involvement of dorsal hippocampus glutamatergic and nitrergic neurotransmission in autonomic...

Neuroscience 258 (2014) 364–373

INVOLVEMENT OF DORSAL HIPPOCAMPUS GLUTAMATERGIC ANDNITRERGIC NEUROTRANSMISSION IN AUTONOMIC RESPONSESEVOKED BY ACUTE RESTRAINT STRESS IN RATS

T. B. MORAES-NETO, a,b A. A. SCOPINHO, a

C. BIOJONE, a,b F. M. A. CORREA a ANDL. B. M. RESSTEL a,b*

aDepartment of Pharmacology, School of Medicine of Ribeirao

Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil

bCenter for Interdisciplinary Research on Applied Neurosciences

(NAPNA), University of Sao Paulo, Brazil

Abstract—The dorsal hippocampus (DH) is a structure of the

limbic system that is involved in emotional, learning and

memory processes. There is evidence indicating that the

DH modulates cardiovascular correlates of behavioral

responses to stressful stimuli. Acute restraint stress (RS)

is an unavoidable stress situation that evokes marked and

sustained autonomic changes, which are characterized by

elevated blood pressure (BP), intense heart rate (HR)

increase and a decrease in cutaneous temperature. In the

present study, we investigated the involvement of an

N-methyl-D-aspartate (NMDA) glutamate receptor/nitric oxide

(NO) pathway of the DH in the modulation of autonomic

(arterial BP, HR and tail skin temperature) responses evoked

by RS in rats. Bilateral microinjection of the NMDA receptor

antagonist AP-7 (10 nmol/500 nL) into the DH attenuated

RS-evoked autonomic responses. Moreover, RS evoked an

increase in the content of NO2/NO3 in the DH, which are

products of the spontaneous oxidation of NO under physio-

logical conditions that can provide an indirect measurement

of NO production. Bilateral microinjection of N-propyl-L-argi-

nine (0.1 nmol/500 nL; N-propyl, a neuronal NO synthase

(nNOS) inhibitor) or carboxy-PTIO (2 nmol/500 nL; c-PTIO,

an NO scavenger) into the DH also attenuated autonomic

responses evoked by RS. Therefore, our findings suggest

that a glutamatergic system present in the DH is involved

in the autonomic modulation during RS, acting via NMDA

receptors and nNOS activation. Furthermore, the present

0306-4522/13 $36.00 � 2013 IBRO. Published by Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.neuroscience.2013.11.022

*Correspondence to: L. B. M. Resstel, Department of Pharmacology,School of Medicine of Ribeirao Preto, USP, Bandeirantes Avenue,3900, Ribeirao Preto, Sao Paulo 14049-900, Brazil. Tel: +55-16-3602-0441; fax: +55-16-3633-2301.

E-mail address: [email protected] (L. B. M. Resstel).Abbreviations: ACSF, artificial cerebrospinal fluid; ANOVA, analysis ofvariance; AP-7, 2-amino-7-phosphonoheptanoic acid; BNST, bednucleus of stria terminalis; BP, blood pressure; BSA, bovine serumalbumin; c-PTIO, carboxy-PTIO(S)-3-carboxy-4-hydroxyphenylglicine;DH, dorsal hippocampus; HR, heart rate; L-glu, glutamate; LSA, lateralseptal area; MAP, mean arterial pressure; MPFC, medial prefrontalcortex; NADPH, reduced form of nicotinamide adenine dinucleotidephosphate; NMDA, N-methyl-D-aspartate; nNOS, neuronal nitric oxidesynthase; NO, nitric oxide; N-propyl, Nx-propyl-L-arginine; RS, restraintstress; SEM, standard error of mean.

364

results suggest that NMDA receptor/nNO activation has a

facilitatory influence on RS-evoked autonomic responses.

� 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: hippocampus, restraint stress, glutamate, nitric

oxide.

INTRODUCTION

The hippocampus is part of the limbic system and is

directly involved in the modulation of emotional, learning

and memory processes (Maclean, 1952; Siegel and

Flynn, 1968; Papez, 1995). Anatomically, the

hippocampus includes CA1, CA2, CA3, and dentate

gyrus sub regions with the pattern of efferent and

afferent connectivity changing between dorsal and

ventral hippocampus (Brodal, 1998). Petrovich and co-

workers have divided the hippocampus in terms of

afferent connectivity into five parallel zones with Zone 1

encompassing the dorsal half of CA1 and Zones 2–5

the ventral CA1/subiculum (Petrovich et al., 2001).

In many mammals, electrical stimulation of the

hippocampus induces behavioral changes and

defensive reactions, such as attack responses (Siegel

and Flynn, 1968), attention (Kaada et al., 1953),

agitation, and growl escape reactions (Maclean and

Delgado, 1953), as well as lightheadedness and

confusion (Maclean, 1957). It has been reported that

autonomic responses caused by electrical stimulation

of the dorsal hippocampus (DH), which are

characterized by respiratory inhibition (Kaada and

Jasper, 1952; Andy and Akert, 1955; Liberson and

Akert, 1955; Anand and Dua, 1956), either increased

blood pressure (BP) or decreased heart rate (HR)

(Smith, 1944; Anand and Dua, 1956) and increased

sympathetic activity (Carlson et al., 1941; Andy and

Akert, 1953). In addition, DH chemical stimulation

using glutamate caused a reduction in BP and HR that

was similar to those evoked by its electrical stimulation

in rats (Ruit and Neafsey, 1988), suggesting that these

cardiovascular responses are due to activation of

hippocampal neurons and not to stimulation of fibers of

passage. Thus, these studies indicate an involvement

of the hippocampus, especially its dorsal portion, in the

modulation of autonomic activity, and in particular in

effects on the cardiovascular system.

Restraint stress (RS) is a widely used experimental

model of acute stress, being an inescapable stressor

d.

http://dx.doi.org/10.1016/j.neuroscience.2013.11.022

mailto:[email protected]

http://dx.doi.org/10.1016/j.neuroscience.2013.11.022

T. B. Moraes-Neto et al. / Neuroscience 258 (2014) 364–373 365

stimulus where animals are placed into a tube of plastic or

metal, which restricts their movements (Conti et al., 2001;

Yoshino et al., 2005), resulting in several autonomic

responses. These autonomic responses include: mean

arterial pressure (MAP) and HR increases (Taylor et al.,

1989; Kubo et al., 2002; Tavares et al., 2007; Busnardo

et al., 2010), skeletal muscle vasodilatation and

cutaneous vasoconstriction, which are accompanied by

a rapid skin temperature drop and are followed by body

temperature increases (Blessing and Seaman, 2003;

Vianna and Carrive, 2005).

The DH has connections to several brain structures

that are involved in the modulation of responses

associated with RS, such as the medial prefrontal cortex

(MPFC), the bed nucleus of stria terminalis (BNST), the

lateral septal area (LSA) and the medial amygdala

(MeA) (Kubo et al., 2002; Tavares and Correa, 2006;

Fortaleza et al., 2009; Crestani et al., 2010). Previous

work has shown that during RS, neurons are activated

in the DH (Chen et al., 2006), suggesting its

involvement in the modulation of these RS-evoked

responses. Furthermore, results from our laboratory (un-

published) indicate that inhibition of neurotransmission

in the DH reduces the magnitude of RS-evoked

autonomic responses, such as increased BP and HR,

as well as the decreased skin temperature caused by

RS, suggesting that the DH plays an important role in

the modulation of autonomic responses associated with

RS. However, which neurotransmitters in the DH are

involved in the modulation of RS-evoked autonomic

responses has not yet been identified.

Glutamate (L-glu) is an important central nervous

system (CNS) neurotransmitter (Fleck et al., 1993;

Khodorov, 2004), which is involved in the modulation of

the autonomic system. It has been demonstrated that

administration of L-glu into the DH of Wistar-Kyoto

(WKY) and spontaneously hypertensive rats (SHR)

caused decrease in BP, which was blocked by prior

administration of AP-5 (N-methyl-D-aspartate (NMDA)

receptors antagonist). Furthermore, administration of

CNQX (non-NMDA receptors antagonist), did not

change the depressor response caused by L-glu (Wang

and Ingenito, 1994) suggesting that the glutamatergic

system into the DH through the NMDA receptors

modulates cardiovascular responses. In addition,

Moghaddam (1993) demonstrated that during RS there

is an increased release of L-glu in the DH of rats. These

results support a possible involvement of the DH

glutamatergic system in the modulation of responses

associated with RS.

Nitric oxide (NO), a free radical gas, is synthesized

from L-arginine by different enzymes (Garthwaite et al.,

1988, 1989). In the CNS the major isoform of these

enzymes is the neuronal form (neuronal nitric oxide

synthase (nNOS)), which is present in hippocampal

neurons (Blackshaw et al., 2003), and whose activity is

regulated by Ca2+ influx induced by the activation of

glutamate receptors, mainly of NMDA receptors

(Garthwaite et al., 1989). Joca and Guimaraes (2006)

demonstrated that inhibition of nNOS in the DH of rats

caused an anxiolytic effect, which was characterized by

a reduction in the immobility observed in the Forced

Swim Test model. In the hippocampus, the activation of

NMDA receptors caused increased formation of NO

(Garthwaite et al., 1989), suggesting an interaction

between NMDA receptors and NO formation in this

structure.

Considering the above evidence, an involvement of

both the glutamatergic and nitrergic systems of the DH

in the modulation of RS-evoked cardiovascular

responses can be proposed. In the present study, we

used the RS model to test the hypothesis that the

glutamatergic/nitrergic systems in the DH modulate

stress-evoked autonomic responses.

EXPERIMENTAL PROCEDURES

Animals

Male Wistar rats weighing 230–250 g were used (total

n= 57). The animals were kept in the animal care unit

of the Department of Pharmacology, School of Medicine

of Ribeirao Preto, University of Sao Paulo. The rats

were housed individually in plastic cages with free

access to food and water under a 12-h light/dark cycle

(lights on at 06:30 h). Experimental procedures were

carried out following protocols approved by the Ethics

Review Committee of the School of Medicine of

Ribeirao Preto (protocol n. 063/2010), which complies

with the guidelines laid down by the National Institutes

of Health (NIH, Guide for the Care and Use of

Laboratory Animals).

Surgery procedure

Five days before the experiment, the rats were

anesthetized with 2,2,2-tribromoethanol (Sigma, St.

Louis, MO, USA) (250 g/kg, i.p.). After scalp anesthesia

with 2% lidocaine, the skull was surgically exposed and

stainless steel guide cannulae (0.55 mm) were

implanted bilaterally into the DH using a stereotaxic

apparatus (Stoelting, Wood Dale, IL, USA). Stereotaxic

coordinates for cannulae implantation in the DH were

chosen based on the rat brain atlas of Paxinos and

Watson (1997): AP: �4 mm from bregma, L: +2.6 mm

from the medial suture, V: �2.1 mm from the skull. The

incisor bar position was set at �2.5 mm. Cannulae were

fixed to the skull with dental cement and one metal screw.

Twenty-four hours before the RS session, a

polyethylene catheter was implanted into the left femoral

artery for BP recording. The arterial catheter consisted

of a piece of PE-10 tubing (4.0 cm) heat-bonded to a

longer segment of PE-50 tubing (10–12 cm). The

catheter was filled with 0.3% heparin (5000 UI/ml) in

sterile saline (0.9% NaCl). The PE-10 piece was

introduced into the femoral artery until the tip reached

the aorta. The catheter was secured in position with

thread, and the PE-50 part was passed under the skin

to be extruded at the dorsum of the animals. The

catheter was extruded at the dorsum and attached to

the skin. After each surgery, animals were treated with

a poly-antibiotic combination of streptomycins and

penicillins i.m. (Pentabiotico, Fort Dodge, Brazil) to

366 T. B. Moraes-Neto et al. / Neuroscience 258 (2014) 364–373

prevent infection and with the non-steroidal anti-

inflammatory flunixine meglumine (2.5 mg/kg s.c.;

Banamine, Schering Plough, Brazil) for post-operative

analgesia.

Drugs

2-Amino-7-phosphonoheptanoic acid (DL-AP-7, Tocris,

Westwoods Business Park, Ellisville, MO, USA); Nx-

propyl-L-arginine (N-propyl, Tocris, Westwoods Business

Park, Ellisville, MO, USA); carboxy-PTIO(S)-3-carboxy-

4-hydroxyphenylglicine (c-PTIO, Tocris, Westwoods

Business Park, Ellisville, MO, USA) were dissolved in

sterile vehicle artificial cerebrospinal fluid (ACSF –

composition: NaCl 100 mM; Na3PO4 2 mM; KCl 2.5 mM;

MgCl2 1 mM; NaHCO3 27 mM; CaCl2 2.5 mM; Sigma,

St. Louis, MO, USA; pH = 7.4; The mixture of

compounds was prepared in our laboratory).

Tribromoethanol (Sigma, St. Louis, MO, USA), and

urethane (Sigma, St. Louis, MO, USA) were dissolved in

sterile vehicle saline (0.9% NaCl). Flunixine meglumine

(Banamine�, Schering Plough, Brazil) and poly-antibiotic

preparation of streptomycins and penicillins

(Pentabiotico�, Fontoura-Wyeth, SP, Brazil) were used

as provided.

Acute restraint

In the morning period (07–12 h), the animals were

transported to the experimental room in their home

cages. For recording MAP and HR, the catheters were

connected to a transducer and a HP-7754A amplifier

(Hewlett Packard, Palo Alto, CA, USA) coupled to a

signal acquisition board (Biopac M-100, Goleta, CA,

USA) connected to a computer. After 15 min of baseline

recording, bilateral microinjections of drugs into the DH

were performed in a volume of 500 nL, using a 1 lLsyringe (7001KH; Hamilton, Reno, NV, USA) connected

to a microinjection needle (gauge 33 Small Parts, Miami

Lakes, FL, USA) through a piece of PE-10 polyethylene

tubing. The microinjection needle was 1 mm longer than

the guide cannula. Microinjections were performed

within a 15-s period. After the microinjection, the needle

was left in the guide cannula for 30 s before being

removed, to avoid reflux. The drug microinjection was

performed without any manipulation or restraint of the

animals. The microinjection was performed whenever

BP and HR were considered to be stable. Ten minutes

later, rats were submitted to a 60-min restraint period in

a plastic cylindrical restraining tube (diameter 6.5 cm

and length 15 cm). After restraint, the animals were

returned to their cages. Each animal was submitted to

only one restraint session.

Tail temperature measurement

The tail cutaneous temperature was recorded using a

thermal camera Multi-Purpose Thermal Imager IRI4010

(InfraRed Integrated Systems Ltd., Northampton, UK) at

a distance of 50 cm (Reis et al., 2011; Busnardo et al.,

2013). For analyzing the images, the temperature

measurement was performed on five points of the

animal’s tail and the mean value was calculated for

each recording.

Detection of intra-hippocampus level of products ofNO, nitrite and nitrate (NOx)

The aim of this experiment was to quantify the levels of

NOx generated in the DH during RS. For these

protocols, animals (which had not undergone any

surgical procedures) were taken to a room where they

remained for 60 min in room ambiance and were then

submitted to RS. The animals were divided into four

groups: unstressed and stressed for 10, 30 or 60 min.

After the RS session, the animals were anesthetized

with urethane (1.25 g/kg, i.p.), decapitated and the

hippocampus dissected and separated into ventral and

dorsal regions. The tissue was homogenized in lysis

buffer (20 mM Tris–HCl, pH 7.6, 0.9% NaCl, 10%

glycerol) and centrifuged at 20,000g for 15 min, 4 �C.The supernatant was collected and subjected to

quantification of total protein by the Bradford method

and NOx by the Griess method.

Total protein was quantified by the colorimetric

reaction of Bradford, in which the Bradford reagent (1�;Bio Rad, Hercules, CA, USA) was added to an

aliquot of sample or standard protein (purified bovine

serum albumin, BSA, Sigma). The color produced by

the dye-binding protein was quantified at 595 nm

(VictorX3 spectrophotometer, Perkin Elmer, Waltham,

Massachusetts, USA). The equation of the line provided

by the standard curve (BSA) was used to calculate the

amount of total protein present in each sample.

Samples for quantifying levels of NO2/NO3 (products

from the spontaneous oxidation of NO under physio-

logical conditions, used as an indirect measurement of

NO production) were incubated (1:1, v/v) overnight at

37 �C in the presence of nitrate reductase and NADPH

(0.4 U/ml and 1 mg/mL, respectively, diluted in 0.2 M

KH2PO4 buffer, pH = 7.5) for conversion of NO3 to

NO2. Subsequently, NO2 present in these samples was

quantified by the Griess colorimetric reaction based on

the reaction of NO2 with a primary aromatic amine in

acid medium to form a diazonium salt. This in turn is

reacted with an aromatic compound forming an azo

compound that can be detected spectrophotometrically

at 540 nm. A kit was used for the determination of nitrite

(Invitrogen, Carlsbad, CA, USA) according to the

manufacturer’s instructions. The results were calculated

based on the standard curve of sodium nitrite (NaNO2)

and were expressed as percentage relative to the non-

stressed group.

Histological procedure

At the end of the experiments, rats were anesthetized with

urethane (1.25 g/kg, i.p.) and 500 nL of 1% Evans Blue

dye was bilaterally injected into the DH to stain the

injection sites. The chest was surgically opened, the

descending aorta occluded, the right atrium severed and

the brain perfused with 10% formalin through the left

ventricle. Brains were postfixed for 24 h at 4 �C, and 40-

lm sections were cut using a cryostat (CM-1900, Leica,


Wetzlar city, Hesse state, Germany). Sections were

stained with 1% Cresyl Violet and injection sites were

identified.

Data analysis

All autonomic responses were continuously recorded for

15 min before and during the 60-min RS period. Data

were expressed as mean ± standard error of mean

(SEM) changes (respectively MAP, HR or temperature)

and were sampled at 2-min intervals as a mean of the

changes during each 2 min. Points sampled during the

10 min before restraint were used as control baseline

values. The autonomic values changes during restraint

were analyzed using two-way analysis of variance

(ANOVA) followed by the Bonferroni’s post hoc test,

with treatment as independent factor and time as

repeated measurement factor. The basal value changes

were analyzed before and after vehicle or drug

administration by Student’s t test. Levels of NOx

generated in the DH during RS were analyzed using

one-way ANOVA followed by the Bonferroni’s post hoc

test.

Values of P< 0.05 were taken as showing

statistically significant differences between means.

Fig. 1. (A) Diagrammatic representation modified from the rat brain atlas of

experiments. Dots (filled circles) indicate the position of microinjection sites

coronal section of a rat brain depicting the site of bilateral microinjection in

distance from interaural line; DH – dorsal hippocampus.

RESULTS

A diagrammatic representation showing bilateral sites of

microinjection in the DH of all animals used in the study

is presented in Fig. 1. Fig. 2 shows a representative

recording of cardiovascular responses from an animal

during the RS and representative infrared images of the

tail skin temperature before and during the RS session.

Effect of bilateral microinjection of ACSF or AP-7 intothe DH on restraint-related cardiovascular and tailtemperature changes

ACSF. ACSF (n= 6) microinjection into the DH did

not change basal levels of MAP (before: 87.1 ±

2 mmHg and after: 87.2 ± 2 mmHg, t= 0.05, P> 0.05),

HR (before: 302.2 ± 16.7 bpm and after: 309.8 ±

19.1 bpm, t= 0.2, P> 0.05) or tail temperature (before:

31.2 ± 0.37 bpm and after: 30.8 ± 0.31 bpm, t= 0.26,

P> 0.05).

The RS-evoked MAP and HR increases in ACSF-

treated animals, which were similar to those observed in

DH-untreated control animals. There were no significant

differences between these two groups (data not shown)

in restraint-evoked MAP increases (F35,360 = 1.4,

P> 0.05) and HR increases (F35,360 = 1.6, P> 0.05)

and tail temperature decreases (F16,170 = 0.7, P< 0.05).

Paxinos and Watson, (1997) for the brains of rats used in the present

in the DH of the rats (n= 6/each group). (B) Photomicrograph of a

the DH. The center of the microinjection is indicated by arrows. IA –

Fig. 2. (Left) A representative recording of mean arterial pressure (MAP), pulsatile arterial pressure (PAP), and heart rate (HR) showing the pressor

and tachycardic responses 10 min before and during the 60 min when the animal was submitted to RS. (Right) Thermography photo showing

variation in skin temperature of the rat tail. (A) Photo before the start of RS with the animal in the home box. (B) Photo of the tail of the rat placed in

the restraint tube after 60 min of RS.


AP-7. Bilateral AP-7 (n= 6) microinjections into the

DH did not change basal levels of MAP (before:

101 ± 2 mmHg and after: 98 ± 3 mmHg, t= 0.7,

P> 0.05), HR (before: 368 ± 16 bpm and after:

349 ± 12 bpm, t= 1.75, P> 0.05) or tail temperature

(before: 32.1 ± 0.41 bpm and after: 32.1 ± 0.4 �C,t= 0.19, P> 0.05).

For the RS-evoked MAP and HR increases and tail

temperature decreases, however, AP-7 administration

into the DH was able to reduce MAP (F1,360 = 274,

P< 0.01) and HR (F1,360 = 236, P< 0.01) increases,

and tail temperature decreases (F1,170 = 180.1,

P< 0.01) when compared to ACSF-treated animals

(n= 6) (Fig. 3).

Detection of intra-hippocampus level of NOx

When compared to unstressed control animals (n= 6),

animals subjected to RS had increased levels of NOx in

the DH (F3,17 = 27, P< 0.01). However, this increase

of Nox level in the DH was detected only after 10 min

(n= 5, P< 0.05) and 30 min (n= 5, P< 0.05) of RS

exposure. After 60 min of RS, there were no significant

differences in the concentration of NOx (n= 5,

P> 0.05) (Fig. 4).

Effect of bilateral microinjection of N-propyl orc-PTIO into the DH on restraint-related cardiovascularand tail temperature changes

N-Propyl. Bilateral N-propyl (n= 6) microinjection into

the DH did not change basal levels of MAP (before:






temperature decreases, however, N-propyl adminis-

tration into the DH was able to reduce MAP (F1,360 = 334,


and tail temperature decreases (F1,170 = 72.4,

P< 0.01) when compared to ACSF-treated animals

(n= 6) (Fig. 5).

c-PTIO. Bilateral c-PTIO (n= 6) microinjection into

the DH did not change basal MAP levels (before:






temperature decreases, however, c-PTIO administration

into the DH was able to reduce MAP (F1,360 = 235,


and tail temperature decreases (F1,170 = 348, P< 0.01)

when compared to ACSF-treated animals (n= 6)

(Fig. 5).

DISCUSSION

Our results have demonstrated that blockade of NMDA

glutamate receptors in the DH reduces hypertension,

tachycardia and the fall in skin temperature observed

during RS. Moreover, microinjection of the selective

nNOS inhibitor N-propyl or the NO scavenger c-PTIO

into the DH also reduces these autonomic responses

associated with RS.

Studies have shown increases in c-Fos protein

expression in the DH during stress situations

(Chowdhury et al., 2000), suggesting that this area is

involved in the modulation of stress responses.

Supporting this role of the DH in modulation of the

autonomic nervous system, previous findings from our

group indicated that DH synaptic inhibition is able to

reduce the hypertension and tachycardia evoked by the

model of fear conditioning to context, showing a

possible role of the DH in the modulation of autonomic

responses associated with defensive reactions (Resstel

et al., 2008). Moreover, the DH has extensive

connections with limbic structures (Swanson, 1981;

Fig. 3. Time-course of mean arterial pressure (DMAP), heart rate (DHR) and tail cutaneous temperature (D Temperature) changes before and

during restraint of animals that received bilateral administration of vehicle (500 nL, n= 6) or AP-7 (10 nmol/500 nL, n= 6) in the DH. Points

represent the mean and bars the SEM, ⁄P< 0.01, two-way ANOVA followed by the Bonferroni’s post-test.

Fig. 4. Variation in concentrations of DH nitrite and nitrate (NOx) of non-stressed animals (n= 6) or stressed animals after 10, 30 and 60 min

(n= 5/each group) of RS. The bars represent mean ± SEM, ⁄P< 0.01, one-way ANOVA followed by Dunnetts post-test, compared to the non-

stressed group.


Herman et al., 1994; Pacak et al., 1995; Carr and Sesack,

1996; Davis et al., 1997) that are involved with autonomic

responses induced by stress situations, such as the

MPFC (Resstel et al., 2006), LSA (Kubo et al., 2002;

Reis et al., 2011), the medial amygdaloid nucleus

(Fortaleza et al., 2009) and the BNST (Crestani et al.,

2009). These results indicate that the DH could be part

of a central pathway involved in modulation of

Fig. 5. (A) Time-course of mean arterial pressure (DMAP), heart rate (DHR) and tail cutaneous temperature (D Temperature) changes before and

during restraint of animals that received bilateral administration of vehicle (500 nL, n= 6) or N-propyl (0.1 nmol/500 nL, n= 6) in the DH. (B) Time-

course of mean arterial pressure (DMAP), heart rate (DHR) and tail cutaneous temperature (D Temperature) changes before and during restraint of

animals that received bilateral administration of vehicle (500 nL, n= 6) or c-PTIO (2 nmol/500 nL, n= 6) in the DH. Points represent the mean and

bars the SEM, ⁄P< 0.01, two-way ANOVA followed by the Bonferroni’s post-test.


autonomic nervous system activity during stress

situations.

Glutamate is the major excitatory neurotransmitter in

the brain, and it is a critical mediator of synaptic and

behavioral plasticity. Dysfunction of the glutamatergic

system is associated with the pathophysiology and

treatment of mood and anxiety disorders (Conn et al.,

2009; Skolnick et al., 2009). The hippocampus is rich in

glutamatergic receptors and stress situations promote

increased release of glutamate in this area (Moghaddam,

1993), suggesting the existence of a functional gluta-

matergic system in this structure that is active during the

exposure to threat situations.

Given the evidence described above, we investigated

the possible involvement of the DH glutamatergic system,

via NMDA receptor activation, in the modulation of

cardiovascular responses during RS. The microinjection

of AP-7, a NMDA glutamate receptor antagonist, evoked

reduction of hypertension and tachycardia caused by

RS. These findings suggest that the activation of NMDA

receptors in the DH modulates autonomic responses

associated with RS. Our findings are in agreement with

previous studies reporting that microinjection of

glutamate into the DH evoked increases in both MAP

and HR (Ruit and Neafsey, 1988). Moreover, the

bilateral injection of AP-7 into the DH was able to

reduce total immobility time in the forced swimming test,

an anti-aversive response, thus suggesting that

glutamate NMDA receptors located in the DH are

involved in the behavioral changes observed in this

model (Padovan and Guimaraes, 2004). Together,

these findings support an important role of NMDA

receptors in DH on modulation of responses evoked by

aversive situations.

NMDA receptors are located in close proximity to

nNOS (Brenman and Bredt, 1997) and generation of

NO has been described after the activation of NMDA

receptors (Garthwaite et al., 1988). NO acts as a

messenger that diffuses through the CNS and is

involved in several neuronal responses (Garthwaite and

Boulton, 1995; Hawkins et al., 1998). Blackshaw et al.

(2003) demonstrated the expression nNOS in the DH of

rats. Moreover, Lourenco et al. (2011) observed that

after administration of L-glu in the DH, there was an

increase in NO concentration, a response that was

inhibited by the administration of NMDA receptor

antagonists, suggesting that activation of NMDA

receptors stimulates nNOS with a consequent NO

production in the DH.

NO is a gas that spreads easily and is converted into

nitrite and nitrate within seconds. This characteristic

complicates the direct determination of changes in its

concentration in biological systems. However,

measurements of the most stable products of NO, nitrite

and nitrate (NOx), provide qualitative measurements of

NOS activity and NO production (Marzinzig et al., 1997).

Activation of glutamate receptors in the DH promotes

cardiovascular responses, and the increase in MAP and


HR is similar to that observed during RS. Cardiovascular

responses to microinjection of L-glu into the DH depend

on the NMDA/NO pathway, and the blockade of NMDA

receptors with AP-7 promotes a reduction in the

magnitude of the autonomic responses during RS. We

therefore evaluated whether during RS there are

changes in NO levels evidenced in the concentrations of

NOx in the DH. The concentration of NOx was found to

be increased when samples were collected 10 and

30 min after the animals were submitted to RS, but no

significant differences were observed after 60 min of

stress. Moghaddam (1993) reported an increase in

glutamate levels in the DH during RS in rats, which

lasted up to 40 min, but that 60 min later, glutamate

levels were back to baseline values. Thus, since the

stimulation of nNOS and consequent NO synthesis

depends on the activation of glutamate NMDA

receptors, NO levels must be reduced due to a

decrease of glutamate release in the rat DH.

Because NO levels are increased in the DH during

RS, we also investigated the direct involvement of NO in

the cardiovascular responses evoked by RS.

Microinjection of the selective nNOS inhibitor N-propylinto the DH evoked a reduction in the hypertension and

tachycardia caused by RS, showing that nNOS present

in the DH modulates these responses.

In the same way, cardiovascular responses evoked by

RS were also inhibited by c-PTIO, a NO scavenger. NO

acts primarily as an intercellular messenger (Guix et al.,

2005). NO can influence neuronal or glial cells far away

from its neuronal source (Wood and Garthwaite, 1994;

Lancaster, 1997). Because c-PTIO is a cell membrane-

impermeable compound (Ko and Kelly, 1999), the

present findings indicate that both NOS activation and

extracellular release of NO in the DH are involved in

RS-evoked autonomic responses. Our findings suggest

that glutamatergic and nitrergic systems in the DH play

a facilitatory role in modulation of cardiovascular

responses to aversive situations.

In agreement with the decreased cardiovascular

responses, pretreatment of the DH with AP-7, N-propyl

or c-PTIO also reduced stress-induced changes in

cutaneous temperature. These findings indicate that

during RS, the DH could be modulating the activity of

spinal sympathetic cardiomotor neurons and

sympathetic neurons controlling temperature changes.

These latter neurons include those that control

cutaneous vascular tone in the tail skin and those that

innervate brown adipose tissue (BAT). In rats,

cutaneous temperature depends on blood flow and

sympathetic vasoconstrictor tone in skin arteries

(Blessing and Seaman, 2003; Vianna and Carrive,

2005). Aversive situations cause a reduction in the tail

blood flow in rats (Blessing, 2003; Blessing and

Seaman, 2003). The decrease in tail blood flow would

occur to prevent blood loss due to injuries, by keeping

low amounts of blood in the skin. Another function

would be the redistribution of blood to more important

organs during a stress situation. Tail cutaneous

temperature can be used as an indirect measurement of

blood flow redistribution by the sympathetic nervous

system in the rat (Blessing, 2003; Blessing and

Seaman, 2003; Vianna and Carrive, 2005). Therefore,

our results suggest that the activation of NMDA

receptors and NO formation in the DH are important

modulators of the sympathetic tonus in the tail artery

during exposure to an aversive situation.

The DH presents connections to the BNST and LSA,

structures that are known to modulate cardiovascular

responses during RS (Kubo et al., 2002; Crestani et al.,

2010; Reis et al., 2011). The BNST sends projections to

areas of the brainstem that are involved in autonomic

regulation, such as the dorsal nucleus of the vagus

(Gray and Magnuson, 1987), the caudal ventrolateral

medulla (Giancola et al., 1993) and the nucleus of the

solitary tract (Holstege et al., 1985; Gray and

Magnuson, 1987). The DH also sends projections to the

MPFC, an important limbic structure involved in

learning, memory, behavioral and autonomic modulation

(Sesack et al., 1989; Takagishi and Chiba, 1991; Conde

et al., 1998; Resstel et al., 2004; Resstel and Correa,

2006). Lesions in the MPFC reduce the cardiovascular

responses caused by administration of L-glu in the DH

(Ruit and Neafsey, 1988), suggesting that these

responses require participation of this area. Moreover,

the MPFC also sends projections to bulbar structures

involved in autonomic modulation (Fisk and Wyss, 2000;

Vertes, 2004), suggesting that the DH modulates

autonomic responses through other structures involved

in cardiovascular regulation.

Therefore, our findings support the idea that a

glutamatergic system present in the DH, through

activation of NMDA receptors followed by NO release,

is involved in modulation of RS-evoked autonomic

responses.

Acknowledgments—The authors wish to thank Laura H.A. de

Camargo, Ivanilda A.C. Fortunato, Simone S. Guilhaume and

Idalia I.B. Aguiar for technical help. Moraes-Neto, T.B. has a

PhD student fellowship, FAPESP (2012/00390-0), and Scopinho,

A.A. has a postdoctoral fellowship, CAPES (PNPD0176087). The

present research was supported by grants from the FAPESP

(2011/07332-3; 2012/09300-4; 2012/17626-7), CNPq and FAEPA.

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(Accepted 10 November 2013)(Available online 22 November 2013)














































































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