Drosophila neurons can be activated or inhibited using different methods, and some are more appropriate under specific conditions.

Neuron activation#

The activation of neurons can be achieved through the following methods:

1) Using an LED of appropriate wavelength to drive activation of Chrimson in the neurons of interest.#

Chrimson is a red shifted opsin that can be expressed in neurons of interest. When shining a light of appropriate wavelength, this depolarizes the neurons.

This is probably the easiest activation method, since shining the light is enough to activate the neurons, and if doing only behavior, there is no need to open the cuticle to expose the fly’s brain. However, it might be problematic when:

1. We don’t have a line that is specific enough to the neurons we want to activate.

2. We want to activate neurons during imaging, since the light might bleed through the objective and compromise our SNR.

To carry out this method you will need:

1. Chrimson flies to cross your line of interest to. We currently have the following in our stocks: …
2. An LED of 660 nm. If using it for behavior, this mounted LED can be used, attaching this condenser lens in front of it, and be controlled by this LED driver. If using it for imaging, you will instead want this LED, and this fiber, to help target the stimulation closer to the animal.
3. You will have to raise your flies on ATR-containing German food in vials covered with foil, because ATR is a necessary co-factor for all channelrhodopsin variants.  The protocol for making ATR-containing German food can be found here. When starving them before experiment, you will want to wet the kimwipe with an ATR solution of …

2) Using localized ATP to activate neurons expressing P2X2#

See full description here.

3) Using perfusion of ATP into the bath to activate neurons expressing P2X2#

If you don’t need the level of spatial or temporal precision achievable using method (2), an easier way of activating P2X2-expressing neurons is to rapidly wash ATP into and out of the bath saline solution using a perfusion pump (Watson Marlow 120U). To achieve reasonable temporal specificity a relatively high flow rate (e.g. ~5 mL/min, 60 rpm on that pump with the default tubing diameter) is recommended. Based on previous experience, a 30-60 sec pulse of 5mM ATP might be expected to activate your neurons of interest for ~3-4 min after the ATP solution first reaches the bath, but YMMV. Ideally the ATP aliquot should be kept on ice to avoid degradation and then added to the appropriate container of saline solution immediately before use.

NOTE: to ensure consistency in the timing, it’s a good idea to stop the perfusion pump briefly while you switch the intake tubing from one bottle of saline to another if you are using two separate containers, to avoid getting an air bubble in the line at the boundary of the two solutions in the tubing.

4) Using the 2P laser#

On the Berg2 scope, we have a Galvo-Galvo-Res scanner system that allows us to conduct near-simultaneous photostimulation and imaging despite the lack of a dedicated photostimulation laser and optical path (with some important limitations). Detailed instructions for using this technique can be found here. Photostimulation-only behavioral experiments can also be done using those instructions as a starting point and leaving out the parts that are specific to imaging.

5) Using current injections when patching#

Neuron inhibition#

1) Using Gt-ACR#

2) Using current injections when patching?#

3) Expressing UAS-Kir2.1#

• (+) The effect of expressing Kir on the target neuron can be monitored via patching (e.g. Kazama & Wilson, 2008; Fig. 4D on the effect on input resistance).
• (+) Compatible with visual behavior.
• (-) Chronic inhibition. Might lead to developmental compensation etc.
• (-) Most (all?) Kir2.1 is tagged with GFP so be careful when combining with GCaMP imaging.