Decision letter: A discrete parasubthalamic nucleus subpopulation plays a critical role in appetite suppression

Field: Neuroscience (Life Sciences)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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 (PSTNTac1 neurons) and those that express corticotropin-releasing hormone (PSTNCRH neurons), and use a panel of genetically encoded tools in mice to show that PSTNTac1 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 PSTNTac1, but not PSTNCRH neurons, reduces the appetite-suppressing effects of these hormones. Consistently, optogenetic and chemogenetic stimulation of PSTNTac1 neurons, but not PSTNCRH neurons, reduces food intake in hungry mice. PSTNTac1 and PSTNCRH neurons project to distinct downstream brain regions, and stimulation of PSTNTac1 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 PSTNTac1 neurons in the hormonal and central regulation of appetite. Editor's evaluation This work has identified a previously unrecognized role of the parasubthalamic nucleus in the regulation of feeding behavior. The combination of genetic and pharmacological approaches nicely demonstrates the physiological role of this group of neurons in regulating appetite. These studies will be of interest to the field and more broadly to the readers of eLife. https://doi.org/10.7554/eLife.75470.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The brain regulates food intake behavior through the coordinated activity of several distinct neuronal populations (Andermann and Lowell, 2017; Sternson and Eiselt, 2017). Activity in orexigenic (appetite-inducing) populations, such as agouti-related peptide (AgRP)-expressing neurons in the hypothalamic arcuate nucleus, can rapidly and reversibly promote an increase in food intake (Aponte et al., 2011; Krashes et al., 2011). In contrast, activity in anorexigenic (appetite-suppressing) populations, such as pro-opiomelanocortin (POMC)-expressing neurons in the hypothalamus (Aponte et al., 2011) or calcitonin gene related peptide (CGRP)-expressing neurons in the brainstem parabrachial nucleus (PBNCGRP neurons) (Campos et al., 2016; Carter et al., 2013), suppresses feeding. In recent years, the parasubthalamic nucleus (PSTN), a relatively understudied population of neurons in the posterolateral hypothalamus, has been hypothesized to regulate feeding behavior (Shah et al., 2022); however, the gene expression patterns within the PSTN and the roles of individual cell types in food intake behavior have remained relatively uncharacterized. The few studies that have explored the PSTN demonstrate a potential role for these neurons in feeding. Anatomically, PSTN neurons receive afferent projections from orexigenic AgRP neurons (Livneh et al., 2017; Wang et al., 2015) and anorexigenic populations including arcuate POMC neurons (Wang et al., 2015), PBNCGRP neurons (Huang et al., 2021), and the central nucleus of the amygdala (CeA) (Barbier et al., 2017). In turn, these neurons project to anorexigenic brain regions including the PBN (Goto and Swanson, 2004; Zséli et al., 2016), CeA (Barbier et al., 2020; Zséli et al., 2018), nucleus of the solitary tract (NST) (Ciriello et al., 2008; Goto and Swanson, 2004; Holt et al., 2019), and paraventricular thalamic nucleus (PVT) (Zhang and van den Pol, 2017). PSTN neurons express Fos, an immediate early gene that serves as an indirect marker of neuronal activation, following a large meal (Barbier et al., 2020; Chometton et al., 2016; Zséli et al., 2016; Zséli et al., 2018), during a learned conditioned flavor aversion (Yasoshima et al., 2006), and in response to dietary amino acid deficiency that causes a decrease in appetite (Zhu et al., 2012). Optogenetic stimulation of glutamatergic projections from the PSTN to the PVT reduces food intake (Zhang and van den Pol, 2017). Finally, chemogenetic inhibition of PSTN neurons attenuates the appetite suppressing effects of the hormone cholecystokinin (Sanchez et al., 2022), and chemogenetic inhibition of PSTN neurons that express tachykinin-1 (Tac1) decreases taste neophobia (Barbier et al., 2020). Taken together, these initial studies suggest a role for the PSTN in feeding behavior. However, the individual subpopulations that make up the PSTN remain relatively uncharacterized, as do their functional roles during food consumption. We therefore sought to characterize the cell types that make up the PSTN, determine their activity patterns in response to various appetitive stimuli, perturb their function in freely behaving mice, and map their connectivity with other brain regions. We identify two discrete populations of PSTN neurons, each with distinct influences on feeding behavior. Results Identification of distinct PSTN cell populations We initially identified the PSTN as a candidate anorexigenic population by investigating sources of afferent input to PBNCGRP neurons, a population that has previously been shown to suppress appetite and control meal termination (Campos et al., 2017; Campos et al., 2016; Carter et al., 2013; Essner et al., 2017). To retrogradely label neurons that project to PBNCGRP neurons, we used a Cre-inducible modified rabies virus system in CalcaCre/+ mice (Calca is the gene that encodes CGRP; Figure 1A and B). Retrograde expression was observed in populations previously shown to project to the PBN, including the bed nucleus of the stria terminalis (BNST) (Wang et al., 2019), central nucleus of the amygdala (CeA) (Cai et al., 2014), arcuate nucleus of the hypothalamus (Arc) (Carter et al., 2013; Essner et al., 2017; Wu et al., 2009), and nucleus of the solitary tract (NTS) (Roman et al., 2016; Figure 1C–H). We also observed retrogradely labeled cells in the PSTN, just medial to the cerebral peduncle in the posterior hypothalamus (Figure 1G and I). Similar to previous studies (Barbier et al., 2020; Chometton et al., 2016; Zséli et al., 2016; Zséli et al., 2018), we found that refeeding following 18 hr food deprivation induced substantial expression of Fos in the PSTN region (Figure 1J and K), suggesting that PSTN neurons play a role in appetite suppression. Therefore, to better characterize these Fos-expressing neurons, we analyzed the cell types that make up the PSTN and explored their roles in food intake behavior. Figure 1 Download asset Open asset PSTN neurons project to anorexigenic PBNCGRP neurons and are activated by refeeding. (A) Modified rabies viral strategy used to identify afferent input to PBNCGRP neurons. (B) Confirmation of modified rabies virus reporter transgenes in PBNCGRP neurons. Experiments repeated in N=6 mice. Scale bar, 250 µm. (C) Sagittal mouse brain diagram showing locations of retrograde eGFP expression in (D–H). (D–H) eGFP-expressing neurons in (D) the bed nucleus of the stria terminalis (BNST); (E) the central nucleus of the amygdala (CeA); (F) the hypothalamic arcuate nucleus (Arc); (G) the parasubthalamic nucleus (PSTN); and (H) the nucleus of the solitary tract (NTS). Scale bar, 250 µm. (I) Coronal mouse brain diagram depicting the bilateral location of the PSTN medial to the cerebral peduncle (cp). Blue arrows point to the location of the PSTN. (J) Immunolabelling of Fos expression following 18 hr food deprivation. Scale bar, 500 µm. (K) Immunolabelling of Fos expression following 18 hr food deprivation followed by 30 min refeeding. Right, higher magnification image. To identify potential genetic markers for PSTN neurons, we consulted the Allen Brain Explorer (http://musebrain-map.org) (Ng et al., 2009) and searched for genes enriched in the PSTN. Additionally, previous studies reported expression of Tac1 (Barbier et al., 2020; Wallén-Mackenzie et al., 2020) and Crh (Zhu et al., 2012) in the PSTN region. Indeed, the Allen Mouse Brain Atlas (Lein et al., 2007) demonstrates expression of Tac1 and Crh within the PSTN region, with expression of Slc17a6 (the gene that encodes VGlut2), Calb1, Calb2, and Pvalb more diffusely located throughout the local area (Figure 2A). Expression of the GABAergic markers Gad1 and Gad2 are notably absent from the PSTN region (Shah et al., 2022). Figure 2 Download asset Open asset Characterization of PSTN cell types. (A) In situ hybridization images of selected mRNA transcripts from the Allen Mouse Brain Expression Atlas. cp, cerebral peduncle. Scale bar, 500 µm. (B) Two-color fluorescent in situ hybridization shows near distinct expression of Tac1 and Crh in PSTN neurons. Right, higher magnification image. Scale bar, 200 µm. (C) Quantification of overlap between PSTNTac1 and PSTNCRH neurons. N=4 mice. (D–K) Three-color fluorescent in situ hybridization comparing spatial overlap between neurons expressing Tac1 and Crh with (D,E) Slc17a6; (F,G) Calb1; (H,I) Calb2; and (J,K) Pvalb. Insets show higher magnification images from middle column. Data at right represent mean ± standard deviation. Dots represent individual experimental animals; N=4 mice. Scale bar, 100 µm. Figure 2—source data 1 This file contains the raw data for the visualization of data presented in Figure 2. https://cdn.elifesciences.org/articles/75470/elife-75470-fig2-data1-v2.xlsx Download elife-75470-fig2-data1-v2.xlsx To quantify the specificity and co-expression of genetic markers in the PSTN, we performed fluorescent in situ hybridization (FISH), initially focusing on Tac1 and Crh due to the apparent specificity of these markers within the PSTN. As expected, Tac1- and Crh-expressing cells were located within the PSTN region (Figure 2B). Interestingly, there was nearly distinct expression of each marker in the PSTN, with 85.1% of labeled PSTN cells expressing Tac1, 14.9% of labeled PSTN cells expressing Crh, and only 1.9% of cells co-expressing both markers (Figure 2C). Therefore, PSTNTac1 and PSTNCRH neurons constitute two nearly distinct cell populations in the PSTN. To determine the co-expression of PSTNTac1 and PSTNCRH neurons with other potential genetic markers, we performed triple-label FISH experiments. Every Tac1- and Crh-expressing cell also expressed Slc17a6, indicating that these populations are glutamatergic (Figure 2D and E). 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. A majority of Tac1- and Crh-expressing cells co-expressed Calb1 and Calb2, although expression of these markers was diffuse and extended medially far beyond the PSTN region (Figure 2F–I). Less than 2% of Tac1 and Crh-expressing cells co-expressed Pvalb (Figure 2J and K). A Pvalb-expressing population appeared dorsomedial to the PSTN, while two other Pvalb populations appeared medially and laterally to the PSTN in the same horizontal plane. Therefore, Tac1 and CRH neurons are specific and discrete glutamatergic subpopulations within the PSTN. PSTN neurons increase activity in response to feeding and anorexigenic hormones To determine whether PSTNTac1 and/or PSTNCRH neurons are active in response to feeding, we first pursued a histological approach using in situ hybridization to measure co-expression of Fos following a large meal. Mice were food deprived for 18 hr; half the animals were then allowed to consume a large meal while the other half remained fasted, and all animals were perfused 45 min later to allow time for Fos mRNA expression dynamics. Animals that were not allowed to eat following the food deprivation period exhibited relatively low Fos expression in the PSTN (Figure 3A). In contrast, animals that were allowed to refeed showed robust co-expression of Fos in both Tac1 and Crh populations (Figure 3B and C; See also Supplementary file 1 for detailed statistical analysis). Although a greater percentage of Crh neurons co-expressed Fos, a significantly higher total number of Tac1 neurons co-expressed Fos (Figure 3D). Taken together, these results demonstrate that refeeding increases activity as measured by Fos expression in both PSTNTac1 and PSTNCRH neurons. Figure 3 Download asset Open asset Refeeding and food consumption activate PSTN neurons. (A–B) Three-color fluorescent in situ hybridization comparing co-expression of Fos in neurons expressing Tac1 or Crh following (A) 18 hr food deprivation or (B) 18 hr food deprivation followed by 30 min refeeding. Insets show higher magnification images from middle column. Scale bar, 200 µm. (C) Quantification of the percentage of Tac1- or Crh-expressing neurons co-expressing Fos in 18 hr food deprivation versus refeeding conditions. N=4 mice. (D) Quantification of the number of Tac1- or Crh-expressing neurons co-expressing Fos following refeeding. N=4 mice. (E) Diagrams showing (left) viral injection strategy to unilaterally target PSTNTac1 neurons with GCaMP6s and (right) subsequent fiber photometry recording in freely moving mice. (F) Representative photomicrograph showing PSTNTac1 neurons expressing GCaMP6s. Scale bar, 100 µm. (G–J) Top, fiber photometry traces in PSTNTac1 neurons following exposure to (G) standard mouse chow, (H) peanut butter, or (I) a novel object from animals food deprived for 18 hr (magenta) or fed ad libitum (grey). (J) Fiber photometry trace in PSTNTac1 neurons following exposure to a water port in water-deprived mice. Data represent the mean ± standard error. Vertical dashed lines depict time of exposure. Bottom, heat maps depicting changes in fluorescence intensity in individual animals. (K) Quantification of maximum values of fluorescence intensity in conditions (G–J). N=6 mice. (L) Quantification of area under the curve of fluorescence intensity among conditions in (G–J). N=6 mice. Data represent mean ± standard error. Dots represent individual experimental animals. Post hoc comparisons: **p<0.01, ***p<0.001, ****p<0.0001. See Supplementary file 1 for additional statistical information. Figure 3—source data 1 This file contains the raw data for the visualization of data presented in Figure 3. https://cdn.elifesciences.org/articles/75470/elife-75470-fig3-data1-v2.xlsx Download elife-75470-fig3-data1-v2.xlsx To determine acute activity patterns in PSTNTac1 or PSTNCRH neurons upon food exposure, we transduced PSTN neurons in Tac1Cre/+ or CrhCre/+ mice with the GCaMP6s calcium indicator and measured real-time fluorescence intensity using fiber photometry (Figure 3E and F). Although we detected post-hoc GCaMP6s fluorescent expression in CrhCre/+ mice, we were not able to record reliable fluorescent signals from these mice following a variety of conditions, potentially due to the relatively low number of CRH neurons in the PSTN. Therefore, we focused on Tac1Cre/+ mice, recording from mice either food deprived for 18 hr or fed ad libitum. Exposure to standard mouse chow or palatable peanut butter produced a significant increase in PSTNTac1 activity in 18 hr food deprived mice compared with exposure to a novel object or exposure to water in 16 hr water deprived mice (Figure 3G–L). This increase in activity persisted for approximately the first 60 s upon exposure to food. These effects were diminished in ad libitum fed mice that were less motivated to consume food. Taken together, these results suggest that PSTNTac1 neurons increase activity during the initial stages of food consumption in hungry mice, but do not respond to water in thirsty mice or other salient stimuli. We next examined the effects of anorexigenic hormone administration on activity in PSTNTac1 and PSTNCRH neurons. To measure neural activity using Fos expression, animals were food deprived for 18 hr to reduce endogenous anorexigenic hormone levels, and then injected intraperitoneally with saline, amylin, cholecystokinin (CCK), or peptide YY (PYY). Administration of these anorexigenic hormones caused an increase in Fos expression in both Tac1 and Crh neurons compared with animals injected with saline (Figure 4A–E), indicating that these anorexigenic hormones cause increased activation of PSTNTac1 and PSTNCRH neurons. To measure neural activity using fiber photometry, Tac1Cre/+ mice transduced with GCaMP6s were implanted with an intraperitoneal catheter (Figure 4F), allowing for real-time measurement of PSTNTac1 activity during hormone administration without the need to inject mice and potentially cause stress by physically handling animals. Administration of amylin, CCK, and PYY caused a significant increase in PSTNTac1 activity compared with administration of saline (Figure 4G–L). Therefore, PSTNTac1 neurons increase activity during food consumption and in response to elevated levels of anorexigenic hormones. Figure 4 Download asset Open asset Anorexigenic hormones cause transient activation of PSTN neurons. (A–D) Three-color fluorescent in situ hybridization comparing co-expression of Fos in neurons expressing Tac1 or Crh following administration of (A) saline, (B) amylin, (C) CCK, or (D) PYY. Insets show higher magnification images from middle column. Scale bar, 200 µm. (E) Quantification of the percentage of Tac1- or Crh-expressing neurons co-expressing Fos across conditions in (A–D). N=4 mice. (F) Diagram showing (left) viral injection strategy to unilaterally target PSTNTac1 neurons with GCaMP6s and (right) subsequent fiber photometry recording in freely moving mice coupled with intraperitoneal infusion of anorexigenic hormones. (G–J) Top, fiber photometry traces in PSTNTac1 neurons following intraperitoneal infusion of (G) saline, (H) amylin, (I) CCK, or (J) PYY. Data represent the mean ± standard error. Vertical dashed lines depict time of injection. Bottom, heat maps depicting changes in fluorescence intensity in individual animals. (K) Quantification of maximum values of fluorescence intensity in conditions (G–J). N=6 mice. (L) Quantification of area under the curve of fluorescence intensity among conditions in (G–J). N=6 mice. Data represent mean ± standard error. Dots represent individual experimental animals. Post hoc comparisons: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Supplementary file 1 for additional statistical information. Figure 4—source data 1 This file contains the raw data for the visualization of data presented in Figure 4. https://cdn.elifesciences.org/articles/75470/elife-75470-fig4-data1-v2.xlsx Download elife-75470-fig4-data1-v2.xlsx Inhibition of PSTNTac1 neurons attenuates the effects of anorexigenic hormones To determine whether activity in PSTNTac1 or PSTNCRH neurons is necessary for normal food intake behavior, we bilaterally injected AAV carrying either Cre-inducible hM4Di-mCherry or mCherry transgenes into the PSTN of Tac1Cre/+ and CrhCre/+ mice (Figure 5A–C). Figure 5 Download asset Open asset Inhibition of PSTNTac1 neurons attenuates the anorexigenic effects of appetite-suppressing hormones. (A) Diagram showing (left) viral injection strategy to unilaterally target PSTNTac1 or PSTNCRH neurons with hM4Di-mCherry or mCherry transgenes and (right) subsequent food intake measurements following administration of CNO and anorexigenic hormones. (B) Representative photomicrograph showing PSTNTac1 neurons expressing hM4Di-mCherry. Scale bar, 100 µm. (C) Representative photomicrograph showing PSTNCRH neurons expressing hM4Di-mCherry. (D–G) Cumulative food intake in Tac1Cre/+ animals administered CNO followed with (D) saline, (E) amylin, (F) CCK, or (G) PYY. (H–J) Quantification of (H) meal size, (I) meal duration, (J) and meal frequency in conditions (D–G). (K–N) Cumulative food intake in CrhCre/+ animals administered CNO followed with (K) saline, (L) amylin, (M) CCK, or (N) PYY. Data represent mean ± standard error. Dots represent individual experimental animals; N=6 mice in all experiments. Post hoc comparisons: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Supplementary file 1 for additional statistical information. Figure 5—source data 1 This file contains the raw data for the visualization of data presented in Figure 5. https://cdn.elifesciences.org/articles/75470/elife-75470-fig5-data1-v2.xlsx Download elife-75470-fig5-data1-v2.xlsx In Tac1Cre/+ mice, there was no difference in food consumption between animals transduced with hM4Di-mCherry and mCherry following intraperitoneal administration of clozapine N-oxide (CNO) and inert saline solution (Figure 5D). Therefore, inhibition of PSTNTac1 neurons is insufficient to affect baseline food intake. Because anorexigenic hormones increased activity in PSTNTac1 neurons (Figure 4), we assessed whether this activity is necessary for their anorexigenic effects. As expected, administration of amylin, CCK, and PYY significantly reduced food intake relative to administration of saline (Figure 5D–G). Intriguingly, co-administration of CNO significantly attenuated these anorexigenic effects in hM4Di-transduced animals (Figure 5E–G), demonstrating the necessity of PSTNTac1 neuronal activity for the full anorexigenic effects of amylin, CCK, and PYY. To better understand how PSTNTac1 neuron inhibition increases food intake in these conditions, we analyzed individual meal parameters in the first 3 hr following injection of CNO. Administration of CNO did not affect meal size or meal duration, but increased meal frequency in hM4Di-mCherry-transduced mice (Figure 5H–J). This analysis suggests that inhibition of PSTNTac1 neurons attenuates the reduction in food intake caused by anorexigenic hormones by increasing the frequency of meals. In contrast to Tac1Cre/+ mice, there were no differences between hM4Di-mCherry and mCherry-transduced CrhCre/+ mice following administration of CNO with saline (Figure 5K) or anorexigenic compounds (Figure 5L–N). Therefore, activity in PSTNTac1 neurons, but not PSTNCRH neurons, is necessary for the suppression of meal frequency caused by amylin, CCK, and PYY. Activation of PSTNTac1 neurons suppresses food intake To determine whether a gain of activity in PSTNTac1 or PSTNCRH neurons is sufficient to suppress feeding, we unilaterally injected AAV carrying either Cre-inducible mCherry or ChR2-mCherry transgenes into the PSTN of Tac1Cre/+ and CrhCre/+ mice for optogenetic stimulation experiments (Figure 6A–C). Mice transduced with ChR2-mCherry in PSTNTac1 neurons consumed significantly less food during the photostimulation period than mice transduced with mCherry (Figure 6D and E). In contrast, photostimulation of PSTNCRH neurons caused no observable effects on food intake (Figure 6F and G). To independently verify these effects and assess the impact of longer term stimulation, we also examined the effects of chemogenetic stimulation of PSTNTac1 or PSTNCRH neurons on food intake behavior by transducing these neurons with mCherry or hM3Dq-mCherry transgenes (Figure 6H–J). Following administration of CNO, mice transduced with hM3Dq-mCherry in PSTNTac1 neurons consumed significantly less food over a 3 hr period than mice transduced with mCherry (Figure 6K). There was no effect of CNO administration between mice transduced with hM3Dq-mCherry or mCherry in PSTNCRH neurons (Figure 6L). Therefore, these gain-of-function experiments suggest that optogenetic or chemogenetic stimulation of PSTNTac1 neurons, but not PSTNCRH neurons, reduces food consumption. Figure 6 Download asset Open asset Stimulation of PSTNTac1 neurons suppresses feeding. (A) Diagram showing (top) viral injection strategy to unilaterally target PSTNTac1 or PSTNCRH neurons with ChR2-mCherry or mCherry transgenes and (bottom) subsequent optogenetic manipulation during food intake recordings. (B) Representative photomicrograph showing PSTNTac1 neurons expressing ChR2-mCherry. Scale bar, 100 µm. (C) Representative photomicrograph showing PSTNCRH neurons expressing ChR2-mCherry. (D–E) Quantification of (D) cumulative food intake and (E) total food consumed during pre-stimulation, stimulation, and post-stimulation of PSTNTac1 neurons. Blue background represents the 60-min photostimulation period. (F–G) Quantification of (F) cumulative food intake and (G) total food consumed during pre-stimulation, stimulation, and post-stimulation of PSTNCRH neurons. Blue background represents the 60-min photostimulation period. (H) Diagram showing (top) viral injection strategy to unilaterally target PSTNTac1 or PSTNCRH neurons with hM3Dq-mCherry or mCherry transgenes and (bottom) subsequent chemogenetic manipulation during food intake recordings. (I) Representative photomicrograph showing PSTNTac1 neurons expressing hM3Dq-mCherry. (J) Representative photomicrograph showing PSTNCRH neurons expressing hM3Dq-mCherry. (K–L) Quantification of cumulative food intake in (K) Tac1Cre/+ or (L) CrhCre/+ mice following administration of CNO. Data represent mean ± standard error. Dots represent individual experimental animals; N=5 mice in all experiments. Post hoc comparisons: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Supplementary file 1 for additional statistical information. Figure 6—source data 1 This file contains the raw data for the visualization of data presented in Figure 6. https://cdn.elifesciences.org/articles/75470/elife-75470-fig6-data1-v2.xlsx Download elife-75470-fig6-data1-v2.xlsx PSTNTac1 and PSTNCRH neurons exhibit different projection patterns The necessity and sufficiency of PSTNTac1 neurons, but not PSTNCRH neurons, in appetite suppression suggests that these neural populations have different projection targets throughout the brain. To identify and distinguish between downstream projections of PSTNTac1 and PSTNCRH neurons, we transduced each population with mCherry and surveyed the brain for anterograde fluorescence (Figure 7). PSTNTac1 neurons uniquely projected to the PVT, PBN, NTS, and intermediate reticular nucleus (IRT), while PSTNCRH neurons uniquely projected to the lateral reticular nucleus (LRN). Both populations projected to the BNST, CeA, medial reticular nucleus (MRN), and tegmental reticular nucleus Figure Download asset Open asset projections from PSTNTac1 and PSTNCRH neurons throughout the brain. (A–B) diagram showing viral injection strategy to unilaterally target (A) PSTNTac1 or (B) PSTNCRH neurons with the mCherry Right, images of mCherry N=4 mice in each Scale bar, 100 µm. Representative and higher magnification images of mCherry expression in PSTNTac1 projections and PSTNCRH projections throughout the brain. Scale 500 µm. (L) Sagittal mouse brain diagram locations of PSTNTac1 and PSTNCRH projections throughout the brain. BNST, bed nucleus of the stria CeA, central nucleus of the intermediate reticular lateral reticular reticular NTS, nucleus of the solitary PBN, parabrachial PVT, paraventricular thalamic tegmental reticular Because stimulation of PSTNTac1 neurons feeding, we the effects of their projections to neuronal populations to regulate feeding behavior. Optogenetic stimulation of projections from PSTNTac1 neurons to the did not cause changes in food intake (Figure In contrast, stimulation of projections from PSTNTac1 neurons to the CeA (Figure PVT (Figure PBN (Figure and (Figure caused decreases in food consumption during the stimulation period. Because it is whether PSTN neurons projections to brain regions, it is that stimulation in a projection target causes activation to or more other target Therefore, these results that PSTNTac1 neurons with projections to the CeA, PVT, PBN, and can suppress food although the functional role of each downstream target region on food intake behavior Figure Download asset Open asset Stimulation of PSTNTac1 neuron projections suppresses feeding. (A) Diagram showing optogenetic strategy to unilaterally target PSTNTac1 neurons with ChR2-mCherry or mCherry transgenes with fiber the (B) Representative photomicrograph showing PSTNTac1 projections expressing ChR2-mCherry in the Quantification of (C) cumulative food intake and (D) total food consumed during pre-stimulation, stimulation, and post-stimulation of PSTNTac1 projections to the (E) Diagram showing optogenetic strategy to unilaterally target PSTNTac1 neurons