Cardiovascular and behavioral effects produced by administration of liposome-entrapped GABA into the rat central nervous system

Neuroscience 285 (2015) 60–69

CARDIOVASCULAR AND BEHAVIORAL EFFECTS PRODUCED BY ADMINISTRATION OF LIPOSOME-ENTRAPPED GABA INTO THE RAT CENTRAL NERVOUS SYSTEM G. C. VAZ, a A. P. C. O. BAHIA, a F. C. DE FIGUEIREDO MU¨LLER-RIBEIRO, a C. H. XAVIER, b K. P. PATEL, c R. A. S. SANTOS, a F. A. MOREIRA, a F. FRE´ZARD a AND M. A. P. FONTES a*

compared with GS and EL groups. These results indicate that liposome-entrapped GABA can be a potential tool for exploring the chronic effects of GABA in specific regions and pathways of the central nervous system. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

a National Institute of Science and Technology in Nanobiopharmaceutics (INCT – Nanobiofar), Department of Physiology & Biophysics, Federal University of Minas Gerais, Belo Horizonte, Brazil b Department of Physiological Sciences, Federal University of Goia´s, Goiania, Brazil

Key words: liposomes, GABA, cardiovascular, behavior.

c

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, United States

INTRODUCTION Abstract—Liposomes are nanosystems that allow a sustained release of entrapped substances. Gammaaminobutyric acid (GABA) is the most prevalent inhibitory neurotransmitter of the central nervous system (CNS). We developed a liposomal formulation of GABA for application in long-term CNS functional studies. Two days after liposome-entrapped GABA was injected intracerebroventricularly (ICV), Wistar rats were submitted to the following evaluations: (1) changes in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) to ICV injection of bicuculline methiodide (BMI) in anesthetized rats; (2) changes in cardiovascular reactivity to air jet stress in conscious rats; and (3) anxiety-like behavior in conscious rats. GABA and saline-containing pegylated liposomes were prepared with a mean diameter of 200 nm. Rats with implanted cannulas targeted to lateral cerebral ventricle (n = 5–8/group) received either GABA solution (GS), empty liposomes (EL) or GABA-containing liposomes (GL). Following (48 h) central microinjection (2 lL, 0.09 M and 99 g/L) of liposomes, animals were submitted to the different protocols. Animals that received GL demonstrated attenuated response of RSNA to BMI microinjection (GS 48 ± 9, EL 43 ± 9, GL 11 ± 8%; P < 0.05), blunted tachycardia in the stress trial (DHR: GS 115 ± 14, EL 117 ± 10, GL 74 ± 9 bpm; P < 0.05) and spent more time in the open arms of elevated plus maze (EL 6 ± 2 vs. GL 18 ± 5%; P = 0.028)

Despite the enormous progress made by neuroscience in the last few decades, the development of appropriate methodology for assessing the chronic actions of different drugs within the brain is still a challenge (Frezard et al., 2011). In this regard the technique of sustained drug delivery using liposomes has emerged as a promising new tool. Liposomes are spherical vesicles consisting of one or several concentric lipid bilayers that isolate one or more internal aqueous compartments from the external environment (Frezard et al., 2005). Liposomes can encapsulate either hydrophilic or lipophilic substances allowing for its sustained release (Batista et al., 2007). GABA (c-aminobutyric acid) is the major inhibitory neurotransmitter in the central nervous system (CNS) (Beleboni et al., 2004). It exerts its inhibitory effects by acting on two distinct classes of receptors: ionotropic GABAA and metabotropic GABAB receptors (Vithlani et al., 2011). It is well known that GABA is implicated in autonomic regulation. Imbalance in central GABAergic transmission has been associated with development of cardiovascular diseases including hypertension (Allen, 2002) and heart failure (Li and Patel, 2003). In rats, site-specific activation of GABAA receptors decreases sympathetic activity and blood pressure (Zhang et al., 1997; Allen, 2002; Dampney et al., 2003) and attenuates the cardiovascular response to emotional stress (StotzPotter et al., 1996; Xavier et al., 2009). Microinjection of GABAA agonist, muscimol, into the lateral ventricle results in marked decreases in mean arterial pressure (MAP), heart rate (HR) and sympathetic activity in spontaneously hypertensive rats (Unger et al., 1984). Conversely, intracerebroventricular (ICV) injections of bicuculline methiodide (BMI), a GABAA, antagonist, into the cerebral ventricles increase blood pressure, heart rate and sympathetic outflow (DiMicco, 1982; Schmidt and DiMicco,

*Corresponding author. Address: Laborato´rio de Hipertensa˜o, Departamento de Fisiologia e Biofı´ sica – ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270 901, Brazil. Tel: +5531-3499-2953; fax: +55-31-3499-2924. E-mail address: [email protected] (M. A. P. Fontes). Abbreviations: ANOVA, analysis of variance; CHOL, cholesterol; CNS, central nervous system; DiI, 1,10 -Dioctadecyl-3,3,30 ,30 Tetramethylindocarbocyanine Perchlorate; DMH, dorsomedial hypothalamic region; DSPC, L-a-distearoyl-phosphatidylcholine; DSPE-PEG2000, distearoyl-phosphatidylethanolamine-polyethylene glycol 2000; EPM, elevated plus maze; FATMLVs, frozen and thawed multilamellar vesicles; GABA, gamma-aminobutyric acid; HR, heart rate; ICV, intracerebroventricular; MAP, mean arterial pressure; PVN, paraventricular nucleus; RSNA, renal sympathetic nerve activity. http://dx.doi.org/10.1016/j.neuroscience.2014.10.067 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 60

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1984; Karson et al., 1999). Remarkably, acute disruption of GABAergic inhibition in brain sites involved with control of emotional stress leads to panic-like behavior and increased sympathetic and cardiorespiratory responses (Shekhar et al., 2003; Fontes et al., 2011). The development of a technique to chronically deliver GABA in the CNS would be helpful to understand autonomic cardiovascular control as well as other brain functions under normal conditions and disease conditions such as hypertension and heart failure. In this study, we developed a liposomal formulation of GABA for application in long-term CNS functional studies. In order to evaluate its efficiency, we tested the effects produced by ICV microinjection of GABA-containing liposomes (GL) on; (1) the cardiovascular response produced by ICV injection of the GABAA antagonist, BMI, (2) the cardiovascular responses to acute emotional stress, and (3) anxiety modulation. Part of these results has been previously transmitted in an abstract form (Vaz et al., 2011, 2012).

EXPERIMENTAL PROCEDURES Ingredients: drug and lipids GABA and BMI were obtained from Sigma - Aldrich (Brazil, SP). L-a-distearoyl-phosphatidylcholine (DSPC), cholesterol (CHOL) and distearoyl-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were obtained from Lipoid (Ludwigshafen, Germany). 1,10 -Dioctadecyl-3,3,30 ,30 -Tetramethylindocarbocyanine Perchlorate (DiI) was obtained from Invitrogen, Ltd (Carlsbad, California, USA).

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calibration curve in the presence of empty liposomes (EL), but known amount of GABA, was used. In vitro release of liposome-encapsulated GABA The ability of liposomes to retain encapsulated GABA was evaluated after incubation for different time intervals at 37 °C (0, 5, 10, 15 days). Aliquots were removed and submitted to ultrafiltration through Microcon YM-50 (50.000 NMWL, Millipore Corp, Brazil, SP). The amount of GABA recovered in the filtrate was analyzed by fluorimetric method as described above. Animals Male Wistar rats (280 ± 20 g) from the animal facility of the Biological Sciences Institute (CEBIO, UFMG, Belo Horizonte, MG, Brazil) were used. The animals had free access to water and food before and after surgical procedures and were kept in a room with controlled light and temperature. All procedures conform to the regulation set by the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996 and were approved by our local committee for ethics in animal experimentation (CEUA UFMG protocol number: 150-2010). Surgical procedures

Liposomal formulations were made from DSPC, CHOL and DSPE-PEG2000 at 5:4:0.3 M ratio. The encapsulation of GABA (0.3 M in 0.9% NaCl) or saline (0.9% NaCl) was first carried out within frozen and thawed multilamellar vesicles (FATMLVs), as described previously (Mayer et al., 1985). FATMLVs (lipid concentration of 99 g/L) were then repeatedly extruded at 65 °C through two stacked polycarbonate membranes of 200-nm pore size (Nayar et al., 1989) and finally submitted to dialysis against saline (0.9% NaCl) to remove non-encapsulated GABA. The mean hydrodynamic diameter and polydispersity index of the vesicles were determined by photon correlation spectroscopy (Malvern Zetasizer Nano ZS90; Malvern Instruments, United Kingdom).

All animals in the anesthetized and conscious experimental protocols were submitted to stereotaxic surgical procedure. Under tribromoethanol (Sigma, USA) anesthesia (250 mg/kg i.p.), rats were placed in a stereotaxic frame (Stoelting, USA) and instrumented with unilateral stainless steel guide cannula (22 gauge, 12 mm length) targeted to the lateral ventricle (anteroposterior 1.0, latero-lateral +1.5, dorso-ventral 4.0; (Paxinos and Watson, 1986). The guide cannula was fixed to the skull by dental acrylic cement anchored with stainless steel screws. After surgical procedures, animals were allowed to recover in their home cages for at least 3 days. After this recovery period, rats were separated in groups (n = 5–8 each) and received ICV injection of a 2 lL volume of the following preparations: GABA solution (GS, 20 mM); empty liposomes (EL, 99 g/L lipid) or liposome-entrapped GABA (GL, 0.09 M GABA and 99 g/L lipid). Rats were submitted to ICV injection of only one compound. After a period of 48 h, groups were submitted to one of the protocols described below.

Incorporation efficiency of GABA into liposomes

Effect of the ICV injection of GABAA antagonist, BMI

GABA was determined exploiting its specific interaction with fluorescamine as previously described (Udenfriend et al., 1972) and the following modifications. Briefly, 5 lL of liposome-encapsulated GABA was added in a 5 mL of the assay solution of methanol (0.5 mL), water (1.5 mL), borate buffer (20 mM, pH 8.2, 2.5 mL) and fluorescamine (0.3 mg/mL diluted in acetone). The amount of encapsulated GABA was determined using the fluorescence of GABA-fluorescamine complex at maximum excitation of 388 nm and maximum emission of 486 nm. A

Three groups of rats (GS, EL, GL) were anesthetized with urethane (Sigma, USA) (1.2–1.4 g/kg i.p.), and the trachea was cannulated to maintain the airways open. The adequacy of anesthesia was verified by the absence of a withdrawal response to nociceptive stimulation of a hindpaw. Supplemental doses of urethane (0.1 g/kg i.v.) were given as necessary. Body temperature was maintained in the range of 36.5–37.0 °C with a heating pad. Using a retroperitoneal approach, the left renal nerve was isolated and covered with mineral oil, and put

Preparation of liposomal formulations

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in contact with a silver bipolar electrode. Renal sympathetic nerve activity (RSNA) signal was amplified by 10 K, filtered (100–1000 Hz), displayed on an oscilloscope and monitored by means of an audio amplifier. The filtered nerve activity signal was rectified; integrated (resetting every 2s), displayed online and acquired using Powerlab 4/20 – Chart 7.2 (ADInstruments, Sydney, Australia). All data were digitized at 1 kHz. The noise level of the RSNA recording system was determined postmortem and subtracted from initial RSNA values. After the stabilization, cardiovascular parameters were recorded for 30 min. Following this initial control period each of the rats received an ICV microinjection of BMI (0.25 mM, 2 lL) through the same ICV guide cannula. Acute stress paradigm Three groups of rats (GS, EL, GL) were anesthetized with tribromoethanol (250 mg/kg i.p.). A polyethylene catheter (0.011 ID, Clay Adams, Parsippany, NJ, USA) filled with heparinized saline was inserted into the abdominal aorta through the femoral artery for recording of MAP and HR. The catheter was routed s.c. to the nape of the neck where it was exteriorized and secured. Rats were then allowed to recover in their home cages. All animals remained in good health conditions throughout the course of surgical procedures and experimental protocols as assessed by appearance, behavior, and maintenance of body weight. On the day of experiment, after 30 min of heart rate and blood pressure monitoring, rats were then placed into a plastic restrainer (60-mm inner diameter) and subjected to a 10-min air jet stress – a stream of air (10 L/min) directed to the head. After the air jet stress the animals were left into the plastic restrainer and MAP and HR were recorded for additional 20 min. In another two separate groups of rats, the cardiovascular reactivity to air stress was also evaluated 120 h after ICV injection of EL (n = 8) and GL (n = 7). Elevated plus maze (EPM) Two groups of rats (EL, GL) were submitted to EPM. The wood EPM used to perform the experiments was located in a sound attenuated and temperature controlled room (23 °C), with one incandescent light (40 W) placed 1.3 m away from the maze. The apparatus consists of two opposing open arms (50  10 cm) without side walls, perpendicular to two enclosed arms (50  10  40 cm), with a central platform common to all arms (10  10 cm). The apparatus is elevated 50 cm above the ground and has an acrylic edge (1 cm) in the open arms to prevent the fall of the animal. In this model, rodents naturally avoid the open arms, exploring more extensively the enclosed arms. Anxiolytic drugs increase the exploration in open arms without affecting the number of enclosed arms entries, which is usually used to assess general exploratory activity (File, 1992). The rat was placed on the central platform of the maze with the head facing one of the enclosed arms. The test lasted for 5 min and was recorded. The animal behavior was analyzed with

the help of the Anymaze Software (version 4.5, Stoelting). This software indicates the location of the animal in the EPM and automatically calculates the percentage of entries (Peo) and time spent in the open arms (Pto) and the number of entries in the enclosed arms (EA). Animals were only considered to enter an open or enclosed arm when 90% of their bodies were inside the region. All experiments were performed in the morning period (8 a.m.–12 p.m.) (Moreira et al., 2007). Histological analysis and localization of liposomes To determine the localization of liposomes in the brain, 3 rats received ICV injection of liposomes labeled with the nonexchangeable fluorescent probe, DiI. DiI-labeled liposomes were prepared essentially according to the method of (Claassen, 1992). Briefly, DiI dissolved in ethanol at 2 mg/mL was added to the empty liposome suspension at a final concentration of 10 g/mL and incubated for 60 min at 60 °C. The preparation of labeled liposomes was then dialyzed against saline (0.9% NaCl) for 24 h at 25 °C to eliminate residual ethanol. After 48 h of the DiI-labeled liposome injection into the lateral ventricle, rats were deeply anesthetized with pentobarbital sodium (80 mg/kg ip). The animals were subjected to transcardial perfusion with 60 mL ice-cold saline followed by 60 mL 4% buffered paraformaldehyde in 0.1 M phosphate-buffered saline. The brain was then removed and coronal sections (60 lm; ranging from +1.68 to 13.2 mm from bregma) were cut on a cryostat and mounted on slides for labeling identification on a fluorescent microscope (Zeiss). Sections were then counterstained with Neutral Red for histological confirmation of the surrounding structures that were identified according to the atlas of Paxinos and Watson (1986). Statistical analysis The kinetics of GABA release by liposomes (data shown in Fig. 1) was analyzed by a one-way ANOVA (analysis of variance) with Bonferroni post hoc test. In all other protocols, (bicuculline, acute stress or elevated plus maze) comparison between groups was analyzed by a one-way ANOVA followed by Newman–Keuls post hoc

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Days Fig. 1. Time course of GABA release from liposomes in 0.9% NaCl at 37 °C. Data are shown as mean ± SEM (n = 5–8). ⁄P < 0.05 compared with day 0, #P < 0.05 compared with day 1 (one-way ANOVA and Bonferroni).

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test or unpaired t test when only two groups were compared. Control values of MAP, HR or RSNA were measured as the average values of these variables for the 2 min period immediately preceding injection of BMI or air jet stress. For calculation of maximal changes in HR, MAP or RSNA of the ICV BMI group and the air jet stress group, the maximal change occurring within 10 min after injection of BMI or onset of air jet stress was used. Level of significance was set as P < 0.05. All values are presented as means ± SEM.

RESULTS Characterization of the preparation of liposome-encapsulated GABA The encapsulation of GABA was achieved with an efficiency of 30% and a ratio of encapsulated GABA/ lipid of 0.093 (w/w). The resulting liposomal formulations showed mean hydrodynamic vesicle diameters of 207 nm for liposomal GABA (GL) and 202 nm for EL, with polydispersity indexes below 0.2 evidencing monodisperse populations. The time required to achieve 50% of GABA release was estimated in the range of 4–5 days, indicating also the marked sustained release property of the liposome formulation (Fig. 1). Effect of the ICV injection of GABAA antagonist, BMI The general strategy for these experiments was to evaluate the effects of ICV administration of GL (48 h earlier) on the sympathetic and cardiovascular responses evoked by acute ICV injection of the GABAA antagonist BMI. In the GS and EL groups, ICV injection of BMI increased RSNA (Fig. 2A). However, the increase in RSNA evoked by BMI was abolished in rats previously injected with GL (maximum changes = GS 48 ± 9%, EL 43 ± 9%, GL 11 ± 8%; P = 0.009 for GL vs. GS, and P = 0.020 for GL vs. EL) (Fig 2B). In all three groups, ICV injection of BMI resulted in similar increases in heart rate (maximum changes = GS 45 ± 12, EL 50 ± 15, GL 27 ± 10 bpm, respectively). No changes in blood pressure were observed (Fig. 2). Conscious rats Acute stress paradigm. Air jet stress evoked immediate increases in MAP and HR in rats that received ICV injection of GS, EL or GL 48 h earlier. The effect was sustained for the entire period of stress and during the recovery period as well (Fig. 3A). The magnitude of the change in MAP evoked by air stress was not different among groups (maximum changes =GS 12 ± 3 vs. EL 17 ± 4 vs. GL 12 ± 5 mmHg, P > 0.05 for comparison between groups). However, the tachycardia induced by air stress was attenuated in rats that received ICV injection of GL (maximum changes = GS 115 ± 14 bpm, EL 117 ± 10 bpm, GL 74 ± 9 bpm; P = 0.036 for GL vs. GS; and P = 0.010 for GL vs. EL) (Fig. 3B). In two separate groups, the cardiovascular reactivity to air stress was additionally evaluated in rats that received ICV injections of GL or EL 120 h earlier

(Fig. 4). In this case, however, the pressor and the tachycardic responses evoked by air jet stress were greatly attenuated in the GL group when compared to the EL group (maximum changes EL 18 ± vs. GL 10 ± mmHg P = 0.014; EL 102 ± vs. GL 39 ± bpm, P = 0.003). It is interesting to note that, after air jet stress, the cardiovascular parameters in the GL group showed a faster recovery to the pre-stress values when compared to the EL group (Fig. 4). EPM. Animals that received GL 48 h earlier spent a higher percentage of time in the open arms (Fig. 5A) when compared to EL animals (EL 6 ± 2 vs. GL 18 ± 5% of time spent, P = 0.028). No significant differences were observed between groups for the number of entries into the open (Fig. 5B) or enclosed arms (Fig. 4C), indicating that the drug effect is not secondary to changes in motor activity. Localization of liposomes The analysis of liposome localization by fluorescent microscopy showed that, 48 h after ICV injection, DiI-labeled liposomes remained mostly at the site of injection, within the ventricular system and did not migrate to areas other than the ventricles. In the rostrocaudal axis, liposomes were found 1.68 mm rostral to 2.52 mm caudal to the bregma (Fig. 6A) in the lateral ventricle and the third ventricle. In the lateral ventricle, the labeling was always observed ipsilateral to the injection site in all sections analyzed (right side). Interestingly, in some sections, liposomes were distributed along the entire contour of the ventricle (Fig. 6B, bottom panel).

DISCUSSION The major findings of this study are that ICV administration of liposome-entrapped GABA (1) blocked the increases in renal sympathetic activity evoked by ICV microinjection of BMI, (2) reduced the tachycardia evoked by acute stress, and (3) reduced anxiety, in rats. These effects were observed at least 48 h after ICV administration and are consistent with the sustained release properties of the liposomal formulation. These functional findings are supported by anatomical experiments with the nonexchangeable lipophilic fluorescent marker, showing that liposomes can remain in the ventricular system for a considerable period of time. Utility of liposome-encapsulated GABA was evaluated previously by (Loeb et al., 1982, 1986) after intravenous administration in epileptic rats. Authors reported a reduction in the epileptic activity which lasted for 104 min and hypothesized that the liposomal carrier may enhance the penetration of GABA across the blood–brain barrier. It is important to note that Loeb et al. used only natural phosphatidylserine for the preparation of liposomes (Loeb et al., 1982), which is expected to form less stable and more leaky vesicles than those used in the current study (Frezard et al., 2011) and to favor the capture of liposomes by macrophages (Tempone et al., 2004). Indeed, the size of liposomes and the kinetic of GABA release

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Time (minutes) Fig. 2. Time courses of changes (A) and maximum changes (B) in renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) evoked by ICV microinjection of BMI in three different groups of rats. Groups were subjected to previous ICV injection (48 h earlier) of different compounds: white bars, GABA solution (GS), gray bars empty liposomes (EL) and black bars GABA liposomes (GL). ⁄P = 0.009 for GL vs. GS, and #P = 0.020 for GL vs. EL (one-way ANOVA and Newman–Keuls).

were not determined in this previous work. In the present study, the liposome membrane was made from highphase transition temperature phosphatidylcholine (DSPC) and cholesterol, to achieve a very low membrane permeability (Silva-Barcellos et al., 2001). Accordingly, in vitro results indicate a very slow release of GABA from liposomes, with only 60% of GABA release after 5 days of incubation at 37 °C. As estimated, the maximum concentration of GABA released from liposomes into the lateral ventricle on a five-day period is approximately 73 lM. Other important characteristics of these liposomes are their mean diameter of 200 nm and the incorporation of a pegylated lipid, to slow down their capture by cells, by endocytosis/phagocytosis and the cell-mediated release of encapsulated drug (Frezard et al., 2011). Acute ICV injection of the selective GABAA antagonist bicuculline (Chebib and Johnston, 1999), produced marked increases in RSNA that were accompanied by tachycardia, as previously reported (Karson et al., 1999).

The increases in RSNA were markedly attenuated in the liposomal GABA group. The exact mechanism of action for both bicuculline and GABA (released from liposomes) after ICV injection remains to be determined. However, it is known that the sympathetic output to the kidney and heart is strongly modulated by GABAergic projections acting on sympathetic premotor neurons via GABAA receptors (Dampney et al., 2000; Allen, 2002). These inhibitory projections feature the sympathetic control in the paraventricular nucleus (PVN) (Martin et al., 1991; Allen, 2002) and dorsomedial hypothalamic region (DMH) (Fontes et al., 2001; Cao et al., 2004), brain regions that surround the third ventricle. Blockade of GABAA receptors in these hypothalamic nuclei results in marked increases in sympathetic activity to the heart and kidney (Zhang et al., 1997; Fontes et al., 2001; Chen and Toney, 2003; Cao et al., 2004) that are accompanied by changes in heart rate and blood pressure. Another possibility is the rostralventrolateral medulla, which is easily accessible from the 4th brain

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Time (min) Fig. 3. Time courses of changes (A) and maximum changes (B) in mean arterial pressure (MAP) and heart rate (HR) evoked by air jet stress in three groups of rats. Groups were subjected to previous ICV injection (48 h earlier) of different compounds: white bars are GABA solution (GS), gray bars are empty liposomes (EL, n = 5) and black bars are GABA liposome (GL, n = 5). ⁄P = 0.036 for GL vs. GS; and #P = 0.010 for GL vs. EL (one-way ANOVA and Newman–Keuls).

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Fig. 4. Time courses of changes (A) and maximum changes (B) in mean arterial pressure (MAP) and heart rate (HR) evoked by air jet stress in two groups of rats. Groups were subjected to previous ICV injection (120 h earlier) of different compounds: gray bars are empty liposomes (EL, n = 8) and black bars are GABA liposome (GL, n = 7). DMAP ⁄P = 0.014 and DHR ⁄P = 0.003, respectively (Student’s t test).

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Fig. 5. Percentage of time spent (A), numbers of entries in open arms (B) and numbers of entries of closed arms (C) in the elevated plus maze. Gray bars are empty liposomes (EL, n = 6) and black bars are GABA liposome (GL, n = 5). ⁄P = 0.028 (Student’s t test).

ventricle (Guertzenstein, 1973), is a key region for maintenance of sympathetic tone and it is strongly modulated by GABAergic projections (Dampney et al., 2000). These brain regions are possible candidates for the effect evoked by bicuculline injected ICV but actions in other brain regions involved in cardiovascular control cannot be excluded. Therefore, it is reasonable to conclude that GABAA receptors in brain regions where bicuculline exerted its effects were occupied by a sustained GABA release from liposomes. This conclusion is supported by data showing that none of the effects evoked by bicuculline were altered in the group injected ICV with GABA solution 48 h earlier. We found that rats that received liposomal GABA ICV presented attenuated cardiovascular response when submitted to air jet stress. Acute emotional stress is characterized by activation of the sympathetic nervous system (Pacak and Palkovits, 2001). The tachycardic and pressor responses evoked by emotional stress depends on integration of different brain regions (Fontes et al., 2011) including periventricular structures such as the PVN (Dampney, 1994) and DMH (Xavier et al., 2009; Fontes et al., 2011), the mesencephalic periaqueductal gray (Bandler et al., 2000) and the nucleus of solitary tract (Sevoz-Couche et al., 2003, 2013). Neuronal activation of DMH, PVN or the lateral/dorsolateral region of periaqueductal gray results in an integrated pattern of

autonomic and behavioral responses that resembles the ‘‘defense reaction’’ and this includes a marked sympathetic mediated tachycardia. Site-specific injections of the GABAA agonist, muscimol, into the DMH and PAG attenuates the tachycardia provoked by air jet stress (Lisa et al., 1989; de Menezes et al., 2008; Xavier et al., 2009). Therefore, these regions could be targets for action of GABA released from liposomes. It could be argued as to why the tachycardia evoked by bicuculline injected ICV was not attenuated in the liposomal GABA group. The current data do not allow for an explanation of this finding, however, it is important to mention that the tachycardia evoked by air stress involves pathways, nuclei and mechanisms different from those activated by bicuculline injected ICV. Even with very low doses, bicuculline evokes marked changes in heart rate when injected in specific brain regions containing groups of neurons involved in sympathetic control (Martin et al., 1991; Zhang et al., 1997; Fontes et al., 2001; Cao and Morrison, 2003). In this regard, it is also possible that the concentration of GABA released from liposomes was not enough to attenuate the effects of bicuculline on neurons controlling heart rate. Rats that received liposomal GABA presented less anxious behavior when submitted to EPM. The EPM has been used as a tool to screen anxio-selective effects of various drugs (Carobrez and Bertoglio, 2005). The test is based on a tendency exhibited by rats to avoid open spaces; typically the closed arms are more explored than the open arms. Such tendency can be altered by anxiolytic and anxiogenic drugs, respectively, increasing or decreasing open arms exploration (Pellow et al., 1985). Several studies implicate the use of benzodiazepines and the anxiolytic behavior. Benzodiazepines act modulating actions of GABA through their binding to GABAA receptor in the brain. A marked reduction in anxiety levels is observed when GABAA receptors are stimulated by benzodiazepines (Argyropoulos and Nutt, 1999). As explained above, GABA released from liposomes could potentially activate GABAA receptors in specific regions involved in anxiety behavior. In this regard, several brain regions could be targets, including amygdala and medial prefrontal cortex. In addition, the DMH and the periaqueductal gray must be highlighted as potential candidate targets because, aside from their involvement in cardiovascular control, they are also involved in anxiety and panic syndrome (Bandler and Shipley, 1994; Johnson et al., 2008) and also represent easy targets for GABA released from liposomes administered ICV. Using different experimental approaches, our current data show that liposome-entrapped GABA was able to attenuate cardiovascular changes and anxiety like behavior 2–5 days after ICV microinjection. Further studies are necessary to elucidate the exact mechanisms and sites of action for GABA released from ICV liposomes. Experiments evaluating the long term physiological effects produced by injection of liposomeentrapped GABA in other brain regions are obviously lacking. We conclude that liposome-entrapped GABA is a promising new tool to be used in neuroscience and all fields of physiology.

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MPOM

-1.08 mm

3V

500 ì m

ec

LV LV

D3v

LV

D3V

500 ì m DMD VMH ArcD

-2.52 mm

Fig. 6. Schematic drawings of the coronal sections of the rat brain (A) and photomicrograph showing the localization of fluorescent DiI-labeled liposomes in the ventricular system (B). The rectangle in A corresponds to the area shown in the photomicrograph in (B). AcbC, accumbens nucleus; ArcD, arcuate hypothalamic nucleus, dorsal part; CPu, caudate putamen; DMD, dorsomedial hypothalamic nucleus, dorsal part; D3v, dorsal third ventricle; ec, external capsule; gcc, genus of the corpus callosum; HDB, nucleus of the horizontal limb of the diagonal band; LV, lateral ventricle; MPOM, medial preoptic nucleus, medial part; MS, medial septal nucleus; SHi, septo hippocampal nucleus; VMH, ventromedial hypothalamic nucleus; 3V, third ventricle; (Paxinos and Watson, 1986).

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Acknowledgements—We thank Fundac¸a˜o de Amaparo a` Pesquisa de Minas Gerais (FAPEMIG GRANTS CBB – APQ-01097-11 APQ-00353-13), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, CNPq (PQ PQ 306000/2013-0) and CAPES.

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(Accepted 29 October 2014) (Available online 13 November 2014)