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No description available.

The majority of rapid cell-to-cell communication mechanisms and information processing within the nervous system makes use of chemical synapses. Fast neurotransmission on these sites not only requires very close apposition of pre- and postsynaptic partners, but also depends on an effective structural arrangement of cellular components on both sides of the synaptic cleft. Synaptic vesicles fuse at active zones (AZs), characterized by an electron-dense protein mesh of insufficiently characterized composition and function. EM analysis of synapses identified electron dense structures thought (but not proven) to play an important role for vesicle release efficacy. The molecular organization of presynaptic AZs during Ca2+ influx–triggered neurotransmitter release is currently a focus of intense investigation. Due to its appearance in electron micrographs, dense bodies at Drosophila synapses were named T-bars. Together with the lab of Erich Buchner, we recently showed that Bruchpilot (BRP) of the Drosophila melanogaster, homologous to the mammalian CAST/ERC family in its N-terminal half, is essential for the T-bar assembly at AZs and efficient neurotransmitter release respectively. The question, in which way BRP contributes to functional and structural organization of the AZ, was a major focus of this thesis. First, stimulated emission depletion microscopy (STED), featuring significantly increased optical resolution, was used to achieve first insights into ‘cytoarchitecture’ of the AZ compartment. In addition, in vivo live imaging experiments following identified populations of synapses over extended periods were preformed to address the trafficking of protein at forming synapses and thereby providing a temporal sequence for the AZ assembly process. Apart from BRP, two additional AZ proteins, DLiprin-α and DSyd-1, were included into the analysis, which were both shown to contribute to efficient AZ assembly. Drosophila Syd-1 (DSyd-1) and Drosophila Liprin-α (DLiprin-α) clusters initiated AZ assembly, finally forming discrete ‘quanta’ at the AZ edge. ELKS-related Bruchpilot, in contrast, accumulated late from diffuse pools in the AZ center, where it contributed to the electron dense specialization by adopting an extended conformation vertical to the AZ membrane. We show that DSyd-1 and DLiprin-α are important for efficient AZ formation. The results of this thesis describe AZ assembly as a sequential protracted process, with matured AZs characterized by sub-compartments and likely quantal building blocks. This step-wise, in parts reversible path leading to mature AZ structure and function offers new control possibilities in the development and plasticity of synaptic circuits. / Durch Ca2+ abhängige Neurotransmitterfreisetzung vermitteln chemische Synapsen die schnelle Informationsübertragung zwischen Nervenzellen. Vorausetzung hierfür sind gewisse zelluläre Eigenschaften, wie eine enge Korrelation zwischen der Prä- und Postsynapse und eine hoch spezialisierte Zusammensetzung von Proteinen. Synaptische Vesikel fusionieren mit der präsynaptischen aktiven Zone (AZ), welche sich aus einem dichten Netzwerk an vielfach noch unerforschter synaptischer Proteine zusammensetzt, das im Transmissionselektronenmikroskop elektronendicht erscheint. Des Weiteren sind ultrastrukturell elektronendichte präsynaptische Spezialisierungen erkennbar (dense bodies), die vermutlich (aber nicht nachweislich) bei der Freisetzung synaptischer Vesikel eine tragende Rolle spielen. Der molekulare Aufbau der AZ ist zurzeit ein weitverbreitetes Studienthema. Die Synapsen der Fruchtfliege Drosophila melanogaster sind präsynaptisch gekennzeichnet durch eine elektronendichte Struktur, welche aufgrund ihrer charakteristischen Form auch als „T-bar“ bezeichnet wird. Durch die Kooperation mit dem Labor von Erich Buchner gelang es uns, das synaptische Protein Bruchpilot (BRP) zu identifizieren. BRP weist im N-terminalen Bereich Homologien zu der in Säuger gefundenen CAST/ERC Proteinfamilie auf, und ist essenziell für die Ausbildung der elektronendichten T-bars an den AZs und für eine effiziente Ausschüttung von Neurotransmitter. In wie weit BRP für die funktionelle und strukturelle Organisation der AZ verantwortlich ist, sollte in der vorliegenden Arbeit erläutert werden. Durch die neu entdeckte „stimulated emission depletion“ Mikroskopie (STED), ist es nun möglich, dank der erhöhten optischen Auflösung, neue Einsichten in die Architektur der AZ zu erlangen. Zusätzlich wurden mit Hilfe von in vivo Experimenten an lebenden Tieren Populationen von Synapsen über längere Zeiträume verfolgt, um so die Synapsenentstehung und den Proteintransport zu untersuchen. Auf diesem Weg sollte eine Abfolge der an der AZ Assemblierung beteiligten Proteine erstellt werden. Neben BRP wurden daher noch zwei weitere AZ Proteine berücksichtigt (DLiprin-α und DSyd-1), welche ebenfalls bei der Bildung neuer synaptischer Kontakten mitwirken. Es konnte gezeigt werden, dass Proteincluster aus Drosophila Syd-1 (DSyd-1) und Drosophila Liprin-α (DLiprin-α) sehr früh während der Bildung neuer synaptischer Kontakte erscheinen und hierbei diskrete ‚Quanta‘ ausbilden, welche sich am Rand der AZ anlagerten. BRP hingegen erreichte die AZ zu einem späteren Zeitpunkt, wahrscheinlich aus diffusen Reservoirs und akkumulierte schließlich im Zentrum der AZ. Mit Hilfe der STED und konfokalen Mikroskopie konnte gezeigt werden, dass sich BRP in einer getreckten, vertikal zur Membran stehenden Orientierung in die elektronendichte Stuktur, den T-bar, einfügt. Zudem sind DSyd-1 und DLiprin-α für eine effiziente Entstehung neuer AZs erforderlich. Die in dieser Arbeit vorgestellten Ergebnisse deuten auf ein länger andauerden sequenziellen Assemblierungsprozess der AZ hin, in dem aus quantalen Baueinheiten Subkompartimente an ausgereiften AZs gebildet werden. Dieser gestaffelte, teils reversible Reifungsablauf der AZ eröffnet neue Möglichkeiten zur Kontrolle der Entwicklung und Plastizität neuronaler Netzwerke durch einen noch nicht beschriebenen Mechanismus.

Acetylcholine modulates neuronal activity in the brain with different responses in activity depending on the region of the brain. Our study was focused on the cholinergic modulation of excitatory synaptic transmission in the monkey anterior cingulate cortex (ACC) and lateral prefrontal cortex (LPFC), with specific focus on the effects of carbachol, a cholinergic agonist, on spontaneous excitatory postsynaptic currents (sEPSCs) and on the expression the muscarinic cholinergic type II (M2) receptor in these regions. We used electrophysiology to analyze the effects of carbachol on sEPSC of layer 3 (LIII) pyramidal neurons from each area. We used confocal microscopy to study the M2 colocalization with axon terminals labeled with vesicular glutamate transporter 1 (VGLUT1) in the ACC and LPFC, and the colocalization of M2 with specific axon terminals from the amygdala labeled with tracer and terminating in the ACC. Results from the electrophysiological experiments showed that both the ACC and LPFC L3 neurons responded to carbachol by decreasing the frequency of sEPSCs. Cells from the LPFC showed a decrease in sEPSC frequency after 4 minutes in carbachol, an earlier timepoint than ACC neurons, which showed a decrease in sEPSCs frequency after 6 minutes in carbachol. In the confocal studies, M2 expression and colocalization with VGLUT1 terminals in the ACC and LPFC were observed. However, we observed a greater total area of M2 expression in the ACC versus the LPFC in layer 1. We found minimal colocalization of the M2 receptor with axon terminals from the amygdala in the ACC. Together, our data show that acetylcholine has distinct interactions with neurons and pathways in ACC and LPFC, which may be related to the distinct function of the two areas in cognition, learning and memory.

Successful synaptic transmission at the neuromuscular junction depends on the precise alignment of the nerve terminals with the postsynaptic specialization of the muscle fiber. It is increasingly apparent that this precision is achieved during development and maintained in the adult through signals exchanged between motoneurons and their target muscle fibers that serve to coordinate their spatial and temporal differentiation. Several aspects of neuronal differentiation appear to be dependent on retrograde signals from the target and studies about synaptic modulation have now focused attention on the characterization of proteins that mediate retrograde signals regulating the organization and function of nerve terminals. According to the published evidences, we find Insulin-like growth factor-I (IGF-I ) might be one of these potential factors. The acute application of IGF-I, a factor which has been addressed to widely express in developing myocyte, dose-dependently enhances the spontaneous acetylcholine secretion at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch clamp recording. The IGF-I-induced potentiating effect is not abolished when calcium is eliminated from culture medium or bath application of pharmacological calcium channel blocker cadmium, indicating calcium influx through voltage-activated calcium channels are not required. We further define the roles of intracellular Ca2+ stores in IGF-I-induced synaptic potentiation. To approach this problem, Ca2+-ATPase inhibitor thapsigargin were initially used to deplete internal Ca2+ stores. IGF-I no longer elicited any changes in SSC frequency in thapsigargin-treated synapses suggesting that an increase in [Ca2+]i due to Ca2+ release from intracellular Ca2+ stores may contribute to the facilitation of transmitter release induced by IGF-I. Application of membrane-permeable inhibitors of IP3-induced Ca2+ release 2-aminoethoxydiphenyl borate (2-APB) or Xestospongin C (XeC) effectively occluded the increase of SSC frequency elicited by IGF-I. Furthermore, pretreatment of the cultures with ryanodine receptor antagonist 8-(dethylamino) octyl 3, 4, 5-trimethoxybenzoate (TMB-8) also blocked the IGF-I effects indicating that IGF-I activates IP3 and/or ryanodine pathway to initiate calcium release from intracellular stores which subsequently potentiate transmitter release. Treating cells with inhibitors of phosphoinositide-3 kinase (wortmannin and LY294002) and Phospholipase C-g (U73122), but not inhibitor of MAP kinase (PD98059) abolishes IGF-1-induced potentiation of synaptic transmission. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by KN-62 effectively blocks the effect of IGF-I. Taken collectively, our results obtained suggest that IGF-I potentiates neurotransmitter secretion by stimulating Ca2+ release from IP3 and ryanodine sensitive intracellular calcium stores via activate PI3 and/or PLC-g signaling cascades, which leading to an activation of CaMKII-dependent transmitter release.

Long-term potentiation (LTP) is a widely studied cellular mechanism of learning and memory. LTP occurs at excitatory synapses on dendritic spines. Two hours after LTP induction in mature rat hippocampal slices, a reduction in spine number that is perfectly balanced by enlargement of the remaining synapses was previously observed. The sequence of events by which mature synapses enlarge is not well understood, but potential pre- and postsynaptic ultrastructural correlates of synapse growth have been identified. Vesicle-free transition zones (VFTZs) are postsynaptic thickenings contiguous with the PSD that have no apposing presynaptic vesicles perpendicular to the presynaptic membrane. VFTZs could be regions where synapses have expanded postsynaptically, but to which presynaptic vesicles have not yet been recruited. Presynaptic 80-nm dense core vesicles (DCVs) transport active zone proteins to the synapse during synaptogenesis, and may perform the same function during synaptic plasticity. 3-D reconstructions from ssTEM were used to investigate changes in VFTZs, DCVs, and presynaptic vesicles following LTP induction. By 30 minutes, VFTZ area and docked vesicle counts decreased, suggesting mobilization of additional vesicles to the synapse and enhanced release or delayed recycling. By two hours, VFTZs enlarged, suggesting VFTZ assembly contributes to synapse enlargement. DCV counts at 2 hours decreased relative to that at 30 minutes in both control and LTP conditions, suggesting DCVs were inserted at existing synapses to enlarge potentiated synapses in the LTP condition and to support ongoing spinogenesis in the control condition. The overall vesicle count in presynaptic boutons decreased at 2 hours following LTP induction, but docked vesicle count did not. Docked vesicle count was elevated at 2 hours relative to 30 minutes, suggesting that the depletion of docked vesicles observed at 30 minutes was followed by a replenishment and enhancement by 2 hours supplied by the non-docked vesicle pool. That the largest spines had more and larger VFTZs and recruited more DCVs and docked vesicles, and that the ratio of the sum of VFTZ area to the sum of PSD area is constant, provide further evidence that dendritic segments serve as functional units that manage resources in a coordinated and homeostatic way. / text

University, Diss., 2005--Würzburg.

Universiẗat, Diss., 2004--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2003.

Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn plays a key role in the development and maintenance of pathological pain states and chronic pain diseases. Important role in this process play Transient receptor potential Vanilloid 1 receptors (TRPV1), present on presynaptic endings of primary afferents in the superficial spinal cord dorsal horn. Changes in TRPV1 activity have significant impact on nociceptive transmission. There are number of processes that influence the function of spinal TRPV1 receptors. This work is focused on the role of protease-activated receptors type 2 (PAR2), C-C motif chemokine ligand 2 (CCL2) and the effect of chemotherapeutic drug paclitaxel in modulation of synaptic nociceptive transmission and activation of TRPV1 receptors. PAR2 receptors belong to a family of four G-protein-coupled receptors activated by proteases. The role of PAR2 receptors in pain perception is closely related to their presence in a population of dorsal root ganglion neurons, where they are also co-expressed with TRPV1. Activation of PAR2 may lead to peripheral and central sensitization. Chemokine CCL2 and its main receptor CCR2 were suggested to be an important factor in the development of neuropathic pain after peripheral nerve injury. In our study we focused on the...

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