Defensive coping strategies are behavioral adaptations elicited when individuals are exposed to aversive stimuli. In general, these adaptations involve neural processing underlying a continuum of behavioral domains ranging from acute (e.g. perception, motivation, activation), to long-term processes (e.g. learning and memory stabilization, behavioral flexibility). These behavioral alterations are responsible for a well-adjusted defensive reaction, prompt to be recalled in anticipation or confrontation of similar situations, contexts, or cues.

In rodents, exposure to aversive stimuli such as foot shock, predator, or predator odor, activates the mesencephalic periaqueductal gray matter (PAG) (Babai et al., 2001, Canteras and Goto, 1999, Dielenberg and McGregor, 2001). Beyond the well-described role in pain processing, PAG presents itself as an anatomical and functional interface connected to different brain areas responsible for regulating the behavioral and autonomic responses associated with aversive situations (Benarroch, 2012). In addition to coordinating the expression of overt defensive responses (DR) such as fight, flight, or freezing, the PAG participates in the modulation of more subtle behaviors associated with DR including risk assessment, crouch-sniffing, avoidance, which may be associated with dynamic association processes for the acquisition of aversive conditioning and the expression of learned DR (Back and Carobrez, 2018, Kincheski et al., 2012, Teixeira and Carobrez, 1999).

Whereas the majority of studies involve the role of PAG on defensive behavior expression following acute chemical, electrical or optogenetic stimulation (Assareh et al., 2016, Back and Carobrez, 2018, Johansen et al., 2010, Kincheski et al., 2012, Pavesi et al., 2011), some studies demonstrated the influence of dorsolateral PAG (dlPAG) in aversive learning and memory formation (Back and Carobrez, 2018, Johansen et al., 2010, Kincheski et al., 2012). This approach renewed the interest in the anatomic-functional organization of dlPAG for trafficking information both during descending defensive behavior expression and ascending aversive instruction to prosencephalic structures.

The glutamatergic system in dlPAG is involved in the descending pathways for defensive behavior expression and the control of ascending prosencephalic aversive instruction (Motta et al., 2017). Previous studies have used NMDA infusion into the dorsal PAG, demonstrating that this chemical stimulation elicits DR (Carvalho-Netto et al., 2009, Miguel and Nunes-de-Souza, 2006, Nunes-de-Souza et al., 2010). During the olfactory aversive conditioning (OAC) task, a dose-response dlPAG-NMDA curve determined that 50-100 pmol dose range was able to elicit an immediate defensive response, able to further support CS-US association. (Back and Carobrez, 2018, Kincheski et al., 2012).

The activation of postsynaptic NMDA receptors induces regulatory systems including neuronal isoform of nitric oxide synthase (nNOS) activation, which results in a local increase in nitric oxide (NO) production (Garthwaite, 2008). In fact, the rostral region of dlPAG has abundant NOS immunoreactive neurons (Onstott et al., 1993), which indicates that local production of NO could act as a regulator of learning and memory expression processes. NO is a molecule of great importance for the maintenance of synaptic homeostasis as it plays a dual role in the regulation of glutamatergic transmission. Stimulation of dlPAG through the infusion of lower doses of NMDA or SP activates a presynaptic positive feedback mechanism induced by NO acting as a retrograde messenger. The NO generated at post-synaptic neurons after activation of NMDARs diffuses across the plasma membrane and activates guanylate cyclases (GCs) at the presynaptic terminal, facilitating the release of glutamate necessary for the processes of synaptic plasticity (Garthwaite, 2008). In addition, GCs activate other pathways mediated by PKG, PKA (Müller, 2000), and AD-ribosylation (Schuman et al., 1994) that also participate in learning and memory processes.

The role of dlPAG-NO in defensive reaction has been studied under several paradigms such as the predator exposure (Beijamini & Guimarães, 2006), contextual fear conditioning (Kelley et al., 2011, Kelley et al., 2010), visual or auditory aversive conditioning (Schafe et al., 2005) and chemical stimulation of defensive neural circuit (De Oliveira et al., 2001, Overeem et al., 2010, Pavesi et al., 2013). Owing to its diffusion characteristics, NO can act at postsynaptic sites (where it is synthesized), in neighboring cells, or in the pre-synapse, where it can modulate the release of other neurotransmitters (Garthwaite, 2008). Additionally, NO is known to actively participate in all phases of the memory process by acting as a promiscuous messenger in different circuits ((Bal et al., 2017, Kendrick et al., 1997, Latini, 2018) for a review, consider (Medeiros et al., 2022).

However, the participation of the nitrergic system in learning and memory processes arising from dlPAG stimulation has not been fully explored. Therefore, the present study was outlined to investigate how NO modulates the immediate DR and aversive memory formation produced by the chemical stimulation of dlPAG during an aversive conditioning task.