N-methyl-D-aspartate Receptor-mediated Excitatory Postsynaptic Potential Research Articles

Memory in aged mice is rescued by enhanced expression of the GluN2B subunit of the NMDA receptor

The GluN2B subunit of the N-methyl-d-aspartate (NMDA) receptor shows age-related declines in expression across the frontal cortex and hippocampus. This decline is strongly correlated to age-related memory declines. This study was designed to determine if increasing GluN2B subunit expression in the frontal lobe or hippocampus would improve memory in aged mice. Mice were injected bilaterally with either the GluN2B vector, containing cDNA specific for the GluN2B subunit and enhanced green fluorescent protein (eGFP); a control vector or vehicle. Spatial memory, cognitive flexibility, and associative memory were assessed using the Morris water maze. Aged mice, with increased GluN2B subunit expression, exhibited improved long-term spatial memory, comparable to young mice. However, memory was rescued on different days in the Morris water maze; early for hippocampal GluN2B subunit enrichment and later for the frontal lobe. A higher concentration of the GluN2B antagonist, Ro 25-6981, was required to impair long-term spatial memory in aged mice with enhanced GluN2B expression, as compared to aged controls, suggesting there was an increase in the number of GluN2B-containing NMDA receptors. In addition, hippocampal slices from aged mice with increased GluN2B subunit expression exhibited enhanced NMDA receptor-mediated excitatory post-synaptic potentials (EPSP). Treatment with Ro 25-6981 showed that a greater proportion of the NMDA receptor-mediated EPSP was due to the GluN2B subunit in these animals, as compared to aged controls. These results suggest that increasing the production of the GluN2B subunit in aged animals enhances memory and synaptic transmission. Therapies that enhance GluN2B subunit expression within the aged brain may be useful for ameliorating age-related memory declines.

Estradiol increases delayed, N-methyl- d-aspartate receptor-mediated excitation in the hippocampal CA1 region

Hippocampal functions, e.g. synaptic plasticity and hippocampal-dependent behavior, are influenced by the circulating levels of ovarian steroids in adult, female rats. The mechanisms underlying this estradiol-dependent modulation, however, are poorly understood. One possibility is that estradiol alters N-methyl- d-aspartate (NMDA)-receptor functioning in the hippocampus. Here, using the in vitro hippocampal slice preparation, we evaluate estradiol-dependent changes in the NMDA receptor- and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated components of excitatory postsynaptic potentials (EPSPs) evoked in CA1 by Schaffer collateral test stimulation. Using established experimental conditions [J Neurosci 17 (1997) 1848], we replicate the observation that estradiol pretreatment of ovariectomized rats increases a pharmacologically isolated NMDA receptor-mediated EPSP evoked by Schaffer collateral stimulation. However, using different conditions that optimize study of this evoked response, the estradiol-dependent increase in the monosynaptic NMDA receptor-mediated EPSP is eliminated. Low-intensity test stimulation of the Schaffer collaterals in this optimized medium reveals a novel, late NMDA receptor-mediated EPSP in CA1 from estradiol-pretreated rats. The mechanism(s) underlying this estradiol-dependent increase in a late, NMDA receptor-mediated EPSP is not known, but enhanced CA1–CA1 excitatory circuitry and glutamate spillover could contribute to this response. We conclude that estradiol pretreatment enhances NMDA receptor function in the female hippocampus by increasing not the monosynaptic, but rather a late NMDA receptor-mediated response. Variations in the magnitude of this late response may well contribute to ovarian steroid-dependent modulation of hippocampal synaptic plasticity.

Enhancement of N-methyl- D-aspartate receptor-mediated excitatory postsynaptic potentials in the neostriatum after methamphetamine sensitization. An in vitro slice study.

It has been suggested that behavioral methamphetamine sensitization involves changes in cortical excitatory synaptic inputs to neostriatal (Str) projection neurons. To test this, we performed blind whole-cell recording of medium spiny neurons in Str slice preparations. In Str neurons of naive rats, the amplitude of the subcortical white matter stimulation-induced N-methyl- D-aspartate receptor-mediated excitatory postsynaptic potentials (NMDA-EPSPs) was decreased upon hyperpolarization, owing to the voltage-dependent Mg(2+) blockade of NMDA receptor channels. In contrast, the amplitude of the NMDA-EPSPs in Str neurons of rats undergoing methamphetamine withdrawal (MW) did not show the Mg(2+) blockade and was nearly voltage independent over the membrane potential range of -70 to -110 mV. Application of the specific protein kinase C (PKC) activator, phorbol 12, 13- DL-acetate, blocked the voltage-dependent Mg(2+) blockade of NMDA receptor channels in Str neurons of naive rats. Application of the specific activator of cAMP-dependent protein kinase A (PKA), Sp-cAMPS-triethylamine salt, increased the amplitude of the NMDA receptor-mediated EPSPs at the rest but not at hyperpolarized potentials. Coapplication of the PKC and PKA activators yielded NMDA-EPSPs similar to those seen in Str neurons of MW rats. In Str slices of naive rats, tetanic subcortical white matter stimulation induced long-term depression of field potentials. In Str slices treated with the PKC and/or PKA, the same stimulation induced long-term potentiation of field potentials similar to those observed in slices obtained from MW rats. These results suggest that the enhancement of the NMDA receptor-mediated corticostriatal synaptic transmission plays an important role in behavioral methamphetamine sensitization. This enhancement is probably associated with phosphorylation of NMDA receptors mediated by the simultaneous activation of PKC and PKA.

17beta-estradiol enhances NMDA receptor-mediated EPSPs and long-term potentiation.

Gonadal steroid hormones influence CNS functioning through a variety of different mechanisms. To test the hypothesis that estrogen modulates synaptic plasticity in the hippocampus, in vitro hippocampal slices from 2-mo-old Sprague-Dawley male rats were used to determine the effect of 17beta-estradiol on both N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic potentials (EPSPs) through intracellular recordings and long-term potentiation (LTP) through extracellular recordings. Intracellular EPSPs and extracellular field EPSPs (fEPSPs) were recorded from CA1 pyramidal cells by stimulating Schaffer collateral fibers. In intracellular experiments, slices were perfused with medium containing bicuculline (5 microM) and low Mg2+ (0.1 mM) to enhance the NMDA receptor-mediated currents and 6, 7-dinitroquinoxaline-2,3-dione (DNQX) (10 microM) to block the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprianate (AMPA) receptor-mediated component. The effects of 17beta-estradiol on NMDA receptor-mediated activity were excitatory; concentrations >10 nM induced seizure activity, and lower concentrations (1 nM) markedly increased the amplitude of NMDA-mediated EPSPs (both the first and second responses increased during paired pulse stimulation by 180 and 197%, respectively). In extracellular experiments, slices perfused with 17beta-estradiol (100 pM) exhibited a pronounced, persisting, and significant enhancement of LTP of both the fEPSP slope (192%) and fEPSP amplitude (177%) compared with control slices (fEPSP slope = 155%; fEPSP amplitude = 156%) 30 min after high-frequency stimulation. These data demonstrate that estrogen enhances NMDA receptor-mediated currents and promotes an enhancement of LTP magnitude.

In rat hippocampal slices, NMDA receptor-mediated EPSPs are more sensitive to hypoxia than AMPA receptor-mediated EPSPs

In slices kept at 33°C, N-methyl- d-aspartate (NMDA) receptor- and (±)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated field excitatory post-synaptic potentials (EPSPs) were pharmacologically isolated in CA1. Both types of EPSPs were reversibly blocked by 3 min of hypoxia (95% N 2/5% CO 2); but NMDA receptor-mediated EPSPs were consistently blocked earlier and recovered later than AMPA receptor-mediated EPSPs, recorded in the same slice. This difference may be due to inactivation of NMDA receptors by hypoxia-induced acidity and/or rise in internal [Ca 2+].

Evidence that heterosynaptic depolarization underlies associativity of long-term potentiation in rat hippocampus.

1. Whole-cell patch-clamp recording has been used to study the effect of heterosynaptic depolarization on pure N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission in the CA1 region of rat hippocampal slices. 2. In neurones voltage clamped at -60 mV, paired-pulse stimulation of one set of Schaffer collateral-commissural fibres resulted in homosynaptic paired-pulse facilitation of the NMDA receptor-mediated excitatory postsynaptic current (EPSCN). In contrast, stimulation of one set of fibres prior to stimulation of a second set of fibres (i.e. heterosynaptic paired-pulse stimulation) did not result in any heterosynaptic interactions. 3. However, under current-clamp conditions, heterosynaptic paired-pulse stimulation resulted in heterosynaptic 'paired-pulse facilitation' of the NMDA receptor-mediated excitatory postsynaptic potential (EPSPN). 4. In neurones held at -50 or -40 mV, perfusion of nominally Mg(2+)-free medium converted the response to heterosynaptic paired-pulse stimulation from 'heterosynaptic facilitation' to 'heterosynaptic depression' of EPSPN. 5. When neurones were held at potentials of between -30 and +40 mV then heterosynaptic paired-pulse stimulation, in normal Mg(2+)-containing medium, resulted in 'paired-pulse depression' of EPSPN. Under voltage-clamp conditions (tested at +40 mV) no heterosynaptic interactions were seen. 6. The time course of 'heterosynaptic facilitation' at -60 mV and of 'heterosynaptic depression' at +40 mV of EPSPN was similar to the time course of EPSCN. 7. We conclude, firstly, that the voltage clamp is able to prevent any voltage breakthrough associated with the synaptic activation of NMDA receptors from influencing neighbouring synapses. Secondly, when the neurone is not voltage clamped these same synapses are strongly influenced by the spreading depolarization generated by the synaptic activation of their neighbours. The time course and direction of this influence are compatible with the hypothesis that spreading synaptic depolarization, leading to a reduction of the voltage-dependent Mg2+ block of synaptic NMDA receptor channels, underlies the property of associativity.

229 Selective formation of silent synapse on immature postsynaptic cells in coculture of hetero-aged neurons

The effects of 1-methyl-4-phenylpyridinium ion (MPP+) on N-methyl-d-aspartate (NMDA) receptor-channel complex were studied in rat hippocampal neurons using intracellular- and whole-cell voltage clamp-recording techniques. Intracellular recordings were made from CA1 pyramidal cells of rat hippocampal slices in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM) and picrotoxin (PTX; 50 μM) which block non-NMDA and GABAA receptors, respectively. Superfusion of MPP+ reversibly decreases the pharmacologically isolated NMDA receptor-mediated excitatory postsynaptic potential (EPSPNMDA) in a concentration-dependent manner. In other experiments, we observed that MPP+ attenuated NMDA-evoked whole-cell currents in a voltage- and use-dependent manner and was not dependent on the extracellular glycine or spermine concentration on neurons freshly dissociated from rat hippocampi CA1 region. These results suggest that MPP+, applied at micromolar concentrations, is a non-competitive NMDA receptor antagonist in rat hippocampal neurons.

The role of glycine in anoxia/aglycaemia-induced potentiation of N-methyl- d-aspartate receptor-mediated postsynaptic potentials in the rat hippocampus

During brain ischaemia there is a sustained increase in extracellular glycine levels, and the potential role of these changes in modulating N-methyl- d-aspartate (NMDA) receptor-mediated activity following an anoxic/aglycaemic insult was studied in the rat hippocampal slice. Addition of large saturating concentrations of glycine (100 μM and 1 mM) to the superfusate resulted in increased extracellularly recorded NMDA receptor-mediated components of excitatory postsynaptic potentials (EPSPs) recorded in area CA1. The effects of added glycine were strychnine-insensitive and blocked by a competitive NMDA antagonist. Anoxic/aglycaemic insults to the tissue caused persistent increases in NMDA receptor-mediated EPSPs, but the magnitude of the observev upregulation was uaffected by the presence of added saturating concentrations of glycine. The data suggests that alterations in glycine levels after oxygen deprivation are not responsible for the long term modulation of NMDA receptor activity.

On the mechanism of long-term potentiation induced by (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) in rat hippocampal slices

We have reported previously that transient application of a specific metabotropic glutamate receptor (mGluR) agonist (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD) can induce a slow-onset form of long-term potentiation (LTP) of synaptic transmission in the CA1 region of rat hippocampal slices [Bortolotto Z. A. and Collingridge G. L. (1993) Neuropharmacology 32, 1–9]. Here we have investigated further the mechanisms involved in the induction and expression of ACPD-induced LTP. Unless otherwise stated, field excitatory postsynaptic potentials (EPSPs) were recorded in stratum radiatum in response to low frequency (0.033 Hz stimulation) of the Schaffer collateral-commissural pathway and 10 μM ACPD was added for 20 min to the perfusate. ACPD-induced LTP was still observed following blockade of GABA A receptor-mediated synaptic inhibition using picrotoxin (50 μM) and was not the result of a change in the presynaptic fibre volley. Intracellular recording from area CA1 revealed an increase in the size of the EPSP but no associated change in membrane potential or input resistance. However, ACPD-induced potentiation was never seen when intracellular electrodes contained the Ca 2+-chelating agent 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA; 0.5 M). In area CA3, ACPD elicited a slow-onset LTP of the intracellularly recorded EPSP, evoked by stimulation of associational fibres. In contrast to area CA1, 10 μM ACPD depolarized CA3 neurones. Unlike certain other forms of tetanus- and chemically-induced potentiation, ACPD-induced LTP was not affected by the L-type Ca 2+ channel antagonist nimodipine (50 μM). It was, however, prevented by delivering low frequency stimulation (900 shocks at 1 Hz) immediately following termination of the application of ACPD; an effect which was inhibited by the specific N-methyl- d-aspartate (NMDA) receptor antagonist d-2-amino-5-phosphonopentanoate (AP5; 50 μM). ACPD failed to induce LTP of pharmacologically-isolated NMDA receptor-mediated EPSPs. The induction of ACPD-induced LTP was blocked by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), in a reversible manner. In slices in which area CA3 had been removed ACPD failed to induce LTP when applied alone or together with AMPA. However, a slow-onset form of LTP was induced, in slices lacking area CA3, when a tetanus (100 Hz, 1 sec) was delivered in the presence of ACPD and 50 μM AP5 (the latter applied to prevent conventional tetanus-induced LTP). ACPD-induced LTP was associated with a parallel increase in the sensitivity of CA1 neurones to AMPA. Considered together, these data suggest that ACPD-induced LTP is due to a direct increase in the AMPA receptor-mediated synaptic conductance and involves postsynaptic induction and expression mechanisms.

Voltage- and use-dependent block by 1-methyl-4-phenylpyridinium ion (MPP +) of N-methyl- d-aspartate-activated currents in rat hippocampal neurons

The effects of 1-methyl-4-phenylpyridinium ion (MPP +) on N-methyl- d-aspartate (NMDA) receptor-channel complex were studied in rat hippocampal neurons using intracellular- and whole-cell voltage clamp-recording techniques. Intracellular recordings were made from CA1 pyramidal cells of rat hippocampal slices in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM) and picrotoxin (PTX; 50 μM) which block non-NMDA and GABA A receptors, respectively. Superfusion of MPP + reversibly decreases the pharmacologically isolated NMDA receptor-mediated excitatory postsynaptic potential (EPSPNMDA) in a concentration-dependent manner. In other experiments, we observed that MPP + attenuated NMDA-evoked whole-cell currents in a voltage- and use-dependent manner and was not dependent on the extracellular glycine or spermine concentration on neurons freshly dissociated from rat hippocampi CA1 region. These results suggest that MPP +, applied at micromolar concentrations, is a non-competitive NMDA receptor antagonist in rat hippocampal neurons.

A selective LTP of NMDA receptor-mediated currents induced by anoxia in CA1 hippocampal neurons

1. The possibility of long-lasting modifications of glutamatergic responses after anoxic-aglycemic (AA) episodes was investigated in CA1 hippocampal neurons of adult slices. Bicuculline (10 microM) was continuously bath applied to block GABAA receptor-mediated currents. AA episodes were induced by brief (1.30-3 min) perfusions with a glucose free artificial-cerebro-spinal-fluid (ACSF) saturated with 95% N2-5% CO2. 2. In presence of (0.6 mM) Mg2+ and a low concentration of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 1 microM), the Schaffer collateral field EPSPs consisted of an early AMPA receptor-mediated component and a late N-methyl-D-aspartate (NMDA) receptor-mediated component. The former was blocked by (10 microM) CNQX and the latter by (50) microM D-2-amino-5-phosphonovalerate (D-APV). The AA episode induced a selective long-term potentiation (LTP) of the NMDA receptor-mediated component [+70 +/- 13% (mean +/- SE), P < or = 0.008, n = 9] without affecting significantly the AMPA receptor-mediated component (+2 +/- 4, P < or = 0.86 n = 9). This selective LTP is due to an enhanced efficacy of synaptic transmission and will be referred to as anoxic LTP. 3. In slices perfused with an ACSF containing a physiological concentration of (1.3 mM) Mg2+ and no CNQX, the intracellularly recorded excitatory postsynaptic potential (EPSP) was mixed (AMPA/NMDA) at -65 mV and exclusively mediated by AMPA receptors at -100 mV. At -65 mV, the AA episode induced a persistent potentiation of the EPSP (peak amplitude potentiated by 43 +/- 6%, P < or = 0.008, n = 9, 1 h after return to control ACSF). This potentiated component of the EPSP was fully sensitive to (50 microM) D-APV. The CNQX-sensitive AMPA receptor-mediated component was not affected by the AA episode (-5.7 +/- 6%, P < or = 0.123, n = 9). Furthermore, at -100 mV a large APV-sensitive component appeared after the AA episode (+58 +/- 18% of the peak amplitude, P < or = 0.018, n = 9). Therefore, the AA episode induced a selective LTP of the NMDA receptor-mediated component of the EPSP. 4. A robust LTP (+50.0 +/- 7.5%, P < or = 0.008, n = 12) of the NMDA receptor-mediated intracellular EPSP was also observed when AMPA receptors were fully and continuously blocked by (15 microM) CNQX.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine inhibits the NMDA receptor-mediated excitatory postsynaptic potential in the hippocampus

The effect of the stable adenosine analogue 2-chloroadenosine (CADO) on the component of the field excitatory postsynaptic potential (fepsp) mediated by the N-methyl- d-aspartate (NMDA) type of glutamate receptor was studied in the hippocampal CA 1 area of the rat. CADO inhibited the NMDA receptor-mediated component of the fepsp (EC 50 = 0.10±0.02 μM), more efficiently than it inhibited the fepsp (EC 50 = 0.40±0.08 μM). The results suggest that adenosine may modulate phenomena associated with the NMDA receptor, such as synaptic plasticity and excitotoxicity.

Arylamine toxins from funnel-web spider (Agelenopsis aperta) venom antagonize N-methyl-D-aspartate receptor function in mammalian brain.

The venom of the North American funnel-web spider Agelenopsis aperta contains a variety of arylamine toxins (the alpha-agatoxins) that paralyze insects by blocking glutamatergic neuromuscular transmission. We have tested six synthetic alpha-agatoxins for their ability to antagonize glutamate receptor function in mammalian brain. These compounds produce, at submicromolar concentrations, noncompetitive inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated elevations in the concentration of cytosolic free calcium in cultured rat cerebellar granule neurons. In contrast, the alpha-agatoxins are relatively weak antagonists of elevations in the cytosolic free calcium concentration induced by non-NMDA receptor agonists. The alpha-agatoxins also produce reversible suppression of the NMDA receptor-mediated excitatory postsynaptic potential in rat hippocampal slices at concentrations that have little effect on the non-NMDA receptor-mediated population spike. We conclude that the alpha-agatoxins are selective and reversible noncompetitive antagonists at NMDA receptors in mammalian brain.

Long-term Potentiation of NMDA Receptor-mediated EPSP in Guinea-pig Hippocampal Slices.

Hippocampal slices from guinea-pigs were used to examine the long-term potentiation (LTP) of the N-methyl-d-aspartate (NMDA)-mediated excitatory postsynaptic potential (EPSP). Intracellular recordings were performed from CA1 pyramidal neurons in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 - 10 microM) and picrotoxin (50 microM). In these experimental conditions test stimuli applied at low frequency (0.1 Hz) to the Schaffer collateral - commissural pathway evoked a prolonged EPSP (150 - 200 ms). To obtain this CNQX-resistant EPSP, stimulus intensities had to be raised above the level required to evoke an EPSP of comparable amplitude in physiological solution. Tetanic stimulation (two trains of 100 Hz, 1 s every 20 s) led to a potentiation of the CNQX-resistant EPSP, and this potentiated response was abolished with d-(-)-2-amino-5-phosphonovaleric acid (50 microM). The potentiation of the NMDA receptor-mediated EPSP was more pronounced for strong than for weak test stimuli, and was suppressed when test EPSPs were evoked during membrane hyperpolarization. These results suggest that NMDA receptor-mediated responses can undergo LTP, and hence can contribute to the maintenance of LTP.

Developmental changes in the susceptibility to long-term potentiation of neurones in rat visual cortex slices

We investigated with intracellular recordings from rat visual cortex slices whether the susceptibility to undergo long-term potentiation (LTP) is age-dependent and whether it is correlated with the expression of synaptic responses mediated by N- methyl- d-aspartate (NMDA) receptors. Test and tetanic stimuli were applied to the white matter and post-tetanic modifications of the amplitude of postsynaptic potentials (PSPs) were assessed in regular spiking cells of supragranular layers. At 2 weeks of age, the amplitudes of early (8–10 ms post-stimulus) and late (20 ms post-stimulus) PSP-components increased after tetanic stimulation to 137.1 ± 13.4% and 141.3 ± 12.1% of the pretetanic controls, respectively. At 3 weeks, potentiation of both PSP-components was less pronounced but still significant, the late component being on average more potentiated than the early one. At 4 weeks, PSPs were no longer potentiated. Bath application of 25 μM dl-2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, blocked LTP induction both at 2 and at 3 weeks. We also studied developmental changes of two synaptic responses known to influence the susceptibility of cortical neurones to LTP, the NMDA receptor-mediated excitatory PSP (EPSP) and the initial inhibitory PSP (iIPSP). The amplitude of the APV-sensitive EPSP decreased with age and reached adult values in 4-week-old animals. The iIPSPs were pronounced already at 2 weeks and showed no marked change during further development. The results suggest a close correlation between the susceptibility to undergo LTP and the extent to which NMDA receptor-gated conductances contribute to the synaptic response. We propose that the decline of experience-dependent malleability that is observed in vivo during postnatal development is causally related to the age-dependent reduction of NMDA receptor-mediated responses.

Postnatal maturation of the GABAergic system in rat neocortex.

1. The postnatal maturation of intracortical inhibitory circuitry and the development of responses to applied gamma-aminobutyric acid (GABA) and baclofen were studied in pyramidal and nonpyramidal neurons from layers II and III of the rat primary somatosensory and primary visual cortex, in vitro. 2. Depolarizing spontaneous inhibitory postsynaptic potentials (IPSPs) could be recorded in approximately 70% of the young (postnatal day 4-10; P4-10), juvenile (P11-16), and adult cells (P28-41), respectively, when they were loaded with nitrate. At all ages these spontaneous events could be blocked by application of the GABAA receptor antagonist bicuculline methiodide (BMI), indicating that they were mediated by activation of GABAA receptors. 3. In 122 of the 130 adult cells tested, standardized electrical stimulation of the white matter or layer VI evoked a brief excitatory postsynaptic potential (EPSP), followed by both a fast (f-) and a long-latency (l-)IPSP. Similar stimuli evoked a biphasic IPSP in only 51 of the 98 juvenile and in only 1 of the 56 young neurons studied. The mean peak conductance of the f-IPSP and the l-IPSP increased significantly from 50.2 and 7.5 nS, respectively, in juvenile cells to 84.2 and 18.0 nS, respectively, in adult neurons. 4. Application of the N-methyl-D-aspartate (NMDA) receptor antagonist D-amino-phosphonovaleric acid (D-APV) to juvenile cells induced a significant negative shift in the reversal potential of both the f-IPSP and l-IPSP. This effect was accompanied by a reduction in the peak conductance during these events by 31 and 48%, respectively, indicating that a prominent long-lasting NMDA receptor-mediated EPSP occurs concurrent with the early and late IPSP in immature neurons. In adult neurons, D-APV had no significant effect on the reversal potential of the f- and l-IPSP, although the peak conductance decreased by 20 and 5%, respectively, suggesting that there was a smaller concurrent activation of NMDA receptors in this age group. 5. The functional maturation of GABAA and GABAB receptors was studied using focal applications of GABA to the soma and the apical dendrite. Somatic GABA applications to adult neurons held at depolarized membrane potentials evoked a triphasic response, consisting of 1) a GABAA-mediated hyperpolarizing fast component (GABAhf; reversal potential, -76 mV), 2) a GABAA-mediated depolarizing phase (GABAd; -54 mV), and 3) a hyperpolarizing late response (GABAhl; -80 mV). The GABAd response could be demonstrated at all ages in almost every neuron.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurotransmitter receptors mediating excitatory input to cells in the cat lateral geniculate nucleus. I. Lagged cells

1. Synaptic mechanisms that might explain the functional properties of the recently discovered class of lagged cells in the dorsal lateral geniculate nucleus (LGN) were analyzed with electrophysiological and pharmacologic techniques. To study the type of excitatory amino acid (EAA) receptor that mediates visual responses of lagged cells, we recorded the response of single cells to a stationary flashing light spot before, during, and after microiontophoretic application of antagonists and agonists to EAA receptors. 2. The visual response of the lagged cells could be almost completely blocked by an antagonist to the N-methyl-D-aspartate (NMDA) receptor. The degree of suppression was dose dependent, and the average maximum degree of suppression for all the cells was 94%. NMDA enhanced the response, and this enhancement was antagonized by NMDA antagonists. A quisqualate/kainate receptor antagonist had no significant effect on the lagged cells. 3. These findings indicate that the visual response in lagged cells is dependent upon activation of NMDA receptors, which may directly result from activation of retinal inputs. 4. No pharmacologic difference was seen between lagged X- and Y-cells, or between lagged ON- and OFF-center cells. 5. gamma-Aminobutyric acid-A (GABA-A) receptor antagonists were used to study whether the characteristic lag of the visual response and the suppression of the initial transient response component of the lagged cells are dependent on geniculate inhibition. Beside enhancement of the visual response, the GABA antagonists strongly reduced the lag of the visual response, and an initial transient response component occurred instead of the initial suppression. The lag remained slightly longer than for nonlagged cells, and the peak firing rate of the transient was below values typical for nonlagged cells, indicating that the lagged cell properties are dependent on other factors beside GABA-A receptor-mediated inhibition. 6. The enhanced visual response during iontophoresis of GABA antagonists could be completely blocked by simultaneous iontophoresis of an NMDA-receptor antagonist. This gives further support to the hypothesis that the retinal input to these cells is mediated by NMDA receptors. 7. The NMDA-receptor/ionophore complex mediates excitatory postsynaptic potentials (EPSPs) characterized by slow rise and decay times and long duration. The ionophore is characterized by a voltage-dependent blockade that makes these receptors particularly sensitive to inhibitory input. The temporal interplay between the slow NMDA receptor-mediated EPSPs and the fast GABA receptor-mediated inhibitory postsynaptic potentials (IPSPs) may explain the characteristic response properties of the lagged cells.

The Involvement of N-Methyl-D-Aspartate Receptors in Induction and Maintenance of Long-Term Potentiation in Rat Visual Cortex.

Pyramidal neurons from layers II and III of rat visual cortex slices were studied with intracellular recordings. The involvement of N-methyl-D-aspartate (NMDA) receptors was investigated: (1) in the synaptic response to white matter stimulation; (2) in the induction of long-term potentiation (LTP); and (3) in the maintenance of LTP. Bath application of 25 microM of 2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, caused a slight (< 10%) reduction of the amplitude of the synaptic response elicited by white matter stimulation. The APV-sensitive excitatory postsynaptic potential (EPSP) had a longer peak latency and duration than the APV-resistant EPSP. Bath application of 10 microM of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, revealed a CNQX-resistant EPSP in response to white matter stimulation which was APV-sensitive. The time course of the CNQX-resistant EPSP was similar to that of the APV-sensitive EPSP and its onset latency was similar to that of the synaptic response in normal medium. Bath application of the GABA-A antagonist bicuculline (0.1 to 0.5 microM) led to a progressive enhancement of the amplitude of the APV-sensitive EPSP. At bicuculline concentrations above 0.3 microM the amplitude of this EPSP increased with membrane depolarization as was the case for the CNQX-resistant EPSP implying that the NMDA receptors were located on the recorded neuron. The susceptibility of the cells to undergo LTP was tested at various concentrations of bicuculline. The effectiveness of bicuculline treatment was quantified by comparing the amplitudes of the synaptic response to just subthreshold stimuli at two post-stimulus delays: (i) at 22 ms, which corresponds to the time to peak of both the initial inhibitory postsynaptic potential and the APV-sensitive EPSP; and (ii) at 8 - 11 ms post-stimulus, which corresponds to the peak of the postsynaptic potential (PSP) in normal medium. Bath application of APV, 20 min after the conditioning tetanus, allowed the authors to measure the amplitude of the APV-sensitive EPSP in the potentiated response. In normal medium, the ratio of the late over the early PSP amplitude was 33.6 +/- 4.1% and tetanic stimulation failed to induce LTP. The conditions remained the same at bicuculline concentrations of 0.1 to 0.2 microM. At higher concentrations of bicuculline the amplitude ratio of late versus early PSP increased and tetanic stimulation induced LTP. In cells, in which bicuculline had caused small ratio increases, only the APV-sensitive EPSP underwent LTP. In cells in which bicuculline had caused large ratio changes, both the APV-resistant and the APV-sensitive EPSP showed LTP. Together with the previous finding that blockade of NMDA receptors prevents LTP (Artola and Singer, 1987) these results suggest that there is a threshold for LTP induction, which is only reached if NMDA receptor-gated channels are sufficiently activated. The data indicate further that the NMDA receptor-mediated EPSP is itself susceptible to LTP whereby its LTP threshold is lower than that of the APV-resistant EPSP. Given the different LTP thresholds of the APV-resistant and APV-sensitive EPSPs, the possibility is raised that their potentiation depends on different mechanisms.

Glycine enhances NMDA-receptor mediated synaptic potentials in neocortical slices

One class of excitatory amino-acid receptors, the N-methyl-D-aspartate (NMDA) receptors, mediates transmission at a small, but important, group of synapses in the neocortex. These receptors are implicated in neuronal plasticity during development in young mammals and in memory acquisition in adults. Recently, responses of isolated membrane patches to NMDA were shown to be greatly enhanced by glycine. This, together with the demonstration that the strychnine-insensitive glycine-binding site is distinct from, but linked to, the NMDA receptor has excited intense interest in glycine as a synaptic modulator. Before proposing a physiological function, however, it is important to determine whether glycine could enhance synaptic responses to NMDA receptor activation in intact, adult tissue. An earlier study failed to demonstrate enhancement of NMDA responses when glycine was applied and it was proposed that in intact tissue the high-affinity glycine site was already saturated by endogenous glycine. It remained possible that glycine concentrations can be maintained at low levels close to synaptic receptors. We have examined responses of neurons in slices of adult neocortex to focal applications of excitatory amino acids and glycine and report enhancement by glycine of NMDA receptor-mediated excitatory postsynaptic potentials.