@article{LichterPaulPaulietal.2022, author = {Lichter, Katharina and Paul, Mila Marie and Pauli, Martin and Schoch, Susanne and Kollmannsberger, Philip and Stigloher, Christian and Heckmann, Manfred and Sir{\´e}n, Anna-Leena}, title = {Ultrastructural analysis of wild-type and RIM1α knockout active zones in a large cortical synapse}, series = {Cell Reports}, volume = {40}, journal = {Cell Reports}, number = {12}, doi = {10.1016/j.celrep.2022.111382}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300913}, year = {2022}, abstract = {Rab3A-interacting molecule (RIM) is crucial for fast Ca\(^{2+}\)-triggered synaptic vesicle (SV) release in presynaptic active zones (AZs). We investigated hippocampal giant mossy fiber bouton (MFB) AZ architecture in 3D using electron tomography of rapid cryo-immobilized acute brain slices in RIM1α\(^{-/-}\) and wild-type mice. In RIM1α\(^{-/-}\), AZs are larger with increased synaptic cleft widths and a 3-fold reduced number of tightly docked SVs (0-2 nm). The distance of tightly docked SVs to the AZ center is increased from 110 to 195 nm, and the width of their electron-dense material between outer SV membrane and AZ membrane is reduced. Furthermore, the SV pool in RIM1α\(^{-/-}\) is more heterogeneous. Thus, RIM1α, besides its role in tight SV docking, is crucial for synaptic architecture and vesicle pool organization in MFBs.}, language = {en} } @article{DannhaeuserMrestaniGundelachetal.2022, author = {Dannh{\"a}user, Sven and Mrestani, Achmed and Gundelach, Florian and Pauli, Martin and Komma, Fabian and Kollmannsberger, Philip and Sauer, Markus and Heckmann, Manfred and Paul, Mila M.}, title = {Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation}, series = {Frontiers in Cellular Neuroscience}, volume = {16}, journal = {Frontiers in Cellular Neuroscience}, issn = {1662-5102}, doi = {10.3389/fncel.2022.1074304}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-299440}, year = {2022}, abstract = {Introduction Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. Methods We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Results and discussion Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13\(^{GFSTF}\) 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13\(^{GFSTF}\), Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13\(^{GFSTF}\) subclusters that move toward the AZ center during PHP with unaltered Unc-13\(^{GFSTF}\) protein levels.}, language = {en} } @article{ScherzerHuangIosipetal.2022, author = {Scherzer, S{\"o}nke and Huang, Shouguang and Iosip, Anda and Kreuzer, Ines and Yokawa, Ken and Al-Rasheid, Khaled A. S. and Heckmann, Manfred and Hedrich, Rainer}, title = {Ether anesthetics prevents touch-induced trigger hair calcium-electrical signals excite the Venus flytrap}, series = {Scientific reports}, volume = {12}, journal = {Scientific reports}, doi = {10.1038/s41598-022-06915-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300411}, year = {2022}, abstract = {Plants do not have neurons but operate transmembrane ion channels and can get electrical excited by physical and chemical clues. Among them the Venus flytrap is characterized by its peculiar hapto-electric signaling. When insects collide with trigger hairs emerging the trap inner surface, the mechanical stimulus within the mechanosensory organ is translated into a calcium signal and an action potential (AP). Here we asked how the Ca\(^{2+}\) wave and AP is initiated in the trigger hair and how it is feed into systemic trap calcium-electrical networks. When Dionaea muscipula trigger hairs matures and develop hapto-electric excitability the mechanosensitive anion channel DmMSL10/FLYC1 and voltage dependent SKOR type Shaker K\(^{+}\) channel are expressed in the sheering stress sensitive podium. The podium of the trigger hair is interface to the flytrap's prey capture and processing networks. In the excitable state touch stimulation of the trigger hair evokes a rise in the podium Ca2+ first and before the calcium signal together with an action potential travel all over the trap surface. In search for podium ion channels and pumps mediating touch induced Ca\(^{2+}\) transients, we, in mature trigger hairs firing fast Ca\(^{2+}\) signals and APs, found OSCA1.7 and GLR3.6 type Ca\(^{2+}\) channels and ACA2/10 Ca\(^{2+}\) pumps specifically expressed in the podium. Like trigger hair stimulation, glutamate application to the trap directly evoked a propagating Ca\(^{2+}\) and electrical event. Given that anesthetics affect K\(^+\) channels and glutamate receptors in the animal system we exposed flytraps to an ether atmosphere. As result propagation of touch and glutamate induced Ca\(^{2+}\) and AP long-distance signaling got suppressed, while the trap completely recovered excitability when ether was replaced by fresh air. In line with ether targeting a calcium channel addressing a Ca\(^{2+}\) activated anion channel the AP amplitude declined before the electrical signal ceased completely. Ether in the mechanosensory organ did neither prevent the touch induction of a calcium signal nor this post stimulus decay. This finding indicates that ether prevents the touch activated, glr3.6 expressing base of the trigger hair to excite the capture organ.}, language = {en} } @article{TianYangNageletal.2022, author = {Tian, Yuehui and Yang, Shang and Nagel, Georg and Gao, Shiqiang}, title = {Characterization and modification of light-sensitive phosphodiesterases from choanoflagellates}, series = {Biomolecules}, volume = {12}, journal = {Biomolecules}, number = {1}, issn = {2218-273X}, doi = {10.3390/biom12010088}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254769}, year = {2022}, abstract = {Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future.}, language = {en} } @article{Kirmse2022, author = {Kirmse, Knut}, title = {Non-linear GABA\(_{A}\) receptors promote synaptic inhibition in developing neurons}, series = {Pfl{\"u}gers Archiv - European Journal of Physiology}, volume = {474}, journal = {Pfl{\"u}gers Archiv - European Journal of Physiology}, number = {2}, issn = {1432-2013}, doi = {10.1007/s00424-021-02652-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-267674}, pages = {181-183}, year = {2022}, abstract = {No abstract available.}, language = {en} } @article{GrafRahmatiMajorosetal.2022, author = {Graf, J{\"u}rgen and Rahmati, Vahid and Majoros, Myrtill and Witte, Otto W. and Geis, Christian and Kiebel, Stefan J. and Holthoff, Knut and Kirmse, Knut}, title = {Network instability dynamics drive a transient bursting period in the developing hippocampus in vivo}, series = {eLife}, volume = {11}, journal = {eLife}, doi = {10.7554/eLife.82756}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300906}, year = {2022}, abstract = {Spontaneous correlated activity is a universal hallmark of immature neural circuits. However, the cellular dynamics and intrinsic mechanisms underlying network burstiness in the intact developing brain are largely unknown. Here, we use two-photon Ca\(^{2+}\) imaging to comprehensively map the developmental trajectories of spontaneous network activity in the hippocampal area CA1 of mice in vivo. We unexpectedly find that network burstiness peaks after the developmental emergence of effective synaptic inhibition in the second postnatal week. We demonstrate that the enhanced network burstiness reflects an increased functional coupling of individual neurons to local population activity. However, pairwise neuronal correlations are low, and network bursts (NBs) recruit CA1 pyramidal cells in a virtually random manner. Using a dynamic systems modeling approach, we reconcile these experimental findings and identify network bi-stability as a potential regime underlying network burstiness at this age. Our analyses reveal an important role of synaptic input characteristics and network instability dynamics for NB generation. Collectively, our data suggest a mechanism, whereby developing CA1 performs extensive input-discrimination learning prior to the onset of environmental exploration.}, language = {en} } @article{HeckmannPauli2022, author = {Heckmann, Manfred and Pauli, Martin}, title = {Visualizing presynaptic active zones and synaptic vesicles}, series = {Frontiers in Synaptic Neuroscience}, volume = {14}, journal = {Frontiers in Synaptic Neuroscience}, issn = {1663-3563}, doi = {10.3389/fnsyn.2022.901341}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-274687}, year = {2022}, abstract = {The presynaptic active zone (AZ) of chemical synapses is a highly dynamic compartment where synaptic vesicle fusion and neurotransmitter release take place. During evolution the AZ was optimized for speed, accuracy, and reliability of chemical synaptic transmission in combination with miniaturization and plasticity. Single-molecule localization microscopy (SMLM) offers nanometer spatial resolution as well as information about copy number, localization, and orientation of proteins of interest in AZs. This type of imaging allows quantifications of activity dependent AZ reorganizations, e.g., in the context of presynaptic homeostatic potentiation. In combination with high-pressure freezing and optogenetic or electrical stimulation AZs can be imaged with millisecond temporal resolution during synaptic activity. Therefore SMLM allows the determination of key parameters in the complex spatial environment of AZs, necessary for next generation simulations of chemical synapses with realistic protein arrangements.}, language = {en} } @article{vomDahlMuellerBerishaetal.2022, author = {vom Dahl, Christian and M{\"u}ller, Christoph Emanuel and Berisha, Xhevat and Nagel, Georg and Zimmer, Thomas}, title = {Coupling the cardiac voltage-gated sodium channel to channelrhodopsin-2 generates novel optical switches for action potential studies}, series = {Membranes}, volume = {12}, journal = {Membranes}, number = {10}, issn = {2077-0375}, doi = {10.3390/membranes12100907}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288228}, year = {2022}, abstract = {Voltage-gated sodium (Na\(^+\)) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na\(^+\) influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na\(^+\) channel (Na\(_v\)1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Na\(_v\)1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K\(^+\) channels on the AP shape, we co-expressed either K\(_v\)1.2 or hERG with one of the Na\(_v\)1.5-ChR2 fusions. As expected, both delayed rectifier K\(^+\) channels shortened AP duration significantly. K\(_v\)1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na\(^+\) current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na\(^+\) channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering.}, language = {en} }