Dpysl2 (CRMP2) is required for the migration of facial branchiomotor neurons in the developing zebrafish embryo.

Dihydropyrimidinase-like family proteins (Dpysls) are relevant in several processes during nervous system development; among others, they are involved in axonal growth and cell migration. Dpysl2 (CRMP2) is the most studied member of this family; however, its role in vivo is still being investigated. Our previous studies in zebrafish showed the requirement of Dpysl2 for the proper positioning of caudal primary motor neurons and Rohon-Beard neurons in the spinal cord.In the present study, we show that Dpysl2 is necessary for the proper migration of facial branchiomotor neurons during early development in zebrafish. We generated a dpysl2 knock-out (KO) zebrafish mutant line and used different types of antisense morpholino oligonucleotides (AMO) to analyze the role of Dpysl2 in this process. Both dpysl2 KO mutants and morphants exhibited abnormalities in the migration of these neurons from rhombomers (r) 4 and 5 to 6 and 7. The facial branchiomotor neurons that were expected to be at r6 were still located at r4 and r5 hours after the migration process should have been completed. In addition, mutant phenotypes were rescued by injecting dpysl2 mRNA into the KO embryos. These results indicate that Dpysl2 is involved in the proper migration of facial branchiomotor neurons in developing zebrafish embryos.

A Rapid Protocol for Intraoperative Assessment of Peripheral Nerve Myelinated Axon Count and Its Application to Cross-Facial Nerve Grafting.

BACKGROUND: Donor nerve myelinated axon counts correlate with functional outcomes in reanimation procedures; however, there exists no reliable means for their intraoperative quantification. In this article, the authors report a novel protocol for rapid quantification of myelinated axons from frozen sections, and demonstrate its applicability to surgical practice. METHODS: The impact of various fixation and FluoroMyelin Red staining strategies on resolved myelin sheath morphology from cryosections of rat and rabbit femoral and sciatic nerves was assessed. A protocol comprising fresh cryosection and rapid staining was developed, and histomorphometric results were compared against conventional osmium-postfixed, resin-embedded, toluidine blue-stained sections of rat sciatic nerve. The rapid protocol was applied for intraoperative quantification of donor nerve myelinated axon count in a cross-facial nerve grafting procedure. RESULTS: Resolution of myelinated axon morphology suitable for counting was realized within 10 minutes of tissue harvest. Although mean myelinated axon diameter appeared larger using the rapid fresh-frozen as compared to conventional nerve processing techniques (mean ± SD; rapid, 9.25 ± 0.62 µm; conventional, 6.05 ± 0.71 µm; p < 0.001), no difference in axon counts was observed on high-power fields (rapid, 429.42 ± 49.32; conventional, 460.32 ± 69.96; p = 0.277). Whole nerve myelinated axon counts using the rapid protocol herein (8435.12 ± 1329.72) were similar to prior reports using conventional osmium processing of rat sciatic nerve. CONCLUSIONS: A rapid protocol for quantification of myelinated axon counts from peripheral nerves using widely available equipment and techniques has been described, rendering possible intraoperative assessment of donor nerve suitability for reanimation.

Constriction of the buccal branch of the facial nerve produces unilateral craniofacial allodynia.

Despite pain being a sensory experience, studies of spinal cord ventral root damage have demonstrated that motor neuron injury can induce neuropathic pain. Whether injury of cranial motor nerves can also produce nociceptive hypersensitivity has not been addressed. Herein, we demonstrate that chronic constriction injury (CCI) of the buccal branch of the facial nerve results in long-lasting, unilateral allodynia in the rat. An anterograde and retrograde tracer (3000MW tetramethylrhodamine-conjugated dextran) was not transported to the trigeminal ganglion when applied to the injury site, but was transported to the facial nucleus, indicating that this nerve branch is not composed of trigeminal sensory neurons. Finally, intracisterna magna injection of interleukin-1 (IL-1) receptor antagonist reversed allodynia, implicating the pro-inflammatory cytokine IL-1 in the maintenance of neuropathic pain induced by facial nerve CCI. These data extend the prior evidence that selective injury to motor axons can enhance pain to supraspinal circuits by demonstrating that injury of a facial nerve with predominantly motor axons is sufficient for neuropathic pain, and that the resultant pain has a neuroimmune component.

Amino acid specificity of fibers of the facial/trigeminal complex innervating the maxillary barbel in the Japanese sea catfish, Plotosus japonicus.

The Japanese sea catfish, Plotosus japonicus, possesses taste and solitary chemoreceptor cells (SCCs) located on the external body surface that detect specific water-soluble substances. Here, we identify two major fiber types of the facial/trigeminal complex that transmit amino acid information to the medulla. Both single and few fiber preparations respond to amino acid stimulation in the 0.1 µM to mM range. One fiber type responds best to glycine and l-alanine (i.e. Gly/Ala fibers) whereas the other fiber type is best stimulated by l-proline and glycine betaine (hereafter referred to only as betaine) (i.e. Pro/Bet fibers). We demonstrate that betaine, which does not alter the pH of the seawater and therefore does not activate the animals' highly sensitive pH sensors (Caprio et al., Science 344:1154-1156, 2014), is sufficient to elicit appetitive food search behavior. We further show that the amino acid specificity of fibers of the facial/trigeminal complex in P. japonicus is different from that in Ariopsis felis (Michel and Caprio, J. Neurophysiol. 66:247-260, 1991; Michel et al., J. Comp. Physiol. A. 172:129-138, 1993), a representative member of the only other family (Ariidae) of extant marine catfishes.

Possible neural network mediating jaw opening during prey-catching behavior of the frog.

The prey-catching behavior of the frog is a complex, well-timed sequence of stimulus response chain of movements. After visual analysis of the prey, a size dependent program is selected in the motor pattern generator of the brainstem. Besides this predetermined feeding program, various direct and indirect sensory inputs provide flexible adjustment for the optimal contraction of the executive muscles. The aim of the present study was to investigate whether trigeminal primary afferents establish direct contacts with the jaw opening motoneurons innervated by the facial nerve. The experiments were carried out on Rana esculenta (Pelophylax esculentus), where the trigeminal and facial nerves were labeled simultaneously with different fluorescent dyes. Using a confocal laser scanning microscope, close appositions were detected between trigeminal afferent fibers and somatodendritic components of the facial motoneurons. Quantitative analysis revealed that the majority of close contacts were encountered on the dendrites of facial motoneurons and approximately 10% of them were located on the perikarya. We suggest that the identified contacts between the trigeminal afferents and facial motoneurons presented here may be one of the morphological substrate in the feedback and feedforward modulation of the rapidly changing activity of the jaw opening muscle during the prey-catching behavior.

Rest represses maturation within migrating facial branchiomotor neurons.

The vertebrate brain arises from the complex organization of millions of neurons. Neurogenesis encompasses not only cell fate specification from neural stem cells, but also the terminal molecular and morphological maturation of neurons at correct positions within the brain. RE1-silencing transcription factor (Rest) is expressed in non-neural tissues and neuronal progenitors where it inhibits the terminal maturation of neurons by repressing hundreds of neuron-specific genes. Here we show that Rest repression of maturation is intimately linked with the migratory capability of zebrafish facial branchiomotor neurons (FBMNs), which undergo a characteristic tangential migration from hindbrain rhombomere (r) 4 to r6/r7 during development. We establish that FBMN migration is increasingly disrupted as Rest is depleted in zebrafish rest mutant embryos, such that around two-thirds of FBMNs fail to complete migration in mutants depleted of both maternal and zygotic Rest. Although Rest is broadly expressed, we show that de-repression or activation of Rest target genes only within FBMNs is sufficient to disrupt their migration. We demonstrate that this migration defect is due to precocious maturation of FBMNs, based on both morphological and molecular criteria. We further show that the Rest target gene and alternative splicing factor srrm4 is a key downstream regulator of maturation; Srrm4 knockdown partially restores the ability of FBMNs to migrate in rest mutants while preventing their precocious morphological maturation. Rest must localize to the nucleus to repress its targets, and its subcellular localization is highly regulated: we show that targeting Rest specifically to FBMN nuclei rescues FBMN migration in Rest-deficient embryos. We conclude that Rest functions in FBMN nuclei to inhibit maturation until the neurons complete their migration.

Mouse hindbrain ex vivo culture to study facial branchiomotor neuron migration.

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.

Facial motor neuron migration advances.

During development, the migration of specific neuronal subtypes is required for the correct establishment of neural circuits. In mice and zebrafish, facial branchiomotor (FBM) neurons undergo a tangential migration from rhombomere 4 caudally through the hindbrain. Recent advances in the field have capitalized on genetic studies in zebrafish and mouse, and high-resolution time-lapse imaging in zebrafish. Planar cell polarity signaling has emerged as a critical conserved factor in FBM neuron migration, functioning both within the neurons and their environment. In zebrafish, migration depends on specialized 'pioneer' neurons to lead follower FBM neurons through the hindbrain, and on interactions with structural components including pre-laid axon tracts and the basement membrane. Despite fundamental conservation, species-specific differences in migration mechanisms are being uncovered.

Bone marrow stem cells in facial nerve regeneration from isolated stumps.

INTRODUCTION: Severe lesions in the facial nerve may have extensive axonal loss and leave isolated stumps that impose technical difficulties for nerve grafting. METHODS: We evaluated bone marrow stem cells (BMSC) in a silicone conduit for rat facial nerve regeneration from isolated stumps. Group A utilized empty silicone tubes; in groups B-D, the tube was filled with acellular gel; and, in groups C and D, undifferentiated BMSC (uBMSC) or Schwann-like cells differentiated from BMSC (dBMSC) were added, respectively. Compound muscle action potentials (CMAPs) were measured, and histology was evaluated. RESULTS: Groups C and D had the highest CMAP amplitudes. Group C had shorter CMAP durations than groups A, B, and D. Distal axonal number and density were increased in group C compared with groups A and B. CONCLUSIONS: Regeneration of the facial nerve was improved by both uBMSC and dBMSC in rats, yet uBMSC was associated with superior functional results.

Recurrent facial taste neurons of sea catfish Plotosus japonicus: morphology and organization in the ganglion.

This study investigated the morphology of the recurrent facial taste neurons and their organization in the recurrent ganglion of the sea catfish Plotosus japonicus. The recurrent ganglion is independent of the anterior ganglion, which consists of trigeminal, facial and anterior lateral line neurons that send peripheral fibres to the head region. The recurrent taste neurons are round or oval and bipolar, with thick peripheral and thin central fibres, and completely wrapped by membranous layers of satellite cells. Two peripheral nerve branches coursing to the trunk or pectoral fin originate from the recurrent ganglion. The results presented here show that the trunk and pectoral-fin neurons are independently distributed to form various sizes of groups, and the groups are intermingled throughout the ganglion. No distinct topographical relationship of the two nerve branches occurs in the ganglion. Centrally, the trunk and pectoral-fin branches project somatotopically in the anterolateral and intermediate medial regions of the trunk tail lobule of the facial lobe, respectively.

Degeneration and regeneration of motor and sensory nerves: a stereological study of crush lesions in rat facial and mental nerves.

The aim of this study was to evaluate the degeneration and regeneration of a sensory nerve and a motor nerve at the histological level after a crush injury. Twenty-five female Wistar rats had their mental nerve and the buccal branch of their facial nerve compressed unilaterally against a glass rod for 30s. Specimens of the compressed nerves and the corresponding control nerves were dissected at 3, 7, and 19 days after surgery. Nerve cross-sections were stained with osmium tetroxide and toluidine blue and analysed using two-dimensional stereology. We found differences between the two nerves both in the normal anatomy and in the regenerative pattern. The mental nerve had a larger cross-sectional area including all tissue components. The mental nerve had a larger volume fraction of myelinated axons and a correspondingly smaller volume fraction of endoneurium. No differences were observed in the degenerative pattern; however, at day 19 the buccal branch had regenerated to the normal number of axons, whereas the mental nerve had only regained 50% of the normal number of axons. We conclude that the regenerative process is faster and/or more complete in the facial nerve (motor function) than it is in the mental nerve (somatosensory function).

Stimulation of trigeminal afferents improves motor recovery after facial nerve injury: functional, electrophysiological and morphological proofs.

Recovery of mimic function after facial nerve transection is poor: the successful regrowth of axotomized motoneurons to their targets is compromised by (1) poor axonal navigation and excessive collateral branching, (2) abnormal exchange of nerve impulses between adjacent regrowing axons, and (3) insufficient synaptic input to facial motoneurons. As a result, axotomized motoneurons get hyperexcitable and unable to discharge. Since improvement of growth cone navigation and reduction of the ephaptic cross talk between axons turn out be very difficult, we concentrated our efforts on the third detrimental component and proposed that an intensification of the trigeminal input to axotomized electrophysiologically silent facial motoneurons might improve specificity of reinnervation. To test our hypothesis we compared behavioral, electrophysiological, and morphological parameters after single reconstructive surgery on the facial nerve (or its buccal branch) with those obtained after identical facial nerve surgery but combined with direct or indirect stimulation of the ipsilateral infraorbital (ION) nerve. We found that in all cases, trigeminal stimulation was beneficial for the outcome by improving the quality of target reinnervation and recovery of vibrissa! motor performance.

Axon tracts guide zebrafish facial branchiomotor neuron migration through the hindbrain.

Appropriate localization of neurons within the brain is a crucial component of the establishment of neural circuitry. In the zebrafish hindbrain, the facial branchiomotor neurons (FBMNs) undergo a chain-like tangential migration from their birthplace in rhombomere (r) 4 to their final destination in r6/r7. Here, we report that ablation of either the cell body or the trailing axon of the leading FBMN, or 'pioneer' neuron, blocks the migration of follower FBMNs into r5. This demonstrates that the pioneer neuron and its axon are crucial to the early migration of FBMNs. Later migration from r5 to r6 is not dependent on pioneer neurons but on the medial longitudinal fasciculus (MLF), a bundle of axons lying ventral to the FBMNs. We find that MLF axons enter r5 only after the pioneer neuron has led several followers into this region; the MLF is then contacted by projections from the FBMNs. The interactions between FBMNs and the MLF are important for migration from r5 to r6, as blocking MLF axons from entering the hindbrain can stall FBMN migration in r5. Finally, we have found that the adhesion molecule Cdh2 (N-cadherin) is important for interactions between the MLF and FBMNs, as well as for interactions between the trailing axon of the pioneer neuron and follower FBMNs. Interestingly, migration of pioneer neurons is independent of both the MLF and Cdh2, suggesting pioneer migration relies on independent cues.

What determines motor neuron number? Slow scaling of facial motor neuron numbers with body mass in marsupials and primates.

How does the number of motor neurons in the brain correlate with the muscle mass to be controlled in the body? Numbers of motor neurons are known to be adjusted during development by cell death, but the change in the percentage of surviving motor neurons in response to experimental changes in target muscle mass is relatively small. Here we address the quantitative matching between final numbers of motor neurons in the facial nucleus and body mass (which we use as a proxy for the muscle mass). In 22 marsupial species, we found that the number of facial motor neurons is strongly correlated with body mass, and scales across species as a power function of body mass with a very small exponent of 0.184, which is close to the exponent found in primates from previously published data. With such an exponent, doubling the body mass is accompanied by a modest increase of only 14% in numbers of facial motor neurons, while halving body mass results in a decrease of only 12%. These numbers are remarkably similar to the 15-20% increase or 8% decrease in the number of spinal cord motor neurons that results from experimental or natural doubling or reducing by half the target muscle field of birds and amphibians. The scaling rule presented here might thus account for the quantitative matching of motor neurons to their target muscle mass in evolution. With this small scaling exponent, our data also raise the possibility that larger animals will have larger motor units.

Planar cell polarity and the developmental control of cell behavior in vertebrate embryos.

Planar cell polarity (PCP), the orientation and alignment of cells within a sheet, is a ubiquitous cellular property that is commonly governed by the conserved set of proteins encoded by so-called PCP genes. The PCP proteins coordinate developmental signaling cues with individual cell behaviors in a wildly diverse array of tissues. Consequently, disruptions of PCP protein functions are linked to defects in axis elongation, inner ear patterning, neural tube closure, directed ciliary beating, and left/right patterning, to name only a few. This review attempts to synthesize what is known about PCP and the PCP proteins in vertebrate animals, with a particular focus on the mechanisms by which individual cells respond to PCP cues in order to execute specific cellular behaviors.

Nociceptive afferents to the premotor neurons that send axons simultaneously to the facial and hypoglossal motoneurons by means of axon collaterals.

It is well known that the brainstem premotor neurons of the facial nucleus and hypoglossal nucleus coordinate orofacial nociceptive reflex (ONR) responses. However, whether the brainstem PNs receive the nociceptive projection directly from the caudal spinal trigeminal nucleus is still kept unclear. Our present study focuses on the distribution of premotor neurons in the ONR pathways of rats and the collateral projection of the premotor neurons which are involved in the brainstem local pathways of the orofacial nociceptive reflexes of rat. Retrograde tracer Fluoro-gold (FG) or FG/tetramethylrhodamine-dextran amine (TMR-DA) were injected into the VII or/and XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the caudal spinal trigeminal nucleus (Vc). The tracing studies indicated that FG-labeled neurons receiving BDA-labeled fibers from the Vc were mainly distributed bilaterally in the parvicellular reticular formation (PCRt), dorsal and ventral medullary reticular formation (MdD, MdV), supratrigeminal nucleus (Vsup) and parabrachial nucleus (PBN) with an ipsilateral dominance. Some FG/TMR-DA double-labeled premotor neurons, which were observed bilaterally in the PCRt, MdD, dorsal part of the MdV, peri-motor nucleus regions, contacted with BDA-labeled axonal terminals and expressed c-fos protein-like immunoreactivity which induced by subcutaneous injection of formalin into the lip. After retrograde tracer wheat germ agglutinated horseradish peroxidase (WGA-HRP) was injected into VII or XII and BDA into Vc, electron microscopic study revealed that some BDA-labeled axonal terminals made mainly asymmetric synapses on the dendritic and somatic profiles of WGA-HRP-labeled premotor neurons. These data indicate that some premotor neurons could integrate the orofacial nociceptive input from the Vc and transfer these signals simultaneously to different brainstem motonuclei by axonal collaterals.

Planar polarity pathway and Nance-Horan syndrome-like 1b have essential cell-autonomous functions in neuronal migration.

Components of the planar cell polarity (PCP) pathway are required for the caudal tangential migration of facial branchiomotor (FBM) neurons, but how PCP signaling regulates this migration is not understood. In a forward genetic screen, we identified a new gene, nhsl1b, required for FBM neuron migration. nhsl1b encodes a WAVE-homology domain-containing protein related to human Nance-Horan syndrome (NHS) protein and Drosophila GUK-holder (Gukh), which have been shown to interact with components of the WAVE regulatory complex that controls cytoskeletal dynamics and with the polarity protein Scribble, respectively. Nhsl1b localizes to FBM neuron membrane protrusions and interacts physically and genetically with Scrib to control FBM neuron migration. Using chimeric analysis, we show that FBM neurons have two modes of migration: one involving interactions between the neurons and their planar-polarized environment, and an alternative, collective mode involving interactions between the neurons themselves. We demonstrate that the first mode of migration requires the cell-autonomous functions of Nhsl1b and the PCP components Scrib and Vangl2 in addition to the non-autonomous functions of Scrib and Vangl2, which serve to polarize the epithelial cells in the environment of the migrating neurons. These results define a role for Nhsl1b as a neuronal effector of PCP signaling and indicate that proper FBM neuron migration is directly controlled by PCP signaling between the epithelium and the migrating neurons.

Effects of corticosteroids on functional recovery and neuron survival after facial nerve injury in mice.

OBJECTIVES: To assess the effects of corticosteroid administration on functional recovery and cell survival in the facial motor nucleus (FMN) following crush injury in adult and juvenile mice and to evaluate the relationship between functional recovery and facial motoneuron survival. METHODS: A prospective blinded analysis of functional recovery and cell survival in the FMN after crush injury in juvenile and adult mice was carried out. All mice underwent a unilateral facial nerve crush injury and received 7 doses of daily injections. Adults received normal saline or low-dose or high-dose corticosteroid treatment. Juveniles received either normal saline or low-dose corticosteroid treatment. Whisker function was monitored to assess functional recovery. Stereologic analysis was performed to determine neuron and glial survival in the FMN following recovery. RESULTS: Following facial nerve injury, all adult mice recovered fully, while juvenile mice recovered slower and incompletely. This corresponded to a significantly greater neuron loss in the FMN of juveniles compared with adults. Corticosteroid treatment slowed functional recovery in adult mice. This corresponded with significantly greater neuron loss in the FMN in corticosteroid-treated mice. In juvenile mice, corticosteroid treatment showed a trend, which was significant at several time points, toward a more robust functional recovery compared with controls. CONCLUSIONS: Corticosteroid treatment slows functional recovery and impairs neuron survival following facial nerve crush injury in adult mice. The degree of motor neuron survival corresponds with functional status. In juvenile mice, crush injury results in overall poor functional recovery and profound cell loss in the FMN. With low-dose corticosteroid treatment, there is a significantly enhanced functional recovery after injury in these mice (P < .05).

Macrophage-colony stimulating factor as an inducer of microglial proliferation in axotomized rat facial nucleus.

We analyzed the mechanism of microglial proliferation in rat axotomized facial nucleus (axotFN). In immunoblotting analysis for possible mitogens, we noticed that the amounts of macrophage-colony stimulating factor (M-CSF) increased in the axotFN for 3-7 days after transection. In contrast, the amounts of granulocyte macrophage-CSF and interleukin-3 did not significantly increase. A potential source for M-CSF was immunohistochemically verified to be microglia. Immunoblotting showed that the amounts of receptor for M-CSF (cFms) increased in the axotFN for 3-14 days after injury, and immunohistochemical staining showed that cFms is expressed in microglia. Proliferating cell nuclear antigen as a marker of proliferation was immunohistochemically identified in microglia in axotFN, and the level was found to peak 3 days after transection in immunoblotting. Hypothesizing that up-regulated M-CSF triggers the above phenomena, we investigated the effects of M-CSF on cFms and proliferating cell nuclear antigen levels in primary microglia. The biochemical experiments revealed that M-CSF induces cFms and drives the cell cycle in microglia. The neutralization of M-CSF in microglia derived from axotFN significantly reduced the proliferation. These results demonstrate that up-regulated M-CSF triggers the induction of cFms in microglia and causes the microglia to proliferate in the axotFN.

Some results of our research on composite facial allograft transplantation in dogs.

Composite facial allograft transplantation is a complicated surgical procedure, requiring evaluation of several questions. How should the facial allograft be preserved? What are the proper surgical skills to reduce surgery duration? Can composite facial allograft transplantation rebuild the motor functions of the facial muscles? In our research, University of Wisconsin (UW) solution was used to preserve canine facial tissue. We manipulated the surgical procedures of complete facial allograft transplantation on cadavers to improve surgery skills Canine facial allograft models with neuromuscular motor units were performed to evaluate nerve regeneration after allotransplantation. We observed that canine facial allografts could be preserved in UW solution for at least 18 hours. Superficial temporal artery and facial artery bipedicle flaps were safer and saved more time than single pedicle flaps from the external carotid artery. Facial neuromuscular activities were shown in the canine model by the blinking reflex.