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Arcuate nucleus and lateral hypothalamic CART neurons in the mouse brain exert opposing effects on energy expenditure

  1. Aitak Farzi
  2. Jackie Lau
  3. Chi Kin Ip
  4. Yue Qi
  5. Yan-Chuan Shi
  6. Lei Zhang
  7. Ramon Tasan
  8. Günther Sperk
  9. Herbert Herzog  Is a corresponding author
  1. Garvan Institute of Medical Research, Australia
  2. Medical University of Graz, Austria
  3. Medical University Innsbruck, Austria
Research Article
Cite this article as: eLife 2018;7:e36494 doi: 10.7554/eLife.36494
12 figures and 2 additional files

Figures

Validation of the expression sites of Cre-dependent AAV-hM3Dq-mCherry for CNO-mediated neuronal activation.

Fluorescence micrographs of coronal brain sections from respective Arc→hM3Dq (A) and LHA→hM3Dq (B) Cartptcre/+ mice, showing the mCherry reporter being specifically expressed in the respective areas. Selective CNO-mediated neuronal activation marked by c-Fos expression in hM3Dq-mCherry-positive CART neurons at the Arc (C) and the LHA (D) (white arrows). Scale bar = 75 μm. Arc, arcuate nucleus; fx, fornix; LHA, lateral hypothalamic area; V3, third ventricle.

https://doi.org/10.7554/eLife.36494.002
Figure 2 with 1 supplement
CNO-mediated activation of hM3Dq-containing Arc CART neurons leads to a reduction in energy expenditure and physical activity in Cartptcre/+ mice.

Indirect calorimetric assessments following saline or CNO injection for the 24 hr time course of energy expenditure, VO2 and VCO2 (A, B), physical activity (C, D), and respiratory exchange ratio (E, F) in Arc→hM3Dq Cartptcre/cre and Arc→hM3Dq Cartptcre/+ mice, respectively. Open and filled horizontal bars indicate the light and dark photoperiods, respectively. Dotted line indicates the time of i.p. injection of saline or CNO. Data are means ±SEM. n = 10–12; *p≤0.05 for saline versus CNO treatments.

https://doi.org/10.7554/eLife.36494.003
Figure 2—figure supplement 1
CNO does not exert any effects on physical activity, energy expenditure and respiratory exchange ratio in the absence of hM3Dq-expression in Cartptcre/cre, Cartptcre/+and wild type mice.

Indirect calorimetric assessments following saline or CNO injection for the 24 hr time course of energy expenditure (A, B, C), physical activity represented as total count (D, E, F), and respiratory exchange ratio (G, H, I) in Cartptcre/cre (A, D, G), Cartptcre/+ (B, E, H) without hM3Dq-injection and wild type mice receiving bilateral LHA→hM3Dq but lacking Cre-recombinase expression (C, F, I), respectively. Open and filled horizontal bars indicate the light and dark photoperiods, respectively. Dotted lines indicate the time of i.p. injection of saline or CNO. Data are means ±SEM. n = 8.

https://doi.org/10.7554/eLife.36494.004
CNO-mediated activation of hM3Dq-containing Arc CART neurons leads to a decrease in body skin temperature in Cartptcre/+ mice.

Temperatures of the interscapular brown adipose tissue (BAT) and the lumbar back region, as well as the temperature differences between the inter-scapular and lumbar areas 2 hr following saline or CNO injection, measured by high-sensitivity infrared imaging in Arc→hM3Dq Cartptcre/cre (A) and Arc→hM3Dq Cartptcre/+ mice (B). Data are means ±SEM. n = 10–12; *p≤0.05, **p≤0.01 for saline versus CNO treatments.

https://doi.org/10.7554/eLife.36494.005
CNO-induced activation of hM3Dq-positive CART neurons in the Arc increases 24 hr food intake and results in weight gain in homozygous Cartptcre/cre mice.

Food intake during fed state 24 hr following saline or CNO injection in Arc→hM3Dq mice, expressed both as absolute amounts of food intake (A, C) and as a percentage of body weight (B, D), for Cartptcre/cre and Cartptcre/+ mice, respectively. The corresponding body weight change at 24 hr in proportion to pre-treatment body weight (E, F) measured in Cartptcre/cre and Cartptcre/+ Arc→hM3Dq mice receiving saline or CNO injection. Data are means ±SEM. n = 10–12; *p≤0.05 for saline versus CNO treatments.

https://doi.org/10.7554/eLife.36494.006
CNO-mediated activation of hM3Dq-containing LHA CART neurons increases energy expenditure and physical activity in Cartptcre/+ mice.

Indirect calorimetric assessments following saline or CNO injection for the 24 hr time course of energy expenditure, VO2 and VCO2 (A, B), physical activity (C, D), and respiratory exchange ratio (E, F) in LHA→hM3Dq Cartptcre/cre and LHA→hM3Dq Cartptcre/+ mice, respectively. Open and filled horizontal bars indicate the light and dark photoperiods, respectively. Dotted line indicates the time of i.p. injection of saline or CNO. Data are means ±SEM. n = 10–12; *p≤0.05, **p≤0.01 for saline versus CNO treatments.

https://doi.org/10.7554/eLife.36494.007
CNO-mediated unilateral activation of hM3Dq-containing LHA CART neurons does not affect body temperature of Cartptcre/cre and Cartptcre/+ mice.

Temperatures of the BAT and the lumbar back region, as well as the temperature differences between the inter-scapular and lumbar areas of Cartptcre/cre (A) and Cartptcre/+ (B) mice measured by high-sensitivity infrared imaging 2 hr after i.p. injection of saline or CNO. Data are means ±SEM. n = 10–12.

https://doi.org/10.7554/eLife.36494.008
CNO-induced activation of hM3Dq-positive CART neurons in the LHA increases 24 hr food intake and body weight gain in heterozygous Cartptcre/+ mice.

Food intake during fed state 24 hr following saline or CNO injection in LHAhM3Dq mice, expressed both as absolute amounts (A, C) and as a percentage of body weight (B, D) for Cartptcre/cre and Cartptcre/+ mice, respectively. The corresponding body weight change at 24 hr in proportion to pre-treatment body weight (E, F) measured in Cartptcre/cre and Cartptcre/+ LHA→hM3Dq mice receiving saline or CNO injection. Data are means ±SEM. n = 10–12; *p≤0.05, **p≤0.01 for saline versus CNO treatments.

https://doi.org/10.7554/eLife.36494.009
CNO-mediated bilateral activation of hM3Dq-containing LHA CART neurons leads to elevation in physical activity, energy expenditure and respiratory exchange ratio in Cartptcre/+ mice.

Indirect calorimetric assessments following saline or CNO injection for the 24 hr time course of energy expenditure, VO2 and VCO2 (A, B), physical activity represented as total count (C, D), and respiratory exchange ratio (E, F) in LHA→hM3Dq Cartptcre/cre and LHA→hM3Dq Cartptcre/+ mice, respectively. Open and filled horizontal bars indicate the light and dark photoperiods, respectively. Dotted lines indicate the time of i.p. injection of saline or CNO. Data are means ± SEM. n = 6–7; *p≤0.05, **p≤0.01, ***p≤0.001 for saline versus CNO treatments.

https://doi.org/10.7554/eLife.36494.010
Figure 9 with 1 supplement
CNO-mediated bilateral activation of hM3Dq-containing LHA CART neurons leads to increased body skin temperature in Cartptcre/cre and Cartptcre/+ mice.

Temperatures of the interscapular brown adipose tissue (BAT) (A, B), the lumbar back (D,E), the tail (G,H) and differences between BAT and lumbar temperatures (J, K) measured by high-sensitivity infrared imaging 1, 3 and 6 hr after i.p. injection of saline or CNO. The difference of these parameters between Cartptcre/cre and Cartptcre/+ mice was assessed by subtracting the value of CNO from that of saline at the corresponding time point for each mouse (C,F, I, L). Data are means ± SEM. n = 6–7; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001 for saline versus CNO treatments.

https://doi.org/10.7554/eLife.36494.011
Figure 9—figure supplement 1
CNO does not exert any effects on body skin temperature in the absence of hM3Dq-expression in Cartptcre/cre, Cartptcre/+ and wild type mice.

Temperatures of the interscapular brown adipose tissue (BAT) (A, B, C), the lumbar back (D, E, F), the tail (G, H, I) measured by high-sensitivity infrared imaging 1, 3 and 6 hr after i.p. injection of saline or CNO of Cartptcre/cre (A, D, G), Cartptcre/+ (B, E, H) without hM3Dq-injection and wild type mice receiving bilateral LHA→hM3Dq but lacking Cre-recombinase expression (C, F, I). Data are means ± SEM. n = 8.

https://doi.org/10.7554/eLife.36494.012
Figure 10 with 1 supplement
CNO-induced bilateral activation of hM3Dq-positive CART neurons in the LHA leads to an increase in ingestive behavior and body weight in heterozygous Cartptcre/+mice.

Cumulative food intake during fed state 6 hr following saline or CNO injection in bilateral LHA→hM3Dq Cartptcre/cre and Cartptcre/+ mice (A, B) as well as interaction with the food hopper (C, D). Cumulative water intake during fed state 6 hr following saline or CNO injection in bilateral LHA→hM3Dq Cartptcre/cre and Cartptcre/+ mice (E, F) and interaction with the water bottle (G, H). Body weight before and after injection of saline or CNO (indicated by dotted line) and corresponding body weight change at 1, 3 and 6 hr in proportion to pre-treatment body weight (I, J) measured in Cartptcre/cre and Cartptcre/+ LHA→hM3Dq mice receiving saline or CNO injection. Open and filled horizontal bars indicate the light and dark photoperiods, respectively. Dotted lines indicate the time of i.p. injection of saline or CNO. Data are means ± SEM. n = 6–7; *p≤0.05, **p≤0.01, ***p≤0.001 for saline versus CNO treatments.

https://doi.org/10.7554/eLife.36494.013
Figure 10—figure supplement 1
CNO does not exert any effects on ingestive behavior and body weight in the absence of hM3Dq-expression in Cartptcre/cre, Cartptcre/+ and wild type mice.

Cumulative food intake and food hopper interaction during fed state 6 hr following saline or CNO injection Cartptcre/cre (A, D) and Cartptcre/+ mice (B, E) without hM3Dq-injection and wild type mice (C, F) receiving bilateral LHA→hM3Dq but lacking Cre-recombinase expression. Cumulative water intake and interaction with the water bottle during fed state 6 hr following saline or CNO injection in Cartptcre/cre (G, J) and CARTcre/+ mice (H, K) and wild type mice (I, L). Body weight before and after injection of saline or CNO (indicated by dotted line) measured in Cartptcre/cre (M) and Cartptcre/+ (N) as well as wild type mice (O). Open and filled horizontal bars indicate the light and dark photoperiods, respectively. Data are means ± SEM. n = 8.

https://doi.org/10.7554/eLife.36494.014
hM3Dq-mCherry-positive CART neurons co-express GAD67 and MCH, but not orexin.

Fluorescence micrographs of coronal brain sections from respective LHA→hM3Dq Cartptcre/+ mice, showing partial co-expression of the mCherry reporter with GAD67 (A), and MCH (B) (white arrows, right panels), while no co-expression could be observed with orexin (C) (right panel). Scale bar = 75 μm.

https://doi.org/10.7554/eLife.36494.015
CART neurons in the LHA are highly heterogeneous and co-localise with various neurotransmitters involved in energy homeostasis.

Representative fluorescence micrograph showing fluorescent CART neurons in the LHA of a CART-TRAP mouse (A). Enrichment (>1) or derichment (<1) of various neurotransmitter transcripts in the immunoprecipitated (IP) mRNA relative to input mRNA of the LHA of CART-TRAP mice (B). Data are means ± SEM. n = 3. Opt, optical tract; V3, third ventricle.

https://doi.org/10.7554/eLife.36494.016

Data availability

All original data generated or analysed are included in the manuscript

Additional files

Supplementary file 1

Sequences of oligonucleotide primers used in qPCR

https://doi.org/10.7554/eLife.36494.018
Transparent reporting form
https://doi.org/10.7554/eLife.36494.019

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