Full text of DOI:10.1038/sj.bjp.0704739

British Journal of Pharmacology (2002) 136, 764 ± 772
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Central injections of nocistatin or its C-terminal hexapeptide exert
anxiogenic-like e€ect on behaviour of mice in the plus-maze test
1
3
1Elaine C. Gavioli, Giles A. Rae, 2,3Girolamo Calo', Remo Guerrini & *,1Thereza C.M. De Lima
2
1Department of Pharmacology, CCB, Universidade Federal de Santa Catarina, 88015-420, Florianopolis, Brazil; Department
of Experimental and Clinical Medicine, Section of Pharmacology and Neuroscience Center and Department of Pharmaceutical
Sciences and Biotechnology Center, University of Ferrara, 44100 Ferrara, Italy
3
1
Nocistatin (NST) antagonizes several actions of nociceptin/orphanin FQ (N/OFQ), but acts on
distinct receptors. As N/OFQ exerts anxiolytic-like actions in various tests, its behavioural actions in
the elevated plus-maze (EPM) test were compared with those of bovine NST.
2
Five minutes after i.c.v. treatment, mice were placed on the EPM for 5 min and entries into and
time spent on open and closed arms were recorded alongside other parameters.
NST (0.1 ± 3 pmol) reduced percentages of entries into (control 39.6+3.1%, peak e€ect at
3
1 pmol NST 8.5+2.9%) and time spent on open arms (control 30.8+2.3%, NST 2.7+1.5%). The
C-terminal hexapeptide of NST (NST-C6; 0.01 ± 10 pmol) closely mimicked these actions of NST,
with peak e€ects at 0.1 pmol.
4
N/OFQ (1 ± 100 pmol) increased percentages of entries into (control 38.5+3.4%; peak e€ect at
10 pmol N/OFQ 67.9+4.9%) and time spent on open arms (control 32.0+3.8%; N/OFQ
74.9+5.8%). Closed arm entries, an index of locomotor activity, were unchanged by all peptides.
5
E€ects of NST or NST-C6, but not N/OFQ, were still detectable 15 min after injection.
Behaviour of animals co-injected with NST (1 pmol) or NST-C6 (0.1 pmol) plus N/OFQ (10 pmol)
was indistinguishable from that of controls.
6
These results reveal potent anxiogenic-like actions of NST and NST-C6, and con®rm the
anxiolytic-like properties of N/OFQ. As NST and N/OFQ both derive from preproN/OF, anxiety
may be modulated in opposing directions depending on how this precursor is processed.
British Journal of Pharmacology (2002) 136, 764 ± 772
Keywords: Anxiety; plus-maze; mice; nociceptin/orphanin FQ; nocistatin; NOP receptor; nocistatin C-terminal hexapeptide
Abbreviations: DZP, diazepam; EPM, elevated plus-maze; N/OFQ, nociceptin/orphanin FQ; NST, nocistatin; NST-C6,
nocistatin C-terminal hexapeptide; PBS, phosphate-bu€ered saline; ppN/OFQ, prepronociceptin/orphanin FQ;
PTZ, pentylenetetrazol
Introduction
Prepronociceptin/orphanin FQ (ppN/OFQ) contains the
sequences of at least three distinct mature peptides,
nociceptin/orphanin FQ (N/OFQ), nociceptin II (or orphanin
FQ2) and nocistatin (NST) (Florin et al., 1997; Okuda-
Ashitaka et al., 1998; for review see Calo' et al., 2000).
Though structurally similar to dynorphin A, the hepta-
decapeptide N/OFQ does not bind to the classical m-, d- and
k-opioid receptors, but acts as a highly selective ligand for
the opioid-like G protein-coupled NOP receptor (also known
as ORL1 or OP4 receptor; Meunier et al., 1995; Reinscheid et
al., 1995; Cox et al., 2000). To the best of our knowledge, the
binding sites/receptors underlying the actions of the hepta-
decapeptide nociceptin II have not yet been identi®ed. NST,
on the other hand, has been shown to associate with speci®c
binding sites in membranes from mouse brain and spinal
chord which are clearly distinct from the NOP receptors
(Okuda-Ashitaka et al., 1998). The length of NST varies
considerably among species (comprising 17, 30, 35 and 41
amino-acid residues in oxen, humans, rats and mice,
respectively), but the last six residues of the C-terminal are
fully conserved in all variants (Okuda-Ashitaka & Ito, 2000).
Importantly in this regard, synthetic NST C-terminal
hexapeptide (NST-C6) has been shown to retain the
biological agonistic activity of full-length bovine NST
(Okuda-Ashitaka et al., 1998).
Intracerebroventricular injections of N/OFQ cause various
behavioural e€ects in rodents, including stimulation of food
intake (Pomonis et al., 1996), inhibition or stimulation of
spontaneous locomotion (Reinscheid et al., 1995; Florin et
al., 1996), antagonism of the reinforcing properties of ethanol
or morphine (Ciccocioppo et al., 1999; Murphy et al., 1999)
and impairment of spatial learning (Sandin et al., 1997),
among other actions. The peptide also a€ects nociceptive
transmission/perception, inducing either hyperalgesia (Meu-
nier et al., 1995; Reinscheid et al., 1995) or reversal of
analgesia mediated or induced by opioids (Mogil et al., 1996;
Grisel et al., 1996). Given intrathecally, however, N/OFQ
appears to exert a dual e€ect comprised of hyperalgesia/
allodynia at low doses and a clear antinociceptive in¯uence at
higher doses (Okuda-Ashitaka et al., 1996; Erb et al., 1997;
Sakurada et al., 1999). Both of these spinal e€ects of N/OFQ
*Author for correspondence at: Department of Pharmacology
±
CCB, Universidade Federal de Santa Catarina, Rua Ferreira Lima,
82 ± Centro, 88015-420 ± Florianopolis ± SC ± Brazil;
E-mail: thereza@farmaco.ufsc.br

E.C. Gavioli et al
Anxiogenic-like effect of nocistatin in mice
765
are absent in NOP receptor null mutant mice (Inoue et al.,
1999). Interestingly, although the other ppN/OFQ-derived
peptide, NST, does not bind to the NOP receptor, it seems to
act as a functional antagonist of N/OFQ. Indeed, several
biological e€ects of N/OFQ are antagonized by NST, such as
nociception in various experimental conditions (Martin et al.,
1998; Okuda-Ashitaka et al., 1998; Yamamoto & Sakashita,
1999; Nakano et al., 2000), morphine analgesia (Zhao et al.,
1999), N/OFQ impairing e€ects on learning/memory (Hir-
amatsu & Inoue, 1999), food intake (Olszewski et al., 2000)
and glutamate release (Nicol et al., 1998).
Jenck et al. (1997) have described anxiolytic-like e€ects of
i.c.v. N/OFQ administration in various rodent models of
anxiety, such as light-dark place preference, elevated plus-
maze (EPM), novel environment exploration and operant
con¯ict tests. A similar pro®le of action was observed with a
non-peptidic synthetic agonist for the NOP receptor (Jenck et
al., 2000). To date, the in¯uence of NST on anxiety-related
behaviour has not yet been examined.
(1986) and employed previously by our group (Teixeira et al.,
1996; Ribeiro & De Lima, 1998). In brief, under light ether
anaesthesia (i.e. just sucient for loss of the postural re¯ex),
a 27 gauge needle attached to a 10 ml Hamilton syringe was
inserted perpendicularly 3 mm deep through the skull, at a
position 2 mm lateral from the midline on the line drawn
through the anterior base of the ears. Each animal only
received one i.c.v. injection. Upon termination of the
experiment (including testing on the rota-rod apparatus, see
below), each mouse was decapitated and its brain examined a
fresco. Results from mice presenting cannula misplacement or
any signs of cerebral haemorrhage were discarded from the
statistical analysis (less than 5% of the animals overall).
The EPM test
The putative anxiolytic or anxiogenic activity of the various
drug treatments was assessed using the EPM test, as adapted
for the mouse by Lister (1987). This test is based on the natural
aversion of rodents for open spaces. The EPM was made of
Plexiglas and consisted of two opposed open arms (30656
0.25 cm) and two opposed closed arms (3065615 cm), all
facing a central platform (565 cm), elevated 45 cm from the
¯oor. The apparatus was placed in a small closed room lit by a
15 W red light and could be adequately viewed by the
experimenter through a glass window. Five, 15 or 30 min after
the i.c.v. treatment, each mouse was placed on the central
platform, facing a closed arm, and observed for a 5 min period.
The frequencies of entry into either open or enclosed arms, as
well as the times spent in each arm type were recorded (in s). An
entry was scored as such only when the animal placed all four
limbs into any given arm. The incidence of ethological
parameters such as time spent on the central platform,
grooming, unprotected head-dipping (i.e. an exploratory
forward head/shoulder movement over the side an open arm
of the maze directed down towards the ¯oor) and protected
stretch-attend postures (i.e. when animal stretches forward and
retracts to original position without actually walking forward,
a behaviour which occurs in or from the relative security of the
closed arms or central platform of the maze) were also recorded
(Cole & Rodgers, 1994).
The ratios `time spent in the open arms/time spent in all (i.e.
open and closed) arms' and `frequency of entries into open arms/
total entries into all arms' were calculated and multiplied by
100, to yield the percentages of time spent in and of frequency
of entries into open arms, respectively. Both parameters are
considered to re¯ect fear-induced inhibition from entering the
open arms and can be related to the `anxiety' level experienced
by the animal (Rodgers & Dalvi, 1997). Drugs with anxiolytic-
like activity usually increase the time spent in and/or frequency
of entries into open arms, whereas the reverse holds true for
anxiogenic-like drugs. Furthermore, the number of entries into
closed arms was used and an index of general activity. All
sessions were also videotaped using a an infra-red video camera
(Philco, model PVC-4H10, Manaus, Brazil), to enable play-
back when necessary.
The present study has therefore investigated the e€ects of
i.c.v. injections of bovine NST and NST-C6 on behaviour of
mice submitted to the EPM test, comparing them with those
elicited by N/OFQ over a broad range of doses. The EPM is
a widely accepted paradigm of anxiety states (for review see
Rodgers & Dalvi, 1997).
Methods
Animals
Experiments were conducted with male Swiss mice (30 ± 40 g),
raised under controlled environmental conditions (12 h light ±
dark cycle, with lights on from 700 h; room temperature set at
22+28C). Food and water were available ad libitum, except
during the experiments. Animals were transferred to the
laboratory (at the Universidade Federal de Santa Catarina)
at least 24 h before testing, and all experimental observations
were carried out between 1300 and 1700 h. Animal housing
conditions and all experimental procedures were previously
approved by the Ethics Committee on Use of Animals in
Research of the Universidade Federal de Santa Catarina and
were carried out in accordance with the Guiding Principles for
the Care and Use of Animals approved by the Brazilian Society
of Neuroscience and Behaviour (1992).
Treatments
NST (10 fmol to 1 nmol), NST-C6 (1 fmol to 1 nmol) or N/
OFQ (100 fmol to 3 nmol) were injected i.c.v. into the right
brain lateral ventricle in
Alternatively, in some experiments, mice were given
a constant volume of 2 ml.
a
combined i.c.v. injection of N/OFQ (10 pmol) plus either
NST (1 pmol) or NST-C6 (0.1 pmol). Other animals received
i.c.v. injections of either diazepam (DZP; 7 nmol) or
pentylenetetrazol (PTZ; 200 nmol), as reference anxiolytic
and anxiogenic treatments, respectively (Teixeira et al., 1996).
Control mice were always similarly treated with vehicle alone
(phosphate-bu€ered saline; PBS) and tested in parallel with
drug-treated animals. All i.c.v. injections were carried out
Motor co-ordination
Immediately after completion of the elevated plus-maze test
session, mice treated with NST, NST-C6, N/OFQ or vehicle
were placed on the revolving bar (diameter 2.5 cm, 12 r.p.m.)
using the `free hand' technique proposed by Haley
McCormick (1957), as modi®ed by Laursen & Belknap
&
British Journal of Pharmacology vol 136 (5)

766
E.C. Gavioli et al
Anxiogenic-like effect of nocistatin in mice
of a rota-rod apparatus for 1 min, and both the latency to
fall and number of falls from the revolving bar were recorded
and used as indices of motor coordination.
concentrations in PBS just prior to use. NST was dissolved in
PBS plus 5% NaHCO3 (50:1) as stock solution.
Data analysis
Results
Results were analysed by Graphpad INSTAT1 version 2.05
software. All data presented are expressed as the mean+
s.e.m., and each value re¯ects the mean of six to eight
animals per group. In all cases, the means were compared by
a one-way analysis of variance (ANOVA), followed by
Bonferroni's test. In the co-injection experiments, additional
statistical evaluation was carried out using the two-tailed
Student t-test for unpaired samples, in which the means of
each co-injected group were compared to those of the
corresponding vehicle-treated control groups. Di€erences
were considered signi®cant when P50.05.
Effects of NST and NST-C6 on behaviour in the EPM
test
The i.c.v. administration of NST (0.1 to 3 pmol) induced
signi®cant decreases in the percentages of time spent in open
arms and of entries into open arms, in addition to reductions in
the number of head-dippings (F(7,62)=6.20, P50.0001;
F(7,62)=7.09, P50.0001; F(7,62)=1.77, P40.001, respectively).
As shown in Figure 1, the dose-response curves for each of
these e€ects of NST were clearly U-shaped, with peak
reductions at 1 pmol and higher doses (up to 100 pmol)
evoking progressively lesser e€ects. In contrast, no signi®cant
e€ects on locomotor activity, as assessed by the number of
closed arm entries, were detected over the full range of NST
doses tested (F(7,62)=1.04, P40.05). As shown in Figure 2,
qualitatively similar results were obtained with NST-C6, which
also decreased the percentages of time spent in and of entries
into the open arms, as well as reduced the number of head-
dipping, at doses ranging from 0.01 to 1 pmol (F(8,63)=9.40,
P50.0001; F(8,63)=16.57, P50.0001; F(8,63)=3.70, P50.001,
respectively). The dose-response curves for these NST-C6-
induced e€ects were U-shaped, as seen with NST, but peak
Drugs
The following drugs were used: bovine NST (from Tocris
Cookson Ltd., Bristol, U.K.), NST-C6 (from American
Peptide Company, Sunnyvale, CA, U.S.A.), N/OFQ (synthe-
sized by Dr R. Guerrini, Department of Pharmaceutical
Sciences, University of Ferrara), DZP and PTZ hydrochlor-
ide (from Sigma Chemical Co., St. Louis, MO, U.S.A.). All
drugs were dissolved in PBS, and stock solutions of NST-C6
and N/OFQ were stored at 7208C and diluted to the desired
Figure 1 E€ects of nocistatin (NST) on the percentages of time spent in (a) and entries into (b) open arms, and on the number of
head-dippings (c) and entries into enclosed arms (d) in mice placed, for 5 min, in the elevated plus-maze 5 min after i.c.v. injection.
Control mice were treated with vehicle only (PBS). Results obtained following similar injection of pentylenetetrazol (PTZ; 200 nmol)
are also shown for comparison. Each value represents the mean+s.e.m. of 6 ± 8 animals. *P50.05 as compared to the PBS-treated
control group (one-way ANOVA followed by Bonferroni's test).
British Journal of Pharmacology vol 136 (5)

E.C. Gavioli et al
Anxiogenic-like effect of nocistatin in mice
767
reductions were obtained at a lower dose (0.1 pmol). The
reference anxiogenic drug, PTZ (200 nmol, i.c.v.), also
decreased the percentages of time spent in and of entries into
open arms (Figure 1). NST and NST-C6, over the full range of
doses tested, failed to in¯uence either locomotor activity, as
assessed by the number of closed arm entries (F(7,62)=1.04,
P40.05; F(8,62)=1.42, respectively; P40.05; Figures 1 and 2).
In addition, NST (but not NST-C6) increased the time spent on
the central platform, NST-C6 (but not NST) augmented the
time animals spent in the closed arms, and both peptides
reduced the number of total arm entries into and the time spent
in open arms (Table 1 shows only results for 1 and 0.1 pmol of
NST and NST-C6, respectively).
percentages of time spent in open arms and, at 10 pmol
only, also increased the frequency of open arm entries
(F(7,44)=10.96 and F(7,44)=5.95, respectively; P50.0001 in
both cases), as well as the incidence of head-dipping
behaviour (F(7,44)=2.18, P50.05). The dose-response
curves for each of these e€ects of N/OFQ were typically
bell-shaped, such that no signi®cant changes were detected
following injection of 1 nmol of this peptide. Moreover,
none of the doses of N/OFQ tested (0.1 pmol ± 1 nmol)
a€ected the number of closed arm entries (F(7,44)=1.15;
P40.05), but at 10 pmol the peptide signi®cantly
augmented the number of entries into and time spent in
open arms, as well as reduced both the time animals spent
in the closed arms and the incidence of stretch-attend
postures (Table 1 shows results for 10 pmol dose only).
The reference anxiolytic drug DZP (7 nmol, i.c.v.), like N/
OFQ, also increased the percentage of time spent in open
arms (Figure 3).
Effects of N/OFQ on behaviour in the EPM test
As shown in Figure 3, i.c.v. administration of N/OFQ, at
either 10 or 100 pmol, induced signi®cant increases in the
Figure 2 E€ects of nocistatin C-terminal hexapeptide (NST-C6) on the percentages of time spent in (a) and entries into (b) open
arms, and on the number of head-dippings (c) and entries into enclosed arms (d) in mice placed, for 5 min, in the elevated plus-maze
5 min after i.c.v. injection. Control mice were treated with vehicle only (PBS). Each value represents the mean+s.e.m. of 6 ± 8
animals. *P50.05 as compared to the PBS-treated control group (one-way ANOVA followed by Bonferroni's test).
Table 1 In¯uence of i.c.v. injection of nocistatin (NST, 1 pmol), its C-terminal hexapeptide (NST-C6, 0.1 pmol) and nociceptin/
orphanin FQ (N/OFQ, 10 pmol) on various behavioural parameters displayed by mice submitted to the EPM test. All values are
expressed as mean+s.e.m. of 7 to 12 observations. For e€ects on other parameters see Figures 1, 2 and 3
Behavioural parameter
Vehicle
NST
NST-C6
N/OFQ
Total arm entries
Closed arm time (s)
Open arm entries
Open arm time (s)
Time in central platform (s)
Stretched-attend postures
Grooming
15.5+1.1
191+11
6.0+0.7
83.0+5.7
26.7+7
9.6+1.7
1.5+0.4
9.0+1.6*
216+16
0.8+0.3*
5.0+2.6*
78+15*
12.6+2.0
0.9+0.2
8.2+1.4*
273+8*
0.8+0.4*
8.4+6.1*
18+6
12.0+1.4
0.6+0.2
20.7+2.9
61+12*
14.0+2.3*
193.5+21.7*
45+12
4.3+0.6*
1.1+0.3
*P50.05 relative to vehicle-treated control (one-way ANOVA followed by Bonferroni's test).
British Journal of Pharmacology vol 136 (5)

768
E.C. Gavioli et al
Anxiogenic-like effect of nocistatin in mice
Figure 3 E€ects of nociceptin/orphanin FQ (N/OFQ) on the percentages of time spent in (a) and entries into (b) open arms, and
on the number of head-dippings (c) and entries into enclosed arms (d) in mice placed, for 5 min, in the elevated plus-maze 5 min
after i.c.v. injection. Control mice were treated with vehicle only (PBS). Results obtained following similar injection of diazepam
(DZP; 7 nmol) are also shown for comparison. Each value represents the mean+s.e.m. of 6 ± 8 animals. *P50.05 as compared to
the PBS-treated control group (one-way ANOVA followed by Bonferroni's test).
Time-course of behavioural actions of NST, NST-C6 and
N/OFQ
(Figure 5). Co-injection of either NST or NST-C6 with N/
OFQ also did not a€ect locomotor activity (i.e. number of
closed arm entries) or the incidence of any of the ethological
parameters recorded (results not shown).
The doses of NST, NST-C6 or N/OFQ that induced the
most prominent behavioural e€ects (i.e. at 1, 0.1 and
10 pmol, respectively) were selected to assess the time-courses
of their actions on performance in the EPM. As shown in
Figure 4, both NST and NST-C6 still exerted pronounced
anxiogenic-like actions in animals placed in EPM up to
15 min after i.c.v. injection (per cent time spent in open arms
Influence of NST, NST-C6 and N/OFQ on performance
in the rota-rod test
None of the doses of NST or NST-C6 tested a€ected motor
performance of mice submitted to the rota-rod test (Table 2
shows only results obtained with the maximally-e€ective
anxiogenic-like doses and highest doses tested). Likewise, N/
OFQ also failed to decrease motor performance at doses
causing anxiolytic-like e€ects (10 or 100 pmol), but at 1 or
3 nmol this peptide signi®cantly increased the number of falls
from and decreased the time spent on the revolving bar.
Furthermore, at both of these higher doses, N/OFQ induced
sporadic muscle jerks and severe alterations in the locomotor
activity, as has been previously described (Reinscheid et al.,
1995).
F(3,28)=29.8 and F(3,37)=18.64; per cent frequency of open
arm entries (3,28)=24.3 and F(3,37)=10.83, respectively;
F
P50.0001 in all cases). The intensity of these e€ects at
15 min was similar to that seen 5 min after injection of either
peptide, whereas no residual behavioural e€ects were
detected in animals tested 30 min after administration. In
contrast, the anxiolytic-like e€ects of N/OFQ were only
evident in animals tested 5 min after injection (per cent time
spent in open arms
frequency of open arm entries
F
(3,26)=8.85, P50.005; per cent
(3,26)=6.39, P50.01),
F
subsiding completely in animals tested in the apparatus 15
or 30 min after administration.
Discussion
Effects of co-injections of NST or NST-C6 together with
N/OFQ on behaviour in the EPM test
The results of the current study demonstrate, to the best of
our knowledge for the ®rst time, that NST is a remarkably
potent anxiogenic-like compound in mice. In addition, we
have found that NST-C6, a sequence which is fully conserved
in the distinct NST isoforms of all species studied to date
(Okuda-Ashitaka & Ito, 2000), shares the agonistic properties
of the full-length bovine peptide in the EPM test. Conversely,
we also con®rmed the previously reported anxiolytic-like
Animals given co-injections of either NST (1 pmol) or NST-
C6 (0.1 pmol) together with N/OFQ (10 pmol) and tested in
the plus-maze 5 min after administration failed to exhibit
alterations in the percentages of time spent in open arms or
in the frequency of open arm entries, when compared to
performance of their respective vehicle-treated control groups
British Journal of Pharmacology vol 136 (5)

E.C. Gavioli et al
Anxiogenic-like effect of nocistatin in mice
769
Figure 5 E€ects of single or combined treatment with nocistatin
(NST, 1 pmol), nocistatin C-terminal hexapeptide (NST-C6,
0.1 pmol) and/or nociceptin/orphanin FQ (N/OFQ, 10 pmol) on
the percentages of time spent in (a) and of entries into (b) open arms
in mice placed, for 5 min, in the elevated plus-maze 5 min after i.c.v.
(co-injection). Control mice were treated with vehicle only (PBS).
Each value represents the mean+s.e.m. of 6 ± 8 animals. *P50.05 as
compared to the PBS-treated control group (non-paired two-tailed
Student's t-test).
Figure 4 Time-course of the e€ects of nocistatin (NST, 1 pmol),
nocistatin C-terminal hexapeptide (NST-C6, 0.1 pmol) and nocicep-
tin/orphanin FQ (N/OFQ, 10 pmol) on the percentages of time spent
in (a) and of entries into (b) open arms in mice placed, for 5 min, in
the elevated plus-maze at the times after i.c.v. injection indicated.
Vehicle-treated control mice were evaluated at 5 min after injection
only. Each value represents the mean+s.e.m. of 6 ± 8 animals.
*P50.05 as compared to PBS-treated control group (one-way
ANOVA followed by Bonferroni's test).
Table 2 In¯uence of i.c.v. injection of nocistatin (NST),
nocistatin C-terminal hexapeptide (NST-C6) and nociceptin/
orphanin FQ (N/OFQ) on performance of mice in the rota-
rod test
pro®le of action of N/OFQ in mice in the EPM test (Jenck et
al., 1997). As NST and N/OFQ are both derived from ppN/
OFQ, these ®ndings strongly suggest that anxiety levels in the
mouse can be modulated, in opposing directions, depending
on how the precursor is processed or on which of the two
systems is more prevalent.
Rota-rod performance
Number of falls Time on the
from revolving bar revolving bar (in s)
Treatment
Dose
Very little is known about the processing mechanisms of
ppN/OFQ. However, pro-hormone convertase 2 appears to
play a key role in N/OFQ generation, as hypothalamic or
amygdala extracts from mice gene-targeted to produce
defective enzyme contain only about 10 ± 30% of the N/
OFQ seen in wild-type controls (Allen et al., 2001).
Furthermore, although ppN/OFQ and biosynthetic inter-
mediates were undetectable in extracts from hypothalamus
of wild-type mice, both entities were present in those from
defective pro-hormone convertase 2 animals. Unfortunately,
the in¯uence of this genetic manipulation on production of
NST was not examined. It is believed that the main sites
of cleavage of ppN/OFQ by pro-hormone convertase 2 lie
between Lys-Arg residues, especially those which separate
NST from N/OFQ and the later peptide from the
remaining N-terminal ppN/OFQ fragment, which also
contains the bioactive nociceptin II sequence (Reinscheid
et al., 2000; Mathis et al., 2001). However, the relative
bio-availability of endogenous NST and N/OFQ in
di€erent brain areas and elsewhere can only be estimated
Control
NST
±
1.0+0.4
1.2+0.5
1.2+0.4
0.8+0.4
0.4+0.2
1.0+0.4
3.2+0.9*
3.3+0.5*
56.2+2.9
54.8+2.4
55.3+1.9
52.2+4.3
58.0+1.0
55.8+1.7
39.8+5.8*
42.3+3.0*
1 pmol
1 nmol
0.1 pmol
1 nmol
10 pmol
1 nmol
3 nmol
NST-C6
N/OFQ
Values are mean+s.e.mean of at least six mice. *P50.05 as
compared to the corresponding PBS-treated control group
(one-way ANOVA followed by Bonferroni's test).
once the various processing steps, including the separation
of NST from the rest of the C-terminal fragment of ppN/
OFQ and the mechanisms of breakdown of both peptides,
are understood.
The anxiogenic-like pro®les of action of NST (and NST-
C6) and the anxiolytic-like ones of N/OFQ were detectable
using three distinct behavioural parameters, namely the
British Journal of Pharmacology vol 136 (5)

770
E.C. Gavioli et al
Anxiogenic-like effect of nocistatin in mice
percentages of time spent on and of entries into the open
arms, as well as the incidence of head-dipping behaviour. All
three peptides appeared to in¯uence anxiety in a selective
fashion, since neither a€ected the number of entries into the
closed arms of the EPM, a parameter which is loaded with
locomotor activity in factorial analysis studies (Wall &
Messier, 2000), even though NST-6C and N/OFQ also
a€ected in opposing ways the time animals spent in closed
arms. Moreover, neither NST nor NST-C6 modi®ed motor
performance on the rota-rod test over the full range of doses
examined, including those causing peak anxiogenic-like
e€ects. On the other hand, N/OFQ signi®cantly depressed
performance in this test only at doses 51 nmol, a ®nding
which is in agreement with previous reports (Reinscheid et
al., 1995; Rizzi et al., 2001a). Indeed, overall, the in¯uences
of both NST-C6 and N/OFQ on anxiety levels were more
clear cut than those of the two reference anxiogenic and
anxiolytic drugs tested, i.e. PTZ and DZP, at least at the
doses of 200 and 7 nmol (i.c.v.), respectively.
It is noteworthy that the dose-response curves demonstrat-
ing the anxiogenic- and anxiolytic-like e€ects of NST (and
NST-C6) and N/OFQ were U- and bell-shaped, respectively.
Similarly, the curve for i.c.v. NST for reversal of the
antagonism of morphine-induced analgesia by N/OFQ in
the rat is also bell-shaped (Zhao et al., 1999), as is that for
the allodynic e€ect of i.t. N/OFQ in mice (Okuda-Ashitaka et
al., 1998). Bell-shaped curves have also been reported for
anxiolytic-like e€ects of i.c.v. N/OFQ (and also for p.o. DZP)
in rats submitted to the elevated plus-maze test and in mice
exposed to a novel environment (following pre-treatment
with urocortin) or submitted to light ± dark aversion or
con¯ict tests (Jenck et al., 1997).
The abilities of both NST and N/OFQ to modulate anxiety
correlate well with histochemical, autoradiographical and
analytical evidence showing high levels of expression of ppN/
OFQ, N/OFQ, NST peptides and NOP binding (receptor)
sites in several brain areas related to emotionality. In situ
hybridization for ppN/OFQ mRNA in mouse brain has
revealed particularly intense labelling of neurones of the
septum and septo-hippocampal and central amygdaloid
nuclei, as well as less pronounced levels in hippocampus
(Boom et al., 1999; Koster et al., 1999). These ®ndings are in
good agreement with the labelling of both ppN/OFQ mRNA
and mature N/OFQ seen in rat brain (Neal et al., 1999a) and
with the detection of relatively high levels of mature NST in
bovine septum and hippocampus (Lee et al., 1999). The
distribution of NOP receptor mRNA or receptor-like binding
sites in the brain appears to be somewhat more widespread
than that of ppN/OFQ mRNA or of N/OFQ, but also
encompasses the septum, hippocampus and amygdala (Neal
et al., 1999b). On the other hand, there are as yet no reports
on the relative distribution of NST binding sites throughout
the brain, although selective binding of bovine NST has been
demonstrated to occur in membranes obtained from mouse
brain and spinal cord (Okuda-Ashitaka et al. 1998).
tively. On the other hand, i.t. administration of an antibody
against NST markedly potentiates (by over 100 fold) i.t. N/
OFQ-induced allodynia in mice (Okuda-Ashitaka et al.,
1998). However, the evidence for a physiological role of
ppN/OFQ-derived peptides in anxiety is controversial. Mice
null-mutated for ppN/OFQ display increased anxiety-related
behaviour when exposed to
a novel environment and
impaired adaptation to repeated stress (Koster et al., 1999),
whereas animals lacking the NOP receptor fail to exhibit any
di€erences in behaviour in the EPM test, relative to wild-type
controls (Mamiya et al., 1998). Several putative reasons could
account for such discrepant ®ndings, including possible
contribution of other ppN/OFQ-derived peptides (NST and
nociceptin II) towards the modulation of anxiety, mediation
of the anxiolytic-like actions of N/OFQ via an as yet
uncharacterized additional receptor type and/or di€erences
in the genetic background of each knockout lineage, among
others. In light of such considerations, it is unclear if the
anxiogenic-like e€ect of NST-C6 seen in the current study
re¯ects functional antagonism of an ongoing anxiolytic-like
in¯uence of endogenous N/OFQ, or is brought about by a N/
OFQ- independent mechanism.
The maximally e€ective anxiogenic-like doses of NST
(1.0 pmol) and NST-C6 (0.1 pmol) were 10 and 100 fold
lower, respectively, than that causing the maximal anxiolytic-
like e€ect of N/OFQ (10 pmol). Furthermore, we also
observed that the behavioural e€ects of both NST and
NST-C6 were longer-lasting than those of N/OFQ. Con-
sidering that ppN/OFQ contains only single copies of the
NST and N/OFQ sequences (Mollereau et al., 1996) and that
levels of mature NST and N/OFQ in brain are similar (Lee et
al., 1999; Quigley et al., 1998), this could be simplistically
interpreted to indicate that NST may be physiologically more
important than N/OFQ in modulating anxiety. However, this
may well depend on their relative bio-availabilities (i.e. rates
of formation and degradation) in the appropriate brain areas.
Mice co-injected with maximally e€ective i.c.v. doses of
either NST or NST-C6 together with N/OFQ failed to
display any behavioural alterations in the EPM test. It thus
seems that the e€ects of NST and N/OFQ on anxiety are
mutually exclusive. Similar antagonistic pro®les of action of
NST have been observed against the e€ects of N/OFQ on
food intake (Olszewski et al., 2000), learning and memory
(Hiramatsu & Inoue, 1999), formalin-evoked pain (Yama-
moto & Sakashita, 1999), prostaglandin E2-induced allodynia
(Okuda-Ashitaka et al., 1998) and glutamate release (Nicol et
al., 1998). It is important to mention that NST can per se
inhibit carrageenan/kaolin-induced hyperalgesia (Nakagawa
et al., 1999) and food intake (Olszewski et al., 2000), but it is
unknown if these e€ects are due to antagonism of
endogenous N/OFQ actions. Moreover, the ®ring rate of
most rat thalamic neurones in vivo is decreased by N/OFQ,
and these same cells are excited by NST (Albrecht et al.,
2001). Nevertheless, this same study also detected a small
proportion of neurones which were excited by N/OFQ, but
unresponsive to NST. Thus, although most e€ects of N/OFQ,
including its e€ects on anxiety, are amenable to antagonism
by NST, some clearly are not.
There is evidence that endogenous N/OFQ and NST might
be physiologically active, at least in certain conditions.
Analgesia induced by morphine or stress in mice, or by
electro-acupuncture in rats, is potentiated by i.c.v. injections
of either the highly selective NOP receptor antagonist
[Nphe1]nociceptin(1-13)NH2 (Rizzi et al., 2000; 2001b) or of
an antibody against N/OFQ (Tian & Han, 2000), respec-
It is important to mention that the current results were
obtained using bovine NST hepta-decapeptide, which
markedly di€ers from the murine 41-residue isoform. Thus,
one cannot rule out entirely the possibility that murine NST
British Journal of Pharmacology vol 136 (5)

E.C. Gavioli et al
Anxiogenic-like effect of nocistatin in mice
771
might display distinct biological properties in this (and other)
species not shared by its bovine counterpart. However, the
fact that the murine and bovine isoforms of NST have been
shown to be roughly equipotent at inhibiting spinal allodynia
induced by i.t. injections of either N/OFQ or PGE2 (Okuda-
Ashitaka et al., 1998), allied to our ®nding that the
anxiogenic-like e€ects seen with bovine NST were mimicked
closely by NST-C6, a sequence which is fully conserved in all
NST isoforms known, would seem to argue against major
di€erences in their bioactivity. Nonetheless, this issue remains
to be adequately addressed by studies which e€ectively
compare the binding characteristics of the various NST
isoforms (and NST-C6) to speci®c binding (receptor) sites, as
well as their biological properties. On the other hand, our
®nding that NST-C6 was actually slightly more potent (3 ± 10
fold) than NST in causing anxiogenic-like e€ects in the EPM
test con®rms previous evidence reported by Okuda-Ashitaka
In conclusion, the results of the current study reveal
remarkably potent anxiogenic-like actions of NST and
con®rm the anxiolytic-like properties of N/OFQ in mice
submitted to the elevated plus-maze test. They also indicate a
potential role of endogenous NST in controlling central
functions related to fear and anxiety. Since ppN/OFQ is the
common precursor of both NST and N/OFQ, the balance
between processing of either peptide may well contribute to
modulation of anxiety. This hypothesis is currently under
investigation in our laboratory. Finally, although receptors
for NST have not yet been characterized, it seems pertinent
to speculate that their blockade may constitute a potential
target for a new class of anxiolytic drugs, and that NST-C6
might be an interesting pharmacological tool to study actions
related to the NST system.
et al. (1998), obtained in
a very di€erent behavioural
paradigm, that this fragment appears to retain the agonistic
properties of bovine NST. Together, both studies present
functional evidence which highlights the usefulness of NST-
C6 as a valuable additional pharmacological tool to study the
NST system.
This study was supported by the Brazilian National Council of
Research (CNPq), who supplied fellowships to E.C. Gavioli. G.A.
Rae and T.C.M. De Lima are recipients of research grants and
fellowships from CNPq.
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(Received November 28, 2001
Revised March 13, 2002
Accepted March 26, 2002)
British Journal of Pharmacology vol 136 (5)