GLP-1 Increases pre-ingestive satiation via hypothalamic circuits in mice and humans
Data files
Jun 10, 2024 version files 372.73 MB
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README.md
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Source_Data_and_Codes.zip
Jul 16, 2024 version files 372.64 MB
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README.md
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Source_Data_and_Codes.zip
Abstract
GLP-1 receptor agonists (GLP-1RAs) are effective anti-obesity drugs. However, the precise central mechanisms of GLP-1RAs remain elusive. We administered GLP-1RAs to obese patients and observed heightened sense of pre-ingestive satiation. Analysis of human and mouse brain samples pinpointed GLP-1R neurons in the dorsomedial hypothalamus (DMH) as candidates for encoding pre-ingestive satiation. Optogenetic manipulation of DMHGLP-1R neurons caused satiation. Calcium imaging demonstrated that these neurons are actively involved in encoding pre-ingestive satiation. GLP-1RA administration increased the activity of DMHGLP-1R neurons selectively during eating behavior. We further identified an intricate interplay between DMHGLP-1R neurons and arcuate NPY/AgRP neurons (ARCNPY/AgRP), to regulate food intake. Our findings reveal a hypothalamic mechanism through which GLP-1RAs control pre-ingestive satiation, offering novel neural targets for obesity and metabolic diseases.
README: GLP-1 Increases Pre-ingestive Satiation via Hypothalamic Circuits in Mice and Humans
Description of data
Figure 1, Figure S1
Path: Source Data and Codes\Figure 1, Figure S1
RAW_Control.xlsx : Raw data for Control test.
RAW_GLP1.xlsx : Raw data for GLP1 test.
Group: The group each person was designated, either A or B. Group number: The number each person was designated inside each group. Participant number: Number designated for those who participated in the test.
P1_1 to P4_29: Each participant’s answers to Questionnaires 1-29. Questionnaires were given to participants in Korean language, and may be provided upon request. P1_1 to P1_29: Baseline, P2_1 to P2_29: Pre-oral, P3_1 to P3_29: Oral, P4_1 to P4_29: Ingestion. N/A refers to not available data including the name and email of each participant, as well as missing data in the questionnaires performed. Missing data were omitted for analysis.
RAW_ingestion.xlsx
Sheet1: Total ingestion amount of food by each participant in control and GLP-1
Sheet Baseline Char&Withdrawal:
Group, Group number, Participant number
Control_ingestion, GLP-1_ingestion: Total ingestion amount of food by each participant in control and GLP-1
Ingestion change: GLP-1_ingestion - Control_ingestion. Participants over +30g were excluded for analysis, colored in red.
Initial_Weight, GLP1_Weight : Body weight of each participant that was measured initially, and post GLP-1 injection
% Weight change: (GLP1_Weight- Initial_Weight)/ Initial_Weight *100. Participants over +5% were excluded for analysis, colored in red.
Compliance: Non-compliant 0, Compliant 1. Participants who were non-compliant were excluded for analysis, colored in red.
Sex: Male 1, Female 2
Age, Baseline_BMI : Baseline characteristics of participants
Excluded from analysis: Colored in red.
Figure 2
Figure 2F-N
Path: Source Data and Codes\Figure 2\Fig 2F-N
NF#: # is number of individual mouse
- Use only Column 8 & 12. Other columns are not used for analysis
- Laser on: Timepoint of start and end of the laser on period or the laser off period
Figure 2O-R
Path: Source Data and Codes\Figure 2\Fig 2O-R
NF#: # is number of individual mouse
- Column 1: Time of laser on
- Column 2: Length of laser
- Column 3: Time of ingestion bout start
- Column 4: Length of ingestion bout
- Cells that contain NaN were removed during analysis (Matlab, rmmissing).
#laser_shuffle: # is number of individual mouse
- Column 1: Trial sequence,
- Column 2: 1 = real laser, 0 = sham laser
#JKEN: # is number of individual mouse
- Column 2: 1 is laser on, 0 is laser off
- Column 3: Systematic pulse sending signal to start laser pulse delivery, 1 is system on, 0 is system off
Figure 2S-U
Path: Source Data and Codes\Figure 2\Fig 2S-U
Labels are identical to Figure 2O-R
Figure 3
Figure 3D
Path: Source Data and Codes\Figure 3\Fig3A-H\Fig.3D.m
dod1, dod2 : Non-food-directed locomotion start, Food-directed seeking start
md1, md2 : Ingestion Start
Data for Pre-Conditioning (Day 1)
Path: Source Data and Codes\Figure 3\Fig3A-H\Day0\#ready_csv file
- #: the individual mouse id
Source Data and Codes\Figure 3\Fig3A-H\Day0\#Observer file
- #: the individual mouse id
- DO = Food Accessibility / SS = Non-food-directed locomotion start / ES = Ingestion Start / EE = Ingestion End
Data for Post-Conditioning (Day 2)
Path: Source Data and Codes\Figure 3\Fig3A-H\Day1\#ready_csv file
- #: the individual mouse id
Source Data and Codes\Figure 3\Fig3A-H\Day1\#Observer file
- #: the individual mouse id
- DO = Food Accessibility / SS = Food-directed seeking start / ES = Ingestion Start / EE = Ingestion End
Figure 3I-L: Data for T-maze test
Behavior files
- # means the individual mouse id
- W = Neutral Side, R = Red Side, B = Blue Side, E = Condition when mice ate food
Ex: EWB: White side to Blue side decision moment, mice ate food after decision, WR: White side to Red side decision moment
Ready# files
1. # means the individual mouse id
2. 1st column, time, 2nd column 405nm, 3rd column 465nm
3. T-maze_learn.xlsx, file for Figure 3L
Pre-conditioning
Path: Source Data and Codes\Figure 3\Fig3I-L\Habituation
Post-conditioning
Path: Source Data and Codes\Figure 3\Fig3I-L\Test
Post-extinction
Path: Source Data and Codes\Figure 3\Fig3I-L\Extinction
Figure 3M-T
Path: \Source Data and Codes\Figure 3\Fig3M-T\Behavior
ChoiceProbabilityREV.mat: file that id for discriminated pre-ingestion neurons and ingestion neurons
- Use only Column 8 & 12. Other columns are not used for analysis
- Column 8: Event Time from recording start
- Column 12: Behavior Label:
i. closeopen = door close to put mice in shelter, food accessibility
ii. search = seeking start to food zone in
iii. eat = ingestion start to ingestion end
iv. fz = food zone in to food zone out
v. back = food zone out to before hitass
vi. hitass = pushing mouse into shelter
Path: \Source Data and Codes\Figure 3\Fig3M-T\ RAW_celltrace
- k# : # is id of individual mice
- Row 1, C## : ## number of cell
- Row 2, manually accepted or rejected cell after cnmf-e
- Column1 : Time, Column2 ~ : raw neural activity value after cnmf-e
Path: \Source Data and Codes\Figure 3\Fig3M-T\Fig 3N contourmaps
- K6cells: individual cell images used for contours
- ChoiceprobabilityREV.mat << cells discriminated from Fig3_glp1_ms_ChoiceProbability.m
- REVindbeh_Z.mat << z-score from Fig3_glp1_new_ms_repheatmap_Traces
- indbeh_data.mat << behavior data preprocessed for use
Figure 4
Behavior
Path: \Source Data and Codes\Figure 4\GLP1_behavior : Behavior for GLP-1 injection
Path: \Source Data and Codes\Figure 4\Saline_behavior : Behavior for saline injection
- Use only Column 8 & 12. Other columns are not used for analysis
- POKE: moment of injection, DO: Food Accessibility, SS: Seeking Start, ES: Ingestion Start, EE: Ingestion End, NES = Food contact without ingestion, NESE, Food contact end without ingestion
Photometry neural activity traces
Path: \Source Data and Codes\Figure 4\GLP1_injection : Photometry neural activity for GLP-1 injection
Path: \Source Data and Codes\Figure 4\Saline_injection : Photometry neural activity for Saline injection
Figure 5
Figure 5B
Path: Source Data and Codes\Figure 5\B
- Sheet1
WCC: Whole-cell capacitance
SR: Series resistance
RMP: Resting membrane potential
Figure 5D
Path: Source Data and Codes\Figure 5\D
CNO injection sheet
BW: Body weight
Columns D to G: The weight of chow at 8am, 11am, 2pm, and 5 pm, respectively.
Columns K to M: Food intake (g) in between 8am to 11am, 11am to 2pm, and 2pm to 5pm, respectively.
Columns Q to S: Cumulative Food intake for 3 hours, 6 hours, and 9 hours, respectively.
Saline injection sheet
BW: Body weight
Columns D to G: The weight of chow at 8am, 11am, 2pm, and 5 pm, respectively.
Columns K to M: Food intake (g) in between 8am to 11am, 11am to 2pm, and 2pm to 5pm, respectively.
Columns Q to S: Cumulative Food intake for 3 hours, 6 hours, and 9 hours, respectively.
Figure 5E
Path: \Source Data and Codes\Figure 5\E
Liraglutide or Leptin treat sheet
WCC: Whole-cell capacitance
SR: Series resistance
RMP: Resting membrane potential
Figure 5F
Path: \Source Data and Codes\Figure 5\F
Liraglutide or Leptin ip inject sheet
WCC: Whole-cell capacitance
SR: Series resistance
RMP: Resting membrane potential
Figure 5G
Path: \Source Data and Codes\Figure 5\G
Saline injection sheet
FI: Food intake (The weight of chow diet on the measurement day)
BW: Body weight
Saline was injected on day 0
Column H (Food intake on day 0): food intake (g) from day -1 to day 0 - before injection
Column I (Food intake on day 1): food intake (g) from day 0 to day 1 - after injection
Column K: percentage change of food intake on day 1 compare to day 0
Column L: percentage change of body weight on day 1 compare to day 0
Liraglutide + CNO injection sheet
FI: Food intake (The weight of chow diet on the measurement day)
BW: Body weight
Liraglutide was injected on day 0
Column H (Food intake on day 0): food intake (g) from day -1 to day 0 - before injection
Column I (Food intake on day 1): food intake (g) from day 0 to day 1 - after injection
Column K: percentage change of food intake on day 1 compare to day 0
Column L: percentage change of body weight on day 1 compare to day 0
Figure 5H
Path: \Source Data and Codes\Figure 5\H
CNO, Liraglutide bath apply sheet
WCC: Whole-cell capacitance
SR: Series resistance
RMP: Resting membrane potential
Fig. S2
Figure S2G
Path: Source Data and Codes\Figure S2\G
DMH: Dorsomedial hypothalamus
Columns H, line 4 to 6: percentage of LepR mRNA coexpressed GLP-1R expressing neurons in each mouse of DMH.
Columns H, line 8: Average of percentage of LepR mRNA coexpressed GLP-1R expressing neurons in the DMH.
Fig. S3
Figure S3A-G
Path: Source Data and Codes\Figure S3\Fig S3A-G
NF#: # is individual number of mice
1. Column 1: Time of laser on,
2. Column 2: Length of laser
3. Column 3: Time of eating bout start,
4. Column 4: Length of eating bout
- Cells that contain NaN were removed during analysis (Matlab, rmmissing).
Figure S3H-N
Path: Source Data and Codes\Figure S3\Fig S3H-N
NF#: # is individual number of mice
1. Column 1: Time of laser on (1st laser time on is used)
2. Column 2: Length of laser (Not used importantly in the code)
3. Column 3: Time of eating bout start
4. Column 4: Length of eating bout
- Cells that contain NaN were removed during analysis (Matlab, rmmissing).
Fig. S4
Path: Source Data and Codes\Figure S4
S4X.xlsx: X corresponds to alphabet for the figure
Fig. S5
Figure S5A-F
Path: Source Data and Codes\Figure S5 \FigS5A-F
Read, Figure 3 Data for Post-Conditioning (Day 2)
Figure S5G-H
Path: Source Data and Codes\Figure S5 \FigS5G-H
Read, Data for T-Maze Test
Figure S5I-L
Path: Source Data and Codes\Figure S5 \FigS5G-H
beh#: # is individual mouse for the Behavior data
ready#: # is individual mouse number,
1. Column 1: Time, Column 2: 405nm, Column 3: 465nm\
Figure S5R
Path: Source Data and Codes\Figure S5 \FigS5R
#ready: # is individual mouse number
1. Column 5: time of behavior
2. Column 6: behavior label
- C: lego contact, CE: lego contact end
Figure S5S-T
Path: Source Data and Codes\Figure S5 \FigS5S-T
#ready: # is individual mouse number
#observer: # is individual mouse number
1. Column 1: time of behavior
2. Column 2: behavior label
- C: Ingestion start, CE: ingestion End
Figure S5U-Y
Path: Source Data and Codes\Figure S5 \FigS5U-Y
#ready: # is individual mouse number
#observer: # is individual mouse number
1. Column 1: time of behavior
2. Column 2: behavior label
- Eat in odd row: ingestion start, Eat in even row: ingestion end
Fig. S6-S7
Path: Source Data and Codes\Figure S6_S7
Read Figure 3M-T
Fig. S8
Path: Source Data and Codes\Figure S8
Fig S8A-D
Path: Source Data and Codes\Figure S8\Fig S8A-D
Day0
ready#: # is individual mouse number
observer#: # is individual mouse number
1. Use only Column 8 & 12. Other columns are not used for analysis
a. Column 8: Event Time from recording start
b. Column 12: Behavior Label: DO = Food Accessibility / SS = Food-directed seeking start / ES = Ingestion Start / EE = Ingestion End
Day1
ready#: # is individual mouse number
a. Column 6: Event Time
b. Column 7: Behavior Label: DO = Food Accessibility / SS = Food-directed seeking start / ES = Ingestion Start / EE = Ingestion End, NES = Food contact without ingestion, NESE, Food contact end without ingestion
Fig S8E-H
Path: Source Data and Codes\Figure S8\Fig S8E-H
NF#: # is number of individual mouse
1. Use only Column 8 & 12. Other columns are not used for analysis
- a. Laser on: Timepoint of start and end of the laser on period or the laser off period
Fig S8I-L
Path: Source Data and Codes\Figure S8\Fig S8I-L
Read Figure 2O-R
Fig S8M-U
Path: Source Data and Codes\Figure S8\Fig S8M-U
Read Figure 4
Fig. S9
Fig S9A
Path: Source Data and Codes\Figure S9\A
Liraglutide or Leptin treat sheet
RMP: Resting membrane potential
delta RMP: Change in membrane potential during Liraglutide or leptin bath application compared to baseline
Figure S9B
Path: Source Data and Codes\Figure S9\B
Liraglutide or Leptin ip inject sheet
RMP: Resting membrane potential
Firing: the number of action potential during recording
Fig. S10
Figure S10A
Path: Source Data and Codes\Figure S10\A
Liraglutide with blockers sheet
RMP: Resting membrane potential
SB: Synaptic blocker
Figure S10C
Path: Source Data and Codes\Figure S10\C
GLP-1R GFP Liraglutide sheet
RMP: Resting membrane potential
SB: Synaptic blocker
Figure S10D
Path: Source Data and Codes\Figure S10\D
GLP-1R GFP-cre Liraglutide sheet
RMP: Resting membrane potential
SB: Synaptic blocker
Figure S10E
Path: Source Data and Codes\Figure S10\E
Liraglutide or Saline injection sheet
RMP: Resting membrane potential
Figure S10F
Path: Source Data and Codes\Figure S10\F
Saline injection sheet
FI: Food intake (The weight of chow diet on the measurement day)
BW: Body weight
Saline was injected on day 0
Column H (Food intake on day 0): food intake (g) from day -1 to day 0 - before injection
Column I (Food intake on day 1): food intake (g) from day 0 to day 1 - after injection
Column K: percentage change of food intake on day 1 compare to day 0
Column L: percentage change of body weight on day 1 compare to day 0
Liraglutide + CNO injection sheet
FI: Food intake (The weight of chow diet on the measurement day)
BW: Body weight
Liraglutide was injected on day 0
Column H (Food intake on day 0): food intake (g) from day -1 to day 0 - before injection
Column I (Food intake on day 1): food intake (g) from day 0 to day 1 - after injection
Column K: percentage change of food intake on day 1 compare to day 0
Column L: percentage change of body weight on day 1 compare to day 0
Fig. S11
Figure S11A-C
Path: Source Data and Codes\Figure S11\Fig S11A-C
1. ready# : # is id of individual mice
2. Column 6 and 7: behavior time and behavior, respectively.
- C = Drinking Start / CE = Drinking End
Figure S11D-G
Path: Source Data and Codes\Figure S11\Fig S11D-G
1. NF_# : # is id of individual mice
2. Column 1: Time of laser on,
Column 2: Length of laser,
Column 3: Time of drinking bout start,
Column 4: Length of drinking bout
Figure. S11H-M
Path: Source Data and Codes\Figure S11\Fig S11H-M
1. 10on10off test: t# : # is id of individual mice
2. 10off10on test: t# : # is id of individual mice
3. Use only Column 8 & 12. Other columns are not used for analysis
Column 12: Behavior Label:
Laseron: Timepoint of start and end of the laser on period or the laser off period
Drink: Start and end of drinking bout
Figure. S11N-O
Behavior
Path: \Source Data and Codes\Figure S11\FigS11N-O\food\beh_event
Path: \Source Data and Codes\Figure S11\FigS11N-O\water\beh_event
Path: \Source Data and Codes\Figure S11\FigS11N-O\object\beh_event
1. Use only Column 8 & 12. Other columns are not used for analysis
a. Column 8: Event Time from recording start
b. Column 12: Behavior Label:
i. closeopen = door close to put mice in shelter, food accessibility
ii. search = seeking start to food zone in
iii. eat = eating start to consumption end
iv. fz = food zone in to food zone out
v. back = food zone out to before hitass
vi. hitass = pushing mouse into shelter
Signal
Path: \Source Data and Codes\Figure S11\FigS11N-O\food\signal
Path: \Source Data and Codes\Figure S11\FigS11N-O\water\signal
Path: \Source Data and Codes\Figure S11\FigS11N-O\object\signal
1. k# : # is id of individual mice
a. Row 1, C## : ## number of cell
b. Row 2, manually accepted or rejected cell after cnmf-e
c. Column1 : Time, Column2 ~ : raw neural activity value after cnmf-e
Fig. S12
Figure. S12A-C
Behavior
Path: Source Data and Codes\Figure S12\Fig S12A-C\beh
1. k# : # is id of individual mice
2. Use only Column 8 & 12. Other columns are not used for analysis
a. Column 8: Event Time from recording start
b. Column 12: Behavior Label:
Waterin: Timepoint when water was exposed to mice
Drink: Start and end of drinking bout
Signal
Path: Source Data and Codes\Figure S12\Fig S12A-C\signal
1. ready# : # is id of individual mice
Figure. S12D-I
Path: Source Data and Codes\Figure S12\Fig S12D-I
1. 5on5off test: t# : # is id of individual mice
2. 5off5on test: t# : # is id of individual mice
3. Use only Column 8 & 12. Other columns are not used for analysis
Figure. S12J-N
Path: Source Data and Codes\Figure S12\Fig S12J-N
1. 10on10off test: t# : # is id of individual mice
2. 10off10on test: t# : # is id of individual mice
3. Use only Column 8 & 12. Other columns are not used for analysis
Sharing/Access information
The current dataset is for Science Manuscript ID adj2537.
Code/Software
Data and codes used for this paper are available in excel files and can be run by MATLAB.
Methods
Analysis
Analysis was done using custom MATLAB code otherwise stated.
Clinical trials
72 patients were assessed for eligibility, and 44 patients were screened, with 4 patients meeting the exclusion criteria. 40 obese individuals were enrolled, who were allocated randomly into two groups (Group A n=20, Group B n=20). The participants were prescribed liraglutide and received a weekly escalating dose of 0.6mg, 1.2mg, 1.8mg, 2.4mg daily over a four-weeks period. Patients were asked to write a diary to record the dose and date daily. Group A participants underwent control clinical tests prior to liraglutide injection, got 4 weeks of injection, and performed the same structured test scheme. Group B participants got 4 weeks of injection and underwent clinical tests, and after 2 weeks of washout period they performed the control clinical tests. A total of 28 patients were analyzed for study. (Withdrawn from trial: Group A n=0, Group B n=2. Excluded from analysis: Group A n=5, Group B n=5) Individual characteristics have been reported in Table S3. Clinical tests were performed as previously described as a structured test scheme, broken down into four distinct phases with a survey at the end of each phase. The survey was reconstructed based on the following questionnaires: VAS (34), RISE-Q (35), RED-13 (36), PFS-K (37), DEBQ (38). Missing values were omitted for analysis. This study was approved by the institutional review board (IRB) of Seoul National University College of Medicine/Seoul National University Hospital (IRB No. 2208-049-1349). Written informed consent was obtained from participants. Inclusion, exclusion, withdrawal, exclusion from analysis criteria have been reported in Table S2.
Optogenetics
Laser stimulation (473 nm for activation and 532 nm for inhibition, Shanghai DPSS Laser) was delivered through an FC-FC fiber patch cord (Doric Lenses) connected to the rotary joint, followed by the FC-ZF 1.25 fiber patch cord delivered stimulation to the cannula (200 µm core, NA 0.37, Doric Lenses or Inper). The laser intensity was approximately 10 mW at the tip. For open-loop stimulation, mice received 10-minute or 2-minute intervals of lasers at wavelengths of 473nm at 10hz, 50ms for neural activation or 532nm continuously for neural inhibition experiments. For closed-loop stimulation, mice received manual laser stimulation when they started to eat a high-fat diet (D12492, Research diets).
Fiber photometry
Fiber photometry signal data were acquired using the Doric Studio software. 465 nm calcium and 405 nm isosbestic signals (for artifact correction), were obtained. 405 nm signals were linearly fitted to 465 signals. △F/F0 signals were corrected as follows to minimize artifact recordings △F/F0 = (465 nm signal − fitted 405 nm signal)/fitted 405 nm signal. Signals were decimated to obtain approximately 20, 25 or 30 data points in 1 s. The mean of the baseline (m) and standard deviation (σ) of the baseline were computed to normalize the corrected signals into Z-scores (Z = (corrected 465 nm − m)/σ). The behavior time points for each test were manually annotated. For the heatmap visualization, Z-Score was used or each trial was normalized as follows: normalized Z = (Z − minimum Z)/(maximum Z − minimum Z). In the GLP-1RAs injection test baseline was designated before injection of drugs to account for the stable state of mice (Fig. 4). To account for signal changes near initiation of the first behavior, when the dosage effect was strongest, mean △F/F0 signals were quantified regarding the initial 5% of time between the initiation of behavior (Food Accessibility, Seeking Start, Consumption Start) and the next behavior (Seeking Start, Consumption Start, Consumption End, respectively) and was compared with baseline. Cumulative probability was quantified by first extracting a section of the Z-score from the behavior moment of interest to the next behavior moment. The section was divided into bins of 1 second and averaged. The averaged values were used for further quantification. Rate of change was quantified by computing the gradient at each behavior moment. Z-scored signals were smoothed using moving average function in MATLAB (movmean) by a sliding window of 1 second (Fig. 4K and R). Afterwards, the gradient of two moments near a behavior moment (before and after 1 second of a behavior moment) was computed.
Micro-endoscope
The raw signal output was preprocessed and computed into calcium dynamics (craw) using CNMF-E by using Inscopix Data Acquisition Software (IDAS ver.1.8). Afterwards, craw was computed into Z-scores (Z = (Craw − m)/σ), according to the mean (m) and standard deviation (σ) of the baseline for each trial (Start of mouse going into shelter until food accessibility). Tuning of each cell was computed using choice probability (CP), defined as how well a single cell’s neural activity could predictively discriminate between two behavioral phases as described from previous reports (44). All frames from the pre-consumption and consumption behavior bouts were used to compute the histogram for each cell. These distributions are computed into a cumulative distribution which are integrated to generate a ROC (receiver operating characteristics) curve. The area under the curve is then computed for each unit regarding the two behavior conditions. The significance of a cell’s CP was determined using shuffled bout timings. Shuffling was repeated 100 times in which the mean and standard deviation was acquired. CP that had a value of over 2 standard deviations above the mean was considered significant and used for analysis (Fig. 3 and fig. S7). To account for visualization, quantification and the responsive cells in different contexts, Z-score was acquired for each trial from each cell (baseline -10 ~ -5 from behavior of interest) (fig. S7 and S9). Representative traces were smoothed using movmean function in MATLAB with a sliding window of 1 second (Fig. 3S). For responsive cell analysis, cells were determined responsive if Z-score surpassed a value of 4 or -4 after behavior of interest (fig. S10). Heatmap visualization was done as stated above (Fig. 3O, T, fig. S10). For visualization of the whole population recordings from all mice, the whole trace of the individual cell itself was used as the baseline (fig. S6).
Statistical Analysis
Statistical data were analyzed using MATLAB, Graphpad Prism 8.0 software and figures were visualized using MATLAB or CorelDrawC8 (64bit). Paired t-tests or unpaired t-tests were used to compare data between two groups. One-way or two-way repeated-measures analyses of variance (ANOVA) were used for multiple comparisons. P-values for comparisons across multiple groups were corrected using the Greenhouse–Geisser, the Tukey, and the Sidak method. Cumulative probability distribution was analyzed with two sample Kolmogorov-Smirnov tests. Results are reported as mean ± SEM, including shades, unless indicated otherwise. Levels of significance were as follows: *p < 0.05. **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistic methods are listed in Table S1.
Electrophysiology studies
Slice preparation
Brain slices were prepared from mice as previously described (13, 14). Briefly, male mice were deeply anesthetized with i.p. injection of 7% chloral hydrate and transcardially perfused with a modified ice-cold artificial CSF (ACSF) (described below). The mice were then decapitated, and the entire brain was removed and immediately submerged in ice-cold, carbogen-saturated (95% O2 and 5% CO2) ACSF (126 mM NaCl, 2.8 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 5 mM glucose). Coronal sections (250 μm) were cut with a Leica VT1000S Vibratome and then incubated in oxygenated ACSF (32 °C–34 °C) for at least 1 h before recording. The slices were bathed in oxygenated ACSF (32 °C–34 °C) at a flow rate of ∼2 ml/min. All electrophysiology recordings were performed at room temperature.
Whole-cell recordings
The pipette solution for whole-cell recording was modified to include an intracellular dye (Alexa Fluor 350 hydrazide dye) for whole-cell recording: 120 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 5 mM EGTA, 1 mM CaCl2, 1 mM MgCl2, and 2 mM MgATP, 0.03 mM Alexa Fluor 350 hydrazide dye (pH 7.3). K-gluconate was replaced with equimolar Cs-gluconate for recording of spontaneous inhibitory postsynaptic currents (IPSCs) in response to photostimulation (2 pulses 50ms interval) upon the ARC. Epifluorescence was used to target fluorescent cells, at which time the light source was switched to infrared differential interference contrast imaging to obtain the whole-cell recording (Zeiss Axioskop FS2 Plus equipped with a fixed stage and a QuantEM:512SC electron-multiplying charge-coupled device camera). Electrophysiological signals were recorded using an Axopatch 700B amplifier (Molecular Devices); low-pass filtered at 2–5 kHz and analyzed offline on a PC with patch-clamp (pCLAMP) electrophysiology data acquisition and analysis program (Molecular Devices). Membrane potentials and action potential were determined from GLP-1R, LepR and NPY expressing neurons in brain slices. Membrane potential values were not compensated to account for junction potential (-8 mV). Recording electrodes showed resistances of 2.5–5 MΩ when filled with the K-gluconate internal solution. Input resistance (IR) was assessed by measuring voltage deflection at the end of the response to a hyperpolarizing rectangular current pulse step (500 ms of −10 to −50 pA).