The female urine sniffing test: a novel approach for assessing reward-seeking behavior in rodents

Abstract

Background: Abnormal hedonic behavior is a key feature of many psychiatric disorders. Several paradigms measure reward-seeking behavior in rodents, but each has limitations. We describe a novel approach for monitoring reward-seeking behavior in rodents: sniffing of estrus female urine by male mice, along with number of ultrasonic vocalizations (USVs) emitted during the test.

Methods: The female urine sniffing test (FUST) was designed to monitor reward-seeking activity in rodents together with tests of helplessness and sweet solution preference. USVs and dopamine release from the nucleus accumbens (NAc) were recorded. Sniffing activity was measured in 1) manipulation-naive C57BL/6J and 129S1/SVImJ mice and Wistar-Kyoto rats; 2) stressed mice; 3) two groups of mice that underwent the learned helplessness paradigm-one untreated, and one treated with the SSRI citalopram; and 4) GluR6 knockout mice, known to display lithium-responsive, mania-related behaviors.

Results: Males from all three strains spent significantly longer sniffing female urine than sniffing water. Males emitted USVs and showed significantly elevated NAc dopamine levels while sniffing urine. Foot-shock stress significantly reduced female urine sniffing time. Compared with mice that did not undergo the LH paradigm, LH males spent less time sniffing female urine, and citalopram treatment alleviated this reduction. Compared with their wildtype littermates, GluR6KO males sniffed female urine longer and showed enhanced saccharin preference.

Conclusions: In rodents, sniffing female urine is a preferred activity accompanied by biological changes previously linked to reward-seeking activities. The FUST is sensitive to behavioral and genetic manipulation and to relevant drug treatment.

Figure 1

Illustration of the female urine sniffing test (FUST). A male C57BL/6J mouse sniffs a cotton-tipped applicator dipped into urine from a female mouse in estrus to assess reward-seeking behavior in rodents. The behavior counted as sniffing duration only when the mouse or rat nose was adjacent to the cotton-tipped applicator (as seen in the figure) and the researcher could detect that the rodent was sniffing.

Figure 2

Cumulative time spent sniffing for male C57BL/6J and 129S1/SVImJ mice and Wistar-Kyoto (WKY) rats exposed to female urinary pheromone odors. Different strains of manipulation-naive male mice (C57BL/6J; 129S1/SVImJ) and rats (WKY) underwent the female urine sniffing test (FUST) paradigm. Male mice and rats were introduced for 3 min to a cotton-tipped applicator dipped in water (as a control). After 45 min, they were introduced for 3 min to a cotton-tipped applicator dipped into estrus female urine from the same strain. All male rodents spent significantly more time sniffing estrus female urine than water during the 3-min FUST (**p<.01; n = 10 – 14).

Figure 3

Ultrasonic vocalizations (USVs) in male C57BL/6J and 129S1/SVImJ mice sniffing water or female urine. Different strains of manipulation-naive male mice (C57BL/6J; 129S1/SVImJ) underwent the female urine sniffing test paradigm. Male mice and rats were introduced for 3 min to a cotton-tipped applicator dipped in water (as a control). After 45 min, they were introduced for 3 min to a cotton-tipped applicator dipped into estrus female urine from the same strain. All male mice displayed significantly more USVs during exposure to estrus female urine than water (*p < .05; n = 12–14).

Figure 4

Dopamine levels in 129S1/SVImJ mice during the female urine sniffing test (FUST). 129S1/SVImJ male mice underwent microdialysis surgeries and then underwent the FUST paradigm. Male mice and rats were introduced for 3 min to a cotton-tipped applicator dipped in water (as a control). After 45 min, they were introduced for 3 min to a cotton-tipped applicator dipped into estrus female urine from the same strain. Extracellular NAc dopamine samples were collected during the entire paradigm. During the FUST, 129S1/SVImJ male mice displayed significantly elevated levels of extracellular dopamine released in the NAc after exposure to estrus female urine but not water (**p < .01; n = 5). NAc, nucleus accumbens.

Figure 5

Time spent sniffing by C57BL/6J mice before and after foot-shock stress. C57BL/6J male mice underwent a foot-shock stress paradigm. The female urine sniffing test (FUST) paradigm was conducted twice: once before stress induction and once afterward. Male mice and rats were introduced for 3 min to a cotton-tipped applicator dipped in water (as a control). After 45 min, they were introduced for 3 min to a cotton-tipped applicator dipped into estrus female urine from the same strain. After foot-shock stress exposure, C57BL/6J male mice had significantly decreased mean sniffing duration of estrus female urine but not water in the FUST (**p< .01; n = 13).

Figure 6

(A) After undergoing the learned helplessness (LH) paradigm, both LH and non-LH (NLH) male C57BL/6J mice underwent the female urine sniffing test (FUST) paradigm. Both the LH and NLH male C57BL/6J mice spent significantly more time sniffing estrus female urine than water (**p < .01). NLH C57BL/6J mice spent significantly longer sniffing the estrus female urine than the LH mice (*p < .05; n = 8–12). No significant differences were found between these groups for time spent sniffing the water-dipped applicator. (B) The LH and NLH C57BL/6J mice displayed significantly more ultrasonic vocalizations during exposure to urine than water in the FUST paradigm (*p < .05; n = 7).

Figure 7

Time spent sniffing female urinary pheromone odors in learned helplessness (LH) 129S1/SVImJ mice treated with saline or citalopram. LH 129S1/SVImJ male mice were treated with the selective serotonin reuptake inhibitor citalopram or saline for 14 days and then underwent the female urine sniffing test (FUST) paradigm. LH 129S1/SVImJ male mice treated with either citalopram or saline both spent significantly more time sniffing estrus female urine than water (**p < .01). However, LH 129S1/SVImJ mice treated with citalopram spent significantly longer sniffing the urine-dipped applicator than the saline-treated LH mice in this paradigm (*p < .05; n = 7–8). No significant differences were found between these two groups for time spent sniffing the water-dipped applicator.

Figure 8

Male GluR6 knockout (KO) mice and their wild-type (WT) littermates underwent the saccharin preference test. Animals were given one bottle of tap water and one of saccharin diluted in tap water to a final concentration ranging from .005% to .35% overnight, and the percentage of saccharin preference per day was calculated. GluR6 KO male mice displayed a significantly higher preference for saccharin solution than water compared to WT mice across the range of saccharin concentrations (**p < .01; *p < .05; n = 13).

Figure 9

(A) GluR6 knockout (KO) and wildtype (WT) male mice spent significantly more time sniffing estrus female urine than water in the female urine sniffing test (FUST) paradigm (**p < .01). Genotype did not affect sniffing per se; however, whereas GluR6KO mice spent significantly less time sniffing water than their WT controls, they spent significantly more time sniffing estrus female urine than the WT mice (**p< .01; n = 11). (B) GluR6KO and WT mice displayed significantly more ultrasonic vocalizations (USVs) during exposure to estrus female urine than water in the FUST paradigm (**p < .01; n = 12). There were no significant differences between the groups in number of USVs after exposure to the water. GluR6KO mice exhibited more USVs during the FUST than their WT controls (*p < .05). (C) GluR6KO mice that underwent the FUST paradigm displayed longer bouts of USVs than their WT controls during exposure to estrus female urine (**p < .01; n = 12). (D) GluR6KO mice displayed a significantly higher peak call frequency during bouts of USVs than WT mice during exposure to estrus female urine (*p < .05; n = 12).