Opioidergic Systems Modulate Anxiety Behaviors in Rats in a Noninteractive Manner

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Provided by Elsevier - Publisher Connector Kaohsiung Journal of Medical Sciences (2011) 27, 485e493

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ORIGINAL ARTICLE

Dorsal hippocampal N-methyl-D-aspartate glutamatergic and d-opioidergic systems modulate anxiety behaviors in rats in a noninteractive manner

Jalal Solati*

Islamic Azad University, Karaj Branch, Department of Biology, Karaj, Iran

Received 2 December 2010; accepted 25 February 2011 Available online 18 August 2011

KEYWORDS Abstract The present study aimed to investigate the effects of N-methyl-D-aspartate (NMDA)- Anxiety; type glutamate receptor agonist, NMDA, on anxiety-like behavior induced by d-opioid receptor Dorsal hippocampus; agents in rats, using the elevated plus maze instrument. The dorsal hippocampus (CA1) is known NMDA; to play an important role in anxiety formation and modulation. Bilateral administration of d-opioid; different doses of d-opioid receptor agonist, [D-pen2,5] enkephalin acetate hydrate (1 mg/rat, Rat 2 mg/rat, 5 mg/rat, and 10 mg/rat; 1 mL/rat; 0.5 mL/rat in each side), into CA1 area induced an anxiolytic-like effect, demonstrated by substantial increases in the percent of open arm time (OAT%) and percent of open arm entries (OAE%). Intra-CA1 injection of different doses of d-opioid receptor antagonist, naltrindole hydrochloride (0.25 mg/rat, 0.5 mg/rat, 1 mg/rat, and 2 mg/rat), produced significant anxiogenic-like behavior. Furthermore, intra-CA1 administration of NMDA glutamate receptor agonist, NMDA (0.125 mg/rat, 0.25 mg/rat, 0.5 mg/rat, and 0.75 mg/rat), increased the OAT% and OAE%, indicating anxiolytic-like behavior. However, administration of different doses of NMDA glutamatergic antagonist, MK801 (0.125 mg/rat, 0.25 mg/rat, 0.5 mg/ rat, and 1 mg/rat), showed no significant effect on the OAT% but decreased the OAE% significantly. The ineffective dose of NMDA (0.125 mg/rat), when coadministered with enkephalin (1 mg/rat, 2 mg/rat, 5 mg/rat, and 10 mg/rat), did not decrease the anxiety behavior significantly. An effective dose of NMDA (0.5 mg/rat), in combination with different doses of naltrindole hydrochloride (0.25 mg/rat, 0.5 mg/rat, 1 mg/rat, and 2 mg/rat), demonstrated no significant interaction with the OAT%, OAE%, and locomotor activity. These results suggest that CA1 d-opioid and NMDA glutamatergic systems modulate anxiety behaviors in a noninteractive manner. Copyright ª 2011, Elsevier Taiwan LLC. All rights reserved.

* Department of Biology, School of Science, Islamic Azad University, Karaj Branch, PO Box 31485-313, Karaj, Iran. E-mail address: [email protected].

Introduction repeated swim stress-induced thermal hyperalgesia [19].In the present study, possible interactions of the two systems The hippocampus plays an important role in the modulation in the anxiety-like behaviors were investigated. and regulation of behaviors, such as learning and memory [1]. In addition, role of dorsal hippocampus (CA1) in the Methods modulation of anxiety is crucial [2]. Previous studies have shown that the serotonergic system of the dorsal hippo- The study was carried out at the Biology Department of campus mediates anxiogenic responses, whereas the dorsal Islamic Azad University, Karaj Branch, Karaj, Iran, between hippocampal nicotinic cholinergic system mediates anxio- December 2008 and June 2009. The University Ethics lytic responses [2]. Committee approved the research protocol. All the treat- Glutamate, a main excitatory neurotransmitter of brain, is ments and behavioral tests were conducted between 3 PM of great significance in the modulation of behaviors by means and 6 PM. of several glutamate receptors. At least three subtypes of excitatory amino acid receptors, named after their prefer- Animals ential agonists, that is, N-methyl-D-aspartate (NMDA), quis- qualate, and kinate have been identified [3]. The NMDA Male Wistar rats from Pasteur Institute (Tehran, Iran), receptor, as an ionotropic glutamate receptor, plays an weighing 180e230 g at the time of surgery, were used. The important role in the modulation of behavioral effects of animals were housed four per cage in a room with glutamate. Studies demonstrated that the NMDA receptor is a 12:12 hour light/dark cycle (lights on 07:00 hours) and involved in the control of anxiety-related behaviors. In controlled temperature (23 1C), and they had access to several studies conducted in animal models of anxiety, after food and water ad libitum and were allowed to adapt to the the administration of agonists of the NMDA receptor, behav- laboratory conditions for at least 1 week before surgery. The ioral and anxiolytic effects have been reported [4]. rats were handled about 3 minutes each day before behav- Opioid peptides are involved in diverse functions, including ioral testing. All the experiments were performed between pain perception, respiration, homeothermy, nutrient intake, 12:00 hours and 15:00 hours, and each rat was tested only and in immune response. Several researches have under- once. Seven animals were used in each experiment. scored the role of opioid receptors in regulating baseline anxiety states and related behaviors [5]. Our previous study showed that dorsal hippocampal opioid receptors are involved Stereotaxic surgery and microinjections in the modulation of anxiety-associated behaviors [6]. Furthermore, neurotransmitter systems in different sites The rats were anesthetized intraperitoneally with ketamine across the central nervous system are also involved in the hydrochloride (50 mg/kg) and xylazine (4 mg/kg) and placed modulation of opioid receptor agonisterelated effects on in a Stoelting stereotaxic instrument (Wood Dale, IL, USA). anxiety and behaviors [7]. Recently, the regulating role of the A stainless steel guide cannula (22 gauge) was implanted in endogenous opioid system on emotional-like behavior has the right and left CA1 regions according to Paxinos [20]. been tested. Investigations on m-andd- but not k-opioid Stereotaxic coordinates for the CA1 regions were as follows: receptor knockout mice have indicated that the basal levels 3.8 mm posterior to bregma, 2 mm lateral to the midline, of anxiety in different models of anxiety are prone to change and 3 mm ventral of the dorsal surface of the skull. The [8]. Although the d-opioid receptor knockout mice showed cannula was fixed to the skull with acrylic dental cement. anxiogenic-like responses in the elevated plus maze (EPM) and The animals were allowed 5 days before the test to recover the light-dark box, the mice lacking m-opioid receptors from surgery. The left and right CA1 were infused by means of showed lower levels of anxiety in the EPM [8]. an internal cannula (27 gauge), terminating 1 mm below the Interactions between opioidergic and glutamatergic tip of the guides, connected by polyethylene tubing to a 1-mL systems are reported in the different areas of the brain, Hamilton syringe (Bonaduz, GR, Switzerland). On each side, such as sensorimotor cortex [9], striatum [10], dorsal raphe 0.5-mL solution was injected (1 mL/rat) over a 60-second nucleus [11], and finally in spinal cord [12]. Opioids inhibit period. The inner cannula was left in place for an additional glutamate release in rat periaqueductal gray cells, CA3 60 seconds to allow diffusion of the solution and to reduce the area [13], and also in the CA1 region of the hippocampus possibility of reflux. Intra-CA1 injections were made [12]. Enkephalin and glutamate regulate the expression of 5 minutes before testing. d-opioid receptors in rat cortical astrocytes [14]. Moreover, the opioidergic system inhibits capsaicin-evoked release of Plus maze glutamate from rat spinal dorsal horn slices [15]. þ The opioidergic system inhibits K -evoked glutamate This wooden, plus-shaped apparatus was elevated to the release from rat cerebrocortical slices [16]. Current height of 50 cm. This apparatus was composed of two evidences suggest that long-term d-opioid receptor activa- 50 10-cm open arms and two 50 10 50-cm enclosed tion may cause alteration in the glial glutamate uptake arms, each with an open roof. The maze was placed in the properties [17]. Furthermore, blocking of d-opioid recep- center of a quiet and dimly lit room. The rats’ behaviors tors induces an NMDA receptoredependent excitotoxicity in were directly observed using a mirror, suspended at an anoxic turtle cortex [18]. On the other hand, some studies angle above the maze. Behavioral data were collected by have reported an interaction between d-opioid and NMDA an observer who quietly sat 1 m behind one of the closed receptors in the development and maintenance of the arms of the maze, using a time recorder. Five minutes after Effects of NMDA and opioids of CA1 on anxiety 487 their respective drug treatment, rats were placed individ- (1 mL/ratdintra-CA1), and the other groups received four ually in the center of the plus maze, facing one of the closed different doses of naltrindole (0.25 mg/rat, 0.5 mg/rat, arms. The observer measured (1) time spent in the open 0.75 mg/rat, and 1 mg/ratdintra-CA1). Five other groups of arms, (2) time spent in the closed arms, (3) number of entries rats received an intra-CA1 injection of NMDA (0.5 mg/ into the open arms, and (4) number of entries into the ratdintra-CA1) 3 minutes before injection of saline (1 mL/ closed arms during the 5-minute test period. An entry was ratdintra-CA1) or naltrindole (0.25 mg/rat, 0.5 mg/rat, defined as all four paws in the arm. The maze was cleaned 0.75 mg/rat, and 1 mg/ratdintra-CA1). with distilled water after each rat was tested. For the purpose of analysis, open arm activity was quantified as the Histology amount of time that the rat spent in the open arms relative to the total amount of time spent in any arm (open/ Figure 1 shows the approximate point of the drug injection total 100), and the number of entries into the open arms in CA1. The histological results were plotted on the repre- was quantified relative to the total number of entries into sentative section taken from the rat brain atlas of Paxinos any arm (open/total 100). The total number of arms [20]. After the completion of behavioral testing, the entered, as well as the total number of closed arms entered, animals were killed with an overdose of chloroform. Ink was used as an index of general locomotor activity [21]. (0.5 mL of 1% aquatic methylene blue solution) was injected into the guide cannulae using 27-gauge injection cannulae Drugs that projected a further 0.5 mm ventral to the tip of the guides to aid in histological verification. Brains were

The drugs used in the present study were [D-pen2,5]- removed and ﬁxed in a 10% formalin solution for 48 hours enkephalin acetate hydrate (d-opioid receptor agonist) and before sectioning. The sections were taken through the naltrindole hydrochloride (d-opioid receptor antagonist) brain areas of cannulae placements, and the cannulae (Tocris Co., Ballwin, MO, USA); NMDA (NMDA glutamatergic receptor agonist); and MK801(NMDA glutamatergic receptor antagonist) (Sigma Chemical Co., St. Louis, MO, USA).

Drug treatments Experiment 1: effects of NMDA receptor agonist and antagonist on anxiety behavior Four groups of rats received bilateral dorsal hippocampus injection (intra-CA1) of NMDA receptor agonist NMDA (0.125 mg/rat, 0.25 mg/rat, 0. 5 mg/rat, and 0.75 mg/rat) [22,23] and four other groups received NMDA receptor antagonist MK801 (0.25 mg/rat, 0.5 mg/rat, 0.75 mg/rat, and 1 mg/rat) [22] and were compared with saline control group.

Experiment 2: effects of d-opioid receptor agonist and antagonist on anxiety-like behavior Four groups of rats received bilateral dorsal hippocampus injection (intra-CA1) of selective d receptor agonist enkephalin (1 mg/rat, 2 mg/rat, 5 mg/rat, and 10 mg/rat) [24,25], and four other groups received d receptor antagonist naltrindole hydrochloride (0.25 mg/rat, 0.5 mg/rat, 1 mg/rat, and 2 mg/ratdintra-CA1) [26,27]. These rats were thereafter compared with a saline control group.

Experiment3:effectsofNMDAincombinationwith d receptor agonist on anxiety behavior First, ﬁve groups of rats received saline (1 mL/ratdintra-CA1). Three minutes later, the ﬁrst group received saline (1 mL/ ratdintra-CA1), and the other groups received four different doses of enkephalin (1 mg/rat, 2 mg/rat, 5 mg/rat, and 10 mg/ ratdintra-CA1). Five other groups of rats received an intra- CA1 injection of NMDA (0.125 mg/ratdintra-CA1) 3 minutes before injection of saline (1 mL/ratdintra-CA1) or enkephalin (1 mg/rat, 2 mg/rat, 5 mg/rat, and 10 mg/ratdintra-CA1).

Experiment4:effectsofNMDAincombinationwith Figure 1. A sample of the drug injection site in dorsal d receptor antagonist on anxiety behavior hippocampus (CA1). The sections were taken through the brain The first five groups of rats received saline (1 mL/ratdintra- areas of cannulae placements, and the cannulae placements CA1). Three minutes later, the first group received saline were verified using the atlas of Paxinos. 488 J. Solati placements were verified using the atlas of Paxinos. Data from the rats with injection sites located outside the hippocampal CA1 area were not used in the analyses. There were seven rats with correct injection site per group.

Statistical analysis

Because data displayed normal distribution and homoge- neity of variance, one-way analysis of variance (ANOVA) was used for comparison among the effects of different doses of drugs with vehicle. Two-way ANOVA was used for the evaluation of interactions between drugs. In case the difference was significant, the post hoc analysis (least significant difference) was performed to assess specific group comparisons. Differences with p < 0.05 between experimental groups at each point were considered statis- tically significant.

Results

Effects of NMDA receptor agonist and antagonist on anxiety behavior

Figure 2 shows the effect of intra-CA1 injection of NMDA (0.125 mg/rat, 0.25 mg/rat, 0.5 mg/rat, and 0.75 mg/rat) in the EPM in rats. One-way ANOVA revealed that NMDA increased the percent of open arm time (OAT%) [F (4,30) Z 12.636, p < 0.05] and percent of open arm entries (OAE%) [F (4,30) Z 6.545, p < 0.05] at the doses of 0.25 mg/rat, 0.5 mg/rat, and 0.75 mg/rat, indicating the induction of anxiolytic response by NMDA. No change in the locomotor activity was observed [F (4,30) Z 0.690, p > 0.05]. However, the rats infused with MK801 (0.25 mg/rat, 0.5 mg/rat, 0.75 mg/rat, and 1 mg/ratdintra-CA1) showed no significant decrease in OAT% [F (4,30) Z 2.294, p > 0.05] but OAE% [F (4,30) Z 4.194, p < 0.05] decreased significantly at doses 0.125 mg/rat and 0.25 mg/rat in comparison with the saline-infused controls. No significant change in the locomotor activity was observed [F (4,30) Z 1.814, p > 0.05] (Fig. 3).

Effects of d-opioid receptor agonists and antagonists on anxiety-like behavior

One-way ANOVA revealed that intra-CA1 injection of d- Figure 2. Effects of bilateral intra-CA1 injection of NMDA in opioid receptor agonist, enkephalin (1 mg/rat, 2 mg/rat, the elevated plus maze. Rats were treated with either saline m m m m 5 mg/rat, and 10 mg/rat; 1 mL/rat) increased OAT% [F (3,24) (1 L/rat) or with NMDA (0.125 g/rat, 0.25 g/rat, 0.5 g/rat, m Z 4.421, p < 0.05] and OAE% [F (3,24) Z 5.599, p < 0.05] at and 0.75 g/rat). Each bar is mean standard error of the doses of 1 mg/rat, 2 mg/rat, and 5 mg/rat, indicating the mean of percent of open arm time (A), percent of open arm d Z < induction of anxiolytic effects. No signiﬁcant change in entries (B), or locomotor activity (C) n 7. *p 0.05, < < the locomotor activity was observed [F (3,24) Z 0.946, **p 0.01, and ***p 0.001 when compared with the saline- Z Z p > 0.05] (Fig. 4). treated rats. CA1 dorsal hippocampus region; NMDA The intra-CA1 infusions of d-opioid receptor antagonist N-methyl-D-aspartate. naltrindole hydrochloride decreased OAT% [F (4,30) Z 5.532, p < 0.05] at doses of 0.5 mg/rat, 1 mg/rat, and 2 mg/rat, and decreased OAE% [F (4,30) Z 5.961, p < 0.05] Effects of enkephalin alone or with NMDA on at doses of 0.25 mg/rat, 0.5 mg/rat, 1 mg/rat, and 2 mg/rat, anxiety-like behavior indicating the induction of anxiogenic effects by naltrindole. No signiﬁcant change in the locomotor activity The effects of intra-CA1 injections of the d receptor was observed [F (4,30) Z 0.431, p > 0.05] (Fig. 5). agonist, enkephalin (1 mg/rat, 2 mg/rat, 5 mg/rat, and Effects of NMDA and opioids of CA1 on anxiety 489

Figure 3. Effects of bilateral intra-CA1 injection of MK801 in the elevated plus maze. Rats were treated with either saline (1 mL/rat) or with MK801 (0.125 mg/rat, 0.25 mg/rat, 0.5 mg/ rat, and 1 mg/rat). Each bar is mean standard error of the Figure 4. Effects of bilateral intra-CA1 injection of mean of percent of open arm time (A), percent of open arm enkephalin in the elevated plus maze. Rats were treated with entries(B), or locomotor activity (C)dn Z 7. *p < 0.05, when either saline (1 mL/rat) or with enkephalin (1 mg/rat, 2 mg/rat, compared with the saline-treated rats. CA1 Z dorsal hippo- 5 mg/rat, and 10 mg/rat). Each bar is mean standard error of campus region. the mean of percent of open arm time (A), percent of open arm entries (B), or locomotor activity (C)dn Z 7. *p < 0.05 and **p < 0.01, when compared with the saline-treated rats. m m m 10 g/rat; 1 L/rat) and NMDA (0.125 g/rat), on the CA1 Z dorsal hippocampus region. response induced by enkephalin are shown in Fig. 6. Two- way ANOVA indicated that the combination of enkephalin (Factor A) with NMDA (Factor B) showed no interactions for OAT% [Factor A: F (4,60) Z 7.488, p < 0.001; Factor B: F Effects of naltrindole alone or with NMDA on (1,60) Z 3.12, p > 0.05; Factor (A B): F (4,60) Z 0.068, anxiety-like behavior p > 0.05]; for OAE% [Factor A; F (4,60) Z 7.951, p < 0.001; Factor B; F (1,60) Z 1.350, p > 0.05; Factor (A B): F (4,60) Figure 7 shows the effects of intra-CA1 injections of the Z 0.250, p > 0.05]; and for locomotor activity [Factor A: F d-opioid receptor antagonist naltrindole (0.25 mg/rat, (4,60) Z 0.48, p > 0.05; Factor B: F (1,60) Z 2.14, p > 0.05; 0.5 mg/rat, 0.75 mg/rat, and 1 mg/ratdintra-CA1) in the Factor (A B): F (4,60) Z 0.930, p > 0.05]. presence or absence of NMDA (0.5 mg/rat) on anxiety-like 490 J. Solati

Figure 6. The effect of bilateral injection of enkephalin in the presence or absence of NMDA in the elevated plus maze. Rats were injected saline (1 mL/ratdintra-CA1) or enkephalin (1 mg/rat, 2 mg/rat, 5 mg/rat, and 10 mg/ratdintra-CA1) 3 minutes after injection of either saline (1 mL/ratdintra-CA1) or NMDA (0.125 mg/ratdintra-CA1). Each bar is mean standard error of the mean of percent of open arm time (A), percent of open arm entries (B), or locomotor activity (C)dn Z 7. *p < 0.05, **p < 0.01, and ***p < 0.001 when compared with the saline-treated rats. þp < 0.05 and þþp < 0.01 when compared with the saline- and NMDA-treated rats. CA1 Z dorsal hippo- Figure 5. Effects of bilateral intra-CA1 injection of nal- campus region; NMDA Z N-methyl-D-aspartate. trindole in the elevated plus maze. Rats were treated with either saline (1 mL/rat) or with naltrindole (0.25 mg/rat, F (4,60) Z 0.696, p > 0.05]; and on locomotor activity 0.5 mg/rat, 1 mg/rat, and 2 mg/rat). Each bar is mean [Factor A: F (4,60) Z 0.725, p > 0.05; Factor B: F (1,60) standard error of the mean of percent of open arm time (A), Z 3.226, p > 0.05; Factor (A B): F (4,60) Z 0.984, percent of open arm entries (B), or locomotor activity (C)d p > 0.05], indicating no interaction between naltrindole n Z 7. *p < 0.05 and ** p < 0.01, when compared with the and NMDA on anxiety-related behaviors. The post hoc saline-treated rats. CA1 Z dorsal hippocampus region. analysis showed that naltrindole induced anxiogenic-like effects, and NMDA induced anxiolytic-like effects. behavior. Two-way ANOVA showed no interactions between Discussion the effects of naltrindole (Factor A) and NMDA (Factor B) on OAT% [Factor A: F (4,60) Z 2.821, p < 0.05; Factor B: F In this study, effects of hippocampal NMDA glutamatergic (1,60) Z 186.06, p < 0.001; Factor (A B): F (4,60) Z 0.25, system and d-opioidergic receptor agents, and also the p > 0.05]; on OAE% [Factor A: F (4,60) Z 5.261, p < 0.001; possible interactions between these agents in the EPM were Factor B: F (1,60) Z 16 .068, p < 0.001; Factor (A B): investigated. Effects of NMDA and opioids of CA1 on anxiety 491

Studies suggest that the hippocampus may be involved in these anxiolytic-like actions of glutamatergic system, because the intrahippocampal injections of LY354740, an mGLU2/3 receptor agonist, produced anxiolytic-like effects in rat models [30,31]. An investigation carried out by Linden et al. [32] showed that the anxiolytic-like activity of LY354740 was associated with the suppression of EPM- induced c-Fos in the hippocampus of mice. So far, many efforts have been made to develop compounds that can modulate the function of NMDA receptors by acting within the NMDA receptor complex. Nonetheless, using NMDA antagonists appears to be greatly hampered by their associated adverse effects on anxiety because of interference with receptors throughout the central nervous system and the body [28]. Our results demonstrate that the NMDA receptor antagonist MK801 decreases OAE% without significant change in the OAT% and locomotor activity, indicating an anxiogenic effect by MK801, whereas the anxiolytic-like effects of NMDA receptor antagonists have been exhibited in some previous studies. Studies conducted by Jardim and Stephens demonstrated that the systemic or intra-dorsolateral periaqueductal gray administration of NMDA receptor antagonists produces anxiolytic-like effects in animal anxiety models, such as the EPM, brain aversive electrical stimulation, and the Vogel’s punished licking test [33,34]. Studies showed that the administration of antagonists of NMDA and non-NMDA-type receptors into the basolateral amygdala reduces anxiety in animal models [35].There- fore, it seems that the major target for systemic injections is likely amygdala rather than the hippocampus region. Moreover, controversial results obtained in different investigations may be attributed to different sites of injections or also the different animal models used. Our results show that the NMDA antagonist, MK801, had Figure 7. The effect of bilateral injection of naltrindole in only partial effects on anxiety, whereas NMDA agonist the presence or absence of NMDA in the elevated plus maze. clearly affected anxiety-related behaviors. Because the Rats were injected saline (1 mL/ratdintra-CA1) or naltrindole NMDA agonist is more effective than its antagonist, it might (0.25 mg/rat, 0.5 mg/rat, 1 mg/rat, and 2 mg/ratdintra-CA1) 3 be suggested that there is no active anxiety-related minutes after injection of either saline (1 mL/ratdintra-CA1) endogenous NMDA receptor activity in the CA1. or NMDA (0.5 mg/ratdintra-CA1). Each bar is mean standard Other parts of our experiments indicate that intra-CA1 error of the mean of percent of open arm time (A), percent of injection of enkephalin increased both OAT% and OAE% open arm entries (B), or locomotor activity (C)dn Z 7. without locomotor impairment in the EPM, indicating *p < 0.05, **p < 0.01, and ***p < 0.001 when compared with the the induction of an anxiolytic response by enkephalin. saline-treated rats. þp < 0.05 when compared with the saline- Furthermore, intra-CA1 administration of the d-receptor and NMDA-treated rats. CA1 Z dorsal hippocampus region; antagonist naltrindole produced substantial anxiogenic-like NMDA Z N-methyl-D-aspartate. effects in the plus maze, without any effect on the locomotor activity. The data show that the opioid receptor The results obtained from the present study show that system in the hippocampus is involved in the anxiolytic-like intraedorsal hippocampus (intra-CA1) injection of NMDA behavior. increased OAT% and OAE%, without any impairment in The important role of the opioidergic system in the locomotor functioning in the EPM, indicating the induction modulation of anxiety has been shown recently [36]. of an anxiolytic response by NMDA. Thus, the involvement Because opioid-deficient mice are more anxious than the of NMDA receptor mechanism of dorsal hippocampus in the normal controls [37], the anxiety disorders may be related, anxiety-like behavior of rats seems possible. at least in part, to the deficiency of the endogenous opioid Several recent investigations about the neurobiology of system [36]. Also, d-opioid receptors have been associated anxiety have highlighted the role of neurotransmitter with anxiety and depression, because d agonists reduce the glutamate as an important element in anxiety and anxious immobility of the rats in the forced swimming test [38,39]. behavior. The effects of glutamate are mediated through A study carried out by Filliol et al. demonstrated that different combinations of ionotropic and metabotropic d-opioid receptor knockout mice show anxiogenic-like glutamate receptors and potentially different subunit response in the EPM and the light-dark box. However, the combinations [28,29]. mice lacking m-opioid receptors exhibit lower levels of 492 J. Solati anxiety in the EPM [8]. The study by Sher in 2001 [37] glutamate release and attenuates the exercise pressor reflex. showed that intracerebroventricular microinjections of Brain Res 2000;865:177e85. d-opioid receptor antagonist cause a significant anxiogenic [13] Scanziani M, Gahwiler B, Thompson S. 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