06-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]. 

```{r heterologous-expression-systems, echo=FALSE, warning=FALSE, message=FALSE}

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 

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. 

<!-- This paper also explains why Xenopus and voltage clamp may not be the best for measuring EC50 for these receptors. tis paper also lists EC50 from many papers! note that choline at 15 uM could activate native receptor (predicted to be alpha7 based on characteristics) in human monocyte-derived macrophages [@baez-pagan2015].  -->

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. 

<!-- Functional expression of insect receptors has been largely unsuccessful. Common problems associated with the heterologous expression of proteins in Xenopus or cell lines are low yields and incorrect processing.  -->

<!-- @jensen2012 -->

<!-- tudies. x-ray studies, radiolabelled.  -->



<!-- Note that alpha7 is abundandtly exprssed in the hypocapus [@].  -->


<!-- [@toro1994] tHIS PAPER SHOWS THAT ALPHA7 IS EXPRESSED IN VIRTUALLY ALL REGIONS OF TE BRAIN, CORRELATED WITH THE GBTX RADIOGRAPH [@clarke1985] - USSED IMMUNOCYTOCHEMISTRY Radiolabelled bgtx bind to sites in the brain sections consistent with ACh and nicotine.  -->

<!-- , gold-bgtx labelling and electron micrographs [doi:10.1016/j.jneumeth.2003.11.001] and immunoreactivity [@10.1523/JNEUROSCI.21-20-07993.2001].  -->

<!-- Radiolabelled bgtx bind to native receptors in the brain [@chen1997].  -->

#### 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. 
<!-- Heterologous co-expression of  RIC-3 with mammalian $\alpha7$ and Drosophila $\alpha5$ resulted in formation of binding sites, as shown by labelling with radiolabelled ligand binding of classical nAChR ligands [@lansdell2008; @lansdell2012]. Co-expression of *C. elegans* RIC-3 protein with heterolously expressed mammalian $\alpha7$ resulted in increased functional expression of receptors [@williams2005]. Therefore, RIC-3 is important in the maturation of worm, mammalian and insect 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]. 

<!-- [125I]a-Bgt autoradiography of brain slices and immunoblotting as shown by of extracted membranes with NACHO-specific antibodies shown Expressed ,  (The nAChR-current was detectable in transfected with alpha7 cells was detectable, upon co-expression of NACHO. Co-transefction of cells with tagged Extraceulllular domain resulted in surface labelling in co-transfected with NACHO cells, and not in the absence of NACHO. Thus, NACHO promoted cell-surface expression. . Note that NACHO does not assembly with alpah7 as shown by immunoprecipitation with Abs specific to the receptor (no detection of NACHO by Abs). Conversly, pulldown assay with Abs specific to NACHO - no bgtx binding. NACHO also involved in maturation of  -->

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 maturaton, stechiometry, 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* expresses proteins important in nAChR maturation ###{#cematnachr}

Heterologous expression of receptor channels requires an appropriate cellular environment to enable receptor maturation and functioning at the cell surface. Expression of nAChRs is a complex process during which individual subunits come together, assemble into a correctly folded pentamer and are transported to the plasma membrane where they function. *C. elegans* expresses many proteins involved in these processes. 

Identification and heterologous expression of *C. elegans* nAChRs greatly contributed to the understanding of proteins involved in the biogenesis of these ion channels. There are two nAChRs expressed at the NMJ of the body wall muscle of *C. elegans*: ACR-16 homopentamic receptor and a heteropentameric receptor composed of UNC-29, UNC-38, UNC-63, LEV-1 and LEV-8. These receptors are classified based on pharmacology into nicotine- and levamisole- sensitive and named N-type and L-type, respectively. DEG-3/DES-2 nAChRs are the neuronal receptors [@treinin1998], whereas EAT-2 containing nAChRs are expressed in the pharyngeal muscle [@mckay2004]. 

<!-- *C. elegans* is a powerfull tool in which the function and pharmacological properties of proteins can be studied by a combination of genetic, behavioural and cellular approaches (Section \@ref(genmanip) \@ref(analytical_behaviour)). -->

#### RIC-3 ####{#ric-3celegans}

RIC-3 (resistant to inhibitors of cholinesterase-3) is an evolutionary conserved, ER-residing [@roncarati2006; @alexander2010] transmembrane protein [@wang2009]. In *C. elegans*, it is ubiquitously expressed in most (if not all) neurons, pharyngeal and body wall muscle in worms [@halevi2002]. The predicted topology of *C. elegans* RIC-3 has 2 transmembrane domains and 3 coiled-coils. The *C. elegans ric-3* mutant has impaired locomotor behaviour, resistance to levamisole [@miller1996] and has an impaired nAChR responses, as measured by electrophysiological recording from the body wall muscle [@halevi2002]. The *C. elegans ric-3* mutant has impaired cholinergic nerurotransmission, as shown by the lack of the cholinergic component of the EPG recording resulting in significantly retarded pharyngeal pumping and starved appearance [@halevi2002].

Heterologous expression of *C. elegans* nAChR in *Xenopus* oocytes provides evidence for their function in receptor maturation. Choline-evoked currents of neuronal DEG-3/DES-2 receptors increased by 5-fold upon RIC-3 co-expression [@halevi2002]. 

This was markedly improved when acr-16 was co-expressed with RIC-3 [@ballivet1996]. The role of RIC-3 in the maturation of this receptor type was also demonstrated *in-vivo*. The nicotine induced current at the body wall muscle was markedly reduced in ric-3 mutant, in comparison to wild-type [@halevi2002]. 

*C. elegans* RIC-3 can also promote maturation of mammalian $\alpha7$ channels. RIC-3 co-expression improved $\alpha-7$ function in *Xenopus* oocytes as shown by enhanced choline- and acetylcholine-evoked currents and cell-surface binding of radiolabelled $\alpha$-Bgtx [@lansdell2005; @williams2005]. RIC-3 also enabled the expression of $\alpha-7$ in otherwise non-permissive insect cell lines [@lansdell2008]. It not only promotes the heterologous cell-surface expression of mammalian, but it also increases the expression of insect chimera nAChRs [@lansdell2012].

Successful heterologous expression of L-type *C. elegans* receptor in *Xenopus* oocytes upon co-expression of RIC-3 and two other proteins, viz. UNC-50 and UNC-74, revealed other components important in the process of maturation of nAChRs [@boulin2008]. 

#### UNC-50 ####{#unc50}

UNC-50 is an ortholog of evolutionary conserved GMH1 protein. In *C. elegans* it was first identified in behavioural and pharmacological screens of *C. elegans* mutants. Several phenotypes have been described including: uncoordinated movement [@lewis1980] reduced binding of radiolabelled levamisole to the membrane fractions [@lewis1987], resistance to levamisole in behavioural assays [@lewis1987; @abiusi2017] and no responses of L-type nAChRs at the body wall muscle to levamisole [@eimer2007]. The lack of cell-surface staining from antibodies against UNC-29 [@eimer2007] in *unc-50* mutant comfirmed the role of UNC-50 in nAChR maturation. *unc-50* mutant is also characterised by an increased lysozyme-dependent degradation of nAChRs, suggesting its preventative role in this process. UNC-50 is predicted to be expressed in the Golgi. Expression of GFP::UNC-50 fusion protein resulted in fluorescence typical of the localisation to this organelle [@eimer2007].

#### UNC-74 ####{#unc74}

UNC-74 is closely related to the human TMX3 protein which is thought to be ER-associated [@haugstetter2005]. Reduced radiolabelled meta-aminolevamisole binding to membrane fraction of *C. elegans* mutant [@lewis1987] combined with its role in expression of L-type receptor in *Xenopus* oocytes [@boulin2008] confirms its role in receptor maturation. 

#### EAT-18 ####{#eat18}

EAT-18 is thought to be required for the function of pharyngeal nAChRs. It consists of a single transmembrane and an extracellular domain. Transgenic worms expressing EAT-18::GFP fusion protein reveal fluorescence in the pharynx with the strongest signal in the muscle, but also in the pharyngeal neuron M5 and unidentified 5 to 6 extrapharyngeal neurons [@mckay2004]. *eat-18* mutants are deficient in pumping and resistant to high concentraton of nicotine, supporting the function of EAT-18 in cholinergic neurotransmission of the pharynx [@raizen1995]. The association of *eat-18* with pharyngeal nAChR was indicated by comparison of the staining in the wild-type and *eat-18* mutant strains. Injection into the pseudocoelom of radiolabelled $\alpha$-bgtx resulted in straining of the pharynx. This was however abolished in the mutant strain [@mckay2004]. In addition, the expression of EAT-2 in *eat-2* was normal, suggesting EAT-18 is not involved in the trafficing of this receptor. It has been proposed that EAT-18 co-assembles with EAT-2 due to their common pharyngeal phenotypes in mutant strains and common cellular localisation in the pharyngeal muscle [@mckay2004]. Recently, successful expression of eat-2 co-assembled with eat-18 has been shown in *Xenopus* oocytes (personal communication).

### Biochemical methods to assess expression of the $\alpha7$ nAChR transgene in *C. elegans*

The cellular localisation of nAChRs expressed in *C. elegans* can be detected by an array of methods, such as 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.

<!-- Xenopus oocyte was the first expression system used for the production of recombinant nAChRs. Expression of proteins in Xenopus oocytes provided an invaluable insight into the stechiometry and pharmacological properties of a number of receptors, including *Torpedo* muscle and human neuronal $\alpha$ 7 nAChRs (reviewed in @millar2009). Whilst the expression of some invertebrate subunits has been succesful, such as *C. elegans* ACR-16 [@ballivet1996] and acr-2 receptor co-expressed with UNC-38 [@squire1995], the expression of insect receptors opposed difficulties.  -->

<!-- A major drawback of nAChR expression in Xenopus oocytes is that the ionic properties of formed channels can be altered [@lewis1997] and hence not reflect their functionality in the native environment. Additionally, Xenopus is a single cell technique, so the preparation is short lived and cannot be propagated for future experiment. The process of recombinant protein expression is typically slow and laborious. -->

<!-- ### Cell lines  -->

<!-- Cell lines are an attractive platform for heterologous expression of proteins for many reasons. They allow for temporal control of expression. Protein can be expressed either constitutively or trasiently (ref). Cell lines may also provide a more favourable cellular environemnt than the Xenopus oocytes for the expession of nAChRs (ref). In addition, larger quantitities of recombinant protein can be generated and hence heterologous expression quantified biochemically by immunoprecipitation and radiolabelled ligand binding studies, whereas function scored using electrophysiological approaches. The expression of full lenth vertebrate and invertebrate subunits in cell lines has been difficult. For example, expression of mammalian $\alpha$ 7 in human HEK-293 and tsA-201 cells was unsuccesful [@lansdell1997; @lansdell2008]. @lansdell2012 introduced 76 Drosophila subunit into the Drosophila S2 cell lines singularly and in different combinations with no success. Expression of Myzus $\alpha$ 1 - 4 in S2 failed [@huang2000], so did the expression of Nl $\alpha$ 1 - 4 [@liu2005], and Cat flee $\alpha$ 1 - 2 and 7, $\beta$ 1 [@bass2006].  -->

<!-- Over the years, several approaches have been adopted to improve functional expression of nAChR in cell lines, expanding the knowledge of important factor for receptor folding and assembly.  -->

<!-- Typically, HEK-293 or tsA-201 human cells or S2 Drosophila cells are used for the expression of nAChRs. The choice of cell lines is crucial as the expression of nAChR is host specific and varies between preparations. For example, mammalian $\alpha$ 7 was succesfully expressed in 9 different cell lines, but the function was only detected in 2 cell line types [@cooper1997]. -->

<!-- The expression of subunits is also temperature sensitive. Expression of Drosophila $\alpha$ 1 or 2 was achieved in HEK-293 cells at 25 but not at 37 &deg;C [@lansdell1997].  -->

<!-- Difficulties in expression of vertebrate and invertebrate subunits could be overcome by generating chimera genes. Detectable expression of human $\alpha$ 7, Drosophila $\alpha$ 6 or $\alpha$ 5 nAChRs was achieved by the expression of a fusion protein containing N-terminal extracellular domain of the nAChR with transmembrane and C terminal domain from the 5-HT~3~ recepor [@eisele1993; @lansdell2004; @lansdell2012].  -->

<!-- The expression of receptors is also improved by the co-expression of nAChR subunits with other proteins. This includes chaperon proteins and $\beta$ subunits of nAChRs.  -->


<!-- check this paper out [@bao2018] -->
<!-- @lansdell1997 succesfully detetced heterologous expression of receptors following co-transfection of cell line with Drosophila $\alpha$ 1 and 2 with rat $\beta$ 2. Ligand binding was also achieved following co-transfection of cells with Myzus $\alpha$ 1 1 3 or Nl $\alpha$ 1 - 3 with rat $\beta$ 2 [@huang2000; @liu2005]. Binding of nAChR compounds to chimera proteins co-expressed with beta subunit was also achieved. Acetylcholine bound to receptors formed from cat flee N-terminal $\alpha$ 1 or 3 /  C-terminal Drosophila $\alpha$ 2 chimera and rat $\beta$ 2 subunit. [@bass2006].  -->

<!-- ###Whole animal preparations -->

<!-- Mice are used in knock out and knock in studies. Knock out mice do not express a functional protein; this approach is useful when studying the function of a native individual nAChRs (reviewed in @drago2003). Knock in techniques allow for the investigation of the effects of mutations in nAChRs on mice models. These mutations can be gain of function or diseaese-associating [@drago2003]. More recently, a technique for cell specific re-expression of nAChRs in knock out has been developed and used to study the function of receptors in specific cholineric circuits [@maskos2005]. This technique is useful for studying the function of native nAChR and for economical and ethical reasons, not used to characterise foreign receptors. -->

<!-- Both Drosophila and *C. elegans* are an attractive invertebarate models for heterologous protein expression. They are both cheap to cultivate, have fast life cycles, their genomes have been sequenced and annotated and there is no ethical issues associated with the genetic manipulation.  -->
<!-- In addition, there are well described genetic techniques used for the generation of transgenic or mutant strains. The phenotypical analysis of trangenic lines is simple, because many of the typical wild-type behaviours have been characterised. An additional advantage of *C. elegans* is that it expresses RIC-3 protein, important for nAChR folding and assembly (ref) which aids succesful heterologous expression of nAChRs (ref).  -->

<!-- Drosophila and *C. elegans* have been succesfully used for the expression and pharmacological characterisation of recombinant nAChR. For example, intoduction of nAChRs from house fly or the cabbage moth into Drosophila mutant, rescued resistance to insecticide sponosyd [@sloan2015]. Whereas expression of nAChRs from parasitic worms in *C. elegans* mutant rescued levamisole-resistance [@sloan2015]. -->

<!-- Transformation with exhogenous protein typically uses plasmid DNA containing a gene of interest. However, studies in which *C. elegans* gene function is studied use cosmids of YACs. -->
<!-- There are several elements of the bacterial plasmid enabling amplification in *E. coli* and purificiation. One of the most important elements is the promoter. Promoter sequence determines the expression patter on the gene of interest.   -->

<!-- Use of phenotypic markers for trasnformation to enable easy identification of the tranformed worms under the microscope. Typically, a marker that the expression will not be interfered with is chosen. First co-marker used was rol-6, a dominant gene encoding for cuticular collagen. Injection into the *C. elegans* wild-type induces a roller phenotype in which the worm has altered movement [@mello1991] easily identified under the dissecting microscope. Other commonly used markers are fluorescent reporter genes. One such gene is green-flourescent protein (GFP). *C. elegans* is transparent, therefore alive organism can be assayed. Therefore by either fusing the gene of interest to the marker, or co-injecting another plasmid DNA this can be achieved. Worms appear green, when viewed under the fluorescence microscope.  -->

<!-- 90% of animals expressig the marker, also express the co-injected gene. [@mello1991] as detected by phenotypical and biochemical techniques. Rollers were stained for beta-galactosidase. -->

#### *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. 

<!-- *C. elegans* pharynx is an attractive platform for investigation of drug sensitivity due to the availbailabiloty of assays scoring the pharyngeal function and the genetic knowledge of the pharyngeal muscle function.  -->

<!-- The effects of drug application on the pharyngeal function can be investigated in the behavioural or cellular assays (Chapter 3 and 4). -->

<!-- Whereas function of the pharynx is mediated by nAChR containing EAT-2 subunit. EAT-2 nAChR subunit is present at the NMJ between the MC neuron and pharyngeal muscles of the metacorpus (pm4) and isthmus (pm5) [@mckay2004]. *Eat-2 C. elegans* knockout has profound behavioral and pharmacological phenotype (Chapter 4). Therefore, selective expression of nAChRs at the post-synapse of the MC neuron of *eat-2 C. elegans* mutant should be a useful platform for the nAChR functional expression. -->

### Chapter aim

The aim of this chapter is to develop the *C. elegans'* pharynx as a platform to heterologously expression and pharmacologically characterise exogenous nAChRs.

## Results

```{r echo=FALSE, results="hide", include=FALSE}
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. 

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]. 

<!-- Receptors were expressed in nAChR mutant worms, to determine if mutant phenotype can be reversed. *Eat-2* was used because it is deficient in expression of a single EAT-2 nAChR in the pharynx and exhibits marked behavioural and pharmacological phenotype (ref to chapter 2). Native EAT-2 nAChRs were expressed in the pharyngeal muscle of *eat-2* mutant to determine if myo-2 is an appropriate promoter and if the *eat-2* phenotype can be rescued. -->

### 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).

```{r eat2-pcr-label, fig.cap="(ref:eat2-pcr)", fig.align='center', out.height = '80%', fig.scap = "Amplification of \\textit{eat-2} gene.", echo=FALSE}
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.

```{r eat-topo-label, fig.cap="(ref:eat-topo)", fig.scap = "Insertion of eat-2 into the TOPO vector.", fig.align='center', out.height = '80%', echo=FALSE}
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).

```{r topo-eat2-pcr-label, fig.cap="(ref:topo-eat2-pcr)", fig.scap = "PCR of eat-2 from the TOPO vector.", fig.align='center',out.height = '80%', echo=FALSE}
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.

```{r pdest-eat2-label, fig.cap="(ref:pdest-eat2)", fig.scap ="The generation of the vector for the expression of EAT-2 nAChR in the \\textit{C. elegans} pharynx.", out.width='70%', fig.align='center', echo=FALSE}
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.

```{r transgenic-feeding-label, fig.cap="(ref:transgenic-feeding)", fig.scap = "Pharyngeal pumping of \\textit{C. elegans} nicotinic acetylcholine receptor mutant and rescue strains.", fig.align='center', echo=FALSE}
# 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 $\mu$M 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 $\mu$M 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.

```{r eat-2-rescue-label, fig.cap = "(ref:eat-2-rescue)", fig.scap = "The effects of 5-HT on wild-type, \\textit{eat-2} mutant and \\textit{eat-2} rescue strains.", fig.align='center', echo=FALSE}
#   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).

```{r CHRNA7-amplification2-label, fig.cap="(ref:CHRNA7-amplification2)", fig.scap = "Amplification of the gene encoding for human $\\alpha7$ subunit of nAChR.", fig.align='center', out.height = '80%', echo=FALSE}
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).

```{r PCR8-CHRNA7-label, fig.cap="(ref:PCR8-CHRNA7)", fig.scap = "Insertion of CHRNA7 into the TOPO vector.", fig.align='center', echo=FALSE}
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).

```{r pdest-chrna7-cloning-label, fig.cap="(ref:pdest-chrna7-cloning)", fig.scap = "Generation of the vector for the expression of $\\alpha7$ nAChR in the \\textit{C. elegans} pharynx.", fig.align='center', out.height = '80%', echo=FALSE, message=FALSE}
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.

```{r feeding-chrna7-transgenic-label, fig.cap="(ref:feeding-chrna7-transgenic)", fig.scap = "Effects of human $\\alpha7$ nAChR expression on the feeding phenotype of \\textit{C. elegans}.", fig.align='center', echo=FALSE}
# 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 $\mu$M 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 $\mu$M 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.

```{r human-transgenic-rescue-label, fig.cap = "(ref:human-transgenic-rescue)", fig.scap = "Effects of human $\\alpha7$ nAChR expression on the 5-HT induced pharyngeal pumping of \\textit{C. elegans}.", fig.align='center', echo=FALSE}
#   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 $\mu$M 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 $\mu$M, 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 $\mu$M, 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 $\mu$M 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.

```{r nicotine-label, fig.cap="(ref:nicotine)", fig.scap = "The effects of human $\\alpha7$ nAChR expression on the nicotine-induced inhibition of 5-HT evoked pumping.", fig.align='center', echo=FALSE}

# 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 $\mu$M 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 $\mu$M. The IC50 of choline on transgenic line was 15 $\mu$M.

(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 $\mu$M 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.

```{r 5HTcholine-label, fig.cap="(ref:5HTcholine)", fig.scap = "The effects of human $\\alpha7$ nAChR expression on the choline-induced inhibition of 5-HT evoked pumping.", fig.align='center', echo=FALSE}
# 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 $\mu$M (Figure \@ref(fig:cytisine-label) a left panel). The two lowest doses had no effect on 5-HT induced pumping. 10 $\mu$M 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 $\mu$M 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 $\mu$M 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.

```{r cytisine-label, fig.cap="(ref:cytisine)", fig.scap = "The effects of human $\\alpha7$ nAChR expression on the cytisine-induced inhibition of 5-HT evoked pumping.", fig.align='center', echo=FALSE, fig.asp= 1.2}
# 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")
  
```


<!-- (ref:nicotineDR) The effects of human $\alpha$ 7 nAChR expression in the pharynx on the nicotine-induced pharyngeal pumping of *C. elegans*. N2 wild-type, N2 transgenic (N2::alpha7) and N2 transgenic control (N2::gfp) cut heads were exposed to varying concentrations of nicotine. The number of pharyngeal pumps over 30 s at 30 minute time points were taken and expressed in Hz. Data are mean $\pm$ SEM of 4-14 individual worms collected from paired experiemnts done on 2 days. For comparison, the maximum pumping achieved by 5-HT is shown in dashed line. -->

<!-- ```{r nicotineDR-label, fig.cap="(ref:nicotineDR)", echo=FALSE} -->
<!-- knitr::include_graphics("fig/results4/PNG/Nicotine_DR_30_mins.png") -->
<!-- ``` -->

<!-- (ref:choline) The effects of heterologous human nAChR expression in the pharynx on choline induced pharyngeal pumping. N2 wild-type, N2 transgenic (N2::alpha7) and N2 transgenic control (N2::gfp) cut heads were exposed to 1 mM choline. The number of pharyngeal pumps pre- (time point 0) and post-choline exposure was recorded over time. Pharyngeal pumps were scored by visual counting the number of pumps in 30 expressed in Hz. Data are mean $\pm$ SEM of 9-17 individual worms collected from paired experiemnts done on 2 days. -->

<!-- ```{r choline-label, fig.cap="(ref:choline)", echo=FALSE} -->
<!-- plot10 <- filter(stats, experiment == "choline-cuthead") %>%  -->
<!--   group_by(Strain, Time)%>%  -->
<!--   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_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")) -->
<!-- plot10 -->
<!-- ``` -->

<!-- Application of 5-HT prior to exposure to drugs may musk the effects of drugs. Thus, in the second set of experiments, drugs were applied on ustimulated pharynx. The efefects of nicotine and acetylcholine on unstimulated pharynx of N2 wild-type and $\alpha$ 7 expressing worms were tested in electrophysiological experiments. Cut heads were perfused with drugs for 5 minutes and the effects on peak pumping frequency was tested.  -->

<!-- It was previously shown that acetylcholine stimulated pumping frequency with the EC50 of 22 $\mu$M (Figure \@ref(fig:ach-train-label)). 1 $\mu$M is a dose close to the EC~50~ and it was chosen to determine if introduction of $\alpha7$ into the wild-type pharynx alters pharyngeal responses to ACh. There was no difference between the pumping frequency of N2 wild type and transgenic strain (Figure \@ref(fig:epg-acetylcholine-transg-label)). The pumping frequency was 0.67 and 0.47, respectively. -->

<!-- (ref:epg-acetylcholine-transg) The effects of human $\alpha7$ nAChR expression on acetylcholine induced EPG frequency. N2 wild-type and N2 transgenic (N2::alpha7) cut heads were perfused with 1 $\mu$M acetylcholine for 5 minutes. Data are mean $\pm$ SEM of 3 individual worms collected from paired experiemnts done on 2 days. -->

<!-- ```{r epg-acetylcholine-transg-label, fig.cap="(ref:epg-acetylcholine-transg)", echo=FALSE} -->
<!-- plot11 <- stats %>%  -->
<!--   filter(experiment == "ach_epg") %>%  -->
<!--   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("peak pumping (HZ)") + -->
<!--   ylim(0, 1) + -->
<!--   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)) -->
<!-- plot11 -->
<!-- ``` -->

<!-- Nicotine induces stimulation of the EPG frequency of wild-type worms with the EC~50~ of 2.7 $\mu$M (Rifure \@ref(fig:epg-nic-graphs-label)). 1 $\mu$M is close to EC~50~ and it weakly stimulates pumping for the duration of the EPG recording (Figure \@ref(fig:nic-epg-traces-label)). Therefore N2 wild-type and $\alpha$ 7 expressing cut heads were exposed to 1 $\mu$M nicotine to determine if expression of human receptor alters pharyngeal responses to this drug. There was no difference between nicotine-induced pumping frequency between the strains (Figure \@ref(fig:nicotine-epg-transg-label). The peak frequency was 1.1 $\pm$ 0.4 in wild-type and 2.1  $\pm$ 0.6 Hz in the transgenic worms.  -->

<!-- (ref:nicotine-epg-transg) The effects of human $\alpha$ 7 nAChR expression on nicotine induced EPG frequency. N2 wild-type and N2 transgenic (N2::alpha7) cut heads were perfused with 1 $\mu$M nicotine for 5 minutes. The peak pumping rate in a 10s window was ploted. Data are mean $\pm$ SEM of 5-8 individual worms collected from experiemnts done on $\ge$ 3 days. -->

<!-- ```{r nicotine-epg-transg-label, fig.cap = "(ref:nicotine-epg-transg)", echo=FALSE} -->
<!-- plot12 <- stats %>%  -->
<!--   filter(experiment == "nic_epg") %>%  -->
<!--   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("peak 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)) -->
<!-- plot12 -->
<!-- ```
The work above utilized 3 distinct functional criteria to address the possible expression. Based on phenotypic rescue of the pharmacological sensitivity, there is little indication that the mammalian $\alpha7$ is expressed. However, the cytisine effects hint at change in sensitivity of the system. 

To better determine if the introduced human receptor is expressed in *C. elegans*, staining of pharynxes with FITC-$\alpha$-Bgtx was carried out. $\alpha$-Bgtx is a high affinity toxin that acts as a selective antagonist of human $\alpha7$ nAChRs and it binds to the extracellular domain of this receptor. $\alpha$-Bgtx conjugated with a fluorescent reagent can be utilized to histologically define expression and resolve sub-cellur localisation of $\alpha7$ receptor in mammalian tissue and *C. elegans* [@barnard1971, @jensen2012]. This technique has been used to shown that $\alpha$-Bgtx binds to receptors at the body wall muscle [@jensen2012] and to the nAChR axillary subunit EAT-18 in the pharynx [@mckay2004]. 

 However, it has little antagonistsic effect on the body wall receptors [@boulin2008, @abongwa2016] and on the pharyngeal receptors (preliminary data not shown). -->

#### $\alpha$-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 $\mu$M) is in the second top image on the left.

```{r staining-label, fig.cap="(ref:staining)", fig.scap = "The staining of \\textit{C. elegans} pharynxes with FITC-bungarotoxin (FITC-bgtx).", fig.align='center', echo = FALSE}
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. 
<!-- Specifically, it activates MC and M4 neurons [@raizen1995; @niacaris2003]. These neurons in turn release ACh which acts on EAT-2 nAChR to stimulate pumping [@mckay2004].  -->
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 EC~50~ 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 EC~50~ of ACh on heterologously expressed $\alpha7$ is 173 $\mu$M [@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 EC~50~ on the heterologously expressed  $\alpha7$ receptors at 14.3 and 565 $\mu$M, 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 $\mu$M. 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.

<!-- Bgtx-staining and pharyngeal pumping in the presence of cytisine experiments hint at the successful expression and function of endogenous receptor in *C. elegans*. However, it is also possible that $\alpha7$ is expressed in the muscle, but not functional.  -->

<!-- <!-- Another chaperon in Nacho which is a NACHO - a TM neuronal ER protein important in receptor folding, assembly, maturation and surface expression - [@gu2016] - (read! Brain a 7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO) --> 

<!-- <!-- Another possibility is that $\alpha$ 7 interacts with native nAChR subunits to form non-functional complexes [@millar2007]. Indeed, in human cell lines, heterologous expression of recombinant forms of heterometic $\alpha$ 7/$\beta$ 2 was noted, whereas in a native environment $\alpha$ 7 forms homopentameric channels.  --> 

<!-- Evidence suggests that the heterologous expression of mammalian $\alpha7$ varies between many types of cell lines [@cooper1997] and even between the isolates of the same cell lines [@blumenthal1997]. Similarly to results obtained here, @cooper1997 and @blumenthal1997 showed that the expression of nAChRs could be detected by staining with antibodies, but there was no binding of nAChR-compounds and thus no functional read-out was obtained. This highlights a complex nature of receptor function.  -->
<!-- <!-- processing and function. Sequences within N terminus, TM domains and N terminus all seem to play a role in nAChR folding and assembly (ref). Improved expression of functional nAChRs upon fusion of N-teminal domain of nAChR to C-termal domain of 5-HT receptor was achieved. This indicates that the interactions between the receptor and cellular proteins are crucial for the formation of biologically active receptor.  --> 
<!-- Proteomics analysis identified 55 potential $\alpha7$ 7 interacting partners [@paulo2009]. There proteins regulate many functions including receptor responsiveness, phosphorylation, clustering, folding and assembly (reviewed in @jones2010a). Some of these interacting partners may not be present in the *C. elegans* pharynx.  -->

<!-- Expression of the full lenght human $\alpha7$ on the cell surface without the function was also noted in mammalian cell lines. This was due to the innapropriate disulphide bond formation [@rakhilin1999].  -->

<!-- Taken together,  there are several reasons for which the functional $\alpha$ 7 expression in the pharynx may have failed. These data highlights the complexity of nAChR processing and assembly and the importance of the cellular environment in these processes. The succesfulness of the heterologous nAChR expression depends on the host and the receptor subunit. Alhough there is no data suggesting functionality of human nAChR in the pharynx, other receptors may exhibit function in this environment. For this reason, efforts into expression of invertebrate receptors in the *C. elegans* pharynx should be made.  -->