Motor neuron pathfinding following rhombomere reversals in the chick embryo hindbrain.

Motor neurons are segmentally organised in the developing chick hindbrain, with groups of neurons occupying pairs of hindbrain segments or rhombomeres. The branchiomotor nucleus of the trigeminal nerve occupies rhombomeres 2 and 3 (r2 and r3), that of the facial nerve r4 and r5, and that of the glossopharyngeal nerve r6 and r7. Branchiomotor neuron cell bodies lie within the basal plate, forming columns on either side of the ventral midline floor plate. Axons originating in rhombomeres 2, 4 and 6 grow laterally (dorsally) towards the exit points located in the alar plates of these rhombomeres, while axons originating in odd-numbered rhombomeres 3 and 5 grow laterally and then rostrally, crossing a rhombomere boundary to reach their exit point. Examination of the trajectories of motor axons in odd-numbered segments at late stages of development (19-25) showed stereotyped pathways, in which axons grew laterally before making a sharp turn rostrally. During the initial phase of outgrowth (stage 14-15), however, axons had meandering courses and did not grow in a directed fashion towards their exit point. When r3 or r5 was transplanted with reversed rostrocaudal polarity prior to motor axon outgrowth, the majority of axons grew to their appropriate, rostral exit point, despite the inverted neuroepithelial polarity. In r3 reversals, however, there was a considerable increase in the normally small number of axons that grew out via the caudal, r4 exit point. These findings are discussed with relevance to the factors involved in motor neuron specification and axon outgrowth in the developing hindbrain.     

Efferent vestibular neurons in the guinea pig by horseradish peroxidase and fluorochrome labeling techniques

Neurons with projections into the vestibular receptor apparatus (efferent vestibular neurons) were identified in different medullary regions by retrograde labeling with horseradish peroxidase and transport-specific fluorochromes in the guinea pig. Two groups of efferent vestibular neurons could be distinguished, located dorsally and ventrally to the facial nerve fiber pathway. The dorsal group of efferent vestbular neurons consisted of small cells located close to the genu and the root of the facial nerve and the subependymal granular layer of the 4th ventricle floor. The ventral group was primarily composed of medium-sized cells, usually with only slight tracer accumulation; these were scattered over an extensive area of the lateral tegmental field within nucleus reticularis lateralis parvocellularis. The question of whether the test cells belong to the system of true vestibular efferents and satellite cells is discussed in the light of findings on cell location, morphology, and pattern of tracer accumulation.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 738–747, November–December, 1986.     

Development of the inner ear efferent system across vertebrate species

Inner ear efferent neurons are part of a descending centrifugal pathway from the hindbrain known across vertebrates as the octavolateralis efferent system. This centrifugal pathway terminates on either sensory hair cells or eighth nerve ganglion cells. Most studies of efferent development have used either avian or mammalian models. Recent studies suggest that prevailing notions of the development of efferent innervation need to be revised. In birds, efferents reside in a single, diffuse nucleus, but segregate according to vestibular or cochlear projections. In mammals, the auditory and vestibular efferents are completely separate. Cochlear efferents can be divided into at least two distinct, descending medial and lateral pathways. During development, inner ear efferents appear to be a specific motor neuron phenotype, but unlike motor neurons have contralateral projections, innervate sensory targets, and, at least in mammals, also express noncholinergic neurotransmitters. Contrary to prevailing views, newer data suggest that medial efferent neurons mature early, are mostly, if not exclusively, cholinergic, and project transiently to the inner hair cell region of the cochlea before making final synapses on outer hair cells. On the other hand, lateral efferent neurons mature later, are neurochemically heterogeneous, and project mostly, but not exclusively to the inner hair cell region. The early efferent innervation to the ear may serve an important role in the maturation of afferent responses. This review summarizes recent data on the neurogenesis, pathfinding, target selection, innervation, and onset of neurotransmitter expression in cholinergic efferent neurons.     

Pathfinding by cranial nerve VII (facial) motorneurons in the chick hindbrain.

Cranial nerve VII (facial) motorneurons begin extending axons through rhombomeres 4 and 5 (R4 and R5) in the chick hindbrain on the second day of incubation. Without crossing the midline, facial motorneuron axons extend laterally from a ventromedial cell body location. All facial motorneuron axons leave the hindbrain through a discrete exit site in R4. To examine the importance of the exit site in R4 on motorneuron pathfinding, we ablated R4 before motorneuron axonogenesis. We find that mechanisms intrinsic to R5 direct the initial lateral orientation of R5 motorneuron axons. Upon reaching a particular lateral position, all R5 motorneuron axons must turn. In normal embryos the axons all turn rostrally to reach the nerve exit in R4. In embryos with R4 ablated, sometimes the axons turn rostrally and sometimes they turn caudally. A model combining permissive fields and chemotropic cues is presented to account for our observations.     

Diencephalic neuronal populations projecting axons into the basal plate in a lizard (Gallotia galloti).

Lizard diencephalic populations sending axons into the basal plate were studied by the in vitro HRP technique in the lizard Gallotia. Retrograde labeled cells were concentrated in distinct neuronal groups within alar plates of prosomeres p1 and p3, whereas the alar plate of p2 was poorly labeled. Efferent fibers from alar p1 and p3 populations entered the basal plate of the diencephalon along topologically dorsoventral courses, bifurcating thereafter into longitudinal ascending (rostral) and descending (caudal) trajectories. Thus, diencephalic segments p1 and p3 have alar cell populations contributing to the longitudinal premotor connectivity of the neural axis , whereas the alar p2 segment projects via the fasciculus retroflexus, the efferent tract of the epithalamus. However, the axons from the habenular complex bifurcate within or adjacent to the floor plate and not within the basal plate.     

Cochlear and lagenar ganglia of the chicken

Cochlear and lagenar components of the statoacoustical ganglion in the inner ear of one chicken were studied quantitatively in the TEM. Both myelinated and unmyelinated nerve fibers were present in these two parts of the ganglion and in a putative efferent bundle within the ganglion. The cochlear portion had the lowest, the efferent bundle the highest percentage of unmyelinated fibers. Compared to the other parts of the ganglia, the cochlear fibers had a high degree of homogeneity, especially in fiber size. Some gradients in the baso-apical direction were found, such as an increase in the size of myelinated cochlear fibers from the base to the apex. Based on the ultrastructure of cellular components, no distinct populations of cell bodies within the statoacoustical ganglion were definable. The ganglion contained some 8,000 cochlear and about 1,200–2,000 lagenar neurons. The putative efferent bundle had only 150–200 fibers. This cannot be the total number of efferents to the hair cells in both the basilar papilla and the lagena. A large number of efferent fibers to the auditory papillae presumably run mingled among the afferent fibers. © 1994 Wiley-Liss, Inc.     

Brainstem neurons projecting to the ampullae of anterior,lateral, and posterior semicircular canals in the guinea pig

Neurons projecting to the ampullae of anterior, lateral, and posterior semicircular canals were identified in the guinea pig brainstem using horseradish peroxidase labeling techniques. Two groups of neurons forming bilateral connections were found, one located dorsally and the other ventrally to facial nerve trajectories. The dorsal group of vestibular efferent neurons projecting to all three canals was detected in the subependymal granular layer of the floor of the 4th ventricle lateral to the facial nerve genu and in the abducent nerve nucleus. Efferent neurons belonging to the ventral group were unevenly distributed through different areas of the parvocellularis nucleus and the rostral portion of the pontine caudal reticular nucleus. The morphological characteristics and distribution pattern of these cells are taken as confirmation of their heterogeneity of neuronal and functional organization in the vestibular efferent system of semicircular canal ampullae.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 4, 1988, pp. 526–532.     

Afferent and efferent components of the facial nerve in a frog,Rana pipiens

Summary The seventh cranial nerve in Rana pipiens is a slender nerve with limited peripheral distribution. We investigated the afferent and efferent components of this nerve by labeling its major branch, the hyomandibular, with horseradish peroxidase. The efferent portion of the seventh nerve originates from a small cell group in the upper medulla which contains two subdivisions. Afferent fibers carried in nerve VII travel in the solitary tract and the dorsolateral funiculus. The solitary component consists of a small number of ascending fibers that reach the level of the trigeminal nucleus and a large descending component that terminates slightly caudal to the obex in the commissural nuclei of the solitary complex. Afferent fibers also descend in the dorsolateral funiculus; many of these fibers cross dorsal to the central canal in the lower medulla. Most of the fibers in the dorsolateral funiculus terminate in the ipsilateral and contralateral dorsal horns and in nuclei of the dorsal column. A few ipsilateral fibers reach lower thoracic levels of the spinal cord.     

Corticotrigeminal motor pathway in the rat--II. Anterio- and retrograde HRP labeling

1. Horseradish peroxidase (HRP) injection into the rat cortical jaw motor area (JMA) disclosed direct projection fibers in and around the motor trigeminal nucleus (MTN), primarily contralaterally. 2. Descending axons were found in the ventromedial half of the cerebral peduncle and MTN-projecting axons were concentrated near the descending facial nerve root. 3. HRP injection into the peduncle could label 1257 cells in the JMA, 128 cells in the taste area and 1409 cells in the neck-forelimb motor area ipsilaterally. 4. Some MTN-terminated axons could be traced from the peduncle in serial sections.     

Autonomous and nonautonomous functions for Hox/Pbx in branchiomotor neuron development

The vertebrate branchiomotor neurons are organized in a pattern that corresponds with the segments, or rhombomeres, of the developing hindbrain and have identities and behaviors associated with their position along the anterior/posterior axis. These neurons undergo characteristic migrations in the hindbrain and project from stereotyped exit points. We show that lazarus/pbx4, which encodes an essential Hox DNA-binding partner in zebrafish, is required for facial (VIIth cranial nerve) motor neuron migration and for axon pathfinding of trigeminal (Vth cranial nerve) motor axons. We show that lzr/pbx4 is required for Hox paralog group 1 and 2 function, suggesting that Pbx interacts with these proteins. Consistent with this, lzr/pbx4 interacts genetically with hoxb1a to control facial motor neuron migration. Using genetic mosaic analysis, we show that lzr/pbx4 and hoxb1a are primarily required cell-autonomously within the facial motor neurons; however, analysis of a subtle non-cell-autonomous effect indicates that facial motor neuron migration is promoted by interactions amongst the migrating neurons. At the same time, lzr/pbx4 is required non-cell-autonomously to control the pathfinding of trigeminal motor axons. Thus, Pbx/Hox can function both cell-autonomously and non-cell-autonomously to direct different aspects of hindbrain motor neuron behavior.     

Transplantation of Xenopus laevis ears reveals the ability to form afferent and efferent connections with the spinal cord

Previous comparative and developmental studies have suggested that the cholinergic inner ear efferent system derives from developmentally redirected facial branchial motor neurons that innervate the vertebrate ear hair cells instead of striated muscle fibers. Transplantation of Xenopus laevis ears into the path of spinal motor neuron axons could show whether spinal motor neurons could reroute to innervate the hair cells as efferent fibers. Such transplantations could also reveal whether ear development could occur in a novel location including afferent and efferent connections with the spinal cord. Ears from stage 24-26 embryos were transplanted from the head to the trunk and allowed to mature to stage 46. Of 109 transplanted ears, 73 developed with otoconia. The presence of hair cells was confirmed by specific markers and by general histology of the ear, including TEM. Injections of dyes ventral to the spinal cord revealed motor innervation of hair cells. This was confirmed by immunohistochemistry and by electron microscopy structural analysis, suggesting that some motor neurons rerouted to innervate the ear. Also, injection of dyes into the spinal cord labeled vestibular ganglion cells in transplanted ears indicating that these ganglion cells connected to the spinal cord. These nerves ran together with spinal nerves innervating the muscles, suggesting that fasciculation with existing fibers is necessary. Furthermore, ear removal had little effect on development of cranial and lateral line nerves. These results indicate that the ear can develop normally, in terms of histology, in a new location, complete with efferent and afferent innervations to and from the spinal cord.     

Anterograde tracing of retinal axons in the avian embryo with low molecular weight derivatives of biotin

Several reactive biotin esters were injected into the eyes of chick and quail embryos at various stages of development. Four of the biotin esters reacted with molecules of the eye tissue and were detected with light and electron microscopy in fluorescein isothiocyanate and peroxidase-avidin incubated sections and whole mounts. Intra and extracellular components of the lens, the vitreous body, and the retina were labeled to different degrees. Three of the biotin esters (biotin-N-hydroxysuccinimidester, biotin-epsilon-aminocaproic acid-N-hydroxysuccinimidester, and desthiobiotin-N-hydroxysuccinimidester) prominently marked the optic fiber layer in the retina and the biotin labels were transported along the optic pathway. The tracers were detected up to the growth cone of axons 24 to 36 hr after injection. Explants from biotin marked retinas were cultured on collagen or basal laminae. During culturing axons grew out from these explants into the substratum showing that labeled tissue and nerve fibers were viable. The development of the optic pathway at the chiasma of quail embryos was studied using the biotin/avidin tracing. The bulk of fibers emerging from the retina crossed as shown by double labeling of both optic nerves in a complex pattern of segregated and interdigitizing axon bundles at the chiasma toward the contralateral side of the brain. From stage 25 onward a minor ipsilateral projection was found. At the same developmental stage a few fibers traveled into the contralateral optic nerve and grew retrogradely toward the contralateral eye. The percentage of specimens having this retino-retinal projection increased during development from 53% (stage 24 to 27; E3.5-E5.5) to 89% (stage 29 to 35; E6-E8) and declined to 40% at late embryogenesis (stage 37 to 41; E9-E12). The fact that all retinal axons were found within predictable pathways with some of them running in the wrong direction suggests that nerve fiber pathways provide accurate positional information, but at best weak directional information for growing nerve fibers.     

Neurosecretory cells in the circadian-clock system of the scorpion,Androctonus australis

Summary The somata of the efferent neurosecretory fibers that control the circadian sensitivity rhythm in the median eyes of the scorpion, Androctonus australis, were detected in the brain by retrograde labeling with Lucifer Yellow CH. A total of 20–40 neurons are arranged in two groups displaying a bilaterally symmetrical, marginal position near the circumesophageal connectives. Half the cells in each group send fibers into the ipsilateral optic nerve; the fibers from the other half enter the contralateral optic nerve.     

F-Spondin is required for accurate pathfinding of commissural axons at the floor plate.

The commissural axons project toward and across the floor plate. They then turn into the longitudinal axis, extending along the contralateral side of the floor plate. F-spondin, a protein produced and secreted by the floor plate, promotes adhesion and neurite extension of commissural neurons in vitro. Injection of purified F-spondin protein into the lumen of the spinal cord of chicken embryos in ovo resulted in longitudinal turning of commissural axons before reaching the floor plate, whereas neutralizing antibody (Ab) injections caused lateral turning at the contralateral floor plate boundary. These combined in vitro and in vivo results suggest that F-spondin is required to prevent the lateral drifting of the commissural axons after having crossed the floor plate.     

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.     

Role of hindbrain in inner ear morphogenesis: analysis of Noggin knockout mice

Signaling from rhombomeres 5 and 6 of the hindbrain is thought to be important for inner ear patterning. In Noggin −/− embryos, the gross anatomy of the inner ear is distorted and malformed, with cochlear duct outgrowth and coiling most affected. We attributed these defects to a caudal shift of the rhombomeres caused by the shortened body axis and the kink in the neural tube. To test the hypothesis that a caudal shift of the rhombomeres affects inner ear development, we surgically generated chicken embryos in which rhombomeres 5 and 6 were similarly shifted relative to the position of the inner ears, as in Noggin mutants. All chicken embryos with shifted rhombomeres showed defects in cochlear duct formation indicating that signaling from rhombomeres 5 and 6 is important for cochlear duct patterning in both chicken and mice. In addition, the size of the otic capsule is increased in Noggin −/− mutants, which most likely is due to unopposed BMP signaling for chondrogenesis in the peri-otic mesenchyme.     

Neuro-Anatomical Evidence Indicating Indirect Modulation of Macrophages by Vagal Efferents in the Intestine but Not in the Spleen

Background

Electrical stimulation of the vagus nerve suppresses intestinal inflammation and normalizes gut motility in a mouse model of postoperative ileus. The exact anatomical interaction between the vagus nerve and the intestinal immune system remains however a matter of debate. In the present study, we provide additional evidence on the direct and indirect vagal innervation of the spleen and analyzed the anatomical evidence for neuroimmune modulation of macrophages by vagal preganglionic and enteric postganglionic nerve fibers within the intestine.

Methods

Dextran conjugates were used to label vagal preganglionic (motor) fibers projecting to the small intestine and spleen. Moreover, identification of the neurochemical phenotype of the vagal efferent fibers and enteric neurons was performed by immunofluorescent labeling. F4/80 antibody was used to label resident macrophages.

Results

Our anterograde tracing experiments did not reveal dextran-labeled vagal fibers or terminals in the mesenteric ganglion or spleen. Vagal efferent fibers were confined within the myenteric plexus region of the small intestine and mainly endings around nNOS, VIP and ChAT positive enteric neurons. nNOS, VIP and ChAT positive fibers were found in close proximity of intestinal resident macrophages carrying α7 nicotinic receptors. Of note, VIP receptors were found on resident macrophages located in close proximity of VIP positive nerve fibers.

Conclusion

In the present study, we show that the vagus nerve does not directly interact with resident macrophages in the gut or spleen. Instead, the vagus nerve preferentially interacts with nNOS, VIP and ChAT enteric neurons located within the gut muscularis with nerve endings in close proximity of the resident macrophages.     

Guidance of commissural growth cones at the floor plate in embryonic rat spinal cord

The floor plate of the embryonic rat spinal cord has been proposed to act as an intermediate target that plays a role in the pattern of extension of commissural axons. To begin to examine the role of the floor plate in axon guidance at the midline, we have studied the precision of the commissural axon projection to and across the floor plate during development. To delineate the pathway, the fluorescent carbocyanine dye, Di-I, has been used as a probe. We show that commissural axons traverse the floor plate and turn rostrally at its contralateral border with remarkable precision. Axons were not observed to turn ipsilaterally and turned only upon reaching the contralateral edge of the floor plate. Virtually all commissural axons follow this route. The morphology of commissural growth cones was also examined. As they encountered the floor plate, commissural growth cones became larger and increased in complexity. The reorientation of axons in register with the floor plate boundary and the change in the morphological properties of commissural growth cones as they traverse the midline suggest that the floor plate may act as a guidepost with functions similar to cells that have been implicated in axon guidance in invertebrates.     

Normal and abnormal pathfinding of facial nerve fibers in the chick embryo.

Development of the facial nerve was studied in normal chicken embryos and after surgical disruption of ingrowing sensory facial nerve fibers at 38-72 h of incubation. Disruption of facial nerve fibers by otocyst removal often induced a rostral deviation of the facial nerve and ganglion to the level of the trigeminal ganglion. Cell bodies of the geniculate ganglion trailed their deviating neurites and occupied an abnormal rostral position adjacent to the trigeminal ganglion. Deviating facial nerve fibers were labeled with the carbocyanine fluorescent tracer DiI in fixed tissue. Labeled fibers penetrated the cranium adjacent to the trigeminal ganglion, but they did not follow the trigeminal nerve fibers into the brain stem. Rather, after entering the cranium, they projected caudally to their usual site of entrance and proceeded towards their normal targets. This rostral deviation of the facial nerve was observed only after surgery at 48-72 h of incubation, but not in cases with early otocyst removal (38-48 h). A rostral deviation of the facial nerve was seen in cases with partial otocyst removal when the vestibular nerve was absent. The facial nerve followed its normal course when the vestibular nerve persisted. We conclude that disruption of the developing facial pathway altered the routes of navigating axons, but did not prevent pathfinding and innervation of the normal targets. Pathfinding abilities may not be restricted to pioneering axons of the facial nerve; later-developing facial nerve fibers also appeared to have positional information. Our findings are consistent with the hypothesis that navigating axons may respond to multiple guidance cues during development. These cues appear to differ as a function of position of the navigating axon.