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NEUROPHARMACOLOGY
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (F.H., N.S., S.M., Y.U.Y., A.Y.A.R., K.F.); Research Laboratory, Zenyaku Kogyo Co., Ltd., Tokyo, Japan (Y.O.Y., M.H.); and Tohoku University 21st Century COE Program "CRESCENDO," Sendai, Japan (K.F.)
Received for publication
February 3, 2008
Accepted
April 2, 2008.
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Abstract |
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; Serby et al., 1985; Koss, 1986). For example, impaired performance on learning and memory tasks, such as passive avoidance and spatial reference memory, was observed in olfactory bulbectomized (OBX) mice and rats (Kovács et al., 2001; Hozumi et al., 2003; Attems et al., 2005; Han et al., 2008). In addition, elevated levels of β-amyloid peptide are seen in extracts from neocortex and hippocampus in OBX mice (Aleksandrova et al., 2004). It is interesting that significant impairment of the olfactory system is observed in humans in the early stages of Alzheimer's disease (AD) (Esiri and Wilcock, 1984; Doty, 1991). OBX injury causes retrograde degeneration of cholinergic neurons from the medial septum, leading to decreased cholinergic innervation of the cortex and hippocampus (Moriguchi et al., 2006; Nakajima et al., 2007). Indeed, levels of acetylcholinesterase (AChE) (Nakajima et al., 2007) and choline acetyltransferase (ChAT) (Han et al., 2008), both presynaptic markers of cholinergic neurons, significantly decrease in the cortex and hippocampus of OBX mice. Consistent with these observations, OBX-induced memory deficits are improved by stimulation of the cholinergic system (Yamamoto et al., 1997; Hozumi et al., 2003). We recently reported that a novel neuroprotective vanadium compound rescues cholinergic neurons in the medial septum from OBX-induced cholinergic neurodegeneration and restores hippocampal cholinergic activity, thereby improving hippocampus-dependent memory deficits as in seen OBX mice (Han et al., 2008).
Glutamatergic neurons in the central nervous system play important roles in memory processes (Staubli et al., 1994; Weisskopf and Nicoll, 1995; Gray et al., 1996). Hippocampal long-term potentiation (LTP) is thought to be a model reflecting synaptic plasticity changes seen in learning and memory (Frey and Morris, 1998; Fukunaga and Miyamoto, 2000). In addition, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity is essential for LTP induction as well as maintenance (Fukunaga and Miyamoto, 2000; Lisman et al., 2002). Likewise, protein kinase C (PKC), protein kinase A, and extracellular signal-regulated kinase (ERK) play pivotal roles in hippocampal LTP induction (Sweatt et al., 1998; Collingridge et al., 2004; Xia and Storm, 2005). We recently found that hippocampal LTP was impaired in OBX mice through N-methyl-D-aspartate (NMDA) receptor hypofunction, with a concomitant decrease in CaMKII autophosphorylation and PKC phosphorylation but without changes in PKA activity (Moriguchi et al., 2006).
To develop a novel cognitive enhancer that does not inhibit AChE, we synthesized a new azaindolizinone derivative, ZSET1446 (ST101), which improves learning and memory by potentiating nicotine-induced acetylcholine (ACh) release in the hippocampus of amyloid-β(1-40)-infused rats (Yamaguchi et al., 2006). ZSET1446 treatment also rescues decreases in ChAT activity seen in the medial septum and hippocampus of the same model (Yamaguchi et al., 2006). In addition, MK-801 treatment significantly blocked the improving effects of ZSET1446 on methamphetamine-induced memory impairment, suggesting that NMDA receptor activation is required for ZSET1446 effects (Ito et al., 2007). Thus, for conditions such as AD, ZSET1446 is a candidate therapeutic agent to improve cognitive impairment without affecting AChE.
Little is known about precise molecular mechanisms in vivo underlying ZSET1446-mediated improvement of learning and memory in the central nervous system. Thus, we asked whether oral ZSET1446 administration protects septo-hippocampal cholinergic neurons from OBX-induced neurodegeneration in mice. We also investigated whether the drug improves impaired hippocampal LTP as well as decreased CaMKII and PKC activity in OBX mice. Our data suggest that stimulation of CaMKII and PKC activities in the hippocampal CA1 region by ZSET1446 treatment underlies its cognition-enhancing effects in OBX mice.
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Materials and Methods |
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Bilateral Olfactory Bulbectomy and Drug Treatment. After an acclimatization period of 1 week, bilateral olfactory bulbectomy was performed. OBX mice were prepared as described by Hozumi et al. (2003). In brief, mice anesthetized with sodium pentobarbital (50 mg/kg i.p.; Dainippon, Osaka, Japan) were placed in a stereotaxic instrument. After exposure of the skull, 1-mm-diameter holes were drilled on either side of the olfactory bulbs. Olfactory bulbs were removed through the hole by gentle aspiration with a suction pump, and care was taken not to damage the frontal cortex. Holes were filled with a hemostatic sponge to avoid further bleeding, and the skin was closed. Sham-operated mice were treated similarly but bulbs were left intact. Drug administration began after a 3-day recovery period. ZSET1446/ST101 was kindly provided by Zenyaku Kogyo Co. Ltd. (Tokyo, Japan). ZSET1446 (0.001–1 mg/kg, dissolved in distilled water or vehicle alone) was administered p.o. daily for 10 consecutive days until mice were analyzed. After sacrifice by decapitation, brains were checked to confirm complete removal of olfactory bulbs and lack of cortical damage to exclude incomplete OBX mice from analysis. Hippocampal CA1 region and the medial septum were dissected out for further analyses.
Behavioral Analyses. Spontaneous alternation behavior in a Y-maze task was recorded and evaluated as a spatial memory task. The apparatus consisted of three identical black Plexiglas arms (length x width x height, 50 x 16 x 32 cm). Each mouse was placed at the end of one fixed arm and allowed to move freely through the maze during an 8-min session. The sequence of arm entries was recorded visually, and three consecutive choices were defined as an alternation. The percentage of alternation was calculated as (actual alternations/maximal alternations) x 100. In addition, the total number of arms entered during the session was determined.
An object recognition task used to evaluate recognition memory (Ennaceur and Aggleton, 1997). This task is based on the tendency of rodents to discriminate a familiar from a new object. Mice were individually habituated to an open-field box (35 x 25 x 35 cm) for 2 consecutive days. The experimenter scoring behavior was blinded to the treatment. During the acquisition phases, two objects of the same material were placed in a symmetric position in the center of the chamber for 10 min. One hour after the acquisition phase training, one of the objects was replaced by a novel object, and exploratory behavior was again analyzed for 5 min. After each session, objects were thoroughly cleaned with 75% ethanol to prevent odor recognition. Exploration of an object was defined as rearing on the object or sniffing it at a distance of less than 1 cm, touching it with the nose, or both. Successful recognition of a previously explored object was reflected by preferential exploration of the novel object. Discrimination of spatial novelty was assessed by comparing the difference between time of exploration of the novel (right) and familiar object (left) and the total time spent exploring both objects, which made it possible to adjust for differences in total exploration time.
Immunohistochemistry. Mice were anesthetized and transcardially perfusion-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). Whole brains were immediately removed and postfixed overnight at 4°C in the same fixative. Then, coronal brain sections at the coordinates of anterior or posterior to bregma (35 µm in thickness) were prepared using a microslicer system (Vibratome, St. Louis, MO). Sections were incubated at room temperature with 0.01% Triton X-100 in PBS for 30 min and for another hour in 3% bovine serum albumin in PBS. For immunolabeling, slices were probed with anti-ChAT (goat polyclonal antibody, 1:500; Millipore Bioscience Research Reagents, Temecula, CA) overnight at 4°C. After washing, sections were incubated with biotinylated anti-goat IgG (1:5000) in TNB buffer (0.1 M Tris-HCl, 0.15 M NaCl, 0.5% blocking reagent; pH 7.4; PerkinElmer Life and Analytical Sciences, Boston, MA) for 1 h, followed by streptavidin-horseradish peroxidase (1:5000) labeled for 2 h. Sections were then stained with tetramethylrhodamine tyramide for 10 min using the TSA-Direct kit (PerkinElmer Life and Analytical Sciences). Immunofluorescent images were taken with a confocal laser-scanning microscope (TCS SP; Leica, Wetzlar, Germany). To normalize immunoreactivity with the anti-ChAT antibody, we analyzed cortical slices without changing confocal laser intensity and aperture, and we confirmed that the intensity of immunoreactivity with anti-ChAT antibody was the same between control and bulbectomized cortical slices. Slices were prepared from the medial septum region according to the nomenclature of stereotaxic mouse brain atlas (Franklin and Paxinos, 1997). From each animal, the number of ChAT-positive neurons was assessed in two 35-µm sections obtained from anterior 1.0 mm relative to bregma. The amounts of ChAT-positive neurons in identical fields were analyzed with acquisition software QCapture 6.0 (QImaging, Burnaby, BC, Canada) and Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA). Changes in amounts of ChAT-positive neurons were expressed as percentages of amounts seen in sham-operated animals.
Electrophysiology. Hippocampal slices were prepared as described previously (Liu et al., 1999). Transverse slices (400 µmin thickness) prepared using a Vibratome microslicer DTK-1000 were incubated 2 h in continuously oxygenized (95% O2, 5% CO2) artificial cerebrospinal fluid at room temperature. After a 2-h recovery period, a slice was transferred to an interface recording chamber and perfused at a flow rate of 2 ml/min with artificial cerebrospinal fluid warmed to 34°C. Field excitatory postsynaptic potentials (fEPSPs) were evoked by a 0.05-Hz test stimulus through a bipolar stimulating electrode placed on the Schaffer collateral/commissural pathway and recorded from the stratum radiatum of CA1 using a glass electrode filled with 3 M NaCl. Recording was performed using a single-electrode amplifier (CEZ-3100; Nihon Kohden, Tokyo, Japan), and the maximal value of the initial fEPSP slope was recorded and averaged every 1 min (three traces) using an A/D converter (PowerLab 200; ADInstruments Pty Ltd., Castle Hill, Australia) and a personal computer. Stimulus intensity was adjusted to evoke a fEPSPs of 1.0-mV amplitude. After a stable baseline was obtained, high-frequency stimulation (HFS) of 100-Hz frequency of 1-s duration was applied twice with a 10-s interval, and test stimuli were continued for the indicated periods.
Western Blotting Analysis. Hippocampal CA1 samples were homogenized in 70 µl of buffer containing 50 mM Tris-HCl, pH 7.4, 0.5% Triton X-100, 4 mM EGTA, 10 mM EDTA, 1 mM Na3VO4, 40 mM sodium pyrophosphate, 50 mM NaF, 100 nM calyculin A, 50 µg/ml leupeptin, 25 µg/ml pepstatin A, 50 µg/ml trypsin inhibitor, and 1 mM dithiothreitol. Insoluble material was removed by a 10-min centrifugation (15,000 rpm). After determining supernatant protein concentration using Bradford's solution, samples were boiled 3 min in Laemmli's sample buffer. Samples containing equivalent amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to an Immobilon polyvinylidene diflouride membrane for 2 h at 70 V. After blocking with TTBS solution (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 2.5% bovine serum albumin for 1 h at room temperature, membranes were incubated overnight at 4°C with anti-phospho-CaMKII (1:5000) (Fukunaga et al., 1988), anti-CaMKII (1:5000) (Fukunaga et al., 1988), anti-phospho-synapsin I (site 3) (1:2000; Millipore Bioscience Research Reagents), anti-synapsin I (1:2000) (Fukunaga et al., 1992), anti-phospho-GluR1 (Ser-831) (1:1000; Millipore, Billerica, MA), anti-GluR1 (1:1000; Millipore Bioscience Research Reagents), anti-phospho-PKC
(1:2000; Millipore), anti-PKC (1:2000; Millipore), anti-phospho-mitogen-activated protein kinase (ERK 1/2) (1:2000; Sigma-Aldrich, St. Louis, MO), anti-ERK (1:2000; Sigma-Aldrich), and anti-β-tubulin (1:10,000; Sigma-Aldrich). Bound antibodies were visualized using the enhanced chemiluminescence detection system (GE Healthcare, Chalfont St. Giles, UK) and analyzed semiquantitatively using the ImageJ program (National Institutes of Health, Bethesda, MD).
Statistical Analysis. Data were represented as means ± S.E.M. Statistical significance was performed by one-way analysis of variance, followed by a Dunnett's test for multigroup comparisons. P < 0.05 indicated statistically significant differences.
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Results |
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ZSET1446 Treatment Does Not Prevent Cholinergic Neuron Degeneration in the Medial Septum after OBX Treatment. We recently reported that OBX treatment induces neurodegeneration of cholinergic neurons in the medial septum (Han et al., 2008). Others have shown that learning and memory-related behavior as assessed by a passive avoidance task is impaired by 14 days after OBX treatment, with a concomitant decrease in ChAT immunoreactivity in the hippocampus (Hozumi et al., 2003). To determine whether ZSET1446 elicits a neuroprotective effect on OBX-induced neurodegeneration, we performed immunohistochemical analyses using an anti-ChAT antibody in the medial septum in OBX mice after chronic ZSET1446 treatment and compared the number of ChAT-positive cholinergic neurons in the medial septum with untreated OBX animals. Consistent with our previous observations (Han et al., 2008), the number of ChAT-positive cholinergic neurons in the medial septum was reduced by 53% 14 days after OBX (Fig. 2) compared with sham-operated animals. Chronic oral administration of ZSET1446 (0.01–1 mg/kg) did not prevent loss of ChAT-positive cholinergic neurons in the septum of OBX mice (Fig. 2), indicating that ZSET1446 does not have a neuroprotective effect on these neurons following OBX.
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ZSET1446 Treatment Significantly Rescues Impaired LTP in the Hippocampal CA1 Region of OBX Mice. LTP in the CA1 region is important for hippocampus-dependent spatial reference memory. We recently documented impaired LTP in the CA1 region of OBX mice (Moriguchi et al., 2006). To understand how memory is improved by ZSET1446 treatment, we evaluated hippocampal LTP in the CA1 region of ZSET1446-treated and -untreated mice. Evaluation of hippocampal slices obtained from sham-operated mice showed that HFS (100 Hz, two times) of Schaffer collateral/commissural pathways induces long-lasting LTP in the hippocampal CA1 region (225.0 ± 38.1% of baseline at 60 min; n = 5; Fig. 3, B and C). Consistent with our previous observation (Moriguchi et al., 2006), markedly reduced LTP was observed in vehicle-treated OBX mice (114.6 ± 9.2% of the baseline at 60 min; n = 5; Fig. 3, B and C). ZSET1446 treatment at 0.1 mg/kg significantly improved LTP in hippocampal CA1 regions (164.4 ± 15.7% of the baseline at 60 min; n = 5; Fig. 3, B and C). ZSET1446 treatment (0.1 mg/kg/day; 10 days p.o.) alone in sham mice did not affect the LTP induction (217.1 ± 21.1% baseline at 60 min; n = 5; Fig. 3C). Thus, in OBX mice, improved LTP associated with chronic ZSET1446 treatment is consistent with improved behavior seen in Y-maze and novel objective recognition tasks. These findings strongly suggest that treatment restores memory deficits.
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phosphorylation in cells of the hippocampal CA1 region of OBX mice (Moriguchi et al., 2006). Thus, we asked whether chronic ZSET1446 treatment would stimulate CaMKII autophosphorylation or PKC phosphorylation in these cells in OBX mice. We also evaluated ERK phosphorylation by immunoblotting with phosphospecific antibodies. Consistent with our previous observation (Moriguchi et al., 2006), decreases in both CaMKII autophosphorylation (40.3 ± 9.2% sham; n = 6; Fig. 4A) and PKC phosphorylation (63.8 ± 9.1% sham; n = 6; Fig. 4B) were observed in OBX mice. ERK phosphorylation was also significantly decreased in OBX compared with sham-operated mice (62.4 ± 4.2% sham; n = 6; Fig. 4C). However, when mice were chronically treated with ZSET1446, decreased CaMKII autophosphorylation was significantly rescued in a dose-dependent manner (0.01 mg/kg, 60.1 ± 6.9%; 0.1 mg/kg, 94.9 ± 9.8%; 1 mg/kg, 94.8 ± 12.9% of sham; n = 6; Fig. 4A), without a change in total CaMKII protein levels. Likewise, PKC phosphorylation also significantly increased in the ZSET1446-treated group at 0.1 mg/kg (81.3 ± 5.1% sham; n = 6; Fig. 4B). However, decreased ERK phosphorylation was not significantly restored by chronic ZSET1446 treatment (Fig. 4C). In addition, ZSET1446 treatment (0.1 mg/kg/day; 10 days p.o.) alone had no apparent effect on phosphorylation of CaMKII, PKC, and ERK in hippocampus of sham-operated animals, respectively (Fig. 4). Thus, rescue of CaMKII and PKC activities, but not that of ERK, by ZSET1446 is correlated with ameliorated LTP seen in the OBX mouse hippocampus.
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Effects of ZSET1446 on Phosphorylation of GluR1 and Synapsin I in OBX Mice. In hippocampal LTP, CaMKII activation is closely associated with stably increased phosphorylation of the GluR1 receptor (Derkach et al., 1999) and synapsin I (Fukunaga et al., 1995). To evaluate the physiological relevance of CaMKII and PKC activation following ZSET1446 treatment, we assessed phosphorylation of GluR1 and synapsin I by immunoblotting with phosphospecific antibodies. Consistent with our previous observation (Moriguchi et al., 2006), we found marked reduction in GluR1 (Ser-831) phosphorylation in cells of the hippocampal CA1 region in OBX mice (48.7 ± 5.8% sham; n = 6). Chronic ZSET1446 treatment at 0.1 and 1 mg/kg significantly restored GluR1 (Ser-831) phosphorylation in OBX mice (0.1 mg/kg, 91.8 ± 4.4%; 1 mg/kg, 76.9 ± 6.4% of sham; n = 6; Fig. 5A) without changing total GluR1 protein levels. However, no significant change in synapsin I (site 3) phosphorylation was observed between sham-operated and OBX mice, and ZSET1446 did not affect synapsin I (site 3) phosphorylation in the hippocampal CA1 region in OBX mice (Fig. 5B). Taken together, in OBX mice, chronic administration of ZSET1446 is required for restored CaMKII and PKC phosphorylation, which is associated with concomitantly restored GluR1 phosphorylation.
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Discussion |
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), and in mice with methamphetamine-induced memory deficits (Ito et al., 2007). Consistent with those observations, this study confirmed a cognition-enhancing effect of ZSET1446 using Y-maze and novel object recognition tasks in OBX mice. Here, we defined the neurochemical and electrophysiological mechanisms underlying this effect. The cognition-enhancing effect of ZSET1446 in OBX mice was closely correlated with recovery of NMDA-dependent LTP in the hippocampal CA1 region. Our data also suggest that increased phosphorylation of CaMKII and PKC in the CA1 region probably mediates improved LTP and underlies in part cognitive improvements seen in OBX mice.
The septo-hippocampal cholinergic system plays a crucial role in memory processes (Galey et al., 1989; Izquierdo and Medina, 1995). For example, lesioning the medial septum impairs acquisition and retention performance in several learning behaviors (Mitchell et al., 1982; Fibiger et al., 1991). The function of the medial septum is particularly essential for hippocampus-dependent learning and memory (Brioni et al., 1990; Durkin, 1994), and degeneration of cholinergic neurons in the basal forebrain correlates with cognitive impairment in AD patients (Lehéricy et al., 1993). OBX probably causes retrograde degeneration of cholinergic neurons in the septum, thereby eliciting anterograde degeneration of cholinergic neurons from the septum to the hippocampus (Serby et al., 1985; Koss, 1986; Bobkova et al., 2001). These observations led us to evaluate whether the neuroprotective effect of ZSET1446 on degeneration of the septal cholinergic neurons contributes to improved memory deficits in OBX mice. Consistent with our previous study (Han et al., 2008), we found that 14 days after OBX treatment, significantly reduced numbers of cholinergic neurons in the medial septum were associated with memory deficits in OBX mice. Treatment of these mice with ZSET1446 did not block degeneration of cholinergic neurons in the medial septum. However, impaired LTP in the hippocampal CA1 region was partly recovered by ZSET1446 treatment (0.1 mg/kg). Since impaired LTP in the OBX hippocampus is correlated with reduced CaMKII and PKC activities (Moriguchi et al., 2006), we tested the effect of ZSET1446 treatment on phosphorylation of CaMKII and PKC, which is required for activation of these kinases. Chronic ZSET1446 treatment preferentially stimulated CaMKII autophosphorylation at all of the doses tested (0.01, 0.1, and 1 mg/kg). Significant stimulation of PKC phosphorylation was also seen at 0.1 and 1 mg/kg doses. ERK phosphorylation was slightly increased by 0.1 mg/kg treatment, but the increase was not significant. Stimulatory effects on CaMKII autophosphorylation were closely correlated with increased GluR1 (Ser-831) phosphorylation but not with synapsin I phosphorylation. Both -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and synapsin I phosphorylation sites are known to be preferentially phosphorylated by CaMKII (Fukunaga et al., 1995; Derkach et al., 1999). Because CaMKII (Thr-286) autophosphorylation and GluR1 phosphorylation function as molecular switches underlying LTP and explicit memory (Lisman et al., 2002), improved NMDA-dependent LTP seen in OBX mice after ZSET1446 treatment is likely due to rescue of CaMKII and GluR1 phosphorylation in the hippocampus CA1 region.
In contrast to increased GluR1 (Ser-831) phosphorylation, which occurs postsynaptically, ZSET1446 treatment did not stimulate presynaptic synapsin I (site 3) phosphorylation. Although OBX treatment largely reduced CaMKII autophosphorylation in the hippocampus, it did not affect synapsin I (site 3) phosphorylation, which is preferentially phosphorylated by CaMKII. OBX treatment caused degeneration of presynaptic terminals of cholinergic neurons (Hozumi et al., 2003; Nakajima et al., 2007; Han et al., 2008). Lack of an effect on synapsin I phosphorylation by OBX suggests that synapsin I is mainly expressed in the other excitatory nerve terminals. Given that ZSET1446 reportedly promotes nicotine-induced ACh release in the hippocampus of amyloid-β(1-40)-infused rats, we hypothesized that ZSET1446 stimulates glutamate release presynaptically by Ca2+ elevation due to presynaptic nicotinic ACh receptor stimulation. A lack of a stimulatory effect on synapsin I (site 3) phosphorylation by ZSET1446 treatment suggests that the drug does not affect Ca2+ levels and/or CaMKII activity in excitatory presynaptic terminals. However, given that postsynaptic receptors or mechanisms underlying the stimulatory effects of ZSET1446 on CaMKII and PKC remain unclear, further studies are required to determine the specific nicotinic ACh receptors functioning in CaMKII activation in the hippocampus.
Recently, Ito et al. (2007) reported that improvement of methamphetamine-induced memory impairment by ZSET1446 treatment is associated with indirect activation of ERK. MK-801 treatment significantly inhibited ZSET1446-induced improvement of METH-induced memory impairment, suggesting involvement of NMDA in the ZSET1446 effect (Ito et al., 2007). Because CaMKII activation is totally dependent on NMDA receptor activation (Fukunaga et al., 1993), the evidence of inhibitory action of MK-801 on ZSET1446-induced improvement of memory impairment is consistent with our results. In the present study, however, ZSET1446 treatment did not restore ERK activity in OBX mice. This discrepancy may be due to different time points measured or different protocols of ZSET1446 administration.
Moreover, a previous study has shown that p.o. administration of ZSET1446 (0.1–1 mg/kg) enhances nicotine-stimulated ACh release in the hippocampus in normal and amyloid-β(1-40)-infused rats (Yamaguchi et al., 2006), but the mechanism of action of ZSET1446 remains unclear. ZSET1446 elicits cognition-enhancing action in scopolamine-induced amnesia at 0.1 to 1 mg/kg p.o. (Yamaguchi et al., 2006). The effective doses on nicotine-induced ACh release are closely correlated with cognition-enhancing action in scopolamine-induced amnesia in the previous study (Yamaguchi et al., 2006) and in the novel object recognition task reported here. It is noteworthy that treatment with similar doses of ZSET1446 markedly elevated CaMKII in the OBX mouse hippocampus. Thus, ZSET1446 preferentially stimulates NMDA receptor/CaMKII pathways in vivo to enhance cognitive function in memory impairment models. An alternative possibility is that ZSET1446 directly acts on postsynaptic machineries affecting channel activities of NMDA and ACh receptors. Therefore, in the future, we will investigate the effect of ZSET1446 on NMDA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and ACh receptor ion channels using electrophysiological experiments.
In conclusion, we have documented that peripheral chronic administration of ZSET1446 (0.1–1 mg/kg p.o.) stimulates NMDA receptor/CaMKII signaling, thereby ameliorating impaired LTP in OBX mice. In the same dose range, ZSET1446 rescued cognitive impairment in novel recognition and Y-maze tasks. In addition to a stimulatory effect of ZSET1446 on ACh release in the hippocampus, enhancement of LTP via CaMKII and/or PKC stimulation probably underlies the improvement of function. These multiple actions on ACh and NMDA receptor functions suggest that ZSET1446 could be an attractive candidate for Alzheimer's drug therapy in the clinic.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: OBX, olfactory bulbectomy; AD, Alzheimer's disease; AChE, acetylcholinesterase; ChAT, choline acetyl transferase; LTP, long-term potentiation; CaMKII, calcium/calmodulin-dependent protein kinase II; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; NMDA, N-methyl-D-aspartate; ZSET1446, spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one; ACh, acetylcholine; PBS, phosphate-buffered saline; fESPS, field excitatory postsynaptic potential; HFS, high-frequency stimulation; GluR1, glutamate receptor 1; MK-801, 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); Veh, vehicle.
Address correspondence to: Dr. Kohji Fukunaga, Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki-Aoba Aoba-ku, Sendai 980-8578. E-mail: fukunaga{at}mail.pharm.tohoku.ac.jp
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