Schwann cell-seeded guidance channels have been exploited to bridge and guide axonal re-growth across gaps in lesioned nerves. Mis-orientation of Schwann cells in the channels can however distort axonal growth within the lesion. We therefore propose to orient the growth of Schwann cells on aligned nanofibers such that axonal growth can be guided along the designated direction towards the target. Chitosan was the choice scaffold material given its biocompatibility and the tunable susceptibility to biodegradation. To be suitable for electrospinning, chitosan was dissolved in trifluoroacetic acid/methylene chloride solution. By replacing the grounded plate collector of the conventional electrospinning setup with parallel collector plates placed 1.6 cm apart, the positively charged chitosan fibersbecame alternately attracted to the parallel plates and ended up uniaxially aligned as fiber suspension across the plates. Stability of the chitosan fibers in aqueous, physiological environment was achieved with the use of sodium carbonate to neutralize residual acidity in the chitosan fiber preparation. Schwann cells seeded onto these stabilized aligned chitosan nanofibers aligned uniaxially with the chitosan nanofibers. In addition, by seeding dissociated cells of dorsal root ganglia (DRG, E14/15 rats) onto the uniaxially aligned nanofibers, both neurons and Schwann cells were aligned with uniaxial arrangement of nanofibers, and the Schwann cells showed myelination ofthe axons. A model of the chitosan nerve conduit was constructed with a core nanofiberbundle, and seeding of Schwann cells. Thesein vitro results provide proof-of-principle for pursuing improvement in post-traumatic recovery from nerve injury with use of uniaxially aligned chitosan nanofibers in Schwann cell-seeded nerve guidance channels. / published_or_final_version / Biochemistry / Master / Master of Philosophy

During neural development, axons rely on extracellular cues to reach their target regions. Although extracellular signaling is one of the principal determinants for the growth of developing axons, only a small handful of known signaling cues has been identified. The existence of some 86 billion neurons of different subtypes, which ultimately form numerous functional circuits in the human nervous system, means an enormous number of extracellular cues would be required during development. Current views hold that even if more extracellular cues were to be discovered, they would never number large enough to account for the complexity of the human nervous system. Rather, intracellular signaling pathways and other cell-intrinsic mechanisms expand the ways in which a neuron can respond to extracellular cues by tuning the degree of responsiveness to them. Cell-intrinsic signaling pathways also give axons the ability to actively control their own development. These pathways can operate independently of the extracellular environment or even independently of the cell body, where the majority of protein synthesis takes place. For example, the local translation of proteins in the axon gives it autonomous control to immediately respond to changing demands in the environment. Local translation also occurs in other cell types, but the compartmentalized control over growth is especially important for neurons since the axon can extend up to a meter away from the cell body. In addition to local translation, axonally derived transcription factors, which can be locally synthesized in or localized to the axon, provide another means to control axon development. Axonally derived transcription factors act as physiological sensors and relay information about events happening in the periphery back to the cell body in order to effectuate a global response. It has recently been shown that transcription factors belonging to the OASIS family are activated by proteolysis in axons. Following their activation by proteolytic cleavage, the transcriptionally active N-terminus of these factors is transported to the cell body to activate global transcriptional pathways. For at least one OASIS family member, CREB3L2, this cleavage event simultaneously produces the C-terminus, which is capable of undergoing secretion. The secreted C-terminus of CREB3L2 acts as an accessory ligand for the activation of Hh pathways in chondrocytes. The generation of two bioactive proteins from one transcription factor, a transcriptionally active portion and a secreted portion, raised the question of whether there was a local function for OASIS transcription factors in axons. Through my research, I identified a mechanism in which DRG axons secrete the C-terminus of CREB3L2, which promotes axon growth in a paracrine manner. CREB3L2 is a transcription factor whose translation is induced by physiological ER stress. For CREB3L2 to be active, it must be cleaved by S2P, which I found is expressed in developing axons. Following proteolysis of CREB3L2 by S2P, the secreted C-terminus of CREB3L2 promotes the formation of Shh and Ptch1 complexes along axons. I found that upon depletion of the secreted CREB3L2 C-terminus, binding of Shh to the Ptch1 receptor is diminished. Returning the CREB3L2 C-terminus to the cultures exogenously was sufficient to rescue the formation of these complexes. These results highlight an intrinsic role for Shh signaling in developing DRG axons. Moreover, these results demonstrate how ER stress machinery is recruited to axons and promotes axon outgrowth. Finally, these results illustrate a novel, neuron-intrinsic mechanism by which developing axons actively regulate their own growth.

Thesis (M.Sc.). / Written for the Dept. of Neurology and Neurosurgery. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

Yra žinoma, jog nervinės ląstelės gali regeneruoti savo aksonus po periferinės, o tam tikrais atvejais ir po centrinės nervų sistemos pažeidimų. Tačiau aksonų augimas yra sudėtingas, o jo reguliacija turi kritinę įtaką tiek neuronų vystymęsi, tiek regeneracijoje. Vekiami aplinkinių ląstelių, išskiriamų pritraukiančiųjų ir atstumiančiųjų molekulių, aksonai augdami nuolat keičia augimo kryptis iki kol pasiekia galutinius taikinius. Dorsalinių ragų ganglijų (DRG) aksonų augimas priklauso nuo semaforinų klasės molekulių. Sekretuojantys, ar su membrana surišti semaforinai dalyvauja įvairiuose biologiniuose procesuose, tokiuose, kaip centrinės ir periferinės nervų sistemos (CNS ir PNS) vystymęsi ir regeneracijoje, širdies ir kraujagyslių vystymęsi ir imuninės sistemos funkcijose. DRG aksonų vystymasis ir išlikimas smarkiai priklauso nuo nervų augimo faktoriaus (NGF). Darbo tikslas buvo įvertinti NGF koncentracijos įtaką DRG aksonų augimo atsakams į semaforiną 3A. 15 parų pelių embrionų DRG buvo preparuojami iš C57/Bl linijos pelių embrionų. DRG neuronų auginimui naudoti sterilūs dengiamieji stikleliai buvo padengiami poli-L-lizino 0,01 mg/ml ir laminino 0,01 mg/ml tirpalu, pagamintu GBSS terpėje. Aksonų augimo kūgelių vertinimas buvo atliekamas praėjus 60 minučių, o aksonų ilgio vertinimas - praėjus 16 valandų po DRG pasodinimo. Tam, kad nustayti DRG apoptozės lygį, DRG neuronuose priklausomai nuo NGF koncentracijos buvo įvertinta Bcl-2, Bax, c-jun genų raiška, naudojant RT - PGR... [toliau žr. visą tekstą] / It is known that nerve cells can regenerate their axons after damage to peripheral and in some cases central nervous system (PNS and CNS). However, axon growth over longer distances, especially in central nervous system, is complicated. Regulation of axon growth is a critical event both in neuronal development and regeneration. To reach their proper targets, axons rely upon the actions of attractive and repulsive guidance molecules. It is known that growth of dorsal root ganglion (DRG) axons depend on guidance molecules of semaphorin class. Secreted and membrane bound semaphorins participate in diverse biological processes, including development and regeneration of central and peripheral nervous system, cardiovascular development, and immune system functioning. In addition to regulation of DRG axon growth by semaphorin class molecules, DRG axon growth and survival is strongly dependent on nerve growth factor (NGF). The aim of this study was to evaluate responses of DRG axons to semaphorin 3A in dependence of NGF concentration. DRG were dissected from C57/Bl mice E15 embryos in dissection HBSS/glucose medium. DRG were plated on cover slips coated with laminin and poly-L-lysine and grown in Neurobasal medium supplemented with 2% of B27 supplement. To evaluate collapse rate the morphology of axons growth cones were evaluated after 60 minutes and axons length were evaluated 16 hours after DRG plating. To evaluate DRG survival and level of apoptosis in dependence of NGF... [to full text]

The early postnatal period is marked by the rapid acquisition of sensorimotor processing capabilities. Initially responding to a limited set of environmental stimuli with a restricted repertoire of behaviors, mammals exhibit a remarkable proliferation of sensorimotor abilities in the early postnatal period. Central to action selection, reinforcement, and contingency learning are a subcortical set of evolutionarily conserved nuclei called the basal ganglia. The striatum, which is the primary input nucleus of the basal ganglia, receives afferent innervation from throughout the CNS. Its projection neurons (SPNs) integrate these diverse inputs, regulating movement and encoding salient cue-outcome contingencies. Here, using electrophysiological, electrochemical, imaging, and behavioral approaches in mice, I will explore the postnatal maturation of the striatal cholinergic interneuron (ChI), a critical modulator of dopamine signaling, afferent excitation, and SPN excitability. In Chapter 1, I will set the stage for this exploration by reviewing the current literature on striatal postnatal development, including cellular physiology, axonal elaboration and synapse formation, and plasticity expression. I will survey striatal deficits observed in clinical neurodevelopmental conditions such as autism, ADHD, tic disorders, and substance use disorders. I will additionally summarize evidence that the striatum is uniquely vulnerable to physiological and immunological insult, as well as early life adversity. In Chapter 2, I turn my focus specifically to the striatal ChI, uncovering fundamental cell-intrinsic changes that occur postnatally in this population. I will also elaborate on the postnatal maturation of dopamine release properties and regulation thereof by cholinergic signaling from the ChI. In Chapter 3, I investigate the circuit connectivity and circuit-driven firing dynamics of ChIs as they mature postnatally. I utilize a brain slice preparation retaining thalmostriatal afferents in order to assay the ChI pause, a synchronized transient quiescence in ChIs thought to facilitate cue learning and behavioral flexibility. I find that the ChI pause is refined postnatally, dependent on developmental changes in thalamic input strength and the cell- intrinsic expression of specific ionic conductances. Finally, in Chapter 4, I present preliminary evidence that ChI circuit maturation as defined in preceding chapters is delayed by chronic stress exposure postnatally. Following the maternal separation model of early life stress, ChI intrinsic characteristics mature normally, but they retain heightened thalamic innervation and thalamus-driven pause expression.