Animals
All studies and procedures involving mice were approved by the Rutgers University Institutional Animal Care and Use Committee (IACUC) and by the National Institute of Health (NIH) guidelines. The animals used in this study were 5-7 weeks old, housed, and bred in the Child Health Institute of New Jersey facility. We used cell-type-specific expression of Cre-recombinase mouse lines, GLP-1R-ires-Cre [35], Gcg-Cre [18], and POMC-Cre [40]. For the whole-brain mapping and analyses, GLP-1R-ires-Cre was crossed with Ai14 JaxMice (#007914), Rosa-CAG-LSL-tdTomato-WPRE::ΔNeo [36] to obtain GLP-1R-ires-Cre::Ai14 mice. In all cases, mice were randomized according to body weight in each experimental group. The investigators were blinded to the treatment. The sample size required n= 7-12 per group was based on the previous studies examining the effects of GLP-1 signaling on feeding behavior [32].
Virus injection
The AAV virus (Adeno-Associated Virus) used include pAAV-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA (Catalog #20298-AAV1), pAAV-FLEX-tdTomato (Catalog #28305-AAV5) and pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Catalog #44361-AAV9).
Electrophysiology
Brain slice electrophysiology was conducted, as described elsewhere [20]. Mice were anesthetized, decapitated, and brains were removed and quickly immersed in cold (4oC) oxygenated cutting solution containing (in mM): 50 sucrose, 2.5 KCl, 0.625 CaCl2, 1.2 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, and 2.5 glucose. Coronal cerebral cortex slices, 300 mm in thickness, were cut using a vibratome (VT 1200S, Leica). Brain slices were collected in artificial cerebrospinal fluid (ACSF) and bubbled with 5%CO2 and 95%O2. The ACSF contained (in mM): 125 NaCl, 2.5 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 2.5 glucose. After 1 hour of recovery, slices were transferred to a recording chamber and constantly perfused with bath solution (30oC) at a flow rate of 2 ml/min. To record EPSCs, picrotoxin (50 mM, Sigma) was added to block IPSCs mediated by GABAa receptors. Exn-4 (10 nM) was added to the bath solution to stimulate the ARC GLP-1R neurons. Patch pipettes with a resistance of 8-10MΩ were made from borosilicate glass (World Precision Instruments) with a pipette puller (PC-10, Narishige) and filled with the pipette solution containing (in mM): 126 K-Gluconate, 4 KCl, 10 HEPES, 4 Mg-ATP, 0.3 Na2-GTP, 10 phosphocreatine (pH to 7.2 with KOH) for current and voltage-clamp recordings. After the whole-cell patch-clamp was achieved, spontaneous EPSCs were recorded under voltage clamp at -70 mV. All data were analyzed offline using ClampFit 10.2 (Molecular Devices, USA) software.
Histology and Immunohistochemistry assay
Mice were anesthetized with Euthasol and transcardially perfused with 4% PFA in PBS, pH 7.4. The brains were kept in 4% PFA overnight and then moved to 30% sucrose. Coronal brain slices (50 μm) were prepared thereafter.
For the c-Fos staining, only slices with the ARC region were prepared. The standard IHC protocol was followed. The primary antibodies used were anti-c-Fos (1:1000, Santa Cruz, SC271243). AlexaFluor secondary antibodies (488-goat anti-rabbit, 1:500, Life Technologies) were used to visualize the signals, and images were acquired through confocal microscopy (Zeiss, LSM700).
C-Fos quantification: Total GLP-1R (RFP) and total c-Fos (GFP) labeled cells were counted using the Cell Counter plugin in ImageJ. Then the overlapping signals between RFP and GFP were measured as c-Fos + cells. This quantification was done for the control and target (hM3Dq) group, and the comparison was presented accordingly.
Behavioral Experiments
Excitatory DREADDs
Male mice were caged into groups of either 3 or 4. For the stimulation of the GLP-1 receptors in the ARC, we injected 300 nL of pAAV-FLEX-tdTomato (Catalog #28305-AAV5) and pAAV-hSyn-DIO-hM3D (Gq)-mCherry (Catalog #44361-AAV9) in ARC at coordinates: AP: -1.3 mm from bregma, lateral (L): +/- 0.2 mm, ventral (V): 6.2-6.1 mm. After four weeks of recovery, glucose tolerance tests (GTT) and food intake behavior experiments were conducted after an i.p. DREADD ligand injection.
Glucose Tolerance Test
The mice were fasted overnight (0.5g fed). The same protocol was used as described before [62]. At Time 0, blood glucose was measured to set a baseline. 0.1 mg/kg body weight CNO was injected intra-peritoneally. After 30 minutes, 1.0 g/kg body weight of 20% dextrose solution was injected i.p., and blood samples were collected from the tail vein at 15,30,60 and 120 minutes. For GTT, injection volume was calculated as mentioned below:
Food Intake
Mice were singly housed before the experiment in a 12 h light/dark cycle with ad libitum access to water and were fasted overnight for 12 hours (9 pm-9 am), access to only 0.5 g of food was given. The following day, each animal's weight was measured, CNO was prepared, and 0.1mg/kg BW CNO was injected i.p. into each mouse. After 30 minutes, food was added to their cage. Standard chow intake was measured at t=0,1,2,3,4,5 and 24 hours. After the experiment, animals were grouped together into their respective cages.
Open Field Locomotor Activity
Mice were single housed the night before the experiment. To test whether there are any side effects of the chemogenetic agent used, 0.1 mg/kg BW CNO was i.p. injected. After 30 minutes, mice were allowed to explore a novel environment for 6 minutes. They were put in the open field “exploratory stage,” and their activity and movement were recorded via the center cage software. These experiments were performed under dim light during the light cycle [63].
Insulin measurements
After overnight fasting, CNO was injected as described before. After 120 minutes, Mice were anesthetized with Euthasol, and 1 ml of blood was collected from the heart and stored at -80oC. Plasma was separated by centrifugation at 6,000 rpm for 10 mins at 4°C. Insulin levels were then analyzed using a mouse supersensitive insulin ELISA (ALPCO, Salem, NH, USA) [64].
Quantification and Statistical Analysis
Statistical analysis was performed using Excel (version 2019). In addition, statistical analysis for blood glucose and food intake behavior was also analyzed using GraphPad Prism 8.0. All the data is presented as mean ± standard error of the mean (SEM). Data were analyzed using Student’s t-test and Two-way ANOVA using Geisser-Greenhouse’s epsilon correction. A p-value of less than 0.05 was considered statistically significant.