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# Pharmacological characterisation of the *C. elegans* pharynx {#results-3}

## Introduction

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Previous chapter describes the effects of nicotine, 5-HT and neonicotinoids on pharyngeal pumping of the cut-head worm, scored by visual observation (Chapter 4). An alternative pharyngeal assay is an extracellular recording of an electrical function of the pharynx, or electropharyngeogram (EPG). Unlike scoring pharyngeal pumping by visual observation, EPG recordings allow for resolution of early time points. Additionally, investigations into drug-induced changes of the EPG waveform may inform on the potential mode of action of compounds. 
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In an EPG assay, a worm's head is placed in a recording chamber. The chamber can be perfused, exposing the pharynx to different drug solutions. A tight seal between the electrode and the tip of the *C. elegans* nose is made. Contracting pharyngeal muscle produces currents which flows out of the worm's mouth; this is detected by the electrode. Each cycle of contraction and relaxation gives rise to a single waveform on the trace, known as an EPG. There are several phases constituting an individual EPG (Figure \@ref(fig:example-epg-label)). The beginning of the EPG signal marks excitatory phase. This phase mirrors depolarisation and contraction of the corpus and the terminal bulb and constitutes from 2 spikes. e spike arises due to the release of ACh from MC neurons and an activation of of nAChRs [@raizen1995]. E peak is due to a subsequent calcium channels activation [@lee1997; @shtonda2005]. e is often unseen on the EPG trace due to merging with the larger E. I or inhibitory spikes are diverse in number and amplitude and arise as a result of the inhibitory currents. These currents are produced by ligand-gated chloride channels in response to glutamate release from M3 neurons [@dent1997; @li1997]. Lastly, R and r reflect relaxation of the corpus and the terminal bulb, due to a repolarisation of the terminal bulb muscle cells caused by the flow of potassium through the potassium ion channel [@shtonda2005]. r spikes are frequently merged with larger in amplitude R. 

<!-- Simultaneous video and EPG recording of the pharynx revealed that the electrical transients correspond with the motion of a particular section of the pharynx. E phase corresponds to the contraction, whereas the R phase to the relaxation of the pharynx [@raizen1994]. p and I no visual consequences, whereas R mirrors corpus relaxation and r terminal bulb relaxation [@raizen1994]. -->

The shape and amplitude of a single EPG varies between worms even of the same genetic make-up. Therefore caution should be taken when the effects of drugs on EPG characteristics are made. Typically, three parameters are quantified: the frequency of EPGs (in Hz), the duration of a a single pump (measured by a time distance between E and R spike) and E/R ratio. E and R spikes are present on each EPG waveform, therefore they can be used to quantify mentioned parameters [@dillon2009]. 

(ref:example-epg) **EPG of *C. elegans* pharynx.** A single pharyngeal activity recorded extracellularly from the wild-type cut head of *C. elegans*. E peaks arise due to contraction, whereas I and R peaks due to relaxation of the pharynx [@dillon2009].

```{r example-epg-label, fig.cap="(ref:example-epg)", fig.scap = "EPG of \\textit{C. elegans} pharynx.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/basa-epg-individual.png")
```

## Chapter aims
In this chapter, the effects of the cholinergic compounds on EPG. These results are compared to the effects elicited by nicotine and neonicotinoids to further inform on their mode of action.

## 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)
```

To assess the effects of exogenous drug application on the pharynx an EPG - an extracellular recordings - from the *C. elegans* pharynx in cut head preparation were made. The pharynx was perfused for 5 minutes in Dent's saline to record basal pumping rate, for 5 minutes in the drug treatment to record changes in pharyngeal activity due to drug application and again for 5 minutes in Dent's saline to determine if the pharynx recovers from drug-induced pharyngeal alterations. The effects of 5-HT, cholinergic compounds including nicotine and neonicotinoids were determined. 

```{r echo=FALSE, fig.width=10, include=FALSE}
EPG_5ht <- magick::image_read("fig/results3/5ht_epg.png")
EPG_5ht1 <- ggdraw() + draw_image("fig/results3/5ht_epg.png")
```

```{r include=FALSE, echo=FALSE}
#read in EPG data 
EPG_data <- readRDS("Analysis/Data/Transformed/EPG/summarydata")

EPG_5HT <- EPG_data %>% 
  filter(Experiment==1)
#create new variable dose which will contain doses as character 
EPG_5HT_1 <-  EPG_5HT %>% 
  mutate (Dose = factor(Conc,
         levels = c("1e-11", "1e-09", "1e-08", "1e-07", "5e-07", "1e-06", "1e-05"), 
         labels = c("Ctr", "1nM", "10nM", "100nM", "500nM", "1uM", "10uM"))) 
#plot 5HT data 
EPG_5HT_plot <- EPG_5HT_1 %>% 
  group_by(Dose) %>% 
  ggplot(aes(x = Dose,
             y = mean_readout, fill = Dose)) +
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) +
  scale_fill_manual(values=c('#000000','#330033', '#660066', '#660033', '#990099', '#CC0099', '#FF66CC')) +  
  ylab("peak response") +
    ylim(0, 5) +
  theme(text=element_text(size=12,  family="sans"),
        legend.position = "none") + 
  ggsave("fig/results3/figure-test.pdf", width = 9, height = 10, units = "cm")
```

### Effects of 5-HT

Application of 5-HT concentrations ranging from 1 nM to 10 $\mu$M led to dose dependent stimulation of the pharyngeal pumping, sustained throughout the 5-minute perfusion (Figure \@ref(fig:epg-5ht-label)). The maximum response achieved was 4.4 Hz by 10 $\mu$M. The EC~50~ was 255 nM. Washing the pharynx for 5 minutes was sufficient to observe recovery from the 5-HT induced stimulation of pumping. 

To determine whether 5-HT had an effect on the shape of an EPG, individual EPG waveforms were examined closely (Figure \@ref(fig:5ht-epg-ind-label)a). A visible decrease in duration of the pump in response to 5-HT was noted. To quantify this, the pump duration of EPGs during the basal and treatment recording as well as after 5 minute wash were derived (Figure \@ref(fig:5ht-epg-ind-label)). The measurements were done by quantifying the time taken from E to R peak. 1 $\mu$M 5-HT reduced the latency by 27 %. This effect was reversible and returned to basal duration after 5 minutes of washing.

(ref:epg-5ht) **The concentration dependence for the effects of 5-HT on the EPG frequency.** *C. elegans* cut heads were perfused with Dent's saline and 5-HT at indicated concentrations. The effects of varying concentrations of 5-HT on pharyngeal pumping was scored by extracting peak response in a 10 second window. Data are mean $\pm$ SEM from 3-6 individual worms collected on $\ge$ 2 days. b) Dose-response curve for the effects of 5-HT on the pharynx. Responses are normalised to basal pumping rate and expressed as a % maximum response. c) Example EPG recording showing basal activity, stimulated activity upon perfusion with 1 $\mu$ of 5-HT and recovery post 5-HT exposure. The response to 1 $\mu$M 5-HT represents the sustained excitatory effects of 5-HT on the pharynx.

```{r epg-5ht-label, fig.cap="(ref:epg-5ht)", fig.scap = "The concentration dependence for the effects of 5-HT on the EPG frequency.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/5HT_EPG-all.png")
```

(ref:5ht-epg-ind) **Effects of 5-HT on the pump duration of *C. elegans*. Cut heads were perused with Dent's saline, 1 $\mu$M 5-HT and again with Dent's to wash off the 5-HT effects.** Basal and 5-HT exposed EPG waveforms were taken from the 10s peak pumping activity of the 5-minute perfusion period, whereas the wash EPG waveform was taken from the 10s peak pumping activity of the last minute of wash. Representative EPGs are shown in (a). Pump duration was derived by measuring the average time from E to R peaks (red arrow in a) of all EPGs over the peak response period (if there were less than 10 EPG in 10s window, 10 consecutive peaks were taken) (b). Data are mean $\pm$ SEM from 4 individual worms collected on 3 days. Two-tailed paired t-test with Welch correction. $***$P $\le$ 0.001.

```{r 5ht-epg-ind-label, fig.cap="(ref:5ht-epg-ind)", fig.scap = "Effects of 5-HT on the pump duration of \\textit{C. elegans}.", fig.align='center', echo=FALSE}
HT_epg_duration <- EPG_data %>% 
  filter(Experiment == 7) %>% 
  group_by("Cond") %>%
  ggplot(aes(x = Cond, y = mean_readout, fill = Cond)) +
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin=mean_readout-se, ymax=mean_readout+se, width = 0.4))+
  scale_fill_manual(values=c('#000000', '#990099', '#000000')) +
  ylab("pump duration (ms)")+
  ylim(0,120) +
  xlab("") +
  theme(text=element_text(size=12,  family="sans"),
        legend.position = "none") +
  ggsave("fig/results3/epg-HT-dur.pdf", width = 13, height = 10, units = "cm")

knitr::include_graphics("fig/results3/5HT_ind_EPG.png")
```

### Effects of Acetylcholine 

Acetylcholine was applied at concentrations ranging from 1 to 100 $\mu$M. This led to concentration and time dependent EPG alterations (Figure \@ref(fig:ach-epg-traces-label)). 10 $\mu$M acetylcholine stimulated pumping. This stimulation was sustained for 5 minutes of ACh perfusion and reversed to basal activity upon washing (Figure \@ref(fig:ach-epg-traces-label) a). Exposure of the pharynx to 15, 25, 50 or 100 $\mu$M acetylcholine led to potent stimulation of the pharyngeal activity before blocking its activity completely. Following, two types of activities were recorded: the pharynges remained blocked even after 5 minutes of washing (Figure \@ref(fig:ach-epg-traces-label) b), or began pumping again whilst being perfused with acetylcholine (Figure \@ref(fig:ach-epg-traces-label) c). The ratio of pharynges exhibiting the first or the second type of response to acetylcholine concentrations $\ge$ 15 $\mu$M was 1 : 1 and this was not concentration dependent (data not shown). 

A closer look at the primary response of the pharynx to acetylcholine concentrations $\ge$ 15 $\mu$M was taken (Figure \@ref(fig:ach-train-label)). The observed stimulation was characterised by a train of EPG spikes, progressively increasing in frequency and deceasing in amplitude (Figure \@ref(fig:ach-train-label) b and c) until the pharyngeal activity was completely inhibited. To determine the potency of acetylcholine on the spike frequency, the stimulatory effect of acetylcholine was scored. The EC~50~ was 22 $\mu$M.

```{r include=FALSE}
EPG_ach <- EPG_data %>% 
  filter(Experiment==9)
#create new variable dose which will contain doses as character 
EPG_Ach_trans <-  EPG_ach %>% 
  mutate (Dose = factor(Conc,
         levels = c("1e-08", "1e-06", "1e-05", "2.5e-05", "5e-05", "1e-04"), 
         labels = c("Ctr", "1uM", "10uM", "25uM", "50uM", "100uM"))) 
#plot 5HT data 
EPG_Ach_trans %>% 
  group_by(Dose) %>% 
  ggplot(aes(x = Dose,
             y = mean_readout)) +
  geom_bar(stat="identity",colour = "black", fill = "grey", size=0.5) + 
  geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) +
  ylab("peak pumping (Hz)") +
    ylim(0, 5) +
  theme(text=element_text(size=12,  family="sans"),
        legend.position = "none") +
ggsave("fig/results3/epg_ach.pdf", width = 9, height = 10, units = "cm")
```

(ref:ach-epg-traces) **Effects of acetylcholine on EPG.** Cut heads of *C. elegans* were perfused for 5 minutes in each Dent’s saline (basal), acetylcholine and again in Dent’s saline for recovery. Example EPG traces from the pharynx exposed to 10 $\mu$M (a), 25 (b), and 100 $\mu$M acetylcholine (c). Traces from 25 and 100 $\mu$M exposure represent variable responses of the pharynx to acetylcholine concentrations ranging from 15 to 100 $\mu$M. Each vertical line represents an EPG.

```{r ach-epg-traces-label, fig.cap="(ref:ach-epg-traces)", fig.scap= "Effects of acetylcholine on EPG.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/ach-epg-traces_3.png")
```

(ref:ach-train) **Effects of acetylcholine on EPG frequency and waveform.** Example EPG recordings from the pharynx exposed to 25 $\mu$ acetylcholine representing effects of acetylcholine at $\ge$ 25 $\mu$M. Exposure of the pharynx to acetylcholine results in stimulation of pharyngeal activity (a) characterised by a train of spikes, (b), of decreasing amplitude (c). The stimulatory period shown in a represents trace boxed in (a). Example EPG waveforms from the basal pharyngeal activity (1), and activity at the beginning (2), in the middle (3) and at the end (1) of train of EPG spikes elicited by acetylcholine.

```{r ach-train-label, fig.cap="(ref:ach-train)", fig.scap = "Effects of acetylcholine on EPG frequency and waveform.", fig.align='center', echo = FALSE}
knitr::include_graphics("fig/results3/ach_train_properties.png")
```

(ref:ach-epg) **The concentration dependence for the effects of acetylcholine on EPG frequency.** Cut-heads were perfused for 5 minutes in each Dent's saline and indicated acetylcholine concentrations. Pumping rates were derived by taking maximum pumping rate in each condition over the 10s time window, (or the entire stimulatory period, if the response to nicotine was under 10s). Data are mean $\ge$ SEM for 2-8 individual worms done on ≥ 2 days. b) Dose-response curve for the effects of nicotine on the pharynx. Data are mean $\ge$ SEM, normalised to the basal activity and expressed as a % maximum response.

```{r ach-epg-label, fig.cap="(ref:ach-epg)", fig.scap = "The concentration dependence for the effects of acetylcholine on EPG frequency.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/epg_ach_graph.png")
```

### Effects of nicotine 

Nicotine elicited concentration and time dependent changes to the EPG. At 1 $\mu$M it led to moderate but sustained stimulation of the pharyngeal activity. At higher doses (i.e. 5, 10, 25 and 50 $\mu$M) it caused a potent but transient elevation of pumping frequency followed by an inhibition of the pharyngeal activity (Figure \@ref(fig:epg-nicotine-2-label)). The stimulation by nicotine concentrations ranging from 5 to 50 $\mu$M consisted of a train of EPG spikes which decreased in amplitude with time (Figure \@ref(fig:epg-nicotine-2-label)). 
To better understand the nicotine-induced pharyngeal events, a video recording and photos of pharynges perfused with nicotine at 10 $\mu$M was taken (Supplementary video 3, Figure \@ref(fig:nicotine-photo-label)). In agreement with EPG data, nicotine induced a short-lived train of pharyngeal contractions. Cycles of simultaneous contraction-relaxation of the corpus and the grinder in the terminal bulb can be seen. This suddenly ceases. Following, asynchronous contraction of the isthmus and the grinder began which led to contraction and hence opening of the grinder and isthmus. In addition, twitching of the terminal bulb muscle could be observed throughout. 

To score for the stimulatory effects of nicotine of pumping, the peak pumping rates pre and post-application of nicotine were derived and the dose-response curves was plotted (Figure \@ref(fig:epg-nic-graphs-label)). The EC~50~ for the effects of nicotine on the pharyngeal activity was 2.7 $\mu$M.

Investigations into the recovery from nicotine-induced pharynx stimulation were made. Following stimulation by 1 $\mu$M nicotine, pharynges returned to the basal pumping rate within 5 minute wash, those exposed to higher nicotine concentration did not  (Figure \@ref(fig:nic-epg-traces-label)). To see if pharynges begin to pump after longer wash, experiment were repeated. Cut heads were exposed to nicotine for 5 minutes, but this time the washing period was extended until the pharyngeal activity was restored. To score for recovery, the time taken from the beginning of wash to the first EPG spike was taken (Figure \@ref(fig:epg-nicotine-3-label)). It takes 8 minutes or longer to recover from the effects induced by 5 and 10 $\mu$M nicotine. The time needed to recover from 50 $\mu$M perfusion was 18 minutes, suggesting longer washing is required to remove residual nicotine after exposure to higher drug concentrations.

(ref:epg-nic-traces) **Concentration dependent effects of nicotine on EPG frequency.** Cut heads were perfused for 5 minutes in each Dent's saline (basal), nicotine and again in Dent's saline for recovery. Example EPG traces from pharynges exposed to 1 $\mu$M (a) and 25 $\mu$M nicotine (b). Response to 25 $\pm$M represent responses to nicotine concentrations ranging from 5 to 50 $mu$M.

```{r nic-epg-traces-label, fig.cap="(ref:epg-nic-traces)", fig.scap = "Concentration dependent effects of nicotine on EPG frequency.", fig.align= 'center', echo=FALSE}
knitr::include_graphics("fig/results3/epg-nicotine-traces.png")
```

(ref:epg-nicotine-2) **Effects of nicotine on EPG frequency and waveform.** Example EPG recordings from the pharynx exposed to 50 $\mu$ nicotine representing effects of nicotine at $\ge$ 5 $\mu$M. Exposure of the pharynx to nicotine results in stimulation of pharyngeal activity (a) characterised by a train of spikes, (b), of decreasing amplitude (c). The stimulatory period shown in a represents trace boxed in (a). Example EPG waveforms from the basal pharyngeal activity (1), and activity at the beginning (2), in the middle (3) and at the end (1) of train of EPG spikes elicited by nicotine.

```{r epg-nicotine-2-label, fig.cap="(ref:epg-nicotine-2)", fig.scap = "Effects of nicotine on EPG frequency and waveform.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/50um-nic-epg-properties.png")
```

(ref:nicotine-photo) **The effects of nicotine on the pharynx morphology.** Appearance of the pharynx pre- and post exposure to nicotine at 10 $\mu$M. Images were taken immediately prior to nicotine exposure and post nicotine-induced inhibition of pumping.

```{r nicotine-photo-label, fig.cap="(ref:nicotine-photo)", fig.scap="The effects of nicotine on the pharynx morphology.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/nicotine-exposure-photo.png")
```

(ref:epg-nic-graphs) **The concentration dependence for the effects of nicotine on EPG frequency.** Cut-heads were perfused for 5 minutes in each Dent's saline and indicated nicotine concentrations. Pumping rates were derived by taking maximum pumping rate in each condition over the 10s time window, (or the entire stimulatory period, if the response to nicotine was under 10s). Data are mean $\ge$ SEM for 2-13 individual worms done on ≥ 2 days. b) Dose-response curve for the effects of nicotine on the pharynx. Data are mean $\ge$ SEM, normalised to the basal activity and expressed as a % maximum response.

```{r epg-nic-graphs-label, fig.cap="(ref:epg-nic-graphs)", fig.scap="The concentration dependence for the effects of nicotine on EPG frequency.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/epg-nic-graphs.png")
```

```{r include=FALSE, echo=FALSE}
EPG_nic <- EPG_data %>% 
  filter(Experiment == 2) %>% 
mutate(Dose=factor(Conc, 
        levels = c(1.0e-09, 1.0e-07, 1.0e-06, 5.0e-06, 1.0e-05, 2.5e-05, 5.0e-05),
        labels = c("Ctr", "100nM", "1uM", "5uM", "10uM","25uM", "50uM")))

EPG_nic_plot <- EPG_nic %>% 
  group_by(Dose) %>% 
  ggplot(aes(x = Dose,
             y = mean_readout, fill = Dose)) +
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) +
  scale_fill_manual(values=c('#000000', '#333333', '#666666','#999999', '#CCCCCC', '#D3D3D3', '#DCDCDC')) + 
  ylab("peak response") +
    ylim(0, 5) +
  theme(text=element_text(size=12,  family="sans"),
        legend.position = "none") 
ggsave("fig/results3/epg-nic.pdf", width = 9, height = 10, units = "cm") 
```

(ref:epg-nicotine-3) **Recovery from nicotine-induced inhibition of EPG of dissected *C. elegans*.** Example trace showing recovery from the effects of 50 $\mu$M nicotine on EPG of cut head (a). Pharynges were perfused with nicotine for 5 minutes before being flushed with Dent's saline. The time taken to recovery 5, 10 and 50 $\mu$M nicotine was derived (b) by measuring the time period from the beginning of wash to the first EPG. Data are mean $\pm$ SEM from 2 - 3 individual worms collected from 2 paired experiments.

```{r epg-nicotine-3-label, fig.cap="(ref:epg-nicotine-3)", fig.scap= "Recovery from nicotine-induced inhibition of EPG of dissected \\textit{C. elegans}.", fig.align='center', echo=FALSE}
EPG_nic_des <- EPG_data %>% 
  filter(Experiment == 6) %>% 
mutate(Dose=factor(Conc, 
        levels = c(5, 10, 50),
        labels = c("5uM", "10uM", "50uM")))

EPG_nic_des_plot <- EPG_nic_des %>% 
  group_by(Dose) %>% 
  ggplot(aes(x = Dose,
             y = mean_readout, fill = Dose)) +
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) +
    scale_fill_manual(values=c('#666666','#999999', '#CCCCCC', '#DCDCDC')) +
    ylab("time to recover (mins)") +
    ylim(0, 30) +
  theme(text=element_text(size=12,  family="sans"),
        legend.position = "none") +
ggsave("fig/results3/epg-nic-des.pdf", width = 9, height = 10, units = "cm")
knitr::include_graphics("fig/results3/NIC-RECOVERY.png")
```

Comparing the effects of nicotine on the pharynx as revealed by EPG and visual scoring reveled a discrepancy of the effects seen when pharynges exposed to 10 and 20 $\mu$M. In EPGs, perfusion of the pharynx for 5 minutes with either 10 or 20 $\mu$M elicited potent stimulation followed by a blockage of the pharyngeal activity (Figure \@ref(fig:nic-epg-traces-label) b). In contrast, no potent stimulation of the pharynx by these doses was observed when the pharyngeal pumping scored visually. Instead, a moderate stimulation of the pharynx was seen after ~20 minutes of incubation which increased in frequency over time (Figure \@ref(fig:cuthead-agonist-label). To determine if this effect can be replicated in the EPG experiments, pharynges were perfused with 10 $\mu$M nicotine for 1 hour (Figure \@ref(fig:prolonged-exp-nic-label)). After 10 minutes of perfusion, little EPG spikes began to emerge which increased in amplitude over time.

(ref:prolonged-exp-nic) **Effects of prolonged nicotine exposure on EPG.** a) Example EPG recording from the *C. elegans* cut head showing the period of basal activity (Dent's), and the activity in response to 10 $\mu$M nicotine over the 1 hour perfusion period. The effects of nicotine on EPG waveform (b) showing the differences between the basal EPG (1), EPG at the beginning (1) and after 10 (2) and 20 minute (3) perfusion with nicotine.

```{r prolonged-exp-nic-label, fig.cap="(ref:prolonged-exp-nic)", fig.scap = "Effects of prolonger nicotine exposure on EPG.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/nic-epg-trace-long-exposure-comb.png")
```

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### Effects of cytisine

The effects of cytisine, an agonist of nAChR was tested. Cytisine was applied at concentrations ranging from 1 to 100 $\mu$M. As in case of acetylcholine and nicotine, two types of responses were observed. Moderate but sustained stimulation of the pharyngeal activity was elicited by 5 $\mu$M, whereas at concentrations $\ge$ 10 $\mu$M, the pharynx was stimulated and subsequently inhibited (Figure \@ref(fig:cyt-epg-label)). The EC~50~ of cytisine on EPG was 3 $\mu$M (Figure \@ref(fig:cyt-epg-graph-label)).

```{r include=FALSE}
EPG_cyt <-  EPG_data %>%
  filter(Experiment==10) %>%
  mutate (Dose = factor(Conc,
         levels = c("1e-08", "1e-06", "5e-06", "1e-05", "5e-05", "1e-04"),
         labels = c("Ctr", "1uM", "5uM", "10uM", "50uM", "100uM")))
#plot 5HT data
EPG_cyt %>%
  group_by(Dose) %>%
  ggplot(aes(x = Dose,
             y = mean_readout)) +
  geom_bar(stat="identity",colour = "black", fill = "grey", size=0.5) +
  geom_errorbar(aes(ymin = mean_readout-se, ymax = mean_readout+se), width=0.4) +
  ylab("peak pumping (Hz)") +
    ylim(0, 5) +
  theme(text=element_text(size=12,  family="sans"),
        legend.position = "none") +
ggsave("fig/results3/epg_cyt.pdf", width = 9, height = 10, units = "cm")
```

(ref:cyt-epg) **Effects of cytisine on EPG.** Cut heads of *C. elegans* were perfused for 5 minutes in each Dent’s saline (basal), cytisine and again in Dent’s saline for recovery. Example EPG traces from the pharynx exposed to 5 $\mu$M (a) and 10 $\mu$M cytisine (b). Trace from 10 $\mu$M exposure represent response of the pharynx to cytisine concentrations ranging from 10 to 100 $\mu$M.

```{r cyt-epg-label, fig.cap="(ref:cyt-epg)", fig.scap= "Effects of cytisine on EPG.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/cyt-traces.png")
```

(ref:cyt-epg-graph) **The concentration dependence for the effects of cytisine on EPG frequency.** Cut-heads were perfused for 5 minutes in each Dent's saline and indicated cytisine concentrations. Pumping rates were derived by taking maximum pumping rate in each condition over the 10s time window, (or the entire stimulatory period, if the response to nicotine was under 10s). Data are mean $\ge$ SEM for 2-5 individual worms done on ≥ 2 days. b) Dose-response curve for the effects of cytisine on the pharynx. Data are mean $\ge$ SEM, normalised to the basal activity and expressed as a % maximum response.

```{r, cyt-epg-graph-label, fig.cap="(ref:cyt-epg-graph)", fig.scap = "The concentration dependence for the effects of cytisine on EPG frequency.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/epg-cyt.png")
```

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### Effects of neonicotinoids 

The effects of neonicotinoids on EPG were also examined. Pharynges were exposed to 100 $\mu$M nitenpyram, 50 $\mu$M thiacloprid and 75 $\mu$M clothianidin. Neither nitenpyram (Figure \@ref(fig:Nit-EPG-label)), nor thiacloprid had an effect on the frequency of pharyngeal activity (Figure \@ref(fig:epg-thia-label)). In contract, clothianidin stimulated pharyngeal activity. The frequency increased from 0.6 to 1.1 Hz and returned to basal following 5 minute wash (Figure \@ref(fig:clo-epg-label)). EPGs from clothianidin-perfused pharynges were examined and a reduction of R peak in relation to E peak was noted. Clothianidin significantly increased the E/R ratio from 1.3 to 1.8 (Figure \@ref(fig:clo-er-ratio-label) a and b). 5-minute wash did not reverse this effect. A change in duration of pumping activity was also observed. The latency of EPG decreased from 130 ms to 110 ms when pharynges exposed to clothianidin (Figure \@ref(fig:clo-er-ratio-label) a and c). This effect was not reversed upon 5 minute wash. 

(ref:Nit-EPG) **The effects of nitenpyram on EPG.** Cut heads were perfused for 5 minutes with solvent (Dent's) and 100 $\mu$ nitenpyram. Peak pharyngeal response over 10-second window pre- and post exposure period were derived. Data are mean $\pm$ SEM from 8 individual worms collected from 3 experiments. For comparison, the maximum pumping achieved by 5-HT is shown in dashed line. b) Example EPG recording showing basal, treatment with 100 $\mu$ nitenpyram and wash periods.

```{r Nit-EPG-label, fig.cap="(ref:Nit-EPG)", fig.scap = "The effects of nitenpyram on EPG.", fig.align='center', echo=FALSE}
Nit_EPG <- EPG_data %>% 
  filter(Experiment == 3) 
Nit_EPG_1 <- Nit_EPG %>%
  mutate(Cond2 = factor(Cond, levels=c("Pre", "Niten", "Wash"),
         labels = c("Basal", "Nitenpyram", "Wash")))

Nit_EPG_plot <- Nit_EPG_1 %>% 
  group_by("Cond2")%>%
  filter(Cond2 == "Basal" | Cond2 == "Nitenpyram") %>% 
  ggplot(aes(x = Cond2, y = mean_readout, fill = Cond2))+
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin=mean_readout-se, ymax=mean_readout+se, width = 0.4)) +
scale_fill_manual(values=c('#000000','#339900')) +  
  ylab("peak pumping (Hz)")+
  ylim(0, 5) +
  xlab("") +
  theme(text=element_text(size=12,family="sans"),
        legend.position = "none") + 
  ggsave("fig/results3/epg-niten.pdf", width = 13, height = 10, units = "cm")
knitr::include_graphics("fig/results3/Niten-EPG-comb.png")
```

(ref:epg-thia) **The effects of thiacloprid on EPG.** Cut heads were perfused for 5 minutes with solvent (Dent's + 0.01% DMSO) and 50 $\mu$ thiacloprid. Peak pharyngeal response over 10-second window pre- and post drug treatment were derived. Data are mean $\pm$ SEM from 8 individual worms collected from 3 experiments. For comparison, the maximum pumping achieved by 5-HT is shown in dashed line. (b) Example EPG recording showing basal, treatment with 50 $\mu$ thiacloprid and wash periods.

```{r epg-thia-label, fig.cap="(ref:epg-thia)", fig.scap = "The effects of thiacloprid on EPG.", fig.align='center', echo=FALSE}
Thia_EPG <- EPG_data %>% 
  filter(Experiment == 4) %>% 
  group_by("Cond") %>%
  filter(Cond== "Pre"| Cond == "Thiacloprid") %>% 
  ggplot(aes(x = Cond, y = mean_readout, fill = Cond)) +
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin=mean_readout-se, ymax=mean_readout+se, width = 0.4))+
  scale_fill_manual(values=c('#000000','#000066')) +
  ylab("peak pumping (Hz)")+
  ylim(0, 5) +
  xlab("") +
  theme(axis.text = element_text(size=12),
        axis.title = element_text(size=12),
        text= element_text(size=12,  family="sans"),
        legend.position ="none") +
  ggsave("fig/results3/epg-thia.pdf", width = 13, height = 10, units = "cm")
knitr::include_graphics("fig/results3/Thia-epg-combined.png")
```

(ref:clo-epg) **The effects of clothianidin on EPG.** Cut heads were perfused for 5 minutes with solvent (Dent's + 0.01% DMSO) and 75 $\mu$ clothianidin (a). Peak pharyngeal response over 10-second pre- and post drug treatment were derived. Data are mean $\pm$ SEM from 9 individual worms collected from 3 experiments. For comparison, the maximum pumping achieved by 5-HT is shown in dashed line. (b) Example EPG recording showing basal, treatment with 75 $\mu$ clothianidin and wash periods.

```{r clo-epg-label, fig.cap="(ref:clo-epg)", fig.scap ="The effects of clothianidin on EPG.", fig.align='center', echo=FALSE}
Clo_EPG <- EPG_data %>% 
  filter(Experiment == 5) %>% 
  group_by("Cond") %>%
  ggplot(aes(x = Cond, y = mean_readout, fill = Cond)) +
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin=mean_readout-se, ymax=mean_readout+se, width = 0.4)) +
  scale_fill_manual(values=c('#000000','#993300', '#000000')) +
  ylab("peak pumping (Hz)")+
  ylim(0, 5) +
  xlab("") +
  theme(axis.text = element_text(size=12),
        axis.title = element_text(size=12),
        text= element_text(size=12,  family="sans"),
        legend.position ="none") +
  ggsave("fig/results3/epg-clo.pdf", width = 13, height = 10, units = "cm")

knitr::include_graphics("fig/results3/Clo-epg-comb.png")
```

```{r, echo=FALSE, include=FALSE}
clo_er_plot <- EPG_data %>% 
  filter(Experiment == 8) %>% 
  group_by("Cond") %>%
  ggplot(aes(x = Cond, y = mean_readout, fill = Cond)) +
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin=mean_readout-se, ymax=mean_readout+se, width = 0.4))+
  scale_fill_manual(values=c('#000000','#993300', '#000000')) +
  ylab("E/R ratio")+
  xlab("") +
  theme(axis.text = element_text(size=12),
        axis.title = element_text(size=12),
        text= element_text(size=12,  family="sans"),
        legend.position ="none") +
  ggsave("fig/results3/clo-er-ratio.pdf", width = 8, height = 10, units = "cm")

clo_er_plot <- EPG_data %>% 
  filter(Experiment == 11) %>% 
  group_by("Cond") %>%
  ggplot(aes(x = Cond, y = mean_readout, fill = Cond)) +
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin=mean_readout-se, ymax=mean_readout+se, width = 0.4))+
  scale_fill_manual(values=c('#000000','#993300', '#000000')) +
  ylab("pump duration (ms)")+
  xlab("") +
  ylim(0,150) +
  theme(axis.text = element_text(size=12),
        axis.title = element_text(size=12),
        text= element_text(size=12,  family="sans"),
        legend.position ="none") +
  ggsave("fig/results3/clo-duration.pdf", width = 8, height = 10, units = "cm")
```

(ref:clo-er-ratio) **The effects of clothianidin on EPG waveform.** Example individual basal (left), clothianidin stimulated (middle) and recovery EPG (right) (a). The effects of 75 $\mu$ clothianidin on E/R ratio (b) and pump duration (c). The amplitude of E relative to R was measured to derive E/R ration, and the time taken from  E to R to derive pump latency. The values are the average pump duration / E/R ratio all EPGs in the period of the maximum pumping. If there were less then 10 EPGs, 10 consecutive peaks were taken. Data are mean $\pm$ SEM of 9 individual worms collected from 3 experiments. Two-tailed t-test, $*$P $\le$ 0.05. $**$P $\le$ 0.01.

```{r clo-er-ratio-label, fig.cap="(ref:clo-er-ratio)", fig.scap = "The effects of clothianidin on EPG waveform.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/clo-ind-epg.png")
```

### Effects of acetylcholine in the presence of nitenpyram and thiacloprid
Neither nitenpyram at 100 $\mu$M nor thiacloprid at 50 $\mu$M impaired on EPG frequency. To determine whether they inhibit the stimulatory effect of acetylcholine on EPG frequency, pharynges were pre-incubated with nitenpyram or thiacloprid and then exposed to both thiacloprid or nitenpyram and acetylcholine (Figure \@ref(fig:ach-nit-label) and Figure \@ref(fig:ach-thia-label)). Acetylcholine at 10 $\mu$M was tested, because this dose is close to the EC~50~ on EPG frequency. The response of the pharynx to acetylcholine in the presence or absence of neonicotinoids was compared. Pre-exposure of the pharynx to either neonicotinoid did not influence the EPG spike frequency elicited by acetylcholine. In both cases, upon application of acetylcholine, the pumping frequency increased from 0.2 to ~ 1 Hz. 

(ref:ach-nit) **Effects of acetylcholine on the EPG frequency in the presence and absence of nitenpyram.** Pharynges were pre- exposed to 100 $\mu$M nitenpyram. 5 minutes later, 100 $\mu$M nitenpyram and 10$\mu$M acetylcholine were applied. Responses to acetylcholine in the presence of nitenpyram were compared to responses elicited by acetylcholine. Pharyngeal pumping rates were derived by extracting peak response in a 10 second window. Data are mean $\pm$ SEM from 3-8 individual worms collected from paired experiments done on $\ge$ 2 days. Example EPG traces of the pharyngeal responses in the presence or absence of nitenpyram (b and c respectively).

```{r ach-nit-label, fig.cap="(ref:ach-nit)", fig.scap = "Effects of acetylcholine on the EPG frequency in the presence and absence of nitenpyram.", fig.align='center', echo=FALSE}
Nit_ach_EPG <- EPG_data %>%
  filter(Experiment == 12)
Nit_ach_EPG_1 <- Nit_ach_EPG %>%
mutate(Cond2 = factor(Cond, levels=c("Basal", "ACh+Nit", "ACh"),
         labels = c("Basal", "ACh+Nit", "ACh")))

Nit_ach_EPG_plot <- Nit_ach_EPG_1 %>%
  group_by("Cond2")%>%
  ggplot(aes(x = Cond2, y = mean_readout, fill = Cond2))+
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin=mean_readout-se, ymax=mean_readout+se, width = 0.4)) +
scale_fill_manual(values=c('#000000', '#339900', 'grey')) +
  ylab("peak pumping (Hz)")+
  ylim(0, 5) +
  xlab("") +
  theme(text=element_text(size=12,family="sans"),
        legend.position = "none") +
  ggsave("fig/results3/epg-nit+ach.pdf", width = 13, height = 10, units = "cm")
knitr::include_graphics("fig/results3/nit+ACH+combined.png")
```

(ref:ach-thia) **Effects of acetylcholine on the EPG frequency in the presence and absence of clothianidin.** Pharynges were pre- exposed to 75 $\mu$M thiacloprid. 5 minutes later, 75 $\mu$M thiacloprid and 10$\mu$M acetylcholine were applied. Responses to acetylcholine in the presence of thiacloprid were compared to responses elicited by acetylcholine. Pharyngeal pumping rates were derived by extracting peak response in a 10 second window. Data are mean $\pm$ SEM from 3-6 individual worms collected from paired experiments done on $\ge$ 2 days. Example EPG traces of the pharyngeal responses in the presence or absence of thiacloprid (b and c respectively).

```{r ach-thia-label, fig.cap="(ref:ach-thia)", fig.scap = "Effects of acetylcholine on the EPG frequency in the presence and absence of clothianidin.", fig.align='center', echo=FALSE}
thia_ach_EPG <- EPG_data %>%
  filter(Experiment == 13)

thia_ach_EPG <- thia_ach_EPG[!thia_ach_EPG$Cond=="Wash" ,]

thia_ach_EPG_1 <- thia_ach_EPG %>%
mutate(Cond2 = factor(Cond, levels=c("Basal", "Thia + ACh", "ACh"),
         labels = c("Basal", "Thia + ACh", "ACh")))

thia_ach_EPG__plot <- thia_ach_EPG_1 %>%
  group_by("Cond2")%>%
  ggplot(aes(x = Cond2, y = mean_readout, fill = Cond2))+
  geom_bar(stat = "identity") +
  geom_errorbar(aes(ymin=mean_readout-se, ymax=mean_readout+se, width = 0.4)) +
scale_fill_manual(values=c('#000000', '#000066', 'grey')) +
  ylab("peak pumping (Hz)")+
  ylim(0, 5) +
  xlab("") +
  theme(text=element_text(size=12,family="sans"),
        legend.position = "none") +
ggsave("fig/results3/epg-thia+ach.pdf", width = 13, height = 10, units = "cm")
knitr::include_graphics("fig/results3/thia+ACH+combined.png")
```

### Effects of nicotine on worms deficient in nAChR

EPG analysis of the effects of nicotine on the pharynx revealed a nicotinic effect not seen in the visual observation experiments. To determine whether the effects of nicotine on EPG on nAChR *eat-2* mutant differs from wild-type, EPG recordings from both strains were obtained. The stimulatory effect of nicotine concentrations ranging from 100 nM to 50 $\mu$M was scored (Figure \@ref(fig:nicotine-epg-eat2-label)). No marked differences in nicotine-sensitivity of *eat-2* mutant vs wild-type worms were noted. The EC~50~ on wild-type was 3 $\mu$M, in comparison to 5 $\mu$M in *eat-2* mutant. 

(ref:nicotine-epg-eat2) **Effects of nicotine on EPG frequency.** Cut heads were perfused for 5 minutes in Dent's and nicotine at indicated concentrations. Peak response in 10s window was derived and normalised to the basal pumping. Data are mean SEM of paired experiments done on $\ge$ 2 worms on a single day.

```{r nicotine-epg-eat2-label, fig.cap="(ref:nicotine-epg-eat2)", fig.scap = "Effects of nicotine on EPG frequency.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results3/DR+TRACENic_N2+eat2.png")
```

## Discussion

*C. elegans* expresses at least 29 different nAChRs. Only a few of these subunits have been linked to the function of the pharynx [@mckay2004]. Cholinergic compounds have been applied on the cut-head *C. elegans* and EPG recording were made to characterise the pharmacological profile of the pharynx, and therein the expressed nAChRs. Results are summarised in Table \@ref(tab:pharynx-summary2). 

### Nicotine inhibits pumping by tetinising pharyngeal muscle

EPG analysis revealed complex responses of the pharynx to nicotine. 1 $\mu$M induced sustained stimulatory effect. At concentrations ranging from 10 to 100 $\mu$M a dual response was observed: a potent stimulation characterised by a train of EPGs. The amplitude of spikes progressively decreased with time until a complete inhibition of pharyngeal activity occurred. This inhibition of pharyngeal activity coincided with a sustained contraction of grinder and isthmus of the pharynx. This suggests that nicotine induces potent contraction of the pharyngeal muscle and that a sustained stimulation of the pharynx by nicotine tetinizes a muscle, leading to pumping inhibition. 

### *Eat-2* is either not involved, or redundant, in the nicotine-induced pharyngeal responses 

The EPG responses elicited by nicotine are reminiscent of the responses achieved by the cholinergic compounds such as acetylcholine, cytisine and choline. This suggests nicotine acts at the pharynx in a similar way to classical nAChR agonist and exogenously applied neurotransmitter targeting nAChRs. EPG recordings from wild-type and *eat-2* nAChR mutant worms show no difference in nicotine-induced responses. This is different from the results obtained in the visual observation experiments. However, the effects were observed at different time points. In EPG experiments, the recording of the events occurring in the 5-minute window of perfusion with nicotine. In contrast, pharyngeal pumping in visual experiments was measured 30-minutes post-exposure to nicotine. EPG recordings show that during the 1-hour incubation of the pharynx with 10 $\mu$ of nicotine, the pharynx is tetinised rapidly, but then after 20 minutes of perfusion, the pharyngeal activity re-emerges. The lack of difference in sensitivity of the pharynx recorded by EPG suggests the function of *eat-2* is redundant in nicotine-driven initial stimulation of pumping. Therefore, in the absence of EAT-2 nicotine acts on other receptors to elicit the same response. Alternatively, nicotine does not act on *eat-2* containing nAChRs.

### Distinct effects of neonicotinoids on the pharyngeal system

The electrophysiological effects achieved by neonicotinoids on insect neuronal preparation and *C. elegans* pharynx differ. In *C. elegans*, clothianidin transiently stimulated pharynx for 5 minutes. EPG analysis revealed the effects of clothianidin on the shape of an EPG waveform. In the presence of 50 $\mu$M, the amplitude of E spikes relative to R spike increased leading to a decrease of RE ratio. E spikes arise due to simultaneous contraction of the pharyngeal muscle syncytium [@franks2006]. Therefore a reduction in E spike amplitude could be due to reduced synchronisation of the muscle syncytium depolarisation. 50 $\mu$M clothianidin also reduced a latency of pump duration. In contrast, thiacloprid and nitenpyram had no effect. In insects, neonicotinoids typically achieve biphasic effects on post-synaptic neurons: excitation followed by an inhibition (Section \@ref(electrophysevidence)). Such effects was not observed in *C. elegans*. This supports the divergent nature of insect and pharyngeal *C. elegans* nAChR families. 

### *C. elegans* pharyngeal nAChRs are more closely related to human than insect nAChRs. 

Investigation of the effects of nicotine and neonicotinoids on *C. elegans* pharyngeal system revealed high efficacy of nicotine, and low efficacy of neonicotinoids, which is also seen in mammals [@tomizawa2003]. In contrast, neonicotinoids are more efficacious on insects in comparison to nicotine [@tomizawa2003]. This suggests that mammalian and *C. elegans* nAChRs are more closely related than insect and *C. elegans* receptors. This is supported by the similarities in the effective doses of cholinergic compounds on the *C. elegans* pharynx and on the mammalian nAChRs. Acetylcholine, nicotine and cytisine all stimulated the pharynx with the EC~50~ of low $\mu$M. These concentrations are also effective against mammalian neuronal preparations and isolated nAChRs. Based on the $\alpha7$ receptors expressed in Xenopus oocytes, the EC50 of acetylcholine, nicotine and cytisine is 21 $\mu$M, 12.6 $\mu$M and 5.6 $\mu$M, respectively [@ballivet1996; @papke2002]. 

Table: (\#tab:pharynx-summary2) Summary of the effects of compounds on the pharyngeal activity of *C. elegans*.

+--------------+--------------------------------+-----------------------------------+
|              |          Pump  frequency       |      EPG waveform                 |
+==============+================================+===================================+
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|5-HT          |1. Dose-dependent, sustained    | 1. Reduced pump duration          |
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|              |stimulation                     |                                   |
|              |2. EC~50~=255$\mu$              |                                   |
+--------------+--------------------------------+-----------------------------------+
|Acetylcholine |1. Sustained stimulation        |1. The train of spikes elicited    |
|              |by 10 $\mu$M                    |by by $\ge$ 10$\mu$M characterised |
|              |2. Potent stimulation followed  |EPG spikes decreasing in amplitude |
|              |by inhibition by $\ge$ 25$\mu$M |                                   |
|              |3. EC~50~=22 $\mu$M             |                                   |
+--------------+--------------------------------+-----------------------------------+
|Cytisine      |1. Sustained stimulation        |1. The train of spikes elicited    |
|              |by 5 $\mu$M                     |by by $\ge$ 10$\mu$M characterised |
|              |2. Potent stimulation followed  |EPG spikes decreasing in amplitude |
|              |by inhibition by $\ge$ 10$\mu$M |                                   |
|              |3. EC~50~=3 $\mu$M              |                                   |
+--------------+--------------------------------+-----------------------------------+
|Nicotine      |1. Sustained stimulation        |1. The train of spikes elicited    |
|              |by 1 $\mu$M                     |by by $\ge$ 10$\mu$M characterised |
|              |2. Potent stimulation followed  |EPG spikes decreasing in amplitude |
|              |by inhibition by $\ge$ 10$\mu$M |                                   |
|              |3. EC~50~=2.7 $\mu$M            |                                   | 
+--------------+--------------------------------+-----------------------------------+
|Nitenpyram    |1. No effect at 0.1 mM          |1. No effect at 0.1 mM             |
+--------------+--------------------------------+-----------------------------------+
|Thiacloprid   |1. No effect at 50 $\mu$M       |1. No effects at 50 $\mu$M         |
+--------------+--------------------------------+-----------------------------------+
|Clothianidin  |1. Weak and sustained           |1. Increased E/R ratio             |
|              |stimulation by 75$\mu$          |2. Decrease in pump duration       |
+--------------+--------------------------------+-----------------------------------+
|Alpha-bgtx    |1. No effects on ACh-evoked     |NA                                 |
|              |pharyngeal response             |                                   |
+--------------+--------------------------------+-----------------------------------+
|Clothianidin  |1. No effects on ACh-evoked     |NA                                 |
|              |pharyngeal response             |                                   |
+--------------+--------------------------------+-----------------------------------+
|Thiacloprid   |1. No effects on ACh-evoked     |NA                                 |
|              |pharyngeal response             |                                   |
+--------------+--------------------------------+-----------------------------------+