Synaptic Orb2A Bridges Memory Acquisition and Late Memory Consolidation in Drosophila

Summary To adapt to an ever-changing environment, animals consolidate some, but not all, learning experiences to long-term memory. In mammals, long-term memory consolidation often involves neural pathway reactivation hours after memory acquisition. It is not known whether this delayed-reactivation schema is common across the animal kingdom or how information is stored during the delay period. Here, we show that, during courtship suppression learning, Drosophila exhibits delayed long-term memory consolidation. We also show that the same class of dopaminergic neurons engaged earlier in memory acquisition is also both necessary and sufficient for delayed long-term memory consolidation. Furthermore, we present evidence that, during learning, the translational regulator Orb2A tags specific synapses of mushroom body neurons for later consolidation. Consolidation involves the subsequent recruitment of Orb2B and the activity-dependent synthesis of CaMKII. Thus, our results provide evidence for the role of a neuromodulated, synapse-restricted molecule bridging memory acquisition and long-term memory consolidation in a learning animal.

Head extracts from w+;tubGal80ts, UAS-orb2BGFP;orb2 mCPEBRBD adult flies were analyzed by IP and WB with Abs against the GFP tag at indicated time points after temperature shift either from 22°C to 27°C or back to 22°C after 7 hour induction at 27°C.  Courtship indices of the n Canton-S males fed with sucrose only (DA-) or supplemented with dopamine (DA+) after being trained with a mated female for 1 hr (Train +) or staying alone (Train-) as indicated in Fig. 1, and tested in singlepair assays with mated or virgin females when indicated. P values determined by permutation test for the null hypothesis that learning equals 0 (H 0 : LI = 0) or for the null hypothesis that specific experimental and control males learn equally well (H 0 : LI n = LI c ) Courtship indices of males of the indicated genotypes, retained at 22°C or warmed to 32°C for 2 hrs at the time points according to Fig. 2A after either 1 hour training with a mated female (Train+) or staying alone (Train-) as indicated above and tested in single-pair assays with mated females. P values determined by permutation test for the null hypothesis that learning equals 0 (H 0 : LI = 0) or for the null hypothesis that specific experimental and control males learn equally well (H 0 : LI n = LI c ) Courtship indices of males of the indicated genotypes, retained at 22°C or warmed to 32°C for 2 hrs according to Fig. 2B after either 1 hour training with a mated female (Train+) or staying alone (Train-) as indicated above and tested in singlepair assays with mated females. P values determined by permutation test for the null hypothesis that learning equals 0 (H 0 : LI = 0) or for the null hypothesis that specific experimental and control males learn equally well (H 0 : LI n = LI c ) Courtship indices of males of the indicated genotypes, retained at 22°C or warmed to 32°C according to Fig. 2C after 7 hour training with a mated female (Train+) or staying alone (Train-) as indicated above and tested in single-pair assays with mated females. P values determined by permutation test for the null hypothesis that learning equals 0 (H 0 : LI = 0) or for the null hypothesis that specific experimental and control males learn equally well (H 0 : LI n = LI c ) Courtship indices of males of the indicated genotypes either trained for 7 hrs with a mated female (Train+) or remaining alone (Train-) as indicated in Fig. 3A and tested in single-pair assays with mated females. Indicated males were fed for 6 hrs with DopR1&2 antagonist, SCH23390, after 7 hrs training on water only. P values determined by permutation test for the null hypothesis that learning equals 0 (H 0 : LI = 0) or for the null hypothesis that specific experimental and control males learn equally well (H 0 : LI n = LI c ) Courtship indices of males of the indicated genotypes fed with sucrose supplemented with dopamine (DA+) after being starved for 16 hrs and trained with a mated female for 1 hr (Train+) or remaining alone (Train-) as indicated in Fig. 3B and tested in single-pair assays with mated females. Indicated males were fed with dopamine supplemented with DopR1&2 antagonist SCH23390. P values determined by permutation test for the null hypothesis that learning equals 0 (H 0 : LI = 0) or for the null hypothesis that specific experimental and control males learn equally well (H 0 : LI n = LI c ) Courtship indices of males of the indicated genotypes either trained for 7 hrs with a mated female (Train+) or remaining alone (Train-) as indicated in Fig. 3C and tested in single-pair assays with mated females. P values determined by permutation test for the null hypothesis that learning equals 0 (H 0 : LI = 0) or for the null hypothesis that specific experimental and control males learn equally well (H 0 : LI n = LI c ) Courtship indices of males of the indicated genotype fed with sucrose supplemented with dopamine (+ DA) after being trained with a mated female for 1 hr (Train+) or remaining alone (Train-) as indicated in Fig. 4A and tested in single-pair assays with mated females. P values determined by permutation test for the null hypothesis that learning equals 0 (H 0 : LI = 0) or for the null hypothesis that specific experimental and control males learn equally well (H 0 : LI n = LI c ) WB signal corresponding to the Orb2-GFP oligomers (Fig. 4B) has been quantified using Fiji-ImageJ (Fig. 4C). Mean intensity was normalized to the wild type not treated with dopamine (wt -DA). Courtship indices of males of the indicated genotypes either trained for 7 hrs with a mated female (CI exp) or remaining alone (CI naive), treated as indicated in Fig. 6 and tested in single-pair assays with mated females. P values determined by permutation test for the null hypothesis that learning equals 0 (H 0 : LI = 0) or for the null hypothesis that rescue flies with the wild type isoform learns equally well as rescue flies with the mutated isoform in the same conditions (H 0 : LI n = LI * )  Medium intensity of the fluorescence measured in the gamma lobe of the MB of the indicated genotype according to Fig. 7C. P values determined by 2-sided t-test for the null hypothesis that the fluorescence intensity at time xhr equals the intensity at 0hr (H 0 : ftx=ft0) or 24 hrs (H 0 : ftx=ft24)

Courtship Conditioning Paradigm
Flies were maintained on conventional cornmeal-agar medium under a 12 hrs light: dark cycle at 25°C and 60% relative humidity. Courtship assays were performed at variable circadian clock of the flies. Males were assayed for courtship conditioning as described (Siwicki and Ladewski, 2003). For training, individual males were placed in food chambers either with (trained) or without (naive) a single premated female. After training, each male was recovered, transferred to a fresh food vial and kept in isolation until testing. For long-term memory, males were trained for 6-7 hrs and tested after 24 hrs. For short-term memory, the training period was 1 hr and the test was performed after 30 min. Tests were performed in a 10 mm diameter courtship chamber and videotaped for 10 min (JVC handycam, 30 GB HD). Videos were scored manually and blind to the genotype for CI, which is the percentage of time each male spent courting during the test. Courtship index (CI) was used to calculate the Learning Index (LI): CI naive -CI trained /CI naive × 100.

Immunohistochemistry
Immunohistochemistry on adult brains was performed as described (Yu et al., 2010). Fly brains were dissected (between 5 to 8 days after eclosion) in PBS and fixed using 4% paraformaldehyde in PBST (PBS with 0.3% Triton X-100) for 20 min at 24°C. After washing in PBST, the tissue was blocked in 5% normal goat serum in PBST for at least 2 hrs. The primary antibody and secondary antibody were incubated for 48 hrs at 4°C. The brains were washed with PBST 3 × 10 min and then overnight at 4°C between the primary and secondary antibody incubations. After the secondary antibody incubation, samples were washed 3 × 10 min and overnight at 4°C before mounting in Vectashield (VectorLabs). Antibodies used: rabbit polyclonal anti-GFP (1:5,000, Torri Pines); secondary Alexa-488 antibodies (1:1,000, Invitrogen).

Confocal Microscopy
For imaging and measurement of the fluorescence intensity of the EYFP+/-CaMKII-3'UTR, the fly brains immunostained as described above, were scanned using a Zeiss LSM 710 with a Zeiss Multi Immersion Plan NeoFluar 63× objective. Scanning parameters were set to image the entire mushroom body. Images were taken at 785 × 785 pixels. Images were processed in Imaris for fluorescence quantification. Briefly a cuboid of similar size was set as surface into each MB gamma lobe and the mean YFP fluorescence quantified.

Immunoprecipitation and Western Blot
Adult heads of the indicated genotype were lysed in homogenization buffer (PBS,150mM NaCl, 0.1mM CaCl2, 3mM MgCl2, 5% Glycerol, 1mM DTT, 0.1% TritonX100, 0.1% NP40, EDTA free protease inhibitor cocktail from Roche). The lysate was cleared by centrifugation prior to incubation with Chromotek GFPtrap beads (according to the manufacturer protocol). The proteins were transferred to a PVDF membrane (Millipore) overnight in the cold room at 35mv. Membrane was blocked in 5% milk prior to incubation for 1 hr with a primary antibody. After 3 washes in PBST (PBS+ 0,05%Tween20) membrane was incubated for 1 hr in a secondary antibody. The membrane was developed using SuperSignal West Femto Maximum Sensitivity Substrate (ThermoScientific). Antibodies used: anti-GFP (Abcam 6556 rabbit polyclonal, 1:2,000).

Immuno-EM on adult brains
The brains of 6-7 day old adult flies were dissected in cold fly saline and fixed for 3 hours on ice with 0.1% glutaraldehyde/4% formaldehyde in 0.07M sodium phosphate buffer, pH 7.4, rinsed with 0.1 M phosphate buffer containing 0.1% saponin, and incubated overnight with an HRPconjugated rabbit anti-GFP polyclonal antibody (Life Technologies, A10260, anti-GFP, rabbit IgG fraction, horseradish peroxidase conjugate) at 1:200 dilution in 0.1 M phosphate buffer containing 5% normal goat serum/1% BSA at 4° C. The brains were then rinsed with 0.1 M phosphate/0.1% saponin buffer and reacted with 0.5 mg/ml DAB in 0.1 M phosphate buffer containing 0.1% saponin for 45 minutes following the addition of 10 µl 0.03% H 2 O 2 . The brains were then rinsed with 0.1 M Na-cacodylate buffer, followed by OTOT enhancement (10 min cycles of 0.01% OsO4 in 0.1M Na-cacodylate buffer followed by 0.1% thiocarbohydrazid1M Na-