hydrogen peroxide attenuates the dipsogenic, renal and pressor responses induced by cholinergic...
TRANSCRIPT
Neuroscience 284 (2015) 611–621
HYDROGEN PEROXIDE ATTENUATES THE DIPSOGENIC, RENAL ANDPRESSOR RESPONSES INDUCED BY CHOLINERGIC ACTIVATION OFTHE MEDIAL SEPTAL AREA
M. R. MELO, J. V. MENANI, E. COLOMBARI *� ANDD. S. A. COLOMBARI *�
Department of Physiology and Pathology, School of Dentistry,
Sao Paulo State University, UNESP, Araraquara, SP, Brazil
Abstract—Cholinergic activation of the medial septal area
(MSA) with carbachol produces thirst, natriuresis, antidiure-
sis and pressor response. In the brain, hydrogen peroxide
(H2O2) modulates autonomic and behavioral responses. In
the present study, we investigated the effects of the combi-
nation of carbachol and H2O2 injected into the MSA on water
intake, renal excretion, cardiovascular responses and the
activity of vasopressinergic and oxytocinergic neurons in
the hypothalamic paraventricular (PVN) and supraoptic
(SON) nuclei. Furthermore, the possible modulation of car-
bachol responses by H2O2 acting through K+ATP channels
was also investigated. Male Holtzman rats (280–320 g) with
stainless steel cannulas implanted in the MSA were used.
The pre-treatment with H2O2 in the MSA reduced carbachol-
induced thirst (7.9 ± 1.0, vs. carbachol: 13.2 ± 2.0 ml/
60 min), antidiuresis (9.6 ± 0.5, vs. carbachol: 7.0 ± 0.8 ml/
120 min,), natriuresis (385 ± 36, vs. carbachol: 528 ±
46 lEq/120 min) and pressor response (33 ± 5, vs. carbachol:
47 ± 3 mmHg). Combining H2O2 and carbachol into the MSA
also reduced the number of vasopressinergic neurons
expressing c-Fos in the PVN (46.4 ± 11.2, vs. carbachol:
98.5 ± 5.9 c-Fos/AVP cells) and oxytocinergic neurons
expressing c-Fos in the PVN (38.5 ± 16.1, vs. carbachol:
75.1 ± 8.5 c-Fos/OT cells) and in the SON (57.8 ± 10.2, vs.
carbachol: 102.7 ± 7.4 c-Fos/OT cells). Glibenclamide (K+ATP
channel blocker) into the MSA partially reversed H2O2 inhibi-
tory responses. These results suggest that H2O2 acting
through K+ATP channels in the MSA attenuates responses
http://dx.doi.org/10.1016/j.neuroscience.2014.10.0240306-4522/� 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding authors. Address: Department of Physiology andPathology, Dentistry School, Sao Paulo State University (UNESP),Rua Humaita, 1680, Araraquara 14801-903, SP, Brazil. Tel: +55-16-3301-6460 (E. Colombari). Tel: +55-16-3301-6483 (D. S. A. Colom-bari).
E-mail addresses: [email protected] (E. Colombari),[email protected] (D. S. A. Colombari).
� E. Colombari and D. S. A. Colombari are co-senior authors.Abbreviations: ANG II, angiotensin II; ANOVA, analysis of variance;ANP, atrial natriuretic peptide; AVP, vasopressin; DAB,diaminobenzidine; H2O2, hydrogen peroxide; HR, heart rate; i.c.v.,intracerebroventricularly; IgG, immunoglobulin G; MAP, mean arterialpressure; mPVN, magnocellular region of the PVN; MSA, medial septalarea; NGS, normal goat serum; OT, oxytocin; PBS, phosphate-buffered saline; PFA, paraformaldehyde; pPVN, parvocellular PVN;PVN, paraventricular nucleus; ROS, reactive oxygen species; SON,supraoptic nucleus; w/v, weight/volume.
611
induced by cholinergic activation in the same area.
� 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: carbachol, c-Fos, vasopressin, forebrain,
oxidative stress.
INTRODUCTION
Cholinergic mechanisms in the forebrain are involved with
drinking induced by dehydration (Block and Fisher, 1970)
and hormone release during volume expansion or hyper-
osmolality (Antunes-Rodrigues et al., 2004). The medial
septal area (MSA), a subnucleus of the septal area or
septum, is an important forebrain area involved in cardio-
vascular regulation and in the control of fluid and electro-
lyte balance (Negro-Vilar et al., 1967; Donevan and
Ferguson, 1988; Tanaka et al., 1988; Luiz et al., 1991;
Colombari et al., 1992; Paulin et al., 2009). Cholinergic
cells and receptors are present in the MSA (Rouse and
Levey, 1996; Jones and Yakel, 1997) and cholinergic acti-
vation of the MSA induces water intake, antidiuresis,
natriuresis and pressor responses (Saad et al., 1975;
Colombari et al., 1992).
The MSA projects to the hypothalamic paraventricular
(PVN) and supraoptic nuclei (SON) which contains
neurons that secrete vasopressin (AVP) and oxytocin
(OT) (Oldfield et al., 1985). Vasopressin release is possi-
bly involved with the antidiuresis produced by carbachol
injected into the MSA, whereas oxytocin, a hormone that
induces natriuresis (Huang et al., 1995) and release of
atrial natriuretic peptide (ANP) (Antunes-Rodrigues
et al., 2004), might be involved with the natriuresis
induced by central injections of carbachol. Although the
anatomical connections suggest this possibility, the
effects of MSA cholinergic stimulation on the activity of
vasopressinergic and oxytocinergic neurons in the PVN
and SON are still unknown.
Reactive oxygen species (ROS) like the free radical
superoxide anion (O2��), hydroxyl radical (HO�) and
hydrogen peroxide (H2O2) can be produced
endogenously acting as intra- and intercellular signaling
molecules to regulate biological functions (Weinberg,
1990; Rhee et al., 2003). The O2�� is suggested to mediate
the responses produced by the classical mediator angio-
tensin II (ANG II) acting centrally (Zimmerman and
Davisson, 2004). Conversely, intracerebroventricular
612 M. R. Melo et al. / Neuroscience 284 (2015) 611–621
(i.c.v.) administration of H2O2 reduces the pressor
response induced by i.c.v. ANG II, suggesting an inhibi-
tory role for this particular ROS on ANG II pressor
response (Lauar et al., 2010).
In addition to inhibiting ANG II pressor responses,
H2O2 also affects cholinergic mechanisms, decreasing
carbachol-induced phosphoinositide hydrolysis by
phospholipase C in human neuroblastoma cells (Li
et al., 1996) and amylase secretion (Mata et al., 2008).
Therefore, H2O2 may negatively modulate the actions of
carbachol by affecting muscarinic receptor function or
intracellular pathways. However, it is not known if H2O2
might affect the responses to central cholinergic
activation.
Therefore, in this present study, we sought to
investigate the changes in water intake, renal excretion,
cardiovascular response and c-Fos expression in the
vasopressinergic and oxytocinergic cells of the SON
and PVN produced by carbachol injected alone or
combined with H2O2 into the MSA in rats. Different
mechanisms have been proposed to explain the
inhibitory effect of H2O2 on the neuronal excitability.
H2O2 may inhibit glutamate action, increase GABA
release or induce K+ATP channel opening (Zoccarato
et al., 1990, 1995; Avshalumov et al., 2005; Bao et al.,
2005). Considering that the MSA is an area rich in K+ATP
channels (Stefani and Gold, 1998; Allen and Brown,
2004), we also tested if H2O2 might modulate carbachol-
induced responses in the MSA by activating K+ATP channels.
EXPERIMENTAL PROCEDURES
Animals
Male Holtzman rats weighing 280–320 g were used. The
animals were housed individually in stainless steel
cages in a room with controlled temperature (23 ± 2 �C)and humidity (55 ± 10%). Lights were on from 7:00 am
to 7:00 pm. Standard rat chow (BioBase Rat Chow,
Basequımica Produtos Quımicos LTDA, Aguas Frias,
Santa Catarina, Brazil) and tap water were available adlibitum. The experimental protocols used in the present
study were approved by the Ethics Committee for
Animal Care and Use of the Dental School of
Araraquara, UNESP, (proc. CEUA 02/2012 and
30/2014) and also by Ethics Committee for Animal Care
and Use of the Federal University of Sao Paulo/School
of Medicine (proc. 0371/12).
Brain surgery
Rats were anesthetized with intraperitoneal ketamine
[Uniao Quımica Farmaceutica Nacional S/A,
Embu-Guacu, SP, Brazil, 80 mg/kg body weight (wt.)]
combined with xylazine (Uniao Quımica Farmaceutica
Nacional S/A, Embu-Guacu SP, Brazil, 7 mg/kg body
wt.) and placed in a stereotaxic apparatus (Kopf,
Tujunga, CA, USA). The skull was leveled between the
bregma and lambda. Stainless steel 23-gauge cannulas
(12 � 0.6 mm) were implanted in the direction of the
MSA using the following coordinates: 0.7 mm rostral to
the bregma, in the midline and 3.6 mm below the
surface of the skull. The cannulas were fixed to the
cranium using dental acrylic resin and jeweler screws. A
prophylactic dose of penicillin (benzylpenicillin – 30,000
IUs plus streptomycin – 16 mg; Pentabiotico Veterinario
– Pequeno Porte, Fort Dodge Saude Animal Ltda,
Campinas, Brazil) and the anti-inflammatory Ketoflex
(ketoprofen 1%–0.03 ml/rat; Ketoflex, Mundo Animal,
Sao Paulo, Brazil) were given intramuscularly post
surgically. After the surgery, rats were allowed to
recover for 1 week before starting the experiments.
Drugs
Carbachol chloride, H2O2 and glibenclamide were
purchased from Sigma Chemical Co. (St. Louis, MO,
USA). Carbachol was used at the dose of 4 nmol/0.5 ll,and was dissolved in isotonic saline (NaCl 0.15 M).
H2O2 was used at the dose of 2.5 lmol/0.5 ll, and was
dissolved in phosphate-buffered saline (PBS, pH = 7.4).
Glibenclamide was used at the dose of 5 nmol/0.5 lland was dissolved in 5% of anhydrous alcohol and
suspended in a mix of propylene glycol and water 2:1
(vehicle). The injections were made using 5-ll-Hamilton
syringe connected by PE-10 polyethylene tubing to a
needle introduced into the brain through the guide
cannula. The needles for injection into the MSA were
2 mm longer than the guide cannula. The volume
injected was 0.5 ll.
Water and food intake tests
Rats were tested in their home cages. Water intake was
measured using glass burets with 0.1-ml divisions fitted
with a metal drinking spout. For food intake, a pre-
weighted amount of regular chow pellets was given to
the animals. At 30, 60, 90 and 120 min of the test, the
ingested food was calculated by subtracting the
remaining amount of chow from the pre-weighted
amount. All chow spillage under the cages was
recovered at every measurement to calculate food intake.
Renal excretion test
Animals were housed in metabolic cages and urine was
collected by gravity in graduated tubes with 0.1-ml
divisions. The urine samples were analyzed by Na+/K+
analyzer (NOVA 1, Nova Biomedical, Waltham, MA,
USA). The Na+ e K+ total excretion was calculated as
Na+ e K+ concentration multiplied by urinary volume.
Arterial pressure and heart rate (HR) recordings
On the day before the experiments, under xylazine and
ketamine anesthesia (as described above), a
polyethylene tubing (PE-10 connected to a PE-50) was
inserted into the abdominal aorta through the femoral
artery. The arterial catheter was tunneled
subcutaneously and exposed on the back of the rat to
allow access in unrestrained, freely moving rats. To
record pulsatile arterial pressure (PAP), MAP and HR,
the arterial catheter was connected to a Statham Gould
(P23 Db) pressure transducer coupled to a pre-amplifier
(model ETH-200 Bridge Bio Amplifier) that was
M. R. Melo et al. / Neuroscience 284 (2015) 611–621 613
connected to a Powerlab computer data acquisition
system (model Powerlab 16SP, AD Instruments, Castle
Hill, NSW, Australia).
Histology and immunohistochemistry
The animals were deeply anesthetized with sodium
thiopental (70 mg/kg of body wt., i.p.) and received an
injection of 0.5 ll of 2% Evans Blue into the MSA.
Thereafter, they were transcardially perfused with 300 ml
of 0.1 M (PBS, pH 7.4), followed by 500 ml of 4% weight/
volume (w/v) paraformaldehyde (PFA, Sigma, St. Louis,
USA) solution in 0.1 M PBS, pH 7.4. The brains were
removed, fixed for 4 h in 4% (w/v) PFA solution and
stored at 4 �C in 0.1 M PBS containing 20% (w/v)
sucrose. The MSA was cut coronally (30-lm sections) in
a cryostat (Leica CM 1850 UV), stained with Giemsa and
analyzed by light microscopy to confirm MSA injection
site. For immunohistochemistry procedures, four sets of
coronal sections (30 lm) of the hypothalamus were
sectioned on a cryostat (Leica CM 1850 UV) and
the free-floating sections were collected in 24-well tissue
culture plates containing PBS. Two out four rat
hypothalamic sections were pre-incubated for 10 min in
3% (v/v) H2O2 (Sigma, St. Louis, MO, USA) in 0.1 M
PBS followed by rinses in PBS (3 � 10 min). Sections
were then incubated in 15 min in a blocking solution
comprising 10% (v/v) normal goat serum (NGS, Sigma,
St. Louis, MO) and 0.3% (v/v) Triton X-100 (Sigma, St.
Louis, MO, USA) in 0.1 M PBS followed by rinses in PBS
(3 � 10 min). Sections were then incubated in a rabbit
polyclonal immunoglobulin G (IgG) anti-Fos primary
antibody (1:4000 Ab-4; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) in PBS containing 1% (v/v) NGS and
0.3% (v/v) Triton X-100 for 24 h at 4 �C. After the
primary antibody incubation the sections were rinsed in
PBS (3 � 10 min) prior to 1-h incubation with biotinylated
goat anti-rabbit IgG (1:500 Vector Laboratories Inc.,
Burlingame, CA, USA), followed by further rinses in PBS
(3 � 10 min), and incubation with Streptavidin HRP
(1:500, Vector Laboratories Inc., Burlingame, CA,
USA) for 1-h. Sections were rinsed (3 � 10 min) and
diaminobenzidine (DAB, Sigma, St. Louis, MO) with
0.5% (w/v) cobalt chloride and 0.5% (w/v) nickel
ammonium sulfate was used to intensify the cell
nucleus. After concluding immunohistochemistry for Fos
described above, the same sections were rinsed
(3 � 5 min), and then incubated for 48 h with either a
rabbit polyclonal antibody against vasopressin (1:20,000;
Peninsula, San Carlos, CA, USA) or a rabbit polyclonal
antibody against oxytocin (1:30,000; Peninsula, San
Carlos, CA, USA) and DAB reaction (without cobalt and
nickel) was performed as above to produce a detectable
brown product in the cytoplasm which indicates either
AVP or OT, accordingly to the antibody used. Sections
were mounted onto slides in 0.5% (w/v) gelatin and
allowed to air-dry, dehydrated in a series of alcohols,
cleared in xylene and cover slipped. Cells expressing
positive nuclear c-Fos and AVP or OT immunoreactivity
were counted bilaterally (5–6 sections for PVN and
7–8 sections for SON) by hand each 60 lm, in
matched, representative sections of the tissue, with a
magnification of 20�. The numbers shown in Figs. 5 and
6 represent the double labeling counting in the sections
divided by the number of sections of each region.
Statistical analysis
All data are expressed as the mean ± standard error of
mean (SEM). Water intake, urine excretion and arterial
pressure were analyzed by a one-way analysis of
variance (ANOVA) or two-way ANOVA for repeated
measures, followed by Student–Newman–Keuls post
hoc. Immunohistochemistry data were analyzed by
Kruskal–Wallis test followed by Student–Newman–Keuls
post hoc. Differences were considered significant at
p< 0.05.
Experimental protocols
For drinking and renal excretion experiments, each group
of rats was submitted to 2–4 different tests with an interval
of at least 3 days between them. In each test, the group of
rats was divided into two and half of the group received
one of the combined treatments and the other half
received another combined treatment described below.
The sequence of the treatments in the different tests
was randomized.
Drinking responses induced by carbachol injected intothe MSA alone or combined with H2O2 in the same
area. In a group of rats (n= 9), H2O2 (2.5 lmol/0.5 ll) orPBS (0.5 ll) was injected into the MSA 1 min before
carbachol (4 nmol/0.5 ll) or saline (0.5 ll) injected in the
same area. Water intake was measured at 15, 30, 45
and 60 min, starting immediately after carbachol or
saline injection. Animals received four combinations of
treatments into the MSA: PBS + saline, PBS+
carbachol, H2O2 + saline and H2O2 + carbachol.
To exclude nonspecific effects of H2O2 on ingestive
behaviors, in two other groups of rats (n= 9/group)
were tested the effects of H2O2 injected into the MSA
on 2% (w/v) sucrose intake or food intake. To test the
ingestion of sucrose, a group of rats was trained to drink
2% sucrose during 2 h/day for 7 days. On the 8th day,
H2O2 (2.5 lmol/0.5 ll) or PBS (0.5 ll) was injected into
the MSA 1 min before rats having access to 2%
sucrose. Water and 2% sucrose were measured at 30,
60, 90 and 120 min, starting immediately after both
fluids being available for the rats. Another group of rats
was deprived of food, but not water for 24 h. After this
period, animals received injections of H2O2 (2.5 lmol/
0.5 ll) or PBS (0.5 ll) into the MSA 1 min before the
access to pre-weighted amount of regular chow pellets.
Food intake and meal-associated drinking were
measured at 30, 60, 90 and 120 min, starting
immediately after the access to food.
Changes in urine volume and urinary excretion of
sodium and potassium induced by carbachol injected intothe MSA alone or combined with H2O2 in the samearea. A group of rats (n= 6) was deprived of food, but
not water for 14 h before the experiment. After this
period, rats received two water loads (10 ml each)
614 M. R. Melo et al. / Neuroscience 284 (2015) 611–621
intragastrically with 1-h interval between them.
Immediately after the 2nd load, H2O2 (2.5 lmol/0.5 ll)or PBS was injected into the MSA, followed 1 min later
by the injection of carbachol (4 nmol/0.5 ll) or saline
(0.5 ll) into the same area. Water load decreases
plasma osmolarity and increases diuresis. Urine was
collected at 30, 60, 90 and 120 min, starting
immediately after carbachol or saline injection. The
animals received four combinations of treatments into
the MSA: PBS+ saline, PBS+ carbachol, H2O2 +
saline and H2O2 + carbachol. During the experimental
session, rats had no access to water or food.
Cardiovascular responses produced by carbacholinjected in MSA combined or not with H2O2 injected in
the same area. Two different groups of rats were used
(n= 8–9/group). In each experiment, around 20 min
after starting the recordings of MAP and HR in
conscious freely moving rats, one group of rats received
injection of H2O2 (2.5 lmol/0.5 ll) and the other group
received injection of PBS into the MSA 1 min before the
injection of carbachol (4 nmol/0.5 ll) into the same area.
Double-staining in the PVN and SON in rats treatedwith carbachol alone or combined with H2O2 into the
MSA. Rats (n= 3/treatment) received injections of H2O2
(2.5 lmol/0.5 ll) or PBS (0.5 ll) into the MSA 1 min
before the injection of carbachol (4 nmol/0.5 ll) or saline(0.5 ll) into the same area. In different groups of rats,
four combinations of treatments into the MSA were
tested: PBS + saline, PBS+ carbachol, H2O2 + saline
or H2O2 + carbachol. After 90 min, the animals were
deeply anesthetized and perfused as described in the
Section ‘Histology and immunohistochemistry’ of the
Experimental procedures.
Dipsogenic response to carbachol injected into theMSA in rats treated with glibenclamide combined withH2O2 in same area. In a group of rats (n= 12),
glibenclamide (5 nmol/0.5 ll) or vehicle (0.5 ll) was
injected into the MSA 14 min before the injection of
H2O2 (2.5 lmol/0.5 ll) or PBS (0.5 ll) into the MSA.
Carbachol (4 nmol/0.5 ll) was also injected into the
MSA 1 min after the injection of H2O2 or PBS.
The animals received four combinations of treatments
into the MSA: glibenclamide + PBS+ carbachol;
glibenclamide + H2O2 + carbachol; vehicle + H2O2 +
carbachol; vehicle + PBS+ carbachol. Water intake
was measured at 15, 30, 45 and 60 min, starting
immediately after carbachol injection.
Natriuresis and antidiuresis to carbachol injected into
the MSA in rats treated with glibenclamide combined withH2O2 in same area. A group of rats (n= 8) received
injection of glibenclamide (5 nmol/0.5 ll) or vehicle
(0.5 ll) into the MSA 45 min after the first intragastric
water load (10 ml). Fourteen minutes after the first
injection into the MSA, H2O2 (2.5 lmol/0.5 ll) or PBS
(0.5 ll) was injected into the MSA. Carbachol (4 nmol/
0.5 ll) was also injected into the MSA 1 min after the
injection of H2O2 or PBS. Immediately after carbachol
injection, the second intragastric water load (10 ml) was
administered. Animals received four combinations of
treatments into the MSA: glibenclamide + PBS+
carbachol; glibenclamide+ H2O2 + carbachol; vehicle +
H2O2 + carbachol; vehicle + PBS+ carbachol. Urine
was collected at 30, 60, 90 and 120 min, starting
immediately after the second water load. During these
tests, the rats had no access to water or food.
Cardiovascular responses produced by carbachol
injected in MSA in rats treated with glibenclamide com-bined with H2O2 in same area. Four different groups were
used (n= 5–7/group). In each experiment, 20 min after
starting the recordings of MAP and HR in conscious
freely moving rats, glibenclamide (5 nmol/0.5 ll) or
vehicle (0.5 ll) was injected into the MSA, followed
14 min later by H2O2 (2.5 lmol/0.5 ll) or PBS
(0.5 ll) injection into the MSA. One min after H2O2 or
PBS injection, carbachol (4 nmol/0.5 ll) was also
injected in the MSA. In different groups of rats, four
combinations of treatments were tested into the
MSA: vehicle + PBS+ carbachol; vehicle + H2O2 +
carbachol; glibenclamide + H2O2 + carbachol and
glibenclamide + PBS+ carbachol.
RESULTS
Histological analysis
Fig. 1 shows the typical injection site into the MSA in a rat
representative of the animals tested in the present study.
The MSA injections were considered properly positioned
if they were placed in the midline, above the dorsal
border of the diagonal band of Broca, with no spread to
the lateral septal area or ventrally in the diagonal band
of Broca or in the median preoptic nucleus. The diagram
in the Fig. 1 shows the spread of the dye pooled from
all animals.
Water intake, urinary volume, natriuresis andkaliuresis in rats treated with carbachol combinedwith H2O2 into the MSA
The injection of carbachol (4 nmol/0.5 ll) combined with
PBS into the MSA induced water intake (13.2 ± 2.0, vs.
PBS + saline: 1.2 ± 0.4 ml/60 min) [F(3,15) = 19.18;
p< 0.05], natriuresis (528 ± 46, vs. PBS + saline:
74 ± 6 lEq/120 min) [F(3,15) = 49.73; p< 0.05],
kaliuresis (160 ± 12, vs. PBS+ saline: 68 ± 3 lEq/120 min) [F(3,15) = 45.52; p< 0.05] and antidiuresis
(7.0 ± 0.8, vs. PBS+ saline: 10.9 ± 0.4 ml/120 min)
[F(3,15) = 13.20; p< 0.05] (Figs. 2 and 3). The
previous injection of H2O2 (2.5 lmol/0.5 ll) into the MSA
reduced MSA carbachol-induced water intake
(7.9 ± 1.0 ml/60 min), natriuresis (385 ± 36 lEq/120 min), kaliuresis (128 ± 7 lEq/120 min) and
antidiuresis (9.6 ± 0.5 ml/120 min) (Figs. 2 and 3).
The injection of H2O2 (2.5 lmol/0.5 ll) combined with
saline into the MSA increased urinary volume
(13.8 ± 1.1 ml/120 min), without changing urinary
sodium (81.5 ± 6.4 lEq/120 min) or potassium
(88.3 ± 6.8 lEq/120 min) (Fig. 3).
Fig. 1. Photomicrographs of sequential coronal rostro-caudal sections of the brain (A–C) from a rat representative of those tested in the present
study, showing (arrows) the typical site of injection into the MSA. The diagrams below each section represents the spread of the dye at that level.
LV, lateral ventricle; ac, anterior commissure, LS, lateral septal area, DB, diagonal band of Broca, MnPO, median preoptic nucleus.
Fig. 2. Cumulative water intake in rats that received injections of
H2O2 or PBS combined with carbachol or saline into the MSA. The
results are expressed as mean ± SEM. Two-way repeated mea-
sures ANOVA combined with Student–Newman–Keuls test;
n= number of rats; carbachol (4 nmol/0.5 ll); saline (0.5 ll); H2O2
(2.5 lmol/0.5 ll); PBS (0.5 ll), phosphate-buffered saline.
M. R. Melo et al. / Neuroscience 284 (2015) 611–621 615
Cardiovascular responses in rats treated withcarbachol combined with H2O2 into the MSA
Baseline MAP and HR were 101 ± 2 mmHg and
387 ± 8 bpm (n= 17), respectively. Injections of H2O2
(2.5 lmol/0.5 ll) into the MSA reduced the pressor
response induced by carbachol (4 nmol/0.5 ll) injected
into the same area (33 ± 5 mmHg, vs. PBS+
carbachol: 47 ± 3 mmHg) [F(3,30) = 59.67; p< 0.05]
(Fig. 4). Bradycardia was observed only in rats treated
with PBS + carbachol (Fig. 4, inset).
c-Fos expression in the PVN and SONvasopressinergic and oxytocinergic neurons in ratstreated with carbachol combined with H2O2 into theMSA
The injection of carbachol (4 nmol/0.5 ll) combined with
PBS into the MSA increased the number of
vasopressinergic neurons expressing c-Fos in the
magnocellular region of the PVN (mPVN:98.5 ± 5.9, vs.
PBS + saline: 1.1 ± 0.4 cells/section – each 60 lm)
[H= 10.421; p= 0.015], the parvocellular PVN (pPVN:
24.3 ± 4.2, vs. PBS+ saline: 0.2 ± 0.1) [H= 9.388;
p= 0.025] and in the SON (113.9 ± 18.6 vs.
PBS + saline: 1.4 ± 0.6) [H= 8.538; p= 0.036],
(Fig. 5). The injections of H2O2 (2.5 lmol/0.5 ll) into the
MSA previously to carbachol in the same area attenuated
the number of vasopressinergic neurons expressing
c-Fos in the mPVN (46.4 ± 11.2 cells/section), without
changing the number of vasopressinergic neurons
expressing c-Fos in the pPVN (12.8 ± 4.6 cells/section)
and in the SON (92.3 ± 19.3 cells/section) (Fig. 5).
Carbachol combined with PBS injected into the MSA
also increased the number of neurons expressing
double c-Fos/OT labeling in the mPVN (75.1 ± 8.5, vs.
PBS + saline: 0.2 ± 0.1 cells/section – each 60 lm)
[H= 9.585; p= 0.022], pPVN (18.5 ± 3.2, vs.
PBS + saline: 0.1 ± 0.05 cells/section) [H= 8.658;
p= 0.034] and SON (102.7 ± 7.4, vs. PBS + saline:
0.6 ± 0.1 cells/section) [H= 9.359; p= 0.025] (Fig. 6).
The injection of H2O2 previously to carbachol into
the MSA also decreased the number of neurons
expressing double c-Fos/OT labeling in the mPVN
(38.5 ± 16.1 cells/section) and SON (57.8 ± 10.2 cells/
Fig. 3. Cumulative (A) sodium excretion, (B) potassium excretion, and (C) urinary volume in rats that received injections of H2O2 or PBS combined
with carbachol or saline into the MSA. The results are expressed as means ± SEM. Two-way repeated measures ANOVA combined with Student–
Newman–Keuls test; (p< 0.05); n= number of rats; carbachol (4 nmol/0.5 ll); saline (0.5 ll); H2O2 (2.5 lmol/0.5 ll); PBS (0.5 ll), phosphatebuffered saline.
Fig. 4. Changes in mean arterial pressure (DMAP) and heart rate
(DHR, inset) in rats that received injections of H2O2 or PBS combined
with carbachol into the MSA. One-way ANOVA combined with
Student–Newman–Keuls test; (p< 0.05); n= number of rats; car-
bachol (4 nmol/0.5 ll); H2O2 (2.5 lmol/0.5 ll); PBS (0.5 ll), phos-phate-buffered saline.
616 M. R. Melo et al. / Neuroscience 284 (2015) 611–621
section) without changing the number of neurons
expressing double c-Fos/OT labeling in the pPVN
(9.4 ± 6.5 cells/section) (Fig. 6).
Dipsogenic response, natriuresis and antidiuresis tocarbachol injected into the MSA in rats treated withglibenclamide combined with H2O2 in same area
The treatment with H2O2 into the MSA decreased
carbachol induced-water intake (8.6 ± 1.4, vs.
vehicle + PBS+ carbachol: 14.5 ± 1.4 ml/60 min)
[F(3,33) = 8.11; p< 0.05], antidiuresis (8.9 ± 0.3,
vs. vehicle + PBS+ carbachol: 6.4 ± 0.4 ml/120 min)
[F(3,21) = 1.03; p< 0.05] and natriuresis (399.5 ±
31.9, vs. vehicle + PBS+ carbachol: 605.8 ± 48.2 lEq/120 min) [F(3,21) = 3.15; p< 0.05] (Fig. 7A–C). The
pre-treatment with glibenclamide (5 nmol/0.5 ll) injectedinto the MSA abolished the inhibitory effects of H2O2 on
carbachol-induced water intake (12.2 ± 1.5 ml/60 min),
antidiuresis (7.4 ± 0.5 ml/120 min) and natriuresis
(549.3 ± 37.2 lEq/120 min) (Fig. 7A–C). The injection of
glibenclamide alone into the MSA did not change water
intake, antidiuresis and natriuresis induced by carbachol
injected into the MSA (Fig. 7A–C).
Cardiovascular responses to carbachol injected intothe MSA in rats treated with glibenclamide combinedwith H2O2 in same area
Baseline MAP and HR were 106 ± 2 mmHg and
383 ± 8 bpm (n= 24), respectively. The treatment
with H2O2 into the MSA decreased carbachol
induced-pressor response (24 ± 5, vs. vehicle +
PBS+ carbachol: 41 ± 5 mmHg) [F(11,60) = 18.25;
p< 0.05], (Fig. 7D). The pre-treatment with
glibenclamide (5 nmol/0.5 ll) injected into the MSA
Fig. 5. Upper panel: photomicrographs of coronal brain sections showing c-Fos expression in the vasopressinergic cells in the PVN and SON.
Lower panel: number of double-staining (c-Fos/AVP) in the magnocellular region of the PVN (mPVN), parvocellular region of the PVN (pPVN) and in
the SON in rats that received injections of saline or carbachol combined with PBS or H2O2 into the MSA. The results in the lower panel are
expressed as mean ± SEM. Kruskal–Wallis followed by Student–Newman–Keuls test (p< 0.05); n= number of rats; carbachol (4 nmol/0.5 ll);saline (0.5 ll); H2O2 (2.5 lmol/0.5 ll); PBS (0.5 ll), phosphate-buffered saline. Scale bar = 100 and 200 lm, respectively, higher magnification
and inset.
M. R. Melo et al. / Neuroscience 284 (2015) 611–621 617
abolished the inhibitory effects of H2O2 on carbachol-
induced pressor response (39 ± 3 mmHg) (Fig. 7D).
Changes in HR were not significantly different in rats
treated with vehicle + PBS+ carbachol (�81 ±
13 bpm), vehicle + H2O2 + carbachol (�38 ± 26 bpm),
glibenclamide + H2O2 + carbachol (�57 ± 18 bpm) or
glibenclamide + PBS+ carbachol (�54 ± 7 bpm) into
the MSA. The injection of glibenclamide or H2O2 alone
into the MSA did not change MAP or HR.
Sucrose and food intake in rats treated with H2O2 intothe MSA
To investigate if the injections of H2O2 into the MSA
produce nonspecific inhibitory effects, the effects of the
injections of H2O2 into the MSA on 2% sucrose intake
and food intake were also tested. Injections of H2O2
(2.5 lmol/0.5 ll) into the MSA did not affect 2% sucrose
intake (6.3 ± 1.0, vs. PBS: 5.9 ± 0.9 ml/120 min),
[F(1,8) = 0.14; p> 0.05], (Table 1) or food intake
(9 ± 3, vs. 10 ± 3 g/120 min), [F(1,8) = 2.32; p> 0.05]
(Table 2). However the injections of H2O2 (2.5 lmol/
0.5 ll) into the MSA decreased meal associated-water
intake (9.3 ± 1.1, vs. PBS: 12.1 ± 1.2 ml/120 min),
[F(1,8) = 8.52; p< 0.05)], (Table 2).
DISCUSSION
The data show that previous treatment with H2O2 in the
MSA attenuated the dipsogenic, antidiuretic, natriuretic
and pressor responses produced by carbachol also
injected into the MSA. The cholinergic activation of
the MSA increased c-Fos expression in the
vasopressinergic cells of the PVN and in the
oxytocinergic cells of the PVN and SON, responses that
were also reduced by the injection of H2O2 into the MSA.
Reduction of cholinergic-induced responses, like
carbachol-induced phosphoinositide hydrolysis by
Fig. 6. Upper panel: photomicrographs of coronal brain sections showing the c-Fos expression in oxytocinergic cells in the PVN and SON and
Lower panel: number of double-staining (c-Fos/OT) in the magnocellular region of the PVN (mPVN), parvocellular region of the PVN (pPVN), and in
the SON in rats that received injections of saline or carbachol combined with PBS or H2O2 into the MSA. The results in the lower panel are
expressed as mean ± SEM. Kruskal–Wallis combined with Student–Newman–Keuls test (p< 0.05); n= number of rats; carbachol (4 nmol/
0.5 ll); saline (0.5 ll); H2O2 (2.5 lmol/0.5 ll); PBS (0.5 ll), phosphate-buffered saline. Scale bar = 100 and 200 lm, respectively, higher
magnification and inset.
618 M. R. Melo et al. / Neuroscience 284 (2015) 611–621
phospholipase C in human neuroblastoma cells or the
secretory response in the isolated rat parotid gland was
seen with the pre-treatment with H2O2 in in vitropreparations (Li et al., 1996; Mata et al., 2008). The pres-
ent results showing that the treatment with H2O2 into the
MSA reduced the responses to cholinergic stimulation in
the MSA are comparable to those from previous studies,
and further demonstrate a central action of H2O2 reducing
the responses to central cholinergic activation, particularly
in the MSA.
The present study clearly demonstrated that the pre-
treatment with H2O2 in the MSA reduced water intake
induced by carbachol injected into the same area.
However, in order to test the specificity of H2O2 in
reducing cholinergic-induced responses into the MSA,
the effects of H2O2 injected into the MSA on other
ingestive behaviors like 2% sucrose or food intake were
also tested. The results showed that the injections of
H2O2 into the MSA did not modify 2% sucrose or food
intake, suggesting that the injections of H2O2 into the
MSA do not cause nonspecific inhibition of behavioral
responses. Although H2O2 injected into the MSA did not
affect food intake, meal associated water intake, a
response that is suggested to depend on cholinergic
pathways of the MSA (De Luca Junior et al., 1988), was
reduced by the injection of H2O2 into the MSA. Therefore,
the reduction of meal-associated water intake by H2O2
pre-treatment into the MSA is in accordance with the
reduction of carbachol-induced water intake in rats pre-
treated with H2O2 in the same area.
Studies have demonstrated projections from the
MSA to the PVN and SON and the involvement of
the septum in the regulation of paraventricular
vasopressinergic neurons by the subfornical organ in the
Fig. 7. Cumulative (A) water intake, (B) urinary volume (C) urinary sodium excretion and (D) changes in mean arterial pressure (DMAP) in animals
treated with the combination of glibenclamide, H2O2 and carbachol into the MSA. The results are expressed as mean ± SEM. Two-way repeated
measures ANOVA associated to Student–Newman–Keuls or One-way ANOVA associated to Student–Newman–Keuls (p< 0.05); n= number of
rats; glibenclamide (5 nmol/0.5 ll); carbachol (4 nmol/0.5 ll); H2O2 (2.5 lmol/0.5 ll); vehicle (0.5 ll).
Table 1. Cumulative 2% sucrose intake (ml) in rats treated with H2O2
(2.5 lmol/0.5 ll) into the MSA
Treatment/
time (min)
30 60 90 120
PBS 5.3 ± 0.7 5.7 ± 0.8 5.7 ± 0.9 5.9 ± 0.9
H2O2 3.7 ± 0.4 5.3 ± 0.8 5.9 ± 0.9 6.3 ± 1.0
Values are means ± SEM; n= 9; PBS, phosphate-buffered saline. (Two-away
ANOVA, followed by Student–Newman–Keuls, p> 0.05.)
Table 2. Food intake and meal-associated water intake after 24-h food-
deprivation in rats treated with H2O2 (2.5 lmol/0.5 ll) injected into the
MSA
Treatment/
time
30 min 60 min 90 min 120 min
Cumulative meal-associated water intake (ml)
PBS 3.8 ± 0.5 8.0 ± 0.7 11.0 ± 0.9 12.1 ± 1.2
H2O2 1.2 ± 0.5⁄ 5.5 ± 0.5⁄ 8.1 ± 0.9⁄ 9.3 ± 1.1⁄
Cumulative food intake (g)
PBS 4 ± 1 7 ± 2 9 ± 3 9 ± 3
H2O2 4 ± 1 7 ± 2 9 ± 3 10 ± 3
Values are means ± SEM; n= 9; PBS, phosphate-buffered saline.* Different from PBS (Two-away ANOVA, followed by Student–Newman–
Keuls, p< 0.05).
M. R. Melo et al. / Neuroscience 284 (2015) 611–621 619
rat (Oldfield et al., 1985; Tanaka et al., 1988). The
present study shows for the first time that cholinergic
activation in the MSA increases c-Fos expression in the
vasopressinergic cells of the PVN and in the oxytocinergic
cells of the PVN and SON, responses that were also
reduced by the treatment with H2O2 injected into the
MSA. Increased activity of vasopressinergic and oxytocin-
ergic cells in the PVN and/or SON and the release of the
respective hormones is probably the mechanism activated
by carbachol in the MSA to produce antidiuresis and natri-
uresis. Central cholinergic activation increases plasma
AVP and part of the antidiuresis induced by central
cholinergic stimulation, including that produced by MSA
activation, is attributed to AVP secretion, whereas the
natriuresis induced by central cholinergic stimulation is
suggested to depend on direct renal effects of OT and also
on OT action stimulating the secretion of ANP by the car-
diac myocytes (Hoffman et al., 1977; Tanaka et al., 1988;
Imai et al., 1989; Huang et al., 1995; Antunes-Rodrigues
et al., 2004). The pre-treatment with H2O2 in the MSA
reduced c-Fos expression in the vasopressinergic and
oxytocinergic cells in the magnocellular PVN and/or
SON. The reduced activity in these neurons probably
causes reduced antidiuresis and natriuresis to carbachol
injected into the MSA. In addition, H2O2 injected alone into
the MSA increased the diuresis, suggesting that H2O2
acting in the MSA may decrease the baseline secretion
of vasopressin.
Similar to previous studies (Colombari et al., 1992;
Barbosa et al., 1995), the present results show that injec-
tions of carbachol into the MSA increase MAP. The pres-
sor response to central cholinergic activation is suggested
to depend on AVP release and sympathoexcitation
620 M. R. Melo et al. / Neuroscience 284 (2015) 611–621
(Hoffman et al., 1977; Imai et al., 1989). Cholinergic acti-
vation of the MSA increased the activity of the vasopress-
inergic neurons in the PVN and the pre-treatment of the
MSA with H2O2, similarly reduced MSA carbachol-
induced pressor response and activation of vasopressin-
ergic neurons in the mPVN, which suggests that reduction
of vasopressin secretion is a possible reason for the
reduced pressor response in rats pre-treated with H2O2
in the MSA. Besides the secretion of AVP, the PVN is also
involved in the control of sympathetic activity. The pPVN
projects to the rostroventrolateral medulla (RVLM), which
is the main pre-motor nucleus that controls the sympa-
thetic activity, and also to the intermediolateral column
(IML) (Shafton et al., 1998; Antunes et al., 2006;
Guyenet, 2006). Cholinergic activation of the MSA also
increased the activity of the vasopressinergic neurons in
the pPVN, a response not modified by the pre-treatment
of the MSA with H2O2. Therefore, the reduction of AVP
secretion as a consequence of the reduced activity of
the mPVN is a possible explanation for the reduction of
carbachol-induced pressor response produced by the
pre-treatment with H2O2. However, with the present
results it is not possible to exclude also changes in sym-
pathetic activity as a cause for the reduced pressor
response when carbachol is combined with H2O2 in the
MSA.
Acting centrally, H2O2 may reduce the neuronal
excitability because of the inhibition of glutamate action,
increase of GABA release or K+ATP channel opening
(Zoccarato et al., 1990, 1995; Avshalumov et al., 2005;
Bao et al., 2005). The K+ATP channels are present in MSA
(Stefani and Gold, 1998; Allen and Brown, 2004) and the
previous treatment with glibenclamide (K+ATP channel
blocker) injected into the MSA partially reversed the inhib-
itory responses produced by H2O2 on water intake, antidiu-
resis, natriuresis and pressor response to carbachol. This
suggests that H2O2 may have an inhibitory action by open-
ing K+ channels. The K+ATP channel opening culminates in
K+ efflux, leaving the cell membrane hyperpolarized, which
opposes the excitation induced by carbachol, reducing the
responses to carbachol in the MSA.
CONCLUSION
The present results show that H2O2 injected into the MSA
inhibits the dipsogenic, natriuretic, antidiuretic and
pressor response induced by carbachol injected into the
same area by acting through K+ATP channels, which
attenuates central mechanisms activated by carbachol.
Acknowledgments—The authors thank Reginaldo C. Queiroz,
Silas P. Barbosa, Silvia Foglia for expert technical assistance,
Silvana A. D. Malavolta and Carla Molina for secretarial assis-
tance and Adriano P. de Oliveira for animal care. This research
was supported by public funding from Conselho Nacional de
Pesquisa (CNPq) and Fundacao de Amparo a Pesquisa do
Estado de Sao Paulo (FAPESP 2011/15340-6). This work is part
of the requirements to obtain a PhD degree by Mariana Rosso
Melo in the Graduate Program in Pharmacology at the Federal
University of Sao Paulo – SP/Brazil.
REFERENCES
Allen TG, Brown DA (2004) Modulation of the excitability of
cholinergic basal forebrain neurones by KATP channels.
J Physiol 554:353–370.
Antunes VR, Yao ST, Pickering AE, Murphy D, Paton JF (2006) A
spinal vasopressinergic mechanism mediates hyperosmolality-
induced sympathoexcitation. J Physiol 576:569–583.
Antunes-Rodrigues J, de CM, Elias LL, Valenca MM, McCann SM
(2004) Neuroendocrine control of body fluid metabolism. Physiol
Rev 84:169–208.
Avshalumov MV, Chen BT, Koos T, Tepper JM, Rice ME (2005)
Endogenous hydrogen peroxide regulates the excitability of
midbrain dopamine neurons via ATP-sensitive potassium
channels. J Neurosci 25:4222–4231.
Bao L, Avshalumov MV, Rice ME (2005) Partial mitochondrial
inhibition causes striatal dopamine release suppression and
medium spiny neuron depolarization via H2O2 elevation, not
ATP depletion. J Neurosci 25:10029–10040.
Barbosa SP, de Gobbi JI, Zilioli L, Camargo LA, Saad WA, Renzi A,
De Luca Junior LA, Menani JV (1995) Role of cholinergic and
adrenergic pathways of the medial septal area in the water intake
and pressor response to central angiotensin II and carbachol in
rats. Brain Res Bull 37:463–466.
Block ML, Fisher AE (1970) Anticholinergic central blockade of salt-
aroused and deprivation-induced drinking. Physiol Behav
5:525–527.
Colombari E, Saad WA, Camargo LA, Renzi A, De Luca Junior LA,
Menani JV (1992) AV3V lesion suppresses the pressor,
dipsogenic and natriuretic responses to cholinergic activation of
the septal area in rats. Brain Res 572:172–175.
De Luca Junior LA, Diniz DL, Antunes-Rodrigues J (1988) Effect of
atropine injection into the medial septal area on food-associated
drinking. Braz J Med Biol Res 21:573–575.
Donevan SD, Ferguson AV (1988) Subfornical organ and
cardiovascular influences on identified septal neurons. Am
J Physiol 254:R544–R551.
Guyenet PG (2006) The sympathetic control of blood pressure. Nat
Rev Neurosci 7:335–346.
Hoffman WE, Philips MI, Schmid PG, Falcon J, Weet JF (1977)
Antidiuretic hormone release and the pressor response to central
angiotensin II and cholinergic stimulation. Neuropharmacology
16:463–472.
Huang W, Lee SL, Sjoquist M (1995) Natriuretic role of endogenous
oxytocin in male rats infused with hypertonic NaCl. Am J Physiol
268:R634–R640.
Imai Y, Abe K, Sasaki S, Minami N, Munakata M, Yumita S,
Nobunaga T, Sekino H, Yoshinaga K (1989) Role of vasopressin
in cardiovascular response to central cholinergic stimulation in
rats. Hypertension 13:549–557.
Jones S, Yakel JL (1997) Functional nicotinic ACh receptors on
interneurones in the rat hippocampus. J Physiol 504(Pt
3):603–610.
Lauar MR, Colombari DS, De Paula PM, Colombari E, Cardoso LM,
De Luca LAJ, Menani JV (2010) Inhibition of central angiotensin
II-induced pressor responses by hydrogen peroxide.
Neuroscience 171:524–530.
Li X, Song L, Jope RS (1996) Cholinergic stimulation of AP-1 and NF
kappa B transcription factors is differentially sensitive to oxidative
stress in SH-SY5Y neuroblastoma: relationship to
phosphoinositide hydrolysis. J Neurosci 16:5914–5922.
Luiz AC, Saad WA, Camargo LA, Renzi A, De Luca Junior LA,
Menani JV (1991) Pressor, dipsogenic, natriuretic and kaliuretic
response to central carbachol in rats with lesion of the medial
septal area. Neurosci Lett 132:195–198.
Mata A, Marques D, Martinez-Burgos MA, Silveira J, Marques J,
Mesquita MF, Pariente JA, Salido GM, Singh J (2008) Effect of
hydrogen peroxide on secretory response, calcium mobilisation
and caspase-3 activity in the isolated rat parotid gland. Mol Cell
Biochem 319:23–31.
M. R. Melo et al. / Neuroscience 284 (2015) 611–621 621
Negro-Vilar A, Gentil CG, Covian M (1967) Alterations in sodium
chloride and water intake after septal lesions in rats. Physiol
Behav 2:167–170.
Oldfield BJ, Hou-Yu A, Silverman AJ (1985) A combined electron
microscopic HRP and immunocytochemical study of the limbic
projections to rat hypothalamic nuclei containing vasopressin and
oxytocin neurons. J Comp Neurol 231:221–231.
Paulin RF, Menani JV, Colombari E, De Paula PM, Colombari DS
(2009) Role of the medial septal area on pilocarpine-induced
salivary secretion and water intake. Brain Res 1298:145–152.
Rhee SG, Chang TS, Bae YS, Lee SR, Kang SW (2003) Cellular
regulation by hydrogen peroxide. J Am Soc Nephrol
14:S211–S215.
Rouse ST, Levey AI (1996) Expression of m1–m4 muscarinic
acetylcholine receptor immunoreactivity in septohippocampal
neurons and other identified hippocampal afferents. J Comp
Neurol 375:406–416.
Saad WA, Camargo LA, Netto CR, Gentil CG, Antunes-Rodrigues J,
Covian MR (1975) Natriuresis, kaliuresis and diuresis in the rat
following microinjections of carbachol into the septal area.
Pharmacol Biochem Behav 3:985–992.
Shafton AD, Ryan A, Badoer E (1998) Neurons in the hypothalamic
paraventricular nucleus send collaterals to the spinal cord and to
the rostral ventrolateral medulla in the rat. Brain Res
801:239–243.
Stefani MR, Gold PE (1998) Intra-septal injections of glucose and
glibenclamide attenuate galanin-induced spontaneous alternation
performance deficits in the rat. Brain Res 813:50–56.
Tanaka J, Saito H, Seto K (1988) Involvement of the septum in the
regulation of paraventricular vasopressin neurons by the
subfornical organ in the rat. Neurosci Lett 92:187–191.
Weinberg ED (1990) Cellular iron metabolism in health and disease.
Drug Metab Rev 22:531–579.
Zimmerman MC, Davisson RL (2004) Redox signaling in central
neural regulation of cardiovascular function. Prog Biophys Mol
Biol 84:125–149.
Zoccarato F, Cavallini L, Deana R, Alexandre A (1990) The action of
the glutathione transferase substrate, 1-chloro-2,4-dinitrobenzene
on synaptosomal glutathione content and the release of hydrogen
peroxide. Arch Biochem Biophys 282:244–247.
Zoccarato F, Valente M, Alexandre A (1995) Hydrogen peroxide
induces a long-lasting inhibition of the Ca(2+)-dependent
glutamate release in cerebrocortical synaptosomes without
interfering with cytosolic Ca2+. J Neurochem 64:2552–2558.
(Accepted 14 October 2014)(Available online 22 October 2014)