Modulation of anxiety-like behaviour by Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels located in the dorsolateral periaqueductal gray

The endocannabinoid anandamide is a possible agonist at the Transient Receptor Potential Vanilloid Type 1 (TRPV1) channel, in addition to its agonist activity at cannabinoid type 1 (CB1) receptor. In the midbrain dorsolateral periaqueductal gray (dlPAC) our previous data showed that CB1 activation induces anxiolytic-like effects. However, the rote of TRPV1 has remained unclear. Thus, in the present study we tested the hypothesis that this channel would contribute to the modulation of anxiety-like behaviour in the dlPAG. Mate Wistar rats received local injections of the TRPV1 antagonist capsazepine (10-60 nmol) and were submitted to the elevated plus-maze (EPM) and to the Vogel test. In addition, animals received local injections of capsaicin (0.01-1nmol), a TRPV1 agonist, and were tested in the same models. In accordance with our hypothesis, capsazepine produced anxiolytic-like effects both in the EPM and in the Vogel test. Capsaicin mimicked these results, which might be attributed to its ability to quickly desensitize the channel. Altogether, our data suggest that, while CB1 receptors seem to inhibit aversive responses in the dlPAG, TRPV1 could facilitate them. Thus, CB1 and TRPV1 may have opposite functions in modulating anxiety-like behaviour in this region. (C) 2008 Elsevier B.V. and ECNP. 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.

Role of IL-18 in overt pain-like behaviour in mice

There are evidences that targeting IL-18 might be beneficial to inhibit inflammatory symptoms, including hypernociception (decrease in nociceptive threshold). The mechanism of IL-18 mechanical hypernociception depends on endothelin in rats and mice. However, the role of IL-18 in overt pain-like behaviour remains undetermined. Therefore, we addressed the role of IL-18 in writhing response induced by intraperitoneal (i.p.) injection of phenyl-p-benzoquinone (PBQ) and acetic acid in mice. Firstly, it was detected that PBQ and acetic acid i.p. injection induced a dose-dependent number of writhes in Balb/c mice. Subsequently, it was observed that the PBQ- but not the acetic acid-induced writhes were diminished in IL-18 deficient ((-/-)) mice. Therefore, considering that IFN-gamma, endothelin and prostanoids mediate IL-18-induced mechanical hypernociception, we also investigated the role of these mediators in the same model of writhing response in which IL-18 participates. It was noticed that PBQ-induced writhes were diminished in IFN-gamma(-/-) mice and by the treatment with bosentan (mixed enclothelin ETA/ETB receptor antagonist), BQ 123 (cyclo[DTrp-DAsp-Pro-DVal-Leu], selective enclothelin ETA receptor antagonist), BQ 788 (N-cys-2,6-dimethylpiperidinocarbonyl-L-methylleucyl-D-1 -methoxycarboyl-D-norleucine, selective endothelin ETB receptor antagonist) or indomethacin (cycloxigenase inhibitor). Thus, IL-18, IFN-gamma, endothelin acting on endothelin ETA and ETB receptors, and prostanoids mediate PBQ-induced writhing response in mice. To conclude, these results further advance the understanding of the physiopathology of overt pain-like behaviour, and suggest for the first time a role for IL-18 in writhing response in mice. (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.

Acute reversible inactivation of the ventral medial prefrontal cortex induces antidepressant-like effects in rats

The ventral medial prefrontal cortex (vMPFC) has direct connections to subcortical, diencephalic and brainstem structures that have been closely related to depression. However, studies aimed at investigating the role of the vMPFC in the neurobiology of depression have produced contradictory results. Moreover, the precise involvement of vMPFC anatomic subdivisions, the prelimbic(PL) and the infralimbic (IL) cortices, in regulating depressive-like behavior have been poorly investigated. The forced swimming test (FST) is a widely employed animal model aimed at detecting antidepressant-like effects. Therefore, to further investigate a possible involvement of the vMFPC in depressive-like behavior, rats bilaterally implanted with cannulae aimed at the PL or IL prefrontal cortices were submitted to 15 min of forced swimming (pre-test) followed, 24 h later, by a 5-min swimming session (test), where immobility time was registered. Synaptic transmission in these regions was temporarily inhibited using local microinjection of cobalt chloride at different periods of the experimental procedure (before or after the pre-test or before the test). PL inactivation decreased immobility time independently of the time of the injection. In the IL inactivation induced a significant antidepressant-like effect when performed immediately before the pre-test or before the test, but not after the pre-test. These results suggest that activation of the vMPFC is important for the behavioral changes observed in rats submitted to the FST. They further indicate that, although both the PL and IL cortices are involved in these effects, they may play different roles. (C) 2010 Elsevier B.V. All rights reserved.

Dorsal raphe nucleus regulation of a panic-like defensive behavior evoked by chemical stimulation of the rat dorsal periaqueductal gray matter

Electrical or chemical stimulation of the dorsal periaqueductal gray matter (DPAG) evokes escape, a defensive behavior that has been related to panic attacks. Injection of 5-HT(1A) or 5-HT(2A) receptor agonists into this midbrain area inhibits this response. It has been proposed that the impairment of 5-HT mechanisms controlling escape at the level of the DPAG may underlie the susceptibility to panic attacks that characterizes the panic disorder. In this study we evaluated the effects of the pharmacological manipulation of the dorsal raphe nucleus (DRN), which are the main source of 5-HT input to the DPAG, on the escape response evoked in rats by the intra-DPAG injection of the nitric oxide donor SIN-1. The results showed that DRN administration of the 5-HT(1A) receptor agonist 8-OH-DPAT which inhibits the activity of 5-HT neurons favored the expression of escape induced by SIN-1. Intra-DRN injection of the excitatory amino acid kainic acid or the 5-HT(1A) receptor antagonist WAY-100635 did not change escape expression. However, both compounds fully blocked the escape reaction generated by intra-DPAG injection of the excitatory amino acid D,L-homocysteic acid (DLH). Overall, the results indicate that 5-HT neurons in the DRN exert a bidirectional control upon escape behavior generated by the DPAG. Taking into account the effect of WAY-100635 on DLH-induced escape, they also strengthen the view that DRN 5-HT(1A) autoreceptors are under tonic inhibitory influence by 5-HT. (C) 2010 Elsevier B.V. All rights reserved.

Intra-dorsal periaqueductal gray administration of cannabidiol blocks panic-like response by activating 5-HT1A receptors

Activation of 5-HT1A receptors in the dorsal periaqueductal gray (dPAG) impairs escape behavior, suggesting a panicolytic-like effect. Cannabidiol (CBD), a major non-psychotomimetic compound present in Cannabis sativa, causes anxiolytic-like effects after intra-dPAG microinjections by activating 5-HT1A receptors. In the present work we tested the hypothesis that CBD could also impair escape responses evoked by two proposed animal models of panic: the elevated T-maze (ETM) and electric stimulation of dPAG. In experiment 1 male Wistar rats with a single cannula implanted in the dPAG received a microinjection of CBD or vehicle and, 10 min later, were submitted to the ETM and open field tests. In experiment 2 escape electrical threshold was measured in rats with chemitrodes implanted in the dPAG before and 10 min after CBD microinjection. In experiment 3 similar to experiment 2 except that the animals received a previous intra-dPAG administration of WAY-100635, a 5-HT1A receptor antagonist, before CBD treatment. In the ETM microinjection of CBD into the dPAG impaired inhibitory avoidance acquisition, an anxiolytic-like effect, and inhibited escape response, a panicolytic-like effect. The drug also increased escape electrical threshold, an effect that was prevented by WAY-100635. Together, the results suggest that CBD causes panicolytic effects in the dPAG by activating 5-HT1A receptors. (C) 2010 Elsevier B.V. All rights reserved.

Panicolytic-like effect of BDNF in the rat dorsal periaqueductal grey matter: the role of 5-HT and GABA

A wealth of evidence suggests a role for brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) in the aetiology of depression and in the mode of action of antidepressant drugs. Less clear is the involvement of this neurotrophin in other stress-related pathologies such as anxiety disorders. The dorsal periaqueductal grey matter (DPAG), a midbrain area rich in BDNF and TrkB receptor mRNAs and proteins, has been considered a key structure in the pathophysiology of panic disorder. In this study we investigated the effect of intra-DPAG injection of BDNF in a proposed animal model of panic: the escape response evoked by the electrical stimulation of the same midbrain area. To this end, the intensity of electrical current that needed to be applied to DPAG to evoke escape behaviour was measured before and after microinjection of BDNF. We also assessed whether 5-HT- or GABA-related mechanisms may account for the putative behavioural/autonomic effects of the neurotrophin. BDNF (0.05, 0.1, 0.2 ng) dose-dependently inhibited escape performance, suggesting a panicolytic-like effect. Local microinjection of K252a, an antagonist of TrkB receptors, or bicuculline, a GABA(A) receptor antagonist, blocked this effect. Intra-DPAG administration of WAY-100635 or ketanserin, respectively 5-HT(1A) and 5-HT(2A/2c) receptor antagonists, did not alter BDNF`s effects on escape. Bicuculline also blocked the inhibitory effect of BDNF on mean arterial pressure increase caused by electrical stimulation of DPAG. Therefore, in the DPAG, BDNF-TrkB signalling interacts with the GABAergic system to cause a panicolytic-like effect.

FACILITATION OF ENDOCANNABINOID EFFECTS IN THE VENTRAL HIPPOCAMPUS MODULATES ANXIETY-LIKE BEHAVIORS DEPENDING ON PREVIOUS STRESS EXPERIENCE

Although several pieces of evidence indicate that the endocannabinoid system modulates anxiety-like behaviors and stress adaptation, few studies have investigated the brain sites of these effects. The ventral hippocampus (VHC) has been related to anxiety behaviors and has a high expression of cannabinoid-1 (CBI) receptors. Moreover, endocannabinoid signaling in the hippocampus is proposed to regulate stress adaptation. In the present study we investigated the role of previous stressful experience on the effects of AM404, an anandamide uptake inhibitor, microinjected into the VHC of rats submitted to the elevated plus maze (EPM), a widely used animal model of anxiety. Stressed animals were forced restrained for two h 24 h before the test. AM404 (5-50 pmol) microinjection promoted an anxiogenic-like effect in non-stressed rats but decreased anxiety in stressed animals. AM251 (0.01 to 1000 pmol), a CBI receptor antagonist, failed to change behavior in the EPM over a wide dose range but prevented the effects of AM404. Anxiolytic-like effects of AM404 (5 pmol) intra-VHC injection were also observed in the Vogel conflict test (VCT), another model of anxiety that involves previous exposure to stressful situations (48 h of water deprivation). These results suggest that facilitation of endocannabinoid system neurotransmission in the ventral hippocampus modulates anxiety-like behaviors and that this effect depends on previous stress experience. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Antidepressant-like effects of cannabidiol in mice: possible involvement of 5-HT(1A) receptors

Background and purpose: Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa that induces anxiolytic- and antipsychotic-like effects in animal models. Effects of CBD may be mediated by the activation of 5-HT(1A) receptors. As 5-HT(1A) receptor activation may induce antidepressant-like effects, the aim of this work was to test the hypothesis that CBD would have antidepressant-like activity in mice as assessed by the forced swimming test. We also investigated if these responses depended on the activation of 5-HT(1A) receptors and on hippocampal expression of brain-derived neurotrophic factor (BDNF). Experimental approach: Male Swiss mice were given (i.p.) CBD (3, 10, 30, 100 mg.kg(-1)), imipramine (30 mg.kg(-1)) or vehicle and were submitted to the forced swimming test or to an open field arena, 30 min later. An additional group received WAY100635 (0.1 mg.kg(-1), i.p.), a 5-HT(1A) receptor antagonist, before CBD (30 mg.kg(-1)) and assessment by the forced swimming test. BDNF protein levels were measured in the hippocampus of another group of mice treated with CBD (30 mg.kg(-1)) and submitted to the forced swimming test. Key results: CBD (30 mg.kg(-1)) treatment reduced immobility time in the forced swimming test, as did the prototype antidepressant imipramine, without changing exploratory behaviour in the open field arena. WAY100635 pretreatment blocked CBD-induced effect in the forced swimming test. CBD (30 mg.kg(-1)) treatment did not change hippocampal BDNF levels. Conclusion and implications: CBD induces antidepressant-like effects comparable to those of imipramine. These effects of CBD were probably mediated by activation of 5-HT(1A) receptors. British Journal of Pharmacology (2010) 159, 122-128; doi:10.1111/j.1476-5381.2009.00521.x; published online 4 December 2009

GABA(A) receptor blockade in dorsomedial and ventromedial nuclei of the hypothalamus evokes panic-like elaborated defensive behaviour followed by innate fear-induced antinociception

Dysfunction in the hypothalamic GABAergic system has been implicated in panic syndrome in humans. Furthermore, several studies have implicated the hypothalamus in the elaboration of pain modulation. Panic-prone states are able to be experimentally induced in laboratory animals to study this phenomenon. The aim of the present work was to investigate the involvement of medial hypothalamic nuclei in the organization of panic-like behaviour and the innate fear-induced oscillations of nociceptive thresholds. The blockade of GABA(A) receptors in the neuronal substrates of the ventromedial. or dorsomedial hypothalamus was followed by elaborated defensive panic-like reactions. Moreover, innate fear-induced antinociception was consistently elicited after the escape behaviour. The escape responses organized by the dorsomedial and ventromedial hypothalamic nuclei were characteristically more elaborated, and a remarkable exploratory behaviour was recorded during GABA(A) receptor blockade in the medial hypothalamus. The motor characteristic of the elaborated defensive escape behaviour and the patterns of defensive alertness and defensive immobility induced by microinjection of the bicuculline either into the dorsomedial. or into the ventromedial hypothalamus were very similar. This was followed by the same pattern of innate fear-induced antinociceptive response that lasted approximately 40 min after the elaborated defensive escape reaction in both cases. These findings suggest that dysfunction of the GABA-mediated neuronal system in the medial hypothalamus causes panic-like responses in laboratory animals, and that the elaborated escape behaviour organized in both dorsomedial and ventromedial hypothalamic nuclei are followed by significant innate-fear-induced antinociception. Our findings indicate that the GABA(A) receptor of dorsomedial and ventromedial hypothalamic nuclei are critically involved in the modulation of panic-like behaviour. (C) 2009 Elsevier B.V. All rights reserved.

Fructose-1,6-bisphosphate reduces inflammatory pain-like behaviour in mice: role of adenosine acting on A(1) receptors

Background and purpose: D-Fructose-1,6-bisphosphate (FBP) is an intermediate in the glycolytic pathway, exerting pharmacological actions on inflammation by inhibiting cytokine production or interfering with adenosine production. Here, the possible antinociceptive effect of FBP and its mechanism of action in the carrageenin paw inflammation model in mice were addressed, focusing on the two mechanisms described above. Experimental approach: Mechanical hyperalgesia (decrease in the nociceptive threshold) was evaluated by the electronic pressure-metre test; cytokine levels were measured by elisa and adenosine was determined by high performance liquid chromatography. Key results: Pretreatment of mice with FBP reduced hyperalgesia induced by intraplantar injection of carrageenin (up to 54%), tumour necrosis factor alpha (40%), interleukin-1 beta (46%), CXCL1 (33%), prostaglandin E(2) (41%) or dopamine (55%). However, FBP treatment did not alter carrageenin-induced cytokine (tumour necrosis factor alpha and interleukin-1 beta) or chemokine (CXCL1) production. On the other hand, the antinociceptive effect of FBP was prevented by systemic and intraplantar treatment with an adenosine A(1) receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine), suggesting that the FBP effect is mediated by peripheral adenosine acting on A(1) receptors. Giving FBP to mice increased adenosine levels in plasma, and adenosine treatment of paw inflammation presented a similar antinociceptive mechanism to that of FBP. Conclusions and implications: In addition to anti-inflammatory action, FBP also presents an antinociceptive effect upon inflammatory hyperalgesia. Its mechanism of action seems dependent on adenosine production but not on modulation of hyperalgesic cytokine/chemokine production. In turn, adenosine acts peripherally on its A(1) receptor inhibiting hyperalgesia. FBP may have possible therapeutic applications in reducing inflammatory pain.

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.