Live-cell imaging of endocytosed synaptophysin around individual hippocampal presynaptic active zones.

In presynaptic terminals 4 types of endocytosis, kiss-and-run, clathrin-mediated, bulk and ultrafast endocytosis have been reported to maintain repetitive exocytosis of neurotransmitter. However, detailed characteristics and relative contribution of each type of endocytosis still need to be determined. Our previous live-cell imaging study demonstrated individual exocytosis events of synaptic vesicle within an active-zone-like membrane (AZLM) formed on glass using synaptophysin tagged with a pH-sensitive fluorescent protein. On the other hand, individual endocytosis events of postsynaptic receptors were recorded with a rapid extracellular pH exchange method. Combining these methods, here we live-cell imaged endocytosed synaptophysin with total internal reflection fluorescence microscopy in rat hippocampal culture preparations. Clathrin-dependent and -independent endocytosis, which was seemingly bulk endocytosis, occurred within several seconds after electrical stimulation at multiple locations around AZLM at room temperature, with the locations varying trial to trial. The contribution of clathrin-independent endocytosis was more prominent when the number of stimulation pulses was large. The skewness of synaptophysin distribution in intracellular vesicles became smaller after addition of a clathrin inhibitor, which suggests that clathrin-dependent endocytosis concentrates synaptophysin. Ultrafast endocytosis was evident immediately after stimulation only at near physiological temperature and was the predominant endocytosis when the number of stimulation pulses was small.

Relaxin-3 Innervation From the Nucleus Incertus to the Parahippocampal Cortex of the Rat.

Spatial learning and memory processes depend on anatomical and functional interactions between the hippocampus and the entorhinal cortex. A key neurophysiological component of these processes is hippocampal theta rhythm, which can be driven from subcortical areas including the pontine nucleus incertus (NI). The NI contains the largest population of neurons that produce and presumably release the neuropeptide, relaxin-3, which acts via the G i/o -protein-coupled receptor, relaxin-family peptide 3 receptor (RXFP3). NI activation induces general arousal including hippocampal theta, and inactivation induces impairment of spatial memory acquisition or retrieval. The primary aim of this study was to map the NI/relaxin-3 innervation of the parahippocampal cortex (PHC), including the medial and lateral entorhinal cortex, endopiriform cortex, perirhinal, postrhinal, and ectorhinal cortex, the amygdalohippocampal transition area and posteromedial cortical amygdala. Retrograde tracer injections were placed in different parts of the medial and lateral entorhinal cortex, which produced prominent retrograde labeling in the ipsilateral NI and some labeling in the contralateral NI. Anterograde tracer injections into the NI and immunostaining for relaxin-3 produced fiber labeling in deep layers of all parahippocampal areas and some dispersed fibers in superficial layers. Double-labeling studies revealed that both hippocampal projecting and calcium-binding protein-positive (presumed GABAergic) neurons received a relaxin-3 NI innervation. Some of these fibers also displayed synaptophysin (Syn) immunoreactivity, consistent with the presence of the peptide at synapses; and relaxin-3-positive fibers containing Syn bouton-like staining were frequently observed in contact with hippocampal-projecting or calcium-binding protein-positive neuronal somata and more distal elements. Finally, in situ hybridization studies revealed that entorhinal neurons in the superficial layers, and to a lesser extent in deep layers, contain RXFP3 mRNA. Together, our data support functional actions of the NI/relaxin-3-parahippocampal innervation on processes related to memory, spatial navigation and contextual analysis.

Clinicopathological heterogeneity between primary and metastatic sites of gastroenteropancreatic neuroendocrine neoplasm.

BACKGROUND: Chromogranin A (CgA), synaptophysin (Syn) and the Ki-67 index play significant roles in diagnosis or the evaluation of the proliferative activity of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). However, little is known about whether these biological markers change during tumor metastasis and whether such changes have effect on prognosis. METHODS: We analyzed 35 specimens of both primary and metastatic tumor from 779 patients who had been diagnosed as GEP-NENs at Wuhan Union Hospital from August 2011 to October 2019. The heterogeneity of CgA, Syn and Ki-67 index was evaluated by immunohistochemical analysis. RESULTS: Among these 779 patients, the three most common sites of NENs in the digestive tract were the pancreas, rectum and stomach. Metastases were found in 311 (39.9%) patients. Among the 35 patients with both primary and metastatic pathological specimens, differences in the Ki-67 level were detected in 54.3% of the patients, while 37.1% showed a difference in CgA and only 11.4% showed a difference in Syn. Importantly, due to the difference in the Ki-67 index between primary and metastatic lesions, the WHO grade was changed in 8.6% of the patients. In addition, a Kaplan-Meier survival analysis showed that patients with Ki-67 index variation had a shorter overall survival (p = 0.0346), while neither Syn variation nor CgA variation was related to patient survival (p = 0.7194, p = 0.4829). CONCLUSIONS: Our data indicate that primary and metastatic sites of GEP-NENs may exhibit pathological heterogeneity. Ki-67 index variation is closely related to the poor prognosis of patients with tumor metastasis, but neither Syn variation nor CgA variation is related to patient prognosis. Therefore, clinicopathologic evaluation of the primary tumor and metastatic sites could be helpful for predicting the prognosis.

Effect of Buyang Huanwu Tang on Synaptic Structural Plasticity After Cerebral Ischemia-reperfusion in Rats / 中国实验方剂学杂志

<b>Objective::To investigate the mechanism of Buyang Huanwu Tang (BYHWT) in improving synaptic structural plasticity after cerebral ischemia-reperfusion in rats. <b>Method::Middle cerebral artery occlusion and reperfusion model was established. SD rats were randomly divided into sham-operated group, model group, BYHWT group, BYHWT+ Gap26(connexin43 inhibitor)groups. BYHWT was given twice a day(16 g·kg^-1), Gap26 was intraperitoneally injected once a day since the third day after surgery (25 g·kg^-1). Brain was taken out at the 7^th day. The changes of neuronal synaptic and gap junction ultrastructure were observed by transmission electron microscopy. Synaptophysin (SYN) and growth-associated protein-43 (GAP-43) protein expression were detected by Western blot and immunofluorescence. <b>Result::The structure of synapses was integrated, and the gap junctions were clear in sham-operated group. In the hippocampus of model group, the structure was destroyed, and the gap junctions disappeared. Compared with the sham-operated group, model group up-regulated the expressions of SYN and GAP-43 (<italic>P</italic><0.05, <italic>P</italic><0.01). In the hippocampus of BYHWT group, the structure was close to the normal. Furthermore, BYHWT up-regulated the expressions of SYN and GAP-43 (<italic>P</italic><0.05, <italic>P</italic><0.01). However, after the combined administration with Cx43 inhibitor (Gap26), the damage of synaptic structural decreased, only a small number of gap junctions with the structural integrity can be seen, and the effect of BYHWT on SYN and GAP-43 was inhibited (<italic>P</italic><0.05, <italic>P</italic><0.01). <b>Conclusion::BYHWT could improve the hippocampal synaptic structural plasticity obviously after the CIRI. The mechanism may be related to the increase of the expression of Cx43 and the promotion of the intervention of SYN and GAP-43.

NSCs promote hippocampal neurogenesis, metabolic changes and synaptogenesis in APP/PS1 transgenic mice.

Adult neurogenesis and synaptic remodeling persist as a unique form of structural and functional plasticity in the hippocampal dentate gyrus (DG) and subventricular zone (SVZ) of the lateral ventricles due to the existence of neural stem cells (NSCs). Transplantation of NSCs may represent a promising approach for the recovery of neural circuits. Here, we aimed to examine effects of highly neuronal differentiation of NSCs transplantation on hippocampal neurogenesis, metabolic changes and synaptic formation in APP/PS1 mice. 12-month-old APP/PS1 mice were used for behavioral tests, immunohistochemistry, western blot, transmission electron microscopy and proton magnetic resonance spectroscopy (1H-MRS). The results showed that N-acetylaspartate (NAA) and Glutamate (Glu) levels were increased in the Tg-NSC mice compared with the Tg-PBS and Tg-AD mice 10 weeks after NSCs transplantation. NSC-induced an increase in expression of synaptophysin and postsynaptic protein-95, and the number of neurons with normal synapses was significantly increased in Tg-NSC mice. More doublecortin-, BrdU/NeuN- and Nestin-positive neurons were observed in the hippocampal DG and SVZ of the Tg-NSC mice. This is the first demonstration that engrafted NSCs with a high differentiation rate to neurons can enhance neurogenesis in a mouse model of AD and can be detected by 1H-MRS in vivo. It is suggested that engraft of NSCs can restore memory and promote endogenous neurogenesis and synaptic remodeling, moreover, 1H-MRS can detect metabolite changes in AD mice in vivo. The observed changes in NAA/creatine (Cr) and glutamate (Glu)/Cr may be correlated with newborn neurons and new synapse formation.