Neurite outgrowth and selection of new electrical connections by adult Helisoma neurons

Abstract

Predictable connectivity changes can be evoked in an adult molluscan ganglion normally characterized by stereotyped interconnections. Growing neurons of Helisoma snails were studied using intracellular recording and intracellular staining with lucifer yellow, neuritic outgrowth being evoked by isolating buccal ganglia and maintaining them cultured in vivo (i.e., in host snails). New electrical connections were routinely established between neuron 5 and a specific population of other neurons. One of these connections (between the bilaterally symmetric pair of neurons 5) was stable, being present in all preparations (n = 64) cultured for 4-90 days. All the other new connections were transient, the strength of such interactions peaking after 1 day of culture and decreasing to undetectable levels thereafter. The new 5-5 connection has characteristics of a direct electrotonic synapse, its physiological properties being comparable to those of an extant direct electrical connection. Morphological studies demonstrate that newly formed central neurites from neuron 5 rapidly traverse the commissure between the paired buccal ganglia. The degree of central sprouting is dependent on the location at which the neurons 5 are axotomized. In ganglia in which these neurons were axotomized distally, the generation of central neurites is sparse and 5-5 coupling is weak (mean coupling coefficient, 0.04 .+-. 0.01). When the axons of both neurons are removed by axotomy close to the soma, prolific central sprouting is evoked and 5-5 coupling is significantly stronger (mean coupling coefficient, 0.17 .+-. 0.05; P < 0.01). The fidelity and apparent inevitability of the 5-5 connection was further demonstrated by crushing and ligating the buccal commissure. Preparations with crushed commissures exhibited strong coupling (mean coupling coefficient, 0.19) and prolific neuritic growth across the commissure. Even in 3 of 4 preparations with ligated commissures, weak 5-5 coupling (mean coupling coefficient, 0.03) was established. Lucifer yellow stain of neurons in such ganglia revealed neurites exiting 1 ganglion and entering the contralateral neuropil after traversing the ligature and exterior ganglion surface. The cellular basis of the new, stable and transient connections was compared to an extant connection by examining their susceptibility to uncoupling by low-pH saline (pH 5.3 .+-. 0.1). Both the 5-5 and 5-4 connections were uncoupled by this treatment to the same degree as the extant connection, suggesting a common physiological basis. Neuron 5 exhibits a high degree of specificity in establishing both transient and stable electrical connections. The new connections are physiologically indistinguishable from an exant connection and are due to newly grown central neurites that exhibit a remarkable ability to locate targets even in the presence of disrupted or foreign terrain.