Involvement of Denervated Midbrain-Derived Factors in the Formation of Ectopic Cortico-Mesencephalic Projection after Hemispherectomy.

Neuronal remodeling after brain injury is essential for functional recovery. After unilateral cortical lesion, axons from the intact cortex ectopically project to the denervated midbrain, but the molecular mechanisms remain largely unknown. To address this issue, we examined gene expression profiles in denervated and intact mouse midbrains after hemispherectomy at early developmental stages using mice of either sex, when ectopic contralateral projection occurs robustly. The analysis showed that various axon growth-related genes were upregulated in the denervated midbrain, and most of these genes are reportedly expressed by glial cells. To identify the underlying molecules, the receptors for candidate upregulated molecules were knocked out in layer 5 projection neurons in the intact cortex, using the CRISPR/Cas9-mediated method, and axonal projection from the knocked-out cortical neurons was examined after hemispherectomy. We found that the ectopic projection was significantly reduced when integrin subunit ß three or neurotrophic receptor tyrosine kinase 2 (also known as TrkB) was knocked out. Overall, the present study suggests that denervated midbrain-derived glial factors contribute to lesion-induced remodeling of the cortico-mesencephalic projection via these receptors.SIGNIFICANCE STATEMENT After brain injury, compensatory neural circuits are established that contribute to functional recovery. However, little is known about the intrinsic mechanism that underlies the injury-induced remodeling. We found that after unilateral cortical ablation expression of axon-growth promoting factors is elevated in the denervated midbrain and is involved in the formation of ectopic axonal projection from the intact cortex. Evidence further demonstrated that these factors are expressed by astrocytes and microglia, which are activated in the denervated midbrain. Thus, our present study provides a new insight into the mechanism of lesion-induced axonal remodeling and further therapeutic strategies after brain injury.

Suppression of DNA Double-Strand Break Formation by DNA Polymerase ß in Active DNA Demethylation Is Required for Development of Hippocampal Pyramidal Neurons.

Genome stability is essential for brain development and function, as de novo mutations during neuronal development cause psychiatric disorders. However, the contribution of DNA repair to genome stability in neurons remains elusive. Here, we demonstrate that the base excision repair protein DNA polymerase ß (Polß) is involved in hippocampal pyramidal neuron differentiation via a TET-mediated active DNA demethylation during early postnatal stages using Nex-Cre/Polß fl/fl mice of either sex, in which forebrain postmitotic excitatory neurons lack Polß expression. Polß deficiency induced extensive DNA double-strand breaks (DSBs) in hippocampal pyramidal neurons, but not dentate gyrus granule cells, and to a lesser extent in neocortical neurons, during a period in which decreased levels of 5-methylcytosine and 5-hydroxymethylcytosine were observed in genomic DNA. Inhibition of the hydroxylation of 5-methylcytosine by expression of microRNAs miR-29a/b-1 diminished DSB formation. Conversely, its induction by TET1 catalytic domain overexpression increased DSBs in neocortical neurons. Furthermore, the damaged hippocampal neurons exhibited aberrant neuronal gene expression profiles and dendrite formation, but not apoptosis. Comprehensive behavioral analyses revealed impaired spatial reference memory and contextual fear memory in adulthood. Thus, Polß maintains genome stability in the active DNA demethylation that occurs during early postnatal neuronal development, thereby contributing to differentiation and subsequent learning and memory.SIGNIFICANCE STATEMENT Increasing evidence suggests that de novo mutations during neuronal development cause psychiatric disorders. However, strikingly little is known about how DNA repair is involved in neuronal differentiation. We found that Polß, a component of base excision repair, is required for differentiation of hippocampal pyramidal neurons in mice. Polß deficiency transiently led to increased DNA double-strand breaks, but not apoptosis, in early postnatal hippocampal pyramidal neurons. This aberrant double-strand break formation was attributed to active DNA demethylation as an epigenetic regulation. Furthermore, the damaged neurons exhibited aberrant gene expression profiles and dendrite formation, resulting in impaired learning and memory in adulthood. Thus, these findings provide new insight into the contribution of DNA repair to the neuronal genome in early brain development.

Dynamic erectile responses of a novel penile organ model utilizing TPEM†.

Male penis is required to become erect during copulation. In the upper (dorsal) part of penis, the erectile tissue termed corpus cavernosum (CC) plays fundamental roles for erection by regulating the inner blood flow. When blood flows into the CC, the microvascular complex termed sinusoidal space is reported to expand during erection. A novel in vitro explant system to analyze the dynamic erectile responses during contraction/relaxation is established. The current data show regulatory contraction/relaxation processes induced by phenylephrine (PE) and nitric oxide (NO) donor mimicking dynamic erectile responses by in vitro CC explants. Two-photon excitation microscopy (TPEM) observation shows the synchronous movement of sinusoidal space and the entire CC. By taking advantages of the CC explant system, tadalafil (Cialis) was shown to increase sinusoidal relaxation. Histopathological changes have been generally reported associating with erection in several pathological conditions. Various stressed statuses have been suggested to occur in the erectile responses by previous studies. The current CC explant model enables to analyze such conditions through directly manipulating CC in the repeated contraction/relaxation processes. Expression of oxidative stress marker and contraction-related genes, Hypoxia-inducible factor 1-alpha (Hif1a), glutathione peroxidase 1 (Gpx1), Ras homolog family member A (RhoA), and Rho-associated protein kinase (Rock), was significantly increased in such repeated contraction/relaxation. Altogether, it is suggested that the system is valuable for analyzing structural changes and physiological responses to several regulators in the field of penile medicine.

Rho Guanine Nucleotide Exchange Factors Regulate Horizontal Axon Branching of Cortical Upper Layer Neurons.

Axon branching is a crucial process for cortical circuit formation. However, how the cytoskeletal changes in axon branching are regulated is not fully understood. In the present study, we investigated the role of RhoA guanine nucleotide exchange factors (RhoA-GEFs) in branch formation of horizontally elongating axons (horizontal axons) in the mammalian cortex. In situ hybridization showed that more than half of all known RhoA-GEFs were expressed in the developing rat cortex. These RhoA-GEFs were mostly expressed in the macaque cortex as well. An overexpression study using organotypic cortical slice cultures demonstrated that several RhoA-GEFs strongly promoted horizontal axon branching. Moreover, branching patterns were different between overexpressed RhoA-GEFs. In particular, ARHGEF18 markedly increased terminal arbors, whereas active breakpoint cluster region-related protein (ABR) increased short branches in both distal and proximal regions of horizontal axons. Rho kinase inhibitor treatment completely suppressed the branch-promoting effect of ARHGEF18 overexpression, but only partially affected that of ABR, suggesting that these RhoA-GEFs employ distinct downstream pathways. Furthermore, knockdown of either ARHGEF18 or ABR considerably suppressed axon branching. Taken together, the present study revealed that subsets of RhoA-GEFs differentially promote axon branching of mammalian cortical neurons.

Genome Stability by DNA Polymerase ß in Neural Progenitors Contributes to Neuronal Differentiation in Cortical Development.

DNA repair is crucial for genome stability in the developing cortex, as somatic de novo mutations cause neurological disorders. However, how DNA repair contributes to neuronal development is largely unknown. To address this issue, we studied the spatiotemporal roles of DNA polymerase ß (Polß), a key enzyme in DNA base excision repair pathway, in the developing cortex using distinct forebrain-specific conditional knock-out mice, Emx1-Cre/Polß fl/fl and Nex-Cre/Polß fl/fl mice. Polß expression was absent in both neural progenitors and postmitotic neurons in Emx1-Cre/Polß fl/fl mice, whereas only postmitotic neurons lacked Polß expression in Nex-Cre/Polß fl/fl mice. We found that DNA double-strand breaks (DSBs) were frequently detected during replication in cortical progenitors of Emx1-Cre/Polß fl/fl mice. Increased DSBs remained in postmitotic cells, which resulted in p53-mediated neuronal apoptosis. This neuronal apoptosis caused thinning of the cortical plate, although laminar structure was normal. In addition, accumulated DSBs also affected growth of corticofugal axons but not commissural axons. These phenotypes were not observed in Nex-Cre/Polß fl/fl mice. Moreover, cultured Polß-deficient neural progenitors exhibited higher sensitivity to the base-damaging agent methylmethanesulfonate, resulting in enhanced DSB formation. Similar damage was found by vitamin C treatment, which induces TET1-mediated DNA demethylation via 5-hydroxymethylcytosine. Together, genome stability mediated by Polß-dependent base excision repair is crucial for the competence of neural progenitors, thereby contributing to neuronal differentiation in cortical development.SIGNIFICANCE STATEMENT DNA repair is crucial for development of the nervous system. However, how DNA polymerase ß (Polß)-dependent DNA base excision repair pathway contributes to the process is still unknown. We found that loss of Polß in cortical progenitors rather than postmitotic neurons led to catastrophic DNA double-strand breaks (DSBs) during replication and p53-mediated neuronal apoptosis, which resulted in thinning of the cortical plate. The DSBs also affected corticofugal axon growth in surviving neurons. Moreover, induction of base damage and DNA demethylation intermediates in the genome increased DSBs in cultured Polß-deficient neural progenitors. Thus, genome stability by Polß-dependent base excision repair in neural progenitors is required for the viability and differentiation of daughter neurons in the developing nervous system.

Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging.

Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMP-response element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons. To visualize CREB at the single-molecule level, fluorescent-tagged CREB in living dissociated cortical neurons was observed by highly inclined and laminated optical sheet microscopy. We found that a significant fraction of CREB spots resided in the restricted locations in the nucleus for several seconds (dissociation rate constant: 0.42 s-1). In contrast, two mutant CREBs, which cannot bind to the cAMP-response element, scarcely exhibited long-term residence. To test the possibility that CREB dynamics depends on neuronal activity, pharmacological treatments and an optogenetic method involving channelrhodopsin-2 were applied to cultured cortical neurons. Increased neuronal activity did not appear to influence the residence time of CREB spots, but markedly increased the number of restricted locations (hot spots) where CREB spots frequently resided with long residence times (>1 s). These results suggest that neuronal activity promotes CREB-dependent transcription by increasing the frequency of CREB binding to highly localized genome locations. SIGNIFICANCE STATEMENT: The transcription factor, cAMP response element-binding protein (CREB) is known to regulate gene expression in neuronal activity-dependent processes. However, its spatiotemporal interactions with the genome remain unknown. Single-molecule imaging in cortical neurons revealed that fluorescent-tagged CREB spots frequently reside at fixed nuclear locations in the time range of several seconds. Neuronal activity had little effect on the CREB residence time, but increased the rapid and frequent reappearance of long-residence CREB spots at the same nuclear locations. Thus, activity-dependent transcription is attributable to frequent binding of CREB to specific genome loci.

The role of T-cadherin in axonal pathway formation in neocortical circuits.

Cortical efferent and afferent fibers are arranged in a stereotyped pattern in the intermediate zone (IZ). Here, we studied the mechanism of axonal pathway formation by identifying a molecule that is expressed in a subset of cortical axons in the rat. We found that T-cadherin (T-cad), a member of the cadherin family, is expressed in deep-layer cell axons projecting to subcortical structures, but not in upper layer callosal axons projecting to the contralateral cortex. Ectopic expression of T-cad in upper layer cells induced axons to project toward subcortical structures via the upper part of the IZ. Moreover, the axons of deep-layer cells in which T-cad expression was suppressed by RNAi projected towards the contralateral cortex via an aberrant route. These results suggest that T-cad is involved in axonal pathway formation in the developing cortex.

Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures.

BACKGROUND: CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging. RESULTS: Dissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity. CONCLUSIONS: Detection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

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.

Repetitive CREB-DNA interactions at gene loci predetermined by CBP induce activity-dependent gene expression in human cortical neurons.

Neuronal activity-dependent transcription plays a key role in plasticity and pathology in the brain. An intriguing question is how neuronal activity controls gene expression via interactions of transcription factors with DNA and chromatin modifiers in the nucleus. By utilizing single-molecule imaging in human embryonic stem cell (ESC)-derived cortical neurons, we demonstrate that neuronal activity increases repetitive emergence of cAMP response element-binding protein (CREB) at histone acetylation sites in the nucleus, where RNA polymerase II (RNAPII) accumulation and FOS expression occur rapidly. Neuronal activity also enhances co-localization of CREB and CREB-binding protein (CBP). Increased binding of a constitutively active CREB to CBP efficiently induces CREB repetitive emergence. On the other hand, the formation of histone acetylation sites is dependent on CBP histone modification via acetyltransferase (HAT) activity but is not affected by neuronal activity. Taken together, our results suggest that neuronal activity promotes repetitive CREB-CRE and CREB-CBP interactions at predetermined histone acetylation sites, leading to rapid gene expression.

Clinical utility of QRS duration normalized to left ventricular volume for predicting cardiac resynchronization therapy efficacy in patients with "mid-range" QRS duration.

BACKGROUND: Cardiac resynchronization therapy (CRT) is effective for patients with heart failure with QRS duration (QRSd) ≥150 ms. However, its beneficial effect seems to be limited for those with "mid-range" QRSd (120-149 ms). Recent studies have demonstrated that modifying QRSd to left ventricular end-diastolic volume (LVEDV)-modified QRSd-improves the prediction of clinical outcomes of CRT. OBJECTIVE: The purpose of this study was to investigate the clinical impact of the modified QRSd on the efficacy of CRT in patients with "mid-range" QRSd. METHODS: We conducted a retrospective, multicenter, observational study, with heart failure hospitalization (HFH) after CRT as the primary endpoint. Modified QRSd is defined as QRSd divided by LVEDV, determined through the Teichholtz method of echocardiography. RESULTS: Among the 506 consecutive patients considered, 119 (mean age 61 ± 15 years; 80% male, QRSd 135 ± 9 ms) with a "mid-range" QRSd who underwent de novo CRT device implantation were included for analysis. During median follow-up of 878 days [interquartile range 381-1663 days], HFH occurred in 45 patients (37%). Fine-Gray analysis revealed modified QRSd was an independent predictor of HFH (hazard ratio [HR] 0.97; 95% confidence interval [CI] 0.96-0.99; P <.01). Receiver operating characteristic curve analysis revealed a cutoff value of 0.65 ms/mL for the modified QRSd in predicting HFH. Patients above the threshold exhibited a significantly lower incidence of HFH than patients below the threshold (HR 0.46; 95% CI 0.25-0.86; P = .01). CONCLUSION: Modified QRSd can effectively predict the efficacy of CRT in patients with a "mid-range" QRSd.

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.

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.

Adaptive phenotype of microglial cells during the normal postnatal development of the somatosensory "Barrel" cortex.

Accumulative evidence indicates that microglial cells influence the normal development of central nervous system (CNS) synapses. Yet, the functional properties of microglia in relation with synapse development remain unclear. We recently showed that in layer 4 of the whisker-related barrel field of the mouse somatosensory cortex, microglial cells are recruited only after postnatal day (P)5 in the center of the barrels where thalamo-cortical synapses are concentrated and begin their maturation. In the present study, we analyzed the phenotype of microglia during this developmental process. We show that between P5 and P7 microglial cells acquire a more ramified morphology with a smaller soma, they express classical markers of microglia (Iba1, CD11b, and CD68) but never markers of activation (Mac-2 and MHCII) and rarely the proliferation marker Ki67. Electrophysiological recordings in acute cortical slices showed that at P5 a proportion of layer 4 microglia transiently express voltage-dependant potassium currents of the delayed rectifier family, mostly mediated by Kv1.3 subunits, which are usually expressed by activated microglia under pathological conditions. This proportion of cells with rectifying properties doubles between P5 and P6, in concomitance with the beginning of microglia invasion of the barrel centers. Finally, analysis of the responses mediated by purinergic receptors indicated that a higher percentage of rectifying microglia expressed functional P2Y6 and P2Y12 receptors, as compared with nonrectifying cells, whereas all cells expressed functional P2X7 receptors. Our results indicate that during normal cortical development distinct microglia properties mature differentially, some of them being exquisitely influenced by the local environment of the maturating neuronal network.

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.

Shaping brain connections through spontaneous neural activity.

An overwhelming number of observations demonstrate that neural activity and genetic programs interact to specify the composition and organization of neural circuits during all stages of development. Spontaneous neuronal activities have been documented in several developing neural regions in both invertebrates and vertebrates, and their roles are mostly conserved among species. Among these roles, Ca(2+) spikes and levels of electrical activity have been shown to regulate neurite growth, axon extension and axon branching. Here, we review selected findings concerning the role of spontaneous activity on circuit development.

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.

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.

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.

Neuronal Activity Patterns Regulate Brain-Derived Neurotrophic Factor Expression in Cortical Cells via Neuronal Circuits.

During development, cortical circuits are remodeled by spontaneous and sensory-evoked activity via alteration of the expression of wiring molecules. An intriguing question is how physiological neuronal activity modifies the expression of these molecules in developing cortical networks. Here, we addressed this issue, focusing on brain-derived neurotrophic factor (BDNF), one of the factors underlying cortical wiring. Real-time imaging of BDNF promoter activity in organotypic slice cultures revealed that patterned stimuli differentially regulated the increase and the time course of the promoter activity in upper layer neurons. Calcium imaging further demonstrated that stimulus-dependent increases in the promoter activity were roughly proportional to the increase in intracellular Ca2+ concentration per unit time. Finally, optogenetic stimulation showed that the promoter activity was increased efficiently by patterned stimulation in defined cortical circuits. These results suggest that physiological stimulation patterns differentially tune activity-dependent gene expression in developing cortical neurons via cortical circuits, synaptic responses, and alteration of intracellular calcium signaling.