Different serotonergic neurons regulate appetite for sucrose and hunger for proteins

Reviewer #1 (Public review):

Summary:

Dorn et al. investigate the role of specific serotonergic cell types in fed appetite and starved hunger. They show that neurons labeled by the Sert3-GAL4 line modulate sucrose appetite and that neurons labeled by R50H05-GAL4 and Tph-GAL4 modulate yeast hunger, by expressing a non-functioning serotonin transporter. Similarly, activating these neurons leads to the same effects - a decrease in sucrose appetite and an increase in yeast hunger, respectively. Manipulation of the serotonin transporter in Sert3 neurons impairs appetitive sugar-odor conditioning, however aversive shock-odor conditioning is intact. The authors further tested the role of insulin signaling in this paradigm and the Sert3 neurons. Expressing either constitutively active or non-function insulin receptor impaired sucrose appetite. The expression of the different modulated insulin receptors affects the anatomy of the cells and the distribution of serotonin transporters. It seems that overexpression of the serotonin transporter can rescue the sugar appetite phenotype caused by the constitutively active insulin receptor. Additional experiments reveal that CG9911 and CG10029 RNAi - genes potentially involved in the insulin-serotonin pathway - knockdown does not affect sugar appetite, however Sec24AB RNAi - required for proper serotonin transporter localization - knockdown also leads to sugar appetite reduction. Finally, the authors show that IR60b taste receptor neurons potentially get modulated by Sert 3 and thereby influence sucrose appetite.

Strengths:

The authors provide a more detailed description of the multiple roles that serotonin neurons can take on. Manipulating specific subsets of serotonergic cells, they can distinguish cells that are involved in sucrose feeding in fed animals, whereas other cells are involved in regulating yeast hunger in starved animals. Thus, further cell-type specific dissections and manipulations are required to understand the full functional repertoire of different serotonergic neurons in the brain. The authors further describe that insulin seems to modulate serotonergic neurons and starts to elucidate the underlying complex neuromodulatory mechanisms.

Weaknesses:

Even though the authors provide evidence for behavioral phenotypes due to manipulations of serotonin and insulin cells, the evidence for the required molecular mechanism and connectivity is not convincing and requires further investigation. The authors expand their findings to play a role in sugar conditioning, however, according to the authors flies were starved for these experiments - thus these results rather contradict the innate phenotype.

Reviewer #2 (Public review):

Summary:

This study by Dorn et al. from Dr. Henrike Scholz's group investigates the function of serotonin signaling in state-dependent feeding control for protein and sugar intake. Using a dominant-negative serotonin transporter to block serotonin reuptake and optogenetics to activate serotoninergic neurons, the authors identified that serotonin released from a small group of Sert3-expressing neurons specifically reduces sucrose consumption of the fed files but not in the starved flies. Conversely, blocking serotonin reuptake in broad serotonergic neurons increases yeast consumption only in starved flies but does not affect fed flies. These results suggest prolonged serotonin signals may suppress sucrose appetite in fed flies while promoting protein intake in starved flies.

Although the phenotypes presented are intriguing and fundamental to animal fitness, the data in its current form is insufficient to support the proposed mechanisms underlying the state-dependent diet control by serotonin signals. Specifically, the authors should carefully analyze the requirement of serotonin by showing the efficiency of the serotonin reuptake blockade caused by the dominant-negative serotonin and validating the requirement of serotonin in the optogenetic activation of Sert3-expressing neurons. Additionally, the conclusions based on the overexpressed Sert3::gfp transgene should be retrieved as the overexpression affects sucrose consumption. Therefore, I recommend some alternative interpretations and approaches below for authors to verify the current form of conclusions.

Strengths:

The authors identified the state-dependent diet control for sucrose and yeast intake regulated by a restricted population of serotonin neurons expressing Sert3.

Weaknesses:

The data only partially supports most conclusions. Specifically, findings based on the use of the transgene Sert3::GFP lack sufficient rigor, as the authors overlooked potential overexpression effects.

Major issues

(1) The authors try to distinguish the motivation to feed on sucrose or protein in fed or starved conditions using "sucrose appetite" and "protein hunger", respectively. However, appetite and hunger should be synonymous in the current context. When specifying protein hunger, readers will expect the craving for protein in the protein-deprived situation. In the current study, starved flies were prepared by starvation on wet tissues so the flies are supposed to be hungry for sugar and protein. To avoid confusion, "sucrose appetite in fed flies" and "protein appetite in starved flies" are better descriptions.

(2) In Figure 1A-1I (lines 141-142), it remains unclear whether additional serotonergic neurons are required or if the serotonergic neurons labeled exclusively by R50H05-Gal4 and Tph-Gal4 are necessary to regulate yeast consumption in starved flies. The overlapping expressions of these two drivers with the Sert3-Gal4 make it hard to distinguish these two possibilities.

(3) The data in Figure 1L-1M suggests that the serotonin-dependent regulation in yeast consumption of starved flies is suppressed by sucrose supplementation. However, the neurons required for yeast consumption remain undefined due to the overlapping expression. This result implies that the neurons labeled by R50H05-Gal4 and Tph-Gal4 influence both sucrose and yeast consumption but not specific to yeast.

(4) The regulatory relationship between insulin receptors and serotonin signaling in sucrose appetite remains unclear. How do the authors interpret the result that both the constitutively active and dominant-negative forms of the insulin receptor (InR) reduce sucrose appetite in Figure 4? One possibility is that insulin receptors are involved in two parallel pathways to regulate sucrose consumption that converge to the same phenotype. However, the insulin receptor (InR) pathways can still be independent of the serotonin signaling pathway despite showing a comparable reduction of sucrose consumption in fed flies. This issue should be discussed further following lines 229-231.

(5) The quantification of Figure 5 should be revised. First, as the transgene Sert3::GFP affects sucrose consumption, quantifying the transgene signals may not explain its endogenous function. Second, the quantification lacks a Gal4 expression control using an untagged fluorescent marker, preferably a different color so that the authors can quantify it in the same individual as the comparison. Lastly, it is hard to be convinced that the distance between two layers represents the broad expression of Sert3::GFP in response to insulin receptor alterations. Quantifying the area size of each layer with fixed imaging conditions such as the intervals of brain sections and the laser intensity may be a better alternative approach.

(6) The conclusions drawn based on the Sert3::GFP transgene failed to explain the endogenous function of the serotonin transporter Sert3 in regulating sucrose consumption. Expressing the constitutive-active form of the insulin receptor in Sert3-expressing neurons reduces the total sucrose consumption of fed flies in Figure 4A, which appears inconsistent with the fly line with an additional Sert3::GFP expression shown in Figures 6F, where the suppression of sucrose consumption is not shown for the normalized sucrose intake. This inconsistency suggests that Sert3::GFP transgene itself affects sucrose consumption.

(7) In lines 324-326, the authors should investigate whether IR60b neurons are indeed the downstream of serotoninergic neurons SE1 to regulate sucrose consumption in fed flies. First, synaptic connections could be confirmed by additional approaches. Following this, the authors could demonstrate that the knockdown of serotonin receptors in IR60b neurons eliminates the suppression in sucrose consumption induced by the activation of Sert3-expressing neurons or by the expression of the dominant-negative serotonin transporter in fed flies.