Shun Nakamura

Requirement of Ras for the Activation of Mitogen-Activated Protein Kinase by Calcium Influx, cAMP, and Neurotrophin in Hippocampal Neurons

Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term potentiation both in vitroand in living animals. MAP kinase is activated in response to a broad range of stimuli, including calcium influx through NMDA receptor and L-type calcium channel, cAMP, and neurotrophins. To investigate the role of Ras in the activation of MAP kinase and cAMP response element-binding protein (CREB) in hippocampal neurons, we inhibited Ras function by overexpressing a Ras GTPase-activating protein, Gap1m, or dominant negative Ras by means of adenovirus vectors. Gap1m expression almost completely suppressed MAP kinase activation in response to NMDA, calcium ionophore, membrane depolarization, forskolin, and brain-derived neurotrophic factor (BDNF). Dominant negative Ras also showed similar effects. On the other hand, Rap1GAP did not significantly inhibit the forskolin-induced activation of MAP kinase. In contrast to MAP kinase activation, the inactivation of Ras activity did not inhibit significantly NMDA-induced CREB phosphorylation, whereas BDNF-induced CREB phosphorylation was inhibited almost completely. These results demonstrate that Ras transduces signals elicited by a broad range of stimuli to MAP kinase in hippocampal neurons and further suggest that CREB phosphorylation depends on multiple pathways.

Demarcation of early mammalian cortical development by differential expression of fringe genes.

Fringe has originally been found in Drosophila as a gene encoding a putative secreted protein which regulates the sensitivity of Notch signaling pathway to different ligands. We show that three members of murine fringe gene family, Lunatic fringe (L-fng), Manic fringe (M-fng) and Radical fringe (R-fng), show related patterns of expression in the developing cerebral wall. L-fng is expressed in immature cells in the ventricular zone. M-fng is upregulated transiently in maturing neurons when they leave the ventricular zone (VZ). R-fng is upregulated in more mature neurons when they enter the preplate and cortical plate. These patterns suggest that the transition from immature to mature neurons involves sequential changes in the member of fringe family genes expressed. More detailed expression analyses of fringe genes and immunohistochemistry for neuron-specific class III beta-tubulin suggest a mode of neurogenesis which might underlie the histogenesis of the cerebral cortex. A proliferative population situated outside of the VZ is defined as M-fng-positive/BrdU-positive cells, which constitutes about 10-20% of the total S-phase cells in the cerebral wall of embryonic day 10.5-12.5. We found that M-fng is expressed in mitotic figures outside the VZ and some of them react with the antibody against class III beta-tubulin. These observations suggest that a significant number of proliferative cells exist outside the VZ, which supply neurons during early cortical development.

Brain-derived neurotrophic factor requirement for activity-dependent maturation of glutamatergic synapse in developing mouse somatosensory cortex

The maturation of cortical circuitry critically depends on experience. Recently, a model of silent synapse has been proposed as a mechanism of activity-mediated transition of immature synapse to mature synapse. It is not clear, however, how activity could regulate this transition. Here, we show the evidence that endogenous brain-derived neurotrophic factor (BDNF) is required for the maturation of glutamatergic synapse in developing mouse somatosensory cortex. Field potential recordings of thalamocortical glutamatergic synaptic activity with brain slices from the BDNF mutant mice showed that AMPA receptor responses are low, but NMDA receptor responses remain high in layer 4, thus, the relative contribution of AMPA receptor response is significantly lower compared to the age-matched wild-type mouse. Furthermore, optical images of development of thalamocortical connectivity with a voltage-sensitive dye showed that NMDA receptor-dominant synapse is established first in layer 4 and layer 5/6 then AMPA receptor response appears later in concomitant with reduction of NMDA receptor response in layer 4 and that the maturation of the silent synapse is impaired in the BDNF mutant mice. In layer 5/6, NMDA receptor response was suppressed without upregulation of AMPA receptor response. This process also required BDNF function. Interestingly, whisker-trimming of the wild-type mouse from just after birth showed quite similar results with the homozygous mutant of their whiskers left intact. Therefore, we would propose that BDNF is a critical mediator for the maturation of glutamatergic synapse in developing mouse somatosensory cortex.

Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells.

The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal–binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin–CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.

Altered whisker patterns induced by ectopic expression of Shh are topographically represented by barrels

Barrels in the somatosensory cortex are segregated columns, which somatotopically relate to facial whiskers. The barrel pattern is assumed to be determined by an extrinsic mechanism (the domino theory). This theory is based on whisker lesion experiments and developmental observations regarding the serial establishment of the somatotopic pattern in which pattern formations are relayed from the periphery to the central nervous system. However, the barrel pattern is possibly determined by an intrinsic mechanism, especially in its primitive form. In order to investigate the definitive mechanism, we established an experimental system in which the cortical barrel pattern can be altered, not by using a lesion paradigm, but by epigenetically changing the whisker pattern. Sonic hedgehog (Shh) plays a pivotal role in whisker development. We transfected an adenovirus harboring chicken Shh (Ad-cShh) to mouse embryos (E9.5-E11.5) using an in utero surgical technique. When Ad-cShh was expressed in the epidermis, Bmp4, Ptch, Ptch2 and Gli1 were induced ectopically in the interfollicular region. In contrast, the expression of Bmp2 and Shh itself was unaltered. At a suitable dose of Ad-cShh, some pups displayed supernumerary whiskers or a disordered whisker pattern. The barrel patterns of these mice after the critical period were topographic representations of the contralateral side of the new whisker patterns when visualized by a cytochrome oxidase or Nissle staining method, supporting the instructive role of the extrinsic mechanism.

Improved data processing for optical imaging of developing neuronal connectivity in the neonatal mouse barrel cortex

Optical recording methods using voltage-sensitive dyes have proven valuable for the analysis of neuronal networks both in vivo and in vitro. This technique detects membrane potential changes as changes in the absorption or fluorescence of voltage-sensitive dyes incorporated into the cellular plasma membranes. The reliability of the optical recording technique is dependent on the dye-related response being fast enough to follow the electrical activity and of the response being more or less proportional to the amplitude of the membrane potential change. A high spatial resolution can be achieved using an appropriate imaging system and a dye with a response of sufficiently high signal-to-noise ratio. Thus, it is now anticipated that this method will be able to shed more light on the spatio-temporal information processing of neocortical circuitry. While the FUJIX HR Deltaron 1700 optical imaging system offers a reasonably high time (0.6 ms) and space-resolution (7 microm at 10x magnification), one drawback of this system, however, is its relatively poor data processing capabilities. We have therefore developed a protocol to improve the signal-to-noise ratio by modifying the calculation algorithm of the optical data. Consequently, we characterized optical responses in thalamocortical slices to find developmental landmarks of thalamocortical and intracortical connectivity in the neonatal mouse barrel cortex. Successful application of this method has been published on the analysis of thalamocortical glutamatergic connectivity [8].

The Ras-like small GTP-binding protein Rin is activated by growth factor stimulation.

A novel 25 K Ras-like protein, Rin, binds calmodulin in a Ca2+-dependent manner and is considered to participate in a calcium/calmodulin-mediated signaling pathway. However, little is known about Rin signaling mechanism. Here we examined the signal transduction pathway through Rin protein using pull down assay system. When we stimulated Rin-expressed Cos-7 cells with ionomycin, phorbol 12-myristate 13-acetate or epidermal growth factor (EGF), Rin protein was rapidly activated. Moreover, cells cotransfected with Rin and mSos showed a similar Rin activation profile, and Rin protein was coimmunoprecipitated with mSos protein in vivo. When cells were cotransfected with Rin and Ras-GTPase-activating proteins, basal Rin activity was decreased. Association with Rin and calmodulin was potentiated after stimulation and Rin activation was regulated by both calcium ion and calmodulin. These studies suggest that Rin may be involved in EGF receptor and mSos-mediated signaling pathway and may participate in calcium/calmodulin-mediated cellular processes.

Loss of Neurofibromin in the Leptomeningeal Astroglial Heterotopia of NF-1

Neurofibromin, the protein product of the neurofibromatosis type 1 (NF-1) gene, has important roles in tumor suppression or normal embryogenesis. Cerebellar leptomeningeal astroglial heterotopia (LAH) is a proliferation of heterotopic astroglial cells and fibers in the cerebellar leptomeninges, which is characteristically demonstrated in the NF-1 patients. In this study, neurofibromin expression was investigated in NF-1 and non-NF-1 human tissues, especially in the cerebellum of NF-1 patients. Neurofibromin was found by immunoblotting in the CNS but not in the heart, liver, and kidney. Immunohistochemistry in the normal areas of the brains with NF-1 demonstrated neurofibromin immunoreactivity as did the brains of unaffected controls. Cerebellar LAH showed no neurofibromin immunoreactivity. The results of this study suggest that neurofibromin expression remains unchanged in the nonproliferated region of the CNS of the NF-1 patient but changes occur in the abnormally proliferated region, resulting in cerebellar LAH. Loss of neurofibromin may result in the excessive migration and growth of astrocytes in the early fetal period.

Current topics in comparative developmental biology of vertebrate brains

Little is known about how unique features in a species can emerge along the conserved body plan beyond species. For example, mammals, including human beings, have acquired the neocortex with distinct function and morphology. Here we review current topics in comparative developmental biology of vertebrate brains, especially focusing on the cerebral neocortex as a suitable model for considering species-specific aspects.