Future directions for epilepsy research.

The authors propose that epilepsy research embark on a revitalized effort to move from targeting control of symptoms to strategies for prevention and cure. The recent advances that make this a realistic goal include identification of genes mutated in inherited epilepsy syndromes, molecular characterization of brain networks, better imaging of sites of seizure origin, and developments in seizure prediction by quantitative EEG analysis. Research directions include determination of mechanisms of epilepsy development, identification of genes for common epilepsy syndromes through linkage analysis and gene chip technology, and validation of new models of epilepsy and epileptogenesis. Directions for therapeutics include identification of new molecular targets, focal methods of drug delivery tied to EEG activity, gene and cell therapy, and surgical and nonablative therapies. Integrated approaches, such as coupling imaging with electrophysiology, are central to progress in localizing regions of epilepsy development in people at risk and better seizure prediction and treatment for people with epilepsy.

A comparison of lorazepam, diazepam, and placebo for the treatment of out-of-hospital status epilepticus.

BACKGROUND: It is uncertain whether the administration of benzodiazepines by paramedics is an effective and safe treatment for out-of-hospital status epilepticus. METHODS: We conducted a randomized, double-blind trial to evaluate intravenous benzodiazepines administered by paramedics for the treatment of out-of-hospital status epilepticus. Adults with prolonged (lasting five minutes or more) or repetitive generalized convulsive seizures received intravenous diazepam (5 mg), lorazepam (2 mg), or placebo. An identical second injection was given if needed. RESULTS: Of the 205 patients enrolled, 66 received lorazepam, 68 received diazepam, and 71 received placebo. Status epilepticus had been terminated on arrival at the emergency department in more patients treated with lorazepam (59.1 percent) or diazepam (42.6 percent) than patients given placebo (21.1 percent) (P=0.001). After adjustment for covariates, the odds ratio for termination of status epilepticus by the time of arrival in the lorazepam group as compared with the placebo group was 4.8 (95 percent confidence interval, 1.9 to 13.0). The odds ratio was 1.9 (95 percent confidence interval, 0.8 to 4.4) in the lorazepam group as compared with the diazepam group and 2.3 (95 percent confidence interval, 1.0 to 5.9) in the diazepam group as compared with the placebo group. The rates of respiratory or circulatory complications (indicated by bag valve-mask ventilation or an attempt at intubation, hypotension, or cardiac dysrhythmia) after the study treatment was administered were 10.6 percent for the lorazepam group, 10.3 percent for the diazepam group, and 22.5 percent for the placebo group (P=0.08). CONCLUSIONS: Benzodiazepines are safe and effective when administered by paramedics for out-of-hospital status epilepticus in adults. Lorazepam is likely to be a better therapy than diazepam.

Differential regulation of basic helix-loop-helix mRNAs in the dentate gyrus following status epilepticus.

In various chemoconvulsant models of human temporal lobe epilepsy, the induction of epileptogenesis by a prolonged period of continuous seizure activity is accompanied by significant changes in hippocampal structure. These changes include an increase in neurogenesis within the proliferative subgranular zone (SGZ) of the dentate gyrus and induction of mossy fiber sprouting in mature dentate granule cells. As dentate granule cell neurogenesis and axon outgrowth are also hallmarks of hippocampal development, we hypothesized that molecules involved in normal development may also play a role in similar changes associated with epileptogenesis. To begin to test this hypothesis, we have analyzed the expression patterns of multiple members of the basic helix-loop-helix (bHLH) family of transcription factors in both normal and epileptic adult rats. bHLH protein expression has been found recently in dentate granule cells at specific developmental stages, and analysis of developmental models suggests specific neural differentiation functions for these molecules. We show that mRNA expression of all seven bHLH family members examined in this study, as well as the divergent homeobox protein Prox1, is present in the adult. Patterns of expression varied considerably between family members, ranging from the limited expression of Mash1 in the neurogenic SGZ of the dentate gyrus to the scattered, widespread profile of Hes5 throughout the dentate gyrus and the hippocampus proper. Moreover, these varied profiles of expression were differentially regulated following status epilepticus, with some increasing (Mash1, Id2), some falling (Hes5, Prox1), and others remaining mostly unchanged (NeuroD/BETA2, NeuroD2/NDRF, Id3, Rath2/Nex1). While the function of these molecules in the adult brain remains to be characterized, our findings support the idea that molecules controlling cell-fate decisions in the developing dentate gyrus are also operative during seizure-induced neurogenesis and plasticity.

Hippocampal heterotopia lack functional Kv4.2 potassium channels in the methylazoxymethanol model of cortical malformations and epilepsy.

Human cortical malformations often result in severe forms of epilepsy. Although the morphological properties of cells within these malformations are well characterized, very little is known about the function of these cells. In rats, prenatal methylazoxymethanol (MAM) exposure produces distinct nodules of disorganized pyramidal-like neurons (e.g., nodular heterotopia) and loss of lamination in cortical and hippocampal structures. Hippocampal nodular heterotopias are prone to hyperexcitability and may contribute to the increased seizure susceptibility observed in these animals. Here we demonstrate that heterotopic pyramidal neurons in the hippocampus fail to express a potassium channel subunit corresponding to the fast, transient A-type current. In situ hybridization and immunohistochemical analysis revealed markedly reduced expression of Kv4.2 (A-type) channel subunits in heterotopic cell regions of the hippocampus of MAM-exposed rats. Patch-clamp recordings from visualized heterotopic neurons indicated a lack of fast, transient (I(A))-type potassium current and hyperexcitable firing. A-type currents were observed on normotopic pyramidal neurons in MAM-exposed rats and on interneurons, CA1 pyramidal neurons, and cortical layer V-VI pyramidal neurons in saline-treated control rats. Changes in A-current were not associated with an alteration in the function or expression of delayed, rectifier (Kv2.1) potassium channels on heterotopic cells. We conclude that heterotopic neurons lack functional A-type Kv4.2 potassium channels and that this abnormality could contribute to the increased excitability and decreased seizure thresholds associated with brain malformations in MAM-exposed rats.

The prehospital treatment of status epilepticus (PHTSE) study: design and methodology.

Status epilepticus is a neurological emergency that is typically first encountered and managed in the prehospital environment. Although aggressive pharmacological treatment of status epilepticus is well established in the emergency department and hospital settings, the relative risks and benefits of active therapy for status epilepticus in the prehospital setting are not known. The Prehospital Treatment of Status Epilepticus (PHTSE) study is a prospective, randomized, double-blind, placebo-controlled study designed to address the following aims: (1) to determine whether administration of benzodiazepines by paramedics is an effective and safe means of treating status epilepticus in the prehospital setting and whether this therapy influences longer-term patient outcome, (2) to determine whether lorazepam is superior to diazepam for the treatment of status epilepticus in the prehospital setting, and (3) to determine whether control of status epilepticus prior to arrival to the emergency department influences patient disposition. The initial phase of the PHTSE study began in January 1994 and was completed in February 1999 after the successful enrollment of 205 patients into the three treatment arms. In this paper, we describe the rationale for the conceptualization of the study and details of the study design and methodology, and emphasize some aspects of study implementation that are unique to research involving the emergency medical system.

Wnt receptors and Wnt inhibitors are expressed in gradients in the developing telencephalon.

The caudomedial margin of the medial pallium, known as the cortical hem, expresses several Wnt genes that have been shown to be crucial for cortical development. We examined the expression of members of the Frizzled (mFz) family of Wnt receptors and the Secreted Frizzled Related Protein (SFRP) family of Wnt inhibitors during telencephalic development. We found that mFz-5 and mFz-8 are specifically expressed in the neocortical neuroepithelium and excluded from the hippocampal neuroepithelium in early telencephalic development, whereas mFz-9 and mFz-10 have expression domains confined to the medial pallium. In addition, SFRP-1 and SFRP-3 are expressed in opposing anterolateral to caudomedial gradients within the telencephalic ventricular zone throughout corticogenesis.

Pax-6 regulates expression of SFRP-2 and Wnt-7b in the developing CNS.

Wnt signaling regulates a wide range of developmental processes such as proliferation, cell migration, axon guidance, and cell fate determination. In this report, we studied the expression of secreted frizzled related protein-2 (SFRP-2), which codes for a putative Wnt inhibitor, in the developing nervous system. SFRP-2 is expressed in several discrete neuroepithelial domains, including the diencephalon, the insertion of the eminentia thalami into the caudal telencephalon, and the pallial-subpallial boundary (PSB). We also noted that Wnt-7b expression was similar to SFRP-2 expression. Because many of these structures are disrupted in Pax-6 mutant mice, we examined SFRP-2 and Wnt-7b expression in the forebrains of Pax-6 Sey/Sey mice. We found that Pax-6 mutants lack SFRP-2 expression in the PSB and diencephalon. Interestingly, Pax-6 mutants also lack Wnt-7b expression in the PSB, but Wnt-7b expression in the diencephalon is preserved. Furthermore, in the spinal cord of Pax-6 mutants, SFRP-2 and Wnt-7b expression was greatly reduced. Our results suggest that by virtue of its apposition to Wnt-7b expression, SFRP-2 may modulate its function, particularly at boundaries such as the PSB, and that changes in Wnt signaling contribute to the phenotype of Pax-6 mutants.

X-irradiation causes a prolonged reduction in cell proliferation in the dentate gyrus of adult rats.

The effects of X-irradiation on proliferating cells in the dentate subgranular zone were assessed in young adult Fisher 344 rats exposed to a range of X-ray doses and followed for up to 120 days. Apoptosis was quantified using morphology and end-labeling immunohistochemistry, and cell proliferation was detected using antibodies against the thymidine analog BrdU and the cyclin-dependent kinase p34(cdc2). Radiation-induced apoptosis occurred rapidly, with maximum morphological and end-labeling changes observed 3-6h after irradiation. Twenty-four hours after irradiation cell proliferation was significantly reduced relative to sham-irradiated controls. The number of apoptotic nuclei increased rapidly with radiation dose, reaching a plateau at about 3Gy. The maximum number of apoptotic nuclei was substantially higher than the number of proliferating cells, suggesting that non-proliferating as well as proliferating cells in the subgranular zone were sensitive to irradiation. Subgranular zone cell proliferation was significantly reduced relative to age-matched controls 120 days after doses of 5Gy or higher. These findings suggest that neural precursor cells of the dentate gyrus are very sensitive to irradiation and are not capable of repopulating the subgranular zone at least up to 120 days after irradiation. This may help explain, in part, how ionizing irradiation induces cognitive impairments in animals and humans.

Unique expression patterns of cell fate molecules delineate sequential stages of dentate gyrus development.

The dentate gyrus of the hippocampus is uniquely organized with a displaced proliferative zone that continues to generate dentate granule cells throughout life. We have analyzed the expression of Notch receptors, Notch ligands, and basic helix-loop-helix (bHLH) genes during dentate gyrus development to determine whether the need to maintain a pool of undifferentiated precursors is reflected in the patterns of expression of these genes. Many of these genes are expressed diffusely throughout the cortical neuroepithelium at embryonic days 16 and 17 in the rat, just preceding the migration of newly born granule cells and dentate precursor cells into the dentate anlage. However, at this time, Mash1, Math3, and Id3 expression are all concentrated in the area that specifically gives rise to granule cells and dentate precursor cells. Two days later, at the time of migration of the first granule cells and dentate precursor cells, cells expressing Mash1 are seen in the migratory route from the subventricular zone to the developing dentate gyrus. Newly born granule cells expressing NeuroD are also present in this migratory pathway. In the first postnatal week, precursor cells expressing Mash1 reside in the dentate hilus, and by the third postnatal week they have largely taken up their final position in the subgranular zone along the hilar side of the dentate granule cell layer. After terminal differentiation, granule cells born in the hilus or the subgranular zone begin to express NeuroD followed by NeuroD2. This study establishes that the expression patterns of bHLH mRNAs evolve during the formation of the dentate gyrus, and the precursor cells resident in the mature dentate gyrus share features with precursor cells found in development. Thus, many of the same mechanisms that are known to regulate cell fate and precursor pool size in other brain regions are likely to be operative in the dentate gyrus at all stages of development.

Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections.

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.

Loss of BETA2/NeuroD leads to malformation of the dentate gyrus and epilepsy.

BETA2/NeuroD is a homologue of the Drosophila atonal gene that is widely expressed during development in the mammalian brain and pancreas. Although studies in Xenopus suggest that BETA2/NeuroD is involved in cellular differentiation, its function in the mammalian nervous system is unclear. Here we show that mutant mice homozygous for a deletion at the BETA2/NeuroD locus fail to develop a granule cell layer within the dentate gyrus, one of the principal structures of the hippocampal formation. To understand the basis of this abnormality, we analyzed dentate gyrus development by using immunocytochemical markers in BETA2/NeuroD-deficient mice. The early cell populations in the dentate gyrus, including Cajal-Retzius cells and radial glia, are present and appear normally organized. The migration of dentate precursor cells and newly born granule cells from the neuroepithelium to the dentate gyrus remains intact. However, there is a dramatic defect in the proliferation of precursor cells once they reach the dentate and a significant delay in the differentiation of granule cells. This leads to malformation of the dentate granule cell layer and excess cell death. BETA2/NeuroD null mice also exhibit spontaneous limbic seizures associated with electrophysiological evidence of seizure activity in the hippocampus and cortex. These findings thus establish a critical role of BETA2/NeuroD in the development of a specific class of neurons. Furthermore, failure to express BETA2/NeuroD leads to a stereotyped pattern of pathological excitability of the adult central nervous system.

Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons.

GABAergic interneurons have major roles in hippocampal function and dysfunction. Here we provide evidence that, in mice, virtually all of these cells originate from progenitors in the basal telencephalon. Immature interneurons tangentially migrate from the basal telencephalon through the neocortex to take up their final positions in the hippocampus. Disrupting differentiation in the embryonic basal telencephalon (lateral and medial ganglionic eminences) through loss of Dlx1/2 homeobox function blocks the migration of virtually all GABAergic interneurons to the hippocampus. On the other hand, disrupting specification of the medial ganglionic eminence through loss of Nkx2.1 homeobox function depletes the hippocampus of a distinct subset of hippocampal interneurons. Loss of hippocampal interneurons does not appear to have major effects on the early development of hippocampal projection neurons nor on the pathfinding of afferrent tracts.

Prehospital stability of diazepam and lorazepam.

Injectable benzodiazepines are commonly stocked on ambulances for use by paramedics. We evaluated the stability of lorazepam and diazepam as a function of storage temperature. Diazepam (5 mg/mL) and lorazepam (2 mg/mL) injectable solutions were stored for up to 210 days in clear glass syringes at three conditions: 4 degrees C to 10 degrees C (refrigerated); 15 degrees C to 30 degrees C (on-ambulance ambient temperature); and 37 degrees C (oven-heated). High-performance liquid chromatography (HPLC) analyses of syringe contents were performed at 30-day intervals. After 210 days, the reduction in diazepam concentration was 7% refrigerated, 15% at ambient temperature, and 25% at 37 degrees C. The reduction in lorazepam concentration was 0% refrigerated, 10% at ambient temperature, and 75% at 37 degrees C. Whereas diazepam retained 90% of its original concentration for 30 days of on-ambulance storage, lorazepam retained 90% of its original concentration for 150 days. The decrease in lorazepam concentration correlated with an increase in the maximum ambient temperature in San Francisco. These results suggest that diazepam and lorazepam can be stored on ambulances. When ambient storage temperatures are 30 degrees C or less, ambulances carrying lorazepam and diazepam should be restocked every 30 to 60 days. When drug storage temperatures exceed 30 degrees C, more frequent stocking or refrigeration is required.

Status epilepticus: an overview of the clinical problem.

Status epilepticus has been recognized since antiquity. The terms état de mal and "status epilepticus" are derived from the slang used by epilepsy patients housed in Salpêtrière and Bicêtre hospitals in Paris during the 1800s. The definition of status epilepticus has been evolving, and is still not precise. In 1903-04 it was described as a development of epilepsy in which seizures are so frequent that "coma and exhaustion are continuous between seizures." In 1964 the International League Against Epilepsy adopted the definition "a seizure [that] persists for a sufficient length of time or is repeated frequently enough to produce a fixed and enduring epileptic condition." 30 min has been the most common specified duration of seizures for the diagnosis of status epilepticus, although a duration of 10 or 20 min has been suggested as well. However, a new set of definitions for generalized, convulsive status epilepticus in adults has been proposed and includes an operational definition (specifying a seizure duration of a least 5 min) and a mechanistic definition. In the future, laboratory tests will provide the means for detecting and defining the critical factors that distinguish a single epileptic seizure from status epilepticus. Recent epidemiological studies suggest status epilepticus occurs in 100,000 to 150,000 people in the US each year, and is associated with substantial morbidity and mortality. Etiology, duration of the seizures, and the patient's age seem to be important determinants of the outcome in status epilepticus.

Inhibition of dentate granule cell neurogenesis with brain irradiation does not prevent seizure-induced mossy fiber synaptic reorganization in the rat.

Aberrant reorganization of dentate granule cell axons, the mossy fibers, occurs in human temporal lobe epilepsy and rodent epilepsy models. Whether this plasticity results from the remodeling of preexisting mossy fibers or instead reflects an abnormality of developing dentate granule cells is unknown. Because these neurons continue to be generated in the adult rodent and their production increases after seizures, mossy fibers that arise from either developing or mature granule cells are potential substrates for this network plasticity. Therefore, to determine whether seizure-induced, mossy fiber synaptic reorganization arises from either developing or mature granule cell populations, we used low-dose, whole-brain x-irradiation to eliminate proliferating dentate granule cell progenitors in adult rats. A single dose of 5 Gy irradiation blocked cell proliferation and eliminated putative progenitor cells in the dentate subgranular proliferative zone. Irradiation 1 d before pilocarpine-induced status epilepticus significantly attenuated dentate granule cell neurogenesis after seizures. Two irradiations, 1 d before and 4 d after status epilepticus, essentially abolished dentate granule cell neurogenesis but failed to prevent mossy fiber reorganization in the dentate molecular layer. These results indicate that dentate granule cell neurogenesis in the mature hippocampal formation is vulnerable to the effects of low-dose ionizing irradiation. Furthermore, the development of aberrant mossy fiber remodeling in the absence of neurogenesis suggests that mature dentate granule cells contribute substantially to seizure-induced network reorganization.

Status epilepticus: new concepts.

Generalized convulsive status epilepticus is a neurological emergency characterized by abnormally prolonged seizures. This review emphasizes recent developments that bear on our understanding of the pathophysiology and management of status epilepticus. Topics include GABAA receptor modulation during prolonged seizures, the role of genetics in susceptibility to status epilepticus, neuron-specific enolas, the Veterans Administration Cooperative Study Group trial comparison of various drug regimens, utility of the electroencephalogram in patient monitoring, emerging drug therapies and patient management in out-of-hospital settings.

The role of diversity in the health care needs of California.

Socioeconomic, racial, and ethic diversity influence many facets of health care, including access to health services, the morbidity and mortality of disease, and the process of training health care professionals. California has an increasingly diverse population, and it is essential that the University of California medical schools carefully address the role of diversity in medical education and in education for the many other professionals who have a key role in the provision of care and services to Californians. In this paper, we provide evidence in support of the belief that the University of California system must train a physician workforce that reflects the racial, ethnic, and socioeconomic characteristics of California's population in order to provide equitable health care to all segments of the state's people.