- Email: [email protected]
Choline acetyltransferase expressed by radial neuroglia cells in the development of elencephalon: A validated study
NEURAL REGENERATION RESEARCH Volume 2, Issue 2, February 2007 Online English edition of the Chinese language journal
Cite this article as: Neural Regen Res,2007,2(2),70-3
Basic Medicine
Choline acetyltransferase expressed by radial neuroglia cells in the development of telencephalon: A validated study* Li Zhou, Lingling Ding, Zhisuo Xiao, Yuanyuan Qin, Guibin Li Department of Histology and Embryology, Basic Medical College of Jilin University, Changchun
130021, Jilin Province, China
Abstract BACKGROUND: Cholinergic neuron directly participants in human motion, learning and memory and is a target cell for multiple degenerative diseases of central nervous system. OBJECTIVE: To investigate whether the mitotic cell is the radial glial cell expressing choline acetyltransferase (ChAT) in ventricle zone (VZ) of telencephalon and whether cholinergic neuron is derived from radial glial cell in ventricle zone of telencephalon. DESIGN: Observational study. SETTING: Department of Histology and Embryology, Basic Medical College of Jilin University. MATERIALS: Nine healthy Wistar rats included 6 females and 3 male. Male and female rats were mated routinely and the day when spermatozoa or vaginal plug were found was regarded as embryonic 0 (E0). Primary monoclonal antibodies ChAT and vimentin were provided respectively by Wuhan Boster Company, and Biogenex Company, USA. METHODS: The experiment was carried out in the Laboratory of Cell Culture and Immunohistochemistry, Department of Histology and Embryology from march 2002 to January 2003. Firstly, fluorescence-activated cell sorting (FACS) was used to confirm the time of generation of cholinergic neuron; secondly, telencephalons of rats at embryonic 14 days (E14) were performed coronary sections, then immunohistochemistry double staining for vimentin (a protein marker of radial neuroglia cell) and ChAT (a protein marker of cholinergic neuron) were used to test whether ChAT was expressed in the radial neuroglia cells. Fluorescence-activated cell numbers of ChAT in telencephalon; MAIN OUTCOME MEASURES: results of immunohistochemistry double staining. RESULTS: It is confirmed using by flow cytometer that embryogenesis time of cholinergic neuron was at E12 and shown the population of cells in VZ of dorsal telencephalon of E14 rat co-expressed vimentin and ChAT through immunohistochemistry double staining. A lot of vimentin-positive cells and ChAT-positive cells respectively were observed in VZ of lateral ganglionic eminence. CONCLUSION: Cholinergic neuron in cerebral cortex is derived from radial glial cells in VZ of dorsal telencephalon; meanwhile, cholinergic neuron of striatum is derived from radial glial cells in VZ of lateral ganglionic eminence. Key Words: telencephalon; choline o-acetyltransferase; vimentin; immunohistochemistry
INTRODUCTION With the further study, it was found that radial glial cells in
Li Zhou , Doctor, Professor, Department of Histology and Embryology, Basic Medical College of Jilin University, Changchun 130021, Jilin Province, China Supported by: the Scientific Research Foundation of the Higher Education Institutions, No. 20030183048* Zhou L, Ding LL, Xiao ZS, Qin YY, Li GB. Choline acetyltransferase expressed by radial neuroglia cells in development of the telencephalon: A validated study.Neural Regen Res 2007;2(2):70-3 www.sjzsyj.com/Journal/ 0702/07-02-70.html
relationship between embryogenesis of cholinergic neuron and differentiation of neural stem cells.
ventricular zone could generate cortical pyramidal neurons in developing telencephalon. And then, the researchers paid more and more attention to property of neural progenitor of radial glial cell. The purpose of this experiment is to investigate whether cholinergic neuron is derived from radial glial cell in developing telencephalon, and discuss the
MATERIALS AND METHODS Materials The experiment was carried out in the Laboratory of Cell Culture and Immunohistochemistry, Department of Histology and Embryology from March 2002 to January 2003. Nine
Received:2006-10-28;Accepted:2006-12-20 (06-S-12-1192/H) Corresponding author: Li Zhou, Doctor, Professor, Department of Histology and Embryology, Basic Medical College of E-mail:[email protected] Jilin University, Changchun 130021, Jilin Province, China
70
Zhou L, et al. / Neural Regeneration Research,2007,2(2):70-3
endogenous peroxydase was blocked with 0.03 volume fraction of methanol-hydrogen peroxide at room temperature for 30 minutes. Additionally, the specimen were blocked with non-immune goat serum, incubated with rabbit-anti-rat monoclonal antibody ChAT (1:200) and mouse-anti-rat monoclonal antibody vimentin (1:200) at 4 overnight, maintained in secondary biotinylation-IgG antibody at room temperature for 10 minutes, maintained in streptomycete-avidin-peroxydase at room temperature for 10 minutes, stained with DAB and AEC, and re-stained with haematoxylin and methyl green. Except for adding primary antibody, the sections were washed with PBS for 5 minutes per time for three times in total before each step. During the period of double staining, double blocking agent was added after coloring the first antibody and then the specimen were incubated with the second antibody and treated with the operations mentioned above. Finally, the sections were mounted, and observed with Olympus microscope and photographed with digital camera. Statistical analysis: SPSS 11.0 software was used by the first author in this study, and data were checked with t test.
RESULTS Detection of flow cytometry Flow cytometer was used to determine embryogenesis time of cholinergic neuron in the developing telencephalon through collecting fluorescence-activated ChAT cells in telencephalon of E10, E11 and E12 rats. It was shown that there were no significant differences of percentage of ChAT positive cells between E10 and E11 rats and negative control rats; however, there were significant differences between E12 rats and negative control rats (P < 0.05). Percentage of fluorescence-positive ChAT cells was 22.49% (Figure 1). This suggested that embryogenesis time of cholinergic neuron was at embryonic 12 days; therefore, E14 rats were used in Immunohistochemistry. 200 160
Counts
healthy white Wistar rats including 6 females and 3 male of clean grade were provided by our Experimental Animal Center [certification: SCXK (Ji) 2003-0001]. The body weight of female is (220 10) g and the one of male was (250 10) g. Then, male and female rats were mated routinely. On the next morning, vaginal smear was tested under microscope, and the day when spermatozoa or vaginal plug were found was regarded as embryonic 0 (E0). Gestational age was recorded and rats were fed in a barrier environment. Primary monoclonal antibodies ChAT and vimentin respectively were provided by Wuhan Boster Company and Biogenex Company, USA. TRITC-IgG antibody was provided by Beijing Zhongshan Biological Reagent Company. Methods FACS: E1012 rats were taken from an anesthetized pregnant rat with aether, based on the modified method of Ignacio Lopez-Coviella in 2005[1]. Meninges were stripped, and washed with Hank's solution. Then, the tissues were added with 1.25 g/L trypsin, incubated at 37 for 30 minutes, added with 0.1 g/L DNA enzyme, incubated at 37 for 15 minutes, blown and hit with pipette, filtered with nylon net, and centrifuged at the speed of 1 000 r/min for 8 minutes to make cell suspension. Congo blue staining showed that living cells were accounted for 98% and the concentration was 5 109 L 1. 0.5 mL cell suspension was respectively added in 10 centrifuge tubes. Among them, there were three negative control tubes. 5 L primary monoclonal antibody ChAT(1:100) was dripped into other 7 tubes. Then, the mixture was maintained in iced bath for 8 minutes, washed with phosphate buffer solution (PBS) for twice, added with secondary TRITC-IgG antibody (1:100), maintained in iced bath for 45 minutes, and washed with PBS for twice. Fluorescence-activated cell sorting (FACS) of flow cytometer and its matched software were used to analyze 2-dimensional spot map based on forward and lateral scattering light; meanwhile, cell groups were obtained based on doorframe. The fluorescence-positive expression rate was limited less than 2% to regulate voltage of electron multiplier. And then, fluorescence-expressed percentage of samples was tested and the data were treated statistically. Immunohistochemistry: 14 days of pregnant rats were perfused with 0.1 volume fraction of formaldehyde solution, and brain of E14 rats were fixed with the same solution at 4 for 12 hours, treated with upward gradient alcohol dehydration, clearing of xylene, paraffin embedding and cerebral coronary sections with the thickness of 20 m. Continuously, the sections were baked at 58 for 2 hours and treated with deparaffinage of xylene and downward gradient alcohol dehydration. Antigen was repaired under hypertensive condition and biotin blocking agent was used to block endogenous biotin. And then, sections were dipped in 1 g/L TritonX-100 at room temperature for 25 minutes and
120 80 M1 40 0 100
101
102 FL2-H
103
104
Figure 1 Results of telencephalon in embryonic 12-day rats detecting with flow cytometry
71
Zhou L, et al. / Neural Regeneration Research,2007,2(2):70-3
Immunohistochemistry double staining The population of ChAT-positive cells was observed in VZ of E14 dorsal telencephalon, which was anlage for development of neocortex, by immunohistochemical staining in brain of coronary sections. Vimentin-positive cells widely existed in the same site of adjacent sections (Figures 2a, b). To confirm that the mitotic cells which expressed ChAT in dorsal telencephalon were radial glial cells found by U.B.Schrmbra in 1989 immunohistochemistry double staining was used. The result showed that population of vimentin-positive cells co-expressed ChAT in VZ of dorsal telencephalon of E14 rats (Figure 2c), the radial glial cells underwent dissymmetry mitosis, produced neuroblasts which had cholinergic phenotype, and migrated to cortical plate along their long processes. In addition to, a lot of vimentin-positive cells were seen in VZ of lateral ganglionic eminence (Figure 2d); while, the population of ChAT-positive cell were also seen in the same site (Figure 2e). The region was an anlage for development of striatum, suggesting that cholinergic neuron in striatum was derived from radial glial cells.
glia cells in VZ, while non- pyramidal neurons were generated at other sites[9-12]. Otherwise, results in our experiment suggested that radial glial cells could not only generate pyramidal -neurons but also non- pyramidal neurons in cerebral cortex.
LV
a: Immunohistochemical staining of vimentin cells in dorsal telencephalon: Positive reaction and brown in cerebral ventricular zone (DAB staining, Methyl 400) green re-staining,
LV
b: Immunohistochemical staining of choline acetyltransferase (ChAT) cells in dorsal telencephalon: Positive reaction and dark blue in cerebral ventricular zone (NBT staining, 400)
LV
DISCUSSION Neocortical pyramidal neurons are generated in VZ and SVZ of dorsal telencephalon. About 96%98% cells in the germinal zone are radial glia cells which are derived from neuroepithelium in E1218 rats. These cells can generate neuroblasts through dissymmetric mitosis and the neuroblasts migrates radially to neocortex along processes of radial glial cell[2,3]. Interneuron of neocortex is generated in medial ganglion eminence of ventral telencephalon and migrates tangentially to cerebral cortex. Generation of neurons in brain prior to neuroglial cell[4-6]. The striatum is the largest part in basal ganglia and contains two main kinds of cholinergic neurons, i.e., interneurons in local circulation of striatum and projection neurons of basal forebrain. Although the two kinds of cholinergic neurons are distributed in various neuronuclei and have different functions, both of them are derived from ventral telencephalon. The migrations of model remain radial migration of projection neuron to nucleus basalis tangential migration of interneuron to striatum[7,8]. In this experiment, the population of double-positive cells for vimentin and ChAT appear to be in VZ of dorsal telencephalon and lateral ganglionic eminence in brain coronary sections of E14 rats, suggesting that cholinergic neuron in cerebral cortex was derived from the population of radial glia cells in dorsal telencephalon and cholinergic neuron in striatum was derived from radial glia cells in VZ of lateral ganglionic eminence. Cholinergic neuron was distributed in the IIV layers in cerebral cortex and it was a non- pyramidal neuron. However, previous viewpoints demonstrated that pyramidal neurons in cerebral cortex were derived from radial
72
LV
c: Compound expression of Vimentin and ChAT in a region of cerebral ventricular zone: Dark violet ( 400)
d: Vimentin-positive reaction in cerebral ventricular zone of lateral ganglionic eminence (DAB staining, haematine re-staining, 400)
LV
e: ChAT-positive reaction in cerebral ventricular zone of lateral ganglionic eminence (AEC staining, haematine re-staining,
400)
LV: Lateral cerebral ventricle
Figure 2 Immunohistochemical staining results of coronary sections of telencephalon in embryonic 14-day rats
It is suggested that the cell fate of the population of radial glial cell has been decided in the asymmetrical cell division, and then synthesizes into neurotransmitter acetylcholine. Radial glial cells started to express the protein marker of cholinergic neuron before the generation of neuroblasts. That was to say, structure-program pattern of cerebral cortex widely existed in VZ before the migration of neurons along
Zhou L, et al. / Neural Regeneration Research,2007,2(2):70-3
processes of radial glial cells to cortical plate[13]. On the other hand, the results brought a challenge to traditional theory of neural stem cells. In a fact, embryonic neural stem cells not only contain neuroepithelial cells before generation of neurons, also contain radial glial cell subsets whose fate has been decided yet. After radial glial cells produce neuroblasts during the embryonic period they differentiate into B type astrocytes, which are real neural stem cells in adult subependyma and express glial fibrillary acidic protein. When neuroblasts are lack in subependyma, the astrocytes can rapidly generate C type neuroblasts[14-16]. This procedure indicates the correlation between embryo and adult neural stem cells. In 2001, Arturo Alvarez-Buylla suggests a hypothesis of neural stem cell lineage. In this hypothesis, embryonic neuroepithelial cells, radial glial cells and astrocytes in adult subependyma are regarded as the neural stem cell lineage[17]. Especially, although radial glial cells and astrocytes in of adult subependyma have a potency of self-renew and continuous differentiation, their cell fate has restricted. Whether they can change their previously cell fate after changing their growth environment is still unclear based on the present reports[18-20]. However, understanding differentiation of mechanism of functional neurons during the embryonic period is very important as if changing cell fate of the neural stem cells. What is the significance of radial glial cells generating neuroblasts and synthesizing acetylcholine during the dissymmetry mitosis is still not known. Some reports demonstrate that long processes of radial glial cell touches with migrating neuroblasts and provides differentiated molecules so as to promote regional specialization[21,22]. For example, radial glial cell from lateral ganglionic eminence of telencephalon can product retinoic acid, which can increase numbers of DARPP-32 positive projection neurons in striatum[23]. Otherwise, migrating neuroblasts also can express neurotransmitter receptor, such as glutamic acid receptor and -amino-butyric acid receptor. The evidences indicate that blocking GABA receptor can promote the migration of neuroblasts to cortical plate, but blocking GABAA/C receptor can completely block the migration[24].
REFERENCES Lopez-Coviella I, Follettie MT, Mellott TJ, et al.Bone 1. morphogenetic protein 9 induces the transcriptome of basal forebrain cholinergic neurons. Proc Natl Acad Sci U S A 2005;102(19):6984-9 2. Kriegstein AR. Constructing circuits: neurogenesis and migration in the developing neocortex. Epilepsia 2005;46 Suppl 7:15-21
3. Kanatani S, Tabata H, Nakajima K. Neuronal migration in cortical development. J Child Neurol 2005;20(4):274-9 4. Noctor SC, Flint AC, Weissman TA, et al.Neurons derived from radial glial cells establish radial units in neocortex. Nature 2001;409(6821):714-20 5. Gotz M, Huttner WB. The cell biology of neurogenesis. Nat Rev Mol Cell Biol 2005;6(10):777-88 6. Anthony TE, Klein C, Fishell G, et al.Radial glia serve as neuronal progenitors in all regions of the central nervous system. Neuron 2004;41(6):881-90 7. Marin O, Anderson SA, Rubenstein JL. Origin and molecular specification of striatal interneurons. J Neurosci 2000;20(16):6063-76 8. Zhuravin IA, Tumanova NL, Ozirskaya EV, et al.Formation of the structural and ultrastructural organization of the striatum in early postnatal ontogenesis of rats in altered conditions of embryonic development. Neurosci Behav Physiol 2006;36(5):473-8 9. Lopez-Coviella I, Berse B, Thies RS, et al.Upregulation of acetylcholine synthesis by bone morphogenetic protein 9 in a murine septal cell line. J Physiol Paris 2002;96(1-2):53-9 10. Tseng HC, Ruegg SJ, Maronski M, et al.Injuring neurons induces neuronal differentiation in a population of hippocampal precursor cells in culture. Neurobiol Dis 2006;22(1):88-97 11. Hevner RF. From radial glia to pyramidal-projection neuron: transcription factor cascades in cerebral cortex development. Mol Neurobiol 2006;33(1):33-50 12. Anthony TE, Klein C, Fishell G, et al.Radial glia serve as neuronal progenitors in all regions of the central nervous system. Neuron 2004;41(6):881-90 13. Rakic P. Specification of cerebral cortical areas. Science 1988;241(4862):170-6 14. Parnavelas JG, Nadarajah B. Radial glial cells. are they really glia? Neuron 2001;31(6):881-4 15. Merkle FT, Tramontin AD, Garcia-Verdugo JM, et al.Radial glia give rise to adult neural stem cells in the subventricular zone. Proc Natl Acad Sci U S A 2004;101(50):17528-32 16. Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A. Regeneration of a germinal layer in the adult mammalian brain. Proc Natl Acad Sci U S A 1999;96(20):11619-24 17. Alvarez-Buylla A, Garcia-Verdugo JM, Tramontin AD. A unified hypothesis on the lineage of neural stem cells. Nat Rev Neurosci 2001;2(4):287-93 18. Wichterle H, Lieberam I, Porter JA, et al. Directed differentiation of embryonic stem cells into motor neurons. Cell 2002;110(3):385-97 19. Trounson A. The production and directed differentiation of human embryonic stem cells. Endocr Rev 2006;27(2):208-19 20. Emsley JG, Mitchell BD, Magavi SS, et al.The repair of complex neuronal circuitry by transplanted and endogenous precursors. NeuroRx 2004;1(4):452-71 21. Sutor B, Hagerty T. Involvement of gap junctions in the development of the neocortex. Biochim Biophys Acta 2005;1719(1-2):59-68 22. Ever L, Gaiano N. Radial 'glial' progenitors: neurogenesis and signaling. Curr Opin Neurobiol 2005;15(1):29-33 23. Campbell K. Signaling to and from radial glia. Glia 2003;43(1):44-6 24. Sobeih MM, Corfas G. Extracellular factors that regulate neuronal migration in the central nervous system. Int J Dev Neurosci 2002;20(3-5):349-57
(Edited by Wang X/Ji H/Wang L)
73
Cite this article as: Neural Regen Res,2007,2(2),70-3
Basic Medicine
Choline acetyltransferase expressed by radial neuroglia cells in the development of telencephalon: A validated study* Li Zhou, Lingling Ding, Zhisuo Xiao, Yuanyuan Qin, Guibin Li Department of Histology and Embryology, Basic Medical College of Jilin University, Changchun
130021, Jilin Province, China
Abstract BACKGROUND: Cholinergic neuron directly participants in human motion, learning and memory and is a target cell for multiple degenerative diseases of central nervous system. OBJECTIVE: To investigate whether the mitotic cell is the radial glial cell expressing choline acetyltransferase (ChAT) in ventricle zone (VZ) of telencephalon and whether cholinergic neuron is derived from radial glial cell in ventricle zone of telencephalon. DESIGN: Observational study. SETTING: Department of Histology and Embryology, Basic Medical College of Jilin University. MATERIALS: Nine healthy Wistar rats included 6 females and 3 male. Male and female rats were mated routinely and the day when spermatozoa or vaginal plug were found was regarded as embryonic 0 (E0). Primary monoclonal antibodies ChAT and vimentin were provided respectively by Wuhan Boster Company, and Biogenex Company, USA. METHODS: The experiment was carried out in the Laboratory of Cell Culture and Immunohistochemistry, Department of Histology and Embryology from march 2002 to January 2003. Firstly, fluorescence-activated cell sorting (FACS) was used to confirm the time of generation of cholinergic neuron; secondly, telencephalons of rats at embryonic 14 days (E14) were performed coronary sections, then immunohistochemistry double staining for vimentin (a protein marker of radial neuroglia cell) and ChAT (a protein marker of cholinergic neuron) were used to test whether ChAT was expressed in the radial neuroglia cells. Fluorescence-activated cell numbers of ChAT in telencephalon; MAIN OUTCOME MEASURES: results of immunohistochemistry double staining. RESULTS: It is confirmed using by flow cytometer that embryogenesis time of cholinergic neuron was at E12 and shown the population of cells in VZ of dorsal telencephalon of E14 rat co-expressed vimentin and ChAT through immunohistochemistry double staining. A lot of vimentin-positive cells and ChAT-positive cells respectively were observed in VZ of lateral ganglionic eminence. CONCLUSION: Cholinergic neuron in cerebral cortex is derived from radial glial cells in VZ of dorsal telencephalon; meanwhile, cholinergic neuron of striatum is derived from radial glial cells in VZ of lateral ganglionic eminence. Key Words: telencephalon; choline o-acetyltransferase; vimentin; immunohistochemistry
INTRODUCTION With the further study, it was found that radial glial cells in
Li Zhou , Doctor, Professor, Department of Histology and Embryology, Basic Medical College of Jilin University, Changchun 130021, Jilin Province, China Supported by: the Scientific Research Foundation of the Higher Education Institutions, No. 20030183048* Zhou L, Ding LL, Xiao ZS, Qin YY, Li GB. Choline acetyltransferase expressed by radial neuroglia cells in development of the telencephalon: A validated study.Neural Regen Res 2007;2(2):70-3 www.sjzsyj.com/Journal/ 0702/07-02-70.html
relationship between embryogenesis of cholinergic neuron and differentiation of neural stem cells.
ventricular zone could generate cortical pyramidal neurons in developing telencephalon. And then, the researchers paid more and more attention to property of neural progenitor of radial glial cell. The purpose of this experiment is to investigate whether cholinergic neuron is derived from radial glial cell in developing telencephalon, and discuss the
MATERIALS AND METHODS Materials The experiment was carried out in the Laboratory of Cell Culture and Immunohistochemistry, Department of Histology and Embryology from March 2002 to January 2003. Nine
Received:2006-10-28;Accepted:2006-12-20 (06-S-12-1192/H) Corresponding author: Li Zhou, Doctor, Professor, Department of Histology and Embryology, Basic Medical College of E-mail:[email protected] Jilin University, Changchun 130021, Jilin Province, China
70
Zhou L, et al. / Neural Regeneration Research,2007,2(2):70-3
endogenous peroxydase was blocked with 0.03 volume fraction of methanol-hydrogen peroxide at room temperature for 30 minutes. Additionally, the specimen were blocked with non-immune goat serum, incubated with rabbit-anti-rat monoclonal antibody ChAT (1:200) and mouse-anti-rat monoclonal antibody vimentin (1:200) at 4 overnight, maintained in secondary biotinylation-IgG antibody at room temperature for 10 minutes, maintained in streptomycete-avidin-peroxydase at room temperature for 10 minutes, stained with DAB and AEC, and re-stained with haematoxylin and methyl green. Except for adding primary antibody, the sections were washed with PBS for 5 minutes per time for three times in total before each step. During the period of double staining, double blocking agent was added after coloring the first antibody and then the specimen were incubated with the second antibody and treated with the operations mentioned above. Finally, the sections were mounted, and observed with Olympus microscope and photographed with digital camera. Statistical analysis: SPSS 11.0 software was used by the first author in this study, and data were checked with t test.
RESULTS Detection of flow cytometry Flow cytometer was used to determine embryogenesis time of cholinergic neuron in the developing telencephalon through collecting fluorescence-activated ChAT cells in telencephalon of E10, E11 and E12 rats. It was shown that there were no significant differences of percentage of ChAT positive cells between E10 and E11 rats and negative control rats; however, there were significant differences between E12 rats and negative control rats (P < 0.05). Percentage of fluorescence-positive ChAT cells was 22.49% (Figure 1). This suggested that embryogenesis time of cholinergic neuron was at embryonic 12 days; therefore, E14 rats were used in Immunohistochemistry. 200 160
Counts
healthy white Wistar rats including 6 females and 3 male of clean grade were provided by our Experimental Animal Center [certification: SCXK (Ji) 2003-0001]. The body weight of female is (220 10) g and the one of male was (250 10) g. Then, male and female rats were mated routinely. On the next morning, vaginal smear was tested under microscope, and the day when spermatozoa or vaginal plug were found was regarded as embryonic 0 (E0). Gestational age was recorded and rats were fed in a barrier environment. Primary monoclonal antibodies ChAT and vimentin respectively were provided by Wuhan Boster Company and Biogenex Company, USA. TRITC-IgG antibody was provided by Beijing Zhongshan Biological Reagent Company. Methods FACS: E1012 rats were taken from an anesthetized pregnant rat with aether, based on the modified method of Ignacio Lopez-Coviella in 2005[1]. Meninges were stripped, and washed with Hank's solution. Then, the tissues were added with 1.25 g/L trypsin, incubated at 37 for 30 minutes, added with 0.1 g/L DNA enzyme, incubated at 37 for 15 minutes, blown and hit with pipette, filtered with nylon net, and centrifuged at the speed of 1 000 r/min for 8 minutes to make cell suspension. Congo blue staining showed that living cells were accounted for 98% and the concentration was 5 109 L 1. 0.5 mL cell suspension was respectively added in 10 centrifuge tubes. Among them, there were three negative control tubes. 5 L primary monoclonal antibody ChAT(1:100) was dripped into other 7 tubes. Then, the mixture was maintained in iced bath for 8 minutes, washed with phosphate buffer solution (PBS) for twice, added with secondary TRITC-IgG antibody (1:100), maintained in iced bath for 45 minutes, and washed with PBS for twice. Fluorescence-activated cell sorting (FACS) of flow cytometer and its matched software were used to analyze 2-dimensional spot map based on forward and lateral scattering light; meanwhile, cell groups were obtained based on doorframe. The fluorescence-positive expression rate was limited less than 2% to regulate voltage of electron multiplier. And then, fluorescence-expressed percentage of samples was tested and the data were treated statistically. Immunohistochemistry: 14 days of pregnant rats were perfused with 0.1 volume fraction of formaldehyde solution, and brain of E14 rats were fixed with the same solution at 4 for 12 hours, treated with upward gradient alcohol dehydration, clearing of xylene, paraffin embedding and cerebral coronary sections with the thickness of 20 m. Continuously, the sections were baked at 58 for 2 hours and treated with deparaffinage of xylene and downward gradient alcohol dehydration. Antigen was repaired under hypertensive condition and biotin blocking agent was used to block endogenous biotin. And then, sections were dipped in 1 g/L TritonX-100 at room temperature for 25 minutes and
120 80 M1 40 0 100
101
102 FL2-H
103
104
Figure 1 Results of telencephalon in embryonic 12-day rats detecting with flow cytometry
71
Zhou L, et al. / Neural Regeneration Research,2007,2(2):70-3
Immunohistochemistry double staining The population of ChAT-positive cells was observed in VZ of E14 dorsal telencephalon, which was anlage for development of neocortex, by immunohistochemical staining in brain of coronary sections. Vimentin-positive cells widely existed in the same site of adjacent sections (Figures 2a, b). To confirm that the mitotic cells which expressed ChAT in dorsal telencephalon were radial glial cells found by U.B.Schrmbra in 1989 immunohistochemistry double staining was used. The result showed that population of vimentin-positive cells co-expressed ChAT in VZ of dorsal telencephalon of E14 rats (Figure 2c), the radial glial cells underwent dissymmetry mitosis, produced neuroblasts which had cholinergic phenotype, and migrated to cortical plate along their long processes. In addition to, a lot of vimentin-positive cells were seen in VZ of lateral ganglionic eminence (Figure 2d); while, the population of ChAT-positive cell were also seen in the same site (Figure 2e). The region was an anlage for development of striatum, suggesting that cholinergic neuron in striatum was derived from radial glial cells.
glia cells in VZ, while non- pyramidal neurons were generated at other sites[9-12]. Otherwise, results in our experiment suggested that radial glial cells could not only generate pyramidal -neurons but also non- pyramidal neurons in cerebral cortex.
LV
a: Immunohistochemical staining of vimentin cells in dorsal telencephalon: Positive reaction and brown in cerebral ventricular zone (DAB staining, Methyl 400) green re-staining,
LV
b: Immunohistochemical staining of choline acetyltransferase (ChAT) cells in dorsal telencephalon: Positive reaction and dark blue in cerebral ventricular zone (NBT staining, 400)
LV
DISCUSSION Neocortical pyramidal neurons are generated in VZ and SVZ of dorsal telencephalon. About 96%98% cells in the germinal zone are radial glia cells which are derived from neuroepithelium in E1218 rats. These cells can generate neuroblasts through dissymmetric mitosis and the neuroblasts migrates radially to neocortex along processes of radial glial cell[2,3]. Interneuron of neocortex is generated in medial ganglion eminence of ventral telencephalon and migrates tangentially to cerebral cortex. Generation of neurons in brain prior to neuroglial cell[4-6]. The striatum is the largest part in basal ganglia and contains two main kinds of cholinergic neurons, i.e., interneurons in local circulation of striatum and projection neurons of basal forebrain. Although the two kinds of cholinergic neurons are distributed in various neuronuclei and have different functions, both of them are derived from ventral telencephalon. The migrations of model remain radial migration of projection neuron to nucleus basalis tangential migration of interneuron to striatum[7,8]. In this experiment, the population of double-positive cells for vimentin and ChAT appear to be in VZ of dorsal telencephalon and lateral ganglionic eminence in brain coronary sections of E14 rats, suggesting that cholinergic neuron in cerebral cortex was derived from the population of radial glia cells in dorsal telencephalon and cholinergic neuron in striatum was derived from radial glia cells in VZ of lateral ganglionic eminence. Cholinergic neuron was distributed in the IIV layers in cerebral cortex and it was a non- pyramidal neuron. However, previous viewpoints demonstrated that pyramidal neurons in cerebral cortex were derived from radial
72
LV
c: Compound expression of Vimentin and ChAT in a region of cerebral ventricular zone: Dark violet ( 400)
d: Vimentin-positive reaction in cerebral ventricular zone of lateral ganglionic eminence (DAB staining, haematine re-staining, 400)
LV
e: ChAT-positive reaction in cerebral ventricular zone of lateral ganglionic eminence (AEC staining, haematine re-staining,
400)
LV: Lateral cerebral ventricle
Figure 2 Immunohistochemical staining results of coronary sections of telencephalon in embryonic 14-day rats
It is suggested that the cell fate of the population of radial glial cell has been decided in the asymmetrical cell division, and then synthesizes into neurotransmitter acetylcholine. Radial glial cells started to express the protein marker of cholinergic neuron before the generation of neuroblasts. That was to say, structure-program pattern of cerebral cortex widely existed in VZ before the migration of neurons along
Zhou L, et al. / Neural Regeneration Research,2007,2(2):70-3
processes of radial glial cells to cortical plate[13]. On the other hand, the results brought a challenge to traditional theory of neural stem cells. In a fact, embryonic neural stem cells not only contain neuroepithelial cells before generation of neurons, also contain radial glial cell subsets whose fate has been decided yet. After radial glial cells produce neuroblasts during the embryonic period they differentiate into B type astrocytes, which are real neural stem cells in adult subependyma and express glial fibrillary acidic protein. When neuroblasts are lack in subependyma, the astrocytes can rapidly generate C type neuroblasts[14-16]. This procedure indicates the correlation between embryo and adult neural stem cells. In 2001, Arturo Alvarez-Buylla suggests a hypothesis of neural stem cell lineage. In this hypothesis, embryonic neuroepithelial cells, radial glial cells and astrocytes in adult subependyma are regarded as the neural stem cell lineage[17]. Especially, although radial glial cells and astrocytes in of adult subependyma have a potency of self-renew and continuous differentiation, their cell fate has restricted. Whether they can change their previously cell fate after changing their growth environment is still unclear based on the present reports[18-20]. However, understanding differentiation of mechanism of functional neurons during the embryonic period is very important as if changing cell fate of the neural stem cells. What is the significance of radial glial cells generating neuroblasts and synthesizing acetylcholine during the dissymmetry mitosis is still not known. Some reports demonstrate that long processes of radial glial cell touches with migrating neuroblasts and provides differentiated molecules so as to promote regional specialization[21,22]. For example, radial glial cell from lateral ganglionic eminence of telencephalon can product retinoic acid, which can increase numbers of DARPP-32 positive projection neurons in striatum[23]. Otherwise, migrating neuroblasts also can express neurotransmitter receptor, such as glutamic acid receptor and -amino-butyric acid receptor. The evidences indicate that blocking GABA receptor can promote the migration of neuroblasts to cortical plate, but blocking GABAA/C receptor can completely block the migration[24].
REFERENCES Lopez-Coviella I, Follettie MT, Mellott TJ, et al.Bone 1. morphogenetic protein 9 induces the transcriptome of basal forebrain cholinergic neurons. Proc Natl Acad Sci U S A 2005;102(19):6984-9 2. Kriegstein AR. Constructing circuits: neurogenesis and migration in the developing neocortex. Epilepsia 2005;46 Suppl 7:15-21
3. Kanatani S, Tabata H, Nakajima K. Neuronal migration in cortical development. J Child Neurol 2005;20(4):274-9 4. Noctor SC, Flint AC, Weissman TA, et al.Neurons derived from radial glial cells establish radial units in neocortex. Nature 2001;409(6821):714-20 5. Gotz M, Huttner WB. The cell biology of neurogenesis. Nat Rev Mol Cell Biol 2005;6(10):777-88 6. Anthony TE, Klein C, Fishell G, et al.Radial glia serve as neuronal progenitors in all regions of the central nervous system. Neuron 2004;41(6):881-90 7. Marin O, Anderson SA, Rubenstein JL. Origin and molecular specification of striatal interneurons. J Neurosci 2000;20(16):6063-76 8. Zhuravin IA, Tumanova NL, Ozirskaya EV, et al.Formation of the structural and ultrastructural organization of the striatum in early postnatal ontogenesis of rats in altered conditions of embryonic development. Neurosci Behav Physiol 2006;36(5):473-8 9. Lopez-Coviella I, Berse B, Thies RS, et al.Upregulation of acetylcholine synthesis by bone morphogenetic protein 9 in a murine septal cell line. J Physiol Paris 2002;96(1-2):53-9 10. Tseng HC, Ruegg SJ, Maronski M, et al.Injuring neurons induces neuronal differentiation in a population of hippocampal precursor cells in culture. Neurobiol Dis 2006;22(1):88-97 11. Hevner RF. From radial glia to pyramidal-projection neuron: transcription factor cascades in cerebral cortex development. Mol Neurobiol 2006;33(1):33-50 12. Anthony TE, Klein C, Fishell G, et al.Radial glia serve as neuronal progenitors in all regions of the central nervous system. Neuron 2004;41(6):881-90 13. Rakic P. Specification of cerebral cortical areas. Science 1988;241(4862):170-6 14. Parnavelas JG, Nadarajah B. Radial glial cells. are they really glia? Neuron 2001;31(6):881-4 15. Merkle FT, Tramontin AD, Garcia-Verdugo JM, et al.Radial glia give rise to adult neural stem cells in the subventricular zone. Proc Natl Acad Sci U S A 2004;101(50):17528-32 16. Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A. Regeneration of a germinal layer in the adult mammalian brain. Proc Natl Acad Sci U S A 1999;96(20):11619-24 17. Alvarez-Buylla A, Garcia-Verdugo JM, Tramontin AD. A unified hypothesis on the lineage of neural stem cells. Nat Rev Neurosci 2001;2(4):287-93 18. Wichterle H, Lieberam I, Porter JA, et al. Directed differentiation of embryonic stem cells into motor neurons. Cell 2002;110(3):385-97 19. Trounson A. The production and directed differentiation of human embryonic stem cells. Endocr Rev 2006;27(2):208-19 20. Emsley JG, Mitchell BD, Magavi SS, et al.The repair of complex neuronal circuitry by transplanted and endogenous precursors. NeuroRx 2004;1(4):452-71 21. Sutor B, Hagerty T. Involvement of gap junctions in the development of the neocortex. Biochim Biophys Acta 2005;1719(1-2):59-68 22. Ever L, Gaiano N. Radial 'glial' progenitors: neurogenesis and signaling. Curr Opin Neurobiol 2005;15(1):29-33 23. Campbell K. Signaling to and from radial glia. Glia 2003;43(1):44-6 24. Sobeih MM, Corfas G. Extracellular factors that regulate neuronal migration in the central nervous system. Int J Dev Neurosci 2002;20(3-5):349-57
(Edited by Wang X/Ji H/Wang L)
73