Progress in Neuro-Psychopharmacology & Biological Psychiatry 45 (2013) 11–20

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Progress in Neuro-Psychopharmacology & Biological

Psychiatry
journal homepage: www.elsevier.com/locate/pnp

Cognitive improvement by acute growth hormone is mediated by

NMDA and AMPA receptors and MEK pathway
Margarita Ramis a, Fiorella Sarubbo a, Jessica Sola a, Sara Aparicio a, Celia Garau b,
Antonio Miralles a, Susana Esteban a,⁎
a
Laboratorio de Neurofisiología, Departamento de Biología, Universitat de les Illes Balears (UIB), Cra. Valldemossa km 7.5, E-07122 Palma de Mallorca, Spain
b
Department of Pharmaceutical Sciences, University of California, Irvine, CA, United States

a r t i c l e i n f o a b s t r a c t

Article history: It has been reported that Growth hormone (GH) has an immediate effect enhancing excitatory postsynaptic
Received 15 February 2013 potentials mediated by AMPA and NMDA receptors in hippocampal area CA1. As GH plays a role in adult
Received in revised form 5 April 2013 memory processing, this work aims to study the acute effects of GH on working memory tasks in rodents
Accepted 9 April 2013
and the possible involvement of NMDA and AMPA receptors and also the MEK/ERK signalling pathway. To
Available online 13 April 2013
evaluate memory processes, two different tests were used, the spatial working memory 8-arm radial maze,
Keywords:
and the novel object recognition as a form of non-spatial working memory test. Acute GH treatment
AMPA (1 mg/kg i.p., 1 h) improved spatial learning in the radial maze respect to the control group either in
ERK young rats (reduction of 46% in the performance trial time and 61% in the number of errors), old rats (reduction
Growth hormone of 38% in trial time and 48% in the number of errors), and adult mice (reduction of 32% in the performance time
NMDA and 34% in the number of errors). GH treatment also increased the time spent exploring the novel object respect
Object recognition to the familiar object compared to the control group in young rats (from 63% to 79%), old rats (from 53% to 70%),
Radial maze and adult mice (from 61 to 68%). The improving effects of GH on working memory tests were blocked by the
NMDA antagonist MK801 dizocilpine (0.025 mg/kg i.p.) injected 10 min before the administration of GH, in
both young and old rats. In addition, the AMPA antagonist DNQX (1 mg/kg i.p.) injected 10 min before the
administration of GH to young rats, blocked the positive effect of GH. Moreover, in mice, the MEK inhibitor SL
327 (20 mg/kg i.p.) injected 30 min before the administration of GH, blocked the positive effect of GH on radial
maze and the novel object recognition. In conclusion, GH improved working memory processes through both
glutamatergic receptors NMDA and AMPA and it required the activation of extracellular MEK/ERK signalling
pathway. These effects could be related to the enhancement of excitatory synaptic transmission in the hippocampus
reported by GH.
© 2013 Elsevier Inc. All rights reserved.

1. Introduction WM declines in late adulthood (Bopp and Verhaeghen, 2005;

Working memory (WM) is the ability to maintain and manipulate
information over short periods of time, a key function for several
higher-order cognitive functions from childhood to old age (for review
see Klingberg, 2010).
Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
DNQX, 6,7-dinitroquinoxaline-2,3-dione; ERK, extracellular signal-regulated kinase;
GABAA, γ-aminobutyric acid type A; GH, growth hormone; MAPK, mitogen activated
protein kinase; MEK, MAPK/ERK kinase; MK801, dizocilpine; NMDA, N-methyl-D-aspartate;
SL 327, α-amino-(4-aminophenyl-thio)-methylene-2-(trifluoromethyl)-benzene-
acetonitrile; WM, working memory.
⁎ Corresponding author.
E-mail addresses: margaramis87@hotmail.com (M. Ramis),
fioresarubbo@hotmail.com (F. Sarubbo), jessik_ss@hotmail.com (J. Sola),
sara.aparicio@alvarez.es (S. Aparicio), cgarauga@uci.edu (C. Garau), amiralles@uib.es
(A. Miralles), susana.esteban@uib.es (S. Esteban).
Borella et al., 2008; Payer et al., 2006) and is considered to be one
of the main contributing factors of cognitive impairment in older
adults (Park et al., 2002) and many neuropsychiatric disorders. It is
thus not surprising that attempts to improve WM have been far
explored and a large number of studies have been investigating the
trainability of WM across the lifespan (Morrison and Chein, 2011;
Shipstead et al., 2010; Takeuchi et al., 2010 among others).
Growth hormone (GH, somatotropin) plays an important role in
the central nervous system throughout life, being essential for normal
growth and development of the nervous system (Harvey and Hull,
2003; Noguchi, 1996; Scheepens et al., 2000). There is growing
evidence that GH might influence some functions of the central
nervous system (CNS) including its effects on cognitive functions,
memory, mood, neuroprotection, sleep, and well-being (Nyberg, 2000).
Decreased GH secretion in some physiological or pathophysiological
conditions has been associated with cognitive impairments (Aleman
et al., 2000; de Boer et al., 1995; Deijen et al., 1996; Hoffman et al.,

0278-5846/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.pnpbp.2013.04.005
12 M. Ramis et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 45 (2013) 11–20
1992; Nyberg, 2000), which was often resolved following GH replace-
ment (Arwert et al., 2005a, 2005b, 2006). GH deficiency during adoles-
cence has negative effects on learning and memory that emerge by
middle age unless prevented by GH supplementation (Nieves-Martinez
et al., 2010). The age-related decline in GH is one of the most robust
endocrine markers of biological ageing and has been hypothesized to
contribute to the physiological deficits observed in aged subjects.
Consequently, GH treatment may attenuate effects of ageing on
memory (Esteban et al., 2010b; Ramsey et al., 2004; Thornton et al.,
2000) and improves attention performance in adult patients suffering
from hypopituitarism with GH deficiency (Oertel et al., 2004). Although
the primary source of GH is the anterior pituitary, there are other sites
for GH production, including the hippocampus (Gossard et al., 1987;
Hojvat et al., 1982). Moreover the hippocampus contains GH receptors
(Burton et al., 1992; Lobie et al., 1993; Mustafa et al., 1994), and hippo-
campal function is affected by GH administration or deficiency (Le
Greves et al., 2002; Ramsey et al., 2004; van Dam et al., 2000). Therefore,
the effects of GH on memory process may be mediated by the hippo-
campus (Nyberg, 2000).
The effects of GH on hippocampal function may occur as a result of
modulating glutamatergic synaptic transmission in as much as facili-
tation of glutamatergic transmission causes a general improvement in
memory encoding (Staubli et al., 1994). Glutamate is the primary
excitatory neurotransmitter in the hippocampus and glutamatergic
synaptic transmission involves the activation of ionotropic receptors
which include the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) and the N-methyl-D-aspartate (NMDA) types of glutamate
receptors (Hollmann and Heinemann, 1994). NMDA receptor is
known to be essential for the induction of synaptic plasticity and mem-
ory formation (Cao et al., 2007; Tang et al., 1999; Tsien et al., 1996) and
the levels of these hippocampal receptors are related to performance on
the WM task (Adams et al., 2001). In the same way, activation of AMPA
receptors selectively increases hippocampal cell firing and it improves
both acquisition performance and memory retention (Granger et al.,
1996; Lynch, 2006).
Recent studies show that GH improved the excitatory postsynap-
tic potentials mediated by both AMPA and NMDA receptors in hippo-
campal slices, without alter the γ-aminobutyric acid type A (GABAA)
receptor-mediated inhibitory synaptic transmission (Mahmoud and
Grover, 2006; Molina et al., 2012). As this enhanced signal detection
by glutamate receptors should improve learning and memory, the
present study aims to investigate whether the GH inducing effects
on hippocampal synaptic excitability through AMPA and NMDA
receptors positively correlate with functional effects on working mem-
ory processes. Accordingly, the present study assessed the acute effects
of GH on working memory tasks in young and old rats and middle-age
mice. The promising involvement of AMPA and NMDA receptors on the
acute effects of GH on working memory was investigated in young rats.
Also, the involvement of NMDA receptors on the acute effects of GH was
explored in old rats. In addition, the implication of the ERK (extracellu-
lar signal-regulated kinase, subfamily of MAPS kinase) signalling path-
way was evaluated in mice because several behavioural memory
paradigms have implicated ERK as an essential component of the signal
transduction mechanisms involved in memory formation (Sweatt,
2004). To evaluate memory processes, two different hippocampal-
dependent tests were used, the spatial working memory 8-arm radial
maze, and the novel object recognition test as a form of non-spatial
working memory.
2. Methods and materials
2.1. Animals
Animals were initially housed in the animal facilities in transparent
methacrylate cages (Panlab s.l., Barcelona) with wood shavings for
nesting material (Ultrasorb, Panlab) under controlled environmental
conditions of temperature (22 ± 2 °C), humidity (70%) and light/dark
cycle (light period: 8:00 a.m. to 20:00 p.m.). They had free access to a
standard diet (Panlab A04) and tap water. The animals were handled
daily for several days before the behavioural experiments (see below)
to reduce stress and aggressiveness that may otherwise arise during
testing. Every effort was made to minimize the number of animals
used and their discomfort. Experimental procedures were conducted
according to standard ethical guidelines (European Communities
Council Directive 86/609/EEC) and approved by the Local Bioethical
Committee of the University of the Balearic Islands (UIB).
2.2. GH treatment and effect of AMPA and NMDA receptor blockade
Young and old male Wistar rats (weighing 280 ± 15 and 500 ± 20 g,
respectively) from Charles River (Barcelona, Spain) were used. GH
hormone can cross the blood–brain barrier in specific regions (Nyberg,
2000). Thus, rats were treated with saline or GH (1 mg/kg dissolved in
0.9% saline, i.p.) 1 h before the working memory tests. The dose of GH
and route for the administration was selected from previous works
(Jaworek et al., 2009). Different groups of rats received the AMPA antag-
onist DNQX (1 mg/kg dissolved in dimethyl sulfoxide, i.p.) or the NMDA
antagonist MK801 dizocilpine (0.025 mg/kg dissolved in 0.9% saline i.p.)
10 min before the administration of GH in order to blockade the respec-
tive receptors. Also, the acute effect of DNQX and MK801 alone were
analysed on memory tests 70 min after the treatment.
2.3. GH treatment and in vivo inhibition of ERK activation
Male Swiss albino CD1 middle-age mice (weighing 30 ± 6 g)
(Charles River, Barcelona) were used. Groups of mice were treated
with saline and GH (1 mg/kg i.p.) 1 h before the working memory
tests. A different group of mice was injected with the MEK inhibitor
SL 327 (20 mg/kg dissolved in dimethyl sulfoxide, i.p.) 30 min before
the administration of GH to compare with the effect of GH alone in
mice. The dose of SL327 and route for the administration were select-
ed from previous works performed to assess the optimal dose of SL
327 that would block substantially ERK1/2 phosphorylation without
altering other proteins in the rat brain (Moranta et al., 2007). We
observed previously that SL 327 (10, 20 and 30 mg/kg, i.p., 90 min)
decreased dose-dependently the content of p-ERK1/2 and increased
that of p-MEK1/2 (Ramos-Miguel et al., 2010). In addition, SL 327
alone (at the dose of 20 mg/kg) produced no changes in behaviour
or motor activity (data not shown).
2.4. Radial maze task
Radial maze test was measured as previously published to test
spatial working memory in rats (Esteban et al., 2010a, 2010b). The
8-arm radial maze (Panlab, S.L., Barcelona, Spain) consisted of an
octagonal central platform (32 cm diameter) with eight equally
spaced radial arms (50 cm long, 12 cm wide). To test memory on
radial maze, animals were allowed to visit the arms to obtain food
pellets until the eight arms had been visited or 20 min had elapsed.
Animals were previously submitted to 48 h fasting to achieve a con-
venient motivational level. Thus, trials were judged complete when
animals had visited all eight arms or spent 20 min in the trial
(maxim time to achieve performance). The maze was set in an exper-
imental room with several external visual cues. The experimenter
monitored the movements of the animals via a digital video tracking
system (LE 8300 with software SEDACOM v 1.3, Panlab, SL, Barcelona
Spain). The sum of non-visited arms and re-entry into arms was
scored as a working memory error. The apparatus was cleaned with
ethanol solution between trials. All procedures were performed dur-
ing the light period.
 M. Ramis et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 45 (2013) 11–20
2.5. Novel object recognition test
The novel object recognition, based on spontaneous exploratory
activity, is a test to measure a form of non-spatial working memory
in rats and mice (Clark et al., 2000; Okamura et al., 2011). The novel
object recognition test was performed in the open field device (cylinder
diameter: 78 cm, height walls: 60 cm). Animals were habituated to the
open field without objects for 10 min daily for four consecutive days. On
day 5, each animal was placed in the apparatus and allowed to explore
for 1 min for re-habituation. Then, in the training phase, animals were
placed in the centre of the apparatus with two identical objects placed
16 cm away from the walls and allowed to explore both objects until
to accumulate 30 s of exploration time or 10 min had elapsed. Object
exploration was recorded by an experienced observer only when the
animal's nose or mouth was in contact with the object. After the delay
(10 min, short term memory), in the test phase, the animals were
placed back in the open field with one object familiar (the same as the
previous phase) and one novel object. The time spent exploring the
objects was recorded until each animal accumulated 30 s of object
exploration or 10 min had elapsed. The test phase reflects the prefer-
ence for the novel object. Recognition index for the novel object was
calculated as the ratio between time spent exploring the novel object
with respect to overall exploring time, expressed by TN/(TN + TF)
ratio [TF = time spent exploring familiar object; TN = time spent
exploring the novel object]. During training session both objects are
novel, the time spent on both objects should be similar and the recogni-
tion index should be around 0.5.
The objects were always placed in the same location, 16 cm from the
wall and 37 cm apart. Velcro into the base of the objects was used to
secure the objects in place. In addition, the objects to be explored
were made of plastic in three different shapes that had no significance
for animals and had never been associated with reinforcement. The
objects and the apparatus were cleaned with ethanol solution between
trials. All procedures were performed during the light period.
2.6. Statistical analysis
All series of data were analysed with the program Graph-Pad
Prism, version 3.0. Results are expressed as mean ± SEM. One-way
and two-way analyses of variance (ANOVA), followed by post hoc
test were used for the statistical evaluations. The level of significance
was p b 0.05.
2.7. Drugs
AMPA receptor antagonist DNQX and NMDA receptor antagonist
(+)-MK801 maleate dizocilpine (Sigma-Aldrich chemical company,
St. Louis, MO, USA). Growth hormone (recombinant human GH; Saizen,
Serono, Spain). The selective MEK (MAPK/ERK kinase) inhibitor SL327 α-
amino-(4-aminophenyl-thio)-methylene-2-(trifluoromethyl)-benzene-
acetonitrile (Tocris Cookson Ltd. Avonmouth, United Kingdom). Other
chemicals were from Sigma.
3. Results
3.1. Acute effects of GH on working memory tasks in rodents
The effect of acute treatment with GH on working memory pro-
cesses was investigated through two different tests, the eight-arm
radial maze for spatial memory, and the novel object recognition
test as a form of non-spatial working memory task. The radial maze
performance is shown in Figs. 1, 3 and 5, and results from the novel
object recognition test in Figs. 2, 4 and 6. A significant improvement
in memory processes was observed after the acute administration of
GH (1 mg/kg, i.p., 1 h) in young and old rats, and mice (Figs. 1 to 6).
13
First, impairment of working memory was observed in old control
rats compared to younger animals in the radial maze (Fig. 1).
Two-way ANOVA for time performance on the radial maze detected sig-
nificant differences between ages (F(1,63) = 162, p b 0.0001) and treat-
ments (saline, GH and MK801 + GH) (F(2,63) = 31.04, p b 0.0001), but
not for the interaction between ages and treatments (F(2,63) = 2.57,
p = 0.0846). Similarly, two-way ANOVA for errors number on the
radial maze detected significant differences between ages (F(1,63) =
22.78, p b 0.0001) and treatments (saline, GH and MK801 + GH)
(F(2,63) = 17.08, p b 0.0001) but not for the interaction between ages
and treatments (F(2,63) = 1.15, p = 0.3224). The analysis indicated
that the effect of GH was independent of age. Old control rats need
more time to complete the trial and commit more errors than young
control animals in the maze test (p b 0.0001) (Fig. 1). The acute admin-
istration of GH significantly improved the spatial memory in the radial
maze respect to the control group in both young and old rats (Fig. 1).
Two-way ANOVA followed by Bonferroni test showed that GH reduced
the time required to complete the test respect to the control in both
young (46% reduction, p b 0.001, Fig. 1A) and aged rats (38% reduction,
p b 0.001, Fig. 1B). Also, GH-treated rats reduced the number of errors
in the radial maze (no visited plus repeated visited arms) respect to
the corresponding control group in both young (61% reduction,
p b 0.001, Fig. 1C), and aged rats (48% error reduction, p b 0.001,
Fig. 1D).
Furthermore, deterioration in performance on the object recogni-
tion task was observed with age (Fig. 2). When compare the time
spent exploring the novel object (novel-familiar), two-way ANOVA
detected significant differences between ages (F(1,70) = 11.58, p b
0.0011) and treatments (saline, GH and MK801 + GH) (F(2,70) =
16.04, p b 0.0001), but not for the interaction between ages and
treatments (F(2,70) = 1.58, p = 0.2127) (Fig. 2C, D). The analysis in-
dicated that the effect of GH was independent of age. Young rats
spent more time exploring the novel object than old rats in the test
session (p = 0.0013) (Fig. 2A, B). As observed in Table 1, during
training session, the time spent on both objects was similar and the
recognition index was around 0.5. An increase in the object recogni-
tion index from training phase to test session is observed in all ani-
mals except to the old control group. The novel object recognition
test was significantly improved by acute administration of GH
(1 mg/kg, i.p., 1 h) in young and old rats (Table 1, Fig. 2). Thus,
two-way ANOVA followed by Bonferroni test showed that GH admin-
istered sixty minutes prior to the training session, significantly in-
creased the time spent exploring the novel object in both young
(from 63% to 79% of total exploring time, p b 0.001, Fig. 2A,C) and
old animals (from 53% to 70% of total exploring time, p b 0.001,
Fig. 2B,D).
3.2. The effect of blockade of NMDA receptors on GH-induced working
memory improvement
The improving effects of GH (1 mg/kg, i.p., 1 h) on spatial memory
in the radial maze were blocked by the administration of MK801
(0.025 mg/kg, i.p.) before GH (1 mg/kg, 10 min apart). Accordingly
the time spent to complete the trial was similar to control rats but
significantly higher respect to the rats treated with GH alone in
both young (p b 0.001; two-way ANOVA followed by Bonferroni
test, Fig. 1A) and old rats (p b 0.001; two-way ANOVA followed by
Bonferroni test, Fig. 1B). Also, the number of errors during the perfor-
mance of task was higher in rats treated with MK801 plus GH than in
rats treated with GH alone in both young (p b 0.001; two-way
ANOVA followed by Bonferroni test, Fig. 1C) and old rats (p b 0.001;
two-way ANOVA followed by Bonferroni test, Fig. 1D). The adminis-
tration of MK801 alone (0.025 mg/kg, 70 min before the test) did
not cause any effect on radial maze test compared to the correspond-
ing control young or old group (Table 2). Table 2 shows the effects of
different doses of MK801 in young and old rats on the radial maze.
14 M. Ramis et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 45 (2013) 11–20

A) B)
Young rats Old rats
20 20

Time performance (min)

Time performance (min)

15 15

10 10

5 5

0 0
Control GH MK+GH Control GH MK+GH

C) D)
20 Young rats 20 Old rats

Total errors (number)

15 15
Total errors (number)

10 10

5 5

0 0
Control GH MK+GH Control GH MK+GH

Fig. 1. Acute effects of growth hormone treatment (GH, 1 mg/kg, i.p., 1 h before the test) in young and old rats on working memory task by means of the eight-arm radial maze test,
compared with the corresponding young or old control group. The effect of GH was blocked by previous administration of the NMDA receptor antagonist MK801 (MK, 0.025 mg/kg
dissolved in saline). The drugs were injected (i.p.) alone or in combination 10 min apart (MK followed by GH). Control rats received vehicle (1 ml/kg of saline i.p.). MK801 administered
alone did not induce significant changes at the mentioned dose (data not shown, see Table 1 for the effects of MK801 at different doses). More details in the Methods and materials section.
Bars represent means ± SEM derived from different animals of the time spent to complete the test (A, B), and the sum of non-visited arm or re-visited arm which was considered as total
errors made during the task (C, D). Two-way ANOVA detected significant differences between ages and treatments but not detected significant differences for the interaction between
ages and treatments. Two-way ANOVA followed by Bonferroni test was used for statistical evaluation, ***p b 0.001 when compared with the corresponding control group;
†††p b 0.001 when compared with the GH group.

From the results obtained in this previous study, it was selected the
dose of 0.025 mg/kg for analysing the effect of NMDA receptor block-
ade on the improvement of working memory induced by GH.
Moreover, the improving effects of GH on the novel object recognition
test were blocked by previous administration of MK801 (0.025 mg/kg).
Accordingly, the time spent exploring the novel and the familiar objects
was similar in the group of rats treated with MK801 plus GH than in con-
trol rats, but significantly lower respect to the rats treated with GH alone
in both young (p b 0.001; two-way ANOVA followed by Bonferroni
test, Fig. 2A, C) and old rats (p b 0.001; two-way ANOVA followed by
Bonferroni test, Fig. 2B, D). Accordingly, as observed in Table 1, no signif-
icant differences respect to the corresponding control group were
observed on the object recognition index after MK801 (0.025 mg/kg,
70 min before the training) or MK801 followed by GH.
These results suggest an implication of NMDA receptors in medi-
ating the effects of GH on working memory.
3.3. The effect of blockade of AMPA receptors on GH-induced working
memory improvement
The effect of blockade of AMPA receptors on working memory was
analysed in a group of young rats. The improving effects of GH (1 mg/kg,
i.p., 1 h) on spatial memory in the radial maze were blocked by the
administration of DNQX (1 mg/kg, i.p.) 10 min before GH. As previously
observed, the acute administration of GH (1 mg/kg, i.p., 1 h) significantly
improved the spatial memory in the radial maze respect to the control
group (Fig. 3). Rats treated with GH showed a reduced performance
time to complete the trial (40% reduction, p b 0.05; one-way ANOVA
[F(2,14) = 12.3, p = 0.0008] followed by Scheffé's test; Fig. 3A). Also
rats treated with GH reduced the number of errors in the radial maze
(no visited plus repeated visited arms) respect to the control group
(49% error reduction, p b 0.05; one-way ANOVA [F(2,14) = 5.8, p =
0.0145] followed by Scheffé's test; Fig. 3B). Noteworthy, the improving
 M. Ramis et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 45 (2013) 11–20 15

A) B)
Young rats Old rats
25 25

20 20
Time spent exploring (sec)

Time spent exploring (sec)

15 15

#
10 10
#

5 5

0 0
new familiar new familiar new familiar new familiar new familiar new familiar
Control GH MK+GH Control GH MK+GH

C) D)

15 15
Time spent exploring (sec)

Time spent exploring (sec)

10 10

5 5

0 0
New - Familiar New - Familiar
Control GH MK+GH Control GH MK+GH

Fig. 2. Acute effects of GH treatment (1 mg/kg, i.p., 1 h before the test) in young and old rats on the novel object recognition test, compared with the corresponding young or old
control group. The effect of GH was blocked by previous administration of the NMDA receptor antagonist MK801 (MK, 0.025 mg/kg dissolved in saline). The drugs were injected
(i.p.) alone or in combination 10 min apart (MK followed by GH). Control rats received vehicle (1 ml/kg of saline i.p.). MK801 administered alone did not induce significant changes
at the mentioned dose (data not shown, see Table 1 for the effects of MK801 at different doses). More details in the Methods and materials section. Bars represent means ± SEM
derived from different animals of the time spent exploring the novel and the familiar objects (A, B), and the time spent exploring the novel object respect to the familiar (C, D).
Two-way ANOVA detected significant differences between ages and treatments but not detected significant differences for the interaction between ages and treatments.
Two-way ANOVA followed by Bonferroni test was used for statistical evaluation, ***p b 0.001 when compared with the novel object of control group; †††p b 0.001 when compared
with the novel object of GH group. #p b 0.001 when compared new vs familiar.

effects of GH on spatial memory in the radial maze were blocked in rats
that received DNQX (1 mg/kg, i.p.) before GH (1 mg/kg, 10 min apart).
Accordingly the time spent to complete the trial was similar to control
rats but significantly higher respect to the rats treated with GH alone
(p b 0.01; one-way ANOVA [F(2,14) = 12.3, p = 0.0008] followed by
Scheffé's test; Fig. 3A). Also, the number of errors during the perfor-
mance of trial was higher in rats injected with DNQX before GH than in
rats treated with GH alone (p b 0.05; one-way ANOVA [F(2,14) = 5.8,
p = 0.0145] followed by Scheffé's test; Fig. 3B).
The single administration of DNQX (1 mg/kg, i.p., 70 min before
the test) did not cause any effect on radial maze compared to control
rats (Table 3). However, Table 3 shows the effects of different doses of
DNQX on the radial maze in rats. From the results obtained in this
previous study, it was selected the dose of 1 mg/kg for analysing
the effect of AMPA receptor blockade on the improvement of working
memory induced by GH.
As it was expected, the recognition memory in the novel object
recognition test was significantly improved by the acute administra-
tion of GH (1 mg/kg, i.p.) sixty minutes before the training in a
group of young rats (Fig. 4). Control group spent more time exploring
the novel object than the familiar. Two-way ANOVA followed by
Bonferroni test for time spent exploring the novel and the familiar
object detected significant differences between treatments (saline,
GH and DNQX + GH) (F(2,36) = 6.28, p = 0.0046), object preference
16 M. Ramis et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 45 (2013) 11–20

A) A)
15 25
Time performance (min)

20

Time spent exploring (sec)

10

15

5 10 #
# #

5
0
Control GH DNQX+GH
0
new familiar new familiar new familiar
B) Control GH DNQX+GH
10
B)
Total errors (number)

7.5
15
Time spent exploring (sec)
5

10

2.5

5
0
Control GH DNQX+GH

Fig. 3. Acute effects of GH treatment (1 mg/kg, i.p., 1 h before the test) in young rats on
the eight-arm radial maze test, compared with the corresponding control group. The
effect of GH was blocked by previous administration of the AMPA receptor antagonist
DNQX (1 mg/kg dissolved in DMSO). The drugs were injected (i.p.) alone or in combi-
nation 10 min apart (DNQX followed by GH). Control rats received vehicle (1 ml/kg of
DMSO i.p.). DNQX administered alone did not induce significant changes at the men-
tioned dose (data not shown, see Table 2 for the effects of DNQX at different doses).
Other details as for Fig. 1. One-way ANOVA followed by Scheffé or Fisher LSD were
used for statistical evaluation, *p b 0.05 when compared with the corresponding control
group; †p b 0.05 and ††p b 0.01 when compared with GH group.
(F(1,36) = 46.15, p b 0.0001), and for the interaction between treat-
ments and object preference (F(2,36) = 7.61, p = 0.0018) (Fig. 4A).
Fig 4B represents the difference of time spent exploring the new
object and the familiar. It shows that the time spent exploring the
novel object minus the time spent exploring the familiar object was
increased notably after GH treatment (from 64% to 75% of total
exploring time, p b 0.05; one-way ANOVA [F(2,18) = 3.58, p = 0.0488]
followed by Fisher test). Thus GH increased the percent time spent
exploring the novel object respect to the familiar object (2.8 fold) respect
to the control group (p b 0.05; one-way ANOVA [F(2,18) = 7.18, p =
0.0051] followed by Scheffé's test). Remarkable, the improving effects
of GH on recognition memory were blocked by DNQX (1 mg/kg). Thus,
results observed in the group of rats injected with DNQX (1 mg/kg,
i.p.) before GH (1 mg/kg, 10 min apart) were similar to those of the con-
trol group (Fig. 4) and the group of rats treated with DNQX alone (see
Table 1 for object recognition index).
These results suggest an implication of AMPA receptors in the
effects of GH on working memory.
0
new familiar
Control GH DNQX+GH
Fig. 4. Acute effects of GH treatment (1 mg/kg, i.p., 1 h before the test) in young rats on
the novel object recognition test, compared with the corresponding control group. The
effect of GH was abolished by previous administration of the AMPA receptor antagonist
DNQX (1 mg/kg dissolved in DMSO). The drugs were injected (i.p.) alone or in combi-
nation 10 min apart (DNQX followed by GH). Control rats received vehicle (1 ml/kg of
DMSO i.p.). DNQX administered alone did not induce significant changes at the mentioned
dose (data not shown, see Table 2 for the effects of DNQX at different doses). Other details
as for Fig 2. A) Two way ANOVA followed by Bonferroni test was used for statistical
evaluation between treatment (Control, GH and DNQX + GH) and object preference
(new vs familiar). Panel B) shows the difference of the time spent exploring the new
object and the familiar; one-way ANOVA followed by Scheffé test was used for statistical
evaluation. *p b 0.05, **p b 0.01, ***p b 0.001 when compared with the corresponding
control group; †p b 0.05 when compared with the GH group; #p b 0.001 when compared
the novel object vs the familiar.
3.4. The effect of in vivo inhibition of ERK activation on GH-induced
working memory improvement
The effect of in vivo inhibition of ERK activation on the improve-
ment of working memory induced by GH was analysed in a group
of mice. First, it was observed that the acute administration of GH
(1 mg/kg, i.p., 1 h) significantly improved the spatial memory in the
radial maze in mice respect to the control group. Mice treated with
GH reduced the time required to complete the test respect to the con-
trol group (32% reduction, p b 0.05; one-way ANOVA [F(3,25) = 3.55,
 M. Ramis et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 45 (2013) 11–20 17

A) A)
Mice Mice
25
10

20

Time spent exploring (sec)

7.5
Time performance (min)

15
# # #
#
5
10

2.5
5

0
0 new fam new fam new fam new fam
Control SL GH SL+GH
Control SL GH SL+GH

B) B)
25
15

20
Time spent exploring (sec)
Total errors (number)

10
15

10

0
Control SL GH SL+GH
Fig. 5. Acute effects of GH treatment (1 mg/kg, i.p., 1 h before the test) in mice on the
eight-arm radial maze test, compared with the corresponding control group. The effect
of GH was abolished by previous administration of the MEK inhibitor SL 327 (20 mg/kg
i.p. dissolved in DMSO) 30 min before the administration of GH (1 mg/kg). The drugs
were injected (i.p.) alone or in combination 30 min apart (SL 327 followed by GH).
Control rats received vehicle (1 ml/kg of DMSO i.p.). Other details as for Fig. 1.
One-way ANOVA followed by Fisher LSD was used for statistical evaluation, *p b 0.05
when compared with the control group; †p b 0.05 when compared with the GH group.
p = 0.0286] followed by Fisher's test; Fig. 5A). Mice treated with GH
also reduced the number of errors in the radial maze (no visited arm
or re-visited arm) respect to the control group (34% error reduction,
p b 0.05; one-way ANOVA [F(3,25) = 3.35, p = 0.035] followed by
Fisher's test; Fig. 5B). However, the injection of MEK inhibitor SL
327 (20 mg/kg, i.p.) 30 min before GH administration, blocked the
positive effects of GH on radial maze test (Fig. 5A, B), since results
in the radial maze were similar in control group and mice injected
with SL 327 (20 mg/kg) before GH, but significantly different to
those observed in mice treated with GH alone for time (p b 0.05;
one-way ANOVA [F(3,25) = 3.55, p = 0.0286] followed by Fisher's
test; Fig. 5A) and errors (p b 0.05; one-way ANOVA [F(3,25) = 3.35,
p = 0.035] followed by Fisher's test; Fig. 5B). No significant changes
0
New - Familiar
Control SL GH SL+GH
Fig. 6. Acute effects of GH treatment (1 mg/kg, i.p., 1 h before the test) in mice on the
novel object recognition test, compared with the corresponding control group. The
effect of GH was abolished by previous administration of the MEK inhibitor SL 327
(20 mg/kg i.p. dissolved in DMSO) 30 min before the administration of GH (1 mg/kg).
The drugs were injected (i.p.) alone or in combination 30 min apart (SL 327 followed by
GH). Control rats received vehicle (1 ml/kg of DMSO i.p.). Other details as for Fig. 2. A)
Two way ANOVA followed by Bonferroni test was used for statistical evaluation between
treatment (Control, SL, GH, and SL + GH) and object preference (new vs familiar). Panel
B) shows the difference of the time spent exploring the new object and the familiar;
one-way ANOVA followed by Scheffé test was used for statistical evaluation. **p b 0.01
and ***p b 0.001 when compared with the corresponding control group; †p b 0.05
when compared with the GH group; #p b 0.001 when compared the novel object vs the
familiar.
were observed on working memory in the radial maze after the single
administration of SL 327 at the used dose (Fig. 5A, B).
Also the recognition memory in the novel object recognition
test was significantly improved by the acute administration of GH
(1 mg/kg, i.p., 1 h) to mice (Fig. 6). Two-way ANOVA followed by
Bonferroni test for time spent exploring the novel and the familiar
object detected significant differences between treatments (saline, SL,
GH and SL + GH) (F(3,22) = 16.82, p = 0.0001), object preference
18 M. Ramis et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 45 (2013) 11–20

Table 1 Table 3
Object recognition index. Comparative effect of GH injected alone or GH injected after Effects of several doses of DNQX on working memory task by means of the eight-arm
MK801, DNQX or SL327 on the novel object recognition test. radial maze test, compared with the corresponding control group. No visited arm or
re-visited arms were considered as an error.
Group and treatment Training session Test session
Group and treatment Performance time (min) Errors (number)
Young-saline, n = 10 0.52 ± 0.09 0.63 ± 0.04
Young-GH, n = 10 0.49 ± 0.05 0.79 ± 0.04⁎ Young control (n = 6) 9.2 ± 0.91 6.17 ± 0.87
Young-MK801 + GH, n = 7 0.50 ± 0.01 0.56 ± 0.03†† DNQX (1 mg/kg, n = 4) 9.4 ± 1.14 5.00 ± 1.08
Young-MK801, n = 5 0.49 ± 0.01 0.64 ± 0.02 DNQX (2 mg/kg, n = 4) 12.7 ± 2.05⁎ 12.25 ± 1.11⁎
Old-saline, n = 13 0.49 ± 0.07 0.53 ± 0.04 DNQX (2.5 mg/kg, n = 5) 20 ± 0.00⁎ 7.50 ± 0.26
Old-GH, n = 13 0.51 ± 0.02 0.70 ± 0.04⁎
⁎ p b 0.05 when compared with control group by one-way ANOVA followed by Scheffé's
Old-MK801 + GH, n = 13 0.52 ± 0.02 0.53 ± 0.05†
test.
Old-MK801, n = 5 0.51 ± 0.01 0.50 ± 0.04
Young-saline, n = 7 0.50 ± 0.04 0.64 ± 0.03
Young-GH, n = 9 0.48 ± 0.05 0.75 ± 0.03 ⁎
Young-DNQX + GH, n = 5 0.51 ± 0.02 0.62 ± 0.04†
Young-DNQX, n = 5 0.50 ± 0.03 0.64 ± 0.021 4. Discussion
Adult-vehicle mice, n = 6 0.49 ± 0.03 0.61 ± 0.017
Adult-GH mice, n = 3 0.50 ± 0.02 0.68 ± 0.003⁎ The two main findings of this study are, first, the single administra-
Adult-SL 327 + GH mice, n = 3 0.51 ± 0.01 0.60 ± 0.007†
tion of growth hormone (GH, 1 h) induces immediate positive effects
Adult-SL 327, n = 3 0.49 ± 0.017 0.60 ± 0.003
on working memory (WM) and secondly, that both glutamate receptor
⁎ p b 0.05 when compared with control group.
†
activation as MEK/ERK pathway plays an essential role in mediating the
p b 0.05 when compared with GH-treated group by one-way ANOVA followed by
acute effect of GH on WM. In this way, the acute administration of GH
Scheffé's test.
††
p b 0.01 when compared with GH-treated group by one-way ANOVA followed by produced favourable effects on two different hippocampal-dependent
Scheffé's test. tests used for evaluating WM. Thus, GH improved both spatial memory
and recognition memory through both glutamatergic receptors NMDA
and AMPA, probably by enhancing excitatory synaptic transmission in
(F(1,22) = 388.32, p b 0.0001), and for the interaction between treat- the hippocampus.
ments and object preference (F(3,22) = 11.90, p = 0.0001) (Fig. 6A). WM is generally considered to involve the brief online storage and
Fig 6B represents the difference of time spent exploring the new object manipulation of stored information (Baddeley, 1998; Cowan, 2008). A
and the familiar. It shows that the time spent exploring the novel object WM operation involves a short-term memory storage component and
minus the time spent exploring the familiar object was increased nota- executive functions, and is considered one of the main contributing
bly by GH administered sixty minutes prior to the training session com- factors of cognitive impairments in older adults and many neuropsy-
pared to controls (from 61% to 68% of total exploring time, p b 0.05; chiatric disorders (Park et al., 2002). In this work, WM was analysed
one-way ANOVA [F(3,11) = 5.39, p = 0.0158] followed by Scheffé's by two different hippocampal-dependent tests to evaluate spatial
test; Table 1). The acute treatment with GH increased the percent memory and recognition memory. Hippocampus is a brain area
time exploring the novel object respect to the familiar object related known to play an essential role in cognitive processes, especially
to the control group (1.8 fold, p b 0.01; one-way ANOVA [F(3,11) = memory and learning. The radial maze widely used in rodents was
8.67, p = 0.0031] followed by Scheffé's test; Fig. 6B). Striking, the used to test spatial memory which is dependent on hippocampus
MEK inhibitor SL 327 (20 mg/kg, i.p.) administered in mice 30 min be- (for review see Martin and Clark, 2007). On the other hand, recogni-
fore GH, significantly blocked the favourable effects of GH on the novel tion memory was assessed by the novel object recognition test, which
object recognition test (Fig. 6A, B), since results in the test were similar deals with the natural ability of animals to explore novelties (Clark et
in control group and mice that received SL 327 (20 mg/kg) before GH, al., 2000) and is considered a common model for non-aversive declar-
but significantly different to those observed in mice treated alone ative memory (Okamura et al., 2011). The acute administration of GH
with GH (p b 0.05; one-way ANOVA [F(3,11) = 8.67, p = 0.0031] caused positive effects on spatial memory and recognition memory in
followed by Scheffé's test; Fig 6B). It can be seen in Fig. 6 as mice treated young and adult rats, and mice.
alone with SL 327 showed no difference with the control group (Fig. 6 A range of observations support a role for GH in development and
and Table 1). function of the brain, and both the GH receptor and GH itself are
Together these results suggest that the observed effects of GH on expressed widely in the CNS. GH is involved in neurogenesis,
working memory are under the control of the MEK/ERK pathway. neuroprotection, plasticity as well as cognitive function (Aberg,
2010; Aberg et al., 2006; Schneider-Rivas et al., 1995). Memory defi-
ciency including both short- and long-term memories is a condition
in GH-deficient patients (Aleman et al., 2000). Also GH levels are
Table 2
high during development and puberty, and then decline with advanc-
Effects of several doses of MK 801 on working memory task by means of the eight-arm
radial maze test, compared with the corresponding control group. No visited arm or ing age (Sonntag et al., 1980). In correlation with a GH decline is the
re-visited arms were considered as an error. decrease in cognitive function (Aleman et al., 2000; de Boer et al., 1995;
Deijen et al., 1996; Hoffman et al., 1992; Nyberg, 2000). Thereby, the
Group and treatment Performance time Errors
(min) (number)
chronic treatment with GH has been shown to be effective in reversing
deficits in working memory of GH-deficient patients (Arwert et al.,
Young-saline (n = 8) 8.2 ± 0.9 7.4 ± 0.9
2005a, 2005b; Arwert et al., 2006) as well as the age-related working
Young-MK 801 (0.025 mg/kg, n = 12) 7.9 ± 0.4 8.5 ± 0.5
Young-MK 801 (0.05 mg/kg, n = 5) 9.7 ± 0.5 9.8 ± 1.2 memory impairment in rats (Esteban et al., 2010b; Ramsey et al.,
Young-MK 801 (0.1 mg/kg, n = 5) 10.5 ± 0.6 10.4 ± 0.9 2004). Several memory impairments in old rats may be partially due
Old-saline (n = 6) 16.8 ± 0.9 11.8 ± 1.2 to abnormal glutamate function, and it was suggested that GH can mit-
Old-MK 801 (0.025 mg/kg, n = 6) 16.2 ± 0.9 11.5 ± 0.3
igate the age-related changes in hippocampal function that underlie
Old-MK 801 (0.05 mg/kg, n = 5) 19.8 ± 0.2⁎ 10.4 ± 0.2
Old-MK 801 (0.1 mg/kg, n = 5) 20.0 ± 0.0⁎ 16.0 ± 2.2
this cognitive impairment. In this context Molina et al. (2012) reported
that old animals treated for 6–8 months with GH restored NMDA-
⁎ p b 0.05 when compared with control group by one-way ANOVA followed by Scheffé's
test; ANOVA F(3, 26) = 1.78, p = 0.1746 for performance time in young rats; ANOVA
receptor-dependent basal synaptic transmission to young adult levels
F(3, 26) = 2.19, p = 0.113 for errors in young rats; ANOVA F(3, 18) = 7.66, p = 0.0017 and enhanced both AMPA-receptor-dependent basal synaptic trans-
for performance time in old rats; ANOVA F(3, 18) = 1.47, p = 0.256 for errors in old rats. mission and long-term potentiation.
 M. Ramis et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 45 (2013) 11–20
Interestingly, some reports demonstrate that hippocampal function
is affected by GH over a short timescale — minutes to hours (Mahmoud
and Grover, 2006). In good agreement, the results of the current
work suggest an immediate effect of GH modulating hippocampal-
dependent memory function. In this manner, the acute administration
of GH caused positive effects on two different tests used to evaluate
working memory in rats and mice. Thus, the single administration of
GH improved spatial memory on the 8-arm radial maze, the animals
treated with GH reduced the time required to the performance on the
radial maze, and also reduced the number of errors. Also, GH induced
short-term effects on recognition memory on the novel object recogni-
tion test as it is increased the percent time spent exploring the novel
object respect to the familiar object compared to controls. One expla-
nation for this fast improvement could be attributed to an enhance-
ment of excitatory synaptic transmission in hippocampus. Thus,
GH enhanced both NMDA- and AMPA-receptor-mediated excitatory
synaptic transmission in area CA1 of rat hippocampus (Mahmoud
and Grover, 2006). Given the paramount importance of both the
NMDA and AMPA receptors in memory formation and the spatial
learning, we investigated whether the acute positive effects of GH on
memory process were mediated by NMDA and/or AMPA receptors,
the effects of GH were assessed in rats injected with the NMDA antag-
onist MK801 (0.025 mg/kg) and also in other group of rats injected
with the AMPA antagonist DNQX (1 mg/kg) 10 min before GH injec-
tion. The results revealed that both NMDA and AMPA receptors were
involved in the effects of GH on working memory since in rats injected
with MK801 or DNQX, GH injection did not modify significantly spatial
memory and recognition memory.
Further, mitogen-activated protein kinases (MAPKs) have been
implicated as playing crucial role in hippocampal synaptic plasticity
and hippocampus-dependent memory formation (Sweatt, 2004). In
particular, a wide variety of studies during the past years indicate
the clear importance of the extracellular signal-regulated kinase
(ERK, subfamily of MAPKs) in synaptic plasticity and memory forma-
tion in general, across many species, brain areas, and types of synap-
ses (for a review see Sweatt, 2004). The above mentioned effects of
GH increasing working memory in the present work required the
activation of MEK/ERK signalling pathway. In this manner, the MEK
inhibitor SL 327 (20 mg/kg, i.p.) blocked the positive effect of GH on
radial maze and the novel object recognition in mice indicating that
activation of MEK/ERK pathway has an important role in mediating
the acute effects of GH on working memory. It is likely that GH
administration initiates a chain of events resulting in enhancement
of glutamate signalling which leads to sustained activation of signal-
ling cascades such as ERK1/2 during memory task. In good agreement,
the enhancement induced by GH on hippocampal excitatory synaptic
transmission mediated by AMPA and NMDA receptors required MEK
pathway activation (Mahmoud and Grover, 2006).
Moreover, ageing is associated with a decline of subunits that
comprise the NMDA receptors in the hippocampus (Adams et al.,
2008; Foster, 2002; Newton et al., 2008; Shi et al., 2007), and NMDA
receptor-mediated synaptic transmission is reduced as well (Barnes,
1990; Barnes et al., 1997). Chronic systemic treatment with GH alters
the pattern of expression of NMDA receptor subunits in the hippo-
campus (Le Greves et al., 2002) and this could at least partially
explain the effects of GH on memory function. Glutamatergic fast syn-
aptic transmission is known to be altered with age in a specific man-
ner in hippocampus of old rats. In the current study, impairment of
working memory was observed in old control rats compared to youn-
ger animals on the 8-arm radial maze test, as previously reported
(Esteban et al., 2010a, 2010b). Old rats need more time to complete
the trial in the radial maze and also increase the number of errors.
Impairment of working memory was also observed in the current
work in old control rats on the novel object recognition test. Neverthe-
less, acute GH treatment improved performance on memory tasks in
young and aged rats, being remarkable that GH treatment significantly
19
reversed the age-related decline in working memory since the old
GH-treated rats showed data similar to those of young control rats.
These findings are consistent with previous observations revealing
that there are fewer NMDA receptors in aged rats (possibly because of
fewer total synaptic contacts) but the NMDA receptors that remain
appear to function normally under certain conditions (Barnes et al.,
1997) and drugs enhancing glutamate receptors selectively increase
hippocampal cell firing and it improves both acquisition performance
and memory retention in middle-aged rats to levels equivalent to
those found in young adult animals (Granger et al., 1996). Also, GH
ameliorated hippocampal-dependent cognitive decline by regulating
NMDA and AMPA receptors, and improved age dependent changes in
spatial learning (Ramsey et al., 2004). In good agreement GH has been
found to regulate the NMDA receptor subunit transcript levels in old
rats (Le Greves et al., 2002, 2006).
5. Conclusion
Our data showed that GH acutely administered in a single dose
promoted improvement on working memory as a cognitive enhancer.
Moreover, the results of this study indicate that the activation of
NMDA, AMPA and MEK/ERK pathway have, together, a major role in
mediating the acute effect of GH on working memory in rodents.
These effects of GH on memory may be mediated by the hippocampus
which contains GH receptors, suggesting a potential role for GH sig-
nalling in the regulation of hippocampal dependent behaviours.
Contributors
A. Miralles, C. Garau and S. Esteban designed the research.
M. Ramis, F. Sarubbo, J. Sola and S. Aparicio performed the exper-
iments. The manuscript was written by A. Miralles and S. Esteban. All
authors discussed results and commented on the manuscript.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank the financial contribution of the UIB. The authors
also thank Dr. JA García Sevilla and Dr. JA Fernández for kindly supply-
ing research materials. M.R. was supported by a predoctoral FPU fellow-
ship from MICINN (Spain), and F.S. was supported by a predoctoral
fellowship from Postgraduate Studies Center (University of Balearic
Islands, Spain).
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