Cholinergic agonist reverses H1-induced memory deficit in mice

    Cholinergic agonist reverses H1-induced memory deficit in mice C.E.M. Fernandes, K.R. Serafim, A.C.L. Gianlorenco, R. Mattioli PII: DOI: Reference:

S0278-5846(16)30130-0 doi: 10.1016/j.pnpbp.2016.08.007 PNP 8960

To appear in:

Progress in Neuropsychopharmacology & Biological Psychiatry

Received date: Revised date: Accepted date:

24 May 2016 28 July 2016 11 August 2016

Please cite this article as: Fernandes CEM, Serafim KR, Gianlorenco ACL, Mattioli R, Cholinergic agonist reverses H1-induced memory deficit in mice, Progress in Neuropsychopharmacology & Biological Psychiatry (2016), doi: 10.1016/j.pnpbp.2016.08.007

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Title: Cholinergic agonist reverses H1-induced memory deficit in mice. C.E.M. Fernandesa; K.R. Serafima; A.C.L. Gianlorencoa and R. Mattiolia* Laboratory of Neuroscience, Physiotherapy Department, Center of Biological Sciences and

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Carlos, Brazil. E-mail addresses:

[email protected] (K.R. Serafim)

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[email protected] (C.E.M. Fernandes)

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Health, Federal University of Sao Carlos, Rod. Washington Luis, Km 235, 13565-905 Sao

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[email protected] (A.C.L. Gianlorenco)

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[email protected] (R. Mattioli) *

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* Corresponding author at: Physiotherapy Department, Federal University of Sao Carlos, Rodovia Washington Luiz, 235. Bairro Monjolinho, CEP: 13565-905, São Carlos, SP, Brazil. 16

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+55

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Tel.:

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fax:

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2081.

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1. Introduction Emotions play a crucial role in human behavior since there is a strong motivational

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component in memorizing an experience that presents an emotional aspect with the propose of instruct the brain how to modulates its behavioral strategy in similar future situations

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(Strata, 2015). The amygdala has been known to play an important regulatory role in the acquisition of emotionally based learning and memory (Galvis-Alonso et al., 2010; Serafim et

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al., 2012). Addionally, moderate densities of histaminergic fibers supply the amygdaloid complex (Haas et al., 2008; Watanabe et al., 1984), which is a set of nuclei with functional

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and anatomical distinction (Ehrlish et al., 2009).

Histamine functions are mediated through the stimulation of four different G-protein receptors subtypes: H1, H2, H3 an H4 (Leurs et al., 2009). High density of postsynaptically

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located. Histamine H1 receptors are significantly expressed in the amygdala and these receptors are related to cognitive functions since their activation leads to the mobilization of

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intracelular Ca2+ by activating the Gq family of G-proteins (Hill, 1990), regulating intracellular signaling pathways that modulate neuroplasticity (Sadek; Stark, 2016). Studies have suggested the involvement of these receptors in anxiety states (Zarrindast et al., 2005),

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learning and memory (Serafim et al., 2013). The study by Zarrindast et al. (2005) showed that an H1 antagonist (pyrilamine) could significantly reverse the anxiogenic effect of histamine in rats using the elevated plus-maze test. In relation to participation of H1 receptor in emotional memory, a study showed that H1 receptor antagonist (chlorpheniramine - CPA) induced memory deficit in mice re-exposed to elevated plus-maze testing (Serafim et al., 2013). Furthermore, currently, several groups of researchers have studied H3 histaminergic receptors to better elucidated the role of these receptors in learning and emotional memory (Bahi et al., 2014; Serafim et al., 2103). It has been shown that the H3 receptors also participated in the emotional memory acquisition since H3Rs acts as an autoreceptor inhibiting the synthesis and release of histamine upon its activation; further, these receptors Abbreviations: HA, histamine; HNS, histaminergic neural system; ACh, acetylcholine; EPM, elevated plus maze; CPA, chlorpheniramine maleate; SAL, saline; T1, Trial 1; T2, Trial 2; %OAE, percentage of open arm entries; %OAT, percentage of open arm time; OAE, open arm entries; EAE, enclosed arm entries; OAT, open arm time; EAT, enclosed arm time; TE, total arm entries; CT, central area time; NBM, nucleus basalis magnocellularis; BLA, basolateral complex of the amygdala; IA, inhibitory avoidance.

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occurs also as heteroreceptors modulating the release other neurotransmitters including acetylcholine (Sadek; Stark, 2016).

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Besides, the participation of the histaminergic system in the processes of learning

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and memory, experimental and clinical evidence has showed to the hypothesis that cerebral

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acetylcholine plays a crucial role in mnemonic processes since there are a strong correlation between cholinergic transmission and cognitive function improvement (McLean et al., 2016; Kitagawa et al., 2003). The two main classes of cholinergic receptors are recognized by

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acetylcoline, muscarinic receptors (a G protein coupled receptor) and nicotinic receptors (ligand-gated ion channels) (Albuquerque et al., 2009). The most prevalent subtypes of the

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nicotinic acetylcholine receptors (nAChRs) in the brain are comprised of α4, β2 and α7 subunits (Dani; Bertrand, 2007) and it has been suggested that they play an important role in

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cognition (Levin et al., 2006; Boccia et al., 2010). A study conducted by Vicens et al. (2016)

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showed that administration of PNU, an alfa nicotinic cholinergic receptor agonist did not show effect on acquisition of a spatial learning task but a reversal of stress effects on

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retention in the Morris water maze was observed in mice with susceptibility to Alzhermer´s disease. Acute and chronic administration of nicotinic cholinergic agonists into the

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hippocampus and frontal cortex facilitates learning and memory in rats in the inhibitory avoidance task (Levin; Simon, 1998), while the administration of the nicotinic cholinergic agonist GTS-21 over 5 days facilitates performance related to memory and attention tasks in humans (Kitagawa et al., 2003). A study in our laboratory (Serafim et al., 2012) demonstrated that microinjection of CPA (0.16 nmol) into the amygdala impairs emotional memory in mice that are re-exposed to the EPM, which suggests that the inhibitory effect of this dose might be caused by the actions of this antagonist at the H1 receptors that are present in the amygdala. This inhibitory effect could induce a decrease in cholinergic activity in this structure and could thus compromise the expression of emotional memory. Based on these results, the present study is the first to investigate whether an agonist of the alpha 7 nicotinic cholinergic

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receptor (PNU-282987) reverses the emotional memory deficits induced by an H1 receptor

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antagonist (CPA) in mice that are re-exposed to the EPM.

Animals

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2.1

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2. Material and Methods

Male Swiss mice weighing 25 - 40 g at testing were used. The mice were housed in groups of 5 per cage (28 × 18 × 11 cm) and maintained under a 12-h light cycle (lights on at

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7 a.m.) in a controlled environment at a temperature of 23±1°C and a relative humidity of 50±5%. Food and drinking water were provided ad libitum. All mice were experimentally

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naïve at the beginning of the study. The experimental sessions were conducted during the light period of the cycle (9 a.m. – 2 p.m.). All of the tests were performed under illumination

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nearly 100 lux, as measured on the central platform of the EPM.

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All procedures were approved by the Ethics Committee on Animal Experimentation of the Federal University of Sao Carlos (Process #009/13) and were compliant with the US

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National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Chlorpheniramine maleate salt (Sigma Chemical Co., St. Louis, MO), an H1 receptor

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antagonist, was dissolved in sterile 0.9 % saline solution (SAL). The CPA solution was microinjected at a dose of 0.16 nmol in a volume of 0.1 μl. The dose used was based on a previous study conducted in our laboratory (Serafim et al., 2012). PNU-282987[N-(3R)-1-Azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide],

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agonist of the alpha 7 nicotinic cholinergic receptor (nAChRα7; Sigma-Aldrich), was microinjected at dose of 0.1 nmol in a volume of 0.1 μl (Ishida et al., 2011). 2.3

Surgery and microinjection

The mice were implanted with a 7-mm stainless steel guide cannula (25-gauge) under ketamine chlorhydrate and xylazine solution anesthesia (100 mg/kg and 10 mg/kg, respectively, delivered via i.p. injection). The surgical procedure was performed in a stereotaxic frame (Stoelting Co., Illinois, USA). The stereotaxic coordinates (Franklin;

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Paxinos, 2001) for the amygdala were the following: antero-posterior (AP)= −0.8 mm; lateral (L)= ±3.0 mm; and ventral (V)= −3.5 mm from the skull surface. The guide cannula was fixed

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to the skull using dental acrylic and jeweler's screws. During implantation, the guide cannula

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was aimed to terminate 1 mm above the target site. A dummy cannula (33-gauge stainless

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steel wire) was inserted into each guide cannula immediately after surgery to reduce the incidence of occlusion. Post-operative analgesia was provided via subcutaneous injection of flunixin meglumine (Banamine; Veterinary Therapeutic Guide).

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After five days of recovery from the surgery, the experimental solutions were bilaterally microinjected into the amygdala using microinjection units (33-gauge) that

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extended 1 mm beyond the tip of the guide cannula. Each microinjection unit was attached to a 5-μl Hamilton microsyringe via polyethylene tubing (PE-10), and the administration was

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controlled by an infusion pump (BI2000, Insight Equipamentos Cientificos Ltda.)

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programmed to deliver a volume of 0.1 μl over a period of 60 s. The microinjection procedure consisted of gently restraining the animal, removing the dummy cannula, inserting

60 s. Apparatus

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the injection unit, infusing the solution and keeping the injection unit in situ for an additional

The apparatus used for EPM testing was similar to those developed by Lister (1987). The acrylic maze was elevated to a height of 38.5 cm and consisted of four arms, two of which were open (30 x 6 x 0.6 cm) and two of which were enclosed (30 x 6 x 15.5 cm). The arms extended from a common central platform (6 x 6 cm). Classically, the EPM evaluates anxiety because the animals behavior associated with the emotional components during the test, expressed the conflict between the motivation to explore the maze and the natural tendency to avoid open spaces, behavior inherent in rodents (Lister, 1990). Additionally, this model was also proposed to evaluate memory. Carobrez and Bertoglio (2005) suggested that the use of the EPM has been extended to the understanding of the biological basis of emotional components related to learning and memory. Therefore,

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anxiety can be inferred by the activity in the open arms during the exposure and emotional learning it can be inferred by reducing the exploration of open arms (open arm entries and

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time spent in the open arms) during re-exposure (File, 1993; Dal-Col et al., 2003). Galvis-

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Alonso et al., (2010), suggested that during the test, the animals acquired information about

acquisition and retention. 2.5

Experimental procedure and behavioral analysis

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safe and dangerous areas of the maze and the use of re-exposure allows inferring memory

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Experiments 1 and 2 were performed on two consecutive days termed Trial 1 (T1) and Trial 2 (T2). In Experiment 1, the mice received bilateral intra-amygdalar microinjections

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of SAL or PNU (0.1 nmol) administered before T1 and T2. After five minutes of these procedures, the animals were exposed (T1) and reexposed (T2) to the EPM (Figure 1a). In

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Experiment 2, on T1, the mice received bilateral intra-amygdalar combined microinjections of

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SAL, CPA (0.16 nmol) or PNU (0.1 nmol). After five minutes, the animals were exposed to the EPM. T2 involved only re-exposure to the EPM (Figure 1b). The procedure for the

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amygdaloid complex microinjections was performed as described by Baptista et al. (2009) and Barbalho et al. (2009).

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Five days after the surgical procedures, the experiment was initiated with the transfer of the mice to the test room, where they were allowed to rest for 1h before being placed individually in the center of the plus-maze facing an open arm and allowed five minutes of free exploration. The maze was cleaned with ethanol (20 %) between tests to avoid possible biases due to odors and/or residues left by the previously tested mice. All sessions were video-recorded using a camera positioned above the maze that was linked to a computer in an adjacent room. The behavioral parameters were defined according to previous studies (Lister, 1987; Rodgers; Johnson, 1995) and included the following: the frequencies of openand enclosed-arm entries (OAE and EAE, respectively; arm entry= all four paws within an arm); total arm entries (TE); total time spent in the open arms (OAT); total time spent in the enclosed arms (EAT); and total time spent in the central area (CT). These data were used to

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calculate the percentages of OAE [%OAE; (OAE/TE) x 100] and OAT [%OAT; (OAT/300) x 100]. Enclosed arm entries (EAE) are considered a locomotor activity measurement in the

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EPM.

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The videotapes were scored using an ethological analysis software package

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developed at the Laboratory of Exploratory Behavior, USP/Ribeirao Preto (Becerra-Garcia et al., 2005). Experimental design

2.6.1

Experiment 1. Effects of bilateral intra-amygdalar microinjections of SAL or PNU-282987 on the

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mice exposed and re-exposed to the EPM.

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This experiment examined the effects of pre-T1 and pre-T2 administrations of PNU on the EPM test. Mice received a bilateral intra-amygdalar infusion of SAL or PNU (0.1 nmol) and were subjected to EPM testing five minutes later (Figure 1a). Experiment 2. Effects of bilateral intra-amygdalar injection of CPA in combination with PNU-

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2.6.2

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282987 on the mice exposed and re-exposed to the EPM.

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This experiment examined the effects of pre-T1 administration of CPA or PNU on the EPM test. The mice received bilateral intra-amygdalar infusions of SAL, CPA (0.16 nmol) or PNU (0.1 nmol). Five minutes later, the mice received bilateral intra-amygdalar infusions of

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SAL or PNU (0.1 nmol) and were subjected to the EPM five minutes after the microinjections. Twenty-four hours later, the animals were re-exposed to the EPM (Figure 1b). 2.7

Histology

At the end of the experiments, each animal received a 0.1-μl infusion of a 1% methylene blue solution into the amygdala using the procedure described above. The animals then received an anesthetic overdose, and their brains were removed and placed in formaldehyde solution (10%). Later, the brain tissues were coronally sectioned using a cryostat microtome 300 (ANCAP, Brazil). The injection sites were microscopically (Olympus B202) verified. Data from the animals with injection sites outside the amygdala were excluded from analysis.

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The histological examinations confirmed that the cannula placements in the amygdala were accurate in 79 mice and that the sample sizes of the different experiments

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cohorts were as follows: Experiment 1: SAL-SAL (n= 10), SAL- PNU (n= 10), PNU-SAL (n=

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9) and PNU-PNU (n= 10); Experiment 2: SAL-SAL (n= 8), PNU-SAL (n= 9), CPA-SAL (n=

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11) and CPA-PNU (n= 12). These individuals were used to investigate the effects of bilateral intra-amygdalar microinjections of CPA and PNU- 282987 on emotional behaviors. Microinjection tips were positioned in the amygdala and the methylene blue was within the

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amygdaloid complex (Figure 2). Therefore, the present results can be interpreted as chemical stimulation of the amygdala. Statistics

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All results were submitted to Levene’s tests for homogeneity of variance. The data

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were analyzed using two-way repeated measures ANOVA. Differences indicated by

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significant F values were further verified by post hoc Duncan’s multiple range tests. In all

3 Results 3.1 Experiment 1.

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cases, p < 0.05 was considered significant.

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Thirty-nine animals with histologically verified correct cannula placements were used in the first experiment. Figure 3AB shows the treatment effects of pre-T1 and pre-T2 intraamygdalar infusions of SAL and PNU-282987 (0.1 nmol) on the EPM behavioral measures. A two-way ANOVA revealed no significant treatment effects in terms of the %OAE [F(3,35)= 1.27, p > 0.05], the %OAT [F(3,35)= 0.32, p > 0.05], the OAE [F(3,35)= 1.31, p > 0.05] or the OAT [F(3,35)= 0.27, p > 0.05]. Post hoc tests did not reveal differences between the SALSAL, PNU-SAL and PNU-PNU groups at T1 (Table 1) in these measures. These findings indicate that the drugs did not induce changes in anxiety levels. Additionally, an ANOVA followed by Duncan´s tests revealed no significant differences between the groups at T1 in terms of TE, EAE, EAT or CT (p > 0.05 for all; Table 1).

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Regarding T2, an ANOVA confirmed statistically significant effects of trial on exploration in terms of %OAE [F(1,35)= 67.94, p < 0.05], %OAT [F(1,35)= 38.00, p < 0.05]

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(Figure 3AB), OAE [F(1,35)= 57.16, p < 0.05] and OAT [F(1,35)= 38.61, p < 0.05] (Table 1).

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Duncan’s tests indicated decreased open arm exploration (%OAE and %OAT) for all

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experimental groups (SAL-SAL, SAL-PNU, PNU-SAL and PNU-PNU) as illustrated in Figure 3AB and Table 1. Furthermore, as indicated by an ANOVA followed by post hoc comparisons, there was an increase in EAT [F(1,35)= 51.06, p < 0.05], but EAE and CT did

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not differ significantly between trials for any experimental group (p > 0.05). Duncan´s tests also indicated that the TE of the SAL-PNU and PNU-PNU groups decreased [F(1,35)=

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20.34, p < 0.05] (Table 1). 3.2 Experiment 2.

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Forty animals with histologically verified cannula placements were used in the

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second experiment. Figure 4AB shows the treatment effects of the intra-amygdalar microinjections of CPA (0.16 nmol) or PNU (0.1 nmol) on the EPM behavioral measures. A

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two-way ANOVA followed by Duncan's post hoc tests revealed no significant treatment effects in terms of %OAE [F(3,36)= 6.51, p > 0.05], %OAT [F(3,36)= 1.27, p > 0.05], OAE

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[F(3,36)= 3.93, p > 0.05] or OAT [F(3,36)= 1.27, p > 0.05] between the SAL-SAL, PNU-SAL, CPA-SAL and CPA-PNU groups at T1 (Table 2). These findings indicate that the drugs did not induce changes in anxiety states. Additionally, post hoc tests revealed no significant differences between groups at T1 in terms of TE, EAE, EAT or CT (p > 0.05) as shown in Table 2. Regarding T2, an ANOVA confirmed statistically significant effects of trial on exploration in terms of %OAE [F (1,36)= 37.87, p < 0.05], %OAT [F(1,36)= 16.32, p < 0.05] (Figure 4AB), OAE [F(1,36)= 15.38, p < 0.05] and OAT [F(1,36)= 16.32, p < 0.05] (Table 2). We also observed an interaction between the factors of treatment and test day on the %OAE [F(1,36)= 5.77, p < 0.05]. The post hoc test indicated significant decreases in activity in the open arms (%OAE, %OAT, OAE and OAT) in the SAL-SAL, PNU-SAL and CPA-PNU

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groups, but not the CPA-SAL group (Figure 4AB and Table 2). Additionally, as shown in Table 2, there were increases in EAT [F(1,36)= 23.78, p < 0.05] at T2 in the SAL-SAL, PNU-

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SAL and CPA-PNU groups, while EAE [F(1,36)= 0.33, p > 0.05] exhibited no significant

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changes across any of the experimental groups.

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A two-way ANOVA followed by Duncan’s post hoc tests indicated significant changes in the TE measure [F(1,36)= 6.98, p < 0.05]; i.e., a decrease in TE in the CPA-PNU group was observed, and the CT measures [F(1,36)= 2.57, p > 0.05] of all of the experimental

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groups at T2 compared to T1 were significantly decreased in all groups (Table 2).

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4. Discussion

The main results obtained in this study demonstrated that microinjections of the

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nicotinic cholinergic agonist PNU-282987 or the H1 histaminergic antagonist CPA into the

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amygdala induced no effect on anxiety in mice exposed to the EPM. As for emotional memory, however, the combined microinjections of PNU-282987 and CPA were able to

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reverse the deficit in memory that was induced by CPA, which suggests an interaction between the histaminergic and cholinergic systems in the amygdala in the mice re-exposed

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to the EPM. Importantly, the drugs used did not induce changes in locomotor activity, as indicated by the entries into the enclosed arms by the animals that were exposed to the EPM, a parameter considered a valid measure of locomotor activity in this model (Cruz et al., 1994). There were no effects of CPA on anxiety-related responses, because there were no differences between the groups CPA and SAL on the first day of test. These results corroborate those of a study developed in our laboratory and reported by Serafim et al. (2012) that used the microinjection of the same dose into the amygdala of mice prior to exposure to the EPM. Intra-amygdala infusion of PNU also did not appear to affect anxiety because there was no significant difference between the SAL and the group that received a PNU infusion on the first day of test. In relation to the effects induced by nicotinic cholinergic

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agonists in anxiety states, studies in rodents have suggested that the administration of these agonists can induce anxiogenic effects, anxiolytic effects or no effects on anxiety depending

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on the species, the dose, the experimental paradigm and the number of sessions used (File,

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pharmacological and behavioral responses they elicit.

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2002; Tucci et al., 2002). Moreover, different classes of nicotinic agonists might differ in the

Our results show that Intra-amygdala microinjection of PNU at dose of 0.1 nmol did not affect emotional memory in mice re-exposed to EPM. Similar results were found by

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Pandya and Yakel (2013) who demonstrated that the acute administration of PNU-282987 alone did not produce any cognitive improvements in spatial-learning or episodic memory;

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however, this drug was able to restore memory impairments induced by scopolamine in adult rats in behavioral tests like: 12-Arm radial maze test, novel object recognition test, open field

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exploration test and Rotarod test. The activation of nicotinic cholinergic receptors in the

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amygdala facilitates synaptic transmission between this structure and the cerebral cortex and improves aversive learning performance (Jiang; Role, 2008). It should be noted that

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nAChRα7s are present on both inhibitory and excitatory basolateral complex of the amygdala (BLA) neurons, their in vivo activation in the functioning BLA may promote either

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excitatory or inhibitory activity depending on a number of factors, such as the concentration of acetylcholine in the vicinity of the nAChRα7s (Pidoplichko et al. ,2013). In the present study the dose used of PNU did not induce effect in the emotional memory acquisition since this dose used did not induce excitatory effect in the amygdaloid complex. Nevertheless, another possible interpretation is that the pro-cognitive effects of nicotinic cholinergic agonists are typically observed in tasks related to fear, such as inhibitory avoidance. The study by Boccia et al. (2010) suggests that hippocampal alpha 7 nicotinic cholinergic receptors play a critical role in the reconsolidation of inhibitory avoidance responses in mice and might also have important implications for dynamic memory processes. Therefore, the microinjections of PNU did not elucidate effects on emotional memory acquisition in the EPM because the emotional nature of this task is related to

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anxiety rather than fear, since this model is based on the innate avoidance of potentially threatening areas acquired on the first test, is related to anxiety.

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Central amygdala nicotinic mediates the reversal effect of PNU on CPA-induced

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amnesia of mice re-exposed to the EPM. Perfusion of the nucleus basalis magnocellularis

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(NBM) with H1 antagonists (mepyramine or triprolidine) failed to alter the spontaneous release of ACh from the cortex in freely moving rats (Cecchi et al., 2001). However, in another study by Cecchi et al. (1998), the histaminergic projections from the NBM were

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found to exert a tonic influence on cortical cholinergic activity via the depolarization of cholinergic neurons through the activation of H1 receptors, which increased the release of

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ACh from the cerebral cortex. Furthermore, the administration of the highest dose of PNU282987 (10mg/kg) enhanced formation and recognition memory in rats (McLean et al.,

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2016). We hypothesize that the activation of the nicotinic cholinergic neurons into the

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amygdala via PNU increase the excitatory cholinergic transmission to the cerebral cortex and hippocampus, reverting the amnesia induced by the antagonist H1. On the other hand, it

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is known that CPA possess antimuscarinic properties and anticholinergic effects were suggested as part of the clinical effects of antihistamines (Yasuda; Yasuda, 1999). However,

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the hypothese of antimuscarinic effects of CPA is unlikely to justify the reversal effect of PNU on H1-induced memory deficit in mice. The amygdaloid complex receives cholinergic innervation from the basal forebrain and nicotine, via activation of neuronal nicotinic acetylcholine receptors (Jiang; Role, 2008). The study conducted by Barros et al. (2005)

indicate that nAChRs in the amygdala

participate in the regulation of work memory formation and short term memory and long-term memory acquisition and consolidation, while infusion of the α4 antagonist impair working memory performance rat basolateral amygdala. The deletion of this receptor in knockout mice induces cognitive deficits, indicating that this cholinergic system is directly related to cognitive processes (Young et al., 2011). These finding support the contention that nACHRs

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amygdala activation may constitute an important strategy for the modulation of emotional memory.

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Nicotine has been widely reported to improve cognition function in humans and

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experimental animals (Levin et al., 2006) and cholinergic dysfunction has been associated to

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the pathophysiology of Alzheimer´s disease (Nordberg, 2001). Our results demonstrate the efficacy of nicotinic agonist to reverse CPA-induced deficit on emotional memory acquisition in mice, suggesting that the interaction between these systems may be useful to detect

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target for cognitive deficits and pharmacological strategy to enhance cognitive performance. The results of this study indicate that an interaction between the histaminergic and

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nicotinic cholinergic systems in the amygdala of the mice that were re-exposed to the EPM modulated emotional memory acquisition. Thus, we suggest that the histaminergic H1

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5. Acknowledgements

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deficits in emotional learning.

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receptor antagonist reduced the selective cholinergic activation in the amygdala, which led to

The authors are grateful for the financial support provide by FAPESP (grant proc.

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2012/10913-0) and CNPq (grant proc.303088/2014-1).

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agents in the central amygdala of rats in the elevated plus-maze test of anxiety.

Figures legends:

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Figure 1ab. Schematic representation of the experimental design for the [a] Experiment 1

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and [b] Experiment 2 in the elevated plus maze (EPM). SAL saline; CPA chlorpheniramine.

Figure 2. Schematic representation of amygdala microinfusion sites in mice. Sections are

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between −1.06 and −1.46 mm from the bregma in the atlas of Franklin and Paxinos (2001).

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There are fewer sites than total mice because of overlaps.

Figure 3AB. Effects of intra-amygdalar infusion of SAL or PNU-282987 (0.1 nmol) on the percentage of open arm entries (%OAE) (A) and the percentage of time spent in the open arms (%OAT) (B) during Trial 1 (T1) and Trial 2 (T2) in the EPM. Drug was administered prior to each trial. Groups: SAL–SAL (n=10), SAL–PNU (n=10), PNU–SAL (n=9) and PNU– PNU (n=10).*p < 0.05 for T1 compared to T2 (ANOVA followed by Duncan’s post hoc test).

Figure 4AB. Effects of intra-amygdalar infusion of SAL, CPA (0.16 nmol) or PNU-282987 (0.1 nmol) on the percentage of open arm entries (%OAE) (A) and the percentage of time spent in the open arms (%OAT) (B) during Trial 1 (T1) and Trial 2 (T2) in the EPM. Both

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drugs were administered prior to T1. Groups: SAL–SAL(n=8), PNU–SAL(n=9), CPA– SAL(n=11) and CPA–PNU(n=12).*p<0.05 for T1 compared to T2 (ANOVA followed by

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Duncan’s post hoc test).

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Table 1. Effects of intra-amygdalar infusion of SAL or PNU-282987 (0.1 nmol) prior to Trial 1 (T1) and prior to Trial 2 (T2) on behavioral measures of mice that had been re-exposed to

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EPM testing.

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Data are reported as mean ± SEM. TE= total entries; EAE= enclosed arm entries; OAE= open arm entries; OAT= time spent in open arms; EAT= time spent in enclosed arms; CT= time spent in the central area; *p < 0.05 for T1 compared to T2 (ANOVA, followed by

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Duncan’s post hoc test).

Table 2. Effects of intra-amygdalar infusion of SAL, CPA (0.16 nmol) or PNU-282987 (0.1 nmol) prior to Trial 1 (T1) and prior to Trial 2 (T2) on behavioral measures of mice that had

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been re-exposed to EPM testing.

Data are reported as mean ± SEM. TE= total entries; EAE= enclosed arm entries; OAE= open arm entries; OAT= time spent in open arms; EAT= time spent in enclosed arms; CT= time spent in the central area; *p < 0.05 for T1 compared to T2 (ANOVA, followed by Duncan’s post hoc test).

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Highlights: • -282987 did not induce effects on anxiety in elevated plus maze. • -282987 per se did not produce effects on emotional memory. • -282987 reverse memory deficits induced by chlorpheniramine.