Lower motor neurons of the peripheral nervous system are responsible for innervating skeletal muscle and controlling all voluntary movements of the body. Degeneration of motor neurons underlies conditions such as amyotrophic lateral sclerosis and spinal muscular atrophy. The identification of genetic factors that influence the form and function of the peripheral nervous system in vivo will be important for our understanding of the neuromuscular system in health and disease. Here, I have studied the effects of three different genes and their respective protein products on the peripheral nervous system: yellow fluorescent protein (YFP), apolipoprotein E (apoE) and Ercc1 (excision repair cross-complementing group 1). YFP has been used as a reporter protein in many fields of research, including as a powerful tool for visualising neurons in mice. It is used under the assumption that it is biologically inert. However, my findings have revealed that YFP expressed in neurons in mice is not inert: it induces a cell stress response at both the RNA and the protein level and alters the time course of dying-back neuropathy. ApoE is a lipid transport protein with three distinct isoforms in humans (apoE2, apoE3 and apoE4), which are known to differentially affect risk and outcome in a number of central nervous system disorders. However, the effects of different apoE isoforms on the peripheral nervous system have yet to be established. I have shown that apoE4 delays peripheral nerve regeneration and subsequent neuromuscular junction reinnervation compared to apoE3, in the absence of any effects on normal form or function, degeneration or developmental plasticity. Ercc1 protein is involved in several DNA repair systems. Ercc1Δ/- mice have reduced levels of functional Ercc1 protein, which leads to a reduced life span and motor abnormalities, potentially due to a build of up DNA damage. Here I have shown that Ercc1Δ/- mice also have increased abnormalities at the neuromuscular junction (an early pathological target in neurodegeneration) with age. These findings contribute significantly to our understanding of the influence of specific genes on the form and function of the peripheral nervous system in health and disease.

The crayfish claw opener neuromuscular junction (NMJ) is a biological model for studying presynaptic neuromodulation by serotonin and synaptic vesicle recycling. Serotonin acts on crayfish axon terminals to increase the release of the neurotransmitter glutamate, but a complete understanding of its mechanisms of action are unknown. In order to sustain enhanced neurotransmission over long periods of time, it was hypothesized that serotonin recruits (activates) a population of previously non-recycling vesicles to become releasable and contribute to neurotransmission. To determine if serotonin activates a distinct population of synaptic vesicles, FM1-43 fluorescence unloading experiments were performed on crayfish excitatory opener axon terminals. These experiments could not resolve a serotonin-activated population of synaptic vesicles, but instead revealed that synaptic vesicles change behaviour in axon terminals independent of serotonin, with vesicles becoming less likely to exocytose and unload FM1-43 dye over time. The change in behaviour was hypothesized to be due to conversion of vesicles from a recycling (releasable) status to a reserve (reluctant to release) status. Synaptic vesicle pool localization was then tested using photoconversion of FM1-43 and transmission electron microscopy techniques. The spatial location of FM1-43-labeled vesicles fixed 2 minutes following 20 Hz stimulation did not reveal retention of vesicles specifically near release sites and the distribution of FM1-43-labeled vesicles was not significantly different between early (2 min) and late (180 min) time points. Terminals fixed 30 seconds following stimulation, however, contained numerous endosome-like structures - the most frequently observed structures resembled large vesicles, which were the equivalent of 2-5 regular vesicle sizes. These results suggest that crayfish axon terminals recycle vast amounts of membrane in response to sustained 20-Hz stimulation and endocytosis appears to occur via multiple routes with the most common being through large vesicle intermediates. / Graduate

No description available.

Polymer Light-Emitting Electrochemical Cells (PLECs) are solid state devices based on the in situ electrochemical doping of the luminescent polymer and the formation of a p-n junction where light is emitted upon the application of a bias current or voltage. PLECs answer the drawbacks of polymer light-emitting diodes as they do not require an ultra-thin active layer nor are they reliant on low work function cathode materials that are air unstable. However, because of the dynamic nature of the doping, they suffer from slow response times and poor stability over time. Frozen-junction PLECs offer a solution to these drawbacks, yet they are impractical due to their sub-ambient operation temperature requirement. Our work presented henceforth aims to achieve room temperature frozen-junction PLECS. In order to do that we removed the ion solvating/transporting polymer from the active layer, resulting in a luminescent polymer combined solely with a salt sandwiched between an ITO electrode and an aluminum electrode. The resulting device was not expected to operate like a PLEC due to the absence of an ion-solvating and ion-transporting medium. However, we discovered that the polymer/salt devices could be activated by applying a large voltage bias, resulting in much higher current and luminance. More important, the activated state is quasi static. Devices based on the well-known orange-emitting polymer MEH-PPV displayed a luminance storage half-life of 150 hours when activated by forward bias (ITO biased positively with respect to the aluminum) and 200 hours when activated by reverse bias. More remarkable yet, devices based on a green co-polymer displayed no notable decay in current density or luminance even after being stored for 1200 hours at room temperature! PL imaging under UV excitation demonstrates the presence of doping. These devices are described herein along with an explanation of their operating mechanisms. / Thesis (Master, Physics, Engineering Physics and Astronomy) -- Queen's University, 2013-11-29 17:40:19.152

Transforming growth factor-βs (TGF-βs) are highly expressed in neural development but why the adult nervous system continues to express them is unclear. TGF-β2 is concentrated at mature neuromuscular junctions (NMJs) of mammalian skeletal muscle fibres, and the nerve terminal expresses TβR-II receptors. To test the role of TGF-β2 at mammalian NMJs, I performed four experiments. The first study tested whether TGF-β2 acutely modulates synaptic transmission at mature mammalian NMJs. Second, I asked if chronically reduced TGF-β2 expression disrupts synaptic transmission. Third, I asked if TGF-β2’s effects differ in terminals adapted to different activity patterns in vivo. Lastly, I asked whether TGF-β1, a related peptide to TGF-β2, is distinct in terms of its effects on transmitter release. Using single electrode potential recording, I found TGF-β2 significantly increased the amplitude of spontaneous released single neurotransmitter vesicles (miniature endplate potentials, MEPPs) and nerve stimulation evoked multi-vesicular release (endplate potentials, EPPs). These effects were blocked by L-vesamicol, a vesicular acetylcholine transporter inhibitor, and bafilomycin, a proton pump inhibitor, suggesting the increase in MEPP/EPP amplitude is due to increased vesicle filling presynaptically. These effects were also blocked by the MARK inhibitors, UO126 and PD98059, suggesting TGF-β2 acts via a MARK-dependent pathway. Postsynaptically, two electrode recording showed postsynaptic potential amplitude was enhanced by an increased fibre input resistance, suggesting TGF-β2 also acts postsynaptically. TGF-β2 reduced the number of vesicles released per stimulus (quanta content, QC) but this was blocked by atropine, showing this was indirect through autoreceptor negative feedback. Voltage clamp recording showed TGF-β2 significantly increased the miniature end plate currents (MEPCs), but not the end-plate currents (EPCs), supporting my initial hypothesis that TGF-β2 acts mainly presynaptically to increase vesicle filling. In TGF-β2+/- mice, I found greater MEPP amplitude variability. This supports my previous findings that TGF-β2 modulates vesicle filling. Unexpectedly, there was an excess in larger MEPP sizes (>0.88 mV), perhaps reflecting upregulation of either presynaptic signalling or another synaptic mediator. Two MEPP amplitude populations were induced in TGF-β2-treated TGF-β2+/- mouse NMJs, similar to the bimodal vesicle population in electroplaques. The extensor digitorum longus (EDL, ~95% fast fibres) and soleus (SOL, ~95% slow fibres) were used to investigate whether the TGF-β2-mediated effect differed between fibre types. Overall, TGF-β2 increased the quantal size (MEPP amplitude) in NMJs of both muscles, suggesting this effect is not fibre-type specific and, together with results in mice, that the TGF-β2-mediated increase in vesicle filling is common to all mammalian neuromuscular terminals. With respect to EPP amplitude and QC, the results differed between muscles. In EDL, the EPP amplitude was not significantly changed, whereas it increased in SOL. In EDL, QC was reduced but not in SOL. These difference compared to diaphragm perhaps do reflect muscle fibre-type dependent differences. TGF-β1, at 0.1 ng/ml, significantly reduced quantal size – the opposite of TGF-β2 at any concentration. One explanation would be that a receptor inhibition by TGF-β1 at low concentration interferes with endogenous TGF-β2 binding/receptor activation at the NMJ. However, when the TGF-β1 concentration was raised to 1 ng/ml, like TGF-β2, it significantly increased MEPP amplitude. This suggests that perhaps sufficient binding of TGF-β1 results in the receptor activation of TGF-β2 like signalling. Overall, I conclude that TGF-β2 enhances the size of spontaneous synaptic potentials in all types of muscle fibres, and this is much more rapid (1 hr vs 1 day) than at central neurone synapses in culture. Detailed study in the rat diaphragm shows it increased the evoked EPP amplitude, reduced QC and increased postsynaptic input resistance. Together, TGF-β2 would therefore enhance the postsynaptic depolarisation increasing synaptic strength, and by reducing QC, increase the efficiency of neurotransmission at mammalian NMJs. While unimportant for single stimuli in healthy terminals, by conserving vesicle use, it may help maintain release during stimulus trains, especially during neuromuscular disease.

Vor 2 Jahren wurden erstmals die wahrscheinlich wichtigsten Tight Junction Proteine, Claudin-1 und -2 in der Maus beschrieben. An Hand von Sequenzhomologien konnten bis heute dieser 4 Transmembrandomänen Proteinfamilie 18 Mitglieder mit unterschiedlichen Gewebeverteilungen zugeordnet werden. Parallel zum murinen Claudin-1 wurde von (Swisshelm et al. 1999) das humane Claudin-1 mit einer 91 prozentigen Sequenzhomologie zum murinen Protein isoliert und molekulargenetisch beschrieben. In der überwiegenden Mehrzahl von Brusttumorzellinien ist die Expression von hu-CLDN1 verloren gegangen. In der vorliegenden Arbeit sollte deshalb die biologische Funktion des humanen Claudin-1 (hu-CLDN1) und dessen Relevanz während der Tumorgenese untersucht werden. Für diese Aufgabenstellung mußten monoklonale Antikörper mittels DNA Vakzinierung und hu-CLDN1 Retroviren zur effektiven Transduktion von Tumorzellen entwickelt werden. Die Antikörper wurden in ELISA, kompetitiven hu-CLDN1 Peptid ELISA und Western Blot auf ihre hu-CLDN1 Spezifität und Epitoperkennungsstelle überprüft. Für biologische Untersuchungen wurden für die neuen Antikörper optimale immunzytochemische und immunhistochemische Methoden etabliert. Mit Hilfe der monoklonalen Antikörper wurde die zelluläre Proteinexpression und die Lokalisation, als auch die Expression von hu-CLDN1 in Gewebeschnitten von Tumoren- und Normalgewebe untersucht. Vor 2 Jahren wurden erstmals die wahrscheinlich wichtigsten Tight Junction Proteine, Claudin-1 und -2 in der Maus beschrieben. An Hand von Sequenzhomologien konnten bis heute dieser 4 Transmembrandomänen Proteinfamilie 18 Mitglieder mit unterschiedlichen Gewebeverteilungen zugeordnet werden. Parallel zum murinen Claudin-1 wurde von (Swisshelm et al. 1999) das humane Claudin-1 mit einer 91 prozentigen Sequenzhomologie zum murinen Protein isoliert und molekulargenetisch beschrieben. In der überwiegenden Mehrzahl von Brusttumorzellinien ist die Expression von hu-CLDN1 verloren gegangen. In der vorliegenden Arbeit sollte deshalb die biologische Funktion des humanen Claudin-1 (hu-CLDN1) und dessen Relevanz während der Tumorgenese untersucht werden. Für diese Aufgabenstellung mußten monoklonale Antikörper mittels DNA Vakzinierung und hu-CLDN1 Retroviren zur effektiven Transduktion von Tumorzellen entwickelt werden. Die Antikörper wurden in ELISA, kompetitiven hu-CLDN1 Peptid ELISA und Western Blot auf ihre hu-CLDN1 Spezifität und Epitoperkennungsstelle überprüft. Für biologische Untersuchungen wurden für die neuen Antikörper optimale immunzytochemische und immunhistochemische Methoden etabliert. Mit Hilfe der monoklonalen Antikörper wurde die zelluläre Proteinexpression und die Lokalisation, als auch die Expression von hu-CLDN1 in Gewebeschnitten von Tumoren- und Normalgewebe untersucht. Für die Reexpression von hu-CLDN1 in Brusttumorzellen wurden retrovirale Genshuttlesysteme hergestellt. Die retroviralen Genshuttlesysteme basieren auf dem MoMuLV Grundgerüst und als Besonderheit wurde der l-NGFR Rezeptor zur schnellen und sicheren Identifkation transduzierter Zellen einkloniert. Hergestellt wurde ein Mock Kontrollvektor (nur l-NGFR) und der hu-CLDN1 Vektor mit l-NGFR. Die hu-CLDN1 retroviralen Überstände wurden verwendet, um verschiedene Brusttumorzellinien zu transduzieren. Die Expression von hu-CLDN1 wurde mittels eigens entwickelter quantitativer gekoppelter Reverser Transkription Polymerase Ketten Reaktion (qRT PCR) in verschiedenen Brusttumorzellinien untersucht. Wie aus der vorliegenden Arbeit ersichtlich, konnten erstmalig monoklonale Antikörper gegen hu-CLDN1 entwickelt werden. Diese sind spezifisch und haben eine hohe Sensitivität gegenüber hu-CLDN1. Desweiteren gelang es erstmals Antikörper gegen alle 4 extra- und intrazellulären Domänen zu gewinnen. Mit diesen Antikörpern gelang es nachzuweisen, daß es sich bei hu-CLDN1 um ein ausschließlich membranständiges Protein handelt. Die Expression des Proteins findet sich nur in konfluenten Zellkulturen von natürlicherweise hu-CLDN1 exprimierenden Brusttumorzellen (T47D, MCF7), wobei die Lokalisation ausschließlich auf die Zell-Zell Kontaktstelle beschränkt ist. Bei der hu-CLDN1 Transduktion in hu-CLDN1 negativen Brusttumorzellinen zeigte sich eine konstitutive mRNA Expression in subkonfluenten und konfluenten Zellkulturen. Allerdings ist fluoreszenzmikroskopisch hu-CLDN1 Protein nur in konfluenten Zellkulturen nachweisbar, wobei diese Tumorzellen das Protein korrekt nur an der Zell-Zell Kontaktstelle einbauen. Offensichtlich haben die hu-CLDN1 negativen Brusttumorzellen noch den intakten Signalweg zur korrekten Expression mit einer noch unbekannten posttranskriptionellen Kontrolle. Bemerkenswert ist in diesem Zusammenhang, daß in Brusttumorzellen, die weder Occludin noch ZO-1 exprimieren (MDA-MB-435) die physiologisch korrekte Expression von hu-CLDN1 existiert. Offensichtlich ist die Lokalisation von hu-CLDN1 von beiden Proteinen unabhängig. Die Analyse von unterschiedlichen hu-CLDN1 positiven und negativen Brusttumorzellen zeigte, daß der Verlust der hu-CLDN1 Expression besser zur in vitro Invasivität von Brusttumorzellen korreliert als der Expressionsverlust von Occludin und ZO-1. Die klinische Relevanz des Verlustes von hu-CLDN1 während der Tumorgenese wurde bei den Expressionsanalysen auf normalen Brust- und Darmgewebe gegenüber transformierten Brust- und Darmgewebe untersucht. Bei der Analyse von normalem Brustgewebe konnte festgestellt werden, daß hu-CLDN1 ein rein epthelial/endotheliales Protein mit eindeutiger Membranlokalisation darstellt. In den untersuchten transformierten Geweben zeigte keines der transformierten Gewebe mehr die membranständige Färbung, sondern nur noch zytoplasmatische Färbung. Desweiteren war eine signifikant reduzierte oder nicht vorhandene hu-CLDN1 Färbung in einer größeren Anzahl von Tumoren zu beobachten. Diese Ergebnisse zeigen, daß der Verlust der Expression zumindest bei der Brust- und Darmtumorentwicklung offensichtlich mit der in vivo Tumorprogression korreliert. Um die Bedeutung der hu-CLDN1 Relokalisation bzw. verringerten Expression während der Tumorgenese zellphysiologisch zu verstehen, wurden in vitro Zellkulturstudien mit hu-CLDN1 negativen Zellinien und ihren hu-CLDN1 transduzierten Tochterpopulationen durchgeführt. In den adhärenten Zellkulturen hat die hu-CLDN1 Expression keinen Einfluß auf Zellwachstum und Apoptose. Allerdings zeigte sich in drei dimensional wachsenden Tumorzellaggregaten, daß die Reexpression von hu-CLDN1 zu einer drastischen Erhöhung der Apoptose führt. Die parazellulären Fluxstudien ergaben, daß bei der Reexpression von hu-CLDN1 in Brusttumorzellen der parazelluläre Flux zwischen den Zellen deutlich zurückgeht. Somit könnte die reduzierte Wachstumskapazität bzw. erhöhte Apoptose in 3 D Kulturen mit einer reduzierten Zugänglichkeit von Nährstoffen und Wachstumsfaktoren in hu-CLDN1 transduzierten Zellen verursacht sein. Dies würde auch erklären, warum bei den untersuchten transformierten noch hu-CLDN1 positiven Brust- und Darmtumorgeweben sich ausschließlich eine zytoplasmatische Lokalisation in den Tumorzellen findet. Während in Organen und Drüsengeweben Epithelien einschichtig vorkommen und die Versorgung der Zellen mit Wachstumsfaktoren basolateral oder apikal durch Diffusion und Mikro-/Makropinozytose erfolgen kann, wachsen Tumorepithelzellen mehrschichtig. Wären die Tight Junctions im Tumor noch intakt, so könnte es zu einer Mangelversorgung und somit zur Apoptose kommen. Für das in vivo Wachstum eines Tumors ist es also notwendig, Membranproteine, die den parazellulären Flux inhibieren, zu verringern. Wie die zellphysiologischen in vitro Studien der vorliegenden Arbeit zeigen, ist es aber möglich, einen tumorinhibierenden Effekt allein durch die Reexpression von hu-CLDN1 unabhängig von Occludin und ZO-1 zu erreichen. In diesem Sinne kann zumindest zellphysiologisch hu-CLDN1 als Tumorsuppressorprotein betrachtet werden. / Two years ago probably the most important tight junction proteins in mice were identified: claudin-1 and -2 . By sequence homology up to 18 members could be assigned to this 4 transmembrane protein family. The human claudin-1 with a 91 per cent sequence similarity to the murine claudin-1 was isolated in parallel (Swisshelm et al. 1999) and it has been shown that most breast cancer cell lines lost the expression of hu-CLDN1. The goal of this Ph. D. thesis was the evaluation of the cell physiological function of the human Claudin-1 (hu-CLDN1) and its relevance during tumorigenesis. To investigate the protein expression and cellular homing monoclonal antibodies using DNA vaccination were generated. The antibodies were analyzed for hu-CLDN1 specifity by Western blot and the epitope binding sites were identified by a competetive hu-CLDN1 peptide ELISA. Using the monoclonal antibodies optimal immunocytochemical and immunohistochemical methods for biological methods were established. With these antibodies the cellular expression and localization, and the expression of hu-CLDN1 in tissue slices of tumor versus normal tissues was analyzed. For the reexpression of hu-CLDN1 in breast tumor cells, retroviral shuttle systems were generated, based on a MoMuLV backbone using the l-NGFR receptor for efficient and rapid transduction of breast cancer cells with hu-CLDN1. For this purpose a mock vector (containing l-NGFR only ) and a hu-CLDN1 vector with l-NGFR were cloned. The expression of hu-CLDN1 in various breast cancer cell lines was analysed using quantitative reverse transcription polymerase chain reaction (qRT PCR). The monoclonal antibodies against hu-CLDN1 are specific against hu-CLDN1. In addition antibodies for all 4 extra- and cytosolic domains were generated. Using these antibodies it was shown by immunofluorescence microscopic analysis that hu-CLDN1 is an exclusively membrane located protein. The expression of the protein is detectable only in confluent cell cultures of naturally hu-CLDN1 expressing cell lines (T47-D, MCF7) and only in the areas of direct cell cell contact. The transduction of hu-CLDN1 negative cell lines analyzed by qRT PCR displayed a mRNA expression in subconfluent and confluent cell cultures, while the protein was only detectable in confluent cell cultures. This indicates that the hu-CLDN1 negative breast tumor cell lines still have the intact signal transduction pathway for the correct expression with an up to now unknown posttranscriptional control mechanism of protein expression. In this context it is noteworthy that even occludin and ZO-1 negative breast tumor cell lines (e. g. MDA-MB-435) still have the physiological homing mechanism of hu-CLDN1. Therefore it can be suggested that expression and homing at hu-CLDN1 might be independent of occludin and ZO-1. The analysis of different hu-CLDN1 positive and negative breast tumor cells showed that the loss of expression of hu-CLDN1 correlates more significantly to the tumorigenesis of cells in comparison to occludin and ZO-1. The clinical relevance of the downregulation and loss of hu-CLDN1 was analyzed using expression analysis of breast and colon tissue versus tumor tissue on tissue microarrays. Normal breast tissue displayed a epithelial/endothelial hu-CLDN1 membrane localization only. None of the breast tumor tissue displayed a membrane localization of the hu-CLDN1 however a relocalization to the cytoplasm was evident. Additionally a significant reduction or loss of expression could be detected in the majority of tumor tissues. These results show that the loss of expression of hu-CLDN1 in breast and colon tumors correlates with tumor progression. In order to analyze the relevance of hu-CLDN1 relocalization and reduction of expression during tumorigenesis on a cellphysiological level, in vitro cell culture studies were performed using hu-CLDN1 negative cell lines (MDA-MB-361) and their hu-CLDN1 transduced counterparts. Hu-CLDN1 transduced cells growing in 2 D cell cultures displayed no altered growth or increased levels of apoptosis. However in three dimensional growing tumor cell aggregates the reexpression of hu-CLDN1 results in a significantly increased induction of apoptosis. In addition paracellular flux analysis revealed that reexpression of hu-CLDN1 decreased the paracellular flux significantly. This data suggests that the reduced growth and increased apoptosis in hu-CLDN1 positive tumor cell aggregates could be due to reduced accessibility of growth factors in hu-CLDN1 transduced cell aggregates. This would explain the necessity for the cytoplasmic homing and loss of expression of hu-CLDN1 during tumorprogression in breast and colon tumors. Most epithelia exist as single layers in organs and lobular structures resulting in a good accessibility of growth factors via diffusion or micro-/macropinocytosis. However, carcinoma tumor cells grow in multilayers. Intact tight junction complexes in carcinoma multi layers would result in a reduced accessibility of growth factors and nutrients. Therefore it is suggested that it is essential for the in vivo growth of a tumor to decrease the expression of tight junctions regulating the paracellular flux. According to the molecular and cell physiological studies presented it might be possible to achieve a tumor inhibiting effect by re-expression of hu-CLDN1. Therefore at least from a cell physiological point of view hu-CLDN1 can be considered to be a tumor suppressor protein.

The conserved human protein KANK1 has been identified as a tumour suppressor and its expression is down-regulated in several tumour types. Roles for this protein in actin regulation, cell migration and cell polarity have been documented in cultured mammalian cells. In C. elegans the KANK1 orthologue, VAB-19, is required for normal development as it helps stabilise attachment structures between muscle and epidermal cells. Despite these studies, the precise cellular role of KANK remains elusive. It was found that the Drosophila KANK orthologue binds directly to EB1, a crucial regulator of microtubule plus-end dynamics. I aimed to determine the role of KANK with respect to this indirect microtubule interaction using Drosophila. I identified residues which mediate the interaction between KANK and EB1, and showed they are essential for localisation of KANK to microtubule plus-ends in Drosophila culture cells. I found that KANK expression increases during embryogenesis and peaks in the late embryonic development when KANK is shown to localise to sites of attachment between muscle and epidermal cells. This suggests a role for the protein in stabilisation of muscle attachment during embryonic development, a process previously shown to require EB1. I generated a KANK deletion mutant and found they are viable and fertile but show a mild neuronal phenotype, specifically early branching of the neurons and less organised neuron bundles. My results suggest previously unknown roles for KANK in myogenesis and neurogenesis in Drosophila embryogenesis.

No description available.

In vitro models of the neuromuscular junction (NMJ) are emerging as a valuable tool to study synaptogenesis, synaptic maintenance, and pathogenesis of neurodegenerative diseases. Many models have previously been developed using a variety of cell sources for muscle and motoneurons, but the models can be further improved by integrating beneficial features to better mimic the native milieu of NMJ development. We created a functional in vitro model of NMJ by bioreactor cultivation of C2C12 myoblasts, transdifferentiated myocytes and stem cell-derived motoneurons with electrical stimulation. Proper coculture medium and electrical stimulation led to improved functional coupling between the emerging motoneurons and myocytes, as evidenced by mature cellular structures, increased expression of neuronal and muscular genes, clusterization of acetylcholine receptors (AChRs) in the vicinity of motoneurons, and the response of the coculture to glutamate stimulation. To validate the models and demonstrate utility for pharmacological testing, we analyzed the potency of the drugs that affect key pathways during NMJ signal transduction, including acetylcholine (ACh) synthesis, ACh vesicular storage, ACh synaptic release, AChR activation, and ACh inactivation in the synaptic cleft. The models properly responded to the drugs in a concentration-dependent manner. The proposed in vitro NMJ model could thus be used in pharmacological screening and controlled studies of neuromuscular diseases.

Untersuchung des transepithelialen Widerstands, der Transkriptionsmenge und der fluoreszenzmikroskopischen Verteilung der Proteine Occludin und ZO-1 in Monolayern in Reaktion auf exogene Faktoren. / Trials of Occludin and ZO-1 in an epithelial model by measure the transepithelial resistance, the transcriptionlevel and the microskopic distibution.