SUMMARY

γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. GABAA receptors are heteropentamers formed by assembly of multiple subunits that generate a wide array of receptors with particular distribution and pharmacological profiles. Malfunction of these receptors has been associated with the pathophysiology of epilepsy and contribute to an imbalance of excitatory and inhibitory neurotransmission. The process of epilepsy development (epileptogenesis) is associated with changes in the expression and function of a large number of gene products. One of the major challenges is to effectively determine which changes directly contribute to epilepsy development versus those that are compensatory or not involved in the pathology. Substantial evidence suggests that changes in the expression and function of GABAA receptors are involved in the pathogenesis of epilepsy. Identification of the mechanisms involved in GABAA receptor malfunction during epileptogenesis and the ability to reverse this malfunction are crucial steps towards definitively answering this question and developing specific and effective therapies.

Objective

To analyze the spectrum of neurological manifestations in children hospitalized with pandemic influenza A H1N1 virus of 2009 (pH1N1).

Design

Retrospective case series of children hospitalized from May 1, 2009, through November 30, 2009.

Setting

Tertiary-care children’s hospital in Colorado.

Patients

All hospitalized patients with pH1N1 with neurological consult or diagnosis, lumbar puncture, electroencephalogram, or neuroimaging were selected as suspected cases. These were systematically reviewed and selected for final analysis if confirmed by pre-established definitions as a neurological complication.

Results

Of 307 children with pH1N1, 59 were selected as having suspected cases of neurological complications. Twenty-three children were confirmed to have a neurological complication. Of these 23, 15 (65%) required intensive care monitoring. The median length of stay was 4 days. Seventeen (74%) had a preexisting neurological diagnosis. The most common manifestation was seizure with underlying neurological disease (in 62% of cases) followed by encephalopathy with or without neuroimaging changes (in 26% of cases). Results from a lumbar puncture showed elevated protein levels in 3 of 6 patients but no significant pleocytosis. Seven of the 9 electroencephalograms showed diffuse slowing, and findings from magnetic resonance imaging were abnormal in 5 of 6 children. Deaths occurred in 13% of patients, and short-term disability in 22%.

Conclusions

Children infected with pH1N1 presented with a wide spectrum of neurological manifestations, which occurred primarily in individuals with preexisting neurological conditions. These individuals had a severe disease course, evidenced by need for intensive care services and relatively high rates of mortality or neurological disability. Children with underlying neurological conditions should be particularly targeted for influenza prevention and aggressive supportive treatment at the onset of influenzalike symptoms.

Major depression is a debilitating psychiatric disease that may be precipitated by a dysregulation of stress neurocircuitry caused by chronic or severe stress exposure. Moreover, hyperresponsivity to stressors correlates with depressed mood and may contribute to the etiology of major depression. The serotonergic dorsal raphe nucleus (DRN) is an important site in the neurocircuitry underlying behavioral responses to stressors, and is tightly regulated, in part, by a combination of intrinsic cell properties, autoinhibition, and GABAergic synaptic transmission. The stress-related neurotransmitter corticotropin-releasing factor (CRF) modulates DRN neuronal excitability and subsequent 5-HT release in the forebrain. Wistar Kyoto (WKY) rats exhibit exaggerated behavioral responses to stressors, that is, stress hyperresponsivity, and are considered an animal model of depression. To better understand the neurobiological basis of the stress hyperresponsivity, we used a combination of mRNA analysis and whole-cell electrophysiological techniques to measure differences in intrinsic activity and receptor response, in 5-HT- and non-5-HT-containing neurons of the DRN in WKY rats compared with Sprague-Dawley controls. In the WKY rat, there was a decrease in the neuronal excitability of 5-HT neurons coupled with decreased TPH2 production. Additionally, we found that CRF did not increase GABAergic activity in 5-HT neurons as is normally seen in 5-HT neurons of Sprague-Dawley controls. The CRF modulation of 5-HT DRN neurotransmission at the single-cell level is selectively disrupted in the WKY animal model of depression and may be one of the cellular correlates underlying depression.

Summary

Intellectual and developmental disabilities (IDD) such as Autistic Spectrum Disorders (ASD) and epilepsies are heterogeneous disorders that have diverse etiologies and pathophysiologies. The high rate of co-occurrence of these disorders, however, suggest potentially shared underlying mechanisms. A number of well-known genetic disorders share epilepsy, intellectual disability and autism as prominent phenotypic features, including tuberous sclerosis, Rett syndrome, and fragile X. In addition, mutations of several genes involved in neurodevelopment, including ARX, DCX, neuroligins and neuropilin2 have been identified in children with epilepsy, IDD, ASD or a combination of thereof. Finally, in animal models, early life seizures can result in cellular and molecular changes that could contribute to learning and behavioral disabilities. Increased understanding of the common genetic, molecular and cellular mechanisms of IDD, ASD and epilepsy may provide insight into their underlying pathophysiology and elucidate new therapeutic approaches for these conditions.

The perineuronal net (PN), a component of the neural extracellular matrix (ECM), is a dynamic structure whose expression decreases following diminished physiological activity. Here, we analyzed the effects of increased neuronal activity on the development of aggrecan, a component of the PN, in the hippocampus. We show aggrecan expression to be prominent around parvalbumin (PV) interneurons in the postnatal hippocampus. Moreover, after seizure induction in early life there was a significant increase in aggrecan expression in a region specific manner during the course of development. We conclude that increased neuronal activity leads to accelerated expression of PNs in the hippocampus that attenuates in the adult hippocampus. This study shows the dynamic nature of the PN component of the ECM and the role neuronal activity has in molding the extracellular milieu of inhibitory interneurons.

Because epilepsy often occurs during development, understanding the mechanisms by which this process takes place (epileptogenesis) is important. In addition, the age-specificity of seizures and epilepsies of the neonatal, infancy, and childhood periods suggests that the processes and mechanisms that culminate in epilepsy might be age specific as well. Here we provide an updated review of recent and existing literature and discuss evidence that neuronal loss may occur during epileptogenesis in the developing brain, but is not required for the epileptogenic process. We speculate about the mechanisms for the resilience of neurons in immature limbic structures to epileptogenic insults, and propose that the type, duration and severity of these insults influence the phenomenology of the resulting spontaneous seizures.

Mutations in the X-linked aristaless-related homeobox gene (ARX) have been linked to structural brain anomalies as well as multiple neurocognitive deficits. The generation of Arx-deficient mice revealed several morphological anomalies, resembling those observed in patients and an interneuron migration defect but perinatal lethality precluded analyses of later phenotypes. Interestingly, many of the neurological phenotypes observed in patients with various ARX mutations can be attributed, in part, to interneuron dysfunction. To directly test this possibility, mice carrying a floxed Arx allele were generated and crossed to Dlx5/6CRE-IRES-GFP(Dlx5/6CIG) mice, conditionally deleting Arx from ganglionic eminence derived neurons including cortical interneurons. We now report that Arx−/y;Dlx5/6CIG (male) mice exhibit a variety of seizure types beginning in early-life, including seizures that behaviourally and electroencephalographically resembles infantile spasms, and show evolution through development. Thus, this represents a new genetic model of a malignant form of paediatric epilepsy, with some characteristics resembling infantile spasms, caused by mutations in a known infantile spasms gene. Unexpectedly, approximately half of the female mice carrying a single mutant Arx allele (Arx−/+;Dlx5/6CIG) also developed seizures. We also found that a subset of human female carriers have seizures and neurocognitive deficits. In summary, we have identified a previously unrecognized patient population with neurological deficits attributed to ARX mutations that are recapitulated in our mouse model. Furthermore, we show that perturbation of interneuron subpopulations is an important mechanism underling the pathogenesis of developmental epilepsy in both hemizygous males and carrier females. Given the frequency of ARX mutations in patients with infantile spasms and related disorders, our data unveil a new model for further understanding the pathogenesis of these disorders.

Summary

Retrospective studies suggest that precipitating events such as prolonged seizures, stroke or head trauma increase the risk of developing epilepsy later in life. The process of epilepsy development, known as epileptogenesis, is associated with changes in the expression of a myriad of genes. One of the major challenges to the epilepsy research community has been to determine which of these changes contributes to epileptogenesis, which may be compensatory, and which may be non-contributory. Establishing this for any given gene is essential if it is to be considered a therapeutic target for the prevention or treatment of epilepsy. Our laboratories have examined alterations in gene expression related to inhibitory neurotransmission that have been proposed as contributing factors in epileptogenesis. The GABAA receptor (GABAR) mediates most fast synaptic inhibition, and changes in GABAR subunit expression and function have been reported in adult animals beginning immediately after prolonged seizures (status epilepticus (SE)) and continue as animals become chronically epileptic. Prevention of GABAR subunit changes after SE using viral gene transfer inhibits development of epilepsy in an animal model, suggesting that these changes directly contribute to epileptogenesis. The mechanisms that regulate differential expression of GABAR subunits in hippocampus after SE have recently been identified, and include the CREB/ICER, JAK/STAT, BDNF and Egr3 signaling pathways. Targeting signaling pathways that alter the expression of genes involved in epileptogenesis may provide novel therapeutic approaches for preventing or inhibiting the development of epilepsy after a precipitating insult.

Objective

Neonatal seizures occur frequently, are often refractory to anticonvulsants, and are associated with considerable morbidity and mortality. Genetic and electrophysiological evidence indicates that KCNQ voltage-gated potassium channels are critical regulators of neonatal brain excitability. This study tests the hypothesis that selective openers of KCNQ channels may be effective for treatment of neonatal seizures.

Methods

We induced seizures in postnatal day 10 rats with either kainic acid or flurothyl. We measured seizure activity using quantified behavioral rating and electrocorticography. We compared the efficacy of flupirtine, a selective KCNQ channel opener, with phenobarbital and diazepam, two drugs in current use for neonatal seizures.

Results

Unlike phenobarbital or diazepam, flupirtine prevented animals from developing status epilepticus (SE) when administered prior to kainate. In the flurothyl model, phenobarbital and diazepam increased latency to seizure onset, but flupirtine completely prevented seizures throughout the experiment. Flupirtine was also effective in arresting electrographic and behavioral seizures when administered after animals had developed continuous kainate-induced SE. Flupirtine caused dose-related sedation and suppressed EEG activity, but did not result in respiratory suppression or result in any mortality.

Interpretation

Flupirtine appears more effective than either of two commonly used anti-epileptic drugs, phenobarbital and diazepam, in preventing and suppressing seizures in both the kainic acid and flurothyl models of symptomatic neonatal seizures. KCNQ channel openers merit further study as potential treatments for seizures in infants and children.

During the past decade, substantial progress has been made in delineating clinical features of the epilepsies and the basic mechanisms responsible for these disorders. Eleven human epilepsy genes have been identified and many more are now known from animal models. Candidate targets for cures are now based upon newly identified cellular and molecular mechanisms that underlie epileptogenesis. However, epilepsy is increasingly recognized as a group of heterogeneous syndromes characterized by other conditions that co-exist with seizures. Cognitive, emotional and behavioral co-morbidities are common and offer fruitful areas for study. These advances in understanding mechanisms are being matched by the rapid development of new diagnostic methods and therapeutic approaches. This article reviews these areas of progress and suggests specific goals that once accomplished promise to lead to cures for epilepsy.

The γ-aminobutyric acid (GABA) type A receptor (GABAAR) is the major inhibitory neurotransmitter receptor in the brain. Its multiple subunits show regional, developmental, and disease-related plasticity of expression; however, the regulatory networks controlling GABAAR subunit expression remain poorly understood. We report that the seizure-induced decrease in GABAAR α1 subunit expression associated with epilepsy is mediated by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway regulated by brain-derived neurotrophic factor (BDNF). BDNF- and seizure-dependent phosphorylation of STAT3 cause the adenosine 3′,5′-monophosphate (cAMP) response element–binding protein (CREB) family member ICER (inducible cAMP early repressor) to bind with phosphorylated CREB at the Gabra1:CRE site. JAK/STAT pathway inhibition prevents the seizure-induced decrease in GABAAR α1 abundance in vivo and, given that BDNF is known to increase the abundance of GABAAR α4 in a JAK/STAT-independent manner, indicates that BDNF acts through at least two distinct pathways to influence GABAAR-dependent synaptic inhibition.

Summary

Purpose

To define the changes in gene and protein expression of the neuronal glutamate transporter (EAAT3/EAAC1) in a rat model of temporal lobe epilepsy as well as in human hippocampal and neocortical epilepsy.

Methods

The expression of EAAT3/EAAC1 mRNA was measured by reverse Northern blotting in single dissociated hippocampal dentate granule cells from rats with pilocarpine-induced temporal lobe epilepsy (TLE) and age-matched controls, in dentate granule cells from hippocampal surgical specimens from patients with TLE, and in dysplastic neurons microdissected from human focal cortical dysplasia specimens. Immunolabeling of rat and human hippocampi and cortical dysplasia tissue with EAAT3/EAAC1 antibodies served to corroborate the mRNA expression analysis.

Results

The expression of EAAT3/EAAC1 mRNA was increased by nearly threefold in dentate granule cells from rats with spontaneous seizures compared with dentate granule cells from control rats. EAAT3/EAAC1 mRNA levels also were high in human dentate granule cells from patients with TLE and were significantly elevated in dysplastic neurons in cortical dysplasia compared with nondysplastic neurons from postmortem control tissue. No difference in expression of another glutamate transporter, EAAT2/GLT-1, was observed. Immunolabeling demonstrated that EAAT3/EAAC1 protein expression was enhanced in dentate granule cells from both rats and humans with TLE as well as in dysplastic neurons from human cortical dysplasia tissue.

Conclusions

Elevations of EAAT3/EAAC1 mRNA and protein levels are present in neurons from hippocampus and neocortex in both rats and humans with epilepsy. Upregulation of EAAT3/EAAC1 in hippocampal and neocortical epilepsy may be an important modulator of extracellular glutamate concentrations and may occur as a response to recurrent seizures in these cell types.