Primary Cultures Of Cortical Neurons Research Articles

Involvement of superoxide in malaoxon-induced toxicity in primary cultures of cortical neurons

ABSTRACTOrganophosphorus compounds (OP) represent a class of insecticides that are used most globally. The neurotoxic effects are attributed mainly to acetylcholinesterase (AChE) enzyme inhibition, which is responsible for cholinergic manifestations in individuals acutely exposed to OP. However, AChE inhibition alone cannot account for the wide range of symptoms that were reported following OP exposures. In agreement with this, evidence shows that non-cholinergic events may be mechanistically linked to OP-induced neurotoxicity. The aim of this study was to investigate the potential occurrence of oxidative stress as a critical step in the toxicity induced by the OP malaoxon(MAL) using primary cultures of mouse cortical neurons, as well as to distinguish MAL-induced oxidative stress and cell toxicity from an action on AChE blockade. Primary cultures of mouse cortical neurons were treated with MAL (0.01; 0.1; 1; 10; or 100 µM) at varying time points (1, 3, 6, 24, 48, or 144 hr) and the following biochemical parameters determined including cell viability, AChE activity, and superoxide production. MAL significantly reduced cell viability in a concentration- and time-dependent manner. Of note, 1 µM MAL significantly inhibited (approximately 75%) AChE activity after 48 hr incubation. Pralidoxime (PRAL) (600 µM), a classical AChE reactivator, significantly protected against MAL-induced AChE blockade; however, PRAL did not affect MAL-mediated fall in cellular viability, indicating that AChE inhibition is not necessarily correlated with insecticide-induced decrease in cell survival. MAL-induced diminished cell viability was preceded by a significant increase in superoxide anion production. The antioxidant agent ascorbic acid (AA) (200 µM), which significantly protected against MAL-induced superoxide anion production, did not alter MAL-induced AChE inhibition and significantly prevented insecticide-mediated fall in cell survival. Data show that increased superoxide anion production is an event that precedes MAL-induced cell toxicity in primary cultures of mouse cortical neurons. Based on the preventative effects of AA against MAL-mediated superoxide anion production and reduced cell viability, evidence indicates that oxidative stress represents an important step mediating MAL-induced toxicity in neurons and that AChE inhibition is not necessarily correlated with lowered cell survival noted in insecticide-exposed cells.

Reactive Oxygen Species Produced by a Photodynamic Effect Induced Calcium Signal in Neurons and Astrocytes.

Photodynamic therapy (PDT) leads to production of reactive oxygen species (ROS) and cell destruction due to oxidative stress. We used photodynamic effect of photosensitizer radachlorin to unravel the effect of photo-induced oxidative stress on the calcium signal and lipid peroxidation in primary culture of cortical neurons and astrocytes using live cell imaging. We have found that irradiation in presence of 200nM of radachlorin induces calcium signal in primary neurons and astrocytes. Photo-induced neuronal calcium signal depends on internal calcium stores as it was still observed in calcium-free medium and could be blocked by depletion of endoplasmic reticulum (ER) stores with inhibitor of sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) thapsigargin. Both inhibitors of phospholipase C activity U73122 and water-soluble analogue of vitamin E Trolox suppressed calcium response activated by PDT. We have also observed that the photodynamic effect of radachlorin induces lipid peroxidation in neurons and astrocytes. This data demonstrate that lipid peroxidation induced by PDT in neurons and astrocytes leads to activation of phospholipase C that results in production of inositol 1,4,5-trisphosphate (IP3).

Inflammatory cytokine-induced changes in neural network activity measured by waveform analysis of high-content calcium imaging in murine cortical neurons

During acute neuroinflammation, increased levels of cytokines within the brain may contribute to synaptic reorganization that results in long-term changes in network hyperexcitability. Indeed, inflammatory cytokines are implicated in synaptic dysfunction in epilepsy and in an array of degenerative and autoimmune diseases of the central nervous system. Current tools for studying the impact of inflammatory factors on neural networks are either insufficiently fast and sensitive or require complicated and costly experimental rigs. Calcium imaging offers a reasonable surrogate for direct measurement of neuronal network activity, but traditional imaging paradigms are confounded by cellular heterogeneity and cannot readily distinguish between glial and neuronal calcium transients. While the establishment of pure neuron cultures is possible, the removal of glial cells ignores physiologically relevant cell-cell interactions that may be critical for circuit level disruptions induced by inflammatory factors. To overcome these issues, we provide techniques and algorithms for image processing and waveform feature extraction using automated analysis of spontaneous and evoked calcium transients in primary murine cortical neuron cultures transduced with an adeno-associated viral vector driving the GCaMP6f reporter behind a synapsin promoter. Using this system, we provide evidence of network perturbations induced by the inflammatory cytokines TNFα, IL1β, and IFNγ.

Diamond microelectrode arrays for in vitro neuronal recordings

A novel microfabrication technique for microelectrode arrays (MEAs) with a full diamond-cell interface is demonstrated. Boron-doped nano-crystalline diamond (BNCD) is used as a conductive electrode material on metal tracks insulated by intrinsic NCD. MEAs successfully recorded spontaneous electrical activity in rat primary cortical neuronal cultures. Patch-clamp measurements show no alterations to cell membrane passive properties or active firing response, for cell developing ex vivo on diamond. Impedance analysis revealed low impedance magnitude of BNCD electrodes, suitable for multi-unit neuronal recordings. Additionally, the impedance phase of the fabricated electrodes shows a high degree of capacitive coupling, ideal for neuron stimulation.

Schisantherin A attenuates ischemia/reperfusion-induced neuronal injury in rats via regulation of TLR4 and C5aR1 signaling pathways

Toll-like receptor 4 (TLR4) and C5aR1 (CD88) have been recognized as potential therapeutic targets for the reduction of inflammation and secondary damage and improvement of outcome after ischemia and reperfusion (I/R). The inflammatory responses which induce cell apoptosis and necrosis after I/R brain injury lead to a limited process of neural repair. To further comprehend how these targets function in I/R state, we investigated the pathological changes and TLR4 and C5aR1 signaling pathways in vitro and in vivo models of I/R brain injury in this study. Meanwhile, we explored the roles of schisantherin A on I/R brain injury, and whether it exerted neuroprotective effects by regulating the TLR4 and C5aR1 signaling pathways or not. The results showed that schisantherin A significantly reduced the neuronal apoptosis induced by oxygen and glucose deprivation and reperfusion (OGD/R) injury in primary culture of rat cortical neurons. Also, schisantherin A alleviated neurological deficits, reduced infarct volume, attenuated oxidation stress, inflammation and apoptosis in ischemic parietal cortex of rats after middle cerebral artery occlusion and reperfusion (MCAO/R) injury. Moreover, the activated TLR4 and C5aR1 signaling pathways were inhibited by schisantherin A treatment. In conclusion, TLR4 and C5aR1 played a vital role during I/R brain injury in rats, and schisantherin A exhibited neuroprotective effects by TLR4 and C5aR1 signaling pathways. These findings also provided new insights that would aid in elucidating the effect of schisantherin A against cerebral I/R and support the development of schisantherin A as a potential treatment for ischemic stroke.

Endothelial Nitric Oxide Synthase Is Present in Dendritic Spines of Neurons in Primary Cultures.

Nitric oxide exerts important regulatory functions in various brain processes. Its synthesis in neurons has been most commonly ascribed to the neuronal nitric oxide synthase (nNOS) isoform. However, the endothelial isoform (eNOS), which is significantly associated with caveolae in different cell types, has been implicated in synaptic plasticity and is enriched in the dendrites of CA1 hippocampal neurons. Using high resolution microscopy and co-distribution analysis of eNOS with synaptic and raft proteins, we now show for the first time in primary cortical and hippocampal neuronal cultures, virtually devoid of endothelial cells, that eNOS is present in neurons and is localized in dendritic spines. Moreover, eNOS is present in a postsynaptic density-enriched biochemical fraction isolated from these neuronal cultures. In addition, qPCR analysis reveals that both the nNOS as well as the eNOS transcripts are present in neuronal cultures. Moreover, eNOS inhibition in cortical cells has a negative impact on cell survival after excitotoxic stimulation with N-methyl-D-aspartate (NMDA). Consistent with previous results that indicated nitric oxide production in response to the neurotrophin BDNF, we could detect eNOS in immunoprecipitates of the BDNF receptor TrkB while nNOS could not be detected. Taken together, our results show that eNOS is located at excitatory synapses where it could represent a source for NO production and thus, the contribution of eNOS-derived nitric oxide to the regulation of neuronal survival and function deserves further investigations.

FUNCTIONAL CHANGES IN MEMORY ASSOCIATED WITH TDP-43 EXPRESSION IN AN APP/PSEN1 MOUSE MODEL

Recent studies have shown that phosphorylated TDP-43 pathology is present in up to 50% of sporadic Alzheimer's disease (AD) cases. Although mislocalized and phosphorylated TDP-43 has been observed in several neurodegenerative diseases, little is known about the role TDP-43 plays in AD pathology. We describe a mouse model of selective human TDP-43 and TDP-43ΔNLS (defective nuclear localization signal) expression in the hippocampus and cortex in an APP/PSEN1 mouse. We utilize a cell-specific inducible system to express human TDP-43 and nuclear localization signal defective TDP-43ΔNLS in hippocampal and cortical neuronal populations using the Camk2a tetracycline transactivator (Camk2a-tTA) in an APP/PSEN1 background. The effect of TDP-43 on cognition and neurotoxicity was evaluated through novel object recognition, Y-maze, pathology, electron microscopy, immunofluorescence, primary cortical neuronal cultures, proteomics, and Western blotting. We describe functional changes in short and long-term memory associated with TDP-43 expression. In addition, we describe pathological changes in AB plaques, tau aggregation, and phosphorylated TDP-43. Collectively, our data lead us to conclude that TDP-43 contributes to functional changes in memory, Ab plaque formation, tau aggregation, and accumulation of phosphorylated TDP-43. Our current data suggest that TDP-43 could be a putative target of therapeutic intervention in AD affecting both AB plaques and tauopathy.

PROTECTIVE PROPERTY OF P3-ALCβ AGAINST A NEUROTOXICITY INDUCED BY Aβ OLIGOMER

Neuronal p3-Alcβ peptide is generated from the precursor protein Alcadeinβ (Alcβ) by a cleavage of α- and γ-secretases. Alcβ generates the C-terminal variant peptides including a major p3-Alcβ37, as does APP. It is understood that Aβ42 level tends to decrease in AD patients in cerebrospinal fluid (CSF) by deposition of Aβ aggregate in brain compared to age-matched non-demented subjects. Although, p3-Alcβ levels are comparable to those of Aβ levels, the trend of non-aggregatable p3-Alcβ levels in CSF of AD subjects has not been analyzed. Moreover, function of p3-Alcβ remains unclear. In this study, we analyzed the change of p3-Alcβ in CSF of AD and explored the function of p3-Alcβ, especially in the role to prevent Aβ oligomer-induced neurotoxicity. We developed a new sandwich ELISA to quantify p3-Alcβ37. The primary cultures of mixed mouse cortical and hippocampal neurons were used to assess functions of p3-Alcβ in Aβ oligomer-induced neurotoxicity. Partial sequence peptides of p3-Alcβ were examined to identify functional regions of p3-Alcβ in vivo and in vitro. To analyze pharmacokinetics of the short peptide including functional amino acid sequence in mouse, we developed another sELISA. CSF p3-Alcβ level significantly decreased in Alzheimer's disease (AD) and/or mild cognitive impairment, compared to age-matched non-demented subjects (see Poster by Hata et al). Interestingly, a synthetic p3-Alcβ37 preserved primary cultured neurons from an impairment by Aβ42 oligomers in dose-dependent manner. Independent study identified the functional amino acid sequence in p3-Alcβ, which restored a cognitive impairment induced by Aβ oligomers in mouse. To develop a new drug therapy with this short peptide/a lead peptide including the functional amino acid sequence, we examined levels of the lead peptide in blood and CSF with sELISA. Following the subcutaneous administration of the lead peptide into mice, the peptide was detected in blood quickly and followed in CSF, indicating the brain transport of the peptide by a peripheral administration. The p3-Alcβ shows a protective effect against neurotoxicity induced by Aβ oligomer. Shorter peptide of p3-Alcβ is a drugable lead with an ability of brain transport by peripheral administration.

VDR SUPPRESSION AND CYP24 INDUCTION BY AMYLOID BETA MIGHT INVOLVE MIR-27B-3P, LET-7A-5P, MIR-125B-5P IN AMYLOID BETA-TREATED CORTICAL NEURON CULTURES

In 2011, a study by our group demonstrated that Aβ induces neurodegeneration also by significantly suppressing the expression of VDR. We also reported that the expression of mRNA for 25-hydroxyvitaminD3–24-hydroxylase (24OHase) encoded by CYP24, the enzyme that accelerates vitamin D catabolism, is induced by Aβ in hippocampal neurons. This finding suggests that the involvement of vitamin D with AD includes also the disruption of vitamin D metabolism and action. The present study investigated the expression levels of miRNAs that were suggested to regulate VDR (hsa-mir-27b-3p, hsa-let-7a-5p, hsa-mir-125b-5p), CYP24 (hsa-mir-125b-5p) in order to evaluate whether alteration of the expressions of VDR or 24(OH)ase by amyloid beta involve these miRNAs or not? 24, 48 and 72 hours of Aβ, vitamin D, vitamin D+Aβ and Aβ+vitamin D treated groups and untreated control groups were established in the primary cortical neuron cultures of Sprague Dawley rat embryos. Amyloid pathology was established with the treatment of 10 μM of Aβ1–42. Vitamin D groups were treated with 10-8M 1α-25dihydroxyvitamin D3 (1,25(OH)2D3). The expressions of let-7a, miR-27b-3p, miR-125b-5p, miR-26b-5p, miR-192–5p, were investigated with qRT-PCR. Our results indicated that only let7a-5p expression was significantly increased in amyloid beta treated cortical neurons at 24 hours of treatments. miR125b-5p expression decreased in both amyloid beta or vitamin D treated ones, yet the attenuations were not statistically significant. miR125b-5p expression also decreased in amyloid beta, amyloid beta+vitamin D, vitamin D+amyloid beta treated groups in 48 hours of treatments compared to control groups. miR27b-3p expression induced in vitamin D treated neurons at the 24 and 48 hours of treatments compared with the control groups. Consequently, our preliminary results indicate that miR27b-3p might be involved in regulation of vitamin D action, by regulating VDR expression. The more likely small RNA that is involved in VDR suppression by amyloid beta might be let7a-5p. CYP24 induction by amyloid beta might involve the suppression of miR125b-5p by amyloid beta. (The present work was supported by the Research Fund of Istanbul University, Project no:21585 and by the Scientific and Technological Research Council of Turkey-TUBITAK, Project no:214S585.)

Evidence that activation of P2X7R does not exacerbate neuronal death after optic nerve transection and focal cerebral ischemia in mice

Conflicting data in the literature about the function of P2X7R in survival following ischemia necessitates the conductance of in-depth studies. To investigate the impacts of activation vs inhibition of the receptor on neuronal survival as well as the downstream signaling cascades, in addition to optic nerve transection (ONT), 30min and 90min of middle cerebral artery occlusion (MCAo) models were performed in mice. Intracellular calcium levels were assessed in primary cortical neuron cultures. Here, we show that P2X7R antagonist Brilliant Blue G (BBG) decreased DNA fragmentation, infarct volume, brain swelling, neurological deficit scores and activation of microglial cells after focal cerebral ischemia. BBG also significantly increased the number of surviving retinal ganglion cells (RGCs) after ONT and the number of surviving neurons following MCAo. Importantly, receptor agonist BzATP resulted in increased activation of microglial cells and induced phosphorylation of ERK, AKT and JNK. These results indicated that inhibition of P2X7R with BBG promoted neuronal survival, not through the activation of survival kinase pathways, but possibly by improved intracellular Ca2+ overload and decreased the levels of Caspase 1, IL-1β and Bax proteins. On the other hand, BzATP-mediated increased number of activated microglia and increased survival kinase levels in addition to increased caspase-1 and IL-1β levels indicate the complex nature of the P2X7 receptor-mediated signaling in neuronal injury.

Abnormal dendritic maturation of developing cortical neurons exposed to corticotropin releasing hormone (CRH): Insights into effects of prenatal adversity?

Corticotropin releasing hormone (CRH) produced by the hypothalamus initiates the hypothalamic-pituitary-adrenal (HPA) axis, which regulates the body’s stress response. CRH levels typically are undetectable in human plasma, but during pregnancy the primate placenta synthesizes and releases large amounts of CRH into both maternal and fetal circulations. Notably, placental CRH synthesis increases in response to maternal stress signals. There is evidence that human fetal exposure to high concentrations of placental CRH is associated with behavioral consequences during infancy and into childhood, however the direct effects on of the peptide on the human brain are unknown. In this study, we used a rodent model to test the plausibility that CRH has direct effects on the developing cortex. Because chronic exposure to CRH reduces dendritic branching in hippocampal neurons, we tested the hypothesis that exposure to CRH would provoke impoverishment of dendritic trees in cortical neurons. This might be reflected in humans as cortical thinning. We grew developing cortical neurons in primary cultures in the presence of graded concentrations of CRH. We then employed Sholl analyses to measure dendritic branching and total dendritic length of treated cells. A seven-day exposure to increasing levels of CRH led to a significant, dose-dependent impoverishment of the branching of pyramidal-like cortical neurons. These results are consistent with the hypothesis that, rather than merely being a marker of prenatal stress, CRH directly decreases dendritic branching. Because dendrites comprise a large portion of cortical volume these findings might underlie reduced cortical thickness and could contribute to the behavioral consequences observed in children exposed to high levels of CRH in utero.

Novel insights into the antioxidant role of tauroursodeoxycholic acid in experimental models of Parkinson's disease

Impaired mitochondrial function and generation of reactive oxygen species are deeply implicated in Parkinson's disease progression. Indeed, mutations in genes that affect mitochondrial function account for most of the familial cases of the disease, and post mortem studies in sporadic PD patients brains revealed increased signs of oxidative stress. Moreover, exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial complex I inhibitor, leads to clinical symptoms similar to sporadic PD.The bile acid tauroursodeoxycholic acid (TUDCA) is an anti-apoptotic molecule shown to protect against MPTP-induced neurodegeneration in mice, but the mechanisms involved are still incompletely identified.Herein we used MPTP-treated mice, as well as primary cultures of mice cortical neurons and SH-SY5Y cells treated with MPP+ to investigate the modulation of mitochondrial dysfunction by TUDCA in PD models. We show that TUDCA exerts its neuroprotective role in a parkin-dependent manner. Overall, our results point to the pharmacological up-regulation of mitochondrial turnover by TUDCA as a novel neuroprotective mechanism of this molecule, and contribute to the validation of TUDCA clinical application in PD.

Neuroprotective effects of sulfiredoxin-1 during cerebral ischemia/reperfusion oxidative stress injury in rats

As an endogenous antioxidant protein, Sulfiredoxin1 (Srxn1) can prevent cell oxidative stress damage. However, its role in cerebral ischemia/reperfusion (I/R) injury and the underlying signaling mechanisms remain largely unknown. Here, we explored effects of Srxn1 knockdown on oxidative stress using in vitro and in vivo I/R models and investigated related neuroprotective mechanisms. For in vitro studies, primary cortical neuronal cultures were transfected with an interfering lentivirus targeting Srxn1. Oxygen-glucose deprivation (OGD) was conducted after Srxn1 knockdown. MTS and lactate dehydrogenase assays indicated that knockdown of Srxn1 increased cell death and reduced cell viability. Similarly, superoxide dismutase (SOD) and reduced glutathionekits assays showed that knockdown of Srxn1 worsened oxidative stress injury. For in vivo studies, siRNA for Srxn1 or negative control siRNA was injected intracerebroventricularly 24h before middle cerebral artery occlusion (MCAO). Data shows silencing Srxn1 resulted in a significant increase in cerebral infarction, neurological deficits, histological injury, and oxidative stress injury 24h after ischemic stroke. Moreover, immunoblot analysis assessed the relationship between Srxn1 levels and Prdx1–4 as well as Prdx-SO3 activity both in vitro and in vivo models. We found that decreased Srxn1 reduced Prdx1–4 and enhanced Prdx-SO3 protein levels. In addition, knockdown of Nrf2 was performed; immunoblot analysis was used to measure Srxn1 and NQO1 protein levels. We further found that interference of Nrf2 reduced Srxn1 and NQO1 protein levels. In summary, Srxn1 can protect neurons from I/R oxidative stress injury and the mechanism involves Prdx activity. Srxn1, which might be downstream of Nrf2, can prevent cerebral ischemia reperfusion by reversing overoxidized Prdx and restoring antioxidant activity of Prdx.

The Marine Guanidine Alkaloid Crambescidin 816 Induces Calcium Influx and Cytotoxicity in Primary Cultures of Cortical Neurons through Glutamate Receptors.

Crambescidin 816 is a guanidine alkaloid produced by the sponge Crambe crambe with known antitumoral activity. While the information describing the effects of this alkaloid in central neurons is scarce, Cramb816 is known to block voltage dependent calcium channels being selective for L-type channels. Moreover, Cramb816 reduced neuronal viability through an unknown mechanism. Here, we aimed to describe the toxic activity of Cramb816 in cortical neurons. Since calcium influx is considered the main mechanism responsible for neuronal cell death, the effects of Cramb816 in the cytosolic calcium concentration of cortical neurons were studied. The alkaloid decreased neuronal viability and induced a dose-dependent increase in cytosolic calcium that was also related to the presence of calcium in the extracellular media. The increase in calcium influx was age dependent, being higher in younger neurons. Moreover, this effect was prevented by glutamate receptor antagonists, which did not fully block the cytotoxic effect of Cramb816 after 24 h of treatment but completely prevented Cramb816 cytotoxicity after 10 min exposure. Therefore, the findings presented herein provide new insights into the cytotoxic effect of Cramb816 in cortical neurons.

Intra-arterial verapamil post-thrombectomy is feasible, safe, and neuroprotective in stroke

Large vessel ischemic stroke represents the most disabling subtype. While t-PA and endovascular thrombectomy can recanalize the occluded vessel, good clinical outcomes are not uniformly achieved. We propose that supplementing endovascular thrombectomy with superselective intra-arterial (IA) verapamil immediately following recanalization could be safe and effective. Verapamil, a calcium channel blocker, has been shown to be an effective IA adjunct in a pre-clinical mouse focal ischemia model. To demonstrate translational efficacy, mechanism, feasibility, and safety, we conducted a group of translational experiments. We performed in vivo IA dose–response evaluation in our animal stroke model with C57/Bl6 mice. We evaluated neuroprotective mechanism through in vitro primary cortical neuron (PCN) cultures. Finally, we performed a Phase I trial, SAVER-I, to evaluate feasibility and safety of administration in the human condition. IA verapamil has a likely plateau or inverted-U dose–response with a defined toxicity level in mice (LD50 16–17.5 mg/kg). Verapamil significantly prevented PCN death and deleterious ischemic effects. Finally, the SAVER-I clinical trial showed no evidence that IA verapamil increased the risk of intracranial hemorrhage or other adverse effect/procedural complication in human subjects. We conclude that superselective IA verapamil administration immediately following thrombectomy is safe and feasible, and has direct, dose–response-related benefits in ischemia.

In vitro α-synuclein neurotoxicity and spreading among neurons and astrocytes using Lewy body extracts from Parkinson disease brains

Synucleinopathies are a group of diseases characterized by the presence of intracellular protein aggregates containing α-synuclein (α-syn). While α-syn aggregates have been shown to induce multimodal cellular dysfunctions, uptake and transport mechanisms remain unclear. Using high-content imaging on cortical neurons and astrocytes, we here define the kinetics of neuronal and astrocytic abnormalities induced by human-derived α-syn aggregates grounding the use of such system to identify and test putative therapeutic compounds. We then aimed at characterizing uptake and transport mechanisms using primary cultures of cortical neurons and astrocytes either in single well or in microfluidic chambers allowing connection between cells and cell-types. We report that astrocytes take up α-syn-aggregates far more efficiently than neurons through an endocytic event. We also highlight that active α-syn transport occurs between cells and any cell-types. Of special interest regarding the disease, we also show that uptake and spreading of α-syn from astrocytes to neurons can lead to neuronal death. Altogether, we here show that patients-derived α-synuclein aggregates, which are taken up by neurons and astrocytes, induce a differential endogenous response in the two cell types including a peculiar astrocytic toxic gain-of-function that leads to neuronal death.

The involvement of BDNF-CREB signaling pathways in the pharmacological mechanism of combined SSRI- antipsychotic treatment in schizophrenia

Previous studies into the mechanism of SSRI-antipsychotic synergism in our laboratory identified unique changes in the brain, particularly in the γ-aminobutyric acid (GABA)-A receptor and its modulators. This study examined the role of brain derived neurotrophic factor (BDNF)-cAMP response element binding (CREB) protein signaling pathways, including protein kinase B (AKT), glycogen synthase kinase (GSK)-3β and related molecules in the molecular response to haloperidol, fluvoxamine, combined haloperidol+fluvoxamine and clozapine treatments in rat frontal cortex, hippocampus and primary cortical neuronal cultures. The effect of fluvoxamine augmentation on BDNF-CREB pathways in peripheral mononuclear cells (PMC׳s) of medicated schizophrenia patients was also studied. Chronic haloperidol (1mg/kg) +fluvoxamine (10mg/kg) treatment increased TrkB receptor and BDNF expression levels, and the phosphorylation of AKT/CREB/GSK-3β, compared to the individual drugs in rat brain. In addition, haloperidol+fluvoxamine treatment improved cognitive functions in rats, indicating that the molecular changes may have a role in behavioral improvement. In primary neuronal cell cultures, pretreatment with a selective PI3K inhibitor abolished the haloperidol+fluvoxamine-induced phosphorylation of AKT and GSK-3β, but did not affect the upregulation of CREB phosphorylation. In the clinic, PMC׳s of treated patients showed upregulation of mRNA expression and protein levels of BDNF, CREB and AKT after addition of fluvoxamine. Analyses of PMC genes and proteins showed significant inter-correlations and some gene changes correlated with improvement in negative and cognitive symptoms. Our study provides new knowledge of the molecular mechanisms of symptom amelioration in schizophrenia and may advance development of new drugs for this disease and other neuropsychiatric disorders.

Temporal relation between neural activity and neurite pruning on a numerical model and a microchannel device with micro electrode array

Synapse elimination and neurite pruning are essential processes for the formation of neuronal circuits. These regressive events depend on neural activity and occur in the early postnatal days known as the critical period, but what makes this temporal specificity is not well understood. One possibility is that the neural activities during the developmentally regulated shift of action of GABA inhibitory transmission lead to the critical period. Moreover, it has been reported that the shifting action of the inhibitory transmission on immature neurons overlaps with synapse elimination and neurite pruning and that increased inhibitory transmission by drug treatment could induce temporal shift of the critical period. However, the relationship among these phenomena remains unclear because it is difficult to experimentally show how the developmental shift of inhibitory transmission influences neural activities and whether the activities promote synapse elimination and neurite pruning. In this study, we modeled synapse elimination in neuronal circuits using the modified Izhikevich's model with functional shifting of GABAergic transmission. The simulation results show that synaptic pruning within a specified period like the critical period is spontaneously generated as a function of the developmentally shifting inhibitory transmission and that the specific firing rate and increasing synchronization of neural circuits are seen at the initial stage of the critical period. This temporal relationship was experimentally supported by an in vitro primary culture of rat cortical neurons in a microchannel on a multi-electrode array (MEA). The firing rate decreased remarkably between the 18–25 days in vitro (DIV), and following these changes in the firing rate, the neurite density was slightly reduced. Our simulation and experimental results suggest that decreasing neural activity due to developing inhibitory synaptic transmission could induce synapse elimination and neurite pruning at particular time such as the critical period. Additionally, these findings indicate that we can estimate the maturity level of inhibitory transmission and the critical period by measuring the firing rate and the degree of synchronization in engineered neural networks.

Neuroprotective effects of gabaergic phenols correlated with their pharmacological and antioxidant properties

AimsVarious investigations have demonstrated the protective capacity of general anesthetics as neuroprotective agents. The effects of propofol against ischemia are known to reside in its antioxidant properties and its GABAergic activity. Other aromatic alcohols have also been reported as able to protect neurons against oxidative damage. The aim of this work is to evaluate the potential neuroprotective effect of some phenols, structurally analogues of propofol, with proven GABAergic activity. These phenols include the naturally occurring compounds thymol, carvacrol and eugenol, the synthetic product chlorothymol, and the most widely used intravenous anesthetic, propofol, as a reference compound. Materials and methodsTaking primary cultures of cortical neurons as a suitable model to evaluate cellular protection against oxidative damage, we developed an injury model to test potential neuroprotective activity. The intracellular hydroperoxides were also determined. Key findingsThe results showed that no compound decreased cell viability at concentrations where they were active on the GABAA receptor. In neuroprotection tests, some phenols and Vit E showed a partial protective effect against the oxidative injury. These compounds induced a clear tendency to reduce H2O2 damage, comparing production of hydroperoxides, although these last changes were statistically non-significant. SignificanceTesting the intracellular oxidation levels suggests that this partial protection exerted by propofol, thymol and chlorothymol may be mediated in some way by their antioxidant activities. However, this neuroprotection is not completely correlated with the antioxidant capacity, but it approaches their relative pharmacological potency, which could be interpreted as a final effect that would involve both activities.

Neuroprotective Effects of Stiripentol on Oxygen-Glucose Deprivation in Primary Culture of Fetal Mice Cortical Neurons

Neuroprotective Effects of Stiripentol on Oxygen-Glucose Deprivation in Primary Culture of Fetal Mice Cortical Neurons