ㄽ ᩥ ෆ ᐜ せ ⣙Plastic changes in medial amygdalar neurons
defined by genetic tracing of taste representation
after conditioned taste aversion learning炷൅ਫ਼޽ָదφϪʖεϱήͶΓΕՆࢻԿ͠Η಼ͪଈἹ౩ର͹ັְ‫ܨ‬࿑ωϣʖϫϱͶ͕͜Ζັְ‫ݑ‬ѳָसͶ൒͑Նા੓รԿ炸୺ᣦᑟᩍဨ㸸➉ᮏ ಇఙ ᩍᤵ㸦་⣔⛉Ꮫ◊✲⛉ ཱྀ⭍ಖ೺⟶⌮Ꮫ㸧๪ᣦᑟᩍဨ㸸ෆ⸨ ┿⌮Ꮚ ᩍᤵ㸦་⣔⛉Ꮫ◊✲⛉ ཱྀ⭍ಖ೺␿Ꮫ㸧๪ᣦᑟᩍဨ㸸ᮡ⏣ ㄔ ᩍᤵ㸦་⣔⛉Ꮫ◊✲⛉ ཱྀ⭍⏕⌮Ꮫ㸧ᙇ ⤞፽㸦་ṑ⸆ಖ೺Ꮫ◊✲⛉ ཱྀ⭍೺ᗣ⛉Ꮫᑓᨷ㸧

Introduction
Animals learn to reject a palatable, sweet tastant (conditioned stimulus[CS]) if it is associated with subsequent visceral malaise (unconditionedstimulus [US]). This phenomenon called as conditioned taste aversion (CTA)
can be studied in the animal model, where an animal tasting saccharin, anovel sweet tastant (CS), and followed by intraperitoneal injection oflithium 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 ofCTA. The association of the CS and the US appears to occur in thebasolateral amygdala (BLA) to get aversive to the CS. It is reported thatduring the CTA conditioning, subsets of neurons in the BLA respond to boththe CS taste and the US. The CTA acquisition appears to be disrupted if thestimulus convergence in the basolateral amygdalar neurons is blocked.
Therefore, it is likely that the association of taste information andaversive viscerosensory information that occurs in the BLA may lead tosubsequent plastic changes in downstream neurons, resulting in theacquisition of CTA memory. However, it remains largely unknown how thebasolateral amygdalar neurons receiving both the CS and US induce theplastic changes in downstream neurons at a molecular, cellular or systemlevel.
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 tovisualize the spatial distribution in the brain of bitter taste-relying neurons
labeled by tWGA-DsRed originating from bitter taste receptor cells. Themice were housed on a 12h light/dark cycle. Food and water were available
ad libitum before the behavioral experiments. The animals were treated inaccordance with the Guide for Animal Experiment, Hiroshima University.
The CTA procedure
Mice were deprived from water for 12 hr. Then they were adapted tothe water intake schedule for 4 days. The mice were divided into threegroups: “control”, “conditioning”, and “extinction”. The “control” mice werewithout conditioning while “conditioning”, and “extinction” were given withthe 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 tothe mice, and the AI was calculated as (water consumption)/(water +saccharin consumption) and used as an index for learned aversion tosaccharin.
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 ofZif-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, andfixed 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 analysisStatistical significance was determined using Chi-square test with
Bonferoni-correction. P values <0.05 were considered statisticallysignificant.

Results and discussion
Changes in the AI after CTA conditioning
After CTA conditioning, all the tested mice become aversive tosaccharin 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 amongindividuals, 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 theperinuclear region of the labeled MeA neurons. The cell somata of tWGADsRed-labeled neurons were found in the MeA from Bregma -0.70 toBregma -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 memoryin 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 asignificant 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-activatedneurons 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 theanterior 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 theiractivities after acquiring CTA, which remain unchanged after the extinctionof CTA memory.