Male Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan) weighting 200–250
g were housed one per cage under the standard laboratory conditions (23 ± 1°C, 55 ± 5% humidity) with free access to standard pellet diet (MEQ, Oriental Yeast Co., Tokyo) and drinking water ad libitum with lights on at 08:00 and off at 20:00. Animal handling and procedures were conducted in accordance with the Animal Welfare Act and with the Guide for the Care and Use of Laboratory Animals approved by the Animal Experiment Committee in Iwate University, Japan. Five rats were used in each group.
All reagents used were analytical grade. MSF was purchased from Sigma-Aldrich (Milwaukee, USA) and (R,S-1-benzyl-4-[(5,6-dimethoxy-1-indanon-2-yl)] methylpiperidine hydrochloride (donepezil) was a gift from Eisai Co., Ltd., (Tokyo, Japan).
Microdialysis experiments were conducted according to Hossain et al. [15
]. Briefly, the rats were anesthetized with sodium pentobarbital (50
mg/kg, i.p.) and then placed in a stereotaxic apparatus (Kopf instrument). The microdialysis guide cannula (AG-8, Eicom, Kyoto, Japan) was implanted into the left hippocampus with the following coordination (from the bregma): A‒
mm and, V‒
mm. Following surgery, the animals were returned to their home cage and allowed to recover for at least 3 days before the beginning microdialysis.
ACh and choline content in the dialysate from the different animals were quantified by high-performance liquid chromatograph (HPLC) with electrochemical detection (ECD). The day of the experiment, the microdialysis probe (A-1-8-02, Eicom, Kyoto) was carefully inserted into the hippocampus through the guide cannula. The inlet of the microdialysis probe was connected to a 2.5
mL gastight syringe and perfused with Ringer's solution (NaCl 147
mM, KCl 4.0
mM and, CaCl2
mM) containing 1μ
M eserine salicylate at a constant flow 2μ
L/min using a microperfusion pump, allowing the rats to move freely in a cubic Plexiglas box (30
cm × 30
cm × 40
The dialysate collected during the first 30
min was discarded to ensure a stable baseline of ACh release. Thereafter, 20μ
L samples of perfusate were collected at 10
min intervals. Upon collection, 20μ
L of 1μ
M ethylhomocholine containing 10
mM EDTA 2Na was added to each sample as an internal standard. Levels of ACh and choline in the dialysate (20μ
L/injection) were determined by electrochemical detection with HPLC (Eicom, Kyoto, Japan) equipped with an enzyme column (AC-ENZ, Eicom, Kyoto, Japan). A 20μ
L sample of the perfusate/ethylhomocholine solution was then injected into a HPLC equipped with ECD (HPLC-ECD, Eicom, Kyoto) and enzyme column (AC-ENZ, Eicom, Kyoto).
ACh and choline were separated on a cation exchange column (EICOMPAK AC-GEL, Eicom, Kyoto) with sodium lauryl sulfate (0.5
mg/mL). The mobile phase consisted of 0.05
M phosphate buffer (Na2
O) pH 8.2 containing 0.13
mM EDTA 2Na, 0.6
mM tetramethylammonium chloride and, 1.2
mM SDS pumped at 1
mL/min. The retention times for choline and ACh were 7.3 and 13.2
The basal efflux was defined as the average output of three samples prior to drug administration, and the results were calculated as the percentage of the baseline choline and ACh.
After establishing the basal efflux, the animals received one of the following IP injections: MSF (1 or 2
mg/kg) or donepezil (1 or 2
mg/kg) dissolved in vehicle (80μ
L ethanol + 88μ
L Tween 20) and prepared to a 1
mL total volume with isotonic sodium chloride. All control animals were injected with the same isotonic sodium chloride/vehicle solution by the same route and volume (1
mL/kg) as the drug.
At the end of the microdialysis experiment, the rats were euthanized with chloroform, the brains were removed, and the position of the probe in the hippocampus was verified by visual examination of 20μ
m frozen sections.
For acetylcholinesterase assays, rats in a parallel group received the same injections of MSF, donepezil, or vehicle on the same schedule as the animals used in microdialysis experiment. Three brain regions, hippocampus, striatum, and cerebral cortex (cortex) were quickly dissected on ice at 180
min and 24
hr after injection of MSF or donepezil and then homogenized in 0.1
M phosphate buffer solution (0.1
, pH 8.0), followed by dilution with the same buffer to 200, 400, and 200 times of tissue weight, respectively, for the analysis of AChE activity by the method of Ellman et al. [16
] with 0.48
mM acetylthiocholine iodide as substrate for 2
min at 25°C using UV-240 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at 412
The extracellular levels of ACh and choline from individual rats were calculated relative to the mean basal release (the average of three 10
min sequential samples before drug administration was taken as 100% basal release). Analysis of variance, followed by Dunnett's post hoc test for repeated measurements (treatment versus time), was used to analyze changes from ACh and choline baselines as well as for tests of significant differences over time. A level of P
< 0.05 was taken to indicate a statistically significant effect.