Divergent effects of l-arginine-NO pathway modulators on diazepam and flunitrazepam responses in NOR task performance

1. Behavioural Brain Research 284 (2015) 179–186
Contents lists available at ScienceDirect
Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr
Research report
Divergent effects of l-arginine-NO pathway modulators on diazepam
and flunitrazepam responses in NOR task performance
Jolanta Orzelska∗
, Sylwia Talarek, Joanna Listos, Sylwia Fidecka
Chair and Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, Chodzki 4A, 20-093 Lublin, Poland
h i g h l i g h t s
• Diazepam and flunitrazepam impaired recognition memory of rats.
• l-Arginine prevented diazepam-induced memory impairment.
• 7-Nitroindazole induced the amnesic effects of diazepam.
• 7-Nitroindazole inhibited the amnesic effects of flunitrazepam.
a r t i c l e i n f o
Article history:
Received 23 October 2014
Received in revised form 2 February 2015
Accepted 6 February 2015
Available online 16 February 2015
Keywords:
Diazepam
Flunitrazepam
Nitric oxide
Novel object recognition
Rat
a b s t r a c t
The goal of the study was an evaluation of the degree, in which nitric oxide (NO) is involved in the
benzodiazepines (BZs)-induced recognition memory impairment in rats. The novel object recognition
(NOR) test was used to examine recognition memory. The current research focused on the object memory
impairing effects of diazepam (DZ; 0.5 and 1 mg/kg, sc) and flunitrazepam (FNZ; 0.1 and 0.2 mg/kg; sc) in
1-hour delay periods in rats. It was found that acute ip injection of l-arginine (l-arg; 250 and 500 mg/kg;
ip), 5 min before DZ administration (0.5 mg/kg, sc) prevented DZ-induced memory deficits. On the other
hand, it was also proven that l-arg (125, 250 and 500 mg/kg; ip) did not change the behaviour of rats
in the NOR test, following a combined administration with FNZ at a threshold dose (0.05 mg/kg; sc). It
was also found that 7-nitroindazole (7-NI; 10, 20 and 40 mg/kg; ip) induced amnesic effects in DZ in
rats, submitted to the NOR test, following a combined administration of 7-NI with a threshold dose of DZ
(0.25 mg/kg; sc). However, following a combined administration of 7-NI (10, 20 and 40 mg/kg; ip) with
FNZ (0.1 mg/kg; sc), it was observed that 7-NI inhibited the amnesic effects of FNZ on rats in the NOR test.
Those findings led us to hypothesize that NO synthesis suppression may induce amnesic effects of DZ,
while preventing FNZ memory impairment in rats, submitted to NOR tasks.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Although benzodiazepines (BZs) are commonly used in
therapeutic practice for their anxiety-relieving, tranquilising, som-
niferous, anticonvulsant and myorelaxant properties, they disrupt
memory performance [1]. The amnesic effects of BZs are regarded
to be rather detrimental in their harmful impact. For instance, a
number of elderly patients on BZ therapy are wrongly diagnosed as
dementia patients, whereas, in fact, they suffer from the amnesic
effects of the therapy [1]. Generally, there is no doubt that memory
is a key cognitive skill for work and life. Therefore, it is impor-
tant to determine what aspects of memory are affected by BZs and
∗ Corresponding author. Tel.: +48 81 4487256.
E-mail address: jolanta.orzelska@umlub.pl (J. Orzelska).
what neural mechanisms/processes are involved in their adverse,
amnesic effects.
BZs have been reported to induce anterograde amnesia, both
in humans and in rodents (mice and rats) with selective mem-
ory deficits caused by impaired acquisition of new information
[1–4]. What is more, it is a well-established fact that acute BZs,
such as diazepam (DZ) or triazolam impair the episodic mem-
ory (a type of declarative memory) encoding in humans [5].
Recognition is thought to be a critical component of declara-
tive memory – a judgement of the prior occurrence [6]. While a
substantial amount of research has shown that BZs impair mem-
ory paradigms in various rodents, e.g., radial maze [4], passive
avoidance [7], T-maze [8], water maze [3,7] and the modi-
fied elevated plus-maze (mEPM) task [2], there are only a few
reports on the effects of BZs on recognition memory in rodents
[9–11].
http://dx.doi.org/10.1016/j.bbr.2015.02.014
0166-4328/© 2015 Elsevier B.V. All rights reserved.

2. 180 J. Orzelska et al. / Behavioural Brain Research 284 (2015) 179–186
The amnesic effect of BZs is mediated by the activation of specific
receptor sites on the gamma-aminobutyric acid (GABAA) receptor
complex, being thereby enhanced in the GABAergic transmission
in the central nervous system (CNS) [12,13]. Moreover, at the
cellular level, there is some evidence for the plastic mechanism,
e.g., long-term potentiation (LTP) involved in BZ-induced memory
impairment [10,14]. However, the exact mechanism of BZ amnesic
effects has not yet been fully unveiled.
Nitric oxide (NO) has been found to be a retrograde transmit-
ter which, via LTP, affects the learning and memory processes [15].
Current reports indicate that both NO donors and NOS inhibitors
(INOS) are involved in the object recognition memory (for review,
see [16]). For instance, it has been demonstrated that NOS block
by different INOS, e.g., NG-nitro-l-arginine methyl ester (l-NAME;
non-selective NOS inhibitors), 7-nitroindazole (7-NI; selective neu-
ronal NOS inhibitor) induce acquisition deficits in recognition
memory tasks in rats and these learning deficits are counteracted
by molsidomine, a NO donor [17,18]. Moreover, NO modulates
the release and the retrograde uptake of many neurotransmitters,
including GABA (for review, see [15]). For instance, at the cellular
level, Szabadits et al. [19] have provided evidence for NO playing a
certain role in the control of hippocampal GABAergic transmission.
The researchers have demonstrated that nNOS is present in hip-
pocampal GABAergic synapses in adult rodents. What is more, our
behavioural studies also suggest some role of NO in anticonvulsant
[20], hypnotic [21] or antinociception [22] effects of BZs, as well as
in the development of tolerance to the coordination disturbing [23]
and sedative [24] effects of BZs.
Our previous studies demonstrated that, in a spatial memory
task–mEPM–acquisition was impaired by DZ and flunitrazepam
(FNZ). But it should be noted that INOS enhanced DZ-induced,
while preventing FNZ-induced recognition memory impairment
[2]. However, the interactions between NO and BZs in recognition
performance has not yet been elucidated.
Taking into account the above-mentioned, somewhat contro-
versial results, the goal of the study was to evaluate the degree, in
which NO is involved in the BZ-induced recognition impairment
in rats. A novel object recognition (NOR) test was employed to
examine recognition memory. Memory performance in the NOR is
based on the natural tendency of animals to explore novel objects.
An important advantage of this task is that no aversive/stressful
stimuli are needed [9]. In order to design a possible interaction
between NO activity and BZ responses in NOR tasks, performed by
rats, l-arginine (l-arg)–a precursor of NO and 7-NI – selective nNOS
inhibitor – were applied [25]. DZ and FNZ were chosen as represen-
tative members of BZ family. In addition, pilot study in NOR task was
conducted, to choose doses appropriate for further experiments.
For this purpose, the effects of BZ and NO-related compounds (l-
arg and 7-NI), given alone, on rats performance in NOR task were
investigated. Moreover, in an attempt to exclude the possible seda-
tive effect of BZ, l-arg or 7-NI, given alone or in combination with
BZ, motility of rats after administration of all the substances, was
evaluated.
2. Materials and methods
2.1. Animals
The examinations were carried out on 2-month-old male albino
Wistar rats (The Farm of Labolatory Animals, Z. Lipiec, Brwinow,
Poland), weighing 200–250 g each. They were housed in groups of
five and maintained on a 12 h light-dark cycle (lights on at 6:00 h)
at controlled temperature (21 ◦C). The experiments were per-
formed between 9:00 h and 17:00 h. They received standard food
(Agropol, Motycz, Poland) and tap water ad libitum. All behavioural
experiments were carried out, according to the National Institute
of Health Guidelines for the Care and Use of Laboratory Animals
and to the European Community Directive for the Care and Use of
Laboratory of 24 November 1986 (86/609/EEC), and approved by
the Local Ethics Committee (37/2010).
2.2. Drugs
l-arg and FNZ were purchased from Sigma Chemicals (St. Louis,
USA). 7-NI (RBJ, Natick, USA) and FNZ were dissolved in 0.5%
Tween-80 (1–2 drops), gently warmed and diluted with saline
solution (0.9% NaCl). DZ (Relanium, Polfa, Poland) was diluted in
0.9% saline. l-arg was dissolved in saline solution. All drug sus-
pensions/solutions were prepared immediately prior to use. l-arg
and 7-NI were given intraperitoneally (ip), whereas DZ and FNZ
subcutaneously (sc). All the drugs were injected in a volume of
0.2 ml/100 g body weight. Control animals were administred a cor-
responding vehicle.
2.3. Novel object recognition test
The apparatus included a square open box, made of plexiglass
(63 cm long × 44.5 cm high × 44 cm wide) and illuminated by a
lamp (light intensity–10 lx), suspended 50 cm above the box. The
objects to be discriminated, made either of wood or plastic, were in
two different shapes: block and ball and too heavy to be displaced
by the animals.
The object recognition test was performed as described else-
where [9,26]. The day before the test, each rat was placed in the
empty box for 2 min to get used to the environment. On the exper-
imental day, the animals were submitted to two trials, spaced by
a 1-h interval. The first trial (acquisition trial, T1) lasted 5 min and
the second one (test trial, T2) was 3 min long. During T1, the appa-
ratus contained two identical objects (wooden blocks), placed in
two opposite corners, 10 cm from the sidewall. A rat was always
placed in the middle of the box. After T1, the rat was put back
into its home cage. Subsequently, after 1 h, T2 was performed. Dur-
ing T2, a new object (N) replaced one of the samples presented in
T1, therefore, the rats were re-exposed to two objects: familiar (F)
and new (N). In order to avoid the presence of olfactory trails, the
apparatus and the objects were cleaned after each rat. The explo-
ration looked as follows: directing the nose toward the object at
a distance of no more than 2 cm and/or touching the object with
nose. Turning around or sitting on the object was not considered as
exploratory behaviour. The time periods, spent by rats in exploring
each object during T1 and T2 tests, were recorded manually with
a stopwatch. The discrimination between F and N during T2 was
measured by comparing the time period, spent for exploration of F
with that, spent for exploration of N. Memory was evaluated with
the discrimination index (DI), calculated for each animal by the
following formula: (N − F)/(N + F), corresponding to the difference
between exploration time periods for N and F, adjusted for the total
exploration time period of both objects in T2. A higher discrimina-
tion index is considered to reflect stronger memory retention for
familiar objects.
2.4. Locomotor activity test
Locomotor activity of individual rats was recorded, using a
photocell device (plexiglass boxes - square cages, 60 cm on each
side; Porfex, Bialystok, Poland) at a sound-attenuated experi-
mental room, under moderate illumination (10 lux). Ambulatory
activity (distance travelled) was measured by two rows of infrared
light-sensitivity photocells, installed along the long axis, 45 and
100 mm above the floor. The animals were placed individually into
cages, 30 min after DZ or FNZ injection and 35 min after l-arg or

3. J. Orzelska et al. / Behavioural Brain Research 284 (2015) 179–186 181
Table 1
Effect of DZ or FNZ treatment on locomotor activity in rats.
Treatment Mean of the
distance
travelled ± SEM
[m] within
15 min
A Saline 17.77 ± 2.913
B DZ 0.25 mg/kg 19.72 ± 2.669
DZ 0.5 mg/kg 20.43 ± 2.497
DZ 1 mg/kg 13.24 ± 3.559
C FNZ 0.05 mg/kg 16.67 ± 3.012
FNZ 0.1 mg/kg 13.50 ± 2.922
FNZ 0.2 mg/kg 12.25 ± 2.07
DZ and FNZ were injected sc 30 min before the test. The data are expressed as
mean ± SEM of total distance travelled in meters within 15 min.
7-NI injection. Total horizontal activity (the distance travelled in
meters) was recorded for a 15-minute time period [26,27].
2.5. Treatment
Different doses of DZ (0.25, 0.5 and 1 mg/kg, sc) [9] and FNZ (0.05,
0.1 and 0.2 mg/kg, sc) [28] were administered on 30 min before T1.
l-arg (125, 250 and 500 mg/kg, ip) [29,30] and 7-NI (10, 20 and
40 mg/kg, ip) [18,30] were administered 35 min before T1, alone.
In order to evaluate the influence of l-arg or 7-NI on DZ or FNZ-
treated rats, l-arg or 7-NI were administered 5 min prior to DZ or
FNZ injections. T2 was carried out always 1 h after T1.
2.6. Statistical analysis
The data, relative to DI values and the distance, travelled for DZ
and FNZ, given alone, were analysed by the one-way analysis of
variances (ANOVA). DI values for the co-administration of “l-arg
and DZ or FNZ”, and also of “7-NI and DZ or FNZ”, were made by
two-way ANOVA, with the drug treatment (saline and DZ or FNZ)
as factor 1 and the drug pre-treatment (saline, l-arg or 7-NI) as fac-
tor 2. In those cases that the interaction between treatment and
pre-treament was significant or quite signicant, Bonferroni’s post
hoc test was applied. The level of p < 0.05 was considered statisti-
cally significant. The data are presented as means ± standard errors
of means (S.E.M.) of DI values or distance segments, travelled in
meters. Each group of animals consisted of 8 rats. All figures were
prepared using GraphPad Prism version 5.00 for Windows, Graph-
Pad Software (San Diego, California, USA), www.graphpad.com.
3. Results
No difference was observed in any group during Test 1 (T1),
when exploration time periods were compared for location of two
identical objects in two opposite corners (the data not shown).
3.1. Effects of treatments on the locomotor activity of rats
No significant difference was observed between the groups,
regarding the effects of either a single DZ (0.25, 0.5, and 1 mg/kg) or
FNZ (0.05, 0.1 and 0.2 mg/kg) injection on the total distance, trav-
elled in meters within 15 minutes by rats, using the photocell device
[one-way ANOVA; F(3,25) = 1.168; p = 0.3416 for DZ, see Table 1A and
B; F(3,28) = 0.8881; p = 0.4593 for FNZ, see Table 1A and C].
Two-way ANOVA revealed that a treatment with DZ (0.5 mg/kg)
or saline [F(1,49) = 1.62] and pre-treatment with l-arg (125, 250 and
500 mg/kg) or saline [F(3,49) = 0.74] did not affect the total distance
travelled by rats (Table 2A, B, D and E).
Table 2
Effect of treatments on locomotor activity in rats treated with l-arg and DZ or FNZ.
Treatment Mean of the
distance
travelled ± SEM
[m] within
15 min
A Saline 16.87 ± 2.48
B DZ 0.5 mg/kg 20.32 ± 2.16
C FNZ 0.05 mg/kg 19.71 ± 2.17
D l-arg 125 mg/kg 24.53 ± 1.47
l-arg 250 mg/kg 21.99 ± 3.30
l-arg 500 mg/kg 20.98 ± 3.86
E DZ 0.5 + l-arg 125 mg/kg 15.72 ± 2.25
DZ 0.5 + l-arg 250 mg/kg 22.28 ± 2.58
DZ 0.5 + l-arg 500 mg/kg 15.72 ± 3.43
F FNZ 0.05 + l-arg 125 mg/kg 22.14 ± 2.88
FNZ 0.05 + l-arg 250 mg/kg 21.68 ± 4.26
FNZ 0.05 + l-arg 500 mg/kg 19.59 ± 3.51
l-arg was injected ip 5 min prior to administration of BZ, whereas BZ were injected
sc 30 min prior to the test. The data are expressed as mean ± SEM of total distance
travelled in meters within 15 min.
Two-way ANOVA revealed that a treatment with FNZ
(0.05 mg/kg) or saline [F(1,48) = 0.02] and pre-treatment with l-arg
(125, 250 and 500 mg/kg) or saline [F(3,48) = 0.89] did not affect the
total distance travelled by rats (Table 2A, C, D and F).
Two-way ANOVA revealed that a treatment with DZ
(0.25 mg/kg) or saline [F(1,46) = 3.34] and pre-treatment with
7-NI (10, 20 and 40 mg/kg) or saline [F(3,46) = 1.47] did not affect
the total distance travelled by rats (Table 3A, B, D and E).
Two-way ANOVA revealed that a treatment with FNZ
(0.1 mg/kg) or saline [F(1,52) = 0.4] did not affect locomotor activ-
ity of rats whereas pre-treatment with 7-NI (10, 20 and 40 mg/kg)
or saline [F(3,52) = 5,48] affected the total distance travelled by rats
(Table 3A, C, D and F). The post hoc Bonferroni’s test showed that
co-administration of FNZ with 7-NI (40 mg/kg) decreased the total
distance travelled by rats compared to FNZ-treated group (p < 0.05).
3.2. Effects of diazepam and flunitrazepam administration on rat
performance in the object recognition task
One-way ANOVA revealed statistically significant effects of
acute sc doses of DZ (0.5 and 1 mg/kg) on DI values [F(3,22) = 4,043;
p = 0.0075]. The applied post hoc Bonferroni’s test revealed a
Table 3
Effect of treatments on locomotor activity in rats treated with 7-NI and DZ or FNZ.
Treatment Mean of the
distance
travelled ± SEM
[m] within 15 min
A Saline 15.01 ± 3.08
B DZ 0.25 mg/kg 19.62 ± 2.31
C FNZ 0.1 mg/kg 21.70 ± 2.35
D 7-NI 10 mg/kg 16.41 ± 1.66
7-NI 20 mg/kg 18.55 ± 1.89
7-NI 40 mg/kg 14.52 ± 2.65
E DZ 0.25 + 7-NI 10 mg/kg 21.49 ± 5.20
DZ 0.25 + 7-NI 20 mg/kg 22.56 ± 0.79
DZ 0.25 + 7-NI 40 mg/kg 15.39 ± 2.38
F FNZ 0.1 + 7-NI 10 mg/kg 19.85 ± 1.28
FNZ 0.1 + 7-NI 20 mg/kg 14.40 ± 2.09
FNZ 0.1 + 7-NI 40 mg/kg 9.49 ± 1.36*
7-NI was injected ip 5 min prior to administration of BZ, whereas BZ were injected
sc 30 min prior to the test. The data are expressed as mean ± SEM of total distance
travelled in meters within 15 min. *
p < 0.05 vs. FNZ 0.1 mg/kg (Bonferroni’s test).

4. 182 J. Orzelska et al. / Behavioural Brain Research 284 (2015) 179–186
Fig. 1. Effects of DZ (0.25, 0.5 and 1 mg/kg, sc) administration on rats performance in the object recognition task (discrimination index (A) and total exploration time of both
objects in T2 (B)). BZs or saline were injected 30 min before the first trial. Data are expressed as mean ± SEM values. *
p < 0.05 vs. saline control group (Bonferroni’s test).
significant decrease of DI by DZ (0.5 and 1 mg/kg), as compared
with the saline-treated control group [p < 0.05], confirming that DZ
did disrupt memory processes (see Fig. 1A), while the lowest dose
of DZ (0.25 mg/kg) did not affect DI.
Similarly, one-way ANOVA revealed statistically significant
effects of the acute sc doses of FNZ (0.1 and 0.2 mg/kg) on DI
values [F(3,38) = 6,481; p = 0.0012]. The post hoc Bonferroni’s test
revealed that FNZ (0.1 and 0.2 mg/kg) significantly decreased DI,
as compared with the saline-treated control group (p < 0.05), thus
confirming FNZ to be a memory process disrupting factor (Fig. 2A).
FNZ, when given at the dose of 0.05 mg/kg, did not impair DI in the
NOR test.
As shown in Fig. 1B and Fig. 2B in this set of experiments, data
analysis indicated that all doses of DZ and FNZ did not change total
exploration time in T2.
3.3. l-Arginine effects on DZ (0.5 mg/kg, sc)-induced memory
impairment of rats in the NOR test
Two-way ANOVA revealed statistically significant effects of
DZ or saline treatment [F(1.37) = 4.68; p = 0.037] and interaction
between l-arg pre-treatment and DZ treatment [F(3.37) = 3.13;
p = 0.037].
The acute ip injection of l-arg (250 and 500 mg/kg), 5 min before
DZ administration (0.5 mg/kg, sc) prevented DZ-induced memory
deficits, as the “l-arg (250 or 500 mg/kg) and DZ (0.5 mg/kg)”-
treated rats discriminated much better the novel objects vs. the
familiar objects during T2, when juxtaposed with their coun-
terparts on saline and DZ (0.5 mg/kg) (p < 0.05, the post hoc
Bonferroni’s test; see Fig. 3A).
As shown in Fig. 3B in this set of experiments total exploration
time was unchanged.
3.4. l-Arginine effects on FNZ (0.05 mg/kg, sc)-treated rats in the
NOR test
Two-way ANOVA revealed no statistically significant effect,
either of FNZ or saline treatment and no interaction between l-arg
pre-treatment and FNZ treatment or between FNZ or saline pre-
treatment (Fig. 4A). As shown in Fig. 4B in this set of experiments
total exploration time was unchanged.
3.5. 7-NI effects on DZ (0.25 mg/kg, sc)-treated rats in the NOR
test
Two-way ANOVA revealed statistically significant effects of DZ
or saline treatment [F(1.49) = 38.05; p < 0.0001] and DZ or saline pre-
treatment [F(3.49) = 4.53; p = 0.007]. The interaction between 7-NI
pre-treatment and DZ treatment was considered not quite signifi-
cant [F(3.49) = 2.6; p = 0.0623].
The acute ip injection of 7-NI (10, 20 and 40 mg/kg), 5 min before
DZ administration (0.25 mg/kg) induced memory deficits, as “7-NI
(10, 20 or 40 mg/kg) and DZ (0.25 mg/kg)”-treated rats did not dis-
criminate between novel and familiar objects during T2 test with
respect to their counterparts on saline and DZ (0.25 mg/kg) (p < 0.05
for 7-NI 10 and 20 mg/kg, p < 0.01 for 7-NI 40 mg/kg, the post hoc
Bonferroni’s test; see Fig. 5A).
As shown in Fig. 5B in this set of experiments total exploration
time was unchanged.
Fig. 2. Effects of FNZ (0.05, 0.1 and 0.2 mg/kg, sc) administration on rats performance in the object recognition task (discrimination index (A) and total exploration time of
both objects in T2 (B)). BZs or saline were injected 30 min before the first trial. Data are expressed as mean ±SEM values. *
p < 0.05 vs. saline control group (Bonferroni’s test).

5. J. Orzelska et al. / Behavioural Brain Research 284 (2015) 179–186 183
Fig. 3. The influence of l-arg pretreatment (125, 250 and 500 mg/kg, ip) on DZ
(0.5 mg/kg, sc)-induced deficits in the object recognition task (A). Total exploration
time displayed by different groups of rats in the object recognition task in T2 (B).
l-arg was injected 5 min prior to administration of DZ, whereas DZ was injected
30 min prior to testing in the first trial. The data are expressed as mean ± SEM val-
ues. **
p < 0.01 vs. saline control group; #
p < 0.05 vs. DZ-treated group (Bonferroni’s
test).
3.6. 7-NI effects on FNZ (0.1 mg/kg, sc)-induced memory
impairment of rats in the NOR test
Two-way ANOVA revealed statistically significant effects of
FNZ or saline treatment [F(1.47) = 6.47; p = 0.0143]. The interaction
between the 7-NI pre-treatment and FNZ treatment [F(3.47) = 2.79;
p = 0.0508] was not quite significant.
The acute ip injection of 7-NI (10, 20 and 40 mg/kg) 5 min
before FNZ administration (0.1 mg/kg, sc) prevented FNZ-induced
memory deficits, as the “7-NI (10, 20 or 40 mg/kg) and FNZ
(0.1 mg/kg)”-treated rats discriminated better the novel objects
vs. the familiar objects during T2 test, when compared with their
counterparts on saline and FNZ (0.1 mg/kg) (p < 0.05, the post hoc
Bonferroni’s test; see Fig. 6A).
As shown in Fig. 6B in this set of experiments total exploration
time was unchanged.
4. Discussion
The reported research indicated object memory impairing
effects of either DZ (0.5 and 1 mg/kg) or FNZ (0.1 and 0.2 mg/kg)
in a 1-h delay in rats. In the reported experiments, the animals
were injected with BZ before the first trial, therefore, BZ could
have disrupted the acquisition phase of memory. Those results are
consistent with the prior findings of BZ amnesic effects in rodents
during NOR tasks [9–11]. Additionally, BZ-impaired memory was
shown in different rodent models (models for spatial [2–4,7] and
Fig. 4. The influence of l-arg pretreatment (125, 250 and 500 mg/kg, ip) on rats
performance in the object recognition task after FNZ (0.05 mg/kg, sc) treatment (A).
Total exploration time displayed by different groups of rats in the object recognition
task in T2 (B). l-arg was injected 5 min prior to administration of FNZ, whereas
FNZ was injected 30 min prior to testing in the first trial. The data are expressed as
mean ± SEM values.
for recognition memory [9–11], therefore, the present data confirm
the amnesic effects of BZs to be not specific for one learning type.
What is more, the observed object memory impairing activi-
ties of BZs were not associated with any changes in the locomotor
activity, since BZs did not alter the total distance, travelled by rats
in performed test. The data are in line with the reports, in which
the amnesic effects of BZs are not related to their sedative action
[9,11].
NOR task has been considered to be a pure memory test in which
animals are able to discriminate between familiar and novel objects
after some retention interval [26]. Since the NOR paradigm, in com-
parison to other animal learning and memory models, does not
require lengthy training nor does it induce high arousal and stress
levels, it provides closer conditions to those under which human
recognition memory is measured [26]. In our experiments, 1-hour
delays were employed, according to literature data, indicating good
object memory performance of rats with 1-hour intervals between
subsequent trials [9].
The reported investigations examined the effects of l-arginine
and 7-NI, a NO precursor and a nNOS inhibitor, respectively, on the
performance of rats, subjected to DZ and FNZ injections in NOR
tasks. Acute ip injections of l-arg (250 and 500 mg/kg), admin-
istered in 5 min before DZ (0.5 mg/kg, sc), prevented DZ-induced
memory deficits. On the other hand, it was also proven that l-arg
(125, 250 and 500 mg/kg) did not change the behaviour of rats in
the NOR test, following it’s combined administration with FNZ at a
threshold dose (0.05 mg/kg). It was also found that 7-NI (10, 20 and
40 mg/kg) induced amnesic effects of DZ in rats, participating in the
NOR test, following it’s combined administration with a threshold
dose of DZ (0.25 mg/kg). However, following the combined admin-
istration of 7-NI (10, 20 and 40 mg/kg) with FNZ (0.1 mg/kg), it was

6. 184 J. Orzelska et al. / Behavioural Brain Research 284 (2015) 179–186
Fig. 5. The influence of 7-NI pretreatment (10, 20 and 40 mg/kg, ip) on rats per-
formance in the object recognition task after DZ (0.25 mg/kg, sc) treatment (A).
Total exploration time displayed by different groups of rats in the object recog-
nition task in T2 (B). 7-NI was injected 5 min prior to administration of DZ, whereas
DZ was injected 30 min prior to testing in the first trial. The data are expressed as
mean ± SEM values. #
p < 0.05, ##
p < 0.01 vs. DZ-treated group (Bonferroni’s test).
observed that 7-NI inhibited the amnesic effects of FNZ on rats in
the NOR test.
The presented results, while indicating a different orientation of
the interactions between NO and DZ or FNZ are consistent with the
previous data [2]. Our earlier studies demonstrated acquisition to
be impaired by DZ and FNZ in a mEPM task and that INOS (l-NAME
and 7-NI) enhanced DZ-induced, but prevented FNZ-induced mem-
ory impairment [2]. An mEPM task evaluates spatial and emotional
learning performance because it is based on the natural aversion
of rodents to open and elevated spaces [31]. Scientists suggested a
dichotomy in the temporal lobe and in the prefrontal structures,
mediating object and spatial memory. It is, therefore, plausible
that recognition memory and spatial memory processes activate
different parts of rat brain [32].
Our findings of the interactions between l-arg and DZ are
in agreement with a lot of literature data, indicating that NO
donors (molsidomine, S-nitroso-N-acetylpenicillamine (SNAP), the
novel NO donor (3-(4-hydroxy-3-methoxyphenyl)-2-propenoic
acid 5-(nitrooxy) butyl ester) NCX-2057) counteract the memory
acquisition deficits induced by other compounds, shown in rats in
the NOR task [33–36]. For instance, it was shown that systemic
administration (ip) of molsidomine reversed scopolamine [33] -
and baclofen [34] - induced acquisition deficits in NOR. Moreover,
Yildiz Akar et al. [25] showed that l-arg (200 mg/kg, ip) counter-
acted the negative effects of 7-NI (5 mg/kg, ip) on memory in rats
in a three-panel runway task. In our experimental conditions, l-arg
(125, 250 and 500 mg/kg), when given alone, had no impact on the
NOR behaviour.
Similarly, our findings of the interactions between 7-NI and DZ
are comparable with a number of previous reports, indicating that
7-NI did inhibit the acquisition phase of learning in different rodent
models (passive avoidance, mEPM, 14-unit T-maze, three-panel
Fig. 6. The influence of 7-NI pretreatment (10, 20 and 40 mg/kg, ip) FNZ (0.1 mg/kg,
sc)-induced deficits in the object recognition task (A). Total exploration time dis-
played by different groups of rats in the object recognition task in T2 (B). 7-NI was
injected 5 min prior to administration of FNZ, whereas FNZ was injected 30 min prior
to testing in the first trial. The data are expressed as mean ± SEM values. **
p < 0.01
vs. saline control group; #
p < 0.05 vs. FNZ-treated group (Bonferroni’s test).
runway test, Y-maze and object recognition test) [25,30,37–39].
What is more, Hölscher [40] showed that a systematic administra-
tion of 7-NI exerts an almost complete block of LTP.
Moreover, the present proamnesic effect of 7-NI on DZ-treated
rats and the suppressed effect of l-arg on the amnesic action of
DZ are consistent with the results of earlier experiments, car-
ried out at our Laboratory. Previous studies have indicated that
some effects of BZs, such as anticonvulsant, hypnotic or antinoci-
ceptive, seem to be modulated by the NO system. Talarek et al.
[20–22] showed the administration of both non-selective INOS: l-
NAME, NG-nitro-l-arginine (l-NOARG) and selective NOS inhibitor:
7-NI, to significantly increase the duration of DZ-, chlordiazepoxide
(CDZ)- and clonazepam (CZ)-induced sleep, in the anticonvulsant
effect of DZ and in the antinociceptive activity of DZ, CDZ and CZ.
What is more, most of those effects were reversed by l-arg.
Our findings, showing that 7-NI successfully (at all used doses)
reversed the FNZ (0.1 mg/kg)-induced memory deficit in the rats
subjected to NOR task, are difficult to explain. That effect was
completely opposite to the interactions between 7-NI and DZ, as
presented in this paper. But, this is consistent with the recent evi-
dence reporting that peripheral acute administration of l-NAME,
at a low dose range (1–3 mg/kg, ip) reversed recognition mem-
ory deficits produced by the NMDA receptor antagonists MK-801
and ketamine in the NOR task in rats [41]. Also, both l-NAME (1
and 3 mg/kg, ip) and 7-NI (1 and 3 mg/kg, ip) antagonized perfor-
mance deficits-induced by dopamine agonist apomorphine in NOR
test in rats [42]. Importantly, pre-test administration of l-NAME,
at the low dose (0.25 ␮g/mouse, intra-CA1), reversed the mem-
ory impairment induced by muscimol - GABAA receptor agonist
in mice, in the passive avoidance test [43]. In addition, it has shown
that l-NAME decreased the memory impairment of mice induced

7. J. Orzelska et al. / Behavioural Brain Research 284 (2015) 179–186 185
by morphine, in the water maze task in mice [44] and in rats, in
the inhibitory avoidance task [45]. Also, it was determined that l-
NAME (10 mg/kg, ip) reversed the beneficial effects of pioglitazone
on acquisition memory impaired by morphine in mice, in both Y-
maze and passive avoidance tasks [46] and by scopolamine [47] in
a passive avoidance test.
However, there is a controversy over the precise role of INOS
in memory processes. It should be underlined that INOS were
reported to disrupt acquisition of new tasks but these findings
are discrepant in this context. Some studies have found that INOS
(l-NAME, 7-NI, l-NOARG) given systemically prior to training, do
not change learning performance in different rodents models of
memory [48–50]. In our experimental conditions, 7-NI (10, 20 and
40 mg/kg), when given alone, had no impact on the NOR behaviour.
The recent evidence reporting that peripheral acute administration
of INOS, at a low dose range (1, 3 and 10 mg/kg) did not affect
animal’s performance in the NOR test [41]. Interestingly, a pre-
test administration of l-NAME, at the low dose (0.25 ␮g/mouse,
intra-CA1), alone cannot affect memory, whereas at the higher
doses (0.5 and 1 ␮g/mouse, intra-CA1) impaired learning perfor-
mance of mice [43]. In this context, research into the role of INOS
in the induction of LTP is also discrepant. Block of NOS has been
reported to impair the induction of LTP in some studies. Other
studies observed a partial block of LTP, while yet other stud-
ies did not find and effect of INOS under any conditions tested
[51]. Despite considerable evidence for NO involvement in at least
some forms of memory processing its required function is not
known.
It should be noted that much remains unknown, regarding the
exact role of the NO signalling system in the CNS [15]. Some reports
assume the release of GABA to be biphasically dependent on NO
concentrations. Getting et al. [52] showed that l-NAME at low con-
centration increased, whereas at higher concentration decreased
basal GABA release. On the contrary, high concentrations of the
NO donor (SNAP) enhanced GABA outflow [53]. In addition, the
interaction between BZs and NO seems to be a more complex pro-
cess, as NO also modulates the release of other neurotransmitters
involved in memory processes, such as acetylcholine and glutamate
(for review, see [51]).
The above-mentioned discrepancy between NO and DZ or FNZ
might have been associated with their selective actions at spe-
cific GABAA receptor subunits. Each GABAA receptor subtype has
got a distinct pattern of expression within the mammalian brain,
suggesting a predefined physiological role. The ␣5 subunit of the
GABAA receptor is found mainly in the hippocampus and it is sug-
gested to be involved in memory process. Genetically modified
mice with a partial or full deficit of ␣5GABAA receptors showed an
improved performance in various associative learning and mem-
ory tasks [54,55]. Other results have indicated that DZ-induced
anterograde amnesia is mediated by ␣1GABAA receptors [56].
FNZ is very potent agonist of GABAA receptor with a high ability
to cause anterograde amnesia, thus called a date-rape drug [57,58].
In addition, our previous results concerning FNZ were also unex-
pected because of increased locomotor activity of mice after the
first injection of FNZ (1 mg/kg), whereas the first injection of DZ
(10 mg/kg) caused sedative effect in mice, in the same experiment
[2]. Moreover, Hauser et al. [59] suggested that FNZ could act as
either an agonist or inverse agonist, depending on the GABAA recep-
tor configuration.
It is important to note the lack of l-arg effect on rat performance
in the NOR task after FNZ injection (0.05 mg/kg). A subthreshold
dose of FNZ was used because, based on previous results [2], l-arg
effects were expected.
What is more, the pharmacokinetic interaction between the
used modulator of NO action and BZs may be one contributing
factor in the incongruities of experimental findings [60]. However
future studies are needed, to check BZ concentration in rat blood
after co-administration of l-arg or 7-NI with DZ and FNZ.
Even more, our results rule out the possibility that the inter-
actions between NO -related compounds with BZs in NOR task
might be caused by motivational factors. Total exploration times
displayed by all groups during the choice trail (T2) were unchanged.
Additionally, it is important to mention that l-arg and 7-NI given
alone and in combination with BZs did not affect rats motility.
Therefore, interactions between l-arg or 7-NI with BZs in NOR task,
do not seem to be modified by non-specific factors. But, it is one
exception–the inhibitory effect of 7-NI at a dose of 40 mg/kg on
the amnesic action of FNZ (0.1 mg/kg). The combination of 7-NI
(40 mg/kg) with FNZ (0.1 mg/kg) decreased locomotor activity of
rats. In this case, it could not be excluded that other factors might
have affected animals’ performance in NOR test.
One may consider that the effect of 7-NI on DZ or FNZ perfor-
mance in the NOR task is due to its influence on blood pressure but
it has been shown that 7-NI does not affect blood pressure at doses
up to 80 mg/kg [61].
To sum up, these findings led us hypothesize that the inhibition
of NO synthesis may induce the amnesic effects of DZ, while pre-
venting FNZ memory impairment of rats, submitted to NOR task
activities.
Acknowledgement
The reported study was supported by Grant No. NN 405091740
from the Polish Ministry of Science and Higher Education.
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