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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

ETH evokes sequential activation of kinin and CAMB neurons.(A) Immunohistochemical staining to verify Gal4 expression in both kinin and CAMB neurons (Scale bars = 50μm). Kinin neurons (Kinin-Gal4, far left), CAMB neurons (Pburs-Gal4, left), and double Gal4 (right) were labeled by GFP using pbur-Gal4, kinin-Gal4 or pburs;kinin combination Gal4 and UAS-GFP. Far right: Schematic diagram showing relative position of CAMB neurons (AN1-4) and kinin neurons (AN 1–7). Note that kinin neurons project axons to a terminal plexus (TP, neuropil) in AN9 (arrow). Kinin neurons project axons posteriorly to TP and then turn anteriorly along the ventral midline. SN: subesophageal neuromeres; TN: thoracic neuromeres; AN: Abdominal neuromeres. (B) Ca2+ dynamics in kinin and CAMB neurons by ETH. (B1) Representative recordings of intracellular Ca2+ dynamics in kinin neurons (AN7, TP) and CAMB (AN3, 4) following exposure to ETH 1 & 2 (300 nM each) applied at time 0 (downward arrows). Following ETH application, kinin cell bodies in AN 7 and TP show robust and highly synchronized calcium oscillations after characteristic delays. CAMB neurons become active shortly after termination of kinin neuron activity. (B2) Video image shows locations of cell bodies and TP where Ca2+ dynamics were recorded (Top). Time-lapse video images captured during Ca2+ responses (bottom): timing of video image recordings (a-h) are indicated by vertical arrows in B1 (faint red). (C) Onset and termination of Ca2+ responses in kinin and CAMB neurons induced by ETH 1 & 2. Upon exposure to ETH1 and ETH2 (300 nM each; left), kinin and CAMB neurons are activated sequentially at 8.5 min and 20.0 min respectively. Doubling ETH concentration (600 nM each of ETH1 and ETH2, right) accelerates kinin and CAMB neuron activation, but sequential activity is maintained (6.0 min and 12.0 min respectively). Note that CAMB neuron activity lasts more than 40 min.
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pgen.1005513.g002: ETH evokes sequential activation of kinin and CAMB neurons.(A) Immunohistochemical staining to verify Gal4 expression in both kinin and CAMB neurons (Scale bars = 50μm). Kinin neurons (Kinin-Gal4, far left), CAMB neurons (Pburs-Gal4, left), and double Gal4 (right) were labeled by GFP using pbur-Gal4, kinin-Gal4 or pburs;kinin combination Gal4 and UAS-GFP. Far right: Schematic diagram showing relative position of CAMB neurons (AN1-4) and kinin neurons (AN 1–7). Note that kinin neurons project axons to a terminal plexus (TP, neuropil) in AN9 (arrow). Kinin neurons project axons posteriorly to TP and then turn anteriorly along the ventral midline. SN: subesophageal neuromeres; TN: thoracic neuromeres; AN: Abdominal neuromeres. (B) Ca2+ dynamics in kinin and CAMB neurons by ETH. (B1) Representative recordings of intracellular Ca2+ dynamics in kinin neurons (AN7, TP) and CAMB (AN3, 4) following exposure to ETH 1 & 2 (300 nM each) applied at time 0 (downward arrows). Following ETH application, kinin cell bodies in AN 7 and TP show robust and highly synchronized calcium oscillations after characteristic delays. CAMB neurons become active shortly after termination of kinin neuron activity. (B2) Video image shows locations of cell bodies and TP where Ca2+ dynamics were recorded (Top). Time-lapse video images captured during Ca2+ responses (bottom): timing of video image recordings (a-h) are indicated by vertical arrows in B1 (faint red). (C) Onset and termination of Ca2+ responses in kinin and CAMB neurons induced by ETH 1 & 2. Upon exposure to ETH1 and ETH2 (300 nM each; left), kinin and CAMB neurons are activated sequentially at 8.5 min and 20.0 min respectively. Doubling ETH concentration (600 nM each of ETH1 and ETH2, right) accelerates kinin and CAMB neuron activation, but sequential activity is maintained (6.0 min and 12.0 min respectively). Note that CAMB neuron activity lasts more than 40 min.

Mentions: We have implicated kinin neurons in regulation of pre-ecdysis behavior and CAMB neurons in the switch to ecdysis behavior. To determine whether they become active at times corresponding to these sequential behaviors, we recorded timing of calcium mobilization in flies that express the calcium reporter GCaMP-3 in both kinin and CAMB neurons. GCaMP expression was confirmed by immunohistochemical staining (Fig 2A); besides staining in cell bodies, axonal projections of kinin neurons into the terminal plexus (TP[11]) of abdominal neuromere 9 (AN9) were observed (Fig 2A, arrow). Since the peptides ETH1 and ETH2 are released from Inka cells under natural conditions prior to onset of these behaviors [2,14], we used a cocktail of ETH1 and ETH2 (each at 300 nM or 600 nM) in all experiments on the isolated CNS. The CNS prepared from pharate pupal flies 3–5 hr prior to ecdysis onset showed low-to-moderate levels of GCaMP fluorescence in cell bodies and TP (Fig 2B2a). Following exposure to ETH, cell bodies of kinin neurons and their TP projections showed robust oscillatory fluorescence activity patterns, indicating fluctuations in cytoplasmic [Ca2+]i levels (Fig 2B1 and 2C and S1 Movie). Notably, kinin and CAMB neurons mobilized calcium in sequential, non-overlapping fashion. When exposed to a cocktail of ETH1 and ETH2 (300 nM each to make a total of 600 nM), kinin neurons mobilize calcium within an average of 8.4 ± 1.4 min following ETH exposure and remain active for 9.1 ± 3.6 (n = 19) min. The duration of kinin neuron activity under these conditions corresponds well with that of pre-ecdysis behavior in vivo (Fig 1). In contrast, calcium mobilization in CAMB neurons was delayed, starting only after termination of kinin neuron activity. These activity patterns are consistent with activation of pre-ecdysis behavior by kinin neurons and ecdysis behavior by CAMB neurons.


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)

ETH evokes sequential activation of kinin and CAMB neurons.(A) Immunohistochemical staining to verify Gal4 expression in both kinin and CAMB neurons (Scale bars = 50μm). Kinin neurons (Kinin-Gal4, far left), CAMB neurons (Pburs-Gal4, left), and double Gal4 (right) were labeled by GFP using pbur-Gal4, kinin-Gal4 or pburs;kinin combination Gal4 and UAS-GFP. Far right: Schematic diagram showing relative position of CAMB neurons (AN1-4) and kinin neurons (AN 1–7). Note that kinin neurons project axons to a terminal plexus (TP, neuropil) in AN9 (arrow). Kinin neurons project axons posteriorly to TP and then turn anteriorly along the ventral midline. SN: subesophageal neuromeres; TN: thoracic neuromeres; AN: Abdominal neuromeres. (B) Ca2+ dynamics in kinin and CAMB neurons by ETH. (B1) Representative recordings of intracellular Ca2+ dynamics in kinin neurons (AN7, TP) and CAMB (AN3, 4) following exposure to ETH 1 & 2 (300 nM each) applied at time 0 (downward arrows). Following ETH application, kinin cell bodies in AN 7 and TP show robust and highly synchronized calcium oscillations after characteristic delays. CAMB neurons become active shortly after termination of kinin neuron activity. (B2) Video image shows locations of cell bodies and TP where Ca2+ dynamics were recorded (Top). Time-lapse video images captured during Ca2+ responses (bottom): timing of video image recordings (a-h) are indicated by vertical arrows in B1 (faint red). (C) Onset and termination of Ca2+ responses in kinin and CAMB neurons induced by ETH 1 & 2. Upon exposure to ETH1 and ETH2 (300 nM each; left), kinin and CAMB neurons are activated sequentially at 8.5 min and 20.0 min respectively. Doubling ETH concentration (600 nM each of ETH1 and ETH2, right) accelerates kinin and CAMB neuron activation, but sequential activity is maintained (6.0 min and 12.0 min respectively). Note that CAMB neuron activity lasts more than 40 min.
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pgen.1005513.g002: ETH evokes sequential activation of kinin and CAMB neurons.(A) Immunohistochemical staining to verify Gal4 expression in both kinin and CAMB neurons (Scale bars = 50μm). Kinin neurons (Kinin-Gal4, far left), CAMB neurons (Pburs-Gal4, left), and double Gal4 (right) were labeled by GFP using pbur-Gal4, kinin-Gal4 or pburs;kinin combination Gal4 and UAS-GFP. Far right: Schematic diagram showing relative position of CAMB neurons (AN1-4) and kinin neurons (AN 1–7). Note that kinin neurons project axons to a terminal plexus (TP, neuropil) in AN9 (arrow). Kinin neurons project axons posteriorly to TP and then turn anteriorly along the ventral midline. SN: subesophageal neuromeres; TN: thoracic neuromeres; AN: Abdominal neuromeres. (B) Ca2+ dynamics in kinin and CAMB neurons by ETH. (B1) Representative recordings of intracellular Ca2+ dynamics in kinin neurons (AN7, TP) and CAMB (AN3, 4) following exposure to ETH 1 & 2 (300 nM each) applied at time 0 (downward arrows). Following ETH application, kinin cell bodies in AN 7 and TP show robust and highly synchronized calcium oscillations after characteristic delays. CAMB neurons become active shortly after termination of kinin neuron activity. (B2) Video image shows locations of cell bodies and TP where Ca2+ dynamics were recorded (Top). Time-lapse video images captured during Ca2+ responses (bottom): timing of video image recordings (a-h) are indicated by vertical arrows in B1 (faint red). (C) Onset and termination of Ca2+ responses in kinin and CAMB neurons induced by ETH 1 & 2. Upon exposure to ETH1 and ETH2 (300 nM each; left), kinin and CAMB neurons are activated sequentially at 8.5 min and 20.0 min respectively. Doubling ETH concentration (600 nM each of ETH1 and ETH2, right) accelerates kinin and CAMB neuron activation, but sequential activity is maintained (6.0 min and 12.0 min respectively). Note that CAMB neuron activity lasts more than 40 min.
Mentions: We have implicated kinin neurons in regulation of pre-ecdysis behavior and CAMB neurons in the switch to ecdysis behavior. To determine whether they become active at times corresponding to these sequential behaviors, we recorded timing of calcium mobilization in flies that express the calcium reporter GCaMP-3 in both kinin and CAMB neurons. GCaMP expression was confirmed by immunohistochemical staining (Fig 2A); besides staining in cell bodies, axonal projections of kinin neurons into the terminal plexus (TP[11]) of abdominal neuromere 9 (AN9) were observed (Fig 2A, arrow). Since the peptides ETH1 and ETH2 are released from Inka cells under natural conditions prior to onset of these behaviors [2,14], we used a cocktail of ETH1 and ETH2 (each at 300 nM or 600 nM) in all experiments on the isolated CNS. The CNS prepared from pharate pupal flies 3–5 hr prior to ecdysis onset showed low-to-moderate levels of GCaMP fluorescence in cell bodies and TP (Fig 2B2a). Following exposure to ETH, cell bodies of kinin neurons and their TP projections showed robust oscillatory fluorescence activity patterns, indicating fluctuations in cytoplasmic [Ca2+]i levels (Fig 2B1 and 2C and S1 Movie). Notably, kinin and CAMB neurons mobilized calcium in sequential, non-overlapping fashion. When exposed to a cocktail of ETH1 and ETH2 (300 nM each to make a total of 600 nM), kinin neurons mobilize calcium within an average of 8.4 ± 1.4 min following ETH exposure and remain active for 9.1 ± 3.6 (n = 19) min. The duration of kinin neuron activity under these conditions corresponds well with that of pre-ecdysis behavior in vivo (Fig 1). In contrast, calcium mobilization in CAMB neurons was delayed, starting only after termination of kinin neuron activity. These activity patterns are consistent with activation of pre-ecdysis behavior by kinin neurons and ecdysis behavior by CAMB neurons.

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