Opioid neurotransmission modulates defensive behavior and fear-induced antinociception in dangerous environments
Graphical abstract
Panic attack-like behavioral responses displayed by Wistar rats threatened by Crotalus durissus terrificus (Reptilia, Viperidae) wild venomous snakes are reverted to fearlessness by the peripheral treatment with naloxone. Background: Photomicrograph of a transverse section of the dorsal midbrain of a Wistar rat showing a fast blue neurotracer-labeled neuron situated in the dorsolateral column of the periaqueductal gray matter, which is rich in opioid peptide neurons and receptors.
Introduction
Polygonal arenas and complex labyrinths with snakes have been used as experimental models of panic attacks (Coimbra et al., 2017b) and are suitable for the study of the antiaversive effects of new potential panicolytic drugs (Uribe-Mariño et al., 2012, Twardowschy et al., 2013). Although both constrictor (Guimarães-Costa et al., 2007, Lobão-Soares et al., 2008) and Viperidae venomous (Almada and Coimbra, 2015) serpents have been used as threatening stimuli to elicit defensive responses in small rodents in these behavioral paradigms, rattlesnakes have not been consistently used as a source of innate aversion-inducing stimuli. However, some animals use sonorous clues to evaluate a dangerous environment. For example, rattlesnakes signal an imminent attack by vibrating their rattles, and some mammals can mimic these rattle sounds to repel other predators from their territory (Owings et al., 2002). Rodents assume a biped posture and hesitate to engage in exploratory behavior or approach an area associated with a snake-produced audible signal (Swaisgood et al., 1999a, Swaisgood et al., 1999b). Therefore, the use of rattlesnakes in prey-versus-predator paradigms may be suitable because rattlesnakes provide threatening visual, olfactory and auditory clues that result in aversion. An instinctive or learned stimulus signaling danger activates the encephalic circuits responsible for generating and elaborating these aversion-inducing states (Blanchard and Blanchard, 1972, Blanchard et al., 1991, Fanselow and Kim, 1994), which are interpreted as a motivational state of fear in humans (Nashold et al., 1969).
Defensive behaviors may consequently be expressed as behavioral inhibition and defensive attention (Hilton, 1982, Coimbra and Brandão, 1993, Guimarães-Costa et al., 2007, Lobão-Soares et al., 2008, Uribe-Mariño et al., 2012), defensive immobility or freezing (Uribe-Mariño et al., 2012, Almada et al., 2015a), escape behavior (Coimbra and Brandão, 1993, Uribe-Mariño et al., 2012, Almada et al., 2015b), or tonic immobility (Leite-Panissi et al., 2003, Leite-Panissi CRA et al., 2012). Affective defense, which is a set of threatening postures, vocalizations, and eventual attacks (when escape is impossible) (Hess and Bruger, 1943), can also be elicited by preys in a dangerous situation.
The expression of defensive behavior is followed by neurovegetative and endocrine alterations, which have been extensively studied (Mancia and Zanchetti, 1981, Hilton, 1982, Carrive, 1993), as well as by antinociception (Fanselow and Bolles, 1979, Fanselow, 1986, Coimbra et al., 2006), a phenomenon in which both opioid (Terman et al., 1986, Nichols et al., 1989) and non-opioid (Coimbra et al., 1992, Coimbra and Brandão, 1997) mechanisms have been implicated. Interestingly, both opioid (Miczek et al., 1982, Thompson et al., 1988) and non-opioid (Griesel et al., 1993) antinociception have been implicated in behavioral responses evoked during resident-intruder interactions.
Some animals assume appeasing postures of submission to avoid ulterior offensive behaviors by dominant opponents (Miczek and Thompson, 1984). However, the behavioral responses displayed by preys confronted by a potential predator are completely different and are rich in risk assessment behavior, defensive immobility and oriented and non-oriented escape behaviors (Almada et al., 2015b, Almada and Coimbra, 2015, Coimbra et al., 2017b). However, the threatening/defensive and offensive responses displayed by predators in snake-versus-rodent confrontation paradigms must be more carefully ethologically assessed in laboratory environments.
Recently, a new controversy has emerged in the literature regarding the proposal of two new coadjutant treatments for panic syndrome using either opioid agonists at low doses (Roncon et al., 2015, Maraschin et al., 2016, Roncon et al., 2017) or opioid receptors antagonists at high concentrations (Eichenberger et al., 2002, Ribeiro et al., 2005, Castellan-Baldan et al., 2006). Endogenous opioid peptides, which act presynaptically on GABAergic terminals, have been proposed to modulate the activity of the dorsal mesencephalon neural networks involved in the organization of defensive behavior (Eichenberger et al., 2002, Osaki et al., 2003, Ribeiro et al., 2005, Castellan-Baldan et al., 2006, Twardowschy and Coimbra, 2015). There is evidence that endogenous opioid peptides inhibit the GABA-mediated synaptic transmission in the periaqueductal gray matter and other encephalic regions by reducing the probability of presynaptic neurotransmitter release (Vaughan et al., 1997, Kishimoto et al., 2001, Tongjaroenbungam et al., 2004).
Considering these findings, a study of the GABAergic and opioid peptidergic pathways is required to identify the neural substrates responsible for fear-induced behavior, panic-like reactions and anticipatory anxiety. Several investigations in the behavioral neurosciences have focused on the neurochemical mechanisms evoked by instinctive or learned stimuli that signal danger (new environments, silhouettes of predators, emotional expressions indicating rage and imminent attacks, odors or sounds of a given predator, threatening vocalizations of intra-specific dominants in a social conflict, or any other factor that may indicate the occurrence of noxious or painful stimuli) (Blanchard et al., 1989, Blanchard et al., 1991, Blanchard et al., 2003a, Blanchard et al., 2003b, Griebel et al., 1996, Guimarães-Costa et al., 2007, Almada et al., 2015b).
Most studies focusing on the neurophysiological bases of behavior use invasive techniques in experimental animal models in which only segmental divisions of the limbic system are targeted. These structures are related to the elaboration of emotional states and are usually accessed either by local microinjections of pharmacologic agents or by induction of specific neurotoxic lesions in nuclei rich with a given neurotransmitter (Coimbra and Brandão, 1993, Ribeiro et al., 2005, Biagioni et al., 2013, Almada et al., 2015a, Almada et al., 2015b). However, the entire limbic system is commonly activated when animals are faced with situations that threaten their survival, such as those characterized by the presence of a natural predator.
The neural bases of attentional behavior related to aversion-inducing events, fear and antinociception must be clarified. In controlled environments, these behaviors are usually displayed by experimental animals exposed to imminent danger or elicited by stimulation of the brainstem and the pathways that elaborate or modulate panic-like reactions (Coimbra and Brandão, 1993, Borelli et al., 2005, Ribeiro et al., 2005, Coimbra et al., 2017a), and their underlying neural bases must be clarified. Furthermore, the precise role played by the opioid system in the modulation of panic-like responses, which has been studied using preclinical and clinical approaches, remains controversial (Colasanti et al., 2011), and the neural bases of the antinociception that follows defensive behavior requires further characterization. The aim of this work was to investigate the possible anxiolytic and panicolytic effects of systemic opioid peptide receptor inhibition using two classical models of unconditioned fear-induced responses, including the elevated plus- and T-maze (EPM and ETM, respectively) tests, and an experimental setup based on confrontations between rodents and rattlesnakes (Coimbra et al., 2017b). The prey-versus-serpent paradigm in a polygonal arena was designed as a new model of panic attacks. Innate fear-induced antinociception and both unconditioned and conditioned fear-induced defensive behavioral responses were also recorded.
Section snippets
Animals
Male Wistar rats (Rattus norvegicus, Rodentia, Muridae) weighing 200–250 g (n = 6–15 per group) were obtained from the animal facility of the Ribeirão Preto School of Pharmaceutical Sciences, University of São Paulo (FCFRP-USP), for use in this study. The rats were housed (4 per cage) in an experimental room (for at least 48 h prior to the experiments) under a 12:12-h light/dark cycle (lights on at 7:00 am) at 22–23 °C. The rats were provided free access to water and food. The predators used in this
Antinociceptive procedure
Immediately after the EPM, ETM and confrontation tests, which are described below, the nociception thresholds of the animals were compared using the tail-flick test. Each animal was placed in a restraining apparatus (Insight, Ribeirão Preto, SP, Brazil) with acrylic walls, and its tail was placed on a heating sensor (Tail-Flick Analgesia Instrument; Insight, Brazil). The progressive heat elevation was automatically interrupted when the animal removed its tail from the apparatus. The current
Experiment 1: Effects of different doses of naloxone on rats subjected to the EPM test
The effects of different doses of naloxone on the behavioral responses displayed by rats subjected to the EPM test are illustrated in Fig. 1A–D. One-way ANOVA revealed that naloxone had a significant effect on the number of open-arm entries and the percentage of time spent in the arms (F(3,24) = 6.57, p < 0.01 and F(3,24) = 14.95, p < 0.001, respectively; Fig. 1A, C, respectively). However, naloxone had no significant effects on the number of closed-arm entries (F(3,24) = 0.91, p > 0.05; Fig. 1B). Compared
Discussion
Exposing rats to the EPM elicited anxiety-like responses, which were expressed as the discrete number of entries into and the time spent in the open arms of the EPM test. These responses were significantly attenuated after systemic treatment with naloxone, which also exerted anxiolytic-like effects on the complementary behaviors displayed by rats subjected to the EPM test. These findings support those of a previous report demonstrating that naloxone induced potentiation of the anxiolytic
Acknowledgments
This study was supported by the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq, Brazil) (process 470119/2004-7), Fundação de Apoio ao Ensino, Pesquisa e Assistência do HC-FMRP-USP (FAEPA, Brazil) (processes 1291/97, 355/2000, 68/2001 and 15/2003), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil) (processes 03/07202-6, 03/01768-8, 03/01794-9, 07/01174-1, and 2012/03798-0), and a Pro-Rectory of the University of São Paulo (USP) Research grant (IaPQ2012;
References (91)
- et al.
The anterior cingulate cortex is a target structure for the anxiolytic-like effects of benzodiazepines assessed by repeated exposure to the elevated plus maze and Fos immunoreactivity
Neuroscience
(2009) - et al.
Medial prefrontal cortex serotoninergic and GABAergic mechanisms modulate the expression of contextual fear: intratelencephalic pathways and differential involvement of cortical subregions
Neuroscience
(2015) - et al.
Endocannabinoid signaling mechanisms in the substantia nigra pars reticulata modulate GABAergic nigrotectal pathways in mice threatened by urutu-cruzeiro venomous pit viper snake
Neuroscience
(2015) - et al.
Naloxone potentiates the effects of subeffective doses of anxiolytic agents in mice
Eur J Pharmacol
(1997) - et al.
Serotonergic neural links from the dorsal raphe nucleus modulate defensive behaviours organised by the dorsomedial hypothalamus and the elaboration of fear-induced antinociception via locus coeruleus pathways
Neuropharmacology
(2013) - et al.
Neuroethological validation of an experimental apparatus to evaluate oriented and non-oriented escape behaviours: comparison between the polygonal arena with a burrow and the circular enclosure of an open-field test
Behav Brain Res
(2016) - et al.
5-Hydroxytryptamine1A receptors in the dorsomedial hypothalamus connected to dorsal raphe nucleus inputs modulate defensive behaviours and mediate innate fear-induced antinociception
Eur Neuropsychopharmacol
(2016) - et al.
Twenty-two kHz alarm cries to presentation of a predator, by laboratory rats living in visible burrow systems
Physiol Behav
(1991) - et al.
Mouse defensive behaviours: pharmacological and behavior assays for anxiety and panic
Neurosci Biobehav Rev
(2001) - et al.
The mouse defensive test battery: pharmacological and behavioral assays for anxiety and panic
Eur J Pharmacol
(2003)