Hippocampal Slice Cultures Research Articles

Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to ∼10 Hz. However, to resolve glutamate dynamics during high-frequency bursts, faster indicators are required. Here, we report the development of fast (iGlu f ) and ultrafast (iGlu u ) variants with comparable brightness but increased Kd for glutamate (137 μM and 600 μM, respectively). Compared with iGluSnFR, iGlu u has a sixfold faster dissociation rate in vitro and fivefold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGlu u is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGlu u responses during 100-Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high-frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.

VOLTAGE‐GATED SODIUM CHANNEL ACTIVATION TRIGGERS CALCIUM INFLUX AND HIPPOCAMPAL NEURON COMPLEXITY

Background and significanceAfter stroke, the molecular mechanisms underlying recovery are similar to the development of the nervous system. During development, neuronal activity regulates both the morphology and connectivity of neurons. Neuronal activity primarily involves voltage‐gated sodium channel (VGNaC) activation. Calcium signaling through the N‐methyl‐D‐aspartate receptor (NMDAR) drives morphological changes such as, dendritic arborization, synaptogenesis, and spinogenesis. VGNaC activation influences NMDAR function making this pathway an attractive therapeutic target for stroke recovery.HypothesisActivation of VGNaCs by PbTx‐2 augments NMDAR signaling resulting in and increased Ca2+ influx leading to dendritic arborization and spinogenesis in hippocampal neurons.Experimental design and MethodsWe used the VGNaC gating modifier, brevetoxin (PbTx‐2), to assess the effects of VGNaC activation on neuronal morphology. We defined the calcium‐signaling pathway triggered by PbTx‐2 in a population of hippocampal neurons (HNs), and evaluated PbTx‐2‐induced effects on dendritic arborization and spinogenesis in organotypic hippocampal slice culture (OHSC).HNscells were prepared from E18 Swiss Webster mouse embryos and plated in 96‐well plates. All experiments were run on (10 Days in vitro). A FlexStation2 was used to monitor PbTx‐2‐induced Ca2+ influx.OHSCSlices were prepared from P5, Thy‐1 YFP transgenic, mouse pups. Slices were treated with Pbtx‐2 18H after plating. A Leica SP8 confocal microscope was used to generate Z‐stack images of neurons or dendrites. Imaris‐XT software was used to reconstruct 3D‐images of the dendritic arbor, run Sholl analysis, and measure spine density.Results and ConclusionIn HNs, the EC50 for PbTx‐2‐induced Ca2+ influx was 376 nM (95% CI, 281–504 nM). The PbTx‐2 triggered Ca2+ influx in HNs was through the reverse mode of operation of Na+/Ca2+ exchanger, the NMDAR and the L‐type Ca2+ channel pathways. In OHSC, four‐day treatment with PbTx‐2 (100 nM) produced significant increases in dendritic arbor complexity as determined by Sholl analysis (p<0.005, ANOVA). PbTx‐2 treatment (300 nM) produced significant increases in spine density (p<0.001, ANOVA). Modifying the gating properties of VGNaC using PbTx‐2 enhanced the morphology of hippocampal neurons. Overall, these studies are consistent with the hypothesis that VGNaC activation may represent a novel pharmacological strategy to regulate NMDAR function and aid in recovery post‐stroke.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuroprotective effect of dexmedetomidine in a murine model of traumatic brain injury

No FDA approved pharmacological therapy is available that would reduce cell death following traumatic brain injury (TBI). Dexmedetomidine (Dex) is a highly selective agonist of alpha-2 adrenergic receptors and has demonstrated neuroprotective effects in hippocampal slice cultures undergoing direct impact. However, no one has tested whether Dex, in addition to its sedative action, has neuroprotective effects in an animal model of TBI. Thus, in the present study, we investigated the effects of Dex on an animal model of TBI. Mice received different doses of Dex (1, 10, or 100 µg/kg bodyweight, n = 10 each group) or saline as control at 1 hour and 12 hours following TBI. The mice treated with Dex lost less cortical tissue than the control mice. Further analysis found that Dex treatment reduced cell death in the cortex and the hippocampus measured by Fluoro-Jade B (FJB) staining, prevented axonal degeneration detected by immunostaining with antibody against β-amyloid precursor protein (β-APP), and protected synapses from elimination with synaptophysin staining. Taken together, in an in vivo murine model of TBI, Dex at the dose of 100 µg/kg not only prevented tissue lesion and cell death, but also reduced axonal injury and synaptic degeneration caused by TBI.

Estrogen receptor beta modulates permeability transition in brain mitochondria

Recent evidence highlights a role for sex and hormonal status in regulating cellular responses to ischemic brain injury and neurodegeneration. A key pathological event in ischemic brain injury is the opening of a mitochondrial permeability transition pore (MPT) induced by excitotoxic calcium levels, which can trigger irreversible damage to mitochondria accompanied by the release of pro-apoptotic factors. However, sex differences in brain MPT modulation have not yet been explored. Here, we show that mitochondria isolated from female mouse forebrain have a lower calcium threshold for MPT than male mitochondria, and that this sex difference depends on the MPT regulator cyclophilin D (CypD). We also demonstrate that an estrogen receptor beta (ERβ) antagonist inhibits MPT and knockout of ERβ decreases the sensitivity of mitochondria to the CypD inhibitor, cyclosporine A. These results suggest a functional relationship between ERβ and CypD in modulating brain MPT. Moreover, co-immunoprecipitation studies identify several ERβ binding partners in mitochondria. Among these, we investigate the mitochondrial ATPase as a putative site of MPT regulation by ERβ. We find that previously described interaction between the oligomycin sensitivity-conferring subunit of ATPase (OSCP) and CypD is decreased by ERβ knockout, suggesting that ERβ modulates MPT by regulating CypD interaction with OSCP. Functionally, in primary neurons and hippocampal slice cultures, modulation of ERβ has protective effects against glutamate toxicity and oxygen glucose deprivation, respectively. Taken together, these results reveal a novel pathway of brain MPT regulation by ERβ that could contribute to sex differences in ischemic brain injury and neurodegeneration.

Effects on Neurons and Hippocampal Slices by Single and Multiple Primary Blast Pressure Waves From Detonating Spherical Cyclotrimethylenetrinitramine (RDX) Explosive Charges.

Threshold shock-impulse levels required to induce cellular injury and cumulative effects upon single and/or multiple exposures are not well characterized. Currently, there are few in vitro experimental models with blast pressure waves generated by using real explosives in the laboratory for investigating the effects of primary blast-induced traumatic brain injury. An in vitro indoor experimental platform is developed using real military explosive charges to accurately represent battlefield blast exposure and to probe the effects of primary explosive blast on dissociated neurons and tissue slices. Preliminary results indicate that physical insults altered membrane permeability, impacted cellular viability, created axonal beadings, and led to synaptic protein loss in hippocampal slice cultures. Injuries from blast under the conditions that were examined did not appear to cause immediate or sustained damage to the cells. Three consecutive primary blasts failed to disrupt the overall cellular integrity in the hippocampal slice cultures and produced a unique type of pathology comprised with distinct reduction in synaptic proteins before cellular deterioration set in. These observed changes might add to the challenges in regard to enhancing our understanding of the complex biochemical and molecular mechanisms caused by primary blast-induced injury.

20-HETE synthesis inhibition promotes cerebral protection after intracerebral hemorrhage without inhibiting angiogenesis

20-HETE, an arachidonic acid metabolite synthesized by cytochrome P450 4A, plays an important role in acute brain damage from ischemic stroke or subarachnoid hemorrhage. We tested the hypothesis that 20-HETE inhibition has a protective effect after intracerebral hemorrhage (ICH) and then investigated its effect on angiogenesis. We exposed hippocampal slice cultures to hemoglobin and induced ICH in mouse brains by intrastriatal collagenase injection to investigate the protective effect of 20-HETE synthesis inhibitor N-hydroxy-N′-(4-n-butyl-2-methylphenyl)-formamidine (HET0016). Hemoglobin-induced neuronal death was assessed by propidium iodide after 18 h in vitro. Lesion volume, neurologic deficits, cell death, reactive oxygen species (ROS), neuroinflammation, and angiogenesis were evaluated at different time points after ICH. In cultured mouse hippocampal slices, HET0016 attenuated hemoglobin-induced neuronal death and decreased levels of proinflammatory cytokines and ROS. In vivo, HET0016 reduced brain lesion volume and neurologic deficits, and decreased neuronal death, ROS production, gelatinolytic activity, and the inflammatory response at three days after ICH. However, HET0016 did not inhibit angiogenesis, as levels of CD31, VEGF, and VEGFR2 were unchanged on day 28. We conclude that 20-HETE is involved in ICH-induced brain damage. Inhibition of 20-HETE synthesis may provide a viable means to mitigate ICH injury without inhibition of angiogenesis.

Intrinsic Inflammation Is a Potential Anti-Epileptogenic Target in the Organotypic Hippocampal Slice Model

Understanding the mechanisms of epileptogenesis is essential to develop novel drugs that could prevent or modify the disease. Neuroinflammation has been proposed as a promising target for therapeutic interventions to inhibit the epileptogenic process that evolves from traumatic brain injury. However, it remains unclear whether cytokine-related pathways, particularly TNFα signaling, have a critical role in the development of epilepsy. In this study, we investigated the role of innate inflammation in an in vitro model of post-traumatic epileptogenesis. We combined organotypic hippocampal slice cultures, representing an in vitro model of post-traumatic epilepsy, with multi-electrode array recordings to directly monitor the development of epileptiform activity and to examine the concomitant changes in cytokine release, cell death, and glial cell activation. We report that synchronized ictal- and interictal-like activities spontaneously evolve in this culture. Dynamic changes in the release of the pro-inflammatory cytokines IL-1β, TNFα, and IL-6 were observed throughout the culture period (3 to 21 days in vitro) with persistent activation of microglia and astrocytes. We found that neutralizing TNFα with a polyclonal antibody significantly reduced ictal discharges, and this effect lasted for 1 week after antibody washout. Neither phenytoin nor an anti-IL-6 polyclonal antibody was efficacious in inhibiting the development of epileptiform activity. Our data show a sustained effect of the anti-TNFα antibody on the ictal progression in organotypic hippocampal slice cultures supporting the critical role of inflammatory mediators in epilepsy and establishing a proof-of-principle evidence for the utility of this preparation to test the therapeutic effects of anti-inflammatory treatments.

Magnolol protects against ischemic-reperfusion brain damage following oxygen-glucose deprivation and transient focal cerebral ischemia.

In the present study, the neuroprotective potential of magnolol against ischemia-reperfusion brain injury was examined via invivo and invitro experiments. Magnolol exhibited strong radical scavenging and antioxidant activity, and significantly inhibited the production of interleukin‑6, tumor necrosis factor‑a and nitrite/nitrate (NOX) in lipopolysaccharide-stimulated BV2 and RAW264.7 cells when applied at concentrations of 10 and 50µM, respectively. Magnolol (100µM) also significantly attenuated oxygen‑glucose deprivation‑induced damage in neonatal rat hippocampal slice cultures, when administered up to 4h following the insult. In a rat model of stable ischemia, compared with a vehicle‑treated ischemic control, pretreatment with magnolol (0.01‑1mg/kg, intravenously) significantly reduced brain infarction following ischemic stroke, and post‑treatment with magnolol (1mg/kg) remained effective and significantly reduced infarction when administered 2h following the onset of ischemia. Additionally, magnolol (0.3 and 1mg/kg) significantly reduced the accumulation of superoxide anions at the border zones of infarction and reduced oxidative damage in the ischemic brain. This was assessed by measuring the levels of NOX, malondialdehyde and myeloperoxidase, the ratio of glutathione/oxidized glutathione and the immunoreactions of 8‑hydroxy‑2'‑deoxyguanosine and 4‑hydroxynonenal. Thus, magnolol was revealed to protect against ischemia‑reperfusion brain damage. This may be partly attributed to its antioxidant, radical scavenging and anti‑inflammatory effects.

Genetically Encoded Glutamate Indicators with Altered Color and Topology.

Glutamate is one of the 20 common amino acids and of utmost importance for chemically mediated synaptic transmission in nervous systems. To expand the color palette of genetically encoded indicators for glutamate, we used protein engineering to develop a red intensity-based glutamate-sensing fluorescent reporter (R-iGluSnFR1). Manipulating the topology of R-iGluSnFR1, and a previously reported green fluorescent indicator, led to the development of noncircularly permutated (ncp) variants. R- and Rncp-iGluSnFR1 display glutamate affinities of 11 μM and 0.9 μM, respectively. We demonstrate that these glutamate indicators are functional when targeted to the surface of HEK-293 cells. Furthermore, we show that Gncp-iGluSnFR enabled reliable visualization of extrasynaptic glutamate in organotypic hippocampal slice cultures, while R-iGluSnFR can reliably resolve action potential-evoked glutamate transients by electrical field stimuli in cultures of dissociated hippocampal neurons.

The Critical Role of IL-10 in the Antineuroinflammatory and Antioxidative Effects of Rheum tanguticum on Activated Microglia.

Rheum tanguticum Maxim. ex Balf. (Rt), a traditional Tibetan medicine, is known to exert various bioactivities, including anti-inflammatory and antioxidative activities. The present study was conducted to investigate anti-inflammatory and antioxidative effects of Rt on activated microglia. Rt (10 μg/ml) significantly inhibited the mean protein level of interleukin-1β (IL-1β) in the organotypic hippocampal slice cultures following treatment with chromogranin A (CGA, 10 nM) and pancreastatin (10 nM), endogenous microglial activators present in senile plaques. Rt also significantly inhibited the expression and production of inflammatory and oxidative molecules, including IL-1β, tumor necrosis factor-α, and nitric oxide, by cultured microglia after treatment with CGA. These effects of Rt are considered to be mediated by the secretion of interleukin-10 (IL-10) from microglia, because neutralizing antibodies against IL-10 significantly canceled these effects. To explore the causative components of Rt responsible for inducing the secretion of IL-10, the effects of seven components of Rt on the IL-10 expression in microglia were examined. Among them, aloe-emodin (10 μM) and (+)-catechin (30 μM) were able to induce the secretion of IL-10 from cultured microglia. Therefore, aloe-emodin and (+)-catechin are deemed responsible for the antineuroinflammatory and antioxidative effects of Rt through the secretion of IL-10 from microglia. Accordingly, Rt is considered potentially useful for the treatment of AD.

Fluorescence detection of Europium-doped very small superparamagnetic iron oxide nanoparticles in murine hippocampal slice cultures.

Fluorescence detection of Europium-doped very small superparamagnetic iron oxide nanoparticles in murine hippocampal slice cultures.

Effects of α7 Nicotinic Receptor Activation on Cell Survival in Rat Organotypic Hippocampal Slice Cultures.

Glutamatergic signaling via N-methyl-D-aspartate receptors (NMDARs) is important for physiological functioning, but can also induce cell death via excitotoxic mechanisms in many neuropathological diseases, such as stroke. Altering the cellular response to excitotoxic insults by modulating the downstream effects of NMDAR activation represents a promising therapeutic approach. For example, α7 nicotinic acetylcholine receptors (α7 nAChRs) signaling has been shown to be able to change NMDA-induced neurotoxicity in some models. However, both neuroprotective and neurotoxic effects have been reported. In this study, we examined the effect of co-activation of α7 nAChRs on NMDA-mediated cell death in rat organotypic hippocampal slice cultures (OHSCs). Our results show that α7 nAChR stimulation did not significantly influence NMDA-induced excitotoxic cell damage as measured by propidium iodide uptake. However, treatment of OHSCs with the α7 nAChR agonist choline alone induced an increase in the propidium iodide signal. Both the α7 nAChR antagonist methyllycaconitine (MLA) and the NMDAR antagonist (RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were able to block this effect in the dentate gyrus and hippocampal subfield CA3.

Activation of EP2 receptor suppresses poly(I: C) and LPS-mediated inflammation in primary microglia and organotypic hippocampal slice cultures: Contributing role for MAPKs.

Brain inflammation is a critical factor involved in neurodegeneration. Recently, the prostaglandin E2 (PGE2 ) downstream members were suggested to modulate neuroinflammatory responses accompanying neurodegenerative diseases. In this study, we investigated the protective effects of prostaglandin E2 receptor 2 (EP2 ) during TLR3 and TLR4-driven inflammatory response using in vitro primary microglia and ex vivo organotypic hippocampal slice cultures (OHSCs). Depletion of microglia from OHSCs differentially affected TLR3 and TLR4 receptor expression. Poly(I:C) induced the production of prostaglandin E2 in OHSCs by increasing cyclooxygenase (COX-2) and microsomal prostaglandin E synthase (mPGES)-1. Besides, stimulation of OHSCs and microglia with Poly(I:C) upregulated EP2 receptor expression. Co-stimulation of OHSCs and microglia with the EP2 agonist butaprost reduced inflammatory mediators induced by LPS and Poly(I:C). In Poly(I:C) challenged OHSCs, butaprost almost restored microglia ramified morphology and reduced Iba1 immunoreactivity. Importantly, microglia depletion prevented the induction of inflammatory mediators following Poly(I:C) or LPS challenge in OHSCs. Activation of EP2 receptor reversed the Poly(I:C)/LPS-induced phosphorylation of the mitogen activated protein kinases (MAPKs) ERK, p38 MAPK and c-Jun N-terminal kinase (JNK) in microglia. Collectively, these data identify an anti-inflammatory function for EP2 signaling in diverse innate immune responses, through a mechanism that involves the mitogen-activated protein kinases pathway.

Preparation of Rat Hippocampal Organotypic Cultures and Application to Study Amyloid β-Peptide Toxicity.

Hippocampal organotypic cultures constitute a very easy but delicate method widely used to study amyloid β-peptide toxicity. This ex vivo technique is performed on tissues isolated from newborn rats. Here, we describe a protocol for the preparation and culture of hippocampal organotypic slices that can be maintained for 14-21days and their application to the study of amyloid β-peptide toxicity.

Anti-aggregant tau mutant promotes neurogenesis

BackgroundThe microtubule-associated protein Tau plays a role in neurodegeneration as well as neurogenesis. Previous work has shown that the expression of the pro-aggregant mutant Tau repeat domain causes strong aggregation and pronounced neuronal loss in the hippocampus whereas the anti-aggregant form has no deleterious effects. These two proteins differ mainly in their propensity to form ß structure and hence to aggregate.MethodsTo elucidate the basis of these contrasting effects, we analyzed organotypic hippocampal slice cultures (OHSCs) from transgenic mice expressing the repeat domain (RD) of Tau with the anti-aggregant mutation (TauRDΔKPP) and compared them with slices containing pro-aggregant TauRDΔK. Transgene expression in the hippocampus was monitored via a sensitive bioluminescence reporter gene assay (luciferase).ResultsThe expression of the anti-aggregant TauRDΔKPP leads to a larger volume of the hippocampus at a young age due to enhanced neurogenesis, resulting in an increase in neuronal number. There were no signs of activation of microglia and astrocytes, indicating the absence of an inflammatory reaction. Investigation of signaling pathways showed that Wnt-5a was strongly decreased whereas Wnt3 was increased. A pronounced increase in hippocampal stem cell proliferation (seen by BrdU) was observed as early as P8, in the CA regions where neurogenesis is normally not observed. The increase in neurons persisted up to 16 months of age.ConclusionThe data suggest that the expression of anti-aggregant TauRDΔKPP enhances hippocampal neurogenesis mediated by the canonical Wnt signaling pathway, without an inflammatory reaction. This study points to a role of tau in brain development and neurogenesis, in contrast to its detrimental role in neurodegeneration at later age.

Long-Term Culture of Organotypic Hippocampal Slice from Old 3xTg-AD Mouse: An ex vivo Model of Alzheimer's Disease.

Objective Conventional methods for organotypic hippocampal tissue slice culture (OHSC) have shown several disadvantages or limitations regarding age of animals used, duration of culture and difficulty using neurodegenerative models. Therefore, we tried to establish OHSC from old 3xTg-Alzheimer’s disease (AD) mice for longer period (over 4 weeks) and to validate utility of this system as a valid platform for translational neuroscience of AD. Methods OHSC was performed with old 3xTg-AD mice (12–14 months), old wild type mice (12–14 months) and young 3xTg-AD mice (2–4 months) using serum-free medium for 4 weeks. Hippocampal structure was evaluated by 4’, 6-diamidino-2-phenylindole (DAPI) intensity and neuronal metabolism was measured by Alamarblue assay. Pathologic characteristics of AD were also investigated; β-amyloid levels by ELISA, amyloid plaque deposition by Thioflavin-S staining, and glial activation by immunohistochemistry. Results Following 4-week culture in serum-free media, hippocampal cells and layers were well preserved in cultured slices from old AD mice as was in those from young AD and old wild type mice. On the contrary, excessive regression of total visible cells was observed in conventional serum-containing medium regardless of genotype of mice. In parallel with this well preserved structure, major pathologic characteristics of AD were also well manifested in hippocampal slices from old AD mice. Conclusion Our findings suggest that long-term OHSC from old 3xTg-AD mouse can serve as a promising ex vivo system for studies on pathophysiology of AD, especially with the minimum number of sacrifice of experimental animals.

Mitochondrial GSH Systems in CA1 Pyramidal Cells and Astrocytes React Differently during Oxygen-Glucose Deprivation and Reperfusion.

Pyramidal cells and astrocytes have differential susceptibility to oxygen-glucose deprivation and reperfusion (OGD-RP). It is known that excessive reactive oxygen species (ROS) in mitochondria initiates cell death, while glutathione (GSH) is one of the major defenses against ROS. Although it is known that astrocytes contain a higher concentration of GSH than neurons, and that astrocytes can provide neurons with GSH, we are unaware of a detailed and quantitative examination of the dynamic changes in the mitochondrial GSH system in the two cell types during OGD-RP. Here, we determined mitochondrial membrane potential and the degrees of oxidation of the mitochondrially targeted roGFP-based sensors for hydrogen peroxide (OxDP) and GSH (OxDG). We also developed a method to estimate the mitochondrial GSH (mGSH) concentration in single cells in the CA1 region of organotypic hippocampal slice cultures at several time-points during OGD-RP. We find that mitochondrial membrane potential drops in pyramidal cells during OGD while it is relatively stable in astrocytes. In both types of cell, the mitochondrial membrane potential decreases during RP. During OGD-RP, mitochondrial peroxide levels are the same. Astrocytic mGSH is more than four times higher than pyramidal cell mGSH (3.2 vs 0.7 mM). Astrocytic mGSH is drained from mitochondria during OGD, whereas in pyramidal cells it remains fairly constant. OxDGSH prior to and during OGD is lower (less oxidized) in pyramidal cells than in astrocytes, but the two nearly converge during RP. The larger changes of redox status in the GSH system in pyramidal cells than astrocytes is an upstream sign of the higher mortality of the pyramidal cells after facing an insult. The pattern of [mGSH] changes in the two cell types could be recognized as another mechanism by which astrocytes protect neurons from transient, extreme conditions.

Endogenous ghrelin-O-acyltransferase (GOAT) acylates local ghrelin in the hippocampus.

Ghrelin is an appetite-stimulating peptide. Serine 3 on ghrelin must be acylated by octanoate via the enzyme ghrelin-O-acyltransferase (GOAT) for the peptide to bind and activate the cognate receptor, growth hormone secretagogue receptor type 1a (GHSR1a). Interest in GHSR1a increased dramatically when GHSR1a mRNA was demonstrated to be widespread in the brain, including the cortex and hippocampus, indicating that it has multifaceted functions beyond the regulation of metabolism. However, the source of octanoylated ghrelin for GHSR1a in the brain, outside of the hypothalamus, is not well understood. Here, we report the presence of GOAT and its ability to acylate non-octanoylated ghrelin in the hippocampus. GOAT immunoreactivity is aggregated at the base of the dentate granule cell layer in the rat and wild-type mouse. This immunoreactivity was not affected by the pharmacological inhibition of GHSR1a or the metabolic state-dependent fluctuation of systemic ghrelin levels. However, it was absent in the GHSR1a knockout mouse hippocampus, pointing the possibility that the expression of GHSR1a may be a prerequisite for the production of GOAT. Application of fluorescein isothiocyanate (FITC)-conjugated non-octanoylated ghrelin in live hippocampal slice culture (but not in fixed culture or in the presence of GOAT inhibitors) mimicked the binding profile of FITC-conjugated octanoylated ghrelin, suggesting that extracellularly applied non-octanoylated ghrelin was acylated by endogenous GOAT in the live hippocampus while GOAT being mobilized out of neurons. Our results will advance the understanding for the role of endogenous GOAT in the hippocampus and facilitate the search for the source of ghrelin that is intrinsic to the brain.

Trajectory Analysis Unveils Reelin's Role in the Directed Migration of Granule Cells in the Dentate Gyrus.

Reelin controls neuronal migration and layer formation. Previous studies in reeler mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in reeler, but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated proopiomelanocortin-EGFP-expressing dentate granule cells in slice cultures from reeler, reeler-like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from reeler and reeler-like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in reeler slices cocultured to wild-type dentate gyrus showed that the reeler neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of reeler slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer.SIGNIFICANCE STATEMENT Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the present study, we took advantage of the expression of proopiomelanocortin-EGFP by newly generated, migrating granule cells to analyze their migratory trajectories in hippocampal slice cultures from wild-type mice and mutants deficient in Reelin signaling. We show that the compartmentalized presence of Reelin is essential for the directionality, but not the actual migratory process or speed, of migrating granule cells leading to their characteristic lamination in the dentate gyrus.

Dendritic Polyglycerol Sulfates in the Prevention of Synaptic Loss and Mechanism of Action on Glia.

Dendritic polyglycerols (dPG), particularly dendritic polyglycerol sulfates (dPGS), have been intensively studied due to their intrinsic anti-inflammatory activity. As related to brain pathologies involving neuroinflammation, the current study examined if dPG and dPGS can (i) regulate neuroglial activation, and (ii) normalize the morphology and function of excitatory postsynaptic dendritic spines adversely affected by the neurotoxic 42 amino acid amyloid-β (Aβ42) peptide of Alzheimer disease (AD). The exact role of neuroglia, such as microglia and astrocytes, remains controversial especially their positive and negative impact on inflammatory processes in AD. To test dPGS effectiveness in AD models we used primary neuroglia and organotypic hippocampal slice cultures exposed to Aβ42 peptide. Overall, our data indicate that dPGS is taken up by both microglia and astrocytes in a concentration- and time-dependent manner. The mechanism of action of dPGS involves binding to Aβ42, i.e., a direct interaction between dPGS and Aβ42 species interfered with Aβ fibril formation and reduced the production of the neuroinflammagen lipocalin-2 (LCN2) mainly in astrocytes. Moreover, dPGS normalized the impairment of neuroglia and prevented the loss of dendritic spines at excitatory synapses in the hippocampus. In summary, dPGS has desirable therapeutic properties that may help reduce amyloid-induced neuroinflammation and neurotoxicity in AD.