Melanocortin 4 receptor signaling in dopamine 1 receptor neurons is required for procedural memory learning

Abstract

It is now widely recognized that exposure to palatable foods engages reward circuits that promote over-eating and facilitate the development of obesity. While the melanocortin 4 receptor (MC4R) has previously been shown to regulate food intake and energy expenditure, little is known about its role in food reward. We demonstrate that MC4R is co-expressed with the dopamine 1 receptor (D1R) in the ventral striatum. While MC4R-null mice are hyperphagic and obese, they exhibit impairments in acquisition of operant responding for a high fat reinforcement. Restoration of MC4R signaling in D1R neurons normalizes procedural learning without affecting motivation to obtain high fat diet. MC4R signaling in D1R neurons is also required for learning in a non-food-reinforced version of the cued water maze. Finally, MC4R signaling in neostriatal slices increases phosphorylation of the Thr34 residue of DARPP-32, a protein phosphatase-1 inhibitor that regulates synaptic plasticity. These data identify a novel requirement for MC4R signaling in procedural memory learning.

Figure 1. Expression of MC4R in D1R neurons of the ventral striatum

Mice expressing GFP under the control of the MC4R promoter (green) were bred to mice expressing Cre-recombinase under control of the dopamine 1 receptor promoter (red) and brain sections were processed for immunohistochemistry. Representative photomicrographs from the A. NAc core, B. lateral NAc shell, C. dorsomedial NAc shell, and D. olfactory tubercle are presented. Double-labeled neurons are highlighted by white arrows.

Figure 2. Restoration of MC4R expression in D1R neurons

Mice in which a transcriptional/translational blocking cassette flanked by lox-P sites was inserted into the MC4R gene (MC4R-TB) were crossed to mice expressing Cre-recombinase under the control of the dopamine 1A receptor (D1R-Cre) and MC4R expression was assessed by in situ hybridization. Expression of Cre-recombinase restores expression of MC4R specifically in the ventral striatum (A, B, and C), paraventricular nucleus of the hypothalamus (D, E, and F), and lateral olfactory tract (G, H, and I). vNAc-sh- ventral nucleus accumbens shell; PVH- paraventricular nucleus of the hypothalamus; 3v- third ventricle; LOT- lateral olfactory tract. N = 4/group. Representative darkfield image presented.

Figure 3. Loss of MC4R signaling affects operant responding for high fat diet

Mice were trained to nosepoke for a HFD pellet on a fixed ratio schedule and then advanced to a progressive ratio schedule to measure effortful responding for a HFD reward during both restricted feeding (P1–P4, shaded) and ad lib feeding (P5–P8). A. Body weights during testing period. B. Locomotor activity during restricted feeding (RF) and ad lib feeding (AL). C. Food intake during restricted feeding at 1, 2 and 4 hours. After completion of training, mice were advanced to progressive ratio testing. Both groups of MC4R-TB achieved fewer D. total nosepokes, E. rewards earned, and F. 30-minute breakpoint than wild-type mice. Statistically different groups are indicated (*p<0.05, **p<0.01, ***p<0.001). Data presented as mean ± S.E.M.

Figure 4. Restoration of MC4R signaling in D1R neurons rescues learning of operant responding

A. Schematic representation of nosepoke activity during FR1 training. Spectrum ranges from red (0 nosepokes) to green (30 nosepokes). B. MC4R-TB mice took longer to pass FR1 training than wild-type mice or mice with MC4R-TB/D1R-Cre mice. Statistically different groups are indicated (*p<0.05, **p<0.01). Data presented as mean ± S.E.M.

Figure 5. Restoration of MC4R signaling in D1R neurons rescues deficits in cued water maze learning

Mice were trained to find a hidden platform in a modified version of the water maze task that measures procedural memory formation. No differences in swim speed were noted during either probe trial on days A. four and B. eight. C. No preference for the ‘target’ is observed during the probe trial on day #4 as measured by time spent in the location of the ‘target’ cue. Restoration of MC4R expression in D1R neurons rescues learning of cued water maze by day #8 as measured by D. time at ‘target’ cue. Statistically different groups are indicated (*p<0.05, **p<0.01). Data presented as mean ± S.E.M.

Figure 6. MC4R signaling increases phosphorylation of DARRP-32 in neocortical slices

The melanocortin agonist melanotan II (MT II, 10 μM) was administered to freshly prepared neocortical slices. A. Representative western blot of phospho-Thr34 DARRP-32 and total DARRP-32 after MT II administration. B. MT II induces a significant increase in phosphorylation of DARRP-32 on Thr34 thirty seconds after administration. (*p<0.05). Data presented as mean ± S.E.M.