When the stress gets too much

It’s not easy being an electrophysiologist. Most of the time we’re under a lot of pressure and not just from our supervisors and managers, but from ourselves too. Sometimes we go for weeks, even months, without a single successful experiment. We know that it has worked for us before, but now that that all important deadline is coming up, electrophysiology refuses to play ball.

Very true

Very true

When asked “what’s wrong?”, most of the time the answer will be “I just don’t know”. This is not because electrophysiologists are lazy and have no idea what they’re doing. On the contrary, we’re very acutely aware that many, many different things have to be just right in order for an experiment to work – and sometimes even that is not enough.

Disaster can strike at any time

Disaster can strike at any time

Going through the list of things that have to be absolutely perfect can be time consuming, but any electrophysiologist worth their salt will have a vague idea of what the weak links are likely to be.

Some days are a bit 'meh'

Annoying

Some days the electrodes  are not absolutely perfect

Sometimes the problem can be really obvious.

Another experiment ruined and a day wasted

My experiment may not have worked, but I do enjoy a bit of DIY

I hate it when the perfusion system doesn't co-operate

Occasionally stress can be released in the form of exclamation marks and profanities.

Sometimes the tools you work with can let you down and spoil your day

And right after I put on some drugs too

Sometimes it's the computer program that can let you down

But when the stress gets too much, sometimes the best thing to do is walk away, have a cup of tea and calm down.

Next time you see an electrophysiologist alone in the corner with a cup of tea, approach with caution

Next time you see an electrophysiologist alone in the corner with a cup of tea, approach with caution

 

Be careful! Sometimes pent up stress can erupt when you least expect it:

A group of PhD students were in the breakout space one afternoon, taking a break from work. Everyone was happily chatting away and conversation turned to favourite Family Guy sketches. Someone quoted a particularly funny phrase and everyone laughed. One person in particular laughed so hard, he could barely breathe. Everyone starts laughing again and this time two more people laughed so much that they struggled to speak. The uncontrollable laughing continued, but not everyone was doing it anymore. Only three people were laughing, but their sounds of laughter started to sound more and more like sobs. There was a bit of an awkward silence and one by one people started to leave and return to the lab. The PhD students wiping their eyes and taking deep breaths looked round at each other and realised that they were electrophysiologists.

Like I said before: if we didn’t laugh, we’d cry and believe me, we’ve cried a lot.

You have been warned

 

“So what do you do?”…

We’ve all been there. Whether it’s at a family gathering, a friend-of-a-friend’s party or in the queue for the beer festival. At some point we’ve been asked by someone we vaguely know (or a complete stranger) “so what do you do?”.

Sometimes we’re waiting for it, as sooner or later the small talk will turn to what we do on a day-to-day basis. Sometimes the question is completely unexpected and results in a slightly blank expression as we’re trying to gather our thoughts.

I’m always ready to answer this question.

“So what do you do?” Well, the general area of my research is using cannabis to treat epilepsy.

Photo courtesy of Marcus Haag

Presenting my research in the form of a Science Slam. Photo courtesy of Marcus Haag

As you can probably imagine, people tend to latch on to the ‘cannabis’ part of that statement and I’ve found that I usually get two types of responses. I either get something like “Wow! That’s really cool” or “Hmm, ok”. To be honest, I’m not surprised that some people are sceptical about using an illicit drug to treat a disease – I certainly am! But the thing is, there’s so much more to it than simply giving cannabis to a person with epilepsy.

The cannabis plant contains around 500 chemicals. Of those ~500 chemicals, approximately 100 are unique to the plant and are known as cannabinoids. Of those ~100 cannabinoids, only 1 produces the euphoric effects associated with taking cannabis. This chemical is known as ‘delta-9-tetrahydrocannabinol’, or simply ‘THC’ for short. It’s described as being ‘psycho-active’ because it binds to, and activates, cannabinoid receptors that are expressed within the brain (CB1 receptors).

What about the other 100 or so cannabinoids found in the plant? Well, some of these have been studied in a variety of disease states including cancer, diabetes, multiple sclerosis, ulcerative colitis, rheumatoid arthritis, schizophrenia and epilepsy. The main one studied is known as ‘cannabidiol’, or simply ‘CBD’ for short. But why is CBD so special? Well, it’s NOT psycho-active. It doesn’t produce the euphoric effects that THC is famous for because it simply doesn’t bind to, and activate, CB1 receptors. It’s also the second most prevalent cannabinoid found in the cannabis plant, which makes growing and extracting it relatively easy.

“If CBD doesn’t bind to CB1 receptors then how does it work?” The thing is we just don’t know. There are many other receptors that have been reported to be implicated in the mechanism of action of CBD, such as the ‘capsaicin receptor’ (TRPV1), the most common serotonin receptor subtype (5-HT1A) and the ‘orphan cannabinoid receptor’ (GPR55). However, none of the effects of CBD at these receptors are convincing enough for the world’s leading cannabinoid scientists to stand up and say “CBD is a potent agonist/antagonist at this receptor and that is how CBD mediates all of its pharmacological effects”.

“What has all this got to do with your PhD?” Over the last couple of years there’s been a surge in interest, particularly the USA, in people with epilepsy using whole cannabis extracts to reduce the frequency of their seizures. This isn’t anything particularly new as cannabis has been used as a medicine for thousands of years. However, the thing that has made people sit up and notice is the fact that some children with severe epilepsy, in which their seizures cannot be controlled and is therefore life-threatening, have been given extracts of cannabis, in which CBD is the main component, and their seizures have dramatically reduced in both severity and frequency. You can read a couple of articles from CNN here: Jayden David and Charlotte Figi.

Unfortunately there are some issues with using homegrown cannabis preparations such as medical marijuana.

Firstly, the preparations still contain a significant amount of THC, despite growers’ best efforts to produce plants that predominantly contain CBD. Also, we still have no idea what the impact of regular THC exposure has on an adult’s brain, let alone a child’s brain and their neurological development.

Secondly, the preparations are unregulated and inconsistent. The relative proportions of cannabinoids present will vary depending on the growing conditions, the methods of extraction and the preparation of the final product. This is a major problem for people with epilepsy. I spoke to someone from California, who sometimes takes medical marijuana for their epilepsy, and he told me that whenever he went to the dispensary to get medical marijuana he had no idea what he was getting in terms of THC levels. He wants to be able to lead a normal life and have a job, but he can’t do that when he’s high on medical marijuana with too much THC in it. Also, in terms of being able to control seizures, it’s extremely important for medication to be consistent. Even a slight change in regime, dose or drug can potentially result in rebound seizures and do more harm than good.

Finally, there are legal restrictions on the use of cannabis-derived medicine in research. Currently in the USA, CBD is classified as a Schedule 1 controlled substance and federal law prohibits its use, even for research, despite the fact that it is NOT psycho-active. Yet, paradoxically, medical marijuana containing THC is available for ‘medical’ uses in about a third of the states in the USA. When I attended a conference in San Diego, California last year, I was astounded by the sheer number of American researchers telling me how incredibly lucky I was to be able to do research on plant-derived cannabinoids (particularly CBD).

I personally think that we need to move away from medical marijuana and pay more attention to regulated botanical drug substances (BDSs) derived from cannabis, in addition to isolated and purified non-psycho-active phytocannabinoids. Thankfully this has already been going on for many years by the pharmaceutical company who sponsor my PhD (GW Pharmaceuticals).

Presenting my research at Neuroscience 2013. Photo courtesy of Immy Smith

Presenting my research at Neuroscience 2013. Photo courtesy of Immy Smith

“OK, but I still don’t get what your PhD is all about…” Right, so I’ve already explained that (anecdotally) cannabis and components of the cannabis plant (eg. CBD) can treat people with epilepsy. For over a decade now, scientists have been conducting pre-clinical studies in order to put together enough evidence that justifies putting non-psychoactive plant-derived cannabinoids through clinical trials for epilepsy. This basically means that several PhD students, like myself, have been doing experiments on live animals or tissue taken from dead animals, in order to justify giving these drugs to people who have a life-threatening disease. I must also point out here that the animals used in these experiments are rats and mice, and that every person doing experiments with live animals MUST hold a personal Home Office license – I will go into more detail about this another time.

Preparations for electrophysiological experiments. Photos courtesy of Tom Hill

Preparations for my electrophysiological experiments on fresh rat brain. Photos courtesy of Tom Hill

So, clinical trials are currently underway at GW, which means that we know at least 2 non-psycho-active phytocannabinoids (CBD and its propyl analogue CBDV) definitely work in animal models of seizure and epilepsy.

But the burning question is “How do these compounds work as anti-convulsant and anti-epileptic drugs?” Well, that’s where my PhD project steps in. I’ve been doing electrophysiological experiments for the last 3 years in an attempt to figure out how these compounds, CBDV in particular, work as anti-convulsant and anti-epileptic drugs. I do this by taking recordings from individual brain cells, as well as networks of brain cells, from fresh rat brain slices. I record what brain cells do under ‘normal’ conditions and then record them again under ‘drug’ conditions and see if there’s a difference. I also record the ‘normal’ and ‘drugged’ activity of brain cells from rats that were epileptic. To then add another level of complexity, I compare the differences between non-epileptic brain cells and epileptic brain cells in terms of their response to the drug.

“Yikes! Now my brain hurts” If you want something that’s a little easier to understand (and A LOT more fun) then you can watch my Science Slam.

“So what have you discovered?” That there are no easy answers in science. Every time I think I’m getting close to answering a particular question, more questions spring up in its place. I suppose in that regard, conducting scientific research is a bit like fighting the Lernaean Hydra. I’ve also discovered that electrophysiology is a cruel and fickle mistress.

In terms of actually discovering something that makes a substantial contribution to science, I’m afraid I can’t say at this point. This is not necessarily because I haven’t found something, it’s more about keeping everything under wraps until I am permitted to reveal all. But don’t worry, there are a few papers in the pipeline.

You can read more about the case for medical marijuana in epilepsy, the case for assessing cannabidiol in epilepsy and a critical review about CBD and its therapeutic role in epilepsy and other neurological disorders for free in the journal Epilepsia.

 

05/08/2014 edit: It’s been brought to my attention that the Epilepsia articles are no longer free to view. If you really want to read them, but can’t get access – let me know and I’ll see what I can do 🙂

For the love of brain (well, the hippocampus) II

So I’ve already described how much I love being able to look at live, functional neurons in real time. But there are other things I enjoy seeing everyday too, in particular my favourite part of the brain; the hippocampus.

The rodent hippocampus in stained, coronal sections

The rodent hippocampus in stained, sequential coronal sections

At the start of every patch clamp experiment, when I look down the microscope at a brain slice, I need to locate a specific structure before I can focus on finding individual neurons. This structure is called the hippocampus (so called because a cross section of it looks like a seahorse) and every brain has a pair of them (one in each hemisphere). The hippocampus is very special because it has a well-defined and distinctive structure, which makes it instantly recognisable to anyone who has studied neuroanatomy.

Freshly cut transverse brain slices. I store them in a plastic tea strainer, submerged in  carboxygentated aCSF. The arrow points to where the hippocampus is loacted

Freshly cut transverse brain slices. I store them in a plastic tea strainer, submerged in carboxygentated aCSF. The arrow points to where the hippocampus is located

 

Acute, transverse hippocampus as seen during my patch experiments - notice the patch electrode emerging from the CA1 region in the right-side image

Acute, transverse hippocampus as seen during my patch experiments – notice the patch electrode emerging from the top of the CA1 region in the image on the right

As I’ve said before, the brain is not a homogenous blob, but contains many intricate and beautiful structures. Many of these structures look pretty much the same across the majority of species, no matter what size or shape the brain may be, and the hippocampus is no exception.

Drawing by Camillo Golgi of a hippocampus stained using silver nitrate

Drawing by Camillo Golgi of a hippocampus stained using silver nitrate

Why do I love the hippocampus? Well, for one thing I love how ‘organised’ the anatomy is (I won’t go into detail here, but I do find the Wikipedia page extremely useful). The hippocampus is a network and I love how you can stimulate a particular pathway and get other neurons to fire in response (ie. stimulate the axons of the CA3 pyramidal neurons and the CA1 pyramidal neurons will produce a response). I also love the fact that it’s involved in lots of different aspects of learning and memory.

So, going back to the point about being able to stimulate pathways in the hippocampus, I also do a lot of experiments using multi-electrode arrays (MEAs). Being able to stimulate particular pathways and measuring evoked responses can tell you a lot about the synapse involved and the neurons on either side. For my experiments, I stimulate the Schaffer collaterals (axons of CA3 pyramidal neurons) and record the responses of the CA1 pyramidal neurons. Instead of observing signals from individual neurons, I look at the combined response of a whole population of neurons, which all fire in unison when stimulated by an extracellular electrode.

I take a photograph (before and after!) of every hippocampus I use for my MEA experiments. It allows me to select which electrodes to stimulate so that I can record evoked field potentials

I take a photograph of every hippocampus I use for my MEA experiments so that I can select which electrodes to stimulate in order to produce evoked responses. This is not a stained section – it’s the actual colour of a ‘living’ hippocampus!

I’d love to go into more detail about MEA experiments, but I’ll save it for another post 🙂

For the love of brain

What gets me out of bed in the morning and into the lab by 8.30am?

Answer: The love of neuroscience.

 

I freeze my aCSF in the shape of little hearts <3

I freeze my aCSF in the shape of little hearts

 

I absolutely love the brain. It’s such a complicated, yet incredibly delicate, structure and was the last major organ I learnt about during my 20+ years of full-time education. Of all the things that humans have discovered and achieved in the last few thousand years, the brain still holds a lot of secrets.

For every experiment I did during my PhD I had to cut very thin slices of fresh rat brain. The slices were 300 micrometres thin, which is just under a third of a millimetre (0.3 mm). They had to be this thin in order for light to shine through them so that I could see individual neurons for my patch clamp experiments.

 

This is what I see when I do my patch clamp experiments

This is what I see when I do my patch clamp experiments – note the patch clamp electrode approaching the middle cell from above. The cells are pyramidal neurons (because they are triangular in shape) from the hippocampus.

 

I really enjoy seeing actual neurons in situ. There’s nothing quite like it in my opinion. It got even better when I would successfully ‘patch’ a cell and see the wiggly lines on my computer screen form the distinctive shapes of action potentials, which are the electrical signals that neurons use to communicate with each other.

In the very beginning I found it slightly odd that the smooth spiky structure on the black and white screen was able to produce the electrical signals on the oscilloscope without looking as though it had changed in some way. In other experiments I’ve done that involved living tissue, there was always something that moved to show that it was still alive and functioning properly. But when it came to successfully patching a neuron in a fresh brain slice, I suppose I was half expecting the neuron to contract or flash or something. But no, it just stays there, unmoving until you’re finished with it (unless it’s very unhappy with having a hole in its membrane and decides to swell and die).

Neurons aren’t the only things I see in brain slices. A lot of the time I see red blood cells, usually in capillaries. I think it’s really cool and it also gives me a better sense of scale when comes to visualising how big neurons are (as it turns out, they’re bigger than red blood cells).

 

A capillary in a brain slice - pretty cool huh?

A capillary in a brain slice – pretty cool huh?

 

Being able to see living, functioning neurons in real time isn’t the only thing that gets me out of bed in the morning, but that’s another story 🙂

 

What not to say to a patch clamper

1. “It’s character building”

Often said when you’ve been banging your head against a brick wall for weeks, trying to get your experiment to work.

I agree, it is character building when you’re just starting out – I think it’s really important to instil the notion that patch clamp is incredibly difficult and frustrating. But when you’ve been doing it for at least a year with virtually no data to show for all those weeks and months of “character building”, then the person saying this is in danger of getting stabbed in the eyes with patch electrodes.

 

Death by patch electrodes

Death by patch electrodes

 

2. “I can see lots of cells”

The person saying this is either looking over your shoulder at a screen, or looking down a microscope at a cover-slip.

Yes, they think  they can see lots of cells. They assume that all those perfectly round, transparent blobs are cells that can be patched. But the truth is is that all those “cells” are in fact dead. Some are so dead, that all that’s left are ghosts of cells. When hunting around for that one reasonable-looking cell; a cell that can temporarily alleviate your misery, the last thing you need is someone with an untrained eye making completely pointless, unhelpful and incorrect observations.

 

3. “Are those miniature currents?”

Similar to the above, where someone (usually a PI) looks over your shoulder at a computer screen showing the latest trace of your cell’s electrical activity.

Once again, they think they can see miniature currents and yes, those currents are quite tiny. What they fail to notice, however, are the signs of a crappy cell, let alone the fact that the cell isn’t being perfused with any drugs (in order to isolate the miniature currents).

The same can be said for the relatively huge spontaneous currents. No, they are not inhibitory postsynaptic currents because I haven’t actually isolated them yet. I’m waiting for you to go away so that I can dash around the lab and prepare my solution before this cell dies.

 

4. “When will you have this dataset complete?”

How long is a piece of string? In an ideal world I can get 6 replicates (the minimum amount I need to run a statistical test) in a couple of weeks. The reality? A couple of months.

 

5. “Can you just do a quick ‘look-see’ experiment for Dr So-and-so?”

Seriously? You think that I can just do a ‘quick’ patch clamp experiment? For someone else? In a different part of the brain? With a completely different cell type and shape? On top of all the other experiments I have to do?

How about….. no.

Patch clamp