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The survival and outgrowth of neurons in culture has usually required conditioning factors. We now report that crustacean neurons, taken from the peptidergic neurosecretory system of the eyestalk of crabs (Cardisoma carnifex) and lobsters (Panulirus marginatus), show immediate outgrowth, sustained for a week or more, in defined medium as simple as physiological saline with glucose and glutamine.The neurons show peptide hormone immunoreactivity that is prominent at growth cones, exhibit differences in form correlated with their immunoreactivity, release peptides to the medium, and have voltage-dependent currents, including a well-sustained Ca current. Cd blocks secretion, growth, and the Ca current. Peptidergic secretory neurons may be able to utilize existing membrane from their store of granules and already active synthetic, transport, and secretory mechanisms for immediate outgrowth.The mechanisms governing the outgrowth of neuronal processes and the mature form of neurons remain largely unknown. Isolation of neurons in culture may provide the possibility of undertaking experimental manipulations under controlled conditions. However, although outgrowth in lowdensity cultures has now been demonstrated for vertebrate (1-5), annelid (6, 7), and molluscan (8-11) preparations, the need for addition of undefined factors has frustrated rigorous studies of the control of outgrowth and form. We report that crustacean neuroendocrine cells show immediate, vigorous outgrowth on a variety of substrates in defined medium as simple as physiological saline and glucose. We propose that this capability for immediate outgrowth is made possible by the utilization of already active synthetic, transport, and secretory mechanisms for growth. Different forms of outgrowth are consistently obtained from the heterogeneous group of neurons cultured, and reactivity with antisera raised against peptide hormones suggests correlations with the biosynthetic capabilities of the neurons. These neurons in culture, with their differences in form, thus provide a promising defined starting point for testing hypotheses about mechanisms governing the control of growth and form in regenerating neurons. We are unaware of any report of crustacean neurons in culture (12). Abstracts describing some of our work have appeared (13)(14)(15).The neurons cultured in these studies form the major neuroendocrine system of crustaceans, the X-organ-sinus gland system of the eyestalk (ref. 16 and, for review, see ref. 17). For the tropical land crab Cardisoma carnifex used for most of the work to be discussed, there have been studies of the morphology of nerve terminals (18), electrophysiological characterization of somata, axons, and terminals (19)(20)(21)(22), studies of secretory capabilities (22,23), and characterization of the hormonal peptides present (24) and their biosynthesis (25). These provide a background against which the cultured cells can be evaluated.The X-organ of crabs is a discrete cluster of about 200 iridescent-white neuronal somata (26)...

1. The X-organ sinus gland is a major peptidergic neurosecretory system in Crustacea, analogous to the vertebrate hypothalamoneurohypophyseal system. Neuronal somata isolated from the crab (Cardisoma carnifex) X-organ and maintained in primary culture in unconditioned, fully defined medium show immediate regenerative outgrowth. Outgrowth occurring as broad lamellipodia ("veiled") distinguishes neurons consistently showing crustacean hyperglycemic hormone immunoreactivity. Neurons that are immunoreactive against molt-inhibiting hormone and red pigment concentrating hormone antisera give rise to branched neurites ("branched"). 2. The whole-cell variation of the patch-clamp technique was used to study the electrophysiology of these two cell types 24-48 h after plating. Under current clamp, only veiled neurons fired overshooting action potentials either spontaneously or in response to depolarization. 3. Under voltage clamp, net current was predominantly outward. When solutions that suppressed outward current were used, only veiled neurons showed significant inward current. These included a tetrodotoxin (TTX)-sensitive Na current and a slow (time to peak 6-10 ms at 0 mV) Cd-sensitive Ca current (ICa) that was activated at potentials less than -30 mV, was maximal at 0 to +20 mV, and did not reverse at potentials up to +60 mV. 4. In TTX, the form of the Ca current I(V) curve was unchanged by changes of holding potential between -40 and -80 mV, and 75-100% of ICa was available from -40 mV. 5. ICa inactivated slowly and incompletely. Analysis with two-pulse regimes suggested that both inactivation and facilitation mechanisms were present. 6. Outward current was examined in the presence and absence of 0.5 mM Cd2+ (1 microM TTX was always present in the external medium). Cd2+ ions slightly reduced the peak outward current, usually by less than 10% (Vc = -10 to +20 mV; Vh = -80 mV). All additional observations were in the presence of TTX and Cd2+. 7. Both cell types expressed a 4-aminopyridine (4-AP)-sensitive transient current, analogous to IA, and a slower-rising (minimum time to peak 20 ms), sustained current that was partially sensitive to tetraethylammonium, analogous to IK. 8. The mean Vh at which IA was half inactivated was -46 mV, and the mean time constant for removal of inactivation was 46 ms.(ABSTRACT TRUNCATED AT 400 WORDS)

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Encounters by growth cones or neurites of motor neurons with target muscle cells evoke prolonged elevations in the concentrations of neuronal cytosolic free calcium ([Ca²⁺]_c). These calcium elevations are initiated at the point of contact and spread throughout the neuron over a period of tens of minutes. In this study, we addressed how target muscle cells initiate this unique presynaptic response. Primary questions regarding the nature of the muscle signal are whether it is diffusible and whether it must first be induced by a growth cone as part of reciprocal interaction. We addressed whether the signal was strictly target-contact dependent by fixing C2 mouse myotubes with formaldehyde, rinsing extensively and then allowing processes of chick ciliary ganglion neurons to interact with them. We observed frequent sustained elevations in [Ca²⁺]_c in ciliary ganglion processes contacting the fixed myotubes. As a control, ciliary neurons were allowed to interact with fixed myotubes of the S27 variant line. S27 cells were isolated from the parent C2 line on the basis of a defect in glycosaminoglycan biosynthesis and previously shown to be defective in supporting synaptic vesicle localization in contacting neurites. Few elevations in [Ca²⁺]_c were detected in encounters between ciliary processes and fixed S27 cells.In addition, neuron-neuron encounters never elicited prolonged increases in [Ca²⁺]_c. These observations demonstrate contact dependence in the neuronal response and rule out reciprocal cellular interactions, diffusible factors or electrical activity in the muscle. The defect in carbohydrate biosynthesis in S27 cells further suggests that cell surface carbohydrates are essential to the signal on the myotube surface that triggers the presynaptic elevation in [Ca²⁺]_c. We conclude that growth cone contact with preexisting cell surface structures on target muscle cells induces changes in presynaptic [Ca²⁺]_c that are associated with retrograde signaling, and that proper carbohydrate biosynthesis is required for this signal.