A Mutation in the Transmembrane Domain of Adenylate Cyclase 3 Impairs Enzymatic Function to Cause Sex-Specific Depression- and Anxiety-Like Behaviors and Food Seeking in a Rat Model

Abstract

We have previously demonstrated that a transmembrane domain mutation in Adenylate cyclase 3 (Adcy3) causes increased adiposity and negative emotion-like behaviors in a rat model. We set out to replicate and expand upon our previous study by conducting comprehensive behavioral testing, and we also investigated the molecular changes that result from this mutation. Rats with a mutation in the second transmembrane helix of ADCY3 (Adcy3^mut/mut) and wild-type rats were fed a high-fat diet for 12 weeks. We measured body weight, body composition, and depression-like and anxiety-like behaviors using the following tests: sucrose splash test, sucrose preference test, forced swim test, open field test, elevated plus maze, successive alleys test, and novelty-suppressed feeding. We also measured serum leptin levels, hypothalamic cyclic AMP (cAMP) production, and membrane fraction ADCY3 content. Adcy3^mut/mut male and female rats had increased adiposity. Adcy3^mut/mut males showed increased despair- and anxiety-like behaviors, food seeking, and higher leptin levels relative to wild-type males. Adcy3^mut/mut females showed only mildly increased anxiety-like behaviors relative to wild-type females. Adcy3^mut/mut rats of both sexes had decreased cAMP production in the hypothalamus, with no changes in ADCY3 content in the membrane fraction. We conclude that the transmembrane domain of ADCY3 plays a critical role in regulating adiposity and behavior, as well as cAMP production. There were key differences between males and females for the observed phenotypes. This study supports the idea that Adcy3 contributes to emotion-like behaviors and potentially mental health disorders, and that the transmembrane domain of ADCY3 is important for protein function.

Keywords: anxiety; cyclic AMP; depression; genetics; obesity; rat models.

FIGURE 1

Study design, body weight, and fat and lean mass in Adcy3^mut/mut. (A) Timeline of the study in WOD. Rats were given an EchoMRI and seven behavioral tests across two buildings before necropsy (sac) at 12 WOD. (B) Adcy3^mut/mut males (M) gain more weight than wild‐type (WT) M over the course of the study. (C) There were no significant differences in body weight between Adcy3^mut/mut and WT females (F). (D) After seven WOD, both Adcy3^mut/mut M and F have more fat mass than WT rats. (E) Adcy3^mut/mut F tend to have less lean mass than WT F. (F) Adcy3^mut/mut M and F have larger retroperitoneal (RetroFat), gonadal, and omental (OmenFat) fat pads at sac than WT. EPM, elevated plus maze; FST, forced swim test; NSF, novelty‐suppressed feeding test; OFT, open field test; SAT, successive alleys test; SPT, sucrose preference test. Mean ± SEM. t test or rmANOVA, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

Adcy3^mut/mut behavior in the sucrose splash test. (A) No differences between Adcy3^mut/mut and wild‐type (WT) rats in splash test latency to groom. (B) No differences between Adcy3^mut/mut and WT rats in splash test total grooming time over the full 5 min, although males (M) did groom significantly more than females (F). (C) Adcy3^mut/mut M show a significantly different pattern of grooming in the splash test relative to WT M, grooming more than WT M in the first 2 min of the test. (D) No differences between Adcy3^mut/mut F and WT F in splash test grooming. Mean ± SEM. Two‐way ANOVA or rmANOVA, *p < 0.05.

FIGURE 3

Adcy3^mut/mut behavior in the sucrose preference test (SPT). All four groups significantly preferred sucrose over water, but there were no significant differences between Adcy3^mut/mut and WT rats in sucrose preference rate. Mean ± SEM. Two‐way ANOVA, ****p < 0.0001.

FIGURE 4

Adcy3^mut/mut behavior in the forced swim test (FST). Adcy3^mut/mut males (M) were less mobile and more immobile in the FST than wild‐type (WT) M. There were no differences between Adcy3^mut/mut and WT females (F) in the FST, although F were significantly more mobile than M. Mean ± SEM. Two‐way ANOVA or t test, *p < 0.05, **p < 0.01.

FIGURE 5

Adcy3^mut/mut behavior in the open field test (OFT). (A) Females (F) have increased OFT total distance relative to males (M), although there are no differences between Adcy3^mut/mut and wild‐type (WT) rats in OFT total distance. (B) Adcy3^mut/mut M appears to have decreased OFT center time, although this does not reach significance. (C) No differences between Adcy3^mut/mut and WT F in OFT center time. (D) Adcy3^mut/mut M spend less time vertically rearing in the OFT than WT M. (E) No differences between Adcy3^mut/mut and WT F in OFT vertical rearing. (F) No differences between Adcy3^mut/mut and WT M in OFT grooming activity. (G) Adcy3^mut/mut F groom more than WT F in the first 5 min of the OFT. Mean ± SEM. t test, two‐way ANOVA, or rmANOVA, *p < 0.05, ****p < 0.0001.

FIGURE 6

Adcy3^mut/mut behavior in the elevated plus maze (EPM) and successive alleys test (SAT). (A–G) No differences between Adcy3^mut/mut and wild‐type (WT) rats in any variable measured in the EPM or SAT, although there are sex differences in several variables. Both WT and Adcy3^mut/mut rats spend most of their time in the EPM junction and the enclosed arm of the SAT, suggesting a floor effect is present. Mean ± SEM. Two‐way ANOVA, *p < 0.05, **p < 0.01.

FIGURE 7

Adcy3^mut/mut behavior in the novelty‐suppressed feeding test (NSF). (A) Adcy3^mut/mut males (M) spend significantly more time eating than wild‐type (WT) M in the NSF, while Adcy3^mut/mut females (F) spend significantly less time eating than WT F. F also spend significantly more time eating than M. (B) No significant differences between Adcy3^mut/mut and WT rats in food approach frequency or eating frequency. (C) F have a significantly lower food approach latency than M, but Adcy3^mut/mut F have increased eating latency relative to WT F. N = 7–11 due to removal of rats that did not eat at all during the test. Mean ± SEM. t test or two‐way ANOVA, *p < 0.05, **p < 0.01.

FIGURE 8

Serum leptin in Adcy3^mut/mut. Adcy3^mut/mut males (M) have significantly higher serum leptin levels than wild‐type (WT) M. There are no significant differences in serum leptin between Adcy3^mut/mut and WT females (F). Mean ± SEM. t test, **p < 0.01.

FIGURE 9

cAMP production and ADCY3 membrane localization in Adcy3^mut/mut. (A) Adcy3^mut/mut males (M) have less cAMP in the hypothalamus than wild‐type (WT) M, both with and without forskolin stimulation. Adcy3^mut/mut females (F) have significantly less cAMP production than WT F only with forskolin stimulation. (B) No differences in membrane or cytosolic ADCY3 content between Adcy3^mut/mut rats and WT rats. (C) Representative Western blot image of ADCY3 in the membrane and cytosol fractions. (D) Representative images of ADCY3‐ and DAPI‐stained sections of the arcuate nucleus of the hypothalamus in male rats. Adcy3^+/− rats show less ADCY3 staining qualitatively than WT rats, while Adcy3^mut/mut rats do not. Mean ± SEM. t test, **p < 0.01.