Sensory-motile cells fulfill various biological functions ranging from immune activity or wound healing to the formation of the highly complex nervous systems of vertebrates. In the case of neurons, a dynamic structure at the tip of outgrowing processes navigates towards target cells or areas during the generation of neural networks. These fan shaped growth cones are equipped with a highly complex molecular machinery able to detect various external stimuli and to translate them into directed motion. Receptor and adhesion molecules trigger signaling cascades that regulate the dynamics of an internal polymeric scaffold, the cytoskeleton. It plays a crucial role in morphology maintenance as well as in the generation and distribution of growth cone forces. The two major components, actin and microtubules (MTs) connect on multiple levels through interwoven biochemical and mechanical interactions. Actin monomers assemble into semiflexible filaments (F-actin) which in turn are either arranged in entangled networks in the flat outer region of the growth cone (lamellipodium) or in radial bundles termed filopodia. The dynamic network of actin filaments extends through polymerization at the front edge of the lamellipodium and is simultaneously moving towards the center (C-domain) of the growth cone. This retrograde flow (RF) of the actin network is driven by the polymerizing filaments themselves pushing against the cell membrane and the contractile activity of motor proteins (myosins), mainly in the more central transition zone (T-zone). Through transmembrane adhesion molecules, a fraction of the retrograde flow forces is mechanically transmitted to the cellular substrate in a clutch-like mechanism generating traction and moving the GC forward. MTs are tubular polymeric structures assembled from two types of tubulin protein subunits. They are densely bundled in the neurite and at the growth cone “neck” (where the neurite opens out into the growth cone) they splay apart entering the C-domain and more peripheral regions (P-domain). Their advancement is driven by polymerization and dynein motor protein activity. The two subsystems, an extending array of MTs and the centripetal moving actin network are antagonistic players regulating GC morphology and motility. Numerous experimental findings suggest that MTs pushing from the rear interact with actin structures and contribute to GC advancement. Nevertheless, the amount of force generated or transmitted through these rigid structures has not been investigated yet. In the present dissertation, the deformation of MTs under the influence of intracellular load is analyzed with fluorescence microscopy techniques to estimate these forces. RF mechanically couples to MTs in the GC periphery through friction and molecular cross-linkers. This leads to MT buckling which in turn allows the calculation of the underlying force. It turns out that forces of at least act on individual MT filaments in the GC periphery. Compared to the relatively low overall protrusion force of neuronal GCs, this is a substantial contribution. Interestingly, two populations of MTs buckle under different loads suggesting different buckling conditions. These could be ascribed to either the length-dependent flexural rigidity of MTs or local variations in the mechanical properties of the lamellipodial actin network. Furthermore, the relation between MT deformation levels and GC morphology and advancement was investigated. A clear trend evolves that links higher MT deformation in certain areas to their advancement. Interactions between RF and MTs also influence flow velocity and MT deformation. It is shown that transient RF bursts are related to higher MT deformation in the same region. An internal molecular clutch mechanism is proposed that links MT deformation to GC advancement. When focusing on GC dynamics it is often neglected that the retraction of neurites and the controlled collapse of GCs are as important for proper neural network formation as oriented outgrowth. Since erroneous connections can cause equally severe malfunctions as missing ones, the pruning of aberrant processes or the transient stalling of outgrowth at pivotal locations are common events in neuronal growth. To date, mainly short term pausing with minor cytoskeletal rearrangements or the full detachment and retraction of neurite segments were described. It is likely that these two variants do not cover the full range of possible events during neuronal pathfinding and that pausing on intermediate time scales is an appropriate means to avoid the misdetection of faint or ambiguous external signals. In the NG108-15 neuroblastoma cells investigated here, a novel type of collapse was observed. It is characterized by the degradation of actin network structures in the periphery while radial filopodia and the C-domain persist. Actin bundles in filopodia are segmented at one or multiple breaking points and subsequently fold onto the edge of the C-domain where they form an actin-rich barrier blocking MT extension. Due to this characteristic, this type of collapse was termed fold collapse. Possible molecular players responsible for this remarkable process are discussed. Throughout fold collapse, GC C-domain area and position remain stable and only the turnover of peripheral actin structures is abolished. At the same time, MT driven neurite elongation is hindered, causing the GC to stall on a time scale of several to tens of minutes. In many cases, new lamellipodial structures emerge after some time, indicating the transient nature of this collapse variant. From the detailed description of the cytoskeletal dynamics during collapse a working model including substrate contacts and contractile actin-myosin activity is derived. Within this model, the known and newly found types of GC collapse and retraction can be reduced to variations in local adhesion and motor protein activity. Altogether the results of this work indicate a more prominent role of forward directed MT-based forces in neuronal growth than previously assumed. Their regulation and distribution during outgrowth has significant impact on neurite orientation and advancement. The deformation of MT filaments is closely related to retrograde actin flow which in turn is a regulator of edge protrusion. For the stalling of GCs it is not only required that actin dynamics are decoupled from the environment but also that MT pushing is suppressed. In the case of fold collapse, this is achieved through a robust barrier assembled from filopodial actin bundles.

Das Ziel der vorliegenden Arbeit war es, nach einer irreversiblen Schädigung des Sehnervs zu zeigen, ob retinale Ganglienzellen zu Umstrukturierungsprozessen an ihren Axonen ohne neuroprotektive Unterstützung fähig sind. Das besondere Interesse galt der licht- und elektronenmikroskopischen Analyse. Verwendet wurden 37 adulte Albinoratten (WISTAR-Prob). Zuerst wurde der Nervus opticus für 30 s 5 mm hinter dem Bulbus oculi gequetscht. Ein zweiter invasiver Eingriff, der zur retrograden Markierung von aberranten Neubildungen an den Axonen der Nervenfaserschicht durchgeführt wurde, fand zu den postoperativen Überlebenszeiten (ÜLZ) von drei und zehn Tagen, zwei, drei,vier, acht und zwölf Wochen sowie nach sechs und zwölf Monaten statt. Für die retrograde Markierung wurde das biotinylisierte Dextranamin (BDA) eingesetzt, das unter Verwendung des Chromogens Diaminobenzidin visualisiert wurde.Die folgenden Befunde konnten auf lichtmikroskopischer Ebene ermittelt werden: · axonale Schwellungen, · dornenförmige Fortsätze, · intraretinale Axonsprosse mit Wachstumskegeln, · zwei Formen von intraretinalen Axonkollateralen und · aberrante axonale Trajektorien in Form von Schleifenaxonen. Basierend auf dem lichtmikroskopischen Befund wurden ausgewählte Bereiche der Netzhaut ultrastrukturell untersucht.Axonale Schwellungen konnten hinsichtlich ihrer Gestalt in spindelförmig, ballonierend und breitbasig polypös unterschieden werden. Dornenförmige Fortsätze an den Nervenfasern stellen vermutlich ein morphologisches Erscheinungsbild zur Herstellung eines funktionellen Gleichgewichts dar. Die intraretinalen Axonsprosse mit ihren birnenförmigen Wachstumskegeln konnten erstmalig elektronenmikroskopisch in einer adulten Rattennetzhaut nach einer Schädigung des Nervus opticus beschrieben werden. Die nach den ÜLZ von zehn Tagen, zwei, drei und vier Wochen dokumentierten aberranten Fortsätze orientierten sich hauptsächlich vom Discus nervi optici zur Netzhautperipherie. Zu allen ÜLZ besaßen sie eine rundliche oder ovoide Gestalt und wiesen eine Größe von 5 bis 10 µm auf. Sie gingen aus einem Hauptfaszikel in der Nervenfaserschicht hervor und existierten in enger Korrelation zu benachbarten Axonen und Blutgefäßen. Die Definition des Wachstumskegels wurde durch den ultrastrukturellen Befund der Akkumulation von Mitochondrien und wachstumskegeltypischen Vesikeln verifiziert. Die Axonsprosse mit ihren Wachstumskegeln stellen das morphologische Substrat von temporären Reorganisationen der RGC nach einer Unterbrechung ihrer axonalen Efferenz dar. Es waren zwei Formen von intraretinalen Axonkollateralen sichtbar. Bei der ersten Form handelt es sich um eine axonale Kollateralisierung nach einem dreiwöchigen Versuch, die unmittelbar hinter dem Axonhügel einer Typ-III-RGC abzweigte. Diese Form der Kollateralisierung könnte vermutlich im Zusammenhang mit regenerativen Leistungen in der Netzhaut stehen, die unter dem Begriff axon-like processes definiert wurden. Die zweite Kollateralisierungsform zwei Wochen nach der Läsion bildete sich in einem orthogonalen Winkel von einer Nervenfaser in einem Axonfaszikel und entsendete mehrere Kollateralzweige. Acht Wochen nach einer Nervus opticus-Axotomie konnte eine Axonkollaterale dokumentiert werden, die sich in einem fortgeschrittenen Degenerationsprozess befand. Die beschriebene intraretinale Axonkollaterale konnte erstmalig bei der adulten Albinoratte beschrieben werden. Zwei und drei Wochen post lesionem konnten in zwei Versuchen Nervenfasern dokumentiert werden, die durch eine auffallende Schleifenbildung gekennzeichnet waren. Resümierend konnte auf licht- und elektronenmikroskopischer Ebene nachgewiesen werden,dass geschädigte retinale Ganglienzellen in der adulten Ratte zu axonalem Wachstum ohne neuroprotektive und neuropermissive Unterstützung fähig sind. Die beobachteten regenerativen Leistungen sind vermutlich auf das Wirken von Neurotrophinen in der Retina oder im Sehnerv selbst zurückzuführen. / The present light and electron microscopic study was undertaken to determine whether axotomized retinal ganglion cells are able to reestablish intraretinal axons without experimental neuroprotective support. 37 adult albino rats (WISTAR-Prob) were used. In a first step, the optic nerve was intraorbitally exposed and crushed for 30 s at about 5 mm from the ocular bulb. A second experiment was conducted in order to stain newly formed intraretinal axonal elements after postlesion times of either three and ten days, two, three, four, eight, and twelve weeks, or six and twelve month. Retrograde labelling was achieved using biotinylated dextran amine (BDA),followed by visualization with diaminobenzidine as chromogen. The following structures were detected light microscopically: · axonal swellings, · spine-like processes, · intraretinal axonal sprouts showing growth cones, · two types of axonal collaterals, and · aberrant axonal fibers forming so-called looping axons. On the basis of light microscopy selected areas of the retina were examined electron microscopically. Axonal swellings were typified by their shape as spindle-shaped, ballon-shaped or broad-basic polypous. Also spine-like processes, which might serve the reestablishment of a functional balance within the retinal network, were detected. Intraretinal axonal sprouts, showing pear-shaped growth cones at their endings, could be demonstrated for the first time on the ultrastructural level. Aberrant processes, most of them orientated from the optic disc to the retinal periphery, were found at survival times of ten days as well as after two, three and four weeks. At all survival stages investigated the growth cones showed a plump or ovoid morphology and ranged in size between 5 to 10 µm. Usually, they were found to originate from nerve fiber fascicles located in close neigbourhood to the axons but also to blood vessels of the inner retina. The light microscopical typification of growth cones was confirmed at the ultrastructural level, particularly as accumulated mitochondria and growth cone-specific vesicles were detected. Probably, axonal sprouts and growth cones represent a temporal attempt of retinal ganglion cells to regenerate after transection of the optic nerve. Two axon collaterals were detected in the inner retina. In the first case, three weeks postlesion, an axon was found to branch immediately behind the axon hillhock of a type III ganglion cell. This kind of collateralization is indicative of a regenerative response as it has been previously reported by other authors who found so-called axon-like processes. In a second case, two weeks postlesion, an axon appeared to bifurcate orthogonally from a fiber fascicle sending off several collaterals on its way. Furthermore, a degenerating axon collateral was seen eight weeks after optic nerve lesion. The types of axon collaterals presented in this study were described for the first time in the albino rat. So-called looping axons typically characterized by their circular course were found in the inner retina two and three weeks postlesion. In conclusion, the light and electron microscopical results demonstrate that axotomized retinal ganglion cells of the adult rat retain the capability for axonal outgrowth without any neuroprotective and neuropermissive support. Since no experimental growth-promoting measures had been taken, it might be speculated whether the observed regenerative processes were due to intrinsic neurotrophic factors in the retina or the optic nerve themselfes.

Sensory-motile cells fulfill various biological functions ranging from immune activity or wound healing to the formation of the highly complex nervous systems of vertebrates. In the case of neurons, a dynamic structure at the tip of outgrowing processes navigates towards target cells or areas during the generation of neural networks. These fan shaped growth cones are equipped with a highly complex molecular machinery able to detect various external stimuli and to translate them into directed motion. Receptor and adhesion molecules trigger signaling cascades that regulate the dynamics of an internal polymeric scaffold, the cytoskeleton. It plays a crucial role in morphology maintenance as well as in the generation and distribution of growth cone forces. The two major components, actin and microtubules (MTs) connect on multiple levels through interwoven biochemical and mechanical interactions. Actin monomers assemble into semiflexible filaments (F-actin) which in turn are either arranged in entangled networks in the flat outer region of the growth cone (lamellipodium) or in radial bundles termed filopodia. The dynamic network of actin filaments extends through polymerization at the front edge of the lamellipodium and is simultaneously moving towards the center (C-domain) of the growth cone. This retrograde flow (RF) of the actin network is driven by the polymerizing filaments themselves pushing against the cell membrane and the contractile activity of motor proteins (myosins), mainly in the more central transition zone (T-zone). Through transmembrane adhesion molecules, a fraction of the retrograde flow forces is mechanically transmitted to the cellular substrate in a clutch-like mechanism generating traction and moving the GC forward. MTs are tubular polymeric structures assembled from two types of tubulin protein subunits. They are densely bundled in the neurite and at the growth cone “neck” (where the neurite opens out into the growth cone) they splay apart entering the C-domain and more peripheral regions (P-domain). Their advancement is driven by polymerization and dynein motor protein activity. The two subsystems, an extending array of MTs and the centripetal moving actin network are antagonistic players regulating GC morphology and motility. Numerous experimental findings suggest that MTs pushing from the rear interact with actin structures and contribute to GC advancement. Nevertheless, the amount of force generated or transmitted through these rigid structures has not been investigated yet. In the present dissertation, the deformation of MTs under the influence of intracellular load is analyzed with fluorescence microscopy techniques to estimate these forces. RF mechanically couples to MTs in the GC periphery through friction and molecular cross-linkers. This leads to MT buckling which in turn allows the calculation of the underlying force. It turns out that forces of at least act on individual MT filaments in the GC periphery. Compared to the relatively low overall protrusion force of neuronal GCs, this is a substantial contribution. Interestingly, two populations of MTs buckle under different loads suggesting different buckling conditions. These could be ascribed to either the length-dependent flexural rigidity of MTs or local variations in the mechanical properties of the lamellipodial actin network. Furthermore, the relation between MT deformation levels and GC morphology and advancement was investigated. A clear trend evolves that links higher MT deformation in certain areas to their advancement. Interactions between RF and MTs also influence flow velocity and MT deformation. It is shown that transient RF bursts are related to higher MT deformation in the same region. An internal molecular clutch mechanism is proposed that links MT deformation to GC advancement. When focusing on GC dynamics it is often neglected that the retraction of neurites and the controlled collapse of GCs are as important for proper neural network formation as oriented outgrowth. Since erroneous connections can cause equally severe malfunctions as missing ones, the pruning of aberrant processes or the transient stalling of outgrowth at pivotal locations are common events in neuronal growth. To date, mainly short term pausing with minor cytoskeletal rearrangements or the full detachment and retraction of neurite segments were described. It is likely that these two variants do not cover the full range of possible events during neuronal pathfinding and that pausing on intermediate time scales is an appropriate means to avoid the misdetection of faint or ambiguous external signals. In the NG108-15 neuroblastoma cells investigated here, a novel type of collapse was observed. It is characterized by the degradation of actin network structures in the periphery while radial filopodia and the C-domain persist. Actin bundles in filopodia are segmented at one or multiple breaking points and subsequently fold onto the edge of the C-domain where they form an actin-rich barrier blocking MT extension. Due to this characteristic, this type of collapse was termed fold collapse. Possible molecular players responsible for this remarkable process are discussed. Throughout fold collapse, GC C-domain area and position remain stable and only the turnover of peripheral actin structures is abolished. At the same time, MT driven neurite elongation is hindered, causing the GC to stall on a time scale of several to tens of minutes. In many cases, new lamellipodial structures emerge after some time, indicating the transient nature of this collapse variant. From the detailed description of the cytoskeletal dynamics during collapse a working model including substrate contacts and contractile actin-myosin activity is derived. Within this model, the known and newly found types of GC collapse and retraction can be reduced to variations in local adhesion and motor protein activity. Altogether the results of this work indicate a more prominent role of forward directed MT-based forces in neuronal growth than previously assumed. Their regulation and distribution during outgrowth has significant impact on neurite orientation and advancement. The deformation of MT filaments is closely related to retrograde actin flow which in turn is a regulator of edge protrusion. For the stalling of GCs it is not only required that actin dynamics are decoupled from the environment but also that MT pushing is suppressed. In the case of fold collapse, this is achieved through a robust barrier assembled from filopodial actin bundles.

Das Ziel der vorliegenden Arbeit war es, nach einer irreversiblen Schädigung des Sehnervs zu zeigen, ob retinale Ganglienzellen zu Umstrukturierungsprozessen an ihren Axonen ohne neuroprotektive Unterstützung fähig sind. Das besondere Interesse galt der licht- und elektronenmikroskopischen Analyse. Verwendet wurden 37 adulte Albinoratten (WISTAR-Prob). Zuerst wurde der Nervus opticus für 30 s 5 mm hinter dem Bulbus oculi gequetscht. Ein zweiter invasiver Eingriff, der zur retrograden Markierung von aberranten Neubildungen an den Axonen der Nervenfaserschicht durchgeführt wurde, fand zu den postoperativen Überlebenszeiten (ÜLZ) von drei und zehn Tagen, zwei, drei,vier, acht und zwölf Wochen sowie nach sechs und zwölf Monaten statt. Für die retrograde Markierung wurde das biotinylisierte Dextranamin (BDA) eingesetzt, das unter Verwendung des Chromogens Diaminobenzidin visualisiert wurde.Die folgenden Befunde konnten auf lichtmikroskopischer Ebene ermittelt werden: · axonale Schwellungen, · dornenförmige Fortsätze, · intraretinale Axonsprosse mit Wachstumskegeln, · zwei Formen von intraretinalen Axonkollateralen und · aberrante axonale Trajektorien in Form von Schleifenaxonen. Basierend auf dem lichtmikroskopischen Befund wurden ausgewählte Bereiche der Netzhaut ultrastrukturell untersucht.Axonale Schwellungen konnten hinsichtlich ihrer Gestalt in spindelförmig, ballonierend und breitbasig polypös unterschieden werden. Dornenförmige Fortsätze an den Nervenfasern stellen vermutlich ein morphologisches Erscheinungsbild zur Herstellung eines funktionellen Gleichgewichts dar. Die intraretinalen Axonsprosse mit ihren birnenförmigen Wachstumskegeln konnten erstmalig elektronenmikroskopisch in einer adulten Rattennetzhaut nach einer Schädigung des Nervus opticus beschrieben werden. Die nach den ÜLZ von zehn Tagen, zwei, drei und vier Wochen dokumentierten aberranten Fortsätze orientierten sich hauptsächlich vom Discus nervi optici zur Netzhautperipherie. Zu allen ÜLZ besaßen sie eine rundliche oder ovoide Gestalt und wiesen eine Größe von 5 bis 10 µm auf. Sie gingen aus einem Hauptfaszikel in der Nervenfaserschicht hervor und existierten in enger Korrelation zu benachbarten Axonen und Blutgefäßen. Die Definition des Wachstumskegels wurde durch den ultrastrukturellen Befund der Akkumulation von Mitochondrien und wachstumskegeltypischen Vesikeln verifiziert. Die Axonsprosse mit ihren Wachstumskegeln stellen das morphologische Substrat von temporären Reorganisationen der RGC nach einer Unterbrechung ihrer axonalen Efferenz dar. Es waren zwei Formen von intraretinalen Axonkollateralen sichtbar. Bei der ersten Form handelt es sich um eine axonale Kollateralisierung nach einem dreiwöchigen Versuch, die unmittelbar hinter dem Axonhügel einer Typ-III-RGC abzweigte. Diese Form der Kollateralisierung könnte vermutlich im Zusammenhang mit regenerativen Leistungen in der Netzhaut stehen, die unter dem Begriff axon-like processes definiert wurden. Die zweite Kollateralisierungsform zwei Wochen nach der Läsion bildete sich in einem orthogonalen Winkel von einer Nervenfaser in einem Axonfaszikel und entsendete mehrere Kollateralzweige. Acht Wochen nach einer Nervus opticus-Axotomie konnte eine Axonkollaterale dokumentiert werden, die sich in einem fortgeschrittenen Degenerationsprozess befand. Die beschriebene intraretinale Axonkollaterale konnte erstmalig bei der adulten Albinoratte beschrieben werden. Zwei und drei Wochen post lesionem konnten in zwei Versuchen Nervenfasern dokumentiert werden, die durch eine auffallende Schleifenbildung gekennzeichnet waren. Resümierend konnte auf licht- und elektronenmikroskopischer Ebene nachgewiesen werden,dass geschädigte retinale Ganglienzellen in der adulten Ratte zu axonalem Wachstum ohne neuroprotektive und neuropermissive Unterstützung fähig sind. Die beobachteten regenerativen Leistungen sind vermutlich auf das Wirken von Neurotrophinen in der Retina oder im Sehnerv selbst zurückzuführen. / The present light and electron microscopic study was undertaken to determine whether axotomized retinal ganglion cells are able to reestablish intraretinal axons without experimental neuroprotective support. 37 adult albino rats (WISTAR-Prob) were used. In a first step, the optic nerve was intraorbitally exposed and crushed for 30 s at about 5 mm from the ocular bulb. A second experiment was conducted in order to stain newly formed intraretinal axonal elements after postlesion times of either three and ten days, two, three, four, eight, and twelve weeks, or six and twelve month. Retrograde labelling was achieved using biotinylated dextran amine (BDA),followed by visualization with diaminobenzidine as chromogen. The following structures were detected light microscopically: · axonal swellings, · spine-like processes, · intraretinal axonal sprouts showing growth cones, · two types of axonal collaterals, and · aberrant axonal fibers forming so-called looping axons. On the basis of light microscopy selected areas of the retina were examined electron microscopically. Axonal swellings were typified by their shape as spindle-shaped, ballon-shaped or broad-basic polypous. Also spine-like processes, which might serve the reestablishment of a functional balance within the retinal network, were detected. Intraretinal axonal sprouts, showing pear-shaped growth cones at their endings, could be demonstrated for the first time on the ultrastructural level. Aberrant processes, most of them orientated from the optic disc to the retinal periphery, were found at survival times of ten days as well as after two, three and four weeks. At all survival stages investigated the growth cones showed a plump or ovoid morphology and ranged in size between 5 to 10 µm. Usually, they were found to originate from nerve fiber fascicles located in close neigbourhood to the axons but also to blood vessels of the inner retina. The light microscopical typification of growth cones was confirmed at the ultrastructural level, particularly as accumulated mitochondria and growth cone-specific vesicles were detected. Probably, axonal sprouts and growth cones represent a temporal attempt of retinal ganglion cells to regenerate after transection of the optic nerve. Two axon collaterals were detected in the inner retina. In the first case, three weeks postlesion, an axon was found to branch immediately behind the axon hillhock of a type III ganglion cell. This kind of collateralization is indicative of a regenerative response as it has been previously reported by other authors who found so-called axon-like processes. In a second case, two weeks postlesion, an axon appeared to bifurcate orthogonally from a fiber fascicle sending off several collaterals on its way. Furthermore, a degenerating axon collateral was seen eight weeks after optic nerve lesion. The types of axon collaterals presented in this study were described for the first time in the albino rat. So-called looping axons typically characterized by their circular course were found in the inner retina two and three weeks postlesion. In conclusion, the light and electron microscopical results demonstrate that axotomized retinal ganglion cells of the adult rat retain the capability for axonal outgrowth without any neuroprotective and neuropermissive support. Since no experimental growth-promoting measures had been taken, it might be speculated whether the observed regenerative processes were due to intrinsic neurotrophic factors in the retina or the optic nerve themselfes.

No description available.

No description available.