Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors

A series of ω-phosphono-α-car☐ylic acids were tested as antagonists of excitatory amino acid depolarizations and long-term potentiation (LTP) in region CA1 of rat hippocampal slices. The 5- and 7-phosphono compounds (±AP5and±AP7) blocked N-methyl-D-aspartate (NMDA) depolarizations and prevented the induction of LTP of the synaptic field potential and population spike components of the Schaffer collateral response.±AP5and±AP7 did not reduce kainate or quisqualate depolarizations and did not affect unpoten synaptic response amplitude.±AP5, ±AP6and±AP8 did not block amino acid excitant responses or LTP.These results demonstrate that NMDA receptors present in hippocampal region CA1 are not necessary for normal synaptic transmission, but are involved in the initiation of long-term synaptic plasticity.     

Presynaptic kainate and N-methyl-d-aspartate receptors regulate excitatory amino acid release in the olfactory cortex

The potassium-evoked release of endogenous aspartate, glutamate and GABA from olfactory cortex slices has been monitored. Release of aspartate alone is significantly reduced by N-methyl-d-aspartate (NMDA) whilst kainate significantly increases release of both aspartate and glutamate. These effects, which are blocked by appropriate receptor antagonists, suggest that presynaptic NMDA and kainate receptors regulate excitatory amino acid release in the olfactory cortex.     

N-methyl-D-aspartate receptors coexist with kainate and quisqualate receptors on single isolated catfish horizontal cells

Horizontal cells enzymatically isolated from catfish retina were exposed to the putative neurotransmitters aspartate (Asp) or N-methyl-D-aspartate (NMDA). Under voltage clamp conditions, inward currents were recorded when the holding potential was more negative than zero and outward currents were recorded when the membrane potential was more positive than zero. The current voltage curve was highly non-linear in the range of membrane potential between −30 and −100 mV. This non-linearity was largely removed in zero magnesium solution. 2-Amino-phosphonovaleric acid selectively blocked Asp and NMDA responses. These response characteristics are consistent with the presence of NMDA receptors in these cells.     

An intracellular study of the interactions of N-methyl-dl-aspartate with ketamine in the mouse hippocampal slice

Intracellular recordings were made from dentate and CA1 pyramidal cells of the mouse hippocampal slice preparation. N-methyl-dl-aspartate (NMDLA), quisqualate and kainate and the anaesthetic agent, ketamine, were applied by microelectrophoresis. Excitation by NMDLA but not by the other amino acids, was associated with increased outward rectification. Ketamine had no effect on the resting potential or current/voltage relation of the cells, but selectively antagonised the responses to NMDLA. Action potentials evoked by NMDLA were characteristically broader than those evoked by the other amino acids or by the passage of depolarising current through the electrode.     

Potassium-induced long-term potentiation in area CA1 of the hippocampus involves phospholipase activation

Previous studies have shown that potassium-induced long-term potentiation (LTP) of the Schaffer collateral/commissural synapses in area CA1 of the hippocampus shares common properties with tetanus-induced LTP. In the present investigation, we performed electrophysiological and binding experiments on CA1 hippocampal slices to evaluate the location and nature of the changes underlying potassium-induced LTP. Paired-pulse facilitation, which represents an index of transmitter release, was markedly reduced by potassium-induced LTP. In addition, KC1-induced LTP was associated with an increase in ³H-AMPA ([³H]-amino-3-hydroxy-5-methylisoxazole-4-propionate) binding to CA1 synaptic membranes when measured 40 min after high-potassium exposure; however, no changes were detected in binding of an antagonist ([³H]-6-cyano-7-nitroquinoxaline-2,3-dione; ³H-CNQX) to AMPA receptors in slices expressing KC1-induced LTP. Administration of the phospholipase A₂ (PLA₂) inhibitor bromophenacyl bromide (BPB) prior to potassium application prevented LTP formation as well as the changes in paired-pulse facilitation and ³H-AMPA binding that characterized this type of potentiation. Taken together, these data indicate that potassium-in-duced LTP may be related to modifications in both pre-and postsynaptic properties and confirm the hypothesis that PLA₂ activation is an important mechanism in long-term changes of synaptic operation. © 1994 Wiley-Liss, Inc.     

Synaptic correlates of associative potentiation/depression: an ultrastructural study in the hippocampus

Brief high-frequency trains delivered to the monosynaptic entorhinal cortical input to the dentate gyrus result in both increases and decreases of synaptic strength as a function of whether a particular afferent is active during conditioning (associative potentiation/depression^{15, 30}). The present report concerns the effect such brief, high-frequency conditioning trains upon the asymmetric synapses of the rat dentate gyrus molecular layer. Only those animals whose responses increased at least 50% following conditioning stimulation were included in the study. Additional animals were used for one-dimensional current source density analyses to localize the activated synaptic region. Double blind scoring procedures were used to classify and quantify electron micrographic data. Asymmetric synapses were scored as a function of their position in the molecular layer, spine head size and shape, and postsynaptic density length. All data were treated as inherently matched comparisons between the conditioned and control sides of each animal. The number of large, concave spine synapses with large postsynaptic densities significantly increases in the central zone of synaptic activation. Bordering this zone are regions with increases in synaptic number following conditioning, primarily due to an increased number of small spine synapses. The increased number of large, concave spine synapses in the central zone is postulated to mediate associative potentiation. The many small spine heads just adjacent to the zone of strongest synaptic activation may reflect synaptic depression evoked at synapses inactive during conditioning.     

Strontium supports synaptic transmission and long-lasting potentiation in the hippocampus

(1) Synaptic transmission was studied in isolated transverse hippocampal slices from guinea pigs. Extracellular evoked potentials were recorded in the region CA1. (2) Changing the normal perfusion solution (containing 2 mM Ca2+) to calcium-free Ringer abolished synaptic transmission which was again restored by adding strontium. A synaptic efficacy of 25--50% ofn normal was obtained for 10 mM Sr2+. (3) Two different synaptic inputs to CA1 pyramidal cells were tested with respect to their ability to produce long-lasting synaptic potentiation after tetanization in strontium Ringer. Following a brief tetanus the field EPSP and, especially, the population spike were greatly enhanced. (4) The potentiation so produced was similar to the long-lasting potentiation seen in the normal slice, because it (i) had a very long duration (hours), (ii) was specific for the tetanized pathway, (iii) showed potentiation of both 'volley-EPSP' and 'EPSP-spike' relations, and (iv) was accompanied by short-lasting (less than 5 min) generalized depression.     

Chemically-induced long-term potentiation in rat motor cortex involves activation of extracellular signal-regulated kinase cascade

The involvement of the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade in long-lasting potentiation of synaptic transmission, induced by tetraethylammonium (TEA) or by elevated extracellular calcium concentration, was investigated in layer V horizontal connections within motor cortex in rat brain slices. Brief application of TEA (25 mM) resulted in a long-lasting potentiation of field potentials by 54+/-12%. A transient exposure of slices to elevated extracellular calcium (5 mM) induced long-lasting potentiation of responses reaching 30+/-8%. The induction of both forms of potentiation was prevented by the exposure of slices to inhibitors of the upstream activator of ERK 1/2, MEK (ERK kinase), U0126 (20 microM) and PD 98059 (50 microM). PhosphoERK2 immunoreactivity was transiently increased above baseline levels 15 min after termination of the exposure of slices to either TEA or elevated calcium concentration. Both forms of potentiation were partially occluded by Sp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Sp-cAMPS; 100 microM), an activator of cAMP-dependent protein kinase (PKA), and they were blocked after preincubation with Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS; 100 microM), a specific inhibitor of PKA activation by cAMP. It has previously been shown that TEA-induced potentiation represents a N-methyl-d-aspartate (NMDA) receptor-independent form of persistent synaptic enhancement, and, on the contrary, calcium-induced potentiation depends on NMDA receptors. Thus, the activation of PKA and the ERK1/2 cascade are required for two forms of chemically induced long-lasting increases of synaptic efficacy in slices of rat motor cortex.     

Long-term enhancement of CA1 synaptic transmission is due to increased quantal size, not quantal content.

Quantal components of Schaffer collateral synaptic transmission recorded intracellularly from CA1 pyramidal cells were examined using 2 methods: simultaneous recordings of CA3-CA1 cell-pairs, and minimal electrical stimulation in stratum radiatum. Quantal parameters estimated by the method of failures and by a computer algorithm that optimized parameter estimates using deconvolution of background noise were highly correlated. EPSP-amplitude histograms of CA3-CA1 cell pairs (N = 10) and minimal electrical stimulation (N = 33) could be adequately described either by Poisson or binomial statistics, or by both, and exhibited similar estimates of unit quantal size (q) and mean quantal content (m). Paired-pulse stimulation with 50 msec between stimuli resulted in an expected facilitation in the EPSP amplitude and increase in m during the second response, as estimated by noise deconvolution, by the decrease in apparent failures, and by a decrease in the coefficient of variation of the EPSP. Tetanization of the Schaffer collaterals that induced long-term enhancement (LTE/LTP) of the population response was associated with an average increase in q for minimal-stimulation responses, with no significant change in any estimate of m. Taken together, these data indicate that, under the present experimental conditions, LTE is expressed as an increase in quantal size, rather than an increase in the number of quanta released per presynaptic impulse. Although this is not definitive evidence for a postsynaptic mechanism, these findings do further restrict the classes of possible presynaptic mechanisms that may be proposed to account for LTE expression.     

Receptor types mediating the excitatory actions of exogenousl-aspartate andl-glutamate in rat olfactory cortex

Changes in potential between the pial and cut surfaces of rat olfactory cortex slices evoked by N-methyl-d-aspartate (NMDA), quisqualate, kainate,l-glutamate andl-aspartate and also by γ-aminobutyric acid (GABA) have been monitored using extracellular electrodes. All agonists produced a pial-negative potential response when superfused onto the pial surface, GABA,l-aspartate andl-glutamate being less potent than the others. Repeated applications of NMDA, but not of the other agonists, led to a progressive reduction in response to approximately 30% of the initial depolarization. The responses to NMDA (100 μM) were selectively abolished by(±)2-amino-5-phosphonopentanoic acid (APP; 100 μM) while depolarizations evoked byl-glutamate andl-aspartate (both at 10 mM) were only antagonized by21 ± 2 (n = 12) and36 ± 3 (n = 12) percent respectively (means ± S.E.M.). γ-d-Glutamylglycine (γ-DGG; 1 mM) and(±)cis-2,3-piperidine dicarboxylate (cis-PDA; 2 and 5 mM), in addition to antagonizing responses to NMDA, also partially blocked quisqualate- and kainate-evoked depolarizations. When a mixture of APP (100 μM), γ-DGG (1 mM) and cis-PDA (5 mM) was applied to preparations, although NMDA receptors were completely blocked and responses to both quisqualate and kainate antagonized by approximately 80%,l-glutamate andl-aspartate evoked depolarizations were only reduced by51 ± 7 (n = 4) and 49 ± 4 (n = 4) percent respectively (means ± S.E.M.). The results are discussed in terms of the contributions made by NMDA, quisqualate and kainate receptors to the composite responses evoked byl-aspartate andl-glutamate.     

N-methyl-D-aspartate receptor-mediated signaling in the supraoptic nucleus involves activation of a nitric oxide-dependent pathway

N-Methyl-D-aspartate (NMDA) microinjection (1 mM, 0.2 μl) into the hypothalamic supraoptic nucleus (SON) stimulated heart rate in urethane-anaesthetized rats. This effect was inhibited by coinjection of a competitive blocker of NMDA receptors, CPP (20 nmol) or by pretreatment with a sympathetic ganglionic blocker, chlorisondamine chloride (5 mg/kg i.p.), but not by prior hypophysectomy. Furthermore, the cardioexcitatory effect of intra-SON NMDA was inhibited by prior intra-SON injection of a competitive blocker of nitric oxide (NO) synthesis, N^G-nitro-L-arginine methyl ester (40 nmol) or a blocker of the soluble guanylate cyclase, Methylene blue (20 nmol), and was mimicked by intra-SON injection of a calcium ionophore, A23187 (10 nmol), which stimulates NO production by raising intracellular free calcium levels. Finally, intra-SON microinjection of a membrane-permeating cGMP analog, 8-bromo-cGMP (20 nmol) stimulated heart rate in urethane-anaesthetized rats. The results point to a functional link between a sympathetically mediated cardiophysiological effect of NMDA receptor stimulation in the SON and activation of the NO/cGMP signal transduction pathway.     

Molecular and functional characterization of Xenopus laevis N-methyl-d-aspartate receptors

N-methyl-d-aspartate (NMDA) receptors of many different vertebrates have been characterized in the past. However, little information is available about amphibian NMDA receptors. Here, we investigated the South African clawed frog Xenopus laevis NR1 subunit at the molecular and functional level. In this subunit, which is obligatory for functional NMDA receptor complexes, we found three exons, the N1, C1, and C3 cassettes, being alternatively spliced. Combinations of these cassettes generated six different splice variants, which were functionally characterized in oocytes. The Xenopus NR1 isoforms generally showed the same functional properties as their mammalian homologs when coexpressed with rat NR2B. In coexpression with Xenopus NR2B, however, some properties changed significantly. This included a Zn²⁺-mediated potentiation of current amplitudes for some subunit combinations which lasted for several minutes. This mechanism presents a novel form of Xenopus NMDA receptor modulation, possibly mediating a form of short-term potentiation in the Xenopus central nervous system.     

Inhibition of an N-methyl-d-aspartate induced short-term potentiation in the rat hippocampal slice

The effects of the phorbol ester 4ß-phorbol-12,13 dibutyrate (PDBu) and the protein kinase (PK) inhibitors H-7 and sphingosine were investigated on the short-term potentiation (STP) of the population excitatory postsynaptic potential (EPSP) induced by perfusion of N-methyl-d-aspartate (NMDA) in the stratum radiatum of CA1 of the rat hippocampal slice. Bath perfusion of 130 μM NMDA for 10 s caused an initial depression of the population EPSP followed by a STP, which averaged 46% and lasted 16 min. PDBu (100 nM) perfused for 2 h completely inhibited the NMDA induced STP, suggesting that the stimulation of PKC inhibited an NMDA receptor activated process which induced the STP. The protein kinase inhibitors H-7 and sphingosine did not alter the NMDA induced STP.     

Large-scale study of phosphoproteins involved in long-term potentiation in the rat dentate gyrus in vivo

The mechanisms underlying the induction of synaptic plasticity and the formation of long-term memory involve activation of cell-signalling cascades and protein modifications such as phosphorylation and dephosphorylation. Based on a protein candidate strategy, studies have identified several protein kinases and their substrates, which show an altered phosphorylation state during the early phases of long-term potentiation (LTP), yet only a limited number of synaptic phosphoproteins are known to be implicated in LTP. To identify new phosphoproteins associated with LTP, we have undertaken a proteomic study of phosphoproteins at different time points following the induction of LTP in the dentate gyrus in vivo (0, 15 and 90 min). For each time point, proteins from the dentate gyrus were separated by two-dimensional gel electrophoresis and stained with Pro−Q^® Diamond, a fluorescent stain specific for phosphoproteins. Fourteen proteins whose phosphorylation state varied significantly following LTP were identified using matrix-assisted laser desorption ionization/time of flight mass spectrometry and electrospray ionization-Orbitrap tandem mass spectrometry (MS/MS). They are involved in various cellular functions implicated in synaptic plasticity, such as intracellular signalling, axonal growth, exocytosis, protein synthesis and metabolism. Our results highlight new proteins whose phosphorylation or dephosphorylation is associated with LTP induction or maintenance. Further studies focusing on the regulation of specific phosphorylation sites will lead to greater understanding of the individual implications of these proteins in LTP as well as of their molecular interactions.     

N-methyld-aspartate acts as an antagonist of the photoreceptor transmitter in the carp retina

Glutamate, aspartate, and their agonists, kainate, quisqualate, cysteine sulfinate and N-methyl-d-aspartate (NMDA), were applied to the isolated carp retina while recording from horizontal cells. All these agents, except NMDA, depolarized horizontal cells membrane and reduced responses to light, thus mimicking the effect of the endogenous photorecepto transmitter. Application of NMDA, on the other hand, caused a membrane hyperpolarization of horizontal cells in the dark, an effect different from its depolarizing effect as observed elsewhere in the central nervous system. NMDA also reduced or blocked the light responses of these cells as well as the depolarizing responses to applications of glutamate, aspartate or kainate. Effects of NMDA on the spectral properties of the horizontal cell responses were identical to the effects of the acidic amino acid receptor antagonists α-methyl glutamate, and α-amino adipate. Thus, NMDA appears to act as a weak antagonist to the photoreceptor transmitter, whose receptors on the horizontal cell membrane interact with a glutamate-like substance but appear atypical of glutamate receptors described elsewhere in the brain.     

l-aspartate binding sites in rat cerebellum: A comparison of the binding ofl-[3H]aspartate andl-[3H]glutamate to synaptic membranes

The binding ofl-[³H]aspartate to sonicated, extensively washed and preincubated cerebellar synaptic membranes was investigated. Binding was optimal under physiological conditions of pH and temperature, and attained equilibrium within 10 min. Binding was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (K_d = 874nM and B_max = 44pmol/mg protein), which displayed no cooperativity (Hill coefficient approx.= 1). Specific [³H]aspartate was readily and reversibly displaced by unlabelledl-aspartate (thed-isomer being less than half as active) with a half-life of dissociation of 32 sec. Quisqualate, 4-fluoroglutamate and 2-amino-4-phosphonobutyrate, which are good displacers of [³H]glutamate binding, were only weakly active against the aspartate system. The excitatory amino acid antagonists,dl-α-aminoadipate,dl-α-aminosuberate and HA-966 were effective displacers, but the proposed aspartate receptor-preferring agonist, N-methyl-d-aspartate was inactive. Kainic acid exhibited negligible affinity for the aspartate binding site, in common with that for glutamate.While freezing or cold storage of membranes resulted in diminished [³H]-aspartate binding, lyophilization was not only able to confer substantial stability, but induced a marked increase in affinity of the binding site.Differential effects of various cations on [³H]aspartate binding were observed — monovalent cations reduced, while divalent cations enhancedl-[³H]aspartate binding.     

Some effects of excitatory amino acid receptor antagonists on synaptic transmission in the rat olfactory cortex slice

A study has been made of the effects of a series of excitatory amino acid receptor antagonists on the field potentials evoked on electrical stimulation of the lateral olfactory tracts of olfactory cortex slices perfused in vitro. The antagonists studied included (+/-)-2-amino-5-phosphonovaleric acid, a potent, specific antagonist of N-methyl-D-aspartate (NMDA) receptors, gamma-D-glutamylglycine, an antagonist of NMDA and kainate receptors and (+/-)-cis-2,3-piperidine dicarboxylic acid and 2-amino-4-phosphonobutyric acid, drugs which in addition to antagonizing NMDA and kainate receptors also block responses to quisqualic acid. From the patterns of effects of the drugs it is proposed that quisqualate and NMDA but not kainate receptors are involved in mediating excitatory transmission in the olfactory cortex; quisqualate receptors are located at the lateral olfactory tract - superficial pyramidal cell synapse whereas NMDA receptors are present at the synapses of the superficial pyramidal cell collaterals with the deep pyramidal cell dendrites and/or at the synapses of the pyramidal cell collaterals and inhibitory interneurones. The results are discussed in terms of possible presynaptic and/or postsynaptic sites of antagonist action.     

Interference of chronically ingested copper in long-term potentiation (LTP) of rat hippocampus

The objective of our study was to find the evidence of copper interaction in LTP, motivated by copper involvement in neurodegenerative illness, like Parkinson, Alzheimer and Amyotrophic Lateral Sclerosis, and we initiated the study of this element in the LTP. For this purpose we used hippocampus slices of rats chronically consuming copper dissolved in water (CuDR; n=26) and non-copper-consuming rats (CR; n=20). The CuDR rats received 8--10 mg/day during 20--25 days. Electrophysiological tests showed absence of LTP in CuDR slices, contrary to CR slices. The stimulus-response test applied before and after LTP showed significant increases of synaptic potential in the CR group. This did not occur in the CuDR group, except for the initial values, which probably seem associated to an early action of copper. The paired-pulse (PP) test, applied to CR and CuDR prior to tetanic stimulation, showed a significant reduction in PP, for the 20-, 30- and 50-ms intervals in CuDR. At the end of the experiments, copper concentration was 54.2 times higher in CuDR slices, compared to the concentration present in CR slices. Our results show that copper reduces synaptic sensibility and also the facilitation capability. These effects represent a significant disturbance in the plasticity phenomenon associated with learning and memory.     

Simultaneous blockade of non-NMDA ionotropic receptors and NMDA receptor-associated ionophore partially protects hippocampal slices from protein synthesis impairment due to simulated ischemia

A large body of evidence exists to demonstrate that excitatory amino acids (EAA) and their receptors are involved in the pathophysiological mechanisms linking several acute brain insults, such as cerebral ischemia, to neuronal degeneration and death. Accordingly, the use of EAA receptor antagonists can be beneficial in attenuating or preventing the neuronal irreversible damage subsequent to various neuropathological syndromes. We have investigated the effect of 15 min of simulated ischemic conditions, i. e., oxygen/glucose deprivation, on hippocampal slices preparation measuring, as neurotoxicity indexes, both the amino acids efflux in the incubation medium, detected by HPLC, and the inhibition of protein synthesis, evaluated as ³H-Leucine incorporation into proteins. Accumulation of neurotransmitter amino acids was measured in the medium during the “ischemic” period. Glutamate increased 30-fold over the basal level while aspartate was sevenfold and GABA 12-fold higher than in normal conditions. After a reoxygenation period of 30 min, the rate of protein synthesis of hippocampal slices subjected to “ischemia” was reduced to 35–50% of controls. The non-competitive NMDA antagonist MK-801 (100 μM) and the competitive NMDA antagonist CGP 39551 (100–250 μM) as well as the non-NMDA receptor antagonist NBQX (100 μM) and AP3 (300 μM) were unable to counteract the metabolic impairment when they were present alone in the incubation fluid during simulated “ischemia.” An incomplete, but highly significant (p < 0.001), protection from protein synthesis impairment was achieved in the presence of an equimolar concentration (100 μM) of MK-801 and NBQX. A similar protective effect could be reproduced using 100 μM NBQX in concomitance with a high Mg⁺⁺ (20 μM) voltage-dependent block of the NMDA receptor-associated channel but not exposing the slices to a NBQX (100 μM) and CGP 39551 (100–250 μM) mixture. The recovery of protein synthesis in the presence of the MK-801/NBQX effective combination was not paralleled by a detectable decrease in the amount of amino acids released in the incubation medium during the “ischemic” period. Taken together, the present data allow new insights into neurotoxicity-mediating mechanisms, suggesting that multiple additive processes are involved and that antagonists acting at different sites on excitatory amino acid receptor subtype can show different neuroprotective potency. © 1995 Wiley-Liss, Inc.