Zinc transporter 3 immunohistochemical tracing of sprouting mossy fibres

Zinc transporter 3 (ZNT3) has been shown to transport zinc ions from the cytosol into presynaptic vesicles in the mammalian brain. Several studies have stated that the zinc ion containing synaptic vesicles of zinc-enriched neurons (ZEN) are loaded with ZNT3 proteins in their membranes. This fact makes it possible to trace sprouting mossy fibres in the temporal lobe epileptic hippocampus. In the present study, we examined the expression and distribution patterns of ZNT3 protein and chelatable zinc ions in the mouse hippocampus after pilocarpine treatment. Our results demonstrate that both ZNT3 immunostaining and autometallography reveal identical patterns of sprouting mossy fibres in the inner molecular layer in the mouse hippocampus. Using ZNT3 immuno-electron microscopic analysis we confirmed the presence of ectopic mossy fibre terminals in the inner molecular layer and found additionally by immuno-blotting a significant increase of ZNT3 in the pilocarpine-treated mouse hippocampi compared to age-matched controls. The increase of ZNT3 after pilocarpine treatment was time-dependent. The results support the notion that ZNT3 immunohistochemistry provides an excellent tool for tracing sprouting of ZEN terminals. The progressive increase of ZNT3 immunostaining in the temporal lobe epileptic hippocampus may relate to the increased levels of vesicular zinc ions during seizure.     

Changes in TWIK-related Acid Sensitive K+-1 and -3 Channel Expressions from Neurons to Glia in the Hippocampus of Temporal Lobe Epilepsy Patients and Experimental Animal Model

In the present study, we analyzed expressions of tandem of P domains in a Weak Inwardly rectifying K⁺ channel (TWIK)-related Acid-Sensitive K⁺ (TASK) channel-1 and -3 in the hippocampus of patients with temporal lobe epilepsy (TLE) and in rat model. In the control human subjects, TASK-1, and -3 immunoreactivity was observed in pyramidal neurons and dentate granule cells. In TLE patients, TASK-1 and -3 immunoreactivity was rarely observed in neurons. However, TASK-1 immunoreactivity was observed in astrocytes, and TASK-3 immunoreactivity was detected in both astrocytes and microglia. In the rat hippocampus, TASK-1 immunoreactivity was observed in astrocytes within normal and epileptic hippocampus. The alterations in TASK-3 immunoreactivity in the rat hippocampus were similar to those in the human hippocampus. These findings reveal that TASK-1 and -3 are differentially expressed in the normal and epileptic hippocampus, and suggest that TASK channels may contribute to the properties of the epileptic hippocampus.     

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca²⁺ transients in astrocytes, and these Ca²⁺ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4⁺ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4⁺ astrocytes. After activation, both TRPV4⁺ and TRPV4⁻ astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4⁺ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4⁺ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.     

Deletion of <Emphasis Type="Italic">mTOR</Emphasis> in Reactive Astrocytes Suppresses Chronic Seizures in a Mouse Model of Temporal Lobe Epilepsy

Germline and somatic mutations in key genes of the mammalian target of rapamycin (mTOR) pathway have been identified in seizure-associated disorders. mTOR mutations lead to aberrant activation of mTOR signaling, and, although affected neurons are critical for epileptogenesis, the role of mTOR activation in glial cells remains poorly understood. We previously reported a consistent activation of the mTOR pathway in astrocytes in the epileptic foci of temporal lobe epilepsy. In this study, it was demonstrated that mTOR deletion from reactive astrocytes prevents increases in seizure frequency over the disease course. By using a tamoxifen-inducible mTOR conditional knockout system and kainic acid, a model was developed that allowed astrocyte-specific mTOR gene deletion in mice with chronic epilepsy. Animals in which mTOR was deleted from 44 % of the astrocyte population exhibited a lower seizure frequency compared with controls. Down-regulation of mTOR significantly ameliorated astrogliosis in the sclerotic hippocampus but did not rescue mossy fiber sprouting. In cultured astrocytes, the mTOR pathway modulated the stability of the astroglial glutamate transporter 1 (Glt1) and influenced the ability of astrocytes to remove extracellular glutamate. Taken together, these data indicate that astrocytes with activated mTOR signaling may provide conditions that are favorable for spontaneous recurrent seizures.     

Dysbalance of Astrocyte Calcium under Hyperammonemic Conditions

Increased brain ammonium (NH₄ ⁺/NH₃) plays a central role in the manifestation of hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH₄ ⁺/NH₃ on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH₄ ⁺/NH₃-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH₄ ⁺/NH₃ for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼90%) experienced a persistent calcium increase by ∼50 nM. This persistent increase was already evoked at concentrations of 1–2 mM NH₄ ⁺/NH₃, developed within 10–20 minutes and was maintained as long as the NH₄ ⁺/NH₃ was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH₄ ⁺/NH₃, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of sodium/calcium exchange (NCX), AMPA- or mGluR5-receptors did not dampen NH₄ ⁺/NH₃-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH₄ ⁺/NH₃ causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE.     

Reduced D2/D3 Receptor Binding of Extrastriatal and Striatal Regions in Temporal Lobe Epilepsy

Objective

Dopamine is an endogenous neuromodulator in cortical circuits and the basal ganglia. In animal models of temporal lobe epilepsy (TLE), seizure threshold is modulated to some extent by dopamine, with D1-receptors having a pro- and D2-receptors an anticonvulsant effect. We aimed to extend our previously reported results on decreased D2/D3 receptor binding in the lateral epileptogenic temporal lobe and to correlate them with demographic and seizure variables to gain a more comprehensive understanding of the underlying involvement of the dopaminergic system in the epileptogenesis of TLE.

Methods

To quantify D2/D3 receptor binding, we studied 21 patients with TLE and hippocampal sclerosis (13 left- and eight right-sided) and 18 controls using PET with the high-affinity dopamine D2/D3-receptor ligand ¹⁸F-Fallypride to image striatal and extrastriatal binding. TLE was defined by interictal and ictal video-EEG, MRI and ¹⁸F-Fluorodeoxyglucose PET. Voxel-based statistical and regions-of-interest analyses were performed.

Results

¹⁸F-Fallypride binding potential was significantly reduced in the affected temporal lobe and bilateral putamen. A positive correlation between age at onset of epilepsy and [¹⁸F]FP BP_nd (binding potential non-displaceable) in temporal regions on the epileptogenic side was found, as well as a negative correlation between epilepsy duration and [¹⁸F]FP BP_nd in the temporal pole on the epileptogenic side and a positive correlation between the estimated number of lifetime GTCS and [¹⁸F]FP BP_nd in the hippocampus on the epileptogenic side.

Significance

The areas of reduced D2/D3 receptor availability correspond to “the irritative zone” surrounding the epileptogenic area. Moreover, reduced D2/D3 receptor availability was detectable in the basal ganglia, which are suspected to be involved in a control circuit for epileptic seizures. The correlational analysis additionally suggests that increased epilepsy duration leads to increasing impairment of the dopaminergic system.     

The Recurrent Mossy Fiber Pathway of the Epileptic Brain

The dentate gyrus is believed to play a key role in the pathogenesis of temporal lobe epilepsy. In normal brain the dentate granule cells serve as a high-resistance gate or filter, inhibiting the propagation of seizures from the entorhinal cortex to the hippocampus. The filtering function of the dentate gyrus depends in part on the near absence of monosynaptic connections among granule cells. In humans with temporal lobe epilepsy and in animal models of temporal lobe epilepsy, dentate granule cells form an interconnected synaptic network associated with loss of hilar interneurons. This recurrent mossy fiber pathway mediates reverberating excitation that can reduce the threshold for granule cell synchronization. Factors that augment activity in this pathway include modest increases in [K⁺]_o; loss of GABA inhibition; short-term, frequency-dependent facilitation (frequencies of 1–2 Hz); feedback activation of kainate autoreceptors; and release of zinc from recurrent mossy fiber boutons. Factors that diminish activity include short-term, frequency-dependent depression (frequencies <1 Hz); feedback activation of type II metabotropic glutamate receptors; and the potential release of GABA, neuropeptide Y, adenosine, and dynorphin from recurrent mossy fiber boutons. The axon sprouting and reactive synaptogenesis that follow seizure-related brain damage can also create or strengthen recurrent excitation in other brain regions. These changes are expected to facilitate participation of these regions in seizures. Thus, reactive processes that are often considered important for recovery of function after most brain injuries probably contribute to neurological dysfunction in epilepsy.     

NeuroKinect: A Novel Low-Cost 3Dvideo-EEG System for Epileptic Seizure Motion Quantification

Epilepsy is a common neurological disorder which affects 0.5–1% of the world population. Its diagnosis relies both on Electroencephalogram (EEG) findings and characteristic seizure−induced body movements − called seizure semiology. Thus, synchronous EEG and (2D)video recording systems (known as Video−EEG) are the most accurate tools for epilepsy diagnosis. Despite the establishment of several quantitative methods for EEG analysis, seizure semiology is still analyzed by visual inspection, based on epileptologists’ subjective interpretation of the movements of interest (MOIs) that occur during recorded seizures. In this contribution, we present NeuroKinect, a low-cost, easy to setup and operate solution for a novel 3Dvideo-EEG system. It is based on a RGB-D sensor (Microsoft Kinect camera) and performs 24/7 monitoring of an Epilepsy Monitoring Unit (EMU) bed. It does not require the attachment of any reflectors or sensors to the patient’s body and has a very low maintenance load. To evaluate its performance and usability, we mounted a state-of-the-art 6-camera motion-capture system and our low-cost solution over the same EMU bed. A comparative study of seizure-simulated MOIs showed an average correlation of the resulting 3D motion trajectories of 84.2%. Then, we used our system on the routine of an EMU and collected 9 different seizures where we could perform 3D kinematic analysis of 42 MOIs arising from the temporal (TLE) (n = 19) and extratemporal (ETE) brain regions (n = 23). The obtained results showed that movement displacement and movement extent discriminated both seizure MOI groups with statistically significant levels (mean = 0.15 m vs. 0.44 m, p<0.001; mean = 0.068 m³ vs. 0.14 m³, p<0.05, respectively). Furthermore, TLE MOIs were significantly shorter than ETE (mean = 23 seconds vs 35 seconds, p<0.01) and presented higher jerking levels (mean = 345 ms⁻³ vs 172 ms⁻³, p<0.05). Our newly implemented 3D approach is faster by 87.5% in extracting body motion trajectories when compared to a 2D frame by frame tracking procedure. We conclude that this new approach provides a more comfortable (both for patients and clinical professionals), simpler, faster and lower-cost procedure than previous approaches, therefore providing a reliable tool to quantitatively analyze MOI patterns of epileptic seizures in the routine of EMUs around the world. We hope this study encourages other EMUs to adopt similar approaches so that more quantitative information is used to improve epilepsy diagnosis.     

Time-irreversible Subconductance Gating Associated with Ba2+ Block of Large Conductance Ca2+-activated K+ Channels

Ba²⁺ block of large conductance Ca²⁺-activated K⁺ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba²⁺ ion would dissociate to the external solution (150 mM N-methyl-d-glucamine⁺ _o, 500 mM K⁺ _i, 10 μM Ba²⁺ _i, +30 mV, and 100 μM Ca²⁺ _i to fully activate the channel), Ba²⁺ blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba²⁺ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ∼0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ∼10,000 Ba²⁺ blocks. Thus, the preopenings represent Ba²⁺-induced time-irreversible subconductance gating. The fraction of Ba²⁺ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba²⁺]_i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba²⁺ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba²⁺ _o and no added Ba²⁺ _i. Adding K⁺, Rb⁺, or Na⁺ to the external solution decreased the fraction of Ba²⁺ blocks with preopenings, with K⁺ and Rb⁺ being more effective than Na⁺. These results are consistent with models in which the blocking Ba²⁺ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba²⁺ blocking site and acts directly as the preopening gate.     

NMR solution structures of Runella slithyformis RNA 2′-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO₄ to NAD⁺ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD⁺. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD⁺-bound states. ITC measurements of RslTpt1 binding to NAD⁺ (K_D ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD⁺-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP⁺, which contains an adenylate 2′-PO₄ (mimicking the substrate RNA 2′-PO₄), binds with lower affinity (K_D ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD⁺ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD⁺ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.     

Control of voltage-gated K+ channel permeability to NMDG+ by a residue at the outer pore

Crystal structures of potassium (K⁺) channels reveal that the selectivity filter, the narrow portion of the pore, is only ∼3-Å wide and buttressed from behind, so that its ability to expand is highly constrained, and the permeation of molecules larger than Rb⁺ (2.96 Å in diameter) is prevented. N-methyl-d-glucamine (NMDG⁺), an organic monovalent cation, is thought to be a blocker of Kv channels, as it is much larger (∼7.3 Å in mean diameter) than K⁺ (2.66 Å in diameter). However, in the absence of K⁺, significant NMDG⁺ currents could be recorded from human embryonic kidney cells expressing Kv3.1 or Kv3.2b channels and Kv1.5 R487Y/V, but not wild-type channels. Inward currents were much larger than outward currents due to the presence of intracellular Mg²⁺ (1 mM), which blocked the outward NMDG⁺ current, resulting in a strong inward rectification. The NMDG⁺ current was inhibited by extracellular 4-aminopyridine (5 mM) or tetraethylammonium (10 mM), and largely eliminated in Kv3.2b by an S6 mutation that prevents the channel from opening (P468W) and by a pore helix mutation in Kv1.5 R487Y (W472F) that inactivates the channel at rest. These data indicate that NMDG⁺ passes through the open ion-conducting pore and suggest a very flexible nature of the selectivity filter itself. 0.3 or 1 mM K⁺ added to the external NMDG⁺ solution positively shifted the reversal potential by ∼16 or 31 mV, respectively, giving a permeability ratio for K⁺ over NMDG⁺ (P_K⁺/P_NMDG⁺) of ∼240. Reversal potential shifts in mixtures of K⁺ and NMDG⁺ are in accordance with P_K⁺/P_NMDG⁺, indicating that the ions compete for permeation and suggesting that NMDG⁺ passes through the open state. Comparison of the outer pore regions of Kv3 and Kv1.5 channels identified an Arg residue in Kv1.5 that is replaced by a Tyr in Kv3 channels. Substituting R with Y or V allowed Kv1.5 channels to conduct NMDG⁺, suggesting a regulation by this outer pore residue of Kv channel flexibility and, as a result, permeability.     

Pore dimensions and the role of occupancy in unitary conductance of Shaker K channels

K channels mediate the selective passage of K⁺ across the plasma membrane by means of intimate interactions with ions at the pore selectivity filter located near the external face. Despite high conservation of the selectivity filter, the K⁺ transport properties of different K channels vary widely, with the unitary conductance spanning a range of over two orders of magnitude. Mutation of Pro475, a residue located at the cytoplasmic entrance of the pore of the small-intermediate conductance K channel Shaker (Pro475Asp (P475D) or Pro475Gln (P475Q)), increases Shaker’s reported ∼20-pS conductance by approximately six- and approximately threefold, respectively, without any detectable effect on its selectivity. These findings suggest that the structural determinants underlying the diversity of K channel conductance are distinct from the selectivity filter, making P475D and P475Q excellent probes to identify key determinants of the K channel unitary conductance. By measuring diffusion-limited unitary outward currents after unilateral addition of 2 M sucrose to the internal solution to increase its viscosity, we estimated a pore internal radius of capture of ∼0.82 Å for all three Shaker variants (wild type, P475D, and P475Q). This estimate is consistent with the internal entrance of the Kv1.2/2.1 structure if the effective radius of hydrated K⁺ is set to ∼4 Å. Unilateral exposure to sucrose allowed us to estimate the internal and external access resistances together with that of the inner pore. We determined that Shaker resistance resides mainly in the inner cavity, whereas only ∼8% resides in the selectivity filter. To reduce the inner resistance, we introduced additional aspartate residues into the internal vestibule to favor ion occupancy. No aspartate addition raised the maximum unitary conductance, measured at saturating [K⁺], beyond that of P475D, suggesting an ∼200-pS conductance ceiling for Shaker. This value is approximately one third of the maximum conductance of the large conductance K (BK) channel (the K channel of highest conductance), reducing the energy gap between their K⁺ transport rates to ∼1 kT. Thus, although Shaker’s pore sustains ion translocation as the BK channel’s does, higher energetic costs of ion stabilization or higher friction with the ion’s rigid hydration cage in its narrower aqueous cavity may entail higher resistance.     

Phe-Met-Arg-Phe-amide Activates a Novel Voltage-dependent K+ Current through a Lipoxygenase Pathway in Molluscan Neurones

The neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) dose dependently (ED₅₀ = 23 nM) activated a K⁺ current in the peptidergic caudodorsal neurones that regulate egg laying in the mollusc Lymnaea stagnalis. Under standard conditions ([K⁺]_o = 1.7 mM), only outward current responses occurred. In high K⁺ salines ([K⁺]_o = 20 or 57 mM), current reversal occurred close to the theoretical reversal potential for K⁺. In both salines, no responses were measured below −120 mV. Between −120 mV and the K⁺ reversal potential, currents were inward with maximal amplitudes at ∼−60 mV. Thus, U-shaped current–voltage relations were obtained, implying that the response is voltage dependent. The conductance depended both on membrane potential and extracellular K⁺ concentration. The voltage sensitivity was characterized by an e-fold change in conductance per ∼14 mV at all [K⁺]_o. Since this result was also obtained in nearly symmetrical K⁺ conditions, it is concluded that channel gating is voltage dependent. In addition, outward rectification occurs in asymmetric K⁺ concentrations. Onset kinetics of the response were slow (rise time ∼650 ms at −40 mV). However, when FMRFa was applied while holding the cell at −120 mV, to prevent activation of the current but allow activation of the signal transduction pathway, a subsequent step to −40 mV revealed a much more rapid current onset. Thus, onset kinetics are largely determined by steps preceding channel activation. With FMRFa applied at −120 mV, the time constant of activation during the subsequent test pulse decreased from ∼36 ms at −60 mV to ∼13 ms at −30 mV, confirming that channel opening is voltage dependent. The current inactivated voltage dependently. The rate and degree of inactivation progressively increased from −120 to −50 mV. The current is blocked by internal tetraethylammonium and by bath- applied 4-aminopyridine, tetraethylammonium, Ba²⁺, and, partially, Cd²⁺ and Cs⁺. The response to FMRFa was affected by intracellular GTPγS. The response was inhibited by blockers of phospholipase A₂ and lipoxygenases, but not by a cyclo-oxygenase blocker. Bath-applied arachidonic acid induced a slow outward current and occluded the response to FMRFa. These results suggest that the FMRFa receptor couples via a G-protein to the lipoxygenase pathway of arachidonic acid metabolism. The biophysical and pharmacological properties of this transmitter operated, but voltage-dependent K⁺ current distinguish it from other receptor-driven K⁺ currents such as the S-current- and G-protein-dependent inward rectifiers.     

The influence of potassium concentration on epileptic seizures in a coupled neuronal model in the hippocampus

Experiments on hippocampal slices have recorded that a novel pattern of epileptic seizures with alternating excitatory and inhibitory activities in the CA1 region can be induced by an elevated potassium ion (K⁺) concentration in the extracellular space between neurons and astrocytes (ECS-NA). To explore the intrinsic effects of the factors (such as glial K⁺ uptake, Na⁺–K⁺-ATPase, the K⁺ concentration of the bath solution, and K⁺ lateral diffusion) influencing K⁺ concentration in the ECS-NA on the epileptic seizures recorded in previous experiments, we present a coupled model composed of excitatory and inhibitory neurons and glia in the CA1 region. Bifurcation diagrams showing the glial K⁺ uptake strength with either the Na⁺–K⁺-ATPase pump strength or the bath solution K⁺ concentration are obtained for neural epileptic seizures. The K⁺ lateral diffusion leads to epileptic seizure in neurons only when the synaptic conductance values of the excitatory and inhibitory neurons are within an appropriate range. Finally, we propose an energy factor to measure the metabolic demand during neuron firing, and the results show that different energy demands for the normal discharges and the pathological epileptic seizures of the coupled neurons.     

Inhibition of cAMP-Activated Intestinal Chloride Secretion by Diclofenac: Cellular Mechanism and Potential Application in Cholera

Cyclic AMP-activated intestinal Cl⁻ secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl⁻ secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl⁻ secretion in human intestinal epithelial (T84) cells with IC₅₀ of ∼20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl⁻ current showed that diclofenac reversibly inhibited CFTR Cl⁻ channel activity (IC₅₀∼10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na⁺-K⁺ ATPases and Na⁺-K⁺-Cl⁻ cotransporters, but inhibited cAMP-activated basolateral K⁺ channels with IC₅₀ of ∼3 µM. In addition, diclofenac suppressed Ca²⁺-activated Cl⁻ channels, inwardly rectifying Cl⁻ channels, and Ca²⁺-activated basolateral K⁺ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl⁻ secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca²⁺-activated Cl⁻ secretion by inhibiting both apical Cl⁻ channels and basolateral K⁺ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal hypersecretion of Cl⁻.     

Dynamic Volume Changes in Astrocytes Are an Intrinsic Phenomenon Mediated by Bicarbonate Ion Flux

Astrocytes, the major type of non-neuronal cells in the brain, play an important functional role in extracellular potassium ([K⁺]_o) and pH homeostasis. Pathological brain states that result in [K⁺]_o and pH dysregulation have been shown to cause astrocyte swelling. However, whether astrocyte volume changes occur under physiological conditions is not known. In this study we used two-photon imaging to visualize real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were observed to swell by 19.0±0.9% in response to a small physiological increase in the concentration of [K⁺]_o (3 mM). Astrocyte swelling was mediated by the influx of bicarbonate (HCO₃₋) ions as swelling was significantly decreased when the influx of HCO₃₋ was reduced. We found: 1) in HCO₃₋ free extracellular solution astrocytes swelled by 5.4±0.7%, 2) when the activity of the sodium-bicarbonate cotransporter (NBC) was blocked the astrocytes swelled by 8.3±0.7%, and 3) in the presence of an extracellular carbonic anhydrase (CA) inhibitor astrocytes swelled by 11.4±0.6%. Because a significant HCO₃₋ efflux is known to occur through the γ-amino-butyric acid (GABA) channel, we performed a series of experiments to determine if astrocytes were capable of HCO₃₋ mediated volume shrinkage with GABA channel activation. Astrocytes were found to shrink −7.7±0.5% of control in response to the GABA_A channel agonist muscimol. Astrocyte shrinkage from GABA_A channel activation was significantly decreased to −5.0±0.6% of control in the presence of the membrane-permeant CA inhibitor acetazolamide (ACTZ). These dynamic astrocyte volume changes may represent a previously unappreciated yet fundamental mechanism by which astrocytes regulate physiological brain functioning.     

Impact of Corticosterone Treatment on Spontaneous Seizure Frequency and Epileptiform Activity in Mice with Chronic Epilepsy

Stress is the most commonly reported precipitating factor for seizures in patients with epilepsy. Despite compelling anecdotal evidence for stress-induced seizures, animal models of the phenomena are sparse and possible mechanisms are unclear. Here, we tested the hypothesis that increased levels of the stress-associated hormone corticosterone (CORT) would increase epileptiform activity and spontaneous seizure frequency in mice rendered epileptic following pilocarpine-induced status epilepticus. We monitored video-EEG activity in pilocarpine-treated mice 24/7 for a period of four or more weeks, during which animals were serially treated with CORT or vehicle. CORT increased the frequency and duration of epileptiform events within the first 24 hours of treatment, and this effect persisted for up to two weeks following termination of CORT injections. Interestingly, vehicle injection produced a transient spike in CORT levels – presumably due to the stress of injection – and a modest but significant increase in epileptiform activity. Neither CORT nor vehicle treatment significantly altered seizure frequency; although a small subset of animals did appear responsive. Taken together, our findings indicate that treatment of epileptic animals with exogenous CORT designed to mimic chronic stress can induce a persistent increase in interictal epileptiform activity.     

Decreased Astroglial Monocarboxylate Transporter 4 Expression in Temporal Lobe Epilepsy

Efflux of monocaroxylates like lactate, pyruvate, and ketone bodies from astrocytes through monocarboxylate transporter 4 (MCT4) supplies the local neuron population with metabolic intermediates to meet energy requirements under conditions of increased demand. Disruption of this astroglial-neuron metabolic coupling pathway may contribute to epileptogenesis. We measured MCT4 expression in temporal lobe epileptic foci excised from patients with intractable epilepsy and in rats injected with pilocarpine, an animal model of temporal lobe epilepsy (TLE). Cortical MCT4 expression levels were significantly lower in TLE patients compared with controls, due at least partially to MCT4 promoter methylation. Expression of MCT4 also decreased progressively in pilocarpine-treated rats from 12 h to 14 days post-administration. Underexpression of MCT4 in cultured astrocytes induced by a short hairpin RNA promoted apoptosis. Knockdown of astrocyte MCT4 also suppressed excitatory amino acid transporter 1 (EAAT1) expression. Reduced MCT4 and EAAT1 expression by astrocytes may lead to neuronal hyperexcitability and epileptogenesis in the temporal lobe by reducing the supply of metabolic intermediates and by allowing accumulation of extracellular glutamate.     

Consequences of Converting Graded to Action Potentials upon Neural Information Coding and Energy Efficiency

Information is encoded in neural circuits using both graded and action potentials, converting between them within single neurons and successive processing layers. This conversion is accompanied by information loss and a drop in energy efficiency. We investigate the biophysical causes of this loss of information and efficiency by comparing spiking neuron models, containing stochastic voltage-gated Na⁺ and K⁺ channels, with generator potential and graded potential models lacking voltage-gated Na⁺ channels. We identify three causes of information loss in the generator potential that are the by-product of action potential generation: (1) the voltage-gated Na⁺ channels necessary for action potential generation increase intrinsic noise and (2) introduce non-linearities, and (3) the finite duration of the action potential creates a ‘footprint’ in the generator potential that obscures incoming signals. These three processes reduce information rates by ∼50% in generator potentials, to ∼3 times that of spike trains. Both generator potentials and graded potentials consume almost an order of magnitude less energy per second than spike trains. Because of the lower information rates of generator potentials they are substantially less energy efficient than graded potentials. However, both are an order of magnitude more efficient than spike trains due to the higher energy costs and low information content of spikes, emphasizing that there is a two-fold cost of converting analogue to digital; information loss and cost inflation.     

Decreased GABA receptor in the cerebral cortex of epileptic rats: effect of Bacopa monnieri and Bacoside-A

Abstact

Background

Gamma amino butyric acid (GABA), the principal inhibitory neurotransmitter in the cerebral cortex, maintains the inhibitory tones that counter balances neuronal excitation. When this balance is perturbed, seizures may ensue.

Methods

In the present study, alterations of the general GABA, GABA_A and GABA_B receptors in the cerebral cortex of the epileptic rat and the therapeutic application of Bacopa monnieri were investigated.

Results

Scatchard analysis of [³H]GABA, [³H]bicuculline and [³H]baclofen in the cerebral cortex of the epileptic rat showed significant decrease in B_max (P < 0.001) compared to control. Real Time PCR amplification of GABA receptor subunits such as GABA_Aά1, GABA_Aγ, GABA_Aδ, GABA_B and GAD where down regulated (P < 0.001) in epileptic rats. GABA_Aά5 subunit and Cyclic AMP responsible element binding protein were up regulated. Confocal imaging study confirmed the decreased GABA receptors in epileptic rats. Epileptic rats have deficit in radial arm and Y maze performance.

Conclusions

Bacopa monnieri and Bacoside-A treatment reverses epilepsy associated changes to near control suggesting that decreased GABA receptors in the cerebral cortex have an important role in epileptic occurrence; Bacopa monnieri and Bacoside-A have therapeutic application in epilepsy management.