Amygdalar neuronal activity mediates the cardiovascular responses evoked from the dorsolateral periaqueductal gray in conscious rats
Introduction
It is generally agreed that specific neural circuits have developed in several organisms, that allow for a quick and strategic response to environmental threats. Specific pathways in the central nervous system (CNS) of mammals are activated during emotional or environmental stress, and induce various autonomic and endocrine responses (DiMicco et al., 2006, Szczepanska-Sadowska, 2008). These changes are accompanied by defensive behaviors including flight, freezing, defensive attack, and risk assessment (Blanchard and Blanchard, 1989a, Blanchard and Blanchard, 1989b), which favor survival.
One of the brain regions involved in the integration of specific physiological changes associated with emotional behavior is the periaqueductal gray matter (PAG) (Bandler et al., 2000). Studies have shown that chemical stimulation of glutamatergic neurons in the caudal portion of the lateral/dorsolateral periaqueductal gray (l/dlPAG) induces tachycardia and increases locomotor activity, blood pressure, and body temperature; responses which are similar to those produced during emotional stress (Carrive, 1993, Bandler and Shipley, 1994, de Menezes et al., 2009). Such tachycardic and pressor responses are mediated by efferent neuronal projections from the PAG to autonomic brain centers (Lovick, 1993, McNaughton and Corr, 2004).
Furthermore, the PAG has been defined as a lower center of defensive reactions influenced by downstream afferent projections arriving from the hypothalamus (Bandler and Keay, 1996, Bernard and Bandler, 1998). However, studies show that the PAG could also be a source of excitatory ascending projections to the hypothalamus during defensive reactions. In this regard, responses induced by stimulated l/dlPAG neurons are dependent on neuronal activity in the dorsomedial hypothalamus (DMH), suggesting that the PAG can produce changes in physiological parameters through upstream centers of neuronal activation, such as the hypothalamus (de Menezes et al., 2009, Horiuchi et al., 2009, Fontes et al., 2011).
Anatomical studies have also identified that both the dorsolateral (dlPAG) and the DMH form connections with the amygdala. Even though these connections appear to be indirect they play an important role in cardiovascular control during defense reactions. In fact, these three structures are believed to form an aversive system in the brain associated with anxiety (Graeff, 2004). The basolateral nucleus of the amygdala (BLA) has been identified as a site where GABAergic mechanisms play an important role in the regulation of cardiovascular function and behavior (Shekhar, 1993, Sanders and Shekhar, 1995). Additionally, it has been suggested that the BLA acts as an integration center for the anxiety states, and that glutamatergic neurotransmission is critical to the regulation of this condition (Campeau and Davis, 1995, Ledoux, 2012). Finally, glutamatergic stimulation of the BLA produces effects similar to those observed for the activation of l/dlPAG (Soltis et al., 1998). Altogether, these data suggest that the excitatory pathway between the dlPAG and the DMH could depend on the activation of BLA neurons.
There is ample evidence that the amygdala is critically involved in the regulation of innate and conditioned reactions to threatening stimuli (Ledoux, 1994, Campeau and Davis, 1995). Furthermore, it is frequently proposed that the PAG is downstream of the amygdala, directing motor outputs toward the proper defensive behavior (Ledoux, 2012). On the other hand, it is not well established to what extent the amygdala regulates the upstream functional connection from the l/dlPAG. Indeed, activation of the dlPAG leads to increased expression of c-Fos protein, a marker for neuronal activation, in the BLA (Ferreira-Netto et al., 2005).
Here, we evaluated role of the functional connection between the l/dlPAG and the BLA on the cardiovascular response evoked by the dlPAG. For that we examined the influence of acute inhibition of the BLA, by injecting muscimol, on responses evoked by the microinjection of the excitatory amino acid NMDA (N-methyl D-Aspartate) into l/dlPAG. We also evaluated the role of BLA ionotropic glutamate receptors in these responses by injecting 2-amino-5-phosphonopentanoate (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione) (CNQX), antagonists of NMDA and AMPA/kainate receptor subtypes, respectively, into the BLA. To establish the anatomical specificity of the effect of muscimol in the BLA, we also tested the consequence of identical injection into the central amygdala (CEA) in parallel experiments. Characterizing the interaction between PAG and amygdala in triggering the defense reaction will lead to a better understanding of these structures’ roles within a neural circuit that modulates reactions to threatening stimuli.
Section snippets
Ethical approval
All procedures were performed according to the regulations set forth by the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and according to the journal policies and regulations on animal experimentation and were approved by the ethics committee for animal research of the Federal University of Ouro Preto (CEUA-UFOP; # 2010/26). All efforts were made to minimize the number of animals used in the present study, and to avoid any unnecessary distress to the
Stimulation of the BLA produces cardiovascular responses similar to those obtained by l/dlPAG activation
To confirm that the activation of BLA neurons is capable of producing increases in HR and MAP comparable to those induced by PAG activation, we injected an NMDA receptor agonist into the BLA. Injection of NMDA into the BLA produced increases in HR and MAP, relative to vehicle treatment (for treatment, HR: F(1,96) = 16.89, p < 0.05; and MAP: F(1,96) = 10.59, p < 0.05) (Fig. 1), as expected (Soltis et al., 1998). These changes were accompanied by increases in locomotor activity as previously reported (
Discussion
In this study, we evaluated the influence of BLA on the cardiovascular response produced by the activation of l/dlPAG. Our results showed that neuronal excitation in the l/dlPAG produces cardiovascular changes through the activation of glutamate receptors present in the BLA. Our data provide evidence that the BLA is downstream of the l/dlPAG, and directs the autonomic components of the defense reaction resulting from the activation of l/dlPAG.
First, we confirmed that the activation of glutamate
Conclusion
In this study, we showed that BLA activity plays a critical role in the modulation of physiological responses caused by l/dlPAG activation, and that these responses specifically depend on the glutamatergic activity of BLA neurons. Importantly, the same role was not observed for another region of the amygdala, the CEA. Thus, knowing that the autonomic responses evoked by l/dlPAG activation depends on neuronal activity in the DMH (de Menezes et al., 2009, Horiuchi et al., 2009), and that the
Acknowledgments
This work was supported by FAPEMIG, PRONEN, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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