Lightning-firing interneurons

Thursday, 12 January, 2023
Tags: News

Balázs Rózsa and his team, researchers from the BrainVisionCenter (BVC), in collaboration with the Friedrich Miescher Institute in Basel and Pázmány Péter University, have discovered previously unknown features of neuronal plasticity in the hippocampus of awake animals. Their work was published in Nature Communications.


Popularity and visibility have long been important to many people, and since the advent of the internet, it seems to have become a prerequisite for human existence. If you're not on the internet, you don't exist, some people joke. Popularity is measured by likes, which is only a matter of time before it becomes the eighth basic unit in the International System of Units (SI). However, there are other indicators of popularity. 

For example, there is Einstein, one of the world's best-known and most respected scientists. Not only is the theory of relativity relatively widely associated with his name, but stories, trump cards, and famous photographs also contribute to his popularity. Who has ever seen a quote from Marie Curie - who is also well known - on the wall of a shopping center shop? But I have read Einstein myself: 'Everyone knows that certain things cannot be achieved until someone who does not know about them comes along and achieves them. "

Since the world began, there have always been people who have ignored what the general opinion is, and if they needed something that did not exist, they invented it. Even things as simple as the hoist, the pulley, or the more complex telescope, microscope, pendulum clock - and, speaking of Einstein, the refrigerator - can remind us of how much we owe to those who sought and found new ways instead of the well-trodden, well-trodden paths.

Even today, despite the countless companies that produce experimental devices, some people still set out to create their own experimental devices because they know best what they need. This is what Balázs Rózsa and his team do. Often they don't get it right the first time, and they don't always get it right quickly, but they get what they want in the end. 

They started an "impossible" project back around 2016. 

Balázs Rózsa recalls. 

"I had a dual goal in organizing the Three-Dimensional Neural Network and Dendritic Activity Research Group. One was to be able to measure the complete, branch-like structure of the "activity fork" several orders of magnitude faster and spatially more accurately than previous methods, and the other was to be the first in Hungary to be able to perform these two-photon measurements in vivo, i.e. on a living animal. It was already assumed at that time that the brain of living animals functions completely differently when it comes to complex, regenerative activity patterns, i.e. activity patterns that spread along the extensions, than was previously possible to detect in vitro on brain slice preparations. A multidisciplinary team of biologists, engineers, physicists, and mathematicians with diverse backgrounds was needed to achieve this goal."

A lot of work and countless improvements were needed. The amount and variety of tasks are also shown by the fact that five of them - Linda Judák, Balázs Chiovini, Gábor Juhász, Botond Roska, Balázs Rózsa - are listed as co-authors of equal standing in the Nature Communications publication of November 2022.

Among them, Linda Judák has taken it upon herself to share with us some of the important events of the long journey that has led her to this point.

- Our basic question was how hippocampal cells and their extensions integrate incoming information and how cellular encoding of short-term memory takes place.

- A lot of people have spent a lot of time answering this 'tiny' question!

 - It is indeed a huge question, and for a long time, it could only be studied in brain slices, in vitro. Initially, Balázs Chiovini and Dénes Pálfi did this series of experiments, and this is the basis of our 2014 Neuron article, which is now the basis of our publication. (*SPW or SPW-R, sharp waves and ripples: oscillatory patterns generated by strictly synchronized activity of neurons in the mammalian hippocampus)

- What is your task?

In addition to our work on somatic and dendritic phenomena during hippocampal sharp waves, we are also involved in the development of microscopy and in vitro and in vivo techniques. The latter includes, for example, the development of chemical "cage molecules" carrying stimulatory and inhibitory neurotransmitters, also known as neurotransmitters.

And from the beginning, it was expected that we would extend hippocampal research to in vivo experiments. 

- An incredible number and variety of tasks! Who started the in vivo developments?

- Since I had considerable in vivo experience by then, Gábor Juhász and I started it. I worked on the refinement of the deep brain adapter, Gábor perfected the production of the LFP* electrodes, which are located ipsilaterally (on the same side). 

Thus, our big dream of side-by-side imaging and LFP abstraction was realized. This was a major step forward, as it had never been shown in the literature before! 

Meanwhile, Gergely Katona and Gergely Szalay were working on the development of the 3D two-photon microscope, so we could make measurements with more and more technical support and obtain more results. We now had 2D and 3D two-photon microscopes, and as a result of Katalin Ócsai's work, we were able to apply motion correction. 

- In a large number of experiments, animals had to be injected, operated on, trained, and the measurements taken and evaluated. Who was responsible for this?

- The three first authors! We worked with Balazs Chiovini and Gábor Juhász to develop the basis for the analysis of the results. The evaluation of the population activity was done by me, and the analysis of the single-cell activity patterns by Balázs. Unfortunately, the evaluation of single events could not be automated, Benedek Szmola helped me.

- They did not give that first prize easily! What was the result, the answer?

- Although we had already measured in vivo, we still did not know how the parvalbumin-expressing (PV) interneuron is able to summarise the multitude of information coming to its extensions. Moreover, we have measured surprising and interesting events in in vitro experiments

- And?

- One interesting finding was that while the apical extensions of the PV neurons were active during SPW-R, the basal extensions were not, so we could not measure them. We thought this was due to the preparation itself, but our hypothesis had to be proven. 

It also remained a question whether PV neurons are capable of generating regenerative activity at all, i.e. whether calcium spikes can form on the extensions. This required in vivo measurements, which proved that PV neuronal spines are able to become "active". 

- How were you able to do this technically?

- In addition to adapting to the daily changes in 3D two-photon microscopy techniques, we also had to improve our in vivo surgical and measurement techniques.

- What exactly was the task here?

- To measure the thin stretches of deep hippocampal PV interneurons in high volume and to solve for simultaneous LFP drainage as close as possible to the site of calcium activity capture.

- Please explain this overly simple sentence!

- We did not derive calcium signals from PV neurons from a single point but measured the whole stretch network. This means measuring hundreds, sometimes thousands of points simultaneously. In addition, this relentlessly large database had to be organized into a meaningful format for evaluation. We did it.

- Is that all right?

- Not at all! Initially, there was no motion correction and no automatic selection system. Once we had found the active cells, we had to map out the network of stretches and then lay out the "measuring tapes" along the stretches. Meanwhile, the measurement frequency could not fall below a certain level. This was a rather time-consuming task. Moreover, if it turned out that the cell and the surrounding tissue had moved in the process, we could start all over again! 

And let's not forget that the whole experiment required well-trained animals.

- Was it worth all the hard work, lots of rework, perseverance, skill, and who knows what else?

- Definitely! When it all came together, the result was amazing! We were able to see the entire basal lamina network of the cell and watch it shoot lightning bolts. This proved that the basal spines of hippocampal PV interneurons can be active during SPW-R.

No one has ever seen and measured the spine-level activity patterns of hippocampal PV cells in awake animals! We have seen where the information packets arrive and how they spread along the axon. Our broad spectrum analysis allowed us to validate and systematize the signals. We saw that the signals in the spines are SPW-R dependent and well categorized along the sharp wavelengths. The icing on the cake was the discovery of a never-before-demonstrated signal-summation mechanism in the thin strands of PV interneurons!

- Congratulations, but please explain!

- Think about it! Up until now, everyone thought of PV cell extensions as passive cables, and now we've discovered that they are actually mini processors, where three levels of logic circuitry from the prehistoric days of computers are created! No one expected this! This is something brand new!

- Your enthusiasm is perfectly understandable, and you all deserve this reward, as well as congratulations on the article. Because it's great to be an author of a Nature Communications article, but for a researcher, it's the moment of discovery that means the most. What measurement has confirmed the presence of that signal summation mechanism? 

- We knew from the literature about the so-called "SPW-R doublets". Their importance was already being touted, but we were able to reveal the mechanisms of these SWP-R duplets at the level of the stretch!! 

- What is their role and importance?

These doublets can open a "time gateway", which helps the different information packets to bind together. We have demonstrated that the interneuron spine of the PV can also encode and summarise information. This is a big deal because until now no one would have thought that PV neurons could produce such complex and important signal summation mechanisms at the spine level as pyramidal cells. 

- For such a major discovery, the acceptance of the manuscript was hopefully only a question of how it was accepted!

- Although the reviews were very favorable, the acceptance of the manuscript took a long time. The editor anticipated this and explained it through the difficulties caused by the epidemic situation. The correspondence with the reviewers also took a long time because we had a lot of work to do. In hindsight, we do not regret this, and we are grateful for the advice because it made the manuscript clearer and easier to follow.

- The article has been published, but the research is ongoing. How?

- Our intention is to follow population- and scaffold-level processes along the entire learning cascade in awake, behaving animals, and we hope the results will contribute significantly to a better understanding of memory processes.