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The necessity of a big picture science

Juan García Ruiz
July 26th, 2022 · 12 min read ·
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The hippie movement that seeked to alter consciousness with drugs such as LSD and marijuana coincided in time with the most prodigious research on highness. The discovery of the receptors involved in cannabis mechanism of action not only helped us understanding what happens when people get high, but also led to the discovery of a crucial nervous modulation system involved in multiple physiological functions like learning, memory, sleep and food intake. The endocannabinoid system involves receptors such as CB1 (remember this, it will be helpful in a little bit) and little lipids synthetized by our system that are able to bind to those CB1 receptors. These lipids are known as endocannabinoids and are very similar in structure to tetrahydrocannabinol (THC), the psychoactive component of marijuana (Figure 1).

This is the alt text small image
Figure 1. Chemical structures of cannabinoids. Exogenous on the left and endogenous on the right.

Let’s now focus on the endogenous cannabinoid system, the physiological one that functions in our system by default with no need of exogenous cannabinoids like THC. How does it work? CB1 receptors are not the only cannabinoid receptors expressed in the brain, but are the most numerous. They are spread across all brain regions, and depending on where they are activated the final output can be different (regulation of food intake, pain, temperature, etc). However, the molecular mechanism of action remains essentially the same. What is that mechanism? CB1 receptors are located at the presynaptic level, in the membrane of a neuron A that communicate with a postsynaptic neuron B by releasing neurotransmitters. When the neurotransmitters are released and bind to the receptors present at the postsynaptic neuron, this one will respond to that by opening or closing different ionic channels, so that its electric potential will change. This will result in subsequent postsynaptic effects: action potential triggering, release of other neurotransmitters, intracellular changes, etc. Among the cellular processes that can take place at the postsynaptic level after neurotransmitter binding there is the release of endocannabinoids, that can go back to the presynaptic neuron. What happens next? When the endocannabinoids bind to presynaptic CB1 receptors they reduce calcium influx by blocking some voltage-gated calcium channels, an essential element for neurotransmitter release. In addition, the binding also leads to an increase in potassium efflux through a channel known as GIRK (G protein-coupled inward rectifier K+), so the neuron is hyperpolarized and the probability of spontaneous action potential occurrence is reduced. In other words, neuron A sends neurotransmitters to neuron B, and B responds back by sending endocannabinoids and telling the neuron to calm down, to be less excitable and to send less neurotransmitters. This mechanism of retrograde transmission helps the neuron to finely regulate the amount of transmission. It is a way of properly tuning the communication between neurons.

Now you understand the basics of the endocannabinoid system. But there are so many interesting things to know about this exciting field. If you want to become a little expert on one of the most important systems in our brain, you should definitely meet Giovanni Marsicano. Luckily for you, we have interviewed him so you just need to carry on the reading. And do not worry, the molecular part is over!

Giovanni Marsicano has a Veterinary background. However, he realized very early that he wanted to do research instead of following the clinical path. At the very beginning, he worked for some time with embryonic stem cells in animals. Later on, he started a neuroscience PhD in the lab of genetics and behaviour at the Max Planck Institute of Psychiatry in Munich in the mid-nineties. At that time conditional mutagenesis was quite trendy as a tool to study cognition. His boss at that moment was Beat Lutz. He asked Giovanni to pick a gene to study its role in learning and memory. But neuroscience was a new thing for Giovanni, so he spent a whole summer reading scientific articles to pick a proper candidate. Most of publications were something like this: gene A is deleted, learning and memory are impaired, paper in Nature; gene B is deleted, cognition is affected, paper in Science; etc. Lots of papers published back then consisted of a loss of function of genes that were important to maintain memory, like the ones coding for kinases. Giovanni started to wonder about genes that worked in the opposite way, namely regulating memory by decreasing it. He had two candidates: phosphatases (working in the opposite direction as the mentioned kinases) and cannabinoid receptors. Then he thought that by deleting one of those genes cognitive abilities of mice may be improved. The phosphatase he was interested in was MAP kinase phosphatase (MKP), and the cannabinoid receptor candidate was CB1. How did he make the choice? Using a very scientific method (note the irony). Car plates in Munich have an “M” on them plus two other characters. He told himself that if he saw a license plate with the letters MKP on it, he would pick the MAP kinase phosphatase. He did not see it, and this made him one of the most important researchers studying cannabinoid receptors around the world.

Juan García Ruiz: Your lab is internationally known for the study of cannabinoid receptors. What are these receptors, in a nutshell?

Giovanni Marsicano: They are regulatory receptors. You could survive without them but they are very important for the fine tuning. I will explain it with an analogy. When you use a microscope you have a coarse-grained wheel that allows you to change the depth at which you position the focus quickly but with low precision, and then you have a finer wheel that allows you to position the focus exactly at the depth layer you want. Cannabinoid receptors are like this fine wheel, and they tune the general functioning of the body.

JGR: When did CB1 receptors appear in evolution?

GM: The first elements of this system that appeared were the endocannabinoids, which are lipids that act as ligands of cannabinoid receptors. Early species showed the ligands I just mentioned but not the receptors. Reptiles are the first species to show something that really looks like a cannabinoid receptor. There is the idea that one of the main functions of cannabinoid system is favoring energy accumulation. Energy accumulation is a feature that allow species to prepare to face an uncertain future in which energy will be needed and maybe the sources will not be available. The specialization of energy accumulation comes with fat. The adipose tissue turns out to be present already in reptiles. Otherwise said, cannabinoid receptors appear in synchrony to adipose tissue and their specific ability to accumulate energy.

JGR: Are CB1 receptors expressed out of the nervous system?

GM: Yes. It is expressed in the adipose tissue, the liver, the lungs, the kidneys, the skin, etc. A little bit everywhere. In the brain, CB1 receptors is the most abundant GPCR (editor’s note: G protein-coupled receptor). The levels of CB1 are similar to NMDA receptors and GABA-A receptors. Some people propose that there is a big excitatory system that is glutamate, a big inhibitory that is GABA and a big modulatory that is CB1.

JGR: What are the main discoveries about CB1 receptors in the recent times around the world?

GM: Benjamin Cravatt, a professor in the Department of Chemistry at The Scripps Research Institute in California, has been working on signalling lipids like cannabinoids. He made great contributions to the field of cannabinoid receptors by proposing enzymes involved in their synthesis and degradation. CB1 are lipid receptors, and lipids are very difficult to study for technical reasons.

JGR: What kind of questions are you trying to answer in your lab?

GM: Our lab has a specific philosophical background. The brain is a complex, redundant and connected machine. This means that by using the typical scientific approach that is specialization, there is a risk of loosing some part of information. If you go too much into details then you do not see the general picture, which is very important in this field. If you take a fruit fly and a human, there are no enormous differences at the smallest levels. The great differences come when you consider the big picture, and that is why it is so important to find a good balance in specialization.

The good thing for us is that CB1 is involved in so many things in the brain that we are highly specialized since we study just a few different proteins, but at the same time we go in many different directions, so we keep the “big-picture mindset”. For instance, CB1 have different functions in neurons, astrocytes, and microglia. So another way to see it is that we use CB1 to understand the complexity of the brain.

JGR: Now that you mention it, what is the role of CB1 in astrocytes?

GM: Astrocytes are very interesting cells. They have always been believed to have structural, protective and feeding roles in the brain. But some years ago researchers realized that astrocytes were much more than passive neuron helpers. This came with the idea of the tripartite synapse that includes a presynaptic neuron, a postsynaptic neuron and an astrocyte that can also take part in the signalling. Neurons release neurotransmitters that can bind to astrocyte receptors, and these cells can respond by releasing gliotransmitters such as D-serine. The work of Alfonso Araque was key to understand that CB1 is expressed in astrocytes and is one of the important actors in the tripartite synapse. This had great implications because it contributed to the idea that astrocytes are not passive and can be at the origin of behaviour through interaction with neurons.

JGR: What is the mechanism of astrocyte-neuron interaction you are talking about?

GM: One of the effects of CB1 receptor in astrocytes is the increase of calcium. This leads to the release of gliotransmitters. The interesting thing is that now there are two worlds coexisting: the world of metabolic astrocytes, and the world of synaptic astrocytes. These two worlds used to be a little bit disconnected, they did not talk to each other, and this was a mistake! What I am trying to do at this moment is to put these things together without excluding anything.

JGR: Did your lab made a discovery that you are particularly proud of?

GM: Yes, the discovery of the mitochondrial CB1 receptor. I am proud in the sense that it was very hard to defend, and it is an example on how science can be the result of serendipity. First, I need to give you a little bit of background. THC, the psychoactive component of marijuana, was discovered in the forties and became very famous in the sixties. For almost 20 years we did not know how THC was working in brain cells. If you look at the publication rate related to this topic, you have a peak in 1964. It was something very cool at that time to put THC on everything and say you were working on highness. Some of the publications that came out suggested a mitochondrial effect of THC. Then in 1990 the receptor of THC, which is a GPCR, was discovered. But something weird was going on: GPCRs are not present in mitochondrial membrane and are by definition present at the plasmic membrane. So the results were explained as a non-specific effect of THC: mitochondrial membrane is sensitive to lipids, and cannabinoids are lipids, so if you use a lot of lipids you can alter mitochondrial membrane and therefore mitochondrial functioning. Fine. So the data suggesting that THC affected specifically the mitochondria were discarded.

JGR: But then you proved that CB1 could actually be expressed on mitochondria. How?

GM: I met Pedro Grandes, a neuroanatomist from Achucarro Basque Center for Neuroscience, I asked him: have you ever seen CB1 on mitochondria? And he told me: yes, but everything we see is just background staining. He explained that what people in the field were doing was to normalize the CB1 expression in the plasma membrane to its expression in the mitochondria, considered to be noise because it was believed to be impossible to have this receptor expressed there. Then I sent him some knock out animals (editor’s note: animals modified genetically so that CB1 receptor was not expressed) just to see if this background staining he saw in the wild-type animals (editor’s note: non-genetically modified animals) was still present. After several months, he came back to me with images that he took with electron microscopy by using immunogold staining on CB1 receptors. I did not even remembered about this. And surprisingly we found out that knock out animals did not show this background, suggesting that the signal we observed at the beginning was not just noise, but actual mitochondrial CB1 receptors! We received a lot of criticism. A paper was written to directly attack our discoveries by saying that everything we saw was background, and then we replied with another methodology paper comparing our methods with theirs, and we concluded that their method was not sensitive enough to detect the receptor, and the difference of the results came from this methodological problem. The way we proceed nowadays is by trying to refute the existence of this mitochondrial CB1 receptor, but so far we have not managed to do that! So this is the discovery I am the most proud of.

JGR: What philosophy do you try to foster in your lab?

GM: One idea I like to promote is the one we talked about before: in neuroscience we have to be specialized, but at the same time open to understand other subfields and try to have a general vision. As for the human aspect, when people come to my lab to work, I always tell them I do not care how many hours they do, how many holidays they take. I really do not know what they do, when I have to validate something I do it automatically. I want people in my lab to be aware that they do not work for me, but for themselves (with me). If I tell you to do this and that because I like it, but turns out you do not like it, then it will not work! So telling people to do everything for themselves makes them more happy to work. And it is important because our work is crazy. We have very few satisfactions, but it seems to be enough. This small thing you get from time to time, a stupid graph that shows you something worked, makes you happy for months. So you need to be a bit crazy to do science.

JGR: What are the most valuable features that a good scientist should have according to you?

GM: The first one is curiosity for sure. One needs to read a lot, and not only scientific stuff. The second is rigour, self-criticism. I surround myself of science-policemen. For instance I was very lucky to start working with Francis Chaouloff fifteen years ago because he is a real policemen. Whenever I am enthousiastic about something he will make me calm down, check the statistics, the controls, and every kind of detail that we could overlook by mistake. When you follow something you do not see the rest. Cristopher Stevens, a student from the neurocampus was working on this, on the confirmation bias. You see physically only the things that confirm what you think at the beginning. But it is also important to keep the enthousiasm or if you prefer the craziness, and this would be the third feature. There are some jobs that people choose for convenience: when you work in a bank, or when you are a lawyer. Even if you do not like your job, you can do it for convenience and then do nice stuff with your life. But other jobs are actually your life: when you are an actor, or a musician. You do not choose this for convenience. I think being a researcher is more similar to the second type of job, where you need some vocation to overcome all the stressing situations and so on. So to sum up a scientist needs to have curiosity, rigour and enthousiasm.

JGR: Would you give an advice to the readers?

GM: For young people, what I always say is: understand what you like. It takes time to know what one likes. I know it is not their fault, but I sometimes see students that come and say: so now I am in my first year of master, next year I will do the second one and I will do my internship this lab or this other one, then I want to do a PhD here or there, and they have everything figured out from the beginning! All I can tell them is: good luck. I am sure that nothing will go as it was planned. A lot of things happen by chance. What I can suggest them is to try to understand what they like and to be as open-minded as possible, to keep eyes and ears as open as possible, and not to be afraid to ask questions.

JGR: Would you like to recommend a scientific book that changed the way you see things?

GM: I would like to recommend a book by Robert Sapolsky called Behave. He also made a series of courses that he recorded with the University of Standford almost 15 years ago now. They are available online. The idea is that he takes a behaviour and he tries to analyze the causes of this behaviour. There is a cause occurring some milliseconds before the observable behaviour (neurons that fire), another cause occuring minutes before, one hour before, twenty-four hours before, years before, and even one-million years before (how the behaviour evolved). We are all talking about the same behaviour, but we approach it differently. He says something really interesting about the rainbows, related to how we categorize differently the same things. In some countries and cultures they see some bands of colours that for instance in Italy we do not see. Depending on our culture and how we name things, we use different boundaries to distinguish things, and this applies also to the understanding of a behaviour. We create these boundaries to give sense so the world and understand it, but they are not real.

JGR: Do you have a final comment you would like to share?

GM: Something I would like to explore more deeply and encourage people to explore as well is evolution. In my lab we always go out to celebrate the birthday of Charles Darwin. This is not only to have fun. In my opinion to understand how things work we should not forget about how they evolved to get to that point. I am not saying that Charles Darwin is like the Holy Bible, but we celebrate its birthday because his character is like a symbol for evolution. Nowadays you hear about Lamarck and you almost laugh. The explanation of natural selection of Darwin has been key for us. But I realized after reading The Origin of Species that Darwin was not so in disagreement with Lamarck ideas of transmission of acquired traits. And nowadays we have epigenetics, which is actually not going against Lamarck. The problem with human is that we like to build churches and create dogma even in science. My last comment for the readers is to avoid building churches.

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