Antiseizure and neuroprotective effects of delayed treatment with midazolam in a rodent model of organophosphate exposure.

OBJECTIVE: Exposure to organophosphates (OPs) and OP nerve agents (NAs) causes status epilepticus (SE) and irreversible brain damage. Rapid control of seizure activity is important to minimize neuronal injury and the resulting neurological and behavioral disorders; however, early treatment will not be possible after mass release of OPs or NAs. METHODS: We utilized a delayed-treatment model of OP exposure in adult rats by administration of diisopropyl fluorophosphate (DFP) to study the relationship between the antiseizure and neuroprotective effects of the "standard-of-care" benzodiazepine, midazolam (MDZ), when given at 30, 60, and 120 minutes after SE onset. After electroencephalography (EEG) recordings, neural damage in serial brain sections was studied with Fluoro-Jade B staining. RESULTS: MDZ-induced seizure suppression was equivalent in magnitude regardless of treatment delay (ie, seizure duration). When assessed globally (ie, normalized across 10 different brain regions) for each treatment delay, MDZ administration resulted in only nonsignificant reductions in neuronal death. However, when data for MDZ treatment were combined from all three delay times, a small but significant reduction in global neuronal death was detected when compared to vehicle treatment, which indicated that the substantive MDZ-induced seizure suppression led to only a small reduction in neuronal death. SIGNIFICANCE: In conclusion, MDZ significantly reduced DFP-induced SE intensity when treatment was delayed 30, 60, and even up to 120 minutes; however, this reduction in seizure intensity had no detectable effect on neuronal death at each individual delay time. These data show that although MDZ suppressed seizures, additional neuroprotective therapies are needed to mitigate the effects of OP exposure.

Staged anticonvulsant screening for chronic epilepsy.

OBJECTIVE: Current anticonvulsant screening programs are based on seizures evoked in normal animals. One-third of epileptic patients do not respond to the anticonvulsants discovered with these models. We evaluated a tiered program based on chronic epilepsy and spontaneous seizures, with compounds advancing from high-throughput in vitro models to low-throughput in vivo models. METHODS: Epileptogenesis in organotypic hippocampal slice cultures was quantified by lactate production and lactate dehydrogenase release into culture media as rapid assays for seizure-like activity and cell death, respectively. Compounds that reduced these biochemical measures were retested with in vitro electrophysiological confirmation (i.e., second stage). The third stage involved crossover testing in the kainate model of chronic epilepsy, with blinded analysis of spontaneous seizures after continuous electrographic recordings. RESULTS: We screened 407 compound-concentration combinations. The cyclooxygenase inhibitor, celecoxib, had no effect on seizures evoked in normal brain tissue but demonstrated robust antiseizure activity in all tested models of chronic epilepsy. INTERPRETATION: The use of organotypic hippocampal cultures, where epileptogenesis occurs on a compressed time scale, and where seizure-like activity and seizure-induced cell death can be easily quantified with biomarker assays, allowed us to circumvent the throughput limitations of in vivo chronic epilepsy models. Ability to rapidly screen compounds in a chronic model of epilepsy allowed us to find an anticonvulsant that would be missed by screening in acute models.

Inhibition of the prostaglandin receptor EP2 following status epilepticus reduces delayed mortality and brain inflammation.

Prostaglandin E2 is now widely recognized to play critical roles in brain inflammation and injury, although the responsible prostaglandin receptors have not been fully identified. We developed a potent and selective antagonist for the prostaglandin E2 receptor subtype EP2, TG6-10-1, with a sufficient pharmacokinetic profile to be used in vivo. We found that in the mouse pilocarpine model of status epilepticus (SE), systemic administration of TG6-10-1 completely recapitulates the effects of conditional ablation of cyclooxygenase-2 from principal forebrain neurons, namely reduced delayed mortality, accelerated recovery from weight loss, reduced brain inflammation, prevention of blood-brain barrier opening, and neuroprotection in the hippocampus, without modifying seizures acutely. Prolonged SE in humans causes high mortality and morbidity that are associated with brain inflammation and injury, but currently the only effective treatment is to stop the seizures quickly enough with anticonvulsants to prevent brain damage. Our results suggest that the prostaglandin receptor EP2 is critically involved in neuroinflammation and neurodegeneration, and point to EP2 receptor antagonism as an adjunctive therapeutic strategy to treat SE.

Unique signaling profiles of positive allosteric modulators of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity.

BACKGROUND: Metabotropic glutamate receptor subtype 5 (mGlu5) activators have emerged as a novel approach to the treatment of schizophrenia. Positive allosteric modulators (PAMs) of mGlu5 have generated tremendous excitement and fueled major drug discovery efforts. Although mGlu5 PAMs have robust efficacy in preclinical models of schizophrenia, preliminary reports suggest that these compounds may induce seizure activity. Prototypical mGlu5 PAMs do not activate mGlu5 directly but selectively potentiate activation of mGlu5 by glutamate. This mechanism may be critical to maintaining normal activity-dependence of mGlu5 activation and achieving optimal in vivo effects. METHODS: Using specially engineered mGlu5 cell lines incorporating point mutations within the allosteric and orthosteric binding sites, as well as brain slice electrophysiology and in vivo electroencephalography and behavioral pharmacology, we found that some mGlu5 PAMs have intrinsic allosteric agonist activity in the absence of glutamate. RESULTS: Both in vitro mutagenesis and in vivo pharmacology studies demonstrate that VU0422465 is an agonist PAM that induces epileptiform activity and behavioral convulsions in rodents. In contrast, VU0361747, an mGlu5 PAMs optimized to eliminate allosteric agonist activity, has robust in vivo efficacy and does not induce adverse effects at doses that yield high brain concentrations. CONCLUSIONS: Loss of the absolute dependence of mGlu5 PAMs on glutamate release for their activity can lead to severe adverse effects. The finding that closely related mGlu5 PAMs can differ in their intrinsic agonist activity provides critical new insights that is essential for advancing these molecules through clinical development for treatment of schizophrenia.

The effect of the cannabinoid-receptor antagonist, SR141716, on the early stage of kainate-induced epileptogenesis in the adult rat.

Pretreatment with the endocannabinoid-receptor antagonist, SR141716, has been reported to suppress the long-lasting hyperexcitability and increased seizure susceptibility present after 30 min of hyperthermia-induced convulsions in immature rats, an animal model of complex febrile seizures in children, which may be a cause of temporal lobe epilepsy. The present experiments tested the hypothesis that SR141716 suppresses epileptogenesis in the adult kainate model, an animal model of temporal lobe epilepsy. Adult male rats (n = 35), implanted for electroencephalography (EEG) recordings, were treated with kainate. Immediately after the first acute electrographic seizure during kainate-induced status epilepticus, either vehicle or SR141716 (10 mg/kg) was injected intraperitoneally. Chronic video-EEG data were collected for the first 2-week period after kainate-induced status epilepticus. More than one-half of both the vehicle- and drug-treated animals showed spontaneous recurrent seizures. Similarly, mean seizure frequency did not differ significantly for the drug- and vehicle-treated animals during the first 2 weeks (n = 9 and 8, respectively). Therefore, no significant differences were found between SR141716-treated and control animals during the first 2 weeks of epileptogenesis. These results suggest that the endocannabinoid-receptor antagonist, SR141716, had no detectable effect on the early stages of epileptogenesis in the adult kainate model. We discuss several potential explanations for the differences in the effects of SR141716 in the adult-rat, kainate versus immature-rat, hyperthermia models.

Detection and localization of an estrogen receptor beta splice variant protein (ERbeta2) in the adult female rat forebrain and midbrain regions.

Estrogens regulate neural processes such as neuronal development, reproductive behavior, and hormone secretion, and signal through estrogen receptor (ER) alpha and ERbeta (here called ERbeta1). Recent studies have found variations in ERalpha and ERbeta1 mRNA splicing in rodents and humans. Functional reporter gene assays suggest that these splicing variations alter ER-mediated transcriptional regulation. Estrogen receptor beta 2 (ERbeta2), an ERbeta1 splice variant containing an 18 amino acid (AA) insert in the ligand binding domain, binds estradiol with approximately 10-fold lower affinity than ERbeta1, suggesting that it may serve as a low-affinity ER. Moreover, ERbeta2 reportedly acts in a dominant-negative fashion when heterodimerized with ERbeta1 or ERalpha. To explore the function of ERbeta2 in brain, an antiserum (TwobetaER.1) targeting the 18 AA insert was developed and characterized. Western blot analysis and transient expression of ERbeta2 in cell lines demonstrated that TwobetaER.1 recognizes ERbeta2. In the adult female rat brain, ERbeta2 immunoreactivity is localized in the cell nucleus and is expressed with a distribution similar to that of ERbeta1 mRNA. ERbeta2 immunoreactive cell numbers were high in, for example, piriform cortex, paraventricular nucleus, supraoptic nucleus, arcuate nucleus, and hippocampal CA regions, whereas it was low in the dentate gyrus. Moreover, ERbeta2 is coexpressed in gonadotropin-releasing hormone and oxytocin neurons. These studies demonstrate ERbeta splice variant proteins in brain and support the hypothesis that ER signaling diversity depends not only on ligand or coregulatory proteins, but also on regional and phenotypic selectivity of ER splice variant proteins.

Epilepsy and reproductive disorders: the role of the gonadotropin-releasing hormone network.

Individuals with temporal lobe epilepsy have an increased incidence of reproductive dysfunction. The comorbidity may be due to the acute effects of the seizures, the chronic effects of the epilepsy, and/or the use of antiepileptic drugs on the gonadotropin-releasing hormone network and the hypothalamic-pituitary-gonadal axis. This review provides a brief overview of evidence from experimental animal and clinical studies exploring the basis for epilepsy-associated reproductive abnormalities.

Evidence that androgen acts through NMDA receptors to affect motoneurons in the rat spinal nucleus of the bulbocavernosus.

In adult male rats, spinal nucleus of the bulbocavernosus (SNB) motoneurons shrink after castration and are restored in size after androgen treatment. Sixty-day-old Sprague Dawley males were castrated and implanted with SILASTIC capsules containing testosterone (T) or nothing, and osmotic minipumps continuously infusing MK-801, a noncompetitive NMDA receptor antagonist, or saline. Twenty-five days later, bulbocavernosus muscles were injected with the retrograde tracer cholera toxin-horseradish peroxidase conjugate (CT-HRP) to label SNB cells. As seen previously, among saline-treated rats, SNB somata of T-treated castrates were significantly larger than those of castrates receiving blank capsules (p < 0.0001). MK-801 treatment blocked this effect of T on the SNB. MK-801 had no effect on non-androgen-responsive spinal motoneurons in the neighboring retrodorsolateral nucleus (RDLN), nor did the drug affect SNB soma size in the absence of androgen treatment. Motoneuronal soma size in Nissl stain revealed the same pattern of results seen with CT-HRP fills. In situ hybridization indicated that SNB motoneurons express mRNA for the NMDA receptor subunits R1, R2a, and R2b. Castration reduced the expression of R1 mRNA in SNB motoneurons, an effect that was blocked by androgen replacement in castrates. R2A and R2B mRNA expression in SNB cells was not affected by androgen manipulations. Likewise, androgen manipulations had no effect on the expression of any NMDA receptor subtypes in RDLN motoneurons. These results suggest that androgen affects the size of SNB motoneurons by influencing their expression of the NMDA receptor, and therefore the response of the motoneurons to endogenous glutamate.

Episodic bursting activity and response to excitatory amino acids in acutely dissociated gonadotropin-releasing hormone neurons genetically targeted with green fluorescent protein.

The gonadotropin-releasing hormone (GnRH) system, considered to be the final common pathway for the control of reproduction, has been difficult to study because of a lack of distinguishing characteristics and the scattered distribution of neurons. The development of a transgenic mouse in which the GnRH promoter drives expression of enhanced green fluorescent protein (EGFP) has provided the opportunity to perform electrophysiological studies of GnRH neurons. In this study, neurons were dissociated from brain slices prepared from prepubertal female GnRH-EGFP mice. Both current- and voltage-clamp recordings were obtained from acutely dissociated GnRH neurons identified on the basis of EGFP expression. Most isolated GnRH-EGFP neurons fired spontaneous action potentials (recorded in cell-attached or whole-cell mode) that typically consisted of brief bursts (2-20 Hz) separated by 1-10 sec. At more negative resting potentials, GnRH-EGFP neurons exhibited oscillations in membrane potential, which could lead to bursting episodes lasting from seconds to minutes. These bursting episodes were often separated by minutes of inactivity. Rapid application of glutamate or NMDA increased firing activity in all neurons and usually generated small inward currents (<15 pA), although larger currents were evoked in the remaining neurons. Both AMPA and NMDA receptors mediated the glutamate-evoked inward currents. These results suggest that isolated GnRH-EGFP neurons from juvenile mice can generate episodes of repetitive burst discharges that may underlie the pulsatile secretion of GnRH, and glutamatergic inputs may contribute to the activation of endogenous bursts.