NEURITE OUTGROWTH AND SYNAPSE FORMATION BY LYMNAEA NEURONS: TOWARDS A CHARACTERIZATION OF MOLLUSCAN NEUROTROPHIC FACTORS

by

A.G.M. BULLOCHa, N.I. SYEDa and R.L. RIDGWAYb (aNeuroscienceResearch Group, Faculty of Medicine,Health SciencesCentre, The Universityof Calgary, Calgary,AB, Canada T2N 4N1; bDepartmentof Biology,Seattle Pacific University, Seattle,Washington, USA)

ABSTRACT

For the last several years we have endeavoured to characterize the endogenous neuro- trophic factors of the pulmonate snail, Lymnaeastagnalis. When plated in brain condi- tioned medium, motoneurons, interneurons and neurosecretory cells of Lymnaeaall exhibited neurite outgrowth. We showed earlier that the motoneurons and interneurons respond to a Nerve Growth Factor-like (NGF-like) component of conditioned medium. Neurosecretory cells, however, appear to be under the control of a factor derived from the endocrine dorsal body cells. Most recently, we were surprised to learn that motoneurons and interneurons also respond to another vertebrate neurotrophic factor, i.e., Ciliary Neurotrophic Factor (CNTF). The outgrowth in CNTF differs both in appearance and in functional outcome. Specifically, neurites in CNTF are thin, have small growth cones and elongate more quickly than their counterparts in NGF. Regard- ing functional consequences, both dopaminergic and peptidergic interneurons fail to form synapses on target neurons when cultured in CNTF, but do so in NGF, as expected from our previous studies in conditioned medium. These data shed new light on the functions of neurotrophic factors and provide the first evidence that outgrowth and synaptogenesis can be differentially regulated. KEY WORDS:mollusc, neurotrophic factor, culture, regeneration, sprout, synapse, plasticity.

INTRODUCTION

This essay summarizes our knowledge regarding the endogenous neu- rotrophic factors in the pulmonate nervous system. A number of groups have pursued an interest in the plasticity of the adult molluscan neurons for some years, and have studied a number of different aspects of plasticity. For example, various models have been developed from Helisoma and Lymnaea to study axonal regeneration and reformation of synapses in the nervous system, sprouting from intact regions of neu- rons, and neurite outgrowth and synapse formation between isolated neurons in vitro (for previous reviews see BULLOCH, 1985; BULLOCH & JONES, 1988; BULLOCH & RIDGWAY, 1989; RIDGWAY & BULLOCH, 1991; J BULLOCH & SYED, 1992; RIDGWAY et al., 1993). Here we summarize our 318 findings regarding the nature and actions of putative endogenous neurotrophic factors in Helisoma and Lymnaea. Neurotrophic factors are believed to play key roles in development, maintenance and regeneration of the nervous system. Pre-eminent in studies of the vertebrate nervous system is the molecule Nerve Growth Factor (NGF), a member of a small gene family now known as the neurotrophins (KORSCHING, 1993). This family includes Brain Derived Neurotrophic Factor (BDNF), Neurotrophin-3 and Neurotrophin-4/5 (NT-3 and NT-4/5). Another distinct class of neurotrophic factor is Ciliary Neurotrophic Factor (CNTF) (KORSCHING, 1993). The field of neurotrophic factors is becoming one of increasing complexity. Rela- tively few functions of these molecules, however, are known. In this context it is important to distinguish developing versus adult neurons, survival versus neurite outgrowth, and in vivo versus in vitro studies. Regarding development, NGF is clearly a survival factor for periph- eral sympathetic and neural crest-derived sensory neurons. Key to understanding this phenomenon are observations that NGF and its receptors arc synthesized in the target area and responsive sensory neurons at the time, but not before, growing axons reach and innervate their targets. Thus, target derived NGF is not likely to be responsible for axon outgrowth in sensory neurons, which may rather be influ- enced by as yet unidentified guidance factors (TES SIER- LAVIGNE& PLACZEK, 1991). In vitro studies of NGF induced outgrowth of PNS neurons may, therefore, not be of direct physiological relevance. Studies of the other neurotrophins indicate they can support overlap- ping, but distinct, populations of PNS and CNS neurons in vitro. In vivo studies are few in number, but indicate, e.g., that BDNF and CNTF can prevent naturally occurring death of specific neuronal populations (CLATTERBUCK et al., 1993; HAGG & VARON, 1993; MASU et al., 1993). Regarding the adult nervous system, NGF is well known to be required for survival of cholinergic neurons of the forebrain (EBENDAL, 1992). Limited regeneration of these neurons can also be induced by transplantation of genetically engineered fibroblasts that release NGF (KAWAJA et al., 1992). It is possible, however, to distinguish between support for the survival and outgrowth in some cases, e.g., adult sensory neurons depend upon NGF for neurite outgrowth but not for survival (LINDSAY, 1988). The role of neurotrophic factors in regeneration of the vertebrate PNS is, however, surprisingly controversial. For exam- ple, NGF is thought to be responsible for collateral sprouting, but not regeneration, of rat cutaneous sensory neurons (DIAMOND et al., 1987, 1992). The role of CNTF in regeneration in the PNS is even more mysterious since this molecule lacks a signal peptide for release, but is synthesized in large quantities by Schwann cells. One suggestion is that