An excitatory ventromedial hypothalamus to paraventricular thalamus circuit that suppresses food intake

It is well recognized that ventromedial hypothalamus (VMH) serves as a satiety center in the brain. However, the feeding circuit for the VMH regulation of food intake remains to be defined. Here, we combine fiber photometry, chemo/optogenetics, virus-assisted retrograde tracing, ChR2-assisted circuit mapping and behavioral assays to show that selective activation of VMH neurons expressing steroidogenic factor 1 (SF1) rapidly inhibits food intake, VMH SF1 neurons project dense fibers to the paraventricular thalamus (PVT), selective chemo/optogenetic stimulation of the PVT-projecting SF1 neurons or their projections to the PVT inhibits food intake, and chemical genetic inactivation of PVT neurons diminishes SF1 neural inhibition of feeding. We also find that activation of SF1 neurons or their projections to the PVT elicits a flavor aversive effect, and selective optogenetic stimulation of ChR2-expressing SF1 projections to the PVT elicits direct excitatory postsynaptic currents. Together, our data reveal a neural circuit from VMH to PVT that inhibits food intake.

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.
Nov 09, 2020 Fiber photometry data were collected using a two-channel dual-wavelength FP system (Doric Lenses); The images were taken using a Leica SP8 Confocal microscopy and/or an inverted microscopy (Olympus IX51); ChR2-assisted circuit mapping was performed by using an electrophysiology rig (Molecular Devices, MultiClamp 700B) quipped with photostimulation (CrystLaser 473 nm); Indirect calorimetry was performed using the Columbus Labs Comprehensive Lab Animal Monitoring Systems (CLAMS); Transgenic mice were genotyped using the Gel Documentation Systems (Corning). ANY-maze behavior tracking software 5.1 (Stoelting) was used to perform and analyze open field and plus maze behavioral tests.
All data were analyzed using Prism 7.0 (GraphPad Software). Student's t-tests were used to analyze differences between two groups of the same or different mice when appropriate, respectively. One-way ANOVA with post hoc test was used to compare group data from more than two groups of mice. Repeated measures (RM) two-way ANOVA with the within-subject factors of time segment and treatment (vehicle vs J60; PS off vs PS on) or mixed ANOVA with the within-subject factor of time segment and the between-subjects factor of viral injections type (control fluorescent proteins vs. hM3Dq) were used to analyze data from more than two groups across various time points. Sidak's post hoc test was used to test from significant effects at various time segments following the detection of a significant effects main effect or interaction. Fiber photometry data were analyzed using a software (Doric Studio V5.3.3.14). Electrophysiology data were analyzed using a software (pClampfit 10.7, Molecular Devices) as stated in the Method section. All experiments were repeated at least twice for each mouse and average values were calculated for each individual mouse for statistical analysis. All attempts at replication were successful.
Male and female mice (half and half for each group unless where noted in the text and figure legends) were randomly assigned to experimental (DREADDs; ChR2) or control groups (control fluorescent protein) before viral injections and animal behavioral experiments.
All behavioral experiments were performed and analyzed by an individual blind to the identification of pharmacological reagents (i.e. J60 vs vehicle) or vectors (ChR2 vs mCherry). Animal genotype was not performed in a blind manner as we need to screen transgenic-positive mice (e.g. Cre-expression mice).

Validation
The Fos antibody is reactive to mouse tissue, and has been cited in 63 publications, including Li R et al 2018; Xue Y et al 2019, which used this antibody in mouse brain tissues. In this study we used this antibody to stain Fos signals in the extended data Figure 3. We used the vGluT2 antibody to stain vGluT2 in the Figure 4, the detailed information of this antibody is provided on the manusfacture's website (https://www.thermofisher.com/antibody/product/VGLUT2-Antibody-clone-S29-29-Monoclonal/ MA5-27613). The secondary goat-anti-mouse antibody Alexa 680 has been cited in 51 publications, including Wang P et al 2019.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals

Wild animals
Field-collected samples

Ethics oversight
Male wild-type C57BL/6J (Jax Stock No: 000664), SFl-Cre (Jax Stock No: 012462), and vGluT2-ires-Cre (Jax Stock No: 016963) have been described previously and purchased from The Jackson Laboratory. Transgenic mice were genotyped for Cre. Both male and female mice (age 5-8 weeks) were used at the start of experiments, unless otherwise noted. Mice were group-housed 3-5 mice per cage in temperature -and humidity-controlled rooms on a 12-h light:12-h dark cycle, with lights on from 8:00 a.m. to 8:00 p.m., and with ad libitum access to water and mouse regular chow (Picolab Rodent Diet 20, 5058, LabDiet). Mice were single-caged after they received viral transductions with or without guide cannula insertion until all experimental procedures were finished. The virally transduced mice were randomly assigned to experimental and control groups at the start of experiments. Mice were also randomly assigned and evenly age and sex-matched for the different viral injections and treatments as described in the text and figure legends.
No wild animals were used in this study.
No field-collected samples were used in this study.
Experimental protocols were approved by the Institutional Animal Care and Use Committees at the Albert Einstein College of Medicine and conducted following the US National Institutes of Health guidelines for animal research.
Note that full information on the approval of the study protocol must also be provided in the manuscript.