Diapositive 1

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Construction article scientifique
Contexte Générale (Thème de recherche)
Contexte Spécifique (Sujet de la recherche)
Univers du discours théorique
Hypothèse de Travail
Hypothèse opérationnelle
Modèle expérimental
Univers Observables
Protocole expérimental
Expériences programmées
Et Résultats
Univers du discours théorique
Conclusion
Implication et Interprétation
Perspective
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Genetic Tracing Shows Segregation of Taste
Neuronal Circuitries for Bitter and Sweet
Makoto Sugita and Yoshiki Shiba
Science 2005, 309 781 - 785
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• Contexte général
• (discours théorique)

The gustatory system is primarily devoted to a
quality check of food, while at the same time
detecting nutrients and avoiding toxic
substances. The initial step in taste perception
takes place at the apical end of taste receptor
cells, tightly packed into taste buds of the oral
epithelium. The cells express taste receptors,
which are responsible for detecting and
distinguishing among sweet, bitter, salty, sour,
and umami stimuli (1). In mammals, bitter and
sweet and/or umami are the two main taste
modalities evoking aversion and attraction,
respectively. Humans also express pleasure for
sweet taste but displeasure for bitter taste. On
the other hand, mammals learn to reject a tastant
if this tastant is associated with subsequent
visceral malaise (2). Therefore, it is likely
that the mammalian gustatory system is an
excellent system to address the question of how
emotion interacts with cognition and memory. To
decipher rationally the underlying molecular,
cellular, and system mechanisms, it is first
necessary to understand and to compare precisely
the contrastive neuronal circuitries that process
and integrate the information of aversive and
attractive taste modalities in the whole brain.
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2. Problème spécifique (discours théorique)
Bitter tastants are detected by members of a
family of 30 different G protein-coupled
receptors (GPCRs), the T2Rs (3-5). Sweet and
umami tastes are substantially mediated by a
small family of three GPCRs (T1R1, T1R2, and
T1R3). T1R2 and T1R3 combine to function as a
sweet receptor, whereas T1R1 and T1R3 form the
umami receptor, which detects glutamate (6, 7).
Sweet, umami, and bitter receptors appear to be
expressed in distinct populations of taste cells
that operate independently of each other to
trigger taste recognition (6, 8-10). The receptor
cells are innervated by afferent fibers that
transmit information to the gustatory cortex
through synapses in the brain stem and thalamus
(11).
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3. Hypothèse de travail (discours théorique)
How is taste information processed in the central
nervous system, while it is discriminated and as
it evokes the emotional and behavioral responses
such as aversion and attraction?
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4. Hypothèse opérationnelle (discours des
observables)
We applied a genetic approach to visualize the
neuronal circuitries of bitter and sweet-umami
taste by using the taste receptor genes and the
plant lectin WGA as molecular tools. Injected
lectin proteins are an effective tracer for
transsynaptically delineating the wiring patterns
in the central nervous system (12-14).
Furthermore, the genetic approach using the WGA
transgene, expressed under the control of
specific promoter elements, is a powerful tool
for tracing selective and functional neuronal
circuitries originating from a specific type of
neuron (15, 16).
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Un parcours banalisé (Nature, 2001, équipe de
Linda Buck)
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Partie 1. Generation of the mT2R5-WGA mouse to
visualize bitter taste neuronal circuitries.
Schematic diagram indicating the structure of the
transgene to trace bitter taste neuronal
circuitries. Blue boxes represent the homologous
regions, found in the 5' upstream sequences of
mT2R5 and human T2R10. (B) Expression of
mT2R5-GFP and tWGA-DsRed in cultured cells.
Subcellular distribution of mT2R5-GFP and
tWGA-DsRed, transiently expressed under the
control of the CMV promoter in Cos7 cells and
HEK293 cells, was directly visualized by the GFP
and DsRed fluorescence. The expression levels of
mT2R5-GFP and tWGA-DsRed were monitored with
immunoblotting by using antibodies against GFP
and WGA, respectively.
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In situ hybridization demonstrated concordance in
the expression pattern of the endogenous mT2R5
gene (red) and the transgene (green).
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Direct fluorescence detection of mT2R5-GFP and
tWGA-DsRed, expressed in taste receptor cells.
Arrows indicate the transgene-expressing taste
buds.
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Spatial distribution of tWGA-DsRed in coronal
sections of the mT2R5-WGA mouse brain, clarified
by direct fluorescence detection (right).
Darkfield images at the same magnification
(middle) and at the lower magnification (left)
were also shown. The distance to the posterior
end of the fasciculus retroflexus (pfr) was
calculated and denoted in each section.
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Partie 2. Generation of the mT1R3-WGA mouse to
visualize sweet-umami taste neuronal circuitries.
Schematic diagram indicating the structure of the
transgene to trace sweet-umami taste neuronal
circuitries.
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Direct fluorescence detection of mT1R3-GFP and
tWGA-DsRed, expressed in taste receptor cells.
Arrows indicate the transgene-expressing taste
buds.
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Confocal images showing localization of
mT1R3-GFP, tWGA-DsRed,     -gustducin, PGP-9.5,
and 5-HT in taste buds of mT1R3-WGA mice.
Location of mT1R3-GFP and tWGA-DsRed was
clarified by detecting GFP and DsRed
fluorescence. Locations of     -gustducin,
PGP-9.5, and 5-HT were detected using the primary
antibodies against those proteins and
Alexa-633-conjugated secondary antibodies.
However, Alexa-633 fluorescence was replaced by
the pseudocolor blue and overlaid. White arrows
indicate the mT1R3-GFP- and tWGA-DsRed-expressing
cells without immunoreactivity for
    -gustducin, PGP-9.5, or 5-HT. Red arrows
indicate the mT1R3-GFP- and tWGA-DsRed-expressing
cells with immunoreactivity for     -gustducin,
PGP-9.5, or 5-HT.
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Partie 3. Spatial distribution of tWGA-DsRed in
the mT2R5-WGA and mT1R3-WGA mouse brains,
revealed by immunohistochemical detection of WGA.
Visualizing the spatial distribution of
tWGA-DsRed-labeled neurons in geniculate
ganglions of mT2R5-WGA mice (left) and mT1R3-WGA
mice (right). Arrows indicate the
tWGA-DsRed-labeled nerve fibers.
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Locations of tWGA-DsRed-labeled neurons in the
coronal sections of the mT2R5-WGA mouse brain.
Arrows in the seventh panel indicate the
tWGA-DsRed-labeled neurons in the amygdala.
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Locations of tWGA-DsRed-labeled neurons in the
coronal sections of the mT1R3-WGA mouse brain.
The distance to pfr was calculated and denoted in
each section.
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