.P Abs binding to recombinant-ribosomal-P0, indicating mimicry between the ribosomal-P0 protein and GAP43. Furthermore, anti-Ribos.P Abs reduced neuronal cell proliferation activity in vitro (P < 0.001), whereas GAP43 decreased this inhibitory activity by a factor of 7.6. The last was related to Akt and Erk dephosphorylation. Anti-Ribos.P Abs penetrate neuronal cells in vitro by targeting GAP43. Anti -Ribos.P Abs inhibit neuronal-cell proliferation via inhibition of Akt and Erk. Our data contribute to deciphering the mechanism for anti-Ribos.P Abs' pathogenic activity in NPSLE.

614. Microfluidic Manufacture of RNA-Lipid Nanoparticles Leads to Highly Efficient Delivery of Potent Nucleic Acid Therapeutics for Controlling Gene Expression

Lipid nanoparticles (LNPs) have demonstrated efficient nucleic acid delivery in vitro and in vivo, as well as in clinical development. They exploit endogenous delivery pathways, by co-opting apolipoprotein E (apoE), to mediate effective delivery of the encapsulated nucleic acids into cells via the low-density lipoprotein receptor (LDLR). However, use of LNPs from the bench to the clinic has been considerably limited by challenges in manufacturing at both small and large scales. Here, we bridge that gap by describing the robust manufacture and use of clinical-grade lipid-based nanoparticles for highly efficient delivery of nucleic acids at scales suitable for both in vitro screening and in vivo applications.RNA-LNPs manufactured using an optimized microfluidic platform enables efficient encapsulation of nucleic acids (e.g. siRNA, mRNA, pDNA) into biocompatible “solid-core” nanoparticles (~50 nm). The resultant nanoparticles can then be applied to cell cultures in vitro or administered in vivo. The following reports a comprehensive set of studies conducted to evaluate the merits of the technology and further provide insights for delivering short interfering RNA (siRNA) and mRNA in difficult-to-transfect cells both in vitro and in vivo.RNA-LNPs were formulated to encapsulate a potent siRNA directed against PTEN - a clinically relevant gene associated with neural regeneration. Exceptional cellular uptake (>98%) with minimal toxicity was observed in both primary rat hippocampal and mixed cortical cell cultures. High transfection efficency (>95%) of the encapsulated material resulted in concomitant high-level (>85%) PTEN knockdown within the first 4 hours of a low dose (100 ng/ml) treatment; that level of knockdown was further sustained for 21 days. Similarly, RNA-LNPs encapsulating mRNA were also found to mediate early ( 75% for 7 days) following a single (500 ng/ml) treatment in primary rat mixed cortical cultures.Strategies for locally administering RNA-LNPs into the brain and spinal cord of adult Sprague Dawley rats were also investigated. Controlled localized injections of PTEN-encaspulated siRNA into the motorcortex resulted in significant and sustained (7 days) knockdown. Similarly, local administration at the site of a cervical spinal cord injury significantly reduced target PTEN expression, 10 days later. Visible uptake of RNA-LNPs characterized by their presence in the soma of neurons found in the red nucleus provides further insights into a regtrograde transport mechanism involving the axons.Collectively, these studies reflect the simplicity and efficacy of this commercially available technology in presenting a cost-effective and advantageous avenue for screening and validating new targeted nucleic acid therapies.

Network burst activity in hippocampal neuronal cultures: the role of synaptic and intrinsic currents.

The goal of this work was to define the contributions of intrinsic and synaptic mechanisms toward spontaneous network-wide bursting activity, observed in dissociated rat hippocampal cell cultures. This network behavior is typically characterized by short-duration bursts, separated by order of magnitude longer interburst intervals. We hypothesize that while short-timescale synaptic processes modulate spectro-temporal intraburst properties and network-wide burst propagation, much longer timescales of intrinsic membrane properties such as persistent sodium (Nap) currents govern burst onset during interburst intervals. To test this, we used synaptic receptor antagonists picrotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP) to selectively block GABAA, AMPA, and NMDA receptors and riluzole to selectively block Nap channels. We systematically compared intracellular activity (recorded with patch clamp) and network activity (recorded with multielectrode arrays) in eight different synaptic connectivity conditions: GABAA + NMDA + AMPA, NMDA + AMPA, GABAA + AMPA, GABAA + NMDA, AMPA, NMDA, GABAA, and all receptors blocked. Furthermore, we used mixed-effects modeling to quantify the aforementioned independent and interactive synaptic receptor contributions toward spectro-temporal burst properties including intraburst spike rate, burst activity index, burst duration, power in the local field potential, network connectivity, and transmission delays. We found that blocking intrinsic Nap currents completely abolished bursting activity, demonstrating their critical role in burst onset within the network. On the other hand, blocking different combinations of synaptic receptors revealed that spectro-temporal burst properties are uniquely associated with synaptic functionality and that excitatory connectivity is necessary for the presence of network-wide bursting. In addition to confirming the critical contribution of direct excitatory effects, mixed-effects modeling also revealed distinct combined (nonlinear) contributions of excitatory and inhibitory synaptic activity to network bursting properties.

The role of parvalbumin-containing interneurons in the regulation of spontaneous synchronous activity of brain neurons in culture

Subtypes of inhibitory GABAergic neurons containing Ca2+-binding proteins play a pivotal role in the regulation of spontaneous synchronous [Ca2+]i transients in a neuronal network. In this study it is shown that: (1) the interneurons that containing Ca2+-binding proteins at buffer concentration can be identified by the shape of Ca2+-signa1 in response to depolarization or activation of ionotropic glutamate receptors; (2) Ca2+-binding proteins are involved in desynchronization of spontaneous Ca2+ transients. At low frequencies of spontaneous synchronous [Ca2+]i transients (less than 0.2 Hz) neurons show quasi-synchronous pulsations. At higher frequencies, synchronization of spontaneous synchronous [Ca2+]i transients occurs in all neurons; (3) it is established that several synchronous oscillations with different frequencies coexist in the network and the amplitude of their depolarizing pulse also varies. This phenomenon is apparently the mechanism that selectively directs information in separate neurons using the same network; and (4) in one population of interneurons at high frequencies of spontaneous synchronous [Ca2+]i transients the inversion of Cl– concentration gradient is observed. In this case, the inhibition of GABA(A) receptors suppresses the activity of neurons in this population and excites other neurons in the network. Thus, the GABAergic neurons that contain Ca-binding proteins show different mechanisms to regulate the synchronous neuronal activities in cultured rat hippocampal cells.