C.M. Roncon

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.

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.

5-Hydroxytryptamine(1A) receptors in the dorsomedial hypothalamus connected to dorsal raphe nucleus inputs modulate defensive behaviours and mediate innate fear-induced antinociception

The dorsal raphe nucleus (DRN) is an important brainstem source of 5-hydroxytryptamine (5-HT), and 5-HT plays a key role in the regulation of panic attacks. The aim of the present study was to determine whether 5-HT1A receptor-containing neurons in the medial hypothalamus (MH) receive neural projections from DRN and to then determine the role of this neural substrate in defensive responses. The neurotracer biotinylated dextran amine (BDA) was iontophoretically microinjected into the DRN, and immunohistochemical approaches were then used to identify 5HT1A receptor-labelled neurons in the MH. Moreover, the effects of pre-treatment of the dorsomedial hypothalamus (DMH) with 8-OH-DPAT and WAY-100635, a 5-HT1A receptor agonist and antagonist, respectively, followed by local microinjections of bicuculline, a GABAA receptor antagonist, were investigated. We found that there are many projections from the DRN to the perifornical lateral hypothalamus (PeFLH) but also to DMH and ventromedial (VMH) nuclei, reaching 5HT1A receptor-labelled perikarya. DMH GABAA receptor blockade elicited defensive responses that were followed by antinociception. DMH treatment with 8-OH-DPAT decreased escape responses, which strongly suggests that the 5-HT1A receptor modulates the defensive responses. However, DMH treatment with WAY-100635 failed to alter bicuculline-induced defensive responses, suggesting that 5-HT exerts a phasic influence on 5-HT1A DMH neurons. The activation of the inhibitory 5-HT1A receptor had no effect on antinociception. However, blockade of the 5-HT1A receptor decreased fear-induced antinociception. The present data suggest that the ascending pathways from the DRN to the DMH modulate panic-like defensive behaviours and mediate antinociceptive phenomenon by recruiting 5-HT1A receptor in the MH.

ENDOCANNABINOID SIGNALING MECHANISMS IN THE SUBSTANTIA NIGRA PARS RETICULATA MODULATE GABAergic NIGROTECTAL PATHWAYS IN MICE THREATENED BY URUTU-CRUZEIRO VENOMOUS PIT VIPER

The substantia nigra pars reticulata (SNpr) is rich in γ-aminobutyric acid (GABA)-ergic neurons and connected to the mesencephalic tectum (MT) structures, such as the superior colliculus and dorsal periaqueductal gray matter. The SNpr presents a high density of cannabinoid receptors (CBRs), suggesting a possible regulatory role that is played by endocannabinoids (eCBs) in the ventral mesencephalon. The present study investigated the involvement of SNpr eCB mechanisms in nigrotectal pathways in the expression of defensive behavior associated with instinctive fear and panic reactions in mice that are confronted with the venomous Viperidae snake Bothrops alternatus. The localization of CB1 receptors (CB1RS) and synaptophysin glycoprotein in the SNpr was also evaluated. Administration of the GABAA receptor antagonist bicuculline in the MT increased defensive responses to the snake that are related to panic, such as freezing and non-oriented escape reactions, sometimes toward the snake itself. Mice that were pretreated with anandamide (5 or 50pmol) in the SNpr, followed by an injection of physiological saline or bicuculline in the MT, exhibited significant decreases in the expression of alertness, freezing, and escape responses. Immunofluorescence showed the presence of fibers that were rich in CB1RS and synaptophysin in the SNpr, indicating that these receptors appear to be located mainly in presynaptic terminals in the striatonigral pathway. These findings suggest that eCB mechanisms in the SNpr facilitate the activity of nigrotectal GABAergic pathways, modulating the activity of striatonigral links during the elaboration and organization of innate fear and panic-like responses in threatening situations.

Interaction between μ-opioid and 5-HT1A receptors in the regulation of panic-related defensive responses in the rat dorsal periaqueductal grey

A wealth of evidence indicates that the activation of 5-HT1A and 5-HT2A receptors in the dorsal periaqueductal grey matter (dPAG) inhibits escape, a panic-related defensive behaviour. Results that were previously obtained with the elevated T-maze test of anxiety/panic suggest that 5-HT1A and μ-opioid receptors in this midbrain area work together to regulate this response. To investigate the generality of this finding, we assessed whether the same cooperative mechanism is engaged when escape is evoked by a different aversive stimulus electrical stimulation of the dPAG. Administration of the μ-receptor blocker CTOP into the dPAG did not change the escape threshold, but microinjection of the μ-receptor agonist DAMGO (0.3 and 0.5 nmol) or the 5-HT1A receptor agonist 8-OHDPAT (1.6 nmol) increased this index, indicating a panicolytic-like effect. Pretreatment with CTOP antagonised the anti-escape effect of 8-OHDPAT. Additionally, combined administration of subeffective doses of DAMGO and 8-OHDPAT increased the escape threshold, indicating drug synergism. Therefore, regardless of the aversive nature of the stimulus, μ-opioid and 5-HT1A receptors cooperatively act to regulate escape behaviour. A better comprehension of this mechanism might allow for new therapeutic strategies for panic disorder.

Pindolol potentiates the panicolytic effect of paroxetine in the elevated T-maze.

AIMS the β-adrenergic and 5-HT(1A) receptor antagonist pindolol has been used in combination with antidepressant drugs, to shorten the time of onset of clinical efficacy and/or increase the proportion of responders in depressive and anxiety disorders. The aim of this study was to examine the interaction between pindolol and the selective serotonin reuptake inhibitor (SSRI), paroxetine in rats submitted to the elevated T-maze (ETM). MAIN METHODS for assessing the drug combination effect, rats were administered with pindolol before paroxetine, using oral or intraperitoneal (i.p.) routes of acute administration, and were submitted to the ETM model. KEY FINDINGS the highest dose of pindolol used (15.0mg/kg, i.p.) increased both inhibitory avoidance and escape latencies in the ETM, probably due to nonspecific motor deficit, since locomotion in a circular arena was also significantly decreased. The highest dose of paroxetine (3.0mg/kg, i.p.) selectively impaired escape, considered a panicolytic effect. Combination of pindolol (5.0mg/kg, i.p.) with an ineffective dose of paroxetine (1.5mg/kg, i.p.) impaired escape, indicating a potentiation of the panicolytic effect of paroxetine. By the oral route, neither paroxetine (3.0mg/kg) nor pindolol (5.0mg/kg) alone were effective, but the combination treatment had a marked panicolytic effect, again indicating drug potentiation. SIGNIFICANCE the present results show that the combination of the ineffective doses of pindolol and paroxetine significantly increased escape latency, indicating a selective panicolytic effect. These findings give preclinical support for the use of this drug combination in the treatment of panic disorder (PD).

mu-Opioid and 5-HT1A receptors in the dorsomedial hypothalamus interact for the regulation of panic-related defensive responses

The dorsomedial hypothalamus (DMH) and the dorsal periaqueductal gray (DPAG) have been implicated in the genesis and regulation of panic-related defensive behaviors, such as escape. Previous results point to an interaction between serotonergic and opioidergic systems within the DPAG to inhibit escape, involving µ-opioid and 5-HT1A receptors (5-HT1AR). In the present study we explore this interaction in the DMH, using escape elicited by electrical stimulation of this area as a panic attack index. The obtained results show that intra-DMH administration of the non-selective opioid receptor antagonist naloxone (0.5 nmol) prevented the panicolytic-like effect of a local injection of serotonin (20 nmol). Pretreatment with the selective μ-opioid receptor (MOR) antagonist CTOP (1 nmol) blocked the panicolytic-like effect of the 5-HT1AR agonist 8-OHDPAT (8 nmol). Intra-DMH injection of the selective MOR agonist DAMGO (0.3 nmol) also inhibited escape behavior, and a previous injection of the 5-HT1AR antagonist WAY-100635 (0.37 nmol) counteracted this panicolytic-like effect. These results offer the first evidence that serotonergic and opioidergic systems work together within the DMH to inhibit panic-like behavior through an interaction between µ-opioid and 5-HT1A receptors, as previously described in the DPAG.

Panicolytic-like effect of tramadol is mediated by opioid receptors in the dorsal periaqueductal grey

HighlightsAcute and systemic administration of tramadol causes panicolytic‐like effect in the elevated T maze.The panicolytic‐like effect of tramadol depends on activation of opioid receptors located in the dPAG.WAY100635 associated to tramadol promoted an anxiogenic effect in the elevated T‐maze. Abstract Tramadol is a synthetic opioid prescribed for the treatment of moderate to severe pain, acting as agonist of &mgr;‐opioid receptors and serotonin (5‐HT) and noradrenaline (NE) reuptake inhibitor. This study evaluated the effects of tramadol in rats submitted to the elevated T‐maze (ETM), an animal model that evaluates behavioural parameters such as anxiety and panic. Male Wistar rats were intraperitoneally (i.p.) treated acutely with tramadol (16 and 32 mg/kg) and were submitted to the ETM. Tramadol (32 mg/kg) promoted a panicolytic‐like effect. Considering that dorsal periaqueductal grey (dPAG) is the main brain structure related to the pathophysiology of panic disorder (PD), this study also evaluated the participation of 5‐HT and opioid receptors located in the dPAG in the panicolytic‐like effect of tramadol. Seven days after stereotaxic surgery for implantation of a cannula in the dPAG, the animals were submitted to the test. To assess the involvement of 5‐HT1A receptors on the effect of tramadol, we combined the 5‐HT1A receptor antagonist, WAY100635 (0.37 nmol), microinjected intra‐dPAG, 10 min prior to the administration of tramadol (32 mg/kg, i.p.). WAY100635 did not block the panicolytic‐like effect of tramadol. We also associated the non‐selective opioid receptor antagonist, naloxone, systemically (1 mg/kg, i.p.) or intra‐dPAG (0.5 nmol) administered 10 min prior to tramadol (32 mg/kg, i.p.). Naloxone blocked the panicolytic‐like effect of tramadol in both routes of administrations, showing that tramadol modulates acute panic defensive behaviours through its interaction with opioid receptors located in the dPAG.

Participation of dorsal periaqueductal gray 5-HT1A receptors in the panicolytic-like effect of the κ-opioid receptor antagonist Nor-BNI

HighlightsNor‐BNI causes panicolytic‐like effect in rat models of panic.5‐HT1A receptor activation is implicated in the panicolytic‐like effect of Nor‐BNI.Nor‐BNI effect does not depend on &mgr;‐opioid receptor activation. Abstract Panic patients may have abnormalities in serotonergic and opioidergic neurotransmission. The dorsal periaqueductal gray (dPAG) plays an important role in organizing proximal defense, related to panic attacks. The 5‐HT1A receptor (5‐HT1A‐R) is involved in regulating escape behavior that is organized in the dPAG. Activation of &kgr;‐opioid receptor (KOR) in this region causes anxiogenic effects. In this study, we investigated the involvement of KOR in regulating escape behavior, using systemic and intra‐dPAG injection of the KOR antagonist Nor‐BNI. As panic models, we used the elevated T‐maze (ETM) and the dPAG electrical stimulation test (EST). We also evaluated whether activation of the 5‐HT1A‐R or the &mgr;‐opioid receptor (MOR) in the dPAG contributes to the Nor‐BNI effects. The results showed that systemic administration of Nor‐BNI, either subcutaneously (2.0 and 4.0 mg/kg) or intraperitoneally (2.0 mg/kg), impaired escape in the EST, indicating a panicolytic‐like effect. Intra‐dPAG injection of this antagonist (6.8 nmol) caused the same effect in the EST and in the ETM. Association of ineffective doses of Nor‐BNI and the 5‐HT1A‐R agonist 8‐OH‐DPAT caused panicolytic‐like effect in these two tests. Previous administration of the 5‐HT1A‐R antagonist WAY‐100635, but not of the MOR antagonist CTOP, blocked the panicolytic‐like effect of Nor‐BNI. These results indicate that KOR enhances proximal defense in the dPAG through 5‐HT1A‐R modulation, independently of MOR. Because former results indicate that the 5‐HT1A‐R is involved in the antipanic action of antidepressants, KOR antagonists may be useful as adjunctive or alternative drug treatment of panic disorder.