 Vol.2, No.4, 161-170 (2013) Advances in Alzheimer’s Disease http://dx.doi.org/10.4236/aad.2013.24021 Donepezil rescues the medical septum cholinergic neurons via nicotinic ACh receptor stimulation in olfactory bulbectomized mice Yui Yamamoto, Kohji Fukunaga* Department of Pharmacology, Graduate School of Pharmaceutical Science, Tohoku University, Sendai, Japan; *Corresponding Author: kfukunaga@m.tohoku.ac.jp Received 12 September 2013; revised 20 October 2013; accepted 1 November 2013 Copyright © 2013 Yui Yamamoto, Kohji Fukunaga. This is an open access article distributed under the Creative Commons Attribu- tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Olfactory bulbectomy (OBX) causes cognitive dysfunction by degeneration of cholinergic neur- ons in the medial septum. Here, we define an involvement of nicotinic acetylcholine receptor (nAChR) in neuroprotective effect of donepezil in the septum neurons of OBX mice. Neuropro- tective effects on the medial septal cholinergic neurons were assessed after chronic donepezil administration in OBX mice. We also measured Akt and ERK phosphorylation to define the neur- oprotective mechanism of donepezil. We found that treatment with donepezil (1 - 3 mg/kg) for 15 consecutive days completely rescued choliner- gic neurons in the OBX mice with concomitant improved memory. Reduction of both protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) phosphorylation were restored by chronic donepezil administration (1 - 3 mg/kg) in OBX mouse medial septum. Both phosphory- lated Akt and ERK immunoreactivities were lo- calized in cell bodies of choline acetyltransfer- ase (ChAT)-positive cholinergic cells in the me- dial septum. Enhancement of Akt and ERK phos- phorylation seen following donepezil admini- stration was totally blocked by pre-administra- tion of mecamylamine (10 μM), a nicotinic ace- tylcholine receptor antagonist. Donepezil increas- es phosphorylation of Akt and ERK via nAChR stimulation in the medial septum cholinergic neurons. The Akt and ERK stimulation by do- nepezil is associated with its ability of neuropro- tection in the medial septum and memory im- provement. Keywords: Donepezil; Neuroprotection; Medial Septum; Nicotinic Acetylcholine Receptors 1. INTRODUCTION Destruction of olfactory system reportedly impairs learning and memory [1-3] and Alzheimer’s disease (AD) patients show severe impairment of olfactory bulb func- tions even in the early stage [4,5]. The cholinergic neu- rons in the basal forebrain and olfactory bulb are origi- nated from the medial septum, thereby providing cho- linergic innervation to the hippocampus, olfactory blub, amygdala and overall neocortex [6]. Interestingly, olfac- tory bulbectomy (OBX) causes degeneration of choliner- gic neurons not only in the medial septum but also in the hippocampus, thereby eliciting hippocampus -dependent memory deficits in mice [7]. In support to this idea, OBX treatment elicits reduction of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) levels in the hip- pocampus [8,9] and nicotine-induced acetylcholine (ACh) release in the hippocampus was largely impaired in OBX mice [10]. In addition, significant elevation of beta- amyloid (Aβ) levels is seen in extracts from neocortex and hippocampus by OBX in mice [11]. Thus, OBX mouse could be a useful model to test AD-like cognitive dysfunction following degeneration of cholinergic neu- rons in the septum. Donepezil, an AChE inhibitor, is prototyped therapeu- tics for Alzheimer’s disease (AD) patients in the world- wide [12]. Donepezil elevates extracellular ACh levels in the cortex [13,14]. Interestingly, neuroprotective effects of donepezil were also found both in vitro and in vivo. For example, donepezil blocked glutamate-induced ex- citotoxicity in rat primary cultured neurons and oxy- gen-glucose deprivation-induced cell death in HEK293 Copyright © 2013 SciRes. OPEN ACCESS  Y. Yamamoto, K. Fukunaga / Advances in Al zheimer’s Disease 2 (2013) 161-170 162 cells and cortical neurons [15-18]. Donepezil also at- tenuates streptozotocin-induced or traumatic brain in- jury-induced neuronal death in hippocampal CA1 region, thereby improving cognitive impairment [19,20]. In hu- man AD patients, donepezil slowed inhibition of hippo- campal atrophy [21]. However, the mechanism underly- ing donepezil-mediated neuroprotection remains unclear. Donepezil treatment activated phosphatidylinositol 3- kinase (PI3K)/protein kinase B (Akt) and increased ex- pression of Bcl-2, an anti-apoptotic protein, in rat corti- cal neurons [22]. Donepezil also potentiates the nerve growth factor (NGF)-induced phosphorylation of ex- tracellular signal-regulated kinase (ERK) in PC12 cells [23]. Furthermore, neuroprotective effects of donepezil were abolished by mecamylamine, the non-selective ni- cotinic acetylcholine receptors (nAChRs) inhibitor, but not with scopolamine, the muscarinic AChRs (mAChRs) inhibitor, in rat cortical neurons [22]. Increased Akt phos- phorylation following donepezil treatment in Aβ42-ex- posed cortical cultured neurons was blocked by meca- mylamine pre-treatment [24]. Nicotine-induced activa- tion of ERK was also blocked by mecamylamine in cul- tured spinal cord neurons [25]. Here, we found that chronic donepezil administration rescue degeneration of medial septum cholinergic neu- rons observed in OBX mice. Furthermore, we defined the expression of nAChR in the medial septum choliner- gic neurons and possible neuroprotective mechanisms of donepezil in OBX mouse medial septum. 2. MATERIALS AND METHODS 2.1. Animals Adult male DDY mice 6 weeks old (Nippon SLC, Hamamatsu, Japan) housed in cages with free access to food and water at a constant temperature (23˚C ± 1˚C) and humidity (55% ± 5%) with a 12-h light/dark cycle (09:00 - 21:00 hours). All experimental procedures using animals were approved by the Committee on Animal Experiments at Tohoku University Graduate School of Pharmaceutical Sciences. 2.2. Establishment of Bilateral Olfactory Bulbectomy Mice and Drug Treatment OBX mice were prepared as described [9,10,26]. Briefly, 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, which were then removed by gentle aspiration by a suction pump. Care was taken not to damage the frontal cortex. Holes were filled with a hemostatic sponge to avoid bleeding and the skin was closed. Sham-operated mice were treated similarly but bulbs were left intact. Drug administrations were begun 3 days after recovery period. Donepezil (0.3, 1 or 3 mg/kg, dissolved in dis- tilled water or vehicle alone) was administered orally daily for 15 consecutive days until mice were sacrificed, or one time intraperitoneally 30 min before sacrificed at 18 days after OBX surgery. Mecamylamine (10 μM) or saline were injected into the left brain lateral cerebroven- tricle 60min before acute donepezil administration. Fol- lowing sacrificed by decapitation, medial septum was dissected out for further analyses. 2.3. Behavioral Analyses Spontaneous alternation behavior in a Y-maze served as a spatial reference memory task. The maze was made of black acryl plate. Each arm was 40 cm long, 8 cm high, 3 cm wide at the bottom and all three arms con- verged at an equal angle. Each mouse was placed at the end of a fixed arm and allowed to move freely through the maze during an 8-min session. The sequence of arm entries was recorded manually. An alternation was de- fined as entries into all three arms on consecutive choices. The maximum number of alternations was defined as the total number of arms entered minus two, and the per- centage of alternation was calculated as (actual alterna- tions/maximum alternations) × 100. The total number of arms entered during the session was also determined. The novel object recognition task used to evaluate recognition memory [27]. This task is based on the ten- dency of rodents to discriminate a familiar from a new object. Mice were individually habituated to an open- field box (35 × 25 × 35 cm) for 2 consecutive days. The experimenter scoring behaviors were blinded to the treatment. During the acquisition phases, two objects of the same material were placed symmetrically in the cen- ter of the chamber for 10 min. One hour after acquisition phase training, one object was replaced by 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. Explora- tion 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 pre- viously explored object was reflected by preferential exploration of the novel object. Discrimination of spa- tial 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. 2.4. Immunohistochemical Studies Sham and OBX mice were treated with vehicle or donepezil (1 mg/kg) from 3 days to 17 days after surgery. Copyright © 2013 SciRes. OPEN ACCESS  Y. Yamamoto, K. Fukunaga / Advances in Al zheimer’s Disease 2 (2013) 161-170 163 At 18 days after surgery, mice anesthetized and perfu- sion-fixed with 4% paraformaldehyde. Briefly, after fixation, 50 μm thick coronal sections were cut using a vibrating microtome (Dosaka EM Co. Ltd., Kyoto, Ja- pan). Sections were incubated as follows: for 30 min in 0.1 M PBS (pH 7.4) containing 0.1% Triton X-100; for 1 h in PBS with 3% BSA (blocking solution); overnight with various primary antibodies in blocking solution at 4˚C. For ChAT-immunostaining, sections were incubated with goat anti-ChAT polyclonal antibody (1:1000) (Mil- lipore Bioscience Research Reagents, Temecula, CA). After washing, sections were incubated with biotinylated anti-goat IgG (1:5000) (Jackson Immuno Research La- boratories, West Grove, PA, USA) in TNB buffer for 1 h, followed by streptavidin-horseradish peroxidase (1:5000) (NEN Life Science Products, Boston, MA) labeled for 2 h. Sections were then stained with tetramethylrhodamine tyramide for 10 min using the TSA-Direct kit (NEN Life Science Products). For double staining with ChAT and phosphorylated Akt or ERK, sections were incubated with goat anti-ChAT polyclonal antibody (1:1000) (Mil- lipore), a rabbit anti-phosphorylated Akt (Thr-308) poly- clonal antibody (1:1000) (Millipore) or a rabbit anti- phosphorylated ERK (Thr-202/Tyr-204) polyclonal an- tibody (1:1000) (Cell Signaling Technology, Beverly, MA, USA). After thorough washing, sections were in- cubated 3 h with Alexa 594-labeled anti-goat IgG or Al- exa 488-labeled anti-rabbit IgG. To detect α7 nAChR, sections were incubated in α-bungarotoxin, tetramethyl- rhodamine conjugate (α-BTX) (Molecular Probes, Eugene, Oregon, USA) for 2 h at 37˚C, followed by incubation in antibodies for ChAT, phospho-Akt or phospho-ERK over- night at 4˚C. After several washes in PBS, sections were mounted on slides with Vectashield (Vector Laboratories, Burlingame, CA). Immunofluorescent images were ana- lyzed using a confocal laser scanning microscope (Leica TCS, Olympus, Tokyo, Japan). 2.5. Immunoblotting Analysis In Figures 1 an d 2, sham and OBX mice were treated with vehicle or donepezil (1 mg/kg) from 3 day to 17 days after OBX. And at 18 days, Medial septum was dissected out and stocked until homogenization. In Fig- ure 3, sham and OBX mice were administrated donepe- zil (1 mg/kg) and mechamylamine as indicated. One hr after administration, the septum is dissected out from each mouse. The septum 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 pep- statin A, 50 µg/ml trypsin inhibitor, and 1 mM dithio- threitol. Insoluble material was removed by a 10 min centrifugation (15,000 rpm). Samples containing equiva- Figure 1. Effects of donepezil administration on cholinergic neurodegeneration in OBX mouse medial septum. (A) Repre- sentative brain sections showing changes in numbers of cho- linergic neurons. (a), cholinergic neurons were identified by ChAT immunostaining in sham (b), donepezil treated (1 mg/kg daily for 15 days) sham mice (c), vehicle (d), or donepezil treated (1, 3 mg/kg daily for 15 days) (e), (f) OBX mice. Con- focal laser scanning images showed predominant ChAT ex- pression in cell bodies of cholinergic neurons in the medial septum indicating in box (a). Scale bar, 150 μm. (B) Quantita- tive analysis of ChAT levels was undertaken by counting ChAT-positive neurons. Data are expressed as percentage of values of sham-operated animals (mean ± S.E.M.) (n = 6). *P < 0.05 versus sham-operated animals and #P < 0.05 versus vehi- cle-treated mice. Dunnett’s multiple comparison tests was used for data analysis. (C) ChAT levels were detected by immu- noblotting with or without treatment with donepezil. Quan- titative analysis of 68 KDa ChAT levels were performed by densitometric analysis of immunoblots. Immunoblots with β- tubulin showed equal protein loading. Data are expressed as percentage of values of sham-operated animals (mean ± S.E.M.) (n = 6). **P < 0.01 versus sham-operated animals and #P < 0.05 versus vehicle-treated OBX mice. Data was analyzed using Dunnett’s multiple comparison test. Veh, vehicle treatment. lent amounts of protein based on Bradford analysis were boiled 3 min in Laemmli sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to an Immobilon polyvinylidene difluoride membrane for 2 h at 70 V. After blocking with TTBS so- lution (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 over- night at 4˚C with anti-ChAT, anti-phospho-Akt (Thr-308), anti-Akt, anti-phospho-ERK (Thr-202/Tyr-204) (1:2000; Sigma-Aldrich, St. Louis, MO), and anti-ERK (1:1000; Sigma-Aldrich). Bound antibodies were visualized using the enhanced chemiluminescence detection system (GE Healthcare, Chalfont St. Giles, UK) and analyzed semi- quantitatively using the Image J program (National In- stitutes of Health, Bethesda, MD). Copyright © 2013 SciRes. OPEN ACCESS  Y. Yamamoto, K. Fukunaga / Advances in Al zheimer’s Disease 2 (2013) 161-170 164 Figure 2. Effects of donepezil on phosphorylation of Akt and ERK in OBX mouse medial septum. Immunoblotting was car- ried out using antibodies recognizing phospho- or total protein. (A) Representative immunoblots of Akt-Ser-473 phosphoryla- tion is indicated. Quantitative analysis of relative Akt-Ser-473 phosphorylation in indicated groups was performed by densi- tometry. Total amounts of Akt protein were unchanged follow- ing OBX. (B) Representative immunoblots of ERK phosphory- lation is indicated. Quantitative analysis of relative ERK phos- phorylation in indicated groups was performed by densitometry. Total amounts of ERK protein were unchanged following OBX. Data are expressed as percentage of values versus sham-operat- ed animals (mean ± S.E.M.). *P < 0.05, **P < 0.01 versus sham- operated mice and #P < 0.05, ##P < 0.01 versus vehicle-treated mice. Data was analyzed using Dunnett’s multiple comparison tests. Veh, vehicle treatment. Donepezil administration stimu- lates Akt on ERK phosphorylation in the cholinergic neurons. (C) Confocal microscopy images of double staining in the me- dial septum for phospho-Akt (green), ChAT (red), and merged images are shown. Enlarged images indicate boxed inset. Scale bar, 20 μm. (D) Confocal microscopy images of double staining in the medial septum for phospho-ERK (green), ChAT (red), and merged images. Enlarged images indicate boxed area. Scale bar, 20 μm. Done, donepezil (1 mg/kg) treatment. 2.6. Statistical Analysis Data were represented as means ± S.E.M. For the re- sult of novel object recognition test was analyzed using the unpaired Student’s t-test. Statistical significance for differences among groups was tested by one-way analy- sis of variance (ANOVA), followed by a Dunnett’s test for multigroup comparisons. P < 0.05 indicated statisti- cally significant differences. Figure 3. Expression of nicotinic acetylcholine receptor and its functions in the medial septal cholinergic neurons (A) Expres- sion of nAChR is found at cholinergic neruron in the medial septum. Double stained-immunohistochemistry in the medial septum for ChAT (green) and α-Bungarotoxin (red) (upper) is performed. Enlarged images in square boxes were shown in the lower panels. Scale bars, 50 μm in low magnification and 20 μm in high magnification images. (B) Representative band of immunoblots using antibodies recognizing phosphor- or total protein of Akt (left) and ERK (right) are indicated. Quantitative analyses of phospho-Akt and ERK levels were performed by densitometry of immunoblots. Data are expressed as percentage of values of sham-operated animals (mean ± S.E.M.) (n = 6). *P < 0.05, **P < 0.01 versus OBX-operated animals and ##P < 0.01 versus donepezil-treated OBX mice. Data was analyzed using Dunnett’s multiple comparison tests. Done, donepezil (1 mg/kg) treatment. Meca, mecamylamine (10 μM) treatment. 3. RESULTS 3.1. Effects of Chronic Donepezil Administration on Impaired Memory-Related Behaviors in OBX Mice As expected, chronic donepezil administration im- proved the spatial working memory and cognitive func- tion in OBX mice in Y-maze task test and a novel object recognition task. OBX mice exhibited a significant de- crease in alternation behaviors compared with sham- operated mice in the Y-maze task (Sham, 66.7% ± 1.9%; OBX, 46.1% ± 2.1%; Figure 4(A)). Donepezil (1 mg/kg) treatment alone did not alter spatial working memory of sham-operated mice (60.8% ± 1.9%; Figure 4(A)). OBX- induced alternation behavior deficit was dose-depen- dently improved by donepezil administration at dose of 1 and 3 mg/kg (0.3 mg/kg, 51.6% ± 0.7%; 1 mg/kg, 55.5% Copyright © 2013 SciRes. OPEN ACCESS  Y. Yamamoto, K. Fukunaga / Advances in Al zheimer’s Disease 2 (2013) 161-170 165 ± 2.7%; 3 mg/kg, 55.5% ± 1.7%; Figure 4) compared with vehicle-treated OBX mice. Similarly, in the novel object recognition task, none of the groups showed a preference for left or right objects in the trial session. In the test session, OBX mice showed significant impairment by failing to discriminate between familiar and novel objects (50.6% ± 3.6%; Figure 4(B)), while sham-operated groups spent more exploratory time for the novel object (61.1% ± 2.2%; Figure 4(B)). Do- nepezil (1 mg/kg) treatment alone to sham mice did not affect the discrimination index (60.2% ± 2.6%; Figure 4(B)) compared to vehicle-treated sham mice. When OBX mice were treated with donepezil (1 or 3 mg/kg, but not 0.3 mg/kg), a higher exploratory preference for the novel object was observed in a dose-dependent manner (0.3 mg/kg, 52.1% ± 1.7%; 1 mg/kg, 58.1% ± 4.3%; 3 mg/kg, 63.3% ± 3.0%; Figure 4(B)). 3.2. Effects of Donepezil Administration on Cholinergic Neurodegeneration in OBX Mouse Medial Septum Since OBX induces cholinergic neurodegeneration in the medial septum [7], we determined whether donepezil elicits the neuroprotective effects on OBX-induced cho- linergic neurodegeneration in medial septum. Consistent with previous observations, ChAT-immunoreactive neu- rons was largely and significantly decreased in the me- dial septum of OBX mice (45% of that in Sham-operated animals) (Figures 1(A) and (B)). Notably, chronic ad- ministration of donepezil (1 and 3 mg/kg; 14 days p.o.) significantly inhibited reduction of ChAT-positive cho- linergic neurons (Figures 1(A) and (B)). Donepezil ad- ministration alone (1 mg/kg) did not affect the number of ChAT positive neurons in sham-operated animals (94.9% ± 3.0%; Figures 1(A) and (B)). Consistent with immu- nohistochemical observations, reduced ChAT protein le- vels were dose-dependently restored by chronic donepe- zil administration in OBX mouse medial septum (OBX, 66.3% ± 5.3%; 0.3 mg/kg, 77.7% ± 11.1%; 1 mg/kg, 103.5% ± 14.7%; 3 mg/kg, 101.6% ± 18.3% of that in sham-operated animals; Figure 1(C)). The maximal ef- fect of donepezil on ChAT protein levels was obtained by 1 mg/kg administration. ChAT protein levels did not differ significantly between groups of sham mice treated by donepezil alone (89.9% ± 7.6%; Figure 1(C)). 3.3. Effects of Donepezil on Phospho-Rylation of Akt and ERK in OBX Mouse Medial Septum Previous study reported that donepezil activates PI3K/Akt pathways in brain, thereby preventing lipo- polysaccharide-induced neuroinflammation [28]. Done- Figure 4. Effects of chronic donepezil administration on im- paired memory-related behaviors in OBX mice. (A) Effect of donepezil OBX-induced impairment of spontaneous alternation behavior, and number of arm entries in the Y-maze. Each bar represents the mean ± S.E.M. **P < 0.01 versus sham-operated animals; #P < 0.05, ##P < 0.01 versus OBX group; data was analyzed using Dunnett’s multiple comparison test. n = 5 - 6 in each group. (B) Novel object recognition behaviors were tested in sham, OBX and donepezil (0.3 - 3.0 mg/kg) treated groups. Differences in exploratory preference were assessed between groups either in the training or test session. Bars indicate means ± S.E.M. (n = 8). *P < 0.05 in the same treatment group and in novel group (right) versus familiar (left). Data was analyzed using Student’s t-test. Veh, vehicle treatment. pezil also promotes NGF-induced ERK phosphorylation coincident with NGF-stimulated neurite outgrowth in PC12 cells [23]. Therefore, we speculated activation of PI3K/Akt and ERK pathways are involved in neuropro- tection of cholinergic neurons by donepezil. Therefore, we first examined changes in Akt and ERK phosphoryla- tion in the extracts from the medial septum by immu- noblotting analyses using phospho-specific antibodies against Akt and ERK (Figure 2). Consistent with our previous observations, OBX resulted in a significant re- duction of Akt phosphorylation after OBX without changes in Akt protein levels (51.0 ± 9.6%; Figure 2(A)). Chronic donepezil administration significantly and com- pletely restored the reduced Akt phosphorylation by the same levels of sham mice (0.3 mg/kg, 72.0% ± 12.9%; 1 mg/kg, 100.2% ± 11.7%; 3 mg/kg, 89.7% ± 11.8%; Fig- ure 2(A)). Similarly, ERK phosphorylation in the medial septum of OBX was markedly reduced (63.4% ± 7.8%; Figure 2(B)) without changes in ERK protein levels. Donepezil administration also dose-dependently restored ERK phosphorylation comparable to levels seen in sham mice (0.3 mg/kg, 74.9% ± 2.4%; 1 mg/kg, 98.9% ± 14.7%; Copyright © 2013 SciRes. OPEN ACCESS  Y. Yamamoto, K. Fukunaga / Advances in Al zheimer’s Disease 2 (2013) 161-170 166 3mg/kg, 92.5% ± 9.2%; Figure 2(B)). Taken together, donepezil chronic administration stimulated both Akt and ERK signaling pathways, thereby likely preventing OBX-induced cholinergic neurodegeneration in the me- dial septum. 3.4. Effects of Donepezil on Akt and ERK Phosphorylation in the Medial Septal Cholinergic Neurons We next confirmed that donepezil-induced enhance- ment of Akt and ERK phosphorylation occurs in the me- dial septal cholinergic neurons by immunohistochemical analyses. Interestingly, phosphorylated Akt immunoreac- tivity was predominantly localized in cell bodies of ChAT-positive cholinergic neurons in the medial septum with or without donepezil administration (1 mg/kg) in sham-operated mice (Figure 2(C)). In OBX mice, the number of phosphorylated Akt/ChAT double-positive neurons was remarkably decreased and the immunofluo- rescence against phosphorylated Akt relatively weak in ChAT-positive neurons. Chronic donepezil administra- tion (1 mg/kg) restored the number of double-positive neurons and immunofluorescence against phosphorylated Akt in ChAT-positive neurons. Similarly, phosphorylated ERK (p-ERK) immunore- activity was predominantly localized in cell bodies of ChAT-positive cholinergic neurons in the medial sep- tum. Coincident with decrease in the number of ChAT- positive neurons, phosphorylated ERK immunofluo- rescence was also decreased following OBX surgery and restored by donepezil administration in the medial septum as compared with sham-operated animals (Fig- ure 2(D)). 3.5. Expression of Nicotinic Acetylcholine Receptor in the Medial Septal Cholinergic Neurons ChAT-positive cholinergic neurons of the medial sep- tum is known to express α7 nAChR mRNA in rat [29]. Recent study also suggests that medial septal/diagonal band neurons express functional nAChRs [30]. To de- termine whether α7 nAChR is expressed in ChAT-posi- tive cholinergic neuron in the medial septum, we per- formed double staining of the medial septum sections with anti-ChAT antibody and fluorescence-conjugated α-bungarotoxin (a selective ligand for α7 nAChR) in a normal mouse. α-bungarotoxin-labeled α7 nAChR was highly expressed in the most ChAT-positive cholinergic neurons in the medial septum (Figure 3(A)). However, other ChAT-negative cells were moderately stained with α-bungarotoxin, suggesting that other neurons or astro- cytes may also express α7 nAChR. 3.6. Function of nAChR in Donepezil- Induced Akt and ERK Activation in Medial Septum of OBX Mice We next evaluated whether donepezil-induced ele- vated phosphorylation of Akt and ERK is mediated by nAChR stimulation in the medial septum. Consistent with increased phosphorylation of Akt and ERK by chronic administration of donepezil, acute donepezil ad- ministration caused significant elevation of Akt and ERK phosphorylation compared with saline-treated OBX mice (Akt, 142.4% ± 7.2%; ERK, 132.2% ± 7.4%; Figure 3(B)). Importantly, mecamylamine pre-treatment com- pletely abolished the elevated phosphorylation Akt and ERK (Akt, 63.97% ± 11.5%; ERK, 99.0% ± 7.4%; Fig- ure 3(B)). These results suggest that donepezil-induced activation of Akt and ERK is mediated by nAChR sti- mulation. 3.7. Effects of Donepezil on CREB Phosphorylation and BDNF Expression in OBX Mouse Medial Septum We finally defined the physiological relevance of Akt and ERK activation by donepezil administration in the medial septum. We measured downstream signaling tar- gets of Akt and ERK pathways. As previously reported [31], CREB phosphorylation levels in OBX mice sig- nificantly reduced compared with sham mice (59.4% ± 8.0%; Figure 5). Decreased level of CREB phosphoryla- tion was restored by repeated donepezil treatment (1 mg/kg) (102.5% ± 8.8%; Figure 5). Unlike CREB phos- phorylation, BDNF protein levels in OBX mice showed no significant changes by OBX treatment (118.0% ± 3.0%; Figure 5). However, BDNF protein level was significantly increased by donepezil treatment (141.3% ± 6.4%; Fig ur e 5). Thus, donepezil-induced CREB phosphorylation may in part mediate the BDNF ex- pression. 4. DISCUSSION Here, we for the first time demonstrated that an AChE inhibitor, donepezil, protects the septo-hippocampal cho- linergic neurons against OBX-induced neurodegenera- tion. Previous studies reported that chronic donepezil treatment improved learning deficits in AD model mice and AD patients [12,32-34]. The septo-hippocampal cho- linergic innervation was also impaired in the CA3 hip- pocampal regions following OBX [7], suggesting that the reduced septo-hippocampal cholinergic innervation ac- counts for impairment of learning and memory in OBX mice. Consistent with those observations, we confirmed that donepezil-induced neuroprotection of the cholinergic neurons is associated with improved cognitive behaviors in OBX mice. Other investigators have shown that Copyright © 2013 SciRes. OPEN ACCESS  Y. Yamamoto, K. Fukunaga / Advances in Al zheimer’s Disease 2 (2013) 161-170 167 Figure 5. Effects of done- pezil on CREB phosphory- lation and BDNF expres- sion in OBX mouse medial septum. Phospho-CREB and BDNF levels were meas- ured after OBX by immu- noblotting with or without administration of donepezil (1 mg/kg). Quantitative ana- lyses of phospho-CREB and BDNF levels were perform- ed by densitometry of im- munoblots. Data are express- ed as percentage of values of sham-operated animals (mean ± S.E.M.) (n = 6). *P < 0.05, **P < 0.01 versus sham-operated animals and #P < 0.05 versus vehicle- treated OBX mice. Data was analyzed using Dunnett’s mul- tiple comparison tests. Done, donepezil (1 mg/kg) treat- ment. donepezil have neuroprotective effects against gluta- mate- or Aβ-induced neuronal cell death in cultured neurons [35,36]. We previously documented that Akt and ERK activi- ties are decreased in the medial septum 14 days after OBX, with a concomitant reduce the number of ChAT- positive neurons [7]. We here confirmed that decreased Akt and ERK occur in the ChAT-positive neurons in the medal septum. Donepezil prevents neurotoxicity in cul- tured cortical neurons through stimulation of Akt and ERK pathways via nAChRs [22,24,37,38]. We also de- fined that α7 nAChR is highly expressed in ChAT-posi- tive cholinergic neurons in addition to ChAT-negative neurons in the medial septum as shown in Figure 3. Al- though the total protein levels of Akt and ERK were un- changed in the medial septum by OBX, the basal phos- phorylated Akt and ERK immunoreactivities were pre- dominantly observed in ChAT-positive neurons, sug- gesting that Akt and ERK activities are higher than those in ChAT-negative neurons in the medial septum. Fur- thermore, the enhancements of phosphorylated Akt and ERK immunoreactivity were seen predominantly in ChAT-positive neurons in the medial septum, suggesting that donepezil primary activates Akt and ERK the cho- linergic neurons in the medial septum. Neuroprotective effect of donepezil was blocked by co-administration of PI3K/Akt inhibitor, LY294002, and mitogen-activated protein kinase kinase (MAPKK) inhibitor, PD98059 in rat cortical cultured neurons and/or SH-SY5Y neuro- blastoma cells [17,38,39]. CREB phosphorylation is like- ly mediated by the increased ERK and/or PI3K/Akt sig- naling [40,41]. Furthermore, BDNF expression as down- stream targets of Akt and/or ERK signaling mediates donepezil-induced neuroprotection in the medial septum. Indeed, BDNF level in the serum decreased in AD pa- tients and it elevates after 15 months of treatment with donepezil [42]. We also demonstrated that α7 nAChR expressed in medial septal cholinergic neurons. Among nAChR, het- eromeric α4β2- and homomeric α7-nAChRs are the most abundant isoforms in the mammalian brain [43]. Indeed, anti-α4nAChR immunoreactivity was observed in the medal septum of CBA/J mice [44]. Recently, novel het- eromeric α7β2 nAChR is found in the rodent basal fore- brain cholinergic neurons [45]. The α7β2 nAChR was major composition in the rat medial septum. Interestingly Aβ (1 - 42) strongly inhibits the α7β2 nAChR current in the cholinergic neurons isolated from the medial septum [45]. Taken together with our studies, α7-containing nAChR is major isoforms and function in the medial septum. Notably, a neuroprotective effect of nicotine and do- nepezil were mediated by activation of non-receptor- type tyrosine kinase, Janus-activated kinase 2 (JAK2) and Fyn via α7 nAChR in rat cortical neurons [22]. This effect leads to phosphorylation of PI3K/Akt and ERK signaling, and facilitates transcription of target genes, including those involved in neuronal survival, such as bcl-2 and BDNF [46-48]. Other group also showed that acute donepezil administration in mice activates neuro- trophin receptor, Trk receptors in the hippocampus [49], suggesting that neuroprotective effects of donepezil through nAChR induce BDNF elevation in the hippo- campus. Thus nAChR likely accounts for the BDNF ex- pression not only in the hippocampus but also in the me- Copyright © 2013 SciRes. OPEN ACCESS  Y. Yamamoto, K. Fukunaga / Advances in Al zheimer’s Disease 2 (2013) 161-170 168 dial septum, where the cholinergic neurons innervate the hippocampus. In conclusion, our findings indicate that cholinergic neurodegeneration in the septo-hippocampal system part- ly contributes to OBX-induced memory-related behavior deficits. Donepezil treatment to OBX mice significantly rescued cholinergic neurons in the medial septum through Akt/ERK survival signals activation, including CREB phosphorylation and BDNF expression. Donepezil-in- duced protection of septo-hippocampal cholinergic neu- rons is potential mechanism to prevent or delay cognitive impairments in AD. Further studies are required to define cells expressing CREB phosphorylation and BDNF in the medial septal neurons, because α7 nAChR is ubiqui- tously expressed in the medial septal neurons. 5. ACKNOWLEDGEMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education Culture, Sports, Science and Technology, Japan (22390109, K.F.) and the Smoking Research Foundation (K.F.). REFERENCES [1] Sieck, M.H. (1972) The role of the olfactory system in avoidance learning and activity. Physiology and Behavior, 8, 705-710. http://dx.doi.org/10.1016/0031-9384(72)90099-6 [2] Serby, M., Corwin, J., Conrad, P. and Rotrosen, J. (1985) Olfactory dysfunction in Alzheimer’s disease and Park- inson’s disease. The American Journal of Psychiatry, 142, 781-782. [3] Koss, E. 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