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.