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Neurite outgrowth from explanted Xenopus retina: an effect of prior optic nerve section.

Experimental conditions are described for the in vitro study of explanted eyes from embryos of Xenopus laevis and of retinas from older larvae. When eyes are explanted from embryos of stages 25-34, a rapid outgrowth of fibroblast-like and pigment cells is observed, upon which a neuritic outgrowth is eventually superimposed. Outgrowth from the retina of later stage tadpoles (50-54) is not seen until about a week following explantation and resembles the mixed cell outgrowth observed in whole eye explants from early stage embryos. If, however, the optic nerve of an older tadpole is cut 7 days prior to explantation, a purely neuritic outgrowth is seen from the previously denervated retina within 1-3 days.

Age Factors

Developmentally regulated induction of neurite outgrowth from immature chick sensory neurons (DRG) by homogenates of avian or mammalian heart, liver and brain.

Neurite outgrowth is elicited from whole explants or dissociated neurons of 8--10-day-old chick embryo sensory, dorsal root ganglia when cultured in the presence of a high speed supernatant fraction (105,000 g) from homogenates of chick or rat heart, liver or brain. The neurite promoting activity is not identical to mouse nerve growth factor (NGF) and is non-dialyzable. Expression of this neurogenic factor would appear to be developmentally regulated as its activity is barely detectable in organs from 6--9-day-old embryos but specific activity rises dramatically in homogenates of organs from embryos of greater than 11 days incubation. Greatest activity is found in chick heart and rat brain with only trace levels in lung or kidney and none in spinal cord.

Animals

Neurotransmitter modulation, phosphodiesterase inhibitor effects, and cyclic AMP correlates of afterdischarge in peptidergic neurites.

The neuroendocrine bag cells in the abdominal ganglion of Aplysia generate a long-lasting synchronous afterdischarge upon brief stimulation of an afferent pathway. After this afterdischarge the cells become refractory to further synaptic stimulation. We find that synchrony, afterdischarge, and prolonged refractoriness are properties that can be expressed in the isolated asomatic neurites of the bag cells. We have distinguished two independent types of refractoriness. The first (type I) is seen as a failure of action potentials generated in the tips of bag cell neurites to invade cell somata. The second form of refractoriness (type II) controls the duration of afterdischarge such that stimuli after the first afterdischarge produce only very short afterdischarges or fail to elicit an afterdischarge. Type II refractoriness is sensitive to serotonin and certain of its analogues, and to dopamine and the methylxanthine phosphodiesterase inhibitors. Extracellularly applied serotonin suppresses an ongoing afterdischarge while dopamine and the phosphodiesterase inhibitors, when applied at the end of the first afterdischarge, generate a subsequent afterdischarge of long duration without further electrical stimulation. None of these compounds influenced the degree of type I refractoriness. We have shown that both serotonin and dopamine stimulate the formation of cyclic AMP in the bag cell clusters and in the pleurovisceral connectives and that the occurrence of an afterdischarge is associated with a specific increase in total cyclic AMP in bag cell bodies. Moreover, afterdischarges can be generated in unstimulated preparations by extracellular application of the cyclic AMP analogues, 8-benzylthio-cyclic AMP or 8-methylthio-cyclic AMP. Our data suggest that serotonin and/or dopamine may control bag cell activity and that activation of adenylate cyclase is linked to bag cell afterdischarge.

Action Potentials

Clockwise growth of neurites from retinal explants.

When retinal explants from goldfish are grown on a polycation substratum, a marked tendency for directionality of neurite outgrowth is observed. While the direct relevance to nerve growth in vivo is not known, the phenomenon is interpreted as reflecting an inherent helicity of the neurites.

Animals

Carboxymethyl cellulose stimulation of neurite outgrowth of neuroblastoma cells in culture.

The addition of 1% (w/v) carboxymethyl cellulose to the culture medium induces the formation of neurites of clone N18 neuroblastoma cells even in the presence of normal (5-10%) serum supplement concentrations, which rivals that previously observed by growth in low 0.1% serum. Heavy metal ions associated with the carboxymethyl cellulose were responsible for small increases in the sizes of cell bodies during treatment. Pretreatment of the PC12 pheochromocytoma line of neuroblasts with carboxymethyl cellulose for 1 day prior to their stimulation with nerve growth factor resulted in an acceleration in the rate, but not extent, of neurite outgrowth.

Animals

Neurite outgrowth elicited by embryonic chick heart: partial purification of the active factor.

Chick heart explants stimulate a dense outgrowth of neurites in co-cultured spinal, sympathetic and ciliary ganglia. A factor sharing these properties was partially purified by gel filtration of extract prepared from lyophilized 18-day embryonic hearts. The active factor has an apparent molecular weight of roughly 40,000 and is evidently distinct from nerve growth factor (NGF) by stimulating the parasympathetic ciliary neurons and by lack of cross-reactivity with antibodies to NGF.

Animals

Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture.

Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasmalemma near the close contact site; their apparent contribution of a region of postsynaptic membrane with undercoating was considered to be the first definitive sign of synapse formation. Tracer work with peroxidase and ferritin confirmed that the traffic of coated vesicles within the neuronal soma is largely from Golgi region to somal surface. Subsequent to the appearance of postsynaptic density, the form and content of the growth cone was altered by the loss of filopodia and the appearance of synaptic vesicles which gradually became clustered opposite the postsynaptic density. As the synapse matured, synaptic vesicles increased in number, cleft width and content increased, presynaptic density appeared, branched membranous reticulum became greatly diminished, and most lysosomal structures disappeared. Coated vesicles continued to be associated with the postsynaptic membrane at all stages of maturation. The incorporation of Golgi-derived vesicles into discrete regions of the cell membrane could provide the mechanism for confining specific characteristics of the neuronal membrane to the synaptic region.

Animals

Differentiating limb tissue affects neurite growth in spinal cord cultures.

Limb bud mesenchyme enhances and directs the growth of tadpole spinal cord nerve fibers in tissue culture. This effect on elongating neurites may involve alterations in nerve-substratum interactions by the presence of undifferentiated target tissues. The relationship between nerve fibers and their potential innervation sites can explain directed nerve growth to the developing limb.

Age Factors

Neuritic growth cone and ependymal gap junctions in the feline subfornical organ during early development.

Intercellular contacts in the subfornical organ (SFO) of kittens 3, 16, and 29 days old were studied in thin sections and by the freeze-etch method. Gap junctions appeared between growing nerve processes and target cells. The junctions were interspersed between immature synapses lacking mitochondria as well as full pre- and postsynaptic membrane specializations. Gap junctions were seen on filopodia as well as on more mature processes. The morphology of these junctions was typical of those described earlier but they were of small size (0.2-0.3 micron). Gap junctions of peculiar form were also seen between ependymal elements in the SFO at 16 days. These were of large size (0.5-0.8 micron) and were often of segmented character. This segmentation consisted of bands 3-4 particles in width with a center-to-center spacing of 90 nm with particle free corridors between corresponding to the width of about two rows of particles. The margin of the group might be circumscribed by a row of particles. Although gap junctions of large size were seen between ependymal cells in thin section, features corresponding to the particle free corridors have not been observed to date.

Aging