Hippocampal-entorhinal Cortex Slices Research Articles

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

L-Theanine Reduces Epileptiform Activity In Brain Slices

L-Theanine Reduces Epileptiform Activity In Brain Slices ABSTRACT Objective: L-theanine is one of the main amino acid of tea plant which can cross the blood brain barrier. In the central nervous system, L-theanine has certain effects on a number of neurotransmitter systems. In this study we tested whether acute application of L-theanine could induce any seizure like or inhibitory activity in the brain slice. Materials and Methods: Effects of L-theanine on epileptiform activity were investigated in two different brain regions. Horizontal hippocampal-entorhinal cortex slices were obtained from 30-35 days old C57BL/6 mice. Extracellular field potentials were recorded from medial entorhinal cortex (EC) and CA3 region of hippocampus. Epileptiform activity was induced by application of 4 Aminopyridine (4AP, 100 µM) in the brain slices and 50 µM L-theanine was applied. From the recordings, the duration and frequencies of ictal like events as well as frequency and amplitude of inter-ictal like activities were calculated. Results: L-theanine alone did not initiate any synchronized activity. Although, bath application of L-theanine after 4AP induces epileptiform acitivities, did not significantly alter the duration and frequency of ictal discharges, L-theanine attenuated the interictal discharges. In EC the frequencies of interictal discharges were significantly decreased. In CA3, the amplitudes of interictal activities were significantly reduced. Conclusion: The present findings suggest that L-theanine do not induce any seizure like event but rather had a suppressive effect on ongoing epileptiform activity. Key Words: Theanine, 4 Aminopyridine, Brain Slice, Epileptiform activity, CA3, Entorhinal cortex

Phoenixin-14 reduces the frequency of interictal-like events in mice brain slices

Phoenixin-14 (PNX-14) has a wide bioactivity in the central nervous system. Its role in the hypothalamus has been investigated, and it has been reported that it is involved in the regulation of excitability in hypothalamic neurons. However, its role in the regulation of excitability in entorhinal cortex and the hippocampus is unknown. In this study, we investigated whether i. PNX-14 induces any synchronous discharges or epileptiform activity and ii. PNX-14 has any effect on already initiated epileptiform discharges. We used 350 µm thick acute horizontal hippocampal-entorhinal cortex slices obtained from 30- to 35-day-old mice. Extracellular field potential recordings were evaluated in the entorhinal cortex and hippocampus CA1 region. Bath application of PNX-14 did not initiate any epileptiform activity or abnormal discharges. 4-Aminopyridine was applied to induce epileptiform activity in the slices. We found that 200nM PNX-14 reduced the frequency of interictal-like events in both the entorhinal cortex and hippocampus CA1 region which was induced by 4-aminopyridine. Furthermore, PNX-14 led to a similar suppression in the total power of local field potentials of 1-120Hz. The frequency or the duration of the ictal events was not affected. These results exhibited for the first time that PNX-14 has a modulatory effect on synchronized neuronal discharges which should be considered in future therapeutic approaches.

Processing of Hippocampal Network Activity in the Receiver Network of the Medial Entorhinal Cortex Layer V.

The interplay between hippocampus and medial entorhinal cortex (mEC) is of key importance for forming spatial representations. Within the hippocampal-entorhinal loop, the hippocampus receives context-specific signals from layers II/III of the mEC and feeds memory-associated activity back into layer V (LV). The processing of this output signal within the mEC, however, is largely unknown. We characterized the activation of the receiving mEC network by evoked and naturally occurring output patterns in mouse hippocampal-entorhinal cortex slices. Both types of glutamatergic neurons (mEC LVa and LVb) as well as fast-spiking inhibitory interneurons receive direct excitatory input from the intermediate/ventral hippocampus. Connections between the two types of excitatory neurons are sparse, and local processing of hippocampal output signals within mEC LV is asymmetric, favoring excitation of far projecting LVa neurons over locally projecting LVb neurons. These findings suggest a new role for mEC LV as a bifurcation gate for feedforward (telencephalic) and feedback (entorhinal-hippocampal) signal propagation.SIGNIFICANCE STATEMENT Patterned network activity in hippocampal networks plays a key role in the formation and consolidation of spatial memories. It is, however, largely unclear how information is transferred to the neocortex for long-term engrams. Here, we elucidate the propagation of network activity from the hippocampus to the medial entorhinal cortex. We show that patterned output from the hippocampus reaches both major cell types of deep entorhinal layers. These cells are, however, only weakly connected, giving rise to two parallel streams of activity for local and remote signal propagation, respectively. The relative weight of both pathways is regulated by local inhibitory interneurons. Our data reveal important insights into the hippocampal-neocortical dialogue, which is of key importance for memory consolidation in the mammalian brain.

Prenatal betamethasone exposure increases corticotropin-releasing hormone expression along with increased hippocampal slice excitability in the developing hippocampus

BackgroundThe objective of this study was to determine whether prenatal exposure to betamethasone alters hippocampal expression of corticotropin-releasing hormone (CRH) and resultant hippocampal circuit excitability. MethodsReal time (RT)-PCR and western blots were used to determine CRH mRNA and protein expression levels, respectively, in hippocampal extracts of two-week old rat pups prenatally primed with betamethasone or saline on gestational day 15. The data were compared to changes in epileptiform activity induced by kainic acid (KA) or depletion of [Mg2+]0 in combined hippocampus-entorhinal cortex slices. ResultsRT-PCR analysis showed 3-fold increased levels of CRH mRNA in hippocampal extracts from prenatally betamethasone-primed pups compared to saline controls (p < 0.05), but no changes in mRNA expression of CRH receptors (1 and 2). Changes in CRH protein isoform ratio in hippocampal extracts suggest 30 % increase in mature CRH levels in betamethasone-primed hippocampi (p < 0.05). No changes in mRNA expression in CRH feedback loop associated genes, GR and FKBP51, were found. Compared to saline-exposed pups, slices from betamethasone-primed pups had faster onset of epileptiform-like activity (inter-ictal discharges and seizure-like-events) after bath application of 4 μM KA (p < 0.05) suggesting a “more hyperexcitable” state. The epileptiform-like activity after KA application was significantly reduced following bath application of a CRH R2 antagonist (p < 0.05) but CRH R1 antagonist had no effect (p > 0.05). Also in the low-Mg2+-induced epileptiform activity, there was increased excitability, in the form of enhanced inter-ictal discharges, in slices from betamethasone primed compared to saline exposed rat pups (p < 0.05). ConclusionsOur study suggests a possible mechanistic link to prenatal betamethasone priming-induced increase in postnatal hippocampal excitability that involves enhanced expression of CRH acting at CRH R2. This is important in regards to the links between prenatal stress/corticosteroid-exposure and syndromes, such as epilepsy, autism spectrum disorders and other psychiatric disorders associated with neuronal hyperexcitability.

Mathematical model of Na-K-Cl homeostasis in ictal and interictal discharges.

Despite big experimental data on the phenomena and mechanisms of the generation of ictal and interictal discharges (IDs and IIDs), mathematical models that can describe the synaptic interactions of neurons and the ionic dynamics in biophysical detail are not well-established. Based on experimental recordings of combined hippocampal-entorhinal cortex slices from rats in a high-potassium and a low-magnesium solution containing 4-aminopyridine as well as previous observations of similar experimental models, this type of mathematical model has been developed. The model describes neuronal excitation through the application of the conductance-based refractory density approach for three neuronal populations: two populations of glutamatergic neurons with hyperpolarizing and depolarizing GABAergic synapses and one GABAergic population. The ionic dynamics account for the contributions of voltage-gated and synaptic channels, active and passive transporters, and diffusion. The relatively slow dynamics of potassium, chloride, and sodium ion concentrations determine the transitions from pure GABAergic IIDs to IDs and GABA-glutamatergic IIDs. The model reproduces different types of IIDs, including those initiated by interneurons; repetitive IDs; tonic and bursting modes of an ID composed of clustered IID-like events. The simulations revealed contributions from different ionic channels to the ion concentration dynamics before and during ID generation. The proposed model is a step forward to an optimal mathematical description of the mechanisms of epileptic discharges.

7,8-Dihydroxyflavone potentiates ongoing epileptiform activity in mice brain slices

In the central nervous system, Tropomyosin-receptor-kinase B (TrkB) signaling is involved in neuronal survival, differentiation as well as in regulation of synaptic transmission and excitability. As its powerful potential to modulate neuronal functions, TrkB pathway is an attractive target for novel drugs and treatment of common neurological disorders. 7,8-Dihydroxyflavone (DHF), a TrkB receptor agonist, has similar properties with neurotrophin Brain Derived Neurotropic Factor (BDNF). DHF is reported to have a number of beneficial effects in neuroprotection, against depression and improving learning and memory. However, the outcome of acute application of DHF on the excitability of neuronal circuits is not clear. Especially the effects of DHF on synchronized epileptiform activity are not known. In this study, we investigated whether DHF induces epileptiform activity in brain slices and DHF has any effect on already initiated epileptiform discharges. We used acute horizontal hippocampal-entorhinal cortex slices obtained from 30 to 35 days of mice. Extracellular field potential recordings were obtained from entorhinal cortex (EC) and hippocampus CA1 region. DHF did not initiate any epileptiform activity or abnormal discharges. However, DHF increased the frequency of 4 aminopyridine (4AP) induced ictal and interictal events in both EC and CA1. The duration of induced ictal charges were also prolonged upon DHF application. In a number of slices, both EC and CA1, DHF led to ictogenesis. These results suggest that the acute activation of TrkB by DHF has a powerful potential on synchronized neuronal discharges which should be considered in future therapeutical approaches.

Curcumin reduces development of seizurelike events in the hippocampal-entorhinal cortex slice culture model for epileptogenesis.

Inhibition of the mammalian target of rapamycin (mTOR) pathway could be antiepileptogenic in temporal lobe epilepsy (TLE), possibly via anti-inflammatory actions. We studied effects of the mTOR inhibitor rapamycin and the anti-inflammatory compound curcumin-also reported to inhibit the mTOR pathway-on epileptogenesis and inflammation in an invitro organotypic hippocampal-entorhinal cortex slice culture model. Brain slices containing hippocampus and entorhinal cortex were obtained from 6-day-old rat pups and maintained in culture for up to 3weeks. Rapamycin or curcumin was added to the culture medium from day 2 invitro onward. Electrophysiological recordings revealed epileptiformlike activity that developed over 3weeks. In week 3, spontaneous seizurelike events (SLEs) could be detected using whole cell recordings from CA1 principal neurons. The percentage of recorded CA1 neurons displaying SLEs was lower in curcumin-treated slice cultures compared to vehicle-treated slices (25.8% vs 72.5%), whereas rapamycin did not reduce SLE occurrence significantly (52%). Western blot for phosphorylated-S6 (pS6) and phosphorylated S6K confirmed that rapamycin inhibited the mTOR pathway, whereas curcumin only lowered pS6 expression at one phosphorylation site. Real-time quantitative polymerase chain reaction results indicated a trend toward lower expression of inflammatory markers IL-1β and IL-6 and transforming growth factor β after 3weeks of treatment with rapamycin and curcumin compared to vehicle. Our results show that curcumin suppresses SLEs in the combined hippocampal-entorhinal cortex slice culture model and suggest that its antiepileptogenic effects should be further investigated in experimental models of TLE.

Early Appearance and Spread of Fast Ripples in the Hippocampus in a Model of Cortical Traumatic Brain Injury.

Fast ripples (FRs; activity of >250 Hz) have been considered as a biomarker of epileptic activity in the hippocampus and entorhinal cortex; it is thought that they signal the focus of seizure generation. Similar high-frequency network activity has been produced in vitro by changing extracellular medium composition, by using pro-epileptic substances, or by electrical stimulation. Here we study the propagation of these events between different subregions of the male rat hippocampus in a recently introduced experimental model of FRs in entorhinal cortex-hippocampal slices in vitro By using a matrix of 4096 microelectrodes, the sites of initiation, propagation pathways, and spatiotemporal characteristics of activity patterns could be studied with unprecedented high resolution. To this end, we developed an analytic tool based on bidimensional current source density estimation, which delimits sinks and sources with a high precision and evaluates their trajectories using the concept of center of mass. With this methodology, we found that FRs can arise almost simultaneously at noncontiguous sites in the CA3-to-CA1 direction, underlying the spatial heterogeneity of epileptogenic foci, while continuous somatodendritic waves of activity develop. An unexpected, yet important propagation route is the propagation of activity from CA3 into the hilus and dentate gyrus. This pathway may cause reverberating activation of both regions, supporting sustained pathological network events and altered information processing in hippocampal networks.SIGNIFICANCE STATEMENT Fast ripples (FRs) have been considered as a biomarker of epileptic activity and may signal the focus of seizure generation. In a model of traumatic brain injury in the rat, FRs appear in the hippocampus within a couple of hours after an extrahippocampal, cortical lesion. We analyzed the origin and dynamics of the FRs in the hippocampus using massive electrophysiological recordings, allowing an unprecedented high spatiotemporal resolution. We show that FRs originate in distinct and noncontiguous locations within the CA3 region and uncover, with high precision, the extent and dynamics of their current density. This activity propagates toward CA1 but also backpropagates to the hilus and the dentate gyrus, suggesting activation of defined microcircuits that can sustain recurrent excitation.

A Comparison of Different Slicing Planes in Preservation of Major Hippocampal Pathway Fibers in the Mouse.

The hippocampus plays a critical role in learning and memory and higher cognitive functions, and its dysfunction has been implicated in various neuropathological disorders. Electrophysiological recording undertaken in live brain slices is one of the most powerful tools for investigating hippocampal cellular and network activities. The plane for cutting the slices determines which afferent and/or efferent connections are best preserved, and there are three commonly used slices: hippocampal-entorhinal cortex (HEC), coronal and transverse. All three slices have been widely used for studying the major afferent hippocampal pathways including the perforant path (PP), the mossy fibers (MFs) and the Schaffer collaterals (SCs). Surprisingly, there has never been a systematic investigation of the anatomical and functional consequences of slicing at a particular angle. In the present study, we focused on how well fiber pathways are preserved from the entorhinal cortex (EC) to the hippocampus, and within the hippocampus, in slices generated by sectioning at different angles. The postmortem neural tract tracer 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI) was used to label afferent fibers to hippocampal principal neurons in fixed slices or whole brains. Laser scanning confocal microscopy was adopted for imaging DiI-labeled axons and terminals. We demonstrated that PP fibers were well preserved in HEC slices, MFs in both HEC and transverse slices and SCs in all three types of slices. Correspondingly, field excitatory postsynaptic potentials (fEPSPs) could be consistently evoked in HEC slices when stimulating PP fibers and recorded in stratum lacunosum-moleculare (sl-m) of area CA1, and when stimulating the dentate granule cell layer (gcl) and recording in stratum lucidum (sl) of area CA3. The MF evoked fEPSPs could not be recorded in CA3 from coronal slices. In contrast to our DiI-tracing data demonstrating severely truncated PP fibers in coronal slices, fEPSPs could still be recorded in CA1 sl-m in this plane, suggesting that an additional afferent fiber pathway other than PP might be involved. The present study increases our understanding of which hippocampal pathways are best preserved in the three most common brain slice preparations, and will help investigators determine the appropriate slices to use for physiological studies depending on the subregion of interest.

ProNGF Drives Localized and Cell Selective Parvalbumin Interneuron and Perineuronal Net Depletion in the Dentate Gyrus of Transgenic Mice.

ProNGF, the precursor of mature Nerve Growth Factor (NGF), is the most abundant NGF form in the brain and increases markedly in the cortex in Alzheimer's Disease (AD), relative to mature NGF. A large body of evidence shows that the actions of ProNGF and mature NGF are often conflicting, depending on the receptors expressed in target cells. TgproNGF#3 mice, expressing furin-cleavage resistant proNGF in CNS neurons, directly reveal consequences of increased proNGF levels on brain homeostasis. Their phenotype clearly indicates that proNGF can be a driver of neurodegeneration, including severe learning and memory behavioral deficits, cholinergic deficits, and diffuse immunoreactivity for A-beta and A-beta-oligomers. In aged TgproNGF#3 mice spontaneous epileptic-like events are detected in entorhinal cortex-hippocampal slices, suggesting occurrence of excitatory/inhibitory (E/I) imbalance. In this paper, we investigate the molecular events linking increased proNGF levels to the epileptiform activity detected in hippocampal slices. The occurrence of spontaneous epileptiform discharges in the hippocampal network in TgproNGF#3 mice suggests an impaired inhibitory interneuron homeostasis. In the present study, we detect the onset of hippocampal epileptiform events at 1-month of age. Later, we observe a regional- and cellular-selective Parvalbumin interneuron and perineuronal net (PNN) depletion in the dentate gyrus (DG), but not in other hippocampal regions of TgproNGF#3 mice. These results demonstrate that, in the hippocampus, the DG is selectively vulnerable to altered proNGF/NGF signaling. Parvalbumin interneuron depletion is also observed in the amygdala, a region strongly connected to the hippocampus and likewise receiving cholinergic afferences. Transcriptome analysis of TgproNGF#3 hippocampus reveals a proNGF signature with broad down-regulation of transcription. The most affected mRNAs modulated at early times belong to synaptic transmission and plasticity and extracellular matrix (ECM) gene families. Moreover, alterations in the expression of selected BDNF splice variants were observed. Our results provide further mechanistic insights into the vicious negative cycle linking proNGF and neurodegeneration, confirming the regulation of E/I homeostasis as a crucial mediating mechanism.

Microglial-derived miRNA let-7 and HMGB1 contribute to ethanol-induced neurotoxicity via TLR7

BackgroundToll-like receptor (TLR) signaling is emerging as an important component of neurodegeneration. TLR7 senses viral RNA and certain endogenous miRNAs to initiate innate immune responses leading to neurodegeneration. Alcoholism is associated with hippocampal degeneration, with preclinical studies linking ethanol-induced neurodegeneration with central innate immune induction and TLR activation. The endogenous miRNA let-7b binds TLR7 to cause neurodegeneration.MethodsTLR7 and other immune markers were assessed in postmortem human hippocampal tissue that was obtained from the New South Wales Tissue Bank. Rat hippocampal-entorhinal cortex (HEC) slice culture was used to assess specific effects of ethanol on TLR7, let-7b, and microvesicles.ResultsWe report here that hippocampal tissue from postmortem human alcoholic brains shows increased expression of TLR7 and increased microglial activation. Using HEC slice culture, we found that ethanol induces TLR7 and let-7b expression. Ethanol caused TLR7-associated neuroimmune gene induction and initiated the release let-7b in microvesicles (MVs), enhancing TLR7-mediated neurotoxicity. Further, ethanol increased let-7b binding to the danger signaling molecule high mobility group box-1 (HMGB1) in MVs, while reducing let-7 binding to classical chaperone protein argonaute (Ago2). Flow cytometric analysis of MVs from HEC media and analysis of MVs from brain cell culture lines found that microglia were the primary source of let-7b and HMGB1-containing MVs.ConclusionsOur results identify that ethanol induces neuroimmune pathology involving the release of let-7b/HMGB1 complexes in microglia-derived microvesicles. This contributes to hippocampal neurodegeneration and may play a role in the pathology of alcoholism.

Glutamate/NMDA excitotoxicity and HMGB1/TLR4 neuroimmune toxicity converge as components of neurodegeneration

Neurodegeneration in brain is linked to both excitotoxicity and neuroimmune gene induction,although the mechanisms are poorly understood. High-mobility group box 1 (HMGB1) is a cytokine like molecule released in brain by glutamate that has been found to enhance neuronal excitability through Toll-like receptor 4 (TLR4). To explore the role of HMGB1 in glutamate/NMDA excitotoxicity or neuroimmune-induced neurodegeneration we used an <em>ex vivo</em> model of organotypic hippocampal-entorhinal cortex (HEC) slice culture. Concentration response and time course studies find release of HMGB1 precedes neuronal death induced by glutamate,NMDA,TNFα and LPS. Blockade of glutamate receptors with antagonist MK-801 prevents glutamate/NMDA stimulation release of HMGB1 and neuronal death as well as blocking neuroimmune (LPS and TNFα) induced neuronal death. Similarly,HMGB1 neutralizing antibodies or inhibitor glycyrrhizin block glutamate/NMDA as well as neuroimmune (LPS and TNFα) induced neuronal death. Further,delayed neuronal cell death mediated by LPS and TNFα was rescued by NR2B inhibitor ifenprodil. Together,these findings suggest HMGB1 contributes a critical element of both glutamate/NMDA as well as neuroimmune induced neurodegeneration indicating HMGB1 may be a novel target crossing multiple neurodegeneration pathologies.

Seizure-induced reduction in PIP3 levels contributes to seizure-activity and is rescued by valproic acid

Phosphatidylinositol (3–5) trisphosphate (PIP3) is a central regulator of diverse neuronal functions that are critical for seizure progression, however its role in seizures is unclear. We have recently hypothesised that valproic acid (VPA), one of the most commonly used drugs for the treatment of epilepsy, may target PIP3 signalling as a therapeutic mode of action. Here, we show that seizure induction using kainic acid in a rat in vivo epilepsy model resulted in a decrease in hippocampal PIP3 levels and reduced protein kinase B (PKB/AKT) phosphorylation, measured using ELISA mass assays and Western blot analysis, and both changes were restored following VPA treatment. These finding were reproduced in cultured rat hippocampal primary neurons and entorhinal cortex–hippocampal slices during exposure to the GABA(A) receptor antagonist pentylenetetrazol (PTZ), which is widely used to generate seizures and seizure-like (paroxysmal) activity. Moreover, VPA's effect on paroxysmal activity in the PTZ slice model is blocked by phosphatidylinositol 3-kinase (PI3K) inhibition or PIP2 sequestration by neomycin, indicating that VPA's efficacy is dependent upon PIP3 signalling. PIP3 depletion following PTZ treatment may also provide a positive feedback loop, since enhancing PIP3 depletion increases, and conversely, reducing PIP3 dephosphorylation reduces paroxysmal activity and this effect is dependent upon AMPA receptor activation. Our results therefore indicate that PIP3 depletion occurs with seizure activity, and that VPA functions to reverse these effects, providing a novel mechanism for VPA in epilepsy treatment.

Gonadal status-dependent effects of in vivo β-estradiol administration to female rats on in vitro epileptiform activity induced by low [Mg2+]o in combined hippocampus-entorhinal cortex slices

There are controversial data regarding estrogen effects on neuronal excitability. We investigated whether β-estradiol (EB) administration to ovariectomized (OVX) or gonadally intact female rats alters epileptiform activity within the dentate gyrus network induced in vitro by removing [Mg2+]o in combined hippocampus-entorhinal cortex slices. In vivo EB administration significantly influenced the epileptiform activity in gonadal status-dependent manner. The onset of epileptiform discharges was modestly delayed in slices from OVX rats replaced with physiologically relevant doses of EB but the number of discharges was not affected. In contrast, EB administration to gonadally intact rats had robust effects such that: EB delayed the onset of discharges but significantly increased their number within the dentate gyrus network. Our data suggest that EB in physiologically relevant concentrations does not seem to negatively affect hippocampal neuronal excitability, nevertheless supraphysiological EB levels may enhance seizure severity.

Reduced ictogenic potential of 4-aminopyridine in the hippocampal region in the pilocarpine model of epilepsy

It was previously shown that the ictogenic potential of 4-aminopyridine (4-AP) was reduced in the parahippocampal region of kainate treated chronic epileptic rats. In the actual study we investigated the potential of 4-aminopyridine (50 and 100μM) to induce seizure like events (SLEs) in combined entorhinal cortex hippocampal slices from Wistar rats following pilocarpine induced status epilepticus. The potential of 4-AP to induce SLEs in the entorhinal cortex was reduced in the latent period and in slices of chronic epileptic animals with a high seizure incidence in vivo (>2seizures/24h). 4-AP induced SLEs in slices from animals with a low incidence of seizures in vivo (<2seizures/24h) in a similar manner as compared to controls. The hippocampal formation displayed no SLEs, instead short recurrent epileptiform discharges (REDs) were evoked by application of 4-AP in areas CA3 and CA1. The incidence of REDs was largest in slices from control animals. This study shows that the reduced ictogenic potential of 4-AP is not restricted to kainate treated chronic epileptic animals as it can be found in the pilocarpine model as well. The underlying mechanisms may relate to altered expression and editing of voltage gated potassium channels.

ATP‐P2X7 receptor signaling controls basal and TNFα‐stimulated glial cell proliferation

Activation and proliferation of glial cells and their progenitors is a key process of neuroinflammation associated with many neurodegenerative disorders. Under neuropathological conditions where glial cell activation and proliferation is evident, controlling the population of glia might be of therapeutic importance. The proliferative action of the cytokine tumor necrosis factor alpha (TNFα) on microglia has been reported, but the molecular mechanism of TNFα regulation of glial cell proliferation is largely unknown. Using a model of organotypic hippocampal-entorhinal cortex (HEC) slice culture, we investigated the role of ATP-P2X(7) receptor signaling in glial proliferation by TNFα. Populations of proliferating cells in HEC culture were labeled with 5-bromo-2'-deoxyuridine (BrdU). Treatment with TNFα induced strong expression of P2X(7) receptor mRNA and immunoreactivity in BrdU+ cells while markedly increasing proliferation of BrdU+ cells. In addition, TNFα increased aquaporin 4 (AQP4) expression, an ion channel involved in glial proliferation. The proliferative action of TNFα was attenuated by blocking the P2X(7) receptors with the specific antagonists oxATP, BBG, and KN62, or by lowering extracellular ATP with ATP hydrolysis apyrase. Basal proliferation of BrdU+ cells was also sensitive to blockade of ATP-P2X(7) signaling. Furthermore, TNFα activation of P2X(7) receptors appear to regulate AQP4 expression through protein kinase C cascade and down regulation of AQP4 expression can reduce TNFα-stimulated BrdU+ cell proliferation. Taken together, these novel findings demonstrate the importance of ATP-P2X(7) signaling in controlling proliferation of glial progenitors under the pathological conditions associated with increased TNFα.

Status epilepticus induces increasing neuronal excitability and hypersynchrony as revealed by optical imaging

In the wake of acquired brain insults such as status epilepticus (SE), time-dependent neuronal network alterations may occur resulting in cortical hyperexcitability and enhanced synchrony merging into chronic epilepsy. To better understand the underlying processes, we performed electrophysiological and optical imaging studies on combined hippocampal-entorhinal cortex slices. These were prepared from rats 1, 4 and 8weeks after electrically-induced SE. Non-invasive imaging using intrinsic optical signal changes allowed detailed analysis of onset and spread patterns of seizure-like events (SLE) since coverage of the entire preparation is possible. The latency to occurrence of first SLEs after omission of Mg2+ from the artificial cerebrospinal fluid was significantly reduced at 4 and 8weeks after SE compared with all other groups indicating increased brain excitability. Optical imaging displayed multiregional onset and discontiguous propagation of SLEs 8weeks after SE. Such patterns indicate neuronal hypersynchrony and are not encountered in naïve rodents in which SLEs commonly begin in the entorhinal cortex and display contiguous spread to invade adjacent regions. The electrophysiological and optical findings of the current study indicate evolving fundamental brain plasticity changes after the detrimental event predisposing to chronic epilepsy. The current results should be incorporated in any strategies aiming at prevention of chronic epilepsy.

Age- and region-specific effects of anticonvulsants and bumetanide on 4-aminopyridine-induced seizure-like events in immature rat hippocampal-entorhinal cortex slices

Seizure-like events (SLEs) induced by 4-aminopyridine in rat organotypic slices cultures, which are prepared early after birth, are resistant to standard antiepileptic drugs. In this study we tested the hypothesis that pharmacoresistance may be an intrinsic property of the immature brain. Frequently recurring SLEs presumably representing status epilepticus were induced by 4-aminopyridine in acute rat hippocampal-entorhinal cortex slices obtained from postnatal day 3-19 (P3-P19), and the effects of carbamazepine, phenytoin, valproic acid, and phenobarbital were examined. In addition, bumetanide was tested, which blocks the Na(+) -K(+) -2Cl(-) (NKCC1) cotransporter, and also acetazolamide, which blocks the carbonic anhydrase and thereby the accumulation of bicarbonate inside neurons. The efficacy of all antiepileptic drugs in blocking SLEs was dependent on postnatal age, with low efficacy in P3-P5 slices. Antiepileptic drugs suppressed SLEs more readily in the medial entorhinal cortex (ECm) than in the CA3. In P3-P5 slices, valproic acid and phenobarbital increased both tonic and clonic seizure-like activities in the CA3, whereas phenytoin and carbamazepine blocked tonic-like but prolonged clonic-like activity. In P3-P5 slices, bumetanide often blocked SLEs in the CA3, but was not as effective in the ECm. Like with other antiepileptic drugs, the seizure-suppressing effects of acetazolamide increased with postnatal age. We conclude that pharmacoresistance may be inherent to very immature tissue and suggest that expression of the NKCC1 cotransporter might contribute to pharmacoresistance.