| BackgroundAs one type of senior activities in the central nervous system, learning and memory of higher animals including human are key to survival and evolutionary of animals. Learning is a neurological process for new information and knowledge acquisition, whereas memory is a neurological process for storing and "reading" such information. Learning and memory thus become interconnected and inseparable. The neurobiological basis of learning and memory lies on the plasticity of the central nervous system, which includes the plasticity at different levels such as neural networks, neural circuits and synaptic connections, whereas synaptic connection is most important. Synaptic plasticity refers to the capability of the synapse to adjust its function, change the morphology and increase or decrease its number under certain conditions, comprising the change in the efficiency of synaptic transmission as well as in the morphological structure of the synapse. Such change mainly involves:(1)Presynaptic modification: including the synthesis, storage, release of neurotransmitter and the change of their receptors; (2)Postsynaptic modification: including activation of second messenger by their receptors and G proteins, membrane ionic current as well as phosphorylation and dephosphorylation,etc.(3) plasticity of presynaptic or postsynaptic structures: curvature of synaptic interface, postsynaptic density, dendritic spines, etc.(4)The repair of non-neurons. Long-term potentiation and long-term depression both of which represent the major forms of synaptic plasticity have been recognized as the biological basis of learning and memory at the cellular level. It is in the CA1of hippocampus and dentate gyrus of mammals that LTP was observed for the first time. LTP refers to the excitatory postsynaptic potential (EPSP) potentiation which is rapidly generated at the postsynaptic neurons for a long period of time upon brief, rapid and repetitive stimulation of the presynaptic neurons. The EPSP is characterized by shortened latency, elevated amplitude and increased slope..The mechanism of LTP can be divided into three stages:induction phase, which induces the generation of LTP; expression phase, at which LTP is initially occurred lasting minutes to hours; maintenance phase, which is the sustained expression of EPSP.There are three major types of opioid peptides: enkephalin, endorphin and dynorphin. Enkephalin possesses the highest concentration in the central nervous system, which were closely related to learning and memory. In1975, Hughes et al. reported the isolation and purification of two pentapeptides with opiate activity from porcine brain, which they named methionine enkephalin (MET-ENK) and leucine enkephaline (LEU-ENK). A series of studies indicate that enkephlains exert dramatic influences on learning and memory. Kastin et al. first reported that MET-ENK given systemically to rats enhanced acquisition to rats in a complex maze task. Martinez J et al. found that D-ala2-[D-leu5] enkephalin enhanced acquisition of a passive avoidance response and impaired retention of an appetitively motivated Y-maze task. Post-training administration of both enkephalins attenuates CO2-induced amnesia. Others found that administration of LEU-ENK(400ug/kg) or D-ala2-[D-leu5]enkephalin (4ug/kg) by intraperitoneal injection to rats impairs acquisition of active avoidance response. Sweis et al. found that stressed animals which showed little to no impairments in memory exhibited significantly up regulated of enkephalin when compared to stressed animals that showed large deficits in memory. Findings suggest that such neurochemical marker (enkephalin) may be novel targets for pharmacological interventions that can prevent or ameliorate the negative effects of stress on memory. Meilandt WJ et al. found that MET-ENK levels were elevated in entorhinal cortex and the dentate gyrus of transgenic mice of Alzheimer’s disease, and they concluded that enkephalin elevations may contribute to cognitive impairments in transgenic mice of Alzheimer’s disease and possibly in humans with Alzheimer’s disease.Since1957Scoville reported that a patient who underwent bilateral medial temporal-lobe resection developed a grave loss of recent memory, Lashley’s theory that learning and memory are dispersively stored in the brain gradually lost its dominance. More than150years of research on learning and memory-related brain regions found that different learning and memory-related brain regions play different roles in different types of learning and memory. It is now believed that the important brain regions that are associated with learning and memory comprise hippocampus, prefrontal cortex, cerebellum, amygdale, Meynet basal ganglia and cerebral cortex et al. The anterograde and retrograde amnesia may be induced when the medial temporal-lobe comprising hippocampus and the adjacent cortex becomes impaired. Closely related to working memory, the Prefrontal cortex contains three neural circuits, dorsal lateral prefrontal neuronal loop which mediates execution behavior, orbital frontal cortex neuronal loop which mediates social behavior and frontal medial surface neuronal loop which is involved in the formation of motivation. Cerebellum plays an irreplaceable role in learning motor skills and spatial cognitive ability. Cerebral cortex is responsible for early learning and memory dysfunction, but Huntington’s disease and Parkinson’s disease and other diseases whose lesions do not reside in the cerebral cortex also suffer from memory disorder, suggesting that subcortical structures are important neural basis of high cognitive functions. It is later found that lesions of the amygdale prevented the formation of new conditioned reflex and the expression of fear conditioning which had formed in human or animals subsequently. Meynet basal nucleus is involved in the pathogenesis of Alzheimer’s disease, and Meynet basal nucleus was must be damaged in patient with Alzheimer’s disease.The marginal division (MrD) that is a newly discovered area in ventromedial border of the striatum, which locates at the center of the brain. In the year1988, Shu SY at,al. using biotinylation combined immunoperoxidase staining method discovered a significant packed band of fusiform cell bodies in the ventromedial border of the neostriatum, surrounding the rostral and lateral border of the globus pallidus in the brains of rat. It exhibits special cellular organization, neurochemistry and ultra microstructure which is distinctly different from those of the main body of the striatum. Thereafter, MrD was also found in the corresponding sites of cat, monkey and human brains, and it was demonstrated that MrD is an ubiquitous structure in the mammalian brain. The more advanced of the mammalian, the more developed of such structure. This MrD is more densely filled with enkephalin, dynorphin B, substance P Immunoreactive terminals and high density of zinc-containing fiber than the rest of the striatum. The MrD also contains high level of substance associated with learning and memory, such as N-methyl-D-aspartate (NMDA)-receptor, protein kinase A(PKA), calcineurin, cAMP response element binding protein(CREB). There are structural and functional connection between MrD and learning and memory-related brain areas including hippocampus, amygdale, Meynet basal nuclei and prefrontal cortex.etc.. Rats had serious learning and memory dysfunction by double blind Y maze test after damaged bilateral MrD by chemical drug. It has been proved the MrD related to learning and memory function by behavior test and magnetic resonance of brain in healthy human and patient with MrD damaged. MrD is involved in the working memory capacity for numbers by received through hearing, the Chinese paired-word associated learning and memory, both Chinese and English writing, digital computing and spatial memory.On such basis, the present study is intended to investigate the characteristics of synaptic ultrastructure of MET-ENK immunoreactive fibers connected with neurons in the marginal division of the striatum in the monkey brains, providing ultrastructural basis for further exploring the mechanisms of learning and memory function in MrD.Objective1. To observe anatomical position and morphology of of MET-ENK immunoreactive terminals and neurons in the MrD of monkey brain;2. Using immunohistochemical combined with Glucose oxidase-Diaminobenzidine-Nickel ammonium sulfate(GOD) staining method, we observe the synaptic ultrastructure characteristics of MET-ENK immunoreactive terminals in MrD by electron microscope to further study the ultrastructure of synapses in the MrD associated with learning and memory and provide a new approach for learning and memory dysfunction of diagnosis and treatment.MethodsSix male rhesus monkeys were anesthetized with pentobarbital and perfused through the heart with0.9%saline followed by O.lmol/L phosphate buffer (PB) containing4%paraformaldehyde,0.1%glutaraldehyde and0.2%picric acid. Brains were then removed from the skull and fixed in4%paraformaldehyde in4℃refrigerator for about28hours. The brains were immersed in20%sucrose in PB, allowing them to sink to the bottom. The brains were sectioned in the coronal plane on a cryostat microtome to a thickness of50μm. The sections were washed in PB briefly, then they were incubated free-floatingly4℃in an1:3000antiserum against MET-ENK generated in the rabbit for36hours. The sections were incubated for4-8hours at room temperature in a biotinylated goat anti-rabbit IgG diluted in PB. Then they were incubated for4hours in the avidinbiotin-horseradish peroxidase complex solution using a rabbit anti-goat ABC kit from the Vector laboratories. Immunoreactivity was revealed by Glucose oxidase-Diaminobenzidine-Nickel ammonium sulfate staining method. After GOD reaction, the sections were fixed in1%glutaraldehyde for30mins, washed again, and transferred into1%osmium tetroxide. The brain tissues were dehydrated by alcohol gradient and processed with propylene oxide, then they were immersed in Epon816overnight at room temperature. They were mounted on teflon-coated slides, covered by teflon-coated cover slips, and polymerized for three days at37℃,45℃and60℃respectively each day. The sections were examined under light microscope and the regions of the marginal division containing darkly stained of MET-ENK immunochemical products were identified. MrD was removed under a macroscope. Small pieces from the labeled marginal division were cut from the sections and mounted onto the top of an Epon block with cyanoacrylate glue. Ultrathin sections were cut on an ultratome, stained with1%lead citrate and viewed under a JEOL-2000EX electron microscope. Results1. Immunohistochemical staining of MET-ENK expression in the rhesus monkey striatum The putamen was the outer segment of striatum medially connected with globus pallidus. A dark band of MET-ENK positive immunoreacted products was observed at the ventromedial margin pf the putamen with no positive imunoreactivity expressed in the inner segment of globus pallidus under microscope. There were a band of abundant MET-ENK immunoreactive neural fibers, terminals and scattered neurons located between the putamen and globus pallidus, along with the medial border of the putamen. The band with purple blue color in the MrD stained lighter than the outer segment of globus pallidus, but darker than other parts of putamen striatum MrD (Figure1).2. Electric morphology and ultrastructure of spindle cells in the marginal division of striatum observed by electron microscope The neuronal cells of MrD had an elongated spindle or fusiform shape and the perikaryon of the cell body was scanty in organelles by electron microscope. The cells occupied mostly by a large nucleus with pale, oval in shape. A few mitochondria, ribosomes and granular endoplasmic reticulum were unevenly scattered in the cytoplasm. Indicated by the arrows to the left side were a few MET-ENK immunoreactive Axo-somatic synapses. There were five types of synapses of MET-ENK immunoreactive dendrites, Axons, cell body and spines in the MrD.(1) Axo-dendritic synapses Most of the immunoreactive MET-ENK Axo-dendritic synapses were symmetric with mainly medium-sized, round or pleomorphic vesicles, and mitochondria were often observed (Fig3A). A single labeled synaptic terminal was seen making contact with two or more dendrites, which were asymmetric (Fig3B). Dendrites were occasionally received synapses with two or more active zones (Fig3C).(2) Axo-spinous synapses All immunoreactive MET-ENK Axo-spinous synapses were asymmetric (Fig4). The vesicles of the Axo-spinous synapses were small to medium-sized, round or pleomorphic and clear. A single labeled synaptic terminal was often found forming synapses onto several spinous, which were asymmetric (Fig4A). In some cases an Axon was observed forming two asymmetric synapses on a spine (Fig4B).(3) Axo-somatic synapses Both symmetric (Fig5A) and asymmetric (Fig5B) Axon(labeled)-somatic synapses were observed in the MrD.(4) Axo-axonic synapses Two types of axo-axonic synapses were observed in MrD. There was an immunoreactive MET-ENK axon-axonic synapse with two active zones, and they two axon terminals made symmetric synapses onto an unlabeled cell body(Fig6A). An immunoreactive MET-ENK terminal formed symmetric synapses with two unlabeled terminal (Fig6B).(5) Compound synapses Compound synapse is a synapse with more than three neural elements. We found that there were mass compound synapses in MrD. An unlabeled spine contacted onto a labeled Axon and the labeled Axon contacted onto unlabeled soma, which were asymmetric, thus forming an spine-Axo-soma serial synapse (Fig7A). An immunoreactive MET-ENK formed symmetric synapses with two dendritics and a spine(Fig7B).Conclusion:The immunoreactive MET-ENK synapses of the MrD which is a newly discovered region of learning and memory were diverse and complex, mainly containing Axo-dendritic synapses, Axo-spinous synapses Axo-somatic synapses, Axo-axonic synapses and a variety of compound synapses. The complex ultrastructural characteristics of sunapses in the striatum of MrD may be responsiblefor the mechanism of the learning and memory function of MrD, which is differentfrom the role of striatum. Further investigation into the mechanism of enkephalin inlearning and memory function MrD is needed. |
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