Psychopharmacology(1991) 103:473-479 003331589100054P

Psychopharmacology © Springer-Verlag 1991

Anxiolytic effects of benzodiazepines in amygdala-lesioned rats Elna Yadin, Earl Thomas, Craig E. Strickland, and Holly L. Grishkat Department of Psychology,Bryn Mawr College, Bryn Mawr, Pennsylvania,PA 19010, USA Received April 5, 1990 / Final version August 3, 1990

Abstract. The role of the amygdala in the anxiolytic action of benzodiazepines was examined. Performance on a water-licking conflict paradigm was tested in rats with localized damage to the central nucleus of the amygdala (ACE) or with general damage to the entire amygdaloid complex. The effects of the benzodiazepine chlordiazepoxide (2.5-20.0 mg/kg) on conflict behavior in these animals was also examined. Electrolytic lesions of either ACE or of the entire amygdaloid complex resulted in a pronounced increase of punished responding, an effect that persisted for at least 12 sessions postoperatively. After shock levels were adjusted in the lesioned groups to match their baseline punished behavior to that of the controls, various doses of chlordiazepoxide were administered. Not only did the lesioned animals show an increase in punished behavior in response to the drug, they were more sensitive than controls to the lower drug doses. A complete model of anxiolytic action may have to include both mechanisms that block anxiogenic regions and those that activate anxiolytic regions. Key words: Amygdala - Septum - Chlordiazepoxide Anxiogenesis - Anxiolysis

There is an emerging model of the role of specific brain structures in anxiety and their relation to the mechanism of action of benzodiazepine (BZD) anxiolytics. The presumption of this model is that there are structures in the brain, the activity of which mediate anxiety. Benzodiazepines are presumed to work via the BZDGABA-C1- ionophore complex such as to enhance the inhibitory effects of GABA upon these anxiety-mediating structures. A number of such brain regions have been proposed as putative sites for the action of BZDs, including the Off'print requests to: E. Yadin, Department of Psychiatry, The Medical College of Pennsylvania, 3200 Henry Avenue, Philadelphia, PA 19129, USA

nuclei of the raphe (e.g., Wise et al. 1972; see Soubri6 1986 for a review), the septo-hippocampal system (Gray 1982), and the amygdala (Scheel-Kruger and Petersen 1982; Costall et al. 1989). In order for a brain region to serve as a site of BZD action under this model at least three criteria should be met. First, direct application of BZD into the proposed site should have anxiolytic effects. Secondly, lesioning the proposed site should mimic, to a large extent, the direct action of the BZD. This would follow from the presumed inhibitory role of BZDs upon the activity of these structures. Thirdly, if the structure is lesioned, since the critical structure that is inhibited by BZDs is removed, the effects of peripherally administered BZDs should be diminished or abolished. One of the most well established tests for the anxiolytic action of drugs is the ability to increase punished responding in a conflict procedure such as that of Geller and Seifter (1960) and Vogel et al. (1971). Using this procedure, a number of sites have been shown to satisfy some of the criteria outlined above. For instance, direct application of BZDs into the central nucleus of the amygdala has been reported to have anticonflict effects (Shibata et al. 1986). Similarly, long lasting, anticonflict effects have also been reported resulting from lesions of the central nucleus of the amygdala (Nagy et al. 1979; Shibata et al. 1986). Direct application of BZDs into the raphe region has been demonstrated to have anticonflict effects (Thi6bot et al. 1982), and lesions of the raphe also release some forms of punished behavior (Thibbot et al. 1983). The data cited above, especially for the amygdala and the raphe, are consistent with the current model of BZD action. A major problem arises, however, when the effects of lesions upon drug action are considered. While lesions of the dorsal raphe may themselves yield increases in punished behavior, anxiolytic drugs maintain their anticonflict ability even after raphe lesions (Thi6bot et al. 1984), thus casting doubt on the role of the raphe in the primary mechanism of BZD action. It becomes important, therefore, to identify structures which, when lesioned, will substantially reduce the efficacy of BZD anxiolytics.

474 R e p o r t e d here are the effects o f B Z D anxiolytics on conflict b e h a v i o r in animals lesioned in one m a j o r candidate for the site o f action o f BZDs, the amygdata.

Experiment 1: Central nucleus lesions There is a large b o d y o f evidence gathered f r o m a m y r i a d o f experimental a p p r o a c h e s including stimulation, lesions, and unit recording, strongly implicating the a m y g d a l a , and especially the central nucleus, in fear (see Davis et al. 1987 for a review). The first experiment in this series therefore investigated the effects o f specific electrolytic lesions aimed at the central nucleus o f the a m y g dala on conflict b e h a v i o r in the rat, and subsequently examined the effects o f high doses o f the benzodiazepine chtordiazepoxide ( C D P ) o n this behavior.

Materials and methods Subjects. Subjects were 21 male albino Sprague-Dawley rats obtained from Ace Animals Inc. They were 90 days old at the start of the experiment and their weights ranged from 350 to 500 g. Animals were individually housed in stainless steel suspended cages in a ventilated, temperature-controlled colony with the light portion of the light/dark cycle from 8:00 A.M. to 8:00 P.M. All behavioral testing was conducted between 9: 00 A.M. and 12:00 P.M. Animals were maintained with food continuously available. Apparatus. The testing apparatus consisted of a 34 cm x 26 cm x 22 cm Plexiglas chamber with a grid floor. A glass water-bottle was mounted on the outside of the front panel, with its water-tube extending into the chamber, at a height of 5 cm from the grid floor. The chamber was contained within a ventilated sound-attenuating cubicle equipped with a houselight and a speaker. White noise (70 db SPL) was fed into the cubicle throughout the experiment. Contact relays (BRS/LVE model 4t= 221-05) controlled by solid state circuitry were used to detect and count the animals' licking behavior. Electric shock was provided by a constant current shock generator and was delivered between the floor grids.

Procedure. All animals were put on a water-deprivation schedule in which they received water for half an hour each day, provided after each daily training or testing session. This type of deprivation schedule did not result in any weight loss. The conflict procedure was a modification of the version used by McCown et al. (1983). Daily sessions consisted of four 2-min periods, the second and fourth of which were signalled by an 85 db, 1000 Hz tone. Animals were first habituated to the apparatus and to the tone by exposing them to the four 2-min sequence and recording the number of spout-licks during each of those time periods with the aid of a drinkometer. After three habituation sessions conflict training began with the introduction of a 60 Hz AC shock (0.5 s, 0.5 mA) delivered every fifth lick to the grids during the signalled time periods. In this phase alternating unpunished (U) and signalled punished (P) periods were presented. Each of the periods lasted 2 min and the U/P sequence cycled twice. For adequate training and for the purpose of showing that the behavior is under stimulus control, it was necessary to have the four-period sequence. However, the relevant data are found in the first two time periods because effects of satiation may contaminate the results, particularly in the last punished time period. The raw data consisting of number of licks were converted into logs (number of licks+ 1). Well-trained animals in this conflict procedure learned to lick freely during the unpunished periods and to suppress their licking during the signalled punished periods. Animals were then subjected

to bilateral electrolytic lesions under sodium pentobarbital anesthesia (Nembutal, 40 mg/kg ip). All lesions were performed using a single 0.18 mm stainless steel wire, insulated to the tip. A 2 mA DC constant current was delivered by a Grass stimuIator and associated constant current unit for 20 s. One group of animals (n= 12) received amygdaloid lesions (AMYG), another (n= 11) received sham lesions (SHAM), in which the electrode was inserted into the amygdala but no current was passed through. The coordinates for the central nucleus of the amygdala were: -2.2 mm from bregma, + 4.0 mm from the midline, and 8.0 mm from the surface of the skull. All the coordinates were taken with the animal in a level-head position. Postoperative testing of conflict behavior began 5 days after surgery and continued for 12 sessions. The effects of chlordiazepoxide (CDP) were subsequently examined. For each dose tested, animals first received a session in which the vehicle was injected 15 min before conflict testing. The following day drug was administered intraperitoneally 15 min before conflict testing. Chlordiazepoxide (purchased from SIGMA) was tested at 10 and 20 mg/kg. At least 6 days were allowed to elapse between each drug sequence. After termination of drug testing animals were sacrificed with an overdose of Nembutal, perfused through the heart with saline and 10% formalin, and brains were removed and sectioned. The 40 pin wet sections were used as negatives and photographed according to the method described by Guzman-Flores et al. (1958).

Results Histology. The extent o f the a m y g d a l o i d lesions is depicted in Fig. 1. U p o n histological examination, m o s t animals were f o u n d to have bilateral d a m a g e to the central nucleus o f the amygdala, and some animals showed primarily unilateral d a m a g e to this nucleus.

Lesion effects. All analyses o f conflict b e h a v i o r were d o n e o n the first u n p u n i s h e d and punished time periods. Figure 2A presents a c o m p a r i s o n between the animals' m e a n conflict b e h a v i o r during three sessions just prior to lesioning ( P R E ) and their behavior during the first three ( P O S T O P I ) a n d last three ( P O S T O P 2 ) testing sessions after lesions were performed. These sessions p r o v i d e d sampling o f b e h a v i o r 1 and 2 weeks after the lesion. The difference between the g r o u p s in their punished responding was a persistent effect [ P O S T O P I : t ( 2 2 ) = 4 . 9 3 , P < 0 . 0 1 ; P O S T O P 2 : t(22)=2.99, P < 0 . 0 1 ] .

Drug effects. The effect o f C D P in a m y g d a l a - d a m a g e d animals a n d in their sham-lesioned controls is depicted in Fig. 2B. A t w o - w a y analysis o f variance on punished responding (with repeated measures on one factor) s h o w e d a significant G r o u p s effect [ F ( 1 , 2 t ) = 4 . 4 4 , P < 0 . 0 5 ] , a n d a significant D o s e effect [F(2,42)= 11.39, P < 0.001]. Consistent with the parallel curves for the two g r o u p s seen in Fig 2B, there was no significant G r o u p s by D o s e interaction. T h e releasing effect o f the a m y g d a l a lesion itself is a persistent one resulting in significantly elevated baseline punished responding in the a m y g daloid-lesioned g r o u p [t(21)=2.46, P < 0 . 0 5 ] . T h u s the higher m a g n i t u d e o f d r u g effect in the lesioned g r o u p might be a function o f the higher baseline condition. The next experiment was designed to test the drug effect u n d e r a c o n d i t i o n o f m a t c h e d baselines.

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Fig. 1. A schematic representation of the extent of lesions when the target was the central nucleus of the amygdala. The black areas mark the most restricted damage, the dotted areas mark the most extensive damage. Plates redrawn from K6nig and Klippel (1963)

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CDP DOSE (rng/kg) E x p e r i m e n t 2: C e n t r a l n u c l e u s l e s i o n s w i t h b a s e l i n e adjustments

I n e x p e r i m e n t 1 licking b e h a v i o r d u r i n g the p u n i s h e d c o m p o n e n t o f the conflict test p r i o r to l e s i o n i n g was s u p p r e s s e d a l m o s t m a x i m a l l y b o t h in the a m y g d a l a a n d in the c o n t r o l g r o u p s . This s i t u a t i o n m a y h a v e c o n t r i b u t e d to the m o d e r a t e a n x i o l y t i c effect seen w i t h the benz o d i a z e p i n e c h l o r d i a z e p o x i d e . In a d d i t i o n , the effect o f a m y g d a l a lesions o n p u n i s h e d licking b e h a v i o r c r e a t e d different baseline c o n d i t i o n s a g a i n s t w h i c h d r u g effects were tested. T h e r e f o r e , in e x p e r i m e n t 2 the s h o c k intensities used d u r i n g the p u n i s h e d c o m p o n e n t s were those t h a t p r o d u c e d a m o d e r a t e s u p p r e s s i v e effect o n p u n i s h e d licking b e h a v i o r d u r i n g t r a i n i n g , b e f o r e the lesioning phase. F o r the test o f d r u g effects three doses o f the d r u g were used to get a d o s e - r e s p o n s e function. F u r t h e r m o r e , after the first series o f d r u g a d m i n i s t r a t i o n was c o m pleted the s h o c k intensities o f t h e a m y g d a l o i d - l e s i o n e d a n i m a l s were a d j u s t e d so t h a t the s u p p r e s s i o n o f p u n i s h ed r e s p o n d i n g in these a n i m a l s w o u l d m a t c h the suppression o f p u n i s h e d r e s p o n d i n g in their s h a m - o p e r a t e d c o n t r o l c o u n t e r p a r t s for a s e c o n d series o f C D P a d ministration. Materials and methods Subjects. Subjects were similar to the ones used in experiment 1. Eighteen naive rats were housed and maintained under conditions which were identical to the ones described above.

Fig. 2A, B. Performance on the water-licking conflict test in animals with sham lesions (SHAM) and central amygdaloid nucleus lesions (AMYG). A Effects of the lesion itself. Open column sham; hatched column amygdala * marks a significant difference from preoperative levels. B A dose-response function of the effects ofchlordiazepoxide (CDP) in these lesioned animals. * marks a significant difference between the two groups. U= Unpunished; P = Punished; PRE= training sessions immediately prior to lesioning; POSTOPl=first training sessions after recovery from surgery; POSTOP2 = training sessions 2 weeks after surgery

Apparatus. The apparatus was the same one used in the previous experiment• Procedure. The procedure for this experiment was similar to the one for the previous experiment with a few important exceptions. Shock levels were individually adjusted to produce licking behavior during the punished component of 1040% of that seen in the unpunished component. When consistent baseline behavior was obtained amygdala (n = 8) or sham (tl = 10) lesions were produced according to the procedure described in the previous experiment• After 1 week recovery, conflict testing was resumed• Subsequently, the effects of various doses of chlordiazepoxide (2.5, 5.0, and 10.0 mg/kg, given in random order) injected intraperitoneally were tested• After completion of the first series of drug injections the baseline licking rates were adjusted (by changing shock levels) so that punished responding in both amygdaloid- and sham-lesioned animals would be comparable. The mean shock level for the amygdaloid-lesioned group was 0.41 mA (range 0.30-0.55) and for the control group it was 0.36 mA (range 0.25~.50). Once matching was achieved a second series of drug administrations was initiated. At the end of the experiment animals were sacrificed with an anesthetic overdose and brain sections were prepared for lesion examination as described above•

476 Results

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Lesion effects. Figure 3A presents the conflict behavior of animals with amygdaloid and sham lesions immediately before the lesions (PRE), during an early stage after lesioning (POSTOP1), and after several subsequent sessions (POSTOP2). Each point is a mean of three consecutive sessions. As can be seen, animals with amygdaloid damage show an increase in punished licking behavior, the effect reaching significance here approximately 2 weeks after receiving the lesion [POSTOP2: t(7)= 3.46, P

Anxiolytic effects of benzodiazepines in amygdala-lesioned rats.

The role of the amygdala in the anxiolytic action of benzodiazepines was examined. Performance on a water-licking conflict paradigm was tested in rats...
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