Long-term changes of synaptic transmission in slices of rat visual cortex were induced by intracellular tetanization: bursts of short depolarizing pulses applied through the intracellular electrode without concomitant presynaptic stimulation. Long-term synaptic changes after this purely postsynaptic induction were associated with alterations of release indices, thus providing a case for retrograde signalling at neocortical synapses. Both long-term potentiation and long-term depression were accompanied by presynaptic changes, indicating that retrograde signalling can achieve both up-and down-regulation of transmitter release. The direction and the magnitude of the amplitude changes induced by a prolonged intracellular tetanization depended on the initial properties of the input. The inputs with initially high paired-pulse facilitation (PPF) ratio, indicative of low release probability, were most often potentiated. The inputs with initially low PPF ratio, indicative of high release probability, were usually depressed or did not change. Thus, prolonged postsynaptic activity can lead to normalization of the weights of nonactivated synapses. The dependence of polarity of synaptic modi®cations on initial PPF disappeared when plastic changes were induced with a shorter intracellular tetanization, or when the NO signalling pathway was interrupted by inhibition of NO synthase activity or by application of NO scavengers. This indicates that the NO-dependent retrograde signalling system has a relatively high activation threshold. Long-term synaptic modi®cations, induced by a weak postsynaptic challenge or under blockade of NO signalling, were nevertheless associated with presynaptic changes. This suggests the existence of another retrograde signalling system, additional to the high threshold, NO-dependent system. Therefore, our data provide a clear case for retrograde signalling at neocortical synapses and indicate that multiple retrograde signalling systems, part of which are NO-dependent, are involved.
Using a systematic, whole-genome analysis of enhancer activity of human-specific endogenous retroviral inserts (hsERVs), we identified an element, hsERV PRODH , that acts as a tissue-specific enhancer for the PRODH gene, which is required for proper CNS functioning. PRODH is one of the candidate genes for susceptibility to schizophrenia and other neurological disorders. It codes for a proline dehydrogenase enzyme, which catalyses the first step of proline catabolism and most likely is involved in neuromediator synthesis in the CNS. We investigated the mechanisms that regulate hsERV PRODH enhancer activity. We showed that the hsERV PRODH enhancer and the internal CpG island of PRODH synergistically activate its promoter. The enhancer activity of hsERV PRODH is regulated by methylation, and in an undermethylated state it can up-regulate PRODH expression in the hippocampus. The mechanism of hsERV PRODH enhancer activity involves the binding of the transcription factor SOX2, whch is preferentially expressed in hippocampus. We propose that the interaction of hsERV PRODH and PRODH may have contributed to human CNS evolution.human-specific endogenous retrovirus | DNA methylation | central nervous system | human speciation | retroelement U nderstanding the molecular basis of phenotypic differences between humans and chimpanzees can provide important clues to human-specific behavioral peculiarities and neurological disorders. For this purpose we conducted a genome-wide analysis of human-specific endogenous retroviral (hsERV) inserts that may induce new regulatory pathways by acting as promoters and enhancers (1, 2). HsERVs of the HERV-K(HML-2) group are one of the four families of transposable elements that were able to transpose at the time of the radiation of human lineage from the lineage of its most closely related species, chimpanzee (3). At least 50% of all hsERV elements exhibit promoter activity in human tissues (4). We found only six hsERV inserts in the upstream regions of known human genes, close to transcription start sites. Three of them displayed strong enhancer activity in transient transfection experiments; of these three, only one-near the PRODH gene-matched the transcriptional activity pattern of its endogenous genomic copy. This copy of hsERV is a full-length, almost intact betaretrovirus belonging to the HERV-K(HML-2) group. PRODH encodes a mitochondrial enzyme proline, dehydrogenase (oxidase), that converts proline to D-1-pyrroline-5-carboxylate (5). PRODH regulates proline catabolism, which is vital for normal CNS functioning. Several PRODH mutations are associated with neuropsychiatric disorders, such as schizophrenia (6). Gene knockouts in mice cause severe changes in the executive functioning of the brain (7). Given the potential importance of PRODH in brain functioning and disease, we attempted to characterize its newly recognized hsERV PRODH enhancer. We showed that hsERV PRODH enhancer activity is regulated by methylation and that the hsERV PRODH enhancer and PRODH internal CpG island act syne...
Background SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, a relatively low brightness of the indicator limits its use. Methods Here we designed a new version of pH-sensor - SypHer-2, that has up to three times brighter fluorescence signal in cultured mammalian cells compared to the SypHer. Results Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent temporal neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop which occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. Conclusions SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. General significance The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.
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