Methods xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Measuring the effects of high CO2 levels in Caenorhabditis elegans Zuela Noam, Friedman Nurit, Zaslaver Alon, Gruenbaum Yosef ⇑ Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel
a r t i c l e
i n f o
Article history: Received 26 December 2013 Revised 5 March 2014 Accepted 7 March 2014 Available online xxxx Keywords: Avoidance C. elegans Fertility Hypercapnia Pharyngeal pumping
a b s t r a c t Carbon dioxide (CO2) is an important molecule in cell metabolism. It is also a byproduct of many physiological processes. In humans, impaired lung function and lung diseases disrupt the body’s ability to dispose of CO2 and elevate its levels in the body (hypercapnia). Animal models allow further understanding of how CO2 is sensed in the body and what are the physiological responses to high CO2 levels. This information can provide new strategies in the battle against the detrimental effects of CO2 accumulation in lung diseases. The nematode Caenorhabditis elegans provides us with such a model animal due to its natural ability to sense and navigate through varying concentrations of CO2, as well as the fact that it can be genetically manipulated with ease. Here we describe the different methods used to measure the effects elevated levels of CO2 have on the molecular sensing mechanism and physiology of C. elegans. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
1.2. High CO2 levels in flies
Maintaining biosphere homeostasis, buffering pH and being involved in cell signaling in most organisms are only a few of the many roles attributed to carbon dioxide. In humans, its normal levels in the blood range from 20 to 29 milliequivalent per liter of blood (5%). An increase in CO2 blood levels (hypercapnia) causes a host of symptoms, which include elevated blood pressure, flushed skin, muscle twitches, headaches, lethargy and reduced brain and nerve function [14]. Prolonged elevation of CO2 in the blood leads to internal organ damage.
CO2 can be sensed by a subpopulation of olfactory sensory neurons that express the gustatory receptors Gr21a and Gr63a [4,9]. In addition, the existence of a non-neuronal CO2-sensing system was demonstrated by showing that in CO2 levels as low as 7%, both wild-type and adult flies lacking the Gr63a receptor become immune suppressed and lay fewer eggs [8]. Further, 13% CO2 suppresses the expression of anti-microbial peptides in the immuneresponsive S2 cell line. This non-neuronal CO2 response pathway appears to be a novel response pathway because its activity is independent of pH, nitrogen monoxide (NO), heat shock and hypoxia responses.
1.1. High CO2 levels in disease states 1.3. High CO2 levels in C. elegans An elevation in the levels of CO2 in the blood is present in many lung diseases, including Chronic Obstructive Pulmonary Disease (COPD), Asthma and Acute Respiratory Distress Syndrome (ARDS), and is correlated with a rapid deterioration and elevated mortality rates of these patients. Patients with COPD show a reduction in muscle mass, peripheral muscle dysfunction as well as increased proteolysis [10,11]. These phenotypes could, in part, be a direct result of exposure to elevated levels of CO2, as chronic exposure of Caenorhabditis elegans to elevated levels of CO2 results in muscle deterioration and fiber disorganization [13]. ⇑ Corresponding author. Fax: +972 2 5637848. E-mail address: [email protected] (Y. Gruenbaum).
Chronic exposure to CO2 levels above 5% causes reduced fertility, a slower developmental rate, impaired motility, muscle deterioration and changes in mitochondria structure. The severity of these phenotypes correlates with the increase in CO2 concentrations that the worms are exposed to [13]. In addition, accute exposure of C. elegans to higher CO2 levels causes a reduction in the pharyngeal pumping rate, which returns to normal when exposing the animals back to air. The similarity of phenotypes between humans and model organisms such as Drosophila and C. elegans suggest that lower organisms are useful models for the study of human pulmonary diseases. Acute exposure of C. elegans at a threshold concentration of 0.5– 1% causes their retraction from the source of CO2 (also known as
http://dx.doi.org/10.1016/j.ymeth.2014.03.008 1046-2023/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: N. Zuela et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.03.008
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N. Zuela et al. / Methods xxx (2014) xxx–xxx
CO2 avoidance) with no dependence on pH. The avoidance behavior is mediated by the BAG neurons [1,2,5,6,7]. Tax-2 and tax-4 are genes encoding subunits of the cGMP gated ion channel and are involved in C. elegans sensing of CO2. Expression of tax-4 in the BAG neurons alone is sufficient to rescue the CO2 avoidance defect present in the tax-4 mutants [7]. CO2 sensing is also regulated by metabolism; Animals defective in key regulators of metabolism, such as DAF-2, IGF-1 or TGF-b, or starved animals do not avoid high CO2 levels [1,5]. Feeding behavior varies between different strains of C. elegans and is linked to CO2 avoidance. Animals with solitary feeding strongly avoid CO2, whereas animals with social feeding do not [5]. Eliminating npr1, a neuropeptide Y receptor, or glb-5 genetically alters the worms feeding behavior from solitary to social, therefore changing their avoidance response. These genes are also involved in C. elegans response to oxygen, suggesting a shared pathway for sensing CO2 and O2 [1,7,12].
1.4. High CO2 levels affect global gene expression Some of the phenotypes mentioned above are accompanied by profound, specific and dynamic changes in transcription [13]. One hour after exposure to 19% CO2 there is a >2-fold up-regulation in 429 genes and down regulation in 59 genes. These genes are most likely responsible for coordinating the initial response to high CO2 levels. After 6 h of exposure to 19% CO2, 374 genes are up-regulated and 283 down-regulated and after 72 h, over 6% of C. elegans genes show at least a 2-fold change in their expression profile.
1.5. High CO2 levels can affect lifespan Exposure of C. elegans to elevated levels of CO2 also has an unexpected result; it significantly extends the lifespan of C. elegans. This phenomenon probably occurs independent of the IGF-1 pathway, mitochondria-regulated aging, fertility or food deprivation [13]. This review will bring to light the various methods that are used to study the different physiological and molecular effects of acute and prolonged exposure to CO2 on the nematode C. elegans.
2. Chronic exposure of C. elegans to elevated levels of CO2 2.1. The experimental setup of chronic exposure of C. elegans to elevated levels of CO2 In order to achieve chronic exposure of worms to elevated CO2 levels, worms are incubated in a Perspex chamber located in a room with a controlled temperature. The levels of CO2 in the chamber are controlled using a DYNAMENT CO2 controller (United Kingdom) equipped with a mini infrared sensor, which can sense CO2 concentrations up to 20%. The CO2 controller has 2 inlets, one that is connected to a 100% CO2 tank, and another connected to an internal air pump that pumps air from the environment. CO2 enters the chamber until the desired level is reached, if CO2 levels increase they are balanced by air pumped into the chamber. Since CO2 is heavier than air, which can lead to a CO2 gradient in the chamber, a fan is located at the bottom of the Perspex chamber (termed from now on the ‘‘CO2 chamber’’) to allow for a uniform concentration of gases throughout the chamber (Fig. 1). 2.2. Measurement of fecundity under varying concentrations of CO2 These experiments are aimed at measuring the fertility of worms in response to prolonged exposure to varying concentrations of CO2 (Fig. 2A). Nematode growth media (NGM) plates are seeded a day or two before the start of the experiment with 50 ll of an overnight culture of Escherichia coli OP50 bacteria (NGM/OP50) (for further information see http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html). To obtain embryos, gravid adult worms are bleached using a fresh commercial bleach solution (0.5 N NaOH, 0.5% sodium hypochlorite) for 10 min and washed several times with M9. This procedure dissolves adult worms and larvae; but does not affect the eggs. The isolated embryos are grown on NGM/OP50 plates in the CO2 chamber (Fig. 1) at the desired CO2 concentrations until they reach the late L4 stage. Single L4 animals are then transferred, each to a separate NGM/OP50 plate, and placed back in the CO2 chamber. A typical experiment consists of at least 30 animals per genotype tested. During the first 3 days, each worm is transferred daily to a fresh plate, while keeping the older plates in the chamber. On the third day, the worms are given 24 h to lay eggs and then discarded. Due to the fact that high CO2 levels can cause a delay in egg laying and
Fig. 1. An experimental setup for prolonged exposure of C. elegans to elevated CO2 levels. (A) Schematic representation of the setup. A transparent Perspex chamber is used to keep the worms at the desired CO2 concentration. The chamber is kept in a temperature-controlled room and connected to a DYNAMENT CO2 controller (2), which has an infrared sensor (4) that detects CO2 concentrations of up to 20%. The infrared sensor is located on the side panel of the Perspex chamber, and can detect the CO2 concentration in the incubator, which it then reports to the controller. CO2 is flowed into the controller from a CO2 gas tank (1), and in order to achieve the desired CO2 concentration it is balanced with normal air, which is flowed into the controller via an air pump (3). The air mixture containing the desired CO2 concentration is then flowed into the chamber. On the bottom of the Perspex chamber there is a fan (5), which is used to mix the air inside the chamber. As CO2 is a heavy gas this helps to maintain a uniform concentration of CO2 throughout the Perspex chamber. (B) Photograph of the system for prolonged exposure of C. elegans to CO2.
Please cite this article in press as: N. Zuela et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.03.008
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Fig. 2. Phenotypes associated with chronic exposure of C. elegans to high CO2 levels. (A) Exposure of wildtype C. elegans to varying CO2 concentrations results in reduced fertility. Worms were maintained at 20 or 25 °C and experiment performed as described [13], ⁄P < 0.05; ⁄⁄⁄P < 0.0005. (B) Exposure of wild-type C. elegans to 19% CO2 results in permanent reduction in motility. Worms were grown in 20 °C and analyzed at the L4 stage either on NGM plates (n = 21) or a drop of ddH2O (n = 21) as described [13]. Average head movements/minute of worms grown in 19% CO2 was divided by average head movements/minute of animals grown in air. (C) Exposure of wild-type C. elegans to 19% CO2 results in a slower developmental rate. (D) Exposure of wild-type C. elegans to 19% CO2 results in the significant (P < 0.0001) extension of their average life span. Continuous line represents worms grown at 20 °C in normal air, broken line = worms grown at 20 °C in 19% CO2, continuous line with squares represents worms grown at 25 °C in normal air, broken line with squares represents worms grown at 25 °C in 19% CO2. (E) Exposure of wild-type C. elegans to 19% CO2 results in deterioration and misorganization of muscle fibers which gradually worsen with exposure time, as demonstrated by thin-section electron micrographs (Scale bars, 500 nm).
to follow the development of the laid eggs into larvae, the progeny are counted only 48–72 h after the worm is removed from the plate, compared to the typical 24–48 h commonly used in other protocols. The progeny are scored as the mean number of laid eggs per worm. Progeny survival rate is calculated as the mean of the percentage of eggs that developed into larvae from the total eggs laid. Fecundity experiments are usually performed at 20 °C. During the experiment, the worms are briefly removed from the CO2 chamber just for the purpose of transferring them to new plates or for counting the progeny. In control experiment a similar number of single worms are grown at the same temperature in the same temperature room/incubator outside the CO2 chamber (air control).
synchronization of the worms is achieved by bleaching adult worms and mildly shaking the isolated embryos over night in a tube containing M9 buffer at 20 °C until they reach the L1 stage. In order to obtain statistically significant results, each experiment consists of at least 90 worms grown in 3 plates. The developmental stage of the worms is assessed daily under a high quality binocular microscope until the worms start to lay eggs. The effect of high CO2 on the developmental rate is normally studied either at 20 or at 25 °C, while the worms are incubated in the CO2 chamber. The worms are briefly removed from the chamber only for the purpose of scoring their developmental stage. Again, worms grown next to the CO2 chamber in air serve as control.
2.3. Measurement of motility under varying concentrations of CO2
2.5. Measurement of lifespan under varying concentrations of CO2
In order to measure the motility of worms following chronic (>24 h) exposure to varying concentrations of CO2 (Fig. 2B), adult worms are bleached and the isolated embryos are grown under the desired CO2 concentration in the CO2 chamber until they reach the desired stage of development. Normally, 30 worms at the L4 stage are placed each on a separate NGM/OP50 plate or in a drop of M9 buffer (for preparation instructions see http://www. wormbook.org/chapters/www_strainmaintain/strainmaintain. html). These worms are subsequently filmed using an Axiocam CCD webcam mounted on an Olympus binocular microscope. Motility is counted as the number of bends of the worm’s body, just behind the pharynx, in the opposite direction of the former movement. Bends can be counted with a manual cell counter or analyzed using movies and WormTracker (http://www.mrc-lmb. cam.ac.uk/wormtracker/). Motility experiments are normally performed at 20 °C. The worms are briefly removed from the chamber for the purpose of counting their movements. The controls for these experiments are measurements of motility of worms grown next to the CO2 chamber in air.
These experiments measure C. elegans lifespan under continuous exposure to CO2 levels of 15% and above (Fig. 2D). Adult worms are bleached and the isolated embryos are placed on NGM/OP50 plates in the CO2 chamber at the desired CO2 concentration and grow until they reach the late L4 stage. Normally, 90 animals at the L4 stage are transferred to 3 NGM/OP50 plates (30 worms per plate). Initially, the worms are transferred daily to new NGM/ OP50 plates until they no longer lay eggs. Animal that die due to the transfer are discarded from the experiment. Viability is scored as worm movement. Worms that do not move are prodded with a platinum wire. Worms that do not respond to the prodding are considered dead. The experiments continue until all worms die. Lifespan experiments are normally performed at 20 °C while the worms are incubated in the CO2 chamber, and briefly removed only for the purpose of scoring their viability. The control for this experiment is to use the same number of worms grown next to the CO2 chamber in normal air.
2.4. Measurement of developmental rates under varying concentrations of CO2 In order to measure the rate of development of C. elegans under prolonged exposure to varying CO2 concentrations (Fig. 2C), synchronized L1 stage worms are placed on NGM/OP50 plates under the desired CO2 concentration in the CO2 chamber. The
2.6. Electron microscopy morphology analysis of C. elegans exposed to high levels of CO2 Adult worms are bleached and the resulting embryos are grown on NGM/OP50 plates under the desired CO2 concentrations in the CO2 chamber until they reach the desired stage. Preparation of worms for electron microscopy analysis is performed as described [3]. Worms from different developmental days are collected from the NGM plates using phosphate buffer saline (PBS) and washed
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twice in PBS. In order to allow for maximal penetration of the various solution used in this protocol, animals are cut behind the tail or the pharynx with sharp forceps. The nicked animals are then immediately fixed overnight at 4 °C in PBS (pH 7.5) containing 2.5% paraformaldehyde and 2.5% glutaraldehyde. The following day animals are washed three times with PBS. In order to maintain horizontal positioning of the worm in the final block of Epon, animals are mounted in 3.4% low melting point agarose (Sigma) near its solidifying point (30 °C). After setting ON at 4 °C, 2 mm3 agarose blocks carrying the animals are cut from the solidified gel. These agarose blocks are incubated in a 0.1 M sodium cacodylate (pH 7.5) solution for 10 min at 22 °C followed by 1 h in a 0.1 M sodium cacodylate based solution consisting 1% OsO4 and 1.5% reduced K4Fe(CN)6. The next step is a 10 min wash of the agarose blocks, four times with a 0.1 M sodium cacodylate solution. The samples are washed twice again with double distilled H2O for 10 min, then incubated for an additional 10 min in 30%, 50%, 70%, 80%, 90%, and 95% ethanol followed by three 20 min washes in 100% ethanol. The agarose blocks are then moved to a propylene oxide solution for two additional 10 min washes. The next step is to incubate the agarose samples in a series of propylene oxide based solutions containing scaled amounts of Epon (1:4, 1:1, 1:3) overnight each at 4 °C followed by an overnight incubation in fresh 100% Epon at 4 °C. The newly formed Epon blocks are left to polymerize for 3 days at 60 °C. The Epon blocks are then sectioned horizontally using a microtome with a Diatome diamond knife to give 80– 90 nm thick slices with longitudinal head to tail view of each worm. The sections are mounted on 200 mesh thin bar copper grids and stained with Uranyl acetate and lead citrate. An electron microscope equipped with a charge-coupled device camera is used to view the mounted grids. The controls for this experiment are worms from the same developmental stages grown in air. An example of adult muscle cells exposed to 19% CO2 is shown in Fig. 2E.
gas. Before entering the chamber the gas flows through water for humidity. A mechanical valve controls the entrance of the gas mixture into the chamber. An outlet allows air to flow out of the chamber. The increase in CO2 concentration in the gas mixture comes at the expense of the nitrogen concentration, while keeping a constant concentration of 21% O2. 3.2. Measurement of pharyngeal pumping rates under varying concentrations of CO2 NGM plates are seeded 5 h before the start of the experiment with 20 ll of E. coli OP50 placed in the middle of the plate. While allowing normal feeding, the small bacterial lawn restricts the worms to a small area on the plate, which allows an easier microscopic analysis. Normally, pumping is measured one day after the worms have reached the L4 stage. Individual worms are placed on the NGM plate covered by a lid chamber (Fig. 3). To allow adjustment to gas flow, normal air mixture (21% O2, 79% N2) is flowed for 1 min into the lid shaped chamber. Next, the number of pharynx muscle contractions in air is counted for 1 min under a binocular microscope. Then, the air mixture is switched to a gas mixture containing the desired CO2 concentration. The worm is then allowed 10–15 s of adjustment and the pharynx muscle contractions are measured for 1 min in the gas mixture. The experiment is performed one worm at a time. A typical experiment consists of at least 30 worms. In order to measure the pumping rate of food-deprived worms, well-fed early adult worms are collected and washed 5 times with M9 buffer. Individual worms are subsequently placed each on a separate NGM plate with no bacteria for a period of 4 h. Control worms are placed on NGM/OP50 plates. Pumping measurements of individual worms are performed as described above. Pumping assays are usually performed at 22 °C. 3.3. Measurement of motility under varying concentrations of CO2
3. Acute exposure of C. elegans to elevated levels of CO2
3.1. A system of acute C. elegans exposure to CO2 In order to expose C. elegans worms to rapid changes in CO2 concentrations and to measure their response, worms are placed on a NGM plate and covered with a lid shape chamber (Fig. 3). The chamber is connected via inlet to a tank filled with pre-mixed
In order to measure the motility of worms following acute exposure (
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Measuring the effects of high CO2 levels in Caenorhabditis elegans Zuela Noam, Friedman Nurit, Zaslaver Alon, Gruenbaum Yosef ⇑ Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel
a r t i c l e
i n f o
Article history: Received 26 December 2013 Revised 5 March 2014 Accepted 7 March 2014 Available online xxxx Keywords: Avoidance C. elegans Fertility Hypercapnia Pharyngeal pumping
a b s t r a c t Carbon dioxide (CO2) is an important molecule in cell metabolism. It is also a byproduct of many physiological processes. In humans, impaired lung function and lung diseases disrupt the body’s ability to dispose of CO2 and elevate its levels in the body (hypercapnia). Animal models allow further understanding of how CO2 is sensed in the body and what are the physiological responses to high CO2 levels. This information can provide new strategies in the battle against the detrimental effects of CO2 accumulation in lung diseases. The nematode Caenorhabditis elegans provides us with such a model animal due to its natural ability to sense and navigate through varying concentrations of CO2, as well as the fact that it can be genetically manipulated with ease. Here we describe the different methods used to measure the effects elevated levels of CO2 have on the molecular sensing mechanism and physiology of C. elegans. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
1.2. High CO2 levels in flies
Maintaining biosphere homeostasis, buffering pH and being involved in cell signaling in most organisms are only a few of the many roles attributed to carbon dioxide. In humans, its normal levels in the blood range from 20 to 29 milliequivalent per liter of blood (5%). An increase in CO2 blood levels (hypercapnia) causes a host of symptoms, which include elevated blood pressure, flushed skin, muscle twitches, headaches, lethargy and reduced brain and nerve function [14]. Prolonged elevation of CO2 in the blood leads to internal organ damage.
CO2 can be sensed by a subpopulation of olfactory sensory neurons that express the gustatory receptors Gr21a and Gr63a [4,9]. In addition, the existence of a non-neuronal CO2-sensing system was demonstrated by showing that in CO2 levels as low as 7%, both wild-type and adult flies lacking the Gr63a receptor become immune suppressed and lay fewer eggs [8]. Further, 13% CO2 suppresses the expression of anti-microbial peptides in the immuneresponsive S2 cell line. This non-neuronal CO2 response pathway appears to be a novel response pathway because its activity is independent of pH, nitrogen monoxide (NO), heat shock and hypoxia responses.
1.1. High CO2 levels in disease states 1.3. High CO2 levels in C. elegans An elevation in the levels of CO2 in the blood is present in many lung diseases, including Chronic Obstructive Pulmonary Disease (COPD), Asthma and Acute Respiratory Distress Syndrome (ARDS), and is correlated with a rapid deterioration and elevated mortality rates of these patients. Patients with COPD show a reduction in muscle mass, peripheral muscle dysfunction as well as increased proteolysis [10,11]. These phenotypes could, in part, be a direct result of exposure to elevated levels of CO2, as chronic exposure of Caenorhabditis elegans to elevated levels of CO2 results in muscle deterioration and fiber disorganization [13]. ⇑ Corresponding author. Fax: +972 2 5637848. E-mail address: [email protected] (Y. Gruenbaum).
Chronic exposure to CO2 levels above 5% causes reduced fertility, a slower developmental rate, impaired motility, muscle deterioration and changes in mitochondria structure. The severity of these phenotypes correlates with the increase in CO2 concentrations that the worms are exposed to [13]. In addition, accute exposure of C. elegans to higher CO2 levels causes a reduction in the pharyngeal pumping rate, which returns to normal when exposing the animals back to air. The similarity of phenotypes between humans and model organisms such as Drosophila and C. elegans suggest that lower organisms are useful models for the study of human pulmonary diseases. Acute exposure of C. elegans at a threshold concentration of 0.5– 1% causes their retraction from the source of CO2 (also known as
http://dx.doi.org/10.1016/j.ymeth.2014.03.008 1046-2023/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: N. Zuela et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.03.008
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N. Zuela et al. / Methods xxx (2014) xxx–xxx
CO2 avoidance) with no dependence on pH. The avoidance behavior is mediated by the BAG neurons [1,2,5,6,7]. Tax-2 and tax-4 are genes encoding subunits of the cGMP gated ion channel and are involved in C. elegans sensing of CO2. Expression of tax-4 in the BAG neurons alone is sufficient to rescue the CO2 avoidance defect present in the tax-4 mutants [7]. CO2 sensing is also regulated by metabolism; Animals defective in key regulators of metabolism, such as DAF-2, IGF-1 or TGF-b, or starved animals do not avoid high CO2 levels [1,5]. Feeding behavior varies between different strains of C. elegans and is linked to CO2 avoidance. Animals with solitary feeding strongly avoid CO2, whereas animals with social feeding do not [5]. Eliminating npr1, a neuropeptide Y receptor, or glb-5 genetically alters the worms feeding behavior from solitary to social, therefore changing their avoidance response. These genes are also involved in C. elegans response to oxygen, suggesting a shared pathway for sensing CO2 and O2 [1,7,12].
1.4. High CO2 levels affect global gene expression Some of the phenotypes mentioned above are accompanied by profound, specific and dynamic changes in transcription [13]. One hour after exposure to 19% CO2 there is a >2-fold up-regulation in 429 genes and down regulation in 59 genes. These genes are most likely responsible for coordinating the initial response to high CO2 levels. After 6 h of exposure to 19% CO2, 374 genes are up-regulated and 283 down-regulated and after 72 h, over 6% of C. elegans genes show at least a 2-fold change in their expression profile.
1.5. High CO2 levels can affect lifespan Exposure of C. elegans to elevated levels of CO2 also has an unexpected result; it significantly extends the lifespan of C. elegans. This phenomenon probably occurs independent of the IGF-1 pathway, mitochondria-regulated aging, fertility or food deprivation [13]. This review will bring to light the various methods that are used to study the different physiological and molecular effects of acute and prolonged exposure to CO2 on the nematode C. elegans.
2. Chronic exposure of C. elegans to elevated levels of CO2 2.1. The experimental setup of chronic exposure of C. elegans to elevated levels of CO2 In order to achieve chronic exposure of worms to elevated CO2 levels, worms are incubated in a Perspex chamber located in a room with a controlled temperature. The levels of CO2 in the chamber are controlled using a DYNAMENT CO2 controller (United Kingdom) equipped with a mini infrared sensor, which can sense CO2 concentrations up to 20%. The CO2 controller has 2 inlets, one that is connected to a 100% CO2 tank, and another connected to an internal air pump that pumps air from the environment. CO2 enters the chamber until the desired level is reached, if CO2 levels increase they are balanced by air pumped into the chamber. Since CO2 is heavier than air, which can lead to a CO2 gradient in the chamber, a fan is located at the bottom of the Perspex chamber (termed from now on the ‘‘CO2 chamber’’) to allow for a uniform concentration of gases throughout the chamber (Fig. 1). 2.2. Measurement of fecundity under varying concentrations of CO2 These experiments are aimed at measuring the fertility of worms in response to prolonged exposure to varying concentrations of CO2 (Fig. 2A). Nematode growth media (NGM) plates are seeded a day or two before the start of the experiment with 50 ll of an overnight culture of Escherichia coli OP50 bacteria (NGM/OP50) (for further information see http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html). To obtain embryos, gravid adult worms are bleached using a fresh commercial bleach solution (0.5 N NaOH, 0.5% sodium hypochlorite) for 10 min and washed several times with M9. This procedure dissolves adult worms and larvae; but does not affect the eggs. The isolated embryos are grown on NGM/OP50 plates in the CO2 chamber (Fig. 1) at the desired CO2 concentrations until they reach the late L4 stage. Single L4 animals are then transferred, each to a separate NGM/OP50 plate, and placed back in the CO2 chamber. A typical experiment consists of at least 30 animals per genotype tested. During the first 3 days, each worm is transferred daily to a fresh plate, while keeping the older plates in the chamber. On the third day, the worms are given 24 h to lay eggs and then discarded. Due to the fact that high CO2 levels can cause a delay in egg laying and
Fig. 1. An experimental setup for prolonged exposure of C. elegans to elevated CO2 levels. (A) Schematic representation of the setup. A transparent Perspex chamber is used to keep the worms at the desired CO2 concentration. The chamber is kept in a temperature-controlled room and connected to a DYNAMENT CO2 controller (2), which has an infrared sensor (4) that detects CO2 concentrations of up to 20%. The infrared sensor is located on the side panel of the Perspex chamber, and can detect the CO2 concentration in the incubator, which it then reports to the controller. CO2 is flowed into the controller from a CO2 gas tank (1), and in order to achieve the desired CO2 concentration it is balanced with normal air, which is flowed into the controller via an air pump (3). The air mixture containing the desired CO2 concentration is then flowed into the chamber. On the bottom of the Perspex chamber there is a fan (5), which is used to mix the air inside the chamber. As CO2 is a heavy gas this helps to maintain a uniform concentration of CO2 throughout the Perspex chamber. (B) Photograph of the system for prolonged exposure of C. elegans to CO2.
Please cite this article in press as: N. Zuela et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.03.008
N. Zuela et al. / Methods xxx (2014) xxx–xxx
3
Fig. 2. Phenotypes associated with chronic exposure of C. elegans to high CO2 levels. (A) Exposure of wildtype C. elegans to varying CO2 concentrations results in reduced fertility. Worms were maintained at 20 or 25 °C and experiment performed as described [13], ⁄P < 0.05; ⁄⁄⁄P < 0.0005. (B) Exposure of wild-type C. elegans to 19% CO2 results in permanent reduction in motility. Worms were grown in 20 °C and analyzed at the L4 stage either on NGM plates (n = 21) or a drop of ddH2O (n = 21) as described [13]. Average head movements/minute of worms grown in 19% CO2 was divided by average head movements/minute of animals grown in air. (C) Exposure of wild-type C. elegans to 19% CO2 results in a slower developmental rate. (D) Exposure of wild-type C. elegans to 19% CO2 results in the significant (P < 0.0001) extension of their average life span. Continuous line represents worms grown at 20 °C in normal air, broken line = worms grown at 20 °C in 19% CO2, continuous line with squares represents worms grown at 25 °C in normal air, broken line with squares represents worms grown at 25 °C in 19% CO2. (E) Exposure of wild-type C. elegans to 19% CO2 results in deterioration and misorganization of muscle fibers which gradually worsen with exposure time, as demonstrated by thin-section electron micrographs (Scale bars, 500 nm).
to follow the development of the laid eggs into larvae, the progeny are counted only 48–72 h after the worm is removed from the plate, compared to the typical 24–48 h commonly used in other protocols. The progeny are scored as the mean number of laid eggs per worm. Progeny survival rate is calculated as the mean of the percentage of eggs that developed into larvae from the total eggs laid. Fecundity experiments are usually performed at 20 °C. During the experiment, the worms are briefly removed from the CO2 chamber just for the purpose of transferring them to new plates or for counting the progeny. In control experiment a similar number of single worms are grown at the same temperature in the same temperature room/incubator outside the CO2 chamber (air control).
synchronization of the worms is achieved by bleaching adult worms and mildly shaking the isolated embryos over night in a tube containing M9 buffer at 20 °C until they reach the L1 stage. In order to obtain statistically significant results, each experiment consists of at least 90 worms grown in 3 plates. The developmental stage of the worms is assessed daily under a high quality binocular microscope until the worms start to lay eggs. The effect of high CO2 on the developmental rate is normally studied either at 20 or at 25 °C, while the worms are incubated in the CO2 chamber. The worms are briefly removed from the chamber only for the purpose of scoring their developmental stage. Again, worms grown next to the CO2 chamber in air serve as control.
2.3. Measurement of motility under varying concentrations of CO2
2.5. Measurement of lifespan under varying concentrations of CO2
In order to measure the motility of worms following chronic (>24 h) exposure to varying concentrations of CO2 (Fig. 2B), adult worms are bleached and the isolated embryos are grown under the desired CO2 concentration in the CO2 chamber until they reach the desired stage of development. Normally, 30 worms at the L4 stage are placed each on a separate NGM/OP50 plate or in a drop of M9 buffer (for preparation instructions see http://www. wormbook.org/chapters/www_strainmaintain/strainmaintain. html). These worms are subsequently filmed using an Axiocam CCD webcam mounted on an Olympus binocular microscope. Motility is counted as the number of bends of the worm’s body, just behind the pharynx, in the opposite direction of the former movement. Bends can be counted with a manual cell counter or analyzed using movies and WormTracker (http://www.mrc-lmb. cam.ac.uk/wormtracker/). Motility experiments are normally performed at 20 °C. The worms are briefly removed from the chamber for the purpose of counting their movements. The controls for these experiments are measurements of motility of worms grown next to the CO2 chamber in air.
These experiments measure C. elegans lifespan under continuous exposure to CO2 levels of 15% and above (Fig. 2D). Adult worms are bleached and the isolated embryos are placed on NGM/OP50 plates in the CO2 chamber at the desired CO2 concentration and grow until they reach the late L4 stage. Normally, 90 animals at the L4 stage are transferred to 3 NGM/OP50 plates (30 worms per plate). Initially, the worms are transferred daily to new NGM/ OP50 plates until they no longer lay eggs. Animal that die due to the transfer are discarded from the experiment. Viability is scored as worm movement. Worms that do not move are prodded with a platinum wire. Worms that do not respond to the prodding are considered dead. The experiments continue until all worms die. Lifespan experiments are normally performed at 20 °C while the worms are incubated in the CO2 chamber, and briefly removed only for the purpose of scoring their viability. The control for this experiment is to use the same number of worms grown next to the CO2 chamber in normal air.
2.4. Measurement of developmental rates under varying concentrations of CO2 In order to measure the rate of development of C. elegans under prolonged exposure to varying CO2 concentrations (Fig. 2C), synchronized L1 stage worms are placed on NGM/OP50 plates under the desired CO2 concentration in the CO2 chamber. The
2.6. Electron microscopy morphology analysis of C. elegans exposed to high levels of CO2 Adult worms are bleached and the resulting embryos are grown on NGM/OP50 plates under the desired CO2 concentrations in the CO2 chamber until they reach the desired stage. Preparation of worms for electron microscopy analysis is performed as described [3]. Worms from different developmental days are collected from the NGM plates using phosphate buffer saline (PBS) and washed
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twice in PBS. In order to allow for maximal penetration of the various solution used in this protocol, animals are cut behind the tail or the pharynx with sharp forceps. The nicked animals are then immediately fixed overnight at 4 °C in PBS (pH 7.5) containing 2.5% paraformaldehyde and 2.5% glutaraldehyde. The following day animals are washed three times with PBS. In order to maintain horizontal positioning of the worm in the final block of Epon, animals are mounted in 3.4% low melting point agarose (Sigma) near its solidifying point (30 °C). After setting ON at 4 °C, 2 mm3 agarose blocks carrying the animals are cut from the solidified gel. These agarose blocks are incubated in a 0.1 M sodium cacodylate (pH 7.5) solution for 10 min at 22 °C followed by 1 h in a 0.1 M sodium cacodylate based solution consisting 1% OsO4 and 1.5% reduced K4Fe(CN)6. The next step is a 10 min wash of the agarose blocks, four times with a 0.1 M sodium cacodylate solution. The samples are washed twice again with double distilled H2O for 10 min, then incubated for an additional 10 min in 30%, 50%, 70%, 80%, 90%, and 95% ethanol followed by three 20 min washes in 100% ethanol. The agarose blocks are then moved to a propylene oxide solution for two additional 10 min washes. The next step is to incubate the agarose samples in a series of propylene oxide based solutions containing scaled amounts of Epon (1:4, 1:1, 1:3) overnight each at 4 °C followed by an overnight incubation in fresh 100% Epon at 4 °C. The newly formed Epon blocks are left to polymerize for 3 days at 60 °C. The Epon blocks are then sectioned horizontally using a microtome with a Diatome diamond knife to give 80– 90 nm thick slices with longitudinal head to tail view of each worm. The sections are mounted on 200 mesh thin bar copper grids and stained with Uranyl acetate and lead citrate. An electron microscope equipped with a charge-coupled device camera is used to view the mounted grids. The controls for this experiment are worms from the same developmental stages grown in air. An example of adult muscle cells exposed to 19% CO2 is shown in Fig. 2E.
gas. Before entering the chamber the gas flows through water for humidity. A mechanical valve controls the entrance of the gas mixture into the chamber. An outlet allows air to flow out of the chamber. The increase in CO2 concentration in the gas mixture comes at the expense of the nitrogen concentration, while keeping a constant concentration of 21% O2. 3.2. Measurement of pharyngeal pumping rates under varying concentrations of CO2 NGM plates are seeded 5 h before the start of the experiment with 20 ll of E. coli OP50 placed in the middle of the plate. While allowing normal feeding, the small bacterial lawn restricts the worms to a small area on the plate, which allows an easier microscopic analysis. Normally, pumping is measured one day after the worms have reached the L4 stage. Individual worms are placed on the NGM plate covered by a lid chamber (Fig. 3). To allow adjustment to gas flow, normal air mixture (21% O2, 79% N2) is flowed for 1 min into the lid shaped chamber. Next, the number of pharynx muscle contractions in air is counted for 1 min under a binocular microscope. Then, the air mixture is switched to a gas mixture containing the desired CO2 concentration. The worm is then allowed 10–15 s of adjustment and the pharynx muscle contractions are measured for 1 min in the gas mixture. The experiment is performed one worm at a time. A typical experiment consists of at least 30 worms. In order to measure the pumping rate of food-deprived worms, well-fed early adult worms are collected and washed 5 times with M9 buffer. Individual worms are subsequently placed each on a separate NGM plate with no bacteria for a period of 4 h. Control worms are placed on NGM/OP50 plates. Pumping measurements of individual worms are performed as described above. Pumping assays are usually performed at 22 °C. 3.3. Measurement of motility under varying concentrations of CO2
3. Acute exposure of C. elegans to elevated levels of CO2
3.1. A system of acute C. elegans exposure to CO2 In order to expose C. elegans worms to rapid changes in CO2 concentrations and to measure their response, worms are placed on a NGM plate and covered with a lid shape chamber (Fig. 3). The chamber is connected via inlet to a tank filled with pre-mixed
In order to measure the motility of worms following acute exposure (
