Anxiolytic effect of methylene blue microinjected into the dorsal periaqueductal gray matter

The dorsal periaqueductal gray (DPAG) has been implicated in the behavioral and autonomic expression of defensive reactions. Several results suggest that, along with GABA, glutamate and serotonin, nitric oxide (NO) may play a role in defense reactions mediated by this region. To further investigate this possibility we microinjected methylene blue (MB; 10, 30 or 100 nmol/0.5 µl) into the DPAG of rats submitted to the elevated plus-maze test, an animal model of anxiety. MB has been used as an inhibitor of soluble guanylate cyclase (sGC) to demonstrate cGMP-mediated processes, and there is evidence that NO may exert its biological effects by binding to the heme part of guanylate cyclase, causing an increase in cGMP levels. The results showed that MB (30 nmol) significantly increased the percent of time spent in the open arms (saline = 11.57 ± 1.54, MB = 18.5 ± 2.45, P<0.05) and tended to do the same with the percentage of open arm entries (saline = 25.8 ± 1.97, MB = 33.77 ± 3.07, P<0.10), but did not change the number of enclosed arm entries. The dose-response curve, however, had an inverted U shape. These results indicate that MB, within a limited dose range, has anxiolytic properties when microinjected into the DPAG.

The inactivation of the basolateral nucleus of the rat amygdala has an anxiolytic effect in the elevated T-maze and light/dark transition tests

Pharmacological evidence indicates that the basolateral nucleus of the amygdala (BLA) is involved in the mediation of inhibitory avoidance but not of escape behavior in the elevated T-maze test. These defensive responses have been associated with generalized anxiety disorder (GAD) and panic disorder, respectively. In the present study, we determined whether the BLA plays a differential role in the control of inhibitory avoidance and escape responses in the elevated T-maze. Male Wistar rats (250-280 g, N = 9-10 in each treatment group) were pre-exposed to one of the open arms of the maze for 30 min and 24 h later tested in the model after inactivation of the BLA by a local injection of the GABA A receptor agonist muscimol (8 nmol in 0.2 µL). It has been shown that a prior forced exposure to one of the open arms of the maze, by shortening latencies to withdrawal from the open arm during the test, improves the escape task as a behavioral index of panic. The effects of muscimol in the elevated T-maze were compared to those caused by this GABA agonist in the avoidance reaction generated in the light/dark transition test. This defensive behavior has also been associated with GAD. In the elevated T-maze, intra-BLA injection of muscimol impaired inhibitory avoidance (control: 187.70 ± 14.90 s, muscimol: 37.10 ± 2.63 s), indicating an anxiolytic effect, without interfering with escape performance. The drug also showed an anxiolytic effect in the light/dark transition test as indicated by the increase in the time spent in the lighted compartment (control: 23.50 ± 2.45 s, muscimol: 47.30 ± 4.48 s). The present findings point to involvement of the BLA in the modulation of defensive responses that have been associated with GAD.

Concurrent nociceptive stimulation impairs the anxiolytic effect of midazolam injected into the periaqueductal gray in mice

This study investigated whether the opportunity to avoid or escape the open arms of an elevated plus-maze (EPM) affects the antinociceptive response observed when mice are subjected to open arm confinement. Furthermore, in order to better characterize the relationship between emotion and antinociception in the EPM, we examined the behavioral effects of midazolam injection into the midbrain periaqueductal gray matter (PAG). As our main aim was to evaluate the relevance of different levels of approach-avoid conflict (i.e. The presence of open and closed arms) to maze-induced antinociception, mice were exposed to one of three types of EPM-a standard EPM (sEPM), an open EPM (oEPM: four open arms) or, as a control condition, an enclosed EPM (eEPM: four enclosed arms). Nociception was assessed using the formalin test. Twenty minutes after formalin injection (50 mu l, 2.5% formalin) into the dorsal right hind paw, mice received an intra-PAG injection of saline or midazolam (10-20 nmol). Five minutes later, they were individually exposed to one of the mazes for 10 min (25-35 min after formalin injection). Videotapes of the test sessions were scored for a variety of behavioral measures including time spent licking the formalin-injected paw. To examine whether the effects of midazolam on anxiety-like behavior may have been influenced by concurrent nociceptive stimulation (i.e. formalin pretreatment), naive mice were submitted to a similar procedure to that described above for the sEPM test but without formalin pretreatment. Results showed that mice exposed to the oEPM spent significantly less time licking the injected paw compared to groups exposed to either the sEPM or eEPM. Although exposure to the sEPM induced anxiety-like behaviors (i.e. open arm avoidance), it did not result in antinociception. Intra-PAG infusions of midazolam failed to block oEPM-induced antinociception or to alter sEPM-induced anxiety in mice that had received formalin injection. However, under normal test conditions (i.e. in the absence of formalin-induced nociceptive stimulation), intra-PAG midazolam produced clear anti-anxiety effects in mice exposed to the sEPM. Findings are discussed in terms of different emotional states induced by the oEPM and sEPM and the influence of concurrent nociceptive stimulation on the anti-anxiety effect of intra-PAG midazolam. (c) 2005 Elsevier B.V. All rights reserved.

Anxiolytic effect of Rubus brasilensis in rats and mice

The aim of the present work was to investigate if infuse and ethanolic extracts (aqueous, butanolic and wax fractions) of Rubus brasiliensis Martius (Rosaceae) induce anxiolytic effect. The extracts were administered to male Wistar rats and Swiss mice per oral route, at 50, 100 and 150 mg/kg, 30 min before the behavioral evaluation in the elevated plus maze (EPM). Both infuse and wax ethanolic fraction at the dosage 150 mg/kg, vo, increased the number and the percentage of open arm entries of rats and mice. The aqueous and butanolic fractions, obtained from ethanolic extract, failed to induce anxiolytic effect. The treatment of mice with flumazenil (Ro 15-1788), 1.5, 2.0 and 2.5 mg/kg, i.p., 15-min before the administration of infuse or wax fraction, 150 mg/kg, vo, blocked the infuse or wax fraction-induced anxiolytic effect. The LD50 for the wax fraction was 1000 mg/kg. In conclusion, the infuse and wax ethanolic fraction of R. brasiliensis present anxiolytic effect in rats and mice. In addition, it is suggested that the anxiolytic effect may be attributed at least to one liposoluble principle with low acute toxicity which may be acting as an agonist on GABA(A)-benzodiazepine receptor complex. (C) 1998 Elsevier B.V. Ireland Ltd. All rights reserved.

Involvement of GABA(A)-benzodiazepine receptor in the anxiolytic effect induced by hexanic fraction of Rubus brasiliensis

To investigate the ability of hexanic ethanolic fraction of Rubus brasiliensis Martius (Roseceae), to induce anxiolytic effect and also the possible involvement of the GABA(A)-benzodiazepine receptor complex, male Wistar rats and Swiss mice behaviour were tested in the elevated plus maze (EPM). All the doses of the extract, 50, 100 and 150 mg/kg, administered per gavage (vo), 30 min before the behavioural evaluation, induced an anxiolytic effect expressed by: increased number of entries in and time spent in the open arms and percentage of open arm entries: and decreased number of entries and time spent in the closed arms. The treatment of mice with flumazenil (Ro 15-1788), 0.5, 1.0 and 1.5 mg/kg, i.p., 15-min before the administration of hexanic fraction, 100 mg/kg, vo, blocked the hexanic fraction-induced anxiolytic effect. The LD50 for the hexanic fraction was 1512 mg/kg. In conclusion, it was shown that the hexanic fraction of R. brasiliensis induced an anxiolytic effect in rats and mice. This effect can be attributed to a liposoluble principle with low toxicity which may be acting as an agonist on GABA(A)-benzodiazepine receptor complex. (C) 1998 Elsevier B.V. Ireland Ltd. All rights reserved.

Anxiolytic effect of estradiol in the median raphe nucleus mediated by 5-HT1A receptors

Estrogen deficiency has been associated with stress, anxiety and depression. Estrogen receptors have been identified in the median raphe nucleus (MRN). This structure is the main source of serotonergic projections to the hippocampus, a forebrain area implicated in the regulation of defensive responses and in the resistance to chronic stress. There is evidence showing that estrogen modulates 5-HT1A receptor functions. In the MRN, somatodendritic 5-HT1A receptors control the activity of serotonergic neurones by negative feedback. The present study evaluated the effect of intra-MRN injection of estradiol benzoate (EB) (600 or 1200 ng/0.2 mu l) on the performance of ovariectomised rats submitted to the elevated plus-maze test of anxiety and to the open-field test. Additionally, the same effect was evaluated with a previous intra-MRN injection of WAY 100635 (100 ng/0.2 mu l), an antagonist of 5-HT1A receptors. The results showed that both doses of EB increased the percentage of entries and the percentage of time spent into the open arms, suggestive of an anxiolytic effect. The highest dose of the drug also increased the number of entries into the enclosed arm and locomotion in the open field, indicating a stimulatory motor effect. WAY 100635 antagonised the effect of estradiol in the elevated plus-maze and in the open-field. The results show that estrogen receptors of the MRN are implicated in the regulation of anxiety-related behaviour. The results also support claims that the effect of estrogen involves a change in 5-HT1A receptor function. (C) 2005 Elsevier B.V. All rights reserved.

Stimulation of 5-HT1A or 5-HT2A receptors in the ventrolateral periaqueductal gray causes anxiolytic-, but not panicolytic-like effect in rats

Evidences from studies using electrical or chemical stimulation of the midbrain periaqueductal gray (PAG) suggest that whereas the dorsal PAG is critical for the regulation of panic-related defensive behaviors, the ventrolateral PAG (vlPAG) modulates generalized anxiety-related responses. In the present study we evaluated whether the activation of 5-HT1A and 5-HT2A/2C receptors in the ventrolateral column of the periaqueductal gray (vlPAG) causes differential effects on an anxiety- and a panic-related defensive behavior, respectively, inhibitory avoidance and escape, in male Wistar rats submitted to the elevated T-maze. Our results showed that intra-vlPAG injection of the endogenous agonist serotonin, the 5-HT1A/7 agonist 8-OH-DPAT or 5-HT2A/2C agonist DOI impaired the acquisition of inhibitory avoidance, without interfering with escape performance. The same selective anxiolytic effect was also observed after local administration of the benzodiazepine receptor agonist midazolam. Moreover, as shown by the results of antagonism studies, 5-HT2A receptors are recruited for the anxiolysis caused by serotonin and DOI. while 5-HT1A receptors account for the effect of 8-OH-DPAT. In conclusion, our data show that the activation of 5-HT1A and 5-HT2A receptors in the vlPAG affects defensive responses related to generalized anxiety, but not panic disorder. (C) 2008 Elsevier B.V. All rights reserved.

Anxiolytic-like effect of way-100635 microinfusions into the median (but not dorsal) raphe nucleus in mice exposed to the plus-maze: influence of prior test experience

Studies in several laboratories have confirmed the anxiolytic potential of a wide range of 5-HT1A receptor antagonists in rats and mice, with recent evidence pointing to a postsynaptic site of action in the ventral hippocampus. It would, therefore, be predicted that blockade of 5-HT1A somatodendritic autoreceptors in the midbrain raphe nuclei should produce anxiogenic-like effects. To test this hypothesis, we investigated the effects of WAY-100635 microinfusions (0, 1.0 or 3.0 mug in 0.1 mul) into the dorsal (DRN) or median (MRN) raphe nuclei on behaviours displayed by male Swiss-Webster mice in the elevated plus-maze. As this test is sensitive to prior experience. The effects of intra-raphe infusions were examined both in maze-naive and maze-experienced subjects. Sessions, were videotaped and subsequently scored for conventional indices of anxiety (open arm avoidance) and locomotor activity (closed arm entries), as well as a range of ethological measures (e.g. risk assessment). In maze-naive mice, intra-MRN (but not intra-DRN) infusions of WAY-100635 (3.0 mug) increased open arm exploration and reduced risk assessment. Importantly, these effects could not be attributed to a general reduction in locomotor activity. A similar, though somewhat weaker, pattern of behavioural change was observed in maze-experienced animals. This unexpected anxiolytic effect of 5-HT1A autoreceptor blockade in the MRN cannot be accounted fur by a disinhibition of 5-HT release in forebrain targets (e.g. hippocampus and amygdala), where stimulation of postsynaptic 5-HT1A receptors enhances anxiety-like responses. However, as the MRN also projects to the periaqueductal gray matter (PAG), an area known to be sensitive to the anti-aversive effects or 5-HT, it is argued that present results may reflect increased 5-HT release at this crucial midbrain locus within the neural circuitry of defense. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Effect of early malnutrition and environmental stimulation in the performance of rats in the elevated plus maze

Malnourished rats since birth (mothers fed on 6% of protein) or controlled ones (16% of protein), half of each group received environmental stimulation (ES) from the age of 0-35th day, were studied. The performance in the elevated plus maze (EPM) was assessed on the last day. ES increased time spent and also the entries into open arms of EPM, but malnourished non-stimulated rats visited more segments near the central area than the distant ones. Data suggests an anxiolytic effect of ES which is less evident in malnourished rats. (C) 2009 Elsevier B.V. All rights reserved.

Effect of estradiol benzoate microinjection into the median raphe nucleus on contextual conditioning

Estrogen deficiency has been associated with stress, anxiety and depression. Estrogen receptors have been identified in the median raphe nucleus (MRN). This structure is the main source of serotonergic projections to the hippocampus, a forebrain area implicated in the regulation of defensive responses and in the resistance to chronic stress. There is reported evidence indicating that estrogen modulates 5-HT(1A) receptor function. In the MRN, somatodendritic 5-HT(1A) receptors control the activity of serotonergic neurones by negative feedback. The present study has evaluated the effect of intra-MRN injection of estradiol benzoate (EB, 600 or 1200 ng/0.2 mu l) on the performance of ovariectormized rats submitted to contextual conditioning. Additionally, the same treatment was given after intra-MRN injection of Way 100635 (100 ng/0.2 mu l). a 5-HT(1A) receptor antagonist. Both doses of EB decreased freezing and increased rearing, indicating an anxiolytic effect. Pretreatment with Way 100635 antagonized the anxiolytic effect of estradiol. On the basis of these results, it may be suggested that estrogens modulate anxiety by acting on 5-HT(1A) receptors localized in the MRN. (C) 2009 Elsevier B.V. All rights reserved.

Cannabidiol inhibitory effect on marble-burying behaviour: involvement of CB1 receptors

Cannabidiol (CBD) is a major nonpsychotomimetic component of Cannabis sativa that has been shown to have an anxiolytic effect in human and animal models. Earlier studies suggest that these effects involve facilitation of serotonin, a neurotransmitter that has also been related to obsessive-compulsive disorder. On the basis of this evidence, this study investigated the effects of CBD in C57BL/6J mice submitted to the marble-burying test (MBT), an animal model proposed to reflect compulsive behaviour. CBD (15, 30 and 60 mg/kg) induced a significant decrease in the number of buried marbles compared with controls (34, 41 and 48%, respectively). A similar, although larger, decrease was also found after the serotonin selective reuptake inhibitor paroxetine (10 mg/kg, 77% decrease) and the benzodiazepine diazepam (2.5 mg/kg, 84% decrease). The effect of CBD (30 mg/kg) was still significant after 7 days of daily repeated administration, whereas the effect of diazepam disappeared. Pretreatment with WAY100635 (3 mg/kg), a 5HT1A receptor antagonist, prevented the effects of paroxetine but failed to alter those of CBD. These latter effects, however, were prevented by pretreatment with the CB1 receptor antagonist AM251 (1 mg/kg). These results indicated that CBD and paroxetine decrease the number of buried marbles in the MBT through distinct pharmacological mechanisms. They also suggest a potential role of drugs acting on the cannabinoid system in modulating compulsive behaviour. Behavioural Pharmacology 21: 353-358 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Anxiolytic and panicolytic effects of escitalopram in the elevated T-maze

Escitalopram is a highly selective inhibitor of serotonin re-uptake that is used to treat anxiety disorders. In the present study, we investigated the effects of acute, sub-chronic ( 14 days) and chronic ( 21 days) administration of escitalopram ( 2, 4 and 8 mg/kg, P0) on the performance of rats in the elevated T-maze. For comparison, imipramine ( 15 mg/ kg, P0) was also studied. The apparatus is made of three elevated arms of equal dimension, one enclosed transversal to the two open arms. Inhibitory avoidance of the open arms, trained in the enclosed arm, has been related to generalised anxiety disorder, while one-way escape from one open arm, to panic disorder. After acute administration, the three doses of escitalopram impaired avoidance ( anxiolytic effect), while imipramine was ineffective. Escape was unaffected by either drug. With sub-chronic administration, both drugs were ineffective on either avoidance or escape. After chronic treatment, avoidance was impaired by imipramine and by the two highest doses of escitalopram. In addition, escape was impaired (panicolytic effect) by imipramine and by the highest dose of escitalopram. Locomotion measured in a square arena was increased by the three doses of escitalopram, given chronically. Therefore, both imipramine and escitalopram had anxiolytic and panicolytic-like effects after chronic administration, but acutely only escitalopram decreased anxiety. Since no such effect was observed following subchronic administration, it is likely that the mechanisms of the early and late anxiolytic actions of escitalopram are different.

Effect of estradiol benzoate microinjection into the median raphe nucleus on contextual conditioning

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of electrolytic and neurotoxic lesions of the median raphe nucleus on anxiety and stress

To study the role played by 5-HT mechanisms of the MRN, behavioural and physiological parameters were presently measured in rats having either electrolytic or 5,7-dihydroxytryptamine (5,7-DHT) lesion of the MRN made 7 days before testing. Half the animals were submitted to 2-h restraint 24 h before the test. In the elevated plus-maze, the electrolytic lesion increased the percentage of open-arm entries and of time spent on open arms - an anxiolytic effect - in both restrained and nonrestrained rats. The neurotoxic lesion had a similar effect, but only on restrained rats. Restraint had anxiogenic effect. The electrolytic lesion increased transitions between the light and dark compartments and the time spent in the bright compartment of the light-dark box in both restrained and nonrestrained rats. The neurotoxic lesion only increased bright time in restrained rats. The incidence, number and size of gastric ulcers were increased by either the electrolytic or the neurotoxic lesion in both restrained and nonrestrained animals. Both types of lesion depleted 5-HT in the hippocampus in restrained and nonrestrained rats. Restraint increased 5-HT levels. These results implicate 5-HT mechanisms of the median raphe nucleus in the regulation of anxiety and in the genesis of gastric stress ulcers. (C) 2001 Elsevier B.V. All rights reserved.

Anxiolytic-like effects produced by bilateral lesion of the periaqueductal gray in mice: Influence of concurrent nociceptive stimulation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)