An axon growth associated antigen is also an early marker of neuronal determination.

We have previously described the generation of a monoclonal antibody (DSS-3) that binds to all neurons in cockroach embryos at 50% development and to only a small subset of interneurons in the adult nervous system. This developmental stage-specific antigen was observed to reappear in all axotomized adult neurons that were undergoing axonal regeneration. In the present study the time course of the appearance of this growth-associated antigen during embryonic development was determined. Unexpectedly, the antigen was observed to be present in embryonic neurons long before axon growth. In addition, all cells in the CNS neuronal lineage (neuroblasts, ganglion mother cells, and neurons) bind the antibody as soon as they can be morphologically identified. However, the antigen is also transiently present in all neuroepithelial cells at a stage prior to the morphological differentiation of some of them to neuroblasts. Analogous patterns of DSS-3 binding to cells involved in the development of sensory neurons and leg pioneer neurons are observed. The DSS-3 antigen is therefore a very early marker for the capacity of ectodermal epithelial cells to develop along a neuronal lineage.

The normal human optic nerve. Axon count and axon diameter distribution.

Computerized image analysis was used to determine the normal axonal count and axon diameter distribution in 12 normal human eyes. Mean axon count per nerve was 969,279 +/- 239,740 and mean axon diameter was 0.72 +/- 0.07 micron. Multiple linear regression disclosed 4909 axons lost yearly (P = 0.08). Statistical analysis did not show a relationship between axon diameter and age or time to fixation. The inferotemporal sector of the nerve had the highest fiber density (P = 0.02). The superonasal nerve had higher mean diameters (P = 0.02). This study may provide a baseline for future pathologic studies.

Vasopressin and oxytocin systems in the brain and upper spinal cord of Macaca fascicularis.

This paper describes the vasopressin (VP) and oxytocin (OXT) immunoreactive structures in the brain and upper spinal cord of the adult male and female Macaca fascicularis. Immunocytochemistry following intraventricular application of colchicine displayed VP neurons in the diagonal band of Broca (DBB), bed nucleus of the stria terminalis (BST), medial amygdaloid nucleus, dorsomedial hypothalamic nucleus, area of the locus coeruleus (LC), solitary tract nuclei (NTS), and the dorsal horn of the cervical spinal cord in addition to those known to exist in the paraventricular, supraoptic, and suprachiasmatic hypothalamic nuclei. Furthermore, a dense accumulation of VP fibers was observed in areas such as the DBB, medial septum, BST, amygdala, hippocampus, ventral tegmental area, periaquaductal gray, dorsal and ventral raphe, area of Forel, LC region, parabrachial nuclei, and NTS. The lateral septum and lateral habenula displayed no and very few VP fibers, respectively. No extrahypothalamic OXT neurons were found in the brain of this macaque monkey. Dense concentrations of OXT fibers were demonstrated in the amygdala, NTS, and marginal layer of the cervical spinal cord. No sexual dimorphism was found in this primate VP or OXT system. The results show a distribution of the central VP and OXT systems in this primate which is quite different from that in the rat. However, in various aspects it agrees with current data on the VP and OXT systems of the human brain. The present results suggest, therefore, that this monkey might serve as a better model for the human VP system than the rat.

Adducin: Ca++-dependent association with sites of cell-cell contact.

Adducin is a protein recently purified from erythrocytes and brain that has properties in in vitro assays suggesting a role in assembly of a spectrin-actin lattice. This report describes the localization of adducin to plasma membranes of a variety of tissues and the discovery that adducin is concentrated at sites of cell-cell contact in the epithelial tissues where it is expressed. Adducin in tissues and cultured cells always was observed in association with spectrin and actin, although spectrin and actin were evident in the absence of adducin. In sections of intestinal epithelial cells spectrin was present on all plasma membrane surfaces while adducin was restricted to the lateral cell borders. Adducin also was not detected in association with actin stress fibers in cultured cells. The presence of adducin at cell-cell contact sites of cultured epithelial cells requires extracellular Ca++ and occurs within 15 min of addition of 0.3 mM Ca++. Redistribution of adducin after addition of extracellular Ca++ is independent of formation of desmosomal and adherens junctions since assembly of adducin at contact sites requires lower concentrations of Ca++ and occurs more rapidly than redistribution of desmoplakin or vinculin. Treatment of keratinocytes and MDCK cells with nanomolar concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) induces redistribution of adducin away from contact sites. The effect of TPA may be a direct consequence of phosphorylation of adducin, since adducin is phosphorylated in TPA-treated cells and the phosphorylation of adducin occurs before disassembly of adducin from sites of cell-cell contact. Spectrin and adducin are both present in a detergent-insoluble form at cell-cell contact sites of cultured cells. These observations are consistent with the idea that adducin recognizes and associates with specific "receptors" localized at regions of cell-cell contact and promotes assembly of spectrin into a more stable structure, perhaps analogous to the highly organized spectrin-actin network of erythrocyte membranes.

Developmental and comparative aspects of nonsynaptic release by the egg-laying controlling caudodorsal cells of basommatophoran snails.

In an immunoelectron microscope study the postembryonic development of the cerebral caudodorsal cells (CDC) in the freshwater snail Lymnaea stagnalis was studied as well as the development of similar neurons in other basommatophoran families. The CDC of adult L. stagnalis control egg-laying and associated behaviors by releasing various peptides, including the ovulation hormone CDCH. The CDC release peptides from neurohemal axon terminals and from nonsynaptic release sites of axon collaterals. During postembryonic development the collateral system develops synchronously with the neurohemal area. The first collaterals appear in the cerebral commissure of juvenile snails (10 mm shell height; S = 10). Up to S = 30 they gradually increase in size and length and eventually run through the entire inner compartment. Secretory granules in both collaterals and neurohemal axon terminals increase in size as well. Immunoelectron microscopy combined with the TARI-method for the demonstration of exocytosis indicates that CDCH-release from collaterals and neurohemal terminals occurs already in S = 10; exocytosis of immunoreactive granule contents takes place from nonsynaptic release sites, unspecialized areas of the axolemma of the collaterals. Release activity in the collaterals gradually increases up to S greater than or equal to 20. Neurohemal release activity shows a similar picture except for a steep increase in adult snails. A distinct glial sheath, separating the neurohemal area from the collateral system, appears around S = 15. Representatives of three families of Basommatophora, viz. the lymnaeid L. ovata, the planorbid Planorbis planorbis, and the bulinid Bulinus truncatus possess a well-developed collateral system showing many signs of exocytosis. A glial sheath separates the collaterals from the neurohemal area. Secretory granules of the CDC in L. ovata stain weakly positive with the anti-CDCH antiserum. Since the other Basommatophora did not show immunoreactivity, the chemical structure of egg laying peptides in Basommatophora seems to be genus specific. Apparently the secretory activity of both the neurohemal area and the collateral system is not only important in the sexually mature animal, being involved in the control of egg laying and egg-laying behavior, but also in the juvenile snail. The finding of a collateral system in representatives of three basommatophoran families strongly indicates the importance of the system for the control of reproduction in basommatophoran snails in general.

Terminal arbors of individual "feedback" axons projecting from area V2 to V1 in the macaque monkey: a study using immunohistochemistry of anterogradely transported Phaseolus vulgaris-leucoagglutinin.

In the present study, the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into area V2 in order to demonstrate the precise morphology of individual axons from area V2 to V1. On the basis of 28 complete axon reconstructions, several characteristic features have been identified. 1) Individual axons arborize in multiple layers: 1, 2, 5, and (inconstantly) 3. A single axon may have numerous terminal clusters in layers 1 and 2, but at most one in layer 3. 2) Axons typically ascend to layer 1, turn asymmetrically in one direction, and travel for long distances in this layer (1.10-4.30 mm; dimensions uncorrected for shrinkage). A few axons (three of 28 reconstructed) were found to have a single terminal cluster (0.3-0.5 mm wide) in layers 1 and 2. 3) Collaterals in layer 5 seem to extend over shorter distances (0.60 mm or less). 4) Delicate sprays of boutons (both beads and spines) are clustered along the main trunk. Spacing is variable but usually ranges from 0.35 mm to 0.65 mm. 5) In addition to clustered boutons, there can be linear collaterals, continuously studded with boutons, parallel to the main axon in layer 1. These results indicate that axons from V2 have complex radial and tangential distributions in V1. Terminations in different layers may be directed to different sets of neurons or to different portions of the dendritic tree (for example, distal portions of pyramidal neuron apical dendrites in layers 1 and 2, but more proximal portions in layer 3). Clustered terminations over wide tangential areas may imply a divergent innervation by a single axon of multiple compartmental structures, such as ocular dominance columns or cytochrome oxidase patches.

GABA innervation in adult rat oculomotor nucleus: a radioautographic and immunocytochemical study.

GABA innervation in the adult rat oculomotor nucleus (n.III) was investigated using two complementary approaches: radioautography after incubation of brain slices with tritiated GABA ([3H]GABA) along with local in vivo microinjections of the tracer, and GABA immunocytochemical procedures involving antibodies directed against a GABA-glutaraldehyde-protein conjugate. As determined by radioautography after in vitro or in vivo labelling, the [3H]GABA uptake sites in the n.III mainly involved axon terminals. These were distributed throughout the neuropil and were often closely apposed to unlabelled motoneuron somata. A small number of glial cells also showed preferential accumulation of the tracer. The GABA-immunostaining likewise involved axon terminals throughout the nucleus, but no glial cells were immunopositive. In the dorsal region of the structure, occasional GABA-immunostained internuclear neurons were observed among unstained motoneuron cell bodies. Electron microscopic examination of [3H]GABA-labelled or GABA-immunostained profiles in n.III revealed axon terminals of around 1 micron in diameter, always filled with small, round synaptic vesicles homogeneously distributed throughout the axoplasm. These boutons frequently contained mitochondria and one or more large granular vesicles. In single thin sections, 35% of [3H]GABA-labelled, and 19% of GABA-immunostained varicose profiles exhibited a synaptic differentiation, suggesting the existence of a predominantly if not entirely junctional innervation. These synapses mostly involved dendritic trunks or dendritic branches and were usually of the symmetrical type. A few, which were always symmetrical, were also observed on large somata of motoneurons. Some of the dendrites synaptically contacted by GABA-immunostained axon terminals were themselves GABA immunoreactive. These data substantiate the idea that GABA is involved in the control of motoneuron activity in n.III, and provide a structural basis for the inhibitory role of this transmitter in oculomotor function.

The morphogenesis of the posterior neural tube and tail in Monodelphis domesticus.

The process of secondary neuralation has been studied in the Brazilian opossum, Monodelphis domesticus. Secondary neuralation in this mammal was found to have qualities of secondary neuralation that were present in both the chick and the mouse. In this study, four stages of secondary neuralation were found beginning with the medullary cord stage. Other stages in the differentiation of the secondary neural tube were: differentiation of the neuroepithelium, cavitation of the medullary cord, and proliferation of the neural tube. The process of secondary neuralation proceeded in a rostral-to-caudal direction and was found to be independent of age. Diastematomyelia (doubling of the tube) was found in several animals. The process of cavitation was completed by the joining of several small, focal cavities in a rostral-to-caudal direction. The most distinctive feature of secondary neuralation in this animal was the finding of axons within the secondary neural tube, a feature not characteristic of either the chick or the mouse.

Calcium channels that are required for secretion from intact nerve terminals of vertebrates are sensitive to omega-conotoxin and relatively insensitive to dihydropyridines. Optical studies with and without voltage-sensitive dyes.

Extrinsic absorption changes exhibited by potentiometric dyes have established the ionic basis of the action potential in synchronously activated populations of nerve terminals in the intact neurohypophyses of amphibia and mammals (Salzberg et al., 1983; Obaid et al., 1983, 1985b). Also, large and rapid changes in light scattering, measured as transparency, have been shown to follow membrane depolarization and to be intimately associated with the release of neuropeptides from the nerve terminals of the mouse neurohypophysis (Salzberg et al., 1985; Gainer et al., 1986). We report some experiments that help to define the pharmacological profile of the calcium channels present in intact neurosecretory terminals of vertebrates. For these, we used the peptide toxin omega-conotoxin GVIA (1-5 microM) and the dihydropyridine compounds Bay-K 8644 and nifedipine (2-5 microM), together with the after-hyperpolarization of the nerve terminal action potential. This undershoot depends upon the activation of a calcium-mediated potassium channel, as suggested by its sensitivity to [Ca++]o and charybdotoxin. omega-conotoxin GVIA substantially reduced the after-hyperpolarization in neurosecretory terminals of Xenopus, while neither of the dihydropyridine compounds had any effect under conditions that mimic natural stimulation. The effects of these calcium channel modifiers on the action potential recorded optically from the terminals of the Xenopus neurohypophysis were faithfully reflected in the behavior of the light-scattering changes observed in the neurohypophysis of the CD-1 mouse. omega-conotoxin GVIA (5 microM) reduced the size of the intrinsic optical signal associated with secretion by 50%, while the dihydropyridines had little effect. These observations suggest that the type of calcium channel that dominates the secretory behavior of intact vertebrate nerve terminals is at least partially blocked by omega-conotoxin GVIA and is insensitive, under normal conditions, to dihydropyridines.

[Effect of low temperature on the axons of chick embryo in in vitro culture]. / Azione delle basse temperature su neuriti di embrioni di pollo nelle colture in vitro.

Hanging drop cultures of rhombencephalon of 6-11 day chick embryos were placed for 15'-24 hours at low temperatures (0 degree, 10 degrees, 15 degrees, 20 degrees, 25 degrees). Phase contrast time-lapse cinemicrography were made of the axons outgrowth from the explants, during recovery of normal temperature (38 degrees). A number of cultures were also filmed during cooling from 38 degrees to 15 degrees or 20 degrees. It was thus possible follow the sequence of the axon changes, especially of its growth cone, and demonstrate the reversible nature of the modifications observed at some subnormal temperatures.

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.

An anterograde tracer study on the development of corticospinal projections from the medial prefrontal cortex in the rat.

The aim of the present study is to investigate, both qualitatively and quantitatively, the development of corticospinal (CS) projections from the medial prefrontal cortex of the rat. This study was carried out with the use of anterogradely transported wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) after iontophoretic injections in the medial prefrontal cortex. For comparison similar injections are made in the sensorimotor cortex. The CS axons of neurons situated in the medial prefrontal cortex have reached the first thoracic segment (T1) at postnatal day 3 (P3) and reach their most caudal extension in the spinal cord sixth thoracic segment (T6) at postnatal day 7 (P7) and then gradually disappear during the second postnatal week. Quantitative results revealed that after labelling of the medial prefrontal cortex no peaks in labelling density, neither at the cervical nor at the lumbar intumescence, were present. Furthermore, the CS axons of medial prefrontal neurons never showed any outgrowth into the spinal grey matter at any age studied. Concludingly, the extension and subsequent elimination of CS axons originating in the medial prefrontal cortex follow a similar time course as those from the occipital cortex (Dev. Brain Res., 36 (1987) 121-130).

[Trigeminal motor nuclei in the mouse: effects of microstimulation and sources of afferent projections]. / Troinichnoe motornoe iadro myshi: effekty mikrostimuliatsii i istochniki afferentnykh proektsii.

Motor responses of low jaw to the microstimulation of the motor nucleus of the trigeminal nerve (MNTN) of albino mice have been recorded. They had the latency ranged of 2.5 to 13.5 ms. After microinjections of horseradish peroxidase into the MNTN, retrogradely labeled neurons were revealed ipsilaterally in both sensory and mesencephalic trigeminal nuclei, in the medullary reticular formation, etc.

The organization of prolactin-like-immunoreactive neurons in the rat central nervous system. Light- and electron-microscopic immunocytochemical studies.

The localization and distribution of prolactin-like-immunoreactive perikarya and nerve fibers in the rat central nervous system have been studied by a preembedding immunoperoxidase method using well-characterized specific immunsera to rat prolactin. Although the localization of labeled neuronal structures in a number of brain areas correlates with the data of previous immunocytochemical studies, we found prolactin-immunoreactive neurons in various regions not previously reported. In untreated animals, the highest concentrations of prolactin-fibers were observed: (i) in the external layers of the median eminence where they exhibited close contact with blood vessels, and (ii) in the bed nucleus of the stria terminalis and in the central nucleus of the amygdala where they closely surrounded unlabeled perikarya. Dense networks of finely varicose prolactin fibers were also observed in the organum vasculosum of the lamina terminalis, in the subfornical organ, and in the dorsolateral regions of the medulla oblongata and the spinal cord. Lastly, a number of large, varicose, intensely immunoreactive fibers were found in the olfactory bulb, the cingulum, and the periventricular regions of the hypothalamus and central gray, whereas isolated fibers could be detected in the caudate nucleus and in the cerebral cortex. In animals treated with colchicine, prolactin-immunoreactive perikarya were essentially located within the periventricular and perifornical regions of the hypothalamus, and within the bed nucleus of the stria terminalis. Although corticotropin (ACTH 17-39)-immunoreactive fibers could be detected in several regions found to contain prolactin fibers, the distribution and organization of both fiber types clearly differed in numerous brain regions, and the regions containing the corresponding perikarya did not overlap.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

A quantitative analysis of the development of the pyramidal tract in the cervical spinal cord in the rat.

A quantitative electron microscopic analysis was undertaken of the development of the pyramidal tract, at the level of the third cervical spinal segment, in rats ranging in age from the day of birth to three months old. The axon number was calculated as the product of axon density, determined in a systematic random sample of electron micrographs, and tract area. During the first postnatal week the tract contains thin unmyelinated axons and growth cones. Growth cones are abundant in neonatal rats, but can still be observed occasionally at the end of the first postnatal week, indicating a continuous addition of pyramidal tract axons during the first postnatal week. Myelination starts around P10. By the end of the first postnatal month approximately 50% of the axons have already been myelinated. Myelination proceeds during further maturation, but in the three month old rat 28% of the axons are still unmyelinated. The total number of axons increases rapidly after birth up to 153,000 at the fourth postnatal day. Subsequently, the number of axons is reduced by nearly 50% to 79,000 in the adult rat. The axon loss is most prominent during the second postnatal week, when 32,000 axons are eliminated, but continues for several weeks at a slower rate.

[Convergence of projections from various thalamic nuclei on the rat somatosensory cortex columns]. / Konvergentsiia proektsii razlichnykh talamicheskikh iader k kolonkam somatosensornoi kory krysy.

The method of primuline fluorochrome retrograde transport was used to study sources of thalamo-cortical projections on a separate somatic cortical neuronal column connected with C3 vibrissae of albino rat. Labeled cells were found in 8 thalamic nuclei: tv, tvd, tpo, pf, rh, tvm, tvl, tr. The intensity of neuron staining and cell quantity and density varied in different nuclei. Hence their axon branching in the rat cortex was also different. The majority of intensively stained and densely packed cells have been observed in tv nucleus. The observed convergence of different thalamo-cortical inputs on single somatic cortex column explains heterogeneity in functional properties of the same column neurons and makes it possible for the column to form several neuronal assemblies with different functions.

Experimental in vivo regeneration of peripheral nerve axons and perineurium guided by resorbable collagen film.

In our previous paper, we reported a marked advantage in using collagen gel matrix available for cell culture in combination with a silicone tube as an effective environment for axonal sprouting during peripheral nerve regeneration. In the present experiment, collagen film was substituted for the silicone tube because of its non-toxicity, biocompatibility and better availability. Also, the surgical procedure was simplified, using a fibrin adhesive system instead of suturing. In the second postoperative week, severed proximal and distal stumps became joined together concomitantly with absorption of the collagen matrix and film. On size-frequency histograms, the diameters of both myelinated and unmyelinated axons at 8 weeks after surgery had recovered to their normal ranges. These findings demonstrate that this procedure of enveloping a collagen matrix and severed nerve stumps in bioresorbable collagen film would be an ideal way of forming a perfect perineurium. The regeneration of peripheral nerve axons resulted in normal thickness of the original nerve bundle without exception, unlike the axons on the control side.