Download 2. Aim of the thesis

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
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identified Lymnaea nAChR subunits in Lymnaea stagnalis. Using real-time quantitative
polymerase chain reaction (qPCR) we show that the LnAChR subunits are predominantly
expressed in the CNS. In situ hybridization (ISH) on sections of the Lymnaea CNS
demonstrates that the LnAChR subunits are expressed exclusively in neurons. Therefore,
we concluded that the identified LnAChR subunits all represent subunits of neuronal-type
nAChRs. Expression of LnAChR subunits is observed in all ganglia of the Lymnaea CNS. The
expression level of most LnAChR subunits differs considerably between ganglion types. We
estimated that at least 10% of the neurons in the Lymnaea CNS expresses nAChR subunits.
As a marker of cholinergic neurons we also investigated the expression of the Lymnaea
vesicular acetylcholine transporter (LVAChT). Also the expression of LVAChT is detected in
all ganglia and involves approximately 10% of the neurons of the CNS. The relatively high
expression levels of many LnAChR subunits and of LVAChT in the pedal ganglia suggest that
the pedal ganglia may represent prominent sites of fast cholinergic transmission. Finally,
using qPCR on cDNA preparations of caudodorsal cells, light green cells, light yellow cells,
pedal cluster IB cells or anterior lobe neurons we demonstrated that these neuroendocrine
cell populations express distinct subsets of LnAChR subunits. In addition, heterogeneity of
LnAChR expression within the neurons of these neuroendocrine populations is suggested
by ISH. This suggests that the neuropeptide release of many neuroendocrine cells may be
under intricate cholinergic control.
In chapter 4 we specifically investigated the ion selectivity of nAChR subtypes
formed by the newly identified LnAChR subunits. Based on molecular features of previous
mutagenesis studies, we hypothesized that among the LnAChR subunits are subunits that
participate in cation-selective (LnAChR A, -C, -D, -E, -G, -H, -J) and anion-selective (LnAChR
B, -F, -I, -K) Lymnaea nAChR subtypes. In order to investigate the contribution of LnAChR
subunits to the ion selectivity of functional receptors we expressed LnAChR subunits
individually or in combination with others receptor subunits in Xenopus oocytes. Only the
individual expressions of the LnAChR A, -B and -I subunits resulted in functional receptors.
We investigated the pharmacological properties of receptors consisting of LnAChR A or
LnAChR B subunits. In these experiments we showed that receptors consisting of the
LnAChR A or -B subunit are sensitive to typical agonists and antagonists of nAChRs. Most
importantly, we found that the LnAChR A receptor is permeable to sodium, whereas the
LnAChR B receptor is permeable to chloride. Phylogenetic comparison suggests that
anion-selective LnAChR subunits evolved from a nAChR, rather than a GABAR or GlycR
subunit ancestor. In view of a correct prediction of ion selectivity we concluded that anionselective LnAChR subunits evolved by mutations analogous to artificial mutations shown
to convert cation-selective nAChRs to anion-selective channels.