No description available.

Degeneration of nociceptive afferent axons and terminals in the heart is associated with painless sudden cardiac death. However, innervation, distribution and morphological structures of sympathetic cardiac nociceptive afferent axons and terminals have not yet been fully characterized. The aim of the present study is to characterize the density, arrangement, and structural features of differentiated sympathetic afferent axons and terminals in whole-mount FVB mouse atria. FVB mice (3-6 months old) were perfused and the tissues were fixed. The right and left atria were processed with immunohistochemistry. Calcitonin gene-related peptide (CGRP) and substance P (SP) are two neuropeptides which have been widely used to label sympathetic nociceptive afferent axons in many tissues. CGRP (rabbit anti-CGRP) and SP (Goat anti-SP) primary antibodies were applied, followed by Alexa Fluor 594 and 660 conjugated secondary antibodies. Whole-mount preparations of right and left atria were examined using a laser scanning confocal microscope. We found that 1) CGRP immunoreactive (IR) axon bundles innervated the right and left atria including the auricle and entrance area of the superior vena cava, the inferior vena cava, left precaval vein and pulmonary veins. Large axon bundles entered the area from the major veins and bifurcated into smaller axon bundles and single axon fibers to form terminal end-nets and free endings in the epicardium at each region with a similar pattern. In the atrial muscle layer, varicose CGRP-IR axons had close contacts with muscle fibers. In addition, CGRP-IR axons iv terminated in the intrinsic cardiac ganglia (ICGs) with varicosities surrounding individual ganglionic principle neurons (PNs). In the aortic arch, the CGRP-IR fibers exhibited similar terminal structures to those seen in the atria. 2) SP-IR axons also projected to the right and left atria and aorta. Similar to CGRP-IR axons, these SP-IR axons also formed end-nets and free endings in these areas. In cardiac ganglia, SP-IR axons formed varicose endings around many individual PNs. However, a salient difference was found: There appeared to be fewer SPIR axons and terminals than CGRP-IR axons and terminals in the atria. 3) None of the cardiac PNs in ICG were CGRP-IR or SP-IR. 4) Many SP-IR axon terminals around PNs within ICGs and atrial muscles were found to have colocalized expression of CGRP-IR. Collectively, our data for the first time documented the distribution patterns and morphology of sympathetic afferent axons and terminals in each region of the atria in the mouse model. This will provide a foundation for future analysis of the pathological changes of sympathetic afferent nerves in the atria in different disease models (e.g., diabetes, sleep apnea, and aging). This study was supported by NIH R01 HL- 79636.

Complex genetic mechanisms both endow developing neuronal subtypes with distinct molecular identities and translate those identities into the signatures of cell surface axon guidance molecules that direct neural circuit assembly. The final steps of this process, where axon guidance molecules instruct circuit outcomes, are well-understood. However, the upstream identity molecules that define guidance molecule signatures, and the molecular mechanisms by which cell type identity is transformed into these signatures, remain enigmatic. The murine olfactory system contains nearly 1,5000 olfactory sensory neuron (OSN) subtypes which are intermixed in the olfactory epithelium (OE). Each OSN subtype expresses a unique olfactory receptor (OR) protein which both tunes its response properties to odorants in the environment and acts as an identity molecule that ensures all axons of a given OSN type converge to a single set of target glomeruli in the olfactory bulb (OB). Using a combination of bioinformatic and mouse genetic approaches, we have discovered an unanticipated role for endoplasmic reticulum stress (ER stress) and the unfolded protein response (UPR) in the translation of OR identity to OSN axon guidance molecule expression and glomerular targeting. We find that slight differences in OR amino acid sequences lead to differential activation of the ER stress sensor PERK in different OSN subtypes. Graded patterns of the UPR are then interpreted through a master regulator transcription factor, Ddit3, which controls a set of stress-responsive axon guidance molecules that orchestrate the process of glomerular segregation in the OB. Our results define a novel paradigm for axon guidance in which graded activation of a canonical stress response pathway is leveraged towards the conversion of discrete neuronal identities into discrete circuit formation outcomes. These findings may be widely relevant for the formation of neural circuits across a variety of systems.

No description available.

<p>The Slit morphogen is a secreted glycoprotein that is naturally cleaved into two fragments. The amino fragment (N-Slit) contains Leucine Rich Repeats (LRR) that are recognised by Robo receptors, and is sufficient to mediate attractive or repellent signalling in <em>Drosophila </em>tissues, for example, during growth cone guidance at the midline of the nervous system. The carboxy fragment (C-Slit) is composed of EGF repeats and a Laminin-like globular domain. Although C-Slit expression does not restore repellent signalling, it does rescue other morphogenetic defects in <em>slit</em> mutants.</p> <p>Formation of the dorsal vessel (or heart) requires function of <em>slit</em> and <em>robo</em>. Slit is required for coordinated migration of heart cell precursors, cell polarisation and the formation of a lumen in the heart tube. We have characterised the morphogenetic activities of N- Slit and C-Slit during assembly of the heart.</p> <p>Our laboratory has shown that Slit transgenes lacking the LRR region fail to rescue the mutant phenotype in the nervous system. However, <em>slit</em> trangenes lacking the LRR results in a partial rescue phenotype in the heart suggesting that C-Slit might have functional significance in the heart. Therefore, Slit function in heart vasculogenesis has different requirements compared to the nervous system. For example, Slit –Robo2 interaction may have an adhesive function in addition to a signalling function during vasculogenesis.</p> <p>Our results indicate that C-Slit funtions as a heart morphogen. In <em>slit</em> mutants, over-expression of C-Slit results in a partial rescue phenotype with several features such as cell clumping, overlapping of cells and cells which are elongated. Together, these data suggest alternative functions for Slit during heart morphogenesis.</p> / Master of Science (MSc)

L’architecture polarisée des neurones est mise en place est maintenue grâce à un adressage hautement contrôlé de protéines vers l’axone ou vers le compartiment somatodendritique. Parmi ces protéines, les récepteurs aux protéines G (RCPG) neuronaux sont des cibles pharmacologiques clés. Cependant, leur pharmacologie est généralement étudiée dans des lignées cellulaires non polarisées et les résultats obtenus dans ces systèmes ne caractérisent pas correctement les effets physiologiques de l’activation des RCPG présents dans le cerveau. Par conséquent, un des principaux sujets de recherche de notre équipe est de comprendre comment la polarité neuronale influe sur la pharmacologie des RCPG, en étudiant l’un des RCPG les plus abondants dans le cerveau : le récepteur cannabinoïque de type-1 (CB1R). Les études précédentes de notre groupe ont suggéré que CB1R acquiert une polarisation axonale grâce à un adressage transcytotique : après leur synthèse, ces récepteurs apparaissent sur la membrane plasmique somatodendritique d’où ils sont rapidement enlevés par endocytose constitutive puis adressés à la membrane plasmique axonale où ils s’accumulent du fait d’une endocytose réduite. Au début de ma thèse, nous avons directement mesuré cette endocytose différentielle et le transport transcytotique de CB1R en utilisant des neurones de rats mis en culture dans des dispositifs microfluidiques. De plus, nous avons montré que des traitements pharmacologiques prolongés peuvent fortement changer la distribution de RCPG à la surface neuronale. Ces résultats démontrent que l’équilibre endocytotique dépendant du compartiment neuronal, qui est contrôlable pharmacologiquement, est important pour la distribution des RCPG neuronaux. Dans une seconde partie, nous avons étudié si le trafic différentiel de CB1R entre axones et dendrites est corrélé avec une pharmacologie différentielle. CB1R est majoritairement couplé à des protéines de type Gi/o et est connu pour inhiber la production d’AMPc. Nous avons donc développé l’imagerie par Föster Resonance Energy Transfer (FRET) appliqué aux cultures de neurones d’hippocampe de rats afin de mesurer la modulation de la voie de signalisation AMPc/PKA en aval de CB1R endogènes dans l’ensemble des compartiments neuronaux : somata, dendrites, mais aussi dans les axones matures très fins. Nos résultats montrent que CB1R possède une pharmacologie différente entre les dendrites et les axones. Notamment, son activation conduit à une diminution plus forte de l’activité basale de la PKA dans les axones comparé aux dendrites, lié au plus grand nombre de récepteurs présents sur la membrane de ce compartiment. De plus, nous démontrons que, contrairement aux récepteurs axonaux, les CB1R somatodendritiques inhibent constitutivement la voie AMPc/PKA. Cette différence est due à la distribution polarisée de la DAGLipase, l’enzyme synthétisant l’endocannabinoïde principal, le 2-arachidonoyglycerol (2-AG). De plus, l’inhibition pharmacologique de la DAGL modifie l’efficacité de plusieurs agonistes de CB1R dans le compartiment somatodendritique mais pas dans l’axone. Cet effet pourrait être dû à une modulation allostérique. Dans une troisième partie, nous avons étudié si les résultats ci-dessus peuvent être généralisés à d’autres RCPG. Etant donné que l’adressage axonal et la pharmacologie in vitro des récepteurs sérotoninergiques 5-HT1B montrent de fortes similitudes avec ceux de CB1R, nous avons étudié la pharmacologie de ces récepteurs en utilisant la technique de FRET développée précédemment. De façon similaire, nous avons trouvé une pharmacologie différentielle entre l’axone et les dendrites. / Polarized neuronal architecture is achieved and maintained mainly through highly controlled targeting of proteins to axons versus to the somatodendritic compartment. Among these proteins, neuronal G protein coupled receptors (GPCRs) are key therapeutic targets. However, their pharmacology is generally studied in non-polarized cell lines, and results obtained in such systems likely do not fully characterize the physiological effects of brain GPCR activation. Therefore, a main research subject of our group is to understand how neuronal polarity influences GPCR pharmacology, by studying one of the most abundant GPCR in the brain: the type-1 cannabinoid receptor (CB1R). Previous studies of the group suggested that CB1Rs achieve axonal polarization through transcytotic targeting: after their synthesis, these receptors appear on the somatodendritic plasma membrane from where they are removed rapidly by constitutive endocytosis and then targeted to the axonal plasma membrane where they accumulate due to relatively reduced endocytosis rate. At the beginning of my PhD project we directly demonstrated this differential endocytosis and transcytotic transport of CB1Rs by using cultured neurons in microfluidic devices. Moreover, we showed that chronic pharmacological treatments may strongly change neuronal GPCR distribution on the neuronal surface. These results demonstrate that subdomain-dependent steady-state endocytosis, which is pharmacologically controllable, is important for GPCR distribution in neurons. In a second part, we asked if differential traffic of CB1Rs between axons and dendrites is correlated with differential pharmacology. CB1R is predominantly coupled to Gi/o proteins and is known to inhibit cAMP production. Thus, we developed live Föster Resonance Energy Transfer (FRET) imaging in cultured hippocampal neurons in order to measure basal cAMP/PKA pathway modulation downstream of endogenous CB1Rs in all neuronal compartments: in somata, in dendrites but also in the very thin mature axons. Our results show that CB1R displays differential pharmacology between axon and dendrites. Notably, its activation leads to a stronger decrease of PKA activity in axons compared to dendrites, due to increased number of membrane receptors in this compartment. Moreover, we demonstrate that somatodendritic CB1Rs constitutively inhibit cAMP/PKA pathway, while axonal receptors do not. This difference is due to polarized distribution of DAGLipase, the enzyme that synthesizes the major endocannabinoid 2-arachidonoylglycerol (2-AG). Moreover, blocking DAGL by pharmacological treatment modifies somatodendritic, but not axonal effects of several CB1R agonists, possibly through allosteric action. In a third part, we asked if the above results may be generalized to other GPCRs. Because the axonal targeting and in vitro pharmacology of 5-HT1B serotonin receptors demonstrate strong similarities with CB1Rs, we studied their neuronal pharmacology by using the previously developed FRET technique. We found similar differential responses to pharmacological treatments between axon and dendrites. In a fourth part, we investigated the role of the threonine 210 (T210) residue in the constitutive activity of neuronal CB1R. We showed that the hypoactive mutant T210A-CB1R do not constitutively recruit signaling pathways even in somatodendritic compartment, where 2-AG is present. This result demonstrates that T210 is necessary for constitutive CB1R activation by 2-AG.Finally, previous results of our group demonstrated the involvement of CB1R in neuronal development. Notably, CB1R activation was shown to have an overall inhibitory effect on the development of polarized neuronal morphology. We established a bibliographic review on this subject. The published literature data suggest that not only neuronal polarization influences both CB1R traffic and pharmacology but CB1Rs also contribute to the achievement of neuronal polarization. (...)

This study examines the prospects of using the electrospinning process to fabricate tissue engineering scaffolds targeting a variety of regenerative applications, with a primary focus on the production of nerve guides for the treatment of long-defect nerve injuries in the peripheral nervous system. A basic overview of the conventional electrospinning process is provided, and the utility of this fabrication scheme in the production of collagen-based tissue engineering scaffolds is demonstrated. Next, a novel modification of the basic electrospinning process is presented. This process, called two pole air gap electrospinning, was developed to produce nerve guides that exhibit an anisotropic structure that mimics the extracellular matrix of native peripheral nerve tissue. This electrospinning process makes it possible to produce macroscopic nerve guides that are cylindrical in shape and composed of dense arrays of nano- to micron-scale diameter fibers. Unlike, conventional hollow core nerve guides, these electrospun constructs lack a central lumen, hence the designation 3D (for three-dimensional) nerve guide. The fibers are nearly exclusively arrayed in parallel with the long axis of the construct. This architectural feature provides thousands of individual channels, and aligned fibers that provide guidance cues that are designed to drive regenerating axons to grow in a highly directed fashion down the longitudinal axis of the guide. To supplement the structural cues provided by the fibrillar arrays of the electrospun 3D nerve guides, an alginate-based platform designed to deliver therapeutic reagents was developed and characterized. This platform makes it possible to fabricate gradients of therapeutic reagents within the fibrillar arrays of an electrospun nerve guide. Functional and structural analyses of these constructs supplemented with or without a gradient of NGF, in a long-defect nerve injury in the rodent sciatic nerve indicate that the 3D design is superior to the gold standard treatment, the autologous nerve graft. Animals treated with the 3D grafts recovered motor and sensory function faster and exhibited far higher nerve-to-nerve and nerve-to-muscle signal amplitudes in electrophysiological studies than animals treated with autologous grafts or conventional hollow core cylindrical grafts.

Im Gehirn gemessene Extrazelluläre Feldpotentiale (EFPs) sind ein wichtiges Maß für neuronale Aktivität. In vielen Fällen ist der genaue physiologische Ursprung dieser Potentiale unbekannt oder umstritten. Der auditorische Hirnstamm der Schleiereule bietet eine ausgezeichnete Möglichkeit, die EFPs und ihren Ursprung zu untersuchen. Der Hirnstamm der Eule ist ideal, weil das Feldpotential in ihm sehr stark ist, weil die zugrundeliegende Anatomie wohl-untersucht ist, und weil das Potential sehr einfach durch auditorische Stimulation gesteuert werden kann. In dieser Arbeit präsentiere ich zwei Beispiele, in welchen ich mir die einzigartigen Eigenschaften der Schleiereule zunutze mache, um das EFP zu erforschen. Das erste Beispiel behandelt Axone, und ich zeige, dass neuronale Aktivität in Axonbündeln, welche eine charakteristische Endzone besitzen, ein starkes Dipolmoment erzeugen kann. Im zweiten Beispiel behandele ich Synapsen. Aus den EFPs der Synapsen konnte ich die Merkmale der synaptischen Kurzzeitplastizität extrahieren. Die Methoden und Erkenntnisse die ich entwickelt habe sind auf andere Organismen übertragbar und erweitern das Verständnis vom Einfluss unterschiedlicher anatomischer Strukturen auf das EFP. / Extracellular field potentials (EFPs) recorded in the brain are an important indicator of neural activity for neuroscientists. In many cases, their physiological basis is unknown or debated. The barn owl auditory brainstem provides an excellent opportunity to study these EFPs and their origins. The barn owl auditory brainstem is ideal because the field potentials are very large and very easily controlled by the auditory stimulus, and the underlying anatomy is well known. Here I present two examples of exploiting the unique properties of the EFP in the barn owl auditory brainstem. The first is concerned with axons, where I show that activity in axon bundles with characteristic termination zones generates strong dipole moments. The second example is concerned with synaptic currents, from which I was able to extract a signature of short-term plasticity. The methods and insights I developed are applicable to other organisms as well, and contribute to the general understanding of the roles different anatomical structures can play in the generation of EFPs.

Su Huan Xing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 79-89). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.iii / Acknowledgements --- p.iv / Abbreviations Frequently Used --- p.v / Table of contents --- p.vi / Chapter Chapter1 --- General Introduction --- p.1 / Chapter Chapter2 --- Effects of an intravitreal optic nerve graft on the sprouting and regeneration of axotomized retinal ganglion cells --- p.17 / Chapter Chapter3 --- Effects of an intravitreal pre-injured optic nerve graft on the sprouting and regeneration of axotomized retinal ganglion cells --- p.44 / Chapter Chapter4 --- Effects of co-transplantation of an optic nerve graft and a peripheral nerve graft into the vitreous body on the sprouting and regeneration of axotomized retinal ganglion cells --- p.60 / Chapter Chapter5 --- General discussion --- p.74 / References --- p.79 / Tables --- p.90

The mig-10 gene in C. elegans is required for proper axon guidance and/or cell migration of certain neurons during development. In mig-10 (ct41) mutant worms, there is incomplete migration of the anterior lateral microtubule cells (ALMs), hermaphrodite specific neuron (HSN), left coelomocyte cells (ccL), and canal associated neuron (CAN) (Manser and Wood, 1990). The mig-10 (ct41) mutation also causes axon guidance defects in the IL2 neurons, and it enhances unc-6 defects in the axon guidance of the anterior ventral microtubule cell (AVM) (Rusiecki, 1999; C. Quinn, personal communication). mig-10's function in axon guidance and neuronal migration is unknown, but is believed to be involved in a signal transduction pathway that uses a G-protein, such as ras. The two mig-10 transcripts discussed in this thesis, mig-10 A and mig-10 B, encode proteins that are similar to Grb-7 and Grb-10 proteins, which are also believed to function in a signal transduction pathway (Manser et al., 1997). One of these similarities is the presence of a proline-rich region, which may be used to bind another protein (Manser et al., 1997). The MIG-10 A protein has an additional proline region, compared to MIG-10 B, which may indicate that the MIG-10 A and B proteins are utilized in different cells, or at different developmental stages. As a first step in learning where MIG-10 is expressed, mig-10 (ct41) mutant worms containing a wild-type mig-10 B::GFP fusion were constructed. Rescue of the mutant phenotype would indicate that the expression pattern of the transgene was similar to that of the endogenous gene. As this experiment did not allow for rescue, even after integration of the construct, a strain of worms containing a mig-10 promoter::GFP transgene was used. Preliminary observations of this strain indicated that mig-10 is expressed in neuronal tissue. The AIY neurons were observed in wild-type and mig-10 (ct41) worms to determine if they are affected by the mig-10 mutation as previously reported (O. Hobert, personal communication). As no difference was detected, the AIYs were not used in any further experiments. In order to determine which cells require functional MIG-10 protein for the proper development/migration of neurons to occur, mig-10 (ct41) worms containing mec-3 promoter::mig-10 A or B cDNA transgenes were constructed. The mec-3 promoter drives expression of the mig-10 cDNA in the ALM neurons and other touch cells early in the development of the embryo. If these transgenes rescued the ALM migration defect, then mig-10 would be acting cell autonomously in ALM. Partial rescue was obtained, which may be due to the need for both of the mig-10 transcripts to be expressed in the same cell; alternatively, one or both transcripts may need to be expressed in a cell nonautonomous fashion in addition to being expressed cell autonomously. Low production of the rescuing protein, or expression of the protein at a later developmental stage than is needed for rescue to occur, may also have been the cause of the partial rescue. Future work in this area includes putting mig-10 promoter::mig-10 A or B cDNA in mig-10 (ct41) background to investigate if the different transcripts rescue different aspects of the mig-10 phenotype. The mig-10 A and mig-10 B cDNA constructs could also be expressed in the same worm in an attempt to correct for partial rescue that may be due to the lack of both MIG-10 proteins.