Sleeping Astrocytes: Failures and Successes on the Journey to Publication

Laura Bojarskaite

Laura Bojarskaite

Today I will tell you a story filled with hope and frustration, a little bit of patience and yet a lot of impatience,and cosily sleeping mice studied by a very tired scientist in the dark. My name is Laura and I am a neuroscientist-in-progress from the University of Oslo.

I want to be honest and tell you the story of how science actually happened for me and not how science should have happened according to all the rules, supposed-to’s, and textbooks. I want to talk about all of the details and nuances that nobody talks about (or at least doesn’t include in the paper), but that constitute 90% of time sciencing. I want to give randomness and simple curiosity credit because that’s a huge part of science – at least it was for me.

This was even how I found my passion for studying neuroscience, since what I started out studying before my PhD was actually quite different from what I do now. My Bachelor’s degree is in biochemistry and my Master’s degree is in pharmaceutical sciences. I was fascinated by the fundamental inner workings of our cells and all the tiny molecules inside, how they interact with one another, and how we can affect them from the outside. When I first started working in science, I was in a bionanotechnology lab trying to isolate various proteins and quantify their interactions on different surfaces to understand whether those materials were compatible with biology. I absolutely loved working at a university. By the end of my Master’s, I was sure I wanted a career in academia, but I was missing the spark of a topic that seriously motivated me to begin a PhD.

Call it luck, simple randomness, or holy intervention, but a book by Norman Doidge called “The Brain That Changes Itself” found its way into my hands and changed everything. In those pages, I learned about brain plasticity and its ability to change peoples’ lives. It soon became clear as day that neuroscience was where I would find my scientific inspiration. As my boyfriend was living in Oslo, the math was clear: neuroscience + Oslo = happiness.

I am very grateful for my background in basic science, which focuses on gaining a better understanding of a specific subject by focusing on the advancement of knowledge rather than solving a specific problem.

In other words, my early days in science allowed me to be driven by my curiosity about the unknown and explore the unexplored for the sake of exploration.

My journey towards understanding the role of astrocytes – the star-shaped glial cells of the brain – in sleep started exactly like that.

I began my PhD studies in the GliaLab research group at the University of Oslo, where we study astrocytes and what these beautiful “brain stars” (they are actually shaped like stars) do in the brain. The cool thing about astrocytes is that for a really long time they were thought of as mere passive supporters of the neurons, but only recently, new advances in technology have allowed us to discover that they do so much more than that. Needless to say, it has been very exciting to work on a cell type whose “true powers” are yet to be discovered.

One day, my supervisors and I were discussing all things astrocyte when we started brainstorming about what could I study for my PhD thesis. One topic that kept coming up was sleep. The science of sleep is a topic that seems to ignite the interest of the public and scientists alike.

At the time, the topic of sleep was again becoming “sexy” because a new piece of very exciting research had come out that suggested that sleep could literally “clean your brain”¹. Waste products from our body are continuously removed with the help of lymphatic system. However, lymphatic vessels are not found in our brain. Instead, the brain utilizes a specialized system made up of astrocytes that hug around the blood vesselsin the brain to form a tunnel system. A special fluid runs in these tunnels and washes cellular waste out of the brain. Here’s the catch – this “brainwashing” system appears to only be on during sleep.

But why is this happening during sleep? Which cells in the brain drive this? Could it be astrocytes? How is it all regulated? These questions formed the foundations of my PhD project, which aimed to map uncharted territory and explore what astrocytes were up to at night. Thinking about these questions gave me chills of excitement, and they still do. Interestingly enough, we were not a sleep lab at that time. But I thought that it couldn’t be toohard to become one, right?

First, the mice needed to sleep. Since we need microscopes and special lasers to look at living astrocytes in the brain, the mouse needs to sleep under a microscope while being head-immobilized by a metal bar that is glued to their skull. Since we can’t see brain cells through skin or the bones of the skull, these mice have a piece of their skull replaced by glass so that we have an actual window into their brains. I guess you can also call me a mouse neurosurgeon!

And so began my 2 year long low-period of absolute failure trying to achieve just this.

For a while I tried to get the mice to sleep by placing them in plastic transparent tubes, hoping that inactivity or my lullabies would bore them to sleep. Unfortunately, both of these tactics seemed to only stress the mice out. After that followed a very long period of time where we tried to have the mice sit on an air-pressure driven floating styrofoam ball with the idea that their ability to move freely might reduce their stress and help them sleep.

The styrofoam ball quickly became my “curse-in-disguise”. At first, the mice seemed to be able to nap while sitting on the ball. I remember the first time one of my mice closed its eyes for more than a few seconds for the first time. The surge of excitement I felt is difficult to put in words. I had so much hope, and in that moment, I saw that I would make it through to my graduation.

Little did I know it would still take 4 years from that time for me to see my name printed on the Nature Communications journal website.

I soon discovered the problem was that after falling asleep, the mice would arouse instantly as their muscles relaxed, causing the ball to move very slightly and wake them up. “Hope is the mother of fools” they say, and this time the thought “but maybe next time the mouse won’t wake up?” cost me half a year of doing the same thing while hoping for a different result.

These were dark times of my life, both literally – as I spent months upon months of my life in a dark microscope room – and metaphorically. After some guidance from my mentor, I was encouraged to literally “drop the ball”, and finally I found my happiness in (and the mice found their sleep on) a horizontally spinning disc.

I remember witnessing my first slow wave sleep episode followed by rapid-eye-movement sleep. The big beautiful delta waves turning into marching strict theta activity in the EEG signal, a clearly visible release of mouse’s muscle tone, and the whiskers twitching in the dark. The amazing part was that we expected astrocytes to be completely silent during sleep, as they seemed to actively signal only during wakefulness and were silent during anesthesia, but that was not the case. What I saw was not an abolishment of activity, but a bunch of tiny beautiful flashes continuing throughout sleep (astrocytes, unlike neurons, are electrically non-excitable and instead of electrical signals, communicate through intracellular calcium ion signals that can be captured by molecules that turn green (or other colours) when binding calcium. Hence “flashes”).

I simultaneously felt feelings of awe, humbleness, and grandeur as I knew I was seeing something no one else in the world had yet seen. And yet, it was still just me in a dark microscopy room lit up by computer screen, clicking shutter sounds, and the little mouse in slumber, as things had been for months before this eureka moment.

In the following years I watched cosily sleeping mice for countless hours and also made some other interesting discoveries: 1) Sleeping mice are more sensitive towards the smells of coffee or a banana rather than you sneezing; 2) The voices of some colleagues seem to put the mice to sleep while mere presence of others wakes the mice up completely; 3) You can actually watch Netflix for 12 hours straight while waiting for mice to sleep; 4) If you hang a sign on the microscope room door saying “Sleeping mice. Please be silent and DON’T come in!”, people will definitely not be silent and will absolutely come in.

Our resulting paper on astrocytic calcium signaling during sleep only scratched the surface regarding astrocytic regulation of slow wave sleep and sleep spindle dynamics. But it also opened a Pandoras box of questions and future projects which drives us further and will do so for many years to come. It was a long and very bumpy (and sleepy) ride, but looking back, I am certain all my struggle was totally worth it.

I graduated on November 11th 2020, and now continue my sleeping astrocyte journey as a postdoc.

Reference:

1. Xie L, Kang H, Xu Q, Chen MJ, Liao Y, Thiyagarajan M, O’Donnell J, Christensen DJ, Nicholson C, Iliff JJ, Takano T, Deane R, Nedergaard M. Sleep drives metabolite clearance from the adult brain. Science. 2013 Oct 18;342(6156):373-7. doi: 10.1126/science.1241224. PMID: 24136970; PMCID: PMC3880190.