Behavioural and neurochemical features of olfactory bulbectomized rats resembling depression with comorbid anxiety

Behavioural Brain Research 178 (2007) 262–273

Research report

Behavioural and neurochemical features of olfactory bulbectomized rats resembling depression with comorbid anxiety Dayong Wang a,c , Yukihiro Noda a,b , Hiroko Tsunekawa a , Yuan Zhou a , Masayuki Miyazaki a , Koji Senzaki a , Toshitaka Nabeshima a,∗ a

Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8560, Japan b Division of Clinical Science of Neuropsychopharmacology in Clinical Pharmacy Practice, Management and Research, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan c Division of Scientific Affairs, Japanese Society of Pharmacopoeia, Tokyo 150-0002, Japan Received 1 November 2006; received in revised form 22 December 2006; accepted 2 January 2007 Available online 7 January 2007

Abstract In order to probe the nature and validity of olfactory bulbectomized (OB) rats as a model of depression, we reevaluated their behavioural and neurochemical deficits in relation to the symptoms and neurochemical abnormalities of depression using our protocols, which distinguish anhedoniaresembling behaviour in sexual behavioural test, the hippocampus (Hip)-dependent long-term memory and anxiety-resembling behaviour specially. Besides exploratory hyperactivity in response to a novel environmental stress resembling the psychomotor agitation, OB rats showed a decrease of libido, and a deficit of long-term explicit memory, resembling loss of interest and cognitive deficits in depressive patients, respectively. OB rats also exhibited the anxiety symptom-resembling behaviour in social interaction and plus-maze tests. In the OB rats, we found degenerated neurons in the piriform cortex, decreased protein expression of NMDA receptor subunit 1 (NR1), but not NR2A or NR2B, in the prefrontal cortex (PFC), Hip and amygdala (Amg), and decreased phosphorylation of cAMP-response element-binding protein (CREB) in the PFC and Hip, but not Amg. The behavioural and neurochemical abnormalities in OB rats, except for the performance in the plus-maze task and neuronal degeneration, were significantly attenuated by repeated treatment with desipramine (10 mg/kg), a typical antidepressant. The present study indicated that OB rats may be a model of depression with comorbid anxiety, characterized by agitation, sexual and cognitive dysfunction, neuronal degeneration, decreased protein expression of NR1, and decreased phosphorylation of CREB. © 2007 Elsevier B.V. All rights reserved. Keywords: Olfactory bulbectomy; Depression; Anxiety; NMDA receptor subunits; CREB; Desipramine

1. Introduction The olfactory bulbs have extensive neural connections with the structures of the limbic system and other parts of the brain, and influence many emotional aspects of behavioural and other brain output functions [31]. Bilateral olfactory bulbectomy (OB) in rodents results in behavioural and neurochemical abnormalities that can only be normalized by chronic, not by acute, treatments with antidepressants, which simulates human disease conditions [31]. OB itself is analogous to the cortical/allocortical degeneration in depressive patients that is general, not depend-

∗

Corresponding author. Tel.: +81 52 744 2674; fax: +81 52 744 2682. E-mail address: [email protected] (T. Nabeshima).

0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2007.01.003

ing on a particular structure [25]. Another potential link between the OB model and depression derives from the psychopathological consequences of traumatic brain injury, which commonly results in depression [25]. In the 1970s and early 1980s, anxiety and depression were considered as two distinct entities in clinical practice as was put forth in DSM-III (American Psychiatric Association, 1980). Previously hierarchical and exclusionary concepts of anxiety and depression are now viewed as overlapping entities and the depressive disorders have been extended with two new research diagnoses: mixed anxiety-depressive disorder and minor depression in DSM-IV (1994) [41]. About 58% of depressive patients suffer from comorbid anxiety, which is characterized by a more malignant course of depression and is a predictor of a poor response to antidepressant treatments [8,13]. OB rats have been

D. Wang et al. / Behavioural Brain Research 178 (2007) 262–273

supposed as a model of major depression until now. In the present study, we proposed for the first time that OB rats are a model of anxiety-comorbid depression. Until now no body has investigated the performance of OB rats in the social interaction test, which has long been used as an anxiety test [62]. In addition, although the performance of OB rats in plus-maze test has been investigated in the experiment of Hozumi et al. [29], however, in their study, the plus-maze test was used to evaluate learning and memory. In the present study, a different protocol of plus-maze test was adopted to evaluate the anxiety-like behaviour in OB rats. Loss of interest is a core symptom of depression. There are several reports on sexual dysfunction of OB rats used as an alternative indicator of the symptom of loss of interest [10,19,45], in which copulation and ejaculation, but not genital probing, were observed. In order to discriminate the decrease in libido from the sexual functional inability that may not correlate with depression, in the present study we observed both genital-probing and thrusting behaviour, and compared the differential effects of desipramine on these behaviours. In addition, major depression and anxiety-comorbid depression are associated with greater cognitive impairments than dysthymia, whereas minor depression proved to be unrelated to cognitive performance [1,50,66]. There are reports on the performance of OB rats in foot-shock-induced freezing test [60,61] which involving short-term memory. Because the formation of long-term memory, but not short-term memory, depends on the function of the hippocampus (Hip) which has been supposed to be involved in the pathophysiology of depression, in the present study, we investigated the performance of OB rats in the cued and contextual conditioning tests which are tests for long-term memory depending on the function of the Hip and amygdala (Amg). Many studies on depression have focused on alterations in the levels of monoamines. Present studies have investigated postsynaptic targets such as the expression of the NMDA receptor and phosphorylation of CREB. The clinical requirement of repeated treatment for antidepressants to take effect has indicated the involvement of neuroplasticity in the pathology of depression. The N-methyl-d-aspartate (NMDA) receptor plays an important role in neuroplasticity. Previous autoradiographic research has indicated a deficit of NMDA receptors in OB rats [63]. The density of NMDA receptors or amount of mRNA of NR1 is decreased in the prefrontal cortex (PFC) or Hip of depressive patients [40,54,55]. It is recognized that long-term use of a NMDA receptor antagonist, phencyclidine (PCP), induces symptoms of acute anxiety and depression in humans [15,42]. Depression is associated with neuroplasticity, and recent studies have revealed the role of CREB as a mediator of neuroplasticity and survival [18]. Yamada et al. found a decrease in the level of phosphorylated CREB in the frontal cortex of patients with major depression [74]. The phosphorylation of CREB in neurons may begin with a strong influx of Ca2+ through NMDA receptors [27]. Following this influx, a Ca2+ -CaM complex is formed to phosphorylate CaMKs which are upstream activators of CREB [40]. Chronic, but not acute, antidepressant treatment increases gene transcription and phosphorylation of CREB at Ser133 in

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limbic structures, particularly the Amg, Hip and hypothalamus [69]. The changes in CREB function at the molecular level can produce persistent behavioural changes. Overexpression of CREB in the hippocampus produces an antidepressant effect [11]. CREB is not only involved in the pathology of depression but also in conditioned fear and anxiety [16]. In order to probe the behavioural similarity between the OB rat model and depression, symptomatology-based research was performed by reconstructing the symptoms of depression such as psychomotor agitation, loss of interest and cognitive dysfunction, and the symptoms of anxiety in anxiety-comorbid depression. In addition, the neuronal degeneration, the protein expression of NR1, NR2A and NR2B, and the phosphorylation of CREB in several regions of the brain in OB rats were investigated to find the neurochemical similarity between the OB rat model and depression. 2. Materials and methods 2.1. Animals Male Sprague–Dawley rats, 170–200 g, were purchased from Japan SLC (Shizuoka, Japan). All rats were housed, in cages put on automatically washed shelves in the clean environment of the Animal Experimental Center of Nagoya University, 3–4 rats per cage, at room temperature 25 ± 1 ◦ C and a relative humidity of 40–60%. The rooms were illuminated from 9:00 a.m. to 9:00 p.m. All experiments were performed following the guidelines for animal experiments of Nagoya University, which conform to the international guidelines set out in “Principles of Laboratory Animal Care” (NIH publication no. 85-23, revised 1985).

2.2. Medicine and reagents Desipramine hydrochloride was purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-phospho-CREB antibody and anti-CREB antibody were obtained from Cell Signaling Technology Inc. (Hertfordshire, England). Horseradish-conjugated goat anti-rabbit IgG antibody was purchased from Funakoshi (Tokyo, Japan). Anti-NR1, NR2A and NR2B antibodies and horseradish-conjugated donkey anti-goat IgG antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-NR1 antibody has an epitope mapping at the C-terminus of NR1011 (938 amino acids in rat brain, about 103 kDa), which is also named as ␨1, NR1a, or NMDAR1-1a and is the predominant form of the eight splices in adult rat brain [39,76]. Fluoro-Jade B was purchased from Chemicon International (Temecula, CA).

2.3. Surgical procedure The rats were anesthetized with pentobarbital Na (60 mg/kg), and fixed on stereotactic instruments (Narishige, Tokyo, Japan). A midline sagittal incision was made to expose the skull overlying the olfactory bulbs. A 4-mm diameter hole was made through the skull 6 mm anterior to the bregma. The olfactory bulbs were cut with a micro-knife, and aspirated out using a pipette tip connected with a water suction pump, care being taken not to damage the frontal cortex. The cavity of the olfactory bulbs was filled with a 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 animals were allowed to recover for 14 days following the surgery. The rats were separated into the following groups: (1) saline (Sal)treated, sham group, (2) desipramine at 10 mg/kg (Des 10)-treated, sham group, (3) Sal-treated OB group, (4) Des 5-treated, OB group and (5) Des 10-treated, OB group. The success of the operation was anatomically confirmed after all of the behavioural tests, and the data from the maloperated rats were excluded from subsequent data analysis.

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Fig. 1. Schedule of drug treatment and behavioural tests. The rats were sacrificed 24 h after plus-maze test, and brain samples were dissected out for Western-blotting analysis.

2.4. Schedule of drug administration and behavioural tests The schedule of drug administration and behavioural tests is shown in Fig. 1. The administration of saline or desipramine (5 or 10 mg/kg, s.c. daily) was started 2 weeks after the surgery and continued until 1 day before the last second behavioural test, the fear-conditioning. After the rats were treated with saline or desipramine for 14 days, the first behavioural test, an open field test, was started.

2.5. Behavioural tests To avoid the direct effect of desipramine on behaviour, drug treatment was performed after each behavioural test, although drug treatment was continued from the 14th to the 30th day after the operation. 2.5.1. Open field test The present protocol was adapted from those of Kameyama et al. [33] and Kelly and Leonard [34]. The open field apparatus, painted gray, consisted of a square arena (60 cm × 60 cm) divided into 15-cm squares by black lines. The wall of the arena was 30 cm high. A 60-W light bulb was positioned at the center 90 cm above the base of the arena. A red lamp (60 W) was used for the convenience of working, being placed at a position that could not be seen directly by the rats. On the 29th day after the operation, each rat was put into the center of the open field arena. The ambulation and rearing frequency were recorded in the first 3 min immediately after the rat entered the arena. After the test for 3 min, the rats were allowed to stay in the arena for another 7 min (the rats were kept in the arena for 10 min in total) as the habituation session for the social interaction test. After each test, the apparatus was sprayed with alcohol and wiped thoroughly to eliminate the residual odor. 2.5.2. Social interaction test The present protocol was adapted from those of File and Hyde [21] and Nabeshima et al. [51]. The same apparatus and testing environment as those of the open field test were used for the social interaction test, except that the illumination was milder (8 W) than that in the open field test. On the 30th day after the operation, pairs of rats of the same treatment group housed in different cages were put into two different corners of the open field arena. The social interaction behaviour including the running toward, probing, grooming, mounting and crawling under the other rat were recorded for 10 min after placement of the animals into the apparatus. After each test, the apparatus was sprayed with alcohol and wiped thoroughly to eliminate the residual odor. 2.5.3. Sexual behavioural test The present protocol was adapted from that of Breigeiron et al. [9]. The apparatus for the sexual behaviour test consisted of a transparent plexiglas box [45 cm (L) × 27 cm (W) × 39.5 cm (H)] with a black plastic base, illuminated with a red lamp (60 W). On the 30th day after the operation, sexual behaviour was observed from 22:00 to 3:00 h in the dark phase of the illumination cycle.

A male rat was first placed into the plexiglas box to be habituated to the environment for 3 min. Then, a sexually receptive female rat which had received subcutaneous injections of 0.14 mg of estradiol 72 and 48 h before the test and 0.7 mg of progesterone 4 h before the test for estrus was introduced. The sexual behaviour of the male rats was observed for 30 min. The following parameters of sexual behaviour were recorded: starting latency of genital-probing and thrusting, frequency of genital-probing and thrusting, and the percentage of rats that probed the female genitals or showed thrusting behaviour. After each test, the apparatus was sprayed with alcohol and wiped thoroughly to eliminate the residual odor. 2.5.4. Cued and contextual fear-conditioning tests The present protocol was adapted from those of Mamiya et al. [47] and Phillips and LeDoux [59]. The apparatus consisted of a transparent Plexiglas box [45 cm (L) × 27 cm (W) × 39.5 cm (H), the neutral cage] with a black plastic base and a Perspex box [32 cm (L) × 26 cm (W) × 48 cm (H), the conditioning cage] with a steel grid floor which was connected to an electric shock generator (Neuroscience Idea Co. Ltd., Osaka, Japan) and was enclosed in a opaque compartment. The neutral cage was illuminated with a red lamp (60 W) and the conditioning box was illuminated with a fluorescent lamp (6 W). For measuring basal levels of the freezing response (preconditioning phase), on the 31st day after the operation, rats were placed individually in the neutral cage for 1 min and then in the conditioning cage for 2 min. For conditioning (conditioning phase), each rat was placed in the conditioning cage, and then 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 through a shock generator. It should be noted that the unconditioned stimulus, 0.5 mA electric current lasting for 0.5 s, was not strong enough to produce a stable conditioned response in all of the rats, hence a difference in learning and memory ability among rats could be observed. Cued and contextual tests were carried out 24 h after fear-conditioning on the 32nd day after the operation. For the cued test, the freezing response was measured in the neutral cage for 1 min in the presence of a continuous-tone stimulus identical to the conditioned stimulus. For the contextual test, rats were placed in the conditioning cage, and the freezing response was measured for 2 min in the absence of the tone and conditioned stimulus. The freezing response was defined as follows: all four paws of the rats are remaining still and the animal stooped down with fear. After each test, the apparatus was sprayed with alcohol and wiped thoroughly to eliminate the residual odor. 2.5.5. Elevated plus-maze test The protocol of Yamada et al. was adjusted for rats [73]. The plus-maze consisted of two open (50 cm × 10 cm) arms and two enclosed (50 cm × 10 cm) arms surrounded by 30-cm high walls. The four arms were joined by a central platform (10 cm × 10 cm) open to all the arms, to form a plus shape. The entire apparatus was elevated to a height of 60 cm above the floor. The apparatus was indirectly illuminated with a ceiling-fronting lamp (54 W) which was

D. Wang et al. / Behavioural Brain Research 178 (2007) 262–273 placed 90 cm above the apparatus and 90 cm away from ceiling. The test was performed on the 32nd day after the operation. At the beginning of the test, the animal was placed in the central platform facing an open arm. The time that the rats spent in the open arms was recorded for 5 min. After each test, the apparatus was sprayed with alcohol and wiped thoroughly to eliminate the residual odor.

2.6. Neurochemical analysis 2.6.1. Fluoro-Jade B staining After all the behavioural tests, the rats were perfused with saline containing 1% heparin and then with a 2% paraformaldehyde solution (PFA, in 0.1 M PBS, pH 7.4). The brains of the rats were taken out and dissected transversely into sections at a thickness of 4 mm, and postfixed with the 2% PFA solution at 4 ◦ C overnight. After postfixation, the sections were kept in a 10% and 15% sucrose–2% PFA solution at 4 ◦ C for 4 h, respectively, and in a 20% sucrose–2% PFA solution at 4 ◦ C overnight. The sections were next frozen in O.C.T. compound at −80 ◦ C, and cut on a freezing sliding microtome at a thickness of 25 ␮m. The slices were collected in 0.1 M PBS, mounted on silanized slides, and stained according to the Fluoro-Jade B-staining protocol suggested by Chemicon International. The Fluoro-Jade B-stained neurons were visualized using a Zeiss epifluorescence microscope equipped with a fluorescein/FITC filter (515–565 nm), at an excitation wavelength of 485 nm. The images were captured with a digital camera (AxioCam HRc, Carl Zeiss) attached to the microscope. The number of Fluoro-Jade B-stained neurons on the images of same brain areas of the rats in different groups (five rats per group) was counted manually. 2.6.2. Western blotting After all the behavioural tests, the rats were sacrificed by decapitation and their brains were removed immediately. 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 rat’s brain [56], frozen, and stored at −80 ◦ C until used. The brain samples were homogenized in 150 ␮l of lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM sodium orthovanadate, 10 mM EDTA, 10 mM NaF, 0.1% sodium dodecyl sulfate (SDS), 1% Igepal CA-630, 1% sodium deoxycholate, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 10 ␮g/ml pepstatin, and 0.5 mM dl-dithiothreitol (DTT)) using an Astrason ultrasonic processor (Farmingdale, NY). After homogenization, the lysates were kept in an ice bath for 20 min, then centrifuged at a speed of 13,000 rpm for 20 min at 4 ◦ C. The total protein concentration of supernatants was analyzed by the Lowry method [44]. Samples of equal protein concentration were made by mixing the supernatants with lysis buffer, diluting 1:1 with sample-loading buffer (100 mmol/l Tris, 200 mmol/l DTT, 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 ␮g/10 ␮l were electrophoresed by SDS-PAGE (6% or 10% resolving gel), transferred to PVDF membranes (Millipore, Bedford, MA), and blocked with KPL DetectorTM block solution (Kirkegaard & Perry Lab., Gaithersburg, MD). For detecting the amount of phospho-CREB, NR1, NR2A, and NR2B protein, the membranes were incubated with anti-phospho-CREB (1:1000), anti-NR1 (1:1000), antiNR2A (1:1000), or anti-NR2B antibody (1:1000) at 4 ◦ C overnight. After a wash with buffer (50 mM Tris–HCl, 0.05% Tween 20 and 150 mM NaCl, pH 7.4), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and washed thoroughly. The membranes were then incubated in ECL (enhanced chemiluminescence) horseradish peroxidase substrate solution (Amersham Pharmacia Biotech Ltd., Bjorkgatan, Sweden), exposed to X-ray film, and developed in a medical image-developing machine (Fuji Photo Film Co. Ltd., Tokyo, Japan) to visualize immune complex bands. The absorbance of the bands on the films was analyzed using an ATTO Densitograph Software Library Lane Analyzer (Atto Co., Tokyo, Japan). For detecting the total amount of CREB (phospho- and non-phosphoCREB) protein, the membranes were incubated in stripping buffer (100 mM 2-melcaptoehanol, 2% SDS, and 62.5 mM Tris–HCl, pH6.7) at 50 ◦ C for 30 min, washed thoroughly, blocked with KPL blocking solution, incubated with antiCREB antibody (1:1000) at 4 ◦ C overnight, visualized, and analyzed as described above.

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The relative amount of target proteins on a sheet of film was calculated as follow: Ri =

A

n i i=1

Ai

Ri is the relative amount of protein in target band i on a sheet of film; Ai is the absorbance of target band i on a sheet of film; n is the number of target bands on a sheet of film.

2.7. Statistical analysis Statistical differences were evaluated with a one-way analysis of variance (ANOVA) followed by the modified Tukey test for multiple comparisons. The difference of the percentage of rats that showed genital-probing and thrusting behaviour among the groups in the sexual behavioural test was tested by Chisquare test. The criterion for a statistically significant difference was p < 0.05.

3. Results 3.1. Analysis of behaviour in OB rats resembling the symptoms of depression 3.1.1. Open field test The open field test is the behavioural test most commonly used to evaluate the antidepressive effects of medicines using the OB model. In the present study, this test was used to verify whether the OB operation was successful. As shown in Fig. 2a and b, OB rats showed increased ambulation (F(4, 55) = 9.190,

Fig. 2. Performance of OB rats in the open field test: (a) ambulation counts in the first 3 min of the test and (b) rearing counts in the first 3 min of the test. Results are expressed as means ± S.E., n = 12 in each group. ** p < 0.01 vs. Saltreated OB rats; ## p < 0.01 vs. Sal-treated sham-operated rats. Sal: saline; Dsp: desipramine; Sham: sham-operated rats; OB: olfactory bulbectomized rats.

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p < 0.01) and rearing (F(4, 55) = 6.411, p < 0.01) behaviour for 3 min after being put into the open field arena. Both increased frequencies of ambulation and rearing were dose-dependently ameliorated by the repeated treatment with desipramine (5 and 10 mg/kg). Desipramine at high dose significantly ameliorated the behavioural abnormality in OB rats. However, the ambulation and rearing in the sham group were not significantly affected by the treatment with desipramine. 3.1.2. Sexual behaviour test The loss of interest shown by depressive patients is also a core symptom of depression as depicted in the DSM-IV. We examined the sexual behaviour and the effect of desipramine on sexual behaviour in OB rats. The starting latency of genital probing (F(4, 55) = 23.367, p < 0.01) and thrusting (F(4, 55) = 8.198, p < 0.01) in OB rats was increased (Fig. 3a and d), whereas the number of genital probing (F(4, 55) = 7.899, p < 0.01) and thrusting events (F(4, 55) = 6.098, p < 0.01) (Fig. 3b and e) and the percentage of rats that showed genital-probing or thrusting behaviour (Fig. 3c and f) were decreased, compared to those in sham rats. OB rats showed sexual deficits in the sexual behaviour test. The treatment with desipramine (10 mg/kg) ameliorated the deficit of genital-probing behaviour in OB rats without affecting

the behaviour in sham rats (Fig. 3a–c). However, the thrusting deficit in OB rats was not significantly ameliorated by desipramine at 5 or 10 mg/kg (Fig. 3d–f). 3.1.3. Cued and contextual fear-conditioning tests Major depression and comorbid anxiety-depressive patients exhibit symptoms of significant cognitive dysfunction, to which minor depression is not related [1,50,64,66]. In the conditioning phase of the test, all the rats had shown similar immobility time either in the neutral cage or in the conditioning cage (data not shown). Twenty-four hours after the conditioning, OB rats showed significantly shortened freezing time in both the cued (F(4, 55) = 7.545, p < 0.01) and contextual (F(4, 55) = 8.409, p < 0.01) fear-conditioning tests compared with sham rats (Fig. 4a and b). The treatment with desipramine (10 mg/kg) significantly improved the performance of OB rats in both of the tests without significantly affecting the behaviour in sham rats (Fig. 4a and b). In previous preliminary experiments, no change in the nociceptive response threshold was found in OB-control and drug-treated rats: the minimal current intensity required to elicit flinching/running, jumping, or vocalization in the OB-control and drug-treated rats was the same as that in sham-operated rats (data not shown).

Fig. 3. Sexual behaviour of OB rats: (a) starting latency of genital probing; (b) number of genital probing; (c) percentage of rats that probed female genitals; (d) starting latency of thrusting; (e) number of thrusting; (f) percentage of rats that showed penis-thrusting behaviour. Results are expressed as means ± S.E. or percentage, n = 12 in each group. * p < 0.05, ** p < 0.01 vs. Sal-treated OB rats; ## p < 0.01 vs. Sal-treated sham-operated rats. Statistical difference in percentage terms among the groups was analyzed with the Chi-square test. Sal: saline; Dsp: desipramine; Sham: sham-operated rats; OB: olfactory bulbectomized rats.

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Fig. 4. Performance of OB rats in cued and contextual fear-conditioning tests of associative learning and memory. (a) Post-conditioning freezing time of rats in the cued conditioning test and (b) post-conditioning freezing time of rats in the contextual conditioning test. Results are expressed as means ± S.E., n = 12 in each group. ** p < 0.01 vs. Sal-treated OB rats; ## p < 0.01 vs. Sal-treated sham-operated rats. Sal: saline; Dsp: desipramine; Sham: sham-operated rats; OB: olfactory bulbectomized rats.

3.2. Behaviour of OB rats in anxiety tests 3.2.1. Social interaction test The social interaction test has been used as a valid test of anxiety. As shown in Fig. 5a, unfamiliar pairs of OB rats showed decreased social interactive time compared with sham rats (F(4, 43) = 21.575, p < 0.01). The deficit was ameliorated by the repeated treatment with desipramine (10 mg/kg) without significant effect on the performance of sham rats. 3.2.2. Elevated plus-maze test The elevated plus-maze test is also commonly used as an anxiety test. As shown in Fig. 5b, OB rats showed a decreased ratio of exploring time in the open arms of the plus-maze to the total time of 5 min (F(4, 49) = 3.442, p < 0.01). The repeated treatment with desipramine, at 5 and 10 mg/kg, failed to improve the performance of OB rats. Although there was no significant difference, these results still showed the specificity of the effect of repeated treatment with desipramine (10 mg/kg) on the deficit in OB rats, since desipramine did not tend to improve the performance of sham-operated rats in the plus-maze test. 3.3. Distinct degeneration of neurons in the brains of OB rats Fluoro-Jade B is a polyanionic fluorescein derivative that specifically and unequivocally binds to neurons that have distinctly degenerated. The degenerated neurons were found in the piriform cortex of OB rats (Fig. 6). The piriform cortex may play very important roles in different behaviours, since it is structurally adjacent to the agranular insula cortex (orbital or sulcal PFC [71]), Amg, oriens layer of Hip, and lateral entorhinal cortex, and has remarkably extensive axonal collaterals with multiple cortical and subcortical areas, such as the prefrontal cortex and amygdala. The repeated treatment with desipramine

Fig. 5. Performance of OB rats in social interaction and elevated plus-maze tests. (a) Social interaction test and (b) elevated plus-maze test. Results are expressed as means ± S.E., n = 8–12. ** p < 0.01 vs. Sal-treated OB rats; # p < 0.05, ## p < 0.01 vs. Sal-treated sham-operated rats. Sal: saline; Dsp: desipramine; Sham: sham-operated rats; OB: olfactory bulbectomized rats.

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Fig. 6. Neuronal degeneration in the piriform cortex of OB rats. The degenerated neurons were identified by Fluoro-Jade B staining, since healthy neurons could not be stained by Fluoro-Jade B. The degenerated neurons on the slice were visualized using a Zeiss epifluorescence microscope equipped with a fluorescein/FITC filter, at the excitation wavelength of 485 nm.

did not significantly reverse the neuronal degeneration in the piriform cortex (data not shown). 3.4. Protein expression of NR1 in the PFC, Hip, and Amg The pyramidal neurons, mainly glutamatergic neurons in the piriform cortex, project to the granule cells of the dentate gyrus of the Hip. NMDA receptors that play an important role in neuroplasticity can be formed with NR1 and at least one of the NR2 subunits. As shown in Fig. 7, the levels of NR1 protein decreased in the PFC (F(2, 30) = 28.825, p < 0.01), Hip (F(2, 30) = 3.362, p < 0.05) and Amg (F(2, 24) = 17.938, p < 0.01) of OB rats compared with sham rats. The decrease in the expression of NR1 protein was reversed by the treatment with desipramine (10 mg/kg). There was no significant difference in the levels of NR2A and NR2B proteins in these regions of the brain between sham and OB rats (data not shown). 3.5. Phosphorylation level of CREB in the PFC, Hip and Amg CREB is also related with neuroplasticity in the brain. The phosphorylation of CREB in neurons may begin with a strong influx of Ca2+ through NMDA receptors [27]. As shown in Fig. 8, the phosphorylation of CREB decreased in the PFC (F(2, 30) = 7.522, p < 0.01) and Hip (F(2, 30) = 1.125, p < 0.01), but not Amg (F(2, 24) = 19.399, p = 0.341) of OB rats. The repeated treatment with desipramine (10 mg/kg) reversed the decrease in the phosphorylation of CREB in the PFC and Hip of OB rats. 4. Discussion 4.1. Antidepressants used in the present study In the present study, the typical antidepressant desipramine was used, which is a tricyclic compound that inhibits the reuptake of norepinephrine and serotonin. In the study, we also treated rats with imipramine at the dose of 20 mg/kg which has been commonly used in numerous behavioural studies of antidepression. However, the subcutaneous injection of imipramine induced severe inflammation on the back of rats. Therefore, imipramine-treated groups were not fit for data analysis, espe-

cially in the emotional studies, and related data were not shown. Selective serotonin reuptake inhibitors (SSRIs) were not preferred in the present study, based on the facts that they increase the risk of suicide-related behaviour in human especially in adolescents [20]. 4.2. Behavioural deficits in OB rats resembling the symptoms of depression The OB rat is considered to be an animal model of depression [32,35,45,72]. The open field test is the behavioural test most widely employed to investigate emotion using OB rats as a model of depression. In a stressful experimental environment, OB rats show agitated hyperactivity, which resembles the psychomotor agitation symptom of depressive patients, the extreme of which is the psychological state that leads a person to commit suicide [25,45]. In the present study, OB rats showed increased frequencies of ambulation and rearing, and the behavioural deficit was ameliorated by repeated treatment with desipramine. It was confirmed by the open field test that the olfactory bulbectomy was successful in the present study. OB rats also showed decreased libido or sexual interest indicated by genital probing, which resembles the loss of interest that is a core symptom of depression and was ameliorable by the treatment with desipramine. The presence of decreased libido in OB rats adds to the rationality of using this model of depression. Since the typical antidepressant desipramine improved genitalprobing behaviour, but failed to improve thrusting behaviour, it suggested that genital-probing behaviour in the OB rat model of depression may fit better with the loss of interest in depression than thrusting behaviour which may also involve a sexual functional inability rather than only a decreased libido, and that genital-probing behaviour may be a more proper index than thrusting behaviour using OB rat model of depression to evaluate antidepression-like effects of medicines. Several studies have reported spared functions of majordepressive patients in tests tapping implicit memory [14,24], explicit memory [4], and attention [38]. Unlike the learning and memory tests relying on the short-term memory that is independent to the Hip, in the present study, we performed the cued and contextual conditioning tests involving the formation of long-

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Fig. 7. Protein expression of NR1 in the prefrontal cortex (PFC), hippocampus (Hip), and amygdala (Amg) of OB rats. (a) In the PFC, n = 11 in each group; (b) in the Hip, n = 11 in each group; (c) in the Amg, n = 9 in each group. Results are expressed as means ± S.E. * p < 0.05, ** p < 0.01 vs. Sal-treated OB rats; # p < 0.05, ## p < 0.01 vs. Sal-treated sham-operated rats. NR1: NMDA receptor subunit 1; Sal: saline; Dsp: desipramine; Sham: sham-operated rats; OB: olfactory bulbectomized rats.

term memory that depends on the function of the Hip and Amg which have been supposed to be correlated with the pathophysiology of depression. It was found that there is an impairment of associative learning and memory in OB rats 24 h after conditioning. The performance of OB rats in both cued and contextual tests was improved by desipramine. These results indicated that OB rats should not be used as a model of minor depression, since major depression and anxiety-comorbid depression have been associated with cognitive impairments, whereas minor depression has been proved to be unrelated to cognitive performance [1,50,66].

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Fig. 8. Phosphorylation of CREB in the prefrontal cortex (PFC), hippocampus (Hip), and amygdala (Amg) of OB rats. (a) In the PFC, n = 11 in each group; (b) in the Hip, n = 11 in each group; (c) in the Amg, n = 9 in each group. Results are expressed as means ± S.E. * p < 0.05, ** p < 0.01 vs. Sal-treated OB rats; # p < 0.05, ## p < 0.01 vs. Sal-treated sham-operated rats. p-CREB: phosphorylated CREB; t-CREB: total CREB including phosphorylated and nonphosphorylated CREB; Sal: saline; Dsp: desipramine; Sham: sham-operated rats; OB: olfactory bulbectomized rats.

4.3. Behaviour of OB rats in the anxiety tests The social interaction and elevated plus-maze tests have long been established as valid models of anxiety [62]. In the present study, the OB rats showed anxiety-like behaviour in the social interaction and elevated plus-maze tests. The repeated treatment with desipramine at the dose of 10 mg/kg ameliorated the impairment of social behaviour in the social interaction test, but not anxiety-like behaviour in the plus-maze test. The present results suggest that OB rats may be used as a model of anxiety-comorbid

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depression, rather than only a model of major depression, and that desipramine may have a limited effect on the symptoms of anxiety in patients with anxiety-comorbid depression. As depicted in DSM-IV (1994), the depressive disorders have been extended with a new diagnosis: comorbid anxiety-depressive disorder [41]. Howland and Thase have reported that the comorbid anxiety in depressive patients may predict poor response to conventional treatments [28]. In spite of the availability of several types of antidepressant and anxiolytic drugs, the prevalence of depression and anxiety is constantly on the rise [2]. Since a majority of patients suffering from unipolar major depression also have comorbid anxiety [75], it seems to be important to develop new drugs exhibiting both anxiolytic and antidepressant properties. OB rats may serve as a model of anxiety-comorbid depression for this purpose. The social interaction test in OB rats is preferable as a model of comorbid anxiety than the elevated plus-maze test, since the individual difference of the rats’ performance in the elevated plus-maze test is much higher than that in the social interaction test. 4.4. Neurobiochemical similarities between the OB rat model and depressive patients It has been found that there is the cortical/allocortical degeneration in diverse brain regions in depressive patients [25]. Similarly, we found that there was also a distinctly degenerated brain region in OB rat model of depression, which was identified to be the piriform cortex by Fluoro-Jade B staining. The piriform cortex has extensive associational axon collaterals in multiple higher order behavioural/cognitive/contextual-related cortical and subcortical areas, including the prefrontal cortex, amygdaloid nuclei, agranular insula cortex, entorhinal cortex, and hippocampus, and functions like an associative area rather than only a typical primary sensory area [12,23,26,67,68]. The fact that the treatment with desipramine did not significantly reverse the neuronal degeneration in the piriform cortex of OB rats indicated that desipramine may not take antidepressive effect by directly ameliorating cortical degeneration and other brain regions may be involved in the behavioural deficits in OB rats as direct acting targets of desipramine. Research on depression has long been focused on changes in monoamine concentrations in several regions of the brain. The concentration can be increased by acute antidepressant treatments, however, in most cases, the therapeutic effect of antidepressants requires chronic administration [18]. The requirement of repeated antidepressant treatments has been thought to be due to neuroplasticity, which may be mediated by the coupling of receptors to their respective intracellular signal transduction pathways. The NMDA receptor plays an important role in regulating fundamental processes in the mammalian nervous system including neurotransmitter release and neuronal plasticity [3,5,7,46,49]. Dysfunction of CNS glutamatergic pathways may be one of the mechanisms involved in the pathophysiology of depression [55]. In the present study, the decrease in the amount of NR1 in the PFC, Hip, and Amg of OB rats further indicated a deficit of glutamatergic

neurotransmission through NMDA receptors, which was consistent with clinical reports that magnetic resonance imaging showed reduced glutamate levels in the PFC that returned to normal following antidepressant treatments [6]. In the study of Mohn et al. [49], mice expressing 5–10% NR1 exhibited hyperlocomotion and social and sexual dysfunction, which supports the present results of the social interaction and sexual behavioural tests. The pharmacological experiments also support our findings that acute treatment with NMDA receptor antagonists increases spontaneous activity in animals, and chronic treatment with MK-801, a NMDA receptor antagonist, prevents both the neurochemical and behavioural consequences of antidepressant treatments [30,48,52,53,58]. Moreover, the density of NMDA receptors decreases in the frontal cortex of suicide victims of depression, and the authors have ruled out the adaptation of the receptor to repeated antidepressant treatments [54]. In addition, Law and Deakin have reported that the expression of NR1 is decreased in the hippocampus of depressive patients [40], and the density of NMDA receptors and the immunoreactivity of NR1 are also decreased in other brain structures, such as the superior temporal cortex, in depressive patients [55]. A molecular and cellular homeostasis or balance is critical to the psychological state of humans. The decrease in NR1 protein expression is a deviation from the molecular and cellular homeostasis in OB rats and in depressive patients, which can be restored by repeated treatment with desipramine. Besides the function of NR1 in animal emotion, NR 1-regional knock out in the hippocampus totally inhibits the learning ability of animals within a specific context [22]. Further, NMDA receptor dysfunction is involved in the behavioural deficit in fear-conditioned learning [57]. These reports support the idea that the decreased protein expression of NR1 in OB rats represents a neurobiochemical similarity to depression. NR1 is indispensable for the assembly of functional NMDA receptors, and is functional in expression systems in a homomeric form, however, NR2 subunits require NR1 to form a functional complex [76]. Most native NMDA receptors appear to function as heterotetrameric assemblies composed of two NR1 and two NR2 subunits [37]. Although there are eight functional splice variants of NR1, they arise from one gene. NR1 is ubiquitously distributed in the brain. Unlike NR1, the NR2 subunits region-restrictedly distribute in the brain. The NR2A distributes widely, but the levels of its expression are higher in the cerebral cortex, the Hip and cerebellar granule cells. The NR2B expresses selectively in the forebrain, with high levels of expression in the cerebral cortex, hippocampal formation, septum, caudateputaman, olfactory bulbs and the thalamus. The NR2C subunit is found predominantly in the cerebellum. Strong expression of NR2C is observed in the granule cell layer of the cerebellum, while weak expression is detected in the olfactory bulbs and the thalamus. The levels of NR2D are much lower than other subunits, and are found in the thalamus, brainstem and olfactory bulbs. Recently, it was reported that there was NR2D in the Hip at extremely lower level compared to NR2A and NR2B [43,70]. Since NR1 is indispensable for all the constructions of NMDA receptors and distributes ubiquitously in the brain, the deficit

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with the protein expression of NR1 in the brain of OB rats indicates the decreased density and function of NMDA receptors [35,63]. Duman et al. have suggested that CREB plays a critical role in the pathology of depression [17]. A number of the signaling pathways governing neural plasticity are intimately linked by CREB [65]: the activation of which can begin with strong Ca2+ entry through specific plasma membrane channels including NMDA receptors [27]. The phosphorylation of CREB has been reported to be a molecular state marker for the response to antidepressant treatments in patients with major-depressive disorder [36]. The upregulation of CREB function in regions of the brain that implicated in emotional and cognitive behaviour contributes to the molecular and cellular response to antidepressant treatments, and is a convergent point integrating responses from various signal transduction pathways triggered by diverse classes of antidepressants [18]. The decrease in the phosphorylation of CREB and its reverse by desipramine in the PFC and Hip of OB rats also bears neurobiochemical similarity to that in depressed patients. 5. Conclusion OB rats showed long-lasting anxiety-comorbid depressionlike behavioural changes accompanied by a deficit of NR1 protein expression and CREB phosphorylation. Through the comparison between the characteristics of OB rat model and depression, the present study adds face validity and rationality for using the model. Acknowledgements This work was supported, in part, by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (14370031) (15922139) (16922036) (17390018), by a Grant-in-aid for Scientific Research on Priority Areas on “Elucidation of glia-neuron network-mediated information processing systems” from the Ministry of Education, Culture, Sports, Science and Technology (16047214), by Funds from Integrated Molecular Medicine for Neuronal and Neoplastic Disorders (21st Century COE Program), by the Japan Brain Foundation, by the Mitsubishi Pharma Research Foundation, by an SRF Grant for Biomedical Research, and by Brain Research Center of the 21st Century Frontier Research Program of the Ministry of Science and Technology, Republic of Korea. We also thank Hiroyuki Mizoguchi, Akihiro Mori, and Rina Murai in our laboratory for providing assistance. References [1] Airaksinen E, Larsson M, Lundberg I, Forsell Y. Cognitive functions in depressive disorders: evidence from a population-based study. Psychol Med 2004;34:83–91. [2] Andrews G, Sanderson K, Slade T, Issakidis C. Why does the burden of disease persist? Relating the burden of anxiety and depression of effectiveness of treatment. Bull World Health Organ 2000;78:446–54. [3] Augustine GJ, Charlton MP, Smith SJ. Calcium action in synaptic transmitter release. Annu Rev Neurosci 1987;10:633–93.

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