An Illuminating Journey

Back in 2016, I decided to make the leap towards my first optogenetics study. A couple of years previously, I had helped set up targeted intracranial nanoinjections for the lab, which meant we were routinely doing experiments with AAV’s (mostly DREADD’s) and retrotracers. And it was only a few years before that that our lab had acquired our first cre line.

So, while the use of transgenic mice in this way was relatively new to us, we were learning quickly and were keen to advance our in vivo capabilities. More and more it was becoming difficult to publish in good journals without showing manipulation of complex behaviours by identified neuronal populations (either with DREADD’s or optotenetics) and demonstrating the circuits involved.

However, optogenetics was still quite new, and totally novel to me, and as I’ve said before one of my failings is my reticence to seek help, so I was figuring this out myself. Not that I was completely alone, I did have a great PhD student to help me, particularly with the in vivo aspects. So anyway, I started by looking at what others had done, focussing on some of the early, high impact work; I was particularly drawn to work from Scott Sternson and Denis Burdakov, as well as the original pioneers of optogenetics including Karl Diesseroth. Picking out the common factors in their methodologies, I wrote up the following list of requirements for my first optogenetics study:

• Use lasers to produce blue light (~470 nm)
• Light is pulsed at a maximum 20% duty cycles to limit heat damage and phototoxicity; typically 10 ms ON at 10-20 Hz
• Light is delivered via fibre optics with a rotary joint to a 200 µm fibre into the mouse’s brain
• Typical light power from the end of the fibre optic cannula (ie. what is actually entering the mouse’s brain) is around 10-15 mW

If I’m honest, setting up one of the laser systems for my first optogenetics study scared me a little. They’re big, expensive, dangerous and difficult to use. Or at least, so it appeared to someone who’s never used them, and I would be facing a mountain of paperwork if I wanted to get a laser system approved for use at the University.

It was around this time that we started seeing LED-based optogenetics systems coming on the market, which definitely appealed to me. The problem with LED’s is that the light scatters (Figure 1), making it challenging to get sufficient light through an optic fibre.

If you want to use LED’s to provide sufficient light output for in vivo optogenetics, you need to have an extremely high power light source with very good lensing and/or reduce the number of optical connections to reduce the light lost along the delivery path (Figure 2).

Looking at the possible applications of LED’s, I could safely discount implanting micro-LED’s into the brain (Figure 2D) due to the highly advanced nature of that method and the fact that nobody sells them, as well as having head-attached LED’s (Figure 2C) because there don’t seem to be any trustworthy versions for sale, although the latter does lend to doing wireless optogenetics which does appeal to me but not for my first optogenetics study.

So, between the “normal” desktop-mounted LED’s (Figure 2A) and the intermediate rotating LED’s (Figure 2B), there were 2 options on the market that seemed likely to work. I say this, because it was very rare for any of these manufacturers to actually state what the light output from the fibre cannula would be in an experiment; hats off to Plexon and Prizmatix as the only ones that seemed to do this.

So, I had narrowed down my options to the Prixmatix desktop LED¹ and the Plexon rotary Plexbright system². However, my distrust of having optical connections, for fear of excessive loss of light, led me to pick the latter. I had already tested an AAV ChR2 construct in vitro, so, together with my experience doing targeted AAV-DREADD injections and cementing ICV cannulae into mouse brains, I was ready for my first optogenetics study.

As Ed (my PhD student) was already working on NPY/AgRP neurones and feeding behaviour, we had the AgRP-cre mouse and we both thought that stimulating AgRP neurones would be the best initial experiment. I maintain you always want to go for the low-hanging fruit when starting anything new.

Ed and I assembled a half dozen AgRP-cre mice, injected them with AAV-DIO-ChR2-mCherry into the arcuate and cemented an optic fibre pointing at the same place. Then came the waiting game – 2 weeks while the transfected neurones ramped up expression of ChR2. We rigged up the Plexon rotary LED’s (we stuck them to a shelf above a bench using electrical tape), wrestled with the Plexon Radiant software to produce a nice stimulation pattern, and finally connected some mice to the ends of the optic fibres.

I can still remember the day we first switched on the LED’s – without a doubt the best moment I’ve ever had in science, and to be honest one of my best in general. How can a simple wedding, or the birth of a child, compare to watching a mouse gorge itself because you flicked a switch on an LED. Absolutely magnificent.

1. https://www.prizmatix.com/Optogenetics/optogenetics-led-Blue.htm

2. https://plexon.com/products/plexbright-optogenetic-stimulation-system/