Jalal Solati, PhD, Department of Biology, School of Science, Islamic Azad University, Karaj Branch, P.O. Box 31485-313, Karaj, Iran. Email: firstname.lastname@example.org
Aims: In the present study, we investigated the possible influence of the opioidergic system of the dorsal hippocampus on anxiety-like behaviors.
Methods: Elevated plus-maze, which is one of the methods used for testing anxiety, was used in the present study. Rats were anesthetized with ketamine and xylazine and special cannulas were inserted stereotaxically into the CA1 region of the dorsal hippocampus. After 1 week of recovery, the effects of intra-CA1 administration of morphine (0.25, 0.5, 1 and 2 µg/rat; 1 µl/rat; 0.5 µl/in each side), naloxone (2, 4, 6 and 8 µg/rat), enkephalin (1, 2, 5 and 10 µg/rat) and naltrindole (0.25, 0.5, 1 and 2 µg/rat) on percentage open arm time (%OAT) and percentage open arm entries (%OAE) were determined.
Results: Bilateral administration of morphine into CA1 decreases %OAT and %OAE, indicating an anxiogenic-like effect. Intra-CA1 injection of naloxone, an opioid receptor antagonist, increased both %OAT and %OAE, parameters of anxiolytic-like behavior. Bilateral administration of δ-opioid receptor agonist, [D-Pen2,5]-enkephalin acetate hydrate into the CA1, induced an anxiolytic-like effect. Furthermore, intra-CA1 injection of δ-opioid receptor antagonist, naltrindole hydrochloride, increased anxiety-related behaviors.
Conclusions: The results of the present study demonstrate that activation of μ-opioid receptors in this area produce an anxiogenic response while activation of δ-opioid receptors produces an anxiolytic response.
THE HIPPOCAMPUS HAS been shown to play an important role in the process of behaviors, such as learning and memory.1,2
Theories of hippocampal function have typically treated the structure as a whole; to the extent that they have differentiated between different regions of the structure, such as ventral and dorsal areas, and focused on the different cell subfield regions.3,4
Considerable data indicate that the ventral hippocampus is primarily involved in emotional processes, such as fear and anxiety, while CA1 is primarily involved in cognitive processes, such as memory and spatial analysis.3,5,6 However, there are several published data that have demonstrated the role of CA1 neural systems in the modulation of anxiety behaviors.1,7–9
Previous studies have indicated that the serotonergic system of the dorsal hippocampal and in particular, the postsynaptic 5-HT1A receptors, mediate an anxiogenic response, whereas the dorsal hippocampal cholinergic tone mediates an anxiolytic response.1,2
One report shows that opioids are involved in diverse functions, e.g. pain perception, respiration, homeothermy, nutrient intake and immune response.10 Recently, the role of the endogenous opioid system in regulating emotional behavior and related behaviors has been suggested.11,12 It has been shown that the peripheral injection of morphine or other μ-opioid receptor agonists elicits an anxiolytic effect, while the opioid receptor antagonists tend to induce an anxiogenic response.13–15
Studies with μ- and δ- but not κ-opioid receptor knockout mice indicated that the basal levels of anxiety in different models of anxiety were changed.16 However, other investigations showed that opioid-receptor-deficient mice are more anxious than normal mice.17 Many neurotransmitter systems in different sites in the central nervous system have been suggested to be involved in the modulation of effects of morphine on anxiety and behaviors.15,18 In the present study, possible mechanism(s) of μ- and δ-opioid systems involved in anxiety-like behaviors have been investigated.
Male Wistar rats obtained from Pasteur Institute (Tehran, Iran), weighing 180–230 g at the time of surgery, were used. Animals were housed four per cage in a room with an ambient temperature of 23 ± 1°C on a daily cycle of 12 h of light/12 h of darkness (7.00 hours to 19.00 hours). Animals had access to food and water ad libitum and were allowed to adapt to the laboratory conditions for at least 1 week before surgery. Rats were handled about 3 min each day prior to behavioral testing. All experiments were performed between 12.00 and 15.00 hours and each rat was tested only once. Seven animals were used in each experiment.19–21
Stereotaxic surgery and microinjections
Rats were anesthetized intraperitoneally with ketamine hydrochloride (50 mg/kg) and xylazine (4 mg/kg) and placed in a Stoelting stereotaxic instrument. The stainless steel guide cannula (22-gauge) was implanted in the right and left CA1 regions according to Paxinos and Watson.22 Stereotaxic coordinates for the CA1 regions were: −3.8 mm posterior to bregma, ±2 mm lateral to the midline and −3 mm ventral of the dorsal surface of the skull. The cannula was fixed to the skull with acrylic dental cement. The animals were allowed 5 days before the test to recover from surgery. The left and right CA1 were infused by means of an internal cannula (27-gauge), terminating 1 mm below the tip of the guides, connected by polyethylene tubing to a 1-µl Hamilton syringe. On each side, 0.5 µl solution was injected (1 µl/rat) over a 60-s period. The inner cannula was left in place for an additional 60 s to allow diffusion of the solution and to reduce the possibility of reflux. Intra-CA1 injections were made 5 min before testing.
This wooden, plus-shaped apparatus was elevated to a height of 50 cm, and consisted of two 50 × 10-cm open arms, and two 50 × 10 × 50-cm enclosed arms, each with an open roof. The maze was in the center of a quiet and dimly lit room. The rats' behavior was directly observed using a mirror that was suspended at an angle above the maze. Behavioral data were collected by a ‘blind’ observer who sat quietly 1 m behind one of the closed arms of the maze, using a chronometer. Five minutes following their respective drug treatment, rats were placed individually in the center of the plus-maze, facing one of the closed arms. The observer measured: (i) time spent in the open arms; (ii) time spent in the closed arms; (iii) number of entries into the open arms; and (iv) number of entries into the closed arms during the 5-min test period. An entry was defined as all four paws in the arm. The maze was cleaned with distilled water after each rat was tested. For the purpose of analysis,23–25 open-arm activity was quantified as the amount of time that the rat spent in the open arms relative to the total amount of time spent in any arm (open/total × 100), and the number of entries into the open arms was quantified relative to the total number of entries into any arm (open/total × 100). The total number of arms entered, as well as the total numbers of closed arms entered were used as indices of general locomotor activity.21,23,25,26
The drugs used in the present study were morphine sulphate (Temad, Tehran, Iran), naloxone hydrochloride (Tolid-Daru, Tehran, Iran), [D-Pen2,5]-enkephalin acetate hydrate and naltrindole hydrochloride (Tocris, Ellisville, MO, USA). All the drugs were dissolved in saline and were injected in a volume of 1 µl/rat (0.5 µl/each side of the dorsal hippocampus).
Experiment 1: Effects of μ-opioid receptor agonist and antagonist on anxiety behavior
Four groups of rats received bilateral dorsal hippocampus injection (intra-CA1) of μ-opioid receptor agonist morphine (0.25, 0.5, 1 and 2 µg/rat; 1 µl/rat) (Fig. 2). The other three groups received μ-opioid receptor antagonist naloxone (2, 4, 6 and 8 µg/rat, intra-CA1) (Fig. 2) and were compared with the saline control group.
Experiment 2: Effects of δ-opioid receptor agonists and antagonists on anxiety-like behavior
Four groups of rats received bilateral dorsal hippocampus injection (intra-CA1) of δ-receptor agonist [D-Pen2,5]-Enkephalin acetate hydrate (1, 2, 5 and 10 µg/rat) (Fig. 3). The other four groups received δ-receptor antagonist naltrindole hydrochloride (0.25, 0.5, 1 and 2 µg/rat intra-CA1) (Fig. 3) and were compared with the saline control group.
Figure 1 shows the approximate point of the drug injection in the dorsal hippocampus (CA1). The histological results were plotted on the representative section taken from the rat brain atlas of Paxinos and Watson.22 After completion of behavioral testing, animals were killed with an overdose of chloroform. Ink (0.5 µl of 1% aquatic methylene blue solution) was injected into the guide cannula, using a 25-gauge injection cannula that projected a further 0.5 mm ventral to the tip of the guides, to aid in histological verification. Brains were removed and fixed in a 9% formalin solution for 48 h before sectioning. Sections were taken through the brain areas of cannula placements, and the cannula placements were verified using the atlas of Paxinos and Watson.22 Data from rats with injection sites located outside the hippocampal CA1 area were not used in the analyses.
As data displayed normality of distribution and homogeneity of variance, one-way anova was used for comparison between the effects of different doses of drugs with vehicle. Two-way anova was used for evaluation of interactions between drugs. Following a significant F-value, post hoc analysis (least significant difference [LSD]) was performed for assessing specific group comparisons. Differences with P < 0.05 between experimental groups at each point were considered statistically significant.
Effects of μ-opioid receptor agonist and antagonist on anxiety behavior
Figure 2 shows the effect of intra-CA1 injection of morphine (0.23, 0.5, 1 and 2 µg/rat) in the elevated plus-maze in rats. One-way anova revealed that morphine decreased percentage open arm time (%OAT) (F[4,30] = 7.636, P < 0.05) and percentage open arm entries (%OAE) (F[4,30] = 6.545, P < 0.05). No change in the locomotor activity was observed (F[4,30] = 0.690, P > 0.05).
However, rats infused intra-CA1 with naloxone (2, 4, 6 and 7 µg/rat) showed significant increase in percentage of %OAT (F[4,30] = 5.294, P < 0.05) and %OAE (F[4,30] = 4.194, P < 0.05). No change in the locomotor activity was observed (F[4,30] = 1.814, P > 0.05) (Fig. 2).
Effects of δ-opioid receptor agonists and antagonists on anxiety-like behavior
One-way anova revealed that intra-CA1 injection of δ-opioid receptor agonist, [D-Pen2,5]-enkephalin acetate hydrate (1, 2, 5 and 9 µg/rat; 1 µl/rat) increased %OAT (F[4,30] = 4.421, P < 0.05) and %OAE (F[4,30] = 5.599, P < 0.05) at doses of 1, 2 and 5 µg/rat, indicating the induction of anxiolytic effects. No significant change in the locomotor activity was observed (F[4, 30] = 0.946, P > 0.05) (Fig. 3).
Intra-CA1 infusions of δ-opioid receptor antagonist, naltrindole hydrochloride, decreased %OAT (F[4,30] = 5.532, P < 0.05) at doses of 0.5, 1 and 2 µg/rat and decreased %OAE (F[4,30] = 5.961, P < 0.05) at doses of 0.23, 0.5, 1 and 2 µg/rat, indicating the induction of anxiogenic effects by naltrindole. No significant change in the locomotor activity was observed (F[4,30] = 0.431, P > 0.05) (Fig. 3).
In the present study, the effects of μ- and δ-opioidergic receptor agents in the elevated plus-maze have been investigated. We used a limited number of animals in each study group. The working objective and ethical requirement for the use of animals in research dictates the need to use the best methods, on the smallest number of appropriate animals, in order to obtain valid scientific information. On the other hand, methods used in the present study are time-consuming and there are difficulties with using large samples in this type of study.
It is widely believed that the hippocampus is involved in the neural circuitry of fear and anxiety. Previous studies showed the role of the dorsal hippocampus in the modulation of anxiety-related behaviors.
Our data showed that intra-dorsal hippocampus (intra-CA1) injection of morphine decreased %OAT and %OAE, without locomotor impairment in the elevated plus-maze, indicating the induction of anxiogenic response by this opioid. Moreover, intra-CA1 injection of naloxone increased %OAT and %OAE, but not locomotor in the elevated plus-maze, showing increased anxiety behaviors.
Other parts of our experiments indicated that intra-CA1 injection of [D-Pen2,5]-enkephalin increased %OAT and %OAE, without locomotor impairment in the elevated plus-maze, showing the induction of anxiolytic response by [D-Pen2,5]-enkephalin. Furthermore, intra-CA1 administration of δ-receptor antagonist, naltrindole, produced clear anxiogenic-like effects in the plus-maze, without any significant effect on locomotor activity.
Our data may show that μ- and δ-opioid receptors in the dorsal hippocampus are involved in anxiolytic-like behavior. The important role of the opioidergic system in the modulation of anxiety has been shown recently.17,27 Opioid-deficient mice are more anxious than normal controls,17,28 so the anxiety disorders may be related, at least in part, to a deficiency of the endogenous opioid system.17
Results of this study showed that activation of dorsal hippocampal μ-receptors by morphine produce an anxiogenic response, while blocking of the opioid receptors by naloxone reduce anxiety-related behaviors.
Studies by Zarrindast and his co-workers have documented that bilateral microinjection of morphine into the ventral hippocampus increased the percentage of open arm time and open arm entries, indicating induction of anxiolytic responses by morphine. However, our study showed that bilateral injection of morphine into the dorsal hippocampus produces an anxiogenic response. Numerous studies report the morphological, neurological, neurochemical and functional differences between dorsal and ventral zones from the hippocampus and these two distinct regions of the hippocampus modulate behaviors in different ways.29,30
The role of μ-receptors on anxiogenic responses has been reported in some previous studies.16,31–33δ-opioid receptor knockout mice showed anxiogenic-like responses, while the mice lacking μ-opioid receptors showed lower levels of anxiety in the elevated plus-maze.16,32
The anxiogenic response produced by morphine may be related to the inhibitory influence of μ-receptors upon GABAergic neurons. The opioid receptors activation elicit an inhibitory effect in GABAergic activity in several brain areas.34–36 Moreover, there are other reports indicating that modest doses of the opioid receptor antagonists, naloxone and naltrexone, which can block μ-receptors, produce anxiolytic recponces.31,37–39 The mechanism by which naloxone could exert such an anxiolytic effect may be the blockade of the tonic inhibitory influence of μ-opioid receptors upon GABAergic system.40
Enkephalin and δ-opiate receptors are extensively distributed inside the brain as a kind of endogenous opioid peptide and play several roles in analgesia, cardiovascular, respiratory and body temperature and regulating the immune functions.41δ-Opioid receptors have also been associated with anxiety and depression, as the δ-agonists reduced the immobility of rats in a forced swimming test.42,43 Intra-cerebroventricular microinjection of δ-opioid receptor antagonist caused a significant anxiogenic effect in mice.41 In contrast, the δ-opioid receptor knockout mice showed anxiogenic-like responses in the elevated plus-maze and the light–dark box.16,41
The present results indicate that the dorsal hippocampal opioidergic system has a different role in the modulation of anxiety. Activation μ-opioid receptors in this area produces an anxiogenic response while activation of δ-opioid receptors produces an anxiolytic response.
This research was supported by financial aid of the Islamic Azad University, Karaj Branch. We would like to thank Mr Masoud Asiaei for his English editing.