Relation of addiction genes to hypothalamic gene changes subserving genesis and gratification of a classic instinct, sodium appetite

Abstract

Sodium appetite is an instinct that involves avid specific intention. It is elicited by sodium deficiency, stress-evoked adrenocorticotropic hormone (ACTH), and reproduction. Genome-wide microarrays in sodium-deficient mice or after ACTH infusion showed up-regulation of hypothalamic genes, including dopamine- and cAMP-regulated neuronal phosphoprotein 32 kDa (DARPP-32), dopamine receptors-1 and -2, α-2C- adrenoceptor, and striatally enriched protein tyrosine phosphatase (STEP). Both DARPP-32 and neural plasticity regulator activity-regulated cytoskeleton associated protein (ARC) were up-regulated in lateral hypothalamic orexinergic neurons by sodium deficiency. Administration of dopamine D1 (SCH23390) and D2 receptor (raclopride) antagonists reduced gratification of sodium appetite triggered by sodium deficiency. SCH23390 was specific, having no effect on osmotic-induced water drinking, whereas raclopride also reduced water intake. D1 receptor KO mice had normal sodium appetite, indicating compensatory regulation. Appetite was insensitive to SCH23390, confirming the absence of off-target effects. Bilateral microinjection of SCH23390 (100 nM in 200 nL) into rats' lateral hypothalamus greatly reduced sodium appetite. Gene set enrichment analysis in hypothalami of mice with sodium appetite showed significant enrichment of gene sets previously linked to addiction (opiates and cocaine). This finding of concerted gene regulation was attenuated on gratification with perplexingly rapid kinetics of only 10 min, anteceding significant absorption of salt from the gut. Salt appetite and hedonic liking of salt taste have evolved over >100 million y (e.g., being present in Metatheria). Drugs causing pleasure and addiction are comparatively recent and likely reflect usurping of evolutionary ancient systems with high survival value by the gratification of contemporary hedonic indulgences. Our findings outline a molecular logic for instinctive behavior encoded by the brain with possible important translational-medical implications.

Fig. 1.

Gene expression study from hypothalamus. (A) Regulated genes either linked to monoaminergic signaling and/or gratification or belonging to the addiction gene sets. The far right column with numerals next to the heat map indicates maximum fold regulation (log₂) for the given gene. Note that all shown genes were regulated between at least one Na⁺ appetite group vs. control, and the regulation is apparently less tight in the gratified state; in some cases, it is reverted (e.g., PENK for furosemide group and PPP1R1B and ADRA2C for ACTH group) (Fig. S1). (B) RNA validation of gene expression by qRT-PCR. Fold regulation was calculated using the ΔΔCt method (44). Note that all fold regulations showed P values < 0.01 for Na⁺ appetite (blue bars). Red bars represent fold regulation of genes whose expression was stimulated with appetite that reversed after gratification (P < 0.05); white bars represent genes regulated by appetite that do not reverse with gratification (P > 0.05). (C) Coregulation of genes as illustrated by enrichment scores resulting from GSEA for individual Na⁺ appetite groups and in combination (i.e., common genes regulated by both stimuli); normalized enrichment scores (allowing comparison of enrichment between sets) and nominal P values (statistical significance of the enrichment score for a single gene set) are shown. Left shows Na⁺ appetite, and Right shows the gratified state. Note the striking loss of normalized enrichment score > 1.5 and nominal P value < 0.05 in gratified animals for both addiction gene sets with significant enrichment when using the combined (furosemide plus ACTH common genes) dataset. (D) Detailed analysis of gene expression in gratified animals showed a loss of significant gene regulation, which is illustrated in Left; it shows the P value of fold regulation vs. control for the top 100 genes. Right shows the proportion of the top 600 genes whose expression is regulated during appetite (blue bars) that revert their initial direction of regulation in response to gratification (red bars) and the number of those genes with a statistically significant difference gratified vs. Na⁺ appetite (green bars).

Fig. 2.

Orexinergic lateral hypothalamic neurons up-regulate DARPP-32 and ARC in sodium-depleted rats. (A Left) Panels of micrographs show orexinergic labeling, colocalization with DARPP-32 immunoreactivity, and colocalization with ARC. Note the increased immunoreactivity of lateral hypothalamic neurons in sodium-depleted animals. (Scale bar: 30 μm.) (Right) An anatomic orientation of the hypothalamus with the target area depicted in A. (B) Representative confocal micrographs showing partial subcellular colocalization of ARC and DARPP-32 in orexin-expressing lateral hypothalamic neurons. (Scale bar: 30 μm.) (C) Morphometry (densitometry) of DARPP-32 and ARC expression in lateral hypothalamic orexinergic neurons. DARPP-32 and ARC are regulated in sodium depletion; differences are statistically significant at levels of P < 0.01 (t test; n = 3 animals per group and n = 17–40 orexin-expressing neurons per animal). (D) Morphometry of colabeling in lateral hypothalamic neurons. The blue bar indicates, based on data from three sodium-deprived rats (n = 19, 32, and 33 lateral hypothalamic neurons positive for orexin), the percentage of colocalization of DARPP-32. The threshold was set at three times the average of nondepleted control animals. The red bar indicates the 100% colocalization of DARPP-32 with ARC in the orexin-DARPP-32–coexpressing neurons.

Fig. 3.

Modulation of reward pathways in sodium appetite. A shows dose-dependent effects of specific inhibition of D1(5) and D2(3) receptors with specific compounds, SCH23390 and raclopride, on sodium appetite of sodium-depleted mice. Cumulative 0.3 M NaCl was assessed every 15 min. Note the striking effect of both compounds at higher doses and attenuation at lower doses, with raclopride-treated mice showing higher variation. B shows cumulative intake of water in response to osmotic dehydration in mice, the attenuating effects of raclopride (lower dose), mGlu5 antagonist, and MTEP, and the complete lack of effect of SCH23390 (higher dose). C illustrates normal depletion-evoked sodium appetite in Drd1^−/− mice; however, there is complete lack of effect of SCH23390 (again, higher dose) in these mice, reiterating the specificity and complete absence of off-target effects of this compound at 0.2 mg/kg. (D) The bar diagram shows averaged sodium appetite for four rats with intrahypothalamic catheters on injection of 200 nL aCSF and the striking reduction when they were infused with 200 nL SCH23390 at 100 nM; P < 0.01 (t test for paired samples) for this dataset (Materials and Methods and Fig. S3). E shows a neuroanatomic schematic, with red circles indicating the location of the injection needle in case of complete elimination of sodium appetite and green circles indicating attenuation (Fig. 2A and Fig. S3B).