Xenopus sonic hedgehog guides retinal axons along the optic tract

The role of classic morphogens such as Sonic hedgehog (Shh) as axon guidance cues has been reported in a variety of vertebrate organisms (Charron and Tessier‐Lavigne [ 2005 ] Development 132:2251–2262). In this work, we provide the first evidence that Xenopus sonic hedgehog (Xshh) signaling is involved in guiding retinal ganglion cell (RGC) axons along the optic tract. Xshh is expressed in the brain during retinal axon extension, adjacent to these axons in the ventral diencephalon. Retinal axons themselves express Patched 1 and Smoothened co‐receptors during RGC axon growth. Blocking Shh signaling causes abnormal ventral pathfinding, and targeting errors at the optic tectum. Misexpression of exogenous N‐Shh peptide in vivo also causes pathfinding errors. Retinal axons grown in culture respond to N‐Shh in a dose‐dependent manner, either by decreasing extension at lower concentrations, or retracting axons in the presence of higher doses. These data suggest that Shh signaling is required for normal RGC axon pathfinding and tectal targeting in the developing visual system of Xenopus. We propose that Shh serves as a ventral optic tract repellent that helps to define the caudal boundary for retinal axons in the diencephalon, and that this signaling is also required for initial target recognition at the optic tectum. Developmental Dynamics 239:2921–2932, 2010. © 2010 Wiley‐Liss, Inc.     

双色荧光示踪鸡胚脊髓两侧连合纤维投射研究方法的建立

目的建立1种双色荧光示踪鸡胚脊髓两侧连合纤维投射的实验方法。方法鸡胚孵育至胚龄2.5~3d,通过鸡胚活体原位电转基因技术将携带有报告基因绿色荧光蛋白(GFP)的质粒(p CAGGS-GFP)准确注射到鸡胚脊髓腔,实现定时、定位活体电转基因。转染后继续孵育至6d,取GFP阳性表达的胚胎,部分做脊髓横向切片,部分利用open-book技术将脊髓展开观察连合纤维的发育情况,每组至少取3个标本。其后在脊髓非转染侧连合神经元所在之处,点状注射Di I乙醇溶液,封片后于4℃避光孵育3d,在荧光显微镜下观察脊髓连合纤维投射情况。结果脊髓横切及open-book结果显示,鸡胚脊髓GFP阳性转染侧的神经元轴突穿过底板投射到脊髓对侧;同时在open-book结果中还可观察到,转染侧轴突穿过底板后分别沿腹索和外侧索向头尾部投射;Di I标记的非转染侧连合神经元轴突也同样穿过底板投射到对侧,并在侧索白质内延伸。结论本实验成功建立了1种双色荧光示踪鸡胚脊髓两侧连合纤维投射的研究方法,为研究脊髓神经发育提供技术保障。     

Transmitter phenotypes of commissural interneurons in the lamprey spinal cord

The fundamental network for locomotion in all vertebrates contains a central pattern generator or CPG that produces the required motor output in the spinal cord. In the lamprey spinal cord different classes of interneuron's forming the core CPG circuitry have been characterized based on their morphological and electrophysiological features. The commissural interneuron's (C-INs) represent one essential component of CPG that have been implicated in controlling left–right alternation of the motor activity during swimming. However, it is still unclear if the C-INs displays a homogenous neurotransmitter phenotype and how they are distributed. In this paper we investigated the segmental distribution of glycine, glutamate and GABA-immunoreactive (ir) C-INs by combining retrograde Neurobiotin tracing with specific antibodies for these transmitters. The C-INs were more abundant in caudal and rostral segments adjacent to the injection site and their number gradually decreased in more distal segments, suggesting that these interneurons project over a short distance. The glycine-ir neurons represented around 50% of the total C-INs, while glutamate-ir neurons represented only 29%. Both types of C-INs were homogenously distributed over different segments along the spinal cord. Finally, no Neurobiotin labeled C-INs displayed GABA-ir, although many interneurons were ir to GABA, suggesting that GABAergic interneurons are not directly responsible for controlling left–right alternation of activity during locomotion in lamprey. Overall, these results show that the C-INs display a gradual rostrocaudal distribution and consist of both glycine- and glutamate-ir neurons. The difference in the proportion of inhibitory and excitatory C-INs represents an anatomical substrate that can ensure the predominance of alternating activity during locomotion.     

Growth of axons in organotypical spinal cord culture

Laboratory of Neuromorphology, I. S. Beritashvili Institute of Physiology, Academy of Sciences of the Georgian SSR, Tbilisi. (Presented by Academician of the Academy of Medical Sciences of the USSR M. M. Khananashvili.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 2, pp. 186–188, February, 1990.     

Depolarizing afterpotentials in myelinated axons of mammalian spinal cord

Microelectrode recordings were made from 5-10 micron dia axons of adult rat spinal cord in vitro. Action potentials in response to electrical stimulation were recorded intracellularly and electrical characteristics of the axons were examined by injecting current pulses through a bridge circuit. All action potentials larger in amplitude than 80 mV were followed by depolarizing afterpotentials, similar to those recorded in peripheral axons [Barrett and Barrett (1982) J. Physiol., Lond. 323, 117-144]. The afterpotential could be described as the sum of three exponential components, the time constants of which (tau 1, tau 2 and tau 3) were 25.2 +/- 5.6, 3.1 +/- 0.8 and 0.8 +/0 0.3 ms, respectively, at 25 degrees C and a membrane potential of -80 mV. The maximal amplitudes of the afterpotential components, obtained by extrapolating to the peak of the action potential, were 3.8 +/- 1.0, 6.4 +/- 5.2 and 21.7 +/- 9.8 mV, for action potential amplitudes of 102 +/- 11 mV. The amplitude of the longest component of the afterpotential decreased with depolarization and increased with hyperpolarization at the recording site. The amplitude decreased markedly with increase of temperature to physiological levels, in conjunction with the expected decrease in action potential duration. Similar afterpotential components were present in the response of the axon to injected hyperpolarizing current pulses. The observations are consistent with the suggestion [Barrett and Barrett (1982) J. Physiol., Lond. 323, 117-144] that the afterpotential results from charging of the axolemmal capacitance by current passing through the myelin sheath during the action potential. They are inconsistent with a number of calculations of electrical characteristics of peripheral axons derived from voltage clamp experiments in isolated fibers. It is argued that the electrical resistance of the myelin lamellae is relatively low, though within the range calculated for other glial membranes. This suggestion is found more compatible with the available morphological data than the alternative proposal that a leakage pathway under the myelin sheath might be responsible for the afterpotential [Barrett and Barrett (1982) J. Physiol., Lond. 323, 117-144]. The significance of this organization for the function of myelinated axons and the electrical basis of the afterpotential are examined further in the accompanying paper [Blight (1985) Neuroscience 15, 13-31].     

Serotonin modulates the properties of ascending commissural interneurons in the neonatal mouse spinal cord

The interneuron populations that constitute the central pattern generator (CPG) for locomotion in the mammalian spinal cord are not well understood. We studied the properties of a set of commissural interneurons whose axons cross and ascend in the contralateral cord (aCINs) in the neonatal mouse. During N-methyl-D-aspartate (NMDA) and 5-HT-induced fictive locomotion, a majority of lumbar (L2) aCINs examined were rhythmically active; most of them fired in phase with the ipsilateral motoneuron pool, but some fired in phase with contralateral motoneurons. 5-HT plays a critical role in enabling the locomotor CPG to function. We found that 5-HT increased the excitability of aCINs by depolarizing the membrane potential, reducing the postspike afterhyperpolarization amplitude, broadening the action potential, and decreasing the action potential threshold. Serotonin had no significant effect on the input resistance and sag amplitude of aCINs. These results support the hypothesis that aCINs play important roles in coordinating left-right movements during fictive locomotion and thus may be component neurons in the locomotor CPG in neonatal mice.     

Patterns of schwann cell myelination of axons within the spinal cord

Patterns of Schwann cell myelination of long-projecting axons in the spinal cord were studied. The goal was to determine if such axons arising from neurons whose somata and processes are normally confined to the central nervous system can interact effectively with Schwann cells, the myelinating cells of the peripheral nervous system. In one paradigm Schwann cells develop in the dorsal funiculi of the lumbar spinal cord subsequent to radiation-induced alterations in development of the glial populations. Light and electron microscopic evaluations were made in the region of the corticospinal tracts (CSTs), which in the rat occupy the base of the dorsal funiculi. At 90 days following irradiation, larger axons of these tracts (> 1.5 μm in diameter) were myelinated by Schwann cells, and smaller axons were ensheathed by them. In the second paradigm cultured Schwann cells were injected into the medial portions of the ventral funiculi at 13 days post-irradiation when the glial population was markedly reduced. Earlier investigations from this laboratory demonstrated that Schwann cells do not develop in the irradiated ventral funiculi, as they do dorsally. When placed in proximity to long-projecting axons in the medial portion of the ventral funiculi, the Schwann cells either formed compact myelin sheaths or ensheathed axons, depending upon their diameter. Fasciculation and presence of collagen were characteristic of this paradigm but were absent from the Schwann cell-occupied regions of the CSTs. This probably relates to the presence of fibroblasts in the injected cultures. These studies demonstrate that long-projecting axons of the spinal cord are capable of interacting with Schwann cells, irrespective of the mechanism by which these cells gain access to the central nervous system.     

Differential expression of Hoxa-2 protein along the dorsal-ventral axis of the developing and adult mouse spinal cord.

We have used synthetic oligopeptides derived from the coding sequence of the murine Hoxa-2 protein to produce polyclonal antibodies that specifically recognize the Hoxa-2 recombinant protein. Immunohistochemical studies reveal a distinct pattern of spatial and temporal expression of Hoxa-2 protein within the mouse spinal cord which is concomitant with the cytoarchitectural changes occurring in the developing cord. Hoxa-2 protein is predominantly detected in the nuclei of cells in the ventral mantle region of 10-day-old mouse embryos. Islet-1, a marker for motor neurons was also shown to be co-localized with Hoxa-2 in nuclei of cells in this region. As development progresses from 10-days to 14-days of gestation, Hoxa-2 protein expression gradually extends to the dorsal regions of the mantle layer. The Hoxa-2 protein expression pattern changes at 16-days of embryonic development with strong expression visible throughout the dorsal mantle layer. In 18-day-old and adult mouse spinal cords, Hoxa-2 protein was expressed predominantly by cells of the dorsal horn and only by a few cells of the ventral horn. Double labeling studies with an antibody against glial fibrillary acidic protein (GFAP, an astrocyte-specific intermediate filament protein) showed that within the adult spinal cord, astrocytes rarely expressed the Hoxa-2 protein. However, Hoxa-2 and GFAP double-labeled astrocytes were found in the neopallial cultures, although not all astrocytes expressed Hoxa-2. Hoxa-2 expressing oligodendrocyte progenitor cells were also identified after double-labeling with O4 and Hoxa-2 antibodies; although cells in this lineage that have begun to develop a more extensive array of cytoplasmic processes were less likely to be Hoxa-2 positive. The early pattern of Hoxa-2 protein expression across transverse sections of the neural tube is temporally and spatially modified as each major class of neuron is generated. This congruence in the expression of the Hoxa-2 protein and the generation of neurons in the cord suggests that the Hoxa-2 protein may contribute to dorsal-ventral patterning and/or to the specification of neuronal phenotype. Dev Dyn 1999;216:201-217.     

Growing corticospinal axons by-pass lesions of neonatal rat spinal cord

The anterograde transport of horseradish peroxidase was used to label newly growing corticospinal axons after they had entered lesioned regions of the neonatal rat spinal cord. Two types of lesions were made at thoracic and lumbar levels before the arrival of the first corticospinal axons. (1) Thermal lesions were produced by the brief application of a heated rod to the vertebral column and could destroy the normal growth path over several spinal segments. Corticospinal axons, when successful in growing distal to thermal lesions, did so at the same rate as in normal controls and retaining their normal relative positions and morphology, especially fasciculation. (2) Surgical lesions were produced by cutting the spinal cord and were limited to one segment but could result in a barrier in the normal growth path composed of a cyst or glial scar. Corticospinal axons that succeeded in growing distal to a surgical lesion did so by being deflected to unusual positions, became defasciculated, and sometimes their normal growth rate was slowed. That corticospinal axons could in many instances grow past the two types lf lesion suggests that a morphologically stereotyped glial scaffolding is not necessary for axon growth. The role of fasciculation in normal axon growth is highlighted by the disparate effects of the two types of lesion.     

Control of membrane sealing in injured mammalian spinal cord axons

The process of sealing of damaged axons was examined in isolated strips of white matter from guinea pig spinal cord by recording the "compound membrane potential," using a sucrose-gap technique, and by examining uptake of horseradish peroxidase (HRP). Following axonal transection, exponential recovery of membrane potential occurred with a time constant of 20 +/- 5 min, at 37 degrees C, and extracellular calcium activity ([Ca(2+)](o)) of 2 mM. Most axons excluded HRP by 30 min following transection. The rate of sealing was reduced by lowering calcium and was effectively blocked at [Ca(2+)](o) 相似文献   

Multiple axon collaterals of single corticospinal axons in the cat spinal cord

To investigate intraspinal branching patterns of single corticospinal neurons (CSNs), we recorded extracellular spike activities from cell bodies of 408 CSNs in the motor cortex in anesthetized cats and mapped the distribution of effective stimulating sites for antidromic activation of their terminal branches in the spinal gray matter. To search for all spinal axon branches belonging to single CSNs in the "forelimb area" of the motor cortex, we microstimulated the gray matter from the dorsal to the ventral border at 100-micron intervals at an intensity of 150-250 microA and systematically mapped effective stimulating penetrations at 1-mm intervals rostrocaudally from C3 to the most caudal level of their axons. From the depth-threshold curves, the comparison of the antidromic latencies of spikes evoked from the gray matter and the lateral funiculus, and the calculated conduction times of the collaterals, we could ascertain that axon collaterals were stimulated in the gray matter rather than stem axons in the corticospinal tract due to current spread. Virtually all CSNs examined in the forelimb area of the motor cortex had three to seven branches at widely separated segments of the cervical and the higher thoracic cord. In addition to terminating at the brachial segments, they had one to three collaterals to the upper cervical cord (C3-C4), where the propriospinal neurons projecting to forelimb motoneurons are located. About three quarters of these CSNs had two to four collaterals in C6-T1. This finding held true for both fast and slow CSNs. About one third of the CSNs in the forelimb area of the motor cortex projected to the thoracic cord below T3. These CSNs also sent axon collaterals to the cervical spinal cord. CSNs in the "hindlimb area" of the motor cortex had three to five axon branches in the lumbosacral cord. These branches were mainly observed at L4 and the lower lumbosacral cord. None of these CSNs had axon collaterals in the cervical cord. CSNs terminating at different segments of the cervical and the thoracic cord were distributed in a wide area of the motor cortex and were intermingled. To determine the detailed trajectory of single axon branches, microstimulation was made at a matrix of points of 100 or 200 micron at the maximum intensity of 30 microA, and their axonal trajectory was reconstructed on the basis of the location of low-threshold foci and the latency of antidromic spikes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conduction properties of spinal cord axons in the myelin-deficient rat mutant.

Spinal cords of myelin-deficient and normal age-matched (control) rats were removed and their conduction and pharmacological properties studied in an in vitro brain slice chamber. The conduction velocity of the myelin-deficient dorsal column axons was reduced to about 25% of control axons; however, the amyelinated myelin-deficient axons displayed refractory periods and the ability to sustain high-frequency action potential discharge similar to that of dorsal column axons in control rats. Pharmacological results suggest that the myelin-deficient dorsal column axons qualitatively express a normal complement of ion channels and receptors. The demonstration of a normal representation of channels and receptors on these axons supports the proposal that the oligodendrocyte, and not the axon, is the site of the primary defect in the myelin-deficient rat mutant. It is concluded that, unlike acutely demyelinated axons which display marked frequency-dependent conduction block, amyelinated axons of the myelin-deficient rat spinal cord develop compensatory mechanisms to stabilize action potential conduction.     

Dual projections of secondary vestibular axons in the medial longitudinal fasciculus to extraocular motor nuclei and the spinal cord of the squirrel monkey

Summary Recordings were made from secondary vestibular axons in the medial longitudinal fasciculus (MLF) of barbiturate-anesthetized squirrel monkeys. Antidromic stimulation techniques were used to identify the axons as belonging to one of three classes of neurons: vestibulo-oculo-collic (VOC) neurons project both to the extraocular motor nuclei and to the spinal cord; vestibulo-ocular (VO) neurons do not have a spinal projection; and vestibulocollic (VC) neurons do not have an oculomotor projection. Galvanic stimulation was used to show that axons of all three classes received excitatory inputs from one labyrinth and inhibitory inputs from the other. VOC axons were confined to the MLF contralateral to the labyrinth from which they were excited. They made up more than half of the vestibular axons descending in the contralateral medial vestibulospinal tract (MVST), but less than one-quarter of those ascending in the contralateral MLF to the level of the oculomotor nucleus. Spinal projections were restricted to cervical segments with about half of the axons reaching segment C6. Conduction velocities, measured for Co-projecting axons, were similar for VOC and VC axons and were typically 25–50 m/s. Unlike the situation in the rabbit (Akaike et al. 1973) and cat (Akaike 1983), none of the MVST axons had conduction velocities > 75 m/s. The morphology of VOC neurons was studied by injection of horseradish peroxidase (HRP) into 60 physiologically identified axons in the MLF. Since individual axons were only stained for short distances, it was not possible to ascertain their complete branching patterns. Labeled fibers could be traced to an origin in and around the ventral lateral vestibular nucleus. This localization was confirmed by comparing the distributions within the vestibular nuclei of neurons retrogradely labeled from the upper cervical spinal cord (this study) and from the oculomotor nucleus (McCrea et al. 1987a; Highstein and McCrea 1988). VOC axons reached the contralateral MLF at the level of the abducens nucleus and immediately divided into an ascending and a descending, usually thicker, branch. Seven VOC axons could be traced to the extraocular motor nuclei; three terminated in the medial aspect of the oculomotor nucleus bilaterally and four terminated in the medial aspect of the contralateral abducens nucleus. The former axons may be part of a crossed, excitatory anterior-canal pathway; the latter, part of a similar horizontal-canal pathway. There were no terminations in the trochlear nucleus even though 12 labeled fibers passed close to it. VOC axons projected to several brainstem nuclei, including the contralateral interstitial nucleus of Cajal, cell groups in the region of the medial longitudinal fasciculus rostral to the abducens nucleus, the nucleus prepositus, the nucleus raphe obscuris, Roller's nucleus, and the paramedian medullary reticular formation. Virtually all of the above connections, except for the bilateral projection to the oculomotor nucleus, were contralateral to the cells of origin. The results in the squirrel monkey are compared with previous studies of VOC neurons in the cat (Isu and Yokota 1983; Uchino and Hirai 1984; Isu et al. 1988). In both species, VOC neurons make up a large proportion of contralaterally projecting MVST fibers. On the other hand, such dual-projecting neurons may provide a considerably smaller fraction of the secondary vestibular axons reaching the oculomotor nucleus in the monkey than they do in the cat.     

Left cardiac isomerism in the Sonic hedgehog null mouse

Sonic hedgehog (Shh) is a secreted morphogen necessary for the production of sidedness in the developing embryo. In this study, we describe the morphology of the atrial chambers and atrioventricular junctions of the Shh null mouse heart. We demonstrate that the essential phenotypic feature is isomerism of the left atrial appendages, in combination with an atrioventricular septal defect and a common atrioventricular junction. These malformations are known to be frequent in humans with left isomerism. To confirm the presence of left isomerism, we show that Pitx2c, a recognized determinant of morphological leftness, is expressed in the Shh null mutants on both the right and left sides of the inflow region, and on both sides of the solitary arterial trunk exiting from the heart. It has been established that derivatives of the second heart field expressing Isl1 are asymmetrically distributed in the developing normal heart. We now show that this population is reduced in the hearts from the Shh null mutants, likely contributing to the defects. To distinguish the consequences of reduced contributions from the second heart field from those of left–right patterning disturbance, we disrupted the movement of second heart field cells into the heart by expressing dominant‐negative Rho kinase in the population of cells expressing Isl1. This resulted in absence of the vestibular spine, and presence of atrioventricular septal defects closely resembling those seen in the hearts from the Shh null mutants. The primary atrial septum, however, was well formed, and there was no evidence of isomerism of the atrial appendages, suggesting that these features do not relate to disruption of the contributions made by the second heart field. We demonstrate, therefore, that the Shh null mouse is a model of isomerism of the left atrial appendages, and show that the recognized associated malformations found at the venous pole of the heart in the setting of left isomerism are likely to arise from the loss of the effects of Shh in the establishment of laterality, combined with a reduced contribution made by cells derived from the second heart field.     

Properties of axons of sympathetic preganglionic neurons of the lower thoracic spinal cord

Sympathetic preganglionic fibers arising in spinal cord segments T7-T10. were investigated. Conduction velocity, excitability and refractory period were obtained by recording the antidromic wave in the ventral roots and the antidromic discharge in sympathetic preganglionic lateral horn neurons. Conduction along the spinal part of the axons is slower than along the peripheral. In four sympathetic preganglionic neurons inhibition of subsequent antidromic responses took place after antidromic stimulation. This suggests that axons of these neurons have recurrent collaterals.