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: ocram@psicobio.epm.br (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.