Long-term Potentiation in the Striatum is Unmasked by Removing the Voltage-dependent Magnesium Block of NMDA Receptor Channels

We have studied the effects of tetanic stimulation of the corticostriatal pathway on the amplitude of striatal excitatory synaptic potentials. Recordings were obtained from a corticostriatal slice preparation by utilizing both extracellular and intracellular techniques. Under the control condition (1.2 mM external Mg2+), excitatory postsynaptic potentials (EPSPs) evoked by cortical stimulation were reversibly blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) ionotropic glutamate receptors, while they were not affected by 30 - 50 microM 2-amino-5-phosphonovalerate (APV), an antagonist of N-methyl-d-aspartate (NMDA) glutamate receptors. In the presence of 1.2 mM external Mg2+, tetanic activation of cortical inputs produced long-term depression (LTD) of both extracellularly and intracellularly recorded synaptic potentials. When Mg2+ was removed from the external medium, EPSP amplitude and duration increased. In Mg2+-free medium, cortically evoked EPSPs revealed an APV-sensitive component; in this condition tetanic stimulation produced long-term potentiation (LTP) of synaptic transmission. Incubation of the slices in 30 - 50 microM APV blocked striatal LTP, while it did not affect LTD. In Mg2+-free medium, incubation of the slices in 10 microM CNQX did not block the expression of striatal LTP. Intrinsic membrane properties (membrane potential, input resistance and firing pattern) of striatal neurons were altered neither by tetanic stimuli inducing LTD and LTP, nor by removal of Mg2+ from the external medium. These findings show that repetitive activation of cortical inputs can induce long-term changes of synaptic transmission in the striatum. Under control conditions NMDA receptor channels are inactivated by the voltage-dependent Mg2+ block and repetitive cortical stimulation induces LTD which does not require activation of NMDA channels. Removal of external Mg2+ deinactivates these channels and reveals a component of the EPSP which is potentiated by repetitive activation. Since the striatum has been involved in memory and in the storage of motor skills, LTD and LTP of synaptic transmission in this structure may provide the cellular substrate for motor learning and underlie the physiopathology of some movement disorders.     

Regulation of hippocampal NMDA receptors by magnesium and glycine during development.

N-Methyl-D-aspartate (NMDA) receptors play an important role in the development of neuronal connections in the retina and visual cortex, and in synaptic plasticity in the hippocampus. The objective of this study was to determine whether the sensitivity of hippocampal NMDA receptors to magnesium, glycine or NMDA changes during development. Xenopus oocytes were injected with mRNA prepared from hippocampi from rats of different ages, and NMDA receptor properties studied under voltage clamp. Voltage-dependent block of the NMDA receptor by magnesium was studied with voltage steps of -90 mV to -30 mV, in increments of 10 mV, during application of 100 microM NMDA, 3 microM glycine and 0-1000 microM Mg2+. The IC50 of Mg2+ for blocking NMDA receptor-mediated currents varied e-fold (2.72-fold) for approximately every 15 mV of membrane potential in the middle range of membrane potential (-70 to -50 mV), but the relationship between log[IC50] for Mg2+ and membrane potential was not linear, as would be expected for simple channel block. The slopes of the curves did not change with development, indicating no change in the voltage-dependence of Mg2+ block with age. However, the IC50 of Mg2+ block did change with age at every membrane potential tested. NMDA receptors expressed from mRNA isolated from 14-15 day old rats were nearly 2-fold less sensitive to block by Mg2+ (IC50 = 33 microM at -60 mV) than those from 1-2 day old rats (IC50 = 18 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

NMDA receptors as targets of heavy metal interaction and toxicity

The N-methyl-D-aspartate (NMDA) receptor (NR) is a ligand-gated channel that carries the slow component of the glutamate-activated postsynaptic current. Divalent metal ions can affect the NR channel activity in a voltage-dependent [Mg(II)-like] or voltage-independent [Zn(II)-like] manner. We have studied the effect of two toxic metals, lead [Pb(II)] and nickel [Ni(II)] on recombinant NR1a-NR2A and NR1a-NR2B channels expressed in RNA-injected Xenopus laevis oocytes or in transiently transfected mammalian HEK293 cells. Pb(II) caused a dose-dependent, but voltage-independent reversible inhibition of NMDA-activated channel activity similar for NR2A and NR2B-containing receptors; it did not modify the single channel conductance, indicating that its binding site is located out of the ionic pathway of permeation. On the contrary, Ni(II) had multiple and complex effects on NR channels. It determined a voltage-dependent, Mg(II)-like block by which the single channel amplitude and the mean open time were reduced in both NR2A and NR2B-containing channels. While high (>100 microM) concentrations caused a dose-dependent reduction of the activity in both channel types, 30 microM determined a voltage-independent decrease in the frequency of NR1a-NR2A channel openings, but an increase in the frequency of NR1a-NR2B channel openings, confirming previous observations of a subunit-dependent effect of this metal. These results were interpreted under the hypothesis that Pb(II) mediates a Zn(II)-like voltage-independent allosteric modulation that, different from Zn(II), is subunit-independent. In contrast, Ni(II) has different modes of action, which are dependent on the NR2 subunit type present in the receptor and are likely to be related to different interaction sites. The NR2B-dependent facilitation bears close similarities with the polyamine-mediated potentiation.     

A quantitative description of NMDA receptor-channel kinetic behavior.

Currents evoked in neurons of the vertebrate CNS by the glutamate agonist N-methyl-D-aspartate (NMDA) exhibit a marked voltage dependence in the presence of extracellular Mg. At the single-channel level, the addition of external Mg alters single-channel openings from long-lived events to many very short events grouped into bursts of openings. These bursts apparently result from short interruptions of current flow during periods when the channel is in the open configuration. Single-channel currents evoked by NMDA have been studied in outside-out patches of membrane taken from hippocampal CA 1 neurons grown in dissociated cell culture. The effects of changing external Mg concentration and holding potential on the single-channel parameters of open time, closed time, and burst length have been successfully described assuming a 3- or 4-state model with 1 open state, 1 or 2 "blocked" states, and 1 absorbing closed state. Evaluation of the blocking rates over Mg concentrations from 0.2-200 microM indicate that a single "blocking" mechanism cannot account for the short closed states and that a second voltage-dependent but Mg-independent "blocked" state is necessary to explain the data especially at low Mg concentrations.     

The anticonvulsant retigabine potently suppresses epileptiform discharges in the low Ca ++ and low Mg++ model in the hippocampal slice preparation

Retigabine (N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester, D-23129) is a broad acting anticonvulsant currently undergoing phase II clinical trials. An opening effect on leakage conductance K+ channels, potentiation of GABA induced currents and a weak blocking effect on Na+ and Ca++ channels were previously reported. The goal of this study was to investigate whether retigabine is capable of blocking epileptiform discharges in the low Ca++ and low Mg++ model in the hippocampal slice preparations and whether the anti-burst activity can be related to the K+ channel opening effect. In the low Ca++ model, synaptic transmission is blocked and discharges evolve from ephaptically-coupled neurons. Compounds which directly interfere with the threshold for action potential induction via alteration of ion channel function (i.e. Na+ channel blocker) may alter the discharges, while compounds interfering with synaptic transmission are not active. Retigabine suppressed the discharges in a concentration-dependent manner. A significant reduction in frequency without effect on amplitude was observed after application of 1 microM, and a full block of all discharges after application of 25 microM. The opener of the ATP sensitive K+ channels cromakalim was also active. Application of 300 microM cromakalim yielded to a lower frequency with no effects on the amplitude of discharges. Treatment with phenytoin and carbamazepine resulted in a marked reduction in amplitude accompanied by a rise in frequency; only at higher concentrations was a full block observed. The effect of retigabine therefore differs from sodium channel blockers and can be related to the K+ channel opening effect. In the low Mg++ model, excitatory neurotransmission is augmented by reducing the Mg++ block of NMDA channels. This results in development of interictal-like epileptiform activity in area CA1 in isolated hippocampal slices. Treatment with retigabine 10 microM resulted in a significant reduction of the discharges, and discharges were fully blocked after application of 25 microM. Qualitatively similar effects were observed with cromakalim and valproate, albeit at higher concentrations. The data indicate that retigabine exerts potent broad spectrum activity making it an interesting candidate for treatment of drug resistant patients.     

Spontaneous Openings of NMDA Receptor Channels in Cultured Rat Hippocampal Neurons

Spontaneous and N-methyl-D-aspartate (NMDA)-evoked single-channel currents were studied in outside-out patches isolated from cultured rat hippocampal neurons. Both spontaneous and NMDA-evoked single-channel currents reversed at potentials close to 0 mV and exhibited multiple amplitude levels of similar amplitude. Both spontaneous and NMDA-evoked single-channel currents were inhibited by Mg²⁺ in a voltage-dependent manner and by 7-chlorokynurenic acid. The activity of spontaneous single-channel currents was reduced by the competitive NMDA receptor antagonists, but by one to three orders of magnitude less than expected assuming that the spontaneous activity is due to an ambient NMDA receptor agonist present in the extracellular solution. Our results suggest that, similar to other ligand-gated ion channels, NMDA receptor channels have a dual mode of activation - spontaneous and agonist induced.     

Subunit-dependent effects of nickel on NMDA receptor channels

Nickel (Ni2+) is a transition metal that affects different neuronal ionic channels. We investigated its effects on glutamate channels of the NMDA-type in the presence of saturating concentration of glutamate or NMDA (50 microM), in 0 external Mg and in the continuous presence of saturating glycine (30 microM). In neonatal rat cerebellar granule cells, Ni2+ inhibited the current evoked by NMDA at -60 mV with an IC50 close to 40 microM. The inhibition was weakly voltage-dependent and the current at +40 mV was inhibited with IC50=86 microM. Wash out of the metal unmasked a stimulatory effect which persisted for a few seconds. In HEK293 cells transiently transfected with recombinant NR1a-NR2A receptors, Ni2+ inhibited the current elicited by glutamate with an IC50=52 microM at -60 mV and 90 microM at +40 mV. In HEK293 expressing NR1a-NR2B receptors, 0.1-100 microM Ni2+ caused a potentiation of the current, with EC50=4 microM, while with 300 microM, a voltage-dependent block became apparent (IC50=170 microM). As previously reported, the current through both classes of recombinant receptors was steeply dependent on external pH, and in both cases the protonic block had an IC50 close to pH 7.2. Application of Ni2+ showed that stimulation of NR1a-NR2B receptor channels was dependent on external pH, while voltage-independent inhibition of NR1a-NR2A was less sensitive to pH change. These results indicate that Ni2+ has multiple and complex effects on NMDA channels, which are largely dependent on the NR2 subunit.     

Zinc (Zn2+) blocks voltage gated calcium channels in cultured rat dorsal root ganglion cells.

Dorsal root ganglion cells (DRGs) exhibit 3 types of voltage-dependent calcium channels. We have cultured DRGs from 2- to 4-day-old rat pups and obtained whole-cell patch-clamp recordings of calcium-channel currents after 1-5 days in culture. The calcium-channel currents (carried by barium) were recorded with tetrodotoxin (TTX) in the external solution. A cesium-based solution containing Na-ATP, HEPES and EGTA was used in the recording pipette. Cells were held at -80 mV and calcium channel currents were evoked by stepping to depolarized voltages. The divalent cation zinc (Zn2+) blocked sustained and transient voltage sensitive calcium channel currents. Onset of the blockade was fast and a steady-state was reached within 5-15 min, depending upon the concentration used. The IC50 for inhibition of the peak current evoked by a step depolarization from -80 mV to 0 mV (N plus L channels) for 80 ms was 69 microM Zn2+ and the Hill slope about 1. The calcium current evoked by a voltage step from -80 mV to voltages between -40 mV and -15 mV (T-type current) was more sensitive (> 80% block with 20 microM Zn2+). During wash the effect was only partly reversible in 50% of the neurons. Thus, Zn2+ is a potent blocker of voltage dependent calcium currents in mammalian neurons, especially of T-type currents.     

Properties of NMDA receptors in rat spinal cord motoneurons

Postnatal development and properties of N-methyl-d-aspartate (NMDA) receptors were studied with whole-cell and outside-out patch-clamp techniques in interneurons and fluorescence-labelled motoneurons in rat spinal cord slices. Both the absolute amplitude of NMDA-induced currents and currents normalized with respect to the motoneuron capacitance increased significantly at postnatal days 10-13 when compared to the responses evoked at postnatal days 2-3. The mean amplitude of the responses to kainate also increased in motoneurons of postnatal days 10-13. Single-channel currents induced by low concentrations of glutamate, exhibited four distinct amplitude levels corresponding to 19.2 +/- 2.4 pS, 38.4 +/- 3.5 pS, 56.3 +/- 2. 4 pS and 69.6 +/- 3.7 pS. In contrast, the conductance of single channels, recorded under identical conditions, in rat spinal cord interneurons was less, 15.3 +/- 3.2 pS, 29.9 +/- 5.4 pS, 46.7 +/- 4. 8 pS and 62.4 +/- 3.9 pS. The high (56/70 pS) conductance single-channel openings in motoneuron patches were sensitive to NMDA receptor inhibitors D-2-amino-5-phosphonovalerate, 7-chlorokynurenic acid and ifenprodil. Whole-cell NMDA-evoked currents were blocked in a voltage-dependent manner by extracellular Mg2+ with an apparent dissociation constant for Mg2+ binding at 0 mV of 1.8 +/- 0.5 mm. The conductance and relative distribution of NMDA receptor channel openings induced by 1 micrometer glutamate in patches isolated from the motoneurons were independent of age from postnatal day 4 to 14. The results suggest that the properties of NMDA receptor channels in motoneurons differ from those in spinal cord interneurons and cells transfected with NR1/NR2 subunits.     

Carbamazepine blocks NMDA-activated currents in cultured spinal cord neurons.

The antiepileptic agents, carbamazepine and phenytoin, suppress seizures in man and convulsant-induced hyperactivity in spinal cord nerve cell cultures. In the present study, we have shown by whole cell recording that carbamazepine, in contrast to phenytoin, blocks N-methyl-D-aspartate (NMDA)-activated membrane currents in cultured neurons in a dose-dependent fashion. The NMDA receptor-activated channel, which is blocked at physiological concentrations of Mg2+ at resting membrane potential, can be activated by glutamate in depolarized neurons and thus be involved in epileptogenesis. Therefore, the block of NMDA-evoked membrane currents in cultured neurons may contribute to the clinical effectiveness of carbamazepine.     

The Influence of Magnesium Ions on the NMDA Mediated Responses of Ventral Rhythmic Neurons in the Spinal Cord of Xenopus Embryos

Xenopus embryos immobilized in tubocurarine respond to natural skin stimulation with fictive swimming. This can also occur in saline without Mg2+ and is blocked by NMDA antagonists. Ventral spinal cord neurons which are rhythmically active during swimming are depolarized by bath applied N-methyl-D-aspartate (NMDA) (in 1 microM tetrodotoxin (TTX) to block indirect effects). By using current clamp techniques this depolarization is shown to be partially blocked by 0.5 and 1 mM Mg2+ in a voltage-dependent manner similar to that described in cultured neurons. Mg2+ partially and reversibly reduces the slow NMDA-mediated component of excitatory post-synaptic potentials (EPSPs) in ventral neurons. However, in 1 mM Mg2+ fictive swimming can still be evoked by natural stimulation. The frequency of swimming is slightly lower than in nominally 0 mM Mg2+, but the pattern of ventral root activity and synaptic drive to ventral neurons seems little affected. Fictive swimming can also be induced by applying NMDA to spinal preparations. In 0 mM Mg2+, such rhythmic activity is unstable and transient over a narrow NMDA concentration range. In 0.5 mM Mg2+, continuous rhythmic activity is induced over a wide range of NMDA concentrations. Lower spinal preparations need higher NMDA concentrations to induce activity. We conclude that the neurons rhythmically active in swimming have NMDA receptor channels which show a voltage dependent block in the presence of Mg2+. However, while Mg2+ exerts a powerful stabilizing influence on rhythmic activity induced in spinal embryos by exogenous NMDA, its influence on 'naturally' evoked fictive swimming is less clear. The fictive swimming machinery in the brain and spinal cord can produce stable swimming with or without Mg2+ induced voltage dependency of the NMDA channels.     

Evidence for N-methyl-D-aspartic acid receptor-mediated modulation of the commissural input to central vestibular neurons of the frog

We have investigated the role of N-methyl-D-aspartate (NMDA) receptors in the excitatory synaptic transmission to central vestibular neurons in the isolated superfused brainstem of the frog. In superfusate containing 1 mM Mg2+ field potentials in the vestibular nuclei evoked by electrical stimulation of either the ipsi- or the contralateral VIIIth nerve were not affected by bath-applied D-2-amino-5-phosphonovaleric acid (D-APV, 25-50 microM), a selective NMDA antagonist. In a low Mg2+ solution postsynaptic field potential components were larger than control but still unaffected by D-APV. Ipsi- and contralaterally evoked excitatory postsynaptic potentials (EPSPs) differed in their shape parameters as well as in their pharmacological sensitivity. Ipsilaterally evoked EPSPs were not affected by D-APV and has a rise time that was faster than that of contralaterally evoked EPSPs. The peak amplitude of hte latter was reduced by D-APV (25-50 microM) to about 65% of the control value in the presence of 1 mM Mg2+. During bath application of NMDA (100 microM) an increased input resistance and repetitive de- and hyperpolarizing membrane potential shifts were observed. Similar events were observed during a reduction of the Mg2+ concentration. Bath application of NMDA (0.1-1 microM) resulted in an enhanced size of the recorded EPSPs. Dendritic and somatic EPSPs were simulated on a computer with the assumption of a constant NMDA receptor activation and a pulse-like non-NMDA receptor activation. The results of these simulations are consistent with the hypothesis that the efficacy of non-NMDA-mediated vestibular commissural synaptic transmission is modulated through tonically activated NMDA receptors.     

Glutamate-induced inhibition of paired pulse facilitation of monosynaptic excitatory post-synaptic potentials in frog spinal motoneurons.

To evaluate actions of glutamate on excitatory synaptic transmission in the central nervous system, we examined glutamate-induced changes in the paired pulse facilitation of monosynaptic excitatory post-synaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Glutamate (1 mM) depolarized motoneurons both in the presence and absence of Mg2+. In most cells perfused with Mg(2+)-free or high Ca(2+)-Mg2+ solutions, the glutamate potential was accompanied by a reduction in peak amplitude of EPSPs, although the degree of change varied with the cells. Glutamate enhanced the EPSP amplitude in a few cells with Mg(2+)-free and high Ca(2+)-Mg2+ solutions, and in most cells with high Mg2+ medium. In 3/5 cells tested, the paired pulse facilitation of EPSPs was reduced by glutamate when the EPSP amplitude either increased or decreased. NMDA (50 microM), kainate (50-100 microM), quisqualate (5-50 microM) and L-2-amino-4-phosphonobutyrate (L-AP4, 1 mM) also decreased the facilitation in about half of the cells tested. The glutamate-induced decrease in the facilitation was observed in both the presence and absence of Mg2+ and was not affected by the concomitant application of glutamate and antagonists for non-NMDA or NMDA receptors, such as 6-cyano-7-nitro-quinoxalinediones (CNQX, 60 microM) or 2-amino-5-phosphonovalerate (APV, 250 microM). Glutamate reduced the facilitation of excitatory post-synaptic currents (EPSCs) recorded at a constant membrane potential under voltage clamp, when the EPSC amplitude either increased or decreased and when the input conductance either increased or decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two distinct types of repetitive bursting activity mediated by NMDA in hypothalamic neurons in vitro

Hypothalamic magnocellular dorsal nucleus neurons were recorded from adult guinea pig brain slices with the whole-cell patch-clamp technique to determine the effects of N-methyl-D-aspartate (NMDA) applied in the bath or by iontophoresis. In a majority of cells (59 of 77, 76.6%), rhythmic bursting discharges were evoked by specific activation of NMDA receptors when the membrane was more negative than -60 mV. This endogenous rhythmic activity was resistant to tetrodotoxin. It was suppressed by removal of extracellular Mg2+, indicating the involvement of the voltage-dependent block of the NMDA channel by Mg2+. Application of thapsigargin showed that rhythmic activity did not depend on the release of Ca2+ from reticulum stores. Blockers of Ca2+ conductances Ni2+ and nifedipine had no effects on the bursts. Their repolarization did not involve the activation of a strophantidin- or ouabain-sensitive pump, but partly depended on an apamine-sensitive Ca2+-dependent K+current. In a small subset of cells (9 of 69, 13%), specific activation of NMDA receptors induced another type of bursting activity which consisted of repetitive low-threshold spikes sustaining bursts of action potentials. Rhythmic low-threshold spikes subsisted in the presence of tetrodotoxin but were suppressed by Ni2+. Increasing the amount of NMDA brought about a switch from the rhythmic low-threshold spike burst firing to the rhythmic bursting activity observed for the majority of cells. The present data show for the first time that NMDA receptor activation can induce two independent rhythmic bursting behaviours in the same neuron, probably depending on the strength of the glutamatergic drive.     

Routes of zinc entry in mouse cortical neurons: role in zinc-induced neurotoxicity

Exposure of central neurons to Zn2+ triggers neuronal death. The routes of Zn2+ entry were investigated in living cortical neurons from the mouse using the specific Zn2+ fluorescent dye N-(6-methoxy-8-quinolyl)-p-toluene sulphonamide (TSQ), which preferentially detects membrane-bound Zn2+. Exposure of cortical neurons to increasing concentrations of Zn2+ (1-100 microM) induced a progressive increase in the fluorescence of TSQ. This fluorescence signal was not attenuated by the permeation of plasma membrane with digitonin. Accordingly, the major part of TSQ fluorescence (two-thirds) was associated to the particulate fraction of cortical neurons exposed to Zn2+. These results suggest that Zn2+ detected with TSQ in neurons is mainly bound to membranes. TSQ fluorescence measured in neurons exposed to 3 microM Zn2+ was enhanced by Na+-pyrithione, a Zn2+ ionophore, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate (NMDA) or KCl-induced depolarization. However, in the absence of any treatment, TSQ labelling of neurons exposed to 3 microM Zn2+ was only decreased by NMDA receptor antagonists, whereas it remained unaltered in the presence of antagonists of AMPA receptors or L-type voltage-gated Ca2+ channels. Zn2+ entry through NMDA receptors did not contribute to Zn2+-induced neuronal death, as it was prevented by antagonists of NMDA receptors only when they were added after the Zn2+ exposure. Finally, Zn2+ induced a delayed accumulation of extracellular glutamate which might be responsible for the delayed NMDA receptor activation that leads to neuronal death.     

Activation of central muscarinic receptors inhibit Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ accumulation activity, with a Km for Ca2+ congruent to 0.5 microM, close to the cytosolic concentration of Ca2+. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1-20 microM), both Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5-1.0 microM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca2+ channels or Na+-Ca2+ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca2+ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca2+ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.     

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.     

Expression of membrane currents in rat neocortical neurons in serum-free culture. II. Outward currents

The timing of expression and properties of outward membrane currents in cultured neocortical pyramidal-shaped neurons have been investigated using the gigaseal whole-cell voltage clamp and single-channel recording techniques. Dissociated primary cultures of synchronized (same cell cycle), growth arrested (G1 phase) and birth-dated cells from fetal rat (E18) were maintained in a serum-free medium. The earliest appearing membrane current in the soma is a voltage-dependent outward current carried by K+. The current consists of two components, one rapidly rising component, resembling those associated with the transient outward current (IA) and the other similar to the delayed rectifier current (IK). The ratio between the peak IA and IK was about 0.3 at all membrane voltages. The magnitude of both IA and IK increased with time in culture but the ratio remained unchanged. Direct measurements of unitary currents showed the presence of two voltage-activated outward conductances, 32 pS and 120 pS. The small conductance channel was sparse. The large conductance channel is K+-selective and was sensitive to both voltage and internal Ca2+.     

Developmental increase in the sensitivity to magnesium of NMDA receptors on CA1 hippocampal pyramidal cells

The N-methyl-D-aspartate (NMDA) receptor is involved in processes, such as associative learning, that are particularly important during early postnatal development. It has been suggested that the activity and regulation of this receptor changes during development. Activation of the NMDA receptor is normally limited by Mg2+ present in the extracellular fluid of brain. We have found that Mg2+ less potently antagonizes the depolarizing action of NMDA in developing rats than in adults. A grease-gap method was used to record depolarizations evoked in CA1 hippocampal pyramidal cells by the excitants NMDA and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate). In the adult CA1 area, Mg2+ shifted the NMDA concentration-response curve to the right in a manner consistent with voltage-dependent open channel block (uncompetitive antagonism) in a preparation with significant receptor reserve. The potency of Mg2+ increased during development; a greater than two-fold change in the EC50 for Mg2+ was observed between 10-15 days of age and adulthood. A concentration of 10 mM reduced the maximum response of CA1 pyramidal cells to NMDA in adult rats, but not in developing rats. In addition, Mg2+ often enhanced the maximum depolarizations evoked by NMDA in 10- to 15-day-old rats, but very seldom in adults. No significant developmental changes in AMPA-induced depolarizations were observed in the presence or absence of Mg2+. These results suggest that synaptically released glutamate will readily activate NMDA receptors during early development and that its ability to do this declines with the maturation of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitatory amino acid neurotoxicity at the N-methyl-D-aspartate receptor in cultured neurons: role of the voltage-dependent magnesium block

Results of the present report show that cerebellar neurons in primary culture are resistant to glutamate concentrations as high as 5 mM in the presence of glucose and Mg2+, but sensitive to glutamate concentrations lower than 35 microM when the neurons are deprived of glucose. Glutamate toxicity is also potentiated when Mg2+ is removed but glucose and EDTA are present; in this case, higher concentrations of glutamate (1 mM) are required for full toxicity. Glucose concentrations as low as 50 microM are fully protective against the toxicity of 100 microM glutamate; pyruvate and, to a lesser extent, lactate are also protective. Significantly, increasing concentrations of extracellular Mg2+ are fully protective against the toxicity of 100 microM glutamate in the absence of glucose and against the toxicity of 1 mM glutamate in the presence of glucose and EDTA. We interpret these results as support for our hypothesis that the pivotal event in glutamate's transition to neurotoxin is relief of the Mg2+ block of the N-methyl-D-aspartate (NMDA) receptor channel, which is known to be voltage-dependent. Partial depolarization in response to depletion of high-energy phosphates relieves the voltage-dependent block enabling glutamate to stimulate an excessive ion influx which results in the death of the neuron by a mechanism which is not yet understood. We propose that this mechanism may be operative in the neuronal damage associated with a variety of neurodegenerative disorders.