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\begin{document}

\title{Sex-dependent effect of inflammatory pain on negative affective states
is prevented by kappa opioid receptors blockade in the nucleus accumbens
shell}



\author[1]{Jesús Lorente}%
\author[1]{Javier Cuitavi}%
\author[2]{Laura Rullo}%
\author[3]{Sanzio Candeletti}%
\author[4]{patrizia romualdi}%
\author[1]{Lucia Hipolito}%
\affil[1]{University of Valencia}%
\affil[2]{Università degli Studi di Bologna}%
\affil[3]{Universita degli Studi di Bologna}%
\affil[4]{University of Bologna}%


\vspace{-1em}



  
  \date{May 16, 2024}


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\selectlanguage{english}
\begin{abstract}
Background and purpouse Psychological disorders, such as anxiety and
anhedonia are pain comorbidities, however how pain affects male and
female individuals and through which mechanism is not well understood.
Previous research show pain-induced alterations in the dynorphinergic
pathway in the mesocorticolimbic system (MCLS) together with a
relationship between corticotropin-releasing system and dynorphin
release in the MCLS. Here, we analyse the sex and time course-dependent
effects of pain on negative affect. Additionally, we study the
implication of dynorphinergic and corticotropin releasing factor
involvement in these pain related behaviours. Experimental approach We
used behavioural pharmacology and biochemical tools to characterize
negative affective states induced by inflammatory pain in male and
female rats, and the alterations in dynorphinergic and corticotropin
systems in the MCLS. Key results. Female rats showed a persistent
anxiety-like together with a reversible anhedonia-like behaviours
derived from inflammatory pain. Additionally, we found alterations of in
both dynorphin and corticotropin releasing factor in NAc and amygdala
that suggest sex-dependent dynamic adaptations. Finally blockade on the
kappa opioid receptor in the NAc confirmed its role in pain-induced
anxiety-like behaviour in female rats. Conclusions and implications Our
results show sex and time dependent anxiety- and anhedonia-like
behaviours induced by the presence of pain in female rats. Furthermore,
we replicated previous data pointing to the KOR/dyn recruitment in the
NAc as key neurological substrate mediating these behaviours. This
research encourages the study the mechanisms underlying these
behaviours, to better understand the emotional dimension of pain.%
\end{abstract}%



\sloppy


\textbf{1. Introduction.}

Pain is suffered by 30\% of adults in developed countries (S\selectlanguage{ngerman}á et al.,
2019). The therapeutical plan of chronic pain has traditionally been
focused on alleviating the symptomatology. In fact, psychological
comorbidities have been overlooked and, in the vast majority, no
treatment has been applied. During the last years, pain management has
become a multidisciplinary area including different specialist. By
applying different psychological therapies in combination with
pharmacotherapy and other pain-relief techniques, patients have improve
their quality of life, easing their pain sensation, diminishing the
possibility of disability, and reducing fear-avoidance behaviours
(Petrucci et al., 2022). Interestingly, men and women show different
pain physiopathology resulting in discrepancies in pain feeling,
response to pain killers and psychological comorbidities (Edwards et
al., 2003; Pieh et al., 2012; Popescu et al., 2010; Riley et al., 1998).
Nevertheless, since females have traditionally been excluded in research
(Burek et al., 2022), the lack of sex-based studies could have led to
undertreat pain-related conditions in women, promoting chronic pain and
the development of neuropsychiatric comorbidities (Cáceda et al., 2021;
Jakubczyk et al., 2016). Moreover, a metanalysis performed by Burek and
collaborators showed that the classical behavioural tests of anxiety-
and depression-like behaviours in the presence of pain lead to
contradictory results depending on the strain, source, sex of the animal
and testing time after the induction of pain (Burek et al., 2022)

Recent reports highlighted that pain induces alterations in the
mesocorticolimbic system (MCLS) in both, kappa opioid receptor
(KOR)/dynorphin (DYN) and corticortropin-releasing factor (CRF) systems
(Fu \& Neugebauer, 2008; Ji et al., 2007; Markovic et al., 2021; Massaly
et al., 2019; Mazzitelli et al., 2022; Navratilova et al., 2019). Along
with other effects within the MCLS, the KOR/DYN system recruitment in
the nucleus accumbens (NAc) has shown to mediate negative affective
states induced by inflammatory and neuropathic pain (Liu et al., 2019;
Massaly et al., 2019). Additionally, presence of inflammatory pain has
led to alterations in the CRF 1 receptors (CRF1R) transmission in
central amygdala (CeA) (Fu \& Neugebauer, 2008; Ji et al., 2007). In
fact, CRF1R activation in CeA is necessary to develop pain-related
anxiety-like behaviour, and the activation of KORs in CeA disinhibits
CRF1R producing pain-related anxiety-like behaviour (Hein et al., 2021;
Ji et al., 2007; Mazzitelli et al., 2022). Furthermore, the
optoinhibition of CRF containing neurons in CeA is sufficient to impair
anxiety-like behaviour induced by pain (Hein et al., 2021; Mazzitelli et
al., 2022). Interestingly, KOR blockade in CeA controls pain-related
behaviour in a model of functional pain by restoring synaptic inhibition
of CeA-CRF neurons, demonstrating that KOR and CRF systems are closely
related in pain and its comorbidities (Yakhnitsa et al., 2022). However,
most of these cited papers have obtained these results only in male
rodents leaving females understudied.

In order to shed light on the time course and sex-dependent effects of
pain-induced comorbidities, here we will use male and female rats to be
tested in classical anxiety/depression behavioural assays for a total
period of three weeks. Additionally, we will study the CRF and KOR/DYN
systems alterations in the MCLS that may be implicated in the observed
behaviours.

\textbf{2. Methodology.}

\textbf{2.1. Animals and pain model.}

A total of 124 Sprague-Dawley rats (Envigo, Barcelona, Spain) of 7-8
weeks old at the beginning of the behavioural experiments, 100 females
(170-200 g) and 24 males (260-300 g) were used. Animals were housed
individually in standard plastic cages (42 x 27 x 18
cm\textsuperscript{3}) provided with shredded aspen bedding (Teklad,
Barcelona, Spain) and cotton enrichment (iso-BLOXTM; Teklad) and were
maintained in 12/12 hours light/dark cycles (light off at 07:00 am,
light on at 07:00 pm), at 23 ± 1 º C and 60\% humidity. All behavioural
tests were conducted during the dark cycle and at least 2 hours after
the lights turn off. Food and tap water were available \emph{ad
libitum}throughout the experimental period. The protocols used were
approved by the Animal Care Committee from the University of Valencia
and authorised by the regional government, and the studies were
performed in strict accordance with Spanish laws (RD 53/2013) and
European Directives (EC 2010/63). Animals were sacrificed by isoflurane
overdose for further biochemistry analysis.

Rats received a subcutaneous injection of 0.1 mL of the complete Freund
adjuvant (CFA; diluted at 1:2 in sterile saline to create an emulsion)
in the hindpaw (Hipolito et al., 2015). This animal model of pain based
on the CFA administration has been broadly used for replicating aspects
of arthritis (Fischer et al., 2017).

\textbf{2.2. Experimental design.}

\textbf{2.2.1. Experiment 1: inflammatory pain effect on negative
affective states, corticortropin releasing factor system and
Kappa/dynorphin system in the Nucleus Accumbens and amygdala.}

\textbf{2.2.1.1. Behavioural experiments design and brain collection.}

To study the time course and the gender effect of negative affective
states induced by inflammatory pain we have selected a combination of
different behavioural approaches. One week prior to CFA administration,
we obtained basal nociceptive measures by means of Von Frey test (VFT)
together with basal measures of the sucrose preference test (SPT).
During the following 18 days after complete Freund Adjuvant (CFA)
administration, animals were repetitively tested in the VFT, light-dark
box test (LDB) and SPT (n=12-14/group). Finally, 18 days after the CFA
administration, rats were sacrificed to collect their brains. Details of
this protocol and timeline are summarized in figure 1A and figure S1.

\textbf{2.2.1.2. Biochemicals analysis.}

\emph{Blood and brain samples collection and preparation.}

Immediately after the overdose of isoflurane, blood samples, were
obtained by heart puncture and placed in plastic tubes, which were flash
frozen in contact with dry ice. Immediately, after collecting blood
samples, we extracted brains which were also flash frozen in dry ice.

The plasma collection was performed the same day the ELISA assays were
performed. To extract plasma, we first defrosted the blood samples in
contact with ice. Once all the samples were thawed, we kept them 30 min
in the ice. Next, we centrifugated them for 10 minutes at 33500 G, and
we collected the supernatant.

Tissue from amygdala and NAc was collected by punching in brain slices
of 61.2 and 61.6 \selectlanguage{greek}µ\selectlanguage{english}m respectively. The bregma coordinates of the brain
slices were from 2.52 to 0.96 mm for NAc, and from -2.04 to -3.24 mm for
amygdala (Figure S2).

\emph{RNA isolation and qRT-PCR.}

Total RNA was isolated using TRIZOL reagent (Life Technologies, USA)
according to the method of Chomczynski and Sacchi (1987). The integrity
of the samples was verified by 1\% agarose gel electrophoresis and the
RNA amount in each sample was measured by spectrophotometry, as
described (Caputi, et al., 2021a).

RNA samples were subsequently subjected to DNase treatment and converted
to cDNA using the GeneAmp RNA PCR kit (Life Technologies, Italy)
according to the manufacturer's protocol.

The qRT-PCR analysis was performed on a StepOne Real-Time PCR System
(Life Technologies, Monza, Italy) using SYBR Green PCR MasterMix (Life
Technologies, Italy), as previously reported (Caputi, et al., 2021b).
Relative expression of the different gene transcripts was calculated by
the Delta-Delta Ct (DDCt) method and converted to relative expression
ratio (2-DDCt) for statistical analysis (Livak \& Schmittgen, 2001). All
data were normalised to the housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). The primers, designed using Primer 3 (Rozen \&
Skaletsky, 2000) were used for PCR amplification.

pDYN forward 5'- CCTGTCCTTGTGTTCCCTGT-3'; pDYN reverse 5'-
AGAGGCAGTCAGGGTGAGAA-3'; KOR forward 5'- TTGGCTACTGGCATCATCTG-3'; KOR
reverse 5'- ACACTCTTCAAGCGCAGGAT-3'; CRF forward
5'-GCAGCGGGACTTCTGTTGA-3'; CRF reverse 5'-CGCAGCCGTTGAATTTCTTG-3'; CRFR1
forward 5'- TGCCAGGAGATTCTCAACGAA-3'; CRFR1 reverse 5'-
AAAGCCGAGATGAGGTTCCAG-3'; GAPDH forward 5'- AGACAGCCGCATCTTCTTGT-3';
GAPDH reverse 5'- CTTGCCGTGGGTAGAGTCAT-3'.

\emph{Protein extraction.}

For protein analysis, we used a previous published protocol of protein
extraction (Lorente et al., 2022). 0.5 mL of cold lysis buffer (1\%
IGEPAL CA-630, 20mMTris-HCl pH 8, 130MNaCl, 10mMNaF, and 1\% proteases
inhibitor cocktail {[}Sigma, Darmstad, Germany{]}) was used to
homogenate 250 mg of brain tissue, thus the volume of the lysis buffer
was adapted to the real quantity of tissue in each sample. Then we
maintained the samples in ice for 30 minutes, before centrifugate at
13000 G for 15 minutes at 4 \selectlanguage{ngerman}º C and collect the supernatant.
Furthermore, we determined the concentration of the lysates by using a
Bradford protein assay kit (Bio-Rad).

\emph{ELISAs.}

Two different ELISA kits (Cusabio, Houston, USA) were used to detect the
levels of DYN in amygdala and NAc samples (\#REF CSB-E13294r) and
cortisol in plasma (\#REF CSB-E05112r), according to kit protocols.

\emph{Western blot.}

We used a protocol previously used in our laboratory (Cuitavi et al.,
2021). 1-mm acrylamide gels at 10\% with 15 wells were used for the
electrophoresis. We mixed 20 \selectlanguage{greek}µ\selectlanguage{english}g of total protein, loading buffer (350 mM
Tris pH 6.8, 30\% glycerol, 30\% mercaptoethanol, 100g/L sodium dodecyl
sulphate, and 200 mg/L bromophenol blue) and water to obtain the same
total protein and volume per sample, and they were heated at 70\selectlanguage{ngerman}ºC for 20
minutes. We used the Bio-Rad Mini-PROTEAN buffer system (6 g/L Trizma
base, 2.88 g/L glycine, and 20 g/L sodium dodecyl sulphate) to perform
the electrophoresis at 120 V for 80 to 90 minutes. Once, the proteins
were separated by sodium dodecyl sulphatepolyacrilamide gel
electrophoresis (SDS-PAGE), we transferred it to nitrocellulose
membranes (Bio-Rad) with the appropriate buffer (3 g/L Trizma base, 1.44
g/L glycine, and 20\% methanol) and a semidry system (Bio-Rad Trans-Blot
TurboTM) for 25 minutes at 25 V buffer.

Then, membranes were blocked using 5\% non-fat dried milk in TBS-Tween
20 (TBS-T) 0.1\% (20 mM Tris and 500 mM NaCl pH 7.5) for 1 hour, and
just before they were incubated with the corresponding primary antibody
(rabbit anti-KOR 1:2000, REF \#44-302G, Thermo Fisher; goat anti-CRFR1
1:1800, REF \#PA5-18801, Thermo Fisher; rabbit anti-GAPDH 1:2000, Thermo
Fisher) overnight at 4º C. After that, membranes were washed 3 times
with TBS-T 0.1\% and were incubated for an hour at room temperature with
the secondary antibody (Goat anti-Rabbit IgG (H+L) HRP conjugated at
1:2000 for GAPDH and KOR (REF \#31460, Thermo Fisher; Donkey anti-Goat
IgG (H+L) HRP conjugated 1:2000 for CRF1R, REF \#A15999, Thermo Fisher).
Finally, we developed the membranes with chemiluminescence using
CheLuminate-Horseradish peroxidase (HRP) PicoDetect (Panreac, Barcelona,
Spain), and images were captured with the ChemiDoc XRS1 System (Bio-Rad)
and quantified using ImageJ software. The grey intensity of the bands
was normalised dividing the values of the protein of interest by the
GAPDH values. The relative protein levels were expressed as mean ± SEM.

\textbf{2.2.2. Experiment 2: effect of norbinaltorphimine administration
in the nucleus accumbens medial shell on complete Freund
adjuvant--induced negative affective states in female rats.}

\textbf{2.2.2.1. Behavioural experiment protocol.}

To study the influence of KOR/DYN system in pain-induced negative
affective states in NAc of female rats, the same protocol of experiment
1 was used except for the additional bilateral infusion of the kappa
opioid receptor antagonist norbinaltorphimine (NorBNI) into the NAc
medial shell. The week prior to the CFA administration after the basal
measures for VFT and SPT were obtained, animals were injected with
NorBNI or artificial cerebral spinal fluid (aCSF) into the NAc medial
shell. The following 14 days after CFA administration animals were
tested following the VFT, LDB and SPT paradigms. The timeline followed
is summarised in figure 4A and figure S3A.

\textbf{2.2.2.2. Surgery and norbinaltorphimine intra-accumbal
administration.}

All surgeries were performed under isoflurane anaesthesia (1.5-2 minimum
alveolar concentration, MAC) and under aseptic conditions. Before
anesthetising the animals for the surgeries, they received injections
(s.c.) of 1.8 mg/kg enrofloxacin (Bayer) and 2.5 mg/kg of carprofen
(Pfizer). Then, we anesthetised animals in a induction chamber and used
0.1\% topical lidocaine in the surgical area and in the ears before
fixing animals to the stereotaxic frame (Stoelting, Wood Dale, USA). The
skull of the rats were exposed, and a craniotomy was bilaterally
performed above the posteromedial NAc shell. By means of a stainless
steel microinjector (33-gauge) attached to a PE-10 tubing and a 25-mL
Hamilton syringe mounted on a syringe pump (Kd Scientific, Holliston,
USA) we injected aCSF (0.5 uL per side) or NorBNI (2 ug per hemisphere
in 0.5 uL, REF \#N1771, SigmaAldrich, St. Louis, USA) in the following
coordinates: +0.96 mm anteroposterior, ±0.8 mm mediolateral, and -6.2 mm
dorsoventral from bregma in a flat skull position (Massaly et al.,
2019). Finally, we covered the craniotomies with bone wax (Ethicon,
Cincinnati, USA) and the skin of each animal was sutured with a nylon
monofilament suture (Ethicon, Cincinnati, USA). Rats were allowed to
recover in a box provided with a heat pad and were closely monitored
until they fully recovered from the anaesthesia. During the days
following the surgery, rats were daily examined and received injections
(s.c.) of 1.8 mg/kg enrofloxacin and 2.5 mg/kg of carprofen once a day
the following 2 days.

\textbf{2.2.2.3. Microinjection placement assessment.}

At the end of the protocol, rats were sacrificed under isoflurane
overdose and brains were removed and rapidly frozen in dry ice; 40
\selectlanguage{greek}µ\selectlanguage{english}m-thick coronal slices of the NAc shell were obtained using a cryostat
(Micron, Boise, USA) and stained with a cresyl violet (Sigma, St. Louis,
USA) protocol to verify proper probe placement as we have used
previously (Campos-Jurado et al., 2020). Only animals that received a
bilateral injection into the NAc medial shell were included in the
analysis (figure S3B).

\textbf{2.3. Behavioural procedures.}

\emph{Sucrose preference test.}

For SPT, a slightly modified protocol of a reported procedure (Schalla
et al., 2020) has been used. Briefly, animals had free access to 5\%
sucrose diluted in tap water along with tap water twice a week, in two
different types of sessions. The first session lasted 48 hours (divided
in 2 consecutive sessions of 24h, since bottles needed to be weighted
and refilled), and was followed by a period of 24h without access to
sucrose. Following, the second session started and lasted for 2 hours.
After obtaining a basal measure the week prior to the pain induction, we
repeated the protocol every week until the end of the experiment. All
measurements were expressed as the mean \selectlanguage{ngerman}± SEM in mg of sucrose/rat
weight in kg/24h or mg/kg/2h.

\emph{Light-dark box test.}

The LDB apparatus consists of a box (64 x 48 x 24 cm\textsuperscript{3})
that is divided in 2 different chambers: light chamber (two-thirds of
the total size) and dark chamber (one-third of the total size). The
light and dark chambers are connected by a squared opening (8 x 8
cm\textsuperscript{2}). The light chamber, which is uncovered, has white
walls, and is illuminated by white light (400-lux). The dark chamber has
black walls and has no appreciable illumination at the centre of the
chamber (\selectlanguage{english}[?] 2 lux). After at least 5 min of habituation, animals were
gently placed at the centre of the light box, with the head facing
opposite to the squared opening, and then animals explored freely for 5
minutes. Spontaneous behaviours of the rats were videotaped in the light
box during the test for its posterior analysis. The test was performed
during the dark cycle, at least 2 hours after the lights went off. The
analysed parameters were the time spend in the light box (seconds, s)
and the number of transitions between chambers. All the measurements
were expressed as the mean +- SEM.

\emph{Von Frey test.}

The VFT was used to assess the mechanical nociceptive thresholds before
(basal) and after the CFA injection. After 5-10 min of habituation to
the apparatus, Von Frey filaments (Aesthesio(r)) were manually applied to
the injected hind paw with the simplified up-down method (Bonin et al.,
2014). Results were expressed as the mean +- SEM of mechanical
sensitivity threshold (in grams, g).

\textbf{2.4. Statistical analysis.}

After testing the normality of distribution data (Shapiro-Wilk test) and
homogeneity of variance, unpaired Student's t test, Welch's t test,
Mann-Whitney U test were used to analyse gene expression, ELISAs and
western blot results. Statistical analysis was performed using GraphPad
Prism 9 software (San Diego, USA) or IBM SPSS statistics v24 software.
For the behavioural data, we used the ANOVA for repeated measures
followed by Bonferroni multiple comparisons as \emph{post hoc} test when
appropriate. Results are expressed as mean +- SEM and the statistical
significance was set at p \textless{} 0.05. All the p values of the
statistic tests are reported in Table 1 and the p values from
the\emph{post hoc} analysis are described in the figure legends.

\textbf{3. Results.}

\textbf{3.1. Sex- and time-dependent anhedonia and anxiety-like
behaviours induced by inflammatory pain.}

Pain highly impacts patients' quality of life by promoting negative
affective states (Pieh et al., 2012; Popescu et al., 2010). Thereby, in
this study we assess the time course of the development of these
negative affective states in an inflammatory pain model based on CFA
injection. To this end, we have monitored anhedonia and anxiety-like
behaviours (SPT and LDB respectively) for several weeks (fig. 1A and
S1).

We selected the SPT as a behavioural approach to measure anhedonia.
First, we analysed the total sucrose consumption (mg/kg/24h) in the 48h
test on post-CFA injection days 1-2, 8-9 and 15-16, followed by the
analysis of the total sucrose consumption in the 2h test on post-CFA
injection days 4,11 and 18 (fig 1A). In male rats, the statistical
analysis of the 48h test revealed significant time (F (3, 66) = 6.636, p
= 0.001, partial \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.966) and the pain x time
interaction effect (F (3, 66) = 3.709, p = 0.016, partial
\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.783; table 1) but no effect for the pain
variable (F (1, 22) = 0.69, p=0.795, partial \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}=
0.057). Thus, the development of the inflammatory condition decreased
the sucrose consumption along the experimental procedure for both pain
and no pain group. Their sucrose intake at days 1-2 for pain group, and
at days 8-9 and 15-16 for control group compared with their basal
measures in the 48h test was significantly lower (fig 1B, Bonferroni
multiple comparisons intra-subjects: p= 0.04; p= 0.17 and p= 0.034,
respectively). The analysis of the 2h access to sucrose sessions by the
repeated measures ANOVA only detected time dependent effect (F (3, 66) =
8.886; p \textless{} 0.001, partial \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.993), but no
differences between groups were found in the Bonferroni multiple
comparisons test (fig. 1D). However, female rats have shown a different
pain-induced anhedonia-like behaviour profile (fig. 1C). Indeed, the
ANOVA detected pain (F (1, 25) = 7.688, p=0.01, partial
\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.760), time (F (3, 75) = 3.496, p=0.02, partial
\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.759) and the interaction pain x time (F (3, 75)
= 7.819, p= 0.001, partial \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.986) significant
effects. In fact, the post-hoc analyses showed that CFA-females reduced
their sucrose consumption during days 1-2 compared with saline-females
(p= 0.0001) and with their own basal intake (p= 0.003). Interestingly,
CFA-females recovered from the anhedonia showed in the 1-2 days post-CFA
injection; indeed, no changes in total sucrose intake were observed in
the next two sessions in comparison with the respective controls
(comparisons with basal: p\textsubscript{8-9}= 1.000 and
p\textsubscript{15-16}= 1.000; comparisons with 1-2 days after
CFA-injection: p\textsubscript{8-9}= 0.001 and p\textsubscript{15-16}=
0.0001). The 2 h tests showed no alterations in the sucrose intake
derived from the presence of pain during the experimental procedure
since no statistical differences were observed in none of the studied
variables (fig.1E, table 1).

Second, anxiety-like behaviours induced by inflammatory pain were
studied with the LDB test by analysing the time spent in the light
chamber (Figures 1F and 1G). The ANOVA for repeated measures detected
only an effect of time in both measures, time in light (F (1, 22) =
19.284; p= 0.0001, partial \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.987) and number of
transitions (F (1, 22) = 19.284; p= 0.0001, partial
\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.987) in the case of males (Fig 1F and 1H),
meaning that inflammatory pain has no effect on anxiety-like behaviour.
Indeed, males showed an increase of the time in the light box and the
number of transitions in the second week when compared to the first week
probably due to habituation.

Interestingly, the analysis of data from female rats, revealed an effect
for pain (F (1, 25) = 7.287; p = 0.012) and time (F (1, 25) = 4.355; p =
0.047, \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.519) but not for the interaction pain x
time (F (1, 25) = 0.278; p = 0.603, \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.080; fig
1G). Thus, the post-hoc analysis revealed that CFA-treated females
reduced their time spent in the light chamber during the second week
compared with non-pain group (p= 0.05). The analysis of the number of
transitions in the LDB, showed no differences in pain, time or the
interaction of these two variables for male and female rats, indicating
no differences in the general motor activity and exploration ability of
the CFA-treated rats.

Finally, the mechanical sensitivity threshold was assessed along the
experimental procedure by using the Von Frey test. Figures 1J and 1K
show that CFA-treated male and female rats maintained a low threshold
after the CFA injection. The statistical analysis showed an effect for
pain, time and pain x time interaction in both sexes (Male rats: pain
F(1,22)= 6.223, p= 0.665, \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= ; time F(2,44)= 4.148,
p= 0.022, \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.932; pain x time F(2,44)= 11.538, p=
0.0001, \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.639; female rats: pain F(1,25)= 56.140,
p=0.0001, \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 1; time F(2,50)= 3.252, p=0.047,
\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.265; pain x time F(2,50)= 11.783, p= 0.0001,
\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.975). Post-hoc analyses revealed that both male
and female rats in pain showed significant lower threshold at day 6 and
13 when compared with their own basal and with no pain group (Male rats:
p\textsubscript{6}\textless{} 0.027 and p\textsubscript{13}\textless{}
0.006; Female rats: p\textsubscript{6}\textless{} 0.001 and
p\textsubscript{13}\textless{} 0.001)

\textbf{3.2. Inflammatory pain influence on corticotropin releasing
factor (CRF), CRF receptor 1 (CRFR1) and blood cortisol levels.}

CRF signalling is increased as a result of the presence of stress
activating the hypothalamus-pituitary-adrenal axis (HPA) to produce a
neurohormonal response. Chronic stress and negative affect derived from
this stress affects the HPA and the functioning of some brain areas such
as amygdala or nucleus accumbens. Activation of the HPA system leads to
increased corticosteroids blood levels to respond to a threat or
stressful event, but finally this increase exerts negative feedback to
the HPA system to limit this response (Herman et al., 2016; Koob et al.,
2014). Thereby, we measured mRNA and protein levels of CRF and CRFR1 in
the NAc and the Amy by qRT-PCR and western blot. In addition, we also
measured blood cortisol levels by ELISA (fig. 2).

mRNA levels of CRF in NAc and Amy where significantly increased in
CFA-treated male rats (fig 2A, p= 0.045; 2B, p=0.038) together with an
increase in the mRNA of CRFR1 only in Amy and not in NAc (fig 2C, p=
0.075; fig 2D, p= 0.046) at the endpoint of the experimental protocol.
However, further analysis of the CRFR1 protein expression showed that
this altered expression in the mRNA had no effect on the local receptor
expression (Figure 2E, p= 0.999 and figure 2F, p= 0.093). Finally, blood
cortisol levels, at this time point, were not altered in CFA-treated
male rats (fig 1G, p= 0.589).

Very interestingly, we did not observe any changes in the levels of the
CRF or CRFR1 mRNAs or proteins in the NAc or in the Amy of CFA-female
rats (figure 2H, p= 0.445; figure 2I, p= 0.177; figure 2J, p= 0.394;
figure 2K, p= 0.42; figure 2L, p= 0.818; figure 2M, p= 0.590). However,
as shown in figure 2N, females with inflammatory pain presented a
significant lower blood cortisol level than saline-treated females (p =
0.033).

\textbf{3.3. Dynorphin/kappa opioid receptors mRNA and protein levels
alterations in the mesocorticolimbic system induced by inflammatory
pain.}

The dynorphinergic system, which comprises the KOR and their ligand DYN,
are tightly related to pain induced negative affect. Moreover, the MCLS
seems to be involved in the modulation of negative affective states
occurring in pain conditions. (Caputi et al., 2019; Jes\selectlanguage{ngerman}ús David Lorente
et al., 2020; Massaly et al., 2019) Thereby, we measured the mRNA levels
of pDYN \emph{and} KOR in the NAc and the Amy by qRT-PCR together with
its protein expression in these areas by ELISA and western blot.

On one hand, as observed in Figures 3A to 3D, the presence of
inflammatory pain in male rats led to some alterations in the pDYN and
KOR mRNA expression in the Amy and NAc at the end point of the
behavioural characterisation. However, the protein levels remained
unaltered (figure 3E to 3H). Indeed, inflammatory pain in male rats
increased pDYN mRNA expression in the Amy (figure 3B, p= 0.05), and
interestingly reduced KOR mRNA in the NAc (figure 3C, p= 0.026).

On the other hand, mRNA expression of pDYN and KOR 18 days
\emph{after}the inflammatory pain induction, were not significantly
altered in the NAc or in the Amy (figure 3I to 3L) of female rats,
although some tendencies to increase the mRNA can be perceived. Protein
expression also remained unaltered (figure 3N to 3P) regardless of the
DYN in the NAc that was significantly reduced in the presence of
inflammatory pain (fig 3M, p = 0.003).

\textbf{3.4. Kappa opioid receptor blockade rescues inflammatory
pain-induced anxiety-like behaviours in female rats.}

Previous data have shown that the blockade of the KOR/DYN signalling in
the NAc shell was an appropriate strategy to impair the development of
negative affect and alcohol-relapse-like behaviour induced by pain
(Massaly 2019, Lorente 2021). Thus, we found interesting to
pharmacologically analyse the role of NAc KOR in the inflammatory
pain-induced negative affective states reported in female rats in the
present study (figure 1). The blockade of the KOR locally in the nucleus
accumbens shell of female rats (figure 5A and S3A) impacted the
behaviour in the LDB without altering the SPT. In fact, in the case of
the SPT 48h, the ANOVA only detected differences for the time variable
(F(2,130)= 15.259; p= 0.0001; \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.19) and the
interaction time x pain (F(2,130)= 11.576; p= 0.0001;
\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.151). Further post-hoc analyses revealed that
CFA groups decreased their sucrose consumption compared with saline
groups at days 1-2 (p= 0.010), while no differences were found for the
other variables and interactions (Figure 4B) including the treatment
variable and its interactions (see table 1). The analysis of the SPT of
2h, revealed that the consumption of sucrose increases over the time and
the animals whose received NorBNI infusion showed a high sucrose
consumption than aCSF groups. Indeed, the ANOVA showed differences for
the time and treatment variables (F(2,130)= 51.364; p=0.0001;
\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.597; and F(1,65)= 5.054; p= 0.028;
\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.072). However, we did not detect differences for
pain variable and the interaction between variables (Figure 4C).

Finally, when analysing the LDB test we observed that NorBNI reverse the
decrease in the time spent in light box of CFA female rats without
altering the transition between boxes. For the time in light, we
detected differences in the time variable (F(1,65)= 12.969, p= 0.001;
\selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.166), showing that animals spent more time in
light during the second week. Furthermore, we detected differences in
the pain variable (F(1,65)= 11.470, p= 0.001; \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}=
0.15) and in the interaction between pain x treatment (F(1,65)= 3.954,
p= 0.05; \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.057). The treatment variable and the
other interactions showed no significative differences, and their F
values are showed in table 1. Post-hoc analyses of the significant
interaction and variables revealed that pain-suffering female rats that
received aCSF spent a shorter time in light box, compared to the
pain-free animals at 7 and 14 days (p\textsubscript{7}= 0.001,
p\textsubscript{14}= 0.012) while the pain-suffering female rats
receiving NorBNI spent in the light compartment the same time as saline
groups (Figure 4D, p\textsubscript{7}= 0.181; p\textsubscript{14}=
0.974). Thus, the administration of NorBNI counteracted the anxiety-like
behaviour induced by CFA injection in female rats. The analysis of the
transitions between boxes detected a significant effect for the time
(F(1,65)= 45.153; p= 0.0001) but not for the other variables and their
interactions (see table1). Thus, the transitions at 7 days were lower
than the transitions at 14 days either after saline or after CFA
injection.

Finally, we analysed the mechanical sensitivity thresholds with the VFT
to confirm that all CFA-treated rats maintained values until the end of
the protocol. We detected differences in pain (F(1,65)= 69.774; p=
0.0001; \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.518) and time (F(1,65)= 55.861; p=
0.0001; \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}= 0.521) variables and in the interaction
between time x pain (F(1,65)= 36.695); p= 0.0001; \selectlanguage{greek}η\selectlanguage{english}\textsuperscript{2}=
0.41), while no differences were detected for treatment variables and
for their interactions (see table 1). Post-hoc analyses revealed that
female rats showed lower paw withdrawal thresholds after 7 and 14 days
following CFA injection compared with their own basal values (before the
injection, p\textsubscript{7}= 0.0001); p\textsubscript{14}= 0.0001) and
with their saline counterparts (p\textsubscript{7}= 0.0001;
p\textsubscript{14}= 0.0001).

\textbf{4. Discussion.}

Here, we provide new details of the sex- and time-dependent effect of
inflammatory pain on the development of negative affective states.
Inflammatory pain induces persistant negative affective states in
females. Very interestingly, we found biochemical alterations in the HPA
axis and in the mRNA of pDYN and KOR in NAc and amygdala in male rats
that did not correlate with the behaviour observed. Alterations in
protein levels were found only in the case of CFA-female rats, showing a
reduction of DYN in NAc, although the pharmacological blockade of the
KOR in the NAc effectively blunted the anxiety-like behaviour showed by
CFA-female rats. Thus, we propose that alterations in how these systems
respond to the presence of inflammatory pain in males
\emph{versus}females, might underlie the observed sex-dependent
pain-induced negative affect. Although our biochemical analysis is not
compelling, the blockade of the KOR within the NAc shell shows a
potential target to treat psychological comorbidities of pain
conditions.

Many studies highlighted that pain induces anxiety- and anhedonia-like
behaviours as well as low motivation for natural reinforcement under a
goal-directed behaviour (Lorente et al., 2020; Markovic et al., 2021;
Massaly et al., 2019). However, unfortunately, most of these studies
used only male subjects and limited observations only to specific time
points after pain induction. Therefore, the aim of this study was to
fill this gap in the literature by exploring whether inflammatory pain
affects anxiety- and anhedonia-like behaviours in both sexes separately
to avoid the overseeing of small differences. In addition, we added
longer periods of time to study the time course of these pain-induced
behaviours. In this framework, the involvement of the HPA axis and
KOR/DYN signalling has also been explored in relevant brain areas. Here,
we shown that both male and female rats developed anhedonia-like
behaviour at the early phase of CFA-induced pain. Indeed, we observed a
reduction of sucrose intake on day 1 and 2 after CFA-administration,
being the effect more pronounced in female rats. This first finding
agrees with previous results indicating a reduction of sucrose
consumption a few days after pain induction both in non-operant and
operant procedures in rats (Hipolito et al., 2015; Markovic et al.,
2021; Massaly et al., 2019; Schwartz et al., 2014). Together with this
decrease in the motivation for sucrose consumption, we also found that
development of anxiety-like behaviour was affected by pain. However,
this specific behaviour was only observed 2 weeks after the induction of
inflammatory pain in female rats. Contradictory data regarding pain
induced anxiety-like behaviour in rodents are present in literature. A
detailed review by Kremer and co-workers, reported that CFA-inflammatory
pain induces anxiety-like behaviour, at the onset of the pain condition
(1-2 weeks) and after becoming chronic ( \textgreater{} 2 weeks);
however a high number of studies did not report these anxiety-like
behaviour (Kremer et al., 2021). In this regard, it is very important to
highlight that most of the examined studies only used male rodents and,
the few studies that used female animals showed that CFA was sufficient
to induce anxiety-like behaviour (Liu et al., 2019; Pitzer et al., 2019;
Refsgaard et al., 2016). In addition, Liu and collaborators also showed
sex-dependent differences of pain effects not only at behavioural but
also at biochemical level.

It has been well documented that stress and anxiety alter cortisol
plasma levels, usually inducing an increase of this hormones. However,
in the presence of chronic stress cortisol levels have also been
reported to be decreased (Adzic et al., 2009; Costache et al., 2020;
Gong et al., 2015; Kim et al., 2018). In the last years, pain has been
defined as a stressor that can participate in the development of
psychological diseases such as anxiety and depressive disorders (Csupak
et al., 2018; Tsang et al., 2008). Accordingly, we analysed cortisol
plasma levels after 18 days of pain induction. Interestingly, we did not
observe differences in male rats; instead, pain-suffering female rats
showed a significant reduction compared with their control counterparts.
It is known that plasma cortisol levels increase after acute pain,
however a prolonged or exaggerated stress condition in response to pain-
and non-pain-related stressors can aggravate pain and promote further
disabilities (Edwards et al., 2008; Hall et al., 2011; Heim et al.,
2000; Tak \& Rosmalen, 2010). Therefore, a desirable response of the
stress system in the presence of prolonged pain would be the reduction
of its activation towards a homeostatic level, to avoid worsening of the
pain condition. This regulation might explain data here observed in male
rats that despite of showing some gene expression changes, did not
display alterations in the CRF and CRFR1 protein levels in NAc and Amy.
In addition, cortisol plasma levels of CFA-male rats were not different
from controls 18 days after pain induction.

On the other hand, the chronic reactivation of stress responses leads to
exhaustion of HPA axis and induces hypocortisolaemia (Penninx et al.,
2007), that may explain the cortisol dysfunction that has been related
with idiopathic pain and inflammation responses (Hannibal \& Bishop,
2014). Furthermore, hypocortisolism has been also related to chronic
stress and pain disorders such as fibromyalgia among others (Ehlert et
al., 2001; Tak \& Rosmalen, 2010; Tsigos \& Chrousos, 2002). Therefore,
our data suggest that pain when suffered by female rats produces a
higher response of the HPA axis than in males leading to a
hypocortisolism condition together with a lack of adaptation of CRF and
CRFR1 18 days after the CFA administration, in the NAc and the Amygdala.

In the research framework of pain related behaviours, several studies
investigated CRF and KOR/DYN systems and their relationships, in
different areas of the MCLS (Hein et al., 2021; Massaly et al., 2019;
Mousa et al., 2007; Navratilova et al., 2019; Palmisano et al., 2019).
Since these studies reported the ability of KOR antagonism to reduce CRF
release and to blunt pain-related behaviors, we decided to further
investigate the biochemical adaptations on the KOR/DYN system in the NAc
and Amy, in our experimental conditions. Although we are aware of
limitation entailed by analysing the gene and protein expression only 18
days after pain induction, our pharmacological data confirm the
involvement of KOR/DYN signalling in NAc in pain-induced anxiety-like
behaviour. Here reported data showing alterations in the gene and
protein expression of DYN and KOR in both males and females. In the case
of males, alterations in gene expression of KOR without effect in the
total protein have been observed. Accordingly, the downregulation of KOR
gene expression has been previously described in the sciatic nerve
chronic constriction model (Palmisano et al., 2019). Two explanations
are plausible; first, the downregulation of KOR could be related with
the dynorphin hyperactivity induced by pain of in this area (Massaly et
al., 2019) at early stages when animals showed a tendency to decrease in
the sucrose intake. Secondly, it should be underlined that gene
expression changes are not invariably related with protein levels (Maier
et al., 2009), and this ability to regulate the final expression of the
functional protein might explain the lack of pain-induced anxiety-like
behaviours in male rats.

Interestingly at 18 days after CFA injection, pain-suffering female rats
showed a reduction in dynorphin peptide content in the NAc compared with
control counterparts. It has been demonstrated that pain induces an
increase in DYN levels in NAc 2 days after the pain induction and that
KOR blockade with NorBNI prevents pain-related behaviour (Massaly et
al., 2019); accordingly, the reported results here of pharmacological
KOR inactivation support this notion. Therefore, DYN reduction in the
NAc of CFA-treated female rats could result from of a late
neuroadaptation mechanism aiming the recovery of homeostasis.
Nonetheless, further experiments detailing the dynamics of the
alterations in both the CRFR1/CRF and KOR/DYN signalling and the HPA
axis underlying the occurrence of anhedonia and negative affect in the
context of pain in male and female animals are warranted.

In conclusion, we describe anhedonia- and anxiety-like behaviours
derived from the development of an inflammatory pain condition that
depends on the sex and the time course of the pain condition. Indeed,
anhedonia-like behaviour is detected in both male and female rats at
early phase, whereas anxiety-like behaviour is only observed in female
rats when the chronic inflammatory pain is stablished. Dynamic
alteration of KOR/DYN and CRFR1/CRF systems, along with cortisol plasma
levels, might underlie these observed behavioural adaptations,
nonetheless further detailed studies are warranted. Finally, the
pharmacological blockade of KOR in the NAc shell of CFA-treated female
rats impairs the development of anxiety-like behaviours in these
animals, supporting the role of this system in pain-induced negative
affective state in female rats.

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\textbf{6. Figure legends}

\textbf{Table 1. P values of the graphs presented.}

\textbf{Figure 1. Anhedonia- and anxiety-like behaviour induced by
inflammatory pain in male and female rats.} A) Timeline of the
experimental design B) Sucrose consumption during 48 hours in male,
expressed as mean +- SEM of mg/kg/24 hours C) Sucrose consumption during
48 hours in female, expressed as mean +- SEM of mg/kg/24hours D) Sucrose
consumption during 2 hours in male, expressed as mean +- SEM of mg/kg/2
hours E) Sucrose consumption during 2 hours in female, expressed as mean
+- SEM of mg/kg/2 hours F) Time (seconds) in light chamber in the light
dark box test (LDB) in male, expressed as mean +- SEM G) Time (seconds)
in light chamber in the LDB in male, expressed as mean +- SEM H) Number
of transitions between chambers in the LDB in male, expressed as mean +-
SEM I) Number of transitions between chambers in the LDB in female,
expressed as mean +- SEM J) Mechanical sensitivity threshold in male,
expressed as mean +- SEM of paw withdrawal in grams K) Mechanical
sensitivity threshold in female, expressed as mean +- SEM of paw
withdrawal in grams. Black and red bars and symbols represent saline-
and CFA-treated male respectively, and grey and orange bars and symbols
represent saline- and CFA-treated female respectively. *, ** and ***
denotes significant differences between groups (two-way ANOVA for
repeated measures followed by Bonferroni multiple comparisons test,
p\textless{} 0.05, p\textless{} 0.01 and p\textless{} 0.001,
respectively), and \#, \#\# and \#\#\# denotes significant differences
within groups compared with the basal measures (two-way ANOVA for
repeated measures followed by Bonferroni multiple comparisons test,
p\textless{} 0.05, p\textless{} 0.01 and p\textless{} 0.001,
respectively). Abbreviations: SPT, sucrose preference test; VFT, Von
Frey test; CFA, Complete Freund's Adjuvant; SAL, saline; LDB, light-dark
box test.

\textbf{Figure 2. Corticotropin-releasing factor system alterations
induced by inflammatory pain.} A) Mean +- SEM of CRF mRNA relative gene
expression in NAc of male rats. B) Mean +- SEM of CRF mRNA relative gene
expression in Amy of male rats. C) Mean +- SEM of CRF1R mRNA relative
gene expression in NAc of male rats. D) Mean +- SEM of CRF1R mRNA
relative gene expression in Amy of male rats. E) Mean +- SEM of CRF1R
protein expression in NAc of male rats (CRF1R/GAPDH arbitrary units). F)
Mean +- SEM of CRF1R protein expression in Amy of male rats (CRF1R/GAPDH
arbitrary units). G) Plasma cortisol levels in male rats, expressed as
mean +- SEM of ng/mL. H) Mean +- SEM of CRF mRNA relative gene expression
in NAc of female rats I) Mean +- SEM of CRF mRNA relative gene expression
in Amy of male rats. C) Mean +- SEM of CRF1R mRNA relative gene
expression in NAc of female rats. J) \emph{CRF1R} expression in NAc in
female, expressing as mean +- SEM of relative gene expression. K) Mean +-
SEM of CRF1R mRNA relative gene expression in Amy of female rats. L)
Mean +- SEM of CRF1R protein expression in NAc of female rats
(CRF1R/GAPDH arbitrary units). M) Mean +- SEM of CRF1R protein expression
in Amy of female rats (CRF1R/GAPDH arbitrary units). O) Plasma cortisol
levels in female rats, expressed as mean +- SEM of ng/mL. Black and red
bars represent saline- and CFA-treated male respectively, and grey and
orange bars represent saline- and CFA-treated female respectively. *
Denotes significant differences between groups (t-student for
independent samples or Welch's t test p \textless{} 0.05).

\textbf{Figure 3. Kappa opioid receptor/Dynorphinergic system
alterations induced by inflammatory pain in male and female rats.} A)
Mean +- SEM of pDYN mRNA relative gene expression in NAc of male rats. B)
Mean +- SEM of pDYN mRNA relative gene expression in Amy of male rats. C)
Mean +- SEM of KOR mRNA relative gene expression in NAc of male rats. D)
Mean +- SEM of KOR mRNA relative gene expression in Amy of male rats. E)
Mean +- SEM of DYN protein expression (ug/tissue) in NAc of male rats. F)
Mean +- SEM of DYN protein expression (ug/tissue) in Amy of male rats. G)
Mean +- SEM of KOR protein expression in NAc of male rats (KOR/GAPDH
arbitrary units). H) Mean +- SEM of KOR protein expression in Amy of male
rats (KOR/GAPDH arbitrary units). I) Mean +- SEM of pDYN mRNA relative
gene expression in NAc of female rats. J) Mean +- SEM of pDYN mRNA
relative gene expression in Amy of female rats. K) Mean +- SEM of KOR
mRNA relative gene expression in NAc of female rats. L) Mean +- SEM of
KOR mRNA relative gene expression in Amy of female rats. M) Mean +- SEM
of DYN protein expression (ug/tissue) in NAc of female rats. N) Mean +-
SEM of DYN protein expression (ug/tissue) in Amy of female rats. O) Mean
+- SEM of KOR protein expression in NAc of female rats (KOR/GAPDH
arbitrary units). P) Mean +- SEM of KOR protein expression in Amy of
female rats (KOR/GAPDH arbitrary units). * Denotes significant
differences between groups (t-student for independent samples or Welch's
t test p \textless{} 0.05).

\textbf{Figure 4. Anhedonia- and anxiety-like behaviour induced by
inflammatory pain in female rats is blocked by NorBNI intra-NAc
administration.} A) Timeline of the experimental design. B) Sucrose
consumption during 48 hours in female rats, expressed as mean +- SEM of
mg/kg/24 hours mean +- SEM. C) Sucrose consumption during 2 hours in
female rats, expressed as mean +- SEM of mg/kg/2 hours. D) Time in light
chamber (seconds) in the light-dark box test (LDB). E) Number of
transitions between chambers in the LDB, expressed as mean +- SEM. F)
Mechanical sensitivity threshold (g) in female rats, expressed as mean +-
SEM of paw withdrawal threshol. Grey and orange bars represent saline-
and CFA-treated female rats respectively, and empty and full bars
represents the intracranial treatments of aCSF and NorBNI respectively.
*, ** and *** denotes significant differences between groups (three-way
ANOVA for repeated measures followed by Bonferroni multiple comparisons
test, p \textless{} 0.05, p \textless{} 0.01 and P \textless{} 0.001,
respectively), and \#, \#\# and \#\#\# denotes significant differences
within groups compared with the basal measures (two-way ANOVA for
repeated measures followed by Bonferroni multiple comparisons test, p
\textless{} 0.05, p \textless{} 0.01 and P \textless{} 0.001,
respectively). Abbreviation: SPT, sucrose preference test; VFT, Von Frey
test; CFA, Complete Freund's Adjuvant; SAL, saline; LDB, light-dark box
test; NorBNI, Norbinaltorphimine.

.

\textbf{Figure S1. Daily calendar of the behavioural protocol.} CFA,
complete Freund adjuvant; SAL, saline; LDB, light--dark box test; SPT,
sucrose preference test; VFT, von Frey test.

\textbf{Figure S2. Schematic of the brain areas dissection.} A) Area of
the NAc dissection B) Area of the amygdala dissection.

\textbf{Figure S3. Daily calendar of the behavioural protocol of the
Experiment 2 and intracranial administration verification.} A) Daily
calendar of the behavioural protocol. B) Dots represent the site where
aCSF and NorBNI were injected: Black represent saline/aCSF treated
female rats, grey represent saline/NorBNI treated female rats, red
represents CFA/aCSF treated female rats, and orange represents
CFA/NorBNI treated female rats. Abbreviatures: CFA, complete Freund
adjuvant; SAL, saline; LDB, light--dark box test; SPT, sucrose
preference test; VFT, von Frey test.\selectlanguage{english}
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