Modulation of sprouting in organ culture after axotomy of an identified molluscan neuron

Abstract

We examined a variety of factors that might modulate the initiation of neurite outgrowth in an attempt to identify means by which its initiation might be accelerated. We examined this initiation from an identified molluscan neuron, Helisoma trivolvis buccal neuron B5 after axotomy, and determined whether the site of injury, temperature, ion channel blockers, pH, the second messenger cAMP, and protein synthesis affect the initiation of neurite outgrowth. Neurite outgrowth was assayed from axotomized neurons by filling the neurons intracellularly with Lucifer Yellow and examining the percentage of axons that extended (sprouted) new process after 9 or 24 h in organ culture. About one-third (31%) of axotomized neurons sprouted from the site of injury after 9 h (n = 22), and 88% (n = 20) sprouted after 24 h in saline at 22 degrees-24 degrees C when the injury was located 800 microns from the soma. Elevating the temperature to 32 degrees C or moving the lesion site to 400 or 1500 microns from the soma did not significantly alter the incidence of sprouting. Blocking sodium channels with tetrodotoxin [TTX (2 x 10(-5) M)] did not significantly reduce the incidence of sprouting, whereas the sodium channel agonist, veratridine (10(-5) M) did. The calcium channel blocker lanthanum (10(-6)-10(-4) M), stimulated neurite outgrowth; however, the organic calcium channel blocker verapamil (10(-3)-10(-5) M), and the calcium ionophore A23187 (10(-5) M), had no effect on sprouting. Exposure of neurons to the potassium channel blocker tetraethylammonium [TEA (20 mM)], elevation of intracellular pH with NH4Cl (5 mM), or treatment with the adenylate cyclase activator forskolin (10(-5) M) reduced the incidence of sprouting, whereas dideoxy-forskolin (10(-5) M) had no effect. Inhibition of protein synthesis with anisomycin (2 x 10(-4) to 2 x 10(-6) M) did not significantly suppress sprouting 24 h after axotomy. Both D and L isomers of glutamate (300 microM) stimulated sprouting. The present results suggest that the initiation of sprouting is regulated locally at or near the site of injury, and that blocking specific ion channels may either inhibit or enhance the initiation of neurite outgrowth.

Aplysia hemolymph promotes neurite outgrowth and synaptogenesis of identifiedHelix neurons in cell culture

“…In other words, the cell retains its unipolar configuration. These findings support the hypothesis that the initiation of sprouting is regulated locally at or near the site of injury (Berdan and Easaw, 1992).…”

Voltage gated calcium channels in molluscs: classification, Ca2+ dependent inactivation, modulation and functional roles

“…HVA calcium currents have been recorded from growth cones of Helix neurons (Marom and Dagan, 1987) and of neuron B5 of Helisoma (Haydon and Man-Son-Hing, 1988). The calcium channel blocker La 3+ stimulates neurite outgrowth in B5 (Berdan and Easaw, 1992;Williams and Cohen, 1995). Berdan et al (1993) reported that chronic (4-6 days) depolarization by high K + slightly increases neurite outgrowth, but strongly reduces the amplitude of both LVA and HVA currents recorded in the somata, suggesting that the expression of LVA and HVA channels is suppressed.…”

Dopamine suppresses neuronal activity of Helisoma B5 neurons via a D2-like receptor, activating PLC and K channels

“…They have been shown to contribute to different phases of action potential repolarization (Sakakibara et al, 2005). The perfusion of a cocktail of 20 mM TEA and 5 mM 4AP, concentrations that have been suggested to be effective to block both K channels in Helisoma and Lymnaea (Berdan and Easaw, 1992;Staras et al, 2002), caused a decrease in firing frequency by 34.5 ± 2.7% (n = 7, P < 0.05) combined with an increase in action potential amplitude by 21.5 ± 0.3% (n = 7, P < 0.05) (Fig. 5D).…”