Antinociceptive effect of aqueous extract of Origanum vulgare L. in male rats: possible involvement of the GABAergic system

The objective of the present investigation was to assess the possible involvement of GABAergic mechanism in analgesic effect of aqueous extract of Origanum Vulgare [ORG] in a rat model of acute pain test. Sixty-three anaesthetized male Wistar rats [200-250 g] were cannulated into the left ventricle. Five to seven days after the recovery from surgery, ORG extract was intraventricularly injected at dose of 3 ?g/rat i.c.v. Then, baclofen [10 mg/Kg, IP], CGP35348 [100 nmol/Kg, i.c.v], muscimol [1 mg/Kg IP] and bicuculline [5 mg/Kg IP] were separately injected 20 min before the injection of ORG. The experimental groups were compared with intact [control] group [n = 7]. The response latency of rats to thermal stimulation was recorded using Tail-Flick test. Injection of ORG extract resulted in a significant and dose-dependent increase in the response latency. There was also a significant increase in the response latency after co-administration of ORG extract with baclofen when compared with control group. However, following co-administration of ORG extract/bicuculline, a significant decrease in the response latency was observed compared to control group. In conclusion, the results of the present study suggest that aqueous extract of Origanum vulgare L. ssp. viridis possesses antinociceptive activity in a dose-dependent manner and ORG-induced antinociception might be mediated, at least in part, by both GABA receptors

Dorsal periaqueductal gray stimulation facilitates anxiety-, but not panic-related, defensive responses in rats tested in the elevated T-maze

The escape response to electrical or chemical stimulation of the dorsal periaqueductal gray matter (DPAG) has been associated with panic attacks. In order to explore the validity of the DPAG stimulation model for the study of panic disorder, we determined if the aversive consequences of the electrical or chemical stimulation of this midbrain area can be detected subsequently in the elevated T-maze. This animal model, derived from the elevated plus-maze, permits the measurement in the same rat of a generalized anxiety- and a panic-related defensive response, i.e., inhibitory avoidance and escape, respectively. Facilitation of inhibitory avoidance, suggesting an anxiogenic effect, was detected in male Wistar rats (200-220 g) tested in the elevated T-maze 30 min after DPAG electrical stimulation (current generated by a sine-wave stimulator, frequency at 60 Hz) or after local microinjection of the GABA A receptor antagonist bicuculline (5 pmol). Previous electrical (5, 15, 30 min, or 24 h before testing) or chemical stimulation of this midbrain area did not affect escape performance in the elevated T-maze or locomotion in an open-field. No change in the two behavioral tasks measured by the elevated T-maze was observed after repetitive (3 trials) electrical stimulation of the DPAG. The results indicate that activation of the DPAG caused a short-lived, but selective, increase in defensive behaviors associated with generalized anxiety.

Panic-like defensive behavior but not fear-induced antinociception is differently organized by dorsomedial and posterior hypothalamic nuclei of Rattus norvegicus (Rodentia, Muridae)

The hypothalamus is a forebrain structure critically involved in the organization of defensive responses to aversive stimuli. Gamma-aminobutyric acid (GABA)ergic dysfunction in dorsomedial and posterior hypothalamic nuclei is implicated in the origin of panic-like defensive behavior, as well as in pain modulation. The present study was conducted to test the difference between these two hypothalamic nuclei regarding defensive and antinociceptive mechanisms. Thus, the GABA A antagonist bicuculline (40 ng/0.2 µL) or saline (0.9% NaCl) was microinjected into the dorsomedial or posterior hypothalamus in independent groups. Innate fear-induced responses characterized by defensive attention, defensive immobility and elaborate escape behavior were evoked by hypothalamic blockade of GABA A receptors. Fear-induced defensive behavior organized by the posterior hypothalamus was more intense than that organized by dorsomedial hypothalamic nuclei. Escape behavior elicited by GABA A receptor blockade in both the dorsomedial and posterior hypothalamus was followed by an increase in nociceptive threshold. Interestingly, there was no difference in the intensity or in the duration of fear-induced antinociception shown by each hypothalamic division presently investigated. The present study showed that GABAergic dysfunction in nuclei of both the dorsomedial and posterior hypothalamus elicit panic attack-like defensive responses followed by fear-induced antinociception, although the innate fear-induced behavior originates differently in the posterior hypothalamus in comparison to the activity of medial hypothalamic subdivisions.

Effect of Sildenafil on Neuropathic Pain and Hemodynamics in Rats

PURPOSE: The inhibition of phosphodiesterase 5 produces an antinociception through the increase of cyclic guanosine monophosphate (cGMP), and increasing cGMP levels enhance the release of gamma-aminobutyric acid (GABA). Furthermore, this phosphodiesterase 5 plays a pivotal role in the regulation of the vasodilatation associated to cGMP. In this work, we examined the contribution of GABA receptors to the effect of sildenafil, a phosphodiesterase 5 inhibitor, in a neuropathic pain rat, and assessed the hemodynamic effect of sildenafil in normal rats. MATERIALS AND METHODS: Neuropathic pain was induced by ligation of L5/6 spinal nerves in Sprague-Dawley male rats. After observing the effect of intravenous sildenafil on neuropathic pain, GABAA receptor antagonist (bicuculline) and GABAB receptor antagonist (saclofen) were administered prior to delivery of sildenafil to determine the role of GABA receptors in the activity of sildenafil. For hemodynamic measurements, catheters were inserted into the tail artery. Mean arterial pressure (MAP) and heart rate (HR) were measured over 60 min following administration of sildenafil. RESULTS: Intravenous sildenafil dose-dependently increased the withdrawal threshold to the von Frey filament application in the ligated paw. Intravenous bicuculline and saclofen reversed the antinociception of sildenafil. Intravenous sildenafil increased the magnitude of MAP reduction at the maximal dosage, but it did not affect HR response. CONCLUSION: These results suggest that sildenafil is active in causing neuropathic pain. Both GABAA and GABAB receptors are involved in the antinociceptive effect of sildenafil. Additionally, intravenous sildenafil reduces MAP without affecting HR.

GABA in the central amygdaloid nucleus modulates the electrolyte excretion and hormonal responses to blood volume expansion in rats

We investigated the involvement of GABAergic mechanisms of the central amygdaloid nucleus (CeA) in unanesthetized rats subjected to acute isotonic or hypertonic blood volume expansion (BVE). Male Wistar rats bearing cannulas unilaterally implanted in the CeA were treated with vehicle, muscimol (0.2 nmol/0.2 µL) or bicuculline (1.6 nmol/0.2 µL) in the CeA, followed by isotonic or hypertonic BVE (0.15 or 0.3 M NaCl, 2 mL/100 g body weight over 1 min). The vehicle-treated group showed an increase in sodium excretion, urinary volume, plasma oxytocin (OT), and atrial natriuretic peptide (ANP) levels compared to control rats. Muscimol reduced the effects of BVE on sodium excretion (isotonic: 2.4 ± 0.3 vs vehicle: 4.8 ± 0.2 and hypertonic: 4.0 ± 0.7 vs vehicle: 8.7 ± 0.6 µEq·100 g-1·40 min-1); urinary volume after hypertonic BVE (83.8 ± 10 vs vehicle: 255.6 ± 16.5 µL·100 g-1·40 min-1); plasma OT levels (isotonic: 15.3 ± 0.6 vs vehicle: 19.3 ± 1 and hypertonic: 26.5 ± 2.6 vs vehicle: 48 ± 3 pg/mL), and ANP levels (isotonic: 97 ± 12.8 vs vehicle: 258.3 ± 28.1 and hypertonic: 160 ± 14.6 vs vehicle: 318 ± 16.3 pg/mL). Bicuculline reduced the effects of isotonic or hypertonic BVE on urinary volume and ANP levels compared to vehicle-treated rats. However, bicuculline enhanced the effects of hypertonic BVE on plasma OT levels. These data suggest that CeA GABAergic mechanisms are involved in the control of ANP and OT secretion, as well as in sodium and water excretion in response to isotonic or hypertonic blood volume expansion.

Agonistic-like responses from the torus semicircularis dorsalis elicited by GABA A blockade in the weakly electric fish Gymnotus carapo

Findings by our group have shown that the dorsolateral telencephalon of Gymnotus carapo sends efferents to the mesencephalic torus semicircularis dorsalis (TSd) and that presumably this connection is involved in the changes in electric organ discharge (EOD) and in skeletomotor responses observed following microinjections of GABA A antagonist bicuculline into this telencephalic region. Other studies have implicated the TSd or its mammalian homologue, the inferior colliculus, in defensive responses. In the present study, we explore the possible involvement of the TSd and of the GABA-ergic system in the modulation of the electric and skeletomotor displays. For this purpose, different doses of bicuculline (0.98, 0.49, 0.245, and 0.015 mM) and muscimol (15.35 mM) were microinjected (0.1 æL) in the TSd of the awake G. carapo. Microinjection of bicuculline induced dose-dependent interruptions of EOD and increased skeletomotor activity resembling defense displays. The effects of the two highest doses showed maximum values at 5 min (4.3 ± 2.7 and 3.8 ± 2.0 Hz, P < 0.05) and persisted until 10 min (11 ± 5.7 and 8.7 ± 5.2 Hz, P < 0.05). Microinjections of muscimol were ineffective. During the interruptions of EOD, the novelty response (increased frequency in response to sensory novelties) induced by an electric stimulus delivered by a pair of electrodes placed in the water of the experimental cuvette was reduced or abolished. These data suggest that the GABA-ergic mechanisms of the TSd inhibit the neural substrate of the defense reaction at this midbrain level.

Effect of cyclooxygenase-2 (COX-2) inhibitors in various animal models (bicuculline, picrotoxin, maximal electroshock-induced convulsions) of epilepsy with possible mechanism of action.

Enzyme cyclooxygenase (COX) is reported to play a significant role in neurodegeneration and may play a significant role in the pathogenesis of epilepsy. Bicuculline (4 mg/kg; ip), picrotoxin (8 mg/kg; ip) and electroshock (60 mA for 0.2 sec) significantly induced convulsions in male Laka mice. COX-inhibitors viz. nimesulide (2.5 mg/kg; ip) and rofecoxib (2 mg/kg, ip) administered 45 minutes prior to an epileptic challenge prolonged mean onset time of convulsions, decreased duration of clonus and decreased % mortality rate against bicuculline- and picrotoxin-induced convulsions in mice. COX-2 inhibitors were ineffective towards maximal electroshock-induced convulsions. Nimesulide (1 mg/kg) and rofecoxib (1 mg/kg) also enhanced the effect of subprotective dose of muscimol against picrotoxin-induced convulsions. The result of the present study strongly suggests for a possible role of cyclooxygenase isoenzymes particularly, COX-2 in the pathophysiology of epilepsy and its GABAergic modulation.

Attenuation of the effect of progesterone and 4'-chlordiazepam on stress-induced immune responses by bicuculline.

The present study investigates the effect of progesterone, a pregnane precursor of neurosteroids, and 4'-chlordiazepam (4'-CD), a specific ligand for mitochondrial diazepam binding inhibitor receptor (MDR) involved in neurosteroidogenesis, on restraint stress (RS)-induced modulation of humoral and cell-mediated immune responses. RS produced a significant reduction in anti-sheep red blood cells (SRBC) antibody titre, a measure of humoral immune response, and % leucocyte migration inhibition (LMI) and foot-pad thickness test, measures of cell-mediated immune responses. These effects of RS on immune responses were effectively blocked by pretreating the animals with progesterone (10 mg/kg, sc) or 4'-CD (0.5 mg/kg, sc) administered just before subjecting the animal to RS. The effect of both progesterone and 4'-CD on RS-induced immune modulation was significantly attenuated by bicuculline (2 mg/kg, ip) but not by flumazenil (10 mg/kg, ip). Unlike its effect on RS-induced immune responsiveness, progesterone (5, 10 mg/kg, sc) when administered to non-stressed animals produced a significant suppression of both humoral and cell-mediated immune responses which was not reversed by bicuculline. However, 4'-CD failed to modulate immune response in naive non-stressed animals. These results suggest that progesterone and 4'-CD affect stress-induced immune responses by modulating GABA-ergic mechanism. However, GABA-A receptor system does not appear to be involved in progesterone-induced immunosuppression in nonstressed animals.

Evidence for presence of GABA-ergic receptor mediated dispersion in isolated scale melanophores of a carp, Cirrhinus mrigala Ham.

Effects of GABA-ergic agonists and antagonists were examined on the melanophores of a carp C. mrigala in vitro. GABA and baclofen both induced concentration - related dispersion in fish melanophores. Denervation of the melanophores by reserpine treatment potentiated the sensitivity of the melanophores to GABA. While denervation by cooling treatment inhibited the sensitivity of the melanophores to GABA, atropine, bicuculline and pentylenetetrazole all inhibited the dispersal responses of the melanophores induced by higher concentrations of GABA. 5-aminovaleric acid also significantly inhibited the dispersion of the melanophores induced either by GABA or baclofen. It is concluded that GABA-ergic agonist induced dispersal responses in C mrigala melanophores are mediated through specific GABA receptors. The presence of both GABAA and GABAB receptors in this fish melanophores has been indicated.

Acute phenobarbital administration induces hyperalgesia: pharmacological evidence for the involvement of supraspinal GABA-A receptors

The aim of the present study was to determine if phenobarbital affects the nociception threshold. Systemic (1-20 mg/kg) phenobarbital administration dose dependently induced hyperalgesia in the tail-flick, hot-plate and formalin tests in rats and in the abdominal constriction test in mice. Formalin and abdominal constriction tests were the most sensitive procedures for the detection of hyperalgesia in response to phenobarbital compared with the tail-flick and hot-plate tests. The hyperalgesia induced by systemic phenobarbital was blocked by previous administration of 1 mg/kg ip picrotoxin or either 1-2 mg/kg sc or 10 ng icv bicuculline. Intracerebroventricular phenobarbital administration (5 æg) induced hyperalgesia in the tail-flick test. In contrast, intrathecal phenobarbital administration (5 æg) induced antinociception and blocked systemic-induced hyperalgesia in this test. We suggest that phenobarbital may mediate hyperalgesia through GABA-A receptors at supraspinal levels and antinociception through the same kind of receptors at spinal levels

Effects of GABA on pancreatic exocrine secretion of rats

Since GABA and its related enzymes had been determined in beta-cells of pancreas islets, effects of GABA on pancreatic exocrine secretion were investigated in the isolated perfused rat pancreas. GABA, given intra-arterially at concentrations of 3, 10, 30 and 100 microM, did not exert any influence on spontaneous or secretin (12 pM)-induced pancreatic exocrine secretion. However, GABA further elevated cholecystokinin (10 pM)-, gastrin-releasing peptide (100 pM)- or electrical field stimulation-induced pancreatic secretions of fluid and amylase, dose-dependently. The GABA-enhanced CCK-induced pancreatic secretions were completely blocked by bicuculline (10 microM), a GABAA receptor antagonist but not affected by saclofen (10 microM), a GABA(B) receptor antagonist. The enhancing effects of GABA (30 microM) on CCK-induced pancreatic secretions were not changed by tetrodotoxin (1 microM) but partially reduced by cyclo-(7-aminoheptanonyl-Phe-D-Trp-Lys-Thr[BZL]) (10 microM), a somatostatin antagonist. In conclusion, GABA enhances pancreatic exocrine secretion induced by secretagogues, which stimulate enzyme secretion predominantly, via GABA(A) receptors in the rat pancreas. The enhancing effect of GABA is partially mediated by inhibition of islet somatostatin release. GABA does not modify the activity of intrapancreatic neurons.

The primate frontal eye field and the generation of saccadic eye movements: comparison of lesion and acute inactivation/activation studies

The frontal eye field (FEF) of monkeys has been repeatedly implicated in the generation of saccadic eye movements by various experimental approaches. Electrical stimulation of most of the FEF produces saccadic eye movements, many cells have activities related to saccades, and it has anatomical connections with many other oculomotor ares. Surprisingly, complete lesions of the FEF have remarkably little effect on oculomotor behavior. Only when more cognitive aspects are tested is a deficit clearly detected. In contrast, acute inactivation of the FEF on monkeys with the GABA agonist muscimol produced much more severe oculomotor impairment. This difference is probably due to the acute nature of the muscimol effect, which does not allow time for reorganization of the control of eye movements before testing begins. In addition, acute activation of the FEF with the GABA antagonist bicuculline caused the monkey to make irrepressible saccades of the same dimensions as those electrically elicited at the site. These experiments further confirm the strong involvement of the FEF in the control of saccadic eye movements and fixation.

Neuropharmacology of amide derivatives of P-GABA.

Three lipophilic amide derivatives of phthaloyl-GABA (P-GABA), namely gamma-phthalimido N-amyl butyramide (PGA), gamma-phthalimido-N-hexylbutyramide (PGH) and gamma-phthalimido N-phenylbutyramide (PGP), were synthesized and evaluated for their hypnotic and anticonvulsant activities in mice. Both PGA and PGH showed moderate hypnotic activity but PGP had no such action. Picrotoxin (0.08 mg/kg) a non-specific GABA antagonist completely abolished the hypnotic action of PGA in subconvulsive doses. Bicuculline (0.04 mg/kg) a GABAA antagonist, 3-mercaptopropionic acid (6 mg/kg) a GAD inhibitor at subconvulsive doses failed to neutralise the hypnotic action of PGA. On the other hand, PGA showed significant protection only against picrotoxin-induced convulsions, but was inactive against other convulsants tested. PGP which has no hypnotic activity, and has a mild anticonvulsant action in all the models except picrotoxin. A definite correlation was observed between the brain GABA and the hypnotic activity of PGA. However the present data indicate that the hypnotic and anticonvulsant activities are mediated probably through different brain GABA-ergic mechanisms.

Behavioral effects of neurotensin applied to periventricular structures of rats

Neurotensin (NT), n active neuropeptide, and bicuculline, a GABA-A receptor antagonist, were microinjected into the rat hypothalaamus (MH) or the dorsal periaqueductal gray matter (DPAG). Bicuculline (80 pmol) produced behavioral activation which included jumping and NT (1-20 nmol) caused a dose-dependent behavioral activation accompanied by catalepsy rather than jumping. These results suggest that the behavioral activation produced by NT may be due to an interaction of the neuropeptide with specific receptors while its cataleptic effect may be attributed to the blockade of dopamine receptors

Increased susceptibility of detelencephalated rats to audiogenic seizures induced by microinjection of bicuculline into the inferior colliculus

The participation of telencephalic forebrain structures in the induction of audiogenic seizure (AGS) susceptibility and in the behavioral expression of AGS was investigated in rats. Rats that were initially susceptible (N = 12) or non-susceptible (N = 28) to audiogenic seizure were surgically detelencephalated. A unilateral microinjection of a low dose (30 pmol) of the GABA antagonist bicuculline methiodide (BM) was applied to the inferior colliculus (IC) before the animals were exposed to a 120-dB acoustic stimulus. All susceptible rats still exhibited all components of audiogenic seizure after removal of the telencephalon. After BM microinjection, a higher incidence (66% vs 41%) and shorter latencies (6-20 s vs 9-55) s) of occurrence of tonic seizures were observed in the detelencephalated non-susceptible rats when compared to non-operated non-susceptible rats(N = 12). These results suggest that the induction of the behavioral expression of audiogenic seizures issubserved by brain stem neuronal networks but does not require the telencephalon and that telencephalic structures may exert control over audiogenic seizures by inhibiting IC cells through GABAergic neurons

Gabaergic fibers from substantia nigra para reticulata modulate escape bahavior induced by midbrain central gray stimulation

Eletrical stimulation or microinjection of GABA antagonists into the dorsal periaqueductal gray (DPAG) produces escape behavior. In order to determine whether the nigrocollicular gabaergic fibers exert some control over this behavior, rats bearing kainic acid lesion of the substantia nigra pars reticulata were submitted to microinjections of bicuculline or electrical stimulation of the DPAG at the escape threshold. Rats thus treated exhibited a significant decrease in the escape threshold while bicuculline increased the expression of flight behavior. These results suggest an inhibitory control of gabaergic fibers from the substantia nigra pars reticulata on aversive behavior induced by DPAG stimulation

Citrate Synthase ande lactate dehydrogenase activities in rat cerebral cortex following the administration of the convulsants bicuculline and 3-mercaptopropionic acid

La administración intraperitoneal de bicuculina (Bic) y ácido 3-mercaptopropiónico (MP) produce convulsiones generalizadas en animales de laboratorio. En este trabajo se estudió el efecto de estos convulsivantes sobre la actividad de la lactato deshidrogenasa y de citrato sintasa de corteza cerebral de rata. La Bic se administró en dosis de1.0 mg/Kg (subconvulsiva) y 7.5 mg/Kg (convulsiva) y el MP en dosis de 150 mg/Kg (convulsiva). La actividad de lactato deshidrogenasa en fracciones solubre y particulada de corteza cerebral no se modificó por la administración de Bic o MP. La actividad de citrato sintasa en homogeneizados de corteza cerebral aumentó alrededor del 40% por la administración de Bic en dosis subconvulsiva y convulsiva; un aumento semejante se encontró por la administración de MP. No se encontró modificación en la actividad de la enzima hepática, sugiriendo especificidad de tejido. La mayor actividad de citrato sintasa en homogeneizados de corteza cerebral encontrada luego de la administración de los convulsivantes se correlaciona con el aumento en los niveles de citrato cerebral descriptos en estados convulsivos