Following the wildly successful first instalment of “An Illuminating Journey”, a sequel was all but inevitable. We pick up the story during the giddy highs induced by the first successful optogenetic test experiment, by our protagonist Nic and his sidekick Ed. This orgasmic euphoria was unfortunately shortlived, when their ignorance was bound to collide with the harsh reality of optimising challenging new techniques.
I think, in hindsight, that using AgRP neurones for our initial optogenetics test may have been reaching for fruit that was hanging too low, so to speak. Essentially, we had outrageously good behavioural responses from that experiment, even with very low (<1 mW) light stimulation, almost no acclimatisation for the animals, and no testing/optimisation of stimulating protocols. So when it came time for the next opto experiment, this time using a different cre line to target a neurone population I’d been investigating for a few years, we didn’t have such good fortune.
We kept the experimental protocol very similar (measuring food intake responses to opto stimulation), but initially saw no effect whatsoever. But then, we had used a low light intensity for the AgRP experiment, so we tried ramping the light output up as high as it would go, and this gave us an inkling of a (non-significant) trend towards a feeding response. Ok, maybe if the mice were less stressed? Unfortunately, they had to be in an open cage to allow for the optic fibre tethers, but we could implement a rigorous acclimatisation protocol, to get the animals used to being both in that stressful open environment and to having their head tethered.
It was during this acclimatisation period that the implanted optic fibre cannulae starting coming out; essentially the small, smooth steel fibre cannulae weren’t adhering to the dental cement well enough to resist the tight optical connections. And I won’t even start on the ceramic cannulae we tried at the same time – they seized up with the connectors and got ripped out on the first connection every time. Any remaining ceramic fibres were rapidly disposed of.
We have since improved our opto techniques, especially with regards to the surgery, to almost entirely negate the chance of lost fibres (the most important step was improving our dental cement; we now use modern light-curable stuff which is super sticky, and turns rock solid in a matter of seconds so works much better than the old mix-and-cure stuff we had). However, at the time Ed and I were getting more and more frustrated with these failing and partially successful opto experiments.
It was then that an event happened that would alter the course of my life, career and professional interests. I remember distinctly when and where it happened. We were just clearing away after another partially successful/partially disastrous opto experiment; I had been thinking how much of a problem it was to have to tether the mice in open cages, and how likely that was to be the root cause of our experimental issues, and my various Google searches had shown me zero viable fibre-free alternatives on the market. I turned to Ed, and voiced my hitherto private thoughts that it would be so great to do away with the optic fibres for these experiments, but there were no wireless opto systems available, and in my frustration said that I could bloody well make a system myself. Ed looked at me and said, “Yeah, let’s do it. I’ll front the cash and you develop it.”
I laughed it off at the time, but his serious suggestion that we do this stuck with me, and a few days later we sat down with a coffee and came up with a serious plan of how we might go about doing this. I brought along a new Lego Batman notebook dedicated to this project, and we sketched out some ideas for how our fibre-free optogenetics system might work. We also came up with a realistic timeline to develop a prototype (alongside doing our actual lab projects, obviously), and we conservatively came up with a target of 6 months till were able to test in vivo, and if all went well with the tests we could start production and begin sales of our ground-breaking product to other like-minded neuroscientists a few months later. Spoiler alert: 3 years later, we still haven’t made it to the in vivo testing stage.
Now, it is important to note at this point that I was our resident electrical engineer, having obtained a C-grade in GCSE electronics a mere 15 years previously. So, after a swift Google to catch me up on the advances in micro-electronics since then, I came up with a plan for how to achieve fibre-free opto’s. We ordered some hobbyist electronics sets off eBay, and I built a circuit on a breadboard that would flash a blue LED in specified patterns under infra-red control. Great. Next step, I downloaded a free electronics circuit mapping software (“Fritzing”), designed a printed circuit board (PCB), and we got it printed from a company in China for a few quid. Ed bought me a soldering iron, and then when my PCB’s arrived, I was able to solder in the components to recreate the breadboard circuit. And that worked! But, this was about the size of a credit card, so next step was to shrink the circuit down to something we could attach to a mouse’s head.
So I redesigned and shrunk the PCB’s, and we got them printed from the same company in China, but this time we paid them to solder the micro-components to the boards (my newfound soldering skills weren’t up to that particular task). It was a very exciting day when our new circuits arrived. I managed to solder in some contacts to connect batteries, and we had yet more success – these mini circuits also worked as I had planned. So, we were now almost ready to take our prototypes in vivo, we just needed to find a way to connect the LED on the circuit to an optic fibre implanted in a mouse.
Tune in next time to find out just how terribly it all went wrong.