Limits...
PPL2ab neurons restore sexual responses in aged Drosophila males through dopamine.

Kuo SY, Wu CL, Hsieh MY, Lin CT, Wen RK, Chen LC, Chen YH, Yu YW, Wang HD, Su YJ, Lin CJ, Yang CY, Guan HY, Wang PY, Lan TH, Fu TF - Nat Commun (2015)

Bottom Line: We find that increasing DA levels in a subset of cells in the PPL2ab neuronal cluster is necessary and sufficient for increased sustained courtship in both young and aged male flies.Our results indicate that preventing the age-related decline in DA levels in PPL2ab neurons alleviates diminished courtship behaviours in male Drosophila.These results may provide the foundation for deciphering the circuitry involved in sexual motivation in the male Drosophila brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, National Chi Nan University, 54561 Nantou, Taiwan.

ABSTRACT
Male sexual desire typically declines with ageing. However, our understanding of the neurobiological basis for this phenomenon is limited by our knowledge of the brain circuitry and neuronal pathways controlling male sexual desire. A number of studies across species suggest that dopamine (DA) affects sexual desire. Here we use genetic tools and behavioural assays to identify a novel subset of DA neurons that regulate age-associated male courtship activity in Drosophila. We find that increasing DA levels in a subset of cells in the PPL2ab neuronal cluster is necessary and sufficient for increased sustained courtship in both young and aged male flies. Our results indicate that preventing the age-related decline in DA levels in PPL2ab neurons alleviates diminished courtship behaviours in male Drosophila. These results may provide the foundation for deciphering the circuitry involved in sexual motivation in the male Drosophila brain.

No MeSH data available.


An intersectional genetic approach identified two subtypes of TH-positive PPL2ab neurons that enhance courtship sustainment.(a) Diagrams explaining the genetic targeting strategies to restrict the driver expression specifically to PPL2ab neurons using an intersectional LexA-induced FLP recombinase (FLP) and Gal4 intersectional methods. The two grey boxes represent the extent of the murashka-1-Gal4 or LG121-LexA expression pattern. The drivers/cell types targeted are illustrated in the overlapping boxes from left to right. The resulting overlapping cell populations that express GFP are indicated in the intersecting green square. (b) eGFP expression is limited to the regions where both FLP and Gal4 are expressed. The scenario indicated by the green shaded box indicates that GFP is expressed when LexA induces LexAop-FLP expression, as the transcriptional stop cassette is removed allowing for GAL4-induced expression (the Right green square). (c) A representative image showing the GFP expression pattern resulting from the intersectional murashka-1-Gal4 and LG121-LexA drivers. Note, the UAS>eGFP reporter is only expressed in the subset of PPL2ab neurons that co-expressed both GAL4 and LexA (a single calyx is magnified in the inset). (c1-3) Demonstration that the cell bodies of PPL2ab neurons in LG121-LexA∩murashka-1-Gal4 (green in c1) are dopaminergic, as indicated by TH-antibody immunostaining (magenta in c2; merged in c3). Scale bar, 20 μm. (d,e) The genetic intersectional approach was applied to restrict TH overexpression in these two pairs of PPL2ab neurons. Courtship sustainment of 40-day-old males towards 3-day-old intact CS females was analysed by determining the courtship index (d) and courtship bout length (e). Significant differences in the courtship index of the LexAop-FLP;+; murashka-1-Gal4 X UAS>*>TH; LG121-LexA flies compared with the corresponding parental heterozygous flies were observed. Each column represents the mean of 17 tests. The error bars indicate+s.e.m. The means for each column followed by the same letters were not significantly different at the threshold of P<0.05 by a one-way ANOVA followed by Tukey's test per grouped columns (separated by a dashed line).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4491191&req=5

f5: An intersectional genetic approach identified two subtypes of TH-positive PPL2ab neurons that enhance courtship sustainment.(a) Diagrams explaining the genetic targeting strategies to restrict the driver expression specifically to PPL2ab neurons using an intersectional LexA-induced FLP recombinase (FLP) and Gal4 intersectional methods. The two grey boxes represent the extent of the murashka-1-Gal4 or LG121-LexA expression pattern. The drivers/cell types targeted are illustrated in the overlapping boxes from left to right. The resulting overlapping cell populations that express GFP are indicated in the intersecting green square. (b) eGFP expression is limited to the regions where both FLP and Gal4 are expressed. The scenario indicated by the green shaded box indicates that GFP is expressed when LexA induces LexAop-FLP expression, as the transcriptional stop cassette is removed allowing for GAL4-induced expression (the Right green square). (c) A representative image showing the GFP expression pattern resulting from the intersectional murashka-1-Gal4 and LG121-LexA drivers. Note, the UAS>eGFP reporter is only expressed in the subset of PPL2ab neurons that co-expressed both GAL4 and LexA (a single calyx is magnified in the inset). (c1-3) Demonstration that the cell bodies of PPL2ab neurons in LG121-LexA∩murashka-1-Gal4 (green in c1) are dopaminergic, as indicated by TH-antibody immunostaining (magenta in c2; merged in c3). Scale bar, 20 μm. (d,e) The genetic intersectional approach was applied to restrict TH overexpression in these two pairs of PPL2ab neurons. Courtship sustainment of 40-day-old males towards 3-day-old intact CS females was analysed by determining the courtship index (d) and courtship bout length (e). Significant differences in the courtship index of the LexAop-FLP;+; murashka-1-Gal4 X UAS>*>TH; LG121-LexA flies compared with the corresponding parental heterozygous flies were observed. Each column represents the mean of 17 tests. The error bars indicate+s.e.m. The means for each column followed by the same letters were not significantly different at the threshold of P<0.05 by a one-way ANOVA followed by Tukey's test per grouped columns (separated by a dashed line).

Mentions: Given that the expression patterns of the four PPL2ab drivers we used in this study are quite broad and considering previous findings implying that PPL2ab neurons function in the regulation of male sexual sustainment, the evidence provided by the experiments to this point was merely correlative. Therefore, we used an intersectional genetic strategy to precisely manipulate DA levels of PPL2ab neurons to establish cause-and-effect relationships between the DA levels of PPL2ab neurons and courtship behaviour in male flies. Specifically, we applied a LexA-induced FLP/frt recombination method that was optimized to induce targeted TH misexpression in an intersectional region defined by overlapping GAL4 and FLPase activities (Fig. 5a). This strategy works as follows: FLPase driven by LexA removes either the frt-stop-frt (>*>) from UAS-frt-stop-frt-mCD8::GFP (UAS>*> GFP) or the frt-stop-frt (>*>) from UAS-frt-stop-frt-TH (UAS>*>TH) in LG121 expressing neurons (Fig. 5b). Then, UAS-mCD8::GFP is activated by GAL4 only in PPL2ab neurons that are located in the regions targeted by both LG121-LexA and murashka-1-Gal4 (LG121∩murashka-1) (Fig. 5c). We then used these flies for further behavioural studies. After applying intersectional targeting to specific PPL2ab neurons, we found only two pairs of TH-positive PPL2ab neurons were labelled by UAS-mCD8::GFP. Overexpression of TH in these two pairs of PPL2ab neurons enhanced the courtship index and courtship bout length in 40-day-old males (one-way ANOVA followed by Tukey's test, P<0.05) (Fig. 5d,e). These results identified the subset of PPL2ab neurons that are necessary and sufficient for DA-mediated courtship.


PPL2ab neurons restore sexual responses in aged Drosophila males through dopamine.

Kuo SY, Wu CL, Hsieh MY, Lin CT, Wen RK, Chen LC, Chen YH, Yu YW, Wang HD, Su YJ, Lin CJ, Yang CY, Guan HY, Wang PY, Lan TH, Fu TF - Nat Commun (2015)

An intersectional genetic approach identified two subtypes of TH-positive PPL2ab neurons that enhance courtship sustainment.(a) Diagrams explaining the genetic targeting strategies to restrict the driver expression specifically to PPL2ab neurons using an intersectional LexA-induced FLP recombinase (FLP) and Gal4 intersectional methods. The two grey boxes represent the extent of the murashka-1-Gal4 or LG121-LexA expression pattern. The drivers/cell types targeted are illustrated in the overlapping boxes from left to right. The resulting overlapping cell populations that express GFP are indicated in the intersecting green square. (b) eGFP expression is limited to the regions where both FLP and Gal4 are expressed. The scenario indicated by the green shaded box indicates that GFP is expressed when LexA induces LexAop-FLP expression, as the transcriptional stop cassette is removed allowing for GAL4-induced expression (the Right green square). (c) A representative image showing the GFP expression pattern resulting from the intersectional murashka-1-Gal4 and LG121-LexA drivers. Note, the UAS>eGFP reporter is only expressed in the subset of PPL2ab neurons that co-expressed both GAL4 and LexA (a single calyx is magnified in the inset). (c1-3) Demonstration that the cell bodies of PPL2ab neurons in LG121-LexA∩murashka-1-Gal4 (green in c1) are dopaminergic, as indicated by TH-antibody immunostaining (magenta in c2; merged in c3). Scale bar, 20 μm. (d,e) The genetic intersectional approach was applied to restrict TH overexpression in these two pairs of PPL2ab neurons. Courtship sustainment of 40-day-old males towards 3-day-old intact CS females was analysed by determining the courtship index (d) and courtship bout length (e). Significant differences in the courtship index of the LexAop-FLP;+; murashka-1-Gal4 X UAS>*>TH; LG121-LexA flies compared with the corresponding parental heterozygous flies were observed. Each column represents the mean of 17 tests. The error bars indicate+s.e.m. The means for each column followed by the same letters were not significantly different at the threshold of P<0.05 by a one-way ANOVA followed by Tukey's test per grouped columns (separated by a dashed line).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4491191&req=5

f5: An intersectional genetic approach identified two subtypes of TH-positive PPL2ab neurons that enhance courtship sustainment.(a) Diagrams explaining the genetic targeting strategies to restrict the driver expression specifically to PPL2ab neurons using an intersectional LexA-induced FLP recombinase (FLP) and Gal4 intersectional methods. The two grey boxes represent the extent of the murashka-1-Gal4 or LG121-LexA expression pattern. The drivers/cell types targeted are illustrated in the overlapping boxes from left to right. The resulting overlapping cell populations that express GFP are indicated in the intersecting green square. (b) eGFP expression is limited to the regions where both FLP and Gal4 are expressed. The scenario indicated by the green shaded box indicates that GFP is expressed when LexA induces LexAop-FLP expression, as the transcriptional stop cassette is removed allowing for GAL4-induced expression (the Right green square). (c) A representative image showing the GFP expression pattern resulting from the intersectional murashka-1-Gal4 and LG121-LexA drivers. Note, the UAS>eGFP reporter is only expressed in the subset of PPL2ab neurons that co-expressed both GAL4 and LexA (a single calyx is magnified in the inset). (c1-3) Demonstration that the cell bodies of PPL2ab neurons in LG121-LexA∩murashka-1-Gal4 (green in c1) are dopaminergic, as indicated by TH-antibody immunostaining (magenta in c2; merged in c3). Scale bar, 20 μm. (d,e) The genetic intersectional approach was applied to restrict TH overexpression in these two pairs of PPL2ab neurons. Courtship sustainment of 40-day-old males towards 3-day-old intact CS females was analysed by determining the courtship index (d) and courtship bout length (e). Significant differences in the courtship index of the LexAop-FLP;+; murashka-1-Gal4 X UAS>*>TH; LG121-LexA flies compared with the corresponding parental heterozygous flies were observed. Each column represents the mean of 17 tests. The error bars indicate+s.e.m. The means for each column followed by the same letters were not significantly different at the threshold of P<0.05 by a one-way ANOVA followed by Tukey's test per grouped columns (separated by a dashed line).
Mentions: Given that the expression patterns of the four PPL2ab drivers we used in this study are quite broad and considering previous findings implying that PPL2ab neurons function in the regulation of male sexual sustainment, the evidence provided by the experiments to this point was merely correlative. Therefore, we used an intersectional genetic strategy to precisely manipulate DA levels of PPL2ab neurons to establish cause-and-effect relationships between the DA levels of PPL2ab neurons and courtship behaviour in male flies. Specifically, we applied a LexA-induced FLP/frt recombination method that was optimized to induce targeted TH misexpression in an intersectional region defined by overlapping GAL4 and FLPase activities (Fig. 5a). This strategy works as follows: FLPase driven by LexA removes either the frt-stop-frt (>*>) from UAS-frt-stop-frt-mCD8::GFP (UAS>*> GFP) or the frt-stop-frt (>*>) from UAS-frt-stop-frt-TH (UAS>*>TH) in LG121 expressing neurons (Fig. 5b). Then, UAS-mCD8::GFP is activated by GAL4 only in PPL2ab neurons that are located in the regions targeted by both LG121-LexA and murashka-1-Gal4 (LG121∩murashka-1) (Fig. 5c). We then used these flies for further behavioural studies. After applying intersectional targeting to specific PPL2ab neurons, we found only two pairs of TH-positive PPL2ab neurons were labelled by UAS-mCD8::GFP. Overexpression of TH in these two pairs of PPL2ab neurons enhanced the courtship index and courtship bout length in 40-day-old males (one-way ANOVA followed by Tukey's test, P<0.05) (Fig. 5d,e). These results identified the subset of PPL2ab neurons that are necessary and sufficient for DA-mediated courtship.

Bottom Line: We find that increasing DA levels in a subset of cells in the PPL2ab neuronal cluster is necessary and sufficient for increased sustained courtship in both young and aged male flies.Our results indicate that preventing the age-related decline in DA levels in PPL2ab neurons alleviates diminished courtship behaviours in male Drosophila.These results may provide the foundation for deciphering the circuitry involved in sexual motivation in the male Drosophila brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, National Chi Nan University, 54561 Nantou, Taiwan.

ABSTRACT
Male sexual desire typically declines with ageing. However, our understanding of the neurobiological basis for this phenomenon is limited by our knowledge of the brain circuitry and neuronal pathways controlling male sexual desire. A number of studies across species suggest that dopamine (DA) affects sexual desire. Here we use genetic tools and behavioural assays to identify a novel subset of DA neurons that regulate age-associated male courtship activity in Drosophila. We find that increasing DA levels in a subset of cells in the PPL2ab neuronal cluster is necessary and sufficient for increased sustained courtship in both young and aged male flies. Our results indicate that preventing the age-related decline in DA levels in PPL2ab neurons alleviates diminished courtship behaviours in male Drosophila. These results may provide the foundation for deciphering the circuitry involved in sexual motivation in the male Drosophila brain.

No MeSH data available.