Thalamocortical axons control the cytoarchitecture of neocortical layers by area-specific supply of VGF.

Neuronal abundance and thickness of each cortical layer are specific to each area, but how this fundamental feature arises during development remains poorly understood. While some of area-specific features are controlled by intrinsic cues such as morphogens and transcription factors, the exact influence and mechanisms of action by cues extrinsic to the cortex, in particular the thalamic axons, have not been fully established. Here, we identify a thalamus-derived factor, VGF, which is indispensable for thalamocortical axons to maintain the proper amount of layer 4 neurons in the mouse sensory cortices. This process is prerequisite for further maturation of the primary somatosensory area, such as barrel field formation instructed by a neuronal activity-dependent mechanism. Our results provide an actual case in which highly site-specific axon projection confers further regional complexity upon the target field through locally secreting signaling molecules from axon terminals.

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures.

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures. This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

Nucleocytoplasmic Shuttling of Histone Deacetylase 9 Controls Activity-Dependent Thalamocortical Axon Branching.

During development, thalamocortical (TC) axons form branches in an activity-dependent fashion. Here we investigated how neuronal activity is converted to molecular signals, focusing on an epigenetic mechanism involving histone deacetylases (HDACs). Immunohistochemistry demonstrated that HDAC9 was translocated from the nucleus to the cytoplasm of thalamic cells during the first postnatal week in rats. In organotypic co-cultures of the thalamus and cortex, fluorescent protein-tagged HDAC9 also exhibited nuclueocytoplasmic translocation in thalamic cells during culturing, which was reversed by tetrodotoxin treatment. Transfection with a mutant HDAC9 that interferes with the translocation markedly decreased TC axon branching in the culture. Similarly, TC axon branching was significantly decreased by the mutant HDAC9 gene transfer in vivo. However, axonal branching was restored by disrupting the interaction between HDAC9 and myocyte-specific enhancer factor 2 (MEF2). Taken together, the present results demonstrate that the nucleocytoplasmic translocation of HDAC9 plays a critical role in activity-dependent TC axon branching by affecting transcriptional regulation and downstream signaling pathways.

Netrin-4 regulates thalamocortical axon branching in an activity-dependent fashion.

Axon branching is remodeled by sensory-evoked and spontaneous neuronal activity. However, the underlying molecular mechanism is largely unknown. Here, we demonstrate that the netrin family member netrin-4 (NTN4) contributes to activity-dependent thalamocortical (TC) axon branching. In the postnatal developmental stages of rodents, ntn4 expression was abundant in and around the TC recipient layers of sensory cortices. Neuronal activity dramatically altered the ntn4 expression level in the cortex in vitro and in vivo. TC axon branching was promoted by exogenous NTN4 and suppressed by depletion of the endogenous protein. Moreover, unc-5 homolog B (Unc5B), which strongly bound to NTN4, was expressed in the sensory thalamus, and knockdown of Unc5B in thalamic cells markedly reduced TC axon branching. These results suggest that NTN4 acts as a positive regulator for TC axon branching through activity-dependent expression.

Regulation of thalamocortical axon branching by BDNF and synaptic vesicle cycling.

During development, axons form branches in response to extracellular molecules. Little is known about the underlying molecular mechanisms. Here, we investigate how neurotrophin-induced axon branching is related to synaptic vesicle cycling for thalamocortical axons. The exogenous application of brain-derived neurotrophic factor (BDNF) markedly increased axon branching in thalamocortical co-cultures, while removal of endogenous BDNF reduced branching. Over-expression of a C-terminal fragment of AP180 that inhibits clathrin-mediated endocytosis affected the laminar distribution and the number of branch points. A dominant-negative synaptotagmin mutant that selectively targets synaptic vesicle cycling, strongly suppressed axon branching. Moreover, axons expressing the mutant synaptotagmin were resistant to the branch-promoting effect of BDNF. These results suggest that synaptic vesicle cycling might regulate BDNF induced branching during the development of the axonal arbor.

Thalamus-derived molecules promote survival and dendritic growth of developing cortical neurons.

The mammalian neocortex is composed of various types of neurons that reflect its laminar and area structures. It has been suggested that not only intrinsic but also afferent-derived extrinsic factors are involved in neuronal differentiation during development. However, the role and molecular mechanism of such extrinsic factors are almost unknown. Here, we attempted to identify molecules that are expressed in the thalamus and affect cortical cell development. First, thalamus-specific molecules were sought by comparing gene expression profiles of the developing rat thalamus and cortex using microarrays, and by constructing a thalamus-enriched subtraction cDNA library. A systematic screening by in situ hybridization showed that several genes encoding extracellular molecules were strongly expressed in sensory thalamic nuclei. Exogenous and endogenous protein localization further demonstrated that two extracellular molecules, Neuritin-1 (NRN1) and VGF, were transported to thalamic axon terminals. Application of NRN1 and VGF to dissociated cell culture promoted the dendritic growth. An organotypic slice culture experiment further showed that the number of primary dendrites in multipolar stellate neurons increased in response to NRN1 and VGF, whereas dendritic growth of pyramidal neurons was not promoted. These molecules also increased neuronal survival of multipolar neurons. Taken together, these results suggest that the thalamus-specific molecules NRN1 and VGF play an important role in the dendritic growth and survival of cortical neurons in a cell type-specific manner.

Deficiency of the microglial receptor CX3CR1 impairs postnatal functional development of thalamocortical synapses in the barrel cortex.

Accumulative evidence indicates that microglial cells influence the normal development of brain synapses. Yet, the mechanisms by which these immune cells target maturating synapses and influence their functional development at early postnatal stages remain poorly understood. Here, we analyzed the role of CX3CR1, a microglial receptor activated by the neuronal chemokine CX3CL1 (or fractalkine) which controls key functions of microglial cells. In the whisker-related barrel field of the mouse somatosensory cortex, we show that the recruitment of microglia to the sites where developing thalamocortical synapses are concentrated (i.e., the barrel centers) occurs only after postnatal day 5 and is controlled by the fractalkine/CX3CR1 signaling pathway. Indeed, at this developmental stage fractalkine is overexpressed within the barrels and CX3CR1 deficiency delays microglial cell recruitment into the barrel centers. Functional analysis of thalamocortical synapses shows that CX3CR1 deficiency also delays the functional maturation of postsynaptic glutamate receptors which normally occurs at these synapses between the first and second postnatal week. These results show that reciprocal interactions between neurons and microglial cells control the functional maturation of cortical synapses.

The action of Semaphorin7A on thalamocortical axon branching.

Developing axons form extensive branches to make synaptic contacts with their target cells. Despite the important role of axon branching in neural circuit formation, its underlying molecular mechanism is still largely unknown. In this study, we investigated the involvement of Semaphorin7A (Sema7A) in thalamocortical (TC) axon branching. In situ hybridization demonstrated that sema7a was expressed specifically in layer 4, the TC recipient layer, when TC axons form extensive arbors. A similar protein expression pattern was observed by immunohistochemistry with an anti-Sema7A antibody. The effect of Sema7A on axon branching was investigated in dissociated cell cultures from embryonic rat thalamus. TC axon branching increased dramatically on Sema7A-coated dishes. We further studied the activity of Sema7A in vivo using loss- and gain-of-function analyses. The number of vesicular glutamate transporter 2-positive puncta was markedly reduced in the Sema7A-deficient cortex. In contrast, their number increased significantly when Sema7A was over-expressed in layer 4 cells by in utero electroporation. Taken together, these findings suggest that Sema7A acts as a positive regulator for TC axon branching and/or pre-synaptic puncta formation.

Laminar and areal expression of unc5d and its role in cortical cell survival.

The UNC-5 family of netrin receptors is known to regulate axon guidance, cell migration, and cell survival. We have previously demonstrated that unc5d, one of the UNC-5 family member genes, is specifically expressed in layer 4 of the developing rat neocortex (Zhong Y, Takemoto M, Fukuda T, Hattori Y, Murakami F, Nakajima D, Nakayama M, Yamamoto N. 2004. Identification of the genes that are expressed in the upper layers of the neocortex. Cereb Cortex. 14:1144-1152). However, the role of UNC5D in cortical development is still unknown. In this study, we revealed that unc5d was highly expressed in the primary sensory areas of the mouse neocortex at around postnatal day 7. Netrin-4 was also found to be predominantly expressed in layer 4 of the sensory cortex and sensory thalamic nuclei. Cell surface binding assay showed that netrin-4 protein bound to UNC5D-expressing cells. An in vitro study further demonstrated that cell death of unc5d-expressing layer 4 cells was reduced by exogenous application of netrin-4 protein, whereas UNC5D is not sufficient to mediate the effect of netrin-4 in deep layer cells. Taken together, these results suggest that UNC5D is primarily expressed by layer 4 cells in the primary sensory areas of the developing neocortex and may mediate the effect of netrin-4 on cortical cell survival in a lamina-specific manner.

Role of pre- and postsynaptic activity in thalamocortical axon branching.

Axonal branching is thought to be regulated not only by genetically defined programs but also by neural activity in the developing nervous system. Here we investigated the role of pre- and postsynaptic activity in axon branching in the thalamocortical (TC) projection using organotypic coculture preparations of the thalamus and cortex. Individual TC axons were labeled with enhanced yellow fluorescent protein by transfection into thalamic neurons. To manipulate firing activity, a vector encoding an inward rectifying potassium channel (Kir2.1) was introduced into either thalamic or cortical cells. Firing activity was monitored with multielectrode dishes during culturing. We found that axon branching was markedly suppressed in Kir2.1-overexpressing thalamic cells, in which neural activity was silenced. Similar suppression of TC axon branching was also found when cortical cell activity was reduced by expressing Kir2.1. These results indicate that both pre- and postsynaptic activity is required for TC axon branching during development.

Activity-dependent thalamocortical axon branching.

The thalamocortical (TC) projection in the mammalian brain involves fundamental aspects in branch formation during development. TC axons are known to form branches not only in a genetically defined but also in an activity-dependent fashion. Recent evidence indicates that TC axon branching is generated by positive and negative regulators that are expressed with laminar specificity in the developing cortex. Moreover, in vitro studies using organotypic cocultures demonstrate that neural activity, including firing and synaptic activity, controls lamina-specific TC axon branching by altering its remodeling process with addition and elimination. Taken together, activity-dependent mechanisms can contribute to branch formation, affecting expression of branch-promoting and inhibiting factors and/or their receptor molecules.

[Molecular mechanisms of thalamocortical circuit formation].

Thalamocortical (TC) projection is one of the major neural circuitries in the brain. TC projection has characteristic aspects of cortical area and laminar specificities, and provides a suitable model system to investigate the developmental mechanisms of neural circuit formation in the mammalian brain including human beings. Recent studies with genetic, molecular and cellular biological approaches reveal area and lamina-specific gene expressions in the developing cortex, regulation mechanisms of TC axon growth and branching by these molecules, and activity-dependence of the mechanisms.

Cooperative activity of multiple upper layer proteins for thalamocortical axon growth.

During development, sensory thalamocortical (TC) axons grow into the neocortex and terminate primarily in layer 4. To study the molecular mechanism that underlies lamina-specific TC axon termination, we investigated the responsiveness of TC axons to ephrin-A5, semaphorin-7A (Sema7A) and kit ligand (KL), which are expressed in the upper layers of the developing cortex. Dissociated cells of the dorsal thalamus from embryonic rat brain were cultured on dishes that were coated with preclustered Fc-tagged extracellular domains of these molecules. Each protein was found to promote TC axon growth in a dose-dependent fashion of a bell-shaped curve. Any combination of the three proteins showed a cooperative effect in lower concentrations but not in higher concentrations, suggesting that their growth-promoting activities act in a common pathway. The effect of spatial distributions of these proteins was further tested on a filter membrane, in which these proteins were printed at a size that recapitulates the scale of laminar thickness in vivo, using a novel protein-printing technique, Simple-To-mAke Micropore Protein-Printing (STAMP2) method. The results demonstrated that TC axons grew massively on the laminin-coated region but were prevented from invading the adjacent ephrin-A5-printed region, suggesting that TC axons detect relative differences in the growth effect between these regions. Moreover, the inhibitory action of ephrin-A5 was enhanced by copresence with KL and Sema7A. Together, these results suggest that the lamina-specific TC axon targeting mechanism involves growth-inhibitory activity by multiple molecules in the upper layers and detection in the molecular environments between the upper and deep layers.

Interplay between laminar specificity and activity-dependent mechanisms of thalamocortical axon branching.

Target and activity-dependent mechanisms of axonal branching were studied in the thalamocortical (TC) projection using organotypic cocultures of the thalamus and cortex. TC axons were labeled with enhanced yellow fluorescent protein (EYFP) by a single-cell electroporation method and observed over time by confocal microscopy. Changes in the firing activity of cocultures grown on multielectrode dishes were also monitored over time. EYFP-labeled TC axons exhibited more branch formation in and around layer 4 of the cortical explant during the second week in vitro, when spontaneous firing activity increased in both thalamic and cortical cells. Time-lapse imaging further demonstrated that branching patterns were generated dynamically by addition and elimination with a bias toward branch accumulation in the target layer. To examine the relationship between neural activity and TC branch formation, the dynamics of axonal branching was analyzed under various pharmacological treatments. Chronic blockade of firing or synaptic activity reduced the remodeling process, in particular, branch addition in the target layer. However, extension of branches was not affected by this treatment. Together, these findings suggest that neural activity can modify the molecular mechanisms that regulate lamina-specific TC axon branching.

Molecular mechanisms of thalamocortical axon targeting.

The thalamocortical (TC) projection in the mammalian brain is a well characterized system in terms of laminar specificity of neocortical circuits. To understand the mechanisms that underlie lamina-specific TC axon targeting, we studied the role of extracellular and cell surface molecules that are expressed in the upper layers of the developing cortex in in vitro culture techniques. The results demonstrated that multiple upper layer molecules co-operated to produce stop behaviour of TC axons in the target layer. Activity dependency of TC axon branching was also investigated in organotypic co-cultures of the thalamus and cortex. TC axon branches were formed dynamically by addition and elimination during the second week in vitro, when spontaneous firing increased in thalamic and cortical cells. Pharmacological blockade of firing or synaptic activity reduced the remodelling process, in particular branch addition, in the target layer. Together, these findings suggest that TC axon targeting mechanisms involve the regulation with multiple lamina-specific molecules and modification of the molecular mechanisms via neural activity.

Formation of the thalamocortical projection regulated differentially by BDNF- and NT-3-mediated signaling.

During development thalamocortical (TC) axons establish lamina-specific connections with cortical cells, and in later developmental stages TC projections are modified by activity-dependent processes. Recent studies have demonstrated that brain-derived neurotrophic factor and neurotrophin-3 are expressed in the cortex with distinct developmental time courses, and are involved not only in the formation of the TC projection but also in the subsequent refinement processes. Evidence further suggests that these actions of neurotrophins are achieved in cooperation with membrane-associated molecules expressed in cortical cells.

BDNF and NT-3 promote thalamocortical axon growth with distinct substrate and temporal dependency.

The role of neurotrophins in thalamic axon growth was studied by culturing embryonic rat thalamus on collagen-coated substrate or fixed cortical slices in the presence of either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). Both BDNF and NT-3 promoted axonal growth, but the axonal growth-promoting activity depended on culture substrates. Axonal growth on collagen-coated membrane was accelerated by BDNF, but not by NT-3. In contrast, axonal outgrowth on fixed cortex was significantly enhanced by NT-3, but not by BDNF. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of cultured thalamic cells demonstrated that culture substrates did not alter the expression of their receptors, trkB and trkC. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining further demonstrated that axonal growth promoted by neurotrophins was not due to reduction of cell death. Measurement of the developmental changes in BDNF and NT-3 levels revealed that, in contrast to the rapid elevation of BDNF after the arrival of thalamocortical axons to their target layer, the regulation of NT-3 protein accompanies the phase of their outgrowth in neocortex. These findings suggest that BDNF and NT-3 promote thalamic axon growth in different manners in terms of substrate dependency and developmental stage.

Development of functional thalamocortical synapses studied with current source-density analysis in whole forebrain slices in the rat.

We analysed the laminar distribution of transmembrane currents from embryonic (E) day 17 until adulthood after selective thalamic stimulation in slices of rat forebrain to study the development of functional thalamocortical and cortico-cortical connections. At E18 to birth a short-latency current sink was observed in the subplate and layer 6, which was decreased, but not fully abolished in a cobalt containing solution or after the application of glutamate receptor blockers (APV and DNQX). This indicated that embryonic thalamic axons were capable of conducting action potentials to the cortex and some of them had already formed functional synapses there. Between birth and P3, when thalamic axons were completing their upward growth, a sink gradually appeared more superficially in the dense cortical plate and synchronously, a current source aroused in layer 5. Both sinks and sources completely disappeared after blocking synaptic transmission. The adult-like distribution of CSDs became apparent after P7. The component in layer 6 cannot be blocked completely after this age suggesting antidromic activation. This study demonstrated that cells of the lowest layers of the cortex received functional thalamic input before birth and that thalamocortical axons formed synapses with more superficial cells as they grew into the cortical plate.