066-results-04.Rmd

C. elegans pharynx as a platform for heterologous nAChR expression {#results-4}

Introduction

nAChRs are the major site of action of neonicotinoids. Several lines of evidence suggest that even within the same species, different neonicotinoid-compounds target distinct nAChRs. They also have a distinct modes of action; some neonicotinoid are true-, partial- or super- agonist whilst other are antagonists of nAChRs (Section @ref(moaneonicsinsects)). The pharmacological characterisation of insect nAChRs is needed to better understand the interactions between the nAChRs and neonicotinids and to identify subunits sensitive to different members of this class of insecticides.

Biological systems for heterologous protein expression ###{#biologicalsystemfornachrexpression}

To pharmacologically characterise the receptor ion channel, a recombinant protein can be heterologously expressed in a number of different systems (reviewed in @millar2009a). The two most commonly used are mammalian cells, insect cells or Xenopus oocytes. Each presents advantages but also disadvantages (summarised in Table @ref(tab:heterologous-expression-systems)). These systems have been extensively used to characterise mammalian and C. elegans nAChRs [@millar2009a].

library(kableExtra)
library(dplyr)

exp_sstms <- data.frame(
  System = c(rep("Xenopus oocytes", 5), rep("Cell lines", 3)),
  Advantages = c("Cheap and easy to maintain", "Easy to inject", "Express a low number \nof endogenous membrane proteins", "Can be transfected with muliple \nmRNA species simultaneously", "Amendable to electrophysiological techniques", "Temporal control of expression", "Favourable cellular environment \nfor many proteins", "Amendable to electrophysiological \nand biochemical \ntechniques"),
  Disadvantages = c("Functional properties \nmay be altered", "Preparation short lived", "Single-cell technique", " ", " ", "High cost", " ", " "))

exp_sstms  %>%
  mutate_all(linebreak) %>% 
  kable("latex", booktabs = T, escape = F,
        col.names = linebreak(c("System", "Advantages", "Disadvantages")),
  caption = "Advantages and disadvantages of heterologous expression systems.") %>%
    collapse_rows(columns = 1, valign = "top", latex_hline = "major") %>% 
    kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position")

Properties of vertebrate

$\alpha7$

nAChR ###{propertiesofalpha7}

One of the well-studied nAChR subunit is the vertebrate homomeric

$\alpha7$

. Heterologous expression of these receptors in Xenopus occytes and mammalian cell lines.

$\alpha7$

nAChR is rapidly desensitising

Acetylcholine and nicotine are classical nAChR agonists that activate vertebrate

$\alpha7$

and many other nAChR types. In addition, there are selective compounds that bind to

$\alpha7$

receptors, such as cytisine and choline. The rank order of potency of these agonists is: cytisine > nicotine > ACh > choline [@papke2000]. The EC50 values vary depending on the method of measure and the expression system [@papke2002]. Nicotine is generally at least 5 times more potent than ACh [@couturier1990], whereas the potency of choline is at least 10 times lower than that of ACh [@papke2002]. The EC50 of the most potent compound cytisine is between 5.6 and 7.1

$\mu$

M [@wonnacott2007].

The kinetics of agonist-evoked nAChR responses are reminiscent to those of other nAChR types. Briefly, in the presence of agonist, receptor channels open rapidly allowing flux of ions, which gradually declines [@corrie2011] until a full depolarisation and desentisation occurs.

Extremely rapid desensitising kinetics is the unique feature of the

$\alpha7$

receptor. In 1990, @couturier1990 heterologously expressed

$\alpha7$

in Xenopus oocyte and recorded the macroscopic current in response to acetylcholine. In the presence of acetylcholine, receptors desensitised in under a millisecond [@couturier1990; @papke2002]. A more precise temporal characterisation of this response was obtained in 2008. Using patch-clamp, a single channel recording transfected human cell lines was obtained [@bouzat2008]. In the presence of 1

$\mu$

M acetylcholine,

$\alpha7$

receptor opens and desensitises in 0.4 ms. This is much faster than other receptor channels. For example,

$\alpha8$

typically desensitises in hundreds of millisecond [@gerzanich1994], whereas C. elegans L-type and N-type nAChRs in tens of seconds [@boulin2008; @touroutine2005].

Single channel recordings revealed another striking difference between

$\alpha7$

and other nAChRs. In response to agonist, nAChR channel typically display several bursts of channel opening flanked by period of inactivity [@mishina1986; @weltzin2019]. In contrast,

\alpha7

typically opens once before entering the inactive form [@bouzat2008]. This feature combined with rapid desensitising kinetics suggest that

\alpha7

receptors are primarily involved in the phasic and not tonic responses to ACh in the physiological conditions.

Heterologous expression of

\alpha7

also allowed for a detailed analysis of the recovery kinetics for receptor channels. Following desensitisation and removal of the agonist by washing, receptor returns to the resting state, allowing for the subsequent activation upon agonist application. Although the activation and desensitisation kinetics of

\alpha7

evoked by many agonists are almost identical, the recovery kinetics are compound, time and concentration -dependent [@mike2000]. After acetylcholine-evoked desensitisation and a 5-minute wash, the subsequent response of

\alpha7

receptors to ACh seemed unaffected [@briggs1998]. In contrast, subsequent response to ACh following nicotine desensitisation was reduced [@briggs1998]. Thus, recovery kinetics following acetylcholine application are faster. Recovery time is also more rapid for choline than for acetylcholine [@mike2000].

Sensitive to

\alpha

-Bungarotoxin (

\alpha

-Bgtx)

A distinct pharmacological feature of

\alpha7

receptors is their high sensitivity to

\alpha

-Bgtx. Interaction between

\alpha

-Bgtx and

\alpha7

receptors was first revealed by biochemical techniques.

\alpha7

receptor was isolated from membrane fraction of transformed bacterial cells with

\alpha

-Bgtx-affinity chromatography [@schoepfer1990]. Whereas, radiolabeled

\alpha

-Bgtx bound to

\alpha7

receptors immunoprecipitated from the chick retina [@keyser1993]. Binding to mammalian brain receptors was also revealed by radiography of mammalian brain slices incubated with labelled

\alpha

-Bgtx [@clarke1985; @segal1978]. The signal was consistent with the expression profile of

\alpha7

receptor, as shown by immunocytochemistry [@toro1994]. In contrast, there was no

\alpha

-Bgtx binding in mice deficient in

\alpha7

expression [@orr-urtreger1997]. Finally, the crystal structure of

\alpha7

receptor showed binding of

\alpha

-Bgtx to the extracellular domain of the receptor [@dellisanti2007].

Electrophysiological evidence provided mechanistic details of the interaction between

\alpha

-Bgtx and

\alpha7

receptor. Incubation with nM concentrations of

\alpha

-Bgtx prevented responses to classical nAChR agonists of heterologous receptors in Xenopus oocytes [@couturier1990] as well as native receptors in PC12 cells [@blumenthal1997] and hippocampal neurons [@alkondon1991]. Upon removal and wash, the receptor responds to ACh normally [@couturier1990], thus the interaction between

\alpha

-Bgtx and

\alpha7

are of high affinity, competitive and reversible.

Highly permeable to calcium ions ####{#capermeability}

\alpha7

receptors are highly permeable to calcium ions [@seguela1993; @bertrand1993]. Historically, calcium ion permeability of nAChRs was measured by establishing the reversal potential of the agonist-evoked current by changing the concentration of calcium ions in the buffer and representing calcium ion flux as a function of sodium ion flux. Using this methods, is was established that there is up to 20-fold difference between calcium and sodium permeability of heterologously expressed

\alpha7

receptors [@seguela1993]. More recently fluorescent calcium indicators [@neher1995] were used to measure calcium ion flux as a function of the entire transmembrane current. Calcium ions account for 11 % of the entire ionic conductance of the heterologously expressed

\alpha7

receptors [@fucile2000]. In comparison to other nAChRs,

\alpha7

has up to a 200-fold difference in calcium ion permeability when the channels are expressed heterologously [reviewed in @fucile2004].

Matured with the aid of NACHO and RIC-3 ####{#ric-3nacho}

Mammalian RIC-3 is a small protein with 2 transmembrane domains and a single coiled-coil domain [@wang2009]. It is expressed in most brain regions, enriched in the regions common to the

\alpha7

expression, namely the hippocampus and the cerebellum, as shown by in-situ hybridisation [@halevi2003]. Fluorescently tagged-RIC-3 localised to the ER and not the surface when expressed heterologously [@roncarati2006], whereas immunostaining of native RIC-3 in PC12 and hippocampal neurons showed co-localisation with neuronal and ER markers [@alexander2010], providing evidence that RIC-3 is an ER residing protein.

RIC-3 has a role in receptor maturation. Co-expression of RIC-3 protein with heterologously expressed mammalian

\alpha7

resulted in increased functional expression of this receptor, as measured by ACh-evoked current [@williams2005] and radiolabelled ligand binding [@lansdell2008]. Additionally, RIC-3 promotes association of nAChRs with proteins involved in post-translational modification, receptor trafficking and transport [@mulcahy2015]. Thus, RIC-3 promotes cell-surface expression of nAChRs.

NACHO is an 18-kDa multi-pass protein ER protein expressed in neurons of hippocampus, cerebral cortex and the olfactory bulb [@gu2016]. ACh-evoked current and cell-surface labelling of heterologously expressed

\alpha7

receptor were elicited upon co-transfection of cells with NACHO [@gu2016]. Absence of

\alpha7

mediated current in the hippocampus of NACHO-knock-out mice, the lack of binding of classical antagonists as well as behavioural phenotype, consistent with the disruption of cholinergic neurotransmission [@matta2017] supports the role of NACHO in maturation of

\alpha7

and other nAChRs including

\alpha4\beta2

,

\alpha3\beta2

and

\alpha3\beta4

[@matta2017].

Further experiments by @gu2016 and @matta2017, provided details of the interactions between the receptor, RIC-3 and NACHO. Transfection of HEK cells with \alpha7 and RIC-3 resulted in no surface expression, based on the lack of \alpha-bgtx or epibatidine binding. Surface expression was achieved when cells were co-transfected with \alpha7 and NACHO, and augmented by RIC-3. Based on these observatins it was proposed that was NACHO promotes early events in the receptor assembly, whereas RIC-3 in synergy with NACHO aids receptor maturation. [@matta2017].

RIC-3 may also aid interactions with many other proteins in the cells, as shown by the enhanced interactome of nAChR \alpha7 and other proteins in the cell upon co-expression of RIC-3 [@mulcahy2015].

RIC-3 and NACHO are the two must studied proteins involved in the maturation of \alpha7 nAChRs, but there are also many other proteins of less defined role involved in the biogenesis of nAChRs (reviewed by @crespi2018). For example, evolutionary coserved CRELD and EMC-6, which are ubiquitonously expressed and ER membrane-bound [@dalessandro2018; @richard2013], as well as NRA-2/nicalin (nicastrin-like protein) and NRA-4/nodal modulator (NOMO) involved in the regulation of receptor stoichiometry [@almedom2009].

Taken together, Xenopus oocytes and eukaryotic cell lines can be used as an heterologous expression platform for vertebral nAChRs. They have been used to described the receptor maturation, stoichiometry, pharmacological and kinetic properties of vertebrate nAChRs, such as \alpha7. However, the expression of many invertebrate receptors in these systems has failed [@huang1999; @liu2005; @liu2009; @yixi2009; @bass2006], hindering their functional characterisation. Thus, other approaches need to be considered.

C. elegans pharynx as a platform for heterologous protein expression

C. elegans pharynx can be used as an alternative biological system for nAChR expression for several reasons.

Eat-2 is a suitable genetic background for functional expression

Many of worm's behaviours are underpinned by the cholinergic neurotransmission (Section @ref(cholinergicneurotransmissioninworms)). Pharyngeal pumping is a measure of the feeding behaviour of the worm, regulated by acetylcholine. It can be easily quantified in whole organisms or cellular assays. Thus, the activity of the pharynx can be used a platform to investigate the performance and sensitivity of this organ to cholinergic drugs in the wild-type and in strains deficient in cholinergic transmission.

The activity of the pharynx is regulated by acetylcholine, thus this organ can be also suitable for the heterologous expression of nAChRs. Acetylcholine acting on EAT-2 containing nAChRs in the pharynx is a main driver of fast pumping (Chapter 3 and @mckay2004). EAT-2 is expressed in pm4 and pm5 muscle cells [@mckay2004], which make synaptic connections with the MC [@albertson1976]. The feeding response is markedly hindered in eat-2 C. elegans mutant [@raizen1995; @mckay2004]. Thus, selective expression of nAChRs in the pharyngeal muscle of eat-2 C. elegans mutant at the MC synapse should be a suitable platform for functional expression.

The differences between the wild-type, mutant and tragenic worms can be scored using behavioural or cellular assays (Section @ref(analytical_behaviour)), whereas receptor expression can be detected using biochemical approaches.

One of the methods used to detect the cellular localisation of nAChRs in C. elegans is by using protein-specific pharmacological agents. \alpha-bgtx is a high affinity antagonist of nAChRs [@blumenthal1997], widely used to label expression on native and heterologous channels. Audioradiography of tissues incubated with radiolabelled \alpha-bgtx visualised mammalian nAChRs at the post-synatic membrane of the end-plate [@barnard1971], and in the peripheral [@clarke1985] and central nervous system [@carbonetto1979]. Flourescently labelled \alpha-bgtx was utilised to show successful expression of heterologous proteins such as mammalian \alpha7 in HEK, P12 and SH SY5Y cell lines [@cooper1997; @gu2016]. In C. elegans, conjugated-α-Bgtx injected into the pseudocoelom, labelled native nAChRs of the pharyngeal [@mckay2004] and body wall muscle nAChRs [@jensen2012]. It also allowed for the identification of heterologously expressed ACR-16 in the body wall muscle of C. elegans [@jensen2012]. \alpha-Bgtx is used to demonstrate cell surface expression, because it binds to the extracellular domain of the nAChR [@dellisanti2007] and does not permeate membranes. There are methods used to label heterologous proteins intracellularly. For example, @salom2012 and @gu2016 used detergents to permeabilised membrane to allow protein-specific antibodies or \alpha-Bgtx to access protein sites inside the cell.

Taken together, C. elegans pharynx is considered a suitable biological system for nAChR expression for several reasons.

Chapter aim

This chapter describes efforts into development of the C. elegans' pharynx as a platform for heterologous expression of nAChRs, with the aim to use it as a platform to characterise their interactions with neonicotinoids. As a first step, an expression of endogenous and exhogenous protein in the wild-type and mutant C. elegans pharynx was carried out. The endogenous receptor was chosen based on the fact it is the key receptor in the function of the pharynx. With regards to the exhogenous protein, since the stiochiometry of insect receptors is unknow (Section), human \alpha7 was selected. Mammalian $\alpha7& is a good candidate because this its subunit composition, pharmacology, kinetics and molecular chaperons are known (Section @ref(propertiesofalpha7). As a second step, behavioural and biochemical assays were utilised with the aim to identify assays suitable for the detection of the cell surface expression and functionality of heterologous expression.

Results

library(grid)
library(cowplot) #plot_grid
library(tidyverse)
library(ggpubr) 
library(readr) #read_csv
library(ggplot2) #ggplot
library(scales)
library(curl)
library(devtools)
library(extrafont)

This chapter describes the development of the method for the heterologous expression of nAChRs in the C. elegans pharynx.

As a positive control, EAT-2 rescue experiment was carried out. The literature suggests EAT-2 is a single molecular determinant of the fast pharyngeal pumping. The Eat-2 knock-out strain has been shown to have reduced pharyngeal response to food and 5-HT [@mckay2004], which can be rescued by the expression of EAT-2 in the pharyngeal muscle, however no data was provided to support this claim. Additionally, the rescue strains are no longer available (personal communication). Therefore, the first step was to generate these strains and to confirm the function of EAT-2.

Transgenic lines were generated by the process of microinjection (Section @ref(microinjection)). eat-2 C. elegans worms were injected with DNA construct containing nAChR cDNA downstream of the myo-2 promoter, which drives expression in all muscle cells of the pharyngeal musculature [@altun2009a].

Heterologous expression of native EAT-2 nAChRs in C. elegans pharyngeal muscle

Generation of the expression vector

The expression vector was cloned using Gateway cloning method (Section @ref(gatewaycloning)). Briefly, eat-2 coding DNA was PCR-amplified from the pTB207 vector (a gift from Dr. Cedric Neveu) Table @ref(tab:eat2-amplification), (Figure @ref(fig:eat2-pcr-label)). Adenosine overhangs were added to the PCR-product (Table @ref(tab:a-overhangs-addition)), which was subsequently cloned into the TOPO vector (Table @ref(tab:TA-reaction), Figure @ref(fig:eat-topo-label)). Cloning success was tested by performing PCR with one gene specific and one insert specific primers (Figure @ref(fig:topo-eat2-pcr-label)). Eat-2 was then inserted into the expression vector by the recombination cloning (Section @ref(lr-reaction-section) and Figure @ref(fig:pdest-eat2-label)). This was authenticated by the analytical digestion (Figure @ref(fig:pdest-eat2-label)) and sequencing (Appendix B).

(ref:eat2-pcr) Amplification of eat-2 gene. Eat-2 cDNA was amplified from pTB207 vector, gel excised and purified for downstream cloning. (a) Cartoon representation of the process of amplification of the gene by PCR including the expected PCR product size (b) Agarose gel of the PCR product with the corresponding size, against DNA ladder (M).

knitr::include_graphics("fig/results4/PNG/PCR_of_eat2.png")

(ref:eat-topo) Insertion of eat-2 into the TOPO vector. (a) Cartoon representation of the process of generation TOPO-eat-2 vector. 3’ A overhangs were added to the purified eat-2 cDNA to enable TOPO cloning. Eat-2 containing TOPO vector was digested with EcoRI which cuts at sites flanking the inserted eat-2 gene yielding (b) 2 linear DNA fragments. (c) Agarose gel of non-digested TOPO-eat-2 plasmid and the two bands following EcoRI digestion.

knitr::include_graphics("fig/results4/PNG/Generation-of-PCR-8-eat2.png")

(ref:topo-eat2-pcr) PCR of eat-2 gene from the TOPO vector. (a) Cartoon representation showing the positioning of primers used to PCR amplify Eat-2 from TOPO vector using insert specific (eat2-Fw) and vector specific (TOPO-Rev) primers. Only clone containing eat-2 inserted in the right direction should produce a PCR product of 1436 bp. (b) Picture of agarose gel of PCR product against DNA ladder (M).

knitr::include_graphics("fig/results4/PNG/PCR_of_eat2_from_TOPO_vector.png")

(ref:pdest-eat2) The generation of the vector for the expression of EAT-2 nAChR in the C. elegans pharynx. (a) Cartoon representation of the cloning process and location of EcoRI restriction sites within plasmids. Eat-2 was cloned into the expression backbone vector downstream of the myo-2 promoter. To identify successful clones, expression vector with and without the cloned eat-2 were analytically digested with EcoRI. EcoRI cuts the backbone plasmid in two places producing 2 DNA fragments. In contrast, digestion of the plasmid containing the eat-2 sequence yields 3 DNA fragments. (b) Picture of the agarose gel containing the digested plasmids against DNA ladder (M). Plasmid names and DNA fragment sizes are given on the gel.

knitr::include_graphics("fig/results4/PNG/Generation_of_pDEST-pmyo2-eat2.png")

Generation of the transgenic strain

Transgenic strains were generated by the process of microinjection (Section @ref(microinjection)). A DNA mix of vector containing EAT-2 gene and vector containing the selectivity marker was prepared. Selectivity marker was a vector containing GFP under the myo-3 promoter (pmyo-3). Endogenous myo-3 promoter drives the expression of myosin in the body wall muscle. Therefore by driving the expression of GFP with pmyo-3, worms will appear green under the fluorescence microscope. This enables identification of successfully transfected worms. Worms expressing GFP are also predicted to express another injected gene, in this case EAT-2.

64 adult eat-2 worms were injected. 6 of those generated 33 green progeny. Only 2 of the 6 F1 produced green progeny- these were kept as stable lines. The genotype of these lines is eat-2::pmyo3::GFP;pmyo2::eat-2, but for simplicity, they will be referred to as eat-2::eat-2 or eat-2 rescue.

Alongside, a control line was generated in which GFP was expressed at the body wall muscle of eat-2 mutant. Worms were injected with a plasmid DNA containing GFP gene. 32 worms were injected. There were 5 green progeny present on a single plate, one of which produced green offspring. This transgenic line was kept and used as a control. The genotype of this line is eat-2::pmyo3::GFP, but for simplicity, they will be referred to as eat-2::GFP or eat-2 transgenic control line.

Feeding phenotype of eat-2 expressing transgenic lines ####{#behaviourofeat2rescue}

Eat-2 mutant has an overt feeding phenotype (Figure @ref(fig:mutant-pumping-label)), @mckay2004]). To determine whether the expression of eat-2 under myo-2 promoter rescues the feeding retardation of eat-2 mutant, behavioural assays were carried out. The feeding phenotype of generated strains, eat-2 mutant and wild-type C. elegans were assayed (Figure @ref(fig:transgenic-feeding-label)). Worms were placed on an agar plate containing an OP50 food patch and pharyngeal pumping on food of adult worms was scored. Wild-type worms pumped at a rate of 4.65 Hz. This dropped to 0.94 and 0.89 Hz in eat-2 mutant and eat-2 transgenic control strain, respectively. The feeding phenotype of two eat-2 rescue lines was assayed. Their feeding phenotypes did not differ (data not shown), therefore results were pooled. Expression of eat-2 in eat-2 mutant restored feeding rate to 3.12 Hz.

(ref:transgenic-feeding) Pharyngeal pumping of C. elegans nicotinic acetylcholine receptor mutant and rescue strains. Pharyngeal pumping on food of N2 wild-type, eat-2 mutant, eat-2 transgenic control strain (eat-2::GFP) and eat-2 rescue (eat-2::eat-2) strains. Pharyngeal pumps of worms present on food were counted by visual observation for 30 seconds and expressed in Hz. Data are mean \pm SEM, collected from 10-46 individual worms on 3 days. One way ANOVA (Kruskal-Wallis test) with Sidak Corrections, ****P \le 0.0001.

# data <- read_csv("Analysis/Data/Transformed/chapter_4_data.csv") %>% 
#   select(-1)
# 
# data1 <- mutate(data, Strain = factor(Strain,
# levels= c("N2", "acr-7", "N2::pmyo3::GFP", "N2::pmyo3::GFP_pmyo2::CHRNA7", "eat-2", "eat-2::pmyo3::GFP", "eat-2::pmyo3::GFP_pmyo2::CHRNA7", "eat-2::pmyo3::GFP_pmyo2::eat-2"),
# labels= c("N2", "acr-7", "N2::GFP", "N2::alpha7", "eat-2", "eat-2::GFP", "eat-2::alpha7", "eat-2::eat-2")))
# 
# 
# data2 <- mutate(data1, experiment = factor(experiment, levels = c("pumping_on_food", "5-HT", "nic_cuthead_transg", "choline_cuthead_transg", "cytisine_cuthead_transg", "choline-cuthead", "ach_epg", "nic_epg")))
# 
# data3 <- mutate(data2, Concentration = factor(Concentration,
#                    levels=c("not", "5-HT", "100nM", "1uM", "10uM", "20uM", "50uM", "100uM", "1mM"),
#                    labels=c("not", "0", "0.1", "1", "10", "20", "50", "100", "1000")))
# 
# stats <- data3 %>% 
#   group_by(experiment, Strain, Concentration, Time) %>%
#   summarise(mean_pumping = mean(Pumps30s),
#             n=n(),
#             sd = sd (Pumps30s),
#             se = sd/sqrt(length(Pumps30s))) 
# 
# 
# #plot <- 
# stats1 <- stats %>% 
#   filter(experiment == "pumping_on_food" & Strain %in% c("N2", "eat-2", "eat-2::GFP", "eat-2::eat-2")) #here i am selecting by mutliple variables. %in% means select listed oservations in Strain variable % only include specified experiment  
# 
# plot <- ggplot(stats1, aes(x=Strain, y=mean_pumping)) + 
#   geom_bar(stat = "identity", fill= "grey" ) +
#   geom_errorbar(aes(ymin=mean_pumping-se, ymax=mean_pumping+se, width = 0.4)) +
#   ylab("Pumping (HZ)") +
#   ylim(0, 5) +
#   theme(axis.title.x = element_blank(),
#         axis.title = element_text(size=12),
#         axis.text = element_text(size=12),
#         axis.text.x=element_text(angle=90)) +
#   ggsave("fig/results4/eat2rescue_feeding.pdf", width = 15, height = 8, units = "cm")
knitr::include_graphics("fig/results4/PNG/eat2rescue_feeding_2.png")

Effects of 5-HT on pharyngeal pumping

The effects of 5-HT on pharyngeal pumping was scored to determine if expression of eat-2 in eat-2 mutant worms rescues their 5-HT insensitivity (Figure @ref(fig:DR-5HT-cuthead-2-label)). Cut-heads were used in this experiment and worms were exposed to 1 \muM 5-HT because this dose elicits maximum response in the wild-type strain (Figure @ref(fig:DR-5HT-cuthead-label)). After 30 minutes of incubation, the effects of 5-HT on pumping was scored (Figure @ref(fig:eat-2-rescue-label)). Wild-type pharynxes pumped at a rate of 3.00 Hz. This was reduced to 0.89 and 0.91 Hz in eat-2 mutant and eat-2 mutant expressing GFP. Transgenic lines were scored separately. The pharyngeal pumping rate induced by 5-HT did not differ between lines, hence the results were pooled. Rescue eat-2 worms pumped at an average rate of 2.97 Hz.

(ref:eat-2-rescue) The effects of 5-HT on wild-type, eat-2 mutant and eat-2 rescue strains. Cut heads of wild-type, eat-2 mutant, transgenic control and rescue strains were incubated with 1 \muM 5-HT or control vehicle. 30 minutes later, the effects of 5-HT on pumping was scored. Pharyngeal pumping was counted by visual observation for 30 seconds and expressed in Hz. Data are mean \pm SEM of 7 - 30 worms collected from paired experiments done on 2 days. ****P \le 0.0001.

#   plot2 <- filter(stats, experiment == "5-HT" & Strain %in% c("N2", "eat-2", "eat-2::GFP", "eat-2::eat-2")) %>%
# ggplot(aes(x=Strain, y=mean_pumping)) + 
#   geom_bar(stat = "identity", fill= "grey" ) +
#   geom_errorbar(aes(ymin=mean_pumping-se, ymax=mean_pumping+se, width = 0.4)) +
#   ylab("Pumping (HZ)") +
#   ylim(0, 5) +
#   theme(axis.title.x = element_blank(),
#         axis.title = element_text(size=12),
#         axis.text = element_text(size=12)) +
#   ggsave("fig/results4/eat2rescue_5ht.pdf", width = 15, height = 8, units = "cm")
  knitr::include_graphics("fig/results4/PNG/eat2rescue_5ht_2.png")

Heterologous expression of human \alpha7 nAChRs in C. elegans pharyngeal muscle

To test whether successful expression of non-native nAChRs can be achieved, human \alpha7 receptor was introduced into the C. elegans pharynx. This receptor was chosen because it is homopentameric [@couturier1990; @cooper1997; @gu2016], therefore does not need to interact with other subunits to form a functional receptor. Additionally, its pharmacology has been thoroughly studied [@papke2002].

Two approaches were taken as they provide alternative ways of assessing the functionality of the introduced receptor.

First, human \alpha7 encoding gene (CHRNA7) was introduced into the eat-2 background to determine whether the eat-2 mutant behavioural and pharmacological phenotype can be reversed.

Alongside, CHRNA7 was introduced into the wild-type background of C. elegans to determine whether the pharmacology of \alpha7 receptor can be imposed on the pharynx.

Generation of the expression vector

A cDNA sequence was inserted downstream of myo-2 promoter by LR recombination. Briefly, CHRNA7 was PCR amplified from pDNA3.1 vector (AddGene plasmid #62276) using flanking primers (Table @ref(tab:primer-seq1), Table @ref(tab:CHRNA7-amplification) and Figure @ref(fig:CHRNA7-amplification2-label)). Amplified PCR product was gel-purified and incubated with non-proofreading PCR polymerase to add 3' A-overhangs. This enabled cloning into the TOPO vector (Figure @ref(fig:PCR8-CHRNA7-label) a). Produced plasmid was analytically digested with EcoRI (Figure @ref(fig:PCR8-CHRNA7-label) b).

(ref:CHRNA7-amplification2) Amplification of the gene encoding for human \alpha7 subunit of nAChR. (a) Cartoon representation of the process of amplification of CHRNA7 by PCR. CHRNA7 was amplified from pcDNA3.1 vector, gel excised and purified for downstream cloning. (b) Picture of the agarose gel of the PCR products of 1509 bp against DNA ladder (M).

knitr::include_graphics("fig/results4/PNG/PCR_of_CHRNA7.png")

(ref:PCR8-CHRNA7) Insertion of CHRNA7 into the TOPO vector. (a) Cartoon representation of generation of TOPO-CHRNA7 vector. 3’ A overhangs were added to the purified PCR product to enable TOPO cloning. CHRNA7 containing TOPO vector was digested with EcoRI which cuts at sites flanking and within the inserted gene, yielding 3 linear DNA fragments. (b) Schematic representation (left) and an agarose gel (right) of DNA fragments generated upon EcoRI digestion against DNA ladder (M).

knitr::include_graphics("fig/results4/PNG/Generation-of-PCR-8-CHRNA7.png")

CHRNA7 was then cloned into the pDEST expression vector downstream of the myo-2 promoter by LT recombination (Figure @ref(fig:pdest-chrna7-cloning-label)). Four clones were analysed by digestion. A single positive clone (clone number 4 in lane 5 of the agarose gel in Figure @ref(fig:pdest-chrna7-cloning-label) b) was selected, sequenced and used in downstream experiments. The entire pmyo-2::CHRNA7 sequence can be found in the Appendix C.

(ref:pdest-chrna7-cloning) Generation of the vector for the expression of \alpha7 nAChR in the C. elegans pharynx. (a) CHRNA7 gene was cloned into the pDEST expression vector downstream of the myo-2 promoter. Four clones of the generated plasmid and the backbone pDEST plasmid were digested with EcoRI. The EcoRI restriction sites within the backbone plasmid and the cloned plasmid are shown in a. (b) The resulting DNA fragments were run on the agarose gel. Digestion of pDEST backbone results in the generation of 2 fragments (Line 1 on the gel), where digestion of CHRNA7-containing plasmid yields 4 fragments (Lines 2-5).

knitr::include_graphics("fig/results4/PNG/Generation_of_pDEST-pmyo2-CHRNA7_2.png")

Generation of eat-2 transgenic strain ####{#eat2charn2microinjection}

Transgenic worms were generated by microinjection. DNA mix containing two plasmids: CHRNA7 downstream of myo-2 promoter and GFP downstream of myo-3 promoter was prepared. 64 adult eat-2 worms were injected. 3 plates contained a total of 23 green progeny. These 23 worms were separated and allowed to propagate. Green offspring were found on three plates. These plates were kept separately and treated as individual worm lines. The genotype of these lines is eat-2::pmyo3::GFP;pmyo2::\alpha7, but for simplicity, they will be referred to as eat-2::\alpha7.

The three eat-2::\alpha7 lines were assayed separately. The behavioural output did not differ (data not shown), therefore the results were pooled.

A control strain in which GFP is expressed at the body wall muscle was generated previously.

Generation of N2 transgenic strain

59 adult N2 worms were injected with the DNA mix used previously (Section @ref(eat2charn2microinjection)). 12 plates contained a total of 74 green progeny. These 74 worms were separated and allowed to propagate. Green offspring were found on two plates. These plates were kept separately and treated as individual worm lines. The genotype of these lines is N2::pmyo3::GFP;pmyo2::\alpha7, but for simplicity, they will be referred to as N2::\alpha7, or N2 transgenic. Their behavioural output did not differ (data not shown), therefore the results were pooled.

A control strain in which GFP is expressed at the body wall muscle was also generated. 8 worms were injected with the GFP containing vector. 2 generated 8 green offspring. 1 line was stable. The genotype of this line is N2::pmyo3::GFP, simply N2::GFP or N2 transgenic control line.

Feeding phenotype of transgenic lines ####{#feedingalpha7celegans}

The feeding phenotype of wild-type, eat-2 mutant, \alpha7-expressing and control lines were assayed. Worms were placed on agar plate containing a food patch and the pharyngeal pumping was scored (Figure @ref(fig:feeding-chrna7-transgenic-label)).

The feeding phenotype of worms did not change upon introduction of human \alpha7 in the pharynx. N2 wild-type, and transgenic C. elegans pumped at an average rate of 4.64 - 4.68 Hz. Pharyngeal pumping of eat-2 mutant and eat-2 transgenic worms varied between 0.89 and 0.98 Hz.

(ref:feeding-chrna7-transgenic) Effects of human \alpha7 nAChR expression on the feeding phenotype of C. elegans. Pharyngeal pumping of N2 wild-type, eat-2 mutant, transgenic strains expressing human \alpha7 nAChR in the pharyngeal muscle (N2::alpha7 and eat-2-alpha7) and transgenic control worms (N2::GFP and eat-2-GFP). Pharyngeal pumps of worms present on food were counted by visual observation for 30 seconds and expressed in Hz. Data are mean \pm SEM, collected from 7-30 individual worms on 3 days. One way ANOVA (Kruskal-Wallis test) with Sidak Corrections.

# stats2 <- stats %>% 
#   filter(experiment == "pumping_on_food" & Strain %in% c("N2", "N2::GFP", "N2::alpha7", "eat-2", "eat-2::GFP", "eat-2::alpha7")) # here i am selecting by mutliple variables. %in% means select listed oservations in Strain variable % only include specified experiment  
# 
# plot3 <- ggplot(stats2, aes(x=Strain, y=mean_pumping)) + 
#   geom_bar(stat = "identity", fill= "grey" ) +
#   geom_errorbar(aes(ymin=mean_pumping-se, ymax=mean_pumping+se, width = 0.4)) +
#   ylab("Pumping (HZ)") +
#   ylim(0, 5) +
#   theme(axis.title.x = element_blank(),
#         axis.title = element_text(size=12),
#         axis.text = element_text(size=12),
#         axis.text.x=element_text(angle=90)) + 
#     ggsave("fig/results4/rescuechrna7_feeding.pdf", width = 15, height = 8, units = "cm")
knitr::include_graphics("fig/results4/PNG/rescuechrna7_feeding_2.png")
  

Effects of 5-HT on pharyngeal pumping ####{#htandtransgenicalpha7}

The effects of 5-HT on the pharyngeal pumping of wild-type, eat- mutant, transgenic and transgenic control worms expressing human receptor was assayed. (Figure @ref(fig:human-transgenic-rescue-label)). Cut-heads were bathed in 1 \muM 5-HT. 30 minutes later, the effects on pharyngeal pumping were scored.

Introduction of human \alpha7 receptor into the pharynx had no effect on the pharyngeal responses to 5-HT. After 30 minutes of incubation, the pharyngeal pumping of transgenic and control C. elegans did not differ.

(ref:human-transgenic-rescue) Effects of human \alpha7 nAChR expression on the 5-HT induced pharyngeal pumping of C. elegans. The effects of 5-HT on N2 wild-type, eat-2 mutant, transgenic strains expressing human \alpha7 7 nAChR in the pharyngeal muscle (N2::alpha7 and eat-2-alpha7) and transgenic control worms (N2::GFP and eat-2-GFP). Cut heads were incubated with 1 \muM 5-HT or control vehicle. 30 minutes later, the effects of 5-HT on pumping was scored. Pharyngeal pumping was measured by counting the number of pharyngeal pumps/30s and expressed in Hz. Data are mean \pm SEM of 7 - 30 worms collected from paired experiments done on 3 days.

#   plot4 <- filter(stats, experiment == "5-HT" & Strain %in% c("N2", "N2::GFP", "N2::alpha7", "eat-2", "eat-2::GFP", "eat-2::alpha7")) %>%
# ggplot(aes(x=Strain, y=mean_pumping)) + 
#   geom_bar(stat = "identity", fill= "grey" ) +
#   geom_errorbar(aes(ymin=mean_pumping-se, ymax=mean_pumping+se, width = 0.4)) +
#   ylab("Pumping (HZ)") +
#   ylim(0, 5) +
#   theme(axis.title.x = element_blank(),
#         axis.title = element_text(size=12),
#         axis.text = element_text(size=12),
#         axis.text.x=element_text(angle=90)) +
#     ggsave("fig/results4/rescuechrna7_5ht.pdf", width = 15, height = 8, units = "cm")
  knitr::include_graphics("fig/results4/PNG/rescuechrna7_5ht_2.png")

Pharmacological characterisation of \alpha7 expressing worms ####{#pharmaalpha7transegnicworms}

The experiments utilizing the pharyngeal pump phenotype on food and in the presence of 5-HT suggest lack of functionality of the introduced receptor. However, the experiments that we used may not be sensitive enough to be able to detect functional expression of the \alpha7 receptors. Thus, we extended the analysis to investigate the pharmacological sensitivity with the aim of determining if the transgenic lines heterologously expressing \alpha7 exhibit known \alpha7 pharmacology.

To asses whether the introduction of receptor into the pharynx of wild-type worms imposes \alpha7 pharmacology on the pharyngeal system, a series of pharyngeal assays were performed. nAChR agonists were tested to determine if there is a differential sensitivity between the N2 wild-type and transgenic worms. Compounds tested were acetylcholine, nicotine, choline and cytisine.

Effects of nAChR agonist on 5-HT induced pharyngeal pumping on cut-heads were tested. Cut heads were exposed to 1 \muM 5-HT for 10 minutes. Following this the activated pharynxes were transferred to a dish containing 5-HT and nAChR agonist and the effects of an agonist on 5-HT induced pumping was scored for 50 minutes (Figure @ref(fig:nicotine-label), @ref(fig:5HTcholine-label), @ref(fig:cytisine-label)).

Exposure to 5-HT results in dose and time dependent elevation of the pharyngeal pumping that is indifferent in the wild-type and the transgenic lines. Exposure to nAChR agonists leads to dose-dependent inhibition of this response.

Exposure of cut heads to nicotine from 1 to 50 \muM, resulted in concentration-dependent inhibition of pumping in both wild-type and transgenic strains (Figure @ref(fig:nicotine-label)). The IC~50 values were comparable: 13 and 11 \muM, respectively indicating no shift in the sensitivity to nicotine upon introduction of human receptor in the C. elegans pharynx.

(ref:nicotine) The effects of human \alpha7 7 nAChR expression on the nicotine-induced inhibition of 5-HT evoked pumping. Cut heads of wild-type (N2) and transgenic worms expressing human \alpha7 in the pharynx (N2::alpha7) were exposed for 10 minutes to 1 \muM 5-HT to stimulated pumping. They were then transferred to 5-HT + indicated concentration of nicotine or vehicle control. a) The effects on pharyngeal pumping pre- (time point 0) and post- nicotine exposure were scored by visual observation for 30 seconds and expressed in Hz. b) 35-minute time points were taken, and normalised to the maximal (5-HT induced) and minimal response. Data are mean \pm SEM from 5 - 10 individual worms collected from paired experiments done on 2 days.

# plot5 <- filter(stats, experiment == "nic_cuthead_transg") %>% 
#   group_by(Strain, Concentration, Time) %>% 
#     filter(Time <= 50) %>% 
#   ggplot(aes(Time, mean_pumping, colour = Concentration)) +
#            geom_line(size=1) +
#            geom_point() +
#            geom_errorbar(aes(ymin = mean_pumping-se, ymax = mean_pumping+se)) +
#            facet_wrap(~Strain) +
#            xlab("Time (mins)") +
#            ylab("Pumping (Hz)") +
#            scale_y_continuous(breaks = seq(0, 6, by = 1)) +
#            labs(colour=expression(Nicotine~mu*M), parse = TRUE) +
#            theme(legend.position = "top",
#                  axis.text = element_text(size=12),
#                  axis.title = element_text(size=12),
#                  strip.text.x = element_text(size = 12),
#                  legend.text=element_text(size=11),
#                  text= element_text(size=12,  family="sans")) + ggsave("fig/results4/raw_images/nic_cuthead.pdf", width = 15, height = 9, units = "cm")

knitr::include_graphics("fig/results4/PNG/nicotine_cuthead.png")

The responses of both strains to choline concentrations ranging from at 1 \muM to 1 mM were also indiscernible (Figure @ref(fig:5HTcholine-label)). Choline inhibited 5-HT evoked pumping of the wild-type worms with the IC50 of 22 \muM. The IC50 of choline on transgenic line was 15 \muM.

(ref:5HTcholine) The effects of human \alpha7 nAChR expression on the choline-induced inhibition of 5-HT evoked pumping. Cut heads of wild-type (N2) and transgenic worms expressing human \alpha7 in the pharynx (N2::alpha7) were exposed for 10 minutes to 1 \muM 5-HT to stimulated pumping. They were then transferred to 5-HT + indicated concentration of choline or vehicle control. a) The effects on pharyngeal pumping pre- (time point 0) and post- nicotine exposure were scored by visual observation for 30 seconds and expressed in Hz. b) 2-minute time points were taken, and normalised to the maximal (5-HT induced) and minimal response. Data are mean \pm SEM from 3 - 12 individual worms collected from paired experiments done on 2 days.

# plot6 <- filter(stats, experiment == "choline_cuthead_transg") %>%
#   group_by(Strain, Concentration, Time) %>%
#     filter(Time <= 50) %>%
#   ggplot(aes(Time, mean_pumping, colour = Concentration)) +
#            geom_line(size=1) +
#            geom_point() +
#            geom_errorbar(aes(ymin = mean_pumping-se, ymax = mean_pumping+se)) +
#            facet_wrap(~Strain) +
#            xlab("Time (mins)") +
#            ylab("Pumping (Hz)") +
#            scale_y_continuous(breaks = seq(0, 6, by = 1)) +
#            labs(colour=expression(Choline~mu*M), parse = TRUE) +
#            theme(legend.position = "top",
#                  axis.text = element_text(size=12),
#                  axis.title = element_text(size=12),
#                  strip.text.x = element_text(size = 12),
#                  legend.text=element_text(size=11),
#                  text= element_text(size=12,  family="sans")) + ggsave("fig/results4/raw_images/choline_cuthead.pdf", width = 15, height = 9, units = "cm")

knitr::include_graphics("fig/results4/PNG/choline_cuthead.png")

Wild-type cut-head were also exposed to cytisine at 100 nM, 1, 10 and 50 \muM (Figure @ref(fig:cytisine-label) a left panel). The two lowest doses had no effect on 5-HT induced pumping. 10 \muM inhibited pumping almost completely and transiently after 2 minutes of incubation. After 10 minutes, the pumping rate was comparable to the control. Comparing the effects of cytisine on 5-HT evoked pumping of N2 wild-type to the effects on transgenic worms revealed a difference at a single dose of 50 \mu M (Figure @ref(fig:cytisine-label) a and b). In wild-type, cytisine rapidly inhibited pumping. Pharynxes remained paralysed for 10 minutes. They began to progressively recover, however the rate of the control was not reached. Transgenic pharynxes also remained paralysed for 10 minutes, however, pumping returned to a rate comparable to control after 20 minutes.

(ref:cytisine) The effects of human \alpha7 nAChR expression on the cytisine-induced inhibition of 5-HT evoked pumping. (a) Cut heads of wild-type (N2) and transgenic worms expressing human \alpha7 in the pharynx (N2::alpha7) were exposed for 10 minutes to 1 \muM 5-HT to stimulate pumping. They were then transferred to 5-HT + indicated concentration of cytisine or vehicle control. The effects on pharyngeal pumping pre- (time point 0) and post- nicotine exposure were scored by visual observation for 30 seconds and expressed in Hz. b) Comparison of the 50 \muM cytisine to show the difference in pharyngeal response between N2 and N2 transgenic worms. Data are mean \pm SEM from 5 - 14 individual worms collected from paired experiments done on 2 days.

# plot7 <- filter(stats, experiment == "cytisine_cuthead_transg") %>%
#   group_by(Strain, Concentration, Time) %>%
#     filter(Time <= 50) %>%
#   ggplot(aes(Time, mean_pumping, colour = Concentration)) +
#            geom_line(size=1) +
#            geom_point() +
#            geom_errorbar(aes(ymin = mean_pumping-se, ymax = mean_pumping+se)) +
#            facet_wrap(~Strain) +
#            xlab("Time (mins)") +
#            ylab("Pumping (Hz)") +
#            scale_y_continuous(breaks = seq(0, 6, by = 1)) +
#            labs(colour=expression(Cytisine~mu*M), parse = TRUE) +
#            theme(legend.position = "top",
#                  axis.text = element_text(size=12),
#                  axis.title = element_text(size=12),
#                  strip.text.x = element_text(size = 12),
#                  legend.text=element_text(size=11),
#                  text= element_text(size=12,  family="sans"))
# 
# 
# stats4 <- filter(stats, experiment == "cytisine_cuthead_transg") %>%
#   group_by(Strain, Concentration, Time) %>%
#     filter(Time == 20) %>%
#   filter(Concentration != "0")


# stats4$Concentration <- as.numeric(as.character(stats4$Concentration))
# 
# plot9 <- stats4 %>%
#   ggplot(aes(Concentration, mean_pumping, colour = Strain)) +
#            geom_line(size=1) +
#            geom_point() +
#            geom_errorbar(aes(ymin = mean_pumping-se, ymax = mean_pumping+se)) +
#            xlab(label=expression(Cytisine~mu*M)) +
#            ylab("Pumping (Hz)") +
#            scale_y_continuous(breaks = seq(0, 5.5, by = 1)) +
#            theme(legend.position = "top",
#                  axis.text = element_text(size=12),
#                  axis.title = element_text(size=12),
#                  strip.text.x = element_text(size = 12),
#                  legend.text=element_text(size=11),
#                  text= element_text(size=12,  family="sans"))

# plot9 <- filter(stats, experiment == "cytisine_cuthead_transg") %>%
#   group_by(Time, Strain) %>%
#     filter(Time <= 50) %>%
#   filter(Concentration == 50) %>% 
#   ggplot(aes(Time, mean_pumping, colour = Strain)) +
#            geom_line(size=1) +
#            geom_point() +
#            geom_errorbar(aes(ymin = mean_pumping-se, ymax = mean_pumping+se)) +
#            xlab("Time (mins)") +
#            ylab("Pumping (Hz)") +
#            scale_color_manual(values=c('#333333','#CCCCCC')) +
#            scale_y_continuous(breaks = seq(0, 6, by = 1)) +
#            labs(colour=expression(Strain), parse = TRUE) +
#            theme(legend.position = "top",
#                  axis.text = element_text(size=12),
#                  axis.title = element_text(size=12),
#                  strip.text.x = element_text(size = 12),
#                  legend.text=element_text(size=11),
#                  text= element_text(size=12,  family="sans"))
# 
# a <-  plot_grid(plot7, plot9, nrow =2, labels = c("a", "b"))
#   a <- ggsave("fig/results4/raw_images/cytisine_cuthead.pdf", width = 15, height = 18, units = "cm")

knitr::include_graphics("fig/results4/PNG/cytisine_cuthead_2.png")
  

\alpha-Bgtx staining ####{#bgtxstaining}

To determine if FITC-\alpha-bgtx binds to native nAChRs, an isolated pharynx was used (Section @ref(fitcmethod) and Figure @ref(fig:exposed-pharynx-label)). In this preparation, there is no cuticular barrier, therefore, \alpha-Bgtx should access to the extracellular nAChR binding sites in the cell membrane of the pharyngeal muscle. Pharynxes were incubated with \alpha-Bgtx for 1 hour before being washed to remove unbound toxin.

Images of N2 wild-type and transgenic as well as eat-2 mutant and transgenic pharynxes were taken (Figure @ref(fig:staining-label)). There was either no fluorescence (12 out or 21 preparations) or very weak fluorescence in the corpus and/or terminal bulb of the wild-type and mutant strains. In comparison, exposure of transgenic pharynxes in which human \alpha7 was expressed in both C. elegans N2 wild-type and eat-2 mutant strains led to strong fluorescence in the pharynx. Comparison of the fluorescence localisation to the localisation of muscle cells in the pharynx (Figure @ref(fig:pharyngeal-muscle-label)) showed that the fluorescence was selectively present in the terminal bulb and largely localised to pm7 and pm8 muscle cells. Weak fluorescence was also observed in the isthmus and corpus, however this was inconsistent among preparations (6 out of 14).

(ref:staining) The staining of C. elegans pharynxes with FITC-bungarotoxin (FITC-bgtx). Representative images of the isolated pharynxes of N2 wild-type, eat-2 mutant, and human \alpha7 nAChR expressing transgenic worms (N2::CHRNA7 and eat-2::CHRNA7). Isolated pharynxes were incubated with FITC-\alpha-Bgtx. 1 hour later, they were washed for 1 hour and imaged immediately after. Bright field (left column) and fluorescent images (middle column) were taken and superimposed (right column). Note that fluorescence posterior to the terminal bulb may represent staining of the extrapharyngeal structures [@mckay2004]. Scale bar (5 \muM) is in the second top image on the left.

knitr::include_graphics("fig/results4/PNG/bgtx_1.png")

Discussion

Understanding the molecular basis of the mode of action of insecticides is the first step towards development of more selective compounds. Neonicotinoids act by targeting nAChRs, but the receptor subunit specificity remains to be elucidated. Several other heterologous systems have been used for the expression of exogenous nAChR [@millar2009a]. However, to enable pharmacological characterisation of nAChRs, we focused on using the C. elegans system.

C. elegans' pharynx is an attractive platform firstly because it has low relative sensitivity to neonicotinoids (Chapter 4 and 5). Secondly, it expresses chaperone proteins including RIC-3 and UNC-50 that are known to be important in aiding the maturation of nAChRs [@millar2008a; @eimer2007]. Additionally, C. elegans has been previously used for the recombinant expression of ion channels, including potassium-activated calcium channels [@crisford2011] and nAChRs [@sloan2015]. Lastly, it allows for scoring of the functionality of introduced receptors in behavioural assays.

Eat-2 as a genetic background for functional nAChR expression

EAT-2 nAChR subunit is the single determinant of the fast pharyngeal function in the pharynx. Literature suggest that the eat-2 C. elegans mutant has profoundly altered feeding and disrupted cholinergic neurotransmission in the pharynx [@raizen1995]. These can be rescued by the re-introduction of the EAT-2 receptor into the pharyngeal muscle [@mckay2004]. Thus, the selective expression of the nAChR at the muscle should be a platform for the functional expression of the introduced receptor. However, eat-2 transgenic strain used in @mckay2004 are no longer available and data supporting rescue was not provided in the publication. Therefore, the first step in this study was to generate eat-2 rescue of C. elegans. Worms were injected with a DNA construct containing eat-2 cDNA downstream of myo-2 promoter. As a result, the expression of the nAChR was driven in the pharyngeal muscle of C. elegans.

The eat-2 mutant and rescue strains were analysed behaviourally and pharmacologically. The intact mutant worms pump on food 70 % less than the wild-type. Eat-2 worms are also relatively insensitive to the application of 5-HT in the cut-head pharyngeal pumping assays. Introduction of eat-2 partially rescued the feeding phenotype of the mutant as measured by counting the number of pharyngeal pumps of the intact worms on the food patch. The insensitivity of the pharynx to 5-HT was reversed.

These results support the argument from Chapter 2, that EAT-2 nAChR is a major driver of the feeding response and the 5-HT induced pharyngeal response in worms. Whilst the 5-HT sensitivity was restored fully (5-HT induced pumping rate in wild-type was 3.00 Hz in comparison to 2.97 in transgenic lines), the pumping rate on food was not. Rescue lines pumped at rate slightly lower in comparison to the wild-type (3.12 vs 4.65 Hz). This lack of full rescue in feeding assay may suggest there are some differences in which pharyngeal pumping is elevated in response to food and 5-HT. Indeed, exogenous application of 5-HT acts directly onto pharyngeal nervous system to stimulate pumping.

In contrast, food stimulates MC and M4 indirectly. Initially it was thought that food evoked response was mediated by 5-HT released from NSM neurons [@horvitz1982]. More recently it was shown that the release of 5-HT selectively from ADF neurons is sufficient to drive the feeding response [@cunningham2012]. 5-HT released from ADF activates ADJ neurons and MC and M3 neurons. MC and M3 release ACh leading to pharyngeal response. Therefore the 5-HT elevation caused by exogenous 5-HT application is likely to be more diffused in comparison to the 5-HT levels in response to food. 5-HT application in the presence of food causes a small increase in the pumping rate, indicative of the presence of circuits regulating feeding in response to 5-HT but not activated in the presence of food.

Although the reversal of the feeding deficiency and 5-HT insensitivity in rescue lines suggests that the myo-2 promoter is suitable for the heterologous nAChR expression in the C. elegans pharynx, this promoter may also have some limitations. Transgenic strain carrying myo2-GFP reporter gene, expresses GFP in all muscles of the pharynx [@altun2009a and (Figure @ref(fig:pharyngeal-muscle-label)]. In contrast, the EAT-2 receptor in native worms is expressed at the NMJ of the MC and pm4 muscle (reference). Therefore, cellular EAT-2 expression driven by the native promoter, may be much more restricted in comparison to the expression driven by myo-2 promoter.

No apparent functionality of human \alpha7 receptors in the eat-2 mutant pharynx

To determine whether exogenous nAChR can be successful expressed in C. elegans, human \alpha7 receptor was introduced into the pharynx. This receptor protein was chosen because it is known to function as a homopentamer, its pharmacology has been well studies, and there are a number of \alpha7 selective compounds which could be used to detect their functionality [@Mazurov2006].

A DNA construct containing \alpha7 nAChR cDNA was generated and authenticated by sequencing. This was used to generate transgenic lines that should drive expression in the pharyngeal muscle of N2 and eat-2 strains. Introduction of human receptor into the eat-2 pharynx did not rescue the feeding phenotype, nor did it reverse the 5-HT insensitivity. This could suggest that \alpha7 may not be capable of performing the function of EAT-2 possibly due to the biological and molecular distinct nature of the pharynx.

One of the unique feature of the pharynx is the presence of acetylcholine-gated chloride channels, expressed in cholinergic and glutamatergic neurons [@pereira2015; @takayanagi-kiya2016], which are involved in the regulation of the synaptic release and inhibition of the neurons they are expressed in.

The properties of EAT-2 channel differ from those of \alpha7. In contrast to \alpha7, EAT-2 is classified as a non-\alpha subunit, due to the lack of vicinal cysteine in the extracellular domain. Typically, non- \alpha subunit must assembly with \alpha subunit to form a functional receptor, however, EAT-2 may function as a homooligomer, as observed by a current was elicited in response to application of nAChR agonist on the Xenopus oocyte injected with eat-2 (Lindy Holden-Dye, personal communication).

The function of the EAT-2 is dependant on EAT-18 [@mckay2004]. EAT-18 is a single pass transmembrane protein of unclear function, but it seems critical in eliciting feeding response [@mckay2004]. The native EAT-2 channels are localised to the pm4 muscle of the pharynx [@mckay2004] in the juxtaposition to cholinergic MC neuron [@albertson1976]. Expressed \alpha7 is mainly localised to pm7 and pm8 which make synaptic connections with cholinergic motor neuron M5 [@albertson1976].

The EC50 for ACh acting on EAT-18-containing receptors is not known, but these receptors are likely activated by the phasically and synaptically released agonist. The EC50 of ACh on heterologously expressed \alpha7 is 173 \muM [@papke2002]. The concentration of ACh in the pharynx may not be adequate to activate human receptors in response to 5-HT or food.

Taken together, the cellular environment, the differences in structure and functional properties as well as the likely different localisation of EAT-2 and \alpha7 receptors in the pharynx, may account for inability of the human receptor to perform the function of EAT-2 in the pharynx. If so, the feeding and 5-HT assays are not appropriate for the detection of \alpha7 functionality.

Distinct response of the N2 transgenic worms to cytisine.

An alternative approach was taken, in which human receptor was introduced into the pharynx of the wild-type worm. \alpha7 is Ca^2+^ specific channel, so one might expect its ability to couple to muscle upon ACh activation. In addition, wide expression of \alpha7 driven by the myo-2 promoter might result in the wide activation of ACh receptor, resulting in feeding behaviour that which could result in abnormal rate of growth [@halevi2002]. However, development of transgenic lines was normal and there was no subtle feeding phenotype observed.

Further experiments were carried out to determine whether pharmacology of \alpha7 receptor can be imposed on the C. elegans pharynx. In the presence of exogenous receptor that is selectively disrupted upon the application of the pharmacological agent, a change in pharyngeal response is expected. The function of the pharynx was scored in the presence of a series of nAChR agonist: acetylcholine, nicotine, cytisine and choline. The latter are selective \alpha7 agonists with the EC50 on the heterologously expressed \alpha7 receptors at 14.3 and 565 \muM, respectively [@chavez-noriega1997; @briggs1996]. The ability of these compounds to inhibit 5-HT induced pharyngeal responses of cut heads were examined.

No differences between the wild-type and transgenic lines were noted in response to acetylcholine, nicotine and choline. The intrinsic sensitivity of the pharynx to these compounds precludes these experiment from being diagnostic for human \alpha7 expression.

Investigating the effects of cytisine on wild-type and transgenic lines shows an interesting difference in the kinetics of the response at a single dose of 50 \muM. Following rapid inhibition of pumping, a pumping recovery was observed in the continual presence of cytisine in both genetic backgrounds. In wild-type, the recovery was slow. In contrast, transgenic lines recovered rapidly. However, it is unclear whether the recovery is due to cytisine acting on \alpha7, since these receptors desensitise rapidly in the presence of agonist and do not recover until the drug is washed off [@briggs1998].

Overall the intrinsic sensitivity of the pharynx to nAChR agonists confounds this approach to determine expression of endogenous receptor but it hints at distinct signature in terms of dynamic of the receptor activation in response to cytisine. Further experiments should be carried out to characterise the responses of the wild-type and transgenic lines in experiments emitting the 5-HT stimulation. The EPG approach should be taken to observe the early time points effects, prior to receptor desensitisation. The effects of choline and other selective nAChRs should be investigated on the wild-type and transgenic worms.

\alpha7 receptors are expressed on the surface of the pharyngeal muscle.

The lack of significant behavioural and pharmacological differential between the transgenic and control strains suggests lack of functionality of these receptors in the C. elegans pharynx, or the inability to resolve the functional human receptor. To independently assess whether \alpha7 is expressed, staining of isolated pharynxes with conjugated \alpha7 nAChR selective antagonist FITC-\alpha-bgtx was carried out.

The lack of intense staining in wild-type suggest that pharynx does not endogenously express \alpha7-Bgtx sensitivity. This is in contrast with the body wall muscle where ACR-16 is readily detected by \alpha-bgtx [@jensen2012]. This reinforces the distinct nature of pharyngeal cholinergic receptors. In contrast, the robust detection of \alpha-bgtx-binding in excised transgenic heads was observed. \alpha-Bgtx can bind to partially assembled receptors. X-ray crystal structure of human \alpha1 shows \alpha-Bgtx binding to the extracellular domain of a single subunit, suggesting, a fully assembled receptor is not necessary for the binding of this antagonist [@dellisanti2007]. This may suggest that the expressed \alpha7 subunits are monomeric. However, nAChR assembly is strictly regulated in the cell [@crespi2018]. Incorrectly assembled receptors are degraded [@brodsky1999] or accumulate inside the cell [@han2000. In this study, there was no detergent permeabilization, thus, the robust \alpha-Bgtx staining is likely concentrated to extracellular cell surface. This favours the idea that staining is to the cell surface homopentamers.

The \alpha-bgtx staining of \alpha7 receptors is consistently localised to pm7 and pm8 muscle cells, and inconsistently in other muscle cells of the pharynx. The receptor expression is driven by the myo-2 promoter, which should result in protein production in all muscle cells of the pharynx [@altun2009a], however selectivity has been observed previously (Anna Crisford, personal communication). In the endogenous system, EAT-2 functions at pm4, suggesting eat-2 promoter should be used to avoid restriction of endogenous promoter expression.