The α4 Nicotinic Acetylcholine Receptor Is Necessary for the Initiation of Organophosphate-Induced Neuronal Hyperexcitability.

Acute intoxication with organophosphorus (OP) cholinesterase inhibitors can produce seizures that rapidly progress to life-threatening status epilepticus. Significant research effort has been focused on investigating the involvement of muscarinic acetylcholine receptors (mAChRs) in OP-induced seizure activity. In contrast, there has been far less attention on nicotinic AChRs (nAChRs) in this context. Here, we address this data gap using a combination of in vitro and in vivo models. Pharmacological antagonism and genetic deletion of α4, but not α7, nAChR subunits prevented or significantly attenuated OP-induced electrical spike activity in acute hippocampal slices and seizure activity in mice, indicating that α4 nAChR activation is necessary for neuronal hyperexcitability triggered by acute OP exposures. These findings not only suggest that therapeutic strategies for inhibiting the α4 nAChR subunit warrant further investigation as prophylactic and immediate treatments for acute OP-induced seizures, but also provide mechanistic insight into the role of the nicotinic cholinergic system in seizure generation.

p-tau Ser356 is associated with Alzheimer's disease pathology and is lowered in brain slice cultures using the NUAK inhibitor WZ4003.

Tau hyperphosphorylation and aggregation is a common feature of many dementia-causing neurodegenerative diseases. Tau can be phosphorylated at up to 85 different sites, and there is increasing interest in whether tau phosphorylation at specific epitopes, by specific kinases, plays an important role in disease progression. The AMP-activated protein kinase (AMPK)-related enzyme NUAK1 has been identified as a potential mediator of tau pathology, whereby NUAK1-mediated phosphorylation of tau at Ser356 prevents the degradation of tau by the proteasome, further exacerbating tau hyperphosphorylation and accumulation. This study provides a detailed characterisation of the association of p-tau Ser356 with progression of Alzheimer's disease pathology, identifying a Braak stage-dependent increase in p-tau Ser356 protein levels and an almost ubiquitous presence in neurofibrillary tangles. We also demonstrate, using sub-diffraction-limit resolution array tomography imaging, that p-tau Ser356 co-localises with synapses in AD postmortem brain tissue, increasing evidence that this form of tau may play important roles in AD progression. To assess the potential impacts of pharmacological NUAK inhibition in an ex vivo system that retains multiple cell types and brain-relevant neuronal architecture, we treated postnatal mouse organotypic brain slice cultures from wildtype or APP/PS1 littermates with the commercially available NUAK1/2 inhibitor WZ4003. Whilst there were no genotype-specific effects, we found that WZ4003 results in a culture-phase-dependent loss of total tau and p-tau Ser356, which corresponds with a reduction in neuronal and synaptic proteins. By contrast, application of WZ4003 to live human brain slice cultures results in a specific lowering of p-tau Ser356, alongside increased neuronal tubulin protein. This work identifies differential responses of postnatal mouse organotypic brain slice cultures and adult human brain slice cultures to NUAK1 inhibition that will be important to consider in future work developing tau-targeting therapeutics for human disease.

Substantially improved gene transfer to interneurons with second-generation glutamate receptor-targeted DART-AAV vectors.

BACKGROUND: Adeno-associated viral vectors (AAVs) are a widely used gene transfer platform in neuroscience. Although naturally AAV serotypes can have preferences for certain tissues, selectivity for particular cell types in the CNS does not exist. Towards interneuron targeting, capsid engineering of AAV2 including display of the designed ankyrin repeat protein (DARPin) 2K19 specific for the glutamate receptor subunit 4 (GluA4) at the N-terminus of the VP2 capsid protein has been established. The resulting AAV-VP2N is highly specific for interneurons, but exhibits rather moderate transduction efficiencies. METHODS: Two alternative insertion sites for 2K19 in the GH2/GH3 loop of capsid proteins VP1 (AAV-VP1L) or VP2 (AAV-VP2L) were exploited to yield second generation GluA4-AAVs. Having packaged reporter genes under ubiquitous promoters, the vectors were characterized for biochemical properties as well as gene delivery into cell lines and rat hippocampal slice cultures. Electrophysiological recordings monitored the functional properties of transduced cells. RESULTS: Compared to AAV-VP2N, the second-generation vectors, especially AAV-VP1L, achieved about 2-fold higher genomic titers as well as a substantially improved GluA4 binding. Improvements in gene transfer activities were 18-fold on GluA4-overexpressing A549 cells and five-fold on rat hippocampal organotypic slice cultures reaching approximately 60 % of all parvalbumin positive interneurons upon a single administration. The spiking behaviour of transduced cells was unaltered and characteristic for a heterogeneous group of interneurons. CONCLUSION: The substantially improved gene transfer activity of the second generation GluA4-targeted AAV combined with low toxicity makes this vector an attractive tool for interneuron-directed gene transfer with unrestricted promotor and transgene choice.

Alteration of Neural Network and Hippocampal Slice Activation through Exosomes Derived from 5XFAD Nasal Lavage Fluid.

Exosomes, key mediators of intercellular transmission of pathogenic proteins, such as amyloid-beta and tau, significantly influence the progression and exacerbation of Alzheimer's disease (AD) pathology. Present in a variety of biological fluids, including cerebrospinal fluid, blood, saliva, and nasal lavage fluid (NLF), exosomes underscore their potential as integral mediators of AD pathology. By serving as vehicles for disease-specific molecules, exosomes could unveil valuable insights into disease identification and progression. This study emphasizes the imperative to investigate the impacts of exosomes on neural networks to enhance our comprehension of intracerebral neuronal communication and its implications for neurological disorders like AD. After harvesting exosomes derived from NLF of 5XFAD mice, we utilized a high-density multielectrode array (HD-MEA) system, the novel technology enabling concurrent recordings from thousands of neurons in primary cortical neuron cultures and organotypic hippocampal slices. The ensuing results revealed a surge in neuronal firing rates and disoriented neural connectivity, reflecting the effects provoked by pathological amyloid-beta oligomer treatment. The local field potentials in the exosome-treated hippocampal brain slices also exhibited aberrant rhythmicity, along with an elevated level of current source density. While this research is an initial exploration, it highlights the potential of exosomes in modulating neural networks under AD conditions and endorses the HD-MEA as an efficacious tool for exosome studies.

Activation of adenosine A1 receptor potentiates metabotropic glutamate receptor 1-mediated Ca2+ mobilization in the rat hippocampal marginal zone.

Two subtypes of group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, participate in the regulation of cell excitability and synaptic plasticity in the central nervous system. They couple to the Gq/11 protein and release Ca2+ from the intracellular stores. In the marginal zone of the neonatal hippocampus, Cajal-Retzius (CR) cells, which control radial migration of neurons, express the subtype mGluR1. The adenosine A1 receptor (A1R) is also G-protein coupled and is extensively expressed in the central nervous system. The interactions among G-protein-coupled receptors have been predicted previously, however, there is insufficient evidence of functional interactions between naturally occurring receptors. In this study, potentiation of the mGluR1-mediated response by A1R activation was demonstrated in hippocampal CR cells. Fluorescence imaging revealed that the application of A1R agonists intensified mGluR1-induced elevation of intracellular Ca2+ concentration ([Ca2+]i). Activation of A1R did not change [Ca2+]i. The potentiated responses were independent of extracellular Ca2+ and prevented by the Gi inhibitor. The potentiation of mGluR1-induced [Ca2+]i. elevation was also enhanced by mGluR2/3 activation. These results suggest that mGluR1 and A1R cooperatively influence postnatal hippocampal development by facilitating Ca2+ mobilization in CR cells.

Living-Neuron-Based Autogenerator.

We present a novel closed-loop system designed to integrate biological and artificial neurons of the oscillatory type into a unified circuit. The system comprises an electronic circuit based on the FitzHugh-Nagumo model, which provides stimulation to living neurons in acute hippocampal mouse brain slices. The local field potentials generated by the living neurons trigger a transition in the FitzHugh-Nagumo circuit from an excitable state to an oscillatory mode, and in turn, the spikes produced by the electronic circuit synchronize with the living-neuron spikes. The key advantage of this hybrid electrobiological autogenerator lies in its capability to control biological neuron signals, which holds significant promise for diverse neuromorphic applications.

Long-term depression induction and maintenance across regions of the apical branch of CA1 dendrites.

Well known as the center for learning and memory, hippocampus is the crucial brain region to study synaptic plasticity in the context of cellular fundamental mechanisms such as long-term depression (LTD) and long-term potentiation (LTP). However, despite years of extensive research, the key to our LTD queries and their induction mechanisms has not been fully understood. Previously, we reported the induction of late-LTD (L-LTD) in the distally located synapses of apical branch of hippocampal CA1 dendrites using strong low-frequency stimulation (SLFS). In contrast synapses at the proximal site could not express L-LTD. Thus, in the present study, we wanted to investigate whether or not synapses of apical dendritic branch at the proximal location could induce and maintain LTD and its related properties in in vitro rat hippocampal slices. Results indicated that the SLFS in the distal and proximal region triggered the plasticity related proteins (PRP) synthesis in both regions, as evident by the induction and maintenance of L-LTD in the distal region by virtue of synaptic and cross-tagging. In addition, the application of emetine at the time of proximal input stimulation prevented the transition of early-LTD (E-LTD) into L-LTD at the distal region, proving PRP synthesis at the proximal site. Further, it was observed that weak low-frequency stimulation (WLFS) could induce E-LTD in the proximal region along with LTD-specific tag-setting at the synapses. In conclusion, the current study suggests unique findings that the synaptic and cross-tagging mediate L-LTD expression is maintained in the proximal location of hippocampus apical CA1 dendrites.

Upconverting Nanoparticles as a New Bio-Imaging Strategy-Investigating Intracellular Trafficking of Endogenous Processes in Neural Tissue.

In recent years, rare-earth-doped upconverting nanoparticles (UCNPs) have been widely used in different life sciences due to their unique properties. Nanoparticles have become a multifunctional and promising new approach to neurobiological disorders and have shown extraordinary application potential to overcome the problems related to conventional treatment strategies. This study evaluated the internalization mechanisms, bio-distribution, and neurotoxicity of NaYF4:20%Yb3+,2%Er3+ UCNPs in rat organotypic hippocampal slices. TEM results showed that UCNPs were easily internalized by hippocampal cells and co-localized with selected organelles inside neurons and astrocytes. Moreover, the UCNPs were taken into the neurons via clathrin- and caveolae-mediated endocytosis. Propidium iodide staining and TEM analysis did not confirm the adverse effects of UCNPs on hippocampal slice viability and morphology. Therefore, UCNPs may be a potent tool for bio-imaging and testing new therapeutic strategies for brain diseases in the future.

A Neurospheroid-Based Microrobot for Targeted Neural Connections in a Hippocampal Slice.

Functional restoration by the re-establishment of cellular or neural connections remains a major challenge in targeted cell therapy and regenerative medicine. Recent advances in magnetically powered microrobots have shown potential for use in controlled and targeted cell therapy. In this study, a magnetic neurospheroid (Mag-Neurobot) that can form both structural and functional connections with an organotypic hippocampal slice (OHS) is assessed using an ex vivo model as a bridge toward in vivo application. The Mag-Neurobot consists of hippocampal neurons and superparamagnetic nanoparticles (SPIONs); it is precisely and skillfully manipulated by an external magnetic field. Furthermore, the results of patch-clamp recordings of hippocampal neurons indicate that neither the neuronal excitabilities nor the synaptic functions of SPION-loaded cells are significantly affected. Analysis of neural activity propagation using high-density multi-electrode arrays shows that the delivered Mag-Neurobot is functionally connected with the OHS. The applications of this study include functional verification for targeted cell delivery through the characterization of novel synaptic connections and the functionalities of transported and transplanted cells. The success of the Mag-Neurobot opens up new avenues of research and application; it offers a test platform for functional neural connections and neural regenerative processes through cell transplantation.

Investigating the Mitoprotective Effects of S1P Receptor Modulators Ex Vivo Using a Novel Semi-Automated Live Imaging Set-Up.

In multiple sclerosis (MS), mitochondrial alterations appear to contribute to disease progression. The sphingosine-1-phosphate receptor modulator siponimod is approved for treating secondary progressive MS. Its preceding compound fingolimod was shown to prevent oxidative stress-induced alterations in mitochondrial morphology. Here, we assessed the effects of siponimod, compared to fingolimod, on neuronal mitochondria in oxidatively stressed hippocampal slices. We have also advanced the model of chronic organotypic hippocampal slices for live imaging, enabling semi-automated monitoring of mitochondrial alterations. The slices were prepared from B6.Cg-Tg(Thy1-CFP/COX8A)S2Lich/J mice that display fluorescent neuronal mitochondria. They were treated with hydrogen peroxide (oxidative stress paradigm) ± 1 nM siponimod or fingolimod for 24 h. Afterwards, mitochondrial dynamics were investigated. Under oxidative stress, the fraction of motile mitochondria decreased and mitochondria were shorter, smaller, and covered smaller distances. Siponimod partly prevented oxidatively induced alterations in mitochondrial morphology; for fingolimod, a similar trend was observed. Siponimod reduced the decrease in mitochondrial track displacement, while both compounds significantly increased track speed and preserved motility. The novel established imaging and analysis tools are suitable for assessing the dynamics of neuronal mitochondria ex vivo. Using these approaches, we showed that siponimod at 1 nM partially prevented oxidatively induced mitochondrial alterations in chronic brain slices.

Glioblastoma-Derived Three-Dimensional Ex Vivo Models to Evaluate Effects and Efficacy of Tumor Treating Fields (TTFields).

Glioblastoma (GBM) displays a wide range of inter- and intra-tumoral heterogeneity contributing to therapeutic resistance and relapse. Although Tumor Treating Fields (TTFields) are effective for the treatment of GBM, there is a lack of ex vivo models to evaluate effects on patients' tumor biology or to screen patients for treatment efficacy. Thus, we adapted patient-derived three-dimensional tissue culture models to be compatible with TTFields application to tissue culture. Patient-derived primary cells (PDPC) were seeded onto murine organotypic hippocampal slice cultures (OHSC), and microtumor development with and without TTFields at 200 kHz was observed. In addition, organoids were generated from acute material cultured on OHSC and treated with TTFields. Lastly, the effect of TTFields on expression of the Ki67 proliferation marker was evaluated on cultured GBM slices. Microtumors exhibited increased sensitivity towards TTFields compared to monolayer cell cultures. TTFields affected tumor growth and viability, as the size of microtumors and the percentage of Ki67-positive cells decreased after treatment. Nevertheless, variability in the extent of the response was preserved between different patient samples. Therefore, these pre-clinical GBM models could provide snapshots of the tumor to simulate patient treatment response and to investigate molecular mechanisms of response and resistance.

Ethanol sustains phosphorylated tau protein in the cultured neonatal rat hippocampus: Implications for fetal alcohol spectrum disorders.

Fetal Alcohol Spectrum Disorders (FASDs) are comprised of developmental, behavioral, and cognitive abnormalities caused by prenatal alcohol exposure, affecting an estimated 2%-5% of children and costing $4 billion annually in the United States. While some behavioral therapies help, the neurobiological mechanisms that underpin FASDs need further elucidation for development of effective pharmacotherapeutics. The role of the tau protein in the hippocampus is likely to be involved. Tau catalyzes microtubule polymerization in developing neurons. However, this function can become disrupted by hyperphosphorylation. Many of the cognitive deficits observed in neurodegenerative tauopathies overlap to some degree with what is observed in juvenile developmental disabilities, such as FASDs (e.g., selective memory, executive dysfunction). Thus, tau protein phosphorylation may be one important mechanism of dysfunction in FASDs. The purpose of this study is to provide an empirical basis for a tauopathic characterization of FASDs. To do so, hippocampal slices were extracted from rats at postnatal day 10 (PND10); hippocampal slices were then exposed to 5 days of 50-mM ethanol between 6 days in vitro (DIV) and 11DIV. Immunoblots were taken for Total and p-Tau (Threonine231) at 12DIV and 24DIV. Immunohistochemical fluorescent images were taken for p-Tau (Threonine231) at 12DIV and 24DIV. Separate p-Tau measures were taken for the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG). Total Tau protein expression remained unchanged between 12DIV and 24DIV regardless of ethanol condition. In the control group, longer DIV was associated with decreased p-Tau. However, in the ethanol-exposed group, p-Tau was sustained across DIV. This is the first study to show that ethanol exposure sustains tau Threonine231 phosphorylation in the perinatal hippocampus regardless of Total Tau expression. These findings could lead to innovative pharmacotherapeutic targets for the treatment of cognitive deficits seen in FASDs.

Putrescine Intensifies Glu/GABA Exchange Mechanism and Promotes Early Termination of Seizures.

Endogenous anticonvulsant mechanisms represent a reliable and currently underdeveloped strategy against recurrent seizures and may recall novel original therapeutics. Here, we investigated whether the intensification of the astroglial Glu-GABA exchange mechanism by application of the GABA precursor putrescine (PUT) may be effective against convulsive and non-convulsive seizures. We explored the potential of PUT to inhibit spontaneous spike-and-wave discharges (SWDs) in WAG/Rij rats, a genetic model of absence epilepsy. Significant shortening of SWDs in response to intraperitoneally applied PUT has been observed, which could be antagonized by blocking GAT-2/3-mediated astrocytic GABA release with the specific inhibitor SNAP-5114. Direct application of exogenous GABA also reduced SWD duration, suggesting that PUT-triggered astroglial GABA release through GAT-2/3 may be a critical step in limiting seizure duration. PUT application also dose-dependently shortened seizure-like events (SLEs) in the low-[Mg2+] in vitro model of temporal lobe epilepsy. SNAP-5114 reversed the antiepileptic effect of PUT in the in vitro model as well, further confirming that PUT reduces seizure duration by triggering glial GABA release. In accordance, we observed that PUT specifically reduces the frequency of excitatory synaptic potentials, suggesting that it specifically acts at excitatory synapses. We also identified that PUT specifically eliminated the tonic depolarization-induced desynchronization of SLEs. Since PUT is an important source of glial GABA and we previously showed significant GABA release, it is suggested that the astroglial Glu-GABA exchange mechanism plays a key role in limiting ictal discharges, potentially opening up novel pathways to control seizure propagation and generalization.

Neuronal and Astrocytic Morphological Alterations Driven by Prolonged Exposure with Δ9-Tetrahydrocannabinol but Not Cannabidiol.

Cannabis derivatives are largely used in the general population for recreational and medical purposes, with the highest prevalence among adolescents, but chronic use and abuse has raised medical concerns. We investigated the prolonged effects of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in organotypic hippocampal slices from P7 rats cultured for 2 weeks. Cell death in the CA1 subregion of slices was quantified by propidium iodide (PI) fluorescence, pre-synaptic and post-synaptic marker proteins were analysed by Western blotting and neurodegeneration and astrocytic alterations by NeuN and GFAP by immunofluorescence and confocal laser microscopy. The statistical significance of differences was analysed using ANOVA with a post hoc Dunnett w-test (PI fluorescence intensities and Western blots) or Newman-Keuls (immunohistochemistry data) for multiple comparisons. A probability value (P) of < 0.05 was considered significant. Prolonged (72 h) THC or CBD incubation did not induce cell death but caused modifications in the expression of synaptic proteins and morphological alterations in neurons and astrocytes. In particular, the expression of PSD95 was reduced following incubation for 72 h with THC and was increased following incubation with CBD. THC for 72 h caused disorganisation of CA1 stratum pyramidalis (SP) and complex morphological modifications in a significant number of pyramidal neurons and in astrocytes. Our results suggest that THC or CBD prolonged exposure induce different effects in the hippocampus. In particular, 72 h of THC exposure induced neuronal and glia alterations that must draw our attention to the effects that relatively prolonged use might cause, especially in adolescents.

Ferrostatin-1 Polarizes Microglial Cells Toward M2 Phenotype to Alleviate Inflammation After Intracerebral Hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is one of the most lethal stroke types and lacks effective therapeutic regimens. Recently, evidence has suggested the involvement of the ferroptosis inhibitor ferrostatin-1 (Fer-1) in the pathophysiological process of ICH. In this study, we examined the underlying mechanism. METHODS: We induced an in vitro apoptosis model in organotypic hippocampal slice (OHS) using hemoglobin (Hb) and an in vivo ICH model using collagenase. OHSs were treated with MK-801, Fer-1, glutamate, and Hb to assess the impacts of Fer-1 on neuron apoptosis, glutathione peroxidase-4 activity, reactive oxygen species production, inflammation-related factors, expression of M1 markers and M2 markers, and the phagocytic function of microglial cells in vitro. Then, ICH mice were treated with Fer-1 and ruxolitinib to evaluate the effects of Fer-1-orchestrating janus kinase 1/signal transducer and activator of transcription 6 pathway on neurological function, brain water content, hematoma volume, the anti-inflammatory factor, M1 and M2 markers, and the phagocytic function of microglial cells in vivo. RESULTS: Hb or glutamate facilitated glutathione peroxidase dysfunction, reactive oxygen species production, and neuronal apoptosis in OHSs, which was nullified by Fer-1. Fer-1 polarized microglial cells to the M2 phenotype, enhanced their phagocytic function, and prevented inflammation in Hb-induced OHSs. In the ICH mouse model, Fer-1 was found to improve neurological function and promote hematoma absorption. In addition, Fer-1 activated the Fer-1-orchestrating janus kinase 1/signal transducer and activator of transcription 6 pathway, which accelerated microglial M2 polarization, enhanced the phagocytic function of microglial cells, and restrained inflammation in ICH mice. CONCLUSIONS: Overall, our findings suggest that Fer-1 may be a novel mechanism underlying microglial M2 polarization and inflammation after ICH.

Cordycepin suppresses glutamatergic and GABAergic synaptic transmission through activation of A1 adenosine receptor in rat hippocampal CA1 pyramidal neurons.

Cordycepin (known as 3-deoxyadenosine, CRD), a natural product from the valuable traditional Chinese medicine Cordyceps militaris, has been reported to improve cognitive function and modulate neuroprotective effects on the central nervous system (CNS). However, the modulating mechanisms of cordycepin on information processing in hippocampal CA1 pyramidal neurons are not fully understood. To clarify how cordycepin modulates synaptic responses of pyramidal neurons in rat hippocampal CA1 region, we conducted an electrophysiological experiment using whole-cell patch-clamp technique. The spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs, respectively) and the spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs, respectively) recorded by this technique evaluated pure single or multi-synapse responses and enabled us to accurately quantify how cordycepin influenced the pre and postsynaptic aspects of synaptic transmission. The present results showed that cordycepin significantly decreased the frequency of both glutamatergic and GABAergic postsynaptic currents without affecting the amplitude, while these inhibitory effects were antagonized by the A1 adenosine receptor antagonist (DPCPX), but not the A2A (ZM 241385), A2B (MRS1754) and A3 (MRS1191) adenosine receptor antagonists. Taken together, our results suggested that cordycepin had a clear presynaptic effect on glutamatergic and GABAergic transmission, and provided novel evidence that cordycepin suppresses the synaptic transmission through the activation of A1AR.

Variants of Oxidized Regenerated Cellulose and Their Distinct Effects on Neuronal Tissue.

Based on oxidized regenerated cellulose (ORC), several hemostyptic materials, such as Tabotamp®, Equicel® and Equitamp®, have been developed to approach challenging hemostasis in neurosurgery. The present study compares ORC that differ in terms of compositions and properties, regarding their structure, solubility, pH values and effects on neuronal tissue. Cytotoxicity was detected via DNA-binding fluorescence dye in Schwann cells, astrocytes, and neuronal cells. Additionally, organotypic hippocampal slice cultures (OHSC) were analyzed, using propidium iodide, hematoxylin-eosin, and isolectin B4 staining to investigate the cellular damage, cytoarchitecture, and microglia activation. Whereas Equicel® led to a neutral pH, Tabotamp® (pH 2.8) and Equitamp® (pH 4.8) caused a significant reduction of pH (p < 0.001). Equicel® and Tabotamp® increased cytotoxicity significantly in several cell lines (p < 0.01). On OHSC, Tabotamp® and Equicel® led to a stronger and deeper damage to the neuronal tissue than Equitamp® or gauze (p < 0.01). Equicel® increased strongly the number of microglia cells after 24 h (p < 0.001). Microglia cells were not detectable after Tabotamp® treatment, presumably due to an artifact caused by strong pH reduction. In summary, our data imply the use of Equicel®, Tabotamp® or Equitamp® for specific applications in distinct clinical settings depending on their localization or tissue properties.

Cerebral excitability in pup rats prenatally exposed to 1-bromopropane is suppressed by bromide accumulated in the brain.

Previously, we reported that prenatal exposure to 1-bromopropane (1-BP) causes the accumulation of bromide (Br-) in the brain of rat pups. Here, we aimed to investigate the effects of Br- accumulation in rat pups prenatally exposed to 1-BP vapor. Dam rats were exposed to 1-BP (400 or 700 ppm; 1-BP group) by inhalation, or to NaBr (20 mM; Br- group) in drinking water during gestation days 1-20. We also analyzed pentylenetetrazole (PTZ, 60 mg/kg, ip)-induced behavioral changes in pups prenatally exposed to 1-BP or Br- on postnatal day (PND) 14. PTZ-induced epileptic convulsions were inhibited in both 1-BP (700 ppm) and Br- groups. The inhibition of neuronal excitability induced by Br- was evaluated electrophysiologically using the hippocampal slices obtained from PND14-16 pups. PTZ (2 mM) failed to induce epileptiform discharge in the presence of 1.2 mM Br- in the slices obtained from the control group. However, it induced epileptiform discharge following the removal of Br-, by perfusing artificial cerebrospinal fluid into the slices obtained from the Br- group. Our results indicate that Br- accumulates in the brain of neonatal rat pups prenatally exposed to 1-BP vapor suppressed neuronal excitability.

GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices.

Persistent acidosis occurs in ischemia and multiple neurological diseases. In previous studies, acidic stimulation leads to rapid increase in intracellular calcium in neurons. However, it remains largely unclear how a prolonged acidosis alters neuronal signaling. In our previous study, we found that GPR68-mediated PKC activities are protective against acidosis-induced injury in cortical slices. Here, we first asked whether the same principle holds true in organotypic hippocampal slices. Our data showed that 1-h pH 6 induced PKC phosphorylation in a GPR68-dependent manner. Go6983, a PKC inhibitor worsened acidosis-induced neuronal injury in wild type (WT) but had no effect in GPR68-/- slices. Next, to gain greater insights into acid signaling in brain tissue, we treated organotypic hippocampal slices with pH 6 for 1-h and performed a kinome profiling analysis by Western blot. Acidosis had little effect on cyclin-dependent kinase (CDK) or casein kinase 2 activity, two members of the CMGC family, or Ataxia telangiectasia mutated (ATM)/ATM and RAD3-related (ATR) activity, but reduced the phosphorylation of MAPK/CDK substrates. In contrast, acidosis induced the activation of CaMKIIα, PKA, and Akt. Besides these serine/threonine kinases, acidosis also induced tyrosine phosphorylation. Since GPR68 is widely expressed in brain neurons, we asked whether GPR68 contributes to acidosis-induced signaling. Deleting GPR68 had no effect on acidosis-induced CaMKII phosphorylation, attenuated that of phospho-Akt and phospho-PKA substrates, while abolishing acidosis-induced tyrosine phosphorylation. These data demonstrate that prolonged acidosis activates a network of signaling cascades, mediated by AGC kinases, CaMKII, and tyrosine kinases. GPR68 is the primary mediator for acidosis-induced activation of PKC and tyrosine phosphorylation, while both GPR68-dependent and -independent mechanisms contribute to the activation of PKA and Akt.

The Neuroprotective Beta Amyloid Hexapeptide Core Reverses Deficits in Synaptic Plasticity in the 5xFAD APP/PS1 Mouse Model.

Alzheimer's disease (AD) is the most common cause of dementia in the aging population. Evidence implicates elevated soluble oligomeric Aß as one of the primary triggers during the prodromic phase leading to AD, effected largely via hyperphosphorylation of the microtubule-associated protein tau. At low, physiological levels (pM-nM), however, oligomeric Aß has been found to regulate synaptic plasticity as a neuromodulator. Through mutational analysis, we found a core hexapeptide sequence within the N-terminal domain of Aß (N-Aßcore) accounting for its physiological activity, and subsequently found that the N-Aßcore peptide is neuroprotective. Here, we characterized the neuroprotective potential of the N-Aßcore against dysfunction of synaptic plasticity assessed in ex vivo hippocampal slices from 5xFAD APP/PS1 mice, specifically hippocampal long-term potentiation (LTP) and long-term depression (LTD). The N-Aßcore was shown to reverse impairment in synaptic plasticity in hippocampal slices from 5xFAD APP/PS1 model mice, both for LTP and LTD. The reversal by the N-Aßcore correlated with alleviation of downregulation of hippocampal AMPA-type glutamate receptors in preparations from 5xFAD mice. The action of the N-Aßcore depended upon a critical di-histidine sequence and involved the phosphoinositide-3 (PI3) kinase pathway via mTOR (mammalian target of rapamycin). Together, the present findings indicate that the non-toxic N-Aßcore hexapeptide is not only neuroprotective at the cellular level but is able to reverse synaptic dysfunction in AD-like models, specifically alterations in synaptic plasticity.