Mapping neuronal activities using Hr38 FISH.

(A) Illustrated summary of this study for mapping Hr38 expression induced by social behavior or artificial activation of specific neuronal populations. (B) Representative images of Hr38 expression before (fru-No PS) and after photo-stimulation of fru-GAL4>Chrimson neurons (fru-PS). BDPG4uw-PS represents the images after Photo-stimulation of empty-GAL4>Chrimson neurons. magenta: Chrimson::tdT (native fluorescence), green: Hr38 HCR signals. Scale bar: 100μm. (C) Magnified confocal section images of Hr38 expression (left three columns) from the boxed region (far right column) after photo-stimulation of fru-GAL4>Chrimson neurons. SMP, superior medial protocerebrum; LH, lateral horn; KC, Kenyon cells. Scale bar: 20µm (left three columns) or 100µm (far right column).

Investigation of neurons activated by social behavior with Hr38 FISH.

(A) Hr38 expression in the neurons labeled with P1a-split GAL4, Tk-GAL4, and aSP2-split GAL4 drivers. magenta: myr::GFP, yellow: Hr38 HCR signals. Dotted lines depicted the outlines of myr::GFP labeled cell bodies. Arrows: cell bodies of Hr38 positive neurons. Scale bar: 10µm. (B) (D) Upper, scatter plots indicate expression level (relative intensity) of Hr38 HCR signals; lower bar plots indicate percentage of Hr38 positive neurons (“positive” defined as neurons with a relative signal intensity > 3σ above the average signal intensity of the control condition) in P1a (B), Tk (C), and aSP2 cells (D) after different behavioral episodes. Aggression: aggressive behavior against another male, Dead: interaction with a dead male, No Food: interaction with another male without food (no fighting), Mesh: interaction with another male, separated with mesh (no physical contact), Courtship: courtship behavior with a virgin female. Bar plots indicate the percentage of Hr38+ neurons among GFP-labeled cells. Bars with the same letter are not statistically significantly different; bars with no common letter are significantly different (p < 0.05, chi-square test). Number of neurons and individuals analyzed; P1a-Control: 135 (N = 8), P1a-Aggression: 84 (N = 5), P1a-Dead Male: 126 (N = 7), P1a-No Food: 115 (N = 6), P1a-Mesh: 87 (N = 6), P1a-Courtship: 114 (N = 6), Tk-Control: 45 (N = 4), Tk-Aggression: 69 (N = 6), Tk-Dead Male: 61 (N = 6), Tk-No Food: 63 (N = 6), Tk-Mesh: 82 (N = 8), Tk-Courtship: 66 (N = 6), aSP2-Control: 20 (N = 3), aSP2-Aggression: 36 (N = 4), aSP2-Dead Male: 23 (N = 3), aSP2-No Food: 49 (N = 4), aSP2-Mesh: 36 (N = 4), aSP2-Courtship: 24 (N = 3).

Investigation of downstream neurons activated by optogenetic activation of P1a neurons.

(A) Illustration of the experimental set up. (B) Expression pattern of Chrimson::tdT driven by P1a split GAL4. (C) Scheme of the data analysis procedure. (D) Time-course study of Hr38 HCR expression after P1a photo-stimulation in P1a labeled neurons and the surrounding area. By 30-60 mins, most of the P1a labeled neurons expressed Hr38 (96.8% of P1a labeled neurons at 30 min). Scale bars: 10µm. (E) Average image of Hr38 expression of control (BDPG4Uw) and P1a-activated brains. 5 individual brains from each conditions were registered into a template brain and averaged. Green: Hr38 HCR signals, Magenta: Brp. Scale bar: 100µm. (F) Heat map analysis of Hr38 expression. Areas depicted by dotted lines represent the areas of PAM neurons (PAM), mushroom bodies (MB), and pC neurons (pC). Scale bar: 100µm. (G) Confocal images of Hr38 expressions in pCd neurons (R41A01), Kenyon cells (OK107), and PAM neurons (0273 or Ddc) of control and P1a activated brains. Green: myr::GFP, Magenta: Hr38. Dotted lines depicted the outlines of cell bodies (pCd) or cluster of neurons (KC, PAM). Scale bar: 10µm.

Drosophila catFISH reveals P1a subpopulations activated by two different social behaviors.

(A) Experimental design of the fly catFISH. Each fly experienced the 1st behavior (aggression or courtship) followed by 2nd behavior (aggression or courtship), and the HCR was performed with the Hr38 probes targeted for the exon or the intron sequence. (B) Time-course of Hr38 signals detected by the exon- or the intron-targeted Hr38 HCR probes and expected Hr38 signals in each neurons. (Top) The intron-targeted Hr38 HCR signals (magenta) induced by 1st behavior disappear at the timing of HCR analysis while the exon-targeted Hr38 signals (blue) remain in the cytoplasmic area. (Middle) Both intron-targeted and exon-targeted signals are present in the nucleus. (Bottom) Both intron-targeted and exon-targeted signals induced by the 2nd behavior are present in the nucleus, and exon-targeted Hr38 signals induced by the 1st behavior are present in the cytoplasmic area. In ‘Neurons active during both’, the exon probe signals from the 1st behavior decline (dotted blue line *1) after 60 mins (also see Fig. 4B-supplement). However, it is canceled out as the signals from 2nd behavior are increased (dotted blue line *2). As a result, the combined Hr38 exon probe signals (solid blue line) remain high at the time point for Hr38 HCR. (C) Representative images of Hr38 HCR signals in P1a neurons after six different behavioral conditions. Green: myr::GFP, Magenta: intron-targeted Hr38 HCR signals: exon-targeted Hr38 signals. Scale bar: 10µm. The images shown are for illustration purposes only and represent single optical sections covering only a portion of the P1a neuronal cluster. They exclude P1a cells present in other Z-planes. Quantification (D) was based on signals measured in all P1a cells contained within an entire Z-stack. See also Figure 4 - figure supplement D. (D) Fraction of responsive P1a neurons across indicated conditions. N.R.: not responded, Nuc: nuclear signal only (responded to only second target), Cyto: cytoplasmic signal only (responded to only first target), Both: nuclear and cytoplasmic signal (responded to both first and second target).