Experimental models and subject details
Animal studies
All animal protocols were approved by the Animal Care Committee of the University of Paris (APAFIS #2015062611174320), or the Institut Biologie Paris Seine of Sorbonne University (C75-05-24). Twelve to fifteen-week-old male Aldh1-L1-Cre (Tg(Aldh1l1-cre) JD1884Htz, Jackson laboratory, Bar Harbor, USA), male C57BL/6J (Janvier, Le Genest St-Isle, France) or male GCaMP6f/Glast-CreERT2 (Pham et al., 2020) mice were individually housed at constant temperature (23± 2°C) and submitted to a 12/12h light/dark cycle. All mice had access to regular chow diet (Safe, Augy, France) and water ad libitum, unless stated otherwise. Additionally, age matched C57BL/6J, GCaMP6f/Glast-CreERT2 or Aldh1-L1-Cre mice groups were fed with either chow diet or high-fat high-sugar diet (HFHS, cat n. D12451, Research Diets, New Brunswick, USA) for twelve to sixteen weeks. Body weight was measured every week and body weight gain was estimated as the difference of body weight in week one of HFHS diet consumption to twelve to sixteen weeks after HFHS diet exposure.
Viral constructs
Designer receptor exclusively activated by designer drugs (DREADD) and GCaMP6f viruses were purchased from http://www.addgene.org/, unless stated otherwise. pAAV-EF1α-DIO-hM3Dq-mCherry (2.4x1012 vg/ml, Addgene plasmid #50460-AAV5; http://www.addgene.org/50460/; RRID: Addgene_50460), pAAV-EF1α-DIO-mCherry (3.6x1012 vg/ml, Addgene plasmid #50462-AAV5; http://www.addgene.org/50462/; RRID: Addgene_50462), pAAV-EF1a-DIO-hM3D(Gq)-mCherry was a gift from Bryan Roth (Addgene plasmid # 50460; http://n2t.net/addgene:50460; RRID: Addgene_50460). pAAV-CAG-Flex.GCaMP6f.WPRE (3.15x1013 vg/ml, working dilution 1:10, Addgene plasmid #100835-AAV5; http://www.addgene.org/100835/; RRID:Addgene_100835) was a gift of Douglas Kim and GENIE Project. pAAV-GfaACC1D.Lck-GCaMP6f.SV40 (1.53x1013 vg/ml, working dilution 1:5, Addgene plasmid #52925-AAV5; http://www.addgene.org/52295/; RRID: Addgene_52925) was a gift of Baljit Khak. pAAV-CAG-dLight1.1 was a gift from Lin Tian (Addgene viral prep # 111067-AAV5; http://n2t.net/addgene:111067; RRID: Addgene_111067)
Surgical procedures
For all surgical procedures, mice were first intraperitoneal (ip) injected with the analgesic Buprenorphine (Buprecare, 0.3 mg/kg, Recipharm, Lancashire, UK). 30 minutes after the injection mice were rapidly anesthetized with isoflurane (3%), intraperitoneal (ip) injected with the analgesic Buprenorphine (Buprecare, 0.3 mg/kg, Recipharm, Lancashire, UK) and Ketoprofen (Ketofen, 10 mg/kg, France) and maintained under 1.5% isoflurane anesthesia throughout the surgery.
Stereotaxic surgery. Male Aldh1-L1-Cre+/-, Aldh1-L1-Cre-/- and male C57BL/6J mice were placed on a stereotactic frame (David Kopf Instruments, California, USA) and bilateral viral injections were performed with 0.6ul in DS (stereotaxic coordinates: L = +/-1.75; AP = +0.6; V = -3.5, and -3 in mm), or 0.3ul in NAc (L=+/- 1; AP=+1.55, V=-4.5) at a rate of 50 nl.min-1. The injection needle was carefully removed after 5 min waiting at the injection site and 2 min waiting half way to the top. Mice recovered for at least 3 weeks after the surgery before being involved in experimental procedures.
Behavioral assays
Haloperidol-induced catalepsy. Mice were injected with haloperidol (0.5 mg.kg-1, i.p.). Catalepsy was measured at several time points, 45-180 min after haloperidol injection. Animals were taken out of their home cage and placed in front of a 4-cm elevated steel bar, with the forelegs upon the bar and hind legs remaining on the ground surface. The time during which animals remained still was measured. A behavioral threshold of 180 seconds was set so the animals remaining in the cataleptic position for this duration were put back in their cage until the next time point.
T-maze. Mice were tested for learning and cognitive flexibility in a gray T maze (arm 35-cm length, 25-cm height, 15-cm width). All mice were mildly food deprived (85-90 % of original weight) for 3 days prior to starting the experiment. The first day mice were placed in the maze for 15 min for habituation. Then, mice underwent 3 days of training with one arm reinforced with a highly palatable food pellet (HFHS, cat n. D12451 Research Diet). Each mouse was placed at a start point and allowed to explore the maze. It was then blocked for 20 seconds in the explored arm and then placed again in the starting arm. This process was repeated 10 times per day. At the end of the learning phase all mice showed a > 70 % preference for the reinforced arm. The average number of entries in each arm over 5 trials was plotted. Two days of reversal learning followed the training phase during which the reinforced arm was changed and the mice were subjected to 10 trials per day with the reward in the arm opposite to the previously baited one.
SKF-induced locomotor activity. Mice were placed in an automated online measurement system using an infrared beam-based activity monitoring system (Phenomaster, TSE Systems GmbH, Bad Homburg, Germany). After 1 day of habituation, mice were first i.p. injected with CNO (0.6 mg/Kg) and 30 minutes after with SKF-81297 (3 mg/kg), and placed back in the chamber for at least 80 minutes. Locomotion was recorded using an infrared beam-based activity monitoring system Phenomaster, TSE Systems GmbH, Bad Homburg, Germany).
Fiber photometry
Aldh1-L1-Cre mice were anaesthetized with isoflurane and received 10 mg.kg-1 intraperitoneal injection (i.p.) of Buprécare® (buprenorphine 0.3 mg) diluted 1/100 in NaCl 9 g.L-1 and 10 mg.kg-1 of Ketofen® (ketoprofen 100 mg) diluted 1/100 in NaCl 9 g.L-1, and placed on a stereotactic frame (Model 940, David Kopf Instruments, California). We unilaterally injected 0.6 µl of virus (pAAV.Syn.Flex.GCaMP6f.WPRE.SV40, Addgene viral prep #100833-AAV9, titer ≥ 1013 genome copy (GC).mL-1, working dilution 1:5) or d-Light1 (pAAV-CAG-dLight1.1, Addgene viral prep # 111067-AAV5, titer ≥ 7×10¹² vg/mL, working dilution 1:1) into the DS (L = +/-1.5; AP = +0.86; V = -3.25, in mm) at a rate of 50 nl.min-1. The injection needle was carefully removed after 5 min waiting at the injection site and 2 min waiting half way to the top. Optical fiber for calcium imaging into the striatum was implanted 100 µm above the viral injection site. A chronically implantable cannula (Doric Lenses, Québec, Canada) composed of a bare optical fiber (400 µm core, 0.48 N.A.) and a fiber ferrule was implanted 100 µm above the location of the viral injection site in the DS (L = +/-1.75; AP = +0.6; V = -3.5, and -3 in mm). The fiber was fixed onto the skull using dental cement (Super-Bond C&B, Sun Medical). Real time fluorescence emitted from the calcium sensor GCaMP6f expressed by astrocytes with the Aldh1-L1-Cre receptor was recorded using fiber photometry as described in (Berland et al., 2020). Fluorescence was collected in the DS using a single optical fiber for both delivery of excitation light streams and collection of emitted fluorescence. The fiber photometry setup used 2 light emitting LEDs: 405 nm LED sinusoidally modulated at 330 Hz and a 465 nm LED sinusoidally modulated at 533 Hz (Doric Lenses) merged in a FMC4 MiniCube (Doric Lenses) that combines the 2 wavelengths excitation light streams and separate them from the emission light. The MiniCube was connected to a fiber optic rotary joint (Doric Lenses) connected to the cannula. A RZ5P lock-in digital processor controlled by the Synapse software (Tucker-Davis Technologies, TDT, USA), commanded the voltage signal sent to the emitting LEDs via the LED driver (Doric Lenses). The light power before entering the implanted cannula was measured with a power meter (PM100USB, Thorlabs) before the beginning of each recording session. The light intensity to capture fluorescence emitted by 465 nm excitation was between 25-40 µW, for the 405 nm excitation this was between 10-20 µW at the tip of the fiber. The fluorescence emitted by the GCaMP6f activation in response to light excitation was collected by a femtowatt photoreceiver module (Doric Lenses) through the same fiber patch cord. The signal was then received by the RZ5P processor (TDT). On-line real time demodulation of the fluorescence due to the 405 nm and 465 nm excitations was performed by the Synapse software (TDT). A camera was synchronized with the recording using the Synapse software. Signals were exported to MATLAB R2016b (Mathworks) and analyzed offline. After careful visual examination of all trials, they were clean of artifacts in these time intervals. The timing of events was extracted from the video. For each session, signal analysis was performed on two-time intervals: one extending from –4 to 0 sec (before entering the reinforced arm) and the other from 0 to +4 sec (reinforced arm). From a reference window (from -180 to -60 sec), a least-squares linear fit was applied to the 405 nm signal to align it to the 465 nm signal, producing a fitted 405 nm signal. This was then used to calculate the ∆F/F that was used to normalize the 465 nm signal during the test window as follows: ∆F/F = (465 nm signal_test - fitted 405 nm signal_ref)/fitted 405 nm signal_ref. To compare signal variations between the two conditions (before vs after entering the reinforced arm), for each mouse, the value corresponding to the entry point of the animal in the reinforced arm was set at zero.
Indirect calorimetry analysis
All mice were monitored for metabolic efficiency (Labmaster, TSE Systems GmbH, Bad Homburg, Germany). After an initial period of acclimation in the calorimetry cages of at least two days, food and water intake, whole energy expenditure (EE), oxygen consumption and carbon dioxide production, respiratory quotient (RQ=VCO2/VO2, where V is volume) and locomotor activity were recorded as previously described83. Additionally, fatty acid oxidation was calculated as previously reported83. Reported data are the results of the average of the last three days of recording. Before and after indirect calorimetry assessment, body mass composition was analyzed using an Echo Medical systems’ EchoMRI (Whole Body Composition Analyzers, EchoMRI, Houston, USA).
Ex-vivo calcium imaging
Male Aldh1-L1-Cre+/- or C57BL/6J mice previously injected with GCamP6f and DREADDs viral constructs, and GCaMP6f/Glast-CreERT2 mice were terminally anaesthetized using isoflurane. Brains were removed and placed in ice-cold oxygenated slicing artificial cerebrospinal solution (aCSF, 30mM NaCl, 4.5mM KCl, 1.2mM NaH2PO4, 1mM MgCl2, 26mM NaHCO3, and 10mM D-Glucose and 194mM Sucrose) and subsequently cut into 300-µm thick PVN coronal slices using a vibratome (Leica VT1200S, Nussloch, Germany). Next, brain slices were recovered in aCSF (124mM NaCl, 4.5mM KCl, 1.2mM NaH2PO4, 1mM MgCl2, 2mM CaCl2, 26mM NaHCO3, and 10mM D-Glucose) at 37 °C for 60 minutes. Imaging was carried out at room temperature under constant perfusion (~3 ml/min) of oxygenated aCSF. The overall cellular fluorescence of astrocytes expressing GCaMP6f was collected by epifluorescence illumination. A narrow-band monochromator light source (Polychrome II, TILL Photonics, Germany) was directly coupled to the imaging objective via an optical fiber. Fluorescence signal was collected with a 40x 0.8NA or a 63x 1.0NA water immersion objective (Zeiss, Germany) and a digital electron-multiplying charge-coupled device (EMCCD Cascade 512B, Photometrics, Birmingham, UK) as previously described (Pham et al., 2020). A double-band dichroic/filter set was used to reflect the excitation wavelength (470 nm) to slices and filter the emitted GCaMP6 green fluorescence (Di03-R488/561-t3; FF01-523/610, Semrock). The same filter was used for slices expressing both GCaMP6f and DREADD-mCherry. Striatal slices were transferred to the imaging chamber, where 3-minute astrocyte spontaneous activity recordings were performed in slices of GCaMP6f/Glast-CreERT2 mice. In the case of striatal slices of Aldh1-L1-Cre+/- and C57BL/6J mice, we performed a basal epifluorescence recording (60 seconds), followed by a 120 second bath application of CNO (10µM) or Glutmate (30µM) and 240 seconds recording over the washing of the compounds.
The responsive regions displaying Ca2+ signals were scrutinized by the three-dimensional spatio-temporal correlation screening method (Pham et al., 2020). Background signal was subtracted from the raw images by using the minimal intensity projection of the entire stack. Ca2+ signals of individual responsive regions were normalized as dF/F0, with F0 representing the baseline intensity and quantified using Matlab (The MathWorks, France) and Igor Pro (Wavemetrics, USA). We gauged signal strength of Ca2+ traces of single responsive regions by calculating their temporal integration and normalizing per minute. The global temporal synchronization of detected Ca2+ signals was determined by the temporal Pearson’s correlation coefficients of all combinations between single Ca2+ regions (Pham et al., 2020).
Brain tissue Immunofluorescence
Mice were euthanized with pentobarbital (500 mg/kg, Dolethal, Vetoquinol, France) and transcardially perfused with 0.1 M sodium phosphate buffer (PBS, pH 7.5) followed by 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.2). Brains were removed and post-fixed overnight in 4% paraformaldehyde. Afterwards, the brains were transferred to 30% sucrose in PBS for 2 days for cryoprotection. Next, 30 µm brain sections were cut in a freezing cryostat (Leica, Wetzlar, Germany) and further processed for immunofluorescence following the procedure previously described (Berland et al., 2020). Free-floating brain sections were incubated at 4°C overnight with mouse anti-Glial fibrillary acidic protein (GFAP, 1:1000, Sigma-Aldrich, Saint-Louis, USA) or mCherry (ab125096; 1:1000, Abcam, Cambridge, MA) primary antibodies. The next day, sections were rinsed in Tris-buffered saline (TBS, 0.25M Tris and 0.5M NaCl, pH 7.5) and incubated for 2 hours with secondary antibodies (1:1000, Thermo fisher Scientific, MA, USA) conjugated with fluorescent dyes: goat anti-chicken Alexa 488, donkey anti-rabbit Alexa 594, donkey anti-mouse Alexa 488 and donkey anti-rabbit Alexa 647. After rinsing, the sections were mounted and coverslipped with DAPI (Vectashield, Burlingade, California, USA) and examined with a confocal laser scanning microscope (Zeiss LSM 510, Oberkochen, Germany) with a color digital camera and AxioVision 3.0 imaging software.
Statistical analyses
Compiled data are always reported and represented as mean ± s.e.m., with single data points plotted. Data were statistically analyzed with GraphPad Prism 9. Normal distribution was tested with Shapiro-Wilk test. When n was > 7 and normality test passed, data were analyzed with Student’s t test, one-way ANOVA, two-way ANOVA or repeated-measures ANOVA, as applicable and Holm-Sidak’s post-hoc tests for two by two comparisons. Otherwise non-parametric Mann-Whitney test. All tests were two-tailed. Significance was considered as p < 0.05.