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

Prior experience with the elevated plus maze (EPM) increases the avoidance of rodents to the open arms and impairs the anxiolytic-like effects of benzodiazepines on the traditional behaviors evaluated upon re-exposure to the maze, a phenomenon known as one-trial tolerance. Risk assessment behaviors are also sensitive to benzodiazepines. During re-exposure to the maze, these behaviors reinstate the information-processing initiated during the first experience, and the detection of danger generates stronger open-arm avoidance. The present study investigated whether the benzodiazepine midazolam alters risk assessment behaviors and Fos protein distribution associated with test and retest sessions in the EPM. Naive or maze-experienced Wistar rats received either saline or midazolam (0.5 mg/kg i.p.) and were subjected to the EPM. Midazolam caused the usual effects on exploratory behavior, increasing exploratory activity of naive rats in the open arms and producing no effects on these conventional measures in rats re-exposed to the maze. Risk assessment behaviors, however, were sensitive to the benzodiazepine during both sessions, indicating anxiolytic-like effects of the drug in both conditions. Fos immunohistochemistry showed that midazolam injections were associated with a distinct pattern of action when administered before the test or retest session, and the anterior cingulate cortex, area 1 (Cg1), was the only structure targeted by the benzodiazepine in both situations. Bilateral infusions of midazolam into the Cg1 replicated the behavioral effects of the drug injected systemically, suggesting that this area is critically involved in the anxiolytic-like effects of benzodiazepines, although the behavioral strategy adopted by the animals appears to depend on the previous knowledge of the threatening environment. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Neural basis of anxiolytic effects of cannabidiol (CBD) in generalized social anxiety disorder: a preliminary report

Animal and human studies indicate that cannabidiol (CBD), a major constituent of cannabis, has anxiolytic properties. However, no study to date has investigated the effects of this compound on human pathological anxiety and its underlying brain mechanisms. The aim of the present study was to investigate this in patients with generalized social anxiety disorder (SAD) using functional neuroimaging. Regional cerebral blood flow (rCBF) at rest was measured twice using (99m)Tc-ECD SPECT in 10 treatment-naive patients with SAD. In the first session, subjects were given an oral dose of CBD (400 mg) or placebo, in a double-blind procedure. In the second session, the same procedure was performed using the drug that had not been administered in the previous session. Within-subject between-condition rCBF comparisons were performed using statistical parametric mapping. Relative to placebo, CBD was associated with significantly decreased subjective anxiety (p < 0.001), reduced ECD uptake in the left parahippocampal gyrus, hippocampus, and inferior temporal gyrus (p < 0.001, uncorrected), and increased ECD uptake in the right posterior cingulate gyrus (p < 0.001, uncorrected). These results suggest that CBD reduces anxiety in SAD and that this is related to its effects on activity in limbic and paralimbic brain areas.

Anxiolytic-like effect of noradrenaline microinjection into the dorsal periaqueductal gray of rats

The brain noradrenergic system has been implicated in the expression of defensive behaviors elicited by acute stress. The dorsal periaqueductal gray area (dPAG) is a key structure involved in the behavioral and cardiovascular responses elicited by fear and anxiety situations. Although there are noradrenergic terminals in the dPAG, few studies have investigated the role of noradrenaline (NA) in the dPAG on anxiety modulation. The aim of this study was to evaluate the effect of NA microinjection into the dPAG of rats subjected to two animal models of anxiety, the elevated plus-maze and the Vogel conflict test. Male Wistar rats implanted with a guide cannula aimed at the dPAG received microinjections of NA (3, 15, or 45 nmol/0.05 mu l) or artificial cerebral spinal fluid into the dPAG immediately before being exposed to the elevated plus-maze or the Vogel conflict test. NA increased the exploration of the open arms and the number of enclosed arm entries in the elevated plus-maze. The increase in open arm exploration remained significant after being subjected to an analysis of covariance using the latter variable as covariate. Moreover, the NA microinjection into the dPAG did not increase general exploratory activity of animals subjected to the open-field test, indicating that the increase in open arm exploration cannot be attributed to a nonspecific increase in exploratory activity. In the Vogel test, the NA microinjection into the dPAG increased the number of punished licks without changing the number of nonpunished licks or interfering with the tail-flick test. The results, therefore, indicate that the NA microinjection into the dPAG produces anxiolytic-like effects, suggesting its possible involvement in the anxiety modulation. Behavioural Pharmacology 20:252-259 (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

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.

Activation of cannabinoid CB, receptors in the dorsolateral periaqueductal gray induces anxiolytic effects in rats submitted to the Vogel conflict test

There are contradictory results concerning the effects of systemic injections of cannabinoid agonists in anxiety-induced behavioral changes. Direct drug administration into brain structures related to defensive responses could help to clarify the role of cannabinoids in these changes. Activation of cannabinoid CB, receptors in the dorsolateral periaqueductal gray induces anxiolytic-like effects in the elevated plus maze. The aim of this work was to verify if facilitation of endocannabinoid-mediated neurotransmission in this region would also produce anxiolytic-like effects in another model of anxiety, the Vogel conflict test. Male Wistar rats (n = 5-9/group) with cannulae aimed at the dorsolateral periaqueductal gray were water deprived for 24 h and pre-exposed to the apparatus where they were allowed to drink for 3 min. After another 24 h-period of water deprivation, they received the microinjections and, 10 min later, were placed into the experimental box. in this box an electrical shock (0.5 nnA, 2 s) was delivered in the spout of a drinking bottle at every twenty licks. The animals received a first microinjection of vehicle (0.2 mu l) or AM251 (a cannabinoid CB1 receptor antagonist; 100 pmol) followed, 5 min later, by a second microinjection of vehicle, anandamide (an endocannabinoid, 5 pmol), AM404 (an inhibitor of anandamide uptake, 50 pmol) or URB597 (an inhibitor of Fatty Acid Amide Hydrolase, 0.01 or 0.1 nmol). Anandamide, AM404 and URB597 (0.01 nmol) increased the total number of punished licks. These effects were prevented by AM251. The results give further support to the proposal that facilitation of CB1 receptor-mediated endocannabinoid neurotransmission in the dorsolateral periaqueductal gray modulates defensive responses. (C) 2008 Elsevier B.V. All rights reserved.

Anxiolytic-like effects induced by acute reversible inactivation of the bed nucleus of stria terminalis

There is conflicting evidence concerning the role of the bed nucleus of the stria terminalis (BNST) in fear and anxiety-elicited behavior. Most of the studies investigating this role, however, employed irreversible lesions of this nucleus. The objective of the present study was to investigate the effects of an acute and reversible inactivation of the BNST in rats submitted to the Vogel conflict test (VCT) and contextual fear conditioning, two widely employed animal models that are responsive to prototypal anxiolytic drugs. Male Wistar rats were submitted to stereotaxic surgery to bilaterally implant cannulae into the BNST. Ten minutes before the test they received bilateral microinjections of cobalt chloride (COCl(2)) (1 mM/100 nL), a nonselective synapse blocker. COCl(2) produced anxiolytic-like effects in tests, increasing the number of punished licks in the VCT and decreasing freezing behavior and the increase in mean arterial blood pressure and heart rate of animals re-exposed to the context where they had received electrical foot shocks 24 h before. The results indicate that the BNST is engaged in behavioral responses elicited by punished stimuli and aversively conditioned contexts, reinforcing its proposed role in anxiety. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Nitric oxide-mediated anxiolytic-like and antidepressant-like effects in animal models of anxiety and depression

The effects of microinjection of the nitric oxide (NO) precursor L-arginine (L-Arg), the NO synthase (NOS) inhibitors N-methyl-L-arginine (L-NAME) and 7-nitroindazole (7-NI), and the cyclic guanosine 3`,5`-monophosphate (cGMP) analog 8-Br-cGMP into the dorsal raphe nucleus (DRN) were assessed in rats using the elevated plus maze (EPM) and the forced swim test (FST). L-Arg (100 and 200 nmol) produced an anxiolytic-like effect in the EPM. 8-Br-cGMP (25 and 50 nmol) dose-dependently increased locomotor activity. In the FST, antidepressant-like effects were produced by L-Arg (50 and 100 nmol) and 8-Br-cGMP (12.5 and 25 nmol). Dual effects were observed with NOS inhibitors L-NAME and 7-NI in both the EPM and FST. While low doses of L-NAME (25 nmol) or 7-NI (1 nmol) induced a selective increase in EPM open arm exploration and a decrease in immobility time in the FST, high doses (L-NAME 400 nmol, 7-NI 10 nmol) decreased locomotor activity. These results show that interference with NO-mediated neurotransmission in the DRN induced significant and complex motor and emotional effects. Further studies are needed to elucidate the mechanisms involved in these effects. (C) 2007 Elsevier Inc. All rights reserved.

The anxiolytic-like effects of cannabidiol injected into the bed nucleus of the stria terminalis are mediated by 5-HT1A receptors

Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa that induces anxiolytic-like effects in rodents and humans after systemic administration. Previous results from our group showed that CBD injection into the bed nucleus of the stria terminalis (BNST) attenuates conditioned aversive responses. The aim of this study was to further investigate the role of this region on the anxiolytic effects of the CBD. Moreover, considering that CBD can activate 5-HT1A receptors, we also verified a possible involvement of these receptors in those effects. Male Wistar rats received injections of CBD (15, 30, or 60 nmol) into the BNST and were exposed to the elevated plus-maze (EPM) or to the Vogel conflict test (VCT), two widely used animal models of anxiety. CBD increased open arms exploration in the EPM as well as the number of punished licks in the VCT, suggesting an anxiolytic-like effect. The drug did not change the number of entries into the enclosed arms of the EPM nor interfered with water consumption or nociceptive threshold, discarding potential confounding factors in the two tests. Moreover, pretreatment with the 5-HT1A receptor antagonist WAY100635 (0.37 nmol) blocked the effects of CBD in both models. These results give further support to the proposal that BNST is involved in the anxiolytic-like effects of CBD observed after systemic administration, probably by facilitating local 5-HT1A receptor-mediated neurotransmission.

Anxiolytic-like effects induced by blockade of transient receptor potential vanilloid type 1 (TRPV1) channels in the medial prefrontal cortex of rats

The endocannabinoid anandamide, in addition to activating cannabinoid type 1 receptors (CB1), may act as an agonist at transient receptor potential vanilloid type 1 (TRPV1) channels. In the periaqueductal gray, CB1 activation inhibits, whereas TRPV1 increases, anxiety-like behavior. In the medial prefrontal cortex (mPFC), another brain region related to defensive responses, CB1 activation induces anxiolytic-like effects. However, a possible involvement of TRPV1 is still unclear. In the present study, we tested the hypothesis that TRPV1 channel contributes to the modulation of anxiety-like behavior in the mPFC. Male Wistar rats (n = 5-7 per group) received microinjections of the TRPV1 antagonist capsazepine (1-60 nmol) in the ventral portion of the mPFC and were exposed to the elevated plus maze (EPM) or to the Vogel conflict test. Capsazepine increased exploration of open arms in the EPM as well as the number of punished licks in the Vogel conflict test, suggesting anxiolytic-like effects. No changes in the number of entries into the enclosed arms were observed in the EPM, indicating that there were no changes in motor activity. Moreover, capsazepine did not interfere with water consumption or nociceptive threshold, discarding potential confounding factors for the Vogel conflict test. These data suggest that TRPV1 in the ventral mPFC tonically inhibits anxiety-like behavior. TRPV1 could facilitate defensive responses opposing, therefore, the anxiolytic-like effects reported after local activation of CB1 receptors.

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.

The GABAergic system contributes to the anxiolytic-like effect of essential oil from Cymbopogon citratus (lemongrass)

Ethnopharmacological relevance: The essential oil (EO) from Cymbopogon citratus (DC) Stapf is reported to have a wide range of biological activities and is widely used in traditional medicine as an infusion or decoction. However, despite this widely use, there are few controlled studies confirming its biological activity in central nervous system. Materials and methods: The anxiolytic-like activity of the EO was investigated in light/dark box (LDB) and marble-burying test (MBT) and the antidepressant activity was investigated in forced-swimming test (FST) in mice. Flumazenil, a competitive antagonist of benzodiazepine binding and the selective 5-HT(1A) receptor antagonist WAY100635 was used in experimental procedures to determine the action mechanism of EO. To exclude any false positive results in experimental procedures, mice were submitted to the rota-rod test. We also quantified some neurotransmitters at specific brain regions after EO oral acute treatment. Results: The present work found anxiolytic-like activity of the EO at the dose of 10 mg/kg in a LDB. Flumazenil, but not WAY100635, was able to reverse the effect of the EO in the LOB, indicating that the EO activity occurs via the GABA(A) receptor-benzodiazepine complex. Only at higher doses did the EO potentiate diethyl-ether-induced sleeping time in mice. In the FST and MBT, EO showed no effect. Finally, the increase in time spent in the light chamber, demonstrated by concomitant treatment with ineffective doses of diazepam (DZP) and the EO, revealed a synergistic effect of the two compounds. The lack of activity after long-term treatment in the LDB test might be related to tolerance induction, even in the DZP-treated group. Furthermore, there were no significant differences between groups after either acute or repeated treatments with the EO in the rota-rod test. Neurochemical evaluation showed no amendments in neurotransmitter levels evaluated in cortex, striatum, pons, and hypothalamus. Conclusions: The results corroborate the use of Cymbopogon citratus in folk medicine and suggest that the anxiolytic-like effect of its EO is mediated by the GABA(A) receptor-benzodiazepine complex. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

5-HT1A autoreceptor modulation of locomotor activity induced by nitric oxide in the rat dorsal raphe nucleus

The dorsal raphe nucleus (DRN) is the origin of ascending serotonergic projections and is considered to be an important component of the brain circuit that mediates anxiety- and depression-related behaviors. A large fraction of DRN serotonin-positive neurons contain nitric oxide (NO). Disruption of NO-mediated neurotransmission in the DRN by NO synthase inhibitors produces anxiolytic- and antidepressant-like effects in rats and also induces nonspecific interference with locomotor activity. We investigated the involvement of the 5-HT1A autoreceptor in the locomotor effects induced by NO in the DRN of male Wistar rats (280-310 g, N = 9-10 per group). The NO donor 3-morpholinosylnomine hydrochloride (SIN-1, 150, and 300 nmol) and the NO scavenger S-3-carboxy-4-hydroxyphenylglycine (carboxy-PTIO, 0.1-3.0 nmol) were injected into the DRN of rats immediately before they were exposed to the open field for 10 min. To evaluate the involvement of the 5-HT1A receptor and the N-methyl-D-aspartate (NMDA) glutamate receptor in the locomotor effects of NO, animals were pretreated with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 8 nmol), the 5-HT1A receptor antagonist N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY-100635, 0.37 nmol), and the NMDA receptor antagonist DL-2-amino-7-phosphonoheptanoic acid (AP7, 1 nmol), followed by microinjection of SIN-1 into the DRN. SIN-1 increased the distance traveled (mean ± SEM) in the open-field test (4431 ± 306.1 cm; F7,63 = 2.44, P = 0.028) and this effect was blocked by previous 8-OH-DPAT (2885 ± 490.4 cm) or AP7 (3335 ± 283.5 cm) administration (P < 0.05, Duncan test). These results indicate that 5-HT1A receptor activation and/or facilitation of glutamate neurotransmission can modulate the locomotor effects induced by NO in the DRN.

Dose-response effects of systemic anandamide administration in mice sequentially submitted to the open field and elevated plus-maze tests

The endocannabinoid system is involved in the control of many physiological functions, including the control of emotional states. In rodents, previous exposure to an open field increases the anxiety-like behavior in the elevated plus-maze. Anxiolytic-like effects of pharmacological compounds that increase endocannabinoid levels have been well documented. However, these effects are more evident in animals with high anxiety levels. Several studies have described characteristic inverted U-shaped dose-response effects of drugs that modulate the endocannabinoid levels. However, there are no studies showing the effects of different doses of exogenous anandamide, an endocannabinoid, in animal models of anxiety. Thus, in the present study, we determined the dose-response effects of exogenous anandamide at doses of 0.01, 0.1, and 1.0 mg/kg in C57BL/6 mice (N = 10/group) sequentially submitted to the open field and elevated plus-maze. Anandamide was diluted in 0.9% saline, ethyl alcohol, Emulphor® (18:1:1) and administered ip (0.1 mL/10 g body weight); control animals received the same volume of anandamide vehicle. Anandamide at the dose of 0.1 mg/kg (but not of 0.01 or 1 mg/kg) increased (P < 0.05) the time spent and the distance covered in the central zone of the open field, as well as the exploration of the open arms of the elevated plus-maze. Thus, exogenous anandamide, like pharmacological compounds that increase endocannabinoid levels, promoted a characteristic inverted U-shaped dose-response effect in animal models of anxiety. Furthermore, anandamide (0.1 mg/kg) induced an anxiolytic-like effect in the elevated plus-maze (P < 0.05) after exposing the animals to the open field test.

Facilitation of 5-HT(1A)-mediated neurotransmission in dorsal periaqueductal grey matter accounts for the panicolytic-like effect of chronic fluoxetine

Chronic administration of antidepressants such as fluoxetine and imipramine increases the responsiveness of 5-HT(1A) receptors in dorsal periaqueductal grey matter (DPAG), a midbrain area consistently implicated in the pathogenesis of panic disorder. This effect has been related to the clinically relevant anti-panic action of these drugs. In this study we determined whether long-term administration of fluoxetine also affects 5-HT efflux in DPAG. As a comparison, the effect of chronic treatment with the anxiolytic 5-HT(1A) receptor agonist buspirone on DPAG 5-HT levels was assessed. We also investigated whether the inhibitory effect of chronic fluoxetine on escape behaviour in the rat elevated T-maze, considered as a panicolytic-like effect, is counteracted by intra-DPAG injection of the 5-HT(1A) receptor antagonist WAY 100635. Male Wistar rats were treated (1 or 21 d, i.p.) with fluoxetine, buspirone or vehicle, once daily. After treatment, 5-HT in DPAG was measured by in-vivo microdialysis coupled to HPLC. In another study, rats treated (21 d, i.p.) with either fluoxetine or vehicle also received intra-DPAG injection of WAY 100635 or saline 10 min before being tested in the elevated T-maze. Chronic, but not acute, administration of fluoxetine significantly raised extracellular levels of 5-HT in DPAG. Long-term treatment with buspirone was ineffective. In the elevated T-maze, intra-DPAG injection of WAY 100635 fully blocked the anti-escape effect of chronic administration of fluoxetine. Therefore, chronic fluoxetine facilitates 5-HT(1A)-mediated neurotransmission within DPAG and this effect accounts for the panicolytic-like effect of this antidepressant in the elevated T-maze.

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.