Anxiolytic-like effect of losartan injected intoamygdala of the acutely stressed rats

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Anxiolytic-like effect of losartan injected into amygdala of the acutely stressed rats

Luis H. Llano López¹, Fernando Caif¹, Sebastián García¹, Miriam Fraile¹, Adriana I. Landa¹, Gustavo Baiardi², José V. Lafuente³, Jan J. Braszko⁴, Claudia Bregonzio⁵, Pascual A. Gargiulo¹

1Laboratory of Neurosciences and Experimental Psychology, IMBECU-CONICET, Department of Pathology, Faculty of Medical Sciences, National University of Cuyo, Mendoza (5500), Argentina

2Laboratory of Neuropharmacology, Faculty of Chemical Sciences, Catholic University of Córdoba, Institute of Biological and Technological Research (IIBYT-CONICET), Córdoba, Argentina

3Laboratory of Clinical and Experimental Neurosciences (LaNCE), Department of Neurosciences, University of the Basque Country, Apdo. 699, 48080-Bilbao, Spain

4Department of Clinical Pharmacology, Medical University of Bialystok, Waszyngtona 15A, PL 15-274 Bia³ystok, Poland

5Institute of Experimental Pharmacology (IFEC-CONICET), National University of Córdoba, Argentina Correspondence: Pascual A. Gargiulo, e-mail: gargiulo@lab.cricyt.edu.ar

Abstract:

It has been recognized that the stress-related peptides are involved in anxiety states. Angiotensin II receptor blockade by systemic administration of the AT₁receptor antagonists has been proposed as a new treatment possibility for anxiety disorders. For better un- derstanding of the related mechanisms, in this study we evaluated effects of bilateral intraamygdaloid injections of 2 (LOS 2) and 4 (LOS 4) µg of losartan (LOS), a selective AT₁receptor antagonist, on the behavior of the not stressed and acutely stressed rats in an elevated “plus” maze. Under non-stress conditions, LOS 4 increased time spent in the open arms (p < 0.01), number of extreme open arm arrivals (p < 0.05), time per entry (p < 0.01), and the number of total arm entries (p < 0.05) showing thus considerable anxiolytic activity. The open arm extreme arrivals were increased by LOS 4 in both not stressed (p < 0.05) and stressed (p < 0.05) rats. When no stressed and stressed LOS 4 animals were compared, time per entry and the number of closed arm entries (p < 0.05, both) were de- creased in the latter group. Moreover, the LOS 4 stressed rats had significantly increased open/closed arm quotient (p < 0.05) as compared to the both control and LOS 4 non-stress group (p < 0.05, both). These findings suggest that the AT₁receptor blockade in amygdala is important for the anxiolytic action of LOS (and probably other AT₁receptor blockers) under both non-stress and stress conditions.

Key words:

losartan, angiotensin, amygdala, anxiety, plus maze

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Introduction

Stress related peptides increase blood pressure. This parameter is an important component of the prepara- tion for “fight or flight” condition as an adaptation to adverse or dangerous situations [see 46]. In this way, corticotropin releasing hormone (CRH) is an important mediator of the stress response [26]. It increases anxiety [13], and is also relevant for pathophysiology of hypertension [18, 19]. Arginine vasopressin (AVP), a peptide classically linked to increases in blood pressure, also responds to stress [25]. In contrast to the acute stress, which increases CRH and AVP secretion, chronic stress leads to a CRH secretion reduction and major increases of AVP release [26]. In relation to this, the genetic selection of high anxiety (HAB/LAB) rats and mice were proposed as an interesting animal model of anxiety [25]. They were described as inborn extremes in anxiety-related behavior, but they also have, as a biological correlate, differential expression of AVP within hypothalamic paraventricular nucleus (PVN) [25].

Ang II was initially considered a vasoconstrictive hormone, acting also locally within brain [38]. Most of its effects are mediated by AT₁ and AT₂ receptors [1]. We have previously reported AT₁ receptor binding in the subfornical organ (SFO), PVN, median eminence, piriform cortex, basolateral amygdaloid nucleus, median preoptic nucleus, area postrema, and the nucleus of the solitary tract [5]. Stress increases production of circulating Ang II and the expression of the AT₁receptors in PVN. It also stimulates CRH for- mation [5]. The stress level influences the expression of AT₁ receptors and the release of Ang II, in brain and in peripheral tissues [5]. AT₁ receptors are involved in stress-induced hormone secretion (CRH, ACTH, corticosteroids and vasopressin) and stimula- tion of the central sympathetic activity [35, 37]. How- ever, alterations in AT₁receptor binding during stress are not limited to hypothalamic structures. In this way, we previously found that acute cold-restraint stress induces a decrease in AT₁ receptor binding in the rat basolateral amygdaloid nucleus [5]. AT₁receptor antagonists reduce stress responses and anxiety in rodents. They prevent sympathoadrenal response to stress and its gastric consequences, confirming the role of Ang II as a stress hormone [37, 38]. Peripheral administration of losartan (LOS), a selective AT₁receptor antagonist, attenuated motor hyperactivity and anxiogenic behavior in hypertensive rats [45]. This

drug also induced anxiolysis in normotensive rats, showing a behavioral profile very similar to diazepam [45]. These findings were observed in two classical and recognized models of anxiety, the elevated “plus”

maze and social interaction test [45]. Valsartan, another AT1 antagonist, has shown anxiolytic-like effects in the plus maze test [4]. Blockade of the AT₁receptors or angiotensin-converting enzyme inhibition decreases hypothalamus-pituitary-adrenal (HPA) axis reactivity [35]. These effects are independent of the blood pressure decrease [35]. The AT₂ receptors are less abundant than AT₁receptors in adult tissues, and they appear to counteract several effects of the AT₁receptors [1]. It has been proposed that AT₂ receptors exert their modulator actions after an up-regulation under certain pathologic conditions [1].

The above data led us to suppose that Ang II in amygdala could play a role in basal conditions and in stress-related anxiety. The aim of the present study was to answer the question whether anxiolytic effects of LOS are mediated through an inhibition of the AT₁ receptors in this structure. Amygdala is a brain part classically related to emotional states, such as fear processing and adaptation to danger [15, 16, 41, 42].

Furthermore, amygdala projects to nucleus accumbens septi (NAS) [2, 14, 20]. This structure has been found to be related to anxiety [14, 23, 27], and it con- tains AT₁ receptors [49]. For the evaluation of the amygdala contribution to the anxiolytic effects of LOS we employed the elevated “plus” maze paradigm (EPM) [4, 33]. This test has previously shown to be a useful tool aiming to detect anxiogenic effects of in- tracerebroventricular (icv) Ang II and its prevention by icv LOS [4]. In the present study, the drug was in- jected directly within the amygdala and tested in the plus maze test, a behavioral method that has been ex- tensively used in rats [13, 16, 27] and mice [17].

Materials and Methods

Animals

Male rats from an inbred Holtzman-derived colony aged 90 days and weighing 240–290 g were used (n = 89).

They were maintained under controlled temperature (22–24°C) and lighting (lights on 07.00 – 19.00 h).

Animals, housed in groups of 5 or 6 rats in each home

Losartan, amygdala and anxiety

Luis H. Llano López et al.

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procedures were carried out in accordance with the guidelines set by European Community Council (Di- rective 86/609/EEC), in accordance with the bioethi- cal rules established by the Faculty of Medicine of the National University of Cuyo, and according to Argen- tine law.

Surgery

Animals were anesthetized with ether (Andes Labora- tories, Mendoza, Argentina) and stereotaxically im- planted with bilateral stainless-steel cannulae fixed to the skull with dental cement (Subiton, Argentina), aiming to reach the amygdala [2, 27]. Coordinates in respect to bregma were: A = –1 mm; L = ±4.5 mm;

V = –6.5 mm. The cannulae were double barreled and the set was composed of an outer guiding stainless- steel tubing (made from BD Precision Glide Needles, 21-gauge, 15 mm in length), cemented to the skull, provided with a removable stylet (made from Dental Cartridge Needles, 27 gauge, 15 mm in length) to avoid its obstruction. After surgery, rats were housed individually and maintained undisturbed for a week of recovery period.

Apparatus

The elevated “plus” maze was made of wood, and con- sisted of two open arms, 50 × 10 cm (length × width), and two enclosed arms 50 × 10 × 50 cm (length × width

× height), arranged such that the two arms of each type were opposite to each other [8, 9, 27, 28]. The maze was elevated to a height of 50 cm. The room was illuminated by a 60 W bulb 1.5 m above the apparatus. The restraint was performed in a Plexiglass tube of 7 cm in diameter and 17 cm long. The rats were un- able to escape from the cylinder because of a perpen- dicular sliding door placed at its end.

Experimental procedure and treatment

All preparations, handling and experimental procedures were done according to the protocols used in our laboratory and described earlier [13, 15, 24, 27].

All tests were conducted in the light phase. Rats were located in the experimental room and maintained un-

Basal NON-STRESS condition

Rats were manually injected 15 min before the behavioral test aiming at bilaterally reaching amygdala using a 27 gauge, 17 mm long stainless steel injection cannula positioned to precisely reach the amygdala.

This cannula was attached to a 10 µl microsyringe (Hamilton). Volumes of 1 µl of saline or LOS solution in saline were gradually injected over 1 min periods into both left and right amygdala. The injection cannula was left in place for an additional 1 min to allow liquid diffusion.

STRESS condition

Immediately before the 15 min restraint the rats of the STRESS group were bilaterally injected into the amygdala with the same procedure as that used in the NON-STRESS condition group. Then the rats were placed for 15 min in the restrainer described above.

Restraint stress

Animals in the stress groups were subjected to a single acute 15-min restraint session in the above described Plexiglass tube. Animals were transferred from the holding room to a separate testing room where they were placed in the Plexiglass tube. The stress sessions were carried out immediately before testing.

Elevated “plus” maze test

All experiments were carried out between 08:00 and 12:00 h (during light cycle). Fifteen minutes after the injection of the drug or saline, each rat was placed in the center of the elevated “plus” maze facing an open arm, and allowed 5 min free exploration.

The following 6 parameters were measured: time spent in open arms, extreme open arm arrivals (defined as number of times the rat reached the end of an open arm), time per open arm entry (defined as the time spent in the open arms divided by the number of entries therein), open/closed arm quotient (ratio between open and closed arm entries), closed arm entries, and total arm entries (arm entry defined as all four paws in an arm) [6, 10, 11, 24, 27].

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Drugs

Rats were injected bilaterally into the amygdala with either saline (1 µl) or LOS (Sigma-Aldrich). Doses used were 2 µg (LOS 2) and 4 µg (LOS 4), dissolved in 1 µl of saline solution, immediately before the injection.

Histological analysis

After the end of testing, rats were submitted to an ex- cess of ether and injected with saturated methylene blue solution (1 µl). The brains were removed from the skull and fixed in 20% formaline solution. The brains were then sectioned and the block faces were examined with a 10× magnifying lens. The sites con- taining the injections were translated to a copy of the Atlas and transferred to standard sections [32]. Micro- scopic inspection of the sections served to precisely ascertain the location of the ends of cannulae. We only report statistical data for those rats with correct placements of cannulae (Fig. 1). The diffusion area of the methylene blue solution in amygdala was checked in all cases.

Statistical data analysis

The Kolmogorov Smirnov test was used to ascertain Gaussian distribution of data. The data were analyzed using a two-way ANOVA (drug × condition) with LOS as drug factor and stress vs. non-stress as condi-

tion factor. If an interaction and/or main effect were observed, pairwise comparisons following ANOVA were made using Bonferroni post-hoc test [30]. In all cases, a p < 0.05 (two tailed) was considered significant. The results are reported as the means ± SEM at n

= 10–18 for each group.

Results

All parameters gave significant results, with the ex- ception of the open arm entries (data not shown).

Time in open arms

Two-way ANOVA revealed a significant drug × condition interaction [F (2, 83) = 3.4, p < 0.05] indicating that the LOS effect depends on stress condition. Sub- sequent pair-wise comparisons by Bonferroni post- hoctest revealed that LOS 2 µg did not have significant effect (p = 0.25), but 4 µg significantly increased time spent in the open arms (t = 3.02, p < 0.01) compared to saline under the non-stress condition. No effects of the drug administration were found in the stress condition, saline vs. LOS 2 µg (t = 1.14, p = 0.25) and saline vs. LOS 4 µg (t = 0.58, p = 0.6) (Fig. 2A).

Fig. 1. Histological placement of can- nuli of rats used in the plus maze test.

Frontal brain sections showing the location of the injection site. Schematic representation of histological findings.

– Injection cannula placement

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Extreme open arm arrivals

Two-way ANOVA showed a significant effect of treatment (saline, LOS) [F (2, 83) = 8.67, p < 0.001] and a non-significant drug × condition interaction [F (2, 83)

= 1.37, p = 0.29]. In this case LOS effects were independ- ent of the stress condition. Bonferroni post-hoc test com-

parisons indicated that application of the higher dose of LOS resulted in significantly more extreme arm arrivals than application of saline under both non-stress (t = 2.66, p < 0.05) and stress (t = 3.09, p < 0.01) conditions.

Moreover, in stress condition, the higher dose of LOS produced significantly different results than that of the lower dose (t = 3.09, p < 0.01; Fig. 2B).

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Time per entry

Two-way ANOVA revealed a significant drug × condition interaction [F (2, 83) = 4.5, p < 0.01], indicating joint influence of the condition (non-stress, stress) and drug treatment (saline, LOS). Subsequent pair- wise comparisons indicated that LOS (4 µg) resulted in a significantly higher time per entry than saline in non-stress conditions (t = 2.98, p < 0.01). The stress condition blunted this response, and the time per entry in the LOS 4 stress group was significantly shorter than that in the non-stress LOS 4 group (t = 2.54, p <

0.05; Fig. 2C), and similar to that in the saline treated animals.

Open/closed arm quotient

An analysis using two-way ANOVA revealed a significant effect of the condition [F (1, 83) = 4.5, p <

0.05] but a non-significant interaction between the condition and drug factors [F (2, 83) = 1.35, p = 0.26].

Subsequent pair-wise comparisons by Bonferroni post-hoc test indicated that stress condition increases this variable in the higher dose group when compared to the non-stress higher dose group (t = 2.41, p < 0.05;

Fig. 2D).

Closed arm entries

Two-way ANOVA revealed a significant interaction (condition × drug) [F (2, 83) = 3.2, p < 0.05]. This re- sult indicated joint influence of the condition (stress;

non-stress) and drug administration (saline, LOS) and, therefore, the LOS 4 µg effects appear dependent on stress condition. Bonferroni post-hoc test indicated that the effect of LOS on this variable at a dose of 4 µg in non-stress condition was significantly different from that in the stress condition (t = 2.51, p < 0.05, Fig. 2E).

Total entries

Regarding total entries, non-stressed rats receiving the higher dose of LOS entered both maze arms significantly more often than those receiving saline (t = 2.4, p < 0.05; Fig. 2F). There were no statistically significant differences between not stressed and stressed groups receiving saline (t = 1.63, p = 0.18) and the lower dose of LOS (t = 0.79, p = 0.43).

Discussion

The results of the present experiments give support to the idea that AT₁ Ang II receptors within amygdala are relevant for the control of anxiety in normal and stressed rats.

A brief 15-min restraint stress did not essentially change control saline injected rats behavior in EPM.

Interestingly however, non-stressed animals tended to move more within the open arms of the maze as ex- plicitly shown by the higher number of the total arm entries in the non-stressed rats. Higher dose (4 µg) of intraamygdala losartan brought statistically significant changes in every of the six parameters measured in the “plus” maze. Under such treatment not stressed rats spent more time in open arms both total and after each single entry in comparison to not stressed saline-injected controls. Time per entry was different between non-stressed and stressed rats with the LOS higher dose (4 µg). Extreme open arm arrivals revealed the disinhibitory component of the anxiolytic effect of intraamygdala LOS (4 µg) in the stressed and not stressed rats. This activity increase may also be explained by a curiosity not inhibited by fear, making them walking along the whole length of an open arm until its end. Interestingly, extreme open arm arrivals appeared to be enhanced by LOS (4 µg) more in stressed than in not stressed animals. A greater component of the anxiolytic effect was observed with the higher dose of LOS in the stressed rats compared with non stressed animals in the significant much higher value of the open/closed arm quotient. It could be considered as a preference parameter. Of course, closed arm entries, a parameter classically related to locomotor activity [4, 33], showed the reversed pro- portions between the non stressed and stressed rats treated with 4 µg of intraamygdala LOS. However, neither non stress nor stress group showed a significant difference with their respective saline groups. It could be indicating that present results cannot be merely explained by a non specific locomotor activity increase. Furthermore, the total entries increase observed in the non-stress rats with the higher LOS dose could be explained by the additive effect of a non significant increase of closed arm entries plus the significant extreme open arm arrivals. It appears to reflect more an anxiolytic disinhibitory profile than a mere increase in locomotor activity. Although the stressed rats of the LOS 4 group showed a tendency to spend

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a greater disinhibitory component.

Previous studies have focused on parameters considered in the plus maze test [24, 27; for a review see 50]. We have here used seven parameters aiming to have a multidimensional record of variables interven- ing in this study. A single parameter could be influ- enced by methodological artifacts. If several parameters are modified in a same way, the approach to the phenomenon could be more convincing. Six of them are present in the graphic, and one was omitted because its not significant value (open arm entries). Dif- ferent parameters have been signaled as related to different facts. In this way, time spent in the open arm is the classical parameter related to anxiety. Here we also used time per entry, as previously [6, 24, 27], aiming to have an anxiolytic parameter not merely in- fluenced by motor variables, measuring time employed in open arm exploration each entrance. In our present conditions, modification of this parameter in non-stressed rats group was significant. The stressed group did not show a modification in this parameter, and the expression of anxiolytic effect had a different profile. Extreme open arm arrivals will be pointing a decrease in fear to the most anxiogenic part of the apparatus; rats are at this moment in the longer dis- tance of the walls protection. It was here clearly modified by higher LOS dose in both stressed and non-stressed groups. Open/closed arm quotient is ex- pressing a modification of preference, from the less dangerous or exposed position (protected by the walls) to the higher risk possibility (open arms). This parameter is here the most convincing manifestation of the anxiolytic effect of LOS higher dose in stressed rats. An increase in closed arm entries is a parameter classically linked to an effect due to locomotor activity [33, 34]. In present results, we have only a decrease in stressed LOS higher dose group when compared to non-stressed corresponding group. This find- ing could be explained by a displacement to another behavior, like open arm exploration in this case. Total entries are increased in higher dose group of non- stressed rats, as previously said. They could be showing the addition of a non significant increase in closed arm entries and a significant increase in extreme open arms arrivals. This parameter could be showing here an anxiety decrease, giving a global idea about both disinhibitory and anxiolytic components.

mologous anxiolytic like effect. The use of an important number of behavioral parameters allows us to register these different profiles in a rich and extensive way. In non-stress condition LOS effect suggests an important basal angiotensin II tone. The fact that LOS effect was different in stress conditions could be explained because additional anxiogenic stress systems could be involved in this part of the schedule, and they are not blocked by this drug. Such a behavioral profile presented by our rats after intraamygdala injection of LOS indicates the ability of this AT₁receptor blocker to modify emotional processes controlled by amygdala under both non-stress and stress conditions. To explain these differences and their potential therapeutic significance more fully another stress and anxiety models should be used.

It was initially proposed that Ang II antagonists exert their central action through circumventricular or- gans, which are not protected by the blood-brain bar- rier (BBB) [5]. Area postrema, SFO [8], and the angiotensinergic projection from PVN to the rostral ventrolateral medulla [9] were proposed as the action sites. LOS is able to cross BBB, and it has been observed that its peripheral administration leads to central actions suggesting that its effects could well be mediated by another brain structures controlling these actions [5, 7, 45]. An example is amygdala, a structure classically involved in anxiety [15, 16, 47], examined in the present study.

Regarding the intraamygdaloid injections, the volume here used (1 µl) diffused within the whole studied structure reaching all neighboring nuclei. Conse- quently, our injections did not only affect specific nuclei near the injection site but whole amygdalar complex involved in emotional processing. Previ- ously, LOS effects were studied only after systemic administration [45]. Therefore, our present intrastruc- tural injections make considerable progress in such studies. Several nuclei have been described within amygdala, and the presence of AT₁ receptors have been reported in medial, basomedial, lateral [49] and basolateral nuclei [5, 48, 49]. The basolateral amygdala has been implicated not only in the emotional response to stress and anxiety [28, 29, 40, 44, 47], but also in memory, and the central nucleus in anxiety [28, 44, 47]. Our previous results, showing that the AT₁ receptors in the basolateral amygdala are in-

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volved in an acute response to major stress [5], are in line with another report on the Ang II-mediated activation of the basolateral amygdala during stress [43].

In our present conditions, the volume here used (1 µl) diffused to both amygdala nuclei, as shown in histological study. Since here both nuclei could be involved, present results should be considered to be produced by the AT₁blockade in the whole amygdalar complex. Future studies, limited to sub-regions and discrete nuclei of the amygdalar complex with smaller injections, may elucidate the role of AT₁receptors in individual amygdalar nuclei and their relation to anxiety parameters here studied.

Recent clinical uses have suggested the possible treatment of anxiety disorders and related illness with angiotensin antagonists. Depressive disorders have been classically related to stress and anxiety disorders and a common etiology have been suggested [36]. In this way, angiotensin receptor blockers (ARB) have therapeutic potency substantiated by basic and clinical findings as recently reviewed [39]. In a previous study, LOS exhibited antidepressant-like properties in the forced swim test in mice [12], and the transla- tional value of this fact has been suggested [39]. In this sense, it is well recognized the fact that ARB drugs improve the life quality of patients with arterial hypertension [51]. It has been found in diabetic patients that these drugs also decrease anxiety and depression [31]. As an additional evidence in the same way, the blockade of angiotensin II synthesis induced by angiotensin converting enzyme inhibitors showed similar therapeutic properties. These drugs appear to improve the efficacy of antidepressant treatments [21]. Additionally, these drugs decrease anxiety and the levels of depression in normotensive subjects [3].

All these facts, in concordance to our present findings, give support to the idea that these compounds could have psychiatric applications, independently of their well recognized effects on blood pressure.

Conclusion

Present findings suggest that the AT₁ receptor blockade within amygdala leads to anxiolytic effects in both, normal and stress conditions, with different behavioral patterns in each case. They give additional support to the idea that AT₁ receptor inhibitors could potentially be used as anxiolytic drugs.

Acknowledgments:

Present study was granted by the Secretary of Science and Technology of the National University of Cuyo (Grants 06/J-255 and 06/J-310), and the First Neuroscience Grant of the Latin American Technological Corporation Foundation (FUCOTEL).

We thank Mrs. Sara Roitman for her invaluable contribution.

We thank Mrs. Patricia Grant de Gargiulo for style corrections in the last version of the manuscript.

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Received: January 1, 2011; in the revised form: July 23, 2011;

accepted: September 1, 2011.