A Novel Role for Ecdysone in <i>Drosophila</i> Conditioned Behavior: Linking GPCR-Mediated Non-canonical Steroid Action to cAMP Signaling in the Adult Brain

<div><p>The biological actions of steroid hormones are mediated primarily by their cognate nuclear receptors, which serve as steroid-dependent transcription factors. However, steroids can also execute their functions by modulating intracellular signaling cascades rapidly and independently of transcriptional regulation. Despite the potential significance of such “non-genomic” steroid actions, their biological roles and the underlying molecular mechanisms are not well understood, particularly with regard to their effects on behavioral regulation. The major steroid hormone in the fruit fly <i>Drosophila</i> is 20-hydroxy-ecdysone (20E), which plays a variety of pivotal roles during development via the nuclear ecdysone receptors. Here we report that DopEcR, a G-protein coupled receptor for ecdysteroids, is involved in activity- and experience-dependent plasticity of the adult central nervous system. Remarkably, a courtship memory defect in <i>rutabaga</i> (Ca²⁺/calmodulin-responsive adenylate cyclase) mutants was rescued by <i>DopEcR</i> overexpression or acute 20E feeding, whereas a memory defect in <i>dunce</i> (cAMP-specific phosphodiestrase) mutants was counteracted when a loss-of-function <i>DopEcR</i> mutation was introduced. A memory defect caused by suppressing dopamine synthesis was also restored through enhanced DopEcR-mediated ecdysone signaling, and rescue and phenocopy experiments revealed that the mushroom body (MB)—a brain region central to learning and memory in <i>Drosophila</i>—is critical for the DopEcR-dependent processing of courtship memory. Consistent with this finding, acute 20E feeding induced a rapid, DopEcR-dependent increase in cAMP levels in the MB. Our multidisciplinary approach demonstrates that DopEcR mediates the non-canonical actions of 20E and rapidly modulates adult conditioned behavior through cAMP signaling, which is universally important for neural plasticity. This study provides novel insights into non-genomic actions of steroids, and opens a new avenue for genetic investigation into an underappreciated mechanism critical to behavioral control by steroids.</p></div

DopEcR is required for the 30-minute courtship memory induced by 1-hour courtship conditioning.

<p>(A) Thirty-minute courtship memory in wild-type flies (control) and flies heterozygous (<i>DopEcR^PB1</i>/+), homozygous (<i>DopEcR^PB1</i>/<i>DopEcR^PB1</i>), and hemizygous (<i>DopEcR^PB1</i>/Df(3L)ED4341) for <i>DopEcR</i>. <i>DopEcR^PB1</i> homozygotes and hemizygotes were defective for 30-minute courtship memory. (B) Time course of courtship memory in <i>DopEcR^PB1</i> homozygotes. Significant memory was observed immediately after conditioning, but not 15 or 30 minutes after conditioning. (C) A defect in 30-minute courtship memory in flies that ubiquitously express the <i>DopEcR</i> RNAi after eclosion, in response to RU486 stimulation of the <i>tub</i>-GS-Gal4 driver. The presence or absence of courtship memory was evaluated by applying the Mann–Whitney U-test to naïve and conditioned males. Statistical significance is shown above each bar as NS, no significant difference, **, <i>P</i><0.01 or ***, <i>P</i><0.001. Sample numbers for naïve and conditioned flies are shown under each graph. PIs were analyzed using Krustal-Wallis One-Way ANOVA, followed by Dunn's pairwise test for multiple comparisons. #, <i>P</i><0.05; ##, <i>P</i><0.01. Error bars (s.e.m.).</p

A <i>DopEcR</i> hypomorphic allele and the <i>DopEcR</i>-Gal4 expression pattern.

<p>(A) Schematic representation of the <i>Drosophila DopEcR</i> gene. <i>DopEcR</i> contains 5 exons and spans a 12.7 kb genomic region in the 64B2-64B3 cytological interval (Flybase). The <i>DopEcR</i> exons are represented by boxes, and the coding regions are indicated with purple color. A green arrow indicates the direction of transcription of <i>DopEcR</i>. The <i>DopEcR</i> locus is completely uncovered by deficiency Df(3L)ED4341. The PBac(PB)c02142 (<i>DopEcR^PB1</i>) allele harbors an insertion of the <i>piggyBac</i> transposable element within the second intron. The insertion site is indicated as a red arrow with an inverted triangle. (B) <i>DopEcR</i> transcript levels in <i>DopEcR^PB1</i> homozygotes and trans-heterozygotes, as assessed by RT-PCR analysis. (C–G) Expression of the GFP reporter gene (UAS-CD4-tdGFP <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003843#pgen.1003843-Han2" target="_blank">[70]</a>) driven by <i>DopEcR</i>-Gal4 (green) and synaptic neuropil labeled with the nc82 antibody against presynaptic marker protein Bruchpilot (magenta). Anterior view of the adult brain displaying <i>DopEcR</i>-Gal4 expression pattern (C) and the nc82 staining (E). A merged image is shown in (D). (F) Adult brain with the thoracicoabdominal ganglion (ventral view). (G) Dorsal view of the thoracicoabdominal ganglion. Scale bars represent 100 µm. ABD: abdominal ganglion; AL: antennal lobe; AMMC: antennal mechanosensory and motor center; MB: mushroom body; OL: optic lobe; SEG: subesophageal ganglion; T1: prothoracic ganglion; T2: metathoracic ganglion; T3: mesothoracic ganglion; VLP: ventrolateral protocerebrum; wing: wing neuropil.</p

Impaired ecdysone synthesis causes a courtship memory defect.

<p>(A) Rescue of courtship memory in <i>DTS-3</i> males by feeding flies 20E prior to conditioning. (B) A defect in 30-minute courtship memory when the <i>dib</i> RNAi was expressed ubiquitously in adults, in response to RU486 application (<i>tub5</i>-GS-Gal4 driver). The <i>dib</i> RNAi-induced memory defect was not observed when flies were fed 20E, but was observed when 20E feeding was accompanied by the expression of the <i>DopEcR</i> RNAi. (C) Rescue of courtship memory in 3-iodo tyrosine (3-IY)-treated males by feeding flies 20E prior to conditioning. (D) Courtship memory in 3-IY-treated males was not restored by 20E when the <i>DopEcR</i> RNAi was expressed ubiquitously in adults. For 20E and RU486 feeding experiments, control flies received vehicle. The presence or absence of courtship memory was evaluated by applying the Mann–Whitney U-test to naïve and conditioned flies. Statistical significance is shown above each bar. NS, no significant difference. **, <i>P</i><0.01; ***, <i>P</i><0.001. Sample numbers for naïve and conditioned flies are shown under each graph. PIs were analyzed using Student's t-test or Krustal-Wallis One-Way ANOVA. #, <i>P</i><0.05. Error bars (s.e.m.).</p

The mushroom body is critical for the DopEcR-dependent processing of courtship memory.

<p>(A) Rescue of the <i>DopEcR^PB1</i> memory defect by expression of wild-type <i>DopEcR</i> transgene using the <i>DopEcR</i>-Gal4 driver. (B) Rescue of <i>DopEcR^PB1</i> memory defect by expression of wild-type <i>DopEcR</i> transgene under control of the MB-specific c772, c739 and 201 y drivers. Note that MB-Gal4 lines drive reporter expression in different subsets of MB neurons (see text). (C) Courtship memory defect induced by MB-specific expression of the <i>DopEcR</i> RNAi using the c772 and c739 drivers. The presence or absence of courtship memory was evaluated by applying Mann–Whitney U-test to naïve and conditioned flies. Statistical significance is shown above each bar. NS, no significant difference. **, <i>P</i><0.01; ***, <i>P</i><0.001. Sample numbers for naïve and conditioned flies are shown under each graph. PIs were analyzed using Student's t-test or Krustal-Wallis One-Way ANOVA, followed by Dunn's pairwise test for multiple comparisons. #, <i>P</i><0.05; ###, <i>P</i><0.001. Error bars (s.e.m.).</p

<i>DopEcR</i> loss-of-function mutants show slow habituation of the giant-fiber escape circuit.

<p>(A) Schematic representation of the giant-fiber (GF) pathway. GF afferent neurons (aff.). Tergotrochanteral muscle (TTM), TTM motor neuron (TTMn), dorsal longitudinal muscle (DLM), peripherally synapsing interneuron (PSI), and DLM motor neuron (DLMn). (B) The long-latency threshold (LLT), short-latency threshold (SLT) and refractory period (RP) for <i>DopEcR</i> mutants and wild-type flies. The data are presented as box plots. (C) Representative traces of muscle responses to 5-Hz brain stimulation. Vertical bars and dots denote successful and failed responses, respectively. An arrowhead represents the time at which a gentle air puff was applied to trigger dishabituation, which confirms that the failure to respond is due to habituation. (D) Cumulative muscle responses to 5-Hz brain stimulations. (E) The average numbers of 5-Hz stimuli delivered before the fly experiences five consecutive failures (criteria for habituation). Crosses with arrows represent flies that did not show habituation within the observation period (2 minutes). Error bars (s.e.m). Data were analyzed by Krustal-Wallis One-Way ANOVA, followed by Dunn's pairwise test for multiple comparisons. NS, no significant difference. *, <i>P</i><0.05.</p

Bacteriological Assessment of Healthcare-Associated Pneumonia Using a Clone Library Analysis

<div><p>Background</p><p>The causative pathogens of healthcare-associated pneumonia (HCAP) remain controversial, and the use of conventional cultivation of sputum samples is occasionally inappropriate due to the potential for oral bacterial contamination. It is also sometimes difficult to determine whether methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is a true causative pathogen of HCAP.</p><p>Methods</p><p>We evaluated the bacterial diversity in bronchoalveolar lavage fluid (BALF) using molecular and cultivation methods in 82 HCAP patients. BALF specimens were obtained from the lesions of pneumonia using bronchoscopy. The bacterial flora was analyzed according to the clone library method using amplified fragments of the 16S ribosomal RNA gene with universal primers. In addition, sputum cultures and the above specimens were assessed.</p><p>Results</p><p>Eighty (97.6%) of the 82 BALF samples obtained from the patients with HCAP showed positive polymerase chain reaction results. The predominant phylotypes detected in the BALF in this study included bacteria common in cases of community- and hospital-acquired pneumonia. In addition, the phylotypes of streptococci and anaerobes were detected in 19 (23.2%) and 8 (9.8%) cases, respectively. In particular, phylotypes of streptococci were highly detected among the patients 75 of age or older. <i>Staphylococcus aureus</i> was cultured in 23 (28.0%) cases using conventional cultivation methods and detected in only 6 (7.3%) cases as predominant phylotypes according to the clone library method.</p><p>Conclusions</p><p>The clone library analysis of BALF in the HCAP patients detected heterogeneous bacteria and a high incidence of streptococci compared with that observed using cultivation methods. In addition, the results of our study may indicate a lower incidence of MRSA than previously expected in HCAP patients.</p></div

Decrease in the chaining behavior.

<p>(A) Retrieval test. Flies exposed to the continuous pulse song for 30 min were maintained in silence for 30 min (bluish-purple) or 60 min (red-purple), and returned to sound-exposure. Dotted vertical lines indicate the start and restart of the sound playback (Left panel). Blue vertical line indicates cessation of the first playback. The chain index for the initial phase (I) was significantly higher than those in later (II) and after the restart (III-a and III-b) temporal phases (Middle and Right panels). (B) GCaMP3 fluorescent change to the continuous pulse song. Left panel shows a ventral view of the AMMC. The AMMC is the primary center for JO neurons and is anatomically subdivided into zones A (magenta), B (light green), C (light blue), D (orange), and E (blue). Pseudocolor images at 0, 10, and 20 min after stimulus onset are shown (middle panels). The region of interest (ROI) was set in the AMMC zone A. 1-min averages of the ΔF/F intensity at 0, 10, and 20 min after stimulus onset was plotted (right panel). No significant difference was observed between them (N = 4, Friedman's test followed by Scheffe's multiple comparison). P, posterior; L, lateral. Scale bar  = 20 µm. (C) Dishabituation tests. Light blink (blue), vortex (red), bang (green), or a short silent period (light blue) does not restore the behavioral suppression to the continuous pulse song (Left panel). A dishabituation stimulus was applied at the start of a 40-s silent interval (arrow). Dotted vertical lines indicate when the sound playback was started (at 5 min) and restarted (at 35 min 40 s). A blue vertical line indicates cessation of the first playback. Right panels show the cumulative chain indices for four temporal phases. **p<0.01; *p<0.05; n. s., not significant.</p

Comparison of the predominat phylotype and the rates of phylotypes of streptococci or anaerobes.

<p>A) With respect to streptococci, the rate of a predominant phylotype was significantly higher in the patients equal to or older than 75 years of age (the rates of streptococci in the patients younger than 75 years old and equal to or older than 75 years of age were 12.1% and 31.9%, respectively; P<0.05); B) The rates of the phylotypes in each microbiota were significantly higher in the patients equal to or older than 75 years of age (<75 vs ≥75 were 11.0 ± 27.0% and 29.5 ± 34.1%, respectively; P<0.05); C-D) Regarding anaerobes, the rate of the predominant phylotype (<75 vs ≥75 were 11.8% vs 8.7%, respectively; P = 0.46) and the phylotypes in each microbiota (<75 vs ≥75 were 12.7 ± 26.5% vs 10.2 ± 23.1%, respectively; P = 0.66) were not significantly different between the two age groups.</p

Systematic counting of the chain index by ChaIN.

<p>(A) Detection of chaining behavior in fruit flies. In the top panel, green line shows the body axis of each fly. Passive recipients (the leader of the train of flies) are included in the score <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074289#pone.0074289-Eberl1" target="_blank">[11]</a>, so that the chain index in the top panel is defined as 2. Bottom panel shows a histogram for the body length of <i>D. melanogaster</i> (mean ± standard deviation 2.27 mm ±0.44 mm; n = 17,184 silhouettes of files). Inset image shows the flies forming chains. (B) Flowchart of ChaIN. Chain indices in images were exported as a time-series dataset. (C) Length and angle constants. Change in the length constant (<i>L</i>) varies the scale of the chain region (Left panel). Change in the angle constant (<i>k</i>) varies the expanse of the chain region (Right panel). Note that ChaIN_13 pixels (px) in the left panel and ChaIN_0.66 in the right panel show the same chain region (<i>L</i> = 13, <i>k</i> = 0.66). (D) Evaluation of ChaIN. Blue and red traces show the time-course of the chain index counted by ChaIN and an experimenter's manual inspection, respectively (Top panel). Sound playback starts at 5 min (dotted vertical line). Thick and thin lines represent mean ± standard error. Time windows for three temporal phases are hatched in gray. The box plot depicts the cumulative chain indices during three temporal phases of 1 min each (Bottom panel). (E) Change in the length and angle constants had less impact on the chain index. Length constant <i>L</i>, originally set as 13 pixels, was shifted to 12 or 14 pixels (Top left panel). Angle constant <i>k</i>, originally set as 0.66, was shifted to 0.5 or 0.75 (Top right panel). Box plot shows the cumulative chain indices during three temporal phases (Bottom panel). n. s., not significant.</p