A comparative immunocytochemical study of development and regeneration of chemosensory neurons in the rat vomeronasal system.

Vomeronasal neurons undergo continuous neurogenesis during development and after neuronal injury. We used immunocytochemical methods to compare different stages of the vomeronasal organ development to those of regeneration following vomeronasal nerve transection. At E15 and at 6 to 10 days after injury, nestin-positive cells were observed throughout the sensory epithelium. We did not find nestin immunoreactivity to be localized to the boundary region of the epithelium. The early appearance and wide distribution of nestin-positive cells suggests that they represent chemosensory precursor cells that develop and migrate vertically in the epithelium. Vomeronasal receptor cells degenerated 6 to 8 days after nerve transection, but axon terminals in the accessory olfactory bulb (AOB) continued to show the presence of the chemosensory specific marker (OMP) for up to ten days, a significant finding observed in this study. It is likely that the distance from the site of nerve transection may contribute to differences in the time course of anterograde and retrograde axon degradation. OMP-positive neurons were observed in the normal adult epithelium and to a much lesser extent 10-60 days after recovery from nerve transection. Axons from regenerated receptor cells did not reach the AOB during this time period. This failure to reestablish connections with target cells in the AOB could explain why OMP-positive cells were rarely observed among the regenerated cells in the vomeronasal epithelium.

Forked end: a novel transmembrane protein involved in neuromuscular specificity in drosophila identified by gain-of-function screening.

The Drosophila neuromuscular connectivity provides an excellent model system for studies on target recognition and selective synapse formation. To identify molecules involved in neuromuscular recognition, we conducted gain-of-function screening for genes whose forced expression in all muscles alters the target specificity. We report here the identification of a novel transmembrane protein, Forked end (FEND), encoded by the fend gene, by the said screening. When the FEND expression was induced in all muscles, motoneurons that normally innervate muscle 12 formed ectopic synapses on a neighboring muscle 13. The target specificity of these motoneurons was also altered in the loss-of-function mutant of fend. During embryonic development, fend mRNA was detected in a subset of cells in the central nervous system and in the periphery. These results suggest that FEND is a novel axon guidance molecule involved in neuromuscular specificity.

Differential fasciculation of follicular nerves for transferring specifically localized cues of the vibrissa rudiments to the central trigeminal sensory system in mice, as exploited with DiI and DiA labeling.

Vibrissa connections play a decisive role in setting the somatotopic coordinates in the trigeminal sensory system. Although previous studies have examined the development of peripheral patterning, certain questions are still in dispute, for example, the way vibrissa connections are structured, and the relationship between periphery and central organization, and ganglion cell organization. In order to fill the blanks left by previous studies, the extension of ganglionic branches and the formation of vibrissa connections were reexamined by using fluorescent carbocyanin dyes, DiI and DiA, during embryonic days 10 to 14 in mouse. Whole-mount preparations satisfactorily demonstrated the ganglionic fiber system, which allowed detailed analysis at both macroscopic and microscopic levels. We show here that the differential fasciculation of follicular nerves is the critical process for organizing vibrissa connections which modulate the initially extending fiber pattern. Follicular nerves developed by fine fibers arising from initially ordered root fascicles, so as to connect with vibrissa rudiments that developed on the facial prominences. During differential fasciculation, ganglion cells were segregated into distinct groups by the vibrissa connections, whereas central fiber terminals did not yet develop specific structures in the nuclear region. In the primary order of the trigeminal sensory system, vibrissa connections in the periphery were organized before those of the central structure. These results indicate that trigeminal ganglion cells have a critical binomial function in order to transfer the somatotopic relations among vibrissa rudiments into the topographic coordinates of the central system.

Development of dynorphin-like immunoreactive auditory nerve terminals in the chick.

The novel discovery that auditory nerve terminals in the chick cochlear nucleus magnocellularis (NM) are immunoreactive for the opioid peptide dynorphin (DYN) was recently reported [3]. The present study examines the development of DYN-immunoreactivity (DYN-I) in auditory nerve terminals in NM from embryos through young post-hatch chicks. No DYN-I was observed in NM at embryonic day 13 (E13). DYN-I first appeared at E16 as short flat structures partially surrounding NM cell bodies. Around post-hatch day 1 (P1), these structures had a more rounded, chalice-type of morphology reminiscent of the specialized auditory nerve terminals found in birds, the end-bulbs of Held. At P6, most NM neurons were circumscribed by a prominent DYN-I calyceal-type of ending. By P13, fewer NM cells were ringed by this DYN-I and by the third post-hatch week, there was very little DYN-I in NM. There were no obvious differences in the density of DYN-I terminals across either the rostrocaudal length or the mediolateral width of NM at any age examined. These results suggest that during a restricted time of development, end-bulbs of Held in the chick NM contain DYN.

Early prenatal critical period for chorda tympani nerve terminal field development.

In order to determine whether the developing central gustatory system responds to dietary manipulation during restricted developmental periods, terminal fields of the chorda tympani nerve within the nucleus of the solitary tract were investigated via anterograde transport of horseradish peroxidase in control rats and in rats in which a low sodium diet was systematically fed during specific periods of development. Rats fed a low sodium diet (0.03% NaCl) from embryonic day 3 (E3) to day E12 and then fed a sodium replete diet to at least 60 days postnatal exhibited enlarged and irregularly shaped chorda tympani terminal fields. Specifically, the dorsal zone of the field was the smallest in controls, whereas it was the largest in restricted rats, occupying more territory within the nucleus. This alteration in the terminal field was apparent in all groups of rats fed the low-NaCl diet beginning at E3, and continuing beyond E12. In contrast, no effects of the dietary manipulation on the developing chorda tympani field was evident when it occurred from E3 to day E9, from E0 to day E9 or when it occurred at adulthood only. Therefore, only 9 days of maternal exposure to a sodium-restricted diet is required for a permanent expansion of the chorda tympani terminal field in the offspring. Moreover, a brief period from E9 to E12 must be included within the 9-day dietary restriction to yield the expanded field. Since this period is before taste receptors appear on the tongue, it is likely that nonactivity-dependent factors determine the formation of the chorda tympani terminal field during later development.

Retrograde signaling in the formation and maintenance of the neuromuscular junction.

The neuromuscular junction is characterized by precise alignment between the nerve terminal and the postsynaptic apparatus formed by the muscle fiber. Organization of the neuromuscular junction during embryonic development, growth, and maintenance is coordinated by signals exchanged between motor neurons and their target muscle fibers. Identification of proteins such as agrin, likely to represent neuronal agents that direct the organization of the postsynaptic apparatus, has focused attention on characterization of proteins that mediate retrograde signals that regulate the organization and function of the nerve terminal. The results of these studies implicate a role for both adhesive and diffusible signals in coordinating the development, maturation, and maintenance of the motor nerve terminal. The diversity of molecules identified to date that appear to play a role in these processes implies a considerable level of redundancy in the transduction pathway. However, studies of early nerve-muscle interactions suggest that a common feature of many of these retrograde agents is activation of a protein kinase coupled with an increase in cytosolic Ca2+ concentration. While the molecular signals that regulate growth and maintenance of neuromuscular junctions are less well understood it seems likely that similar adhesive and diffusible factors will be involved.

PC12 cells as a window for the differentiation of neural crest into adrenergic nerve ending and adrenal medulla.

Studies on PC12 and isolated adrenal chromaffin cells have revealed that PC12 cells have a closer identity to the adrenergic nerve ending than do the chromaffin cells. This is revealed by the presence of monoamine oxidase (MAO) A and tyramine-released pool of catecholamines in PC12, resembling that in adrenergic neurones, and their absence in adrenal chromaffin cells. Indeed, chromaffin cells possess primarily MAO-B activity. Like the observations on adrenergic neurones, non-selective and selective MAO-A inhibitors potentiate the catecholamine-releasing property of tyramine in PC12 cells. This property has clearly been demonstrated to be associated with selective inhibition of MAO-A and not MAO-B. The fact that MAO-A and MAO-B are different proteins and under separate gene product control suggests that their regulation may be highly differentiated. Indeed, it has been shown that while steroids such as progesterone and hydrocortisone induce and estrogen diminishes MAO-A activity in PC12 cells, no such regulatory mechanism has been identified for MAO-B activity in chromaffin cells. In the final analysis the inter-relationship between MAO-A activity and the presence of tyramine-releasable pool of catecholamines in adrenergic neurons and PC12 cells may have a genetic basis and could be important in illuminating the differentiation of neural crest into adrenergic neurones and adrenal medulla on the one hand and chromaffin cells to PC12 cells on the other.

[Ultrastructure of the sympathetic paravertebral ganglia in rat embryogenesis. II. The development of the neuropil and the interneuronal connections]. / Ul'trastruktua simpaticheskikh paravertebral'nykh gangliev v émbriogeneze u krys. II. Razvitie neiropilia i mezhneironnykh sviazei.

Electron microscopy was used to study the process of ingrowth of nerve terminals in the primordia of sympathetic ganglia and the formation of specialized contacts. Nerve terminals appeared first in 12 day old embryos. In the forming ganglia of 13 day old embryos there are many preganglionic nerve terminals and processes of principal neuroblasts. The growth cones of nerve endings are usually distended and with transparent cytoplasm. The plasmalemmas of growth cones are lacking often the trilaminar structure. Synapses were observed first in 16 day old fetuses. They are axo-dendritic and axo-somatic ones. The number of synaptic contacts does not increase much during prenatal period. Presumptive afferent nerve terminals were found in the late fetuses.

Ontogeny of neuropeptidergic systems: luteinizing hormone releasing hormone (LHRH); somatostatin (SRIF) and neurophysin (NF) in the hypothalamus of the domestic pig by immunocytochemistry.

The ontogenic development of some hypothalamic neuropeptides: luteinizing hormone releasing hormone (LHRH); somatostatin (SRIF) and neurophysin (NF) and their localization in the hypothalamus of fetuses in different stages of the fetal life were studied by immunoperoxidase method. It was found that differentiation of the neurons which produce the examined hormones begins in the midstage of pregnancy. LHRH is stored in the nerve terminals of the median eminence (ME) and organum vasculosum of the lamina terminalis (OVLT) since 72 day of gestation and its amount gradually increases with the development of the embryo. In this stage a few immunoreactive (ir) LHRH perikarya appear but they are most numerous in the last days of pregnancy (110 day). They are localized in the most anterior periventricular parts of the hypothalamus, area preoptica, diagonal band of Broca and very rare in the medial-basal hypothalamus. Somatostatin is produced in the separate neuronal system and appears in the last days of fetal life. Neurophysin is present in both magnocellular nuclei in 72 day-old fetuses, but at the end of gestation it is seen also in some preoptico-septal region.

Early development of vestibular receptors in human embryos. An electron microscopic study.

The development of the vestibular receptors in 7 to 9-week-old human embryos was investigated by light, scanning and transmission electron microscopy. The greater part of the vestibular epithelium is undifferentiated in the 7-week-old embryo. It is composed of polystratified epithelial cells. Some afferent endings are found at the base of the epithelial cells but no synaptic specializations are detected. A few afferent endings reach the upper part of the epithelium. Thus, at this stage the fibers are present in the vestibular epithelium before the differentiation of the sensory cells. The apical pole of the epithelial cells presents basal bodies linked with striated rootlets and other typical vestibular epithelium cell structures. Short hair bundles are found in a very small part of the vestibular epithelium. At 8-9 weeks of gestation, numerous nerve endings surround the base of the sensory cells. Densifications of the pre- and postsynaptic membranes and synaptic bodies are seen. The newly afferented cells present polarized hair bundles. We suspect that the hair cells are important for the guidance of afferent terminals perhaps even before their morphological differentiation.

Acetylcholinesterase in developing embryos of Hyalomma rufipes Koch, 1844 (Ixodoidea: Ixodidae).

Acetylcholinesterase (AChE) is demonstrated, apparently for the first time, in developing embryos of the tick Hyalomma rufipes. The enzyme was detected in the neuropile of the oesophageal ganglia and in the nerve endings of the appendages; it was absent from the neurons and neurolemma of the nerve ganglia.

The nerve endings of the glycogen body of embryonic and adult avian spinal cord: on the existence of two different varieties of nerve fibers.

The innervation of the enigmatic glycogen body in both embryonic and adult spinal cords in Gallus domesticus was investigated using frozen sections stained with the Del Rio Hortega silver method and the procedure of Cajal. Two distinct types of neural terminals were found, one appearing to be sensory associated with neurovascular glomi and the other related to the periependimary glycogen cells. The presence of nests of argentaphin cells indicates the probability of a neurosecretory function.

Nerve endings in the pulmonary trunk, ductus arteriosus and aorta of intact and decapitated pig fetuses.

Nerve fibres reactive to acetyl-thiocholine, and tissues showing catecholamine fluorescence were examined in the pulmonary trunk, ductus arteriosus and aorta of 28 pig fetuses between 31 and 113 days of gestation (term = 114 +/- 1 days). Eight additional fetuses, which had been decapitated in utero at 40-43 days, were also studied at ages between 51 and 114 days of gestation. Spherical micro-networks of nervous tissue reactive to acetyl-thiocholine are present in the adventitia on the cranial aspect of the pulmonary trunk and ductus arteriosus, between the aorta and pulmonary trunk, and on the caudal aspects of the pulmonary trunk and the pulmonary arteries. These fibres invest spherical clusters of catecholamine containing cells which are well supplied with blood vessels. Nerve fibres which fluoresce are also found in association with these cells. Decapitation in utero does not appear to affect the distribution of morphology of these structures. The observations show that structures are present in the major arteries of the fetal pig which may act as sensory receptors, and that these structures are unaffected by chronic vagotomy of the fetus produced by decapitation early in gestation.

Histochemical study of lamellar cell development of Meissner corpuscles.

The development of the lamellar cells of mouse digital corpuscles (Meissner corpuscle) was studied by light and electron microscopic histochemistry for cholinesterase (ChE) The materials used were the hind limbs taken from fetuses at 14, 17 and 20 days of gestation, and from young mice at 1, 5, 7, 15 and 20 days after birth. Embryonal Schwann cells had non-specific ChE activity in the cisternae of the rough endoplasmic reticulum and the nuclear envelope, suggesting that they had the ability of synthesizing the enzyme. After birth, such ability gradually decreased and by five days of age non-specific ChE activity was no longer demonstrable in Schwann cells at the time when myelin sheath formation began. However, Schwann cells which were associated with the axonal tips penetrating into the epidermis still had an intense non-specific ChE activity. Such Schwann cells surrounded the axons in gradually increasing numbers of cytoplasmic processes, which later became the lamellae around the axon terminals; thus by 20 days after birth they had differentiated into mature lamellar cells of Meissner corpuscles. These lamellar cells had, as in the embryonal Schwann cells, an intense ChE activity in the cytoplasm. These findings indicate that the lamellar cell is a specialized form of Schwann cell which still retains the embryonal characteristics for synthesizing non-specific ChE.

Tooth morphogenesis: the role of the innervation during induction and pattern formation.

The hypothesis is discussed that the innervation of the early mandibular and maxillary processes influences the initiation and patterning of tooth germs. Silver staining of embryonic mouse tissue supports the notion that the innervation is present before tooth buds appear. Data from other experimental studies is discussed in support of this hypothesis. The importance of the early events of tooth morphogenesis in initiating and patterning the dentition is discussed in the context of tooth morphogenesis as a whole. The processes involved in odontogenesis are categorized as Phase I: Initiating events; Phase II: Histogenetic events; and Phase III: Cytodifferentiative events. The complications of the interrelationship of these three seemingly discrete segments of tooth development are discussed in the context of inductive tissue interaction.

Development of Meissner corpuscle of mouse toe pad.

The cytologic development of Meissner corpuscles of the mouse toe pad has been studied using light and electron microscopy, and correlated with silver impregnations of frozen sections. By 18 days of gestation, neurites are seen near the epidermis, but intraepidermal neurites are few. One day after birth, the number of intraepidermal neurites increases, and some accompanying Schwann cells extend their cytoplasmic processes penetrating the basal lamina of the epidermis. Four days after birth, Schwann cells invade the epidermis further, extending many cytoplasmic processes which are intimately associated with basal cells of epidermis. These specialized Schwann cells which contact the epidermis proper also begin to develop cytoplasmic lamellae and thus denote the onset of lamellar cell development. By eight days after birth, the developing lamellar cells become more elaborated, and their cytoplasmic processes contain caveolae and filaments, characteristic features of lamellar cells. This developmental sequence supports the concept that lamellar cells are derived from Schwann cells. Through all stages of development, neurites and Schwann cells interact closely with epidermal cells. Epidermal cells may be essential for corpuscle formation. By 20 to 25 days after birth, mouse toe pad Meissner corpuscles are cytologically mature.