Anti-panic effect of fluoxetine during late diestrus in female rats is mediated through GABAergic mechanisms in the dorsal periaqueductal gray.

Panic disorder is more frequent in women than in men. In women, vulnerability to panic is enhanced during the late luteal phase of the menstrual cycle. At this time secretion of progesterone and its neuroactive metabolite allopregnanolone (ALLO), which acts as a positive allosteric modulator of the actions of GABA at GABAA receptors, decline sharply. In female rats, responsiveness to a hypoxic panicogenic challenge increases during the late diestrus (LD) phase as ALLO concentration in the brain falls. During LD, short-term treatment with fluoxetine at a low dose (1.75 mg/kg i.p.) blocked panic-related escape behavior in response to hypoxia. At this dose fluoxetine increases brain concentration of ALLO without affecting 5-HT levels, thereby stabilizing brain ALLO concentration. We here report that the panicolytic-like effect of fluoxetine during LD is prevented by microinjection of the GABAA receptor antagonist bicuculline (5 pmol) into the dorsal periaqueductal gray (dPAG), a key panic-related area. This result suggests that fluoxetine's effect is indirectly mediated via a GABAergic mechanism in the dPAG and highlights the important role of changes in GABAergic tone in regulating neuronal excitability in the panic circuitry during the estrous cycle. It also points to the potential for using short-term, low dose fluoxetine as an anti-panic medication in women.

Local effect of allopregnanolone in rat ovarian steroidogenesis, follicular and corpora lutea development.

Allopregnanolone (ALLO) is a known neurosteroid and a progesterone metabolite synthesized in the ovary, CNS, PNS, adrenals and placenta. Its role in the neuroendocrine control of ovarian physiology has been studied, but its in situ ovarian effects are still largely unknown. The aims of this work were to characterize the effects of intrabursal ALLO administration on different ovarian parameters, and the probable mechanism of action. ALLO administration increased serum progesterone concentration and ovarian 3ß-HSD2 while decreasing 20α-HSD mRNA expression. ALLO increased the number of atretic follicles and the number of positive TUNEL granulosa and theca cells, while decreasing positive PCNA immunostaining. On the other hand, there was an increase in corpora lutea diameter and PCNA immunostaining, whereas the count of TUNEL-positive luteal cells decreased. Ovarian angiogenesis and the immunohistochemical expression of GABAA receptor increased after ALLO treatment. To evaluate if the ovarian GABAA receptor was involved in these effects, we conducted a functional experiment with a specific antagonist, bicuculline. The administration of bicuculline restored the number of atretic follicles and the diameter of corpora lutea to normal values. These results show the actions of ALLO on the ovarian physiology of the female rat during the follicular phase, some of them through the GABAA receptor. Intrabursal ALLO administration alters several processes of the ovarian morpho-physiology of the female rat, related to fertility and oocyte quality.

Alpha1- and Beta-norepinephrinergic receptors of dorsomedial and ventromedial hypothalamic nuclei modulate panic attack-like defensive behaviour elicited by diencephalic GABAergic neurotransmission disinhibition.

Gamma-aminobutyric acid (GABA) disinhibition in medial hypothalamus (MH) nuclei of rats elicits some defensive reactions that are considered panic attack-like behaviours. Recent evidence showed that the norepinephrine-mediated system modulates fear-related defensive behaviours organised by MH neurons at least in part via noradrenergic receptors recruitment on midbrain tegmentum. However, it is unknown whether noradrenergic receptors of the MH also modulate the panic attack-like reactions. The aim of this work was to investigate the distribution of noradrenergic receptors in MH, and the effects of either α1-, α2- or ß-noradrenergic receptors blockade in the MH on defensive behaviours elaborated by hypothalamic nuclei. Defensive behaviours were evaluated after the microinjection of the selective GABAA receptor antagonist bicuculline into the MH that was preceded by microinjection of either WB4101, RX821002, propranolol (α1-, α2- and ß-noradrenergic receptor selective antagonists, respectively), or physiological saline into the MH of male Wistar rats. The α1-, α2- and ß-noradrenergic receptors were found in neuronal perikarya of all MH nuclei, and the α2-noradrenergic receptor were also found on glial cells mainly situated in the ventrolateral division of the ventromedial hypothalamic nucleus. The α1- and ß-noradrenergic receptors blockade in the MH decreased defensive attention and escape reactions elicited by the intra-MH microinjections of bicuculline. These findings suggest that, despite the profuse distributions of α1-, α2- and ß-noradrenergic receptors in the MH, both α1- and ß-noradrenergic receptor- rather than α2-noradrenergic receptor-signalling in MH are critical for the neuromodulation of panic-like behaviour.

Prepulse inhibition of the acoustic startle reflex impairment by 5-HT2A receptor activation in the inferior colliculus is prevented by GABAA receptor blockade in the pedunculopontine tegmental nucleus.

The relationship between serotonin dysfunction and schizophrenia commenced with the discovery of the effects of lysergic acid diethylamide (LSD) that has high affinity for 5-HT2A receptors. Activation of these receptors produces perceptual and behavioural changes such as illusions, visual hallucinations and locomotor hyperactivity. Using prepulse inhibition (PPI) of the acoustic startle, which is impaired in schizophrenia,we aimed to investigate:i) the existence of a direct and potentially inhibitory neural pathway between the inferior colliculus (IC) and the pedunculopontine tegmental nucleus (PPTg) involved in the mediation of PPI responses by a neural tract tracing procedure;ii) if the microinjection of the 5-HT2A receptors agonist DOI in IC would activate neurons in this structure and in the PPTg by a c-Fos protein immunohistochemistry study;iii) whether the deficits in PPI responses, observed after the administration of DOI in the IC, could be prevented by the concomitant microinjection of the GABAA receptor antagonist bicuculline in the PPTg.Male Wistar rats were used in this study. An IC-PPTg reciprocated neuronal pathway was identified by neurotracing. The number of c-Fos labelled cells was lower in the DOI group in IC and PPTg, suggesting that this decrease could be due to the high levels of GABA in both structures. The concomitant microinjections of bicuculline in PPTg and DOI in IC prevented the PPI deficit observed after the IC microinjection of DOI. Our findings suggest that IC 5-HT2A receptors may be at least partially involved in the regulation of inhibitory pathways mediating PPI response in IC and PPTg structures.

GABAergic Regulation of Astroglial Gliotransmission through Cx43 Hemichannels.

Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. It is produced by interneurons and recycled by astrocytes. In neurons, GABA activates the influx of Cl- via the GABAA receptor or efflux or K+ via the GABAB receptor, inducing hyperpolarization and synaptic inhibition. In astrocytes, the activation of both GABAA and GABAB receptors induces an increase in intracellular Ca2+ and the release of glutamate and ATP. Connexin 43 (Cx43) hemichannels are among the main Ca2+-dependent cellular mechanisms for the astroglial release of glutamate and ATP. However, no study has evaluated the effect of GABA on astroglial Cx43 hemichannel activity and Cx43 hemichannel-mediated gliotransmission. Here we assessed the effects of GABA on Cx43 hemichannel activity in DI NCT1 rat astrocytes and hippocampal brain slices. We found that GABA induces a Ca2+-dependent increase in Cx43 hemichannel activity in astrocytes mediated by the GABAA receptor, as it was blunted by the GABAA receptor antagonist bicuculline but unaffected by GABAB receptor antagonist CGP55845. Moreover, GABA induced the Cx43 hemichannel-dependent release of glutamate and ATP, which was also prevented by bicuculline, but unaffected by CGP. Gliotransmission in response to GABA was also unaffected by pannexin 1 channel blockade. These results are discussed in terms of the possible role of astroglial Cx43 hemichannel-mediated glutamate and ATP release in regulating the excitatory/inhibitory balance in the brain and their possible contribution to psychiatric disorders.

Presynaptic nigral GPR55 receptors stimulate [3 H]-GABA release through [3 H]-cAMP production and PKA activation and promote motor behavior.

Striatal medium-sized spiny neurons express mRNA and protein of GPR55 receptors that stimulate neurotransmitter release; thus, GPR55 could be sent to nigral striatal projections, where it might modulate GABA release and motor behavior. Here, we study the presence of GPR55 receptors at striato-nigral terminals, their modulation of GABA release, their signaling pathway, and their effect on motor activity. By double immunohistochemistry, we found the colocation of GPR55 protein and substance P in the dorsal striatum. In slices of the rat substantia nigra, the GPR55 agonists LPI and O-1602 stimulated [3 H]-GABA release induced by high K+ depolarization in a dose-dependent manner. The antagonists CID16020046 and cannabidiol prevented agonist stimulation in a dose-dependent way. The effect of GPR55 on nigral [3 H]-GABA release was prevented by lesion of the striatum with kainic acid, which was accompanied by a decrement of GPR55 protein in nigral synaptosomes, indicating the presynaptic location of receptors. The depletion of internal Ca2+ stores with thapsigargin did not prevent the effect of LPI on [3 H]-GABA release, but the remotion or chelation of external calcium did. Blockade of Gi, Gs, PLC, PKC, or dopamine D1 receptor signaling proteins did not prevent the effect of GPR55 on release. However, the activation of GPR55 stimulated [3 H]-cAMP accumulation and PKA activity. Intranigral unilateral injection of LPI induces contralateral turning. This turning was prevented by CID16020046, cannabidiol, and bicuculline but not by SCH 23390. Our data indicate that presynaptic GPR55 receptors stimulate [3 H]-GABA release at striato-nigral terminals through [3 H]-cAMP production and stimulate motor behavior.

The blockade of µ­opioid receptors in the lateral hypothalamus enhances panic attack­like behaviour and diminishes defensive antinociception.

The lateral hypothalamus (LH) sends neural pathways to structures involved on predator­related defensive behaviours, escape and antinociception. The aim of this study was to investigate the role played by µ-opioid receptors located on LH neurons in defensive behaviour and unconditioned fear­induced antinociception elicited by electric stimulation of LH. To achieve the goals, the µ1-opioid receptor selective antagonist naloxonazine was administered at different concentrations in the LH, and the defensive behaviour and fear­induced antinociception elicited by electrical stimulation of LH were evaluated. The electrical stimulation of LH caused escape behaviour followed by defensive antinociception. Microinjections of naloxonazine in a concentration of 5.0 µg/0.2 µL in the LH decreased the aversive stimulus­induced escape behaviour thresholds, but diminished defensive antinociception. These findings suggest that µ-opioid receptors of LH can be critical to panic attack­related symptoms and facilitate the unconditioned fear­induced antinociception produced by LH neurons activation.

Relative Contribution of Blood Pressure and Renal Sympathetic Nerve Activity to Proximal Tubular Sodium Reabsorption via NHE3 Activity.

We examined the effects of an acute increase in blood pressure (BP) and renal sympathetic nerve activity (rSNA) induced by bicuculline (Bic) injection in the paraventricular nucleus of hypothalamus (PVN) or the effects of a selective increase in rSNA induced by renal nerve stimulation (RNS) on the renal excretion of sodium and water and its effect on sodium-hydrogen exchanger 3 (NHE3) activity. Uninephrectomized anesthetized male Wistar rats were divided into three groups: (1) Sham; (2) Bic PVN: (3) RNS + Bic injection into the PVN. BP and rSNA were recorded, and urine was collected prior and after the interventions in all groups. RNS decreased sodium (58%) and water excretion (53%) independently of BP changes (p < 0.05). However, after Bic injection in the PVN during RNS stimulation, the BP and rSNA increased by 30% and 60% (p < 0.05), respectively, diuresis (5-fold) and natriuresis (2.3-fold) were increased (p < 0.05), and NHE3 activity was significantly reduced, independently of glomerular filtration rate changes. Thus, an acute increase in the BP overcomes RNS, leading to diuresis, natriuresis, and NHE3 activity inhibition.

Bicuculline regulated protein synthesis is dependent on Homer1 and promotes its interaction with eEF2K through mTORC1-dependent phosphorylation.

The regulation of protein synthesis is a vital and finely tuned process in cellular physiology. In neurons, this process is very precisely regulated, as which mRNAs undergo translation is highly dependent on context. One of the most prominent regulators of protein synthesis is the enzyme eukaryotic elongation factor kinase 2 (eEF2K) that regulates the elongation stage of protein synthesis. This kinase and its substrate, eukaryotic elongation factor 2 (eEF2) are important in processes such as neuronal development and synaptic plasticity. eEF2K is regulated by multiple mechanisms including Ca2+ -ions and the mTORC1 signaling pathway, both of which play key roles in neurological processes such as learning and memory. In such settings, the localized control of protein synthesis is of crucial importance. In this work, we sought to investigate how the localization of eEF2K is controlled and the impact of this on protein synthesis in neuronal cells. In this study, we used both SH-SY5Y neuroblastoma cells and mouse cortical neurons, and pharmacologically and/or genetic approaches to modify eEF2K function. We show that eEF2K activity and localization can be regulated by its binding partner Homer1b/c, a scaffolding protein known for its participation in calcium-regulated signaling pathways. Furthermore, our results indicate that this interaction is regulated by the mTORC1 pathway, through a known phosphorylation site in eEF2K (S396), and that it affects rates of localized protein synthesis at synapses depending on the presence or absence of this scaffolding protein.

Anticonvulsant, Anxiolytic and Antidepressant Properties of the ß-caryophyllene in Swiss Mice: Involvement of Benzodiazepine-GABAAergic, Serotonergic and Nitrergic Systems.

BACKGROUND: Central nervous system disorders such as anxiety, depression and epilepsy are characterized by sharing several molecular mechanisms in common and the involvement of the L-arginine/NO pathway in neurobehavioral studies with ß-caryophyllene is still little discussed. OBJECTIVES: One of the objectives of the present study was to demonstrate the anxiolytic behavioral effect of ß-caryophyllene (ß-CBP) in female Swiss mice, as well as to investigate the molecular mechanisms underlying the results obtained. METHODS: This study evaluated the neurobehavioral effects of ß-CBP using the open field test, rota- rod test, elevated plus maze test, novelty suppressed feeding test, tail suspension test and forced swim test, as well as pilocarpine, pentylenetetrazole and isoniazid-induced epileptic seizure models. RESULTS: The results demonstrated that the neuropharmacological activities of ß-CBP may involve benzodiazepine/GABAergic receptors, since the pre-treatment of ß-CBP (200 mg/kg) associated with flumazenil (5 mg/kg, benzodiazepine receptor antagonist) and bicuculline (1 mg/kg, selective GABAA receptor antagonist) reestablished the anxiety parameters in the elevated plus-maze test, as well as the results of reduced latency to consume food in the novelty suppressed feeding test. In addition to benzodiazepine/GABAergic receptors, the neuropharmacological properties of ß-CBP may be related to inhibition of nitric oxide synthesis, since pre-treatment with L-arginine (500-750 mg/kg) reversed significantly the anxiolytic, antidepressant and anticonvulsant activities of ß-CBP. CONCLUSION: The results obtained provide additional support in understanding the neuromolecular mechanisms underlying the anxiolytic, antidepressant and anticonvulsive properties of ß-CBP in female Swiss mice.

Adolescent Exposure to WIN 55212-2 Render the Nigrostriatal Dopaminergic Pathway Activated During Adulthood.

BACKGROUND: During adolescence, neuronal circuits exhibit plasticity in response to physiological changes and to adapt to environmental events. Nigrostriatal dopaminergic pathways are in constant flux during development. Evidence suggests a relationship between early use of cannabinoids and psychiatric disorders characterized by altered dopaminergic systems, such as schizophrenia and addiction. However, the impact of adolescent exposure to cannabinoids on nigrostriatal dopaminergic pathways in adulthood remains unclear. The aim of this research was to determine the effects of repeated activation of cannabinoid receptors during adolescence on dopaminergic activity of nigrostriatal pathways and the mechanisms underlying this impact during adulthood. METHODS: Male Sprague-Dawley rats were treated with 1.2 mg/kg WIN 55212-2 daily from postnatal day 40 to 65. Then no-net flux microdialysis of dopamine in the dorsolateral striatum, electrophysiological recording of dopaminergic neuronal activity, and microdialysis measures of gamma-aminobutyric acid (GABA) and glutamate in substantia nigra par compacta were carried out during adulthood (postnatal days 72-78). RESULTS: Repeated activation of cannabinoid receptors during adolescence increased the release of dopamine in dorsolateral striatum accompanied by increased population activity of dopamine neurons and decreased extracellular GABA levels in substantia nigra par compacta in adulthood. Furthermore, perfusion of bicuculline, a GABAa antagonist, into the ventral pallidum reversed the increased dopamine neuron population activity in substantia nigra par compacta induced by adolescent cannabinoid exposure. CONCLUSIONS: These results suggest that adolescent exposure to cannabinoid agonists produces disinhibition of nigrostriatal dopamine transmission during adulthood mediated by decreased GABAergic input from the ventral pallidum.

Anandamide in the anterior hypothalamus diminishes defensive responses elicited in mice threatened by Epicrates cenchria constrictor serpents.

The purpose of this study was to investigate whether the panicolytic­like effect of different doses of anandamide microinjected into the anterior hypothalamus (AH) follows the same pattern of a bell­shaped dose­response curve observed with the same dose treatment in dorsomedial and ventromedial hypothalamus. We investigated this assumption by administering the cannabinoid and vanilloid receptor agonist anandamide into the anterior hypothalamus of mice and exposing them to the real threatening situation by using our experimental model based on confrontations between rodents and wild snakes. Our findings showed a gradual decay of response, with a significant attenuation of the panic attack­like responses with anandamide at the highest dose but no effect was found after anandamide at the lowest or intermediate doses. An immunohistochemical procedure showed a lower degree of TRPV1 receptor and moderate to higher degree of Cb1 receptors in anterior hypothalamus. In conclusion, the pattern of dose­response curve of anandamide microinjected in the AH does not seem to be the same classical pattern compared with other hypothalamic nuclei.

Endocannabinoid neuromodulation in the neostriatum decreases the GABAergic striato-nigral disinhibitory function and increases the nigro-collicular inhibitory pathway activity.

We previously reported the involvement of neostriato-nigral projections in the organisation of innate fear and panic attack-like responses organised by dorsal midbrain neurons, such as the periaqueductal grey matter and the deep layers of the superior colliculus (dlSC). In addition, several lines of evidence have demonstrated that cannabinoid receptor type 1 is found in the neostriatum (caudate nucleus and putamen; CPu). In the present study, we investigated the role of endocannabinoid neuromodulation in CPu in the expression of unconditioned fear-related behavioural responses elicited by microinjections of the γ-aminobutyric acid (GABA)A receptor selective antagonist bicuculline (BIC) in the dlSC. Wistar rats received injection of vehicle or anandamide (AEA) at 0.5, 5, 50, 100 pmol in CPu, followed by injections of BIC in a dose of 40 ng in the dlSC. The treatment of the CPu with AEA in a dose of 5 and 50 pmol attenuated the unconditioned fear-related behaviour, such as defensive alertness, defensive immobility and escape, induced by GABAA receptor blockade in dlSC. These findings suggest that endogenous cannabinoids acting on CPu neurons exert an indirect modulatory influence on the activity of superior colliculus neurons, possibly through an inhibitory activity on neostriato-nigral disinhibitory connections that modulate the nigro-collicular inhibitory GABAergic pathways.

Interaction between angiotensin II and GABA in the spinal cord regulates sympathetic vasomotor activity in Goldblatt hypertension.

Previous studies have been described changes in brain regions contributing to the sympathetic vasomotor overactivity in Goldblatt hypertension (2K1C). Furthermore, changes in the spinal cord are also involved in the cardiovascular and autonomic dysfunction in renovascular hypertension, as intrathecal (i.t.) administration of Losartan (Los) causes a robust hypotensive/sympathoinhibitory response in 2K1C but not in control rats. The present study evaluated the role of spinal γ-aminobutyric acid (GABA)-ergic inputs in the control of sympathetic vasomotor activity in the 2K1C rats. Hypertension was induced by clipping the renal artery. After six weeks, a catheter (PE-10) was inserted into the subarachnoid space and advanced to the T10-11 vertebral level in urethane-anaesthetized rats. The effects of i.t. injection of bicuculline (Bic) on blood pressure (BP), renal and splanchnic sympathetic nerve activity (rSNA and sSNA, respectively) were evaluated over 40 consecutive minutes in the presence or absence of spinal AT1 antagonism. I.t. Bic triggered a more intense pressor and sympathoexcitatory response in 2K1C rats, however, these responses were attenuated by previous i.t. Los. No differences in the gene expression of GAD 65 and GABA-A receptors subunits in the spinal cord segments were found. Thus, the sympathoexcitation induced by spinal GABA-A blockade is dependent of local AT1 receptor in 2K1C but not in control rats. Excitatory angiotensinergic inputs to sympathetic preganglionic neurons are tonic controlled by spinal GABAergic actions in Goldblatt hypertension.

Involvement of GABAergic system in the antidepressant-like effects of chrysin (5,7-dihydroxyflavone) in ovariectomized rats in the forced swim test: comparison with neurosteroids.

RATIONALE: The absence of ovarian hormones that is characteristic of natural and surgical postmenopause in women is frequently related to such disorders as depression and anxiety. Chronic treatment with the flavonoid chrysin was previously shown to exert antidepressant-like effects in rodents subjected to validate behavioral models. Chrysin has also been shown to have anxiolytic-like properties, but its antidepressant-like effects and mechanism of action in the absence of ovarian hormones remain unknown. OBJECTIVES: To compare the effects of the flavonoid chrysin with the effects of the neurosteroids progesterone and allopregnanolone on depression-like behavior in ovariectomized rats and evaluate the participation of γ-aminobutyric acid-A (GABAA) receptors in these actions. METHODS: Ovariectomized female Wistar rats were subjected to the locomotor activity test and forced swim test. The animals were assigned to eight treatment groups: vehicle, chrysin (1 mg/kg), progesterone (1 mg/kg), allopregnanolone (1 mg/kg), bicuculline (1 mg/kg), and pretreatment with bicuculline followed by chrysin, progesterone or allopregnanolone, respectively. After the treatments, the rats underwent the behavioral tests. RESULTS: Chrysin, progesterone, and allopregnanolone increased the latency to the first immobility and decreased the total immobility time in the forced swim test. The number of crossings and the time spent rearing and grooming decreased from the pretest to test sessions in the locomotor activity test. Chrysin, progesterone, and allopregnanolone only prevented the decreases in rearing and grooming. Bicuculline blocked the effects of chrysin, progesterone, and allopregnanolone in both behavioral tests. CONCLUSIONS: These results show that the GABA-binding site at GABAA receptors participates in the acute antidepressant-like effects of chrysin, similar to neurosteroids, in ovariectomized rats.

Pattern of sympathetic vasomotor activity induced by GABAergic inhibition in the brain and spinal cord.

BACKGROUND: Knowledge of the central areas involved in the control of sympathetic vasomotor activity has advanced in the last few decades. γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammal nervous system, and a microinjection of bicuculline, an antagonist of GABA type A (GABA-A) receptors, into the paraventricular nucleus of the hypothalamus (PVN) alters the pattern of sympathetic activity to the renal, splanchnic and lumbar territories. However, studies are needed to clarify the role of GABAergic inputs in other central areas involved in the sympathetic vasomotor activity. The present work studied the cardiovascular effects evoked by GABAergic antagonism in the PVN, RVLM and spinal cord. METHODS AND RESULTS: Bicuculline microinjections (400 pMol in 100 nL) into the PVN and rostral ventrolateral medulla (RVLM) as well as intrathecal administration (1.6 nmol in 2 µL) evoked an increase in blood pressure, heart rate, and renal and splanchnic sympathetic nerve activity (rSNA and sSNA, respectively), inducing a higher coherence between rSNA and sSNA patterns. However, some of these responses were more intense when the GABA-A antagonism was performed in the RVLM than when the GABA-A antagonism was performed in other regions. CONCLUSIONS: Administration of bicuculline into the RVLM, PVN and SC induced a similar pattern of renal and splanchnic sympathetic vasomotor burst discharge, characterized by a low-frequency (0.5 Hz) and high-amplitude pattern, despite different blood pressure responses. Thus, the differential control of sympathetic drive to different targets by each region is dependent, in part, on tonic GABAergic inputs.

The Blockade of µ1- and κ-Opioid Receptors in the Inferior Colliculus Decreases the Expression of Panic Attack-Like Behaviours Induced by Chemical Stimulation of the Dorsal Midbrain.

BACKGROUND: Gamma-aminobutyric acid (GABA)ergic and opioid systems play a crucial role in the neural modulation of innate fear organised by the inferior colliculus (IC). In addition, the IC is rich in GABAergic fibres and opioid neurons, which are also connected to other mesencephalic structures, such as the superior colliculus and the substantia nigra. However, the contribution of distinct opioid receptors (ORs) in the IC during the elaboration and expression of innate fear and panic-like responses is unclear. The purpose of the present work was to investigate a possible integrated action exerted by ORs and the GABAA receptor-mediated system in the IC on panic-like responses. METHODS: The effect of the blockade of either µ1- or κ-ORs in the IC was evaluated in the unconditioned fear-induced responses elicited by GABAA antagonism with bicuculline. Microinjections of naloxonazine, a µ1-OR antagonist, or nor-binaltorphimine (nor-BNI), a κ-OR antagonist, were made into the IC, followed by intramesencephalic administration of the GABAA-receptor antagonist bicuculline. The defensive behaviours elicited by the treatments in the IC were quantitatively analysed, recording the number of escapes expressed as running (crossing), jumps, and rotations, over a 30-min period in a circular arena. The exploratory behaviour of rearing was also recorded. RESULTS: GABAA-receptor blockade with bicuculline in the IC increased defensive behaviours. However, pretreatment of the IC with higher doses (5 µg) of naloxonazine or nor-BNI followed by bicuculline resulted in a significant decrease in unconditioned fear-induced responses. CONCLUSIONS: These findings suggest a role played by µ1- and κ-OR-containing connexions and GABAA receptor-mediated neurotransmission on the organisation of panic attack-related responses elaborated by the IC neurons.

GABAA/benzodiazepine receptors in the dorsal periaqueductal gray mediate the panicolytic but not the anxiolytic effect of alprazolam in rats.

Although the etiology of panic disorder (PD) remains elusive, accumulating evidence suggests a key role for the dorsal periaqueductal gray matter (dPAG). There is also evidence that this midbrain area is critically involved in mediation of the panicolytic effect of antidepressants, which with high potency benzodiazepines (e.g. alprazolam and clonazepam) are first line treatment for PD. Whether the dPAG is also implicated in the antipanic effect of the latter drugs is, however, still unknown. We here investigated the consequences of blocking GABAA or benzodiazepine receptors within the dPAG, with bicuculline (5 pmol) and flumazenil (80 nmol), respectively, on the panicolytic and anxiolytic effects of alprazolam (4 mg/kg). Microinjection of these antagonists fully blocked the anti-escape effect, considered as a panicolytic-like action, caused by a single systemic injection of alprazolam in male Wistar rats submitted to the elevated T-maze. These antagonists, however, did not affect the anxiolytic effect of the benzodiazepine on inhibitory avoidance acquisition and punished responding, measured in the elevated T-maze and Vogel conflict tests, respectively. Altogether, our findings show the involvement of GABAA/benzodiazepine receptors of the dPAG in the panicolytic, but not the anxiolytic effect caused by alprazolam. They also implicate the dPAG as the fulcrum of the effects of different classes of clinically effective antipanic drugs.

Modulation of tonic immobility by GABAA and GABAB receptors of the medial amygdala.

Tonic immobility (TI) is a temporary state of profound motor inhibition associated with great danger as the attack of a predator. Previous studies carried out in our laboratory evidenced high Fos-IR in the posteroventral region of the medial nucleus of the amygdala (MEA) after induction of the TI response. Here, we investigated the effects of GABAA and GABAB of the MEA on TI duration. Intra-MEA injections of the GABAA agonist muscimol and GABAB agonist baclofen reduced TI response, while intra-MEA injections of the GABAA antagonist bicuculline and GABAB antagonist phaclofen increased the TI response. Moreover, the effects observed with muscimol and baclofen administrations into MEA were blocked by pretreatment with bicuculline and phaclofen (at ineffective doses per se). Finally, the activation of GABAA and GABAB receptors in the MEA did not alter the spontaneous motor activity in the open field test. These data support the role of the GABAergic system of the MEA in the modulation of innate fear.

GABAA Modulation of S100B Secretion in Acute Hippocampal Slices and Astrocyte Cultures.

Astrocytes are the major glial cells in brain tissue and are involved, among many functions, ionic and metabolic homeostasis maintenance of synapses. These cells express receptors and transporters for neurotransmitters, including GABA. GABA signaling is reportedly able to affect astroglial response to injury, as evaluated by specific astrocyte markers such as glial fibrillary acid protein and the calcium-binding protein, S100B. Herein, we investigated the modulatory effects of the GABAA receptor on astrocyte S100B secretion in acute hippocampal slices and astrocyte cultures, using the agonist, muscimol, and the antagonists pentylenetetrazol (PTZ) and bicuculline. These effects were analyzed in the presence of tetrodotoxin (TTX), fluorocitrate (FLC), cobalt and barium. PTZ positively modify S100B secretion in hippocampal slices and astrocyte cultures; in contrast, bicuculline inhibited S100B secretion only in hippocampal slices. Muscimol, per se, did not change S100B secretion, but prevented the effects of PTZ and bicuculline. Moreover, PTZ-induced S100B secretion was prevented by TTX, FLC, cobalt and barium indicating a complex GABAA communication between astrocytes and neurons. The effects of two putative agonists of GABAA, ß-hydroxybutyrate and methylglyoxal, on S100B secretion were also evaluated. In view of the neurotrophic role of extracellular S100B under conditions of injury, our data reinforce the idea that GABAA receptors act directly on astrocytes, and indirectly on neurons, to modulate astroglial response.