Detection of white spot syndrome virus in Brazil using negative staining, immunoelectron microscopy and immunocytochemistry techniques / Detección del virus del síndrome de mancha blanca en el Brasil utilizando inmunomicroscopía e inmunomarcación con partículas de oro coloidal

In this study thirty shrimp samples from commercial marine shrimp (L. vannamei) farms of southern region of Brazil were obtained. Hepatopancreas and shell scrapings fragments collected in these animals were processed by transmission electron microscopy using negative staining (rapid preparation), immunoelectron microscopy and immunocytochemistry (immunolabelling with colloidal gold particles) techniques. On the transmission electron microscopy a great number of white spot virus particles, ovoid or bacilliform-to-ellipsoid, measured 230-290 nm in length and 80-160 nm in diameter with intra-nuclear projections were visualized by the negative staining technique in 27 (90 percent) out of 30 samples examined. Using immunoelectron microscopy technique, the anti-VP 664 serum agllutinated a large number of particles formed by antigen-antibody interaction. In the immunocytochemistry technique, the antigen-antibody reaction was styrongly marked by the particles of colloidal gold over the virus. Notably, this is the first report, to our knowledge, describing use of these microscopy techniques to study Brazilian L. vannamei marine shrimp samples; moreover, this methodology also appears to be a viable complementary tool for diagnosing the presence of the white spot virus within shrimp tissues. Importantly, these are the first photoelectron micrographs of the WSSV in Brazil.

Se obtuvieron para el estudio 30 muestras de camarones marinos comerciales (L. vannamei) de las granjas de la región sur de Brasil. Fueron procesados fragmentos de hepatopáncreas y raspados internos del cefalotórax recogidos en estos animales por microscopía electrónica de transmisión con tinción negativa (preparación rápida), inmunomicroscopía y técnicas de inmunocitoquímica (inmunomarcación con partículas de oro coloidal). En la microscopía electrónica de transmisión de un gran número de partículas de virus de la mancha blanca, ovoide o elipsoidal a baciliformes, medían 230-290 nm de longitud y 80-160 nm de diámetro. En 27 (90 por ciento) de las 30 muestras examinadas intra-nuclear proyecciones se visualizaron mediante la técnica de tinción negativa. Utilizando una técnica de inmunomicroscopía electrónica, el anti-suero VP 664 reunió a un gran número de partículas formadas por la interacción antígeno-anticuerpo. En la técnica de inmunocitoquímica, la reacción antígeno-anticuerpo fue fuertemente reforzada por las partículas de oro coloidal en los virus. En particular, en Brasil este es el primer informe, a nuestro entender, que describe el uso de estas técnicas de microscopía en muestras de camarón marino L. vanamei. Además, esta metodología también parece ser una herramienta complementaria viable para diagnosticar la presencia del virus de la mancha blanca en tejidos de camarón. Es importante destacar que estas son las primeras fotos en microscopia electrónica del WSSV obtenidas en Brasil.

Expression and ultrastructure location of PPAR-γ and COX-2 in endometrial carcinoma / 解剖学报

Objective To investigate the development and prognosis of peroxisome proliferator-activated recepor-γ(PPAR-γ) and cyclooxygenase-2(COX-2) in endometrial carcinoma. Methods The electron microscopic immunohistochemical technique was used to observe the ultrastructure location of COX-2 protein labeled by colloidal gold in the endometrial carcinoma. Results 1.There were negative immunohistochemical staining signals of PPAR-γ in both the normal endometrium and hyperplasia endometrium, whereas, was positive staining in the endometrial carcinoma;2.The intensities of the COX-2 immunohistochemical staining were significantly statistical difference among the normal endometrium, the hyperplasia endometriu and the endometrial carcinoma(P<0.01);3. The COX-2 labelled by colloidal gold granules was observed in the endoplasmic reticulum, nuclear membrane and nuclei in the endometrial carcinoma. Conclusion Both PPAR-γ and the COX-2 might play an important role in the development of the endometrial carcinoma.

Changes of behavior, Ca~(2+)/CaMKⅡ in hippocampus of rats with chronic forced swimming stress model / 解剖学报

Objective To observe the changes of behavior, intracellular free calcium and the expression of calmodulin dependent protein kinase Ⅱ(CaMKⅡ) in the hippocampal neurons of chronic forced swimming stress rats. Methods Male Wistar rats were randomly divided into control group and chronic forced swimming stress group. The behavior was examined using sucrose preference test, open-filed test and Morris water maze. The intracellular free calcium was examined by fluorescence spectrophotometer. The expression of CaMKⅡ was detected using colloidal gold immunoelectron microscopy technique, Western blotting and RT-PCR. Results The consumption of sucrose and erect quantity of chronic forced swimming stress group were lower than those of control group(P<0.01, P<0.05). The escape latency time in Morries water maze test of chronic forced swimming stress group was higher than that of control group(P<0.01). The intracellular free calcium level and the expression of CaMKⅡ in the hippocampus was higher than that of control group(P<0.01).Conclusion The lasting dysfunction of Ca~(2+)/CaMKⅡ signaling cascades in hippocampus may play important roles in the pathogenesis of chronic forced swimming stress rats.

Application of Tyramide Signal Amplification (TSA) Both to Biochip Platform and to the Immunoelectron Microscopy to Label Proteins within the Organelle / 대한해부학회지

The tyramide signal amplification (TSA) technique, based on the ability of HRP to catalyze the deposition of tyramide onto the surrounding proteins, has been proved to detect scarce tissue antigens. In this study we applied this technique to a biochip platform and an immunocytochemistry at the electron microscopic level. First, in the optical fluorescence sensing, the signal was amplified by Dako Envision(TM) (goat anti-mouse immunoglobulins IgG conjugated to peroxidase labelled-dextran polymer) and tyramide-Cy3, which was then compared to the non-amplified control using goat antimouse IgG-Cy3 conjugate instead. The result showed that the tyramide method produced a more sensitive signal than the control method. Secondly, in the pre-embedding immunocytochemistry, we investigated to see whether it is possible to label proteins within a organelle in the cell using the TSA method. The signal was amplified by a primary antibody, a biotinylated secondary antibody, streptavidin-HRP, biotinyl-tyramide, and streptavidinnanogold followed by silver enhancement and gold toning. Then, this protocol was compared to the non-amplified or simple protocol that does not include the steps of streptavidin-HRP and biotinyl-tyramide. With the TSA protocol, the labeling for a membrane bound antigen (gp100) that is known to be exclusively localized to melanosomes in melanocyte, was tested in a melanoma cell line (G361) and found to be highly sensitive and more enhanced than with the simple protocol. Moreover, the gold particles were well localized to the subcellular structures or melanosomes both in the TSA and simple protocols, which indicates that resolution of the signals remains high. Control experiment with omission of the primary antibody demonstrated that background levels or nonspecific bindings are negligible. This result showed that the TSA method can be successfully applied to label the intra-organelle protein that is known to be labeled only in the specific fixation condition with the optimal permeability.

Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues

The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione-S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein.

PARVALBUMIN-IMMUNOREACTIVE INTERNEURONS ARE CONTROLLED BY AN INHIBITORY NEURONAL NETWORK IN BASOLATERAL NUCLEUS OF THE RAT AMYGDALA / 神经解剖学杂志

As the elements of local neuronal circuits, parvalbumin (PV)-containing interneurons in the basolateral nucleus (BL) of the amygdala play an important role in the amygdaloid functions of emotion, learning and memory. In order to investigate how the PV-containing interneurons in the BL are controlled, the synapses established on PV- containing interneurons in the BL of the rat amygdala were examined under immunoelectron microscopy using the double labeling methods with anti-PV and anti-dopamine (DA) antibodies for a reference of dopaminergic axon terminals. The results show that the PV immunoreactive (IR) neurons formed the synapses mainly on the dendritic structures from shafts of the dendrites to median and small dendritic branches. 68% of the synapses on the PV-IR profiles were formed by unlabeled axon terminals, and 32 % of them were formed by DA- (21 % ) and PV- (11 % )IR axon terminals. Majority of the synapses on the PV-IR neurons formed by unlabeled axon terminals were symmetric type, and only a small a mount of them were asymmetric that were observed between the PV-IR spines and unlabeled axon terminals and in the serial synapses in which an unlabeled axon terminal symmetrically contacted to another unlabeled axon terminal that, in turn, synapsed asymmetrically to the PV-IR dendritic profiles. The synapses formed between the PV-IR profiles and DA- or PV-IR axon terminals were exclusively symmetric. The present results suggest that the PV-containing interneurons in the BL of the rat amygdala were controlled by an inhibitory network formed by the symmetric synapses around them, among which the DA system was included.

Apoptosis and Expression of Nitric Oxide Synthase in Hypoxic Ischemic Cerebral Injury of Rats

PURPOSE: Recently, it was reported that apoptosis in hypoxic ischemic cerebral injury is involved in neuronal injury while nitric oxide synthase(NOS) is involved in neuronal apoptosis. The aim of the present study is to investigate the relationship between the expression pattern of NOS and the apoptosis in hypoxic ischemic cerebral injury of rats. METHODS: To investigate the expression pattern of nitric oxide synthase and the relationship between apoptosis and activity of NOS, immunoelectron microscopic examination and in situ apoptosis detection(TUNEL) were performed in male Sprague Dawley rats. Ischemic injury was induced by permanent ligation of left common carotid artery and hypoxic injury by exposure of a mixture of 10% oxygen+90% nitrogen gas. Unicryl embedding method was used for immunoelectron microscopy and Apoptag kit for apoptosis. RESULTS: The number of apoptotic cells reached the highest at 24 hr, decreased after 72 hr and maintained the expression level until 168 hr. nNOS was expressed in neurons of the cortex, peaked at 24 hr and decreased after 72 hr. However, nNOS was not detected in the hippocampus. eNOS was expressed at 12 hr and at 24 hr in the hippocampus and the cortex, respectively, and persisted at each time point. iNOS was expressed after 72 hr in the cerebral cortex and the hippocampus. CONCLUSION: The expression of three isoforms of NOS in hypoxic ischemic cerebral injury was different in time. nNOS seems to be involved in cortical damage in the early phase of hypoxic ischemic cerebral injury and iNOS is related to apoptotic cell deaths in the late phase, but further study on their mechanisms will be needed.

Apoptosis and Expression of Nitric Oxide Synthase in Hypoxic Ischemic Cerebral Injury of Rats

PURPOSE: Recently, it was reported that apoptosis in hypoxic ischemic cerebral injury is involved in neuronal injury while nitric oxide synthase(NOS) is involved in neuronal apoptosis. The aim of the present study is to investigate the relationship between the expression pattern of NOS and the apoptosis in hypoxic ischemic cerebral injury of rats. METHODS: To investigate the expression pattern of nitric oxide synthase and the relationship between apoptosis and activity of NOS, immunoelectron microscopic examination and in situ apoptosis detection(TUNEL) were performed in male Sprague Dawley rats. Ischemic injury was induced by permanent ligation of left common carotid artery and hypoxic injury by exposure of a mixture of 10% oxygen+90% nitrogen gas. Unicryl embedding method was used for immunoelectron microscopy and Apoptag kit for apoptosis. RESULTS: The number of apoptotic cells reached the highest at 24 hr, decreased after 72 hr and maintained the expression level until 168 hr. nNOS was expressed in neurons of the cortex, peaked at 24 hr and decreased after 72 hr. However, nNOS was not detected in the hippocampus. eNOS was expressed at 12 hr and at 24 hr in the hippocampus and the cortex, respectively, and persisted at each time point. iNOS was expressed after 72 hr in the cerebral cortex and the hippocampus. CONCLUSION: The expression of three isoforms of NOS in hypoxic ischemic cerebral injury was different in time. nNOS seems to be involved in cortical damage in the early phase of hypoxic ischemic cerebral injury and iNOS is related to apoptotic cell deaths in the late phase, but further study on their mechanisms will be needed.

IMMUNOGOLD LOCALIZATION OF GFAP AND MDR1 IN SURGICALLY RESECTED BRAIN TISSUES FROM CLINICAL INTRACTABLE EPILEPSY PATIENTS BY TRANSMISSION ELECTRON MICROSCOPY / 解剖学报

Objective To determine the ultrastructural localization of MDR1 and GFAP in the surgically resected brain tissues from intractable epilepsy patients. Methods Expression of MDR1 and GFAP in brain tissues was examined by using PAG immunolabeling technique for electron microscopy. Results The MDR1 and GFAP labeled by gold particles were only detected at some reactive astrocytes. The positive gold particles were mainly located in the astrocytic cytoplasm and their membrane, but not in the nucleus.Conclusion The expression of MDR1 and GFAP in the brain of patients with clinically intractable epilepsy were mainly located at the cytoplasm and membrane of certain reactive astrocytics.;

Ultrastructural localization of 28 kDa glutathione S-transferase in adult Clonorchis sinensis

Glutathione S-transferase (28GST) with molecular mass of 28 kDa is an antioxidant enzyme abundant in Clonorchis sinensis. In adult C. sinensis, 28GST was localized in tegumental syncytium, cytons, parenchyma, and sperm tails examined by immunoelectron microscopy. C. sinensis 28GST was earlier found to neutralize bioreactive compounds and to be rich in eggs. Accordingly, it is suggested that 28GST plays important roles in phase II defense system and physiological roles in worm fecundity of C. sinensis.

Detection of Helicobacter pylori by Pre-embedding Immunoelectron Microscopy: Comparison with Immunoblotting Method

PURPOSE: We tried to evaluate whether the detection rate of Helicobacter pylori in gastric biopsy specimens could be improved by using pre-embedding immunoelectron microscopy. METHODS: A total of 119 children who complained of upper gastrointestinal symptoms were endoscoped at the Gyeongsang National University Hospital from July, 1996 to July, 1999. Five biopsy specimens(three for urease test, one for hematoxylin-eosin(H and E) staining, and one for pre- embedding immunoelectron microscopy) were obtained from each antrum and body. Immunoblotting analysis were also performed. RESULTS: Among the 119 patients, H. pylori were found in 116 patients(97.5%) by the immunoelectron microscopy. Among three patients who were found H. pylori negative in immunoelectron microscopy, two patients showed H. pylori in H and E stained slides and one patient was urease test positive(color change within six hours). Urease tests were positive in 107 patients(89.9 %). The positive rate of immunoblotting tests was 81.5%. However, only 13 patients(10.9%) showed H. pylori on the H and E stained antrum or body tissue. CONCLUSION: In this study, we found H. pylori histopathologically in most of the pediatric patients who complained of upper gastrointestinal symptoms. This study showed that pre-embedding immunoelectron microscopic examinations can be used as a gold standard in the diagnosis of childhood H. pylori infection. However, this method also has limited capacity to detect widely scattered H. pylori compared to the other histopathologic diagnostic methods.

Double Labeling Immunoelectron Microscopic Study on the Synaptic Connections between Glutamic Acid Neurons and GABA Neurons in the Hippocampus of Rats / 华中科技大学学报（医学）（英德文版）

In order to explore the roles of different neurotransmitters in epileptic pathogenesis,the synaptic connections between glutamic acid (Glu) neurons and GABA neurons in normal rat hippocampus were studied by pre-embedding double labeling immunoelectron microscopy. The GABA immunoreaction was first demonstrated by chromogen DAB, then the Glu immunoreaction was demonstrated by molybdic acid-TMB method. After being stabilized by DAB-cobalt chloride,the sections were processed for electron microscopic embedding. Under electron microscope, there were many Glu immunoreaction-positive neurons in the pyramidal layer of hippocampal CA1 area and some GABA immunoreaction-positive neurons with pyramidal or polygonal perikarya in the pyramidal, polymorphic and radiant layer of CA1 area. There were also symmetric dendro-axonic synapses formed by GABA-positive dendrites and Glu-positive axons in the polymorphic layer and symmetric axo-dendritic synapses formed by GABA-positive axons and Glu-positive dendrites in the radiant layer. In addition, there were symmetric autoregulatory axo-dendritic synapses between Glu-positive axons and dendrites and autoregulatory axo-axonic synapses (both symmetric and asymmetric) between GABA-positive axons. Above mentioned results, for the first time,showed that there were complex synaptic regulatory relationships between excitatory Glu neurons and inhibitory GABA neurons in the hippocampal CA1 area, thereby, providing ultrastructural evidence for different neurotransmitters participating in epileptic pathogenesis.

Double Labeling Immunoelectron Microscopic Study on the Synaptic Connections between Glutamic Acid Neurons and GABA Neurons in the Hippocampus of Rats / 华中科技大学学报（医学）（英德文版）

In order to explore the roles of different neurotransmitters in epileptic pathogenesis,the synaptic connections between glutamic acid (Glu) neurons and GABA neurons in normal rat hippocampus were studied by pre-embedding double labeling immunoelectron microscopy. The GABA immunoreaction was first demonstrated by chromogen DAB, then the Glu immunoreaction was demonstrated by molybdic acid-TMB method. After being stabilized by DAB-cobalt chloride,the sections were processed for electron microscopic embedding. Under electron microscope, there were many Glu immunoreaction-positive neurons in the pyramidal layer of hippocampal CA1 area and some GABA immunoreaction-positive neurons with pyramidal or polygonal perikarya in the pyramidal, polymorphic and radiant layer of CA1 area. There were also symmetric dendro-axonic synapses formed by GABA-positive dendrites and Glu-positive axons in the polymorphic layer and symmetric axo-dendritic synapses formed by GABA-positive axons and Glu-positive dendrites in the radiant layer. In addition, there were symmetric autoregulatory axo-dendritic synapses between Glu-positive axons and dendrites and autoregulatory axo-axonic synapses (both symmetric and asymmetric) between GABA-positive axons. Above mentioned results, for the first time,showed that there were complex synaptic regulatory relationships between excitatory Glu neurons and inhibitory GABA neurons in the hippocampal CA1 area, thereby, providing ultrastructural evidence for different neurotransmitters participating in epileptic pathogenesis.

Expression of Antigenic Surface Molecules of Pneumocystis Carinii by Immunoelectron Microscopic Examination

This study was carried out to investigate the morphologic characteristics and localization of antigenic molecules of Pneumocystis carinii in experimentally induced P. carinii pneumonia in rats. After six weeks of administration of low protein diet and dexamethasone, Sprague-Dawley rats were sacrificed to submit lungs or bronchoalveolar lavage for the study. Monoclonal (092, 900, 902, and 904) and polyclonal (SP-D) antibodies were used for immunohistochemistry and immunoelectron microscopy (ITEM and ISEM). Immunohistochemically P. carinii organisms were well identified as clusters or separated forms in the alveolar spaces being frequently attached to the alveolar walls. Immunoelectron microscopically the adherences of gold particles were observed on the surface of all stages of the P. carinii. Occasionally positive immunogold labeling was observed in the cytoplasm of the trophozoites and on the pellicle of the intracystic bodies within the cysts. The monoclonal antibodies 092, 900, 902, and 904 reacted mainly with pellicles of P. carinii, whereas SP-D labeled on the pellicles, intracystic bodies, cytoplasms of the alveolar macrophages, and free floated surfactant material in the alveolar spaces. The immunogold particles were observed more diffusely and intensely in the cysts than in the trophozoites. These results indicate that antigen is mainly localized on the pellicles, and accumulated during development from the trophozoite to the cyst stages.

Mammotrope heterogeneity in the pituitary gland of European ferret, M ustela putorius Juro: An immunoelectron-microscopic study.

Employing the superimposition technique of electron-microscopic immunocytochemistry ultrastructural heterogeneity of the mammοtropes in the pituitary gland of the European ferret, Mustela putorius furo, was studied. On the basis of the size of their secretory granules, the mammotropes were classified into three subtypes, type-I, type-II and type-Ill, which may correspond to different developmental or physiological states of a single cell type. Simultaneous study of mammotropes and somatotropes in several pairs of serial semithin sections demonstrated the occasional occurrence of bihormonal somatomammotropes/ mammosomatotropes which may represent a transitional stage of the progenitor stem-somatotrope during its differentiation into mammotrope; alternatively it may be a functional intermediate during the cross-transformation of somatotrope into mammotrope or vice versa.

The Effect of Ginseng Saponin on the Dopaminergic Neurons in the Parkinson's Disease Model in Mice

Saponin has been known to be a major antioxidant component in panax ginseng. Recent experimental study suggests that some antioxidant materials prevent Parkinson's disease caused by 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) in an animal model. The present study was performed to demonstrate the effect of ginseng saponins in the Parkinson's disease model induced by MPTP. To verify the effect of ginseng saponin on dopaminergic neurons in the mice brain, the tyrosine hydroxylase-immunoreactive (TH-ir) neurons were observed by immunohistochemical stain and immunoelectron microscopy (preembedding method). Also, in order to estimate the immunoreactivity of dopaminergic neuropils, they were quantified by image analysis. The number of TH-ir neurons of substantia nigra was significantly increased in the high-dose (0.46 mg/kg) ginseng saponin group compared with the MPTP injected group. The immunoreactivity of TH-ir neuropils in striatum was significantly increased in both high and low-dose (0.1 mg/kg) ginseng saponin groups compared with the MPTP injected group. In immunoelectron microscopic observation, TH-ir neurons of the control and both ginseng saponin injected group showed normal nuclei and well preserved cytoplasmic organelles. In the MPTP injected group, dying dopaminergic neurons showed destroyed nuclei and cytoplasmic organelles. These results suggest that ginseng saponin has a protective effect on the Parkinson's disease model induced by MPTP.

Fibronectin in the Trabecular Meshwork: Immunofluorescent and Immunoeledron Microscopic Findings

We examined the fibronectin(FN) secretion by cultured trabecular meshwork(TM) cells from a normal human eye by indirect immunofluorescent technique using mouse anti-human FN monoclonal antibody and FITC-conjugated goat anti-mouse IgG. To localize FN on frozen sections of normal TM which were obtained from 7 enucleated eyes owing to traumatic eyeball rupture, the same indirect immunofluorescent method was used. Immunoelectron microscopy was applied to demonstrate the distribution pattern of FN in the normal TM of 2 human eyes using an avidin-biotin-peroxidase complex method. In tissue culture of TM, TM cell walls and extracellular matrices showed an intense staining with antibody to FN. Indirect immunofluorescent staining of FN on frozen sections of TM showed strong positive reactions in the subendothelial region. There was no reaction in the central core of the trabecular beam. Immunoelectron microscopy revealed the reaction products to FN in the areas lining the trabecular endothelial cells.

IMMUNOELECTRON-MICROSCOPIC LOCALIZATION OF A 54-kDa PROTEIN OVEREXPRESSED BY CHLOROQUINE-RESISTANT PLASMODIUM BERGHEI ANKA STRAIN / 中国寄生虫学与寄生虫病杂志

A 54-kDa protein overexpressed by chloroquine-resistant Plasmodium berghei ANKA strain was first reported by us. In this paper, the localization of this protein by immunoelec-tron microscopy is presented. The results showed that the protein was mainly scattered inside the cytoplasm of the early date trophozoites and schizonts of erythrocytic stage of P. berghei ANKA strain , and some of it was also found in cytoplasm of erythrocytes infected with parasites. The protein content was much higher in chloroquine-resistant P. berghei ANKA strain than in chloroquine-sensitive P. berghei ANKA strain, suggesting the importance of this protein in understanding mechanism of chloroquine resistance in malaria parasites .

LOCALIZATION OF NEUROTENSIN-LIKE IMMUNOREACTIVE AMACRINE CELLS IN THE CHICK RETINA:A LIGHT AND ELECTRON MICROSCOPIC STUDY / 解剖学报

Immunohistochemistry was utilized to investigate the light and electron micros-copic localization of neurotensin-like (NT) immunoreactive amacrine cells in thechicken retina.The results showed that the NT-immunoreactive cell bodies wereoval and situated in either the second or third row of cells from the inner borderof the inner nuclear layer.The processes of such cells extended into the innerplexiform layer where they ramified as a fine plexus in sublamina 1 and as a denseplexus in sublamina 3 and 4.At the ultrastructural level,NT positive soma exhibited a rather dense and evenlydistributed immune reaction product throughout their cytoplasm.The nucleus ofNT-amacrine cells possessed a round,unindented nuclear membrane.NT positiveprocesses of such cells receive synaptic input from processes of unlabeled amacrineand bipolar cells.They formed synaptic output onto processes of nonimmuno-reactive amacrine cells and bipolar cells.Moreover,each of the above synapticrelationships were identified in each of sublamina 1 and 3 to 4 of the inner plexiform layer.In addition,NT positive processes fromed synaptic output to processesdevoid of synaptic vesicles,which may originated from ganglion cells.They alsoformed synaptic output to somas situated in the innermost cell row of the innernuclear layer.Identification of synaptic elements and retinal circuitry were also discussed.

ORIGINS AND ULTRASTRUCTURAL DISTRIBUTION OF SEROTONERGIC FIBERS OF THE SPINAL DORSAL HORN IN THE RAT——A STUDY WITH COMBINED METHOD OF HRP AND IMMUNOCYTOCHEMISTRY AND IMMUNOELECTRON MICROSCOPIC OBSERVATION / 解剖学报

Origin and ultrastructural characteristics of serotonergic fibers of the spinal dorsal horn in the rat have been confirmed by means of a combined method of HRP and immunocytochemistry and immunoelectron microscopic observation. The results showed that serotonergic axonal terminals in the spinal dorsal horn come mainly from nucleus raphe magnus and the ventral part of the reticular formation of medulla oblongata. Serotonin immunoreactive positive structures of the spinal dorsal horn have been found in lamina Ⅰ (marginal zone) and lamina Ⅱ (substantia gelatinosa) as fine myelinated and unmylinated fibers. There were mainly axo-axonic synapses between the labeled and nonlabeled terminals. The labeled terminals were presynaptic or postsynaptic element. Axo-dendritic synapses were rarely found. The non-synaptic releasing figures have not been found. Based on the ultrastructural characteristics the authors suggest that in performing analgesia role the serotonergic system in the spinal dorsal horn might influence directly or indirectly the excitability of interneurons and inhibit directly the nerve impulses of primary afferents by means of synaptic connections instead of non-synaptic releasing manner.