EphA4 signaling promotes axon segregation in the developing auditory system

Precision of synaptic connections within neural circuits is essential for the accurate processing of sensory information. Specificity is exemplified at cellular and subcellular levels in the chick auditory brainstem, where nucleus magnocellularis (NM) neurons project bilaterally to nucleus laminaris (NL). Dorsal dendrites of NL neurons receive input from ipsilateral, but not contralateral, branches of NM axons whereas ventral dendrites are innervated by contralateral NM axons. This organization is analogous to that of the mammalian medial superior olive (MSO) and represents an important component of the circuitry underlying sound localization. However, the molecular mechanisms that establish segregated inputs to individual regions of NL neurons have not been identified. During synapse formation in NL, the EphA4 receptor is expressed in dorsal, but not ventral NL, neuropil, suggesting a potential role in targeting synapses to appropriate termination zones. Here, we directly tested this role by ectopically expressing EphA4 and disrupting EphA4 signaling using in ovo electroporation. We found that both misexpression of EphA4 and disruption of EphA4 signaling resulted in an increase in the number of NM axons that grow aberrantly across NL cell bodies into inappropriate regions of NL neuropil. EphA4 signaling is thus essential for targeting axons to distinct subsets of dendrites. Moreover, loss of EphA4 function resulted in morphological abnormalities of NL suggestive of errors in cell migration. These results suggest that EphA4 has multiple roles in the formation of auditory brainstem nuclei and their projections.     

Receptor protein tyrosine phosphatases regulate retinal ganglion cell axon outgrowth in the developing Xenopus visual system

Receptor protein tyrosine phosphatases (RPTPs) are regulators of axon outgrowth and guidance in a variety of different vertebrate and invertebrate systems. Three RPTPs, CRYP-alpha, PTP-delta, and LAR, are expressed in overlapping but distinct patterns in the developing Xenopus retina, including expression in retinal ganglion cells (RGCs) as they send axons to the tectum (Johnson KG, Holt CE. 2000. Expression of CRYP-alpha, LAR, PTP-delta, and PTP-rho in the developing Xenopus visual system. Mech Dev 92:291-294). In order to examine the role of these RPTPs in visual system development, putative dominant negative RPTP mutants (CS-CRYP-alpha, CS-PTP-delta, and CS-LAR) were expressed either singly or in combination in retinal cells. No effect was found on either retinal cell fate determination or on gross RGC axon guidance to the tectum. However, expression of these CS-RPTP constructs differentially affected the rate of RGC axon outgrowth. In vivo, expression of all three CS-RPTPs or CS-PTP-delta alone inhibited RGC axon outgrowth, while CS-LAR and CS-CRYP-alpha had no significant effect. In vitro, expression of CS-CRYP-alpha enhanced neurite outgrowth, while CS-PTP-delta inhibited neurite outgrowth in a substrate-dependent manner. This study provides the first in vivo evidence that RPTPs regulate retinal axon outgrowth.     

VEGF signaling through neuropilin 1 guides commissural axon crossing at the optic chiasm

During development, the axons of retinal ganglion cell (RGC) neurons must decide whether to cross or avoid the midline at the optic chiasm to project to targets on both sides of the brain. By combining genetic analyses with in vitro assays, we show that neuropilin 1 (NRP1) promotes contralateral RGC projection in mammals. Unexpectedly, the NRP1 ligand involved is not an axon guidance cue of the class 3 semaphorin family, but VEGF164, the neuropilin-binding isoform of the classical vascular growth factor VEGF-A. VEGF164 is expressed at the chiasm midline and is required for normal contralateral growth in vivo. In outgrowth and growth cone turning assays, VEGF164 acts directly on NRP1-expressing contralateral RGCs to provide growth-promoting and chemoattractive signals. These findings have identified a permissive midline signal for axons at the chiasm midline and provide in vivo evidence that VEGF-A is an essential axon guidance cue.     

Glutamic acid decarboxylase in different areas of the developing chick central nervous system

The temporal course of the development of GAD activity in GABAergic neurons was studied in the chick retina, optic lobe and cerebellum. The developmental pattern of GAD activity was similar in the three areas studied, showing typical sigmoideal curves, which reached a maximal value at the 3^rd post-hatching day. Kinetic studies during development revealed that K_m remained unchanged while V_max increased 3-fold in the retina (48.99±0.84 nmol/hr/mg protein), almost 4-fold in the optic lobe (162.77±4.32 nmol/hr/mg protein) and 3.5 fold in the cerebellum (69.30±1.26 nmol/hr/mg protein). The developmental pattern of GAD activity in homogenates of the three areas studied from dark-reared and light-reared chicks with respect to normal light-dark cycle animals showed no significant differences. These results indicate that the increase in GAD activity during development are not due to a change in the affinity for its substrate but rather to changes in the concentration of the enzyme. The developmental pattern of GAD activity in the chick visual system was not affected by environmental conditions suggesting that the developmental profile is lightindependent.     

Effects of L1 blockade on sensory axon outgrowth and pathfinding in the chick hindlimb

In the developing chick hindlimb, sensory axons, which grow together in bundles as they extend distally, and the motoneuron axons they encounter express the cell adhesion molecule L1. Following injection of function-blocking anti-L1 antibodies into the limb at stage 25, some sensory axons choose inappropriate peripheral nerves even though motoneuron pathfinding is unaffected. Here, to further elucidate L1's role, we assessed the effects of this perturbation using pathway tracing, immune labeling, confocal microscopy, and electron microscopy. After L1 blockade, sensory axons were still bundled and closely apposed. However, clear signs of decreased adhesion were detectable ultrastructurally. Further, sensory axons grew into the limb more slowly than normal, wandering more widely, branching more frequently, and sometimes extending along inappropriate peripheral nerves. Sensory axons that ultimately projected along different cutaneous nerves showed increased intermixing in the spinal nerves, due to errors in pathfinding and also to a decreased ability to segregate into nerve-specific fascicles. These results suggest that, in the highly complex in vivo environment, as in tissue culture, L1 stimulates axon growth and enhances fasciculation, and that these processes contribute to the orderly, timely, and specific growth of sensory axons into the limb.     

Identification of chick frizzled-10 expressed in the developing limb and the central nervous system

We have identified chick frizzled (Fz)-10, encoding a Wnt receptor, and examined the expression pattern during embryogenesis. Fz-10 is expressed in the region posterior to the Hensen's node at stage 6. Fz-10 expression is detected in the dorsal domain of the neural tube and the central nervous system of the developing embryo. In the developing limb, Fz-10 expression starts at stage 18 in the posterior-dorsal region of the distal mesenchyme, and gradually expands to the anterior-distal region. Fz-10 is also expressed in the feather bud and branchial arch. Implantation of Sonic hedgehog (Shh)-expressing cells into the anterior margin of the limb bud resulted in the induction of Fz-10 expression in anterior-dorsal mesenchyme.     

Amitriptyline inhibits neurite outgrowth in chick cerebral neurons: A possible mechanism

Previous studies showed that amitriptyline (AMI), a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants and inhibited adenylyl cyclase activity in cerebral membrane preparations. In the present study, we have investigated the possibility that AMI may have additional effects on cellular metabolism and signal transduction that underlie AMI-mediated inhibition of neurite outgrowth. In vitro AMI inhibited phospholipase C in a dose- and GTP-dependent manner in membranes from 8-day-old chick forebrain. Brain homogenates from 8-day-old chick embryos, treated in vivo for 6 days with AMI (20 μg/g/day), showed significant reductions in (1) phosphorylation of two polypeptides (49 and 105 kD), and (2) levels of three polypeptides (43, 53, and 92 kD). Western blots showed that the 43- and 53-kD polypeptides corresponded to actin and tubulin, respectively. Diolein and dilinolein, potent activators of protein kinase C, stimulated neurite outgrowth and reversed the inhibitory effects of AMI. Sphingosine, a protein kinase C inhibitor, significantly inhibited neurite outgrowth and eliminated the stimulatory effects of diolein and dilinolein on neurite outgrowth. These data suggest that AMI-mediated inhibition of neurite outgrowth involves multiple effects on cellular metabolism and signal transduction. A hypothesis consistent with our data is that AMI interferes in some manner with the action of G proteins in the signal transduction cascade. © 1993 John Wiley & Sons, Inc.     

Development of axon pathways in the zebrafish central nervous system

The zebrafish has a number of distinct advantages as an experimental model in developmental biology. For example, large numbers of embryos can be generated in each lay, development proceeds rapidly through a very precise temporal staging which exhibits minimal batch-to-batch variability, embryos are transparent and imaging of wholemounts negates the need for tedious histological preparation while preserving three-dimensional spatial relationships. The zebrafish nervous system is proving a convenient model for studies of axon guidance because of its small size and highly stereotypical trajectory of axons. Moreover, a simple scaffold of axon tracts and nerves is established early and provides a template for subsequent development. The ease with which this template can be visualized as well as the ability to spatially resolve individual pioneer axons enables the role of specific cell-cell and molecular interactions to be clearly deciphered. We describe here the morphology and development of the earliest axon pathways in the embryonic zebrafish central nervous system and highlight the major questions that remain to be addressed with regard to axon guidance.     

Ectoderm removal prevents cutaneous nerve formation and perturbs sensory axon growth in the chick hindlimb

Target tissues are thought to provide important cues for growing axons, yet there is little direct evidence that they are essential for axonal pathfinding. Here we examined whether target ectoderm is necessary for the formation of cutaneous nerves, and for the normal growth and guidance of cutaneous axons as they first enter the limb plexus. To do this, we removed a patch of ectoderm from the chick hindlimb at various times during early axon outgrowth. We find there is a critical period when cutaneous nerve formation requires target ectoderm. When the ectoderm is absent during this time, axons progress into the limb more slowly and, although a few sensory axons occasionally diverge a short distance from the plexus, they do not form a discrete nerve that travels to the skin. A few days later, when the nerve pattern is mature, axons normally destined for the 'deprived' cutaneous nerve are not segregated appropriately within the plexus. Some cutaneous axons are instead misdirected along an inappropriate cutaneous nerve, while others have seemingly failed to reach their correct target, or a suitable alternative, and died. These results demonstrate that the target ectoderm is necessary for normal sensory axon growth and guidance in the hindlimb.     

DNA synthesis in the embryonic chick central nervous system

Summary DNA synthesis has been studied in chick embryos age between 2 and 10 days, using labelling with tritiated thymidine and stripping film autoradiography. The observations made earlier in the literature on a premitotic migration of the nuclei in the neural epithelium have been verified. In young stages (before day 7) peripherally migrated cells do not synthesize DNA, but after day 7 such a synthesis occurs. In spite of this, few mitoses are seen. The interpretation of these facts is discussed.The costs of this investigation were defrayed by grants from the Swedish Medical Research Council, the Medical Faculty of Lund, and the Royal Physiographic Society.     

Specific immunolabeling of brain macrophages and microglial cells in the developing and mature chick central nervous system.

The present study showed that the HIS-C7 monoclonal antibody, which recognizes the chick form of CD45, is a specific marker for macrophages/microglial cells in the developing and mature chick central nervous system (CNS). HIS-C7-positive cells were characterized according to their morphological features and chronotopographical distribution patterns within developing and adult CNS, similar to those of macrophages/microglial cells in the quail CNS and confirmed by their histochemical labeling with Ricinus communis agglutinin I, a lectin that recognizes chick microglial cells. Therefore, the HIS-C7 antibody is a valuable tool to identify brain macrophage and microglial cells in studies of the function, development, and pathology of the chick brain. CD45 expression differed between chick microglia (as revealed with HIS-C7 antibody) and mouse microglial cells (as revealed with an antibody against mouse form of CD45). Thus, a discontinuous label was seen on mouse microglial cells with the anti-mouse CD45 immunostaining, whereas the entire surface of chick microglial cells was labeled with the anti-chick CD45 staining. The functional relevance of these differences between species has yet to be determined.     

中枢神经系统内神经元信号处理(英文)

一种新颖的轴突断端(axon bleb)膜片钳记录方法大力促进了中枢神经系统轴突功能的研究。我们的工作应用这一方法揭示了大脑皮层锥体神经元的数码信号(具全或无特性的动作电位)的爆发和传播机制。在轴突始段(axon initial segment,AIS)远端高密度聚集的低阈值Na+通道亚型Nav1.6决定动作电位的爆发;而在AIS近端高密度聚集的高阈值Na+通道亚型Nav1.2促进动作电位向胞体和树突的反向传播。应用胞体和轴突的同时记录,我们发现胞体阈下膜电位的变化可以在轴突上传播较长的距离并可到达那些离胞体较近的突触前终末。进一步的研究证明了胞体膜电位的变化调控动作电位触发的突触传递,该膜电位依赖的突触传递是一种模拟式的信号传递。轴突上一类特殊K+通道(Kv1)的活动调制动作电位的波形,特别是其波宽,从而调控各种突触前膜电位水平下突触强度的变化。突触前终末的背景Ca2+浓度也可能参与模拟信号的传递。这些发现深化了我们对中枢神经系统内神经信号处理基本原理的认识,进而帮助我们理解脑如何工作。     

JAK-STAT信号转导途径与中枢神经系统

作为一条新型信号转导通路,JAK－STAT广泛参与细胞的生长、分化等过程。但目前对该通路的研究主要集中在造血及免疫系统,对其在中枢神经系统（CNS）内的功能及作用机制的没有完全阐明。本文对JAK－STAT途径各成员在CNS内的表达、分布情况,以及该途径在CNS发育及病理状态下的功能变化进行了简要介绍。     

JAK-STAT信号转导途径与中枢神经系统

作为一条新型信号转导通路 ,JAK STAT广泛参与细胞的生长、分化等过程。但目前对该通路的研究主要集中在造血及免疫系统 ,对其在中枢神经系统 (CNS)内的功能及作用机制尚没有完全阐明。本文对JAK STAT途径各成员在CNS内的表达、分布情况 ,以及该途径在CNS发育及病理状态下的功能变化进行了简要介绍     

Superimposing spatially coherent electromagnetic noise inhibits field-induced abnormalities in developing chick embryos

Living cells exist in an electrically noisy environment. This has led to the so-called “signal-to-noise” problem whereby cells are observed to respond to extremely-low-frequency (ELF) exogenous fields that are several orders of magnitude weaker than local endogenous fields associated with thermal fluctuations. To resolve this dilemma, we propose that living cells are affected only by electromagnetic fields that are spatially coherent over their surface. The basic idea is that a significant number of receptors must be simultaneously and coherently activated (biological cooperativity) to produce effects on the biochemical functioning of the cell. However, like all physical detection systems, cells are subject to the laws of conventional physics and can be confused by noise. This suggests that a spatially coherent but temporally random noise field superimposed on a coherent ELF signal will defeat the mechanism of discrimination against noise, and any observed field-induced bioeffects would be suppressed. An experimental test of this idea was conducted using morphological abnormalities in developing chick embryos caused by electromagnetic field exposure as the endpoint. At an impressed noise amplitude comparable to the ELF field strength (but roughly one-thousandth of the thermal noise field), the increased abnormality rate observed with only the ELF field present was reduced to a level essentially the same as for the control embryos. © 1994 Wiley-Liss, Inc.