Steven M. Rothman

Excitotoxity and the NMDA receptor

Abstract The same receptors for excitatory amino acids (EAA) that mediate direct neuronal depolarization can also be responsible for neuronal injury. Prolonged stimulation of EAA receptors of either the N-methyl-d-aspartate (NMDA) or non-NMDA types eventually results in the death of most central neurons. The exact mechanism(s) of cell injury is complicated, since depolarization and neuronal swelling, calcium influx, and possibly second messengers all contribute. Evidence is accumulating that the brain damage associated with anoxia, stroke, hypoglycemia, epilepsy, and perhaps neurodegenerative illnesses such as Huntingtons disease may be at least partially produced by excessive activation of NMDA receptors. To the extent that the pathophysiology can be explained by this mechanism, it may be amenable to rational therapies now under development.

Intracellular calcium concentrations during "chemical hypoxia" and excitotoxic neuronal injury

Because hypoxic/ischemic neurodegeneration appears to be in part linked to glutamate neurotoxicity, we measured intracellular calcium (Ca2+i) levels in cultured hippocampal neurons during exposure to toxic doses of glutamate (GLU) and to an anoxic environment simulated by sodium cyanide (NaCN). Changes in Ca2+i produced by cyanide greatly exceeded those induced by GLU. The NaCN response was mimicked when oxidative metabolism was also disrupted by sodium azide, oligomycin, or dinitrophenol. Noncompetitive NMDA receptor antagonists and enzymatic GLU degradation abolished the GLU-induced Ca2+i increases and attenuated those produced by NaCN. Only NaCN-induced increases were blocked when dantrolene and ruthenium red were applied to prevent release from intracellular pools. All responses were reduced proportionally in the absence of added external calcium. These results suggest that extracellular GLU accumulation and subsequent activation of GLU receptors were involved in the NaCN response. During such metabolic compromise, however, GLU-induced elevations of Ca2+i were enormously amplified. In parallel toxicity studies, NaCN was not neurotoxic despite the large elevations in Ca2+i, indicating that a general elevation in cytoplasmic calcium does not necessarily predict neurodegeneration.

Ionized Intracellular Calcium Concentration Predicts Excitotoxic Neuronal Death: Observations with Low-Affinity Fluorescent Calcium Indicators

Cytosolic calcium ([Ca2+]i) is an important mediator of neuronal signal transduction, participating in diverse biochemical reactions that elicit changes in synaptic efficacy, metabolic rate, and gene transcription. Excessive [Ca2+]i also has been implicated as a cause of acute neuronal injury, although measurement of [Ca2+]i in living neurons by fluorescent calcium indicators has not consistently demonstrated a correlation between [Ca2+]i and the likelihood of neuronal death after a variety of potentially lethal insults. Using fluorescence videomicroscopy and microinjected calcium indicators, we measured [Ca2+]i in cultured cortical neurons during intense activation with either NMDA (300 μm) or AMPA (450 μm). At these concentrations NMDA killed >80% of the cultured neurons by the next day, whereas neuronal death from AMPA was <20%. Using the conventional calcium indicator, fura-2/AM, we estimated [Ca2+]i elevations to be ∼300–400 nm during exposure to either glutamate agonist. In contrast, indicators with lower affinity for calcium, benzothiazole coumarin (BTC), and fura-2/dextran reported [Ca2+]i levels >5 μmduring lethal NMDA exposure, but [Ca2+]i levels were <1.5 μm during nonlethal activation of AMPA receptors or voltage-gated calcium channels. Fura-2 reported [Ca2+]i responses during brief exposure to glutamate, NMDA, AMPA, kainate, and elevated extracellular K+ between 0.5 and 1 μm. With the use of BTC, only NMDA and glutamate exposures resulted in micromolar [Ca2+]i levels. Neurotoxic glutamate receptor activation is associated with sustained, micromolar [Ca2+]i elevation. The widely used calcium indicator fura-2 selectively underestimates [Ca2+]i, depending on the route of entry, even at levels that appear to be within its range of detection.

Multiple mechanisms of picrotoxin block of GABA‐induced currents in rat hippocampal neurons.

1. We have examined the effect of picrotoxin on GABA‐induced currents in dissociated rat hippocampal neurons. In addition, we used the putative picrotoxin receptor antagonist, alpha‐isopropyl‐alpha‐methyl‐gamma‐butyrolactone (alpha IMGBL), and the picrotoxin agonist, beta‐ethyl‐beta‐methyl‐gamma‐butyrolactone (beta EMGBL) to explore the mechanisms of picrotoxins interaction with the GABA‐Cl‐ receptor‐ionophore complex. 2. The picrotoxin block of GABA current was use dependent, suggesting that the site of picrotoxin block is exposed by the conformational change initiated by GABA binding to the receptor. 3. The alkyl‐substituted butyrolactone antagonist, alpha IMGBL, selectively blocked the use‐dependent mechanism of picrotoxin effect. After the apparent complete inhibition of the use‐dependent effect, there was a residual picrotoxin effect that was independent of the time or concentration of GABA application. This indicates that the picrotoxin block of the GABA current is mediated by two different mechanisms. alpha IMGBL influences just one of these mechanisms. 4. The picrotoxin receptor agonist, beta EMGBL, exclusively blocked the GABA current in a use‐dependent manner. Consistent with a use‐dependent mechanism, the rate of onset of block increased with GABA concentration. Surprisingly, the fraction of GABA current block decreased with increasing GABA concentration. 5. These results suggest that the relationship of picrotoxin and gamma‐butyrolactones with the GABA‐Cl‐ receptor‐ionophore is quite complex. They are consistent with at least two possible models of agonist‐antagonist interactions. Both cases require different antagonist affinities for the various kinetic states of the GABA‐Cl‐ receptor‐ionophore. However, there is no need to require that either picrotoxin or beta EMGBL acts as an open channel blocker.

Outcomes after allogeneic hematopoietic cell transplantation for childhood cerebral adrenoleukodystrophy: the largest single-institution cohort report

Cerebral adrenoleukodystrophy (cALD) remains a devastating neurodegenerative disease; only allogeneic hematopoietic cell transplantation (HCT) has been shown to provide long-term disease stabilization and survival. Sixty boys undergoing HCT for cALD from 2000 to 2009 were analyzed. The median age at HCT was 8.7 years; conditioning regimens and allograft sources varied. At HCT, 50% demonstrated a Loes radiographic severity score ≥ 10, and 62% showed clinical evidence of neurologic dysfunction. A total of 78% (n = 47) are alive at a median 3.7 years after HCT. The estimate of 5-year survival for boys with Loes score < 10 at HCT was 89%, whereas that for boys with Loes score ≥ 10 was 60% (P = .03). The 5-year survival estimate for boys absent of clinical cerebral disease at HCT was 91%, whereas that for boys with neurologic dysfunction was 66% (P = .08). The cumulative incidence of transplantation-related mortality at day 100 was 8%. Post-transplantation progression of neurologic dysfunction depended significantly on the pre-HCT Loes score and clinical neurologic status. We describe the largest single-institution analysis of survival and neurologic function outcomes after HCT in cALD. These trials were registered at www.clinicaltrials.gov as #NCT00176904, #NCT00668564, and #NCT00383448.

Diazoxide blocks glutamate desensitization and prolongs excitatory postsynaptic currents in rat hippocampal neurons

1. The effects of diazoxide (DZ) on synaptic transmission and upon responses to exogenously applied glutamate agonists were examined in cultured hippocampal neurons. 2. DZ reversibly increased the peak amplitude of evoked excitatory postsynaptic currents (EPSCs) to 150 +/‐ 100% of control and prolonged the EPSC decay time constant (tau) from 5.9 +/‐ 1.2 ms to 14 +/‐ 6.2 ms (240% of control). 3. Peak and steady‐state glutamate (Glu) and quisqualate (QA) currents activated by exogenous application were dramatically increased by DZ at concentrations which did not influence N‐methyl‐D‐aspartate (NMDA), kainate (KA), or GABA currents. These effects were rapidly and completely reversible. Active and passive membrane properties were unaffected by DZ. 4. Inhibitory postsynaptic currents (IPSCs) were unaffected by the same DZ concentrations. 5. These experiments indicate that desensitization plays an important role in terminating excitatory transmission between mammalian central neurons. DZ and perhaps related compounds will ultimately help us identify the regions of the AMPA/KA receptor responsible for desensitization.

Kainate Seizures Cause Acute Dendritic Injury and Actin Depolymerization In Vivo

Seizures may cause brain injury via a variety of mechanisms, potentially contributing to cognitive deficits in epilepsy patients. Although seizures induce neuronal death in some situations, they may also have “nonlethal” pathophysiological effects on neuronal structure and function, such as modifying dendritic morphology. Previous studies involving conventional fixed tissue analysis have demonstrated a chronic loss of dendritic spines after seizures in animal models and human tissue. More recently, in vivo time-lapse imaging methods have been used to monitor acute changes in spines directly during seizures, but documented spine loss only under severe conditions. Here, we examined effects of secondary generalized seizures induced by kainate, on dendritic structure of neocortical neurons using multiphoton imaging in live mice in vivo and investigated molecular mechanisms mediating these structural changes. Higher-stage kainate-induced seizures caused dramatic dendritic beading and loss of spines within minutes, in the absence of neuronal death or changes in systemic oxygenation. Although the dendritic beading improved rapidly after the seizures, the spine loss recovered only partially over a 24 h period. Kainate seizures also resulted in activation of the actin-depolymerizing factor, cofilin, and a corresponding decrease in filamentous actin, indicating that depolymerization of actin may mediate the morphological dendritic changes. Finally, an inhibitor of the calcium-dependent phosphatase, calcineurin, antagonized the effects of seizures on cofilin activation and spine morphology. These dramatic in vivo findings demonstrate that seizures produce acute dendritic injury in neocortical neurons via calcineurin-dependent regulation of the actin cytoskeleton, suggesting novel therapeutic targets for preventing seizure-induced brain injury.

Leptin inhibits 4-aminopyridine– and pentylenetetrazole-induced seizures and AMPAR-mediated synaptic transmission in rodents

Leptin is a hormone that reduces excitability in some hypothalamic neurons via leptin receptor activation of the JAK2 and PI3K intracellular signaling pathways. We hypothesized that leptin receptor activation in other neuronal subtypes would have anticonvulsant activity and that intranasal leptin delivery would be an effective route of administration. We tested leptins anticonvulsant action in 2 rodent seizure models by directly injecting it into the cortex or by administering it intranasally. Focal seizures in rats were induced by neocortical injections of 4-aminopyridine, an inhibitor of voltage-gated K+ channels. These seizures were briefer and less frequent upon coinjection of 4-aminopyridine and leptin. In mice, intranasal administration of leptin produced elevated brain and serum leptin levels and delayed the onset of chemical convulsant pentylenetetrazole-induced generalized convulsive seizures. Leptin also reduced neuronal spiking in an in vitro seizure model. Leptin inhibited alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor-mediated synaptic transmission in mouse hippocampal slices but failed to inhibit synaptic responses in slices from leptin receptor-deficient db/db mice. JAK2 and PI3K antagonists prevented leptin inhibition of AMPAergic synaptic transmission. We conclude that leptin receptor activation and JAK2/PI3K signaling may be novel targets for anticonvulsant treatments. Intranasal leptin administration may have potential as an acute abortive treatment for convulsive seizures in emergency situations.

Decision-making for termination of pregnancies with fetal anomalies: analysis of 53,000 pregnancies.

OBJECTIVE To evaluate the degree to which prenatal knowledge of fetal anomalies and sociodemographic characteristics determined outcome of 53,000 pregnancies. METHODS Pregnancies were consecutively evaluated at a university hospital between 1984 and 1997. The severity of anomalies was graded by using an ordinal scale, in which 0 was no anomalies, 1 was no impact on quality of life, 2 was little impact but possibly requiring medical therapy, 3 was serious impact on quality of life even with optimal medical therapy, and 4 was incompatible with life. RESULTS The abortion rates for grades 1 and 3 anomalies increased from 0.9% to 72.5%, and 0.9% to 37.1% for central nervous system and non–central nervous system anomalies, respectively (P < .001). Multiple logistic regression showed that mothers without a high school education were more likely than those who completed high school to abort a normal pregnancy (odds ratio [OR] 1.62, 95% confidence interval [CI] 1.07, 2.45). In the 452 pregnancies in which there was one grade 3 anomaly, logistic regression also showed that the abortion rate decreased by 6% per year as maternal age decreased (OR 0.94, 95% CI 0.91, 0.97). CONCLUSIONS The severity of anomalies directly correlates with abortion rates, but at similar degrees of severity, central nervous system anomalies are more likely to lead to abortion. Maternal level of education inversely correlates with likelihood of termination of a normal pregnancy, whereas maternal age directly correlates with pregnancy termination when serious anomalies are present. Serious congenital anomalies may disproportionately affect children from families with the youngest mothers because these mothers are likely to continue these pregnancies.

Prolonged exposure to levetiracetam reveals a presynaptic effect on neurotransmission.

Summary:  Purpose: The antiepileptic drug levetiracetam (LEV) is an enigma. Despite the fact that it specifically binds to the presynaptic vesicle protein, SV2A, no satisfactory mechanism of action has yet been identified. Using a combination of electrophysiological and cellular imaging techniques, we carefully tested the hypothesis that LEV directly interferes with neurotransmitter release.