An Illuminating Journey 2

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.

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.

Laser vs LED light into 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.

Number of optical connections in different in vivo optogenetics setups.

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.



A Modern Classic, Part II

In 1900, Lord Kelvin addressed an assembly of physicists and claimed, “There is nothing new to be discovered in physics now. All that remains is more and more precise measurement.” Then just 5 years later, Einstein shook up the scientific world with his theory of relativity. Dare I compare this situation to the second paper in my Modern Classic series? No, that would be exaggeration on a disgusting scale. However, hyperbole aside, the findings from today’s paper were a revelation to me and, I’m sure, to many others, and has opened up a new way of thinking about the neuronal control of energy balance.

We saw last time that we can drive voracious feeding in mice by activating AgRP neurones, with DREADD’s or optogenetics. The thinking at the time was that, in a fasted mouse, Ghrelin is high (and satiety hormones such as PYY3-36 are low), so AgRP neurones are actively firing to drive food seeking and consumption. Then as the mouse eats and the balance of hormones switch, the AgRP neurone activity drops, and so does feeding drive. However, this thinking was soon to be upended, when Chen et al. used in vivo photometry recordings to measure the activity of AgRP and POMC neurones in awake behaving animals1.

Photometry is probably my favourite in vivo method, because it allows you to investigate the behaviour of an identified group of neurones in an awake behaving animal. Chen et al. transfected AgRP and POMC neurones with the calcium indicator GCaMP, which closely mimicked the electrical activity of the cell (Figure 1A-E). They then put it into live mice, which allowed them to record the activity of AgRP or POMC neurones in awake behaving animals (Figure 1F-H).

They next showed that Ghrelin injection in their AgRP or POMC photometry recordings caused a rapid increase or decrease, respectively, in activity in those neurone populations. No surprises so far. However, the revelation came when they fasted the animals (to drive up AgRP neurone activity), then presented food (Figure 2). The change in neuronal activity came rapidly after presentation of food. In fact they found, as stated in the name of the paper, that the AgRP and POMC neurone activity was modulated upon sensory detection of food – in fact they had already altered their behaviour before the mice had taken a bite of food (Figure 2H). This shows that the classic theories of AgRP and POMC neurone regulation by hormones and other homeostatic methods are too slow to drive actual feeding. The question then becomes, how do AgRP neurons drive food intake if they are switched off during the time that the animals are actually eating?

Chen et al. go on to show that the degree of response to food presentation is relative to the caloric content of the food, ie. they have a bigger response to peanut butter than to regular chow. There has since been a lot of work to investigate further the control of feeding behaviours by these neurone populations, but I won’t go into detail here. However, it is worthy to note two papers came out soon after the Chen paper, confirming the rapid modulation of AgRP neurones by sensory detection of food, using different methods (Betley et al. used GCaMP and head-mounted miniscopes; Mandelblat-Cerf et al. used optetrode ephys recordings to provide actual spiking data)2,3.

1. Chen et al., Cell 160, 829-841 (2015) Sensory detection of food rapidly modulates arcuate feeding circuits.

2. Betley et al., Nature 521, 180-185 (2015) Neurons for hunger and thirst transmit a negative-valence teaching signal.

3. Mandelblat-Cerf et al., eLife 4, e07122 (2015) Arcuate hypothalamic AgRP and putative POMC neurons show opposite changes in spiking across multiple timescale.

A Modern Classic, Part I

Today I’ll be revisiting a paper that had a massive impact both on what we know about control of energy balance, but also how I think about and approach my experiments. I’m talking about Atasoy’s 2012 Nature paper with the pretentious title1, in the study that first introduced me to optogenetics. Or at least to the possibility of controlling awake mouse behaviour using optogenetics.

This paper came out not long after the seminal work by Krashes et al., where he used DREADDs to drive to activity of AgRP neurones in vivo, and show the direct effect on feeding when these neurones are activated2. These papers were published towards the end of my PhD, and I was very keen to use these exciting new tools in my own research.

In fact, one of the first things I did in my postdoc was to help set up the use of targeted nanoinjections of AAV DREADD’s in our transgenic mice. It was only after a couple of successful experiments with DREADD’s that I even began to think about using optogenetics – I really wanted to develop the easier stages before jumping straight into the more advanced stuff.

Anyway, back to Atasoy’s paper. After some initial testing to make sure they can express ChR2 in AgRP neurones, and to demonstrate inhibitory input onto POMC neurones with electrophysiology, they take the optogenetic stimulation in vivo. They show, firstly, that you get increased food intake with coincident stimulation of AgRP and POMC neurones, demonstrating for the first time that the feeding drive for AgRP neurones is outside the Arcuate nucleus, ie. that the acute feeding action of AgRP neurones was not mediated by the suppression of POMC neurone activity (Figure 1).

But, if the acute feeding effects of AgRP neurones are not mediated by action on POMC neurones in the Arcuate, where are they mediated? Atasoy demonstrates the power of optogenetics for investigating neuronal circuits, by activating AgRP neurone terminals in awake behaving animals (Figure 2). Picking 2 areas with dense AgRP neurone terminals and known for controlling food intake, they target the PVH and the PBN. The results speak for themselves, with such a drastic response from the PVH, and nothing at all from the PBN.

There are more figures in this paper, but for me these were the most important findings. I think the power of optogenetics comes down to several factors, allowing us to overcome a number of the most challenging aspects of studying the brain:

  • High temporal precision – can get physiological responses instantly, and influence behaviours that rely on millisecond response times
  • Circuits – optogenetics allows us to investigate the neuronal circuitry involved in complex behaviours by stimulating neurotransmitter release in target areas
  • Stimulation patterns – the flashing light can be patterned to mimic neuronal firing patterns, which can produce differing behaviours even from an otherwise identical experiment

For those interested to read more, there is another early paper that really influenced my thoughts on optogenetics, which was a 2013 paper from Betley et al.3 For Part II, I’ll be investigating the next big advance in in vivo technology which has changed my approach to understanding energy balance.

1. Atasoy et al., Nature 488, 172-177 (2012) Deconstruction of a neural circuit for hunger.

2. Krashes et al., J Clin Invest 121(4), 1424-1428 (2011) Rapid, reversible activation of AgRP neurones drives feeding behaviour in mice.

3. Betley et al., Cell 155, 1337-1350 (2013) Parallel, redundant circuit organisation for homeostatic control of feeding behaviour.