ูグᵝᘧ➨  ྕ㸦➨  ᮲➨  㡯㸪➨  ᮲➨  㡯㛵ಀ㸧ㄽ ᩥ ᑂ ᰝ ࡢ ⤖ ᯝ ࡢ せ ᪨༤ኈࡢᑓᨷศ㔝ࡢྡ⛠ ༤ኈ㸦ཱྀ⭍೺ᗣ⛉Ꮫ㸧ẶྡᏛ ఩ ᤵ ୚ ࡢ ᮲ ௳ Ꮫ఩つ๎➨  ᮲➨‫ۑ‬ ࣭ 㡯ヱᙜᙇ⤞፽
CHANG CHIEH MEIㄽ ᩥ 㢟 ┠
Plastic changes in medial amygdalar neurons defined by genetic tracing of taste
representation after conditioned taste aversion learning㸦Ⓨ⏕ᕤᏛⓗࢺ࣮ࣞࢩࣥࢢ࡟ࡼࡾྍど໬ࡉࢀࡓෆഃᡥ᱈యࡢ࿡ぬ⤒㊰ࢽ࣮ࣗࣟࣥ࡟࠾ࡅࡿ࿡ぬ᎘ᝏᏛ⩦࡟క࠺ྍረᛶኚ໬㸧ㄽᩥᑂᰝᢸᙜ⪅୺  ᰝ   ᩍᤵ  ኴ⏣ ⪔ྖ             ༳ᑂᰝጤဨ   ᩍᤵ  ஧ᕝ ᾈᶞ             ᑂᰝጤဨ   ᩍᤵ  ᑎᒣ 㝯ྖ             ˭媽㕮⯐㟢͆䴷㞃͆奨㗏ˮ
Conditioned taste aversion (CTA) occurs as a memory paradigm whenanimals learn to reject a flavorsome sweet tastant (conditioned stimulus [CS])with the subsequent visceral malaise (unconditioned stimulus [US]). CTA is anextremely adaptive, powerful form of conditioning that species do not need aspecial training to obtain the restricted responses for. It can be robustlyacquired only in a single trial, making animals avoid the similar tastes. Andthe 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 andthe 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 indownstream neurons at a molecular, cellular or system level. The specific aimof this study was to identify the amygdalar neurons which induce the plasticchanges in activities during CTA acquisition. In this study, the author
examined whether subpopulations of the amygdalar neurons processing
aversive bitter taste information represent the plastic changes in responses tothe CS during CTA acquisition.
By using the transgenic mice that express the transsynaptic tracer tWGADsRed in mT2R5-expressing bitter taste receptor cells, the author visualized
the spatial distribution in the brain of bitter taste-relying neurons labeled bytWGA-DsRed originating from bitter taste receptor cells. For the importantpart, CTA procedure, mice were deprived from water for 12 hr and wereadapted to the water intake schedule for 4 days. The mice were divided into
three groups: “control”, “conditioning”, and “extinction”. The “control” mice werewithout conditioning while “conditioning”, and “extinction” were given with theconditioning 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 themice, and the AI was calculated as (water consumption)/(water + saccharinconsumption) and used as an index for learned aversion to saccharin.
To examine whether the amygdalar neurons were activated by oral
application of saccharin, the author detected the saccharin-induced expressionof 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 10min. Nonspecific binding sites 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 ofsecondary antibodies in the Zenon labeling system. Location of tWGA-DsRed
was determined by direct fluorescence detection. The subcellular distribution ofZif268 was visualized by detecting Alexa-647 fluorescence.
After CTA conditioning, all the tested mice became aversive to saccharin
and establish strong CTA. The mice that acquired CTA memory were subjectedto 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%, used as the“extinction” group. The author examined the spatial distribution of medial
amygdalar (MeA) neurons labeled by tWGA-DsRed that originated from bitter
taste receptor cells in the transgenic mice. tWGA-DsRed was mainly located inthe 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. Zif268 expression was induced by oral stimulation with the sweet tastantsaccharin 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 ofneurons with Zif268-immunoreactivity were found to be 63% (“conditioning”)
versus 37% (“control”). The ratio of the saccharin-activated, WGA-DsRedlabeled 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. To characterize the spatial distributionof the saccharin-activated neurons in the MeA, the author further divided theMeA 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 tothat in the whole MeA.
From the above results, this paper clarified that the subsets of WGADsRed-lebeled neurons in the MeA were activated by the CS only after, but not
before, CTA acquisition and concluded that the specific population of MeA
neurons inherently processing aversive bitter information may induce theplastic changes in their activities after acquiring CTA, which remainunchanged after the extinction of CTA memory. Therefore, all the committee
members admitted the sufficient value to confer the Doctor of philosophy of oral
health science to the author.
௨ୖࡢ⤖ᯝ࠿ࡽ㸪ᮏㄽᩥࡣ࿡ぬ᎘ᝏᏛ⩦࡟క࠺ෆഃᡥ᱈యࡢ࿡ぬ⤒㊰ࢽ࣮ࣗࣟࣥࡢኚ໬࡟ࡘ࠸࡚᪂つࡢ▱ぢ࡟ࡘ࠸࡚㏙࡭ࡓࡶࡢ࡜ㄆࡵࡽࢀࡓࠋࡼࡗ࡚ᑂᰝጤဨ఍ጤဨ඲ဨࡣ㸪ᮏㄽᩥࡀᙇ⤞፽࡟༤ኈ㸦ཱྀ⭍೺ᗣ⛉Ꮫ㸧ࡢᏛ఩ࢆᤵ୚ࡍࡿ࡟༑ศ࡞౯್ࡢ࠶ࡿࡶࡢ࡜ㄆࡵࡓࠋ