N-甲基-D-天冬氨酸型受体亚单位-1在大鼠第三脑室室管膜细胞上的表达
作者：张琳 杨诺 王淑玲
单位：
关键词：室管膜细胞；N-甲基-D-天冬氨酸受体；第三脑室；免疫组织化学
解剖学报000220
【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
, 百拇医药
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】 Objective To explore the expression of N-methyl-D-aspartate receptor subunit-1 (NMDAR1) in the ependymal cells of the third ventricle of rat. Methods The 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. Conclusion The present investigation provided the morphological evidence supporting that glutamate of CSF might regulate ependymal cells via NMDAR.
, 百拇医药
【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 NMDAR2A-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.
, 百拇医药
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.
, 百拇医药
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.
, 百拇医药
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.
, 百拇医药
图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
图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
图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
图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
, 百拇医药
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 NMDAR2A-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]. Ependymal cells are known to have functions of supporting, secreting and sensing, and may also play a role in the exchange between CSF and brain tissue[5]. Neurotransmitters and neuromodulators, such as vasoactive intestinal polypeptide, nitric oxide, the angiotensin Ⅱ receptor and prolactin have been shown to be present in ependymal cells.
, 百拇医药
In the present study, we demonstrated that NMDAR1 was expressed in ependymal cells of the third ventricle. Furthermore, evidences have shown that CSF contains glutamate and it may play an important role in transporting information between CSF and brain tissue[1]. Investigation demonstrated that metabotropic glutamate receptor subtype-1α was expressed strongly in the ependymal cells of caudal medulla oblongata and T2-3 segments of the spinal cord of rat[6]. The present investigation provided morphological evidence supporting that glutamate of CSF might regulate ependymal cells via NMDAR.
, http://www.100md.com
Although some parts of the thalamus show sex dimorphism and tanycyte, a kind of specialized ependymal cell, also revealed morphological changes during the estrous cycle in rat, the present study revealed no significant sex difference in the morphology and distribution of the NMDAR1-IR ependymal cells between the male and the female.
Acknowledgement:We thank the Hengsheng Limited Corporation of Electronics (Hangzhou) for great help and financial aid to this projection.
, http://www.100md.com
【Foundation item】This work is supported by the National Nature Science Foundation of China (39470309) and the Science Research Foundation of the Health Ministry of China(98-1-263)
【Biography】 ZHANG Lin, Birthdate:(1971—).Sex:male; Place of Birth: Changsha, Hunan Province; Degree:Bachelor of Medicine; Title:Assistant
Reference
[1]Davson H, Segal MB. Physiology of the CSF and Blood-brain Barriars[M]. Boca Roton: CRC Press, 1996:328-357.
, 百拇医药
[2]Brose N, Huntley GW, Stern-Bach Y, et al. Differential assembly of coexpressed glutamate receptor subunits in neurons of rat cerebral cortex[J]. J Biol Chem, 1994, 269(24):16780-16784.
[3]Shen L, Zhang L, Nan Y. NMDA receptor and development of the central nervous system[J]. Prog Physiol Sci, 1999, 30(1):60-62.
[4]Steinhauser C, Gallo V. News on glutamate receptors in glial cells[J]. Trends Neurosci, 1996,19(8):339-345.
, http://www.100md.com
[5]Parent A. Carpenter's Human Neuroanatomy[M]. 9th edition. London: Williams and Wilkins, 1996:218-224.
[6]Tang FR, Sim MK. Metabotropic glutamate receptor subtype-1 alpha (mGluR1 alpha) immunoreactivity in ependymal cells of the rat caudal medulla oblongata and spinal cord[J]. Neurosci Lett, 1997, 225(3):117-180.
【Received date】1999-01-12, http://www.100md.com
单位：
关键词：室管膜细胞；N-甲基-D-天冬氨酸受体；第三脑室；免疫组织化学
解剖学报000220
【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
, 百拇医药
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】 Objective To explore the expression of N-methyl-D-aspartate receptor subunit-1 (NMDAR1) in the ependymal cells of the third ventricle of rat. Methods The 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. Conclusion The present investigation provided the morphological evidence supporting that glutamate of CSF might regulate ependymal cells via NMDAR.
, 百拇医药
【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 NMDAR2A-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.
, 百拇医药
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.
, 百拇医药
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.
, 百拇医药
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.
, 百拇医药
图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
图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
图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
图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
, 百拇医药
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 NMDAR2A-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]. Ependymal cells are known to have functions of supporting, secreting and sensing, and may also play a role in the exchange between CSF and brain tissue[5]. Neurotransmitters and neuromodulators, such as vasoactive intestinal polypeptide, nitric oxide, the angiotensin Ⅱ receptor and prolactin have been shown to be present in ependymal cells.
, 百拇医药
In the present study, we demonstrated that NMDAR1 was expressed in ependymal cells of the third ventricle. Furthermore, evidences have shown that CSF contains glutamate and it may play an important role in transporting information between CSF and brain tissue[1]. Investigation demonstrated that metabotropic glutamate receptor subtype-1α was expressed strongly in the ependymal cells of caudal medulla oblongata and T2-3 segments of the spinal cord of rat[6]. The present investigation provided morphological evidence supporting that glutamate of CSF might regulate ependymal cells via NMDAR.
, http://www.100md.com
Although some parts of the thalamus show sex dimorphism and tanycyte, a kind of specialized ependymal cell, also revealed morphological changes during the estrous cycle in rat, the present study revealed no significant sex difference in the morphology and distribution of the NMDAR1-IR ependymal cells between the male and the female.
Acknowledgement:We thank the Hengsheng Limited Corporation of Electronics (Hangzhou) for great help and financial aid to this projection.
, http://www.100md.com
【Foundation item】This work is supported by the National Nature Science Foundation of China (39470309) and the Science Research Foundation of the Health Ministry of China(98-1-263)
【Biography】 ZHANG Lin, Birthdate:(1971—).Sex:male; Place of Birth: Changsha, Hunan Province; Degree:Bachelor of Medicine; Title:Assistant
Reference
[1]Davson H, Segal MB. Physiology of the CSF and Blood-brain Barriars[M]. Boca Roton: CRC Press, 1996:328-357.
, 百拇医药
[2]Brose N, Huntley GW, Stern-Bach Y, et al. Differential assembly of coexpressed glutamate receptor subunits in neurons of rat cerebral cortex[J]. J Biol Chem, 1994, 269(24):16780-16784.
[3]Shen L, Zhang L, Nan Y. NMDA receptor and development of the central nervous system[J]. Prog Physiol Sci, 1999, 30(1):60-62.
[4]Steinhauser C, Gallo V. News on glutamate receptors in glial cells[J]. Trends Neurosci, 1996,19(8):339-345.
, http://www.100md.com
[5]Parent A. Carpenter's Human Neuroanatomy[M]. 9th edition. London: Williams and Wilkins, 1996:218-224.
[6]Tang FR, Sim MK. Metabotropic glutamate receptor subtype-1 alpha (mGluR1 alpha) immunoreactivity in ependymal cells of the rat caudal medulla oblongata and spinal cord[J]. Neurosci Lett, 1997, 225(3):117-180.
【Received date】1999-01-12, http://www.100md.com