(A) Thirst-induced sequence of behavior. (B) Two-chambered open field assay with a vertical divider at the center. Thirsty flies avidly seek and occupy a discrete water source (dyed blue for …
(A) Dopaminergic neuron activation effect on water seeking. (B) R48B04-Gal4, previously implicated in thirsty humidity preference, contains water seeking neurons that are different from those in …
(A) Subtraction of R65D05 neurons (R65D05>Gal80) from the Durstig pattern decreases Durstig activation water seeking. One-way ANOVA/Dunnett's compared to Durstig>TrpA1. (B) Activation of R65D05 …
(A) Otd-nlsFLPo expresses Flippase predominantly in the central brain, effectively limiting R65D05-Gal4-driven GFP to the central brain. High gain to highlight absence of GFP positive neuron cell …
(A) Subtraction of subsets of neurons from Durstig>TrpA1 with patterns of GAL80 expression, occupancy of an inaccessible water source. Cha-Gal80: choline acetyltransferase enhancer, VGlut-Gal80: …
(A) Janu activation limited to central brain neurons promotes occupancy of open water source. One-way ANOVA/Dunnett's compared to Janu>UAS-STOP-TrpA1,otd-nlsFLPo. (B) Janu neuron inactivation …
Stochastic labeling of Janu neurons using a flip-out technique, detected with anti-FLAG (green), and counterstained with anti-VGAT (magenta). (A) Janu-AstA, arrowhead points to cell body (micrograph …
(A–D) Janu-GABA1 interneuron morphology in the SEZ of a Janu>multicolor flip-out brain, compressed z-stack. Arrowhead in (A) points to the cell body. Boxed area is enlarged in (B–D), showing …
(A) Decreased thirsty water seeking when VGAT is knocked down by a second, independent RNAi in Janu neurons. (B) No change in pharyngeal pumping rate with Gad1 knockdown in Janu neurons in thirsty …
(A–G) Janu-AstA neuron morphology and AstA expression. (A) Janu-AstA neuron morphology obtained from a multicolor flip-out brain. Green: anti-FLAG; magenta: anti-VGAT. Arrowhead points to cell body. …
(A, B) Colocalization of Janu>myrGFP with AstA immunoreactivity, in the central brain (A) and in the ventral nervous system (B). Colocalization is limited to the Janu-AstA neurons. (C) AstA …
(A) Examples of TRIC detection of neuronal activity (green) and the pattern of cells expressing the calcium indicator (magenta) under water replete and thirsty conditions in Janu neurons, detected …
(A) Positional preference of Janu > Chrimson flies without ATR. Positive values indicate attraction to optogenetic stimulation. (B) Positional preference for R65D05>Chrimson with and without ATR. (C)…
(A) Janu-AstA presynaptic endings in boxed area imaged for immunohistochemistry. (B) Janu-AstA presynaptic endings partially overlap with NPF immunolabeling. Single 1 μm confocal section. Yellow …
(A) AstA-R1 receptor RNAi in NPF neurons had no effect on thirst or feeding behavior. (B) No change in pharyngeal pumping rate in thirsty NPF>AstA-R2 RNAi flies. (C) Immunohistochemical detection of …
Figure | Question | Approach | Result | Interpretation |
---|---|---|---|---|
3K | Are Janu-GABA neurons important for neural activation driven seeking? | We co-expressed a GABA neurotransmitter RNAi with TrpA1 activation. | Water seeking is suppressed. | Janu-GABA neuron activation drives replete water seeking. |
3L,M 3S1B,C | Are Janu-GABA neurons important for water ingestion? | We used well-established behavioral assays to specifically address ingestive behaviors. | Water ingestion does not change. But, the first bout of water intake decreases. Pumping rate is unchaged. | The first bout of water intake is mediated by Janu-GABA neurons. Other downstream neurons may be responsible for the maintenance of persistent ingestive behaviors. |
3N | Are Janu-GABA neurons important for fed and hungry feeding behavior? | We expressed a GABA neurotransmitter RNAi and assessed dry sucrose occupancy in fed and hungry flies. | Fed and hungry dry sucrose occupancy is unaltered. | Janu-GABA neurons are specific for thirst and do not contribute to feeding behavior. |
4L | Are Janu-AstA neurons important for neural activation driven seeking? | We co-expressed an AstA neurotransmitter RNAi with TrpA1 activation. | Water seeking is suppressed | Janu-AstA neuron activation drives replete water seeking. |
4M,N | Are Janu-AstA neurons important for water ingestion? | We used well-established behavioral assays to specifically address ingestive behaviors. | Water ingestion and first bout of water intake does not change. Pumping rate is unchanged. | Janu-AstA neurons are dispensable for thirsty water ingestion. |
5A,B,C | Are Janu-GABA and Janu-AstA activated by states of thirst and hunger? | We used TRIC, a calciumdependent fluorescent reporter, to assess neuronal activity. | Only the Janu-GABA1 subtype was highly active in thirsty flies. Janu-AstA was not active in thirsty or hungry states. | The Janu-GABA1 neuron is thirst activated. It may be regulated by the osmosensory ISNs. |
5D,E 5S1A,B,C | Does the activity of Janu and R65D05 neurons encode positive or negative valence? | We optogenetically activated Janu and R65D05 neurons to assess positional preference. | Flies exhibit positional preference for activation of Janu or R65D05 neurons. | Janu and R65D05 neurons encode positive valence. This is distinct from mammalian hunger and thirst neurons which encode negative valence. |
6EF 6S1B | Is AstA-R2 signaling on NPF neurons important for thirsty water ingestion? | We expressed an AstA-R2 RNAi in NPF neurons. We used well-established behavioral assays to specifically address ingestive behaviors. | Total water consumption and first bout water consumption are decreased. Pumping rate is unaffected | AstA signaling onto NPF neurons is important for water intake. |
6H | Is NPF signaling important for thirsty water seeking? | We expressed a NPF RNAi in thirsty flies to assess water seeking. | Water seeking is increased. | NPF released by NPF neurons suppresses water seeking. Thus, NPF can reciprocally modulate hunger and thirst. |
6I 6S1C | Are NPF neurons activated by thirst? | We used TRIC, a calcium-dependent fluorescent reporter, to assess neuronal activity. | Dorsomedial P1 and lateral L1 NPF neurons both are significantly activated in thirsty states. | NPF neurons encode thirst and are a critical convergence node for the integration of thirst and hunger signals. |
InSITE strains and seeking index.
Strains and resources.
Experimental conditions and statistical analyses.