A specific prelimbic-nucleus accumbens pathway controls resilience versus vulnerability to food addiction

Food addiction is linked to obesity and eating disorders and is characterized by a loss of behavioral control and compulsive food intake. Here, using a food addiction mouse model, we report that the lack of cannabinoid type-1 receptor in dorsal telencephalic glutamatergic neurons prevents the development of food addiction-like behavior, which is associated with enhanced synaptic excitatory transmission in the medial prefrontal cortex (mPFC) and in the nucleus accumbens (NAc). In contrast, chemogenetic inhibition of neuronal activity in the mPFC-NAc pathway induces compulsive food seeking. Transcriptomic analysis and genetic manipulation identified that increased dopamine D2 receptor expression in the mPFC-NAc pathway promotes the addiction-like phenotype. Our study unravels a new neurobiological mechanism underlying resilience and vulnerability to the development of food addiction, which could pave the way towards novel and efficient interventions for this disorder.


Supplementary Figures
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Supplementary Tables
Supplementary Table 1

Kolmogorov-Smirnov
Late period

Late period
Persistence to response U = 1043.50 Chi-square Genotype C-S = 7.06 P < 0.01         Fig. 3 and Supplementary Figure 6 Inhibition of PL-NAc core projection pathway leads to compulsivity          Kolmogorov-Smirnov

Kolmogorov-Smirnov
PL mPFC AAV-D2R     The differentially expressed genes have a fold change > 1.5 fold, P < 0.01 and average read counts > 40. Pressing on the active lever resulted in a food pellet delivery paired with a stimulus-light (associated-cue), located above the active lever, and while pressing on the inactive lever had no consequences. A food dispenser equidistant between the two levers permitted the delivery of food pellets when required. The floor of the chambers was a grid floor that served to deliver electric food shocks in the session of shock-test and served as a contextual cue in the session of shock-associated cue the day after the shock session. During the rest of selfadministration sessions, a metal sheet with holes was placed above the grid floor. Thus, mice could discriminate between different contexts. The chambers were made of aluminum and acrylic and were housed in sound-and light-attenuated boxes equipped with fans to provide ventilation and white noise.  genes were normalized using Tbp as housekeeping gene, although the same significant changes were also found using the other two housekeeping genes (Usp11, actin).
Furthermore, we measured the Drd2 mRNA levels in the mPFC of control and D2Roverexpressing mice by qPCR. qPCR analysis was carried out using designed specific forward (TGCAGACCACCACCAACTAC) and reverse (GGAGGTGGTAGGTGAGTGGAAA) primers to target both mouse and human Drd2 coding sequence and specific designed specific forward (CTCTGCTCCTCCCTGTTCC) and reverse (TCCCTAGACCCGTACAGTGC) primers for mouse Gapdh coding sequence.
Designed primers and cDNA extracted from brain samples were used to carry the qPCR experiments following the same procedure and experimental conditions as described above, except that here SYBR Green methodoloy was applied (Life Technologies). Relative mRNA levels were determined after normalization with Gapdh as housekeeping gene using the ΔΔCt method.
The DNA template for Drd2 probe was originally generated by RT-PCR from cDNA derived from total mouse brain, previously reported 7 . GenBank accession number, primer sequences and length of probe are listed therein. For a riboprobe specific for Cre recombinase RNA, we isolated the stretch of cDNA from Cre recombinase sequence of the AAV-retrograde-Cre (Addgene vector AAV pmSyn1-EBFP-Cre) using a forward primer which contains at the 5' end also the EcoR1 recognition sequence as well as 5 nucleotides at the very 5'end (fw primer 5′-ACTATGAATTCCGAGTGATGAGGTTCGCAAG-3′) and the reverse primer containing at the 5' end the XhoI recognition sequence preceded by 5 nucleotides (rev primer 5'-AACTACTCGAGCCGGTATTGAAACTCCAGCG-3') resulting in a 867 bp product.
PCR products were cloned into pBluescript KSand used as templates for riboprobe synthesis as described. The identity of subcloned fragments was checked by sequencing. Linearized template DNA was column purified (PCR purification kit, Invitrogen), resuspended in diethyl pyrocarbonate (DEPC)-treated H2O at a concentration of 1 µg/µl, and stored at -20°C.
For both probes in vitro transcription was carried out for 3 h at 37°C in a total volume of 20 µl containing 2 µg of linearized plasmid with inserts of desired genes Drd2 or Cre recombinase. Restriction enzymes (New England Biolabs) used for linearization and RNA polymerases used for each probe were as described 7 : Cre recombinase antisense: EcoRI, T3; Cre recombinase sense: XhoI, T7; Drd2 antisense: BamHI, T3; Drd2 sense: Eco RI, T7.
Pretreatment, hybridization and visualization of signals in fluorescent in situ hybridization procedure was carried out as described 8