Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N.E. Brown essential oil

The objective of this study was to identify the possible involvement of the GABAergic system in the anesthetic effect of Lippia alba essential oil (EO). We propose a new animal model using silver catfish (Rhamdia quelen) exposed to an anesthetic bath to study the mechanism of action of EO. To observe the induction and potentiation of the anesthetic effect of EO, juvenile silver catfish (9.30 ± 1.85 g; 10.15 ± 0.95 cm; N = 6) were exposed to various concentrations of L. alba EO in the presence or absence of diazepam [an agonist of high-affinity binding sites for benzodiazepinic (BDZ) sites coupled to the GABAA receptor complex]. In another experiment, fish (N = 6) were initially anesthetized with the EO and then transferred to an anesthetic-free aquarium containing flumazenil (a selective antagonist of binding sites for BDZ coupled to the GABAA receptor complex) or water to assess recovery time from the anesthesia. In this case, flumazenil was used to observe the involvement of the GABA-BDZ receptor in the EO mechanism of action. The results showed that diazepam potentiates the anesthetic effect of EO at all concentrations tested. Fish exposed to diazepam and EO showed faster recovery from anesthesia when flumazenil was added to the recovery bath (12.0 ± 0.3 and 7.2 ± 0.7, respectively) than those exposed to water (9.2 ± 0.2 and 3.5 ± 0.3, respectively). In conclusion, the results demonstrated the involvement of the GABAergic system in the anesthetic effect of L. alba EO on silver catfish.

Lippia alba (Mill.) N.E. Brown, known as "false-melissa" in Brazil, is an aromatic shrub whose great chemical variability also leads to variations in its pharmacological activity. In Brazil, three main chemotypes are reported and classi-fied according to their major constituent as citral, carvone and linalool (7). This plant has been commonly used for its sedative properties, which have been demonstrated in some rodent studies (7,8). Recently, the anesthetic effect of L. alba essential oil (EO) was demonstrated on silver catfish (Rhamdia quelen). The EO of L. alba is considered to be a novel natural sedative and anesthetic agent that can be potentially used in aquaculture practices due to its ability to reduce stress in fish with a consequent reduction of economic losses in fish culture (9).
Most anesthetics exert their effects by regulating the gamma-aminobutyric acid (GABA) receptor complex, as www.bjournal.com.br Braz J Med Biol Res 45 (5) 2012 GABA is the main inhibitory neurotransmitter in the central nervous system (10). Synaptic GABA release activates GABA A and GABA C ionotropic receptors as well as the metabotropic receptor GABA B (11,12). GABA A receptors have been implicated in the majority of GABAergic signaling in mammals. High-affinity binding sites for benzodiazepines (BDZ) are known as "central BDZ receptors", and these compounds exert their pharmacological action by allosteric modulation in these regions. The activation of these sites enhances GABA-induced chloride ion flux, resulting in hyperpolarization and central depression. Expression of the GABAergic system seems to be conserved among vertebrates, from fishes to mammals (13). This system has been detected in certain parts of the zebrafish brain (13,14) and could be involved in their response to anesthetics. The mechanism of action of L. alba EO has not yet been elucidated. In order to determine the mechanism of the anesthetic effect of EO, we propose a new animal model using silver catfish to examine the involvement of the GABAergic mechanism in fish exposed to an anesthetic bath.

Material and Methods
Plant material L. alba was grown at the CESNORS/UFSM Campus, Frederico Westphalen, southern Brazil (geographically situated at 27°23'26" South, 53°25'43" West and 461 m above sea level). The species was identified by Gilberto Dolejal Zanetti, Department of Industrial Pharmacy, UFSM, and a voucher specimen (SMDB No. 10050) was deposited in the herbarium of the Department of Biology, UFSM.

EO extraction and analysis
EO was obtained from fresh L. alba leaves using the hydrodistillation process with a Clevenger type apparatus (15) for 2 h. The yield was calculated as w/w (%). The EO was stored at -20°C until composition analysis and testing. For comparison purposes, the composition of the EO used in this study (EO1) and the corresponding data for the EO used by Cunha et al. (9) (EO2) are presented in Table 1. The compositions of both EOs were analyzed by GC-MS.
The constituents of the EOs were identified by comparison of the mass spectra with a mass spectral library (16) and by comparison of the Kovats retention index with literature data (17).

Animals
Juvenile silver catfish (9.30 ± 1.85 g; 10.15 ± 0.95 cm) were housed in continuously aerated 250-L tanks (18.46 ± 0.51°C; pH 7.47 ± 0.073; total ammonia levels, 0.0883 mg/L; dissolved oxygen levels, 8.84 ± 0.37 mg/L). A semistatic system was used, and 50% of the water volume was changed daily. The fish received a commercial diet (Vicente Alimentos S.A., Brazil) with 3.5% Ca 2+ , 28.0% crude protein and 3500 kcal/kg digestible energy, as stated by the manufacturer. Juveniles were fed once a day and were fasted for a period of 24 h prior to the experiments. The water parameters were measured before the experiments. Dissolved oxygen and temperature were measured with a YSI oxygen meter (Model Y5512; YSI Inc., USA). The pH was verified with a DMPH-2 pH meter (Digimed, Brazil) and nesslerization was used to verify total ammonia levels according to the method of Greenberg et al. (18).
The experiments were approved by the Ethics and Animal Welfare Committee of Universidade Federal de Santa Maria (protocol No. 23081.014553/2009-39).

Standardization of the test conditions
The depressor effect of anesthetics in fish was evaluated by the method of Schoettger and Julin (19). The method involves six stages, in which the following parameters were observed: light and deep sedation (stages 1 and 2, respectively), partial and total loss of equilibrium (stages 3a and b, respectively), deep anesthesia (stage 4) and medullar collapse (stage 5).
Prior to the potentiation test, diazepam concentration (BDZ) (DEG, Brazil) was standardized. Fish were transferred to 1-L aquaria containing 25, 100, and 150 µM BDZ, previously solubilized in 0.033% Tween 80. To evaluate the time required to reach the different stages of anesthesia induction, three juveniles were used for each concentration tested, and each juvenile was used only once, according to the method of Schoettger and Julin (19). The maximum time of observation was 30 min. After induction, animals were transferred to an anesthetic-free aquarium to assess the recovery time.
For tests of the reversal of anesthetic effects, the flumazenil (Flumazil ® , Cristália, Brazil) concentration in the recovery bath was also standardized and used to observe the involvement of the GABA-BDZ receptor in the mechanism of action of EO. Fish (N = 6) were subjected to sedation induction with 150 µM BDZ in 1-L aquaria. After induction, the animals were divided into two groups: one group was transferred to an anesthetic-free aquarium with water while the second group was placed in an aquarium containing flumazenil. In both groups, fish behavior was scored after 1, 5, 10, 15, and 20 min, as described in Table 2. After 20 min, the sum of scores for each fish was calculated. When agitation was verified in a given fish, we added 0.5 to its score. Each observation included an external stimulus while observing the fish; undisturbed animals tend to remain www.bjournal.com.br Braz J Med Biol Res 45(5) 2012  stationary. This stimulation consisted of successive hits with a glass rod on the bottom of the aquarium.

Experiment 1: Potentiation with BDZ
Various concentrations (50, 100, and 300 μL/L) of L. alba EO (previously diluted 1:10 in ethanol) were added along with BDZ (150 μM) to the 1-L aquaria. Six fish were tested individually for each concentration. Evaluation of BDZ with ethanol (EO vehicle), EO and both vehicles alone (Tween 80 and ethanol at the same proportions used) was also performed. The stages of anesthesia induction and recovery time were evaluated according to the protocol described in the standardization of the test conditions.

Experiment 2: Reversal of anesthetic effects
The same protocol described in the standardization of the test conditions was repeated with L. alba EO at 300 μL/L. After induction, fish (N = 6) were divided into two groups: one group was transferred to an anesthetic-free aquarium with water, and the second group was placed in an aquarium containing 5 μM flumazenil. The results were scored (Table 2)

Statistical analyses
All data were subjected to a Levene test to determine the homogeneity of variances. One-way ANOVA followed by the Tukey test or Kruskal-Wallis and Mann-Whitney tests were used in the potentiation test when appropriate (SPSS version 17.0). Results of the study of reversal of the anesthetic effects were compared by two-way ANOVA and the Tukey test with Sigma Plot (version 11.0). Significance was set at the level of 95% (P < 0.05). Data are reported as means ± SEM.

EO composition
The EO yield (w/w) was 0.35% based on its mass in relation to the mass of plant material. The chemical composition of the EO used in this study (EO1) is similar to the corresponding data of the EO used by Cunha et al. (

Standardization of the test conditions
Fish exposed to 25 μM BDZ reached deep sedation (stage 2), whereas those exposed to 100 and 150 μM presented total loss of equilibrium (stage 3a). Silver catfish exposed to the concentrations of diazepam used in this study did not reach stage 4 of anesthesia. The concentration of 150 μM was chosen for the subsequent tests because fish reached stage 3a within a relatively short period of time (about 9 min) and showed a longer recovery time (about 18 min) compared to those exposed to 100 μM BDZ (induction time approximately 22 min; recovery time approximately 5 min). A longer recovery time was required to better determine the efficacy of the antagonist in the reversal of the anesthetic effects.
The flumazenil concentration was standardized by testing 0.05, 1.0, and 5.0 µM of this GABA receptor antagonist. Scores of recovery from anesthesia in fish exposed to 1 µM (10.2 ± 0.6) and 5 µM flumazenil (9.3 ± 0.6) were significantly different from those of control animals (8.2 ± 0.4 and 7.8 ± 0.4, respectively) with P < 0.05. Fish that recovered in water with 0.05 µM flumazenil showed scores (8.5 ± 0.3) that were not significantly different from animals that recovered in water alone (7.8 ± 0.3). In this test, higher scores indicate faster recovery from the anesthesia.
The application of ethanol alone did not produce an anesthetic effect.

Experiment 1: Potentiation with BDZ
The combination of BDZ+EO of L. alba significantly reduced the time required to reach anesthesia compared to the EO alone. BDZ potentiated deep sedation (stage 2) and total loss of equilibrium (stage 3a) of the animals exposed to L. alba EO, except in the case of fish exposed to 100 μL/L regarding stage 2 ( Figure 1A and B). Given alone, 50 μL/L EO only led to stage 3a anesthesia, but, in combination with BDZ, it led to stage 4 anesthesia. The highest EO concentrations evaluated (100 and 300 μL/L) also led to stage 4, and combined treatment with BDZ decreased the time required to reach this stage ( Figure 1C). The time of recovery from L. alba EO anesthesia was significantly Table 2. Scores of recovery from anesthesia in fish.

Scores Behavior 0
No sign of recovery 0.5 Reaction only after a caudal peduncle stimulus 1 First sign of recovery but without posture 1.5 Stopped after erratic swimming 2 Normal swimming, but without reflex after an external stimulus 2.5 Stopped after normal swimming, but without reflex after an external stimulus 3 Normal swimming with reflex after an external stimulus longer in fish exposed to 300 μL/L than to the lower EO concentrations, and the combination of BDZ+EO of L. alba did not change the recovery time ( Figure 1D).

Experiment 2: Reversal of the anesthetic effects
Fish anesthetized with L. alba EO (300 μL/L) or exposed to BDZ presented significantly higher recovery score sums when recovering in water with flumazenil than in water alone (P < 0.05; Figure 2). Therefore, recovery was faster in water containing flumazenil.

Discussion
The EO used in this study, as well as that used by Cunha et al. (9), was obtained from L. alba chemotype linalool, which was previously described by Frighetto et al. (20) and Duarte et al. (21). When comparing the composition of the EO used in this study to that used by Cunha et al. (9), we observed similar chemical compositions. Although there were discrepancies between our results and previous results regarding the percentages of the major EO components, the effects observed for the natural anesthetic mixture without BDZ were as expected, and we demonstrated that the EO used in our assays can reproduce the effect observed by Cunha et al. (9). However, the time to achieve stage 4 was longer in the present study than in the study by Cunha et al. (9). This disparity can be explained by the different percentage of some compounds, including the major compound (linalool).
GABA complex ligands include agonists, antagonists and modulators. Typically, some behavioral effects that result from positive allosteric modulators of GABA A receptors are anxiolysis, cessation of convulsions, sedation, and general anesthesia (22)(23)(24). All of these activities have been described for L. alba EO (7)(8)(9)25,26). Additionally, Vale et al. (7) verified that EOs of L. alba belonging to limonene and citral chemotypes have similar pharmacological profiles to those of BDZ, indicating that they might interact with the binding sites for benzodiazepines located adjacent to the GABA A receptor complex (11,12).
This potential GABAergic involvement was evaluated by the potentiation of the anesthetic effect of L. alba EO with BDZ. The best result observed in the present study was produced by the induction of anesthesia with the lowest concentration of EO tested. This clearly indicates the synergism of the compounds and the possibility for the combined use of these products to reduce the concentration of both in fish anesthesia. Additionally, the results indicate that the EO of L. alba has some effect on the GABA-receptor complex. This finding was confirmed by the faster recovery period observed with flumazenil treatment after the EO bath. In the animal model developed in the present study, the recovery scores recorded after 20 min of observation were summed and the values were converted to percentages. In these Figure 1. Time required for the induction of and recovery from anesthesia using the essential oil of Lippia alba (EO1), diazepam (BDZ) and the essential oil + diazepam (EO+BDZ) combination in silver catfish juveniles. Stages according to Schoettger and Julin (19). Maximum observation time was 30 min. Time to reach different stages of anesthesia: A, stage 2 (deep sedation); B, stage 3a (partial loss of equilibrium); C, stage 4 (deep anesthesia); D, recovery time. Different letters indicate significant differences between concentrations in the same group: EO, BDZ or EO+BDZ. *P < 0.05 compared to the EO group tested at the same concentration (one-way ANOVA followed by the Tukey test or Kruskal-Wallis and Mann-Whitney tests). calculations, 100% corresponded to the maximum score possible (17.5). Fish exposed to EO showed percent recovery scores of 40.91% with flumazenil and 20.0% in water. Similarly, for the standard drug (BDZ), we observed percent recovery scores of 68.57% in the presence of flumazenil and of 52.34% under control conditions (water).
Flumazenil, a GABA A competitive inhibitor, significantly accelerated recovery from BDZ-and EO-induced depression, suggesting the involvement of GABA A receptors in the anesthetic effect of L. alba EO in silver catfish. However, the existence of other possible neurochemical mechanisms, such as anti-nicotinic (27) and anti-glutamatergic (28) activity, cannot be excluded on the basis of the current results.
The literature describes the central nervous system activity of some essential oils, including their effect on the GABAergic system (29)(30)(31)(32). Some natural products, such as terpenoids, which are the main components of the EO tested, have exhibited effects on GABA A receptors (26). According to the literature, some of these constituents, such as α-pinene (33), nerolidol (34), linalool (34,35), borneol (36), and viridiflorol (37) are positive modulators of GABA A receptors. This result was also observed for EO1 and EO2, which contain these substances, as shown in Table 1. However, given that EOs are complex mixtures of components and that some of their constituents can act by synergism, more studies are necessary to discover the active compound(s) responsible for the anesthetic effect of L. alba EO.
We showed for the first time the effect of BDZ on the potentiation of the anesthetic effects of EO in silver catfish. In addition, the anesthetic effects of the EO of L. alba were reversed sooner by flumazenil, suggesting the involvement of the GABAergic system in this effect of the EO.