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The development of immunoreactivity (IR) in the nervous system of asexually reproducing Microstomum lineare has been studied by a combination of simultaneous and double immunostaining with antisera to 5-HT and RF-amide, as well as with monoclonal antibodies to SCPB (molluscan small cardioactive peptide). Immunoreactivity appears in a distinct sequential order. 5-HT antigenicity in the postpharyngeal commissure indicates the initiation of the development of a new zooid. The development of a new brain and pharyngeal plexus always starts in connection to the parental nerve cords. Significantly different developmental patterns are observed for the IR to 5-HT and RF-amide, whereas IR to SCPB has the same localization as that to RF-amide, but appears both weaker and later during the development. Influences of the immunoreactive substances on the asexual reproduction and the feeding behaviour are discussed.

Rat cortical explants were cultured in the presence or absence of the mitotic inhibitor cytosine arabinoside to determine whether or not it affects the composition of the nonneuronal cell population within the outgrowth zone. Ultrastructural morphometric analysis of the incidence of fibroblasts, fibrous astrocytes, and protoplasmic astrocytes-epithelial cells at 18 days in vitro, revealed statistically significant decreases in the incidence of fibroblasts and fibrous astrocytes in the explants treated with the inhibitor compared with control explants. Coupled with earlier findings of enhanced neurite outgrowth and decreased nonneuronal cell proliferation that follows such treatment, it appears that cytosine arabinoside may potentiate neurite outgrowth by altering the composition, as well as the number, of nonneuronal cells in the outgrowth zone. These data indicate that fibroblasts and fibrous astrocytes may limit the regenerative response of severed axons in the mammalian central nervous system.

In many cases, in a fixed neuronal circuit, the complete morphological and biochemical development of a neuron depends on the impulses it receives from the neuron before. The possible involvement of this phenomenon to learning is studied in this work.

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.

Postnatal development of catecholaminergic and gamma-aminobutyric acid (GABA)-ergic neurons in the periglomerular region of the rat main olfactory bulb was studied immunohistochemically using antisera against tyrosine hydroxylase (TH), glutamic acid decarboxylase and GABA. TH-like immunoreactive neurons almost always contained GABA-like immunoreactivity in the first postnatal week, but about 10% of them did not contain GABA-like immunoreactivity in older animals.

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4-5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1-2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor (NGF)-untreated (naive) and neurite-bearing NGF-treated ("primed") PC12 rat pheochromocytoma cells were used as model system to study the role of phospholipid methylation in the NGF mechanism of action. The neurite-bearing cultures were deprived of NGF for 3 h before experimentation. Under both experimental conditions, the cells were labelled with [methyl-3H]methionine and then challenged with NGF for time periods ranging from 5 s to 30 min. Methylated phospholipids were extracted and then resolved and identified by TLC as phosphatidyl mono-, di-, and trimethyl ethanolamine. Quantification of the amount of radioactivity incorporated into each of the phospholipids indicated that NGF does not significantly alter phospholipid methylation either in naive or in neurite-bearing cells. Furthermore, using a methyltransferase inhibitor, it was found that neurite outgrowth still occurs when phospholipid methylation is almost completely blocked. These results indicate that phospholipid methylation does not play a primary role in the mechanism of action of NGF.

Neural and non-neural cellular processes have been studied in organotypic cultures of spinal cord and olfactory bulb by means of the freeze-fracturing technique. Identification of specific cellular elements in replicas has been achieved by comparison with thin-sectioned material in which differences in shape and contents are evident. Freeze-fracturing reveals that neural growth cones may be distinguished from glial pseudopodia by the low number of intramembranous particles within their plasma membrane; the counts of particles within the growth cone membrane average 85/microm(2) (for the inner leaflet) as opposed to hundreds per square micrometer in glial pseudopodia. Whereas the intramembranous particle number in glial pseudopodia is only slightly lower than in their perikaryal plasmalemma, the number of particles in outgrowing axons increases about eightfold from the periphery towards the perikaryon. Furthermore, with prolonged time of growth in culture, the particle density in the young nerve fibers increases by about the same factor. The same phenomenon, i.e. a low intramembranous particle level at earlier stages and an increase in numbers as the nerve fiber matures, is observed in fetal nerve tissue in vivo. These findings suggest that the plasmalemma of the outgrowing nerve, and especially of the growth cone, is immature and that maturation is accompanied by the insertion of intramembranous particles. Furthermore, these data indicate that the chemistry of the growth cone membrane is distinct from that of the neuron soma which may be significant for the mechanisms of guidance and recognition in the growing nerve tip.

Colchicine, injected into the amniotic cavity of 3 to 10 day-old chick embryos, has the following effects on the developing retina: 1. Cells in arrested metaphase accumulate in the ventricular portion of the matrix region. If colchicine is applied at the end of the first week of incubation, the kinetic migration of the cell nuclei is inhibited. 2. The cells lose their typical slender, bipolar shape to become ovoid to spherical. These morphological changes are more obvious when colchicine acts during the first week of incubation. 3. The formation of temporary gap junctions between the matrix cells of the retina occurs during the first week of development. When colchicine is administered around the 3rd day of incubation, the assembly of intramembranous particles (IMPs) during gap junctions formation is incomplete. These results suggest that an intact microtubular system within the submembranous cytoskeleton is essential for the assembly of these intercellular contacts.

Nucleus laminaris (NL) is a third-order auditory nucleus in the avian brain stem which receives spatially segregated binaural inputs from the second-order nuclei magnocellularis. The examination of the development of dendritic structure in NL revealed a number of events: In the initial period of dendritic growth (E 8--9) there is no gradient of dendritic morphology or apparent size. Starting about E 9--10, there is a spatiotemporal gradient of proliferation of numerous fine dendritic processes, from rostromedial to caudolateral, corresponding with the morphological lamination of NL, and possibly with the onset of cell death. This is followed by a spatiotemporal gradient of the elimination of the overproliferated processes, from rostromedial to caudolateral possibly coinciding with the cessation of cell death. A result of the spatiotemporal gradients of dendritic process proliferation and elimination is a spatial gradient in the morphology (extension, branching) of the remaining "mature" dendrites. At E 15 there is only a slight spatial gradient of total dendritic size across NL; this gradient is larger at E 19, and by P 25 there is a 13-fold change in dendritic size from rostromedial to caudolateral. Regression analyses suggest the size gradient begins to form when NL activity becomes driven by cochlear activity, at about E 14. The progressive formation of the size gradient is largely the result of two factors: the growth of dendritic trees, and the loss or primary dendrites. The growth rate of the dendritic trees of NL cells was found to be very highly correlated with the intensities of the sound frequencies to which the cells respond. From E 15 to P 25 there is a 50% loss of the "mature" primary dendrites of NL neurons. The separate dorsal and ventral dendritic size gradients seen at E 15 realign to coincide at E 19, and the moderate correlation of dorsal and ventral dendritic sizes seen at E 15 and E 19 is significantly increased at P 25, indicating a developmental process of sharpening in the relationship of the dorsal and ventral dendritic organizations in the nucleus. The data suggest that a key element in the regulation of dendritic size and structure in n. laminaris may be the activity of the afferents to the cells.

The early development of descending pathways from the brain stem to the spinal cord has been studied in Xenopus laevis tadpoles. The relatively protracted development of this permanently aquatic amphibian as well as its transparency during development make this animal particularly attractive for experimental studies. Between the 5th and 10th myotome the spinal cord was crushed with a thin needle and dry horseradish peroxidase (HRP) crystals were applied. After a survival time of one day the tadpoles were fixed and the brain and spinal cord were stained as a whole according to a modification of the heavy metal intensification of the DAB-reaction, cleared in cedarwood oil and examined as wholemounts. At stage 28 (the neural tube has just closed) the first brain stem neurons projecting to the spinal cord were found in what appear to be the nucleus reticularis inferior and -medius. At this stage of development the first, uncoordinated swimming movements can be observed. At stage 30/31 (the tailbud is visible) both Mauthner cells project to the spinal cord as well as the interstitial nucleus of the fasciculus longitudinalis medialis situated in the mesencephalon. Towards stage 35/36 (the tail is now clearly visible), a more extensive reticulospinal innervation of the spinal cord appears, now including cells of the nucleus reticularis superior. At this stage also the first vestibulospinal and raphespinal projections were found. At stage 43/44 (the tadpoles have now a well-developed tail) the pattern of reticulospinal projections appears to be completed with the presence of labeled neurons in the nucleus reticularis isthmi. From stage 43/44 on, the number of HRP-positive cells is steadily increasing. At stage 47/48, when the hindlimb buds appear, the descending projections to the spinal cord are comparable with the adult situation except for the absence of a rubrospinal and a hypothalamospinal projection. The observations demonstrate that already very early in development reticulospinal fibers and, somewhat later, Mauthner cell axons and vestibulospinal fibers innervate the spinal cord. Furthermore, a caudorostral gradient appears to exist with regard to the development of descending projections to the spinal cord. However, the interstitial nucleus of the fasciculus longitudinalis medialis forms an exception to this rule.

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.

The cerebellum presents the best site in the central nervous system for defining fundamental problems concerning the origin and differentiation of neurones, and their growth and development. The many recent experimental investigations of these problems are reviewed, and hypotheses based upon them are developed in relation to neurogenesis, morphogenesis, and synaptogenesis.

The effects of four concentrations of colchicine (2.5 x 10(-7), x 10(-5), x 10(-3), and x 10(-2)M) on the cell cycle of grasshopper neuroblasts have been determined by direct observations on living cells. The lowest concentration, 2.5 x 10(-7)M, does not completely disorganize the spindle but does retard its action. The three higher concentrations disorganize the spindle, so that all cells reaching metaphase are blocked in a c-mitotic condition throughout the period of observations (308 min at 38 degrees C, the minimum duration of the cell cycle in untreated neuroblasts). Continuous treatment with all concentrations reduces the rate at which neuroblasts enter metaphase, the extent of the reduction being a function of increasing concentration and time of exposure. After a short exposure to 2.5 x 10(-5)M colchicine, the neuroblasts recover from the inhibiting effects on progression through the cycle to metaphase, but they show no recovery from the inhibiting effects on spindle formation for more than 3 hr. Apparent stimulation of progression rate occurs early in exposure to all concentrations and during recovery from a short exposure to 2.5 x 10(-5)M. Morphological alterations in the chromatin of telophase, interphase, and prophase cells are induced by the higher concentrations of colchicine. The data indicate that caution should be exercised in the use of colchicine for determining cell cycle duration and/or the effects of physical and chemical agents on the cycle.