Critical neuropsychobiological analysis of panic attack- and anticipatory anxiety-like behaviors in rodents confronted with snakes in polygonal arenas and complex labyrinths: a comparison to the elevated plus- and T-maze behavioral tests

Objective: To compare prey and snake paradigms performed in complex environments to the elevated plus-maze (EPM) and T-maze (ETM) tests for the study of panic attack- and anticipatory anxiety-like behaviors in rodents. Methods: PubMed was reviewed in search of articles focusing on the plus maze test, EPM, and ETM, as well as on defensive behaviors displayed by threatened rodents. In addition, the authors’ research with polygonal arenas and complex labyrinth (designed by the first author for confrontation between snakes and small rodents) was examined. Results: The EPM and ETM tests evoke anxiety/fear-related defensive responses that are pharmacologically validated, whereas the confrontation between rodents and snakes in polygonal arenas with or without shelters or in the complex labyrinth offers ethological conditions for studying more complex defensive behaviors and the effects of anxiolytic and panicolytic drugs. Prey vs. predator paradigms also allow discrimination between non-oriented and oriented escape behavior. Conclusions: Both EPM and ETM simple labyrinths are excellent apparatuses for the study of anxiety- and instinctive fear-related responses, respectively. The confrontation between rodents and snakes in polygonal arenas, however, offers a more ethological environment for addressing both unconditioned and conditioned fear-induced behaviors and the effects of anxiolytic and panicolytic drugs.

Evaluation of neurotransmitters involved in the anxiolytic and panicolytic effect of the aqueous fraction of Paullinia cupana (guaraná) in elevated T maze

This study investigated the effects of repeatedly administration of an aqueous fraction of Paullinia cupana Kunth, Sapindaceae (guaraná) seeds (8 mg/kg) on rats submitted to the elevated T-maze, model of generalized anxiety and panic disorders. The selective serotonin reuptake inhibitor paroxetine (3 mg/kg), was used as a positive control. To evaluate possible neurotransmissions involvement, ineffective doses of metergoline (3 mg/kg - non-selective serotonin receptor antagonist), sulpiride (20 mg/kg - non-selective dopaminergic receptor antagonist) or ketamine (0.125 mg/kg - non-selective glutamate receptor antagonist) were acutely administered in association with the aqueous fraction of P. cupana. Both aqueous fraction and paroxetine decrease the inhibitory avoidance latencies of the elevated T-maze, indicating anxiolytic effect and increased one-way escape latencies from the open arm of the elevated T-maze, indicating a panicolytic effect. The pre-treatment with metergoline, sulpiride and ketamine blocked the anxiolytic effect of aqueous fraction. The panicolytic effect of aqueous fraction was blocked by both metergoline and sulpiride. These results show that the serotonergic, dopaminergic and glutamatergic neurotransmission systems are involved in anxiolytic effect promoted by aqueous fraction, whereas only the serotonergic and the dopaminergic neurotransmission systems are involved in the panicolytic effect promoted by aqueous fraction of P. cupana. The effects produced by paroxetine, were blocked only by metergoline, validating this experimental procedure.

The effects of swimming exercise on recognition memory for objects and conditioned fear in rats / Os efeitos do exercício de natação sobre a memória para reconhecimento de objetos e de medo condicionados em ratos

Experiments conducted in animals have repeatedly demonstrated the ability of exercise to enhance cognitive function. This study examines the effects of chronic swimming exercise on non-spatial memory in adult rats after 12 weeks of swimming exercise in object recognition and elevated T-maze tests. In the object recognition test, repeated measures analysis of variance revealed a group effect (F1,42 = 26,093; p < 0.001), control rats had lower discrimination ratios than the exercise group. However, the swimming exercise did not affect the performance of inhibitory avoidance and escapes, when memory was tested in elevated T-maze. Analysis of variance showed a significant reduction in inhibitory avoidance 24h after the first training (F1,42 = 14,552; p < 0.001). Results indicated that regular swimming exercise significantly increased non-spatial memory in object recognition behavior, but did not affect the performance of inhibitory avoidance and escape on elevated T-maze test in adult rats. These findings suggest that the perirhinal cortex plays a role in memory consolidation and storage in addition to that of the amygdala, which could be regarded as the center of a second memory system, separate from those governed by the perirhinal cortex.

As experiências realizadas em animais mostram a capacidade do exercício em melhorar as funções cognitivas. Este estudo analisa os efeitos do exercício crônico de natação sobre a memória não-espacial em ratos adultos após 12 semanas de exercício de natação nos testes de reconhecimento de objetos e labirinto em T elevado. O teste de reconhecimento de objetos, pelas repetidas análises de variância revelaram um efeito de grupo (F1,42 = 26.093; p < 0,001), os ratos controles discriminaram uma razão inferior ao do grupo de exercício. Entretanto, o exercício de natação não afetou o desempenho de esquiva inibitória e escape, quando a memória foi testada no labirinto em T elevado. Análise de variância mostrou redução significativa na esquiva inibitória 24h após o primeiro treino (F1,42 = 14.552; p < 0,001). Os resultados indicam que o exercício regular de natação aumenta significativamente a memória não-espacial no comportamento de reconhecimento de objetos, mas não afeta o medo condicionado no teste do labirinto em T elevado em ratos adultos. Estes resultados sugerem que o córtex peririnal desempenha papel nos processos de consolidação e armazenamento de memória além da amígdala, podendo esta ser encarada como um segundo centro de sistema de memória, separada dos regidos pelo córtex peririnal.

Dorsal periaqueductal gray stimulation facilitates anxiety-, but not panic-related, defensive responses in rats tested in the elevated T-maze

The escape response to electrical or chemical stimulation of the dorsal periaqueductal gray matter (DPAG) has been associated with panic attacks. In order to explore the validity of the DPAG stimulation model for the study of panic disorder, we determined if the aversive consequences of the electrical or chemical stimulation of this midbrain area can be detected subsequently in the elevated T-maze. This animal model, derived from the elevated plus-maze, permits the measurement in the same rat of a generalized anxiety- and a panic-related defensive response, i.e., inhibitory avoidance and escape, respectively. Facilitation of inhibitory avoidance, suggesting an anxiogenic effect, was detected in male Wistar rats (200-220 g) tested in the elevated T-maze 30 min after DPAG electrical stimulation (current generated by a sine-wave stimulator, frequency at 60 Hz) or after local microinjection of the GABA A receptor antagonist bicuculline (5 pmol). Previous electrical (5, 15, 30 min, or 24 h before testing) or chemical stimulation of this midbrain area did not affect escape performance in the elevated T-maze or locomotion in an open-field. No change in the two behavioral tasks measured by the elevated T-maze was observed after repetitive (3 trials) electrical stimulation of the DPAG. The results indicate that activation of the DPAG caused a short-lived, but selective, increase in defensive behaviors associated with generalized anxiety.

Anxiolytic effects of repeated treatment with an essential oil from Lippia alba and (R)-(-)-carvone in the elevated T-maze

Lippia alba (Mill.) N.E. Brown (Verbenaceae) is widely used in different regions of Central and South America as a tranquilizer. The plant’s anxiolytic properties, however, merit investigation. The present study evaluated the effects of repeated daily (14 days) intraperitoneal (ip) treatment with an essential oil (EO) from a chemotype of L. alba (LA, chemotype II, 12.5 and 25 mg/kg; N = 6-8) and (R)-(-)-carvone (25 mg/kg; N = 8-12), the main constituent of this chemotype, on male Wistar rats (weighing 250 g at the beginning of the experiments) submitted to the elevated T-maze (ETM). The ETM allows the measurement of two defensive responses: inhibitory avoidance and one-way escape. In terms of psychopathology, these responses have been related to generalized anxiety and panic disorder, respectively. Treatment with the EO impaired ETM avoidance latencies, without altering escape, in a way similar to the reference drug diazepam (P < 0.05) (avoidance 2: control = 84.6 ± 35.2; EO 12.5 mg/kg = 11.8 ± 3.8; EO 25 mg/kg = 14.6 ± 2.7; diazepam = 7 ± 2.1). (R)-(-)-carvone also significantly altered this same response (P < 0.05; avoidance 1: control = 91.9 ± 31.5; carvone = 11.6 ± 1.8; diazepam = 8.1 ± 3.3). These results were not due to motor changes since no significant effects were detected in an open field. These observations suggest that LA exerts anxiolytic-like effects on a specific subset of defensive behaviors that have been implicated in generalized anxiety disorder, and suggest that carvone is one of the constituents of LA responsible for its action as a tranquilizer.

Effects of chronic corticosterone and imipramine administration on panic and anxiety-related responses

It is known that chronic high levels of corticosterone (CORT) enhance aversive responses such as avoidance and contextual freezing. In contrast, chronic CORT does not alter defensive behavior induced by the exposure to a predator odor. Since different defense-related responses have been associated with specific anxiety disorders found in clinical settings, the observation that chronic CORT alters some defensive behaviors but not others might be relevant to the understanding of the neurobiology of anxiety. In the present study, we investigated the effects of chronic CORT administration (through surgical implantation of a 21-day release 200 mg pellet) on avoidance acquisition and escape expression by male Wistar rats (200 g in weight at the beginning of the experiments, N = 6-10/group) tested in the elevated T-maze (ETM). These defensive behaviors have been associated with generalized anxiety and panic disorder, respectively. Since the tricyclic antidepressant imipramine is successfully used to treat both conditions, the effects of combined treatment with chronic imipramine (15 mg, ip) and CORT were also investigated. Results showed that chronic CORT facilitated avoidance performance, an anxiogenic-like effect (P < 0.05), without changing escape responses. Imipramine significantly reversed the anxiogenic effect of CORT (P < 0.05), although the drug did not exhibit anxiolytic effects by itself. Confirming previous observations, imipramine inhibited escape responses, a panicolytic-like effect. Unlike chronic CORT, imipramine also decreased locomotor activity in an open field. These data suggest that chronic CORT specifically altered ETM avoidance, a fact that should be relevant to a better understanding of the physiopathology of generalized anxiety and panic disorder.

Effects of reversible inactivation of the dorsomedial hypothalamus on panic- and anxiety-related responses in rats

The medial hypothalamus is part of a neurobiological substrate controlling defensive behavior. It has been shown that a hypothalamic nucleus, the dorsomedial hypothalamus (DMH), is involved in the regulation of escape, a defensive behavior related to panic attacks. The role played by the DMH in the organization of conditioned fear responses, however, is less clear. In the present study, we investigated the effects of reversible inactivation of the DMH with the GABA A agonist muscimol on inhibitory avoidance acquisition and escape expression by male Wistar rats (approximately 280 g in weight) tested in the elevated T-maze (ETM). In the ETM, inhibitory avoidance, a conditioned defensive response, has been associated with generalized anxiety disorder. Results showed that intra-DMH administration of the GABA A receptor agonist muscimol inhibited escape performance, suggesting an antipanic-like effect (P < 0.05), without changing inhibitory avoidance acquisition. Although a higher dose of muscimol (1.0 nmol/0.2 µL; N = 7) also altered locomotor activity in an open field when compared to control animals (0.2 µL saline; N = 13) (P < 0.05), the lower dose (0.5 nmol/0.2 µL; N = 12) of muscimol did not cause any motor impairment. These data corroborate previous evidence suggesting that the DMH is specifically involved in the modulation of escape. Dysfunction of this regulatory mechanism may be relevant in the genesis/maintenance of panic disorder.

The hypothalamic-pituitary-adrenal axis in anxiety and panic

This review article focuses on the differential activation of the hypothalamic-pituitary-adrenal (HPA) axis in generalized anxiety and panic. The results of experimental studies that assayed adrenocorticotropic hormone, cortisol and prolactin show that real-life panic attacks as well as those induced by selective panicogenic agents, such as lactate and carbon dioxide, do not activate the HPA axis. Accordingly, experiments carried out in two animal models of panic, namely electrical stimulation of the dorsal periaqueductal gray matter of the rat and the escape from the open arm of the elevated T maze, have shown that in neither case stress hormones are increased in the plasma. Also in humans, reported results have shown that neither cortisol nor prolactin levels were increased following simulated public speaking, an experimental task that has been related to panic, in either healthy volunteers or patients with panic disorder diagnosis. Therefore, although the panic attack causes a major sympathetic stimulation, it has little effect on the HPA axis. In contrast, anticipatory or generalized anxiety activates both the HPA and the sympatho-adrenal axes.

The effects of diazepam on the elevated T-maze are dependent on the estrous cycle of rats

In order to determine the modulation of anxiolytic and panicolytic-like effects of diazepam by the hormonal cycle of female rats, male and female rats - the latter divided per estrous cycle phase (estrus, diestrus, metaestrus and proestrus) - were tested in the elevated T-maze, a behavioral model of panic and anxiety. Diazepam (0.5, 1.0 and 2.0 mg/kg) or saline solution was injected in individual animals that were submitted to one session in the elevated T-maze 25 min after drug/saline administration. The test consisted of three avoidance trials and one escape trial, separated by a 30 s interval, during which the animals were isolated in individual cages. The avoidance trials began with the animal being placed at the end of the maze's enclosed arm. The time necessary for the animal to leave the central square was considered as the response's latency. The trials that exceeded 300 s were considered as failures. Results demonstrate a decrease in the effects of diazepam in inhibitory avoidance (anxiety) trials in females in diestrus and proestrus, but no relation of gender or estrous cycle on diazepam effects on escape trials (fear). The results support the hypothesis that down-regulation of GABA A receptors by activation of nuclear estrogen receptors and induction of PKC-mediated GABA A receptor phosphorylation by activation of surface estrogen receptors in raphe neurons underlie the modulation of diazepam sensitivity by estrogen.