Ectoderm removal prevents cutaneous nerve formation and perturbs sensory axon growth in the chick hindlimb

Target tissues are thought to provide important cues for growing axons, yet there is little direct evidence that they are essential for axonal pathfinding. Here we examined whether target ectoderm is necessary for the formation of cutaneous nerves, and for the normal growth and guidance of cutaneous axons as they first enter the limb plexus. To do this, we removed a patch of ectoderm from the chick hindlimb at various times during early axon outgrowth. We find there is a critical period when cutaneous nerve formation requires target ectoderm. When the ectoderm is absent during this time, axons progress into the limb more slowly and, although a few sensory axons occasionally diverge a short distance from the plexus, they do not form a discrete nerve that travels to the skin. A few days later, when the nerve pattern is mature, axons normally destined for the 'deprived' cutaneous nerve are not segregated appropriately within the plexus. Some cutaneous axons are instead misdirected along an inappropriate cutaneous nerve, while others have seemingly failed to reach their correct target, or a suitable alternative, and died. These results demonstrate that the target ectoderm is necessary for normal sensory axon growth and guidance in the hindlimb.     

Ephrin-As play a rhombomere-specific role in trigeminal motor axon projections in the chick embryo

In this study, we investigate the possible role of ephrin-Eph signaling in trigeminal motor axon projections. We find that EphA receptors are expressed at higher levels by rhombomere 2 (r2) trigeminal motor neurons than by r3 trigeminal motor neurons in the chick embryo. Mapping of rhombomere-specific axon projections shows that r2 and r3 trigeminal motor neurons project to different muscle targets, including the mandibular adductor and the intermandibularis muscles respectively. Ephrin-A5 is expressed in these muscles, especially in some regions of the intermandibularis muscle, and can cause growth cone collapse of both r2 and r3 motor axons in vitro. We demonstrate that in vivo overexpression of ephrin-A5 in the intermandibularis muscle, or overexpression of dominant-negative EphA receptors in trigeminal motor neurons leads to a reduction in branching of r3-derived motor axons specifically. Overexpression of full-length EphA receptors impairs the formation of r3 projections to the intermandibularis muscle. These findings indicate that ephrins and their Eph receptors play a role in trigeminal motor axon topographic mapping and in rhombomere 3-derived projections in particular.     

Ultrastructural evidence for adrenergic nerve degeneration in the guinea pig uterus during pregnancy

Summary In the guinea pig myometrium, the adrenergic nerves selectively demonstrated at the ultrastructural level after treatment with 5-OH-DA, show varying degree of degeneration during pregnancy. The changes are more extensive in a late gestational stage (40–45 days) than in an early one (20–25 days), and are particularly evident in the uterus overlying the conceptus as compared to the regions between the fetuses. Scattered degenerative changes were also observed in myometrial specimens from virgin animals, but probably reflect the normal continuous turnover of axons.     

Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.     

ERK/MAPK and PI3K/AKT signal channels simultaneously activated in nerve cell and axon after facial nerve injury

BackgroundThe in-vitro study indicated that ERK/MAPK and PI3K/AKT signal channels may play an important role in reparative regeneration process after peripheral nerve injury. But, relevant in-vivo study was infrequent. In particular, there has been no report on simultaneous activation of ERK/MAPK and PI3K/AKT signal channels in facial nerve cell and axon after facial nerve injury.ResultsThe expression of P-ERK enhanced in nerve cells at the injury side on the 1 d after the rat facial nerve was cut and kept on a higher level until 14 d, but decreased on 28 d. The expression of P-AKT enhanced in nerve cells at the injury side on 1 d after injury, and kept on a higher level until 28 d. The expression of P-ERK enhanced at the near and far sections of the injured axon on 1 d, then increased gradually and reached the maximum on 7 d, but decreased on 14 d, until down to the level before the injury on 28 d. The expression of P-AKT obviously enhanced in the injured axon on 1 d, especially in the axon of the rear section, but decreased in the axon of the rear section on 7 d, while the expression of axon in the far section increased to the maximum and kept on till 14 d. On 28 d, the expression of P-AKT decreased in both rear and far sections of the axon.ConclusionThe facial nerve simultaneously activated ERK/MAPK and PI3K/AKT signal channels in facial nerve cells and axons after the cut injury, but the expression levels of P-ERK and P-AKT varied as the function of the time. In particular, they were quite different in axon of the far section. It has been speculated that two signal channels might have different functions after nerve injury. However, their specific regulating effects should still be testified by further studies in regenerative process of peripheral nerve injury.     

Death of an axon: studying axon loss in development and disease

The loss of axon branches is a common feature of both the developing and the diseased nervous system. Despite its fundamental importance, a clear mechanistic understanding is lacking on how axonal loss occurs. However, the first molecular inroads into post-traumatic (Wallerian) axon degeneration have recently been made. In parallel, imaging techniques that allow visualizing single axons in vivo are providing a first glimpse at the cellular mechanisms of active dismantling of superfluous or diseased axons. This gives hope that soon a clearer mechanistic understanding of axon loss will emerge: comparing different forms of axon loss will reveal the spectrum of axon loss mechanisms; studies aimed at integrating the known molecular and cellular players during axon loss will provide mechanistic insight into axon dismantling; finally—by understanding how axons are normally lost—we will hopefully find ways to protect them during neurological disease or after trauma. Note: Robert Feulgen Prize Lecture 2005 presented at the Joint meeting of The Histochemical Society and The Society for Histochemistry in Noordwijkerhout, The Netherlands. After completion of this review, a comprehensive and highly informative overview of axon loss events in development and disease was published (Luo and O’Leary 2005).     

Development of the axon cap neuropil of the Mauthner cell in the goldfish

Summary Development of the axon cap neuropil of the Mauthner neuron in post-hatching larval goldfish brains was observed electron-microscopically. The axonal initial segment of newly hatched (day-4) larvae is completely covered with synaptic terminals containing clear spherical synaptic vesicles. Profiles of thin terminal axons, the spiral fibers, containing similar synaptic vesicles, rapidly increase in number around the initial segment and form glomerular neuropil similar to the central core of the adult axon cap by day 7. Three types of synapses are formed in the core neuropil. Bouton-type synapses contacting the initial segment are most abundant in day-4 to-14 larvae; they decrease thereafter and are rare on the distal half of the initial segment of day-40 larvae. Asymmetric axo-axonic synapses are commonly observed between spiral fibers in the core neuropil of day-7 to -19 larvae, but become fewer by day 40. Unique symmetrical axo-axonic synapses showing accumulation of synaptic vesicles on either side of apposed membrane thickenings first appear in day-14 core neuropil, gradually increase in number, and become the predominant type in day-40 core neuropil. Thick myelinated axons, which lose their myelin sheaths in the glial cap cell layer, start to penetrate into the axon cap on day 10. They gradually increase in number and form the peripheral part of the axon cap together with the cap dendrites, which finally grow into the axon cap from the axon hillock region of the Mauthner cell by day 40.     

The depth of the lingual fossa in permanent incisors of Norwegians. I. Method of measurement, statistical distribution and sex dimorphism

The depth of the incisor lingual fossa in permanent extracted incisors and plaster casts of Norwegians was examined. It was shown that plaster casts are well suited for measurements of lingual fossa depth, and that the measurements can be performed with great accuracy. Skewness values showed the symmetry of the distribution to decrease with decreasing mean lingual fossa depth. Kurtosis was found small in maxillary incisors, in mandibular outside the limits for normal theory. The distribution of the depth of the lingual fossa cannot generally be described as normal. No sex differences were found. The inheritance of this trait in Norwegians is probably not sex linked.     

Histochemical and ultrastructural features of the lingual epithelium of the rat snake (Elaphe climacophora)

The histological, ultrastructural and histochemical characteristics of the lingual epithelium of the rat snake (Elaphe climacophora) were investigated by light and transmission electron microscopy. The cells in the beta-layer of the epithelium of the bifurcated apex were filled with beta-keratin fibers and an amorphous matrix. Round projections covering the surface of the epithelial cells, namely, microfacets which contained pale granules, were clearly visible on the outer faces of Oberh?utchen cells on the epithelium, and they were identified as fine granules filled with lipid. These granules might play an important role as a coating on the surface of the bifurcated lingual apex. The lipid on the surface of the lingual apex might also serve to trap and retain odorant molecules. Keratohyalin-like granules were distributed within the a-layer of the epithelium of the bifurcated apex of the tongue in the resting phase and cellular interdigitation was well developed in this region. Evidence of a shedding line was apparent under the light microscope in the cleft between outer and inner epithelial generations. The epithelial surface of the body of the tongue appeared suitable for retention of odorant and other molecules.     

Global evaluation of growth in a rural population: comparative analysis of shape and size

In this work the analogies and differences in shape and size between a rural school population (6–14 aged) of the Lozoya-Somosierra region (Madrid) and several recent and past Spanish populations from different environments have been studied. The results show the growth trend as well as the influences of ecological and socioeconomical factors. Paper presented at the 4^th congress of the European Anthropology Association (Florence, Sept. 1984).     

Effects of exposure to static magnetic fields on the morphology and morphometry of mouse epididymal sperm

Morphologic and morphometric sperm characteristics of mouse epididymal extracts from animals exposed to static magnetic fields were evaluated. For this purpose, animals were exposed for 35 days to a field of 0.7 T generated by a commercial permanent magnet for either 1 or 24 h per day. The values of morphometric parameters were obtained using the morphometric module of the Sperm Class Analyzer® computerized image analysis system, and percentages of abnormalities were calculated. The size of sperm heads was unaffected by exposure to static magnetic fields. Lack of hook was a sperm head abnormality found significantly more frequently in animals exposed continually than in nonexposed animals, showing a possible alteration to the spermatogenic process after exposure to static magnetic fields. The percentage of sperm with coiled tails or of sperm with abnormal midpiece or tail was not altered by exposure. Bioelectromagnetics 19:377–383, 1998. © 1998 Wiley-Liss, Inc.     

The depth of the lingual fossa in permanent incisors of Norwegians. II. Differences between central and lateral incisors correlations, side asymmetry and variability

The depth of the lingual fossa in permanent incisors of Norwegians (plaster casts and extracted teeth) was studied. In the plaster casts the depth of the lingual fossa in the right side incisors was (in mm): I,sup=0.51,I(2)sup= 0.30, I(1)inf =0.07 and I (2)inf =0.08. For 1(1)sup significant bilateral asymmetry was found (p<0.00l). All incisors were positively correlated in the depth of the lingual fossa, mandibular higher than maxillary. In both jaws centrals were better correlated across the midline than laterals, and centrals to laterals were less correlated than centrals to centrals and laterals to laterals. Interjaw correlations of 1(1)sup were higher than interjaw correlations with I(2)sup. Dental field theory is confirmed: The upper lateral and lower central were the most variable incisors in each jaw for lingual fossa depth. The values for this trait in Norwegians are within the Caucasoid range.     

Synaptic input to the axon hillock and initial segment of inhibitory interneurons in the cerebellar cortex of the rat

Summary The axon hillock (AH) and initial segment (IS) of 10 Golgi neurons and 6 basket cells in the cerebellar cortex of the rat were investigated by electron microscopy using serial sections. An average of 10.4 and 11.3 synaptic terminals were observed to establish synaptic contact with the axon hillock region of Golgi and basket cells, respectively. Most of these terminals were identified as the varicosities of the ascending parallel fibers. It is suggested that the focal innervation of AH regions represents an excitatory input pattern which is basically different from the randomly distributed, huge, parallel-fiber input onto the dendritic trees of Golgi and basket cells. In contrast to Golgi and basket neurons, no accumulation of parallel-fiber synapses was observed around the AH of stellate cells. The IS proper of the three neuronal types were devoid of true axo-axonal synapses.     

Patched regulation of axon guidance is by specifying neural identity in the<Emphasis Type="Italic"> Drosophila</Emphasis> nerve cord

Within an axon bundle, one or two are pioneering axons and the rest are follower axons. Pioneering axons are projected first and the follower axons are projected later but follow a pioneering axon(s) pathway. It is not clear whether the pioneering axons have a guidance role for follower axons. In this paper, we have investigated the role of Patched (Ptc) in regulating the guidance of medial tract, one of the longitudinal tracts in the nerve cord. In patched mutants the medial longitudinal tract fails to fasciculate on its own side along the nerve cord, instead it abnormally crosses the midline and fasciculates with the contralateral tract. Interestingly, the medial tracts cross the midline ignoring the axon-repellant Slit on the midline and Roundabout on growth cones. The medial tract is pioneered by neurons pCC and vMP2. Our results show that guidance defects of this tract are due to loss and mis-specification of vMP2, which results in the projection from pCC to either stall or project outward near the location of vMP2. Thus, both pioneering neurons are necessary for the proper guidance of pioneering and follower axons. We also show that the loss of Ptc activity in the neuroectoderm prior to the formation of S1 and S2 neuroblasts causes the majority of axon guidance defects. These results provide insight into how mis-specification and loss of neurons can non-autonomously contribute to defects in axon pathfinding.     

Anatomy of the tongue and microstructure of the lingual papillae in the fallow deer Dama dama (Linnaeus, 1758)

The function of mouth organs in ruminants is connected with the process of rumination. To study morphofunctional relations, microstructures in tongues of 4- to 5-year old adult fallow deer were examined using scanning electron microscopy.When analyzing the tongue of the fallow deer, i.e. a ruminant classified as an intermediate mixed feeder between grass and roughage eaters, two processes were taken into account: (i) foraging and forage selecting, and (ii) chewing the cud during rumination to reduce particle size and improve digestibility.Microstructural results show that the above mentioned processes in fallow deer are important selection factors, which in the anterior part of tongue led to the development of clusters of fungiform papillae connected with preselection of food as well as a specific pattern of filiform papillae promoting increased adhesion of transported food. Massive and flattened conical papillae on the torus are arranged according to sideways jaw movements and are co-localized with flattened fungiform papillae and two rows of vallate papillae. Such an arrangement of papillae on the lingual torus presumably facilities distribution of ruminated food, with simultaneous transferring of taste signals about masticated food particles.     

Noradrenergic axon terminals in the substantia gelatinosa of the rat spinal cord

Summary The noradrenergic terminals in the substantia gelatinosa of the dorsal horn of the cervical spinal cord of the rat were investigated by means of the histofluorescence technique and electron-microscopic cytochemistry using the glyoxylic acid-KMnO₄ fixation technique. In accordance with the topographical distribution of fluorescent catecholaminergic fibers, noradrenergic terminals containing small granular vesicles were frequently observed electron microscopically in the outer layer of the substantia gelatinosa. These terminals were most frequently found to appose without showing typical synaptic features, small-caliber dendrites, spine apparatus, and rarely, large caliber dendrites. Only in a few cases, the noradrenergic terminals exhibited typical synaptic contacts with dendritic elements of small size. In addition, noradrenergic terminals apposed non-noradrenergic terminals containing small agranular vesicles. In rats bearing surgical lesions of the dorsal roots, no noradrenergic terminal were found in contact with the degenerated axon terminals in the substantia gelatinosa. These findings suggest that the noradrenergic afferents to the substantia gelatinosa may exert their influence on sensory transmission via dorsal horn cells.     

Microdomain-forming proteins and the role of the reggies/flotillins during axon regeneration in zebrafish

The two proteins reggie-1 and reggie-2 (flotillins) were identified in axon-regenerating neurons in the central nervous system and shown to be essential for neurite growth and regeneration in fish and mammals. Reggies/flotillins are microdomain scaffolding proteins sharing biochemical properties with lipid raft molecules, form clusters at the cytoplasmic face of the plasma membrane and interact with signaling molecules in a cell type specific manner. In this review, reggie microdomains, lipid rafts, related scaffolding proteins and caveolin—which, however, are responsible for their own microdomains and functions—are introduced. Moreover, the function of the reggies in axon growth is demonstrated: neurons fail to extend axons after reggie knockdown. Furthermore, our current concept of the molecular mechanism underlying reggie function is presented: the association of glycosyl-phophatidyl inositol (GPJ)-anchored surface proteins with reggie microdomains elicits signals which activate src tyrosine and mitogen-activated protein kinases, as well as small guanosine 5′-triphosphate-hydrolyzing enzymes. This leads to the mobilization of intracellular vesicles and to the recruitment of bulk membrane and specific cargo proteins, such as cadherin, to specific sites of the plasma membrane such as the growth cone of elongating axons. Thus, reggies regulate the targeted delivery of cargo—a process which is required for process extension and growth. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Novel mutations affecting axon guidance in zebrafish and a role for plexin signalling in the guidance of trigeminal and facial nerve axons

In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial (VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord.