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Infusion of Neuropeptide Y Into CA3 Region of Hippocampus Produces Antidepressant-Like Effect via Y1 Receptor Hisahito Ishida, Yukihiko Shirayama,* Masaaki Iwata, Seiji Katayama, Ayaka Yamamoto, Ryuzou Kawahara, and Kazuyuki Nakagome ABSTRACT: A couple of papers indicate that patients with depression show a decrease in serum neuropeptide Y (NPY). To study the role of NPY in depression, we examined the effects of infusion of NPY into the hippocampus of learned helplessness (LH) rats (an animal model of depression). Infusion of NPY into the cerebral ventricle of LH rats showed antidepressant-like effects. Infusion of NPY into the CA3 region, but not the dentate gyrus (DG), produced antidepressant-like effects in the LH paradigm. Infusion of NPY did not affect locomotor activity or aversive learning ability. Coadministration of BIBO3304 (a Y1 receptor antagonist) with NPY to the CA3 region blocked the antidepressant-like effects of NPY, whereas coadministration of NPY with BIIE0246 (a Y2 receptor antagonist) to the CA3 region failed to block antidepressantlike effects. Furthermore, infusions of [Leu31 Pro34]PYY (a Y1 and Y5 receptor agonist) alone and BIIE0246 alone into the CA3 region produced the antidepressant-like effects in LH rats. These results suggest that infusion of NPY into the CA3 region of hippocampus of LH rats produces antidepressant-like activity through Y1 receptors and attenuating effects through Y2 receptors. V 2007 Wiley-Liss, Inc. C
KEY WORDS: learned helplessness (LH); neuropeptide Y (NPY); depression; hippocampus; behavior
INTRODUCTION Neuropeptide Y (NPY) might be involved in the mechanism of depression. Depressed patients showed lower plasma NPY levels than controls (Nilsson et al., 1995; Hashimoto et al., 1996), and one study found that the concentrations of NPY in the cerebrospinal fluid (CSF) of depressed patients were decreased (Heilig et al., 2004), although another study reported an unchanged concentrataion (Gjerris et al., 1992). In contrast, antidepressant treatments and electroconvulsive treatment increased NPY levels in the CSF of depressive patients (Mathe´ et al., 1996). These studies demonstrate the possible involvement of NPY in the pathophysiology of depression. Recently, some reviews described the relationship of NPY with depression (Redrobe et al., 2002b; Holmes et al., 2003; Obuchowicz et al., 2004). Several studies have demonstrated that chronic antidepressant treatment changed NPY, its mRNA, and its receptors in the hippocampus of rats and mice (Widdowson and Halaris, 1991; Caberlotto et al., 1998; Husum et al., 2000). Furthermore, repeated electroconvulsive seizure (ECS), which is also an effective therapy for depression, increases NPY and its mRNA in Department of Neuropsychiatry, Faculty of Medicine, Tottori University, Yonago, Japan *Correspondence to: Yukihiko Shirayama MD, PhD, Department of Psychiatry, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba, Chiba 260-8677, Japan. E-mail:
[email protected] Accepted for publication 20 December 2006 DOI 10.1002/hipo.20264 Published online 30 January 2007 in Wiley InterScience (www.interscience. wiley.com). C 2007 V
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the hippocampus of rats (Wahlestedt et al., 1990; Stenfors et al., 1992; Kragh et al., 1994; Mikkelsen et al., 1994; Zachrisson et al., 1995a; Mathe´ et al., 1997; Ma et al., 2002). In addition, the mood stabilizer lithium was found to increase hippocampal NPY (Zachrisson et al., 1995b). These reports indicate that mechanisms of action of antidepressants could involve hippocampal NPY. However, other studies reported that chronic antidepressant treatment does not affect NPY immunoreactivity and NPY mRNA in the hippocampus (Bellmann and Sperk, 1993; Heilig and Ekman, 1995). Learned helplessness (LH) is an animal model of depression. In this paradigm, an animal is initially exposed to uncontrollable stress. When the animal is later placed in a situation in which shock is controllable (escapable), the animal fails to acquire the escape and avoidance responses (Overmier and Seligman, 1967). This escape deficit is reversed by chronic antidepressant treatment (Anisman et al., 1980; Kametani et al., 1983). LH rats showed decreases in the numbers of NPY-positive cells in the hilus of the hippocampus (Ishida et al., 2005). Compatible with the above reports, infusion of NPY into the cerebral ventricle of rats and mice produced antidepressant-like effects in a forced swimming test (Stogner and Holmes, 1999; Redrobe et al., 2002a). Furthermore, subchronic treatment of LH rats with imipramine ameliorated the decrease in the number of NPY-positive cells in the hilus of the hippocampus (Ishida et al., 2005). Maternal deprivation, another animal model of depression, produced a reduction in NPY levels in the hippocampus (Jimenez-Vasquez et al., 2001; Husum and Mathe´, 2002; Lim et al., 2003). Two genetic animal models of depression, Finders Sensitive Line (FSL) rats and Fawn Hooded (FH) rats, showed decreases in the concentration of NPY in the hippocampus compared with control rats (Mathe´ et al., 1998; JimenezVasquez et al., 2000; Husum et al., 2001). Previous studies also reported that the Y1 receptor controlled anxiety (Wahlestedt et al., 1993) and voluntary alcohol consumption (Thiele et al., 2002). In addition, a recent study reported that overexpression of NPY in the amygdala of rats reduced anxiety-related behaviors through Y1 receptors (Primeaux et al., 2005). Thus, NPY may have antidepressant effects. The present study examined the effects of infusion of NPY into the DG or CA3 region of the hippocampus of LH rats, an animal model of depression, on the con-
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ditional active avoidance test. The second goal was to elucidate the mechanism by which NPY exerts antidepressant-like effects in the hippocampus of LH rats.
MATERIALS AND METHODS Animal and Treatments Animals use procedures were in accordance with the Tottori University Guide for the Care and Use of Laboratory Animals and were approved by the Tottori University Animal Care and Use Committee. Male Sprague-Dawley rats (225–300 g) were used. The animals were housed under 12 h light/dark cycle with free access to food and water. Surgery was performed using a stereotaxic apparatus (Narishige, Tokyo) under anesthesia with pentobarbital sodium solution (50 mg/kg, intraperitoneal injection, Abbott Laboratories) next day after the acquisition of LH. Rats received bilateral microinjection of different amounts of NPY (0.25, 2.5, 25, 250 ng/side), NPY and BIBO3304 (a Y1 receptor antagonist, 0.5 ng/ side), NPY and BIIE0246 (a Y2 receptor antagonist, 0.5 ng/side), [Leu31 Pro34]PYY (a Y1 and Y5 receptor agonist, 0.05, 0.5, 2.5, 25 ng/side), NPY13–36 (a Y2 receptor agonist, 2.5, 25 ng/side), or saline (0.9%) into the DG or CA3 region of the hippocampus. BIBO3304 and BIIE0246 were generously provided by Boeringer Ingelheim (Germany). A total volume of 1.0 ll was infused into each side over 15 min and the injection syringe was left in place for an additional 5 min to allow for diffusion. The coordinates for the cerebral ventricle, DG, and CA3 relative to the bregma according to the atlas of Paxinos and Watson (1997) were as follows: 20.3 anteroposterior (AP), 61.2 lateral, 23.4 dorsoventral (DV) from dura (cerebral ventricle); 23.8 AP, 62.0 lateral, 23.2 DV from dura (DG); and 23.6AP, 63.8 lateral, 23.0 DV from dura (CA3). Brains were sectioned at 15 lm and stained with cresyl violet. Sections were examined by light microscopy for the placement of NPY- and related compound infusions. The sites of the infusions are shown in Figure 1.
average 20–40 s)] were preceded by a 3 s conditioned stimulus tone that remained on until the shock was terminated. Rats with more than 20 escape failures in the 30 trials were regarded as having reached criterion and were used for further experiments. Approximately 60–70% of the rats reached this criterion. It is well established that subchronic treatment with imipramine significantly improves the ability of the animals to escape in the avoidance test (Shirayama et al., 2002, 2004). Thus, the LH paradigm is responsive to antidepressant treatment. On Day 4, rats received bilateral microinjections of NPY and/or other chemicals (BIBO3304, BIIE0246, [Leu31 Pro34]PYY, NPY13–36) as described above. On Day 7 (three days after surgery), a two-way conditioned avoidance test was performed. This test session consisted of 30 trials in which electric foot shocks [0.65 mA, 30 s duration, at random intervals (mean 30 s, average 20–40 s)] were preceded by a 3 s conditioned stimulus tone that remained on until the shock was terminated. The numbers of escape failures and the latency to escape in each 30 trial were recorded by the Gemini Avoidance System.
Open Field Test Three days after surgery, an open field test was performed in a square area (76.5 3 76.5 3 49 cm3) using a standard procedure (Lacroix et al., 1998). The open field was divided into
LH Paradigm To create the LH paradigm, animals are initially exposed to uncontrollable stress. When the animal is later placed in a situation in which shock is controllable (escapable), the animal not only fails to acquire the escape responses but also often makes no efforts to escape the shock at all. This escape deficit is reversed by subchronic antidepressant treatment (Chen et al., 2001; Iwata et al., 2006). LH behavioral tests were performed using the Gemini Avoidance System (San Diego Instruments, San Diego, CA). This apparatus was divided into two compartments by a retractable door. On Day 1 and 2, rats were subjected to 60 inescapable electric footshock [0.65 mA, 30 s duration, at random intervals (mean 30 s, average 20–40 s)]. On Day 3, a two-way conditioned avoidance test was performed as a postshock test to determine if the rats would show the predicted escape deficits. This screening session consisted of 30 trials in which electric foot shocks [0.65 mA, 6 s duration, at random intervals (mean 30 s, Hippocampus DOI 10.1002/hipo
FIGURE 1. Location of microinjection sites. Top is the DG; bottom is the CA3 region.
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Twenty-four hours later, each rat was placed in the lighted safe compartment, and the latency until re-entry into the darkened shock compartment was recorded as the measure of retention. In the current experiment, this test was performed for three consecutive days as a learning task based on retention memory.
Statistical Analysis Statistical differences among more than three groups were estimated by a one-way analysis of variance (ANOVA), fol-
FIGURE 2. Infusion of NPY into the cerebral ventricle decreases escape failure in the LH paradigm. NPY or saline was administered via bilateral infusion into the cerebral ventricle, and animals were subjected to a conditioned avoidance test three days later. Escape failure and latency to escape were determined, and the results are expressed as mean 6 standard error of mean (SEM). The number of animals is listed under each column. Top, t 5 2.276, P 5 0.0325; bottom, t 5 2.190, P 5 0.0389. *P < 0.05 when compared to saline-injected controls (Student’s t-test).
two areas, a peripheral area and a square center (40 3 40 cm2). The test room was dimly illuminated (60 W light, indirect). Rats were allowed to explore for 30 min. A computer software program (Be Trace: Behavioral and Medical Sciences Research Consortium, Hyogo, Japan) calculated the velocity of movement, the distance traveled and the time spent in the center of the open field. These parameters are thought to reflect locomotor activity and fear or anxiety, respectively.
Repeated Passive Avoidance Test A passive avoidance test was conducted according to standard procedures with the following modifications (Ferry et al., 1999). The apparatus was divided into two compartments by a retractable door: a lighted safe compartment and a darkened shock compartment (Gemini Avoidance System). Three days after surgery, animal received a single inescapable foot shock (0.65 mA; 3 s duration).
FIGURE 3. Infusion of NPY into the DG of the hippocampus in the LH paradigm. After exposure to IES, NPY or saline was infused into the DG of the hippocampus at the doses indicated, and the conditioned avoidance test was conducted three days later. Escape failure and latency to escape were determined, and the results are expressed as mean 6 SEM. The number of animals is listed under each column. Top, F(4,55) 5 1.131, P 5 0.3513; bottom, F(4,55) 5 1.349, P 5 0.2635. Hippocampus DOI 10.1002/hipo
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RESULTS
Infusion of NPY Into the Cerebral Ventricle of LH Rats The effect of bilateral microinjections of NPY into the cerebral ventricle was determined. LH rats that received bilateral microinjections of NPY into the cerebral ventricle demonstrated a significant improvement in the conditioned avoidance test relative to saline-treated controls (Fig. 2).
FIGURE 4. Infusion of NPY into the CA3 region of the hippocampus decreased escape failure, but the effect of NPY was reversed by coinfusion with BIBO3304 (Y1 antagonist) in the LH paradigm. After exposure to IES, NPY, NPY plus BIBO3304, NPY plus BIIE0246 (Y2 antagonist) or saline was infused into the CA3 of hippocampus at the doses indicated, and the conditioned avoidance test was conducted three days later. Escape failure and latency to escape were determined, and the results are expressed as mean 6 SEM. The number of animals is listed under each column. Top, F(4,45) 5 9.860, P < 0.0001; bottom, F(4,45) 5 8.041, P < 0.0001. *P < 0.05; **P < 0.01; ***P < 0.001 when compared to salineinjected animals (ANOVA followed by Scheffe’s test).
lowed by Scheffe’s test. For comparison of the mean values between the two groups, statistical evaluation was done using the two-tailed Student’s t-test. For the passive avoidance learning test, two-way repeated-measures ANOVA was performed to Hippocampus DOI 10.1002/hipo
FIGURE 5. Infusion of [Leu31 Pro34] PYY (Y1 agonist) into the CA3 of the hippocampus produced improvement in the LH paradigm. After exposure to inescapable foot shock, [Leu31 Pro34] PYY (Y1 agonist), NPY13–36 (Y2 agonist), saline was infused into the CA3 of the hippocampus at the doses indicated, and the conditioned avoidance test was conducted three days later. Escape failure and latency to escape were determined, and the results are expressed as mean 6 SEM. The number of animals is listed under each column. Top, F(6,65) 5 3.903, P 5 0.0023; bottom, F(6.65) 5 4.103, P 5 0.0017. *P < 0.05 when compared with saline-injected animals (ANOVA followed by Scheffe’s test).
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FIGURE 6. Effects of BIIE0246 (Y2 antagonist) infusion into the DG of the hippocampus in the LH paradigm. The results are expressed as mean 6 SEM of the number of animals listed under each column. Left top, F(2,16) 5 0.601, P 5 0.5600; left bottom,
F(2,16) 5 0.758, P 5 0.4849; right top, F(2,18) 5 3.728, P 5 0.0442; right bottom, F(2,18) 5 3.993, P 5 0.0367. *P < 0.05 when compared to saline-injected animals (ANOVA followed by Scheffe’s test).
Effects of NPY Infusion Into DG or CA3 Region of LH Rats
NPY into the CA3 region. Coadministration blocked the antidepressant-like effect of NPY (Fig. 4). However, coadministration of BIIE0246 (Y2 antagonist) with NPY into the CA3 did not prevent the antidepressant-like effects (Fig. 4). This indicates that NPY exerts antidepressant-like effects through Y1 receptors of the CA3 region of the hippocampus.
LH rat that received bilateral microinjection of NPY into the DG of the hippocampus did not demonstrate a significant improvement in the conditioned avoidance test relative to saline-treated controls (Fig. 3). In contrast, infusion of NPY into the CA3 region of the hippocampus of LH rats significantly decreased escape failure and latency to enter in the conditioned avoidance test (Fig. 4).
Effects of Coadministration of Y1 Antagonist or Y2 Antagonist With NPY Into CA3 Region of LH Rats To investigate which subtypes of NPY receptors were affected by NPY infusion, BIBO3304 (Y1 antagonist) was infused with
Infusion of Y1 Agonist or Y2 Agonist Alone Into CA3 Region of LH Rats After infusion of [Leu31 Pro34]PYY (Y1 and Y5 agonist) into the CA3 region, LH rats showed a significant improvement in performance of the conditioned avoidance test relative to vehicle-treated controls (Fig. 5). However, infusion of NPY13–36 (Y2 agonist) alone into the CA3 region of the hippocampus Hippocampus DOI 10.1002/hipo
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Infusion of Y2 Antagonist Alone Into DG or CA3 Region of LH Rats Infusion of BIIE0246 (Y2 antagonist) alone into the DG of hippocampus failed to improve performance in the avoidance test (Fig. 6). In contrast, infusion of BIIE0246 alone into the CA3 region of hippocampus of LH rats significantly decreased escape failure and latency to enter in the conditioned avoidance test (Fig. 6).
Effect of NPY on Open Field Tests Studies were conducted to determine the behavioral specificity of NPY infusion into the hippocampus. First, the effect of NPY infusion into the hippocampus on activity in an open
FIGURE 7. Effects of NPY infusion into the hippocampus on locomotor activity. NPY or saline (SAL) was infused into the DG or CA3, and three days later, the time spent in center, distance traveled, and velocity in an open field were determined. The results are the mean 6 SEM of the number of animals indicated under each column. Left top, Time in the center, t 5 0.171, P 5 0.8663; left middle, Distance, t 5 0.264, P 5 0.7956; left bottom, Velocity, t 5 0.824, P 5 0.4229; right top, Time in the center, t 5 0.011, P 5 0.9912; right middle, Distance, t 5 0.144, P 5 0.8872; right bottom, Velocity, t 5 0.899, P 5 0.3804.
failed to improve performance in the conditioned avoidance test (Fig. 5). These indicate that activation of Y1 receptors in the CA3 region produces the antidepressant-like effects. Hippocampus DOI 10.1002/hipo
FIGURE 8. Effect of NPY infusion into the hippocampus on passive avoidance learning. NPY or saline (SAL) was infused into the DG or CA3 region, and three days later, passive avoidance learning tests were conducted for three consecutive days. The results are the mean 6 SEM of the number of animals indicated under each column. Top, DG, treatment, F(1,45) 5 1.123, P 5 0.3060; time, F(3,45) 5 73.335, P < 0.0001. Bottom, CA3 region, treatment, F(1,51) 5 1.056, P 5 0.3185; time F(3,52) 5 25.153, P < 0.0001. There were no significant treatment 3 time interactions.
HIPPOCAMPAL NPY AND LEARNED HELPLESSNESS field was determined. The distance traveled and velocity as well as the time spent in the center, which is recognized as a marker of anxiety, was determined. Infusions of NPY into the DG or CA3 region of the hippocampus failed to affect time spent in the center, distance traveled, or velocity (Fig. 7). This would not be the result expected if a general increase in locomotor activity were to contribute to the effect of NPY on conditioned avoidance in the LH models of depression.
Effect of NPY on Repeated Passive Avoidance Test We also examined the effect of NPY infusion on the ability to learn using a passive avoidance test. Infusion of NPY into the DG failed to change the efficiency with which rats learned the aversive condition (Fig. 8). Similarly, infusion of NPY into the CA3 region of the hippocampus did not alter the length of time spent in the darkened compartment in the consecutive retention tests (Fig. 8). These results suggest that NPY infusion does not cause a deficit in learning that could result in the effects observed in the LH paradigms.
DISCUSSION The primary finding of the present study is that infusion of NPY into the CA3 region, but not the DG, produced antidepressant-like effects in LH rats, an animal model of depression, as well as it did when it was infused into the cerebral ventricle. The antidepressant effect of injection of NPY into the cerebral ventricle was seen in the forced swimming test in a previous study (Stogner and Holmes, 2000). Therefore, it is conceivable that NPY exerts antidepressant effects, at least in part, through the CA3 region of the hippocampus. This result is supported by previous studies demonstrating that maternal deprivation, an animal model of depression, reduced NPY levels in the hippocampus (Husum and Mathe´, 2002; Lim et al., 2003) and that FSL rats, a genetic animal model of depression, show decreased levels of NPY in the hippocampus compared with control rats (Husum et al., 2001). In addition, LH decreased the numbers of NPY-positive cells in the hilus of the hippocampus (Ishida et al., 2005), indicating the possibility of the involvement of NPY in learned despair. A neuroplasticity hypothesis has been proposed for depression (Duman et al., 1997; Kemperman and Kronenberg, 2003). Our previous study demonstrated that infusion of brain-derived neurotrophic factor (BDNF) into the DG or CA3 region of the hippocampus produced an antidepressant effect on depression-like behavioral deficits (Shirayama et al., 2002). It is noteworthy that expression of NPY is increased in hippocampal neurons following administration of BDNF (Croll et al., 1994; Marty and Onteniente, 1999). Furthermore, widespread infusion of BDNF into the hippocampus was reported to increase the numbers of NPYpositive cells in the hilus (Reibel et al., 2000). Taken together, the results may indicate that NPY produces antidepressant-like
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effects by the similar mechanism with BDNF. However, the present study demonstrated that NPY infusion into the CA3, but not the DG, display the antidepressant-like effects, whereas BDNF exerts antidepressants effects in both the DG and the CA3 region (Shirayama et al., 2002). Therefore, some difference in the mechanisms of BDNF and NPY must exist. Both the mossy fiber-pathway from the granule cell layer to the CA3 region and GABAergic interneurons coexpressing NPY in the CA3 region could be involved in the present result. Since NPY is synthesized by granule cells and transferred to the CA3 region through mossy fiber (McCarthy et al., 1998), it is likely that NPY released from the terminal of mossy fiber exerts the antidepressant effects in the CA3 region. In addition, NPYpositive interneurons in the CA3 region are connected with the end of mossy fibers through Y2 autoreceptors (Vezzani et al., 1999). Although NPY is considered as a type of neurotransmitter (or neuromodulator), NPY is known to have a neuroproliferative effect on hippocampal precursor cells (Howell et al., 2003). Similarly, administration of NPY in the cerebral ventricules blocked methamphetamine-induced apoptosis in the mouse striatum (Thiriet et al., 2005). Therefore, it could be that neuroproliferative effects are involved in the NPY-produced antidepressant-like effects. Future study is needed to address this question. The second finding is that coadministration of a Y1 antagonist blocked the antidepressant-like effects of NPY infusion into the CA3 region, and activation of Y1 receptors in the CA3 region exerted the antidepressant-like effects. Therefore, it could be that Y1 receptors in the CA3 region are involved in the antidepressant-like action of NPY. This is partially supported by a previous study that intracerebroventricular administration of NPY and a Y1 agonist reduced the time spent in immobility in the forced swimming test, another model of depression (Redrobe et al., 2002a). These antidepressant effects were found to be sensitive to serotonin, and Y1 receptors in the hippocampus as well as the amygdala and cortex seemed to be involved in the beneficial properties of NPY (Redrobe et al., 2005). Other studies reported that FSL rats, a genetic model of depression, showed decreases in the expressions of NPY mRNA and increases in Y1, but not Y2, binding sites in the CA regions of the hippocampus (Caberlotto et al., 1998, 1999). In contrast, antidepressant fluoxetine treatment increased NPY mRNA in the CA region and DG and decreased Y1 receptor mRNA expression in the DG of FSL rats (Caberlotto et al., 1998). Other type of study reported that lithium inhibits Y1 receptor internalization (Parker et al., 2005). Although electroconvulsive shock is the effective treatment for depression, kainic acid-induced seizures, which cause increases in NPY expression, increased Y1 receptor mRNA in the CA2 pyramidal neurons that are adjacent to the CA3 region (Kofler et al., 1997). The precise mechanism remains to be proven. The third finding is that infusion of a Y2 antagonist alone into the CA3 region exerted antidepressant-like effects. Amelioration of LH-behavior by a Y2 antagonist suggests a tonic inhibitory action through Y2 receptors at the CA3 region of LH rats. However, this is unlikely because NPY levels have been Hippocampus DOI 10.1002/hipo
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reported to be decreased in animal models of depression (see introduction section). For a while, it is likely that Y2 receptors activation reduces the antidepressant-like effects of NPY. Y2 receptors are autoreceptors that inhibit NPY secretion and glutamate release. Thus, antagonism of the Y2 receptors may prompt NPY release, producing a similar condition with exogenous infusion of NPY. A recent study reported that mice lacking Y2 receptors displayed less immobility in the forced swimming test (Tschenett et al., 2003). It could be that Y2 autoreceptors modulate the NPY concentration as a feedback system, such that NPY concentration remains within a normal range. It is likely that the antidepressant effects by NPY infusion into the CA3 region are attenuated through the subsequent activation of Y2 receptors, although this speculation remains to be verified. Therefore, a Y2 antagonist could be a therapeutic drug for the treatment of depression. Several roles of NPY in the hippocampus have been proposed. For example, reduced hippocampal NPY levels are associated with increased ethanol consumption, suggesting that enhancing ethanol consumption might constitute a compensatory antianxiety strategy (Ehlers et al., 1998; Thiele et al., 1998), and it is often seen in patients suffering from depression. A recent study reported that rats overexpressing NPY in the hippocampus displayed an attenuated sensitivity to restraint stress (Thorsell et al., 2000), and acute stress was found to alter NPY expression in the hippocampus (Conrad and McEwen, 2000). In human studies, acute stress increased plasma NPY levels, and there was a significant negative relationship between psychological distress and stress-released NPY, indicating that NPY confers anxiolytic activity (Morgan et al., 2002). However, open field test in the present study did not show any significant differences in time spent in the center, which is taken as one measure of anxiety (Lacroix et al., 1998), suggesting that NPY’s antidepressant-like effects contribute to the modulation of anxiety to a small extent. In addition, rats overexpressing NPY in the hippocampus displayed impairment of spatial learning (Thorsell et al., 2000). However, repeated passive avoidance test in the current study did not indicate that NPY has the ability to enhance learning or induce a learning deficit. Therefore, the antidepressant-like effects produced by NPY infusion into the CA3 region were not due to impairments in learning and memory. A deficit in GABA function is hypothesized in depression (Cryan and Kaupmann, 2005). Thus, reductions in GABAergic transmission were seen in depressed patients, and antidepressants raised cortical GABA levels in patients with depression (reviewed by Krystal et al., 2002). One report showed that the neuroactive drug topiramate, which enhances GABAergic transmission, normalized hippocampal NPY levels in FSL rats (Husum et al., 2003). Furthermore, previous studies demonstrated that chronic stress alters mRNA expression of a GABAsynthesizing enzyme, glutamic acid decarboxylase in the hippocampus (Bowers et al., 1998) and that chronic exposure to stress levels of corticosterone altered GABAA receptor subunit mRNA levels in the hippocampus (Orchinik et al., 1995). Since NPY is colocalized with GABA interneurons in the hipHippocampus DOI 10.1002/hipo
pocampus (Kohler et al., 1986; Pascual et al., 1999), NPY could produce the antidepressant-like effects through enhancement of GABAergic neurotransmission. The alternative mechanism might be that NPY injection attenuates GABAergic transmission, producing in turn mild enhancement of glutamatergic transmission, although this is a speculation. In support of this, AMPA-R potentiators, mGluR1/5 or 2/3 antagonists are known to produce antidepressant-like effects (Alt et al., 2005; Karasawa et al., 2005; Belozertseva et al., 2007). The reason why [Leu31Pro34]PYY (Y1 and Y5 agonist) were not dose-dependent in the current study might be explained. NPY mediates the anticonvulsant effects through Y5 receptors (Woldbye et al., 1997; Marsh et al., 1999). Y5 receptors are assumed to inhibit glutamate release in the hippocampus, suggesting the existence of Y5 receptors at the dendrites. Even if antidepressant effects of NPY are exerted through enhancement of glutamate transmission, the activation of Y5 receptors might reduce the antidepressant action of [Leu31Pro34]PYY, producing the biphasic curve. The precise mechanism remains to be elucidated. In summary, infusion of NPY into the CA3 region, but not into the DG, of the hippocampus produced antidepressant-like effects in LH rats. Coadministration of BIBO3304 (Y1 antagonist) with NPY into the CA3 region blocked the effect of NPY, whereas coadministration of BIIE0246 (Y2 antagonist) with NPY into the CA3 region failed to block antidepressant-like effects. Furthermore, infusions of [Leu31 Pro34]PYY (Y1 and Y5 agonist) alone and BIIE0246 (Y2 antagonist) alone into the CA3 region produced the antidepressant-like effects in the LH rats. Finally, infusion of NPY did not alter locomotor activity or aversive learning ability. These results demonstrate that NPY produces antidepressant-like activity through Y1 receptors and attenuating effects through Y2 receptors at the CA3 region of the hippocampus of LH rats.
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