Proc. Natl. Acad. Sci. USA Vol. 94, pp. 14048–14053, December 1997 Neurobiology Glucocorticoid enhancement of memory storage involves noradrenergic activation in the basolateral amygdala (dexamethasoneyRU 28362yb-adrenergic antagonistsyinhibitory avoidance) GINA L. QUIRARTE*†,BENNO ROOZENDAAL*‡, AND JAMES L. MCGAUGH*§ *Center for the Neurobiology of Learning and Memory and §Department of Psychobiology, University of California, Irvine, CA 92697-3800; and †Centro de Neurobiologı´a, Universitat Nacional Autonome de Mexico, Queretaro, Qro., 76001 Mexico Contributed by James L. McGaugh, October 7, 1997 ABSTRACT Evidence indicates that the modulatory ef- and opioid peptidergic systems influence the training-induced fects of the adrenergic stress hormone epinephrine as well as release of norepinephrine (13, 14). several other neuromodulatory systems on memory storage Findings of a series of recent experiments from our labo- are mediated by activation of b-adrenergic mechanisms in the ratory indicate that the amygdala is involved in mediating the amygdala. In view of our recent findings indicating that the memory-modulating effects of glucocorticoids (15). Excito- amygdala is involved in mediating the effects of glucocorti- toxically induced lesions of the basolateral (BLA), but not the coids on memory storage, the present study examined whether central (CEA), nucleus of the amygdala blocked the enhancing the glucocorticoid-induced effects on memory storage depend effects of systemic posttraining injections of dexamethasone, a on b-adrenergic activation within the amygdala. Microinfu- synthetic glucocorticoid, on memory for inhibitory avoidance sions (0.5 mgin0.2ml) of either propranolol (a nonspecific training (16). Furthermore, infusions of the specific glucocor- b b -adrenergic antagonist), atenolol (a 1-adrenergic antago- ticoid receptor (GR or type II) agonist RU 28362 (11b,17b- b nist), or zinterol (a 2-adrenergic antagonist) administered dihydroxy-6,21-dimethyl-17a-pregna-4,6-trien-20yn-3-one) bilaterally into the basolateral nucleus of the amygdala (BLA) into the BLA enhanced memory storage (17). Such findings of male Sprague–Dawley rats 10 min before training blocked suggest that glucocorticoid effects on memory storage are the enhancing effect of posttraining systemic injections of mediated, at least in part, by GRs in the BLA. Although the y dexamethasone (0.3 mg kg) on 48-h memory for inhibitory specific mode of action of glucocorticoids differs from that of b avoidance training. Infusions of these -adrenergic antago- the hormones and neurotransmitters just described (i.e., glu- nists into the central nucleus of the amygdala did not block the cocorticoids are highly lipophylic and bind directly to intra- dexamethasone-induced memory enhancement. Furthermore, cellular receptors in the brain), the involvement of the amyg- m atenolol (0.5 g) blocked the memory-enhancing effects of the dala in mediating the effects of all these compounds appears specific glucocorticoid receptor (GR or type II) agonist RU to be strikingly similar. These findings suggest that glucocor- 28362 infused concurrently into the BLA immediately post- ticoid-induced effects on memory storage may also depend on b training. These results strongly suggest that -adrenergic intact noradrenergic neurotransmission in the amygdala. In activation is an essential step in mediating glucocorticoid support of this view, there is extensive evidence from bio- effects on memory storage and that the BLA is a locus of chemical and electrophysiological experiments indicating in- interaction for these two systems. teractions between glucocorticoids and the noradrenergic system in several brain structures, including the hypothalamus, Extensive evidence from studies of memory of inhibitory hippocampus, and cerebral cortex (18, 19). avoidance training in rats indicates that several neuromodu- The present study examined the involvement of b- latory systems interact with the noradrenergic system in the adrenoceptors in the amygdala in the modulating effects of amygdala in modulating memory storage (1–8). Intra- glucocorticoids on memory storage. In a first experiment, rats b amygdala infusions of the -adrenergic antagonist propranolol received microinfusions of b-adrenergic antagonists with ei- b b or depletion of norepinephrine in the amygdala by the neu- ther a selective affinity for 1-or 2-adrenoceptors or with a rotoxin N-2-chloroethyl-N-ethyl-bromobenzylamine (DSP-4) nonspecific affinity for both receptor types into the BLA or block the memory-enhancing effect of the adrenergic stress CEA before training in an inhibitory avoidance task. Dexa- hormone epinephrine (5, 6). Moreover, intra-amygdala infu- methasone was injected subcutaneously immediately after b b sions of propranolol as well as specific 1-or 2-adrenergic training. In a second experiment, rats received concurrent antagonists also block the effects, on memory, of drugs b immediate posttraining infusions of the 1-adrenergic antag- affecting opioid peptidergic and GABAergic systems (4, 7). b onist atenolol and the specific GR agonist RU 28362 into the Norepinephrine and other -adrenergic agonists administered BLA. In both experiments the animals were tested on retention to the amygdala after training dose-dependently enhance 48 h after training. retention (5, 9–11). These effects are also time-dependent; they affect retention when given shortly after training but have no effect if given several hours later. These findings are MATERIALS AND METHODS consistent with evidence from recent experiments using in vivo Subjects. Male Sprague–Dawley rats (270–300 g at time of microdialysis indicating that norepinephrine is released in the surgery) from Charles River Laboratories were used. They amygdala by footshock stimulation of the kind typically used in were individually housed in a temperature-controlled (22°C) inhibitory avoidance training (12). Furthermore, adrenergic colony room and maintained on a standard 12-h lighty12-h dark cycle (0700–1900 h lights on) with food and water The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: BLA, basolateral amygdala; CEA, central amygdala; accordance with 18 U.S.C. §1734 solely to indicate this fact. GR, glucocorticoid receptor; RU 28362, 11b,17b-dihydroxy-6,21- © 1997 by The National Academy of Sciences 0027-8424y97y9414048-6$2.00y0 dimethyl-17a-pregna-4,6-trien-20yn-3-one. PNAS is available online at http:yywww.pnas.org. ‡To whom reprint requests should be addressed. 14048 Downloaded by guest on September 23, 2021 Neurobiology: Quirarte et al. Proc. Natl. Acad. Sci. USA 94 (1997) 14049 available ad libitum. Training and testing were performed delivered. For the animals given pretraining infusions of drug between 1000 and 1500 h. or control solution into the BLA, the footshock level used was Surgery. The animals were adapted to the vivarium for at 0.45 mA with a duration of 1 s. Preliminary data revealed that least 1 week before surgery. They were anesthetized with comparable retention latencies in CEA-infused animals were sodium pentobarbital (50 mgykg of body weight, i.p.) and given obtained with a slightly lower footshock intensity. Therefore, atropine sulfate (0.4 mgykg, i.p.). The skull was fixed to a CEA-infused animals received a footshock intensity of 0.40 stereotaxic frame (Kopf Instruments, Tujunga, CA), and mA for 1 s. The animals were removed from the shock stainless-steel guide cannulae (15 mm; 23 gauge) were im- compartment 15 s after termination of the footshock and, after planted bilaterally with the cannula tips 2 mm above the BLA drug treatment, returned to their home cages. On the retention [coordinates: anteroposterior (AP), 22.8 mm from bregma; test 48 h after training, the rat was placed in the starting mediolateral (ML), 65.0 mm from midline; dorsoventral compartment, as in the training session, and the latency to (DV), 26.5 mm from skull surface] or CEA [coordinates: AP, re-enter the dark compartment (maximum latency of 600 s) 22.2 mm; ML, 64.3 mm; DV, 26.0 mm] according to the atlas was recorded. Longer latencies were interpreted as indicating of Paxinos and Watson (20). The cannulas and two anchoring better retention. Shock was not administered on the retention screws were affixed to the skull with dental cement. Stylets (15 test trial. mm long 00 insect dissection pins) were inserted into the Histology. The rats were anesthetized with an overdose of cannulae to maintain patency and were removed only for the sodium pentobarbital (100 mgykg, i.p.) and perfused intra- infusion of drugs. The rats were allowed to recover 7 days cardially with a 0.9% saline solution followed by 10% form- before training and were handled three times for 1 min during aldehyde (volyvol). Following decapitation, the brains were this recovery period. removed and placed in 10% formaldehyde. At least 24 h before Drugs and Infusion Procedures. The nonspecific b- sectioning, the brains were submerged in a 20% sucrose adrenergic antagonist dl-propranolol (0.5 mg per side; Sigma) solution (wtyvol) for cryoprotection. Sections of 40 mm were b b m or the specific 1-or 2-adrenergic antagonists atenolol (0.5 g made (using a freezing microtome) and stained with cresyl per side; Sigma) or zinterol (0.5 mg per side; Bristol-Myers), violet. The sections were examined under a light microscope. respectively, were dissolved in 0.9% saline and infused either Determination of the