Lab report: Optogenetics – A screening with the channelrhodopsin Chrimson

on Tuesday, November 24th, 2015 7:17 | by Lena Danyeli

Optogenetics is a technique in which light is used to control cells in living tissue, typically neurons that have been genetically modified to express light-sensitive ion channels. The technique is used to modify the activity of a given set of neurons, even within freely-moving animals.

In the course of Christian Rohrsen’s project “Dopamine neuronal populations involved in reward and punishment” we performed a screening to assess avoidance and appetitive behaviour of flies by monitoring the escape or approach from or towards the illuminated arm. We assessed distinct types of neurons by means of two different optogenetic setups– the T-maze and a custom-built platform, which are presented in the following:

Experiment 1: T-maze

First part of the internship was to further optimize parameters in the already established method T-maze (as introduced by Christian Rohrsen: see scheme below) for an efficient use in optogenetic experiments with the channelrhodopsin “chrimson” (spectral peak at 590 nm).

tmaze

The general setup consisted of one illuminated arm and one non-illuminated dark arm. Following parameters were monitored to select the most convenient conditions for the screening:

  1. Light source:

→ pulsed vs. constant light, light intensity and positioning of the light source

  1. Time parameters:

→ duration of trials and time between repetitions

  1. Food supplement:

→ ATR vs. no ATR

  1. Genetic drift:

→ separate breeding of flies with different genetic background

The screening was performed with following fly lines (all crossed with NorpA and therefore blind):

NorpA;Or42a>Chrimson

NorpA;58E02>Chrimson

NorpA;TrpA1>Chrimson

NorpA;Gr66a>Chrimson

NorpA;UAS-Chrimson and wtb; UAS-Chrimson (control for leaky expression) as genetic controls

To control for the genetic drift, we used two groups of flies of the same line but reared separately for several generations (group “yellow” and group “white”, referring to the color used for labeling).

Results:

The most suitable light condition for the screening was pulsed light (20 Hz, 10 ms pulse width, 1500-2000 lx), which was favored since neurons seem to be activated more efficiently in a phasic than in a tonic way (Inagaki et al., 2013). Furthermore we assumed that constant light could induce abnormal levels of neuronal activity, which could lead to excitotoxicity. The best position of the light source was at the outside of a short transparent tube, one on top and one above, which guaranteed homogeneous illumination. 1 min trials with repetition and 1 day between repetitions worked best to rule out possible effects of the CO2 anesthesia on the experimental outcome. The genetic background did not seem to play a big role in the results of this setup, which is shown in the graph below (yellow vs. white group; screening performed before optimal parameters were found).

NorpA_Chrimson_all2

With described parameters we observed an escape behaviour towards the illuminated arm, indicating Gr66a, TrpA1 and 58E02 being neurons eliciting the avoidance behaviour when activated (we did not observe any effect for Or42a; data not shown). We furthermore found a strong enhancing effect of this behaviour due to the administration of ATR. The graph shows the mean PI of the two repetitions for each fly line with (ATR) or without (Co) ATR as food supplement (n = 6).

Screening_total

Experiment 2: Platform

The second part of the internship aimed to optimize the experimental parameters of a custom-built platform, which represents a second method to assess preference/ avoidance behaviour, tested in an operant behaviour experiment by means of optogenetics. The platform is connected to a fiberglass, which allows light to be directly placed on top of a fly’s head to excite the assessed neurons. By walking to one or the other side, the flies can move the platform and thereby switch the light on and off. The pictures below show the platform holding a fly hooked with a copper hook on the neck (left) and a fly hooked on the thorax with a “double-L”-shaped wolfram hook (middle and right).

flies

The experiment was performed with variation in following parameters:

  1. Light intensity:

→ 15 lx – 100 lx

  1. Fixation of the flies:

→ hooks glued on the thorax or the neck, facing either to the back or the front of the fly

→ different material used for the hooks: copper vs. wolfram

→ different shapes of hooks: triangle vs. “double-L”-shape

Since Gr66a showed promising results in the T-maze, this fly line was used to further optimize experimental parameters with the aim of confirming the observed avoidance effect.

Results:

platform

Most promising outcome was observed at 80 lx – 100 lx with “double-L”-shaped wolfram hooks facing to the back of the fly, as can be seen in the graphs for the PI by time (left side: 80 lx, right side: 100 lx; n = 3). In this combination the light reached the fly’s head and therefore the assessed neurons in the most efficient way. We found the thicker wolfram hooks to be more useful than the copper hooks since they were more tightly attached to the body and allowed us to position the flies very accurately under the light. Furthermore, we found a change in walking behaviour due to the positioning of the hooks on the flies. Hooks facing to the back of the flies, and therefore covering the wings, unfortunately seemed to make the flies more immobile. Also the bodypart on which the hooks were glued seemed to play a role, with hooks glued on the thorax being the most stable condition, since it allowed us to use more glue, which was needed due to the increased weight of the wolfram hooks.

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Category: Optogenetics

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