Björn Brembs

I was very excited when our [latest research
paper](https://doi.org/10.12688/f1000research.146347.2) came out, after
all, I was confident our [30-year-long
search](https://bjoern.brembs.net/2024/01/the-speed-or-lack-thereof-of-science/)
for the sites of plasticity in the form of motor learning we study was
coming to an end. In this work, we were fairly confident that underlying
the type of learning we study was a novel form of plasticity in a very
specific set of motor neurons in the ventral nerve cord of the flies we
use for our research. The reason for our confidence was the convergence
of several lines of genetic evidence:

- inhibiting all PKCs (protein kinase C) in motor neurons abolished
  motor learning
- knocking-out just aPKC (atypical PKC) in motor neurons also abolished
  motor learning
- knocking out aPKC in neurons that also express the gene FoxP abolished
  motor learning (and FoxP is also expressed in motor neurons and itself
  necessary for motor learning)
- knocking-out FoxP outside of the ventral nerve cord had no effect

In addition to these genetic lines of evidence, we looked at the
expression patterns of the two genes we know are required for motor
learning: aPKC and FoxP.
[\#3](https://bjoern.brembs.net/tag/3/){.hashtag .u-tag .u-category
rel="tag"} in the list of genetic evidence above suggests that aPKC is
required in FoxP neurons, so we looked for neurons in the fly nervous
system that co-express both aPKC and FoxP. The first finding was that
there don't seem to be any such co-expressing neurons in the brain, just
in the ventral nerve cord. We (and by 'we' I mean our collaborator in
Mainz, motor neuron expert Carsten Duch) identified some of those
co-expressing neurons in the ventral nerve cord to be the steering motor
neurons that control the muscles that position the wings during turning
maneuvers. This last bit made us all excited: in our motor learning
task, we train flies to make specific turning maneuvers, i.e., left or
right turns, respectively. In our experiments, before we train the
animals, they make just as many left turns as they make right turns.
Then we train them to just do one kind of turns, such that, after
training, the flies preferentially turn into the, say, right direction,
if that was the side they've been trained to do. Finding that the motor
neurons involved in these kinds of behaviors also expressed both aPKC
and FoxP supported the genetic evidence that these neurons may be where
the plasticity underlying the learning may reside. However, there aren't
the genetic tools available at the moment (they are being created as I
type this) to specifically manipulate plasticity in just these neurons
and no other neurons, so while all the evidence was pointing towards
them, we couldn't be quite sure, yet. There may be other neurons
involved that we just didn't have on our radar, yet.

There is another behavior that also involved these steering motor
neurons and that is the "optomotor response" (OMR). Almost all animals
show OMRs to moving stimuli: if you sit in a car or train and look
outside to the passing landscape, your eyes will show the typical rapid
movements (nystagmus) when trying to stabilize the moving image on your
retina. Other animals move not just their eyes, but also their heads to
stabilize the image, again others move their eyes, heads and sometimes
also the rest of the body to, say, a rotating stimulus around them (or
if they are rotated). Flies flying stationarily at a tether presented
with vertical stripes moving around them either towards the left (i.e.,
counterclockwise, if seen from above) or right (clockwise, from above),
respectively, will try to follow the stripes and generate turning
maneuvers in the same direction: the optomotor response. It's known that
in order to execute these elicited turning maneuvers, there are neurons
in the brains of the animals that send the information about the
rotating stimuli directly to the steering motor neurons, bypassing all
other neurons on the way. This means that they also bypass those neurons
that are used to generate the spontaneous turning maneuvers we train the
flies to perform in our motor learning paradigm.

Essentially, the only part of the nervous system, where the OMRs overlap
with the behaviors we train are these steering motor neurons. This means
that if these steering motor neurons are modified by our motor learning,
as all the evidence described above suggests, we should se some
modulation of the OMR after motor learning. Our
[paper](https://doi.org/10.12688/f1000research.146347.2) shows the data
where exactly such a modulation can be observed. So with this data we
were fairly convinced that the plasticity we have been searching for in
all these decades was indeed in the steering motor neurons. But we
weren't quite certain, yet. What was missing? Because we had so far only
observed the modulation of the OMR in wild type animals, we didn't know
if the manipulations of the FoxP and aPKC that abolish motor learning
would also abolish the modulation of the OMR. Now we finally also have
that piece of the puzzle!

We again used the CRISPR/Cas9 technique to knock out aPKC on
FoxP-positive neurons. These flies were first tested for their OMR
before training, then subjected to motor learning of either left or
right turning maneuvers, respectively, and finally tested for their OMR
again. The genetic control flies that carried the same genetic
constructs, but in a dysfunctional way so aPKC expression would not be
affected were subjected to the same OMR-ML-OMR regime. The initial OMR
between both groups was identical. The motor learning phase showed the
same defiicit as we had observed with these flies in our paper:

<figure class="wp-block-image size-large is-resized">
<a
href="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled.png"><img
src="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-896x1024.png"
class="wp-image-2860" style="width:840px;height:auto"
data-fetchpriority="high" decoding="async"
srcset="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-896x1024.png 896w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-263x300.png 263w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-768x878.png 768w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-44x50.png 44w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled.png 1344w"
sizes="(max-width: 896px) 100vw, 896px" width="896" height="1024" /></a>
</figure>

The two control groups (blue and green) show a preference for the
unpunished turning direction that is significant both in frequentist
(Wilcoxon) and Bayesian (Bayes Factor, bf) statistics against zero (no
preference), while the aPKC knock-outs (yellow) show no such preference.
We replicated our finding that knocking-out aPKC in FoxP-neurons
abolishes motor learning.

To compare the controls with the knock-outs, we pooled the controls and
separately analyzed the OMR of those flies that were punished for
left-turning maneuvers and those that were punished for right-turning
maneuvers, respectively:

<figure class="wp-block-image size-large is-resized">
<a
href="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-1.png"><img
src="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-1-1024x614.png"
class="wp-image-2861" style="width:840px;height:auto" decoding="async"
srcset="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-1-1024x614.png 1024w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-1-300x180.png 300w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-1-768x461.png 768w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-1-1536x922.png 1536w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-1-50x30.png 50w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-1.png 1920w"
sizes="(max-width: 1024px) 100vw, 1024px" width="1024"
height="614" /></a>
</figure>

The control flies (above) show the modulation of the OMR that we had
already seen in wild-type flies: animals punished for left turns showed
a reduced OMR for left-turning stimuli and vice versa for flies punished
for left turns. Quantifying this asymmetry shows us that flies punished
on left turns show an asymmetry shifted to the right (i.e., positive
values), compared to flies punished on right turns, who are relatively
more negative (i.e., shifted to the left):

<figure class="wp-block-image size-large">
<a
href="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-2.png"><img
src="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-2-1024x512.png"
class="wp-image-2862" decoding="async"
srcset="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-2-1024x512.png 1024w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-2-300x150.png 300w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-2-768x384.png 768w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-2-1536x768.png 1536w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-2-2048x1024.png 2048w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-2-50x25.png 50w"
sizes="(max-width: 1024px) 100vw, 1024px" width="1024"
height="512" /></a>
</figure>

In contrast, this quantification in the knock-outs looks a lot more
symmetrical:

<figure class="wp-block-image size-large">
<img
src="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-4-1024x512.png"
class="wp-image-2864" loading="lazy" decoding="async"
srcset="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-4-1024x512.png 1024w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-4-300x150.png 300w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-4-768x384.png 768w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-4-1536x768.png 1536w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-4-2048x1024.png 2048w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/Untitled-4-50x25.png 50w"
sizes="(max-width: 1024px) 100vw, 1024px" width="1024" height="512" />
</figure>

If one now takes this measure of asymmetry and changes the signs such
that positive doesn't mean "right turns" any more, but "unpunished
turning direction", then we can compare the OMR modulation directly to
the trained turning preference in a single figure:

<figure class="wp-block-image size-large is-resized">
<img
src="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/image-1024x818.png"
class="wp-image-2865" style="width:840px;height:auto" loading="lazy"
decoding="async"
srcset="https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/image-1024x818.png 1024w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/image-300x240.png 300w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/image-768x614.png 768w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/image-1536x1228.png 1536w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/image-50x40.png 50w, https://bjoern.brembs.net/wp/wp-content/uploads/2024/07/image.png 1629w"
sizes="(max-width: 1024px) 100vw, 1024px" width="1024" height="818" />
</figure>

The pooled genetic controls (A) show both a significant OMR modulation
(blue, left) and a significant turning preference (yellow, right), while
the aPKC knock-outs (B) show neither.

So with this data, we are now really quite sure that plasticity in
steering motor neurons is one way in which this type of motor learning
manifests itself in the nervous system.


I was very excited when our latest research paper came out, after all, I was confident our 30-year-long search for the sites of plasticity in the form of motor learning we study was coming to an end. In this work, we were fairly confident that underlying the type of learning we study was a novel form of plasticity in a very specific set of motor neurons in the ventral nerve cord of the flies we use for our research.

Rogue Scholar Posts

Whodathunk? Motor learning in motor neurons, huh?