The authors have declared that no competing interests exist.
The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: AS SH PS AM MP MJP. Performed the experiments: AS SH PS AM MP MZ. Analyzed the data: AS SH PS AM MP MZ MJP. Contributed reagents/materials/analysis tools: RS ASC. Wrote the paper: AS MJP.
This study reveals that a cluster of neurons expressing the neuropeptide hugin transmit inputs from higher brain centers to motor centers, thereby regulating feeding and locomotion in fruit fly larvae.
Central mechanisms by which specific motor programs are selected to achieve meaningful behaviors are not well understood. Using electrophysiological recordings from pharyngeal nerves upon central activation of neurotransmitter-expressing cells, we show that distinct neuronal ensembles can regulate different feeding motor programs. In behavioral and electrophysiological experiments, activation of 20 neurons in the brain expressing the neuropeptide hugin, a homolog of mammalian neuromedin U, simultaneously suppressed the motor program for food intake while inducing the motor program for locomotion. Decreasing hugin neuropeptide levels in the neurons by RNAi prevented this action. Reducing the level of hugin neuronal activity alone did not have any effect on feeding or locomotion motor programs. Furthermore, use of promoter-specific constructs that labeled subsets of hugin neurons demonstrated that initiation of locomotion can be separated from modulation of its motor pattern. These results provide insights into a neural mechanism of how opposing motor programs can be selected in order to coordinate feeding and locomotive behaviors.
In the animal kingdom, two of the most essential behaviors are locomotion and feeding. The motor programs underlying these behaviors are controlled by higher-order circuits in the central nervous system. However, how an organism selects a particular motor program based on inputs from the information-processing higher brain centers to generate an adaptable behavior is not well understood. Here, we analyze the behavior of
The recruitment of appropriate motor programs to changing environmental conditions is an essential aspect of animal behavior
In vertebrates, the motor system for locomotion has been extensively studied with various methods including pharmacological, electrophysiological, and more recently genetic tools
In addition to the highly conserved locomotor motor behaviors, those related to feeding are critical for growth and survival. These encompass movements involving the whole body for searching and getting access to food sources, local parts of the body for actual food intake, as well as organ-specific movements for post-ingestive phases of feeding. In invertebrates, the rhythmic nature of swallowing and food transport has been utilized as a model to study the structure of CPGs that generate oscillating motor patterns
Studies in
Recently, an electrophysiological approach was used in semi-intact preparations to monitor the rhythmic motor patterns that comprise the
In this study, we used behavioral, genetic, imaging, and electrophysiological approaches to study central mechanisms that modulate feeding-related behaviors. We first identified neurotransmitter and neuropeptide clusters that modulate subsets of motor programs for feeding. This revealed that a small neuronal cluster can oppositely regulate feeding and locomotive motor programs. The cells of this cluster express the gene
We previously characterized the major muscles and the nerves driving the movements that underlie feeding behavior
(A) Experimental setup for AN, MN, and PaN recordings at the deafferented CNS; dTrpA1 was activated by a Peltier-driven heating device. (B) Single extracellular recordings of AN, MN, and PaN revealed differential alteration of feeding-related motor patterns by dTrpA1 activation. Red blocks on top of the control recordings denote motor output. For the experimental recordings, an up arrow (↑) indicates significant acceleration of motor pattern, down arrow (↓) indicates significant deceleration of motor pattern and a dash (–) indicates no significant difference in the motor pattern (exception: Glu (Ok371-Gal4 showed no rhythmic motor pattern by dTrpA1 activation [no]). (C) Statistical data from AN, MN, and PaN motor patterns quantified as relative change in cycle frequency (mean ± standard error). Significance was tested by Mann-Whitney Rank Sum Test (***p≤0.001). 5-HT, serotonin; ACh, acetylcholine; DA, dopamine; Glu, glutamate; Hug, hugin neuropeptide; MHD, mouth hook depressor; MHE, mouth hook elevator; ProdoA, dorsal protractor A.
Neuronal activation of the Glu population resulted in a reversible state of tonic excitation in the motor patterns of all three pharyngeal nerves (
Food intake studies further confirmed the roles of these neurones in feeding behavior. A short-term yeast intake assay was used in order to minimize longer-acting peripheral influence on the feeding response (
(A) Experimental setup: yeast intake of larvae (% of body stained) was determined after 20 min of dTrpA1 activation (upper picture). All tested Gal4-lines showed a decreased relative change in yeast intake except 5-HT (TRH-Gal4) line, which showed an increase (lower panel; Mann-Whitney Rank Sum Test: ***p≤0.001). (B) CDM contractions were tracked by measuring the length difference of pharyngeal lumen (Δd) at 32°C relative to the maximal contractions at 18°C (upper picture). Tracking of the CDM contractions correspond to deduced muscle activity based on the AN recordings (lower left panel). CDM contractions were quantified as relative change in contractions/min (lower right panel). Significance was tested by Mann-Whitney Rank Sum Test (*p≤0.05, ***p≤0.001). 5-HT, serotonin; ACh, acetylcholine; DA, dopamine; Glu, glutamate; Hug, hugin neuropeptide.
The combined electrophysiological and behavioral analyses opened up several avenues to pursue, as all the lines revealed interesting features relating to selection and modulation of motor patterns. For example, the unique finding that the serotonergic line, when activated, was the only one of 11 lines tested which resulted in increased food intake. The dopaminergic and hugin lines were interesting since they affected only a subset of the motor programs (i.e., the AN, but not MN or PaN motor programs), thus demonstrating a specificity in recruitment of different motor programs that comprise the feeding system. For the current study, we decided to focus on the hugin neuronal cluster for one critical reason, namely the relative simplicity of the expression pattern generated by the HugS3-Gal4 line in both numerical and spatial terms. Previous studies showed that this line drives reporter gene expression precisely in 20 cells, all tightly clustered in the subesophageal ganglion (SOG)
We first wanted to verify the effect of hugin on the feeding motor system using an independent method to activate neurons. Thus, we used NaChBac and recorded the AN motor pattern
Based on these observations, we next wanted to analyze the alterations in feeding behavior when hugin neurons were activated in more detail. Specifically, we wanted to determine if the suppressed food intake was accompanied by alterations in a food-related locomotory behavior, namely the wandering-like behavior. This is a behavior that is observed in certain mutant larvae which are defective in food intake, where they move away from the food source and wander about the surrounding area
(A–B) Photographs of OrgR (A) and HugS3>dTrpA1 (B) larvae (upper panel) and crawling tracks (lower panel) after 20 min at 18°C (no dTrpA1 activation) and 32°C (dTrpA1 activation), displaying the yeast intake and wandering-like behavior. Compared with OrgR, HugS3>dTrpA1 larvae showed reduced yeast intake and increased wandering-like behavior. (C) Activation of the hugin neurons by dTrpA1 significantly reduced the relative change in yeast intake compared with OrgR. Data is presented as a box plot (Mann-Whitney Rank Sum Test: ***p≤0.001). (D) Analysis of the locomotory activity showing that HugS3>dTrpA1 had a significantly increased wandering-like behavior (max. larvae outside the yeast/min [%]) relative to OrgR on the restrictive temperature (32°C).
Due to the alteration in locomotive behavior, we next asked if the activity of the abdominal segmental muscles that underlie locomotion were affected by activating the hugin neurons. The
(A) Single intracellular muscle recording of M6 (experimental setup). (B) Representative muscle recordings of OrgR and HugS3>dTrpA1 at 18°C (before dTrpA1 activation) and 32°C (during dTrpA1 activation); activation of the hugin neurons leads to an acceleration of the M6 motor pattern (colored bars indicate bursts of PSPs). (C) Increased acceleration effect of dTrpA1 induced activation of the hugin neurons on the motor pattern (indicated by colored bars) for individual muscle recordings. (D) Activation of the hugin neurons significantly increased cycle frequency (presented as box plot) of the M6 motor pattern (Mann-Whitney Rank Sum Test: ***p≤0.001). (E) Double intracellular muscle recording of the CDM and M6 (experimental setup). (F) Representative CDM/M6 recordings of OrgR and HugS3>dTrpA1 at 18°C (before dTrpA1 activation), at 32°C (during dTrpA1 activation), and after shift down to 18°C. Note the opposite effect on the CDM and M6 motor patterns at 32°C. (G) Temporal progression of CDM and M6 motor activity for OrgR- and HugS3>dTrpA1 recordings (F) upon temperature stimulation. The graph shows the number of cycles per bin (bin size: 20 s) over the recording. (H) Temperature shift from 18°C to 32°C increased the cycle frequency of the CDM and M6 motor pattern of OrgR in the same manner, whereas in the case of HugS3>dTrpA1 the CDM cycle frequency decreased and the M6 cycle frequency increased (symbols indicate the mean, whiskers indicate the standard error). Significance was tested by Mann-Whitney Rank Sum Test (*p≤0.05, ***p≤0.001). AbN, abdominal nerve.
We then asked if pharyngeal pumping and abdominal activity could be coordinately regulated. Therefore, we performed double intracellular recordings of the CDM and the abdominal muscle M6 (
Since the results described above indicated that activation of the hugin neurons leads to suppression of feeding, we next wanted to determine if the hugin neuropeptide is required for this suppression. The strategy was to decrease hugin neuropeptide levels in the hugin neurons through RNA interference (RNAi) and see if activating the hugin neurons would still result in suppression of feeding behavior. First we determined the effectiveness of several RNAi lines to decrease hugin neuropeptide levels (
(A) Antibody staining of CNS from HugS3>dTrpA1 larva with hugin antibody (left panel). Double staining of CNS from HugS3>dTrpA1,HugRNAi1A larva (middle two panels); this
Animals which only expressed the
The hugin neuronal cluster comprises just 20 cells, whose soma are all located in the SOG. Earlier work showed that the hugin neurons form four distinct subclasses, each having different projection targets
(A–C) Double antibody staining of Hug0.8: fluorescence expression driven by Hug0.8-Gal4 (C). Cell bodies and aborizations labelled by hugin antibody (B); merge of B and C (A). (A′–C′) Magnification of labeled somata in the SOG (magnified region indicated by dashed box in the original image (A–C)). Hug0.8 lacks the four hugin cells (marked in B′ and C′) which project to the VNC (indicated by arrows in B and C). (D–F) Double antibody staining of HugVNC: fluorescence expression driven by HugVNC-Gal4 (F). Cell bodies and aborizations labelled by hugin antibody (E); merge of E and F (D). (D′–F′) Magnification of labeled somata in the SOG (magnified region indicated by dashed box in the original image (D–F)). Only the four cells that project to the VNC are labelled. Arrows mark the missing projections to protocerebrum (A–F: 50 µm, A′–F′: 10 µm). (G) Schematic summary of the three different hugin promoter constructs. HugS3 drives target gene expression in all 20 hugin cells; Hug0.8 lacks the four cells that project to the VNC; HugVNC drives expression only in the four cells that project to the VNC. (H) At activating temperature (32°C), HugVNC>dTrpA1 animals displayed no wandering-like behavior whereas Hug0.8>dTrpA1 animals displayed increased wandering-like behavior similar to HugS3>dTrpA1 (see
We then performed the converse experiment: to determine the function of the 4-cell hugin cluster that projects to the VNC. We therefore made a promoter construct from a region that was deleted in Hug0.8 construct relative to the HugS3 construct. This line drove target gene expression in precisely the four hugin cells that project down the VNC (
Next we measured cycle frequency of the AN motor pattern after dTrpA1 activation of these two nonoverlapping neuronal clusters. The hugin-0.8 line suppressed the AN motor pattern, whereas the VNC-line could not (
(A) Experimental setup of AN recording for dTrpA1 activation. (B) Representative AN recordings of control (OrgR), Hug0.8>dTrpA1, and HugVNC>dTrpA1 at 18°C (before dTrpA1 activation) and 32°C (during dTrpA1 activation). Activation of dTrpA1 in Hug0.8-Gal4 significantly decreased the cycle frequency of the AN-motor pattern, but not in HugVNC-Gal4 (colored bars indicate the motor pattern). (C) Relative change in cycle frequency of the AN-motor pattern by dTrpA1 activation in control, HugS3>dTrpA1, Hug0.8>dTrpA1, and HugVNC>dTrpA1, illustrated as box plots (Mann-Whitney Rank Sum Test: n.s., nonsignificant; ***p≤0.001). The effect of 20-cell hugin cluster on the CDM motor pattern was verified by a second genetic tool to activate neurons (tubGal80ts; NaChBac; for details see
Taken together, these results indicated that food intake (motor program for pharyngeal pumping) and initiation of wandering-like behavior can be decoupled from modulation of the speed of abdominal muscle contraction. The 4-cell hugin VNC cluster can thus regulate locomotion speed separately from pharyngeal pumping. Therefore, although activation of the entire 20-cell hugin cluster coordinately suppresses feeding and enhances locomotion speed, the two motor programs are under the control of distinct hugin neuronal subclasses. Both the suppression of food intake and the induction of wandering-like behavior are performed by the 16-cell cluster, whereas the 4-cell VNC cluster is required to increase the cycle frequency of the locomotor motor pattern.
The above results indicated that the 16 cell hugin cluster mediates the suppressive effect of hugin neurons on the AN motor pattern. These comprise three different subclasses of hugin neurons
(A) AN recording of the intact CNS (experimental setup, upper panel). At 18°C, OrgR×dTrpA1 and HugS3>dTrpA1 show a rhythmic motor output. At 32°C (dTrpA1 activation), the motor pattern of HugS3>dTrpA1 is decelerated (middle recordings). Analysis of the AN motor pattern during dTrpA1 activation of both genotypes quantified as fold change in mean cycle frequency (lower panel). (B) AN recording after removal of VNC (experimental setup, upper panel). Representative AN recording of OrgR×dTrpA1 and HugS3>dTrpA1 at 18°C and 32°C (dTrpA1 activation). During dTrpA1 activation, the deceleration of motor pattern effected by HugS3>dTrpA1 was still observed after removing the VNC (middle recordings). Analysis of the AN motor pattern during dTrpA1 activation of both genotypes quantified as fold change in mean cycle frequency (lower panel). (C) AN recording after removal of the brain hemispheres (experimental setup, upper panel). In HugS3>dTrpA1, lesion of the brain hemispheres resulted in no deceleration of the AN motor pattern during dTrpA1 activation (representative AN recordings of both genotypes; middle recordings). Analysis of the AN motor pattern during dTrpA1 activation of both genotypes quantified as fold change in mean cycle frequency (lower panel: Mann-Whitney Rank Sum Test: n.s, nonsignificant; **p≤0.01, ***p≤0.001).
At 18°C, when dTrpA1 is not activated, lesioning the VNC or the brain hemispheres (H) still resulted in a rhythmic motor pattern from the AN (
Behavioral modules can be seen to be composed of distinct motor programs that are differentially recruited based on adaptive needs
The behavioral module that comprises
For both feeding and locomotion, the cellular identities of the CPGs remain largely unknown. Previous studies have demonstrated the existence of feeding CPG(s) in the
In this context, a major issue is that of cellular specificity: which of the cells targeted by these neurotransmitter Gal4 lines are responsible for the observed effects on the feeding and locomotor program? For example, serotonin is expressed in about 84 cells in the larval CNS: ∼56 in the VNC, ∼8 in the SOG, and ∼20 in the protocerebrum
(A) Illustration of effect of neuronal populations on different motor programs. Hugin neurons affect a subset (pharyngeal pumping), whereas serotonergic neurons affect all feeding motor patterns (head tilting, mouth hook movement, and pharyngeal pumping). Hugin neurons also regulate in an opposite manner the motor program for segmental contraction, whereas serotonergic neurons affect segmental contraction in the same manner as the feeding motor pattern. (B) Activation of the 20-cell hugin cluster simultaneously suppresses feeding and initiates locomotion motor programs (see text for details). 5-HT, serotonergic neurons.
A striking finding from our study is the fact that activating a small cluster of 20 neurons in the SOG, all expressing the neuropeptide hugin, leads to a simultaneous suppression of a motor program for feeding and induction of one for locomotion. This is observed both at the behavioral and electrophysiological level. Thus, the hugin cluster can regulate two essentially competing programs since larvae, as with most animals, do not feed and move at the same time. A notable feature of the hugin neuronal cluster is that we have not been able to observe any difference to the control situation when hugin neuronal activity is decreased. For both pharyngeal pumping and wandering-like behavior, it is only when the hugin neurons are activated that we see a modulatory effect. Similarly, the increase in the frequency of M6 abdominal muscle contraction is observed only under activation of hugin neurons. We believe these observations provide insights into the mechanism by which the hugin neurons act. This can be illustrated in terms of how a brake and gas pedal function to coordinate two mutually exclusive operations of a car. Activating hugin neurons decreases feeding, but inhibiting them does not increase feeding: applying a brake causes deceleration, but removing it does not cause acceleration. Similarly, activating the hugin neurons enhance abdominal muscle contraction, but their inhibition does not slow down contraction: stepping on the gas pedal increases speed, but taking it off does not actively decrease speed. This scenario can be used to explain the requirement of hugin neuropeptide in our RNAi experiments. Lowering the level of hugin neuropeptide in activated hugin neurons no longer affected the motor patterns underlying food intake and locomotion, indicating that hugin neuropeptide is necessary for the hugin neurons to suppress feeding and induce wandering-like behavior.
It is of interest to note that hugin neuropeptide does not seem to be required for speeding up the motor program for locomotion. This could be because of the residual quantity of hugin neuropeptide or to some compensation mechanism; more likely, the accelerating effect is due to a different neurotransmitter. At this point, we do not know which classical neurotransmitters are expressed in the hugin cells. In mammals, it has been shown that serotonergic and cholinergic systems influence the speed of motor neuron firing in the spinal cord that underlies locomotion
In many vertebrates, the center for swallowing is thought to be localized in the brainstem
It has recently been postulated that the insect central complex might play an analogous role to the basal ganglia
A major future challenge will be to determine how the different neuronal components of the feeding motor hierarchy are interconnected. One essential effort will be to analyze the receptor for the hugin neuropeptides. Two putative receptors have already been identified and it would be necessary to determine the cells that express the receptors
The following Gal4 driver and UAS effector lines were used: OK371-Gal4 (Bloomington #26160), Cha-Gal4 (Bloomington #6798), GAD-Gal4
Adult flies and larvae were reared on standard fly food and kept at 25°C unless otherwise stated. All experiments were performed with third instar larvae 98±2 h AEL (after egg laying). Four hours egg collections were made on apple juice-agar plates with yeast-water paste. After 48 h, larvae were transferred into vials containing standard fly food. For experiments with shibirets larvae were raised at 18°C to avoid temperature-induced developmental defects
For Hug0.8-Gal4 line, a 793 bp hugin promoter fragment was amplified by primer1:
Reduced semi-intact preparations were made of third instar larva consisting of the CNS, CPS, and associated pharyngeal nerves and muscles. Detailed description of the dissection has been described earlier
For
For intracellular muscle recordings, semi-intact CDM/M6 preparations of third instar larvae were used. PSPs of the muscle M6 of 4th abdominal segment and CDM were recorded using glass microelectrodes filled with 3 M KCl solution (tip resistance: 20–30 MΩ) connected to an intracellular amplifier (BRAMP-01R, npi electronic GmbH). All recordings were digitally sampled by a Micro3 1401 or Power 1401 mk2 A/D board (Cambridge Electronic Design) at 20 kHz. Data was acquired with Spike2 software (Cambridge Electronic Design).
For analysis, data pairs of successive 60 s or 120 s recording-sections under unstimulated and stimulated conditions were analyzed. Processing of the electrophysiological recordings was performed with a modified script of Spike2 (provided by Cambrigde Electronic Design). For a pair of successive recording-sections, fold change in cycle frequency was calculated. The dTrpA1-experiments revealed an endogenous temperature effect which could mask the impact of dTrpA1-activated GAL4-driver lines on the rhythmic motor output. Due to this, the mean fold change in cycle frequency of the respective control experiments was subtracted for each data point, termed relative change in cycle frequency.
For dTrpA1-experiments (nerve/muscle recording and CDM tracking) thermal stimuli were applied to the dorsal side of CNS. The custom-made stimulator consisted of a silver wire (diameter: 4 mm) attached to a Peltier element with thermally conductive adhesive. Peltier element was driven by a voltage-regulated power supply (VSP 2405, Voltcraft) connected to an A/D board. The end of the thermal stimulator was filed to a tip and insulated with nail polish. Applied temperature was measured by digital thermometer (GMH 3210, Greisinger electronic). The sensor for the thermometer was placed 5 mm from the tip (for temperature calibration see
For measurement of yeast ingestion, larvae were first washed and then starved in a Petri dish lined with tap water-moistened tissue for 30 min on RT. Afterwards they were transferred on colored yeast (colored with crimson red powder) on pre-warmed (30 min at 32°C) apple juice-agar plates and incubated for 20 min at 32°C. Afterwards the larvae were removed from the yeast and placed in 65°C hot water. For analysis larvae were photographed using a digital camera (Axiocam ICc 1, Zeiss) mounted on a binocular (Stemi 2000-CS, Zeiss). For each individual, the amount of yeast ingested was calculated as area of the alimentary tract stained by colored yeast divided by body surface area using the software ImageJ (Fiji). Data on the feeding assay is represented as percentage of ingested yeast relative to the body surface.
For simultaneous investigation of feeding and wandering-like behavior, five larvae were placed on a pre-heated/-cooled apple juice agar plate (18°C or 32°C). 20 min videos at 18°C and 32°C were acquired using a digital camera (Quickcam 9000 Pro, Logitech) and the software VirtualDub. The measurement of yeast ingestion was performed as listed in the previous paragraph. The locomotion data was analyzed using the tracking software MTrack2 (Fiji). Analysis of larvae leaving the yeast spot was carried out using a custom-made macro for ImageJ (Fiji).
CDM contractions were studied in semi-intact larvae. The preparation consisted of the CNS, the abdominal body wall, and the feeding apparatus (CPS including associated muscles). Thermal stimulation was applied directly to the CNS. Consecutive videos of 60 s at 18°C and 60 s at 32°C were recorded using a digital camera (Axiocam ICc 1, Zeiss) mounted on a binocular (Stemi 2000-CS, Zeiss). CDM contractions were tracked by measuring the length-difference of pharyngeal lumen (Δd) over time relative to the maximal contractions at 18°C. The measurements were performed using the software ImageJ (Fiji).
Dissected larval brains were fixed in paraformaldehyde (4%). For the antibody staining of hug-eYFP, primary antibody was rabbit-antiGFP (1∶500, Abcam plc) and the secondary antibody was rabbit-antiGFP Cy2 (1∶200, Dianova GmbH). The antibody staining of HugVNC>Cam2.1 was performed with chicken anti-GFP (1∶500, Abcam plc) and as secondary antibody anti-chicken Alexa488 (1∶200, Invitrogen) was used. Antibody staining of hugin was performed with guinea pig anti-Hugin (1∶200, Pankratz laboratory; for hug0.8>rpr/hid) or rabbit anti-Hugin (1∶500, Pankratz laboratory; hug0.8>eYFP). Antibody stainings for RNAi experiments were done using rabbit anti-Hugin (1∶500).Secondary antibodies were: anti-rabbit Cy3, anti-guinea pig Cy3 (1∶200, Jackson ImmunoResearch), and mouse anti-GFP (1∶500, Sigma-Aldrich). Nuclei were counter stained with DAPI or Draq5. Labeled larval brains were mounted in Mowiol. Imaging was carried out using Laser Scanning Microscope (ZEISS LSM780). The obtained images were arranged using Zen LE and Photoshop CS5 (Adobe) (for detailed staining procedures see
All images were obtained by using a confocal microscope Zeiss LSM 780; non-specific background fluorescence of the in vivo images was reduced by the Median Filter of the Zeiss Zen Software.
Hugin cDNA PCR fragment flanked by a
For the lesion experiments we used the standard reduced semi-intact preparations of third instar larvae as mentioned above (see Electrophysiology). VNC or brain hemispheres were removed by a microdissecting scissor (Fine Science Tools). Five minutes after the lesion of the neuronal tissue, extracellular recording of antennal nerve was started. Thermal stimuli were applied by the above described protocol for temperature stimulation. Consecutive 60 s sections of the AN motor output at 18°C and 32°C were analyzed. The cycle frequency of AN motor pattern at 18°C showed no significant difference between OrgR×dTrpA1 and HugS3>dTrpA1 for each lesion. Therefore the data is presented as fold change in cycle frequency of AN motor pattern between both genotypes at 32°C (during dTrpA1 activation) for each experiment.
All electrophysiological and behavioral experiments were tested for significance with the Mann-Whitney-Rank-Sum-test (*p≤0.05, **p≤0.01, ***p≤0.001).
(A) Experimental set up of heating device calibration. (B) Calibration curve of the heating device (x-axis – Theat element [°C], y-axis – Tenvironment [°C]). At Theat element 18°C the measured Tenvironment was 19.8+/−0.48°C and at Theat element 32°C the measured Tenvironment was 26.9+/−0.3°C.
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(A) Experimental setup: yeast intake of larvae [% of body stained] was determined after 20 min of dTrpA1-activation. The following major neurotransmitter systems were used for the initial screening: glutamatergic (Glu), cholinergic (ACh), GABAergic (GABA), serotonergic (5-HT), dopaminergic (DA), combined serotonergic/dopaminergic (5-HT/DA) and combined octopaminergic/tyraminergic (OA/TYR) neuronal populations. We also tested four neuropeptide genes shown in earlier studies to be involved in some aspect of feeding response:
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(A–C) Antibody staining of HugS3>10×GFP expression pattern in the CNS; Magnification (A′–C) of hugin cell cluster (20 cells) in the SOG (A: scale bar: 50 µm; A′: scale bar: 10 µm). Schematic summary of the projection pattern HugS3-Gal4 line in the larval CNS (right side). Target region of the projections are: PC, RG, SOG, VNC and periphery via PaN. Abbr.: CNS – central nervous system; PaN – prothoracic accessory nerve; PC – protocerebrum; RG – ring gland; SOG – subesophageal ganglion; VNC – ventral nerve cord.
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(A) Experimental set up of AN recordings at the isolated CNS. Larvae of both genotypes were 166+/−2 h old (raised on 18°C) and kept for at least 8–12 h on 30°C before recording. (B) Original AN recordings of HugS3>tubGal80ts;eYFP and HugS3>tubGal80ts;NaChBac (colored boxes represent the CDM activity). (C) Box plot of the cycle frequency [Hz] of HugS3>tubGal80ts;eYFP (mean (std. dev.): 0.423 (+/−0.121); number of larvae (number of experiments): 29(10)) and HugS3>tubGal80ts;NaChBac (mean (std. dev.): 0.196 (+/−0.192); number of larvae (number of experiments): 30(10)). HugS3>tubGal80ts;NaChBac was significant different to HugS3>tubGal80ts;eYFP (p-value≤0.001). Abbr.: AN – antennal nerve; CDM – cibarial dilator muscle; CNS – central nervous system.
(TIF)
(A) Motor pattern recorded from CDM (presented as box plot for OrgR, OrgR>shits, HugS3>shits). CDM motor patterns showed no significant difference in fold change of cycle frequency between OrgR, OrgR>shits, HugS3>shits (performed Mann-Whitney Rank Sum Test (n.s. – not significant)). (B) Left side: Experimental setup for the nerve recordings of HugS3>rpr/hid (upper panel) and HugS3>Kir2.1 (lower panel). Right side: Graph shows the cycle frequency of the AN motor pattern after ablation of the hugin neurons by the apoptotic factors rpr and hid and during inhibition of hugin neurons using Kir2.1 (lower panel). Compared to the control (OrgR) inhibiting and ablating the hugin neurons showed no significant difference (performed Mann-Whitney Rank Sum Test (n.s. – not significant)). (C) Motor pattern recorded from M6 (presented as box plot for OrgR, OrgR>shits, HugS3>shits). M6 motor output showed no significant difference in fold change of cycle frequency between OrgR, OrgR>shits, HugS3>shits (performed Mann-Whitney Rank Sum Test (n.s. – not significant)).
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(A,B) Statistical analysis of food intake and wandering-like behavior assay of OrgR×shits compared to HugS3>shits under starved (A) and fed (B) conditions. The graph (left) shows the intake of yeast (area of the alimentary tract stained by colored yeast divided by body surface area) after 20 min at 32°C. Graph (right) illustrates the statistical data of the wandering-like behavior of OrgR×shits compared to HugS3>shits measured as larvae outside the yeast/min [%] over a time period of 20 min. In both nutritional conditions HugS3>shits showed no significant difference in food intake and wandering-like behavior relative to OrgR×shits at 32°C.
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Hugin antibody staining of the genotypes: OrgR>dTrpA1, HugS3>dTrpA1, HugS3>dTrpA1, HugRNAi1A and HugS3>dTrpA1, TRiP.JF03122. Images show the subesophageal ganglion of the larval CNS as indicated in the schematic drawing (left side, scale bar: 20 µm).
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(A) Hugin antibody staining of hug0.8>rpr/hid showing four remaining cells in the SOG that project to the VNC (A, scale bar: 50 µm; A′, scale bar: 10 µm).
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Graphs show the relative change in cycle frequency of M6- and CDM-motor pattern of HugS3>dTrpA1 and TRH>dTrpA1 compared to the control lines (Mann-Whitney Rank Sum Test: *p≤0.05, **p≤0.01, ***p≤0.001).
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We thank Rachel Wilson and Ansgar Büschges for help with electrophysiology, Loren Looger, Stefan Pulver, Hermann Aberle, Leslie Griffith, Paul Garrity, Ping Shen, Julie Simpson, Jay Hirsh, Olga Alekseyenko, David Krantz, Gero Miesenböck, Hiromu Tanimoto, and the Bloomington and Kyoto Stock Centers for reagents, Anja Nagel and Anette Preiss for RNAi vector, Silvana Opp and Thor Kastilan for RNAi construct, SFB 645 and 704, NRW LIMES graduate school, DFG Cluster of Excellence ImmunoSensation, DFG grant PA 787 for financial support, and Ingo Zinke, Ravi Allada, Valerie Kilman, and Frank Hirth for valuable discussions, and Claire McKellar for suggesting the SOG–brainstem analogy. We also thank Gaia Tavosanis and R.W. for critical comments on earlier versions of this manuscript.
serotonergic
cholinergic
after egg laying
antennal nerve
cibarial dilator muscles
central nervous system
central pattern generator
dopaminergic
transient receptor potential ion channel
glutamatergic
hemisphere
hugin
maxillary nerve
abdominal muscle M6
prothoracic accessory nerve
post-synaptic potential
RNA interference
subesophageal ganglion
ventral nerve cord