Enhanced cortical extracellular levels of cholecystokinin-like material in a model of anticipation of social defeat in the rat

Abstract

The involvement of cholecystokinin (CCK) in the mechanisms of stress and/or anxiety was assessed by in vivo microdialysis in rats subjected to a social stress paradigm. During the initial 30 min period of each conditioning session, a male Sprague Dawley rat (intruder) was placed in a protective cage inside the cage of a male Tryon Maze Dull rat (resident), allowing unrestricted visual, olfactory, and auditory contacts but precluding close physical contact between them. During the following 15 min period, both the protective cage and the resident were removed (nondefeated intruders) or only the protective cage was removed allowing the resident to attack the intruder (defeated rats). This procedure was repeated once daily for 4 d. On the fifth day, a guide cannula was implanted into the prefrontal cortex of intruders. During a single 30 min test session, performed 4 d later, intruders were subjected to only the 30 min protected confrontation to the resident. Anxiety-like behavior (immobility, ultrasonic vocalizations, and defensive postures), associated with an increase (approximately +100% above baseline) in cortical outflow of CCK-like material (CCKLM), were observed in defeated intruders. Pretreatment with diazepam (5 mg/kg, i.p.), but not buspirone (0.5-2 mg/kg, i.p.), prevented both the anxiety-related behavior and CCKLM overflow. The selective CCK-B receptor antagonist CI-988 (2 mg/kg, i.p.) reduced the anxiety-like behavior without affecting the increase in CCKLM outflow. These data indicate that anticipation of social defeat induces a marked activation of cortical CCKergic neurons associated with anxiety-related behaviors in rats.

Fig. 1.

Social defeat stress procedure. A, Conditioning sessions. During period I (30 min), the intruder was placed in a protective cage inside the home cage of resident. The protective cage allowed unrestricted visual, auditory, and olfactory contacts with the resident but precluded close physical contact. Then the protective cage was removed, allowing the resident to attack the intruder (period II, 15 min). B, The experimental procedure consisted of four daily conditioning sessions (D1–D4) and one test session (D9) that involved the same pairs of residents and intruders. A microdialysis guide cannula was implanted on day 5. On day 9 in the course of the microdialysis study, intruders were subjected to a single test session that consisted of a 30 min protected confrontation to resident (period I only).

Fig. 2.

A, Behavioral responses of defeated rats during four consecutive conditioning sessions of social stress. Comparison with nondefeated intruders. Behavior of intruders was quantified during the 30 min period I of each conditioning session, when they were placed in a protective cage inside the home cage of residents (i.e., before removing the protective cage for 15 min, period II). The duration of immobility and USV and the number of defensive–submissive postures, rearings, and wall-climbings were measured for the entire period I (30 min). No significant changes were noted between the successive sessions for nondefeated intruders; only the data corresponding to the fourth session [(4)] are shown. Data are the means ± SEM of parameters recorded in eight defeated and six nondefeated intruders. *p < 0.01 versus defeated intruders on day 1; ‡p < 0.05 versus defeated intruders on day 2; †p < 0.01 versus nondefeated intruders on day 4. B, Effects of a 30 min protected exposure to resident rats during the microdialysis test session on the behavioral responses of defeated or nondefeated intruders. During the microdialysis experiment, i.e., on the ninth day of the protocol illustrated in Figure 1B, behavior of intruders was recorded for the 30 min protected exposure to residents. The five parameters were quantified as described above. Each bar is the mean ± SEM of data obtained in eight defeated and six nondefeated intruders. *p < 0.01 versus nondefeated intruders.

Fig. 3.

Effects of a 30 min protected exposure to residents on extracellular cortical levels of CCKLM in defeated or nondefeated intruders. Cortical CCKLM levels were measured for 120 min before, during (fraction 5), and 180 min after the 30 min exposure (arrow) of defeated (n = 8; ▪) or nondefeated (n = 6; ■) intruders to residents. Data are the means ± SEM of CCKLM contents of collected fractions, expressed as percentages of basal values, taken as the mean of fractions 1–3. Fraction 1 is the first fraction collected after a 90 min washout period after probe insertion. *p < 0.001 with respect to basal values.

Fig. 4.

Effects of diazepam (± flumazenil), buspirone, or CI-988 on cortical CCKLM outflow in defeated intruders subjected to a 30 min protected exposure to residents. A, Diazepam (5 mg/kg, i.p.; n = 8; ●), CI-988 (2 mg/kg, i.p.;n = 10; ■), or acacia gum (n= 7; ▪) was administered (small filled arrow) 30 min before the 30 min protected exposure to residents (corresponding to fraction 5; large filled arrow). Flumazenil (10 mg/kg, i.p.; n = 6; ○) was administered 5 min before diazepam. B, Buspirone [0.5 mg/kg (n = 6; ●), 1.0 mg/kg (n = 10; ○), or 2.0 mg/kg (n = 6; ■)] or saline (n = 7; ▪) was injected intraperitoneally 30 min before the 30 min protected exposure to residents. Data are the means ± SEM of CCKLM contents of collected fractions, expressed as percentages of basal values, taken as the mean of fractions 1–3. Fraction 1 is the first sample collected after a 90 min wash-out period after probe insertion. *p < 0.01; **p < 0.001 with respect to basal values (100%). †p < 0.05; ††p < 0.001 with respect to corresponding values for defeated intruders given acacia gum.