numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos.

Neurons and support cells of each sensory organ in Drosophila embryos are most likely derived from a single precursor cell. This cell lineage is affected in numb mutants. Morphological alterations of sensory structures, as well as changes in the number of cells expressing cell type-specific markers, indicate that sensory neurons in numb mutant embryos are transformed into lineage-related nonneuronal support cells. Thus the numb gene controls the fate of progeny derived from sensory organ precursors. The numb gene has been isolated by the plasmid rescue method. The structure of its predicted product is discussed.

Three-dimensional scanning electron microscopic study of the normal hamster olfactory epithelium.

The olfactory epithelium of the adult hamster (Mesocricetus auratus) was studied using the scanning electron microscope. A method that produced fractures in the epithelium exposed structures below the surface and made it possible to examine the morphological and structural relationships among cells. Three cell types were studied: supporting cells, olfactory neurons (receptor cells) and basal cells. Supporting cells were observed spanning the full extent of the epithelium, and had basal foot processes that terminated at or near the basal lamina. Along the lateral margin of supporting cells, cellular processes were observed extending outwards, reaching olfactory neurons and adjacent supporting cells. These cellular contacts among supporting cells and olfactory neurons were present at different levels of the epithelium. Olfactory neurons were located primarily in the middle and lower epithelial regions. Their dendritic processes reached the epithelial surface in a straight or tortuous manner, passing between the supporting cells. Olfactory axons were observed as thin unbranched processes that emerged from a conical hillock region, passed basally, and fasciculated into larger sensory bundles within the lamina propria. Basal cells were observed adjacent to the basal lamina as a row of single cells or clustered in groups. Within the lamina propria connective tissue, blood vessels, axon bundles and Bowman's glands were examined. Bowman's glands were composed of pyramidal secretory cells arranged about a single duct that extended to the epithelial surface. Scanning electron microscopy provided a unique three-dimensional analysis of cell structure within the olfactory epithelium. The results provide new and different observations on the detailed morphology and intimate relationships that exist among epithelial cells, and complement previous light and transmission EM observations.

T cell function and expression are dramatically altered in T cell receptor V gamma 1.1J gamma 4C gamma 4 transgenic mice.

We have characterized transgenic mice carrying a functional T cell receptor (TCR) C gamma 4 (V gamma 1.1J gamma 4C gamma 4) gene. Results indicate that active transcription of the C gamma 4 transgene can influence expression of the endogenous C gamma 4, C gamma 1 (V gamma 3-, V gamma 4-, V gamma 2-, or V gamma 5J gamma 1C gamma 1) and C gamma 2 (V gamma 1.2J gamma 2C gamma 2) genes, while the ultimate expression of other TCR delta, alpha, and beta chain genes, as well as the adult T cell response, are relatively unaltered. Cells expressing transgenic C gamma 4 and endogenous delta TCR transcripts can migrate to the skin as dendritic epithelial cells (DEC) even though C gamma 4 cells are rarely, if at all, found in the skin. Transgenic and control mice were compared at 2 weeks, 6-7 weeks, and older. At 2 weeks, the thymus of transgenic mice, particularly the medulla, was much larger than control. Moreover, peripheral lymphoid tissues of younger mice were markedly (as much as 100-fold) more immunoreactive (both Con A response and alloreactivity). These differences, although persistent, became smaller in older mice. The data suggest that transgene expression has a major effect on T cell development and reactivity.

Dendritic morphology of identified retinal ganglion cells in Xenopus laevis: a comparison between the results of horseradish peroxidase and cobaltic-lysine retrograde labelling.

Following the application either of cobaltic-lysine complex or a 30% solution of horseradish peroxidase (HRP) in a sealed tube to the cut end of the optic nerve of young adult Xenopus frogs, the dendritic morphology of large ganglion cells was studied in retinal wholemount preparations, and compared with that in animals of the same size as revealed by the short time administration of HRP crystals. In the former two groups of animals, after 24 h survival, the size of the dendritic arborization of characterized ganglion cell types, Types I and III, were found to be significantly larger (61-79% and 180-187%, respectively) than those which survived 3 days after the administration of HRP crystals. These findings suggest that the very fine dendritic branches of large ganglion cells may remain unlabelled after a short-time exposure to HRP.

Projection neurons in the rat dorsolateral septal nucleus possess recurrent axon collaterals.

Projection neurons in the rat dorsolateral septal nucleus (DLSN) were labeled intracellularly with horseradish peroxidase (HRP) in an in vitro slice preparation. The labeled neurons exhibited widespread 'isodendritic' type dendritic fields. Each of the neurons was identified as a projection neuron by the tracing of its main axon out of DLSN. The axons of these neurons gave rise to intrinsic collaterals which branched to form an extensive axon plexus which was confined to DLSN. These axon collaterals exhibited numerous en passant swellings suggestive of boutons. It is proposed that the recurrent axon collaterals of DLSN projection neurons may form an anatomical substrate for local inhibition within DLSN.

Characteristics of microtubules at the different stages of neuronal differentiation and maturation.

The developing nervous system has proved to be a very powerful tool to analyze how MT are involved in basic biological processes such as cell proliferation, cell migration, cell shaping, and transport. A better knowledge of the basic events occurring during neurogenesis also affords us the possibility of establishing the basis of experiments and trying to solve unanswered and important questions. Despite the considerable value of cell culture, we need to use more discrete regions of the developing brain in situ in order to analyze the MT and their modifications into cells developing their "natural" environment. One major problem remains the question of the mode of assembly and disassembly, that is, the behavior of MT in living cells. Dynamic instability and/or treadmilling are accurate interpretations of the dynamics of MT at least in vitro or in cell culture, but we do need more information on what happens in situ and in vitro. One of the main tasks of cell biologists is to devise satisfactory tests to approach this fundamental question. In this view, pharmacological manipulation of embryos treated in whole-embryo culture systems might be a possible way. Microtubules are ubiquitous cell components. However, the extensive heterogeneity of MAP and tubulin in the CNS confers on the neurons a wide range of capabilities of assembly of these proteins and suggests that the neuron has a unique potential of a relation between MT composition and cell function. We have seen that each major event during neurogenesis is related to a specific series of modifications of the MT components. It remains to be determined if there is a causal or just a correlative relationship between the appearance of specific isotypes and the occurrence of specific events and/or functions. We have also to determine the exact spatial and temporal relations among the different isotypes of MT proteins, tubulin, and MAP. Is there a close correspondence between a tubulin and a MAP isotype? Can the appearance of one isotype of tubulin influence the appearance and the assembly of a specific MAP, or vice versa? Recent results obtained with the Tyr- and Glu-MT shed light on these questions and suggest a whole series of possibilities for cells to modulate the structure, behavior, and function of MT in specific domains of the neuron or in specific regions of the brain, by only a minute modification of the molecule of tubulin. Microtubule protein heterogeneity raises also a number of questions.(ABSTRACT TRUNCATED AT 400 WORDS)

Polarity orientation of microtubules in hippocampal neurons: uniformity in the axon and nonuniformity in the dendrite.

We have analyzed the polarity orientation of microtubules in the axons and dendrites of cultured rat hippocampal neurons. As previously reported of axons from other neurons, microtubules in these axons are uniform with respect to polarity; (+)-ends are directed away from the cell body toward the growth cone. In sharp contrast, microtubules in the mid-region of the dendrite, approximately 75 microns from the cell body, are not of uniform polarity orientation. Roughly equal proportions of these microtubules are oriented with (+)-ends directed toward the growth cone and (+)-ends directed toward the cell body. At distances within 15 micron of the growth cone, however, microtubule polarity orientation in dendrites is similar to that in axons; (+)-ends are uniformly directed toward the growth cone. These findings indicate a clear difference between axons and dendrites with respect to microtubule organization, a difference that may underlie the differential distribution of organelles within the neuron.

Induction of process outgrowth in vertebrate and invertebrate cell lines by a 2-pyridinyl thiosemicarbazone.

Regulation of differentiation in cells of disparate origin is often mediated by widely differing molecular signals and receptor mechanisms. For example, two neuron-like cell lines used extensively as models for molecular control of differentiation, the steroid-sensitive Kc line from Drosophila and the polypeptide- and cyclic nucleotide-sensitive PC12 line from rat, share no obvious growth factor or hormone receptors. However, we have found that a thiosemicarbazone, 1-pyrrolidinecarbothioic acid [1-(2-pyridinyl)ethylidene] hydrazide, one of a class of synthetic antineoplastic agents, induces process outgrowth - a marker of cellular differentiation - in cells of both of these lines. Moreover, the thiosemicarbazone induces process outgrowth in cells of mutant clones of these lines that are refractory to treatment with growth factors or hormones. Activity of the thiosemicarbazone is dependent upon the alpha-(N)-heterocyclic ring. These findings show that the 2-pyridinyl thiosemicarbazone mimics the effects of diverse epigenetic factors in inducing process outgrowth similar to that seen in cellular differentiation of these cell lines induced by natural regulators. Regulation may be by a mechanism, common to both invertebrate and vertebrate cells, which occurs downstream from the receptors that have been previously shown to mediate epigenetically induced differentiation.

[Neuronal organization of the periamygdaloid cortex in the cat brain]. / Neironnaia organizatsiia periamigdaliarnoi kory mozga koshki.

Neuronal organization of the fields Pmm, Pml2, Pe and epm of the periamygdaloid cortex of the cat brain has been studied by means of Golgi and Nissl methods. The field Pmm essentially differs from other fields of this cortex by primitiveness of its cytoarchitectonic an neuronal organization (two layers uniform by the composition of their neurons are distinguished, the structure of the latter is relatively primitive). In the medial part of this field long axonal rarely branching short dendritic, and in the lateral part--poorly differentiating pyramidal and spindle-like cells predominate. The field Pmm can be considered as a transitional formation between the subcortex (the medial nucleus of the amygdaloid body) and other fields of the periamygdaloid cortex. The fields Pml2, Pe and epm are built more complexly: the cells are organized in 4 layers, more complexly differentiated by their form and size than in the field Pmm and correspondingly more various (long axonal densely branching cells are observed: pyramidal and spindle-like--of the cortical type and bushy--of the subcortical type, as well as long axonal rarely branching reticular cells). The short axonal cells in the fields Pml2, Pe and epm are rather variable in their form, size and direction of axons; in the field Pmm they are less numerous. The field Pmm and the complex of the fields Pml2, Pe and epm are perhaps different in their function, this is evident from different projection of their neurons. Axons of the cells in the field Pmm get into less differentiated and the most ancient medial nucleus of the amygdaloid body and into the ancient system of connections of the latter--terminal strip, and neurons of the fields Pml2, Pe and epm are projected into the basolateral part of the amygdaloid body and into the external capsule--phylogenetically younger structures. Besides, poverty of the axonal collateralies in the long axonal neurons and a small amount and uniformity of the forms of the short axonal cells in the field Pmm and contrary, rich collateralies and variety of short axonal cells in the composition of other fields demonstrate more complex internal integrative function, performing in that composition.

[Neuronal organization of field 4 of the motor cortex of the cat brain]. / Neironnaia organizatsiia polia 4 motornoi kory mozga koshki.

By means of the silver nitrate impregnation method after Golgi-Kopsch in kittens and young cats the field 4 in the cerebral motor cortex has been studied. The motor cortex of the field 4 possesses certain heteromorphism. Besides usual stellate and pyramidal neurons, that differ from real ones by some morphological signs: their body is often round, the apical dendrite is much thinner than the corresponding dendrite of a pyramidal neuron, it does not produce oblique branches along the course, never gets into the I layer, the spines arrange less densely. According to the mode of dendrites setting off, the atypical pyramidal neurons can be divided into multipolar and spindle-like with horizontal or vertical branching of the dendrites. According to the spines distribution, the multipolar atypical neurons can be divided into spinous, rare-spinous and aspinous. With respect to various cellular forms and distribution of various types of neurons in layers, every of the areas (gamma, alpha, sfu, fu) possesses specific peculiarities. The greatest variability of the neurons have the field 4 gamma and 4 alpha, where, besides stellate and pyramidal, atypical neurons can be found. The stellate neurons of the field 4 gamma are characterized with a deep arrangement, their number is essentially less, than in other areas of the field 4. In the field 4 alpha they are situated in the layers II-III. Suprafundal and fundal parts of the field do not possess pyramidal atypical neurons and are characterized with presence of large amount of the stellate neurons. In respect to the axonal branching in the suprafundal part of the field 4, 2 types of the stellate cells are distinguished.

Synaptic connections of an interneuron with axonal arcades in the cat visual cortex.

A single, isolated interneuron with axonal arcades in the cat visual cortex was analysed in detail by both light and electron microscopy. The neuron was impregnated by the Golgi-Kopsch method, gold-toned, and processed for electron microscopy using the ethanolic phosphotungstic acid (PTA) staining method of Bloom & Aghajanian (1968). These methods, in combination, resulted in the successful identification of a large number of synaptic boutons arising from the axon of the cell under study. We examined serially at the electron microscope level 210 boutons of the axonal arborization of the cell. Of these, 152 formed identifiable symmetrical synaptic contacts with a variety of postsynaptic elements. The vast majority of the postsynaptic targets were dendritic profiles, which represented 95.7% of all the synaptic contacts identified. Only one example was observed of two labelled boutons making contacts with the same postsynaptic element; the rest were apparently on different elements. This distribution of synapses, characterized by the lack of convergence, is very similar to that reported by other authors for a certain kind of double bouquet cell which, in turn, shares some morphological features with the neurons with axonal arcades. It is suggested that fine details of the geometry of the axonal arborization of a given cell are an important reflection of the distribution of its synapses.

Shapes of nerve cells in the myenteric plexus of the guinea-pig small intestine revealed by the intracellular injection of dye.

The shapes of myenteric neurons in the guinea-pig small intestine were determined after injecting living neurons with the dye Lucifer yellow via a microelectrode. The cells were fixed and the distribution of Lucifer yellow rendered permanent by an immunohistochemical method. Each of 204 nerve cells was examined in whole-mount preparations of the myenteric plexus and drawn using a camera lucida at 1250 x magnification. Four cell shapes were distinguished: (1) neurons with several long processes corresponding to type II of Dogiel; (2) neurons with a single long process and lamellar dendrites corresponding to type I of Dogiel; (3) neurons with numerous filamentous dendrites; and (4) small neurons with few processes. About 15% of the neurons could not be placed into these classes or into any single class. The type II neurons (39% of the sample) had generally smooth somata and up to 7 (average 3.3) long processes, most of which ran circumferentially. Dogiel type I neurons (34% of sampled neurons) had characteristic lamellar dendrites, i.e., broad dendrites that were flattened in the plane of the plexus. The filamentous neurons (7% of the sample), had, on average, 14 fine processes up to about 50 microns in length. Small neurons with smooth outlines and a few fine processes made up 5% of the neurons encountered. We conclude that myenteric neurons that have been injected with dye can be separated into morphologically distinct classes and that the different morphological classes probably correspond to different functional groupings of neurons.

Alterations in the morphology of ganglion cell dendrites in the adult rat retina after optic nerve transection and grafting of peripheral nerve segments.

Transected ganglion cell axons from the adult retina are capable of reinnervating their central targets by growing into transplanted peripheral nerve (PN) segments. Injury of the optic nerve causes various metabolic and morphological changes in the retinal ganglion cell (RGC) perikarya and in the dendrites. The present work examined the dendritic trees of those ganglion cells surviving axotomy and of those whose served axons re-elongated in PN grafts to reach either the superior colliculus (SC), transplanted SC, or transplanted autologous thigh muscle. The elaboration of the dendritic trees was visualized by means of the strongly fluorescent carbocyanine dye DiI, which is taken up by axons and transported to the cell bodies and from there to the dendritic branches. Alternatively, retinofugal axons regrowing through PN grafts were anterogradely filled from the eye cup with rhodamine B-isothiocyanate. The transection of the optic nerve resulted in characteristic changes in the ganglion cell dendrites, particularly in the degeneration of most of the terminal and preterminal dendritic branches. This occurred within the first 1 to 2 weeks following axotomy. The different types of ganglion cells appear to vary in their sensitivity to axotomy, as reflected by a rapid degeneration of certain cell dendrites after severance of the optic nerve. The most vulnerable cells were those with small perikarya and small dendritic fields (type II), whereas larger cells with larger dendritic fields (type I and III) were slower to respond and less dramatically affected. Regrowth of the lesioned axons in peripheral nerve grafts and reconnection of the retina with various tissues did not result in a significant immediate recovery of ganglion cell dendrites, although it did prevent some axotomized cells from further progression toward posttraumatic cell death.

Dendritic maturation in cat retinal ganglion cells: a Lucifer yellow study.

The dendritic morphology of developing cat alpha- and beta-retinal ganglion cells was investigated by intracellular injection of Lucifer yellow. In both cell classes the basic pattern of adult morphology was present at birth. However, the presence of transient small spiny protrusions along the dendrites was characteristic of early postnatal cells. Many alpha-cells were further distinguished by a small degree of dendritic bi-stratification which disappeared within the first 5 postnatal days. Therefore during the period before the eyes opened (P7-P10) there was a considerable degree of modification and maturation in dendritic morphology in both classes of retinal ganglion cells. alpha- and beta-cells exhibited differing temporal patterns of dendritic growth, which argues against a 'passive-stretching' hypothesis that explains dendritic field enlargement solely as an effect of retinal areal growth.

Effect of tetraploidy on dendritic branching in neurons and glial cells of the frog, Xenopus laevis.

Morphological aspects of four different groups of Golgi impregnated brain cells from a tetraploid strain of Xenopus laevis frogs were compared to analogous cells in comparably sized diploid frogs. The cells examined included neurons from the telencephalon, caudal hypothalamus, and optic tectum, and radial glial cells from the optic tectum. The brains of tetraploid frogs appeared grossly normal and were the same size and contained similar cell types as diploid brains. As observed in previous studies on polyploid amphibia, somal diameters increased significantly in tetraploid cells for each of the four groups of cells examined. Also, the total length of the dendritic arbors in tetraploid brain cells increased significantly by factors ranging from 1.4 to 2.4 times the total length of the analogous processes in diploid cells. Tetraploid neurons in the telencephalon and hypothalamus increased their arbor lengths predominantly by increasing the number of dendritic branches, while maintaining the average distance between branch points in the dendritic segments. In contrast, the tetraploid large pear-shaped neurons in the optic tectum had significantly longer terminal dendritic segments than the analogous diploid neurons, although these tetraploid neurons maintained their average number of dendritic segments per cell. Tetraploid tectal radial glial cells appeared to increase both their number of branches and the lengths of their terminal segments. Thus, the mode by which tetraploid brain cells achieved longer dendritic arbors varied from cell type to cell type. These results suggest a hypothetical basis for possible effects of genomic size on vertebrate brain structure and evolution at the cellular level.

Stages in the structural differentiation of retinal ganglion cells.

Using a cultured wholemount technique we have studied the morphological differentiation of ganglion cells in the retina of the rat and cat, during normal development. In both species the differentiation of ganglion cells begins in embryonic life, before embryonic day (E) 17 in the rat and E36 in the cat. It is useful to describe the morphological differentiation of ganglion cells as occurring in three stages. In the first stage, each germinal cell becoming a ganglion cell extends an axon into the fibre layer of the retina and towards the optic disc, and the soma of the cell moves towards the ganglion cell layer. As the soma approaches the ganglion cell layer, the processes that attach its poles to the inner and outer surfaces of the retina are withdrawn. When the soma reaches the ganglion cell layer, a stage of active dendritic growth begins, which lasts until shortly before birth in the cat and until several days after birth in the rat. The cell extends stem dendrites that branch profusely and are commonly tipped by growth cones. The major morphological classes of ganglion cell become distinct in the latter part of stage 2, as do the centroperipheral gradients in ganglion cell size apparent in the cat. During the third stage, the dendritic trees of ganglion cells no longer branch or extend by means of active growth cones. Very considerable growth of all parameters of the cell (soma size, dendrite calibre and length, axon calibre) occurs nevertheless, presumably by interstitial addition of membrane throughout the cell.