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

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.

Fig 1. 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 (Lkr ^f02594 / Lkr ^f02594). 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 Lkr ^f02594. Kinin receptor mutant Lkr ^f02594 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).

Fig 2. 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.

Fig 3. Altered ETHR expression in central ensembles modifies scheduling of the ecdysis FAP.

(A) Knockdown of ETHR expression using three independent ETHR-RNAi lines: UAS-ETHR-IR1, UAS-ETHR-IR2, and UAS-ETHR-sym carrying UAS-Dicer2. ETHR knockdown in kinin neurons reduced pre-ecdysis duration. ETHR knockdown in CAMB neurons (Pburs-Gal4) delays the switch to ecdysis behavior. A model below depicts how ETHR knockdown in kinin neurons or CAMB neurons changes pre-ecdysis duration. Bars represent mean time (± SEM, min) of the switch from pre-ecdysis to ecdysis onset relative to pre-ecdysis initiation (time zero). Data was analyzed using Mann-Whitney test (* p < 0.01; ** p < 0.001; *** p < 0.0001.) (B) ETHR over-expression in kinin neurons causes increased pre-ecdysis duration due to premature onset of pre-ecdysis. On the other hand, over-expression of ETHR in CAMB neurons accelerates the switch to ecdysis behavior due to increased sensitivity to ETH. See model below depicting how ETHR overexpression in kinin neurons or CAMB neurons affects pre-ecdysis duration. Error-bars represent standard error of mean (S.E.M). Data was analyzed using Mann-Whitney test (** P < 0.001, *** P < 0.0001).

Fig 4. ETHR expression levels affect timing of calcium mobilization in CAMB neurons.

(A) Changes in timing of calcium mobilization in CAMB neurons caused by manipulation of ETHR expression. Latency to onset of calcium mobilization in ETHR-RNAi knockdown preparations (ETHR-KD) is delayed in excess of 40 min. Conversely, onset timing of calcium mobilization is accelerated as a consequence of ETHR overexpression (ETHR-OE). (B) Over-expression of ETH receptor increased spontaneous responses in the neurons in absence of ETH. Under conditions of ETHR knockdown, no spontaneous response was observed.

Fig 5. Role of G-Protein-mediated signal transduction in timing of the switch to ecdysis behavior.

(A) Inhibition of Gαo signaling using two different fly lines expressing the pertussis toxin gene (PTX(2), PTX(3) on 2^nd and 3^rd chromosomes, respectively). (B) Enhancement of Gαo signaling by overexpression of a constitutively active form (Gαo ^Q205L). (C) Overexpression of Gαq signaling in CCAP and kinin neurons by expression of a wild type Gαq. Error-bars represent standard error of mean (S.E.M). Data was analyzed using Mann-Whitney test (** P < 0.001, *** P < 0.0001).

Fig 6. Activation of CCAP and CAMB neurons initiates ecdysis behavior.

(A) Held at a constant 24°C, the natural ecdysis FAP observed in puparium-free control (w ¹¹¹⁸) and two test groups (CCAP-Gal4>UAS-TRPM8, Pburs-Gal4>UAS-TRPM8) shows normal scheduling of behavioral steps. (B) Induction of ecdysis behavior within minutes upon reducing temperature from 24°C to 18°C in CCAP (CCAP>TRPM8) and CAMB (Pburs>TRPM8) flies. Upon returning to 24°C, the natural ecdysis FAP ensues with occasional bouts of ecdysis behavior (red strips in the pre-ecdysis bar) occurring during pre-ecdysis. Head eversion is delayed and highly variable in CAMB-activated flies, whereas it occurs within a few minutes of ecdysis behavior onset in CCAP-activated flies. (C) Ecdysis swing frequency during natural behavior or temperature-induced behavior. At 18°C, CAMB flies show ecdysis contraction frequency significantly low than control, whereas CCAP-induced behavior is similar to the natural ecdysis swing frequency. Overall, ecdysis swing frequency is decreased significantly at 18°C relative to 24°C.

Fig 7. A model depicting functional roles of kinin and CAMB neurons in scheduling of the ecdysis FAP.

ETH release from Inka cells activates ETHR neurons (ETHR-A and ETHR-B). ETHR-B neurons are more sensitive to ETH and become active immediately following ETH release. These neurons release signal(s) that engage Gαo signaling in CAMB neurons. ETH activates kinin neurons directly governing pre-ecdysis and CAMB neurons via ETHR-A and Gαq signaling. Initially, pre-ecdysis is induced, whereas CAMB neurons remain silent due to relatively low sensitivity to ETH and Gαo-mediated inhibitory input. Upon reaching adequate ETH levels in the hemolymph, ETH-mediated Gαq signaling overrides Gαo signaling in CAMB neurons, leading to co-release of CCAP, AstCC, MIP, and bursicon. This results in pre-ecdysis inhibition and the switch to ecdysis behavior and post-ecdysis behavior. Additional excitatory inputs from non-CAMB CCAP neurons contribute to vigorous ecdysis swings, resulting in head eversion. The dashed arrow represents hypothetical input to CAMB neurons from as yet unidentified ETHR-B neurons.