Invited Symposium: Neuronal Histamine Systems and Behavior



Materials & Methods


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Involvement of Central Histamine in Amygdaloid Kindled Seizures in Rats

Contact Person: Chiaki Kamei (kamei@pheasant.pharm.okayama-u.ac.jp)

Materials and Methods


Male Wistar strain rats, 7-8 weeks old and weighing 200-250 g, were used (Oriental Bioservice, Kyoto, Japan). All animals were maintained in an air conditioned room controlled for temperature (24-25C) and humidity (50 60%). The animals were given food and water ad libitum.


Under sodium pentobarbital (35 mg/kg, i.p., Dainippon, Osaka, Japan), the animals were fixed to a stereotaxic apparatus and bipolar electrodes were implanted unilaterally into the right frontal cortex (Anterior, A: 6.9, Lateral, L: 3.0), right dorsal hippocampus (A: 3.0, L: 2.5, Horizontal, H: 2.5) and right amygdala (A: 5.0, L: 5.0, H: -2.5) according to the atlas of de Groot (5). The cortical electrodes were made of stainless steel screws (1.0 mm in diameter and 1.5 mm in length) and were screwed into the skull of the motor area. The subcortical electrodes were bipolar twisted stainless steel wires 200 microns in diameter (tip distance of 0.5-1.0 mm) and insulated except for 0.5 mm at the tip. A guide cannula made of stainless steel tubing 700 m in outside diameter was implanted into the right lateral ventricle (A: 5.4, L: 1.5, H: 3.0). Electrodes were connected to a miniature receptacle, which was embedded in the skull with dental cement. At least 2 weeks were allowed for recovery from the surgery.

Experimental procedures in kindled seizures

The procedure to cause kindled seizures was similar to that described in a previous paper (6). The animals were placed in a plexiglas observation chamber (20X35X25 cm). An EEG was recorded bipolarly with an electroencephalograph (San-Ei, 1A74), stimulation of the amygdala was applied bipolarly every day by a constant current stimulator (Nihon Kohden, SEN-3301, SS-102J) and continued until a generalized convulsion was obtained. Stimulating parameters were 1.0 msec pulse duration, 60 Hz frequency and 1.0 sec train duration at an intensity just sufficient to induce after discharge (AD, 100-200 A). The developmental patterns of clinical manifestations of amygdaloid kindling were classified using the method described by Racine (7). i.e., convulsive behavior was divided into 5 stages: (1) Jaw movement. (2) Head nodding. (3) Forelimbs clonus. (4) Kangaroo posture. (5) Kangaroo posture and falling back. After the animals developed the final stage of generalized seizures, the stimulation was repeated for 5 more days, and the animals were used for drug study. Seizure stage (motor seizure, stage 1-5) and AD duration (electroencephalographic seizure in frontal cortex, hippocampus and amygdala) were used as indices of kindled seizures.

Determination of brain histamine

The histamine content of the brain was determined by means of the method described previously (8). Ten animals were used in each group and the brain was quickly removed and placed on ice. Brain regions were dissected in accordance with the procedure of Glowinski and Iversen (9). The brain specimens were homogenized in 0.4 N perchloric acid and centrifuged at 10,000 X for 60 min, and 20 l of the supernatant was injected into a high performance liquid chromatography (HPLC) column. The histamine content was determined using an HPLC equipped with a fluorometric detector (CCP & 8010 Series, Tosoh, Tokyo, Japan).


The drugs used were thioperamide hydrochloride (provided by Eisai), (R)-alpha methylhistamine oxalate (Funakoshi), diphenhydramine hydrochloride (Wako), dl chlorpheniramine maleate (Wako), cimetidine (Sigma), ranitidine hydrochloride (Glaxo), and metoprine (provided by Burroughs Wellcome). Thioperamide was dissolved in saline and when the drug was to be injected intracerebroventricularly, the pH of the drug solution was adjusted to 7.4. I.c.v. injection was administered using the same method as described previously (10). All drugs except metoprine were dissolved in saline whereas metoprine was suspended in 0.025% Tween 80. These drugs were administered i.p. at a volume of 1.0 ml/kg of body weight. Doses of drugs were expressed in terms of the free base. Control rats were administered saline for i.c.v. injection and solvent for i.p. injection.

Histological examinations

After the experiments, the animals were sacrificed and localization of the electrodes in the brain was verified histologically.

Statistical analysis

The statistical significance of difference in the AD duration was determined by ANOVA with Dunnett's test. In case of seizure stage, Mann-Whitney U test was used.

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Kamei, C.; (1998). Involvement of Central Histamine in Amygdaloid Kindled Seizures in Rats. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/huston/kamei0281/index.html
© 1998 Author(s) Hold Copyright