Sex differences in specific aspects of two animal tests of anxiety-like behavior

Abstract

Rationale: Anxiety, a negative state of high arousal and vigilance, is especially prevalent in women, making identification of underlying mechanisms critical for developing effective therapies. With the challenge of disentangling biological and social factors in humans, animal tests can provide valuable insights, although such tests, developed in males, have unclear validity for females.

Objective: To better understand patterns of sex differences across multiple measures within two classical rodent anxiety tests.

Methods: We examined female and male adult Wistar rats (n = 15-18/group) that were single-housed in the novelty suppression of feeding test (NSFT) that involves food under a bright light in food-restricted animals, and light-dark test (LDT), which reflects innate aversion to bright light. To further validate these tests in females, we also examined the impact of 1 mg/kg diazepam.

Results: NSFT measures of the most direct interaction with food, latency to grab food and food consumed, indicated increased anxiety-like behavior in females versus males, with diazepam altering these behaviors in females but not males. Most other measures showed more similar effects of diazepam across the sexes, with some evidence of reduced anxiety-like behavior in LDT for females. Principal component analyses indicated limited relationships across behavioral factors, underscoring previous suggestions of the importance of assessing multiple measures to maximize information and ethological relevance.

Conclusions: Combining our findings and previous studies, we speculate that increased anxiety-like behavior in females manifests especially when there is a specific, life-relevant condition (e.g., food in the NSFT). Our findings also validate NSFT and LDT use in females.

Keywords: Anxiety; Diazepam; Females; Light–dark box; Novelty-suppressed feeding; Sex difference.

Fig1. Timeline schematic for behavioral tasks

All rats were 5–6 months old, after single-housing since ~2 months old, and thus potential effects of differences in animal age should have limited impact for our findings. During the testing period, 30 minutes after diazepam or vehicle injection, rats were tested in NSFT for 15 minutes. Rats were removed to home cage for 5 minutes, then placed in Open Field (OField) for 10 minutes, then returned to the home cage for 30 minutes feeding assessment. One week later, 30 minutes after diazepam or saline injection, rats were tested in LDT for 10 minutes. DZP: diazepam; sal: saline.

Fig2. Significant interactions of sex and drug in NFST for measures most directly related to food interaction

(A) Latency to initially grab food was significantly longer under saline in females than males, and significantly reduced by diazepam in females but not males. (B) Food consumed during the test period under saline was significantly lower in females than males, and increased by diazepam in females but not males. DZP: diazepam; Sal: saline vehicle. *** indicates p<0.001 for diazepam within a particular sex; ##,### indicate p<0.01, p<0.001 across sexes for a given treatment condition (saline or drug).

Fig3. Putative anxiety-like NFST measures with more distal interaction with food showed less clear sex differences

(A) Total time spent in the brightly lit center was similar under saline in females and males, and was similarly and significantly enhanced by diazepam in both sexes. (B) Number of approaches to the center was not significantly different between sexes under saline or diazepam, although diazepam only significantly reduced number of approaches in males. DZP: diazepam; Sal: saline vehicle. **,*** indicate p<0.01, p<0.001 for diazepam within a particular sex.

Fig4. Overall similar locomotor, body weight, and non-test-day intake across sexes, although home cage intake on test day after NSFT showed sex effects

(A) Total distance traveled in the open field was significantly greater in females under both saline and diazepam. (B) Velocity in open field was slower in females under saline, with a similar trend under diazepam. (C) Home cage intake after the NSFT test showed similar responding as intake during the test (Fig. 2A), with less intake in saline females, and diazepam enhancement of intake in females but not males. (D,E) No differences in saline- vs diazepam-treated rats in (D) body weight, or (E) home cage intake not on the NSFT test day, although females weighed less and ate less (Online Resource 1). DZP: diazepam; Sal: saline vehicle. *** indicates p<0.001 for diazepam within a particular sex; ### indicates p<0.001 across sexes for a given treatment condition (saline or drug).

Fig5. Behavioral measures in LDT overall suggest reduced female anxiety-like responding relative to males

(A) Latency to enter the dark chamber was slowed in males but not females. (B-E) Latency to re-enter the lit chamber (B), time in the lit chamber (C), transitions between the lit and dark chambers (D), and rearing (E) had significant effects of sex and drug but no interaction (see Results). DZP: diazepam; Sal: saline vehicle. ** indicates p<0.01 for diazepam within males.