【摘要】目的研究N-甲基-D-天冬氨酸型受体亚单位-1(NMDAR1)在大鼠第三脑室室管膜细胞上的表达。方法免疫组织化学技术。结果(1)NMDAR1在第三脑室室管膜细胞中的表达呈强阳性；(2)NMDAR1阳性室管膜细胞的形态与分布，在雌、雄大鼠间不存在明显的性别差异。结论为脑脊液中的谷氨酸可通过NMDA受体介导调节室管膜细胞提供了形态学证据。

【CLC number】R322.【Document cade】A

【Article ID】0529-1356(2000)02-174

EXPRESSION OF N-METHYL-D-ASPARTATE RECEPTOR SUBUNIT-1 IN EPENDYMAL CELLS OF THE THIRD VENTRICLE OF RAT

ZHANG Lin, WANG Shu-ling,NAN Yan, YU En-hua, SHEN Li

（Division of Neurochemistry, Department of Anatomy）

YANG Nuo

（Student Majored in Basic Medicine ′95 Beijing Medical University, Beijing 100083,China）

【Abstract】ObjectiveTo explore the expression of N-methyl-D-aspartate receptor subunit-1 (NMDAR1) in the ependymal cells of the third ventricle of rat.MethodsThe immunohistochemistry technique was used.Results(1)NMDAR1-immunoreaction (NMDAR1-IR) was strongly expressed in the ependymal cells of the third ventricle; (2)There was no significant sex difference in morphology and distribution of the NMDAR1-IR ependymal cells between male and female.ConclusionThe present investigation provided the morphological evidence supporting that glutamate of CSF might regulate ependymal cells via NMDAR.

【Key words】Ependymal cell; N-methyl-D-aspartate receptor; The third ventricle;Immunohistochemistry

Glutamate is a major excitatory neurotransmitter in the mammalian brain. Meanwhile, evidences have shown that CSF contains glutamate and it may play an important role in transporting information between the cerebrospinal fluid(CSF) and brain tissue^［1］. Therefore, focal problem is that whether the glutamate receptor exists in the ependymal cells, which directly contact with CSF. The ependymal cell of the third ventricle has become a hot point of these researches, because it borders on the hypothalamus, the center of neuroendocrine. N-methyl-D-aspartate receptor(NMDAR) is one of the ionotropic glutamate receptors and several NMDAR subunits have been cloned, including NMDAR1 and NMDAR2_A-D. NMDAR1 is the main functional subunit of NMDAR^［2,3］. In the present study, we demonstrated that NMDAR1 was expressed in ependymal cells of the third ventricle.It provided the morphological evidence supporting that glutamate of CSF might regulate ependymal cells via NMDAR.

Materials and Methods

1. Animals

Eight Wistar rats weighing between 200 to 250g(laboratory animal center of BMU), four males and four females, were used in this study. The animals were anesthetized with chloroform anesthetic (3ml/kg i.p.). Then the animals were perfused through the left ventricle with 100ml of 0.85% sodium chloride followed by ice-cold 400ml of 4% paraformaldehyde in 0.1mol/l phosphate buffer saline (PBS pH 7.4) within 30 minutes. After perfusion, the brains were removed from the skulls and postfixed with the same fixative for 2 hr at 4℃. Then they were immersed in PBS containing 20% sucrose for 6-8 hr and then stored in 30% sucrose overnight. The frozen brains were sectioned in the coronal plan at 42μm with a freezing microtome. The serial free-floating sections were collected in 0.1mol/l PBS and divided into two groups. The first group was used for NMDAR1 immunohistochemistry, the second group for Nissl stain and control experiments.

2. Immunohistochemistry

NMDAR1 immunohistochemistry was performed by the labeled streptavidin biotin method. The sections were incubated in 5% normal horse serum for 1 hr at room temperature. They were subsequently incubated in 1:500 NMDAR1 monoclonal antibody(Pharmingen) for 1 hr at 37℃ and then overnight at 4℃. Afterwards, the sections were incubated in biotinylated horse anti-mouse secondary antibody (Zymed) for 1 hr at 37℃, followed by the enzyme conjugated streptavidin-peroxidase (Zymed) for 1 hr at 37℃. The sections were stained in 0.01 PBS (pH 7.4) containing 0.1%3，3'-diaminobenzidine and 0.01%H₂O₂ for a few minutes. The sections were also washed in PBS for 10 minutes trice after each step. Sections were finally mounted on gelatin-chrome-coated glass slides, dried in air, dehydrated and coverslipped. Preabsorption controls were also performed.

Results and Discussion

Strong NMDAR1-immunoreactive (NR1-IR) ependymal cells were noted in the ependymal lining of the lateral walls (Fig.1-3), the floor (Fig.4) and the roof of the third ventricle. Most of NR1-IR ependymal cells were aligned tightly (Fig.1,2), while several others were arranged separately (Fig.3,4). And intracellular NR1-IR product was located around the nucleus (Fig.1-4). There was no significant sex difference in morphology and distribution of the NMDAR1-IR ependymal cells between the male and the female. No NMDAR1-IR cell was observed when the primary antibody was substituted with the vehicle of the antibody in the control experiment.

1箭头所示为位于第三脑室侧壁的NMDAR1免疫反应性室管膜细胞。标尺示30μm

2箭头所示为位于第三脑室侧壁呈紧密排布的NMDAR1免疫反应性室管膜细胞。标尺示30μm

3箭头所示为位于第三脑室侧壁呈分散排布的NMDAR1免疫反应性室管膜细胞。标尺示30μm

4箭头所示为位于第三脑室室底的NMDAR1免疫反应性室管膜细胞。标尺示30μm

Fig.1 Arrows show NMDAR1-IR ependymal cells in the lateral wall of the third ventricle. Bar=30μm

Fig.2 Arrows show NMDAR1-IR ependymal cells packed tightly in the lateral wall of the third ventricle. Bar=30μm

Fig.3 Arrows with tails show the NMDAR1-IR ependymal cells were arranged separately in the lateral wall of the third ventricle. Arrow without tail shows the NMDAR1-IR neuron in the subependymal layer. Bar=30μm

Fig.4 Arrows show the NMDAR1-IR ependymal cells in the floor of the third ventricle. Bar=30μm

Glutamate is a major excitatory neurotransmitter in the mammalian brain. Glutamatergic neurotransmission is mediated by a family of glutamate receptors. They can be grouped into two classes, ionotropic and metabotropic receptors. The ionotropic glutamate receptors can be divided into two subclasses, NMDA and non-NMDA receptors, based on physiological studies and affinity of different glutamate agonists. At least two NMDAR subunit classes have been cloned: NMDAR1 and NMDAR2_A-D. NMDAR1 is required for the formation of functional NMDAR^［2］. Recent studies have revealed that glutamate receptors not only mediate excitatory neurotransmission and play an important role in the development of the nervous system and in neurotoxicity^［3］, but are also involved in the physiology of glia and neuro-glia interaction^［4］