Plastic changes in medial amygdalar neurons defined by genetic tracing of taste representation after conditioned taste aversion learning

概要

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Plastic changes in medial amygdalar neurons
defined by genetic tracing of taste representation
after conditioned taste aversion learning
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ᙇ ⤞፽
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Introduction
Animals learn to reject a palatable, sweet tastant (conditioned stimulus
[CS]) if it is associated with subsequent visceral malaise (unconditioned
stimulus [US]). This phenomenon called as conditioned taste aversion (CTA)
can be studied in the animal model, where an animal tasting saccharin, a
novel sweet tastant (CS), and followed by intraperitoneal injection of
lithium chloride that induces illness (US) can acquire one-trial learning of
CTA to that particular sweet tastant.
The amygdala plays a primary role in acquisition and expression of
CTA. The association of the CS and the US appears to occur in the
basolateral amygdala (BLA) to get aversive to the CS. It is reported that
during the CTA conditioning, subsets of neurons in the BLA respond to both
the CS taste and the US. The CTA acquisition appears to be disrupted if the
stimulus convergence in the basolateral amygdalar neurons is blocked.
Therefore, it is likely that the association of taste information and
aversive viscerosensory information that occurs in the BLA may lead to
subsequent plastic changes in downstream neurons, resulting in the
acquisition of CTA memory. However, it remains largely unknown how the
basolateral amygdalar neurons receiving both the CS and US induce the
plastic changes in downstream neurons at a molecular, cellular or system
level.
The specific aim of this study was to identify the amygdalar neurons
which induce the plastic changes in activities during CTA acquisition. In
this study, we examined whether subpopulations of the amygdalar neurons
processing aversive bitter taste information represent the plastic changes
in responses to the CS during CTA acquisition.

Materials and methods
Breeding, housing, and genotyping of transgenic mice
The transgenic mice that express the transsynaptic tracer tWGADsRed in mT2R5-expressing bitter taste receptor cells were used to
visualize the spatial distribution in the brain of bitter taste-relying neurons
labeled by tWGA-DsRed originating from bitter taste receptor cells. The
mice were housed on a 12h light/dark cycle. Food and water were available
ad libitum before the behavioral experiments. The animals were treated in
accordance with the Guide for Animal Experiment, Hiroshima University.
The CTA procedure
Mice were deprived from water for 12 hr. Then they were adapted to
the water intake schedule for 4 days. The mice were divided into three
groups: “control”, “conditioning”, and “extinction”. The “control” mice were
without conditioning while “conditioning”, and “extinction” were given with
the conditioning schedule to acquire CTA.
For CTA conditioning, the mice were applied with two bottles filled with
0.2 % saccharin (CS) for 10 min, and then treated with an intraperitoneal
injection of 0.15 M LiCl (2 % of body weight) as the US 15 min after the CS.
Testing for aversion to saccharin was performed daily after the conditioning.
One bottle filled with water and the other with saccharin were presented to
the mice, and the AI was calculated as (water consumption)/(water +
saccharin consumption) and used as an index for learned aversion to
saccharin.
Sensory stimulation and detection of Zif268 expression in tWGA-DsRedlabeled neurons
To examine whether the amygdalar neurons were activated by oral
application of saccharin, we detected the saccharin-induced expression of
Zif-268, an immediate early gene. Mice were orally applied with saccharin.
The brain was isolated after mice were euthanized with sodium
pentobarbital, and fixed with 2% formaldehyde solution (1x PBS, 2%
formaldehyde). The sections are made by the film-transfer method, and
fixed with 3.7% formaldehyde solution (1x PBS, 3.7% formaldehyde, 0.18%
Triton X-100) for 10 min. Nonspecific binding side were blocked with 1%
bovine serum albumin (BSA) for 30 min.
The sections were treated with primary antibodies against Zif268
which were pre-bound with the Alexa-647-labeled Fab fragments of

secondary antibodies in the Zenon labeling system. Location of tWGADsRed was determined by direct fluorescence detection. The subcellular
distribution of Zif268 was visualized by detecting Alexa-647 fluorescence.
Statistical analysis
Statistical significance was determined using Chi-square test with
Bonferoni-correction. P values <0.05 were considered statistically
significant.

Results and discussion
Changes in the AI after CTA conditioning
After CTA conditioning, all the tested mice become aversive to
saccharin and establish strong CTA. The mice that acquired CTA memory
were subjected to the continued two-bottle test to monitor the daily changes
in the AI. Although the time courses for the AI changes were varied among
individuals, the mice had the AI declining to the value less than 50%. Those
mice were used as the “extinction” group.
Location of bitter-taste relaying neurons labeled by tWGA-DsRed
We examined the spatial distribution of medial amygdalar (MeA)
neurons labeled by tWGA-DsRed that originated from bitter taste receptor
cells in mT2R5-WGA mice. tWGA-DsRed was mainly located in the
perinuclear region of the labeled MeA neurons. The cell somata of tWGADsRed-labeled neurons were found in the MeA from Bregma -0.70 to
Bregma -2.06.
The subsets of WGA-DsRed-lebeled neurons in the MeA were activated by
the CS only after, but not before, CTA acquisition
Zif268 expression was induced by oral stimulation with the sweet
tastant saccharin in more tWGA-DsRed-labeled neurons which inherently
process aversive bitter taste information after acquiring the CTA memory
in the “conditioning” group, compared with those in the “control” group. The
ratios of neurons with Zif268-immunoreactivity were found to be 63%
(“conditioning”) versus 37% (“control”). The ratio of the saccharin-activated,

WGA-DsRed-labeled neurons in the “extinction” group was 65%, showing a
significant difference between the “control” and “extinction” groups, but not
between the “conditioning” and “extinction” groups
Comparison of Zif268 induction in tWGA-DsRed-lebeled neurons divided
into the MeA subregions
To characterize the spatial distribution of the saccharin-activated
neurons in the MeA, we further divided the MeA into anterodorsal medial
amygdala, anteroventral medial amygdala, posterodorsal medial amygdala
and posteroventral medial amygdala. However, the observed patterns in the
anterior and posterior MeA were found similar to that in the whole MeA.

Conclusion
We conclude that the specific population of MeA neurons inherently
process aversive bitter information may induce the plastic changes in their
activities after acquiring CTA, which remain unchanged after the extinction
of CTA memory.