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.

Whodathunk? Motor learning in motor neurons, huh?

I just sent the poster for this year's Society for Neuroscience meeting
to the printer. As our graduate student is preparing his defense and our
postdoc did not get a visa (no thanks, US!), we just have a single
poster this year and I will present it myself on Monday, November 14,
2022, 8:00 AM -- 12:00 PM, on poster board WW53.

Contrary to our plans, this poster will not just contain the work of
postdoc Dr. Radostina Lyutova, but also some PhD thesis results from
graduate student Andreas Ehweiner. The general topic is how animals
learn from the consequences of their actions. To this end, we train
*Drosophila* fruit flies in different situations, with or without
genetically manipulated nervous system. In the first situation, the
tethered animals control a punishing heat beam with their yaw torque
(roughly corresponding to turning attempts around the vertical body
axis). In such experiments, wild type animals form a motor memory after
8 minutes of training. We have called the type of learning giving rise
to this memory self-learning, because the animals are exclusively
learning about their own behavior. This is in contrast to a second, very
similar experiment, where we have made one seemingly small, but highly
consequential modification. In this 'composite' situation, the animal is
controlling the heat as before, but now the environment of the fly is
colored in either green or blue, depending on the attempted turning
direction. In other words, now the animal can not only form a motor
memory about the un/punished turning directions, it can also learn which
color is associated with heat. We call this form of learning
world-learning, because the animal is learning about relationships in
the world around it.

More then ten years ago now, we developed tests to probe which learning
mechanism is engaged under which circumstances.

For instance, both after yaw torque training (where the self-learning
mechanism is engaged) and after composite training, we test for torque
preference (i.e., whether a motor memory has been formed) without
colors. After eight minutes of torque training, wild type flies show
motor memory, while after eight minutes of composite training, no such
motor memory can be detected. We are hypothesizing that the
world-learning mechanism is engaged in composite training and inhibits
the self-learning mechanism. Without colors, the self-learning mechanism
is free to operate, so motor memory is formed during torque training.

On the poster, we now show the most recent results on the molecular
components of the self-learning mechanism and how the world-learning
mechanism inhibits self-learning. We found that the gene for the
atypical protein kinase C (aPKC) is necessary for self-learning and that
it likely acts in motor neurons that also express the *FoxP* gene. When
we overexpress a constitutively active form of aPKC, this improves
self-learning so much, that such flies not only can form a motor memory
after 4 minutes of training (not sufficient for learning in wild type
flies), but also can overcome the inhibition of motor learning by the
world-learning mechanism, such that motor memory is detectable after
eight minutes of composite training. Because we knew that this
inhibition acts via neurons in the neuropil called the mushroom body, we
also screened mushroom body output neurons (MBONs) to find out where the
inhibition is transmitted from the mushroom bodies. We silenced these
MBONs one category at a time, looking for lines which showed motor
memory, similar to the flies with the improved self-learning. we found
three candidate lines and the rescreen of these lines is still in
progress.

The poster can be downloaded by clicking on the picture below:

<figure class="wp-block-image size-full is-resized">
<a href="bjoern.brembs.net/download/sfn_2022/?wpdmdl=2486"><img
src="https://bjoern.brembs.net/wp/wp-content/uploads/2022/11/sfn22-2.png"
class="wp-image-2493" decoding="async"
srcset="https://bjoern.brembs.net/wp/wp-content/uploads/2022/11/sfn22-2.png 1000w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/11/sfn22-2-300x177.png 300w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/11/sfn22-2-768x452.png 768w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/11/sfn22-2-50x29.png 50w"
sizes="(max-width: 750px) 100vw, 750px" width="750" height="442"
alt="Poaster" /></a>
</figure>


I just sent the poster for this year’s Society for Neuroscience meeting to the printer. As our graduate student is preparing his defense and our postdoc did not get a visa (no thanks, US!), we just have a single poster this year and I will present it myself on Monday, November 14, 2022, 8:00 AM – 12:00 PM, on poster board WW53.

Interacting learning systems at SfN22

The first conference after the Sars CoV2 pandemic! We're headed for
Paris, France tomorrow and our lab will present two posters, the work of
graduate student Andreas Ehweiner and postdoc Radostina Lyutova.

Andreas has been working on the cellular and genetic mechanisms
underlying operant self-learning (a form of motor learning). On his
poster, he presents evidence that one crucial site of plasticity for
this type of motor learning is motor neurons of the fly ventral nerve
cord co-expressing both *FoxP* and *aPKC*. Interestingly, *FoxP*
expression in the brain seems to be dispensable for self-learning and
knocking-out *FoxP* in all neurons in the adult only seems to show an
effect on learning 14 days after the knock-out. Overexpressing a
constitutively active form of *aPKC* improves motor learning, such that
these flies still learn when training has been reduced to a level where
wild-type flies no longer show a significant learning score. I'm
particularly happy that we now seem to have found which of the five
*Drosophila* PKC genes is involved in operant self-learning. We had been
trying to identify the right PKC for a few years now. Surprisingly, it
is not the same PKC as the one that is required for self-learning in
*Aplysia*, for instance. Andreas is writing up his thesis at the moment,
so he sure would appreciate job offers. Andreas is smart, resourceful
and persistent. Here is his poster (link opens A0 PDF):

<figure class="wp-block-image size-large">
<a href="https://bjoern.brembs.net/wp/?wpdmdl=2448"><img
src="https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-1024x684.png"
class="wp-image-2455" loading="lazy" decoding="async"
srcset="https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-1024x684.png 1024w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-300x200.png 300w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-768x513.png 768w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-1536x1026.png 1536w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-2048x1368.png 2048w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-50x33.png 50w"
sizes="(max-width: 1024px) 100vw, 1024px" width="1024"
height="684" /></a>
</figure>

Radostina studies the regulation of self-learning by other forms of
learning. Operant self-learning only requires eight minutes of training
(see Andreas' poster) when no other stimuli can be learned. If such
stimuli are present, self-learning requires overtraining of 16 minutes.
This procedure is reminiscent of habit formation in vertebrates.
Radostina found that habit formation in flies is regulated by a single
cell receiving input from a prominent insect neuropil, the mushroom
body. If she inhibits this cell, flies show a habit already after eight
minutes, suggesting that the inhibition of self-learning is mediated via
this mushroom body output neuron (MBON 02). Connectome analysis revealed
that MBON 02 input is provided mainly by Kenyon cells from the
little-studied lateral and dorsal accessory calyx, which, in turn,
receive thermosensory and visual input, respectively. This has been a
massive effort over several years, clouded by a case of sabotage, that
Radostina has picked up and breathed new life into. Here is her poster:

<figure class="wp-block-image size-large">
<a href="https://bjoern.brembs.net/wp/?wpdmdl=2446"><img
src="https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-1-1024x724.png"
class="wp-image-2456" loading="lazy" decoding="async"
srcset="https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-1-1024x724.png 1024w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-1-300x212.png 300w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-1-768x543.png 768w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-1-1536x1085.png 1536w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-1-2048x1447.png 2048w, https://bjoern.brembs.net/wp/wp-content/uploads/2022/07/image-1-50x35.png 50w"
sizes="(max-width: 1024px) 100vw, 1024px" width="1024"
height="724" /></a>
</figure>


The first conference after the Sars CoV2 pandemic! We’re headed for Paris, France tomorrow and our lab will present two posters, the work of graduate student Andreas Ehweiner and postdoc Radostina Lyutova.

Rogue Scholar Posts

Whodathunk? Motor learning in motor neurons, huh?

Interacting learning systems at SfN22

Off to Paris for #FENS2022 with two posters