Team SUSTC-Shenzhen

Demonstrate

Project

Overview

So far, our project has been almost completed. The whole project is demonstrated in the following parts:

1.Optogenetic results of spectrum determination

2.Microinjection results

3.Neuron activation

4.Behavior experiment (including single worm and population)

5.Addiction inducing results

Spectrum determination

The channelrhodopsins we chose in our project were evolved from the algae, so if we applied these light sensitive channel to the Caenorhabditis elegans as the receptors, we need to check its optical parameters before our behavior experiment. The spectrum of Chrimson and CoCHR have been already measured by other scientists. With the spectrums of two channelrhodopsins, we need to figure out the another two questions :crosstalk and indicator spectrum.

The first question is that whether the channelrhodopsin CoCHR will have the wavelength crosstalk with the indicator protein—GEM-GECO , the excitation wavelength of CoCHR is crossed with the emission wavelength of activated GEM-GECO combined with calcium, which means that we can not activate CoCHR and receive GEM-GECO emission fluorescence in the same time with same wavelengths because of the filter. So we have to do the experiment based on the cell to select another suitable excitation wavelength for CoCHR.

Fig.1 The experiment result have been showed in figure 1a and 1b. In this experiment ,we have expressed CoCHR combined with another indicator R-GECO to avoid the crosstalk. The figure 1a showed that the indicator have been all activated because the KCl had induced plenty of calcium influx inside cells through calcium channel. Compared with the full-state ,we choose the relatively high excitation wavelength 470nm which can activate almost 60% of the indicator protein. Also the sharp and acute peak in this figure proves that the light inducing is much more sensitive than the chemical signal.

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Fig 1-c

The excitation and emission wavelength are the most important parameters for all kinds of optical protein, so if we want to know what happened inside the worms’ neuron after light induing, the spectrum of indicators expressed in the C.elegans must be figured out. So we use the sensitive detector to receive the emission wavelength from the C.elegans AWA neuron after activated by diacetyl inducing. Then we analyze the emission data and select the best two wavelength area for the indicator emission wavelength with and without calcium existing. This part will be shown in the neuron experiment.

System construction in C.elegans

In order to select the worms stable transacted with the system we have constructed, we design a series selection experiments for verification. These figures showed that the result of the selection process using the fluorescence marker selection ,the rescue selection and the negative poison selection. We then do the mapping for the genome of the worms to check the insertion of the system. Finally we use confocal microscope to check the location marker to test the expression of the whole system.

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Fig.2 F1 Negative selection, a poisonous gene which were co-injected with the target gene along with the heatshock promoter, after heatshock the worms with multiple arrays will all died. After 34°C,4h heatshock, these stable transfected worms which were survived with freemoving will be picked out for fluorescence checking.

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Fig.3 Confocal Image for Odr-10::CoCHR::GEM-GECO::mCherry, mCherry is the location marker of AWA neuron (compared with picture from database). This figure can show our circuit both insertion and expression in the C.elegans we selected.

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Fig.4 The left figure is fluorescence image for Str-1::Crimson::GEM-GECO::GFP in AWB neuron. Another figure is TM4063 (wild type) originally with body fluorescence, so the fluorescence noise on above noise should be subtracted.

Activation of C.elegans head neurons

Since the whole system has expressed in the C.elegans, we need to check the function of each part in our project. So we have done many experiments on the head neuron of the worms, we first check the function of the GEM-GECO.

These figures showed the position and the reaction of AWA neuron which expressed GEM-GECO after diacetyl inducing.

Microfluidics

In order to study locomotion of C.elegans populations, we design the Gaussian Chip, a pillar-filled area, where the pillars were designed such that they allow crawling-like behaviors of C.elegans even though worms are immersed in liquid environment.

Fig. 5 Gaussian plate. C.elegans can move award freely in our channel without any difficulties, and then we would get a similar Gaussian distribution ideally. Next, after we injected a kind of chemical (diacetyl or 2- nonanone) in the right (or left) lateral channel, the distribution of worms will be changed, in other words, the peak of this distribution will move towards the right (or the left)

Fig. 6 Immobilized channel. In order to observe a neuronal response in an individual worm, we design four subuliform channels to fix its head in case of the influence of worms’ drastic movement. In addition, we also want to research whether C.elegans will prefer blue light (like diacetyl), and repulse red light (like 2-nonanone). Therefore, we design another four narrow channels that C.elegans can just move forwards and back.

Response to light (Individual)

To verify the response to specific wavelength light inducing, we picked out a single worm to observe the behavior change.

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Fig.7 Control the C. elegans by the light. We used the optical fiber to form a light spot and the worm can follow the spot.

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Fig8. The test of the CoChR using light with different wavelengths and intensity. The lights blue- and blue+ is from the projector whose wavelength is about 480 and the lights whose wavelengths are 395, 440, 470, 560 and 640 are from the LED of fluorescence microscope (Nikon eclipse Ti).

Alcohol addiction

To verify the function of the system we constructed in the C.elegans and give our project a real word meaning, we design this experiment to train the C.elegans be addicted to alcohol.

If our system worked and can control the worms prefer to alcohol after blue light (preference) trained, it can prove that all the function we purposed for this system has been already realized.

Here, we employ the Odr-10::CoChR::GEM-GECO::mCherry worms to measure the preference for the alcohol. We added the alcohol with different concentration on the NGM plate to form a liquid film to stimulate the C.elegans and give the worms blue light induced which is proved can make them show a preference. After 2 hours we washed the plate to recover the worms in M9 buffer and put the mixture (contained worms and M9 buffer) on one side of a new plate. After recovery of the worms, we put the alcohol on the other side of the same plate to find out if the worms have the tropism of the alcohol.

Then we observed that the Odr-10::CoChR::GEM-GECO::mCherry worms could move to the alcohol while the wild types could not (Fig.8). And both of the experimental worms and wild type worms show no response to the buffer after training.By the same time, the Odr-10::CoChR::GEM-GECO::mCherry worms show no response if we do not train the with the blue light.

Fig. 8 A. The Odr-10::CoChR::GEM-GECO::mCherry worms after 2 hours' training. We cycle the area of worms after training in red. B. The worms in A after alcohol inducing. The yellow cycled area represents the alcohol and the worms could crawl towards the alcohol. C. The wild type worms control. The wild types could not crawl towards the alcohol although trained with blue light.

Fig. 9 A. The Odr-10::CoChR::GEM-GECO::mCherry worms after training and alcohol induing (the same as (Fig.8 B). The Odr-10::CoChR::GEM-GECO::mCherry worms after training, then use buffer to induce. The yellow cycled area represents the buffer and the worms could not crawl towards the alcohol. C. The Odr-10::CoChR::GEM-GECO::mCherry worms without training, then use the alcohol to induce them, the worms show no response.

This experiment showed that our worms which constructed the circuit (specific promoter::channelrhodopsin::Calcium indicator) can form the habit we purposed after trained with the light (decided by the channelrhodopsin). In brief, we have proved that our experimental worms have the learning ability inducing by light. Thus, the goal of our experiment has been almost achieved. We get the worms controlled by the light and the neuron activity can also be traced by observing the change of the indicator.

Future Plan

In the future, we will track the neuron activity and do the alcohol inducing at the same time on the Odr-10::CoChR::GEM-GECO::mCherry worms after 2 hours training. By the time, we will build a model to select the downstream neuron of the AWA/AWB neurons and then using the specific promoters of these neurons fused with indicator to verify the relationship between them.Then finally we will figure out the whole neuron network of the learning ability, we can do the simulation on wherever we like to test the network, and we can know the logical order of the neurons when a new ability formed.

Team:SUSTech Shenzhen/Demonstrate