Category: Electrophysiology

Practical uses of 3D printing in an electrophysiology lab

I have mentioned before about my 3D printer, and how useful I have found it. Today, I’ll explain some of the practical uses I’ve found for 3D printing for electrophysiology. The point is that electrophysiology equipment is both extortionately expensive and annoyingly non-compatible. So, it is often quicker, cheaper and easier to design and print a “thing” than try to buy something to fit your particular need.

Build-a-bath workshop

A year or two ago, I was setting up our third (at the time unused) rig for calcium imaging. We had various bits of baths, but no complete set. And it would cost a (relatively) large sum of money to buy a replacement. I found that what I was missing was the “bath” bit (I had the holder). So I measured up one of our existing ones and designed a reasonable copy in Autocad:

3D design for an electrophysiology bath insert.

My printer was able to make it with a very smooth base, which is the crucial aspect for obtaining a watertight seal. I installed it on the calcium imaging rig, it worked well, and is still in use there to this day. Oh, and for anyone who’s interested, I have made the 3D design available on Thingiverse.

Moving an LED

My loyal readers will know that it was around this time that the light source for the calcium rig died. A replacement LED source would cost anywhere from £3k to £15k, depending on how many colours I wanted access to. However, we had animals ready for experiments at the time, and even if we had the money, it could take weeks for new kit to arrive.

Luckily, we had a blue LED of the correct wavelength attached to one other rigs, that was no longer needed there. I had purchased it to do optogenetic stimulation, but we had switched opto’s to the third rig.

Anyway, this seemed like an easy fix, just swap it over. But of course, it’s never that easy. Because, I wanted to move the LED from an Olympus microscope to a Zeiss. And the manufacturers do not make it easy on the consumer by having common fittings.

So, I measured up the fitting on the LED and the back of the Ziess fluorescence port. I then designed a 2-part “sleeve” that would modify the Zeiss port to resemble the back of an Olympus:

3D design for an Olympus-to-Zeiss microscope fluorescence adapter.

I used cable ties to hold it on tight to the Zeiss fluorescence port. The benefit of cable ties over something more permanent like glue is that they can just be cut off if/when the LED wants changing. The LED now fitted snugly onto the back of the microscope, and, after some fiddling with the data and control connections, was now fully functional for calcium imaging.

A “lab things” service

The main point I want readers to take away from this post is the usefulness of 3D printing for electrophysiology labs. I would strongly recommend anyone reading this who performs a practical skill in the lab like electrophysiology to consider investing in a 3D printer. They are actually quite cheap nowadays (mine was about £250 a few years ago), and I’m sure they’ll save you a lot of time and money in the long run.

In fact, the biggest investment to 3D printing things yourself is the time it takes to learn 3D CAD software and optimising the 3D print process itself. So, if you want something custom making, but would prefer not to have to figure it out yourself, just head over to the Services page and send a request. You never know, I might well be able to save you a lot of time, effort and money.

Optogenetic Stimulation Frequencies

Today I’ll be talking about the importance of optimising in vivo optogenetics frequency, having previously looked at the pulse on-times. All too often, I will see papers or talk to colleagues who use an unfeasible stimulation frequency for their in vivo optogenetics. For example, where I work in the hypothalamus, you often see stimulation at 20 Hz. And from my experience of patch clamping multiple neurone types in the hypothalamus, they just don’t fire that fast.

If you’re not an electrophysiologist, it might not be obvious, but action potentials are energetically expensive. So, neurones will only fire quickly if they need to. In fact, they will only be able to fire quickly if it is required for their function. Which it is for cognitive processing, but not for the much simpler processing required in many other brain regions.

Back to the beginning

As usual, first thing we do is go back to the early optogenetics publications from Karl Diesseroth. In their 2012 Nature Methods paper, Mattis et al. performed a thorough investigation of the opsins available at the time1. And, despite being a decade later, the data still stand, and are still very useful. I strongly recommend reading this paper for anyone who plans to perform optogenetic studies. It’s a huge paper with bags of useful info.

Mattis et al. measured spike fidelity, ie. the success rate of the cell to produce an action potential in response to a flash of light. They used a high light intensity, so there is no issue of there being not enough light to activate the opsin. Instead, the loss of fidelity comes from the neurone being unable to keep up. As I’ve mentioned before, the neurone needs to recover its membrane potential below a certain threshold or it won’t be able to trigger another action potential, so if you chronically overstimulate a neurone they become silenced.

I’ve shown here a comparison of ChR2h134r (also called ChR2R) and ChIEF (Figure 1A). The black lines show the spike fidelity to light pulses, and the grey lines show the fidelity to electrical pulses. Essentially, the grey lines show what the cell is intrinsically capable of, whereas the black lines show how it fares under optogenetic control. Notice how the ChR2h134r loses fidelity at 20 Hz, whereas ChIEF only loses it at 40 Hz. This is largely because of the “off kinetics”, which means that ChR2h134r takes a lot longer to close than ChIEF (Figure 1B). And it’s only after the opsin has closed that the cell can recover its membrane potential.

Optogenetic spike fidelity of ChR2 and ChIEF, from Mattis et al.

A self test

Luckily I have access to an electrophysiology rig, so I was able to test spiking fidelity my target neurones. Namely, AgRP neurones of the arcuate nucleus of the hypothalamus (Arc). I transfected AgRP neurones with ChR2h134r, cut ex vivo slices and patched using current clamp. I then flashed the neurone with increasing frequencies of 470 nm light at a high intensity (Figure 2).

Optogenetic spike fidelity in an AgRP neurone

As you can see, the cell responds nicely with big action potentials at low frequency stimulation. But the action potentials disappear even at 10 Hz. Remember that you really need the action potential to get the response you want, whether you are stimulating the soma or the terminal. Otherwise, you really don’t know what you’re doing to the neurone, although I strongly suspect you’ll be silencing the cells. Either way, I don’t recommend flashing faster than you are able to produce action potentials.

In fact, to demonstrate why you need to limit your flashing frequency, I’ve zoomed in on the 5 Hz flashing and aligned the electrical recording with a visual representation of the likely open/closed state of the ChR2 in those cells (Figure 3). I’ve drawn the light pulses and used the published τOFF to estimate the ChR2 channel close time1.

Now imagine that you have additional pulses in between the ones shown (1 extra for 10 Hz or 3 extra for 20 Hz). Between the slow closing of the ChR2 and the slow recovery of the neurone’s membrane potential, it’s easy to see why the neurone loses firing fidelity above 5 Hz.

An important message

One of the other tests done by Mattis et al. was to simply turn the blue light on continuously for 1 second in two different neurone types. A “regular-spiking” neurone fires one action potential before being silenced, whereas a “fast-spiking” neurone fires continuously through the illumination. The point here is that some neurones can fire at 200 Hz under optogenetic stimulation (mainly cortical neurones). And if your research involves them, you probably know that.

But every neurone I’ve ever investigated wasn’t capable of anything close to that rate. So please check the firing rate your neurone is actually capable of before deciding your in vivo optogenetics frequency. Or, if you are not able to do it or get a friend to, be very careful with your stimulation paradigm. And feel free to ask someone, it never hurts to ask for help.

1. Mattis et al. Nat Methods 9(2), 159-172 (2012) Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins

How Bright is Bright?

I have previously written about the importance of brightness for in vivo optogenetics experiments. It’s just as important for in vitro optogenetics, which is what I’ll be looking at today. This came about because we’re planning a publication, and we need quantification of the light irradiance that we get on the brain slices.

When I started in vitro optogenetics, I tested the brightness of the LED system I had bought for the purpose, but not with a light meter – instead I tested directly on ChR2-expressing neurones, and found that 2 % brightness of the 470 nm LED was sufficient to elicit action potentials.

This was enough for me at the time, and I never bothered doing the metred quantification because the light meter didn’t fit under the objective (even after removing the tissue perfusion bath). However, for publishing I wanted a proper irradiance value, which meant me and our ephys technician spent an afternoon trying to dismantle the condenser under the stage. I say trying to, because microscope has been in pretty heavy use for at least a decade without any kind of service, and we found a lot of salt residue from past aCSF leakages.

Hopefully y’all cringed at the thought of that, because salt build-up inevitably means corrosion of expensive microscope parts. And, surprise surprise, we found the screws and bolts holding the condenser together and onto the microscope are all rusted in place. In the end, we managed to unscrew the top part of the condenser’s lens and wiggle the light meter in place under the objective. Phew! So now we went through a range of LED brightness and measured the brightness coming out the bottom (Figure 1).

LED brightness on my electrophysiology rig.

As ever, the brightness is not the important parameter here. What matters for activating opsins is the irradiance hitting the slice (irradiance being intensity of light per unit area). So now comes the difficult bit – how do I know the area that the light is hitting on the slice? It is possible to get a microscope ruler, put that under the objective and measure the diameter of your field of view. However, how do I know the camera or eyepiece are visualising the entirety of the illuminated area?

The answer is to go back to physics, and field of view of the objective in use. I found a useful guide from the makers of my LED’s1:

Diagram for calculating irradiance for in vitro optogenetics.

A quick investigation shows me that the Field Number for my objective is 22. Dividing that by the magnification of 40 gives a diameter of 0.55 mm. I know the area of a circle is πr2, which gives me a surface area of 0.238 mm2. So, adjusting the brightness values obtained earlier gives the irradiance output from the LED’s (Figure 2).

LED irradiance on my electrophysiology rig.

I have also plotted a simple linear regression line on the irradiance graph to give an easy formula to give a rough estimate of the irradiance at any given LED brightness. However, I will still make sure to use actual measured values in any publication, rather than the estimates obtained from the regression. Anyway, this does match up nicely with my earlier test data, as the EC50 for ChR2h134r is 1 mW/mm2, and 2 % brightness on my blue LED gives an irradiance of just over 2 mW/mm2.

For any future in vitro optogenetics studies on this rig, I will aim to use 10 % LED intensity, as this will give a solid irradiance of 10-15 mW/mm2, without going into higher saturating irradiance levels.

1. www.coolled.com

A Musing on Frequencies

This month a paper was published by one of my former colleagues, in Frontiers1. Using acute brain slices on a multi-electrode array (MEA), they investigate neurone burst firing frequency.

The MEA is a piece of recording equipment that I have never used myself, but I have seen its use. And as someone used to doing single-cell patch clamping, I am interested by the type of data you can get from it, in particular the high quantities of recordings in a short space of time.

In case anyone doesn’t know, the MEA uses an array of recording electrodes in 2D matrix, and the brain slice is set on top. This lets you record action potentials from a number of points across a region of interest, which in this paper was the NTS, PVN and SON.

The benefits of the MEA is that lets you record natural firing dynamics from many neurones simultaneously, and this enable Chrobok et al. to identify some neurones in the NTS that exhibit phasic firing behaviours (Figure 1). This was very interesting behaviour, with 5-10 Hz neurone burst firing frequency interspersed with long periods of complete silence.

My particular interest in these results is how it pertains to in vivo optogenetics, in particular trying to mimic natural neuronal firing behaviours with the stimulation pattern. I usually try to keep the experimental paradigm as straightforward as possible, so I go with 5, 10 or 20 Hz continuous stimulation.

However, it is worth noticing that Betley et al. used a phasic stimulation pattern when investigating AgRP neurone-driven behaviours (they did 20 Hz stim for 1 second, then off for 3 seconds)2. The reason they picked this stimulation paradigm is to mimic AgRP firing behaviours as seen by Van den Top et al.3 I have found this to be an interesting approach, to hopefully improve your in vivo behavioural data by trying to closely mimic the natural firing dynamics.

The importance of matching firing dynamics in an experimental setting extends beyond simply trying to mimic any information that might be encoded in such pattern. In fact, it has been known for over 40 years that phasic firing can enhance the release of neuropeptides from the nerve terminal4. This is an important aspect of optogenetics experiments that is often ignored – when stimulating neurones you are likely to be getting fast neurotransmitter release (ie. glutamate and GABA), but depending on your stimulation paradigm you may not be getting commensurate release of neuropeptide.

We have seen in our lab how this can change the animal behaviour, beyond simply increasing the degree of any behavioural response, eg. going from changes to stress hormone levels at low frequency stimulation to freezing and escape behaviour at high frequency stimulation.

So, back to Chrobok et al., who saw much slower phasic behaviour: approx. 2-4 Hz firing for 4-8 seconds, repeated every 10-100 seconds. Next time I (or someone in the lab) want to optogenetically stimulate NTS circuits in vivo, I will point them towards this paper and suggest they try phasic stimulation to see if that produces better behaviour resoponses.

There is one final point I want to take from the Chrobok paper, which is that the phasic behaviour they see in the NTS is not in sync. This is in contrast to nuclei such as the SCN which has strong synchronicity. Anyway, this got me thinking, if we intend to mimic natural firing dynamics as closely as possible, shouldn’t we try to perform unsynchronised phasic stimulation of the NTS neurones?

I envisage an AAV that has multiple opsins, responsive to different colours of light, whose activity is randomly chosen by mixed LoxP sites, similar to the Brainbow construct. For example, if we had an AAV with ChR2(h134r), C1V1TT, and Chrimson (Figure 2), after a stop codon so you get no expression, but under cross-reactive LoxP sites such that cre will randomly switch on one of the variants only, you could produce a selected population of neurones (eg. TH neurones in the NTS) expressing a variety of opsins.

Then you would need to set up an in vivo optogenetics stimulation system that could switch between 450 nm, 530 nm, and 620 nm, allowing you to produce desynchronised phasic behaviour among that single population. You could easily set it up to cycle through flashing blue light for 5 seconds, then green light for 5 seconds, then red light for 5 seconds to produce desynchronised phasic behaviour.

Furthermore, given that you can easily get multiple insertions in a single neurone, you might well have some members of the population expressing more than one opsin variant, which would give you further variety in phasic behaviour, and even some that simply fire continuously. This is unfortunately A LOT of work simply to see what happens to the behaviour when you try to closely mimic natural phasic firing dynamics, so, while someone out there might be brave enough to do something like this, I don’t think I or anyone in my lab is likely to.

1. Chrobok et al., Front Phys 12, 638695 (2021) Phasic neuronal firing in the rodent nucleus of the solitary tract ex vivo.

2. Betley et al., Cell 155, 1337-1350 (2013) Parallel, redundant circuit organization for homeostatic control of feeding behavior.

3. Van den Top et al., Nat Neurosci 7, 493-494 (2004) Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus.

4. Dutton and Dyball, J Physiol 290, 433-440 (1979) Phasic firing enhances vasopressin release from the rat neurohypophysis.