Hippocampal Slice Cultures Research Articles

The fate of hippocampal synapses depends on the sequence of plasticity-inducing events.

Synapses change their strength in response to specific activity patterns. This functional plasticity is assumed to be the brain's primary mechanism for information storage. We used optogenetic stimulation of rat hippocampal slice cultures to induce long-term potentiation (LTP), long-term depression (LTD), or both forms of plasticity in sequence. Two-photon imaging of spine calcium signals allowed us to identify stimulated synapses and to follow their fate for the next 7 days. We found that plasticity-inducing protocols affected the synapse's chance for survival: LTP increased synaptic stability, LTD destabilized synapses, and the effect of the last stimulation protocol was dominant over earlier stimulations. Interestingly, most potentiated synapses were resistant to depression-inducing protocols delivered 24 hr later. Our findings suggest that activity-dependent changes in the transmission strength of individual synapses are transient, but have long-lasting consequences for synaptic lifetime.

Electrokinetic infusions into hydrogels and brain tissue: Control of direction and magnitude of solute delivery

BackgroundDelivering solutes to a particular region of the brain is currently achieved by iontophoresis for very small volumes and by diffusion from a microdialysis probe for larger volumes. There is a need to deliver solutes to particular areas with more control than is possible with existing methods. New MethodElectrokinetic infusions of solutes were performed into hydrogels and organotypic hippocampal slice cultures. Application of an electrical current creates electroosmotic flow and electrophoresis of a dicationic fluorescent solute through organotypic hippocampal tissue cultures or larger hydrogels. Transport was recorded with fluorescence microscopy imaging in real-time. ResultsElectrokinetic transport in brain tissue slice cultures and hydrogels occurs along an electrical current path and allows for anisotropic delivery over distances from several hundred micrometers to millimeters. Directional transport may be controlled by altering the current path. The applied electrical current linearly affects the measured solute fluorescence in our model system following infusions. Comparison with Existing MethodsLocalized drug delivery involves iontophoresis, with diffusion primarily occurring beyond infusion capillaries under current protocols. Pressure-driven infusions for intraparenchymal targets have also been conducted. Superfusion across a tissue surface provides modest penetration, however is unable to impact deeper targets. In general, control over intraparenchymal drug delivery has been difficult to achieve. Electrokinetic transport provides an alternative to deliver solutes along an electrical current path in tissue. ConclusionsElectrokinetic transport may be applied to living systems for molecular transport. It may be used to improve upon the control of solute delivery over that of pressure-driven transport.

Na+, K+-ATPase inhibition induces neuronal cell death in rat hippocampal slice cultures: Association with GLAST and glial cell abnormalities

Na+, K+-ATPase is a highly expressed membrane protein. Dysfunction of Na+, K+-ATPase has been implicated in the pathophysiology of several neurodegenerative and psychiatric disorders, however, the underlying mechanism of neuronal cell death resulting from Na+, K+-ATPase dysfunction is poorly understood. Here, we investigated the mechanism of neurotoxicity due to Na+, K+-ATPase inhibition using rat organotypic hippocampal slice cultures. Treatment with ouabain, a Na+, K+-ATPase inhibitor, increased the ratio of propidium iodide-positive cells among NeuN-positive cells in the hippocampal CA1 region, which was prevented by MK-801 and d-AP5, specific blockers of the N-methyl-d-aspartate (NMDA) receptor. EGTA, a Ca2+-chelating agent, also protected neurons from ouabain-induced injury. We observed that astrocytes expressed the glutamate aspartate transporter (GLAST), and ouabain changed the immunoreactive area of GFAP-positive astrocytes as well as GLAST. We also observed that ouabain increased the number of Iba1-positive microglial cells in a time-dependent manner. Furthermore, lithium carbonate, a mood-stabilizing drug, protected hippocampal neurons and reduced disturbances of astrocytes and microglia after ouabain treatment. Notably, lithium carbonate improved ouabain-induced decreases in GLAST intensity in astrocytes. These results suggest that glial cell abnormalities resulting in excessive extracellular concentrations of glutamate contribute to neurotoxicity due to Na+, K+-ATPase dysfunction in the hippocampal CA1 region.

Epilepsy-on-a-Chip System for Antiepileptic Drug Discovery.

Hippocampal slice cultures spontaneously develop chronic epilepsy several days after slicing and are used as an in vitro model of post-traumatic epilepsy. Here, we describe a hybrid microfluidic-microelectrode array (μflow-MEA) technology that incorporates a microfluidic perfusion network and electrodes into a miniaturized device for hippocampal slice culture based antiepileptic drug discovery. Field potential simulation was conducted to help optimize the electrode design to detect a seizure-like population activity. Epilepsy-on-a-chip model was validated by chronic electrical recording, neuronal survival quantification, and anticonvulsant test. To demonstrate the application ofμflow-MEA in drug discovery, we utilized a two-stage screening platform to identify potential targets for antiepileptic drugs. In Stage I, lactate and lactate dehydrogenase biomarker assays were performed to identify potential drug candidates. In Stage II, candidate compounds were retested withμflow-MEA-based chronic electrical assay to provide electrophysiological confirmation of biomarker results. We screened 12 receptor tyrosine kinases inhibitors, and EGFR/ErbB-2 and cFMS inhibitors were identified as novel antiepileptic compounds. This epilepsy-on-a-chip system provides the means for rapid dissection of complex signaling pathways in epileptogenesis, paving the way for high-throughput antiepileptic drug discovery.

Parvalbumin expression and gamma oscillation occurrence increase over time in a neurodevelopmental model of NMDA receptor dysfunction.

Dysfunction of the N-methyl-d-aspartate receptor (NMDAR) is thought to play a role in the pathophysiology of neurodevelopmental diseases like schizophrenia. To study the effects of NMDAR dysfunction on synaptic transmission and network oscillations, we used hippocampal tissue of NMDAR subunit GluN2A knockout (KO) mice. Field excitatory postsynaptic potentials were recorded in acute hippocampal slices of adult animals. Synaptic transmission was impaired in GluN2A KO slices compared to wild-type (WT) slices. Further, to investigate whether NMDAR dysfunction would alter neurodevelopment in vitro, we used organotypic hippocampal slice cultures of WT and GluN2A KO mice. Immunostaining performed with cultures kept two, seven, 14, 25 days in vitro (DIV) revealed an increasing expression of parvalbumin (PV) over time. As a functional readout, oscillatory activity induced by the cholinergic agonist carbachol was recorded in cultures kept seven, 13, and 26 DIV using microelectrode arrays. Initial analysis focused on the occurrence of delta, theta, beta and gamma oscillations over genotype, DIV and hippocampal area (CA1, CA3, dentate gyrus (DG)). In a follow-up analysis, we studied the peak frequency and the peak power of each of the four oscillation bands per condition. The occurrence of gamma oscillations displayed an increase by DIV similar to the PV immunostaining. Unlike gamma occurrence, delta, theta, and beta occurrence did not change over time in culture. The peak frequency and peak power in the different bands of the oscillations were not different in slices of WT and GluN2A KO mice. However, the level of PV expression was lower in GluN2A KO compared to WT mice. Given the role of PV-containing fast-spiking basket cells in generation of oscillations and the decreased PV expression in subjects with schizophrenia, the study of gamma oscillations in organotypic hippocampal slices represents a potentially valuable tool for the characterization of novel therapeutic drugs.

Melatonin Acts in Synergy with Hypothermia to Reduce Oxygen-Glucose Deprivation-Induced Cell Death in Rat Hippocampus Organotypic Slice Cultures

Background: Hypoxic-ischemic encephalopathy is a major cause of neonatal morbidity. Therapeutic hypothermia, while beneficial, still leaves many treated infants with lifelong disabilities. Thus, adjunctive therapies, such as melatonin, are needed to provide additional neuroprotection. Objectives: The aim of this study was to determine a range of melatonin concentrations that could result in neuroprotective synergy with hypothermia. Methods: Hypoxia-ischemia was simulated by transient oxygen-glucose deprivation (OGD) in organotypic hippocampal slice cultures derived from neonatal rats. Cell damage was quantified by propidium iodide (PI) labeling. Results: Melatonin reduced OGD- induced cell death in a concentration-dependent manner (1–100 μM) with an EC₅₀ of about 25 μM. Hypothermia attenuated cell death in a time-dependent manner, with a nearly full protection upon 24-h exposure (78%) and partial protection (40%) upon 6-h exposure. When submaximal effective concentrations of melatonin (25 or 50 μM, resulting in 54 and 64% protection) were combined with 6 h of hypothermia, nearly full protection (73 and 78%, respectively; p < 0.05 and p < 0.01) was observed. Conclusion: Melatonin acts in synergy with hypothermia in attenuating OGD-induced damage in organotypic hippocampal cultures. This reductionist approach allows the determination of a range of concentrations of melatonin capable of enhancing hypothermic neuroprotection. This information, coupled with pharmacokinetic data, will help to define the therapeutic dosage of melatonin in vivo and, ultimately, in patients.

Inflammatory Cytokines Facilitate the Sensitivity of P2X7 Receptors Toward Extracellular ATP at Neural Progenitor Cells of the Rodent Hippocampal Subgranular Zone

Organotypic hippocampal slice cultures were used to model the effects of neuroinflammatory conditions following an epileptic state on functional P2X7 receptors (Rs) of subgranular zone (SGZ) neural progenitor cells (NPCs). The compound, 4-aminopyridine (4-AP), is known to cause pathological firing of neurons, consequently facilitating the release of various transmitter substances including ATP. Lipopolysaccharide (LPS) and interleukin-1β (IL-1β) both potentiated the dibenzoyl-ATP (Bz-ATP)-induced current amplitudes in NPCs, although via different mechanisms. Whereas LPS acted via promoting ATP release, IL-1β acted via its own receptor to directly influence P2X7Rs. Thus, the effect of LPS was inhibited by the ecto-ATPase inhibitor, apyrase, but not by the IL-1β antagonist, interleukin-1RA (IL-1RA); by contrast, the effect of IL-1β was inhibited by IL-1RA, but not by apyrase. Eventually, incubation with 4-AP upregulated the number of nestin/glial fibrillary acidic protein/P2X7R immunoreactive cells and their appropriate staining intensity, suggesting increased synthesis of P2X7Rs at NPCs. In conclusion, inflammatory cytokines accumulating after epilepsy-like neuronal firing may facilitate the effect of endogenous ATP at P2X7Rs of NPCs, thereby probably promoting necrosis/apoptosis and subsequent cell death.

340 Convection-Enhanced Delivery of Macromolecules to the Brain Using Electrokinetic Transport

Electrokinetic transport in brain tissue represents the movement of molecules due to an applied electric field and the interplay between the electrophoretic and electroosmotic velocities that are developed. This dissertation provides a framework for understanding electrokinetic transport and how it may be utilized for short-distance ejections, relevant to capillary iontophoresis, and long-distance infusions, for the clinical management of malignant brain tumors as a novel convection-enhanced drug delivery system.In particular, electrokinetic transport was first analyzed in a series of poly(acrylamide-co-acrylic acid) hydrogels that demonstrated varying electroosmotic velocities. Moreover, a hydrogel was synthesized to mimic the electrokinetic properties of organotypic hippocampal slice cultures (OHSC), as a surrogate for brain tissue. Short- and long-distance capillary infusions of molecules into the hydrogels and OHSC provided a framework to understand the relevant phenomena, such as the effect of varying the capillary tip size, applied electrical current, ζ-potential of the capillary or the outside matrix, infusion time, tortuosity, and properties of the solute (including molecular weight and electrophoretic mobility). Control of the directional transport of molecules was also demonstrated over a distance of several hundred micrometers to millimeters. Finally, electrokinetic infusions were conducted in vivo in the adult rat brain, with results compared to those of pressure-driven infusions.The experiments and results described in this dissertation provide a foundation for further development, by presenting a methodical means to increase the ejection profile and attain clinically relevant penetration distances while minimizing adverse effects to the brain tissue, including from the electric field itself. The rate of electrokinetic transport is greater than the rate of diffusion, and therefore it represents a novel form of convection-enhanced drug delivery system.

Pyrethroid bifenthrin induces oxidative stress, neuroinflammation, and neuronal damage, associated with cognitive and memory impairment in murine hippocampus

Exposure to synthetic pyrethroid (SPs) pesticides such as bifenthrin (BF) has been associated with adverse neurodevelopmental outcomes and cognitive impairments, but the underlying neurobiological mechanism is poorly understood so far. The present study has been designed to evaluate changes in behavior and in biomarkers of oxidative stress and neuroinflammation in the hippocampus of rats subchronically treated with BF. Rats exposed daily to BF at doses of 0.6 and 2.1 mg/kg b. w. for 60 days exhibited spatial and cognitive impairments as well as memory dysfunction after 60 days. This repeated BF treatment also significantly increased mRNA expression of pro-inflammatory cytokines tumor necrosis factor (TNF-α), interleukin (IL-1β), (IL-6), nuclear factor erythroid-2 (Nrf2), cyclooxygenase-2 (COX-2), nuclear factor-kappaB pathway (NF-kappaB), and prostaglandin E2 (PGE2) in the hippocampus. It further resulted in a significant increase in protein levels of Nrf2, COX-2, microsomal prostaglandin synthase-1 (mPGES-1) and NF-kappaB. This was accompanied by oxidative/nitrosative stress in the hippocampus of treated rats, as shown by increased levels of malondialdehyde (MDA), protein carbonyls (PCO), and nitric oxide (NO), and reduced levels of enzymatic (catalase, superoxide dismutase, and glutathione peroxidase) and non-enzymatic (reduced glutathione) antioxidants. The data are in line with those obtained in organotypic hippocampal slice cultures (OHSCs) isolated from mouse brain and exposed to BF for 72 h, showing neuronal death only at the high dose of 20 μM when compared to controls.These findings suggest that exposure to BF induces neuronal damage, alters redox state, and causes neuroinflammation in the hippocampus, which might lead to cognitive and memory impairment.

Regulation of P2X7 receptor function of neural progenitor cells in the hippocampal subgranular zone by neuronal activity in the dentate gyrus

P2X7 receptors (Rs) mediate apoptosis/necrosis in neuronal and non-neuronal systems. Patch-clamp recordings from dentate gyrus (DG) granule cells in acutely prepared hippocampal slices of mice showed that incubation with 4-aminopyridine (4-AP) causes an excitability increase. This led to an enhanced sensitivity of P2X7Rs of the underlying subgranular zone neural progenitor cells (NPCs) towards dibenzoyl-ATP (Bz-ATP). The glutamatergic agonists NMDA and AMPA, as well as the purinergic agonist ATP also increased the Bz-ATP-induced current amplitudes (IBzATP). Tetrodotoxin as well as the standard antiepileptic drugs phenytoin, valproic acid and gabapentin counteracted the effect of 4-AP, most likely by decreasing the firing rate and/or action potential duration of DG granule cells and in consequence the release of ATP/glutamate onto NPCs. Experiments with organotypic hippocampal slice cultures confirmed these results also under conditions when 4-AP was applied for longer time periods and at much lower concentrations than used in acute slices. It was concluded that pathological firing modelled by 4-AP might trigger a sensitivity increase of P2X7Rs leading to necrosis/apoptosis of NPCs with the subsequent decrease of NPC, and in consequence, granule cell number. Hence, supersensitive P2X7Rs may exert a beneficial counter-regulatory effect by reducing the chances for the evolution of chronic temporal lobe epilepsy by ectopically located granule cells.

Stimulation-induced structural changes at the nucleus, endoplasmic reticulum and mitochondria of hippocampal neurons

Neurons exhibit stimulation-induced ultrastructural changes such as increase of thickness and curvature of the postsynaptic density, decrease in contact area between subsurface cistern and plasma membrane, and formation of CaMKII clusters and synaptic spinules. These structural characteristics help in identifying the activity state of the neuron and should be taken into consideration when interpreting ultrastructural features of the neurons. Here in organotypic hippocampal slice cultures where experimental conditions can be easily manipulated, two additional features are documented in forebrain neurons as reliable benchmarks for stimulation-induced structural changes: (1) The neuronal nucleus showed conspicuous clustering of dark chromatin, and (2) the endoplasmic reticulum formed stacks with a uniform gap of ~ 13 nm filled with dark materials. Both structural changes progressed with time and were reversible upon returning the slice cultures to control medium. These stimulation-induced structural changes were also verified in dissociated hippocampal neuronal cultures and perfusion-fixed brains. In hippocampal slice cultures, the neuronal chromatin clustering was detectable within 30 s of depolarization with high K+ (90 mM) or treatment with NMDA (50 μM). In contrast, the formation of ER cisternal stacks did not become apparent for another 30 s. Importantly, in dissociated neuronal cultures, when the extracellular calcium was chelated by EGTA, treatment with high K+ no longer induced these changes. These results indicate that the stimulation-induced chromatin clustering and formation of ER stacks in neurons are calcium-dependent. Additionally, mitochondria in neuronal somas of tissue culture samples consistently became swollen upon stimulation. However, swollen mitochondria were also present in some neurons of control samples, but could be eliminated by blocking basal activity or calcium influx. This calcium-dependent structural change of mitochondria is specific to neurons. These structural changes may bring insights to the neuron’s response to intracellular calcium rise upon stimulation.

Glycine receptors are involved in hippocampal neuronal damage caused by oxygen-glucose deficiency.

Glycine receptors (GlyRs) belong to the family of ligand-gated cys-loop receptors and effectuate fast inhibitory neurotransmission in central nervous system (CNS). They are involved in numerous physiological processes, such as movement, respiration, and processing of sensory information, as well as in regulation of neuronal excitability in different brain regions. GlyRs play important role in the maintenance of excitatory/inhibitory balance in the hippocampus and participate in the development of various brain pathologies. In the present study, we have examined a surface expression of GlyRs by pyramidal neurons and astrocytes in control and after 30 min of oxygen-glucose deprivation (OGD) in the organotypic culture of hippocampal slices. Our investigation has demonstrated a decrease in GlyR-positive staining associated with pyramidal neurons and relative stability of GlyRs expression at the surface of astrocytes 4 hs after OGD. These data indicate that GlyRs dysfunction may represent a significant additional factor leading to enhanced neuronal damage induced by OGD. Pharmacological modulation of GlyRs is a promising venue of research for the correction of negative consequences of oxygen-glucose deficiency.

The recovery from transient cognitive dysfunction induced by propofol was associated with enhanced autophagic flux in normal healthy adult mice

Propofol is the most widely accepted intravenous anesthetic available for clinical use. However, neurotoxicity of propofol in the developing brain has been reported. This study investigated the effects of propofol on cognitive function in normal healthy adult mice. Thirty-three GFP-LC3 adult mice were included. Propofol was injected for anesthesia (n = 22). The sham control (n = 11) received intralipid injections. The mice completed a Y-maze test on 3 and 7 days after being anesthetized. Western blotting, immunofluorescence staining, and transmission electron microscopic (TEM) analyses were performed with their hippocampi. In addition, we conducted a separate ex vivo experiment using organotypic hippocampal slice cultures (OHSCs) to investigate the effects of propofol on induced autophagy. There was a significantly lower percentage of alternation in the Y-maze test on day 3 after propofol anesthesia than the control, but no difference was observed on day 7. Western blot analyses and immunofluorescence assays showed that the levels of cognitive function-related proteins significantly decreased in the propofol group compared to the control on day 3 but had recovered by day 7. In terms of autophagy-related proteins, western blot analyses and immunofluorescence assays showed that propofol increased autophagic induction, flux, and degradation of autophagosomes. Ex vivo experiments showed that propofol enhanced autophagic flux of the induced autophagy. In conclusion, although transient cognitive dysfunction occurred, adult mice recovered their cognitive function after the administration of propofol anesthesia. And this finding may be associated with enhanced autophagic flux.

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

Bicuculline Reverts the Neuroprotective Effects of Meloxicam in an Oxygen and Glucose Deprivation (OGD) Model of Organotypic Hippocampal Slice Cultures

We previously demonstrated that the non-steroidal anti-inflammatory agent meloxicam has neuroprotective effects in an oxygen and glucose deprivation model (OGD) of rat organotypic hippocampal slice cultures. We wondered if GABAergic transmission changed the neuroprotective effects of meloxicam and if meloxicam was able to modulate endoplasmic reticulum stress (ER stress) in this model. Mortality was measured using propidium iodide. Western blot assays were performed to measure levels of cleaved and non-cleaved caspase-3 to quantify apoptosis, while levels of GRP78, GRP94 and phosphorylated eIF2α were used to detect unfolded protein response (UPR). Transcript levels of GRP78, GRP94 and GABAergic receptor α, β, and γ subunits were measured by real-time quantitative polymerase chain reaction (qPCR). In the present study, we show that the presence of meloxicam in a 30 min OGD assay, followed by 24 h of normoxic conditions, presented an antiapoptotic effect. The simultaneous presence of the GABAA receptor antagonist, bicuculline, in combination with meloxicam blocked the neuroprotective effect provided by the latter. However, in light of its effects on caspase 3 and PARP, bicuculline did not seem to promote the apoptotic pathway. Our results also showed that meloxicam modified the unfolded protein response (UPR), as well as the transcriptional response of different genes, including the GABAA receptor, alpha1, beta3 and gamma2 subunits. We concluded that meloxicam has a neuroprotective anti-apoptotic action, is able to enhance the UPR independently of the systemic anti-inflammatory response and its neuroprotective effect can be inhibited by blocking GABAA receptors.

NMDA-receptor inhibition and oxidative stress during hippocampal maturation differentially alter parvalbumin expression and gamma-band activity

Dysfunction of parvalbumin (PV)-expressing interneurons is thought to underlie the alterations of gamma-band oscillations observed in schizophrenia. Although the pathomechanisms of this disease remain unclear, oxidative stress induced by NMDA receptor (NMDAR) hypofunction and decreased glutathione (GSH) synthesizing capacity have been shown to lead to PV-loss and aberrant oscillatory activity. However, the individual contributions of NMDAR-inhibition and GSH-depletion to the developmental alterations observed in schizophrenia are largely unknown. We therefore investigated each condition in isolation using hippocampal slice cultures wherein interneuron maturation occurs entirely in vitro. Although both treatments caused oxidative stress, NMDAR-inhibition led to an immediate reduction in gamma oscillation frequency and a delayed loss of PV. In contrast, GSH-depletion immediately decreased PV expression and increased power, without affecting frequency. Hence, although disturbances of PV-expression and gamma oscillations coexist in schizophrenia, they can arise from separate pathological processes.

3,4-Dihydroxyphenylethanol Assuages Cognitive Impulsivity in Alzheimer's Disease by Attuning HPA-Axis via Differential Crosstalk of α7 nAChR with MicroRNA-124 and HDAC6.

Cognitive impulsivity, a form of suboptimal cost-benefit decision making, is an illustrious attribute of an array of neurodegenerative diseases including Alzheimer's disease (AD). In this study, a delay discounting paradigm was used to assess the effect of 3,4-dihydroxyphenylethanol (DOPET) on cognitive impulsivity, in an oA42i (oligomeric amyloid β1-42 plus ibotenic acid) induced AD mouse model, using a nonspatial T-maze task. The results depicted that oA42i administration elevated cognitive impulsivity, whereas DOPET treatment attenuated the impulsive behavior and matched the choice of the sham-operated controls. In addition, DOPET treatment has ameliorated the anxiety-like behavior in the oA42i-challenged mice. Probing the molecular signaling cascades underpinning these functional ramifications in the oA42i-challenged mice revealed reduced cholinergic (α7 nAChR; alpha 7 nicotinic acetylcholine receptor) function, dysregulated hypothalamic-pituitary-adrenal (HPA) axis (manifested by amplified glucocorticoid receptor expression and plasma corticosterone levels), and also aberrations in the neuroepigenetic (microRNA-124, HDAC6 (histone deacetylase 6), and HSP90 (heat-shock protein 90) expressions) as well as nucleocytoplasmic (importin-α1 expression and nuclear ultra-architecture) continuum. Nonetheless, DOPET administration ameliorated these perturbations and the observations were in line with that of the sham-operated mice. Further validation of the results with organotypic hippocampal slice cultures (OHSCs) confirmed the in vivo findings. We opine that HPA-axis attunement by DOPET might be orchestrated through the α7 nAChR-mediated pathway. Based on these outcomes, we posit that 3,4-dihydroxyphenylethanol might be a potential multimodal agent for the management of cognitive impulsivity and neuromolecular quagmire in AD.

Improved method for MR microscopy of brain tissue cultured with the interface method combined with Lenz lenses

MR in microscopy can non-invasively image the morphology of living tissue, which is of particular interest in studying the mammalian brain. Many studies use live animals for basic research on brain functions, disease pathogenesis, and drug development. However, in vitro systems are on the rise, due to advantages such as the absence of a blood-brain barrier, predictable pharmacokinetics, and reduced ethical restrictions. Hence, they present an inexpensive and adequate technique to answer scientific questions and to perform drug screenings. Some publications report the use of acute brain slices for MR microscopy studies, but these only permit single measurements over several hours. Repetitive MR measurements in longitudinal studies demand an MR-compatible setup which allows cultivation for several days or weeks, and hence properly functioning in vitro systems. Organotypic hippocampal slice cultures (OHSC) are a well-established and robust in vitro system which still exhibits most histological hallmarks of the hippocampal network in vivo. An MR compatible incubation platform is introduced in which OHSC are cultivated according to the interface method following Stoppini et al. In this cultivation method a tissue slice is placed onto a membrane with nutrition medium underneath and a gas atmosphere above, where the air-tissue interface perpendicular to the B0 field induces strong artefacts. We introduce a handling protocol that suppresses these artefacts and increases signal quality significantly to acquire high resolution images of tissue slices. An additional challenge is the lack of available of MR microscopy equipment suitable for small animal scanners. A Lenz lens with an attached capacitor can dramatically increase the SNR in these cases, and wirelessly bring the detection system in close proximity to the sample without compromising the OHSC system through the introduction of wired detectors. The resultant signal gain is demonstrated by imaging a PFA-fixed brain slice with a 72 mm diameter volume coil without a Lenz lens, and with a broadband and a self-resonant Lenz lens. In our setting, the self-resonant Lenz lens increases the SNR 10-fold over using the volume coil only.

Semaphorin 4D promotes inhibitory synapse formation and suppresses seizures in vivo.

We previously discovered a role for the extracellular domain of the transmembrane protein semaphorin 4D (Sema4D) as a fast-acting, selective, and positive regulator of functional γ-aminobutyric acid (GABA)ergic synapse formation in hippocampal neuronal culture. We also demonstrated that Sema4D treatment increases inhibitory tone and suppresses hyperexcitability in an organotypic hippocampal slice culture model of epilepsy. Here, we investigate the ability of Sema4D to promote GABAergic synapse formation and suppress seizure activity in vivo in adult mice. We performed a 3-hour, intrahippocampal infusion of Sema4D or control protein into the CA1 region of adult mice. To quantify GABAergic presynaptic bouton density, we performed immunohistochemistry on hippocampal tissue sections isolated from these animals using an antibody that specifically recognizes the glutamic acid decarboxylase isoform 65 protein (GAD65), which is localized to presynaptic GABAergic boutons. To assess seizure activity, we employed 2 in vivo mouse models of epilepsy, intravenous (iv) pentylenetetrazol (PTZ) and hippocampal electrical kindling, in the presence or absence of Sema4D treatment. We monitored seizure activity by behavioral observation or electroencephalography (EEG). To assay the persistence of the Sema4D effect, we monitored seizure activity and measured the density of GAD65-positive presynaptic boutons 3 or 48 hours after Sema4D infusion. Sema4D-treated mice displayed an elevated density of GABAergic presynaptic boutons juxtaposed to hippocampal pyramidal neuron cell bodies, consistent with the hypothesis that Sema4D promotes the formation of new inhibitory synapses in vivo. In addition, Sema4D acutely suppressed seizures in both the PTZ and electrical kindling models. When we introduced a 48-hour gap between Sema4D treatment and the seizure stimulus, seizure activity was indistinguishable from controls. Moreover, immunohistochemistry on brain sections or hippocampal slices isolated 3 hours, but not 48 hours, after Sema4D treatment displayed an increase in GABAergic bouton density, demonstrating temporal correlation between the effects of Sema4D on seizures and GABAergic synaptic components. Our findings suggest a novel approach to treating acute seizures: harnessing synaptogenic molecules to enhance connectivity in the inhibitory network.

Inflammatory and cytotoxic effects of bifenthrin in primary microglia and organotypic hippocampal slice cultures

BackgroundPyrethroids, such as bifenthrin (BF), are among the most widely used class of insecticides that pose serious risks to human and wildlife health. Pyrethroids are proposed to affect astrocytic functions and to cause neuron injury in the central nervous system (CNS). Microglia are key cells involved in innate immune responses in the CNS, and microglia activation has been linked to inflammation and neurotoxicity. However, little information is known about the effects of BF-induced toxicity in primary microglial cells as well as in organotypic hippocampal slice cultures (OHSCs).MethodsOxidative stress and inflammatory responses induced by BF were evaluated in primary microglial cells and OHSCs incubated with different concentrations of BF (1–20 μM) for 4 and 24 h. mRNA and protein synthesis of cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), nuclear erythroid-2 like factor-2 (Nrf-2), and microsomal prostaglandin synthase-1 (mPGES-1) was also studied by qPCR and Western blot. Cell viability was analyzed by MTT-tetrazolio (MTT) and lactate dehydrogenase (LDH) assays. Neurotoxicity in OHSCs was analyzed by propidium iodide (PI) staining and confocal microscopy.ResultsExposure of microglial cells to BF for 24 h resulted in a dose-dependent reduction in the number of viable cells. At sub-cytotoxic concentrations, BF increased reactive oxygen species (ROS), TNF-alpha synthesis, and prostaglandin E2 (PGE2) production, at both 4- and 24-h time points, respectively. Furthermore, BF incubation decreased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and increased lipid peroxidation, protein oxidation, and H2O2 formation. In addition, BF significantly induced protein synthesis and mRNA expression of oxidative and inflammatory mediators after 4 and 24 h, including Nrf-2, COX-2, mPGES-1, and nuclear factor kappaB (NF-kappaB). A 24-h exposure of OHSCs to BF also increased neuronal death compared to untreated controls. Furthermore, depletion of microglia from OHSCs potently enhanced neuronal death induced by BF.ConclusionsOverall, BF exhibited cytotoxic effects in primary microglial cells, accompanied by the induction of various inflammatory and oxidative stress markers including the Nrf-2/COX-2/mPGES-1/NF-kappaB pathways. Moreover, the study provided evidence that BF induced neuronal death in OHSCs and suggests that microglia exert a protective function against BF toxicity.