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Rescheduling Behavioral Subunits of a Fixed Action Pattern by Genetic Manipulation of Peptidergic Signaling.

Kim DH, Han MR, Lee G, Lee SS, Kim YJ, Adams ME - PLoS Genet. (2015)

Bottom Line: Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior.Our findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP.Moreover, temporal organization of the FAP is likely a function of ETH receptor density in target neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of California, Riverside, Riverside, California, United States of America.

ABSTRACT
The ecdysis behavioral sequence in insects is a classic fixed action pattern (FAP) initiated by hormonal signaling. Ecdysis triggering hormones (ETHs) release the FAP through direct actions on the CNS. Here we present evidence implicating two groups of central ETH receptor (ETHR) neurons in scheduling the first two steps of the FAP: kinin (aka drosokinin, leucokinin) neurons regulate pre-ecdysis behavior and CAMB neurons (CCAP, AstCC, MIP, and Bursicon) initiate the switch to ecdysis behavior. Ablation of kinin neurons or altering levels of ETH receptor (ETHR) expression in these neurons modifies timing and intensity of pre-ecdysis behavior. Cell ablation or ETHR knockdown in CAMB neurons delays the switch to ecdysis, whereas overexpression of ETHR or expression of pertussis toxin in these neurons accelerates timing of the switch. Calcium dynamics in kinin neurons are temporally aligned with pre-ecdysis behavior, whereas activity of CAMB neurons coincides with the switch from pre-ecdysis to ecdysis behavior. Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior. Our findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP. Moreover, temporal organization of the FAP is likely a function of ETH receptor density in target neurons.

No MeSH data available.


Related in: MedlinePlus

Flies with impaired kinin or CAMB signaling show significant defects in the ecdysis FAP.(A) Roles for kinin in the ecdysis behavioral sequence were investigated by analysis of behavioral defects in kinin cell-killing (CK) flies and homozygous piggyBac-insertional kinin receptor mutant flies (Lkrf02594 / Lkrf02594). In both instances, pre-ecdysis durations were highly variable. Precise flip-out of piggyBac insertion by piggyBac transposase rescued normal pre-ecdysis behavior. Complementation testing with a kinin-receptor-gene deficient line [Df(3L)Exel6105] also showed high variation in pre-ecdysis duration. The small black arrowheads represent pre-ecdysis durations of individual animals. Error-bars represent standard deviation (SD). (B) Relative expression ratio of kinin receptor genes in control and homozygous Lkrf02594. Kinin receptor mutant Lkrf02594 showed significant reduction (26.2%) in gene expression level. Error-bars represent standard error of mean (SEM) (* P < 0.01; Student’s t-test). (C) EGFP staining patterns of kinin and CAMB (Pburs-Gal4) neurons. (D) Flies bearing targeted cell-killing (CK) of CAMB neurons exhibit prolonged pre-ecdysis and complete absence of ecdysis and post-ecdysis. Pre-ecdysis behavior begins with the normal frequency of rhythmic contractions, but the behavior weakens gradually after 10 min, ending at ~26 min. Error-bars represent standard deviation (SD).
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pgen.1005513.g001: Flies with impaired kinin or CAMB signaling show significant defects in the ecdysis FAP.(A) Roles for kinin in the ecdysis behavioral sequence were investigated by analysis of behavioral defects in kinin cell-killing (CK) flies and homozygous piggyBac-insertional kinin receptor mutant flies (Lkrf02594 / Lkrf02594). In both instances, pre-ecdysis durations were highly variable. Precise flip-out of piggyBac insertion by piggyBac transposase rescued normal pre-ecdysis behavior. Complementation testing with a kinin-receptor-gene deficient line [Df(3L)Exel6105] also showed high variation in pre-ecdysis duration. The small black arrowheads represent pre-ecdysis durations of individual animals. Error-bars represent standard deviation (SD). (B) Relative expression ratio of kinin receptor genes in control and homozygous Lkrf02594. Kinin receptor mutant Lkrf02594 showed significant reduction (26.2%) in gene expression level. Error-bars represent standard error of mean (SEM) (* P < 0.01; Student’s t-test). (C) EGFP staining patterns of kinin and CAMB (Pburs-Gal4) neurons. (D) Flies bearing targeted cell-killing (CK) of CAMB neurons exhibit prolonged pre-ecdysis and complete absence of ecdysis and post-ecdysis. Pre-ecdysis behavior begins with the normal frequency of rhythmic contractions, but the behavior weakens gradually after 10 min, ending at ~26 min. Error-bars represent standard deviation (SD).

Mentions: During the pupal ecdysis FAP in Drosophila, pre-ecdysis behavior (air bubble translocation, alternating, anteriorly-directed rolling contractions along the lateral edges of the abdomen) proceeds for ~10 min, whereupon a switch to ecdysis behavior (lateral swinging movements along with anteriorly directed peristaltic contractions) occurs. This leads within ~1–2 min to head eversion, after which rhythmic “ecdysis swinging” contractions continue for ~10 min [2,8]. In our previous study, we assessed functional consequences of ablating three central peptidergic ETHR ensembles defined by peptides they release (FMRFa, eclosion hormone, and CCAP) on the ecdysis FAP. Loss of FMRFa neurons had no effect on scheduling of the FAP, whereas loss of eclosion hormone neurons had a minor effect, producing a ~6 min delay in the switch from pre-ecdysis to ecdysis behavior. Loss of the entire CCAP neuron ensemble caused severe disruption of the FAP by abolishing the switch to ecdysis. Here we expand the dataset to include examination of additional ETHR ensembles in scheduling of the FAP using the Gal4-UAS system to drive expression of apoptosis genes (rpr, hid) for cell killing (CK) and assessed behavioral outcomes (Fig 1 and S1 Fig). Our findings confirm previous results and provide new evidence for disruption of the FAP through ablation of kinin, MIP, Burs, and Pburs ensembles. Kinin neurons are necessary for pre-ecdysis scheduling (see next section), while ablation of several CCAP neuron subsets abolishes the switch to ecdysis behavior. The smallest CCAP neuron subset is characterized by co-expression of myoinhibitory peptide (MIP), allatostatin-CC (Ast-CC), and bursicon, which we refer to as “CAMB” neurons (S2C Fig). Based on these results, we focused on kinin and CAMB ensembles for deeper analysis of the neural basis for pre-ecdysis and ecdysis scheduling.


Rescheduling Behavioral Subunits of a Fixed Action Pattern by Genetic Manipulation of Peptidergic Signaling.

Kim DH, Han MR, Lee G, Lee SS, Kim YJ, Adams ME - PLoS Genet. (2015)

Flies with impaired kinin or CAMB signaling show significant defects in the ecdysis FAP.(A) Roles for kinin in the ecdysis behavioral sequence were investigated by analysis of behavioral defects in kinin cell-killing (CK) flies and homozygous piggyBac-insertional kinin receptor mutant flies (Lkrf02594 / Lkrf02594). In both instances, pre-ecdysis durations were highly variable. Precise flip-out of piggyBac insertion by piggyBac transposase rescued normal pre-ecdysis behavior. Complementation testing with a kinin-receptor-gene deficient line [Df(3L)Exel6105] also showed high variation in pre-ecdysis duration. The small black arrowheads represent pre-ecdysis durations of individual animals. Error-bars represent standard deviation (SD). (B) Relative expression ratio of kinin receptor genes in control and homozygous Lkrf02594. Kinin receptor mutant Lkrf02594 showed significant reduction (26.2%) in gene expression level. Error-bars represent standard error of mean (SEM) (* P < 0.01; Student’s t-test). (C) EGFP staining patterns of kinin and CAMB (Pburs-Gal4) neurons. (D) Flies bearing targeted cell-killing (CK) of CAMB neurons exhibit prolonged pre-ecdysis and complete absence of ecdysis and post-ecdysis. Pre-ecdysis behavior begins with the normal frequency of rhythmic contractions, but the behavior weakens gradually after 10 min, ending at ~26 min. Error-bars represent standard deviation (SD).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581697&req=5

pgen.1005513.g001: Flies with impaired kinin or CAMB signaling show significant defects in the ecdysis FAP.(A) Roles for kinin in the ecdysis behavioral sequence were investigated by analysis of behavioral defects in kinin cell-killing (CK) flies and homozygous piggyBac-insertional kinin receptor mutant flies (Lkrf02594 / Lkrf02594). In both instances, pre-ecdysis durations were highly variable. Precise flip-out of piggyBac insertion by piggyBac transposase rescued normal pre-ecdysis behavior. Complementation testing with a kinin-receptor-gene deficient line [Df(3L)Exel6105] also showed high variation in pre-ecdysis duration. The small black arrowheads represent pre-ecdysis durations of individual animals. Error-bars represent standard deviation (SD). (B) Relative expression ratio of kinin receptor genes in control and homozygous Lkrf02594. Kinin receptor mutant Lkrf02594 showed significant reduction (26.2%) in gene expression level. Error-bars represent standard error of mean (SEM) (* P < 0.01; Student’s t-test). (C) EGFP staining patterns of kinin and CAMB (Pburs-Gal4) neurons. (D) Flies bearing targeted cell-killing (CK) of CAMB neurons exhibit prolonged pre-ecdysis and complete absence of ecdysis and post-ecdysis. Pre-ecdysis behavior begins with the normal frequency of rhythmic contractions, but the behavior weakens gradually after 10 min, ending at ~26 min. Error-bars represent standard deviation (SD).
Mentions: During the pupal ecdysis FAP in Drosophila, pre-ecdysis behavior (air bubble translocation, alternating, anteriorly-directed rolling contractions along the lateral edges of the abdomen) proceeds for ~10 min, whereupon a switch to ecdysis behavior (lateral swinging movements along with anteriorly directed peristaltic contractions) occurs. This leads within ~1–2 min to head eversion, after which rhythmic “ecdysis swinging” contractions continue for ~10 min [2,8]. In our previous study, we assessed functional consequences of ablating three central peptidergic ETHR ensembles defined by peptides they release (FMRFa, eclosion hormone, and CCAP) on the ecdysis FAP. Loss of FMRFa neurons had no effect on scheduling of the FAP, whereas loss of eclosion hormone neurons had a minor effect, producing a ~6 min delay in the switch from pre-ecdysis to ecdysis behavior. Loss of the entire CCAP neuron ensemble caused severe disruption of the FAP by abolishing the switch to ecdysis. Here we expand the dataset to include examination of additional ETHR ensembles in scheduling of the FAP using the Gal4-UAS system to drive expression of apoptosis genes (rpr, hid) for cell killing (CK) and assessed behavioral outcomes (Fig 1 and S1 Fig). Our findings confirm previous results and provide new evidence for disruption of the FAP through ablation of kinin, MIP, Burs, and Pburs ensembles. Kinin neurons are necessary for pre-ecdysis scheduling (see next section), while ablation of several CCAP neuron subsets abolishes the switch to ecdysis behavior. The smallest CCAP neuron subset is characterized by co-expression of myoinhibitory peptide (MIP), allatostatin-CC (Ast-CC), and bursicon, which we refer to as “CAMB” neurons (S2C Fig). Based on these results, we focused on kinin and CAMB ensembles for deeper analysis of the neural basis for pre-ecdysis and ecdysis scheduling.

Bottom Line: Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior.Our findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP.Moreover, temporal organization of the FAP is likely a function of ETH receptor density in target neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of California, Riverside, Riverside, California, United States of America.

ABSTRACT
The ecdysis behavioral sequence in insects is a classic fixed action pattern (FAP) initiated by hormonal signaling. Ecdysis triggering hormones (ETHs) release the FAP through direct actions on the CNS. Here we present evidence implicating two groups of central ETH receptor (ETHR) neurons in scheduling the first two steps of the FAP: kinin (aka drosokinin, leucokinin) neurons regulate pre-ecdysis behavior and CAMB neurons (CCAP, AstCC, MIP, and Bursicon) initiate the switch to ecdysis behavior. Ablation of kinin neurons or altering levels of ETH receptor (ETHR) expression in these neurons modifies timing and intensity of pre-ecdysis behavior. Cell ablation or ETHR knockdown in CAMB neurons delays the switch to ecdysis, whereas overexpression of ETHR or expression of pertussis toxin in these neurons accelerates timing of the switch. Calcium dynamics in kinin neurons are temporally aligned with pre-ecdysis behavior, whereas activity of CAMB neurons coincides with the switch from pre-ecdysis to ecdysis behavior. Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior. Our findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP. Moreover, temporal organization of the FAP is likely a function of ETH receptor density in target neurons.

No MeSH data available.


Related in: MedlinePlus