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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