Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex.

One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using two-photon in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a "protracted" human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in "real time".

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain.

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries unique challenges in children. Similarly, transcranial direct current stimulation (tDCS) can improve motor learning in adults but has only recently been applied to children. The use of tDCS and emerging techniques like high-definition tDCS (HD-tDCS) require special methodological considerations in the developing brain. Robotic TMS motor mapping may confer unique advantages for mapping, particularly in the developing brain. Here, we aim to provide a practical, standardized approach for two integrated methods capable of simultaneously exploring motor cortex modulation and motor maps in children. First, we describe a protocol for robotic TMS motor mapping. Individualized, MRI-navigated 12x12 grids centered on the motor cortex guide a robot to administer single-pulse TMS. Mean motor evoked potential (MEP) amplitudes per grid point are used to generate 3D motor maps of individual hand muscles with outcomes including map area, volume, and center of gravity. Tools to measure safety and tolerability of both methods are also included. Second, we describe the application of both tDCS and HD-tDCS to modulate the motor cortex and motor learning. An experimental training paradigm and sample results are described. These methods will advance the application of non-invasive brain stimulation in children.

Prenatal neural origins of infant motor development: Associations between fetal brain and infant motor development.

Functional circuits of the human brain emerge and change dramatically over the second half of gestation. It is possible that variation in neural functional system connectivity in utero predicts individual differences in infant behavioral development, but this possibility has yet to be examined. The current study examines the association between fetal sensorimotor brain system functional connectivity and infant postnatal motor ability. Resting-state functional connectivity data was obtained in 96 healthy human fetuses during the second and third trimesters of pregnancy. Infant motor ability was measured 7 months after birth using the Bayley Scales of Infant Development. Increased connectivity between the emerging motor network and regions of the prefrontal cortex, temporal lobes, posterior cingulate, and supplementary motor regions was observed in infants that showed more mature motor functions. In addition, females demonstrated stronger fetal-brain to infant-behavior associations. These observations extend prior longitudinal research back into prenatal brain development and raise exciting new ideas about the advent of risk and the ontogeny of early sex differences.

A unique mouse model for investigating the properties of amyotrophic lateral sclerosis-associated protein TDP-43, by in utero electroporation.

TDP-43 is a discriminative protein that is found as intracellular aggregations in the neurons of the cerebral cortex and spinal cord of patients with amyotrophic lateral sclerosis (ALS); however, the mechanisms of neuron loss and its relation to the aggregations are still unclear. In this study, we generated a useful model to produce TDP-43 aggregations in the motor cortex using in utero electroporation on mouse embryos. The plasmids used were full-length TDP-43 and C-terminal fragments of TDP-43 (wild-type or M337V mutant) tagged with GFP. For the full-length TDP-43, both wild-type and mutant, electroporated TDP-43 localized mostly in the nucleus, and though aggregations were detected in embryonic brains, they were very rarely observed at P7 and P21. In contrast, TDP-43 aggregations were generated in the brains electroporated with the C-terminal TDP-43 fragments as previously reported in in vitro experiments. TDP-43 protein was distributed diffusely-not only in the nucleus, but also in the cytoplasm-and the inclusion bodies were ubiquitinated and included phosphorylated TDP-43, which reflects the human pathology of ALS. This model using in utero electroporation of pathogenic genes into the brain of the mouse will likely become a useful model for studying ALS and also for evaluation of agents for therapeutic purpose, and may be applicable to other neurodegenerative diseases, as well.

[Fusion of brain neurons in rat embryos].

Syncytial interneuronal connections were studied in the sensomotor cortex and caudate nucleus of twenty 14-22 day rat embryos. It was shown that with the extremely weak development of glial processes, many neuronal bodies and their processes were in the direct contact with each other. The contacting membranes in these areas formed oblong and dot-like contacts resembling gap and tight junctions. As a result, the intercellular cleft experienced varicose-like deformations. In the area of contacts, barely visible membrane pores were formed that broadened to form large perforations. The perforation margins presented the rounded shape of fused plasma membranes of adjacent neurons. Inside the perforations, residual vesicular membranous bodies were formed. The areas of the paired membranes between perforations were fragmented, thus increasing the number of residual vesicles, until the neurons fused with each other completely by unifying the neuroplasm of contacting cells. The results of these studies suggest that that the fusion of neurons in vertebrate brain cortex and brainstem nuclei could occur not only in pathology, but also in normal animals at the stage of embryonic development.

NrCAM deletion causes topographic mistargeting of thalamocortical axons to the visual cortex and disrupts visual acuity.

NrCAM is a neural cell adhesion molecule of the L1 family that has been linked to autism spectrum disorders, a disease spectrum in which abnormal thalamocortical connectivity may contribute to visual processing defects. Here we show that NrCAM interaction with neuropilin-2 (Npn-2) is critical for semaphorin 3F (Sema3F)-induced guidance of thalamocortical axon subpopulations at the ventral telencephalon (VTe), an intermediate target for thalamic axon sorting. Genetic deletion of NrCAM or Npn-2 caused contingents of embryonic thalamic axons to misproject caudally in the VTe. The resultant thalamocortical map of NrCAM-null mutants showed striking mistargeting of motor and somatosensory thalamic axon contingents to the primary visual cortex, but retinogeniculate targeting and segregation were normal. NrCAM formed a molecular complex with Npn-2 in brain and neural cells, and was required for Sema3F-induced growth cone collapse in thalamic neuron cultures, consistent with a vital function for NrCAM in Sema3F-induced axon repulsion. NrCAM-null mice displayed reduced responses to visual evoked potentials recorded from layer IV in the binocular zone of primary visual cortex (V1), particularly when evoked from the ipsilateral eye, indicating abnormal visual acuity and ocularity. These results demonstrate that NrCAM is required for normal maturation of cortical visual acuity, and suggest that the aberrant projection of thalamic motor and somatosensory axons to the visual cortex in NrCAM-null mutant mice impairs cortical functions.

3D global and regional patterns of human fetal subplate growth determined in utero.

The waiting period of subplate evolution is a critical phase for the proper formation of neural connections in the brain. During this time, which corresponds to 15 to 24 postconceptual weeks (PCW) in the human fetus, thalamocortical and cortico-cortical afferents wait in and are in part guided by molecules embedded in the extracellular matrix of the subplate. Recent advances in fetal MRI techniques now allow us to study the developing brain anatomy in 3D from in utero imaging. We describe a reliable segmentation protocol to delineate the boundaries of the subplate from T2-W MRI. The reliability of the protocol was evaluated in terms of intra-rater reproducibility on a subset of the subjects. We also present the first 3D quantitative analyses of temporal changes in subplate volume, thickness, and contrast from 18 to 24 PCW. Our analysis shows that firstly, global subplate volume increases in proportion with the supratentorial volume; the subplate remained approximately one-third of supratentorial volume. Secondly, we found both global and regional growth in subplate thickness and a linear increase in the median and maximum subplate thickness through the waiting period. Furthermore, we found that posterior regions--specifically the occipital pole, ventral occipito-temporal region, and planum temporale--of the developing brain underwent the most statistically significant increases in subplate thickness. During this period, the thickest region was the developing somatosensory/motor cortex. The subplate growth patterns reported here may be used as a baseline for comparison to abnormal fetal brain development.

Transplantation of embryonic neural stem/precursor cells overexpressing BM88/Cend1 enhances the generation of neuronal cells in the injured mouse cortex.

The intrinsic inability of the central nervous system to efficiently repair traumatic injuries renders transplantation of neural stem/precursor cells (NPCs) a promising approach towards repair of brain lesions. In this study, NPCs derived from embryonic day 14.5 mouse cortex were genetically modified via transduction with a lentiviral vector to overexpress the neuronal lineage-specific regulator BM88/Cend1 that coordinates cell cycle exit and differentiation of neuronal precursors. BM88/Cend1-overexpressing NPCs exhibiting enhanced differentiation into neurons in vitro were transplanted in a mouse model of acute cortical injury and analyzed in comparison with control NPCs. Immunohistochemical analysis revealed that a smaller proportion of BM88/Cend1-overexpressing NPCs, as compared with control NPCs, expressed the neural stem cell marker nestin 1 day after transplantation, while the percentage of nestin-positive cells was significantly reduced thereafter in both types of cells, being almost extinct 1 week post-grafting. Both types of cells did not proliferate up to 4 weeks in vivo, thus minimizing the risk of tumorigenesis. In comparison with control NPCs, Cend1-overexpressing NPCs generated more neurons and less glial cells 1 month after transplantation in the lesioned cortex whereas the majority of graft-derived neurons were identified as GABAergic interneurons. Furthermore, transplantation of Cend1-overexpressing NPCs resulted in a marked reduction of astrogliosis around the lesioned area as compared to grafts of control NPCs. Our results suggest that transplantation of Cend1-overexpressing NPCs exerts beneficial effects on tissue regeneration by enhancing the number of generated neurons and restricting the formation of astroglial scar, in a mouse model of cortical brain injury.

Synaptic circuit abnormalities of motor-frontal layer 2/3 pyramidal neurons in an RNA interference model of methyl-CpG-binding protein 2 deficiency.

Rett syndrome, an autism spectrum disorder with prominent motor and cognitive features, results from mutations in the gene for methyl-CpG-binding protein 2 (MeCP2). Here, to identify cortical circuit abnormalities that are specifically associated with MeCP2 deficiency, we used glutamate uncaging and laser scanning photostimulation to survey intracortical networks in mouse brain slices containing motor-frontal cortex. We used in utero transfection of short hairpin RNA constructs to knock down MeCP2 expression in a sparsely distributed subset of layer (L) 2/3 pyramidal neurons in wild-type mice, and compared input maps recorded from transfected-untransfected pairs of neighboring neurons. The effect of MeCP2 deficiency on local excitatory input pathways was severe, with an average reduction in excitatory synaptic input from middle cortical layers (L3/5A) of >30% compared with MeCP2-replete controls. MeCP2 deficiency primarily affected the strength, rather than the topography, of excitatory intracortical pathways. Inhibitory synaptic inputs and intrinsic eletrophysiological properties were unaffected in the MeCP2-knockdown neurons. These studies indicate that MeCP2 deficiency in individual postsynaptic cortical pyramidal neurons is sufficient to induce a pathological synaptic defect in excitatory intracortical circuits.

Bhlhb5 regulates the postmitotic acquisition of area identities in layers II-V of the developing neocortex.

While progenitor-restricted factors broadly specify area identities in developing neocortex, the downstream regulatory elements involved in acquisition of those identities in postmitotic neurons are largely unknown. Here, we identify Bhlhb5, a transcription factor expressed in layers II-V, as a postmitotic regulator of area identity. Bhlhb5 is initially expressed in a high caudomedial to low rostrolateral gradient that transforms into a sharp border between sensory and rostral motor cortices. Bhlhb5 null mice exhibit aberrant expression of area-specific genes and structural organization in the somatosensory and caudal motor cortices. In somatosensory cortex, Bhlhb5 null mice display postsynaptic disorganization of vibrissal barrels. In caudal motor cortex, Bhlhb5 null mice exhibit anomalous differentiation of corticospinal motor neurons, accompanied by failure of corticospinal tract formation. Together, these results demonstrate Bhlhb5's function as an area-specific transcription factor that regulates the postmitotic acquisition of area identities and elucidate the genetic hierarchy between progenitors and postmitotic neurons driving neocortical arealization.

[Somatic maturation and sensorimotor development of C57BL/6 mice prenatally exposed to cytosine arabinoside].

The topical problem of experimental neurobiology is the development of pharmacological models to search for correlation between induced brain pathology and changes in behavioral phenotype. Cytosine arabinoside (Ara-c) is an antiproliferative agent, exposure to which in the critical period of the embryonic formation of the cortex results in the abnormality of its development. This study was aimed at estimation of the somatic and sensorimotor aspects of the early postnatal maturatrion of behavioral acts in mice with developmental abnormalities of the cortex induced by Ara-c. Pregnant C57BL/6 mice were injected with the substance on the 12.5th 13.5th gestation days. Offspring behavior was studied using a modified Fox battery on the 1st-21st postnatal days. Severe disorders of the sensorimotor development with slight somatic changes were revealed in the offsprings of Ara-c-treated mice. Features of these pathological changes point to a correlation between the developmental changes in behavioral phenotype and irregularities of the cortex formation. This experimental model can be applied to neurobiological and pharmacological studies.

In vitro examination of ontogenesis of developing neuronal cells in vagal nuclei in medulla oblongata in newborns.

The development of neuron cells in vagal nerve nuclei in medulla oblongata was studied in vitro in live newborns and stillborns from different cases. Morphological changes were studied in respiratory nuclei of dorsal motor centre (DMNV) and nucleus tractus solitarius (NTS) in medulla oblongata. The material from medulla oblongata was fixated in 10 micro buffered formalin solution. Fixated material was cut in series of 10mu thickness, with starting point from obex in +/- 4 mm thickness. Special histochemical and histoenzymatic methods for central nervous system were used: cresyl echt violet coloring, tolyidin blue, Sevier-Munger modification and Grimelius coloring. In immature newborns (abortions and immature) in dorsal motor nucleus of the vagus (DMNV) population stages S1, S2, S3 are dominant. In neuron population in vagal sensory nuclei (NTS) stages S1, S2 are dominant. In more advanced stages of development of newborns (premature), in DMNV stages S3 and S4 are seen and in NTS stages S2 and S3 are dominant. In mature phase of newborns (maturity) in vagal nucleus DMNV stages S5 and S6 are dominant, while in sensory nucleus NTS stages S4 and S5 are dominant. These data suggest that neuron population in dorsal motor nucleus of the vagus (DMNV) are more advanced in neuronal maturity in comparison with sensory neuron population of vagal sensory nucleus NTS. This occurrence shows that phylogenetic development of motor complex is more advanced than the sensory one, which is expected to take new information's from the extra uterine life after birth (extra uterine vagal phenotype).

Embryonic and postnatal development of the layer I-directed ("matrix") thalamocortical system in the rat.

Inputs to the layer I apical dendritic tufts of pyramidal cells are crucial in "top-down" interactions in the cerebral cortex. A large population of thalamocortical cells, the "matrix" (M-type) cells, provides a direct robust input to layer I that is anatomically and functionally different from the thalamocortical input to layer VI. The developmental timecourse of M-type axons is examined here in rats aged E (embryonic day) 16 to P (postnatal day) 30. Anterograde techniques were used to label axons arising from 2 thalamic nuclei mainly made up of M-type cells, the Posterior and the Ventromedial. The primary growth cones of M-type axons rapidly reached the subplate of dorsally situated cortical areas. After this, interstitial branches would sprout from these axons under more lateral cortical regions to invade the overlying cortical plate forming secondary arbors. Moreover, retrograde labeling of M-type cell somata in the thalamus after tracer deposits confined to layer I revealed that large numbers of axons from multiple thalamic nuclei had already converged in a given spot of layer I by P3. Because of early ingrowth in such large numbers, interactions of M-type axons may significantly influence the early development of cortical circuits.

COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas.

We used cortex-specific deletion of the transcription factor gene COUP-TFI (also known as Nr2f1) in mice to demonstrate previously unknown fundamental roles for it in patterning mammalian neocortex into areas. The highest COUP-TFI expression is observed in the cortical progenitors and progeny in parietal and occipital cortex that form sensory areas, and the lowest expression was observed in frontal cortex that includes motor areas. Cortical deletion of COUP-TFI resulted in massive expansion of frontal areas, including motor, to occupy most of neocortex, paralleled by marked compression of sensory areas to caudal occipital cortex. These area patterning changes are preceded and paralleled by corresponding changes in molecular markers of area identity and altered axonal projections to maintain patterned area-specific input and output connections. We conclude that COUP-TFI is required for balancing patterning of neocortex into frontal/motor and sensory areas by acting in its expression domain to repress frontal/motor area identities and to specify sensory area identities.

The cellular composition of the cerebral cortex of rat fetuses after fractionated low-dose irradiation.

Histological and morphometric studies were performed to address the characteristics of the morphogenesis of the sensorimotor cortex of the brain in rat fetuses subjected to fractionated gamma irradiation during the period from day 6 to day 18 of antenatal development at doses of 5, 25, 30, and 75 cGy. The results showed that fractionated irradiation at doses of 5-75 cGy had adverse effects on the processes of stem cell proliferation in the tissues of the developing cortex and also increased the intensity of cell destruction proportionally to the radiation dose. All cellular zones of the developing cortex showed increases in the absolute number of macroglial cells, which may be associated on the one hand with reactive increases in their production due to functional deficiencies and, on the other, with accelerated transformation of radial gliocytes into macroglial cells. Irradiation at doses of 5-75 cGy decreased the rate of migration of neuroblasts into the primary cortex, as shown by decreases in the numbers of cells in the neural differon in the neocortical rudiment and increases in their levels in the deep layers of the developing cortex.

Progress in realizing the promise of microarrays in systems neurobiology.

The power of microarrays in neuroscience has been challenged by the cellular heterogeneity and complexity of the central nervous system. In this issue of Neuron, Arlotta, Molyneaux, and colleagues have developed a technique combining retrograde labeling, flow cytometry, and microarrays to purify and molecularly characterize a specific population of neurons from the brain, focusing here on cortical projection neurons. We discuss these findings and the implications of this development for both systems and molecular neuroscience.

EMX2 regulates sizes and positioning of the primary sensory and motor areas in neocortex by direct specification of cortical progenitors.

Genetic studies of neocortical area patterning are limited, because mice deficient for candidate regulatory genes die before areas emerge and have other complicating issues. To define roles for the homeodomain transcription factor EMX2, we engineered nestin-Emx2 transgenic mice that overexpress Emx2 in cortical progenitors coincident with expression of endogenous Emx2 and survive postnatally. Cortical size, lamination, thalamus, and thalamocortical pathfinding are normal in homozygous nestin-Emx2 mice. However, primary sensory and motor areas are disproportionately altered in size and shift rostrolaterally. Heterozygous transgenics have similar but smaller changes. Opposite changes are found in heterozygous Emx2 knockout mice. Fgf8 expression in the commissural plate of nestin-Emx2 mice is indistinguishable from wild-type, but Pax6 expression is downregulated in rostral cortical progenitors, suggesting that EMX2 repression of PAX6 specification of rostral identities contributes to reduced rostral areas. We conclude that EMX2 levels in cortical progenitors disproportionately specify sizes and positions of primary cortical areas.

Topographic motor projections in the limb imposed by LIM homeodomain protein regulation of ephrin-A:EphA interactions.

The formation of topographic neural maps relies on the coordinate assignment of neuronal cell body position and axonal trajectory. The projection of motor neurons of the lateral motor column (LMC) along the dorsoventral axis of the limb mesenchyme constitutes a simple topographic map that is organized in a binary manner. We show that LIM homeodomain proteins establish motor neuron topography by coordinating the mediolateral settling position of motor neurons within the LMC with the dorsoventral selection of axon pathways in the limb. These topographic projections are established, in part, through LIM homeodomain protein control of EphA receptors and ephrin-A ligands in motor neurons and limb mesenchymal cells.

A system for MEA-based multisite stimulation.

The capability for multisite stimulation is one of the biggest potential advantages of microelectrode arrays (MEAs). There remain, however, several technical problems which have hindered the development of a practical stimulation system. An important design goal is to allow programmable multisite stimulation, which produces minimal interference with simultaneous extracellular and patch or whole cell clamp recording. Here, we describe a multisite stimulation and recording system with novel interface circuit modules, in which preamplifiers and transistor transistor logic-driven solid-state switching devices are integrated. This integration permits PC-controlled remote switching of each substrate electrode. This allows not only flexible selection of stimulation sites, but also rapid switching of the selected sites between stimulation and recording, within 1.2 ms. This allowed almost continuous monitoring of extracellular signals at all the substrate-embedded electrodes, including those used for stimulation. In addition, the vibration-free solid-state switching made it possible to record whole-cell synaptic currents in one neuron, evoked from multiple sites in the network. We have used this system to visualize spatial propagation patterns of evoked responses in cultured networks of cortical neurons. This MEA-based stimulation system is a useful tool for studying neuronal signal processing in biological neuronal networks, as well as the process of synaptic integration within single neurons.