(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



A STIM dependent dopamine-neuropeptide axis maintains the larval drive to feed and grow in Drosophila [1]

['Nandashree Kasturacharya', 'National Centre For Biological Sciences', 'Tifr', 'Bellary Road', 'Bengaluru', 'The University Of Trans-Disciplinary Health Sciences', 'Technology', 'Tdu', 'Jasmine Kaur Dhall', 'Gaiti Hasan']

Date: 2023-08

Among other factors, the exit of quiescence and maintenance of neuroblast proliferation at the early second instar stage depends on nutritional intake [ 26 , 29 , 30 ]. The slow growth and delayed exit from quiescence suggested that STIM KO larvae may lack adequate nutritional inputs. As a first step staged larvae were placed on yeast, mixed with a blue dye and tested for ingestion of food. Even as early as 40-44h AEL there was a significant reduction of food intake in STIM KO larvae ( Fig 1I , quantification in Fig 1J ). By 80-84h AEL two classes of STIM KO larvae were evident. One with reduced food intake and others with no food intake ( Figs 1I and S1D Fig ). The proportion of STIM KO larvae with no food intake reached ~70% by 82-86h AEL ( S1E Fig ). The ability to feed was further quantified in STIM KO animals by measuring mouth hook contractions through larval development [ 31 ]. Control larvae (CS) exhibit a steady increase in mouth hook contractions with age, except prior to and during larval molts, indicating greater nutrient intake with age. In contrast, increase in mouth hook movements of STIM KO larvae follows a slower developmental trajectory, with minimal increase as they progress from first to second instar larvae and a cessation at 74h AEL, that is further retarded at 86h ( Fig 1K ; S1 – S4 Videos) . Thus, the acceleration of mouth hook movements observed in CS larvae from first to third instar is retarded in STIM KO larvae before the appearance of growth deficits ( Fig 1I–1K ) suggesting that the consequent nutritional deficits prevent normal growth.

The momentum of larval growth is maintained primarily by cell growth in the endoreplication tissues [ 23 , 24 ]. In a few organs like the brain and the imaginal discs growth is accompanied by constant cell division. Normally, at the end of embryonic development, mitotic cells such as a majority of neuroblasts (NBs) and imaginal disc cells enter a quiescent state [ 4 , 25 , 26 ]. Postembryonic larval development is initiated in the late first instar and early second instar stages by cell growth and renewed cell proliferation in the brain and imaginal discs, where it is nutrient dependent [ 26 ]. Cessation of growth in STIM KO larvae ( Fig 1D and 1E ), suggested a deficit in cell growth and/or cell division. To investigate the status of cell proliferation in STIM KO larvae we chose the well-characterized system of thoracic neuroblasts [ 27 ]. Upon comparison of thoracic segments of WT and STIM KO larvae at 70-74h ( Fig 1F , first two columns) it was evident that NBs exited from quiescence and entered the proliferative state in both genotypes. Both the NB marker Deadpan (red) and the post-mitotic cell marker Prospero (blue) appeared normal in STIM KO larvae of 70-74h AEL. Subsequently, at 82-86h, the number of postmitotic cells (Prospero positive) decreased significantly in STIM KO animals as compared to the controls but the number of thoracic neuroblasts remained unchanged ( Fig 1F , compare third and fourth columns). Upon quantification, the ratio of dividing neuroblasts (Deadpan surrounded by Prospero positive cells) to non-dividing neuroblasts (Deadpan with either no or few Prospero positive cells) changed significantly in 86h aged STIM KO larvae ( Fig 1G and 1H ). To identify the cause underlying the reduced number of postmitotic cells we analyzed different phases of the cell cycle of thoracic neuroblasts in STIM KO larval brains. For this, we used the genetically encoded FUCCI system [ 28 ]. Here, the G1, S, and G2 phases of interphase are marked by green, red and green+red (yellow) fluorescent tags respectively. At 72-76h, both control and STIM KO showed an asynchronous pattern of division. But at 82-86h, control larvae persisted with the asynchronous pattern, whereas a majority of thoracic neuroblasts in STIM KO animals remained in the G2/M state ( S1C Fig ).

(A-C) Number of 1 st instar (L1), 2 nd instar (L2) and 3 rd instar (L3) larvae from CS (grey) and STIM KO (magenta) measured at 6h intervals after egg laying (AEL) at the specified time points (mean ± SEM). Number of sets (N) = 3, number of larvae per replicate (n) = 10. *P < 0.05, Student’s t-test with unequal variances. P values are given in S2 Table . (D) Representative images of larvae from CS and STIM KO at the indicated time. Scale bar = 1 mm. (E) Measurement of larval length (mean ± SEM) from CS (grey) and STIM KO (magenta) larvae at the specified time points. Number of larvae per genotype per time point is (n) ≥ 12. *P < 0.05 for all time points, Student’s t-test with unequal variances. P values are given in S2 Table . (F) Representative images of thoracic neuroblasts marked with Insc>mCD8GFP (green), a neuroblast marker (anti-Deadpan, red) and a marker for post-mitotic cells (anti-Prospero, blue) from control (Insc>mCD8GFP) and STIM KO ; Insc>mCD8GFP animals at the indicated ages. Similar images were obtained from four or more animals. Scale bar = 20μm. (G) Diagrammatic summary of neuroblast proliferation in control (Insc>mCD8GFP) and STIM KO ; Insc>mCD8GFP animals. (H) Stack bar graph showing number of dividing neuroblasts to non-dividing neuroblasts. *P < 0.05, Student’s t-test with unequal variances, n = 4 animals from each genotype. P values are given in S2 Table . (I) Representative images of dye-fed larvae from CS and STIM KO at the indicated times AEL, scale bar = 200μm except for CS (80-84h) where scale = 1mm. (J) Quantification (mean ± SEM) of ingested blue dye in CS and STIM KO larvae at the indicated ages by normalizing optical density (OD) of the dye at 655nm to concentration of protein. Number of feeding plates per time point (N) = 6, number of larvae per plate (n) = 10. *P < 0.05, Student’s t-test with unequal variances. P values are given in S2 Table . (K) Line graph with quantification of larval mouth hook contractions per 30 seconds (mean ± SEM) from CS and STIM KO at indicated developmental time points. Number of larvae per genotype per time point is (n) ≥ 10. *P < 0.05 at all time points, Student’s t-test with unequal variances. All P values are given in S2 Table .

STIM KO larvae appear normal after hatching but their transition from first to second instar stages is slower than wild-type animals ( S1A and S1B Fig ) [ 22 ] and as second instars they die gradually between 86h to 326h after egg laying ( AEL; S1B Fig ). To identify the precise time window when STIM KO larvae become sickly they were observed over 6h time intervals from 36h to 90h AEL. Whereas, wild type (Canton-S or CS) larvae transition from 1 st to 2 nd instar between 42-54h AEL, the same transition in STIM KO larvae occurs between 60-72h AEL, indicating a delay of 18h ( Fig 1A and 1B ). The delay is followed by an inability to transition to 3 rd instar ( Fig 1C ). STIM KO larvae also exhibit retarded growth. At 72h they appear similar to CS larvae of 60h ( Fig 1D ) . After 72h however, there is a complete cessation of growth in STIM KO larvae ( Fig 1D and 1E ), followed by gradual loss of viability after 80-86h ( S1B Fig ). From these results, it became evident that cessation of growth precedes loss of viability in STIM KO larvae.

Ingestion of food and frequency of mouth hook contractions is also rescued by THD’>STIM + expression in STIM KO larvae ( Fig 2G–2I ). To further confirm the relevance of STIM function in THD’ marked dopaminergic neurons we knocked down STIM using a previously characterized STIM RNAi (dsSTIM) [ 37 ]. THD’>dsSTIM animals exhibit delayed larval growth ( Figs 2J and S2B) and reduced feeding ( Fig 2K and 2L ) but no larval lethality. Adults however exhibit reduced body weight ( S2C Fig ). Taken together these data identified a CNS-specific subgroup of dopaminergic neurons that require STIM function for persistent feeding during the early stages of larval growth.

Next, we analysed THD’ driven expression of mCD8GFP and identified two classes of GFP positive cells in the larval brain. All GFP expressing cells in the central brain (three cells of DL1 and two cells of DL2 clusters [ 11 , 34 , 35 ], appear positive for Tyrosine Hydroxylase (TH) but a pair of THD’ cells in the ventral ganglion (VG) appear TH negative (TH -ve ) ( Fig 2C ). To understand the relative contribution of the VG-localised TH -ve cells to THD’>STIM + rescue of STIM KO animals, we restricted THD’GAL4 expression to VG localised THD’ neurons using THGAL80 [ 36 ] ( Fig 2D ). Expression of STIM + in the VG localised THD’ neurons (THD’GAL4, THGAL80) reduced the rescue of STIM KO larvae significantly ( Figs S2A and 2E ) and was absent in adults ( Fig 2F ). Thus, the rescue of viability in STIM KO animals derives to a significant extent from brain-specific THD’ dopaminergic neurons.

(A) Number (mean ± SEM) of 3 rd instar larvae (L3) are restored close to wildtype (CS) levels by expression of STIM + in THD’ cells of STIM KO larvae (rescue). Larvae were monitored at 6h intervals from 66h to 96h AEL. Number of sets (N) = 3, number of larvae per set (n) = 10. Letters represent statistically similar groups for the 90h and 96h time point. Also see S2A Fig for a complete developmental profile of CS, STIM KO and rescue (STIM KO ; THD’>STIM + ) animals with appropriate genetic controls. (B) Stack bars with the number of adults (mean ± SEM) that eclosed at 320 to 326h AEL from the indicated genotypes. The genotype of rescue larvae is STIM KO ; THD’>STIM + . Number of sets (N) = 3, number of organisms per set (n) = 25. (C) Representative confocal images of the larval brain from animals of the genotype THD’>mCD8GFP. Anti-GFP (green) indicates the expression of THD’GAL4 and anti-TH (magenta) marks all dopaminergic cells. Asterisks mark TH +ve cells in CNS whereas arrowheads mark non-TH positive cells in ventral ganglia of larval brain (i and ii). DL1 and DL2 clusters in the central brain of three and two dopaminergic cells respectively are marked (iii). Scale bars = 20μm. (D) Representative confocal images of the larval brain from animals of the genotype THGAL80,THD’>mCD8GFP. THD’GAL4 driven GFP expression (green) is suppressed in DL1 and DL2 clusters in the CNS (asterisk) by THGAL80 but not in the ventral ganglia (arrowheads). Scale bar = 20μm. (E) Line graph shows the number (mean ± SEM) of 3 rd instar larvae from CS, STIM KO , STIM KO ;THD’>STIM + and STIM KO ;THGAL80,THD’>STIM + at 6h intervals between 60 to 96h AEL. Number of sets (N) = 3, number of larvae per set (n) = 10. Different alphabet represent statistically significant groups for 84h, 90h and 96h. Also see S2A Fig for complete larval staging profile and additional genetic controls. (F) Stack bar graph showing the number of adults eclosed (mean ± SEM) at 320 to 326h AEL from CS (wildtype), STIM KO (mutant), STIM KO ;THD’>STIM + (rescue) and STIM KO ;THGAL80,THD’>STIM + (THGAL80—restrictive rescue) genotypes. Different alphabet represent statistically significant groups. Number of sets (N) = 3, number of organisms per set (n) = 25. (G) Representative images of dye-fed larvae of CS, STIM KO and rescue (STIM KO ; THD’>STIM + ) genotypes at 80-84h AEL Scale bar = 1mm. (H) Bar graph with quantification of ingested food containing a blue dye in larvae of the indicated genotypes (CS, STIM KO and STIM KO ; THD’>STIM + rescue) at the indicated developmental times. Mean (mean ± SEM) optical density (655nM) of blue dye in larval lysates after normalizing to larval protein concentration (OD/Protein concentration X 10 −2 ) was obtained from 6 feeding plates (N) each containing 10 larvae (n). Different alphabet represent statistically significant groups. (I) Expression of STIM + in THD’ cells rescues the feeding behaviour deficit of STIM KO larvae. Box graph with quantification of larval mouth hook contractions of the indicated genotypes (CS, STIM KO and STIM KO ; THD’>STIM + rescue). Circles represent single larvae in the box graph of 25 th and 75 th percentiles with the median (bar), and mean (square). Number of larvae per genotype per time point is (n) ≥10. Different alphabet represent statistically significant groups. (J) Number of 3r d instar larvae (mean ± SEM) from RNAi knockdown of STIM + in THD’ neurons (THD’>dsSTIM) along with control genotypes THD’/+ and dsSTIM/+ at 6h intervals between 66h to 96h AEL (After Egg laying). Number of sets (N) = 3, number of larvae per set (n) = 10. Also see S2B and S2C Fig for complete larval staging profile and adult weights. Different alphabet represent statistically significant groups. (K) Representative images of dye-fed larvae of the indicated genotypes at 58-62h and 82-86h AEL. Scale bar = 200μm. (L) Quantification (mean ± SEM) of blue dye containing ingested food (OD/Protein concentration X 10 −2 ; similar to panel D above) in larvae of the indicated genotypes at 58-62h and 82-86h AEL. No. of plates for each time point, (N) = 3, number of larvae per plate (n) = 10. In all graphs and box plots, different alphabets represent distinct statistical groups as calculated by one way ANOVA followed by post-hoc Tukey’s test. P values for individual panels are given in S2 Table .

To understand how the loss of STIM might affect larval feeding we identified specific cells that require STIM function for larval growth and viability. From previous work, we know that knock out of STIM in dopaminergic neurons marked by THGAL4 [ 32 ] leads to larval lethality [ 22 ]. Over-expression of a wildtype UASSTIM + transgene, henceforth referred to as STIM + , in dopaminergic neurons marked by THGAL4 rescued larval lethality of STIM KO animals to a significant extent ( S2A Fig ). Absence of complete rescue by STIM + expression in dopaminergic cells suggests additional requirement for STIM in non-dopaminergic cells of STIM KO larvae, not investigated further in this study. Further to identify specific dopaminergic neurons that require STIM function for growth and viability we tested rescue by overexpression of STIM + in two non-overlapping subsets of dopaminergic neurons marked by THC’GAL4 and THD’GAL4 [ 33 ] and henceforth referred to as THD’ and THC’. Rescue of STIM KO larvae from 2 nd to 3 rd instar (~90%) was evident upon over-expression of STIM + in THD’ marked neurons ( Fig 2A and 2B ). Because developmental profiles of the control genotypes STIM KO ; THD’ and STIM KO ; STIM + are similar to STIM KO at 80-86h and at 168-174h ( S2A Fig ) these were not included in the experiment in Fig 2A . Though unlikely, the developmental profile of rescue larvae (STIM KO THD’; STIM + ) between 72h and 84h may thus in part be due to presence of the THD’ and STIM + transgenes on their own. The rescue by THD’ was equivalent to the rescue by THGAL4 (20±2 and 18±1.5 viable adults eclosed respectively from batches of 25 larvae; S2A Fig ; dark and light green arrows). In contrast rescue by expression of STIM + in THC’GAL4 marked neurons was considerably less (out of batches of 25 animals 5±1 adults eclosed ; S2A Fig ; blue arrow), indicating a greater requirement for STIM function in THD’ marked dopaminergic neurons.

Neuronal excitability and dopamine release requires STIM

The status of THD’ marked central brain dopaminergic cell clusters, DL1 and DL2 was investigated next in STIMKO larval brains at 80-84h, when a few viable organisms are still present despite cessation of growth and feeding (Fig 2G). THD’ cells were marked with GFP in controls, STIMKO and STIMKO animals with STIM+ rescue and the brains were stained with anti-TH sera. THD’>mCD8GFP cells appeared no different in STIMKO as well as STIM+ rescued STIMKO animals at 80-84h AEL when compared to controls at either 58-62h or 80-84h AEL (S3A Fig). Moreover, the numbers of THD’ GFP cells and TH positive cells in the CNS also appeared identical (S3B Fig). Therefore, the loss of STIM does not lead to the loss of dopaminergic neurons in the larval brain.

In order to test if reduced feeding in STIMKO larvae is indeed due to a loss in dopamine signalling we measured larval feeding with knockdown of a key dopamine synthesising enzyme Tyrosine Hydroxylase (TH), in THD’ neurons (THD’>dsTH). Knockdown of TH led to significantly fewer mouth hook contractions in larvae at 80-86h AEL (Fig 3A, S5–S7 Videos), indicating reduced feeding, This was accompanied by slower progression through larval moults and some mortality at each larval stage. Finally out of a total of 25 just 20±1.2 3rd instar larvae pupated, of which 15 adults emerged (S3C Fig). In agreement with lower nutrient intake during larval stages, third instar larvae were smaller in size (Fig 3B–3C), and gave rise to adults with significantly reduced body weight (Fig 3D).

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Dopamine synthesis and excitability in THD’ neurons is important for larval growth. (A) Larval mouth hook movements that correlate with feeding are reduced in larvae with reduced dopamine synthesis in THD’ neurons. Box graph with quantification of mouth hook contractions in larvae with knockdown of Tyrosine Hydroxylase (dsTH) in THD’ neurons and appropriate control genotypes. Number of larvae per genotype per time point is (n) ≥10. (B) Representative images of larvae with knockdown of Tyrosine Hydroxylase (THD’>dsTH) and controls (THD’/+, dsTH/+) at 82-86h. Scale bar = 1mm. (C) Quantification of larval length from the indicated genotypes. n≥15. (D) Quantification of weight of 10 flies from the indicated genotypes. Each circle represents one set of adult flies consisting of 5 females and 5 males, 6-8h post-eclosion. A minimum of 5 sets were measured for each genotype. (E) Representative images of the central brain (left panels) indicating the region of focus (boxed), followed by images of DL1 and DL2 clusters of THD’ cells from two lobes of same brain with Ca2+ transients at the indicated time points before and after addition of a depolarizing agent (KCl– 70mM). Ca2+ transients were measured by changes in the intensity of GCaMP6m fluorescence from THD’>GCaMP6m (control), STIMKO; THD’>GCaMP6m, STIMKO;THD’>GCaMP6m, STIM+ (rescue). Scale bar 20μm. (F) Changes in GCaMP6m fluorescence (mean ± SEM of ΔF/F) from THD’ neurons of the indicated genotypes. Number of brains, (N) ≥ 5, number of cells, (n) ≥ 15. (G) Peak intensities of GCaMP6m fluorescence (ΔF) in THD’ cells from the indicated genotypes. Box plots show 25th and 75th percentiles, the median (bar), and mean (square) of ΔF of each cell (small circles). (H) Number of 3rd instar larvae of CS, STIMKO and STIMKO;THD’>NaChBac genotypes at the indicated times AEL (mean ± SEM). Number of sets (N) = 3, number of larvae per set (n) = 15. (I) Representative images of larvae from CS, STIMKO and STIMKO; THD’>NaChBac at 94-98h AEL, scale bar = 1mm. (J) Quantification of larval length from the indicated genotypes. n = 10. (K) Number of adults eclosed at 320h AEL (mean ± SEM) from wildtype (CS), mutant (STIMKO) and NaChBac rescue (STIMKO; THD’>NaChBac) animals. Numbers were obtained from three experiments (N = 3), number of organisms per experiment, n = 15. In all box plots, circles represent single larvae or flies. The box plots span 25th and 75th percentiles with the median (bar), and mean (square). Alphabets represent distinct statistical groups as calculated by one way ANOVA followed by post-hoc Tukey’s test. P values are given in S2 Table. https://doi.org/10.1371/journal.pgen.1010435.g003

To understand how loss of STIM in THD’ marked neurons might affect their neuronal function, we investigated properties of excitation. For this purpose, Potassium Chloride (KCl, 70mM) evoked cytosolic calcium transients were measured from THD’ neurons using the genetically encoded Ca2+ sensor GCaMP6m in ex vivo preparations of similarly staged control (58-62h AEL) and STIMKO (70-74h AEL) larvae. We chose these time points because at 72h STIMKO larvae appear healthy and developmentally similar to control larvae at 60h (Fig 1D). THD’ cells responded with similar changes in GCaMP intensity, in control and STIMKO larvae at these time points (Fig 3E–3G). However, the ability to evoke and maintain cytosolic Ca2+ transients upon KCl depolarisation was lost in THD’ neurons from the DL1 and DL2 clusters of STIMKO larvae at 76-80h AEL (Fig 3E–3G). Overexpression of STIM+ in THD’ cells of STIMKO larvae rescued the KCl evoked Ca2+ response in larvae as late as 80-84h AEL (Fig 3E–3G).

STIM requirement for maintaining excitability of THD’ neurons was tested further by KCl stimulation of THD’ neurons with STIM knockdown (THD’>dsSTIM) from 2nd instar larvae aged 58-62h. Two classes of responses to depolarisation by KCl were observed. Most cells (70%) responded with normal or greater changes in intensity as compared to control cells, whereas in 30% of cells KCl did not evoke a Ca2+ transient (S3D–S3F Fig). We attribute this heterogeneous response to differential STIM knockdown by the RNAi in individual THD’ cells and a potential effect of STIM knock-down on ER-Ca2+ homeostasis (see below).

These data suggest that loss of STIM affects membrane excitability properties and the ability of THD’ neurons to respond to stimuli. This idea was tested directly by the expression of transgenes that alter membrane potential. Over-expression of an inward rectifier K+ channel, Kir2.1 in THD’ neurons, that is known to hyperpolarise the plasma membrane [38], resulted in developmental delays followed by the lethality of second and third instar larvae (S4A Fig). Further, overexpression of a bacterial Na+ channel NaChBac [39] in THD’ neurons of STIMKO larvae evinced a weak rescue of developmental deficits, including the transition to third instar larvae (4.4±0.4) and adult viability (2.4±0.6 from of batches of 15 animals; Fig 3H–3K). Though weak, NaChBac’s rescue was consistent. We attribute the variability to a stochastic effect of NaChBac in THD’ cells. This is also evident from the variable extent of rescue in growth observed in STIMKO; THD’>NaChBac larvae (Fig 3I and 3J). Alternatively, in addition to neuronal excitability, STIM might affect other cellular functions such as ER stress, that are not alleviated by expression of NaChBac, resulting in the weak rescue. Control animals with overexpression of NaChBac in THD’ neurons exhibit delayed pupariation (S4B Fig).

Neuronal excitability is required for neurotransmitter release at presynaptic terminals. We hypothesized that dopamine release from THD’ neurons might be affected in STIMKO larvae. To test this idea, we identified the pre-synaptic (green) and post-synaptic (red) terminal regions of THD’ neurons by marking them with Syt-eGFP and Denmark respectively (Fig 4A) [40]. Analysis of pre-synaptic regions (Syt-eGFP expression) identified three distinct areas in the CNS. One at the centre of the CNS corresponding to the mushroom body (Fig 4A; asterisk), the second as a branched form in the basomedial protocerebrum of the CNS (Fig 4A; arrowhead) and the third one consisting of punctae spread across the oesophageal regions where brain-gut interactions take place (Fig 4A; hash). Based on the projection patterns observed we speculate that cells marked by THD’ correspond to DL1-2, DL1-5, DL1-6 from the DL1 cluster and DL2-2 and DL2-3 from the DL2 cluster [35].

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. STIM is necessary for dopamine release and ER-Ca2+ homeostasis. (A) Axonal and dendritic projections of THD’ neurons visualized by expression of SyteGFP (green) and Denmark (anti-RFP, magenta) respectively in a representative brain immunostained for anti-Brp (blue). Panels marked as (d) contain magnified images from (c) of presynaptic terminals (green) located at the base of the mushroom body in the CNS (asterisk), as branches extruding into basomedial protocerebrum of the CNS (arrowhead) and as punctae near the oesophageal region (hash). (B) Representative images of dopamine release before and after addition of Carbachol (CCh) as measured by changes in intensity of GRAB DA at the presynaptic terminals of THD’ neurons of control (THD’>GRAB DA ), STIMKO (STIMKO;THD’>GRAB DA ) and rescue (STIMKO;THD’>GRAB DA , STIM+) genotypes. Scale bar = 20μm. (C) Normalized changes in fluorescence (ΔF/F) of GRAB DA measuring dopamine release from THD’ neurons of control (THD’>GRAB DA ), STIMKO (STIMKO;THD’>GRAB DA ) and rescue (STIMKO;THD’>GRAB DA , STIM+) genotypes. Traces show the average change in GRAB DA florescence (mean ± SEM of (ΔF/F)) measured from individual presynaptic regions of interest (≥10) taken from N ≥ 6 brains of each genotype. (D) Representative time series images of ER calcium transients as measured by changes in intensity of ER-GCaMP in THD’ neurons of control (THD’>ER-GCaMP-210), STIMKO (STIMKO;THD’> ER-GCaMP-210) and rescue (STIMKO;THD’> ER-GCaMP-210, STIM+) genotypes. (E) i) Traces of normalized ER-GCaMP (ΔF/F) responses (mean ± SEM) from THD’ neurons of the indicated genotypes. Control (THD’>ER-GCaMP-210) Type 1, indicates cells that exhibit ER-Ca2+ release upon CCh addition followed by refilling of ER-stores. Control Type 2 indicates cells where ER-Ca2+ release was not evident. Rescue indicates STIMKO; THD’>ERGCaMP-210,STIM+. For each genotype, number of cells (n) ≥ 15 and N ≥ 5 brains. (ii) Quantification of control cells that exhibit ER Ca2+ release (Type 1) and cells where ER Ca2+ release was not observed (Type 2). (iii) Boxed region from (i) enlarged to show ER Ca2+ response of Type 1 and Type 2 control cells. https://doi.org/10.1371/journal.pgen.1010435.g004

Next, dopamine release was measured in the most prominent presynaptic areas of THD’ neurons, corresponding to the MB and the basomedial protocerebrum, by change in fluorescence of the genetically encoded fluorescent GPCR-activation-based-DopAmine sensor (GRAB DA ) [41]. Dopamine release in THD’ neurons of STIMKO larvae at 76-80h is significantly attenuated as compared with controls (Fig 4B and 4C). Importantly, overexpression of STIM+ in THD’ neurons rescued dopamine release, though with altered dynamics from control animals (Fig 4B and 4C; see discussion). We chose to measure dopamine release in 76-80h STIMKO larvae because THD’ neurons in their brains no longer responded to KCl evoked depolarization (Fig 3B) even though the larvae appear normal (Fig 1D). Dopamine release was stimulated by Carbachol (CCh), an agonist for the muscarinic acetylcholine receptor (mAChR), that links to Ca2+ release from ER-stores through the ER-localised IP 3 receptor [42] and is expressed on THD’ neurons [43]. CCh-induced changes in ER-Ca2+ were tested directly by introducing an ER-Ca2+ sensor [44] in THD’ neurons (Fig 4D and 4E). Though ER-Ca2+ release, in response to CCh could be measured in just 7 out of 23 cells, the subsequent step of ER-store refilling, presumably after Store-operated Ca2+ entry into the cytosol through the STIM/Orai pathway, could be observed in all control THD’ cells (58-62h AEL), whereas it was absent in THD’ neurons from STIMKO brains (76-80h AEL; Fig 4E). The ER-Ca2+ response was rescued by over-expressing STIM+ in THD’ neurons (Fig 4D, and 4E). Taken together our data establish an important role for STIM-dependent ER-Ca2+ homeostasis in maintaining optimal dopamine release from THD’ neurons in turn required for the larval drive to feed constantly.

Because late third instar larvae stop feeding [3,19,20,45] we hypothesized that CCh-stimulated Ca2+ responses in THD’ cells might change in wandering stage third instar larvae. To test this idea we monitored carbachol-stimulated GCaMP activity in THD’ neurons from wild-type larvae at 96h AEL (mid 3rd instar), 120h AEL (early wandering stage) and 124h AEL (late wandering stage). A robust GCaMP response was observed at 96h, whereas at the beginning of the wandering stage (118-122h AEL), the peak response was both reduced and delayed. A further delay in the peak response time was observed in late wandering stage larvae (122-126h AEL) (S4E and S4F Fig). Thus with gradual cessation of feeding in late third instar larvae, the dynamics of CCh-stimulated Ca2+ responses in THD’ neurons also undergo changes (see discussion).

[END]
---
[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010435

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.