Embryonic development of GABAergic signaling in the mouse spinal trigeminal nucleus interpolaris.

In the mature central nervous system, γ-amino butyric acid (GABA) is an inhibitory neurotransmitter, whereas during development, GABA induces depolarization. To examine the embryonic development of GABAergic transmission in the mouse spinal trigeminal nucleus interpolaris (SpVi), which receives sensory input from the face and is important in survival of rodents, we performed immunohistochemistry for three related molecules: glutamic acid decarboxylase (GAD), a marker of GABAergic neurons; vesicular GABA transporter (VGAT), a marker of GABAergic and glycinergic vesicles; and potassium chloride co-transporter 2 (KCC2), which shifts GABA action from excitatory to inhibitory. GAD-positive longitudinal projection fibers, where VGAT-positive dots were localized, were clearly discernible until embryonic day (E)17, and were markedly decreased in number on postnatal day 0. GAD-positive neurons were detected after E15, and GAD- and VGAT-positive axon varicosities were observed after E17. KCC2 immunolabeling was first localized in the dendrites and cell bodies of several neurons in the lateral part of the SpVi on E13 and throughout the nucleus on E17. These results suggest that the SpVi may first receive GABAergic projection fibers from extra-nuclear area before birth, and GABAergic interneurons may form synapses within the SpVi after E17. In addition, GABA action may gradually shift from excitatory to inhibitory between E13 and E17.

Extensive reorganization of primary afferent projections into the gustatory brainstem induced by feeding a sodium-restricted diet during development: less is more.

Neural development is especially vulnerable to environmental influences during periods of neurogenesis and rapid maturation. In fact, short periods of environmental manipulations confined to embryonic development lead to significant changes in morphology and function. A guiding principal emerging from studies of sensory systems is that experimentally induced effects are most dramatic in higher neural levels (e.g., cortex) and primarily involve postnatal synaptic refinements. In contrast to other sensory systems, the gustatory system is particularly susceptible to the effects of deprivation much earlier and with profound changes evident in the brainstem. Here we show that feeding pregnant rats a custom diet featuring a low-sodium content for 9 d before the tongue appears in the fetus produces extensive restructuring of the gustatory brainstem. Rats born to mothers fed the custom diet from embryonic day 3 (E3) to E12 have terminal field volumes of the greater superficial petrosal, chorda tympani, and glossopharyngeal nerves at adulthood that are expanded as much as 10 times beyond that found in rats fed a standard rat chow. The widespread alterations are not attributable to increased numbers of nerve cells, increased target size, or obvious changes in peripheral taste function. Moreover, we show that the limited period of feeding the custom diet has much larger effects than if rats were fed the diet to postweaning ages. Our results suggest that early periods of altered experience, especially during nucleus of the solitary tract neurogenesis, leads to a restructuring of the gustatory brainstem, which in turn may impact the control of sensory and homeostatic processes.

Immunohistochemical localization of enkephalin and substance P in the nucleus caudalis of the spinal trigeminal V in the medulla oblongata of the human fetus.

The distribution of enkephalin-positive neurons, substance P-positive and enkephalin-positive fibers was studied in the nucleus caudalis of the trigeminal spinal V in the medulla oblongata regions of developing humans (12 weeks gestation to 40 weeks gestation). Enkephalin-positive neurons were identified in all the subnuclei of the nucleus caudalis as early as 12 weeks of gestation and increased in number as the fetus aged. Substance P-positive neurons were absent in this area throughout development. On the other hand, substance P-positive and enkephalin-positive fibers were present in all the subnuclei, again commencing as early as 12 weeks of gestation. These fibers tended to be linked to each other in the different subnuclei and to the reticular formation in this area and to increase significantly in quantity by the latter quarter of pregnancy. These results show the early presence of these neurons and fibers in the first trimester of development.

Ontogeny of afferents to the fetal rat cerebellum.

Afferents to the fetal rat cerebellum have been studied in fixed tissue with the fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate (DiI). The dye was applied to the cerebellar anlage at ages from embryonic day (E) 12 to birth (P0). Central processes of vestibular ganglion cells were found to be the first identifiable afferents to the cerebellum, being present at least by E13 and perhaps as early as E12. Ipsilateral spinocerebellar fibres may be labelled from E15, vestibular nuclei (both ipsi- and contralateral) also from E15, while contralateral inferior olivary nuclei could not be retrogradely labelled until E17. Trigeminocerebellar neurons in the interpolaris subnucleus of the nucleus of the trigeminal spinal tract and neurons of the lateral reticular nucleus were not labelled until E22 and P0, respectively. Finally, contralateral pontine nuclei were retrogradely labelled from the cerebellum after birth.

The human rhombencephalon at the end of the embryonic period proper.

The human rhombencephalon at 8 postovulatory weeks (stage 23) is described and illustrated for the first time with the aid of silver-impregnated sections and graphic reconstructions. The motor and sensory trigeminal nuclei were among those studied, and the latter was found to be almost contiguous to the dentate nucleus. Fibers to the principal sensory nucleus join the mesencephalic trigeminal tract, which also seems to be connected with the motor fibers. Fine fibers from the sensory root join the tractus solitarius, which appears to receive connections from the facial, glossopharyngeal, and vagal nerves. Main and accessory abducent nuclei are evident. A part (the Kappenkern des Facialisknies) of the nucleus funiculi teretis is particularly prominent. The presence of the pyramidal decussation during the embryonic period is noted for the first time. The arrangement of nuclei and tracts at 8 weeks is shown to be closely similar to that present in the newborn, and it is likely that the rapid growth of the rhombencephalon during the embryonic period proper is associated with correspondingly early functional activity.

Development of the peripheral trigeminal system in the chick revealed by an isotype-specific anti-beta-tubulin monoclonal antibody.

Using a monoclonal antibody directed against a class III beta-tubulin isotype, c beta 4, we studied the time course of the expression of this protein, the morphological differentiation of the immunoreactive cells, and the time course of peripheral axon outgrowth in the chick trigeminal (V) system. The neural crest precursors of the V ganglion neurons do not express the antigen until they begin to differentiate as neurons. The placodal precursors of the V ganglion neurons express the antigen while they still are cuboidal epithelial cells. They continue to be immunoreactive as they migrate from the placode and settle in the ganglion, prior to sprouting axons. The V motoneurons express the antigen near the time of their terminal mitotic division. Using this antibody to visualize axons, we demonstrate that both ganglionic and motoneuron axons grow out as individual fibers, much like pioneer axons. Both halt their extension for several hours once they attain the vicinity of their targets. During this pause many other axons join the nerve bundles. Finally, single pioneer axons split from the main trunk to begin local target innervation.

Ontogeny of the calcitonin gene-related peptide in the nervous system of rat brain stem: an immunohistochemical analysis.

The ontogeny of the calcitonin gene-related peptide in the neuron system of the rat brain stem was investigated by means of the indirect immunofluorescence technique. Calcitonin gene-related peptide-like immunoreactivity was first detected in the fibers of the nucleus of spinal tract trigeminal nerve on gestational day 18, and thereafter appeared gradually in various brain stem areas such as in the fibers of the solitary tract, gracile nucleus, cuneate nucleus, inferior colliculus, superior colliculus, medial geniculate nucleus and in the neurons of the hypoglossal nucleus, facial nucleus, superior olive, parabrachial area, superior colliculus and peripeduncular nucleus. In colchicine-untreated animals, the immunoreactive fibers increased in number and reached adult level by postnatal day 14, whereas the number of cells reached a maximum between postnatal days 2 and 6 and then decreased in number and immunoreactivity or disappeared, except in some areas such as the superior olive and peripeduncular nucleus, which showed the same immunoreactivity as for adult animals. With colchicine treatment, calcitonin gene-related peptide-like immunoreactive cells were found in more areas of the brain stem such as the abducens nucleus, parabigeminal nucleus, principal oculomotor nucleus, trochlear nucleus and central gray, along with the nuclei which had shown calcitonin gene-related peptide immunoreactivity in the untreated animals. However, the neurons in the inferior olive showed a different ontogenetical pattern of calcitonin gene-related peptide of immunoreactivity. Immunoreactivity disappeared completely by postnatal day 21 in both colchicine-untreated and -treated animals.

Species specificity in the responsiveness of chick embryo neural tube explants to target-conditioned medium.

Explants from the metencephalic region of 40-h chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured in appropriate target muscle-conditioned media (MCM) derived from chick, quail and rat embryos. Enhanced neuritic outgrowth was found only in the presence of chick MCM, indicating that this early, initial responsiveness to target-released materials within this system is species-specific.

The development of the trigeminal (V) motor nucleus in normal and tubocurare treated chick embryos.

The generation of cells and the naturally occurring neuronal death was studied in the trigeminal motor neuron pool in normal and tubocurare treated chick embryos between the 5th and 18th days of incubation. 3H-thymidine autoradiography revealed that the generation time extends from the 2nd to the 5th day of incubation, wherein about 50% of trigeminal motoneurons are born on the 3rd day. Maximum neuron number was found on the 7th day of incubation which steadily decreased to about 50% of the originally generated neurons by the 13th day. Nuclear pyknosis occurred from the 6th to the 13th day of incubation with a peak of neuron loss on the 7th day. Tubocurare, administered daily from the 5th day of incubation rescued most of the generated motoneurons which would otherwise have died. Cell nuclear area measurements in the motoneuron pool of the tubocurare treated animals showed a marked hypertrophy accompanying the increased neuronal survival. These observations indicate that tubocurare treatment prevents naturally occurring neuron death and causes significant nuclear hypertrophy within the trigeminal motoneuron pool innervating special, branchial arch derived muscles. Thus these neurons respond to tubocurare treatment in a manner similar to motoneurons of the spinal cord.

Embryonic origin of substance P containing neurons in cranial and spinal sensory ganglia of the avian embryo.

The ontogeny of the neurons exhibiting substance P-like immunoreactivity (SPLI) was examined in the spinal and cranial sensory ganglia of chick and quail embryos. It was shown that in dorsal root ganglia (DRG) virtually all neuronal somas occupying the mediodorsal (MD) region of the ganglia are SPLI-positive while the larger neurons of the lateroventral (LV) area are SPLI-negative. In the cranial nerve ganglia, both types of neurons coexist in the trigeminal ganglion but with a different distribution: small neurons with SPLI are proximal while large neurons without SPLI occupy the maxillomandibular and ophthalmic lobes. The distal ganglia of nerves VII and IX (i.e., geniculate, petrosal) do not show cell bodies with SPLI in the two species considered. A few of them only (about 12%) are found in the nodose (distal ganglion of nerve X). The proximal ganglia of nerves IX and X (i.e., superior-jugular complex) are composed of small neurons which virtually all exhibit SPLI. Chimaeric cranial sensory ganglia were constructed by grafting the quail hind-brain primordium into chick embryos. Revelation of SPLI was combined with acridine orange staining on the same sections in order to ascertain the placodal (chick host) or neural crest (quail donor) origin of the SP-positive neurons in each type of ganglion. We found that all the neurons showing SPLI are derived from the neural crest in the trigeminal and in the superior and jugular ganglia. In the geniculate, petrosal, and nodose all the neurons are derived from the placodal ectoderm. The small number of SPLI-positive cells of the nodose ganglia are not an exception to this rule. Therefore, generally speaking, the sensory neurons of the cranial ganglia that express the SP phenotype are derived from the crest, with the exception of some neurons present in the nodose of both quail and chick embryos and which are of placodal origin. The vast majority of placode-derived neurons do not have amounts of SP that can be detected under the conditions of the present study.