Paraoxon suppresses Ca(2+) spike and afterhyperpolarization in snail neurons: Relevance to the hyperexcitability induction

The effects of organophosphate (OP) paraoxon, active metabolite of parathion, were studied on the Ca(2+) and Ba(2+) spikes and on the excitability of the neuronal soma membranes of land snail (Caucasotachea atrolabiata). Paraoxon (0.3 muM) reversibly decreased the duration and amplitude of Ca(2+) and Ba(2+) spikes. It also reduced the duration and the amplitude of the afterhyperpolarization (AHP) that follows spikes, leading to a significant increase in the frequency of Ca(2+) spikes. Pretreatment with atropine and hexamethonium, selective blockers of muscarinic and nicotinic receptors, respectively, did not prevent the effects of paraoxon on Ca(2+) spikes. Intracellular injection of the calcium chelator BAPTA dramatically decreased the duration and amplitude of AHP and increased the duration and frequency of Ca(2+) spikes. In the presence of BAPTA, paraoxon decreased the duration of the Ca(2+) spikes without affecting their frequency. Apamin, a neurotoxin from bee venom, known to selectively block small conductance of calcium-activated potassium channels (SK), significantly decreased the duration and amplitude of the AHP, an effect that was associated with an increase in spike frequency. In the presence of apamin, bath application of paraoxon reduced the duration of Ca(2+) spike and AHP and increased the firing frequency of nerve cells. In summary, these data suggest that exposure to submicromolar concentration of paraoxon may directly affect membrane excitability. Suppression of Ca(2+) entry during the action potential would down regulate Ca(2+)-activated K(+) channels leading to a reduction of the AHP and an increase in cell firing.     

Intrinsic membrane potential oscillations in hippocampal neurons in vitro

Membrane potential oscillations (MPOs) of 2-10 Hz and up to 6 mV were found in almost all stable hippocampal CA1 and CA3 neurons in the in vitro slice preparation. MPOs were prominent for pyramidal cells but less pronounced in putative interneurons. MPOs were activated at threshold depolarizations that evoked a spike and the frequency of the MPOs increased with the level of depolarization. MPOs were distinct from and seemed to regulate spiking, with a spike often riding near the top of a depolarizing MPO wave. Analysis of the periodicity of the oscillations indicate that the period of MPOs did not depend on the afterhyperpolarization (AHP) following a single spike. MPOs persisted in low (0-0.1 mM) Ca2+ medium, with or without Cd2+ (0.2 mM), when synaptic transmission was blocked. Choline-substituted low-Na+ (0-26 mM) medium, 3 microM tetrodotoxin (TTX) or intracellular injection of QX-314 reduced or abolished the fast Na(+)-spike and reduced inward anomalous rectification. About 40% of CA1 neurons had no MPOs after Na+ currents were blocked, suggesting that these MPOs were Na(+)-dependent. In about 60% of the cells, a large depolarization activated Ca(2+)-dependent MPOs and slow spikes. MPOs were not critically affected by extracellular Ba2+ or Cs2+, or by 0.2 mM 4-aminopyridine, with or without 2 mM tetraethylammonium (TEA). However, in 5-10 mM TEA medium, MPOs were mostly replaced by 0.2-3 Hz spontaneous bursts of wide-duration spikes followed by large AHPs. Low Ca2+, Cd2+ medium greatly reduced the spike width but not the spike-bursts. In conclusion, each cycle of an MPO in normal medium probably consists of a depolarization phase mediated by Na+ currents, possibly mixed with Ca2+ currents activated at a higher depolarization. The repolarization/hyperpolarization phase may be mediated by Na+/Ca2+ current inactivation and partly by TEA-sensitive, possibly the delayed rectifier, K+ currents. The presence of prominent intrinsic, low-threshold MPOs in all hippocampal pyramidal neurons suggests that MPOs may play an important role in information processing in the hippocampus.     

Activation of N-methyl-D-aspartate (NMDA) receptors augments repolarizing responses in lamprey spinal neurons

Current- and voltage-clamp techniques were used to analyze the mechanisms underlying the repolarization during N-methyl-D-aspartate (NMDA)-induced, tetrodotoxin-resistant pacemaker-like oscillations in lamprey spinal neurons. Long-lasting depolarizing current pulses (15-40 mV, 50-400 ms, tetrodotoxin and tetraethylammonium present) were followed by hyperpolarizing afterpotentials even when NMDA receptors were blocked, but they were markedly enhanced by application of N-methyl-D,L-aspartate (NM(DL)A). The afterpotentials were depressed by replacing Ca2+ with Ba2+. During voltage-clamp NM(DL)A enhanced a Ba2+-sensitive outward tail current following voltage steps of 15-40 mV. The outward current remained after injection of Cl-, as did the NMDA-induced membrane potential oscillations observed under current-clamp. These results suggest that the repolarization during NMDA-induced oscillations is due to Ca2+ entry both via NMDA-gated channels and conventional voltage-gated Ca2+ channels, leading to an activation of Ca2+-dependent K+ channels. The afterhyperpolarization following single action potentials, which is also due to Ca2+-dependent K+ channels, was not significantly altered by NMDA receptor activation, suggesting a different location of the Ca2+ entry during the two conditions in relation to the location of the activated Ca2+-dependent K+ channels.     

Voltage-clamp analysis of the potentiation of the slow Ca2+-activated K+ current in hippocampal pyramidal neurons

Exploring the principles that govern activity-dependent changes in excitability is an essential step to understand the function of the nervous system, because they act as a general postsynaptic control mechanism that modulates the flow of synaptic signals. We show an activity-dependent potentiation of the slow Ca2+-activated K+ current (sl(AHP)) which induces sustained decreases in the excitability in CA1 pyramidal neurons. We analyzed the sl(AHP) using the slice technique and voltage-clamp recordings with sharp or patch-electrodes. Using sharp electrodes-repeated activation with depolarizing pulses evoked a prolonged (8-min) potentiation of the amplitude (171%) and duration (208%) of the sl(AHP). Using patch electrodes, early after entering the whole-cell configuration (<20 min), responses were as those reported above. However, although the sl(AHP) remained unchanged, its potentiation was markedly reduced in later recordings, suggesting that the underlying mechanisms were rapidly eliminated by intracellular dialysis. Inhibition of L-type Ca2+ current by nifedipine (20 microM) markedly reduced the sl(AHP) (79%) and its potentiation (55%). Ryanodine (20 microM) that blocks the release of intracellular Ca2+ also reduced sl(AHP) (29%) and its potentiation (25%). The potentiation of the sl(AHP) induced a marked and prolonged (>50%; approximately equals 8 min) decrease in excitability. The results suggest that sl(AHP) is potentiated as a result of an increased intracellular Ca2+ concentration ([Ca2+]i) following activation of voltage-gated L-type Ca2+ channels, aided by the subsequent release of Ca2+ from intracellular stores. Another possibility is that repeated activation increases the Ca2+-binding capacity of the channels mediating the sl(AHP). This potentiation of the sl(AHP) could be relevant in hippocampal physiology, because the changes in excitability it causes may regulate the induction threshold of the long-term potentiation of synaptic efficacy. Moreover, the potentiation would act as a protective mechanism by reducing excitability and preventing the accumulation of intracellular Ca2+ to toxic levels when intense synaptic activation occurs.     

Nifedipine blocks calcium-dependent cholinergic depolarization in the guinea pig hippocampus

The possibility that cholinergic stimulation might directly activate a receptor-operated Ca2+ channel was investigated in the CA1 region of guinea pig hippocampus using intracellular recording techniques. Two cholinergic responses were studied: (1) the plateau depolarization evoked by cholinergic stimulation in the presence of Ba2+; and (2) the Ca2(+)-dependent component of membrane depolarization. Both of these responses were blocked by 1-5 microM of nifedipine, a blocker of voltage-dependent L-type Ca2+ channels. In addition, the plateau response was mimicked by direct postsynaptic depolarization in the presence of Ba2+. We conclude that cholinergic stimulation does not directly activate a Ca2+ conductance in these neurons, but rather leads to the indirect activation of L channels which may be located both pre- and postsynaptically.     

Interactive effects involving different classes of excitatory amino acid receptors and the survival of cerebellar granule cells in culture

Differentiating granule cells develop survival requirements in culture which can be met by treatment with high K+ or N-methyl-D-aspartate (NMDA) and, according to our recent findings, also with low concentrations of kainic acid (KA, 50 microM). We have now attempted to elucidate the mechanism(s) underlying the trophic effect of KA. KA rescue of cells was completely suppressed by blockers of voltage-sensitive calcium channels, such as nifedipine in low concentrations (5 x 10(-7) M), indicating that the promotion of cell survival is mediated through the activation of these channels by membrane depolarization. Thus the trophic influences of KA and NMDA share a common mechanism, increased Ca2+ influx (albeit through different routes), a conclusion that is supported by the observation that the effects of these agonists at concentrations causing maximal promotion of cell survival were not additive. Interactive effects involving different classes of excitatory amino acid receptors were revealed by the potentiation of the KA rescue of cells by the NMDA receptor antagonists, 2-amino 5-phosphonovalerate (APV) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate (MK-801), which on their own failed to promote, but rather reduced cell survival. The potentiation of the KA effect by the competitive NMDA antagonist APV was counteracted by the weak NMDA agonist, quinolinic acid. These observations suggest that KA alone has both trophic and toxic effects, the latter being mediated secondarily through an NMDA-like glutamate receptor, which is distinct from the conventional NMDA, KA and quisqualate preferring subtypes.     

Cellular mechanisms underlying the rhythmic bursts induced by NMDA microiontophoresis at the apical dendrites of CA1 pyramidal neurons

This article reports the cellular mechanisms underlying a form of intracellular "theta-like" (theta-like) rhythm evoked in vitro by microiontophoresis of N-methyl-D-aspartate (NMDA) at the apical dendrites of CA1 pyramidal neurons. Rhythmic membrane potential (Vm) oscillations and action potential (AP) bursts (approximately 6 Hz; approximately 20 mV; approximately 2-5 APs) were evoked in all cells. The response lasted approximately 2 s, and the initial oscillations were usually small (< 20 mV) and below AP threshold. Rhythmic bursts were never evoked by imposed depolarization in the absence of NMDA. Block of Na+ conductance with tetrodotoxin (TTX) (1.5 microM), of non-NMDA receptors with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 microM) and of synaptic inhibition by bicuculline (50 microM) and picrotoxin (50 microM) did not prevent NMDA oscillation. Inhibition of the voltage dependence of the NMDA conductance in Mg2+-free Ringer's solution blocked oscillations. Preventing Ca2+ influx with Ca2+-free and Co2+ (2-mM) solutions and block of the slow Ca2+-dependent afterhyperpolarization (sAHP) by carbamilcholine (5 microM), isoproterenol (10 microM), and intracellular BAPTA blocked NMDA oscillations. Inhibition of L-type Ca2+ conductance with nifedipine (30 microM) reduced oscillation amplitude. Block of tetraethylammonium (TEA) (10 mM) and 4AP (10 mM)-sensitive K+ conductance increased the duration and amplitude, but not the frequency, of oscillations. In conclusion, theta-like bursts relied on the voltage dependence of the NMDA conductance and on high-threshold Ca2+ spikes to initiate and boost the depolarizing phase of oscillations. The repolarization is initiated by TEA-sensitive K+ conductance and is controlled by the sAHP. These results suggest a role of interactions between NMDA conductance and intrinsic membrane properties in generating the CA1 theta-rhythm.     

A novel T-type current underlies prolonged Ca(2+)-dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus.

The inhibitory GABAergic projection of thalamic nucleus reticularis (nRt) neurons onto thalamocortical relay cells (TCs) is important in generating the normal thalamocortical rhythmicity of slow wave sleep, and may be a key element in the production of abnormal rhythms associated with absence epilepsy. Both TCs and nRt cells can generate prominent Ca(2+)-dependent low-threshold spikes, which evoke bursts of Na(+)-dependent fast spikes, and are influential in rhythm generation. Substantial differences in the pattern of burst firing in TCs versus nRt neurons led us to hypothesize that there are distinct forms of transient Ca2+ current (I(T)) underlying burst discharges in these two cell types. Using whole-cell voltage-clamp recordings, we analyzed I(T) in acutely isolated TCs and nRt neurons and found three key differences in biophysical properties. (1) The transient Ca2+ current in nRt neurons inactivated much more slowly than I(T) in TCs. This slow current is thus termed I(Ts). (2) The rate of inactivation for I(Ts) was nearly voltage independent. (3) Whole-cell I(Ts) amplitude was increased when Ba2+ was substituted for Ca2+ as the charge carrier. In addition, activation kinetics were slower for I(Ts) and the activation range was depolarized compared to that for I(T). Other properties of I(Ts) and I(T) were similar, including steady-state inactivation and sensitivities to blockade by divalent cations, amiloride, and antiepileptic drugs. Our findings demonstrate that subtypes of transient Ca2+ current are present in two different classes of thalamic neurons. The properties of I(Ts) lead to generation of long-duration calcium-dependent spike bursts in nRt cells. The resultant prolonged periods of GABA release onto TCs would play a critical role in maintaining rhythmicity by inducing TC hyperpolarization and promoting generation of low-threshold calcium spikes within relay nuclei.     

Membrane properties and firing characteristics of rat cardiac neurones in vitro.

Electrophysiological characteristics of neurones in isolated cardiac ganglia from the left atrium and interatrial septum of the rat were studied with intracellular microelectrodes. At rest the neurones were characterized by a membrane potential of -52.6 +/- 0.83 mV, an input resistance of 85.6 +/- 7.6 M omega, a membrane time constant of 4.6 +/- 0.24 ms and an input capacitance of 63.1 +/- 5.25 pF. Removal of Ca2+ ions from the external solution resulted in a membrane depolarisation of 5.5 +/- 0.70 mV and an increase in input resistance of 96 +/- 52% which indicated that a substantial Ca(2+)-sensitive component contributed to resting membrane potential. A prolonged after-hyperpolarization (AHP) was recorded following a train of spikes; this was inhibited in a Ca(2+)-free solution, indicating that a Ca(2+)-sensitive component of potassium conductance contribute to it. On the basis of the duration of the AHP following a single spike, two types of neurones, I and II, were tentatively identified, having short (less than 300 ms) and long (greater than 300 ms) AHPs, respectively. Type I neurones responded to prolonged membrane depolarization with bursts of firing (Ib neurones) or multiple discharges (Im neurones). Type II neurones also responded with single spikes or multiple discharges to prolonged membrane depolarization. In some Im neurones, tonic firing was recorded which was inhibited by a hyperpolarizing current and accelerated by a depolarizing current injected through the recording microelectrode. Thus, neurones of isolated cardiac ganglia of the rat from the region studied here are heterogeneous in their electrical behaviour, suggesting the existence of functionally different groups within the ganglia.     

Effects of methylmercury on perineurial Na+ and Ca(2+)-dependent potentials at neuromuscular junctions of the mouse.

The ability of acute application of the neurotoxicant methylmercury (MeHg) to disrupt the function of presynaptic Ca2+ and Na+ channels at intact neuromuscular junctions was examined using mouse triangularis sterni motor nerves. In Ba(2+)-containing solutions, potential changes arising from Na+ and Ca2+ channel function could be recorded from the perineurial sheath surrounding motor neurons when K+ channels were blocked by tetraethylammonium chloride and 3,4-diaminopyridine. MeHg (100 microM) reduced both Na(+)- and Ba(2+)-dependent components to block within 3-5 min at apparently equivalent rates. Time to block was approximately 7 min after exposure to 50 microM MeHg. In 2 of 5 preparations exposed to 50 microM MeHg, the Ca2+ channel-mediated component was blocked prior to the Na+ channel-mediated component. In the remaining three preparations, Na(+)- and Ba(2+)-dependent potentials were blocked at similar times. Following block by MeHg, neither perfusing the preparation in MeHg-free solutions nor increasing the intensity and/or duration of stimulus to the intercostal nerves resulted in recovery of Na+ or Ca2+ potentials. In the presence of K+ channel blockers, repetitive firing of nerves in response to a single stimulus was observed in 20-30% of the triangularis preparations; in the two preparations treated with MeHg in which repetitive firing was observed, it decreased prior to block of the stimulus-induced Na+/Ba2+ potentials. These results corroborate the results obtained in isolated synaptosomes and pheochromocytoma cells, and suggest that MeHg decreases motor nerve excitability by disrupting Na+ channel function and may block neurotransmitter release by disrupting Na+ and Ca2+ channel function.     

Glutamatergic Induction of CREB Phosphorylation and Fos Expression in Primary Cultures of the Suprachiasmatic Hypothalamus In Vitro is Mediated by Co-Ordinate Activity of NMDA and Non-NMDA Receptors

Exposure of Syrian hamsters to light 1 h after lights-off rapidly (10 min) induced nuclear immunoreactivity (–ir) to the phospho-Ser¹³³ form of the Ca²⁺/cAMP response element (CRE) binding protein (pCREB) in the retinorecipient zone of the suprachiasmatic nuclei (SCN). Light also induced nuclear Fos-ir in the same region of the SCN after 1 h. The glutamatergic N-methyl- d -aspartate (NMDA) receptor blocker MK801 attenuated the photic induction of both factors. To investigate glutamatergic regulation of pCREB and Fos further, tissue blocks and primary cultures of neonatal hamster SCN were examined by Western blotting and immunocytochemistry in vitro. On Western blots of SCN tissue, the pCREB-ir signal at 45 kDa was enhanced by glutamate or a mixture of glutamatergic agonists (NMDA, amino-methyl proprionic acid (AMPA), and Kainate (KA)), whereas total CREB did not change. Glutamate or the mixture of agonists also induced a 56 kDa band identified as Fos protein in SCN tissue. In dissociated cultures of SCN, glutamate caused a rapid (15 min) induction of nuclear pCREB-ir and Fos-ir (after 60 min) exclusively in neurones, both GABA-ir and others. Treatment with NMDA alone had no effect on pCREB-ir. AMPA alone caused a slight increase in pCREB-ir. However, kainate alone or in combination with NMDA and AMPA induced nuclear pCREB-ir equal to that induced by glutamate. The effects of glutamate on pCREB-ir and Fos-ir were blocked by antagonists of both NMDA (MK801) and AMPA/KA (NBQX) receptors. In the absence of extracellular Mg²⁺, MK801 blocked glutamatergic induction of Fos-ir. However, the AMPA/KA receptor antagonist was no longer effective at blocking glutamatergic induction of either Fos-ir or pCREB-ir, consistent with the model that glutamate regulates gene expression in the SCN by a co-ordinate action through both NMDA and AMPA/KA receptors. Glutamatergic induction of nuclear pCREB-ir in GABA-ir neurones was blocked by KN-62 an inhibitor of Ca²⁺/Calmodulin (CaM)-dependent kinases, implicating Ca²⁺-dependent signalling pathways in the glutamatergic regulation of gene expression in the SCN.     

Barium action potentials in regenerating axons of the lamprey spinal cord

Intracellular recordings were obtained from growing tips of regenerating giant axons in the lamprey spinal cord, the recording sites verified by Lucifer yellow injection. In the presence of extracellular Ba++ (3-6 mM), tetraethylammonium (10-15 mM), and 4-aminopyridine (4-6 mM), action potentials showed prolonged plateaus. The fast initial phase of the action potential, but not the plateau (Ba++-spike), was blocked by tetrodotoxin (10(-6) gm/ml). The Ba++ spike was associated with increased membrane conductance and could be terminated with hyperpolarizing current pulses. Normal axons did not generate similar Ba++ spikes. However, TTX-resistant, voltage-dependant conductance changes could be elicited in normal axons if much higher concentrations of Ba++ (18-30 mM) were used. Their rate of rise was slower than in regenerating axons (0.6 V/sec vs 3.2 V/sec; n = 5), and the response did not outlast the current pulse. The Ba++ responses in normal and regenerating axons were blocked by ions known to block voltage-gated Ca++ conductances (Co++, Ni++, or Cd++). Therefore, these spikes probably represent Ba++ entry through voltage-dependent Ca++ channels, suggesting the presence of a higher-than-average voltage-dependent Ca++ conductance in the growing axon. However, Ca++-dependent spikes could not be obtained under any conditions in either normal or regenerating axons. Simultaneous intracellular recordings from growth cones and axons indicated that the Ba++ spike was initiated, in most cases, at the growth cone. The Ba++ spikes were recorded in regenerating axons for as long as 50 days following cord transection and were not correlatable with the "dying-back" phenomenon in cut axons, which usually is over before day 6. The concept of a higher-than-average voltage-dependent Ca++ conductance in growing tips of regenerating axons is in agreement with the hypothesis that Ca++ is important in regeneration and that regeneration may be related to the process of chemical synaptic transmission.     

The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro

Using microspectrofluorimetry and the calcium-sensitive dye fura-2, we examined the effect of excitatory amino acids on [Ca2+]i in single striatal neurons in vitro. N-methyl-D-aspartic acid (NMDA) produced rapid increases in [Ca2+]i. These were blocked by DL-2-amino-5-phosphonovaleric acid (AP5), by Mg2+, by phencyclidine, and by MK801. The block produced by Mg2+ and MK801 could be relieved by depolarizing cells with veratridine. When external Ca2+ was removed, NMDA no longer increased [Ca2+]i. Furthermore, the effects of NMDA were not blocked by concentrations of La3+ that blocked depolarization induced rises in [Ca2+]i. Substitution of Na+o by Li+ did not block the effects of NMDA. Concentrations of L-glutamate greater than or equal to 10(-6) M also increased [Ca2+]i. The effects of moderate concentrations of glutamate were blocked by AP5 but not by La3+ or by substitution of Na+ by Li+. The effects of glutamate were blocked by removal of external Ca2+ but were not blocked by concentrations of Mg2+ or MK801 that completely blocked the effects of NMDA. The glutamate analogs kainic acid (KA) and quisqualic acid also increased [Ca2+]i. The effects of KA were blocked by removal of external Ca2+ but not by La3+, Mg2+, MK801, or replacement of Na+ by Li+. Although AP5 was able to block the effects of KA partially, very high concentrations were required. These results may be explained by considering the properties of glutamate-receptor-linked ionophores. Excitatory amino acid induced increases in [Ca2+]i are consistent with the possibility that Ca2+ mediates excitatory amino acid induced neuronal degeneration.     

The interactions between plasma membrane depolarization and glutamate receptor activation in the regulation of cytoplasmic free calcium in cultured cerebellar granule cells

The complex modulation of cytoplasmic free calcium concentration ([Ca2+]c) in primary cultures of cerebellar granule cells in response to glutamate receptor agonists has been the subject of several contradictory reports. We here show that 3 components of the [Ca2+]c response can be distinguished: (1) Ca2+ entry through voltage-dependent Ca2+ channels, following KCl- or receptor-evoked depolarization, (2) Ca2+ entry through NMDA receptor channels, and (3) liberation of internal Ca2+ via a metabolotropic receptor. Depolarization with KCl induced a transient [Ca2+]c response (subject to voltage inactivation) decaying to a sustained plateau (largely inhibited by nifedipine). The NMDA response was potentiated by glycine, totally inhibited by (+)5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), and blocked by Mg2+ in a voltage-sensitive manner. Polarized cells displayed small responses to quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA). Depolarization enhanced a transient response to QA, but not to AMPA. Trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD), a selective agonist for the metabolotropic glutamate receptor, caused a transient elevation of [Ca2+]c, which was blocked by prior exposure to QA but not AMPA. The prolonged [Ca2+]c response to kainate (KA) can be resolved into 2 major components: an indirect NMDA receptor-mediated response due to released glutamate and a nifedipine-sensitive component consistent with depolarization-mediated entry via Ca2+ channels. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), QA at greater than 10 microM, and AMPA (but not trans-ACPD) reversed the KA response, consistent with an inactivation of the KA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.     

Hyperpolarizing action of enkephalin on neurons in the dorsal motor nucleus of the vagus, in vitro

Intracellular recordings were made from neurons of the dorsomotor vagal nucleus (DMV) in slices of rat medulla oblongata. [D-Ala2, D-Leu5]-enkephalin (DADLE), applied by perfusion (0.01-3 microM) or droplets, dose-dependently hyperpolarized 85% of the DMV neurons tested. The hyperpolarization, associated with a decrease in membrane resistance, persisted after elimination of synaptic activity by perfusion with Ca2(+)-free/high-Mg2+ solution or with 1 microM TTX solution. The opioid antagonist, naloxone, reversibly inhibited DADLE-induced hyperpolarization. The hyperpolarization depended on extracellular K+ concentration and reversed at about -90 mV. DADLE also decreased Ca2(+)-dependent spike duration and after-hyperpolarization (AHP). DAGO (a selective mu-receptor agonist), but not DPLPE (a selective delta-receptor agonist), mimicked DADLE's effects on membrane potential, Ca2(+)-dependent spike duration, and AHP. It is concluded that DADLE, through postsynaptic mu-type opioid receptors, hyperpolarized DMV neurons by increasing K+ conductance, which may have an inhibitory effect on DMV output. DADLE-induced decrease of spike duration and AHP was also mediated by mu-receptors and could have additional effects on functions of the DMV neuron by virtue of reduction in Ca2+ entry.     

Forskolin potentiates the paraoxon-induced hyperexcitability in snail neurons by blocking afterhyperpolarization

One characteristic of organophosphate poisoning is the ability to increase excitability or induce epileptiform activity in nerve cells, but underlying mechanisms are not fully understood. We have previously reported that paraoxon, an organophosphate compound, at submicromolar concentrations effectively suppress Ca(2+) spikes and modulate the activity of snail neurons. This effect was unrelated to acetylcholinesterase (AChE) inhibition but was found to involve the direct or indirect modulation of ion channels [Vatanparast J, Janahmadi M, Asgari AR, Sepehri H, Haeri-Rohani A. Paraoxon suppresses Ca(2+) spike and afterhyperpolarization in snail neurons: relevance to the hyperexcitability induction. Brain Res 2006a;1083(1):110-7]. In the present study, the interaction of paraoxon with cAMP formation on the modulation of Ca(2+) spikes and neuronal excitability was examined. Forskolin, the activators of adenylate cyclase, suppressed afterhyperpolarization (AHP) and increased the activity of snail neurons without any significant effect on the Ca(2+) spike duration. Pretreatment with forskolin, although attenuated the suppressing effect of paraoxon on the duration of Ca(2+) spikes but also potentiated the paraoxon-induced hyperexcitability by enhancing the suppressive effects of paraoxon on AHP. Our findings support the possible involvement of cAMP formation in the paraoxon-induced AHP suppression and neuronal hyperexcitability, although activation of cAMP pathway may attenuates some effects of paraoxon.     

Calcium activates two types of potassium channels in rat hippocampal neurons in culture

Several calcium-dependent potassium currents can contribute to the electrophysiological properties of neurons. In hippocampal pyramidal cells, 2 afterhyperpolarizations (AHPs) are mediated by different calcium-activated potassium currents. First, a rapidly activated current contributes to action-potential repolarization and the fast AHP following individual action potentials. In addition, a slowly developing current underlies the slow AHP, which occurs after a burst of action potentials and contributes substantially to the spike-frequency accommodation observed in these cells during a prolonged depolarizing current pulse. In order to investigate the single Ca2(+)-dependent channels that might underlie these currents, we performed patch-clamp experiments on hippocampal neurons in primary culture. When excised inside-out patches were exposed to 1 microM Ca2+, 2 types of channel activity were observed. In symmetrical bathing solutions containing 140 mM K+, the channels had conductances of 19 pS and 220 pS, and both were permeable mainly to potassium ions. The properties of these 2 channels differed in a number of ways. At negative membrane potentials, the small-conductance channels were more sensitive to Ca2+ than the large channels. At positive potentials, the small-conductance channels displayed a flickery block by Mg2+ ions on the cytoplasmic face of the membrane. Low concentrations of tetraethylammonium (TEA) on the extracellular face of the membrane specifically caused an apparent reduction of the large-channel conductance. The properties of the large- and small-conductance channels are in accord with those of the fast and slow AHP, respectively.     

Topiramate alters excitatory synaptic transmission in mouse hippocampus

Antiepileptic drugs may exert neuroprotective effects by decreasing excessive membrane excitability, neurotransmitter release, or postsynaptic Ca2+ entry. To assess these sites of action, we combined fluorescence Ca2+ imaging with extracellular field recording to analyze axonal excitability, evoked presynaptic Ca2+ entry through presynaptic Ca2+ channels, postsynaptic excitatory field potentials (fEPSP), and postsynaptic Ca2+ buildup ([Capost]) at the mouse hippocampal CA3-CA1 synapse exposed to topiramate (TPM). Topiramate had no effect on presynaptic Ca2+ entry, and produced only a minor inhibition of axonal excitability. Topiramate at concentrations up to 100 microM only slightly reduced the amplitude of the evoked fEPSP, but strongly inhibited the [Capost] evoked by repetitive synaptic activation. Postsynaptically, the action of TPM on the fEPSP and [Capost] was not mediated by an inhibition of the NMDA receptor, or by direct modulation of voltage-dependent Ca2+ channels, but reflected reduced somatic or dendritic membrane depolarization by AMPA and kainate receptors. These results are consistent with the known anticonvulsant properties of TPM. In addition, the ability of TPM to reduce postsynaptic Ca2+ buildup may provide a potential mechanism for neuronal protection during paroxysmal firing associated with epileptic seizures.     

Glutamatergic and morphological alterations associated with early life seizure-induced preconditioning in young rats

Postnatal day (P)20 rats are sensitive to CA1 injury following a single injection of kainic acid (KA) but are resistant to this injury when animals have a history of two neonatal seizures. We hypothesized that the two earlier seizures led to neuroprotection by a preconditioning mechanism. Therefore, morphology, [Ca(2+)](i) and NMDA subunit proteins of the hippocampus were examined after KA was administered once (1 × KA, on P6, P9, P13 or P20), twice (2 × KA, on P6 and P9) or three times (3 × KA, on P6, P9, P13 or P20). After 1 × KA on P20, the Golgi method revealed marked decreases in spine densities and aborization of CA1 and CA3 apical dendrites. After 3 × KA, morphological alterations were attenuated in CA1 neurons and were similar to pruning observed after 1 × KA on P6 or 2 × KA. After 1 × KA at P13, baseline [Ca(2+)](i) was elevated within pyramidal and dentate granule cells. N-methyl-D-aspartate (NMDA) responses were simultaneously enhanced. After 3 × KA, Ca(2+) elevations were attenuated. Immunohistochemistry revealed selective depletion of the NR2A/B subunit modulator in the same areas. NR1 subunit expression was downregulated in the subiculum and increased in the CA3, causing a significant shift in the NR1:NR2A/B ratio throughout the hippocampus. After 1 × KA or 3 × KA at P20, reduced expression was only observed in areas of cell injury. Results indicate that different changes in morphology and excitatory responses occur depending upon when seizures begin. Partial pruning and persistent shift in the NR1:NR2A/B ratio among excitatory synapses of the hippocampus early in life may produce epileptic tolerance and protect against subsequent insults.