Michael W. Oliver

Development of glutamate binding sites and their regulation by calcium in rat hippocampus

The postnasal development of the Na-independent [3H]glutamate binding sites, which exhibit some characteristics of postsynaptic glutamate receptors, has been studied in rat hippocampal membranes. The amount of binding sites (expressed in pmol/hippocampus) represents 4% of the adult level at postnatal day (PND) 4, increases very rapidly until PND 9, and then increases at a slower rate reaching 80% of the adult value at PND 23. In contrast, the density of binding sites (expressed in pmol/mg protein) exhibits a maximum at PND 9 and slowly decreases to reach the adult value at PND 23. These changes seen to be only quantitative since the affinity (about 450nM) and Hill coefficient (about 1.0) of these binding sites remain constant throughout development. Calcium ions have been shown to markedly stimulate [3H]glutamate binding in adult hippocampal membranes. This effect appears on PND 9--10 and increases rapidly until PND 16 when it is similar to that seen in the adult rat. We also determined the minimum age at which long-term potentiation (LTP) of synaptic transmission could be detected in the CA1 field of hippocampal slice preparations following repetitive electrical stimulation of the Schaffer-commissural pathways. LTP was only rarely detected at PND 8 whereas it could be reliably obtained after PND 9. These results indicate that the postnatal development of Na-independent glutamate binding sites closely parallels synapse formation in the hippocampus, further supporting the idea that the binding sites are associated with a physiological receptor. They also show that the appearance of the stimulatory effect of calcium on glutamate binding occurs at a time when several forms of synaptic plasticity appear in the hippocampus. In particular the correlation of the development of LTP with the calcium-stimulation of glutamate binding suggests that these phenomena have similar cellular mechanisms.

Developmental changes in synaptic properties in hippocampus of neonatal rats.

The properties of synaptic responses in area CA1 of hippocampus were analyzed in slices prepared from 7-9 and 12-15 day old neonate rats. As expected from earlier work, only slices of two-week-old animals showed a consistent degree of long-term potentiation (LTP) in response to patterned high frequency stimulation. Several other synaptic properties were found to change during this developmental period. Inhibitory responses were absent in 7-9 day old but not in 12-15 day old neonates. Paired-pulse facilitation and the calcium sensitivity of postsynaptic responses were considerably reduced in 7-9 as compared to 12-15 day old rats. However, phorbol esters and 4-aminopyridine treatment still produced a strong facilitation of field potentials. The N-methyl-D-aspartate (NMDA) component of responses to single pulse stimulation in low magnesium medium was found to be larger in slices of 7-9 than 12-15 day old or adult animals. At the two time periods examined, trains of high frequency stimulation applied in the presence of regular magnesium elicited an NMDA dependent response. It is concluded that the differences in synaptic properties observed between 7-9 and 12-15 day old neonates may not account for the absence of LTP in the younger animals.

Stimulation of NMDA receptors induces proteolysis of spectrin in hippocampus

Stimulation of N-methyl-D-aspartate (NMDA) receptors was found to induce proteolysis of brain spectrin in hippocampal slices. The effect was dependent upon extracellular calcium, blocked by the antagonist 2-amino-5-phosphonovalerate (AP5), and was not reproduced by potassium-induced depolarization. These results are consistent with the hypothesis that the involvement of NMDA receptors in plasticity and excitotoxicity is at least partially mediated by calcium-activated proteolysis of cytoskeletal proteins.

Development of hippocampal long-term potentiation is reduced by recently introduced calpain inhibitors

The effects of two recently synthesized inhibitors of calpains, calpain inhibitor I (CiI) and calpain inhibitor II (CiII) were tested on the development of long-term potentiation (LTP) in region CA1 of rat hippocampus. Slices maintained in 100 microM of CiI or CiII showed an initial degree of potentiation after theta burst stimulation that, in contrast to controls, slowly decayed across time. The effects of CiI and CiII appeared to be independent of possible actions on the physiological mechanisms that take place during the induction stage of LTP. Since these inhibitors are more potent and specific than leupeptin in blocking calpain activity, their effects on LTP can be more convincingly ascribed to a selective blockade of the calcium-sensitive protease. Accordingly, the results favor the idea that a proteolytic event of the kind found after N-methyl-D-aspartate receptor activation is an intermediary step in the development of LTP.

The protease inhibitor leupeptin interferes with the development of LTP in hippocampal slices

The effect of leupeptin, an inhibitor of thiol-proteases, was tested on the induction of long-term potentiation (LTP) in field CA1 of hippocampal slices. Two h of drug application did not produce substantial changes while a greater than 3-h application caused a sizeable reduction in the degree of LTP induced. Leupeptin had no obvious effects on the facilitation of postsynaptic responses occurring within or between the short high frequency bursts used to induce LTP, suggesting that the drug acted on cellular chemistries occurring after the initial physiological events that normally trigger LTP. These results are consistent with the hypothesis that a calcium-activated thiol protease (calpain) is involved in the induction of LTP.

Effect of bromophenacyl bromide, a phospholipase A2 inhibitor, on the induction and maintenance of LTP in hippocampal slices

The effect of bromophenacyl bromide (BPB), a phospholipase A2 (PLA2) inhibitor, on both the induction and the maintenance of long-term potentiation (LTP) was investigated in field CA1 of the hippocampal slice preparation. One hour of BPB application (50 microM) caused a large reduction in the magnitude of LTP induced by a theta burst stimulation (TBS) paradigm. BPB had no significant effect on either the degree of paired-pulse facilitation or the amount of pre-established LTP. Furthermore, the facilitation of postsynaptic responses occurring during TBS and in the first minute following TBS was not reduced by the PLA2 inhibitor. These results indicate that the inhibition of LTP produced by BPB is not due to an effect of the drug on a physiological event that triggers LTP. The data also suggest that PLA2 activation plays a critical role in the expression of LTP, but is not required for the maintenance of the potentiation.

Increase in glutamate receptors following repetitive electrical stimulation in hippocampal slices

Abstract Repetitive electrical stimulation of identified pathways in the hippocampal slice preparation induces long-term potentiation (LTP) of synaptic transmission and is accompanied by a long-lasting (up to 30 minutes) increase in L- 3 H-glutamate accumulation by the slices. This increased accumulation of 3 H-glutamate is restricted to the terminal field of the stimulated fibers and does not seem to represent a non-specific increased accumulation of amino acids. In addition, synaptic membranes prepared from stimulated slices exhibit an increase in the maximal number of the sodium-independent high-affinity binding sites for 3 H-glutamate without changes in their affinity. These results suggest that repetitive electrical stimulation elicits an increased number of glutamate receptors which might be responsible for LTP.

Effects of enkephalin, morphine, and naloxone on the electrical activity of the in vitro hippocampal slice preparation.

The effects of infusion of low concentrations of d-Ala-d-Leu-enkephalin, morphine, or naloxone on electrical responses to stimulation of the in vitro hippocampal slice preparation were investigated. Concentrations of enkephalin as low as 10 nm and morphine as low as 1 μm dramatically increased the magnitude of the population spike response of pyramidal cells to Schaffer-commissural stimulation. The drug response had a rapid onset and was replicable several times after infusion of normal incubation medium. Enhancement of the population spike by either morphine or enkephalin was blocked by the opiate receptor antagonist naloxone. The response to antidromic stimulation was not altered by enkephalin, nor was the slope of the dendritic response, indicating that the enkephalin effect is not mediated by depolarization of the pyramidal cells themselves nor by facilitation of synaptic processes at the dendrites. The possibility that the enkephalin effect might be produced by alteration of either a feed-forward or a feedback mechanism mediated by basket cells was investigated. Feed-forward inhibition was demonstrated, but enkephalin did not alter its efficacy. Feedback inhibition induced by antidromic stimulation was also unaffected by enkephalin. On the basis of these results we suggest that enkephalin acts to alter the efficiency of a mechanism involved in coupling the dendritic EPSP with the spike initiation mechanism of the cell body.

Activation of the glycine site associated with the NMDA receptor is required for induction of LTP in neonatal hippocampus

The role played by the glycine site associated with the NMDA receptor in inducing long‐term potentiation (LTP) in neonatal hippocampus was examined. An antagonist of the glycine site, 7‐chlorokynurenic acid (Cl‐Kyn), completely blocked both the short‐term and the long‐term potentiation associated with theta burst stimulation (TBS) linked to NMDA receptor activation in slices from hippocampus at postnatal days 10–16; this effect was reversed by the glycine agonist, d‐serine. Analysis of the TBS‐evoked responses showed: (1) a developmental alteration in the burst response morphology that may be related to maturation of GABA‐mediated inhibition; and (2) that, unlike 2‐amino‐5‐phosphonovalerate (APS), Cl‐Kyn did not reduce any portion of the burst response. These results suggest that stimulation of the glycine site coupled to the NMDA receptor complex is necessary to induce LTP in neonatal tissue and that two NMDA receptor types may be present in the hippocampus.

The glycine site modulates NMDA-mediated changes of intracellular free calcium in cultures of hippocampal neurons

Recent evidence indicates that the N-methyl-D-aspartate (NMDA) receptor-channel complex contains a glycine subunit whose activation may be necessary for channel operation. It has been previously shown that stimulation of the NMDA receptor leads to an increase in intracellular ionic Ca2+ [( Ca2+]i); therefore, we examined the role of the NMDA receptor-associated glycine site in modulating [Ca2+]i using the fluorescent dye Fura II in hippocampal neuron cultures. A 3-s pulse of 200 microM NMDA resulted in a mean [Ca2+]i increase of 363 nM above the average resting concentration of 122 nM. Perfusion of the glycine site antagonist 7-chlorokynurenate (Cl-Kyn) essentially eliminated the NMDA-induced alteration in [Ca2+]i. Either 40 microM glycine or 50 microM D-serine completely reversed the effect of Cl-Kyn, indicating that the drug was acting at the glycine site. The NMDA receptor antagonists 2-amino-5-phosphonovalerate (AP5) and ketamine, which bind to the glutamate recognition site and the ion channel, respectively, also blocked the NMDA-mediated [Ca2+]i response; however, glycine or D-serine did not reverse this effect. These data show that the glycine binding site coupled to the NMDA receptor modulates the NMDA-mediated increase in [Ca2+]i. Antagonists of the glycine site provide a new tool to investigate and possibly control neuroplasticity and neurotoxicity related to the NMDA receptor complex.