Microglia maintain structural integrity during fetal brain morphogenesis.

Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.

Grey matter heterotopia subtypes show specific morpho-electric signatures and network dynamics.

Grey matter heterotopia (GMH) are neurodevelopmental disorders associated with abnormal cortical function and epilepsy. Subcortical band heterotopia (SBH) and periventricular nodular heterotopia (PVNH) are two well-recognized GMH subtypes in which neurons are misplaced, either forming nodules lining the ventricles in PVNH, or forming bands in the white matter in SBH. Although both PVNH and SBH are commonly associated with epilepsy, it is unclear whether these two GMH subtypes differ in terms of pathological consequences or, on the contrary, share common altered mechanisms. Here, we studied two robust preclinical models of SBH and PVNH, and performed a systematic comparative assessment of the physiological and morphological diversity of heterotopia neurons, as well as the dynamics of epileptiform activity and input connectivity. We uncovered a complex set of altered properties, including both common and distinct physiological and morphological features across heterotopia subtypes, and associated with specific dynamics of epileptiform activity. Taken together, these results suggest that pro-epileptic circuits in GMH are, at least in part, composed of neurons with distinct, subtype-specific, physiological and morphological properties depending on the heterotopia subtype. Our work supports the notion that GMH represent a complex set of disorders, associating both shared and diverging pathological consequences, and contributing to forming epileptogenic networks with specific properties. A deeper understanding of these properties may help to refine current GMH classification schemes by identifying morpho-electric signatures of GMH subtypes, to potentially inform new treatment strategies.

FLNA regulates neuronal maturation by modulating RAC1-Cofilin activity in the developing cortex.

Periventricular nodular heterotopia (PNH), the most common brain malformation diagnosed in adulthood, is characterized by the presence of neuronal nodules along the ventricular walls. PNH is mainly associated with mutations in the FLNA gene - encoding an actin-binding protein - and patients often develop epilepsy. However, the molecular mechanisms underlying the neuronal failure still remain elusive. It has been hypothesized that dysfunctional cortical circuitry, rather than ectopic neurons, may explain the clinical manifestations. To address this issue, we depleted FLNA from cortical pyramidal neurons of a conditional Flnaflox/flox mice by timed in utero electroporation of Cre recombinase. We found that FLNA regulates dendritogenesis and spinogenesis thus promoting an appropriate excitatory/inhibitory inputs balance. We demonstrated that FLNA modulates RAC1 and cofilin activity through its interaction with the Rho-GTPase Activating Protein 24 (ARHGAP24). Collectively, we disclose an uncharacterized role of FLNA and provide strong support for neural circuit dysfunction being a consequence of FLNA mutations.

Early suppression of excitability in subcortical band heterotopia modifies epileptogenesis in rats.

Malformations of cortical development represent a major cause of epilepsy in childhood. However, the pathological substrate and dynamic changes leading to the development and progression of epilepsy remain unclear. Here, we characterized an etiology-relevant rat model of subcortical band heterotopia (SBH), a diffuse type of cortical malformation associated with drug-resistant seizures in humans. We used longitudinal electrographic recordings to monitor the age-dependent evolution of epileptiform discharges during the course of epileptogenesis in this model. We found both quantitative and qualitative age-related changes in seizures properties and patterns, accompanying a gradual progression towards a fully developed seizure pattern seen in adulthood. We also dissected the relative contribution of the band heterotopia and the overlying cortex to the development and age-dependent progression of epilepsy using timed and spatially targeted manipulation of neuronal excitability. We found that an early suppression of neuronal excitability in SBH slows down epileptogenesis in juvenile rats, whereas epileptogenesis is paradoxically exacerbated when excitability is suppressed in the overlying cortex. However, in rats with active epilepsy, similar manipulations of excitability have no effect on chronic spontaneous seizures. Together, our data support the notion that complex developmental alterations occurring in both the SBH and the overlying cortex concur to creating pathogenic circuits prone to generate seizures. Our study also suggests that early and targeted interventions could potentially influence the course of these altered developmental trajectories, and favorably modify epileptogenesis in malformations of cortical development.

Somatic double-hit in MTOR and RPS6 in hemimegalencephaly with intractable epilepsy.

Single germline or somatic activating mutations of mammalian target of rapamycin (mTOR) pathway genes are emerging as a major cause of type II focal cortical dysplasia (FCD), hemimegalencephaly (HME) and tuberous sclerosis complex (TSC). A double-hit mechanism, based on a primary germline mutation in one allele and a secondary somatic hit affecting the other allele of the same gene in a small number of cells, has been documented in some patients with TSC or FCD. In a patient with HME, severe intellectual disability, intractable seizures and hypochromic skin patches, we identified the ribosomal protein S6 (RPS6) p.R232H variant, present as somatic mosaicism at ~15.1% in dysplastic brain tissue and ~11% in blood, and the MTOR p.S2215F variant, detected as ~8.8% mosaicism in brain tissue, but not in blood. Overexpressing the two variants independently in animal models, we demonstrated that MTOR p.S2215F caused neuronal migration delay and cytomegaly, while RPS6 p.R232H prompted increased cell proliferation. Double mutants exhibited a more severe phenotype, with increased proliferation and migration defects at embryonic stage and, at postnatal stage, cytomegalic cells exhibiting eccentric nuclei and binucleation, which are typical features of balloon cells. These findings suggest a synergistic effect of the two variants. This study indicates that, in addition to single activating mutations and double-hit inactivating mutations in mTOR pathway genes, severe forms of cortical dysplasia can also result from activating mutations affecting different genes in this pathway. RPS6 is a potential novel disease-related gene.

Direct and Collateral Alterations of Functional Cortical Circuits in a Rat Model of Subcortical Band Heterotopia.

Subcortical band heterotopia (SBH), also known as double-cortex syndrome, is a neuronal migration disorder characterized by an accumulation of neurons in a heterotopic band below the normotopic cortex. The majority of patients with SBH have mild to moderate intellectual disability and intractable epilepsy. However, it is still not clear how cortical networks are organized in SBH patients and how this abnormal organization contributes to improper brain function. In this study, cortical networks were investigated in the barrel cortex in an animal model of SBH induced by in utero knockdown of Dcx, main causative gene of this condition in human patients. When the SBH was localized below the Barrel Field (BF), layer (L) four projection to correctly positioned L2/3 pyramidal cells was weakened due to lower connectivity. Conversely, when the SBH was below an adjacent cortical region, the excitatory L4 to L2/3 projection was stronger due to increased L4 neuron excitability, synaptic strength and excitation/inhibition ratio of L4 to L2/3 connection. We propose that these developmental alterations contribute to the spectrum of clinical dysfunctions reported in patients with SBH.

Spontaneous epileptiform activity in a rat model of bilateral subcortical band heterotopia.

OBJECTIVE: Malformations of cortical development are common causes of intellectual disability and epilepsy, yet there is a crucial lack of relevant preclinical models associating seizures and cortical malformations. Here, we describe a novel rat model with bilateral subcortical band heterotopia (SBH) and examine whether this model develops spontaneous epileptic seizures. METHODS: To generate bilateral SBH in rats, we combined RNAi-mediated knockdown of Dcx and in utero electroporation with a tripolar electrode configuration enabling simultaneous transfection of the two brain hemispheres. To determine whether bilateral SBH leads to epileptiform activity, rats of various ages were implanted for telemetric electrocorticographic recordings and histopathological examination was carried out at the end of the recording sessions. RESULTS: By 2 months, rats with bilateral SBH showed nonconvulsive spontaneous seizures consisting of spike-and-wave discharges (SWDs) with dominant frequencies in the alpha and theta bands and secondarily in higher-frequency bands. SWDs occurred during both the dark and the light period, but were more frequent during quiet awake state than during sleep. Also, SWDs were more frequent and lasted longer at older ages. No sex differences were found. Although frequencies and durations of SWDs were found to be uncorrelated with the size of SBH, SWDs were initiated in some occasions from brain hemispheres comprising a larger SBH. Lastly, SWDs exhibited absence-like pharmacological properties, being temporarily alleviated by ethosuximide administration. SIGNIFICANCE: This novel model of bilateral SBH with spontaneous epilepsy may potentially provide valuable new insights into causality between cortical malformations and seizures, and help translational research aiming at designing novel treatment strategies for epilepsy.

Correct Laminar Positioning in the Neocortex Influences Proper Dendritic and Synaptic Development.

The neocortex is a 6-layered laminated structure with a precise anatomical and functional organization ensuring proper function. Laminar positioning of cortical neurons, as determined by termination of neuronal migration, is a key determinant of their ability to assemble into functional circuits. However, the exact contribution of laminar placement to dendrite morphogenesis and synapse formation remains unclear. Here we manipulated the laminar position of cortical neurons by knocking down doublecortin (Dcx), a crucial effector of migration, and show that misplaced neurons fail to properly form dendrites, spines, and functional glutamatergic and GABAergic synapses. We further show that knocking down Dcx in properly positioned neurons induces similar but milder defects, suggesting that the laminar misplacement is the primary cause of altered neuronal development. Thus, the specific laminar environment of their fated layers is crucial for the maturation of cortical neurons, and influences their functional integration into developing cortical circuits.

Normotopic cortex is the major contributor to epilepsy in experimental double cortex.

OBJECTIVE: Subcortical band heterotopia (SBH) is a cortical malformation formed when neocortical neurons prematurely stop their migration in the white matter, forming a heterotopic band below the normotopic cortex, and is generally associated with intractable epilepsy. Although it is clear that the band heterotopia and the overlying cortex both contribute to creating an abnormal circuit prone to generate epileptic discharges, it is less understood which part of this circuitry is the most critical. Here, we sought to identify the origin of epileptiform activity in a targeted genetic model of SBH in rats. METHODS: Rats with SBH (Dcx-KD rats) were generated by knocking down the Dcx gene using shRNA vectors transfected into neocortical progenitors of rat embryos. Origin, spatial extent, and laminar profile of bicuculline-induced interictal-like activity on neocortical slices were analyzed by using extracellular recordings from 60-channel microelectrode arrays. Susceptibility to pentylenetetrazole-induced seizures was assessed by electrocorticography in head-restrained nonanesthetized rats. RESULTS: We show that the band heterotopia does not constitute a primary origin for interictal-like epileptiform activity in vitro and is dispensable for generating induced seizures in vivo. Furthermore, we report that most interictal-like discharges originating in the overlying cortex secondarily propagate to the band heterotopia. Importantly, we found that in vivo suppression of neuronal excitability in SBH does not alter the higher propensity of Dcx-KD rats to display seizures. INTERPRETATION: These results suggest a major role of the normotopic cortex over the band heterotopia in generating interictal epileptiform activity and seizures in brains with SBH.

Cell-autonomous and cell-to-cell signalling events in normal and altered neuronal migration.

The cerebral cortex is a complex six-layered structure that contains an important diversity of neurons, and has rich local and extrinsic connectivity. Among the mechanisms governing the cerebral cortex construction, neuronal migration is perhaps the most crucial as it ensures the timely formation of specific and selective neuronal circuits. Here, we review the main extrinsic and extrinsic factors involved in regulating neuronal migration in the cortex and describe some environmental factors interfering with their actions.

Spontaneous epileptic manifestations in a DCX knockdown model of human double cortex.

Previous reports indicate that in utero knockdown of doublecortin (DCX) results in the genesis of a subcortical heterotopia reminiscent of the doublecortex observed in female patients with DCX mutations. It has also been shown that these rats display an increased susceptibility to convulsant agents and increased cortical neurons excitability; but it is presently unknown whether they display spontaneous seizures. Furthermore, the link between the size of heterotopia and the clinical manifestation remained to be elucidated. Using video-electrocorticogram recordings, we now report that DCX knockdown induces frequent spontaneous seizures commonly associated with myoclonic jerks in adult rats. Surprisingly, epilepsy occurred even in rats with very small subcortical heterotopias, as revealed by histological analysis of recorded animals. Moreover, the severity of the epileptic manifestations was positively correlated with both the size of the subcortical heterotopia and the age of recorded animals; thus, epileptic features were not detected in immature affected rats. In conclusion, our data demonstrate for the first time that subtle alterations can yield epilepsy and reveal a strong correlation between thicknesses of subcortical heterotopia, age of affected individuals and clinical impairment.

New and improved tools for in utero electroporation studies of developing cerebral cortex.

In utero electroporation (IUE) has become a method of choice for rapid gain and loss of function studies in embryonic cerebral cortex. In this review we highlight some of the proven and recent advances in IUE technology that make it applicable to an increasingly wide array of experiments requiring spatial and temporal control of gene expression. Recently, cell-type-specific promoters and tamoxifen-gated cre-recombinase have been shown to work effectively with IUE. Experiments can now be designed and carried out to test whether and which cell-type-specific mechanisms operate within defined periods of neuronal migration and maturation. We have recently adapted this conditional expression approach to implement conditional rescue experiments. In conditional rescue, expression of an RNA interference (RNAi) target is restored by tamoxifen-induced cre-mediated recombination. An initial disruption in migration, and resultant malformation, caused by DCX RNAi was reversed by delayed re-expression of Dcx. In the future, combinations of spatially directed, cell-type-specific, and tamoxifen-gated transgene expression can be used to address the complex mechanisms likely to operate during development of cerebral cortex.

Size of Subcortical Band Heterotopia Influences the Susceptibility to Hyperthermia-Induced Seizures in a Rat Model.

Studies conducted in human and rodent models have suggested that preexisting neurodevelopmental defects could predispose immature brains to febrile seizures (FS). However, the impact of the anatomical extent of preexisting cortical malformations on FS susceptibility was never assessed. Here, we induced hyperthermic seizures (HS) in rats with bilateral subcortical band heterotopia (SBH) and found variable degrees of HS susceptibility depending on inter-individual anatomical differences in size and extent of SBH. This indicates that an association exists between the overall extent or location of a cortical malformation, and the predisposition to FS. This also suggests that various predisposing factors and underlying causes may contribute to the etiology of complex FS.

Spared cognitive and behavioral functions prior to epilepsy onset in a rat model of subcortical band heterotopia.

Subcortical band heterotopia (SBH), also known as doublecortex syndrome, is a malformation of cortical development resulting from mutations in the doublecortin gene (DCX). It is characterized by a lack of migration of cortical neurons that accumulate in the white matter forming a heterotopic band. Patients with SBH may present mild to moderate intellectual disability as well as epilepsy. The SBH condition can be modeled in rats by in utero knockdown (KD) of Dcx. The affected cells form an SBH reminiscent of that observed in human patients and the animals develop a chronic epileptic condition in adulthood. Here, we investigated if the presence of a SBH is sufficient to induce cognitive impairment in juvenile Dcx-KD rats, before the onset of epilepsy. Using a wide range of behavioral tests, we found that the presence of SBH did not appear to affect motor control or somatosensory processing. In addition, cognitive abilities such as learning, short-term and long-term memory, were normal in pre-epileptic Dcx-KD rats. We suggest that the SBH presence is not sufficient to impair these behavioral functions.

Antiepileptic drugs and brain maturation: fetal exposure to lamotrigine generates cortical malformations in rats.

Intake of antiepileptic drugs (AEDs) during pregnancy can provoke severe and subtle fetal malformations associated with deleterious sequelae, reflecting the need for experimental investigations on the comparative teratogenic potential of these agents. We recently reported that prenatal exposure to vigabatrin and valproate, two AEDs which act through GABAergic mechanisms, induces hippocampal and cortical dysplasias in rodents. We have now investigated the effects of phenobarbital (PB, 30 mg/kg day) i.p.), a drug also endowed with GABAergic effects, and the new generation AEDs lamotrigine (LTG, 5-20mg/kg/day i.p.), topiramate (TPM, 10mg/kg/day i.p.), and levetiracetam (LEV, 50mg/kg/day i.p.) on brain development. Prenatal exposure to LTG induced hippocampal and cortical malformations in a dose-dependent manner, at maternal plasma concentrations within the clinically occurring range. These abnormalities were not observed after exposure to PB, TP and LEV. These observations raise concerns about potential clinical correlates and call for detailed comparative investigations on the consequences of AED use during pregnancy.

Glutamate acting on AMPA but not NMDA receptors modulates the migration of hippocampal interneurons.

Paracrine GABA and glutamate acting, respectively, on GABAA and NMDA receptors modulate the migration of hippocampal pyramidal cells. Using corticohippocampal organotypic explants from glutamic acid decarboxylase (GAD) 67-enhanced green fluorescent protein (EGFP) knock-in embryos, we now report that, in contrast to pyramidal neurons, the blockade of AMPA but not NMDA receptors exerts important actions on the migration of GABAergic interneurons. In addition, the blockade of GABAA receptors fails to modify the migration rates of GABAergic interneurons. Immunohistochemical analyses of GAD67-EGFP embryos (from embryonic day 14 to birth) reveal that interneurons colonize the hippocampal primordium by embryonic day 15. At that stage, the hippocampal primordium is already composed of pioneer glutamatergic neurons, including (1) Cajal-Retzius cells, immunopositive to calretinin and reelin, and (2) other presumptive pioneer pyramidal cells that are immunopositive to betaIII-tubulin and vesicular glutamate transporter 3 and immunonegative to GABA or GAD67. Therefore, the migrations of pyramidal neurons and GABAergic interneurons are cross-modulated: glutamate released from pioneer glutamatergic neurons facilitates the migration of GABAergic interneurons, which in turn would release GABA, facilitating the migration of glutamatergic neuroblasts. This general sequence may provide a retroactive positive loop needed to construct the hippocampal network. It might constitute a primitive homeostatic mechanism in the developing brain that acts to balance GABA-glutamate contributions to network construction and activity.

Neurotransmitters and brain maturation: early paracrine actions of GABA and glutamate modulate neuronal migration.

Migration of neurons from their birthplace to their final destination is an extremely important step in brain maturation, and cortical migration disorders are the most common brain developmental alteration observed in human patients. Among the mechanisms that govern neuronal migration, the neurotransmitters GABA and glutamate deserve particular attention: 1) neurotransmitters and receptors are expressed early in the developing brain, 2) neurotransmitters may act as paracrine signaling molecules in the immature brain, and 3) neurotransmitters regulate intracellular calcium required for many cellular functions, including cytoskeletal dynamic changes. Thus, many reports reviewed here aimed to demonstrate that the activation of specific GABA and glutamate receptors is instrumental in cell migration by acting as motility promoting, acceleratory, or stop signal. Interestingly, the regulation of migration by neurotransmitters and receptors depends on the type of migration (radial, tangential, or chain migration), the type of cells (principal glutamatergic neurons vs. GABAergic interneurons), and the brain area (neocortex, cerebellum, rostral migratory stream). A hypothesis is proposed that these differential actions in different cell types arise from a "homeostatic-like" regulation that controls final position, timing, and number of cells at destination.

A noncanonical release of GABA and glutamate modulates neuronal migration.

Immature neurons express GABA and glutamate receptors before synapse formation, and both transmitters are released at an early developmental stage. We have now tested the hypothesis that the ongoing release of GABA and glutamate modulates neuronal migration. Using 5-bromo-2'-deoxyuridine labeling and cocultures of hippocampal slices obtained from naive and green fluorescent protein-transgenic mice, we report that migration is severely affected by GABA(A) or NMDA receptor antagonist treatments. These effects were also present in munc18-1 knock-out slices in which soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicular secretion of transmitters has been deleted. GABA(A) antagonists were more efficient than NMDA antagonists to reduce cell migration, in keeping with the earlier maturation of GABAergic mechanisms. We conclude that GABA and, to a lesser degree, glutamate released in a SNARE-independent mechanism exert a paracrine action on neuronal migration.

Glutamate transporters prevent the generation of seizures in the developing rat neocortex.

Glutamate transporters are operative at an early developmental stage well before synapse formation, but their functional significance has not been determined. We now report that blockade of glutamate transporters in the immature neocortex generates recurrent NMDA receptor-mediated currents associated with synchronous oscillations of [Ca2+]i in the entire neuronal population. Intracerebroventricular injections of the blocker to pups generate seizures that are prevented by coinjections of NMDA receptor blockers. Therefore, the early expression of glutamate transporters plays a central role to prevent the activation by local glutamate concentrations of NMDA receptors and the generation of seizures that may alter the construction of cortical networks. A dysfunction of glutamate transporters may be a central event in early infancy epilepsy syndromes.

Causes and consequences of gray matter heterotopia.

The objective of this article is to review the pathophysiological bases of gray matter heterotopia and to appreciate their involvement in brain cortical development and functional consequences, namely epilepsy. The development of the cerebral cortex results from complex sequential processes including cell proliferation, cell migration, cortical organization, and formation of neuronal networks. Disruption of these steps yields different types of cortical malformations including gray matter heterotopia, characterized by the ectopic position of neurons along the ventricular walls or in the deep white matter. Cortical malformations are major causes of epilepsy, being responsible for up to 40% of drug-resistant epilepsy, and the cognitive level of affected patients varies from normal to severely impaired. This review reports data from human patients and animal models highlighting the genetic causes for these disorders affecting not only neuronal migration but also the proliferation of cortical progenitors. Therefore, gray matter heterotopias should not be considered as solely due to an abnormal neuronal migration and classifying them as such may be too restrictive. The review will also summarize literature data indicating that besides ectopic neurons, neighbor cortical areas also play a consistent role in epileptogenesis, supporting the notion that plastic changes secondary to the initial malformation are instrumental in the pathophysiology of epilepsy in affected patients.