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Horseradish peroxidase-wheat germ agglutinin conjugate (HRP) was injected into masticatory and eye muscles of 6- to 15-day-old chick embryos and posthatched chicks to establish the timetable of axonal outgrowth and distribution of central and peripheral terminations of mesencephalic trigeminal neurones (MTN). HRP-labelled MTN first appeared on the 10th day of incubation and by the 14th day most of MTN became labelled, indicating that axonic outgrowth to peripheral targets occurred between the 10th and 14th days of incubation. Peripheral targets included the pterygoideus lateralis and medialis, protractor quadratus, pseudotemporalis superficialis and profundus, and adductor mandibulae of jaw-closing muscles. HRP-filled central axonic processes of MTN were identified first on the 13th day of incubation and they terminated exclusively in the motor nucleus of the trigeminal nerve. Between the 10th and 13th days of incubation, at the peak of naturally occurring cell death in the MTN pool, a consistently lower percentage of neurones could be labelled with HRP than in older embryos and in posthatched chicks. This finding suggests that many MTN die before establishing contact with peripheral targets.

The avian trigeminal ganglion, which is embryonically derived from the neural crest and epidermal placodes, consists of two topographically segregated classes of immature neurons, large and small, during the second week of incubation, and two neuronal cell types, dark and light, interspersed throughout the mature ganglion. In order to establish the times of terminal mitosis of trigeminal sensory neurons, embryos were treated with [3H]thymidine during the first week of incubation and their ganglia fixed on embryonic day 11. The embryonically large, distal, placodal-derived neurons are generated between days 2 and 5, while the small, proximal, neural crest-derived neurons are formed mostly between days 4 and 7. By comparing the locations of labeled cells in ganglia treated with isotope but fixed on day 18 of incubation with their 11-day counterparts, we have proved that there are no morphogenetic rearrangements of neurons during the final week of incubation. Thus, no unique relationship exists between the two neuron types in the mature ganglion and the two cell classes in the immature trigeminal. Therefore, both the light and the dark neurons in the mature trigeminal ganglion arise from neural crest as well as placodal primordia.

The precise site of origin and the cells of origin of the mesencephalic nucleus of the trigeminal nerve in birds have been studied by the method of growing embryonic fore-, mid- and hind brain grafts inside a millipore filter chamber filled with amniotic fluid, upon a host chorioallantoic membrane, and by the method of transplantation of cranial neural crest of the mesencephalon between embryo-donors and duck embryo-hosts. The growth and differentiation of grafts grown in the chambers resemble very closely those of normal embryos at corresponding stages of development. Data obtained from a total of 42 grafts representative of the three species of birds used in this study, show that the cells of the mesencephalic nucleus are present only in grafts of embryonic midbrain and totally absent in forebrain and hindbrain grafts. The presence of cells of the mesencephalic nucleus in midbrain grafts suggests that these neurons arise chiefly from the developing mesencephalon. Histological examination of such grafts has shown that these neurons are observed in the leptomeningeal tissue overlying the midbrain region and extend into the various laminae of the optic tecta. A progressive increase in the size of the cells from an immature state confined to the mesodermal tissue to fully mature neurons in the stratum griseum periventriculare of the optic tectum is observed. This is also indicative of a downward migration of these cells. In a few embryonic midbrain grafts mature cells clearly identifiable as belonging to the mesencephalic nucleus are observed in the leptomeningeal tissue and outside the external limiting membrane of the neural tube. These appear to be neurons which had failed to migrate. The resemblance of these latter neurons to those in the stratum griseum periventriculare of the optic tectum lend further support to the hypothesis that the neurons of the mesencephalic nucleus are derived from neural crest of the developing mesencephalon in birds. In interspecific transplantation experiments of cranial neural crest of the mesencephalon, the quail cells occur in the form of clusters and appear to migrate towards the ventricular surface independent of the migration of the cells of the tectum which takes place simultaneously. Three stages in the development of the grafted neural crest material of the quail are observed based on the perikaryal diameter of the cells, and the accumulation of Nissl material in the cytoplasm. The smallest cells are located more superficially near the pial surface, and the larger mature neurons are observed in the stratum griseum periventriculare of the optic tectum. All the layers of the optic tectum including the neuroepithelium on the experimental side are comprised entirely of duck cells...

The effects of low levels of electric current were determined on organ cultures of 6- to 19-day-old chick embryo trigeminal ganglia. Current levels ranged from 0.00115 to 11.5 nA/mm2; the time of electrical treatment varied from 2 h to 96 h. Low levels of electricity were found to have at least three major effects on this system of mixed cellular types: (1) Outgrowth of nerve fibers from the explant was enriched. This resulted in a greater number of fibers which were longer and more highly branched than those in control cultures. (2) Survival of neurons within the original explanted ganglion was enchanced by treatment with electricity. The presence of healthy neurons was found in 93.1% of the treated cultures and in 53.5% of untreated cultures. (3) Neurons, fibers and non-neural cells were stimulated to grow in the direction of the cathode. The rate of cathodal migration was calculated to be 0.1 mm/h (2.4 mm/day) in an explant from 12-day-old embryo. No differences were observed between surviving treated and control cultures in histochemical tests for acetycholinesterase. A similarity between the action of low levels of electrical current and nerve growth factor (NGF) is suggested.

The development of the ophthalmic, maxillary and mandibular nerves has been followed in Xenopus laevis embryos from the first emergence of growth cones from the trigeminal ganglia until the establishment of functional innervation of the skin or cement gland. The course of each main nerve is highly predictable and follows pre-existing openings between blocks of other tissues. The development of the mandibulary nerve was observed most easily. Like that of the other trigeminal nerves it falls into three stages: (1) A pioneer neurite emerges and a nerve forms as other, later neurites fasciculate with this. (2) On reaching the inside surface of the cement gland the neurites separate and penetrate holes in the basal lamina. (3) The neurites grow between the cells they will innervate and form free nerve endings. The scanning EM observations have been confirmed by electrical recordings from trigeminal neurones. The role of pioneer fibres and substrate guidance are discussed.