ORIGINAL ARTICLE

THE INSULAR CORTEX ROLE IN ANXIETY MODULATION

DAHER, Lucas Abrahão1; ALVES, Fernando H.F1; PACHECO, Túlio Daher1; BUSNARDO, Cristiane2; CRESTANI,
Carlos C3; OMENA, Luana Giatti3

1 Health Sciences Department, Faculty of Medicine; Lavras Federal University (UFLA), Lavras, Minas Gerais, Brazil
2 Pharmacology Department, Ribeirão Preto School of Medicine; University of São Paulo (USP), Ribeirão Preto, São
Paulo, Brazil
3 Drugs and Pharmaceutics Department, School of Pharmaceutical Sciences; São Paulo State University (UNESP),
Araraquara, São Paulo, Brazil
Corresponding author: Ms. Lucas Daher. ORCID: 0000-0002-4892-411X
Adresse: Pedro Sales Avenue nº 650, Lavras, MG, Brazil. CEP: 37207650 E-mail: ldaher1986@gmail.com

ABSTRACT
Introduction: The insular cortex (IC) is a structure that interconnects several brain regions. It has been
reported as an important modulator of emotions due to its connections with limbic structures and has been
associated with the modulation of anxious behavior where the NMDA/NO/cGMP pathway can be an
important target, both for therapy and for understanding the events involved in anxiety modulation.
Objective: To study the glutamatergic synapses and the NMDA/NO/cGMP pathway antagonism effect, in
the IC of rats, through anxiety tests. Method: 63 Wistar rats, weighting of 280 grams, in average,
subdivided into 7 groups, 1 naive, 1 control and 6 treated, submitted to stereotactic surgery for IC
cannulation, use of antagonists and inhibitors of the pathway, administered by direct microinjection,
application of behavioral tests (EPM and OF), euthanasia, removal of the brains and cryostatic cuts for
histological evaluation of the region in question. Results: Inferential statistical analysis showed no
significant difference for the descriptive level in question. Some problems were observed in the final phase
of the experiment and may have affected the results. Conclusion: Antagonism of excitatory pathways in
peri-insular regions does not seem to affect anxiety-related behavior in rats. However, some tangible
descriptive statistics demonstrated the need to renew the study, in order to delineate the agranular area of the
IC, for greater understanding.
KEYWORDS: Anxiety, insular cortex, NMDA, Nitric Oxide, cGMP.

INTRODUCTION

Anxiety can be defined as an aversive state to the unknown, with fear upgrading in the absence of real
danger (SHIN; LIBERZON, 2010), which induces emotional responses to potentially unidentified threats
(MÉNDEZ-RUETTE et al., 2019a), triggering behavioral, hormonal and autonomic reactions (ANDERSON;
ADOLPHS, 2014; DAVIS et al., 2010; DAVIS; WHALEN, 2001; LEDOUX, 2000, 2014). It is a state of
anticipation about the perception of future threats, this anticipation being disproportionate to the risk or danger
represented. Fear results from the perception of imminent danger, arising from a real and recognized stimulus
(CRASKE et al., 2009). Several authors have shown that, despite the similarities, fear and anxiety are different
emotions (SHIN; LIBERZON, 2010; SYLVERS; LILIENFELD; LAPRAIRIE, 2011).
Behavioral patterns related to fear and anxiety can be observed in many animal species, which reflects
their importance as a survival factor, increasing adaptations process that protect them from a potentially
harmful environment (TOVOTE; FADOK; LÜTHI, 2015). Acute anxiety is physiological and necessary,
since it premeditates dangerous situations (GRAEFF, 2007). However, it can occur in a chronic and
disproportionate way, generating inadequate adaptive responses and even psychiatric disorders, becoming
pathological (GRAEFF, 2007).
In adult life, transient fear and anxiety can arise from stressful life periods, but to be diagnosed as
anxiety disorders (AD), they must persist for a long period of time and their diagnostic requirements are
described in the DSM-V (CRASKE et al., 2017; CRASKE; STEIN, 2016; THIBAUT, 2015). It is important
to understand that chronic anxiety has a high prevalence and represents a serious global public health problem
(BAXTER et al., 2013; CRASKE et al., 2017; TOVOTE; FADOK; LÜTHI, 2015). Individuals diagnosed
with AD, such as post-traumatic disorder, panic disorder, agoraphobia or social anxiety disorder, when
exposed to the fear stimulus, presented “safety behaviors”, which resulted in the non- cessation of the neural
mechanisms of fear (LOVIBOND et al., 2007). Studies with rodents demonstrate that conditioned fear
(DUITS et al., 2015; DUNSMOOR; PAZ, 2015) and deficit in controlling the proportion of this fear are, in
hypothesis, contributors to the development of anxiety (MILAD; ROSENBAUM; SIMON, 2014).
Thus, it is necessary to develop new strategies and laboratory tests to chemically differentiate the types
of disorders, in addition to new studies that can elucidate the neural mechanisms of fear and anxiety, through
animal models, for a better understanding of the events involved, contributing to new guidelines therapies
(TOVOTE; FADOK; LÜTHI, 2015).
Emotions emerge from coordinated activities of brain regions interconnected by the limbic system
(CATANI; DELL'ACQUA; THIEBAUT DE SCHOTTEN, 2013), where the behavioral and adaptive basis
for emotional responses comes from substrates provided by cortico-limbic circuits (KOVNER; OLER;
KALIN, 2019).
By definition, the limbic system (SL) can be considered a set of cortical and subcortical structures,
morphologically and functionally interconnected, correlating with memory and emotions (CATANI;
DELL'ACQUA; THIEBAUT DE SCHOTTEN, 2013). Initially the term “limbic” (from Latin, border) was
designated by Thomas Willis, in 1664, to describe a circular cortical border located above the brainstem
(ROLLS, 2015). Paul Broca, in 1878, argued that the large limbic area was a structure common to the brains
of all mammals and its main function was related to smell, although it also performed other functions
(CATANI; DELL'ACQUA; THIEBAUT DE SCHOTTEN, 2013). After Broca's reports, several studies were
conducted associating limbic structures with behavioral and emotional aspects (ROLLS, 2015; WALTER B
CANNON, 1927). Later studies contributed to the formulation of the first unified model that linked action and
perception to emotions, the so-called “Papez circuit”, developed by James Papez in 1937, which proposed that
emotions emerged both from cognitive activities, entering the circuit through the hippocampus, and also
visceral and somatic perception, entering the circuit through the hypothalamus (PAPEZ, 1937). Structurally,
the circuit included the hypothalamus, hippocampus, the anterior nucleus of the thalamus, the fornix, the
mammillary bodies, the anterior cingulate gyrus and their interconnections, however, it did not include the
amygdala (PAPEZ, 1937). It was hypothesized at that time, and later confirmed by new studies, numerous
connections and participation of brain circuits and regions in the processes involved in emotional behavior
(PESSOA; HOF, 2015), including the participation of the amygdala, anterior temporal lobe, orbitofrontal
cortex and insular cortex (IC) as part of the network that delimits emotions and motivation (YAKOVLEV,
1948). The prefrontal cortex, cingulate cortex, entorhinal cortex, hippocampus, periaqueductal gray matter,
amygdala and hypothalamus, have been reported as part of the limbic system by some authors (CHOW et al.,
2018; ROLLS, 2019). Thus, a complex network of interconnected subcortical and cortical structures,
responsible for connecting visceral sensations and emotions to behavioral and cognitive reactions, makes up
the limbic system (MESULAM, 2000).
There is a current tendency to classify it functionally, proposing that different areas and different
connections of this system are associated in the modulation of emotions and episodic memory, differentiating
it into the emotional limbic system (eLS), where the main connections revolve around the anterior cortex
cingulate and limbic memory system (mLS), where the main interconnections revolve around the
hippocampus (ROLLS, 2019). Its interconnections with IC were demonstrated in Paul Yakovlev’s
experiments, in the 40s, and have been studied for a better understanding of its interaction (CHOW et al.,
2018; ROLLS, 2019).
The region that corresponds to the majority part of the cerebral cortex is called the prefrontal cortex.
Evolutionarily, it is the part of the brain that has been greatly developed in humans (SMAERS et al., 2011).
It has interconnections with the entire limbic system, basal ganglia, thalamus, amygdala and brainstem
(ÖNGÜR; PRICE, 2000). Its role is fundamental in the modulation of behavioral stimuli, cognitive actions,
speech and linguistic areas, in addition to regulating autonomic and neuroendocrine functions
(BENARROCH, 2019). Anatomically, we can divide the prefrontal cortex into three regions: medial, ventral
(or orbital), and lateral. The IC is an anatomical region that integrates cortical and subcortical structures
through an extensive connective network, functioning as a cortical center involved in enteroception,
multimodal sensory processing, autonomous control, perceptual self-awareness and emotional orientation of
social behavior (BENARROCH, 2019). It is responsible for modulating behavioral reactions such as fear,
anxiety and depression, as well as responses to external stressors, being an area directly involved in sensitive,
cognitive, motivational and emotional processes (GOGOLLA, 2017).
 In mice and rats, it is located just above the rhinal fissure, laterally to the hemispheres (RANJBAR;
HATAM; NASIMI, 2015; SAPER, 1982). In humans, it is deeply located between the lateral cerebral sulci
(UDDIN et al., 2017) and is delimited by the frontal, parietal and temporal opercula, by the periinsular sulcus
and divided into anterior and posterior lobes by the central insular sulcus, and may have variations regarding
the anatomical markings of the gyri (UDDIN et al., 2017; WYSIADECKI et al., 2018).
Cyto-architectural subdivisions (cell composition) can be morphologically observed, showing three
distinct areas: Granular area, located posteriorly, related to the perception of general body status, such as
temperature, pain and visceral sensation. Disgranular area, located medially, responsible for integrating
enteroception and proprioception, thus connecting action and emotion. Agranular area, located anteriorly,
related to cognitive and emotional control, as well as autonomic control (BENARROCH, 2019).
In both humans and rodents, the IC is often reported to be overactivated in processes of fear
(GEHRLACH et al., 2019), anxiety (MÉNDEZ-RUETTE et al., 2019a) and negative emotions (CHANG et
al., 2015). Recently, the direct involvement of the rodent IC in the modulation of anxiety was demonstrated,
using a selective inhibitor of AMPA receptors, through direct microinjections in different regions of the IC
(granular, agranular and disgranular) and exposing the rodents to the elevated plus maze test (EPM), observing
anxiolytic effects (MÉNDEZ-RUETTE et al., 2019a).
Studies have correlated IC with AD (DAMSA; KOSEL; MOUSSALY, 2009), where it seems to play
a fundamental role in its pathophysiology and, although this correlation is established (DENNIS et al., 2011),
neurotransmission and IC involvement in behavior are not yet elucidated (ALVES et al., 2013b), animal
models are fundamental (MÉNDEZ-RUETTE et al., 2019a).
Neurotransmitters make it possible to send and receive information, whether central or peripheral,
somatic or autonomous, afferent or efferent. For each neurotransmitter, there is a system of receptors,
messengers and cascading events that, directly or indirectly, ensure that actions can be disseminated (KREBS.;
WEINBERG.; AKESSON., 2013).
Glutamate, synthesized from glutamine or alpha-ketoglutarate transamination in the Krebs cycle, is the
main excitatory neurotransmitter in the CNS (FLECK et al, 1993; KHODOROV, 2004). It is associated to
two types of receptors: ionotropic, subdivided into Alpha-Amino-3-Hydroxy-Methyl-5-4-Isoxozolpropionic
(AMPA), Kainato (AK) and N-Methyl D-Aspartate (NMDA), and metabotropic (mGluR), associated with G
protein, subdivided into 3 types: Type 1 (mGluR 1 and 5), type 2 (mGluR 2 and 3) and type 3 (mGluR 4, 6, 7
and 8) (HANSEN et al., 2018; TRAYNELIS et al., 2010).
Nitric oxide (NO) is obtained by metabolizing L-arginine by the enzyme nitric oxide synthase (NOS).
There are three NOS subtypes known in the literature: inducible nitric oxide synthase (iNOS), endothelial
nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) (MICHEL; FERON, 1997;
NAKASHIMA et al., 2003; NATHAN, 1997). nNOS is widely found in mammalian brains, being directly
related to brain neurotransmission and nitrergic signaling as well as brain NO formation (GARTHWAITE,
2016). The NO formed by nNOS acts as a neuronal signalize and also as an atypical transmitter, responsible
for regulating processes of blood flow in the CNS (GARRY et al., 2015). With the diffusion of NO to the
presynaptic terminal, there is activation of soluble guanylate cyclase (sGC) (ESPLUGUES, 2002), which
catalyzes the production of cyclic guanosine monophosphate (cGMP) (GARRY et al., 2015) and can modulate
the release of neurotransmitters and second messengers (TAQATQEH et al., 2009), among other functions.
Glutamatergic neurotransmission is directly linked to the nitrergic system and to the nNOS isoform
(CHACHLAKI; PREVOT, 2019; GARTHWAITE, 2016). The activation of NMDA receptors culminates in
a calcium (Ca 2+) influx which one binds in the calmodulin, activating nNOS, leading to the synthesis of NO
(CHACHLAKI; PREVOT, 2019; GARTHWAITE; CHARLES; CHESS- WILLIAMS, 1988). Once produced
on demand, NO gas will bind to soluble guanylate cyclase (sGC) which will convert guanosine triphosphate
(GTP) into cyclic guanosine monophosphate (cGMP), activating the protein kinase system, acting as second
messengers (ESPLUGUES, 2002; GARRY et al., 2015). Part of this NO will act retrogradely, going back
through the synaptic cleft and increasing the release of glutamate in the cleft (GARTHWAITE, 2019).
The interaction between the glutamatergic and nitrergic systems has been characterized as an active
component in the control of several regulatory systems, both autonomic and behavioral (LISBOA et al., 2015;
ZHOU et al., 2011).
 The presence of glutamatergic terminals in the IC was observed (DORI et al., 1992; RAMÍREZ-LUGO
et al., 2015), as well as modulation of baroreflex activity by NMDA receptors in the IC (ALVES et al., 2009),
demonstrating the presence of these receptors in this region.
Animal models for anxiety tests are based on conflicting situations, which can generate aversive states
(CAMPOS et al., 2013). The elevated plus maze (EPM) is the most used animal model for anxiety test
(CAMPOS et al., 2013). It is an apparatus that is about 50 centimeters from the ground, with 4 arms and 4
sides that cross perpendicularly with each other, one of the sides being closed by a lateral wall and the other
ones are wide open. The test consists in the rodent's natural tendency to remain in closed arms. Thus, the time
spent in the open arms is compared with the closed arms, in addition to the number of crossings between the
sides (LISTER, 1987). External factors such as lighting, odors, luminosity, noise and the animal's previous
experience can directly interfere with the results, being ideal the asepsis of the apparatus before each test, in
addition to a calm environment and the use of animals that have not previously experienced the test (CAMPOS
et al., 2013). The animal must be handled carefully and placed in the center of the maze, with the face facing
the closed side. 5 minutes is then counted and it is indicated that the animal stays alone at that moment, while
being monitored by a camera. This procedure was successfully used in studies that evaluated anxiolytic drugs,
where a longer time of permanence of the animal in the open arm was demonstrated (PELLOW; FILE, 1986).
The open field test (OF) consists of evaluating the increase in the animal's locomotor activity and also
its exploratory capacity (PRUT; BELZUNG, 2003). The rodent is placed in the center of a closed square box
(60 x 60 cm), demarcated by 16 quadrants of 15 x 15 cm. External factors can influence animal behavior, as
well as in EPM. Therefore, the same precautions are necessary, such as asepsis before using each animal,
careful handling, a calm environment and the absence of people in the testing area. There is a tendency for
rodents to remain in the corners of the apparatus. When an anxiolytic effect is observed, the animal spends
more time in the center, but when very agitated, they go through the apparatus in a disorderly way (PRUT;
BELZUNG, 2003), thus serving as a counterpoint to the EPM test, so, if the animal remained for a long time
in the open arm and had a high number of crossings between the arms, the OF is essential to bring evidence
that this behavior may have occurred by mechanisms that led to an excessive excitability of the animal and
not by anxiolytic mechanisms, in fact. The time spent in the center, the number of quadrants explored and the
time spent in the corners of the apparatus are counted for comparison between groups (PRUT; BELZUNG,
2003).

MATERIAL AND METHODS

Sixty-three male Wistar rats weighing between 280 and 320 grams were used, fed granulated ration
and water ad libidum, kept under the same conditions of light and dark cycle 12 hours for 12 hours (with lights
onset at 7:00 am and offset at 7:00 pm) and they were confined in plastic boxes throughout the experimentation
period. Handling and cleaning were performed only by the author.
The 63 animals were divided into 7 groups of 9 animals where each group underwent the same
procedure, including pre and post-surgical protocols, with the exception of the control group. These The
animals were provided by the Central Animal Facility of the Federal University of Lavras and the procedures
were approved by the Ethic Committee in Animal Using (ECAU) protocol 023/21.

Fig.1 Detailing of the experimental groups.

For the surgical procedure, the animals were anesthetized with tribromoethanol at a dose of 250 mg/kg,
intraperitoneally. After performing the head trichotomy, they were immobilized in a stereotaxic for small
animals (Stolelting, Wood Dale, Illinois, USA) and 0.3 ml of lidocaine with vasoconstrictor was injected
subcutaneously in the animal's head, for local anesthesia. Povidine® antiseptic was used for head asepsis and
then the cranial vault was exposed through a skin incision of approximately 1.5 cm. The periosteum was
removed and removed with 10% hydrogen peroxide (H2 O2). Local asepsis was performed with saline solution
(NaCl 0.9%) and cotton. All the coordinates for the implantation of the cannulas in the IC were determined
from the Atlas of Paxinos & Watson (PAXINOS; WATSON, 1997): anteroposterior, +3.2 mm in relation to
the bregma; laterally, +3.75 mm from bregma; vertical, -4.5 mm in relation to bregma, with lateral inclination
of 0° and incisor -3.3 mm. The perforation of the cranium bone (incisure) was performed with an odontology
drill adapted to a micro-grinder, for the bilateral implantation of the guide cannulas in the IC and to insert a
small fixation screw in the cranial vault. These cannulas were made from hypodermic needle segments 13 mm
long and 0.55 mm of external diameter. The cannulas and the screw were fixed in to the cranium with self-
curing acrylic resin. Mandrels with an 0.2 mm of external diameter and 13 mm length were introduced into
the cannulas to prevent possible obstructions during the post-surgical recovery period of the animals. As a
prophylactic measure, after surgery, the animals received 80,000 IU of Veterinary Pentabiotic (Fort Dodge,
São Paulo, Brazil) applied in 0.3 mL intramuscularly and 2.5 mg/Kg of analgesic and anti-inflammatory non-
steroidal inflammatory drug Flunixin Meglumine (Banamine ®, Schering Plow, Brazil), subcutaneously
(ALVES et al., 2010). After 5 days of recovery and verification of the animal's well-being, observing the
absence of pain signals such as apathy, diarrhea and pyloric reflex, the behavioral experimental procedure
could be started.

Description of drugs
The following compounds were used: artificial cerebrospinal fluid ( aCSF), dimethyl sulfoxide
(DMSO), tribromoethanol (SIGMA, USA), urethane (SIGMA, USA), LY235959 (TOCRIS, USA, NMDA
receptor antagonist), NBQX (TOCRIS, USA, non-receptor antagonist -NMDA); N-Propyl-L-arginine (N-
propyl, TOCRIS, USA, selective nNOS inhibitor), Carboxy -PTIO (TOCRIS, USA, NO scavenger); ODQ
(specific inhibitor of guanylate cyclase formation); Veterinary pentabiotic (Fort Dodge, Campinas, SP, Brazil)
and non-steroidal analgesic flunexine meglumine ( Banamine® , Schering Plow , Brazil).

Microinjection of drugs into the insular cortex

The injector needle (12 mm, 33 G, SmallParts, Lagos de Miami, FL, USA) used for microinjection of
drugs into the IC, was made one millimeter longer in relation to the guide cannula fixed to the skull and was
connected to a 2 μL (7002-KH, Hamilton Co., Reno, NV, USA) through a polyethylene tube (PE-10). With
the animal still in its home cage, the injection needle was inserted into the guide cannula for drug or vehicle
injection. Drugs were injected into the two cannulas (one at a time) in a volume of 100 nL for each cannula.
After the injection, 30 seconds were waited and the needle was withdrawn and inserted in the second guide
cannula for microinjection in the contralateral IC. After the microinjection, we waited 10 minutes for the
administered drugs to take effect locally. Only then was the animal inserted into the EPM and then into the
CA, for another 5 minutes (ALVES et al., 2013; HAN et al., 2015; ADAMI et al., 2017; BARRETO-DE-
SOUZA et al., 2017; GOMES-DE-SOUZA et al., 2020).
Elevated Plus Maze
The test was performed on a wooden EPM, raised 50 cm from the ground and formed by two open
arms (50 x 10 x 40 cm) that form a cross with two closed arms (50 x 10 x 40 cm). Ten minutes after
administering the drug, each animal was placed for 5 min in the EPM, with its face facing one of the closed
arms. Only one session per animal was performed to avoid habituation. For behavioral analysis, the session
was filmed by a digital camera and analyzed using the AnyMaze software (Stolelting, USA). The following
parameters were analyzed: the number of entries into the closed arms and the percentage of entries and time
spent in the open arms of the EPM.

Open Field
The test was carried out in a square wooden box, 60 cm high and 60 cm deep, marked with 16 quadrants
measuring 15 x 15 cm. The animals were placed in the center after asepsis of the apparatus, right after being
submitted to EPM, and remained there for 5 minutes, with one session per animal, which was filmed by a
digital camera and analyzed later with the aid of the AnyMaze software (Stolelting, USA).

Anatomical determination of drug injection sites in the insular cortex

At the end of each experiment, each animal was anesthetized with urethane (1.2 mg/kg-1)
intraperitoneally. 100 nL of Evans blue dye (1%) was injected by microinjection, following cannulation of the
insular cortex, through which the drugs were injected. For the perfusion procedure, the heart was exposed
through an incision in the rib cage in order to block the aorta using a hemostat. The perfusion needle was then
introduced into the left ventricle. An incision in the right atrium was made to allow blood and perfusate to
flow out. Initially, 20ml of saline solution (0.9% NaCl) was injected and then a 10% buffered formalin solution
was injected into the left ventricle. With the animal perfused, a guillotine was used to remove the head and
the cranial vault was opened for the removed brain, which, in turn, was placed in a buffered 10% formalin
solution for conservation and fixation. After 1 week, the brains were submitted to coronal sections with the
aid of a cryostat. A microscopic analysis using the atlas of Paxinos and Watson (1997) as a reference was
performed to investigate the injection sites.

Statistical analysis
Data were expressed as mean ± SEM. The confidence interval (CI) adopted was 95%, the significance
level (α) was 5% and the descriptive level (p) was less than or equal to 0.05. Thus, for the theory of hypotheses,
we establish the null hypothesis, that there are no statistical differences between the groups, if p > 0.05, or the
alternative hypothesis, that there are statistical differences between the groups, if p ≤ 0.05.
For data analysis, the GraphPad© Prism ® software was used. A set of descriptive statistics was
performed to verify the data and identify and exclude outliers. The Shapiro-Wilk data normality test was used.
As the data were not parametric, the Kruskal-Wallis H test was adopted, with Dunn's post hoc, to verify if
there were differences between the NAIVE, aCSF and DMSO animals. only control group for comparison
purposes. The same procedure was adopted for comparison between groups (control vs pharmacological
treatment). The data analyzed for the ECL were the number of entries into the open arm, length of stay in the
open arm, and number of crosses and length of stay at the center. For the CA, the length of stay in the center
and the length of stay in the outskirts were analyzed.

Fig. 2 - Chronology of experimental protocols: On the first day, stereotaxic surgeries were performed to
implant cannulas in the insular cortex. On the sixth day, the EPM and OF tests were performed, with the
administration of drugs according to the groups previously described. After the experiment, the animals
were euthanized and the injection sites in the insular cortex were stained and then the brains were extracted,
preserved and fixed. After seven days, histological slides of the brain were made and observed then the data
was analyzed.

RESULTS
After the histological section of the brains, it was verified that the cannulas that were directed to the
IC were positioned in areas close to the IC, but not directly in it. Such an occurrence can arise from several
reasons, including: erroneous fixation of the animal in the stereotaxic, size and weight of the animals, problems
in placing the guide cannula in the mandrel of the stereotaxic, errors in calculations for the correct placement
of the cannulas in the CNS, among others. In this way, the results described below correspond to the animals,
in which the cannulas directed to the IC are 1 to 2 mm outside the area. Unfortunately, it cannot be reliably
stated that the spreading radius has reached the IC, as the injection volume is only 100nl. Therefore, we
decided to carry out joint statistics of all animals whose cannulas were between 1 and 2 mm from the IC.

Fig.3 - Representative photomicrograph of a coronal section of the rat brain demonstrating the microinjection site in the
region close to the insular cortex. Legend: cingulate cortex (Cgl), corpus callosum (CC), orbital cortex (OC), insular
cortex (IC). Representative coronal section of the coordinate 4.20 mm in relation to bregma according to the atlas by
Paxinos and Watson (2006).

Fig.4 – Graphical representation of the results related to the EPM test. Analysis of variance of data obtained in the EPM
test. (a) refers to the number of entries into the open arms. (b) refers to the permanence time, in seconds, that the animal
still in the open arms of the EPM. (c) refers to the complete number of crossings through the center of the EPM. (c)
refers to the time the animal spent, in seconds, in the center of the maze. Data expressed using the standard error of the
mean. The total test time was 5 minutes for each animal.
Fig. 5 – Analysis of variance of the data obtained in the OF test. (a) refers to the time the animal remains, in seconds, in
the center of the apparatus. (b), in turn, refers to the time, in seconds, spent in the peripheral region of the OF. Data
expressed using the standard error of the mean. The total test time was 5 minutes for each animal.

Tab.1 – Comparison between the NAIVE, aCSF and DMSO groups, to form the control group.
KRUSKAL-WALLIS
Multiple comparisons between NAIVE,
p value H value
aCSF and DMSO groups
Entries into the open arm of the ECL 0.7012 0.7636
Permanence in the open arm of the ECL 0.3328 2,289
Crossings in the center of the ECL 0.148 3,852
Staying in the center of the ECL 0.4017 0.1923
Permanence on the periphery of the CA 0.7692 0.4687
Stay in the center of CA 0.7692 0.4687
Caption: Analysis of variance, according to the Kruskal-Wallis test. The purpose of this test was to verify whether the
surgical procedure would interfere with the results of behavioral tests, thus isolating drug administration. It is noted that
there were no statistical differences between the groups that underwent stereotaxis (aCSF and DMSO) and received only
the drug dilution vehicle and the group that did not undergo any procedure (NAIVE).

Tab.2 – Kruskal-Wallis test, for comparison between the control and treated groups.

KRUSKAL-WALLIS
Multiple comparisons between “control”
p value H value
and “treated” groups
Entries into the open arm of the ECL 0.5005 4,348
Permanence in the open arm of the ECL 0.3647 5,439
Crossings in the center of the ECL 0.1449 8.212
Staying in the center of the ECL 0.0808 9,809
Permanence on the periphery of the CA 0.5742 3.83
Stay in the center of CA 0.5868 3,744
Caption: Control group: (NAIVE, aCSF and DMSO). Drug group: (LY235959, NBQX, NPLA, CARBOXY-PTIO and
ODQ). Analysis of variance, according to the Kruskal-Wallis test. For the null hypothesis to be rejected, a descriptive
level (p) less than or equal to 0.05 would be required, for a 95% confidence interval. The H value refers to the difference
of the total rankings made by the test. It is a parameter to be considered when rejecting the null hypothesis.

The Shapiro-Wilk test was used to verify normality, where we confirmed that the data were not
parametric. The comparison between the NAIVE, aCSF and DMSO groups, verified by the Kruskal-Wallis
test, also did not reveal difference between the groups, in any of the tests, as observed in table 1. Therefore,
we considered the combination of these three groups as a control group (NAIVE, aCSF and DMSO), since
both reveal drug-free animal behavior.
The analysis of tab.2 allows us to conclude that, for this confidence interval (CI = 95%), significance
level (α = 5%) and descriptive level (p ≤ 0.05), we must accept the null hypothesis, where we conclude that
there is no statistically significant difference between the groups. Even when using Dunn's post hoc, we found
no difference between any of the groups. That is, it is not possible to statistically state that the drugs in question
modified the behavior of the animals in the EPM and in the OF tests. However, when carefully analyzing the
graphs, we found discrepant values for the ODQ group, selective inhibitor of the soluble guanylate cyclase
enzyme, in the EPM test (fig. 4 a and b), as well as for the LY235959 groups, which is an antagonist of NMDA
receptors , NPLA which is a selective inhibitor of iNOS and ODQ a selective inhibitor of soluble guanylate
cyclase, in the CA test (fig.5 a and b), we observed a discrepancy that can be attributed to the use of the
standard error of the mean instead of the standard deviation.
Tab. 3 – Lowest, median and highest values referring to the number of entries in the open arm of the EPM.
EPM CENTER PERMANENCE TIME (s)
GROUPS NAIVE aCSF DMSO LY235959 NBQX NPLA CPTIO ODQ
Sample size (n) 7 6 4 10 10 6 10 6
Lowest value 41.6 89.4 16.7 50.4 30.7 26.8 43.3 14.4
Median 133.5 128.8 105.3 81.1 88.35 82.6 62.85 78.75
Highest value 162.8 152.1 131.1 140.2 140.3 152.4 144.2 89.2
Caption: There is a large discrepancy between the maximum and minimum value, in the same group, for the LY235959
(difference = 13), NPLA (difference = 12) and carboxy - PTIO (difference = 16) groups. Note: Difference calculated as
maximum value minus minimum value (Dif = Vmax – Vmin).

Tab. 4 – Lowest, median and highest values of time spent in the open arm of the EPM.
EPM OPEN ARM PERMANENCE TIME (s)
GROUPS NAIVE aCSF DMSO LY235959 NBQX NPLA CPTIO ODQ
Sample size (n) 7 6 4 10 10 6 10 6
Lowest value 1.5 0 0 0 0 0 0 0
Median 32.2 15.4 45.65 14.55 27.4 38.6 19.9 6.95
Highest value 58.6 48.4 56.7 96.6 106.4 53.5 141.7 21.3
Caption: The time was measured in seconds (s). There is a discrepancy between the medians of all treated and untreated
groups, where the drug ODQ obtained the most discrepant value.

Tab. 5 – Lowest, median and highest values referring to the number of center crossings in the EPM.
EPM CROSSING THROUGH CENTER
GROUPS NAIVE aCSF DMSO LY235959 NBQX NPLA CPTIO ODQ
Sample size (n) 7 6 4 10 10 6 10 6
Lowest value 7 15 15 3 8 1 6 8
Median 18 22 22 11 15 10 14 15
Highest value 20 25 25 27 33 35 29 23
Caption: There is a large discrepancy between maximum and minimum, in the same group, for groups LY235959
(difference = 24), NBQX (difference = 25) and NPLA (difference = 34). Note: Difference calculated as maximum
value minus minimum value.

Tab. 6 – Lowest, median and highest values referring to the EPM center permanence time.
EPM CENTER PERMANENCE TIME (s)
GROUPS NAIVE aCSF DMSO LY235959 NBQX NPLA CPTIO ODQ
Sample size (n) 7 6 4 10 10 6 10 6
Lowest value 41.6 89.4 16.7 50.4 30.7 26.8 43.3 14.4
Median 133.5 128.8 105.3 81.1 88.35 82.6 62.85 78.75
Highest value 162.8 152.1 131.1 140.2 140.3 152.4 144.2 89.2
Caption: The time was measured in seconds (s). There is a great discrepancy between the medians of the treated and
untreated groups, especially the drug carboxy -PTIO (CPTIO).

Tab. 7 – Lowest, median and highest values referring to the OF peripheral permanence time.
OF PERIPHERAL PERMANENCE TIME (s)
GROUPS NAIVE aCSF DMSO LY235959 NBQX NPLA CPTIO ODQ
Sample size (n) 7 6 4 10 10 6 10 6
Lowest value 298.7 299.6 299 295.4 299.2 292 297.6 292.5
Median 300 300 300 300 300 299.6 300 300
Highest value 300 300 300 300 300 300 300 300
Caption: The time was measured in seconds (s). There are slightly discrepant values between the maximum and
minimum time, in the same group, for the LY235959, NPLA and ODQ groups. Source: From the author (2022).

Tab. 8 – Lowest, median and highest values referring to the OF center permanence time.
OF CENTER PERMANENCE TIME (s)
GROUPS NAIVE aCSF DMSO LY235959 NBQX NPLA CPTIO ODQ
Sample size (n) 7 6 4 10 10 6 10 6
Lowest value 0 0 0 0 0 0 0 0
Median 0 0 0 0 0 0,4 0 0
Highest Value 1.3 0.4 1 4.6 0.3 8 2,4 7.5
Caption: The time was measured in seconds (s). There are slightly discrepant values between the maximum and
minimum time, in the same group, for the LY235959, NPLA and ODQ groups.

DISCUSSION

Studies involving stereotactic surgery for cannulation of brain areas require extreme dexterity, training
and assiduity. Although this technique is consolidated in the field of research, together with micro injections,
many variables directly interfere with the results of these studies. Animal weight, stereotaxic positioning,
mechanical problems with the surgical apparatus, unpredictability of the anesthetic action, clogging of the
cannula already placed in the CNS, are points that make precision and guarantee of results difficult. In
addition, certain intercurrences, such as confirmation of the injection site, are only obtained in the final stage
of the technique, when the histological sections have already been made and the microscopic slides are
analyzed. Studies involving behavior need a larger sample number, since the animal can naturally present
variable behavioral patterns and, therefore, outliers need to be excluded from the sample in question.
By considering the assumptions, history, validation and divergences, we can infer some observation
points based on descriptive statistical analysis.
Table 3 shows a discrepancy between some treated groups regarding the number of entries into the
open arms. These data suggest that there is a pattern break in the behavior of some animals. The LY235959
group showed a difference between the maximum and minimum number of entries in the open arm of the
EPM of 13, that is, one animal did not enter it once while another entered it 13 times. The same occurs in the
NPLA and carboxy -PTIO groups, which respectively showed differences of 12 and 16. That is, there are
animals in these groups that behaved in an anxiolytic way, but this fact cannot be evidenced as a statistical
difference since were exceptions to the group. This fact can be explained if we consider the possibility of
problems in the experimental procedures. Perhaps problems with the injector or even problems with the
cannulation procedure generated discrepant data. A similar fact occurs when observing Table 5, which
considers the number of crossings in the EPM where, for the LY235959, NBQX and NPLA groups, there was
a difference of 24, 25 and 34, respectively. That is, in groups where glutamate receptors were antagonized or
iNOS was inhibited, preventing the production of nitric oxide, some animals seem to have behaved in an
anxiolytic way. This fact corroborates the findings of Méndez- Ruette and collaborators, where non-NMDA
receptors were antagonized in the IC and anxiolytic behavior was observed in rats (MÉNDEZ-RUETTE et
al., 2019b).
In table 4, which deals with the length of stay in the EPM, when we understand that non-parametric
data have the median as the most reliable measure of central tendency, since it is established by the central
value of a reorganization of the data in ascending order, we observe that the ODQ group has a median
substantially lower than the other groups, being the group that spent less time in the open arms of the EPM.
Since ODQ is a specific inhibitor for the formation of sGC, which is the NO receptor, we could infer that this
NO did not bind to its receptor and did not release the second messenger cascade and, in turn, was concentrated
in the postsynaptic regions or it ended up returning through the presynaptic terminal, increasing the release of
glutamate, according to the mechanism described by Garthwaite (GARTHWAITE, 2019). This fact suggests
that glutamate increases anxiogenic activity, causing the animals in this group to reduce their time spent in the
open arm. Unfortunately, the sample number was small due to losses during the surgical procedure, in addition
to the problems already reported in relation to the injector, making the statistics dubious for this level of
confidence. Something similar is observed in Table 6, which deals with the number of crossings in the EPM,
where there is a discrepancy between the medians of the treated groups (lower medians) in relation to the
untreated groups (higher medians). This fact suggests that animals that crossed the EPM less often explored
the EPM less, either because they spent more time in the open arms or because they spent less time in the open
arms. Thus, when we analyze Table 4, which verifies the time spent in the open arms, where we observe that
the ODQ group has the lowest median and the lowest maximum value, being discrepant from the others,
suggesting that when we sequester the sGC, the animal tends to closed arm and also tends not to explore the
CSF, reducing its time spent in the open arm and also the number of crossings, corroborating, once again, with
Garthwaite 's findings , since by increasing NO feedback, there is greater release of glutamate suggesting that
it may be related to anxiogenesis .
Tables 7 and 8, which refer respectively to the length of stay on the outskirts and in the center of the
CA, did not demonstrate abnormal behavior patterns.
Studies relating IC and anxiety are still rare. Paulus and collaborators suggested that IC is associated
with interoception and feelings of danger or threat, playing a crucial role in anxiety (PAULUS; STEIN, 2006).
In a recent study by Mendez-Ruette and collaborators, it was demonstrated that the IC presents different
behaviors according to its region. In the rostrum -caudal and gustatory agranular part, the antagonism of
AMPA receptors culminated in an anxiolytic behavior. The primary and rostral posterior interoceptive regions,
however, did not show behavioral differences to AMPA receptor antagonism (MÉNDEZ-RUETTE et al.,
2019b). Thus, it may be that the cannulation coordinates used in this study affected more posterior areas of
the IC, where effects were not observed in a statistically significant way.
The present study aimed to investigate the relationship between anxiety and ionotropic glutamate
receptors, as well as the NO/cGMP pathway, triggered by activation of the NMDA receptor, in the IC. In the
descriptive analysis, we found grounds suggesting that there is involvement of these receptors in the
neuromodulation of anxiety in rats, however, statistical tests did not show significance for this involvement.
A renewal of this study, with a larger sample size and different coordinates for different regions of the IC, is
necessary for more conclusive results.

FINAL CONSIDERATIONS

The IC is involved in the processing of visceral and vestibular sensory data, attention, pain, emotion,
verbal and motor information, as well as gustatory, olfactory, visual, auditory and tactile processes. Recent
neuroimaging studies have revealed that the insular cortex is involved in several neuropsychiatric diseases,
such as mood disorders and generalized anxiety.
Due to intercurrences in this study, we cannot state that there is significant inferential statistical
evidence to support the theory of the direct involvement of the NMDA/NO/cGMPc pathway, of the insular
cortex, in the modulation of anxiety, as well as that the antagonism of the ionotropic glutamate receptors , the
inhibition of iNOS, the sequestration of NO and the inhibition of sGC, in regions close to the insular cortex
of rats submitted to the ECL and CA test, culminate in significant behavioral differences when comparing the
treated and control groups, although some descriptive statistical data and bases in the literature suggest that
the pathway plays a fundamental role in modulating anxiety-related behavior.

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