Vasoactive intestinal peptide (VIP) increases neuronal survival in dissociated spinal cord cultures during a critical period of development. In the present study, two mechanisms contributing to this action of VIP have been observed: 1) VIP was shown to be a secretagogue for neuron survival-promoting activity; and 2) VIP was found to be an astroglial mitogen. A high molecular weight substance (greater than 30 kDa), which increased neuronal survival in tetrodotoxin (TTX)-treated spinal cord cultures, was detected in the medium from nonneuronal cells incubated for 1 hr with 0.1 nM VIP. In addition, 3H-thymidine autoradiography and glial fibrillary acid protein (GFAP) immunocytochemistry were used to show that a 5 day treatment with (VIP) increased astroglial mitosis. This effect was specific for astroglia, as silver grain-positive cells not exhibiting GFAP immunoreactivity did not increase in number after VIP treatment. The dual action of VIP may regulate glial-derived trophic substances that are important for neuronal survival during the course of development.
The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K+-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K+ depolarization, in the presence of Ca2+, triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A–blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca2+ is required for synaptic vesicle membrane retrieval.
Neurons in dissociated cell culture provide a favorable system for the quantitative analysis of structural changes and the examination of structure-function relationships during development. Fragment C of tetanus toxin was used to label neurons in murine spinal cord cell cultures and dendrite outgrowth was monitored by a number of measures. The dissociated neurons increased in morphologic complexity from approximate spheres to highly branched structures during the first week in culture. Much of the structural complexity of the dendrite arbor, as quantified by fractal dimension, was established within 48 hr after plating, i.e., prior to the development of interneuronal contacts. During the first few days in culture, dendrite branching complexity increased more rapidly than dendrite size, whereas after 4 days, fractal dimension remained relatively constant while dendrites continued to grow. Fractal analysis has provided data which suggest that the early development of dendrite branching complexity is determined intrinsically. Fractal dimension, as an effective index of morphologic complexity, should be a useful tool for the further study of extrinsic signals which might modify the generation or stabilization of dendrite form.
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