Objective detection of microtremors in netrin-G2 knockout mice.

BACKGROUND: Essential tremor is the most prevalent movement disorder and is thought to be caused by abnormalities in the cerebellar system; however, its underlying neural mechanism is poorly understood. In this study, we found that mice lacking netrin-G2, a cell adhesion molecule which is expressed in neural circuits related to the cerebellar system, exhibited a microtremor resembling an essential tremor. However, it was difficult to quantify microtremors in netrin-G2 KO mice. NEW METHOD: We developed a new tremor detector which can quantify the intensity and frequency of a tremor. RESULTS: Using this system, we were able to characterize both the microtremors in netrin-G2 KO mice and low-dose harmaline-induced tremors which, to date, had been difficult to detect. Alcohol and anti-tremor drugs, which are effective in decreasing the symptoms of essential tremor in patients, were examined in netrin-G2 KO mice. We found that some drugs lowered the tremor frequency, but had little effect on tremor intensity. Forced swim as a stress stimulus in netrin-G2 KO mice dramatically enhanced tremor symptoms. COMPARISON WITH EXISTING METHODS: The detection performance even for tremors induced by low-dose harmaline was similar to that in previous studies or more sensitive than the others. CONCLUSIONS: Microtremors in netrin-G2 KO mice are reliably and quantitatively detected by our new tremor detection system. We found different effects of medicines and factors between human essential tremors and microtremors in netrin-G2 KO mice, suggesting that the causations, mechanisms, and symptoms of tremors vary and are heterogeneous, and the objective analyses are required.

Diversification of behavior and postsynaptic properties by netrin-G presynaptic adhesion family proteins.

BACKGROUND: Vertebrate-specific neuronal genes are expected to play a critical role in the diversification and evolution of higher brain functions. Among them, the glycosylphosphatidylinositol (GPI)-anchored netrin-G subfamily members in the UNC6/netrin family are unique in their differential expression patterns in many neuronal circuits, and differential binding ability to their cognate homologous post-synaptic receptors. RESULTS: To gain insight into the roles of these genes in higher brain functions, we performed comprehensive behavioral batteries using netrin-G knockout mice. We found that two netrin-G paralogs that recently diverged in evolution, netrin-G1 and netrin-G2 (gene symbols: Ntng1 and Ntng2, respectively), were responsible for complementary behavioral functions. Netrin-G2, but not netrin-G1, encoded demanding sensorimotor functions. Both paralogs were responsible for complex vertebrate-specific cognitive functions and fine-scale regulation of basic adaptive behaviors conserved between invertebrates and vertebrates, such as spatial reference and working memory, attention, impulsivity and anxiety etc. Remarkably, netrin-G1 and netrin-G2 encoded a genetic "division of labor" in behavioral regulation, selectively mediating different tasks or even different details of the same task. At the cellular level, netrin-G1 and netrin-G2 differentially regulated the sub-synaptic localization of their cognate receptors and differentiated the properties of postsynaptic scaffold proteins in complementary neural pathways. CONCLUSIONS: Pre-synaptic netrin-G1 and netrin-G2 diversify the complexity of vertebrate behaviors and differentially regulate post-synaptic properties. Our findings constitute the first genetic analysis of the behavioral and synaptic diversification roles of a vertebrate GPI protein and presynaptic adhesion molecule family.

Netrin-G/NGL complexes encode functional synaptic diversification.

Synaptic cell adhesion molecules are increasingly gaining attention for conferring specific properties to individual synapses. Netrin-G1 and netrin-G2 are trans-synaptic adhesion molecules that distribute on distinct axons, and their presence restricts the expression of their cognate receptors, NGL1 and NGL2, respectively, to specific subdendritic segments of target neurons. However, the neural circuits and functional roles of netrin-G isoform complexes remain unclear. Here, we use netrin-G-KO and NGL-KO mice to reveal that netrin-G1/NGL1 and netrin-G2/NGL2 interactions specify excitatory synapses in independent hippocampal pathways. In the hippocampal CA1 area, netrin-G1/NGL1 and netrin-G2/NGL2 were expressed in the temporoammonic and Schaffer collateral pathways, respectively. The lack of presynaptic netrin-Gs led to the dispersion of NGLs from postsynaptic membranes. In accord, netrin-G mutant synapses displayed opposing phenotypes in long-term and short-term plasticity through discrete biochemical pathways. The plasticity phenotypes in netrin-G-KOs were phenocopied in NGL-KOs, with a corresponding loss of netrin-Gs from presynaptic membranes. Our findings show that netrin-G/NGL interactions differentially control synaptic plasticity in distinct circuits via retrograde signaling mechanisms and explain how synaptic inputs are diversified to control neuronal activity.

Regulatory factor X transcription factors control Musashi1 transcription in mouse neural stem/progenitor cells.

The transcriptional regulation of neural stem/progenitor cells (NS/PCs) is of great interest in neural development and stem cell biology. The RNA-binding protein Musashi1 (Msi1), which is often employed as a marker for NS/PCs, regulates Notch signaling to maintain NS/PCs in undifferentiated states by the translational repression of Numb expression. Considering these critical roles of Msi1 in the maintenance of NS/PCs, it is extremely important to elucidate the regulatory mechanisms by which Msi1 is selectively expressed in these cells. However, the mechanism regulating Msi1 transcription is unclear. We previously reported that the transcriptional regulatory region of Msi1 is located in the sixth intron of the Msi1 locus in NS/PCs, based on in vitro experiments. In the present study, we generated reporter transgenic mice for the sixth intronic Msi1 enhancer (Msi1-6IE), which show the reporter expression corresponding with endogenous Msi1-positive cells in developing and adult NS/PCs. We found that the core element responsible for this reporter gene activity includes palindromic Regulatory factor X (Rfx) binding sites and that Msi1-6IE was activated by Rfx. Rfx4, which was highly expressed in NS/PCs positive for the Msi1-6IE reporter, bound to this region, and both of the palindromic Rfx binding sites were required for the transactivation of Msi1-6IE. Furthermore, ectopic Rfx4 expression in the developing mouse cerebral cortex transactivates Msi1 expression in the intermediate zone. This study suggests that ciliogenic Rfx transcription factors regulate Msi1 expression through Msi1-6IE in NS/PCs.

Identification of transcriptional regulatory elements for Ntng1 and Ntng2 genes in mice.

BACKGROUND: Higher brain function is supported by the precise temporal and spatial regulation of thousands of genes. The mechanisms that underlie transcriptional regulation in the brain, however, remain unclear. The Ntng1 and Ntng2 genes, encoding axonal membrane adhesion proteins netrin-G1 and netrin-G2, respectively, are paralogs that have evolved in vertebrates and are expressed in distinct neuronal subsets in a complementary manner. The characteristic expression patterns of these genes provide a part of the foundation of the cortical layer structure in mammals. RESULTS: We used gene-targeting techniques, bacterial artificial chromosome (BAC)-aided transgenesis techniques, and in vivo enhancer assays to examine transcriptional mechanisms in vivo to gain insight into how the characteristic expression patterns of these genes are acquired. Analysis of the gene expression patterns in the presence or absence of netrin-G1 and netrin-G2 functional proteins allowed us to exclude the possibility that a feedback or feedforward mechanism mediates their characteristic expression patterns. Findings from the BAC deletion series revealed that widely distributed combinations of cis-regulatory elements determine the differential gene expression patterns of these genes and that major cis-regulatory elements are located in the 85-45 kb upstream region of Ntng2 and in the 75-60 kb upstream region and intronic region of Ntng1. In vivo enhancer assays using 2-kb evolutionarily conserved regions detected enhancer activity in the distal upstream regions of both genes. CONCLUSIONS: The complementary expression patterns of Ntng1 and Ntng2 are determined by transcriptional cis-regulatory elements widely scattered in these loci. The cis-regulatory elements characterized in this study will facilitate the development of novel genetic tools for functionally dissecting neural circuits to better understand vertebrate brain function.

Hippocampal CA3 and CA2 have distinct bilateral innervation patterns to CA1 in rodents.

Ipsilateral and contralateral hippocampal CA3-CA1 and CA2-CA1 projections were investigated in adult male Long-Evans rats by retrograde tracing. Injection of the retrograde tracer cholera toxin subunit B in the strata oriens and radiatum of dorsal CA1 resulted in labeling of predominantly pyramidal cells in ipsilateral and contralateral CA3 and CA2. The contralateral and ipsilateral anterior-posterior extents of CA3 innervation to CA1 were similar. Fifteen to twenty per cent of the hippocampus proper cells that give rise to CA1 stratum oriens innervation were CA2 pyramidal cells, whereas CA2 cells were a mere 3% for CA1 stratum radiatum innervation. The preferred projection of CA2 pyramidal cells to the CA1 stratum oriens was also manifested in transgenic mice that express GFP under the control of the CACNG5 promoter, in which CA2 cells express high amounts of GFP. The ratios of ipsilateral to contralateral projections were compared. For the CA3-CA1 connection, we found that dorsal CA1 stratum radiatum received more ipsilateral projections whereas CA1 stratum oriens received more contralateral innervation. Interestingly, ipsilateral connections dominated for both CA2-CA1 stratum oriens and CA2-CA1 stratum radiatum. These results demonstrate that the primary intrahippocampal target of CA2 pyramidal cells is the ipsilateral CA1 stratum oriens, in contrast to CA3 cells which project more diversely to bilateral CA1 regions. Such innervation patterns may suggest differential dendritic information processing in apical and basal dendrites of CA1 pyramidal cells.

Identification of a novel intronic enhancer responsible for the transcriptional regulation of musashi1 in neural stem/progenitor cells.

BACKGROUND: The specific genetic regulation of neural primordial cell determination is of great interest in stem cell biology. The Musashi1 (Msi1) protein, which belongs to an evolutionarily conserved family of RNA-binding proteins, is a marker for neural stem/progenitor cells (NS/PCs) in the embryonic and post-natal central nervous system (CNS). Msi1 regulates the translation of its downstream targets, including m-Numb and p21 mRNAs. In vitro experiments using knockout mice have shown that Msi1 and its isoform Musashi2 (Msi2) keep NS/PCs in an undifferentiated and proliferative state. Msi1 is expressed not only in NS/PCs, but also in other somatic stem cells and in tumours. Based on previous findings, Msi1 is likely to be a key regulator for maintaining the characteristics of self-renewing stem cells. However, the mechanisms regulating Msi1 expression are not yet clear. RESULTS: To identify the DNA region affecting Msi1 transcription, we inserted the fusion gene ffLuc, comprised of the fluorescent Venus protein and firefly Luciferase, at the translation initiation site of the mouse Msi1 gene locus contained in a 184-kb bacterial artificial chromosome (BAC). Fluorescence and Luciferase activity, reflecting the Msi1 transcriptional activity, were observed in a stable BAC-carrying embryonic stem cell line when it was induced toward neural lineage differentiation by retinoic acid treatment. When neuronal differentiation was induced in embryoid body (EB)-derived neurosphere cells, reporter signals were detected in Msi1-positive NSCs and GFAP-positive astrocytes, but not in MAP2-positive neurons. By introducing deletions into the BAC reporter gene and conducting further reporter experiments using a minimized enhancer region, we identified a region, "D5E2," that is responsible for Msi1 transcription in NS/PCs. CONCLUSIONS: A regulatory element for Msi1 transcription in NS/PCs is located in the sixth intron of the Msi1 gene. The 595-bp D5E2 intronic enhancer can transactivate Msi1 gene expression with cell-type specificity markedly similar to the endogenous Msi1 expression patterns.