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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 cisregulatory 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. [ABSTRACT FROM AUTHOR]

Background: Neuronal activity alters calcium ion (Ca2+) dynamics in astrocytes, but the physiologic relevance of these changes is controversial. To examine this issue further, we generated an inducible transgenic mouse model in which the expression of an inositol 1,4,5-trisphosphate absorbent, "IP3 sponge", attenuates astrocytic Ca2+ signaling. Results: Attenuated Ca2+ activity correlated with reduced astrocytic coverage of asymmetric synapses in the hippocampal CA1 region in these animals. The decreased astrocytic 'protection' of the synapses facilitated glutamate 'spillover', which was reflected by prolonged glutamate transporter currents in stratum radiatum astrocytes and enhanced N-methyl-D-aspartate receptor currents in CA1 pyramidal neurons in response to burst stimulation. These mice also exhibited behavioral impairments in spatial reference memory and remote contextual fear memory, in which hippocampal circuits are involved. Conclusions: Our findings suggest that IP3-mediated astrocytic Ca2+ signaling correlates with the formation of functional tripartite synapses in the hippocampus. [ABSTRACT FROM AUTHOR]

GABAergic inhibition plays a central role in the control of pyramidal cell ensemble activities; thus, any signaling mechanism that regulates inhibition is able to fine-tune network patterns. Here, we provide evidence that the retrograde nitric oxide (NO)-cGMP cascade triggered by NMDAreceptor(NMDAR)activation plays a role in the control of hippocampal GABAergic transmission in mice. GABAergic synapses express neuronal nitric oxide synthase (nNOS) postsynaptically and NO receptors (NO-sensitive guanylyl cyclase) in the presynaptic terminals. We hypothesized that-similar to glutamatergic synapses-the Ca2+ transients required to activate nNOS were provided by NMDA receptor activation. Indeed, administration of 5 μM NMDA induced a robust nNOS-dependent cGMP production in GABAergic terminals, selectively in the CA1 and CA3c areas. Furthermore, using preembedding, postembedding, and SDS-digested freeze-fracture replica immunogold labeling, we provided quantitative immunocytochemical evidence that NMDAR subunits GluN1, GluN2A, and GluN2B were present in most somatic GABAergic synapses postsynaptically. These data indicate that NMDARs can modulate hippocampal GABAergic inhibition viaNO-cGMPsignaling in an activity-dependent manner and that this effect is subregion specific in the mouse hippocampus. [ABSTRACT FROM AUTHOR]

Neurogenesis occurs continuously in the forebrain of adult mammals, but the functional importance of adult neurogenesis is still unclear. Here, using a genetic labeling method in adult mice, we found that continuous neurogenesis results in the replacement of the majority of granule neurons in the olfactory bulb and a substantial addition of granule neurons to the hippocampal dentate gyrus. Genetic ablation of newly formed neurons in adult mice led to a gradual decrease in the number of granule cells in the olfactory bulb, inhibition of increases in the granule cell number in the dentate gyrus and impairment of behaviors in contextual and spatial memory, which are known to depend on hippocampus. These results suggest that continuous neurogenesis is required for the maintenance and reorganization of the whole interneuron system in the olfactory bulb, the modulation and refinement of the existing neuronal circuits in the dentate gyrus and the normal behaviors involved in hippocampal-dependent memory. [ABSTRACT FROM AUTHOR]

S100B is a calcium-binding protein predominantly expressed in astrocytes. Previous studies using gene-manipulated animals have suggested that the protein has a role in synaptic plasticity and learning. In order to assess the physiological roles of the protein in active neural circuitry, we recorded spontaneous neural activities from various layers of the neocortex and hippocampus in urethane-anesthetized S100B knockout (KO) and wildtype (WT) control mice. Typical local field oscillation patterns including the slow (0.5–2 Hz) oscillations in the neocortex, theta (3–8 Hz) and sharp wave-associated ripple (120–180 Hz) oscillations in the hippocampus were observed in both genotypes. Comparisons of the frequency, power and peak amplitude have shown that these oscillatory patterns were virtually indistinguishable between WT and KO. When seizure was induced by intraperitoneal injection of kainic acid, a difference between WT and KO appeared in the CA1 radiatum local field potential pattern, where seizure events were characterized by prominent appearance of hyper-synchronous gamma band (30–80 Hz) activity. Although both genotypes developed seizures within 40 min, the gamma amplitude was significantly smaller during the development of seizures in KO mice. Our results suggest that deficiency of S100B does not have a profound impact on spontaneous neural activity in normal conditions. However, when neural activity was sufficiently raised, activation of S100B-related pathways may take effect, resulting in modulation of neural activities. [ABSTRACT FROM AUTHOR]

Summary: Thalamocortical (TC) connectivity is reorganized by thalamic inputs during postnatal development; however, the dynamic characteristics of TC reorganization and the underlying mechanisms remain unexplored. We addressed this question using dendritic refinement of layer 4 (L4) stellate neurons in mouse barrel cortex (barrel cells) as a model; dendritic refinement of L4 neurons is a critical component of TC reorganization through which postsynaptic L4 neurons acquire their dendritic orientation toward presynaptic TC axon termini. Simultaneous labeling of TC axons and individual barrel cell dendrites allowed in vivo time-lapse imaging of dendritic refinement in the neonatal cortex. The barrel cells reinforced the dendritic orientation toward TC axons by dynamically moving their branches. In N-methyl-D-aspartate receptor (NMDAR)-deficient barrel cells, this dendritic motility was enhanced, and the orientation bias was not reinforced. Our data suggest that L4 neurons have “fluctuating” dendrites during TC reorganization and that NMDARs cell autonomously regulate these dynamics to establish fine-tuned circuits. [Copyright &y& Elsevier]

Summary: The ephrin/Eph system plays a central role in neuronal circuit formation; however, its downstream effectors are poorly understood. Here we show that α-chimerin Rac GTPase-activating protein mediates ephrinB3/EphA4 forward signaling. We discovered a spontaneous mouse mutation, miffy (mfy), which results in a rabbit-like hopping gait, impaired corticospinal axon guidance, and abnormal spinal central pattern generators. Using positional cloning, transgene rescue, and gene targeting, we demonstrated that loss of α-chimerin leads to mfy phenotypes similar to those of EphA4 −/− and ephrinB3 −/− mice. α-chimerin interacts with EphA4 and, in response to ephrinB3/EphA4 signaling, inactivates Rac, which is a positive regulator of process outgrowth. Moreover, downregulation of α-chimerin suppresses ephrinB3-induced growth cone collapse in cultured neurons. Our findings indicate that ephrinB3/EphA4 signaling prevents growth cone extension in motor circuit formation via α-chimerin-induced inactivation of Rac. They also highlight the role of a Rho family GTPase-activating protein as a key mediator of ephrin/Eph signaling. [Copyright &y& Elsevier]

Background: The netrin-G1 (NTNG1) and -G2 (NTNG2) genes, recently cloned from mouse, play a role in the formation and/or maintenance of glutamatergic neural circuitry. Accumulating evidence strongly suggests that disturbances of neuronal development and the N-methyl-d-aspartate receptor–mediated signaling system might represent a potential pathophysiology in schizophrenia. We therefore set out to examine the genetic contribution of human NTNG1 and NTNG2 to schizophrenia. Methods: Twenty-one single nucleotide polymorphisms (SNPs) from NTNG1 and 10 SNPs from NTNG2 were analyzed in 124 schizophrenic pedigrees. All genotypes were determined with the TaqMan assay. The expression levels of NTNG1 and NTNG2 were examined in the frontal (Brodmann’s Area [BA]11 and BA46) and temporal (BA22) cortices from schizophrenic and control postmortem brains. The isoform-specific expression of NTNG1 splice variants was assessed in these samples. Results: Specific haplotypes encompassing alternatively spliced exons of NTNG1 were associated with schizophrenia, and concordantly, messenger ribonucleic acid isoform expression was significantly different between schizophrenic and control brains. An association between NTNG2 and schizophrenia was also observed with SNPs and haplotypes that clustered in the 5′ region of the gene. Conclusions: The NTNG1 and NTNG2 genes might be relevant to the pathophysiology of schizophrenia. [Copyright &y& Elsevier]

Cell–cell interactions are primarily mediated by secreted and transmembrane proteins which play essential roles in the neuronal circuit formation. However, molecular mechanisms underlying neuronal circuit formation, which is mediated by the cell–cell interactions, remain largely elusive. We isolated and characterized a novel gene, Btcl2 (brain-specific transmembrane protein containing CUB [complement subcomponent C1r/C1s, sea urchin protein Uegf, and BMP-1] and LDLa [low-density lipoprotein receptor domain class A] domains 2), using the signal sequence trap (SST) method. The extracellular domain of BTCL2 contains two CUB domains and an LDLa domain. BTCL2 and BTCL1 have similar domain structures, sharing 51% overall identity. The CUB1, CUB2, and LDLa domains of these two proteins share 63%, 72%, and 84% identity, respectively. The CUB domains of BTCL1 and BTCL2 share significant identity with those of neuropilins. Btcl2 mRNA was detected as a single 6-kb transcript in Northern blot analysis. In situ hybridization (ISH) analysis revealed that both Btcl1 and Btcl2 mRNAs were observed restrictively in brain throughout embryonic and postnatal stages. Btcl1 and Btcl2 mRNAs were expressed uniquely in the pontine nucleus and subplate, which are required for establishing the neuronal circuit formation. These results will aid in resolving the mechanisms underlying neuronal circuit formations (e.g., pontocerebellar and thalamocortical axon guidance) and permit more precise studies aimed at understanding the role of BTCL1 and BTCL2 in the central nervous system. [Copyright &y& Elsevier]

• Netrin-G2 KO mice exhibit microtremors, which have been difficult to detect so far. • We developed a tremor detection system to evaluate microtremors quantitatively. • Using the new system, microtremors in netrin-G2 KO mice could be measured. • Microtremors were slightly influenced by anti-tremor drugs and enhanced by stress. • Our system enables us to quantify various types of tremors in model animals. 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. We developed a new tremor detector which can quantify the intensity and frequency of a tremor. 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. The detection performance even for tremors induced by low-dose harmaline was similar to that in previous studies or more sensitive than the others. 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. [ABSTRACT FROM AUTHOR]

Classical transection studies suggest that, in addition to the hypothalamus, the brainstem is essential for non-rapid eye movement (NREM) sleep. The circuits underlying this function, however, have remained largely unknown. We identified a circuit distributed in the midbrain, pons, and medulla that promotes NREM sleep in mice. We focused on the sublaterodorsal tegmentum, an area implicated in dual regulation of REM and NREM sleep. Transcriptomic and genetic analyses revealed that neurons positive for the neuropeptide neurotensin promote NREM sleep. Further analyses identified downstream NREM sleep-promoting neurons in the dorsal deep mesencephalic nucleus, the lateral part of the periaqueductal gray, and the medial vestibular nucleus that were also neurotensinergic. Infusion of neurotensin into the fourth ventricle induced NREM sleep-like cortical activity, whereas mice deficient for neurotensin exhibited increased REM sleep, implicating the involvement of the neuropeptide itself. These findings identify a widely distributed NREM sleep-regulating circuit in the brainstem with a common molecular property. • SubLDT neurons positive for the neuropeptide neurotensin promote NREM sleep • Putative target neurons identified in dDpMe, lPAG, and MVe also promote NREM sleep • These neurons in dDpMe, lPAG, and MVe are also neurotensinergic • Neurotensin is involved in sleep regulation Kashiwagi et al. identify a novel NREM sleep-promoting neural circuit distributed in the midbrain, pons, and medulla. All the neurons of this circuit are positive for the neuropeptide neurotensin, and neurotensin itself is involved in sleep regulation. [ABSTRACT FROM AUTHOR]