Female preproenkephalin-knockout mice display altered emotional responses

Abstract

The endogenous opioid system has been implicated in sexual behavior, palatable intake, fear, and anxiety. The present study examined whether ovariectomized female transgenic preproenkephalin-knockout (PPEKO) mice and their wild-type and heterozygous controls displayed alterations in fear and anxiety paradigms, sucrose intake, and lordotic behavior. To examine stability of responding, three squads of the genotypes were tested across seasons over a 20-month period. In a fear-conditioning paradigm, PPEKO mice significantly increased freezing to both fear and fear + shock stimuli relative to controls. In the open field, PPEKO mice spent significantly less time and traversed significantly less distance in the center of an open field than wild-type controls. Further, PPEKO mice spent significantly less time and tended to be less active on the light side of a dark-light chamber than controls, indicating that deletion of the enkephalin gene resulted in exaggerated responses to fear or anxiety-provoking environments. These selective deficits were observed consistently across testing squads spanning 20 months and different seasons. In contrast, PPEKO mice failed to differ from corresponding controls in sucrose, chow, or water intake across a range (0.0001-20%) of sucrose concentrations and failed to differ in either lordotic or female approach to male behaviors when primed with estradiol and progesterone, thereby arguing strongly for the selectivity of a fear and anxiety deficit which was not caused by generalized and nonspecific debilitation. These transgenic data strongly suggest that opioids, and particularly enkephalin gene products, are acting naturally to inhibit fear and anxiety.

Figure 1

Alterations in fear conditioning in WT mice, PPEKO mice with both alleles eliminated, and preproenkephalin HZ knockout mice with only one allele eliminated (HZ). The amount of time in a 30-sec period in which the animal exhibited freezing behavior (mean ± SEM) was assessed in animals during an auditory stimulus before (Pre Shock) and after (Post Shock) foot shock on 2 consecutive days (Days 1 and 2). Freezing behavior observed during the first day of auditory stimulation that served as the neutral stimulus (Pre Shock, Day 1) did not differ from the first day of shock, which served as the shock stimulus (Post Shock, Day 1). The PPEKO and HZ groups displayed significantly greater freezing responses during the second day of auditory stimulation, which served as a measure of fear conditioning (Pre Shock, Day 2), and all three groups displayed significantly greater freezing responses during the second day of shock, which served as the fear + shock condition (Post Shock, Day 2) (data not shown). PPEKO mice displayed significantly greater freezing responses during the second day of auditory stimulation both before (Pre Shock, Day 2, fear condition) and after (Post Shock, Day 2, fear + shock condition) shock relative to either WT (*) or HZ (✚) mice. Significant differences were observed among genotypes [ANOVA F(2,28) = 22.32; Tukey's t test, P < 0.0001], across conditions [F(3,18) = 184.08, P < 0.0001], and for the interaction between genotypes and conditions [F(6,84) = 12.54, P < 0.0001]. Variability as a function of squads of testing failed to account for any significant results.

Figure 2

Alterations (mean ± SEM) in either the amount of distance traversed (A) or the amount of time spent (B) in the center of an open field by WT, PPEKO, and HZ mice. PPEKO mice traversed significantly less distance [ANOVA, F(2,28) = 5.52; Tukey's t test, P < 0.01] and spent significantly less time (F = 3.49, P < 0.044) in the center of the open field than WT mice (*). HZ mice also spent significantly less time in the center of the open field than WT mice. Although the second squad of animals displayed significant increases in the traversed distances [F(2,12) = 6.88, P < 0.01] and elapsed time [F(2,12) = 14.98, P < 0.0005] in the center of the open field than the first and third squads, these effects occurred consistently over all genotypes and failed to account for any significant genotype results.

Figure 3

Alterations (mean ± SEM) in either the amount of time spent in the light compartment (A) or the amount of activity occurring in the light (B) and dark (C) compartments in WT, PPEKO, and HZ mice. PPEKO mice spent signicantly less time [ANOVA, F(2,28) = 3.89; Tukey's t test, P < 0.032] in the light compartment than either WT or HZ mice (*). The activity of the three groups in either the light (F = 2.50, P < 0.10) or dark [F = 0.44, not significant (n.s.)] compartments failed to differ significantly from each other. Significant differences were not observed either among squads [light time, F(2,12) = 2.97, n.s.; light activity, F = 1.99, n.s.; dark activity, F = 0.28, n.s.] or for the interaction between genotypes and squads [light time, F(4,24) = 2.12, n.s.; light activity, F = 2.21, n.s.; dark activity, F = 1.80, n.s.].

Figure 4

Alterations (mean ± SEM) in sucrose (A), chow (B), or water (C) intake across a range (0.0001–20%) of sucrose concentrations in WT (dark circles), PPEKO (open squares), and HZ (closed squares) mice. As expected, significant increases in sucrose intake [ANOVA, F(8,48) = 334.38; Tukey's t test, P < 0.0001] and significant decreases in chow (F = 319.57, P < 0.0001) and water (F = 47.47, P < 0.0001) intakes were observed as a function of sucrose concentration. However, the patterns of each of the three types of intake across the sucrose concentrations failed to differ among genotypes [sucrose, F(2,30) = 2.76, P < 0.07; chow, F = 2.71, P < 0.083; water, F = 2.44, n.s.]. Although the second squad of animals consumed significantly more sucrose [F(2,12) = 29.42, P < 0.0001] and chow (F = 4.12, P < 0.044) and drank significantly less water (F = 27.71, P < 0.0001) than the first and third squads, these effects occurred consistently over all genotypes and failed to alter the genotype results.

Figure 5

Alterations (mean ± SEM) in the lordosis quotient (A) and approach behaviors of female (B) and male (C) mice in sexual encounters involving WT, PPEKO, and HZ female mice. Although the three groups failed to differ in lordotic behavior [F(2,30) = 0.07, n.s.], HZ mice produced significantly more approach behaviors (*) than the other two groups (F = 4.66, P < 0.017). In contrast, male approach behavior failed to differ as a function of female genotype (F = 0.39, n.s.). Although the third squad of animals displayed significantly less lordotic behavior [ANOVA: F(2,12) = 22.76, Tukey's t test: P < 0.0001] and the first squad of animals displayed significantly more female approach behavior (F = 18.86, P < 0.0002) relative to the other squads, these effects occurred consistently over all genotypes and failed to alter the genotype results.