Preferential distribution of nuclear MAPK signal in α/β core neurons during long-term memory consolidation in Drosophila

Neuronal signal relay from synapse to nucleus, which is evokedbybehavioral training, playsa vital part in consolidation of protein synthesis-dependent long-term memory (LTM) from invertebrates to vertebrates (Kandel et al., 2014). Among different training-induced neuronal signals, activation of MAPK (mitogen-activated protein kinase) is extensively studied and widely believed to be essential and critical for LTM consolidation from invertebrates to vertebrates (Alberini and Kandel, 2015). Extensive studies contribute to two fundamental questions that how behavioral training activates synaptic signaling molecules and how nuclear signaling molecules initiate new transcription of genes (Alberini and Kandel, 2015). However, relatively slower progress has been made on how behavioral training-induced synaptic signals translocate into nucleus, which is a critical step to bridge the former two questions together. In a recent study, we found that DIM-7, an importin in Drosophila, plays a critical role in mediating nuclear translocation of pMAPK to initiate LTM consolidation (Li et al., 2016). In that study, we found that Kenyon cells (KCs), neurons of mushroom body (MB), are critical places for nuclear translocation of pMAPK signal in determining LTM consolidation. Since the MB, which is a center of associative memory in Drosophila (Davis, 2005), contains about 2,000 neurons (Aso et al., 2009), it is interesting and useful to know whether such pMAPK nuclear translocation occurs evenly in all these neurons or preferentially in a specific group of KCs. In the current study,wecombinedbehavioral trainingparadigmwith confocal imaging to address this question. What we found is that consolidation-related pMAPK nuclear translocation occurs preferentially in a small group ofMBneurons (α/βcKCs), which are reported to be necessary and specific for LTM consolidation (Huang et al., 2012). According to our previous study (Li et al., 2016), we found that LTM training (spaced training, four repeated training sessions with 15-min interval) significantly induces more nuclear translocation of pMAPK at a representative time point of consolidation (8-h after spaced training), compared with naive flies and flies subjected to non-LTM training (massed training, four repeated and consecutive training sessions). This data indicates that LTM training specifically causes pMAPK nuclear translocation in KCs. In the current work, by using the same method, we employ more specific Gal4 lines to study the distribution of such training-induced nuclear pMAPK signal in subgroups of KCs. We first checked the distribution of nuclear pMAPK signal in threemajor classesofMBneurons (α/β, γ, andα’/β’) at 8-h after spaced training, a representative time point during LTM consolidation (Li et al., 2016). To distinguish these classes, we employed three specificGal4 lines: c739, VT44966, VT57244. These lines were reported to be specific drivers of different MB drivers (Aso et al., 2009; Wu et al., 2013; Yang et al., 2016). By crossing these Gal4 lines with UAS-mCD8::GFP; MB247-DsRed flies, we confirmed their specific expression patterns in MB lobes (Fig. S1). Relative to all MB lobes labeled by DsRed signal (red color), c739-Gal4, VT44966-Gal4, and VT57244-Gal4 showed strong and specific expression respectively in α/β lobe, γ lobe, andα’/β’ lobe (SeeGFP signal, greencolor). TheseGal4 tools allowus todetect pMAPKsignal in each specific type of KCs during LTM consolidation. The data were shown in Fig. 1. We crossed these Gal4 lines with UAS-nlsGFP flies to label the nuclei of specific KCs (GFP signal, green color). All nuclei in MBwere labeled by TO-PRO3 (blue color), while pMAPK signals were detected by its specific antibody (red color). From the representative images, we could see a clearly preferential distribution of pMAPK inMB nuclei (Fig. 1A). In contrast to γ KCs (VT44966) and α’/β’ KCs (VT57244), nuclear translocation of pMAPK occurred more likely in nuclei of α/β KCs (c739). Then we analyzed all the imaging data by measuring the mean intensity of nuclear pMAPK relative to calyx (the dendritic area of MB) and by counting the number of nuclei with strong pMAPK signal in different types of KCs. As Figure 1B showed, pMAPK mean intensity in nuclei of α/β KCs (c739) were significantly higher than γ KCs (VT44966) and α’/β’ KCs (VT57244). Consistently, the number of nuclei with strong pMAPK signal in α/β KCs (c739) was also apparently more than other two types of KCs (Fig. 1C). Interestingly, there were more nuclei with strong pMAPK signal in γ KCs (VT44966) compared with α’/β’ KCs (VT57244) (Fig. 1C), despite that there were no significant differences of nuclear pMAPK mean intensity between these

Immunofluorescence. The procedures are also described previously (Li et al., 2016). Flies were quickly anesthetized on ice and dissected in ice-cold PBS within 5 minutes. Brains were fixed in 4% paraformaldehyde in PBS for 30 min on ice. All solutions added after fixation in pMAPK staining were treated with 1% phosphatase inhibitor cocktail (Thermo Fisher Scientific). After being washed three times in PBS, brains were treated in blocking solution (PBS with 2% Triton X-100 and 10% goat serum) for 60 min at room temperature. Then the brains were transferred into primary antibody solution (PBS with primary antibody, 0.2% Triton X-100 and 1% goat serum) and incubated for at least 24 hours at 4°C. Rabbit anti-pMAPK antibody (1:50, Cell Signaling Technology), mouse anti-nc82 antibody (1:10, DSHB), rabbit anti-DsRed (1:400, CloneTech) or chicken anti-GFP antibody (1:2000, Abcam) was used as primary antibody depending on the experiment. Brains were washed three times again in PBS and transferred into secondary antibody solution (PBS with secondary antibody, 0.2% Triton X-100 and 1% goat serum) and incubated overnight at 4°C. Goat anti-rabbit IgG Cy3 antibody (1:200, Jackson ImmunoResearch), goat anti-mouse IgG Cy3 antibody (1:200, Jackson ImmunoResearch) or goat anti-chicken IgG Alexa Fluor 488 antibody (1:200, Invitrogen) was used as secondary antibody depending on the experiment. Then TO-PRO-3 iodide in diluent buffer (1:500) was used to stain nuclei by incubating for 30 min at room temperature and finally washing with PBS three times. Brains were mounted in VECTASHIELD mounting medium (Vector Laboratories). Images were taken using confocal microscopy (Zeiss LSM710META).

Quantification of images.
All images used for quantifications were carefully acquired avoiding overexposure or underexposure. For each genotype, data from flies were acquired at the same time and under the same microscope parameters. Four consecutive slices of each fly, which contained dendritic region of mushroom body (calyx) surrounded by Kenyon cells, were selected and calculated using Imaris software. Mean intensity of pMAPK in nuclei was divided by that in calyx to get the mean intensity ratio. For ratio of nuclear number, two consecutive slices of each fly, which contained dendritic region of mushroom body (calyx) surrounded by Kenyon cells, were selected and calculated. All statistics were calculated from at least six brains.
Statistics. All data were analyzed using Graphpad Prism 6.0 software. Comparisons between two groups used two-tailed t-test. Comparisons of multiple groups used one-way ANOVA followed by Bonferroni post hoc comparisons. Statistically significance was showed with *, if P value < 0.05. All data in bar graphs are showed as means ± SEM.

Supplemental figures
Fig. S1 Expression pattern of three different MB Gal4 lines. Indicated Gal4 lines were crossed to flies with the genotype UAS-mCD8::GFP; MB247-DsRed and identified by confocal imaging of whole adult central brain. Gal4 expression is displayed by green color. Structure of MB lobes is shown by red color. c739-Gal4, VT44966-Gal4 and VT57244-Gal4 showed strong expression in α/β lobe, γ lobe and α'/β' lobe respectively. Scale bar is 20 μm.

Fig. S2
Expression pattern of two different Gal4 lines of α/β KCs. Indicated Gal4 lines were crossed to flies with the genotype UAS-mCD8::GFP; MB247-DsRed and identified by confocal imaging of whole adult central brain. Gal4 expression is displayed by green color. Structure of MB lobes is shown by red color. VT26665-Gal4 and NP7175-Gal4 showed strong expression in α/βs lobe and α/βc lobe respectively. Scale bar is 20 μm.