Jonas I. Addae

The pharmacological manipulation of glutamate receptors and neuroprotection

The overactivation of glutamate receptors is a major cause of Ca(2+) overload in cells, potentially leading to cell damage and death. There is an abundance of agents and mechanisms by which glutamate receptor activation can be prevented or modulated in order to control these effects. They include the well-established, competitive and non-competitive antagonists at the N-methyl-D-aspartate (NMDA) receptors and modulators of desensitisation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. More recently, it has emerged that some compounds can act selectively at different subunits of glutamate receptors, allowing a differential blockade of subtypes. It is also becoming clear that a number of endogenous compounds, including purines, can modify glutamate receptor sensitivity. The kynurenine pathway is an alternative but distinct pathway to the generation of glutamate receptor ligands. The products of tryptophan metabolism via the kynurenine pathway include both quinolinic acid, a selective agonist at NMDA receptors, and kynurenic acid, an antagonist at several glutamate receptor subtypes. The levels of these metabolites change as a result of the activation of inflammatory processes and immune-competent cells, and may have a significant impact on Ca(2+) fluxes and neuronal damage. Drugs which target some of these various sites and processes, or which change the balance between the excitotoxin quinolinic acid and the neuroprotective kynurenic acid, could also have potential as neuroprotective drugs.

Activation of thermogenesis of brown fat in rats by baclofen

Injection of baclofen (0.5-5 micrograms) into the ventromedial hypothalamus (VMH) of anaesthetized rats produced marked increases in the temperature (over 2 degrees C) and thermogenic activity of brown adipose tissue (BAT). These effects were abolished by ganglionic or beta-adrenergic blockade, or denervation of the tissue, but unaffected by hypophysectomy, adrenalectomy or vagotomy. Injections into the hypothalamus close to, but outside the ventromedial hypothalamus did not affect brown adipose tissue. Intravenous administration of baclofen produced similar increases in the temperature of brown adipose tissue, but at larger doses (50-1000 micrograms), and a subcutaneous injection stimulated the metabolic rate in conscious rats. Chronic treatment with baclofen suppressed weight gain and stimulated activity of brown adipose tissue without affecting food intake. These effects of baclofen, which were not mimicked by injections of gamma-aminobutyric acid (GABA), indicate a novel action of baclofen in the ventromedial hypothalamus, leading to marked increases in metabolic rate and body temperature by stimulating sympathetic outflow to brown fat.

NMDA-induced preconditioning attenuates synaptic plasticity in the rat hippocampus

It was recently demonstrated that glutamate could precondition hippocampal slices against the damaging effects of hypoxia, and we have now extended this observation by investigating (i) the ability of glutamate receptor agonists to act as preconditioning agents and (ii) the effects of preconditioning on synaptic plasticity. Using rat hippocampal slices, 15 microM NMDA applied for 10 min (chemical insult) caused abolition of the population spike potentials (PS) followed by approximately 33% recovery at 60 min post-insult. In comparison, a 5 min preconditioning exposure of 10 microM NMDA given 30 min prior to the insult significantly improved the recovery to 69%. Preconditioning did not alter paired pulse facilitation; however, it significantly enhanced paired pulse depression and reduced population spike long-term potentiation (PS-LTP) and LTP in field recordings. This effect on PS-LTP appeared to be NMDA receptor dependent and was blocked by the nitric oxide synthase inhibitors nitro-L-arginine methyl ester (L-NAME) and 7-nitro indazole (7-NI) but not by the adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). We conclude that preconditioning by NMDA can improve recovery following acute insults but may have deleterious effects on neuronal plasticity.

Effects of topically applied excitatory amino acids on evoked potentials and single cell activity in rat cerebral cortex

The effect of topically applied excitatory amino acids and related compounds on the somatosensory evoked potential and single cell responses to iontophoretically applied compounds have been studied in the neocortex of urethane anaesthetised rats. N-methyl-aspartate (NMDLA), DL-homocysteic acid (DLH), kainic acid, quinolinic acid and carbachol produced a depression of evoked potential amplitude. L-glutamate and quisqualic acid had no effect. Tetrodotoxin and 2-amino-5-phosphonovaleric acid prevented the effects of NMDLA, but bicuculline and theophylline derivatives did not. There was an apparent desensitisation of the depression of evoked potentials following topical application of NMDLA or DLH, the potentials recovering to control levels during the amino acid application. A second application of amino acid was then ineffective unless applied about 1 h later. Single cell responses to iontophoretically applied NMDLA behaved similarly. There was an apparent cross-desensitisation between NMDLA, DLH and quinolinic acids. The results may indicate a desensitising receptor for amino acids on superficial neocortical neurones.

Effects of anticonvulsants on responses to excitatory amino acids applied topically to rat cerebral cortex

1. Five clinically used anticonvulsants were examined to find out if they block the effects of excitatory amino acids in the cerebral cortex of urethane anaesthetised rats. 2. Compounds were tested by topical application to the cortical surface and following their intraperitoneal injection at anticonvulsant doses. 3. Pentobarbital and diphenylhydantoin blocked the effect of quisqualic acid but only at concentrations higher than the therapeutically relevant levels. 4. Pentobarbital and diphenylhydantoin did not alter the effects of N-methyl-D,L-aspartic acid (NMDLA) or kainic acid. 5. Diazepam prevented the kainic acid-induced development of distorted somatosensory evoked potentials (SEPs) at therapeutically relevant levels. 6. Diazepam had no effect on NMDLA or quisqualic acid. 7. Carbamazepine and chlormethiazole had no effect on NMDLA, kainic acid or quisqualic acid. 8. The anticonvulsive effects of these drugs, with the exception of diazepam, probably do not involve antagonism of endogenous EAAs.

Long-term potentiation protects rat hippocampal slices from the effects of acute hypoxia

We have previously shown that long-term potentiation (LTP) decreases the sensitivity of glutamate receptors in the rat hippocampal CA1 region to exogenously applied glutamate agonists. Since the pathophysiology of hypoxia/ischemia involves increased concentration of endogenous glutamate, we tested the hypothesis that LTP could reduce the effects of hypoxia in the hippocampal slice. The effects of LTP on hypoxia were measured by the changes in population spike potentials (PS) or field excitatory post-synaptic potentials (fepsps). Hypoxia was induced by perfusing the slice with (i) artificial CSF which had been pre-gassed with 95%N2/5% CO2; (ii) artificial CSF which had not been pre-gassed with 95% O2/5% CO2; or (iii) an oxygen-glucose deprived (OGD) medium which was similar to (ii) and in which the glucose had been replaced with sucrose. Exposure of a slice to a hypoxic medium for 1.5-3.0 min led to a decrease in the PS or fepsps; the potentials recovered to control levels within 3-5 min. Repeat exposure, 45 min later, of the same slice to the same hypoxic medium for the same duration as the first exposure caused a reduction in the potentials again; there were no significant differences between the degree of reduction caused by the first or second exposure for all three types of hypoxic media (P>0.05; paired t-test). In some of the slices, two episodes of LTP were induced 25 and 35 min after the first hypoxic exposure; this caused inhibition of reduction in potentials caused by the second hypoxic insult which was given at 45 min after the first; the differences in reduction in potentials were highly significant for all the hypoxic media used (P<0.01; paired t-test). The neuroprotective effects of LTP were not prevented by cyclothiazide or inhibitors of NO synthetase compounds that have been shown to be effective in blocking the effects of LTP on the actions of exogenously applied AMPA and NMDA, respectively. The neuroprotective effects of LTP were similar to those of propentofylline, a known neuroprotective compound. We conclude that LTP causes an appreciable protection of hippocampal slices to various models of acute hypoxia. This phenomenon does not appear to involve desensitisation of AMPA receptors or mediation by NO, but may account for the recognised inverse relationship between educational attainment and the development of dementia.

Students’ perception of a modified form of PBL using concept mapping

Background: Problem-based learning (PBL) and concept mapping have been shown to promote active and meaningful learning. Aim: To design a method of PBL that includes concept mapping and examine students’ perceptions of this form of PBL. Methods: We designed a 5-phase method of PBL which produced three clearly identifiable mapping phases that reflected the learning activities during the tutorial: (1) the initial understanding of the clinical problem, (2) students’ prior knowledge of the problem, (3) the final understanding of the problem following self-directed study. The process of developing the second and third phases of the map involved the students answering questions that they generated on two occasions to give the entire process a 5-phase approach. Each student was exposed to both methods of PBL: a conventional 7-step method (Maastricht type) and the modified PBL (5-phase) method. We used a questionnaire to evaluate the students’ perceptions of the two methods in four learning domains. Result: The students’ ratings for the 5-phase method were significantly higher than for the 7-step method (paired t-test) on all items on the questionnaire. Conclusion: The students perceived the 5-phase method as promoting their passion for learning, and developing their cognitive, metacognitive and interpersonal skills.

Interactions of glutamate receptor agonists with long-term potentiation in the rat hippocampal slice.

Previous work has described the apparent desensitisation of neuronal networks in the rat neocortex to amino acid agonists, following prior exposure several minutes earlier. Since long-term potentiation is believed to involve activation of amino acid receptors, we have now sought to determine whether long-term potentiation can modify the sensitivity of neurones to glutamate receptor agonists in rat hippocampal slices. Responses were measured as the change in population spike or postsynaptic potential (e.p.s.p.) size. Two applications of N-methyl-D-aspartate (NMDA), quinolinic acid, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainate, 45 min apart, did not exhibit any apparent desensitisation. However, the induction of long-term potentiation produced a marked loss of sensitivity to quinolinic acid, with smaller effects on NMDA, AMPA and kainate responses. No marked changes were obtained of e.p. s.p. size. In order to localise the cellular sites of these changes, agonists were also applied by microiontophoresis to the cell bodies or dendritic regions of CA1 neurones. Responses to quinolinic acid showed apparent desensitisation at both sites, whereas no decrease was observed in responses to NMDA or AMPA application. The induction of long-term potentiation again produced a decrease in the size of responses to NMDA and AMPA. Inhibition of nitric oxide (NO) synthase prevented the long-term potentiation-induced loss of responsiveness to NMDA, but not AMPA, implying a role for NO in the loss of NMDA sensitivity. Recordings of single cell activity during the iontophoretic application of agonists and induction of long-term potentiation showed that responses to NMDA were often suppressed to a greater extent than to quinolinic acid. The results indicate that long-term potentiation can modify the sensitivity of hippocampal neurones to glutamate receptor agonists, and that differences exist in the pharmacology of NMDA and quinolinic acid.

NMDA-induced changes in a cortical network in vivo are prevented by AMPA

Analogues of glutamic acid including N-methyl-D-aspartic acid (NMDA) depolarise neurones of the cerebral cortex in vivo and thus change the size of the somatosensory evoked potentials (SEPs). The potentials recover rapidly despite maintained superfusion with NMDA, suggesting a form of neuronal desensitisation or network adaptation. In this study potentials were evoked at the cortical surface by electrical stimulation of the contralateral forepaw and compounds applied topically to the cortical surface by a cortical cup. NMDA at 50-250 microM caused a concentration-dependent decrease in the amplitude of the SEPs, with the highest concentration always abolishing them. AMPA at 50 microM did not affect evoked potentials when applied alone, but prevented the NMDA. Such AMPA-NMDA interactions were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and enhanced by cyclothiazide (which prevents AMPA desensitisation). Superfusion with potassium did not change sensitivity to NMDA. These results suggest that, in the rat cerebral cortex in vivo, activation of AMPA receptors can induce a loss of the network response to activation of NMDA receptors. Such a phenomenon may have physiological and therapeutic implications.

Injection of baclofen into the ventromedial hypothalamus stimulates gastric motility in the rat.

Injection of 2 micrograms (+/-)-baclofen into the ventromedial hypothalamus (VMH) of urethane-anaesthetized rats resulted in an increase in gastric tone and amplitude of contractions. This effect was curtailed by administration of atropine methyl nitrate (20 mg/kg i.p.) or bilateral cervical vagotomy. These results provide evidence for a hypothalamic modulation of gastric motility by the vagus. Baclofen, possibly acting on receptors insensitive to gamma-aminobutyric acid (GABA), may be mimicking a vagal activation system, located within the ventro-medial hypothalamus.