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Corazonin neurons function in sexually dimorphic circuitry that shape behavioral responses to stress in Drosophila.

Zhao Y, Bretz CA, Hawksworth SA, Hirsh J, Johnson EC - PLoS ONE (2010)

Bottom Line: Silencing and activation of corazonin neurons lead to differential lifespan under stress, and these effects showed a strong dependence on sex.Additionally, altered corazonin neuron physiology leads to fundamental differences in locomotor activity, and these effects were also sex-dependent.These results underscore the importance of central peptidergic processing within the context of stress and place corazonin signaling as a critical feature of neuroendocrine events that shape stress responses and may underlie the inherent sexual dimorphic differences in stress responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Wake Forest University, Winston-Salem, North Carolina, United States of America.

ABSTRACT
All organisms are confronted with dynamic environmental changes that challenge homeostasis, which is the operational definition of stress. Stress produces adaptive behavioral and physiological responses, which, in the Metazoa, are mediated through the actions of various hormones. Based on its associated phenotypes and its expression profiles, a candidate stress hormone in Drosophila is the corazonin neuropeptide. We evaluated the potential roles of corazonin in mediating stress-related changes in target behaviors and physiologies through genetic alteration of corazonin neuronal excitability. Ablation of corazonin neurons confers resistance to metabolic, osmotic, and oxidative stress, as measured by survival. Silencing and activation of corazonin neurons lead to differential lifespan under stress, and these effects showed a strong dependence on sex. Additionally, altered corazonin neuron physiology leads to fundamental differences in locomotor activity, and these effects were also sex-dependent. The dynamics of altered locomotor behavior accompanying stress was likewise altered in flies with altered corazonin neuronal function. We report that corazonin transcript expression is altered under starvation and osmotic stress, and that triglyceride and dopamine levels are equally impacted in corazonin neuronal alterations and these phenotypes similarly show significant sexual dimorphisms. Notably, these sexual dimorphisms map to corazonin neurons. These results underscore the importance of central peptidergic processing within the context of stress and place corazonin signaling as a critical feature of neuroendocrine events that shape stress responses and may underlie the inherent sexual dimorphic differences in stress responses.

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Manipulations of corazonin neurons alter levels of corazonin immunoreactivity.(A) Representative image of an adult brain stained with anti-corazonin expressing a GFP transgene in corazonin cells showing co-localization of immunosignals and GFP expression. (B) Representative image of an adult brain expressing the ork transgene. (C) Lack of corazonin immunosignals in brains expressing the reaper construct which induces apoptosis. For this image, we increased the brightness of the background to show that no cell bodies were stained with the corazonin antibody. (D) Representative brain from fly expressing the NaChBac transgene in corazonin neurons, which is aimed to facilitate peptide secretion, and note less intense staining in the soma of corazonin expressing neurons. (E). Quantification of immunosignals in each of these different treatments was performed on ten brains per genotype per sex. Images were collected under the same microscope settings. For quantification, a region of interest was drawn to include corazonin neuronal cell bodies and divided by the pixel intensity of a similar sized area of the brain lacking corazonin immunosignals. For animals expressing the reaper construct, there was no evidence of any corazonin immunolabels, preventing this analysis. Asterisks indicate significant differences (p<0.05) between the Crz-GFP ratios as determined by ANOVA.
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pone-0009141-g001: Manipulations of corazonin neurons alter levels of corazonin immunoreactivity.(A) Representative image of an adult brain stained with anti-corazonin expressing a GFP transgene in corazonin cells showing co-localization of immunosignals and GFP expression. (B) Representative image of an adult brain expressing the ork transgene. (C) Lack of corazonin immunosignals in brains expressing the reaper construct which induces apoptosis. For this image, we increased the brightness of the background to show that no cell bodies were stained with the corazonin antibody. (D) Representative brain from fly expressing the NaChBac transgene in corazonin neurons, which is aimed to facilitate peptide secretion, and note less intense staining in the soma of corazonin expressing neurons. (E). Quantification of immunosignals in each of these different treatments was performed on ten brains per genotype per sex. Images were collected under the same microscope settings. For quantification, a region of interest was drawn to include corazonin neuronal cell bodies and divided by the pixel intensity of a similar sized area of the brain lacking corazonin immunosignals. For animals expressing the reaper construct, there was no evidence of any corazonin immunolabels, preventing this analysis. Asterisks indicate significant differences (p<0.05) between the Crz-GFP ratios as determined by ANOVA.

Mentions: We tested the possibility that corazonin neurons were part of the central circuitry mediating responses to various stressors by employing the GAL4-UAS binary system [20] to alter corazonin neuronal function and evaluate stress responses. We introduced the pro-apoptotic gene reaper to ablate corazonin cells using a specific corazonin-GAL4 line, thereby producing a corazonin deficient animal [9], [21]. We also introduced modified ion channel constructs, which have been used to silence and activate (Δork and NaChBac, respectively) discrete neuronal populations in Drosophila [22]–[24]. We used these constructs to alter excitability with the expectation of modulating subsequent corazonin peptide release. To evaluate the impact of transgene expression within corazonin neurons with respect to expected neuronal alterations, we employed an immunocytochemical evaluation of peptide quantity using a specific antibody against the corazonin neuropeptide (Figure 1A). We observed a complete loss of corazonin immunolabeling in flies expressing the reaper transgene (Figure 1C), a depletion of corazonin immunolabeling in NaChBac expressing (Figure 1D), and a slight increase in corazonin immunolabeling in ork expressing flies (Figure 1B) compared to flies expressing a GFP reporter (P<0.0001, Two-way ANOVA) (Figure 1E). Therefore, we conclude that these transgenes are producing phenotypes consistent with heightened secretion (NaChBac expressing), silenced secretion (ork-expressing), and ablated (reaper) corazonin neurons. However, we note that immunosignals derived from adult female brains expressing the ork channel in corazonin neurons did not significantly differ from females expressing GFP in corazonin neurons, which may reflect saturation of immunosignals in these two backgrounds.


Corazonin neurons function in sexually dimorphic circuitry that shape behavioral responses to stress in Drosophila.

Zhao Y, Bretz CA, Hawksworth SA, Hirsh J, Johnson EC - PLoS ONE (2010)

Manipulations of corazonin neurons alter levels of corazonin immunoreactivity.(A) Representative image of an adult brain stained with anti-corazonin expressing a GFP transgene in corazonin cells showing co-localization of immunosignals and GFP expression. (B) Representative image of an adult brain expressing the ork transgene. (C) Lack of corazonin immunosignals in brains expressing the reaper construct which induces apoptosis. For this image, we increased the brightness of the background to show that no cell bodies were stained with the corazonin antibody. (D) Representative brain from fly expressing the NaChBac transgene in corazonin neurons, which is aimed to facilitate peptide secretion, and note less intense staining in the soma of corazonin expressing neurons. (E). Quantification of immunosignals in each of these different treatments was performed on ten brains per genotype per sex. Images were collected under the same microscope settings. For quantification, a region of interest was drawn to include corazonin neuronal cell bodies and divided by the pixel intensity of a similar sized area of the brain lacking corazonin immunosignals. For animals expressing the reaper construct, there was no evidence of any corazonin immunolabels, preventing this analysis. Asterisks indicate significant differences (p<0.05) between the Crz-GFP ratios as determined by ANOVA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2818717&req=5

pone-0009141-g001: Manipulations of corazonin neurons alter levels of corazonin immunoreactivity.(A) Representative image of an adult brain stained with anti-corazonin expressing a GFP transgene in corazonin cells showing co-localization of immunosignals and GFP expression. (B) Representative image of an adult brain expressing the ork transgene. (C) Lack of corazonin immunosignals in brains expressing the reaper construct which induces apoptosis. For this image, we increased the brightness of the background to show that no cell bodies were stained with the corazonin antibody. (D) Representative brain from fly expressing the NaChBac transgene in corazonin neurons, which is aimed to facilitate peptide secretion, and note less intense staining in the soma of corazonin expressing neurons. (E). Quantification of immunosignals in each of these different treatments was performed on ten brains per genotype per sex. Images were collected under the same microscope settings. For quantification, a region of interest was drawn to include corazonin neuronal cell bodies and divided by the pixel intensity of a similar sized area of the brain lacking corazonin immunosignals. For animals expressing the reaper construct, there was no evidence of any corazonin immunolabels, preventing this analysis. Asterisks indicate significant differences (p<0.05) between the Crz-GFP ratios as determined by ANOVA.
Mentions: We tested the possibility that corazonin neurons were part of the central circuitry mediating responses to various stressors by employing the GAL4-UAS binary system [20] to alter corazonin neuronal function and evaluate stress responses. We introduced the pro-apoptotic gene reaper to ablate corazonin cells using a specific corazonin-GAL4 line, thereby producing a corazonin deficient animal [9], [21]. We also introduced modified ion channel constructs, which have been used to silence and activate (Δork and NaChBac, respectively) discrete neuronal populations in Drosophila [22]–[24]. We used these constructs to alter excitability with the expectation of modulating subsequent corazonin peptide release. To evaluate the impact of transgene expression within corazonin neurons with respect to expected neuronal alterations, we employed an immunocytochemical evaluation of peptide quantity using a specific antibody against the corazonin neuropeptide (Figure 1A). We observed a complete loss of corazonin immunolabeling in flies expressing the reaper transgene (Figure 1C), a depletion of corazonin immunolabeling in NaChBac expressing (Figure 1D), and a slight increase in corazonin immunolabeling in ork expressing flies (Figure 1B) compared to flies expressing a GFP reporter (P<0.0001, Two-way ANOVA) (Figure 1E). Therefore, we conclude that these transgenes are producing phenotypes consistent with heightened secretion (NaChBac expressing), silenced secretion (ork-expressing), and ablated (reaper) corazonin neurons. However, we note that immunosignals derived from adult female brains expressing the ork channel in corazonin neurons did not significantly differ from females expressing GFP in corazonin neurons, which may reflect saturation of immunosignals in these two backgrounds.

Bottom Line: Silencing and activation of corazonin neurons lead to differential lifespan under stress, and these effects showed a strong dependence on sex.Additionally, altered corazonin neuron physiology leads to fundamental differences in locomotor activity, and these effects were also sex-dependent.These results underscore the importance of central peptidergic processing within the context of stress and place corazonin signaling as a critical feature of neuroendocrine events that shape stress responses and may underlie the inherent sexual dimorphic differences in stress responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Wake Forest University, Winston-Salem, North Carolina, United States of America.

ABSTRACT
All organisms are confronted with dynamic environmental changes that challenge homeostasis, which is the operational definition of stress. Stress produces adaptive behavioral and physiological responses, which, in the Metazoa, are mediated through the actions of various hormones. Based on its associated phenotypes and its expression profiles, a candidate stress hormone in Drosophila is the corazonin neuropeptide. We evaluated the potential roles of corazonin in mediating stress-related changes in target behaviors and physiologies through genetic alteration of corazonin neuronal excitability. Ablation of corazonin neurons confers resistance to metabolic, osmotic, and oxidative stress, as measured by survival. Silencing and activation of corazonin neurons lead to differential lifespan under stress, and these effects showed a strong dependence on sex. Additionally, altered corazonin neuron physiology leads to fundamental differences in locomotor activity, and these effects were also sex-dependent. The dynamics of altered locomotor behavior accompanying stress was likewise altered in flies with altered corazonin neuronal function. We report that corazonin transcript expression is altered under starvation and osmotic stress, and that triglyceride and dopamine levels are equally impacted in corazonin neuronal alterations and these phenotypes similarly show significant sexual dimorphisms. Notably, these sexual dimorphisms map to corazonin neurons. These results underscore the importance of central peptidergic processing within the context of stress and place corazonin signaling as a critical feature of neuroendocrine events that shape stress responses and may underlie the inherent sexual dimorphic differences in stress responses.

Show MeSH
Related in: MedlinePlus