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.     

Tetanic stimulation of schaffer collaterals induces rhythmic bursts via NMDA receptor activation in rat CA1 pyramidal neurons

Exploring the principles that regulate rhythmic membrane potential (Vm) oscillations and bursts in hippocampal CA1 pyramidal neurons is essential to understanding the theta rhythm (theta). Recordings were performed in vitro in hippocampal slices from young rats, and a group of the recorded CA1 pyramidal cells were dye-filled with carboxifluorescein and immunolabeled for the R1 subunit of the NMDA receptor. Tetanic stimulation of Schaffer collaterals (SCs) and iontophoresis of glutamate evoked rhythmic Vm oscillations and bursts (approximately 10 mV, approximately 7 Hz, 2-5 spikes per burst) in cells (31%) placed close to the midline ("medial cells"). Rhythmic bursts remained under picrotoxin (10 microM) and Vm oscillations persisted with tetrodotoxin (1.5 microM), but bursts were blocked by AP5 (25 microM) and Mg2+-free solutions. Depolarization and AMPA never induced rhythmic bursts. The rest of the neurons (69%), recorded closer to the CA3 region ("lateral cells"), discharged rhythmically single repetitive spikes under SC stimulation and glutamate in control conditions, but fired rhythmic bursts under similar stimulation, both when NMDA was applied and when non-NMDA receptors were blocked with CNQX (20 microM). Medial cells exhibited a larger NMDA current component and a higher NMDAR1 density at the apical dendritic shafts than lateral cells, suggesting that these differences underlie the dissimilar responses of both cell groups. We conclude that the "theta-like" rhythmic oscillations and bursts induced by glutamate and SC stimulation relied on the activation of NMDA receptors at the apical dendrites of medial cells. These results suggest a role of CA3 pyramidal neurons in the generation of CA1 theta via the activation of NMDA receptors of CA1 pyramidal neurons.     

N-methyl-d-aspartate receptor-mediated processing of beta-amyloid precursor protein in rat hippocampal slices: in vitro--superfusion study

Abnormal proteolytic degradation of the beta amyloid precursor protein (beta-APP) may result in accumulation of potentially neurotoxic beta amyloid (betaA). The role of various receptors in the regulation of beta-APP processing has been suggested. This study aimed to determine how NMDA receptors and Ca2+ ions regulate proteolysis of beta-APP in rat hippocampus in vitro. Adult rat hippocampal slices were superfused with NMDA-containing media, and immunoreactivity of soluble beta-APP derivatives was detected in dialysates. Application of 100 microM and 250 microM NMDA for 20 min in Ca2+-containing medium induced dose-dependent release of aminoterminal beta-APP derivatives, and a fragment of Abeta sequence, whereas carboxy-terminal fragments of beta-APP were only slightly detected. This indicates activation of beta-APP processing, and release of its soluble cleavage products. This effect was inhibited by NMDA receptor antagonist 1 microM MK-801 and 100 microM CPP in Ca2+-free medium, thus indicating that NMDA receptors and calcium ions mediate proteolytic non-amyloidogenic degradation of the beta-APP.     

Effects of the N-methyl-D-aspartate antagonists on the rise in [Ca2+]i following depolarization in aged rat brain synaptosomes.

The effects of non-competitive NMDA antagonists, MK-801 and dextrorphan in relation to the rise in intracellular Ca2+ concentrations ([Ca2+]i) after stimulation with 15 mM K+ in whole brain synaptosomes from young (3 months old) and aged (24 months old) Fisher344 rats were examined. A fluorescent chelating agent, Rhod-2, was employed to monitor any alterations of K(+)-evoked [Ca2+]i. In young rats, the rise in [Ca2+]i following depolarization was affected by neither dextrorphan (1, 10, 100 microM) nor MK-801 (0.1, 1, 10 microM), while in aged rats, 1 microM dextrorphan and 0.1 microM MK-801 brought about a significant increase in [Ca2+]i following depolarization. In low Mg2+ medium, 10 microM MK-801 and 100 microM dextrorphan significantly inhibited the rise in [Ca2+]i after stimulation with 15 mM K+ in young rats, while neither dextrorphan nor MK-801 could affect the rise in [Ca2+]i significantly in aged rats. When 100 microM NMDA was applied in a medium containing 1.2 mM Mg2+, the rise in [Ca2+]i following depolarization was slightly inhibited by 1 microM MK-801 in young rats, but it was not inhibited significantly by dextrorphan. In aged rats, both 100 microM dextrorphan and 10 microM MK-801 strongly inhibited the rise in [Ca2+]i following depolarization in the presence of 100 microM NMDA. Instead of NMDA, when 100 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a non-NMDA receptor agonist, was applied, dextrorphan did not inhibit the rise in [Ca2+]i. In low Mg2+ medium, 100 microM NMDA potentiated the inhibitory effect of 10 microM dextrorphan in young rats, while 100 microM dextrorphan or MK-801 did not show any further inhibition by adding 100 microM NMDA. The addition of 100 microM AMPA did not affect the effect of dextrorphan in a low Mg2+ medium in young rats. These results suggest that NMDA antagonist-mediated [Ca2+]i homeostatic system may alter through aging. In addition, the findings that NMDA potentiated the inhibitory effect of NMDA antagonist, which being further potentiated by aging or lowered extrasynaptosomal Mg2+, indicate the possibility that the Mg2+ block to NMDA receptors might be attenuated through aging.     

Induction of delayed synchronized bursts in hippocampal slices by weak sine-wave stimulation; role of the NMDA receptor

Weak (20-50 microA) sine-wave stimulation at 60 Hz (SWS) of either the mossy fibers or the Schaffer collaterals promoted epileptiform synchronized bursts in the CA2/3 area of rat hippocampal slices in the absence of epileptogenic agents. Following brief SWSs (2-10 sec every 5 min), delayed synchronized bursts (DSBs) were triggered by weak test pulses in either pathway and transmitted to CA1. The long (2-10 sec) refractory periods which followed synchronized bursts in CA2/3 limited their rate of occurrence. Furthermore, SWS decreased the activity for several minutes in slices that exhibited frequent bursts. DSBs were reversibly blocked by perfusion with the N-methyl-D-aspartate (NMDA) specific antagonist DL-2-amino-5-phosphono-valeric acid (APV). The involvement of NMDA receptors was further suggested by the facilitation of CA2/3 synchronized bursts in medium with NMDA (5 microM) or lacking magnesium, and by iontophoresis of NMDA in the CA2/3 stratum radiatum. The findings that SWS-induced DSBs persisted for hours in undisturbed slices, and that bursts abolished by APV reappeared during washout in control solution, suggest long-term changes in the CA2/3 synaptic region.     

NMDA-induced increase in [Ca2+]i and 45Ca2+ uptake in acutely dissociated brain cells derived from adult rats.

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-D-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca2+]i measured with fluo3 and 45Ca2+ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 microM) and N-methyl-DL-aspartate (200 microM) increased [Ca2+]i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated 45Ca2+ uptake about 16-10% in the same regions. The increases in [Ca2+]i and 45Ca2+ uptake were inhibited by 40% in the presence of 1 mM MgCl2 and by 90-50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca2+]i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca2+]i increase in adult age.     

NMDA receptor-mediated synaptic excitation selectively inhibited by ethanol in hippocampal slice from adult rat

The effect of ethanol (EtOH) on synaptic transmission mediated by N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors was investigated in slices from adult rat hippocampus. Synaptic responses were elicited by stimulation of stratum radiatum and were recorded in CA1 stratum radiatum or stratum pyramidale. Population EPSPs (pEPSPs) mediated by NMDA receptor activation were isolated by application of a solution containing the kainate/quisqualate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione and either low (0.1 mM) Mg2+ or 100 microM bicuculline. Increasing concentrations of EtOH produced increasing inhibition of NMDA receptor-mediated pEPSPs with EtOH concentrations between 1 and 50 mM. At a concentration of 50 mM, EtOH inhibited NMDA receptor-mediated pEPSPS by 43%; the inhibition by 100 mM EtOH was not significantly different from that produced by 50 mM. Methanol and 1-butanol also inhibited the NMDA receptor-mediated pEPSPs; the potency of the alcohols for inhibition of NMDA receptor-mediated pEPSPs was 1-butanol greater than ethanol greater than methanol. pEPSPs mediated by non-NMDA glutamate receptors were isolated by the application of the NMDA receptor antagonist d,1-2-amino-5-phosphonovaleric acid in the presence of 1.5 mM Mg2+. These pEPSPs were not significantly affected by 50 mM EtOH, whereas 100 mM EtOH reduced the amplitude of these pEPSPs by 9%. The observations indicate that synaptic excitation mediated by NMDA receptors in tissue from adult rat is inhibited by intoxicating concentrations of EtOH. The data are consistent with the hypothesis that EtOH-induced inhibition of EPSPs mediated NMDA receptors may contribute to the intoxicating effects of EtOH.     

Redox modulation of NMDA receptor-mediated synaptic activity in the hippocampus.

The sulfhydryl reducing agent dithiothreitol (DTT) and the oxidizing agent 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) reversibly modulate the component of synaptic potentials mediated by N-methyl-D-aspartate (NMDA) receptors in slices of hippocampal area CA1. DTT (1 mM) reversibly potentiates NMDA receptor-mediated synaptic potentials while DTNB (1 mM) has the opposite effect. However, treatment of the slices with the irreversible sulfhydryl alkylating agent N-ethylmaleimide (300 microM) prevents DTNB from reversing the potentiation induced by DTT. These results further implicate the redox modulatory site as a regulator of the NMDA receptor-channel complex in vivo.     

Reparatory effects of nicotine on NMDA receptor-mediated synaptic plasticity in the hippocampal CA1 region of chronically lead-exposed rats

Activation of neuronal nicotinic acetylcholine receptors (nAChRs) modulates the induction of long-term potentiation (LTP): a possible cellular mechanism of learning. To investigate the effect of nicotine on synaptic plasticity in chronically lead-exposed rats, field excitatory postsynaptic potentials and paired-pulse facilitation (PPF) were recorded in the CA1 area of hippocampal slices from chronically lead-exposed 23-30-day-old rats. The results showed the following. (1) Nicotine (1 microm) facilitated the induction of LTP in CA1 by a weak tetanic stimulation (100 Hz, 20 pulses), which does not by itself produce LTP in lead-exposed rats. This effect was significantly suppressed by mecamylamine, a nicotinic antagonist, suggesting that the facilitation of LTP was through nAChRs. (2) The nicotine-facilitated LTP was blocked by dihydro-beta-erythroidine (DHbetaE), a non-alpha7 nAChR antagonist, whereas long-term depression (LTD) was produced by the combination of nicotine and methyllycaconitine, a alpha7-nAChR antagonist. This type of LTD was blocked by DHbetaE. This suggested that several nAChR subtypes were involved in the nicotine-facilitated synaptic plasticity. (3) Nicotine enhanced PPF in the hippocampal CA1 region, and the nicotine-facilitated LTP in lead-exposed rats was blocked by either d-(-)-2-amino-5-phosphonopentanoic acid, the N-methyl-d-aspartate (NMDA) receptor antagonist, or picrotoxin, an antagonist of gamma-aminobutyric acid(A) receptors. We suggest that nicotine-facilitated synaptic plasticity was due to the activation of NMDARs by disinhibition of pyramidal cells through presynaptic nAChRs. This may represent the cellular basis of nicotine-facilitated cognitive enhancement observed in chronically lead-exposed rats.     

Differential responses to NMDA receptor activation in rat hippocampal interneurons and pyramidal cells may underlie enhanced pyramidal cell vulnerability

Hippocampal interneurons are generally more resistant than pyramidal cells to excitotoxic insults. Because NMDA receptors play a crucial role in neurodegeneration, we have compared the response to exogenous NMDA in CA1 pyramidal cells and interneurons of the stratum oriens using combined whole-cell patch-clamp recording and ratiometric Ca2+ imaging. In voltage-clamp, current-clamp or in nominally Mg2+-free medium, NMDA (10 microM; 3-5 min exposure in the presence of tetrodotoxin) induced a markedly larger inward current and Ca2+ rise in pyramidal cells than in interneurons. Pyramidal cells also showed a more pronounced voltage dependence in their response to NMDA. We hypothesized that this enhanced response to NMDA receptor activation in pyramidal cells could underlie their increased vulnerability to excitotoxicity. Using loss of dye as an indicator of degenerative membrane disruption, interneurons tolerated continuous exposure to a high concentration of NMDA (30 microM) for longer periods than pyramidal cells. This acute neurodegeneration in pyramidal cells was independent of intracellular Ca2+, because high intracellular BAPTA (20 mM) did not prolong survival time. Thus, a plausible explanation for the enhanced sensitivity of pyramidal neurons to excitotoxic insults associated with cerebral ischemia is their greater response to NMDA receptor activation, which may reflect differences in NMDA receptor expression and/or subunit composition.     

Characteristics of protective effects of NMDA antagonist and calcium channel antagonist on ischemic calcium accumulation in rat hippocampal CA1 region

Effects of excitatory amino acid receptor antagonists and voltage-dependent Ca(2+) channel antagonists on ischemia-induced intracellular free Ca(2+) accumulation in rat hippocampal slices were examined. Ischemia caused a large Ca(2+) accumulation in CA1 region but a small Ca(2+) accumulation in CA3 and dentate gyrus regions. When applied during ischemia, the NMDA receptor antagonist MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate) inhibited the ischemic Ca(2+) accumulation only in the CA1, but the non-NMDA receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) inhibited it in all the three regions. The L-type Ca(2+) channel antagonists nifedipine and verapamil inhibited the ischemic Ca(2+) accumulation only in the CA1 region, but omega-conotoxin, a N- and L-type Ca(2+) channel antagonist inhibited the Ca(2+) accumulation in all the three regions of the hippocampus. When applied after 5-min ischemia, nifedipine but not MK-801, inhibited sustained postiscehmic Ca(2+) elevation in the CA1 region but not in the CA3 and dentate gyrus regions. These findings suggest that the enhanced ischemia-induced Ca(2+) accumulation in the CA1 region is mediated via activation of both NMDA receptors and L-type-like Ca(2+) channels. It appears that sustained postischemic Ca(2+) elevation in the CA1 region is mediated via activation of L-type-like Ca(2+) channels, but not of NMDA receptors.     

Differential induction of LTP and LTD is not determined solely by instantaneous calcium concentration: an essential involvement of a temporal factor

Two opposite types of synaptic plasticity in the CA1 hippocampus, long-term potentiation (LTP) and long-term depression (LTD), require postsynaptic Ca2+ elevation. To explain these apparently contradictory phenomena, the current view assumes that a moderate postsynaptic increase in Ca2+ leads to LTD, whereas a large increase leads to LTP. No detailed study has so far been attempted to investigate whether the instantaneous Ca2+ elevation level differentially induces LTP or LTD. We therefore used low-frequency (1 Hz) stimulation of Schaffer collateral/commissural fibers in rat hippocampal slices, during a Mg2+-free period, as the conditioning stimulus to investigate this. This allowed low-frequency afferent stimulation to cause a postsynaptic Ca2+ influx because the voltage-dependent block of N-methyl-D-aspartate (NMDA) receptor-channels by Mg2+ was removed. When delivered during the Mg2+-free period, a single pulse, as well as 2-600 pulses, induced LTP that was occluded with tetanus-induced LTP. To decrease the Ca2+ influx, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors were completely blocked by the addition of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the conditioning medium, in which 1 Hz afferent stimuli (1-600 pulses) induced less LTP and never induced LTD. To further reduce the Ca2+ influx, NMDA receptors were partially blocked with D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5). A small number of 1 Hz stimuli, however, never induced LTD. Only when the conditioning stimuli exceeded 200 pulses was LTD induced. The present findings provide definitive evidence that protracted conditioning is a prerequisite for the induction of LTD. Thus, not only the amplitude but also the duration of postsynaptic Ca2+ elevation could be essential factors for differentially inducing LTP or LTD.     

Ascorbic acid is neuroprotective against global ischaemia in striatum but not hippocampus: histological and voltammetric data

Following reports that ascorbic acid (AA) blocks NMDA receptors, we examined its possible neuroprotective properties in vivo (gerbil bilateral carotid artery occlusion model: BCAO) and in vitro (ischaemia-induced dopamine (DA) release in brain slices). Five minutes of BCAO caused substantial cell loss of 90-95% and 40-50% in gerbil CA1 hippocampus and striatum, respectively, measured in haematoxylin and eosin-stained sections, 5 days post-insult. AA (500 mg kg(-1) day(-1) i.p. for 312 days, first dose 1 h before occlusion) significantly (P<0.05) reduced striatal cell loss (from 40 to 13%) while only reducing CA1 cell loss from 95 to 88%. A lower dose (250 mg kg(-1) day(-1) i.p. for 312 days) was ineffective in either region. AA (750 mg kg(-1) day(-1) i.p. for 312 days) caused significant striatal protection (cell loss reduced from 49 to 20%) if treatment was initiated 1 h before occlusion. Initiation of treatment immediately post occlusion did not cause significant protection. Neither treatment regime protected CA1 hippocampus. In separate experiments we examined the effect of AA on DA release, monitored by voltammetry, in an in vitro model of striatal ischaemia. Four DA release variables were measured: T(on)--time from initiation of ischaemia to the onset of DA release, T(pk)--the time from onset of DA release to maximum, deltaDA/deltat--the mean rate of DA release and [DA](max)-- the maximum extracellular DA concentration. Control values in drug-naive slices were: T(on)=193+/-8 s, T(pk) = 24 +/- 4 s, [DA](max) = 69 +/- 6 microM and deltaDA/deltat = 4.2 +/- 0.7 microM s(-1) (means+/-S.E.M., n=15). 212 h pretreatment with AA (0.4 to 10 mM) did not affect T(on) or [DA](max) but increased T(pk) and decreased deltaDA/deltat (P<0.05) with an EC50 of 1.66 mM. NMDA (100 microM) shortened T(on). N-ethylmaleimide (20 microM) had no effect on the response to AA but potentiated the action of NMDA on T(on). AA (2 or 10 mM) had no effect on the response to NMDA. We conclude that AA is neuroprotective against global ischaemia in the striatum and that some of this action may be due to attenuation of ischaemia-induced DA release. This action is mediated neither by blockade of the NMDA receptor nor modulation of its redox status.     

Glutamate stimulation of tyrosine hydroxylase is mediated by NMDA receptors in the rat striatum.

We have studied the action of glutamate on striatal tyrosine hydroxylase activity and determined which type of glutamate receptors are involved. Glutamate stimulated (EC50 = 4 +/- 2 microM) the activity of tyrosine hydroxylase in slices of rat neostriatum. The selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (10 microM) blocked the stimulation; however, both the non-NMDA receptor antagonist glutamate diethyl ester (10 microM) and the general excitatory amino acid antagonist kynurenate (10 microM) had no effect. NMDA was even more potent than glutamate in stimulating tyrosine hydroxylase activity. Quisqualate (100 microM) only slightly stimulated the enzyme, and kainate had practically no effect. Omission of Mg2+ from the incubation medium potentiated the glutamate stimulation. Neither tetrodotoxin nor atropine prevented the stimulation. These results suggest that glutamate stimulates striatal tyrosine hydroxylase activity via NMDA receptors. The lack of effect of tetrodotoxin and atropine suggests that glutamate acts on NMDA receptors located on the dopaminergic nigrostriatal terminal. The stimulation may involve the entry of Ca2+ into the terminal through the NMDA receptor ionophore, since a Ca(2+)-free medium or cadmium totally blocked the stimulation of the enzyme by glutamate.     

Prenatal exposure to morphine alters kinetic properties of NMDA receptor-mediated synaptic currents in the hippocampus of rat offspring

Whole-cell patch-clamp recordings of pharmacologically isolated N-methyl-D-asparate (NMDA) receptor-mediated evoked excitatory postsynaptic currents (EPSCs) were made, to study whether prenatal exposure to morphine affected functional properties of synaptic NMDA receptors in hippocampal slices of 2-week-old rat offspring from morphine-addicted mothers. The saturated amplitude of synaptic NMDA receptor-mediated EPSCs from morphine-treated offspring was about twofold larger than that from vehicle-control offspring. The apparent dissociation constant (Kd) values of NMDA receptors for Mg2+ at 0 mV were 7.5 +/- 1.4 and 7.9 +/- 1.3 mM in slices from vehicle-control and morphine-treated offspring, respectively. In addition, no distinguishable changes in the voltage-dependent nature and the reversal potential of NMDA receptors occurred in morphine-treated offspring, suggesting no alterations of Mg2+ blockade and ion selectivity to NMDA receptors. The 10-90% rise times of NMDA receptor-mediated EPSCs in morphine-treated offspring became longer than those in vehicle-control offspring. The decay of NMDA receptor-mediated EPSCs in both morphine-treated and vehicle-control offspring could be described by the sum of a fast and a slow exponential function. The slow, but not fast, decay times of synaptic NMDA receptor-mediated currents in morphine-treated offspring became slower than those in vehicle-control offspring. Collectively, these results suggest that prenatal exposure to morphine altered kinetic properties of synaptic NMDA receptors in hippocampal CA1 pyramidal neurons of rat offspring during early life. The extended duration of synaptic NMDA receptor-mediated currents presumably provided more Ca2+ entry through NMDA receptors in morphine-treated offspring, and its further prolongation by depolarization in such young offspring strengthened NMDA receptor-dependent functions. Thus, in light of pathophysiological implications within the central nervous system of morphine-treated offspring during early life, the present study may provide important insights and serve as a basis for therapeutic intervention in conditions under which NMDA receptors become abnormal.     

Age-dependent occurrence of synchronized population oscillation suggestive of a developing functional coupling between NMDA and ryanodine receptors in the neocortex

Synchronized population oscillation of delta to alpha range frequencies was synaptically induced in rat visual cortex neurons, depending on (1) the extent of N-methyl-D-aspartate (NMDA) receptor activation, (2) occurrence of calcium-induced calcium release (CICR), and (3) the age of animals. In adult slices bathed with 0.1 mM Mg(2+), as white matter stimulation continued, solitary synaptic potentials gradually became enlarged and overridden by small wavelets oscillating at alpha range frequencies. The calcium store depeletor thapsigargin prevented appearance of these wavelets. When Mg(2+) block of NMDA receptors was intensified with 0.5 or 2.0 mM Mg(2+), the oscillation failed to be induced, but became inducible by caffeine, which facilitates CICR. NMDA receptor blockade abolished induction of the oscillation even under caffeine application. In immature slices, the induction of oscillation was possible only with 0.1 mM Mg(2+) and bath-applied caffeine, but failed with 0.5 mM Mg(2+) or without caffeine. The oscillation induced in immature tissue was smaller in amplitude and frequency (delta-theta; range) than in adult tissue (alpha range). Immature tissue thus requires more restricted conditions for inducing the oscillation. We propose that NMDA and ryanodine receptors become coupled during maturation to enhance synaptically induced intracellular calcium releases, thereby facilitating induction of the oscillation at later stages.     

Different changes in spontaneous field potential oscillations precede epileptiform bursting in hippocampal slices perfused with penicillin or reduced magnesium

Power spectra were used to analyse spontaneous field potentials (SFPs) recorded in the CA3 distal apical dendritic region of guinea pig hippocampal slices perfused with either penicillin or reduced Mg2+. High concentrations of penicillin (2000 IU/ml) progressively converted the low amplitude, irregular oscillations observed in control medium to higher amplitude, low frequency, rhythmic oscillations at approximately 2-3 Hz just prior to the onset of spontaneous, synchronized bursting. Low concentrations (50-300 IU/ml) increased the power of frequencies below 10 Hz and suppressed higher frequencies in a dose-dependent fashion. Although Mg2(+)-free medium also increased the magnitude of the SFPs prior to the onset of synchronous bursting, the changes were smaller than with penicillin and the frequency distribution was completely different. Low concentrations of Mg2+ (0.0-0.5 mM) increased the power across all frequencies, however, the maximal effect was on frequencies between 5 and 25 Hz. The transition from normal to epileptiform activity may proceed through at least 2 distinct intermediate states. When recurrent inhibition is blocked (penicillin), synchronous synaptic activity precedes the onset of bursting, whereas non-specific increases in excitability and activation of NMDA receptors (reduced Mg2+) produce an asynchronous transition state.     

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)     

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.     

N-methyl-D-aspartate induces recurrent synchronized burst activity in immature hippocampal CA3 neurones in vitro

Slices of hippocampus prepared from rats aged 1-10 days have been used to examine the chemosensitivity of CA3 pyramidal neurones to N-methyl-D-aspartate (NMDA). Superfusion of NMDA excited all neurones tested at all ages including the first day postnatal. In the majority of neurones this excitation was associated with the induction of a period of burst firing which disappeared on removal of NMDA. These bursts took the form of paroxysmal depolarizing shifts (PDSs) with a large amplitude depolarization and a high frequency discharge of spikes. The amplitude but not the frequency of occurrence of the PDSs was influenced by changes in the membrane potential and they could be abolished by either a high divalent cation medium or tetrodotoxin. Their occurrence was synchronous with an extracellularly recorded discharge. The NMDA induced excitation and the induction of the PDSs was attenuated by selective NMDA receptor antagonists D-aminophosphonovalerate (10-50 microM) and D,L-aminophosphonoheptanoate (20-30 microM). The results indicate that chemosensitivity to NMDA develops prenatally and that activation of NMDA receptors can in immature CA3 pyramidals induce recurrent synchronized burst activity.