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The development of the human embryonic spinal cord is very complicated, and many cell types are involved in the process. However, the morphological characteristics of neuronal and glial cells during the development of the human fetal spinal cord have not been described. We investigated the systemic distributions and expression pattern of the cell type-specific markers Neuron-specific nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP) during the development of the human fetal spinal cord, in order to clarify the detailed developmental changes of neuronal and glial cells in chronological and spatial aspects. A total of 35 fetuses, aged 3 weeks to 8 months of gestation (E3W–E8M), were studied. The markers used for immunohistochemical study were NeuN and GFAP. The intracellular makers NeuN and GFAP were widely detected expression in different structures and cells during the development of the human fetal spinal cord, including the following: central canal, neuroepithelial layer, internal limiting membrane, mantle layer, marginal layer, basal plate, alar plate, ependymal layer, gray matter, white matter, neuron, astrocytes, and nerve fibers. However, there was an absence of GFAP in astrocytes during early fetal spinal cord development until E9W, and the appearance of GFAP-positive reactivity was later than that of neurons. We consider that NeuN and GFAP can be used to identify neuronal and glial cells during the development of the human fetal spinal cord, and their distribution differs both chronologically and spatially. These characteristic expression patterns would give us a clue to better understand the developmental characteristics of the human spinal cord.

The spinal cord originates from the primitive neural tube, the dorsal horn arises from the alar plate, and the anterior horn arises from the basal plate. The adult cord is located in the spinal dural sac (dura + arachnoid), is continuous with the medulla at the spinal-medulla junction, and extends to spinal levels L1 to L2. Cervical spinal levels are characteristically oval in shape with large amounts of gray matter and white matter, thoracic levels are round with a very reduced gray matter and less white matter, and lumbosacral levels are also round but with large amounts of gray matter and significantly reduced white matter. Spinal nerves are formed by the joining of the posterior root (two sensory components) and ventral root (two motor components) at about the intervertebral foramen. Spinal reflexes are integral parts of most neurologic examinations; all have an afferent limb, an efferent limb, and various degrees of processing of information within the spinal cord. In addition to the sensor-motor features of the spinal reflex, collaterals of primary sensory fibers ascend in spinal tracts that convey information to higher levels of the central nervous system (CNS) for the perception of the sensory data; much of this sensory input may be transmitted to motor centers that give rise to descending tracts to the spinal cord. A wide variety of spinal-related deficits may results from trauma, neurodegenerative and neurotransmitter diseases, and lesions of the CNS above the spinal-medulla junction.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians. (C) 2017 Elsevier B.V. All rights reserved.

In contrast to mammals, lampreys are capable of recovering apparently normal locomotion after complete spinal cord transection, and the spinal axons regenerate selectively in their correct paths. Descending serotonergic projections to the spinal cord play a role in the modulation of locomotion at spinal levels in both mammals and lampreys. In this study, we used combined immunofluorescence and tract-tracing techniques to show that in the sea lamprey, serotonergic descending neurons of the caudal rhombencephalon (vagal nucleus) regenerate their axons across the lesion site after complete spinal cord transection. The spinal cord of mature larval sea lampreys was transected at the level of the fifth gill, then after a recovery period of 5 months, the spinal cord was exposed again, 1 mm caudal to the injury site, and the tracer Neurobiotin(™) was applied. Double-labeled cells were observed in the caudal portion of the serotonin-immunoreactive vagal nucleus of the caudal rhombencephalon. In order to investigate whether the reinnervation was due to sprouting from axons above the injury site or to regeneration of axotomized axons, the experiments were performed again, but the tracer Fluoro-Gold(™) was applied at the time of transection. Triple-labeled cells were observed in the vagal nucleus, indicating that at least part of the reinnervation corresponds to true regeneration. This study provides a new and interesting model for investigating the intrinsic molecular mechanisms involved in regeneration of the serotonergic descending axons in vertebrates. Use of this model may provide valuable information for proposing new therapies for patients with spinal cord injury.

Mutations in PLA2G6, which encodes calcium-independent phospholipase A2 group VIA (iPLA2-VIA), underlie the autosomal recessive disorder infantile neuroaxonal dystrophy (INAD). INAD typically presents in the first year of life, and leads to optic atrophy and psychomotor regression. We have examined PLA2G6 expression in early human embryonic development by in situ hybridization. At Carnegie Stage (CS) 19 (approximately 7 post conception weeks [PCW]), strong expression is evident in the ventricular zone (VZ) of midbrain and forebrain suggestive of expression in neural stem and progenitor cells. At CS23 (8 PCW) expression is also detectable in the VZ of the hindbrain and the subventricular zone (SVZ) of the developing neocortex, ganglionic eminences and diencephalon. By 9 PCW strong expression in the post-mitotic cells of the cortical plate can be seen in the developing neocortex. In the eye, expression is seen in the lens and retina at all stages examined. PLA2G6 expression is also evident in the alar plate of the spinal cord, dorsal root ganglia, the retina and lens in the eye and and several non-neuronal tissues, including developing bones, lung, kidney and gut. These findings suggest a role for PLA2G6 in neuronal proliferation throughout the developing brain and in maturing neurons in the cortical plate and hindbrain. Although widespread PLA2G6 expression is detected in neuronal tissues, the pattern shows dynamic changes with time and indicates that INAD pathogenesis may begin prior to birth.

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. Developmental Dynamics 236:2993–3006, 2007. © 2007 Wiley-Liss, Inc.

Objective—To analyze the morphology, cytoarchitecture, and lumbosacral spinal cord projections of the red nucleus (RN) in cattle. Animals—8 healthy Friesian male calves. Procedures—Anesthetized calves underwent a dorsal laminectomy at L5. Eight bilateral injections (lateral to the midline) of the neuronal retrograde fluorescent tracer fast blue (FB) were administered into the exposed lumbosacral portion of the spinal cord. A postsurgical calf survival time of 38 to 55 days was used. Following euthanasia, the midbrain and the L5-S2 spinal cord segments were removed. Nissl's method of staining was applied on paraffin-embedded and frozen sections of the midbrain. Results—The mean length of the RN from the caudal to cranial end ranged from 6,680 to 8,640 μm. The magnocellular and parvicellular components of the RN were intermixed throughout the nucleus, but the former predominate at the caudal portion of the nucleus and the latter at the cranial portion with a gradual transitional zone. The FB-labeled neurons were found along the entire craniocaudal extension of the nucleus, mainly in its ventrolateral part. The number of FB-labeled neurons was determined in 4 calves, ranging from 191 to 1,469 (mean, 465). The mean cross-sectional area of the FB-labeled neurons was approximately 1,680 μm2. Conclusions and Clinical Relevance—In cattle, small, medium, and large RN neurons, located along the entire craniocaudal extension of the RN, contribute to the rubrospinal tract reaching the L6-S1 spinal cord segments. Thus, in cattle, as has been shown in cats, the RN parvicellular population also projects to the spinal cord.

We studied the organization of the dorsal column nucleus (DCN) of larval sea lamprey with immunohistochemical and tract-tracing techniques. Texas red-coupled dextran amine was injected into the spinal cord, which allowed tracing the dorsal column fibers and characterizing the DCN. The dorsal column fibers formed a dense tract coursing adjacent to the dorsal midline of the spinal cord to the caudal rhombencephalon alar plate. In larvae, most spinal cord dorsal cells and spinal ganglion perikarya, and many dorsal column fibers, were calretinin-immunoreactive. We delineated the DCN in the dorsomedial portion of the obex and preobecular alar plate. It consists of a periventricular neuronal cell layer and neurons scattered in the lateral neuropil and receives dorsal column fibers. After immunohistochemistry with antibodies against glutamate, glycine, and GABA numerous immunoreactive perikarya were observed in the DCN. In addition to glutamate-, glycine-, and GABA-immunoreactive processes, serotonin- and dopamine-immunoreactive fibers coursed in the neuropil of this nucleus. A few small calretinin-immunoreactive perikarya were also observed in the DCN. Our results reveal the presence of inhibitory and excitatory transmitters in neurons of the DCN, and suggest that dopamine and serotonin modulate the activity of this nucleus.

Metastin, the product of metastasis suppressor gene KiSS-1 , is proposed to be the natural ligand for the G-protein-coupled receptor GPR54, known also as AXOR12. This immunohistochemical study, using a rabbit polyclonal antiserum against the human metastin fragment (45–54)-NH 2 , showed that in rats metastin-like immunoreactivity (MTS-LI) was present in neurons of the nucleus of the solitary tract and caudoventrolateral reticular nucleus, and in cell processes of the spinal trigeminal tract and lateral reticular nucleus. MTS-LI was confined mainly to neurons and fibers at or caudal to the area postrema. In the spinal cord, MTS-LI cell processes formed a dense plexus in superficial layers I and II of the dorsal horn. The pattern of distribution of MTS-LI in the medulla and spinal cord suggests that this novel peptide may participate in autonomic and sensory neural signaling.

The diencephalon is a complex brain area that derives from the caudal region of the prosencephalon. This structure is divided into four longitudinal neuroepithelial zones: roof, alar, basal and floor plates, which constitute its dorso-ventral (DV) columnar domains. Morphogenetic differences between alar and basal plates in the prosencephalon and mesencephalon contribute to the characteristic expansion of alar plate derivatives in the brain and the formation of the cephalic flexure. Although differential histogenesis among DV regions seems to be relevant in understanding structural and functional complexity of the brain, most of our knowledge about DV regionalization comes from the spinal cord development. Therefore, it seems of interest to study the molecular mechanisms that govern DV patterning in the diencephalon, the brain region where strong differences in size and complexity between alar and basal derivatives are evident in all vertebrates. Different morphogenetic signals, which induce specific progenitors fate to the neighboring epithelium, are involved in the spinal cord DV patterning. To study if Wnt1, one of these signaling molecules, has a role for the establishment of the diencephalic longitudinal domains, we carried out gain- and loss-of-function experiments, using mice and chick embryos. Our results demonstrated functional differences in the molecular mechanisms downstream of Wnt1 function in the diencephalon, in relation to the spinal cord. We further demonstrated that Bmp4 signal induces Wnt1 expression in the diencephalon, unraveling a new molecular regulatory code downstream of primary dorsalizing signals to control ventral regionalization in the diencephalon.

The gray matter of the spinal cord comprises 10 laminae (layers) and a number of spinal cord nuclei. These laminae were first described by Rexed in transverse sections of the spinal cord of the cat on the basis of neuronal size, shape, cytological features and density. Neurons within a lamina show distinct dendritic architecture, chemoarchitecture and patterns of connection as well as function. A number of neurons in the spinal cord are organized as spinal cord nuclei. Of these, the lateral spinal and the lateral cervical nuclei are located in the white matter, and the remaining such as the central cervical nucleus, the intermediomedial nucleus, the internal basilar nucleus, the dorsal nucleus, and the intermediolateral and sacral parasympathetic nuclei are located in the gray matter. A large variety of neurochemical substances are found in neurons of Rexed’s laminae and spinal cord nuclei including acetylcholine, calbindin, calcitonin gene-related peptide, glutamate, nitric oxide and substance P.

The putative transcription factor Lbx1 is expressed in the mantle zone of the hindbrain and spinal cord caudal to rhombomere 1, in a specific domain of the alar plate. The Lbx1 domain overlaps with the expression domains for Tlx3 and partially with the domains for Pax2/Lim1. The ventral border of the Lbx1 domain coincides with the ventral border of the dorsalmost Serrate1 stripe in the ventricular zone. The latter borders the intermediate stripe of both Delta and Lunatic fringe expression. The Lbx1 domain contains differentiated interneurons that project into the lateral longitudinal fasciculus.

Functionally useful repair of the mature spinal cord following injury requires axon growth and the re-establishment of specific synaptic connections. We have shown previously that axons from peripherally grafted human embryonic dorsal root ganglion cells grow for long distances in adult host rat dorsal roots, traverse the interface between the peripheral and central nervous system, and enter the spinal cord to arborize in the dorsal horn. Here we show that these transplants mediate synaptic activity in the host spinal cord. Dorsal root ganglia from human embryonic donors were transplanted in place of native adult rat ganglia. Two to three months after transplantation the recipient rats were examined anatomically and physiologically. Human fibres labelled with a human-specific axon marker were distributed in superficial as well as deep laminae of the recipient rat spinal cord. About 36% of the grafted neurons were double labelled following injections of the fluorescent tracers MiniRuby into the sciatic and Fluoro-Gold into the lower lumbar spinal cord, indicating that some of the grafted neurons had grown processes into the spinal cord as well as towards the denervated peripheral targets. Electrophysiological recordings demonstrated that the transplanted human dorsal roots conducted impulses that evoked postsynaptic activity in dorsal horn neurons and polysynaptic reflexes in ipsilateral ventral roots. The time course of the synaptic activation indicated that the human fibres were non-myelinated or thinly myelinated. Our findings show that growing human sensory nerve fibres which enter the adult deafferentated rat spinal cord become anatomically and physiologically integrated into functional spinal circuits.

The avian spinal cord is characterized by an absence of motor nerves and sensory nerves and ganglia at its caudalmost part. Since peripheral sensory neurons derive from neural crest cells, three basic mechanisms could account for this feature: (i) the caudalmost neural tube does not generate any neural crest cells; (ii) neural crest cells originating from the caudal part of the neural tube cannot give rise to dorsal root ganglia or (iii) the caudal environment is not permissive for the formation of dorsal root ganglia. To solve this problem, we have first studied the pattern of expression of ventral (HNF3beta) and dorsal (slug) marker genes in the caudal region of the neural tube; in a second approach, we have recorded the emergence of neural crest cells using the HNK1 monoclonal antibody; and finally, we have analyzed the developmental potentials of neural crest cells arising from the caudalmost part of the neural tube in avian embryo in in vitro culture and by means of heterotopic transplantations in vivo. We show here that neural crest cells arising from the neural tube located at the level of somites 47-53 can differentiate both in vitro and in vivo into melanocytes and Schwann cells but not into neurons. Furthermore, the neural tube located caudally to the last pair of somites (i.e. the 53rd pair) does not give rise to neural crest cells in any of the situations tested. The specific anatomical aspect of the avian spinal cord can thus be accounted for by limited developmental potentials of neural crest cells arising from the most caudal part of the neural tube.

The spinal cords of vertebrates are generally divided into the cord proper and the minute filum terminale. While the spinal cord extends the entire length of the vertebral canal in the adult tiger puffer, Takifugu rubripes, the cord proper is greatly reduced in length and almost all of the canal is occupied by the filum terminale, which is tape-like rather than thread-like. The dorsal and ventral roots of the spinal nerves extend, respectively, above and below the filum terminale; as a whole, these form a massive cauda equina. Supramedullary cells are found in the rostral half of the medulla oblongata caudal to the cerebellum. In 4-mm long tiger puffers, the spinal cord is cylindrical and supramedullary cells are found in the rostral half of the cord. In 7-mm puffers, the longitudinally arranged ventral roots appear ventrally in the middle portion of the spinal cord. In 15-mm puffers, the dorsal and ventral roots run longitudinally along the spinal cord and have noticeably increased in number. Supramedullary cells are located in the rostral 15% of the cord. In 21-mm puffers, the spinal cord in large part becomes dorsoventrally flattened. In 30-mm puffers, the spinal cord becomes much flatter, and supramedullary cells now are located mainly in the medulla oblongata. These observations indicate that formation of the shortened spinal cord proper is due to at least two developmental processes. First, the elongation of the spinal cord proper is remarkably less than that of the vertebral canal. Second, the bulk of the spinal cord proper is translocated to the cranial cavity, where it is transformed into part of the medulla oblongata.

The present study was carried out to characterize anatomically the vestibulospinal (VS) system of lampreys. Cobalt-lysine or Texas Red dextran amines were applied in vitro to the rostral spinal cord. Two distinct populations of VS neurons were labeled in the ventral nucleus of the area octavolateralis. The rostral group, comprising the intermediate octavomotor nucleus (ION), contained between 100 and 150 neurons, having somata of variable size and morphology. Intracellular injections of Lucifer Yellow in single neurons revealed ION VS neurons with dendrites extending in the ventrolateral alar plate as well as medially in the basal plate. The caudal group, comprising the posterior octavomotor nucleus (PON), contained approximately 65 neurons, most of which were unipolar with round or oval somata. To study the projections of VS axons, cobalt-lysine was injected into the ION or PON regions in the brainstem. Axons from the ION projected to the ipsilateral spinal cord, whereas PON axons decussated within the basal plate giving out descending and ascending branches. The descending branch projected to the contralateral spinal cord. Injections of two fluorescent dextran-amines, each restricted to one side of the spinal cord, did not double-label VS cells in either octavomotor nuclei, indicating that the projections of each nucleus are restricted to one side. Injections of horseradish peroxidase further caudally in the spinal cord revealed that VS axons from the ION reached past the gill region. Our results indicate that the organization of the VS system of lampreys is similar to that observed in other vertebrates. J. Comp. Neurol. 407:512‐526, 1999. r 1999 Wiley-Liss, Inc.

Pax6 and Pax7 are transcription factors essential for the development of the CNS. In addition, increasing data, mainly obtained in amniotes, support that they are expressed in subsets of neurons in the adult, likely playing a role in maintaining neuron type identity. In the present study we analyzed the detailed distribution of Pax6 and Pax7 cells in the adult CNS of Xenopus laevis. Immunohistochemistry with antibodies that are required for high-resolution analysis of Pax-expressing cells was conducted. A wide distribution of Pax6 and Pax7 cells throughout the CNS was detected, with distinct patterns that showed only slight overlapping. Only Pax6 was expressed in the telencephalon, including the olfactory bulbs, septum, striatum and amygdaloid complex. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, respectively, of prosomere 3. Large numbers of Pax6 and Pax7 cells were distributed in the pretectal region (alar plate of prosomere 1) but only Pax6 cells extended into basal plate. Pax7 specifically labeled cells in the optic tectum, including the ventricular zone, and Pax6 cells were the only cells found in the tegmentum. Pax6 was found in most granule cells of the cerebellum and Pax7 expression was found only in the ventricular zone. In the rostral rhombomere 1, Pax7 labeling was detected in cells of the ventricular zone of the alar plate, but numerous migrated cells were located in the basal plate, including the griseum centrale and the interpeduncular nucleus. Pax6 cells also formed a column of scattered neurons in the reticular formation and were found in the octavolateral area. The rhombencephalic ventricular zone of the alar plate expressed Pax7. Dorsal Pax7 cells and ventral Pax6 cells were found along the spinal cord separated from the ventricle, which did not show immunoreactivity. Our results show that the expression of Pax6 and Pax7 is widely maintained in the adult brain of Xenopus, like in urodele amphibians and in contrast to the situation described in amniotes. Therefore, in amphibians these transcription factors seem to be needed to maintain specific entities of subpopulations of neurons in the adult CNS.

The formation of laminar-specific projections is a key event in the development of appropriate neuronal connections in many regions of the central nervous system. In order to provide a framework for defining functions of molecules related to spinal laminar targeting of dorsal root ganglion neurons in mice, we have characterized the initial trajectories of sensory axons in relation to the maturation of their target laminae in the spinal cord. We show that morphological and biochemical differentiation of distinct clusters of neurons in the dorsal region of the spinal cord precedes initial collateral branching from sensory axons. Between embryonic day (E)12.5 and E13.5, sensory axons develop swellings (“nodes”) along their entire intraspinal extent and elaborate interstitial collateral branches from these nodes. Collaterals from the different classes of sensory axons then penetrate the gray matter of the spinal cord sequentially. Each class of sensory axons projects directly to its target lamina, never branching into inappropriate laminae en route. Some cutaneous afferents traverse the entire width of the spinal cord to reach superficial laminae on the contralateral side, strictly avoiding both the ventral spinal cord and inappropriate laminae of the deep dorsal horn. The pathways taken by developing sensory afferents are compatible with the idea that cells in inappropriate laminae exert inhibitory influences on sensory axons which regulate their laminar specificity. J. Comp. Neurol. 380:215–229, 1997. © 1997 Wiley-Liss, Inc.

The brainstem is composed of the medulla, pons, and midbrain; the midbrain continues with the diencephalon (thalamus) through the tentorial notch. The cerebellum, while derived from the rhombic lip, is not considered a component of the brainstem but rather as a suprasegmental structure in the posterior fossa. The brainstem is divided into tegmental and basilar regions, the former containing the nuclei of cranial nerves and the latter fiber tracts, most notably the corticospinal tract. The ventricular spaces of the brain are the cerebral aqueduct and fourth ventricle (plus lateral recesses); the caudal part of the fourth ventricle (medullary part) narrows and is continuous with the central canal of the cervical spinal cord. Cranial nerves (CNs) associated with the brainstem are III and IV (midbrain); V (pons); VI, VII, VIII (pons-medulla junction); and IX, X, XII (medulla); CN XI arises from spinal levels C1 to C5/6. The nuclei of these nerves are located in the tegmental area while their roots course through the basilar area to exit the brainstem. The nuclei of motor CNs are derived from the basal plate of the brainstem, the nuclei of sensory CNs from the alar plate of the brainstem, and the nuclei of mixed CNs (motor + sensory) from basal and alar plates. Increased intracranial pressure with resultant brain herniation may result in deficits related to CNs and/or to deficits related to long tract damage.

The vestibular nuclear complex (VNC) consists of a collection of sensory relay nuclei that integrates and relays information essential for coordination of eye movements, balance, and posture. Spanning the majority of the hindbrain alar plate, the rhombomere (r) origin and projection pattern of the VNC have been characterized in descriptive works using neuroanatomical tracing. However, neither the molecular identity nor developmental regulation of individual nucleus of the VNC has been determined. To begin to address this issue, we found that Hoxb1 is required for the anterior-posterior (AP) identity of precursors that contribute to the lateral vestibular nucleus (LVN). Using a gene-targeted Hoxb1-GFP reporter in the mouse, we show that the LVN precursors originate exclusively from r4 and project to the spinal cord in the stereotypic pattern of the lateral vestibulospinal tract that provides input into spinal motoneurons driving extensor muscles of the limb. The r4-derived LVN precursors express the transcription factors Phox2a and Lbx1, and the glutamatergic marker Vglut2, which together defines them as dB2 neurons. Loss of Hoxb1 function does not alter the glutamatergic phenotype of dB2 neurons, but alters their stereotyped spinal cord projection. Moreover, at the expense of Phox2a, the glutamatergic determinants Lmx1b and Tlx3 were ectopically expressed by dB2 neurons. Our study suggests that the Hox genes determine the AP identity and diversity of vestibular precursors, including their output target, by coordinating the expression of neurotransmitter determinant and target selection properties along the AP axis.

In order to determine if reticulospinal neurons involved in the control of locomotion and responsive to exogenously applied 5-hydroxtryptamine (5-HT) are innervated by fibers that contain serotonin, the serotoninergic innervation of reticulospinal neurons, identified by retrograde labeling with fluorescein-conjugated dextran-amine (FDA), was investigated by immunohistochemistry in the lamprey brainstem. A widespread distribution of 5-HT immunoreactive (5-HT-ir) fibers was seen within the basal plate of the brainstem, an area containing reticulospinal somata and dendritic arborizations. Numerous 5-HT varicose fibers were found in close relation to large reticulospinal cell bodies, particularly in the middle and anterior rhombencephalic reticular nuclei (MRRN and ARRN). Some of these reticulospinal somata were surrounded by a very dense pericellular 5-HT innervation. 5-HT-ir fibers were also seen in other brain structures that are known to influence reticulospinal neurons such as the rhombencephalic alar plate containing sensory relay interneurons, cranial nerves (III–X), cerebellum, and tectum. These findings suggest that, as in the spinal cord, motor behavior controlled by reticulospinal neurons may be subject to a serotoninergic modulation. © 1994 Wiley-Liss, Inc.

Interpretation of the new wealth of gene expression and molecular mechanisms in the developing mouse spinal cord requires an accurate anatomical base on which data can be mapped. Therefore, we have assembled a spinal cord atlas of the P4 mouse to facilitate direct comparison with the adult specimens and to contribute to studies of the development of the mouse spinal cord. This study presents the anatomy of the spinal cord of the P4 C57Bl/6J mouse using Nissl and acetyl cholinesterase-stained sections. It includes a detailed map of the laminar organization of selected spinal cord segments and a description of named cell groups of the spinal cord such as the central cervical (CeCv), lateral spinal nucleus, lateral cervical, and dorsal nuclei. The motor neuron groups have also been identified according to the muscle groups they are likely to supply. General features of Rexed's laminae of the P4 spinal cord showed similarities to that of the adult (P56). However, certain differences were observed with regard to the extent of laminae and location of certain cell groups, such as the dorsal nucleus having a more dispersed structure and a more ventral and medial position or the CeCv being located in the medial part of lamina 5 in contrast to the adult where it is located in lamina 7. Motor neuron pools appeared to be more tightly packed in the P4 spinal cord. The dorsal horn was relatively larger and there was more white matter in the P56 spinal cord.

The course and sites of termination of dorsal column fibres in the lamprey brainstem are described along with their brainstem relays projecting to reticulospinal neurons. Dorsal column fibres ascend to the brainstem level where they intermingle with cells located in the alar plate close to the obex, a location that is analogous to that of the dorsal column nucleus in other vertebrates. Some dorsal column fibres continue further rostrally where they reach the octavolateralis and octavomotorii nuclei. Finally, a small contingent of fibres reach the cerebellum. Injections of cobalt-lysine into the posterior rhombencephalic reticular nucleus retrogradely label neurons within the dorsal column nucleus and within the octavolateralis and octavomotorii nuclei. Microstimulation of the dorsal column nucleus on either side elicits monosynaptic inhibitory responses in reticulospinal neurons while stimulation of octavolateralis and octavomotorius nuclei elicits excitation. By using intracellular recordings, it was shown that neurons within these alar plate nuclei receive monosynaptic inputs from the dorsal columns. It is thus proposed that disynaptic inputs from dorsal columns to reticulospinal neurons are relayed by these alar plate neurons: inhibition is relayed mainly by neurons in dorsal column nuclei and excitation by neurons in the octavolateralis and octavomotorii nuclei. © 1993 Wiley-Liss, Inc.

The distribution of nerve growth factor receptor (NGFR) immunoreactive profiles was investigated in the adult human brainstem and spinal cord using a monoclonal antibody directed against the primate low affinity (p75) NGFR. In the human brainstem, p75NGFR immunoreactive profiles were seen within the mesencephalic and descending nucleus of the trigeminal nerve, the nucleus and tractus solitarius, glossopharyngeal nerve, hypoglossal nucleus, nucleus subtrigeminalis, subnucleus ventralis of the central nucleus of the medulla, nucleus cuneatus and gracilis. At the level of the upper cervical spinal cord, p75NGFR immunoreactive profiles were also seen within the incoming dorsal roots, zone of Lissauer and substantia gelatanosa (lamina II). Virtually no immunoreactivity was associated with cervical spinal cord motor neurons. The demonstration of the p75NGFR in brainstem and spinal cord regions associated with the central transmission of peripheral sensory information suggests that these systems may be influenced by the trophic substance nerve growth factor.

The aims of this study were to elucidate the processes of neuronal differentiation and ventrodorsal patterning in the spinal cord of the chick embryo from embryonic day (E) 3 to E17 and to study the effect of a prenatal spinal open neural tube defect (ONTD) on these processes.Expression patterns of neuronal antigens (neuronal nuclear antigen, neurofilament-associated protein (NAP), and synaptophysin) and related ventral markers [sonic hedgehog, paired box gene (PAX)6, and islet-1], and dorsal markers (bone morphogenetic protein, Notch homolog 1, and PAX7) were investigated in the normal spinal cord and in a surgically induced spinal ONTD in chick embryos. Four normal and ONTD chick embryos were used for each antigen group.There were no differences in the expression of neuronal and ventrodorsal markers between the control and ONTD groups. NAP and synaptophysin were useful for identifying dorsal structures in the distorted anatomy of the ONTD chicks.

Publisher Summary Neurons in the spinal cord use a variety of neurotransmitters to carry out their functions, and they in turn receive a variety of neurotransmitter inputs. For the most part these neurotransmitters show distinct patterns of distribution within the spinal cord that are similar across different species. Furthermore, specific neurotransmitter systems in the spinal cord are often associated with specific functions. This chapter focuses on the distribution of acetylcholine (ACh) and substance P (SP) within the spinal cord, and examines the serotoninergic (5-HT), noradrenergic (NA), and dopaminergic (DA) projections from the brain to the spinal cord. Cholinergic neurons are found throughout the longitudinal extent of the spinal cord from cervical to coccygeal segments. In most species, they are localized to the gray matter of the spinal cord. Substance P (SP), a member of the tachykinin neuropeptide family, is an important signaling molecule that has been associated with functionally distinct spinal cord systems. SP is involved in sensory, autonomic, and motor functions of the spinal cord. Noradrenergic (NA) containing fibers to the spinal cord originate from the locus coeruleus (LC), and other NA cell groups, namely the locus subcoeruleus (SC) and the A5 and A7 cell groups of the lateral tegmentum. All NA in the spinal cord comes from these four areas. The serotoninergic projections from ventral medullary raphe nuclei arise from raphe magnus (RMg), raphe pallidus (RPa), and raphe obscurus (ROb) nuclei; they terminate in all laminae of the spinal cord.

This chapter introduces the general anatomy and organization of the spinal cord. The spinal cord is the part of the central nervous system that controls the voluntary muscles of the limbs and trunk, and which receives sensory information from these regions. It also controls most of the viscera and blood vessels of the thorax, abdomen, and pelvis. The cord is described as being made up of a series of segmental components, but in the adult the cord is actually a continuous cylinder of central nervous tissue. The segments are determined by the pattern of emergence of spinal nerves; the nerve rootlets arising from the cord are bundled together so that one pair of spinal nerves emerges from each segment. The spinal cord and its meninges (dura mater, arachnoid, and pia mater) lie within the vertebral canal. The spinal cord is cylindrical, but slightly flattened dorsoventrally. The spinal white matter occupies most of the cross-sectional area of the cervical enlargement, while the spinal cord gray matter is greatly enlarged in the regions where the nerves of the limbs (the brachial and lumbosacral plexuses) arise. The human spinal cord has 12 thoracic segments, 5 lumbar segments, 5 sacral segments, and 1 coccygeal segment, making a total of 31 segments. Pairs of spinal nerves arise from the spinal cord and leave the vertebral column through the intervertebral foramina. The central canal of the spinal cord is a remnant of the embryological ventricular system and is present throughout the whole length of the spinal cord. The spinal cord is supplied by a single ventral spinal artery and two dorsal spinal arteries.

The notochord is probably involved in the development of the neural tube. In this study, a fragment of caudal notochord was extirpated in ovo from chick embryos at 1.5 days of incubation. At 4.5 days a distinct notochord-deficient region at thoracolumbar level was found. Profound effects were seen, especially at the cranial site of this region. Somites were smaller than normal, or even not recognizable, and in some cases the myotomes were fused in the midline. The spinal cord appeared reduced in size and lacked a floor plate. The average amount of spinal cord neurons was 23% of the normal value, the cells being located circularly along the outer margin of the spinal cord, except for the roof plate. Axonal roots left the cord in the ventral midline only. Caudal to this site, neurons or floor plate cells were alternately present in the ventral spinal cord, and axonal roots left bilaterally. In a caudal direction, a normal morphology gradually reappeared. The possibility is discussed that reduction in spinal cord size and amount of neurons is a direct or indirect effect of the absence of the notochord, and that the sclerotome may be involved.(ABSTRACT TRUNCATED AT 250 WORDS)

The labyrinthine-spinal reflexes are influenced by the inputs from the cervical and lumbal propriocepters. We studied the afferent route by the retrograde WGA-HRP method in cats. After the injection of WGA-HRP into either the dorsal or the ventral Deiters' nucleus, labeled neurons were investigated in the spinal cord, the cerebellum, the contralateral vestibular nucleus complex and the brain stem nucleus. In this paper, we report the results in the spinal cord of cats. i) When WGA-HRP was injected into the dorsal Deiters' nucleus, labeled neurons in the spinal cord were found mainly extending from the cervical to the lumbosacral area of the spinal cord. A number of labeled cells were located predominantly in the contralateral cervical segments, while a small number of labeled cells was found ipsilaterally in the lumbosacral segments. ii) In the case of the ventral Deiters' nucleus, labeled neurons were found extending from the cervical to the upper thoracic area of the spinal cord. Localization of labeled neurons in the spinal cord was limited mainly to Rexed's laminae VII and VIII. These results suggest that the afferent fibers from the spinal cord to the Deiters' nucleus are closely related to the labyrinthine-spinal reflex.

The Olig3 gene encodes a bHLH factor that is expressed in the ventricular zone of the dorsal alar plate of the hindbrain. We found that the Olig3+ progenitor domain encompassed subdomains that co-expressed Math1, Ngn1, Mash1 and Ptf1a. Olig3+ cells give rise to neuronal types in the dorsal alar plate that we denote as class A neurons. We used genetic lineage tracing to demonstrate that class A neurons contribute to the nucleus of the solitary tract and to precerebellar nuclei. The fate of class A neurons was not correctly determined in Olig3 mutant mice. As a consequence, the nucleus of the solitary tract did not form, and precerebellar nuclei, such as the inferior olivary nucleus, were absent or small. At the expense of class A neurons, ectopic Lbx1+ neurons appeared in the alar plate in Olig3 mutant mice. By contrast, electroporation of an Olig3 expression vector in the chick hindbrain suppressed the emergence of Lbx1+ neurons. Climbing fiber neurons of the inferior olivary nucleus express Foxd3 and require Olig3 as well as Ptf1a for the determination of their fate. We observed that electroporation of Olig3 and Ptf1a expression vectors, but not either alone, induced Foxd3. We therefore propose that Olig3 can cooperate with Ptf1a to determine the fate of climbing fiber neurons of the inferior olivary nucleus.

Projections from the spinal cord and solitary nucleus to the lateral parabrachial nucleus (PBN1) in the cat were directly compared using double anterograde tracing methods. The two inputs were found to overlap within a well-circumscribed zone in the rostral 2/3 of PBN1. This zone was flanked ventrally by a zone receiving only solitary nucleus input and dorsally by a zone receiving only spinal input. Other authors have shown that neurons within these three recipient zones (overlap area, solitary nucleus and spinal cord) project to different forebrain targets (hypothalamus, amygdala and thalamus, respectively). This orderly input-output organization is likely to provide part of the framework for PBN's complex involvement in the coordination of respiratory and cardiovascular activities and their association with pain, visceral sensation and emotion.

Background The function of the nervous system depends on precise and selective connections of neurons. Various guidance cues instruct axons in choosing their pathway, polarity of growth, termination of growth and also branching. In vitro studies demonstrated that signaling cascades activated by axonal guidance receptors could be modulated by cyclic nucleotides. Previous analyses of cGKI (cGMP dependent protein kinase I) deficient mice in our lab showed that cGMP signaling via cGKI is important in axonal pathfinding and connectivity of sensory neurons. Sensory axons bifurcate upon arrival at the dorsal root entry zone (DREZ) of the spinal cord. Embryonic cGKI knock-out mice lack the bifurcation of sensory axons at the DREZ, i.e. the ingrowing axon either turns rostrally or caudally. Here, we present results of the detailed analysis of axonal pathfinding errors in cGKI deficient mice and of the search for other components of cGMP signaling in dorsal root ganglions (DRG).

Cell death in the developing human spinal cord was investigated in 5-12 week human conceptuses using immunohistochemical and TUNEL methods. Expression of pro-apoptotic (Fas-receptor, caspase-3) and anti-apoptotic (bcl-2) markers and marker for internucleosomal fragmentation (TUNEL) were analysed in the cranial and caudal parts of the human spinal cord. In early developmental stages (5-6 weeks) of the cranial spinal cord, bcl-2 positive cells were seen in the ventricular zone and in the roof plate, while in the caudal part they were seen surrounding the central lumen. Subsequently, bcl-2 expression appeared in the basal plates of the grey matter and in the spinal ganglia, and from the seventh week on they also appeared in the intermediate horn of the grey matter. In the fetal period, bcl-2 expression appeared in the dorsal horns of the grey matter (9 weeks) but ceased in the ventricular zone (12 weeks) . In the trunk region, TUNEL-positive cells were found in ventricular and mantle zones along the whole length of the spinal cord. Caspase-3 positive cells and Fas-receptor positive cells appeared only in the grey matter of the cranial segments (head and trunk) of the spinal cord, but they were missing in the caudal parts. Caspase-3 dependant pathway, probably activated by Fas-receptor, seems to operate only in the cranial part of the human spinal cord. In the caudal (sacrococcygeal and tail) parts, cells seem to die by caspase-3 independent pathway. The interplay of pro-apoptotic and anti-apoptotic factors may be associated with cranial spinal cord morphogenesis, adjustment of cells number and selective survival of neurons, while in the caudal regions these factors cause massive cell death associated with regression of the caudal spinal cord.

Neurons of the dorsal horn integrate and relay sensory information and arise during development in the dorsal spinal cord, the alar plate. Class A and B neurons emerge in the dorsal and ventral alar plate, differ in their dependence on roof plate signals for specification, and settle in the deep and superficial dorsal horn, respectively. We show here that the basic helix-loop-helix (bHLH) gene Olig3 is expressed in progenitor cells that generate class A (dI1-dI3) neurons and that Olig3 is an important factor in the development of these neuronal cell types. In Olig3 mutant mice, the development of class A neurons is impaired; dI1 neurons are generated in reduced numbers, whereas dI2 and dI3 neurons are misspecified and assume the identity of class B neurons. Conversely, Olig3 represses the emergence of class B neurons in the chick spinal cord. We conclude that Olig3 expression distinguishes the two major classes of progenitors in the dorsal spinal cord and determines the distinct specification program of class A neurons.

Early brain and spinal cord regionalization along the dorsoventral axis are thought to be governed by similar mechanisms. Subsequently, the size of the alar plate of the neural tube increases dramatically in the midbrain and anterior forebrain, compared to the spinal cord. This suggests that additional mechanisms may be required to refine A/P and D/V patterning in these structures. The isthmic organizer is a signaling center that controls both growth and patterning in the midbrain and anterior hindbrain through the production of several secreted molecules, in particular FGF8. Several studies have indicated that the isthmic organizer is involved in the positioning and development of the midbrain roof and floor plates, the two structures that respectively mark the dorsal and ventral axis of the neural tube. It remains unclear whether its influence on axis formation in the midbrain is a consequence of a more general function of the isthmic organizer/FGF8 as a modulator of DV patterning or if selection of an axis is a necessary and general by-product of its organizing function not directly related to D/V patterning. In this paper, we review the current data supporting each possibility.

By using in situ hybridization histochemistry the distribution of growth hormone (GH) receptor mRNA was examined in the rat brain stem and spinal cord. Dense labeling was seen in the arcuate nucleus of the hypothalamus, as reported previously, but also in several other areas, including the locus coeruleus, the area postrema, and the commissural part of the nucleus of the solitary tract. Other labeled structures included the superior lateral parabrachial nucleus, the facial, hypoglossal and trigeminal motor nuclei, the nucleus incertus, the dorsal tegmental nucleus, the dorsal raphe nucleus, the nucleus of the trapezoid body, and the superficial layers of the dorsal horn of the spinal cord. These findings provide support for a direct action of GH on brain regions involved in various aspects of homeostatic control. Thus, the distribution of GH receptor mRNA to visceral sensory and motor structures is consonant with a role of GH in the regulation of food intake and energy homeostasis. Its presence in the superficial dorsal horn of the spinal cord indicates a role for GH in the initial processing of fine afferent input, and may help explain the beneficial effects of GH replacement in certain unclear pain conditions.

It is generally believed that the spinal cord and hindbrain consist of a motor basal plate and a sensory alar plate. We now have molecular markers for these territories. The relationship of migrating branchiomotor neurons to molecularly defined alar and basal domains was examined in the chicken embryo by mapping the expression of cadherin-7 and cadherin-6B, in comparison to genetic markers for ventrodorsal patterning (Otp, Pax6, Pax7, Nkx2.2, and Shh) and motoneuron subpopulations (Phox2b and Isl1). We show cadherin-7 is expressed in a complete radial domain occupying a lateral region of the hindbrain basal plate. The cadherin-7 domain abuts the medial border of Pax7 expression; this common limit defines, or at least approximates, the basal/alar boundary. The hindbrain branchiomotor neurons originate in the medial part of the basal plate, close to the floor plate. Their cadherin-7-positive axons grow into the alar plate and exit the hindbrain close to the corresponding afferent nerve root. The cadherin-7-positive neuronal cell bodies later translocate laterally, following this axonal trajectory, thereby passing through the cadherin-7-positive basal plate domain. Finally, the cell bodies traverse the molecularly defined basal/alar boundary and move into positions within the alar plate. After the migration has ended, the branchiomotor neurons switch expression from cadherin-7 to cadherin-6B. These findings demonstrate that a specific subset of primary motor neurons, the branchiomotor neurons, migrate into the alar plate of the chicken embryo. Consequently, the century-old concept that all primary motor neurons come to reside in the basal plate should be revised.

Oxytocin-containing axons project from the hypothalamic paraventricular nucleus to the neurohypophysis and thoracic spinal cord to ultimately influence uterine contractions and autonomic activity, respectively. Whether or not oxytocin-immunoreactive axons project to the female rat lumbosacral spinal cord to influence autonomic outflow to pelvic organs has not been investigated. Thus, the present study was designed to investigate the presence, distribution, and origin of oxytocin-immunoreactive axons in the female rat lumbosacral spinal cord. Immunohistochemistry, spinal cord transections, and axonal tracing with Fluorogold, True Blue, and pseudorabies virus were used. Oxytocin-immunoreactive nerve fibers were present in the L6/S1 segments of the spinal cord. Prominent varicose axons were evident throughout the dorsal horn, along the lateral and medial collateral pathways, in the dorsal intermediate gray area, around the central canal in lamina X, and throughout the sacral parasympathetic nucleus. Injection of retrograde tracer into the L6/S1 spinal cord labeled neurons in the hypothalamic paraventricular nucleus. Transection of the thoracic spinal cord eliminated oxytocin-immunoreactive nerve axons in the L6/S1 spinal cord. In addition, transection of the thoracic spinal cord eliminated transport of retrograde axonal tracer from the L6/S1 spinal cord to the paraventricular nucleus. Pseudorabies virus, a transneuronal retrograde tracer, injected into the uterus and cervix marked uterine-related preganglionic neuronal cell bodies in the sacral parasympathetic nucleus and uterine-related neurons in the hypothalamic paraventricular nucleus. Double immuno-labeling of viral-infected spinal cord sections showed oxytocin-immunoreactive axons closely associated with viral labeled uterine-related preganglionic cell bodies of the sacral parasympathetic nucleus. The results of this study revealed that oxytocin-immunoreactive neurons of the hypothalamic paraventricular nucleus project axons to the lumbosacral spinal cord to areas involved in sensory processing and parasympathetic outflow to the uterus.

The segmental extent and topography of the spinal nucleus of the accessory nerve (SNAN) was investigated in the adult human spinal cord. Transverse sections of segments between the lower medulla and C6 were stained with cresyl violet and the motor cell columns identified according to the numerical locations defined by Elliott (1942). The segmental extent and topography of the cervical part of column 2 resembled that previously described for the SNAN of primates.

Quantitative receptor autoradiography and immunoblotting were used to study the expression and distribution of AMPA, kainate and NMDA receptors in first trimester human spinal cord obtained from elective abortions ranging from 4 to 11.5 weeks of gestational age. Spinal cord tissue sections were processed for receptor autoradiography with the ligands [ 3 H ]AMPA, [ 3 H ]kainate and [ 3 H ]MK-801 and the optical density was measured separately in a dorsal region (alar plate) and ventral region (basal plate) of the autoradiographs. Binding sites for all three ligands were demonstrated already at 4–5.5 weeks of gestation and increased continuously during the first trimester both in the dorsal and ventral regions. [ 3 H ]AMPA binding to both high- and low-affinity sites increased from undetectable levels to about 35 and 400 fmol/mg tissue, respectively, during this period. A temporal difference in the distribution of [ 3 H ]AMPA binding sites was observed. The early homogeneous pattern of [ 3 H ]AMPA binding in both alar and basal plates had changed to a heterogeneous pattern at 11 weeks of gestation with the highest density of [ 3 H ]AMPA binding sites in the superficial layers of the immature dorsal horn. [ 3 H ]kainate and [ 3 H ]MK-801 binding sites were densely and homogeneously distributed already at 4 weeks, and steadily increased six- and two-fold, respectively, to about 100 fmol/mg tissue at 11.5 weeks of gestation. Immunoreactive bands corresponding to the NMDA receptor subunits NR1, NR2A, NR2B, NR2C and NR2D were demonstrated by immunoblotting at the earliest between 4.5 and 7 weeks and increasing concentrations were seen up to 11 weeks of gestation. These results suggest that AMPA, kainate and NMDA receptors are expressed in the human spinal cord early in embryogenesis.

As part of a recent program on the evolution of somatosensory systems in vertebrates, the cytoarchitecture, chemoarchitecture, and fiber connections of the caudal rhombencephalic alar plate were studied in the ribbed newt, Pleurodeles waltl. This part of the brain stem includes ill-defined dorsal column and lateral cervical nuclei. A cytoarchitectonic analysis revealed that the caudal medullary alar plate consists of an inner and an outer cell layer. The dorsomedial part of the outer cell layer at the obex level contains the dorsal column nucleus (DCN), whereas its ventrolateral part constitutes the lateral cervical nucleus (LCN). NADPH-diaphorase histochemistry and calbindin D-28k immunohistochemistry clearly delineate the main components of the compact inner cell layer, i.e. the nucleus of the solitary tract dorsally and the nucleus of the descending trigeminal tract ventrally. Neither NADPH-diaphorase-labeled nor calbindin D-28k positive neurons were observed in the DCN and LCN. With anterograde and retrograde tracing, the DCN and LCN were further delineated. Labeling of ascending dorsal root projections showed that the dorsal column and the DCN are somatotopically arranged: lumbar primary afferent fibers terminate on medial DCN neurons, whereas cervical primary afferent fibers terminate on lateral DCN neurons. The LCN is densely innervated by the dorsolateral funiculus. Retrograde tracing showed extensive, predominantly contralateral projections of both the DCN and LCN to the torus semicircularis and the ventral thalamus. These data show that even in the poorly segregated caudal rhombencephalic alar plate of urodeles a DCN and LCN can be distinguished with afferent and efferent projections comparable to those in anurans and other terrestrial vertebrates.

Brn-3.0, a POU-domain transcription factor, is expressed in specific postmitotic neurons in the dorsal part of the neural tube which are among the first spinal cord neurons to appear in development. In the mature spinal cord, the Brn-3.0 cells form a numerous population of scattered neurons in the intermediate spinal gray. Ablation of the notochord in chick embryos extends the domain of Brn-3.0 expression into the ventral neural tube, while ectopic grafts of notochord tissue suppress Brn-3.0 expression. The notochord effects on Brn-3.0 expression are reproducedin vivoby the implantation of a local source of recombinant Shh protein. The down-regulation of Brn-3.0 expression in the dorsal spinal cord by the notochord and Shh contrasts with the known inductive effects of these ventral signals on the approximately simultaneous development of the spinal motor neurons. In cultured explants of neural plate from the region of the presumptive spinal cord, Brn-3.0 neurons develop in the absence of surface ectoderm and ventral midline tissue, suggesting that the Brn-3.0 phenotype may represent a “default” developmental pathway for early spinal cord neurons. Together these results advance the understanding of the mechanism of the generation of neuronal diversity in the developing vertebrate CNS.

Netrin-1, a diffusible signal secreted by floor plate cells at the ventral midline of the vertebrate CNS, can attract ventrally migrating axons and repel a subset of dorsally migrating axons in the spinal cord and rostral hindbrain in vitro. Whether netrin-1 can act as a global cue to guide all circumferentially migrating axons is, however, unknown. Here, we show that netrin-1 can attract alar plate axons that cross the floor plate along its entire rostrocaudal axis. Dorsally directed axons forming the posterior commissure are, however, repelled by the floor plate by a netrin-independent mechanism. These results suggest that netrin-1 functions as a global guidance cue for attraction to the midline. Moreover, floor plate–mediated chemorepulsion may also operate generally to direct dorsal migrations, but its molecular basis may involve both netrin-dependent and -independent mechanisms.

Early development of the spinal cord was studied in macaque monkey embryos, using light- and electron microscopy, Golgi impregnation and [3H]thymidine radioautography. All neurons engaged in the first reflex arc are generated before E27 in the 165 day gestation period. The earliest-generated cells differentiate into motoneurons while the commissural and association neurons are generated later. Synaptogenesis starts at E27 in the basal plate, and 2 days later in the alar plate. The first synapses appear as symmetrical junctions situated on the neuronal perikarya and proximal dendrites. Closure of the first spinal reflex are is established within 2 days and follows an antidromic pattern as related to the physiological spread of nerve impulses: synapses on motoneuronal somata and primary dendrites in the basal plate appear first and are followed by synapses in the alar plate, between dorsal root axon collaterals and somata of borderline (commissural/association) neurons. Commissural axons grow towards the floor plate, cross the midline and proceed caudo-rostrally, while association fibers remain ipsilateral. The first wave of apoptosis (programmed cell death) thins out dense populations of nerve and glial cells by E30. Some of the early-generated borderine cells that form commissural and association interneurons, seem to play the role of transient target cells and die once the definitive axonal pathways are established. Since transient cells form numerous synapses, deprivation from the afferent impulses is not a likely cause of their elimination. The present results indicate that the initial developmental events, including formation of the first reflex arc in the primate spinal cord, occur considerably earlier in respect to birth than in other mammals, but that the schedule of cellular events and cellular mechanisms seem to be the same.

It is unknown whether neuroepithelial cells in the mammalian central nervous system express neurotransmitter-synthesizing enzymes. In this study, expression of glutamate decarboxylase (GAD), the γ-aminobutyric acid (GABA)-synthesizing enzyme, was examined in proliferative cells and postmitotic neuroblasts in embryonic rat spinal cord. Immunostaining coronal sections of the embryonic spinal cord with K2 antiserum, which recognizes GAD proteins encoded by the GAD67 gene, revealed intensely stained neuroepithelial cells in the basal plate at embryonic day (E) 13, in the intermediate plate between E 13–16, and last seen in the alar plate at E 16. Nissl counterstaining demonstrated that a small number of these GAD-immunoreactive cells adjacent to the neural tube lumen were mitotic. The ventral-to-dorsal gradient of GAD expression in precursor cells and postmitotic neuroblasts correlates anatomically and temporally with the sequential generation of motoneurons, commissural neurons, and interneurons in the dorsal horn. Some of these GAD-immunoreactive neuroepithelial cells may re-enter the mitotic cycle, while others are postmitotic neuroblasts presumably migrating to the intermediate zone to differentiate into young neurons. Double-immunostaining cells acutely dissociated from E 11–18 spinal cords with K2 and anti-bromodeoxyuridine antisera, following a bromodeoxyuridine pulse in vivo, revealed considerable numbers of DNA-synthesizing cells immunoreactive for GAD. The absolute number of double-stained cells peaked during E 12–15, coinciding with terminal cell division in most spinal neurons. These observations suggest that spinal neuronal precursors can synthesize GAD-related proteins prior to, or during, the terminal cell cycle. Although GAD immunoreactivity revealed by K2 antiserum was detected in proliferative cells and in migrating postmitotic neuroblasts, GABA immunoreactivity was never detectable in these cells. These early embryonic GAD-immunoreactive neuroepithelial cells may either synthesize levels of GABA that cannot be detected immunocytochemically, and/or express enzymatically inactive GAD-related proteins. © 1992 Wiley-Liss, Inc.

The localization of CD15 (synonyms: stage-specific embryonic antigen-1 (SSEA-1), 3(alpha)-fucosyl-N-acetyl-lactosamine or FAL), which is implicated in neuronal differentiation, in the developing dorsal rat spinal cord was studied by immunocytochemistry. A embryonal day 9 (E9), SSEA-1 was detected in the neural ectoderm and, at E11, in cells near the ventricle of the matrix layer. This localization indicated that SSEA-1 is present in proliferating premigratory cells of the rat spinal cord. Between E12 and E16, cells of the alar plate expressed SSEA-1. Expression of the antigen was restricted to neuroblasts that will form the dorsal horn. SSEA-1, therefore, can be used at this stage as a marker for a subdivision of the matrix layer. At E14, the dorsal root entrance zone showed SSEA-1. This indicated that SSEA-1 is associated with ingrowing primary afferents. From E16 on, SSEA-1 was present in the dorsal raphe, which suggested a function for SSEA-1 in the guidance of developing fibres. After E17, the antigen was also found within the dorsal mantle layer. SSEA-1 was first present in Rexed's laminae II, IV and V. Later on in development the antigen was detected only in Rexed's laminae II (substantia gelatinosa). These distribution patterns indicated that SSEA-1 is present on migratory and/or postmigratory cells. In addition, SSEA-1 is associated with small-diameter dorsal root fibres, the C fibres and A(sigma) fibres, that terminate within the substantia gelatinosa. After birth, SSEA-1 was present throughout the dorsal horn, probably as a result of the myelination of the fibres.