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Behavioural Brain Research 240 (2013) 26– 28

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research

j ourna l ho me pa ge: www.elsev ier .com/ locate /bbr

hort Communication

elective increase in the association of the �2 adrenergic receptor, � Arrestin-1nd p53 with Mdm2 in the ventral hippocampus one month after underwaterrauma�

apita Sooda,1, Gilad Ritovb,1, Gal Richter-Levinb,c,d, Liza Barki-Harringtona,∗

Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Mt. Carmel, Haifa, 31905, IsraelThe Sagol Department of Neurobiology, Faculty of Natural Sciences, University of Haifa, Mt. Carmel, Haifa, 31905, IsraelDepartment of Psychology, Faculty of Social Sciences, University of Haifa, Mt. Carmel, Haifa, 31905, IsraelThe Institute for the Study of Affective Neuroscience (ISAN), University of Haifa, Mt. Carmel, Haifa, 31905, Israel

i g h l i g h t s

Underwater trauma (UWT) causes heightened anxiety one month after trauma.UWT increases association of Mdm2 with beta2AR, beat Arrestin-1, and p53 in ventral not dorsal CA1.Stress-related events cause changes in ‘emotion-related’ parts of the hippocampus.

r t i c l e i n f o

rticle history:eceived 27 September 2012eceived in revised form 7 November 2012ccepted 11 November 2012vailable online 20 November 2012

eywords:

a b s t r a c t

Chronic infusion of mice with a �2 adrenergic receptor (�2AR) analog was shown to cause long-termDNA damage in a pathway which involves � Arresin-1-mediated activation of Mdm2 and subsequentdegradation of the tumor suppressor protein p53. The objective of the present study was to test whethera single acute stress, which manifests long lasting changes in behavior, affects the interaction of Mdm2with p53, �2AR, and � Arrestin-1 in the dorsal and ventral hippocampal CA1. Adult rats were subjectto underwater trauma, a brief forceful submersion under water and tested a month later for behavioral

tress2 Adrenergic receptor

Arrestin-1, Mdm2ippocampus

and biochemical changes. Elevated plus maze tests confirmed that animals that experienced the threatof drowning present heightened levels of anxiety one month after trauma. An examination of the CA1hippocampal areas of the same rats showed that underwater trauma caused a significant increase in theassociation of Mdm2 with �2AR, � Arrestin-1, and p53 in the ventral but not dorsal CA1. Our resultsprovide support for the idea that stress-related events may result in biochemical changes restricted to

ted’ p

the ventral ‘emotion-rela

Real or perceived threats to homeostasis evoke a stress response,hich initiates a series of adaptive physiologic and behavioral

esponses, among them activation of the sympathetic nervous sys-em to secrete the stress hormones adrenalin/noradrenalin. Albeitritical for survival and well-being, chronic sympathetic activity is

armful in the long-run [1]. The p53 tumor suppressor protein is theajor hub for a complex signaling network, which is rapidly and

fficiently upregulated to induce cell cycle arrest and death. The

� Research support: The Israel Science Foundation grant (no. 1403/07) to G.R.-L., by U.S. Army Medical Research Acquisition Activity (USAMRAA) grant (no. W81XWH-1-20111), and by the Institute for the Study of Affective Neuroscience, Universityf Haifa, which was endowed by the Hope for Depression Research Foundation.∗ Corresponding author. Tel.: +972 4 8288776, fax: +972 4 8288763.

E-mail address: lbarki@psy.haifa.ac.il (L. Barki-Harrington).1 Author Italic footnote text.

166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2012.11.009

arts of the hippocampus.© 2012 Elsevier B.V. All rights reserved.

actions of this extremely potent molecule are reined-in by the neg-ative regulator Mdm2, which is in itself upregulated by p53. Mdm2forms a physical interaction with p53 and by doing so repressesits transcriptional activity and shuttles it out of the nucleus tothe cytosol, where it is subsequently degraded by the proteasomalmachinery [2,3]. A recent study has shown that continuous infusionof mice with a synthetic analog of adrenaline for four weeks causeda marked reduction in the levels of p53 and accumulation of DNAdamage in the thymus and the cerebellum. The researchers showedthat chronic stimulation of �2 adrenergic receptors (�2ARs) leadsto activation of Mdm2 and subsequent degradation of p53 in a path-way which is mediated by the adaptor protein � Arrestin-1 [4]. �Arrestin-1 plays a dual role in regulation of this pathway: in the

cytosol, it mediates catecholamine-induced activation of AKT andMdm2, and in the nucleus it serves as an adaptor for the Mdm2-p53interaction, which promotes p53 ubiquitination and subsequentdegradation.

R. Sood et al. / Behavioural Brain

Table 1UWT causes heightened anxiety one month after exposure.

Control UWT Sig.

Time (s) Center 54.66 ± 7.03 27.16 ± 5.05 p < 0.01**

Open arms 36.75 ± 7.32 17.72 ± 5.06 p < 0.05*

Closed arms 205.14 ± 13.76 253.29 ± 9.97 p < 0.01**

Distance (cm) Center 359.56 ± 40.84 196.15 ± 37.38 p < 0.01**

Open arms 262.40 ± 54.46 140.98 ± 39.72 n.s.Closed arms 1257.70 ± 84.78 1103.02 ± 83.81 n.s.

Data are shown as Mean + S.E.M n = 12 animals per group.

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complex and multifactorial nature of both the trauma itself and

* Student’s t test p < 0.05.** Student’s t test p < 0.01.

While these findings are enlightening with regards to the mech-nism by which sympathetic activity can be harmful in the longun, one might argue that a model of chronic infusion of cate-holamines may not mimic an actual physiological/pathologicaltimulus. The objective of the present study was therefore to testhether a single acute stress, which causes long-lasting effects,ay render changes in the interaction of Mdm2 with p53, �2AR,

nd � Arrestin-1. For this, we used the underwater trauma (UWT)odel, a brief intense experience of stress, which was shown to

ause heightened anxiety and poor performance in spatial mem-ry tasks at both short and long terms time periods [5–7]. Adultale Sprague Dawley rats were randomly assigned to either con-

rol (CONT) or UWT protocols. Five days after acclimation, animalsndergoing the UWT protocol were submerged into a water tanksing a metal net, as described before [6]. The procedure lasted5 s, after which animals were dried briefly and returned to theirousing cage. Rats in the control group were allowed a two-minutecclimation period to the experimental room and returned to theirousing cages. One month after the procedure, the anxiety levelf animals in both groups was tested using the elevated plus mazeEPM) test [6,8]. Each animal was allowed a 5 min period of acclima-ion to the room, after which it was placed in the center of the mazeacing an open arm and allowed to explore the arena for 5 min. Ani-

als in both groups presented similar body weights. As shown inable 1, animals that experienced the UWT spent significantly lessime in the center (t(22) = 3.1, p < 0.01) and open arms (t(22) = 2.1,

< 0.05) of the maze, and traveled shorter distances in these tworeas. Time spent in the closed arms was significantly higher in theWT group (t(22) = 2.8, p < 0.01), along with a smaller effect on theistance traveled in this section. These data implicate that the UWTaused a prolonged effect, with a higher level of anxiety-like behav-or compared to the control group, a month after the exposure tohe stressor, as previously noted [5–7].

Having confirmed that the single acute stress of UWT is man-fested by long term behavioral changes, we next tested whetherhese effects are reflected in biochemical changes occurring in dor-al and ventral segments of the CA1. We chose to study these areasecause while the role of the hippocampus as a cognitive maphich serves primarily to process and store spatial information

s well-established, recent evidence point toward important rolesor the ventral hippocampus in processing inputs related to stress9]. Furthermore, �ARs in the hippocampus play an importantole in regulating synaptic plasticity and memory consolidation,nd almost all of the neuronal nuclei positive cells express �1-nd �2ARs [10–12]. Interestingly however, whereas both type ofeceptors are localized to the cell membrane and cytoplasm, only2ARs are found in the nucleus [13], where they may have a poten-

ially unique role in regulating the Mdm2-p53 transport.To test for changes in the association of Mdm2 with p53, �2AR,

nd � Arrestin-1 in the brains of animals following UWT, the sameats that underwent the procedure were sacrificed 24 h after test-ng for anxiety, and ventral and dorsal CA1 were isolated bilaterally.

Research 240 (2013) 26– 28 27

Preliminary experiments showed no lateral differences thereforetissue from both sides of each animal was pooled. Analysis of thedifferent fractions showed no treatment-dependent differences inthe total levels of Mdm2 or p53 in either dorsal or ventral CA1(Fig. 1A). We then immunoprecipitated Mdm2 and probed for thepresence of �2AR, � Arrestin-1 and p53 on the same sample [14]. Asdepicted in Fig. 1, animals that underwent the UWT showed a sig-nificantly higher association between Mdm2 and �2AR (t(13) = 6.5,p < 0.01; Fig. 1B), Mdm-2 and � Arresin 1 (t(13) = 4.7, p < 0.01;Fig. 1C) and between Mdm2 and p53 (t(6) = 4.3, p < 0.01; Fig. 1D).Strikingly, this effect was restricted to the ventral CA1, while thedorsal area showed no treatment-related changes in the associationof any of these proteins with Mdm2 (Fig. 1B–D, gray bars).

Together the data presented here showed that the brief but acutestress of underwater trauma causes a marked elevation in long-term anxiety-like behavior in rats, and that while there is no changein the total levels of Mdm2 and p53, there is a marked increase inthe association of �2AR, � Arrestin-1 and p53 with Mdm2. Sincethe � Arrestin-1 mediated interaction between p53 and Mdm2was shown to facilitate p53 degradation and subsequently to resultin DNA damage, it is tempting to speculate that our results mayrepresent a possible mechanism that can lead to Mdm2-mediatedneuronal cell damage or death following trauma. As opposed toexperiments with continuous infusion of catecholamines [4], wehave no record of the levels of these hormones in the blood or thebrain of the animals throughout the experiment. However, previousstudies suggest that the stress-induced increase in catecholaminesis temporary and returns to baseline levels within two hours fromthe stressful input [15]. Secretion of adrenalin/noradrenaline haslikely occurred during trauma itself, and may have recurred for anunknown duration and intensity in the course and following theanxiety testing. Therefore, we cannot say with certainty whetherthe observed differences occurred at the time of the trauma andthe testing merely revealed them, or whether these are adaptivechanges that occurred either some time after the trauma, or in thecourse of testing for anxiety.

An interesting observation of this study was that the stresstreatment caused marked changes in the biochemical correlatesonly in the ventral CA1. The dorsal and ventral areas of the hip-pocampus (DH and VH, respectively) are distinct in several aspects.Whereas the DH is primarily connected with the neocortex, the VHis connected to subcortical structures, including the amygdale andhypothalami, which are well-known stress-mediating regions ofthe brain [9,16]. They are also different in the milieu of postsynapticreceptors and in their ability to induce long term potentiation (LTP)[17]. This cellular correlate of learning and memory was found tobe lower in neurons of the VH than DH under resting conditions[18]. However, exposure of animals to high level stressors suchas forced swim or directly to the stress hormone corticosterone,caused a marked reduction of LTP in DH CA1 with a concomitantfacilitation in the magnitude of LTP in VH CA1 [19]. This and otherobservations have raised the recent hypothesis that stress may re-route hippocampal connections such that under normal conditionsthe hippocampus is linked to the rest of the brain primarily throughits dorsal efferents, while a significant stressful event may renderthe dorsal efferents less excitable, and increase connectivity of thehippocampus to the amygdale and hypothalamus [9]. Our resultsprovide support for the idea that stress-related events may resultin biochemical changes restricted to the ventral ‘emotion-related’parts of the hippocampus.

Understanding the causes of long-term changes in behavior dueto severe emotional trauma is extremely challenging due to the

the short and long term changes that follow. Despite the relativelylimited information that can be drawn from a single ‘snapshot’ ofthe brain one month after the traumatic event, we were able to

28 R. Sood et al. / Behavioural Brain

Fig. 1. Increased association of �2AR, � Arrestin-1 and p53 with Mdm2 in the ventralCA1 one month after trauma. No change in the total levels of Mdm2 (top panel) andp53 (bottom panel) in the dorsal or ventral CA1 (A). Mdm2 was immunoprecipitatedfrom ventral (black bars) and dorsal (gray bars) of the CA1. Blots were probed for�2AR, (B), � Arrestin-1 (�Arr1) (C), and p53 (D). Antibodies used were as follows,indicated WB for western blotting, IP for immunoprecipitation. Mdm2 (IP, 2 �g, WB,1:500), Mouse p53 (FL-393; WB, 1:100), � Arrestin-1 (K-16: WB, 1:100), mouse �2-adrenoreceptor (M-20: WB, 1:200), all from Santa Cruz. Anti-mouse IgG–HRP (WB,1:10,000), anti-rabbit IgG–HRP (WB, 1:10,000), anti-goat IgG–HRP (WB, 1:10,000)all from Jackson ImmunoResearch. Insets are representative immunoblots from eachgroup. Graphs represent Mean + S.E.M of n = 6–8 animals per group, * Student’s t testp < 0.05 vs. Control.

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Research 240 (2013) 26– 28

demonstrate region-specific increases in the association of �2AR,� Arrestin-1 and p53 with Mdm2. Since Mdm2 is a master negativeregulator of p53, it remains to be determined whether this interac-tion leads to activation of Mdm2, which may promote DNA damageand partly explain the long-term adverse effects of exposure tostress.

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