Neuronal maturation in the foetal brain in Down's syndrome.

Biochemical indices of neuronal maturation have been examined in postmortem cerebral cortex tissue from Down's syndrome (DS) and control foetuses matched for age. No differences were found in the concentration of neural cell adhesion molecule (N-CAM), or in the proportion of the 'adult' form of N-CAM, in the total concentration or composition of gangliosides, or in the activity of choline acetyltransferase (ChAT). The concentration of major lipid classes was also examined, and the DS specimens differed only in having a small but significantly higher proportion of phosphatidylcholine. The findings suggest that, by the indices examined, there is no delay in neuronal maturation, nor a major abnormality in lipid composition although anomalies in the polyunsaturated fatty acid composition of phosphoglycerides do exist at this stage of brain development in DS (J. Neurochem., 44 (1985) 869-874). Furthermore, the normal activity of ChAT found in the DS foetal cerebral cortex suggests that the impaired cortical cholinergic innervation which is apparent later in life may not be due to initial defects in cholinergic differentiation.

Differentiation of smooth muscle in the genital tract of the female mouse and its temporal relation with the development of the Wolffian nerve.

The development of the smooth muscle in the genital tract of the female mouse was studied by light and electron microscopy before and after birth. These studies showed that: a) between 13 days of fetal development and 2 days after birth the cells surrounding the Mullerian duct were undifferentiated and showed a fibroblast-like appearance; b) between 3 and 10 days after birth the cells acquired several characteristics of smooth muscle but they did not seem fully mature; c) between 30 and 180 days after birth the cells acquired a mature appearance; and d) the Wolffian nerve reached the Mullerian duct surrounding tissue before the start of smooth muscle differentiation.

Delta, a Drosophila neurogenic gene, is transcriptionally complex and encodes a protein related to blood coagulation factors and epidermal growth factor of vertebrates.

Delta (D1) is required for normal segregation of the embryonic ectoderm into neural and epidermal cell lineages in Drosophila melanogaster. Loss-of-function mutations in D1 and other zygotic neurogenic loci lead to expansion of the neuroblast population at the expense of the dermoblast population within the ectoderm. Characterization of the transcriptional organization and maternal/embryonic expression within the chromosomal interval corresponding to D1 reveals that the locus encodes multiple transcripts: a minimum of two maternal transcripts, approximately 4.5 and 3.6 kb in length, and four zygotic transcripts, approximately 5.4 (two distinct species), 3.5, and 2.8 kb in length. These transcripts differ on the bases of differential splicing and differential polyadenylation site choice. The DNA sequence of a cDNA clone representing the predominant transcripts of the locus indicates that D1 encodes a transmembrane protein homologous to blood coagulation factors and epidermal growth factor. The relationship between coding sequences and transcript-specific exons within the locus suggests that D1 encodes multiple translational products.

Cell differentiation features in embryos resulting from interphylum nuclear transplantation: echinoderm nucleus to ascidian zygote cytoplasm.

When an echinoderm nucleus was transplanted into an ascidian zygote cytoplast there was developmental cooperation at the cellular level between nucleus and cytoplasm of these normally nonhybridizable species. A blastula stage nucleus from the sand dollar Echinarachnius parma was injected into an activated but nonnucleate egg fragment of the ascidian Ciona intestinalis. During culture, some of the "hybrid" embryos displayed ultrastructural evidence of cellular differentiation. Two recognizable features were (1) extracellular matrix components, and (2) neural cell characteristics, including elaboration of associated cilia. Nonnucleate zygote fragments alone, and such fragments injected with seawater or punctured by glass needle, did not develop organized subcellular structures. Morphologic expressions resulting from nuclear transplantations between these two phyla (Echinodermata and Chordata) seemingly indicate functional interactions at a gene regulatory level. Creation of such nuclear-cytoplasmic hybrids suggests thereby a means of exploring the nature of the egg cytoplasmic agents in ascidian embryos that appear to determine gene expression related to histospecific differentiation products.

Opiate receptor ontogeny in the rat medial preoptic area is androgen-dependent.

The relationship of opiate receptors in the medial preoptic area of the hypothalamus (MPOA) to the gonadal steroid hormone environment during development was assessed using regional densitometric analysis of [3H]naloxone binding in autoradiographs prepared using brain sections from 5-day-old male and female rats treated postnatally either with tamoxifen (0.5 mg/kg), flutamide (20 mg/kg), dihydrotestosterone (DHT; 12.5 mg/kg), or sesame oil vehicle. Tamoxifen, a specific estrogen receptor antagonist, did not alter MPOA binding density in either males or females. Flutamide, a specific androgen receptor antagonist, and DHT, a nonaromatizable androgenic compound, altered the MPOA binding density in males and females, respectively. No treatment altered the binding density in several other brain regions. The results suggest that androgen, not estrogen, regulates the differentiation of MPOA opiate receptors. Since the neuronal development in the region is thought to be mediated by estrogen, both hormones probably act concurrently to affect the ontogeny of different parameters in the same brain region.

Neurogenesis in the vomeronasal epithelium of adult garter snakes: 3. Use of H3-thymidine autoradiography to trace the genesis and migration of bipolar neurons.

Use of H3-thymidine autoradiography and unilateral vomeronasal (VN) axotomy has permitted us to demonstrate directly the existence of VN stem cells in the adult garter snake and to trace continuous bipolar neuron development and migration in the normal VN and deafferentated VN epithelium in the same animal. The vomeronasal epithelium and olfactory epithelium of adult garter snakes are both capable of incorporating H3-thymidine. In the sensory epithelium of the vomeronasal organ, H3-thymidine-labeled cells were initially restricted to the base of the undifferentiated cell layer in animals surviving 1 day following H3-thymidine injection. With increasing survival time, labeled cells progressively migrated vertically within the receptor cell column toward the apex of the bipolar neuron layer. In both the normal and denervated VN epithelium, labeled cells were observed through the 56 days of postoperative survival. In the normal epithelium, labeled cells were always located within the matrix of the intact receptor cell columns. However, labeled cells of the denervated epithelium were always located at the apical front of the newly formed cell mass following depletion of the original neuronal cell population. In addition, at postoperative days 28 and 56, labeled cells of the denervated VN epithelium achieved neuronal differentiation and maturation by migrating much farther away from the base of the receptor cell column than the labeled cells on the normal, unoperated contralateral side. This study directly demonstrates that basal cells initially incorporating H3-thymidine are indeed stem cells of the VN epithelium in adult garter snakes.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphometric relationships between satellite and nerve cells in the sensory lumbar ganglia of the domestic fowl in different pre-and post-hatching stages

Foi feito um estudo combinado a microscopia óptica e eletrônica em embriöes de 10 a 18 dias e em espécimens de 8, 35, 61, e 120 dias após a eclosäo de galo doméstico. O volume nuclear da célula nervosa, praticamente näo varia após o 18ª dia de incubaçäo enquanto que, considerável aumento do mesmo volume para células satélites foi observado. O volume citoplasmático varia intensa e progressivanebte para ambos os tipos de células, sendo menor para célula nervosa. Há relativa proporcionalidade de aumento de volume para ambos os tipos de células, sendo contudo, mais intenso para células satélites. Säo feitas consideraçöes de ordem morfofuncional

Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo.

In the ventral hindbrain and spinal cord of zebrafish embryos, the first neurones that can be identified appear as single cells or small clusters of cells, distributed periodically at intervals equal to the length of a somite. In the hindbrain, a series of neuromeres of corresponding length is present, and the earliest neurones are located in the centres of each neuromere. Young neurones within both the hindbrain and spinal cord were identified in live embryos using Nomarski optics, and histochemically by labelling for acetylcholinesterase activity and expression of an antigen recognized by the monoclonal antibody zn-1. Among them are individually identified hindbrain reticulospinal neurones and spinal motoneurones. These observations suggest that early development in these regions of the CNS reflects a common segmental pattern. Subsequently, as more neurones differentiate, the initially similar patterning of the cells in these two regions diverges. A continuous longitudinal column of developing neurones appears in the spinal cord, whereas an alternating series of large and small clusters of neurones is present in the hindbrain.

Fetal antigen retained by mature neurons and ependyma studied with a monoclonal antibody (6B9).

A mouse monoclonal antibody (MAb 6B9, isotype IgM) was raised against autopsy tissue samples from the central nervous system (CNS) of multiple sclerosis (MS) patients. By immunofluorescence microscopy, MAb 6B9 intensely stains most or all cells in fetal rats. However, MAb 6B9 differentially stains various cell types in adult rats. Neurons, ependymal cells, and adrenal chromaffin cells are stained intensely, whereas astrocytes and oligodendrocytes are not stained. The 6B9-reactive antigen (6B9 antigen) is sensitive to periodic acid, but insensitive to treatment with protease, RNase, or hyaluronidase. Results from immunofluorescence microscopy on semithin sections and cultured neuroblastoma cells indicate that 6B9 antigen is intracellular. This is supported by immunoelectron microscopy, where labeling for 6B9 antigen appears in the cytoplasm distinct from any identifiable organelle. Further studies on 6B9 antigen should reveal its chemical nature as well as the significance of developmental changes in its distribution.

Developmental-stage-dependent radiosensitivity of neural cells in the ventricular zone of telencephalon in mouse and rat fetuses.

Pregnant ICR mice were treated with single whole-body X-radiation at a dose of 0.24 Gy on day 10, 13, or 15 of gestation. Fetuses were obtained from mothers during 1 and 24 hours after irradiation. Pyknotic cells in the ventricular zone of telencephalon were counted in serial histological sections. Incidence of pyknotic cells peaked during 6 and 9 hours after irradiation in each gestation day group. Then, dose-response curves were obtained 6 hours after 0-0.48 Gy of irradiation. All three dose-response curves showed clear linearity in the dose range lower than 0.24 Gy. Ratios of radiosensitivity estimated from the slopes of dose-response curves in day 10, 13, and 15 groups were 1, 1.4, and 0.4, respectively. These demonstrated that ventricular cells in the day 13 fetal telencephalon were the most radiosensitive among the three different age groups. In order to confirm the presence of the highly radiosensitive stage common to mammalian cerebral cortical histogenesis, pregnant F344 rats were treated with single whole-body gamma-irradiation at a dose of 0.48 Gy on day 13, 14, 15, 17, or 19 of gestation. The incidence of pyknotic cells in the ventricular zone of telencephalon was examined microscopically during 1 and 24 hours after irradiation. The peak incidence was shown 6 hours after irradiation in all the treated groups, and the highest peak incidence was shown in day-15-treated group. The developmental stage of telencephalon of day 15 rat fetuses was comparable to that of day 13 mouse fetuses. Thus, the highest radiosensitivity in terms of acute cell death was shown in the same developmental stage of brain development, i.e., the beginning phase of cerebral cortical histogenesis, in both mice and rats.

Ionic currents in neurones cultured from embryonic cockroach (Periplaneta americana) brains.

Neurones isolated from embryonic cockroach brains were maintained in culture for up to 8 weeks. A single patch electrode was used to record voltage changes in response to injected current, membrane ionic currents under whole-cell voltage-clamp conditions or single-channel currents from isolated membrane patches. The voltage changes in response to injected current that depolarized the cell indicated increases in membrane permeability to calcium and potassium. These observations were confirmed using a voltage clamp. The potassium current observed in the youngest cultures turned on with a delay and was blocked by tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Two kinds of decrease in the outward potassium current were observed. One may be associated with extracellular potassium accumulation, inactivation of the potassium channel or inactivation of a calcium channel. The other appears to be a voltage-dependent inactivation. The magnitude of the calcium permeability appeared to increase as the cultures developed, being most prominent in cultures more than 2 weeks old. Single-channel conductance measured from an analysis of records from six isolated membrane patches ranged from 15 to 110 pS. Except for one channel, the probability of the channels being open did not change appreciably with membrane potential. Our results suggest that much of the increase in potassium permeability may be due an increase in intracellular calcium level.

A freeze-fracture study on the developing satellite cells of spinal ganglia in the chick embryo.

A freeze-fracture analysis of the satellite cells of spinal ganglia of the chick embryo was performed in 8 successive stages of development, from the 5th incubation day to hatching. The characteristic laminar disposition of the cells were first observed on the 7th day. Tight junctions were found at the 20th incubation day. Small groups or irregular aggregates of particles, but not gap junctions, were described on the 7th and 8th days. Pinocytotic vesicles were pointed out in the different stages considered.

Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina.

Despite evidence that glial cell surfaces and components of the extracellular matrix (ECM) support neurite outgrowth in many culture systems, the relative contributions of these factors have rarely been compared directly. Specifically, it remains to be determined which components of peripheral nerve support growth of central nerve fibers. We have directly compared neurite outgrowth from embryonic day 15 rat retinal explants placed onto beds of (1) Schwann cells without ECM, (2) Schwann cells expressing ECM (including a basal lamina), (3) cell-free ECM prepared from neuron-Schwann cell cultures, (4) nonglial cells (fibroblasts), and (5) 2 isolated ECM components, laminin and type I collagen. From the first day in culture, retinal explants extended neurites when placed on Schwann cells without ECM. Outgrowth on Schwann cells expressing ECM was also extensive, but not obviously different form that on Schwann cells alone. Ultrastructural study revealed that 95% of retinal neurites in ECM-containing cultures contacted other neurites and Schwann cell surfaces exclusively. On cell-free ECM prepared from neuron-Schwann cell cultures, neurite extension was poor to nonexistent. No neurite outgrowth occurred on fibroblasts. Retinal explants also failed to extend neurites onto purified laminin and ammoniated type I collagen substrata; however, growth was rapid and extensive on air-dried type I collagen. In cultures containing islands of air-dried type I collagen on a laminin-coated coverslip, retinal explants attached and extended neurites on collagen, but these neurites did not extend off the island onto the laminin substratum. We conclude from these experiments that neurite extension from embryonic rat retina is supported by a factor found on the surface of Schwann cells and that neither organized nor isolated ECM components provide this neurite promotion. These findings are discussed in relation to possible species differences in growth requirements for retinal ganglion cell neurites and to the specificity of response of different CNS neurites to ECM substrata.

The influence of laminin on the initial differentiation of cultured neural tube neurons.

Portions of the metencephalic neural tube containing the trigeminal (V) motor nucleus from 40-hr chick embryos were excised and held freely floating in culture medium for 36-40 hr, so that neuronal generation within motor V could be completed, but precluding neuronal differentiation. The explants were then dissociated and plated either on 1) glass coverslips that had been coated with the extracellular matrix (ECM) glycoprotein, laminin, and subsequently irradiated to produce a grid pattern; or 2) coverslips, one-half of which had been coated with laminin, and the other one-half with collagen, another component of the ECM. The purpose of these studies was to assess possible laminin influences on neuronal adhesion and nerve fiber expression and extension during these periods of initial neuronal differentiation. The early neural tube neurons selectively adhered to the established laminin grid pattern; neuronal survival, elaboration of neurites, and extent of neurites were significantly enhanced on the laminin side of the laminin/collagen preparations. These latter effects were specifically blocked by the application of anti-laminin. In demonstrating these influences during stages of initial neuronal differentiation, the results support the hypothesis that laminin may play a role in normal neurogenesis, presumably by providing an adhesive surface for outgrowing growth cones.

Morphological studies of the neuromuscular mechanism shifting from sucking to biting of mice.

In order to give a neuroanatomical evidence to the mechanism of shifting from sucking to biting, we investigated in prenatal, newborn and postnatal mice whether there is a time difference in the neurogenesis of the neurons relative to sucking and biting or in the histogenesis of their peripheral effector organs by the HRP labeling technique and electron microscopy. The results obtained are as follows. (1) At birth the facial motoneurons exceed the trigeminal motoneurons in cell area and development. (2) After birth, the trigeminal motoneurons grow rapidly and outstrip the growth of the facial motoneurons at the age of 6 days. (3) Thereafter, the cell area of both neuron types continues to increase gradually. (4) The initial sign of the alpha motor end plates is found in the orbicularis oris muscle innervated by the facial nerve in 17-day-old fetuses, while that of the trigeminal nerve is delayed in the masseter muscle of 18-day-old fetuses. (5) The initial sign of the muscle spindle appears with the sensory terminals in the masseter muscle of 17-day-old fetuses and the fundamental structure of the muscle spindle is formed in 4-day-old youngs. (6) Myelination of the facial nerve begins in 3-day-old youngs, while that of the trigeminal nerve becomes apparent in 4- or 5-day-old youngs. From these bases, it is obvious that the facial nerve elements related to sucking are firstly developed at birth and that the differentiation of the trigeminal nerve elements related to biting is rapidly accelerated after birth.

Comparison of the proliferative activity of neuroblasts from chick embryo cerebral hemispheres of different ages in culture.

Dissociated cerebral hemisphere cells from 4- to 7-day-old chick embryos were cultured either on a collagen or a polylysine substrate in a serum-containing medium. Neurons were characterized by the demonstration of acetylcholinesterase, the presence of D2/N-CAM glycoprotein and neurofilament proteins. The proliferation of neuronal precursor cells was shown by morphological observations, autoradiographic analysis and measurements of [3H]-thymidine incorporation. Neuronal precursors derived from the 6-day-old embryos showed the highest proliferative activity. Neuroblast proliferation was found to be dependent on the culture substrates (i.e. polylysine or collagen), which yielded either isolated cells or cell aggregates, and the latter favored the mitogenic effect.

Differential effects of anticonvulsants on developing neurons in vitro.

The effects of four anticonvulsants, phenobarbital, diphenylhydantoin (DPH), carbamazepine and valproic acid, on dissociated cultures of embryonic chick dorsal root ganglia (DRG) neurons were examined. DPH and valproic acid inhibited process formation in a dose-dependent manner and reduced neuronal production of substrate attached neurite promoting activity (SANPA). Less inhibition of process formation occurred with phenobarbital and carbamazepine and these two anticonvulsants had no effect on SANPA production. Substrate attachment of neurons, their survival and receptor interactions of DRG neurons with nerve growth factor were not influenced by any of the four anticonvulsants. These data suggest a rank ordering of direct toxic effects of anticonvulsants on developing neurons. The mechanisms of this neurotoxicity may include decreased neuronal production of autostimulating neurite promoting factors.

Abnormal cutaneous nerves in incontinentia pigmenti.

Cutaneous nerves showed multiple ultrastructural abnormalities in a patient with incontinentia pigmenti. Fetal nerve bundles were seen both in the dermis and entering the epidermis. A cell with some Schwann cell features was identified above the basal layer amid keratinocytes. Dermal melanophages were in close apposition to Schwann cells. All of these features suggest that neural structures may be abnormal in incontinentia pigmenti and play a role in transfer of melanin from epidermis to dermis.

Combined effects of deafferentation and de-efferentation on isthmo-optic neurons during the period of their naturally occurring cell death.

We have studied the effects on the chick embryo's isthmo-optic nucleus of de-efferentation alone or in combination with deafferentation. De-efferentation was achieved by pharmacological destruction of the axonal target cells in the retina at E13, or by colchicine-blockade of axoplasmic transport in the intraocular parts of the isthmo-optic axons at E13; deafferentation was by a tectal lesion at E11 or E12. De-efferentation alone causes all the isthmo-optic neurons to die, and mostly by the "endocytic-autophagic" mode of cell death, which is characterized by pronounced endocytosis (of an intravascularly injected label) and by intense, clumped activity of two lysosomal enzymes (acid phosphatase and N-acetyl-beta-glucosaminidase). Deafferentation plus de-efferentation caused there to be less endocytic-autophagic dying cells in the isthmo-optic nucleus than after de-efferentation alone, but all the neurons still died. Our interpretation is that deafferentation switched many of the isthmo-optic neurons to a completely different (nonendocytic, nonautophagic) mode of cell death.