Team:SUSTech Shenzhen/Design2
Caenorhabditis elegans, a widely used model organism, is the objective in our project, because its whole genome has been sequenced, and it is also the only organism to have its connectome(neuronal ‘wiring diagram’) completed by 2012 [1][2]. Adault C.elegans is about 1mm in length, 45μm in wideth and it is easily observed and manipulated under stereomicroscope[3][4].What more, C.elegans has a phenomenon known as eutely, which means it has fixed ‘genetically determined number of cells’[5].These all are of great help when we doing neural operation.
Compared with other model organism like Mus musculus, which needs approximately two to three months to get matured, C.elegans possesses shorter lifespan. It is a self-fertilizing hermaphrodite lives for two to three weeks[6]. Lifespan-regulating genes in C.elegans,2,16010.Whithin this cycle the genetics is immutable. At the same time, it can also propagate sexually, which means that the genetic phenotype of it is easy to control.
Fig1. The Connectome of C.elegans.
Normally, the formation of a new ability is corresponding with the specific environment signal induc-ing, so in our project we need the specific stimulation to train worms and let them obtain new abilities. In C.elegans, we hope that the stimulation can be localized to the target neurons for the accuracy and efficiency. Different behaviors of the worms are controlled by different neurons, and due to their sensitivity to odors, many behaviors, such as attraction, avoidance, feeding, or mating, can be induced by different chemicals. In our project, we choose the olfactory neurons ( AWA &AWB) to accomplish our design. The attrac-tive odorant diacetyl is detected by the receptor protein ODR- 10, which is normally expressed in the AWA olfactory neurons. The repulsive odorant 2-nonanone is detected by the AWB olfactory neu-rons. With the expression of CoChR and Chrimson in these neurons, worms can get attractive and repulsive response to blue light and red light, so we can use lights to induce them to learn new behav-iors.
Skinner Box is a laboratory apparatus used to study animal behavior invented by Burrhus Frederic Skinner(1904—1990), and it uncloses the secret of the operant behavior. Skinner’s study deeply influenced American education at that time. He divided the behaviors into two types: respondent behavior which induced by some known stimulus like Pavlov's dog, and another is operant behavior caused by a subject itself like Skinner's pigeon. In our life, most behaviors or reflects are operant behaviors and operant reflects, so it’s very important to study the mechanism of Skinner box. The structure forming the shell of it is a chamber large enough to easily accommodate the animal being used as a subject. (Commonly used model animals include rodents—usually lab rats—pigeons, and primates). An operant conditioning chamber permits experimenters to study behavior conditioning (training) by teaching a subject animal to perform certain actions (like pressing a lever) in response to specific stimuli, such as a light or sound signal.[7][8] There are several manipulandums in the box, which can automatically detect the occurrence of a behavioral response or action. Typical operands for primates and rats are response levers; if the subject presses the lever, the opposite end moves and closes a switch that is monitored by a computer or other programmed device. When the lever is pressed, food, water, or some other type of reinforcement (a consequence that will strengthen an organism's future behavior whenever that behavior is preceded by a specific antecedent stimulus.) might be dispensed. In some instances, the floor of the chamber may be electrified. The rats learn to press the lever through several training periods because of the awards or the punishments. [9] The researchers can also control the probability between the lever and the switch, for example, the food will not be given to the rats until the rats press the lever for about 30-40 times. In this experiment, scientists find that although the food will be given after the rats press the lever so many times, the rats will still press the lever ceaselessly. It is just like the gambles among the people. In our project, we train the transgenic C.elegans and control the behavior of them by lights. Expression of two channelrhodopsins in the olfactory receptor neuron pair provides worms with the preference and aversion to specific wavelengths, and the corresponding lights are employed to reinforce their addictive or abstemious attitude to alcohol. Those reflects or behaviors are all operant behaviors and operant reflects which can be helpful for people’s more deep studies about the nerve and behaviors.
Optogenetics is a method which involves the use of light to control cells in living tissue, typically neurons, which have been genetically modified to express light-sensitive ion channels like channelrhodopsin, halorhodopsin, and archaerhodopsin. Optogenetics provides millisecond-scale temporal precision which allows us to attain a quick shift between positive and negative reinforcement(a consequence that will strengthen an organism's future behavior ).Employing light as the input signal instead of chemical signals, optogenetics is leading a methods revolution across all fields of science and engineering("Method of the year",2010, Nature Methods)[10] Optogenetics can stimulate and monitor the activities of individual neurons within freely-moving animals and precisely measure their response in real-time. In our project, it makes it possible to train worms, observe their behaviors and quantify the neuronal signal. In traditional training experiments of worms, however, the simulation (input) is always along with an inevitable residue, from hours to days. Besides, it is also important to have fast readouts(output) that can keep pace with the optical control. This is realized by using our optical device(see hardware-link) to receive the signal from the reporter proteins(mcherry) and indicator(GEM-GECO).
Microfluidics is a powerful technology that can integrate a number of functions in a plate of very tiny scale, which is best matched with the size of C.elegans. Scientists have successfully achieved high-through imaging and screening of worm populations with this technology[11]. Inspired by that, we designed two microfluidic chips to study the development of learning behaviors of C.elegans by observing their phisical response at the group level and measuring the signal of individual neuron after optical stimulation. Despite the fact that worms are confined in chips and pushed by the fluid, the channels are specially designed to effectively simulate their normal movement. The material of the chips PDMS is transparent, and it has no influence on the quantification of light signals in optogenetics experiment. Gas molecules, diacetyl and 2-nonanone, can diffuse in PDMS so that the worms can sense the odor of such molecule. As a result, we can observe their preference to these odors.
In our design, a group of optogenetic proteins and optical devices are used. Channelrhodopsins protein CoChR and Chrimson are coupled with dual-Color fluorescence protein GEM-GECO, expressed in the pair of neurons AWA and AWB. This combination is determined to lower the cross-talk and to get clearer image during experiments. Excited by blue light and red light separately, CoChR or Chrimson can induce the change of calcium ion concentration inside the nerve cells. Then it will causes the blue-shift of fluorescence of GEM-GECO. To enhance preciseness and the ability of expansion, we modified the projector and own software of microscope. New filters are installed in the device so that we can simultaneously control the intensity and period of light when stimulating the individual C.elegans during manipulation.
Fig2. Our optical design of protein and devices
Along with the development of neuroscience, more and more techniques have been used to study the behavior psychology. The original platform of the behavior psychology research, Skinner box, is not suitable for today’s research because its “black-box” model cannot reveal the changes in the organ-isms during the process of their learning new ability. Meanwhile, the former research of learning abil-ity based on C.elegans were more focused on chemical signals like olfactory inducing, heavy mental stimulus. These can demonstrate the existence of the learning behaviors but seems tenable as the relia-ble experimental data, since the residue of the stimuli are hard to erase. Thus, if we aim to study the learning ability associated with neuroscience and behavior psychology, we need to develop a new platform which allows us to explore the inside mechanism. Compared to the traditional methods, light stimulation is easier-controlled and more reliable for the following data analysis. That’s why our project is so important. It employs the optogenetics and neuroimaging to record the change in each neuron during a new abil-ity formed, so we can know what happened actually inside the brain during the ability formed: would the new connection formed between two individual neurons or the logic network changes? Moreover, the application of microfluidics in our platform allows us combine the process of ability training and neuron activity, and makes the research becoming easier to carry out. In the future, our platform can also be applied in different fields, such as the simulation of the Brain-Computer Interface (BCI). The BCI can help the paralysis patient obtain the ability to move and oper-ate things. But the signal from the human brain to which convert to the order to control the computer is so complex and difficult to deal with. If we can decipher the neuronal signals during people learning when the patient’s mind interact with the computer, it is possible to simplify the network and build more connection between them. We are now using our platform to study the alcoholism, in which we train worms to be addicted with alcohol and then study the neuron network during this behavior forming. Hope one day we figure out the logic network of alcoholism of human and help to treat more serious social problem.
The miniMos transposon is used to carry transgenes into the C.elegans'genome by the means of inserting a transgene into a modified Mos1 element (miniMos). The miniMos transposon can carry the inserted transgene (together with a selection marker) into the genome where insertions are generated directly by injecting worms. It will take approximately 1 week to get the result. The insertion site is random and can be identified by inverse PCR on the strain. The miniMos system contains 4 vectors——transposon with target gene, transposase, mcherry and peer-1 markers. Transposon needs transposase to cut and paste in the genome. mcherry and peer-1 are two negative selecter that can help to select the target strains. The method has some advantages: 1)The insertion frequency and fidelity is high. 2)The exact insertion site can be determined. 3)Large transgenes can be inserted. 4)The miniMos element is active in C.elegans. 5)Transgenes are expressed in the germline at high frequency.
Fig3. The MiniMos
As a classic model organism, C.elegans has many advantages.It is one of the simplest organisms with a nervous system. Research has explored the neural and molecular mechanisms that control several behaviors of C.elegans, including chemotaxis, thermotaxis, mechanotransduction, learning, memory, and mating behaviour. Therefore, C.elegans is a suitable model organism which we can easily manipulate and observe. In our program, we aim to change the physical behaviour of C.elegans by using the two specific optogenetic traits. Through microinjection and selection, we are able to get two strains with two phenotypes of the preference to blue light and the aversion to red light, and the next step is to obtain the single worm with the combination of these 2 different traits. To achive our objectives, we choose a mating method, because it is less time-consuming and easier to operate than microinjecting the other array to the existed strain. In general, mating is the pairing of either opposite-sex or hermaphroditic organisms. However, for C.elegans, mating can hybridize two different traits on the next generation by the paring of the hermaphrodite and the male. Besides, one of the greatest advantages of C.elegans is that it can either autocopulation or hybridization, which means that after hybridization, the hermaphrodite can self-fertilize and guarantee to produce a large number of offsprings with identical genetic characteristics. Here is the mating procedures: 1.Heat shock to get males(30℃,8h). 2.Parents mating to get the first filial hybrid generation(：=3:4). 3.Single out each hermaphrodite in a small plate. 4.Test the fluorescence of F1. 5.Single out F1 and self-fertilizing to get F2. 6.Test the fluorescence of F2.
References
[1]Caenorhabditis elegans(WIKIPEDIA). Retrieved September 25,2017 https://en.wikipedia.org/wiki/Caenorhabditis_elegans [2]Brenner, S., Draft, A. N. C., Chklovskii, D., System, F. F. N., Brain, T. M., & Seung, S., et al. . The connectome debate: is mapping the mind of a worm worth it?. Scientific American [3]Chen, Y., Scarcelli, V., & Legouis, R. (2017). Approaches for studying autophagy in Caenorhabditis elegans Cells 6(3) [4]Uno, M., & Nishida, E. (2016) [5]Caenorhabditis elegans(WIKIPEDIA). Retrieved September 25, 2017 https://en.wikipedia.org/wiki/Caenorhabditis_elegans [6]Uno, M., & Nishida, E. (2016) [7]R.Carlson, Neil (2009). Psychology-the science of behavior. U.S: Pearson Education Canada; 4th edition. p. 207. ISBN 978-0-205-64524-4. [8]Krebs, John R.(1983). "Animal behaviour: From Skinner box to the field". Nature. 304 (5922): 117. Bibcode:1983Natur.304..117K. PMID 6866102. doi:10.1038/304117a0 [9] Brembs, Björn. "Operant conditioning in invertebrates". Current Opinion in Neurobiology. 13 (6): 710–717. doi:10.1016/j.conb.2003.10.002 [10] Editorial, N. (2011). Method of the year 2010. Nature Methods. [11] Chung, K., et al. (2008). "Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans." Nature Methods 5(7): 637-643.
