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ORlGINS OF NEUROPEPTIDE Y-LIKE IMMUNOREACTIVE FIBERS IN THE LATERAL AND BASOLATERAL AMYGDALOID NUCLEI OF THE RAT

Gaoge SUN ; Yamano MARIKO ; Tohyama MASAYA ; Qunyuan XU

Chinese Journal of Neuroanatomy.2000;16(4):347-352,58.

The origin of neuropeptide Y(NPY)-like immunoreactive(IR)fibers in the lateral and basolateral amygdaloid nuclei was examined by using indirect immunofluorescence and retrograde tracing method in the rat.1njection of a retrograde tracer.fluorogold(FG),into the lateral and basolateral amygdaloid nuclei,labeled many neurons in the ipsilateral anterior amygdaloid area,and simultaneous treatment with antiserum against NPY stained some of these neurons.Destruction of the anterior amygdaloid area caused an ipsilateral decrease of NPY-IR fibers in the lateral and basolateral amygdaloid nuclei.These findings indicate that NPY-IR neurons in the anterior amygdaloid area project ipsilaterally to the lateral and basolateral amygdaloid nuclei.In addition,the present study also shows that NPY-IR neurons located in the lateral and basolateral amygdaloid nuclei are intrinsic to the amygdaloid complex,since after destruction of the anterior amygdaloid area,some of NPY-IR fibers still can be found in lateral and baso lateral nuclei,and transection of the two major amygdalofugal system,stria terminalis and ventral amygdalofugal pathway,failed to cause the accumulation of NPY-like immunoreactivity in the axons proximal to the section.

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THE EFFECTS OF HYPERTHERMIA ON THE NEUROEPITHELIAL CELLS IN PRIMARY CULTURE

Yingjun GUAN ; Yingmao GAO ; Zhaodong JUAN ; Shouxun WANG ; Rujiang LI

Chinese Journal of Neuroanatomy.2000;16(4):-.

The animal experiment studies have demonstrated that hyperthermia is a strong teratogen to many kinds of animal with high incidence of neural tube defects(NTD).Epidermiological investigation showed that hyperthermia was closely related to the acencephaly and excenphaly in human being,but little was known about the distinct mechanism of NTD induced by hyperthermia.In order to study the developmental mechanism of NTD induced by hyperthermia,we observed the effects of hyperthermia on the neuroepithelial cells in primary culture.The neural tubes of the hamster embryos on 10 d after fertilization were obtained.the neuroepithelia were dissociated and then seeded at the density of 1×106 cells per well.The cells were divided into experimental and control groups randomly.The experimental groups were exposed to 42 C for 20 min,whereas groups exposed to 37 Cserved as control.After treatment of hyperthermia,the cells were incubated continuously at 37 C in a 95%air/5%CO2 humidified incubator,the culture was terminated after different intervals.Phase-contrast microscopy.scanning electron microscopy.transmission electron microscopy,MTT assay,agarose gel electrophoresis analysis,TUNEL detection,immunocytochemistryand image analysis were made.The results of the experimental groups indicated that the floating cells and apoptotic cells increased in number,the neurites of the cells were shortened even disappeared,the survival cells decreased in number,the ultrastructure of the cells demonstrated distinct abnormal changes,the function of mitochondria was impaired,the expressions of both bcl-2 and bax were abnormal.The above results suggest that hyperthermia may induce apoptosis of neuroepithelial cells,duringwhich bcl-2 and bax genes may play important regulatory roles.

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COEXISTENCE OF ADRENOCORTICOTROPIN AND SECRETOGRANIN ⅡIN THE RAT PITUITARY GLAND AFTER BRAIN INJURY

Chunmei WANG ; Xiaofeng HUANG ; Jiaji YANG ; Mei HUANG ; Xiangdang LI ; Dan CHEN

Chinese Journal of Neuroanatomy.2000;16(4):337-340,55.

The localization of adrenocorticotropin(ACTH),secretogranin Ⅱ in rat pituitary gland Was studied by using specific antisera in conjunction with double immunofluorescence staining techniques.Stainings with secretogranin Ⅱ serum showed intense cytoplasmic reaction in the anterior lobe of the pituitary gland.Double immunofluorescence staining was used to identify the cells that expressed secretogranin Ⅱ and ACTH.Confocal laser scanning microscopy revealed that ACTH existed simultaneouslywith secretogranin Ⅱ in quite a number of cells.The results indicate that secretogranin Ⅱ is expressed in corticotrophs of rat pituitary gland,its physiological role may be associated with the regulation of optimal pH in the secretory vesicles for the cleavage of ACTH from its precursor.

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EXPRESSION OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN DEAFFERENT SPINAL CORD DORSAL HORN OF CAT

Lihua CHEN ; Liangfang WU ; Xue ZHOU

Chinese Journal of Neuroanatomy.2000;16(3):269-272.

To investigate the expression change of BDNF in lamina II of spinal cord from partial deafferented cats, L6 segmentsof spinal cord from 20 adult male cats (5 normal cats, 15 unilateral L6 spared roots cats allowed to survive 3 d, 6 d and 12 d re-spectively) were stained with immunohistochemical technique. The results showed: BDNF positive products were mainly dis-tributed in nerve terminals, varicosities and few neurons of spinal cord lamina II in normal cat. After operation, the density ofpositive nerve terminals and varicosities began to decrease on the third day, reached the lowest level on the 6th day and recoveredto normal level on the 12th day on operated side. But the number of BDNF neurons showed no obvious change. The authors sug-gest that the decreased density of BDNF positive products in lamina II on the 3rd and 6th day was related with the degenerationof the nerve fibers and varicosities after section of the adjacent dorsal roots. On the 12th day, the remaining L6 dorsal roots un-derwent collateral sprouting compensatoryly and reestablished functional connection with target neurons. Therefore, BDNF maybe involved in the normal physiological function and the plasticity of spinal cord after damage.

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THE CONNECTION AMONG THE MARGINAL DIVISION OF STRIATUM,AMYGDALOID NUCLEUS AND BED NUCLEUS OF THE STRIA TERMINALIS IN THE RAT BRAIN AS REVEALED BY IMMUNOHISTOCHEMISTRY

Yaoyu LI ; Siyun SHU ; Xinnin BAO ; Wenlong WU

Chinese Journal of Neuroanatomy.2000;16(3):265-268.

To understand the relationship of neurotransmitter between the striatum and limbic system such as amygdaloid nucle-us and bed nucleus of the stria terminalis. 30 male Sprague Dawley rats were used. Immunohistochemical ABC method was per-formed to detect the expression of substance P (SP), calcitonin gene-related peptide (CGRP), leucine-enkephalin (L-enk),cholecyctokinin (CCK) and neuronal nitric oxide synthase (NOS) on seetions of the brain. Some transmitters including substanceP, calcitonin gene-related peptide and cholecyctokinin were mainly distributed at the marginal division of the striatum. Theleucine-enkephalin was mainly distributed at the globus pallidus and was secondly distributed at the marginal division of the stria-tun. The neuronal nitric oxide synthase was mainly distributed at caudate putamen and the marginal division. All these transmit-ters were not only distributed at amygdaloid nucleus and bed nucleus of the stria terminalis, but also had fibers connection amongthe amygdaloid nucleus, marginal division and bed nucleus of the stria terminalis. CONCLUSION: There were special fibersconnection between the marginal division and other basal ganglia nucleus or the limbic system. The marginal division may beplayed some important functions of basal ganglia and limbic system.

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IMMUNOCYTOCHEMIICAL AND MORPHOMETRICAL STUDY OF THE NEURONAL PRECURSOR CELLS DERIVED FROM THE SUBVENTRICULAR ZONE OF THE POSTNATAL RAT FOREBRAIN IN VITRO

Dianshuai GAO ; Fengzhen ZHANG ; Deguang WANG ; Changfu ZHOU

Chinese Journal of Neuroanatomy.2000;16(3):261-264.

In order to investigate the chemical and morphornetrical properties of the neuronal precursor cells derived from thesubventricular zone(SVZ) of the postnatal rat forebrain in vitro. The cell-type specific antibodies were used for the immunocy-tochemical staining ,and the morphometric parameters which were the mean soma diameter and the ellipticity index (i. e. , thesmallest soma diameter divided by the largest soma diameter) of every SVZ-derived cell were measured for identifying the pheno-types of the SVZ cells in vitro. The experiment animals were SD rats (weights: 100～ 150 g), the SVZ cells derived from thepostnatal rats were cultured on poly-D-lysine-coated 24-well glass chamber slides in the Neurobasal Medium supplemented withB27 in 5% CO2 at 37 C. The following results were obtained.. At 1 day in vitro, almost all SVZ cells (〉90%) from the postna-tal rat forebrain expressed Tujl, an antibody that recognizes neuron-specific tubulin. Likewise, the preponderance of the SVZcells expressed the polysialylated neural cell adhesion molecule (PSA-N-CAM) ; The majority of the SVZ Tujl-positive cells cul-tured were the cells that had oval-shaped bodies with two short, unbranched processes protruded from every two poles, theirmean soma diameter were 8.42±1.03μm and their ellipticity index were 0.57±0.12. Meanwhile, there were approximately20% of the SVZ cells in culture that were sphere-shaped cells with mean soma diameter 7.20±l.04 μm , and it might be observed that these cells connected with one another. As the time in culture went on, these sphere-shaped SVZ-derived cells alsotransformed to oval-shaped ones as described above, but it could be observed that the cells were still connected in the processesof them. By 3 and 5 days in culture, the SVZ cells had larger cell somas (average diameter 9. 07±1.07 μm), and often consider-ably longer processes but still with few branches. Immunocytochemical staining revealed that the majority of the SVZ cells in cul-ture remained Tujl-positive, PSA-N-CAM-positive. By 7 days in culture, the Tujl-positive cells in culture showed remarkablemorphological changes, and possessed typical neuronal phenotypes, which had more larger cell somas (average diameter 12.8 ±1.13 μm), and had more longer, branched processes. Our results showed that the SVZ in the postnatal SD rats contained theneuronal precursor cells which were PSA-N-CAM-positive and could differentiate into new neurons in vitro.

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THE PROJECTIONS FROM LAMINA Ⅱ OF MEDULLARY DORSAL HORN TO PARABRACHIAL REGION, CAUDAL VENTROLATERAL MEDULLA AND SPINAL CORD IN THE RAT

Jianhong WU ; Yunqing LI ; Jiwu SHI

Chinese Journal of Neuroanatomy.2000;16(3):251-256.

After injecting retrograde tracer fiuoro-gold (FG) into the parabrachial region(PB), caudal ventrolateral medulla(CVLM) and the fourth segment of cervical spinal cord (C4), respectively, neurons in laminae I ～ Ⅱ of the medullary dorsalhorn projecting to the above mentioned brain areas were observed. PB received projections from bilateral laminae I and Ⅱ withan ipsilateral dominance; CVLM and C4 received projections from ipsilateral laminae I and Ⅱ. Neurons projecting to C4 werevery sparsely distributed in laminae I and Ⅱ of the medullary dorsal horn. The projecting neurons in outer part of lamina Ⅱwere more than those in inner part of lamina Ⅱ . Combined with immunofluorescence histochemistry for calbindin-D28k(CB) andparvalbumin(PV), it was demonstrated that a part of neurons projecting to PB or CVLM showed CB-like immunoreactivity, butnone of them exhibited PV-like immunoreactivity. There were only a few neurons in lamina Ⅱ projecting to C4 and they exhibitedneither CB- nor PV-like immunoreactivity. The present study provides further evidence for the existence of projecting neurons inlamina Ⅱ and suggests that immunostaining against CB and PV may distinguish two neuronal subpopulations in lamina Ⅱ .

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EFFECTS OF CULTURED ASTROCYTES FROM RAT CEREBRAL CORTEX ON THE DEVELOPMENT OF PC12 CELLS

Yongyan MO ; Yuan CHEN ; Mei ZHOU ; Bao ZHANG

Chinese Journal of Neuroanatomy.2000;16(3):247-250.

To investigate effects of cultured astrocytes from Sprague Dawley rat cerebral cortex on the development of PC12 cellsderived from rat pheochromocytoma, PC12 cells were cocultured with astrocyte according to different astrocytes/neurons ratio(50:1～1:1) , or with serum-free conditioned medium of astrocytes(ACM). The vitality of PC12 cells was measured by sensi-tive MTT method and their morphologic features were observed by Olympus light microscope. The results showed: (1) WhenPC12 cells were cultured with ACM, compared with the control group, the vitality of PC12 cells was increased significantly (0.255+0. 012 vs 0. 510±0. 036, P<0. 001) and the morphological changes were not obvious in the experimental group. (2) WhenPC12 cells were cocultured with astrocyte in the ratio of 30: 1～1: 1, not only was the vitality of PC12 cells enhanced, but alsothe neurite-outgrowth of PC12 was observed. (3) When PC12 cells were cocuhured with astrocyte in proportion of 50: 10～40 : 1, the vitality of PC12 cells was also enhanced, but the neurite-outgrowth of PC12 was not found. This study suggested en-hancement of PC12 cell-vitality was mediated by soluble factors produced by astrocytes, while activity of the neurite-promotingwas associated with cell-cell contact and with the ratio of two cells.

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RELATIONSHIP BETWEEN NEURONAL INJURIES AND REACTION OF ASTROCYTES FOLLOWING CEREBRAL ISCHEMIA REPERFUSION IN RAT

Qionglan YUAN ; Ruiaiang LI ; Huijun YANG ; Guangpeng ZHANG ; Changfu QIU

Chinese Journal of Neuroanatomy.2000;16(3):242-246.

The involvement of astrocytes and correlation between neuronal injury and astrocyte response were studied. Blockingmiddle cerebral artery and reperfusing o. 5～48 h, H-E staining, immunoccytochemistry single-and double-labeling, dotble label-ing combined with TUNEL and GFAP immunocytochemistry were used to investigate neuronal injury and astrocyte response.The is chemic area peaked at 24 h of reperfusion. The neurons presented irreversible degeneration at 6 h of reperfusion. At24 h,ischemic area in the preoptic area developed into infarcted area; astrocytes exhibited differential morphological features: reactive,malnourished and degenerative changes. At 48 h of reperfusion, the number of astrocytes began to go up. The astrocytes in is-chemic area didn't proliferate within 48 h. By contrast, a few astrocytes underwent apoptosis. In conclusion, these data indicatethat the reaction of astrocytes is closely connected with the extent of neuronal injuries. The reactive astrocytes imply that astro-cytes positively respond to the neuronal injuries, which might play a role in promoting neuronal survival.

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cGMP POSITIVE CELLS AFTER CEREBRAL ISCHEMIA-REPERFUSION IN THE GERBIL HIPPOCAMPUS AND DENTATE GYRUS

Jingjun ZHANG ; Zuoli XIA ; Mingfeng YANG

Chinese Journal of Neuroanatomy.2000;16(3):239-241.

The purpose of the present work is to observe cGMP positive cells after cerebral ischemia-reperfusion in the gerbil hip-pocampus. Immunofluorescent methods were used in gerbil hippocampal tissue slices. The results showed that after cerebral is-chemia cGMP synthesis in the CAi-a subfields was increased, cGMP positive cells were distributed mainly in CA1 subfield. Mostof cGMP positive cells were astrocytes. The number of small round cGMP positive cells were increased after recirculation follow-ing ischemia in the dentate gyrus. These results indicate that after cerebral ischemia cGMP synthesis was increased in the CA1-asubfield, It is possible that astrocytes play an important role in the regulation of metabolism in the early stage after ischemia-reperfusion.

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