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The natural mutant mouse rolling Nagoya is severely ataxic and frequently shows body roll-overs. A missense mutation was identified in Cacna1a, the gene encoding the α1 pore subunit of CaV2.1 type voltage-gated Ca2+ channels. We here discuss the main phenotypic and neuronal characteristics of this mutant, with relevance to the human neurological disorders associated with CaV2.1 dysfunction (‘Ca2+-channelopathies’).

Neuroinflammatory changes involving neuronal HMGB1 release and astrocytic NF-KB nuclear translocation occur following cortical spreading depolarization (CSD) in wildtype (WT) mice but it is unknown to what extent this occurs in the migraine brain. We therefore investigated in familial hemiplegic migraine type 1 (FHM1) knock-in mice, which express an intrinsic hyperexcitability phenotype, the extent of neuroinflammation without and after CSD. CSD was evoked in one hemisphere by pinprick (single CSD) or topical KCl application (multiple CSDs). Neuroinflammatory (HMGB1, NF-KB) and neuronal activation (pERK) markers were investigated by immunohistochemistry in the brains of WT and FHM1 mutant mice without and after CSD. Effects of NMDA receptor antagonism on basal and CSD-induced neuroinflammatory changes were examined by, respectively, systemically administered MK801 and ifenprodil or topical MK801 application. In FHM1 mutant mice, CSD caused enhanced neuronal HMGB1 release and astrocytic NF-KB nuclear translocation in the cortex and subcortical areas that were equally high in both hemispheres. In WT mice such effects were only pronounced in the hemisphere in which CSD was induced. Neuroinflammatory responses were associated with pERK expression indicating neuronal activation. Upon CSD, contralateral cortical and striatal HMGB1 release was reduced by topical application of MK801 in the hemisphere contralateral to the one in which CSD was induced. This study reveals that neuroinflammatory activation after CSD is widespread and extends to the contralateral hemisphere, particularly in brains of FHM1 mutant mice. Effective blockade of CSD-induced neuroinflammatory responses in the contralateral hemisphere in FHM1 mice by local NMDA receptor antagonism suggests that neuronal hyperexcitability-related neuroinflammation is relevant in migraine pathophysiology, but possibly also other neurological disorders in which spreading depolarization is involved.

Epidemiological estimates indicate that individuals with epilepsy are more likely to experience headaches, including migraine, than individuals without epilepsy. Headaches can be temporally unrelated to seizures, or can occur before, during or after an episode; seizures and migraine attacks are mostly not temporally linked. The pathophysiological links between headaches (including migraine) and epilepsy are complex and have not yet been fully elucidated. Correct diagnoses and appropriate treatment of headaches in individuals with epilepsy is essential, as headaches can contribute substantially to disease burden. Here, we review the insights that have been made into the associations between headache and epilepsy over the past 5 years, including information on the pathophysiological mechanisms and genetic variants that link the two disorders. We also discuss the current best practice for the management of headaches co-occurring with epilepsy and highlight future challenges for this area of research. Headaches and epilepsy frequently co-exist in the same individual, but the pathophysiological mechanisms underlying this relationship are not yet clear. Here, the authors discuss the epidemiological and pathophysiological links between epilepsy and headache, and apply this knowledge to the clinical management of the two disorders.

Background: Epileptic (absence) seizures in the cerebral cortex can be stopped by pharmacological and optogenetic stimulation of the cerebellar nuclei (CN) neurons that innervate the thalamus. However, it is unclear how such stimulation can modify underlying thalamo-cortical oscillations. Hypothesis: Here we tested whether rhythmic synchronized thalamo-cortical activity during absence seizures can be desynchronized by single-pulse optogenetic stimulation of CN neurons to stop seizure activity. Methods: We performed simultaneous thalamic single-cell and electrocorticographical recordings in awake tottering mice, a genetic model of absence epilepsy, to investigate the rhythmicity and synchronicity. Furthermore, we tested interictally the impact of single-pulse optogenetic CN stimulation on thalamic and cortical recordings. Results: We show that thalamic firing is highly rhythmic and synchronized with cortical spike-and-wave discharges during absence seizures and that this phase-locked activity can be desynchronized upon single-pulse optogenetic stimulation of CN neurons. Notably, this stimulation of CN neurons was more effective in stopping seizures than direct, focal stimulation of groups of afferents innervating the thalamus. During interictal periods, CN stimulation evoked reliable but heterogeneous responses in thalamic cells in that they could show an increase or decrease in firing rate at various latencies, bi-phasic responses with an initial excitatory and subsequent inhibitory response, or no response at all. Conclusion: Our data indicate that stimulation of CN neurons and their fibers in thalamus evokes differential effects in its downstream pathways and desynchronizes phase-locked thalamic neuronal firing during seizures, revealing a neurobiological mechanism that may explain how cerebellar stimulation can stop seizures. (c) 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND

Epidemiological estimates indicate that individuals with epilepsy are more likely to experience headaches, including migraine, than individuals without epilepsy. Headaches can be temporally unrelated to seizures, or can occur before, during or after an episode; seizures and migraine attacks are mostly not temporally linked. The pathophysiological links between headaches (including migraine) and epilepsy are complex and have not yet been fully elucidated. Correct diagnoses and appropriate treatment of headaches in individuals with epilepsy is essential, as headaches can contribute substantially to disease burden. Here, we review the insights that have been made into the associations between headache and epilepsy over the past 5 years, including information on the pathophysiological mechanisms and genetic variants that link the two disorders. We also discuss the current best practice for the management of headaches co-occurring with epilepsy and highlight future challenges for this area of research.

Objective To review and discuss the literature on the role of cortical structure and function in migraine. Discussion Structural and functional findings suggest that changes in cortical morphology and function contribute to migraine susceptibility by modulating dynamic interactions across cortical and subcortical networks. The involvement of the cortex in migraine is well established for the aura phase with the underlying phenomenon of cortical spreading depolarization, while increasing evidence suggests an important role for the cortex in perception of head pain and associated sensations. As part of trigeminovascular pain and sensory processing networks, cortical dysfunction is likely to also affect initiation of attacks. Conclusion Morphological and functional changes identified across cortical regions are likely to contribute to initiation, cyclic recurrence and chronification of migraine. Future studies are needed to address underlying mechanisms, including interactions between cortical and subcortical regions and effects of internal (e.g. genetics, gender) and external (e.g. sensory inputs, stress) modifying factors, as well as possible clinical and therapeutic implications.

Dravet syndrome (DS) is an epileptic encephalopathy that still lacks biomarkers for epileptogenesis and its treatment. Dysfunction of NaV1.1 sodium channels, which are chiefly expressed in inhibitory interneurons, explains the epileptic phenotype. Understanding the network effects of these cellular deficits may help predict epileptogenesis. Here, we studied θ-γ coupling as a potential marker for altered inhibitory functioning and epileptogenesis in a DS mouse model. We found that cortical θ-γ coupling was reduced in both male and female juvenile DS mice and persisted only if spontaneous seizures occurred. θ-γ Coupling was partly restored by cannabidiol (CBD). Locally disrupting NaV1.1 expression in the hippocampus or cortex yielded early attenuation of θ-γ coupling, which in the hippocampus associated with fast ripples, and which was replicated in a computational model when voltage-gated sodium currents were impaired in basket cells (BCs). Our results indicate attenuated θ-γ coupling as a promising early indicator of inhibitory dysfunction and seizure risk in DS.

Migraine patients often report (inter)ictal hypersensitivity to light, but the underlying mechanisms remain an enigma. Both hypo‐ and hyperresponsivity of the visual network have been reported, which may reflect either intra‐individual dynamics of the network or large inter‐individual variation in the measurement of human visual evoked potential data. Therefore, we studied visual system responsivity in freely behaving mice using combined epidural electroencephalography and intracortical multi‐unit activity to reduce variation in recordings and gain insight into visual cortex dynamics. For better clinical translation, we investigated transgenic mice that carry the human pathogenic R192Q missense mutation in the α1A subunit of voltage‐gated CaV2.1 Ca2+ channels leading to enhanced neurotransmission and familial hemiplegic migraine type 1 in patients. Visual evoked potentials were studied in response to visual stimulation paradigms with flashes of light. Following intensity‐dependent visual stimulation, FHM1 mutant mice displayed faster visual evoked potential responses, with lower initial amplitude, followed by less pronounced neuronal suppression compared to wild‐type mice. Similar to what was reported for migraine patients, frequency‐dependent stimulation in mutant mice revealed enhanced photic drive in the EEG beta‐gamma band. The frequency‐dependent increases in visual network responses in mutant mice may reflect the context‐dependent enhancement of visual cortex excitability, which could contribute to our understanding of sensory hypersensitivity in migraine., Migraine patients often report hypersensitivity to light, but the underlying network mechanisms are unclear. We here studied visual network responsivity relevant to migraine by simultaneous recording of EEG and multi‐unit activity during visual stimulation paradigms with flashing light in freely behaving wild‐type and familial hemiplegic migraine type 1 (FHM1) R192Q mutant mice. Mutant mice showed intensity‐ and frequency‐dependent changes in visual evoked potentials that indicate context‐dependent visual network hyperexcitability relevant to migraine.

Early onset seizures are a hallmark of Dravet syndrome. Previous studies in rodent models have shown that the epileptic phenotype is caused by loss-of-function of voltage-gated Na(V)1.1 sodium channels, which are chiefly expressed in gamma-aminobutyric acid (GABA)ergic neurons. Recently, a possibly critical role has been attributed to the hippocampus in the seizure phenotype, as local hippocampal ablation of Na(V)1.1 channels decreased the threshold for hyperthermia-induced seizures. However, the effect of ablation of Na(V)1.1 channels restricted to cortical sites has not been tested. Here we studied local field potential (LFP) and behavior in mice following local hippocampal and cortical ablation of Scn1a, a gene encoding the alpha 1 subunit of Na(V)1.1 channels, and we compared seizure characteristics with those of heterozygous global knockout Scn1(-/+) mice. We found a high incidence of spontaneous seizures following either local hippocampal or cortical ablation, notably during a transient time window, similar to Scn1a(-/+) mice. Nonconvulsive seizure activity in the injected area was common and preceded generalized seizures. Moreover, mice were susceptible to hyperthermia-induced seizures. In conclusion, local ablation of Na(V)1.1 channels in the hippocampus and cortex results in focal seizure activity that can generalize. These data indicate that spontaneous epileptic activity may initiate in multiple brain regions in Dravet syndrome.

Here we show, for the first time, spontaneous cortical spreading depolarization (CSD) events – the electrophysiological correlate of the migraine aura – in animals by using the first generated familial hemiplegic migraine type 3 (FHM3) transgenic mouse model. The mutant mice express L263V‐mutated α1 subunits in voltage‐gated NaV1.1 sodium channels (Scn1a L263V). CSDs consistently propagated from visual to motor cortex, recapitulating what has been shown in patients with migraine with aura. This model may be valuable for the preclinical study of migraine with aura and other diseases in which spreading depolarization is a prominent feature.

Seizure-related apnea is common and can be lethal. Its mechanisms however remain unclear and preventive strategies are lacking. We postulate that brainstem spreading depolarization (SD), previously associated with lethal seizures in animal models, initiates apnea upon invasion of brainstem respiratory centers. To study this, we assessed effects of brainstem seizures on brainstem function and respiration in male and female mice carrying a homozygous S218L missense mutation that leads to gain-of-function of voltage-gated CaV2.1 Ca2+channels and high risk for fatal seizures. Recordings of brainstem DC potential and neuronal activity, cardiorespiratory activity and local tissue oxygen were performed in freely behaving animals. Brainstem SD occurred during all spontaneous fatal seizures and, unexpectedly, during a subset of nonfatal seizures. Seizure-related SDs in the ventrolateral medulla correlated with respiratory suppression. Seizures induced by stimulation of the inferior colliculus could evoke SD that spread in a rostrocaudal direction, preceding local tissue hypoxia and apnea, indicating that invasion of SD into medullary respiratory centers initiated apnea and hypoxia rather thanvice versa. Fatal outcome was prevented by timely resuscitation. Moreover, NMDA receptor antagonists MK-801 and memantine prevented seizure-related SD and apnea, which supports brainstem SD as a prerequisite for brainstem seizure-related apnea in this animal model and has translational value for developing strategies that prevent fatal ictal apnea.SIGNIFICANCE STATEMENTApnea during and following seizures is common, but also likely implicated in sudden unexpected death in epilepsy (SUDEP). This underlines the need to understand mechanisms for potentially lethal seizure-related apnea. In the present work we show, in freely behaving SUDEP-prone transgenic mice, that apnea is induced when spontaneous brainstem seizure-related spreading depolarization (SD) reaches respiratory nuclei in the ventrolateral medulla. We show that brainstem seizure-related medullary SD is followed by local hypoxia and recovers during nonfatal seizures, but not during fatal events. NMDA receptor antagonists prevented medullary SD and apnea, which may be of translational value.

Objective To determine whether transgenic mouse models of migraine exhibit upper gastrointestinal dysmotility comparable to those observed in migraine patients. Background There is considerable evidence supporting the comorbidity of gastrointestinal dysmotility and migraine. Gastrointestinal motility, however, has never been investigated in transgenic mouse models of migraine. Methods Three transgenic mouse strains that express pathogenic gene mutations linked to monogenic migraine-relevant phenotypes were studied: CADASIL (Notch3-Tg88), FASP (CSNK1D-T44A), and FHM1 (CACNA1A-S218L). Upper gastrointestinal motility was quantified by measuring gastric emptying and small intestinal transit in mutant and control animals. Gastrointestinal motility was measured at baseline and after pretreatment with 10 mg/kg nitroglycerin (NTG). Results No significant differences were observed for gastric emptying or small intestinal transit at baseline for any of the 3 transgenic strains when compared to appropriate controls or after pretreatment with NTG when compared to vehicle. Conclusions We detected no evidence of upper gastrointestinal dysmotility in mice that express mutations in genes linked to monogenic migraine-relevant phenotypes. Future studies seeking to understand why humans with migraine experience delayed gastric emptying may benefit from pursuing other modifiers of gastrointestinal motility, such as epigenetic or microbiome-related factors.

Objectives: Visual system abnormalities in migraine are linked to symptoms like photophobia and the visual aura. Little is known about the mechanisms contributing to these visual system alterations. Processing of visual input by the brain is a highly nonlinear operation, involving complex interactions among cortical and subcortical neuronal networks. Timing of this process can be estimated by analysing the cortical response to external light input at different frequencies. Using a sum-of-sinusoid light signal, instead of the classic pulse train, as input and novel EEG analyses it is possible to assess the time delay and frequency domain response. Here we investigate nonlinear visual processing in subgroups of migraine patients and headache-free participants. Methods: Migraine patients with aura, without aura and healthy participants (N = 10/group) were subjected to bi-sinusoidal light stimulation for 320 1 sec-epochs, while scalp EEG was recorded at the occipital, parietal and frontal lobes. Light stimulus frequencies were chosen to guarantee no overlap of their harmonic and intermodulation frequencies for different orders of nonlinearity. Nonlinear interactions and time delay from stimulus to cortical EEG response were analysed in the frequency domain using novel phase clustering measures and amplitude spectral measures. Results: Higher harmonic and intermodulation interactions were detected between visual input and cortical responses. Amplitude spectrum and phase clustering responses differed per order and group. Migraine patients with aura showed a decreased time delay only at the occipital lobe compared to healthy controls and migraine patients without aura. Conclusion: Visual processing is altered in migraine patients with aura compared to healthy controls and patients without aura. Furthermore, we demonstrated the potential of quantifying nonlinear interactions and temporal dynamics in the visual system using sum-of-sinusoid light stimulation. We are able to uncover alterations in visual processing in the context of neurological disease.

Cortical spreading depolarization (SD) is the electrophysiological event underlying migraine aura, and a critical contributor to secondary damage after brain injury. Experimental models of SD have been used for decades in migraine and brain injury research; however, they are highly invasive and often cause primary tissue injury, diminishing their translational value. Here we present a non-invasive method to trigger SDs using light-induced depolarization in transgenic mice expressing channelrhodopsin-2 in neurons (Thy1-ChR2-YFP). Focal illumination (470 nm, 1–10 mW) through intact skull using an optical fiber evokes power-dependent steady extracellular potential shifts and local elevations of extracellular [K(+)] that culminate in an SD when power exceeds a threshold. Using the model, we show that homozygous mice are significantly more susceptible to SD (i.e., lower light thresholds) than heterozygous ChR2 mice. Moreover, we show SD susceptibility differs significantly among cortical divisions (motor, whisker barrel, sensory, visual, in decreasing order of susceptibility), which correlates with relative channelrhodopsin-2 expression. Furthermore, the NMDA receptor antagonist MK-801 blocks the transition to SD without diminishing extracellular potential shifts. Altogether, our data show that the optogenetic SD model is highly suitable for examining physiological or pharmacological modulation of SD in acute and longitudinal studies.

Sudden unexpected death in epilepsy (SUDEP) is a fatal complication of epilepsy in which brainstem spreading depolarization may play a pivotal role, as suggested by animal studies. However, patiotemporal details of spreading depolarization occurring in relation to fatal seizures have not been investigated. In addition, little is known about behavioural and neurophysiological features that may discriminate spontaneous fatal from non-fatal seizures. Transgenic mice carrying the missense mutation S218L in the α1A subunit of Cav2.1 (P/Q-type) Ca2+ channels exhibit enhanced excitatory neurotransmission and increased susceptibility to spreading depolarization. Homozygous Cacna1aS218L mice show spontaneous non-fatal and fatal seizures, occurring throughout life, resulting in reduced life expectancy. To identify characteristics of fatal and non-fatal spontaneous seizures, we compared behavioural and electrophysiological seizure dynamics in freely-behaving homozygous Cacna1aS218L mice. To gain insight on the role of brainstem spreading depolarization in SUDEP, we studied the spatiotemporal distribution of spreading depolarization in the context of seizure-related death. Spontaneous and electrically-induced seizures were investigated by video monitoring and electrophysiological recordings in freely-behaving Cacna1aS218L and wild-type mice. Homozygous Cacna1aS218L mice showed multiple spontaneous tonic-clonic seizures and died from SUDEP in adulthood. Death was preceded by a tonic-clonic seizure terminating with hindlimb clonus, with suppression of cortical neuronal activity during and after the seizure. Induced seizures in freely-behaving homozygous Cacna1aS218L mice were followed by multiple spreading depolarizations and death. In wild-type or heterozygous Cacna1aS218L mice, induced seizures and spreading depolarization were never followed by death. To identify temporal and regional features of seizure-induced spreading depolarization related to fatal outcome, diffusion-weighted MRI was performed in anaesthetized homozygous Cacna1aS218L and wild-type mice. In homozygous Cacna1aS218L mice, appearance of seizure-related spreading depolarization in the brainstem correlated with respiratory arrest that was followed by cardiac arrest and death. Recordings in freely-behaving homozygous Cacna1aS218L mice confirmed brainstem spreading depolarization during spontaneous fatal seizures. These data underscore the value of the homozygous Cacna1aS218L mouse model for identifying discriminative features of fatal compared to non-fatal seizures, and support a key role for cortical neuronal suppression and brainstem spreading depolarization in SUDEP pathophysiology.

Cortical spreading depression, which plays an important role in multiple neurological disorders, has been studied primarily with experimental models that use highly invasive methods. We developed a relatively non-invasive optogenetic model to induce cortical spreading depression by transcranial stimulation of channelrhodopsin-2 ion channels expressed in cortical layer 5 neurons. Light-evoked cortical spreading depression in anesthetized and freely behaving mice was studied with intracortical DC-potentials, multi-unit activity and/or non-invasive laser Doppler flowmetry, and optical intrinsic signal imaging. In anesthetized mice, cortical spreading depression induction thresholds and propagation rates were similar for invasive (DC-potential) and non-invasive (laser Doppler flowmetry) recording paradigms. Cortical spreading depression-related vascular and parenchymal optical intrinsic signal changes were similar to those evoked with KCl. In freely behaving mice, DC-potential and multi-unit activity recordings combined with laser Doppler flowmetry revealed cortical spreading depression characteristics comparable to those under anesthesia, except for a shorter cortical spreading depression duration. Cortical spreading depression resulted in a short increase followed by prolonged reduction of spontaneous active behavior. Motor function, as assessed by wire grip tests, was transiently and unilaterally suppressed following a cortical spreading depression. Optogenetic cortical spreading depression induction has significant advantages over current models in that multiple cortical spreading depression events can be elicited in a non-invasive and cell type-selective fashion.

Background Epilepsy and migraine are paroxysmal neurological conditions associated with disturbances of cortical excitability. No useful biomarkers to monitor disease activity in these conditions are available. Phase clustering was previously described in electroencephalographic (EEG) responses to photic stimulation and may be a potential epilepsy biomarker. Objective The objective of this study was to investigate EEG phase clustering in response to transcranial magnetic stimulation (TMS), compare it with photic stimulation in controls, and explore its potential as a biomarker of genetic generalized epilepsy or migraine with aura. Methods People with (possible) juvenile myoclonic epilepsy (JME), migraine with aura, and healthy controls underwent single-pulse TMS with concomitant EEG recording during the interictal period. We compared phase clustering after TMS with photic stimulation across the groups using permutation-based testing. Results We included eight people with (possible) JME (five off medication, three on), 10 with migraine with aura, and 37 controls. The TMS and photic phase clustering spectra showed significant differences between those with epilepsy without medication and controls. Two phase clustering-based indices successfully captured these differences between groups. One participant was tested multiple times. In this case, the phase clustering-based indices were inversely correlated with the dose of antiepileptic medication. Phase clustering did not differ between people with migraine and controls. Conclusion We present methods to quantify phase clustering using TMS–EEG and show its potential value as a measure of brain network activity in genetic generalized epilepsy. Our results suggest that the higher propensity to phase clustering is not shared between genetic generalized epilepsy and migraine.

Endocannabinoids are suggested to control pain, even though their clinical use is not fully validated and the underlying mechanisms are incompletely understood. To clarify the targets of endocannabinoid actions, we studied how activation of the endocannabinoid CB1 receptor (CB1R) affects neuronal responses in two in vitro preparations of rodents, namely the trigeminal sensory ganglion (TG) in culture and a coronal slice of the cerebral cortex. On TG small-medium size neurons, we tested whether submicromolar concentrations of the endogenous CB1R agonist anandamide (AEA) modulated inhibitory GABA A receptors and excitatory ATP-gated P2X3 receptors. AEA reversibly depressed GABA-mediated membrane currents without altering P2X3 receptor responses. The AEA antagonism was non-competitive, prevented by the CB1R antagonist AM-251, mimicked by the other cannabinoids 2-arachidonylglycerol and WIN 55,212-2, and insensitive to TRPV1 blocker capsazepine. AEA inhibited the potentiation of GABAergic responses by the cAMP activator forskolin, in line with the canonical inhibition of cAMP synthesis by CB1Rs. In the cerebral cortex, AEA or WIN 55,212-2 did not affect electrically-evoked local field potentials or characteristics of cortical spreading depolarization (CSD) elicited by high potassium application. The GABA A receptor blocker gabazine, however, strongly enhanced field potentials without affecting CSD properties, suggesting that CSD was not dominantly controlled by GABAergic mechanisms. Our data propose that, despite the widespread expression of CB1Rs peripherally and centrally, the functional effects of AEA are region-specific and depend on CB1R coupling to downstream effectors. Future studies concerned with the mechanisms of AEA analgesia should perhaps be directed to discrete subcortical nuclei processing trigeminal inputs.

Er is toenemend bewijs voor een belangrijke rol voor apneus in de pathofysiologie van sudden unexpected death in epilepsy. Recent preklinisch onderzoek beschrijft een potentieel mechanisme voor (post-)ictale apneus.

OBJECTIVE: Disrupting thalamocortical activity patterns has proven to be a promising approach to stop generalized spike-and-wave discharges (GSWDs) characteristic of absence seizures. Here, we investigated to what extent modulation of neuronal firing in cerebellar nuclei (CN), which are anatomically in an advantageous position to disrupt cortical oscillations through their innervation of a wide variety of thalamic nuclei, is effective in controlling absence seizures. METHODS: Two unrelated mouse models of generalized absence seizures were used: the natural mutant tottering, which is characterized by a missense mutation in Cacna1a, and inbred C3H/HeOuJ. While simultaneously recording single CN neuron activity and electrocorticogram in awake animals, we investigated to what extent pharmacologically increased or decreased CN neuron activity could modulate GSWD occurrence as well as short-lasting, on-demand CN stimulation could disrupt epileptic seizures. RESULTS: We found that a subset of CN neurons show phase-locked oscillatory firing during GSWDs and that manipulating this activity modulates GSWD occurrence. Inhibiting CN neuron action potential firing by local application of the γ-aminobutyric acid type A (GABA-A) agonist muscimol increased GSWD occurrence up to 37-fold, whereas increasing the frequency and regularity of CN neuron firing with the use of GABA-A antagonist gabazine decimated its occurrence. A single short-lasting (30-300 milliseconds) optogenetic stimulation of CN neuron activity abruptly stopped GSWDs, even when applied unilaterally. Using a closed-loop system, GSWDs were detected and stopped within 500 milliseconds. INTERPRETATION: CN neurons are potent modulators of pathological oscillations in thalamocortical network activity during absence seizures, and their potential therapeutic benefit for controlling other types of generalized epilepsies should be evaluated. Ann Neurol 2015;77:1027-1049.

It is not fully understood how seizures terminate and why some seizures are followed by a period of complete brain activity suppression, postictal generalized EEG suppression. This is clinically relevant as there is a potential association between postictal generalized EEG suppression, cardiorespiratory arrest and sudden death following a seizure. We combined human encephalographic seizure data with data of a computational model of seizures to elucidate the neuronal network dynamics underlying seizure termination and the postictal generalized EEG suppression state. A multi-unit computational neural mass model of epileptic seizure termination and postictal recovery was developed. The model provided three predictions that were validated in EEG recordings of 48 convulsive seizures from 48 subjects with refractory focal epilepsy (20 females, age range 15-61 years). The duration of ictal and postictal generalized EEG suppression periods in human EEG followed a gamma probability distribution indicative of a deterministic process (shape parameter 2.6 and 1.5, respectively) as predicted by the model. In the model and in humans, the time between two clonic bursts increased exponentially from the start of the clonic phase of the seizure. The terminal interclonic interval, calculated using the projected terminal value of the log-linear fit of the clonic frequency decrease was correlated with the presence and duration of postictal suppression. The projected terminal interclonic interval explained 41% of the variation in postictal generalized EEG suppression duration (P < 0.02). Conversely, postictal generalized EEG suppression duration explained 34% of the variation in the last interclonic interval duration. Our findings suggest that postictal generalized EEG suppression is a separate brain state and that seizure termination is a plastic and autonomous process, reflected in increased duration of interclonic intervals that determine the duration of postictal generalized EEG suppression.

Familial hemiplegic migraine type 1 (FHM1) is a rare monogenic subtype of migraine with aura caused by mutations in CACNA1A that encodes the α1A subunit of voltage-gated CaV2.1 calcium channels. Transgenic knock-in mice that carry the human FHM1 R192Q missense mutation (‘FHM1 R192Q mice’) exhibit an increased susceptibility to cortical spreading depression (CSD), the mechanism underlying migraine aura. Here, we analysed gene expression profiles from isolated cortical tissue of FHM1 R192Q mice 24 h after experimentally induced CSD in order to identify molecular pathways affected by CSD. Gene expression profiles were generated using deep serial analysis of gene expression sequencing. Our data reveal a signature of inflammatory signalling upon CSD in the cortex of both mutant and wild-type mice. However, only in the brains of FHM1 R192Q mice specific genes are up-regulated in response to CSD that are implicated in interferon-related inflammatory signalling. Our findings show that CSD modulates inflammatory processes in both wild-type and mutant brains, but that an additional unique inflammatory signature becomes expressed after CSD in a relevant mouse model of migraine. Electronic supplementary material The online version of this article (doi:10.1007/s12035-015-9681-5) contains supplementary material, which is available to authorized users.

Migraine is a highly prevalent and disabling neurological disorder with a strong genetic component. Rare monogenic forms of migraine, or syndromes in which migraine frequently occurs, help scientists to unravel pathogenetic mechanisms of migraine and its comorbidities. Transgenic mouse models for rare monogenic mutations causing familial hemiplegic migraine (FHM), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and familial advanced sleep-phase syndrome (FASPS), have been created. Here, we review the current state of research using these mutant mice. We also discuss how currently available experimental approaches, including epigenetic studies, biomolecular analysis and optogenetic technologies, can be used for characterization of migraine genes to further unravel the functional and molecular pathways involved in migraine.

Tissue preparation is the key to a successful matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) experiment. Rapid post-mortem changes contribute a significant challenge to the use of MSI approaches for the analysis of peptides and metabolites. In this technical note we aimed to compare the tissue fixation method ex-vivo heat-stabilization with in-situ funnel-freezing in a middle cerebral artery occlusion (MCAo) mouse model of stroke, which causes profound alterations in metabolite concentrations. The influence of the duration of the thaw-mounting of the tissue sections on metabolite stability was also determined. We demonstrate improved stability and biomolecule visualization when funnel-freezing was used to sacrifice the mouse compared with heat-stabilization. Results were further improved when funnel-freezing was combined with fast thaw-mounting of the brain sections.

MALDI mass spectrometry can simultaneously measure hundreds of biomolecules directly from tissue. Using essentially the same technique but different sample preparation strategies, metabolites, lipids, peptides and proteins can be analyzed. Spatially correlated analysis, imaging MS, enables the distributions of these biomolecular ions to be simultaneously measured in tissues. A key advantage of imaging MS is that it can annotate tissues based on their MS profiles and thereby distinguish biomolecularly distinct regions even if they were unexpected or are not distinct using established histological and histochemical methods e.g. neuropeptide and metabolite changes following transient electrophysiological events such as cortical spreading depression (CSD), which are spreading events of massive neuronal and glial depolarisations that occur in one hemisphere of the brain and do not pass to the other hemisphere , enabling the contralateral hemisphere to act as an internal control. A proof-of-principle imaging MS study, including 2D and 3D datasets, revealed substantial metabolite and neuropeptide changes immediately following CSD events which were absent in the protein imaging datasets. The large high dimensionality 3D datasets make even rudimentary contralateral comparisons difficult to visualize. Instead non-negative matrix factorization (NNMF), a multivariate factorization tool that is adept at highlighting latent features, such as MS signatures associated with CSD events, was applied to the 3D datasets. NNMF confirmed that the protein dataset did not contain substantial contralateral differences, while these were present in the neuropeptide dataset. This article is part of a Special Issue entitled: Imaging Mass Spectrometry: A User’s Guide to a New Technique for Biological and Biomedical Research.

Patterned neuronal activity such as spindle bursts in the neonatal cortex is likely to promote the maturation of cortical synapses and neuronal circuits. Previous work on cats has shown that removal of subplate neurons, a transient neuronal population in the immature cortex, prevents the functional maturation of thalamocortical and intracortical connectivity. Here we studied the effect of subplate removal in the neonatal rat primary somatosensory cortex (S1). Using intracortical EEG we show that after selective removal of subplate neurons in the limb region of S1, endogenous and sensory evoked spindle burst activity is largely abolished. Consistent with the reducedin vivoactivity in the S1 limb region, we find byin vitrorecordings that thalamocortical inputs to layer 4 neurons are weak. In addition, we find that removal of subplate neurons in the S1 barrel region prevents the development of the characteristic histological barrel-like appearance. Thus, subplate neurons are crucially involved in the generation of particular types of early network activity in the neonatal cortex, which are an important feature of cortical development. The altered EEG pattern following subplate damage could be applicable in the neurological assessment of human neonates.

In mass spectrometry imaging (MSI), on-tissue proteolytic digestion is performed to access larger protein species and to assign protein identities through matching the detected peaks with those obtained by LC-MS/MS analyses of tissue extracts. The on-tissue proteolytic digestion also allows the analysis of proteins from formalin-fixed, paraffin-embedded tissues. For these reasons, on-tissue digestion-based MSI is frequently used in clinical investigations, for example, to determine changes in protein content and distribution associated with a disease. In this work, we sought to investigate the completeness and uniformity of the digestion in on-tissue digestion MSI. On the basis of an extensive experiment investigating three groups with varying incubation times: (i) 1.5 h, (ii) 3 h, and (iii) 18 h, we have found that longer incubation times improve the repeatability of the analyses. Furthermore, we discovered morphology-associated differences in the completeness of the proteolysis for short incubation times. These results support the notion that a more complete proteolysis allows better quantitation.

Migraine is a common brain disorder characterized by recurrent attacks of severe headaches and other neurological symptoms. In one-third of patients headaches are accompanied by auras, which consist of transient visual and sensory disturbances, believed to be caused by cortical spreading depression (CSD). CSD is characterized by a wave of neuronal and glial depolarization with concomitant changes in metabolite concentrations in the brain and cerebrospinal fluid. It remains unknown whether CSD-induced brain metabolic changes can be captured outside the central nervous system, i.e., in peripheral fluids. This study investigated plasma metabolic changes in transgenic mice that harbor a gene mutation in voltage-gated CaV2.1 Ca(2+) channels previously identified in patients with familial hemiplegic migraine, a subtype of migraine with auras. The use of a mouse model allows investigation of molecular changes occurring shortly after CSD, which is notoriously difficult in patients. Capillary electrophoresis - mass spectrometry was used for the analysis of plasma samples to obtain, for the first time, a comprehensive view of molecular changes immediately after experimentally induced CSD. Multivariate data analysis showed a clear distinction between profiles of transgenic and wild-type animals after CSD. Two metabolites considered important for this discrimination were tentatively identified as being lysine and its by-product pipecolic acid with additional evidence provided by hydrophilic interaction chromatography combined with tandem mass spectrometry. The changed metabolites suggest a compensatory increase in GABAergic neurotransmission upon enhanced excitatory neurotransmission. These results show that CSD induces metabolic remodeling in transgenic migraine mice that can be captured and measured in plasma.

Summary Purpose: CO2 has been long recognized for its anticonvulsant properties. We aimed to determine whether inhaling 5% CO2 can be used to suppress seizures in epilepsy patients. The effect of CO2 on cortical epileptic activity accompanying behavioral seizures was studied in rats and nonhuman primates, and based on these data, preliminary tests were carried out in humans. Methods: In freely moving rats, cortical afterdischarges paralleled by myoclonic convulsions were evoked by sensorimotor cortex stimulation. Five percent CO2 was applied for 5 min, 3 min before stimulation. In macaque monkeys, hypercarbia was induced by hypoventilation while seizure activity was electrically or chemically evoked in the sensorimotor cortex. Seven patients with drug-resistant partial epilepsy were examined with video-EEG (electroencephalography) and received 5% CO2 in medical carbogen shortly after electrographic seizure onset. Results: In rats, 5% CO2 strongly suppressed cortical afterdischarges, by approximately 75%, whereas responses to single-pulse stimulation were reduced by about 15% only. In macaques, increasing pCO2 from 37 to 44–45 mm Hg (corresponding to inhalation of 5% CO2 or less) suppressed stimulation-induced cortical afterdischarges by about 70% and single, bicuculline-induced epileptiform spikes by approximately 25%. In a pilot trial carried out in seven patients, a rapid termination of electrographic seizures was seen despite the fact that the application of 5% CO2 was started after seizure generalization. Conclusions: Five percent CO2 has a fast and potent anticonvulsant action. The present data suggest that medical carbogen with 5% CO2 can be used for acute treatment to suppress seizures in epilepsy patients.

The entorhinal cortex (EC) plays an important role in temporal lobe epilepsy. Under normal conditions, the enriched cholinergic innervation of the EC modulates local synchronized oscillatory activity; however, its role in epilepsy is unknown. Enhanced neuronal activation has been shown to induce transcriptional changes of key cholinergic genes and thus alter cholinergic responses. To examine cholinergic modulations in epileptic tissue we studied molecular and electrophysiological cholinergic responses in the EC of chronically epileptic rats following exposure to pilocarpine or kainic acid. We confirmed that while the total activity of the acetylcholine (ACh)-hydrolysing enzyme, acetylcholinesterase (AChE) was not altered, epileptic rats showed alternative splicing of AChE pre-mRNA transcripts, accompanied by a shift from membrane-bound AChE tetramers to soluble monomers. This was associated with increased sensitivity to ACh application: thus, in control rats, ACh (10-100 microm) induced slow (< 1Hz), periodic events confined to the EC; however, in epileptic rats, ACh evoked seconds-long seizure-like events with initial appearance in the EC, and frequent propagation to neighbouring cortical regions. ACh-induced seizure-like events could be completely blocked by the non-specific muscarinic antagonist, atropine, and were partially blocked by the muscarinic-1 receptor antagonist, pirenzepine; but were not affected by the non-specific nicotinic antagonist, mecamylamine. Epileptic rats presented reduced transcript levels of muscarinic receptors with no evidence of mRNA editing or altered mRNA levels for nicotinic ACh receptors. Our findings suggest that altered cholinergic modulation may initiate seizure events in the epileptic temporal cortex.

We investigated the potential of 4-AP (50-100 microM) to induce seizure-like events (SLEs) in combined entorhinal cortex-hippocampal slices from Sprague Dawley rats which developed spontaneous limbic seizures following kainic acid induced status epilepticus. Slices from control rats (n=8) displayed SLEs in the entorhinal and perirhinal cortex upon application of 50 or 100 microM 4-AP. By contrast, 4-AP failed to induce SLEs in slices from chronic epileptic rats (n=13) except for one slice from one rat. This animal displayed only minor cell loss in layer III of the entorhinal cortex, in contrast to the other epileptic rats for which layer III neuronal loss was extensive. In all slices from epileptic rats, 4-AP induced recurrent epileptiform discharges similar to the interictal activity observed in control rats. Combined application of 4-AP (100 microM) and bicuculline methiodide (30 microM) induced frequent and prolonged recurrent epileptiform discharges in both control and chronic epileptic rats. 4-AP at 50-100 microM likely affects potassium channels containing Kv1.4, Kv1.5, Kv3.1 or Kv3.2 subunits. Real-time PCR revealed no significant downregulation of Kv1.4, Kv1.5, Kv3.1 or Kv3.2 in the subiculum, entorhinal and perirhinal cortex from chronic epileptic rats compared to controls. However, the expression of Kv3.4, responding to 4-AP in mM range, was significantly reduced. Using sub-unit-specific antibodies, the real-time PCR findings were confirmed by immunocytochemistry. We suggest that after chronic epilepsy, reorganization in the entorhinal cortex is accompanied by adaptations in homeostatic plasticity with anticonvulsant consequences.

Cortical spreading depression (CSD) is the electrophysiological correlate of migraine aura. Transgenic mice carrying the R192Q missense mutation in the Cacna1a gene, which in patients causes familial hemiplegic migraine type 1 (FHM1), exhibit increased propensity to CSD. Herein, mass spectrometry imaging (MSI) was applied for the first time to an animal cohort of transgenic and wild type mice to study the biomolecular changes following CSD in the brain. Ninety-six coronal brain sections from 32 mice were analyzed by MALDI-MSI. All MSI datasets were registered to the Allen Brain Atlas reference atlas of the mouse brain so that the molecular signatures of distinct brain regions could be compared. A number of metabolites and peptides showed substantial changes in the brain associated with CSD. Among those, different mass spectral features showed significant (t-test, P < 0.05) changes in the cortex, 146 and 377 Da, and in the thalamus, 1820 and 1834 Da, of the CSD-affected hemisphere of FHM1 R192Q mice. Our findings reveal CSD- and genotype-specific molecular changes in the brain of FHM1 transgenic mice that may further our understanding about the role of CSD in migraine pathophysiology. The results also demonstrate the utility of aligning MSI datasets to a common reference atlas for large-scale MSI investigations. Graphical Abstract ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-015-1136-8) contains supplementary material, which is available to authorized users.

Migraine is a common multifactorial episodic brain disorder with strong genetic basis. Monogenic subtypes include rare familial hemiplegic migraine, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, familial advanced sleep-phase syndrome (FASPS), and retinal vasculopathy with cerebral leukodystrophy. Functional studies of disease-causing mutations in cellular and/or transgenic models revealed enhanced (glutamatergic) neurotransmission and abnormal vascular function as key migraine mechanisms. Common forms of migraine (both with and without an aura), instead, are thought to have a polygenic makeup. Genome-wide association studies have already identified over a dozen genes involved in neuronal and vascular mechanisms. Here, we review the current state of molecular genetic research in migraine, also with respect to functional and pathway analyses. We will also discuss how novel experimental approaches for the identification and functional characterization of migraine genes, such as next-generation sequencing, induced pluripotent stem cell, and optogenetic technologies will further our understanding of the molecular pathways involved in migraine pathogenesis.

There is considerable need in neuroscience research to reliably measure concentrations of extracellular ions in the living brain as the dynamics of ion levels increasingly is considered to play a key role in the pathophysiology of many brain disorders. Unfortunately, most probes currently used for direct measurement of extracellular ion concentrations in living brain tissue in experimental animals have considerable limitations. They are custom-made glass capillary ion-selective microelectrodes that are fragile, timeconsuming to prepare, and can practically only be used to measure at one depth and one location in the brain. We here present the design and fabrication of a novel type of miniaturized probe that can simultaneously measure, in the brain of a living animal, multiple parameters relevant to the neurological phenomenon of spreading depression (SD) at multiple depths and locations. SD is characterized by a slowly propagating wave of initial neuronal and glial cell depolarization that is followed by depression of activity. SD is accompanied by a massive redistribution of ions, including K+, between intracellular and extracellular compartments and is considered important in brain diseases such as stroke, traumatic brain injury, and migraine with aura. This work focuses on the functionalization of fabricated probes for measuring changes in extracellular K+ concentration (and its associated changes in neuronal activity) using an ion-selective potentiometric sensor. We show that the choice of conductive polymer and the method of electro-deposition is critical to obtain low cross-sensitivity for pH and O-2. The sensor's response to IC is approximately linear between 2 and 40 mM with a voltage-response of 39 mV/log[K+]. Proof-of-principle in vivo brain recordings were performed in the cortex of a wild-type mouse during induction of cortical SD. We reliably measured the theoretically expected rise and fall of brain tissue K+ levels during a locally induced cortical SD. We envisage that our probe can be of great use to reliably measure K+, without being affected by pH or O-2 changes, in the living brain and be widely applicable in neuroscience research. (C) 2014 Elsevier B.V All rights reserved

On-tissue enzymatic digestion is performed in mass spectrometry imaging (MSI) experiments to access larger proteins and to assign protein identities. Most on-tissue digestion MSI studies have focused on method development rather than identifying the molecular features observed. Herein, we report a comprehensive study of the mouse brain proteome sampled by MSI. Using complementary proteases, we were able to identify 5337 peptides in the matrix-assisted laser desorption/ionization (MALDI) matrix, corresponding to 1198 proteins. 630 of these peptides, corresponding to 280 proteins, could be assigned to peaks in MSI data sets. Gene ontology and pathway analyses revealed that many of the proteins are involved in neurodegenerative disorders, such as Alzheimer's, Parkinson's, and Huntington's disease.