Researchers overcome winking, napping pigs to prove brain test works


Date:
July 8, 2021
Source:
University of Illinois College of Agricultural, Consumer and
Environmental Sciences
Summary:
If you've ever been to an eye doctor, there's a good chance
you've felt the sudden puff of air to the eye that constitutes a
traditional test for glaucoma. It's no one's favorite experience,
but the puff is non-invasive and harmless. Scientists use a similar
method to test learning and memory in animals and humans.



FULL STORY ==========================================================================
If you've ever been to an eye doctor, there's a good chance you've felt
the sudden puff of air to the eye that constitutes a traditional test
for glaucoma.

It's no one's favorite experience, but the puff is non-invasive and
harmless.


========================================================================== Scientists use a similar method to test learning and memory in animals
and humans. Like Pavlov's classic experiments linking a neutral stimulus
with a physiological response, the eyeblink test pairs a light or sound
with a quick puff of air to the eye. With repetition, the animal learns
to close its eye, or blink, in response to the light or sound only. It's
called associative learning, and the response is ruled by a brain region
known as the cerebellum.

While the eyeblink test has been around since 1922, it had never been
attempted in pigs until now. In a new study in Frontiers in Behavioral Neuroscience, researchers prove the eyeblink test works in 3-week-old
pigs, a model species for nutritional neuroscience research in human
infants.

"The idea is, if we can improve structural development in the brain
through nutritional interventions, it should take pigs fewer trials to
learn the rule.

We're in the process of assessing the nutrition piece now, but we
had to get the test to work first," says Ryan Dilger, professor in the Department of Animal Sciences at the University of Illinois and co-author
on the study.

Dilger specializes in the effects of nutrition on the developing
brain, with much of his work feeding directly into the infant formula
industry. He uses neonatal pigs because, unlike rodents, their brain
anatomy and structure, gut physiology, and nutritional requirements are strikingly similar to human infants.

Dilger's team typically studies pig brain response to new ingredients
through magnetic resonance imaging, MRI, which focuses on the structure
and size of various brain regions. They also rely on well-validated
behavioral tasks, such as novel object recognition, that reflect activity
in the hippocampus and striatum, some of the brain regions related to
learning and memory.



==========================================================================
But Dilger wanted a tool to specifically assess cognitive processing in
the cerebellum. That's when he turned to Henk-Jan Boele and Sebastiaan Koekkoek, eyeblink specialists and neuroscientists at Erasmus Medical
Center in the Netherlands.

Although humans and all sorts of animals have undergone the eyeblink test,
the behavioral paradigm had never been validated for pigs.

"For human and mouse eyeblink conditioning, we use completely different systems," says Boele, a postdoctoral researcher with dual appointments at Erasmus and Princeton University. "Humans are easy to instruct, usually
are very cooperative, and sit still during the experiment, which makes
it easy to deliver the puff and measure the eyelid. Mice are mostly
head-fixed during the experiment, which makes it easy to deliver the
air puff and measure the eyelid.

Pigs, instead, were a challenge because we did not want to fixate
their head.

It was really difficult to reliably deliver the air puff and measure
the eyelid responses.

"We tried video cameras, diodes, and all kinds of things, to capture
the blink.

And we had to use a piece of equipment to deliver the air puff that was
very close to the eye to avoid any delays. We need really sharp, short
puffs that are not invasive for the animal but are still very precisely
timed. So that was a challenge," he says.

The solution was taping a short piece of air tubing next to one eye,
and pasting magnetic sensors on the forehead and eyelid to record the
blink. The system measured the eyeblink down to the millisecond.



==========================================================================
And yes, Boele puffed himself in the eye to test out the system. It
worked. "Oh yes, I blinked," he laughs.

To ensure the pigs had free head movement but didn't get up and run
around, the researchers placed them in a custom-built sound-dampening
box fitted with a pig-sized hammock.

It was apparently very comfy.

Sangyun Joung, a doctoral student in the Neuroscience Program at Illinois
and co-author on the study, says, "Each pig had five days of training to habituate them to the hammock and the testing environment. By day three,
they were very relaxed, to the point where some of them were literally
falling asleep. That was a little challenge for us during the analysis,
because that definitely influenced their responses. But it also told
us this whole behavior paradigm is not stressful for them. For us, it
was interesting and frustrating at the same time." Once the pigs were
used to the setup, the real work began. The pigs did a series of eight
tests in a row. The first was the air puff only, to measure the eyeblink reflex. The next six tests paired a small blue LED light with the air
puff. The light came on for half a second, 500 milliseconds, and right
at the end, with the light still on, the air puff was delivered. That
last part, with both the light on and the air puffing, lasted a tiny 50 milliseconds. The final test was the light only -- no puff.

The researchers repeated each set of eight tests five times on each of
five consecutive days. The time between tests varied a bit to keep the
pigs guessing.

"It wasn't just on and off, once per second. The system waits until the
eye is in a stable place, and then repeats the test at random times so
the pigs can't anticipate the puff," Dilger says.

But they did learn, over the course of the five-day experiment, to
anticipate the puff. Pretty soon, pigs were closing their eyes at
exactly the 500- millisecond mark during the eighth trial -- the one
with a light but no puff.

"The timing is perfect. If you look at the conditioned eyelid responses,
you can see that the eyelid is closed exactly at the moment the puff
would have been delivered," Boele says. "Just perfect motor timing,
down to the millisecond. That's beautiful." The research team learned something else about pigs they hadn't known before.

"We learned that pigs can just close one eye at a time; they can wink. We weren't sure about that, actually," Dilger says. "But because they are
pigs, they will frustrate you all day long. Some pigs would just lay
there with their one eye closed, which meant we couldn't use that that particular subject.

They're smart creatures." The eyeblink test specifically targets activity
in the cerebellum, the part of the brain responsible for making quick, unconscious predictions. These predictions relate to motor responses,
such as where to step while you're walking; and cognitive processes,
such as predicting what someone is going to say next in a conversation.

"The cerebellum is making short-term predictions all the time,
continuously.

It's essential to interact with our environment. When we think about
learning and memory, often we think about really complex cognitive
things, but most of our daily life behavior is just smooth, automatic interactions with our environment," Boele says. "In eyeblink conditioning,
your cerebellum basically solves the problem for you. You don't have to
think about it. It's making this short-term prediction, and that's what
we are studying." Pigs are born with a more developed cerebellum than
human babies. That's clear from eyeblink conditioning tests Boele has
done with six-to-eight-month-old infants; they don't typically learn
the task at that age.

Unlike humans, pigs need to be able to make motor and cognitive
predictions right away, since they can get up and walk around minutes
after birth. When Dilger studies the pig cerebellum using MRI, which
focuses mostly on structure, he typically doesn't see much change due
to nutritional interventions. That's because the cerebellum is more
developed at birth in the pig, which makes them a precocial species. But nutritional changes might alter the functioning of the brain region. That
is what the eyeblink test will tell him in future studies.

"Often, function follows structure, but not always. Having a nutritional deficiency may show a deficit in eyeblink conditioning, this associative learning task," he says. "We want to be able to use a nutritional
intervention as a relatively non-invasive way of understanding cerebellar development here." The Department of Animal Sciences is in the College
of Agricultural, Consumer and Environmental Sciences at the University
of Illinois.

========================================================================== Story Source: Materials provided by University_of_Illinois_College_of_Agricultural,_Consumer and_Environmental_Sciences. Original written by Lauren Quinn. Note:
Content may be edited for style and length.


========================================================================== Journal Reference:
1. Henk-Jan Boele, Sangyun Joung, Joanne E. Fil, Austin T. Mudd,
Stephen A.

Fleming, Sebastiaan K. E. Koekkoek, Ryan N. Dilger. Young
Domestic Pigs (Sus scrofa) Can Perform Pavlovian Eyeblink
Conditioning. Frontiers in Behavioral Neuroscience, 2021; 15 DOI:
10.3389/fnbeh.2021.690019 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/07/210708134910.htm

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