Our long-range goal is to gain insight into the mechanisms governing the release and actions of neurotransmitters. In recent years it has become clear that most neurons use multiple transmitters, commonly a single fast acting conventional transmitter and one or more peptide transmitters. While much research has focused on conventional transmitters, large gaps exist in our knowledge of the biology of neuropeptides at all stages from release to physiological function. We are currently using a number of tools including single cell biochemistry, electrophysiology, cell culture, and immunocytochemistry to identify novel transmitters and study their actions.

In our studies we have utilized the comparatively simple nervous system of Aplysia, a sea slug. This animal's nervous system consists of relatively few neurons many of which are large and can be individually identified making this preparation amenable for studies on the regulation of synaptic transmission from the molecular to the behavioral level. The results found on the molecular level can often be interpreted in terms of their behavioral significance-an advantage afforded in very few preparations.

To determine the functional roles played by neuropeptide transmitters, three experimental requirements must be satisfied. First, peptide transmitters must be characterized and localized to individual, identified neurons. We have developed procedures that permit biochemical characterization of the neuropeptides present in single identified neurons.

Second, release of the peptide transmitters must be monitored. We use several procedures, such as measuring changes in post-synaptic conductances or second messenger levels, to monitor the release of specific neuropeptides from these neurons. We have also recently succeeded in measuring the release of neuropeptides from single identified neurons placed in primary culture where they regrow axons and varicosities. We have found that each neuron regulates release of its neuropeptide differently and we are beginning to examine the cellular properties of the neurons which regulate this release.

Third, the physiological consequences of the released transmitter must be assessed. Using reduced preparations we have been able to satisfy these requirements for several neuropeptides. We are also exploring the manner in which neuropeptides modulate the effectiveness of conventional transmitters such as acetylcholine which are released from the same terminals. These studies have broad implications in our understanding of the roles of multiple transmitters in individual neurons.

Ongoing projects in the laboratory include the purification and sequencing of novel neuropeptides from Aplysia, the immunocytochemical localization of these peptides to individual neurons, and the analyses of peptide function using a variety of procedures including cyclic nucleotide radioimmunoassays, intracellular recording from identified neurons during behavior and voltage clamp recording.
 
 

References

Fox, L.E. and Lloyd, P. E. (1998) Identified serotonergic neurons differentially modulate the synaptic efficacy of two motor neurons innervating the same muscle fibers in Aplysia. J. Neurophysiol. 80: 647-655.  Fox, L.E. and Lloyd, P. E. (1997) Serotonin and the SCPs differentially modulate two motor neurons that innervate the same muscle fibers in Aplysia. J. Neurosci. 17: 6064-6074.