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

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

Ethanol and gene expression in brain

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Izuru Matusmoto and Peter A. Wilce. The presentations were (1) GABA receptor subunit expression in the human alcoholic brain, by Tracey Buckley and Peter Dodd; (2) NMDAR gene expression during ethanol addiction, by Jorg Puzke, Rainer Spanagel, Walther Zieglgansberger, and Gerald Wolf; (3) Differentially expressed gene in the nucleus accumbens from ethanol-administered rat, by Shuangying Leng; (4) Expression of a novel gene in the alcoholic brain, by Peter A. Wilce; and (5) Investigations of haplotypes of the dopamine Da-receptor gene in alcoholics, by Hans Rommelspacher, Ulrich Finckh, and Lutz G. Schmidt.

Excited to death: different ways to lose your neurones

The selective loss of neurones in a range of neurodegenerative diseases is widely thought to involve the process of excitotoxicity, in which glutamate-mediated neuronal killing is elaborated through the excessive stimulation of cell-surface receptors. Every such disease exhibits a distinct regional and subregional pattern of neuronal loss. so processes must be locally triggered to different extents to account for this. We have studied several mechanisms which could lead to excitotoxic glutamate pathophysiology and compared them in different diseases. Our data suggest that glutamate can reach toxic extracellular levels in Alzheimer disease by malfunctions in cellular transporters, and that the toxicity may be exacerbated by continued glutamate release from presynaptic neurones acting on hypersensitive postsynaptic receptors. Thus the excitotoxicity is direct. In contrast, alcoholic brain damage arises in regions where GABA-mediated inhibition is deficient, and fails properly to dampen trans-synaptic excitation, Thus the excitotoxicity is indirect. A variety of such mechanisms is possible, which may combine in different ways.

Muscimol-induced death of GABAergic neurons in rat brain aggregating cell cultures.

During brain development, spontaneous neuronal activity has been shown to play a crucial role in the maturation of neuronal circuitries. Activity-related signals may cause selective neuronal cell death and/or rearrangement of neuronal connectivity. To study the effects of sustained inhibitory activity on developing inhibitory (GABAergic) neurons, three-dimensional primary cell cultures of fetal rat telencephalon were used. In relatively immature cultures, muscimol (10 microns), a GABAA receptor agonist, induced a transient increase in apoptotic cell death, as evidenced by a cycloheximide-sensitive increase of free nucleosomes and an increased frequency of DNA double strand breaks (TUNEL labeling). Furthermore, muscimol caused an irreversible reduction of glutamic acid decarboxylase activity, indicating a loss of GABAergic neurons. The muscimol-induced death of GABAergic neurons was attenuated by the GABAA receptor blockers bicuculline (100 microns) and picrotoxin (100 microns), by depolarizing potassium concentrations (30 mM KCl) and by the L-type calcium channel activator BAY K8644 (2 microns). As compared to the cholinergic marker (choline acetyltransferase activity), glutamic acid decarboxylase activity was significantly more affected by various agents known to inhibit neuronal activity, including tetrodotoxin (1 micron), flunarizine (5 microns), MK 801 (50 microns) and propofol (40 microns). The present results suggest that the survival of a subpopulation of immature GABAergic neurons is dependent on sustained neuronal activity and that these neurons may undergo apoptotic cell death in response to GABAA autoreceptor activation.

Neuronal in vitro models for the estimation of acute systemic toxicity.

The objective of the EU funded integrated project "ACuteTox" is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA(A) receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro-in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.

Oxytocin selectively gates fear responses through distinct outputs from the central amygdala.

Central amygdala (CeA) projections to hypothalamic and brain stem nuclei regulate the behavioral and physiological expression of fear, but it is unknown whether these different aspects of the fear response can be separately regulated by the CeA. We combined fluorescent retrograde tracing of CeA projections to nuclei that modulate fear-related freezing or cardiovascular responses with in vitro electrophysiological recordings and with in vivo monitoring of related behavioral and physiological parameters. CeA projections emerged from separate neuronal populations with different electrophysiological characteristics and different response properties to oxytocin. In vivo, oxytocin decreased freezing responses in fear-conditioned rats without affecting the cardiovascular response. Thus, neuropeptidergic signaling can modulate the CeA outputs through separate neuronal circuits and thereby individually steer the various aspects of the fear response.

Novel anesthetics and other treatment strategies for refractory status epilepticus

Refractory status epilepticus (RSE)-that is, seizures resistant to at least two antiepileptic drugs (AEDs)-is generally managed with barbiturates, propofol, or midazolam, despite a low level of evidence (Rossetti, 2007). When this approach fails, the need for alternative pharmacologic and nonpharmacologic strategies emerges. These have been investigated even less systematically than the aforementioned compounds, and are often used, sometimes in succession, in cases of extreme refractoriness (Robakis & Hirsch, 2006). Several possibilities are reviewed here. In view of the marked heterogeneity of reported information, etiologies, ages, and comedications, it is extremely difficult to evaluate a given method, not to say to compare different strategies among them. Pharmacologic Approaches Isoflurane and desflurane may complete the armamentarium of anesthetics,' and should be employed in a ''close'' environment, in order to prevent intoxication of treating personnel. c-Aminobutyric acid (GABA)A receptor potentiation represents the putative mechanism of action. In an earlier report, isoflurane was used for up to 55 h in nine patients, controlling seizures in all; mortality was, however, 67% (Kofke et al., 1989). More recently, the use of these inhalational anesthetics was described in seven subjects with RSE, for up to 26 days, with an endtidal concentration of 1.2-5%. All patients required vasopressors, and paralytic ileus occurred in three; outcome was fatal in three patients (43%) (Mirsattari et al., 2004). Ketamine, known as an emergency anesthetic because of its favorable hemodynamic profile, is an N-methyl-daspartate (NMDA) antagonist; the interest for its use in RSE derives from animal works showing loss of GABAA efficacy and maintained NMDA sensitivity in prolonged status epilepticus (Mazarati & Wasterlain, 1999). However, to avoid possible neurotoxicity, it appears safer to combine ketamine with GABAergic compounds (Jevtovic-Todorovic et al., 2001; Ubogu et al., 2003), also because of a likely synergistic effect (Martin & Kapur, 2008). There are few reported cases in humans, describing progressive dosages up to 7.5 mg/kg/h for several days (Sheth & Gidal, 1998; Quigg et al., 2002; Pruss & Holtkamp, 2008), with moderate outcomes. Paraldehyde acts through a yet-unidentified mechanism, and appears to be relatively safe in terms of cardiovascular tolerability (Ramsay, 1989; Thulasimani & Ramaswamy, 2002), but because of the risk of crystal formation and its reactivity with plastic, it should be used only as fresh prepared solution in glass devices (Beyenburg et al., 2000). There are virtually no recent reports regarding its use in adults RSE, whereas rectal paraldehyde in children with status epilepticus resistant to benzodiazepines seems less efficacious than intravenous phenytoin (Chin et al., 2008). Etomidate is another anesthetic agent for which the exact mechanism of action is also unknown, which is also relatively favorable regarding cardiovascular side effects, and may be used for rapid sedation. Its use in RSE was reported in eight subjects (Yeoman et al., 1989). After a bolus of 0.3 mg/kg, a drip of up to 7.2 mg/kg/h for up to 12 days was administered, with hypotension occurring in five patients; two patients died. A reversible inhibition of cortisol synthesis represents an important concern, limiting its widespread use and implying a careful hormonal substitution during treatment (Beyenburg et al., 2000). Several nonsedating approaches have been reported. The use of lidocaine in RSE, a class Ib antiarrhythmic agent modulating sodium channels, was reviewed in 1997 (Walker & Slovis, 1997). Initial boluses up to 5 mg/kg and perfusions of up to 6 mg/kg/h have been mentioned; somewhat surprisingly, at times lidocaine seemed to be successful in controlling seizures in patients who were refractory to phenytoin. The aforementioned dosages should not be overshot, in order to keep lidocaine levels under 5 mg/L and avoid seizure induction (Hamano et al., 2006). A recent pediatric retrospective survey on 57 RSE episodes (37 patients) described a response in 36%, and no major adverse events; mortality was not given (Hamano et al., 2006 Verapamil, a calcium-channel blocker, also inhibits P-glycoprotein, a multidrug transporter that may diminish AED availability in the brain (Potschka et al., 2002). Few case reports on its use in humans are available; this medication nevertheless appears relatively safe (under cardiac monitoring) up to dosages of 360 mg/day (Iannetti et al., 2005). Magnesium, a widely used agent for seizures elicited by eclampsia, has also been anecdotally reported in RSE (Fisher et al., 1988; Robakis & Hirsch, 2006), but with scarce results even at serum levels of 14 mm. The rationale may be found in the physiologic blockage of NMDA channels by magnesium ions (Hope & Blumenfeld, 2005). Ketogenic diet has been prescribed for decades, mostly in children, to control refractory seizures. Its use in RSE as ''ultima ratio'' has been occasionally described: three of six children (Francois et al., 2003) and one adult (Bodenant et al., 2008) were responders. This approach displays its effect subacutely over several days to a few weeks. Because ''malignant RSE'' seems at times to be the consequence of immunologic processes (Holtkamp et al., 2005), a course of immunomodulatory treatment is often advocated in this setting, even in the absence of definite autoimmune etiologies (Robakis & Hirsch, 2006); steroids, adrenocorticotropic hormone (ACTH), plasma exchanges, or intravenous immunoglobulins may be used alone or in sequential combination. Nonpharmacologic Approaches These strategies are described somewhat less frequently than pharmacologic approaches. Acute implantation of vagus nerve stimulation (VNS) has been reported in RSE (Winston et al., 2001; Patwardhan et al., 2005; De Herdt et al., 2009). Stimulation was usually initiated in the operation room, and intensity progressively adapted over a few days up to 1.25 mA (with various regimens regarding the other parameters), allowing a subacute seizure control; one transitory episode of bradycardia/asystole has been described (De Herdt et al., 2009). Of course, pending identification of a definite seizure focus, resective surgery may also be considered in selected cases (Lhatoo & Alexopoulos, 2007). Low-frequency (0.5 Hz) transcranial magnetic stimulation (TMS) at 90% of the resting motor threshold has been reported to be successful for about 2 months in a patient with epilepsia partialis continua, but with a weaning effect afterward, implying the need for a repetitive use (Misawa et al., 2005). More recently, TMS was applied in a combination of a short ''priming'' high frequency (up to 100 Hz) and longer runs of low-frequency stimulations (1 Hz) at 90-100% of the motor threshold in seven other patients with simple-partial status, with mixed results (Rotenberg et al., 2009). Paradoxically at first glance, electroconvulsive treatment may be found in cases of extremely resistant RSE. A recent case report illustrates its use in an adult patient with convulsive status, with three sessions (three convulsions each) carried out over 3 days, resulting in a moderate recovery; the mechanism is believed to be related to modification of the synaptic release of neurotransmitters (Cline & Roos, 2007). Therapeutic hypothermia, which is increasingly used in postanoxic patients (Oddo et al., 2008), has been the object of a recent case series in RSE (Corry et al., 2008). Reduction of energy demand, excitatory neurotransmission, and neuroprotective effects may account for the putative mechanism of action. Four adult patients in RSE were cooled to 31_-34_C with an endovascular system for up to 90 h, and then passively rewarmed over 2-50 h. Seizures were controlled in two patients, one of whom died; also one of the other two patients in whom seizures continued subsequently deceased. Possible side effects are related to acid-base and electrolyte disturbances, and coagulation dysfunction including thrombosis, infectious risks, cardiac arrhythmia, and paralytic ileus (Corry et al., 2008; Cereda et al., 2009). Finally, anecdotic evidence suggests that cerebrospinal fluid (CSF)-air exchange may induce some transitory benefit in RSE (Kohrmann et al., 2006); although this approach was already in use in the middle of the twentieth century, the mechanism is unknown. Acknowledgment A wide spectrum of pharmacologic (sedating and nonsedating) and nonpharmacologic (surgical, or involving electrical stimulation) regimens might be applied to attempt RSE control. Their use should be considered only after refractoriness to AED or anesthetics displaying a higher level of evidence. Although it seems unlikely that these uncommon and scarcely studied strategies will influence the RSE outcome in a decisive way, some may be interesting in particular settings. However, because the main prognostic determinant in status epilepticus appears to be related to the underlying etiology rather than to the treatment approach (Rossetti et al., 2005, 2008), the safety issue should always represent a paramount concern for the prescribing physician. Conclusion The author confirms that he has read the Journal's position on issues involved in ethical publication and affirms that this paper is consistent with those guidelines.

Differential effects of sodium oxybate and baclofen on EEG, sleep, neurobehavioral performance, and memory.

STUDY OBJECTIVES: Sodium oxybate (SO) is a GABA(B) agonist used to treat the sleep disorder narcolepsy. SO was shown to increase slow wave sleep (SWS) and EEG delta power (0.75-4.5 Hz), both indexes of NREM sleep (NREMS) intensity and depth, suggesting that SO enhances recuperative function of NREM. We investigated whether SO induces physiological deep sleep. DESIGN: SO was administered before an afternoon nap or before the subsequent experimental night in 13 healthy volunteers. The effects of SO were compared to baclofen (BAC), another GABA(B) receptor agonist, to assess the role of GABA(B) receptors in the SO response. MEASUREMENTS AND RESULTS: As expected, a nap significantly decreased sleep need and intensity the subsequent night. Both drugs reversed this nap effect on the subsequent night by decreasing sleep latency and increasing total sleep time, SWS during the first NREMS episode, and EEG delta and theta (0.75-7.25 Hz) power during NREMS. The SO-induced increase in EEG delta and theta power was, however, not specific to NREMS and was also observed during REM sleep (REMS) and wakefulness. Moreover, the high levels of delta power during a nap following SO administration did not affect delta power the following night. SO and BAC taken before the nap did not improve subsequent psychomotor performance and subjective alertness, or memory consolidation. Finally, SO and BAC strongly promoted the appearance of sleep onset REM periods. CONCLUSIONS: The SO-induced EEG slow waves seem not to be functionally similar to physiological slow waves. Our findings also suggest a role for GABA(B) receptors in REMS generation. CITATION: Vienne J; Lecciso G; Constantinescu I; Schwartz S; Franken P; Heinzer R; Tafti M. Differential effects of sodium oxybate and baclofen on EEG, sleep, neurobehavioral performance, and memory. SLEEP 2012;35(8):1071-1084.

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.

In vivo co-ordinated interactions between inhibitory systems to control glutamate mediated hippocampal excitability.

We present an overview of the long-term adaptation of hippocampal neurotransmission to cholinergic and GABAergic deafferentation caused by excitotoxic lesion of the medial septum. Two months after septal microinjection of 2.7 nmol a -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), a 220% increase of GABA A receptor labelling in the hippo- campal CA3 and the hilus was shown, and also changes in hippocampal neurotransmission characterised by in vivo microdialysis and HPLC. Basal amino acid and purine extra- cellular levels were studied in control and lesioned rats. In vivo effects of 100 m M KCl perfusion and adenosine A 1 receptor blockade with 1,3-dipropyl- 8-cyclopentylxanthine (DPCPX) on their release were also investigated. In lesioned animals GABA, glutamate and glutamine basal levels were decreased and taurine, adenosine and uric acid levels increased. A similar response to KCl infusion occurred in both groups except for GABA and glutamate, which release decreased in lesioned rats. Only in lesioned rats, DPCPX increased GABA basal level and KCl-induced glutamate release, and decreased glutamate turnover. Our results evidence that an excitotoxic septal lesion leads to increased hippocampal GABA A receptors and decreased glutamate neurotransmis- sion. In this situation, a co-ordinated response of hippocampal retaliatory systems takes place to control neuron excitability.

Effects of centrally acting antihypertensive drugs on the microcirculation of spontaneously hypertensive rats

We investigated the acute effects of centrally acting antihypertensive drugs on the microcirculation of pentobarbital-anesthetized spontaneously hypertensive rats (SHR). The effects of the sympatho-inhibitory agents clonidine and rilmenidine, known to activate both alpha2-adrenoceptors and nonadrenergic I1-imidazoline binding sites (I1BS) in the central nervous system, were compared to those of dicyclopropylmethyl-(4,5-dimethyl-4,5-dihydro-3H -pyrrol-2-yl)-amine hydrochloride (LNP 509), which selectively binds to the I1BS. Terminal mesenteric arterioles were observed by intravital microscopy. Activation of the central sympathetic system with L-glutamate (125 µg, ic) induced marked vasoconstriction of the mesenteric microcirculation (27 ± 3%; N = 6, P < 0.05). In contrast, the marked hypotensive and bradycardic effects elicited by intracisternal injection of clonidine (1 µg), rilmenidine (7 µg) and LNP 509 (60 µg) were accompanied by significant increases in arteriolar diameter (12 ± 1, 25 ± 10 and 21 ± 4%, respectively; N = 6, P < 0.05). The vasodilating effects of rilmenidine and LNP 509 were two-fold higher than those of clonidine, although they induced an identical hypotensive effect. Central sympathetic inhibition elicited by baclofen (1 µg, ic), a GABA B receptor agonist, also resulted in vasodilation of the SHR microvessels. The acute administration of clonidine, rilmenidine and LNP 509 also induced a significant decrease of cardiac output, whereas a decrease in systemic vascular resistance was observed only after rilmenidine and LNP 509. We conclude that the normalization of blood pressure in SHR induced by centrally acting antihypertensive agents is paralleled by important vasodilation of the mesenteric microcirculation. This effect is more pronounced with substances acting preferentially (rilmenidine) or exclusively (LNP 509) upon I1BS than with those presenting important alpha2-adrenergic activity (clonidine).

The inactivation of the basolateral nucleus of the rat amygdala has an anxiolytic effect in the elevated T-maze and light/dark transition tests

Pharmacological evidence indicates that the basolateral nucleus of the amygdala (BLA) is involved in the mediation of inhibitory avoidance but not of escape behavior in the elevated T-maze test. These defensive responses have been associated with generalized anxiety disorder (GAD) and panic disorder, respectively. In the present study, we determined whether the BLA plays a differential role in the control of inhibitory avoidance and escape responses in the elevated T-maze. Male Wistar rats (250-280 g, N = 9-10 in each treatment group) were pre-exposed to one of the open arms of the maze for 30 min and 24 h later tested in the model after inactivation of the BLA by a local injection of the GABA A receptor agonist muscimol (8 nmol in 0.2 µL). It has been shown that a prior forced exposure to one of the open arms of the maze, by shortening latencies to withdrawal from the open arm during the test, improves the escape task as a behavioral index of panic. The effects of muscimol in the elevated T-maze were compared to those caused by this GABA agonist in the avoidance reaction generated in the light/dark transition test. This defensive behavior has also been associated with GAD. In the elevated T-maze, intra-BLA injection of muscimol impaired inhibitory avoidance (control: 187.70 ± 14.90 s, muscimol: 37.10 ± 2.63 s), indicating an anxiolytic effect, without interfering with escape performance. The drug also showed an anxiolytic effect in the light/dark transition test as indicated by the increase in the time spent in the lighted compartment (control: 23.50 ± 2.45 s, muscimol: 47.30 ± 4.48 s). The present findings point to involvement of the BLA in the modulation of defensive responses that have been associated with GAD.

Effect of 4-aminoantipyrine on gastric compliance and liquid emptying in rats

Dipyrone (Dp) delays gastric emptying (GE) in rats. There is no information about whether 4-aminoantipyrine (AA), one of its metabolites, has the same effect. The objectives of the present study were to assess the effects of AA and Dp on GE when administered intravenously (iv) and intracerebroventricularly (icv) (240 µmol/kg and 4 µmol/animal, respectively) and on gastric compliance when administered iv (240 µmol/kg). GE was determined in male Wistar rats weighing 250-300 g (5-10 per group) after icv or iv injection of the drug by measuring percent gastric retention (GR) of a saline meal labeled with phenol red 10 min after administration by gavage. Gastric compliance was estimated in anesthetized rats (10-11 per group), with the construction of volume-pressure curves during intragastric infusion of a saline meal. Compliance was significantly greater in animals receiving Dp (mean ± SEM = 0.26 ± 0.009 mL/mmHg) and AA (0.24 ± 0.012 mL/mmHg) than in controls (0.19 ± 0.009 mL/mmHg). AA and Dp administered iv significantly increased GR (64.4 ± 2.5 and 54.3 ± 3.8%, respectively) compared to control (34 ± 2.2%), a phenomenon observed only with Dp after icv administration. Subdiaphragmatic vagotomy reduced the effect of AA (GR = 31.4 ± 1.5%) compared to sham-treated animals. Baclofen, a GABA B receptor agonist, administered icv significantly reduced the effect of AA (GR = 28.1 ± 1.3%). We conclude that Dp and AA increased gastric compliance and AA delayed GE, with the participation of the vagus nerve, through a pathway that does not involve a direct action of the drug on the central nervous system.

Anandamide injected into the lateral ventricle of the brain inhibits submandibular salivary secretion by attenuating parasympathetic neurotransmission

Our objective was to determine the effect of arachidonylethanolamide (anandamide, AEA) injected intracerebroventricularly (icv) into the lateral ventricle of the rat brain on submandibular gland (SMG) salivary secretion. Parasympathetic decentralization (PSD) produced by cutting the chorda tympani nerve strongly inhibited methacholine (MC)-induced salivary secretion while sympathetic denervation (SD) produced by removing the superior cervical ganglia reduced it slightly. Also, AEA (50 ng/5 µL, icv) significantly decreased MC-induced salivary secretion in intact rats (MC 1 µg/kg: control (C), 5.3 ± 0.6 vs AEA, 2.7 ± 0.6 mg; MC 3 µg/kg: C, 17.6 ± 1.0 vs AEA, 8.7 ± 0.9 mg; MC 10 µg/kg: C, 37.4 ± 1.2 vs AEA, 22.9 ± 2.6 mg). However, AEA did not alter the significantly reduced salivary secretion in rats with PSD, but decreased the slightly reduced salivary secretion in rats with SD (MC 1 µg/kg: C, 3.8 ± 0.8 vs AEA, 1.4 ± 0.6 mg; MC 3 µg/kg: C, 14.7 ± 2.4 vs AEA, 6.9 ± 1.2 mg; P < 0.05; MC 10 µg/kg: C, 39.5 ± 1.0 vs AEA, 22.3 ± 0.5 mg; P < 0.001). We showed that the inhibitory effect of AEA is mediated by cannabinoid type 1 CB1 receptors and involves GABAergic neurotransmission, since it was blocked by previous injection of the CB1 receptor antagonist AM251 (500 ng/5 µL, icv) or of the GABA A receptor antagonist, bicuculline (25 ng/5 µL, icv). Our results suggest that parasympathetic neurotransmission from the central nervous system to the SMG can be inhibited by endocannabinoid and GABAergic systems.

Effects of reversible inactivation of the dorsomedial hypothalamus on panic- and anxiety-related responses in rats

The medial hypothalamus is part of a neurobiological substrate controlling defensive behavior. It has been shown that a hypothalamic nucleus, the dorsomedial hypothalamus (DMH), is involved in the regulation of escape, a defensive behavior related to panic attacks. The role played by the DMH in the organization of conditioned fear responses, however, is less clear. In the present study, we investigated the effects of reversible inactivation of the DMH with the GABA A agonist muscimol on inhibitory avoidance acquisition and escape expression by male Wistar rats (approximately 280 g in weight) tested in the elevated T-maze (ETM). In the ETM, inhibitory avoidance, a conditioned defensive response, has been associated with generalized anxiety disorder. Results showed that intra-DMH administration of the GABA A receptor agonist muscimol inhibited escape performance, suggesting an antipanic-like effect (P < 0.05), without changing inhibitory avoidance acquisition. Although a higher dose of muscimol (1.0 nmol/0.2 µL; N = 7) also altered locomotor activity in an open field when compared to control animals (0.2 µL saline; N = 13) (P < 0.05), the lower dose (0.5 nmol/0.2 µL; N = 12) of muscimol did not cause any motor impairment. These data corroborate previous evidence suggesting that the DMH is specifically involved in the modulation of escape. Dysfunction of this regulatory mechanism may be relevant in the genesis/maintenance of panic disorder.