Amy Arai

Factors regulating the magnitude of long-term potentiation induced by theta pattern stimulation

Electrical stimulation patterned after the hippocampal theta rhythm produces a robust and stable long-term potentiation (LTP) effect. Pharmacological manipulations were used in the present studies in an effort to relate characteristics of the responses occurring during theta stimulation to the magnitude of potentiation which follows it. Comparisons were made using five or ten bursts of stimulation which respectively induce sub-maximal or near maximal degrees of LTP. DPCPX, a drug that increases release by blocking adenosine A1 receptors, was used to enhance the depolarization produced by individual theta bursts. This resulted in a marked increase in the amount of stable LTP induced by five theta bursts but did not affect that resulting from ten bursts. This finding suggested that depolarization occurring during a burst response influences per burst potentiation but not the ceiling on maximum LTP. Aniracetam, a nootropic drug that enhances responses via an action on glutamate (AMPA) receptors, was used to test this conclusion. Like DPCPX, aniracetam increased the size of the burst response and enhanced the degree of LTP caused by five but not ten theta bursts. Forskolin, an activator of adenylate cyclase, was used to test the effects of blocking the hyperpolarization normally present between theta bursts on the induction of LTP. The drug augmented the degree of LTP resulting from five theta bursts and, in contrast to DPCPX and aniracetam, nearly doubled that obtained with ten bursts. Thus the drug affected both per burst potentiation and the ceiling on LTP. These results are discussed in terms of an hypothesis in which the magnitude of NMDA receptor mediated currents affects the degree of potentiation produced by individual theta bursts while the duration of the currents is related to the limit on the maximum LTP induced by a series of bursts. The possible implications of the findings for learning are also considered.

The effects of adenosine on the development of long-term potentiation

In previous work we found that a brief period of hypoxia occurring within 1-2 min of high-frequency stimulation disrupts the development of long-term potentiation (LTP) in the CA1 field of rat hippocampal slices. We now report that extracellular application of adenosine if applied within 1 min but not 5 min after high-frequency stimulation similarly prevents LTP. Adenosine was ineffective if DPCPX (1,3-dipropyl-8-cyclopentyl-xanthine), a selective antagonist of adenosine A1 receptors, was present. The post-stimulation application mode excludes the possibility that adenosine interferes with NMDA receptor activation and its role in initiating LTP. It suggests instead that changes in intracellular control systems linked to adenosine receptors can during a brief vulnerable period interrupt the biochemical processes leading to the expression of long-term potentiation.

Proteolysis of spectrin by calpain accompanies theta-burst stimulation in cultured hippocampal slices

Tests were carried out to determine if repetitive bursts of afferent stimulation activate calpain, a calcium-dependent protease hypothesized to be involved in the production of long-term potentiation. Antibodies against a stable breakdown product that results from proteolysis of spectrin by calpain were used to identify sites of enzyme activation in cultured hippocampal slices. Slices in which theta-burst stimulation was applied to the Schaffer collateral fibers had pronounced accumulations of breakdown product that were restricted to field CA1, the zone innervated by the stimulated axons. Labelling occurred in the form of scattered puncta and was also present in dendritic processes. The extent of these effects was correlated (r = 0.73) with the amount of theta-burst stimulation delivered. Control slices or those receiving low frequency stimulation had variable, but uniformly lower, amounts of breakdown product and were clearly distinguishable from those given theta bursts. Statistical analyses using a six point rating scheme confirmed this point (P < 0.001). These results satisfy an essential prediction of the hypothesis that calpain plays an important role in the induction of long-term potentiation.

A centrally active drug that modulates AMPA receptor gated currents.

Systemic administration of the drug 1-(1,3-benzodioxol-5-ylcarbonyl)-piperidine (1-BCP) has been reported to enhance monosynaptic responses in the hippocampus in vivo and to improve spatial and olfactory memory in rats. The drugs mechanism of action was investigated in the present study using membrane patches excised from cultured hippocampal slices. The decay time of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor mediated inward currents was greatly increased by 1-BCP in a concentration dependent and reversible fashion; peak current was also enhanced but to a lesser degree. In vitro slice experiments indicated that the drug has parallel effects on the field EPSP. It is concluded that 1-BCP is a centrally active modulator of the AMPA receptor.

Anoxia reveals a vulnerable period in the development of long-term potentiation

Transient anoxia occurring 1-2 min after high-frequency stimulation selectively prevented the stable expression of long-term potentiation (LTP). Anoxia occurring after this brief vulnerable period did not reverse LTP. Experiments on the duration of anoxia necessary to block LTP expression indicated that simply reducing synaptic transmission was insufficient but that membrane depolarization was not required. The effects of anoxia on LTP were blocked by antagonists of A1 adenosine receptors. It is concluded that LTP develops in about one minute and that the chemistries operating in this period are easily disrupted by an event triggered by adenosine receptors.

Calpain inhibitors improve the recovery of synaptic transmission from hypoxia in hippocampal slices.

Two inhibitors of calcium activated proteases (calpains) were tested for their effects on hypoxia-induced synaptic dysfunction in hippocampal slices. Hypoxic episodes lasting for either one or two minutes beyond the point at which action potentials (fiber volleys) disappeared were used. Leupeptin and calpain inhibitor I had no reliable effects on the rate at which synaptic transmission declined during hypoxia or the time required for loss of action potentials, but both drugs did substantially improve the degree of recovery. Moreover, the percentage of slices meeting an arbitrary criterion for viability after hypoxic treatment was greatly increased by the drug treatment. These results point to the conclusion that proteolysis triggered by calcium influx during hypoxia contributes to pathophysiology.

A brief period of hypoxia causes proteolysis of cytoskeletal proteins in hippocampal slices

Breakdown products (BDPs) resulting from the partial proteolysis of spectrin were examined in hippocampal slices after periods of hypoxia lasting for 5 or 10 min. The concentration of a approximately 155 kDa BDP increased nearly twofold after 5 min of hypoxia; further increases were not seen with 10 min episodes or 10 min of hypoxia followed by reoxygenation. The hypoxia-induced proteolysis was blocked by prior infusion of a newly introduced inhibitor of calpain (calpain inhibitor I, 200 microM). Together with previously published data showing improved recovery of hippocampal slices from hypoxia in the presence of calpain inhibitors, these data suggest that activation of calpain may contribute significantly to the pathophysiology of ischemia.

Benzamide-Type AMPA Receptor Modulators Form Two Subfamilies with Distinct Modes of Action

CX516 (BDP-12) and CX546, two first-generation benzamide-type AMPA receptor modulators, were compared with regard to their influence on AMPA receptor-mediated currents, autaptic responses in cultured hippocampal neurons, hippocampal excitatory postsynaptic currents, synaptic field potentials, and agonist binding. The two drugs exhibited comparable potencies in most tests but differed in their efficacy and in their relative impact on various response parameters. CX546 greatly prolonged the duration of synaptic responses, and it slowed 10-fold the deactivation of excised-patch currents following 1-ms pulses of glutamate. The effects of CX516 on those measures were, by comparison, small; however, the drug was equally or more efficacious than CX546 in increasing the amplitude of synaptic responses. This double dissociation suggests that amplitude and duration of synaptic responses are governed by different aspects of receptor kinetics, which are differentially modified by the two drugs. These effects can be reproduced in receptor simulations if one assumes that CX516 preferentially accelerates channel opening while CX546 slows channel closing. In binding tests, CX546 caused an approximately 2-fold increase in the affinity for radiolabeled agonists, whereas CX516 was ineffective. More importantly, even millimolar concentrations of CX516 did not influence the dose-response relation for CX546, suggesting the possibility that they bind to different sites. Taken together, the evidence suggests that benzamide modulators from the Ampakine family form two subgroups with different modes and sites of action. Of these, CX516-type drugs may have the greater therapeutic utility because of their limited efficacy in prolonging synaptic responses and in attenuating receptor desensitization.

Effects of a centrally active benzoylpiperidine drug on α-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid receptor kinetics

A newly developed benzoylpiperidine drug that increases the size of fast, excitatory synaptic responses was examined for its effects on the kinetic properties of α-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid-type glutamate receptors. When long pulses of glutamate were applied to excised hippocampal patches of the rat, the compound [1-(1,3-benzodioxol-5-ylcarbonyl)piperidine-20] caused an approximately 15-fold reduction in the rate at which responses desensitized and a similar size increase in steady-state currents. In experiments using 1-ms glutamate pulses, 1-(1,3-benzodioxol-5-ylcarbonyl)piperidine-20 prolonged response deactivation by a factor of about four and greatly reduced the depression in the second response when two consecutive glutamate pulses were given. Two types of equilibrium binding assays indicated that 1-(1,3-benzodioxol-5-ylcarbonyl)piperidine-20 causes a measurable increase in the affinity of α-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid receptors; the ec50 values for this effect were similar to those obtained in excised patch studies. The actions of 1-(1,3-benzodioxol-5-ylcarbonyl)piperidine-20 on physiology and ligand binding could be adequately reproduced in a receptor model by slowing the rate of desensitization and increasing the affinity of the sensitized states. The biochemical and physiological effects of benzoylpiperidine compounds were qualitatively different from those obtained with cyclothiazide, although both types of drug increased α-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid receptor-mediated synaptic responses. Moreover, interactions between the drugs were at most only partially competitive; α-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid receptors may thus have multiple modulatory sites with distinct drug preferences and different effects on receptor kinetics.

Stimulation of NMDA receptors activates calpain in cultured hippocampal slices

The hypothesis that intense stimulation of NMDA receptors activates calpain was tested in long-term cultures of hippocampus. Slices prepared from 10-day-old rats were maintained for periods of up to 6 weeks and then assayed for a stable breakdown product that results from the proteolysis of spectrin by calpain. The breakdown product increased dramatically during the first 24 h after tissue preparation and then decreased to a low level that remained unchanged for weeks. NMDA caused a 2- to 3-fold increase in breakdown product that rose linearly with time (5-30 min) and was blocked by the receptor antagonist MK-801. The effect of NMDA was the same throughout the culture period and was dependent upon the concentration of extracellular calcium with no effect at 2 mM and maximal effect at 4 mM calcium. These results indicate that rapid activation of calpain occurs in undamaged hippocampal neurons following stimulation of NMDA receptors.