Zicong
2012.2.27

• Corresponding Author
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• LTM produced by spaced repetitive training induces cyclic adenosine monophosphate (cAMP) response element –binding protein ( CREB ) –dependent gene transcription followed by de novo protein synthesis .
• Mushroom body (MB) is a prominent neuroanatomical site involved with STM and LTM formation. Studies have shown that each cell type displays a distinct and altered activity at different time after training.
• Nevertheless, de novo protein synthesis required for
LTM consolidation has never been directly visualized and/or manipulated in any targeted brain structures, including the MBs.
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GAL4/UAS system in drosophila
Gal4 lines : express Gal4 gene in cells or tissues where the driver gene is usually active. (e.g. per-Gal4 , express
Gal4 mainly in lateral neurons where period gene is active)
UAS-reporter lines : upstream activation sequence combined a reporter gene. (e.g. UAS-kaede )
Only when Gal4 binds to the UAS , the reporter gene will be expressed. (e.g., per-Gal4>UAS-kaede, this line will express the GFP only in lateral neurons)
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• Drosophila Gal4 lines: express Gal4 in different types of target neurons.
• UAS-kaede lines: KAEDE , a GFP which changes its structure irreversibly to a red fluorescent protein (RFP) upon UV irradiation.
• UAS-ricin cs lines: RICIN , a potent cytotoxic protein that inactivates eukaryotic ribosomes; cold sensitive, works at
30 ℃ .
• Olfactory associative conditioning (T maze; Spaced training and massed training)
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1. Visualizing de novo protein synthesis in target neurons
In per-Gal4>UAS-
kaede flies:
By photoconverting green KAEDE to red every 4 hours, we showed that KAEDE exhibits a diurnal cycle of de novo synthesis in the lateral neurons.
This de novo KAEDE synthesis was reduced in flies fed the protein synthesis inhibitor, cycloheximide (CXM).
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1. Visualizing de novo protein synthesis in target neurons
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KAEDE synthesis in lateral neurons was not inhibited by RICIN CS at 18 ℃ but decreased ~80% by
RICIN CS at 30 ℃ .

2. Behavioral screen for neurons involved in protein synthesis –dependent memory formation
RICIN CS activated immediately after spaced training in Cha-
Gal4 neurons impaired 1-day memory.
Cha-Gal4 neurons: choline acetyltransferase ( Cha ) promoter – driving Gal4 , which likely labels most acetylcholine-producing neurons.
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2. Behavioral screen for neurons involved in protein synthesis –dependent memory formation
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Unexpectedly , 1-day memory retention remained intact when
RICIN CS was expressed in olfactory sensory neurons, olfactory projection neurons, MB modulatory neurons, all MB neurons, αβ neurons,α′β′ neurons, ellipsoid body neurons and dopaminergic neurons.

2. Behavioral screen for neurons involved in protein synthesis –dependent memory formation
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LTM was impaired with other 8 Gal4 drivers, cer -
, Ddc ( Dopa decarboxylase )-, Trh493 , Trh996 -
, cry ( cryptochrome )-, per -, CaMKII -(X), and CaMKII -
Gal4 (III).

2. Behavioral screen for neurons involved in protein synthesis –dependent memory formation
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By limiting RICIN CS expression to neurons outside of MB using a combination of Cha-Gal4 and MB-Gal80 (Gal80 inhibits Gal4), 1-day memory again was impaired when
RICIN CS was activated immediately after spaced training.

• De novo protein synthesis during LTM formation occurs in Cha-Gal4 –expressing neurons outside of the MBs ;
• LTM was impaired with 9 Gal4 drivers, Cha , cer -, Ddc ( Dopa decarboxylase )-, Trh493 -
, Trh996 -, cry ( cryptochrome )-, per -, CaMKII -(X), and CaMKII Gal4 (III).
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3. Identification of individual neurons with protein synthesis during LTM formation
DDC-antibody immunostaining revealed that the DAL neurons are included in the expression patterns of all nine Gal4 driver lines
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… “subtract” expression of RICIN CS in the two DAL neurons from Cha-Gal4 and per-
Gal4 expression patterns. In both cases, activated RICIN CS in the remaining neurons did NOT affect 1-day memory after spaced training.

3. Identification of individual neurons with protein synthesis during LTM formation
Three new Gal4 drivers with relatively limited patterns of expression, but each of which contained the DAL neurons (validated by DDC-antibody immunostaining). In all three cases, we found that RICIN CS , when activated during the first 12 hours after training , disrupted 1-day memory after spaced training.
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3. Identification of individual neurons with protein synthesis during LTM formation
( UAS-shi ts acutely to block neurotransmission from DAL neurons )
Retrieval of LTM was disrupted when neurotransmission was blocked during the test trial 1 day after spaced training.
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• DAL neurons are required for consolidation and retrieval of LTM.
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4. DAL axons are structurally connected with MB calyx
Putative DAL dendrites distributed mainly in the superior dorsofrontal protocerebrum ( SDFP ) region;
DAL axons distributed widely in three brain regions:
SDFP , dorsolateral protocerebrum ( DLP ), and inferior dorsofrontal protocerebrum ( IDFP ).
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GFP labelling

4. DAL axons are structurally connected with MB calyx
• DAL neurons in G0431-Gal4 and the MB pioneer αβ neurons in L5275-LexA (left) were structurally interconnected at the K5 region (right).
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GRASP labelling

5. Identification of newly synthesized proteins in DAL neurons during LTM formation
• RNAi-mediated disruption of
protein expression in DAL neurons impaired LTM formation after spaced training but not after massed training.
• RNAi constructs for
,
,
, or
in DAL neurons also impaired LTM formation after spaced training.
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6. CREB2 activity in DAL neurons, but not MB neurons, is required for LTM formation
• Adult-specific over-expression of UAS-dcreb2-b ( depressor ) or
RNAi-mediated down-regulation of CREB2 expression in DAL neurons impaired 1-day memory after spaced training but not after massed training, and feeding with cycloheximide (CXM) did not exaggerate these impairments.
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7. Visualizing transcriptional activity in identified neurons during LTM formation
• After photoconversion of preexisting KAEDE, spaced training specifically induced new KAEDE (green) in DAL neurons when UAS-kaede was driven by CaMKII -Gal4 and per -Gal4 .
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7. Visualizing transcriptional activity in identified neurons during LTM formation
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• spaced training– induced levels of
KAEDE synthesis driven by CaMKII-
Gal4 or per-
Gal4 in DAL neurons were diminished , when Gal4 driven UASdcreb2-b expression was induced by tub-
Gal80 ts .

7 evidences suggested that the bilaterally paired DAL neurons are a site of LTM storage:
1.
De novo protein synthesis in DAL neurons during the first 12 hours after spaced training was required for normal LTM formation (slide 14);
2.
Several proteins that have previously been shown to be necessary for LTM formation were colocalized in
DAL neurons (figs. S6A and S9A);
3.
Disruptions of PER, dNR1, dNR2, CaMKII, TEQUILA,
DDC, TRH, and CREB2 in DAL neurons impaired 1day memory after spaced (but not massed) training
(slide 19 & 20);
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4. Expression of a repressor form of CREB2 protein in DAL neurons was sufficient to disrupt 1-day memory after spaced training (slide 20);
5. The transcriptional activities of CaMKII and per were elevated in DAL neurons after spaced training (slide 21);
6. The up-regulations of CaMKII and per in DAL neurons were CREB2-dependent (slide 22);
7. Neurotransmission from DAL neurons was required only for LTM retrieval but not for acquisition (LRN) or consolidation of LTM (slide 15).
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