06-results-04.Rmd 90.9 KB
 mk11g11 committed Feb 19, 2020 1 # *C. elegans* pharynx as a platform for heterologous nAChR expression {#results-4} mk11g11 committed Sep 29, 2019 2 3 4 ## Introduction mk11g11 committed Feb 19, 2020 5 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. mk11g11 committed Sep 29, 2019 6 7 8 ### Biological systems for heterologous protein expression ###{#biologicalsystemfornachrexpression} mk11g11 committed Feb 19, 2020 9 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]. mk11g11 committed Sep 29, 2019 10 11 12 13 14 15 16 17 {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)), mk11g11 committed Feb 19, 2020 18 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"), mk11g11 committed Sep 29, 2019 19 20 21 22 23 24 25 26 27 28 29 30 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")  mk11g11 committed Feb 19, 2020 31 ### Properties of vertebrate $\alpha7$ nAChR mk11g11 committed Sep 29, 2019 32 mk11g11 committed Feb 19, 2020 33 One of the well-studied nAChR subunit is the vertebrate homomeric $\alpha7$. Heterologous expression of these receptors in *Xenopus* occytes and mammalian cell lines. mk11g11 committed Sep 29, 2019 34 mk11g11 committed Feb 19, 2020 35 #### $\alpha7$ nAChR is rapidly desensitising mk11g11 committed Sep 29, 2019 36 mk11g11 committed Feb 19, 2020 37 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]. mk11g11 committed Sep 29, 2019 38 mk11g11 committed Feb 19, 2020 39 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. mk11g11 committed Sep 29, 2019 40 mk11g11 committed Feb 19, 2020 41 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]. mk11g11 committed Sep 29, 2019 42 mk11g11 committed Feb 19, 2020 43 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. mk11g11 committed Sep 29, 2019 44 45 46 mk11g11 committed Feb 19, 2020 47 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]. mk11g11 committed Sep 29, 2019 48 49 50 #### Sensitive to $\alpha$-Bungarotoxin ($\alpha$-Bgtx) mk11g11 committed Feb 19, 2020 51 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]. mk11g11 committed Sep 29, 2019 52 mk11g11 committed Feb 19, 2020 53 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. mk11g11 committed Sep 29, 2019 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 #### Highly permeable to calcium ions ####{#capermeability} $\alpha7$ receptors are highly permeable to calcium ions [@seguela1993; @bertrand1993]. Historically, calcium ion permeability of nAChRs was measured by establishing the reversal potential of the agonist-evoked current by changing the concentration of calcium ions in the buffer and representing calcium ion flux as a function of sodium ion flux. Using this methods, is was established that there is up to 20-fold difference between calcium and sodium permeability of heterologously expressed $\alpha7$ receptors [@seguela1993]. More recently fluorescent calcium indicators [@neher1995] were used to measure calcium ion flux as a function of the entire transmembrane current. Calcium ions account for 11 % of the entire ionic conductance of the heterologously expressed $\alpha7$ receptors [@fucile2000]. In comparison to other nAChRs, $\alpha7$ has up to a 200-fold difference in calcium ion permeability when the channels are expressed heterologously [reviewed in @fucile2004]. #### Matured with the aid of NACHO and RIC-3 ####{#ric-3nacho} Mammalian RIC-3 is a small protein with 2 transmembrane domains and a single coiled-coil domain [@wang2009]. It is expressed in most brain regions, enriched in the regions common to the $\alpha7$ expression, namely the hippocampus and the cerebellum, as shown by *in-situ* hybridisation [@halevi2003]. Fluorescently tagged-RIC-3 localised to the ER and not the surface when expressed heterologously [@roncarati2006], whereas immunostaining of native RIC-3 in PC12 and hippocampal neurons showed co-localisation with neuronal and ER markers [@alexander2010], providing evidence that RIC-3 is an ER residing protein. RIC-3 has a role in receptor maturation. Co-expression of RIC-3 protein with heterologously expressed mammalian $\alpha7$ resulted in increased functional expression of this receptor, as measured by ACh-evoked current [@williams2005] and radiolabelled ligand binding [@lansdell2008]. Additionally, RIC-3 promotes association of nAChRs with proteins involved in post-translational modification, receptor trafficking and transport [@mulcahy2015]. Thus, RIC-3 promotes cell-surface expression of nAChRs. NACHO is an 18-kDa multi-pass protein ER protein expressed in neurons of hippocampus, cerebral cortex and the olfactory bulb [@gu2016]. ACh-evoked current and cell-surface labelling of heterologously expressed $\alpha7$ receptor were elicited upon co-transfection of cells with NACHO [@gu2016]. Absence of $\alpha7$ mediated current in the hippocampus of NACHO-knock-out mice, the lack of binding of classical antagonists as well as behavioural phenotype, consistent with the disruption of cholinergic neurotransmission [@matta2017] supports the role of NACHO in maturation of $\alpha7$ and other nAChRs including $\alpha4\beta2$, $\alpha3\beta2$ and $\alpha3\beta4$ [@matta2017]. Further experiments by @gu2016 and @matta2017, provided details of the interactions between the receptor, RIC-3 and NACHO. Transfection of HEK cells with $\alpha7$ and RIC-3 resulted in no surface expression, based on the lack of $\alpha$-bgtx or epibatidine binding. Surface expression was achieved when cells were co-transfected with $\alpha7$ and NACHO, and augmented by RIC-3. Based on these observatins it was proposed that was NACHO promotes early events in the receptor assembly, whereas RIC-3 in synergy with NACHO aids receptor maturation. [@matta2017]. RIC-3 may also aid interactions with many other proteins in the cells, as shown by the enhanced interactome of nAChR $\alpha7$ and other proteins in the cell upon co-expression of RIC-3 [@mulcahy2015]. mk11g11 committed Feb 19, 2020 91 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]. mk11g11 committed Sep 29, 2019 92 mk11g11 committed Feb 19, 2020 93 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. mk11g11 committed Sep 29, 2019 94 95 96 ### *C. elegans* expresses proteins important in nAChR maturation ###{#cematnachr} mk11g11 committed Feb 19, 2020 97 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. mk11g11 committed Sep 29, 2019 98 mk11g11 committed Feb 19, 2020 99 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]. mk11g11 committed Sep 29, 2019 100 101 102 103 104 #### RIC-3 ####{#ric-3celegans} mk11g11 committed Feb 19, 2020 105 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]. mk11g11 committed Sep 29, 2019 106 mk11g11 committed Feb 19, 2020 107 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]. mk11g11 committed Sep 29, 2019 108 mk11g11 committed Feb 19, 2020 109 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]. mk11g11 committed Oct 06, 2019 110 mk11g11 committed Feb 19, 2020 111 *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]. mk11g11 committed Oct 06, 2019 112 mk11g11 committed Feb 19, 2020 113 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]. mk11g11 committed Sep 29, 2019 114 115 116 #### UNC-50 ####{#unc50} mk11g11 committed Feb 19, 2020 117 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]. mk11g11 committed Sep 29, 2019 118 119 120 #### UNC-74 ####{#unc74} mk11g11 committed Feb 19, 2020 121 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. mk11g11 committed Sep 29, 2019 122 123 124 #### EAT-18 ####{#eat18} mk11g11 committed Feb 19, 2020 125 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). mk11g11 committed Sep 29, 2019 126 mk11g11 committed Feb 19, 2020 127 ### Biochemical methods to assess expression of the $\alpha7$ nAChR transgene in *C. elegans* mk11g11 committed Sep 29, 2019 128 mk11g11 committed Feb 19, 2020 129 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]. mk11g11 committed Sep 29, 2019 130 mk11g11 committed Feb 19, 2020 131 $\alpha$-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. mk11g11 committed Sep 29, 2019 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 #### *Eat-2* is a suitable genetic background for functional expression mk11g11 committed Oct 06, 2019 173 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. mk11g11 committed Sep 29, 2019 174 mk11g11 committed Oct 06, 2019 175 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. mk11g11 committed Sep 29, 2019 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 ### 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. mk11g11 committed Feb 19, 2020 203 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. mk11g11 committed Sep 29, 2019 204 mk11g11 committed Feb 19, 2020 205 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]. mk11g11 committed Sep 29, 2019 206 207 208 209 210 211 212 ### Heterologous expression of native EAT-2 nAChRs in *C. elegans* pharyngeal muscle #### Generation of the expression vector mk11g11 committed Feb 19, 2020 213 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). mk11g11 committed Sep 29, 2019 214 215 216 217 218 219 220 (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")  mk11g11 committed Feb 19, 2020 221 (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. mk11g11 committed Sep 29, 2019 222 223 224 225 226 {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")  mk11g11 committed Feb 19, 2020 227 (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). mk11g11 committed Sep 29, 2019 228 229 230 231 232 {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")  mk11g11 committed Feb 19, 2020 233 (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. mk11g11 committed Sep 29, 2019 234 235 236 237 238 239 240 {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 mk11g11 committed Feb 19, 2020 241 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. mk11g11 committed Sep 29, 2019 242 mk11g11 committed Feb 19, 2020 243 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. mk11g11 committed Sep 29, 2019 244 mk11g11 committed Feb 19, 2020 245 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. mk11g11 committed Sep 29, 2019 246 247 #### Feeding phenotype of eat-2 expressing transgenic lines ####{#behaviourofeat2rescue} mk11g11 committed Feb 19, 2020 248 *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. mk11g11 committed Sep 29, 2019 249 mk11g11 committed Feb 19, 2020 250 (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. mk11g11 committed Sep 29, 2019 251 mk11g11 committed Feb 19, 2020 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 {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") mk11g11 committed Sep 29, 2019 289 290 291 292 293 294 295 296 297 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. mk11g11 committed Feb 19, 2020 298 299 300 301 302 303 304 305 306 307 308 {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") mk11g11 committed Sep 29, 2019 309 310 311 312 knitr::include_graphics("fig/results4/PNG/eat2rescue_5ht_2.png")  mk11g11 committed Feb 19, 2020 313 ### Heterologous expression of human $\alpha7$ nAChRs in *C. elegans* pharyngeal muscle mk11g11 committed Sep 29, 2019 314 315 316 317 318 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. mk11g11 committed Feb 19, 2020 319 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. mk11g11 committed Sep 29, 2019 320 321 322 323 324 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 mk11g11 committed Feb 19, 2020 325 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). mk11g11 committed Sep 29, 2019 326 327 328 329 330 331 332 (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")  mk11g11 committed Feb 19, 2020 333 334 (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). mk11g11 committed Sep 29, 2019 335 336 337 338 339 {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")  mk11g11 committed Feb 19, 2020 340 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. mk11g11 committed Sep 29, 2019 341 mk11g11 committed Feb 19, 2020 342 (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). mk11g11 committed Sep 29, 2019 343 344 345 346 347 348 349 {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} mk11g11 committed Feb 19, 2020 350 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::$\alpha7$*. mk11g11 committed Sep 29, 2019 351 mk11g11 committed Feb 19, 2020 352 The three *eat-2::$\alpha7$* lines were assayed separately. The behavioural output did not differ (data not shown), therefore the results were pooled. mk11g11 committed Sep 29, 2019 353 354 355 356 357 A control strain in which GFP is expressed at the body wall muscle was generated previously. #### Generation of N2 transgenic strain mk11g11 committed Feb 19, 2020 358 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. mk11g11 committed Sep 29, 2019 359 mk11g11 committed Feb 19, 2020 360 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. mk11g11 committed Sep 29, 2019 361 362 363 364 365 #### 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)). mk11g11 committed Feb 19, 2020 366 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. mk11g11 committed Sep 29, 2019 367 mk11g11 committed Feb 19, 2020 368 (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. mk11g11 committed Sep 29, 2019 369 370 {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} mk11g11 committed Feb 19, 2020 371 372 373 374 375 376 377 378 379 380 381 382 383 # 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") mk11g11 committed Sep 29, 2019 384 385 386 387 knitr::include_graphics("fig/results4/PNG/rescuechrna7_feeding_2.png")  mk11g11 committed Oct 06, 2019 388 #### Effects of 5-HT on pharyngeal pumping ####{#htandtransgenicalpha7} mk11g11 committed Sep 29, 2019 389 390 391 392 393 394 395 396 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} mk11g11 committed Feb 19, 2020 397 398 399 400 401 402 403 404 405 406 407 # 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") mk11g11 committed Sep 29, 2019 408 409 410 411 knitr::include_graphics("fig/results4/PNG/rescuechrna7_5ht_2.png")  mk11g11 committed Feb 19, 2020 412 ### Pharmacological characterisation of $\alpha7$ expressing worms ####{#pharmaalpha7transegnicworms} mk11g11 committed Sep 29, 2019 413 mk11g11 committed Feb 19, 2020 414 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. mk11g11 committed Sep 29, 2019 415 416 417 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. mk11g11 committed Feb 19, 2020 418 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)). mk11g11 committed Sep 29, 2019 419 420 421 422 423 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. mk11g11 committed Feb 19, 2020 424 (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. mk11g11 committed Sep 29, 2019 425 426 427 {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} mk11g11 committed Feb 19, 2020 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 # 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") mk11g11 committed Sep 29, 2019 446 447 448 449 knitr::include_graphics("fig/results4/PNG/nicotine_cuthead.png")  mk11g11 committed Feb 19, 2020 450 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. mk11g11 committed Sep 29, 2019 451 mk11g11 committed Feb 19, 2020 452 (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. mk11g11 committed Sep 29, 2019 453 454 {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} mk11g11 committed Feb 19, 2020 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 # 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") mk11g11 committed Sep 29, 2019 473 474 475 476 knitr::include_graphics("fig/results4/PNG/choline_cuthead.png")  mk11g11 committed Feb 19, 2020 477 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. mk11g11 committed Sep 29, 2019 478 mk11g11 committed Feb 19, 2020 479 (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. mk11g11 committed Sep 29, 2019 480 481 {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} mk11g11 committed Feb 19, 2020 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 # 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") mk11g11 committed Sep 29, 2019 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 # 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")) mk11g11 committed Feb 19, 2020 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 # 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") mk11g11 committed Sep 29, 2019 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 knitr::include_graphics("fig/results4/PNG/cytisine_cuthead_2.png")  mk11g11 committed Oct 06, 2019 618 mk11g11 committed Sep 29, 2019 624 mk11g11 committed Feb 19, 2020 625 626 #### $\alpha$-Bgtx staining ####{#bgtxstaining} mk11g11 committed Sep 29, 2019 627 628 629 630 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). mk11g11 committed Feb 19, 2020 631 (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. mk11g11 committed Sep 29, 2019 632 mk11g11 committed Feb 19, 2020 633 {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} mk11g11 committed Sep 29, 2019 634 635 636 637 638 639 640 641 642 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. mk11g11 committed Feb 19, 2020 643 ### *Eat-2* as a genetic background for functional nAChR expression mk11g11 committed Sep 29, 2019 644 mk11g11 committed Feb 19, 2020 645 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*. mk11g11 committed Sep 29, 2019 646 mk11g11 committed Feb 19, 2020 647 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. mk11g11 committed Sep 29, 2019 648 649 650 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. mk11g11 committed Feb 19, 2020 651 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. mk11g11 committed Sep 29, 2019 652 mk11g11 committed Feb 19, 2020 653 654 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. mk11g11 committed Sep 29, 2019 655 mk11g11 committed Feb 19, 2020 656 ### No apparent functionality of human $\alpha7$ receptors in the *eat-2* mutant pharynx mk11g11 committed Sep 29, 2019 657 mk11g11 committed Feb 19, 2020 658 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]. mk11g11 committed Sep 29, 2019 659 mk11g11 committed Feb 19, 2020 660 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. mk11g11 committed Sep 29, 2019 661 mk11g11 committed Feb 19, 2020 662 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. mk11g11 committed Sep 29, 2019 663 mk11g11 committed Feb 19, 2020 664 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). mk11g11 committed Sep 29, 2019 665 666 667 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]. mk11g11 committed Feb 19, 2020 668 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. mk11g11 committed Sep 29, 2019 669 670 671 672 673 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. mk11g11 committed Feb 19, 2020 674 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. mk11g11 committed Sep 29, 2019 675 676 677 678 679 680 681 682 683 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. mk11g11 committed Feb 19, 2020 684 ### $\alpha7$ receptors are expressed on the surface of the pharyngeal muscle. mk11g11 committed Sep 29, 2019 685 686 687 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. mk11g11 committed Feb 19, 2020 688 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. mk11g11 committed Sep 29, 2019 689 mk11g11 committed Feb 19, 2020 690 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. mk11g11 committed Sep 29, 2019 691 692 693 mk11g11 committed Oct 06, 2019 694 mk11g11 committed Sep 29, 2019 695 696 697 698 699 700 701 702 703 704 705 706