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Antisense hippocampal knockdown of NMDA-NR1 by HVJ-liposome vector induces deficit of prepulse inhibition but not of spatial memory
Neuroscience Research 45 (2003) 473 /481 www.elsevier.com/locate/neures
Antisense hippocampal knockdown of NMDA-NR1 by HVJliposome vector induces deficit of prepulse inhibition but not of spatial memory Ken Inada a,*, Jun Ishigooka a, Takeshi Anzai b, Eiji Suzuki a, Hitoshi Miyaoka a, Makoto Saji c a Department of Psychiatry, School of Medicine, Kitasato University, Sagamihara, Kanagawa 228-8520, Japan Department of Anesthesiology, School of Medicine, Kitasato University, Sagamihara, Kanagawa 228-8555, Japan c Department of Physiology, School of Allied Health Sciences, Kitasato University, Sagamihara, Kanagawa 228-8555, Japan b
Received 9 October 2002; accepted 27 December 2002
Abstract Considerable evidence suggests that an N -methyl-D-aspartate (NMDA) receptor plays a crucial role in memory and cognitive function. To identify the role of this receptor in higher functions of the brain, we delivered antisense oligonucleotides against an NMDA-NR1 subunit (NR1) to the hippocampus in rats using the HVJ-liposome-mediated gene-transfer method. NR1 hippocampal knockdown was performed by the focal injection of the NR1 antisense /HVJ-liposome complex into the bilateral hippocampus. The blocking effect of NR1-antisense on the expression of NR1 was confirmed by Western blot analysis. Spatial memory was tested by a water maze task, and sensorimotor gating was examined by prepulse inhibition (PPI). Western blot analysis demonstrated that the NR1-antisense treatment specifically provided the down-regulation (about 30%) of NR1 protein levels in the hippocampus. The water maze task showed that the antisense treatment did not affect spatial memory, while the PPI test revealed that NR1 hippocampal knockdown caused a deficit in sensorimotor gating. We conclude that mild dysfunction of hippocampal NMDA receptor causes sensorimotor gating deficit and relatively intact in spatial memory. # 2003 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: NMDA; Hippocampus; Antisense; Rat; Water maze; Prepulse inhibition
1. Introduction The dysfunction or hypofunction of N -methyl-Daspartate (NMDA) receptors in the whole brain, induced experimentally in animals treated with the systemic application of an NMDA receptor antagonist or genetic mutation, causes psychotic symptoms, defective adaptation in a novel environment, and/or defects in cognition similar to those of schizophrenic patients (Duncan et al., 1999a,b; Mohn et al., 1999; Olney et al., 1999, 1995; Toru et al., 1994). On the other hand, functional disruption of hippocampal circuits by intrahippocampal injection of an NMDA receptor antago-
* Corresponding author. Tel.: /81-42-748-9111; fax: /81-42-7653570. E-mail address: [email protected] (K. Inada).
nist or ablation of the hippocampus produces defects in spatial learning memory as measured by the Morris water maze task (Morris et al., 1982, 1986). Furthermore, the focal infusion of a non-competitive NMDA receptor antagonist into the hippocampus causes impaired prepulse inhibition (PPI) that is reflected by defective sensorimotor gating or cognitive dysfunction (Bakshi et al., 1998; Braff et al., 1990; Geyer, 1998). Therefore, it is hypothesized that NMDA receptors in the hippocampus may play a crucial role in higher brain functions such as spatial/working memory or cognitive function. Functional NMDA receptors are composed of an NMDA-NR1 subunit (NR1) subunit and one of four NR2 subunits (NR2A-NR2D) combined in an undetermined ratio to make the heteromeric receptor assembly (Kutsuwada et al., 1992; Monyer et al., 1992). NR1-null
0168-0102/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/S0168-0102(03)00012-9
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mice die perinatally (Forrest et al., 1994), while mutant mice with reduced NR1 expression (5 /10% of the normal level) survive to adulthood and exhibit behavioral abnormalities similar to those observed in experimentally induced animal models of schizophrenia (Mohn et al., 1999). These findings suggest that the experimentally induced down-regulation of NR1 expression produces a marked reduction in NMDA receptormediated synaptic function, including glutamate transmission, synaptic refinement, and neuronal plasticity. To examine whether the dysfunction or hypofunction of NR1 receptors in the hippocampus causes defects in spatial learning memory and/or sensorimotor gating, we performed antisense-induced down-regulation of hippocampal NR1 expression. In rats with long-lasting antisense knockdown of hippocampal NR1 expression, we tested spatial learning memory using the Morris water maze task, and we tested sensorimotor gating function or cognition in the PPI of the acoustic startle response. To achieve long-lasting dysfunction of NR1 for the behavioral tests, repetitive application or chronic infusion by osmotic pump of antisense oligodeoxy nucleotides (ODNs) to NR1 into the brain through intra-ventricular cannulation has been used in previous studies (Roberts et al., 1998; Zapata et al., 1997; Matthies et al., 1995; Standaert et al., 1996). However, for the focal and chronic application of the antisense ODNs into the hippocampus, there are concerns about tissue injury by chronic infusion through an implanted cannula. In the current study, we accepted the combined use of the antisense technique and the HVJ (hemagglutinating virus of Japan)-liposome-mediated gene transfer method (Saeki et al., 1997; Yamada et al., 1996) to achieve the long-lasting dysfunction of hippocampal NR1 by a single intra-hippocampal injection of antisense ODNs.
2. Materials and methods 2.1. Animal care Fifty experimentally naive, 10-week-old, male Wistar rats (Japan SLC, Kanagawa, Japan), each weighing 230 /300 g, were used in the present study. The animals were housed in clear plastic cages in groups of two or three and were allowed to access to food and water throughout the experiment. The animals were maintained in a temperature-, humidity-, and light-controlled environment with a 12-h light: 12-h dark cycle. On arrival in the colony, the rats were handled gently by the experimenter. The handling was continued for 5 min every day until the surgical operation for the intrahippocampal injection of HVJ-liposome containing ODNs against NR-1.
All experiments conformed to Japanese and international guidelines on the ethical use of animals, and every effort was made to minimize the number of animals and their suffering. 2.2. Oligodeoxy nucleotides (ODNs) Phosphotioated ODNs (18 mers) corresponding to a specific segment in the 5?-coding region of NR1 cDNA were designed to selectively decrease the biosynthesis of the NR1 (Wahlestedt et al., 1993) as antisense ODNs to NR1 (NR1-AS: 5?-CAGCAGGTGCATGGTGCT-3?). Sense ODNs (NR1-SE: 5?-AGCACCATGCACCTGCTG-3?) in which the bases of the 18 mers corresponding to the same coding region of NR1 cDNA in sense orientation were synthesized for use as a control. That none of the ODN sequences overlapped with other mammalian sequences was determined by a search of the Genebank/EMBL database. 2.3. Preparation of HVJ-liposome-containing ODNs The detailed preparation of cationic HVJ-liposomecontaining ODNs has been described elsewhere (Saeki et al., 1997). Briefly, five kinds of lipids, phosphatidyl choline (ePC) (Sigma, St. Louis, MO), phosphatidylethanolaminedioleoyl (DOPE) (Wako), sphingomyelin (eSph) (Sigma), cholesterol (Chol) (Sigma), and DCcholesterol (DC-chol) (Avanti) were dissolved in chloroform and mixed in an approximate weight ratio of ePC:DOPE:eSph:Chol:DC-chol /1:1:1:2:0.5. A total of 1 ml of lipid mixture (8.375 mg) was transferred into a glass tube and dried as a thin lipid film using a rotary evaporator filled with nitrogen gas at 40 8C. The lipid thin film layering the bottom of a glass tube was hydrated in 200 ml Balanced Salt solution (BSS: 137 mM NaCl, 5.4 mM KCl, Tris /HCl pH 7.5) containing 100 mg ODNs which were dispersed in the aqueous phase at 37 8C. The mixture of the hydrated lipid thin film and ODNs was agitated by vortexing for 50 s and incubated at 37 8C for 10 s. This procedure was repeated until the fragments of lipid thin film became liposomes in which ODNs were wrapped. Then, 800 ml of BSS was added to the liposome suspension. The liposome suspension was extruded through a cellulose acetate membrane (pore size, 0.45 mm), and the liposomes left in the filter were collected by extruding 0.5 ml of BSS. Two milliliter of the liposome suspension and additional BSS (0.5 ml) were extruded through another membrane filter with 0.2 mm pores to obtain sized unilamellar liposomes (2 ml in total). The liposome suspension prepared above was mixed with a 1 ml HVJ suspension (30 000 hemagglutinating units) whose RNA genome was inactivated by ultraviolet irradiation (198 mJ/cm2), and it was then incubated at 37 8C for 1 h with shaking to facilitate the fusion between the inactivated HVJ and the liposomes.
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The HVJ-liposome complex was loaded onto a discontinuous sucrose gradient and centrifuged at 25 000 rpm at 4 8C for 1.5 h to separate the ODN /HVJ-liposome complex from free HVJ. The purified ODN /HVJliposome suspension was adjusted to OD 0.8 (540 nm) with 1 /1.5 ml BSS. Since 10 /30% of ODNs (10 /30 mg) can be contained in the liposomes (Kaneda et al., 1987), it is estimated that 1 ml of the purified ODN /HVJliposomes (OD 0.8) contains about 20/60 ng ODNs. 2.4. Administration of HVJ-liposomes containing ODNs Rats were anesthetized with an intra-peritoneal injection of pentobarbital sodium (40 mg/kg). The 30 ml of the ODN /HVJ-liposome suspension (0.5 /1.5 mg DNA) was bilaterally injected into 12 sites throughout the entire hippocampus (2.5 ml per site). We first injected antisense /HVJ-liposome with 0.2 ml /6 sites, but we could not find any change or downregulation of target protein by Western blot analysis or immunohistochemistry. Raising the volume of injection (volume of injection per site and the number of injection sites), we finally could find the significant down-regulation (30%) of NR1 protein by Western blot with 2.5 ml /12 sites. When we injected higher dose of HVJliposome suspension than 2.5 ml per site, we observed a small lesion, necrotic aspects of neurons at injection site by Nissl-stain. Then we determined that 2.5 ml of HVJliposome containing ODNs was optimal dose to induce antisense in vivo knockdown of NR1. The sites for intra-hippocampal injection were stereotaxically positioned. The coordinates in mm with respect to ear bar were as follows: (1) anterior 5.2 mm; lateral9/ 1.4 mm; depth 3.2 mm; (2) anterior 5.2 mm; lateral9/2.1 mm; depth 3.2 mm; (3) anterior 5.2 mm; lateral9/2.8 mm; depth 3.2 mm; (4) anterior 3.7 mm; lateral9/5.0 mm; depth 3.5 mm; (5) anterior 3.7 mm; lateral9/5.0 mm; depth 5.8 mm; (6) anterior 3.7 mm; lateral9/5.0 mm; depth 6.5 mm. A silicon-coated glass micropipette (tip size: 30/40 mm; volume: 1 ml/mm) made from disposable micropipette (20 ml, Drummond) that was connected to an air pressure system was used for focal injection of the ODN /HVJ-liposome suspension.
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pus on a freezing microtome. The hippocampal sections were Nissl-stained with thionin. 2.6. Western blot analysis For quantification of protein expression levels, Western blot analysis was used. Rats were anesthetized with ether and killed by decapitation at various days after the intra-hippocampal injection of an HVJ-liposome suspension containing antisense ODNs, sense ODNs, or vehicle (BSS). The hippocampus was quickly dissected out and cut by a tissue chopper. The tissue was placed in a sonicator in 1 ml of 1 mM NaHNO3 buffer (pH 6.8) containing the protease inhibitor PMSF (phenylmethlylsulfonyl fluoride) (Sigma). The homogenate was centrifuged at 3000 rpm for 10 min. The supernatant was stored at /80 8C. The protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL), and protein samples (100 mg) were loaded on 7.5% sodium dodecylsulfate /polyacrylamide gel and separated by electrophoresis. Protein bands were transferred from the gel to a nitrocellulose membrane (Bio-Rad). After being blocked for 18 h with BlockAce (Dainihon, Osaka), the membrane was incubated in the primary antibodies for 45 min at room temperature. Two primary antibodies were used: a rabbit polyclonal antibody against NMDA-NR1 (1:1000: Chemicon, Temecura, CA) and a rabbit polyclonal antibody against GluR2/3 (1:1000: Chemicon). The membrane was then incubated in the secondary antibody for 30 min at room temperature. The secondary antibody used was peroxydase-conjugated goat antirabbit IgG (Vector Lab, Burlingame, CA). Then the membrane was processed with chemiluminescence reagents to visualize the antibody reaction using an ECL detection kit (Amersham, Arlington Heights, IL) and finally exposed to X-ray film (Kodak). For quantification of NR-1 protein levels, Western blots were analyzed with computer densitometry by using NIH image software. Mean optical densities of bands for two samples per animal were determined, and the film background was subtracted. The band densities for NR1 protein in treated rats were represented relatively as the percentages of those in non-treated control rats.
2.5. Histology To assess whether the injection of an ODN /HVJliposome suspension itself causes injury to the hippocampal tissue, some of the rats that received the intrahippocampal injection of the ODN /HVJ-liposome suspension were allowed to survive for 6 days following the injection and were perfused transcardially with 4% phosphate-buffered paraformaldehyde under deep anesthesia. The removed brains were sectioned into 25 mm thicknesses through the various levels of the hippocam-
2.7. Measurement of spontaneous locomotor activity in an open field A rat was put in a white square box (60 /60 cm) as a novel environment. The behavior of the rat was recorded by a video camera during the initial 10 min period following the start of spontaneous locomotion in the open field. The open field was divided into 16 blocks (15 /15 cm). From the 10 min recording of locomotor behavior, the crossing rate was measured as locomotor
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activity by counting the number of crossings over the borderlines of the 16 blocks during the 10 min period. 2.8. Water maze task training for spatial learning memory We used the Morris water maze task to test for spatial learning and memory. The apparatus used for the water maze task was a white circular pool (diameter: 1.5 m; height: 0.5 m) filled to a depth of 20 cm with clear water (249/2 8C). For a hidden platform task, a transparent plastic platform submerged 2 cm below the water surface was located at a fixed position throughout the training sessions. A training session consisted of six consecutive trials with a 60 s inter-trial interval. Rats were trained for 5 days on the hidden platform task, consisting of one session per day (total 30 trials). On the last day of the training session, only a spatial probe test was conducted. The submerged platform was removed from the pool. A rat that had completed the training sessions was given 60 s to search the platform in the pool, and the search pattern was recorded. From the recording, the time spent in each quadrant zone was measured to access the place preference. Each quadrant zone was defined as follows: target, the quadrant in which the hidden platform had been positioned; opposite, the quadrant opposite to the target; right, the quadrant on the right side of the target; left, the quadrant on the left side of the target. 2.9. Prepulse inhibition test Rats were placed in a chamber with a floor equipped with an electric weighing machine and allowed to acclimate for 5 min before the test session was started. Background noise was set at 65 dB of white noise throughout the acclimation period and the session. In a test session, three types of ten trials (total 30 trials) were given in pseudo-random order after initial startle-stimuli (20 ms burst of 120 dB white noise), which were given to avoid the effect of high responses to initial stimulations in the tests. One of the types was a startle pulse alone (P alone) trial, which involved a 20 ms burst of 120 dB white noise, and the other two types consisted of prepulse and startle pulse (PP70&P and PP80&P) trials, which involved a 20 ms burst of 70 or 80 dB of white noise, respectively, followed by the same pulse as in the P alone trial 100 ms later. The inter-trial intervals averaged 40 s (20 /60 s) and were pseudo-randomized. In addition to these three types of trials, no-stimulus (nonstim) trials were inserted between trials to check the baseline amplitude without stimulation. The startle response was measured for 100 ms from the start of the startle pulse presentation, and the average value was defined as the startle amplitude. The startle amplitudes in response to repetitions of each trial type were
averaged across the session. The test schedule was controlled by a microcomputer. The percent of PPI of a startle response was calculated by the following formula: %PPI80[1(PP80&P=P alone)]100 %PPI70[1(PP70&P=P alone)]100:
2.10. Statistics The effects of NR1-AS and NR1-SE treatment were analyzed by one-way analysis of variance (ANOVA) with the treatment as a factor. Post-hoc individual comparison was carried out by using Fisher’s PLSD or the Student’s t -test. A P -value of less than 0.05 was considered to represent a significant difference.
3. Results 3.1. Antisense-induced down-regulation of NR-1 protein levels and its time course We first used Western blot analysis with antibodies against NR1 and GluR2/GluR3 to examine the antisense-induced reduction of NR1 protein levels in the hippocampus. Fig. 1A shows representative blots (upper) and quantification of relative band densities (lower) for NR1 protein from the hippocampus of rats that received hippocampal injections of HVJ-liposomes containing ODNs or BSS at 6 days post-injection. The amount of NR1 protein in the NR1-AS-injected hippocampus was markedly reduced. On the other hand, in the hippocampus injected with BSS or HVJ-liposomes containing NR1-SE, the NR1 protein levels were not changed. As shown in the quantitative analysis, a significant reduction (29.759/6.97%(S.E.)) of NR1 protein levels was found only in the hippocampus of the NR1-AS-treated rats, while the other two treatments did not affect the NR1 protein levels. Fig. 1B indicates representative blots (upper) and quantification of relative band densities (lower) for GluR2/3 protein obtained from the same hippocampus as described above. As shown in Fig. 1B, the levels of AMPA receptor subunit GluR2 and GluR3 protein detected by antibodies against both GluR2 and GluR3 subunits were not changed in the hippocampus of the rats treated with ODNs or BSS at 6 days post-injection. Fig. 1C shows the quantitative analysis of the time course of the antisense-induced reduction of the hippocampal NR1 protein levels. The NR1 protein levels in the hippocampus of rats that received a single injection of HVJ-liposomes containing NR1-AS into the hippocampus started to decrease 3 days after the NR1-AS
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treatment, reached a maximal reduction of 30% by 7 days post-treatment, and recovered to the original levels up to 14 days post-treatment, indicating that the antisense-induced down-regulation of hippocampal NR1 expression by a single injection of an NR1-AS/ HVJ-liposome complex occurred in a transient but longlasting (more than 10 days) manner. The Nissl-stained sections of the hippocampus injected with the NR1-AS /HVJ-liposome complex 6 days after the injection left no lesions on the hippocampal regions, except for small scar found in the core site of the injection (Fig. 2).
3.2. Normal spontaneous locomotor activity in rats with NR1 hippocampal knockdown To examine whether the antisense-induced downregulation of NR1 protein expression in the hippocampus causes an alteration of adaptive behaviors in a novel environment, we measured spontaneous locomotor activity in an open field in rats that received a hippocampal injection of NR1-AS or NR1-SE 6 days prior to the open field test compared with non-treated rats. The spontaneous locomotor activity was estimated by the crossing rate during the initial 10 min period in an open field test. As shown in Fig. 3, there was no significant difference in the spontaneous locomotor activity among the NR1-AS-treated rats (n/5), the
Fig. 1
Fig. 1. Antisense-induced down-regulation of hippocampal NR1 protein levels and its time course following a single intra-hippocampal injection of antisense ODNs to NR1 by using HVJ-liposome mediated gene transfer method. NR1 was detected using antibody against NR1. For measurements of GluR2/3 as control proteins, the membrane used for NR1 was reprobed with polyclonal antibody against GluR2 and GluR3 to confirm equal protein loading in each lane. Level of hippocampal NR1 and GluR2/3 protein treated rats was represented as a percentage of that of non-treated rats (NT). (A), Representative blots (upper) and quantitation of relative band densities (lower) for NR1 protein obtained from the hippocampus of rats that received an intra-hippocampal injection of NR1-AS (AS), NR1-SE (SE) or vehicle (BSS) by using HVJ-liposome method, 6 days prior to the Western bolt analysis. (B), Representative blots (upper) and quantitation of relative band densities (lower) for GluR2/3 proteins obtained from the same hippocampus of rats as described above. Each treatment group: AS, rats with intra-hippocampal injection of NR1-AS (n/4); SE, rats with intra-hippocampal injection NR1-SE (n/4); BSS, rats with intrahippocampal injection of BSS (n/4). The significant down-regulation (30%) of NR1 protein was observed only in the NR1-AS treated group, while the levels of GluR2/3 proteins were not changed in any treatment groups. (C), Time course of antisense-induced downregulation of hippocampal NR1 protein levels. Each value is an average of relative abundance of NR1 protein obtained from two rats that received a single intra-hippocampal injection of NR1-AS by using HVJ-liposome method, 3 days (day 3)(n/4), 7 days (day 7)(n/4), 11 days (day 11)(n/4), and 14 days (day 14) (n/4) prior to the Western blotting, respectively. *, Significantly different from the SE (P B/0.05) and the BSS (P B/0.05) for (A) and different from the day 14 (P B/ 0.05) for (C) by ANOVA and post-hoc Fisher’s test.
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Fig. 2. The Nissl-stained sections of the ventral (A) and dorsal (B) hippocampus of the rat that received an single intra-hippocampal injection of antisense ODNs (NR1-AS) /HVJ-liposome complex 6 days prior to the histological assay. The Nissl-staining patterns in A and B indicate that the treatment with HVJ-liposome containing NR1-AS did not produce any lesions in the neural tissue except for a small scar (arrow). Scale bar represents 1 mm.
Fig. 3. The effect of antisense-induced down-regulation of hippocampal NR1 expression on the spontaneous locomotor activity in an open filed. Treatment groups were: NT, non-treated rats; NR1-AS, rats that received a single intra-hippocampal injection of NR1-AS by using HVJ-liposome method 6 days prior to the open field test; NR1-SE, rats that received an intra-hippocampal injection of sense ONDs (NR1-SE) by using HVJ-liposome method 6 days prior to the open field test. The NR1-SE group was used as a control for the NR1-AS. From the 30 min recording of locomotor behavioral, the crossing rate during the initial 10 min period was measured as locomotor activity by counting the number of crossings over the borderlines of 16 blocks in the field. Each value represents mean9/S.E.M. (n /5 for the NR1-AS, n/6 for the NR1-SE, n/6 for the NT). There was no significant difference between the two treatment groups.
NR1-SE-treated rats (n /6) and non-treated rats (n / 6). 3.3. Effect of NR1 hippocampal knockdown on spatial learning To investigate the effect of NR-1 hippocampal knockdown by antisense on spatial learning, we used the Morris water maze test. The rats received an intrahippocampal injection of HVJ-liposomes containing NR1-AS or NR1-SE 6 days prior to undergoing training in a water maze. All of the rats in both treatment groups learned to swim directly to the hidden platform during five training sessions, well before training was completed
Fig. 4. The effect of antisense-induced down-regulation of hippocampal NR1 expression on the acquisition of spatial memory in the hidden platform water maze. Treatment groups were: NR1-AS, rats (n/5) that received a single intra-hippocampal injection of NR1-AS by using HVJ-liposome method 6 days prior to the water maze training; NR1SE, rats (n/6) that received an intra-hippocampal injection of sense ONDs (NR1-SE) by using HVJ-liposome method to NR1 4 days prior to the water maze training. The NR1-SE group was used as a control for the NR1-AS. (A), Escape latency (mean9/S.E.M.) in water maze training. There was no significant difference in the spatial learning between the two treatment groups. (B), Place preference (mean9/ S.E.M.) in the probe test conducted just after the end of the training session. Both NR1-AS treated and NR1-SE treated rats exhibited strong place preference for the target quadrant where the hidden platform was previously located, indicating that there was no significant difference in the acquisition of spatial memory between the two treatment groups.
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(Fig. 4A). On the last day of training, a spatial probe test was conducted just after the final training session. All of the rats in both treatment groups spent most of the trial search within a target quadrant zone, indicating their place preference in the target zone. There was no significant difference in the place preference in the target zone between the NR1-AS-treated group and NR1-SEtreated group (Fig. 4B). The swimming speeds of each animal through the sessions were also measured by computer. There were no difference between antisense treated and control animals. Also individual animal in both treatment groups did not show any change of swimming speed through the sessions (data not shown). 3.4. Sensorimotor gating deficit and its recovery in rats with NR-1 hippocampal knockdown By using the PPI test, we examined whether NR1 hippocampal knockdown causes a deficit in sensorimotor gating and whether any such deficit can be recovered. Six days prior to the PPI test, intra-hippocampal injection of the HVJ-liposome containing NR1-AS was given. The results showed a significant disruption of PPI upon stimulation at PP80, while they showed no significant disturbance of PPI at PP70 even though a tendency toward a reduced PPI was apparent, compared with the NR1-SE-treated rats (Fig. 5A). On the other hand, 14 days after the NR1-AS treatment, when the down-regulated NR1 protein expression had completely recovered to the original levels, the NR1-AS-treated rats did not exhibit disruption of PPI at either PP70 or PP80 (Fig. 5B), suggesting a recovery from the antisenseinduced PPI deficit. The amplitude of startle response to pulse stimulations were also measured. There was a tendency of increase of startle response to pulse stimulations in animals with NR1-AS or NR1-SE treatment compared with non-treated animals, although the increase was not significant (data not shown). On the other hand, there was no difference between NR1-AS treated and NR1SE treated animals. Thus, it is considered that tendency of increase is due to the stress of operation.
Fig. 5. Deficit of PPI of acoustic startle response and its recovery observed in the rats with antisense knockdown of hippocampal NR1. Treatment groups were: NR1-AS, rats (n /8) that received a single intra-hippocampal injection of antisense ODNs (NR1-AS) by using HVJ-liposome method 6 or 14 days prior to the PPI test; NR1-SE, rats (n/6) that received an intra-hippocampal injection of sense ONDs (NR1-SE) by using HVJ-liposome method 6 or 14 days prior to the PPT test. The PPI test was performed under the two types of stimulation referred as PP70 and PP80. PP70 is an acoustic stimulation consisted of prepulse (20 ms burst of 70 dB) and startle-pulse (20 ms burst of 120 dB), while PP80 is that consisted of prepulse (20 ms burst of 80 dB) and startle-pulse (20 ms burst of 120 dB). (A), Percent of %PPI(mean9/S.E.M.) in the NR1-AS or the NR1-SE, 6 days after the treatment. The rats treated with NR1-AS exhibited a significant disruption of PPI at the stimulation of PP80, although they also showed a tendency toward a reduced PPI at the stimulation of PP70. *, significantly different from PPI in the NR1-SE group as a control (P B/0.05). (B), %PPI (mean9/S.E.M.) in the NR1-AS or the NR1-SE 14 days after the treatment, when the down-regulated NR1 expression had completely recovered to the original levels. The rats with NR1-AS treatment did not exhibit disruption of PPI at either PP70 or PP80, suggesting a recovery from the antisense-induced PPI deficit.
4. Discussion 4.1. Antisense knockdown of hippocampal NR1 expression Western blot analysis revealed that a single administration of antisense ODN /HVJ-liposome complex caused a 30% inhibition of NR1 expression for about 10 days. The blocking effect of this intra-hippocampal injection of antisense ODNs by using the HVJ-liposome method on the biosynthesis of NR1 protein and the long-lasting action of antisense ODNs are equal to those
of repeated applications or chronic infusion of the antisense ODNs to NR1 (Roberts et al., 1998; Zapata et al., 1997; Standaert et al., 1996). Furthermore, because of a single injection of the antisense ODN / HVJ-liposome without any stressful implantation of cannula for repeated or chronic administration, behavioral tests under less biased experimental conditions have been achieved by the treatment with antisense ODN /HVJ-liposomes. Thus, we conclude that the combined use of the antisense technique and HVJ-
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liposome-mediated gene transfer is a suitable way to investigate the functional role of some specific proteins in higher brain function by behavioral analysis. It is considered that the regional extent of antisense knockdown of NR1 is restricted to the hippocampus injected by antisense. Because the cationic HVJ-liposome vectors we used in this study are positively charged, the injected HVJ-liposomes containing antisense ODNs do not diffuse widely from the negatively charged tissue around the injection site and thereby antisense knockdown occurs specifically in the focal area of injection (Kaneda, 1999). 4.2. Behavioral alterations induced by the NR1 hippocampal knockdown In the present study, rats with an antisense-induced knockdown of hippocampal NR1 exhibited normal locomotor activity in an open field test (see Fig. 2). This result is consistent with normal motor activity found in mutant mice in which the targeted knockout of NR1 receptors in pyramidal neurons was restricted to the hippocampal CA1 region (Tsien et al., 1996), but it is inconsistent with abnormal motor activity such as an increase in both locomotor activity and stereotypic behavior observed in mutant mice with reduced NR1 expression in the whole brain (Mohn et al., 1999). Thus, behavioral abnormalities in motor activity may be related to the dysfunction of NMDA receptors in the whole brain. As demonstrated in this study, in a Morris water maze task, rats with antisense-induced knockdown of hippocampal NR1 expression displayed intact spatial memory in both acquisition and retrieval (see Fig. 3). However, this result is inconsistent with the results of studies regarding impaired spatial memory found in mutant mice with a targeted knockout of NR1 in the hippocampal CA1 (Tsien et al., 1996) and normal mice treated with an intra-hippocampal injection of competitive NMDA receptor antagonist AP-5 (Morris et al., 1986). This discrepancy may be due to the partial dysfunction (30%) of hippocampal NR1 in our study, compared with the completely impaired or inactivated NMDA receptor-mediated function in the hippocampus in the previous studies. In this study, we have demonstrated that rats with NR1 hippocampal knockdown display impaired PPI of acoustic startle response. In particular, the recovery from the deficit of PPI in the antisense-treated rats was observed at 14 days after the antisense treatment, when the down-regulated expression of NR1 protein recovered to the original level. This recovery from the deficit of PPI suggests that the disruption of PPI occurs depending on the antisense-induced down-regulation (30%) of NR1 in the hippocampus. It has been reported that focal infusion of a noncompetitive NMDA receptor
antagonist into the amygdala or hippocampus produces the disruption of PPI in normal rats, suggesting that multiple limbic regions mediate the PPI deficit (Bakshi et al., 1998). From our data, only 30% impairment of NR1-related function in one of the multiple limbic regions is sufficient to mediate the disruption of PPI. Since the PPI reflects the function of the sensorimotor gating system, which partly overlaps with cognitive function (Bakshi et al., 1998; Geyer, 1998; Braff et al., 1990, 1992), it has been thought that the disruption of sensorimotor gating might provide a model for a deficit observed in schizophrenia as schizophrenic patients have a significant impairment of PPI (Braff et al., 1990, 1992, 2001; Geyer, 1998). As for memory deficit, schizophrenic patients have impaired episodic memory, but they maintain a relatively intact procedural memory and priming of semantic memory (Goldberg et al., 1989; Schmand et al., 1992; Tamlyn et al., 1992). Recently, mutant mice with reduced NR1 expression (5% of the normal level) in the whole brain exhibited deficits in social and sexual interactions and hypersensitivity to amphetamine (Mohn et al., 1999), treatment of phenciclidine or MK-801, a NMDA receptor antagonist, induces schizophrenia-related behaviors in rodents, include increases in locomotion and stereotypies, deficits in social interactions, deficit of sensorimotor gating and cognitive functions, increase of immobidity in forced swim test (Corbett et al., 1995; Duncan et al., 1999a,b; Swerdlow et al., 1998; Sams Dodd, 1998; Noda et al., 2000; Gainetdinov et al., 2001). Due to those findings, a lot of interest has been focused on the relationship between schizophrenic symptoms and dysfunction of NR1 receptors. Therefore, the combination of impaired PPI with intact spatial memory observed here in rats with the antisense-induced partial knockdown of hippocampal NR1 receptors may provide a possible animal model of schizophrenia, even though the present knockdown rats display only a part of modeling behaviors such as cognitive impairment, defective sensorimotor gating, and relatively intact memory. Further study is needed to demonstrate the rest of modeling behaviors for schizophrenic symptoms, such as hypersensitivity to dopamine, behavioral abnormality in forced swim test and defective social interaction as negative symptoms.
Acknowledgements This study was supported by grants from Inokashira Hospital, Japan, and grants from the Mitsubishi Pharma Research Foundation, Japan. This study was also supported in part by the Grant for Scientific Research from Kitasato University Graduate School of Medical Science, Japan.
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Morris, R.G., Garrud, P., Rawlins, J.N., O’Keefe, J., 1982. Place navigation impaired in rats with hippocampal lesions. Nature 297 (5868), 681 /683. Morris, R.G., Anderson, E., Lynch, G.S., Baudry, M., 1986. Selective impairment of learning and blockade of long-term potentiation by an N -methyl-D-aspartate receptor antagonist, AP5. Nature 319, 774 /776. Noda, Y., Kamei, H., Mamiya, T., Furukawa, H., Nabeshima, T., 2000. Repeated phencyclidine treatment induces negative symptom-like behavior in forced swimming test in mice: imbalance of prefrontal serotonergic and dopaminergic functions. Neuropsychopharmacology 23, 375 /387. Olney, J.W., Farber, N.B., 1995. Glutamate receptor dysfunction and schizophrenia. Arch. Gen. Psychiatry 52 (12), 998 /1007. Olney, J.W., Newcomer, J.W., Farber, N.B., 1999. NMDA receptor hypofunction model of schizophrenia. J. Psychiatr. Res. 33 (6), 523 /533. Roberts, E.B., Meredith, M.A., Ramoa, A.S., 1998. Suppression of NMDA receptor function using antisense DNA block ocular dominance plasticity while preserving visual responses. J. Neurophysiol. 80 (3), 1021 /1032. Saeki, Y., Matsumoto, N., Nakano, Y., Mori, M., Awai, K., Kaneda, Y., 1997. Development and characterization of cationic liposomes conjugated with HVJ (Sendai virus): reciprocal effect of cationic lipid for in vitro and in vivo gene transfer. Hum. Gene Ther. 8 (17), 2133 /2141. Sams Dodd, F., 1998. Effects of continuous D-amphetamine and phencyclidine administration on social behavior, stereotyped behavior, and locomotor activity in rats. Neuropsychopharmacology 19, 18 /25. Schmand, B., Kop, W.J., Kuipers, T., Bosveld, J., 1992. Implicit learning in psychotic patients. Schizophr. Res. 7 (1), 55 /64. Standaert, D.G., Testa, C.M., Rudolf, G.D., Hollingsworth, Z.R., 1996. Inhibition of N -methyl-D-aspartate glutamate receptor subunit expression by antisense oligonucleotides reveals their role in striatal motor regulation. J. Pharmacol. Exp. Ther. 276 (1), 342 / 352. Swerdlow, N.R., Geyer, M.A., 1998. Using an animal model of deficient sensorimotor gating to study the pathophysiology and new treatments of schizophrenia. Schizophr. Bull. 24, 285 /301. Tamlyn, D., McKenna, P.J., Mortimer, A.M., Lund, C.E., Hammond, S., Baddeley, A.D., 1992. Memory impairment in schizophrenia: its extent, affiliations and neuropsychological character. Psychol. Med. 22 (1), 101 /115. Toru, M., Kurumaji, A., Ishimaru, M., 1994. Excitatory amino acids: implications for psychiatric disorders research. Life Sci. 55 (22), 1683 /1699. Tsien, J.Z., Huerta, P.T., Tonegawa, S., 1996. The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell 87 (7), 1327 /1338. Wahlestedt, C., Golanov, E., Yamamoto, S., Yee, F., Ericson, H., Yoo, H., Inturrisi, C.E., Reis, D.J., 1993. Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischaemic infarctions. Nature 363 (6426), 260 /263. Yamada, K., Moriguchi, A., Morishita, R., Aoki, M., Nakamura, Y., Mikami, H., Oshima, T., Ninomiya, M., Kaneda, Y., Higaki, J., Ogihara, T., 1996. Efficient oligonucleotide delivery using the HVJliposome method in the central nervous system. Am. J. Physiol. 271 (5 Pt 2), R1212 /R1220. Zapata, A., Capdevila, J.L., Tarrason, G., Adan, J., Martinez, J.M., Piulats, J., Trullas, R., 1997. Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies. Brain Res. 745 (1 /2), 114 /120.
Antisense hippocampal knockdown of NMDA-NR1 by HVJliposome vector induces deficit of prepulse inhibition but not of spatial memory Ken Inada a,*, Jun Ishigooka a, Takeshi Anzai b, Eiji Suzuki a, Hitoshi Miyaoka a, Makoto Saji c a Department of Psychiatry, School of Medicine, Kitasato University, Sagamihara, Kanagawa 228-8520, Japan Department of Anesthesiology, School of Medicine, Kitasato University, Sagamihara, Kanagawa 228-8555, Japan c Department of Physiology, School of Allied Health Sciences, Kitasato University, Sagamihara, Kanagawa 228-8555, Japan b
Received 9 October 2002; accepted 27 December 2002
Abstract Considerable evidence suggests that an N -methyl-D-aspartate (NMDA) receptor plays a crucial role in memory and cognitive function. To identify the role of this receptor in higher functions of the brain, we delivered antisense oligonucleotides against an NMDA-NR1 subunit (NR1) to the hippocampus in rats using the HVJ-liposome-mediated gene-transfer method. NR1 hippocampal knockdown was performed by the focal injection of the NR1 antisense /HVJ-liposome complex into the bilateral hippocampus. The blocking effect of NR1-antisense on the expression of NR1 was confirmed by Western blot analysis. Spatial memory was tested by a water maze task, and sensorimotor gating was examined by prepulse inhibition (PPI). Western blot analysis demonstrated that the NR1-antisense treatment specifically provided the down-regulation (about 30%) of NR1 protein levels in the hippocampus. The water maze task showed that the antisense treatment did not affect spatial memory, while the PPI test revealed that NR1 hippocampal knockdown caused a deficit in sensorimotor gating. We conclude that mild dysfunction of hippocampal NMDA receptor causes sensorimotor gating deficit and relatively intact in spatial memory. # 2003 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: NMDA; Hippocampus; Antisense; Rat; Water maze; Prepulse inhibition
1. Introduction The dysfunction or hypofunction of N -methyl-Daspartate (NMDA) receptors in the whole brain, induced experimentally in animals treated with the systemic application of an NMDA receptor antagonist or genetic mutation, causes psychotic symptoms, defective adaptation in a novel environment, and/or defects in cognition similar to those of schizophrenic patients (Duncan et al., 1999a,b; Mohn et al., 1999; Olney et al., 1999, 1995; Toru et al., 1994). On the other hand, functional disruption of hippocampal circuits by intrahippocampal injection of an NMDA receptor antago-
* Corresponding author. Tel.: /81-42-748-9111; fax: /81-42-7653570. E-mail address: [email protected] (K. Inada).
nist or ablation of the hippocampus produces defects in spatial learning memory as measured by the Morris water maze task (Morris et al., 1982, 1986). Furthermore, the focal infusion of a non-competitive NMDA receptor antagonist into the hippocampus causes impaired prepulse inhibition (PPI) that is reflected by defective sensorimotor gating or cognitive dysfunction (Bakshi et al., 1998; Braff et al., 1990; Geyer, 1998). Therefore, it is hypothesized that NMDA receptors in the hippocampus may play a crucial role in higher brain functions such as spatial/working memory or cognitive function. Functional NMDA receptors are composed of an NMDA-NR1 subunit (NR1) subunit and one of four NR2 subunits (NR2A-NR2D) combined in an undetermined ratio to make the heteromeric receptor assembly (Kutsuwada et al., 1992; Monyer et al., 1992). NR1-null
0168-0102/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/S0168-0102(03)00012-9
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mice die perinatally (Forrest et al., 1994), while mutant mice with reduced NR1 expression (5 /10% of the normal level) survive to adulthood and exhibit behavioral abnormalities similar to those observed in experimentally induced animal models of schizophrenia (Mohn et al., 1999). These findings suggest that the experimentally induced down-regulation of NR1 expression produces a marked reduction in NMDA receptormediated synaptic function, including glutamate transmission, synaptic refinement, and neuronal plasticity. To examine whether the dysfunction or hypofunction of NR1 receptors in the hippocampus causes defects in spatial learning memory and/or sensorimotor gating, we performed antisense-induced down-regulation of hippocampal NR1 expression. In rats with long-lasting antisense knockdown of hippocampal NR1 expression, we tested spatial learning memory using the Morris water maze task, and we tested sensorimotor gating function or cognition in the PPI of the acoustic startle response. To achieve long-lasting dysfunction of NR1 for the behavioral tests, repetitive application or chronic infusion by osmotic pump of antisense oligodeoxy nucleotides (ODNs) to NR1 into the brain through intra-ventricular cannulation has been used in previous studies (Roberts et al., 1998; Zapata et al., 1997; Matthies et al., 1995; Standaert et al., 1996). However, for the focal and chronic application of the antisense ODNs into the hippocampus, there are concerns about tissue injury by chronic infusion through an implanted cannula. In the current study, we accepted the combined use of the antisense technique and the HVJ (hemagglutinating virus of Japan)-liposome-mediated gene transfer method (Saeki et al., 1997; Yamada et al., 1996) to achieve the long-lasting dysfunction of hippocampal NR1 by a single intra-hippocampal injection of antisense ODNs.
2. Materials and methods 2.1. Animal care Fifty experimentally naive, 10-week-old, male Wistar rats (Japan SLC, Kanagawa, Japan), each weighing 230 /300 g, were used in the present study. The animals were housed in clear plastic cages in groups of two or three and were allowed to access to food and water throughout the experiment. The animals were maintained in a temperature-, humidity-, and light-controlled environment with a 12-h light: 12-h dark cycle. On arrival in the colony, the rats were handled gently by the experimenter. The handling was continued for 5 min every day until the surgical operation for the intrahippocampal injection of HVJ-liposome containing ODNs against NR-1.
All experiments conformed to Japanese and international guidelines on the ethical use of animals, and every effort was made to minimize the number of animals and their suffering. 2.2. Oligodeoxy nucleotides (ODNs) Phosphotioated ODNs (18 mers) corresponding to a specific segment in the 5?-coding region of NR1 cDNA were designed to selectively decrease the biosynthesis of the NR1 (Wahlestedt et al., 1993) as antisense ODNs to NR1 (NR1-AS: 5?-CAGCAGGTGCATGGTGCT-3?). Sense ODNs (NR1-SE: 5?-AGCACCATGCACCTGCTG-3?) in which the bases of the 18 mers corresponding to the same coding region of NR1 cDNA in sense orientation were synthesized for use as a control. That none of the ODN sequences overlapped with other mammalian sequences was determined by a search of the Genebank/EMBL database. 2.3. Preparation of HVJ-liposome-containing ODNs The detailed preparation of cationic HVJ-liposomecontaining ODNs has been described elsewhere (Saeki et al., 1997). Briefly, five kinds of lipids, phosphatidyl choline (ePC) (Sigma, St. Louis, MO), phosphatidylethanolaminedioleoyl (DOPE) (Wako), sphingomyelin (eSph) (Sigma), cholesterol (Chol) (Sigma), and DCcholesterol (DC-chol) (Avanti) were dissolved in chloroform and mixed in an approximate weight ratio of ePC:DOPE:eSph:Chol:DC-chol /1:1:1:2:0.5. A total of 1 ml of lipid mixture (8.375 mg) was transferred into a glass tube and dried as a thin lipid film using a rotary evaporator filled with nitrogen gas at 40 8C. The lipid thin film layering the bottom of a glass tube was hydrated in 200 ml Balanced Salt solution (BSS: 137 mM NaCl, 5.4 mM KCl, Tris /HCl pH 7.5) containing 100 mg ODNs which were dispersed in the aqueous phase at 37 8C. The mixture of the hydrated lipid thin film and ODNs was agitated by vortexing for 50 s and incubated at 37 8C for 10 s. This procedure was repeated until the fragments of lipid thin film became liposomes in which ODNs were wrapped. Then, 800 ml of BSS was added to the liposome suspension. The liposome suspension was extruded through a cellulose acetate membrane (pore size, 0.45 mm), and the liposomes left in the filter were collected by extruding 0.5 ml of BSS. Two milliliter of the liposome suspension and additional BSS (0.5 ml) were extruded through another membrane filter with 0.2 mm pores to obtain sized unilamellar liposomes (2 ml in total). The liposome suspension prepared above was mixed with a 1 ml HVJ suspension (30 000 hemagglutinating units) whose RNA genome was inactivated by ultraviolet irradiation (198 mJ/cm2), and it was then incubated at 37 8C for 1 h with shaking to facilitate the fusion between the inactivated HVJ and the liposomes.
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The HVJ-liposome complex was loaded onto a discontinuous sucrose gradient and centrifuged at 25 000 rpm at 4 8C for 1.5 h to separate the ODN /HVJ-liposome complex from free HVJ. The purified ODN /HVJliposome suspension was adjusted to OD 0.8 (540 nm) with 1 /1.5 ml BSS. Since 10 /30% of ODNs (10 /30 mg) can be contained in the liposomes (Kaneda et al., 1987), it is estimated that 1 ml of the purified ODN /HVJliposomes (OD 0.8) contains about 20/60 ng ODNs. 2.4. Administration of HVJ-liposomes containing ODNs Rats were anesthetized with an intra-peritoneal injection of pentobarbital sodium (40 mg/kg). The 30 ml of the ODN /HVJ-liposome suspension (0.5 /1.5 mg DNA) was bilaterally injected into 12 sites throughout the entire hippocampus (2.5 ml per site). We first injected antisense /HVJ-liposome with 0.2 ml /6 sites, but we could not find any change or downregulation of target protein by Western blot analysis or immunohistochemistry. Raising the volume of injection (volume of injection per site and the number of injection sites), we finally could find the significant down-regulation (30%) of NR1 protein by Western blot with 2.5 ml /12 sites. When we injected higher dose of HVJliposome suspension than 2.5 ml per site, we observed a small lesion, necrotic aspects of neurons at injection site by Nissl-stain. Then we determined that 2.5 ml of HVJliposome containing ODNs was optimal dose to induce antisense in vivo knockdown of NR1. The sites for intra-hippocampal injection were stereotaxically positioned. The coordinates in mm with respect to ear bar were as follows: (1) anterior 5.2 mm; lateral9/ 1.4 mm; depth 3.2 mm; (2) anterior 5.2 mm; lateral9/2.1 mm; depth 3.2 mm; (3) anterior 5.2 mm; lateral9/2.8 mm; depth 3.2 mm; (4) anterior 3.7 mm; lateral9/5.0 mm; depth 3.5 mm; (5) anterior 3.7 mm; lateral9/5.0 mm; depth 5.8 mm; (6) anterior 3.7 mm; lateral9/5.0 mm; depth 6.5 mm. A silicon-coated glass micropipette (tip size: 30/40 mm; volume: 1 ml/mm) made from disposable micropipette (20 ml, Drummond) that was connected to an air pressure system was used for focal injection of the ODN /HVJ-liposome suspension.
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pus on a freezing microtome. The hippocampal sections were Nissl-stained with thionin. 2.6. Western blot analysis For quantification of protein expression levels, Western blot analysis was used. Rats were anesthetized with ether and killed by decapitation at various days after the intra-hippocampal injection of an HVJ-liposome suspension containing antisense ODNs, sense ODNs, or vehicle (BSS). The hippocampus was quickly dissected out and cut by a tissue chopper. The tissue was placed in a sonicator in 1 ml of 1 mM NaHNO3 buffer (pH 6.8) containing the protease inhibitor PMSF (phenylmethlylsulfonyl fluoride) (Sigma). The homogenate was centrifuged at 3000 rpm for 10 min. The supernatant was stored at /80 8C. The protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL), and protein samples (100 mg) were loaded on 7.5% sodium dodecylsulfate /polyacrylamide gel and separated by electrophoresis. Protein bands were transferred from the gel to a nitrocellulose membrane (Bio-Rad). After being blocked for 18 h with BlockAce (Dainihon, Osaka), the membrane was incubated in the primary antibodies for 45 min at room temperature. Two primary antibodies were used: a rabbit polyclonal antibody against NMDA-NR1 (1:1000: Chemicon, Temecura, CA) and a rabbit polyclonal antibody against GluR2/3 (1:1000: Chemicon). The membrane was then incubated in the secondary antibody for 30 min at room temperature. The secondary antibody used was peroxydase-conjugated goat antirabbit IgG (Vector Lab, Burlingame, CA). Then the membrane was processed with chemiluminescence reagents to visualize the antibody reaction using an ECL detection kit (Amersham, Arlington Heights, IL) and finally exposed to X-ray film (Kodak). For quantification of NR-1 protein levels, Western blots were analyzed with computer densitometry by using NIH image software. Mean optical densities of bands for two samples per animal were determined, and the film background was subtracted. The band densities for NR1 protein in treated rats were represented relatively as the percentages of those in non-treated control rats.
2.5. Histology To assess whether the injection of an ODN /HVJliposome suspension itself causes injury to the hippocampal tissue, some of the rats that received the intrahippocampal injection of the ODN /HVJ-liposome suspension were allowed to survive for 6 days following the injection and were perfused transcardially with 4% phosphate-buffered paraformaldehyde under deep anesthesia. The removed brains were sectioned into 25 mm thicknesses through the various levels of the hippocam-
2.7. Measurement of spontaneous locomotor activity in an open field A rat was put in a white square box (60 /60 cm) as a novel environment. The behavior of the rat was recorded by a video camera during the initial 10 min period following the start of spontaneous locomotion in the open field. The open field was divided into 16 blocks (15 /15 cm). From the 10 min recording of locomotor behavior, the crossing rate was measured as locomotor
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activity by counting the number of crossings over the borderlines of the 16 blocks during the 10 min period. 2.8. Water maze task training for spatial learning memory We used the Morris water maze task to test for spatial learning and memory. The apparatus used for the water maze task was a white circular pool (diameter: 1.5 m; height: 0.5 m) filled to a depth of 20 cm with clear water (249/2 8C). For a hidden platform task, a transparent plastic platform submerged 2 cm below the water surface was located at a fixed position throughout the training sessions. A training session consisted of six consecutive trials with a 60 s inter-trial interval. Rats were trained for 5 days on the hidden platform task, consisting of one session per day (total 30 trials). On the last day of the training session, only a spatial probe test was conducted. The submerged platform was removed from the pool. A rat that had completed the training sessions was given 60 s to search the platform in the pool, and the search pattern was recorded. From the recording, the time spent in each quadrant zone was measured to access the place preference. Each quadrant zone was defined as follows: target, the quadrant in which the hidden platform had been positioned; opposite, the quadrant opposite to the target; right, the quadrant on the right side of the target; left, the quadrant on the left side of the target. 2.9. Prepulse inhibition test Rats were placed in a chamber with a floor equipped with an electric weighing machine and allowed to acclimate for 5 min before the test session was started. Background noise was set at 65 dB of white noise throughout the acclimation period and the session. In a test session, three types of ten trials (total 30 trials) were given in pseudo-random order after initial startle-stimuli (20 ms burst of 120 dB white noise), which were given to avoid the effect of high responses to initial stimulations in the tests. One of the types was a startle pulse alone (P alone) trial, which involved a 20 ms burst of 120 dB white noise, and the other two types consisted of prepulse and startle pulse (PP70&P and PP80&P) trials, which involved a 20 ms burst of 70 or 80 dB of white noise, respectively, followed by the same pulse as in the P alone trial 100 ms later. The inter-trial intervals averaged 40 s (20 /60 s) and were pseudo-randomized. In addition to these three types of trials, no-stimulus (nonstim) trials were inserted between trials to check the baseline amplitude without stimulation. The startle response was measured for 100 ms from the start of the startle pulse presentation, and the average value was defined as the startle amplitude. The startle amplitudes in response to repetitions of each trial type were
averaged across the session. The test schedule was controlled by a microcomputer. The percent of PPI of a startle response was calculated by the following formula: %PPI80[1(PP80&P=P alone)]100 %PPI70[1(PP70&P=P alone)]100:
2.10. Statistics The effects of NR1-AS and NR1-SE treatment were analyzed by one-way analysis of variance (ANOVA) with the treatment as a factor. Post-hoc individual comparison was carried out by using Fisher’s PLSD or the Student’s t -test. A P -value of less than 0.05 was considered to represent a significant difference.
3. Results 3.1. Antisense-induced down-regulation of NR-1 protein levels and its time course We first used Western blot analysis with antibodies against NR1 and GluR2/GluR3 to examine the antisense-induced reduction of NR1 protein levels in the hippocampus. Fig. 1A shows representative blots (upper) and quantification of relative band densities (lower) for NR1 protein from the hippocampus of rats that received hippocampal injections of HVJ-liposomes containing ODNs or BSS at 6 days post-injection. The amount of NR1 protein in the NR1-AS-injected hippocampus was markedly reduced. On the other hand, in the hippocampus injected with BSS or HVJ-liposomes containing NR1-SE, the NR1 protein levels were not changed. As shown in the quantitative analysis, a significant reduction (29.759/6.97%(S.E.)) of NR1 protein levels was found only in the hippocampus of the NR1-AS-treated rats, while the other two treatments did not affect the NR1 protein levels. Fig. 1B indicates representative blots (upper) and quantification of relative band densities (lower) for GluR2/3 protein obtained from the same hippocampus as described above. As shown in Fig. 1B, the levels of AMPA receptor subunit GluR2 and GluR3 protein detected by antibodies against both GluR2 and GluR3 subunits were not changed in the hippocampus of the rats treated with ODNs or BSS at 6 days post-injection. Fig. 1C shows the quantitative analysis of the time course of the antisense-induced reduction of the hippocampal NR1 protein levels. The NR1 protein levels in the hippocampus of rats that received a single injection of HVJ-liposomes containing NR1-AS into the hippocampus started to decrease 3 days after the NR1-AS
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treatment, reached a maximal reduction of 30% by 7 days post-treatment, and recovered to the original levels up to 14 days post-treatment, indicating that the antisense-induced down-regulation of hippocampal NR1 expression by a single injection of an NR1-AS/ HVJ-liposome complex occurred in a transient but longlasting (more than 10 days) manner. The Nissl-stained sections of the hippocampus injected with the NR1-AS /HVJ-liposome complex 6 days after the injection left no lesions on the hippocampal regions, except for small scar found in the core site of the injection (Fig. 2).
3.2. Normal spontaneous locomotor activity in rats with NR1 hippocampal knockdown To examine whether the antisense-induced downregulation of NR1 protein expression in the hippocampus causes an alteration of adaptive behaviors in a novel environment, we measured spontaneous locomotor activity in an open field in rats that received a hippocampal injection of NR1-AS or NR1-SE 6 days prior to the open field test compared with non-treated rats. The spontaneous locomotor activity was estimated by the crossing rate during the initial 10 min period in an open field test. As shown in Fig. 3, there was no significant difference in the spontaneous locomotor activity among the NR1-AS-treated rats (n/5), the
Fig. 1
Fig. 1. Antisense-induced down-regulation of hippocampal NR1 protein levels and its time course following a single intra-hippocampal injection of antisense ODNs to NR1 by using HVJ-liposome mediated gene transfer method. NR1 was detected using antibody against NR1. For measurements of GluR2/3 as control proteins, the membrane used for NR1 was reprobed with polyclonal antibody against GluR2 and GluR3 to confirm equal protein loading in each lane. Level of hippocampal NR1 and GluR2/3 protein treated rats was represented as a percentage of that of non-treated rats (NT). (A), Representative blots (upper) and quantitation of relative band densities (lower) for NR1 protein obtained from the hippocampus of rats that received an intra-hippocampal injection of NR1-AS (AS), NR1-SE (SE) or vehicle (BSS) by using HVJ-liposome method, 6 days prior to the Western bolt analysis. (B), Representative blots (upper) and quantitation of relative band densities (lower) for GluR2/3 proteins obtained from the same hippocampus of rats as described above. Each treatment group: AS, rats with intra-hippocampal injection of NR1-AS (n/4); SE, rats with intra-hippocampal injection NR1-SE (n/4); BSS, rats with intrahippocampal injection of BSS (n/4). The significant down-regulation (30%) of NR1 protein was observed only in the NR1-AS treated group, while the levels of GluR2/3 proteins were not changed in any treatment groups. (C), Time course of antisense-induced downregulation of hippocampal NR1 protein levels. Each value is an average of relative abundance of NR1 protein obtained from two rats that received a single intra-hippocampal injection of NR1-AS by using HVJ-liposome method, 3 days (day 3)(n/4), 7 days (day 7)(n/4), 11 days (day 11)(n/4), and 14 days (day 14) (n/4) prior to the Western blotting, respectively. *, Significantly different from the SE (P B/0.05) and the BSS (P B/0.05) for (A) and different from the day 14 (P B/ 0.05) for (C) by ANOVA and post-hoc Fisher’s test.
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Fig. 2. The Nissl-stained sections of the ventral (A) and dorsal (B) hippocampus of the rat that received an single intra-hippocampal injection of antisense ODNs (NR1-AS) /HVJ-liposome complex 6 days prior to the histological assay. The Nissl-staining patterns in A and B indicate that the treatment with HVJ-liposome containing NR1-AS did not produce any lesions in the neural tissue except for a small scar (arrow). Scale bar represents 1 mm.
Fig. 3. The effect of antisense-induced down-regulation of hippocampal NR1 expression on the spontaneous locomotor activity in an open filed. Treatment groups were: NT, non-treated rats; NR1-AS, rats that received a single intra-hippocampal injection of NR1-AS by using HVJ-liposome method 6 days prior to the open field test; NR1-SE, rats that received an intra-hippocampal injection of sense ONDs (NR1-SE) by using HVJ-liposome method 6 days prior to the open field test. The NR1-SE group was used as a control for the NR1-AS. From the 30 min recording of locomotor behavioral, the crossing rate during the initial 10 min period was measured as locomotor activity by counting the number of crossings over the borderlines of 16 blocks in the field. Each value represents mean9/S.E.M. (n /5 for the NR1-AS, n/6 for the NR1-SE, n/6 for the NT). There was no significant difference between the two treatment groups.
NR1-SE-treated rats (n /6) and non-treated rats (n / 6). 3.3. Effect of NR1 hippocampal knockdown on spatial learning To investigate the effect of NR-1 hippocampal knockdown by antisense on spatial learning, we used the Morris water maze test. The rats received an intrahippocampal injection of HVJ-liposomes containing NR1-AS or NR1-SE 6 days prior to undergoing training in a water maze. All of the rats in both treatment groups learned to swim directly to the hidden platform during five training sessions, well before training was completed
Fig. 4. The effect of antisense-induced down-regulation of hippocampal NR1 expression on the acquisition of spatial memory in the hidden platform water maze. Treatment groups were: NR1-AS, rats (n/5) that received a single intra-hippocampal injection of NR1-AS by using HVJ-liposome method 6 days prior to the water maze training; NR1SE, rats (n/6) that received an intra-hippocampal injection of sense ONDs (NR1-SE) by using HVJ-liposome method to NR1 4 days prior to the water maze training. The NR1-SE group was used as a control for the NR1-AS. (A), Escape latency (mean9/S.E.M.) in water maze training. There was no significant difference in the spatial learning between the two treatment groups. (B), Place preference (mean9/ S.E.M.) in the probe test conducted just after the end of the training session. Both NR1-AS treated and NR1-SE treated rats exhibited strong place preference for the target quadrant where the hidden platform was previously located, indicating that there was no significant difference in the acquisition of spatial memory between the two treatment groups.
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(Fig. 4A). On the last day of training, a spatial probe test was conducted just after the final training session. All of the rats in both treatment groups spent most of the trial search within a target quadrant zone, indicating their place preference in the target zone. There was no significant difference in the place preference in the target zone between the NR1-AS-treated group and NR1-SEtreated group (Fig. 4B). The swimming speeds of each animal through the sessions were also measured by computer. There were no difference between antisense treated and control animals. Also individual animal in both treatment groups did not show any change of swimming speed through the sessions (data not shown). 3.4. Sensorimotor gating deficit and its recovery in rats with NR-1 hippocampal knockdown By using the PPI test, we examined whether NR1 hippocampal knockdown causes a deficit in sensorimotor gating and whether any such deficit can be recovered. Six days prior to the PPI test, intra-hippocampal injection of the HVJ-liposome containing NR1-AS was given. The results showed a significant disruption of PPI upon stimulation at PP80, while they showed no significant disturbance of PPI at PP70 even though a tendency toward a reduced PPI was apparent, compared with the NR1-SE-treated rats (Fig. 5A). On the other hand, 14 days after the NR1-AS treatment, when the down-regulated NR1 protein expression had completely recovered to the original levels, the NR1-AS-treated rats did not exhibit disruption of PPI at either PP70 or PP80 (Fig. 5B), suggesting a recovery from the antisenseinduced PPI deficit. The amplitude of startle response to pulse stimulations were also measured. There was a tendency of increase of startle response to pulse stimulations in animals with NR1-AS or NR1-SE treatment compared with non-treated animals, although the increase was not significant (data not shown). On the other hand, there was no difference between NR1-AS treated and NR1SE treated animals. Thus, it is considered that tendency of increase is due to the stress of operation.
Fig. 5. Deficit of PPI of acoustic startle response and its recovery observed in the rats with antisense knockdown of hippocampal NR1. Treatment groups were: NR1-AS, rats (n /8) that received a single intra-hippocampal injection of antisense ODNs (NR1-AS) by using HVJ-liposome method 6 or 14 days prior to the PPI test; NR1-SE, rats (n/6) that received an intra-hippocampal injection of sense ONDs (NR1-SE) by using HVJ-liposome method 6 or 14 days prior to the PPT test. The PPI test was performed under the two types of stimulation referred as PP70 and PP80. PP70 is an acoustic stimulation consisted of prepulse (20 ms burst of 70 dB) and startle-pulse (20 ms burst of 120 dB), while PP80 is that consisted of prepulse (20 ms burst of 80 dB) and startle-pulse (20 ms burst of 120 dB). (A), Percent of %PPI(mean9/S.E.M.) in the NR1-AS or the NR1-SE, 6 days after the treatment. The rats treated with NR1-AS exhibited a significant disruption of PPI at the stimulation of PP80, although they also showed a tendency toward a reduced PPI at the stimulation of PP70. *, significantly different from PPI in the NR1-SE group as a control (P B/0.05). (B), %PPI (mean9/S.E.M.) in the NR1-AS or the NR1-SE 14 days after the treatment, when the down-regulated NR1 expression had completely recovered to the original levels. The rats with NR1-AS treatment did not exhibit disruption of PPI at either PP70 or PP80, suggesting a recovery from the antisense-induced PPI deficit.
4. Discussion 4.1. Antisense knockdown of hippocampal NR1 expression Western blot analysis revealed that a single administration of antisense ODN /HVJ-liposome complex caused a 30% inhibition of NR1 expression for about 10 days. The blocking effect of this intra-hippocampal injection of antisense ODNs by using the HVJ-liposome method on the biosynthesis of NR1 protein and the long-lasting action of antisense ODNs are equal to those
of repeated applications or chronic infusion of the antisense ODNs to NR1 (Roberts et al., 1998; Zapata et al., 1997; Standaert et al., 1996). Furthermore, because of a single injection of the antisense ODN / HVJ-liposome without any stressful implantation of cannula for repeated or chronic administration, behavioral tests under less biased experimental conditions have been achieved by the treatment with antisense ODN /HVJ-liposomes. Thus, we conclude that the combined use of the antisense technique and HVJ-
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liposome-mediated gene transfer is a suitable way to investigate the functional role of some specific proteins in higher brain function by behavioral analysis. It is considered that the regional extent of antisense knockdown of NR1 is restricted to the hippocampus injected by antisense. Because the cationic HVJ-liposome vectors we used in this study are positively charged, the injected HVJ-liposomes containing antisense ODNs do not diffuse widely from the negatively charged tissue around the injection site and thereby antisense knockdown occurs specifically in the focal area of injection (Kaneda, 1999). 4.2. Behavioral alterations induced by the NR1 hippocampal knockdown In the present study, rats with an antisense-induced knockdown of hippocampal NR1 exhibited normal locomotor activity in an open field test (see Fig. 2). This result is consistent with normal motor activity found in mutant mice in which the targeted knockout of NR1 receptors in pyramidal neurons was restricted to the hippocampal CA1 region (Tsien et al., 1996), but it is inconsistent with abnormal motor activity such as an increase in both locomotor activity and stereotypic behavior observed in mutant mice with reduced NR1 expression in the whole brain (Mohn et al., 1999). Thus, behavioral abnormalities in motor activity may be related to the dysfunction of NMDA receptors in the whole brain. As demonstrated in this study, in a Morris water maze task, rats with antisense-induced knockdown of hippocampal NR1 expression displayed intact spatial memory in both acquisition and retrieval (see Fig. 3). However, this result is inconsistent with the results of studies regarding impaired spatial memory found in mutant mice with a targeted knockout of NR1 in the hippocampal CA1 (Tsien et al., 1996) and normal mice treated with an intra-hippocampal injection of competitive NMDA receptor antagonist AP-5 (Morris et al., 1986). This discrepancy may be due to the partial dysfunction (30%) of hippocampal NR1 in our study, compared with the completely impaired or inactivated NMDA receptor-mediated function in the hippocampus in the previous studies. In this study, we have demonstrated that rats with NR1 hippocampal knockdown display impaired PPI of acoustic startle response. In particular, the recovery from the deficit of PPI in the antisense-treated rats was observed at 14 days after the antisense treatment, when the down-regulated expression of NR1 protein recovered to the original level. This recovery from the deficit of PPI suggests that the disruption of PPI occurs depending on the antisense-induced down-regulation (30%) of NR1 in the hippocampus. It has been reported that focal infusion of a noncompetitive NMDA receptor
antagonist into the amygdala or hippocampus produces the disruption of PPI in normal rats, suggesting that multiple limbic regions mediate the PPI deficit (Bakshi et al., 1998). From our data, only 30% impairment of NR1-related function in one of the multiple limbic regions is sufficient to mediate the disruption of PPI. Since the PPI reflects the function of the sensorimotor gating system, which partly overlaps with cognitive function (Bakshi et al., 1998; Geyer, 1998; Braff et al., 1990, 1992), it has been thought that the disruption of sensorimotor gating might provide a model for a deficit observed in schizophrenia as schizophrenic patients have a significant impairment of PPI (Braff et al., 1990, 1992, 2001; Geyer, 1998). As for memory deficit, schizophrenic patients have impaired episodic memory, but they maintain a relatively intact procedural memory and priming of semantic memory (Goldberg et al., 1989; Schmand et al., 1992; Tamlyn et al., 1992). Recently, mutant mice with reduced NR1 expression (5% of the normal level) in the whole brain exhibited deficits in social and sexual interactions and hypersensitivity to amphetamine (Mohn et al., 1999), treatment of phenciclidine or MK-801, a NMDA receptor antagonist, induces schizophrenia-related behaviors in rodents, include increases in locomotion and stereotypies, deficits in social interactions, deficit of sensorimotor gating and cognitive functions, increase of immobidity in forced swim test (Corbett et al., 1995; Duncan et al., 1999a,b; Swerdlow et al., 1998; Sams Dodd, 1998; Noda et al., 2000; Gainetdinov et al., 2001). Due to those findings, a lot of interest has been focused on the relationship between schizophrenic symptoms and dysfunction of NR1 receptors. Therefore, the combination of impaired PPI with intact spatial memory observed here in rats with the antisense-induced partial knockdown of hippocampal NR1 receptors may provide a possible animal model of schizophrenia, even though the present knockdown rats display only a part of modeling behaviors such as cognitive impairment, defective sensorimotor gating, and relatively intact memory. Further study is needed to demonstrate the rest of modeling behaviors for schizophrenic symptoms, such as hypersensitivity to dopamine, behavioral abnormality in forced swim test and defective social interaction as negative symptoms.
Acknowledgements This study was supported by grants from Inokashira Hospital, Japan, and grants from the Mitsubishi Pharma Research Foundation, Japan. This study was also supported in part by the Grant for Scientific Research from Kitasato University Graduate School of Medical Science, Japan.
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