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In these studies, strong HLA-A,B,C or b2-m expression was not detected in any stage of development of normal neurons. These included regenerating cells of the olfactory epithelium, developing neural tissue in the mouse embryo, and normal adult brain. Nor was an obligatory role for HLA-A,B,C or b2-m revealed by in vitro analysis of a growing neuroblastoma cell line.

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.

Based on the staining of Drosophila embryos with neuron-specific monoclonal antibodies we describe the differentiation of the earliest neurons in the central nervous system. The metameric array undergoes a number of changes during development that distinguish several morphological units: metameres, neuromeres and ganglia. Neural landmarks in the CNS and the periphery are identified on the bases of their developmental history, axonal projection and segment specificity. A time sequence of differentiation starting in the posterior gnathal and anterior thoracic anlage has been found.

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.

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.

Cultures of chick dorsal root ganglia were examined with the scanning electron microscope. Fibroblasts, Schwann cells and neurites with their characteristic terminal filamentous growth cones were identified and their surface ultrastructure and inter-relationships described. Although exploratory filopodia of nerve growth cones were observed to pass both over and underneath the edges of wandering fibroblasts present at the periphery of the outgrowth, it was noted that the nerve bundles ultimately came to overlie sheets of fibroblasts. The neurites appeared generally bare along their lengths but were sometimes attached to the substrate or underlying fibroblastic sheet by small lateral projections. Some Schwann cells were observed to migrate freely between the neurites whilst others appeared to have wrapped around the nerve bundles in a manner suggestive of early myelination.

Treponema pallidum (Nichols strain) was incubated with cultured nerve cells derived from rat embryos. Primary cultures were established from dorsal root ganglia, superior cervical ganglia, and spinal cord. Using phase contrast microscopy treponemes were seen to interact with the nerve cells in a similar manner to other cultured mammalian cells. Organisms began to attach within minutes after inoculation, actively motile organisms attached at the tip of one end, higher numbers of organisms attached with continued incubation, and attached organisms survived longer than unattached organisms. T pallidum attached both to nerve cell bodies and to neuronal processes of each of the three nerve cell cultures. As shown by scanning electron microscopy the mechanism of attachment was identical to that of cultured cells derived from rabbits testis, rat skeletal muscle, and human cervical carcinoma. There was no indentation or swelling of the cultured cell surface at the point of attachment, just a close physical proximity of organisms and cells. These techniques provide a biological means of studying the in-vitro detrimental influences of micro-organisms on nerve tissue.

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.

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.

The role of a notochord fragment on the origin of an additional floor plate area in the neural tube is investigated by quantitative morphological methods. In 1.5 to 2 day chick embryos a notochordal fragment was implanted in close apposition to the lateral wall of the neural groove in the region between prospective wing and leg bud. At 4 days, adjacent to the implant a distinct area of the neural wall was present, which resembled the natural floor plate with respect to its thickness, the abluminal location of elongated nuclei and the absence of neuroblasts. The mitotic density of this area was reduced. This "additional floor plate" was distinct when the experiment was performed at 1.5 days but was hardly recognizable when it was carried out at 2 days. From these results it is concluded that a) the notochord induces floor plate like structures and diminishes proliferation, and b) that the period of floor plate induction by the notochord is very restricted.

The development of the optic tectum and the establishment of retinotectal projections were investigated in the quail embryo from day E2 to hatching day (E16) with Cresyl violet-thionine, silver staining and anterograde axonal tracing methods. Both tectal cytodifferentiation and retinotectal innervation occur according to a rostroventral-caudodorsal gradient. Radial migration of postmitotic neurons starts on day E4. At E14, the tectum is fully laminated. Optic fibers reach the tectum on day E5 and cover its surface on day E10. 'Golgi-like' staining of optic fibers with HRP injected in vitro on the surface of the tectum reveals that: growing fronts are formed exclusively by axons extending over the tectal surface; fibers penetrating the outer tectal layers are always observed behind the growing fronts; the penetrating fibers are either the tip of the optic axons or collateral branches; as they penetrate the tectum, optic fibers give off branches which may extend for long distances within their terminal domains; the optic fiber terminal arbors acquire their mature morphology by day E14. The temporal sequence of retinotectal development in the quail was compared to that already established for the chick, thus providing a basis for further investigation of the development of the retinotectal system in chimeric avian embryos obtained after xenoplastic transplantation of quail tectal primordia into the chick neural tube.

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.

We have studied the orientation of neurite growth in the culture of embryonic Xenopus neurons in response to three types of extracellular electric fields: spatially uniform pulsed fields, focally applied steady (DC) fields, and focally applied pulsed fields. Under uniform pulsed fields, neurites showed a preferential orientation toward the cathode pole of the field in a manner similar to that previously found for DC fields. The extent of neurite orientation depended upon the duration, amplitude, and frequency of the pulse but appeared to be similar to that produced by a uniform DC field of an equivalent time-averaged field intensity. For square pulses of 5 msec duration, the minimal amplitude and frequency required to produce a detectable orientation of neurite growth over a period of 24 hr were 2.5 V/cm and 10 Hz, which correspond to a time-averaged field intensity of 125 mV/cm. Steady or pulsed focal fields were applied by passing a current through a micropipette placed near the growth cone of the neurite. Fields of negative polarity (current sink) were found to attract the growth cone, whereas fields of positive polarity (current source) were found to deflect the growth cone away from the pipette. The threshold DC current density needed at the growth cone to perturb its direction of growth within 15 min was 0.2 to 2 pA/micron2 (or 3 to 30 mV/cm); and for focal pulsed currents (pulse duration 5 msec), a typical combination of minimal pulse amplitude and frequency was 4 pA/micron2 and 10 Hz. This threshold focal current is similar to that which occurs at the synaptic cleft during active synaptic activity.

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)

Fetal thalamic cells implanted into the excitotoxically lesioned thalamus of adult rats construct a neo-nucleus. This study shows that the vascular network of this neo-nucleus exhibits normal characteristics considering both the morphology (at the light and ultrastructural levels) and the presence of a blood-brain barrier for macromolecules. A role of the neuronal environment in this reconstruction is suggested.

Dorsal root ganglion nerve cells undergoing axon elongation in vitro have been analyzed ultrastructurally. The growth cone at the axonal tip contains smooth endoplasmic reticulum, vesicles, neurofilaments, occasional microtubules, and a network of 50-A in diameter microfilaments. The filamentous network fills the periphery of the growth cone and is the only structure found in microspikes. Elements of the network are oriented parallel to the axis of microspikes, but exhibit little orientation in the growth cone. Cytochalasin B causes rounding up of growth cones, retraction of microspikes, and cessation of axon elongation. The latter biological effect correlates with an ultrastructural alteration in the filamentous network of growth cones and microspikes. No other organelle appears to be affected by the drug. Removal of cytochalasin allows reinitiation of growth cone-microspike activity, and elongation begins anew. Such recovery will occur in the presence of the protein synthesis inhibitor cycloheximide, and in the absence of exogenous nerve growth factor. The neurofilaments and microtubules of axons are regularly spaced. Fine filaments indistinguishable from those in the growth cone interconnect neurofilaments, vesicles, microtubules, and plasma membrane. This filamentous network could provide the structural basis for the initiation of lateral microspikes and perhaps of collateral axons, besides playing a role in axonal transport.