Differential molecular and behavioural alterations in mouse models of GABRG2 haploinsufficiency versus dominant negative mutations associated with human epilepsy.

Genetic epilepsy is a common disorder with phenotypic variation, but the basis for the variation is unknown. Comparing the molecular pathophysiology of mutations in the same epilepsy gene may provide mechanistic insights into the phenotypic heterogeneity. GABRG2 is an established epilepsy gene, and mutations in it produce epilepsy syndromes with varying severities. The disease phenotype in some cases may be caused by simple loss of subunit function (functional haploinsufficiency), while others may be caused by loss-of-function plus dominant negative suppression and other cellular toxicity. Detailed molecular defects and the corresponding seizures and related comorbidities resulting from haploinsufficiency and dominant negative mutations, however, have not been compared. Here we compared two mouse models of GABRG2 loss-of-function mutations associated with epilepsy with different severities, Gabrg2+/Q390X knockin (KI) and Gabrg2+/- knockout (KO) mice. Heterozygous Gabrg2+/Q390X KI mice are associated with a severe epileptic encephalopathy due to a dominant negative effect of the mutation, while heterozygous Gabrg2+/- KO mice are associated with mild absence epilepsy due to simple haploinsufficiency. Unchanged at the transcriptional level, KI mice with severe epilepsy had neuronal accumulation of mutant γ2 subunits, reduced remaining functional wild-type subunits in dendrites and synapses, while KO mice with mild epilepsy had no intracellular accumulation of the mutant subunits and unaffected biogenesis of the remaining wild-type subunits. Consequently, KI mice with dominant negative mutations had much less wild-type receptor expression, more severe seizures and behavioural comorbidities than KO mice. This work provides insights into the pathophysiology of epilepsy syndrome heterogeneity and designing mechanism-based therapies.

Test order effects in simultaneous protocols.

Simultaneous protocols typically yield poorer stimulus equivalence outcomes than do other protocols commonly used in equivalence research. Two independent groups of three 3-member equivalence sets of stimuli were used in conditional discrimination procedures in two conditions, one using the standard simultaneous protocol and the other using a hybrid simultaneous training and simple-to-complex testing. Participants completed the two conditions in one long session in Experiment 1, but in separate sessions in Experiment 2. The same stimulus sets used in Experiment 1 were randomized for the two conditions in Experiment 2. Overall, accuracy was better with the hybrid than with the standard protocol in both experiments. The equivalence yield was also better under the hybrid than under the standard protocol in each experiment. The results suggest that the order of testing for emergent relations may account for the difficulty often encountered with the standard simultaneous protocol.

Sex differences associated with intermittent swim stress.

Various animal models of depression have been used to seek a greater understanding of stress-related disorders. However, there is still a great need for novel research in this area, as many individuals suffering from depression are resistant to current treatment methods. Women have a higher rate of depression, highlighting the need to investigate mechanisms of sex differences. Therefore, we employed a new animal model to assess symptoms of depression, known as intermittent swim stress (ISS). In this model, the animal experiences 100 trials of cold water swim stress. ISS has already been shown to cause signs of behavioral depression in males, but has yet to be assessed in females. Following ISS exposure, we looked at sex differences in the Morris water maze and forced swim test. The results indicated a spatial learning effect only in the hidden platform task between male and female controls, and stressed and control males. A consistent spatial memory effect was only seen for males exposed to ISS. In the forced swim test, both sexes exposed to ISS exhibited greater immobility, and the same males and females also showed attenuated climbing and swimming, respectively. The sex differences could be due to different neural substrates for males and females. The goal of this study was to provide the first behavioral examination of sex differences following ISS exposure, so the stage of estrous cycle was not assessed for the females. This is a necessary future direction for subsequent experiments. The current article highlights the importance of sex differences in response to stress.

The therapeutic effect of stiripentol in Gabrg2+/Q390X mice associated with epileptic encephalopathy.

Anti-seizure drugs (ASDs) are widely used and known to increase inhibitory tone on neuro-circuits and reduce aberrant synchronous firing in epilepsy. Some ASDs act as agonist at the GABAA receptor. Stiripentol, known to increase GABAA receptor activity as well as the metabolites of GABAA receptor agonists, is often used in the treatment of an epileptic encephalopathy, Dravet syndrome (DS), which is caused by mutations mainly in SCN1A and in other genes such as GABRG2. We have recently generated a Gabrg2+/Q390X knockin mouse model associated with DS in humans. The objective of the study was to explore the effects of stiripentol in DS with GABAA receptor functional deficiency because of the etiology heterogeneity in DS. Monotherapy (stiripentol or Diazepam) and polytherapy (stiripentol and diazepam) treatments were tested in Gabrg2+/Q390X mice challenged with pentylenetetrazol (PTZ) seizure induction in conjunction with video-monitoring synchronized electroencephalogram (EEG) recordings. A combination of stiripentol and diazepam greatly reduced seizure-related events in Gabrg2+/Q390X mice following PTZ administration and increased survival. However, the treatment of stiripentol alone was mostly ineffective in alleviating seizure-related events except that it reduced mortality in PTZ challenged Gabrg2+/Q390X mice. The study suggests that stiripentol could be only used as add-on therapy for DS with GABAA receptor functional deficiency, which is consistent with the most established clinical application of stiripentol. The study highlights the importance of mechanism-based precision treatment for DS considering the highly heterogeneous nature of etiology in DS and the fact that mutations in different genes give rise to the same clinical phenotype.

Altered glutamate clearance in ascorbate deficient mice increases seizure susceptibility and contributes to cognitive impairment in APP/PSEN1 mice.

Ascorbate (vitamin C) is critical as a first line of defense antioxidant within the brain, and specifically within the synapse. Ascorbate is released by astrocytes during glutamate clearance and disruption of this exchange mechanism may be critical in mediating glutamate toxicity within the synapse. This is likely even more critical in neurodegenerative disorders with associated excitotoxicity and seizures, in particular Alzheimer's disease, in which ascorbate levels are often low. Using Gulo-/- mice that are dependent on dietary ascorbate, we established that low brain ascorbate increased sensitivity to kainic acid as measured via behavioral observations, electroencephalography (EEG) measurements, and altered regulation of several glutamatergic system genes. Kainic acid-induced immobility was improved in wild-type mice following treatment with ceftriaxone, which upregulates glutamate transporter GLT-1. The same effect was not observed in ascorbate-deficient mice in which sufficient ascorbate is not available for release. A single, mild seizure event was sufficient to disrupt performance in the water maze in low-ascorbate mice and in APPSWE/PSEN1dE9 mice. Together, the data support the critical role of brain ascorbate in maintaining protection during glutamatergic hyperexcitation events, including seizures. The study further supports a role for mild, subclinical seizures in cognitive decline in Alzheimer's disease.

Heat induced temperature dysregulation and seizures in Dravet Syndrome/GEFS+ Gabrg2+/Q390X mice.

It has been established that febrile seizures and its extended syndromes like generalized epilepsy with febrile seizures (FS) plus (GEFS+) and Dravet syndrome have been associated with mutations especially in SCN1A and GABRG2 genes. In patients, the onset of FS is likely due to the combined effect of temperature and inflammation in genetically vulnerable individuals because fever is often associated with infection. Much effort has been spent to understand the mechanisms underlying fever induction of seizures. In addition to the role of cytokines in FS, previous studies in Scn1a+/- knockout mice, a model of Dravet syndrome, indicated that temperature elevation alone could result in seizure generation, and the effect of elevated temperature inducing seizures was age-dependent. Here, we report the thermal effect in a different mouse model of Dravet syndrome, the Gabrg2+/Q390X knockin mouse. We demonstrated age-dependent dysregulated temperature control and that temperature elevation produced myoclonic jerks, generalized tonic clonic seizures (GTCSs) and heightened anxiety-like symptoms in Gabrg2+/Q390X mice. The study indicated that regardless of other inflammatory factors, brief heat alone increased brain excitability and induced multiple types of seizures in Gabrg2+/Q390X mice, suggesting that mutations like GABRG2(Q390X) may alter brain thermal regulation and precipitate seizures during temperature elevations.

Altered GABAA receptor expression in brainstem nuclei and SUDEP in Gabrg2(+/Q390X) mice associated with epileptic encephalopathy.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause for death in individuals with epilepsy. The frequency of SUDEP correlates with the severity of epilepsies and lack of response to antiepileptic drug treatment, but the underlying mechanisms of SUDEP have not been elucidated fully. GABRG2(Q390X) is a mutation associated with the epileptic encephalopathy Dravet syndrome (DS) and with genetic epilepsy with febrile seizures plus (GEFS+) in patients. The Gabrg2(+/Q390X) knockin (KI) mouse phenocopies the major features of DS and GEFS+ and has SUDEP throughout life. The Gabrg2(+/-) knockout (KO) mouse is associated with infrequent absence seizures and represents a model of mild absence epilepsy syndrome without increased mortality. To explore the basis for SUDEP in DS and GEFS+, we compared mutant γ2 subunit and wild-type α1 and ß2/3 subunit expression in mice in brainstem nuclei associated with respiratory function including the solitary tract, pre-Botzinger complex and Kolliker-Fuse nuclei. We found that synaptic GABAA receptors were reduced while intracellular nonfunctional γ2(Q390X) subunits were increased in the heterozygous DS and GEFS+ KI mice, but not in the heterozygous absence epilepsy KO mice. Given the critical role of these nuclei in cardiorespiratory function, it is likely the impaired GABAergic transmission and neuronal dysfunction in these brainstem nuclei are involved in the cardiorespiratory collapse in SUDEP. The study provides novel mechanistic insights into cardiorespiratory failure of SUDEP.

Low brain ascorbic acid increases susceptibility to seizures in mouse models of decreased brain ascorbic acid transport and Alzheimer's disease.

Seizures are a known co-occurring symptom of Alzheimer's disease, and they can accelerate cognitive and neuropathological dysfunction. Sub-optimal vitamin C (ascorbic acid) deficiency, that is low levels that do not lead the sufferer to present with clinical signs of scurvy (e.g. lethargy, hemorrhage, hyperkeratosis), are easily obtainable with insufficient dietary intake, and may contribute to the oxidative stress environment of both Alzheimer's disease and epilepsy. The purpose of this study was to test whether mice that have diminished brain ascorbic acid in addition to carrying human Alzheimer's disease mutations in the amyloid precursor protein (APP) and presenilin 1 (PSEN1) genes, had altered electrical activity in the brain (electroencephalography; EEG), and were more susceptible to pharmacologically induced seizures. Brain ascorbic acid was decreased in APP/PSEN1 mice by crossing them with sodium vitamin C transporter 2 (SVCT2) heterozygous knockout mice. These mice have an approximately 30% decrease in brain ascorbic acid due to lower levels of SVCT2 that supplies the brain with ASC. SVCT2+/-APP/PSEN1 mice had decreased ascorbic acid and increased oxidative stress in brain, increased mortality, faster seizure onset latency following treatment with kainic acid (10 mg/kg i.p.), and more ictal events following pentylenetetrazol (50 mg/kg i.p.) treatment. Furthermore, we report the entirely novel phenomenon that ascorbic acid deficiency alone increased the severity of kainic acid- and pentylenetetrazol-induced seizures. These data suggest that avoiding ascorbic acid deficiency may be particularly important in populations at increased risk for epilepsy and seizures, such as Alzheimer's disease.

Ultrasonic vocalizations during intermittent swim stress forecasts resilience in subsequent forced swim and spatial learning tests.

The examination of stress resilience has substantially increased in recent years. However, current paradigms require multiple behavioral procedures, which themselves may serve as secondary stressors. Therefore, a novel predictor of stress resilience is needed to advance the field. Ultrasonic vocalizations (USVs) have been observed as a behavioral correlate of stress in various rodent species. It was recently reported that rats that emitted ultrasonic vocalizations during intermittent swim stress (ISS) later showed resilience when tested on an instrumental swim escape test. In the current study, we extend this earlier observation on two additional behavioral endpoints. Rats were subjected to ISS, and USVs were recorded. Twenty-four hours later, behavioral performance was evaluated in either the forced swim test or Morris water maze. Rats that emitted ultrasonic vocalizations were resilient to the effects of ISS as indicated by performance similar to controls on both measures. These results extend the original findings that ISS-induced USVs are associated with resilience and are related to subsequent aversively motivated behavior. Such a non-invasive forecast of stress responsivity will allow future work to utilize USVs to examine the neural correlates of initial stress resistance/resilience, thereby eliminating potential confounds of further behavioral testing. Future studies can utilize USVs to target potentially unappreciated neural systems to provide novel pharmacotherapeutic strategies for treatment-resistant depression.

Resilience in shock and swim stress models of depression.

Experimental models of depression often entail exposing a rodent to a stressor and subsequently characterizing changes in learning and anhedonia, which may reflect symptoms of human depression. Importantly, not all people, and not all laboratory rats, exposed to stressors develop depressed behavior; these "resilient" individuals are the focus of our review. Herein we describe research from the "learned helplessness" and "intermittent swim stress" (ISS) models of depression in which rats that were allowed to control the offset of the aversive stimulus with a behavioral response, and in a subset of rats that were not allowed to control the stressor that appeared to be behaviorally and neurochemically similar to rats that were either naive to stress or had controllability over the stressor. For example, rats exposed to inescapable tailshock, but do not develop learned helplessness, exhibit altered sensitivity to the behavioral effects of GABAA receptor antagonists and reduced in vitro benzodiazepine receptor ligand binding. This pattern suggested that resilience might involve activation of an endogenous benzodiazepine-like compound, possibly an allostatic modulator of the GABAA receptor like allopregnanolone. From the ISS model, we have observed in resilient rats protection from stressor-induced glucocorticoid increases and immune activation. In order to identify the neural mediators of these correlates of resilience, non-invasive measures are needed to predict the resilient or vulnerable phenotype prior to analysis of neural endpoints. To this end, we found that ultrasonic vocalizations (USVs) appear to predict the resilient phenotype in the ISS paradigm. We propose that combining non-invasive predictive measures, such as USVs with biological endpoint measures, will facilitate future research into the neural correlates of resilience.

Intermittent swim stress causes Morris water maze performance deficits in a massed-learning trial procedure that are exacerbated by reboxetine.

Various animal models of depression have been used to seek a greater understanding of stress-related disorders. However, there is still a great need for research in this area, as many unanswered questions remain. Therefore, we sought to employ a novel animal model of depression known as intermittent swim stress (ISS). In this model, the animal experiences 100 trials of cold water swim stress. ISS has already shown subsequent immobility in the forced swim test (FST), deficits in instrumental and spatial (spaced-trial procedure), and responsiveness to norepinephrine. We are now examining how this will translate in the Morris water maze for rats in a massed-learning trial procedure, and further assessing ISS sensitivity toward norepinephrine selective anti-depressant drugs. The results indicated no difference in cued learning when the platform was visible in the water maze, but a hidden platform task revealed poorer spatial learning for ISS-exposed rats versus controls. In terms of spatial memory, there was a notable ISS-induced deficit 1h after the learning trials, regardless of performance on the previous platform task. Interestingly, the administration of reboxetine interfered with the spatial learning and memory trials for both ISS and CC groups. As a result, ISS exposure compromised spatial learning and memory in the Morris water maze, and norepinephrine does not appear to be a mediator of this deficit. The results demonstrate a key difference in the effects of reboxetine in a massed- vs. spaced-learning trial procedure in the Morris water maze following ISS exposure.

Morris water maze performance deficit produced by intermittent swim stress is partially mediated by norepinephrine.

Intermittent swim stress (ISS) exposes a rat to cold water and the effects of the procedure produce detrimental results on activity measures 24h later. The ISS model can be used with the Morris water maze (MWM) to investigate the impact of stress on a spatial learning and memory task, known to involve the hippocampus. We investigated if the ISS model produced performance deficits in the MWM (experiments 1 and 2). We also investigated the role of norepinephrine by using an alpha-2 adrenergic agonist (i.e., clonidine) to exacerbate ISS-induced deficits (experiment 3), and using antidepressants (i.e., desipramine and reboxetine) that enhance the synaptic availability of norepinephrine to reduce ISS-induced deficits (experiments 4 and 5). Results indicated a main effect for stress in all experiments, with the exception of experiment 2, as ISS did induce performance deficits in the MWM. Clonidine enhanced ISS-induced deficits only in the learning trials, while desipramine and reboxetine reduced ISS-induced deficits in the learning trials. Additionally, only reboxetine reduced memory deficits in the MWM. These findings provide evidence that norepinephrine may act as a partial mediator of ISS-induced deficits in MWM performance.

Intermittent and continuous swim stress-induced behavioral depression: sensitivity to norepinephrine- and serotonin-selective antidepressants.

RATIONALE: Intermittent swim stress (ISS) produces deficits in swim escape learning and increases immobility in the forced swim test (FST). A previous attempt to reverse this immobility with the selective serotonin reuptake inhibitor (SSRI), fluoxetine (FLX), was unsuccessful, but the sensitivity of this immobility to other types of antidepressants is unknown. OBJECTIVES: In experiment 1, we evaluate the ability of the norepinephrine (NE) selective reuptake inhibitor (NSRI), desipramine (DES), to reverse the ISS-induced immobility in the FST compared to confined controls (CC), while in experiment 2, we test the efficacy of either the SSRI or NSRI to reverse the immobility produced by either ISS or continuous swim (CS)/FST. METHODS: Rats were exposed to their respective behavioral pretreatment (ISS, CS/FST, or CC) and were then injected with an antidepressant or saline solution 23.5, 5, and 1 h prior to the FST. RESULTS: In experiment 1, DES reduced immobility and increased the climbing behavior in the ISS group without altering these behaviors in the CC, while in experiment 2, the CS/FST-induced immobility was reduced by both antidepressants (i.e., FLX and DES), while the ISS-induced immobility was only affected by DES. CONCLUSIONS: These results suggest that the ISS-induced immobility is mediated through the NE system and may represent a model for atypical depression.