Lestaurtinib (CEP-701) reduces the duration of limbic status epilepticus in periadolescent rats.

BACKGROUND: The timely abortion of status epilepticus (SE) is essential to avoid brain damage and long-term neurodevelopmental sequalae. However, available anti-seizure treatments fail to abort SE in 30% of children. Given the role of the tropomyosin-related kinase B (TrkB) receptor in hyperexcitability, we investigated if TrkB blockade with lestaurtinib (CEP-701) enhances the response of SE to a standard treatment protocol and reduces SE-related brain injury. METHODS: SE was induced with intra-amygdalar kainic acid in postnatal day 45 rats under continuous electroencephalogram (EEG). Fifteen min post-SE onset, rats received intraperitoneal (i.p.) CEP-701 (KCEP group) or its vehicle (KV group). Controls received CEP-701 or its vehicle following intra-amygdalar saline. All groups received two i.p. doses of diazepam, followed by i.p. levetiracetam at 15 min intervals post-SE onset. Hippocampal TrkB dimer to monomer ratios were assessed by immunoblot 24 hr post-SE, along with neuronal densities and glial fibrillary acid protein (GFAP) levels. RESULTS: SE duration was 50% shorter in the KCEP group compared to KV (p < 0.05). Compared to controls, SE induced a 1.5-fold increase in TrkB dimerization in KV rats (p < 0.05), but not in KCEP rats which were comparable to controls (p > 0.05). The KCEP group had lower GFAP levels than KV (p < 0.05), and both were higher than controls (p < 0.05). KCEP and KV rats had comparable hippocampal neuronal densities (p > 0.05), and both were lower than controls (p < 0.05). CONCLUSIONS: Given its established human safety, CEP-701 is a promising adjuvant drug for the timely abortion of SE and the attenuation of SE-related brain injury.

TRPM7 kinase is required for insulin production and compensatory islet responses during obesity.

Most overweight individuals do not develop diabetes due to compensatory islet responses to restore glucose homeostasis. Therefore, regulatory pathways that promote ß cell compensation are potential targets for treatment of diabetes. The transient receptor potential cation channel subfamily M member 7 protein (TRPM7), harboring a cation channel and a serine/threonine kinase, has been implicated in controlling cell growth and proliferation. Here, we report that selective deletion of Trpm7 in ß cells disrupted insulin secretion and led to progressive glucose intolerance. We indicate that the diminished insulinotropic response in ß cell-specific Trpm7-knockout mice was caused by decreased insulin production because of impaired enzymatic activity of this protein. Accordingly, high-fat-fed mice with a genetic loss of TRPM7 kinase activity displayed a marked glucose intolerance accompanied by hyperglycemia. These detrimental glucoregulatory effects were engendered by reduced compensatory ß cell responses because of mitigated protein kinase B (AKT)/ERK signaling. Collectively, our data identify TRPM7 kinase as a potentially novel regulator of insulin synthesis, ß cell dynamics, and glucose homeostasis under obesogenic diet.

Hippocampal injury and learning deficits following non-convulsive status epilepticus in periadolescent rats.

The effects of non-convulsive status epilepticus (NCSE) on the developing brain remain largely elusive. Here we investigated potential hippocampal injury and learning deficits following one or two episodes of NCSE in periadolescent rats. Non-convulsive status epilepticus was induced with subconvulsive doses of intrahippocampal kainic acid (KA) under continuous EEG monitoring in postnatal day 43 (P43) rats. The RKA group (repeated KA) received intrahippocampal KA at P43 and P44, the SKA group (single KA injection) received KA at P43 and an intrahippocampal saline injection at P44. Controls were sham-treated with saline. The modified two-way active avoidance (MAAV) test was conducted between P45 and P52 to assess learning of context-cued and tone-signaled electrical foot-shock avoidance. Histological analyses were performed at P52 to assess hippocampal neuronal densities, as well as potential reactive astrocytosis and synaptic dysfunction with GFAP (glial fibrillary acidic protein) and synaptophysin (Syp) staining, respectively. Kainic acid injections resulted in electroclinical seizures characterized by behavioral arrest, oromotor automatisms and salivation, without tonic-clonic activity. Compared to controls, both the SKA and RKA groups had lower rates of tone-signaled shock avoidance (p < 0.05). In contextual testing, SKA rats were comparable to controls (p > 0.05), but the RKA group had learning deficits (p < 0.05). Hippocampal neuronal densities were comparable in all groups. Compared to controls, both the SKA and RKA groups had higher hippocampal GFAP levels (p < 0.05). The RKA group also had lower hippocampal Syp levels compared to the SKA and control groups (p < 0.05), which were comparable (p > 0.05). We show that hippocampal NCSE in periadolescent rats results in a seizure burden-dependent hippocampal injury accompanied by cognitive deficits. Our data suggest that the diagnosis and treatment of NCSE should be prompt.

Cavß3 Regulates Ca2+ Signaling and Insulin Expression in Pancreatic ß-Cells in a Cell-Autonomous Manner.

Voltage-gated Ca2+ (Cav) channels consist of a pore-forming Cavα1 subunit and auxiliary Cavα2-δ and Cavß subunits. In fibroblasts, Cavß3, independent of its role as a Cav subunit, reduces the sensitivity to low concentrations of inositol-1,4,5-trisphosphate (IP3). Similarly, Cavß3 could affect cytosolic calcium concentration ([Ca2 +]) in pancreatic ß-cells. In this study, we deleted the Cavß3-encoding gene Cacnb3 in insulin-secreting rat ß-(Ins-1) cells using CRISPR/Cas9. These cells were used as controls to investigate the role of Cavß3 on Ca2+ signaling, glucose-induced insulin secretion (GIIS), Cav channel activity, and gene expression in wild-type cells in which Cavß3 and the IP3 receptor were coimmunoprecipitated. Transcript and protein profiling revealed significantly increased levels of insulin transcription factor Mafa, CaMKIV, proprotein convertase subtilisin/kexin type-1, and nitric oxide synthase-1 in Cavß3-knockout cells. In the absence of Cavß3, Cav currents were not altered. In contrast, CREB activity, the amount of MAFA protein and GIIS, the extent of IP3-dependent Ca2+ release and the frequency of Ca2+ oscillations were increased. These processes were decreased by the Cavß3 protein in a concentration-dependent manner. Our study shows that Cavß3 interacts with the IP3 receptor in isolated ß-cells, controls IP3-dependent Ca2+-signaling independently of Cav channel functions, and thereby regulates insulin expression and its glucose-dependent release in a cell-autonomous manner.

A Modified Two-Way Active Avoidance Test for Combined Contextual and Auditory Instrumental Conditioning.

Available two-way active avoidance paradigms do not provide contextual testing, likely due to challenges in performing repetitive trials of context exposure. To incorporate contextual conditioning in the two-way shuttle box, we contextually modified one of the chambers of a standard two-chamber rat shuttle box with visual cues consisting of objects and black and white stripe patterns. During the 5 training days, electrical foot shocks were delivered every 10 s in the contextually modified chamber but were signaled by a tone in the plain chamber. Shuttling between chambers prevented an incoming foot shock (avoidance) or aborted an ongoing one (escape). During contextual retention testing, rats were allowed to freely roam in the box. During auditory retention testing, visual cues were removed, and tone-signaled shocks were delivered in both chambers. Avoidance gradually replaced escape or freezing behaviors reaching 80% on the last training day in both chambers. Rats spent twice more time in the plain chamber during contextual retention testing and had 90% avoidance rates during auditory retention testing. Our modified test successfully assesses both auditory and contextual two-way active avoidance. By efficiently expanding its array of outcomes, our novel test will complement standard two-way active avoidance in mechanistic studies and will improve its applications in translational research.

Dysfunctional cerebrovascular tone contributes to cognitive impairment in a non-obese rat model of prediabetic challenge: Role of suppression of autophagy and modulation by anti-diabetic drugs.

Prediabetes is a highly prevalent stage of early metabolic dysfunction that poses a high risk for cardiovascular and cognitive impairment without a clear pathological mechanism. Here, we used a non-obese prediabetic rat model previously developed in our laboratory to examine this mechanism. These rats were subjected to a mild metabolic challenge leading to hyperinsulinemia without hyperglycemia or obesity. This was associated with impaired hippocampal-dependent cognitive functions together with an augmented cerebrovascular myogenic tone. Consequently, hippocampal expression of hypoxia-inducible factor-1α increased, together with markers of mitochondrial dysfunction and oxidative stress. In parallel, the phosphorylation of Akt and mTOR increased in the prediabetic rat hippocampus alongside increased expression of p62 and LC3 puncta indicating a possible repression of autophagic flux. Neuroinflammation and neuronal apoptosis were detected in the hippocampal CA1 area as increased CD68 and IBA-1 staining, as well as increased TUNEL staining and caspase-3 activity, respectively. Treatment with metformin or pioglitazone, at a previously determined vasculoprotective non-hypoglycemic dose, reversed the cerebrovascular and hippocampal molecular alterations and ameliorated cognitive function. The present study proposes a mechanistic framework whereby prediabetic cerebrovascular impairment potentially leads to a mild hypoxic state that is exacerbated by the metabolic dysfunction-driven suppression of neuronal autophagy leading to cognitive impairment.

Overview on Emotional Behavioral Testing in Rodent Models of Pediatric Epilepsy.

Psychiatric and cognitive disturbances are the most common comorbidities of epileptic disorders in children. The successful treatment of these comorbidities faces many challenges including their etiologically heterogonous nature. Translational neurobehavioral research in age-tailored and clinically relevant rodent seizure models offers a controlled setting to investigate emotional and cognitive behavioral disturbances, their causative factors, and potentially novel treatment interventions. In this review, we propose a conceptual framework that provides a nonsubjective approach to rodent emotional behavioral testing with a focus on the clinically relevant outcome of behavioral response adaptability. We also describe the battery of neurobehavioral tests that we tailored to seizure models with prominent amygdalo-hippocampal involvement, including testing panels for anxiety-like, exploratory, and hyperactive behaviors (the open-field and light-dark box tests), depressive-like behaviors (the forced swim test), and visuospatial navigation (Morris water maze). The review also discusses the modifications we introduced to active avoidance testing in order to simultaneously test auditory and hippocampal-dependent emotionally relevant learning and memory. When interpreting the significance and clinical relevance of the behavioral responses obtained from a given testing panel, it is important to avoid a holistic disease-based approach as a specific panel may not necessarily mirror a disease entity. The analysis of measurable behavioral responses has to be performed in the context of outcomes obtained from multiple related and complementary neurobehavioral testing panels. Behavioral testing is also complemented by mechanistic electrophysiological and molecular investigations.

Methods in Electrode Implantation and Wiring for Long-Term Continuous EEG Monitoring in Rodent Models of Epilepsy and Behavioral Disturbances.

Rodent seizure models that pathologically and behaviorally recapitulate age-tailored epileptic disorders are used by us and others to advance our understanding of the chronobiology and mechanisms of epileptic seizure emergence and their comorbidities and to investigate potential novel treatment strategies. Obtaining prolonged continuous electroencephalogram (EEG) tracings over months is essential in this line of translational research, particularly to assess the relation between electrographic changes and the development of seizures and their various psychiatric and cognitive comorbidities in models where seizures gradually emerge over weeks following brain insults. Here we describe our approach to electrode implantation and wiring in order to successfully obtain high-quality continuous EEG tracings in rats for prolonged periods. A detailed stepwise methodological description is provided with a special focus on the details that help most in avoiding notorious pitfalls such as premature EEG cable disconnections and a poor signal to noise ratio.

Methods in Emotional Behavioral Testing in Immature Epilepsy Rodent Models.

Pediatric epilepsy is associated with prominent comorbid psychiatric and cognitive disturbances. Neurobehavioral testing is employed to characterize the cognitive and emotional behavioral derangements that accompany seizures in age-tailored and clinically relevant immature rodent seizure models. In addition to dissecting the causes of the etiologically multifaceted psychiatric and cognitive comorbidities of the epilepsies, neurobehavioral panels are essential in investigating potential neuroprotective strategies, especially during neurodevelopment. Here we describe a battery of behavioral testing panels that we tailored to our rodent seizure models with prominent amygdalo-hippocampal involvement. The panels include the open field and light-dark box tests for exploratory, hyperactive, and anxiety-like behaviors, the forced swim test for depressive-like behaviors, the Morris water maze for visuospatial navigation, and the modified active avoidance test for emotionally relevant learning and acquisition of adaptive behaviors. The behavioral laboratory setup and the employed methodologies are reviewed in details, with a special focus on the potential pitfalls that should be avoided.

Enhanced setup for wired continuous long-term EEG monitoring in juvenile and adult rats: application for epilepsy and other disorders.

BACKGROUND: The electroencephalogram (EEG) is a widely used laboratory technique in rodent models of epilepsy, traumatic brain injury (TBI), and other neurological diseases accompanied by seizures. Obtaining prolonged continuous EEG tracings over weeks to months is essential to adequately answer research questions related to the chronobiology of seizure emergence, and to the effect of potential novel treatment strategies. Current EEG recording methods include wired and the more recent but very costly wireless technologies. Wired continuous long-term EEG in rodents remains the mainstay approach but is often technically challenging due to the notorious frequent EEG cable disconnections from the rodent's head, and to poor signal-to-noise ratio especially when simultaneously monitoring multiple animals. Premature EEG cable disconnections and cable movement-related artifacts result from the animal's natural mobility, and subsequent tension on the EEG wires, as well as from potential vigorous and frequent seizures. These challenges are often accompanied by injuries to the scalp, and result in early terminations of costly experiments. RESULTS: Here we describe an enhanced customized swivel-balance EEG-cage system that allows tension-free rat mobility. The cage setup markedly improves the safety and longevity of current existing wired continuous long-term EEG. Prevention of EEG cable detachments is further enhanced by a special attention to surgical electrode anchoring to the skull. In addition to mechanically preventing premature disconnections, the detailed stepwise approach to the electrical shielding, wiring and grounding required for artifact-free high signal-to-noise ratio recordings is also included. The successful application of our EEG cage system in various rat models of brain insults and epilepsy is described with illustrative high quality tracings of seizures and electrographic patterns obtained during continuous and simultaneous monitoring of multiple rats early and up to 3 months post-brain insult. CONCLUSION: Our simple-to-implement key modifications to the EEG cage setup allow the safe acquisition of substantial high quality wired EEG data without resorting to the still costly wireless technologies.

Lestaurtinib (CEP-701) modulates the effects of early life hypoxic seizures on cognitive and emotional behaviors in immature rats.

Hypoxic encephalopathy of the newborn is a major cause of long-term neurological sequelae. We have previously shown that CEP-701 (lestaurtinib), a drug with an established safety profile in children, attenuates short-term hyperexcitability and tropomyosin-related kinase B (TrkB) receptor activation in a well-established rat model of early life hypoxic seizures (HS). Here, we investigated the potential long-term neuroprotective effects of a post-HS transient CEP-701 treatment. Following exposure to global hypoxia, 10 day old male Sprague-Dawley pups received CEP-701 or its vehicle and were sequentially subjected to the light-dark box test (LDT), forced swim test (FST), open field test (OFT), Morris water maze (MWM), and the modified active avoidance (MAAV) test between postnatal days 24 and 44 (P24-44). Spontaneous seizure activity was assessed by epidural cortical electroencephalography (EEG) between P50 and 100. Neuronal density and glial fibrillary acidic protein (GFAP) levels were evaluated on histological sections in the hippocampus, amygdala, and prefrontal cortex at P100. Vehicle-treated hypoxic rats exhibited significantly increased immobility in the FST compared with controls, and post-HS CEP-701 administration reversed this HS-induced depressive-like behavior (p < 0.05). In the MAAV test, CEP-701-treated hypoxic rats were slower at learning both context-cued and tone-signaled shock-avoidance behaviors (p < 0.05). All other behavioral outcomes were comparable, and no recurrent seizures, neuronal loss, or increase in GFAP levels were detected in any of the groups. We showed that early life HS predispose to long-lasting depressive-like behaviors, and that these are prevented by CEP-701, likely via TrkB modulation. Future mechanistically more specific studies will further investigate the potential role of TrkB signaling pathway modulation in achieving neuroprotection against neonatal HS, without causing neurodevelopmental adverse effects.