A discrete parasubthalamic nucleus subpopulation plays a critical role in appetite suppression
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Evaluation Summary:
This paper will be of interest to neuroscientists studying ingestive behavior and control of body weight. It reveals two distinct subsets of neurons within a little-studied brain area that are both activated by feeding, but only of them contributes to hormone-mediated suppression of feeding. The combination of molecular profiling and functional modulation of the neurons compellingly support the claims of the paper.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Food intake behavior is regulated by a network of appetite-inducing and appetite-suppressing neuronal populations throughout the brain. The parasubthalamic nucleus (PSTN), a relatively unexplored population of neurons in the posterior hypothalamus, has been hypothesized to regulate appetite due to its connectivity with other anorexigenic neuronal populations and because these neurons express Fos, a marker of neuronal activation, following a meal. However, the individual cell types that make up the PSTN are not well characterized, nor are their functional roles in food intake behavior. Here, we identify and distinguish between two discrete PSTN subpopulations, those that express tachykinin-1 (PSTN Tac1 neurons) and those that express corticotropin-releasing hormone (PSTN CRH neurons), and use a panel of genetically encoded tools in mice to show that PSTN Tac1 neurons play an important role in appetite suppression. Both subpopulations increase activity following a meal and in response to administration of the anorexigenic hormones amylin, cholecystokinin (CCK), and peptide YY (PYY). Interestingly, chemogenetic inhibition of PSTN Tac1 , but not PSTN CRH neurons, reduces the appetite-suppressing effects of these hormones. Consistently, optogenetic and chemogenetic stimulation of PSTN Tac1 neurons, but not PSTN CRH neurons, reduces food intake in hungry mice. PSTN Tac1 and PSTN CRH neurons project to distinct downstream brain regions, and stimulation of PSTN Tac1 projections to individual anorexigenic populations reduces food consumption. Taken together, these results reveal the functional properties and projection patterns of distinct PSTN cell types and demonstrate an anorexigenic role for PSTN Tac1 neurons in the hormonal and central regulation of appetite.
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Author Response:
Reviewer #3 (Public Review):
Two cell types in the parasubthalamic nucleus (a region of the posterior hypothalamus) are activated following food intake. The authors determine that the Tac1 expressing population is sufficient to suppress food intake and the Crh population does not influence food intake. Further, the authors demonstrate that only the Tac1 population projects to the PBN. The Tac1 neurons are transiently activated following food presentation or satiation hormones (for about 1 minute). This transient change in activity is interesting and fits into a lot of other recently published work showing transient neural activity changes that are involved in longer term behavior. Longer term activation of these neurons reduces food intake and the authors begin to explore the circuits/networks that these neurons …
Author Response:
Reviewer #3 (Public Review):
Two cell types in the parasubthalamic nucleus (a region of the posterior hypothalamus) are activated following food intake. The authors determine that the Tac1 expressing population is sufficient to suppress food intake and the Crh population does not influence food intake. Further, the authors demonstrate that only the Tac1 population projects to the PBN. The Tac1 neurons are transiently activated following food presentation or satiation hormones (for about 1 minute). This transient change in activity is interesting and fits into a lot of other recently published work showing transient neural activity changes that are involved in longer term behavior. Longer term activation of these neurons reduces food intake and the authors begin to explore the circuits/networks that these neurons influence. Overall, the work is well done and the experiments support the conclusions. Some minor clarifications could enhance the manuscript and could be addressed through further analysis or adding in text.
1. What % of the overall PSTN neurons are tac1/crh (ie, how many other cell types are there?). Or what % of the vglut2 neurons do they make. This just requires further analysis of the current dataset. And, are there any GABAergic cells (like are the PV GABAergic)?
We thank the Reviewer for suggesting this analysis because it is interesting and other readers are likely to ask the same questions. In our original submission we were hesitant to report these values because they ultimately represent an approximation. Because the neurons that surround the PSTN are also glutamatergic (including the subthalamic nucleus and the lateral hypothalamic area), it is impossible to precisely delineate the border of the PSTN using Slc17a6 as a marker. However, this is an important question and we feel that reporting these values while qualifying them as an estimation will be impactful. Therefore, in the revised manuscript, we now include the following statement:
“Although it is impossible to delineate a precise border for the PSTN using Slc17a6 because adjacent regions are also glutamatergic, we estimate that ~22% of Slc17a6- expressing neurons within the PSTN region do not express either Tac1 or Crh, indicating the presence of glutamatergic PSTN cell types that may express other unique genetic markers.”
We did not examine GABAergic expression in the PSTN because the Allen Brain Atlas and recent RNA-Seq studies (e.g., Wallén-Mackenzie et al., 2020) found an almost complete absence of Gad1- and Gad2-expressing cells in the PSTN region. We report this previous finding within the Results:
“Expression of the GABAergic markers Gad1 and Gad2 are notably absent from the PSTN region (Shah et al., 2022).”
2. The 60 second increase in tac1 neuron activity is interesting. In the discussion, the authors present some plausible arguments for how that may affect feeding for hours. Additionally, it would be nice to point out that this is a recurring theme. This occurs in other neuron populations that influence food intake. Although this is seemingly counterintuitive, I think it is good to mention as these short-term neural activity changes are clearly having large effects on behavior and it is important for everyone to realize this.
This point is an excellent observation and we agree that we could highlight other studies showing transient activation of neural activity controlling food intake. Therefore, we added to our Discussion:
“Indeed, many other neural populations that regulate food intake behavior also show a transient increase in neural activity on the timescale of seconds (Berrios et al, 2021; Luskin et al., 2021; Mohammad et al., 2021; Wu et al., 2022).”
3. Something a little strange with the meal frequency. I thought CCK reduced meal size not frequency. Why does the rescue then increase frequency? Could it be that the rescue to the CCK is by a different means than just blocking the effect of CCK? Adding some language to the discussion about how to interpret the satiation peptide data would be useful.
We thank the Reviewer for bringing up this interesting point. Previous studies do indicate that CCK (and also amylin, to a large extent) reduces meal size and does not have much of an effect on meal frequency. We therefore added a paragraph to the Discussion to note and discuss this point:
“It is also noteworthy that chemogenetic inhibition of PSTN^Tac1 neurons attenuates the effects of amylin, CCK, and PYY by decreasing the frequency of meals as opposed to meal size or meal duration (Figure 5). Previous studies of these anorexigenic hormones, especially amylin and CCK, indicate that they affect food intake primarily by decreasing meal size as opposed to meal frequency (Drazen and Woods, 2003; Lutz et al., 1995; West et al., 1987). Therefore, inhibition of PSTN^Tac1 neurons might attenuate the effects of these hormones indirectly, perhaps by reducing activity in downstream populations such as the NTS or PBN. In this model, infusion of anorexigenic hormones activate PSTN^Tac1 neurons that, in turn, cause sustained activation of downstream populations. Without this sustained activity, downstream populations may not have sufficient activity to cause a reduction in the intermeal interval, leading to increased bouts of feeding. The mechanism by which anorexigenic hormones activate PSTN^Tac1 neurons, as well as how decreases in PSTN^Tac1 neuronal activity affect downstream populations, are important topics for future investigation.”
4. The axonal stimulation data needs qualification - as axons could project to multiple target regions (like the projections to the PVT could also have a collateral to the CEA). For this type of experiment, I prefer to use the phrase "neurons with a projection to region X do behavior Y". Otherwise, the implication in reading the results is that the particular projection is mediating the behavior. Also, the collateral issue, which is qualified in the discussion, should be mentioned here.
We see the Reviewer’s point and have revised the language to highlight this important qualification of our results. Specifically, we added text in the Results section in regard to Figure 8:
“Because it is unknown whether PSTNneurons send collateral projections to multiple brain regions, it is possible that stimulation in a single projection target causes antidromic activation to one or more other target areas. Therefore, these results indicate that PSTNTac1 neurons with projections to the CeA, PVT, PBN, and NTS can suppress food intake, although the exact functional role of each downstream target region on food intake behavior remains undetermined.”
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Evaluation Summary:
This paper will be of interest to neuroscientists studying ingestive behavior and control of body weight. It reveals two distinct subsets of neurons within a little-studied brain area that are both activated by feeding, but only of them contributes to hormone-mediated suppression of feeding. The combination of molecular profiling and functional modulation of the neurons compellingly support the claims of the paper.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors report that neurons expressing tachykinin-1 in the parasubthalamic nucleus (PSTNTac1) play significant roles in anorexic feeding behavior in response to anorexigenic hormones such as amylin, cholecystokinin, and peptide YY. They found that the injections of these peptides dramatically increased neuronal activation in PSTNTac1 neurons, and optogenetic/chemogenetic activation of PSTNTac1 neurons reduced food intake. In addition, they found that chemogenetic inhibition of PSTNTac1 neurons partially reversed the anorexic effects of the peptides. Further, they determined the key downstream sites of PSTNTac1 neurons using optogenetics. The authors also addressed another neuronal population, corticotropin-releasing hormone-expressing neurons in PSTN, but these neurons are unlikely to be involved in food …
Reviewer #1 (Public Review):
The authors report that neurons expressing tachykinin-1 in the parasubthalamic nucleus (PSTNTac1) play significant roles in anorexic feeding behavior in response to anorexigenic hormones such as amylin, cholecystokinin, and peptide YY. They found that the injections of these peptides dramatically increased neuronal activation in PSTNTac1 neurons, and optogenetic/chemogenetic activation of PSTNTac1 neurons reduced food intake. In addition, they found that chemogenetic inhibition of PSTNTac1 neurons partially reversed the anorexic effects of the peptides. Further, they determined the key downstream sites of PSTNTac1 neurons using optogenetics. The authors also addressed another neuronal population, corticotropin-releasing hormone-expressing neurons in PSTN, but these neurons are unlikely to be involved in food intake behavior. In sum, the experiments are well-designed, the manuscript is well-written, and the results support their conclusion.
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Reviewer #2 (Public Review):
In this manuscript the authors examine a relatively understudied brain area, the parasubthalamic nucleus (PSTN) to determine the molecular signatures of resident neurons and how they contribute to regulating ingestive behavior. They use a combination of cre-expressing mice and site-specific injection of cre-dependent AAVs to modulate the PSTN neurons, and specific subsets within this region. This study arises from their finding that the PSTN provides projections to the parabrachial area, a region now well-established to modulate feeding. They then demonstrate that the PSTN contains two separate sets of neurons that are marked by their expression of Tac1 and Crh respectively. Both of these PSTN populations are activated by feeding stimuli and the anorectic hormones amylin, CCK and PYY, as assessed via cFos …
Reviewer #2 (Public Review):
In this manuscript the authors examine a relatively understudied brain area, the parasubthalamic nucleus (PSTN) to determine the molecular signatures of resident neurons and how they contribute to regulating ingestive behavior. They use a combination of cre-expressing mice and site-specific injection of cre-dependent AAVs to modulate the PSTN neurons, and specific subsets within this region. This study arises from their finding that the PSTN provides projections to the parabrachial area, a region now well-established to modulate feeding. They then demonstrate that the PSTN contains two separate sets of neurons that are marked by their expression of Tac1 and Crh respectively. Both of these PSTN populations are activated by feeding stimuli and the anorectic hormones amylin, CCK and PYY, as assessed via cFos and more temporally specific fiber photometry measurement. Moreover, both PSTN subpopulations project to roughly the same brain areas. Intriguingly, only experimental inhibition or activation of the PSTNTac1 subset modulates feeding, while neither activation nor inhibition of the PSTNCrh subset budges feeding.
The major strength of the manuscript is its use of a wide variety of approaches to map the connections of the PSTN neurons, their endogenous regulation and employing both chemogenetic and optogenetic approaches to describe the roles of the PSTN neurons in ingestive behavior. What results is a comprehensive survey of the PSTN neurons, and a mostly complete comparison of the PSTNTac1 vs. PSTNCrh subpopulations. A weakness is that not all of the manipulations provide a 1:1 comparison of the subpopulations, which would be valuable. Given the wealth of methods herein, the data are presented logically, the text is economical, and there is a parsimonious interpretation of what the respective populations do and do not do. This work is exemplary for those studying ingestive behavior of how to combine the current neuroscience tools to characterize the molecular signature, pathways and function of neural populations. It will therefore be of broad interest to the neuroscience community using these tools, and more specifically, to the ingestive behavior field seeking to define which neurons coordinate feeding.
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Reviewer #3 (Public Review):
Two cell types in the parasubthalamic nucleus (a region of the posterior hypothalamus) are activated following food intake. The authors determine that the Tac1 expressing population is sufficient to suppress food intake and the Crh population does not influence food intake. Further, the authors demonstrate that only the Tac1 population projects to the PBN. The Tac1 neurons are transiently activated following food presentation or satiation hormones (for about 1 minute). This transient change in activity is interesting and fits into a lot of other recently published work showing transient neural activity changes that are involved in longer term behavior. Longer term activation of these neurons reduces food intake and the authors begin to explore the circuits/networks that these neurons influence. Overall, the …
Reviewer #3 (Public Review):
Two cell types in the parasubthalamic nucleus (a region of the posterior hypothalamus) are activated following food intake. The authors determine that the Tac1 expressing population is sufficient to suppress food intake and the Crh population does not influence food intake. Further, the authors demonstrate that only the Tac1 population projects to the PBN. The Tac1 neurons are transiently activated following food presentation or satiation hormones (for about 1 minute). This transient change in activity is interesting and fits into a lot of other recently published work showing transient neural activity changes that are involved in longer term behavior. Longer term activation of these neurons reduces food intake and the authors begin to explore the circuits/networks that these neurons influence. Overall, the work is well done and the experiments support the conclusions. Some minor clarifications could enhance the manuscript and could be addressed through further analysis or adding in text.
1. What % of the overall PSTN neurons are tac1/crh (ie, how many other cell types are there?). Or what % of the vglut2 neurons do they make. This just requires further analysis of the current dataset. And, are there any GABAergic cells (like are the PV GABAergic)?
2. The 60 second increase in tac1 neuron activity is interesting. In the discussion, the authors present some plausible arguments for how that may affect feeding for hours. Additionally, it would be nice to point out that this is a recurring theme. This occurs in other neuron populations that influence food intake. Although this is seemingly counterintuitive, I think it is good to mention as these short-term neural activity changes are clearly having large effects on behavior and it is important for everyone to realize this.
3. Something a little strange with the meal frequency. I thought CCK reduced meal size not frequency. Why does the rescue then increase frequency? Could it be that the rescue to the CCK is by a different means than just blocking the effect of CCK? Adding some language to the discussion about how to interpret the satiation peptide data would be useful.
4. The axonal stimulation data needs qualification - as axons could project to multiple target regions (like the projections to the PVT could also have a collateral to the CEA). For this type of experiment, I prefer to use the phrase "neurons with a projection to region X do behavior Y". Otherwise, the implication in reading the results is that the particular projection is mediating the behavior. Also, the collateral issue, which is qualified in the discussion, should be mentioned here.
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