On the verge of a respiratory-type panic attack: Selective activations of rostrolateral and caudoventrolateral periaqueductal gray matter following short-lasting escape to a low dose of potassium cyanide
Graphical abstract
Introduction
Clinical and epidemiological studies showed that panics are either respiratory or non-respiratory depending on the prominence of respiratory symptoms (Roberson-Nay and Kendler, 2011). In turn, increasing evidence suggests that the periaqueductal gray matter (PAG) is crucial in panic attacks (Deakin and Graeff, 1991, Schenberg et al., 2001, Schenberg et al., 2014, Canteras and Graeff, 2014, Schenberg, 2016). Indeed, electrical stimulations of PAG of humans provoke both panic and feelings of severe anxiety, intense discomfort, urge to escape, dyspnea, smothering and ‘hunger for air’ that are typical of clinical panic (Nashold et al., 1969, Amano et al., 1978, Young, 1989, Kumar et al., 1997). As confirmed by X-ray of electrode localization, stimulations of a “medial zone … just lateral to the aqueduct” (up to 5 mm laterally from midline) provoked unbearable fear, whereas stimulations of lateral tegmentum were well tolerated in spite of eliciting intense pain (Nashold et al., 1969). Notably, as well, Young (1989) found that whereas the stimulation of “dorsal PAG often evokes sensations of anxiety, fear or other unpleasant sensations”, stimulations of “ventral PAG evokes a sensation of warmth, floating or generalized well-being”. These studies were supported by positron emission tomography data showing that the PAG is activated in volunteers either experiencing definite symptoms of smothering (Brannan et al., 2001) or escaping from a virtual predator that was otherwise able to inflict real shocks to the subject’s finger (Mobbs et al., 2007). Moreover, Amano et al. (1978) have long reported that during a surgery for pain relief, a patient stimulated in the PAG uttered “somebody is now chasing me, I am trying to escape from him”. These studies show that stimulations of dorsal regions of PAG are able to produce genuine panic feelings. In particular, in a two-point positron emission tomography of cholecystokinin-induced panic attacks, the early scan (30 s) showed an activation of a ‘hypothalamic region… which extended into the brain stem structures’ (Javanmard et al., 1999).
In animals, electrical and chemical stimulations of dorsomedial (DMPAG), dorsolateral (DLPAG) and lateral (LPAG) columns of PAG, herein named the ‘dorsal PAG’ (DPAG), elicit freezing and/or flight behaviors along with marked cardiovascular and respiratory responses (Bandler and Depaulis, 1991, Schenberg et al., 1993, Schenberg et al., 2005, Schenberg and Lovick, 1995, Bittencourt et al., 2004, Subramanian et al., 2008, Subramanian and Holstege, 2013, Deng et al., 2016, Tovote et al., 2016). That these responses reminisce clinical panic was shown by their attenuation by panicolytics given in doses and regimen alike to those employed in panic therapy (Schenberg et al., 2001). Notably, DPAG stimulations that produced robust escape in a 60-cm diameter open-field failed in increasing the ‘stress hormones’ corticotropin, corticosterone and prolactin when the rats were stimulated in a 20-cm diameter cylinder that prevented escape (Armini et al., 2015). The latter data are a fair reproduction of the neuroendocrine unresponsiveness that is a hallmark of clinical panic (Liebowitz et al., 1985, Levin et al., 1987, Woods et al., 1987, Sinha et al., 1999).
Remarkably, as well, Schimitel et al. (2012) presented compelling evidence that the PAG harbors a hypoxia-sensitive alarm system that may be implicated in respiratory-type panic attacks. Thus, whereas the escape to electrical stimulations of DPAG was facilitated by a slow intravenous infusion of potassium cyanide (KCN, 0.7 µg/s), the escape induced by in bolus injections of KCN (20–60 µg, i.v.) alone was virtually suppressed by electrolytic lesions of DPAG. Moreover, whereas 8% and 13% carbon dioxide (CO2) did not elicit escape by its own, they potentiated KCN-evoked escape of rats. Lastly, recent studies showed that escape responses to both KCN and 6% hypoxia are attenuated by panic-relieving drugs (Schimitel et al., 2014, Spiacci et al., 2015). Because KCN-evoked behaviors are strictly dependent on the integrity of carotid sinus nerve (Franchini and Krieger, 1993), the above studies suggest that the PAG harbors a hypoxia-sensitive suffocation alarm system which spontaneous activation could both precipitate a respiratory-type panic attack and render the subject hypersensitive to CO2 (Schimitel et al., 2012, Schenberg et al., 2014, Schenberg, 2016). These studies substantiate Klein’s “suffocation false alarm” theory of panic disorder (Klein, 1993a, Klein, 1993b, Preter and Klein, 2008, Preter and Klein, 2014).
Although Schimitel et al. (2012) suggested that respiratory (suffocation-like) and non-respiratory (proximal threat-like) panics are processed in LPAG and DLPAG, respectively, the lack of specificity of electrolytic lesions hindered the precise localization of KCN-responsive sites within the PAG. The identification of these sites was also compromised in c-fos immunohistochemistry studies showing extensive labeling of DPAG following repeated injections of high doses of KCN (>60 µg) in urethane anesthetized rats (Hayward and Von Reizenstein, 2002). To further localize the KCN-responsive sites, the present study compared the c-Fos protein expression in PAG and brainstem nuclei following either a single injection of a low dose of KCN (40 µg, i.v.), a 2-min exposure to CO2 (13%), or a combined stimulus (CO2/KCN). To rule out the KCN eventual direct action at DPAG, the behavioral effects of KCN microinjections into the PAG were assessed as well. Results suggest that rostrolateral (rLPAG) and caudoventrolateral (cVLPAG) PAG are key components of a hypoxia-sensitive suffocation alarm system which ‘spontaneous’ activation could trigger a respiratory-type panic attack. Data also suggest that KCN-evoked activations of PAG are accompanied by the concomitant activation of laterodorsal tegmental nucleus (LDTg).
Section snippets
Animals
Adult male Wistar rats (n = 70), weighing between 230 and 260 g, were housed in groups of 4–5 rats in polypropylene cages (60 cm × 50 cm × 22 cm) with wood-shave bedding and food and water ad libitum. Cages were kept in a temperature-controlled (20–25 °C) sound-attenuated (46 dB) room under a 12-h light/dark cycle (lights on at 6:00 am). All efforts were made to minimize suffering and number of animals. Experiments conformed to the National Institute of Health Guide for the Care and Use of Laboratory
Behavioral effects of KCN and/or CO2 administrations
Intravenous injections of 40-µg KCN produced short-lasting escape responses (2.7 ± 0.5 s) in all tested rats. Escape responses to 40-µg KCN were predominantly trotting; gallops and jumps being either very rare or totally absent, respectively. Indeed, whereas 40-µg KCN is twice the median effective dose (ED50) of trotting, it is only 0.7 ED50 of galloping (Schimitel et al., 2012). Pre-exposures to 13% CO2 produced significant increases in both range (Δ = 153%, t8 = 3.7, P < 0.01) and duration (Δ = 189%, t8 =
Discussion
Behavioral data showed that although the KCN intravenous injection produced short-lasting escape in all tested rats, KCN microinjections into the DPAG were totally ineffective. Data also showed that although the exposure to 13% CO2 alone did not elicit any defensive behavior, it more than doubled the range (2.5 times) and duration (2.8 times) of KCN-evoked escape. Treatments activated only the cNTS, the LDTg, the rLPAG and the cVLPAG. Most notably, however, the latter two structures were
Acknowledgments
This study was part of the PhD Thesis of CJTM. Authors were recipients of fellowships from CAPES (CJTM) and CNPq (LCS, ST). Histology and microphotographs were performed at the facilities of the Laboratory of Molecular Histology and Immunohistochemistry (LHMI) of the Health Sciences Center of the Federal University of Espirito Santo. The research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico/Fundação de Amparo à Pesquisa do Espírito Santo (CNPq/FAPES convention
References (76)
- et al.
In a rat model of panic, corticotropin responses to dorsal periaqueductal gray stimulation depend on physical exertion
Psychoneuroendocrinology
(2015) - et al.
Midbrain periaqueductal grey region in the cat has afferent and efferent connection with solitary tract nuclei
Neurosci Lett
(1987) - et al.
'Air hunger' arising from increased PCO2 in mechanically ventilated quadriplegics
Respir Physiol
(1989) - et al.
Stimulus-response characteristics of CO2-induced air hunger in normal subjects
Respir Physiol
(1996) - et al.
Postnatal changes in Fos-like immunoreactivity evoked by hypoxia in the rat brainstem and hypothalamus
Brain Res
(2000) - et al.
Map of spinal neurons activated by chemical stimulation in the nucleus raphe magnus of the unanesthetized rat
Neuroscience
(1995) - et al.
Organization of single components of defensive behaviors within distinct columns of periaqueductal gray matter of the rat: role of N-methyl-D-aspartic acid glutamate receptors
Neuroscience
(2004) - et al.
Functional and developmental identification of a molecular subtype of brain serotonergic neuron specialized to regulate breathing dynamics
Cell Rep
(2014) - et al.
Executive and modulatory neural circuits of defensive reactions: Implications for panic disorder
Neurosci Biobehav Rev
(2014) - et al.
Effect of insular cortex inactivation on autonomic and behavioral responses to acute hypoxia in conscious rats
Behav Brain Res
(2013)