Reelin regulates the development and synaptogenesis of the layer-specific entorhino-hippocampal connections

Here we examine the role of Reelin, an extracellular protein involved in neuronal migration, in the formation of hippocampal connections. Both at prenatal and postnatal stages, the general laminar and topographic distribution of entorhinal projections is preserved in the hippocampus of reeler mutant mice, in the absence of Reelin. However, developing and adult entorhinal afferents show severe alterations, including increased numbers of misrouted fibers and the formation of abnormal patches of termination from the medial and lateral entorhinal cortices. At perinatal stages, single entorhinal axons in reeler mice are grouped into thick bundles, and they have decreased axonal branching and decreased extension of axon collaterals. We also show that the number of entorhino-hippocampal synapses is lower in reeler mice than in control animals during development. Studies performed in mixed entorhino-hippocampal co-cultures combining slices from reeler and wild-type mice indicate that these abnormalities are caused by the lack of Reelin in the target hippocampus. These findings imply that Reelin fulfills a modulatory role during the formation of layer-specific and topographic connections in the hippocampus. They also suggest that Reelin promotes maturation of single fibers and synaptogenesis by entorhinal afferents.

Na(+)-K(+)-ATPase gene expression during in vitro development of rat fetal forebrain.

The existence of at least three isoforms of Na(+)-K(+)-ATPase in adult brain tissues [alpha 1, kidney type; alpha 2 [or alpha(+)]; alpha 3] suggests that these genes might be regulated in a cell-specific and time-dependent manner during development. We have studied this question in serum-free aggregating cell cultures of mechanically dissociated rat fetal telencephalon. At the protein level, the relative rate of synthesis of the pool of alpha 1-, alpha 2-, and alpha 3-subunits increased approximately twofold over 15 days of culture, leading to a marked increase in the immunochemical pool of alpha-subunits as measured by a panspecific polyclonal antibody. Concomitantly, Na(+)-K(+)-ATPase enzyme-specific activity increased three- (lower forebrain) to sixfold (upper forebrain). The transcripts of all three alpha-isoforms and beta-subunit were detected in vitro in similar proportion to the level observed in vivo. alpha 3-mRNA (3.7 kb) was more abundant than alpha 1 (3.7 kb) or alpha 2 (5.3 and 3.4 kb). Cytosine arabinoside (0.4 microM) and cholera toxin (0.1 microM) were used to selectively eliminate glial cells or neurons, respectively. It was found that alpha 2-mRNA is predominantly transcribed in glial cell cultures, whereas alpha 3- and beta 1-mRNA (2.7, 2.3, and 1.8 kb) are predominant in neuronal cultures.

Neurons in the corpus callosum of the cat during postnatal development.

The corpus callosum (CC) is a major telencephalic commissure containing mainly cortico-cortical axons and glial cells. We have identified neurons in the CC of the cat and quantified their number at different postnatal ages. An antibody against microtubule-associated protein 2 was used as a marker of neurons. Immunocytochemical double-labelling with neuron-specific enolase or gamma-aminobutyric acid antibodies in the absence of glial fibrillary acidic protein positivity confirmed the neuronal phenotype of these cells. CC neurons were also stained with anti-calbindin and anti-calretinin antibodies, typical for interneurons, and with an anti-neurofilament antibody, which in neocortex detects pyramidal neurons. Together, these findings suggest that the CC contains a mixed population of neuronal types. The quantification was corrected for double counting of adjacent sections and volume changes during CC development. Our data show that CC neurons are numerous early postnatally, and their number decreases with age. At birth, about 570 neurons are found within the CC boundaries and their number drops to about 200 in the adult. The distribution of the neurons within the CC also changes in development. Initially, many neurons are found throughout the CC, while at later ages they become restricted to the boundaries of the CC, and in the adult to the rostrum of the CC close to the septum pellucidum or to the indusium griseum. Although origin and function of transient CC neurons in development and in adulthood remain unknown, they are likely to be interstitial neurons. Some of them have well-developed and differentiated processes and resemble pyramidal cells or interneurons. An axon-guiding function during the early postnatal period can not be excluded.

Building hippocampal circuits to learn and remember: insights into the development of human memory

The hippocampal formation is essential for the processing of episodic memories for autobiographical events that happen in unique spatiotemporal contexts. Interestingly, before 2 years of age, children are unable to form or store episodic memories for recall later in life, a phenomenon known as infantile amnesia. From 2 to 7 years of age, there are fewer memories than predicted based on a forgetting function alone, a phenomenon known as childhood amnesia. Here, we discuss the postnatal maturation of the primate hippocampal formation with the goal of characterizing the development of the neurobiological substrates thought to subserve the emergence of episodic memory. Distinct regions, layers and cells of the hippocampal formation exhibit different profiles of structural and molecular development during early postnatal life. The protracted period of neuronal addition and maturation in the dentate gyrus is accompanied by the late maturation of specific layers in different hippocampal regions that are located downstream from the dentate gyrus, particularly CA3. In contrast, distinct layers in several hippocampal regions, particularly CA1, which receive direct projections from the entorhinal cortex, exhibit an early maturation. In addition, hippocampal regions that are more highly interconnected with subcortical structures, including the subiculum, presubiculum, parasubiculum and CA2, mature even earlier. These findings, together with our studies of the development of human spatial memory, support the hypothesis that the differential maturation of distinct hippocampal circuits might underlie the differential emergence of specific "hippocampus-dependent" memory processes, culminating in the emergence of episodic memory concomitant with the maturation of all hippocampal circuits.

The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development.

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3-deficient mice show several hallmarks of ciliopathies including left-right asymmetry defects and hydrocephalus. Here we show that Rfx3-deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.

Changes in D-serine levels and localization during postnatal development of the rat vestibular nuclei.

The patterns of development of the vestibular nuclei (VN) and their main connections involving glutamate neurotransmission offer a good model for studying the function of the glial-derived neuromodulator D-serine in synaptic plasticity. In this study we show that D-serine is present in the VN and we analyzed its distribution and the levels of expression of serine racemase and D-amino acid oxidase (D-AAO) at different stages of postnatal (P) development. From birth to P21, high levels of D-serine were detected in glial cells and processes in all parts of the VN. This period corresponded to high expression of serine racemase and low expression of D-AAO. On the other hand, in the mature VN D-serine displayed very low levels and was mainly localized in neuronal cell bodies and dendrites. This drop of D-serine in adult stages corresponded to an increasing expression of D-AAO at mature stages. High levels of glial D-serine during the first 3 weeks of postnatal development correspond to an intense period of plasticity and synaptogenesis and maturation of VN afferents, suggesting that D-serine could be involved in these phenomena. These results demonstrate for the first time that changes in D-serine levels and distribution occur during postnatal development in the central nervous system. The strong decrease of D-serine levels and the glial-to-neuronal switch suggests that D-serine may have distinct functional roles depending on the developmental stage of the vestibular network.

Brain Spectrins 240/235 and 240/235E: Differential Expression During Development of Chicken Dorsal Root Ganglia in vivo and in vitro.

Brain spectrin, a membrane-related cytoskeletal protein, exists as two isoforms. Brain spectrin 240/235 is localized preferentially in the perikaryon and axon of neuronal cells and brain spectrin 240/235E is found essentially in the neuronal soma and dendrites and in glia (Riederer et al., 1986, J. Cell Biol., 102, 2088 - 2097). The sensory neurons in dorsal root ganglia, devoid of any dendrites, make a good tool to investigate such differential expression of spectrin isoforms. In this study expression and localization of both brain spectrin isoforms were analysed during early chicken dorsal root ganglia development in vivo and in culture. Both isoforms appeared at embryonic day 6. Brain spectrin 240/235 exhibited a transient increase during embryonic development and was first expressed in ventrolateral neurons. In ganglion cells in situ and in culture this spectrin type showed a somato - axonal distribution pattern. In contrast, brain spectrin 240/235E slightly increased between E6 and E15 and remained practically unchanged. It was localized mainly in smaller neurons of the mediodorsal area as punctate staining in the cytoplasm, was restricted exclusively to the ganglion cell perikarya and was absent from axons both in situ and in culture. This study suggests that brain spectrin 240/235 may contribute towards outgrowth, elongation and maintenance of axonal processes and that brain spectrin 240/235E seems to be exclusively involved in the stabilization of the cytoarchitecture of cell bodies in a selected population of ganglion cells.

Transient neuronal populations are required to guide callosal axons: a role for semaphorin 3C.

The corpus callosum (CC) is the main pathway responsible for interhemispheric communication. CC agenesis is associated with numerous human pathologies, suggesting that a range of developmental defects can result in abnormalities in this structure. Midline glial cells are known to play a role in CC development, but we here show that two transient populations of midline neurons also make major contributions to the formation of this commissure. We report that these two neuronal populations enter the CC midline prior to the arrival of callosal pioneer axons. Using a combination of mutant analysis and in vitro assays, we demonstrate that CC neurons are necessary for normal callosal axon navigation. They exert an attractive influence on callosal axons, in part via Semaphorin 3C and its receptor Neuropilin-1. By revealing a novel and essential role for these neuronal populations in the pathfinding of a major cerebral commissure, our study brings new perspectives to pathophysiological mechanisms altering CC formation.

Notch1 is required for neuronal and glial differentiation in the cerebellum.

The mechanisms that guide progenitor cell fate and differentiation in the vertebrate central nervous system (CNS) are poorly understood. Gain-of-function experiments suggest that Notch signaling is involved in the early stages of mammalian neurogenesis. On the basis of the expression of Notch1 by putative progenitor cells of the vertebrate CNS, we have addressed directly the role of Notch1 in the development of the mammalian brain. Using conditional gene ablation, we show that loss of Notch1 results in premature onset of neurogenesis by neuroepithelial cells of the midbrain-hindbrain region of the neural tube. Notch1-deficient cells do not complete differentiation but are eliminated by apoptosis, resulting in a reduced number of neurons in the adult cerebellum. We have also analyzed the effects of Notch1 ablation on gliogenesis in vivo. Our results show that Notch1 is required for both neuron and glia formation and modulates the onset of neurogenesis within the cerebellar neuroepithelium.

Mutations in tubulin genes are frequent causes of various foetal malformations of cortical development including microlissencephaly.

Complex cortical malformations associated with mutations in tubulin genes are commonly referred to as "Tubulinopathies". To further characterize the mutation frequency and phenotypes associated with tubulin mutations, we studied a cohort of 60 foetal cases. Twenty-six tubulin mutations were identified, of which TUBA1A mutations were the most prevalent (19 cases), followed by TUBB2B (6 cases) and TUBB3 (one case). Three subtypes clearly emerged. The most frequent (n = 13) was microlissencephaly with corpus callosum agenesis, severely hypoplastic brainstem and cerebellum. The cortical plate was either absent (6/13), with a 2-3 layered pattern (5/13) or less frequently thickened (2/13), often associated with neuroglial overmigration (4/13). All cases had voluminous germinal zones and ganglionic eminences. The second subtype was lissencephaly (n = 7), either classical (4/7) or associated with cerebellar hypoplasia (3/7) with corpus callosum agenesis (6/7). All foetuses with lissencephaly and cerebellar hypoplasia carried distinct TUBA1A mutations, while those with classical lissencephaly harbored recurrent mutations in TUBA1A (3 cases) or TUBB2B (1 case). The third group was polymicrogyria-like cortical dysplasia (n = 6), consisting of asymmetric multifocal or generalized polymicrogyria with inconstant corpus callosum agenesis (4/6) and hypoplastic brainstem and cerebellum (3/6). Polymicrogyria was either unlayered or 4-layered with neuronal heterotopias (5/6) and occasional focal neuroglial overmigration (2/6). Three had TUBA1A mutations and 3 TUBB2B mutations. Foetal TUBA1A tubulinopathies most often consist in microlissencephaly or classical lissencephaly with corpus callosum agenesis, but polymicrogyria may also occur. Conversely, TUBB2B mutations are responsible for either polymicrogyria (4/6) or microlissencephaly (2/6).

Characterization of the neuronal microtubule regulator SCG10 in transfected cells and " in vivo "

SUMMARY The ability of neuronal processes to find their way along complex paths and to establish appropriate connections depends on continual rearrangements of the cytoskeletal components. The regulation of microtubules plays an important role for morphological changes underlying nevrite outgrowth, axonal elongation, and growth cone steering. SCG10 (superior cervical ganglion clone 10) is a neuronal growthassociated protein developmentally regulated and highly enriched in the neuronal growth cones. SCG10 presents a microtubule destabilizing activity that could participate to the regulation of microtubule dynamics and thus explain microtubule behaviors in the growth cone during axonal elongation and turning. It is here suggested that a tight control of the opposite effects on microtubules of SCG10 and the stabilizing microtubule-associated protein MAP1B allows a fine tuning of cytoskeletal rearrangement and may provide the required microtubule dynamic instability to promote axonal growth. Moreover, antibodyblockade of SCG10 function, that leads to growth cone pauses similar as those triggered by the guidance molecule EphB, and the modulation of SCG10 activity by the Rho GTPase Rnd1 suggest a potential role for SCG10 in the signal transduction pathways of extracellular guidance cues. The identification of the active zone protein Bassoon as a potential interaction partner for the SCG10-related protein NPC2, using atomic force microscopy as well as COS-7 and neuronal cell cultures, also gives new insights for a role of this protein family into the processes of synapse genesis or plasticity. Finally, SCG10 mutant mice generated by gene targeting and expressing a soluble form of the protein have been characterized during early postnatal development and in the adulthood. Due to the deletion of its membrane binding domain, SCG10 specific subcellular targeting to growth cones is compromised and results in impairments of motor and coordination development. Further histological analysis in the sciatic nerve reveal that these symptoms are associated with neurodegenerative signs. RESUME Une navigation correcte des prolongements cellulaires neuronaux leur permettant de former des connections appropriées repose sur de continuels réarrangements des constituants de leur cytosquelette. La régulation des microtubules joue notamment un rôle important dans les changements morphologiques qui accompagnent la croissance axonale et les réorientations du cône de croissance. SCG10 (superior cervical ganglion clone 10) est une protéine étroitement associée à la croissance neuronale, hautement régulée durant le développement et abondante au niveau du cône de croissance. SCG10 présente une activité déstabilisatrice sur les microtubules qui pourrait permettre une régulation des paramètres dynamiques propres aux microtubules et ainsi expliquer leur comportement durant la navigation du cône de croissance. Il est ici proposé qu'un contrôle précis des effets opposés de SCG10 et d'une autre protéine stabilisante associée aux microtubules (MAP1 B) permette un réglage fin des réarrangements du cytosquelette et puisse ainsi produire l'instabilité dynamique nécessaire à la croissance anale. Par ailleurs, le blocage de la fonction de SCG10 par un anticorps spécifique, conduisant à des pauses du cônes de croissance similaires à celles provoquées par la molécule de guidage EphB, ainsi que la modulation de l'activité de SCG10 par la Rho GTPase Rnd1 suggèrent une potentielle implication de SCG10 dans les voies de transduction des signaux provenant de molécules de guidage extracellulaires. L'identification d'une interaction de la protéine synaptique Bassoon avec la protéine NPC2 apparentée à SCG10, au moyen de la microscopie à force atomique et dans des cultures de cellules neuronales et COS-7, ouvre des perspectives concernant ces protéines dans la formation et la plasticité synaptiques. Finalement, des souris mutantes pour SCG10 produites par ciblage de gène et exprimant une forme soluble de la protéine ont été caractérisées durant la phase précoce du développement et à l'âge adulte. La délétion du domaine permettant l'ancrage de SCG10 aux membranes compromet sa sub-localisation au niveau du cône de croissance et résulte en l'apparition de troubles moteurs et de la coordination. Des analyses histologiques complémentaires au niveau du nerf sciatique montrent que ces symptômes sont associés avec des signes neurodégénératifs.

Regulation of dendritic development by neurotrophic factors

AbstractEstablishment of a functional nervous system occurs through an orchestrated multistep process during embryogenesis. As dendrites are the primary sites of synaptic connections, development of dendritic arborization is essential for the formation of functional neural circuits. Maturation of dendritic arbor occurs through dynamic processes that are regulated by intrinsic genetic factors and external signals, such as environmental stimuli, neuronal activity and growth factors. Among the latter, the neurotrophic factor BDNF is a key regulator of dendritic growth. However, the mechanisms by which BDNF controls dendritic development remain elusive.In this study, we first showed that activation of the MAPK signaling pathway and phosphorylation of the transcription factor CREB are required to mediate the effects of BDNF on dendritic development of cortical neurons. However, phosphorylation of CREB alone is not sufficient to induce dendritic growth in response to BDNF. Thus, by using a mutant form of CREB unable to bind its coactivator CRTC1, we demonstrated that BDNF-induced dendritic elaboration requires the functional interaction between CREB and CRTC1. Consistent with these observations, inhibition of CRTC1 expression by shRNA-mediated knockdown was found to suppress the effects of BDNF on dendritic length and branching of cortical neurons.The nuclear translocation of CRTC1, a step necessary for the interaction between CREB and CRTC1, was shown to result from the activation of NMD A receptors by glutamate, leading to the dephosphorylation of CRTC1 by the protein phosphatase calcineurin. In line with these findings, prevention of CRTC1 nuclear translocation in the absence of glutamate, or by inhibiting NMDA receptors or calcineurin suppressed the promotion of dendritic growth by BDNF.Increasing evidence supports a role for the growth factor HGF in the regulation of dendritic morphology during brain development. Despite these observations, little is known about the cellular mechanisms underlying the effects of HGF on dendritic elaboration of cortical neurons. The second part of this study was aimed at elucidating the cellular processes that mediate the effects of HGF on dendritic differentiation. We found that HGF increases cortical dendritic growth through mechanisms that involve MAPK-dependent phosphorylation of CREB, and interaction of CREB with its coactivator CRTC1. These data indicate that the mechanisms underlying the promotion of dendritic growth by HGF are similar to those that mediate the effects of BDNF, suggesting that the role of CREB and CRTC1 in the regulation of dendritic development may not be limited to HGF and BDNF, but may extend to other neurotrophic factors that control dendritic differentiation.Together, these results identify a previously unrecognized mechanism by which CREB and its coactivator CRTC1 mediate the effects of BDNF and HGF on dendritic growth of cortical neurons. Moreover, these data highlight the important role of the cooperation between BDNF/HGF and glutamate that converges on CREB to stimulate the expression of genes that contribute to the development of dendritic arborization.RésuméL'établissement d'un système nerveux fonctionnel s'accomplit grâce à des mécanismes précis, orchestrés en plusieurs étapes au cours de l'embryogenèse. Les dendrites étant les principaux sites de connexions synaptiques, le développement de l'arborisation dendritique est essentiel à la formation de circuits neuronaux fonctionnels. La maturation de l'arbre dendritique s'effectue grâce à des processus dynamiques qui sont régulés par des facteurs génétiques intrinsèques ainsi que par des facteurs externes tels que les stimuli environnementaux, l'activité neuronale ou les facteurs de croissance. Parmi ces derniers, le facteur neurotrophique BDNF est - connu pour être un régulateur clé de la croissance dendritique. Cependant, les mécanismes par lesquels BDNF contrôle le développement dendritique demeurent mal connus.Au cours de cette étude, nous avons montré dans un premier temps que l'activation de la voie de signalisation de la MAPK et la phosphorylation du facteur de transcription CREB sont nécessaires aux effets du BDNF sur le développement dendritique des neurones corticaux. Toutefois, la phosphorylation de CREB en tant que telle n'est pas sûffisante pour permettre la pousse des dendrites en réponse au BDNF. Ainsi, en utilisant une forme mutée de CREB incapable de se lier à son coactivateur CRTC1, nous avons démontré que l'élaboration des dendrites induite par le BDNF nécessite également une interaction fonctionnelle entre CREB et CRTC1. Ces résultats ont été confirmés par d'autres expériences qui ont montré que l'inhibition de l'expression de CRTC1 par l'intermédiaire de shRNA supprime les effets du BDNF sur la longueur et le branchement dendritique des neurones corticaux.Les résultats obtenus au cours de ce travail montrent également que la translocation nucléaire de CRTC1, qui est une étape nécessaire à l'interaction entre CREB et CRTC1, résulte de l'activation des récepteurs NMDA par le glutamate, entraînant la déphosphorylation de CRTC1 par la protéine phosphatase calcineurine. De plus, le blocage de la translocation nucléaire de CRTC1 en absence de glutamate, ou suite à l'inhibition des récepteurs NMDA ou de la calcineurine, supprime complètement la pousse des dendrites induite par le BDNF.De nombreuses d'évidences indiquent que le facteur de croissance HGF joue également un rôle important dans la régulation de la morphologie dendritique au cours du développement cérébral. Malgré ces observations, peu d'éléments sont connus quant aux mécanismes cellulaires qui sous-tendent les effets du HGF sur la croissance dendritique des neurones corticaux. Le but de la seconde partie de cette étude a eu pour but d'élucider les processus cellulaires responsables des effets du HGF sur la différenciation dendritique des neurones corticaux. Au cours de ces expériences, nous avons pu mettre en évidence que le HGF induit la pousse dendritique par des mécanismes qui impliquent la phosphorylation de CREB par la MAPK, et l'interaction de CREB avec son coactivateur CRTC1. Ces données indiquent que les mécanismes impliqués dans la stimulation de la croissance dendritique par le HGF sont similaires à ceux régulant les effets du BDNF, ce qui suggère que le rôle de CREB et de CRTC1 dans la régulation du développement dendritique n'est vraisemblablement pas limité aux effets du HGF ou du BDNF, mais pourrait s'étendre à d'autres facteurs neurotrophiques qui contrôlent la différenciation dendritique.En conclusion, ces résultats ont permis l'identification d'un nouveau mécanisme par lequel CREB et son coactivateur CRTC1 transmettent les effets du BDNF et du HGF sur la croissance dendritique de neurones corticaux. Ces observations mettent également en évidence le rôle important joué par la coopération entre BDNF/HGF et le glutamate, dans l'activation de CREB ainsi que dans l'expression de gènes qui participent au développement de l'arborisation dendritique des neurones corticaux.

Effects of trimethyltin (TMT) on glial and neuronal cells in aggregate cultures: dependence on the developmental stage.

Long-term effects of trimethyltin (TMT) applied at concentrations below the cytotoxic level were examined in three-dimensional cell cultures of fetal rat telencephalon using biochemical, immunochemical and morphological criteria. It was found that in immature cultures low concentrations of TMT (10(-8) M) specifically induced a gliotic response in astrocytes, with increased immunoreactivity for glial fibrillary acidic protein, and a greater number of astrocytic processes. Significant changes in oligodendrocytic and neuronal parameters were found only at 10(-6) M of TMT. In differentiated cultures, distinct changes in cell type-specific parameters occurred at 10(-6) M of TMT (the lowest effective concentration). In addition, different patterns of responses were found for astrocytes and oligodendrocytes, as compared to immature cultures. These results suggest that among neural cells, astroblasts are most sensitive to TMT, and that the glial responses to this neurotoxicant are development-dependent.

Microtubule-associated protein 1a is involved in the early development of the rat spinal cord.

The expression of microtubule-associated protein 1a (MAP1a) in the developing rat spinal cord was studied using the monoclonal antibody BW6. Immunoblots of microtubule preparations revealed the presence of MAP1a in spinal cord tissue of rats aged embryonal day 16 and postnatal day 0. The spinal cord matrix layer, between embryonal days 12-17, displayed a pattern of MAP1a-positive processes, horizontally oriented in between the membrane limitans interna and externa. The mantle layer stained intensely for MAP1a between embryonal day 12 and postnatal day 2. MAP1a was found in neuronal cell bodies, axons and dendrites, located mainly in the ventral and intermediate mantle layer. In the marginal layer, MAP1a-positive axons could be observed between embryonal days 14-18. During further development, the intensity of the MAP1a staining in the spinal columns gradually decreased. These expression patterns indicate an involvement of MAP1a in the proliferation and differentiation of neuroblasts, and the maturation of the long spinal fiber systems, i.e. early events in spinal cord development

Identification of neuronal network properties from the spectral analysis of calcium imaging signals in neuronal cultures

Neuronal networks in vitro are prominent systems to study the development of connections in living neuronal networks and the interplay between connectivity, activity and function. These cultured networks show a rich spontaneous activity that evolves concurrently with the connectivity of the underlying network. In this work we monitor the development of neuronal cultures, and record their activity using calcium fluorescence imaging. We use spectral analysis to characterize global dynamical and structural traits of the neuronal cultures. We first observe that the power spectrum can be used as a signature of the state of the network, for instance when inhibition is active or silent, as well as a measure of the network's connectivity strength. Second, the power spectrum identifies prominent developmental changes in the network such as GABAA switch. And third, the analysis of the spatial distribution of the spectral density, in experiments with a controlled disintegration of the network through CNQX, an AMPA-glutamate receptor antagonist in excitatory neurons, reveals the existence of communities of strongly connected, highly active neurons that display synchronous oscillations. Our work illustrates the interest of spectral analysis for the study of in vitro networks, and its potential use as a network-state indicator, for instance to compare healthy and diseased neuronal networks.