D. Steven Kerr

Modulation of hippocampal long-term potentiation and long-term depression by corticosteroid receptor activation

Long-term potentiation (LTP) and long-term depression (LTD) are lasting changes in synaptic efficacy which may underlie memory and learning processes. Hippocampal LTP has been shown to vary inversely with the degree of stress or adrenal activity, and adrenal corticosteroids (CORT) have been strongly implicated in this process. The present in vitro study was undertaken to further examine the role of glucocorticoids in LTP, and to extend this analysis to heterosynaptic LTD. Animals were injected twice daily (s.c.) for 2 days prior to sacrifice with either the corticosteroid biosynthesis inhibitor metyrapone (200 mg/kg/day) or CORT (20 mg/kg/day). Noninjected and saline-injected controls were also examined. CA1 population spike amplitudes and field EPSP slopes were assessed in response to activation of two separate stratum radiatum inputs. One input received tetanic stimulation and both inputs were monitored for 30 min following tetanization to determine long-term changes in synaptic efficacy (homosynaptic LTP of tetanized synapses; heterosynaptic LTD of nontetanized synapses). Animals exhibiting low and high serum CORT titers exhibited significantly less LTP and LTD than did animals exhibiting moderate levels of CORT. In addition, in vitro perfusion of RU-28362, a Type II glucocorticoid receptor agonist, markedly reduced both LTP and LTD. There was no correlation between serum CORT and serum glucose titers, nor was there any correlation between serum glucose and either LTP or LTD. These results are consistent with recent reports of an inverted-U function for CORT levels versus primed-burst LTP (Bennett, Diamond, Fleshner, & Rose, 1991; Diamond, Bennett, Fleshner, & Rose, 1992) and further demonstrate that manipulation of corticosterone in adrenal-intact animals affects LTP and LTD in a similar fashion.

Age-related changes in tolerance to the marine algal excitotoxin domoic acid.

During an incident of toxic mussel poisoning, the epileptogenic excitotoxin domoic acid (DOM) was associated with lasting neurological deficits mainly in older patients (), suggesting supersensitivity to excitotoxins is a feature of brain aging. Here, hippocampal slices from young (3 months) and aged (26-29 months) Sprague Dawley rats were assessed by CA1 field potential analysis before and after preconditioning with DOM. In naïve slices from young animals, DOM produced initial hyperexcitability followed by significant dose-dependent reductions in population spike amplitude during prolonged application. Following toxin washout, only small changes in neuronal activity were evident during a second application of DOM, suggesting that a resistance to the effects of DOM occurs in hippocampal slices which have undergone prior exposure to DOM. This inducible tolerance was not antagonized by the NMDA receptor blockers APV or MK-801, nor was it diminished by the group I, II or III mGluR blockers AIDA, CPPG and EGLU. Likewise, neither the AMPA/KA blocker CNQX nor the VSCC blocker nifedipine were effective in blocking tolerance induction in young slices. Field potential analysis revealed significant age-related reductions in CA1 EPSP strength, population spike amplitude and paired-pulse inhibition, but aged slices did not differ in sensitivity to DOM relative to young. However, aged CA1 failed to exhibit any tolerance to DOM following preconditioning, suggesting that a loss of inducible neuroprotective mechanisms may account for increased sensitivity to excitotoxins during aging.

Clomethiazole: mechanisms underlying lasting neuroprotection following hypoxia-ischemia

Damage after hypoxia‐ischemia (HI) is observed in both cortical and subcortical regions. In this study, we employed a “Levine” rat model of HI (left carotid ligation + 1 h global hypoxia on PND‐26) and used histological and electrophysiological paradigms to assess the long‐term neuroprotective properties of clomethiazole (CMZ; a GABAA receptor modulator). Key enzymes involved in inflammation, namely nitric oxide synthase (NOS) and arginase, were also examined to assess potential CMZ mechanisms not involving GABA‐R activation. Assessments were carried out 3 and 90 days post‐HI. Extensive CNS lesions were evident after HI ipsilaterally at both short‐ and long‐term intervals. CMZ significantly decreased the lesion size at 3 and 90 days (P<0.01; P<0.05). Evoked field potential analyses were used to assess hippocampal CA1 neuronal activity ex vivo. Electrophysiological measurements contralateral to the occlusion revealed impaired neuronal function after HI relative to short‐ and long‐term controls (P<0.001, 3 and 14 days; P<0.01, 90 days), with CMZ treatment providing near complete protection (P<0.001 at 3 and 14 days; P<0.01 at 90 days). Both NOS and arginase activities were significantly increased at 3 days (P<0.01), with arginase remaining elevated at 90 days post‐HI (P<0.05) ipsilaterally. CMZ suppressed the HI‐induced increase in iNOS and arginase activities (P<0.001; P<0.05). These data provide evidence of long‐term functional neuroprotection by CMZ in a model of HI. We further conclude that under conditions of HI, functional deficits are not restricted to the ipsilateral hemisphere and are due, at least in part, to changes in the activity of NOS and arginase.

Domoic acid-induced hippocampal CA1 hyperexcitability independent of region CA3 activity

Domoic acid (DOM) is a potent agonist of AMPA and kainic acid (KA) receptors in the CNS and is known to produce seizures acutely, and lasting excitotoxic damage in several brain regions. While the excitotoxic effects of DOM are well documented, its seizurogenic properties are less clear. In this study, we assessed the acute effects of DOM and KA in region CA1 of intact rat hippocampal slices (CA3-on) and in slices lacking region CA3 (CA3-off). Orthodromic Schaffer collateral-evoked CA1 field potentials (population spikes and somal EPSPs) were monitored during DOM and KA (10-500 nM) administration. In CA3-off slices both KA and DOM produced immediate increases in CA1 population spike amplitude. With prolonged exposure, lasting dose-dependent reductions in spike amplitude and EPSP slope were observed, possibly due to depolarising conduction block following excessive AMPA/KA receptor activation; DOM was several-fold more potent than KA in this regard. Population spike threshold did not vary with DOM, but in CA3-on slices a dose-dependent steepening of the I/O curve and increase in maximum spike amplitude was seen. CA1 hyperexcitability, as evidenced by the appearance of prominent second and third population spikes, was equivalently increased across a range of DOM concentrations in both CA3-on and CA3-off slices and, in general, DOM-induced CA1 hyperexcitability was not enhanced by the presence of CA3 for any of the other variables assessed in this study. These findings show that DOM directly promotes neuronal hyperactivity in region CA1, presumably due to tonic AMPA and/or KA-receptor mediated depolarization, and further suggests that DOM-induced hyperactivity in the recurrently networked, AMPA/KA-receptor rich region CA3 does not contribute to the onset and spread of limbic seizures during relatively mild DOM intoxication.

Domoic acid preconditioning and seizure induction in young and aged rats.

Clinical reports suggest that the elderly are hypersensitive to the neurological effects of domoic acid (DOM). In the present study we assessed DOM-induced seizures in young and aged rats, and seizure attenuation following low-dose DOM pretreatment (i.e. preconditioning). Seizure behaviours following saline or DOM administration (0.5-2mg/kg i.p.) were continuously monitored for 2.5h in naïve and DOM preconditioned rats. Competitive ELISA was used to determine serum and brain DOM concentrations. Dose- and age-dependent increases in seizure activity were evident in response to DOM. Lower doses of DOM in young and aged rats promoted low level seizure behaviours. Animals administered high doses (2mg/kg in young; 1mg/kg in aged) progressed through various stages of stereotypical behaviour (e.g., head tics, scratching, wet dog shakes) before ultimately exhibiting tonic-clonic convulsions. Serum and brain DOM analysis indicated impaired renal clearance as contributory to increased DOM sensitivity in aged animals, and this was supported by seizure analysis following direct intrahippocampal administration of DOM. Preconditioning young and aged animals with low-dose DOM 45-90 min before high-dose DOM significantly reduced seizure intensity. We conclude that age-related supersensitivity to DOM is related to reduced clearance rather than increased neuronal sensitivity, and that preconditioning mechanisms underlying an inducible tolerance to excitotoxins are robustly expressed in both young and aged CNS.

Kainate receptor agonists and antagonists mediate tolerance to kainic acid and reduce high-affinity GTPase activity in young, but not aged, rat hippocampus.

Domoic acid acts at both kainic acid (KA) and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)‐sensitive glutamate receptors and induces tolerance against subsequent domoic acid insult in young but not aged rat hippocampus. To determine the receptor specificity of this effect, tolerance induction was examined in hippocampal slices from young and aged rats. Slices were preconditioned by exposure to low‐dose KA to activate kainate receptors, or the AMPA‐receptor selective agonist (S)‐5‐fluorowillardiine (FW), and following washout, tolerance induction was assessed by administration of high concentrations of KA or FW (respectively). FW preconditioning failed to induce tolerance to subsequent FW challenges, while KA‐preconditioned slices were significantly resistant to the effects of high‐dose KA. KA preconditioning failed to induce tolerance in aged CA1. Given the lasting nature of the tolerance effect, we examined G‐protein‐coupled receptor function. A number of ionotropic KA receptor agonists and antagonists significantly reduced constitutive GTPase activity in hippocampal membranes from young but not aged rats. Furthermore, in young CA1, low concentrations of the AMPA/KA blocker GYKI‐52466 also induced tolerance to high‐dose KA. Our findings suggest that tolerance is triggered by a selective reduction in constitutive KA‐sensitive G‐protein activity, and that this potential neuroprotective mechanism is lost with age.

Ischemic Cardiomyopathy Following Seizure Induction by Domoic Acid

Exposure to the excitotoxin domoic acid (DOM) has been shown to produce cardiac lesions in both clinical and animal studies. We have previously shown that DOM failed to directly affect cardiomyocyte viability and energetics, but the development of this cardiomyopathy has remained unexplained. The present study compared effects of high-level seizure induction obtained by intraperitoneal (2 mg/kg) or intrahippocampal (100 pmol) bolus administration of DOM on development of cardiac pathologies in a rat model. Assessment of cardiac pressure derivatives and coronary flow rates revealed a significant time-dependent decrease in combined left ventricular (LV) systolic and diastolic function at 1, 3, 7, and 14 days after intraperitoneal administration and at 7 and 14 days after intrahippocampal DOM administration. LV dysfunction was matched by a similar time-dependent decrease in mitochondrial respiratory control, associated with increased proton leakage, and in mitochondrial enzyme activities. Microscopic examination of the LV midplane revealed evidence of progressive multifocal ischemic damage within the subendocardial, septal, and papillary regions. Lesions ranged from reversible early damage (vacuolization) to hypercontracture and inflammatory necrosis progressing to fibrotic scarring. Plasma proinflammatory IL-1α, IL-1β, and TNF-α cytokine levels were also increased from 3 days after seizure induction. The observed cardiomyopathies did not differ between intraperitoneal and intrahippocampal groups, providing strong evidence that cardiac damage after DOM exposure is a consequence of a seizure-evoked autonomic response.

Domoic Acid Impairment of Cardiac Energetics

Excitatory mediated neuronal injury has been shown to involve a complex cascade of events. However, the associated cardiac damage reported in humans and marine animals following exposure to excitotoxins has not been well characterized. We hypothesized that the excitotoxin domoic acid can traverse cardiac cell membranes and elicit a deleterious effect on cardiac mitochondrial energetics. Domoic acid (0.05-0.25 microM; 10 min) treatment of isolated rat cardiac mitochondria produced a marked decrease of both mitochondrial flavin adenine dinucleotide (FAD)- and nicotinamide adenine linked respiratory control indices (p < 0.001). Enzymatic assays of the mitochondrial electron transport chain (complexes I-V) and the mitochondrial matrix marker enzyme citrate synthase, showed marked concentration-dependent impairment in activity and integrity following exposure to domoic acid (p < 0.01). Similar mitochondrial effects were seen following exposure to the glutamic acid analog, kainic acid (0.5-2 microM). Domoic acid (0.05-10 microM; 40 min) was shown by competitive enzyme-linked immunosorbent assay to traverse the cellular membrane of H9c2 rat cardiac myoblasts. Exposure of intact H9c2 cells to domoic acid (10 microM; 24 h) impaired complex II-III activity but did not compromise cellular viability as assessed using cell quantification or lactate dehydrogenase leakage assays. Assessment of reactive oxygen species (superoxide and hydrogen peroxide) production in both isolated cardiac mitochondria and H9c2 cardiomyocytes failed to show any significant differences following exposure to domoic acid (0.05-5 microM). This is the first study to demonstrate a direct effect of domoic acid on cardiac mitochondrial energetics. However, the absence of substantial damage to intact cardiomyocytes raises questions regarding direct toxicological effects on cardiac energetics or viability under conditions of natural domoic acid exposure.

High-affinity [3H] kainic acid binding to brain membranes: a re-evaluation of ligand potency and selectivity

[3H]Kainic acid ([3H]KA) is a widely used tool for studying the KA class of excitatory amino acid receptors. [3H]KA of significantly higher specific activity has become available permitting use of radioligand concentrations below the dissociation constant (K(D)) of the high-affinity binding site. We employed low radioligand (0.05-0.2 nM) and receptor concentrations (0.01 nM) to gain new insights into the binding characteristics of the high-affinity KA binding site in a standard preparation of lyzed synaptosomal membranes from the cerebral cortex of male Sprague-Dawley rats. Under these conditions, KA binds to a single class of high-affinity sites with a K(D) of 1.0+/- 0.3 nM. The potencies of competing agents are considerably higher than published reports. Specifically, domoic acid, glutamate, and glutamine exhibit IC(50) values for displacing [3H]KA of 0.37+/-0.02, 94+/-13, and 1500+/-500 nM, respectively. Domoate (1 microM) was tested against a panel of 32 central nervous system binding sites and found to be inactive at each, indicating this toxin displays considerable selectivity. This study illustrates the remarkable potency of domoic acid and underlines the importance of performing radioligand binding studies at concentrations of constituents that permit characterization of high-affinity interactions.

NOESY on neurotoxins: NMR and conformational assignments of picrotoxins

Nuclear Overhauser effect spectroscopy (NOESY) gave full assignments of the 1H-NMR spectra of the picrotoxane neurotoxins tutin, hyenanchin, picrotoxinin and picrotin, as well as the solution conformations of these compounds, consistent with molecular modelling. Fully assigned 13C-NMR data are reported.