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 channelrhodopsin we chose in our project were evolved from the algae, so if we applied these channel proteins to the C.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, due to their spectrum we need to figure out the another two questions :crosstalk and indicator spectrum.

The first question is that 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 GEM-GECO combined with calcium, which means that we can not activate one protein and receive another protein’s emission together with same wavelengths because of the filter. So we have 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 the cell 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.

Fig 1-c

The excitation and emission wavelength are the most important parameters for all kinds of optical protein, so if we want 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 show 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.

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 is survived with freemoving will be picked out for fluorescence checking.

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.

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 are designed such that they allow crawling-like behaviors even though worms are immersed in liquid environment.

Fig. 5 Gaussian plate. C.elegans can move award freely in our channel without any influence, and then we will get a Gaussian distribution ideally. Next, after we injected a kind of chemical (diacetyl or 2- nonanone) in the right (or left) optional channel, the distribution will be changed, which means 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 pick out single worm to observe the behavior change.

Fig.1 Control the C. elegans by the light. We used the optical fiber to form a light spot and the worm can follow the spot.

Fig.10 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).

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