Rat Hippocampal CA1 Neurons Research Articles

Calcium-permeable Acid-sensing Ion Channel Is a Molecular Target of the Neurotoxic Metal Ion Lead

Acid-sensing ion channels (ASICs) are emerging as fundamental players in the regulation of neural plasticity and in pathological conditions. Here we showed that lead (Pb2+), a well known neurotoxic metal ion, reversibly and concentration-dependently inhibited ASIC currents in the acutely dissociated spinal dorsal horn and hippocampal CA1 neurons of rats. In vitro expression of ASIC subunits in combination demonstrated that both ASIC1 and -3 subunits were sensitive to Pb2+. Mechanistically, Pb2+ reduced the pH sensitivity of ASICs independent of membrane voltage change. Moreover, Pb2+ inhibited the ASIC-mediated membrane depolarization and the elevation of intracellular Ca2+ concentration. In addition, we compared the effect of Pb2+ with that of Ca2+ or amiloride to explore the possible interactions of Pb2+ and Ca2+ in regulating ASICs, and we found that Pb2+ inhibited ASIC currents independent of the amiloride/Ca2+ blockade. Because ASIC1b and -3 subunits are mainly expressed in peripheral neurons, our data identified ASIC1a-containing Ca2+-permeable ASIC as a novel central target of Pb2+ action, which may contribute to Pb2+ neurotoxicity.

Effect of mild hypothermia on post-traumatic hyperexcitability in the rat hippocampal CA1 neurons

Effect of mild hypothermia on post-traumatic hyperexcitability in the rat hippocampal CA1 neurons

Presynaptic GABA A receptors facilitate spontaneous glutamate release from presynaptic terminals on mechanically dissociated rat CA3 pyramidal neurons

Mossy fiber-derived giant spontaneous miniature excitatory postsynaptic currents have been suggested to be large enough to generate action potentials in postsynaptic CA3 pyramidal neurons. Here we report on the functional roles of presynaptic GABA A receptors on excitatory terminals in contributing to spontaneous glutamatergic transmission to CA3 neurons. In mechanically dissociated rat hippocampal CA3 neurons with adherent presynaptic nerve terminals, spontaneous excitatory postsynaptic currents were recorded using conventional whole-cell patch clamp recordings. In most recordings, unusually large spontaneous excitatory postsynaptic currents up to 500 pA were observed. These large spontaneous excitatory postsynaptic currents were highly sensitive to group II metabotropic glutamate receptor activation, and were still observed even after the blockade of voltage-dependent Na + or Ca 2+ channels. Exogenously applied muscimol (0.1–3μM) significantly increased the frequency of spontaneous excitatory postsynaptic currents including the large ones. This facilitatory effect of muscimol was completely inhibited in the presence of 10μM 6-imino-3-(4-methoxyphenyl)-1(6 H)-pyridazinebutanoic acid HBr, a specific GABA A receptor antagonist. Pharmacological data suggest that activation of presynaptic GABA A receptors directly depolarizes glutamatergic terminals resulting in the facilitation of spontaneous glutamate release. In the current-clamp condition, a subset of large spontaneous excitatory postsynaptic potentials triggered action potentials, and muscimol greatly increased the frequency of spontaneous excitatory postsynaptic potential-triggered action potentials in postsynaptic CA3 pyramidal neurons. The results suggest that presynaptic GABA A receptors on glutamatergic terminals play an important role in the excitability of CA3 neurons as well as in the presynaptic modulation of glutamatergic transmission onto hippocampal CA3 neurons.

Theta bursts set up glutamatergic as well as GABA-ergic plasticity in neonatal rat hippocampal CA1 neurons

γ-Aminobutyric acid (GABA) is inhibitory in adult, but excitatory in neonatal, neurons. The switch from excitatory to inhibitory action is due to a negative shift in the equilibrium potential for the GABA A receptor-mediated postsynaptic current ( E GABA-PSC). Here, we report that, in neonatal rat hippocampal CA1 neurons, presynaptic theta-burst activation induces not only a shift in E GABA-PSC towards that in adult neurons, but also a recruitment of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor-mediated postsynaptic currents.

Effects of extracellular δ‐aminolaevulinic acid on sodium currents in acutely isolated rat hippocampal CA1 neurons

The effects of delta-aminolaevulinic acid (ALA) on voltage-gated sodium channel (VGSC) currents (I(Na)) in acutely isolated hippocampal CA1 neurons from 10- to 12-day-old Wistar rats were examined by using the whole-cell patch-clamp technique under voltage-clamp conditions. ALA from 0.01 microm to 20 microm was applied to the recorded neurons. Low concentrations of ALA (0.01-1.0 microM) increased I(Na) amplitude, whereas high concentrations of ALA (5.0-20.0 microM) decreased it. The average I(Na) amplitude reached a maximum of 117.4 +/- 3.9% (n = 9, P < 0.05) with 0.1 microM ALA, and decreased to 78.1 +/- 3.8% (n = 13, P < 0.05) with 10 microm ALA. ALA shifted the steady-state activation and inactivation curves of I(Na) in the hyperpolarizing direction with different V0.5, suggesting that ALA could depress the opening threshold of the voltage-gated sodium channel (VGSC) and thus increase the excitability of neurons through facilitating the opening of VGSC. The time course of recovery from inactivation was significantly prolonged at both low and high concentrations of ALA, whereas either low or high concentrations of ALA had no significant effect on the attenuation of I(Na) during stimulation at 5 Hz, indicating that the effect of ALA on VGSC is state-independent. Furthermore, we found that application of ascorbic acid, which blocks pro-oxidative effects in neurons, could prevent the increase of I(Na) amplitude at low concentrations of ALA. Baclofen, an agonist of GABAb receptors, induced some similar effects to ALA on VGSC, whereas bicuculline, an antagonist of GABAa receptors, could not prevent ALA-induced effects on VGSC. These results suggested that ALA regulated VGSC mainly through its pro-oxidative effects and GABAb receptor-mediated effects.

Benzodiazepine involvement in LTP of the GABA-ergic IPSC in rat hippocampal CA1 neurons

Benzodiazepine binding sites are present on γ-aminobutyric acid (GABA) receptors in hippocampal neurons. Diazepam is known to potentiate the amplitude and prolong the decay of GABA A receptor-mediated inhibitory postsynaptic currents (IPSCs). In this study, benzodiazepine involvement in long-term potentiation (LTP) of the IPSC was examined. Whole-cell recordings of IPSCs were made from rat hippocampal CA1 neurons in a slice preparation. LTP was induced by a tetanic stimulation in the stratum radiatum (2 trains of 100 Hz for 1 s, 20 s inter-train interval) while pharmacologically blocking ionotropic glutamate receptors. During LTP, the amplitude of the IPSCs was potentiated in the majority of neurons with the IPSC decay and shape unaffected. Diazepam (5 μM) potentiated the IPSC amplitude and prolonged the decay when applied before, but not during, LTP. In neurons in which LTP could not be induced by a tetanic stimulation, diazepam did not increase the amplitude of the pre-tetanic IPSC. Flumazenil, at a concentration (10 μM) that blocked the enhancement of the IPSC by applied diazepam, had no effect on the IPSC amplitude when applied before LTP induction but significantly decreased the IPSC when applied during LTP maintenance. The antagonist, when applied during the tetanic stimulation, did not block LTP, suggesting that benzodiazepine receptors do not participate in LTP induction. These results indicate that the maintenance of LTP of the IPSC involves (a) the release of endogenous benzodiazepine agonist(s) and/or (b) the participation of benzodiazepine binding sites on subsynaptic GABA A receptors.

Effects of copper on A-type potassium currents in acutely dissociated rat hippocampal CA1 neurons

The effects of copper on voltage-gated A-type potassium currents were investigated in acutely dissociated rat hippocampal CA1 neurons using the whole-cell patch-clamp technique. Extracellular application of various concentrations of copper (1-1000 microM) reversibly reduced the amplitude of voltage-gated A-type potassium currents in a dose-dependent manner with a 50% inhibitory concentration value of 130 microM. Copper (300 microM) increased the V1/2 of the activation curve and state-inactivation curve by 17.2 and 9.0 mV, respectively. Thus, copper slowed down the activation and inactivation process of voltage-gated A-type potassium currents. This study indicated that copper reversibly inhibits the hippocampal CA1 neuronal voltage-gated A-type potassium current in a dose-dependent and voltage-dependent manner, and such actions are likely involved in the regulation of the neuronal excitability and the pathophysiology of Wilson's disease.

Antiepileptic effect of acylpolyaminetoxin JSTX-3 on rat hippocampal CA1 neurons in vitro

The Joro spider toxin (JSTX-3), derived from Nephila clavata, has been found to block glutamate excitatory activity. Epilepsy has been studied in vitro, mostly on rat hippocampus, through brain slices techniques. The aim of this study is to verify the effect of the JSTX-3 on the epileptiform activity induced by magnesium-free medium in rat CA1 hippocampal neurons. Experiments were performed on hippocampus slices of control and pilocarpine-treated Wistar rats, prepared and maintained in vitro. Epileptiform activity was induced through omission of magnesium from the artificial cerebrospinal fluid (0-Mg 2+ ACSF) superfusate and iontophoretic application of N-methyl- d-aspartate (NMDA). Intracellular recordings were obtained from CA1 pyramidal neurons both of control and epileptic rats. Passive membrane properties were analyzed before and after perfusion with the 0-Mg 2+ ACSF and the application of toxin JSTX-3. During the ictal-like activity, the toxin JSTX-3 was applied by pressure ejection, abolishing this activity. This effect was completely reversed during the washout period when the slices were formerly perfused with artificial cerebrospinal fluid (ACSF) and again with 0-Mg 2+ ACSF. Our results suggest that the toxin JSTX-3 is a potent blocker of induced epileptiform activity.

Effects of lead on voltage-gated sodium channels in rat hippocampal CA1 neurons

In this study, the effects of lead (Pb 2+) on voltage-gated sodium channel currents (I Na) were investigated in acutely dissociated rat hippocampal CA1 neurons using the conventional whole-cell patch-clamp technique. We found that Pb 2+ reduced the amplitudes of I Na in a concentration-dependent manner, and the effect could be washed out by extracellular application of 3mM EGTA. The results also showed that at the concentration of 100μM, Pb 2+ decreased the activation threshold and the voltage at which the maximum I Na current was evoked and caused negative shifts of I Na steady-state activation curve, and enlarged I Na tail-currents; Pb 2+ induces a left shift of the steady-state inactivation curve, and delayed the recovery of I Na from inactivation, and reduced the fraction of available sodium channels; Pb 2+ delayed the activation of I Na in a concentration- and voltage-dependent manner, and prolonged the time course of the fast inactivation of sodium channels; activity-dependent attenuation of I Na was not altered by Pb 2+. It was suggested that Pb 2+ might exert its effects on sodium channels by binding a specific site on the extracellular side of sodium channels and dragging the IIS4 voltage sensor outwardly. The interaction of Pb 2+ with voltage-dependent sodium channels may lead to change in electrical activity and contribute to worsen the neurotoxicological damage.

Enhancement of sodium metabisulfite on sodium currents in acutely isolated rat hippocampal CA1 neurons

The effect of sodium metabisulfite (SMB) on voltage-gated sodium channel currents ( I Na) was examined in freshly isolated rat hippocampal CA1 neurons using whole-cell patch-clamp technique under voltage-clamp conditions. SMB irreversibly enhanced I Na in a concentration-dependent manner, shifted the inactivation curve to more positive potential, without affecting the current activation curve. In addition, SMB increased the time to peak and the inactivation time constant of I Na. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) could all partly inhibit the effect of SMB on the sodium current. These results suggested that SMB have neuronal toxicity by increasing the excitability of neurons and its mechanism might involve the oxidative damage on ion channels.

Evidence that injury-induced changes in hippocampal neuronal calcium dynamics during epileptogenesis cause acquired epilepsy.

Alterations in hippocampal neuronal Ca(2+) and Ca(2+)-dependent systems have been implicated in mediating some of the long-term neuroplasticity changes associated with acquired epilepsy (AE). However, there are no studies in an animal model of AE that directly evaluate alterations in intracellular calcium concentration ([Ca(2+)](i)) and Ca(2+) homeostatic mechanisms (Ca(2+) dynamics) during the development of AE. In this study, Ca(2+) dynamics were evaluated in acutely isolated rat CA1 hippocampal, frontal, and occipital neurons in the pilocarpine model by using [Ca(2+)](i) imaging fluorescence microscopy during the injury (acute), epileptogenesis (latency), and chronic-epilepsy phases of the development of AE. Immediately after status epilepticus (SE), hippocampal neurons, but not frontal and occipital neurons, had significantly elevated [Ca(2+)](i) compared with saline-injected control animals. Hippocampal neuronal [Ca(2+)](i) remained markedly elevated during epileptogenesis and was still elevated indefinitely in the chronic-epilepsy phase but was not elevated in SE animals that did not develop AE. Inhibiting the increase in [Ca(2+)](i) during SE with the NMDA channel inhibitor MK801 was associated in all three phases of AE with inhibition of the changes in Ca(2+) dynamics and the development of AE. Ca(2+) homeostatic mechanisms in hippocampal neurons also were altered in the brain-injury, epileptogenesis, and chronic-epilepsy phases of AE. These results provide evidence that [Ca(2+)](i) and Ca(2+)-homeostatic mechanisms are significantly altered during the development of AE and suggest that altered Ca(2+) dynamics may play a role in the induction and maintenance of AE and underlie some of the neuroplasticity changes associated with the epileptic phenotype.

Effects of aluminum chloride on sodium current, transient outward potassium current and delayed rectifier potassium current in acutely isolated rat hippocampal CA1 neurons

The effects of aluminum chloride (AlCl 3) on sodium current ( I Na), the transient outward potassium ( I A) and delayed rectifier potassium currents ( I K) in hippocampal CA1 neurons of rats were studied using the whole cell patch-clamp technique. AlCl 3 decreased I Na, I A, and I K in a partly reversible, dose and voltage-dependent manner. AlCl 3 prolonged the time to peak of I Na, and increased the inactivation time constants of I Na and I A. In addition, 1000 μM AlCl 3 shifted the voltage dependence of steady-state activation of I Na, I A and I K toward positive potential, and the voltage dependence of steady-state inactivation of I Na, I A toward negative potential. These results imply that AlCl 3 could affect the activation and inactivation courses of sodium current and potassium current of rat hippocampal CA1 neurons, which may contribute to damage of the central nervous system by aluminum.

Modulation of single channels underlying hippocampal L-type current enhancement by agonists depends on the permeant ion.

The influx of calcium (Ca(2+)) ions through L-type channels underlies many cellular processes, ranging from initiation of gene transcription to activation of Ca(2+)-activated potassium channels. L-type channels possess a diagnostic pharmacology, being enhanced by the dihydropyridine BAY K 8644 and benzoylpyrrole FPL 64176. It is assumed that the action of these compounds is independent of the ion conducted through the channel. In contrast to this assumption, modulation of L-type channel activity in acutely dissociated rat CA1 hippocampal neurons depended on the divalent ion identity. BAY K 8644 and FPL 64176 substantially increased single-channel open time only when barium (Ba(2+)) was the permeant ion. BAY K 8644 increased single-channel conductance when either Ba(2+) or Ca(2+) ions were the charge carrier, an effect not observed with FPL 64176. BAY K 8644 enhanced the whole cell L-type channel Ca(2+)- or Ba(2+)-carried current without a change in deactivation tail kinetics. In contrast, enhancement by FPL 64176 was associated with a dramatic slowing of deactivation kinetics only when Ba(2+) and not Ca(2+) was the charge carrier. Current activation was slowed by FPL 64176 with either charge carrier, an effect arising from a clustering of agonist-modified long-duration openings toward the end of the voltage step. These data indicate that agonists enhanced L-type current by distinct mechanisms dependent on the permeant ion, indicating that care must be considered when used as diagnostic tools.

Activation of protein kinase A contributes to irreversible membrane dysfunction produced by in vitro ischemia in rat hippocampal CA1 neurons.

Activation of protein kinase A contributes to irreversible membrane dysfunction produced by in vitro ischemia in rat hippocampal CA1 neurons.

Failure of Repeated Electroconvulsive Shock Treatment on 5-HT4-Receptor-Mediated Depolarization Due To Protein Kinase A System in Young Rat Hippocampal CA1 Neurons

We previously demonstrated that repeated electroconvulsive shock (ECS) treatment enhanced serotonin (5-HT)1A- and 5-HT3-receptor-mediated responses in hippocampal CA1 pyramidal neurons. The electrophysiological studies were performed to elucidate the effects of ECS treatment on depolarization, which was an additional response induced by 5-HT, and the second messenger system involved in this depolarization of hippocampal CA1 neurons. Bath application of 5-HT (100 μM) induced depolarization of the membrane potential in the presence of 5-HT1A-receptor antagonists. This depolarization was mimicked by 5-HT4-receptor agonists, RS 67506 (1 – 30 μM) and RS 67333 (0.1 – 30 μM), in a concentration-dependent manner. 5-HT- and RS 67333-induced depolarization was attenuated by concomitant application of RS 39604, a 5-HT4-receptor antagonist. H-89, a protein kinase A (PKA) inhibitor, inhibited 5-HT-, RS 67506-, and RS 67333-induced depolarizations, while forskolin (10 μM), an activator of adenylate cyclase, induced depolarization. Furthermore, RS 67333-induced depolarization was not significantly different between hippocampal slices prepared from rats administered ECS once a day for 14 days and those from sham-treated rats. These findings suggest that 5-HT4-receptor-mediated depolarization is caused via the cAMP-PKA system. In addition, repeated ECS-treatment did not modify 5-HT4-receptor functions in contrast to 5-HT1A- and 5-HT3-receptor functions.

Effects of hydrogen peroxide on sodium current in acutely isolated rat hippocampal CA1 neurons

The effects of hydrogen peroxide (H 2O 2) on sodium currents (Na + currents) in freshly dissociated rat hippocampal neurons were studied using the whole-cell patch-clamp techniques. H 2O 2 caused a reversible increase of the voltage-activated Na + currents in a concentration- and voltage-dependent manner. The half-increasing concentration (EC 50) of H 2O 2 on Na + currents was 10.79 μM. In addition, 10 μM H 2O 2 shifted the steady-state inactivation curve of Na + currents toward positive potential (control V h=−64.58±1.22 mV, H 2O 2 V h=−53.55±0.94 mV, n=10, P<0.01 without changing the slope factor). However, the steady-state activation curve was not affected. These results indicated that H 2O 2 could increase the amplitudes of Na + currents and change the inactivation properties of Na + channels even in very low concentration.

Reduced contribution from Na+/H+ exchange to acid extrusion during anoxia in adult rat hippocampal CA1 neurons.

The effect of anoxia on Na+/H+ exchange activity was examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H+-sensitive fluorophore, BCECF. Five-minute anoxia imposed under nominally HCO3-/CO2-free conditions induced a fall in pHi, the magnitude of which was smaller following prolonged exposure to medium in which N-methyl-D-glucamine (NMDG+) was employed as an extracellular Na+ (Na(+)(o)) substitute. Also consistent with the possibility that Na+/H+ exchange becomes inhibited soon after the induction of anoxia, rates of Na(+)(o)-dependent pHi recovery from internal acid loads imposed during anoxia were slowed, compared to rates of Na(+)(o)-dependent pHi recovery observed prior to anoxia. At the time at which rates of pHi recovery were reduced during anoxia, cellular adenosine triphosphate (ATP) levels had fallen to 35% of preanoxic levels, suggesting that ATP depletion might contribute to the observed inhibition of Na+/H+ exchange. In support, incubation of neurons with 2-deoxyglucose and antimycin A under normoxic conditions induced a fall in cellular ATP levels that was also associated with reduced Na(+)(o)-dependent rates of pHi recovery from imposed acid loads; conversely, pre-treatment with 10 mm creatine attenuated the effects of anoxia to reduce both ATP levels and Na(+)(o)-dependent rates of pHi recovery from internal acid loads. Taken together, the results are consistent with the possibility that functional Na+/H+ exchange activity in adult rat CA1 neurons declines soon after the onset of anoxia, possibly as a result of anoxia-induced falls in intracellular ATP.

Functional Mapping and Ca2+Regulation of Nicotinic Acetylcholine Receptor Channels in Rat Hippocampal CA1 Neurons

Diverse subtypes of nicotinic acetylcholine receptors (nAChRs), including fast-desensitizing alpha7-containing receptors thought to be Ca2+-permeable, are expressed in the CNS, where they appear to regulate cognitive processing and synaptic plasticity. To understand the physiological role of nAChRs in regulating neuronal excitability, it is important to know the distribution of functional receptors along the surface of neurons, whether they can increase [Ca2+]i, and/or are regulated by Ca2+. We mapped the distribution of receptors on the membrane of rat hippocampal CA1 stratum radiatum interneurons and pyramidal cells in acute slices by recording nAChR-mediated currents elicited by local UV laser-based photolysis of caged carbachol in patch-clamped neurons. The local application (approximately 7 microm patches) allowed mapping of functional nAChRs along the soma and dendritic tree, whereas the fast uncaging minimized the effects of desensitization of alpha7-containing nAChRs and allowed us to measure the kinetics of responses. The alpha7-containing nAChRs were the predominant subtype on interneurons, and were located primarily at perisomatic sites (<70 microm from the soma; in contrast to the more uniform distribution of glutamate receptors); no currents were detectable on pyramidal neurons. The activation of nAChRs increased [Ca2+]i, indicating that these native receptors in acute slices are significantly Ca2+-permeable, consistent with previous observations made with recombinant receptors. In addition, they exhibited strong desensitization, the rate of recovery from which was controlled by [Ca2+]i. Our results demonstrate the strategic location and Ca2+ regulation of alpha7-containing nAChRs, which may contribute to understanding their involvement in hippocampal plasticity.

Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors.

CA1 pyramidal neurons degenerate after transient forebrain ischemia, whereas neurons in other regions of the hippocampus remain intact. Here we show that in rat hippocampal CA1 neurons, forebrain ischemia induces the phosphorylation of the N-methyl-D-aspartate (NMDA) receptor 2A subunit at Ser1232 (phospho-Ser1232). Ser1232 phosphorylation is catalyzed by cyclin-dependent kinase 5 (Cdk5). Inhibiting endogenous Cdk5, or perturbing interactions between Cdk5 and NR2A subunits, abolished NR2A phosphorylation at Ser1232 and protected CA1 pyramidal neurons from ischemic insult. Thus, we conclude that modulation of NMDA receptors by Cdk5 is the primary intracellular event underlying the ischemic injury of CA1 pyramidal neurons.

Role of protein kinase A in GABAA receptor dysfunction in CA1 pyramidal cells following chronic benzodiazepine treatment.

One-week treatment with the benzodiazepine (BZ) flurazepam (FZP), results in anticonvulsant tolerance, associated with reduced GABAA receptor (GABAR) subunit protein and miniature inhibitory post-synaptic current (mIPSC) amplitude in CA1 neurons of rat hippocampus. Because protein kinase A (PKA) has been shown to modulate GABAR function in CA1 pyramidal cells, the present study assessed whether GABAR dysfunction is associated with changes in PKA activity. Two days after 1-week FZP treatment, there were significant decreases in basal (- 30%) and total (- 25%) PKA activity, and a 40% reduction in PKA RIIbeta protein in the insoluble fraction of CA1 hippocampus. The soluble component of CA1 showed a significant increase in basal (100%) but not total PKA activity. Whole-cell recording in vitro showed a 50% reduction in mIPSC amplitude in CA1 pyramidal cells, with altered sensitivity to PKA modulators. Neurons from FZP-treated rats responded to 8-bromo-cAMP with a significant increase (31%) in mIPSC amplitude. Likewise, vasoactive intestinal polypeptide (VIP), an endogenous PKA activator, caused a significant 36% increase in mIPSC amplitude in FZP-treated cells. Neither agent had a significant effect on mIPSC amplitude in control cells. This study supports a role for PKA in GABAR dysfunction after chronic FZP treatment.