Psychopharmacology

https://doi.org/10.1007/s00213-023-06309-7

ORIGINAL INVESTIGATION

Unravelling the dorsal periaqueductal grey matter NMDA

receptors relevance in the nitric oxide‑mediated panic‑like
behaviour and defensive antinociception organised by the anterior
hypothalamus of male mice
Luiz Luciano Falconi‑Sobrinho1,2,3 · Tayllon dos Anjos‑Garcia1,4 · Paloma Molina Hernandes5 ·
Bruno Mangili de Paula Rodrigues1 · Rafael Carvalho Almada3,5 · Norberto Cysne Coimbra1,2,3 

Received: 6 September 2022 / Accepted: 31 December 2022

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
Rationale  Previousstudies suggested that the dorsal column of the periaqueductal grey matter (dPAG) can be a target of neu-
ral pathways from hypothalamic nuclei involved in triggering fear-related defensive responses. In turn, evidence is provided
suggesting that microinjection of the nitric oxide (NO) donor SIN-1 into the anterior hypothalamus (AH) of mice evokes
panic-like behaviours and fear-induced antinociception. However, it is unknown whether the dPAG of mice mediates these
latter defensive responses organised by AH neurons.
Objectives  This study was designed to examine the role of dPAG in mediating SIN-1-evoked fear-induced defensive behav-
ioural and antinociceptive responses organised in the AH of mice.
Methods  First, neural tract tracing was performed to characterise the AH-dPAG pathways. Then, using neuropharmacologi-
cal approaches, we evaluated the effects of dPAG pretreatment with either the non-selective synaptic blocker cobalt chloride
­(CoCl2; 1 mM/0.1 μL) or the competitive N-methyl-d-aspartate (NMDA) receptor antagonist LY235959 (0.1 nmol/0.1 μL)
on defensive behaviours and antinociception induced by microinjections of SIN-1 in the AH of male C57BL/6 mice.
Results  AlexaFluor488-conjugated dextran-labelled axonal fibres from AH neurons were identified in both dorsomedial and
dorsolateral PAG columns. Furthermore, we showed that pre-treatment of the dPAG with either ­CoCl2 or LY235959 inhibited
freezing and impaired oriented escape and antinociception induced by infusions of SIN-1 into the AH.
Conclusions  These findings suggest that the panic-like freezing and oriented escape defensive behaviours, and fear-induced
antinociception elicited by intra-AH microinjections of SIN-1 depend on the activation of dPAG NMDA receptors.

Keywords  Anterior hypothalamus · Defensive behaviour · Nitric oxide · NMDA receptors · Periaqueductal grey matter ·
Stress · Fear-induced analgesia

* Luiz Luciano Falconi‑Sobrinho of São Paulo (USP), Av. Bandeirantes 3900, Ribeirão Preto,
lucianofalconi@usp.br São Paulo 14049‑900, Brazil
3
* Norberto Cysne Coimbra Behavioural Neurosciences Institute (INeC), Avenida do
nccoimbr@fmrp.usp.br Café, 2450, Ribeirão Preto, São Paulo 14220‑030, Brazil
4
1 Biomedical Sciences Institute of the Federal University
Laboratory of Neuroanatomy and Neuropsychobiology,
of Alfenas (UNIFAL), Alfenas, Minas Gerais, Brazil
Department of Pharmacology, Ribeirão Preto Medical
5
School of the University of São Paulo (USP), Av. Department of Biological Sciences, School of Science,
Bandeirantes 3900, Ribeirão Preto, São Paulo 14049‑900, Humanities and Languages, São Paulo State University
Brazil (UNESP), Assis, São Paulo, Brazil
2
NAP‑USP‑Neurobiology of Emotions (NuPNE) Research
Centre, Ribeirão Preto Medical School of the University

13
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 Psychopharmacology
Introduction
The defensive behaviour exhibited by laboratory rodents
can be not only naturally elicited in dangerous situa-
tions (Anseloni et al. 1999; Blanchard and Blanchard
1989; Lobão-Soares et  al. 2008; Almada et  al. 2015;
Almada and Coimbra 2015; Coimbra et al. 2017; dos
Anjos-Garcia and Coimbra 2019) but also by electri-
cal and chemical stimulation of limbic (Milani and Gra-
eff 1987; Falconi-Sobrinho et al. 2017a; Shekhar and
DiMicco 1987) and paralimbic (Coimbra et  al. 2000,
2006) structures. The periaqueductal grey matter (PAG)
is a brainstem structure anatomically studied into four
longitudinal divisions known as dorsomedial (dmPAG),
dorsolateral (dlPAG), lateral and ventrolateral columns
(Bandler and Shipley 1994), with different functions
(Carrive 1993). In particular, the dorsal PAG columns
(dPAG) have been proposed as one of the main targets of
neural pathways from different diencephalic structures
involved in triggering fear-related defensive responses
(Canteras et  al. 1994; Ullah et  al. 2017; Wang et  al.
2015). Indeed, both dmPAG and dlPAG receive inputs
from the dorsomedial division of the ventromedial hypo-
thalamus (dmVMH) (Canteras et al. 1994; Ullah et al.
2017; Wang et al. 2015) and the hypothalamic dorsal
premammillary nucleus (dPM) (Canteras and Swanson
1992; Wang et  al. 2021), which along with the ante-
rior hypothalamus (AH) integrate a distinct neural cir-
cuit known as the medial hypothalamic defence system
(MHD) (Canteras 2002). Evidence from studies per-
formed in rodents has shown that dPAG is activated
during threatening situations caused by the presence
of a predator (Paschoalin-Maurin et al. 2008; de Paula
et al. 2022) and is recruited by neurons from dmVMH
and the PM to mediate both unconditioned and condi-
tioned fear-induced behaviours (Ullah et al. 2017; Wang
et al. 2015, Wang et al. 2021). In particular, studies per-
formed by Ullah et al. (2017) showed that both freezing
and explosive/non-oriented escape panic-like defensive
behaviours induced by chemical stimulation of dmVMH
with NMDA, an excitatory amino acid analogue, were
inhibited by prior microinjections of cobalt chloride
­(CoCl2) in the dPAG. Wang et al. (2015), in turn, showed
that optogenetic activation of dmVMH projections to
the dlPAG of mice surprisingly elicited freezing but not
escape. In addition, more recently, Wang et al. (2021)
presented evidence that inhibition of the cholecystokinin
(cck) in the dPM, which is expressed by a vast majority
of glutamatergic neurons, decreases the vigorous escape
displayed by mice exposed to threatening situations, via
projections to the dlPAG. Although these latter find-
ings show that connexions between the MHD system
structures and dPAG seem to play an important role in
13
modulating panic-like defensive behaviours, there is
still a lack of studies demonstrating the role of dPAG in
mediating defensive responses organised by AH neurons.
Interestingly, both hypothalamic and dPAG neurons
in addition to orchestrating different panic-like defensive
behaviours have also been implicated in the modulation of
fear-related antinociceptive responses (Biagioni et al. 2016;
Baptista-de-Souza et al. 2018; Miguel and Nunes-de-Souza
2006; Coimbra et al. 2006; Falconi-Sobrinho and Coim-
bra 2018; Falconi-Sobrinho et al. 2017a,b, 2021). Fear-
induced antinociception is long known as a physiological
and adaptive response that is part of the defence (Bolles and
Fansellow 1980). This analgesic phenomenon is mediated
by spinally projecting pain inhibitory pathways (Basbaum
and Fields 1978; Fields and Basbaum 1978) originating
from the cerebral cortex (Falconi-Sobrinho et al. 2017a,
2021; Millan 2002), hypothalamus (Biagioni et al. 2016; de
Freitas et al. 2013; Falconi-Sobrinho et al. 2017a,b; Frei-
tas et al. 2009), amygdala (Harris and Westbrook 1995;
Helmstetter 1992), dPAG (Baptista-de-Souza et al. 2018;
Coimbra et al. 2006; Miguel and Nunes-de-Souza 2006)
and ventrolateral PAG (Tovote et al. 2016; Samineni et al.
2017; Watson et al. 2016). In particular, pain-modulatory
descending pathways from the hypothalamus have been
proposed to relay through the PAG and ultimately through
the RVM, to produce antinociceptive effect via dorsal horn
of the spinal cord (DHSC) inputs (Aimone et al. 1988;
Behbehani et al. 1988; Butler and Finn 2009 for review).
However, it remains unknown whether the dPAG medi-
ates defensive antinociceptive responses triggered by AH
neurons. Evidence from studies performed in rodents has
shown that chemical stimulation of the hypothalamus and
dPAG with excitatory neurotransmitters and neuromodula-
tors, such as N-methyl-D-aspartate (NMDA) (Khan et al.
2020; Miguel and Nunes-de-Souza 2006) and nitric oxide
donors, respectively (Falconi-Sobrinho and Coimbra 2018;
Miguel et al. 2012), elicits defensive behaviours along with
antinociception. In contrast, the latter responses to chemi-
cal stimulation of the posterior and anterior hypothalamus
as well as dPAG were shown to be attenuated in rodents
that received prior microinjections of the NMDA recep-
tor antagonists LY235959 (Falconi-Sobrinho et al. 2017a)
and AP-7 (Falconi-Sobrinho and Coimbra 2018), and the
neuronal nitric oxide synthase (nNOS) inhibitor NPLA
(Miguel and Nunes-de-Souza 2006) in these same struc-
tures, and in independent group of animals. More recently,
Falconi-Sobrinho et  al. (2021) presented evidence that
escape behaviours and antinociception elicited by micro-
injections of SIN-1 into the AH of mice are exacerbated
and impaired, respectively, by increased activity of the
glutamatergic inputs to the AH from the cingulate cortex.
Altogether, these latter pharmacological studies suggest that
the interaction between glutamatergic and nitrergic systems
Psychopharmacology
within the AH and dPAG seems to regulate both defensive
behaviour and fear-induced antinociception.
In particular, it is widely known that NO acts as a retro-
grade transmitter, because it is synthesised in post-synaptic
neurons and diffuses to the pre-synaptic terminal, where
it facilitates the glutamate release (Garthwaite et al. 1988,
1989 for review).
Therefore, to examine whether the dPAG mediates fear-
related defensive behavioural and antinociceptive responses
evoked by SIN-1 microinjected into the AH, we first used
neuroanatomical techniques to identify AH-dPAG pathways.
Then, neuropharmacological approaches were performed
using either the synaptic activity blocker cobalt chloride
­(CoCl2; 1  mM) or the highly selective NMDA receptor
antagonist LY235959 (0.1 nmol) in the dPAG prior to intra-
AH microinjection of SIN-1.
Material and methods
Animals
Male C57BL/6 mice aged 10 weeks and weighing 30–35 g
(N = 53; n = 7 per group for neuropharmacological experi-
ments and additional control experiment of place preference
in the enriched polygonal arena test, and n = 4 for neuroana-
tomical experiments) from the animal facility of Ribeirão
Preto Medical School of the University of São Paulo
(FMRP-USP) were used in this study. They were housed in
4 per cage and maintained in the experimental room for at
least 48 h prior to the experiments with free access to water
and food. The enclosure was maintained under a light/dark
cycle of 12/12 h (lights on from 7 a.m. to 7 p.m.) and at
a constant room temperature of 23 °C ± 1 °C. The experi-
ments were carried out during the light phase of the light/
dark cycle (8:00 a.m.–3:00 p.m.). All efforts were made to
minimise animal suffering. The experiments were performed
in accordance with the recommendations of the Commis-
sion of Ethics in Animal Experimentation of the FMRP-USP
(process 187/2015), which is consistent with the ethical prin-
ciples in animal research adopted by the National Council
for Animal Experimentation Control (CONCEA) and were
approved by the Commission of Ethics in Animal Research
(CETEA) on 2/29/2016.
Stereotaxic surgery
Animals were anaesthetised with ketamine at 92  mg/kg
(Ketamine Agener, União Química Farmacêutica Nacional,
São Paulo, Brazil) and xylazine at 9.2 mg/kg (Calmium,
União Química Farmacêutica Nacional, São Paulo, Bra-
zil) and fixed in a stereotaxic frame (David Kopf, Tujunga,
CA, USA). In neuroanatomical experiments, AlexaFluor
488-conjugated dextran neural tract tracer was deposited
into the diencephalon through a dental needle directed to
the AH according to anteroposterior: − 0.70 mm, mediolat-
eral: − 0.3 mm, and dorsoventral: − 5.0 mm coordinates. In
neuropharmacological experiments, stainless steel guide
cannulae (outer diameter 0.6 mm, inner diameter 0.4 mm)
were implanted in the midbrain tectum and diencephalon,
targeting 1  mm above the dPAG and AH, respectively.
The following coordinates were used for the dPAG and
AH: anteroposterior, − 4.2 mm and − 0.70 mm; mediolat-
eral, − 1.3 mm and − 0.3 mm; and dorsoventral, − 1.8 mm
and − 4.0 mm. The guide cannula targeting the dPAG was
implanted at 26° in relation to the median axis. Acrylic
resin was used to anchor the guide cannulae to the skull
of each mouse. At the end of the surgery, each guide can-
nula was sealed with a stainless-steel wire to protect it from
obstruction. The coordinates used in both experimental sets
were based on Paxinos and Franklin’s mouse brain in stere-
otaxic coordinates atlas (2001), and bregma was used as a
reference.
After the stereotaxic surgery, each mouse was treated
with an intramuscular injection of penicillin G-benzathine
(120,000 UI; 0.1 mL) followed by a subcutaneous injection
of the non-steroidal analgesic and antiinflammatory flunixin
meglumine (2.5 mg/kg) (Schering-Plough, São Paulo, SP,
Brazil). After surgery, the animals were allowed to recover
for 5 and 10 days before the behavioural tests and morpho-
logical study, respectively.
Drugs
Neuroanatomical experiments
The AlexaFluor 488-conjugated dextran neural tract tracer,
10.000 MW (Molecular Probes, Eugene, OR, USA), at 0.1
μL, dissolved in 0.01 M phosphate-buffered saline (PBS),
pH 7.4, was microinjected into the AH.
Neuropharmacological experiments
The NO donor SIN-1 (3-morpholinosydnonimine hydro-
chloride; Tocris Biosciences, Bristol, UK) at 300 nmol/0.1
μL (Falconi-Sobrinho and Coimbra 2018; Falconi-Sobrinho
et al. 2021) or saline (vehicle; 0.9% NaCl/0.1 μL) was micro-
injected into the AH following the intra-dPAG microin-
jections of cobalt chloride ­(CoCl2, 1 mM/0.1 μL; Sigma/
Aldrich, St. Louis, MO), the NMDA receptor antagonist
LY235959 (0.1 nmol/0.1 μL; Tocris Bioscience, Bristol,
UK) or saline (vehicle; 0.9% NaCl/0.1 μL). The doses of
1.0 mM of ­CoCl2 and 0.1 nmol of LY235959 were based
on previous studies performed by Ullah et al. (2017) and
13
 Psychopharmacology
Falconi-Sobrinho et al. (2017a), respectively. The drugs
were dissolved in physiological saline shortly before use.
Experimental procedure
Neuroanatomical experiments: labelling the AH‑dPAG
pathways using a fluorescent neural tract tracer
The AlexaFluor 488-conjugated dextran neural tract
tracer was deposited into the AH (n = 4) at a volume
of 0.1 μL over the course of 5  min. Infusions were
delivered using an infusion pump (Stoelting, Kiel, WI,
USA) through a polyethylene tube (PE10) attached to a
dental needle, for targeting this hypothalamic nucleus.
The dental needle was left in place for 2 min after the
end of each microinjection to allow local drug diffu-
sion. After completing the microinjection procedure, the
dental needle was removed and the skin was sutured.
Ten days after neural tract tracer microinjections, mice
were deeply anaesthetised with ketamine at 92 mg/kg
(Ketamina®) and xylazine at 9.2  mg/kg (Dopaser®)
and perfused intracardially with physiological saline
followed by 4% paraformaldehyde (PFA, Sigma) dis-
solved in 0.1 M phosphate buffer (pH 7.4). The encepha-
lon was removed, post-fixed in PFA for 4 h and then
transferred to 10% and 20% sucrose dissolved in 0.1 M
sodium phosphate buffer, pH 7.3, at 4 °C, for at least
12 h in each solution. The nervous tissue was immersed
in 2-methylbutane (Sigma), frozen on dry ice (30  s),
embedded in Tissue Tek and sectioned (20 mm thick-
ness) using a cryostat (CM 1950, Leica, Wetzlar, Ger-
many) at 20 °C. Then, the forebrain and midbrain slices
were mounted with Fluoromount with DAPI (Electron
Microscopy Sciences, Hatfield, PA, USA) on silanised
slides, and both the AH and PAG were observed under
motorised microscopy (AxioImager Z1 with APOTOME
II, Zeiss, Oberkochen, Germany). The timeline of the
Fig. 1  Timeline of neuroanatomical and neuropharmacological experiments
13
neuroanatomical experiments is shown in Fig. 1a. The
neural pathways were shown in Fig. 2.
Neuropharmacological experiments: effect
of the pretreatment of dPAG with either ­CoCl2 or LY235959
on panic‑like defensive behaviour and fear‑induced
antinociception elicited by microinjections of SIN‑1
in the AH
Before the behavioural test (see Fig. 3, 4, 5, and 6), mice
(n = 7) were habituated for 4 days in the polygonal arena
with free access to water and food. In the habituation period,
the floor of the polygonal arena was covered with sawdust,
and a shelter box and two elevated platforms for escape
with an access ladder (safe places) were arranged inside
the polygonal arena, as shown in Fig. 7a–c. At the end of
habituation, each mouse was removed from the polygonal
arena and submitted to three measures of control tail-flick
latencies to determine the baseline nociceptive threshold,
as described in the ‘Nociceptive testing’ section. At the
day of the experiment, the sawdust was removed, and the
polygonal arena was cleaned to perform the behavioural
test. Then, the following groups were performed: dPAG
vehicle (10 min) + AH vehicle (n = 7) (Veh-Veh); dPAG
­CoCl2 (10 min) + AH vehicle (n = 7) ­(CoCl2-Veh); dPAG
LY235959 (10 min) + AH vehicle (n = 7) (LY-Veh); dPAG
vehicle (10 min) + AH SIN-1 (n = 7) (Veh-SIN-1); dPAG
­CoCl2 (10 min) + AH SIN-1 (n = 7) ­(CoCl2-SIN-1); and
dPAG LY235959 (10 min) + AH SIN-1 (n = 7) (LY-SIN-1).
Considering that ­CoCl2 (10  min) + vehicle ­(CoCl2-Veh)
(n = 7) and LY235959 (10 min) + vehicle (LY-Veh) (n = 7)
treatments did not produce defensive behavioural effects
(zero mean and variance), these data were not included in
Fig. 3. However, data related to the distance travelled by all
groups, which was expressed by the number of crossings,
were included in the study to demonstrate the effect of differ-
ent drugs on general activity, as shown in Table 1. Drugs or
Psychopharmacology

Fig. 2  Photomicrographs of
coronal section of mice brain
at the level of the anterior
hypothalamus (AH) (a) and
periaqueductal grey mat-
ter (PAG) (c–j), showing a
representative site of intra-AH
microinjection of the Alex-
aFluor 488-conjugated dextran
neural tract tracer (white arrow
in a). Histologically confirmed
sites (closed green circles) of
neural tract tracer microinjec-
tions in the AH were depicted
on modified diagrams from
mouse brain in stereotaxic coor-
dinates by Franklin and Paxinos
(2007) (b). Photomicrographs
of transverse sections of mice
midbrain at cranial (c), caudal
(d), and middle (e, f) levels
of the PAG, show neural tract
tracer-labelled perikarya (white
arrow), axonal fibres (closed
arrowheads), varicosities and
appositions suggesting terminal
buttons (open arrowheads) in
lateral (lPAG), in c; dorsolateral
(dlPAG; d on the top); lateral
(lPAG; d at the bottom); and
dorsomedial (dmPAG; e, f) PAG
columns. Photomicrographs
of transverse sections of mice
midbrain at cranial (g) and mid-
dle/caudal (h–j) levels of the
PAG, show neural tract tracer-
labelled axonal fibres (closed
arrowheads), varicosities and
appositions suggesting terminal
buttons (open arrowheads) in
dlPAG. Aq, Aquaeductus Sylvii

13
 Psychopharmacology

13
 Psychopharmacology
◂Fig. 3  Effect of dPAG pretreatment with vehicle (Veh), C
­ oCl2 or
LY235959 (LY) on the number and duration of freezing (a, b), num-
ber and duration of oriented escape (c, d), number of entries and
time spent inside the burrow (e, f), number of access and time spent
on/under the elevated platforms for escape (g, h) after the oriented
escape, and number and duration of non-oriented escape (i, j) induced
by infusions of SIN-1 into the AH. Data are presented as median
and interquartiles; *p < 0.05; ***p < 0.001, compared with the Veh-
dPAG + Veh-AH control group; #p < 0.05; ###p < 0.001 compared with
the Veh-dPAG + SIN-1-AH-treated group, according to the Kruskal–
Wallis H test followed by Dunn’s post hoc test. The position of each
dot indicates the value for an individual data point
vehicle were infused through a polyethylene tube (PE-10) in
a volume of 0.1 μL for 30 s using a 5-µL syringe (Hamilton,
Reno, NV, USA) connected to an infusion pump (Stoelting,
Kiel, WI, USA). To prevent reflux, the dental needle was left
in place for 15 s after the end of each injection.
After the intra-AH microinjections of either vehicle or
SIN-1, mice were placed in the enriched polygonal arena
test, and the behavioural responses were quantitatively ana-
lysed for 10 min. Immediately after the end of the behav-
ioural tests, each mouse was submitted to the tail-flick test.
Tail-flick latencies (TLFs) were recorded at 10-min intervals
for 60 min. Nociceptive data from the control (­ CoCl2-Veh)
and (LY-Veh) groups were also included in Fig. 4, as the
current intensity of the tail-flick test device was adjusted to
obtain baseline latencies between 2.5 and 3.5 s. The time-
line of the neuropharmacological experiments is shown in
Fig. 1b.
Experimental apparatus: the enriched polygonal arena
test  An acrylic semi-transparent parallelepiped-shaped
enriched polygonal arena (140 × 62 × 50 cm) was used for
recording the defensive behaviour. The floor of the polyg-
onal arena is divided into twelve equal sections using a
demarcating black line under a transparent glass surface.
These sections allow us to evaluate general motor activity
by quantifying the number of quadrants that the rodent trav-
elled during the experiment (Falconi-Sobrinho and Coimbra
2018; Falconi-Sobrinho et al. 2021). In the enriched polygo-
nal arena test, there was a shelter box (burrow; black acrylic;
10 × 7 × 5 pol) and two elevated platforms for escape with
an access ladder (safe places), which allows us to discrimi-
nate oriented and non-oriented escape behaviours (Almada
and Coimbra 2015; Almada et al. 2021; de Paula Rodrigues
and Coimbra 2022; dos Anjos-Garcia and Coimbra 2019;
Falconi-Sobrinho and Coimbra 2018; Falconi-Sobrinho
et al. 2021).
Behavioural tests  Behavioural reactions displayed by mice
were recorded for 10 min using a video camera (Handycam,
Sony Corporation, Osaki, Shinagawa-ku, Tokyo, Japan) and
assessed using the X-Plo-Rat software version 1.1.0, devel-
oped by researchers from the Laboratory of Exploratory
Behaviour of Ribeirão Preto School of Philosophy, Sciences
and Literature of the University of São Paulo. This soft-
ware does not automatically estimate behavioural reactions;
instead, the investigator, blinded to the experimental groups,
uses the software to record the number/frequency and
duration of each behaviour (dos Anjos-Garcia et al. 2017;
Falconi-Sobrinho and Coimbra 2018; Falconi-Sobrinho
et al. 2021; Khan et al. 2020). Behavioural defensive reac-
tions comprised freezing and escape responses. Freezing
(recorded as number/frequency of events and duration of
defensive immobility) was characterised by a defensive
immobility for at least 6 s followed by at least one auto-
nomic reaction, such as defecation, exophthalmia or mictu-
rition (Coimbra et al. 2017; Falconi-Sobrinho and Coimbra
2018; Uribe-Mariño et al. 2012). The escape behaviour was
categorised as oriented and non-oriented escape responses.
Oriented escape was defined as running towards the elevated
platforms for escape (climbing the ladder or simply hiding
in the bottom) and/or the burrow (climbing the wall and
reaching the top of the burrow or going to the entrance and
staying inside the shelter). The number of burrow entries and
accesses to the elevated platforms for escape as well as the
time spent inside the burrow and on the elevated platforms
for escape after oriented escape were also recorded. Non-
oriented escape was defined as running in a direction in the
arena other than towards the elevated platforms for escape
and/or burrow (Almada et al. 2022; Falconi-Sobrinho and
Coimbra 2018; Falconi-Sobrinho et al. 2021), with explosive
(i.e., purposeless vigorous running with horizontal jumps
and collisions of the animals with the walls of the arena,
as reported elsewhere by Ullah et al. 2015 and Ullah et al.
2017) or non-explosive (i.e., running towards the safe places,
as reported elsewhere by Falconi-Sobrinho and Coimbra
2018) characteristics. Additionally, the number of crossings
(stepping with four legs within a delimited rectangle on the
floor of the enriched polygonal arena after crossing the bor-
der of each section line) was also recorded and quantified
for general motor activity investigation.
Nociceptive testing  The nociceptive thresholds were meas-
ured using the tail-flick test, specifically recording the tail-flick
latency (TFL). After the habituation period in the enriched
polygonal arena test, each mouse was submitted to three meas-
ures of TFLs to determine the baseline nociceptive threshold.
The TFL to determine the baseline was performed with 5-min
intervals between each test. For this, each animal was placed
in a restraining apparatus (Insight, Ribeirão Preto, SP, Brazil)
with acrylic walls, and its tail was placed on a heating sen-
sor (tail flick Analgesia Instrument; FMRP-USP Workshops;
Ribeirão Preto, SP, Brazil). The amount of heat applied to the
tail was increased until the animal removed its tail from the
apparatus. The coil (Ni/Cr alloy; 26.04 cm in length × 0.02 cm
in diameter) began at room temperature (approximately 20 °C),
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 Psychopharmacology

Fig. 4  (a) Effect of dPAG treat-

ment with vehicle (Veh), C ­ oCl2
or LY235959 (LY) followed by
intra-AH infusions of SIN-1
on tail-flick latencies. Data are
presented as the mean ± S.E.M.;
*p < 0.05, compared with the
Veh-dPAG + Veh-AH control
group; #p < 0.05 compared with
the Veh-dPAG + SIN-1-AH-
treated group; +p < 0.05 com-
pared with the LY-dPAG + SIN-
1-AH-treated group, according
to two-way RM-ANOVA
followed by Tukey’s post hoc
tests. BL, baseline nociceptive
threshold. b Linear regression
curve showing the correlation
between panic-like defensive
behaviours and antinociception

and then current was applied to increase the temperature of
the coil at a rate of approximately 9 °C/s (da Silva Soares
et al. 2019; Falconi-Sobrinho et al. 2017b, 2021; Prado and
Roberts 1985). Small adjustments in the current intensity
were made, if necessary, at the beginning of the experiment
(baseline records) to obtain three consecutive TFLs between
2.5 and 3.5 s. If the animal did not remove its tail from the
heater within 6 s, the apparatus was turned off to prevent
damage to the skin. The day after baseline, mice were sub-
jected to an enriched polygonal arena behavioural test under

13
Psychopharmacology

Fig. 5  Diagrammatic representation of coronal sections of the
C57BL/6 mice brain illustration (a, b): sites of Veh-dPAG + Veh-
AH (○), ­CoCl2-dPAG + Veh-AH (∆), LY-dPAG + Veh-AH (◊),
Veh-dPAG + SIN-1-AH (●), ­CoCl2-dPAG + SIN-1-AH (▲) and LY-
dPAG  + SIN-1-AH (♦) microinjections. Photomicrographs of the
midbrain (bottom) and diencephalic (bottom corner) coronal sections
of the mice showing a representative site of drug microinjections in
both dPAG and AH. Scale bar: 500 μm. Histological staining: haema-
toxylin and eosin

Fig. 6  Schematic diagram
suggesting the mediating role
of dorsal periaqueductal grey
matter (dPAG) on fear-related
defensive behavioural and
antinociceptive responses
triggered by anterior hypothala-
mus (AH) neurons. Chemical
stimulation of the AH with the
nitric oxide (NO) donor SIN-1
possibly activates the AH-
dPAG glutamatergic pathways
to trigger panic-like behaviour
and defensive antinociception.
In contrast, pretreatment of
dPAG with either non-selective
synaptic blocker cobalt chloride
­(CoCl2) or N-methyl-d-aspartate
(NMDA) receptor antagonist
LY235959 impairs the proaver-
sive and antinociceptive effects
elicited by microinjections of
SIN-1 in the AH

13
 Psychopharmacology
Fig. 7  Exploratory behaviour of mice submitted to the habituation
procedure in the enriched polygonal arena test (a–c) for 4  days, in
which mice defensive attention after the exit from the burrow (a),
exploratory behaviour of elevated platform for escape on the top (a,
b) and in the bottom (b), burrow entrance (c, on the top) and feed-
ing area visiting (c, at the bottom). After habituation, each mouse was
submitted to a 10-min re-exposure to the experimental environment
(d–l) without neurostimulation in a place preference control experi-
ment. The place preference and the interaction between rodents and
each component of the enriched polygonal arena test were recorded
as follows: active avoidance from the open area to the safe places
(d), to the first elevated platform for escape (e), rearing under the
first elevated platform for escape (f), exploratory behaviour towards
the second elevated platform for escape (g), climbing the ladder (h),
head dipping at the top of the second elevated platform for escape (i),
exploratory behaviour near the burrow (j), burrow entrance (k) and
exploratory behaviour of the open area (l)
13
pharmacological treatment. Immediately after the end of the
behavioural test, the animals were again submitted to the tail-
flick test. TFLs were measured at 10-min intervals for 60 min.
Histology  Upon completion of the neuropharmacological
experiments, the animals were anaesthetised with keta-
mine at 100 mg/kg (Ketamina®) and xylazine at 10 mg/kg
(Dopaser®) and perfused through the left cardiac ventricle
using an infusion pump (Master Flex® L/S TM, Vernon
Hills, IL, USA). The thoracic descending aorta was clamped,
the pericardial heart wrap was released to allow perfusion
through the left cardiac ventricle, and blood was washed out
with Tyrode buffer (40 mL at 4 °C). The animal was then
perfused with 200 mL ice-cold 4% (w/v) paraformaldehyde
in 0.1 M sodium phosphate buffer (pH 7.3) for 15 min at a
pressure of 50 mmHg. The brain was quickly removed and
maintained in 4% paraformaldehyde for at least 4 h and was
then immersed in a 20% sucrose solution for 24 h followed
by 10% for a further 24 h. Tissue pieces were immersed
in 2-methylbutane (Sigma-Aldrich, St. Louis, USA), frozen
on dry ice (30 min), embedded in Tissue Tek and cut on
a cryostat (CM 1950, Leica, Mannheim, Germany). Slices
were then mounted on glass slides that were coated with
chrome alum gelatin to prevent detachment and stained in a
robotic autostainer (CV 5030 Leica Autostainer) with hae-
matoxylin–eosin. The sections were viewed under a motor-
ised photomicroscope (AxioImager Z1, Zeiss, Oberkochen,
Germany), and the positions of the tips of the guide cannu-
lae were localised according to the Paxinos and Franklin´s
mouse brain in stereotaxic coordinates atlas (2001).
Place preference displayed by control mice
submitted to the enriched polygonal arena test
An independent naïve control group of male mice (n = 7)
was submitted to the habituation procedure in the enriched
polygonal arena test, provided with food and tap water in the
open area, located diametrically opposite the safe area, in
which two elevated platforms for space were disposed, one of
them beside a burrow with a single entrance (see Fig. 7a–c).
After the habituation procedure, the mice returned to their
cage for 2 h, the arena was vacuumed with a vacuum cleaner,
the food and water were removed, and both the wall and floor
were cleaned with 20% alcoholic solution. The elevated plat-
forms for escape and the burrow were washed and cleaned
with 20% alcoholic solution. The roof of the burrow was
replaced by another one similar to the original except for the
colour that was transparent, allowing the recording of the
place preference behaviour of each mouse inside the burrow.
A rectangular piece of white paper was placed on the floor
of the burrow for contrast enhancement.
The place preference experiment was then performed, and
each mouse without any pharmacological treatment was put
Psychopharmacology
Table 1  Effect of each Groups
psychopharmacological
treatment on motor behaviour of Crossings Veh-Veh
mice submitted to the enriched
CoCl2-Veh
polygonal arena test
LY-Veh
Veh-SIN-1*
CoCl2-SIN-1
LY-SIN-1
Data are presented as the mean ± S.E.M.; *p < 0.05, compared with Veh-dPAG + Veh-AH control group
with a net in the middle of the polygonal arena facing the
open area and was allowed the mouse the exploration of
the environment for 10 min. The exploratory behaviour was
recorded by the ANY-maze behavioural tracking software
(Stoelting Co, Wood Dale, IL, USA) with a video feed from
an overhead digital USB camera (60,528 lens for 60,516
camera; The Imaging Source, Charlotte, NC, USA) con-
nected to a computer setup used to record and automatically
track the movement and heatmap of mice exposed to the
enriched polygonal arena test.
The place preference and the interaction between rodents
and each component of the enriched polygonal arena test
were recorded as follow: (a) active avoidance, defined as
exploratory movements displayed from the open area to the
safe places; (b) passive avoidance, defined as permanence
inside the burrow, with the head near to the burrow entrance;
(c) rearing, defined as an elevation of the front paws in the
air or touching either the ladder, the columns of the elevated
platforms for escape or burrow walls; (d) climbing, defined
was exploratory behaviour towards either the ladder or the
burrow walls, reaching the elevated platform and the top of
the burrow, respectively; (e) head dipping, defined as the
behaviour in which the mouse dips its head either below the
level of the elevated platform for escape floor or the roof of
the burrow; and (f) exploratory behaviour of the open area,
defined as motor behaviour for acquiring information about
the open area of the enriched polygonal arena test.
Statistical analysis
Considering that, according to the Shapiro–Wilk test of nor-
mality, the majority of defensive behaviour-related data did
not follow a Gaussian distribution, the behavioural data were
analysed by the Kruskal–Wallis test followed by Dunn’s post
hoc test. These data were reported as median with interquar-
tile range and represented as scatter dot plots in the figures.
Data from the crossings and nociceptive threshold experi-
ments recorded immediately after the end of the defensive
behaviour were submitted to a repeated measures two-way
ANOVA followed by Tukey’s post hoc test. A P value < 0.05
was considered statistically significant. Data from the
MEAN ± S.E.M. Statistic data
64.43 ± 6.917 1st inj. dPAG: F2,36 = 2.132, p >
0.05
101.4 ± 7.047
104.7 ± 4.545 2nd inj. AH: F1,36 = 8.169, p < 0.05
128.7 ± 9.586
88 ± 6.69 Interaction between factors: F2,36 =
120.4 ± 15.44 8.524, p < 0.05
nociceptive threshold experiments recorded immediately
after the end of the defensive behaviour were submitted to
a repeated measures two-way ANOVA followed by Tukey’s
post hoc test. A P value < 0.05 was considered statistically
significant. The software used for statistical analysis and
graph plotting was GraphPad Prism version 7.0.
The total number/frequency of defensive behaviours
(sum of the number of freezing, oriented and non-oriented
escape) and the nociceptive threshold (recorded immediately
after the test—t0) displayed by each animal (i.e., individual
values) were used to perform a linear regression analysis
to study the correlation between panic-like behaviours and
antinociception.
Results
Neuroanatomical experiments: neural tract tracing
of pathways connecting the AH to the dPAG
Microinjections of the fluorescent dextran neural tract tracer
in the diencephalon aiming at the AH of mice showed con-
nections between this hypothalamic nucleus and the PAG
(Fig. 2a–j). Neural tract tracer-labelled axonal fibres, vari-
cosities and appositions suggesting terminal buttons from AH
neurons reaching lateral PAG (Fig. 2c, d), dlPAG (Fig. 2d in
the top) and dmPAG (Fig. 2e, f), at the cranial (Fig. 2c), mid-
dle (Fig. 2e, f) and caudal (Fig. 2d) levels of the rostro-cau-
dal axis were demonstrated. In addition, neural tract tracer-
labelled axonal fibres, varicosities and appositions suggesting
terminal buttons were also found at the cranial (Fig. 2g), mid-
dle, and caudal (Fig. 2h–j) levels of the dlPAG.
Neuropharmacological experiment: effect of dPAG
pretreatment with either ­CoCl2 or LY235959
on defensive responses induced AH microinjections
of the SIN‑1
Behavioural reactions
Treatment in the AH with SIN-1 preceded by administra-
tion of vehicle into the dPAG induced freezing (number;
13
 Psychopharmacology
H3 = 26.46; p < 0.001; duration; H3 = 26.28; p < 0.001),
and oriented (number; H3 = 23.78; p < 0.001; duration;
H3 = 23.2; p < 0.001) and non-oriented (number; H3 = 19.4;
p < 0.001; duration; H3 = 18.5; p < 0.001) escape compared
with intra-AH treatment with vehicle preceded by adminis-
tration of vehicle in the dPAG (control group). In addition,
the treatment in the AH with SIN-1 increased the number
of entries (H3 = 22.68; p < 0.001) and time spent inside
(H3 = 20.74; p < 0.001) the burrow as well as the number of
access (H3 = 24.41; p < 0.001) and the time spent (H3 = 18.26;
p < 0.001) under or on the top of the elevated platforms for
escape, after displaying oriented escape to safe places. In con-
trast, intra-dPAG pretreatment with either ­CoCl2 or LY235959
followed by infusions of SIN-1 in the AH inhibited freez-
ing (p < 0.001 in both number and duration) and attenuated
the duration and number of oriented escape (p < 0.05 in both
cases), respectively, compared with intra-dPAG administra-
tion of vehicle followed by infusions of SIN-1 in the AH.
However, only intra-dPAG pretreatment with LY235959 was
able to reduce the number of burrow entries and elevated
platforms for escape access (p < 0.05 in both cases), com-
pared with intra-dPAG administration of vehicle followed
by infusions of SIN-1 in the AH. Regarding non-oriented
escape behaviour, intra-dPAG pretreatment with either C ­ oCl2
or LY235959 did not produce effects significantly different
(p > 0.05 considering both number and duration of non-ori-
ented escape) compared with intra-dPAG administration of
vehicle followed by infusions of SIN-1 in the AH (Fig. 3a–j).
Intra-dPAG microinjections of either ­CoCl2 or LY235959
followed by vehicle administration in the AH (control
groups) did not produce any defensive behaviour as com-
pared to the control (data not shown).
Regarding the locomotor activity, the treatment of the
AH with SIN-1 preceded by the administration of vehicle
into the dPAG caused an increase in the number of cross-
ings (distance travelled) compared with intra-AH treatment
with vehicle preceded by the administration of vehicle in the
dPAG (control group). In turn, neither ­CoCl2 nor LY235959
microinjections in the dPAG before the AH treatment with
vehicle or SIN-1 changed the locomotor behaviour of the
animals, as shown in Table 1.
Fear‑induced antinociception
According to a two-way RM-ANOVA, there were statisti-
cally significant effects of time (F9,162 = 4.226, p < 0.001),
but neither of treatment (F2,18 = 3.29, p > 0.05) nor of treat-
ment-by-time interaction (F18.162 = 3.29, p > 0.05) on tail-
flick latencies. The pretreatment of dPAG with neither C­ oCl2
nor LY235959 followed by vehicle administration in the AH
(control groups) produced nociceptive changes (p > 0.05)
compared with intra-AH treatment with vehicle preceded
by administration of vehicle in the dPAG (Fig. 4a).
13
According to a two-way RM-ANOVA, there were statisti-
cally significant effects of treatment (F3,24 = 24.8, p < 0.001),
of time (F9,216 = 105.9, p < 0.001), and of treatment-by-time
interaction (F27,216 = 14.28, p < 0.001) on tail-flick latencies.
AH treatment with SIN-1 preceded by the administration of
vehicle into the dPAG increased tail-flick latencies from 0 to
20 min after defensive behaviours compared to control group
(Tukey´s post hoc test; p < 0.05). In contrast, intra-dPAG
pretreatment with either C ­ oCl2 or LY235959 followed by
infusions of SIN-1 into the AH reduced tail-flick latencies
at the same time (p < 0.05 in both cases). In addition, the
intra-dPAG pretreatment with C ­ oCl2 was more effective in
reducing tail-flick latencies than intra-dPAG pretreatment
with LY235959 at time 0 (p < 0.05) after defensive behav-
iours elicited by infusions of SIN-1 in the AH (Fig. 4b).
In addition, a significant positive correlation was
observed between panic-like defensive behaviours and fear-
induced antinociception (r2 = 0.7395; ­df1,26 = 73.3; p < 0.05),
as shown in Fig. 4c.
Sites of drug microinjections into the dPAG and AH
Histologically confirmed sites of microinjections of vehicle,
­CoCl2 or LY235959 in the dPAG (Fig. 5a), and vehicle or
SIN-1 in the AH (Fig. 5b), were shown in schematic draw-
ings of coronal sections of the C57BL/6 mice brain. Repre-
sentative sites of drug microinjections in both dPAG and AH
were shown in photomicrographs of transverse sections of
mice midbrain and diencephalon, respectively (Fig. 5a, b).
A summary of the neuroanatomical and neuropharmaco-
logical findings obtained in the present work was provided
in Fig. 6.
Place preference behaviour displayed by mice
when submitted to the enriched polygonal arena
test
During the habituation procedure in the enriched polyg-
onal arena test for 4 days, mice displayed exploratory
behaviour and investigated uniformly all sections of the
new environment: the open space, in which food and tap
water were provided ad libitum and safe places, as shown
in Fig. 7a–c. Initially, they displayed defensive attention
after exit from the burrow (Fig. 7a), followed by explora-
tory behaviour towards the elevated platform for escape
on the top (Fig. 7a, b) and exploring the place situated
under the elevated platform for escape (Fig. 7d), some-
times returning to the burrow (Fig.  7c, on the top) or
going to the feeding area (Fig. 7c, at the bottom). After
habituation, when each mouse was submitted to a 10-min
re-exposure to the experimental environment (Fig. 7d–l)
without neurostimulation in a place preference control
experiment, they displayed exploratory behaviour of the
Psychopharmacology

Fig. 8  Track plots showing the

path travelled by each mouse
during exposure to the enriched
polygonal arena test (a–g) and
tracking heat map representing
the average movement and place
preference of mice submitted
to the enriched polygonal arena
test (h). The place preference
behaviour displayed by each
mouse was also recorded and
illustrated at the bottom (i).
Open area: non-enriched divi-
sion of the polygonal arena test;
safe places: enriched division
of the polygonal arena test
with two elevated platforms for
escape ­(EPE1 and ­EPE2) and a
burrow

open area (Fig. 7d, e, i) and the safe paces (Fig. 7f–k).
Track plots showing the path travelled by each mouse
during exposure to the enriched polygonal arena test
(Fig. 8a–g) and tracking heat map representing the mean
of the movement and place preference during exposure
of mice to the enriched polygonal arena test (Fig. 8h)
can be visualised in Fig. 8. Mice showed a preference
to explore the safe places (82.63%) rather than the open
area (58.37%), as shown in Fig. 8i. Considering the safe
places, the interactions of the mice with each division of
the first (the nearest to the open area) elevated platform
for escape was 15.69% (place situated under the elevated
platform) and 0% (top); the interactions of the mice with
each division of the second (the nearest to the burrow)
elevated platform for escape was 16.45% (place under the
elevated platform) and 1.4% (top); the interaction of mice
with the adjacency of the burrow was 35.57%, the inner
of the burrow was 13.88% and the roof of the burrow was
0.49%, as shown in Fig. 7i. Mice displayed passive avoid-
ance (time spent inside burrow) during 99 s. The time in
average spent in the open area was relatively short (121 s)
in comparison with the time spent in safe places that was

13
 Psychopharmacology
much higher (458 s), and 42 s was spent in neutral areas
(data not shown).
Discussion
This work aimed to address the role of the dPAG in medi-
ating fear-related defensive responses organised by AH
neurons. When a neural tract tracer was deposited in the
AH, dextran-labelled neural fibres from AH neurons were
found reaching both dmPAG and dlPAG, revealing AH-
dPAG efferent pathways. In turn, the activation of AH
neurons by local microinjections of the NO donor SIN-1
produced freezing, and oriented and non-oriented escape
behaviours along with antinociception in mice submitted
to the enriched polygonal arena test. In addition, SIN-1
injections in AH were also able to produce a significant
increase in locomotor activity, which was represented by
the increase in the number of crossings. Despite the NO
neuromodulation provoked panic-like responses in mice
submitted to the enriched polygonal arena test, the expo-
sure of control laboratory animals to this environment
showed a place preference to safe places, demonstrat-
ing the aversive valence of open areas in comparison to
the elevated platform for escape and burrow. It is known
that NO facilitates the release of glutamate in the syn-
aptic cleft. Once released from the synaptic cleft, gluta-
mate binds to the NMDA receptor, causing N ­ a+ and C­ a++
influx into the postsynaptic cell (Garthwaite et al. 1988,
1989 for review). Considering that glutamate signalling
within the AH is released by local infusion of the NO
donor SIN-1, glutamate could be contributing to triggering
defensive responses related to fear, as already proposed
elsewhere (Falconi-Sobrinho and Coimbra 2018). None-
theless, evidence from rodent studies suggests that neurons
of the MHD system do not independently organise certain
defensive responses, but rather recruit neurons from the
midbrain tectum to trigger them (Gross and Canteras 2012;
Ullah et al. 2017; Wang et al. 2015; 2021). Recent find-
ings by Ullah et al. (2017) showed that pretreatment of
dPAG with ­CoCl2 abolished freezing and explosive/non-
oriented escape behaviour, but failed to inhibit oriented
escape behaviours induced by microinjection of NMDA in
a higher dose into the dmVMH. Conversely, however, our
study showed that the pretreatment of dPAG with either
­CoCl2 or LY235959, while abolishing freezing, impaired
oriented escape rather than non-oriented escape behav-
iour elicited by administration of SIN-1 in the AH. Our
data suggest that dPAG of mice mediates the freezing and
oriented escape rather than non-oriented escape behav-
iours organised by AH neurons. Nevertheless, we observed
that non-oriented escape behaviours provoked by chemi-
cal stimulation of AH with SIN-1 were not characterised
13
by vigorous/explosive running. In this way, in addition to
AH of mice relying on dPAG neurons to organise freez-
ing and oriented escape behaviours, it seems to trigger
less robust defensive behaviours than the dmVMH when
chemically stimulated. Furthermore, the panicolytic effects
caused by pretreatment of the dPAG with either C ­ oCl2 or
LY235959 may be linked to a decrease in the activity of
glutamatergic inputs to this midbrain structure from the
AH. This insight can be evidenced by the fact that the anti-
aversive effects caused by the non-selective blocker of syn-
aptic contacts C
­ oCl2 administrated in the dPAG were also
observed after the pretreatment of dPAG with LY235959,
a highly selective NMDA glutamatergic receptor antago-
nist, which inhibited and impaired freezing and oriented
escape, respectively, induced by intra-AH infusion of the
SIN-1. However, although the AH-dPAG pathway exists,
functional manipulations of this pathway are needed to
support this hypothesis.
Interestingly, recent studies performed by Wang et al.
(2021) have suggested an analogous hypothalamic-PAG
functional pathway for rodents and human beings. These
authors provided evidence from functional magnetic reso-
nance imaging studies showing that hypothalamic-PAG
functional connectivity increases in human beings submit-
ted to aversive images exposure, whereas in rodents the
dPM-dPAG projections when optogenetically stimulated
can mediate defensive responses exhibited by these ani-
mals during exposure to aversive situations. In this respect,
considering that the blockade of the NMDA receptors in
the dPAG attenuated fear-related defensive behaviours
organised by AH neurons of mice as demonstrated herein,
our findings may provide new insights into the hypotha-
lamic-dPAG connection as a potential target for functional
manipulation and mapping studies of neural connections
in rodents and humans, respectively, as a putative pathway
for fear control.
It has been known that panic disorder is more preva-
lent among women than men (Remes et  al. 2016; Steel
et al. 2014). However, recent studies by Ferreira-Sgobbi
et al. (2022) comparing male and female rats did not iden-
tify defensive behavioural differences between them when
submitted to animal models of panic attacks based on
unconditioned threatening stimuli such as the prey versus
predator confrontation paradigm (rodent versus snake) and
acute exposure to hypoxia (7% O ­ 2). Both male and female
confronting a venomous pit viper initially triggered defen-
sive attention, risk assessment, defensive immobility and
escape. In addition, acute hypoxia evoked panic-like jump-
ing behaviour in both sexes. Among females, however,
defensive behaviours were different depending on the phase
of the menstrual cycle. In this sense, although these findings
only demonstrate behavioural differences between females in
different phases of oestrous cycle, but not between gender,
Psychopharmacology
additional studies using female mice submitted to a fear-
related unconditioned experimental model characterised by
intracerebral chemical stimulation could be relevant. This
was acknowledged as a limitation of the present study.
This investigation also showed that the microinjection
of SIN-1 into the AH provoked defensive antinociceptive
responses up to 20 min after the end of the behavioural test.
These data corroborated previous studies performed by our
team demonstrating that the activation of AH excitatory neu-
rons by local microinjections of SIN-1 activates pain-inhib-
itory descending pathways that mediate the antinociceptive
response that follows defensive behaviour (Falconi-Sobrinho
and Coimbra 2018; Falconi-Sobrinho et al. 2021). Further-
more, neuropharmacological data obtained in the present
study suggested that the modulation of spinal cord/spinal
trigeminal nucleus nociceptive transmission by AH activation
is mediated by dPAG neurons. We found herein that dPAG
pretreatment with C­ oCl2 impaired antinociceptive effects pro-
duced by intra-AH microinjections of SIN-1. Most notably,
decreased tail withdrawal latencies during the tail-flick test
were also observed in those animals that received intra-dPAG
microinjections of LY235959 prior to SIN-1 administration
into the AH. These data suggest that the reverse effects on the
fear-induced antinociception caused by the dPAG pretreat-
ment with either C ­ oCl2 or LY235959 were likely due to a
decreased activity of glutamatergic projections from AH to
the dPAG. In particular, evidence from studies performed in
laboratory rats suggest that the midbrain may be a relevant
relay of pain-inhibitory descending pathways from the lateral
hypothalamus, since that microinjection of either lidocaine
or ibotenic acid into the PAG resulted in significant increases
in electrical thresholds for lateral hypothalamus stimulation-
induced inhibition of the tail-flick reflex (Aimone et al. 1988).
Indeed, the findings obtained by Aimone et al. (1988) are in
line with our results that revealed that dPAG is an important
relay centre of descending pain modulatory pathways from
hypothalamic neurons. However, to examine the organisa-
tion in the midbrain tectum of pathways mediating descend-
ing inhibition of the pain-induced tail-flick reflex, unlike our
study, in which mice were submitted to an unconditioned
fear-related experimental model characterised by intracerebral
chemical stimulation, these authors used electrical stimulation
in lightly anaesthetised rats. In turn, other studies have sug-
gested that stress/fear-induced antinociceptive responses can
vary greatly among individuals and that this variability may
be linked to different factors such as sensitivity, species, prior
experience with stressful and type of aversive stimuli (Butler
and Finn 2009 for review; Cornélio et al. 2011). Interestingly,
pharmacological evidence for a dissociation between defen-
sive behaviour and fear-induced antinociception was also
previously reported (Biagioni et al. 2016; Falconi-Sobrinho
et al. 2021). Despite this, our data showed a significant posi-
tive correlation between panic-like defensive behaviours
and antinociceptive responses. The decrease in SIN-evoked
panic-like behaviours after microinjection of either C ­ oCl2
or LY235959 in dPAG was accompanied by a decrease in
defensive antinociception. In fact, considering that increas-
ing activity of glutamatergic pathways from AH to the dPAG
by intra-AH microinjections of SIN-1 facilitates panic-like
behaviours along with antinociception, it is expected that the
decrease in the activity of glutamatergic inputs to dPAG from
AH by blocking NMDA receptors in this midbrain structure
would impair both defensive responses.
In summary, our findings suggest that NMDA receptors
blockade in the dPAG impairs freezing and oriented escape,
but fails to reduce the non-oriented escape behaviour triggered
by the activation of AH neurons. In addition, dPAG NMDA
receptors seem to play a critical role in mediating defensive
antinociception organised by AH neurons.
Author contribution  L.L.F.-S. designed and executed the experiments,
analysed and interpreted the data, designed the figures and wrote the
manuscript. T. dos A.-G. collaborated on pharmacological approaches.
P.M.H and R.C.A collaborated in neuroanatomical approaches. B.M.de
P.R. performed the place preference behaviour-related experiments.
N.C.C designed the experiments, invented the enriched polygonal arena
test, performed the place preference experiments, recorded the neural
tract tracing images, analysed and interpreted the data, and wrote the
manuscript. All authors approved the final version of the manuscript
and are entirely responsible for the scientific content of this article.
Funding  The authors disclosed receipt of the following financial sup-
port for the research, authorship and/or publication of this article: This
work was supported by Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP) (grant numbers 2007/01174–1, 2012/03798–0,
2017/11855–8, and 2020/15050–7) and Conselho Nacional de Pesquisa
e Desenvolvimento Tecnológico (CNPq) (grant numbers 483763/2010–
1, 474853/2013–6 and 427397/2018–9). L.L. Falconi-Sobrinho was
supported by FAPESP (Scientiae Magister grant 2013/10984–8; post-
doctoral grant 2019/05255–3) and CNPq (Sc.M. fellowship grant
134267/2013–3; Scientiae Doctor fellowship grant 145258/2015–7).
T. dos Anjos-Garcia was financially supported by CNPq (Sc.M. fel-
lowship grant 130124/2012–5; Sc.D. fellowship grant 141124/2014–8)
and FAPESP (postdoctoral grant 2017/22647–7). P. M. Hernandes was
supported by CNPq (Sc.D. fellowship grant 142030/2020–1). B.M.
de Paula Rodrigues was financially supported by CAPES (M.Sc.
and Sc.D. fellowships, CAPES–PROEX-Psicobiologia-FFCLRP-
USP grant 0487 and CAPES-PROEX grant 8887.475628/2020–00,
respectively). R.C. Almada was supported by FAPESP (postdoc-
toral fellowship process 2012/22681–7, Young Investigator Program:
research grant process 2018/03898–1 and researcher fellowship pro-
cess 2019/01713–7) and CAPES (postdoctoral fellowship process
PNPD20131680-33002029012P3). N. C. Coimbra is a researcher sup-
ported by CNPq ­(PQ1A grants 301905/2010–0 and 301341/2015–0;
­PQ2 grant 302605/2021–5).
Data availability  The data that support the findings of this study are
available on request from the corresponding authors N.C. Coimbra and
L.L. Falconi-Sobrinho.
Declarations 
Conflict of interests  The authors declare no competing interests.
13
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