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Commit 5d56d2f0 authored by mk11g11's avatar mk11g11
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corrections

parent e74fe8cf
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......@@ -19,7 +19,7 @@ library(kableExtra)
## General bacterial methods
### Transformation of *E. coli* with DNA vectors
Briefly, 25 to 50 $\mu$L of chemically competent Mach1 or DH5$\alpha$ *Escherichia coli (E.coli)* cells were combined and gently mixed with ~10 pg of plasmid DNA. The mix was left on ice for 30 minutes. Cells were placed in 42$^\circ$C water bath and after 45 seconds placed back on ice. Next, 250-500 $\mu$L of growth medium (Luria-Bertani (LB) broth) was added and cells placed in the 37 $^\circ$C shaking incubator for 45 minutes to 1 hour. The entire volume of cells was spread onto 10 cm LB-agar plate containing an appropriate antibiotic for selection. Antibiotics used for each vector selection are listed in Table \@ref(tab:antibiotics-used). Cells were spread on plates were left in the 37$^{\circ}$C incubator overnight to allow for growth of transformed cells into colonies.
Briefly, 25 to 50 $\mu$L of chemically competent Mach1 or DH5$\alpha$ *Escherichia coli (E.coli)* cells were combined and gently mixed with ~10 pg of plasmid DNA. The mix was left on ice for 30 minutes. Cells were placed in 42$^\circ$C water bath and after 45 seconds placed back on ice. Next, 250-500 $\mu$L of growth medium (Luria-Bertani (LB) broth) was added and cells placed in the 37$^\circ$C shaking incubator for 45 minutes to 1 hour. The entire volume of cells was spread onto 10 cm LB-agar plate containing an appropriate antibiotic for selection. Antibiotics used for each vector selection are listed in Table \@ref(tab:antibiotics-used). Cells were spread on plates were left in the 37$^{\circ}$C incubator overnight to allow for growth of transformed cells into colonies.
```{r antibiotics-used, echo=FALSE}
library(kableExtra)
......@@ -36,10 +36,10 @@ knitr::kable(text_tbl, format = "latex", caption = 'Selection pressure for DNA p
\newpage
### Isolation of DNA plasmid from *E. coli* ### {#miniprep}
Transformed *E.coli* colony was picked, placed in 5 mL of LB supplemented with appropriate antibiotic and incubated overnight at 37 $^{\circ}$C whilst shaking. DNA was extracted using the MiniPrep Kit (Thermo Scientific or Qiagen) following the manufacturers instructions. Centrifugation was carried out in table top centrifuge at 12 000 g. Isolated DNA was quantified in a Nanodrop UV-Vis spectophotometer.
Transformed *E.coli* colony was picked, placed in 5 mL of LB supplemented with appropriate antibiotic and incubated overnight at 37$^{\circ}$C whilst shaking. DNA was extracted using the MiniPrep Kit (Thermo Scientific or Qiagen) following the manufacturers instructions. Centrifugation was carried out in table top centrifuge at 12 000 g. Isolated DNA was quantified in a Nanodrop UV-Vis spectophotometer.
### Analytic digestion of DNA plasmids
DNA plasmids were digested with restriction enzyme(s) (Promega). The reaction mix (Table \@ref(tab:RE-reaction) was incubated at 37 $^{\circ}$C for 2-8 hours and the DNA fragments were resolved on the agarose gel (Section \@ref(electrophoresis)).
DNA plasmids were digested with restriction enzyme(s) (Promega). The reaction mix (Table \@ref(tab:RE-reaction) was incubated at 37$^{\circ}$C for 2-8 hours and the DNA fragments were resolved on the agarose gel (Section \@ref(electrophoresis)).
```{r RE-reaction, echo=FALSE}
......@@ -49,7 +49,7 @@ RE_reaction <- data.frame(
names(RE_reaction) <- NULL
knitr::kable(RE_reaction, "latex", escape = FALSE, booktabs = TRUE, caption = "Components assembled to cary out restriction enzyme reaction.") %>%
knitr::kable(RE_reaction, "latex", escape = FALSE, booktabs = TRUE, caption = "Components assembled to carry out restriction enzyme reaction.") %>%
kable_styling(latex_options = "hold_position")
# %>% kable_styling(position = "center") # use the option escape=FALSE to be able to pass greek letters to the table, booktabs = TRUE means there will only be a top and bottom, and not all borders, caption = NA (no caption, but the table will be in the middle, otherwise it is ligned to the left of the page). Note that the styling function does not work here because it is from the extra package
......@@ -60,7 +60,7 @@ knitr::kable(RE_reaction, "latex", escape = FALSE, booktabs = TRUE, caption = "C
### Amplification of DNA fragments by Polymerase Chain Reaction (PCR)
#### Primer design
To enable the amplification of the DNA of interest, appropriate PCR primers were designed applying the following criteria: primers were unique to the annealing site on the designated DNA, 18 to 25 nucleotides long, guanine-cytosine content from 40 to 60 %, melting temperature from 55 to 75 $^{\circ}$C. Where possible, primers rich in guanine and cytosine at 3’ end were selected to facilitate high specificity of primer binding to the target. The primers were ordered from Eurofins Genomics, subsequently diluted in ddH~2~O to the concentration of 100 pmol/$\mu$l and stored at -20 $^{\circ}$C. Sequences of primers used in this study can be found in Table \@ref(tab:primer-seq1).
To enable the amplification of the DNA of interest, appropriate PCR primers were designed applying the following criteria: primers were unique to the annealing site on the designated DNA, 18 to 25 nucleotides long, guanine-cytosine content from 40 to 60 %, melting temperature from 55 to 75$^{\circ}$C. Where possible, primers rich in guanine and cytosine at 3’ end were selected to facilitate high specificity of primer binding to the target. The primers were ordered from Eurofins Genomics, subsequently diluted in ddH~2~O to the concentration of 100 pmol/$\mu$l and stored at -20$^{\circ}$C. Sequences of primers used in this study can be found in Table \@ref(tab:primer-seq1).
```{r primer-seq1, echo=FALSE}
library(kableExtra)
......@@ -281,7 +281,7 @@ kable("latex", booktabs = T, escape = F,
\newpage
### DNA electrophoresis ### {#electrophoresis}
To resolve the size of DNA samples, agarose gel electrophoresis was run using BioRad Wide horizontal electrophoresis system and PowerPac Basic Power Supply. The resolving gel was prepared by addition of agarose (0.6-1.2 % (w/v); Sigma Aldrich) to 1x TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) buffer. This mix was heated in the microwave until agarose completely melted and left on the bench to cool down to ~ 50 $^\circ$C. Subsequently, Nancy-520 DNA Gel Stain (Sigma-Aldrich) at 5 mg/mL was added in 1:1000 (v/v) dilution and the mixture was poured into the gel caster. Meanwhile, the DNA samples were prepared by mixing them with 1 % (v/v) loading dye (Blue/Orange Loading Dye, Promega). Once the gel set, the samples were loaded into wells alongside the indicated molecular weight marker. Markers variously used in this thesis include 1kb Hyperladder (Bioline), 1kb ladder (Promega) or 1 kb Plus DNA ladder (Thermo Scientific). Electrophoresis was run at 70 V until the samples were sufficiently resolved (typically 30 minutes to 2 hours) and gels imaged using Syngenta GBox.
To resolve the size of DNA samples, agarose gel electrophoresis was run using BioRad Wide horizontal electrophoresis system and PowerPac Basic Power Supply. The resolving gel was prepared by addition of agarose (0.6-1.2 % (w/v); Sigma Aldrich) to 1x TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) buffer. This mix was heated in the microwave until agarose completely melted and left on the bench to cool down to ~ 50$^\circ$C. Subsequently, Nancy-520 DNA Gel Stain (Sigma-Aldrich) at 5 mg/mL was added in 1:1000 (v/v) dilution and the mixture was poured into the gel caster. Meanwhile, the DNA samples were prepared by mixing them with 1 % (v/v) loading dye (Blue/Orange Loading Dye, Promega). Once the gel set, the samples were loaded into wells alongside the indicated molecular weight marker. Markers variously used in this thesis include 1kb Hyperladder (Bioline), 1kb ladder (Promega) or 1 kb Plus DNA ladder (Thermo Scientific). Electrophoresis was run at 70 V until the samples were sufficiently resolved (typically 30 minutes to 2 hours) and gels imaged using Syngenta GBox.
### DNA purification following PCR and electrophoresis
Following gel electrophoresis, the band of interest was visualised under the UV light and isolated by cutting with a surgical blade.
......@@ -356,7 +356,7 @@ knitr::kable(topo_reaction_tb, escape = FALSE, align = 'l', booktabs = TRUE, cap
```
#### Generation of the expression vector by LR reaction ####{#lr-reaction-section}
LR reaction was assembled using Gateway LR Clonase II Enzyme Mix (Invitrogen) (Table \@ref(tab:LR-reaction). Reaction was incubated at room temperature for 2 hours. To inactivate the enzyme, 2 $\mu$L of proteinase K was added and the reaction mix was incubated at 37 $^\circ$C for 10 minutes. 1 $\mu$L of reaction mix was used to transform 50 $\mu$L of One Shot OmniMAX 2 T1 phage resistant cells (Invitrogen). Transformed cells were plated and grew overnight. Following, plasmid was isolated from transformed cells and subjected to sequencing to ensure successful formation of the plasmid.
LR reaction was assembled using Gateway LR Clonase II Enzyme Mix (Invitrogen) (Table \@ref(tab:LR-reaction). Reaction was incubated at room temperature for 2 hours. To inactivate the enzyme, 2 $\mu$L of proteinase K was added and the reaction mix was incubated at 37$^\circ$C for 10 minutes. 1 $\mu$L of reaction mix was used to transform 50 $\mu$L of One Shot OmniMAX 2 T1 phage resistant cells (Invitrogen). Transformed cells were plated and grew overnight. Following, plasmid was isolated from transformed cells and subjected to sequencing to ensure successful formation of the plasmid.
```{r LR-reaction, echo=FALSE}
gateway_cloning2 <- data.frame(
......@@ -367,7 +367,7 @@ names(gateway_cloning2) <- NULL
options(knitr.table.format= "latex")
knitr::kable(gateway_cloning2, escape = FALSE, align = 'l', booktabs = TRUE, caption = "Components assembled for the generation of recombinant vector by gateway cloning.") %>%
knitr::kable(gateway_cloning2, escape = FALSE, align = 'l', booktabs = TRUE, caption = "Components assembled for the generation of recombinant vector by Gateway cloning.") %>%
kable_styling(latex_options = "hold_position")
# %>% kable_styling(position = "center")
# use the option escape=FALSE to be able to pass greek letters to the table, booktabs = TRUE means there will only be a top and bottom, and not all borders, caption = NA (no caption, but the table will be in the middle, otherwise it is ligned to the left of the page)
......@@ -384,10 +384,10 @@ knitr::include_graphics("fig/methods/purification-process.png")
```
### Growth of transformed *E. coli* cells
Chemically competent bacterial cells (BL21(DE3)) engineered for high efficiency protein expression were transformed with the authenticated expression vector as described (Section \@ref(miniprep)). Transformed colonies were resuspended and placed in 5 mL of growth medium supplemented with the appropriate antibiotic. Seed culture was placed in the shaking incubator at 37 $^\circ$C and left to grow until OD~600nm~ of 1-2. This starter culture was used to inoculate growth medium supplemented with appropriate antibiotic in 2 L baffled flasks, to the final OD~600nm~ of 0.01-0.05. Inoculated flasks were placed in a shaking incubator at 37 $^\circ$C, 250 RPM. The following protocol was followed, unless otherwise stated: At OD~600nm~ = 0.5, the temperature was lowered to 18 $^\circ$C. When the 18 $^\circ$C culture reached an OD~600nm~ ≈ 1, 0.2 mM isopropyl $\beta$-D-1-thiogalactopyranoside (IPTG) was added and the growth continued overnight at 18 $^\circ$C.
Chemically competent bacterial cells (BL21(DE3)) engineered for high efficiency protein expression were transformed with the authenticated expression vector as described (Section \@ref(miniprep)). Transformed colonies were resuspended and placed in 5 mL of growth medium supplemented with the appropriate antibiotic. Seed culture was placed in the shaking incubator at 37$^\circ$C and left to grow until OD~600nm~ of 1-2. This starter culture was used to inoculate growth medium supplemented with appropriate antibiotic in 2 L baffled flasks, to the final OD~600nm~ of 0.01-0.05. Inoculated flasks were placed in a shaking incubator at 37$^\circ$C, 250 RPM. The following protocol was followed, unless otherwise stated: At OD~600nm~ = 0.5, the temperature was lowered to 18$^\circ$C. When the 18$^\circ$C culture reached an OD~600nm~ ≈ 1, 0.2 mM isopropyl $\beta$-D-1-thiogalactopyranoside (IPTG) was added and the growth continued overnight at 18$^\circ$C.
### Protein purification ### {#purification-general-methods}
*E.coli* were harvested by centrifuging the cell culture at 5000 g for 20 minutes at 4 $^\circ$C and either used immediately or stored at -20 $^\circ$C for further use.
*E.coli* were harvested by centrifuging the cell culture at 5000 g for 20 minutes at 4$^\circ$C and either used immediately or stored at -20$^\circ$C for further use.
Harvested cells were kept on ice throughout the purification procedure. Two methods of purification were tested: HIS-tag purification using Ni-NTA resin and maltose binding protein (MBP) purification with Sepharose-Dextrin Beads (GE Healthcare Life Sciences).
### HIS-tag purification #### {#his}
......@@ -395,7 +395,7 @@ The composition of buffers used is as follows :
Re-suspension buffer: 0.1M TRIS (pH=8), 0.15 M NaCl. Wash buffer 1: as previous. Wash buffer 2: 0.1 M TRIS (pH=8), 1 M NaCl. Wash buffer 3: 0.1 M TRIS, (pH=8), 0.15 M NaCl. Elution buffer: 0.1 M TRIS, (pH=8), 0.15 M NaCl, 0.2 M imidazole (pH=7.5)
Cells harvested from 1 L of culture medium were re-suspended in 40 mL re-suspension buffer supplemented with 2 Pierce™ Protease Inhibitor Mini Tablets (Thermo Fisher Scientific) and sonicated on ice using the following settings: power 7, pulse on: 10 seconds, pulse off: 20 seconds, total time 6 minutes. Sonicated cells were subject to 16000 g spin for 45 minutes at 4 $^\circ$C to sediment cellular debris. The supernatant was collected and spun again at 100 000 g for 1 hour at 4 $^\circ$C to separate non-soluble fraction (e.g. aggregated proteins) in the pellet from the supernatant containing soluble fraction. Supernatant was mixed with 0.5 mL of Ni-NTA resin (previously equilibrated in the resuspension buffer) and equilibrated for 1 hour or overnight at 4 $^\circ$C on the rotating tube rotator (speed 8-9). Following this incubation, the mix was decanted into a low pressure 5 mL chromatography column. Resin was washed with 10 mL of each one of the 3 washing buffers. Lastly, bound to Ni-NTA resin proteins were eluted off by addition of 5 x 0.5 mL of elution buffer. Eluted fractions were stored at 4 $^\circ$C. At each stage, a samples consisting of the pre induction (pre-I; post induction (post-I) Homogenate (H) (Whole cells), high speed supernatant (LOAD), Flow through (FT0) wash (W) and eluate (E) fractions were collected for SDS-PAGE analysis (Section \@ref(samples)).
Cells harvested from 1 L of culture medium were re-suspended in 40 mL re-suspension buffer supplemented with 2 Pierce™ Protease Inhibitor Mini Tablets (Thermo Fisher Scientific) and sonicated on ice using the following settings: power 7, pulse on: 10 seconds, pulse off: 20 seconds, total time 6 minutes. Sonicated cells were subject to 16000 g spin for 45 minutes at 4$^\circ$C to sediment cellular debris. The supernatant was collected and spun again at 100 000 g for 1 hour at 4$^\circ$C to separate non-soluble fraction (e.g. aggregated proteins) in the pellet from the supernatant containing soluble fraction. Supernatant was mixed with 0.5 mL of Ni-NTA resin (previously equilibrated in the resuspension buffer) and equilibrated for 1 hour or overnight at 4$^\circ$C on the rotating tube rotator (speed 8-9). Following this incubation, the mix was decanted into a low pressure 5 mL chromatography column. Resin was washed with 10 mL of each one of the 3 washing buffers. Lastly, bound to Ni-NTA resin proteins were eluted off by addition of 5 x 0.5 mL of elution buffer. Eluted fractions were stored at 4$^\circ$C. At each stage, a samples consisting of the pre induction (pre-I; post induction (post-I) Homogenate (H) (Whole cells), high speed supernatant (LOAD), Flow through (FT0) wash (W) and eluate (E) fractions were collected for SDS-PAGE analysis (Section \@ref(samples)).
<!-- ### MBP purification -->
<!-- The composition of buffers used is as follows : -->
......@@ -414,7 +414,7 @@ Protein content of the eluted samples was measured with NanoDrop 1000 Spectropho
### Analysis of protein molecular weight using denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
#### Sample preparation #### {#samples}
This section described how each sample was prepared. Pre and post induction whole cell samples: 1 mL of *E. coli* cell culture was taken, spun down in tabletop centrifuge at max speed for 5 minutes. Supernatant was discarded, pellet re-suspended in 150 $\mu$L dH~2~O. Total protein content of 2 $\mu$L samples were measured with NanoDrop by measuring protein absorbance at 280 nm. Seventy mg/ml of protein was loaded onto a gel in each sample. Cell lysate: following sonication, 30 $\mu$L of cell lysate was taken. Cell-debris and supernatant: 30 $\mu$L of cell lysate sample taken, spun down in tabletop centrifuge for 10 mins at maximum speed, at 4 $^\circ$C. supernatant was pipetted into another micro centrifuge (labelled supernatant) tube whereas pellet re-suspended in 30 $\mu$L dH~2~O (labelled whole-cell). Supernatant and Pellet samples: 50 $\mu$L of cell lysate spun down in tabletop centrifuge for 10 mins at maximum speed, at 4 $^\circ$C. Thirty $\mu$L of the supernatant taken, spun down in ultracentrifuge at 100 000 g, at 4 $^\circ$C for 1 hour. Supernatant was pipetted into another microcentrifuge tube (ultra-supernatant) whereas debris was re-suspended in 30 $\mu$L of dH~2~0. The same volumes of cell-lysate, cell-debris, supernatant, ultra-supernatant, ultra-pellet and flow-through were loaded onto SDS-PAGE gels. Protein samples were mixed with sample buffer (2 % SDS, 2 mM DTT, 4 % glycerol, 0.04 M Tris pH = 6.8, 0.01 % bromophenol blue) and boiled for 5 – 10 minutes. Next, 4 – 10 $\mu$L samples were loaded onto 8 – 12 % acrylamide SDS-PAGE gel alongside 4 $\mu$L of Protein Marker PageRulerTM Prestained/Unstained Protein (Thermo Fisher Scientific).
This section described how each sample was prepared. Pre and post induction whole cell samples: 1 mL of *E. coli* cell culture was taken, spun down in tabletop centrifuge at max speed for 5 minutes. Supernatant was discarded, pellet re-suspended in 150 $\mu$L dH~2~O. Total protein content of 2 $\mu$L samples were measured with NanoDrop by measuring protein absorbance at 280 nm. Seventy mg/ml of protein was loaded onto a gel in each sample. Cell lysate: following sonication, 30 $\mu$L of cell lysate was taken. Cell-debris and supernatant: 30 $\mu$L of cell lysate sample taken, spun down in tabletop centrifuge for 10 mins at maximum speed, at 4$^\circ$C. supernatant was pipetted into another micro centrifuge (labelled supernatant) tube whereas pellet re-suspended in 30 $\mu$L dH~2~O (labelled whole-cell). Supernatant and Pellet samples: 50 $\mu$L of cell lysate spun down in tabletop centrifuge for 10 mins at maximum speed, at 4$^\circ$C. Thirty $\mu$L of the supernatant taken, spun down in ultracentrifuge at 100 000 g, at 4$^\circ$C for 1 hour. Supernatant was pipetted into another microcentrifuge tube (ultra-supernatant) whereas debris was re-suspended in 30 $\mu$L of dH~2~0. The same volumes of cell-lysate, cell-debris, supernatant, ultra-supernatant, ultra-pellet and flow-through were loaded onto SDS-PAGE gels. Protein samples were mixed with sample buffer (2 % SDS, 2 mM DTT, 4 % glycerol, 0.04 M Tris pH = 6.8, 0.01 % bromophenol blue) and boiled for 5 – 10 minutes. Next, 4 – 10 $\mu$L samples were loaded onto 8 – 12 % acrylamide SDS-PAGE gel alongside 4 $\mu$L of Protein Marker PageRulerTM Prestained/Unstained Protein (Thermo Fisher Scientific).
#### Gel electrophoresis
......@@ -440,7 +440,7 @@ BioRad electrophoresis apparatus was used. Electrophoresis chamber was filled wi
### Western blots ###{#western}
#### Protein transfer
Resolved protein are transferred to polyvinylidene difluoride (PVDF). Membrane cut to the size of the resolving gel was equilibrated for 15 minutes to 1 hour in the transfer buffer (12.1 g Tris, 57.6 g glycine, 800 mL methanol in total volume of 4 L) then washed with dH~2~O followed by methanol. Freshly run polyacrylamide gel was placed on top of the submerged in transfer buffer sponge, 2 x filter paper and PVDF membrane stack and covered with 2 x filter paper. Assembled transfer mount with the gel and PVDF membrane was placed in the BioRad Mini Trans-Blot Module which was in turn inserted into Mini-PROTEAN Tetra Cell tank. The tank was filled with transfer buffer and the protein transferred from the gel onto the membrane at 100 V constant voltage for for 1 - 3 hours at 4 $^\circ$C.
Resolved protein are transferred to polyvinylidene difluoride (PVDF). Membrane cut to the size of the resolving gel was equilibrated for 15 minutes to 1 hour in the transfer buffer (12.1 g Tris, 57.6 g glycine, 800 mL methanol in total volume of 4 L) then washed with dH~2~O followed by methanol. Freshly run polyacrylamide gel was placed on top of the submerged in transfer buffer sponge, 2 x filter paper and PVDF membrane stack and covered with 2 x filter paper. Assembled transfer mount with the gel and PVDF membrane was placed in the BioRad Mini Trans-Blot Module which was in turn inserted into Mini-PROTEAN Tetra Cell tank. The tank was filled with transfer buffer and the protein transferred from the gel onto the membrane at 100 V constant voltage for for 1 - 3 hours at 4$^\circ$C.
#### Antibody binding ####{#abs}
Transfer of his-tagged human $\alpha7$ nAChR ECD-chimera protein were detected using 1 mg/mL monoclonal mouse anti-Hexa-His primary antibodies (Thermo Fisher Scientific) and 1mg/mL IRDye® 680RD Goat anti-Mouse IgG (Li-Cor) used at 1 in 1000 dilution. PVDF membrane was incubated for at least 1 hour in blocking, primary and secondary antibody buffer (Table \@ref(tab:WB-buffers)). To remove residual solution, three 10-minute-long washes were carried out in-between and after last incubations.
......@@ -467,7 +467,7 @@ Immunodecorated PVDF were imaged using Odyssey imaging system (Li-Cor Bioscience
Protein purified and eluted with the Ni-NTA resin were subject to size exclusion chromatography with GE Healthcare Superdex^TM^ 200 10/300GL column with the separation range between 10 and 600 kDa. This methods allows for the molecular weight assessment and separation of proteins present in the sample based on their mobility through the resin-filled column.
#### Sample preparation
Sample prepared with VIVASPIN20 column with the cut off point of 30 kDa (Sartorius) by spinning down in a centrifuge at 36 000 RPM at 4 $^\circ$C.
Sample prepared with VIVASPIN20 column with the cut off point of 30 kDa (Sartorius) by spinning down in a centrifuge at 36 000 RPM at 4$^\circ$C.
#### Buffers
Buffer used : 0.1M TRIS (pH=8), 0.15 M NaCl degassed and ddH~2~0 degassed.
......@@ -516,29 +516,32 @@ FX863 (genotype *acr-7, (alleletm863)II.* ; outcrossed x0
Transgenic strains:
eat-2 (ad465) II Ex; [pDESTgcy32 (Pmyo-3::GFP)]
*eat-2 (ad465) II* Ex; [pDESTgcy32 (Pmyo-3::GFP)]
eat-2 (ad465) II Ex; [[pDESTgcy32 (Pmyo-2::CHRNA7)]; [pDESTgcy32 (Pmyo-3::GFP)]]
*eat-2 (ad465) II* Ex; [[pDESTgcy32 (Pmyo-2::CHRNA7)]; [*pDESTgcy32 (Pmyo-3::GFP)*]]
eat-2 (ad465) II Ex; [[pDESTgcy32 (Pmyo-2::EAT-2); [pDESTgcy32 (Pmyo-3::GFP)]]
*eat-2 (ad465) II* Ex; [[*pDESTgcy32 (Pmyo-2::EAT-2*); [*pDESTgcy32 (Pmyo-3::GFP)*]]
### *C. elegans* culture
*C. elegans* strains were cultured at 20 $^\circ$C on the nematode growth medium (NGM) [@brenner1974] and fed with OP50 strain of *E. coli*. Worms were picked with a platinum wire.
*C. elegans* strains were cultured at 20$^\circ$C on the nematode growth medium (NGM) [@brenner1974] and fed with OP50 strain of *E. coli*. Worms were picked with a platinum wire.
### Preparation of *C. elegans* plates ### {#plates}
NGM was prepared weekly in 4 or 8 L batches as described: 2 % agar (w/v), 0.25 % peptone (w/v), 50 mM NacL (w/v) in dH~2~0. The components were autoclaved and cooled to 55 $^\circ$C, then 1 mM MgSO~4~, 1 mM CaCl~2~, 1 mM K~2~HPO~4~ and 0.1 % cholesterol were added. 10 mL NGM portions were poured into 5.5 cm Petri dishes with a peristaltic pump. Once solidified, NGM were seeded with 50 $\mu$L of OP50. OP50 was applied in the middle of the plate, creating a round food patch for *C. elegans* to feed on. Prepared plates were left overnight to allow bacteria growth.
NGM was prepared weekly in 4 or 8 L batches as described: 2 % agar (w/v), 0.25 % peptone (w/v), 50 mM NacL (w/v) in dH~2~0. The components were autoclaved and cooled to 55$^\circ$C, then 1 mM MgSO~4~, 1 mM CaCl~2~, 1 mM K~2~HPO~4~ and 0.1 % cholesterol were added. 10 mL NGM portions were poured into 5.5 cm Petri dishes with a peristaltic pump. Once solidified, NGM were seeded with 50 $\mu$L of OP50. OP50 was applied in the middle of the plate, creating a round food patch for *C. elegans* to feed on. Prepared plates were left overnight to allow bacteria growth.
### Maintanance and preparation of *E. coli* OP50
Fresh stock of OP50 plates were prepared at monthly intervals. A single colony was picked from the OP50 stock plate and placed in 10 mL of LB. Following overnight growth, cells were streaked on a plate and allowed to form colonies by incubation at 37 $^\circ$C.
Fresh stock of OP50 plates were prepared at monthly intervals. A single colony was picked from the OP50 stock plate and placed in 10 mL of LB. Following overnight growth, cells were streaked on a plate and allowed to form colonies by incubation at 37$^\circ$C.
### *E. coli* OP50 culture
To prepare OP50 culture, a single colony was picked from the OP50 stock plate and placed in 10 mL of LB. Bacterial culture was grown in a shaking incubator at 37 $^\circ$C until OD~600nm~ reached 0.6 to reach the exponentially growing phase. Cultures were stored at 4 $^\circ$C for up to 2 week and used to seed NGM plates.
To prepare OP50 culture, a single colony was picked from the OP50 stock plate and placed in 10 mL of LB. Bacterial culture was grown in a shaking incubator at 37$^\circ$C until OD~600nm~ reached 0.6 to reach the exponentially growing phase. Cultures were stored at 4$^\circ$C for up to 2 week and used to seed NGM plates.
### General *C. elegans* methods
All experiments, with the exception of the development assay, were performed on young hermaphrodite adults (L4 + 1 day). Drugs and reagents were purchased from Sigma Aldrich, unless otherwise stated. Behavioral observations were made using a binocular microscope, unless otherwise stated. Results are expressed as mean $\pm$ SEM of ‘N’ determinations. Graph generation and measurement of EC~50~ or IC~50~ were performed in GraphPad (version 6.07). Concentration response curves were fitted into nonlinear regression sigmoidal dose-response (three parameter logistic) equation [@hill1910].
All experiments, with the exception of the development assay, were performed on young hermaphrodite adults (L4 + 1 day). Drugs and reagents were purchased from Sigma Aldrich, unless otherwise stated. Behavioral observations were made using a binocular microscope, unless otherwise stated. Results are expressed as mean $\pm$ SEM of ‘N’ determinations.
### Dose-response curves ###{#doseresponsecurves}
Dose-response curves and the measurement of EC~50~ or IC~50~ were performed in GraphPad (version 6.07). The curves were fitted into nonlinear regression sigmoidal dose-response (three parameter logistic) equation [@hill1910]. This model was chosen, because it allows for determination of the predicted EC/IC~50~ value even if the entire dose-response curve is not available, as it was the case in many behavioural assays involving neonicotinoids. A potential disadvantage of chosing this model is that it assumes Hill coefficient of 1, in other words, lack of cooperativity among binding sites in nAChRs, which is the predicted target of compounds used in this study. nAChRs have multiple binding sites: from two in the muscle-type receptor [@blount1989] to 5 in homomeric complexes [@@celie2004; @li2011]. Their opening is generally governed by positive cooperativity binding of ligands [@katz1957; @edelstein1996], however some nAChRs do not seem to display this property. For example, a Hill coefficient of 1 was determined for acetylcholine binding to levamisome-type receptor [@boulin2008] as well as many agonists binding to AChBP [@Kaczanowska2014] suggesting, a single agonist is sufficient to open the channel, despite the presence of three and five binding sites, respectively. Although the properites of L- and N- type *C. elegans* receptors have been described [@boulin2008; @touroutine2005], kinetics of most *C. elegans* nAChRs is unknown. Resultantly, it is uncertain whether the three parameter logistic model is appropriate for dose-response curves of the effects of nAChRs agonists on behaviour of *C. elegans*. Whilst IC50/EC50 values derived using this model might not be accurate, they provide an estimation of the potency of agnoists on *C. elegans* nAChRs.
### Drug stocks
5-HT was used in form of serotonin creatinine sulfate monohydrate, ampicillin in form of sodium salt, whereas nicotine was in the form of hydrogen tartrate salt. Stock concentration of FITC-alpha-bungarotoxin (FITC-$\alpha$-Bgtx) at 500 $\mu$g/ml was made in ddH~2~O. Thiacloprid and clothianidin were dissolved in 100 % dimethyl sulfoxide (DMSO). Nitenpyram and nicotine stocks were prepared by dissolving drugs in dH~2~0 and diluted to the indicated final concentrations. Working concentration of 100 $\mu$g/mL FITC-$\alpha$Bgtx was prepared and stored at 4 $^\circ$C for up to 2 weeks. The solution was span down briefly before use to pellet aggregates. Drugs were stored at -18 $^\circ$C for long term storage (>1 month). Once defrosted, they were used within 2 weeks or discarded. Nitenpyram stock was made immediately prior to the experiment and protected from light using foil to prevent photo-degradation. Buffers used for the behavioral assays in liquid were supplemented with 0.1 % (w/v) Bovine Serum Albumin (BSA), which prevents worms from sticking to the bottom of the experimental plate. Therefore, M9 and Dent’s solution refer to buffers supplemented with BSA, unless otherwise stated.
5-HT was used in form of serotonin creatinine sulfate monohydrate, ampicillin in form of sodium salt, whereas nicotine was in the form of hydrogen tartrate salt. Stock concentration of FITC-alpha-bungarotoxin (FITC-$\alpha$-bgtx) at 500 $\mu$g/ml was made in ddH~2~O. Thiacloprid and clothianidin were dissolved in 100 % dimethyl sulfoxide (DMSO). Nitenpyram and nicotine stocks were prepared by dissolving drugs in dH~2~0 and diluted to the indicated final concentrations. Working concentration of 100 $\mu$g/mL FITC-$\alpha$-bgtx was prepared and stored at 4$^\circ$C for up to 2 weeks. The solution was span down briefly before use to pellet aggregates. Drugs were stored at -18$^\circ$C for long term storage (>1 month). Once defrosted, they were used within 2 weeks or discarded. Nitenpyram stock was made immediately prior to the experiment and protected from light using foil to prevent photo-degradation. Buffers used for the behavioral assays in liquid were supplemented with 0.1 % (w/v) Bovine Serum Albumin (BSA), which prevents worms from sticking to the bottom of the experimental plate. Therefore, M9 and Dent’s solution refer to buffers supplemented with BSA, unless otherwise stated.
### Effects of drugs on intact *C. elegans* locomotion and feeding behavior upon acute exposure. {#liquidassay}
All assays were performed in M9 medium. M9 buffer composition is (g/litre): 6 g Na~2~HPO~4~, 3 g KH~2~PO~4~, 5 g NaCl, 0.25 g MgSO~4~.H~2~O. Worms were exposed to varying indicated concentrations of nicotine or neonicotinoids for a maximum period of 2 hours. The effects of these compounds on locomotion and feeding was scored.
......@@ -575,7 +578,7 @@ Size of worms was determined in ImageJ. The scale was set using a graticule and
On-plate assays were carried out to determine the effects of prolonged drug exposure on *C. elegans* behaviour. Worms were placed on NGM plates containing the indicated drug / drug vehicle and a food source in form of *E. coli* OP50 patch. All drugs were added to the NGM at 1 in 200 dilution.
### Plate preparation
NGM was prepared as described in Section \@ref(plates). Fifty $\mu$L of drug solution at appropriate concentration was added to 10 mL of molten NGM at approx 50 $^\circ$C and mixed by gentle inversion. 3 mL of such mix was placed in each of the three successive wells of a 6-well plate (Figure \@ref(fig:on-plate-assay-method)). The medium was left overnight to solidify. One well was then seeded with 50 $\mu$L of OP50 culture, whereas the other two wells remained unseeded. This provided an experimental arena with the food on, the cleaning well and the experimental arena containing no food. In parallel, control plates containing drug solvent (water or 0.5 % DMSO) were prepared.
NGM was prepared as described in Section \@ref(plates). Fifty $\mu$L of drug solution at appropriate concentration was added to 10 mL of molten NGM at approx 50$^\circ$C and mixed by gentle inversion. 3 mL of such mix was placed in each of the three successive wells of a 6-well plate (Figure \@ref(fig:on-plate-assay-method)). The medium was left overnight to solidify. One well was then seeded with 50 $\mu$L of OP50 culture, whereas the other two wells remained unseeded. This provided an experimental arena with the food on, the cleaning well and the experimental arena containing no food. In parallel, control plates containing drug solvent (water or 0.5 % DMSO) were prepared.
Due to heat instability, nitenpyram plates were prepared by pipetting 50 $\mu$L of drug solution onto solidified 3 mL NGM. The plates were left overnight to enable diffusion of the compounds into the solid agar. The appropriate well was then seeded. Nitenpyram-containing plates were covered with aluminium foil at all times, to prevent photodegradation.
(ref:on-plate-assay-fig) **Diagram of the 24-hour “on-plate” assay arena.** Drug or drug solvent was incorporated into the NGM and poured into rows of a 6-well plate. Wells in the first column were seeded with the OP50. Two to four L4 + 1 worms were placed on the experimental arena containing food source. After 24 hours, pumping rate on food and the number of eggs laid per worm were counted. Following, worms were transferred to the cleaning well and left for 5-10 minutes to remove the residual food. Worms were then transferred to the experimental arena containing no food source. After period of acclimatisation (5-10 minutes), their locomotion on food was measured by counting body bends.
......@@ -627,7 +630,7 @@ knitr::include_graphics("fig/methods/WHOLE_AND_CUT_HEAD_2.png")
(ref:cut-head-ctr) **Pharyngeal pumping of dissected *C. elegans* in liquid.** Cut heads were placed in Dent's saline and the pharyngeal pumping was counted over time. Measurements were made by visual observations, counted for 30 seconds and expressed in Hz. Data are $\pm$ SEM collected over $\ge$ 2 observations; number of replicates $\ge$ 4. For comparison, the average pharyngeal pumping in the presence of 1 $\mu$M 5-HT is shown is dashed purple line.
```{r cut-head-ctr-label, fig.cap="(ref:cut-head-ctr)", fig.scap= "Pharyngeal pumping of dissected \\textit{C. elegans*} in liquid.", fig.align='center', echo=FALSE, message=FALSE, fig.pos = 'H', warning = FALSE}
```{r cut-head-ctr-label, fig.cap="(ref:cut-head-ctr)", fig.scap= "Pharyngeal pumping of dissected \\textit{C. elegans} in liquid.", fig.align='center', echo=FALSE, message=FALSE, fig.pos = 'H', warning = FALSE}
#read in cut head data
cut_head <- readRDS("Analysis/Data/Transformed/cut_head/summary_data")
ctr_cut_head_plot <- cut_head %>%
......@@ -664,7 +667,7 @@ Cut heads were exposed to 1 $\mu$M 5-HT for 10 minutes to stimulate pumping. Fol
Cut heads were prepared (Section \@ref(cuthead)) and transferred to the experimental arena by pipetting. Extracellular recordings were made with an electropharyngeogram (EPG) technique (Figure \@ref(fig:EPG-setup-method)).
#### Preparation of a microelectrode
Non-filamented borosillicate capillary tube (Havard apparatus) with outer diameter (OD) of 1.5 mm and internal diameter (ID) of 0.1 mm was pulled with a Narishige puller (model PC:10). The puller was set at 98.2 $^\circ$C for step 1 and 72.8 $^\circ$C for step 2 to make a tip of ~ 10 $\mu$m. The needle was back-filled with Dent’s using a micropipette filler (250 $\mu$m ID, 350 $\mu$m OD, World Precision Instruments).
Non-filamented borosillicate capillary tube (Havard apparatus) with outer diameter (OD) of 1.5 mm and internal diameter (ID) of 0.1 mm was pulled with a Narishige puller (model PC:10). The puller was set at 98.2$^\circ$C for step 1 and 72.8$^\circ$C for step 2 to make a tip of ~ 10 $\mu$m. The needle was back-filled with Dent’s using a micropipette filler (250 $\mu$m ID, 350 $\mu$m OD, World Precision Instruments).
#### Experimental set-up
The microelectrode was inserted into a microelectrode holder containing a silver wire. The microelectrode was inserted into a headstage (HS-2A Asoclamp) and carefully lowered using a micromanipulator (Burleigh) into a recording chamber filled with Dent’s saline and resting on a stage of Axoscope 2 (Zeiss) microscope. The reference electrode was made with a glass capillary filled with 2 % agar in 3 M KCl. The reference electrode was placed in the recording chamber and connected to the amplifier headstage via a dish filled with 3 M KCl solution and a silver wire electrode. The cut head was placed in a recording chamber and a tight seal between the tip of the nose and the microelectrode was made by applying suction. The extracellular electrical signals from the pharynx were amplified by an Axoclamp-2B Microelectrode Amplifier, digitized by Digidata 1322A and recorded with Axoscope 9.2.
......@@ -684,7 +687,7 @@ A single EPG reflects a contruction-relaxation of a pharyngeal muscle. It consis
### Microinjection to generate *C. elegans* transgenic lines ###{#microinjection}
#### Preparation of a needle
Alluminosilicate capillaries SM100F-10 (1 mm external diameter, 0.5 mm internal diameter) needle was pulled with Narishige puller (model PC:10) using the following settings: step 1 at 99 $^\circ$C, step 2 at 79 $^\circ$C. The pulled needle was filled with 1 $\mu$L of injection mix and assembled into Transferman NK2 (Eppendorf) micromanipulator. Microinjection was performed with FemtoJet Microinjector (Eppendorf).
Alluminosilicate capillaries SM100F-10 (1 mm external diameter, 0.5 mm internal diameter) needle was pulled with Narishige puller (model PC:10) using the following settings: step 1 at 99$^\circ$C, step 2 at 79$^\circ$C. The pulled needle was filled with 1 $\mu$L of injection mix and assembled into Transferman NK2 (Eppendorf) micromanipulator. Microinjection was performed with FemtoJet Microinjector (Eppendorf).
#### Generation of transgenic lines
......@@ -707,10 +710,10 @@ knitr::include_graphics("fig/methods/select-transgenic-worms.png")
\newpage
### Determination of human nAChR expression in the *C. elegans* pharynx by staining with conjugated $\alpha7$ selective antagonist FITC-$\alpha$-bungarotoxin(Bgtx) ###{#fitcmethod}
### Determination of human nAChR expression in the *C. elegans* pharynx by staining with conjugated $\alpha7$ selective antagonist FITC-$\alpha$-bungarotoxin (bgtx) ###{#fitcmethod}
#### Worm preparation
Worms were submerged in 3 mL of Dent's saline in 5 cm Petri dish. To ease dissection, they were paralysed by placing the dish at -20 $^\circ$C for 5 minutes. Following this, the tip of the nose was cut perpendicular to the head to allow the cuticle to roll back and expose the pharynx. Next, the cut just below the terminal bulb was made and liberated pharynxes collected (Figure \@ref(fig:exposed-pharynx-label)).
Worms were submerged in 3 mL of Dent's saline in 5 cm Petri dish. To ease dissection, they were paralysed by placing the dish at -20$^\circ$C for 5 minutes. Following this, the tip of the nose was cut perpendicular to the head to allow the cuticle to roll back and expose the pharynx. Next, the cut just below the terminal bulb was made and liberated pharynxes collected (Figure \@ref(fig:exposed-pharynx-label)).
(ref:exposed-pharynx-method) **Exposure of the *C. elegans* pharynx.** Using surgical blade, the cut was made at a tip of the nose and just below the terminal bulb (left image, black lines) to expose the pharynx (right).
......
......@@ -15,14 +15,14 @@ library(knitr)
## Introduction
Neonicotinoids are the most commonly used insecticides worldwide due to their high efficacy against pest insects (Section \@ref(potentpests)), selective toxicity to insect pests over mammals (Section \@ref(seltox)) and advantageous physicochemical attributes (Section \@ref(physchem)). The main disadvantage of these compounds is that they can be toxic to non-target species, including bees (Section \@ref(sublethal)). This undesired ecotoxicological effect spurred a debate over their environmental impact and revealed a necessity to further investigate their effects on other ecologically important organisms such as worms.
## Ecological role of non-parasitic worms
## Ecological role of non-parasitic worms ##{#ecologoicalroleofwormschapter3}
Non parasitic earth worms and nematodes, play an important biological role. They are the most abundant multicellular organisms on earth and are significant biomass contributors. In addition, they cycle nutrients contributing as much as 1/5 of all bioavailable nitrogen in soil [@neher2001], promoting plant growth [@ingham1985] and soil fertility. They are also valuable bioindicators and have been used in the assessment of contaminated soil [@lecomte-pradines2014].
## Residues of neonicotinoids in soil
Neonicotinoids are commonly applied as a seed dressing [@jeschke2011; @alford2017], due to a benefit of extended crops protection resulting in a reduction in the insecticide application frequency. However, on average, only 5 % of the active ingredient is taken up by and distributed throughout the developing plant [@sur2003]. The remainder enters the wider environment, including soils, where they can have a negative effect on inhabiting worm species.
Neonicotinoids are commonly applied as seed dressing [@jeschke2011; @alford2017], due to a benefit of extended crops protection resulting in a reduction in the insecticide application frequency. However, on average, only 5 % of the active ingredient is taken up by and distributed throughout the developing plant [@sur2003]. The remainder enters the wider environment, including soils, where they can have a negative effect on inhabiting worm species.
The levels of neonicotinoids in terrestrial terrains vary depending on the composition and the physical properties of the soil [@moertl2016; @selim2010; @zhang2018] Numerous studies investigated their levels in various soil types, following variable post planting period and generally report the sub $\mu$M concentrations (reviewed in @wood2017). However, they persist in terrestrial terrains from a few days to several years (reviewed in @goulson2013). Nitenpyram and thiacloprid typically remain there for several weeks, clothianidin for just over a year, whereas imidacloprid for several years. Long dissipation half-life and absorption capacity means that the neonicotinoids may come in contact with soil- residing worms for prolonged time periods. Neonicotinoids can also enter the worm’s interior by multiple routes. They may diffuse across the worm’s cuticle, or be ingested with soil particles [@pisa2015]. Exposure to residual neonicotinoids can have a negative impact on many aspects of worm’s biology.
The levels of neonicotinoids in terrestrial terrains vary depending on the composition and the physical properties of the soil [@moertl2016; @selim2010; @zhang2018]. Numerous studies investigated their levels in various soil types, following variable post planting period and generally report the sub $\mu$M concentrations (reviewed in @wood2017). However, they persist in terrestrial terrains from a few days to several years (reviewed in @goulson2013). Nitenpyram and thiacloprid typically remain there for several weeks, clothianidin for just over a year, whereas imidacloprid for several years. Long dissipation half-life and absorption capacity means that neonicotinoids may come in contact with soil- residing worms for prolonged time periods. Neonicotinoids can also enter the worm’s interior by multiple routes. They may diffuse across the worm’s cuticle, or be ingested with soil particles [@pisa2015]. Exposure to residual neonicotinoids can have a negative impact on many aspects of worm’s biology.
<!-- ### **C. elegans** in toxicity testing -->
<!-- *C. elegans* is a simple organism, easy to maintain, with well defined anatomy (Sections @\ref(anatomy)) and behaviour (Section @\ref(analytical_behaviour)). One of its major advantages is that it amenable to genetic manipulations (Section @\ref(genmanip)). Since its isolation in 1950, it has grown into an alternative model for the mode of action and toxicity studies of many compounds, most notably antiparasites [@Holden-Dye2014] such as levamisole, as well as an insect repelent N,N-diethyl-meta-toluamide (DEET). *C. elegans* is an attractive platform for the toxicity and the mode of action studies but it may be also useful in drug development screens. Several studies showed that the lethality rank order of hundreds of comounds tested on *C. elegans* correlated well with the lethality rank order in other species [reviewe in @hunt2017]. -->
......@@ -54,7 +54,7 @@ knitr::include_graphics("fig/intro_2/motility.jpg")
\newpage
#### Egg laying
Egg-laying is controlled by the contraction of vulvar muscles under the influence of the nervous system (Figure \@ref(fig:egg-laying-label)). The main excitatory neurotransmitter is serotonin [@waggoner1998] released from the Hermaphrodite Specific Neurons (HSNs). There are other neurotransmitters involved, such as excitatory acetylcholine [@trent1983] released from the Ventral C neurons (VCs). In addition, there are four uv1 neuroendicrine cells linking uterus and vulva which release tyramine to inhibit egg laying [@alkema2005].
Egg-laying is controlled by the contraction of vulvar muscles under the influence of the nervous system (Figure \@ref(fig:egg-laying-label)). The main excitatory neurotransmitter is 5-HT [@waggoner1998] released from the Hermaphrodite Specific Neurons (HSNs). There are other neurotransmitters involved, such as excitatory acetylcholine [@trent1983] released from the Ventral C neurons (VCs). In addition, there are four uv1 neuroendicrine cells linking uterus and vulva which release tyramine to inhibit egg laying [@alkema2005].
(ref:Egg-laying-fig) **Neuronal circuitry of *C. elegans* vulva.** Lateral image of the *C. elegans* hermaphrodite (top) and positioning of the vulva (bottom). 16 vulval muscles, out of which vm1 and vm2 are the most important, contract to expel eggs out. HNS and VC neurons synapse onto vm2 muscle. Uv1 neuroendocrine cells link the uterus and vulva and inhibit egg-laying. Image taken from [@collins2016].
......@@ -62,37 +62,6 @@ Egg-laying is controlled by the contraction of vulvar muscles under the influenc
knitr::include_graphics("fig/intro_2/vulva.png")
```
### Pharmacological evidence for the role of nAChRs in the regulation of *C. elegans* behaviour ###{#pharmacelegans}
Pharmacological experiments in which nAChR agonists, namely levamisole and nicotine, were tested against *C. elegans* behaviours provide evidence for the important role of these receptors in the regulation of locomotion and egg laying.
#### Levamisole
Levamisole is a synthetic compound used in treatment of parasitic worm infestation in both humans and animals [@miller1980]. It is an agonist of a subset of receptors present at a body wall muscle [@richmond1999]. Levamisole causes spastic paralysis of worms [@lewis1980b] and stimulates egg-laying [@trent1983].
#### Nicotine
Nicotine is an alkaloid naturally occuring in the Tobacco plant [@steppuhn2004]. It is an agonist of the second type receptor at a body wall muscle, namely the N-type [@ballivet1996], but based on the nicotine-intoxication worm phenotype, it is likely to target receptors regulating pharyngeal pumping and vulva muscle. Nicotine inhibits locomotion [@kudelska2017] pharyngeal pumping [@kudelska2018], and egg-laying.
#### Neonicotinoids ####{#chapter3effectsofneonics}
<!-- There is a limited literature regarding the effects of neonicotinoids on nematodes. Studies by [@dong2014; @dong2017] revealed antiparasytic potential of neonicotinoids. Thiacloprid kills plant parasite *Meloidogyne incognita* with the LC50 of 24 $\mu$M [@dong2014] -->
<!-- Thiaclopand inhibits its egg hatching with the EC~50~ of 300 $\mu$M [@dong2014; @dong2017]. -->
Neonicotinoids have variable effects on *C. elegans*. @mugova2018 reports an inhibitory effect on motility of imidacloprid at concentration ranging from 120 $\mu$M to 2 mM. Thiacloprid seems to have an opposite effect. At concentrations ranging from 2 to 40 $\mu$M it elevates locomotion in liquid of mixed developmental stage population of *C. elegans* [@hopewell2017].
Variable effects of neonicotinoids on egg-laying are also reported. Low mM concentrations of clothianidin and thiacloprid inhibit egg-laying [@gomez-amaro2015]. In contrast, imidacloprid at a single concentration of 20 nM, elevates the number of egg-laid, but has no effect at 120 $\mu$M - 2 mM, suggesting this effect is not dose-dependent [@ruan2009].
### Two nAChR types are expressed at the body wall muscle of *C. elegans* nAChRs ####{#muscletypenachr}
Acetylcoline exerts its effects by acting on nAChRs. *C. elegans* expresses at least 29 nAChR subunits (Section \@ref(celegansnacheintro)). To date, four receptor assemblies have been identified [@richmond1999; @treinin1998; @touroutine2005]. Two of the *C. elegans* nAChRs are expressed at the post-synaptic membrane of the neuromuscular junction of the body wall muscle [@richmond1999], where they are involved in the regulation of locomotion. The identity and function of these proteins has been studies using a combination of behavioural, pharmacological and electrophysiological approaches.
##### L-type receptors
Behavioural analysis of *C. elegans* mutants identified several strains in which locomotion was disrupted, including unc-29, unc-38 and unc-63 [@lewis1980b]. unc-29, unc-38 and unc-63, as well as lev-1 and lev-8 were also resistant to levamisole [@lewis1980b]. Expression of lev-1, unc-29 and unc-38 in *Xenopus oocytes* generated a protein with nAChR-like properties: in response to acetylcholine and levamisole, depolarising current was elicited [@fleming1997]. @richmond1999 provided evidence that these receptors are expressed at the NMJ of the body wall muscle. Intracellular recordings from the post-synaptic membrane at the NMJ of the body wall muscle showed that in response to acetylcholine and levamisole inward current is elicited. That current was abolished in unc-29 and unc-38 mutants [@richmond1999]. The identity of the levamisole sensitive nAChRs was revealed by @boulin2008, who showed that eight genes are required for the generation of fully functional receptor in *Xenopus oocytes*. Five genes encode for nAChR subunits UNC-29, UNC-38, UNC-63, LEV-1 and LEV-8, two of which, viz. UNC-29 and LEV-1 are non-$\alpha$. In the absence of any one of the 5 subunits, agonist-evoked currents were abolished, suggesting all subunits are essential for the receptor function. The remaining 3 genes encode for the auxiliary subunits RIC-3, UNC-50, AND UNC-74. Their role is described in Section \@ref(cematnachr).
##### N-type receptors
Work of @richmond1999 identified second type of nAChR at the muscular junction of the body wall muscle. This receptor showed high sensitivity to nicotine, thus was named N-type nAChR. N-type receptor is composed of ACR-16 subunits, which form homomeric receptors in Xenopus oocytes [@ballivet1996].
<!-- ### Effects of neonicotinoids on earthworms -->
<!-- Most ecotoxicological studies focused on the effects of neonicotinoids on behaviours governed by the cholinergic neurotransmission and worms mortility. These were conducted on earthworms, Lumbricus terrestris and Eisenia fetida, which reflects the pivotoal ecological role of these “undrgrdound dwellers”. @basley2017 showed that field realistic concentrations of clothianidin have no effects on Lumbricus terrestris mortality, feeding and worm population [@basley2017], but it is toxic to Eisenia fetida with LC50 value of ~1 mM [@wang2012]. -->
<!-- DEET was discovered in 1944 and was originally intendeed for use in agriculture. It is currently used as an insect repellent effective agains flies, mosquitos, ticks and fleas. -->
<!-- Levamisole, discovered in 1960s, has a potent antihelmintic action [@pinnock1988]. It causes contraction of the body wall muscle of the parasitic worm **A.suum** [@martin1991] and nematode **C. elegans** [@lewis1980b], which leads to their paralysis. Body wall muscle of both **A.suum** and **C. elegans** express N- and L-type nAChR types [@qian2006; @richmond1999]. Genetic studies shed light on the receptor type targeted by levamisole. *C. elegans* mutants deficient in several constituents of L-type, and not N-type nAChR, exihibited markedly reduced levamisole-sensitivity [@lewis1987; @lewis1980], suggesting L-type receptor is the principal site of action of this compound. -->
......@@ -101,6 +70,10 @@ Work of @richmond1999 identified second type of nAChR at the muscular junction o
<!-- Strains useful - When challenged with M. nematophilum, wild-type worm develops characteristic swelling in the tail region. This phenotype is absent in worms with mutated bus (Bacterially UnSwollen) genes. -->
### Effects of neonicotinoids on worms ###{#chapter3effectsofneonics}
There is a limited literature regarding the effects of neonicotinoids on earthworms and nematodesn (Section \@ref(effectsofneonicsonworms)). In earth worms negative effects of neonicotinoid on the reproduction [@gomez-eyles2009; @baylay2012; @kreutzweiser2008; @alves2013], avoidance [@alves2013] and locomotion [@dittbrenner2011] have been described. Studies by [@dong2014; @dong2017] revealed antiparasytic potential of these insecticides. Thiacloprid kills plant parasite *Meloidogyne incognita* with the LC50 of 24 $\mu$M [@dong2014], whereas thiaclopand inhibits its egg-hatching with the EC~50~ of 300 $\mu$M [@dong2014; @dong2017]. They seem to have variable effects on *C. elegans*. @mugova2018 reports an inhibitory effect on motility of imidacloprid at concentration ranging from 120 $\mu$M to 2 mM. Thiacloprid seems to have an opposite effect. At concentrations ranging from 2 to 40 $\mu$M it elevates locomotion in liquid of mixed developmental stage population of *C. elegans* [@hopewell2017]. Variable effects of neonicotinoids on egg-laying are also reported. Low mM concentrations of clothianidin and thiacloprid inhibit egg-laying [@gomez-amaro2015]. In contrast, imidacloprid at a single concentration of 20 nM, elevates the number of egg-laid, but has no effect at 120 $\mu$M - 2 mM, suggesting this effect is not dose-dependent [@ruan2009].
### Chapter aims
The aim of this chapter is to better understand the effects of neonicotinoids on Nematoda representative *C. elegans*. Their impact on defined and well understood behaviors governed by the cholinergic transmission are tested and compared to the effects elicited by a classical nAChR agonist nicotine. This will be used to inform the potential risk of these environmental neurotoxins against Nematoda and on their mode of action.
......@@ -137,7 +110,7 @@ Mutation of *bus-17* genes have no significant effect on the thrashing behavior
(ref:thrashing-cuticle) **The effects of the cuticle on the concentration and time dependence of nicotine inhibition of *C. elegans* thrashing.** N2 wild type (a) and *bus-17* mutant (b) worms were exposed to varying concentrations of nicotine. The number of thrashes were recorded for 30 seconds at the indicated time points. b) Concentration dependence of nicotine dependent inhibition of thrashing on N2 (black) and *bus-17* worms (grey). Dose-response curve were generated by taking the 120-minute time- points; that is when the steady-state of thrashing inhibition was reached, and expressed as % of control thrashing. EC~50~ values are shown on graphs. Data are mean $\pm$ SEM of $\ge$ 15 individual worms collected from paired experiments done on 3 days.
```{r thrashing-cuticle-label, fig.cap= "(ref:thrashing-cuticle)", fig.scap = "The effects of the cuticle on the concentration and time dependence of nicotine inhibition of \\textit{C. elegans} thrashing.", fig.align='center', echo=FALSE}
```{r thrashing-cuticle-label, fig.cap= "(ref:thrashing-cuticle)", fig.scap = " The effects of the cuticle on the concentration and time dependence of nicotine inhibition of \\textit{C. elegans} thrashing.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results2/final/pngpdf/fig2.png")
```
......@@ -177,7 +150,7 @@ In contrast, all worms paralysed by nitenpyram or thiacloprid returned to normal
(ref:recovery-thrashing) **Recovery kinetics of nicotine and neonicotinoid-paralysed *C. elegans*.** N2 wild-type and *bus-17* mutant worms were exposed to indicated concentrations of nicotine (a and b), nitenpyram (c) and thiacloprid (d). Paralysed worms were transformed to drug-free dish and recovery was scored by noting a number of thrashes / 30s. Only worms recovered are included in this analysis. Alongside these, worms were transferred from drug-containing to drug containing dish (+ve ctr ) and from drug-free to drug-free dish (-ve ctr). Data are mean $\pm$ SEM of 8 individual worms collected from experiments done $\ge$ 2 days.
```{r merged-recovery-title, fig.cap= "(ref:recovery-thrashing)", fig.scap= "Recovery kinetics of nicotine and nonicotinoid-induced-paralysed \\textit{C. elegans}.", fig.align='center', echo=FALSE}
```{r merged-recovery-title, fig.cap= "(ref:recovery-thrashing)", fig.scap= "Recovery kinetics of nicotine and nonicotinoid-paralysed \\textit{C. elegans}.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results2/final/pngpdf/recovery.png")
```
......@@ -198,7 +171,7 @@ During acute and chronic exposure experiments, it was noted that a 4 hour incuba
(ref:shrinking) **Effects of nicotinic compounds on *C. elegans* body length.** *Bus-17* mutant was submerged for 4 hours in solution containing 25 mM nicotine, 50 mM nitenpyram, 1.5 mM thiacloprid or 2.5 mM clothianidin or vehicle control (Ctr). 4 hours later, the images of worms were taken and the body length measured. Data are mean $\pm$ SEM. Number of determinations $\ge$ 12 collected over 3 observations. One way ANOVA with Bonferonni corrections, $**$ P $\le$ 0.01.
```{r shrinking-title1, fig.cap="(ref:shrinking)", fig.scap = "Effects of nicotinic compounds on \\textit{C. elegans} body lenght.", fig.align='center', echo=FALSE,}
```{r shrinking-title1, fig.cap="(ref:shrinking)", fig.scap = " Effects of nicotinic compounds on \\textit{C. elegans} body lenght.", fig.align='center', echo=FALSE,}
# cbp1 <- c("#999999", "#E69F00", "#56B4E9", "#009E73",
# "#F0E442", "#0072B2", "#D55E00", "#CC79A7")
......@@ -260,7 +233,7 @@ During the experimentation, an observation that the proportion of worms disappea
(ref:avoid) **The concentration dependence of the effects of nicotine on *C. elegans* avoidance.** 4-10 wild-type worms were placed on agar plate containing indicated nicotine concentrations or drug vehicle (0). 24 hours later, the % of worms remaining on the plate was scored. Data are mean $\pm$ SEM, collected from 2 - 4 individual experiments. One way ANOVA (Kruskal-Wallis test) with Sidak Corrections, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.
```{r avoid-label, fig.cap="(ref:avoid)", fig.scap= "The concentration dependence of the effects of nicotine on \\textit{C. elegans} avoidance.", fig.align='center', echo=FALSE}
```{r avoid-label, fig.cap="(ref:avoid)", fig.scap= " The concentration dependence of the effects of nicotine on \\textit{C. elegans} avoidance.", fig.align='center', echo=FALSE}
# ann_text_avoid_1 <- data.frame(Dose = factor(c(25, 50, 100), levels = c(25, 50, 100)),
# mean_readout=100,
......@@ -329,9 +302,9 @@ Untreated wild-type worms move at a rate of 39 body bends per minute (Figure \@r
<!-- # se=sd/sqrt(length(readout))) -->
<!-- # ``` -->
(ref:BB-plot-capt) **The concentration-dependence for the effects of nicotine and neonicotinoid on body bends of *C. elegans*.** N2 wild-type (left panel) and *bus-17* mutant (right panel) worms were exposed for 24 hours to varying concentrations of nicotine, nitenpyram, thiacloprid, clothianidin or drug vehicle (O), incorporated into solid medium. Body bends were counted by visual observation. Data are mean $\pm$ SEM of $\ge$ 5 individual worms collected from $\ge$ 3 paired experiments. One way ANOVA (Kruskal-Wallis test) with Dunn’s Corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.
(ref:BB-plot-capt) **The concentration dependence for the effects of nicotine and neonicotinoid on body bends of *C. elegans*.** N2 wild-type (left panel) and *bus-17* mutant (right panel) worms were exposed for 24 hours to varying concentrations of nicotine, nitenpyram, thiacloprid, clothianidin or drug vehicle (O), incorporated into solid medium. Body bends were counted by visual observation. Data are mean $\pm$ SEM of $\ge$ 5 individual worms collected from $\ge$ 3 paired experiments. One way ANOVA (Kruskal-Wallis test) with Dunn’s Corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.
```{r BB-plot-label, fig.cap= "(ref:BB-plot-capt)", fig.scap= "The concentration-dependence for the effects of nicotine and neonicotinoid on body bends of \\textit{C. elegans}.", fig.align='center', include=TRUE, echo=FALSE}
```{r BB-plot-label, fig.cap= "(ref:BB-plot-capt)", fig.scap= " The concentration dependence for the effects of nicotine and neonicotinoid on body bends of \\textit{C. elegans}.", fig.align='center', include=TRUE, echo=FALSE}
#Plot body bends graphs
#make a data frame for facet labels
# labelsBB <- c("9" = "Nicotine N2", "10" = "Nicotine bus17", "11"= "Nitenpyram N2", "12" = "Nitenpyram bus17", "13" = "Thiacloprid N2", "14" = "Thiacloprid bus17", "15" = "Clothianidin N2", "16" = "Clothianidin bus17")
......@@ -473,9 +446,9 @@ knitr::include_graphics("fig/results2/final/pngpdf/bodybendsplot.png")
```
(ref:DR-body-bends) **Concentration-dependence curves for the effects of nicotine and neonicotinoids on *C. elegans* body bends.** Concentration-response curves for the effects of nicotine (a), nitenpyram (b), thiacloprid (c) and clothianidin (d) on body-bend rates of wild-type N2 and *bus-17* mutant *C. elegans*. Body bend rates are expressed as a % of control activity. Data are mean $\pm$ SEM. The EC~50~ of thiacloprid on N2 and clothianidin on *bus-17* is an approximation, as at highest concentrations tested (1.5 mM thiacloprid and 3.75 mM clothianidin) the maximum inhibition observed was 21 and 42 %.
(ref:DR-body-bends) **Concentration dependence curves for the effects of nicotine and neonicotinoids on *C. elegans* body bends.** Concentration-response curves for the effects of nicotine (a), nitenpyram (b), thiacloprid (c) and clothianidin (d) on body-bend rates of wild-type N2 and *bus-17* mutant *C. elegans*. Body bend rates are expressed as a % of control activity. Data are mean $\pm$ SEM. The EC~50~ of thiacloprid on N2 and clothianidin on *bus-17* is an approximation, as at highest concentrations tested (1.5 mM thiacloprid and 3.75 mM clothianidin) the maximum inhibition observed was 21 and 42 %.
```{r DR-body-bends-label, fig.cap="(ref:DR-body-bends)", fig.scap= "Concentration-dependence curves for the effects of nicotine and neonicotinoids on \\textit{C. elegans} body bends.", fig.width=10, fig.align='center', fig.asp=1.1, echo=FALSE}
```{r DR-body-bends-label, fig.cap="(ref:DR-body-bends)", fig.scap= " The concentration dependence curves for the effects of nicotine and neonicotinoids on \\textit{C. elegans} body bends.", fig.width=10, fig.align='center', fig.asp=1.1, echo=FALSE}
knitr::include_graphics("fig/results2/DR-body-bends.png")
```
......@@ -483,11 +456,11 @@ knitr::include_graphics("fig/results2/DR-body-bends.png")
\newpage
#### Effects on egg laying
On-plate experiments allow to assay for other aspects of *C. elegans* biology such as egg-laying (Section \@ref(egglayingbehaviour)). The number of eggs laid per worm in the presence of nicotine and neonicotinoids over a period of 24 hours was counted and compared to the control (Figure \@ref(fig:EL-plot-label) and \@ref(fig:egg-laying-lbl)). Both N2 wild-type and *bus-17* mutants lay ~ 95 eggs/day/worm. No effects on wild-type worm of either compound was observed. However, egg-laying rate of *bus-17* mutant was reduced by low mM concentrations of thiacloprid and clothianidin.
On-plate experiments allow to assay for other aspects of *C. elegans* biology such as egg-laying (Section \@ref(egglayingbehaviour)). The number of eggs laid per worm in the presence of nicotine and neonicotinoids over a period of 24 hours was counted and compared to the control (Figure \@ref(fig:EL-plot-capt-label) and \@ref(fig:egg-laying-lbl)). Both N2 wild-type and *bus-17* mutants lay ~ 95 eggs/day/worm. No effects on wild-type worm of either compound was observed. However, egg-laying rate of *bus-17* mutant was reduced by low mM concentrations of thiacloprid and clothianidin.
(ref:EL-plot-capt) **The concentration-dependence for the effects of nicotine and neonicotinoids on egg-laying of *C. elegans*.** Wild type (left panel) and *bus-17* (right panel) worms were exposed for 24 hours to varying concentrations of nicotine, thiacloprid, clothianidin or drug vehicle (0), incorporated into solid medium. Number of eggs laid in 24 hours were counted and expressed as eggs laid per worm. Data are mean $\pm$ SEM collected from $\ge$ 8 individual on $\ge$ 2 days. One way ANOVA (Kruskal-Wallis test) with Dunnett’s Corrections, $*$ P $\le$ 0.05, $**$ P $\le$ 0.01, $***$ P $\le$ 0.001.
(ref:EL-plot-capt) **The concentration dependence for the effects of nicotine and neonicotinoids on egg-laying of *C. elegans*.** Wild type (left panel) and *bus-17* (right panel) worms were exposed for 24 hours to varying concentrations of nicotine, thiacloprid, clothianidin or drug vehicle (0), incorporated into solid medium. Number of eggs laid in 24 hours were counted and expressed as eggs laid per worm. Data are mean $\pm$ SEM collected from $\ge$ 8 individual on $\ge$ 2 days. One way ANOVA (Kruskal-Wallis test) with Dunnett’s Corrections, $*$ P $\le$ 0.05, $**$ P $\le$ 0.01, $***$ P $\le$ 0.001.
```{r EL-plot-label, fig.cap= "(ref:EL-plot-capt)", fig.scap = "The concentration-dependence for the effects of nicotine and neonicotinoids on egg-laying of \\textit{C. elegans}.", fig.align='center', fig.asp=1.2, include=TRUE, results="hide", echo=FALSE}
```{r EL-plot-capt-label, fig.cap= "(ref:EL-plot-capt)", fig.scap = "The concentration dependence for the effects of nicotine and neonicotinoids on egg-laying of \\textit{C. elegans}.", fig.align='center', fig.asp=1.2, include=TRUE, echo=FALSE}
# labelsEL <- c("17" = "Nicotine N2", "18" = "Nicotine bus17", "19"= "Nitenpyram N2", "20" = "Nitenpyram bus17", "21" = "Thiacloprid N2", "22" = "Thiacloprid bus17", "23" = "Clothianidin N2", "24" = "Clothianidin bus17")
# ann_textEL <- data.frame(Dose = factor (c(0.25,0.5,1.5), levels = c("0.25", "0.5", "1.5")), mean_readout = 105,labEL = c("*","**","**"), Exp = 22)
......@@ -589,7 +562,7 @@ knitr::include_graphics("fig/results2/final/pngpdf/egglayingplot.png")
(ref:DR-egg-laying) **Dose-response curves for the effects of nicotine and neonicotinoids on egg-laying of *C. elegans*.** Concentration-response curves for the effects of nicotine (a), nitenpyram (b), thiacloprid (c) and clothianidin (d) on egg-laying of N2 wild-type and *bus-17* mutant *C. elegans*. Egg laying is expressed as a % control activity. The EC~50~ for clothianidin is an approximation, as at the highest concentration tested (3.75 mM), the maximum response observed was 44 %. Data are mean $\pm$ SEM.
```{r, egg-laying-lbl, fig.cap="(ref:DR-egg-laying)", fig.scap = "Dose-response curves for the effects of nicotine and neonicotinoids on egg-laying of \\textit{C. elegans}.", fig.align='center', echo=FALSE}
```{r, egg-laying-lbl, fig.cap="(ref:DR-egg-laying)", fig.scap = " Dose-response curves for the effects of nicotine and neonicotinoids on egg-laying of \\textit{C. elegans}.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results2/DR-egg-laying.png")
```
......@@ -598,9 +571,9 @@ knitr::include_graphics("fig/results2/DR-egg-laying.png")
#### Effects on egg hatching
Eggs laid on the plate hatch after 9 hours of exo-utero development (Figure \@ref(fig:life-cycle-label)). To investigate the effects of nicotine and neonicotinoids on egg-hatching L4 + 1 worms were incubated with nicotine and neonicotinoids. After 24 hours of incubation, they were removed from the experimental plate, leaving the progeny and eggs behind. After a further 24 hours, the number of unchanged eggs and larvae present were counted to derive the % hatching rate. Almost 100 % of eggs laid by N2 and *bus-17* hatched (Figure \@ref(fig:EH-plot-label)). Neither compound had an effect on hatching of N2 worms (Figure \@ref(fig:EH-plot-label), left panel). However, thiacloprid at 1.5 mM and clothianidin at 2 mM reduced the proportion of hatched eggs of *bus-17* worms by 19 and 13 %, respectively (Figure \@ref(fig:EH-plot-label), right panel).
(ref:EH-plot-capt) **The concentration-dependence for the effects of nicotine and neonicotinoids on *C. elegans* egg-hatching.** N2 wild-type (a) and *bus-17* mutant (b) worms laid eggs in the presence of varying concentrations of nicotine, thiacloprid, clothianidin or drug vehicle (0). After 24 hours adult worms were removed and the eggs left behind. Number of unhatched eggs and larvae were counted 24 hours later. Data are mean $\pm$ SEM, of $\ge$ 2 paired experiments performed on $\ge$ days. One way ANOVA (Kruskal-Wallis test) with Dunnett’s Corrections, $*$ P $\le$ 0.05, $**$ P $\le$ 0.01.
(ref:EH-plot-capt) **The concentration dependence for the effects of nicotine and neonicotinoids on *C. elegans* egg-hatching.** N2 wild-type (a) and *bus-17* mutant (b) worms laid eggs in the presence of varying concentrations of nicotine, thiacloprid, clothianidin or drug vehicle (0). After 24 hours adult worms were removed and the eggs left behind. Number of unhatched eggs and larvae were counted 24 hours later. Data are mean $\pm$ SEM, of $\ge$ 2 paired experiments performed on $\ge$ days. One way ANOVA (Kruskal-Wallis test) with Dunnett’s Corrections, $*$ P $\le$ 0.05, $**$ P $\le$ 0.01.
```{r EH-plot-label, fig.cap = "(ref:EH-plot-capt)", fig.align='center', fig.cap= "(ref:EH-plot-capt)", fig.scap = "The concentration-dependence for the effects of nicotine and neonicotinoids on \\textit{C. elegans} egg-hatching.", echo=FALSE}
```{r EH-plot-label, fig.cap = "(ref:EH-plot-capt)", fig.align='center', fig.cap= "(ref:EH-plot-capt)", fig.scap = " The concentration dependence for the effects of nicotine and neonicotinoids on \\textit{C. elegans} egg-hatching.", echo=FALSE}
# labelsEH <- c("25" = "Nicotine N2", "26" = "Nicotine bus17", "27"= "Nitenpyram N2", "28" = "Nitenpyram bus17", "29" = "Thiacloprid N2", "30" = "Thiacloprid bus17", "31" = "Clothianidin N2", "32" = "Clothianidin bus17")
# ann_textEH <- data.frame(Dose = factor (c(1, 1.5), levels=c("1", "1.5")), mean_readout = 105,labelEH = c("*","**"), Exp = 30)
......@@ -702,7 +675,7 @@ To investigate whether compounds hinder the hatching of larvae, images of unhatc
(ref:unhatched-eggs) **Effects of thiacloprid and clothianidin on *C. elegans* egg-hatching.** The appearance of unhatched eggs laid by *bus-17 C. elegans* mutant in the presence of 1.5 mM thiacloprid.
```{r unhatched-eggs-labels, fig.cap="(ref:unhatched-eggs)", fig.scap= "Effects of thiacloprid and clothianidin on \\textit{C. elegans} egg-hatching.", fig.align='center', echo=FALSE}
```{r unhatched-eggs-labels, fig.cap="(ref:unhatched-eggs)", fig.scap= " Effects of thiacloprid and clothianidin on \\textit{C. elegans} egg-hatching.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results2/unhatched-egg-2.png")
```
......@@ -713,7 +686,7 @@ Eggs that are laid on plate with a food source, hatch and develop into adults in
(ref:development-images-capt) **Effects of nicotine and thiacloprid on larval development of *C. elegans*.** Eggs were laid by N2 wild-type worms on medium containing 1 mM nicotine or 1 mM thiacloprid. 72-hours later, the images of the progeny were taken. Worms developing in the presence of treatment are visibly smaller in comparison to the control.
```{r dev-image, fig.cap= "(ref:development-images-capt)", fig.scap = "Effects of nicotine and thiacloprid on larval development of \\textit{C. elegans}.", fig.align='center', echo=FALSE}
```{r dev-image, fig.cap= "(ref:development-images-capt)", fig.scap = " Effects of nicotine and thiacloprid on larval development of \\textit{C. elegans}.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results2/Development_images.png")
```
......@@ -757,7 +730,7 @@ A synchronous population of L4 + 1 worms laid eggs on drug-treated plate. 2 hour
(ref:development-plot-capt) **Effects of nicotine and thiacloprid on the development of *C. elegans*.** N2 wild-type worms laid eggs on plates dosed with 1mM thiacloprid, 1mM nicotine or drug vehicle (Ctr). Larval development in the presence of drugs was monitored over time. Worms were assigned to each one of 5 life-stages, namely L1, L2, L3, L4 and gravid adults. The fraction of worms in each stage as a % of total population at time point: 30, (day 1), 48 hours (day 2), 72 hours (day 3), 144 hours (day 6) was measured. Data are shown as the mean of N $\ge$ 3.
```{r development-selected-plot, fig.cap= "(ref:development-plot-capt)", fig.scap= "Effects of nicotine and thiacloprid on the development of \\textit{C. elegans}.", fig.align='center', echo=FALSE}
```{r development-selected-plot, fig.cap= "(ref:development-plot-capt)", fig.scap= " Effects of nicotine and thiacloprid on the development of \\textit{C. elegans}.", fig.align='center', echo=FALSE}
#select values
# dev_t3 <- dev_dat_t2 %>%
# filter(Comp == "Nic" | Comp == "Thia" | Comp == "Ctr") %>%
......
# The effects of nicotine and neonicotinoids on the pharyngeal pumping of *C. elegans* {#results-2}
# The effects of nicotine and neonicotinoids on the *C. elegans* pharyngeal pumping {#results-2}
## Introduction
......@@ -106,7 +106,7 @@ knitr::include_graphics("fig/results3/pharyngeal_system_2.png")
Genetic studies of worm mutant strains provide evidence for a critical role of neurotransmission in the regulation of feeding. UNC-13 protein is involved in the regulation of neurotransmitter release at the synapse, as shown by the biochemical and behavioural analysis of the *unc-13 C. elegans* strain. Mutants deficient in UNC-13 show severe retention of vesicles in the pre-synapse [@richmond1999] and impaired synaptic transmission [@aravamudan1999]. Their feeding is also affected: 70 % reduction of the pharyngeal pumping rate was noted [@richmond2001]. These data highlight the key role of neurotransmitters in the regulation of pumping. The activity of the pharynx is influenced by acetylcholine, glutamate and 5-HT (Table \@ref(tab:pharynx-neurons) and Figure \@ref(fig:pharyngeal-nervous-system-label)), and potential tyramine and octopamine [@alkema2005].
#### 5-HT
#### 5-HT ####{#pharynx5ht}
5-HT is synthesised in two pharyngeal neurons NSM and I5 [@chase2007]. Mutant deficient in enzyme in the 5-HT biosynthetic pathway has blunted response to food [@sze2000]. Exogenous application of 5-HT induces feeding response in the absence of food whereas competitive 5-HT antagonists inhibit pumping [@horvitz1982; @avery1990]. Collectively, this suggests that serotonergic neurotransmission induce food-evoked feeding response.
......@@ -114,7 +114,7 @@ In the presence of food, 5-HT can be released from multiple sites, including NSM
Detection of food leads to the elevation of pumping rate via 5-HT acting at multiple G-protein coupled receptors [@avery2012]. 5-HT can regulate feeding response by binding to neuronal SER-4 receptors and SER-1 receptors expressed both in the pharyngeal muscle cells and neurons [@tsalik2003]. It also binds to SER-5 receptors expressed on extrapharyngeal interneurons [@cunningham2012]. However, the main driver of the 5-HT driven pharyngeal response it the activation of SER-7 receptors expressed on cholinergic MC and M4 neurons and and on glutametergic M3 neurons [@hobson2003; @song2013]. Acetylcholine released from MC and M4 neurons increases contraction frequency and induce isthmus peristalsis, respectively [@avery1987; @raizen1994]. This leads to an increase in the activity of the pharynx to ~260 pumps/min. Glutamate released from M3 shortens pump duration to <200ms.
#### Glutamate
#### Glutamate ####{#glutamatepharynx}
Glutamate is produced in at least 4 neurons (Table \@ref(tab:pharynx-neurons)), but its function in M3 is the best studied. Laser ablated M3^-^ animals have reduced pumping rate on food [@raizen1995]. Similar phenotype is observed in animals deficient in glutametergic neurotransmission. EAT-4 encodes for vesicular glutamate transporter [@lee1999]. Mutation of this gene in *C. elegans*, leads to reduction of the pharyngeal pumping rate of food [@lee2008].
......@@ -129,21 +129,17 @@ In response to food, released 5-HT activates M3 [@niacaris2003], leading to rele
<!-- There are also other 5-HT receptors, such as MOD-1 (for modulation of locomotion defective). MOD-1 is a 5HT-gated chloride channel [@ranganathan2000] with topology similar to the Cys-loop receptor superfamily. Phenotypical analysis of worms deficient in MOD-1 showed that starved mutant worms move faster in comparison to the wil-type strain upon entry onto the food patch, suggesting MOD-1 is involved in the regulation of locomotion in response to food in food-deprived animals. -->
<!-- https://www.pnas.org/content/116/14/7107 -->
#### ACetylcholine ##{#achpumping}
#### Acetylcholine ##{#achpumping}
The role of acetylcholine in the regulation of feeding was investigated in mutants deficient in proteins essential for the cholinergic neurotransmission (Section \@ref(cholinergicneurotransmissioninworms)). Null mutants die soon after birth due to starvation [@rand1989; @alfonso1993], whereas polymorphic mutants show reduced pharyngeal pumping both in the presence and absence of food [@dalliere2015]. Hindered feeding was also observed in animals in which one of the 6 cholinergic neuron, namely MC neurons, were ablated [@avery1989; @raizen1995].
Pharmacological and genetic studies suggest that acetylcholine stimulates pharynx by acting on nAChRs. Application of nAChR agonists, acetylcholine and nicotine, resulted in the stimulation of the pharyngeal pumping in the absence of food [@raizen1995]. In contrast, nAChR antagonist d-tubacurarine inhibited pharyngeal pumping in the presence of food [@raizen1995].
Although there are at least 29 nAChRs expressed in *C. elegans* (Figure \@ref(fig:seqidentityecd-label)), only one, namely EAT-2 has been identified as essential in mediation of the feeding response [@mckay2004]. EAT-2 is expressed in pm4 and pm5 muscle cells [@mckay2004], which make synaptic connections with the MC [@albertson1976]. *C. elegans eat-2* mutant shows significantly reduced pumping in the presence of food [@raizen1995; @mckay2004]. A similar phenotype was noted in the *eat-18* mutants. EAT-18 however is not a nAChR subunit. Instead, it is predicted to be a single transmembrane protein. Based on the localisation and behavioural phenotype, EAT-18 and EAT-2 are believed to co-assembly to form a functional receptor [@mckay2004].
Pharmacological and genetic studies suggest that acetylcholine stimulates pharynx by acting on nAChRs. Application of nAChR agonists, acetylcholine and nicotine, resulted in the stimulation of the pharyngeal pumping in the absence of food [@raizen1995]. In contrast, nAChR antagonist d-tubacurarine inhibited pharyngeal pumping in the presence of food [@raizen1995]. Although there are at least 29 nAChRs expressed in *C. elegans* (Figure \@ref(fig:seqidentityecd-label)), only one, namely EAT-2 has been identified as essential in mediation of the feeding response [@mckay2004] (section \@ref(eat2atthepharynx)). EAT-2 is expressed in pm4 and pm5 muscle cells [@mckay2004], which make synaptic connections with the MC [@albertson1976]. *C. elegans eat-2* mutant shows significantly reduced pumping in the presence of food [@raizen1995; @mckay2004]. A similar phenotype was noted in the mutant of *eat-18* auxilary protein [@mckay2004].
ACR-7 is also expressed at the pharyngeal muscle [@saur2013], as was shown with a reporter construct, however its function in pharyngeal pumping is unclear as *acr-7* mutant pump normally in the presence of food [@saur2013]. Besides nAChRs, there are other acetylcholine - sensitive receptors in the pharynx.
<!-- ACR-7 is also expressed at the pharyngeal muscle [@saur2013], as was shown with a reporter construct, however its function in pharyngeal pumping is unclear as *acr-7* mutant pump normally in the presence of food [@saur2013]. Besides nAChRs, there are other acetylcholine - sensitive receptors in the pharynx. -->
<!-- nAChR are likely to be expressed not only at the neuromuscular junction, as suggested by the EAT-18 expression pattern. Expression of GFP-tagged eat-18 gene under the native promoter leads to fluorescence in all types of pharyngeal muscle cells and in M5 neuron [@mckay2004]. The identity of other subunits remains to be elucidated. -->
GAR-3 is a ACh-sensitive GPCR receptor [@hwang1999] expressed in metacorpus, isthmus, terminal bulb of the pharyngeal muscle and in the I3 pharyngeal neuron [@steger2004]. Its role may involve regulation of the membrane electrical potential [@steger2004] and control of feeding during starvation [@you2006].
Acetylcholine-gated chloride channels (ACC) are ionotropic channels and like nAChRs, members of Cys-loop receptor family. There are at least 8 members isoforms: ACC-1 to ACC-4, LGC-46 to LGC-49 [@takayanagi-kiya2016], which form homo- and hetero-pentamers [@putrenko2005]. They are generally expressed in a distinct subset of cholinergic and glutametergic neurons (including acc-1 in pharyngeal M1 and M3) [@pereira2015; @takayanagi-kiya2016]. Interestingly, *acc-4* is expressed in almost all cholinergic neurons. Electrophysiological data suggest ACC channels are inhibitory but they may be playing multiple functions. ACC-4 may be acting as autoinhibitory, as most neurons predicted to express it do not receive direct synaptic input from other neurons [@albertson1976]. LGC-49 are expressed in presynaptic specialisations of cholinergic motor neurons where they regulate synaptic vesicle release [@takayanagi-kiya2016]. Based on pharmacologically characterised ACC-1 and ACC-2 *in vivo*, ACC receptors are also pharmacologically distinct from other ACh receptors. Some of the nAChR and GPCR receptor compounds were potent at modulating the activity of ACC channels, but not others. For example, ACC channels are insensitive to nAChR compounds nicotine, cytisine and antagonist $\alpha$-bgtx but are sensitivity to tubocurarine. This mixed pharmacological profile suggest ligand binding site is distinct from the binding site of nAChRs and cholinergic GPCRs.
Except from nAChRs, there are other acetylcholine sensitive receptors in the pharynx. GAR-3 is a ACh-sensitive GPCR receptor [@hwang1999] expressed in metacorpus, isthmus, terminal bulb of the pharyngeal muscle and in the I3 pharyngeal neuron [@steger2004]. Its role may involve regulation of the membrane electrical potential [@steger2004] and control of feeding during starvation [@you2006]. Whereas acetylcholine-gated chloride channels (ACC) are ionotropic channels and like nAChRs, members of Cys-loop receptor family. There are at least 8 members isoforms: ACC-1 to ACC-4, LGC-46 to LGC-49 [@takayanagi-kiya2016], which form homo- and hetero-pentamers [@putrenko2005]. They are generally expressed in a distinct subset of cholinergic and glutametergic neurons (including acc-1 in pharyngeal M1 and M3) [@pereira2015; @takayanagi-kiya2016]. Interestingly, *acc-4* is expressed in almost all cholinergic neurons. Electrophysiological data suggest ACC channels are inhibitory but they may be playing multiple functions. ACC-4 may be acting as autoinhibitory, as most neurons predicted to express it do not receive direct synaptic input from other neurons [@albertson1976]. LGC-49 are expressed in presynaptic specialisations of cholinergic motor neurons where they regulate synaptic vesicle release [@takayanagi-kiya2016]. Based on pharmacologically characterised ACC-1 and ACC-2 *in vivo*, ACC receptors are also pharmacologically distinct from other ACh receptors. Some of the nAChR and GPCR receptor compounds were potent at modulating the activity of ACC channels, but not others. For example, ACC channels are insensitive to nAChR compounds nicotine, cytisine and antagonist $\alpha$-bgtx but are sensitivity to tubocurarine. This mixed pharmacological profile suggest ligand binding site is distinct from the binding site of nAChRs and cholinergic GPCRs.
### Assays for scoring the effects of compounds on pharyngeal pumping
......@@ -557,9 +553,9 @@ To determine whether the effects of 5-HT treatment were reversible, heads were w
\newpage
(ref:DR-5HT-cuthead) **Concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected *C. elegans*.** a) Cut heads were exposed to varying concentrations of 5-HT, or vehicle control (0). The effects on pharyngeal pumping over time was scored by visual observation by counting the number of pharyngeal pumps in 30 seconds and the data was expressed in Hz. Data are mean $\pm$ SEM of 8 - 23 individual worms collected from experiments done on $\ge$ 3 days. Significance levels between the control and treatment are given in a figure legend and refer to 30 minute time points. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001. b) Dose-response curve for the effects of 5-HT on pharyngeal pumping of dissected *C. elegans*. The graph was generated by taking 30-minute time points and fitted into nonlinear regression sigmoidal dose-response (three parameter logistic) equation. Data are mean $\pm$ SEM, normalised to control and a maximum response, and expressed as % maximum pumping.
(ref:DR-5HT-cuthead) **The concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected *C. elegans*.** a) Cut heads were exposed to varying concentrations of 5-HT, or vehicle control (0). The effects on pharyngeal pumping over time was scored by visual observation by counting the number of pharyngeal pumps in 30 seconds and the data was expressed in Hz. Data are mean $\pm$ SEM of 8 - 23 individual worms collected from experiments done on $\ge$ 3 days. Significance levels between the control and treatment are given in a figure legend and refer to 30 minute time points. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001. b) Dose-response curve for the effects of 5-HT on pharyngeal pumping of dissected *C. elegans*. The graph was generated by taking 30-minute time points and fitted into nonlinear regression sigmoidal dose-response (three parameter logistic) equation. Data are mean $\pm$ SEM, normalised to control and a maximum response, and expressed as % maximum pumping.
```{r DR-5HT-cuthead-label, fig.cap="(ref:DR-5HT-cuthead)", fig.asp=1, fig.scap= "Concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected \\textit{C. elegans}", fig.align= 'center', echo=FALSE, message=FALSE, warning=FALSE}
```{r DR-5HT-cuthead-label, fig.cap="(ref:DR-5HT-cuthead)", fig.asp=1, fig.scap= "The concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected \\textit{C. elegans}", fig.align= 'center', echo=FALSE, message=FALSE, warning=FALSE}
knitr::include_graphics("fig/results3/cuthead-5ht-combined.png")
```
......@@ -679,9 +675,9 @@ To score the effects of compounds on a pharynx of dissected animal, cut heads we
Unstimulated pharynxes pump at an average rate of 0.1 Hz. Nicotine was tested at concentrations ranging from 100 nm to 100 $\mu$M (Figure \@ref(fig:cuthead-agonist-label) a). It had a dual action on the pharynx - stimulation at lower and inhibition at higher concentrations. At 10 $\mu$M it can be seen that a transient stimulation of pumping at 2 minute time point was seen. Incubation in 10 $\mu$M and 20 $\mu$M of nicotine led to weak but sustained stimulation of pumping - an effect seen at 20, 30 and 60 minute time points. 50 and 100 $\mu$M nicotine inhibited pumping completely. At these higher concentrations the muscle exhibited a visible twitching that can be described as fibrillation (data not shown). Neither nitenpyram nor thiacloprid had an effect on pharyngeal pumping of cut heads (Figure \@ref(fig:cuthead-agonist-label) b and c). In contrast, clothianidin stimulated pharynx (Figure \@ref(fig:cuthead-agonist-label) d). In response to 50 $\mu$M, an elevated pharyngeal pumping after 20 minutes was seen. whereas 500 and 750 $\mu$M of clothianidin elicited potent but short lived response. This response peaked after 2 minutes and returned to the basal rate after 10 minutes of incubation.
(ref:cuthead-agonist) **Concentration and time dependence for the effects of nicotine and neonicotinoids on pharyngeal pumping of dissected *C. elegans*.** Cut heads were exposed to varying concentrations of (a) nicotine (Nic), (b) nitenpyram (Nit), (c) thiacloprid (Thia), (d) clothianidin (Clo), 1 $\mu$M 5-HT + vehicle control or vehicle control only (Ctr). The effects of treatment on pumping were scored over time by counting the number of pharyngeal pumps in 30 seconds time windows and expressed in Hz. Data are $\pm$ SEM of 5 - 24 worms collected on $\ge$ 3 days. Significance levels between the vehicle control and treatment are given in a figure legend and refer to 30 minute time points. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.
(ref:cuthead-agonist) **The concentration and time dependence for the effects of nicotine and neonicotinoids on pharyngeal pumping of dissected *C. elegans*.** Cut heads were exposed to varying concentrations of (a) nicotine (Nic), (b) nitenpyram (Nit), (c) thiacloprid (Thia), (d) clothianidin (Clo), 1 $\mu$M 5-HT + vehicle control or vehicle control only (Ctr). The effects of treatment on pumping were scored over time by counting the number of pharyngeal pumps in 30 seconds time windows and expressed in Hz. Data are $\pm$ SEM of 5 - 24 worms collected on $\ge$ 3 days. Significance levels between the vehicle control and treatment are given in a figure legend and refer to 30 minute time points. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.
```{r cuthead-agonist-label, fig.cap= "(ref:cuthead-agonist)", fig.scap = "Concentration and time dependence for the effects of nicotine and neonicotinoids on pharyngeal pumping of dissected \\textit{C. elegans}.",fig.align='center', echo=FALSE}
```{r cuthead-agonist-label, fig.cap= "(ref:cuthead-agonist)", fig.scap = "The concentration and time dependence for the effects of nicotine and neonicotinoids on pharyngeal pumping of dissected \\textit{C. elegans}.",fig.align='center', echo=FALSE}
# Nic_plot <- cut_head %>%
# filter(Experiment == "7") %>%
# group_by(Time, Conc) %>%
......@@ -839,9 +835,9 @@ knitr::include_graphics("fig/results3/pumpingmutant.png")
Further experiments were carried out to determine whether pharyngeal responses induced by 5-HT, nicotine and neonicotinoids are dependent on the expression of the EAT-2 nAChR. Cut heads of wild-type and *eat-2* mutant worms were exposed to 5-HT, nicotine, nitenpyram, thiacloprid and clothianidin. The effects of treatment on pharyngeal pumping of mutant strain was scored and compared to the wild-type.
The effects of 5-HT concentrations ranging from 10 nM to 100 $\mu$M elicited dose-dependent stimulatory response in wild-type worm (Figure \@ref(fig:DR-5HT-cuthead-2-label) a). The maximum rate of 3.5 Hz was achieved by 1 $\mu$M 5-HT after 10 minutes of incubation. 5-HT also stimulated pumping of *eat-2* mutant, but the responses were much weaker (Figure \@ref(fig:DR-5HT-cuthead-2-label) b). The maximum rate of 0.78 Hz was achieved by 50 $\mu$M 5-HT after 60 minutes of incubation. To compare the effects of 5-HT on mutant to the effects elicited on the wild-type cut heads, data were plotted on dose-response curve (Figure \@ref(fig:DR-5HT-cuthead-3-label)). The efficacy of 5-HT on the pharyngeal pumping of *eat-2* mutant was markedly reduced. The maximum response achieved by 5-HT was 55 % lower in comparison to the wild-type. The potency was also reduced as reflected in the shift of the EC~50~ from 155 nM in wild-type to 104 $\mu$M in a mutant strain.
(ref:DR-5HT-cuthead-2) **Concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected wild-type and *eat-2 C. elegans*.** a) Wild-type (N2) and mutant *eat-2* cut heads were exposed to varying concentrations of 5-HT, or vehicle control (0). The effects on pharyngeal pumping over time was scored by visual observation by counting the number of pharyngeal pumps in 30 seconds and expressed in Hz. Data are mean $\pm$ SEM of 8 - 23 individual worms collected from paired experiments done on $\ge$ 3 days. Significance levels between the control and treatment are given in a figure legend and refer to 30 minute time points. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.
(ref:DR-5HT-cuthead-2) **The concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected wild-type and *eat-2 C. elegans*.** a) Wild-type (N2) and mutant *eat-2* cut heads were exposed to varying concentrations of 5-HT, or vehicle control (0). The effects on pharyngeal pumping over time was scored by visual observation by counting the number of pharyngeal pumps in 30 seconds and expressed in Hz. Data are mean $\pm$ SEM of 8 - 23 individual worms collected from paired experiments done on $\ge$ 3 days. Significance levels between the control and treatment are given in a figure legend and refer to 30 minute time points. One way ANOVA (Kruskal-Wallis test) with Sidak corrections, $*$P $\le$ 0.05, $**$P $\le$ 0.01, $***$P $\le$ 0.001, $****$P $\le$ 0.0001.
```{r DR-5HT-cuthead-2-label, fig.cap="(ref:DR-5HT-cuthead-2)", fig.scap = "Concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected wild-type and \\textit{eat-2 C. elegans}.", fig.align='center', echo=FALSE}
```{r DR-5HT-cuthead-2-label, fig.cap="(ref:DR-5HT-cuthead-2)", fig.scap = "The concentration and time dependence of the effects of 5-HT on pharyngeal pumping of dissected wild-type and \\textit{eat-2 C. elegans}.", fig.align='center', echo=FALSE}
# eat2_cuthead <- cut_head %>%
# filter(Experiment ==2) %>%
# group_by(Time, Conc) %>%
......
......@@ -16,7 +16,7 @@ The shape and amplitude of a single EPG varies between worms even of the same ge
knitr::include_graphics("fig/results3/basa-epg-individual.png")
```
## Chapter aims
### Chapter aims
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.
## Results
......@@ -68,9 +68,8 @@ To assess the effects of exogenous drug application on the pharynx an EPG - an e
<!-- ggsave("fig/results3/figure-test.pdf", width = 9, height = 10, units = "cm") -->
<!-- ``` -->
### Effects of 5-HT
### Effects of 5-HT on electropharyngeogram
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 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.
......@@ -100,7 +99,7 @@ knitr::include_graphics("fig/results3/5HT_EPG-all.png")
knitr::include_graphics("fig/results3/5HT_ind_EPG.png")
```
### Effects of Acetylcholine
### Effects of acetylcholine on electropharyngeogram
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).
......@@ -146,7 +145,7 @@ knitr::include_graphics("fig/results3/ach_train_properties.png")
knitr::include_graphics("fig/results3/epg_ach_graph.png")
```
### Effects of nicotine
### Effects of nicotine on electropharyngeogram
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.
......@@ -155,9 +154,9 @@ To score for the stimulatory effects of nicotine of pumping, the peak pumping ra
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.
(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.
(ref:epg-nic-traces) **The 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.
```{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}
```{r nic-epg-traces-label, fig.cap="(ref:epg-nic-traces)", fig.scap = "The concentration dependent effects of nicotine on EPG frequency.", fig.align= 'center', echo=FALSE}
knitr::include_graphics("fig/results3/epg-nicotine-traces.png")
```
......@@ -234,7 +233,7 @@ knitr::include_graphics("fig/results3/nic-epg-trace-long-exposure-comb.png")
\newpage
### Effects of cytisine
### Effects of cytisine on electropharyngeogram
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)).
......@@ -272,7 +271,7 @@ knitr::include_graphics("fig/results3/epg-cyt.png")
\newpage
### Effects of neonicotinoids
### Effects of neonicotinoids on electropharyngeogram
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.
......@@ -384,7 +383,8 @@ knitr::include_graphics("fig/results3/Clo-epg-comb.png")
knitr::include_graphics("fig/results3/clo-ind-epg.png")
```
### Effects of acetylcholine in the presence of nitenpyram and thiacloprid
### Effects of acetylcholine in the presence of nitenpyram and thiacloprid on electropharyngeogram
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.
(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).
......@@ -438,7 +438,7 @@ knitr::include_graphics("fig/results3/nit+ACH+combined.png")
knitr::include_graphics("fig/results3/thia+ACH+combined.png")
```
### Effects of nicotine on worms deficient in nAChR
### Effects of nicotine on on electropharyngeogram of 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.
......
......@@ -28,7 +28,7 @@ exp_sstms %>%
```
### Properties of vertebrate $\alpha7$ nAChR
### Properties of vertebrate $\alpha7$ nAChR ###{#propertiesofalpha7}
One of the well-studied nAChR subunit is the vertebrate homomeric $\alpha7$. Heterologous expression of these receptors in *Xenopus* occytes and mammalian cell lines.
......@@ -46,11 +46,11 @@ Single channel recordings revealed another striking difference between $\alpha7$
Heterologous expression of $\alpha7$ also allowed for a detailed analysis of the recovery kinetics for receptor channels. Following desensitisation and removal of the agonist by washing, receptor returns to the resting state, allowing for the subsequent activation upon agonist application. Although the activation and desensitisation kinetics of $\alpha7$ evoked by many agonists are almost identical, the recovery kinetics are compound, time and concentration -dependent [@mike2000]. After acetylcholine-evoked desensitisation and a 5-minute wash, the subsequent response of $\alpha7$ receptors to ACh seemed unaffected [@briggs1998]. In contrast, subsequent response to ACh following nicotine desensitisation was reduced [@briggs1998]. Thus, recovery kinetics following acetylcholine application are faster. Recovery time is also more rapid for choline than for acetylcholine [@mike2000].
#### Sensitive to $\alpha$-Bungarotoxin ($\alpha$-Bgtx)
#### Sensitive to $\alpha$-bungarotoxin ($\alpha$-bgtx)
A distinct pharmacological feature of $\alpha7$ receptors is their high sensitivity to $\alpha$-Bgtx. Interaction between $\alpha$-Bgtx and $\alpha7$ receptors was first revealed by biochemical techniques. $\alpha7$ receptor was isolated from membrane fraction of transformed bacterial cells with $\alpha$-Bgtx-affinity chromatography [@schoepfer1990]. Whereas, radiolabeled $\alpha$-Bgtx bound to $\alpha7$ receptors immunoprecipitated from the chick retina [@keyser1993]. Binding to mammalian brain receptors was also revealed by radiography of mammalian brain slices incubated with labelled $\alpha$-Bgtx [@clarke1985; @segal1978]. The signal was consistent with the expression profile of $\alpha7$ receptor, as shown by immunocytochemistry [@toro1994]. In contrast, there was no $\alpha$-Bgtx binding in mice deficient in $\alpha7$ expression [@orr-urtreger1997]. Finally, the crystal structure of $\alpha7$ receptor showed binding of $\alpha$-Bgtx to the extracellular domain of the receptor [@dellisanti2007].
A distinct pharmacological feature of $\alpha7$ receptors is their high sensitivity to $\alpha$-bgtx. Interaction between $\alpha$-bgtx and $\alpha7$ receptors was first revealed by biochemical techniques. $\alpha7$ receptor was isolated from membrane fraction of transformed bacterial cells with $\alpha$-bgtx-affinity chromatography [@schoepfer1990]. Whereas, radiolabeled $\alpha$-bgtx bound to $\alpha7$ receptors immunoprecipitated from the chick retina [@keyser1993]. Binding to mammalian brain receptors was also revealed by radiography of mammalian brain slices incubated with labelled $\alpha$-bgtx [@clarke1985; @segal1978]. The signal was consistent with the expression profile of $\alpha7$ receptor, as shown by immunocytochemistry [@toro1994]. In contrast, there was no $\alpha$-bgtx binding in mice deficient in $\alpha7$ expression [@orr-urtreger1997]. Finally, the crystal structure of $\alpha7$ receptor showed binding of $\alpha$-bgtx to the extracellular domain of the receptor [@dellisanti2007].
Electrophysiological evidence provided mechanistic details of the interaction between $\alpha$-Bgtx and $\alpha7$ receptor. Incubation with nM concentrations of $\alpha$-Bgtx prevented responses to classical nAChR agonists of heterologous receptors in *Xenopus* oocytes [@couturier1990] as well as native receptors in PC12 cells [@blumenthal1997] and hippocampal neurons [@alkondon1991]. Upon removal and wash, the receptor responds to ACh normally [@couturier1990], thus the interaction between $\alpha$-Bgtx and $\alpha7$ are of high affinity, competitive and reversible.
Electrophysiological evidence provided mechanistic details of the interaction between $\alpha$-bgtx and $\alpha7$ receptor. Incubation with nM concentrations of $\alpha$-bgtx prevented responses to classical nAChR agonists of heterologous receptors in *Xenopus* oocytes [@couturier1990] as well as native receptors in PC12 cells [@blumenthal1997] and hippocampal neurons [@alkondon1991]. Upon removal and wash, the receptor responds to ACh normally [@couturier1990], thus the interaction between $\alpha$-bgtx and $\alpha7$ are of high affinity, competitive and reversible.
<!-- Functional expression of insect receptors has been largely unsuccessful. Common problems associated with the heterologous expression of proteins in Xenopus or cell lines are low yields and incorrect processing. -->
......@@ -90,99 +90,16 @@ RIC-3 may also aid interactions with many other proteins in the cells, as shown
RIC-3 and NACHO are the two must studied proteins involved in the maturation of $\alpha7$ nAChRs, but there are also many other proteins of less defined role involved in the biogenesis of nAChRs (reviewed by @crespi2018). For example, evolutionary coserved CRELD and EMC-6, which are ubiquitonously expressed and ER membrane-bound [@dalessandro2018; @richard2013], as well as NRA-2/nicalin (nicastrin-like protein) and NRA-4/nodal modulator (NOMO) involved in the regulation of receptor stoichiometry [@almedom2009].
Taken together, *Xenopus* oocytes and eukaryotic cell lines can be used as an heterologous expression platform for vertebral nAChRs. They have been used to described the receptor maturaton, stechiometry, pharmacological and kinetic properties of vertebrate nAChRs, such as $\alpha7$. However, the expression of many invertebrate receptors in these systems has failed [@huang1999; @liu2005; @liu2009; @yixi2009; @bass2006], hindering their functional characterisation. Thus, other approaches need to be considered.
Taken together, *Xenopus* oocytes and eukaryotic cell lines can be used as an heterologous expression platform for vertebral nAChRs. They have been used to described the receptor maturation, stoichiometry, pharmacological and kinetic properties of vertebrate nAChRs, such as $\alpha7$. However, the expression of many invertebrate receptors in these systems has failed [@huang1999; @liu2005; @liu2009; @yixi2009; @bass2006], hindering their functional characterisation. Thus, other approaches need to be considered.
### *C. elegans* expresses proteins important in nAChR maturation ###{#cematnachr}
### *C. elegans* pharynx as a suitable biological system for the expression of nAChRs
Heterologous expression of receptor channels requires an appropriate cellular environment to enable receptor maturation and functioning at the cell surface. Expression of nAChRs is a complex process during which individual subunits come together, assemble into a correctly folded pentamer and are transported to the plasma membrane where they function. *C. elegans* expresses many proteins involved in these processes.
Identification and heterologous expression of *C. elegans* nAChRs greatly contributed to the understanding of proteins involved in the biogenesis of these ion channels. There are two nAChRs expressed at the NMJ of the body wall muscle of *C. elegans*: ACR-16 homopentamic receptor and a heteropentameric receptor composed of UNC-29, UNC-38, UNC-63, LEV-1 and LEV-8. These receptors are classified based on pharmacology into nicotine- and levamisole- sensitive and named N-type and L-type, respectively. DEG-3/DES-2 nAChRs are the neuronal receptors [@treinin1998], whereas EAT-2 containing nAChRs are expressed in the pharyngeal muscle [@mckay2004].
<!-- *C. elegans* is a powerfull tool in which the function and pharmacological properties of proteins can be studied by a combination of genetic, behavioural and cellular approaches (Section \@ref(genmanip) \@ref(analytical_behaviour)). -->
#### RIC-3 ####{#ric-3celegans}
RIC-3 (resistant to inhibitors of cholinesterase-3) is an evolutionary conserved, ER-residing [@roncarati2006; @alexander2010] transmembrane protein [@wang2009]. In *C. elegans*, it is ubiquitously expressed in most (if not all) neurons, pharyngeal and body wall muscle in worms [@halevi2002]. The predicted topology of *C. elegans* RIC-3 has 2 transmembrane domains and 3 coiled-coils. The *C. elegans ric-3* mutant has impaired locomotor behaviour, resistance to levamisole [@miller1996] and has an impaired nAChR responses, as measured by electrophysiological recording from the body wall muscle [@halevi2002]. The *C. elegans ric-3* mutant has impaired cholinergic nerurotransmission, as shown by the lack of the cholinergic component of the EPG recording resulting in significantly retarded pharyngeal pumping and starved appearance [@halevi2002].
Heterologous expression of *C. elegans* nAChR in *Xenopus* oocytes provides evidence for their function in receptor maturation. Choline-evoked currents of neuronal DEG-3/DES-2 receptors increased by 5-fold upon RIC-3 co-expression [@halevi2002].
This was markedly improved when acr-16 was co-expressed with RIC-3 [@ballivet1996]. The role of RIC-3 in the maturation of this receptor type was also demonstrated *in-vivo*. The nicotine induced current at the body wall muscle was markedly reduced in ric-3 mutant, in comparison to wild-type [@halevi2002].