General introduction

2. AIM OF THE THESIS As described above the synaptic cholinergic transmission in the molluscan nervous system has an important role in neuronal network activity. First, the molluscan CNS makes frequently use of fast cholinergic synaptic transmission and various pharmacologically distinct types of nAChRs. Second, the cholinergic transmission involves ion channels that can conduct anions or cations. Thereby molluscan nAChRs can control both excitatory and inhibitory network activities. Third, molluscan glial cells express nAChRs that are involved in the modulation of cholinergic synaptic transmission.

Therefore, the molluscan CNS in principle holds the promise to reveal the contribution of nAChRs to neuronal network function, in particular since dissection of network physiology is feasible both in vitro and in situ. In order to obtain a full appreciation of cholinergic transmission in neuronal network function, it is crucial to know the various molecular players in the system, in particular the structure and biophysical properties of nAChRs. However, at the start of my PhD a molecular and functional framework of the receptors contributing to cholinergic transmission in Lymnaea was absent. Therefore, in this thesis I set out (i) to reveal the diversity in structure of Lymnaea nAChR subunits, (ii) to identify cellular expression of subunits as to assess their potential participation in network physiology and (iii) to functionally characterize channel properties in vitro to shed light on the presumed cationic and anionic nicotinic receptors.

3. SUMMARY OF THE THESIS In chapter 2 I describe the identification of nAChR subunits that are expressed in the CNS of Lymnaea stagnalis. For this I applied a PCR-based approach using degenerate primers designed to conserved regions in nAChR subunits known in other species. PCR reactions were performed on cDNA templates derived from the complete Lymnaea CNS and from well-characterized VD4, RPed1 or LPeD1 neurons. In total twelve partial cDNA sequences with sequence similarity to nAChR subunits were identified and were named LnAChR A - L. Full length sequence information was obtained for all of these subunits except for LnAChR L that lacks a large part of the 3’-sequence. Molecular features present in the deduced protein sequences suggest that LnAChR A - I and LnAChR K should be classified as α-type nAChR subunits, whereas LnAChR J should be classified as a β-type subunit. Phylogenetic analysis of deduced protein sequences shows that a number of Lymnaea nAChR subunits are more closely related to human nAChR subunit types, whereas others do not display such a clear relation. In particular, a group of related nAChR subunits of Lymnaea that consists of LnAChR B, -F, -I and -K seems absent in mammals or insects. In chapter 3 I characterized the localization and level of expression of the newly