Posttraining infusion of lidocaine into the amygdala basolateral complex impairs retention of inhibitory avoidance training

BRAIN RESEARCH ELSEVIER

Brain Research 661 (1994) 97-103

Research report

Posttraining infusion of lidocaine into the amygdala basolateral complex impairs retention of inhibitory avoidance training Marise B. Parent *, James L. McGaugh Center,[or ttle Neurobiology of Learning and Memory, and Department of Pwchobiolo~,9', Uniz'ersity (~¢"CahJ'ornia. Ir~'im'. lrt'ine, CA 92717-3800. USA Accepted 19 July 1994

Abstract

The present experiment examined the role of the central nucleus and basolateral complex in the retention of inhibitory avoidance training by reversibly inactivating these regions with lidocaine immediately following training. Male Sprague-Dawley rats were surgically implanted bilaterally with cannulae aimed at the central nucleus or the basolateral complex. One week later, they received one trial inhibitory avoidance training (0.45 mA; 1 s), followed immediately by infusions of lidocainc hydrochloride or buffer (10 p,g/0.25 /zl). Retention was tested 2 days after training. Immediate posttraining infusions of lidocaine into the central nucleus did not affect retention performance; in contrast, immediate posttraining infusions of lidocaine into the basolateral complex significantly impaired retention performance. In addition, the effect of posttraining infusions of lidocainc into the basolateral complex was time-dependent: infusions administered 6 h after training also impaired memory, but infusions administered 24 h after training had no effect. Immediate posttraining infusions of lidocaine also impaired the retention performance of rats trained with a more intense footshock (0.75 mA). However, at the higher footshock intensity, administration of lidocaine 6 h after training had no effect on retention performance. The time- and footshock-dcpendent retrograde impairment of memory produced by posttraining reversible inactivation of the basolateral complex suggests that this region of the amygdala is inw~lved in the consolidation of memory for inhibitory avoidance training.

Keywords: Amygdala; Central nucleus; Basolateral complex; Lidocaine; Inhibitory avoidance; Learning: Memory; Retcntion

1. Introduction

Extensive evidence indicates that retention of aversively motivated learning is influenced by manipulations of the amygdala performed after learning. Posttraining electrical stimulation [16,32], lesions [28], and chemical stimulation [22,35] of the amygdala affect inhibitory avoidance retention performance. The effects of posttraining amygdala manipulations on memory are time-dependent: the treatments affect memory most effectively when administered immediately after learning and are less effective as the training-treatment interval is lengthened. Recently, it was reported that posttraining reversible inactivation of the amygdala with tetrodotoxin (TTX) produces a time-depend-

* Corresponding author. Department of Psychology, Gilmer Hall, University of Virginia, Charlottesville, VA 22901, USA. Fax: (1) (804) 982-4785; e-mail: mbp4m~zvirginia.edu. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 9 0 5 - 8

ent retrograde inhibitory avoidance retention impairment [5]. The use of posttraining reversible inactivation limits the disruption of neural activity to the period immediately following learning: neural activity is intact during learning and retrieval. As a result, these findings are consistent with extensive evidence indicating that the amygdala is involved in the consolidation of inhibitory avoidance learning [33-35]. The amygdala is composed of heterogeneous regions that can be subdivided on the basis of histochemistry, cytoarchitectonics, connectivity, physiology, and function [11,42,50]. The central nucleus and the basolateral complex are two areas within the amygdala that have been extensively implicated in learning and memory [1,50]. The findings of lesion studies suggest that the involvement of the central nucleus and basolateral complex varies with different learning tasks. For example, lesions to areas within the basolateral complex, but not lesions of the central nucleus, impair retention of

98

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~I.B. l'ar{'nt. .I.I. Me(laugh

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e n c e [20] a n d t a s t e - p o t e n t i a t e d o d o r a v e r s i o n c o n d i t i o n i n g [18]. C o n v e r s e l y , l e s i o n s o f t h e c e n t r a l n u c l e u s , b u t n o t b a s o l a t e r a l c o m p l e x l e s i o n s , impair t h e m e m o r y of an association between a spatial location and the m a g n i t u d e o f a r e w a r d [24]. At present, the role of the central nucleus and the b a s o l a t e r a l c o m p l e x in t h e c o n s o l i d a t i o n o f i n h i b i t o r y a v o i d a n c e l e a r n i n g is u n c l e a r . P r e t r a i n i n g l e s i o n s t o a r e a s w i t h i n t h e b a s o l a t e r a l c o m p l e x o f t e n r e s u l t in a n i m p a i r m e n t in t h e a c q u i s i t i o n o r r e t e n t i o n o f i n h i b i t o r y a v o i d a n c e l e a r n i n g [3,40,55]; h o w e v e r , t h e r e a r e ins t a n c e s in w h i c h l e s i o n s to a r e a s w i t h i n t h e b a s o l a t e r a l c o m p l e x h a v e n o e f f e c t [9,55]. L e s i o n s o f t h e c e n t r a l n u c l e u s h a v e b e e n r e p o r t e d t o r e s u l t in r o b u s t [47], m o d e r a t e [55], l i t t l e [23], o r n o [13] i m p a i r m e n t in t h e acquisition or retention of inhibitory avoidance learning. T h e r e a p p e a r s t o b e o n l y o n e s t u d y t h a t h a s examined the effects of posttraining lesions of amygdala nuclei on inhibitory avoidance retention perform a n c e . R o o z e n d a a l , K o o l h a a s , a n d B o h u s [47] r e c e n t l y reported that posttraining electrolytic lesions of the central nucleus have no effect on inhibitory avoidance retention performance. To our knowledge, the effects of posttraining basolateral complex lesions have not been previously examined. The present experiment examined the involvement o f t h e c e n t r a l n u c l e u s a n d b a s o l a t e r a l c o m p l e x in t h e consolidation of inhibitory avoidance learning by reversibly inactivating these regions with lidocaine hydrochloride immediately following inhibitory avoidance training, The effects of delayed posttraining infusions of lidocaine were also investigated. Lidocaine hydrochloride prevents propagated action potentials by temp o r a r i l y b l o c k i n g s o d i u m c h a n n e l s [19]. P r e v i o u s f i n d ings indicate that the use of intracerebral infusion of l i d o c a i n e is a n e f f e c t i v e m e t h o d f o r e x a m i n i n g t h e f u n c t i o n a l i n v o l v e m e n t o f n e u r a l s t r u c t u r e s in b e h a v i o r [41,44,48,49,56].

Brain Research 001 (1994) 97-107

as needed to maintain anesthesia. l h e skull t)l: the ral was fixed to a stereotaxic frame (Kopf Instrs., Tujunga, CA}. Permanent stainless steel guide cannulae (23 gauge; 15 ram), aimed ai either the central nucleus (anterior-posterior {AP) -2.3 mm from bregma: mediallateral (ML)= +4.1 mm from midlinm dorsab-ventral (DV) 5.0 mm from dura, nose bar = -3.3{) mm from inleraural line) or the basolateral complex (AP ..... 3.1 ram: ML :: ~ 5.[ nnn; I)V= 5.0 ram) were implanted bilaterally [39]. The cannulae were affixed 1o the skull with two screws and dental acrylic; stylets were inserted into the cannulae to keep them patent. Immediately following surgery, each rat received penicillin (0.05 cc i.m.; HanR~rd's Penicillin G Procaine 300,000 U / m l ) and saline (3 cc s.c.) and was placed in a temperature-controlled chamber until recovew from anesthesia. 2.1.3. Apparatus and procedure Approximately 1 week following surgery the rats received one-trial inhibitory aw)idance training. The training apparatus was a troughshaped alley (91 cm long, 20 cm wide at the top, 6.4 cm wide at the floor, 15 cm deep) that was separated into two compartments by a guillotine door that opened by retracting into the floor. The starting compartment (31 cm long) was illuminated by a tensor lamp (14 W); whereas, the shock compartment (60 cm) was dark. The apparatus was located in a sound-attenuated, non-illuminated room. For the training, each rat was placed into the lighted compartment with its head facing away from the door. When the rat turned around to face the door, the door was opened. Immediately after the rat entered the dark/shock compartment with all four paws, the door was closed and a footshock (0.45 mA; 1 s) was delivered through stainless steel floor plates. The rat was then immediately removed from the apparatus, given bilateral infusions of lidocaine hydrochloride or vehicle, and returned to its home cage. The lidocaine hydrochloride (Sigma Chemical Co., St. Louis, MO; dissolved in phosphate buffer saline pH 7.0; 10 p-g; 0.25 p.I; 40 p-gSp-I) or vehicle were injected into the cannulae bilaterally through injection needles (30 gauge; 17 mm) attached to 10 p-I syringes (Hamilton Co., Rent), NV) via polyethylene tubing (PE-20). The infusions were delivered at a rate of 0.25 p-l/min for l rain with the use of an automated infusion pump (CMA/100; Carnegie Medicin, Stockholm, Sweden). The injection needles were retained in the cannulae for 1 rain following the infusion in order to maximize diffusion away from the needle tip and to minimize dorsal diffusion. Retention of the inhibitory avoidance training was assessed 48 h after training. Each rat was placed in the lighted/safe compartment with its head facing away from the dark/shock compartment. Thirty seconds later, the door was opened and the latency to enter the dark/shock compartment with all four paws was recorded. Shock was not administered. The rat was then left in the apparatus with the door open and the total time spent in the lighted/safe compartment was recorded for the remainder of the 600-s test.

2. Experiment 1 2.1. Materials and methods 2.1.1. Subjects Male Sprague-Dawley rats (n = 68; 175-200 g upon arrival from Charles River Labs., Wilmington, MA) were used. They were individually housed in a temperature- and light-controlled vivarium (22°C; 12:12-h light dark cycle; lights on at 07.00 h), provided with food and water ad lib, and acclimatized to laboratory conditions for approximately 1 week prior to surgery. 2.1.2. Surgical procedure The rats received atropine sulfate (0.4 mg/kg i.p.) and were then anesthetized with sodium pentobarbital (50 mg/kg i.p.). Supplemental doses of sodium pentobarbital (25 mg/kg i.p.) were administered

2.1.4. Histology After completion of the behavioral tests, the rats were anesthetized with an overdose of sodium pentobarbital (150 mg/kg i.p.) and perfused intracardially with 0.9% saline followed by 10% formalin. The brains were removed and placed in a 10% formalin solution for approximately 1 week, then sectioned into 40 p-m slices with a freezing microtome, and stained with thionin. Cannula location was determined using a light microscope (Olympus BH-2) and standardized atlas plates [39] by an observer blind to the behavioral results. The behavioral results of those rats whose injection needles were located within approximately 0.5 mm of a target nucleus and located approximately 0.5 mm away from the non-targeted nucleus (e.g. for the basolateral complex group within 0.5 mm of the basolateral complex and 0.5 mm distant from the central nucleus) were included in the statistical analysis. These criteria were employed as it was assumed that 0.25 p-I of lidocaine would block neural activity at

M.B. Parent, J.L. McGaugh /Brain Research 661 (1994) 97-103 the infusion site and in a region extending approximately 0.5 m m [2,44,48]•

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2,1.5. Statistics Retention latencies and duration of time spent in the lighted compartment were analyzed using analysis of variance and Fisher post hoc tests. An alpha level of 0.05 was used as a criterion of statistical significance. 2.2• Results

99

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The results of the histological analysis indicated that the cannulae location of 18 rats met the criteria of inclusion for the central nucleus group (see Fig. IA) and the cannulae location of 50 rats met the criteria of inclusion for the basolateral complex group (see Fig. IB). In several cases, vacuoles were observed in the area proximal to the injection needle tip, which suggests that the infusion process produced some neuronal damage in rats given buffer or lidocaine infusions. However, there was no relationship between the presence and degree of damage and retention performance. T h e results of the retention test are shown in Fig. 2. For both retention measures, the performance of rats given infusions of buffer into the central nucleus (Cent-Buff) did not differ significantly from

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that of rats given infusions of buffer into the basolateral complex (Baso-Buff; retention latency: t]~= 0.78; P > 0.05; time in lighted compartment: tls = 0.96; P > 0.05). Consequently, the retention performance of Cent-Buff and Baso-Buff rats was combined for the statistical analyses. Results of the retention test indicated that the effects of immediate posttraining infusions of lidocaine depended upon the injection location (retention latency: F2.44 = 6.0l; P < 0.01; time in lighted compartment: F2.42 = 6.03; P < 0.01). Infusions of lidocaine into the basolateral complex significantly impaired retention performance. In comparison with the retention performance of buffer-control rats, rats that were given infusions of lidocaine into the basolateral complex (Baso-Lido) had significantly shorter retention latencies (see Fig. 2A) and spent less time in the lighted/safe c o m p a r t m e n t (see Fig. 2B; P < 0.05 for both comparisons). However, posttraining infusions of lidocaine into the central nucleus did not affect retention performance: there was no difference between the retention latencies and time spent in the lighted/safe compartment of buffer-control rats and rats given lidocaine infusions into the central nucleus (Cent-Lido; P > 0.05 for both comparisons). In view of the finding that posttraining infusions of lidocaine into the central nucleus did not affect retention performance, the effects of delayed infusions of lidocaine were examined only in rats with cannulae aimed at the basolateral complex. The results indicated that the memory-impairing effects of posttraining infusions of lid()caine into the basolateral complex were time-dependent (retention latency: F4,~,4 = 5.17; P < 0.01; time in lighted compartment: F4~,] = 5.28; P < 0.01). As in the case of immediate posttraining infusions, infusions of lidocaine into the basolateral complex administered 6 h after training also impaired retention performance: rats given infusions of lidocaine into the basolateral complex 6 h after training ( B a s o - L i d o / 6 h) had significantly shorter retention latencies (see

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M.B. Parent, J.l. Mc(;augh ~Brain Research 66l (1994) 97-103

Fig. 2A) and spent less time in the lighted/safe compartment (see Fig. 2B) than did buffer-control rats (P < 0.05 for both comparisons). In addition, the retention performance of Baso-Lido/6 h rats did not differ from that of rats that received immediate posttraining micro infusions of lidocaine into the basolateral complex (P > 0.05 for both comparisons). However, posttraining infusions of lidocaine into the basolateral complex administered 24 h after training (Baso-Lido/24 h) did not affect retention performance: the retention latencies and time spent in the lighted compartment of Baso-Lido/24 h rats were not significantly different from those of buffer-control rats (P > 0.05 for both comparisons). Also, the retention performance of BasoLido/24 h rats was significantly better than that of Baso-Lido and Baso-Lido/6 h rats: Baso-Lido/24 h rats had longer retention latencies and spent more time in the lighted/safe compartment than did Baso-Lido and Baso-Lido/6 h rats (P < 0.[)5 for all comparisons).

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3. Experiment 2 It is well established that the effects of many types of pre- and posttraining treatments on inhibitory avoidance retention performance depend upon the footshock intensity employed during training [8,12,14,15, 27,37,41]. The purpose of the second experiment was to determine whether the retrograde impairment produced by posttraining inactivation of the basolateral complex would be attenuated or blocked by increasing the footshock intensity employed during training. More specifically, the effects of posttraining infusions of lidocaine into the basolateral complex were examined in rats trained with a higher intensity footshock than that used in Expt. 1.

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Fig. 3. The effects of immediate posttraining and delayed infusions of lidocaine hydrochloride or buffer into the basolateral complex of animals given inhibitory avoidance training using a 0.45 mA footshock (Expt. 1) or a 0.75 mA footshock (Expt. 2) on (A) mean latency to enter the dark compartment and (B) mean time spent in the lighted compartment on the 600 s retention test (* P < 0.05 vs. buffer basolateral immediate-0,45 mA; * P < 0,05 vs. buffer basolateral immediate-0.75 mA; n = 9-12 per group for Expt. 2).

3.1. Materials and methods

The same procedure was employed as that used in Expt. 1, with the exception that rats with cannulae aimed at the basolateral complex (n = 31) were trained with a 0.75 mA; 1 s footshock rather than a 0.45 mA footshock.

infusions of lidocaine administered 6 h after training did not impair retention performance: there were no significant differences between the retention latencies and time spent in the lighted compartment of BasoBuff/0.75 mA and Baso-Lido/0.75 mA-6 h rats (P > 0.05 for both comparisons).

3.2. Results

As shown in Fig. 3, the results of Expt. 2 indicated that, as in Expt. 1, posttraining reversible inactivation of the basolateral complex impaired retention performance (retention latency: F2,28= 3.76; P < 0.05; time in lighted compartment: F2,zs = 4.03; P < 0.05). The retention performance of rats trained with a 0.75 mA footshock and given immediate posttraining infusions of lidocaine into the basolateral complex (BasoLido/0.75 mA) was significantly poorer than that of buffer-control rats trained with the same footshock (Baso-Buff/0.75 mA). Baso-Lido/0.75 mA rats had significantly shorter retention latencies and spent less time in the lighted compartment than did BasoBuff/0.75 mA rats (P < 0.05 for both comparisons). However, in animals trained with a 0.75 mA footshock

4. Discussion

The major finding of the present experiment is that posttraining reversible inactivation of the basolateral complex with lidocaine impairs retention of inhibitory avoidance tested 48 h after training. This treatment produces deficits in retention performance as assessed by two behavioral measures: latency to enter the dark/shock compartment and the amount of time spent in the lighted/safe compartment. Furthermore. the memory impairment depends upon both the footshock intensity employed during training and the time of the administration of the lidocaine following training. At a low training footshock intensity (0.45 mA), a retrograde memory impairment was induced by infusions

M.B. Parent, J.L. McGaugh / Brain Research 661 (1994) 97-103

administered either immediately or 6 h following training. Furthermore, inactivation of the basolateral complex induced 24 h after training had no effect on retention performance tested 24 h following the inactivation. However, when a higher footshock intensity (0.75 mA) was used for training, only immediate posttraining infusions of lidocaine into the basolateral complex produced a retrograde impairment: infusions administered 6 h following training did not affect retention performance. These results are consistent with previous findings indicating that posttraining reversible inactivation of a region assumed to encompass the entire amygdala impaired inhibitory avoidance retention performance in a time-dependent manner [5]. In that study, infusions of TTX into the amygdala impaired retention when administered within 90 min after training, were less effective when given 6 h after training, and had no effect when given 24 h after training. The present experiment extends those findings by indicating that the impairment produced by posttraining inactivation of the amygdala appears to be mediated by selective inactivation of the basolateral complex. This interpretation is supported by our findings indicating that posttraining reversible inactivation of the central nucleus has no effect on inhibitory avoidance retention performance as well as by the recent report that posttraining electrolytic lesions of the central nucleus do not affect inhibitory avoidance retention performance [47]. These findings are also consistent with previous findings indicating that the involvement of the central nucleus and basolateral complex varies with different learning tasks [18,20,24]. The present results do not indicate whether the impairing effects of posttraining reversible inactivation of the basolateral complex with lidocaine are due to inactivation of intrinsic neurons or due to inactivation of fibers passing through the basolateral complex. Pretraining neurotoxin-induced lesions of the basolateral complex, which are assumed to spare fibers of passage, impair inhibitory avoidance retention performance [3]. This finding suggests that the lidocaine-induced impairment observed in the present study may also be attributable to inactivation of neurons within the basolateral complex. However, reversible inactivation of the basolateral complex with a substance that does not inactivate fibers of passage, such as the GABA agonist muscimol is needed to directly address this possibility [301. It is possible that the differential effects of reversible inactivation of the central nucleus and basolateral complex reflect differences in the sensitivity of these areas to the effects of lidocaine rather than differences in the involvement of these regions in the consolidation of inhibitory avoidance learning. That is, it may be that the 10/xg dose employed in the present study effectively inactivated the basolateral complex,

1111

but not the central nucleus. For several reasons, this possibility appears unlikely. Using electrophysiological measurements, Sandkuhler, Maisch and Zimmerman [49] demonstrated that a smaller dose of lidocaine effectively blocks conduction in the dorsal columns of the cat spinal cord. In view of the fact that large myelinated fibers were effectively inactivated, Sandkuhler and colleagues [49] concluded that this dose of lidocaine would also completely block other structures in the central nervous system (CNS). Furthermore, the results of studies using behavioral or electrophysiological measurements have demonstrated that the dose of lidocaine employed in the present study, as well as smaller doses, effectively inactivate other regions in the CNS [7,29,52,56,57]. Finally, the present conclusion that posttraining reversible inactivation of the central nucleus has no effect on inhibitory avoidance retention performance is consistent with the finding that posttraining electrolytic lesions of the central nucleus do not affect inhibitory avoidance retention performance [47]. However, lesions of the central nucleus that are induced prior to inhibitory avoidance training do impair subsequent retention performance [17,36,47], even when animals are trained to criterion [55]. Thus, although the central nucleus may be involved in the acquisition or retrieval of inhibitory avoidance learning, our findings suggest that it is not involved in the consolidation of that learning. Results of behavioral and physiological studies suggest that lidocaine blocks neuronal activity for approximately one half hour [10,29,30,31,54]. Thus, in the present study functional inactivation of the basolateral complex was restricted to the period immediately following learning. As a result, the finding that posttraining inactivation of the basolateral complex results in a retrograde memory impairment suggests that the basolateral complex is active immediately following training and is involved in the consolidation of inhibitory avoidance learning. The results of the delayed lidocaine infusions administered following training with the lower footshock intensity suggest that the functional integrity of the basolateral complex is necessary for at least 6 h following inhibitory avoidance training. However, influences mediated by the basolateral complex are no longer needed 24 h later. These results are consistent with other reports indicating that the amygdala is temporarily involved in the retention of inhibitory avoidance learning. Pre-retention infusions of lidocaine into the amygdala impair retention when retention is assessed 2 days after learning, but not when retention is assessed 21 days later [26]. Similarly, posttraining amygdala lesions impair inhibitory avoidance retention performance when the lesions are induced within 2 days after training; however, lesions that are induced 10 days after training have no effect [28]. The finding that the time-dependent characteristics

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of posttraining inactivation are influenced by footshock intensity is in agreement with other results indicating that the gradient of retrograde amnesia produced by some treatments is inversely related to the footshock intensity employed during training [4,43,45]. It may be that increased footshock intensity recruits additional neural structures during acquisition and that these additional neural structures mediate the retention that is observed following the 6 h delayed micro infusions. However, this possibility appears questionable in view of our finding that immediate posttraining inactivation of the basolateral complex results in comparable memory impairment at both training footshock intensities. Alternatively, this finding suggests that the functional integrity of the basolateral complex may be required for a shorter duration in animals trained with a higher footshock. The more intense stimulation produced by the higher footshock may in some way accelerate the consolidation process and decrease the duration of basolateral complex involvement in the retention of inhibitory avoidance learning. Although the findings of the present experiment implicate the basolateral complex in the consolidation of inhibitory avoidance learning, the nature of the role played by the basolateral complex remains unclear. In view of the evidence suggesting that the amygdala may be a site of the neural changes underlying fear conditioning, it is possible that the basolateral complex has a direct and permanent role in inhibitory avoidance memory storage processes [25,51]. The time-dependent nature of the retrograde memory impairment produced by posttraining inactivation of the basolateral complex may reflect a temporary fragility and susceptibility of memory storage processes within the basolateral complex to posttraining manipulations. Alternatively, these results are also consistent with the view that the amygdala plays a modulatory role in the consolidation of inhibitory avoidance learning [33-35]. According to this view, the time-dependent effect of posttraining inactivation of the basolateral complex reflects the amygdala's temporary role in the amplification or finetuning of memory storage occurring elsewhere in the brain.

Acknowledgements This research was supported by a 1967 Natural Science and Engineering Research Council of Canada fellowship to M.B.P. and U S P H S Grant MH12526 from N I M H and N I D A to J.L.M. We thank Eloise Avila and Mary West for their technical assistance, Mark Packard for his comments on an earlier version of this manuscript, and Nancy Collett for her assistance in the preparation of this article.

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