The expression of MC4Rs in D1R neurons regulates food intake and locomotor sensitization to cocaine

Abstract

While it is known that mice lacking melanocortin 4 receptor (MC4R) expression develop hyperphagia resulting in early-onset obesity, the specific neural circuits that mediate this process remain unclear. Here, we report that selective restoration of MC4R expression within dopamine-1 receptor-expressing neurons [MC4R/dopamine 1 receptor (D1R) mice] partially blunts the severe obesity seen in MC4R-null mice by decreasing meal size, but not meal frequency, in the dark cycle. We also report that both acute cocaine-induced anorexia and the development of locomotor sensitization to repeated administration of cocaine are blunted in MC4R-null mice and normalized in MC4R/D1R mice. Neuronal retrograde tracing identifies the lateral hypothalamic area as the primary target of MC4R-expressing neurons in the nucleus accumbens. Biochemical studies in the ventral striatum show that phosphorylation of DARPP-32(Thr) (-34) and GluR1(Ser) (-845) is diminished in MC4R-null mice after chronic cocaine administration but rescued in MC4R/D1R mice. These findings highlight a physiological role of MC4R-mediated signaling within D1R neurons in the long-term regulation of energy balance and behavioral responses to cocaine.

Keywords: Cocaine; dopamine-1 receptor; food intake; locomotor sensitization; melanocortin 4 receptor; obesity.

Figure 1. MC4R signaling in D1R neurons regulates body weight and food intake

(a–d) Restoration of MC4Rs specifically in D1R neurons results in a significant reduction in body weight on chow in male (a) and female (c) mice and on high-fat diet in male (b) and female (d) mice. *P < 0.05, **P< 0.01 and ***P< 0.001 compared with MC4R–TB group by two-way ANOVA with Bonferroni’s post hoc analysis.(e and f) Food intake for genotypes above was measured in separate cohorts of mice at 6–7 weeks of age. Restoration of MC4Rs in D1R neurons decreases intake of both chow and high-fat diet in male (e) and female (f) mice compared with MC4R-TB mice. (g) NMR body composition analysis in 8- to 9-week-old male mice. *P< 0.05, **P< 0.01 and ***P< 0.001 compared between the groups by one-way ANOVA with Tukey’s post hoc analysis. Data are presented as mean±SEM.

Figure 2. MC4R signaling in D1R neurons regulates meal size and acute cocaine-induced anorexia

(a–f) Metabolic cage analysis confirmed reduction of food intake in MC4R/D1R mice (a) and further showed that restoration of MC4Rs in D1R neurons reduces meal size (b), but not meal frequency (c), in the dark cycle. Oxygen consumption (d), carbon dioxide production (e) and daily locomotor activity (f) were not affected in MC4R–TB/D1R mice. (g and h) Acute (g) cocaine- and (h) MTII-induced suppression of 4-h food intake was significantly blocked in MC4R–TB mice but normalized in MC4R/D1R mice. *P< 0.05, **P< 0.01 and ***P < 0.001 compared between the groups by one-way ANOVA with Tukey’s post hoc analysis. Data are presented as mean±SEM.

Figure 3. MC4R–positive neurons in the NAc shell project to the LHA

Representative injection case showing that injection was exactly made into the LHA (a). Double immunohistochemistry for FG and MC4R-GFP showed that retrograde tracer-positive neurons in the ventrolateral part of the NAc shell were colocalized with MC4R-GFP (b). Schematic drawing showing the detailed distribution of FG/GFP double-positive neurons in the NAc shell (c). (d and f) Multichanneled zoom-in pictures for boxes b1 and b2 of (b), respectively. Pictures in (e) and (g) are high magnification from (d) and (f). f, fornix; ac, anterior commissure. Scale bars: 320 µm for (a and b); 80 µm for (d and f) and 20 µm for (e and g).

Figure 4. MC4R signaling in D1R neurons affects locomotor sensitization to cocaine and intracellular signaling in the ventral striatum

(a) Development of locomotor sensitization to repeated cocaine administration was significantly blocked in MC4R-TB mice compared with control mice, which was fully normalized in MC4R/D1R mice. *P < 0.05, **P< 0.01 and ***P< 0.001 are for MC4R-TB vs. control groups; #P <0.01 for MC4R-TB vs. MC4R/D1R groups (two-way ANOVA with Bonferroni’s post hoc analysis). (b) Representative images of the blots of pT³⁴-DARPP-32 and beta-actin. (c) Quantitative analysis showed that pT³⁴-DARPP-32 levels after cocaine exposure were significantly decreased in MC4R-TB mice but normalized in MC4R/D1R mice. (d) Representative images for the blots of pS⁸⁴⁵-GluR1 and beta-actin. (e) Quantitative analysis showed that pS⁸⁴⁵-GluR1 levels after cocaine exposure were significantly decreased in MC4R-TB mice but normalized in MC4R/D1Rmice. *P< 0.05 by one-way ANOVA with Tukey’s post hoc analysis. Data are presented as mean±SEM.