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Brain Research, 585 (1992) 94-104 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 17814

Evidence that cholecystokinin-enhanced retention is mediated by changes in opioid activity in the amygdala James F. Flood, Jon S. Garland and John E. Morley VA Medical Center, Geriatric Research Educational and Clinical Center, St. Louis, MO 63106 (USA) and Division of Geriatric Medicine, St. Louis Unit'ersity Hospital, St. Louis, MO 63104 (USA)

(Accepted 28 January 1992)

Key words: Active avoidance; D-Amphetamine;CCK-8; Cholecystokinin;/3-Endorphin; Epinephrine; Memory; Mouse; Naloxone;

Opioid; Retention

Mice, partially trained to avoid footshock in a T-maze, showed enhanced retention relative to vehicle-injected mice when treated peripherally with arecoline, D-amphetamine, cholecystokinin octapeptide (CCK-8), epinephrine or naloxone. Both intra-amygdaloid and intraventricular injections of/3-endorphin resulted in amnesia. D-amphetamine and arecoline blocked the amnestic effect of/J-endorphin administered into the amygdala but it required higher doses for CCK-8, epinephrine and naloxone to block the amnestic effect of/3-endorphin. The effects of CCK-8, epinephrine and naloxone showed a differential ability to block amnesia induced by ~-endorphin intraventricularly with epinephrine and naloxone preventing amnesia but CCK-8 not improving retention. This data suggests that the memory enhancement produced by peripherally administered CCK-8 involves the amygdala and that both CCK-8 and epinephrine interact with opioid amnestic mechanisms within the amygdala to alter memory processing.

INTRODUCTION Recent evidence suggests that opioid and noradrenergic systems are involved in learning and memory processes, The amygdala has been suggested as a possible site at which the opioid and noradrenergic systems interact in memory processing 23,32,53. Various types of studies indicate that the amygdala is involved in memory processing, Retention is impaired by electrical stimulation of the amygdala45,5~.52. Catecholamine synthesis and protein synthesis inhibitors caused amnesia when injected into the amygdala~4, The opiate agonist levorphanol caused dose-dependent amnesia when injected into the amygdala while naloxone improved retention 2~ as did naltrexone, diprenorphine and levalIorphan 2°. Post-training injections of opiates such as morphine or opioid peptides such as /]-endorphin (B-END), Met- and Leu-enkephalins impair retention for passive and active avoidance and habituation to noveltY 5'21'33'35'38'44'48'49. Opioid antagonists naloxone, naltrexone, diprenorphine and levallorphan block the

amnestic effects of opioid peptides s,21,-~'~.Naloxone and naltrexone have been found to enhance retention in a variety of experimental paradigms 2`6.12`i'~-22,2'~,33,ss,s~' possibly by blocking opioid inhibition of the noradrenergic system when naloxone is 8dministered centrally 2`a. It has been proposed that opioids may exert a tonic inhibition on the noradrenergic system 34. This is supported by biochemical studies that report opioids inhibit the release of norepinephrine and reduce neuronal firing 1,3`43.s~,c'6.e'~, Pharmacological studies have reported that higher doses of the ~-adrenergic agonist, clenbuterol, were required to improve retention when B-END was administered at the same time and lower doses were needed to improve retention when naloxone was administered simultaneously -a°. lntra.amygdaloid administration of the noradre.ergic receptor antagonist, propranolol, or the ~-adrenergic antagonists, atenolol and zinterol, attenuated the memoryfacilitating effect of peripherally administered naloxone 54,

Evidence suggests that modulation of memory processing may occur due to increased visceral and neu-

Correspondence to: F. Flood, VA Medical Center (151/JC), 915 N. Grand Blvd., St. Louis, MO 63109, USA.

95 roendocrine activity in the periphery as well as stimulation of areas of the brain involved in motivation and reinforcement 24,26.4m,57. Consistent with these theories is that feeding after training ~5'27'57or peripheral admin-

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istration of cholecystokinin octapeptide (CCK-8) has been found to improve retention for active and passive footshock avoidance training 7'~°'4°. CCK-8-induced improvement of retention was found to be dependent on

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Fig. 1. A shows a standard brain section indicating the area over which amygdaloid injections were gtven anu n , typ,,,o . . . . . . . . . . . . . . . . . . standard brain section indicating the area over which needle tracts for intracerebroventricular injections were found.

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an intact vagus nerve ts as is CCK-8's inhibitory effect on food intake 6s. The cholecystokinin receptor antagonist, L-364,718, blocked the improvement of retention produced by feeding or CCK-8 tT. CCK-8's effect on retention could be mediated by vagal stimulation through the nucleus of the solitary tract which sends impulses directly to the a m y g d a l a or indirectly t h r o u g h the stria terminalis 6°-63. In this study, we d e t e r m i n e d if the m e m o r y - e n h a n c ing effect o f CCK-8 might be m e d i a t e d by the s a m e o p i o i d - n o r a d r e n e r g i c system in the amygdala t h a t has b e e n p r o p o s e d as m e d i a t i n g the i m p r o v e m e n t r e t e n tion by naloxone and e p i n e p h r i n e . MATERIALS AND METHODS

Subjectsand Drugs After I week in the laboratory, CD-I male mice obtained at 6 weeks of age from Charles River Breeding Laboratories, Wilmington, MA, were individually caged 24-48 h prior to training and remained singly housed until retention was tested one week later. The median body weight was 35 g, with a range of 33-38 g. Animal rooms are maintained on a 12 h light-dark cycle with light on at 06.00. The mice, 15 per group, were trained between 08.00 and 14.00. Mice were assigned randomly to coded groups to prevent experi. menter bias. B-END and CCK-8 were purchased from Peninsula Laboratories, Inc., Behnont CA. Arecoline hydrobromide. D-amphetamine hydro. chloride and ( - ) epinephrine bitartrate were purchased from Sigma Chemical Co., St. Louis, Me. All drug solutions were prepared in saline. The dose is expressed on the basis of the salt.

T.maze apparatusand training The T.maze apparatus consisted of a black plastic start alley (46 cm long) with a start box at one end and two goal boxes (1%5 cm long) at the other and a depth of 12,5 cm and width of 9.8 cm throughout. The floor consisted of stainless steel rods, The start box was separated from the start alley by a plastic guillotine door which prevented the mouse from moving down the alley until the training started, A training trial started when a mouse was placed into the start box. The guillotine door was raised and the buzzer sounded simultaneously, then 5 s later footshock was applied. The goal box that the mouse first entered on this trial was designated as "incorrect" and the footshock was continued until the mouse entered the other goal box, which on all subsequent trials was designated "correct" for the particular mouse. At the end of each trial, the mouse was removed from the goal box and returned to its home cage. A new trial began by placing the mouse in the start box. At which time, the buzzer was sounded and the guillotine door was raised. Footshock was given $ s later if the mouse did not move into its correct goal box. Entry into the correct goal box terminated the buzzer and footshock. Two training conditions were used in these studies. Weak train. ing was used so that the control group would show poor retention (mean trials to criterion of greater than 9) so that enhanced reran. tion could be detected. Under this training condition mice received 4 training trials with an intertrial interval of 30 s. A doorbell-type buzzer (55 dB) served as the conditioned stimulus. The unconditioned stimulus was 030 mA of footsbock (Coulbourn Instruments scrambled grid floor shocker model F.3-08). In the second training condition, the training strength was increased so that the control group showed good retention (mean trials to criterion less than 7). This was accomplished by giving 5 training trials with a 45 s intertrial interval, an unconditioned stimulus of 6:5 dB and 0.35 mA footshock. Under weak or strong training conditions, retention was tested one

week later by continuing to train the mice until they made 5 avoidances in 6 consecutive trials. The overall significance of the drug treatment effect was determined by a one-way or two-way analysis of variance (ANOVA)42.6s. Tukey's t-test was used to test for significant differences between means following a two-way ANOVA. Dunnett's t-test was used to make multiple comparisons between each drug group and the saline control group following a one-way ANOVA4,42.

Drugadministration The surgical procedures used to prepare mice for bilateral injections into the amygdala or for an intracerebroventricular (i.c.v.) injection into the third ventricle have been described in detail previously I~'1s. In brief, mice were anesthetized with methoxyflurane, placed in a stereotaxic instrument and holes were drilled through the skull over the injection sites after deflecting the scalp. The coordinates for the amygdaloid injections were -1.6 mm with respect to bregma, 3.3 mm right and left of the central suture and 4.5 mm deep. The coordinates for the i.c.v, injection were -0.2 mm with respect to bregma, 1.0 mm to the right of the central suture and 2.5 mm deep. The coordinates were confirmed using a stereotaxic atlas~. Mice were trained 24-48 h after surgery. Immediately after training mice were again placed in the stereotaxic under enflurane anesthesia. Within 3 min after training, a 0.5/zl solution of drug or saline was injected into the target structure over 60 s through a 31-gauge needle fixed to a 10-/zl syringe with PE-10 tubing and driven by a Sage Syringe Pump (Model 341A). The reliability of the injections was determined by thionin solution into the sites and determining the location of dye in frozen brain sections. Fig. IA shows the area over which the needle tips were located and Fig. IB a typical brain section, Mice received i.c.v, injections in a similar manner except that 2/~l of drug solution or saline was administered. Fig. IC shows the area over which needle tracts for the i.c.v. injections were detected. The distribution of the needle tracts for the i.c.v, injections indicated that all injections were given into the lateral ventricle where it join,,, the third ventricle, RESULTS

Experiment 1 Effect of intracerebroventricular administration of ~.en. dorphin on retention, T h e p u r p o s e o f this study was to establish the d o s e - r e s p o n s e

curve by which B - E N D

caused a m n e s i a in a dose d e p e n d e n t m a n n e r w h e n a d m i n i s t e r e d i.c.v, a f t e r T - m a z e footshock avoidance training. Mice w e r e t r a i n e d as d e s c r i b e d above so t h a t controls w o u l d show good r e t e n t i o n , B - E N D (0.10, 0,25, 0,50, 1.00 ng in 2 /~! o f saline) o r saline w a s a d m i n i s t e r e d i m m e d i a t e l y a f t e r training, R e t e n t i o n was tested o n e w e e k a f t e r training by continuing training until mice m a d e 5 avoidance r e s p o n s e s in 6 consecutive trials. A o n e - w a y A N O V A r u n o n trials to criterion indic a t e d t h a t t h e post-training a d m i n i s t r a t i o n of B - E N D effected r e t e n t i o n (F4,ss ffi 10.11, P < 0.001). T h e m e a n s for g r o u p s receiving 0.50 a n d 1.0 ng o f B - E N D w e r e significantly g r e a t e r t h a n for the g r o u p receiving saline

(Fig. 2). Experiment 2 Effect of intra-amygdaloid administration of {3-endorphin on retention. T h e a m y g d a l a s e e m s a possible

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Fig. 2. Intracerebroventricular administration of/3-endorphin causes dose-dependent impairment in retention test performance. Statistically significant difference between the mean trials to criterion (:I:S.E.M.) for the control group (0 ng) and the groups receiving /3-endorphin at P < 0.01 are indicated by an *.

site of action as B-END exists within this structure, mu opioids caused amnesia when injected in the amygdala and evidence suggests that the amygdala is involved in memory processing. The purpose of this study was to determine if B.END caused amnesia when injected into the amygdala. To accomplish this, mice were prepared for bilateral intra-amygdaloid injections 24-48 h prior to T-maze training as described above. Immedi. ately after training a total dose of 0.01, 0.05, 0.10, 0.25 ng of B-END or saline was injected bilaterally into the amygdala. Retention was tested 1 week later as described above. A one-way ANOVA run on trials to criterion indicated a significant treatment effect (F4.70- 7.41, P < 0.001) in which groups receiving 0.05-0.25 ng had significantly higher mean trials to criterion than the saline-treated control (Fig. 3).

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immediately after training with 15 mice in each group. Retention was tested one week after training. An analysis of trials to criterion by a one-way ANOVA indicated a significant treatment effect (F6.gs - 5.73, P < 0.001). Only groups receiving 50 and 100 ~ g / k g had means that were significantly (P < 0.01 in both cases, Dunnett's t-test) less than the mean of the

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Experiment 3 Effect of epinephrine on retention. Though there have been numerous reports of post-training peripheral administration of epinephrine improving retention, the effective dose varies considerably with the test situation 25'3t'32'36'46'67. Mice were trained under weak training conditions as described above where the control group has poor retention so that we could detect improvement in retention due to epinephrine administration. Saline or 1 of 6 doses of epinephrine (5, 10, 25, 50, 100, 150/~g/kg) were administered subcutaneously

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Fig. 4. Peripherally administered epinephrine caused a dose.dependent modulation of retention. Statistically significant differences between the mean trials to criterion (4-S.E.M.) for the control group (0 ng) and the groups receiving epinephrine at P < 0.01 are indicated by an *

98 control group ( F i g 4). The lowest mean trials to criterion was obtained by the group receiving 100 ~ g / k g .

Experiment 4 lntra-amygdaloid administration of [3-endorphin selectil'ely blocks effects of peripherally administered memory-enhancing agents. As indicated, we hypothesized that CCK-8 enhancement of retention may be mediated by antagonizing the opioid system in the amygdala. The purpose of this experiment was to determine if B-END would specifically block memory enhancement due to CCK-8 administration. To test this, mice were trained so that the vehicle control would show poor retention making it possible to determine if B-END would block enhancement of retention by CCK-8. To determine if B-END would specifically block enhancement by CCK-8, the effect of B-END on improvement of retention by arecoline, o-amphetamine, epinephrine and naloxone were also determined. The optimal dose for improving retention was based on results of Exp. 3 and previously published studies~-L~.I~. Elalf the mice received a total dose of 0.05 ng of B-END and the other half received saline injected bilaterally into the amygdala immediately after training. Immediately after the intra-amygdaloid injection, separate groups of mice received saline, CCK-8 (1 /zg/kg i.p.), arecolin¢ (1.5 mg/kg s.c.), D-amphetamine (3 mg/kg i.p.), epinephrine (100/zg/kg s.c.) or naloxone (3 mg/kg s.c.) with 15 mice in each group. Retention was t~st~d by continuing training 1 week later to a criteriol~ of 5 avoidances in 6 consecutive trials. The mean trials to criterion were analyzed by a

two-way ANOVA. The main effect of the first injection (Saline versus B-END) was significant (FLi6s = 44.68, P < 0.001) as was the main effect for the second injection (Saline versus memory enhancers, F5.16s = 12.13, P < 0.001). The interaction of the main effects was significant (F5.16s = 5.16, P < 0.005) indicating that the first injection determined the effect, in part, of the second injection. Among the groups first injected with saline, all those given one of the 5 compounds previously found to improve retention had significantly lower mean trial to criterion that the mean of the group given Saline + Saline (Table I). For those groups receiving an injection of B-END first, only the groups receiving a second injection of D-amphetamine or arecoline had significantly low means (P