Selective incorporation of 2-deoxyglucose into the developing spinal cord in vitro in response to target tissue and substratum.

Differential uptake of [3H]deoxyglucose by larval frog spinal cord explants in vitro has demonstrated that metabolic activity is localized to the lateral motor columns in response to co-cultured target tissue or a neurite-promoting substratum. Autoradiographic data suggest that chemokinesis may be an integral component of motor neuron growth activation.

Stimulation of choline acetyltransferase in spinal cord explants by limb mesenchyme.

Choline acetyltransferase (ChAT) activity in developing spinal cord explants in vitro is shown to be dependent on the presence of co-cultured immature limb tissue. Frog tadpole spinal cord explants grown on collagen or polylysine expressed stage-appropriate levels of ChAT activity only when in the presence of the limb mesenchyme target. Neither skeletal muscle nor polylysine, both of which enhance neurite growth accompanied by increases in cord protein, were capable of maintaining the level of ChAT activity characteristic of these spinal cords in vivo. The results demonstrate that developmentally significant levels of ChAT can be maintained in vitro under appropriate conditions that may act in part through the maintenance of cholinergic motor neurons.

Development of presynaptic specializations induced by basic polypeptide-coated latex beads in spinal cord cultures.

The development of the neuromuscular synapse is initiated by an interaction between the motoneuron processes and the skeletal muscle. Previously we showed that basic polypeptide-coated latex beads can mimic the nerve in effecting a postsynaptic differentiation (Peng, H.B. and Cheng, P.-C.: J. Neurosci., 2:1760-1774, 1982). In this study, we examined whether these beads can also induce a presynaptic differentiation along the nerve processes. Explant cultures were prepared from the spinal cords of Xenopus larvae and polyornithine-coated latex beads (0.45-4.5 micron in diameter) were applied. After 1-2 days, the cultures were processed for light and electron microscopy. For light microscopy, the cells were permeabilized and labeled with a monoclonal antibody against a 65-KD antigen on synaptic vesicles. Indirect immunofluorescence revealed that this antigen was concentrated at 60% of the bead-neurite contacts, therefore suggesting the clustering of synaptic versicles at these sites. This phenomenon was not observed at the contacts between neurites and negatively charged (polycarboxylate) beads. However, a concentration of this antigen was also observed at the varicosities along the neurites cultured on polylysine substrate. Thin-section electron microscopy showed the following features: (1) The neurites formed terminal-like varicosities on the bead. (2) Within these varicosities, clusters of 50-60-nm clear vesicles were prominent at the bead-neurite contact. (3) Large (80-100 nm) dense-cored vesicles were also present in the varicosity, but they did not form clusters. (4) Basement membrane did not form at the bead-neurite interface, in contrast to its appearance at the bead-muscle contact from our previous study.(ABSTRACT TRUNCATED AT 250 WORDS)

Target tissue control of nerve fiber growth rate and periodicity in vitro.

Spinal cord and dorsal root ganglion nerve fibers of tadpoles elongate in vitro at an increased rate in the presence of target tissue or target-conditioned medium. These neurites extend in a cyclic fashion with the period between the occurrence of maximal growth rates being under target influence. Rate parameters may be an important feature in the understanding of directed nerve growth during development and regeneration.

Neurotropic influence of mesenchymal limb target tissue on spinal cord neurite growth in vitro.

Peripheral nerve fiber outgrowth from developing spinal cord is proposed to be under the influence of the limb bud target which, at the time of nerve fiber invasion, is in an essentially premuscular, mesenchymal condition. Thus, the true target for elongating spinal nerve fibers in early development is mesenchyme rather than differentiated skeletal muscle. Spinal cord explants derived from stage V larval Rana pipiens were cultured in a defined medium in the presence or absence of mesenchymal limb tissue or limb-conditioned medium (LCM). Analysis of quantified neuritic outgrowth under these conditions demonstrated a dependency on the target tissue for enhanced nerve fiber density and oriented growth. The characteristics of neuritic growth in the presence of limb mesenchyme or LCM changed from the relatively sparse and straight outgrowth of control cords to dense, wavy arborizations. Areas of the cord explants nearest the limb tissue exhibited the greatest increases in nerve fiber density and morphologic complexity. Additionally, an inverse relationship existed between growth enhancement and the cord-to-target distance. Regulation of directed nerve growth in vitro is suggested to result from a diffusible, target-originated growth factor that binds to the attachment substratum as a concentration gradient guidance pathway with implications for mechanisms of in vivo nerve growth.