Upload
rosana-alves
View
214
Download
2
Embed Size (px)
Citation preview
www.elsevier.com/locate/brainres
Brain Research 1058
Research Report
High and low rearing subgroups of rats selected in the open field
differ in the activity of K+-stimulated p-nitrophenylphosphatase
in the hippocampus
Rosana Alves, Jose Gilberto Barbosa de Carvalho, Marco Antonio Campana Benedito*
Universidade Federal de Sao Paulo, Departamento de Psicobiologia, Rua Botucatu 862, 1o˙andar, 04023-900 Sao Paulo, SP, Brazil
Accepted 5 August 2005
Available online 8 September 2005
Abstract
Na+/K+-adenosinetriphosphatase (Na+/K+-ATPase) is of paramount importance for the proper functioning of the organism. The enzyme is
involved in several aspects of brain function, such as the repolarization of the neuronal membranes and neurotransmitters uptake/release.
Therefore, individual differences in the activity of brain Na+/K+-ATPase may result in differences in the functioning of the brain, which, in
consequence, could lead to behavioral divergences. Individual differences in rearing, an exploratory behavior, have been shown to be
genetically determined. In rats, the inhibition of the activity of Na+/K+-ATPase was reported to induce changes in the exploratory behavior.
The aim of this work was to verify if subgroups of rats selected according to the number of rearings (high and low rearing subgroups) in the
open field test differ in the activity of Na+/K+-ATPase in brain regions. Adult, male outbred Wistar rats were selected in the open field test
according to the number of rearings in subgroups of high (HR) and low (LR) rearing responders. After a rest of about 20 days after the open
field session, HR and LR rats were sacrificed. In the first experiment, frontal cortex, striatum, brainstem, hippocampus and the amygdala
(including the overlying limbic cortex) were dissected. The reaction of dephosphorylation of Na+/K+-ATPase (K+ stimulated p-
nitrophenylphosphatase) was assayed in homogenates rich in synaptosomes. The results obtained showed a statistically significant higher
activity of K+ p-nitrophenylphosphatase only in the hippocampus of HR subgroup of rats. This result was replicated in two other subsequent
experiments with different HR and LR subgroups of rats selected at different times of the year. Our data suggest that the difference in the
activity of Na+/K+-ATPase in the hippocampus is innate and is involved in the expression of the rearing behavior.
D 2005 Elsevier B.V. All rights reserved.
Theme: Excitable membranes and synaptic transmission
Topic: Other ion channels
Keywords: Open field; Rearing; Na+/K+-ATPase; Hippocampus, brain region, rat
1. Introduction
Sodium/potassium-adenosinetriphosphatase (Na+/K+-
ATPase) is of paramount importance for the proper
functioning of the organism; for instance, mice lacking
one of the enzyme subunits die just after birth [14,24]. In
the nervous tissue, Na+/K+-ATPase moves Na+ ions out and
K+ ions into the neurons, re-establishing the differences in
0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2005.08.005
* Corresponding author. Fax: +55 11 5572 5092.
E-mail address: [email protected] (M.A.C. Benedito).
ions’ concentrations between intra and extracellular
medium, thus repolarizing the neurons’ membranes and
preparing them to fire upon stimulation [13]. Besides its
role on ions’ translocation, the enzyme is also involved in
other aspects of brain functioning, such as neurotransmitters
release and uptake [16,23,35,37]. On the other hand,
neurotransmitters regulate the activity of Na+/K+-ATPase
[12]. Therefore, differences in the activity of Na+/K+-
ATPase could be reflected in differences in the brain
electrical excitability leading to divergences in the behavior
of the individuals.
(2005) 178 – 182
R. Alves et al. / Brain Research 1058 (2005) 178–182 179
Open field is one of the most common behavioral tests
used in studies with rodents [28]. Behaviors assumed to
reflect distinct brain functions can be scored in an open field
session. Rearing, for instance, is a behavior considered to be
an indication of exploratory behavior [4], whereas larger
time and higher number of visits to the center of the open
field are considered to be measures related to a lower fear/
anxiety [25,26,28]. As already mentioned, differences in
brain excitability due to changes in Na+/K+-ATPase activity
may lead to changes in behavior. For instance, inhibition of
Na+/K+-ATPase with ouabain, a specific inhibitor of the
enzyme [13], induced seizures [1,3] and caused an increase
in the activity of rats in the open field [30]. There is a
paucity of data regarding the role of Na+/K+-ATPase in
behavior. Therefore, the aim of this study was to verify if
high (HR) and low (LR) rearing subgroups of rats selected
in the open field test would differ in the activity of K+-
stimulated p-nitrophenylphosphatase, which reflects K+-
dependent dephosphorylation of phosphorylated Na+/K+-
ATPase, in brain regions.
2. Materials and methods
2.1. Subjects and reagents
Naive adult (3 months old) outbred male Wistar rats from
our own colony were used to select subgroups of rats
showing high (HR) and low (LR) rearings number in the
open field test. The rats after weaning at 21 days old were
kept in polypropylene cages (60 � 50 � 22 cm) large
enough to support 5–6 rats. The rats were kept in a room
with lights (lights on from 07:00 a.m. to 07:00 p. m.) and
temperature (22 T 2 -C)-controlled environment until they
were tested in the open field and sacrificed. Food (Purina\lab chow) and tap water were available ad libitum until the
rats were sacrificed. Besides cage cleaning and food and
water delivery, the rats were not submitted to other
disturbances. In order to avoid possible influences of daily
variations [6], both open field sessions and sacrifice of the
rats were carried out between 01:00 and 05:00 p.m. All
reagents used were obtained from Sigma Chemical Com-
pany, St. Louis, MO, USA. This work was approved by our
institution ethics committee (proc.# 0938/03).
2.2. Open field selection
The open field used in this study has been described in
details elsewhere [6]. Briefly, it consists of a circular arena
(80 cm in diameter and 30 cm high) with the floor divided in
three concentric circles which are, in turn, divided in equal
segments. The open field apparatus is illuminated by 5 bulb
lights (60 W), and it is maintained in a room with no other
apparatus inside it.
On the day of the experiment, the rats were moved in the
morning from the stock room to another room close to the
open field. During the open field session, one rat at a time
was carried in a smaller cage to the open field room and was
submitted to a 3-min session. Ambulation (number of
segments crossed with the four paws) and the total number
of rearings were scored. HR (high rearing, �29 rearings)
and LR (low rearing, �19 rearings) subgroups of rats were
selected based in the mean number of rearings reported in
mice genetically selected for high and low rearing in the
open field [32,33].
2.3. Homogenate preparation
Twenty days after the open field session, the rats were
sacrificed by decapitation in another room. One rat at a time
was moved to the room and decapitated. The brain regions
(frontal cortex, striatum, brainstem, amygdala plus the
overlying limbic cortex and hippocampus) were dissected
rapidly on a cooled petri dish on crushed ice. The tissues
were weighed and kept frozen (�20 -C) until the
preparation of the homogenates.
The homogenates (2.5% W/V) were prepared in ice-cold
0.32 M sucrose (made pH 7.4 with 0.2 M Tris base) using a
glass homogenizer tube and a motor-driven Teflon pestle.
The homogenates were centrifuged at 1200 � g for 5 min at
4 -C. The supernatants were collected and centrifuged at
23,000 � g for 20 min at 4 -C. The supernatants were
discarded and the pellets resuspended in 50 mM Tris/HCl
pH 7.4 containing 1 mM EDTA. The samples were
centrifuged again at 23,000 � g for 20 min at 4 -C and
the supernatants discarded. Finally, the pellets were kept
frozen until assayed. All steps during the homogenate
preparation were carried out keeping solutions and materials
cold in a bath of crushed ice.
2.4. Enzymatic assay
The pellets were resuspended in 50 mM Tris/HCl pH 7.4
using a glass homogenizer tube and a motor-driven Teflon
pestle. K+ p-nitrophenylphosphatase (K+-dependent dephos-
phorylation of phosphorylated Na+/K+-ATPase) was
assayed spectrophotometrically according to Robinson
[29] with slight modifications. The final incubation volume
was 100 Al. Briefly, 50 Al of homogenate in 50 mM Tris/
HCl buffer pH 7.4 was pipetted into a small glass test tube,
and 50 Al of a solution of 50 mM Tris/HCl buffer pH 7.4
containing MgCl2 (5 mM final concentration in the assay),
p-nitrophenylphosphatase (10 mM final concentration in the
assay) and KCl was added. Another set of test tubes was
prepared omitting the KCl. The incubation in a water-
shaking bath lasted 10 min at 37 -C. After adding cold
trichloroacetic acid (10% W/V) to precipitate the proteins,
the test tubes were centrifuged, and an aliquot of the clear
supernatant was transferred into a test tube containing 600
Al of 1 M Tris-base solution. The samples were read in the
spectrophotometer at 410 nm wavelength. K+ p-nitro-
phenylphosphatase activity corresponds to the reaction in
Fig. 1. The activity of K+ p-nitrophenylphosphatase in the hippocampus
from subgroups of rats selected in the open field as high (HR) and low (LR)
rearing responders. The results are expressed as the means T SEM. n = 7 for
each subgroup. *P = 0.02, **P = 0.006, ***P = 0.002, two-tailed.
R. Alves et al. / Brain Research 1058 (2005) 178–182180
the presence of KCl (10 mM final concentration in the
assay) minus that in the absence of KCl. A standard curve
was prepared using p-nitrophenol. We have also assayed
hippocampal K+ p-nitrophenylphosphatase using a 3 mM
KCl final concentration in the assay.
The enzyme activity is expressed as nmol p-nitrophenol
formed/min mg protein. Enzyme activity was assayed in
triplicate and blanks (without KCl) in duplicate. The
reactions were carried out in the linear range for both
incubation time and protein concentration.
2.5. Protein determination
Protein was assayed by the method of Lowry et al. [19]
using bovine serum albumin as standard.
2.6. Statistical analysis
Data were statistically analyzed using the unpaired
Student’s t test for the parametric measures and the
Mann–Whitney U test for non-parametric measures with a
significance level set at P � 0.05, two-tailed.
3. Results
As it can be seen in Table 1, the highly statistically
significant difference in the mean number of rearings
between HR and LR subgroups of rats selected in the open
field was kept constant in the 3 selections (Exp. 1 P =
0.0002, Exp. 2 P = 0.0002, Exp. 3 P = 0.0003, Mann–
Whitney U test, two-tailed), and the number of rearings in
each subgroup was similar in the 3 selections carried out
months apart. In the 3 selections, a difference in ambulation
was also statistically significant between HR and LR
subgroups of rats (Exp. 1 P = 0.04, Exp. 2 P = 0.02, Exp.
3 P = 0.009, Mann–Whitney U test, two-tailed) (Table 1).
Fig. 1 shows the results obtained in the assay of the
enzyme in the hippocampus. As it can be seen in Fig. 1 in all
the 3 experiments carried out, HR subgroups of rats
presented a statistically significant higher enzymatic activity
(Exp. 1 P = 0.02. Exp. 2 P = 0.002, Exp. 3 P = 0.006,
unpaired Student’s t test, two-tailed). The statistical analysis
of the results obtained in the hippocampal enzymatic assay
using the 3 mMKCl concentration did not show a significant
difference between HR and LR subgroups of rats (HR 52.5 T
Table 1
The behavior of high (HR) and low (LR) rearing subgroups of rats selected in th
Exp. 1 Exp. 2
HR LR HR
Ambulation 51.4 T 4.2 (8) 36.6 T 5.4 (8) 67.6 T 6.2
P = 0.04 P = 0.02
Rearings 34.5 T 1.0 (8) 13.5 T 1.8 (8) 34.6 T 1.7
P = 0.0002 P = 0.0002
The data are expressed as the means T SEM. (n) number of rats.
3.1 nmol p-nitrophenol formed/min mg protein, mean TSEM (n = 8), LR 55.5 T 3.3 (n = 8), P > 0.05).
Table 2 shows the results of enzymatic activity in the
other brain regions assayed. As it can be seen in Table 2,
there were no statistically significant differences between
HR and LR subgroups of rats in enzyme activity.
Statistical analysis of the protein content in the resus-
pended pellets showed no statistically significant differences
between HR and LR subgroups of rats for all regions
assayed [brainstem: HR 55.7 T 2.0 Ag protein/50 Al, mean TSEM (n = 8); LR 55.6 T 1.1 (n = 8); striatum: HR 45.7 T 2.4(n = 8), LR 40.9 T 4.1 (n = 6); frontal cortex: HR 59.2 T 0.6
(n = 8), LR 63.6 T 5.0 (n = 6); amygdala: HR 55.0 T 3.5 (n =
8), LR 60.2 T 1.6 (n = 8); hippocampus: Exp. 1: HR 61.1 T1.6 (n = 7), LR 56.2 T 2.7 (n = 7); Exp. 2: HR 72.9 T 1.7
(n = 7), LR 70.7 T 1.7 (n = 7), Exp. 3: HR 57.3 T 2.4 (n =
7), LR 62.7 T 1.7 (n = 7); P > 0.05, unpaired Student’s t test,
two-tailed].
4. Discussion
The data obtained in this study showed a consistent
higher activity of K+-stimulated dephosphorylation of
phosphorylated Na+/K+-ATPase in the hippocampus of HR
subgroup of rats selected in the open field test. The higher
activity of Na+/K+-ATPase in the hippocampus of HR rats
e open field test
Exp. 3
LR HR LR
(8) 44.6 T 5.7 (8) 76.4 T 7.9 (7) 43.5 T 6.8 (8
P = 0.009
(8) 13.3 T 1.2 (8) 38.4 T 2.2 (7) 12.0 T 1.4 (8
P = 0.0003
)
)
Table 2
The activity of K+ p-nitrophenylphosphatase in the brain regions from high
(HR) and low (LR) rearing subgroups of rats selected in the open field test
Frontal cortex Striatum Amygdala Brainstem
HR 174.5 T 4.5 (8) 130.6 T 5.5 (8) 138.3 T 3.7 (8) 126.7 T 4.8 (8)
LR 171.9 T 7.3 (6) 129.4 T 4.2 (6) 136.8 T 3.8 (8) 123.6 T 5.4 (8)
The enzyme activity is expressed as nmol p-nitrophenol formed/min mg
protein (means T SEM). (n) number of rats. The enzymatic assays were
carried out in subgroups of HR and LR rats from Exp. 1.
R. Alves et al. / Brain Research 1058 (2005) 178–182 181
suggests, among other possibilities, a more efficient
repolarization of the cell membrane in HR subgroup of
rats. This result was obtained in 3 different experiments
carried out at different times of the year and using litters of
rats from our outbred Wistar strain born at different times of
the year. Therefore, the data obtained suggest that the
difference in the activity of the enzyme in the hippocampus
is innate and may be related to the rearing behavior and
consequently to exploratory behavior.
The criteria to select the subgroups of HR and LR rats
were based in the rearing number of the strains of mice
selected in the open field for high and low rearing behavior
[32,33]. The mean number of rearings in our subgroups of
rats is very similar to the mean number of rearings in those
selected mice strains. In our selection, as well as in the
selected mice strains, a relation between ambulation and
rearing was also observed, HR subgroup of rats ambulate
more in the open field.
Open field behaviors, such as rearing, have been shown
to be genetically determined [10,32,33]. Data in the
literature have been showing a correlation between rearing
behavior in the open field and hippocampal electrical
activity [34]. Moreover, novelty acquisition enhanced
long-term depression in hippocampal CA1 region of rats
[21], rats highly reactive to novelty have a lower cell
proliferation in the dentate gyrus and less cells within the
granule cell layer of the dentate gyrus than low reactive rats
[17], electrical stimulation of the hippocampus in rats
induces rearings [20], and hippocampal lesion in mice
decreases the number of rearings [5], and strains of selected
mice [11] or not selected [15] differ anatomically in the
hippocampus. All these data put together seem to strengthen
the possibility of an involvement of hippocampal Na+/K+-
ATPase activity in the difference in the rearing behavior in
the open field test between the HR and LR subgroups of
rats.
Na+/K+-ATPase are the assembly of two alpha, two beta
and a gamma subunit [2,13,18]. Based on the affinity to
ouabain, the existence of 3 alpha isoforms which are
highly expressed in the rat’s brain has been reported [18].
Both glia and neurons express more than one alpha
isoform [22]; however, the expression of alpha2-subunit
mRNA was characteristic of glia, whereas alpha3-subunit
transcripts were predominant in neurons [36]. In the
hippocampal neurons, alpha 1 and 3 subunits mRNA and
proteins are densely expressed [22,36], both in axons and
dendrites [27]. The gamma subunit seems to be involved
in the modulation of the activation of Na+/K+-ATPase by
K+ [2]. We have assayed K+ p-nitrophenylphosphatase in
the whole hippocampus; therefore, it is possible that larger
differences in enzyme activity can be circumscribed to
hippocampal regions differing in the expression of the
alpha subunits, which would lead to a higher difference in
enzyme activity between HR and LR subgroups of rats.
This possibility needs further experimentation to be
explored.
Recent quantitative trait loci studies in mice have
suggested the involvement of chromosome 1, which
contains genes for subunits of Na+/K+-ATPase, in the
susceptibility to convulsions [7,8,9] and in the rearing
behavior in the open field [10]. Therefore, these data
indicate a genetic control of the brain excitability promoted
by Na+-K+/ATPase and suggest a relation between rearing
behavior and the activity of Na+-K+/ATPase, indicating that
the difference obtained in our study may involve differ-
ences in the expression of Na+/K+-ATPase catalytic
subunits in the hippocampus of HR and LR subgroups of
rats.
It is worth mentioning that, besides its important role in
the maintenance of differences in the electrical potential
between intra and extracellular medium, Na+-K+/ATPase is
also involved in other important functions in the brain, such
as neurotransmitters uptake and release [23,35,37]. There-
fore, an innate difference in the activity of Na+/K+-ATPase
in the brain, as the one observed in our experiment, would
lead to differences in the concentrations of these ions intra
and extracellularly between HR and LR rats, which could
lead to differences in the uptake/release of neurotransmitters
at the synaptic cleft.
Extracellular potassium concentration [K+]0 has an
important role in the normal function of the central nervous
system (for review, see [31]). The ‘‘resting’’ [K+]0 is
considered to be around 3 mM, and repetitive electrical
stimulation of the tissue, or of an afferent pathway, can
easily drive [K+]0 from its ‘‘resting’’ level to 5–6 mM and
sometimes as high as 8 or even 10 mM. We measured the
activity of K+ p-nitrophenylphosphatase in the hippocampus
of HR and LR subgroups of rats using a 3 or 10 mM KCl
concentration. At 3 mM of K+, a difference in the enzyme
activity which occurred using a 10 mM concentration of K+
was not observed. These data suggest that in the hippo-
campus of HR rats the higher Na+/K+-ATPase activity is
more efficient in pumping extracellular K+ ions back into
the neurons when the [K+]0 reaches higher concentrations
under repetitive depolarization.
To conclude, we have showed a higher hippocampal
activity of Na+/K+-ATPase in the subgroup of rats of higher
rearings. These data suggest the involvement of hippo-
campal Na+/K+-ATPase in the rearing behavior test and
consequently in exploratory behavior. Further work is
necessary to better understand the present findings.
R. Alves et al. / Brain Research 1058 (2005) 178–182182
Acknowledgments
This work was supported by Associacao Fundo de
Incentivo a Psicofarmacologia (AFIP). Rosana Alves is
the recipient of a fellowship from Conselho Nacional de
Desenvolvimento Cientıfico e Tecnologico (CNPq).
References
[1] G. Bagetta, M. Iannone, E. Palma, P. Rodino, T. Granato, G.
Nistico, Lack of involvement of nitric oxide in the mechanisms
of seizures and hippocampal damage produced by kainate and
ouabain in rats, Neurodegeneration 4 (1995) 43–49.
[2] P. Beguin, X. Wang, D. Firsov, A. Puoti, D. Claeys, J.D. Horisberger,
K. Geering, The gamma subunit is a specific component of the Na,K-
ATPase and modulates its transport function, EMBO 16 (1997)
4250–4260.
[3] M.L. Brines, A.O. Dare, N.C. Lanerolle, The cardiac glycoside
ouabain potentiates excitotoxic injury of adult neurons in rat hippo-
campus, Neurosci. Lett. 191 (1995) 145–148.
[4] W.E. Crusio, Genetic dissection of mouse exploratory behavior,
Behav. Brain Res. 125 (2001) 127–132.
[5] R.M.J. Deacon, A. Croucher, J.N.P. Rawlins, Hippocampal cytotoxic
lesion effects on species-typical behaviours in mice, Behav. Brain Res.
132 (2002) 203–213.
[6] D.S. Eidman, M.A.C. Benedito, J.R. Leite, Daily changes in
pentylenetetrazol-induced convulsions and open-field behavior in rats,
Physiol. Behav. 47 (1990) 853–856.
[7] T.N. Ferraro, G.T. Golden, G.G. Smith, P.S. Jean, N.J. Schork, N.
Mulholland, C. Ballas, J. Schill, R.J. Buono, W. Berretini, Mapping
loci for pentylenetetrazol-induced seizure susceptibility in mice,
J. Neurosci. 19 (1999) 6733–6739.
[8] T.N. Ferraro, G.T. Golden, G.G. Smith, R.L. Longman, R.L. Snyder, D.
Demuth, I. Szpilzak, N. Mulholland, E. Eng, F.W. Lohoff, R.J. Buono,
W.H. Berretini, Quantitative genetic study of maximal electroshock
seizure threshold in mice: evidence for a major seizure susceptibility
locus on distal chromosome 1, Genomics 75 (2001) 35–42.
[9] T.N. Ferraro, G.T. Golden, G.G. Smith, J.F. Martin, F.W. Lohhoff,
T.A. Gieringer, D. Zamboni, C.L. Schwebel, D.M. Press, S.O. Kratzer,
H. Zhao, W.H. Berretini, R.J. Buono, Fine mapping of a seizure
susceptibility locus on mouse chromosome 1: nomination of Kcnj10
as a causative gene, Mamm. Genome 15 (2004) 239–251.
[10] H.K. Gershenfeld, P.E. Neumann, C. Mathis, J.N. Crawley, X.
Li, S.M. Paul, Mapping quantitative trait loci for open-field
behavior in mice, Behav. Genet. 27 (1997) 201–210.
[11] Z. Hausheer-Zarmakupi, D.P. Wolfer, M.C. Leisinger-Trigona, H.P.
Lipp, Selective breeding for extremes in open-field activity of mice
entails a differentiation of hippocampal mossy fibers, Behav. Genet.
26 (1996) 167–176.
[12] J.R. Hernandez, Na+/K+-ATPase regulation by neurotransmitters,
Neurochem. Int. 20 (1992) 1–10.
[13] J.D. Horisberger, V. Lemas, J.P. Kraehenbuhl, B.C. Rossier, Structure–
function of Na,K-ATPase, Annu. Rev. Physiol. 53 (1991) 565–584.
[14] K. Ikeda, T. Onaka, M. Yamakado, J. Nakai, T. Ishikawa, M.M. Taketo,
K. Kawakami, Degeneration of the amygdala/piriform cortex and
enhanced fear/anxiety behaviors in sodium pump alpha2 subunit(AT-
P1alpha2)-deficient mice, J. Neurosci. 23 (2003) 4667–4676.
[15] A. Laghmouch, J.Y. Bertholet, W.E. Crusio, Hippocampal morphol-
ogy and open-field behavior in Mus musculus domesticus and Mus
spretus inbred mice, Behav. Genet. 27 (1997) 67–73.
[16] G.L. Lees, Inhibition of sodium–potassium-ATPase: a potentially
ubiquitous mechanism contributing to central nervous system neuro-
pathology, Brain Res. Rev. 16 (1991) 283–300.
[17] V. Lemaire, C. Aurousseau, M. Le Moal, D.N. Abrous, Behavioural
trait of reactivity to novelty is related to hippocampal neurogenesis,
Eur. J. Neurosci. 11 (1999) 4006–4014.
[18] J.B. Lingrel, Na,K-ATPase: isoform structure, function, and expres-
sion, J. Bioenerg. Biomembr. 24 (1992) 263–270.
[19] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein
measurement with the Folin phenol reagent, J. Biol. Chem. 193
(1951) 265–275.
[20] J. Ma, L.W.S. Leung, Medial septum mediates the increase in post-
ictal behaviors and hippocampal gamma waves after an electrically
induced seizure, Brain Res. 833 (1999) 51–57.
[21] D. Manahan-Vaughan, K.H. Braunewell, Novelty acquisition is
associated with induction of hippocampal long-term depression, Proc.
Natl. Acad. Sci. U. S. A. 96 (1999) 8739–8744.
[22] K.M. McGrail, J.M. Phillips, K.J. Sweadner, Immunofluorescent
localization of three Na,K-ATPase isozymes in the rat central nervous
system: both neurons and glia can express more than one Na,K-
ATPase, J. Neurosci. 11 (1991) 381–391.
[23] E.M. Meyer, J.R. Cooper, Correlations between Na+-K+ ATPase
activity and acetylcholine release in rat cortical synaptosomes,
J. Neurochem. 39 (1981) 467–475.
[24] A.E. Moseley, S.P. Lieskes, R.K. Wetzel, P.F. James, S. He, D.A.
Shelly, R.J. Paul, G.P. Boivin, D.P. Witte, J.M. Ramirez, K.J.
Sweadner, J.B. Lingrel, The Na,K-ATPase alpha2 isoform is expressed
in neurons, and its absence disrupts neuronal activity in newborn mice,
J. Biol. Chem. 278 (2003) 5317–5324.
[25] M. Nazar, M. Jessa, A. Plaznik, Benzodiazepine–GABAA receptor
complex ligands in two models of anxiety, J. Neural Transm. 104
(1997) 733–746.
[26] M. Nazar, M. Siemiatkowski, A. Czlonkowka, H. Sienkiewicz-Jarosz,
A. Plaznik, The role of the hippocampus and 5-HT/GABAA
interaction in the central effects of benzodiazepine receptor ligands,
J. Neural Transm. 106 (1999) 369–381.
[27] G. Pietrini, M. Matteoli, G. Banker, M.J. Caplan, Isoforms of the
Na,K-ATPase are present in both axons and dendrites of hippo-
campal neurons in culture, Proc. Natl. Acad. Sci. U. S. A. 89 (1992)
8414–8418.
[28] L. Prut, C. Belzung, The open field as a paradigm to measure the
effects of drugs on anxiety-like behaviors: a review, Eur. J. Pharmacol.
463 (2003) 3–33.
[29] J.D. Robinson, Kinetic studies on a brain microsomal adenosinetri-
phosphatase II. Potassium-dependent phosphatase activity, Biochem-
istry 8 (1969) 3348–3355.
[30] D.J. Ruktanonchai, R.S. El-Mallakh, R. Li, R.S. Levy, Persistent
hyperactivity following a single intracerebroventricular dose of
ouabain, Physiol. Behav. 63 (1998) 403–406.
[31] G.G. Somjen, Extracellular potassium in the mammalian central
nervous system, Annu. Rev. Physiol. 41 (1979) 159–177.
[32] J.H.F. Van Abeelen, Genetic analysis of behavioural responses to
novelty in mice, Nature 254 (1975) 239–241.
[33] J.H.F. Van Abeelen, Rearing responses and locomotor activity in mice:
single locus control, Behav. Biol. 19 (1977) 401–404.
[34] H. Van Lier, A.M.L. Coenen, W.H.I.M. Drinkenburg, Behavioral
transitions modulate hippocampal electroencephalogram correlates of
open field behavior in the rat: support for a sensorimotor function of
hippocampal rhythmical synchronous activity, J. Neurosci. 23 (2003)
1459–1465.
[35] E.S. Vizi, B. Sperlagh, Separation of carrier mediated and vesicular
release of GABA from rat brain slices, Neurochem. Int. 34 (1999)
407–413.
[36] A.G. Watts, G. Sanchez-Watts, J.R. Emanuel, R. Levenson, Cell-
specific expression of mRNA encoding Na+,K+-ATPase alpha- and
beta-subunit isoforms within the rat central nervous system, Proc.
Natl. Acad. Sci. U. S. A. 88 (1991) 7425–7429.
[37] B.H.C. Westerink, G. Damsma, J.B. de Vries, Effect of ouabain applied
by intrastriatal microdialysis on the in vivo release of dopamine,
acetylcholine, and amino acids in the brain of conscious rats, J.
Neurochem. 52 (1989) 705–712.