Male Long-Evans rats (total n=229, Charles River, Raleigh, NC, 250-390 g) were individually housed in a colony room under a reverse 12-h:12-h light-dark cycle with lights off at 9 am. We excluded 13 rats due to equipment failure, misplaced cannula, lost headcap, or illness. The rats were deprived to 85% of their free-feeding body weight at the beginning of the experiment and kept at that weight throughout the experiment, with free access to water. All procedures followed the guidelines outlined in the “Principles of Laboratory Animal Care” (NIH publication no. 85-23) and were approved by the local Animal Care and Use Committee. Experiments were conducted in 12 self-administration chambers (Med Associates, St Albans, VT). Each chamber had two levers 9 cm above the floor, but only one lever (“active,” retractable lever) activated the pellet dispenser, delivering 45-mg food pellets (# F00021, 5.5% fat, 60% carbohydrate, 4.5% fiber; Bioserv, Frenchtown, NJ). The chambers’ grid floors were connected to electric shock generators.
All drugs were prepared fresh before testing. NPY (Bachem, Torrance, CA, catalog number: H-6375), D-Phe CRF(12-41) ([D-Phe12
) (Bachem, Torrance, CA, catalog number: H-3266) and BIBO3304 ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate) (Tocris Bioscience, Ellisville, MO, catalog number: 2412) were each dissolved in saline. MTIP (3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine) (Lilly Research Laboratories, Indianapolis, IN) was dissolved in 10% Tween-80. BIIE0246 ((S)-N(2)-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl] cylopentyl] acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamid) (Tocris Bioscience, Ellisville, MO, catalog number: 1700) was dissolved in 30% polyethylene glycol (Sigma-Aldrich, St. Louis, MO). The injection volume for systemic injections was 1 ml/kg. The injection volume for ventricular (i.c.v.) delivery was 1 μl. D-Phe CRF(12-41) (0 and 10 μg, i.c.v.) and BIBO3304 (0, 20 and 40 μg, i.c.v.) were injected 10 min before testing. MTIP (0, 10 and 20 mg/kg, s.c.) and NPY (0, 5 and 10 μg, i.c.v.) were injected 30 min before testing. BIIE0246 (0, 2.5 and 5 mg/kg, i.p.) was injected 40 min before testing. Doses were chosen based on previous studies in rats showing effects on fear conditioning, ethanol self-administration and stress-induced reinstatement, stress-induced defecation, and blockade of the effects of Y2 agonism on food seeking (Gehlert et al., 2007
; Ghitza et al., 2007
; Gutman et al., 2008
; Kask et al., 2000
; Liu et al., 2002
; Scott et al., 2005
; Valdez et al., 2002
Intracranial surgery and intracranial injections
The rats were anaesthetized with a mixture of sodium pentobarbital and chloral hydrate (60 and 25 mg/kg, respectively, i.p.). They were then implanted with guide cannulae (23-gauge; Plastics One, Roanoke, VA) 2 mm above the right lateral ventricle: antero-posterior: -0.9 mm, medio-lateral: +1.4 mm and dorso-ventral: -2.0 mm (Paxinos et al., 2005
) using a stereotaxic instrument (Kopf, Tujunga, CA). The analgesic buprenorphine (0.1 mg/kg, s.c.) was given after surgery and the rats were allowed to recover for at least 5 days. The rats to be tested 2 days after training were implanted with guide cannulae before training. With the exception of 2 rats that were implanted with guide cannulae prior to training, the rats to be tested 1 month after training were implanted with cannulae 13-19 days after the last training day. Cannulae placements were verified by a positive dipsogenic response to angiotensin II (50 ng in 2 μl; Sigma, St. Louis, MO). Placements were considered accurate if a rat started drinking within 2 min of the injection and sustained drinking for 3-4 min (Sakai et al., 1995
). Ventricular injections of D-Phe CRF(12-41) (0 or 10 μg in 1 μl of physiological saline), NPY (0, 5 or 10 μg in 1 μl of physiological saline) and BIBO3304 (0, 20 or 40 μg in 1 μl of physiological saline) were made with Harvard infusion pumps, using 10-ml Hamilton syringes that were connected to 30-gauge injectors (Plastics One) via polyethylene-50 tubing. Injections lasted 1 min and injectors were left in place for an additional minute before being replaced with cannula blockers. Rats were placed in the operant chambers 10 min (D-Phe CRF(12-41) and BIBO3304) or 30 min (NPY) after the completion of the injection and the test program was then started immediately. One rat with a blocked cannula in Exp. 2 was tested without the vehicle injection and included in the vehicle group.
We used a fear incubation protocol consisting of 6 phases (): magazine training (1 session), lever-press acquisition (5 sessions), fear conditioning (1 or 10 sessions), incubation period (2 days or 1 month), baseline session (1 session), and test for cue-induced fear conditioning (1 session). Rats were trained during the dark cycle. Sessions began with extension of the active lever and illumination of a red houselight. Rats were weighed and fed after the daily sessions.
Experimental timeline and fear conditioning training
Food self-administration training
During the first session, the rats were given 60-min magazine training (pellet delivery every 125 s). The following day, 2 sessions of fixed-interval-1 (FI-1) reinforcement schedule (lever-presses could earn a pellet each sec) were run 2-4 h apart. These sessions ended when rats received 50 pellets (up to 1 h). A third session of up to 3 h was run immediately after the second session for rats that did not earn 50 pellets in the second session. All rats except 2 achieved 50 pellets in this session. These 2 rats were given an additional session the following day after their usual VI-30 session and they both earned 50 pellets within this session. The rats were then given one 90-min session in which pellets were earned under a variable-interval-30 (VI-30) reinforcement schedule (pellet availability for lever-presses ranging from 1 to 59 sec), and 2 daily 90-min sessions on a VI-60 schedule (pellet availability ranging from 1 to 119 sec). Rats were maintained on the VI-60 schedule for the rest of the experiment.
Fear conditioning training occurred over 1 or 10 90-min sessions during which the rats were given ten 30-sec tones (2900 Hz, 20 dB above background), ranging from 3 to 14 min apart and co-terminating with an electric shock (0.5-sec, 0.5-mA, scrambled, shock intensity adjusted for inter-chamber variability) while earning pellets on a VI-60 schedule. Conditioned inhibition of lever-pressing for food pellets was our measure of fear (Estes et al., 1941
; Hunt et al., 1951
; Miczek, 1973
Lever-presses were recorded during the 30-sec prior to tone presentation (Precue) and during the 30-sec tone presentation (Cue), and were converted into a suppression ratio: Suppression ratio = ((Precue-Cue)/(Precue+Cue)). The suppression ratio normalizes lever-pressing during the tone for baseline Precue responding (Annau et al., 1961
; Armony et al., 1997
). A value of 1 indicates total conditioned suppression of lever-pressing during tone presentation (high fear). A value of 0 reflects no lever-press suppression during tone presentation (no fear). In each experiment, the rats assigned to the different treatments were matched for their suppression ratios during training.
Conditioned fear testing
During the incubation periods, the food-restricted rats were weighed and handled 5-7 times per week. On the last day of each incubation period, lever-pressing was re-stabilized in a 90-min baseline session with no tones or shocks. The following day, conditioned fear to the tone was tested by presenting four 30-sec tones, without shock, over 35 min. The first tone occurred after 6.5 min and subsequent tones occurred after inter-trial intervals of 4, 7, and 11 min. The suppression ratios across the first 3 extinction trials were used as our measure of conditioned fear in order to assess the strength of the incubated fear response over repeated trials and to avoid a potential ceiling effect of a high fear response on the first trial. No effects were seen during the fourth trial in any of the experiments in this paper or in our previous report (Pickens et al., 2009
), thus this trial was not included in our analyses. During testing, the rats ate all pellets earned regardless of drug treatment.
Experiment 1: Effect of NPY on conditioned fear
We studied the effect of NPY on the expression of conditioned fear 2 days or 30-32 days (1 month) after long (10 days of 10 tone-shock pairings/day) training. We also studied the effect of NPY on the expression of conditioned fear 2 days after short (1 day of 10 tone-shock pairings) training. All of the rats given 1 day training and half of the rats given 10 day training were tested for responses to the fear tone 2 days after training. The other half of the rats given 10 day training was tested for responses to the tone 1 month after training. In the long-training condition, the experimental design included the between-subjects factors of Incubation Period (2 days or 1 month) and NPY dose (0, 5 or 10 μg) (n=11-16 per dose per incubation period). In the short-training condition, the experimental design included the between-subjects factor of NPY dose (0, 5 or 10 μg, n=8 per dose).
Experiment 2: Effect of NPY Y1 and Y2 receptor antagonists on conditioned fear
We studied the effect of the selective Y1 receptor antagonist BIBO3304 and the selective Y2 receptor antagonist BIIE0246 on the expression of fear conditioning 2 and 32 days (1 month) after long (10 days) training, respectively. The experimental design included the between-subjects factor of BIBO3304 dose (0, 20, or 40 μg, i.c.v., n=7-8 rats per dose) or BIIE0246 dose (0, 2.5 or 5 mg/kg, s.c., n=7-8 rats per dose).
Experiment 3: Effect of CRF receptor antagonists on conditioned fear
We studied the effect of the selective CRF1 receptor antagonist MTIP and the non-selective CRF antagonist D-Phe CRF(12-41) on the expression of fear conditioning 32-36 days (termed 1 month in the rest of the text and figures) after long (10 days) training. We also studied the effect of D-Phe CRF(12-41) on the expression of fear 2 days after short (1 day) training. In the long-training condition, the experimental design included the between-subjects factor of MTIP dose (0, 10, or 20 mg/kg, s.c., n=7-8 rats per dose) or D-Phe CRF(12-41) dose (0 or 10 μg, i.c.v., n=12-13 per dose). In the short-training condition, the experimental design included the between-subjects factor of D-Phe CRF(12-41) dose (0 or 10 μg, i.c.v., n=10 per dose).
Data were analyzed by Statistica 5.1 software (Tulsa, OK). The main dependent measure was suppression ratio that is defined as: ((Precue-Cue)/(Precue+Cue)). The factors used in the statistical analyses are described in the Results section and significant effects (p<0.05) in the different ANOVAs were followed by post-hoc Fischer PLSD tests.