Tg mouse production. The transgene construct contained the gene cassette encoding IL-2Rα/mVenus followed by the SV40 early-gene polyadenylation signal in the place of the initiation codon in exon 3 of the ChAT gene30 (link). The construct was linearized by SfiI digestion, purified by pulse field gel electrophoresis, and microinjected into fertilized C57BL/6J mouse eggs, which were then implanted into pseudopregnant females. The ChAT-IL-2Rα/mVenus Tg mice were identified by Southern blot hybridization or PCR with genomic DNA prepared from tail clips. Tg and non-Tg littermates were used for the following experiments. All animal experiments were approved and performed in accordance with the guidelines for the care and use of laboratory animals established by the Animal Experiments Committee of Fukushima Medical University and Hiroshima University.
Intracranial surgery. Mice (8 weeks old) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and subjected to bilateral intracranial injection of IT solution [20 μg/ml anti-Tac(Fv)-PE38 in PBS containing 0.1% mouse serum albumin]. For targeting of cholinergic neurons in the MS/vDB and NBM, solution was injected into 12 sites (0.2 μl/site) and 6 sites (0.3 μl/site), respectively, through a glass micropipette that was stereotaxically introduced by using the coordinates from an atlas of the mouse brain52 . The anteroposterior, mediolateral and dorsoventral coordinates (mm) from bregma and dura were (1.1, ±0.1, −3.7), (1.1, ±0.1, −4.1), (0.8, ±0.1, −3.8), (0.8, ±0.3, −4.7), (0.6, ±0.1, −3.7), and (0.6, ±0.1, −4.2) for injection into the MS/vDB; and (−0.4, ±1.6, −3.7), (−0.7, ±1.8, −3.8), and (−0.9, ±2.0, −3.8) for injection into the NBM. Injection was carried out at a constant flow rate of 0.1 μl/min with a microinfusion pump, and the micropipette was left in situ for 2 min after each infusion.
Drug treatment. Donepezil hydrochloride (Sequoia Research Products Ltd.) and rivastigmine hydrogen tartrate (provided by Novartis Pharma AG, Basel, Switzerland) were dissolved into saline at a concentration of 0.2 or 0.4 mM. Mice received the intraperitoneal treatment of drug solution (2 or 4 μmol/kg) 30 min before the behavioural testing.
Histology. Fixed brains were cut into sections, and the sections were incubated with primary antibodies for GFP (rabbit, 1:2,000, Life Technologies), ChAT (mouse, 1:1,000, Millipore), parvalbumin (rabbit, 1:1000, Sigma-Aldrich), and then with fluorescein isothiocyanate-conjugated or biotinylated secondary antibodies. The immunoreactive signals were visualized by using a Vectastain Elite ABC kit. For double immunofluorescence histochemistry, the sections were incubated with anti-GFP and anti-ChAT antibodies, and then with species-specific secondary antibodies conjugated to Alexa488 (Molecular Probes) and Cy3 (Jackson ImmunoResearch). 4,6-Diamidino-2-phenylindole (DAPI, 1:1,000, Molecular Probes) was used to label nuclei. For cell counts, the number of immunopositive cells in each area was counted in the representative four sections through the MS/VDB or NBM (the anteroposterior coordinates from bregma: 1.3, 0.9, 0.7, and 0.5 mm for the MS/VDB; and −0.3, −0.5, −0.8, and −1.0 mm for the NBM), and the total number of immunopositive cells was calculated. Cresyl violet staining was processed to check for nonspecific damage on the brain tissue around the injection sites. For AChE staining, brain sections were rinsed in 0.1 M maleic acid buffer (pH = 6.0) and incubated for 10 min in 0.1 M maleic acid buffer containing 340 μM acetylthiocholine iodide, 50 μM sodium citrate, 30 μM cupric sulphate, and 5 μM potassium ferricyanide. To inhibit non-acetylcholinesterases, 10 nM ethopropazine was added to the solution. After the incubation, sections were washed with 50 mM Tris-HCl buffer (pH = 7.6) and soaked in 50 mM Tris-HCl buffer containing 620 nM cobalt chloride. Sections were then incubated for 7 min in 50 mM Tris-HCl buffer containing 190 μM diaminobenzidine, 0.003% H2O2, and 0.1% nickel ammonium sulphate.
Behavioural analysis. Adult naïve male mice were housed in standard lab Plexiglas cages (225 × 338 × 140 mm, length × width × height, four mice per cage) on a 12-h light/12-h dark cycle. The experiments were conducted during the light period. After the surgery, mice were given a 1-week recovery period, followed by the serial object exploration task32 (link) or one-trial object exploration task33 (link)34 (link). Different mice were used for the serial object exploration task, one-trial object recognition task, and the experiment with drug treatments. The open fields for these tasks were positioned in the centere of a room that had overhead lighting and contained various visual cues, including a computer, monitor, and shelves and posters on the wall. The animals’ behaviour was monitored using an overhead colour CCD camera (AVC-636SN; ITS, Co. Ltd.) connected to a digital video cassette recorder. During the tasks, the number of times the mouse snout made contact with an object (i.e., number of contacts) was manually counted. The counted data were confirmed by the video-recorded behaviour. The measurements of exploration were scored by an observer who was blinded to the animal groups and drug treatments.
For the serial object exploration task, a circular, polyvinylchloride open field (70-cm in diameter, 40-cm high) was used, and four positions in the open field were marked as north (N), south (S), east (E), and west (W) (seeFig. 2a ). The wall was equipped with a striped board composed of 2.5-cm-wide vertical black and white lines in the N position. The base of the open field was divided into four quadrants (NW, NE, SW, and SE), and further subdivided into 16 equal-sized areas for the measurement of locomotor activity. All of the objects (A–F) used for the task had different visual and haptic features. The task contained seven successive sessions (S1–S7; 6 min for each session) with an intersession interval of 3 min. These sessions consisted of four different phases for familiarization (S1), object exploration (S2–S4), displaced object exploration (S5/S6), and novel object exploration (S7). During S1, a mouse was placed in the empty open field and familiarized with it. During S2–S4, five different objects (A–E) were used, and four objects (A–D) were placed in the middle of each quadrant, while another object (E) was positioned in the centre of the field. Two objects (B and E) were displaced during S5, and no objects were moved during S6. Another object (A) was replaced by a novel object (F) during S7. A duplicate in the case of a repeating object was used for each phase within a complete trial. The mouse was allowed to explore freely in the open field during S2–S7, and the number of contacts with the objects was counted. In each session, the mouse began its exploration from one of four release points (N, S, E, and W) in a pseudo-random manner. Mice were placed back in their home cages during the 3-min intersession interval. Locomotor activity was assessed by counting the number of unit crossings with the subdivided areas in the base. During S5, the average of number of contacts with non-displaced objects (A, C, and D) or displaced objects (B and D) was calculated. During S7, the average number of contacts with non-displaced objects (C and D) or displaced objects (B and E) and the number of contacts with the novel object (F) were determined. All objects and the open field were washed with 70% ethanol after each trial.
For the one-trial object exploration task, a square, polyvinylchloride open field (35 × 35 cm and 30 cm high) was used (seeFig. 3a ). The task consisted of two sessions (3 min for each session) for the object exploration and displaced/novel object exploration with an intersession delay period of 3 or 30 min. The sequencing of 3-min and 30-min delays after the object exploration was counterbalanced among the mice. During the object exploration session, two identical objects were placed in the open field in a line-shaped spatial configuration, and during the displaced/novel object exploration session one object was displaced or exchanged with a novel one (B). A duplicate in the case of a repeating object was used for each phase within a complete trial. The mice were allowed to explore freely in the open field during the sessions, and the number of contacts with the objects was counted. In each session, the mice began their exploration from one of two release points in a pseudo-random manner and were placed back in their home cages during the 3- or 30-min intersession delay period. The left–right positions of the displaced and non-displaced/novel objects were counterbalanced. The open field and objects were washed in 70% ethanol after each trial.
Statistical analysis. For statistical comparisons, the ANOVA and post hoc Bonferroni test were used with significance set at P < 0.05. All values were expressed as the mean ± s.e.m. of the data. Repeated ANOVA was used for the analysis of within-subjects design.
Intracranial surgery. Mice (8 weeks old) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and subjected to bilateral intracranial injection of IT solution [20 μg/ml anti-Tac(Fv)-PE38 in PBS containing 0.1% mouse serum albumin]. For targeting of cholinergic neurons in the MS/vDB and NBM, solution was injected into 12 sites (0.2 μl/site) and 6 sites (0.3 μl/site), respectively, through a glass micropipette that was stereotaxically introduced by using the coordinates from an atlas of the mouse brain52 . The anteroposterior, mediolateral and dorsoventral coordinates (mm) from bregma and dura were (1.1, ±0.1, −3.7), (1.1, ±0.1, −4.1), (0.8, ±0.1, −3.8), (0.8, ±0.3, −4.7), (0.6, ±0.1, −3.7), and (0.6, ±0.1, −4.2) for injection into the MS/vDB; and (−0.4, ±1.6, −3.7), (−0.7, ±1.8, −3.8), and (−0.9, ±2.0, −3.8) for injection into the NBM. Injection was carried out at a constant flow rate of 0.1 μl/min with a microinfusion pump, and the micropipette was left in situ for 2 min after each infusion.
Drug treatment. Donepezil hydrochloride (Sequoia Research Products Ltd.) and rivastigmine hydrogen tartrate (provided by Novartis Pharma AG, Basel, Switzerland) were dissolved into saline at a concentration of 0.2 or 0.4 mM. Mice received the intraperitoneal treatment of drug solution (2 or 4 μmol/kg) 30 min before the behavioural testing.
Histology. Fixed brains were cut into sections, and the sections were incubated with primary antibodies for GFP (rabbit, 1:2,000, Life Technologies), ChAT (mouse, 1:1,000, Millipore), parvalbumin (rabbit, 1:1000, Sigma-Aldrich), and then with fluorescein isothiocyanate-conjugated or biotinylated secondary antibodies. The immunoreactive signals were visualized by using a Vectastain Elite ABC kit. For double immunofluorescence histochemistry, the sections were incubated with anti-GFP and anti-ChAT antibodies, and then with species-specific secondary antibodies conjugated to Alexa488 (Molecular Probes) and Cy3 (Jackson ImmunoResearch). 4,6-Diamidino-2-phenylindole (DAPI, 1:1,000, Molecular Probes) was used to label nuclei. For cell counts, the number of immunopositive cells in each area was counted in the representative four sections through the MS/VDB or NBM (the anteroposterior coordinates from bregma: 1.3, 0.9, 0.7, and 0.5 mm for the MS/VDB; and −0.3, −0.5, −0.8, and −1.0 mm for the NBM), and the total number of immunopositive cells was calculated. Cresyl violet staining was processed to check for nonspecific damage on the brain tissue around the injection sites. For AChE staining, brain sections were rinsed in 0.1 M maleic acid buffer (pH = 6.0) and incubated for 10 min in 0.1 M maleic acid buffer containing 340 μM acetylthiocholine iodide, 50 μM sodium citrate, 30 μM cupric sulphate, and 5 μM potassium ferricyanide. To inhibit non-acetylcholinesterases, 10 nM ethopropazine was added to the solution. After the incubation, sections were washed with 50 mM Tris-HCl buffer (pH = 7.6) and soaked in 50 mM Tris-HCl buffer containing 620 nM cobalt chloride. Sections were then incubated for 7 min in 50 mM Tris-HCl buffer containing 190 μM diaminobenzidine, 0.003% H2O2, and 0.1% nickel ammonium sulphate.
Behavioural analysis. Adult naïve male mice were housed in standard lab Plexiglas cages (225 × 338 × 140 mm, length × width × height, four mice per cage) on a 12-h light/12-h dark cycle. The experiments were conducted during the light period. After the surgery, mice were given a 1-week recovery period, followed by the serial object exploration task32 (link) or one-trial object exploration task33 (link)34 (link). Different mice were used for the serial object exploration task, one-trial object recognition task, and the experiment with drug treatments. The open fields for these tasks were positioned in the centere of a room that had overhead lighting and contained various visual cues, including a computer, monitor, and shelves and posters on the wall. The animals’ behaviour was monitored using an overhead colour CCD camera (AVC-636SN; ITS, Co. Ltd.) connected to a digital video cassette recorder. During the tasks, the number of times the mouse snout made contact with an object (i.e., number of contacts) was manually counted. The counted data were confirmed by the video-recorded behaviour. The measurements of exploration were scored by an observer who was blinded to the animal groups and drug treatments.
For the serial object exploration task, a circular, polyvinylchloride open field (70-cm in diameter, 40-cm high) was used, and four positions in the open field were marked as north (N), south (S), east (E), and west (W) (see
For the one-trial object exploration task, a square, polyvinylchloride open field (35 × 35 cm and 30 cm high) was used (see
Statistical analysis. For statistical comparisons, the ANOVA and post hoc Bonferroni test were used with significance set at P < 0.05. All values were expressed as the mean ± s.e.m. of the data. Repeated ANOVA was used for the analysis of within-subjects design.
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