M
ATERIALS AND
M
ETHODS
Extraction and isolation of alkaloids from the plant were described in a previous study (Ponglux et al., 1994).
The solution of the MS alkaloid extract was prepared by dissolving the extract in co-solvent (Tween80: propylene
glycol: H
2
O at a 1:4:4 ratio) and adjusted to desired concentrations with distilled water.
Adult male Wistar rats (300-350 g) were anesthetized with intramuscular injection of 60 mg/kg Zoletil® 100
(Virbac, Thailand Co. Ltd.). Stainless steel screw electrodes were stereotaxically implanted in the frontal cortex (AP; +3,
ML; 3) and the parietal cortex (AP; -4, ML; 4) on the left side skull (Fig. 1 A – C). Electrodes placed at midline
overlying the cerebellum were used as reference and ground electrodes. All electrodes were secured in place with acrylic
resin (Unifast trad, Japan).
To induce ethanol-dependent, 7-10 days after surgery, the animals were housed individually and given a
modified liquid diet (MLD) containing ethanol, at a progressively increasing concentrations (from 2.4 % for 3 days, to
4.8 % for 4 days, to 7.2 % for 21 days) to induce dependence as previous described (Uzbay and Kayaalp, 1995). Then,
animals were divided into 3 groups; 1) ethanol non-withdrawal, 2) ethanol withdrawal and 3) ethanol withdrawal with
60 mg/kg MS alkaloid extract. On the testing day, the ethanol containing MLD was replaced with isocaloric ethanol-free
MLD to induce withdrawal. Before the testing day, animals were acclimatized with recording condition for 4 times. The
dimension of recording chamber was 40x40x40 cm
3
.
Fifteen minutes before ethanol withdrawal, ethanol dependent animals received oral administration with either
vehicle (control) or MS alkaloid. Then, EEG signals of individual rats were recorded for 8-h period. EEG signals were
amplified with a low-pass 60 Hz, high-pass 0.1s and digitized at 400 Hz by a PowerLab/4SP system (AD Instruments)
with 12-bit A/D, and stored in a PC through the Chart program software. The EEG signals were processed through 1.25
– 45 Hz bandpass filter. The digitized EEG data were segmented into 1024-point (50% overlap) and the signals were
converted to power spectra by the fast Fourier transform algorithm (Hanning window cosine transform). Then the power
spectra of 2.56-sec sweeps in each 5-min length of selected period of artifact-free signals were averaged to give the
power spectra of the period. Awake periods were summed to reflect arousal levels. EEG power of theta wave (4 – 8 Hz)
was also analyzed to represent CNS hyperactivity. It has been found that theta power was produced during hyperactive
period. The powers of theta band (4-8 Hz) of each group were averaged and expressed as mean ± S.E.M.
One-way analysis of variance (ANOVA) followed by Student-Newman-Keuls method was used for statistical
analysis. The differences were considered statistically significant at
p <
0.05.
The experimental protocols for care and use of experimental animals described in the present study were
approved and guided by the Animals Ethical Committee of the Prince of Songkla University, Hat Yai, Songkhla,
Thailand.
