A Study On The Effect And Mechanism Of Rat Astrocyte On Synaptic Formation And Transmission

In the central nervous system (CNS), astrocytes (AST) envelope the pre- and post-synaptic neuronal components and these "peri-synaptic glial cells" have come to be regarded as a third integral component of synapses. However, their roles at synapses have remained incompletely understood. Recent evidence suggests that astrocytes have important functions in addition to their classical role in the trophic support of neurons. These newly discovered functions include regulation of neuron communication, neurosecretion etc. Wherease less evidence about astrocyte on synaptogenesis and synaptic plasticity was reported. The evidence that astrocyte directly enhance synapse formation has mainly been obtained from the experiments in which Pfrieger and Barres found astrocytes conditioned medium could increase the number of synapse formed on the retinal ganglion cell (RGCs). So far a few studies has been conducted in different neuronal cell lines, in hippocampal neurons generated from stem cells of adult rats, and in cultured neurons from the spinal cord, hippocampus and hypothalamus of embryonic or postnatal rats. Furthermore, In the peripheral nervous system (PNS) Schwann cells participate in the formation or maintenance of synapses at the neuromuscular junction (NMJ). These findings raise important questions regarding just how glial cells enhance synapses formation and regulate synaptic plasticity. Prevenient studies have shown that steroids in the brain not only take part in the development, sexual differentiation, physiology and pathology of the CNS, but also play an part role in the regulation of synapse function. The steroids in the brain, therefore, were termed "neurosteroid". Our recent studies have shown that aromatase p450 is highly expressed in glioma cells and neural stem cells, which is the key enzyme for the synthesis of estrogen from androgen. These findings indicate astrocytes may produce estrogen, furthermore, our studies have also shown that estrogen receptors (ERs) are abundantly expressed by neurons. Consequently, we proposed that astrocytes maybe influence the synaptogenesis and synaptic transmission through ERs which activate the intracellular response cascade. To examine our hypothesis that whether AST participates in the processof synapse formation and synaptic transmission, and that what is the potential mechanism, the experiment was carried out based on the pure neuronal culture through ELISA, immunofluorescence, synaptic puncta count, electrophysiological technology, and presynaptic vesicle release marked by FM 4-64. Now we show our results as follows:1. Pure cultured cortical astrocytes can synthesize and secrete estrogen, however pure cultured cortical neurons can't.In the first part of this study, we opitimized the cultural methods and conditions about the purification of astrocytes and neurons based on those references reported. The purity of these two kinds of nervous cells reached 99% after tests which were cell counting and immunofluorescence against glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP 2) and neurofilament 200 (NF 200). On the 2h, 7th day, 14th day and 21rt day after passage, we detected the concentration of estrogen (E2) in the astrocyte-conditioned medium (ACM) by using ELISA techniques. The content of those time points were (ng/L ):0n117+2K 266 +22 > 252+27, however no estrogen was detected in the control medium. The concenteration of estrogen increased in correspondence with the culturing days and reached the peak around the 14th d, then decreased gradually but kept at a certain high level on the 21rt day. Compared with pure cultured astrocytes, neurons can't produce estrogen because no estrogen was detected in the neuronal culture medium. These results demonstrated that E2 was not from neuronself but mainly from ACM when ACM was added into pure cultured neurons.2. Exogenic estrogen can mimic the effect of ACM (or AST) on enhancing the synaptogenesis and synaptic transmission.In this part of the trial, the experimental groups were designed as follows: pure neuron cultures(Group P), mixed cultures (group M), ACM cultures (Group A), Estrogen cultures(group E). The number of synapse and the mini postsynaptic currents (mPSCs) were measured through synaptic counting and electrophysiological techniques. The results showed the number of synapse formed in the Group P, A > M and E were: 14 + 3, 79 ± 9, 83 ± 111 80 ± 9, and the mean amplitude and frequency of mPSCs were: 20.5 ± 2 pA, 13 ±4 /min; 29.1 ±3 pA, 73 + 16 /min; 31.3±3 pA, 78±20/min; 30.2 + 3 pA, 76+18 /min. From the above data we found that astrocytes-neurons cocultured or when ACM was added into pure cultured neurons, the synapse number and the amplitude and frequency of mPSCsincreased dramatically compared with Group P. This revealed that AST regulate synaptic formation and function not through direct mechanical contact but mainly through some soluble factors secreted from AST. When external estrogen was supplemented to pure cultured neurons, the same effect that E2 can mimic the the effect of ACM or AST appeared. Togetherwith the first experiment, we can deduce that the impact of AST on enhancing synapse formation and strengthening the mPSCs was mediated mainly by soluble factors contained by ACM which maybe the E2 secreted by AST itself. To prove this assumption and explore the potential mechanism, we carried out the third part of the experiment. 3. Astrocyte-derived estrogen may modulate synapse formation and synaptic transmission mainly through estrogen receptor.In the final part of the experiment, estrogen receptor (ER) expressed mostly in the nucleus was found in the pure cultured neurons. As we all know, the key signal pathway of estrogenic biological effect was the so called "genie mechanism" mediated by ER (however, some biological effect of estrogen was mediated by "non-genic mechanism"). If AST-derived E2 was the factor of ACM enhancing synapsegenesis and synaptic activity, the ER antagonist can block the effect of ACM. So the Tamoxifen which is ER antagonist was added into pure cultured neurons (Group A+T), and the control group: Group P and Group T which only Tamoxifen was added in were set up. We found that the number of synapse formation decreased from 79±9 to 32 ±3, and the mean amplitude and frequency of mPSCs also decreased to 24.5+2 pA, 35 + 10/min. Nonetheless, it was very interesting that the number of synapse and the amplitude and frequency of mPSCs in Group T which is 29 ±3, and 22.13+2 pA, 37 + 10/min respectively, were more than the corresponding data of Group P. As a result we can see astrocyte-derived E2 is the main molecule which mediated the effect of ACM on synapse through ER. Finally, to make clear whether astrocyte derived E2 regulate the synaptic transmission via presynapse, the assay of presynaptic vesicle release marked by a dye named FM 4-64 was carried out. The same results were obtained that in Group A, M and E the kinetics of vesicle release were similar, which were faster than in just pure neuronal cultures and in neuronal cultures containing tamoxifen alone. But Tamoxifen couldn't completely inhibited the effect of ACM on synapse vesicle release.In conclusion, our study found that astrocytes not only have important functions fortheir classical role in the CNS, but also enhance the synaptogenesis and synaptic transmission. These newly discovered functions maybe mediated by secreted estrogen that stimulates ER activating a series of signal cascade which causes the increase in synapse formation, the enhancing of presynaptic vesicle release and the positive change in synaptic transmissible efficiency.