In the present study, we aimed to investigate the role of N-methyl-d-aspartate (NMDA) receptors in the antidepressant-like effects of a σ1 receptor agonist, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (SA-4503), in the olfactory bulbectomized (OB) rat model of depression. A symptomatology-based behavioral investigation was made by reconstructing in OB rats the symptoms of depression, such as psychomotor agitation, loss of interest, and cognitive dysfunction, using a typical antidepressant, desipramine, as a positive control. Repeated treatment with SA-4503 ameliorated the behavioral deficits in OB rats resembling depression symptoms in the open-field test, sexual behavior test, and cued and contextual fear-conditioning test. SA-4503 displayed advantages over desipramine in the sexual behavior test. SA-4503 also reversed the decrease in the protein expression of NMDA receptor subunit (NR)1, but not NR2A or NR2B, in the prefrontal cortex, hippocampus, and amygdala of OB rats. The behavioral and neurochemical effects of SA-4503 were blocked by combined treatment with a specific σ1 receptor antagonist, N,N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl)ethylamine monohydrochloride (NE-100). Furthermore, the effects of SA-4503 on the performance of OB rats in the behavioral tests were abrogated by acute treatment with an NMDA receptor antagonist, (–)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). The present study indicated for the first time that the σ1 receptor agonist SA-4503 may have effects on depressive symptoms such as agitation, loss of interest, and impaired cognition, which are mediated by NMDA receptors.
σ1 Receptors are particularly concentrated in the limbic structures of the brain, which play important roles in emotion and cognition (Matsuno et al., 1996; Skuza, 2003; Skuza and Wedzony, 2004; Bermack and Debonnel, 2005; Stahl, 2005). Various antidepressants, including tricyclic compounds, selective serotonin reuptake inhibitors, and monoamine oxidase inhibitors, possess affinity and act as agonists for σ1 receptors (Maurice et al., 2001; Su and Hayashi, 2003). Based on the above-mentioned points, it is hypothesized that σ1 receptor agonists may act as antidepressants. SA-4503 is a highly selective agonist of σ1 receptors, with higher binding affinity than a prototypical σ1 receptor agonist, (+)-SKF-10047 (Matsuno et al., 1996; Guitart et al., 2004). Although it has been reported that SA-4503 facilitates the release of acetylcholine or dopamine and potentiates the function of N-methyl-d-aspartate (NMDA) receptors (Bergeron and Debonnel, 1997; Urani et al., 2002) via the activation of σ1 receptors, the mechanisms underlying the antidepressant-like effects of SA-4503 are not clear.
Although many studies on depression have focused on alterations in the levels of monoamines, recent studies have investigated postsynaptic targets. NMDA receptors play important roles in fundamental functions of neurons. However, the role of NMDA receptors in depression is still not clear. It has been reported that NMDA receptor density or the mRNA expression of NR1 decreases in the prefrontal cortex (PFC) or hippocampus (Hip) of depressive patients (Nowak et al., 1995; Law and Deakin, 2001; Nudmamud-Thanoi and Reynolds, 2004), and long-term use of a specific NMDA receptor antagonist, phencyclidine, induces symptoms of acute anxiety and depression in humans (Liden et al., 1975; De Angelis and Goldstein, 1978). The involvement of NMDA receptors in depression has also been indicated in pharmacological research: repeated treatments with NMDA receptor antagonists, e.g., phencyclidine and MK-801, not only impair performance in the forced swimming test but also prevent the behavioral and neurochemical effects of antidepressant treatments (De Montis et al., 1993; Meloni et al., 1993; Petrie et al., 2000; Javitt, 2004).
The olfactory bulbectomized (OB) rat has been proposed as a model of depression, exhibiting several essential symptomatic isomorphisms, such as psychomotor agitation, loss of interest, and impaired learning and memory (Holmes, 2003). The olfactory bulbs have extensive neural connections with the structures of the limbic system and other parts of the brain, and they influence many emotional aspects of behavioral and other brain output functions (Jesberger and Richardson, 1985). Bilateral olfactory bulbectomy in rodents produces neuroanatomical deficits analogous to the cortical/allocortical degeneration in depressive patients that is, in general, not dependent on particular structures (Holmes, 2003).
The present symptomotology-based study was conducted to investigate the antidepressant-like effects of the σ1 receptor agonist SA-4503 and the role of NMDA receptors in the effects, using OB rats as a model of depression, since OB rats have dysfunctional glutamatergic systems (Kelly et al., 1997). In the present study, a tricyclic antidepressant, desipramine, was selected as a positive control to compare the σ1 receptor- and non-σ1 receptor-mediated effects, based on the fact that desipramine is a typical antidepressant and its affinity for σ1 receptors is the weakest of the antidepressants presently in clinical use and several hundred times weaker than that of SA-4503 (Matsuno et al., 1996; Narita et al., 1996).
Materials and Methods
Animals. Five-week-old male Sprague-Dawley rats (170–200 g), were purchased from Japan SLC (Shizuoka, Japan). All rats were housed in our Animal Experimental Center, at a room temperature of 25 ± 1°C and a relative humidity of 40 to 60%. The rooms were illuminated from 9:00 AM to 9:00 PM. All experiments were performed following the Guidelines for Animal Experiments of Nagoya University, which conformed to the international guidelines set out in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).
Medicines and Reagents. SA-4503 was provided by M's Science Corporation (Kobe, Japan). Desipramine hydrochloride (DES), NE-100, and (+)-MK-801 were purchased from Sigma-Aldrich (St. Louis, MO). Goat polyclonal anti-NR1, NR2A, and NR2B IgG recognizing protein bands of approximately 103, 180, and 200 kDa and horseradish peroxidase-conjugated donkey anti-goat IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-NR1 antibody has an epitope mapping at the C terminus of NR1011 (938 amino acids in rat brain; approximately 103 kDa), which is also known as ζ1, NR1a, or NMDAR1-1a, and is the predominant form of the eight splices of NR1 in adult rat brain (Laurie and Seeburg, 1994; Zukin and Bennett, 1995).
Surgical Procedure. The rats were anesthetized with 60 mg/kg pentobarbital sodium, and then they were fixed on a stereotactic apparatus (Narishige, Tokyo, Japan). A midline sagittal incision was made to expose the skull overlaying the olfactory bulbs. A hole 4 mm in diameter was made through the skull 6 mm anterior to the bregma. The olfactory bulbs were cut with a microknife, and they were aspirated out using a pipette tip connected with a water suction pump, with care being taken not to damage the frontal cortex. The cavity for the olfactory bulbs was filled with hemostatic sponge. The hole in the skull was covered with a piece of gelatin gauze, and the skin was sutured. Sham-operated rats were treated in a similar way, except that the olfactory bulbs were not removed. The success of the operation was anatomically confirmed after all of the behavioral tests, and the data from the maloperated rats were excluded from the subsequent analysis.
Schedule of Drug Administration and Behavioral Tests. The schedule of drug administration and behavioral tests is shown in Fig. 1. Saline, desipramine, SA-4503, and/or NE-100 was s.c. administered daily 2 weeks after the surgery for 16 days. MK-801 was s.c. injected 30 min before testing or training trials.
Open-Field Test. The present protocol was adapted from those of Kameyama et al. (1980) and Kelly and Leonard (1994). The open field apparatus, painted gray, consisted of a square arena (60 × 60 cm) divided into 15-cm squares by black lines. The wall of the arena was 30 cm in height. A 60-watt light bulb was positioned at the center 90 cm above the base of the arena.
On the 29th day after the operation, ambulation and rearing frequencies were recorded in the first 3 min immediately after each rat entered the arena. After each test, the apparatus was sprayed with 70% alcohol, and it was wiped thoroughly to eliminate residual odor.
Sexual Behavior Test. The present protocol was adapted from that of Breigeiron et al. (2002). The apparatus for the sexual behavior test consisted of a transparent Plexiglas box [45 (length) × 27 (width) × 39.5 (height) cm] with a black plastic base, illuminated with a red lamp. On the 30th day after the operation, the sexual behavior of individual male OB rats was observed for 30 min between 10:00 PM and 3:00 AM. A male rat was first placed in the Plexiglas box to habituate to the environment for 3 min. Then, a sexually receptive normal female rat was introduced that had been s.c. administered 0.14 mg of estradiol 72 and 48 h before the test and 0.7 mg of progesterone 4 h before the test. The following parameters of sexual behavior were recorded: starting latency of genital probing and thrusting, count of genital probing and thrusting, and the percentage of the rats that probed the female genitals or showed thrusting behavior. After each test, the apparatus was sprayed with 70% alcohol, and it was wiped thoroughly to eliminate residual odor.
Cued and Contextual Fear-Conditioning Test. The present protocol was adapted from those of Mamiya et al. (2003) and Phillips and LeDoux (1992). The apparatus consisted of a transparent Plexiglas box [45 (length) × 27 (width) × 39.5 (height) cm; the neutral box] with a black plastic base and a Perspex box [32 (length) × 26 (width) × 48 (height) cm; the conditioning box] with a steel grid floor connected to an electric shock generator (Neuroscience-Idea Co., Ltd., Osaka, Japan) and enclosed in an opaque compartment. The neutral box was illuminated with a red lamp, and the conditioning box was illuminated with a fluorescent lamp (6 watt).
For measuring basal levels of the freezing response (preconditioning phase), on the 31st day after the operation, rats were individually placed in the neutral box for 1 min and then in the conditioning box for 2 min. For conditioning (conditioning phase), a 60-s tone (75 dB) was presented as a conditioned stimulus. Just before the end of the tone, a 0.5-mA electric foot-shock lasting for 0.5 s was delivered as an unconditioned stimulus. The tone and the electric foot-shock ceased together. It should be noted that a 0.5-mA electric current lasting for 0.5 s in only one training session was not strong enough to form a stable conditioned response in all of the rats; hence, a difference in the ability to learn and memorize could be observed.
Cued and contextual tests were carried out 24 h after the conditioning. For the cued test, the freezing response was measured in the neutral box for 1 min in the presence of the tone. For the contextual test, rats were placed in the conditioning box, and the freezing response was measured for 2 min in the absence of the tone and foot shock. The freezing response was defined as follows: all four paws of the rat remaining still and the animal stooped down with fear. After each test, the apparatus was sprayed with 70% alcohol and wiped thoroughly to eliminate residual odor.
Western Blot Analysis. After all of the behavioral tests, the rats were sacrificed by decapitation. The dorsal PFC, the CA1-CA3 and dentate gyrus of the Hip, and the posteromedial and posterolateral cortical amygdaloid nuclei of the Amg were rapidly dissected out according to the atlas of the brain of the rat (Paxinos and Watson, 1998). Brain samples were frozen and stored at –80°C until used. The brain samples were homogenized in 150 μl of ice-cold lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 1 mM sodium orthovanadate, 10 mM EDTA, 10 mM NaF, 0.1% SDS, 1% Igepal CA-630 (Sigma-Aldrich), 1% sodium deoxycholate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 0.5 mM dl-dithiothreitol] using an ultrasonic processor (Astrason, Farmingdale, NY). After homogenization, the lysates were kept in an ice bath for 20 min, and then they were centrifuged at 13,000 rpm for 20 min at 4°C. The protein concentration of supernatants was determined by Lowry's method (Lowry et al., 1951). Samples of equal protein concentration were made by mixing the supernatants with lysis buffer, diluting 1:1 with sample-loading buffer (100 mM Tris, 200 mM dl-dithiothreitol, 4% SDS, 0.2% bromphenol blue, and 20% glycerol, pH 6.8), and heating at 95°C for 5 min. Different samples with a protein concentration of 30 μg/10 μl were electrophoresed by SDS-polyacrylamide gel electrophoresis (PAGE) (6–15% step-gradient resolving gel, an upper 6% gel used for the separation of NMDA receptor subunits, and a lower 15% gel for the separation of β-actin), transferred to polyvinylidene difluoride membranes, and incubated in block solution. The membranes were then incubated overnight with anti-NR1, NR2A, or NR2B antibody (1:1000) at 4°C. After wash, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and then they were washed thoroughly. The bands on membranes were then visualized, and the light absorbance was analyzed using an ATTO Densitograph Software Library Lane Analyzer (Atto Co., Tokyo, Japan).
Statistical Analysis. Statistical differences were evaluated with a one-way analysis of variance (ANOVA), except for the evaluation of the time-dependent change in body weight, for which a two-way ANOVA was used. The modified Tukey's test was applied after ANOVA tests for multiple comparisons. The difference in the percentage of rats that showed genital-probing and thrusting behavior among the groups in the sexual behavioral test was evaluated with the χ2 test. The criterion for a statistically significant difference was p < 0.05.
Antidepressant-Like Effects of SA-4503 in OB Rats
Open-Field Test. The open-field test is the behavioral test most commonly used to evaluate the antidepressant-like effects of medicines using the OB rat model. As shown in Fig. 2, a and b, saline-treated OB rats exhibited significantly increased counts of ambulation [F(8,141) = 10.088; p < 0.01] and rearing [F(8,141) = 8.168; p < 0.01] in the first 3 min after being put into the open-field arena. The exploratory hyperactivity in OB rats was reversed by the repeated treatments both with 10 mg/kg desipramine and 0.3 mg/kg SA-4503. The effects of SA-4503 were abolished by the combined treatment with 5 mg/kg NE-100. Desipramine at 10 mg/kg, SA-4503 at 0.3 mg/kg, and NE-100 at 5 mg/kg did not significantly affect behavior in sham-operated rats. Interestingly, the counts of ambulation and rearing decreased in OB rats compared with sham-operated rats from 6 to 9 min after the animals were put into the open-field arena. These data are not shown because these counts are not commonly adopted as experimental indices in the open-field test using OB rats.
Sexual Behavior Test. The loss of interest shown in depressive patients is a core symptom of depression as depicted in the Diagnostic and Statistical Manual, Version IV (Seidman and Roose, 2001). As an alternative measure, we examined the effect of the σ1 receptor agonist SA-4503 on the sexual dysfunction. As shown in Fig. 3, the starting latency of genital probing [F(8,141) = 29.466; p < 0.01] and thrusting [F(8,141) = 6.343; p < 0.01] was increased in saline-treated OB rats (Fig. 3, a and d), whereas the number of genital-probing [F(8,141) = 13.300; p < 0.01] and thrusting events [F(8,141) = 4.178; p < 0.01] (Fig. 3, b and e) and the percentage of the rats that showed genital-probing and thrusting behavior (Fig. 3, c and f) were decreased, compared with values for sham rats. These results showed sexual deficits in OB rats.
Treatment with 0.3 mg/kg SA-4503 reduced the deficits of genital-probing and thrusting behavior in OB rats without affecting the behavior in sham-operated rats (Fig. 3). The effects of SA-4503 on sexual behavior were blocked by NE-100 (Fig. 3). Repeated treatment with desipramine at the dose of 10 mg/kg ameliorated the loss of genital-probing behavior (Fig. 3, a–c), without affecting thrusting behavior (Fig. 3, d–f).
Cued and Contextual Fear-Conditioning Test. Major depressive patients exhibit significant cognitive dysfunction, to which minor depression is not related (Murphy et al., 1998; Airaksinen et al., 2004; Stordal et al., 2004). In the preconditioning phase of the test, all the rats showed a similar freezing time either in the neutral box or in the conditioning box (data not shown). Twenty-four hours after conditioning, OB rats exhibited a significantly shortened freezing time in both the cued [F(8,116) = 8.783; p < 0.01] and contextual [F(8,116) = 10.440; p < 0.01] tests compared with sham-operated rats (Fig. 4, a and b).
In both the cued and contextual tests, the cognitive deficits in OB rats were reversed by repeated treatment with 10 mg/kg desipramine and SA-4503 (0.3 mg/kg in cued test and 0.1 and 0.3 mg/kg in contextual test; Fig. 4, a and b). The effects of SA-4503 were blocked by NE-100 at the dose of 5 mg/kg (Fig. 4, a and b). In preliminary experiments, no change in the response threshold was found in saline- and drug-treated OB rats: the minimal current intensities required to elicit flinching/running, jumping, or vocalization in saline- and drug-treated OB rats were the same as those in sham-operated control rats (data not shown).
Involvement of NMDA Receptors in the Effects of SA-4503
Because 0.1 and 0.3 mg/kg SA-4503 significantly improved the behavioral abnormalities in OB rats in a dose-dependent manner, the dose of 0.3 mg/kg was used in all subsequent experiments.
Effects of SA-4503 on Protein Expression of NRs. As shown in Fig. 5, the protein expression of NR1 decreased in the PFC [F(4,50) = 19.055; p < 0.01], Hip [F(4,50) = 4.274; p < 0.01], and Amg [F(4,50) = 19.399; p < 0.01] of saline-treated OB rats compared with sham-operated control rats. The loss of NR1 was ameliorated by the treatments with 10 mg/kg desipramine and 0.3 mg/kg SA-4503. The improving effects of SA-4503 on the protein expression of NR1 in these regions in OB rats were blocked by 5 mg/kg NE-100. There was no significant difference in the protein expression of NR2A or NR2B in these regions between sham-operated and OB control rats (Fig. 6). These results are consistent with previous publications on the density and function of NMDA receptors in the brain of OB rats, and they fit well with reports that the expression of NR1 decreases in the brain of depressive patients (Kelly et al., 1997; Law and Deakin, 2001; Robichaud et al., 2001; Nudmamud-Thanoi and Reynolds, 2004).
Open-Field Test. As shown in Fig. 7, the treatment with MK-801 at 0.03 mg/kg blocked the effect of 0.3 mg/kg SA-4503 on the exploratory hyperactivity in OB rats. The treatment with MK-801 at 0.03 mg/kg tended to increase the counts of ambulation [F(4,85) = 11.143; p < 0.01] and rearing [F(4,85) = 11.230; p < 0.01] in the sham-operated rats.
Sexual Behavior Test. The effects of 0.3 mg/kg SA-4503 on the latency of genital probing [F(4,75) = 38.257; p < 0.01] and thrusting [F(4,75) = 4.797; p < 0.01] (Fig. 8, a and d), the number of genital-probing [F(4,75) = 6.784; p < 0.01] and thrusting events [F(4,75) = 3.850; p < 0.01] (Fig. 8, b and e), and the percentage of animals that probed the female genitals and showed thrusting behavior (Fig. 8, c and f) were blocked by the treatment with MK-801 at 0.03 mg/kg, a dose that did not significantly affect sexual behavior in the sham-operated rats (Fig. 8).
Cued and Contextual Fear-Conditioning Test. The treatment with MK-801 at 0.03 mg/kg blocked the effects of 0.3 mg/kg SA-4503 in both the cued [F(4,49) = 15.305; p < 0.01] and contextual [F(4,49) = 13.493; p < 0.01] tests (Fig. 9, a and b). At the dose of 0.03 mg/kg, the treatment with MK-801 significantly impaired the performance of sham-operated rats (Fig. 9, a and b).
Changes in Body Weight of Sham-Operated and OB Rats Treated with Saline and Medicines
Olfactory bulbectomy induced the decrease of body weight in rats. Treating OB rats with SA-4503 at the dose of 0.3 mg/kg partially reversed the decrease of the body weight. In contrast to the treatment with SA-4503, repeated treatment with desipramine at the dose of 10 mg/kg decreased the body weight of OB rats [Fgroup (3,2897) = 222.742; p < 0.01; and Ftime (28,2897) = 485.303; p < 0.01] (Fig. 10).
The OB rat is considered to be one of the best animal models of depression in terms of construct validity (Jesberger and Richardson, 1988; Lumia et al., 1992; Kelly et al., 1997; van der Stelt et al., 2005). Chronic deprivation of olfaction, the primary sensory mode in rats, constitutes a stress of high intensity, and the behavioral deficits induced by OB are primarily the result of alterations in neuronal functions, which is supported by the phenomenon that the behavioral deficits can be reversed by antidepressant treatments although the olfactory bulbs are nonexistent (van Riezen et al., 1977; Jesberger and Richardson, 1988; Mar et al., 2000; O'Neil and Moore, 2003). The depression symptom-resembling deficits in OB rats can be normalized by chronic, not acute, antidepressant treatments (Jesberger and Richardson, 1985; Kelly et al., 1997). In previous preliminary study, treating OB rats with SA-4503 for 1 week did not significantly ameliorate the behavioral deficits, which were ameliorated after treating for 2 weeks in the present study.
Desipramine, a conventional tricyclic antidepressant that inhibits the reuptake of norepinephrine and 5-HT, was used as a positive control. The binding affinity of desipramine (Ki ≈ 1987 nM) for σ1 receptors is approximately 450 times weaker than that of SA-4503 (Ki ≈ 4.4 nM; IC50 ≈ 17.4 nM) (Narita et al., 1996; Shiba et al., 2006), and it takes effects mainly by inhibiting the reuptake of norepinephrine (IC50 ≈ 8.3 nM) or 5-HT (IC50 ≈ 17.5 nM) at the present dose (Pi et al., 1986; Hyttel, 1993). We also treated rats with imipramine at the dose of 20 mg/kg; however, the subcutaneous or intraperitoneal treatment induced severe inflammation in the rats. Therefore, imipramine-treated rats were not fit for behavioral analyses in the emotional study. Selective serotonin reuptake inhibitors were not preferred as control agents, given that they increase the risk of suicide-related behavior, especially in adolescents (Fegert and Herpertz-Dahlmann, 2005).
The open-field test is most commonly used for screening antidepressants using OB rat model. Within the initial 3 min in a stressful environment, OB rats show hyperlocomotion, which resembles the psychomotor agitation in depression, the extreme of which is a suicidal impulse (Lumia et al., 1992; Holmes, 2003). Based on the predictive value of the open-field test, it was suggested that SA-4503 may have antidepressant-like effects, which is further supported by the results of the sexual behavioral and the fear-conditioning tests.
Patients with major depression exhibit symptoms of sexual and cognitive dysfunction, which have been proven to be unrelated to minor depression and dysthesia (Murphy et al., 1998; Seidman and Roose, 2001; Airaksinen et al., 2004; Stordal et al., 2004). Alternatively, the sexual dysfunction in OB rats resembles the loss of interest that is a core symptom of depression. The result with the sexual dysfunction in OB rats is consistent with previous publications (Mathew et al., 1980; Mathew and Weinman, 1982). Compared with SA-4503, desipramine had relatively weak effects on sexual behavior in OB rats. It improved genital-probing behavior, rather than thrusting behavior, indicating a combination of positive effects and latent side effects of desipramine and that SA-4503 may have therapeutic advantages over it.
Major depression is associated with cognitive impairments (Murphy et al., 1998; Airaksinen et al., 2004; Stordal et al., 2004). Studies have reported spared functions in depressed patients in tests tapping implicit memory (Hertel and Hardin, 1990; Danion et al., 1995), explicit memory (Bazin et al., 1994), and attention (Landro et al., 2001). In the present study, an impairment of associative learning and long-term memory involving the Hip and Amg was observed in OB rats in the fear-conditioning test, which fits well with some clinical observations demonstrating explicit memory deficits in depressive patients (Vythilingam et al., 2004; Kieseppa et al., 2005). The performance of OB rats in both the cued and contextual tests was improved by desipramine and SA-4503. These results indicated that SA-4503 may ameliorate the cognitive symptoms of depression.
NMDA receptors play fundamental roles in the mammalian nervous system. They also have neurotrophic effects, and NR1 null animals cannot survive (Augustine et al., 1987; Malenka, 1994; Mohn et al., 1999; Balazs, 2006). Dysfunctional central nervous system glutamatergic pathways may be one of pathophysiological factors in depression (Nudmamud-Thanoi and Reynolds, 2004), and magnetic resonance imaging revealed a reduced level of glutamate in the PFC, which returned to normal following treatments with antidepressants (Bermack and Debonnel, 2005). Law and Deakin (2001) have reported that the expression of NR1 decreases in the hippocampus of depressive patients. The NMDA receptor density and the immunoreactivity of NR1 also decrease in other brain structures in depressive patients (Nudmamud-Thanoi and Reynolds, 2004). Furthermore, NMDA receptor density is decreased in the frontal cortex of suicide victims of depression, and the adaptation of the density to repeated antidepressant treatments has been ruled out (Nowak et al., 1995).
NR1 is indispensable for diverse NMDA receptors, and it is functional in a homomeric form; however, NR2 subunits require NR1 to form functional complexes (Zukin and Bennett, 1995). NR1 is distributed ubiquitously in the brain. In contrast, NR2 subunits are region-specifically distributed. NR2A is distributed widely, with relatively high levels in the cerebral cortex, the Hip, and cerebellar granule cells. NR2B is expressed selectively in the forebrain, with high levels in the cerebral cortex, hippocampal formation, septum, caudate-putamen, olfactory bulbs, and thalamus. The NR2C subunit is found predominantly in the cerebellum, whereas weak expression is detected in the olfactory bulbs and the thalamus. NR2D is expressed at much lower levels than the other subunits, and it is found in the thalamus, brainstem, and olfactory bulbs (Liu and Zhang, 2000). The deficit in the protein expression of NR1 underlies the decreased density and function of NMDA receptors in the OB rat brain (Kelly et al., 1997; Robichaud et al., 2001). Hei et al. (2006) have reported that NR1 expression was increased by activation of NMDA receptors that coexist with and are potentiated by σ1 receptors (Bergeron and Debonnel, 1997; Urani et al., 2002), which indicates a mechanism for the effect of SA-4503 on protein expression of NR1.
The affinities of SA-4503 for α1,D2, 5-HT1A, 5-HT2,H1,M1, and M2 receptors are at least 100 times weaker than that for the σ1 receptors (Matsuno et al., 1996). In the present study, all the effects of SA-4503 were blocked by the σ1 receptor antagonist NE-100 at a low dose, which confirmed that the effects of SA-4503 are basically mediated by σ1 receptors.
The behavioral effects of SA-4503 were blocked by an NMDA receptor antagonist, MK-801. This result is at least partially supported by the report that the mice expressing 5–10% NR1 exhibit sexual dysfunction (Mohn et al., 1999). The decrease in the protein expression of NR1 is just one facet of neurodegeneration in the OB rat brain, and NMDA receptors play a crucial role in the emotional effects of SA-4503, because the compensatory phenomena evident in sham-operated rats were not observed in SA-4503-treated OB rats.
Besides having a role in emotion, NMDA receptors are involved in the cognitive effects of SA-4503 in the fear-conditioning test, which is supported by the report that the Hip-regional knockout of NR1 inhibits the ability of animals to learn a new set of tasks within a specific context (Greene, 2005). Because MK-801 at the low dose of 0.03 mg/kg not only blocked the effects of SA-4503 but also impaired the performance of sham-operated rats in the fear-conditioning test, NMDA receptors play little of a compensable role in the performance of this test, unlike in the other behavioral tests. The MK-801-induced behavioral change seems less extensive in the SA-4503-treated group than that in the sham-operated group, indicating that NMDA receptors may be partially involved in the effect of SA-4503 in the test.
Because the dose of MK-801 that we used in the present study (0.03 mg/kg) is very low, it shows a relatively specific antagonizing effect at NMDA receptors. By contrast, NMDA receptor antagonists ketamine and memantine have been reported to have antidepressant-like effects (Berman et al., 2000; Skuza and Rogoz, 2006). Although ketamine (i.v.) and its active metabolite norketamine with 20 to 30% activity have half-lives of approximately 1 and 6 h, respectively, antidepressant-like effect of ketamine reaches a significant level several hours after the intravenous infusion, and it gradually increases for several days (Berman et al., 2000; Zarate et al., 2006). Furthermore, ketamine also has an affinity for the μ opiate receptors, and it is an agonist for the σ receptors (Berman et al., 2000). Besides being a NMDA receptor antagonist, memantine also acts as uncompetitive antagonists at the 5-HT3 and the nicotinic acetylcholine receptors, with potencies similar to or more than that for the NMDA receptors (Buisson and Bertrand, 1998; Rammes et al., 2001; Aracava et al., 2005). Therefore, the antidepressant-like effects of ketamine and memantine may not be mediated by NMDA receptors. The following phenomena best support the conception of the present study that NMDA receptors play fundamental roles in brain function, and although the receptors are used extensively in daily life, people usually do not become depressed after extensive thinking or learning.
We thank Hiroyuki Mizoguchi, Akihiro Mori, and Rina Murai in our laboratory for providing assistance.
This work was supported, in part, by Grants-in-aid for Scientific Research 14370031, 15922139, 16922036, and 17390018 from the Japan Society for the Promotion of Science; by Grant-in-aid for Scientific Research on Priority Areas 16047214 on “Elucidation of glia-neuron network-mediated information processing systems” from the Ministry of Education, Culture, Sports, Science and Technology; by funds from Integrated Molecular Medicine for Neuronal and Neoplastic Disorders (21st Century COE program); by a grant from the Brain Research Center from the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea; by the Japan Brain Foundation; by the Mitsubishi Pharma Research Foundation; and by the Brain Research Center of the 21st Century Frontier Research Program of the Ministry of Science and Technology, Republic of Korea.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: SA-4503, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride; NMDA, N-methyl-d-aspartate; NR, N-methyl-d-aspartate receptor subunit; PFC, prefrontal cortex; Hip, hippocampus; MK-801, (–)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohep-ten-5,10-imine maleate; OB, olfactory bulbectomy/bulbectomized; DES/Des, desipramine; NE-100, N,N-dipropyl-2-(4-methoxy-3-(2-phenyle-thoxy)phenyl)ethylamine monohydrochloride; ANOVA, analysis of variance; Amg, amygdala; 5-HT, 5-hydroxytryptamine (serotonin); Sham, sham-operated; Sal, saline; (+)-SKF-10047, [2S-(2α,6α,11R*)]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol hydrochloride.
- Received April 20, 2007.
- Accepted June 6, 2007.
- The American Society for Pharmacology and Experimental Therapeutics