Psychoneuroendocrinology (2014) 43, 114—125

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Mineralocorticoid receptors in the ventral tegmental area regulate dopamine efflux in the basolateral amygdala during the expression of conditioned fear Amanda R. de Oliveira a,b,*, Adriano E. Reimer a,b, ˜o a,b Marcus L. Branda a

´rio de Psicobiologia, Faculdade de Filosofia, Cie ˜o Preto, Laborato ˆncias e Letras de Ribeira ˜o Paulo, Ribeira ˜o Preto, SP, Brazil Universidade de Sa b ˜o Preto, SP, Brazil Instituto de Neurocie ˆncias e Comportamento (INeC), Ribeira Received 18 November 2013; received in revised form 20 January 2014; accepted 10 February 2014

KEYWORDS Fear conditioning; Mineralocorticoid receptors; Glucocorticoid receptors; Ventral tegmental area; Basolateral amygdala complex; Dopamine

Summary Despite the recognized involvement of corticosteroids in the modulation of emotional behavior, the specific role of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) in the expression of conditioned fear responses is still open to investigation. The present study sought to clarify the involvement of both types of corticosteroid receptors in two different brain regions — the ventral tegmental area (VTA) and the basolateral amygdala complex (BLA) — on the expression of conditioned fear. The first experiment assessed the effects of intra-VTA or intra-BLA administration of spironolactone (MR antagonist) or mifepristone (GR antagonist) on the expression of conditioned freezingtoalight-CSandonmotorperformanceintheopen-fieldtest.Intra-VTAspironolactone,butnot mifepristone, attenuated the expression of the conditioned freezing response whereas intra-BLA spironolactone or mifepristone had no significant effects. These treatments did not affect motor performance in the open-field test. Since dopamine is released in the BLA from the VTA during the expressionofconditionedfear,theanxiolytic-likeeffectofdecreasedcorticosteroidactivityinthefirst experiment could be associated with changes in dopaminergic neurotransmission. The second experiment, using in vivo microdialysis, investigated the role of MRs in the VTA on dopamine levels in the BLA during the expression of conditioned fear. Blocking MRs locally in the VTAwith spironolactone reduced dopamine efflux in the BLA and decreased the expression of conditionedfreezing in response to the CS. Taken together, the data indicate that corticosterone, acting locally on MRs in the VTA, stimulates dopamine efflux in the BLA, which facilitates the expression of conditioned freezing to a light-CS. # 2014 Elsevier Ltd. All rights reserved.

* Corresponding author at: Laborato ˆncias e Letras de Ribeira ´rio de Psicobiologia, Faculdade de Filosofia, Cie ˜o Preto, Universidade de Sa ˜o Paulo. Av. Bandeirantes, 3900, Ribeira ˜o Preto. SP 14090-901, Brazil. Tel.: +55 16 3602 3838; fax: +55 16 3602 4830. E-mail address: [email protected] (A.R. de Oliveira). http://dx.doi.org/10.1016/j.psyneuen.2014.02.010 0306-4530/# 2014 Elsevier Ltd. All rights reserved.

Ventral tegmental area mineralocorticoid receptors regulate the expression of conditioned fear

1. Introduction Considering the complexity of aversive information processing and defensive response expression, a combined action of several stress mediators may be required for optimal performance during threatening situations. With specific regard to fear conditioning, much research has been performed elucidating the involvement of distinct mediators during its acquisition and consolidation phases, but comparatively less is known about the retrieval and expression of conditioned fear memories (Lupien and McEwen, 1997; Rodrigues et al., 2009). Taking into account the adaptive importance of previous experience retrieval for the expression of appropriate defensive responses, studying the neural substrates and mediators involved in these processes is of great interest, especially because of their relevance to different aspects of human anxiety disorders. The hypothalamic-pituitary-adrenocortical (HPA) axis activity, which leads to the release into the bloodstream of corticosteroids (cortisol in primates, corticosterone in rodents), has been considered a key part of the stress reaction and can be triggered either by innate or conditioned aversive stimuli (Cordero et al., 1998; Reis et al., 2012). Corticosteroids are hormones that can easily pass the blood— brain barrier, thus affecting a variety of fear-related brain areas (McEwen et al., 1969; Stevens et al., 1971). In the brain, corticosteroids bind to two types of receptors: mineralocorticoids (MRs) and glucocorticoids (GRs) (Reul and de Kloet, 1986; Lu et al., 2006). Despite the recognized involvement of corticosteroids in modulating emotional behavior, the specific role of MRs and GRs in the expression of conditioned fear responses is still open to investigation. The present study sought to clarify the possible involvement of both types of corticosteroid receptors in two different fearrelated brain regions — the ventral tegmental area (VTA) and the basolateral amygdala complex (BLA) — on the expression of conditioned fear. Previous studies confirmed that both MRs and GRs are present in VTA and BLA neurons (Harfstrand et al., 1986; Ronken et al., 1994; Johnson et al., 2005). So, the first experiment assessed the effects of intra-VTA or intra-BLA administration of spironolactone (MR antagonist) or mifepristone (GR antagonist) on the expression of conditioned freezing to a light-CS and on motor performance in the open-field test. Recently, several laboratories have shown great interest in the interaction between the activation of the HPA axis and dopaminergic neurotransmission during aversive states (Barr et al., 2009; Sapolsky, 2009). Dopamine, although more commonly associated with the reinforcing effects of various stimuli, is one of the most active neuromodulators of fear and anxiety (Reis et al., 2004; de Oliveira et al., 2006; Fadok et al., 2009; Zweifel et al., 2011). In fact, dopamine is released in the BLA (consisting of the lateral, basal and accessory basal nuclei) from neurons of the VTA during the expression of conditioned fear (de Oliveira et al., 2011, 2013). Furthermore, quinpirole (a dopamine D2 receptor agonist) targeting autoreceptors in the VTA or microinjections of sulpiride (a dopamine D2 receptor antagonist) into the BLA decrease the expression of conditioned fear responses (de Oliveira et al., 2009, 2011; de Souza Caetano et al., 2013). These findings suggest that reducing the activity of dopaminergic neurons in

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the VTA-BLA pathway reduces conditioned fear. However, the neurohumoral mechanisms involved in regulating the dopamine efflux in the VTA-BLA pathway triggered by a CS remain to be clarified. In an attempt to determine the extent to which the combined action of the HPA axis and dopaminergic neurotransmission is important for the expression of conditioned fear responses, we observed in a previous study that systemic administration of metyrapone (a corticosterone synthesis blocker) prevented enhanced dopamine release in the BLA during a conditioned fear test and decreased the expression of conditioned freezing (de Oliveira et al., 2013). Thus, HPA axis activation seems to be an important step in an integrated neuroendocrine—neurochemical—behavioral response when the organism evaluates and interprets the threat associated with a specific environmental stimulus and subsequently triggers adaptive defense reactions to cope with this situation. To further clarify this issue, in the second part of the present study using in vivo microdialysis, we examined the influence of MRs in the VTA on modulating the release of dopamine in the BLA during the expression of conditioned fear.

2. Methods 2.1. Animals One-hundred and forty naive male Wistar rats from the animal facility of the Campus of the University of Sa ˜o Paulo at Ribeira ˜o Preto were used. The rats, weighing 270—290 g at the beginning of the experiments, were housed in groups of four in plastic boxes (40 cm  33 cm  26 cm) and maintained under controlled conditions (23  1 8C; 12 h/12 h light/dark cycle, lights on at 0700 h) with food and water freely available. The experiments were carried out during the light phase of the cycle. All the procedures were approved by the Committee for Animal Care and Use of the University of Sa ˜o Paulo at Ribeira ˜o Preto (No. 10.1.595.53.7), and were performed in compliance with the recommendations of the Brazilian Society of Neuroscience and Behavior, which are based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available.

2.2. Surgery The rats were anesthetized with ketamine/xylazine (100/ 7.5 mg/kg, intraperitoneal; Agener Unia ˜o, Embu-Guac¸u, SP, Brazil) and fixed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). The incisor bar was set at 3.0 mm below the interaural line, such that the skull was horizontal between bregma and lambda. For experiment 1, bilateral guide cannulae for drug injections (0.6 mm outer diameter, 0.4 mm inner diameter) were stereotaxically implanted over the VTA or the BLA. For experiment 2, unilateral VTA cannulation for drug injection and unilateral BLA cannulation for the microdialysis probe (CMA/12; CMA/Microdialysis AB, Solna, Sweden) were performed in the right hemisphere. In previous studies, we showed that microdialysis with a

116 probe implanted unilaterally into the BLA accurately measures changes in the extracellular concentrations of dopamine in this structure (de Oliveira et al., 2011, 2013). Taking bregma as the reference point, the following coordinates were used (Paxinos and Watson, 2007): VTA (anterior/posterior, 5.9 mm; medial/lateral, 0.7 mm; dorsal/ventral, 7.6 mm) and BLA (anterior/posterior, 2.3 mm; medial/ lateral, 5.5 mm; dorsal/ventral, 7.0 mm). The cannulae were fixed to the skull with acrylic resin and stainless steel screws. In addition, each guide cannula was sealed with a stainless steel wire to protect it from blockage. At the end of surgery, the rats received an intramuscular injection of a polyvalent veterinary antibiotic (Pentabio ´tico, 0.2 ml; Fort Dodge, Campinas, SP, Brazil) and a subcutaneous injection of the anti-inflammatory and analgesic flunixin meglumine (Banamine, 2.5 mg/kg; Schering-Plough, Cotia, SP, Brazil). Afterwards, the rats were allowed 5 days to recover from the surgical procedure.

2.3. Drugs The following drugs were used: the corticosteroid MR antagonist spironolactone and the corticosteroid GR antagonist mifepristone. Spironolactone and mifepristone (Sigma; St. Louis, MO, USA) were dissolved initially in 100% DMSO, kept in a stock solution at 70 8C, and then diluted in saline shortly before use. The final DMSO concentration was 1%. Both drugs were microinjected into the VTA or BLA (5 or 10 ng/0.2 ml/ site) 10 min before the test session. The drugs, doses, and injection times were based on previous studies (Roozendaal and McGaugh, 1997; Conrad et al., 2004; Yang et al., 2006). Each treatment group had its own control group that received an injection of saline + DMSO (1%) 10 min before the test session.

2.4. Microinjection procedure The injection needle was a thin dental needle (0.3 mm outer diameter) connected to a 10 ml syringe (Hamilton Company, Reno, NV, USA) by means of a polyethylene tube (PE-10; Becton—Dickinson, Franklin Lakes, NJ, USA). The injection needle was introduced through the guide cannula until its lower end reached 1 mm below the cannula. The solutions were injected into the VTA or BLA using an infusion pump (Harvard Apparatus, South Natick, MA, USA). The displacement of an air bubble inside the polyethylene catheter connecting the syringe needle to the intracerebral needle was used to monitor the microinjection. The microinjection lasted 1 min and the needle was held in place for an additional 1 min to maximize diffusion from the needle tip.

2.5. Experiment 1: conditioned fear and openfield tests 2.5.1. Conditioned fear Training: The rats were conditioned to a light-CS in a cage (20 cm  20 cm  25 cm) with stainless steel side and back walls, a transparent Plexiglas ceiling and front door, and a grid floor that consisted of stainless-steel rods spaced 1.0 cm apart. This cage was located within a ventilated and sound-attenuated chamber (45 cm  45 cm  45 cm). After recovery from

A.R. de Oliveira et al. surgery, the rats were placed in the training cage and received 10 CS-US pairings after a habituation phase of 5 min using a 4 s, 6 W light-CS that coterminated with a 1 s, 0.6 mA footshockUS. The footshocks were delivered through the training cage floor by a constant current generator built with a scrambler (Albarsh Instruments, Porto Alegre, RS, Brazil). The intertrial interval varied randomly between 60 and 180 s. Stimulus presentation was controlled by a microprocessor and an input/output board (Insight Equipment, Ribeira ˜o Preto, SP, Brazil). Each animal was removed 2 min after the last footshock and returned to its home cage. The duration of the training session was approximately 25 min, including habituation time. Testing: The conditioned fear test was conducted without footshock presentation in a wire-grid cage (16.5 cm  7.5 cm  7.5 cm) different from the training cage to avoid conditioning to the context and located inside a ventilated, sound-attenuating plywood chamber (64 cm  60 cm  40 cm). Twenty-four hours after training, the rats received intra-VTA or intra-BLA administration of spironolactone, mifepristone or vehicle and, after 10 min, were placed in the test cage. After a habituation phase of 5 min, the rats received 10 CS presentations (4 s, 6 W light-CS). The duration of the test session, including habituation time, was 20 min. The behavior of the rats was recorded by a video camera positioned behind the observation chamber with the signal relayed to a monitor in another room via a closed circuit. The behavioral measure that was used to assess conditioned fear was the time that rats spent freezing during this test session. Freezing was operationally defined as the total absence of movement of the body and vibrissae, except those required for respiration, for at least 6 s. Freezing behavior was monitored during the test and subsequently scored from video recordings by a person who was blind with regard to the experimental condition of each rat. The results are presented as total time rats spent freezing during the test session. Each rat was subjected to only one treatment and to a single conditioned fear test session. 2.5.2. Open-field The same rats used for the conditioned fear experiment were also used to assess the effects of the drug treatments on motor activity in the open-field test. This experiment was conducted in an arena consisting of a circular enclosure made of transparent Plexiglas (60 cm in diameter and 50 cm height). The arena was located in a room with a controlled and indirect illumination of 30 lux on the floor of the apparatus. The rats’ behavior was recorded by a video camera positioned above the arena. Two days after the conditioning testing session, the rats received intra-VTA or intra-BLA administration of spironolactone, mifepristone or vehicle. Ten min after drug injection, rats were placed in the middle of the arena and left for a 20 min period of free exploration. The following behavioral responses were recorded over the course of the session: total distance traveled, distance traveled in the border and in the center of the arena, time spent in the border and in the center of the arena, and total immobility time. Motor activity was monitored during the test and subsequently scored automatically from video recordings using ANY-maze software (version 4.7; Stoelting, Wood Dale, IL, USA).

Ventral tegmental area mineralocorticoid receptors regulate the expression of conditioned fear

2.6. Experiment 2: conditioned fear and in vivo microdialysis Training: For this experiment, additional groups of rats were subjected to fear conditioning to a light-CS as described for experiment 1. Testing: The next day, the rats were placed in a microdialysis bowl (BAS, West Lafayette, IN, USA) and a probe (2 mm membrane; CMA/Microdialysis AB) was inserted into the BLA and perfused with Ringer’s solution (147.0 mM NaCl, 4.0 mM KCl, and 2.2 mM CaCl2) at a constant flow rate of 1.5 ml/min (Microinjection pump; BAS). Following a 2 h equilibrium period, ten dialysate samples (four during baseline, one during the test, five post-test) were collected every 30 min into vials that contained 3 ml of 0.05 M perchloric acid solution. After the baseline samples were collected, the rats received intra-VTA administration of spironolactone or vehicle and were transferred to a Plexiglas cage (20 cm  23 cm  31 cm) for the conditioned fear test. In this cage, after a 10 min habituation period, over the course of the 20 min test session, the 4 s light-CS was presented 10 times with an interstimulus interval of 60—180 s. The behavioral criterion used to assess conditioned fear was again the time rats spent freezing. At the end of the test session, the rats were placed back in the microdialysis bowl and dialysates were collected for an additional 150 min. The mean of the four baseline samples served as the reference for all analyses, which were therefore expressed as percent changes. Each rat was subjected to only one treatment and to a single conditioned fear test session. Dopamine assay: The amount of dopamine in the dialysates was analyzed using high-performance liquid chromatography (HPLC). The reverse-phase ODS column was a HyperClone 150 mm  2.0 mm C-18 with a 3 mm particle size (Phenomenex, Torrance, CA, USA). The HPLC device consisted of a BAS Epsilon electrochemical detector with a glass-carbon electrode and pump (PM-92e). The potential was set at 650 mV (compared with the Ag-AgCl reference electrode). The mobile phase, consisting of 50 mM NaH2PO4, 0.1 mM Na2-EDTA, 0.5 mM n-octyl sodium sulfate, and 10% methanol (pH 5.5), was filtered and pumped through the system at a flow rate of 200 ml/min. The injection volume was 50 ml. This set-up allowed the dopamine levels in each sample to be analyzed in a run that lasted approximately 12 min.

2.7. Histology Upon the conclusion of the experiments, in order to confirm the position of the microinjection and microdialysis sites, the rats were given a lethal dose of urethane (3 g/kg; Sigma— Aldrich, St. Louis, MO, USA) and 0.2 ml of Evans Blue (2%) was microinjected into the VTA or BLA. Afterwards, the rats were perfused transcardially with 0.9% saline and 4% paraformaldehyde solutions. The brains were removed from the skulls, maintained in paraformaldehyde for 2 h, and then cryoprotected in 30% sucrose until soaked. Coronal 60 mm slices were cut, mounted on gelatin-coated slides, and stained with cresyl violet (5%), allowing localization of the microinjection and probe sites according to the atlas of Paxinos and Watson (2007).

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2.8. Statistical analysis The injection and/or microdialysis sites of all animals considered for the statistical analysis in the present study were located inside the VTA and/or BLA, depending on the experiment. The data are reported as mean  S.E.M. For experiment 1, the conditioned freezing response and activity in the open-field test were subjected to one-way analysis of variance (ANOVA). For experiment 2, the conditioned freezing response was subjected to a Student’s t-test. Two-way repeated-measures ANOVA was used for the microdialysis data, with treatment (drug and its control) as the between-subjects factor, and time of sample collection as the within-subjects factor. Significant comparisons were followed by the Newman—Keuls post hoc test. The significance level was set at p < 0.05.

3. Results 3.1. Experiment 1: conditioned fear and openfield tests Only rats with microinjection sites located bilaterally within the VTA were included in this part of the study. We had 17 animals with cannulae outside the VTA. Since these rats were distributed in six distinct groups of the present experiment, the number of rats with misplaced cannulae per group was not sufficient for entering into the statistical analysis. Fig. 1A shows a photomicrograph of representative microinjection sites in the VTA. Fig. 1B shows the histological localization of the injection sites in the VTA, depicted on diagrams from the atlas of Paxinos and Watson (2007). Fig. 2A depicts the timeline of the procedures looking at VTA MR or GR involvement in the expression of conditioned freezing response and motor activity. Fig. 2B shows the mean freezing response for rats that received intra-VTA microinjection of vehicle (control; n = 10), 5 ng/0.2 ml/site of the MR antagonist spironolactone (Spiro5; n = 10) or 10 ng/0.2 ml/ site spironolactone (Spiro10; n = 10). One-way ANOVA revealed significant effects of the treatments on the conditioned freezing response (F 2,27 = 3.98, p < 0.05). The Newman—Keuls post hoc test revealed that 10 ng/0.2 ml/site spironolactone significantly reduced the duration of the conditioned freezing compared with the control group (p < 0.05). Fig. 2C and D displays the data obtained with rats that received intra-VTA vehicle (control; n = 7), 5 ng/ 0.2 ml/site spironolactone (Spiro5; n = 7) or 10 ng/0.2 ml/site spironolactone (Spiro10; n = 7) in the open-field test. Oneway ANOVA did not reveal significant effects of the treatments on the total distance traveled (F 2,18 = 1.27, p > 0.05) or total time spent immobile (F 2,18 = 0.38, p > 0.05). Oneway ANOVA also showed no significant effects on the distance traveled in the border (F 2,18 = 1.68, p > 0.05) or in the center of the arena (F 2,18 = 0.4, p > 0.05), or on time spent in the border (F 2,18 = 0.09, p > 0.05) or in the center of the arena (F 2,18 = 0.72, p > 0.05) — data not shown graphically. Fig. 2E shows the mean freezing response for rats that received intra-VTA microinjection of vehicle (control; n = 10), 5 ng/ 0.2 ml/site of the GR antagonist mifepristone (Mifep5; n = 10) or 10 ng/0.2 ml/site mifepristone (Mifep10; n = 10). One-way ANOVA revealed no significant effects of the treatments on

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Figure 1 Target sites for microinjection into the ventral tegmental area (VTA). Photomicrograph showing representative microinjection sites in the VTA (A). Outline of microinjection locations on cross-sections from the Paxinos and Watson atlas (2007) (B). The number of points in the figure is less than the total number of rats used because of several overlaps. Scale bar = 0.5 mm. DMPAG, dorsomedial periaqueductal gray; CLi, caudal linear nucleus of the raphe; Pn, pontine nuclei.

the conditioned freezing response (F 2,27 = 2.04, p > 0.05). Fig. 2F and G displays the data from the rats treated with intra-VTA vehicle (control; n = 7), 5 ng/0.2 ml/site mifepristone (Mifep5; n = 7) or 10 ng/0.2 ml/site mifepristone (Mifep10; n = 7) in the open-field test. One-way ANOVA did not reveal significant effects of the treatments on the total distance traveled (F 2,18 = 2.12, p > 0.05) or total time spent immobile (F 2,18 = 1.84, p > 0.05). One-way ANOVA also revealed no significant effects on the distance traveled in the border (F 2,18 = 2.75, p > 0.05) or in the center of the arena (F 2,18 = 0.08, p > 0.05), or on time spent in the border (F 2,18 = 2.21, p > 0.05) or in the center of the arena (F 2,18 = 1.08, p > 0.05) — data not shown graphically. Only rats with microinjection sites located bilaterally within the BLA were included in the next part of the study. We had 20 animals with cannulae outside the BLA. Since these rats were distributed in six distinct groups, the number of rats with misplaced cannulae per group was not sufficient for entering into the statistical analysis. Fig. 3A shows a photomicrograph of representative microinjection sites in the BLA. Fig. 3B shows the histological localization of the injection sites in the BLA, depicted on diagrams from the atlas of Paxinos and Watson (2007). Fig. 4A displays the timeline of the procedures looking at BLA MR or GR involvement in the expression of conditioned

A.R. de Oliveira et al. freezing and motor activity. Fig. 4B shows the mean freezing response for rats that received intra-BLA microinjection of vehicle (control; n = 10), 5 ng/0.2 ml/site of the MR antagonist spironolactone (Spiro5; n = 10) or 10 ng/0.2 ml/site spironolactone (Spiro10; n = 10). One-way ANOVA revealed no significant effects of the treatments on the conditioned freezing response (F 2,27 = 0.16, p > 0.05). Fig. 4C and D shows the data for the rats treated with intra-BLA vehicle (control; n = 7), 5 ng/0.2 ml/site spironolactone (Spiro5; n = 7) or 10 ng/0.2 ml/site spironolactone (Spiro10; n = 7) in the open-field test. One-way ANOVA did not reveal significant effects of the treatments on the total distance traveled (F 2,18 = 0.09, p > 0.05) or total time spent immobile (F 2,18 = 0.52, p > 0.05). One-way ANOVA also showed no significant effects on the distance traveled in the border (F 2,18 = 0.21, p > 0.05) or in the center of the arena (F 2,18 = 0.22, p > 0.05), or on time spent in the border (F 2,18 = 0.85, p > 0.05) or in the center of the arena (F 2,18 = 1.05, p > 0.05) — data not shown graphically. Fig. 4E shows the mean freezing response for rats that received intra-BLA microinjection of vehicle (control; n = 10), 5 ng/0.2 ml/site of the GR antagonist mifepristone (Mifep5; n = 10) or 10 ng/0.2 ml/site mifepristone (Mifep10; n = 10). One-way ANOVA revealed no significant effects of the treatments on the conditioned freezing response (F 2,27 = 0.19, p > 0.05). Fig. 4F and G shows the data from the rats treated with intra-BLA vehicle (control; n = 7), 5 ng/ 0.2 ml/site mifepristone (Mifep5; n = 7) or 10 ng/0.2 ml/site mifepristone (Mifep10; n = 7) in the open-field test. One-way ANOVA did not reveal significant effects of the treatments on the total distance traveled (F 2,18 = 0.55, p > 0.05) or total time spent immobile (F 2,18 = 1.80, p > 0.05). One-way ANOVA also showed no significant effects on the distance traveled in the border (F 2,18 = 0.02, p > 0.05) or in the center of the arena (F 2,18 = 2.29, p > 0.05), or on time spent in the border (F 2,18 = 2.44, p > 0.05) or in the center of the arena (F 2,18 = 3.57, p > 0.05) — data not shown graphically.

3.2. Experiment 2: conditioned fear and in vivo microdialysis Only rats with microinjection and probe sites located within the VTA and BLA, respectively, were included in this experiment. Five animals with misplaced cannulae were not considered for the statistical analysis. Photomicrographs of representative microinjection and probe sites into the VTA and BLA are shown in Fig. 5A and B. Fig. 5C and D displays the histological localization of the injection sites in the VTA and probe sites in the BLA, depicted on diagrams from the atlas of Paxinos and Watson (2007). Fig. 6A displays the procedures timeline for testing the effects of intra-VTA administration of the MR antagonist spironolactone on extracellular dopamine concentration in the BLA and the conditioned freezing response. Fig. 6B shows the time-course of extracellular dopamine concentration in the BLA in rats subjected to the conditioned fear test and treated with vehicle (control; n = 10) or 10 ng/0.2 ml spironolactone (Spiro10; n = 10). Two-way repeated-measures ANOVA revealed no significant effect of treatments (F 1,162 = 0.06, p > 0.05), but a significant effect of time (F 10,199 = 3.78, p < 0.05) and a significant treatments  time

Ventral tegmental area mineralocorticoid receptors regulate the expression of conditioned fear

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Figure 2 Mineralocorticoid (MRs) and glucocorticoid (GRs) receptors in the ventral tegmental area (VTA) and conditioned fear. Timeline of the procedures looking at VTA MR and GR involvement in the expression of conditioned fear and motor activity (A). Time of freezing in rats that received intra-VTA vehicle (control; n = 10), 5 ng/0.2 ml/site of the MR antagonist spironolactone (Spiro5; n = 10) or 10 ng/0.2 ml/site spironolactone (Spiro10; n = 10) and were subjected to the conditioned fear test (B). Motor activity of rats treated with intra-VTA vehicle (control; n = 7), 5 ng spironolactone (Spiro5; n = 7) or 10 ng spironolactone (Spiro10; n = 7) (C and D). Time of freezing in rats that received intra-VTA vehicle (control; n = 10), 5 ng/0.2 ml/site of the GR antagonist mifepristone (Mifep5; n = 10) or 10 ng/0.2 ml/site mifepristone (Mifep10; n = 10) and were subjected to the conditioned fear test (E). Motor activity of rats treated with intra-VTA vehicle (control; n = 7), 5 ng mifepristone (Mifep5; n = 7) or 10 ng mifepristone (Mifep10; n = 7) (F and G). *p < 0.05, different from control.

time interaction (F 10,199 = 2.41, p < 0.05). The Newman— Keuls post hoc test revealed an increase in dopamine concentration in the BLA of control rats during the conditioned fear test compared with baseline (p < 0.05). Newman—Keuls post hoc comparison also showed that intra-VTA spironolactone blocked this increase in dopamine concentration in the BLA (p < 0.05). Fig. 6C shows the conditioned freezing response for the same rats for the 20 min test period that corresponded to the fifth microdialysis sample. Student’s ttest revealed that intra-VTA spironolactone significantly decreased the conditioned freezing response (t18 = 2.42, p < 0.05), as seen in our first experiment.

4. Discussion Conditioned freezing is the main response to cues associated with footshock and is a widely used index of conditioned fear in rodents. In the present study, light-CS consistently induced a conditioned freezing response during the test session in all control rats. This behavioral result shows the efficacy of the conditioning protocol used here. The MR antagonist spironolactone decreased the conditioned freezing response when administered intra-VTA

before the test session. Spironolactone’s effect cannot be attributed to nonspecific effects since the same dose did not affect the performance of rats in the open-field test. On the other hand, intra-VTA microinjection of the GR antagonist mifepristone, or intra-BLA administration of spironolactone or mifepristone, caused no significant effects on freezing behavior. The doses of spironolactone and mifepristone injected into the VTA and BLA also did not affect motor performance in the open-field test. In fact, spironolactone and mifepristone did not alter either locomotor (e.g., total distance traveled and distance traveled in the border) or unconditioned fear behaviors, reflected in the distance traveled and time spent in the center of the open-field. Therefore, it may be suggested that the MR and GR antagonists did not have a broad anxiolytic-like action when injected intraVTA or intra-BLA; however, intra-VTA administration of the MR antagonist exhibited a specific anxiolytic-like effect in the conditioned fear test used in the present study. This difference in the effects of intra-VTA spironolactone may reflect distinct modulatory roles of corticosteroids in different defensive responses and it is in agreement with the idea that corticosteroids may be more important for cognitiverelated emotional responses than for instinctive fear behaviors, as pointed out by previous studies from our group

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Figure 3 Target sites for microinjection into the basolateral amygdala complex (BLA). Photomicrograph of representative microinjection sites in the BLA (A). Outline of microinjection locations on cross-sections from the Paxinos and Watson atlas (2007) (B). The number of points in the figure is less than the total number of rats used because of several overlaps. Scale bar = 1.0 mm. CA3, CA3 field of the hippocampus; VPM, ventral posteromedial thalamic nucleus; ec, external capsule; MeP, medial amygdaloid nuclei.

(Albrechet-Souza et al., 2007; Reis et al., 2012; de Oliveira et al., 2013). The intra-VTA spironolactone effect observed here is consistent with other studies that showed anxiolytic-like effects of systemic or intracerebroventricular injections of this MR antagonist on the expression of contextual conditioned freezing responses (Korte et al., 1995; Ninomiya et al., 2010). As the concentration of corticosteroids may be increased after spironolactone administration (Ratka et al., 1989; Otte et al., 2007), we cannot exclude that an enhanced activation of GRs might have contributed to the present results. However, this seems unlikely since in our previous work systemic administration of corticosterone did not change the expression of conditioned freezing (de Oliveira et al., 2013). In fact, we showed in that same study that blocking corticosterone synthesis with metyrapone impaired

A.R. de Oliveira et al. the expression of conditioned freezing to a light-CS in rats. Convergent with this, decreased HPA axis activity has also been associated with impaired emotional memory in humans, while no effects were observed on the recall of neutral information (Marin et al., 2011; Rimmele et al., 2013). Now, the present data suggest that VTA MRs are involved in the expression of conditioned fear. The selectivity of the effect produced by the MR antagonist is further attested by the lack of effect of pre-testing intra-VTA microinjection of the GR antagonist on the conditioned freezing response. The present data also indicate that MRs and GRs in the BLA do not appear to be important for the expression of conditioned fear to a light-CS. The above-mentioned general absence of effects of mifepristone has to be interpreted with caution since only two doses of this GR antagonist were used in the present study. Higher doses were not used because they could act at receptors other than GRs; for instance, mifepristone may act as a progesterone antagonist (Gaillard et al., 1984; Lu et al., 2006). To avoid these side effects, the doses commonly used in studies showing that mifepristone affected fear under other experimental conditions do not go beyond the ones used in the present study (Roozendaal and McGaugh, 1997; Conrad et al., 2004; Yang et al., 2006). Also, when injected systemically or intracerebroventricularly, mifepristone did not affect the expression of conditioned freezing to the context (Korte et al., 1995; Ninomiya et al., 2010). Another important point that should be considered is that, although the classical mechanism of corticosteroid action involves intracellular receptors that regulate gene expression, the rapidity with which corticosteroids exert some of their effects indicates that they also act through nongenomic mechanisms, possibly by binding to neuronal membrane receptors (Schumacher, 1990; Orchinik et al., 1991; Lu et al., 2006; Tasker et al., 2006). Since the GR antagonist mifepristone alters the nuclear translocation of GRs (Tasker et al., 2006; Spiga et al., 2011), one possibility is that it may be specific for genomic mechanisms of receptor signaling, leaving the membrane action of GRs still free to occur. The rapidity of the anxiolytic-like effect of the MR antagonist in the present study also argues against a genomic action, suggesting that corticosterone, via high affinity MRs located in the VTA membrane, modulates the expression of conditioned freezing. MRs and GRs may mediate different effects of corticosteroids in fear memory, since they differ in their function, neuroanatomical distribution, and affinity for corticosterone (Lupien and McEwen, 1997; Lu et al., 2006). For many years it was thought that stress-induced changes were mediated via the low-affinity GRs rather than the MRs. Recently, however, it has become evident that stress-induced rises in corticosterone can also activate MRs located in the neuronal membrane, which lead to rapid nongenomic effects (de Kloet et al., 2008; Joels et al., 2008). According to this view, activation of MRs appears to be involved in the fast/adaptive behavioral response to environmental aversive cues, while GR-mediated effects promote consolidation of recently acquired information (Lupien and McEwen, 1997; de Kloet et al., 2008; Joels et al., 2008). In this way, a blockade or deficiency in MR activation would make it more difficult for an individual to discriminate relevant cues from irrelevant ones.

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Figure 4 Mineralocorticoid (MRs) and glucocorticoid (GRs) receptors in the basolateral amygdala complex (BLA) and conditioned fear. Timeline of the procedures for looking at BLA MR and GR involvement in the expression of conditioned fear and motor activity (A). Time of freezing in rats that received intra-BLA vehicle (control; n = 10), 5 ng/0.2 ml/site of the MR antagonist spironolactone (Spiro5; n = 10) or 10 ng/0.2 ml/site spironolactone (Spiro10; n = 10) and were subjected to the conditioned fear test (B). Motor activity of rats treated with intra-BLA vehicle (control; n = 7), 5 ng spironolactone (Spiro5; n = 7) or 10 ng spironolactone (Spiro10; n = 7) (C—D). Time of freezing of rats that received intra-BLA vehicle (control; n = 10), 5 ng/0.2 ml/site of the GR antagonist mifepristone (Mifep5; n = 10) or 10 ng/0.2 ml/site mifepristone (Mifep10; n = 10) and were subjected to the conditioned fear test (E). Motor activity of rats treated with intra-BLA vehicle (control; n = 7), 5 ng mifepristone (Mifep5; n = 7) or 10 ng mifepristone (Mifep10; n = 7) (F and G).

Altogether, the results of our first experiment suggest an important role of MRs located in the VTA in modulating the aversive memory retrieval and conditioned freezing expression. Since dopamine is released in the BLA from the VTA during the expression of conditioned fear (de Oliveira et al., 2011, 2013), the anxiolytic-like effect of decreased HPA axis activity in the expression of conditioned freezing could be associated to changes in dopamine levels subsequent to the binding of corticosterone to MRs in the VTA. In our second experiment, the role of MRs in the VTA on dopaminergic neurotransmission in the BLA during the expression of conditioned fear was examined in a combined pharmacological/ neurochemical study using in vivo microdialysis. With the second experiment, we demonstrated that the anxiolytic-like effect of intra-VTA spironolactone administration was associated with changes in dopaminergic efflux in the BLA. Similar to what we observed in previous studies (de Oliveira et al., 2011, 2013), rats that received microinjection of vehicle and were subjected to the conditioned fear test exhibited an anxiety-like profile, reflected by freezing behavior and increased dopamine release in the BLA. This pattern, however, was blocked by treatment with intra-VTA spironolactone that prevented the increase in dopamine release in the BLA during the conditioned fear test and also decreased

the expression of the conditioned freezing response. So, these results confirm the influence of HPA axis mobilization upon the expression of conditioned fear. Immediate corticosterone release in response to a stressful situation appears to play a stimulating role in dopaminergic mechanisms in the BLA via MRs in the VTA, facilitating the expression of adaptive responses to cues that signal threatening conditions. Overall, the present data suggest that interference with the ability of the fear-evoking CS to activate VTA-BLA dopaminergic neurons modulated by the action of corticosteroids on MRs in the VTA is associated with a reduction in the expression of conditioned fear. A relationship between corticosteroids and dopamine has already been demonstrated. For example, intraperitoneal administration of metyrapone inhibited the increase in dopamine levels in the BLA in response to an aversive light-CS (de Oliveira et al., 2013). Also, adrenalectomy inhibited the increase in dopamine in the prefrontal cortex induced by restraint stress (Imperato et al., 1989) and prevented the occurrence of behavioral sensitization to amphetamine (Rivet et al., 1989). The VTA is a major source of dopamine in the brain and it is known that aversive CS elicits an activation of VTA forebrain projections to the amygdala, but also to the nucleus accumbens core and shell subregions

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Figure 5 Target sites for microinjections into the ventral tegmental area (VTA) and for microdialysis in the basolateral amygdala complex (BLA). Photomicrographs showing a representative microinjection site in the VTA (A) and a representative probe site for microdialysis in the BLA (B). Outline of microinjection sites in the VTA (C) and probe locations in the BLA (D) on cross-sections from the Paxinos and Watson atlas (2007). The number of points in the figures is less than the total number of rats used because of several overlaps. Scale bar = 0.5 mm. DMPAG, dorsomedial periaqueductal gray; CLi, caudal linear nucleus of the raphe; Pn, pontine nuclei; CA3, CA3 field of the hippocampus; VPL, ventral posterolateral thalamic nucleus; MeP, medial amygdaloid nuclei.

and to different areas of the medial prefrontal cortex (Yoshioka et al., 1996; Martinez et al., 2008; de Oliveira et al., 2011, 2013). Although our results implicate a VTA-BLA dopaminergic pathway in the expression of conditioned fear, determining whether the above mentioned effect may also be observed in other regions of the mesocorticolimbic system after pharmacological manipulation of VTA MRs during conditioned fear remains to be investigated. Further studies using in vivo microdialysis targeting dopamine in these regions will help to elucidate this point. Altogether, the results obtained with the present study show that intra-VTA injections of the MR antagonist spironolactone inhibited the efflux of dopamine in the BLA in response to the light-CS and also decreased the expression of conditioned freezing response. These findings suggest that

corticosteroids, acting through MRs and together with dopaminergic neurotransmission, play an important role in the expression of conditioned fear elaborated via the VTA-BLA pathway. Once more, because of the existence of multiple mediators with distinct but overlapping temporal and mechanistic attributes, more studies are needed in the attempt to clarify the circuits and mechanisms recruited during the expression of conditioned fear. For example, glutamatergic neurons from a variety of cortical, thalamic, and brainstem structures provide excitatory input to the VTA, which is also under the inhibitory control of GABAergic interneurons (Johnson and North, 1992; Bonci and Malenka, 1999). Also, our findings are consistent with a model in which the amygdala receives emotionally arousing inputs from multiple sources and modulates fear expression elaborated

Ventral tegmental area mineralocorticoid receptors regulate the expression of conditioned fear

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Figure 6 Mineralocorticoid receptors (MRs) in the ventral tegmental area (VTA) and dopamine levels in the basolateral amygdala complex (BLA) during conditioned fear. Timeline of the procedures for assessing the effects of intra-VTA administration of the MR antagonist spironolactone on extracellular dopamine concentration in the BLA during the expression of conditioned fear (A). Extracellular dopamine concentration in the BLA in rats that received intra-VTA microinjection of vehicle (control; n = 10) or 10 ng/0.2 ml of the MR antagonist spironolactone (Spiro10; n = 10) and were subjected to the conditioned fear test (B). Time of conditioned freezing in the same rats during the 20 min test session (C). #p < 0.05, different from baseline; *p < 0.05, different from control.

in other brain regions (Maren and Fanselow, 1996; LeDoux, 2003; Pare et al., 2004). Finally, studying the mediators and neural substrates involved in the expression of conditioned fear responses is of great interest because they are thought to be important for different aspects of human anxiety disorders (Phelps and LeDoux, 2005; Indovina et al., 2011). The present findings may have clinical implications by enabling better understanding of the relationship between hormonal and neurochemical events that are important for fear-related behaviors or, more precisely, by highlighting the interplay between corticosteroids and dopamine in the expression of conditioned fear responses. The use of agents targeting dopaminergic neurotransmission as adjunctive therapy in anxiety disorders is beginning to be explored, at least in the treatment of certain individuals that have not responded adequately to current interventions (Houlihan, 2011; Katzman, 2011). The action of agents that decrease HPA axis activation may also be useful in preventing subsequent stress-induced activation of BLA neurons and associated increases in emotionality. Since dopamine plays an important role in the pathogenesis of several psychiatric disorders, such agents could be therapeutic alternatives not only for anxiety but also for other dopamine related disorders. In summary, the present study shows that corticosteroids, acting through MRs in the VTA, upregulate the dopaminergic system in the BLA, enabling a better evaluation of threats associated with aversive stimuli and the expression of appropriate defense reactions to cope with the situation.

Role of the funding source This research was supported by FAPESP (Proc. no. 11/000413) and CNPq (Proc. no. 471325/2011-2). AR de Oliveira and AE Reimer hold Post-Doctoral fellowships from FAPESP (Proc. no. 10/50669-6 and Proc. no. 13/04741-5, respectively). FAPESP and CNPq funded this study but had no further role in the study design, collection, analysis and interpretation of the data, writing of the report, and decision to submit the paper for publication.

Conflict of interest None declared.

Acknowledgements Research was supported by FAPESP (Proc. no. 11/00041-3) and CNPq (Proc. no. 471325/2011-2). AR de Oliveira and AE Reimer hold Post-Doctoral fellowships from FAPESP (Proc. no. 10/50669-6 and Proc. no. 13/04741-5, respectively).

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Mineralocorticoid receptors in the ventral tegmental area regulate dopamine efflux in the basolateral amygdala during the expression of conditioned fear.

Despite the recognized involvement of corticosteroids in the modulation of emotional behavior, the specific role of mineralocorticoid receptors (MRs) ...
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