Elisabeth Aparecida Audi

Role of the amygdala and periaqueductal gray in anxiety and panic

The amygdala (AM) and the periaqueductal gray (PAG) represent the rostral and the caudal pole, respectively, of a longitudinally organized neural system, that is responsible for the integration of behavioral and physiological manifestations of defensive reactions against innate and learned threats. Microinjection of benzodiazepine (BZD) anxiolytics, GABAA receptor agonists or 5-HT receptor antagonists into the AM has anxiolytic effects in conflict tests and other models of conditioned fear, while similar administration of 5-HT or of a 5-HT1A receptor agonist has anxiogenic effects. On the other hand, in the test of electrical stimulation of the PAG, microinjection of 5-HT, 5-HT mimetics, or of drugs that enhance the action of endogenous 5-HT into the same brain area has an antiaversive effect, like BZD and GABAA agonists. Furthermore, microinjection of midazolam, of the NMDA receptor antagonist AP-7, or of the 5-HT1A/1B receptor blocker propranolol increased the exploration of the open arms of the elevated plus-maze, having therefore an anxiolytic effect. These results point to an inhibitory role of the GABA-BZD system in both the AM and the PAG. In contrast, 5-HT seemingly enhances conditioned fear in the AM, while inhibiting unconditioned fear in the PAG. Thus, 5-HT2/1C antagonists reportedly release punished behavior when injected into the AM, whereas they antagonized the antiaversive effect of 5-HT, zimelidine and 5-HT1A/1B receptor blockers in the PAG. Since reported clinical studies revealed that one of such compounds, ritanserin, relieves generalized anxiety but tends to aggravate panic disorder, a relationship may be established between the AM and anxiety and the PAG and panic.

Benzodiazepine receptors in the periaqueductal grey mediate anti-aversive drug action

The microinjection of 80, 160 and 320 nmol chlordiazepoxide (CDP) as well as of 20, 40 and 80 nmol midazolam (MDZ) into the dorsal midbrain of rats bearing chronically implanted chemitrodes raised the threshold electrical current inducing escape behaviour by stimulating the dorsal periaqueductal grey matter (DPAG). Parallel linear regressions were obtained by plotting the log dose against drug-induced increases in escape threshold, MDZ being 3.55 times more potent than CDP (95% confidence limits 1.21 and 8.57). Local pretreatment with 80 nmol of the benzodiazepine antagonist Ro 15-1788 blocked the anti-aversive effect of either 160 nmol CDP or 40 nmol MDZ. The same dose of Ro 15-1788 was ineffective when given alone. These results suggest that the anti-aversive action of CDP and MDZ is due to their combination with benzodiazepine receptors in the DPAG.

Microinjection of propranolol into the dorsal periaqueductal gray causes an anxiolytic effect in the elevated plus-maze antagonized by ritanserin

The 5-HT1A/1B receptor antagonist propranolol was injected into the dorsal periaqueductal gray (DPAG) of rats exposed to the elevated plus-maze in order to investigate the participation in anxiety of 5-HT mechanisms operating in this brain region. Microinjection ofd,l- orl-propranolol into the DPAG increased the percentage of total arm entries without affecting the total number of entries into either open or enclosed arms of the maze, an effect characteristic of anxiolytic drugs injected systemically. The doses of 5 nmoll-propranolol and 10 nmold,l-propranolol caused anxiolytic effects of comparable magnitude, while the doses of 2.5 nmol of the former and 5 nmol of the latter were ineffective. Therefore, thel-isomer is likely to be the main one responsible for the pharmacological activity observed. In addition, the anxiolytic effect of 10 nmold,l-propranolol was antagonized by 10 nmol of the 5-HT2/1C receptor antagonist ritanserin, previously injected into the DPAG. The present as well as previously reported results suggest that the anxiolytic effect of propranolol injected into the DPAG is due to increased release of 5-HT acting on post-synaptic 5-HT2 receptors, resultant from blockade of 5-HT1B autoreceptors that inhibit amine release from serotonergic nerve endings.

Mediation by serotonin of the antiaversive effect of zimelidine and propranolol injected into the dorsal midbrain central grey

Previously reported results indicate that serotonin (5-HT) inhibits the neural sub strate of aversion in the dorsal midbrain central grey (DCG) of the rat. In addition, the present results show that microinjection of the 5-HT uptake inhibitor zimelidine (100 nmol) into the DCG of rats with chronically implanted chemitrodes raised the threshold of aversive electrical stimulation. This antiaversive effect of zimelidine was antagonized by pretreatment with the 5-HT2 receptor blocker ritanserin (10 nmol), also microinjected into the DCG. In contrast, the antiaversive effect of the benzodiazepine agonist midazolam (40 nmol) was unaffected by ritanserin. Propranolol (2.2, 4.4 and 8.8 nmol) raised the aversive threshold in a dose-depen dent way following its injection into the DCG. The antiaversive effect of 4.4 nmol of propranolol was antagonized by previous administration of ritanserin (10 nmol). Moreover, combined administration of zimelidine (100 nmol) followed by propranolol (4.4 nmol) caused an anti aversive effect which was equivalent to the sum of the effect of each drug alone. These results indicate that the antiaversive effect of intracerebrally injected zimelidine and propranolol is mediated by endogenous 5-HT, through activation of 5-HT2 receptors.

Behavioral effects of 5-HT receptor ligands in the aversive brain stimulation, elevated plus-maze and learned helplessness tests

In order to illustrate the use of animal models in the study of the anxiolytic and antidepressant properties of drugs acting on 5-HT receptors, a series of experiments is described. With electrical stimulation of the midbrain central gray (CG), an aversive area of the brain, the 5-HT-1 receptor antagonist propranolol raised the aversive threshold in a dose-dependent way, following its microinjection into the CG. This antiaversive effect of propranolol, which is similar to that of benzodiazepine anxiolytics, was prevented by microinjection into the same brain site of the 5-HT-2 receptor blocker ritanserin. Ritanserin itself and the 5-HT-1A receptor ligand ipsapirone caused either little or no effect. In another animal model of anxiety, the elevated plus-maze, intra-CG propranolol also caused an anxiolytic-like effect, antagonized by ritanserin, indicating a 5-HT mediation. However, systemically injected isamoltane, a congener of propranolol, was ineffective in the elevated plus-maze, whereas ipsapirone caused an anxiolytic effect. Ritanserin was again inactive. Finally, both ipsapirone as well as another 5-HT-1A receptor ligand BAY R 1531, given IP, reversed the learning deficit resulting from exposure to uncontrollable foot-shocks, an effect characteristic of antidepressant drugs.

Cooperative regulation of anxiety and panic-related defensive behaviors in the rat periaqueductal grey matter by 5-HT1A and µ-receptors

Previous results with the elevated T-maze (ETM) test indicate that the antipanic action of serotonin (5-HT) in the dorsal periaqueductal grey (dPAG) depends on the activation endogenous opioid peptides. The aim of the present work was to investigate the interaction between opioid- and serotonin-mediated neurotransmission in the modulation of defensive responses in rats submitted to the ETM. The obtained results showed that intra-dPAG administration of morphine significantly increased escape latency, a panicolytic-like effect that was blocked by pre-treatment with intra-dPAG injection of either naloxone or the 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1 piperazinyl] ethyl] -N- 2- pyridinyl-ciclohexanecarboxamide maleate (WAY-100635). In addition, previous administration of naloxone antagonized both the anti-escape and the anti-avoidance (anxiolytic-like) effect of the 5-HT1A agonist (±)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), but did not affect the anti-escape effect of the 5-HT2A agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI). Moreover, the combination of sub-effective doses of locally administered 5-HT and morphine significantly impaired ETM escape performance. Finally, the µ-antagonist D-PHE-CYS-TYR-D-TRP-ORN-THR-PEN (CTOP) blocked the anti-avoidance as well as the anti-escape effect of 8-OHDPAT, and the association of sub-effective doses of the µ-opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt (DAMGO) and of 8-OHDPAT had anti-escape and anti-avoidance effects in the ETM. These results suggest a synergic interaction between the 5-HT1A and the µ-opioid receptor at post-synaptic level on neurons of the dPAG that regulate proximal defense, theoretically related to panic attacks.

The panicolytic-like effect of fluoxetine in the elevated T-maze is mediated by serotonin-induced activation of endogenous opioids in the dorsal periaqueductal grey.

Serotonin (5-HT), opioids and the dorsal periaqueductal grey (DPAG) have been implicated in the pathophysiology of panic disorder. In order to study 5-HT–opioid interaction, the opioid antagonist naloxone was injected either systemically (1 mg/kg, i.p.) or intra-DPAG (0.2 μg/0.5 μL) to assess its interference with the effect of chronic fluoxetine (10 mg/kg, i.p., daily for 21 days) or of intra-DPAG 5-HT (8 μg/0.5 μL). Drug effects were measured in the one-escape task of the rat elevated T-maze, an animal model of panic. Pretreatment with systemic naloxone antagonized the lengthening of escape latency caused by chronic fluoxetine, considered a panicolytic-like effect that parallels the drug’s therapeutic response in the clinics. Pretreatment with naloxone injected intra-DPAG antagonized both the panicolytic effect of chronic fluoxetine as well as that of 5-HT injected intra-DPAG. Neither the performance of the inhibitory avoidance task in the elevated T-maze, a model of generalized anxiety nor locomotion measured in a circular arena was affected by the above drug treatments. These results indicate that the panicolytic effect of fluoxetine is mediated by endogenous opioids that are activated by 5-HT in the DPAG. They also allow reconciliation between the serotonergic and opioidergic hypotheses of panic disorder pathophysiology.

GABAA receptors in the midbrain central grey mediate the antiaversive action of GABA

Intracerebral injection of the GABAA agonists muscimol (1 nmol), isoguvacine (1 nmol) or THIP (1, 2 and 4 nmol) in rats with chemitrodes implanted in the dorsal midbrain central grey raised the threshold electrical current for inducing escape behaviour. The effect of THIP was dose-dependent. In contrast, the GABAB agonist baclofen (10 and 100 nmol) did not affect the aversive threshold. Furthermore, pretreatment with baclofen (10 nmol and 100 nmol) did not significantly change the effect of THIP (2 nmol). These results indicate that the antiaversive action of GABA in the midbrain central grey is mediated by GABAA but not by GABAB receptors.

Anxiolytic Effects of a Semipurified Constituent of Guaraná Seeds on Rats in the Elevated T-Maze Test

The objective of this study was to investigate the effects of chronic administration of a semi-purified extract (Purified Extract A--PEA; 4, 8, or 16 mg/kg) of PAULLINIA CUPANA (guaraná) seeds on rats submitted to the elevated T-maze (ETM) model of generalized anxiety and panic disorders. The selective serotonin (5-HT) reuptake inhibitor (SSRI) paroxetine (PAR; 3 mg/kg), was used as a positive control. To evaluate possible serotonergic and dopaminergic neurotransmission involvement in the action of PEA during the ETM test, ineffective doses of metergoline (MET; 5-HT (2A/2C) antagonist receptor) or sulpiride (SUL; dopaminergic receptor antagonist) were acutely administered together with the PEA. The locomotion of the rats was assessed in a circular arena following each drug treatment. Both PEA (8 and 16 mg/kg) and PAR (3 mg/kg) increased one-way escape latencies from the open arm of the ETM, indicating a panicolytic effect compared to the control group. MET, in higher doses (1, 2 or 3 mg/kg), produced a panicolytic effect in the ETM test, whereas SUL did not (10, 20 or 40 mg/kg). The panicolytic effect produced by PEA (8 mg/kg) was blocked by both MET (2 mg/kg) and SUL (20 mg/kg), whereas the panicolytic effect produced by PAR (3 mg/kg) was blocked only by MET (2 mg/kg). These results show that chronic treatment with PEA produces a panicolytic effect during the ETM test, and that the dopaminergic and the serotonergic neurotransmission systems are involved in this effect.

Acute and Chronic Toxicity of an Aqueous Fraction of the Stem Bark of Stryphnodendron adstringens (Barbatimão) in Rodents

Stryphnodendron adstringens has a high tannin content and is used as an antiseptic and antimicrobial and in the treatment of leucorrhea, gonorrhea, wound healing, and gastritis. The present study evaluated the toxic effects of the heptamer prodelphinidin (F2) from the stem bark of S. adstringens in rodents. In the acute toxicity test, the mice that received oral doses exhibited reversible effects, with an LD50 of 3.015u2009mg · kg−1. In the chronic toxicity test at 90 days, Wistar rats were treated with different doses of F2 (10, 100, and 200u2009mg · kg−1). In the biochemical, hematological, and histopathological examinations and open-field test, the different dose groups did not exhibit significant differences compared with controls. The present results indicate that F2 from the stem bark of S. adstringens caused no toxicity with acute and chronic oral treatment in rodents at the doses administered.