DISCUSSION
The results obtained in the present study supported the hypothesis that
IC glutamatergic neurotransmission controls cardiovascular and autonomic
responses during restraint stress. Indeed, we observed that blockade of
NMDA glutamate receptors within the IC potentiated the pressor and
tachycardiac responses to acute restraint stress and inhibited the
decrease in tail skin temperature. In addition, we found that IC NMDA
glutamate receptors played a facilitatory role in spontaneous baroreflex
activity. On the other hand, IC glutamatergic neurotransmission mediated
by local non-NMDA glutamate receptors did not seem to control these
adjustments. Additionally, contrary to our initial hypothesis, the
present data do not indicate an involvement of IC glutamatergic
neurotransmission present in restraint-evoked increases in circulating
corticosterone during restraint stress.
Restraint stress in animal models causes autonomic changes, such as
increased blood pressure and HR, sympathetically mediated cutaneous
vasoconstriction resulting in a drop in tail skin temperature, and
modulation of baroreflex function (Crestani, 2016; Dos Reis, Fortaleza,
Tavares, & Correa, 2014). This stressor also activates the HPA axis,
which results in increased circulating corticosterone levels in rodents
(Bali & Jaggi, 2015; Buynitsky & Mostofsky, 2009). These physiological
adjustments are highly reproductible between different laboratories
around the world; thus, restraint is one of most commonly used models of
stress in rodents (Bali & Jaggi, 2015; Buynitsky & Mostofsky, 2009).
Therefore, the restraint stress model is an excellent model for studying
neurobiological mechanisms involved in physiological adjustments to
stress.
Based on the combination of sensory inputs and limbic connectivity, the
IC has been described as an important cortical centre for the
integration of autonomic and behavioural responses during aversive
threats (Gogolla, 2017; Oppenheimer & Cechetto, 2016; Verberne &
Owens, 1998). Indeed, the IC has been implicated in the modulation of
stress responses, such as restraint (Alves, Crestani, & Correa, 2010;
Myers, 2017; Nagai, Hoshide, & Kario, 2010; Oppenheimer & Cechetto,
2016), contextual fear conditioning (Alves et al., 2013), and
fear-induced underestimation (Kamada & Hata, 2018). In the contextual
fear conditioning test, microinjection of cobalt chloride (a
nonselective synapse blocker) into the IC before re-exposure to an
aversive context attenuated the blood pressure and heart rate increases
evoked by the conditioned stimulus (Alves et al., 2013). Accordingly, IC
treatment with CoCl2 also greatly attenuated both
pressor and tachycardiac responses evoked by acute restraint stress
(Alves et al., 2010). We further demonstrated similar effects following
IC treatment with either α1- or
α2-adrenoceptor antagonists (Alves et al., 2014), thus
demonstrating a role of local noradrenergic neurotransmission in the
IC-mediated control of restraint-evoked cardiovascular changes.
A previous study on urethane-anaesthetized rats identified that
glutamate microinjection into the IC produced different types of
cardiovascular responses, including long oscillatory, pressor,
depressor, bradycardiac and tachycardiac responses (Ranjbar, Hatam, &
Nasimi, 2015). These results are intriguing and demonstrate that the IC
generates ambiguous responses when stimulated with glutamate. Aiming to
understand the role of glutamatergic neurotransmission within the IC in
autonomic responses during stress situations, we evaluated the
participation of the glutamatergic ionotropic NMDA and non-NMDA
receptors within the IC using the selective antagonists LY235959 and
NBQX, respectively. IC treatment with LY235959 potentiated the pressor
and tachycardiac responses evoked by restraint stress, confirming the
involvement of NMDA receptors in the IC in the modulation of
cardiovascular responses during restraint. It is important to note that
microinjection of the NMDA receptor antagonist in the IC did not alter
the basal values of blood pressure, heart rate or baroreflex parameters,
suggesting that IC glutamatergic neurotransmission does not tonically
modulate these parameters. In contrast to the effect identified
following non-selective neurotransmission blockade in the IC, which
indicated a facilitatory influence of this cortical structure on
cardiovascular responses induced by restraint stress, the results
reported here indicate that glutamatergic neurotransmission in the IC
has an inhibitory influence on the increases in BP and HR observed
during restraint stress. One explanation for these discrepancies might
be that there are both facilitatory and inhibitory
neurochemical mechanisms within the IC controlling cardiovascular
responses during restraint stress; thus, the nonselective blockade
caused by CoCl2 is not able to reveal the specific role
of the different local mechanisms. IC treatment with NBQX did not affect
the increases in arterial pressure or HR induced by restraint,
suggesting that non-NMDA receptors within the IC do not participate in
the cardiovascular changes observed during acute stress.
The increase in blood pressure observed during restraint is concomitant
with increases in heart rate and sympathetic activity. A previous study
published by our laboratory showed that rats with sinoaortic
baroreceptor denervation presented exacerbated increases in blood
pressure and HR when subjected to restraint stress (Dos Reis et al.,
2014), showing the active role of the baroreflex in controlling
cardiovascular function during this aversive situation. Likewise,
(Crestani, Tavares, Alves, Resstel, & Correa, 2010) demonstrated that
the heart rate reflex curve was shifted upward and to the right during
restraint stress. Based on previous evidence that glutamatergic
neurotransmission within the IC plays an excitatory role in the
modulation of baroreflex function (Alves, Crestani, Resstel, & Correa,
2009), in the present study, we evaluated spontaneous baroreflex
activity during the restraint stress session. We observed a decrease in
BEI during restraint stress in the group treated with the NMDA receptor
antagonist in the IC. These data provide additional evidence that NMDA
receptors in the IC are also involved in the control of reflex responses
during more discrete changes in arterial pressure. Corroborating the
present findings, we demonstrated previously that local IC treatment
with a selective NMDA receptor antagonist (but not with a non-NMDA
glutamate receptor antagonist) decreased the reflex bradycardia response
evoked by an increase in blood pressure caused by intravenous infusion
of phenylephrine (Alves et al., 2009). Therefore, taken together, the
cardiovascular and spontaneous baroreflex findings in the present study
indicate that the inhibitory influence of IC NMDA receptors in the
pressor and tachycardiac responses evoked by restraint stress might be
mediated, at least partly, via facilitation of baroreflex function.
In addition to modulation of baroreflex function, restraint stress also
triggers other autonomic responses, including sympathetically mediated
cutaneous vasoconstriction, which in turn causes a drop in tail skin
temperature (Brasil, Fassini, & Correa, 2018; Busnardo et al., 2019;
Vianna & Carrive, 2005). Previous studies demonstrated that IC
modulates sympathetic nerve activity (Cechetto & Chen, 1990), thus
contributing to cutaneous vasodilation or vasoconstriction and changes
in tail skin temperature. The participation of glutamatergic
neurotransmission in modulating autonomic responses involving a drop in
skin temperature has been described in other areas in animals subjected
to stress (Moraes-Neto, Scopinho, Biojone, Correa, & Resstel, 2014).
Moreover, IC inactivation using bupivacaine potentiated hypothermia and
bradycardic and hypertensive responses to hypoxia (Casanova, Contreras,
Moya, Torrealba, & Iturriaga, 2013). In the present study, we observed
that blockade of NMDA receptors in the IC shifted the drop in tail skin
temperature to an increase (see Fig. 4). The increase in tail
temperature was unexpected, since NMDA receptors in the IC appear to
have an inhibitory influence on cardiovascular responses to restraint.
Thus, we expected that this glutamatergic receptor would have a similar
role in the control of the tail skin temperature response. The present
results provide evidence of the existence of different sympathetic
mechanisms controlling cutaneous and other vascular beds, such as the
muscular, renal, and splanchnic circulations. Another possible
explanation is that glutamatergic neurotransmission through NMDA
receptors within the IC modulates thermoregulatory centres and causes
increased temperature by increasing metabolism, which is reflected as an
increase in the tail skin temperature due to heat dissipation. The IC
has reciprocal connections with several brain areas responsible for
controlling sympathetic activities and thermogenesis, including the
lateral hypothalamus, periaqueductal grey and parabrachial nucleus
(Gogolla, 2017). Additionally, the parabrachial nucleus has been
associated with temperature control related to behaviour via projections
to the dorsal raphe nucleus (Yahiro, Kataoka, Nakamura, & Nakamura,
2017). On the other hand, the lateral hypothalamus participates in body
temperature regulation (de Vrind, Rozeboom, Wolterink-Donselaar,
Luijendijk-Berg, & Adan, 2019) and brown adipose tissue-mediated
thermogenesis (You, Chu, Guo, & Lu, 2020), while the periaqueductal
grey is an important relay in the descending pathways regulating
thermogenesis (de Git et al., 2018). However, more studies are necessary
to clarify the involvement of the IC in the control of temperature
adjustments during acute restraint stress. In the present study, we only
evaluated the autonomic responses evoked by restraint stress and did not
measure thermoregulatory responses.
One of the main responses triggered by exposure to a stressor stimulus
is the activation of the HPA axis and the consequent release of
glucocorticoids into the circulation (Selye, 1951). Glucocorticoids are
essential to preparation for physiological, environmental and
psychological challenges. One of the main functions of glucocorticoids
is energy redistribution to optimize survival when facing a challenge
(Herman et al., 2016). Treatment of the IC with either NMDA or non-NMDA
receptor antagonist did not change the increase in corticosterone levels
during stress. Taken together with the results of the other parameters
analysed in the present study, these findings provide further evidence
indicating that the central pathways and neurochemical mechanisms
controlling circulating corticosterone are distinct from those
regulating autonomic and cardiovascular responses during restraint
stress (Busnardo et al., 2019; Gouveia et al., 2016).
In summary, the results reported in the present study suggest that
glutamatergic neurotransmission present in the IC differentially
modulates cardiovascular and autonomic responses during aversive
threats. Our data indicate that IC NMDA glutamate receptors have an
inhibitory effect on tachycardic and pressor responses to restraint
stress that seems to be mediated, at least partially, by the
facilitation of baroreflex function. Furthermore, the data reported here
suggest that NMDA glutamatergic receptors in the IC are involved in the
drop in tail skin temperature during acute restraint. Finally, the
present findings provide evidence that HPA axis control during stress is
mediated by mechanisms other than glutamatergic neurotransmission in the
IC.