05-results-03.Rmd 46.8 KB
 mk11g11 committed Sep 29, 2019 1 2 3 4 # Pharmacological characterisation of the *C. elegans* pharynx {#results-3} ## Introduction  mk11g11 committed Feb 19, 2020 5 The previous chapter (Chapter 4) describes the effects of nicotine, 5-HT and neonicotinoids on pharyngeal pumping of the "cut-head worm". An alternative pharyngeal assay is an extracellular recording of the electrical action of the pharynx. This is a electropharyngeogram (EPG). Unlike scoring pharyngeal pumping by visual observation, EPG recordings allow for more detailed temporal resolution. Additionally, investigations into drug-induced changes of the EPG waveform may inform on the potential mode of action of compounds.  mk11g11 committed Sep 29, 2019 6   mk11g11 committed Feb 19, 2020 7 In an EPG assay, a worm's head is placed in a recording chamber. 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 characteristic waveform. There are several different 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. Excitatory e spike arises due to the release of ACh from MC neurons and an activation of of nAChRs [@raizen1995]. Excitatory 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.  mk11g11 committed Sep 29, 2019 8 9 10 11 12  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].  mk11g11 committed Feb 19, 2020 13 (ref:example-epg) **Electropharyngeogram (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].  mk11g11 committed Sep 29, 2019 14   mk11g11 committed Feb 19, 2020 15 {r example-epg-label, fig.cap="(ref:example-epg)", fig.scap = "Electropharyngeogram (EPG) of \\textit{C. elegans} pharynx.", fig.align='center', echo=FALSE}  mk11g11 committed Sep 29, 2019 16 17 18 19 knitr::include_graphics("fig/results3/basa-epg-individual.png")  ## Chapter aims  mk11g11 committed Feb 19, 2020 20 In this chapter, the effects of 5-HT and the cholinergic compounds on EPG are investigated. These results are compared to the effects elicited by nicotine and neonicotinoids to further inform on what is their mode of action.  mk11g11 committed Sep 29, 2019 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37  ## 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)   mk11g11 committed Feb 19, 2020 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 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 the spontaneous pharyngeal activity due to drug application and again for 5 minutes in Dent's saline to determine if the pharynx recovers from drug-induced changes in pumping.  mk11g11 committed Sep 29, 2019 70 71 72 73 74  ### 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.  mk11g11 committed Feb 19, 2020 75 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 measured (Figure \@ref(fig:5ht-epg-ind-label)). The direct measurements were made 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.  mk11g11 committed Sep 29, 2019 76   mk11g11 committed Feb 19, 2020 77 (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 $\mu$M 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.  mk11g11 committed Sep 29, 2019 78 79 80 81 82  {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")   mk11g11 committed Feb 19, 2020 83 (ref:5ht-epg-ind) **Effects of 5-HT on the pump duration of *C. elegans*.** Basal and 5-HT exposed EPG waveforms were taken from the time window where maximum pumping activity was noted, whereas the wash EPG waveform was taken from 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.  mk11g11 committed Sep 29, 2019 84 85  {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}  mk11g11 committed Feb 19, 2020 86 87 88 89 90 91 92 93 94 95 96 97 98 # 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")  mk11g11 committed Sep 29, 2019 99 100 101 102 103 104  knitr::include_graphics("fig/results3/5HT_ind_EPG.png")  ### Effects of Acetylcholine  mk11g11 committed Feb 19, 2020 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 Acetylcholine was applied at concentrations ranging from 1 to 100 $\mu$M. This led to concentration and time dependent effects on pumping (Figure \@ref(fig:ach-epg-traces-label)). 10 $\mu$M acetylcholine stimulated pumping. This stimulation was sustained for 5 minute 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 pharynxes 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 pharynxes 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 at 25 $\mu$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. (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 a single EPG.  mk11g11 committed Sep 29, 2019 132 133 134 135 136  {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")   mk11g11 committed Feb 19, 2020 137 (ref:ach-train) **Effects of acetylcholine on EPG frequency and waveform.** Example EPG recordings from the pharynx exposed to 25 $\mu$M 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.  mk11g11 committed Sep 29, 2019 138 139 140 141 142 143 144 145 146 147 148 149 150  {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  mk11g11 committed Feb 19, 2020 151 152 Similarly to ACh, 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 pharynxes 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.  mk11g11 committed Sep 29, 2019 153 154 155  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.  mk11g11 committed Feb 19, 2020 156 Investigations into the recovery from nicotine-induced pharynx stimulation were made. Following stimulation by 1 $\mu$M nicotine, pharynxes returned to the basal pumping rate within 5 minute wash, those exposed to higher nicotine concentrations remained inhibited (Figure \@ref(fig:nic-epg-traces-label)). To see if pharynxes begin to pump after longer wash, experiment were repeated. Cut heads were exposed to nicotine for 5 minutes, but after 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.  mk11g11 committed Sep 29, 2019 157   mk11g11 committed Feb 19, 2020 158 (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 pharynxes 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.  mk11g11 committed Sep 29, 2019 159 160 161 162 163  {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")   mk11g11 committed Feb 19, 2020 164 (ref:epg-nicotine-2) **Effects of nicotine on EPG frequency and waveform.** Example EPG recordings from the pharynx exposed to 50 $\mu$M 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.  mk11g11 committed Sep 29, 2019 165 166 167 168 169 170 171 172 173 174 175  {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")   mk11g11 committed Feb 19, 2020 176 (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 $\pm$ 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.  mk11g11 committed Sep 29, 2019 177 178 179 180 181  {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")   mk11g11 committed Feb 19, 2020 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203  (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). pharynxes 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.  mk11g11 committed Sep 29, 2019 204 205  {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}  mk11g11 committed Feb 19, 2020 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 # 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")  mk11g11 committed Sep 29, 2019 224 225 226 knitr::include_graphics("fig/results3/NIC-RECOVERY.png")   mk11g11 committed Feb 19, 2020 227 Comparing the effects of nicotine on the pharynx as revealed by EPG and by visual scoring revealed a discrepancy in the effects seen when pharynxes were 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, pharynxes 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.  mk11g11 committed Sep 29, 2019 228   mk11g11 committed Feb 19, 2020 229 (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 25 $\mu$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.  mk11g11 committed Sep 29, 2019 230   mk11g11 committed Feb 19, 2020 231 {r prolonged-exp-nic-label, fig.cap="(ref:prolonged-exp-nic)", fig.scap = "Effects of prolonged nicotine exposure on EPG.", fig.align='center', echo=FALSE}  mk11g11 committed Sep 29, 2019 232 233 234 knitr::include_graphics("fig/results3/nic-epg-trace-long-exposure-comb.png")   mk11g11 committed Oct 06, 2019 235 236 \newpage  mk11g11 committed Sep 29, 2019 237 238 239 240 ### 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)).  mk11g11 committed Feb 19, 2020 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259   mk11g11 committed Sep 29, 2019 260 261 262 263 264 265 266 267 268 269 270 271 272  (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")   mk11g11 committed Oct 06, 2019 273 274 \newpage  mk11g11 committed Sep 29, 2019 275 276 ### Effects of neonicotinoids  mk11g11 committed Feb 19, 2020 277 The effects of neonicotinoids on EPG were also examined. Pharynxes 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 a 5 minute wash (Figure \@ref(fig:clo-epg-label)). EPGs from clothianidin-perfused pharynxes 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 pharynxes exposed to clothianidin (Figure \@ref(fig:clo-er-ratio-label) a and c). This effect was not reversed upon 5 minute wash.  mk11g11 committed Sep 29, 2019 278   mk11g11 committed Feb 19, 2020 279 (ref:Nit-EPG) **The effects of nitenpyram on EPG.** Cut heads were perfused for 5 minutes with solvent (Dent's) and 100 $\mu$M nitenpyram. Peak pharyngeal response over 10-second window pre- and post exposure period were derived. Data are mean $\pm$ SEM from eight 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 $\mu$M nitenpyram and wash periods.  mk11g11 committed Sep 29, 2019 280 281  {r Nit-EPG-label, fig.cap="(ref:Nit-EPG)", fig.scap = "The effects of nitenpyram on EPG.", fig.align='center', echo=FALSE}  mk11g11 committed Feb 19, 2020 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 # 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")  mk11g11 committed Sep 29, 2019 301 302 303 knitr::include_graphics("fig/results3/Niten-EPG-comb.png")   mk11g11 committed Feb 19, 2020 304 (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 $\mu$M thiacloprid and wash periods.  mk11g11 committed Sep 29, 2019 305 306  {r epg-thia-label, fig.cap="(ref:epg-thia)", fig.scap = "The effects of thiacloprid on EPG.", fig.align='center', echo=FALSE}  mk11g11 committed Feb 19, 2020 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 # 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")  mk11g11 committed Sep 29, 2019 323 324 325 knitr::include_graphics("fig/results3/Thia-epg-combined.png")   mk11g11 committed Feb 19, 2020 326 (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 $\mu$M clothianidin (a). Peak pharyngeal response over a 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 $\mu$M clothianidin and wash periods.  mk11g11 committed Sep 29, 2019 327 328  {r clo-epg-label, fig.cap="(ref:clo-epg)", fig.scap ="The effects of clothianidin on EPG.", fig.align='center', echo=FALSE}  mk11g11 committed Feb 19, 2020 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 # 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")  mk11g11 committed Sep 29, 2019 344 345 346 347  knitr::include_graphics("fig/results3/Clo-epg-comb.png")   mk11g11 committed Feb 19, 2020 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381  (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 $\mu$M 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.  mk11g11 committed Sep 29, 2019 382 383 384 385 386 387  {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  mk11g11 committed Feb 19, 2020 388 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, pharynxes 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.  mk11g11 committed Sep 29, 2019 389   mk11g11 committed Feb 19, 2020 390 (ref:ach-nit) **Effects of acetylcholine on the EPG frequency in the presence and absence of nitenpyram.** Pharynxes 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).  mk11g11 committed Sep 29, 2019 391 392  {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}  mk11g11 committed Feb 19, 2020 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 # 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")  mk11g11 committed Sep 29, 2019 411 412 413 knitr::include_graphics("fig/results3/nit+ACH+combined.png")   mk11g11 committed Feb 19, 2020 414 (ref:ach-thia) **Effects of acetylcholine on the EPG frequency in the presence and absence of clothianidin.** pharynxes 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).  mk11g11 committed Sep 29, 2019 415 416  {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}  mk11g11 committed Feb 19, 2020 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 # thia_ach_EPG <- EPG_data %>% # filter(Experiment == 13) # # thia_ach_EPG <- thia_ach_EPG[!thia_ach_EPG$Cond=="Wash" ,] # # thia_ach_EPG_1 <- thia_ach_EPG %>% # mutate(Cond2 = factor(Cond, levels=c("Basal", "Thia + ACh", "ACh"), # labels = c("Basal", "Thia + ACh", "ACh"))) # # thia_ach_EPG__plot <- thia_ach_EPG_1 %>% # group_by("Cond2")%>% # ggplot(aes(x = Cond2, y = mean_readout, fill = Cond2))+ # geom_bar(stat = "identity") + # geom_errorbar(aes(ymin=mean_readout-se, ymax=mean_readout+se, width = 0.4)) + # scale_fill_manual(values=c('#000000', '#000066', 'grey')) + # ylab("peak pumping (Hz)")+ # ylim(0, 5) + # xlab("") + # theme(text=element_text(size=12,family="sans"), # legend.position = "none") + # ggsave("fig/results3/epg-thia+ach.pdf", width = 13, height = 10, units = "cm")  mk11g11 committed Sep 29, 2019 438 439 440 441 442 443 444 knitr::include_graphics("fig/results3/thia+ACH+combined.png")  ### Effects of nicotine on worms deficient in nAChR EPG analysis of the effects of nicotine on the pharynx revealed a nicotinic effect not seen in the visual observation experiments. To determine whether the effects of nicotine on EPG on nAChR *eat-2* mutant differs from wild-type, EPG recordings from both strains were obtained. The stimulatory effect of nicotine concentrations ranging from 100 nM to 50$\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$M in *eat-2* mutant.  mk11g11 committed Feb 19, 2020 445 (ref:nicotine-epg-eat2) **Effects of nicotine on EPG frequency of *eat-2* nAChR *C. elegans* mutant.** Cut heads were perfused for 5 minutes in Dent's and nicotine at indicated concentrations. Peak response in 10s window was derived and normalised to the basal pumping (a). Data are mean SEM of paired experiments done on$\ge$2 worms on a single day. Example EPG recording showing response of the wild-type and *eat-2* mutant to 25$\muM nicotine (b).  mk11g11 committed Sep 29, 2019 446   mk11g11 committed Feb 19, 2020 447 {r nicotine-epg-eat2-label, fig.cap="(ref:nicotine-epg-eat2)", fig.scap = "Effects of nicotine on EPG frequency of \\textit{eat-2} nAChR \\textit{C. elegans} mutant.", fig.align='center', echo=FALSE}  mk11g11 committed Sep 29, 2019 448 449 450 451 452 knitr::include_graphics("fig/results3/DR+TRACENic_N2+eat2.png")  ## Discussion  mk11g11 committed Feb 19, 2020 453 *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 were applied to the cut-head *C. elegans* and EPG recording were made to characterise the pharmacological profile of the pharynx, and in order to determine the expressed nAChRs. Results are summarised in Table \@ref(tab:pharynx-summary2).  mk11g11 committed Sep 29, 2019 454   mk11g11 committed Feb 19, 2020 455 ### Nicotine inhibits pumping by contracting pharyngeal muscle  mk11g11 committed Sep 29, 2019 456   mk11g11 committed Feb 19, 2020 457 EPG analysis revealed complex responses of the pharynx to nicotine. 1 and 10\mu$M induced sustained stimulatory effect. At concentrations ranging from 25 to 100$\mu$M a dual response was observed: a potent stimulation characterised by a train of EPGs. The amplitude of spikes progressively decreased with time until a complete inhibition of pharyngeal activity occurred (Figure \@ref(fig:nic-epg-traces-label)). This inhibition of pharyngeal activity coincided with a sustained contraction of grinder and isthmus of the pharynx. This suggests that nicotine induces potent contraction of the pharyngeal muscle and that a sustained stimulation leads to pumping inhibition.  mk11g11 committed Sep 29, 2019 458   mk11g11 committed Feb 19, 2020 459 ### *Eat-2* is not involved in the nicotine-induced pharyngeal responses  mk11g11 committed Sep 29, 2019 460   mk11g11 committed Feb 19, 2020 461 The EPG responses elicited by nicotine are reminiscent of the responses achieved by the cholinergic compounds such as acetylcholine, cytisine and choline. This suggests nicotine acts at the pharynx in a similar way to classical nAChR agonist and exogenously applied neurotransmitter targeting nAChRs. In the preence of food, acetylcholine acts on *eat-2* containing nAChRs to induce pumping [@mckay2004]. EPG recordings from wild-type and *eat-2* nAChR mutant worms show no difference in nicotine-induced responses, suggesting nicotine does not act on *eat-2* containing nAChRs.  mk11g11 committed Sep 29, 2019 462 463 464  ### Distinct effects of neonicotinoids on the pharyngeal system  mk11g11 committed Feb 19, 2020 465 The electrophysiological effects achieved by neonicotinoids on insect neuronal preparation and *C. elegans* pharynx differ. In *C. elegans*, clothianidin transiently stimulated pharynx for 5 minutes. EPG analysis revealed the effects of clothianidin on the shape of an EPG waveform. In the presence of 50$\mu$M clothianidinsax, the amplitude of E spikes relative to R spike decreased leading to an increase of ER ratio. E spikes arise due to simultaneous contraction of the pharyngeal muscle syncytium [@franks2006]. Therefore, a reduction in E spike amplitude could be due to reduced synchronisation of the muscle syncytium depolarisation. 50$\mu$M clothianidin also reduced a latency of pump duration. In contrast, thiacloprid and nitenpyram had no effect. In insects, neonicotinoids typically achieve biphasic effects on post-synaptic neurons: excitation followed by an inhibition (Section \@ref(electrophysevidence)). Such effects was not observed in *C. elegans*. This supports the divergent nature of insect and pharyngeal *C. elegans* nAChR families.  mk11g11 committed Sep 29, 2019 466 467 468  ### *C. elegans* pharyngeal nAChRs are more closely related to human than insect nAChRs.  mk11g11 committed Feb 19, 2020 469 Investigation of the effects of nicotine and neonicotinoids on *C. elegans* pharyngeal system revealed high efficacy of nicotine, and low efficacy of neonicotinoids, which is also seen in mammals [@tomizawa2003]. In contrast, neonicotinoids are more efficacious on insects in comparison to nicotine [@tomizawa2003]. This suggests that mammalian and *C. elegans* nAChRs are more closely related than insect and *C. elegans* receptors. This is supported by the similarities in the effective doses of cholinergic compounds on the *C. elegans* pharynx and on the mammalian nAChRs. Acetylcholine, nicotine and cytisine all stimulated the pharynx with the EC~50~ of low$\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$M, respectively [@ballivet1996; @papke2002]. This suggests there are similarities in the pharmacophore of pharyngeal nAChR and mammalian$\alpha7$receptors.  mk11g11 committed Sep 29, 2019 470 471 472 473 474 475  Table: (\#tab:pharynx-summary2) Summary of the effects of compounds on the pharyngeal activity of *C. elegans*. +--------------+--------------------------------+-----------------------------------+ | | Pump frequency | EPG waveform | +==============+================================+===================================+  mk11g11 committed Oct 06, 2019 476 |5-HT |1. Dose-dependent, sustained | 1. Reduced pump duration |  mk11g11 committed Sep 29, 2019 477 | |stimulation | |  mk11g11 committed Feb 19, 2020 478 | |2. EC~50~=255$\mu$M | |  mk11g11 committed Sep 29, 2019 479 480 481 482 +--------------+--------------------------------+-----------------------------------+ |Acetylcholine |1. Sustained stimulation |1. The train of spikes elicited | | |by 10$\mu$M |by by$\ge$10$\mu$M characterised | | |2. Potent stimulation followed |EPG spikes decreasing in amplitude |  mk11g11 committed Feb 19, 2020 483 | |by inhibition by$\ge$25$\mu$M| |  mk11g11 committed Sep 29, 2019 484 485 486 | |3. EC~50~=22$\mu$M | | +--------------+--------------------------------+-----------------------------------+ |Cytisine |1. Sustained stimulation |1. The train of spikes elicited |  mk11g11 committed Feb 19, 2020 487 | |by 5$\mu$M |by by$\ge$10$\mu$M characterised|  mk11g11 committed Sep 29, 2019 488 | |2. Potent stimulation followed |EPG spikes decreasing in amplitude |  mk11g11 committed Feb 19, 2020 489 | |by inhibition by$\ge$10$\mu$M| |  mk11g11 committed Sep 29, 2019 490 491 492 | |3. EC~50~=3$\mu$M | | +--------------+--------------------------------+-----------------------------------+ |Nicotine |1. Sustained stimulation |1. The train of spikes elicited |  mk11g11 committed Feb 19, 2020 493 | |by 1$\mu$M |by by$\ge$10$\mu$M characterised|  mk11g11 committed Sep 29, 2019 494 | |2. Potent stimulation followed |EPG spikes decreasing in amplitude |  mk11g11 committed Feb 19, 2020 495 | |by inhibition by$\ge$10$\mu$M| |  mk11g11 committed Sep 29, 2019 496 497 498 499 500 501 502 | |3. EC~50~=2.7$\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$M | +--------------+--------------------------------+-----------------------------------+ |Clothianidin |1. Weak and sustained |1. Increased E/R ratio |  mk11g11 committed Feb 19, 2020 503 | |stimulation by 75$\mu\$M |2. Decrease in pump duration |  mk11g11 committed Sep 29, 2019 504 505 506 507 508 509 510 511 512 513 514 +--------------+--------------------------------+-----------------------------------+ |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 | | +--------------+--------------------------------+-----------------------------------+