14898
2015
eng
29
9
article
1
2017-05-22
--
--
Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation
The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp\(^{69}\)). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp\(^{69}\) AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (syt\(^{KD}\)) in wildtype (wt), brp\(^{69}\) and rab3 null mutants (rab3\(^{rup}\)), where Brp is concentrated at a small number of AZs. At wt and rab3\(^{rup}\) synapses, syt\(^{KD}\) lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, syt\(^{KD}\) did not alter EPSC amplitude at brp\(^{69}\) synapses, but shortened delay and rise time. In fact, following syt\(^{KD}\), these kinetic properties were strikingly similar in wt and brp\(^{69}\), which supports the notion that Syt protracts release at brp\(^{69}\) synapses. To gain insight into this surprising role of Syt at brp\(^{69}\) AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At tonic type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after syt\(^{KD}\). Notably, syt\(^{KD}\) boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that syt\(^{KD}\) increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.
Frontiers in Cellular Neuroscience
10.3389/fncel.2015.00029
urn:nbn:de:bvb:20-opus-148988
Frontiers in Cellular Neuroscience 9:29 (2015). DOI: 10.3389/fncel.2015.00029
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Mila M. Paul
Martin Pauli
Nadine Ehmann
Stefan Hallermann
Markus Sauer
Robert J. Kittel
Manfred Heckmann
eng
uncontrolled
neuromuscular junction
eng
uncontrolled
Bruchpilot
eng
uncontrolled
synaptic delay
eng
uncontrolled
dSTORM
eng
uncontrolled
synaptotagmin
eng
uncontrolled
presynaptic differentiation
eng
uncontrolled
neurotransmitter release
eng
uncontrolled
active zone
eng
uncontrolled
synaptic transmission
eng
uncontrolled
fluorescent probes
Medizin und Gesundheit
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14898/052_Paul_Frontiers_in_Cellular_Neuroscience.pdf
14518
2015
eng
1389-1400
8
article
1
2017-03-02
--
--
Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons
Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, we show that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, we uncovered a novel function of RSK in anterograde axonal transport. Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling.
Disease Models & Mechanisms
10.1242/dmm.021246
urn:nbn:de:bvb:20-opus-145185
Disease Models & Mechanisms (2015) 8, 1389-1400. DOI: 10.1242/dmm.021246
Katherina Beck
Nadine Ehmann
Till F. M. Andlauer
Dmitrij Ljaschenko
Katrin Strecker
Matthias Fischer
Robert J. Kittel
Thomas Raabe
eng
uncontrolled
mrsk2 KO mouse
eng
uncontrolled
S6KII RSK
eng
uncontrolled
transmission
eng
uncontrolled
neuromuscular junction
eng
uncontrolled
synapse
eng
uncontrolled
MAPK signaling
eng
uncontrolled
axonal transport
eng
uncontrolled
motoneuron
eng
uncontrolled
RSK
eng
uncontrolled
Drosophila
eng
uncontrolled
mechanisms
eng
uncontrolled
plasticity
eng
uncontrolled
protein kinase
eng
uncontrolled
signal transduction pathway
eng
uncontrolled
mitochondrial transport
eng
uncontrolled
glutamate receptor
Medizin und Gesundheit
open_access
Physiologisches Institut
Institut für Medizinische Strahlenkunde und Zellforschung
Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie
Rudolf-Virchow-Zentrum
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14518/095_Beck_Disease_Models_Mechanisms.pdf
12880
2013
eng
1407-1413
5
3
article
1
2016-03-07
--
--
Hebbian Plasticity Guides Maturation of Glutamate Receptor Fields In Vivo
Synaptic plasticity shapes the development of functional neural circuits and provides a basis for cellular models of learning and memory. Hebbian plasticity describes an activity-dependent change in synaptic strength that is input-specific and depends on correlated pre- and postsynaptic activity. Although it is recognized that synaptic activity and synapse development are intimately linked, our mechanistic understanding of the coupling is far from complete. Using Channelrhodopsin-2 to evoke activity in vivo, we investigated synaptic plasticity at the glutamatergic Drosophila neuromuscular junction. Remarkably, correlated pre- and postsynaptic stimulation increased postsynaptic sensitivity by promoting synapse-
specific recruitment of GluR-IIA-type glutamate receptor subunits into postsynaptic receptor fields. Conversely, GluR-IIA was rapidly removed from synapses whose activity failed to evoke substantial postsynaptic depolarization. Uniting these results with developmental GluR-IIA dynamics provides a comprehensive physiological concept of how Hebbian plasticity guides synaptic maturation and sparse transmitter release controls the stabilization of the molecular composition of individual synapses.
Cell Reports
10.1016/j.celrep.2013.04.003
urn:nbn:de:bvb:20-opus-128804
Cell Reports 3, 1407–1413. doi:10.1016/j.celrep.2013.04.003
Dmitrij Ljaschenko
Nadine Ehmann
Robert J. Kittel
Medizin und Gesundheit
open_access
Physiologisches Institut
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/12880/020_Hebbian Plasticity Guides Maturation.pdf
11818
2015
eng
doctoralthesis
1
2015-08-20
--
2015-07-23
Linking the active zone ultrastructure to function in Drosophila
Struktur-Funktions-Beziehungen an der aktiven Zone in Drosophila
Accurate information transfer between neurons governs proper brain function. At chemical synapses, communication is mediated via neurotransmitter release from specialized presynaptic intercellular contact sites, so called active zones. Their molecular composition constitutes a precisely arranged framework that sets the stage for synaptic communication.
Active zones contain a variety of proteins that deliver the speed, accuracy and plasticity inherent to neurotransmission. Though, how the molecular arrangement of these proteins influences active zone output is still ambiguous. Elucidating the nanoscopic organization of AZs has been hindered by the diffraction-limited resolution of conventional light microscopy, which is insufficient to resolve the active zone architecture on the nanometer scale. Recently, super-resolution techniques entered the field of neuroscience, which yield the capacity to bridge the gap in resolution between light and electron microscopy without losing molecular specificity. Here, localization microscopy methods are of special interest, as they can potentially deliver quantitative information about molecular distributions, even giving absolute numbers of proteins present within cellular nanodomains.
This thesis puts forward an approach based on conventional immunohistochemistry to quantify endogenous protein organizations in situ by employing direct stochastic optical reconstruction microscopy (dSTORM). Focussing on Bruchpilot (Brp) as a major component of Drosophila active zones, the results show that the cytomatrix at the active zone is composed of units, which comprise on average ~137 Brp molecules, most of which are arranged in approximately 15 heptameric clusters. To test for a quantitative relationship between active zone ultrastructure and synaptic output, Drosophila mutants and electrophysiology were employed. The findings indicate that the precise spatial arrangement of Brp reflects properties of short-term plasticity and distinguishes distinct mechanistic causes of synaptic depression. Moreover, functional diversification could be connected to a heretofore unrecognized ultrastructural gradient along a Drosophila motor neuron.
Kommunikation zwischen Nervenzellen ist von grundlegender Bedeutung für die Hirnfunktion. An chemischen Synapsen findet diese an hoch spezialisierten interzellulären Kontaktstellen statt, den aktiven Zonen, welche die Voraussetzung für präzise Neurotransmission schaffen und somit die synaptische Kommunikation gewährleisten.
In aktiven Zonen befindet sich eine Vielzahl von Proteinen dicht gepackt, die Geschwindigkeit, Genauigkeit und Plastizität der Signaltransduktion vermitteln. Bisher ist es jedoch unklar, in welcher Weise die molekularen Organisationsprinzipien dieser Proteine die Funktion der aktiven Zone beeinflussen. Teilweise ist dies dem Auflösungsvermögen konventioneller Lichtmikroskopie geschuldet, das nicht ausreicht um die Architektur der aktiven Zone im Nanometer Bereich aufzuklären. Unlängst jedoch haben neue Methoden der hochaufgelösten Fluoreszenzmikroskopie ihren Weg in die Neurowissenschaften gefunden. Diese sind in der Lage die Lücke zwischen optischer Lichtmikroskopie und Elektronenmikroskopie zu schließen, ohne die Identität der Proteinspezies aus den Augen zu verlieren. Besonderes Interesse kommt hierbei sogenannten Lokalisationsmikroskopie Techniken zu. Diese können neben der Darstellung molekularer Organisationen im Idealfall sogar quantitative Informationen über die absolute Anzahl bestimmter Moleküle in subzellulären Bereichen liefern.
In der vorliegenden Arbeit wurde eine Methode entwickelt, die auf klassischer Immunohistochemie beruht und dSTORM (direct stochastic optical reconstruction microscopy) nutzt, um die endogene Proteinorganisation in situ zu quantifizieren. Fokussierend auf Brp (Bruchpilot), einem Protein an der aktiven Zone von Drosophila melanogaster, zeigen die Ergebnisse, dass die Zytomatrix an der aktiven Zone modular aufgebaut ist, wobei jedes Modul ~137 Brp Moleküle umfasst. Diese sind zum Großteil in etwa 15 Gruppen mit je 7 Untereinheiten angeordnet. Um auf einen quantitativen Zusammenhang zwischen der Ultrastruktur der aktiven Zone und ihrer Funktion zu schließen, wurden Drosophila Mutanten eingesetzt und mittels Elektrophysiologie funktionell untersucht. Die Ergebnisse veranschaulichen, dass sich spezifische Eigenschaften von Kurzzeitplastizität in der präzisen Anordnung von Brp widerspiegeln, was Rückschlüsse auf verschiedene Ursprünge synaptischer Depression zulässt. Darüber hinaus beschrieben dSTORM Experimente erstmals, dass ein funktioneller Gradient entlang des Motoneurons mit der graduellen Veränderung der Anzahl von Bruchpilotmolekülen pro aktive Zone korreliert.
urn:nbn:de:bvb:20-opus-118186
X 126169
Nadine Ehmann
deu
swd
Taufliege
deu
swd
Elektrophysiologie
deu
swd
Fluoreszenzmikroskopie
deu
swd
Synapse
eng
uncontrolled
Drosophila
eng
uncontrolled
active zone
eng
uncontrolled
structure-function relationships
eng
uncontrolled
super-resolution microscopy
eng
uncontrolled
electrophysiology
eng
uncontrolled
Synapses
eng
uncontrolled
Microscopy
Physiologie und verwandte Themen
open_access
Graduate School of Life Sciences
Universität Würzburg
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/11818/PhD_thesis_Nadine_Ehmann.pdf
14899
2015
eng
7
9
article
1
2017-05-22
--
--
Super-resolution microscopy of the synaptic active zone
Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins
Frontiers in Cellular Neuroscience
10.3389/fncel.2015.00007
urn:nbn:de:bvb:20-opus-148997
Frontiers in Cellular Neuroscience 9:7 (2015). DOI: 10.3389/fncel.2015.00007
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Nadine Ehmann
Markus Sauer
Robert J. Kittel
eng
uncontrolled
excitation-secretion coupling
eng
uncontrolled
Ca\(^{2+}\) channels
eng
uncontrolled
structure-function relationships
eng
uncontrolled
super-resolution microscopy
eng
uncontrolled
active zone
eng
uncontrolled
presynaptic calcium
eng
uncontrolled
neurotransmitter release
Medizin und Gesundheit
open_access
Physiologisches Institut
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14899/053_Ehmann_Frontiers_in_Cellular_Neuroscience.pdf
17752
2018
eng
643
12
article
1
2019-02-27
--
--
Synthetic light-activated ion channels for optogenetic activation and inhibition
Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2\(^{2+}\)) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca\(^{2+}\) might be desirable. Moreover, there is need for an efficient light-gated potassium (K\(^{+}\)) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca\(^{2+}\) and K\(^{+}\) in cell physiology, light-activated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca\(^{2+}\) or for K\(^{+}\), respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca\(^{2+}\)-permeant channel, and to body extension when expressing the light-sensitive K\(^{+}\) channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.
Frontiers in Neuroscience
10.3389/fnins.2018.00643
urn:nbn:de:bvb:20-opus-177520
Frontiers in Neuroscience 2018, Volume 12, Article 643. DOI: 10.3389/fnins.2018.00643
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Sebastian Beck
Jing Yu-Strzelczyk
Dennis Pauls
Oana M. Constantin
Christine E. Gee
Nadine Ehmann
Robert J. Kittel
Georg Nagel
Shiqiang Gao
eng
uncontrolled
optogenetics
eng
uncontrolled
calcium
eng
uncontrolled
potassium
eng
uncontrolled
bPAC
eng
uncontrolled
CNG channel
eng
uncontrolled
cAMP
eng
uncontrolled
Drosophila melanogaster motoneuron
eng
uncontrolled
rat hippocampal neurons
Biowissenschaften; Biologie
open_access
Physiologisches Institut
Julius-von-Sachs-Institut für Biowissenschaften
Theodor-Boveri-Institut für Biowissenschaften
Förderzeitraum 2018
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17752/Beck_Frontiers_in_Neuroscience.pdf