Myocardial glycophagy flux dysregulation and glycogen accumulation characterize diabetic cardiomyopathy.

Diabetic heart disease morbidity and mortality is escalating. No specific therapeutics exist and mechanistic understanding of diabetic cardiomyopathy etiology is lacking. While lipid accumulation is a recognized cardiomyocyte phenotype of diabetes, less is known about glycolytic fuel handling and storage. Based on in vitro studies, we postulated the operation of an autophagy pathway in the myocardium specific for glycogen homeostasis - glycophagy. Here we visualize occurrence of cardiac glycophagy and show that the diabetic myocardium is characterized by marked glycogen elevation and altered cardiomyocyte glycogen localization. We establish that cardiac glycophagy flux is disturbed in diabetes. Glycophagy may represent a potential therapeutic target for alleviating the myocardial impacts of metabolic disruption in diabetic heart disease.

Transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor regulatory protein expression during the development of absence seizures in genetic absence epilepsy rats from Strasbourg.

The transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) regulatory proteins (TARPs), γ2 (stargazin), γ3, γ4, γ5, γ7, and γ8, are a family of proteins that regulate AMPAR trafficking, expression, and biophysical properties that could have a role in the development of absence seizures. Here, we evaluated the expression of TARPs and AMPARs across the development of epilepsy in the genetic absence epilepsy rats from Strasbourg (GAERS) model of idiopathic generalized epilepsy (IGE) with absence seizures. Pre-epileptic (7-day-old), early epileptic (6-week-old), and chronically epileptic (16-week-old) GAERS, and age-matched male nonepileptic control rats (NEC) were used. Electroencephalographic (EEG) recordings were acquired from the 6- and 16-week-old animals to quantify seizure expression. Somatosensory cortex (SCx) and whole thalamus were collected from all the animals to evaluate TARP and AMPAR mRNA expression. Analysis of the EEG demonstrated a gradual increase in the number and duration of seizures across GAERS development. mRNA expression of the TARPs γ2, γ3, γ4, γ5, and γ8 in the SCx, and γ4 and γ5 in the thalamus, increased as the seizures started and progressed in the GAERS compared to NEC. There was a temporal association between increased TARP expression and seizures in GAERS, highlighting TARPs as potential targets for developing novel treatments for IGE with absence seizures.

Effects of the T-type calcium channel CaV3.2 R1584P mutation on absence seizure susceptibility in GAERS and NEC congenic rats models.

RATIONALE: Low-voltage-activated or T-type Ca2+ channels play a key role in the generation of seizures in absence epilepsy. We have described a homozygous, gain of function substitution mutation (R1584P) in the CaV3.2 T-type Ca2+ channel gene (Cacna1h) in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). The non-epileptic control (NEC) rats, derived from the same original Wistar strains as GAERS but selectively in-breed not to express seizures, are null for the R1584P mutation. To study the effects of this mutation in rats who otherwise have a GAERS or NEC genetic background, we bred congenic GAERS-Cacna1hNEC (GAERS null for R1584P mutation) and congenic NEC-Cacna1hGAERS (NEC homozygous for R1584P mutation) and evaluated the seizure and behavioral phenotype of these strains in comparison to the original GAERS and NEC strains. METHODS: To evaluate seizure expression in the congenic strains, EEG electrodes were implanted in NEC, GAERS, GAERS-Cacna1hNEC without the R1584P mutation, and NEC-Cacna1hGAERS with the R1584P mutation rats. In the first study, continuous EEG recordings were acquired from week 4 (when seizures begin to develop in GAERS) to week 14 of age (when GAERS display hundreds of seizures per day). In the second study, the seizure and behavioral phenotype of GAERS and NEC-Cacna1hGAERS strains were evaluated during young age (6 weeks of age) and adulthood (16 weeks of age) of GAERS, NEC, GAERS-Cacna1hNEC and NEC-Cacna1hGAERS. The Open field test (OFT) and sucrose preference test (SPT) were performed to evaluate anxiety-like and depressive-like behavior, respectively. This was followed by EEG recordings at 18 weeks of age to quantify the seizures, and spike-wave discharge (SWD) cycle frequency. At the end of the study, the whole thalamus was collected for T-type calcium channel mRNA expression analysis. RESULTS: GAERS had a significantly shorter latency to first seizures and an increased number of seizures per day compared to GAERS-Cacna1hNEC. On the other hand, the presence of the R1584P mutation in the NEC-Cacna1hGAERS was not enough to generate spontaneous seizures in their seizure-resistant background. 6 and 16-week-old GAERS and GAERS-Cacna1hNEC rats showed anxiety-like behavior in the OFT, in contrast to NEC and NEC-Cacna1hGAERS. Results from the SPT showed that the GAERS developed depressive-like in the SPT compared to GAERS-Cacna1hNEC, NEC, and NEC-Cacna1hGAERS. Analysis of the EEG at 18 weeks of age showed that the GAERS had an increased number of seizures per day, increased total seizure duration and a higher cycle frequency of SWD relative to GAERS-Cacna1hNEC. However, the average seizure duration was not significantly different between strains. Quantitative real-time PCR showed that the T-type Ca2+ channel isoform CaV3.2 channel expression was significantly increased in GAERS compared to NEC, GAERS-Cacna1hNEC and NEC-Cacna1hGAERS. The presence of the R1584P mutation increased the total ratio of CaV3.2 + 25/-25 splice variants in GAERS and NEC-Cacna1hGAERS compared to NEC and GAERS-Cacna1hNEC. DISCUSSION: The data from this study demonstrate that the R1584P mutation in isolation on a seizure-resistant NEC genetic background was insufficient to generate absence seizures, and that a GAERS genetic background can cause seizures even without the mutation. However, the study provides evidence that the R1584P mutation acts as a modulator of seizures development and expression, and depressive-like behavior in the SPT, but not the anxiety phenotype of the GAERS model of absence epilepsy.

Integrative genomics reveals pathogenic mediator of valproate-induced neurodevelopmental disability.

Prenatal exposure to the anti-seizure medication sodium valproate (VPA) is associated with an increased risk of adverse postnatal neurodevelopmental outcomes, including lowered intellectual ability, autism spectrum disorder and attention-deficit hyperactivity disorder. In this study, we aimed to clarify the molecular mechanisms underpinning the neurodevelopmental consequences of gestational VPA exposure using integrative genomics. We assessed the effect of gestational VPA on foetal brain gene expression using a validated rat model of valproate teratogenicity that mimics the human scenario of chronic oral valproate treatment during pregnancy at doses that are therapeutically relevant to the treatment of epilepsy. Two different rat strains were studied-inbred Genetic Absence Epilepsy Rats from Strasbourg, a model of genetic generalized epilepsy, and inbred non-epileptic control rats. Female rats were fed standard chow or VPA mixed in standard chow for 2 weeks prior to conception and then mated with same-strain males. In the VPA-exposed rats maternal oral treatment was continued throughout pregnancy. Foetuses were extracted via C-section on gestational Day 21 (1 day prior to birth) and foetal brains were snap-frozen and genome-wide gene expression data generated. We found that gestational VPA exposure via chronic maternal oral dosing was associated with substantial drug-induced differential gene expression in the pup brains, including dysregulated splicing, and observed that this occurred in the absence of evidence for significant neuronal gain or loss. The functional consequences of VPA-induced gene expression were explored using pathway analysis and integration with genetic risk data for psychiatric disease and behavioural traits. The set of genes downregulated by VPA in the pup brains were significantly enriched for pathways related to neurodevelopment and synaptic function and significantly enriched for heritability to human intelligence, schizophrenia and bipolar disorder. Our results provide a mechanistic link between chronic foetal VPA exposure and neurodevelopmental disability mediated by VPA-induced transcriptional dysregulation.

Altered cardiac structure and function is related to seizure frequency in a rat model of chronic acquired temporal lobe epilepsy.

OBJECTIVE: This study aimed to prospectively examine cardiac structure and function in the kainic acid-induced post-status epilepticus (post-KA SE) model of chronic acquired temporal lobe epilepsy (TLE), specifically to examine for changes between the pre-epileptic, early epileptogenesis and the chronic epilepsy stages. We also aimed to examine whether any changes related to the seizure frequency in individual animals. METHODS: Four hours of SE was induced in 9 male Wistar rats at 10 weeks of age, with 8 saline treated matched control rats. Echocardiography was performed prior to the induction of SE, two- and 10-weeks post-SE. Two weeks of continuous video-EEG and simultaneous ECG recordings were acquired for two weeks from 11 weeks post-KA SE. The video-EEG recordings were analyzed blindly to quantify the number and severity of spontaneous seizures, and the ECG recordings analyzed for measures of heart rate variability (HRV). PicroSirius red histology was performed to assess cardiac fibrosis, and intracellular Ca2+ levels and cell contractility were measured by microfluorimetry. RESULTS: All 9 post-KA SE rats were demonstrated to have spontaneous recurrent seizures on the two-week video-EEG recording acquired from 11 weeks SE (seizure frequency ranging from 0.3 to 10.6 seizures/day with the seizure durations from 11 to 62 s), and none of the 8 control rats. Left ventricular wall thickness was thinner, left ventricular internal dimension was shorter, and ejection fraction was significantly decreased in chronically epileptic rats, and was negatively correlated to seizure frequency in individual rats. Diastolic dysfunction was evident in chronically epileptic rats by a decrease in mitral valve deceleration time and an increase in E/E` ratio. Measures of HRV were reduced in the chronically epileptic rats, indicating abnormalities of cardiac autonomic function. Cardiac fibrosis was significantly increased in epileptic rats, positively correlated to seizure frequency, and negatively correlated to ejection fraction. The cardiac fibrosis was not a consequence of direct effect of KA toxicity, as it was not seen in the 6/10 rats from separate cohort that received similar doses of KA but did not go into SE. Cardiomyocyte length, width, volume, and rate of cell lengthening and shortening were significantly reduced in epileptic rats. SIGNIFICANCE: The results from this study demonstrate that chronic epilepsy in the post-KA SE rat model of TLE is associated with a progressive deterioration in cardiac structure and function, with a restrictive cardiomyopathy associated with myocardial fibrosis. Positive correlations between seizure frequency and the severity of the cardiac changes were identified. These results provide new insights into the pathophysiology of cardiac disease in chronic epilepsy, and may have relevance for the heterogeneous mechanisms that place these people at risk of sudden unexplained death.

Inhibition of purinergic P2X receptor 7 (P2X7R) decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in U251 glioblastoma cells.

Glioblastoma is the most aggressive form of primary brain cancer, with a median survival of 12-15 months. The P2X receptor 7 (P2X7R) is upregulated in glioblastoma and is associated with increased tumor cell proliferation. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is also upregulated in glioblastoma and has been shown to have both pro- and anti-tumor functions. This study investigates the potential mechanism linking P2X7R and GM-CSF in the U251 glioblastoma cell line and the therapeutic potential of P2X7R antagonism in this setting. P2X7R protein and mRNA was demonstrated to be expressed in the U251 cell line as assessed by immunocytochemistry and qPCR. Its channel function was intact as demonstrated by live cell confocal imaging using a calcium indicator Fluo-4 AM. Inhibition of P2X7R using antagonist AZ10606120, decreased both GM-CSF mRNA (P < 0.05) and protein (P < 0.01) measured by qPCR and ELISA respectively. Neutralization of GM-CSF with an anti-GM-CSF antibody did not alter U251 cell proliferation, however, P2X7R antagonism with AZ10606120 significantly reduced U251 glioblastoma cell numbers (P < 0.01). This study describes a novel link between P2X7R activity and GM-CSF expression in a human glioblastoma cell line and highlights the potential therapeutic benefit of P2X7R inhibition with AZ10606120 in glioblastoma.

A rat model of valproate teratogenicity from chronic oral treatment during pregnancy.

OBJECTIVE: Sodium valproate (VPA), the most effective antiepileptic drug for patients with genetic generalized epilepsy (GGE), is a potent human teratogen that increases the risk of a range of congenital malformations, including spina bifida. The mechanisms underlying this teratogenicity are not known, but may involve genetic risk factors. This study aimed to develop an animal model of VPA-induced birth defects. METHODS: We used three different rat strains: inbred Genetic Absence Epilepsy Rats From Strasbourg (GAERS), a model of GGE with absence seizures; inbred Non-Epileptic Controls (NEC); and outbred nonepileptic Wistars. Female rats were fed standard chow or VPA (20 g/kg food) mixed in standard chow for 2 weeks prior to conception, and then mated with same-strain males. Treatment continued throughout pregnancy. Fetuses were extracted via C-section on gestational day 21 and examined for birth defects, including external assessment and spinal measurements. RESULTS: VPA-exposed pups showed significant reductions in weight, length, and whole-body development compared with controls of all three strains (P < .0001). Gestational VPA treatment altered intravertebral distances, and resulted in underdeveloped vertebral arches between thoracic region T11 and caudal region C2 in most pups (GAERS, 100%; NEC, 95%; Wistar, 80%), more frequently than in controls (9%, 13%, 19%). SIGNIFICANCE: Gestational VPA treatment results in similar developmental and morphological abnormalities in three rat strains, including one with GGE, indicating that the genetic underpinnings of epilepsy do not contribute markedly to VPA-induced birth defects. This model may be used in future studies to investigate mechanisms involved in the pathogenesis of antiepileptic drug-induced birth defects.

An extraordinary Karenia mikimotoi "beer tide" in Kachemak Bay Alaska.

In autumn of 2013 an immense dinoflagellate bloom developed in Kachemak Bay, AK, USA. Much of the Bay was discolored a dark amber color and raised public concerns as small scale fish kills were reported in a few locations. Light microscopy revealed a monospecific bloom of gymnodinoid dinoflagellates that were previously unknown from the Bay. Gene sequencing of SSU rDNA from cells collected from the bloom confirmed the causative species to be Karenia mikimotoi. This represents the first report of a K. mikimotoi bloom in Alaska. After the bloom organism was confirmed, a K. mikimotoi species-specific qPCR assay was developed and used to assess K. mikimotoi abundances in DNA extracted from phytoplankton samples from Kachemak Bay and Lower Cook Inlet (LCI) obtained over a six-year period. The K. mikimotoi abundances were compared with corresponding time series of environmental variables (water temperature, salinity, water column stability, nutrients, precipitation and wind speed) to assess the factors contributing to the development of the bloom. The results showed early bloom development occurred in August when snow melt reduced salinities and increased water column stability during a period of calm winds. Peak bloom concentrations occurred in late September (107 cell eq. L-1) even as water temperatures were decreasing. The bloom gradually declined over the winter but persisted until April of 2014. Karenia mikimotoi cells were not detected two years prior or three years following the bloom, suggesting cells were introduced to Kachemak Bay at a time when conditions allowed K. mikimotoi to thrive.

Validation of reference genes for gene expression analysis following experimental traumatic brain injury in a pediatric mouse model.

Quantitative polymerase chain reaction (qPCR) is the gold standard method in targeted analysis of messenger RNA (mRNA) levels in a tissue. To minimize methodological errors, a reference gene (or a combination of reference genes) is routinely used for normalization to account for technical variables such as RNA quality and sample size. While presumed to have stable expression, reference genes in the brain can change during normal development, as well as in response to injury, such as traumatic brain injury (TBI). This study is the first to evaluate the stability of reference genes in a controlled cortical impact (CCI) model in the pediatric mouse brain, using two methods of qPCR normalization for optimal reference gene selection. Three week old mice were subjected to unilateral CCI at two severity of injuries (mild or severe), compared to sham controls. At 1 and 8 weeks post-injury, the ipsilateral hemisphere was analyzed to determine reference gene stability. Five commonly-used reference genes were compared: tyrosine 3 monooxygenase/tryptophan 5 monooxygenase activation protein zeta (Ywhaz), cyclophilin A (Ppia), hypoxanthine phosphoribosyl transferase (Hprt), glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and ß-actin (Actb). Ppia and Hprt were chosen as the most stable combination of genes using GeNORM software analysis. These results highlight the instability of several commonly used reference genes after TBI, and provide a selection of validated genes for future gene expression analyses in the injured pediatric mouse brain.

Improving mammography access for women with disabilities: Outcomes of the CDC's right to know campaign.

Women with disabilities share similar risks for breast cancer as other women yet experience a lack of access to cancer screening and are less likely to receive screening mammograms in accordance with recommended guidelines. The present study evaluated mammography centers across the state of Montana in response to the Centers for Disease Control and Prevention's Right to Know campaign, which focused on addressing barriers to breast cancer screening. Mammography centers were originally evaluated in 2009 and were reassessed in 2011 and 2015 after being given action plans to address accessibility barriers. The current study examined changes in accessibility across time in four priority areas: 1) van and standard parking, 2) exterior and interior routes, 3) mammography rooms, and 4) restrooms. Results indicate all mammography centers had a least one mammography machine that lowered for patients in a seated position and that accessibility of the four priority areas improved over time; however, improvements were still needed to encourage health equity for women with disabilities.

The effects of cell therapy on seizures in animal models of epilepsy: protocol for systematic review and meta-analysis of preclinical studies.

BACKGROUND: Epilepsy is one of the most common and serious brain conditions, characterised by recurrent unprovoked seizures. It affects about 1% of the population worldwide. Despite a range of antiepileptic drugs being available, one third of the patients do not achieve adequate seizure control. Only a minority of these patients may be suitable to undergo surgical resection of the seizure focus, but this is an invasive and not always successful procedure. There is an urgent need to develop more effective treatment options for uncontrolled seizures. With the recent advances in regenerative and translational medicine, cell therapies could prove to be beneficial. Here we describe the protocol for a proposed systematic review and meta-analysis to assess the effects for cell transplantation in animal models of epilepsy. METHODS: We will include all preclinical animal models of epilepsy that evaluate the effects of cell transplantation compared to the untreated control. The primary outcome will be the change in frequency and duration of seizures from baseline measured by video electroencephalography (EEG). The secondary outcomes will include histological and neurobehavioural assessments. We will perform an electronic search of MEDLINE via PubMed, Web of Science, and EMBASE. Search results will be screened independently by two reviewers and confirmed by a third reviewer. Data from eligible studies will be extracted and pooled, and the summary estimate of effect size will be calculated using DerSimonian and Laird random effects meta-analysis. Heterogeneity will be explored using sub-group meta-analysis, and meta-regression risk of bias will be assessed by using the CAMARADES checklist for study quality tool. DISCUSSION: The purpose of this systematic review is to assess and summarise the existing literature in the field of cell transplantation as a treatment for epilepsy in animal models. Efficacy will be measured by evaluating the reduction in seizure intervals, number, and duration, within animal models of epilepsy. Analysis of the existing literature will mark the achievement made in the field and locate the existing gaps, a process that will aid in the search for the next needed step. SYSTEMATIC REVIEW REGISTRATION: CRD42018103628.

An animal model of genetic predisposition to develop acquired epileptogenesis: The FAST and SLOW rats.

Epidemiological data and gene association studies suggest a genetic predisposition to developing epilepsy after an acquired brain insult, such as traumatic brain injury. An improved understanding of genetic determinants of vulnerability is imperative for early disease diagnosis and prognosis prediction, with flow-on benefits for the development of targeted antiepileptogenic treatments as well as optimal clinical trial design. In the laboratory, one approach to investigate why some individuals are more vulnerable to acquired epilepsy than others is to examine unique rodent models exhibiting either vulnerability or resistance to epileptogenesis. This review focuses on the most well-characterized of these models, the FAST (seizure-prone) and SLOW (seizure-resistant) rat strains, which were derived by selective breeding for differential amygdala electrical kindling rates. We describe how these strains differ in their seizure profiles, neuroanatomy, and neurobehavioral phenotypes, both at baseline and after a brain insult, with this knowledge proving fruitful to identify common pathological abnormalities associated with seizure susceptibility and psychiatric comorbidities. It is important to note that accruing data on strain differences in multiple biological processes provides insight into why some individuals may be more vulnerable to epileptogenesis, although future studies are evidently needed to identify the precise molecular and genetic risk factors. Together, the FAST and SLOW rat strains, and other similar experimental models, are invaluable neurobiological tools to investigate the effect of genetic background on acquired epilepsy risk, as well as the poorly understood relationship between epilepsy development and associated comorbidities.

Disease-modifying effects of a novel T-type calcium channel antagonist, Z944, in a model of temporal lobe epilepsy.

We evaluated whether pharmacologically targeting T-type Ca2+ channels with Z944, a potent and selective antagonist, has disease-modifying effects in a model of temporal lobe epilepsy (TLE) that exhibits spontaneous recurrent seizures, and manifests behavioral and cognitive comorbidities commonly experienced by patients with this condition. Wistar rats underwent implantation of EEG electrodes and one week later 4 h of kainic acid-induced status epilepticus (SE). Animals were randomly assigned to one of 5 different groups: post-SE + Z944 (60 mg/kg/day, n = 8); post-SE + levetiracetam (200 mg/kg/day, n = 9); post-SE + vehicle (n = 8); sham + vehicle (n = 6) or sham + Z944 (60 mg/kg/day, n = 6). Treatments were delivered by continuous subcutaneous infusion for four weeks during which time continuous video-EEG was acquired. Four weeks after completion of treatment, the animals had two further weeks of continuous video-EEG monitoring to evaluate the effects of the different treatments. Behavioral tests were performed to evaluate anxiety, depression, and cognition. On the video-EEG recordings four-week post-treatment, the Z944 group manifest reduced number of seizures (0.01 ±â€¯0.01seizures/day) compared to vehicle (0.8 ±â€¯0.1) and levetiracetam (0.5 ±â€¯0.1) treated animals (p < 0.0001). Post-SE+ vehicle rats showed elevated depressive-like behavior, and deficits in spatial learning and memory compared to sham+vehicle rats, and these behavioral deficits were significantly improved in post-SE rats treated with Z944 (p < 0.05, for all comparisons). The results of this study show that treatment with Z944 has a disease-modifying effects in the post-SE model of TLE, reducing seizures as well as comorbid depressive-like behavior and cognitive impairment. This indicates that pharmacologically targeting T-type Ca2+ channels may be an effective disease-modifying treatment for temporal lobe epilepsy.

Acquired cardiac channelopathies in epilepsy: Evidence, mechanisms, and clinical significance.

There is growing evidence that cardiac dysfunction in patients with chronic epilepsy could play a pathogenic role in sudden unexpected death in epilepsy (SUDEP). Recent animal studies have revealed that epilepsy secondarily alters the expression of cardiac ion channels alongside abnormal cardiac electrophysiology and remodeling. These molecular findings represent novel evidence for an acquired cardiac channelopathy in epilepsy, distinct from inherited ion channels mutations associated with cardiocerebral phenotypes. Specifically, seizure activity has been shown to alter the messenger RNA (mRNA) and protein expression of voltage-gated sodium channels (Nav 1.1, Nav 1.5), voltage-gated potassium channels (Kv 4.2, Kv 4.3), sodium-calcium exchangers (NCX1), and nonspecific cation-conducting channels (HCN2, HCN4). The pathophysiology may involve autonomic dysfunction and structural cardiac disease, as both are independently associated with epilepsy and ion channel dysregulation. Indeed, in vivo and in vitro studies of cardiac pathology reveal a complex network of signaling pathways and transcription factors regulating ion channel expression in the setting of sympathetic overactivity, cardiac failure, and hypertrophy. Other mechanisms such as circulating inflammatory mediators or exogenous effects of antiepileptic medications lack evidence. Moreover, an acquired cardiac channelopathy may underlie the electrophysiologic cardiac abnormalities seen in chronic epilepsy, potentially contributing to the increased risk of malignant arrhythmias and sudden death. Therefore, further investigation is necessary to establish whether cardiac ion channel dysregulation similarly occurs in patients with epilepsy, and to characterize any pathogenic relationship with SUDEP.

Cav 3.2 T-type calcium channel mutation influences kindling-induced thalamic neuronal firing patterns in Genetic Absence Epilepsy Rats From Strasbourg.

OBJECTIVE: Recent data indicate that amygdala kindling leads to significant changes in interictal neuronal firing patterns of thalamic reticular nucleus (TRN) neurons by decreasing the spontaneous firing rate and increasing burst firing in nonepileptic control (NEC) rats. Genetic Absence Epilepsy Rats From Strasbourg (GAERS) were resistant to these kindling-induced firing changes in TRN neurons, and are also resistant to the progression of kindling. We investigated whether a homozygous, missense, single nucleotide mutation (R1584P) in the Cav 3.2 T-type Ca2+ channel gene, which has been correlated with the expression of absence seizures in GAERS, influenced kindling progression and TRN firing patterns. METHODS: Double-crossed (GAERS vs NEC; F2) rats that were homozygous for the Cav 3.2 mutation (PP) and those negative for the mutation (RR) were implanted with a stimulating electrode in the amygdala. Rats received a total of 30 kindling stimulations at their afterdischarge threshold current twice daily, and kindling progression was evaluated. Thereafter, the extracellular neuronal activity of TRN neurons was recorded in vivo under neuroleptanesthesia to investigate the influence of Cav 3.2 mutation on TRN firing patterns. RESULTS: We found that the R1584P mutation did not affect kindling progression in F2 crosses (P = 0.78). However, it influenced kindling-induced neuronal firing of TRN neurons. After 30 stimulations, RR rats exhibited a lower firing rate and a higher percentage of burst firing compared to PP rats. The decrease in firing frequency was correlated with the increase in the amount of burst firing in RR rats (R2  = 0.497). SIGNIFICANCE: Our findings suggest that mutation in Cav 3.2 T-type Ca2+ channels may play a role in the resistance to kindling-induced changes in TRN neurons to a low-frequency and high-percentage bursting pattern seen in association with the convulsive stages of amygdala kindling, but is not in itself enough to explain the resistance to kindling progression observed in GAERS.

Environmental factors influencing the distribution and abundance of Alexandrium catenella in Kachemak bay and lower cook inlet, Alaska.

Despite the long history of paralytic shellfish poisoning (PSP) events in Alaska, little is known about the seasonal distribution and abundance of the causative organism, Alexandrium, or the environmental factors that govern toxic bloom development. To address this issue, a five year study (2012-2017) was undertaken in Kachemak Bay and lower Cook Inlet Alaska to determine how the occurrence of Alexandrium catenella, the dominant PSP-causing Alexandrium species, was influenced by temperature, salinity, nutrient concentrations, and other environmental factors. Cell concentrations from 572 surface water samples were estimated using quantitative PCR. Monthly sampling revealed a seasonal pattern of A. catenella bloom development that was positively correlated with water temperature. Prevailing salinity conditions did not significantly affect abundance, nor was nutrient limitation a direct factor. Elevated cell concentrations were detected in 35 samples from Kachemak Bay (100-3050 cell eq. L-1) while a maximum abundance of 67 cell eq. L-1 was detected in samples from lower Cook Inlet sites. Monitoring data showed average water temperatures in Kachemak Bay increased by ∼2 °C over the course of the study and were accompanied by an increase in Alexandrium abundance. Based on these findings, 7-8 °C appears to represent a temperature threshold for significant bloom development in Kachemak Bay, with the greatest risk of shellfish toxicity occurring when temperatures exceed 10-12 °C. The role of temperature is further supported by time series data from the Alaska Coastal Current (station GAK1), which showed that summertime shellfish toxicity events in Kachemak Bay generally followed periods of anomalously high winter water temperatures. These data indicate monitoring changes in water temperatures may be used as an early warning signal for subsequent development of shellfish toxicity in Kachemak Bay.

CaV 3.2 drives sustained burst-firing, which is critical for absence seizure propagation in reticular thalamic neurons.

OBJECTIVE: Genetic alterations have been identified in the CACNA1H gene, encoding the CaV 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of CaV 3.2 calcium channels, and we investigated whether a gain-of-function mutation in the Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) contributes to seizure propagation and pacemaking in the TRN. METHODS: Pathophysiological contributions of CaV 3.2 calcium channels to burst firing and absence seizures were assessed in vitro using acute brain slice electrophysiology and quantitative real-time polymerase chain reaction (PCR) and in vivo using free-moving electrocorticography recordings. RESULTS: TRN neurons from GAERS display sustained oscillatory burst-firing that is both age- and frequency-dependent, occurring only in the frequencies overlapping with GAERS SWDs and correlating with the expression of a CaV 3.2 mutation-sensitive splice variant. In vivo knock-down of CaV 3.2 using direct thalamic injection of lipid nanoparticles containing CaV 3.2 dicer small interfering (Dsi) RNA normalized TRN burst-firing, and in free-moving GAERS significantly shortened seizures. SIGNIFICANCE: This supports a role for TRN CaV 3.2 T-type channels in propagating thalamocortical network seizures and setting the pacemaking frequency of SWDs.

Gene therapy mediated seizure suppression in Genetic Generalised Epilepsy: Neuropeptide Y overexpression in a rat model.

Neuropeptide Y (NPY) is an important 36 amino acid peptide that is abundantly expressed in the mammalian CNS and is known to be an endogenous modulator of seizure activity, including in rat models of Genetic Generalised Epilepsy (GGE) with absence seizures. Studies have shown that viral-mediated "gene therapy" with overexpression of NPY in the hippocampus can suppress seizures in acquired epilepsy animal models. This study investigated whether NPY gene delivery to the thalamus or somatosensory cortex, using recombinant adeno-associated viral vector (rAAV), could produce sustained seizure suppression in the GAERS model of GGE with absence seizures. Three cohorts of GAERS were injected bilaterally into the thalamus (short term n = 14 and long term n = 8) or the somatosensory cortex (n = 26) with rAAV-NPY or rAAV-empty. EEG recordings were acquired weekly post-treatment and seizure expression was quantified. Anxiety levels were tested using elevated plus maze and open field test. NPY and NPY receptor mRNA and protein expression were evaluated using quantitative PCR, immunohistochemistry and immunofluorescence. Viral overexpression of human NPY in the thalamus and somatosensory cortex in GAERS significantly reduced the time spent in seizure activity and number of seizures, whereas seizure duration was only reduced after thalamic NPY overexpression. Human and rat NPY and rat Y2 receptor mRNA expression was significantly increased in the somatosensory cortex. NPY overexpression in the thalamus was observed in rAAV-NPY treated rats compared to controls in the long term cohort. No effect was observed on anxiety behaviour. We conclude that virally-mediated human NPY overexpression in the thalamus or somatosensory cortex produces sustained anti-epileptic effects in GAERS. NPY gene therapy may represent a novel approach for the treatment of patients with genetic generalised epilepsies.

Delayed myelination and neurodevelopment in male seizure-prone versus seizure-resistant rats.

OBJECTIVE: Aberrant myelination and developmental delay have been reported in epilepsy. However, it is unclear whether these are linked to intrinsic mechanisms that support a predisposition toward seizures and the development of epilepsy. Thus, we compared rates of myelination and neurodevelopment in male rats selectively bred for enhanced susceptibility to kindling epileptogenesis (FAST) with male rats bred for resistance (SLOW). METHODS: Myelin-specific gene expression was compared in the brainstem, cerebellum, and cerebral hemisphere of FAST and SLOW rats on postnatal days (PNDs) 5, 11, 17, 23, and 90 to determine strain-specific myelination rates. Myelin protein levels were also compared at PNDs 5 and 23 in the brainstem. Relative rates of neurodevelopment were evaluated between PNDs 5 and 21 using physical growth landmarks and neuromotor tests including righting reflex, cliff avoidance, negative geotaxis, and locomotor activity. RESULTS: Myelin-specific mRNA expression was significantly down-regulated in FAST rats on PNDs 5 and 11 in all 3 brain structures, indicating relatively delayed myelination. Likewise, corresponding protein levels were significantly lower in FAST brainstem on PND 5. Developmental delay was evident in the FAST strain such that only 9% of FAST pups, compared to 81% of SLOW, had open eyes by PND 13, locomotor activity was significantly reduced between PNDs 12 and 16, and neuromotor task acquisition was delayed between PNDs 5 and 10. SIGNIFICANCE: Relative delays in myelination and neurodevelopment co-occurred in the seizure-prone FAST strain in the absence of seizures. These findings suggest these symptoms are not seizure-induced and may be mechanistically linked to an underlying pathophysiology supporting a predisposition toward developing epilepsy.

Evaluating whole genome sequence data from the Genetic Absence Epilepsy Rat from Strasbourg and its related non-epileptic strain.

OBJECTIVE: The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbreed Wistar rat strain widely used as a model of genetic generalised epilepsy with absence seizures. As in humans, the genetic architecture that results in genetic generalized epilepsy in GAERS is poorly understood. Here we present the strain-specific variants found among the epileptic GAERS and their related Non-Epileptic Control (NEC) strain. The GAERS and NEC represent a powerful opportunity to identify neurobiological factors that are associated with the genetic generalised epilepsy phenotype. METHODS: We performed whole genome sequencing on adult epileptic GAERS and adult NEC rats, a strain derived from the same original Wistar colony. We also generated whole genome sequencing on four double-crossed (GAERS with NEC) F2 selected for high-seizing (n = 2) and non-seizing (n = 2) phenotypes. RESULTS: Specific to the GAERS genome, we identified 1.12 million single nucleotide variants, 296.5K short insertion-deletions, and 354 putative copy number variants that result in complete or partial loss/duplication of 41 genes. Of the GAERS-specific variants that met high quality criteria, 25 are annotated as stop codon gain/loss, 56 as putative essential splice sites, and 56 indels are predicted to result in a frameshift. Subsequent screening against the two F2 progeny sequenced for having the highest and two F2 progeny for having the lowest seizure burden identified only the selected Cacna1h GAERS-private protein-coding variant as exclusively co-segregating with the two high-seizing F2 rats. SIGNIFICANCE: This study highlights an approach for using whole genome sequencing to narrow down to a manageable candidate list of genetic variants in a complex genetic epilepsy animal model, and suggests utility of this sequencing design to investigate other spontaneously occurring animal models of human disease.