The effect of cannabis on synapses

One of the most cited articles in cannabis research was published in 1995. The experiments of two young researchers, István Katona and Beáta Sperlágh, from the research teams led by Tamás Freund and Szilveszter E. Vizi, proved that"the receptor for the active substance of marijuana/cannabis is located on nerve endings and inhibits the release of neurotransmitters. Based on their results, it was predicted as early as 1999 that the physiological role of cannabinoid receptors is to regulate synaptic feedback processes." As even this simple sentence may contain statements that may not be understood by those unfamiliar with the subject, it is best to consult an expert. It is for sure that Benjámin Barti, a member of István Katona's research team and the first author of a recent article in Science Advances, a highly respected scientific journal, can be called an expert.

- Perhaps it is best to start at the beginning!

- Let's start with the characteristic of our nervous system that the nerve cells can communicate with each other through synapses. In the process, the sending cell releases a transmitter of excitatory material sensed by specialised receptors in the receiving cell. As a result, ions carrying electrical charges enter the receiving cell, resulting in measurable changes in electrical potential. The strength of a synapse is determined by the reliability of the contact between the two cells, the amount of excitatory substances released between them and the magnitude of the resulting measurable electric current.

- This does not sound simple!

- It is not! The strength of individual synapses fundamentally influences many network-level processes, such as learning or the recording of memory traces, so they are therefore under precise temporal and spatial control. The endocannabinoid signalling pathway is one of the most common regulatory systems to fine-tune the synapses. This system propagates from the receiving nerve cell to the sending nerve cell. In other words, it performs backward regulation to synaptic signalling.

- So we have come to understand the meaning of retrograde synaptic signalling, and more specifically, cannabinoids, or more specifically, endocannabinoids. . . 

- The cannabinoid receptors are located on the nerve cell sending the signal, i.e. presynaptically, and the endocannabinoid molecules produced by the body are usually released from the receiving nerve cell, i.e. postsynaptically, for a short period. This signalling pathway helps cells avoid overstimulation, which could lead to neurotoxicity or even epileptic seizures. 

- A brilliant solution! What else do we know about endocannabinoid signalling?

- We already know a lot about the so-called phasic form of endocannabinoid signalling, which is the focus of most research. However, there is another, more specific form, called tonic cannabinoid signalling, which continuously regulates the function of the given synapses. Although damage to this signalling pathway is observed in many neuropathological conditions, very little is known about the exact principles by which it works. So one of our questions was how tonic cannabinoid signalling affects the strength of synapses and which cannabinoid molecules regulate this process. The study of this system is important, among other reasons, because cannabis, also known as marijuana, also acts through the endocannabinoid system.

- What was the starting point for your work?

- One of the two studies published in 2015 by our research group investigated different forms of endocannabinoid signalling and their physiological effects on synapses. The results of a cell-type-specific super-resolution microscopy study published in Nature Neuroscience in 2015 showed that THC, the psychoactive ingredient in marijuana, significantly reduces the number of cannabinoid (CB1) receptors at nerve endings. My colleagues also looked at the effect of THC on the distribution of receptors. 

Immediately, the question arose: what physiological changes in synaptic communication result from a reduction in CB1 receptor number due to cannabis consumption, and whether the ratio of CB1 receptors to molecular complexes regulating the release of synaptic signalling substances affects the strength of synapses? 

- What tools helped to answer this question? 

- First, we needed to develop a method that would allow us to measure the physiological properties of a single identified synapse within brain tissue, both at baseline and after pharmacological treatment, and also to count the number of CB1 receptors in the same synapse. 

- This does not seem to be a simple task, not only because of the size of the synapse, not to mention the much smaller size of the receptors!

- A nerve ending is roughly 1 micrometre in diameter, and a synapse is a fifth of that, about 200 nanometres, not almost incomprehensibly small, but also the limit of the resolution (resolution) of classical light microscopy! Adding to the difficulty of the task, the cannabinoid receptor is one of the most abundant G-protein coupled receptors in the brain, crowding the surface of the investigated nerve endings. Using classical light microscopy methods, accurate quantitative analysis is therefore impossible. For our studies, we used a super-resolution single molecule detection microscope to observe the number and location of receptors around the synapse at the nanometre scale, which we could then couple with physiological data. 

- Another experimental challenge, if I'm not mistaken!

- In the first step, called paired electrophysiological recordings, two thin glass capillaries of roughly 2 micrometres in diameter were used to puncture the pre-and postsynaptic cells in living brain tissue to specifically measure the strength of the synapses between them and the extent to which this is regulated by cannabinoid tone. The two cells were loaded with markers to identify the synapses between them for subsequent morphological reconstruction in the 10-micrometre-thick slices of brain tissue. (This 10 micrometre corresponds to one-tenth of the thickness of the average human air.)

Once this was done, super-resolution imaging and data analysis could be performed. 

- It doesn't seem easy, even when you put it like that!

- The work required extraordinary individual attention from each researcher at every step and precisely coordinated teamwork. The contributions of all the authors and contributors to this article were essential to the study!

- My heartfelt congratulations and sincere appreciation to everyone who contributed to the results of this article! What was the most important and unexpected result? 

- First, I would like to mention something already hinted at in our 2015 Journal of Neuroscience article but is still considered unexpected in cannabinoid research. It is the existence of a form of tonic cannabinoid signalling that works in the absence of the two main endogenous cannabinoid-producing enzymes. 

Second, that cannabinoid tone is set by the amount of active CB1 receptors around the synapse. We proved it using a super-resolution microscope, which can identify the location of individual receptors on the nerve terminals. 

It is also an unexpected observation that THC treatment completely abolishes cannabinoid tone in the synapses studied, while phasic endocannabinoid signalling remains mostly functional.

I will never forget the feeling when, after the THC treatment experiments, I analysed my measured data and saw the difference between the groups studied. I could not believe my eyes!

- These findings can call for further investigations and perhaps a rethinking of what is known. What do you consider the most important?

- In my opinion, the main strength of our study is that we have provided for the first time experimental evidence for the molecular ordering principles of one of the fundamental types of retrograde synaptic signalling. It is not excluded that similar principles govern the function of other G-protein coupled receptors, whose nanoscale organization logic is still poorly understood. 

- In the hope that you have succeeded in clarifying some of the basic concepts of cannabis research and understanding the significance of some of its important issues, please tell us more about the truly picturesque cover of the journal that is your work!

- On a personal note, I feel it is a great achievement and recognition that my drawing illustrating our work has been featured on the cover page of the journal. The relationship between science and art has always been important to me. I often make drawings when I see something aesthetically pleasing in my work. That's why I've always loved checking the morphology of nerve cells under the microscope after electrophysiological measurements in experiments filled with tracers. To see the cells from which I made the recordings is fascinating because each is like a painting. I am so happy that the editors of this prestigious journal found my picture worthy of the front page!

- Probably there is no honest work that cannot be considered a vocation, but there can hardly be a successful researcher who does not consider research a vocation!

- I would also like to stress that on my behalf, participation in such research itself is an important achievement! I grew up in Magyargencs, a village in Transdanubia with less than 500 inhabitants. Although I often felt the difficulties of catching up with colleagues who could attend good schools from an early age, I would not trade my childhood for anything else. I was fortunate to have a loving and supportive family during this period, in an environment where my interest in science could flourish.

This article is the result of 9 years of work. During this time I have also had to overcome many difficulties, which I could not have done without the support of those around me. I am sincerely grateful to them, and I hope they are proud of what they have achieved, all the more so because they deserve credit for it. 

- With this article published in an excellent journal, one period may have come to an end, but the research certainly has not!

- That's right.  We don't yet know the molecular pathway through which the cannabinoid tone just described acts, so the next exciting task may be to uncover it. The other physiological signal to be discovered is the one that sets how many active CB1 receptors there should be in a given synapse! 

- Neither of these questions seems easy to answer, and who knows how many years of diligent work will be required to find the answers? Is there anything that can already be learned from these newly published results that might help in the treatment of cannabis users?

- As long as we have known about cannabis and its effects, we still do not understand many of the mechanisms of action that its consumption exerts on neurobiological processes. Moreover, the THC content of commercial cannabis has increased about tenfold in recent decades compared to the early 2000s. Therefore, I believe that any type of basic research that contributes to understanding the molecular processes influenced by this drug could be clinically relevant.

- Does the work also have implications that could be important besides cannabis research?

- As I mentioned earlier, previous results from our research team have shown that cannabis significantly reduces the number of cannabinoid receptors on nerve endings. In the present study, we investigated the physiological effects of this in different animal models. We were surprised to find that change in the communication between nerve cells occurs only in the case when the number of receptors around the synapses is reduced by a few hundred nanometres distance from the synapse.  More broadly, this could mean that to understand the effects of pharmacological agents, we need to track changes in the brain with nanometre precision!