Currents In CA1 Neurons Research Articles

Anesthesia with sevoflurane in neonatal rats: Developmental neuroendocrine abnormalities and alleviating effects of the corticosteroid and Cl− importer antagonists

Background1.5 million children under 12 months of age are exposed to general anesthesia annually in the United States alone. Human and especially animal studies provide evidence that exposure to general anesthesia during the early postnatal period may lead to long-term neurocognitive abnormalities via poorly understood mechanisms. We investigated whether an immature stress response system and γ-aminobutyric acid (GABA) type A receptor activities are involved in mediating these abnormalities. MethodsSprague–Dawley rats at postnatal days 4, 5 or 6 were anesthetized with 2.1% sevoflurane for 6h; maternally separated and house reared rats served as controls. ResultsSevoflurane anesthesia markedly increased corticosterone levels in rat pups of both genders. In adulthood, these rats responded to stress with heightened secretion of corticosterone and a greater increase in corticosterone levels in males versus females. Only male rats, previously exposed to neonatal sevoflurane, had a higher frequency of miniature inhibitory postsynaptic currents in CA1 neurons, spent a shorter time in open arms of the elevated plus maze (EPM) and exhibited impaired prepulse inhibition (PPI) of startle. Pretreatment of male rats prior to sevoflurane with the Na+-K+-2Cl− cotransporter inhibitor, bumetanide, or the mineralocorticoid receptor antagonist, RU28318, normalized endocrine responses to stress and the EPM behavior in adulthood, while only those pretreated with bumetanide exhibited normalized PPI of startle responses. Neither bumetanide nor RU28318 altered the effect of sevoflurane on synaptic activity. ConclusionsSevoflurane-enhanced neuronal excitation and elevated corticosteroid levels at the time of anesthesia contribute to the mechanisms initiating neonatal sevoflurane-induced long-term endocrine and neurobehavioral abnormalities.

Neuroplasticity of A-type potassium channel complexes induced by chronic alcohol exposure enhances dendritic calcium transients in hippocampus.

Chronic alcohol-induced cognitive impairments and maladaptive plasticity of glutamatergic synapses are well-documented. However, it is unknown if prolonged alcohol exposure affects dendritic signaling that may underlie hippocampal dysfunction in alcoholics. Back-propagation of action potentials (bAPs) into apical dendrites of hippocampal neurons provides distance-dependent signals that modulate dendritic and synaptic plasticity. The amplitude of bAPs decreases with distance from the soma that is thought to reflect an increase in the density of Kv4.2 channels toward distal dendrites. The aim of this study was to quantify changes in hippocampal Kv4.2 channel function and expression using electrophysiology, Ca(2+) imaging, and western blot analyses in a well-characterized in vitro model of chronic alcohol exposure. Chronic alcohol exposure significantly decreased expression of Kv4.2 channels and KChIP3 in hippocampus. This reduction was associated with an attenuation of macroscopic A-type K(+) currents in CA1 neurons. Chronic alcohol exposure increased bAP-evoked Ca(2+) transients in the distal apical dendrites of CA1 pyramidal neurons. The enhanced bAP-evoked Ca(2+) transients induced by chronic alcohol exposure were not related to synaptic targeting of N-methyl-D-aspartate (NMDA) receptors or morphological adaptations in apical dendritic arborization. These data suggest that chronic alcohol-induced decreases in Kv4.2 channel function possibly mediated by a downregulation of KChIP3 drive the elevated bAP-associated Ca(2+) transients in distal apical dendrites. Alcohol-induced enhancement of bAPs may affect metaplasticity and signal integration in apical dendrites of hippocampal neurons leading to alterations in hippocampal function.

Endocrine and neurobehavioral abnormalities induced by propofol administered to neonatal rats.

The authors studied whether neonatal propofol anesthesia affects development of the endocrine and neural systems. Sprague-Dawley rats were anesthetized using intraperitoneal propofol for 5 h on postnatal days (P) 4, 5, or 6. Pups that received either saline or intralipid, but not those in the negative control groups, were also maternally separated for 5 h. Serum levels of corticosterone were measured immediately after anesthesia and in adulthood after prepulse inhibition of acoustic startle testing (≥P80), followed by measurement of hippocampal neuronal activity. Propofol acutely increased corticosterone levels to 146.6 ± 23.5 ng/ml (n = 6) versus 16.4 ± 3.5 ng/ml (n = 6) and 18.4 ± 3.2 ng/ml (n = 6) in saline- and intralipd-treated pups, respectively. In adulthood, the propofol group exhibited exacerbated endocrine responses to stress in a form of increased corticosterone levels (1,171.58 ± 149.17 ng/ml [n = 15] vs. 370.02 ± 36.01 ng/ml [n = 10] in the saline group). The propofol group had increased the frequency of miniature inhibitory postsynaptic currents in CA1 neurons of male and female rats, but reduced prepulse inhibition of startle was detected only in males. The Na-K-2Cl cotransporter inhibitor bumetanide, administered to pups before propofol injection, alleviated long-term endocrine and prepulse inhibition abnormalities. Exogenous corticosterone, administered to naive pups, induced synaptic and endocrine but not prepulse inhibition effects, similar to those of propofol. Propofol-caused acute increases in corticosterone levels and γ-aminobutyric acid type A receptor-mediated excitation at the time of anesthesia may play mechanistic roles in development of exacerbated endocrine responses to stress and neurobehavioral abnormalities.

L-type Ca2+ currents at CA1 synapses, but not CA3 or dentate granule neuron synapses, are increased in 3xTgAD mice in an age-dependent manner

Abnormal neuronal excitability and impaired synaptic plasticity might occur before the degeneration and death of neurons in Alzheimer's disease (AD). To elucidate potential biophysical alterations underlying aberrant neuronal network activity in AD, we performed whole-cell patch clamp analyses of L-type (nifedipine-sensitive) Ca2+ currents (L-VGCC), 4–aminopyridine-sensitive K+ currents, and AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid) and NMDA (N-methyl-D-aspartate) currents in CA1, CA3, and dentate granule neurons in hippocampal slices from young, middle-age, and old 3xTgAD mice and age-matched wild type mice. 3xTgAD mice develop progressive widespread accumulation of amyloid β-peptide, and selective hyperphosphorylated tau pathology in hippocampal CA1 neurons, which are associated with cognitive deficits, but independent of overt neuronal degeneration. An age-related elevation of L-type Ca2+ channel current density occurred in CA1 neurons in 3xTgAD mice, but not in wild type mice, with the magnitude being significantly greater in older 3xTgAD mice. The NMDA current was also significantly elevated in CA1 neurons of old 3xTgAD mice compared with in old wild type mice. There were no differences in the amplitude of K+ or AMPA currents in CA1 neurons of 3xTgAD mice compared with wild type mice at any age. There were no significant differences in Ca2+, K+, AMPA, or NMDA currents in CA3 and dentate neurons from 3xTgAD mice compared with wild type mice at any age. Our results reveal an age-related increase of L-VGCC density in CA1 neurons, but not in CA3 or dentate granule neurons, of 3xTgAD mice. These findings suggest a potential contribution of altered L-VGCC to the selective vulnerability of CA1 neurons to tau pathology in the 3xTgAD mice and to their degeneration in AD patients.

Saikosaponin a Enhances Transient Inactivating Potassium Current in Rat Hippocampal CA1 Neurons

Saikosaponin a (SSa), a main constituent of the Chinese herb Bupleurum chinense DC., has been demonstrated to have antiepileptic activity. Recent studies have shown that SSa could inhibit NMDA receptor current and persistent sodium current. However, the effects of SSa on potassium (K+) currents remain unclear. In this study, we tested the effect of SSa on 4AP-induced epileptiform discharges and K+ currents in CA1 neurons of rat hippocampal slices. We found that SSa significantly inhibited epileptiform discharges frequency and duration in hippocampal CA1 neurons in the 4AP seizure model in a dose-dependent manner with an IC 50 of 0.7 μM. SSa effectively increased the amplitude of I Total and I A, significantly negative-shifted the activation curve, and positive-shifted steady-state curve of I A. However, SSa induced no significant changes in the amplitude and activation curve of I K. In addition, SSa significantly increased the amplitude of 4AP-sensitive K+ current, while there was no significant change in the amplitude of TEA-sensitive K+ current. Together, our data indicate that SSa inhibits epileptiform discharges induced by 4AP in a dose-dependent manner and that SSa exerts selectively enhancing effects on I A. These increases in I A may contribute to the anticonvulsant mechanisms of SSa.

Amyloid β‐protein suppressed nicotinic acetylcholine receptor‐mediated currents in acutely isolated rat hippocampal CA1 pyramidal neurons

Amyloid β protein (Aβ) is responsible for the deficits of learning and memory in Alzheimer's disease (AD). The high affinity between Aβ and nicotinic acetylcholine receptors (nAChRs) suggests that the impairment of cognitive function in AD might be involved in the Aβ-induced damage of nAChRs. This study investigated the effects of Aβ fragments on nAChR-mediated membrane currents in acutely isolated rat hippocampal pyramidal neurons by using whole-cell patch clamp technique. The results showed that: (1) nonspecific nAChR agonist nicotine, selective α7 nAChR agonist choline, and α4β2 nAChR agonist epibatidine all effectively evoked inward currents in CA1 neurons at normal resting membrane potential, with different desensitization characteristics; (2) acute application of different concentrations (pM-μM) of Aβ25-35, Aβ31-35, or Aβ35-31 alone did not trigger any membrane current, but pretreatment with 1 μM Aβ25-35 and Aβ31-35 similarly and reversibly suppressed the nicotine-induced currents; (3) further, choline- and epibatidine-induced currents were also reversibly suppressed by the Aβ pretreatment, but more prominent for the choline-induced response. These results demonstrate that the functional activity of both α7 and α4β2 nAChRs in the membrane of acutely isolated hippocampal neurons was significantly downregulated by Aβ treatment, suggesting that nAChRs, especially α7 nAChRs, in the brain may be the important biological targets of neurotoxic Aβ in AD. In addition, the similar suppression of nAChR currents by Aβ25-35 and Aβ31-35 suggests that the sequence 31-35 in Aβ molecule may be a shorter active center responsible for the neurotoxicity of Aβ in AD.

Sustained K+Outward Currents are Sensitive to Intracellular Heteropodatoxin2 in CA1 Neurons of Organotypic Cultured Hippocampi of Rats

Blocking or regulating K+ channels is important for investigating neuronal functions in mammalian brains, because voltage-dependent K+ channels (Kv channels) play roles to regulate membrane excitabilities for synaptic and somatic processings in neurons. Although a number of toxins and chemicals are useful to change gating properties of Kv channels, specific effects of each toxin on a particular Kv subunit have not been sufficiently demonstrated in neurons yet. In this study, we tested electrophysiologically if heteropodatoxin2 (HpTX2), known as one of Kv4-specific toxins, might be effective on various K+ outward currents in CA1 neurons of organotypic hippocampal slices of rats. Using a nucleated-patch technique and a pre-pulse protocol in voltage-clamp mode, total K+ outward currents recorded in the soma of CA1 neurons were separated into two components, transient and sustained currents. The extracellular application of HpTX2 weakly but significantly reduced transient currents. However, when HpTX2 was added to internal solution, the significant reduction of amplitudes were observed in sustained currents but not in transient currents. This indicates the non-specificity of HpTX2 effects on Kv4 family. Compared with the effect of cytosolic 4-AP to block transient currents, it is possible that cytosolic HpTX2 is pharmacologically specific to sustained currents in CA1 neurons. These results suggest that distinctive actions of HpTX2 inside and outside of neurons are very efficient to selectively reduce specific K+ outward currents.

Relating the Effects of Prenatal Stress in Rodents to the Pathogenesis of Schizophrenia

s t t a s c t T he study by Escobar et al. (1) in this issue shows that exposure of the pregnant rat dam at day 19 of gestation to a bacterial endotoxin, a manipulation used to model maternal immune ctivation (MIA), leads to abnormal postnatal development of synptic long-term depression (LTD) in the CA3-CA1 hippocampal ciruit in the offspring. The form of LTD they examined is induced by ow-frequency stimulation of the glutamatergic efferents from CA3, hich synapse extensively onto the dendrites of the CA1 projection eurons. The study shows that in neonatal brains from control and MIAxposed offspring, LTD in the CA3-CA1 circuit induced by a train of ow-frequency stimuli is prominent and depends on N-methyl-Dspartate (NMDA) receptors and downstream messengers. This LTD ormally decreases across postnatal development; however, MIAxposed offspring show a more rapid developmental reduction, osing this form of plasticity altogether by the late juvenile period. he authors also found a parallel developmental loss of NMDA relative to 2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid AMPA]) currents in CA1 neurons of MIA offspring but do not adress whether this is causally related to the developmental loss of TD. They added a more preliminary finding, that MIA offspring also how abnormal ontogeny, particularly evident in the late juvenile eriod, of a different form of synaptic plasticity regulated in part by etabotropic glutamate receptors. Taken together, the findings upport the hypothesis that MIA leads to changes in mechanisms egulating synaptic plasticity in the CA3-CA1 circuit that may, in urn, be hypothesized to have a profound and persistent impact on he ability of CA1 neurons to integrate input signals from other ippocampal subregions, the subiculum, the entorhinal and paraippocampal cortices, and the limbic basal forebrain (1,2). Many believe that the study by Escobar et al. (1) and similar tudies have important implications for the pathogenesis and exression of schizophrenia. Unpacking the assumptions underlying his belief, however, can reveal critical gaps in knowledge that emain to be filled to build a theory linking prenatal maternal stress xposure to neural circuit dysfunction in schizophrenia. Below we llustrate some of the domains of knowledge and the gaps among hem. We start with the evidence linking prenatal stressors to the risk or schizophrenia in the offspring. Epidemiologic studies have hown that adverse maternal exposures during gestation, includng various nutritional deficiencies, infections, and toxins, are assoiated with increased risk for neurodevelopmental disorders in chilren, and some specifically to disorders, such as schizophrenia, enerally diagnosed after puberty ([3]; see also reference 2 in Escoar et al). Although it is not yet clear which of these findings will

Enriching the environment of αCaMKIIT286A mutant mice reveals that LTD occurs in memory processing but must be subsequently reversed by LTP

alphaCaMKII(T286A) mutant mice lack long-term potentiation (LTP) in the hippocampal CA1 region and are impaired in spatial learning. In situ hybridization confirms that the mutant mice show the same developmental expression of alphaCaMKII as their wild-type littermates. A simple hypothesis would suggest that if LTP is a substrate for learning, then enriching the environment should cause learning-dependent changes in wild-type mice that have LTP. Such changes would not be seen in LTP-deficient alphaCaMKII(T286A) mutants. Excitatory synaptic currents in CA1 neurons, recorded with patch clamp in brain slices, revealed that enrichment induces an increase in glutamate release probability and a decreased miniature current amplitude. Confocal microscopy also showed dendritic spine density to be reduced. However, contrary to the hypothesis above, these enrichment-induced changes occur only in the mutant mice and are not detectable in wild-type littermates. We suggest that enrichment induces alphaCaMKII-independent changes in both wild-type and mutant mice. Such changes may be subsequently reversed in wild-type animals via alphaCaMKII-dependent mechanisms, such as LTP. Reversal of plasticity has long been hypothesized to be essential for the hippocampus to maintain its role in memory processing. The inability to reverse plasticity in alphaCaMKII(T286A) mutant mice would then result in impairment of spatial learning.

Effects of Toluene Exposure during Brain Growth Spurt on GABAA Receptor-Mediated Functions in Juvenile Rats

Toluene is a commonly abused inhalant. Its neurobiological effects are, at least in part, mediated by gamma-aminobutyric acid (GABA(A)) receptors. Since GABA(A) receptor function is critical during brain development, the long-term effects of toluene exposure during brain growth spurt were investigated. Spargue-Dawley male rats were administered with toluene (500 mg/kg, i.p.) on postnatal day (PN) 4-9. Behavioral and electrophysiological measures and the levels of messenger RNA (mRNA) expression of GABA(A) receptor subunits were examined on PN 28-32. The seizure sensitivity induced by bicuculline (a GABA(A) receptor antagonist), methyl beta-carboline-3-carboxylate (inverse agonists of the GABA(A)/benzodiazepine receptor) but not 3-mercaptopropionic acid (a glutamate decarboxylase inhibitor) was enhanced by toluene exposure. Toluene exposure had no effect on the performance in the elevated plus-maze and rotarod test but reduced the responses to diazepam in these two tests. In vitro intracellular electrophysiological recordings employing brain slices from rats treated with toluene demonstrated a significant decrease in GABA(A) receptor-mediated inhibitory postsynaptic currents in CA1 neurons but an increased response to GABA perfusion. The relative abundance of the mRNAs encoding various subunits of GABA(A) receptor (alpha1, alpha2, alpha4, alpha5, alpha6, beta2, beta3, gamma2S, gamma2L) was examined in four brain regions (hippocampus, striatum, cortex, and cerebellum) by semiquantitative reverse transcription-PCR. These results demonstrated that subunit- and brain area-selective alterations in GABA(A) receptors after toluene exposure during brain growth spurt. The alterations in GABA(A) receptors might be associated with the neurobehavioral disturbance in offspring of toluene-abusing women.

Neural Cell Adhesion Molecule-associated Polysialic Acid Potentiates α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid Receptor Currents

The highly negatively charged polysialic acid (PSA) is a carbohydrate predominantly carried by the neural cell adhesion molecule (NCAM) in mammals. NCAM and, in particular, PSA play important roles in cellular and synaptic plasticity. Here we investigated whether PSA modulates the activity of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors (AMPA-Rs). Single channel recordings of affinity-purified AMPA-Rs reconstituted in lipid bilayers revealed that bacterially derived PSA, called colominic acid, prolonged the open channel time of AMPA-R-mediated currents by severalfold and altered the bursting pattern of the receptor channels but did not modify AMPA-R single channel conductance. This effect was reversible, concentration-dependent, and specific, since monomers of sialic acid and another negatively charged carbohydrate, chondroitin sulfate, did not potentiate single channel AMPA-R currents. Recombinant PSA-NCAM also potentiated currents mediated by reconstituted AMPA-Rs. In pyramidal neurons acutely isolated from the CA1 region of the early postnatal hippocampus, l-glutamate or AMPA (applied in the presence of antagonists blocking voltage-gated Na(+) and K(+) currents and N-methyl-d-aspartate and metabotropic glutamate receptors) induced inward currents, which were significantly increased by co-application of colominic acid. Chondroitin sulfate did not affect AMPA-R-mediated currents in CA1 neurons. The effect of colominic acid was age-dependent, since in pyramidal neurons from adult hippocampus, colominic acid failed to potentiate glutamate responses. Thus, our study demonstrates age-dependent potentiation of AMPA receptors by PSA via a mechanism probably involving direct PSA-AMPA-R interactions. This mechanism might amplify AMPA-R-mediated signaling in immature cells, thereby affecting their development.

Blocker-resistant Ca 2+ currents in rat CA1 hippocampal pyramidal neurons

Ca 2+ currents resistant to organic Ca 2+ channel antagonists are present in different types of central neurons. Here, we describe the properties of such currents in CA1 neurons acutely dissociated from rat hippocampus. Blocker-resistant Ca 2+ currents were isolated by combined application of N-, P/Q- and L-type Ca 2+ current antagonists (ω-conotoxin GVIA 2 μM; ω-conotoxin MVIIC 3 μM; ω-agatoxin IVA 200 nM; nifedipine 10 μM) and constituted approximately 21% of the total Ba 2+ current. The blocker-resistant current showed properties similar to R-type currents in other cell types, i.e. voltages of half-maximal inactivation and activation of −76 and −17 mV, respectively, and strong inactivation during the test pulse. In addition, blocker-resistant Ca 2+ currents in CA1 neurons displayed a characteristically rapid deactivation. Application of mock action potentials revealed that charge transfer through blocker-resistant Ca 2+ channels is highly sensitive to action potential shape and changes in resting membrane voltage. Pharmacological experiments showed that these currents were highly sensitive to the divalent cation Ni 2+ (half-maximal block at 28 μM), but were relatively resistant to the spider toxin SNX-482 (8% and 52% block at 0.1 and 1 μM, respectively). In addition to the functional analysis, we examined the expression of pore-forming and accessory Ca 2+ channel subunits on the messenger RNA level in isolated CA1 neurons using quantitative real-time polymerase chain reaction. Of the pore-forming α subunits encoding high-threshold Ca 2+ channels, Ca v2.1, Ca v2.2 and Ca v2.3 messenger RNA levels were most prominent, corresponding to the high proportion of N-, P/Q- and R-type currents in these neurons. In summary, CA1 neurons display blocker-resistant Ca 2+ currents with distinctive biophysical and pharmacological properties similar to R-type currents in other neuron types, and express Ca 2+ channel messenger RNAs that give rise to R-type Ca 2+ currents in expression systems.

Effect of phenytoin on sodium and calcium currents in hippocampal CA1 neurons of phenytoin-resistant kindled rats

About 20–30% of patients with epilepsy continue to have seizures despite carefully monitored treatment with antiepileptic drugs. The mechanisms explaining why some patients' respond and others prove resistant to antiepileptic drugs are poorly understood. It has been proposed that pharmacoresistance is related to reduced sensitivity of sodium channels in hippocampal neurons to antiepileptic drugs such as carbamazepine or phenytoin. In line with this proposal, a reduced effect of carbamazepine on sodium currents in hippocampal CA1 neurons was found in the rat kindling model of temporal lobe epilepsy (TLE), i.e. a form of epilepsy with the poorest prognosis of all epilepsy types in adult patients. To address directly the possibility that neuronal sodium currents in the hippocampus play a crucial role in the pharmacoresistance of TLE, we selected amygdala-kindled rats with respect to their in vivo anticonvulsant response to phenytoin into responders and nonresponders and then compared phenytoin's effect on voltage-activated sodium currents in CA1 neurons. Furthermore, in view of the potential role of calcium current modulation in the anticonvulsant action of phenytoin, the effect of phenytoin on high-voltage-activated calcium currents was studied in CA1 neurons. Electrode-implanted but not kindled rats were used as sham controls for comparison with the kindled rats. In all experiments, the interval between last kindled seizure and ion channel measurements was at least 5 weeks. In kindled rats with in vivo resistance to the anticonvulsant effect of phenytoin (phenytoin nonresponders), in vitro modulation of sodium and calcium currents by phenytoin in hippocampal CA1 neurons did not significantly differ from respective data obtained in phenytoin responders, i.e. phenytoin resistance was not associated with a changed modulation of the sodium or calcium currents by this drug. Compared to sham controls, phenytoin's inhibitory effect on sodium currents was significantly reduced by kindling without difference between the responder and nonresponder subgroups. Further studies in phenytoin-resistant kindled rats may help to elucidate the mechanisms that can explain therapy resistance.

Differential changes of potassium currents in CA1 pyramidal neurons after transient forebrain ischemia.

CA1 pyramidal neurons are highly vulnerable to transient cerebral ischemia. In vivo studies have shown that the excitability of CA1 neurons progressively decreased following reperfusion. To reveal the mechanisms underlying the postischemic excitability change, total potassium current, transient potassium current, and delayed rectifier potassium current in CA1 neurons were studied in hippocampal slices prepared before ischemia and at different time points following reperfusion. Consistent with previous in vivo studies, the excitability of CA1 neurons decreased in brain slices prepared at 14 h following transient forebrain ischemia. The amplitude of total potassium current in CA1 neurons increased approximately 30% following reperfusion. The steady-state activation curve of total potassium current progressively shifted in the hyperpolarizing direction with a transient recovery at 18 h after ischemia. For transient potassium current, the amplitude was transiently increased approximately 30% at approximately 12 h after reperfusion and returned to control levels at later time points. The steady-state activation curve also shifted approximately 20 mV in the hyperpolarizing direction, and the time constant of removal of inactivation markedly increased at 12 h after reperfusion. For delayed rectifier potassium current, the amplitude significantly increased and the steady-state activation curve shifted in the hyperpolarizing direction at 36 h after reperfusion. No significant change in inactivation kinetics was observed in the above potassium currents following reperfusion. The present study demonstrates the differential changes of potassium currents in CA1 neurons after reperfusion. The increase of transient potassium current in the early phase of reperfusion may be responsible for the decrease of excitability, while the increase of delayed rectifier potassium current in the late phase of reperfusion may be associated with the postischemic cell death.

Novel Role for the NMDA Receptor Redox Modulatory Site in the Pathophysiology of Seizures

Redox-active compounds modulate NMDA receptors (NMDARs) such that reduction of NMDAR redox sites increases, and oxidation decreases, NMDAR-mediated activity. Because NMDARs contribute to the pathophysiology of seizures, redox-active compounds also may modulate seizure activity. We report that the oxidant 5, 5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and the redox cofactor pyrroloquinoline quinone (PQQ) suppressed low Mg(2+)-induced hippocampal epileptiform activity in vitro. Additionally, in slices exposed to 4-7 microM bicuculline, DTNB and PQQ reversed the potentiation of evoked epileptiform responses by the reductants dithiothreitol and Tris(2-carboxyethyl)phosphine (TCEP). NMDA-evoked whole-cell currents in CA1 neurons in slices were increased by TCEP and subsequently decreased by DTNB or PQQ at the same concentrations that modulated epileptiform activity. However, DTNB and PQQ had little effect on baseline NMDA-evoked currents in control medium, and PQQ did not alter NMDAR-dependent long-term potentiation. In contrast, in slices returned to control medium after low Mg(2+)-induced ictal activity, DTNB significantly inhibited NMDAR-mediated currents, indicating endogenous reduction of NMDAR redox sites under this epileptogenic condition. These data suggested that PQQ and DTNB suppressed spontaneous ictal activity by reversing pathological NMDAR redox potentiation without inhibiting physiological NMDAR function. In vivo, PQQ decreased the duration of chemoconvulsant-induced seizures in rat pups with no effect on baseline behavior. Our results reveal endogenous potentiation of NMDAR function via mass reduction of redox sites as a novel mechanism that may enhance epileptogenesis and facilitate the transition to status epilepticus. The results further suggest that redox-active compounds may have therapeutic use by reversing NMDAR-mediated pathophysiology without blocking physiological NMDAR function.

Potassium currents in CA1 neurons of rat hippocampus increase shortly after transient cerebral ischemia

Total potassium current in CA1 pyramidal neurons was studied using whole-cell voltage-clamp recording technique in hippocampal slices prepared before and at 6–8 h after transient forebrain ischemia. The total potassium current significantly increased from a control value of 2.17±0.17 to 2.96±0.31 nA (measured at +70 mV, P<0.05) after ischemia. The slope factor V c of activation curve significantly decreased and the half-inactivation voltage V h shifted to more depolarized potentials after ischemia. These results indicate that the increase of potassium current might be responsible for the decreased excitability in CA1 neurons after severe ischemia and may be involved in postischemic cell death in hippocampus.

Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons.

Recent evidence indicates that long-chain polyunsaturated fatty acids (PUFAs) can prevent cardiac arrhythmias by a reduction of cardiomyocyte excitability. This was shown to be due to a modulation of the voltage-dependent inactivation of both sodium (INa) and calcium (ICa) currents. To establish whether PUFAs also regulate neuronal excitability, the effects of PUFAs on INa and ICa were assessed in CA1 neurons freshly isolated from the rat hippocampus. Extracellular application of PUFAs produced a concentration-dependent shift of the voltage dependence of inactivation of both INa and ICa to more hyperpolarized potentials. Consequently, they accelerated the inactivation and retarded the recovery from inactivation. The EC50 for the shift of the INa steady-state inactivation curve was 2.1 +/- 0.4 microM for docosahexaenoic acid (DHA) and 4 +/- 0.4 microM for eicosapentaenoic acid (EPA). The EC50 for the shift on the ICa inactivation curve was 2.1 +/- 0.4 for DHA and > 15 microM for EPA. Additionally, DHA and EPA suppressed both INa and ICa amplitude at concentrations > 10 microM. PUFAs did not affect the voltage dependence of activation. The monounsaturated oleic acid and the saturated palmitic acid were virtually ineffective. The combined effects of the PUFAs on INa and ICa may reduce neuronal excitability and may exert anticonvulsive effects in vivo.

Attenuation of high-voltage-activated Ca2+ current run-down in rat hippocampal CA1 pyramidal cells by NaF.

Calcium currents in CA1 neurons from rat hippocampus were studied with the whole-cell, patch-clamp technique. Under control conditions high-voltage-activated (HVA) calcium currents activated from membrane potentials of -80 mV and -40 mV underwent "run-down". The rate of run-down of the current activated from -40 mV was significantly attenuated by inclusion of the G-protein activator NaF (1 mM) in the pipette and also irreversibly attenuated by brief batch application of NaF (10 mM). This effect was significantly reduced by inclusion of high (10 mM) ethyleneglycoltetraacetate (EGTA) concentrations in the pipette, suggesting an involvement of calcium-dependent processes. It is suggested that activation of guanine nucleotide-binding proteins by NaF leads to a long-lasting attenuation of HVA calcium current run-down in hippocampal CA1 cells.

Patch-clamp study of postnatal development of CA1 neurons in rat hippocampal slices: membrane excitability and K+ currents.

1. The postnatal development of membrane properties and outward K+ currents in CA1 neurons in rat hippocampal slices was studied with the use of whole-cell patch-clamp techniques. 2. Neurons at all postnatal ages (2-30 days; P2-30) were capable of generating tetrodotoxin (TTX)-sensitive action potentials in response to intracellular injection of depolarizing current pulses. There was a gradual increase in the amplitude and a decrease in the duration of these action potentials with age. Stable values for spike duration were reached by P15, whereas spike amplitude increased until P20-25. In P2-5 neurons, the duration of action potentials was greatly prolonged by depolarization from the resting membrane potential, indicating a weak spike repolarizing mechanism at depolarized potentials. In contrast, the duration of spikes evoked in P20-30 neurons was not affected by similar changes in the membrane potential. 3. Application of tetraethylammonium (TEA, 10 mM) had no effect on the duration of spikes in P3-5 neurons, whereas application of 4-aminopyridine (4-AP, 2 mM) produced large increases in spike duration. In contrast, the duration of spikes in P26 neurons was greatly increased after TEA application, whereas 4-AP had smaller effects on spike duration in these neurons. 4. The input resistance and membrane time constant decreased with age from P2 to P15. The values for both parameters were considerably greater than those reported with conventional intracellular recording electrodes in the immature hippocampus. The resting membrane potential became more hyperpolarized with age. When the recording pipettes contained KCl (140 mM), the resting potential of P3-4 neurons was 34 mV depolarized compared with resting potentials observed with potassium gluconate-filled pipettes. Only a 13-mV change in resting potential was observed during similar comparisons in P27-28 neurons. 5. Outward currents activated by depolarization were examined with the use of voltage-clamp techniques in P2-30 neurons. In P2-5 cells, a small, slowly inactivating outward current was evoked with depolarizing commands from holding potentials near -50 mV. By preceding the depolarizing commands with a hyperpolarizing prepulse, an additional early transient outward current was evoked. The sustained and transient outward currents were separated by their kinetic properties and their sensitivity to cobalt (Co2+), TEA, and 4-AP.(ABSTRACT TRUNCATED AT 400 WORDS)