Rat Hippocampal-entorhinal Cortex Research Articles

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.

Release of Neuronal HMGB1 by Ethanol through Decreased HDAC Activity Activates Brain Neuroimmune Signaling

Neuroimmune gene induction is involved in many brain pathologies including addiction. Although increased expression of proinflammatory cytokines has been found in ethanol-treated mouse brain and rat brain slice cultures as well as in post-mortem human alcoholic brain, the mechanisms remain elusive. High-mobility group box 1 (HMGB1) protein is a nuclear protein that has endogenous cytokine-like activity. We previously found increased HMGB1 in post-mortem alcoholic human brain as well as in ethanol treated mice and rat brain slice cultures. The present study investigated the mechanisms for ethanol-induced release of HMGB1 and neuroimmune activation in a model of rat hippocampal-entorhinal cortex (HEC) brain slice cultures. Ethanol exposure triggered dose-dependent HMGB1 release, predominantly from neuronal cells. Inhibitors of histone deacetylases (HDACs) promoted nucleocytoplasmic mobilization of HDAC1/4 and HMGB1 resulting in increased total HMGB1 and acetylated HMGB1 release. Similarly, ethanol treatment was found to induce the translocation of HDAC1/4 and HMGB1 proteins from nuclear to cytosolic fractions. Furthermore, ethanol treatment reduced HDAC1/4 mRNA and increased acetylated HMGB1 release into the media. These results suggest decreased HDAC activity may be critical in regulating acetylated HMGB1 release from neurons in response to ethanol. Ethanol and HMGB1 treatment increased mRNA expression of proinflammatory cytokines TNFα and IL-1β as well as toll-like receptor 4 (TLR4). Targeting HMGB1 or microglial TLR4 by using siRNAs to HMGB1 and TLR4, HMGB1 neutralizing antibody, HMGB1 inhibitor glycyrrhizin and TLR4 antagonist as well as inhibitor of microglial activation all blocked ethanol-induced expression of proinflammatory cytokines TNFα and IL-1β. These results support the hypothesis that ethanol alters HDACs that regulate HMGB1 release and that danger signal HMGB1 as endogenous ligand for TLR4 mediates ethanol-induced brain neuroimmune signaling through activation of microglial TLR4. These findings provide new therapeutic targets for brain neuroimmune activation and alcoholism.

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.

Transition from Interictal to Ictal Activity in Limbic NetworksIn Vitro

The transition from brief bursts of synchronous population activity characteristic of interictal epileptiform discharges (IEDs) to more prolonged epochs of population activity characteristic of seizures (ictal-like activity) was recorded in juvenile rat hippocampal-entorhinal cortex slices and hippocampal slices using multiple-site extracellular electrodes. Epileptiform activity was elicited by either increased extracellular potassium or 4-AP. IEDs originated in the CA3 a-b region and spread bidirectionally into CA1 and CA3c dentate gyrus. The transition from IEDs to ictal-like sustained epileptiform activity was reliably preceded by (1) increase in IED propagation velocity, (2) increase in IED secondary afterdischarges and their reverberation between CA3a and CA3c, and (3) shift in the IED initiation area from CA3 a-b to CA3c. Ictal-like sustained network oscillations (10-20 Hz) originated in CA3c and spread to CA1. The pattern of hippocampal ictal-like activity was unaffected by removal of the entorhinal cortex. These findings indicate that interictal and ictal activity can originate in the same neural network, and that the transition from interictal to ictal-like-sustained activity is preceded by predictable alterations in the origin and spread of IEDs. These findings elucidate new targets for investigating the proximate causes, prediction, and treatment of seizures.

Ketone bodies do not directly alter excitatory or inhibitory hippocampal synaptic transmission.

To determine the effect of the ketone bodies beta-hydroxybutyrate (betaHB) and acetoacetate (AA) on excitatory and inhibitory neurotransmission in the mammalian CNS. The ketogenic diet is presumed to be an effective anticonvulsant regimen for some children with medically intractable seizures. However, its mechanism of action remains a mystery. According to one hypothesis, ketone bodies have anticonvulsant properties. The authors examined the effect of betaHB and AA on excitatory and inhibitory synaptic transmission in rat hippocampal-entorhinal cortex slices and cultured hippocampal neurons. In cultured neurons, their effect was also directly assayed on postsynaptic receptor properties. Finally, their ability to prevent spontaneous seizures was determined in a hippocampal-entorhinal cortex slice model. betaHB and AA did not alter synaptic transmission in these models. The anticonvulsant properties of the ketogenic diet do not result from a direct effect of ketone bodies on the primary voltage and ligand gated ion channels mediating excitatory or inhibitory neurotransmission in the hippocampus.

Effects of bicuculline and different glutamate receptor antagonists on 4-aminopyridine-induced epileptiform discharges in rat hippocampal-entorhinal cortex slices

Application of 4-aminopyridine has previously been reported to produce different patterns of epileptiform discharges in entorhinal cortex-hippocampal-slices. We describe two types of discharges: seizure-like events and negative-going potentials in the medial entorhinal cortex. Using recordings of field potentials, we investigated the pharmacological effects of the GABAA-receptor antagonist bicuculline and blockers of NMDA and non-NMDA glutamate receptors (APV as well as NBQX) on 4-aminopyridine-induced epileptiform activity. Using K+-sensitive microelectrodes we measured changes in extracellular K+ concentrations associated with the discharges. The different discharge patterns could be distinguished by their different pharmacological sensitivity to the different receptor antagonists. We propose that the study of the different activities induced by 4-aminopyridine may provide an in vitro model for the development of new drugs against difficult-to-treat focal epilepsies.

Effects of bicuculline and different glutamate receptor antagonists on 4-aminopyridine-induced epileptiform discharges in rat hippocampal-entorhinal cortex slices

Effects of bicuculline and different glutamate receptor antagonists on 4-aminopyridine-induced epileptiform discharges in rat hippocampal-entorhinal cortex slices

Intrinsic optical signal measurements reveal characteristic features during different forms of spontaneous neuronal hyperactivity associated with ECS shrinkage in vitro.

We induced three different forms of spontaneous synchronous hyperactivity in adult rat hippocampal-entorhinal cortex slices in order to investigate effects on the intrinsic optical signal and associated changes in the extracellular space (ECS) volume. Low-Mg2+ artificial cerebrospinal fluid (ACSF) and the addition of 4-aminopyridine induced synchronous hyperactivity resulting mainly from increased synaptic transmission, while low-Ca2+ ACSF induced hyperactivity in the absence of evoked synaptic transmission. In the two models of enhanced synaptic transmission, spontaneous activity lead to an immediate increase of light transmission. In contrast, a decrease of light transmission took place during low-Ca2+-induced hyperactivity. All three forms of synchronous neuronal hyperactivity were associated with a shrinkage of the ECS volume, as revealed by the tetraethylammonium signal, measured with ion-sensitive microelectrodes. This indicates that the change in the intrinsic optical signal is not simply related to a shrinkage in ECS volume. We conclude that different forms of spontaneous synchronous neuronal hyperactivity are associated with characteristic optical signals and that the direction of the change in intrinsic optical signal does not reflect ECS shrinkage alone.

Neuropeptide Y suppresses epileptiform activity in rat hippocampus in vitro.

Neuropeptide Y (NPY) potently inhibits glutamate-mediated synaptic transmission in areas CA1 and CA3 of the rat hippocampus without affecting other synaptic inputs onto principal cells of the hippocampal formation, suggesting that its biological role may include the regulation of excitability within the hippocampus. Here we examine NPY's actions in three in vitro models of epilepsy [0 Mg2+-, picrotoxin-, and stimulus-train-induced bursting (STIB)] with the use of extracellular and whole cell patch-clamp recordings from rat hippocampal-entorhinal cortex slices. Perfusion of the slice with saline that had Mg2+ omitted (0 Mg2+) or that had picrotoxin (100 microM) added resulted in brief spontaneous bursts (SBs) resembling interictal discharges. SB frequency is significantly reduced in both models by 1 microM NPY and by the Y2-preferring agonists peptide (P)YY(3-36) (1 microM) and 1-4-(6-aminohexanoic acid)-25-36 ([ahx(5-24)] NPY; 3 microM). The Y1-preferring agonist Leu31-Pro34NPY (1 microM) is considerably less potent, but also reduces burst frequency, even in the presence of the selective Y1 receptor antagonist GR231118, suggesting the involvement of a different receptor. In STIB, high-frequency stimulus trains to stratum radiatum of area CA2/CA3 result in clonic or tonic-clonic ictaform primary afterdischarges (primary ADs) as well as longer, spontaneous secondary ictaform discharges and SBs similar to those in the other models. Primary AD duration is greatly reduced or abolished by Y2- but not Y1-preferring agonists. SBs, although variable, were inhibited by both Y1 and Y2 agonists. In single and dual whole cell recordings from CA3 pyramidal cells, we frequently observed spontaneous, rhythmic synchronous events (SRSEs) arising after several STIB stimuli. Once established, SRSEs persist in the absence of further stimuli and are insensitive to the application of NPY. SRSEs in pyramidal cells typically occur at 2-4 Hz, are outward currents when cells are clamped near rest (>100 pA at a holding potential of -55 mV), reverse between -60 and -70 mV, and are inhibited by 100 microM picrotoxin, indicating involvement of gamma-aminobutyric acid-A receptors. They are inhibited by blockers of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) but not N-methyl-D-aspartate receptors. Whole cell patch-clamp recordings from interneurons in CA3 after STIB reveal NPY-insensitive, rhythmic, inward AMPA-receptor-mediated currents that are similar in frequency to SRSEs seen in pyramidal cells. We conclude that NPY, acting predominantly via Y2 receptors, can dramatically inhibit epileptiform activity in three fundamentally different in vitro models of epilepsy without affecting endogenous inhibitory activity. The results also provide support for the hypothesis that endogenous NPY may normally control excitability in the hippocampus and suggest the potential for NPY receptors as targets for anticonvulsant therapy.

Serotonin reduces synaptic excitation of principal cells in the superficial layers of rat hippocampal-entorhinal cortex combined slices

The cells of the entorhinal cortex receive a dense innervation of serotonergic fibres from the Raphe nuclei and express a high density of 5-hydroxytryptamine 1A (5-HT 1A) receptors. We investigated the effects of serotonin on excitatory synaptic transmission in principal cells from entorhinal cortex layers II and III within hippocampal-entorhinal cortex combined slices. Although serotonin had an effect upon the membrane conductance of some, but not all cells, its most pronounced action was to reduce stimulus evoked excitatory synaptic potentials and currents (EPSP/Cs). Both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and N-methyl- d-aspartate receptor-mediated EPSPs were reduced to similar extents over a range of concentrations. Since the principal cells in layer II and layer III are the main projection cells of the entorhinal cortex, these inhibitory effects of serotonin may have implications for the transfer of information to the hippocampus.

Tracing of axonal connections by rhodamine-dextran-amine in the rat hippocampal-entorhinal cortex slice preparation.

In order to demonstrate axonal connections preserved in rat temporal cortex slices the authors used rhodamine-dextran-amine as a tracer. The slices contained the neocortical areas Te2 and Te3, the medial and lateral entorhinal cortices (MEC and LEC), the subicular regions, and the dentate gyrus and hippocampus proper. Rhodamine-dextran-amine crystals were placed by microinjection into a given area. Following this local lesioning the dye was permitted to diffuse and migrate intraaxonally in antero- and retrograde directions for about 8 hours. The slices were then formaldehyde-fixed and analyzed by fluorescence microscopy. Most of the known connections within and between the entorhinal cortex and the hippocampus and dentate gyrus were preserved in the slice preparation, provided that the slices were cut with a near horizontal orientation corresponding to plates 99-108 in Paxinos and Watson (1986). Only the lateral perforant path between the LEC and the hippocampus could not be followed to its full extent. The authors conclude that most aspects of the intrinsic synaptic organization of the temporal lobe can be reliably studied in hippocampal-entorhinal cortex slice preparations.