High-fat diet disrupts a septal control on feeding to promote obesity in male mice methods
Aim. Evidence-backed execution summary for High-fat diet disrupts a septal control on feeding to promote obesity in male mice methods from High-fat diet disrupts a septal control on feeding to promote obesity in male mice.
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mouse
Subject model for the experiment.
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Downregulation of Hcn1 contributes to hypoexcitability of septal neurons and exacerbates HFD-induced obesity
reagent used in the protocol.
- Use
- To evaluate the impact of Hcn1 downregulation on the neuronal excitability of LS GABA neurons and the contribution to HFD-induced obesity, we used short hairpin RNA (shRNA) to knockdown Hcn1 in LS GABA neurons (Fig. ). Immunostaining confirmed effective reduction of Hcn1 protein expression following shRNA expr...
Overexpression of Hcn1 restores LS neuronal excitability and prevents HFD-induced obesity
reagent used in the protocol.
- Use
- Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig. ). Immunofluorescence confirmed successful Hcn1 overexpression following this viral strategy (Fig. ). Overexpression of Hcn1 led to...
Downregulation of Gad2 in the LS contributes to HFD-induced obesity
reagent used in the protocol.
- Use
- Beyond the observed reductions in neuronal excitability, our snRNA-seq data also revealed downregulation of synaptic transmission and neurotransmitter secretion in LS GABA neurons following HFD exposure, suggesting potential alterations in the GABA synthesis or releasing. To investigate this hypothesis, we checked o...
Downregulation of Gad2 in the LS contributes to HFD-induced obesity
reagent used in the protocol.
- Use
- To explore whether restoring GABA levels in the LS could rescue HFD-induced obesity, AAV-DIO-Gad2-EGFP was bilaterally injected into LS of Gad2-Cre mice for LS GABA neurons Gad2 overexpression (Fig. ). Western blot and LC-MS analyses showed a significant increase in Gad2 and GABA protein in LS (Fig. ). I...
Single-nuclei RNA seq and data analysis
reagent used in the protocol.
- Use
- The mice were anesthetized with isoflurane, and the brain tissue from the septal area was extracted. All tissue harvested were transferred to -80 °C freezer for storage. Subsequently, the samples were further processed and subjected to single-nuclei RNA sequencing by OE Biotech Co., Ltd. (Shanghai,...
Electrophysiological recordings
reagent used in the protocol.
- Use
- Procedures for preparing acute brain slices were similar to those described previously. Mice were anesthetized with isoflurane. Under sterile conditions, we perfused the mice with 4 °C slicing solution containing (in mM) 110 choline chloride, 2.5 KCl, 0.5 CaCl 2, 7 MgCl 2, 1.3 NaH 2 PO 4, 1.3 Na-ascor...
LC-MS
reagent used in the protocol.
- Use
- Mice were anesthetized with isoflurane, and septal area were rapidly dissected at a 4 °C environment. For the LC-MS analytical procedure, samples (8-13 mg) were pulverized in a glass container following the addition of 200 µl of ice-cold methanol containing 0.1% formic acid and 100&...
Virus-mediated neural tracing
reagent used in the protocol.
- Use
- To trace the downstream projections of LS GABA neurons, AAV2/9-hSyn-FLEX-tdTomato-T2A-synaptophysin-EGFP-WPRE-pA was injected into the LS. Brain sections from these mice were obtained following the procedures described above. For immunostaining, brain sections were rinsed with PBS (3 × 10 min) and then bl...
Single-nuclei RNA seq and data analysis
The mice were anesthetized with isoflurane, and the brain tissue from the septal area was extracted. All tissue harvested were transferred to -80 °C freezer for storage. Subsequently, the samples were further processed and subjected to single-nuclei RNA sequencing by OE Biotech Co., Ltd. (Shanghai,...
- Use
- The mice were anesthetized with isoflurane, and the brain tissue from the septal area was extracted. All tissue harvested were transferred to -80 °C freezer for storage. Subsequently, the samples were further processed and subjected to single-nuclei RNA sequencing by OE Biotech Co., Ltd. (Shanghai,...
Electrophysiological recordings
Procedures for preparing acute brain slices were similar to those described previously. Mice were anesthetized with isoflurane. Under sterile conditions, we perfused the mice with 4 °C slicing solution containing (in mM) 110 choline chloride, 2.5 KCl, 0.5 CaCl 2, 7 MgCl 2, 1.3 NaH 2 PO 4, 1.3 Na-ascor...
- Use
- Procedures for preparing acute brain slices were similar to those described previously. Mice were anesthetized with isoflurane. Under sterile conditions, we perfused the mice with 4 °C slicing solution containing (in mM) 110 choline chloride, 2.5 KCl, 0.5 CaCl 2, 7 MgCl 2, 1.3 NaH 2 PO 4, 1.3 Na-ascor...
LC-MS
Mice were anesthetized with isoflurane, and septal area were rapidly dissected at a 4 °C environment. For the LC-MS analytical procedure, samples (8-13 mg) were pulverized in a glass container following the addition of 200 µl of ice-cold methanol containing 0.1% formic acid and 100&...
- Use
- Mice were anesthetized with isoflurane, and septal area were rapidly dissected at a 4 °C environment. For the LC-MS analytical procedure, samples (8-13 mg) were pulverized in a glass container following the addition of 200 µl of ice-cold methanol containing 0.1% formic acid and 100&...
Single-cell calcium imaging
Population decoding analysis was performed using a linear support vector machine (SVM) classifier in MATLAB (via the 'fitcsvm' function) to assess whether trial types (chow vs. HFD consumption) could be predicted from trial-by-trial population activities of LS GABA neurons during consumption epochs. For...
- Use
- Population decoding analysis was performed using a linear support vector machine (SVM) classifier in MATLAB (via the 'fitcsvm' function) to assess whether trial types (chow vs. HFD consumption) could be predicted from trial-by-trial population activities of LS GABA neurons during consumption epochs. For...
Statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data were processed and analyzed using MATLAB, R and GraphPad Prism 9.1.0. Three-way ANOVA followed by post hoc test using the two-stage step-up method of Benjamini, Krieger and Yekutieli to compare more than two experimental groups with time, diet and virus variables. Two-way ANOVA followed by the post hoc Sidak...
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Results
To investigate how HFD-induced obesity modulates the transcriptional profiles of cells within the septal region, adult mice were assigned to either a HFD or control chow diet. Both groups had ad libitum access to food. Mice on the HFD exhibited significantly higher energy intake compared to those on the control diet, resulting in a significant increase in bodyweight that surpassed that of control mice as early as the first week (Fig. ). The caloric intake continued to increase, leading to the development of an obesity phenotype (Fig. ). After a 4-week dietary regimen, septal tissues were harvested for single-nucleus RNA sequencing (snRNA-seq) (Fig. ). Fig. 1 The HFD alters the transcriptional profiles of septal area neurons. a Left: quantification of daily energy intake for the normal food and high-fat diet groups (diet: F 1,60 = 157.2; p <...
Overexpression of Hcn1 restores LS neuronal excitability and prevents HFD-induced obesity
Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig. ). Immunofluorescence confirmed successful Hcn1 overexpression following this viral strategy (Fig. ). Overexpression of Hcn1 led to a significantly higher voltage sag amplitude and enhanced excitability of LS GABA neurons (Fig. ). In the liquid food intake assays, overexpression of Hcn1 did not affect the standard liquid food intake, but significantly decreased the lick numbers and total intake of palatable sucrose solution and Ensure (Fig. ). A separate cohort of naïve Gad2-Cre mice were used to test whether Hcn1 overexpression in LS GABA neurons could prevent HFD-induced overeating and obesity. We found mice with Hcn1 overexpression resisted excessive energy intake on an HFD, maintain...
Downregulation of Gad2 in the LS contributes to HFD-induced obesity
To explore whether restoring GABA levels in the LS could rescue HFD-induced obesity, AAV-DIO-Gad2-EGFP was bilaterally injected into LS of Gad2-Cre mice for LS GABA neurons Gad2 overexpression (Fig. ). Western blot and LC-MS analyses showed a significant increase in Gad2 and GABA protein in LS (Fig. ). In the liquid food intake assays, similar to overexpression of Hcn1, overexpression of Gad2 in LS GABA neurons did not affect the standard liquid food intake, but significantly decreased the lick numbers and total intake of sucrose solution and Ensure (Fig. ). In a distinct cohort of mice, 3 weeks after virus injection, the mice were switched to a 4-week HFD or chow diet. The results demonstrated that Gad2 overexpression in LS significantly reduced HFD intake and prevented obesity (Fig. and Supplementary Fig. ). Overexpression of Gad2 did not affect chow in...
Methods
In this study, adult male C57BL/6 J mice were obtained from the Guangdong Medical Laboratory Animal Center, Guangzhou, China. Additionally, the experimental cohorts included Gad2-Cre (male, Jax No. 010802) mice. All mice were housed in a temperature (22-25 °C) and humidity (55-65%) controlled environment, with ad libitum access to food and water, except during experimental sessions. A 12-hour light-dark cycle was maintained (lights on from 7:00 am to 7:00 pm). All efforts were made to minimize animal suffering and the number of animals used. A total of 338 mice were used in the different experiments. All experiments were conducted in accordance with relevant guidelines and regulations, and approved by the IACUC (Institutional Animal Care and Use Committee) of SIAT, Chinese Academy of Sciences (CAS, SIAT-IACUC-230927).
Electrophysiological recordings
Procedures for preparing acute brain slices were similar to those described previously. Mice were anesthetized with isoflurane. Under sterile conditions, we perfused the mice with 4 °C slicing solution containing (in mM) 110 choline chloride, 2.5 KCl, 0.5 CaCl 2, 7 MgCl 2, 1.3 NaH 2 PO 4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO 3 ), then placed the tissue in 4 °C slicing solution saturated with 95% O 2 and 5% CO 2. Coronal slices (250-300 µm) containing the LS, LH or TN were prepared using a vibratome (Leica, VT-1000S). Slices were incubated in 37 °C oxygenated artificial cerebrospinal fluid (in mM: 125 NaCl, 2.5 KCl, 2 CaCl 2, 1.3 MgCl 2, 1.3NaH 2 PO 4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO 3 ) for at least 30 min for recovery. Then the slices were transferred to a recording cham...
Virus-mediated neural tracing
To trace the downstream projections of LS GABA neurons, AAV2/9-hSyn-FLEX-tdTomato-T2A-synaptophysin-EGFP-WPRE-pA was injected into the LS. Brain sections from these mice were obtained following the procedures described above. For immunostaining, brain sections were rinsed with PBS (3 × 10 min) and then blocked with 10% normal goat serum and 0.3% TritonX-100 in PBS for 2 hr at room temperature. Next, sections were incubated with primary antibody (Rabbit anti-GFP, 1:1000, ThermoFisher, Cat# A-11122) diluted in 10% normal goat serum and 0.3% TritonX-100 in PBS for 24-48 hr at 4 °C. After washing with PBS (3 × 10 min), sections were incubated with secondary antibody (Goat anti-Rabbit 488, 1:1000, ThermoFisher, Cat# A-11008) diluted in 10% normal goat serum and 0.3% Triton X-100 in PBS at room temperature for 2 hr and the...
Measurement outputs
What raw and processed outputs should exist?
The expression of Gad2 in HFD group relative to the control group was calculated by the ∆∆CT method using 18sRNA as the reference gene. Data were natural log-transfo...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
To evaluate the impact of Hcn1 downregulation on the neuronal excitability of LS GABA neurons and the contribution to HFD-induced obesity, we used short hairpin RNA (shRNA) to k...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig....
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Beyond the observed reductions in neuronal excitability, our snRNA-seq data also revealed downregulation of synaptic transmission and neurotransmitter secretion in LS GABA neuro...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
To investigate how HFD-induced obesity modulates the transcriptional profiles of cells within the septal region, adult mice were assigned to either a HFD or control chow diet.
from paperScoring or quantification
Quantify the primary readouts for this experiment: The expression of Gad2 in HFD group relative to the control group was calculated by the ∆∆CT method using 18sRNA as the reference gene. Data were natural log-transfo...; To evaluate the impact of Hcn1 downregulation on the neuronal excitability of LS GABA neurons and the contribution to HFD-induced obesity, we used short hairpin RNA (shRNA) to k...; Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig....; Beyond the observed reductions in neuronal excitability, our snRNA-seq data also revealed downregulation of synaptic transmission and neurotransmitter secretion in LS GABA neuro....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
To investigate how HFD-induced obesity modulates the transcriptional profiles of cells within the septal region, adult mice were assigned to either a HFD or control chow diet. B...; To evaluate the impact of Hcn1 downregulation on the neuronal excitability of LS GABA neurons and the contribution to HFD-induced obesity, we used short hairpin RNA (shRNA) to k...; Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig....; Beyond the observed reductions in neuronal excitability, our snRNA-seq data also revealed downregulation of synaptic transmission and neurotransmitter secretion in LS GABA neuro...
from paperReporting output
Report representative outputs alongside summary comparisons for The expression of Gad2 in HFD group relative to the control group was calculated by the ∆∆CT method using 18sRNA as the reference gene. Data were natural log-transfo..., To evaluate the impact of Hcn1 downregulation on the neuronal excitability of LS GABA neurons and the contribution to HFD-induced obesity, we used short hairpin RNA (shRNA) to k..., Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig...., Beyond the observed reductions in neuronal excitability, our snRNA-seq data also revealed downregulation of synaptic transmission and neurotransmitter secretion in LS GABA neuro....
inferred from protocolStructured statistical methods
To investigate how HFD-induced obesity modulates the transcriptional profiles of cells within the septal region, adult mice were assigned to either a HFD or control chow diet. B...; To evaluate the impact of Hcn1 downregulation on the neuronal excitability of LS GABA neurons and the contribution to HFD-induced obesity, we used short hairpin RNA (shRNA) to k...; Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig....; Beyond the observed reductions in neuronal excitability, our snRNA-seq data also revealed downregulation of synaptic transmission and neurotransmitter secretion in LS GABA neuro...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
To investigate how HFD-induced obesity modulates the transcriptional profiles of cells within the septal region, adult mice were assigned to either a HFD or control chow diet. Both groups had ad libitum access to food. Mice on the HFD exhibited significantly higher energy intake compared to those on the control diet, resulting in a significant increase in bodyweight that surpassed that of control mice as early as the first week (Fig. ). The caloric intake continued to increase, leading to the development of an obesity phenotype (Fig. ). After a 4-week dietary regimen, septal tissues were harvested for single-nucleus RNA sequencing (snRNA-seq) (Fig. ). Fig. 1 The HFD alters the transcriptional profiles of septal area neurons. a Left: quantification of daily energy intake for the normal food and high-fat diet groups (diet: F 1,60 = 157.2; p < 0.001; time: F 4,60 = 132.6, p < 0.001; interaction: F 4,60 = 9.5, p < 0.001). Right: quantification of bodyweight between the Chow and HFD groups (diet: F 1,60 = 49.3; p < 0.001; time: F 4,60 &...
Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig. ). Immunofluorescence confirmed successful Hcn1 overexpression following this viral strategy (Fig. ). Overexpression of Hcn1 led to a significantly higher voltage sag amplitude and enhanced excitability of LS GABA neurons (Fig. ). In the liquid food intake assays, overexpression of Hcn1 did not affect the standard liquid food intake, but significantly decreased the lick numbers and total intake of palatable sucrose solution and Ensure (Fig. ). A separate cohort of naïve Gad2-Cre mice were used to test whether Hcn1 overexpression in LS GABA neurons could prevent HFD-induced overeating and obesity. We found mice with Hcn1 overexpression resisted excessive energy intake on an HFD, maintaining bodyweights similar to chow-fed groups and significantly lower than HFD-fed EGFP groups (Fig. ). Crucially, this metabolic protection was diet-dependent. Overexpression of Hcn1 did not alter food intake or body weight in mice maintained on a standard chow diet (Fig, and Supplementary Fig.&...
To explore whether restoring GABA levels in the LS could rescue HFD-induced obesity, AAV-DIO-Gad2-EGFP was bilaterally injected into LS of Gad2-Cre mice for LS GABA neurons Gad2 overexpression (Fig. ). Western blot and LC-MS analyses showed a significant increase in Gad2 and GABA protein in LS (Fig. ). In the liquid food intake assays, similar to overexpression of Hcn1, overexpression of Gad2 in LS GABA neurons did not affect the standard liquid food intake, but significantly decreased the lick numbers and total intake of sucrose solution and Ensure (Fig. ). In a distinct cohort of mice, 3 weeks after virus injection, the mice were switched to a 4-week HFD or chow diet. The results demonstrated that Gad2 overexpression in LS significantly reduced HFD intake and prevented obesity (Fig. and Supplementary Fig. ). Overexpression of Gad2 did not affect chow intake or weight gain (Fig. ). qPCR analysis confirmed that Gad2 expression was significantly higher in the overexpression group after 4 weeks of HFD feeding compared to the Chow+GFP control group (Fig. ). Importantly, neither silencing of LS GABA neurons, Gad2 overexpression, nor 4-week h...
In this study, adult male C57BL/6 J mice were obtained from the Guangdong Medical Laboratory Animal Center, Guangzhou, China. Additionally, the experimental cohorts included Gad2-Cre (male, Jax No. 010802) mice. All mice were housed in a temperature (22-25 °C) and humidity (55-65%) controlled environment, with ad libitum access to food and water, except during experimental sessions. A 12-hour light-dark cycle was maintained (lights on from 7:00 am to 7:00 pm). All efforts were made to minimize animal suffering and the number of animals used. A total of 338 mice were used in the different experiments. All experiments were conducted in accordance with relevant guidelines and regulations, and approved by the IACUC (Institutional Animal Care and Use Committee) of SIAT, Chinese Academy of Sciences (CAS, SIAT-IACUC-230927).
Procedures for preparing acute brain slices were similar to those described previously. Mice were anesthetized with isoflurane. Under sterile conditions, we perfused the mice with 4 °C slicing solution containing (in mM) 110 choline chloride, 2.5 KCl, 0.5 CaCl 2, 7 MgCl 2, 1.3 NaH 2 PO 4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO 3 ), then placed the tissue in 4 °C slicing solution saturated with 95% O 2 and 5% CO 2. Coronal slices (250-300 µm) containing the LS, LH or TN were prepared using a vibratome (Leica, VT-1000S). Slices were incubated in 37 °C oxygenated artificial cerebrospinal fluid (in mM: 125 NaCl, 2.5 KCl, 2 CaCl 2, 1.3 MgCl 2, 1.3NaH 2 PO 4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO 3 ) for at least 30 min for recovery. Then the slices were transferred to a recording chamber and superfused with 2 ml min -1 artificial cerebrospinal fluid. Recording was performed at room temperature (23 °C) with a Multiclamp 700B amplifier and a Digidata 1550B acquisition system (Molecular Devices). Data were sampled at 10 kHz and analyzed with Clamp...
To trace the downstream projections of LS GABA neurons, AAV2/9-hSyn-FLEX-tdTomato-T2A-synaptophysin-EGFP-WPRE-pA was injected into the LS. Brain sections from these mice were obtained following the procedures described above. For immunostaining, brain sections were rinsed with PBS (3 × 10 min) and then blocked with 10% normal goat serum and 0.3% TritonX-100 in PBS for 2 hr at room temperature. Next, sections were incubated with primary antibody (Rabbit anti-GFP, 1:1000, ThermoFisher, Cat# A-11122) diluted in 10% normal goat serum and 0.3% TritonX-100 in PBS for 24-48 hr at 4 °C. After washing with PBS (3 × 10 min), sections were incubated with secondary antibody (Goat anti-Rabbit 488, 1:1000, ThermoFisher, Cat# A-11008) diluted in 10% normal goat serum and 0.3% Triton X-100 in PBS at room temperature for 2 hr and then counterstained with DAPI (1:3000).
Machine-readable layer
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"text": "To investigate how HFD-induced obesity modulates the transcriptional profiles of cells within the septal region, adult mice were assigned to either a HFD or control chow diet. Both groups had ad libitum access to food. Mice on the HFD exhibited significantly higher energy intake compared to those on the control diet, resulting in a significant increase in bodyweight that surpassed that of control mice as early as the first week (Fig. ). The caloric intake continued to increase, leading to the development of an obesity phenotype (Fig. ). After a 4-week dietary regimen, septal tissues were harvested for single-nucleus RNA sequencing (snRNA-seq) (Fig. ). Fig. 1 The HFD alters the transcriptional profiles of septal area neurons. a Left: quantification of daily energy intake for the normal food and high-fat diet groups (diet: F 1,60 = 157.2; p <..."
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"text": "Next, we examine the effect of Hcn1 overexpression on the neuronal excitability of LS GABA neurons and HFD-induced obesity by utilizing adeno-associated virus vectors (Fig. ). Immunofluorescence confirmed successful Hcn1 overexpression following this viral strategy (Fig. ). Overexpression of Hcn1 led to a significantly higher voltage sag amplitude and enhanced excitability of LS GABA neurons (Fig. ). In the liquid food intake assays, overexpression of Hcn1 did not affect the standard liquid food intake, but significantly decreased the lick numbers and total intake of palatable sucrose solution and Ensure (Fig. ). A separate cohort of naïve Gad2-Cre mice were used to test whether Hcn1 overexpression in LS GABA neurons could prevent HFD-induced overeating and obesity. We found mice with Hcn1 overexpression resisted excessive energy intake on an HFD, maintain..."
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"text": "To explore whether restoring GABA levels in the LS could rescue HFD-induced obesity, AAV-DIO-Gad2-EGFP was bilaterally injected into LS of Gad2-Cre mice for LS GABA neurons Gad2 overexpression (Fig. ). Western blot and LC-MS analyses showed a significant increase in Gad2 and GABA protein in LS (Fig. ). In the liquid food intake assays, similar to overexpression of Hcn1, overexpression of Gad2 in LS GABA neurons did not affect the standard liquid food intake, but significantly decreased the lick numbers and total intake of sucrose solution and Ensure (Fig. ). In a distinct cohort of mice, 3 weeks after virus injection, the mice were switched to a 4-week HFD or chow diet. The results demonstrated that Gad2 overexpression in LS significantly reduced HFD intake and prevented obesity (Fig. and Supplementary Fig. ). Overexpression of Gad2 did not affect chow in..."
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"text": "In this study, adult male C57BL/6 J mice were obtained from the Guangdong Medical Laboratory Animal Center, Guangzhou, China. Additionally, the experimental cohorts included Gad2-Cre (male, Jax No. 010802) mice. All mice were housed in a temperature (22-25 °C) and humidity (55-65%) controlled environment, with ad libitum access to food and water, except during experimental sessions. A 12-hour light-dark cycle was maintained (lights on from 7:00 am to 7:00 pm). All efforts were made to minimize animal suffering and the number of animals used. A total of 338 mice were used in the different experiments. All experiments were conducted in accordance with relevant guidelines and regulations, and approved by the IACUC (Institutional Animal Care and Use Committee) of SIAT, Chinese Academy of Sciences (CAS, SIAT-IACUC-230927)."
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"text": "Procedures for preparing acute brain slices were similar to those described previously. Mice were anesthetized with isoflurane. Under sterile conditions, we perfused the mice with 4 °C slicing solution containing (in mM) 110 choline chloride, 2.5 KCl, 0.5 CaCl 2, 7 MgCl 2, 1.3 NaH 2 PO 4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO 3 ), then placed the tissue in 4 °C slicing solution saturated with 95% O 2 and 5% CO 2. Coronal slices (250-300 µm) containing the LS, LH or TN were prepared using a vibratome (Leica, VT-1000S). Slices were incubated in 37 °C oxygenated artificial cerebrospinal fluid (in mM: 125 NaCl, 2.5 KCl, 2 CaCl 2, 1.3 MgCl 2, 1.3NaH 2 PO 4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO 3 ) for at least 30 min for recovery. Then the slices were transferred to a recording cham..."
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"datePublished": "2025",
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"identifier": "10.1038/s41467-025-68010-x"
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