Crest synapses of the substantia gelatinosa have been observed in rat spinal cord by means of electron microscopy. They associate with axe-dendritic or dendrodendritic junctions in this study. The postsynaptic elements come from clubbed finger-like dendritic crest which is characterized by prominent postsynaptic membrane thickening, subjunctional bodies and longer active zone. Two presynaptic terminals contact the wall of crest process side by side and contain complex vesicles: spheric vesicles (40～60 nm in diameter), flat vesicles (75?25 nm), medium-sized granular vesicles (about 70 nm) and large granular vesicles (about 125 nm).Crest synapses differ from spine synapses with respect to that they typically contain two junctions, one on each side of the middle part of the crest and that the postsynaptic boutons are always provided with sub junctional bodies.

Observation on the ultrastructure of ependyma in the central canal of 12 rat spinal cords by means of transmission and scanning electron microscope showed that the ependyma is composed of ependymal cells, tanycytes and neurons. The ependymal cells are columnar or cubic in shape. Their apical surfaces are provided with microvilli and cilia which may contact with Reissner's fiber. Neighbouring cells are connected by zonula adherens or zonula occludens. The cytoplasm contains numerous mitochondria and vesicles. Elongated tanycytes situate between ependymal cells. They are provided with microvilli (but no cilia) at apical surfaces. The basal processes thrust into the grey matter, where they inclose capillaries. Abundant microfilaments and microtubes are embedded in the cytoplasm. This paper reports the cerebrospinal fluid-contacting neurons existing in the ependyma of rat spinal cord for the first time. These belong to supraependymal or hypoendymal neurons with small, multipolarcell bodies. Their dendrites and axons could be found in the lumen of central canal. Occasionally, the contact between axon terminal and dendrite was encountered. Morphological evidence mentioned above suggests that ependyma may perform receptive, absorptive and secretory function.

Somatostatin-containing neurons and nerve fibers in the commissural subnucleus of the nucleus tractus solitarii(NTS) of the rat were studied by means of munoelectron microscopy. The results showed that somatostatin-like immunoreactive positive neurons are medium or small cells, fusiform or elliptical. Somatostatin- like immunoreactive positive axons are unmyelinated,and mainly form passage or terminal synapses. Somatostatin-positive cell bodies themselves have not been obser- ved to form synapses with somatostatin-positive fibers,but their dendrites may receive innervation from immunoreactive-negative fibers by axo-dendritic synapses.

Objective To explore the regulating mechanism of the astrocytes on the expression of ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors(AMPARs) subunit in epileptogenesis. Methods The astrocytic conditional medium(ACM) was collected after being stimulated by glutamate,and then ACM was added to the cultured hippocampal neurons.The expression changes of neuronal GluR2 and protein interacting with C-kinase-1(PICK1) mRNA were detected by RT-PCR. Results In the cultured hippocampal neurons,the GluR2 mRNA expression was significantly decreased at the 2nd,8(th),and 12(th) hours after the administration of ACM compared with that in the control group(P

The chemical nature of spinal ganglionic neurons, the peripheral processes of which project divergently to the somatic and visceral areas, has been identified by means of tri-labeling method of combining fluorescein tracing and immunocytochemistry. Ten rats were used. First, 2?l of 2% fast blue (FB) were injected into the left coeliac ganglion. Two days later, 2% nuclear yellow (NY) was injected into left 9-11th intercostal nerves(l?l for each). On the 4th day, animal was perfused with 10% formalin in 0.1mol/L phosphate buffer.The left Th9-11 spinal ganglia were removed and cut into sections by cryostat. The sections were observed under fluorescence microscope and photographed. The results showed that there were three kinds of neurons in the spinal ganglia: (1) single FB labeled cells with blue fluorescent cytoplasm accounted for 38.8% of total labeled cells; (2) single NY labeled cells with yellow fluorescent nuclei accounted for 52,7%; (3) FB and NY double labeled cells accounted for 8.5% and mostly were small or medium in size. Then, the double labeled cells-containing sections were further processed by substance P-demonstrating PAP immunocytochemical staining. The immunostain and fluorescent photographs in the same section were compared and identified each other. We have found that the labeling ratio of SP/NY was 1.4%; SP/FB was 7% and SP/NY+FB was 28.8%. Present study has not only identified the convergence of somato-vesceral sensation in spinal ganglia but also detected the chemical nature of these neurons(substance P) for the first time. In addition, this result has provided a morphological basis for the mechanisma of referred pain and somato-visceral reflection.

Skin-visceral divergent projections of cholecystokinin(CCK)-containing dorsal root ganglion neurons were studied by combined technique of fluorescent double-labelling and immunohistochemistry. Fast Blue(FB) and Nuclear Yellow (NY) were injected into the coeliac ganglion and the cutaneous branches of left 9th-11th intercostal nerves, respectively. Three kinds of neurons labelled with fluorescein were observed in T_(9-11) dorsal root ganglia under Nikon fluorescence microscope with 365 nm excitation light: FB-labelled neurons with blue-fluorescing cytoplasm; NY-labelled neurons with yellow-fluorescing nucleus and double-labelled neurons with blue cytoplasm and yellow nucleus. The double-labelled neurons were found to be 2.8％ of total labelled neurons.The sections containing fluorescein labelled neurons were then stained by CCKimmunohistochemical procedure. Four kinds of neurons could be identified: NY-neurons, with CCK-immunoreactivity (NY+CCK); FB-neurons with CCK-immunoreactivity(FB + CCK);NY + FB neurons with CCK-immunoreactivity(NY + FB + CCK); Single CCK-positive neurons. NY + FB + CCK triple-labelled neurons accounted for approximately 11.5％ of NY + FB double-labelled neurons,and 0.4％ of all CCK-positive neurons.The findings clearly indicated that the peripheral processes of some sensory dorsal root ganglion neurons project divergently to both skin and visceral structure, and contain CCK. The present results suggest that the peripheral dichotomization of the dorsal root ganglion nearons might converge sensory inputs from both skin and visceral fields, and thus not only provide one of the structural basis for the referred pain but also reveal that CCK might play a mediation role in the skin-visceral reflection and referred pain.

Projections from substance P (SP)-and cholecystokinin (CCK) containing neurons in the periaqueductal gray(PAG)and Edinger-Westphal(E-W)nucleus to the spinal cord were studied by means of the combining method of HRP tracing with immunocytochemistry in rats. The results showed that a few neurons in the ventrolateral region of PAG projected bilaterally to the cervical, thoracic and lumbar segments of the spinal cord, with the predominant projections from the ipsilateral side. The authors reported that these descending projection neurons showed SP-like immunoreactivity for the first time(account for 48%). The neurons of E-W nucleus projected diffusely to all segments of the spinal cord contained SP (70%) or CCK (73%) respectively, suggesting that at least a part of E-W neurons projecting to the spinal cord contain both SP and CCK.

The distribution of GABA-containing neurons in the anterior horn of L_(4～5) segments of rat spinal cord and their relationship with somatic efferent were studied by combined method of HRP and immunocytochemistry and immunoelectron microscopic method. The results showed that under light microscope, the GABA-immunoreactive cell bodies and terminals were seen in all layers of the anterior horn including Rexed′s layer IX which located in the anterolateral part of the anterior horn. The GABA-positive neurons had round or triangle cell body with many processes and could be divided into two (large and medium) types. Under electron microscope, GABA-immunoreactive products appeared as small granular deposits located in perikarya, dendrite and axon. In axon terminals the immunoreactive products located at periphery of the synaptic vesicles and on the outer membrane of the mitochondria. The GABA-positive dendrites received symmetrical afferent synapses from GABA-positive or negative axon terminals. The combined method of HRP and immunocytochemistry showed that in Rexed′s layer IX there were HRP single labeled neurons, GABA single labeled neurons and HRP/GABA double labeled neurons. The double labeled neurons accounted for 79% of total HRP labeled cells. Above mentioned results first identified the neurons in Rexed′s layer IX of the anterior horn contain GABA which participate in somatic efferent and receive autoregulation from GABA-neurons and nonGABA-neurons at synaptic level.

The relationship between CCK-and VP-neurons in the rat retrochiasmatic area was studied at ultrastructural level by means of pre-embedding(PAP) double immunoelectron microscopic labeling technique. First, the VP-immunoreactivity was demonstrated by DAB method. After thoroughly washing, the CCK-immuno- reactivity was revealed by ammonium molybdate-TMB method. Being stabili- zed by DAB-cobalt chloride, the sections were embedded in Epon 812. Under ele- ctron microscope, it was observed that in the retrochiasmatic area, the VP-LI products distributed diffusely as high electron dense granular or flocculent depo- sits, whereas the CCK-LI products distributed sparsely as needle-or mass-like deposits. VP-LI perikarya were small in size with oval shape and CCK-LI peri- karya were medium in size with polygonal shape. CCK-LI perikarya and dendri- tes received afferent synapses from non-CCK- and non-VP-axonal terminals VP- LI axons received afferent synapses from VP and non-VP-axonal terminals It was interesting that the VP-LI axonal terminals formed efferent axoaxonic syna- pses with CCK-LI axonal endings and, vice versa, the CCK-LI axonal terminals established also efferent axoaxonic synapses with VP-LI axonal endings. The above mentioned results identified for the first time that in the rat retrochiasma- tic area not only there were CCK- and VP-neurons, but also there were reciprocal synaptic regulations between above two kinds of peptidergic neuron, providing new ultrastructural basis for the regulatory mechanism of the neuroendocrine in hypothalamus.

Objective:To express the recombinant single chain Fv(scFv) in E.coli and reduce immunogenicity and molecular weight of a monoclonal antibody specific for human platelet.Methods:The variable regions of the heavy and light chains of platelet specific antibody SZ 2 were amplified by reverse transcription and polymerase chain reaction.VH and VL gene segments were cloned into pUC Tm and joined together with a (gly 4ser) 3 linker.The resulting scFv was expressed in PET expression system.The expressed recombinant protein was characterized by its size on SDS PAGE,by Western blot,by flow cytometry and its functions.Results:The VH and VL genes were homologous with the published gene sequences of mouse antibody variable region.The recombinant scFv was expressed mostly in the form of inclusion bodies,and the yield was up to 25% of the total cell proteins.Functional studies showed that SZ 2 scFv could bind to platelet and could suppress platelet aggregation induced by ristocatin and thrombin.Conclusion:A recombinant SZ 2 scFv specific against platelet was developed and characterized.