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J B Tuttle

Publications and source records attributed to J B Tuttle.

64 records · Page 4Linked to original sources
PubMed1988

Cultured smooth muscle targets lack survival activity for ciliary ganglion neurons.

Cultured neurons require specific trophic agents in order to survive. This dependence is thought to resemble the neuron-target interdependence that develops in vivo during synaptogenesis and neuronal cell death. The notion that neurons in general derive trophic support from their synaptic targets is based primarily on studies of peripheral neurons and motor neurons. To assess the general applicability of this nerve-target relationship, we tested the ability of vascular smooth muscle (VSM) to support dissociated neurons from the chick ciliary ganglion. The ciliary ganglion contains 2 distinct neuronal populations, one of which innervates striated muscle, the other VSM. Striated muscle cocultures are known to support all of the neurons in the ganglion for extended periods. Dissociated neurons were therefore cocultured in microwells containing VSM derived from the rat or chick aorta and from the choroid coat of the chick eye. Surviving neurons were counted after 1, 2, 5, and 7 d. Striated muscle is able to support full neuronal survival in the same assay. However, in no case was VSM capable of contributing to neuronal survival in vitro. The neurons in the VSM cocultures were able to form neurites and make contacts with their putative targets, as confirmed by scanning electron and light microscopy. The presence of viable and differentiated smooth muscle cells was demonstrated in the cultures by transmission electron microscopy and analysis of smooth muscle alpha-actin. The failure of VSM and even the choroid target tissue to support the survival of their innervating neurons suggests that novel mechanisms may operate to provide trophic support for neurons innervating VSM targets.

Animals↗
PubMed1987

[3H]acetylcholine synthesis in cultured ciliary ganglion neurons: effects of myotube membranes.

Avian ciliary ganglion neurons in cell culture were examined for the capacity to synthesize acetylcholine (ACh) from the exogenously supplied precursor, choline. Relevant kinetic parameters of the ACh synthetic system in cultured neurons were found to be virtually the same as those of the ganglionic terminals in the intact iris. Neurons were cultured in the presence of and allowed to innervate pectoral muscle; this results in an capacity for ACh synthesis. In particular, the ability to increase ACh synthesis upon demand after stimulation is affected by interaction with the target. This effect is shown to be an acceleration of the maturation of the cultured neurons. Lysed and washed membrane remnants of the muscle target were able to duplicate, in part, this effect of live target tissue on neuronal transmitter metabolism. Culture medium conditioned by muscle, and by the membrane remnants of muscle, was without significant effect. Thus, substances secreted into the medium do not play a major role in this interaction. Neurons cultured with either muscle or muscle membrane remnants formed large, elongate structures on the target membrane surface. These were not seen in the absence of the target at the times examined. This morphological difference in terminal-like structures may parallel the developmental increases in size and vesicular content of ciliary ganglion nerve terminals in the chick iris, and may relate to the increased ACh synthetic activity. The results suggest that direct contact with an appropriate target membrane has a profound, retrograde influence upon neuronal metabolic and morphological maturation.

Acetylcholine↗
PubMed1985

Regulation of cholinergic development by target contact.

Avian ciliary ganglion neurons in cell culture have been used as a model neuronal system to examine the developmental role of direct contact with an appropriate target tissue. Live myotubes, which are immediately innervated, and their membrane remnants, on which neurons form terminal structures, were both found to stimulate or accelerate the acquisition or retention of important parameters of neuronal function. Contact with the target membrane supports survival, aids retention of active nicotinic receptors to acetylcholine, and accelerates the acquisition of adult transmitter synthetic capacity. These results emphasize the multifaceted nature of neurodevelopment, with soluble protein factors, membrane-bound elements, and other functional events all acting in concert in the embryonic nervous system.

Acetylcholine↗
PubMed1983

Interaction with membrane remnants of target myotubes maintains transmitter sensitivity of cultured neurons.

Parasympathetic neurons, when cultured alone, lose sensitivity to acetylcholine, but if striated muscle is included in the culture, neuronal chemosensitivity is maintained. The membrane remnants of myotubes ruptured by osmotic shock also supported the responsiveness of the cultured neurons to transmitter, whereas muscle-conditioned medium or membrane remnants of nonmuscle embryonic skin cells did not support this responsiveness. The regulation of chemosensitivity by contact of neurons with the target cell membrane may be important in the formation and maintenance of neuronal circuitry.

Acetylcholine↗
PubMed1983

Target influences on [3H]ACh synthesis and release by ciliary ganglion neurons in vitro.

The developmental influence of neuron-target interaction upon transmitter synthesis from labeled precursor and the capacity to release labeled transmitter were examined in dispersed cell cultures of embryonic ciliary ganglion neurons by comparing cultures of neurons plated alone and neurons plated upon pectoral myotubes. Of the total ACh synthesized from radiolabeled choline by neurons plated alone, more than half is via a Na+-dependent path, but a larger fraction of the synthesis is Na+ insensitive in culture than in mature neurons in vivo. In addition, at 1 week in culture the neurons lacking target failed to significantly increase ACh synthesis from the labeled choline in response to a previous high [K+]0 depolarization. Synthetic responsiveness to depolarization is a characteristic of mature nerve terminals in this preparation. One week after plating neurons onto myotube cultures, synthesis of ACh from the exogenous precursor is double that of sibling cultures lacking muscle, and prior depolarization with [K+]0 results in an increase in labeled product. Release from the labeled transmitter pool by the neurons with myotubes was also enhanced. [3H]ACh release elicited by depolarization via a Ca2+-dependent mechanism was more than fivefold higher in the cocultures. The influence of coculture with myotubes upon neuronal development is not duplicated by the neurons themselves despite formation of apparent interneuronal synapses (G. Crean, G. Pilar, J. Tuttle, and K. Vaca, 1982, J. Physiol. (London). 331, 87-104), by "fibroblasts" or medium conditioned over myotube cultures. Neurons under these conditions neither increase synthesis of [3H]ACh in response to a prior depolarization nor demonstrate enhanced basal [3H]ACh synthesis and release. Thus, coculture of embryonic ciliary ganglion neurons with a striated muscle target has a somewhat specific inductive effect, enhancing the capacity for neuronal [3H]ACh synthesis and release toward mature levels. This influence of a readily accessible target upon ciliary neuron cholinergic development in vitro may reflect a normal neuromuscular interaction occurring during embryogenesis.

Acetylcholine↗
PubMed1982

Enhanced chemosensitivity of chick parasympathetic neurones in co-culture with myotubes.

1. The influence of target interaction upon the electrophysiological properties of dissociated ciliary ganglion cells was investigated by testing the sensitivity of the neuronal somal membrane to ionophoretically applied acetylcholine (ACh). Variations in the percentage of cells responsive to the transmitter were measured with time in culture. 2. Twenty-four hours after plating, all cells respond to an ionophoretic pulse of ACh with a depolarization. However, 1 week after plating (between 7 and 14 days) most of the neurones are unresponsive, and highly responsive cells (greater than 100 mV peak depolarization/nC) are extremely rare. At even later times in culture, neurones sensitive to the transmitter are again more frequent. 3. When neurones are plated onto pre-formed pectoral myotubes, however, ACh sensitivity is maintained throughout a 3 week culture period. Neuromuscular junctions are formed by the neurones, and when sufficient neurones are present, all the muscle fibres tested show evidence of functional synaptic transmission. Chemosensitivity to ACh is not maintained by neurones in muscle-free microcultures are present on the same cover-slip. 4. Interneuronal synaptic contacts, defined by ultrastructural criteria, are formed in cultures of neurones alone, but evidence of widespread functional synaptic interaction between cells was not found at 7-14 days in culture. 5. It is concluded that the maintenance of ACh sensitivity of cultured ciliary ganglion cells is enhanced by the presence of muscle in co-culture. The interneuronal synaptic contacts observed are apparently not as potent a stimulus as co-culture with muscle for the full expression of the cholinergic phenotype under these culture conditions.

Acetylcholine↗
PubMed1980

Long-term survival and development of dissociated parasympathetic neurons in culture.

Ciliary ganglion neurons from chick embryo were grown in cell culture following enzymatic and mechanical dissociation. The culture conditions necessary for long-term (greater than 21 days) survival of the isolated neurons were investigated. Neurons could be cultured with and without non-neural cells by adjusting the culture substrate. Chick embryo extract was found to be essential in the growth medium, and the inclusion of horse serum had an additional beneficial effect. Also, non-neural cells increased the survival of these neurons in culture. Thus, there appear to be several factors involved in the survival of these neurons in culture. Assay of choline acetyltransferase activity revealed a 100-fold increase in activity over the first two weeks in vitro, and the developmental pattern of the enzyme activity was initially similar to that seen in vivo. The cultured neurons also retained many of the electrophysiological properties of ganglionic neurons in vivo. These included normal resting transmembrane potential, action potential amplitude and an afterpotential. The passive membrane properties of the cultured neurons (membrane resistance, capacitance and time constant) differed somewhat from those of neurons in ganglia. These results suggest that if proper culture conditions are provided, these parasympathetic neurons adapt well to cell culture and develop many of the properties of normal ciliary ganglion neurons in vivo.

Animals↗
PubMed1975

Ionic excitation of a clone of mouse neuroblastoma.

The electrical properties and the possible regulation of these properties were studied by means of intracellular microelectrode recordings in cells of mouse neuroblastoma clone N1E-115. This clone has high levels of tyrosine hydroxylase and regulates this enzyme. Cells treated for 24 h with 4 muM aminopterin followed by at least 5 days in culture developed rhythmic discharge of action potentials when superfused with phosphate-buffered saline containing less than 0.2 mM calcium or less than 0.2 mM calcium and zero potassium. This ionic excitation occurred in no cells at less than 5 days after treatment with aminopterin but at 5 days or more after treatment, 20% of cells responded to low calcium while 52% responded to low calcium and zero potassium. Concomitant with the development of a susceptibility to ionic excitation was an increase in the average resting membrane potential and morphologic maturation. This ionic excitation of cultured mouse neuroblastoma cells may be useful for studying biochemical events associated with repetitive discharge of action potentials.

Action Potentials↗