04-results-02.Rmd 94.3 KB
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# The effects of nicotine and neonicotinoids on the pharyngeal pumping of *C. elegans* {#results-2}

## Introduction

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Results from the previous results chapter show low susceptibility of *C. elegans* to neonicotinoids and that the cuticle is a limiting factor of the efficacy of these compounds on locomotion. This chapter aims to further characterise the concentration-dependent effects of neonicotinoids on worm and the role of the cuticle in their efficacy, by utilising the pharyngeal pumping assay. 
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The pharynx is responsible for feeding. It functions to capture the bacterial suspension, expel the fluid and trap bacteria inside the pharyngeal lumen [@song2012]. The bacteria is then smashed and passed to the gut for digestion [@avery1987].
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These complex pharyngeal functions are performed by two motions: pumping and peristalsis (Figure \@ref(fig:feeding-label) [@avery1989]). Pumping is the contraction and a subsequent relaxation of the corpus, anterior isthmus and the terminal bulb. Peristalsis is the motion of the posterior isthmus that pushes the bacteria to the terminal bulb for grinding. On average, peristalsis occurs every 3.4 pumps [@song2013] and begins after the relaxation of the pharyngeal muscle. Grinding of the food particles is performed by the grinder. The grinder is positioned in the terminal bulb and composed of 3 pairs of muscle cells [@albertson1976]. Contraction of these cells leads to their rotation, resulting in food maceration.
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\newpage
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(ref:feeding) **Feeding of *C. elegans*.** As the corpus and anterior isthmus contract, bacterial suspension enters the pharynx through the mouth (top). Subsequent relaxation expels water out, trapping bacteria in the corpus-anterior isthmus lumen (middle). This pumping movement is synchronised with the terminal bulb contraction-relaxation cycle leading to movement of the grinder and crashing of food trapped between the muscle segments (not shown). This happens on average 3.4 times to allow food accumulation [@song2012] and passage to the intestine. The peristalsis of the posterior isthmus opens the isthmus-terminal bulb lumen and pushes the next portion of food in [@avery1987] for fragmentation (bottom).
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```{r feeding-label, fig.cap="(ref:feeding)", fig.scap='\\textit{C. elegans} feeding.', fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/feeding.png")
```
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<!-- ```{r synapses-celegans-pharynx, echo=FALSE, warning=FALSE, message=FALSE} -->
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<!-- library(kableExtra) -->
<!-- library(dplyr) -->
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<!-- innervation_pharynx_tbl <- data.frame( -->
<!--   Muscle = c("pm1", "pm2", "pm3", "pm4", "pm5", "pm6", "pm7", "pm8"), -->
<!--   Chemical  = c("M1", "M1", "M1", "M2, M3, MC", "M2, M3, M4, I5", "M5 , M4?", "M5", " "), -->
<!--   Gap = c("", "", "", "MC via mc2", " ", "M5 via mc3", "M5 via mc3", "M5 via mc3")) -->
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<!-- innervation_pharynx_tbl %>%  -->
<!-- mutate_all(linebreak) %>%  -->
<!-- kable("latex", booktabs = T, escape = F, -->
<!--   col.names = linebreak(c("Muscle cell", "Chemical synapse", "Gap junction")), -->
<!--   caption = "Connectivity of the pharyngeal neuromuscular system.") %>%  -->
<!--   kable_styling(latex_options = "hold_position") -->

<!-- ``` -->
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### Anatomy of the *C. elegans* pharynx
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Electron micrograph analysis of the *C. elegans* pharyngeal sections [@albertson1976] provides a detailed picture of its anatomy. The pharynx is a tubular feeding organ located in the head of the worm. It is 20 $\mu$M wide and 100 $\mu$M long, encapsulated by the basal membrane which separates the pharyngeal cells from the pseudocoelom. On the apical surface, the basal lamina lies directly below the cuticle. This is in contrast to the body wall muscle, where these two layers are also separated by the hypodermis. The pharynx can be divided into three anatomical features: most anterior corpus, middle isthmus and posterior terminal bulb. There are five different main cell types in the pharynx: muscles, neurons, epithelial, glands and marginal cells (Figure \@ref(fig:pharyngeal-muscle-label)). The main constituents are the 20 muscle cells and 9 marginal cells which wrap around the pharyngeal lumen. Embedded within those cells are 4 glands and 20 neurons.
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There are 8 layers of muscle cells encapsulating the pharynx (pm1-pm8). pm1 is the most anterior and constitutes from a single cell surrounding the pharyngeal lumen and six processes running down the pro-corpus. Posterior to pm1 are the three cells of the pm2 muscle cells which also wrap around the lumen of the pharynx. Both pm1 and pm2 are, relative to other muscles of the pharynx, thin. pm3 together with pm1 and pm2 form pro-corpus, pm4 meta-corpus whereas pm5 the isthmus of the pharynx. These three sections are wedge-shaped and formed from three cells. There are three muscular layers forming the terminal bulb: pm6, pm7 and pm8. pm6 and pm7 are composed of three cells, whereas pm is a single cell. pm8 is the most anterior and it is connected to the intestine by the toroidal valve composed of six cells. 
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<!-- Cells with three fold symmetry : 3 cells in each pm2, pm3, pm4 and pm5 are syncytial. Each contains two nuclei (6 total).  -->
<!-- pm1 six nuclei also syncytial, one cell, syncytial  -->
<!-- pm6 and pm7 three non syncytial cells each -->
<!-- pm8 is one cell and nonsyncytial cell. -->
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There are nine marginal cells in the pharynx: mc1 - mc3 each with three fold symmetry. These cells run along the pharynx from the pm1 to pm8. They receive chemical synapses from M5 and form gap junctions with muscle cells.
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\newpage

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(ref:pharyngeal-muscle) **The musculature of the *C. elegans* pharynx.** Cartoon representation of the muscle cells constituting each of the three anatomical features: the corpus, isthmus and the terminal bulb (a) A single cell of each layer is pictured. Epifluorescence image of pharyngeal muscle cells from the transgenic worm expressing myofilaments GFP reporter gene (b, left panel) and epifluorescence image of the transgenic worm expressing mitochondrial GFP reporter gene (b, right panel), to highlight the muscle cells of the terminal bulb. Images adapted from @albertson1976 (a) and @altun2009a (b).
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```{r pharyngeal-muscle-label, fig.cap="(ref:pharyngeal-muscle)", fig.scap= "The musculature of the \\textit{C. elegans} pharynx.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/general_intro/png/pharynx_muscle.png")
```

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\newpage

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### Sensory regulation of pumping
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There are 302 neurons in a *C. elegans* hermaphrodite, 20 of which are present in the pharynx  [@white1986]. The pharyngeal nervous system is connected to the somatic nervous system a single point: a pair of gap junctions between the extrapharyngeal RIP and pharyngeal I1 neurons [@albertson1976]. Laser ablation of RIP has no effect on pumping rates on or off food [@dalliere2015], suggesting that pharyngeal system is sufficient to drive pharyngeal responses. However, there is evidence that pharyngeal pumping can be influenced by the sensory cues. Animals with abolished sensory neurons and not RIP neurons have blunted pharyngeal response to light touch [@riddle1997], light [@bhatla2015], familial food, [@song2013], or attractive and repellent odours [@li2012]. This evidence supports the sensory regulation of the pharyngeal activity. Numerous sensory cues associated with food are detected by the *C. elegans* to modulate pharyngeal pumping including odour [@li2012] and mechanical stimulation [@chalfie1985]. These cues are detected by the sensory neurons. There are 60 sensory neurons in *C. elegans*. There are also pharyngeal neurons which are likely to serve sensory function. This includes NSM and all I neurons with the exception of I4. The activity of the parynx is also under the influence of the humoral transmission. The pharynx expresses receptors activated by neurotransmitters not synthesised by the pharynx, such as dopamine [@sugiura2005]. Mutation of genes not expressed in the pharynx has an effect on the pharyngeal response to food [@calahorro2018]. 
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\newpage
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```{r pharynx-neurons, echo=FALSE, warning=FALSE, message=FALSE, warning=FALSE}
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library(kableExtra)
library(dplyr)
pharyngeal_nrs <- data.frame(
  Type = c(rep("Motorneuron", 5), rep("Interneurons", 6), rep("Other", 3)),
  Pharyngeal = c("M1", "M2*", "M3*", "M4", "M5", "I1*", "I2*", "I3", "I4", "I5", "I6", "MI", "NSM", "MC*"),
  Neurtras = c("Acetylcholine", "Acetylcholine", "Glutamate", "Acetylcholine", "Acetylcholine", "Acetylcholine", "Glutamate", "Acetylcholine", "?", "Glutamate", "?", "Glutamate", "5-HT", "Acetylcholine"))

pharyngeal_nrs %>% 
mutate_all(linebreak) %>% 
kable("latex", booktabs = T, escape = F,
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  col.names = linebreak(c("Type", "Pharyngeal\nneuron", "Neurotransmitter\nreleased")),
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  caption = "Pharyngeal neurons and the neurotransmitters they release.") %>% 
  kable_styling(latex_options = "hold_position") %>% 
  collapse_rows(columns = 1, valign = "top") %>% 
  footnote(general = "Bilateral neurons are marked with *. Most neurons are either motor (M) or inter -neurons (I), but some have additional functions. NSM function as motor and secretory neuron, MI is a motor-interneurons, whereas MC is a motor and sensory neuron.  Three neurons are sufficient for feeding: MC, M3 and M4.", 
           threeparttable = T)

```

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\newpage
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### Pharyngeal nervous system
There are 20 neurons in the pharynx [@albertson1976] and Table \@ref(tab:pharynx-neurons)). Laser ablation studies combined with behavioural analysis of generated worms provides an explanation of the role of the entire pharyngeal nervous system, and the role of individual neurons in the pharyngeal pumping. 

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The pharyngeal nervous system is not essential for the function of the pharynx, but it is crucial for the regulation and modulation of the pharyngeal function in response to the environment. In the absence of food, the wild-type worm pumps at a low rate. This increases markedly in the presence of food. The laser ablation of the pharyngeal nervous system does not abolish pumping entirely [@avery1989]. In the absence of food, wild-type worm makes 43 pumps per minute. This is reduced to 16 in worms lacking pharyngeal neurons. A stronger phenotype of neuron-ablated worms can be seen upon introduction of food. In the presence of food, pharyngeal pumping rate drops from 224 to 26 in laser ablated worms. In addition, there are abnormalities in the mechanism of pumping of worms lacking the pharyngeal nervous system: little food passes into the intestine resulting in retarded growth and compromised fertility [@avery1989; @avery1993] showing the essential role of the pharyngeal nervous system in the feeding response.
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Three out of 14 pharyngeal neuron types are sufficient to elicit feeding. These neurons are MC, M3 and M4. Laser ablation of MC neuron (MC^-^strain) leads to reduction of pumping rate by 75% on food and 37% off food. Therefore, MC is important in initiation of pumping [@avery1989; @raizen1995]. Laser ablation of M3 or M4 has little effect on the pumping rate [@raizen1995], but it effects the mechanism of pumping. M3^-^ animals have markedly extended latency of a single pump [@avery1993] suggesting M3 governs the timing of pumping. The sensory endings of M3 synapse onto the  M4^-^ animals accumulate bacteria in the corpus suggesting M4 initiates peristalsis [@avery1989; @avery1993] by stimulating isthmus opening via mechanism that is not yet fully understood.

\newpage
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(ref:pharyngeal-nervous-system) **Pharyngeal nervous system.** Simplified pharyngeal nervous system showing the major neurons, synaptic connection and neurotransmitters. Synapses between I1-RIP connect the extrapharyngeal and pharyngeal nervous system.
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```{r pharyngeal-nervous-system-label, fig.cap="(ref:pharyngeal-nervous-system)", fig.scap= "Pharyngeal nervous system.", fig.align= 'center', echo=FALSE}
knitr::include_graphics("fig/results3/pharyngeal_system_2.png")
```
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\newpage

### Neurotransmitters of the pharynx

Genetic studies of worm mutant strains provide evidence for a critical role of neurotransmission in the regulation of feeding. UNC-13 protein is involved in the regulation of neurotransmitter release at the synapse, as shown by the biochemical and behavioural analysis of the *unc-13 C. elegans* strain. Mutants deficient in UNC-13 show severe retention of vesicles in the pre-synapse [@richmond1999] and impaired synaptic transmission [@aravamudan1999]. Their feeding is also affected: 70 % reduction of the pharyngeal pumping rate was noted [@richmond2001]. These data highlight the key role of neurotransmitters in the regulation of pumping. The activity of the pharynx is influenced by acetylcholine, glutamate and 5-HT (Table \@ref(tab:pharynx-neurons) and Figure \@ref(fig:pharyngeal-nervous-system-label)), and potential tyramine and octopamine [@alkema2005]. 

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#### 5-HT
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5-HT is synthesised in two pharyngeal neurons NSM and I5 [@chase2007]. Mutant deficient in enzyme in the 5-HT biosynthetic pathway has blunted response to food [@sze2000]. Exogenous application of 5-HT induces feeding response in the absence of food whereas competitive 5-HT antagonists inhibit pumping [@horvitz1982; @avery1990]. Collectively, this suggests that serotonergic neurotransmission induce food-evoked feeding response. 
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In the presence of food, 5-HT can be released from multiple sites, including NSM, ADF and HSN neurons. NSM are neurosecretory neurons in the pharyngeal nervous system. In the presence of food, they release 5-HT into the pseudocoelomic fluid [@horvitz1982] to inhibit locomotion, induce pumping and alter other behaviours [@horvitz1982]. ADF are chemosensory neurons present on the head of the worm. Release of 5-HT selectively from ADF neurons is sufficient to drive the feeding response [@cunningham2012]. Similarly, selective release of 5-HT from extrapharyngeal HSN neurons when NSM and ADF neurotransmission is defective, results in potent pumping response [@lee2017]. Therefore, there are 3 possible routes by which elevated levels of 5-HT in the presence of food can be achieved. It is possible that these routes co-function or operate at different conditions.
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Detection of food leads to the elevation of pumping rate via 5-HT acting at multiple G-protein coupled receptors [@avery2012]. 5-HT can regulate feeding response by binding to neuronal SER-4 receptors and SER-1 receptors expressed both in the pharyngeal muscle cells and neurons [@tsalik2003]. It also binds to SER-5 receptors expressed on extrapharyngeal interneurons [@cunningham2012]. However, the main driver of the 5-HT driven pharyngeal response it the activation of SER-7 receptors expressed on cholinergic MC and M4 neurons and and on glutametergic M3 neurons [@hobson2003; @song2013]. Acetylcholine released from MC and M4 neurons increases contraction frequency and induce isthmus peristalsis, respectively [@avery1987; @raizen1994]. This leads to an increase in the activity of the pharynx to ~260 pumps/min. Glutamate released from M3 shortens pump duration to <200ms.

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#### Glutamate 
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Glutamate is produced in at least 4 neurons (Table \@ref(tab:pharynx-neurons)), but its function in M3 is the best studied. Laser ablated M3^-^ animals have reduced pumping rate on food [@raizen1995]. Similar phenotype is observed in animals deficient in glutametergic neurotransmission. EAT-4 encodes for vesicular glutamate transporter [@lee1999]. Mutation of this gene in *C. elegans*, leads to reduction of the pharyngeal pumping rate of food [@lee2008]. 

In response to food, released 5-HT activates M3 [@niacaris2003], leading to release of glutamate which in turn contributes to the potent pharyngeal response. Specifically, it leads to shortening of the duration of the pump. M3-released glutamate acts via glutamate-gated chloride channel expressed on pm4 and pm5 pharyngeal muscles [@dent1997]. Activation of these channels during depolarisation phase of the pharyngeal muscle action potential leads to the generation of post-synaptic inhibitory potentials, which bring about the repolarisation and hence relaxation of the muscle. This in turn shortens the pump duration [@avery1993], allowing another muscle depolarisation to occur.
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<!-- The critical role of glutamate in pharyngeal pumping was demonstrated in behavioural studies of *C. elegans* mutants. Laser ablated M3^-^ animals have reduced pumping rate on food [@raizen1995]. Similar phenotype is observed in animals deficient in glutametergic neurotransmission. EAT-4 encodes for vesicular glutamate transporter [@lee1999]. Mutation of this gene leads to reduction of the pharyngeal pumping rate of food [@lee2008].  -->
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<!-- Glutamate may play other roles in the pharynx. @dillon2015 investigated an involvement in another glutamate receptor in pharyngeal response upon acute food removal. MGL-1 is a G-protein coupled receptor widely expressed in the pharynx, including the pharyngeal nervous system. *Mgl-1 C. elegans* mutant shows reduced pumping rate on food upon acute food removal.  -->
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<!-- Note that the food activated and 5-HT activated pathways are different. Check out the figure 6 in @lee2017 -->

<!-- There are also other 5-HT receptors, such as MOD-1 (for modulation of locomotion defective). MOD-1 is a 5HT-gated chloride channel [@ranganathan2000] with topology similar to the Cys-loop receptor superfamily. Phenotypical analysis of worms deficient in MOD-1 showed that starved mutant worms move faster in comparison to the wil-type strain upon entry onto the food patch, suggesting MOD-1 is involved in the regulation of locomotion in response to food in food-deprived animals.  -->
<!-- https://www.pnas.org/content/116/14/7107 -->

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#### ACetylcholine ##{#achpumping}
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The role of acetylcholine in the regulation of feeding was investigated in mutants deficient in proteins essential for the cholinergic neurotransmission (Section \@ref(cholinergicneurotransmissioninworms)). Null mutants die soon after birth due to starvation [@rand1989; @alfonso1993], whereas polymorphic mutants show reduced pharyngeal pumping both in the presence and absence of food [@dalliere2015]. Hindered feeding was also observed in animals in which one of the 6 cholinergic neuron, namely MC neurons, were ablated [@avery1989; @raizen1995]. 
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Pharmacological and genetic studies suggest that acetylcholine stimulates pharynx by acting on nAChRs. Application of nAChR agonists, acetylcholine and nicotine, resulted in the stimulation of the pharyngeal pumping in the absence of food [@raizen1995]. In contrast, nAChR antagonist d-tubacurarine inhibited pharyngeal pumping in the presence of food [@raizen1995]. 
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Although there are at least 29 nAChRs expressed in *C. elegans* (Figure \@ref(fig:seqidentityecd-label)), only one, namely EAT-2 has been identified as essential in mediation of the feeding response [@mckay2004]. EAT-2 is expressed in pm4 and pm5 muscle cells [@mckay2004], which make synaptic connections with the MC [@albertson1976]. *C. elegans eat-2* mutant shows significantly reduced pumping in the presence of food [@raizen1995; @mckay2004]. A similar phenotype was noted in the *eat-18* mutants. EAT-18 however is not a nAChR subunit. Instead, it is predicted to be a single transmembrane protein. Based on the localisation and behavioural phenotype, EAT-18 and EAT-2 are believed to co-assembly to form a functional receptor [@mckay2004]. 
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ACR-7 is also expressed at the pharyngeal muscle [@saur2013], as was shown with a reporter construct, however its function in pharyngeal pumping is unclear as *acr-7* mutant pump normally in the presence of food [@saur2013]. Besides nAChRs, there are other acetylcholine - sensitive receptors in the pharynx.
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<!-- nAChR are likely to be expressed not only at the neuromuscular junction, as suggested by the EAT-18 expression pattern. Expression of GFP-tagged eat-18 gene under the native promoter leads to fluorescence in all types of pharyngeal muscle cells and in M5 neuron [@mckay2004]. The identity of other subunits remains to be elucidated.  -->
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GAR-3 is a ACh-sensitive GPCR receptor [@hwang1999] expressed in metacorpus, isthmus, terminal bulb of the pharyngeal muscle and in the I3 pharyngeal neuron [@steger2004]. Its role may involve regulation of the membrane electrical potential [@steger2004] and control of feeding during starvation [@you2006].
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Acetylcholine-gated chloride channels (ACC) are ionotropic channels and like nAChRs, members of Cys-loop receptor family. There are at least 8 members isoforms: ACC-1 to ACC-4, LGC-46 to LGC-49 [@takayanagi-kiya2016], which form homo- and hetero-pentamers [@putrenko2005]. They are generally expressed in a distinct subset of cholinergic and glutametergic neurons (including acc-1 in pharyngeal M1 and M3) [@pereira2015; @takayanagi-kiya2016]. Interestingly, *acc-4* is expressed in almost all cholinergic neurons. Electrophysiological data suggest ACC channels are inhibitory but they may be playing multiple functions. ACC-4 may be acting as autoinhibitory, as most neurons predicted to express it do not receive direct synaptic input from other neurons [@albertson1976]. LGC-49 are expressed in presynaptic specialisations of cholinergic motor neurons where they regulate synaptic vesicle release [@takayanagi-kiya2016]. Based on pharmacologically characterised ACC-1 and ACC-2 *in vivo*, ACC receptors are also pharmacologically distinct from other ACh receptors. Some of the nAChR and GPCR receptor compounds were potent at modulating the activity of ACC channels, but not others. For example, ACC channels are insensitive to nAChR compounds nicotine, cytisine and antagonist $\alpha$-bgtx but are sensitivity to tubocurarine. This mixed pharmacological profile suggest ligand binding site is distinct from the binding site of nAChRs and cholinergic GPCRs. 
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### Assays for scoring the effects of compounds on pharyngeal pumping

Pharyngeal pumping can be scored in two distinct animal preparations. One is the intact worm preparation, and the other is the cut-head preparation in which the cuticular barrier is removed by cutting the head away from the body, enabling easy drug access to pharyngeal binding sites. The pharyngeal pumping of the worm can be scored by visual observation. This is performed by counting the number of grinder movements in a period of time. Grinder activity is coupled to the contraction and relaxation cycles of the pharynx, therefore is a good indication of the pharyngeal function. 

### Chapter aims 

This chapter describes the effects of 5-HT, nicotine and neonicotinoids on the pharyngeal pumping. The effects of compounds are scored on intact and cut-head worm in which access to the pharyngeal binding sites is increased. The aim of these investigations is to inform on the sensitivity of the pharyngeal system to 5-HT, nicotine and neonicotinoids and to provide an insight into the cholinergic regulation of the pharyngeal system. Additionally, effects of compounds on the pharyngeal pumping of mutants deficient in nAChR expression is investigated to elucidate the molecular basis of drug-induced pharyngeal alterations.

\newpage

## Results 

```{r echo=FALSE, results="hide", include=FALSE}
library(grid)
library(cowplot)
library(tidyverse)
library(ggpubr)
library(readr)
library(ggplot2)
library(scales)
library(curl)
library(devtools)
library(extrafont)
library(magick)
```

### Effects on pharyngeal pumping of intact worms on food

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Pharyngeal pumping is a feeding behaviour of the intact worm mediated by the pharynx. In the presence of food, the pharynx pumps at an approximate rate of 4 pumps/s (4Hz) to ingest food particles Figure \@ref(fig:pump-on-food-plot-label). A typical way of assessing the effects of compounds on feeding is to place *C. elegans* on agar plate soaked with drug solution and laced with *E. coli* OP50 food patch. A drug present on the plate comes in direct contact with foraging worm, but also can enters the worm via ingestion and/or diffusion cross the cuticle. To assess the effects of nicotine and neonicotinoids, wild-type worms were exposed to nicotine, nitenpyram, thiacloprid and clothianidin for 24-hours before scoring their effects on pharyngeal pumping (Figure \@ref(fig:pump-on-food-plot-label) left panel). Nicotine at concentrations $\ge$ 1 mM inhibited pumping in a dose - dependent manner. Nitenpyram and thiacloprid at 1 mM as well as clothianidin at 3.75 mM had no effect.

Analysis of responses to compounds in *bus-17* mutants show that the worm's cuticle can hinder the efficacy of drugs (Chapter 3). To determine whether the cuticle affects the efficacy of nicotine and neonicotinoids on the feeding behaviour, pharyngeal pumping experiments were repeated on the *bus-17*, cuticle-disrupted mutant of *C. elegans* (Figure \@ref(fig:pump-on-food-plot-label) right panel)). As in the wild-type, the pharyngeal pumping of the *bus-17* mutant was inhibited by nicotine at 1 and 10mM, whereas nitenpyram had no effect. In contrast, the dose of thiacloprid and clothianidin was ineffective on wild-type, inhibited pumping of the mutant at low mM concentrations.

To compare the efficacy of compounds on wild-type versus mutant strain, dose-response curves for the effects of treatment on pharyngeal pumping were generated (Figure \@ref(fig:dr-pumping-on-food-label)). Almost one fold difference in the potency of nicotine was observed. The EC~50~ on wild-type worm was 4.3 mM. This decreased to 2.3 mM in a mutant strain. Nicotine was also the most efficacious out of all compounds tested at inhibiting pumping. The estimated EC~50~ of thiacloprid and clothianidin on a mutant strain were 2.6 and 6.2 mM, respectively. Since they had no effect on wild-type it is not possible to estimate the fold-potency change between the wild-type and mutant strain.

<!-- # ```{r echo=FALSE, include=FALSE, message=FALSE, results="hide"} -->
<!-- # on_plate_dat_1 <-readRDS("Analysis/Data/Transformed/combined.RSD") -->
<!-- # ``` -->

(ref:pump-of-food) **The concentration-dependence for the effects of nicotine and neonicotinoids on feeding of *C. elegans*.** Wild-type (left panel) and *bus-17* (right panel) worms were exposed for 24 hours to nicotine, nitenpyram, thiacloprid, clothianidin or vehicle control (O), incorporated into solid medium. Pharyngeal pumps of worms present on food were counted by visual observation for 1 minute and expressed in Hz. Data are mean $\pm$ SEM, collected from 8 - 32 individual worms on $\ge$ 3 days. One way ANOVA (Kruskal-Wallis test) with Dunn’s Corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.

```{r  pump-on-food-plot-label, fig.cap="(ref:pump-of-food)", include=TRUE, echo=FALSE, fig.scap="The concentration-dependence for the effects of nicotine and neonicotinoids on feeding of \\textit{C. elegans}.", fig.align='center'}
# on_plate_dat_trans_1 <- on_plate_dat_1 %>%
#   drop_na() %>%
#   mutate(Dose = factor(Conc,
#                        levels= c(0, 0.001, 0.01, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3.75, 5, 10, 25, 50, 100),
#                        labels = c("0", "0.001", "0.01", "0.1", "0.25", "0.5", "0.75", "1", "1.5", "2", "2.5", "3.75", "5", "10", "25", "50", "100")),
#                 Exp = factor(Experiment,
#                            levels= Experiment,
#                            labels = Experiment))
# 
# on_plate_pump_selected <- on_plate_dat_trans_1 %>%
#   filter(Assay == "Pump")
# 
# on_plate_pump_selected$Hz <- on_plate_pump_selected$readout/60
# 
# on_plate_pump_stats <- on_plate_pump_selected %>%
#     group_by(Assay, Exp, Strain, Comp, Dose) %>%
#   summarise(mean_Hz=mean(Hz),
#             n=n(),
#             sd=sd(Hz),
#             se=sd/sqrt(length(Hz)))
# 
# labels_pump_on_food <- c("1" = "Nicotine N2", "2" = "Nicotine bus17", "3"= "Nitenpyram N2", "4" = "Nitenpyram bus17", "5" = "Thiacloprid N2", "6" = "Thiacloprid bus17", "7" = "Clothianidin N2", "8" = "Clothianidin bus17")
# 
# ann_text_pump_on_food <-data.frame(Dose = factor(c(1, 10, 25), levels = c(1, 10, 25)),
#     mean_Hz = 4.5,
#     lab_pump_on_food = c("****", "****", "****"),
#     Exp = as.factor(1))
# 
# ann_text_pump_on_food_1 <-data.frame(Dose = factor(c(1, 10), levels = c(1, 10)),
#     mean_Hz = 4.5,
#     lab_pump_on_food_1 = c("****", "****"),
#     Exp = as.factor(2))
# 
# ann_text_pump_on_food_2 <-data.frame(Dose = factor(c(0.5, 1, 1.5), levels = c(0.5, 1, 1.5)),
#     mean_Hz = 4.5,
#     lab_pump_on_food_2 = c("****", "****", "****"),
#     Exp = as.factor(6))
# 
# ann_text_pump_on_food_3 <-data.frame(Dose = factor(c(1, 2, 3.75), levels = c(1, 2, 3.75)),
#     mean_Hz = 4.5,
#     lab_pump_on_food_3 = c("****", "**", "****"),
#     Exp = as.factor(8))
# 
# pump_on_food_plot_nic <- on_plate_pump_stats %>%
#     filter(Exp=="1" | Exp == "2") %>%
#   drop_na() %>%
#   ggplot(aes(x = Dose, y= mean_Hz, fill=Dose)) +
#   geom_errorbar(aes(ymin = mean_Hz-se, ymax = mean_Hz+se), width=0.4) +
#     geom_bar(stat = "identity") +
#     theme(legend.position="none") +
#     facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labels_pump_on_food)) +
#     geom_text(data = ann_text_pump_on_food, aes(label = lab_pump_on_food)) +
#   geom_text(data = ann_text_pump_on_food_1, aes(label = lab_pump_on_food_1)) +
#   scale_fill_manual(values=c('#000000','#333333', '#666666','#999999', '#CCCCCC')) +
#    ylim(0, 5) +
#   ylab("Pumping (Hz)") +
#     theme(axis.text = element_text(size=12),
#           axis.title.x=element_blank(),
#           panel.background = element_blank(),
#           axis.line = element_line(colour = "black"),
#           strip.text.x = element_text(size=12),
#            axis.title = element_text(size=12),
#            text = element_text(size=12, family="sans"))
# 
# pump_on_food_plot_nit <- on_plate_pump_stats %>%
#     filter(Exp=="3" | Exp == "4") %>%
#   drop_na() %>%
#   ggplot(aes(x = Dose, y= mean_Hz, fill=Dose)) +
#   geom_errorbar(aes(ymin = mean_Hz-se, ymax = mean_Hz+se), width=0.4) +
#     geom_bar(stat = "identity") +
#     theme(legend.position="none") +
#     facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labels_pump_on_food)) +
#    theme(legend.position="none") +
# scale_fill_manual(values=c('#000000','#339900')) +
#    ylim(0, 5) +
#   ylab("Pumping (Hz)") +
#     theme(axis.text = element_text(size=12),
#           axis.title.x=element_blank(),
#           panel.background = element_blank(),
#           axis.line = element_line(colour = "black"),
#           axis.title = element_text(size=12),
#           strip.text.x = element_text(size=12),
#           text = element_text(size=12, family="sans"))
# 
# pump_on_food_plot_thia <- on_plate_pump_stats %>%
#     filter(Exp=="5" | Exp == "6") %>%
#   drop_na() %>%
#   ggplot(aes(x = Dose, y= mean_Hz, fill=Dose)) +
#   geom_errorbar(aes(ymin = mean_Hz-se, ymax = mean_Hz+se), width=0.4) +
#     geom_bar(stat = "identity") +
#     theme(legend.position="none") +
#     facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labels_pump_on_food)) +
#   scale_fill_manual(values=c('#000000','#000066', '#0000CC','#0000FF', '#0033FF','#3366CC', '#66CCFF')) +
#     geom_text(data = ann_text_pump_on_food_2, aes(label = lab_pump_on_food_2)) +
#    ylim(0, 5) +
#   ylab("Pumping (Hz)") +
#     theme(axis.text = element_text(size=12),
#           axis.title.x=element_blank(),
#           strip.text.x = element_text(size=12),
#           panel.background = element_blank(),
#           axis.line = element_line(colour = "black"),
#           axis.title = element_text(size=12),
#            text = element_text(size=12, family="sans"))
# 
# pump_on_food_plot_clo <- on_plate_pump_stats %>%
#     filter(Exp=="7" | Exp == "8") %>%
#   drop_na() %>%
#   ggplot(aes(x = Dose, y= mean_Hz, fill=Dose)) +
#   geom_errorbar(aes(ymin = mean_Hz-se, ymax = mean_Hz+se), width=0.4) +
#     geom_bar(stat = "identity") +
#     theme(legend.position="none") +
#     facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labels_pump_on_food)) +
#   scale_fill_manual(values=c('#000000','#993300', '#996633','#CC9933', '#FFCC33')) +
#     geom_text(data = ann_text_pump_on_food_3, aes(label = lab_pump_on_food_3)) +
#    ylim(0, 5) +
#   ylab("Pumping (Hz)") +
#     theme(axis.text = element_text(size=12),
#           axis.title.x=element_blank(),
#           panel.background = element_blank(),
#           axis.line = element_line(colour = "black"),
#           axis.title = element_text(size=12),
#           strip.text.x = element_text(size=12),
#           plot.margin = unit(c(5.5,5.5,12,5.5), "pt"),
#           text = element_text(size=12, family="sans"))
# 
# clo_grid <- plot_grid(pump_on_food_plot_nic, pump_on_food_plot_nit, pump_on_food_plot_thia, pump_on_food_plot_clo, nrow=4)
# clo_grid = clo_grid + draw_label("Concentration [mM]", x = 0.4, y = 0, hjust = 0, vjust = 0) +
#   ggsave("fig/results3/pumping_on_food_graph.pdf")

knitr::include_graphics("fig/results3/pumping_on_food_graph.png")
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```

(ref:DR-Pumping-on-food) **Dose-response curves for the effects of nicotine and neonicotinoids on feeding of *C. elegans*.** Concentration-dependence curves for the effects of nicotine (a), nitenpyram (b), thiacloprid (c) and clothianidin (d) on feeding of wild-type and *bus-17* *C. elegans*. Dose-response curves were generated by taking data points from Figure \@ref(fig:pump-on-food-plot-label), and expressed as % control pumping. Data and mean $\pm$ SEM. The EC~50~ for thiacloprid and clothianidin are approximations, because at the highest concentration tested (1.5 and 3.75 mM) they inhibited *bus-17* pumping by 45 and 50 %, respectively.

```{r dr-pumping-on-food-label, fig.cap="(ref:DR-Pumping-on-food)", fig.asp=1.2, echo=FALSE, fig.scap = "Dose-response curves for the effects of nicotine and neonicotinoids on feeding of \\textit{C. elegans}.", fig.align='center', warning=FALSE}
knitr::include_graphics("fig/results3/DR-pumping_on_food.png")
```

\newpage

#### Effects on pharyngeal pumping in liquid 

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During the course of the investigation, it was noted that high concentrations of nicotine had an antimicrobial effect on *C. elegans* food source - the OP50 *E. coli*. This raised a concern that the observed effects of nicotine on feeding could be partially due to the effects of this compound on the density of bacteria. To circumvent this issue, an alternative assay was developed in which the need for bacteria is removed. This alternative assay is performed in liquid, in the presence of 10 mM 5-HT. 5-HT has dual effect: it makes the worms immobile and stimulates their pumping (Figure \@ref(fig:5HT-pumping-label) a). After a 5 minute incubation with 10 mM 5-HT, 45 % of worms were paralysed. This increased to 65 % after 1 hour of incubation. The paralysing effect of 5-HT enabled measurements of the pharyngeal pumping activity of worms. Whilst in the liquid, worms display negligible food intake [@gomez-amaro2015]. 10 mM 5-HT stimulated pumping to 4 Hz - an effect seen after 5-minute exposure and sustained for 1 hour (Figure \@ref(fig:5HT-pumping-label) b).
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\newpage

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(ref:10mM-5HT-pumping-in-liquid) **The effects of 5-HT on *C. elegans* behaviour in liquid.** Worms were exposed to 10mM 5-HT or vehicle control (0). The effects on locomotion over time was scored by counting the number of immobile worms pre- (time point zero) and post-exposure to treatment or vehicle control. Data are expressed as % of worms paralysed (a). Additionally, the effects of 5-HT on pumping of immobile worms was scored. The measurements were made immediately after addition of 5-HT or vehicle control (time point 0) and post-exposure at the indicated time points. Pumping was scored by visual observation by counting the number of pumps over the period of 30 seconds and expressed in Hz. Data are mean $\pm$ SEM of 3 independent repeats. Motility of 10 worms per condition was scored during each experiment.

```{r 5HT-pumping-label, echo=FALSE, fig.cap="(ref:10mM-5HT-pumping-in-liquid)", fig.scap = "The effects of 5-HT on \\textit{C. elegans} behaviour in liquid.", fig.align='center', message=FALSE, echo=FALSE, warning=FALSE}
# ht_pump_in_liq <- read_csv("Analysis/Data/Transformed/intact_worm/5ht_pumping_in_liquid.csv")
# ht_paralysis <- read_csv("Analysis/Data/Transformed/intact_worm/5ht_paralysis")
# ht_paralysis_transf <- ht_paralysis %>%
#   mutate (Dose = factor(Dose,
#                         levels = Dose,
#                         labels = Dose))
# ht_paralysis_plot <- ht_paralysis_transf %>% 
#   group_by(Time, Dose) %>% 
#   na.omit() %>% 
#   summarise(mean_paralysed = mean(paralysed), 
#          sd=sd(paralysed),
#          se = sd/sqrt(length(paralysed))) %>% 
#   ggplot(aes(Time, mean_paralysed, colour = Dose)) +
#   scale_color_manual(values=c("grey","black"))+
#   geom_line(size=1) + 
#   geom_point(size=1) + 
#   geom_errorbar(aes(ymin = mean_paralysed-se, ymax = mean_paralysed+se)) +
#   theme(legend.position="none") +
#   xlab("Time (mins)") +
#   ylab("% Paralysed")  +
#   theme(panel.background = element_blank(), 
#           axis.line = element_line(colour = "black"),
#         text=element_text(size=12,  family="sans"))
# 
# ht_pump_in_liq$Pump <- ht_pump_in_liq$Pump/60
# ht_pump_liq_stats <- ht_pump_in_liq %>% 
#   group_by(Time, Dose) %>% 
#   na.omit() %>% 
#   summarise(mean_pump=mean(Pump),
#   sd=sd(Pump),
#   se = sd/sqrt(length(Pump)),
#   n())
# ht_pump_liq_plot <- ht_pump_liq_stats %>% 
#   ggplot(aes(Time, mean_pump)) +
#   geom_line(size=1) + 
#   geom_point(size=1) + 
#   geom_errorbar(aes(ymin = mean_pump-se, ymax = mean_pump+se)) +
#   xlab("Time (mins)") +
#   ylab("Pumping (Hz)") +
#   ylim(0,5) +
#   theme(panel.background = element_blank(), 
#         axis.line = element_line(colour = "black"),
#         text=element_text(size=12,  family="sans"))
# #+ guides(col = guide_legend(title = "Dose [mM]"))
# plot_grid(ht_paralysis_plot, ht_pump_liq_plot) +
#   ggsave("fig/results3/5htparalysis.pdf")
knitr::include_graphics("fig/results3/5htparalysis.png")
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```

\newpage

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To score the effects of compounds on 5-HT induced pumping, wild-type worms were exposed to 10 mM 5-HT. 30 minutes after, nicotine, nitenpyram, thiacloprid or clothianidin was added at the indicated concentrations, so that the worm was bathed in a solution containing both 5-HT and the drug of interest. The effect of nicotine and neonicotinoids on 5-HT stimulated pumping of paralysed worms was scored 30 minutes after the addition of nicotine or one of the neonicotinoids (Figure \@ref(fig:pumpining-liquid-label) and \@ref(fig:DR-pumping-liq-label)). Nicotine at concentrations ranging from 0.1 to 50 mM inhibited pumping with the EC~50~ of 1.7 mM. Concentration dependent inhibition was also caused by the incubation with nitenpyram. The estimated EC~50~ was 72.9 mM. Neither thiacloprid nor nitenpyram had an effect. 
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Incubation of *bus-17* mutant resulted in a decrease of EC~50~ of nicotine from 1.7 to 3.6 mM. A greater shift in potency of nitenpyram was noted. The EC~50~ increased from 72.9 in wild-type to 41.2 mM in a mutant strain. Estimated EC~50~ of thiacloprid was 4 mM whereas clothianidin at 2.5 mM did not significantly alter pumping. 

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\newpage


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(ref:pumping-liquid) **The effects of nicotine and neonicotinoids on 5-HT stimulated pharyngeal pumping.** Pharyngeal pumping was stimulated by incubation of worms in 10 mM 5-HT for 30 minutes. Following, indicated concentrations of nicotine, nitenpyram, thiacloprid or clothianidin were added. The effect of nicotine and neonicotinoids on 5-HT induced pumping on N2 wild-type (left panel) and *bus-17* (right panel) was scored. Pharyngeal pumping was measured by visual observation for a period of 30 seconds and expressed in Hz. Data are mean $\pm$ SEM of 3-17 individual worms collected on $\ge$ 2 separate days. One way ANOVA (Kruskal-Wallis test) with Sidak Corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.
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```{r pumpining-liquid-label, fig.cap="(ref:pumping-liquid)", fig.scap = "The effects of nicotine and neonicotinoids on 5-HT stimulated pharyngeal pumping.",fig.align='center', echo=FALSE}

# pump_liq_stat <- on_plate_dat_trans_1 %>% 
#   filter(exp_set=="intact") 
# 
# pump_liq_stat$readout <- pump_liq_stat$readout/60 
# 
# pump_liq_stats <- pump_liq_stat %>% 
#   group_by(Assay, Exp, Strain, Comp, Dose) %>% 
#   summarise(mean_readout=mean(readout),
#             n=n(),
#             sd=sd(readout),
#             se=sd/sqrt(length(readout)))
# 
# labels_pump_liq <- c("33" = "Nicotine N2", "34" = "Nicotine bus17", "35"= "Nitenpyram N2", "36" = "Nitenpyram bus17", "37" = "Thiacloprid N2", "38" = "Thiacloprid bus17", "39" = "Clothianidin N2", "40" = "Clothianidin bus17")
# 
# ann_text_pump_liq <- data.frame(Dose = factor (c(5, 10,25, 50), levels=c("5", "10", "25", "50")), mean_readout = 4,label_pump_liq = c("****","****", "****", "****"), Exp = as.factor(33))
# 
# ann_text_pump_liq_1 <- data.frame(Dose = factor (c(5, 10,25, 50), levels=c("5", "10", "25", "50")), mean_readout = 4,label_pump_liq_1 = c("**", "**", "****", "****"), Exp = as.factor(34))
# 
# ann_text_pump_liq_2 <- data.frame(Dose = factor (c(25, 100), levels=c("25", "100")), mean_readout = 4,label_pump_liq_2 = c("***","****"), Exp = as.factor(35))
# 
# ann_text_pump_liq_3 <- data.frame(Dose = factor (c(25, 50, 100), levels=c("25", "50", "100")), mean_readout = 4,label_pump_liq_3 = c("***","***", "****"), Exp = 36)
# 
# ann_text_pump_liq_4 <- data.frame(Dose = factor (1.5), mean_readout = 4,label_pump_liq_4 = "*", Exp = 38)
# 
# pump_liq__plot_nic <- pump_liq_stats %>% 
#   drop_na() %>% 
#   filter (Exp== "33" | Exp== "34") %>% 
#     ggplot(aes(x = Dose, y= mean_readout, fill = Dose)) +
#   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) +
#          geom_bar(stat = "identity") +
#   theme(legend.position="none") +
#     facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labels_pump_liq )) +
#   geom_text (data = ann_text_pump_liq, aes(label = label_pump_liq)) +
#     scale_fill_manual(values=c('#000000','#333333', '#666666','#999999', '#CCCCCC', '#D3D3D3', '#DCDCDC')) +
#    geom_text (data = ann_text_pump_liq_1, aes(label = label_pump_liq_1)) +
#   ylim(0, 5) +
#   ylab("Pumping (Hz)") +
#   theme(axis.title.x = element_blank()) +
#   theme(axis.text = element_text(size=12),
#         panel.background = element_blank(), 
#           axis.line = element_line(colour = "black"),
#            axis.title = element_text(size=12),
#         strip.text.x = element_text(size=12),
#            text = element_text(size=12, family="sans"))
# 
# pump_liq__plot_nit <- pump_liq_stats %>% 
#   drop_na() %>% 
#   filter (Exp== "35" | Exp== "36") %>% 
#     ggplot(aes(x = Dose, y= mean_readout, fill = Dose)) +
#   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) +
#          geom_bar(stat = "identity") +
#   theme(legend.position="none") +
#     facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labels_pump_liq )) +
# scale_fill_manual(values=c('#000000','#003300', '#006600','#009900', '#00FF33', '#CCFF33')) +
#    geom_text (data = ann_text_pump_liq_2, aes(label = label_pump_liq_2)) +
#     geom_text (data = ann_text_pump_liq_3, aes(label = label_pump_liq_3)) +
#   ylim(0, 5) +
#   ylab("Pumping (Hz)") +
#   theme(axis.text = element_text(size=12),
#         axis.title.x = element_blank(),
#         panel.background = element_blank(), 
#           axis.line = element_line(colour = "black"),
#            axis.title = element_text(size=12),
#         strip.text.x = element_text(size=12),
#            text = element_text(size=12, family="sans"))
# 
# pump_liq__plot_thia <- pump_liq_stats %>% 
#   drop_na() %>% 
#   filter (Exp== "37" | Exp== "38")  %>% 
#             filter(Dose == "1.5" | Dose == "0") %>% 
#     ggplot(aes(x = Dose, y= mean_readout, fill = Dose)) +
#   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) +
#          geom_bar(stat = "identity") +
#   theme(legend.position="none") +
#         geom_text (data = ann_text_pump_liq_4, aes(label = label_pump_liq_4)) +
#     facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labels_pump_liq )) +
# scale_fill_manual(values=c('#000000','#000066')) +
#   ylim(0, 5) +
#   ylab("Pumping (Hz)") +
#   theme(axis.text = element_text(size=12),
#         axis.title.x=element_blank(),
#         panel.background = element_blank(), 
#           axis.line = element_line(colour = "black"),
#         axis.title = element_text(size=12),
#         strip.text.x = element_text(size=12),
#         text = element_text(size=12, family="sans"))
# 
# pump_liq__plot_clo <- pump_liq_stats %>% 
#   filter (Exp== "39" | Exp== "40")  %>% 
#             filter(Dose == "2.5" | Dose == "0") %>% 
#     ggplot(aes(x = Dose, y= mean_readout, fill = Dose)) +
#   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) +
#          geom_bar(stat = "identity") +
#   theme(legend.position="none") +
#     facet_wrap(~ Exp, scale = "free", ncol = 2, labeller = labeller(Exp = labels_pump_liq )) +
# scale_fill_manual(values=c('#000000','#993300')) +
#   ylim(0, 5) +
#   ylab("Pumping (Hz)") +
#   theme(axis.text = element_text(size=12),
#         axis.title.x=element_blank(),
#         panel.background = element_blank(), 
#           axis.line = element_line(colour = "black"),
#            axis.title = element_text(size=12),
#         strip.text.x = element_text(size=12),
#            text = element_text(size=12, family="sans"),
#            plot.margin = unit(c(5.5,5.5,12,5.5), "pt"))
# p <- plot_grid(pump_liq__plot_nic, pump_liq__plot_nit, pump_liq__plot_thia, pump_liq__plot_clo, nrow=4)
# p = p + draw_label("Concentration [mM]", x = 0.4, y = 0, hjust = 0, vjust = 0) +
#   ggsave("fig/results3/nicandneonics-intact-pumping.pdf")
knitr::include_graphics("fig/results3/nicandneonics-intact-pumping.png")
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```

(ref:DR-Pumping-in-liquid) **The concentration dependence for the effects of nicotine and neonicotinoids on 5-HT stimulated pharyngeal pumping.** Concentration-dependence curves for the effects of nicotine (a), nitenpyram (b), thiacloprid (c) or clothianidin (d) on 5-HT stimulated pharyngeal pumping of N2 wild-type and *bus-17* mutant *C. elegans*. Data are expressed as % control pumping and are mean $\pm$ SEM. The EC~50~ for thiacloprid is an approximation, because at the highest concentration tested (2.5 mM) it inhibited pumping by 25 % in *bus-17*.

```{r DR-pumping-liq-label, fig.cap="(ref:DR-Pumping-in-liquid)", fig.asp=1, fig.scap = "The concentration dependence for the effects of nicotine and neonicotinoids on 5-HT stimulated pharyngeal pumping.",fig.align='center', echo=FALSE, warning=FALSE}

knitr::include_graphics("fig/results3/DR-pump-intact.png")
```

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<!-- ```{r N2_5HT_cuthead_timecourse_label, fig.cap="(ref:N2_5HT_timecourse)", results = "hide", echo=FALSE, message=FALSE, warning=FALSE} -->

<!-- #plot data -->
<!-- cut_head <- readRDS("Analysis/Data/Transformed/cut_head/summary_data") -->
<!-- N2_5HT_plot <- cut_head %>% -->
<!--   filter(Experiment==1) %>% -->
<!--   group_by(Time,Conc) %>%  #Group data by multiple variables -->
<!--   ggplot(aes(Time, mean_readout, colour = factor(Conc, labels = c("0", "10nM      ns", "100nM   ***","500nM  ****", "1uM      ****", "10uM    ****","50uM    ****", "100uM  ****")), group=Conc)) + #plot graph using mean -->
<!--   geom_line(size=1) + #Modify graph -->
<!--   geom_point(size=1) + -->
<!--     scale_color_manual(values=c('#000000','#330033', '#660066', '#660033', '#990099', '#CC0099', '#FF66CC', '#FF99FF')) + -->
<!--   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) + -->
<!--   xlab("Time (mins)") + -->
<!--   ylab("Pumping (Hz)") + -->
<!--   guides(col = guide_legend(title = "[5-HT]")) + -->
<!--   ylim(0, 5) + -->
<!--   scale_x_continuous(breaks=seq(0,90,15)) + -->
<!--   geom_segment((aes(x = 1, y = 4.5, xend = 60, yend = 4.5))) + -->
<!--   theme(axis.text = element_text(size=12), -->
<!--         axis.title = element_text(size=12), -->
<!--         text = element_text(size=12)) + -->
<!--   ggsave("fig/results3/cuthead_5HT.pdf", width = 15, height = 8, units = "cm") -->
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<!-- ``` -->
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\newpage

### Effects on pharyngeal pumping of dissected animal

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#### 5-HT

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These data show that nicotine and neonicotinoids have an effect on *C. elegans* feeding at mM concentrations and that the potency of compounds on pumping slightly increases in cuticle compromised *bus-17* mutant. This supports the idea that the cuticle presents a barrier for drug entry, limiting bioavailability and hindering the exerted effect. *Bus-17* mutant presents an attractive platform for studying the effects of the cuticle on drug's potency. An important caveat is that *bus-17* cuticle, although more permeable than the wild-type, could still hinder drug entry. To circumvent this, a liquid cut-head assay was performed. In this assay, the effects of nicotine and neonicotinoids on pharyngeal pumping of dissected, rather than the intact worm was performed. 
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#### Nicotine and neonicotinoids ####{#dissectedanimalnicotineandneonics}

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To score the effects of nicotine and neonicotinoids on the pharynx, their effects on 5-HT stimulated pharyngeal pumping was determined. First, the effects of 5-HT were investigated (Figure \@ref(fig:DR-5HT-cuthead-label) a and b). Exposure of cut-heads to 5-HT concentrations ranging from 10 nM to 100 $\mu$M resulted in dose-dependent stimulation of pharyngeal pumping. The maximal effect of effective doses was observed after 10 minutes of incubation and it typically gradually decreased for the next 50 minutes. 1 $\mu$M was the most effective and elicited a maximum average pumping response of 3.35 Hz. The EC~50~ for the effects of 5-HT on pharyngeal pumping of cut-heads was of 169 nM.
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To determine whether the effects of 5-HT treatment were reversible, heads were washed for 15 minutes after indicated 5-HT treatment (Figure \@ref(fig:DR-5HT-cuthead-label) a). Full recovery of all cut heads was noted, with the exception of those exposed to the highest concentration of 5-HT. Following treatment with 100 $\mu$M, only partial recovery of pumping was observed. 

\newpage

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(ref:DR-5HT-cuthead) **Concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected *C. elegans*.** a) Cut heads were exposed to varying concentrations of 5-HT, or vehicle control (0). The effects on pharyngeal pumping over time was scored by visual observation by counting the number of pharyngeal pumps in 30 seconds and the data was expressed in Hz. Data are mean $\pm$ SEM of 8 - 23 individual worms collected from experiments done on $\ge$ 3 days. Significance levels between the control and treatment are given in a figure legend and refer to 30 minute time points. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001. b) Dose-response curve for the effects of 5-HT on pharyngeal pumping of dissected *C. elegans*. The graph was generated by taking 30-minute time points and fitted into nonlinear regression sigmoidal dose-response (three parameter logistic) equation. Data are mean $\pm$ SEM, normalised to control and a maximum response, and expressed as % maximum pumping.
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```{r DR-5HT-cuthead-label, fig.cap="(ref:DR-5HT-cuthead)", fig.asp=1, fig.scap= "Concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected \\textit{C. elegans}", fig.align= 'center', echo=FALSE, message=FALSE, warning=FALSE}
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knitr::include_graphics("fig/results3/cuthead-5ht-combined.png")
```

\newpage

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To examine the effects of nicotine and neonicotinoids on pharyngeal pumping, cut heads were placed in 1 $\mu$M 5-HT for 10 minutes. 1 $\mu$M concentration was chosen because it elicits maximal pharyngeal response, whereas 10 minutes is sufficient for the response to equilibrate. Following 5-HT incubation, cut heads were transferred to a dish containing 1 $\mu$M 5-HT and an indicated concentration of nicotine, nitenpyram, thiacloprid or clothianidin and the effects of nicotine or neonicotinoids on 5-HT induced pumping was measured at multiple time point over 50 minutes. To determine if the response was reversible, cut heads were then transferred to 1 $\mu$M 5-HT and recovery scored over the period of 15 minutes (Figure \@ref(fig:N2-antagonist-timecourse-label)).

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Nicotine at concentration ranging from 1 - 20 $\mu$M inhibited 5-HT induced pumping partially, whereas 100 $\mu$M incubation resulted in a complete seizure of pumping activity (Figure \@ref(fig:N2-antagonist-timecourse-label) a). The maximal effects were typically observed after 10 minutes of incubation. For some concentrations i.e. 10, 20 and 50 $\mu$M, these effects weakened with time. Generally, the inhibitory effects of nicotine were reversible. Cut heads recovered from treatment with nicotine concentrations ranging from 1 - 50 $\mu$M after 15 minutes of washing. Cut heads previously incubated with 100 $\mu$M nicotine, pumped at half a rate of the control. 
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5-HT stimulated pharynges were inhibited by mM concentrations of nitenpyram (Figure \@ref(fig:N2-antagonist-timecourse-label) b). The effects were observed after 10 minutes and sustained throughout the course of the experimentation. Cut heads recovered well from nitenpyram-induced inhibition and returned to control pumping rate within 15 minutes of washing. 

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Thiacloprid at high $\mu$M concentrations had moderate, but not significant inhibitory effect, whereas clothianidin at a single dose of 500 $\mu$M significantly inhibited 5-HT stimulated pumping (Figure \@ref(fig:N2-antagonist-timecourse-label) c and d). 
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To compare the relative potencies, dose-response curves for the effects of nicotine and the three neonicotinoids were plotted (Figure \@ref(fig:cuthead-dr-label)). The order of potency is: nicotine > thiacloprid > clothianidin = nitenpyram. Nicotine is almost a magnitude more potent that neonicotinoids. It inhibits pumping with the EC~50~ of 10 $\mu$M, whereas neonicotinoids act at mM low range (2-3). 

<!-- ```{r N2_5HT-Nic-timecourse-label, fig.cap="(ref:N2-5HT-timecourse)",fig.align='center', echo=FALSE, results="hide", message=FALSE, warning=FALSE} -->
<!-- Nic_5HT_plot <- cut_head %>%  -->
<!--   filter(Experiment == "3")  %>%  -->
<!--   group_by(Time, Conc) %>%   #Group data by multiple variables -->
<!--   ggplot(aes(Time, mean_readout, colour = factor(Conc, labels = c("1 uM 5-HT", "Nic 100nM", "Nic 1uM         *", "Nic 10uM    ***", "Nic 20uM   ****", "Nic 50uM   ****", "Nic 100uM ****"))), group= Conc) + #plot graph using mean -->
<!--   geom_line(size=1) + #Modify graph -->
<!--   geom_point(size=1) + -->
<!--   scale_color_manual(values=c('#cc0099','#333333', '#666666','#999999', '#CCCCCC', '#D3D3D3', '#DCDCDC')) +  -->
<!--   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) + -->
<!--   ylab("Pumping (Hz)") + -->
<!--   guides(col = guide_legend(title = " ")) + -->
<!--   ylim(0, 5) + -->
<!--   geom_segment((aes(x = 1, y = 4.5, xend = 50, yend = 4.5))) + -->
<!--   theme(axis.text = element_text(size=12), -->
<!--         axis.title.x = element_blank(), -->
<!--           axis.title = element_text(size=12), -->
<!--           text = element_text(size=12, family="sans"), -->
<!--           legend.text=element_text(size=12)) -->


<!-- #plot nitenpyram data  -->
<!-- Nit_5HT_plot <- cut_head %>%  -->
<!--   filter(Experiment == "4")  %>%  -->
<!--   group_by(Time, Conc) %>%   #Group data by multiple variables -->
<!--   ggplot(aes(Time, mean_readout, colour = factor(Conc, labels = c("5-HT 1uM", "Nit 100uM", "Nit 1mM", "Nit 25mM  ****")), group= Conc)) + # plot graph using mean -->
<!--   geom_line(size=1) + #Modify graph -->
<!--   geom_point(size=1) + -->
<!--   scale_color_manual(values=c('#CC0099','#003300', '#006600','#009900')) + -->
<!--   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) + -->
<!--   ylab("Pumping (Hz)") + -->
<!--   guides(col = guide_legend(title = " ")) + -->
<!--   ylim(0, 5) + -->
<!--   geom_segment((aes(x = 1, y = 4.5, xend = 50, yend = 4.5))) + -->
<!--   theme(axis.text = element_text(size=12), -->
<!--         axis.title.x = element_blank(), -->
<!--           axis.title = element_text(size=12), -->
<!--           text = element_text(size=12, family="sans"), -->
<!--           legend.text=element_text(size=12)) -->

<!-- Thia_5HT_plot <- cut_head %>%  -->
<!--   filter(Experiment == "5")  %>%  -->
<!--   group_by(Time, Conc) %>%   #Group data by multiple variables -->
<!--   ggplot(aes(Time, mean_readout, colour = factor(Conc, labels=c("5-HT  ", "Thia 100uM", "Thia 250uM", "Thia 500uM")), group= Conc)) + # plot graph using mean -->
<!--   geom_line(size=1) + #Modify graph -->
<!--   geom_point(size=1) + -->
<!--     scale_color_manual(values=c('#CC0099','#000066', '#0000CC','#0000FF')) + -->
<!--   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) + -->
<!--   ylab("Pumping (Hz)") + -->
<!--   guides(col = guide_legend(title = " ")) + -->
<!--   ylim(0, 5) + -->
<!--   geom_segment((aes(x = 1, y = 4.5, xend = 50, yend = 4.5))) + -->
<!--   theme(axis.text = element_text(size=12), -->
<!--         axis.title.x = element_blank(), -->
<!--           axis.title = element_text(size=12), -->
<!--           text = element_text(size=12, family="sans"), -->
<!--           legend.text=element_text(size=12)) -->

<!-- Clo_5HT_plot <- cut_head %>%    -->
<!--   filter(Experiment == "6", Conc != 0.000001) %>% ##exclude conc = 0.000001 from the graph -->
<!--   group_by(Time, Conc) %>% -->
<!--   ggplot(aes(Time, mean_readout, colour = factor(Conc, labels = c("5-HT 1uM      ", "Clo 50uM      ", "Clo 500uM     *", "Clo 750uM")), group= Conc)) + -->
<!--   geom_line(size=1) + -->
<!--   geom_point(size=1) + -->
<!--  scale_color_manual(values=c('#CC0099','#993300', '#996633','#CC9933')) + -->
<!--   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) + -->
<!--   ylab("Pumping (Hz)") + -->
<!--   guides(col = guide_legend(title = " ")) + -->
<!--   ylim(0, 5) + -->
<!--   geom_segment((aes(x = 1, y = 4.5, xend = 50, yend = 4.5))) + -->
<!--     theme(axis.text = element_text(size=12), -->
<!--           axis.title.x = element_blank(), -->
<!--            text = element_text(size=12, family="sans"), -->
<!--           plot.margin = unit(c(5.5,5.5,12,5.5), "pt"), -->
<!--            legend.text=element_text(size=12)) -->
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<!-- ``` -->
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\newpage

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(ref:N2-antagonist-timecourse) **The concentration and time dependence of the effects of nicotine and neonicotinoid on 5-HT stimulated pharyngeal pumping of dissected worm.** Pharyngeal pumping of cut heads pre- and post-exposed to 5-HT + nicotine (a), nitenpyram (b), thiacloprid (c) and clothianidin (d) or vehicle control (dashed purple line). Pharyngeal pumps were counted by visual observation for 30 seconds and expressed in Hz. Data are mean $\pm$ SEM from 3 - 34 individual worms collected on $\ge$ 3 days. Statistic analysis shown in legend refers to the 30 minute time points between 5-HT control and 5-HT + treatment. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.
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```{r N2-antagonist-timecourse-label, fig.cap="(ref:N2-antagonist-timecourse)", echo=FALSE, fig.scap= "The concentration and time dependence of the effects of nicotine and neonicotinoid on 5-HT stimulated pharyngeal pumping of dissected worm.", fig.align='center'}
# cut_head_grid <- plot_grid(Nic_5HT_plot, Nit_5HT_plot, Thia_5HT_plot, Clo_5HT_plot, labels = c("a", "b", "c", "d"), nrow = 4)  
# cut_head_grid = cut_head_grid +  draw_label("Time (mins)", x=0.4, y=0, hjust=0, vjust=0) + 
#    ggsave("fig/results3/cuthead_antagonistic_effect.pdf")
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knitr::include_graphics("fig/results3/cuthead_antagonistic_effect_modified.png")
```

\newpage

(ref:cuthead-dr1) **Dose-response curves for the effects of nicotine and neonicotinoids on 5-HT stimulated pharyngeal pumping of dissected *C. elegans*.** Concentration-dependence curves for the effects of varying concentrations of nicotine, nitenpyram, thiacloprid or clothianidin on 5-HT stimulated pharyngeal pumping of cut heads. 30 minute time-points were taken (Figure \@ref(fig:N2-antagonist-timecourse-label)) and expressed as % control (5-HT) pumping.

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```{r cuthead-dr-label, fig.cap="(ref:cuthead-dr1)", fig.scap="Dose-response curves for the effects of nicotine and neonicotinoids on 5-HT stimulated pharyngeal pumping of dissected \\textit{C. elegans}", fig.align='center', echo=FALSE}
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knitr::include_graphics("fig/results3/DR-cut-head1.png")
```

\newpage

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Cut head assays provide an opportunity to determine the effects of compounds on un-stimulated pharynx. Cut heads are not mobile, hence scoring of pharyngeal activity is possible. 
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To score the effects of compounds on a pharynx of dissected animal, cut heads were incubated in buffer + solvent to record basal activity. Immediately after, cut heads were transferred to a dish containing an indicated concentration of nicotine, nitenpyram, thiacloprid, clothianidin or drug vehicle. The effects of treatment was scored for 1 hour and compared to the effects of 5-HT at 1 $\mu$M (Figure  \@ref(fig:cuthead-agonist-label)).

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Unstimulated pharynxes pump at an average rate of 0.1 Hz. Nicotine was tested at concentrations ranging from 100 nm to 100 $\mu$M (Figure \@ref(fig:cuthead-agonist-label) a). It had a dual action on the pharynx - stimulation at lower and inhibition at higher concentrations. At 10 $\mu$M it can be seen that a transient stimulation of pumping at 2 minute time point was seen. Incubation in 10 $\mu$M and 20 $\mu$M of nicotine led to weak but sustained stimulation of pumping - an effect seen at 20, 30 and 60 minute time points. 50 and 100 $\mu$M nicotine inhibited pumping completely. At these higher concentrations the muscle exhibited a visible twitching that can be described as fibrillation (data not shown). Neither nitenpyram nor thiacloprid had an effect on pharyngeal pumping of cut heads (Figure \@ref(fig:cuthead-agonist-label) b and c). In contrast, clothianidin stimulated pharynx (Figure \@ref(fig:cuthead-agonist-label) d). In response to 50 $\mu$M, an elevated pharyngeal pumping after 20 minutes was seen. whereas 500 and 750 $\mu$M of clothianidin elicited potent but short lived response. This response peaked after 2 minutes and returned to the basal rate after 10 minutes of incubation. 
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(ref:cuthead-agonist) **Concentration and time dependence for the effects of nicotine and neonicotinoids on pharyngeal pumping of dissected *C. elegans*.** Cut heads were exposed to varying concentrations of (a) nicotine (Nic), (b) nitenpyram (Nit), (c) thiacloprid (Thia), (d) clothianidin (Clo), 1 $\mu$M 5-HT + vehicle control or vehicle control only (Ctr). The effects of treatment on pumping were scored over time by counting the number of pharyngeal pumps in 30 seconds time windows and expressed in Hz. Data are $\pm$ SEM of 5 - 24 worms collected on $\ge$ 3 days. Significance levels between the vehicle control and treatment are given in a figure legend and refer to 30 minute time points. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.

```{r cuthead-agonist-label, fig.cap= "(ref:cuthead-agonist)", fig.scap = "Concentration and time dependence for the effects of nicotine and neonicotinoids on pharyngeal pumping of dissected \\textit{C. elegans}.",fig.align='center', echo=FALSE}
# Nic_plot <- cut_head %>% 
#   filter(Experiment == "7")  %>% 
#   group_by(Time, Conc) %>%  
#   filter(Time <= 60) %>% #Group data by multiple variables
#   ggplot(aes(Time, mean_readout, colour = factor(Conc, label = c("5-HT 1uM", "Ctr", "Nic 1um", "Nic 10uM", "Nic 20uM", "Nic 50uM", "Nic 100uM")), group= Conc)) + #plot graph using mean
#   geom_line(size=1) + #Modify graph
#   geom_point(size=1) +
#   scale_color_manual(values=c('#CC0099', '#000000','#333333', '#666666','#999999', '#CCCCCC', '#D3D3D3')) +
#   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) +
#   ylab("Pumping (Hz)") +
#   guides(col = guide_legend(title = " ")) +
#   ylim(0, 5) +
#   scale_x_continuous(breaks = seq(0, 60, by=10)) +
#   theme(axis.text = element_text(size=12),
#         axis.title.x = element_blank(),
#           axis.title = element_text(size=12),
#           text = element_text(size=12, family="sans"))
# 
# Nit_plot <- cut_head %>% 
#   filter(Experiment == "8")  %>% 
#   group_by(Time, Conc) %>% 
#   filter(Time <=60) %>% #Group data by multiple variables
#   ggplot(aes(Time, mean_readout, colour = factor(Conc, label = c("5-HT 1uM", "Ctr", "Nit 100uM", "Nit 1mM", "Nit 25mM")), group= Conc)) + # plot graph using mean
#   geom_line(size=1) + #Modify graph
#   geom_point(size=1) +
#     scale_color_manual(values=c('#CC0099', '#000000','#003300', '#006600','#009900')) +
#   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) +
#   ylab("Pumping (Hz)") +
#   guides(col = guide_legend(title = " ")) +
#   ylim(0, 5) +
#   scale_x_continuous(breaks = seq(0, 60, by=10)) +
#   theme(axis.text = element_text(size=12),
#         axis.title.x = element_blank(),
#           axis.title = element_text(size=12),
#           text = element_text(size=12, family="sans"))
# 
# Thia_plot <- cut_head %>% 
#   filter(Experiment == "9")  %>% 
#   group_by(Time, Conc) %>%  
#   filter(Time <= 60) %>% #Group data by multiple variables
#   ggplot(aes(Time, mean_readout, colour = factor(Conc, label = c("5-HT 1uM", "Ctr", "Thia 100uM", "Thia 250uM", "Thia 500uM")), group= Conc)) + # plot graph using mean
#   geom_line(size=1) + #Modify graph
#   geom_point(size=1) +
#   scale_color_manual(values=c('#CC0099', '#000000','#000066', '#0000CC','#0000FF')) +
#   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) +
#   ylab("Pumping (Hz)") +
#   guides(col = guide_legend(title = " ")) +
#   ylim(0, 5) +
#   scale_x_continuous(breaks = seq(0, 60, by=10)) +
#   theme(axis.text = element_text(size=12),
#         axis.title.x = element_blank(),
#           axis.title = element_text(size=12),
#           text = element_text(size=12, family="sans"))
# 
# Clo_plot <- cut_head %>% 
#   filter(Experiment == "10", Conc != 0.000001)  %>% 
#   group_by(Time, Conc) %>%
#   filter(Time <= 60) %>% #Group data by multiple variables
#   ggplot(aes(Time, mean_readout, colour = factor(Conc, labels = c("5-HT 1uM", "Ctr", "Clo 50uM", "Clo 500uM", "Clo 750 uM")), group= Conc)) +
#   geom_line(size=1) + #Modify graph
#   geom_point(size=1) +
#   scale_color_manual(values=c('#CC0099', '#000000','#993300', '#996633','#CC9933')) +
#   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) +
#   ylab("Pumping (Hz)") +
#   guides(col = guide_legend(title = " ")) +
#   ylim(0, 5) +
#   scale_x_continuous(breaks = seq(0, 60, by=10)) +
#     theme(axis.text = element_text(size=12),
#           axis.title.x = element_blank(),
#            axis.title = element_text(size=12),
#            plot.margin = unit(c(5.5,5.5,12,5.5), "pt"),
#            text = element_text(size=12, family="sans"))
# 
# cut_head_grid2 <- plot_grid(Nic_plot, Nit_plot, Thia_plot, Clo_plot, labels=c("a  ", "b  ", "c  ", "d"), nrow=4)
# cut_head_grid2 = cut_head_grid2 + draw_label("Time (mins)",x=0.4, y=0, hjust=0, vjust=0) + 
#   ggsave("fig/results3/cuthead_agonistic_effect.pdf")
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knitr::include_graphics("fig/results3/cuthead_agonistic_effect_modified.png")
```

\newpage

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To determine the onset of clothianidin induced stimulation of pumping, the experiment was repeated but the measurements were taken every minute for the first 5 minutes and at 10- minute time points (Figure \@ref(fig:clo-opt-cuthead-label)). 500 and 750 $\mu$M clothianidin stimulated pumping with the onset of action at 1 minute. After 1 minute these effects began to gradually weaken.
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\newpage

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(ref:clo-opt-cuthead) **The onset kinetics of clothianidin induced stimulation of pharyngeal pumping of dissected *C. elegans*.** Cut heads were exposed to varying concentrations of clothianidin (Clo), 1 $\mu$M 5-HT + solvent or solvent (Ctr). The effects on pumping were scored over time by counting the number of pharyngeal pumps over a 30 second time window. Data are expressed in Hz and are mean $\pm$ SEM of 7-13 individual worms collected on $\ge$ 3 days. Significance levels between the solvent control and treatment are given in a figure legend and refer to 2 minute time points. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $**$P $\le$ 0.01, $****$P $\le$ 0.0001.

```{r clo-opt-cuthead-label, fig.cap="(ref:clo-opt-cuthead)", fig.scap = "The onset kinetics of clothianidin induced stimulation of pharyngeal pumping of dissected \\textit{C. elegans}.",fig.align='center', echo=FALSE}
# Clo_opt_plot <- cut_head %>%
#   filter(Experiment == "11", Time <= 10)  %>%
#   group_by(Time, Conc) %>%   #Group data by multiple variables
#   ggplot(aes(Time, mean_readout, colour = factor(Conc, labels = c("5-HT 1uM **", "Ctr", "Clo 1uM", "Clo 50uM", "Clo 500uM", "Clo 750 uM ****")), group= Conc)) +
#   geom_line(size=1) + #Modify graph
#   geom_point(size=2) +
#   scale_color_manual(values=c('#CC0099', '#000000', '#993300', '#996633','#CC9933', '#FFCC33')) +
#   geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) +
#   xlab("Time (mins)") +
#   ylab("Pumping (Hz)") +
#   guides(col = guide_legend(title = " ")) +
#   ylim(0, 5) +
#   scale_x_continuous(breaks = seq(0, 10, by = 2)) +
#   theme(text=element_text(size=12,  family="sans"),
#         panel.grid.major = element_blank(), 
#         panel.grid.minor = element_blank(),
#         panel.background = element_blank(), 
#         axis.line = element_line(colour = "black")) +
#   ggsave("fig/results3/raw-images/clo_opt_image.pdf")
knitr::include_graphics("fig/results3/clo_opt_image.png")
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```

\newpage

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### Effects of 5-HT, nicotine and neonicotinoids on pharyngeal pumping in animals deficient in nAChR subunits ###{#nachrutantfeeding}
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An investigation into the potential targets of action of 5-HT, nicotine and neonicotinoids on the pharynx were made. *C. elegans* *eat-2* and *acr-7* nAChR mutants were investigated. Both genes are expressed in the pharynx and have been implicated in the pharyngeal function. Wild-type and mutant worms were placed on agar plate containing a food patch and the pharyngeal pumping rate of those present on food was scored (Figure \@ref(fig:mutant-pumping-label)). Pumping of the *acr-7* mutant was comparable to that of the wild-type strain. In contrast, pumping of the *eat-2* mutant was 70% lower than that of the wild-type.
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(ref:mutant-pumping) **Pharyngeal pumping of *C. elegans* nicotinic acetylcholine receptor mutants.** Pharyngeal pumping on food of N2 wild-type, eat-2 and acr-7 mutants. Pharyngeal pumps of worms present on food were counted by visual observation by counting the number of pharyngeal pumps in 30 seconds and expressed in Hz. Data are mean $\pm$ SEM, collected from $\ge$ 11 individual worms on $\ge$ 3 days. One way ANOVA (Kruskal-Wallis test) with Sidak Corrections, $****$ P $\le$ 0.0001.
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```{r mutant-pumping-label, fig.cap="(ref:mutant-pumping) ", fig.scap = "Pharyngeal pumping of \\textit{C. elegans} nicotinic acetylcholine receptor mutants.",fig.align='center', echo=FALSE, warning=FALSE, message=FALSE}
mutant_pumping <- read_csv("Analysis/Data/Transformed/pumping_on_food")

trans_mutant_pumping <- mutant_pumping %>% 
  mutate(Strain = factor(Strain,
levels= c("N2", "eat-2", "acr-7", "N2::pmyo3::GFP", "N2::pmyo3::GFP_pmyo2::CHRNA7", "eat2-2::pmyo3::GFP", "eat-2::pmyo3::GFP_pmyo2::CHRNA7", "eat-2::pmyo3::GFP_pmyo2::CHRNA7_2", "eat-2::pmyo3::GFP_pmyo2::CHRNA7_3")))

trans_mutant_pumping$Pumpsmin <- trans_mutant_pumping$Pumpsmin/30

mutant_pumping_stats <- trans_mutant_pumping %>% 
  group_by(Strain) %>%
  summarise(mean_pumping = mean(Pumpsmin),
            sd = sd (Pumpsmin),
            se = sd/sqrt(length(Pumpsmin))) #select all columns from rows 1 to 3 

ann_text_mutant_pump <-data.frame(Strain =  factor("eat-2"), levels = ("eat-2"),
    mean_pumping = 4.5,
    lab_mutant_pump = ("****"))

mutant_pumping_plot <- mutant_pumping_stats %>%   
  "["(., 1:3,) %>% 
  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)) +
  geom_text(data = ann_text_mutant_pump, aes(label = lab_mutant_pump)) +
  ylab("Pumping (HZ)") +
  ylim(0, 5) +
  theme(axis.title.x = element_blank(),
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        panel.background = element_blank(), 
        axis.line = element_line(colour = "black"),
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        axis.title = element_text(size=12),
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        axis.text = element_text(size=12)) +
  ggsave("fig/results3/pumpingmutant.pdf")
knitr::include_graphics("fig/results3/pumpingmutant.png") 
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```

Further experiments were carried out to determine whether pharyngeal responses induced by 5-HT, nicotine and neonicotinoids are dependent on the expression of the EAT-2 nAChR. Cut heads of wild-type and *eat-2* mutant worms were exposed to 5-HT, nicotine, nitenpyram, thiacloprid and clothianidin. The effects of treatment on pharyngeal pumping of mutant strain was scored and compared to the wild-type.
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The effects of 5-HT concentrations ranging from 10 nM to 100 $\mu$M elicited dose-dependent stimulatory response in wild-type worm (Figure \@ref(fig:DR-5HT-cuthead-2-label) a). The maximum rate of 3.5 Hz was achieved by 1 $\mu$M 5-HT after 10 minutes of incubation. 5-HT also stimulated pumping of *eat-2* mutant, but the responses were much weaker (Figure \@ref(fig:DR-5HT-cuthead-2-label) b). The maximum rate of 0.78 Hz was achieved by 50 $\mu$M 5-HT after 60 minutes of incubation. To compare the effects of 5-HT on mutant to the effects elicited on the wild-type cut heads, data were plotted on dose-response curve (Figure \@ref(fig:DR-5HT-cuthead-3-label)). The efficacy of 5-HT on the pharyngeal pumping of *eat-2* mutant was markedly reduced. The maximum response achieved by 5-HT was 55 % lower in comparison to the wild-type. The potency was also reduced as reflected in the shift of the EC~50~ from 155 nM in wild-type to 104 $\mu$M in a mutant strain. 
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(ref:DR-5HT-cuthead-2) **Concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected wild-type and *eat-2 C. elegans*.** a) Wild-type (N2) and mutant *eat-2* cut heads were exposed to varying concentrations of 5-HT, or vehicle control (0). The effects on pharyngeal pumping over time was scored by visual observation by counting the number of pharyngeal pumps in 30 seconds and expressed in Hz. Data are mean $\pm$ SEM of 8 - 23 individual worms collected from paired experiments done on $\ge$ 3 days. Significance levels between the control and treatment are given in a figure legend and refer to 30 minute time points. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.

```{r DR-5HT-cuthead-2-label, fig.cap="(ref:DR-5HT-cuthead-2)", fig.scap = "Concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected wild-type and \\textit{eat-2 C. elegans}.", fig.align='center', echo=FALSE}
# eat2_cuthead <- cut_head %>% 
#   filter(Experiment ==2) %>% 
#   group_by(Time, Conc) %>% 
#   ggplot(aes(x= Time, y= mean_readout, colour = factor(Conc, labels = c("0", "10nM", "100nM ","500nM", "1uM", "10uM","50uM", "100uM")), group=Conc)) +
#            geom_line(size=1) +
#          geom_point(size=1) +
# scale_color_manual(values=c('#000000','#330033', '#660066', '#660033', '#990099', '#CC0099', '#FF66CC', '#FF99FF')) +
#            geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se)) +
#   ylab("Pumping (Hz)") +
#   guides(col = guide_legend(title = " ")) +
#   ylim(0, 5) +
#   scale_x_continuous(breaks=seq(0,90,15)) +
#   geom_segment((aes(x = 1, y = 4.5, xend = 60, yend = 4.5))) +
#   theme(axis.text = element_text(size=12),
#         axis.title.x = element_blank(),
#           axis.title = element_text(size=12),
#           text = element_text(size=12, family="sans"),
#           legend.text=element_text(size=12)) +
#   ggsave("fig/results3/cuthead_5HT_eat2.pdf", width = 15, height = 8, units = "cm")
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knitr::include_graphics("fig/results3/eat2-5ht.png")
```

(ref:DR-5HT-cuthead-3) **Dose-response curves for the effects of 5-HT on pharyngeal pumping of N2 and *eat-2* cut heads.** The graphs were generated by taking 30-minute time points (Figure \@ref(fig:DR-5HT-cuthead-2-label)). Data are mean $\pm$ SEM, normalised to control and a maximum response elicited on N2 strain, expressed as a % maximum pumping.

```{r DR-5HT-cuthead-3-label, fig.cap="(ref:DR-5HT-cuthead-3)", fig.scap = "Dose-response curves for the effects of 5-HT on pharyngeal pumping of N2 and \\textit{eat-2} cut heads.", fig.align='center', echo=FALSE, warning=FALSE, message=FALSE}
knitr::include_graphics("fig/results3/DR-5HT-cuthead-n2+EAT-2.png")
```

\newpage

The investigations into the direct effects of nicotine, nitenpyram, thiacloprid and clothianidin on *eat-2* cut-head pumping were carried out in visual observation experiments. These data were compared to the effects induced on the cut heads of wild-type animals (Figure \@ref(fig:cuthead-eat2-dr-label)). 
Nicotine stimulated wild-type pharynx at 10 and 20 $\mu$M. At 50, 100 $\mu$M and 1 mM it inhibited pumping. Nicotine stimulated pumping of *eat-2* mutant at concentrations ranging from 10 - 100 $\mu$M. A dose of 1 mM was required to observe pumping inhibition. Nitenpyram at 100 $\mu$M to 100 mM was with no effect on either of the strains (Figure \@ref(fig:cuthead-eat2-dr-label) b). 
Thiacloprid at 250 and 500 $\mu$M stimulated pumping of both wild-type and *eat-2* strains (Figure \@ref(fig:cuthead-eat2-dr-label) c). However, a greater stimulation of *eat-2* by thiacloprid at 250 $\mu$M was observed in comparison to the wild-type. Clothianidin had a stimulatory effects on the wild-type worm. This was also observed in the mutant (Figure \@ref(fig:cuthead-eat2-dr-label) c).

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(ref:cuthead-eat2-dr) **The effects of the *eat-2* mutation on the concentration dependence of nicotine and neonicotinoid-induced pharyngeal pumping responses.** N2 wild-type and *eat-2* mutant cut heads were exposed to varying concentrations of a) nicotine, b) nitenpyram, c) thiacloprid and d) clothianidin. The number of pharyngeal pumps over 30 s at 60 minute for nicotine, nitenpyram and thiacloprid or 2 minute time-points for clothianidin were taken (Figure \@ref(fig:N2-antagonist-timecourse-label)) and expressed in Hz. Data are mean $\pm$ SEM of 5-25 individual worms collected from paired experiments done on $\ge$ 3 days. For comparison, the maximum pumping achieved by 5-HT is shown in dashed line.
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```{r cuthead-eat2-dr-label, fig.cap="(ref:cuthead-eat2-dr)", fig.scap = "The effects of the \\textit{eat-2} mutation on the concentration dependence of nicotine and neonicotinoid-induced pharygeal pumping responses.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/cuthead_DR_eat2.png")
```

<!-- (ref:cuthead-dr1) Concentration dependence for the effects of nicotine and neonicotinoids on  pharyngeal pumping of dissected *C. elegans*. The effects of varying concentrations of a) nicotine, b) nitenpyram, c) thiacloprid or d) clothianidin on pumping of cut heads. 60 minute for nicotine, nitenpyram and thiacloprid or 2 minute time-points were taken (Figure \@ref(fig:N2-antagonist-timecourse-label)) and expressed as % control (5-HT) pumping, with the exception of graph in a. For nicotine, the response was normalised to the 5-HT pumping and the minimal pumping response achieved by nicotine. Data are mean $\pm$ SEM. -->

<!-- ```{r (ref:cuthead-dr1)-label, fig.cap="(ref:cuthead-dr1)", echo=FALSE} -->
<!-- knitr::include_graphics("fig/results3/cuthead-n2-agonist-dr.png") -->
<!-- ``` -->
\newpage

## Discussion

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In this chapter, the effects of nicotine and neonicotinoids on pharyngeal pumping of intact and dissected, cut-head *C. elegans* are described. Pharyngeal pumping was assayed by visually scoring the frequency of pharyngeal pumps. These investigations were carried out to further investigate the toxicity of nicotine and neonicotinoids on worms, and to explore the role of the worm's cuticle on drug susceptibility.
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## 5-HT induces fast pumping in *C.elegans* by the activation of EAT-2 containing nAChRs 
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Application of 5-HT on cut-head preparation elevated pumping from 0.17 Hz to 3.34 Hz with the EC~50~ of 169 nM (Table \@ref(tab:pharynx-summary)).These data suggest that 5-HT drives a feeding response in worms. The maximum pumping frequency achieved by the exogenous 5-HT is comparable to the pharyngeal pumping rate of the worm in the presence of food (3.34 Hz and 4.33 Hz, respectively). Pumping rate on food of a mutant with defective 5-HT biosynthesis pathway is markedly reduced [@sze2000]. Endogenous 5-HT is released from NSM and ADF neurons in response to the presence of food. It then acts on MC and M4 neurons [@raizen1995; @niacaris2003] to increase pumping frequency and on M3 neurons [@song2013; @niacaris2003] to reduce the pumping latency. Application of 5-HT bypasses the sensory pathway to activate MC, M4 and M3 neurons directly by acting on GPCRs [@hobson2003; @song2013].
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To provide an insight into the mechanism of 5-HT induced *C. elegans* responses, the effects of 5-HT on pharyngeal pumping of cut head *eat-2* nAChR mutant were performed and compared to the wild-type. The maximum response achieved by a 5-HT was reduced by 70 %, whereas the EC~50~ increased from 169 nM in wild-type to 150 $\mu$M in a mutant (Table \@ref(tab:pharynx-summary)). This relative 5-HT insensitivity of *eat-2* strain suggests 5-HT induces pumping by eliciting cholinergic neurotranmission via EAT-2 containing nAChRs. 5-HT acts on cholinergic MC and M4 motorneurons to stimulate ACh release. ACh binds to EAT-2 nAChRs expressed at the MC NMJ to stimulate pumping [@mckay2004]. Hence 5-HT evokes pharyngeal activity by indirectly activating EAT-2 containing nAChRs. 
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##  Cuticle limits efficacy of nicotine and neonicotinoids on *C. elegans* pharynx

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To investigate the role of the cuticle in susceptibility of worms to neonicotinoids, experiments were carried out on intact worm of two strains: wild-type and *bus-17* mutant. *Bus-17* strain has altered surface coat [@gravato-nobre2005] and reduced cuticle integrity [@yook2007]. The pharynx was stimulated with 5-HT and the effects of nicotine and neonicotinoids on 5-HT induced pumping were examined (Figure \@ref(fig:cuticle-pumping-label)). Nicotine and nitenpyram inhibited 5-HT evoked pharyngeal pumping at mM concentrations. The efficacy of nicotine as measured by EC~50~ was comparable on both strains: 2 and 3 mM on wild-type and mutant strain, respectively. In contrast, the EC~50~ of nitenpyram increased from 73 in wild-type to 42 mM in a mutant strain. Further experiments were performed in which the cuticular barrier was removed and the effects of compounds on 5-HT stimulated pumping of dissected, cut-head wild-type *C. elegans* were investigated (Figure \@ref(fig:cuticle-pumping-label)). Nicotine inhibited pumping with the EC~50~ of 10 $\mu$M, whereas the EC~50~ of nitenpyram was 3 mM. By comparing this to the intact worm data, it can be seen that the removal of the cuticle resulted in a 300 and 24-fold increase in efficacy of nicotine and nitenpyram on the 5-HT evoked pumping of wild-type worm, respectively. Due to poor solubility, testable concentrations of clothianidin and thiacloprid were limited. In the presence of high $\mu$M - low mM concentrations of thiacloprid or clothianidin, the 5-HT evoked pumping of intact and cut head worm was slightly reduced. No full dose-response curves were obtained, however, there is a trend towards left shift of partial dose-response curves (Figure \@ref(fig:cuticle-pumping-label)) for both compounds. Taken together this suggested that the cuticular structures are limiting the access of nicotine and nitenpyram to the receptors of the pharyngeal system of *C. elegans*. The greater decrease of EC~50~ for nicotine versus nitenpyram might suggest better permeability of the latter, possibly due to its higher hydrophobicity (Table \@ref(tab:properties)). 
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(ref:cuticle-pumping) **The effects of the cuticle on nicotine and neonicotinoid - induced inhibition of 5-HT induced pumping.**
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```{r cuticle-pumping-label, fig.cap="(ref:cuticle-pumping)", fig.scap = "The effects of the cuticle on nicotine and neoncotinoid- induced inhibition of 5-HT induced pumping.", fig.align='center', echo = FALSE}
knitr::include_graphics("fig/results3/DR of inhibition of 5-HT stimulated pumping.png")
```

## Differential effects of nicotine and neonicotinoids on the pharynx

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To further characterise the effects of compounds on the pharyngeal pumping, nicotine and neonicotinoids were applied on unstimulated wild-type *C. elegans* cut-head (Table \@ref(tab:pharynx-summary)). Differential effects of nicotine and neonicotinoids are noted.
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Nicotine had a concentration dependent effect: a moderate stimulation by 10 and 20 $\mu$M and an inhibition of pumping activity by 50 and 100 $\mu$M. 
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Scoring the effects of clothianidin on pharyngeal pumping revealed potent but transient effect at high $\mu$M concentrations. The maximum stimulatory effect was observed after 2 minutes of incubation and declined progressively to the basal pumping rate within 10 minutes. 

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Thiacloprid at high $\mu$M concentrations moderately stimulated pumping. This effect was sustained throughout the duration of the assay (1 hour). Nitenpyram at doses up to 100 mM had no effect on pharyngeal activity.
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The differences in the pharyngeal effect achieved by nicotine and neonicotinoids could be due to targeting different receptors proteins. To investigate this further, the effects of nicotine and neonicotinoids on pharyngeal pumping of nAChR subunit mutant were investigated. Compounds were applied on cut heads of *eat-2* *C. elegans* mutant and wild-type. Pharyngeal pumping was scored visually after 30 minutes of exposure (Table \@ref(tab:pharynx-summary)). Different responses at 50 and 100 $\mu$M nicotine were noted. A stimulation of pumping occurred in the mutant, whereas pumping was inhibited in wild-type worms. Clothianidin at 250, 500 and 750 $\mu$M stimulated pumping of both strains. A difference at a single concentrations of thiacloprid was noted. At 250 $\mu$M thiacloprid moderately elevated pumping of *eat-2*, but it had no effect on the wild-type. However, there was no difference at 100 and 500 $\mu$M. Nitenpyram was with no effect on either strain. Taken together, these data suggests that EAT-2 may be involved in the pharyngeal responses to nicotine, but there are likely other receptors involved too, whereas neonicotinoids act on receptors other than EAT-2. 
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Differential pharyngeal responses to nicotine and neonicotinoids suggest they have different mode of action in the *C. elegans* pharynx. 
This suggests the existence of multiple types of nAChRs with distinct pharmacology with regards to neonicotinoids. 
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## Neonicotinoids may target different receptor protein in *C. elegans* pharynx

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Neonicotinoids may have a differential mode of action on *C. elegans* pharynx. Clothianidin and thiacloprid have distinct stimulatory effects on pumping. Clothianidin induced transient, whereas thiacloprid induced sustained stimulation. In contrast, nitenpyram has no effect. Differential effects of neonicotinoids on animal behaviour was also observed in bumblebee [@moffat2016], honey and wild-bee [@woodcock2017] and could be underpinned by the differences in targeted receptors. Indeed, imidacloprid and clothianidin target distinct nAChRs in the honey bee mushroom body [@moffat2016] and in cochroach neuronal preparation [@thany2009]. 
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## Alternative sites for the action of nicotine and neonicotinoids

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What are the alternative molecular sites for nicotine and neonicotinoids in worms? The response to nicotine does not depend on pharyngeal neurons [@raizen1995] suggesting nicotine acts directly on the muscle and/or on somatic nervous system. Behavioural and genetic analysis suggests nicotine can act on nAChRs other than EAT-2. The response to food in *lev-8* [@towers2005] mutant is reduced, making LEV-8 a potential target. Nicotine could also act on somatic nervous system, for example on IL1 and/or IL2 labial sensory neurons. IL2 are cholinergic [@pereira2015] and express nAChR DES-2 subunit [@treinin1998]. IL1 neurons are involved in mechanosensation, they express ACR-2 nAChR subunits [@Nurrish1999; @Hallam2000]. Both IL1 and IL2 output onto pharyngeal RIP neurons [@albertson1976; @serrano-saiz2013] which are a point where extrapharyngeal and pharyngeal nervous system connect [@albertson1976].
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Nicotine can also bind to receptors other than nAChRs, such as TRPV channels [@Liu2004; @Talavera2009; @feng2006]. TRPV channels are expressed on IL1 neurons [@Kindt2007], however effective nicotine doses are higher than those used in this study (i.e. typically $\ge$ 100 $\mu$M [@Liu2004; @Talavera2009].

## Relative insensitivity of *C. elegans* to neonicotinoids

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Concentrations of neonicotinoids effective against *C. elegans* feeding, are at least several fold higher than those effective against the feeding of insects. Clothianidin and thiacloprid stimulated pharyngeal pumping at high $\mu$M concentrations. In insects, they impair on feeding at sub $\mu$M concentrations. Imidacloprid inhibits feeding of mayflies [@alexander2007], thiamethoxam impairs on the feeding of bumble bees and some species of wild-bees [@baron2017]. This supports results from the previous chapter that at field realistic concentrations, neonicotinoids have no impact on tested *C. elegans* behaviours. 
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## Pharyngeal nAChRs have low sensitivity to neonicotinoids 

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The low potency of neonicotinoids on the pharynx in cut-head preparation suggest pharyngeal *C. elegans* nAChRs have low sensitivity to neonicotinoids relative to insects. Electrophysiological recordings from insect neuronal preparations show the effects of neonicotinoids at sub $\mu$M doses [@thany2009; @moffat2016; @tan2008; @buckingham1997]. In this study, a dose of at least 250 $\mu$M was required to observe an effect. Therefore, there is at least several fold difference in the susceptibility to neonicotinoids between the worm and insects. This suggests *C. elegans* nAChRs are pharmacologically distinct from those found in insects. 
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<!-- Divergency of insect and worm nAChRs is supported by the low homology of amino-acid sequence similarities. The highest between the worm and bee is less than 40 %. -->

Table: (\#tab:pharynx-summary) Summary of the effects of compound on the pharyngeal pumping of *C. elegans* wild-type (N2) and nAChR mutant *eat-2*.
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+------------+--------------------------------+-----------------------------------+
|            |N2                              |eat-2                              |
+============+================================+===================================+
|5-HT        |1. Dose dependent and sustained |1. Dose dependent and sustained    |
|            |stimulation                     |stimulation                        |
|            |2. Maximum pumping 3.34 Hz      |2. Maximum pumping 0.87 Hz         |
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|            |3. EC~50~=169nM                 |3. EC~50~=150 $\mu$M                |
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+------------+--------------------------------+-----------------------------------+
|Nicotine    |1. Sustained stimulation by     |1. Sustained stimulation by        |
|            |10 and 20 $\mu$M                |concentrations ranging from 10     |
|            |                                |100 $\mu$M                         |
|            |2. Inhibition by concentrations |2. Inhibition by 1 mM              |
|            |from 50 $\mu$M to 1 mM          |                                   |
+------------+--------------------------------+-----------------------------------+
|Nitenpyram  |1. No effects at 0.1-100mM      |As N2                              |
+------------+--------------------------------+-----------------------------------+
|Thiacloprid |1. Weak stimulation             |1. Weak stimulation by 250         |
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|            |by 500 $\mu$M                   |and 500 $\mu$M                       |
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+------------+--------------------------------+-----------------------------------+
|Clothianidin|1. Weak and sustained           | As N2                             |
|            |stimulation by 50 $\mu$M        |                                   |
|            |2. Potent, dose-dependent       |                                   |
|            |and transient stimulation by    |                                   |     
|            |500 and 750 $\mu$M              |                                   |
|            |3. Onset at 2 minutes           |                                   |
+------------+--------------------------------+-----------------------------------+


<!-- --------------------------------------------------------------------------------- -->
<!--                                          Pump frequency                            -->
<!-- ------------- -------------- ---------------- --------------- ------ ------------- -->
<!--                       Visual observation                  EPG        EPG waveform  -->
<!-- ------------- -------------- ---------------- --------------- ------ ------------- -->
<!-- Compound      N2               eat-2          N2              eat-2  N2            -->
<!-- ------------- -------------- ---------------- --------------- ------ ------------- -->
<!-- 5-HT          Dose dependent Dose dependent   Dose dependent  NA     Reduced       -->
<!--               stimulation    stimulation      stimulation            pump          -->
<!--               EC~50~=169nM   EC~50~=150$\mu$  EC~50~=255$\mu$        duration      -->
<!-- Nicotine      Sustained      Sustained        Sustained       As N2  -             -->
<!--               stimulation    stimulation      stimulation by                       -->
<!--               by 10          by 10-100 $\mu$M 1 $\mu$M                             -->
<!--               and 20 $\mu$M  Inhibition by    Potent                               -->
<!--               Inhibition at  1mM              stimulation                          -->
<!--               $\ge$ 50$\mu$M                  followed by                          -->
<!--                                               inhibition by                        -->
<!--                                               $\ge$ 10$\mu$M                       -->
<!--  Nitenpyram   No effects at  As N2            No effects at   NA     No effect     -->
<!--               0.1-100mM                       0.1mM           NA     at 0.1mM      -->
<!--  Thiacloprid  Moderate       Moderate         No effect at    NA     No effect     -->
<!--               stimulation by stimulation by   50$\mu$M               at            -->
<!--               500$\mu$M      250                                     50$\mu$M      -->
<!--                              and 500$\mu$M                                         -->
<!--  Clothianidin Moderate       As N2            Moderate        NA     Reduced       -->
<!--               and sustained                   and sustained          pump          -->
<!--               elevation by                    elevation by           duration      -->
<!--               50$\mu$                         75$\mu$                Decresed      -->
<!--               Potent and                                             E/R ratio     -->
<!--               brief                                                                -->
<!--               stimulation by                                                       -->
<!--               500                                                                  -->
<!--               and 750$\mu$M                                                        -->
<!-- ---------------------------------------------------------------------------------- -->