Involvement of the contralateral hippocampus in ictal-like but not interictal epileptic activities in the kainate mouse model of temporal lobe epilepsy.

OBJECTIVE: Animal and human studies have shown that the seizure-generating region is vastly dependent on distant neuronal hubs that can decrease duration and propagation of ongoing seizures. However, we still lack a comprehensive understanding of the impact of distant brain areas on specific interictal and ictal epileptic activities (e.g., isolated spikes, spike trains, seizures). Such knowledge is critically needed, because all kinds of epileptic activities are not equivalent in terms of clinical expression and impact on the progression of the disease. METHODS: We used surface high-density electroencephalography and multisite intracortical recordings, combined with pharmacological silencing of specific brain regions in the well-known kainate mouse model of temporal lobe epilepsy. We tested the impact of selective regional silencing on the generation of epileptic activities within a continuum ranging from very transient to more sustained and long-lasting discharges reminiscent of seizures. RESULTS: Silencing the contralateral hippocampus completely suppresses sustained ictal activities in the focus, as efficiently as silencing the focus itself, but whereas focus silencing abolishes all focus activities, contralateral silencing fails to control transient spikes. In parallel, we observed that sustained focus epileptiform discharges in the focus are preceded by contralateral firing and more strongly phase-locked to bihippocampal delta/theta oscillations than transient spiking activities, reinforcing the presumed dominant role of the contralateral hippocampus in promoting long-lasting, but not transient, epileptic activities. SIGNIFICANCE: Altogether, our work provides suggestive evidence that the contralateral hippocampus is necessary for the interictal to ictal state transition and proposes that crosstalk between contralateral neuronal activity and ipsilateral delta/theta oscillation could be a candidate mechanism underlying the progression from short- to long-lasting epileptic activities.

Reproducible network changes occur in a mouse model of temporal lobe epilepsy but do not correlate with disease severity.

Studying the development of brain network disruptions in epilepsy is challenged by the paucity of data before epilepsy onset. Here, we used the unilateral, kainate mouse model of hippocampal epilepsy to investigate brain network changes before and after epilepsy onset and their stability across time. Using 32 epicranial electrodes distributed over the mouse hemispheres, we analyzed EEG epochs free from epileptic activity in 15 animals before and 28 days after hippocampal injection (group 1) and in 20 animals on two consecutive days (d28 and d29, group 2). Statistical dependencies between electrodes were characterized with the debiased-weighted phase lag index. We analyzed: a) graph metric changes from baseline to chronic stage (d28) in group 1; b) their reliability across d28 and d29, in group 2; c) their correlation with epileptic activity (EA: seizure, spike and fast-ripple rates), averaged over d28 and d29, in group 2. During the chronic stage, intra-hemispheric connections of the non-injected hemisphere strengthened, yielding an asymmetrical network in low (4-8 Hz) and high theta (8-12 Hz) bands. The contralateral hemisphere also became more integrated and segregated within the high theta band. Both network topology and EEG markers of EA were stable over consecutive days but not correlated with each other. Altogether, we show reproducible large-scale network modifications after the development of focal epilepsy. These modifications are mostly specific to the non-injected hemisphere. The absence of correlation with epileptic activity does not allow to specifically ascribe these network changes to mechanisms supporting EA or rather compensatory inhibition but supports the notion that epilepsy extends beyond the sole repetition of EA and impacts network that might not be involved in EA generation.

Paradoxical neuronal hyperexcitability in a mouse model of mitochondrial pyruvate import deficiency.

Neuronal excitation imposes a high demand of ATP in neurons. Most of the ATP derives primarily from pyruvate-mediated oxidative phosphorylation, a process that relies on import of pyruvate into mitochondria occuring exclusively via the mitochondrial pyruvate carrier (MPC). To investigate whether deficient oxidative phosphorylation impacts neuron excitability, we generated a mouse strain carrying a conditional deletion of MPC1, an essential subunit of the MPC, specifically in adult glutamatergic neurons. We found that, despite decreased levels of oxidative phosphorylation and decreased mitochondrial membrane potential in these excitatory neurons, mice were normal at rest. Surprisingly, in response to mild inhibition of GABA mediated synaptic activity, they rapidly developed severe seizures and died, whereas under similar conditions the behavior of control mice remained unchanged. We report that neurons with a deficient MPC were intrinsically hyperexcitable as a consequence of impaired calcium homeostasis, which reduced M-type potassium channel activity. Provision of ketone bodies restored energy status, calcium homeostasis and M-channel activity and attenuated seizures in animals fed a ketogenic diet. Our results provide an explanation for the seizures that frequently accompany a large number of neuropathologies, including cerebral ischemia and diverse mitochondriopathies, in which neurons experience an energy deficit.

Mouse models characterize GNAO1 encephalopathy as a neurodevelopmental disorder leading to motor anomalies: from a severe G203R to a milder C215Y mutation.

GNAO1 encephalopathy characterized by a wide spectrum of neurological deficiencies in pediatric patients originates from de novo heterozygous mutations in the gene encoding Gαo, the major neuronal G protein. Efficient treatments and even the proper understanding of the underlying etiology are currently lacking for this dominant disease. Adequate animal models of GNAO1 encephalopathy are urgently needed. Here we describe establishment and characterization of mouse models of the disease based on two point mutations in GNAO1 with different clinical manifestations. One of them is G203R leading to the early-onset epileptic seizures, motor dysfunction, developmental delay and intellectual disability. The other is C215Y producing much milder clinical outcomes, mostly-late-onset hyperkinetic movement disorder. The resultant mouse models show distinct phenotypes: severe neonatal lethality in GNAO1[G203R]/ + mice vs. normal vitality in GNAO1[C215Y]/ + . The latter model further revealed strong hyperactivity and hyperlocomotion in a panel of behavioral assays, without signs of epilepsy, recapitulating the patients' manifestations. Importantly, despite these differences the two models similarly revealed prenatal brain developmental anomalies, such as enlarged lateral ventricles and decreased numbers of neuronal precursor cells in the cortex. Thus, our work unveils GNAO1 encephalopathy as to a large extent neurodevelopmental malady. We expect that this understanding and the tools we established will be instrumental for future therapeutic developments.

Slow oscillations open susceptible time windows for epileptic discharges.

OBJECTIVE: In patients with epilepsy, interictal epileptic discharges are a diagnostic hallmark of epilepsy and represent abnormal, so-called "irritative" activity that disrupts normal cognitive functions. Despite their clinical relevance, their mechanisms of generation remain poorly understood. It is assumed that brain activity switches abruptly, unpredictably, and supposedly randomly to these epileptic transients. We aim to study the period preceding these epileptic discharges, to extract potential proepileptogenic mechanisms supporting their expression. METHODS: We used multisite intracortical recordings from patients who underwent intracranial monitoring for refractory epilepsy, the majority of whom had a mesial temporal lobe seizure onset zone. Our objective was to evaluate the existence of proepileptogenic windows before interictal epileptic discharges. We tested whether the amplitude and phase synchronization of slow oscillations (.5-4 Hz and 4-7 Hz) increase before epileptic discharges and whether the latter are phase-locked to slow oscillations. Then, we tested whether the phase-locking of neuronal activity (assessed by high-gamma activity, 60-160 Hz) to slow oscillations increases before epileptic discharges to provide a potential mechanism linking slow oscillations to interictal activities. RESULTS: Changes in widespread slow oscillations anticipate upcoming epileptic discharges. The network extends beyond the irritative zone, but the increase in amplitude and phase synchronization is rather specific to the irritative zone. In contrast, epileptic discharges are phase-locked to widespread slow oscillations and the degree of phase-locking tends to be higher outside the irritative zone. Then, within the irritative zone only, we observe an increased coupling between slow oscillations and neuronal discharges before epileptic discharges. SIGNIFICANCE: Our results show that epileptic discharges occur during vulnerable time windows set up by a specific phase of slow oscillations. The specificity of these permissive windows is further reinforced by the increased coupling of neuronal activity to slow oscillations. These findings contribute to our understanding of epilepsy as a distributed oscillopathy and open avenues for future neuromodulation strategies aiming at disrupting proepileptic mechanisms.

Endogenous erythropoietin signaling regulates migration and laminar positioning of upper-layer neurons in the developing neocortex.

Erythropoietin (EPO), the hypoxia-inducible hematopoietic hormone, has well-established neuroprotective/neurotrophic roles in the developing central nervous system and the therapeutic potential of EPO has been widely explored in clinical studies for the treatment of perinatal hypoxic brain lesion, as well as prematurity. Here, we reveal that both EPO and Epo receptor (EPOR) are expressed in the developing rat somatosensory cortex during radial migration and laminar positioning of granular and supragranular neurons. Experimental deregulation of EPO signaling using genetic approaches results in aberrant migration, as well as permanent neuronal misplacement leading to abnormal network activity and protracted sensory behavioral deficits. We identify ERK as the downstream effector of the EPO signaling pathway for neuronal migration. These findings reveal a crucial role for endogenous EPO signaling in neuronal migration, and offer important insights for understanding how the temporary deregulation of EPO could result in migration defects that lead to abnormal behavior in the adult.

Transplanted Embryonic Neurons Improve Functional Recovery by Increasing Activity in Injured Cortical Circuits.

Transplantation of appropriate neuronal precursors after injury is a promising strategy to reconstruct cortical circuits, but the efficiency of these approaches remains limited. Here, we applied targeted apoptosis to selectively ablate layer II/III pyramidal neurons in the rat juvenile cerebral cortex and attempted to replace lost neurons with their appropriate embryonic precursors by transplantation. We demonstrate that grafted precursors do not migrate to replace lost neurons but form vascularized clusters establishing reciprocal synaptic contacts with host networks and show functional integration. These heterotopic neuronal clusters significantly enhance the activity of the host circuits without causing epileptic seizures and attenuate the apoptotic injury-induced functional deficits in electrophysiological and behavioral tests. Chemogenetic activation of grafted neurons further improved functional recovery, and the persistence of the graft was necessary for maintaining restored functions in adult animals. Thus, implanting neuronal precursors capable to form synaptically integrated neuronal clusters combined with activation-based approaches represents a useful strategy for helping long-term functional recovery following brain injury.

Large-Scale Networks for Auditory Sensory Gating in the Awake Mouse.

The amplitude of the brain response to a repeated auditory stimulus is diminished as compared to the response to the first tone (T1) for interstimulus intervals (ISI) lasting up to hundreds of milliseconds. This adaptation process, called auditory sensory gating (ASG), is altered in various psychiatric diseases including schizophrenia and is classically studied by focusing on early evoked cortical responses to the second tone (T2) using 500-ms ISI. However, mechanisms underlying ASG are still not well-understood. We investigated ASG in awake mice from the brainstem to cortex at variable ISIs (125-2000 ms) using high-density EEG and intracerebral recordings. While ASG decreases at longer ISIs, it is still present at durations (500-2000 ms) far beyond the time during which brain responses to T1 could still be detected. T1 induces a sequence of specific stable scalp EEG topographies that correspond to the successive activation of distinct neural networks lasting about 350 ms. These brain states remain unaltered if T2 is presented during this period, although T2 is processed by the brain, suggesting that ongoing networks of brain activity are active for longer than early evoked-potentials and are not overwritten by an upcoming new stimulus. Intracerebral recordings demonstrate that ASG is already present at the level of ventral cochlear nucleus (vCN) and inferior colliculus and is amplified across the hierarchy in bottom-up direction. This study uncovers the extended stability of sensory-evoked brain states and long duration of ASG, and sheds light on generators of ASG and possible interactions between bottom-up and top-down mechanisms.

Background EEG Connectivity Captures the Time-Course of Epileptogenesis in a Mouse Model of Epilepsy.

Large-scale brain networks are increasingly recognized as important for the generation of seizures in epilepsy. However, how a network evolves from a healthy state through the process of epileptogenesis remains unclear. To address this question, here, we study longitudinal epicranial background EEG recordings (30 electrodes, EEG free from epileptiform activity) of a mouse model of mesial temporal lobe epilepsy. We analyze functional connectivity networks and observe that over the time course of epileptogenesis the networks become increasingly asymmetric. Furthermore, computational modelling reveals that a set of nodes, located outside of the region of initial insult, emerges as particularly important for the network dynamics. These findings are consistent with experimental observations, thus demonstrating that ictogenic mechanisms can be revealed on the EEG, that computational models can be used to monitor unfolding epileptogenesis and that both the primary focus and epileptic network play a role in epileptogenesis.

Large-Scale 3-5 Hz Oscillation Constrains the Expression of Neocortical Fast Ripples in a Mouse Model of Mesial Temporal Lobe Epilepsy.

Large-scale slow oscillations allow the integration of neuronal activity across brain regions during sensory or cognitive processing. However, evidence that this form of coding also holds for pathological networks, such as for distributed networks in epileptic disorders, does not yet exist. Here, we show in a mouse model of unilateral hippocampal epilepsy that epileptic fast ripples generated in the neocortex distant from the primary focus occur during transient trains of interictal epileptic discharges. During these epileptic paroxysms, local phase-locking of neuronal firing and a phase-amplitude coupling of the epileptic discharges over a slow oscillation at 3-5 Hz are detected. Furthermore, the buildup of the slow oscillation begins in the bihippocampal network that includes the focus, which synchronizes and drives the activity across the large-scale epileptic network into the frontal cortex. This study provides the first functional description of the emergence of neocortical fast ripples in hippocampal epilepsy and shows that cross-frequency coupling might be a fundamental mechanism underlying the spreading of epileptic activity.

Whole-Night Continuous Rocking Entrains Spontaneous Neural Oscillations with Benefits for Sleep and Memory.

Sensory processing continues during sleep and can influence brain oscillations. We previously showed that a gentle rocking stimulation (0.25 Hz), during an afternoon nap, facilitates wake-sleep transition and boosts endogenous brain oscillations (i.e., EEG spindles and slow oscillations [SOs]). Here, we tested the hypothesis that the rhythmic rocking stimulation synchronizes sleep oscillations, a neurophysiological mechanism referred to as "neural entrainment." We analyzed EEG brain responses related to the stimulation recorded from 18 participants while they had a full night of sleep on a rocking bed. Moreover, because sleep oscillations are considered of critical relevance for memory processes, we also investigated whether rocking influences overnight declarative memory consolidation. We first show that, compared to a stationary night, continuous rocking shortened the latency to non-REM (NREM) sleep and strengthened sleep maintenance, as indexed by increased NREM stage 3 (N3) duration and fewer arousals. These beneficial effects were paralleled by an increase in SOs and in slow and fast spindles during N3, without affecting the physiological SO-spindle phase coupling. We then confirm that, during the rocking night, overnight memory consolidation was enhanced and also correlated with the increase in fast spindles, whose co-occurrence with the SO up-state is considered to foster cortical synaptic plasticity. Finally, supporting the hypothesis that a rhythmic stimulation entrains sleep oscillations, we report a temporal clustering of spindles and SOs relative to the rocking cycle. Altogether, these findings demonstrate that a continuous rocking stimulation strengthens deep sleep via the neural entrainment of intrinsic sleep oscillations.

Author Correction: PV plasticity sustained through D1/5 dopamine signaling required for long-term memory consolidation.

In the version of this article initially published, the right panel in Fig. 2b was duplicated from the corresponding panel in Fig. 2c, and some data points in Fig. 3b were duplicated from Fig. 3a. None of the conclusions in the paper are affected. The errors have been corrected in the HTML and PDF versions of the article, and source data have been posted for the revised panels. The original and corrected figures are shown in the accompanying Author Correction.

Electrophysiological Evidence for the Development of a Self-Sustained Large-Scale Epileptic Network in the Kainate Mouse Model of Temporal Lobe Epilepsy.

Most research on focal epilepsy focuses on mechanisms of seizure generation in the primary epileptic focus (EF). However, neurological deficits that are not directly linked to seizure activity and that may persist after focus removal are frequent. The recruitment of remote brain regions of an epileptic network (EN) is recognized as a possible cause, but a profound lack of experimental evidence exists concerning their recruitment and the type of pathological activities they exhibit. We studied the development of epileptic activities at the large-scale in male mice of the kainate model of unilateral temporal lobe epilepsy using high-density surface EEG and multiple-site intracortical recordings. We show that, along with focal spikes and fast ripples that remain localized to the injected hippocampus (i.e., the EF), a subpopulation of spikes that propagate across the brain progressively emerges even before the expression of seizures. The spatiotemporal propagation of these generalized spikes (GSs) is highly stable within and across animals, defining a large-scale EN comprising both hippocampal regions and frontal cortices. Interestingly, GSs are often concomitant with muscular twitches. In addition, while fast ripples are, as expected, highly frequent in the EF, they also emerge in remote cortical regions and in particular in frontal regions where GSs propagate. Finally, we demonstrate that these remote interictal activities are dependent on the focus in the early phase of the disease but continue to be expressed after focus silencing at later stages. Our results provide evidence that neuronal networks outside the initial focus are progressively altered during epileptogenesis.SIGNIFICANCE STATEMENT It has long been held that the epileptic focus is responsible for triggering seizures and driving interictal activities. However, focal epilepsies are associated with heterogeneous symptoms, calling into question the concept of a strictly focal disease. Using the mouse model of hippocampal sclerosis, this work demonstrates that focal epilepsy leads to the development of pathological activities specific to the epileptic condition, notably fast ripples, that appear outside of the primary epileptic focus. Whereas these activities are dependent on the focus early in the disease, focus silencing fails to control them in the chronic stage. Thus, dynamical changes specific to the epileptic condition are built up outside of the epileptic focus along with disease progression, which provides supporting evidence for network alterations in focal epilepsy.

Systematic population spike delays across cortical layers within and between primary sensory areas.

The coordinated propagation of activity across cortical layers enables simultaneous local computation and inter-areal interactions. A pattern of upward propagation from deeper to more superficial layers, which has been repeatedly demonstrated in spontaneous activity, would allow these functions to occur in parallel. But it remains unclear whether upward propagation also occurs for stimulus evoked activity, and how it relates to activity in other cortical areas. Here we used a new method to analyze relative delays between spikes obtained from simultaneous laminar recordings in primary sensory cortex (S1) of both hemispheres. The results identified systematic spike delays across cortical layers that showed a general upward propagation of activity in evoked and spontaneous activity. Systematic spike delays were also observed between hemispheres. After spikes in one S1 the delays in the other S1 were shortest at infragranular layers and increased in the upward direction. Model comparisons furthermore showed that upward propagation was better explained as a step-wise progression over cortical layers than as a traveling wave. The results are in line with the notion that upward propagation functionally integrates activity into local processing at superficial layers, while efficiently allowing for simultaneous inter-areal interactions.

Perturbed Wnt signaling leads to neuronal migration delay, altered interhemispheric connections and impaired social behavior.

Perturbed neuronal migration and circuit development have been implicated in the pathogenesis of neurodevelopmental diseases; however, the direct steps linking these developmental errors to behavior alterations remain unknown. Here we demonstrate that Wnt/C-Kit signaling is a key regulator of glia-guided radial migration in rat somatosensory cortex. Transient downregulation of Wnt signaling in migrating, callosal projection neurons results in delayed positioning in layer 2/3. Delayed neurons display reduced neuronal activity with impaired afferent connectivity causing permanent deficit in callosal projections. Animals with these defects exhibit altered somatosensory function with reduced social interactions and repetitive movements. Restoring normal migration by overexpressing the Wnt-downstream effector C-Kit or selective chemogenetic activation of callosal projection neurons during a critical postnatal period prevents abnormal interhemispheric connections as well as behavioral alterations. Our findings identify a link between defective canonical Wnt signaling, delayed neuronal migration, deficient interhemispheric connectivity and abnormal social behavior analogous to autistic characteristics in humans.

PV plasticity sustained through D1/5 dopamine signaling required for long-term memory consolidation.

Long-term consolidation of memories depends on processes occurring many hours after acquisition. Whether this involves plasticity that is specifically required for long-term consolidation remains unclear. We found that learning-induced plasticity of local parvalbumin (PV) basket cells was specifically required for long-term, but not short/intermediate-term, memory consolidation in mice. PV plasticity, which involves changes in PV and GAD67 expression and connectivity onto PV neurons, was regulated by cAMP signaling in PV neurons. Following induction, PV plasticity depended on local D1/5 dopamine receptor signaling at 0-5 h to regulate its magnitude, and at 12-14 h for its continuance, ensuring memory consolidation. D1/5 dopamine receptor activation selectively induced DARPP-32 and ERK phosphorylation in PV neurons. At 12-14 h, PV plasticity was required for enhanced sharp-wave ripple densities and c-Fos expression in pyramidal neurons. Our results reveal general network mechanisms of long-term memory consolidation that requires plasticity of PV basket cells induced after acquisition and sustained subsequently through D1/5 receptor signaling.

Whole-scalp EEG mapping of somatosensory evoked potentials in macaque monkeys.

High-density scalp EEG recordings are widely used to study whole-brain neuronal networks in humans non-invasively. Here, we validate EEG mapping of somatosensory evoked potentials (SSEPs) in macaque monkeys (Macaca fascicularis) for the long-term investigation of large-scale neuronal networks and their reorganisation after lesions requiring a craniotomy. SSEPs were acquired from 33 scalp electrodes in five adult anaesthetized animals after electrical median or tibial nerve stimulation. SSEP scalp potential maps were identified by cluster analysis and identified in individual recordings. A distributed, linear inverse solution was used to estimate the intracortical sources of the scalp potentials. SSEPs were characterised by a sequence of components with unique scalp topographies. Source analysis confirmed that median nerve SSEP component maps were in accordance with the somatotopic organisation of the sensorimotor cortex. Most importantly, SSEP recordings were stable both intra- and interindividually. We aim to apply this method to the study of recovery and reorganisation of large-scale neuronal networks following a focal cortical lesion requiring a craniotomy. As a prerequisite, the present study demonstrated that a 300-mm(2) unilateral craniotomy over the sensorimotor cortex necessary to induce a cortical lesion, followed by bone flap repositioning, suture and gap plugging with calcium phosphate cement, did not induce major distortions of the SSEPs. In conclusion, SSEPs can be successfully and reproducibly recorded from high-density EEG caps in macaque monkeys before and after a craniotomy, opening new possibilities for the long-term follow-up of the cortical reorganisation of large-scale networks in macaque monkeys after a cortical lesion.

Dynamic connectivity among cortical layers in local and large-scale sensory processing.

Cortical processing of sensory stimuli typically recruits multiple areas, but how each area dynamically incorporates activity from other areas is not well understood. We investigated interactions between cortical columns of bilateral primary sensory regions (S1s) in rats by recording local field potentials and multi-unit activity simultaneously in both S1s with electrodes positioned at each cortical layer. Using dynamic connectivity analysis based on Granger-causal modeling, we found that, shortly after whisker stimulation (< 10 ms), contralateral S1 (cS1) already relays activity to granular and infragranular layers of S1 in the other hemisphere, after which cS1 shows a pattern of within-column interactions that directs activity upwards toward superficial layers. This pattern of predominant upward driving was also observed in S1 ipsilateral to stimulation, but at longer latencies. In addition, we found that interactions between the two S1s most strongly target granular and infragranular layers. Taken together, the results suggest a possible mechanism for how cortical columns integrate local and large-scale neocortical computation by relaying information from deeper layers to local processing in superficial layers.

Multi-modal assessment of long-term erythropoietin treatment after neonatal hypoxic-ischemic injury in rat brain.

Erythropoietin (EPO) has been recognized as a neuroprotective agent. In animal models of neonatal brain injury, exogenous EPO has been shown to reduce lesion size, improve structure and function. Experimental studies have focused on short course treatment after injury. Timing, dose and length of treatment in preterm brain damage remain to be defined. We have evaluated the effects of high dose and long-term EPO treatment in hypoxic-ischemic (HI) injury in 3 days old (P3) rat pups using histopathology, magnetic resonance imaging (MRI) and spectroscopy (MRS) as well as functional assessment with somatosensory-evoked potentials (SEP). After HI, rat pups were assessed by MRI for initial damage and were randomized to receive EPO or vehicle. At the end of treatment period (P25) the size of resulting cortical damage and white matter (WM) microstructure integrity were assessed by MRI and cortical metabolism by MRS. Whisker elicited SEP were recorded to evaluate somatosensory function. Brains were collected for neuropathological assessment. The EPO treated animals did not show significant decrease of the HI induced cortical loss at P25. WM microstructure measured by diffusion tensor imaging was improved and SEP response in the injured cortex was recovered in the EPO treated animals compared to vehicle treated animals. In addition, the metabolic profile was less altered in the EPO group. Long-term treatment with high dose EPO after HI injury in the very immature rat brain induced recovery of WM microstructure and connectivity as well as somatosensory cortical function despite no effects on volume of cortical damage. This indicates that long-term high-dose EPO induces recovery of structural and functional connectivity despite persisting gross anatomical cortical alteration resulting from HI.