Distribution of conduction system fibers in the developing and adult rabbit heart revealed by an antineurofilament antibody.

Using an immunological approach, we demonstrated previously that a neurofilament-like protein is expressed in rabbit heart conduction tissue myocytes, and we proposed that these specialized cardiac muscle cells are of neuroectodermal origin. In the present study, we used the expression of the neurofilament-like protein as a marker for identifying conduction tissue cells and studying their distribution in the developing heart. In 11-day-old rabbit embryos, myocytes expressing the neurofilament-like protein were localized at the atrioventricular and the sinoatrial junctions and had a ring-like distribution. At embryonic day 12, reactive myocytes were found also in the subendocardial layer of the dorsal ventricular wall, in continuity with labeled myocytes at the atrioventricular junction. Examination of older embryos and of neonatal and adult hearts revealed that the expression of the neurofilament-like protein was not restricted to myocytes of conduction tissue regions, but it was also detectable in myocytes of the sinoatrial ring bundle, in scattered myocytes localized in the left sinal horn wall, and in the right atrium in proximity to atrioventricular sulcus tissue. Thus, using an intracellular marker, we show that precursors of adult atrial conduction tissue are distributed at the sinoatrial and atrioventricular junctions; at variance, ventricular conduction tissue precursors do not have a ring-like distribution but are localized in the subendocardial layer, in continuity with the atrioventricular junctional myocytes.

A morphometric study of human fetal sural nerve.

A morphometric study was performed on sural nerves from human fetuses at 15 to 36 weeks postovulation. There were no myelinated fibres at 15 and 16 weeks, but by 21 weeks there were 5,000/mm2, rising to 25,000/mm2 at 36 weeks. During the fetal period, the mean myelin lamellar count trebled and the g ratio (axon diameter: total fibre diameter) decreased from 0.90 to 0.75, although the axon diameter of myelinated fibres did not increase. The smallest myelinated axon diameter was 0.63 micron, whereas the largest unmyelinated axon in a 1:1 relationship with a Schwann cell was 2.83 micron, suggesting that axon size is unlikely to be the only stimulus for myelination. The density of unmyelinated axons that were the sole occupants of a Schwann cell fell considerably between 23 and 33 weeks, while the ratio of total unmyelinated axons to myelinated fibres decreased from 82:1 at 21 weeks to 6:1 at 36 weeks. Data for Schwann cell nuclear density and percentages of fibres cut through the nucleus are also presented.

On the development of the pyramidal tract in the rat. II. An anterograde tracer study of the outgrowth of the corticospinal fibers.

An anterograde tracer study has been made of the developing corticospinal tract (CST) in the rat using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Analysis of normal Rager stained material revealed that corticospinal axons reach upper cervical spinal cord levels at the day of birth (PO). Postnatal rats ranging in age from one (P1) to fourteen (P14) days received multiple WGA-HRP injections into the cortex of their left hemisphere and were allowed to survive for 24 h. The first labeled CST fibers caudally extend into the third thoracic spinal cord segment at P1; into the eighth thoracic segment at P3; into the first or second lumbar segment at P7 and into the second to third sacral segment at Pg. Thus the outgrowth of the leading 'pioneer' fibers of the CST is completed at P9 but later developing axons are continuously added even beyond P9. Quantitative analysis of the amount of label along the length of the outgrowing CST revealed a characteristic pattern of labeling varying with age. The most striking features of that pattern are: the formation of two standing peaks at the level of the cervical and lumbar enlargements respectively and the transient presence of a smaller running peak which moves caudally with the front of the outgrowing bundle. The standing peaks are ascribed to the branching of the axon terminals at both intumescences, whereas the running peak probably arises by the accumulation of tracer within the growth cones at the tips of the outgrowing CST axons. Factors such as the number of axons, the varying axon diameters, the branching collaterals, the presence of varicosities, the transport rate of the tracer, the uptake of the tracer at the injection site, which possibly may affect the amount of label present in both the entire bundle and in the individual axons are discussed. Current research is focused upon an analysis of the relation between the site of injection within the cortex and the pattern of labeling of the CST. A delay of two days was found between the arrival of the CST axons at a particular spinal cord level and their outgrowth into the adjacent spinal gray. However, combined HRP and electronmicroscopic experiments are necessary to determine the factors behind the maturation of the CST as well as the maturation of the spinal gray.

Ontogenetical development of the chick and duck subcommissural organ. An immunocytochemical study.

The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day in incubation. Some duck embryos, and adult chickens and ducks were also studied. Immunocytochemistry using an anti-Reissner's fiber (RF) serum as the primary antibody was the principal method used. In the chick embryos the events occurring at different days of incubation were: day 3 morphologically undifferentiated cells in the dorsal diencephalon displayed immunoreactive material (IRM); days 4 to 6 immunoreactive cells proliferated, formed a multilayered structure and developed processes which traversed the growing posterior commissure and ended at the brain surface; day 7 blood vessels penetrated the SCO, scarce hypendymal cells appeared, the first signs of ventricular release of IRM were noticed, appearance of IRM bound to cells of the floor of the Sylvius aqueduct; day 7 to 10 the number of apical granules and amount of extracellular IRM increased progressively; day 11 RF was observed along the Sylvian aqueduct, day 12 RF was present in the lumbar spinal cord; day 13 IRM on the aqueductal floor disappeared; days 10 to 21 hypendymal cells proliferated, developed processes and migrated dorsally, ependymal processes elongated and their endings covered the external limiting membrane. In adult specimens the ependymal cells lacked basal processes and the external membrane was contacted by hypendymal cells. the duck SCO appears to follow a similar pattern of development.

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.

Investigations on the development and topographic order of retinotectal axons: anterograde and retrograde staining of axons and perikarya with rhodamine in vivo.

Rhodamine-B-isothiocyanate (RITC) is shown to be a convenient and advantageous fluorescence tracer both for anterograde staining of retinal ganglion cell axons on the tectum and for retrograde staining of ganglion cell bodies in the retina of chick embryos. After intravitreal injection the dye is taken up by ganglion cells of the retina from the extracellular space and is transported anterogradely at about 10 mm/day up to the axonal growth cones on the tectum. RITC can be taken up by growing axons on the tectum and it is transported retrogradely at about 5 mm/day to the cell bodies in the retina. Local staining can be achieved if RITC is applied in its crystalline form. RITC is nontoxic for the cells and their axons, is resistant to histological fixation procedures, and allows quick observation in vivo and on dissection stained tissue. Local application of RITC to distinct retinal areas allows examination of the position of the corresponding stained fibers along the retinotectal pathway. Fibers which arise from the central temporal retina occupy deeper layers, whereas fibers from the peripheral temporal retina occupy more superficial layers in the optic tract and in the stratum opticum on the anterior tectum. The growth cones of early retinal fibers growing directly on the tectal surface show a different morphology to later growth cones growing on top of the stratum opticum on the tectum.

Development of the human fetal auditory cortex: growth of afferent fibres.

The sequential development and growth of the subcortico-cortical fibres have been studied by means of acetylcholinesterase (AChE; acetylcholine acetylhydrolase; EC 3.1.1.7) histochemistry on the serial sections obtained from brains of human fetuses ranging from 10 to 28 weeks of gestation. It was found that thalamic and basal telencephalic fibres approaching the fetal auditory cortex have very strong AChE reactivity during a prolonged period of growth. In the youngest fetuses (10.5 weeks) a prominent fibre system was seen emerging from the ventroposterior thalamic territory while the auditory neopallium was free of AChE staining. In older fetuses (16-18 weeks) AChE-positive fibres originating in the ventroposterior thalamus and basal telencephalon penetrate the 'subplate layer' of the auditory cortex. On the basis of AChE reactivity of the 'subplate layer' the auditory cortex can be delineated from the surrounding neocortical areas. In the oldest fetuses (22-28 weeks) AChE-positive fibres of thalamic origin penetrate the developing cortical plate of the auditory cortex.

Innervation of the undifferentiated limb bud in rabbit embryo.

The concept that there are no nerves in the limb bud of mammalian embryos prior to differentiation has been re-examined. Rabbit embryos were collected at 260 and 290 hours gestation, which is prior to cartilage formation in the forelimb at 320 hours. Forelimb buds and adjacent neural tube were excised, fixed and embedded for light and electron microscopy. The limb buds were sectioned in two planes by serial 1 micrometer sections and inspected by light microscopy. Bundles of nerve fibres were seen within the proximal third of the limb bud, with distal ramification into adjacent zones of condensing mesenchyme. Electron microscopy confirmed the presence of axons and associated immature Schwann cells. These results demonstrate the existence of an anatomical framework through which a neurotrophic influence might be brought to bear upon mesenchyme prior to early differentiation.

[Development of the dorsal branches of the spinal nerves during the true embryonic period]. / Développement des branches dorsales des nerfs spinaux durant la période embryonnaire vraie.

A study of the Carnegie collection of human embryos has enabled the author to establish the sequence of events in the development of the dorsal rami of the spinal nerves. As the ventral rami enter the muscle anlagen and the rami communicantes appear at stage 14, it is sometimes possible to note the anlage of a dorsal ramus in the cervical region. At stage 15, the dorsal ramus is well distinguished, but seems to correspond with only its lateral division; indeed, the medial division is evident only in the next stage. At stage 17, the lateral and medial branches exist throughout the entire length of the body. It is only at stage 19, that nerves fibres reach beyond the myotome to the fascia. The next stage does not present any specific feature. At stage 21, however, fibres of the lateral ramus pass beyond the b=fascia and reach the cutaneous vascular layer, whereas fibres of the medial ramus have not yet gained the fascia. At the end of the embryonic period proper (stage 23), the cutaneous fibres of the lateral branch begin to cling to the corium (dermis), but the medial fibres seem merely to pass through the vascular layer without any attachment to the chorion. In summary, it can be stated that: 1) the dorsal rami of the spinal nerves are a little late in comparison with the lateral ones, 2) their lateral branches precede the medial ones, 3) they develop steadily from the depth to the surface, from the cervical to the caudal region, and from the lateral to the medial area of the dorsal skin.

[Formation of a system of exogenous tangential fibers in layer I of the cerebral cortex in the ontogeny of the dog]. / Formirovanie sistemy ékzogennykh tangentsial'nykh volokon I sloia kory bol'shogo mozga v ontogeneze sobaki.

During pre- and postnatal periods, development of nuclear projections of the diencephalon into the cortical layer I has been studied in dogs by means of silver nitrate impregnation after Cajal. It has been stated that after the 5th week of the intrauterine development a great number of fibres from the diencephalon, through the germ of the cortical lamina, begin penetrating into the layer 1 within the areas of the paleocortex, the island, the subicular field of the archicotex, the presubicular and entorhinal cortex. A little less fibres have been revealed in the sensomotor and temporoentorhinal areas on the convex surface of the frontal lobe and on the medial surface of the occipital lobe. At the places of their penetrating into the cortical layer 1, by the time of birth, the fibres have sprouted up thickness of the whole area of the hemisphere.

Pre-natal innervation of the human female genital tract.

In the human fetus of 14 weeks, ganglia on either sides of the Müllerian uterovaginal canal contained two types of cells. In the 16th week, axons invaded the basal zone of the stratified squamous epithelium at the sides of the upper vagina. In the 20th week, vesicular nuclei typified the large neurons in the midportion of the cervico-vaginal ganglion. During the 22nd week, capsulated ganglia invaded the wall of the upper vagina forming three concentrically disposed strata. Non-capsulated clusters invaded its lamina propria. At the 24th week, axons were shaded after reaching the superficial zone of the stratified vaginal epithelium. In the 28th week, satellites surrounded the mature neurons and sheath cells enveloped the axons. Ganglia invaded the splitted muscle layer of the upper vagina at 30 weeks. Intraepithelial fibres invaded the whole thickness of the endometrium, the columnar epithelium of the cervix and uterine tube at 40 weeks. Nerve cells were detected among the basal epithelial cells of the lower vagina and its subepithelial plexus.

The early innervation of the developing deciduous teeth.

Ingrowth of dental nerve fibres into the mesenchyme of the tooth-bearing areas of the mouth takes place at an relatively early stage, when the dental laminae are forming but the tooth buds are undeveloped. By the time a developing tooth has reached the cap and bell stages small nerve bundles have begun to enter the mesenchyme from which the dental papilla and sac arise.

Nerve fibers in the human organ of Corti.

In the human cochlea, the following characteristics of nerve fiber arrangements were observed. 1. Where there was only the basilar fiber in the tunnel of Corti, the internal and tunnel spiral bundle were not observed. In such cases, the number of nerve fibers in the external spiral bundle was small. 2. In the tunnel of Corti, the basilar fibers (afferent) took a course at the base and crossing the tunnel slanting to the basal end. 3. The medial fibers were present with the internal spiral bundle and tunnel spiral bundle. These fibers are possibly efferent. 4. The external spiral bundle increases its fiber count when internal and tunnel spiral bundles were present in the organ of Corti. 5. The external spiral bundle was composed of the basilar and medial fibers. The former were less and the latter much more numerous in the bundle. 6. 85-90% of the afferent fibers went to the internal hair cells, while the remainder went to the external hair cells. 7. The nerve fibers were found in the organ of Corti lateral to the external spiral bundles. The fibers were running in the Hensen's and Claudius' cell area. 8. In some cases medial fibers showed a torpedo-like deformity in the tunnel of Corti. When this was observed, it was found distributed throughout the entire cochlea. Hearing disorders due to this pathology of the efferent fiber was suggested.