Dissociable hindbrain GLP1R circuits for satiety and aversion

May combine two claims.

AP and NTS GLP1R neuron tuning to nutritive and aversive stimuli was not absolute; their physiological responses were mixed.

“while APGLP1R neurons generally had greater responses to aversive stimuli and NTSGLP1R neurons had greater responses to nutritive stimuli, this neural tuning was not absolute”

• AssumptionRests on an assumption: Ensure administered intraduodenally is a nutritive stimulus.
• AssumptionRests on an assumption: Lithium chloride administered intravenously is an aversive or nauseogenic stimulus.

May combine two claims.

Optogenetic activation of AP GLP1R→lPBN neurons, but not NTS GLP1R→PVH neurons, reduced time spent in the stimulated environment in a real-time place avoidance assay.

“Optogenetic activation of APGLP1R→lPBN neurons, but not NTSGLP1R→PVH neurons, significantly reduced time spent in the environment where mice received stimulation”

The quote only partly states this claim.

Among drug-accessible GLP1R populations, only hindbrain DVC GLP1R neurons are necessary for the efficacy of GLP1-based obesity drugs.

“Systematic investigation across drug-accessible GLP1R populations revealed that only hindbrain neurons are required for the efficacy of GLP1-based obesity drugs.”

• Also stated in: § Obesity drugs act on hindbrain GLP1R neurons · boosted

  “demonstrating the necessity of DVCGLP1R neurons for the efficacy of this obesity drug”

• AssumptionRests on an assumption: The necessity of each drug-accessible GLP1R neural population for the anorexic and weight-loss effects of obesity therapeutics had not been systematically tested.

May combine two claims.

Reanalysis of three single-cell/nucleus RNA sequencing datasets showed that AP and NTS GLP1R neurons are mixed excitatory and inhibitory populations, with a greater proportion of NTS GLP1R neurons being excitatory.

“we combined and re-analyzed three existing single cell/nucleus RNA sequencing datasets13,16,17 to determine that APGLP1R and NTSGLP1R neurons are mixed excitatory and inhibitory populations, with a greater proportion of NTSGLP1R neurons being excitatory”

• cites [13] Zhang, C. et al. Area postrema cell types that mediate nausea-associated behaviors. Neuron 109, 461–472 (2021). sourceNot checked yet

  Not checked in the free run.

• cites [16] Ludwig, M. Q. et al. A genetic map of the mouse dorsal vagal complex and its role in obesity. Nat. Metab. 3, 530–545 (2021). sourceNot checked yet

  Not checked in the free run.

• cites [17] Adriaenssens, A. et al. Hypothalamic and brainstem glucose-dependent insulinotropic polypeptide receptor neurons employ distinct mechanisms to affect feeding. 8, https://doi.org/10.1172/jci.insight.164921 (2023). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Optogenetic activation of AP GLP1R→lPBN, but not NTS GLP1R→PVH, projections reduced hedonic and increased aversive real-time taste reactivity.

“optogenetic activation of APGLP1R→lPBN, but not NTSGLP1R→PVH, projections reduced hedonic and increased aversive real-time taste reactivity”

May combine two claims.

More AP GLP1R neurons were activated by aversive cinacalcet, whereas more NTS GLP1R neurons were activated by nutritive Ensure.

“Further, more APGLP1R neurons were activated by the aversive stimulus (cinacalcet), whereas more NTSGLP1R neurons were activated by nutrients (Ensure)”

• AssumptionRests on an assumption: Ensure administered intraduodenally is a nutritive stimulus.
• AssumptionRests on an assumption: Lithium chloride administered intravenously is an aversive or nauseogenic stimulus.

May combine two claims.

Ablation of ARC or NG GLP1R neurons did not block food intake suppression by exendin-4 or semaglutide.

“but not the populations in the ARC or NG, completely blocked food intake suppression by both exendin-4”

May combine two claims.

Semaglutide significantly activated both AP and NTS GLP1R neurons compared with vehicle.

“Both APGLP1R and NTSGLP1R neurons were significantly activated by semaglutide in comparison to vehicle administration”

May combine two claims.

Neither AP nor NTS GLP1R deletion affected exendin-4- or semaglutide-induced anorexia.

“Neither APGLP1R nor NTSGLP1R deletion affected exendin-4 or semaglutide-induced anorexia”

May combine two claims.

AP GLP1R neuron activation, but not NTS GLP1R neuron activation, was sufficient to condition a CFA.

“Similarly, APGLP1R neuron13, but not NTSGLP1R neuron, activation was sufficient to condition a CFA”

May combine two claims.

AAV2.2 EYFP tracing showed axon terminals in the lPBN of AP GLP1R-injected mice and in the PVH of NTS GLP1R-injected mice, with virtually no collateralization.

“This analysis revealed axon terminals in the lPBN of APGLP1R-injected mice, and axon terminals in the PVH of NTSGLP1R-injected mice, with virtually no collateralization”

May combine two claims.

Chemogenetic activation of DVC GLP1R neurons paired with a novel flavor reduced conditioned hedonic responses and increased conditioned aversive taste reactivity.

“After pairing, mice displayed fewer conditioned hedonic responses and increased conditioned aversive responses compared to control mice”

May combine two claims.

Only activation of AP GLP1R neurons, not NTS GLP1R neurons, reduced hedonic and increased aversive taste reactivity.

“Strikingly, only activation of APGLP1R neurons, and not NTSGLP1R neurons, reduced hedonic and increased aversive taste reactivity responses”

May combine two claims.

Activation of NTS GLP1R neurons increased inter-meal interval but did not change meal size, indicating increased satiety.

“Activation of NTSGLP1R neurons increased inter-meal interval (Fig. 3i) but had no effect on meal size (Extended Data Fig. 8g), indicating that NTSGLP1R neurons increase satiety.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Alternative motion correction methods did not change the calcium imaging results or conclusions.

“While these corrections did not influence our results or conclusions, they may be necessary for those analyzing frames with substantial uniform or non-uniform distortions, especially for preparations with ROIs along image edges.”

May combine two claims.

DVC GLP1R neurons form at least two subpopulations that are activated by either nutritive or aversive stimuli and are much less likely to be activated by both.

“we also demonstrate that DVCGLP1R neurons form at least two subpopulations that are activated by either nutritive or aversive stimuli (and are much less likely to be activated by both).”

• AssumptionRests on an assumption: Ensure administered intraduodenally is a nutritive stimulus.
• AssumptionRests on an assumption: Lithium chloride administered intravenously is an aversive or nauseogenic stimulus.

May combine two claims. The quote only partly states this claim.

Exendin-4 reduced food intake in fasted control, ARC-ablated, and NG-ablated mice, but not in DVC-ablated mice.

“(d) Food intake following IP injection of saline or exendin-4 in fasted control or GLP1R-ablated mice (n=8–14 mice/group, two-way repeated measures ANOVAs, all ps<0.01 except DVC where p=ns).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Dark period energy expenditure normalized to lean mass did not significantly differ between high-fat diet-fed control mice and mice with chronic NaChBac-mediated DVCGLP1R neuron activation (p=0.06).

“(f) Dark period energy expenditure normalized to lean mass by ANCOVA in high-fat diet-fed control mice or mice with chronic NaChBac-mediated DVCGLP1R neuron activation (n=7–8 mice/group, ANCOVA, p=0.06).”

May combine two claims.

GLP1R neurons in the dorsal vagal complex project densely to the NTS but not to the AP.

“Images depict the dense GLP1R projection into the NTS but not into the AP.”

May combine two claims.

Ensure administration increased average z-scored activity in both AP and NTS GLP1R neurons relative to saline.

“Average z-scored activity responses in APGLP1R neurons (blue) and NTSGLP1R neurons (green) to saline or Ensure administration (n=6 mice, two-way ANOVA, AP: saline vs Ensure: p<0.01, NTS: saline vs. Ensure p<0.001).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Quinine concentration significantly affected composite aversive taste reactivity scores and individual aversive behaviours.

“Composite score (e, n=5–10 mice/group, one-way ANOVA, all ps<0.01) and individual behaviours (f, 5–10 mice/group, two-way ANOVA, all ps<0.05) for aversive taste reactivity responses to quinine concentrations (0, 2.4, 4 mM).”

May combine two claims.

Chemogenetic activation of AP GLP1R neurons changed hedonic taste reactivity behaviours after conditioning, while activation of NTS GLP1R neurons did not.

“Individual hedonic taste reactivity behaviours at baseline and after conditioning with chemogenetic APGLP1R (l, n=8 mice, two-way ANOVA, RMM baseline vs. conditioned p<0.001) or NTSGLP1R (m, n=5 mice, two-way ANOVA, all ps=ns) neuron activation.”

May combine two claims.

Chemogenetic activation of AP GLP1R neurons reduced meal size in fasted mice, whereas activation of NTS GLP1R neurons did not.

“Meal size in fasted mice following chemogenetic activation of APGLP1R or NTSGLP1R neurons (n=10–11 mice/group, two-way ANOVA, APGLP1R-hM3Dq vehicle vs. CNO: p<0.001, NTSGLP1R-hM3Dq vehicle vs. CNO: p=ns).”

May combine two claims.

Semaglutide induced conditioned flavour avoidance in WT mice but not in DVC GLP1R-deleted mice.

“Intake of flavour paired with semaglutide (CS+) in control (WT) or DVC GLP1R-deleted (Glp1rfl/fl) mice before (baseline) and after conditioning (n=6–8 mice/group, two-way ANOVA, WT baseline vs. conditioned: p<0.001, Glp1rfl/fl baseline vs. conditioned: p=ns).”

May combine two claims.

Chemogenetic activation of AP GLP1R neurons changed aversive taste reactivity behaviours after conditioning, while activation of NTS GLP1R neurons did not.

“Individual aversive taste reactivity behaviours at baseline and after conditioning with chemogenetic APGLP1R (n, n=8 mice, two-way ANOVA, CR baseline vs. conditioned: p<0.001) or NTSGLP1R (o, n=5 mice, two-way ANOVA, all ps=ns) neuron activation.”

May combine two claims.

Optogenetic stimulation of APGLP1R→lPBN, but not NTSGLP1R→PVH, altered aversive taste reactivity behaviours.

“Individual aversive taste reactivity behaviours at baseline (pre-stimulation) and during optogenetic stimulation of APGLP1R→lPBN (m, n=6 mice, two-way ANOVA, CR no stim vs. stim: p<0.001) or NTSGLP1R→PVH (n, n=6 mice, two-way ANOVA, all ps=ns) neurons.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Quinine concentration significantly affected composite hedonic taste reactivity scores and individual hedonic behaviours.

“Composite score (c, n=5–10 mice/group, one-way ANOVA, all ps<0.05) and individual behaviours (d, n=5–10 mice/group, two-way ANOVA, all ps<0.001) for hedonic taste reactivity responses to quinine concentrations (0, 2.4, 4 mM).”

May combine two claims.

Optogenetic activation of APGLP1R→lPBN or NTSGLP1R→PVH neurons did not affect 1-h food intake in control mice expressing EYFP.

“Food intake (1 h) in control mice expressing EYFP in APGLP1R→lPBN (a, n=6 mice, paired t-test, p=ns) and NTSGLP1R→PVH (b, n=5 mice, two-sided paired t-test, p=ns) neurons.”

May combine two claims.

Optogenetic activation of APGLP1R→lPBN or NTSGLP1R→PVH did not induce real-time place avoidance in fasted mice expressing ChR2.

“Time spent in stimulation zone during real-time place avoidance assay in fasted mice expressing ChR2 in APGLP1R→lPBN (i, n=11 mice, two-way ANOVA, all ps=ns) or NTSGLP1R→PVH (j, n=7 mice, two-way ANOVA, all ps=ns) neurons.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The mean time to semaglutide-induced activation did not differ between AP and NTS GLP1R neurons.

“Mean time to semaglutide-induced activation in APGLP1R and NTSGLP1R neurons depicted in h (n=5 mice, two-sided unpaired t-test, p=ns).”

May combine two claims.

Optogenetic activation of APGLP1R→lPBN, but not NTSGLP1R→PVH, induced real-time place avoidance in ad libitum-fed mice expressing ChR2.

“Time spent in stimulation zone during real-time place avoidance assay in ad libitum-fed mice expressing ChR2 in APGLP1R→lPBN (g, n=10–11 mice/group, two-way ANOVA, Trials 2&3 no stim vs. stim: ps<0.05) or NTSGLP1R→PVH (h, n=7 mice, two-way ANOVA, all ps=ns).”

May combine two claims.

The number of AP GLP1R neurons expressing hM3Dq correlates with avoidance behaviour, while the number of NTS GLP1R neurons expressing hM3Dq does not.

“Number of APGLP1R, but not NTSGLP1R, neurons expressing chemogenetic receptors correlates with avoidance behaviour.”

May combine two claims.

In ad libitum-fed control mice expressing EYFP, optogenetic stimulation of APGLP1R→lPBN or NTSGLP1R→PVH did not affect time spent in the stimulation zone during a real-time place avoidance assay.

“Time spent in stimulation zone during real-time place avoidance assay in ad libitum-fed control mice expressing EYFP in APGLP1R→lPBN (c, n=5–6 mice/group, two-way ANOVA, all ps=ns) or NTSGLP1R→PVH (d, n=5 mice, two-way ANOVA, all ps=ns) neurons.”

May combine two claims.

AP and NTS GLP1R neurons are generally distinct from other hindbrain cell types and mediate aversion and satiety, respectively.

“Together with physiological and behavioural results, these data indicate that APGLP1R and NTSGLP1R neurons are generally distinct from other hindbrain cell types and mediate aversion and satiety responses, respectively.”

May combine two claims.

The NTS GLP1R→PVH circuit suppresses feeding without inducing aversion.

“highlighting a NTSGLP1R→PVH circuit that suppresses feeding without inducing aversion.”

May combine two claims.

DVC GLP1R neuron activation suppresses food intake through increased satiety, indicated by longer inter-meal intervals and unchanged meal size, rather than through changes in energy expenditure.

“These effects were mediated by suppression of food intake due to increased satiety – indicated by increased inter-meal interval and no change in meal size”

• Also stated in: § Obesity drugs act on hindbrain GLP1R neurons · plain

  “and not by changes in energy expenditure”

May combine two claims.

Two anatomically and functionally distinct GLP1R projections arising from the AP and NTS drive aversion and satiety, respectively.

“reveal two anatomically and functionally distinct GLP1R projections – arising from the AP and the NTS – that drive aversion and satiety, respectively.”

May combine two claims.

GLP1R agonist obesity drugs produce aversive effects such as nausea and vomiting, and these effects may contribute to their therapeutic efficacy.

“The most successful obesity therapeutics – glucagon-like peptide-1 receptor (GLP1R) agonists – cause aversive responses such as nausea and vomiting1,2, effects which may contribute to their efficacy.”

• cites [1] Wadden, T. A. et al. Effect of subcutaneous semaglutide vs placebo as an adjunct to intensive behavioral therapy on body weight in adults with overweight or obesity: The STEP 3 Randomized Clinical Trial. JAMA 325, 1403–1413 (2021). sourceNot checked yet

  Not checked in the free run.

• cites [2] Wilding, J. P. H. et al. Once-weekly semaglutide in adults with overweight or obesity. N. Engl. J. Med. 384, 989–1002 (2021). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

AP GLP1R and NTS GLP1R neurons send projections to largely different downstream brain regions and cell types, potentially mediating distinct anorexic behaviors.

“Therefore, APGLP1R and NTSGLP1R neurons send projections to largely different downstream brain regions and cell types to potentially mediate distinct anorexic behaviours.”

May combine two claims.

lPBN CGRP neurons suppress food intake and drive aversion.

“lPBNCGRP neurons suppress food intake and drive aversion28–30”

• cites [28] Carter, M. E., Han, S. & Palmiter, R. D. Parabrachial calcitonin gene-related peptide neurons mediate conditioned taste aversion. J. Neurosci. 35, 4582–4586 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [29] Campos, C. A., Bowen, A. J., Schwartz, M. W. & Palmiter, R. D. Parabrachial CGRP neurons control meal termination. Cell Metab 23, 811–820 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [30] Carter, M. E., Soden, M. E., Zweifel, L. S. & Palmiter, R. D. Genetic identification of a neural circuit that suppresses appetite. Nature 503, 111–114 (2013). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

NTS GLP1R neurons inhibit food intake and body weight without inducing aversion.

“These data highlight NTSGLP1R neurons as a population that inhibits food intake and body weight without inducing aversion.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Investigators collecting or analyzing data were blinded to experimental conditions.

“Investigators collecting (e.g, performing behavioural measurements) or analyzing (e.g., counting neurons) data were blinded to experimental conditions.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Mice showing excessive food grinding were excluded from all analyses.

“Mice with excessive food grinding behaviours (<5% of all mice tested) were excluded from all analyses.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The adverse gastrointestinal side effects of GLP1-based obesity drugs are more severe at the start of treatment and tend to decrease over time.

“Clinically, the adverse gastrointestinal side effects of GLP1-based obesity drugs are more severe at the start of treatment and tend to decrease over time54.”

• cites [54] Shu, Y. et al. Gastrointestinal adverse events associated with semaglutide: a pharmacovigilance study based on FDA adverse event reporting system. Front. Public Health 10, 996179 (2022). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based).

Before calcium activity analysis, AP and NTS boundaries were drawn using anatomical landmarks to prevent bias.

“Prior to analysis of calcium activity (to prevent any bias), boundaries between the AP and NTS were drawn for each z plane using (1) the differential density of GLP1R neurons in AP vs. NTS which creates a natural boundary”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Because nausea and vomiting are side effects of many treatments, the concept of dissociable circuits could be applied to investigate other therapeutics.

“Because nausea and vomiting are side effects of thousands of treatments for human disease60, this concept could be applied to investigate other therapeutics”

• cites [60] Wickham, R. J. Nausea and vomiting: a palliative care imperative. Curr. Oncol. Rep. 22, 1 (2020). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Mice that did not eat during the 4-6 h post-CNO period were excluded from inter-meal interval analyses.

“Mice that did not eat in this time period (n=2 across all experiments) were excluded from inter-meal-interval analyses.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

NTS serotonin 2C receptors are both necessary and sufficient for the anorexic effects of lorcaserin.

“NTS serotonin 2C receptors (5HT2CR) are both necessary and sufficient for the anorexic effects of a previously-used obesity drug, lorcaserin47.”

• cites [47] D’Agostino, G. et al. Nucleus of the solitary tract serotonin 5-HT2C receptors modulate food intake. Cell Metab. 28, 619–630 (2018). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Technical constraints, including imaging under anesthesia and applying only one trial each of nutritive and aversive stimuli within the same imaging session, limit interpretations of the results.

“We also note that technical constraints, such as imaging under anesthesia and applying only one trial each of nutritive and aversive stimuli within the same imaging session, limit interpretations of results.”

Classified as probably not a substantial claim.

DTA has previously been used to ablate nodose ganglion neurons.

“because DTA has been previously used to ablate NG neurons67”

• cites [67] Ghidewon, M. et al. Growth differentiation factor 15 (GDF15) and semaglutide inhibit food intake and body weight through largely distinct, additive mechanisms. Diabetes Obes. Metab. 24, 1010–1020 (2022). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

There were no significant body weight differences between groups at baseline.

“Although there were no significant body weight differences between groups at baseline”

May combine two claims.

NTS CCK neurons project to the lPBN and PVH; NTS CCK→lPBN projections cause negative valence, whereas NTS CCK→PVH projections do not.

“NTSCCK neurons also project to the lPBN and PVH, and NTSCCK→lPBN projections cause negative valence but NTSCCK→PVH projections do not49–51.”

• cites [49] D’Agostino, G. et al. Appetite controlled by a cholecystokinin nucleus of the solitary tract to hypothalamus neurocircuit., eLife 5, e12225 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [50] Roman, C. W., Sloat, S. R. & Palmiter, R. D. A tale of two circuits: CCKNTS neuron stimulation controls appetite and induces opposing motivational states by projections to distinct brain regions. Neuroscience 358, 316–324 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [51] Cheng, W. et al. Calcitonin receptor neurons in the mouse nucleus tractus solitarius control energy balance via the non-aversive suppression of feeding. Cell Metab. 31, 301–312 (2020). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Fluorescently tagged semaglutide binds in both the AP and the NTS.

“Although fluorescently-tagged semaglutide binds in both the AP and the NTS10”

• cites [10] Gabery, S. et al. Semaglutide lowers body weight in rodents via distributed neural pathways. JCI Insight 5, https://doi.org/10.1172/jci.insight.133429 (2020). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Both Casp3 and DTA methods effectively ablated the targeted GLP1R neurons.

“We verified that both techniques (Casp3 and DTA) effectively ablated our neurons of interest (Extended Data Fig. 1b, c).”

Probably not a substantial claim: may not pass S3 (load-bearing).

All sample sizes reported in the manuscript refer to biological replicates.

“All ns reported throughout the manuscript are biological replicates.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

Exclusions from data were limited to mice with significant spillage, unexpected illness or euthanasia, and no food intake for meal pattern analyses.

“There were no exclusions from the data except for the following: For food intake measurements, mice with significant spillage were excluded from analysis”

May combine two claims.

No significant sex differences were observed across all studies except one experiment, so results for males and females were combined.

“Except for one experiment (Extended Data Fig. 1f) which had a smaller but still significant effect in female compared to male mice, we did not observe any significant sex differences across all studies and therefore we combined results for males and females.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

GLP1R signaling reduces motivational aspects of feeding and other behaviors such as alcohol and drug intake.

“given the role of GLP1R signaling in reducing motivational aspects of feeding and other behaviours such as alcohol and drug intake38,57–59”

• cites [38] Alhadeff, A. L. & Grill, H. J. Hindbrain nucleus tractus solitarius glucagon-like peptide-1 receptor signaling reduces appetitive and motivational aspects of feeding. Am. J. Physiol. Regul. Integr. Comp. Physiol. 307, R465–R470 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [57] Egecioglu, E. et al. The glucagon-like peptide 1 analogue Exendin-4 attenuates alcohol mediated behaviors in rodents. Psychoneuroendocrinology 38, 1259–1270 (2013). sourceNot checked yet

  Not checked in the free run.

• cites [58] Tuesta, L. M. et al. GLP-1 acts on habenular avoidance circuits to control nicotine intake. Nat. Neurosci. 20, 708–716 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [59] Schmidt, H. D. et al. Glucagon-like peptide-1 receptor activation in the ventral tegmental area decreases the reinforcing efficacy of cocaine. Neuropsychopharmacology 41, 1917–1928 (2016). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Mice lacking viral expression or with expression outside the target region were excluded from analyses after post-mortem verification.

“Where appropriate, viral expression was verified post-mortem; any mouse that did not express virus or expressed virus outside of the target region was excluded from analyses.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For between-subject analyses, mice were randomly assigned to experimental condition.

“For between-subject analyses, mice were randomly assigned to experimental condition.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Power analyses were run to ensure that sample sizes were sufficient to determine significant differences between groups.

“Power analyses were run [beta = 0.2 (80% power), alpha =0.05] with effect sizes based on pilot studies (n=3–6) to ensure that sample sizes (number of mice for behavioural studies, number of neurons for physiological/anatomical studies) were sufficient to determine significant differences between groups.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Alternative motion correction strategies did not significantly change the proportions of activated neurons or individual calcium traces in this imaging setup.

“Although these alternate motion correction strategies did not significantly change our data, we note for readers that some imaging preparations (especially those that have more ROIs around the edges of frames) may require such strategies.”

May combine two claims.

Individual AP and NTS GLP1R neurons are specialized, with most biased toward nutritive or aversive stimuli and less overlap than expected by chance.

“That the probability of a cell being activated by both stimuli is generally lower than the product of individual probabilities suggests a level of specialization of individual cells where most are biased toward nutritive or aversive stimuli, with less overlap than would be expected by chance.”