成年樱花钩吻鲑大脑的神经发生

doi:10.3969/j.issn.1673-5374.2013.01.002

摘要/Abstract

摘要：

我们应用免疫组化染色和组织化学染色的方法检测几种神经化学标志物在樱花钩吻鲑生后不同发育阶段的脑内分布。结果显示，鲑鱼大脑室周细胞表现出神经元和胶质细胞，包括放射状胶质细胞的形态，并包含多种神经化学物质。大脑脚背盖背内侧、髓质和脊髓中央灰质层、间脑室周围区的细胞增殖区出现酪氨酸羟化酶、γ氨基丁酸和NADPH黄递酶免疫阳性细胞。Pax6免疫定位检测揭示鲑鱼在生后不同发育阶段脑结构变化，这些变化也得到酪氨酸羟化酶和 γ氨基丁酸免疫组化证实。

关键词: 神经再生, 神经发生, 硬骨鱼类, 成体神经发生, 神经递质信号, 迁移, 酪氨酸羟化酶, γ氨基丁酸, 发育, Pax6, NADPH黄递酶, 增殖, 基金资助文章, 图片文章

Abstract:

We investigated the distribution of gamma aminobutyric acid, tyrosine hydroxylase and nitric oxide-producing elements in a cherry salmon Oncorhynchus masou brain at various stages of postnatal ontogenesis by immunohistochemical staining and histochemical staining. The periventricular region cells exhibited the morphology of neurons and glia including radial glia-like cells and contained several neurochemical substances. Heterogeneous populations of tyrosine hydroxylase-, gamma aminobutyric acid-immunoreactive, аs well as nicotinamide adenine dinucleotide phosphate diaphorase-positive cells were observed in proliferating cell nuclear antigen-immunoreactive proliferative zones in periventricular area of diencephalon, central grey layer of dorsomedial tegmentum, medulla and spinal cord. Immunolocalization of Pax6 in the cherry salmon brain revealed a neuromeric construction of the brain at various stages of postnatal ontogenesis, and this was confirmed by tyrosine hydroxylase and gamma aminobutyric acid labeling.

Key words: neural regeneration, neurogenesis, teleostei, adult neurogenesis, neurotransmitter signaling, migration, tyrosine hydroxylase, gamma aminobutyric acid, development, Pax6, NADPH- diaphorase, proliferation, grant-supported paper, neuroregeneration

. 成年樱花钩吻鲑大脑的神经发生[J]. 中国神经再生研究(英文版), 2013, 8(1): 13-23.

Evgeniya V. Pushchina, Dmitry K. Оbukhov, Anatoly A. Varaksin. Features of adult neurogenesis and neurochemical signaling in the Cherry salmon Oncorhynchus masou brain[J]. Neural Regeneration Research, 2013, 8(1): 13-23.

参考文献

[1] Zupanc GK, Horschke I. Proliferation zones in the brain of adult gymnotiform fish: a quantitative mapping study. J Comp Neurol. 1995;353(2):213-233.http://www.ncbi.nlm.nih.gov/pubmed/7745132

[2] Grandel H, Kaslin J, Ganz J, et al. Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate. Dev Biol. 2006;295(1):263-277.http://www.ncbi.nlm.nih.gov/pubmed/16682018

[3] Zupanc GK. Neurogenesis and neuronal regeneration in the adult fish brain. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2006;192(6):649-670. http://www.ncbi.nlm.nih.gov/pubmed/16463148

[4] Pellegrini E, Mouriec K, Anglade I, et al. Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish. J Comp Neurol. 2007;501(1):150-167. http://www.ncbi.nlm.nih.gov/pubmed/17206614

[5] Pushchina EV, Obukhov DK, Varaksin AA. Neurochemical markers of cells of the periventricular brain area in the masu salmon Oncorhynchus masou (Salmonidae). Ontogenez. 2012;43(1):39-53. http://www.ncbi.nlm.nih.gov/pubmed/22567927

[6] Pushchina EV, Obukhov DK. The participation of radial glia cells into histogenesis of nervous system. In: Loseva E, Loginova N, Sinelnikova V, eds. Neuroscience for Medicine and Psychology. Moscow: MAKS Press, Russia. 2010: 242-243.

http://rd.springer.com/article/10.1134/S1062360412010055

[7] von Bohlen und Halbach O. Immunohistological markers for proliferative events, gliogenesis, and neurogenesis within the adult hippocampus. Cell Tissue Res. 2011;345(1):1-19. http://www.ncbi.nlm.nih.gov/pubmed/21647561

[8] Maekawa M, Takashima N, Arai Y. Pax6 is required for production and maintenance of progenitor cells in postnatal hippocampal neurogenesis. Genes Cells. 2005;10(10):1001-1014. http://www.ncbi.nlm.nih.gov/pubmed/16164600

[9] Osumi N, Shinohara H, Numayama-Tsuruta K, et al. Concise review: Pax6 transcription factor contributes to both embryonic and adult neurogenesis as a multifunctional regulator. Stem Cells. 2008;26(7):1663-1672. http://www.ncbi.nlm.nih.gov/pubmed/18467663

[10] Pushchina EV, Fleishman MY, Timoshin SS. Proliferative zones in the brain of the Amur sturgeon fry interactions with neuromeres and migration of secondary matrix zones. Rus J Dev Biol. 2007;38(5):286-293. http://www.ncbi.nlm.nih.gov/pubmed/18038653

[11] Extröm P, Johnsson CM, Ohlin LM. Ventricular proliferation zones in the brain of an adult teleost fish and their relation to neuromeres and migration (secondary matrix) zones. J Comp Neurol. 2001;436(1):92-110. http://www.ncbi.nlm.nih.gov/pubmed/11413549

[12] Platel JC, Lacar B, Bordey A. GABA and glutamate signaling: homeostatic control of adult forebrain neurogenesis. J Mol Histol. 2007;38(4):303-311. http://www.ncbi.nlm.nih.gov/pubmed/17554632

[13] Cameron HA, McEwen BS, Gould E. Regulation of adult neurogenesis by excitatory input and NMDA receptor activation in the dentate gyrus. J Neurosci. 1995;15(6):4687-4692. http://www.ncbi.nlm.nih.gov/pubmed/7790933

[14] Aniol VA, Stepanichev MY. Nitric oxide and gamma-aminobutyric acid as regulators of neurogenesis in the brain of adult mammals: models of seizure activity. Neurochem J. 2007;1(4):265-274. http://www.springerlink.com/content/a0j57400784h2101/

[15] Platel JC, Stamboulian S, Nguyen I, et al. Neurotransmitter signaling in postnatal neurogenesis: the first leg. Brain Res Rev. 2010;63(1-2):60-71. [PMID] http://www.ncbi.nlm.nih.gov/pubmed/20188124

[16] Extröm P, Ohlin L. Ontogeny of GABA-immunoreactive neurons in the central nervous system in a teleost Gasterosteus aculeatus L. J Chem. Neuroanat. 1995;9(4):271-288. http://www.ncbi.nlm.nih.gov/pubmed/8719276

[17] Overstreet Wadiche L, Bromberg DA, Bensen AL, et al. GABA-ergic signaling to newborn neurons in dentate gyrus. J Neurophysiol. 2005;94(6):4528-4532. http://www.ncbi.nlm.nih.gov/pubmed/16033936

[18] Gascon E, Dayer AG, Sauvain MO, et al. GABA regulates dendritic growth by stabilizing lamellipodia in newly generated interneurons of the olfactory bulb. J Neurosci. 2006;26(50):12956-12966. http://www.ncbi.nlm.nih.gov/pubmed/17167085

[19] Ge S, Goh EL, Sailor KA, et al. GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature. 2006;439(7076):589-593. http://www.ncbi.nlm.nih.gov/pubmed/16341203

[20] Martinoli M-G, Dubourg P, Geffard M, et al. Distribution of GABA immunoreactive neurons in the forebrain of the goldfish Carassius auratus. Cell Tissue Res. 1990;260(1):77-84. http://www.springerlink.com/content/j4m55v76627n3222/abstract/

[21] Medina M, Reperant J, Dufour S, et al. The distribution of GABA-immunoreactive neurons in the brain of the silver eel (Anguilla anguilla L.). Anal Embryol. 1994;189(1):25-39. http://www.ncbi.nlm.nih.gov/pubmed/8192235

[22] Adolf B, Chapouton P, Lam CS, et al. Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon. Dev Biol. 2006;295(1):278-293. http://www.ncbi.nlm.nih.gov/pubmed/16828638

[23] Rakic P. The radial edifice of cortical architecture: from neuronal silhouttes to genetic engineering. Brain Res Rev. 2007;55(2):204-219. http://www.ncbi.nlm.nih.gov/pubmed/17467805

[24] Hebsgaard JB, Nelander J, Sabelström H, et al. Dopamine neuron precursors within the deveoping human mesencephalon show radial glial characteristics. Glia. 2009;57(15):1648-1658. http://www.ncbi.nlm.nih.gov/pubmed/19330857

[25] Ugrumov MV. Developing brain as an endocrine organ: a paradoxical reality. Neurochem Res. 2010;35(6):837-850. http://www.ncbi.nlm.nih.gov/pubmed/20135220

[26] Callier S, Snapyan M, Le Crom S, et al. Evolution and cell biology of dopamine receptors in vertebrates. Biol Cell. 2003;95(7):489-502. http://www.ncbi.nlm.nih.gov/pubmed/14597267

[27] Diaz J, Ridray S, Mignon V, et al. Selective expression of dopamine D3 receptor mRNA in proliferative zones during embryonic development of the rat brain. J Neurosci. 1997;17(11):4282-4292. http://www.ncbi.nlm.nih.gov/pubmed/9151745

[28] Hoglinger GU, Rizk P, Muriel MP, et al. Dopamine depletion impairs precursor cell proliferation in Parkinson disease. Nat Neurosci. 2004;7(7):726-735. http://www.ncbi.nlm.nih.gov/pubmed/15195095

[29] Kapsimali M, Vidal B, Gonzalez A, et al. Distribution of the mRNA encoding the four dopamine D (1) receptor subtypes in the brain of the european eel (Anguilla anguilla): comparative approach to the function of D(1) receptors in vertebrates. J Comp Neurol. 2000;419(3):320-343. http://www.ncbi.nlm.nih.gov/pubmed/10723008

[30] Meek J. Catecholamines in the brains of osteichtyes (bony fishes). In: Smetts WJ, Reiner A, eds. Phylogeny and development of catecholamine systems in the CNS of vertebrates. Cambridge: Cambrifge University Press. 1994.http://books.google.com.hk/books/about/Phylogeny_and_Development_of_Catecholami.html?id=VYPfO4zhpOQC

[31] Wullimann MF, Rink E. Detailed immunohistology of Pax6 protein and tyrosine hydroxylase in the early zebrafish brain suggests role of Pax6 gene in development of dopaminergic diencephalic neurons. Brain Res. Dev Brain Res. 2001;131(1-2):173–191. http://www.ncbi.nlm.nih.gov/pubmed/11718849

[32] Pushchina EV. Tyrosine hydroxylase in telencephalon and diencephalons of the Rhodeus sericeus (Cyprinidae). Tsitologyia. 2009;51(1):63-77. http://www.ncbi.nlm.nih.gov/pubmed/19281050

[33] Götz M, Stoykova A, Gruss P. Pax6 controls radial glia differentiation in the cerebral cortex. Neuron. 1998;21(5):1031-1044. http://www.ncbi.nlm.nih.gov/pubmed/9856459

[34] Puelles L, Kuwana E, Puelles E, et al. Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1. J Comp Neurol. 2000;424(3):409-438. http://www.ncbi.nlm.nih.gov/pubmed/10906711

[35] Kohwi M, Osumi N, Rubenstein JL. Pax6 is required for making specific subpopulations of granule and periglomerular neurons in the olfactory bulb. J Neurosci. 2005; 25(30):6997-7003. http://www.ncbi.nlm.nih.gov/pubmed/16049175

[36] Ma PM. Tanycytes in the sunfish brain: NADPH-diaphorase histochemistry and regional distribution. J Comp. Neurol. 1993;336(1):77-95. http://www.ncbi.nlm.nih.gov/pubmed/8254115

[37] Bicker G. Stop and go with NO: nitric oxide as a regulator of cell motility in simple brains. BioEssays. 2005;27(5):495-505. http://www.ncbi.nlm.nih.gov/pubmed/15832386

[38] Pinto L, Götz M. Radial glial cell heterogeneity – the source of diverse progeny in the CNS. Prog Neurobiol. 2007;83(1):2-23. http://www.ncbi.nlm.nih.gov/pubmed/17580100

[39] Kriegstein A, Alvarez-Buylla A. The glial nature of embryonic and adult neural stem cells. Annu Rev Neurosci. 2009;32:149-184. http://www.ncbi.nlm.nih.gov/pubmed/19555289

[40] Hope ВТ, Vincent SR. Histochemical characterization of neuronal NADPH-diaphorase. J Histochem Cytochem. 1989;37(5):653-661. http://www.ncbi.nlm.nih.gov/pubmed/2703701

[41] Pushchina EV. Nitric oxide-ergic organization of medullar cranial nuclei in teleost fishes. Tsitologyia. 2007;49(6):471-483. http://www.ncbi.nlm.nih.gov/pubmed/17802744

引言

材料方法

Design

A controlled, observational animal experiment.

Time and setting

This study was performed at Laboratory of Cytophysiology, A.V. Zhirmunskii Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Russia, from February 2009 to May 2011.

Materials

The cherry salmon Oncorhynchus masou was obtained from Ryazanka experimental fish hatchery (Peter the Great Bay, Sea of Japan); 20 specimens were taken from each age bracket group: 3-month-old, 6-month-old, 1-year-old and 3-year-old mature fish. The fish were kept in aquaria with aerated fresh water at 17–18°С and anesthetized with a stock solution of 0.1% (w/v) tricaine methane sulphonate (MS-222; Sigma, St. Louis, MO, USA) for 10–15 minutes. The intracranial cavity of the immobilized fish was perfused with 0.1 M PBS (pH 7.2) containing 4% paraformaldehyde solution via a syringe. After prefixation, the brain was removed from the cranial cavity and fixed at 4°С for 2 hours in the same solution. Then, the brain was washed five times in 30% sucrose solution at 4°С during 24 hours. Serial frontal transverse brain sections (50 μm thick) of the cherry salmon were sliced using a freezing microtome.

Methods

Histochemical staining

The NADPH-d activity in the teleost fish brain was investigated according to a modification of conventional histochemical methods^[40-41]. Free-floating sections were incubated in the medium, made up of 1mM β-NADPH, 0.8 mM nitro blue tetrazolium, and 0.06% Triton X-100 in 0.1 M PBS (pH 7.2), at 37°С for 2 hours. All chemicals were purchased from Sigma-Aldrich Chemie GmbH (USA). After incubation, the sections were rinsed in PBS, mounted on a gelatin-coated glass slide, and air-dried overnight. The next day, they were dehydrated according to the standard technique and embedded in balsam. In order to determine the specificity of histochemical reaction, the following controls were performed: incubation without the substrate β-NADPH and incubation without chromogen nitro blue tetrazolium in order to detect possible nonspecific formation of the reaction product. In all experiments, no residual reaction was observed. Control preparations were incubated in the media with addition of nitric oxide synthase inhibitor and 10 mМ N-monomethyl-L-аrginine. The sections were visualized under an Axiovert 200M fluorescent microscope (Carl Zeiss MicroImaging, Munich, Germany).

Immunohistochemical staining of proliferating cell nuclear antigen (PCNA)

Avidin-biotin-peroxidase complex (ABC) immunohistochemical staining of PCNA was performed to investigate the proliferative activity of cerebral cells. The fish brain was fixed using the above described technique and then divided into several blocks. The obtained samples were embedded in paraffin according to the conventional technique^[10]. Serial frontal sections (25 μm thick) were prepared using a microtome, mounted on polylysine-covered glass slides and then deparaffinized. The obtained preparations were subjected to thermal treatment for 45 minutes at 95°С with Target Retrieval Solution (code No. S 3308; Dako Cytomation), citrate buffer (10 mM; pH 6.0) or Tris buffer (10 mM), and ethylenediamine tetraacetic acid (1 mM; pH 9.0; DAKO, Denmark) to increase membrane permeability. After cooling down to room temperature, the glass slides were rinsed in distilled water. To remove nonspecific peroxidase activity, the slices were incubated in 1% hydrogen peroxide solution for 5 minutes at 37°С and washed three times in 0.1 М PBS (pH 7.2). The immunocytochemical detection of PCNA was performed with a commercially available mouse MoAb against PCNA (PC 10, lgG, DAKO, Denmark) (1:400) for 20 minutes at room temperature and washed three times in 0.1 M PBS. The slices were incubated with secondary biotin-conjugated mouse anti-rabbit IgG (1:100 in PBS + NHS 1% LSAB 2 System, HRP; DAKO, Denmark) for 20 minutes at 37°С and then washed three times in 0.1 M PBS (pH 7.2). Subsequently, the sections were incubated with streptavidin-biotin visualization systems (LSAB 2 System, HRP; DAKO, Denmark) under the same condition (20 minutes, 37°С) and washed in 0.1 M PBS. The reaction products were detected with diaminobenzidine dissolved (to 0.5 mg/mL) in PBS; 1- or 2-mL aliquots were prepared. Before application, hydrogen peroxide, based on 0.1 M PBS, was added to aliquots to a final concentration of 0.03%. The obtained preparations were kept for 5–7 minutes in a thermostat at 37°С to reach a clear visualization of the used marker (process was controlled under a microscope). Glass slides were washed in distilled water, stained with hematoxylin (Lilly-Mayer, BioVitrum, Russia) for 30 seconds, washed with running water for 15–20 minutes, dehydrated according to the standard technique, and embedded in balsam.

Immunohistochemical staining for TH, GABA and Pax6

ТН, GABA and transcription factor Pax6 were immunohistochemically identified using the standard avidin-biotin-peroxidase labeling on free-floating sections. The sections were incubated with mouse anti-GABA IgG1 (clone: 5A9; MP Biomedicals, Santa Ana, CA, USA; 1:5 000), mouse anti-TH IgG2a (clone: 1B5; Vector Laboratories, Burlingame, CA, USA; 1:10 000), mouse anti-Pax6 monoclonal antibody IgG1 (clone: AD2.38; Chemicon, Billerica, MA, USA; 1:4 000) at 4°С for 2 days. For visualization of immunohistochemical labeling, a Vectastain Elite ABC kit (Vector Laboratories) was applied. For identification of the reaction products, the substrates of blue (GABA, Pax6) and red (TH) colors (VIP Substrate Kit, Vector Labs, Burlingame, USA) were used. The staining process was controlled under an Axiovert 200M fluorescent microscope (Carl Zeiss MicroImaging). The sections were rinsed in water, mounted on slides, dehydrated according to the standard method, and embedded in balsam. Negative control was used to evaluate the specificity of immunohistochemical reaction. Brain sections were incubated for 1 day with 1% nonimmune horse serum rather than primary antibodies and then stained as described above. In all control experiments, no immunoreactive reactions occur.