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ORIGINAL INVESTIGATION
Evidence for mediation of nociception by injection
of the NK-3 receptor agonist, senktide, into the dorsal
periaqueductal gray of rats
Gabriel S. Bassi &Ana C. Broiz &Margarete Z. Gomes &
Marcus L. Brandão
Received: 17 July 2008 / Accepted: 1 December 2008 / Published online: 18 December 2008
#Springer-Verlag 2008
Abstract
Rationale Ultrasound vocalizations (USVs) at approxi-
mately 22 kHz are usual components of the defensive
response of rats. However, depending on the neural
substrate that is activated, such as the dorsal periaqueductal
gray (dPAG), USV emissions may be reduced. Activation
of neurokinin-1 (NK-1)-mediated mechanisms of the dPAG
causes analgesia, reduced 22 kHz USVs, and anxiogenic-
like effects in rats exposed to the elevated plus maze
(EPM). Involvement of other types of neurokinin receptors
in this activation has not yet been evaluated.
Objectives The present study examined whether local injec-
tions of the selective NK-3 agonist senktide (1-100 pmol/
0.2 μL) into the dPAG can (1) cause anxiogenic effects in the
EPM, (2) influence novelty-induced 22 kHz USVs, or (3)
change nociceptive reactivity in the tail-flick test.
Results Senktide elicited a significant increase in exploratory
behavior, an effect accompanied by hyperalgesia and an
increase in the number of 22 kHz USVs. The nociceptive
effects, increased locomotor activity, and USV emissions
elicited by local injections of senktide (50 pmol/0.2 μL) were
reduced by prior injections of the selective NK-3 receptor
antagonist SB222200 (50 pmol/0.2 μL) into the dPAG.
Conclusions These findings show that NK-3 receptors in
the dPAG mediate nociceptive responses in this area,
contrasting with the known fear-related processes mediated
by NK-1 receptors in the dPAG. Both hyperalgesia and
fear-related processes are accompanied by emissions of 22
kHz USVs.
Keywords Elevated plus maze .Ultrasonic vocalizations .
NK-3 receptors .Dorsal periaqueductal gray .Senktide
Introduction
The dorsal periaqueductal gray (dPAG) is a central structure
of the so-called brain aversion system responsible for
several behavioral and somatic responses characteristic of
high states of fear in rats (Adams 1979; Bandler and
DePaulis 1988; Graeff et al. 1990; Brandão et al. 2005) cats
(Hunsperger 1956), monkeys (Jurgens and Pratt 1979), and
mice (Miczek et al. 1985). This reaction is similar to those
observed in animals confronted by predators or dangerous
environmental cues and has been associated with panic
attacks (Graeff 2004; Vianna et al. 2001; Brandão et al.
2005). Stimulation of the dPAG also produces antinocicep-
tion considered part of the unconditioned defense reaction
(Fanselow and Helmstetter 1988; Brandão et al. 1990;
Castilho et al. 2002).
The association of pain and fear generated in the dPAG
has led many laboratories to explore the involvement of
tachykinin substance P-mediated mechanisms in these
processes. The three main tachykinins, substance P, neuro-
kinin A, and neurokinin B, constitute a family of neuro-
peptides interacting with three distinct neurokinin (NK)
receptors. Substance P binds preferentially to the NK-1
receptor, NKA binds to the NK-2 receptor, and neurokinin
B binds to the NK-3 receptor (Pennefather et al. 2004;
Regoli et al. 1994). In mammals, substance P is the most
abundant tachykinin in the central nervous system, where it
Psychopharmacology (2009) 204:13–24
DOI 10.1007/s00213-008-1434-y
G. S. Bassi :A. C. Broiz :M. Z. Gomes :M. L. Brandão
Instituto de Neurociências & Comportamento-INeC,
Campus USP,
14040-901 Ribeirão Preto, SP, Brasil
G. S. Bassi :A. C. Broiz :M. Z. Gomes :M. L. Brandão (*)
Laboratório de Psicobiologia, FFCLRP,
Campus USP, Avenida Bandeirantes 3900,
14049-901 Ribeirão Preto, SP, Brasil
e-mail: mbrandao@usp.br
is widely distributed in brain regions involved in the
regulation of affective behavior and the mediation of stress
responses, such as the amygdala, septum, hippocampus,
hypothalamus, and periaqueductal gray (PAG; Barbaresi
1998; Commons and Valentino 2002; Hietala et al. 2005;
Maeno et al. 1993; Nagano et al. 2006; Rigby et al. 2005).
Previous animal studies have shown that exposure to a
variety of aversive and stressful situations alter substance
P transmission in various brain regions (Bannon et al. 1986;
Brodin et al. 1994; Ebner et al. 2004;Krameretal.1998;
Rosen et al. 1992; Siegel et al. 1987). Substantial concen-
trations of substance P have been found in the dPAG
(Ljungdahl et al. 1978;Lietal.1990; Barbaresi 1998). NK-1
and NK-2 receptors are mainly involved in the mediation of
depressant- and anxiety-like behaviors in different animal
species (Ebner and Singewald 2006;Dablehetal.2005;
Kramer et al. 1998; Varty et al. 2002,Duarteetal.2004).
Local injections of substance P agonists into the dPAG
elicit a variety of anxiety-like behaviors in animals,
including conditioned place aversion and less time spent in
the open arms of the elevated plus maze (Aguiar and
Brandão 1996; De Araújo et al. 1998,2001a). Injections
of a C-terminal fragment of substance P also induce
behavioral activation with defensive characteristics (De
Araújo et al. 1999,2001b). The anxiogenic-like effects of
substance P in the dPAG appear to be mediated by NK-1
receptors (Mongeau et al. 1998; De Araújo et al. 2001b;
Duarte et al. 2004; Bassi et al. 2007b). The observation that
NK-1 receptor antagonists may be effective in the treatment
of depression in patients with symptoms of appreciable
anxiety has led to research efforts aimed at the development
of therapies for both depression and anxiety (Kramer et al.
1998; Santarelli et al. 2001; Ranga and Krishnan 2002;
Quartara and Altamura 2006).
An important component of the defensive behavior
repertoire of rats and a reliable measure of anxiety-like
behavior in myomorph rodents is the phenomenon of
ultrasonic vocalizations (USVs), more specifically 22 kHz
vocalizations (van der Poel et al. 1989; Blanchard et al. 1991;
Commissaris et al. 2000; Brudzynski and Chiu 1995; Wohr
et al. 2005; Tomazini et al. 2006). Rats emit vocalizations of
approximately 22 kHz only under experimental conditions
of potential or distal threat, not immediate or proximal threat,
suggesting that these vocalizations are associated with
anxiety rather than fear (Jelen et al. 2003). Such vocaliza-
tions may therefore be relevant to intraspecies signaling or
warning in situations of potential danger (Brudzynski and
Holland 2005).
Depending on the neural substrate of fear/anxiety that is
activated, such as the dPAG and inferior colliculus, USVs
may decrease (Nobre et al. 2003; Bassi et al. 2007a).
Interestingly, activation of NK-1-mediated mechanisms of
the dPAG causes analgesia, a reduction in 22 kHz USVs,
and anxiogenic-like effects in rats exposed to the elevated
plus maze (Bassi et al. 2007b). However, the involvement
of NK-3 receptors in the defensive repertoire induced by
activation of the dPAG has not yet been thoroughly
analyzed. To examine this issue in greater detail, we studied
whether injection of the NK-3 agonist senktide (10–
100 pmol/0.2 μL) into the dPAG induces anxiogenic effects
in rats submitted to the elevated plus maze, changes their
nociceptive reactivity, or influences novelty-induced
22 kHz USVs recorded within the 22-kHz frequency range.
Senktide was chosen because of its preferential affinity
for NK-3 receptors (Regoli et al. 1994; Jenkinson et al.
2000; Yip and Chahl 1999,2001; Sculptoreanu and
de Groat 2007).
Materials and methods
General methodology
Animals
One hundred thirty-one male Wistar rats from the Univer-
sity of São Paulo vivarium were used. The animals were
transported to a local vivarium in the laboratory when they
were 45 days old and weighed an average of 230 g. The
room was maintained under constant temperature (23 ± 1°C)
with a 12:12 h light/dark cycle (lights on between 0700 and
1900 hours). They had free access to food and water
throughout the experiment, and care was taken to ensure
minimal handling and stress (Nunes Mamede Rosa et al.
2005). The rats were housed in groups of five per cage
(40×30×25 cm). The experiments were conducted between
900 and 1400 hours and complied with the recommen-
dations of the Brazilian Society for Neuroscience and
Behavior, which are based on the US National Institutes of
Health Guide for the Care and Use of Laboratory Animals.
Surgery
The animals were anesthetized with tribromoethanol
(250 mg/kg, i.p.) and placed in a stereotaxic frame (David
Kopf, Tujunga, CA, USA). The upper incisor bar was set
3.3 mm below the interaural line such that the skull was
horizontal between bregma and lambda. A unilateral
stainless steel guide cannula (12 mm, 24 gauge) aimed at
the dPAG was implanted in each animal. The cannula was
introduced at an angle of 10° with lambda serving as the
reference for each plane: anterior/posterior −0.3 mm,
medial/lateral 1.2 mm, dorsal/ventral 2.2 mm (Paxinos
and Watson 1997). For all groups, the cannulae were fixed
to the skull by means of acrylic resin and two stainless steel
screws. At the end of surgery, each guide cannula was
14 Psychopharmacology (2009) 204:13–24
sealed with a stainless steel wire to protect it from
blockage. Afterward, the animals were transported to the
vivarium and housed in pairs. Seven days after surgery, the
animals were submitted to the testing sessions. Each animal
was subjected to only one of the tests described below.
Microinjection procedure
The animal was gently wrapped in a cloth and held by the
experimenter for 2 min, and a thin dental needle (0.3 mm,
outside diameter) was introduced through the guide cannula
until its lower end was 1 mm below the guide cannula. The
injection needle was connected to a 5 μL Hamilton syringe
by means of a polyethylene tube. A total volume of 0.2 μL
was used for injections into the dPAG. The solutions were
injected into the dPAG (0.2 μL/min), driven by an infusion
pump (Harvard Apparatus, South Natick, MA, USA). The
displacement of an air bubble inside the polyethylene
catheter (PE-10; Becton-Dickinson, Franklin Lakes, NJ,
USA) connecting the syringe needle to the intracerebral
needle was used to monitor the microinjection. The needle
was held in place for an additional 1 min to maximize
diffusion away from the tip of the needle.
Drugs
The NK-3 receptor agonist senktide (succinyl-[Asp
6
, Me-
Phe
8
]SP
6-11
) was obtained from Sigma (Brazil). The drug
was dissolved and diluted to the desired concentration with
phosphate-buffered saline (pH 7.4) shortly before use.
Senktide was injected at doses of 1, 10, 50, and 100 pmol/
0.2 μL (depending on the group of animals) immediately
before the tests. The doses of senktide were selected based on
pilot experiments and on a previous study (Ribeiro et al. 1999).
Histology
Upon completion of the experiments, the animals were
deeply anesthetized with urethane and perfused intra-
cardially with 0.9% saline followed by formalin solution
(10%). Three hours later, the brains were immersed in 30%
sucrose. Seven days later, the brains were frozen. Serial
60 μm brain sections were cut using a microtome to
localize the positions of the cannulae tips according to the
rat brain atlas (Paxinos and Watson 1997).
Experiment I: involvement of NK-3 receptors of the dPAG
in the modulation of anxiety-related behavior
Apparatus
The elevated plus maze was made of wood and had two
open arms (50×10 cm) perpendicular to two enclosed arms
of the same size with 50-cm-high walls, with the exception
of the central part (10×10 cm) where the arms crossed. The
apparatus was elevated 50 cm above the floor (Pellow et al.
1985; Anseloni and Brandão 1997). The behavior of the
animals was recorded with a video camera (Everfocus, Sao
Paulo, Brazil) positioned above the maze. The signal was
relayed to a monitor in another room via a closed-circuit
television camera to discriminate all forms of behavior.
Luminosity at the level of the open arms of the elevated
plus maze was 20 lx. The maze was cleaned thoroughly
after each test using damp and dry cloths.
Procedure
The effects of senktide injected into the dPAG of rats were
assessed with five groups of animals (n= 8 in all groups):
(1) saline, (2) 1 pmol senktide, (3) 10 pmol senktide,
(4) 50 pmol senktide, (5) 100 pmol senktide. Five minutes
after the dPAG injections, the animals were placed in the
maze for 5-min sessions. Experimental sessions were
conducted between 1200 and 1800 hours. Rats were placed
individually in the center of the maze facing an enclosed
arm and allowed 5 min of free exploration of the maze. An
observer trained to measure ethological elevated plus
maze parameters subsequently scored the videotapes. The
behavioral categories were scored using a software (Noldus,
Amsterdam, The Netherlands) which allowed measurement
of the number of entries in both the open and closed arms
and the time spent in different parts of the maze. An arm
entry or exit was defined as all four paws entering or exiting
an arm, respectively. These data were used to calculate the
percentage of open arm entries and percentage of time spent
in the open arms. A thorough description of the use of the
elevated plus maze test in this laboratory can be found
elsewhere (Anseloni and Brandão 1997).
Statistical analysis
The data obtained in the elevated plus maze test were
analyzed using one-way analysis of variance (ANOVA) for
each variable in the study, followed by the Student–
Newman–Keuls post hoc test. Values of p<0.05 were
considered statistically significant.
Experiment II: involvement of NK-3 receptors in the dPAG
in the generation of 22 kHz ultrasonic vocalizations
Recording of ultrasonic vocalizations
The apparatus used for recording and analyzing USVs
consisted of a testing box (25×15×12 cm) made of steel
bars spaced approximately 12 mm apart. This experimental
chamber was situated inside of a larger, padded, echo-free
Psychopharmacology (2009) 204:13–24 15
(sound-attenuated), ventilated box (60 × 40 × 45 cm) with a
28-W red light bulb located at the top of the chamber. For
recording and analyzing USVs, an Electret ultrasound
microphone (Emkay FG-3629; Avisoft Bioacoustics,
Berlin, Germany) that is sensitive to frequencies of 1–
100 kHz with a flat frequency response was used. The
microphone was connected via an Avisoft UltraSoundGate
116 USB audio device (Avisoft Bioacoustics) to a com-
puter, where acoustic data were displayed in real time by
Avisoft Recorder (version 2.7; Avisoft Bioacoustics) and
were recorded with a sampling rate of 214,285 Hz in 16-bit
format. For acoustical analysis, recordings were transferred
to SASLab Pro (version 4.38; Avisoft Bioacoustics), and a
fast Fourier transformation was performed (512 FFT-length,
100% frame, Hamming window, 75% time window
overlap). Spectrograms were produced at a frequency
resolution of 488 Hz and a time resolution of 0.512 ms.
Call detection was provided by an automatic threshold-
based algorithm (threshold, −10 dB; start/end threshold,
−20 dB) and a hold-time mechanism (hold time, 20 ms).
A lower cut-off frequency of 1 kHz was used for the
analysis of the USV parameters. Various parameters derived
from the average spectrum of the entire element were
determined automatically. The number of calls emitted at
each frequency served as the statistical unit in each
subject. Vocalizations recorded at frequencies below
20 kHz are operationally defined in this study as “audible”
vocalizations, although this term refers to the human
capacities.
The sessions consisted of placing the animals individu-
ally inside the experimental chamber for 15 min. During
each testing session, the microphone was placed through a
hole in the middle of the roof of the chamber, 40 cm above
the floor, to record the entire spectrum of USVs. A video
camera linked to a television was used to monitor all
behavior for the 15-min recording period. The animals were
allocated to four groups: (1) saline (n= 8), (2) 1 pmol
senktide (n= 6), (3) 10 pmol senktide (n=9), and (4)
50 pmol senktide (n=10).
The USV emissions obtained from this experiment were
stored on a hard disk and subsequently transferred to tables
in the Microsoft Excel spreadsheet program (Redmond,
WA, USA) for off-line analysis.
Statistical analysis
Data are expressed as mean ± SEM. Vocalizations were
analyzed by two-way repeated-measures ANOVA. The
group factor refers to treatments, and the condition factor
refers to the recorded frequencies. Student–Newman–Keuls
post hoc comparisons were performed whenever significant
overall Fvalues were obtained. Values of p<0.05 were
considered statistically significant.
Experiment III: influence of NK-3 receptors of the dPAG
on the tail-flick latency test
Tail-flick test
This test was based on D’Amour and Smith’s(1941)
method. Rats were placed in a restraining tube, from which
their tails protruded. Radiant heat was focused on the lower
third of the tail of the animal (Ugo Basile, Varese, Italy).
Movement of the tail activates a photocell, turning off both
the light and a reaction timer. The light intensity was
adjusted to achieve baseline latencies of 2.5 and 3.5 s. A
maximum latency of 6 s (i.e., the cutoff) was established to
minimize tail damage. The baseline trial consisted in
determining the average of three individual tests separated
by a 5-min interval before injection of drug or saline into
the dPAG. After the determination of baseline tail-flick
latency, saline or senktide at 1, 10, and 50 pmol (n=8 in all
groups, with the exception of the saline group that had ten
animals) was microinjected into the dPAG according to
each group assignment. Six other tail-flick latencies were
recorded across the experiments at 5-min intervals. This
procedure has been used successfully in this laboratory
(De Luca-Vinhas et al. 2006; Bassi et al. 2007b).
Statistical analysis
Data are expressed as mean ± SEM. Tail-flick latencies
were analyzed by two-way repeated-measures ANOVA.
The group factor refers to treatments, and the condition
factor refers to the time before and after tail-flick latency
recordings. Student–Newman–Keuls post hoc comparisons
were performed whenever significant overall Fvalues were
obtained. Values of p<0.05 were considered statistically
significant.
Results
Histological examination of the midbrain slices indicated
that all cannulae tips were located within the dorsal portion
of the PAG. The upper panels of Fig. 1show outlines of the
injection sites, and a representative site of microinjection
into the dPAG is shown in the lower panel.
Experiment I
The first experiment was designed to evaluate the influence
of senktide on anxiety-like behaviors of rats tested in the
elevated plus maze. Senktide injection led to increased
exploration of the open and closed arms in this animal
model of anxiety. Figure 2shows a significant increase in
the frequency of entries into both arms (F
4,35
=7.23 and
16 Psychopharmacology (2009) 204:13–24
3.00 for the open and closed arms, respectively; p< 0.05 in
both cases). For both open- and closed-arm entries, post
hoc analyses showed that these effects were caused by
doses of 50 and 100 pmol/0.2 μL. As a consequence, the
statistical analysis of the proportion (percentage) of time
spent and entries into the open arms compared with total
time of the test and entries into both open and closed arms
of the maze did not reveal any significant effect.
Experiment II
Figure 3illustrates the effects of senktide injections on
“audible”(Fig. 3a) and ultrasonic (Fig. 3b) vocalizations.
Whereas all saline-injected rats emitted some vocalizations at
the frequencies of 4–12 kHz during the test sessions, only
12% of the animals injected with saline emitted USVs. Two-
way ANOVA revealed a significant main effect of treatment
on USVs (F
3,116
=8.19, p<0.05) and frequencies (F
4,116
=
7.18, p<0.05). A significant interaction was observed
between treatment and frequencies of USVs (F
12,116
=2.66,
p<0.05). Significant changes were observed in the number
of audible vocalizations caused by treatments (F
3,116
=10.61,
p<0.05) and in the frequency of “audible”vocalizations
(F
4,116
=6.71, p<0.05). A significant interaction was observed
between treatment and frequency in the number of calls
(F
12,116
=2.77, p<0.05). Post hoc comparisons revealed that
senktide injection into the dPAG caused an increase in the
number of USVs recorded at 20–24 kHz and a reduction in
this parameter of “audible”vocalizations at the range of 8–
10 kHz compared with saline-injected control animals.
“Audible”vocalizations (∼0.04 s/call) were shorter than
ultrasonic vocalizations (∼0.4 s/call).
Experiment III
Figure 4shows the tail-flick latencies (mean ± SEM) mea-
sured before drug administration (−10, −5, and 0 min) and
over six 5-min intervals after injection into the dPAG. No
differences were detected between groups during the three
baseline tail-flick latencies. Repeated-measures ANOVA
revealed significant effects of treatment (F
3,240
=9.09; p<
0.05) and time (F
8,240
=8.08; p<0.05) and a significant
interaction between treatment and time (F
24,240
=2.90; p<
0.05). Post hoc pairwise comparisons indicated that these
differences were attributable to the groups senktide 10 and
50 pmol 5–20 min after dPAG injections compared with the
saline group. Thermal hyperalgesia peaked 20 min after
injection of drug and returned to normal within 30 min.
Fig. 1 a Outlines of all injec-
tion sites in the dPAG (gray
areas) on cross-sections from
the atlas of Paxinos and Watson
(1997). The numbers below the
brain diagrams represent the
atlas frontal coordinates in
millimeters posterior to bregma.
bRepresentative photomicro-
graph of an injection site in the
dorsal periaqueductal gray
matter (dPAG). Scale bar
represents 400 μm. DR dorsal
raphe nucleus, dPAG dorsal
periaqueductal gray, vPAG
ventrolateral periaqueductal
gray, SC superior colliculus
Psychopharmacology (2009) 204:13–24 17
Experiment IV
After the completion of Experiments I–III, we examined
whether the observed effects of senktide were selectively
mediated by NK-3 receptors. In this additional study, the
effects of senktide were challenged with intra-dPAG
injections of the selective NK-3 receptor antagonist
SB222200 (Massi et al. 2000, Sarau et al. 2000). Using
the same procedures described above, rats were tested in
the elevated plus maze and USVs and tail-flick tests. The
elevated plus maze was first used to preliminarily evaluate
the dose of SB222200 to be used. Intra-dPAG injections of
SB222200 diluted in dimethyl sulfoxide (DMSO 7.5%—
vehicle) at a dose of 50 pmol did not change the
exploratory behavior of animals in the elevated plus maze;
however, a dose of 100 pmol caused motor deficits assessed
by a reduction in closed arm entries. For this study, rats
were also randomly allocated to one of four treatment
groups (n=6 for each group): (1) vehicle + saline, (2)
vehicle + senktide 50 pmol; (3) SB222200 50 pmol +
senktide 50 pmol, (4) SB222200 50 pmol + saline. A 5-min
interval separated the two injections. Immediately after the
second injection, the animals were submitted to one of the
three tests separated by an interval of 24 h.
Similar to the previous experiments, only rats with
cannulae tips located within the dorsal portion of the
PAG were used in experiment IV. The data obtained in
this experiment are shown in Fig. 5. The treatments
increased exploration of the open and closed arms of the
elevated plus maze. Figure 5a shows that these animals
exhibited a significant increase in the frequency of entries
into both open and closed arms (F
3,23
=4.73 and 8.91
for open and closed arms, respectively; p<0.05inboth
cases). For both open- and closed-arm entries, post hoc
analyses revealed that these effects were attributable to
the intra-dPAG senktide injections. These effects were
inhibited by SB222200 treatment. The effects of injections
of this antagonist alone did not produce any significant
effects.
Figure 5b illustrates the effects of combined treatments
on the number of USVs. Two-way ANOVA revealed a
main effect of treatment on USVs (F
3,80
=20.78; p<0.05)
and frequencies (F
4,80
=16.11; p<0.05). A significant
interaction was observed between treatment and frequencies
for number of USVs (F
12,80
=4.98; p<0.05). Post hoc
comparisons revealed that senktide injection into the dPAG
caused an increase in the number of USVs recorded at
22 kHz, which was significantly reduced in animals
challenged with prior injections of SB222200. The effects
of intra-dPAG injections of this antagonist alone did not
produce any significant effects.
Figure 5c shows the tail-flick latencies measured
before drug administration and over six 5-min intervals
after combined injections into the dPAG. Repeated-
measures ANOVA revealed significant effects of treat-
ment (F
3,168
=3.73; p<0.05) and time (F
8,168
=4.46;
p<0.05) and a significant interaction between treatment
and time (F
24,168
=2.30; p<0.05). Post hoc pairwise com-
parisons indicated that these differences were attributable
to the senktide group 5–10 min after dPAG injections
compared with controls. This hyperalgesia was signifi-
cantly reduced in the group of animals that received prior
injections of SB222200. The effects of intra-dPAG
injections of this antagonist alone did not produce any
significant effects.
Fig. 2 Mean (±SEM) number of entries into the open and closed arms
of the elevated plus maze and percentage of entries in the open arms
compared with total entries in both arms in controls and in rats under
the effects of senktide injections into the dPAG. *p<0.05, compared
with the control group (Saline). n=8 animals for each group
18 Psychopharmacology (2009) 204:13–24
Discussion
Substance P has been implicated in the mediation of fear in
certain regions of the brain aversion system, such as the
amygdala, medial hypothalamus, and dPAG (Shaikh et al.
1993; De Araújo et al. 1999,2001a). Local injections of
substance P agonists into the dPAG elicit a variety of
anxiety-like behaviors in animals, including conditioned
place aversion and less time spent in the open arms of the
elevated plus maze, a widely used animal model of anxiety
Fig. 3 Effects of senktide
injections into the dorsal
periaqueductal gray on the
number of vocalizations emitted
at frequencies of 18–26 kHz
during the 15-min test of
novelty exposure. Data are
expressed as mean±SEM.
*p<0.05, compared with 4 kHz
emissions in the control group
(Saline). Pound sign p<0.05,
compared with the same
frequency in the control group.
Two-way ANOVA followed by
Newman–Keuls post hoc test.
Saline (n=8), senktide 1 pmol
(n=6), senktide 10 pmol (n= 9),
senktide 50 pmol (n=10)
Fig. 4 Time-course of tail-flick
latencies during each 5-min
period across the baseline period
(−10, −5, and 0 min) and up to
30 min after injection of saline
or senktide into the dPAG of rats
submitted to the tail-flick test.
Data are expressed as mean±
SEM. *p<0.05 different from
the control group (Saline). Two-
way repeated-measures ANOVA
followed by Student–Newman–
Keuls post hoc test. n=8 in each
group, with the exception of
n=10 for the 50 pmol group.
The arrow indicates the time of
injection into the dPAG
Psychopharmacology (2009) 204:13–24 19
(Aguiar and Brandão 1994; De Araújo et al. 1998,2001b).
NK-1 receptors appear to be involved in the aversive effects
of substance P in the dPAG. However, the role of NK-2 and
NK-3 receptors in the dPAG in the modulation of fear is
still unclear. Despite the differences in the localization of
NK-3 receptors in rodents, a significant density of NK-3
receptors is present in the PAG of rats (Bergstrom et al.
1987; Shughrue et al. 1996, Langlois et al. 2001). The
present study, therefore, sought further evidence of the
involvement of neurokinin mechanisms of the dPAG in the
defense reaction by exploring the effects of injection of the
NK-3 receptor-selective agonist senktide into the dPAG
(Regoli et al. 1994; Jenkinson et al. 2000; Yip and Chahl
1999,2001; Sculptoreanu and de Groat 2007). Hyper-
algesia and 22 kHz USV emissions were found to be the
main effects produced by injection of senktide at doses of
10 and 50 pmol/0.2 μL into the dPAG, without any
detectable anxiogenic-like effects assessed by the elevated
Fig. 5 a Effects of injections of
senktide (50 pmol/0.2 μL) and
SB222200 (50 pmol/0.2 μL)
into the dPAG on the number of
entries into the open and closed
arms of the elevated plus maze.
Data are expressed as mean±
SEM. bEffects of injections of
senktide (50 pmol/0.2 μL) and
SB220200 (50 pmol/0.2 μL)
into the dPAG on the number of
vocalizations emitted by rats at
frequencies of 18–26 kHz
during the 15-min test of
novelty exposure. Data are
expressed as mean±SEM.
cTime-course of tail-flick
latencies during each 5-min
period across the baseline period
(−10, −5, and 0 min) and up to
30 min after combined treat-
ments in rats submitted to the
tail-flick test. Data are expressed
as mean± SEM. *p< 0.05, com-
pared with the control group
(V–Svehicle–saline).
#
p<0.05,
compared with the Vehicle-
senktide (V-Senktide) group.
ANOVA followed by the
Newman–Keuls post hoc test.
n=6 for all groups. Injections
were separated by a 5-min
interval. Rats were placed in the
test apparatus soon after the
second injection
20 Psychopharmacology (2009) 204:13–24
plus maze. These animals did not show any other signs of
distress, such as urination, defecation, struggle, or abnormal
breathing patterns, but senktide produced enhanced
exploratory behavior when injected into the dPAG of rats
tested in the elevated plus maze. These animals entered the
open and closed arms of the maze more often than controls,
which is characteristic of heightened motor activity in this
test. These effects cannot be attributed to anxiolytic-like
effects because the proportion of time spent in the open
arms was not statistically different than control animals.
The present findings contrast with recent data obtained
in a similar study from this laboratory, in which injections
of the NK-1 receptor agonist SAR-Met-SP into the dPAG
caused anxiogenic-like effects in the elevated plus maze,
analgesia in the tail-flick test, and reduced USV emissions
(Bassi et al. 2007b). NK-1-mediated mechanisms have been
associated with anxiety-like behavior in several animal
species (Ebner and Singewald 2006; Dableh et al. 2005;
Kramer et al. 1998; Varty et al. 2002; Duarte et al. 2004).
The anxiogenic-like effects of substance P in the dPAG
may be mediated by NK-1 receptors (Mongeau et al. 1998;
De Araújo et al. 2001b; Duarte et al. 2004; Bassi et al.
2007a,b).
USVs in the present study lasted longer than “audible”
calls in a pattern similar to those of previous studies,
suggesting that these types of calls reflect distinct affective
states (Wohr et al. 2005). Rats emit USVs at frequencies of
20–24 kHz when exposed to moderately stressful condi-
tions, such as when they are exposed to potentially
dangerous situations (Blanchard et al. 1991; Vivian et al.
1994; Brudzynski and Chiu 1995; Commissaris et al. 2000;
Nobre and Brandão 2004). However, a nonmonotonic
function may ensue when rats with past stressful experience
are exposed to threatening conditions of an intense nature
(Nunes Mamede Rosa et al. 2005; Tomazini et al. 2006).
Thus, an intense condition of fear or proximal danger, such
as when the animals are confronted by predators, produces
opposite effects (i.e., a reduction in USVs at 22–24 kHz)
that are not sensitive to the anxiolytic effects of midazolam
(Tomazini et al. 2006). Consistent with this, rats have been
claimed to only emit 22 kHz vocalizations under experi-
mental conditions associated with anxiety and not fear
(Jelen et al. 2003). In fact, uncontrollable stressors, such as
imminent attack by predators or electrical stimulation of the
dorsal midbrain, led to freezing and a reduction in USVs at
22 kHz (Nobre and Brandão 2004; Nunes Mamede Rosa et
al. 2005). In this respect, previous studies in this laboratory
have shown that injection of substance P or NK-1 agonists
into the dPAG leads to a reduction in the number and
duration of 22 kHz USVs (De Araújo et al. 1998,1999,
2001a,b). Moreover, activation of NK-1 mechanisms in the
dPAG also produces anxiogenic-like effects assessed by
the elevated plus maze and antinociception evaluated by the
tail-flick test (Bassi et al. 2007b). Antinociception has been
considered to be an important sensory component of fear
(Fanselow and Helmstetter 1988). Such a secondary
response to the stimulation of the dPAG may activate a
descending inhibitory mechanism of pain associated with
the ventrolateral region of the PAG. Indeed, substance P
administration into this region had antinociceptive effects in
several animal models, including the tail-flick test (Xin
et al. 1997; Rosen et al. 2004). Notably, these effects appear
to be mediated selectively by NK-1 receptors because they
were blocked by selective NK-1 receptor antagonists.
Ultrasonic vocalizations in the 22 kHz range have been
used to address the emotional-affective responses to painful
stimuli under various experimental conditions, including
arthritic pain (Ardid et al. 1993; Vivian and Miczek 1998;
Jourdan et al. 1995,1998; Calvino et al. 1996; Dinh et al.
1999; Han et al. 2005). The hyperalgesia caused by
injections of 10 or 50 pmol/0.2 μL of senktide into the
dPAG was accompanied by a concomitant increase in the
emission of 22 kHz USVs. Interestingly, these effects were
also accompanied by a decrease in “audible”USVs in the
8–10 kHz frequency range. These latter effects may be
reflexive emotional reactivity to the nociceptive effects of
senktide (i.e., aversion to these injections has the effect of
producing higher USVs frequencies). Considering that
when rats are in pain they emit frequencies denoting
suffering (22 kHz), they should reduce the vocalizations
they normally emit (8–10 kHz) in conditions without
distress. In fact, NK-3 receptors are known to be involved
in the processing of information leading to central sensiti-
zation and nociception. This biological function could be
subserved by excitation of a subpopulation of neurons of
the PAG that result from NK-3 receptor activation in this
region, consistent with a recent study that administered
senktide into the PAG (Drew et al. 2005). Another study
showed that intracerebroventricular injections of senktide
induced strong neuronal activation in the PAG measured by
the expression of the protooncogene c-fos (Smith and Flynn
2000). Also supporting the present findings, activation of
NK-3 receptors has been found to increase motor activity
and facilitate the electrically evoked nociceptive flexor
reflex in adult rats (Linden and Seybold 1999; Gaudreau
and Plourde 2003; De Souza Silva et al. 2006). Pretreat-
ment with N(G)-nitro-L-arginine methyl ester (30 nmol), a
nitric oxide synthase inhibitor, blocked the hyperalgesic
effect of senktide, suggesting that senktide-induced thermal
hyperalgesia is also mediated by the production of nitric
oxide (Linden et al. 1999). Unfortunately, these studies did
not clearly distinguish between ventral and dorsal regions
of the PAG, so direct comparisons with the present results
found after intra-dPAG administration cannot be made,
especially when the drug effects on NK receptors depend
on a particular subregion of the PAG (Chahl 2006). Indeed,
Psychopharmacology (2009) 204:13–24 21
some studies have shown an antinociceptive effect of NK-3
receptors after intrathecal injections of NK-3 receptor
agonists (Papir-Kircheli et al. 1987; Laneuville et al. 1988;
Couture et al. 1993; Chahl 2006). The results of experiment
IV confirm that the nociceptive effects, increased locomotor
activity, and USV emissions elicited by local injections of
senktide into the dPAG were attributable to activation of
NK-3 receptors in this region. These effects were all
reduced by prior injections of the selective NK-3 receptor
antagonist SB222200 into the dPAG. It is also important to
mention that the fact that the use of young adult rats in this
study might have had an impact on the reported results
especially because it is known that the spontaneous
behavior of rats at this age is highly influenced by their
emotional reactivity (Adriani and Laviola 2004; Tomazini
et al. 2006). Nonetheless, more studies are needed to firmly
establish the role of NK-3 receptors and its endogenous
agonist neurokinin B.
In summary, the present results show that activation of
NK-3-mediated mechanisms of the dPAG produces a
pattern of effects distinct from those mediated by NK-1
mechanisms found in the dPAG. The vocal and behavioral
profiles are consistent with activation of acute pain
mechanisms that lead to increased psychomotor activation
accompanied by 22 kHz USVs that may reflect the
emotional-affective state of the animal. These data associate
NK-3 mechanisms of the dPAG with the processing of
sensory stimulation in the dPAG. These effects contrast
with those attributed to the NK-1 receptor subtype in the
dPAG in the control of pathological anxiety states.
Acknowledgments This work wassupported by FAPESP (06/06354-5
and 06/03930-5) and CNPq (06/472030-0).
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