Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming.

In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.

Postictal hypoxia involves reactive oxygen species and is ameliorated by chronic mitochondrial uncoupling.

Prolonged severe hypoxia follows brief seizures and represents a mechanism underlying several negative postictal manifestations without interventions. Approximately 50% of the postictal hypoxia phenomenon can be accounted for by arteriole vasoconstriction. What accounts for the rest of the drop in unbound oxygen is unclear. Here, we determined the effect of pharmacological modulation of mitochondrial function on tissue oxygenation in the hippocampus of rats after repeatedly evoked seizures. Rats were treated with mitochondrial uncoupler 2,4 dinitrophenol (DNP) or antioxidants. Oxygen profiles were recorded using a chronically implanted oxygen-sensing probe, before, during, and after seizure induction. Mitochondrial function and redox tone were measured using in vitro mitochondrial assays and immunohistochemistry. Postictal cognitive impairment was assessed using the novel object recognition task. Mild mitochondrial uncoupling by DNP raised hippocampal oxygen tension and ameliorated postictal hypoxia. Chronic DNP also lowered mitochondrial oxygen-derived reactive species and oxidative stress in the hippocampus during postictal hypoxia. Uncoupling the mitochondria exerts therapeutic benefits on postictal cognitive dysfunction. Finally, antioxidants do not affect postictal hypoxia, but protect the brain from associated cognitive deficits. We provided evidence for a metabolic component of the prolonged oxygen deprivation that follow seizures and its pathological sequelae. Furthermore, we identified a molecular underpinning of this metabolic component, which involves excessive oxygen conversion into reactive species. Mild mitochondrial uncoupling may be a potential therapeutic strategy to treat the postictal state where seizure control is absent or poor.

Intermittent vs continuous ketogenic diet: Impact on seizures, gut microbiota, and mitochondrial metabolism.

We have shown previously that the ketogenic diet (KD) is effective in reducing seizures associated with infantile spasms syndrome (ISS) and that this benefit is related to alterations in the gut microbiota. However, it remains unclear whether the efficacy of the KD persists after switching to a normal diet. Employing a neonatal rat model of ISS, we tested the hypothesis that the impact of the KD would diminish when switched to a normal diet. Following epilepsy induction, neonatal rats were divided into two groups: continuous KD for 6 days; and a group fed with KD for 3 days and then a normal diet for 3 days. Spasms frequency, mitochondrial bioenergetics in the hippocampus, and fecal microbiota were evaluated as major readouts. We found that the anti-epileptic effect of the KD was reversible, as evidenced by the increased spasms frequency in rats that were switched from the KD to a normal diet. The spasms frequency was correlated inversely with mitochondrial bioenergetic function and a set of gut microbes, including Streptococcus thermophilus and Streptococcus azizii. These findings suggest that the anti-epileptic and metabolic benefits of the KD decline rapidly in concert with gut microbial alterations in the ISS model.

Proposed anti-seizure medication combinations with rufinamide in the treatment of Lennox-Gastaut syndrome: Narrative review and expert opinion.

Lennox-Gastaut syndrome (LGS) is a severe, chronic, complex form of early childhood-onset epilepsy characterized by multiple seizure types, generalized slow (≤2.5 Hz) spike-and-wave activity and other electroencephalography abnormalities, and cognitive impairment. A key treatment goal is early seizure control, and several anti-seizure medications (ASMs) are available. Due to the low success rate in achieving seizure control with monotherapy and an absence of efficacy data supporting any particular combination of ASMs for treating LGS, a rational approach to selection of appropriate polytherapy should be applied to maximize benefit to patients. Such "rational polytherapy" involves consideration of factors including safety (including boxed warnings), potential drug-drug interactions, and complementary mechanisms of action. Based on the authors' clinical experience, rufinamide offers a well-considered first adjunctive therapy for LGS, particularly in combination with clobazam and other newer agents for LGS, and may be particularly useful for reducing the frequency of tonic-atonic seizures associated with LGS.

kcna1a mutant zebrafish model episodic ataxia type 1 (EA1) with epilepsy and show response to first-line therapy carbamazepine.

OBJECTIVE: KCNA1 mutations are associated with a rare neurological movement disorder known as episodic ataxia type 1 (EA1), and epilepsy is a common comorbidity. Current medications provide only partial relief for ataxia and/or seizures, making new drugs needed. Here, we characterized zebrafish kcna1a-/- as a model of EA1 with epilepsy and compared the efficacy of the first-line therapy carbamazepine in kcna1a-/- zebrafish to Kcna1-/- rodents. METHODS: CRISPR/Cas9 mutagenesis was used to introduce a mutation in the sixth transmembrane segment of the zebrafish Kcna1 protein. Behavioral and electrophysiological assays were performed on kcna1a-/- larvae to assess ataxia- and epilepsy-related phenotypes. Real-time quantitative polymerase chain reaction (qPCR) was conducted to measure mRNA levels of brain hyperexcitability markers in kcna1a-/- larvae, followed by bioenergetics profiling to evaluate metabolic function. Drug efficacies were tested using behavioral and electrophysiological assessments, as well as seizure frequency in kcna1a-/- zebrafish and Kcna1-/- mice, respectively. RESULTS: Zebrafish kcna1a-/- larvae showed uncoordinated movements and locomotor deficits, along with scoliosis and increased mortality. The mutants also exhibited impaired startle responses when exposed to light-dark flashes and acoustic stimulation as well as hyperexcitability as measured by extracellular field recordings and upregulated fosab transcripts. Neural vglut2a and gad1b transcript levels were disrupted in kcna1a-/- larvae, indicative of a neuronal excitatory/inhibitory imbalance, as well as a significant reduction in cellular respiration in kcna1a-/- , consistent with dysregulation of neurometabolism. Notably, carbamazepine suppressed the impaired startle response and brain hyperexcitability in kcna1a-/- zebrafish but had no effect on the seizure frequency in Kcna1-/- mice, suggesting that this EA1 zebrafish model might better translate to humans than rodents. SIGNIFICANCE: We conclude that zebrafish kcna1a-/- show ataxia and epilepsy-related phenotypes and are responsive to carbamazepine treatment, consistent with EA1 patients. These findings suggest that kcna1-/- zebrafish are a useful model for drug screening as well as studying the underlying disease biology.

A clinically relevant selective ERK-pathway inhibitor reverses core deficits in a mouse model of autism.

BACKGROUND: Extracellular signal-regulated kinase (ERK/MAPK) pathway in the brain is hypothesized to be a critical convergent node in the development of autism spectrum disorder. We reasoned that selectively targeting this pathway could reverse core autism-like phenotype in animal models. METHODS: Here we tested a clinically relevant, selective inhibitor of ERK pathway, PD325901 (Mirdametinib), in a mouse model of idiopathic autism, the BTBR mice. FINDINGS: We report that treating juvenile mice with PD325901 reduced ERK pathway activation, dose and duration-dependently reduced core disease-modeling deficits in sociability, vocalization and repetitive behavior, and reversed abnormal EEG signals. Further analysis revealed that subchronic treatment did not affect weight gain, locomotion, or neuronal density in the brain. Parallel treatment in the C57BL/6J mice did not alter their phenotype. INTERPRETATION: Our data indicate that selectively inhibiting ERK pathway using PD325901 is beneficial in the BTBR model, thus further support the notion that ERK pathway is critically involved in the pathophysiology of autism. These results suggest that a similar approach could be applied to animal models of syndromic autism with dysregulated ERK signaling, to further test selectively targeting ERK pathway as a new approach for treating autism. FUNDING: This has beenwork was supported by Alberta Children's Hospital Research Foundation (JMR & NC), University of Calgary Faculty of Veterinary Medicine (NC), Kids Brain Health Network (NC), and Natural Sciences and Engineering Research Council of Canada (NC).

Sudden unexpected death in epilepsy is prevented by blocking postictal hypoxia.

Epilepsy is at times a fatal disease. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality in people with intractable epilepsy and is defined by exclusion; non-accidental, non-toxicologic, and non-anatomic causes of death. While SUDEP often follows a bilateral tonic-clonic seizure, the mechanisms that ultimately lead to terminal apnea and then asystole remain elusive and there is a lack of preventative treatments. Based on the observation that discrete seizures lead to local and postictal vasoconstriction, resulting in hypoperfusion, hypoxia and behavioural disturbances in the forebrain we reasoned those similar mechanisms may play a role in SUDEP when seizures invade the brainstem. Here we tested this neurovascular-based hypothesis of SUDEP in awake non-anesthetized mice by pharmacologically preventing seizure-induced vasoconstriction, with cyclooxygenase-2 or L-type calcium channel antagonists. In both acute and chronic mouse models of seizure-induced premature mortality, ibuprofen and nicardipine extended life while systemic drug levels remained high enough to be effective. We also examined the potential role of spreading depolarization in the acute model of seizure-induced premature mortality. These data provide a proof-of-principle for the neurovascular hypothesis of SUDEP rather than spreading depolarization and the use of currently available drugs to prevent it.

Cannabidiol Impairs Brain Mitochondrial Metabolism and Neuronal Integrity.

Background: The mechanisms underlying the clinical effects of CBD remain poorly understood. Given the increasing evidence for CBD's effects on mitochondria, we sought to examine in more detail whether CBD impacts mitochondrial function and neuronal integrity. Methods: We utilized BE(2)-M17 neuroblastoma cells or acutely isolated brain mitochondria from rodents using a Seahorse extracellular flux analyzer and a fluorescent spectrofluorophotometer assay. Mitochondrial ion channel activity and hippocampal long-term potentiation were measured using standard cellular electrophysiological methods. Spatial learning/memory function was evaluated using the Morris water maze task. Plasma concentrations of CBD were assessed with liquid chromatography-mass spectrometry, and cellular viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction neuronal injury assay. Results: At low micromolar concentrations, CBD reduced mitochondrial respiration, the threshold for mitochondrial permeability transition, and calcium uptake, blocked a novel mitochondrial chloride channel, and reduced the viability of hippocampal cells. These effects were paralleled by in vitro and in vivo learning/memory deficits. We further found that these effects were independent of cannabinoid receptor 1 and mitochondrial G-protein-coupled receptor 55. Conclusion: Our results provide evidence for concentration- and dose-dependent toxicological effects of CBD, findings that may bear potential relevance to clinical populations.

Addition of Prebiotics to the Ketogenic Diet Improves Metabolic Profile but Does Not Affect Seizures in a Rodent Model of Infantile Spasms Syndrome.

The ketogenic diet (KD) is an effective treatment for infantile spasms syndrome (IS). However, the KD has implications for somatic growth, development, and the gut microbiota. The impact of incorporating a prebiotic fiber (PRE, oligofructose-enriched inulin, 0.8 g/dL) into a KD diet on spasms, developmental milestones, fecal gut microbiota, metabolites, and hippocampal mitochondrial metabolism were examined. Following IS induction, animals were randomized to KD or KD + PRE diets. A third group without IS and suckled by dams was included as a normally developing reference group (R). PRE inclusion decreased ketones and increased circulating glucose levels but had no impact on spasms. In the liver, PRE increased triglyceride concentrations, decreased carnitine levels, and downregulated genes encoding enzymes responsible for ketogenesis. In the hippocampus, PRE increased glutathione levels but did not affect the maximal respiratory capacity of mitochondria. Analysis of the gut microbiota showed that KD + PRE increased microbial richness and the relative abundance of Bifidobacterium pseudolongum and Lactobacillus johnsonii. No differences in developmental milestones (i.e., surface righting, negative geotaxis, and open field behavior) were observed between KD and KD + PRE, except for ultrasonic vocalizations that were more frequent in KD + PRE. In summary, PRE did not impact spasms or developmental outcomes, but was effective in improving both metabolic parameters and gut microbiota diversity.

Selective Probiotic Treatment Positively Modulates the Microbiota-Gut-Brain Axis in the BTBR Mouse Model of Autism.

Recent studies have shown promise for the use of probiotics in modulating behaviour through the microbiota-gut-brain axis. In the present study, we assessed the impact of two probiotic strains in mitigating autism-related symptomology in the BTBR T+ Itpr3tf/J mouse model of autism spectrum disorder (ASD). Male juvenile BTBR mice were randomized into: (1) control, (2) Lr probiotic (1 × 109 CFU/mL Lacticaseibacillus rhamnosus HA-114), and (3) Ls probiotic groups (1 × 109 CFU/mL Ligilactobacillus salivarius HA-118) (n = 18-21/group), receiving treatments in drinking water for 4 weeks. Gut microbiota profiling by 16S rRNA showed Lr, but not Ls supplementation, to increase microbial richness and phylogenetic diversity, with a rise in potential anti-inflammatory and butyrate-producing taxa. Assessing serum and brain metabolites, Lr and Ls supplementation produced distinct metabolic profiles, with Lr treatment elevating concentrations of potentially beneficial neuroactive compounds, such as 5-aminovaleric acid and choline. As mitochondrial dysfunction is often observed in ASD, we assessed mitochondrial oxygen consumption rates in the prefrontal cortex and hippocampus. No differences were observed for either treatment. Both Lr and Ls treatment reduced behavioural deficits in social novelty preference. However, no changes in hyperactivity, repetitive behaviour, and sociability were observed. Results show Lr to impart positive changes along the microbiota-gut-brain axis, exhibiting beneficial effects on selected behaviour, gut microbial diversity, and metabolism in BTBR mice.

Circadian Responses to Light in the BTBR Mouse.

Animals with altered freerunning periods are valuable in understanding properties of the circadian clock. Understanding the relationship between endogenous clock properties, entrainment, and influence of light in terms of parametric and non-parametric models can help us better understand how different populations adapt to external light cycles. Many clinical populations often show significant changes in circadian properties that in turn cause sleep and circadian problems, possibly exacerbating their underlying clinical condition. BTBR T+Itpr3tf/J (BTBR) mice are a model commonly used for the study of autism spectrum disorders (ASD). Adults and adolescents with ASD frequently exhibit profound sleep and circadian disruptions, including increased latency to sleep, insomnia, advanced and delayed sleep phase disorders, and sleep fragmentation. Here, we investigated the circadian phenotype of BTBR mice in freerunning and light-entrained conditions and found that this strain of mice showed noticeably short freerunning periods (~22.75 h). In addition, when compared to C57BL/6J controls, BTBR mice also showed higher levels of activity even though this activity was compressed into a shorter active phase. Phase delays and phase advances to light were significantly larger in BTBR mice. Despite the short freerunning period, BTBR mice exhibited normal entrainment in light-dark cycles and accelerated entrainment to both advanced and delayed light cycles. Their ability to entrain to skeleton photoperiods of 1 min suggests that this entrainment cannot be attributed to masking. Period differences were also correlated with differences in the number of vasoactive intestinal polypeptide-expressing cells in the suprachiasmatic nucleus (SCN). Overall, the BTBR model, with their unique freerunning and entrainment properties, makes an interesting model to understand the underlying circadian clock.

Targeted gut microbiota manipulation attenuates seizures in a model of infantile spasms syndrome.

Infantile spasms syndrome (IS) is a devastating early-onset epileptic encephalopathy associated with poor neurodevelopmental outcomes. When first-line treatment options, including adrenocorticotropic hormone and vigabatrin, are ineffective, the ketogenic diet (KD) is often employed to control seizures. Since the therapeutic impact of the KD is influenced by the gut microbiota, we examined whether targeted microbiota manipulation, mimicking changes induced by the KD, would be valuable in mitigating seizures. Employing a rodent model of symptomatic IS, we show that both the KD and antibiotic administration reduce spasm frequency and are associated with improved developmental outcomes. Spasm reductions were accompanied by specific gut microbial alterations, including increases in Streptococcus thermophilus and Lactococcus lactis. Mimicking the fecal microbial alterations in a targeted probiotic, we administered these species in a 5:1 ratio. Targeted probiotic administration reduced seizures and improved locomotor activities in control diet-fed animals, similar to KD-fed animals, while a negative control (Ligilactobacillus salivarius) had no impact. Probiotic administration also increased antioxidant status and decreased proinflammatory cytokines. Results suggest that a targeted probiotic reduces seizure frequency, improves locomotor activity in a rodent model of IS, and provides insights into microbiota manipulation as a potential therapeutic avenue for pediatric epileptic encephalopathies.

Gut-based manipulations spur hippocampal mitochondrial bioenergetics in a model of pediatric epilepsy.

A growing body of evidence supports a role of the gut microbiota in regulating diverse physiological processes, including neural function and metabolism via the gut-brain axis. Infantile spasms syndrome is an early-onset epileptic encephalopathy associated with perturbed brain mitochondrial bioenergetics. Employing a neonatal rat model of infantile spasms, mitochondria respirometry and biochemical analyses, the present study reveals that gut microbiota manipulation by diet, antibiotics and probiotics have the potential to enhance hippocampal mitochondrial bioenergetics. Although preliminary in nature, our data reveal that microbial manipulation that regulates brain mitochondrial function may be a novel strategy for the treatment of epileptic disorders.

The metabolic basis of epilepsy.

The brain is a highly energy-demanding organ and requires bioenergetic adaptability to balance normal activity with pathophysiological fuelling of spontaneous recurrent seizures, the hallmark feature of the epilepsies. Recurrent or prolonged seizures have long been known to permanently alter neuronal circuitry and to cause excitotoxic injury and aberrant inflammation. Furthermore, pathological changes in bioenergetics and metabolism are considered downstream consequences of epileptic seizures that begin at the synaptic level. However, as we highlight in this Review, evidence is also emerging that primary derangements in cellular or mitochondrial metabolism can result in seizure genesis and lead to spontaneous recurrent seizures. Basic and translational research indicates that the relationships between brain metabolism and epileptic seizures are complex and bidirectional, producing a vicious cycle that compounds the deleterious consequences of seizures. Metabolism-based treatments such as the high-fat, antiseizure ketogenic diet have become mainstream, and metabolic substrates and enzymes have become attractive molecular targets for seizure prevention and recovery. Moreover, given that metabolism is crucial for epigenetic as well as inflammatory changes, the idea that epileptogenesis can be both negatively and positively influenced by metabolic changes is rapidly gaining ground. Here, we review evidence that supports both pathophysiological and therapeutic roles for brain metabolism in epilepsy.

Probiotics counteract hepatic steatosis caused by ketogenic diet and upregulate AMPK signaling in a model of infantile epilepsy.

BACKGROUND: Infantile spasms syndrome (IS) is a type of epilepsy affecting 1.6 to 4.5 per 10,000 children in the first year of life, often with severe lifelong neurodevelopmental consequences. Only two first-line pharmacological treatments currently exist for IS and many children are refractory to these therapies. In such cases, children are treated with the ketogenic diet (KD). While effective in reducing seizures, the diet can result in dyslipidemia over time. METHODS: Employing a neonatal Sprague-Dawley rat model of IS, we investigated how the KD affects hepatic steatosis and its modulation by a defined probiotic blend. A combination of multiple readouts, including malondialdehyde, fatty acid profiles, lipid metabolism-related enzyme mRNA expression, mitochondrial function, histone deacetylase activity, cytokines and chemokines were evaluated using liver homogenates. FINDINGS: The KD reduced seizures, but resulted in severe hepatic steatosis, characterized by a white liver, triglyceride accumulation, elevated malondialdehyde, polyunsaturated fatty acids and lower acyl-carnitines compared to animals fed a control diet. The KD-induced metabolic phenotype was prevented by the co-administration of a blend of Streptococcus thermophilus HA-110 and Lactococcus lactis subsp. lactis HA-136. This probiotic blend protected the liver by elevating pAMPK-mediated signaling and promoting lipid oxidation. The strains further upregulated the expression of caspase 1 and interleukin 18, which may contribute to their hepatoprotective effect in this model. INTERPRETATION: Our results suggest that early intervention with probiotics could be considered as an approach to reduce the risk of hepatic side effects of the KD in children who are on the diet for medically indicated reasons. FUNDING: This study was funded by the Alberta Children's Hospital Research Institute and Mitacs Accelerate Program (IT16942).

Seizure modulation by the gut microbiota and tryptophan-kynurenine metabolism in an animal model of infantile spasms.

BACKGROUND: The infantile spasms syndrome is an early-onset epileptic encephalopathy presenting in the first 2 years of life, often with severe developmental consequences. The role of the gut microbiota and metabolism in infantile spasms remains unexplored. METHODS: Employing a brain injury neonatal rat model of infantile spasms intractable to anticonvulsant medication treatments, we determined how the ketogenic diet and antibiotics affected specific microbial communities and the resultant circulating factors that confer spasms protection in the infantile spasms model. To confirm a role of kynurenine metabolism pathway in spasms protection, indoleamine 2,3-dioxygenase 1 was pharmacologically inhibited and comprehensive metabolomics was applied. FINDINGS: We show that antibiotics reduced spasms and improved the effectiveness of the ketogenic diet when given in combination. Examination of the gut microbiota and metabolomics showed the downregulation of indoleamine 2,3-dioxygenase 1 and upregulation of hippocampal kynurenic acid, a metabolite with antiepileptic effects. To further test the involvement of indoleamine 2,3-dioxygenase 1, a specific antagonist 1-methyltryptophan and minocycline, an antibiotic and inhibitor of kynurenine formation from tryptophan, were administered, respectively. Both treatments were effective in reducing spasms and elevating hippocampal kynurenic acid. A fecal microbiota transplant experiment was then performed to examine the contribution of the gut microbiota on spasm mitigation. Transplant of feces of ketogenic diet animals into normal diet animals was effective in reducing spasms. INTERPRETATION: These results highlight the importance of tryptophan-kynurenine metabolism in infantile spasms and provide evidence for new-targeted therapies such as indoleamine 2,3-dioxygenase 1 inhibition or microbiota manipulation to promote kynurenic acid production as a strategy to reduce spasms in infantile spasms. FUNDING: This study was funded by the Alberta Children's Hospital Research Institute and the Owerko Centre.

Abnormal oxidative metabolism in the cuprizone mouse model of demyelination: An in vivo NIRS-MRI study.

Disruptions in oxidative metabolism may occur in multiple sclerosis and other demyelinating neurological diseases. The impact of demyelination on metabolic rate is also not understood. It is possible that mitochondrial damage may be associated with many such neurological disorders. To study oxidative metabolism with one model of demyelination, we implemented a novel multimodal imaging technique combining Near-Infrared Spectroscopy (NIRS) and MRI to cuprizone mouse model. The cuprizone model is used to study demyelination and may be associated with inhibition of mitochondrial function. Cuprizone mice showed reduced oxygen extraction fraction (-39.1%, p ≤ 0.001), increased tissue oxygenation (6.4%, p ≤ 0.001), and reduced cerebral metabolic rate of oxygen in cortical gray matter (-62.1%, p ≤ 0.001). These changes resolved after the cessation of cuprizone exposure and partial remyelination. A decrease in hemoglobin concentration (-34.4%, p ≤ 0.001), but no change in cerebral blood flow were also observed during demyelination. The oxidized state of the mitochondrial enzyme, Cytochrome C Oxidase (CCO) increased (46.3%, p ≤ 0.001) while the reduced state decreased (-34.4%, p ≤ 0.05) significantly in cuprizone mice. The total amount of CCO did not change significantly during cuprizone exposure. Total CCO did decline after recovery both in control (-23.1%, p ≤ 0.01) and cuprizone (-28.8%, p ≤ 0.001) groups which may relate to age. A reduction in the magnetization transfer ratio, indicating demyelination, was found in the cuprizone group in the cerebral cortex (-3.2%, p ≤ 0.01) and corpus callosum (-5.5%, p ≤ 0.001). In summary, we were able to detect evidence of altered CCO metabolism during cuprizone exposure, consistent with a mitochondrial defect. We observed increased oxygenation and reduced metabolic rate associated with reduced myelination in the gray and white matter. The novel multimodal imaging technique applied here shows promise for noninvasively assessing parameters associated with oxidative metabolism in both mouse models of neurological disease and for translation to study oxidative metabolism in the human brain.

Genotype-phenotype correlations in SCN8A-related disorders reveal prognostic and therapeutic implications.

We report detailed functional analyses and genotype-phenotype correlations in 392 individuals carrying disease-causing variants in SCN8A, encoding the voltage-gated Na+ channel Nav1.6, with the aim of describing clinical phenotypes related to functional effects. Six different clinical subgroups were identified: Group 1, benign familial infantile epilepsy (n = 15, normal cognition, treatable seizures); Group 2, intermediate epilepsy (n = 33, mild intellectual disability, partially pharmaco-responsive); Group 3, developmental and epileptic encephalopathy (n = 177, severe intellectual disability, majority pharmaco-resistant); Group 4, generalized epilepsy (n = 20, mild to moderate intellectual disability, frequently with absence seizures); Group 5, unclassifiable epilepsy (n = 127); and Group 6, neurodevelopmental disorder without epilepsy (n = 20, mild to moderate intellectual disability). Those in Groups 1-3 presented with focal or multifocal seizures (median age of onset: 4 months) and focal epileptiform discharges, whereas the onset of seizures in patients with generalized epilepsy was later (median: 42 months) with generalized epileptiform discharges. We performed functional studies expressing missense variants in ND7/23 neuroblastoma cells and primary neuronal cultures using recombinant tetrodotoxin-insensitive human Nav1.6 channels and whole-cell patch-clamping. Two variants causing developmental and epileptic encephalopathy showed a strong gain-of-function (hyperpolarizing shift of steady-state activation, strongly increased neuronal firing rate) and one variant causing benign familial infantile epilepsy or intermediate epilepsy showed a mild gain-of-function (defective fast inactivation, less increased firing). In contrast, all three variants causing generalized epilepsy induced a loss-of-function (reduced current amplitudes, depolarizing shift of steady-state activation, reduced neuronal firing). Functional effects were known for 170 individuals. All 136 individuals carrying a functionally tested gain-of-function variant had either focal (n = 97, Groups 1-3) or unclassifiable (n = 39) epilepsy, whereas 34 individuals with a loss-of-function variant had either generalized (n = 14), no (n = 11) or unclassifiable (n = 6) epilepsy; only three had developmental and epileptic encephalopathy. Computational modelling in the gain-of-function group revealed a significant correlation between the severity of the electrophysiological and clinical phenotypes. Gain-of-function variant carriers responded significantly better to sodium channel blockers than to other anti-seizure medications, and the same applied for all individuals in Groups 1-3. In conclusion, our data reveal clear genotype-phenotype correlations between age at seizure onset, type of epilepsy and gain- or loss-of-function effects of SCN8A variants. Generalized epilepsy with absence seizures is the main epilepsy phenotype of loss-of-function variant carriers and the extent of the electrophysiological dysfunction of the gain-of-function variants is a main determinant of the severity of the clinical phenotype in focal epilepsies. Our pharmacological data indicate that sodium channel blockers present a treatment option in SCN8A-related focal epilepsy with onset in the first year of life.

Pharmacokinetics and central accumulation of delta-9-tetrahydrocannabinol (THC) and its bioactive metabolites are influenced by route of administration and sex in rats.

Up to a third of North Americans report using cannabis in the prior month, most commonly through inhalation. Animal models that reflect human consumption are critical to study the impact of cannabis on brain and behaviour. Most animal studies to date utilize injection of delta-9-tetrahydrocannabinol (THC; primary psychoactive component of cannabis). THC injections produce markedly different physiological and behavioural effects than inhalation, likely due to distinctive pharmacokinetics. The current study directly examined if administration route (injection versus inhalation) alters metabolism and central accumulation of THC and metabolites over time. Adult male and female Sprague-Dawley rats received either an intraperitoneal injection or a 15-min session of inhaled exposure to THC. Blood and brains were collected at 15, 30, 60, 90 and 240-min post-exposure for analysis of THC and metabolites. Despite achieving comparable peak blood THC concentrations in both groups, our results indicate higher initial brain THC concentration following inhalation, whereas injection resulted in dramatically higher 11-OH-THC concentration, a potent THC metabolite, in blood and brain that increased over time. Our results provide evidence of different pharmacokinetic profiles following inhalation versus injection. Accordingly, administration route should be considered during data interpretation, and translational animal work should strongly consider using inhalation models.

The link between brain acidosis, breathing and seizures: a novel mechanism of action for the ketogenic diet in a model of infantile spasms.

Infantile spasms (IS) syndrome is a catastrophic, epileptic encephalopathy of infancy that is often refractory to current antiepileptic therapies. The ketogenic diet (KD) has emerged as an alternative treatment for patients with medically intractable epilepsy, though the prospective validity and mechanism of action for IS remains largely unexplored. We investigated the KD's efficacy as well as its mechanism of action in a rodent model of intractable IS. The spasms were induced using the triple-hit paradigm and the animals were then artificially reared and put on either the KD (4:1 fats: carbohydrate + protein) or a control milk diet (CM; 1.7:1). 31Phosphorus magnetic resonance spectroscopy (31P MRS) and head-out plethysmography were examined in conjunction with continuous video-EEG behavioural recordings in lesioned animals and sham-operated controls. The KD resulted in a peripheral ketosis observed both in the blood and urine. The KD led to a robust reduction in the frequency of spasms observed, with approximately a 1.5-fold increase in the rate of survival. Intriguingly, the KD resulted in an intracerebral acidosis as measured with 31P MRS. In addition, the respiratory profile of the lesioned rats on the KD was significantly altered with slower, deeper and longer breathing, resulting in decreased levels of expired CO2. Sodium bicarbonate supplementation, acting as a pH buffer, partially reversed the KD's protective effects on spasm frequency. There were no differences in the mitochondrial respiratory profiles in the liver and brain frontal cortex measured between the groups, supporting the notion that the effects of the KD on breathing are not entirely due to changes in intermediary metabolism. Together, our results indicate that the KD produces its anticonvulsant effects through changes in respiration leading to intracerebral acidosis. These findings provide a novel understanding of the mechanisms underlying the anti-seizure effects of the KD in IS. Further research is required to determine whether the effects of the KD on breathing and intracerebral acid-base balance are seen in other paediatric models of epilepsy.