2.2. Peripheral leptin treatment

Leptin was obtained from Dr. Parlow (National Hormone and Peptide Program, http://www.humc.edu/hormones). To identify LepRb neurons via leptin-induced pSTAT3, mice were injected intraperitoneally (i.p.) with leptin (5 mg/kg body weight) and perfused after one hour of incubation. For studies investigating leptin-induced cFos (as a surrogate of neuronal activity) in LepRb neurons, LepRb^GFP mice were injected with vehicle (saline) or leptin (5 mg/kg, i.p.) and perfused three hours later.

2.3. Stereotaxic injection of adeno-associated viruses (AAVs) in the DMH/DHA

We used designer-receptors-exclusively activated-by-designer drugs (DREADD) technology to allow neuron-specific activation of DMH/DHA LepRb neurons. For DMH/DHA specific deletion of LepRb we injected a cre-expressing AAV or control virus into LepRb^fl/fl mice. AAV-hSyn-DIO-hM3D(G_q)-mCherry (AAV-DREADD, kindly made available from Dr. Bryan Roth) and AAV-CMV-TR-EGFP (control virus) were obtained from the vector core of the University of North Carolina at Chapel Hill. AAV-CMV-HI-GFP-Cre-WPRE-SV40 virus (AAV-cre) was obtained from the vector core of the University of Pennsylvania. Three separate cohorts of LepRb^cre mice (Group A–C) were used for AAV-DREADD injections targeted to the DMH/DHA as described earlier [23]. Two cohorts of LepRb^fl/fl mice received control or AAV-cre virus injections targeted to the DMH/DHA. Mice were anesthetized with 5% isofluorane and their heads immobilized within the frame of a stereotaxic instrument (M1900 Stereotaxic alignment system; David Kopf Instruments, Tujunga, CA). Following sterilization of the surgical site, anesthesia was maintained at 1% isofluorane and an incision was made to expose the skull. Injections were aimed at −1.7 mm posterior, ±0.25 mm lateral and −4.6 mm ventral to Bregma according to the Paxinos Mouse Brain Atlas [24], and adjusted in LepRb^cre mice to −1.1 mm posterior, ±0.25 mm lateral and −5 mm ventral to Bregma. Access holes were drilled and a bilateral guide cannula (Plastics One C253, Roanoke, VA) was inserted into the brain. A bilateral injector filled with virus (3–6 × 1012 viral molecules/ml) and attached to a 0.5 μl Hamilton syringe (Hamilton Company, Reno, NV) was threaded into the guide cannula and a volume of 200 nl per site was slowly infused into the DMH/DHA tissue at a rate of 20 nl per minute. Subsequently, the guide cannula and injector were removed, the skull access sealed with bone wax, and the incision closed with wound clips. Analgesics were applied once to the incision site (bupivacaine/lidocaine, 5 mg/kg) and subcutaneously during recovery every 12 h for two days (carprofen, 5–10 mg/kg). After the surgery mice were single housed for up to three weeks to allow for recovery and viral expression before further experimentation. Cannula placement and viral spread was verified histochemically at the end of all experiments.

2.4. Chronic intra-DMH/DHA cannulations for central leptin treatment

For DMH/DHA specific leptin or vehicle injections we chronically implanted bilateral cannulas (Plastics One C353, Roanoke, VA), as described in detail earlier [25], into the DMH/DHA. Briefly, procedures were identical to acute AAV injections, described above, but the bilateral guide cannula was secured in place with Loctite 454 (Fisher, Pittsburgh, PA) and dental cement before the incision was closed with wound clips. A dummy was inserted into the guide cannula to prevent contamination or blockage. Analgesics were applied as described above. Mice were allowed to recover for one week after surgery and adjusted to handling, twice daily saline injections, and body weight, food intake, and rectal temperature measurements over five days prior to leptin treatment to minimize stress. Body weight, food intake, and rectal temperature data were collected once per day prior to the second daily injection. After three consecutive days of leptin treatment, all mice were perfused and their brains were analyzed to confirm correct cannula placement.

2.5. Measurement of body temperature, energy expenditure, activity, body weight, and food intake in AAV-DREADD mice

Two cohorts of AAV-DREADD mice (group A, n = 4 and group B, n = 4) were injected once with the designer drug clozapine N-oxide (CNO, 0.3 mg/kg body weight; Sigma–Aldrich, St. Louis, MO) and rectal temperature was measured every 15 min for 90 min. In addition, two AAV-DREADD mice (from group B) were equipped with subcutaneous temperature transmitters (G2 E-Mitter; Respironics, Murrysville, PA). Temperature was continuously recorded in these two animals over three days of twice daily i.p. vehicle (saline) injections followed by three days of twice daily CNO (1.5 mg/kg body weight). Another separate cohort of DREADD mice (group C, n = 8) were shaved from head to tail along their backs to allow for temperature monitoring with an infrared/thermal camera (SC5000; FLIR Inc., Wilsonville, OR). Thermal photographs were taken at 10 min intervals following a single injection of vehicle or CNO (i.p., 1.5 mg/kg body weight). Two regions of interest (ROI) were applied to thermal photographs and average temperatures were calculated using Thermovison ExaminIR Beta software (FLIR Inc., Wilsonville, OR). The BAT ROI covered the shaved area from the back of the skull to the bottom of the shoulder blades, while the Body ROI spanned the remaining shaved area caudal the BAT ROI. Following temperature measurements, mice in groups A and B were adapted to oxymax chambers [Comprehensive Lab Animal Monitoring system (CLAMS); Columbus Instruments, Columbus, OH] and i.p. vehicle injections to minimize stress-induced energy expenditure. Then mice received three days i.p. vehicle treatment (both groups, n = 8), followed by three days CNO treatment [i.p. 0.3 mg/kg body weight (group A, n = 4) or 1.5 mg/kg body weight (group B, n = 4)]. Animals were allowed to recover for one week after the last CNO injection. A number of mice from group A and C (n = 11) were adapted to the BioDAQ system (Research Diets Inc., New Brunswick, NJ) for a week and body weight and food intake was monitored daily during three days of vehicle injections and three days of CNO (0.3 mg/kg body weight) i.p. injections. Group A mice (n = 4) were again recovered for at least one week after the last CNO injection to test the effect of β₃-adrenergic antagonist SR 59230A (Sigma–Aldrich, St. Louis, MO) on CNO-induced energy expenditure. Animals were acclimated to new metabolic chambers (TSE Systems Inc., Chesterfield, MO) and received at least three days of i.p. vehicle, CNO (0.3 mg/kg body weight), and CNO + SR 59230A (0.3 mg/kg + 1 mg/kg body weight, respectively) injections over consecutive days during which energy expenditure was recorded continuously.

2.6. Measurement of body temperature, energy expenditure, activity, body weight/composition, and food intake in control and AAV-cre mice

Beginning at two weeks prior to AAV-cre injection, two cohorts of LepRb^fl/fl mice (control virus, n = 6 and AAV-cre, n = 5) were monitored for daily body weight and food intake over eight weeks. One week prior to AAV-cre injection, mice were adapted to oxymax chambers to minimize stress-induced energy expenditure. Then mice were monitored for baseline energy expenditure and activity across two days. Immediately prior to AAV-cre injection, rectal temperature was recorded and body composition was determined by NMR (minispec-mq series; Bruker, Billerica, MA). Following AAV-cre injection, mice were continually monitored for body weight and food intake. Every other week rectal temperature was recorded, body composition was determined by NMR, and mice were returned to oxymax chambers for energy expenditure and activity recordings. After recovering from the last oxymax recordings, mice were placed in a 4 °C environmental chamber and rectal temperatures were monitored every 30 min for three hours. At the completion of all physiological recordings, mice were fasted for 24 h, injected with leptin (5 mg/kg, i.p.) and perfused after an hours of incubation to determine leptin-induced pSTAT3 by histochemical analysis as an indicator of functional LepRb expression.

2.7. Brain slices, whole-cell patch-clamp recordings and histology

Acute brain slices were prepared as previously described [26,27]. Briefly, under isofluorane anesthesia the brain was removed and immersed in ice-cold oxygenated artificial cerebrospinal fluid (aCSF) containing the following (in mM): 124 NaCl, 26 NaHCO₃, 1.4 NaH₂PO₄, 11 glucose, 3 KCl, 1.3 MgCl₂, 1.5 CaCl₂, pH 7.3–7.4. Coronal hypothalamic slices (300 μm) containing the DHA/DMH were made using a vibrating microtome (Vibratome Series 1000; Warner Instruments, Hamden, CT). The slices were stored in a holding chamber at 34–37 °C, and then transferred to a recording chamber mounted on a fixed stage under an upright microscope (Nikon FN1; Nikon Instrument Inc., Melville, NY).Whole-cell patch-clamp recordings were performed at 34–37 °C from DHA/DMH neurons expressing AAV-DREADD, which were identified by mCherry expression under a 40× water-immersion objective (N.A = 0.8). Epifluorescence was used to visualize mCherry-expressing neurons and infrared differential interference contrast optics (IR-DIC) to target specific cells. For whole-cell patch-clamp recordings, electrodes (3–7 MΩ) were filled with a solution containing with following (in mM): 130 K⁺ gluconate, 10 HEPES, 5 EGTA, 1 NaCl, 1 MgCl₂, 1 CaCl₂, 3 KOH, 2–3 Mg-ATP, 0.2% biocytin, pH 7.3–7.4. Electrophysiological signals were recorded using an Axoclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) and acquired by pClamp (Molecular Devices, Sunnyvale, CA). Action potentials were analyzed offline using pClamp and MiniAnalysis (Synaptosoft, Decatur, GA). CNO (5 μM; Sigma, St. Louis, MO) was dissolved in 0.05% DMSO in aCSF. After recordings brain slices were fixed with 4% paraformaldehyde in 0.15 M sodium phosphate buffer overnight at 4 °C. After several rinse in 0.01 M phosphate buffered saline (PBS), slices were immersed in AMCA Avidin D (1:200; Vector Labs, Burlingame, CA) in 0.01 M PBS containing 1% Triton X-100 for 72 h at 4 °C to visualize the recorded neurons. Slices were then washed with 0.01 M PBS and mounted onto charged slides (Fisher Scientific, Waltham, MA), air dried, covered in an anti-oxidant medium (Vectashield; Vector Labs, Burlingame, CA) and coverslipped.

2.8. Perfusion and immunohistochemistry

Perfusions and immunohistochemistry were performed as described earlier [25]. Briefly, mice were deeply anesthetized with isoflurane and transcardially perfused with ice cold physiological saline for 30 s and ice cold 10% neutral buffered formalin (Sigma–Aldrich, St. Louis, MO) for 5 min. Brains were removed and either post fixed in formalin and cryoprotected in 30% sucrose before cryosectioning into 4 representative series of 30 μm sections or snap frozen on dry ice until cryosectioning. Sections were stained immunohistochemically as described elsewhere [18,23] using rabbit anti-phospho(Tyr705)STAT3 (1:2000, #9131; Cell Signaling, Danvers, MA), rabbit anti-cFos (1:10,000,#PC38; Millipore, Billerica, MA), goat anti-dsRed (1:1000, for detection of mCherry; Santa Cruz Biotechnology Inc, Santa Cruz, CA) and chicken-anti-GFP (1:1000, #ab13970; Abcam, Cambridge, MA) as primary antibodies. Nuclear peptides (cFos or pSTAT3) were stained first and developed with the diaminobenzidine (DAB) method. Staining with additional primary antibodies was performed and detected with fluorescent labeled secondary antibodies, Alexa568 (dsRed) or Alexa488 (GFP) from Life Technologies (Carlsbad, CA).

2.9. Neuronal cell count estimates

Immunohistochemical staining for cFos and GFP (LepRb^GFP mice) was visualized with a fluorescent microscope (Olympus BX51; Center Valley, PA) and images were taken with a digital camera (Olympus DP30BW; Center Valley, PA) using appropriate filters for fluorophores or bright-field illumination for DAB stain. Images for cFos/GFP were taken at identical sites, superimposed, and pseudocolored using Olympus Software and Adobe Photoshop (Adobe Systems, San Jose, CA). Brightness and contrast were modified for all images to enhance visibility of nuclear cFos staining for figure preparation and cell counts, however, care was taken that all images within an experiment were modified with identical settings. For each brain (vehicle group, n = 4; leptin group, n = 3–5) two sections containing the compact DMH (Bregma level −1.7 to −2 mm, coordinates based on Paxinos Mouse Brain atlas [24]) were analyzed. Cell counts were performed in the DMH/DHA based on the distribution of LepRb^GFP cells [18]. The DMH/DHA was defined as the area above the compact DMH up to the mammillary thalamic tract (mt). The total number of LepRb^GFP cells and the number of cFos + LepRb^GFP co-labeled cells was counted and co-localization of cFos was expressed as % cFos co-localization per LepRb^GFP cells. For leptin-induced pSTAT3 cell counts (control and AAV-cre mice) bright-field images were taken from the DMH and the ARC. For each brain (control, n = 6; AAV-cre, n = 4) cell counts were obtained in the ventral, compact and dorsal DMH, as well as DHA, and in the ARC of three anatomically comparable hypothalamic sections. The total number of pSTAT3⁺ neurons was compared between control and AAV-cre mice.

3.2. Pharmacogenetic activation of DMH/DHA LepRb neurons

We next determined whether the activation of DMH/DHA LepRb neurons is sufficient to stimulate BAT thermogenesis and energy expenditure. DREADDs are mutated muscarinic receptors that completely lack responsiveness for their endogenous ligand and instead they potently respond to nanomolar doses of the designer drug CNO [30,31]. CNO stimulation is thought to close KCNQ potassium channels via the Gq coupled designer receptor hM3Dq, resulting in intracellular Ca²⁺ flux and neuronal depolarization [32]. We injected AAV-DREADD [31] into the DMH/DHA of LepRb^cre mice (AAV-DREADD mice) to obtain restricted DREADD expression in DMH/DHA LepRb neurons. Correct AAV-DREADD injection sites and anatomical expression were verified by immunohistochemical detection of mCherry (“HIT” mice, Figure 2A) and distinguished from inaccurate AAV-DREADD injection sites or poor expression, that we labeled as “MISSED” injections (Figure 2B). A schematic drawing of the rostro-caudal viral spread of “HIT” AAV-DREADD mice is also depicted across the three panels of Figure 3C. We confirmed the functionality of AAV-DREADD and show that CNO-induced cFos in mCherry⁺ neurons (Figure 2D–F), and that CNO bath application induced action potentials and depolarization in mCherry⁺ DMH/DHA LepRb neurons using patch-clamp electrophysiological recordings in brain slices (Figure 2G,H).

Open in a separate window

Figure 2

AAV-DREADD expression and functionality in DMH/DHA LepRb neurons. Representative images of AAV-DREADD expression in LepRb^cre mice injected with AAV-hSyn-DIO-hM3D(G_q)-mCherry showing a “HIT” (A.) or “MISSED” (B.) injection into the DMH/DHA. C. Schematic representation of viral/mCherry spread across the DMH/DHA. D. Two hours after an acute i.p. CNO injection (1.5 mg/kg), cFos is robustly induced in mCherry⁺ neurons of “HIT” mice (E.), but not “MISSED” mice (F.). G. Representative image of a successfully patched mCherry⁺ neuron labeled with biocytin. H. CNO bath application depolarized the recorded mCherry⁺ neuron leading to burst firing of action potentials. 3v, third ventricle; DHA, dorsal hypothalamic area; DMH, dorsomedial hypothalamus; fx, fornix; mt, mammillothalamic tract.

Open in a separate window

Figure 3

Activation of DMH/DHA LepRb neurons increases BAT and body temperature. A. Representative temperature images of AAV-DREADD mice after vehicle or CNO injections; immediately after injection (left panel) and 1 h post-injection (right panel). Images were taken every 10 min over 1 h to monitor temperature changes in the brown adipose tissue (BAT, intrascapular level, B. p^ANOVA < 0.05, **p^post-hoc < 0.01, ***p^post-hoc < 0.001) and the lower body (C. p^ANOVA < 0.01, *p^post-hoc < 0.02, **p^post-hoc < 0.01, ***p^post-hoc < 0.001). D. Continuous subcutaneous temperature measurement using implanted minimitters in mice with DREADD expression in DMH/DHA LepRb neurons after a 1.5 mg/kg CNO injection (n = 2). ***p^ANOVA < 0.001. E. Rectal temperature of mice with AAV-DREADD expression in DMH/DHA LepRb neurons after a 0.3 mg/kg CNO injection (n = 8). p^ANOVA < 0.001, ***p^post-hoc < 0.001. GMT, Greenwich Mean Time.

3.3. Activation of DMH/DHA LepRb neurons increases BAT and body temperature

We further tested the thermogenic effect of DMH/DHA LepRb activation by measuring body temperature. First, we monitored skin temperature with an infrared/thermal camera, which does not require handling of the animals and thus prevents stress induced rises in body temperature. Furthermore, we could spatially distinguish skin temperature at the level of the intrascapular BAT and the lower body in AAV-DREADD mice after CNO (1.5 mg/kg BW, i.p.) or vehicle injection. Thermal imaging revealed significant increases in skin temperature with CNO treatment, when compared to vehicle (p < 0.05 and 0.01; Figure 3B and C, respectively). BAT and lower body temperature increased with similar magnitudes and chronology following CNO treatment, suggesting that BAT thermogenesis was neither the initial nor the sole source of thermogenesis. An immediate rise in body temperature after CNO treatment was also demonstrated with subcutaneously implanted transmitters (p < 0.001; Figure 3D) and by measurement of rectal temperature (p < 0.001; Figure 3E). In all cases body temperature did not increase more that 1–1.5 °C, well within normal physiological range and far from eliciting a fever response.

3.4. Activation of DMH/DHA LepRb neurons induces energy expenditure, locomotor activity, and body weight loss

We next evaluated the impact of DMH/DHA LepRb neuronal activation on energy expenditure with indirect calorimetry. CNO injections (i.p. 0.3 or 1.5 mg/kg body weight) raised energy expenditure robustly and dose-dependently within minutes (p < 0.001), peaking after an hour and returning to normal within four to five hours (Figure 4A). Increased energy expenditure was further accompanied by an increase in total locomotor activity (p < 0.02 and p < 0.001, respectively; Figure 4B), further suggesting that the profound induction of energy expenditure does not arise exclusively from BAT thermogenesis but also from increased locomotor activity. Following three days of consecutive CNO treatment, correctly targeted “HIT” mice showed a significant reduction in body weight compared to vehicle treatment (p < 0.001, Figure 4C). CNO dependent changes in energy expenditure and body weight were only observed in “HIT” mice. Mice with “MISSED” injections, showed no changes in energy expenditure or body weight with CNO treatment (Figure 4D and E, respectively). DREADD mediated activation of DMH/DHA LepRb neurons did not affect food intake (Figure 4F), demonstrating that changes in body weight were solely attributable to changes in energy expenditure.

Open in a separate window

Figure 4

Activation of DMH/DHA LepRb neurons induces energy expenditure, locomotor activity, and body weight loss. A. “HIT” mice, percent change in oxygen consumption [after i.p. saline (n = 8) or CNO (0.3 and 1.5 mg/kg body weight, n = 4 per dose) injections]. ***p^ANOVA < 0.001. B. “HIT” mice, locomotor activity [after i.p. saline (n = 8) or CNO (0.3 and 1.5 mg/kg body weight, n = 4 per dose) injections]. *p^ANOVA < 0.02, ***p^ANOVA < 0.001. C. “HIT” mice, body weight change during 3 days of vehicle or CNO (0.3 mg/kg body weight) treatment (n = 8). p^ANOVA < 0.001; *p^post-hoc < 0.05, **p^post-hoc < 0.01. D. “MISSED” mice, percent change in oxygen consumption [after i.p. saline (n = 3) or CNO (0.3 and 1.5 mg/kg body weight, n = 4 per dose) injections]. E. “MISSED” mice, body weight change during three days of vehicle or CNO (0.3 mg/kg body weight) treatment (n = 3). In cases with insufficient AAV-DREADD expression or poor DMH/DHA targeting, CNO was unable to increase energy expenditure or decrease body weight (D. and E.). F. “HIT” mice, 24 h food intake after three days of i.p. vehicle or CNO treatment [0.3 mg/kg body weight (n = 4)]. G. “HIT” mice, average change in oxygen consumption (three hours post-injections) after vehicle, CNO (0.3 mg/kg body weight) and CNO + β₃-antagonist (SR 59230A, 1 mg/kg body weight), n = 4 per group, p^ANOVA < 0.001; *p^post-hoc < 0.02, ***p^post-hoc < 0.001 (vs. vehicle). H. “HIT” mice, average locomotor activity (three hours post-injections) after vehicle, CNO (0.3 mg/kg body weight) and CNO + β₃-antagonist (SR 59230A, 1 mg/kg body weight). n = 4 per group, p^ANOVA < 0.03; *p^post-hoc < 0.05.

Sympathetic induction of BAT thermogenesis is initiated via β₃-adrenergic receptor signaling in BAT adipocytes. To determine the extent to which classic BAT thermogenesis contributes to CNO-induced energy expenditure (compared to total locomotor activity), we tested whether β₃-adrenergic receptor blockade would abolish this effect. β₃-adrenergic blockade attenuated CNO-induced energy expenditure significantly, but incompletely (p < 0.05; Figure 4G); further supporting that CNO-induced energy expenditure is not exclusively mediated by β₃-dependent BAT thermogenesis. Indeed, β₃-adrenergic blockade did not affect CNO-induced locomotor activity (Figure 4H), indicating that CNO-induced energy expenditure results from β₃-dependent induction of BAT thermogenesis and β₃-independent induction of locomotion.

3.5. Intra-DMH/DHA leptin administration corrects hypothermia in ob/ob mice

To test the physiological relevance of DMH/DHA LepRb neurons in leptin-stimulated thermogenesis, we injected leptin or vehicle into the DMH/DHA of leptin-deficient ob/ob mice and control littermates twice daily over three days. Correct cannula placement was verified anatomically after the experiment (Figure 5B). As expected, all ob/ob mice were hypothermic and obese before leptin treatment compared to their control littermates (p < 0.0002, data not shown). Leptin treatment had no measureable effect on body temperature in wildtype littermates at the end of the three day treatment, while intra-DMH/DHA leptin injection in ob/ob mice normalized hypothermia to body temperatures comparable to control littermates (Figure 5C). Leptin also significantly elevated body temperature in ob/ob mice compared to vehicle treated ob/ob mice (p < 0.02; Figure 5C). Leptin treatment also trended towards suppressing body weight gain in ob/ob mice compared to vehicle treated ob/ob mice, even though data did not reach statistical significance (p < 0.07; Figure 5D). In line with our previous observations in Figure 4F, activation of LepRb neurons via intra-DMH/DHA leptin administration did not affect food intake (Figure 5E).

Open in a separate window

Figure 5

Intra-DMH/DHA leptin injections correct hypothermia in leptin-deficient ob/ob mice. A. Schematic drawing of bilateral cannulations of the DMH/DHA area. B. Example of the anatomical verification of correctly cannulated DMH/DHA. C. Rectal body temperature of vehicle treated wildtype mice (n = 8), vehicle treated ob/ob mice (n = 5), leptin treated wildtype mice (n = 8), and leptin treated ob/ob mice (n = 6) after three consecutive days of treatment. *p^post-hoc < 0.02. D. Body weight change after three consecutive days of vehicle or leptin treatment. ^†p^t-test < 0.07. E. 24 h food intake during three days of vehicle injections and during three days of leptin injections. wt, wildtype.

3.6. Blockade of DMH/DHA leptin signaling impairs acute thermoregulatory responses, increases body weight, and augments food intake

To validate that leptin action on DMH/DHA LepRb neurons per se is physiologically relevant for energy homeostasis, we selectively depleted LepRb from DMH/DHA neurons by injecting AAV-cre or control virus into LepRb^fl/fl mice (AAV-cre and control mice, respectively). Spatially confined, accurate AAV-cre injection sites and anatomical AAV-cre expression were verified by IHC detection of GFP (“HIT” mice, Figure 6A). Brains from mice with inaccurate injection sites or poor viral expression were excluded from analysis. Adequate ablation of LepRb in DMH/DHA neurons was further confirmed by quantification of leptin-induced pSTAT3 in the hypothalamus of AAV-cre and control mice. AAV-cre mice showed significant less leptin-induced pSTAT3 specifically in the DMH/DHA compared to control mice, while other hypothalamic sites, like the ARC, remained fully responsive to leptin (Figure 6B–D). Leptin-induced pSTAT3 was reduced by 50% in the DMH (including the ventral, compact and dorsal portion of the DMH, and DHA) of AAV-cre mice compared to controls, while the ARC remained fully responsive to leptin (p < 0.05; Figure 6D). At room temperature AAV-cre mice exhibited no differences in rectal temperatures when compared to controls, but acute 4 °C cold exposure revealed that AAV-cre mice defended their body temperature less efficiently over the first two hours of cold exposure (p < 0.01; Figure 6E), even though rectal temperature was equally low in both groups after three hours in the cold.

Open in a separate window

Figure 6

Blockade of DMH/DHA leptin signaling impairs acute thermoregulatory responses, increases body weight, and augments food intake. A. Representative image of AAV-cre expression/LepRb deletion in the DMH/DHA of a properly targeted mouse. Representative images of hypothalamic pSTAT3 induction following leptin treatment (i.p., 5 mg/kg for an hour) of control (B.) or AAV-cre virus mice (C.). Sections were stained for pSTAT3 as a surrogate for functional LepRb signaling. D. Cell counts of pSTAT3⁺ neurons in the ARC and DMH/DHA of control and AAV-cre injected mice. *p^t-test < 0.05. E. Rectal temperature of control (n = 6) and AAV-cre (n = 5) injected mice during a 4 °C cold challenge over three hours. p^ANOVA < 0.001; *p^post-hoc < 0.02, **p^post-hoc < 0.01. F. Weekly body weight of control and AAV-cre mice over eight weeks. ***p^ANOVA < 0.001. G. Body composition of control and AAV-cre mice over eight weeks. Fat mass: p^ANOVA < 0.001; *p^post-hoc < 0.02, ***p^post-hoc < 0.001; lean mass: *p^t-test < 0.05. H. Daily food intake per lean body mass of control and AAV-cre mice over eight weeks. **p^t-test < 0.01, ***p^t-test < 0.001. 3v, third ventricle; ARC, arcuate nucleus; cDMH, compact part of the dorsomedial hypothalamus; DHA, dorsal hypothalamic area; dDMH, dorsal part of the dorsomedial hypothalamus; DMH, dorsomedial hypothalamus; fx, fornix; mt, mammillothalamic tract; VMH, ventromedial hypothalamus.

Furthermore, AAV-cre mice showed a robust time-dependent weight gain immediately following virus-injection (p < 0.001; Figure 6F). NMR analysis showed that this was mostly due to a significant time-dependent accumulation of fat mass (p < 0.001; Figure 6G), although an increase in lean mass was also apparent five weeks following AAV-cre injection (p < 0.05; Figure 6G). Food intake relative to lean body mass also rose substantially in AAV-cre mice during the first two weeks following viral injections, but tapered off after that and became indistinguishable from control mice after three weeks post-injection (Figure 6H). Therefore, the ongoing rise in body weight at weeks post-injection and beyond is largely food intake-independent.

This work was supported by P/F DK020572-30, NIH P20-RR02195, P/F NORC #2P30-DK072476-06, R01-DK092587 (HM), DK099598 (AZ), P30-GM103337 (AVD), and F32-DK097896 (KRZ). This work utilized the facilities of the Cell Biology and Bioimaging Core and Phenotyping Core, supported in part by COBRE (NIH P20-RR021945), CNRU (NIH 1P30-DK072476) center grants from the National Institutes of Health.