Development of cutaneous and proprioceptive afferent projections in the chick spinal cord.

Muscle and cutaneous nerves were individually labeled with DiI in chick embryos to examine the development of sensory afferent arborizations in the spinal cord. Initially, cutaneous and muscle arbors were similar; both types first entered the spinal gray matter at stage 28-29 (embryonic day (E) 6). Differences in projections were first observed by late stage 34 (E8.5): muscle afferent collaterals extended almost unbranched to the level of motoneuronal dendrites while cutaneous afferents branched frequently and remained within the dorsal horn. Projections of putative small caliber axons into laminae 1 and 2, located laterally in the chick, did not develop until E13-14.     

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。     

The initial appearance of the cranial nerves and related neuronal migration in staged human embryos

The initial development of the cranial nerves was studied in 245 human embryos of stages 10-23 (4-8 postfertilizational weeks). Significant findings in the human embryo include the following. (1) Neuronal migration is a characteristic feature in the development of all the cranial nerves at stages 13-18, with the exception of the somatic efferent group. (2) The somatic efferent and the visceral efferent neurons are arranged respectively in ventrolateral and ventromedial columns (stages 13-17). (3) The ventrolateral column gives rise to somatic efferent nuclei; the neurons of the hypoglossal nerve develop rapidly and show a segmental organization as four roots that innervate three of the four occipital somites (stage 13); the abducent nucleus becomes displaced rostrally by a change in the rhombomeric pattern at stage 16. (4) The ventromedial column, originally continuous in rhombomeres 2-7, gives rise to visceral efferent and pharyngeal efferent nuclei. (5) All the 'true' cranial nerves (III-XII) are recognizable by stage 16. (6) In a primary migration the visceral efferent neurons proceed mediolaterally and accumulate dorsolaterally as nuclei (stages 13, 14); they differentiate into salivatory nuclei (stages 16, 17). (7) A secondary migration involves the pharyngeal efferent neurons (of nerves V and IX-XI), which also proceed mediolaterally and then form ventrolateral nuclei (stages 17, 18). (8) The facial complex shows a distinctive development in that its neural crest arises from the lateral wall of the neural folds/tube. Moreover, the migration of its pharyngeal efferent neurons is delayed, which may be related to the formation of the internal genu, and the motor nucleus begins to appear only at stage 23. (9) The sequence of appearance of afferent constituents is: cranial ganglia (stage 12), mesencephalic trigeminal nucleus (stage 15), vestibular nuclei (stages 18-22), and cochlear nuclei (stage 19). The unsatisfactory term special is avoided and the term pharyngeal for air-breathing vertebrates replaces branchial. The six functional categories used here are vestibulocochlear, somatic afferent, visceral afferent, visceral efferent, pharyngeal efferent, and somatic efferent, together with appropriate abbreviations. The cardiac and hypoglossal neural crests are included, and it is emphasized that all the ectodermal placodes develop within the 'ectodermal ring'.     

The rat renal nerves during development

The prenatal and postnatal development of the innervation of the rat kidney has been investigated using immunocytochemical methods. The efferent innervation was studied using dopamine-beta-hydroxylase and neuropeptide Y antibodies. Calcitonin gene related peptide and substance P antibodies were used to investigate the afferent innervation. Kidneys from embryos of 14 to 20 days, from newborn rats, and from animals of 4, 10, 12, 21, 38, 60, and 90 days of age were studied. Slices of whole kidneys were analyzed, and frozen sections were used to investigate the location of the nerves in more detail. Both afferent and efferent nerves are observed inside the kidney by embryonic day 16. At birth, the afferent nerves are found (1) forming a rich plexus in the renal pelvis; (2) associated with the renal vasculature as far as the interlobular arteries (cortical radial arteries) and (3) in the corticomedullary connective tissue. The efferent innervation appears, at birth, to extend to the interlobular arteries and to the afferent arterioles of the perihilar juxtamedullary nephrons. The efferent innervation increases rapidly during the following days, and by postnatal day 21 a distribution of the innervation similar to that of the adult is observed. While the afferent innervation reaches the major target regions of the kidney by birth, the efferent does most of its expansion into the kidney postnatally. Afferent and efferent fibers are found, extrarenally and intrarenally, in the same nerve bundles. This proximity between afferent and efferent fibers may represent anatomical bases for their interaction in the adult as well as during development.Supported by U.S. Public Health Service Grant Rol 18340 from the National Institute of Health     

The development of functional innervation in the hind limb of the chick embryo.

1. The development of functional motor innervation was studied in the hind limb of chick embryos from Stages 25 to 43 by observing contraction of individual muscles and by recording the resultant tension when individual spinal nerves were electrically stimulated. 2. At later developmental stages (35-43) a given muscle always received functional innervation from specific spinal nerves. This pattern, with respect to the craniocaudal position of motoneurones, was similar to those described for amphibians and mammals. 3. The observed pattern was similar throughout development from the time that movement could first be elicited at Stages 27-28. There was no indication that motoneurones form initial synapses with inappropriate muscles. 4. Recordings from muscle nerves during excitation of individual spinal nerves gave results similar to the tension recordings, showing that even at early developmental stages muscle nerves did not contain substantial numbers of inappropriate axons. 5. Most limb muscles or primitive muscle masses became functionally innervated at the same time with no clearly defined proximo-distal sequence of limb innervation. 6. It appears that chick motoneurones are initially specified with respect to their peripheral destination and grow out selectively to synapse with appropriate muscles from the outset.     

神经生长导向因子Slit2在鸡胚脊髓发育中的表达

目的 观察神经生长导向因子Slit2在鸡胚神经管和脊髓不同发育时期的表达变化。方法 用免疫组织化学方法检测Slit2蛋白在鸡胚原肠期(HH6-HH10)神经管和第3d-17d(E3-E17期)脊髓中的表达和分布情况。结果 Slit2蛋白在鸡胚神经管和脊髓不同发育时期均有阳性表达,在脊髓中线结构区呈优势表达,在第9d(E9期)脊髓中线底板处表达最明显,第11d后Slit2蛋白阳性表达逐渐减弱并呈散在分布。结论 神经生长导向因子Slit2在鸡胚各发育时期神经管和脊髓的阳性表达呈动态变化。Slit2蛋白在脊髓发育过程起着重要作用。     

Rab23 GTPase is expressed asymmetrically in Hensen's node and plays a role in the dorsoventral patterning of the chick neural tube.

The mouse Rab23 protein, a Ras-like GTPase, inhibits signaling through the Sonic hedgehog pathway and thus exerts a role in the dorsoventral patterning of the spinal cord. Rab23 mouse mutant embryos lack dorsal spinal cord cell types. We cloned the chicken Rab23 gene and studied its expression in the developing nervous system. Chick Rab23 mRNA is initially expressed in the entire neural tube but retracts to the dorsal alar plate. Unlike in mouse, we find Rab23 in chick already expressed asymmetrically during gastrulation. Ectopic expression of Rab23 in ventral midbrain induced dorsal genes (Pax3, Pax7) ectopically and reduced ventral genes (Nkx2.2 and Nkx6) without influencing cell proliferation or neurogenesis. Thus, in the developing brain of chick embryos Rab23 acts in the same manner as described for the caudal spinal cord in mouse. These data indicate that Rab23 plays an important role in patterning the dorso-ventral axis by dorsalizing the neural tube.     

Spinal nerve segmentation in higher vertebrates: axon guidance by repulsion and attraction

The development of spinal nerve segmentation in higher vertebrate embryos provides a convenient experimental system for the analysis of axon guidance mechanisms. We review evidence from chick embryo experiments that segmentation of motor and sensory axons results from a combination of contact repulsion of axon growth cones by posterior somite cells and chemoattraction of growth cones by anterior cells. We also review progress in identifying the underlying molecular mechanisms in this system, and suggest a prominent role for carbohydrate groups in mediating growth cone repulsion.     

Patterning spinal nerves and vertebral bones

A prominent anatomical feature of the peripheral nervous system is the segmentation of mixed (motor, sensory and autonomic) spinal nerves alongside the spinal cord. During early development their axon growth cones avoid the developing vertebral elements by traversing the anterior/cranial half of each somite‐derived sclerotome, so ensuring the separation of spinal nerves from vertebral bones as axons extend towards their peripheral targets. A glycoprotein expressed on the surface of posterior half‐sclerotome cells confines growth cones to the anterior half‐sclerotomes by contact repulsion. A closely similar glycoprotein is expressed in avian and mammalian grey matter, where we hypothesize it may have evolved to regulate neural plasticity in birds and mammals.     

Slit2/Robo1信号对鸡胚早期神经管及体节发育的影响

目的: 探讨Slit2/Robo1对鸡胚早期神经管和体节发育的影响。 方法: 显微注射法将质粒注射入HH10期胚胎神经管内,活体胚胎细胞电穿孔方法转染胚胎半侧神经管,以另一侧神经管为对照侧,原位杂交及免疫荧光方法观察转染10 h后神经管的发育和神经嵴细胞迁移至体节的情况。结果: 下调Robo1侧神经管发育较正常对照侧异常,同时发现Slug表达和神经嵴细胞迁移至体节路线发生改变。结论: Slit2/Robo1信号可能通过影响Slug基因表达,对胚胎早期神经管闭合、神经嵴细胞正常产生及迁移方向以及体节分化有重要作用。     

Development of early swimming in Xenopus laevis embryos: myotomal musculature, its innervation and activation

The development of the axial musculature, its innervation and early locomotion in Xenopus laevis embryos are described. Between stages 17 and 40 some 45 myotomes are formed on each side of the body. During this period the animals develop from non-motile to free swimming embryos. Using fluorescein-conjugated bungarotoxin the acquisition of acetylcholine receptor-sites was studied. At stage 25 (early flexure stage) bound bungarotoxin was confined to the first seven intermyotomal clefts, in free swimming embryos (stage 33) to the first 20 clefts. Application of horseradish peroxidase to the intermyotomal clefts in embryos ranging from stages 25 to 37/38 revealed that primary motoneurons were usually situated 100-400 microns, i.e. 0.5-1.5 myotomes, rostral to the cleft they innervated. The motor axons left the spinal cord at the caudal side of each spinal segment where neural crest was present between the cord and the myotomes. At stage 25 ventral root activity could be recorded extracellularly from only the first three intermyotomal clefts, at stage 32/33 from the first 16 clefts. The first spontaneous rhythmic swimming-like activity could be recorded around stage 28. Between stages 27 and 32/33 the initial swimming frequency and the swimming episode duration increased at least three-fold. Comparable results were obtained with high-speed cinematography and measurements with a photoelectric transducer. Between stages 17 and 40 the number of myotomes increased by 0.9 myotome h, approximately 11.4 h later followed by the innervation of the myotomes at 0.7 cleft/h. About 3.6 h after this, ventral root activity appeared at the rate of 0.6 cleft h. This study shows that the early swimming pattern generating neuronal network, located within the rostral spinal cord, reaches a state of "critical mass" around stage 27, at which the first rhythmic swimming activity occurs. At least 6-10 functional spinal segments and adjacent myotomes are required for early swimming.     

Afferent and efferent axons in the medial and posterior articular nerves of the cat

The goal of this study is to determine the average numbers of afferent axons and postganglionic autonomic (sympathetic) efferent axons supplying the cat knee joint through the medial and posterior articular nerves. Interestingly, both nerves are composed primarily of unmyelinated axons. Only 20% of the axons in the medial articular nerve are myelinated, with the overwhelming majority, 80%, being unmyelinated. The posterior articular nerve has 78% unmyelinated and 22% myelinated axons. Neither nerve contains ventral root efferent axons. The sympathetic chain, in both nerves, contributes no myelinated and only 50% of the unmyelinated axons. The medial and posterior articular nerves are therefore predominantly afferent, since all myelinated and the remaining 50% of the unmyelinated axons arise from the dorsal root ganglion cell. The ratio of afferent unmyelinated to myelinated axons is 2:1. The roles of these afferent unmyelinated axons must now be considered in regard to joint kinesthetics and pain.     

The myth of ventrally emigrating neural tube (VENT) cells and their contribution to the developing cardiovascular system

The cardiac neural crest cells are a group of cells that emigrate from the dorsal side of the neural tube during a specific time window and contribute to the pharyngeal arch arteries and the aorticopulmonary septum of the heart. Recent publications have suggested that another group of cells emigrating from the ventral side of the neural tube also contributes to the developing cardiovascular system. The first aim of our study was to define the specific time window of cardiac neural crest cell migration by injecting a retrovirus containing a lacZ reporter gene into a chick embryo at different stages during development. The second aim was to study the contribution of the supposed ventrally emigrating neural tube cells to the cardiovascular system using three approaches. One approach was to inject a lacZ retrovirus into the lumen of the chick hindbrain. Secondly, we injected the retrovirus into the neural tube at the position of the 10-12 somite pair. Finally, we used the chimera technique in which we transplanted a quail neural tube segment into a chick embryo. Cardiac neural crest cells were shown to emigrate from the dorsal side of the neural tube between HH9 and HH13(-). The HH13(+) neural tube has ceased to produce cardiac neural crest cells between the level of the otic placode and the fourth pair of somites. Retroviral injection directly into the chick hindbrain at HH14 resulted in 50% of the embryos with minimal labeling of the hindbrain and intense labeling of the adjacent mesenchyme, suggesting that virus was spilled. This implies that this technique is not useful for confirming the existence of ventrally emigrating cells. Both retroviral injections into the neural tube lumen at HH14 at the position of the 10-12 somite pair and the chimeras showed no signs of ventrally emigrating neural tube cells. We conclude that there is no contribution of ventral neural tube cells to the developing cardiovascular system.     

Distribution of different regions of cardiac neural crest in the extrinsic and the intrinsic cardiac nervous system.

In this study we focused upon whether different levels of postotic neural crest as well as the right and left cardiac neural crest show a segmented or mixed distribution in the extrinsic and intrinsic cardiac nervous system. Different parts of the postotic neural crest were labeled by heterospecific replacement of chick neural tube by its quail counterpart. Quail-chick chimeras (n = 21) were immunohistochemically evaluated at stage HH28+, HH29+, and between HH34-37. In another set of embryos, different regions of cardiac neural crest were tagged with a retrovirus containing the LacZ reporter gene and evaluated between HH35-37 (n = 13). The results show a difference in distribution between the right- and left-sided cardiac neural crest cells at the arterial pole and ventral cardiac plexus. In the dorsal cardiac plexus, the right and left cardiac neural crest cells mix. In general, the extrinsic and intrinsic cardiac nerves receive a lower contribution from the right cardiac neural crest compared with the left cardiac neural crest. The right-sided neural crest from the level of somite 1 seeds only the cranial part of the vagal nerve and the ventral cardiac plexus. Furthermore, the results show a nonsegmented overlapping contribution of neural crest originating from S1 to S3 to the Schwann cells of the cranial and recurrent nerves and the intrinsic cardiac plexus. Also the Schwann cells along the distal intestinal part of the vagal nerve are derived exclusively from the cardiac neural crest region. These findings and the smaller contribution of the more cranially emanating cardiac neural crest to the dorsal cardiac plexus compared with more caudal cardiac neural crest levels, suggests an initial segmented distribution of cardiac neural crest cells in the circumpharyngeal region, followed by longitudinal migration along the vagal nerve during later stages.     

Catecholamine accumulation in neural crest cells and the primary sympathetic chain

Catecholamine accumulation in chick embryos of stages 16 to 24 was investigated using formaldehyde-induced fluorescence. Fluorescence first appeared at stage 21 in the anterior sympathetic chain. After L-DOPA treatment, this fluorescence appeared at stage 18. Noradrenaline could not advance the onset of fluorescence or reconstitute fluorescence after its depletion by reserpine at stages 22 to 24. Under no conditions could fluorescence be identified in neural crest cells prior to their aggregation to form the primary sympathetic chain. Noradrenaline induced fluorescence in the neural tube, notochord, myotome, sclerotome, gut mesenchyme and suprarenal cortical cells. In addition to these structures, the dorsal pancreas and some blood cells were fluorescent after 1-DOPA treatment. The implication of the results for the neural crest origin of APUD (Amine Precursor Uptake Decaboxylase) cells is considered.     

Arrangement and innervation of the iliocostalis and longissimus muscles of the brown caiman (Caiman crocodilus fuscus: Alligatoridae,crocodilia)

The axial musculature of the brown caiman was investigated in detail with particular attention to the nerve supply, using a binocular stereomicroscope. Due to the prominent development of the longissimus (Lo) and the iliocostalis (IC) muscles of the caiman, the pattern of distribution of the spinal nerves in the body wall was unique; there also was less differentiation of the external intercostalis. There were four primary divisions of the spinal nerves in the thoracic region of the caiman, from ventral to dorsal: the intercostal nerve, the IC nerve, the Lo nerve, and the dorsal main trunk. Thus, the classic concept of the organization of the spinal nerves may not be suitable for the caiman. These findings suggest that evolutionary changes in the dorsolateral axial musculature have brought about the rearrangement of the organization of the spinal nerves. In addition, each clearly segmented myotome of the Lo and IC was innervated by more than two segments of the spinal nerves (pluriseg-mental innervation). The manner of formation of the myotome and its innervation is discussed from the viewpoint of comparative and developmental anatomy.     

GAP-43 expression in developing cutaneous and muscle nerves in the rat hindlimb.

The expression of the growth associated protein, GAP-43, in developing rat hindlimb peripheral nerves has been studied using immunocytochemistry. GAP-43, is first detected in lumbar spinal nerves at embryonic day (E)12 as the axons grow to the base of the hindlimb. It is expressed along the whole length of the nerves as well as in the growth cones. GAP-43 staining becomes very intense over the next 36 h while the axons remain in the plexus region at the base of the limb bud before forming peripheral nerves at E14. It remains intense along the length of the growing peripheral nerves, the first of which are cutaneous, branching away from the plexus and growing specifically to the skin, their axon tips penetrating the epidermis of the proximal skin at E15 and the toes at E19. GAP-43-containing terminals form a dense plexus throughout the epidermis which subsequently withdraws subepidermally in the postnatal period. GAP-43 staining is also evident along the growing muscle nerves during muscle innervation, which follows behind that of skin. Axons branch over the surface of proximal muscles at E15 but do not form terminals until E17. As target innervation proceeds, GAP-43 staining declines in the proximal part of the nerve but remains intense in the distal portions. Overall GAP-43 expression in the hindlimb decreases in the second postnatal week as axon growth and peripheral terminal formation decline.     

Efferent synapses return to inner hair cells in the aging cochlea

Efferent innervation of the cochlea undergoes extensive modification early in development, but it is unclear if efferent synapses are modified by age, hearing loss, or both. Structural alterations in the cochlea affecting information transfer from the auditory periphery to the brain may contribute to age-related hearing deficits. We investigated changes to efferent innervation in the vicinity of inner hair cells (IHCs) in young and old C57BL/6 mice using transmission electron microscopy to reveal increased efferent innervation of IHCs in older animals. Efferent contacts on IHCs contained focal presynaptic accumulations of small vesicles. Synaptic vesicle size and shape were heterogeneous. Postsynaptic cisterns were occasionally observed. Increased IHC efferent innervation was associated with a smaller number of afferent synapses per IHC, increased outer hair cell loss, and elevated auditory brainstem response thresholds. Efferent axons also formed synapses on afferent dendrites but with a reduced prevalence in older animals. Age-related reduction of afferent activity may engage signaling pathways that support the return to an immature state of efferent innervation of the cochlea.     

An electron microscope study of vagus nerve composition in the ferret

Summary The total number of axons in the cervical and abdominal vagus nerves of the ferret was counted. The ratio of myelinated to non-myelinated, and afferent to efferent axons was determined. The fibre diameter spectrum of myelinated axons was measured. The total number of axons in the ferret cervical vagus is similar to other mammals (approximately 28,000); the majority of axons are afferent (approx. 24,000) and also the majority of axons are nonmyelinated (approx, 27,000). The dorsal abdominal trunk is about twice the size of the ventral trunk although both trunks have the same number of efferent axons. The abdominal vagal trunks are over 90% afferent.     

The central nervous system efferent control of the organs of balance and equilibrium.

The vestibular labyrinth is innervated by both primary afferent nerves and efferent axons with cell bodies located in the central nervous system. Efferent terminals are found on both hair cells and on primary afferent axons. Acetylcholine is the major efferent transmitter, but enkephalin and calcitonin gene-related peptide (CGRP) have also been localized to efferent terminals and somata. The efferent vestibular nuclei are bilaterally organized in the majority of species. Semicircular canal primary afferents have been classified by their sensitivity and phase in response to rotation. Electrical activation of efferents in monkey and fish increases afferent resting discharge and reduces afferent gain to adequate stimulation. Effects are most profound on high-gain, phase-advanced (re. velocity) afferents. Experiments in alert animals indicate that multiple sensory modalities can activate the efferent system.