Exercise enhances motor skill learning by neurotransmitter switching in the adult midbrain

Welcome to our Monthly Journal Club! Each month I post a paper or two that I have read and find interesting. I use this as a forum for open discussion about the paper in question. Anyone can participate in the journal club, and provide comments/critiques on the paper by leaving a comment below. I picked this months paper because it adds additional evidence for a really cool and understudied type of neural plasticity: neurotransmitter switching. Also, this is the first preprint featured on this website, and therefore you must take the findings in this paper with a grain of salt as they have not been peer reviewed. I do love BioRxiv, because it is a great way of getting your ideas out there before the long publication process has been completed, and it is journal agnostic. Also…I have accepted an assistant professor position at Cold Spring Harbor Laboratory, where BioRxiv was started…so the fit for this month was perfect!

This month’s paper is “Exercise enhances motor skill learning by neurotransmitter switching in the adult midbrain” by Hui-quan Li and Nicholas Spitzer at the Kavli Institute for Brain and Mind located at the University of California - San Diego. On top of his great research program, Dr. Spitzer is undoubtedly the winner of the “Best Moustache in Neuroscience” award. I will provide a brief overview of the techniques/approaches used to make it more understandable to non-expert readers. If I can’t figure something out, I’ll just say so. Check out the video below for a quick summary of how neurotransmitter switching makes up a unique form of neural plasticity.

Hui-quan Li and Nicholas Spitzer were interested in how motor learning occurs, the process by which we become better and better at specific motor tasks via trial and error (e.g., using chopsticks, speaking fluently, and quick reflexes…). This obviously involves a form of neural plasticity, as our brains need to change in some way to strengthen the circuits that improve a behavior, while refining those that detract from it. Motor learning has been intensely studied in the realm of neuroplasticity, involving circuits in the cortex, basal ganglia, brainstem, cerebellum, and spinal cord. However, whether neurotransmitter switching contributes to this form of learning was unknown. Neurotransmitter switching is an under-appreciated form of plasticity, as in most high school and college textbooks, neurons are assigned a neurotransmitter (e.g., dopamine neuron) which sticks with them for life, making the concept of a plastic ‘switchable’ neurotransmitter repertoire foreign to most students. Today I’m going to try and make the case for neurotransmitter switching, using this beautiful study as a template!

The authors started by examining how the brain changes in response to a week of aerobic exercise (a running task, shown below). They trained mice to run on a wheel throughout the week, and then tested their motor coordination on a rotating rod (rotarod) and a balance beam. They demonstrated that after a week of training, mice that ran increased their speed on the running wheel, fell off the rotarod at higher speeds of rotation, and kept better balance on the balance beam than mice that didn’t run. This learning effect lasted for up to 2 weeks following training, suggesting that mice learned the motor behavior, but this ‘motor memory’ can be lost if it is not reinforced with more exercise.

One week of running training induces motor learning, an effect that lasts at least 2 weeks post-training. (Credit: Li & Spitzer, 2019)

One week of running training induces motor learning, an effect that lasts at least 2 weeks post-training. (Credit: Li & Spitzer, 2019)

So now that we have a strong motor learning experimental set up, we can begin to understand what is going on in the brain in response to the running training. To do this in an unbiased fashion, the authors used a technique calledcFos mapping’, where the brain is sectioned following training (or no training as control) and cells are labeled with antibodies against the immediate early gene cFos. This is a proxy of recent neural activity, and lets researchers look for cells in the brain that were activated immediately before the tissue was collected (Approx 30-90 minutes before). By using this method, they found that in response to running training, neurons in a brain structure called the pedunculopontine nucleus showed signs of increased activity (more cFos+ cells detected; see below).

Running induces neural activity (cFos labeling) in multiple brain regions, and most notably in the pedunculopontine nucleus (PPN). Here, we can see that in response to running, higher amounts of cFos are detected in the PPN (red = all neurons, green = cFos (active neurons)). (Credit: Li & Spitzer, 2019)

Running induces neural activity (cFos labeling) in multiple brain regions, and most notably in the pedunculopontine nucleus (PPN). Here, we can see that in response to running, higher amounts of cFos are detected in the PPN (red = all neurons, green = cFos (active neurons)). (Credit: Li & Spitzer, 2019)

This is an interesting finding, but the authors still did not know what type of neurons were being activated by the running. This is important to know, as different neurons (even in the same brain area) can have opposing effects on behavior/learning. As there had been previous work done in this brain area, they labeled cells for some primary neurotransmitter types in the PPN: Acetylcholine and GABA.

Running induces a neurotransmitter switch from acetylcholine to GABA in the caudal pedunculopontine nucleus (cPPN). Above, we can see that the number of acetylcholine producing neurons (ChAT+) decreases in response to running, with a concomitant increase in GABA-production (GAD1+). (Credit: Li & Spitzer, 2019).

Running induces a neurotransmitter switch from acetylcholine to GABA in the caudal pedunculopontine nucleus (cPPN). Above, we can see that the number of acetylcholine producing neurons (ChAT+) decreases in response to running, with a concomitant increase in GABA-production (GAD1+). (Credit: Li & Spitzer, 2019).

Indeed, they found these two types of neurons in the PPN. They observed that in response to running, the number of active acetylcholine-producing neurons (ChAT+ and cFos+) increased dramatically in the caudal region of the PPN. However, this was associated with a decrease in the amount of acetylcholine producing neurons in this area…how could that be? It turns out that these cells were not disappearing, but switching which neurotransmitter they predominantly make (from acetylcholine to GABA) in response to running!

Acetylcholine-expressing neurons in the caudal Pedunculopontine nucleus (cPPN) lose acetylcholine and gain GABA in response to 1 week of running training. (Credit: Li & Spitzer, 2019).

Acetylcholine-expressing neurons in the caudal Pedunculopontine nucleus (cPPN) lose acetylcholine and gain GABA in response to 1 week of running training. (Credit: Li & Spitzer, 2019).

To investigate this more deeply, they used viruses that infect neurons (adeno-associated viruses (AAVs)) that carry transgenic DNA payloads (plasmid DNA; pDNA). This payload is inactive (i.e., it does not do anything by itself), but becomes active in the cell when a certain enzyme is present: Cre-recombinase. The researchers used mice that express this enzyme only in acetylcholine-producing neurons (also known as ChAT-IRES-Cre mice). By combining cre-dependent viruses with ChAT-Cre mice, they are able to express transgenes in a specific brain region in a ‘cell-type specific’ manner. They used this approach to make acetylcholine neurons in the cPPN express mRuby2, a very bright version of red fluorescent protein. This way, they can track how these neurons (which will always be red) change their neurotransmitter components in response to running. Using this technique, they demonstrated that a sizable proportion of neurons in this brain area switch their neurotransmitter of choice from acetylcholine to GABA in response to just a week of running!

Viral-mediated prevention of neurotransmitter switching (via upregulation of ChAT expression) prevents motor learning following a running task. (Credit: Li & Spitzer, 2019).

Viral-mediated prevention of neurotransmitter switching (via upregulation of ChAT expression) prevents motor learning following a running task. (Credit: Li & Spitzer, 2019).

The authors went on to test what other brain regions these cells send projections to, which likely mediates their involvement in motor learning. They used beads that travel backwards along neuron projection axons (retrograde labeling) to label cells in the PPN that project to other brain areas involved in motor behavior/learning. They found that running reduced the number of ChAT+ (acetylcholine) terminals (where neurotransmitter is released) in multiple brain regions, including the ventral tegmental area, substantia nigra, and thalamus, key regions for motor learning.

Now the question moved onto whether this neurotransmitter switching was actually important for the motor learning. In simpler terms, this is the ‘so what?’ question. To test whether the loss of ChAT expression in the cPPN was important for motor learning (or was just a side-effect), they used ChAT-Cre mice as before, but injected a virus that promoted the cells to make a ton of acetylcholine, preventing them from making the switch to GABA (see above). When they did this, the mice no longer were able to display motor learning behavior, and they performed just as badly as non-trained mice on the rotarod and balance beam tests! This demonstrates that loss of acetylcholine expression in the PPN is important for this type of motor learning.

Prevention of the Acetylcholine/GABA switch during training prevents motor learning. Using a short-hairpin targeting GAD1 (shGAD1) to knock down it’s expression in ChAT+ neurons in the PPN, the authors demonstrate that without increased levels of GABA in response to running, the mice no longer learn this task. (Credit: Li & Spitzer, 2019)

Prevention of the Acetylcholine/GABA switch during training prevents motor learning. Using a short-hairpin targeting GAD1 (shGAD1) to knock down it’s expression in ChAT+ neurons in the PPN, the authors demonstrate that without increased levels of GABA in response to running, the mice no longer learn this task. (Credit: Li & Spitzer, 2019)

What about the GABA, though? If loss of acetylcholine expression is necessary for learning, is gaining GABA also necessary? To test this, the authors again used a viral approach. Using ChAT-Cre mice to target only neurons expressing acetylcholine in the PPN, they injected a Cre-dependent short-hairpin RNA against a gene that is important in the synthesis of GABA (shGAD1). When they trained these and control mice (which were injected with ‘scrambled’ shRNA) on the running protocol, only mice with the scrambled construct (which doesn’t affect gene expression) that completed the motor learning task showed a major uptick in the number of GABA-expressing neurons (GAD1+). This suggested that their shRNA technique successfully knocked down GABA and prevented acetylcholine neurons from ‘switching’ in response to running. When these same mice had their motor learning tested on the rotarod and balance beam, those that had GABA knocked down in the PPN showed no signs of learning (see above panel (e) and (f)). Together, over expression of acetylcholine or knockdown of GABA in the cPPN prevented motor learning. This strongly indicates that neurotransmitter switching in the PPN plays a major role in motor learning. Aerobic physical exercise promotes the ability to acquire new motor skills, and it serves as a therapy for many motor disorders including Parkinson’s disease, coordination disorder, and autism. Until this study, how this worked at the neural level was poorly understood. These findings strongly implicate neurotransmitter switching as a form of neuroplasticity that underlies motor learning, and offers a potentially new target for treatment of a large variety of diseases.

That’s it for this post guys! Please share what you think in the comments below, or send me a message on twitter @jborniger! See you soon!