Involvement of nitric oxide-dependent pathways of dorsolateral periaqueductal gray in the effects of ethanol in rats submitted to the elevated plus-maze test

Behavioural Brain Research 153 (2004) 341–349

Research report

Involvement of nitric oxide-dependent pathways of dorsolateral periaqueductal gray in the effects of ethanol in rats submitted to the elevated plus-maze test Gina Struffaldi Morato a,∗ , Roberta Moura Ortiga a , Vania Maria Moraes Ferreira a,b a

b

Departamento de Farmacologia, Centro de Ciˆencias Biológicas, Universidade Federal de Santa Catarina, Campus Universitário Trindade, 88040-900 Florianópolis, SC, Brazil Departamento de Fisiologia, Centro de Ciˆencias Biológicas, Universidade Federal do Pará, Rua Augusto Corrˆea 01, 66075-900 Belém, PA, Brazil Received 15 August 2003; received in revised form 10 December 2003; accepted 12 December 2003 Available online 4 February 2004

Abstract Our previous study showed the microinjection of drugs that influence the nitric oxide (NO)-mediated neurotransmission in the hippocampus impacts upon the anxiolytic-like effect of ethanol. In this study, we examined whether NO-dependent pathways of the dorsolateral periaqueductal gray (dlPAG) participate in the anxiolytic effect of ethanol in rats submitted to the elevated plus-maze test. We evaluated the impact on ethanol effects of the nitric oxide synthase (NOS) inhibitor 7-nitroindazole, the soluble guanylate cyclase inhibitor 1H-(1,2,4)-oxodiazolo (4,3-a) quinoxalin-1-one (ODQ), the cyclic guanylate monophosphate (cGMP) analogue 8-bromo-cGMP and the NO donor sodium nitroprusside. The results showed that ODQ and 7-nitroindazole increased the percentage of open arm entries and of time spent on open arms in the elevated plus maze in rats injected with ethanol at 1.0 g/kg, a dose that did not produce anxiolysis per se. Conversely, 8-bromo-cGMP and sodium nitroprusside blocked the increased exploration of open arms exhibited by rats treated with a higher dose of ethanol (1.2 g/kg). Taken together, the results suggest that the inhibition of NO-dependent pathways of the dlPAG enhances the anxiolytic effect of ethanol, whereas the activation of these pathways results in an opposite effect. © 2003 Elsevier B.V. All rights reserved. Keywords: Anxiety; Elevated plus maze; Ethanol; Periaqueductal gray; Nitric oxide

1. Introduction Nitric oxide (NO) is a neuromodulator and an intercellular and retrograde messenger that mediates several functions in peripheral organs and the central nervous system [44,63,76]. NO is synthesized by the enzyme nitric oxide synthase (NOS), which is found in three distinct isoforms: endothelial (eNOS), inducible (iNOS), and neuronal (nNOS) [26]. Studies indicate that NO activates soluble guanylate cyclase, an enzyme that catalyzes the production of cyclic guanosine monophosphate (cGMP) [12,22,28,65]. Although several effects of NO are independent of cGMP activation [48], it is currently accepted that the NO–cGMP pathway is the main effector of NO actions in the brain [27]. At the central level, this small molecule appears to participate in a variety of mechanisms including: the control of

∗

Corresponding author. Tel.: +55-48-331-9491; fax: +55-48-222-4164. E-mail address: [email protected] (G.S. Morato).

0166-4328/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2003.12.010

sleep [45], processes related to synaptic plasticity [50], neurotoxicity [37], the baroreceptor reflex modulation of heart rate [58], catalepsy [18,42], and anxiety [6,46,51,71]. NOS inhibitors caused anxiolytic-like effects after administration by either intraperitoneal (i.p.) route [71] or microinjection into the dorsal central gray (dPAG) [35]. Neurochemical evidence suggests that NOS immunoreactive neurons are contained predominantly in the dorsolateral portion of the PAG (dlPAG) [54]. Moreover, the expression of nNOS mRNA in the dlPAG is increased after acute restraint stress in rats [51]. It is well-established that stimulation of the dorsal column of the PAG, particularly the dlPAG, results in flight or defensive behavior in several species (for review see [13,32]). NMDA glutamate receptors also have been described in the dlPAG [4], and behavioral studies using the elevated plus maze have shown that microinjection of NMDA agonists and antagonists into the dlPAG causes responses suggestive of anxiogenic and anxiolytic effects, respectively, [34,62,69]. Evidence for the participation of NO in the effects of ethanol was shown, perhaps for the first time, in 1987, when

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the endothelium-dependent tolerance to ethanol-induced contraction of the rat aorta was associated with the release of an endothelium-derived relaxing factor [39]. Although in the last few years several studies have supported the notion that NO is involved in the long-term effects of ethanol [49,60,70,74], a small number of studies have focused on the acute effects of ethanol. A recent in vitro study has shown that NO can modulate the sensitivity of the NMDA receptor to acute ethanol exposure in cultured hippocampal neurons from neonatal rats [16]. Meanwhile, behavioral studies have shown that the inhibition of NOS prolongs the sleeping time induced by ethanol [1], and that drugs acting on NO-dependent pathways in the hippocampus alter the anxiolytic effect of ethanol [25]. The anxiolytic profile of ethanol has been described in several different models of anxiety [10,15,20], including the elevated plus maze [8,24,41] a useful model to study defensive behavior in rodents [7,13,56]. There is a paucity of studies concerning the involvement of the midbrain PAG in ethanol effects. However, some evidence indicates that the ventrolateral PAG may be a component of the circuitry for the seizures observed after interruption of chronic ethanol administration [75]. Nevertheless, to our knowledge there are no studies on the participation of the dlPAG in the acute effects of ethanol. Given that NOS is particularly abundantly expressed in the dlPAG [54], a region involved in modulation of anxiety-related behaviors [32], and that NO-dependent pathways have been implicated in ethanol’s effects [1] including the anxiolytic effect [25], the aim of the present study was to examine the influence over the effects of ethanol, in rats tested in the elevated plus maze, of drugs acting on the NO-dependent pathways, microinjected into the dlPAG. We used 7-nitroindazole, a preferential inhibitor of neuronal NOS [3], the NO precursor l-arginine and its isomer d-arginine [44], the soluble guanylate cyclase inhibitor ODQ [59], the cGMP permeant analog 8-bromo-cGMP [5], and the NO donor sodium nitroprusside [64].

2. Material and methods 2.1. Subjects Experimentally naive male Wistar rats were used, with an age range of between 3 and 3.5 months and weighing between 280 and 320 g. The animals were bred at the Universidade Federal de Santa Catarina animal house and transferred to our department’s facilities at least 4 weeks prior to use, where they were housed in groups of six in plastic cages (42 cm × 34 cm × 17 cm), and maintained at 23 ± 1 ◦ C under artificial illumination (lights on from 06:00 to 18:00 h) with standard laboratory chow and tap water ad libitum. All experiments were conducted between 09:00 and 13:00 h in order to reduce circadian influences. All procedures were carried out in accordance with the Brazilian Society for Neu-

roscience and Behavior animal care guidelines and European Community animal care guidelines, and were approved by our institutional ethics committee (058/CEUA/UFSC). 2.2. Apparatus The apparatus was a wooden, plus-shaped maze, elevated 50 cm from the floor [36]. Two opposite arms were open (50 cm × 10 cm) and the other two opposite arms were enclosed with walls (50 cm × 10 cm × 40 cm). The floor and walls of the maze were painted black. To minimize falls from the open arms, these were surrounded by a 1 cm-high Plexiglas edge. Experiments were carried out in a sound-attenuated room with low luminosity (44 lx at the level of the apparatus). The wooden arena in which rats were placed before exposure to the maze measured 60 cm × 60 cm × 35 cm. 2.3. Drugs The following substances were obtained from Sigma Chemical Co. (St. Louis, MO, USA): 7-nitroindazole (7-NI), l-arginine (l-Arg), d-arginine (d-Arg), 1H-(1,2,4)-oxodiazolo (4,3-a)quinoxalin-1-one (ODQ), 8-bromo cyclic guanylate monophosphate (8-bromo-cGMP), sodium nitroprusside (SNP), propylene glycol, dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), polyoxyethylene sorbitanmonooleate (Tween® -80), and magnesium sulfate heptahydrate (MgSO4 ·7H2 O). Sodium pentobarbital was obtained from Abbott Laboratories (São Paulo, Brazil). Analytical grade ethanol, chloral hydrate, and all other reagents were purchased from Merck (Hawthorne, NY, USA). Ethanol was prepared by dilution in 0.9% NaCl to a concentration of 14% (w/v) and was administered by the i.p. route in volumes adjusted according to the animal’s body weight. Chloral hydrate was prepared as a 50 mM solution in 173 mM magnesium sulfate. Sodium pentobarbital was prepared as a 76.2 mM solution in propylene glycol plus 13% ethanol. 7-NI was dissolved in phosphate buffered saline (PBS) containing 20% DMSO, 50 mM NaOH, and 16% Tween-80. l-Arginine, d-arginine, and SNP were dissolved in PBS. ODQ was dissolved in sterile saline plus 0.01% DMSO. The respective control groups were treated identically with vehicle alone. All solutions were freshly prepared. All drugs, except ethanol, were microinjected into the dlPAG in a volume of 0.5 ␮l. 2.4. Surgery Rats were anesthetized with a mixture of chloral hydrate (127.5 mg/kg) and sodium pentobarbital (35 mg/kg), given i.p. in a volume of 0.3 ml/100 g of body weight. The rats were then transferred to a Stoelting stereotaxic frame for implantation of a 23-gauge stainless steel guide cannula (13 mm), directed to the dlPAG (coordinates: AP −7.6 mm; ML +1.9 mm; DV −2.0 mm). The cannula was secured by

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acrylic dental cement anchored to three stainless steel screws fixed to the skull, and a 30-gauge stainless steel stylet was placed in the lumen of the cannula to ensure patency until the animals were given i.c.v. injections. 2.5. Experimental design After a recovery period of 5–7 days, the animals were randomly assigned to experimental and control groups. In one set of experiments, groups of animals were pretreated with ethanol at 1.0 g/kg or saline, by i.p. route. After 10 min each group was further divided into subgroups that were microinjected into the dlPAG with 7-NI at 40 nmol, ODQ at 1 nmol, 7-NI at 40 nmol plus l-Arg at 600 nmol or 7-NI at 40 nmol plus d-Arg at 600 nmol. In another set of experiments groups of rats were pretreated with ethanol at 1.2 g/kg or saline, by i.p. route and, after 10 min, each group was further divided into subgroups in order to receive microinjections of 8-bromo-cGMP at 40 nmol or SNP (40 or 80 nmol) into the dlPAG. In all experiments parallel control groups received the respective vehicles. The doses used in this study were obtained from the literature [25,59,73,74] and preliminary experiments. Five minutes after dlPAG injections each animal was placed in the center of the arena for 5 min. Then, each rat was gently transferred to the center of the plus maze, facing an enclosed arm, and allowed to explore the maze for 5 min. An observer scored the number of entries into and the time spent in open and/or enclosed arms. After each trial, the maze was cleaned with an alcohol:water (1:10) solution, and completely dried with paper towels. The percentage of open arm entries (%OAE) and the percentage of time spent in the open arms (%OAT) were calculated with respect to the total arm entries and total experimental time [56]. 2.6. Verification of injection site After completion of the experiments, all animals were deeply anesthetized with a mixture of chloral hydrate (127.5 mg/kg) and sodium pentobarbital (35 mg/kg), given i.p. in a volume of 0.4 ml/100 g of body weight. They were then perfused with 0.9% NaCl followed by 10% formaldehyde saline solution. Next, 0.4 ␮l of Evans Blue was injected through the guide cannula in order to stain the injection site and adjacent tissue to which the injection may have spread. Immediately thereafter, the brains were removed and sectioned on a freezing microtome (75 ␮m). Coronal sections were mounted on glass slides and examined under a light microscope. Injection sites were plotted onto coronal diagrams from Paxinos and Watson’s [55] rat brain atlas. 2.7. Statistical analysis The Statistica for Windows 4.5 (Statsoft Inc., USA) software was used to perform the statistical analysis. Data were analyzed by means of analysis of variance (ANOVA), with

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pretreatment and treatment as the independent variables, and with the percentage open arm entries, percentage time spent in open arms and number of enclosed arm entries as the dependent variables. Post hoc comparisons were performed using the LSD test. Values of P < 0.05 were considered significant. Figures were drawn using Graph Pad Prism 3.03 (Graph Pad Software Inc., USA).

3. Results Systemic injection of 1 g/kg ethanol or intra-dlPAG microinjection of 7-NI at 40 nmol did not alter per se the behavior of animals in the elevated plus maze as compared to control groups (vehicle-treated rats) (Fig. 1, panels A–F). However, intra-dlPAG microinjection of 7-NI together with ethanol at 1 g/kg i.p. resulted in increased percentages of OAE and OAT when compared to controls (Fig. 1, panels A and B). For the percentage of OAE overall ANOVA showed a significant effect of pretreatment [F(1, 35) = 8.79; P < 0.01] and treatment [F(1, 35) = 9.90; P < 0.01] and a non-significant pretreatment × treatment interaction (Fig. 1, panel A). For the percentage of OAT ANOVA revealed a significant effect of pretreatment [F(1, 35) = 8.65; P < 0.01] and treatment [F(1, 35) = 8.17; P < 0.01], and a significant pretreatment × treatment interaction [F(1, 35) = 5.68; P < 0.03] (Fig. 1, panel B). The post hoc analysis showed that the association of ethanol and 7-NI significantly increased the percentage of OAT (P < 0.05, LSD test) in comparison to the groups treated with ethanol plus vehicle or 7-NI at 40 nmol plus saline (Fig. 1, panel B). These results suggest that both drugs have an anxiolytic-like action. Moreover, 7-NI or ethanol did not affect the frequency of enclosed arm entries (Fig. 1, panel C) suggesting that neither drug given individually nor the association of both drugs interfered with locomotor activity. Another experiment was performed to evaluate the influence of the inhibition of soluble guanylate cyclase (sGC) with ODQ on the performance of ethanol-treated rats submitted to the plus-maze test. As expected, the dose of 1 g/kg ethanol did not change per se the behavior of animals in comparison to controls. Similarly, ODQ intra-dlPAG did not cause any difference per se between the performances of the animals in comparison with controls (Fig. 1, panels D–F). However, intra-dlPAG ODQ given together with i.p. ethanol significantly increased the percentage of OAE. ANOVA showed an effect of pretreatment [F(1, 36) = 5.65; P < 0.03] and treatment [F(1, 36) = 11.62; P < 0.002], and a significant pretreatment × treatment interaction [F(1, 36) = 6.77; P < 0.02]. The post hoc analysis showed that association of ethanol and ODQ significantly increased the percentage of OAE (P < 0.05, LSD test) in comparison to the groups treated with ethanol plus vehicle or with ODQ plus saline (Fig. 1, panel D). Considering the percentage of OAT ANOVA showed a significant effect of pretreatment [F(1, 36) = 6.96; P < 0.02] and

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Fig. 1. Behavior of rats in the elevated plus-maze apparatus showing the percentage of open arm entries (panels A and D), the percentage of time spent in the open arms (panels B and E) and the number of enclosed arm entries (panels C and F). Rats were injected with saline or ethanol (EtOH, 1.0 g/kg) i.p. and 10 min later they were microinjected in the dlPAG with either vehicle (V) or 7-NI at 40 nmol or ODQ at 1 nmol. Animals were allowed to move freely for 5 min in a wooden arena before being tested in the elevated plus maze for 5 min. The effects of 7-NI and of ODQ are shown on the left and on the right side of the figure, respectively. Each value represents the mean ± S.E.M. of 8–11 animals. ∗ P < 0.05 vs. respective control animals, injected with ethanol + vehicle, saline + 7-NI, or saline + ODQ (LSD test).

treatment [F(1, 36) = 8.56; P < 0.01] but a non-significant pretreatment × treatment interaction. Other experiments were performed to verify if the co-administration of 7-NI and the NO precursor l-Arg would modify the synergistic effect of 7-NI on ethanol anxiolysis. For the percentage of OAE overall ANOVA showed a significant effect of i.p. treatment [F(1, 61) = 6.70; P < 0.02], a significant effect of intra-dlPAG treatment [F(3, 61) = 3.58; P < 0.02] and a significant i.p. treatment × intra-dlPAG treatment interaction [F(3, 61) = 3.12; P < 0.05]. The post hoc analysis confirmed that treatment with ethanol plus 7-NI produced a significant anxiolytic effect and that l-Arg prevented this (Fig. 2, panel A). The analysis of the percentage of OAT revealed a significant effect of treatment i.p. [F(1, 61) = 4.36; P < 0.05] and a non-significant effect of intra-dlPAG treatment (Fig. 2, panel B). When the enantiomer d-Arg was co-administered with 7-NI, ANOVA

revealed that there was no effect on the synergism between 7-NI and ethanol on the behavior of rats submitted to the plus-maze test (Fig. 2, panels D and E), showing only a significant effect of i.p. treatment [F(1, 72) = 12.63; P < 0.001] for percentage of OAT and a non-significant effect of i.p. treatment [F(1, 72) = 3.30; P = 0.07] for the percentage of OAE. The frequency of enclosed arm entries was not affected by the treatments (Fig. 2, panels C and F). In the next experiment, the dose of 1.2 g/kg ethanol caused the expected anxiolytic-like effect suggested by the increased percentage of OAE and of OAT as compared to controls (Fig. 3, panels A, B, D, and E). The administration of 8-bromo-cGMP significantly reduced the effects of ethanol on the percentage of OAE (Fig. 3, panel A). ANOVA revealed an effect of i.p. treatment [F(1, 28) = 10.14; P < 0.01] and intra-dlPAG treatment [F(1, 28) = 6.03; P < 0.02], and a significant i.p. treatment×intra-dlPAG treatment

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Fig. 2. Behavior of rats in the elevated plus-maze apparatus showing the percentage of open arm entries (panels A and D), the percentage of time spent in the open arms (panels B and E) and the number of enclosed arm entries (panels C and F). Rats were injected with saline or ethanol (EtOH, 1.0 g/kg) i.p. and 10 min later they were microinjected in the dlPAG with either vehicle (V) or one of the following: 7-NI at 40 nmol, l-arginine (LA) at 600 nmol, d-arginine (DA) at 600 nmol, 7-NI at 40 nmol plus LA at 600 nmol, or 7-NI at 40 nmol plus DA at 600 nmol. The test protocol was the same as that described in Fig. 1. The influence of l-arginine and d-arginine is shown on the left and on the right side of the figure, respectively. Each value represents the mean ± S.E.M. of 7–10 animals. ∗ P < 0.05 vs. respective control animals, injected with ethanol + vehicle or saline + 7-NI. # P < 0.05 vs. ethanol + LA-treated group or ethanol + 7-NI-treated group (LSD test).

interaction [F(1, 28) = 4.10; P < 0.05]. The post hoc test confirmed that 8-bromo-cGMP significantly influenced the effect of ethanol on this parameter. The effects of treatments on the percentage of OAT were similar to those observed for OAE (Fig. 3, panel B). The ANOVA revealed a significant effect of i.p. treatment [F(1, 28) = 17.50; P < 0.01] and intra-dlPAG treatment [F(1, 28) = 5.20; P < 0.05], and a significant i.p. treatment × intra-dlPAG-treatment interaction [F(1, 28) = 5.40; P < 0.03]. The post hoc analysis confirmed the anxiolysis produced by this dose of ethanol, and that 8-bromo-cGMP blocked this effect (P < 0.05, LSD test). 8-bromo-cGMP did not affect the frequency of enclosed arm entries (Fig. 3, panel C). The influence of intra-dlPAG injection of the NO donor SNP (40 or 80 nmol) on the effect of ethanol at 1.2 g/kg also was evaluated in rats submitted to the elevated plus-maze

test (Fig. 3, panels D–F). For the percentage of OAE the overall ANOVA showed a significant effect of intra-dlPAG treatment [F(2, 42) = 10.37; P < 0.001] and a significant i.p. treatment × intra-dlPAG treatment interaction [F(2, 42) = 3.48; P < 0.04] (Fig. 3, panel D). For the percentage of OAT ANOVA revealed a significant effect of intra-dlPAG treatment [F(2, 42) = 5.35; P < 0.01] and a significant i.p. treatment × intra-dlPAG treatment interaction [F(2, 42) = 4.11; P < 0.03] (Fig. 3, panel E). The post hoc analysis confirmed the anxiolytic effect of ethanol at 1.2 g/kg and the blockade of this effect by SNP at 80 nmol. In all experiments, the frequency of enclosed arms entries presented by the different treatment groups was similar (Fig. 3, panel F). The localization of injection sites in the dlPAG in schemes based on Paxinos and Watson’s rat brain atlas [55] is presented in Fig. 4.

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Fig. 3. Behavior of rats in the elevated plus-maze apparatus showing the percentage of open arm entries (panels A and D), the percentage of time spent in the open arms (panels B and E) and the number of enclosed arm entries (panels C and F). Rats were injected with saline or ethanol (EtOH, 1.2 g/kg) i.p. and 10 min later they were microinjected in the dlPAG with either vehicle (V) or one of the following: 8-bromo-cGMP (8-Br) at 40 nmol or sodium nitroprusside (SNP) at 40 or 80 nmol. The test protocol was the same as that described in Fig. 1. The influence of 8-bromo-cGMP and sodium nitroprusside is shown on the left and on the right side of the figure, respectively. Each value represents the mean ± S.E.M. of 7–9 animals. ∗ P < 0.05 vs. respective control animals, injected with saline + vehicle. # P < 0.05 vs. ethanol + vehicle-treated-rats. ◦ P < 0.05 vs. ethanol + SNP 40 nmol-treated rats (LSD test).

4. Discussion

Fig. 4. Histological localization of injection sites (0.5 ␮l, solid symbols) in the dlPAG in schemes based on the atlas of Paxinos and Watson [55]. Numbers represent atlas coordinates posterior to the bregma line. Due to overlap the number of points in the figure is fewer than the total number of animals used during the study.

Our study shows that the increase in, or the depletion of NO levels in the dlPAG results in modulation of ethanol effects in opposite directions. Intra-dlPAG injection of 7-NI associated with i.p. 1.0 g/kg ethanol resulted in an anxiolytic-like effect that was blocked by l-Arg, but not by its enantiomer d-Arg. These results are in accordance with evidence that microinjection of the NOS inhibitor l-NAME into the dPAG [35] or peripheral injection of 7-NI increased open arm exploration by rats in the elevated plus maze [21]. Additionally, our data agree with the findings that the inhibition of NOS increases the sleeping time [1] and the anxiolytic effect [25] induced by ethanol in rats. However, our results with 7-NI disagree with data showing that mice lacking the neuronal NOS gene were less sensitive to the

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depressant effect of ethanol [66]. This discrepancy could be related to the fact that specific genetic background and the environment may influence the behavioral phenotype of knock-out mice. Another reason for obtaining different results could be that the knock-out animals lack the enzyme in all areas of the body throughout development, while we have only looked at a single area in mature animals in the present study. Moreover, the inhibition of other NOS isoforms may have influenced some of the results obtained in the present study. Our data also show that the soluble guanylate cyclase inhibitor ODQ associated with ethanol caused a significant increase in the percentage of OAE and of OAT in the elevated plus maze as compared to the values obtained for each drug per se. It is noteworthy that previous studies have reported that reduction of cGMP synthesis by intra-dPAG methylene blue injection resulted in an anxiolytic-like effect [53] or reduced the flight reaction [52] in rats. Additionally, in the present study either the cGMP analog 8-bromo-cGMP, or the NO donor SNP significantly attenuated the effects of an anxiolytic dose of ethanol in the elevated plus maze, suggesting opposite effects for ethanol and the other drugs. These findings are in accordance with the detection of flight reactions in rats receiving intra-dlPAG injections of the NO donors SIN-1 and DEA/NO, or 8-bromo-cGMP [17], and with the report that intra-hippocampal injection of 7-NI potentiated, whereas NO donors or 8-bromo-cGMP prevented the acute anxiolytic-like effect of ethanol in rats submitted to the elevated plus-maze test [25]. Thus, our data support the hypothesis that inhibition and activation of NO-dependent pathways in the dlPAG, respectively, increases and reduces the efficacy of ethanol in producing acute anxiolysis. Even though ethanol can affect membrane architecture and several neurotransmitter systems at higher concentrations (more than 100 mM), selectivity in its action is distinguished at physiologically significant concentrations (less than 50 mM). In this concentration range the ethanol molecule attaches to specific binding sites located in the GABA-A, NMDA, AMPA/kainate, nicotinic, and 5HT3 receptors [19,23,40,57]. Numerous pieces of evidence suggest that ethanol can stimulate the transmission mediated by GABA and 5HT3 receptors, and/or block the transmission mediated by glutamate [67]. On the other hand, considerable evidence obtained with chemical or electrical brain stimulation suggests that behaviors correlated to the dPAG may be mediated by distinct neurotransmitter systems that are related to, for example, GABA-A [11,61], NMDA and AMPA/kainate [34,43,47], 5HT1 and 5HT2 [29,31], opioid [30], and to a lesser extent cholinergic [14,33] receptors. The mechanism by which NO-dependent pathways in the dlPAG influence the anxiolytic effect of ethanol is unclear. Considering the main receptor systems involved in ethanol effects in the brain, as well as those systems particularly implicated in the modulation of defensive behaviors in the

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PAG, one could hypothesize that the interaction between ethanol and the drugs injected into the dlPAG in the present study would involve the modulation by NO of the sensitivity of NMDA, AMPA/kainate, and/or GABA-A receptors. For example, it was reported that the aversive reaction produced by intra-dlPAG injection of the NO donor SIN-1 was prevented by the NMDA receptor antagonist AP7, or by the AMPA/kainate receptor antagonist NBQX, suggesting an interaction between NO and glutamate-mediated pathways underlying defensive responses in the dlPAG [47]. In another study Costa et al. [16] have shown that ethanol can reversibly inhibit currents gated by NMDA plus glycine in cultured hippocampal neurons. This effect was significantly reduced in the presence of the NO donors NOC-12 or SNAP, suggesting that ethanol and the NO donors have opposite effects [16]. Therefore, it is possible that the increase in NO concentration by SNP in the present study counteracted the anxiolytic effect of ethanol through an action on NMDA and/or AMPA/kainate receptors. However, since other studies [68] have proposed that cGMP does not mediate the actions of NO on NMDA receptor responses in cultured rat neurons, the effects of ODQ and 8-bromo-cGMP in the present study may be unrelated to this pathway. An alternative mechanism by which NO donors opposed ethanol actions in our study could be some direct blockade by NO of NMDA receptor activity. There is evidence that both the nitrosium ion (NO+ ) and the nitroxyl anion (NO− ) can react with the NMDA receptor thiol to downregulate receptor activity and provide neuroprotection [38]. Moreover, AMPA and kainate receptors can be negatively influenced by cGMP. For example, in retinal cells kainate receptor activity is reduced by NO and cGMP by a mechanism involving PKG, whereas in the hippocampus AMPA receptor activity is reduced by cGMP in a PKG-independent manner (for a review see [2]). Such mechanisms could explain a reduction in the sensitivity of these receptors to ethanol effects in the present study. NO can also influence the GABA-A receptor system. For example, the reduction of NO levels can modulate GABAergic neurotransmission in the internal granule cell layer in rat cerebellum, leading to augmented granule cell GABA-A receptor activation, and part of this effect is mimicked by the blockade of GABA uptake [72]. Conversely, a study with whole cell patch-clamp recording in dorsal root ganglion neurons [9] has shown that NO inhibits the GABA-A mediated inward currents, possibly through a PKG-dependent pathway, an important cellular target for NO signal transduction [27]. Thus, it is possible that the result of inhibition of NOS or soluble guanylate cyclase in the dlPAG together with ethanol administration in this study is a consequence of an increase in GABA-A mediated neurotransmission leading to an anxiolytic effect. The opposite effects of ethanol and NO donors in this study could be also explained by a mechanism involving the GABA-A system. In light of such findings, the current results could be interpreted to suggest that the influence on the anxiolytic effects

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of ethanol of drugs acting on NO-dependent pathways into the dlPAG could be related to the GABA-A and/or the glutamate receptor systems. However, further studies will be needed to consolidate this view.

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