Face-valid phenotypes in a mouse model of the most common mutation in EEF1A2-related neurodevelopmental disorder.

De novo heterozygous missense mutations in EEF1A2, encoding neuromuscular translation-elongation factor eEF1A2, are associated with developmental and epileptic encephalopathies. We used CRISPR/Cas9 to recapitulate the most common mutation, E122K, in mice. Although E122K heterozygotes were not observed to have convulsive seizures, they exhibited frequent electrographic seizures and EEG abnormalities, transient early motor deficits and growth defects. Both E122K homozygotes and Eef1a2-null mice developed progressive motor abnormalities, with E122K homozygotes reaching humane endpoints by P31. The null phenotype is driven by progressive spinal neurodegeneration; however, no signs of neurodegeneration were observed in E122K homozygotes. The E122K protein was relatively stable in neurons yet highly unstable in skeletal myocytes, suggesting that the E122K/E122K phenotype is instead driven by loss of function in muscle. Nevertheless, motor abnormalities emerged far earlier in E122K homozygotes than in nulls, suggesting a toxic gain of function and/or a possible dominant-negative effect. This mouse model represents the first animal model of an EEF1A2 missense mutation with face-valid phenotypes and has provided mechanistic insights needed to inform rational treatment design.

Reversal of cell, circuit and seizure phenotypes in a mouse model of DNM1 epileptic encephalopathy.

Dynamin-1 is a large GTPase with an obligatory role in synaptic vesicle endocytosis at mammalian nerve terminals. Heterozygous missense mutations in the dynamin-1 gene (DNM1) cause a novel form of epileptic encephalopathy, with pathogenic mutations clustering within regions required for its essential GTPase activity. We reveal the most prevalent pathogenic DNM1 mutation, R237W, disrupts dynamin-1 enzyme activity and endocytosis when overexpressed in central neurons. To determine how this mutation impacted cell, circuit and behavioural function, we generated a mouse carrying the R237W mutation. Neurons from heterozygous mice display dysfunctional endocytosis, in addition to altered excitatory neurotransmission and seizure-like phenotypes. Importantly, these phenotypes are corrected at the cell, circuit and in vivo level by the drug, BMS-204352, which accelerates endocytosis. Here, we demonstrate a credible link between dysfunctional endocytosis and epileptic encephalopathy, and importantly reveal that synaptic vesicle recycling may be a viable therapeutic target for monogenic intractable epilepsies.

Abnormal brain state distribution and network connectivity in a SYNGAP1 rat model.

Mutations in the SYNGAP1 gene are one of the common predictors of neurodevelopmental disorders, commonly resulting in individuals developing autism, intellectual disability, epilepsy, and sleep deficits. EEG recordings in neurodevelopmental disorders show potential to identify clinically translatable biomarkers to both diagnose and track the progress of novel therapeutic strategies, as well as providing insight into underlying pathological mechanisms. In a rat model of SYNGAP1 haploinsufficiency in which the exons encoding the calcium/lipid binding and GTPase-activating protein domains have been deleted (Syngap+/Δ-GAP ), we analysed the duration and occurrence of wake, non-rapid eye movement and rapid eye movement brain states during 6 h multi-electrode EEG recordings. We find that although Syngap+/Δ-GAP animals spend an equivalent percent time in wake and sleep states, they have an abnormal brain state distribution as the number of wake and non-rapid eye movement bouts are reduced and there is an increase in the average duration of both wake and non-rapid eye movement epochs. We perform connectivity analysis by calculating the average imaginary coherence between electrode pairs at varying distance thresholds during these states. In group averages from pairs of electrodes at short distances from each other, a clear reduction in connectivity during non-rapid eye movement is present between 11.5 Hz and 29.5 Hz, a frequency range that overlaps with sleep spindles, oscillatory phenomena thought to be important for normal brain function and memory consolidation. Sleep abnormalities were mostly uncorrelated to the electrophysiological signature of absence seizures, spike and wave discharges, as was the imaginary coherence deficit. Sleep spindles occurrence, amplitude, power and spread across multiple electrodes were not reduced in Syngap+/Δ-GAP rats, with only a small decrease in duration detected. Nonetheless, by analysing the dynamic imaginary coherence during sleep spindles, we found a reduction in high-connectivity instances between short-distance electrode pairs. Finally comparing the dynamic imaginary coherence during sleep spindles between individual electrode pairs, we identified a group of channels over the right somatosensory, association and visual cortices that have a significant reduction in connectivity during sleep spindles in mutant animals. This matched a significant reduction in connectivity during spindles when averaged regional comparisons were made. These data suggest that Syngap+/Δ-GAP rats have altered brain state dynamics and EEG connectivity, which may have clinical relevance for SYNGAP1 haploinsufficiency in humans.

New inroads into the brain circuits and network dynamics behind sudden unexpected death in epilepsy.

This scientific commentary refers to 'Hyperexcitable superior colliculus and fatal brainstem spreading depolarization in a model of sudden unexpected death in epilepsy' by Cain et al. (https://doi.org/10.1093/braincomms/fcac006) and 'Ictal neural oscillatory alterations precede sudden unexpected death in epilepsy' by Gu et al. (https://doi.org/10.1093/braincomms/fcac073).

Medial septal GABAergic neurons reduce seizure duration upon optogenetic closed-loop stimulation.

Seizures can emerge from multiple or large foci in temporal lobe epilepsy, complicating focally targeted strategies such as surgical resection or the modulation of the activity of specific hippocampal neuronal populations through genetic or optogenetic techniques. Here, we evaluate a strategy in which optogenetic activation of medial septal GABAergic neurons, which provide extensive projections throughout the hippocampus, is used to control seizures. We utilized the chronic intrahippocampal kainate mouse model of temporal lobe epilepsy, which results in spontaneous seizures and as is often the case in human patients, presents with hippocampal sclerosis. Medial septal GABAergic neuron populations were immunohistochemically labelled and were not reduced in epileptic conditions. Genetic labelling with mRuby of medial septal GABAergic neuron synaptic puncta and imaging across the rostral to caudal extent of the hippocampus, also indicated an unchanged number of putative synapses in epilepsy. Furthermore, optogenetic stimulation of medial septal GABAergic neurons consistently modulated oscillations across multiple hippocampal locations in control and epileptic conditions. Finally, wireless optogenetic stimulation of medial septal GABAergic neurons, upon electrographic detection of spontaneous hippocampal seizures, resulted in reduced seizure durations. We propose medial septal GABAergic neurons as a novel target for optogenetic control of seizures in temporal lobe epilepsy.

Modelling epilepsy in the mouse: challenges and solutions.

In most mouse models of disease, the outward manifestation of a disorder can be measured easily, can be assessed with a trivial test such as hind limb clasping, or can even be observed simply by comparing the gross morphological characteristics of mutant and wild-type littermates. But what if we are trying to model a disorder with a phenotype that appears only sporadically and briefly, like epileptic seizures? The purpose of this Review is to highlight the challenges of modelling epilepsy, in which the most obvious manifestation of the disorder, seizures, occurs only intermittently, possibly very rarely and often at times when the mice are not under direct observation. Over time, researchers have developed a number of ways in which to overcome these challenges, each with their own advantages and disadvantages. In this Review, we describe the genetics of epilepsy and the ways in which genetically altered mouse models have been used. We also discuss the use of induced models in which seizures are brought about by artificial stimulation to the brain of wild-type animals, and conclude with the ways these different approaches could be used to develop a wider range of anti-seizure medications that could benefit larger patient populations.

Rodent genetic models of neurodevelopmental disorders and epilepsy.

Neurodevelopmental disorders (NDDs) are characterised by cognitive, social and motor deficits and are highly comorbid with intractable epilepsies. Through advances in genetic sequencing technologies a vast number of genes have been implicated in NDDs. State-of-the-art gene-editing techniques have led to the generation of hundreds of mouse models of NDDs. As an example, rodent models of Rett and Dravet syndromes as well as the syndromes caused by mutations in CDKL5 and Syngap1 display cognitive deficits in conjunction with seizure phenotypes. These models allow researchers to understand the underlying mechanisms as well as develop novel treatment strategies that can potentially be translated to the clinic. Furthermore, it may be possible to gain insights into the contribution of epilepsy to the progression of cognitive, social and motor phenotypes in NDDs.

Circadian and Brain State Modulation of Network Hyperexcitability in Alzheimer's Disease.

Network hyperexcitability is a feature of Alzheimer' disease (AD) as well as numerous transgenic mouse models of AD. While hyperexcitability in AD patients and AD animal models share certain features, the mechanistic overlap remains to be established. We aimed to identify features of network hyperexcitability in AD models that can be related to epileptiform activity signatures in AD patients. We studied network hyperexcitability in mice expressing amyloid precursor protein (APP) with mutations that cause familial AD, and compared a transgenic model that overexpresses human APP (hAPP) (J20), to a knock-in model expressing APP at physiological levels (APPNL/F). We recorded continuous long-term electrocorticogram (ECoG) activity from mice, and studied modulation by circadian cycle, behavioral, and brain state. We report that while J20s exhibit frequent interictal spikes (IISs), APPNL/F mice do not. In J20 mice, IISs were most prevalent during daylight hours and the circadian modulation was associated with sleep. Further analysis of brain state revealed that IIS in J20s are associated with features of rapid eye movement (REM) sleep. We found no evidence of cholinergic changes that may contribute to IIS-circadian coupling in J20s. In contrast to J20s, intracranial recordings capturing IIS in AD patients demonstrated frequent IIS in non-REM (NREM) sleep. The salient differences in sleep-stage coupling of IIS in APP overexpressing mice and AD patients suggests that different mechanisms may underlie network hyperexcitability in mice and humans. We posit that sleep-stage coupling of IIS should be an important consideration in identifying mouse AD models that most closely recapitulate network hyperexcitability in human AD.

GABAergic projections from the medial septum selectively inhibit interneurons in the medial entorhinal cortex.

The medial septum (MS) is required for theta rhythmic oscillations and grid cell firing in the medial entorhinal cortex (MEC). While GABAergic, glutamatergic, and cholinergic neurons project from the MS to the MEC, their synaptic targets are unknown. To investigate whether MS neurons innervate specific layers and cell types in the MEC, we expressed channelrhodopsin-2 in mouse MS neurons and used patch-clamp recording in brain slices to determine the response to light activation of identified cells in the MEC. Following activation of MS axons, we observed fast monosynaptic GABAergic IPSPs in the majority (>60%) of fast-spiking (FS) and low-threshold-spiking (LTS) interneurons in all layers of the MEC, but in only 1.5% of nonstellate principal cells (NSPCs) and in no stellate cells. We also observed fast glutamatergic responses to MS activation in a minority (<5%) of NSPCs, FS, and LTS interneurons. During stimulation of MS inputs at theta frequency (10 Hz), the amplitude of GABAergic IPSPs was maintained, and spike output from LTS and FS interneurons was entrained at low (25-60 Hz) and high (60-180 Hz) gamma frequencies, respectively. By demonstrating cell type-specific targeting of the GABAergic projection from the MS to the MEC, our results support the idea that the MS controls theta frequency activity in the MEC through coordination of inhibitory circuits.

Hippocampal neuron firing and local field potentials in the in vitro 4-aminopyridine epilepsy model.

Excessive synchronous neuronal activity is a defining feature of epileptic activity. We previously characterized the properties of distinct glutamatergic and GABAergic transmission-dependent synchronous epileptiform discharges in mouse hippocampal slices using the 4-aminopyridine model of epilepsy. In the present study, we sought to identify the specific hippocampal neuronal populations that initiate and underlie these local field potentials (LFPs). A perforated multielectrode array was used to simultaneously record multiunit action potential firing and LFPs during spontaneous epileptiform activity. LFPs had distinct components based on the initiation site, extent of propagation, and pharmacological sensitivity. Individual units, located in different hippocampal subregions, fired action potentials during these LFPs. A specific neuron subgroup generated sustained action potential firing throughout the various components of the LFPs. The activity of this subgroup preceded the LFPs observed in the presence of antagonists of ionotropic glutamatergic synaptic transmission. In the absence of ionotropic glutamatergic and GABAergic transmission, LFPs disappeared, but units with shorter spike duration and high basal firing rates were still active. These spontaneously active units had an increased level of activity during LFPs and consistently preceded all LFPs recorded before blockade of synaptic transmission. Our findings reveal that neuronal subpopulations with interneuron properties are likely responsible for initiating synchronous activity in an in vitro model of epileptiform discharges.

Cellular mechanisms of desynchronizing effects of hypothermia in an in vitro epilepsy model.

Hypothermia can terminate epileptiform discharges in vitro and in vivo epilepsy models. Hypothermia is becoming a standard treatment for brain injury in infants with perinatal hypoxic ischemic encephalopathy, and it is gaining ground as a potential treatment in patients with drug resistant epilepsy. However, the exact mechanism of action of cooling the brain tissue is unclear. We have studied the 4-aminopyridine model of epilepsy in mice using single- and dual-patch clamp and perforated multi-electrode array recordings from the hippocampus and cortex. Cooling consistently terminated 4-aminopyridine induced epileptiform-like discharges in hippocampal neurons and increased input resistance that was not mimicked by transient receptor potential channel antagonists. Dual-patch clamp recordings showed significant synchrony between distant CA1 and CA3 pyramidal neurons, but less so between the pyramidal neurons and interneurons. In CA1 and CA3 neurons, hypothermia blocked rhythmic action potential discharges and disrupted their synchrony; however, in interneurons, hypothermia blocked rhythmic discharges without abolishing action potentials. In parallel, multi-electrode array recordings showed that synchronized discharges were disrupted by hypothermia, whereas multi-unit activity was unaffected. The differential effect of cooling on transmitting or secreting γ-aminobutyric acid interneurons might disrupt normal network synchrony, aborting the epileptiform discharges. Moreover, the persistence of action potential firing in interneurons would have additional antiepileptic effects through tonic γ-aminobutyric acid release.

The 4-aminopyridine in vitro epilepsy model analyzed with a perforated multi-electrode array.

Epileptiform discharges recorded in the 4-aminopyridine (4-AP) in vitro epilepsy model are mediated by glutamatergic and GABAergic signaling. Using a 60-channel perforated multi-electrode array (pMEA) on corticohippocampal slices from 2 to 3 week old mice we recorded interictal- and ictal-like events. When glutamatergic transmission was blocked, interictal-like events no longer initiated in the hilus or CA3/CA1 pyramidal layers but originated from the dentate gyrus granule and molecular layers. Furthermore, frequencies of interictal-like events were reduced and durations were increased in these regions while cortical discharges were completely blocked. Following GABA(A) receptor blockade interictal-like events no longer propagated to the dentate gyrus while their frequency in CA3 increased; in addition, ictal-like cortical events became shorter while increasing in frequency. Lastly, drugs that affect tonic and synaptic GABAergic conductance modulated the frequency, duration, initiation and propagation of interictal-like events. These findings confirm and expand on previous studies indicating that multiple synaptic mechanisms contribute to synchronize neuronal network activity in forebrain structures. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Analgesic effects of Sazetidine-A, a new nicotinic cholinergic drug.

BACKGROUND: The use of nicotinic agonists for analgesia is limited by their unacceptable side effects. Sazetidine-A is a new partial agonist nicotinic ligand that has very high selectivity for beta2-containing nicotinic acetylcholine receptors. It potently and selectively desensitizes alpha4beta2 nicotinic acetylcholine receptors without measurable effects on alpha3beta4 receptors. The authors investigated the analgesic effects of Sazetidine-A using the formalin model of chronic inflammatory pain. METHODS: The formalin test was conducted after rats received intraperitoneal saline, Sazetidine-A (0.125, 0.25, 0.5, 1, 2 mg/kg), or subcutaneous epibatidine (2.5-5-10 mug/kg). In other experiments, Sazetidine-A was preceded by naloxone (0.5 mg/kg) or mecamylamine (10 mg). Effects of Sazetidine-A and epibatidine on locomotor were tested in an open field, and seizure activity was measured using the Racine scale. Locus coeruleus neuron extracellular single-unit spontaneous discharge was recorded in anesthetized animals after Sazetidine-A and epibatidine. RESULTS: Higher doses of Sazetidine-A (0.5, 1, or 2 mg/kg) induced analgesia, with pain scores significantly lower than those seen after saline, lower doses of Sazetidine-A, and epibatidine (P < 0.001). Naloxone did not antagonize the effects of Sazetidine-A, and mecamylamine had partial, dose-dependent antagonistic effects. Epibatidine excited locus coeruleus neurons, whereas Sazetidine-A had no effect on these neurons. Epibatidine and Sazetidine-A affected animals' locomotor activity for the initial 20 min. While analgesic doses of epibatidine caused seizures, no seizure activity or other neurologic complications were seen in animals that received as much as four times the minimum analgesic dose of Sazetidine-A. CONCLUSIONS: Sazetidine-A seems to be a potent analgesic without causing neurologic side effects.

Electrophysiologic effects of systemic and locally infused epibatidine on locus coeruleus neurons.

We evaluated the electrophysiologic response of locus coeruleus neurons to the systemic and local infusion of epibatidine. Rats were anesthetized with 2% halothane and single-unit locus coeruleus discharge was recorded after administration of systemic (2.5, 5 and 10 microg/kg subcutaneously) and intracoerulear (0.03-0.01-0.001 microg) epibatidine. The subcutaneous epibatidine activated locus coeruleus neurons only at the highest dose (10 microg/kg). The 2.5-5 microg/kg doses, previously shown to induce analgesia, did not activate locus coeruleus neurons. The intracoerulear infusion of epibatidine induced excitement of locus coeruleus neurons at every tested dose. Higher doses (0.03 and 0.01 microg) excited 100% of the recorded neurons. A significantly lower number of neurons (50% and 43% respectively) were excited when lower doses (0.005-0.001 microg) were used (P=0.035). The intracoerulear infusion of mecamylamine (1 microg) significantly reduced neuronal discharge rate (45%) and blocked the effects of epibatidine. The intra-dorsal raphe infusion of 0.03 microg epibatidine induced significant excitation of locus coeruleus neurons. These data show that the administration of epibatidine induces excitation of locus coeruleus neurons, which is mediated by nicotinic receptors. This activation occurs after systemic and selective local administration of epibatidine. The response of locus coeruleus neurons to systemic and locally administered epibatidine is dose-related.

Nuclear processing and export of microRNAs in Arabidopsis.

In mammalian cells, the nuclear export receptor, Exportin 5 (Exp5), exports pre-microRNAs (pre-miRNAs) as well as tRNAs into the cytoplasm. In this study, we examined the function of HASTY (HST), the Arabidopsis ortholog of Exp5, in the biogenesis of miRNAs and tRNAs. In contrast to mammals, we found that miRNAs exist as single-stranded 20- to 21-nt molecules in the nucleus in Arabidopsis. This observation is consistent with previous studies indicating that proteins involved in miRNA biogenesis are located in the nucleus in Arabidopsis. Although miRNAs exist in the nucleus, a majority accumulate in the cytoplasm. Interestingly, loss-of-function mutations in HST reduced the accumulation of most miRNAs but had no effect on the accumulation of tRNAs and endogenous small interfering RNAs, or on transgene silencing. In contrast, a mutation in PAUSED (PSD), the Arabidopsis ortholog of the tRNA export receptor, Exportin-t, interfered with the processing of tRNA-Tyr but did not affect the accumulation or nuclear export of miRNAs. These results demonstrate that HST and PSD do not share RNA cargos in nuclear export and strongly suggest that there are multiple nuclear export pathways for these small RNAs in Arabidopsis.