Free Artificial Cerebrospinal Fluid Research Articles

Roles of metabotropic glutamate receptor 5 in low [Mg2+]o-induced interictal epileptiform activity in rat hippocampal slices.

Group I metabotropic glutamate receptors (mGluRs) modulate postsynaptic neuronal excitability and epileptogenesis. We investigated roles of group I mGluRs on low extracellular Mg2+ concentration ([Mg2+]o)-induced epileptiform activity and neuronal cell death in the CA1 regions of isolated rat hippocampal slices without the entorhinal cortex using extracellular recording and propidium iodide staining. Exposure to Mg2+-free artificial cerebrospinal fluid can induce interictal epileptiform activity in the CA1 regions of rat hippocampal slices. MPEP, a mGluR 5 antagonist, significantly inhibited the spike firing of the low [Mg2+]o-induced epileptiform activity, whereas LY367385, a mGluR1 antagonist, did not. DHPG, a group 1 mGluR agonist, significantly increased the spike firing of the epileptiform activity. U73122, a PLC inhibitor, inhibited the spike firing. Thapsigargin, an ER Ca2+-ATPase antagonist, significantly inhibited the spike firing and amplitude of the epileptiform activity. Both the IP3 receptor antagonist 2-APB and the ryanodine receptor antagonist dantrolene significantly inhibited the spike firing. The PKC inhibitors such as chelerythrine and GF109203X, significantly increased the spike firing. Flufenamic acid, a relatively specific TRPC 1, 4, 5 channel antagonist, significantly inhibited the spike firing, whereas SKF96365, a relatively non-specific TRPC channel antagonist, did not. MPEP significantly decreased low [Mg2+]o DMEM-induced neuronal cell death in the CA1 regions, but LY367385 did not. We suggest that mGluR 5 is involved in low [Mg2+]oinduced interictal epileptiform activity in the CA1 regions of rat hippocampal slices through PLC, release of Ca2+ from intracellular stores and PKC and TRPC channels, which could be involved in neuronal cell death.

Zinc‐α2‐glycoprotein relieved seizure‐Induced neuronal glucose uptake impairment via insulin‐like growth factor 1 receptor‐regulated glucose transporter 3 expression

Glucose hypometabolism is observed in epilepsy and promotes epileptogenesis. Glucose hypometabolism in epilepsy may be attributed to decreased neuronal glucose uptake, but its molecular mechanism remains unclear. Zinc-α2-glycoprotein (ZAG) is related to glucose metabolism and is reported to suppress seizures. The anti-epileptic effect of ZAG may be attributed to its regulation of neuronal glucose metabolism. This study explored the effect of ZAG on neuronal glucose uptake and its molecular mechanism via insulin-like growth factor 1 receptor (IGF1R)-regulated glucose transporter 3 (GLUT-3) expression. The ZAG level was modulated by lentivirus in primary culture neurons. Neuronal seizure models were induced by Mg2+ -free artificial cerebrospinal fluid. We assessed neuronal glucose uptake by the 2-NBDG method and Glucose Uptake Colorimetric Assay Kit. IGF1R was activated by IGF1 and blocked by AXL1717. The expression and distribution of IGF1R and GLUT-3, together with IGF1R phosphorylation, were measured by western blot. The binding between ZAG and IGF1R was determined by coimmunoprecipitation. Neuronal glucose uptake and GLUT-3 expression were significantly decreased by seizure or ZAG knockdown, whereas ZAG over-expression or IGF1 treatment reversed this decrease. The effect of ZAG on neuronal glucose uptake and GLUT-3 expression was blocked by AXL1717. ZAG increased IGF1R distribution and phosphorylation possibly by binding. Additionally, IGF1R increased GLUT-3 activity by increasing GLUT-3 expression. In epilepsy/seizure, neuronal glucose uptake suppression may be attributed to a decrease in ZAG, which suppresses neuronal GLUT-3 expression by regulating the activity of IGF1R. ZAG, IGF1R, and GLUT-3 may be novel potential therapeutic targets of glucose hypometabolism in epilepsy and seizures.

Effect of priming low-frequency magnetic fields on zero-Mg2+ -induced epileptiform discharges in rat hippocampal slices

During the process of seizures, the addition of low-frequency magnetic fields has been proved to be an effective method to suppress epileptic discharges. However, whether adding magnetic fields before the appearance of epileptic discharges can produce this inhibition has not been studied. In the present study, we first constructed epilepsy models on brain slices by perfusing them with Mg2+-free artificial cerebrospinal fluid (aCSF). The events of seizures evolved from inter-ictal epileptiform discharges (IIDs) to inter-epileptiform discharges (IDs). Combined with the multi-electrode array platform, we designed a flexible moving coil to generate a 0.5 Hz magnetic field on the brain slices. Using this method, we added the magnetic fields to brain slices for 30 min before epileptiform discharges were induced. The experimental results demonstrated that although the priming magnetic fields could not completely inhibit epileptiform discharges, they can significantly reduce the frequency of IDs and increase the frequency of IIDs in the CA3 region of the hippocampal slices. In the control group, the rates of IDs and IIDs were 0.0024 ± 0.0006 Hz and 0.0138 ± 0.0043 Hz, respectively, while in the magnetic stimulation group, the rates were 0.0012 ± 0.0004 Hz and 0.0251 ± 0.0067 Hz. Moreover, the results indicated that changing the frequency of interictal discharges did not affect ictogenesis. The results demonstrated that the priming magnetic fields had a certain weakening effect on the frequency of IDs, which was achieved by reducing the signal propagation speed and increasing the excitability threshold of hippocampal neurons.

Seizure-induced impairment in neuronal ketogenesis: Role of zinc-α2-glycoprotein in mitochondria.

Ketone bodies (KBs) were known to suppress seizure. Untraditionally, neurons were recently reported to utilize fatty acids and produce KBs, but the effect of seizure on neuronal ketogenesis has not been researched. Zinc‐α2‐glycoprotein (ZAG) was reported to suppress seizure via unclear mechanism. Interestingly, ZAG was involved in fatty acid β‐oxidation and thus may exert anti‐epileptic effect by promoting ketogenesis. However, this promotive effect of ZAG on neuronal ketogenesis has not been clarified. In this study, we performed immunoprecipitation and mass spectrometry to identify potential interaction partners with ZAG. The mechanisms of how ZAG translocated into mitochondria were determined by quantitative coimmunoprecipitation after treatment with apoptozole, a heat shock cognate protein 70 (HSC70) inhibitor. ZAG level was modulated by lentivirus in neurons or adeno‐associated virus in rat brains. Seizure models were induced by magnesium (Mg2+)‐free artificial cerebrospinal fluid in neurons or intraperitoneal injection of pentylenetetrazole kindling in rats. Ketogenesis was determined by cyclic thio‐NADH method in supernatant of neurons or brain homogenate. The effect of peroxisome proliferator–activated receptor γ (PPARγ) on ZAG expression was examined by Western blot, quantitative real‐time polymerase chain reaction (qRT‐PCR) and chromatin immunoprecipitation qRT‐PCR. We found that seizure induced ketogenesis deficiency via a ZAG‐dependent mechanism. ZAG entered mitochondria through a HSC70‐dependent mechanism, promoted ketogenesis by binding to four β‐subunits of long‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase (HADHB) and alleviated ketogenesis impairment in a neuronal seizure model and pentylenetetrazole‐kindled epileptic rats. Additionally, PPARγ activation up‐regulated ZAG expression by binding to promoter region of AZGP1 gene and promoted ketogenesis through a ZAG‐dependent mechanism.

Muscarinic acetylcholine receptor-dependent persistent activity of layer 5 intrinsic-bursting and regular-spiking neurons in primary auditory cortex.

Cholinergic signaling coupled to sensory-driven neuronal depolarization is essential for modulating lasting changes in deep-layer neural excitability and experience-dependent plasticity in the primary auditory cortex. However, the underlying cellular mechanism(s) associated with coincident cholinergic receptor activation and neuronal depolarization of deep-layer cortical neurons remains unknown. Using in vitro whole cell patch-clamp recordings targeted to neurons (n = 151) in isolated brain slices containing the primary auditory cortex (AI), we investigated the effects of cholinergic receptor activation and neuronal depolarization on the electrophysiological properties of AI layer 5 intrinsic-bursting and regular-spiking neurons. Bath application of carbachol (5 µM; cholinergic receptor agonist) paired with suprathreshold intracellular depolarization led to persistent activity in these neurons. Persistent activity may involve similar cellular mechanisms and be generated intrinsically in both intrinsic-bursting and regular-spiking neurons given that it 1) persisted under the blockade of ionotropic glutamatergic (kynurenic acid, 2 mM) and GABAergic receptors (picrotoxin, 100 µM), 2) was fully blocked by both atropine (10 µM; nonselective muscarinic antagonist) and flufenamic acid [100 µM; nonspecific Ca2+-sensitive cationic channel (CAN) blocker], and 3) was sensitive to the voltage-gated Ca2+ channel blocker nifedipine (50 µM) and Ca2+-free artificial cerebrospinal fluid. Together, our results support a model through which coincident activation of AI layer 5 neuron muscarinic receptors and suprathreshold activation can lead to sustained changes in layer 5 excitability, providing new insight into the possible role of a calcium-CAN-dependent cholinergic mechanism of AI cortical plasticity. These findings also indicate that distinct streams of auditory processing in layer 5 intrinsic-bursting and regular-spiking neurons may run in parallel during learning-induced auditory plasticity.NEW & NOTEWORTHY Cholinergic signaling coupled to sensory-driven neuronal depolarization is essential for modulating lasting changes in experience-dependent plasticity in the primary auditory cortex. Cholinergic activation together with cellular depolarization can lead to persistent activity in both intrinsic-bursting and regular-spiking layer 5 pyramidal neurons. A similar mechanism involving muscarinic acetylcholine receptor, voltage-gated Ca2+ channel, and possible Ca2+-sensitive nonspecific cationic channel activation provides new insight into our understanding of the cellular mechanisms that govern learning-induced auditory cortical and subcortical plasticity.

Natural product incarvillateine aggravates epileptic seizures by inhibiting GABAA currents

A natural monoterpene alkaloid incarvillateine isolated from the plant Incarvillea sinensis is known to relieve inflammatory and neuropathic pain. However, the molecular target for the action of incarvillateine remains elusive. Here, we report that incarvillateine exacerbates epileptic seizures by inhibiting subtypes of γ-Aminobutyric acid type A (GABAA) receptors. Two-electrode voltage clamp recordings of α1β3γ2, α2β3γ2, α3β3γ2 and α5β3γ2 subtypes expressed in Xenopus oocytes revealed that incarvillateine inhibited the GABAA currents with IC50 of 25.1 μM, 43.1 μM, 105.1 μM and 93.7 μM, respectively. Whole-cell patch clamp recordings of hippocampal slices confirmed that incarvillateine inhibited spontaneous inhibitory postsynaptic currents (IPSCs), and miniature IPSCs and tonic currents. Moreover, inhibition of GABAA currents and spontaneous IPSCs by incarvillateine persisted even in the presence of blockers of adenosine receptors. In addition, incarvillateine enhanced epileptic discharges induced by Mg2+-free artificial cerebrospinal fluid (ACSF) in hippocampal slices. Furthermore, intracerebral ventricular injections of incarvillateine increased the severity of seizures induced by kainic acid in a dose-dependent manner. Taken together, our data demonstrate that incarvillateine aggravates seizures by inhibition of GABAA currents and GABAergic synaptic transmissions.

GABAA Receptor Activity Suppresses the Transition from Inter-ictal to Ictal Epileptiform Discharges in Juvenile Mouse Hippocampus

Exploring the transition from inter-ictal to ictal epileptiform discharges (IDs) and how GABAA receptor-mediated action affects the onset of IDs will enrich our understanding of epileptogenesis and epilepsy treatment. We used Mg2+-free artificial cerebrospinal fluid (ACSF) to induce epileptiform discharges in juvenile mouse hippocampal slices and used a micro-electrode array to record the discharges. After the slices were exposed to Mg2+-free ACSF for 10min-20min, synchronous recurrent seizure-like events were recorded across the slices, and each event evolved from inter-ictal epileptiform discharges (IIDs) to pre-ictal epileptiform discharges (PIDs), and then to IDs. During the transition from IIDs to PIDs, the duration of discharges increased and the inter-discharge interval decreased. After adding 3μmol/L of the GABAA receptor agonist muscimol, PIDs and IDs disappeared, and IIDs remained. Further, the application of 10μmol/L muscimol abolished all the epileptiform discharges. When the GABAA receptor antagonist bicuculline was applied at 10μmol/L, IIDs and PIDs disappeared, and IDs remained at decreased intervals. These results indicated that there are dynamic changes in the hippocampal network preceding the onset of IDs, and GABAA receptor activity suppresses the transition from IIDs to IDs in juvenile mouse hippocampus.

Effective Connectivity of Hippocampal Neural Network and Its Alteration in Mg2+-Free Epilepsy Model

Understanding the connectivity of the brain neural network and its evolution in epileptiform discharges is meaningful in the epilepsy researches and treatments. In the present study, epileptiform discharges were induced in rat hippocampal slices perfused with Mg2+-free artificial cerebrospinal fluid. The effective connectivity of the hippocampal neural network was studied by comparing the normal and epileptiform discharges recorded by a microelectrode array. The neural network connectivity was constructed by using partial directed coherence and analyzed by graph theory. The transition of the hippocampal network topology from control to epileptiform discharges was demonstrated. Firstly, differences existed in both the averaged in- and out-degree between nodes in the pyramidal cell layer and the granule cell layer, which indicated an information flow from the pyramidal cell layer to the granule cell layer during epileptiform discharges, whereas no consistent information flow was observed in control. Secondly, the neural network showed different small-worldness in the early, middle and late stages of the epileptiform discharges, whereas the control network did not show the small-world property. Thirdly, the network connectivity began to change earlier than the appearance of epileptiform discharges and lasted several seconds after the epileptiform discharges disappeared. These results revealed the important network bases underlying the transition from normal to epileptiform discharges in hippocampal slices. Additionally, this work indicated that the network analysis might provide a useful tool to evaluate the neural network and help to improve the prediction of seizures.

Influence of chronic ethanol intake on mouse synaptosomal aspartyl aminopeptidase and aminopeptidase A: Relationship with oxidative stress indicators

Aminopeptidase A (APA) and aspartyl aminopeptidase (ASAP) not only act as neuromodulators in the regional brain renin-angiotensin system, but also release N-terminal acidic amino acids (glutamate and aspartate). The hyperexcitability of amino acid neurotransmitters is responsible for several neurodegenerative processes affecting the central nervous system. The purpose of the present work was to study the influence of chronic ethanol intake, a well known neurotoxic compound, on APA and ASAP activity under resting and K+-stimulated conditions at the synapse level. APA and ASAP activity were determined against glutamate- and aspartate-β-naphthylamide respectively in mouse frontal cortex synaptosomes and in their incubation supernatant in a Ca2+-containing or Ca2+-free artificial cerebrospinal fluid. The neurotoxic effects were analyzed by determining free radical generation, peroxidation of membrane lipids and the oxidation of synaptosomal proteins. In addition, the bioenergetic behavior of synaptosomes was analyzed under different experimental protocols. We obtained several modifications in oxidative stress parameters and a preferential inhibitor effect of chronic ethanol intake on APA and ASAP activities. Although previous in vitro studies failed to show signs of neurodegeneration, these in vivo modifications in oxidative stress parameters do not seem to be related to changes in APA and ASAP, invalidating the idea that an excess of free acidic amino acids released by APA and ASAP induces neurodegeneration.

Endogenous ACh effects on NMDA‐induced interictal‐like discharges along the septotemporal hippocampal axis of adult rats and their modulation by an early life generalized seizure

Our earlier findings of the modulation of cholinergic neurotransmission by an early life generalized seizure and the reported interaction between muscarinic and N-methyl-d-aspartate (NMDA) receptors prompted us to investigate the effects of endogenous acetylcholine (ACh) on the frequency (Hz) of the epileptiform discharges following NMDA-receptor activation in the hippocampal slice. A sustained (>20 min) generalized convulsion was induced in Sprague-Dawley juvenile rats by intraperitoneal injection with pentylenetetrazole (PTZ, 70-90 mg/kg) at postnatal day (P) 20. Temporal and septal hippocampal slices were prepared of normal (N) and PTZ-treated (PTZ) adult (≥P60) rats, and CA3 field potentials were recorded during perfusion with Mg(2+) -free artificial cerebrospinal fluid (ACSF) or with ACSF containing 50 μm 4-aminopyridine (4-AP). In Mg(2+) -free ACSF, spontaneous interictal-like epileptiform discharges (IEDs) were recorded in all slices, with significantly higher frequencies in temporal (0.46 ± 0.03 Hz, n = 85) versus septal slices (0.20 ± 0.02 Hz, n = 47, p < 0.000001) but no consistent differences in any other group (i.e., male vs. female or N vs. PTZ). The anticholinesterase eserine (10 μm) increased their frequencies by 150-200% in N-septal and in all temporal slices and by 300% in PTZ-septal slices (p = 0.0028). In 60% of the slices the excitatory effect persisted throughout drug perfusion, whereas in the remaining ones it was distinguished in two phases: an early "transient" and a late "steady state." The steady-state frequencies resembled the predrug ones in N slices but remained significantly elevated in PTZ slices, especially in the septal group. The muscarinic antagonist atropine (1 μm) decreased IED frequency in all slices (n = 36, p = 0.005) and also fully reversed the eserine effect (n = 38, p < 0.0001). In 4-AP ACSF, eserine increased spontaneous IED frequency (n = 21) in N and PTZ slices alike; IEDs were subsequently abolished by addition of the NMDA-receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP5; 50 μm, n = 6). These results demonstrate an intrinsic tonic positive muscarinic acetylcholine receptor (mAChR) contribution to the frequency of NMDA receptor-dependent epileptiform discharges that is amplified following an elevation of endogenous ACh and is more pronounced in the septal hippocampus. Moreover, this positive mAChR contribution to the frequency of IEDs is even more pronounced and persistent in the septal extremity after an early life generalized sustained convulsion. This cholinergic enhancement of the excitatory septal hippocampal output may influence cognitive function and performance, and possibly the adult seizure threshold.

Barium Plateau Potentials of CA1 Pyramidal Neurons Elicit All-or-None Extracellular Alkaline Shifts Via the Plasma Membrane Calcium ATPase

In many brain regions, synchronous neural activity causes a rapid rise in extracellular pH. In the CA1 region of hippocampus, this population alkaline transient (PAT) enhances responses from postsynaptic, pH-sensitive N-methyl-d-aspartate (NMDA) receptors. Recently, we showed that the plasma membrane Ca(2+)-ATPase (PMCA), a ubiquitous transporter that exchanges internal Ca(2+) for external H(+), is largely responsible for the PAT. It has also been shown that a PAT can be generated after replacing extracellular Ca(2+) with Ba(2+). The cause of this PAT is unknown, however, because the ability of the mammalian PMCA to transport Ba(2+) is unclear. If the PMCA did not carry Ba(2+), a different alkalinizing source would have to be postulated. Here, we address this issue in mouse hippocampal slices, using concentric (high-speed, low-noise) pH microelectrodes. In Ba(2+)-containing, Ca(2+)-free artificial cerebrospinal fluid, a single antidromic shock to the alveus elicited a large (0.1-0.2 unit pH), "all-or-none" PAT in the CA1 cell body region. In whole cell current clamp of single CA1 pyramidal neurons, the same stimulus evoked a prolonged plateau potential that was similarly all-or-none. Using this plateau as the voltage command in other cells, we recorded Ba(2+)-dependent surface alkaline transients (SATs). The SATs were suppressed by adding 5 mM extracellular HEPES and abolished when carboxyeosin (a PMCA inhibitor) was in the patch pipette solution. These results suggest that the PAT evoked in the presence of Ba(2+) is caused by the PMCA and that this transporter is responsible for the PAT whether Ca(2+) or Ba(2+) is the charge carrying divalent cation.

Regionally Localized Recurrent Excitation in the Dentate Gyrus of a Cortical Contusion Model of Posttraumatic Epilepsy

Posttraumatic epilepsy is a frequent consequence of brain trauma, but relatively little is known about how neuronal circuits are chronically altered after closed head injury. We examined whether local recurrent excitatory synaptic connections form between dentate granule cells in mice 8-12 wk after cortical contusion injury. Mice were monitored for behavioral seizures shortly after brain injury and < or = 10 wk postinjury. Injury-induced seizures were observed in 15% of mice, and spontaneous seizures were observed weeks later in 40% of mice. Timm's staining revealed mossy fiber sprouting into the inner molecular layer of the dorsal dentate gyrus ipsilateral to the injury in 95% of mice but not contralateral to the injury or in uninjured controls. Whole cell patch-clamp recordings were made from granule cells in isolated hippocampal brain slices. Cells in slices with posttraumatic mossy fiber sprouting had an increased excitatory postsynaptic current (EPSC) frequency compared with cells in slices without sprouting from injured and control animals (P < 0.001). When perfused with Mg(2+)-free artificial cerebrospinal fluid containing 100 microM picrotoxin, these cells had spontaneous bursts of EPSCs and action potentials. Focal glutamate photostimulation of the granule cell layer evoked a burst of EPSCs and action potentials indicative of recurrent excitatory connections in granule cells of slices with mossy fiber sprouting. In granule cells of slices without sprouting from injured animals and controls, spontaneous or photostimulation-evoked epileptiform activity was never observed. These results suggest that a new regionally localized excitatory network forms between dentate granule cells near the injury site within weeks after cortical contusion head injury.

Conductances Mediating Intrinsic Theta-Frequency Membrane Potential Oscillations in Layer II Parasubicular Neurons

Ionic conductances that generate membrane potential oscillations in neurons of layer II of the parasubiculum were studied using whole cell current-clamp recordings in horizontal slices from the rat brain. Blockade of ionotropic glutamate and GABA synaptic transmission did not reduce the power of the oscillations, indicating that oscillations are not dependent on synaptic inputs. Oscillations were eliminated when cells were hyperpolarized 6-10 mV below spike threshold, indicating that they are mediated by voltage-dependent conductances. Application of TTX completely eliminated oscillations, suggesting that Na(+) currents are required for the generation of the oscillations. Oscillations were not reduced by blocking Ca(2+) currents with Cd(2+) or Ca(2+)-free artificial cerebrospinal fluid, or by blocking K(+) conductances with either 50 microM or 5 mM 4-aminopyridine (4-AP), 30 mM tetraethylammonium (TEA), or Ba(2+)(1-2 mM). Oscillations also persisted during blockade of the muscarinic-dependent K(+) current, I(M), using the selective antagonist XE-991 (10 microM). However, oscillations were significantly attenuated by blocking the hyperpolarization-activated cationic current I(h) with Cs(+) and were almost completely blocked by the more potent I(h) blocker ZD7288 (100 microM). Intrinsic membrane potential oscillations in neurons of layer II of the parasubiculum are therefore likely driven by an interaction between an inward persistent Na(+) current and time-dependent deactivation of I(h). These voltage-dependent conductances provide a mechanism for the generation of membrane potential oscillations that can help support rhythmic network activity within the parasubiculum during theta-related behaviors.

Lamotrigine inhibits postsynaptic AMPA receptor and glutamate release in the dentate gyrus

The dentate gyrus (DG) is a gateway that regulates seizure activity in the hippocampus. We investigated the site of action of lamotrigine (LTG), an effective anticonvulsant, in the regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptor-mediated excitatory synaptic transmission on DG. Evoked AMPA and NMDA receptor-mediated excitatory postsynaptic currents (eEPSCampa and eEPSCnmda) were recorded by whole-cell patch-clamp recording from the granule cells of DG in brain slice preparation of young Wistar rats (60-120 g). Exogenously applied AMPA and NMDA-induced currents and AMPA receptor-mediated miniature EPSC (mEPSCampa) were recorded in the presence of specific antagonists. LTG inhibited both eEPSCampa and eEPSCnmda, and had no effect on exogenously applied NMDA-induced current indicating LTG inhibited glutamate release. Previous studies demonstrated that alteration in glutamate concentration in synaptic cleft causes parallel changes of eEPSCampa and eEPSCnmda. Our results showed that LTG inhibited eEPSCampa significantly more than eEPSCnmda (p < 0.05), suggesting that LTG may also have blocked the postsynaptic AMPA receptor. The hypothesis is further supported by the facts that; (1) LTG (30-100 microM) inhibited direct exogenously applied AMPA-induced currents (to 90%), (2) LTG significantly reduced the amplitude, but not the frequency of mEPSCampa and asynchronous (EPSC), and (3) LTG-induced reduction of eEPSCampa was not associated with a modification of the paired-pulse ratio. To sum up, LTG exerts a postsynaptic inhibitory mechanism on the AMPA receptor. Our results demonstrate that LTG suppresses postsynaptic AMPA receptors and reduces glutamate release in granule cells of DG. The postsynaptic effect can be one of the underlying mechanisms of LTG's anticonvulsant action.

Glutamate and dopamine receptors contribute to the lateral spread of epileptiform discharges in rat neocortical slices

The effects of AMPA-type glutamate receptor as well as dopamine D1 and D2 receptors on the lateral propagation of epileptiform field potentials (EFP) were studied across adjacent areas of rat neocortical tissues. Epileptiform burst discharges were induced by superfusion of Mg(2+)-free artificial cerebrospinal fluid. Simultaneous field potential recordings of EFP were obtained from four microelectrodes placed 2-3 mm apart across coronal slices in the third layer of the neocortex. The effects of AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), dopamine D1 receptor agonist SKF 81297, and dopamine D2 receptor agonist quinpirole on lateral propagation of burst discharges were investigated. CNQX, applied focally between recording sites, blocked rapid propagation across treated areas and resulted in the emergence of spatially separate, independent pacemakers. Focal application of SKF 81297 between recording sites increased the repetition rate of EFP, but reduced the amplitude as well as the duration of epileptic discharges. However, addition of SKF 81297 to the bath medium abolished EFP. Both local and systemic applications of quinpirole irreversibly enhanced repetition rate of epileptiform burst discharges. The results indicate the prerequisite of AMPA synaptic transmission for synchronized lateral propagation of Mg(2+)-free ACSF-induced epileptic activity and the modulatory effects of dopamine D1 and D2 receptors on both EFP initiation and propagation in epileptic tissues.

The antiepileptic activity of JSTX-3 is mediated by N-methyl-D-aspartate receptors in human hippocampal neurons

We analyzed the effect of the acylpolyaminetoxin JSTX-3 on the epileptogenic discharges induced by perfusion of human hippocampal slices with artificial cerebrospinal fluid lacking Mg2+ or N-methyl-D-aspartate. Hippocampi were surgically removed from patients with refractory medial temporal lobe epilepsy, sliced in the surgical room and taken to the laboratory immersed in normal artificial cerebrospinal fluid. Epileptiform activity was induced by perfusion with Mg2+-free artificial cerebrospinal fluid or by iontophoretically applied N-methyl-D-aspartate and intracellular and field recordings of CA1 neurons were performed. The ictal-like discharges induced by Mg2+-free artificial cerebrospinal fluid and N-methyl-D-aspartate were blocked by incubation with JSTX-3. This effect was similar to that obtained with the N-methyl-D-aspartate receptor antagonist DL (-)2-amino-5 phosphonovaleric acid. Our findings suggest that in human hippocampal neurons, the antiepileptic effect of JSTX-3 is mediated by its action on N-methyl-D-aspartate receptor.

Effect of spermine and N 1-dansyl-spermine on epileptiform activity in mouse cortical slices

N 1-dansyl-spermine is a novel polyamine analogue, which has been demonstrated to have an antagonist action at the stimulatory polyamine site on the N-methyl- d-aspartate (NMDA) receptor macrocomplex. Cortical wedges from genetically epilepsy-prone DBA/2 mice demonstrate spontaneous epileptiform activity when perfused with Mg 2+-free artificial cerebrospinal fluid (aCSF). This epileptiform activity has been demonstrated to be primarily mediated through the NMDA receptor. N 1-dansyl-spermine reduced the spontaneous epileptiform activity at a high dose (100 μM) but had no effect at a lower dose (50 μM). The polyamine, spermine (300 μM) caused an increase in the rate of the spontaneous epileptiform discharges. This effect of spermine was antagonised by administration of the low dose of N 1-dansyl-spermine (50 μM). This further demonstrates the role of the NMDA receptor in the production of spontaneous epileptiform discharges in the cortical wedge preparation and clearly illustrates both the facilitatory action of spermine and the polyamine antagonist action of N 1-dansyl-spermine at the stimulatory polyamine site on the NMDA receptor.

Postsynaptic excitation of prefrontal cortical pyramidal neurons by hypocretin‐1/orexin a through the inhibition of potassium currents

Hypocretins are crucial for the regulation of wakefulness by the excitatory actions on multiple subcortical arousal systems. To date, there is little information about the direct postsynaptic excitatory effects of hypocretins on the neurons in prefrontal cortex (PFC), which is important for higher cognitive functions and is correlated with level of wakefulness. In this study, we tested the excitatory effects of hypocretin-1 on acutely isolated PFC pyramidal neurons of rats and studied the possible ionic mechanisms by using whole-cell patch-clamp techniques. Puff application of hypocretin-1 caused a dose-dependent excitation. Further observations that perfusion of Ca2+-free artificial cerebrospinal fluid did not influence the depolarizing effects of hypocretin-1, in conjunction with the findings that hypocretin-1 could decrease net whole-cell K+ currents, demonstrate that the excitatory effects of hypocretin-1 on PFC neurons are mediated by the inhibition of K+ currents but not Ca2+ influx. Finally, the decrease in K+ currents induced by hypocretin-1 was abolished by a protein kinase C (PKC) inhibitor (BIS II) or a phospholipase C (PLC) inhibitor (D609), suggesting that PKC and PLC appear to be involved in mediating the inhibitory effects of hypocretin-1 on K+ currents. These results indicate that hypocretin-1 exerts a postsynaptic excitatory action on PFC neurons through the inhibition of K+ currents, which probably results from activation of PKC and PLC signaling pathways.

Depolarization Activates ERK and Proline-rich Tyrosine Kinase 2 (PYK2) Independently in Different Cellular Compartments in Hippocampal Slices

In the hippocampus, extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity. Both are also activated under pathological conditions following ischemia, convulsions, or electroconvulsive shock. Although in non-neuronal cells PYK2 activates ERK through the recruitment of Src-family kinases (SFKs), the link between these pathways in the hippocampus is not known. We addressed this question using K(+)-depolarized rat hippocampal slices. Depolarization increased the phosphorylation of PYK2, SFKs, and ERK. These effects resulted from Ca(2+) influx through voltage-gated Ca(2+) channels and were diminished by GF109203X, a protein kinase C inhibitor. Inhibition of SFKs with PP2 decreased PYK2 tyrosine phosphorylation dramatically, but not its autophosphorylation on Tyr-402. Moreover, PYK2 autophosphorylation and total tyrosine phosphorylation were profoundly altered in fyn-/- mice, revealing an important functional relationship between Fyn and PYK2 in the hippocampus. In contrast, ERK activation was unaltered by PP2, Fyn knock-out, or LY294002, a phosphatidyl-inositol-3-kinase inhibitor. ERK activation was prevented by MEK inhibitors that had no effect on PYK2. Immunofluorescence of hippocampal slices showed that PYK2 and ERK were activated in distinct cellular compartments in somatodendritic regions and nerve terminals, respectively, with virtually no overlap. Activation of ERK was critical for the rephosphorylation of a synaptic vesicle protein, synapsin I, following depolarization, underlining its functional importance in nerve terminals. Thus, in hippocampal slices, in contrast to cell lines, depolarization-induced activation of non-receptor tyrosine kinases and ERK occurs independently in distinct cellular compartments in which they appear to have different functional roles.

Pregnenolone sulfate enhances long‐term potentiation in CA1 in rat hippocampus slices through the modulation of N‐methyl‐D‐aspartate receptors

Among the different steroids found in the brain, pregnenolone sulfate (3beta-hydroxy-5-pregnen-20-one-3-sulfate; PREGS) is known to enhance hippocampal-associated memory. The present study employs rat hippocampal slices to investigate the ability of PREGS to modulate long-term potentiation (LTP), a phenomenon considered as a model of synaptic plasticity related to memory processes. LTP (3 x 100 Hz/1 sec within 2 min), implicated essentially glutamatergic transmission, for which the different synaptic events could be pharmacologically dissociated. We show that PREGS enhances LTP in CA1 pyramidal neurons at nanomolar concentrations and exhibits a bell-shaped concentration-response curve. The maximal effect of PREGS on both induction and maintenance phases of LTP is observed at 300 nM and requires 10 min of superfusion. Although PREGS does not change the N-methyl-D-aspartate (NMDA) component of the field potentials (fEPSPs) isolated in the presence of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in Mg2+-free artificial cerebrospinal fluid, PREGS does enhance the response induced by NMDA application (50 microM, 20 sec). PREGS does not modify the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component of the fEPSPs isolated in the presence of 100 microM DL-2-amino-7-phosphopentanoic acid (DL-AP5) or its potentiation induced by a single tetanic stimulation and the response induced by AMPA application (10 microM, 10 sec). Furthermore, PREGS does not affect the recurrent inhibition of the fEPSPs mediated by gamma-aminobutyric acid type A (GABA(A)) receptor. In conclusion, this study shows the ability of PREGS to enhance LTP in CA1 by accentuating the activity of NMDA receptors. This modulation of LTP might mediate the steroid-induced enhancement of memory.