As estruturas oculares dos vertebrados apresentam diversas adaptações relacionadas aos hábitats e atividades das espécies. A infra-ordem Serpentes possui amplo número de espécies distribuídas em quase todas as regiões da Terra e seu sistema visual apresenta variações que apontam para adaptações ecológicas. O presente estudo teve por objetivo fazer uma análise comparativa do sistema visual de diferentes espécies de serpentes Caenophidia, das famílias Dipsadidae e Colubridae, centrada no potencial de visão de cores, na densidade e topografia celular da retina e na acuidade visual. Para tanto, foram identificados os genes de opsinas expressos nas retinas, e analisadas a densidade e distribuição dos diferentes tipos de fotorreceptores e das células da camada de células ganglionares (CCG). As serpentes obtidas junto ao Laboratório de Herpetologia do Instituto Butantan foram sacrificadas com dose letal do anestésico thiopental. Os olhos foram enucleados e as retinas dissecadas para estudos genéticos e morfológicos, com imunohistoquímica e coloração de Nissl. Para sequenciamento dos genes das opsinas SWS1, Rh1 e LWS, dois olhos de 17 espécies foram utilizados. A amplificação por PCR mostrou que os três genes são expressos nas retinas de todas as espécies analisadas; o pico de sensibilidade espectral (max) de cada opsina foi estimado a partir das sequências de aminoácidos. O max do fotopigmento SWS1 foi estimado em 360 nm (UV), para todas as espécies. O fotopigmento Rh1, apresentou três diferentes combinações de aminoácidos que geram picos de sensibilidade em 500 nm, 494 nm e 484 nm. Todas as espécies de serpentes diurnas apresentaram a combinação de aminoácidos que gerou o max 484 nm. O fotopigmento LWS apresentou 4 diferentes combinações de aminoácidos, com max variando entre 543 nm e 560 nm. Para os estudos morfológicos foram utilizadas 86 retinas de 20 diferentes espécies. Retinas íntegras foram marcadas com anticorpos específicos para quantificação e análise topográfica de fotorreceptores. A coloração de Nissl foi empregada em retinas planas para quantificação de células da CCG e cálculo da acuidade visual. As análises morfológicas em retinas de serpentes noturnas mostraram uma grande densidade média de fotorreceptores (82.042 ± 37.945 células/mm2), com predominância de bastonetes, enquanto espécies diurnas apresentaram baixa densidade média de fotorreceptores (11.290 ± 2.810 células/mm2) e ausência de bastonetes. Serpentes noturnas apresentaram densidade média mais baixa de células da CCG (7.653 ± 1.636 4 células/mm2) comparada às diurnas (9.575 ± 2.301 células/mm2). O poder de resolução espacial também foi maior em espécies diurnas (2,3 ± 0,7 cpg) do que nas noturnas (1,45 ± 0,4 cpg). A distribuição de fotorreceptores e células da CCG nas retinas mostrou a presença de area centralis em diferentes regiões das retinas de espécies noturnas, e faixa horizontal em retinas das espécies diurnas, com exceção da serpente aquática e diurna Helicops modestus, que apresentou area centralis. As diferenças de localização das areae centralis variaram de acordo com hábitat e características comportamentais das espécies. Serpentes fossoriais do gênero Atractus, por exemplo, apresentaram area centralis na região dorsal da retina, que favorece o campo de visão inferior e auxilia no comportamento de escavar. Os resultados obtidos neste abrangente estudo apontam para a complexidade das adaptações do sistema visual deste grupo de vertebrados. As variações do padrão de atividade (diurna ou noturna) e uso de hábitat parecem ser fatores de forte influência sobre as características do sistema visual, como a sensibilidade espectral dos pigmentos visuais, a densidade e distribuição de neurônios nas retinas e o poder de resolução espacial do olho / The structures of vertebrate eyes have many adaptations related to the habitats and activities of the species. The infra-order Serpentes has a large number of species distributed in almost all regions of the Earth and its visual system presents variations that point to ecological adaptations. This study aimed to compare the visual system of different species of Caenophidian snakes, from the Dipsadidae and Colubridae families. To do so, the opsin genes expressed in the retinas were identified and the density and distribution of the different types of photoreceptors and the cells of the ganglion cell layer (GCC) were analyzed. The snakes were colected from Butantan Institute and were sacrificed with a lethal dose of the anesthetic thiopental. The eyes were enucleated and the retinas dissected for genetic and morphological studies, using Nissl stainig technique and immunohistochemistry. For the sequencing the opsins genes SWS1, Rh1 and LWS, two eyes of 17 species were colected. PCR amplification showed that the three opsin genes are expressed in the retinas of all species analyzed; the maximum spectral sensitivity (max) of each opsin was estimated based on the amino acid sequences. The max of the SWS1 photopigment was estimated at 360 nm (UV), for all species. The photopigment Rh1 had three different combinations of amino acids that generate max at 500 nm, 494 nm and 484 nm. All diurnal species had the amino acid combination that generate the max at 484 nm. The photopigment LWS had 4 different combinations of amino acids with max ranging from 543 nm to 560 nm. For morphological studies, 86 retinas of 20 different species were analyzed. Wholemounted retinas were stained with specific antibodies for analysis of the photoreceptors density and topography. The Nissl stainig technique was used for quantification of GCL cells in flatmounted retinas and estimation of the spatial resolving power. Nocturnal snakes had retinas with higher photoreceptor densities (82,042 ± 37,945 cells/mm2), with predominance of rods, compared to diurnal species that had low photoreceptors density (11,290 ± 2,810 cells/mm2) and the absence of rods. On the other hand, diurnal snakes had higher density of GCL cells (9,575 ± 2,301 cells/mm2) and spatial resolving power (2.3 ± 0.7 cpd), compared to nocturnal (7,653 ± 1,636 cells/mm2 and 1.45 ± 0.4 cpg). The distribution of cells in the retinas had variations related to the circadian rhythm of the species, with the presence of area centralis in retinas of nocturnal species and horizontal streak in retinas of diurnal snakes, except for the diurnal and aquatic Helicops 6 modestus, that had an area centralis in the ventral retina. The location of the area centralis varies according the habitat and specific behavior of each species. The fossorial snake Atractus, for example, had an area in the dorsal retina, which improves the resolution of the inferior visual field and benefits the digging habit in this snake. The results of this comprehensive study point to the complexity of adaptations of the visual system of this group of vertebrates. The differences in the activity pattern (diurnal or nocturnal) and habitat seem to be of great influence on the characteristics of the visual system, such as the spectral sensitivity of the visual pigments, the density and distribution of neurons in the retina and the spatial resolving power of eye

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

O envelhecimento é uma falha progressiva nos processos fisiológicos celulares, produzindo alterações morfológicas nas células e nos tecidos. No sistema nervoso, produz uma redução no número de neurônios, nas fibras nervosas, principalmente nas arborizações dendríticas e nas espinhas sinápticas, e nas células da glia que, de acordo com sua localização e tipo celular, podem diminuir, permanecer constantes ou mesmo aumentar numericamente. Na presente pesquisa, avaliou-se os efeitos do envelhecimento sobre o gânglio trigeminal (GT) de ratos Wistar em animais jovens (2 meses de vida), adultos (12 meses de vida) e idosos (24 meses de vida). Os GT foram submetidos às técnicas histológicas da hematoxilina e eosina e Picro-sírius, onde avaliou-se, respectivamente, a densidade das células satélites glias (CGS) e o componente colágeno ganglionar. Através da técnica histoquímica da NADH-d, avaliou-se a área do perfil do GT, a área do perfil dos corpos celulares dos neurônios ganglionares e a densidade neuronal. Uma avaliação qualitativa foi também realizada relativamente à imunorreatividade dos neurônios ganglionares à substância P (SP) e ao peptídeo intestinal vasoativo (VIP). A densidade das CGS foi maior nos animais jovens do que nos animais adultos e idosos. Verificou-se, qualitativamente, que à medida que o animal envelhece há uma diminuição das fibras colágenas do tipo III, passando a predominar, nos animais idosos, as fibras do tipo I. A área do perfil celular dos corpos neuronais foi maior nos animais adultos sendo que em todos os grupos predominaram neurônios de tamanho médio, com a área do perfil celular entre 490 e 1100 μm2. A densidade neuronal apresentou-se maior nos animais jovens, e sem variações estatísticas entre os animais adultos e idosos. Em todos os grupos estudados, os neurônios pequenos foram os que apresentaram maior imunorreatividade à SP e ao VIP. / Aging is a progressive failure in cellular physiological processes. It determines morphological changes in cells of different tissues. In the nervous system, a reduction in neuron number and in neuron fibers, mainly in dendritic tree and synaptic, are described. With aging the glial cells may increase or decrease in number or also remain constant. In the present work the effects of aging were evaluated on the trigeminal ganglion (TG) comparing young (2 months age), adult (12 months age) and old rats (24 months age). Histological sections of TG were stained with hematoxilin-eosin technique to determine the density of satellite glial cells and Picro-sirius under polarized light to evaluate the Types I and III of collagen fibers. The NADH-diaphorase technique allowed determining the perycarion area. The immunoreactivity of ganglionar neurons to Substance P (SP) and vasoactive intestinal peptide (VIP) were also qualitatively evaluated. The glial cells density was higher in young and adult animals than in old animals. The type I collagen fibers predominates in ganglia of old animals whereas in the young animals is characteristic the presence of the type III collagen fibers. Although the perycarion area was higher in adult animals the medium-sized neurons predominated in all groups. Their areas ranged from 490 to 1100 μm2. It was also observed that the neuron density was higher in young animals. In the adult and old animals the neuron density was similar. In all groups the immunoreactivity both to SP an VIP was detected mainly in neurons of small perycarion.

O sistema nervoso simpático é escassamente descrito em livros na anatomia veterinária e encontra-se pouca informação a respeito de seu funcionamento em grandes mamíferos. O conceito atual da estrutura e função dos gânglios simpáticos deriva de estudos desenvolvidos no gânglio cervical superior (GCC) em animais de laboratório, devido ao seu grande tamanho, facilidade de acesso e multiplicidade de território de inervação nestas espécies. A ganglionectomia unilateral do GCC causou condição patológica associada à síndrome de Horner com sinais associados à anisocoria, enoftalmia, ptose e hipertermia de orelha. Aplicando-se métodos estereológicos, objetivamos investigar a neuroplasticidade do G CC em condições de exigência funcional em diferentes períodos de tempo. A neuroplasticidade foi investigada em vista dos aspectos morfoquantitativos, tamanho e número total de neurônios. Objetivou-se encontrar variação, ou não, no tamanho dos neurônios, na densidade neuronal, no número de neurônios secundários à ganglionectomia unilateral do GCC. Alterações macroscópicas revelaram um aumento médio de 8%, 3% e 11% em comprimento, largura e espessura para os gânglios operados no grupo I. Para o grupo II encontraram-se aumentos em 4% para comprimento e largura e 5% em espessura, enquanto que para o grupo III, 29% de aumento foram encontrados para comprimento, 4% em largura e 7% em espessura. Os gânglios controle e operados apresentaram diferença significativa (p = 0,0001) na densidade neuronal (Nv). Os gânglios operados apresentaram diminuição na densidade neuronal em média de 89% para o grupo I, 65% para o grupo II e 47% para o grupo III. Esta redução reflete a distribuição heterogênea dos neurônios no gânglio operado. Um aumento no volume neuronal global foi significativamente detectado (p = 0,0001), o gânglio operado apresentou aumentos médios de 13% para o grupo I, 24% para o grupo II e 29% para o grupo III, sugestivo de maior exigência funcional em resposta à ganglionectomia. O numero total de neurônios apresentou diferenças significativas (p = 0,0514) e dois efeitos distinto nos gânglios operados. No grupo I observou-se um aumento de 3% no numero total de neurônios enquanto que uma redução foi determinada para os grupos II (8%) e III (20%). A avaliação global dos resultados leva-nos a inferir duas hipóteses associadas e consecutivas: 1) a hipertrofia neuronal estaria associada a um mecanismo compensatório para a re-inervação contralateral sendo que a maior exigência funcional poderia levar estes neurônios à morte, seguido de hipertrofia de tecido não-neuronal cicatricial refletido na diminuição da densidade neuronal; 2) a hipertrofia neuronal estaria associada a um mecanismo compensatório à morte celular determinada pela maior exigência funcional. As alterações quantitativas principais secundárias à ganglionectomia unilateral do GCC no gânglio remanescente estão associadas à: perda significativa no número total de neurônios a partir da 8ª semana de evolução da doença, perda significativa na densidade neuronal a partir da 8ª semana e aumento significativo na área e volume neuronal. / The sympathetic nervous system is briefly described in veterinary anatomy text-books and there is little information so far concerning its function in large mammal species. The current concept of the structure and function of the sympathetic ganglia is derived from studies on the cranial cervical ganglia (CCG) carried out in laboratory animals due to very attractive characteristics of CCG in these species such as large size, accessibility and multiplicity of target organs. The CCG unilateral ganglionectomy caused a pathological condition associated with Horner´s syndrome which includes anisocoria, enophtalmos, ptosis and increase in the temperature of ear as a result of peripheral vasodilatation. Using stereology-designed methods, we aimed to study CCG neuroplasticity under experimental functional overloading along distinct periods of time. Neuroplasticity was investigated according to morphoquantitative aspects, mostly size and total number of neurons. We wanted to find out whether or not neuron size, numerical density and nerve cell numbers would vary as a result of CCG ganglionectomy. Gross anatomic differences were considered to the increase in the operated ganglia, means of 8%, 3% and 11% for length, width and thickness, respetively in the group I. For group II it was encountered means increases of 4% for length and width and 5% in thickness. Group III showed means values of 29% increase in length, 4% in width and 7% in thickness. Control and operated sheep CCG revealed significant difference (p=0.0001) in the neuronal density (Nv). The operated ganglia revealed reduction in the neuronal density of 89% for group I, 65% for group II and 47% for group III. This reduction in the operated ganglia reflects its inhomogeneous distribution of neurons. An increase in the global neuronal volume was significantly detected (p= 0.0001), the operated ganglia showed increases in the means of 13% for group I, 24% for group II and 29% for group III, suggesting a functional overload response for the unilateral ganglionectomy. The total number of neurons presented significant differences (p = 0.0514) and two distinct effects in the operated ganglia. In the group I, an increase of 3% was encountered meanwhile reduction in the total number was associated to groups II (8%) and group III (20%). An overlook of the results suggests two consecutive and associated hypotheses: 1) the hypertrophy of neurons would be associated to a compensatory mechanism for contralateral re-innervation although the functional overload would drive these neurons to cellular death, followed by hypertrophy of non-neuronal tissue as reflected in the neuronal density; 2) the neuronal hypertrophy would be associated to a compensatory mechanism to cellular death caused by the functional overload The main quantitative changes in the remaining ganglia are: Significant loss in the total number of neurons from the 8th week of evoloution of the disease; Significant decrease in the numerical density (Nv) from the 8th week and significant increase in both neuron area and neuron volume.

Le récepteur activateur de NF-κB (RANK), membre de la famille des récepteurs au TNF, est connu pour son rôle dans l’homéostasie de l’os, mais joue aussi un rôle important dans le système immunitaire. J’ai tout d’abord étudié des outils permettant de cibler RANK/RANKL. J’ai caractérisé et comparé l’activité biologique de deux anticorps anti-RANK. J’ai également criblé une librairie de petites molécules pour identifier des inhibiteurs de l’interaction RANK/RANKL. Dans une deuxième partie, je me suis intéressée au rôle du ligand de RANK (RANKL) dans l’homéostasie du ganglion lymphatique. RANKL joue un rôle dans la différenciation des ostéoclastes mais son rôle dans la différenciation d’autres macrophages n’a pas été étudié. Nous avons étudié des souris déficientes pour RANKL dans les cellules marginales réticulaires (MRC) qui expriment RANKL de manière constitutive dans le ganglion adulte. Nous avons observé une diminution de la population de macrophages sous-capsulaires (SSM). Nous avons également montré que les cellules endothéliales lymphatiques (LEC) expriment l’intégrine alpha 2b (ITGA2b) et que cette expression est sensible à la présence de RANKL. / The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation. Firstly I looked for new molecular tools to target RANK/RANKL axis. I characterized and compared the biological activity of two anti-RANK antibodies. Moreover, I screened the Prestwick Chemical Library® of small molecules in order to identify inhibitors of RANK/RANKL interaction. Secondly, I studied the effect of the RANK/RANKL axis in lymph node homeostasis. RANKL is known to promote osteoclast differentiation but whether it also plays a role in the differentiation of other macrophage subsets is not known. We addressed this question by conditionally deleting RANKL from marginal reticular stromal cells (MRCs) that constitutively express RANKL in the lymph node. We observed impaired differentiation of the subcapsular sinus macrophages (SSMs). We also studied lymph node lymphatic endothelial cells (LECs) and showed that integrin alpha 2b (ITGA2b) is expressed by a lymph node subset of LECs and its expression is sensitive to RANKL.

<p>Our knowledge of the fine structure of the Human Spiral Ganglion (HSG) is still inadequate and new treatment techniques for deafness using electric stimulation, call for further information and studies on the neuronal elements of the human cochlea. This thesis presents results of analyses of human cochlear tissue and specimens obtained during neurosurgical transpetrosal removal of life-threatening meningeomas. The use of surgical biopsies produced a well-preserved material suitable for ultrastructural and immunohistochemical studies on the human inner ear. The SG was studied with respect to fine structure, using TEM technique and the immunostaining pattern of synaptophysin, which is an integral membrane protein of neuronal synaptic vesicles. The immunostaining patterns of the tight junctional protein ZO-1 and the gap junctional proteins Cx26 and Cx43 in the human cochlea were also studied. The ultrastructural morphology revealed an absence of myelination pattern in the HSG, thus differing from that in other species. Furthermore, formation of structural units as well as signs of neural interaction between the type 1 neurons could be observed. Type 1 cells were tightly packed in clusters, sharing the ensheathment of Schwann cells. The cells frequently made direct physical contact in Schwann cell gaps in which membrane specializations appeared. These specializations consisted of symmetrically or asymmetrically distributed filamentous densities along the apposed cell membranes. The same structures were also present between individual unmyelinated nerve fibres and the type 1 cells. Synapses were observed on the type 2 neurons, with nerve fibres originating from the intraganglionic spiral bundle. Such synapses, though rare, were also observed on the type 1 cells. The ultrastructural findings were confirmed by the synaptophysin study. A 3-D model of a Schwann cell gap between two type 1 cells was constructed, describing the distribution pattern of membrane specializations. In the immunohistochemical studies on the human cochlea, ZO-1 was expressed in tissues lining scala media, thus contributing to the formation of a closed compartment system, important for the maintenance of the specific ionic composition of the endolymph. Protein Cx26 could be identified in non-sensory epithelial cells of the organ of Corti, in connective tissue cells of the spiral ligament and spiral limbus, as well as in the basal and intermediate cell layers of stria vascularis. Cx26 in this region may be involved in the recycling of potassium. Protein Cx43 stained weakly in the spiral ligament, but intense staining in the SG may indicate that gap junctions exist between these neurons. A different functional role for the HSG can be assumed from the morphological characteristics and the signs of a neural interaction between the SG cells. This might be relevant for neural processing mechanisms in speech coding and could have implications for cochlear implant techniques.</p>

La biopsie du ganglion sentinelle (GS) est actuellement la technique de référence pour le diagnostic des métastases ganglionnaires du cancer du sein. Cependant, les traceurs utilisés pour la cartographie du GS (colorant bleu et radiocolloïde) ne sont pas idéaux et peuvent occasionner des réactions allergiques et engendrer des coûts importants. Une alternative à l'utilisation de ces traceurs repose sur le repérage du GS par imagerie de fluorescence proche infrarouge à l'aide de nanoparticules. Deux types de nanoparticules ont été étudiés : les Quantum Dots (QDs) à base d'indium et les nanoparticules de silice (NPSi) renfermant de la cyanine 7. Notre étude s'est basée sur des QDs-indium, dont la toxicité est supposée être plus faible que celle des QDs à base de cadmium. La visualisation du GS par les traceurs actuels étant altérée par l'envahissement métastatique du ganglion, un modèle murin de carcinome mammaire présentant des métastases ganglionnaires a été utilisé. La présence de cellules métastatiques dans le ganglion n'affecte pas la migration des QDs puisqu'aucune différence d'intensité de fluorescence n'a pu être détectée entre les ganglions sains et les ganglions envahis. L'étude de biodistribution a mis en évidence une capture majeure des QDs au point d'injection et dans les ganglions ainsi qu'une plus faible concentration dans le foie et la rate. La toxicité des QDs a été évaluée in vitro et a démontré une toxicité fortement réduite des QDs-indium par rapport aux QDs-cadmium. Les NPSi ont été largement appliquée à l'imagerie tumorale mais les études portant sur la visualisation du GS sont peu nombreuses. Les avantages de l'encapsulation de la cyanine 7, fluorophore hydrophobe émettant dans le proche infrarouge, dans des NPSi comprennent notamment une meilleure rétention dans le GS et une toxicité in vitro considérablement limitée par rapport au fluorophore libre. Les souris ont été suivies pendant 3 mois après injection de NPSi et aucun signe de toxicité générale ou hépatique n'a pu être décelé. Ces deux types de nanoparticules fluorescentes sont particulièrement bien adaptés à la cartographie du GS et pourraient avantageusement remplacer les traceurs employés actuellement.

Following microsurgical reconstruction of injured peripheral nerves, severed axons are able to undergo spontaneous regeneration. However, the functional result is always unsatisfactory with poor sensory recovery and reduced motor function. One contributing factor is the retrograde neuronal death, which occurs in the dorsal root ganglia (DRG) and in the spinal cord. An additional clinical problem is the loss of nerve tissue that often occurs in the trauma zone and which requires “bridges” to reconnect separated nerve ends. The present thesis investigates the extent of retrograde degeneration in spinal motoneurons and cutaneous and muscular afferent DRG neurons after permanent axotomy and following treatment with N-acetyl-cysteine (NAC). In addition, it examines the survival and growth-promoting effects of nerve reconstructions performed by primary repair and peripheral nerve grafting in combination with NAC treatment. In adult rats, cutaneous sural and muscular medial gastrocnemius DRG neurons and spinal motoneurons were retrogradely labeled with fluorescent tracers from the homonymous transected nerves. Survival of labeled neurons was assessed at different time points after nerve transection, ventral root avulsion and ventral rhizotomy. Axonal regeneration was evaluated using fluorescent tracers after sciatic axotomy and immediate nerve repair. Intraperitoneal or intrathecal treatment with NAC was initiated immediately after nerve injury or was delayed for 1-2 weeks. Counts of labeled gastrocnemius DRG neurons did not reveal any significant retrograde cell death after nerve transection. Sural axotomy induced a delayed loss of DRG cells, which amounted to 43- 48% at 8-24 weeks postoperatively. Proximal transection of the sciatic nerve at 1 week after initial axonal injury did not further increase retrograde DRG degeneration, nor did it affect survival of corresponding motoneurons. In contrast, rhizotomy and ventral root avulsion induced marked 26- 53% cell loss among spinal motoneurons. Primary repair or peripheral nerve grafting supported regeneration of 53-60% of the motoneurons and 47-49% of the muscular gastrocnemius DRG neurons at 13 weeks postoperatively. For the cutaneous sural DRG neurons, primary repair or peripheral nerve grafting increased survival by 19-30% and promoted regeneration of 46-66% of the cells. Regenerating sural and medial gastrocnemius DRG neurons upregulate transcription of peripherin and activating transcription factor 3. The gene expression of the structural neurofilament proteins of high molecular weight was significantly downregulated following injury in both regenerating and non-regenerating sensory neurons. Treatment with NAC was neuroprotective for spinal motoneurons after ventral rhizotomy and avulsion, and sural DRG neurons after sciatic nerve injury. However, combined treatment with nerve graft and NAC had significant additive effect on neuronal survival and also increased the number of sensory neurons regenerating across the graft. In contrast, NAC treatment neither affected the number of regenerating motoneurons nor the number of myelinated axons in the nerve graft and in the distal nerve stump. In summary, the present results demonstrate that cutaneous sural sensory neurons are more sensitive to peripheral nerve injury than muscular gastrocnemius DRG cells. Moreover, the retrograde loss of cutaneous DRG cells taking place despite immediate nerve repair would still limit recovery of cutaneous sensory functions. Experimental data also show that NAC provides a highly significant degree of neuroprotection in animal models of adult nerve injury and could be combined with nerve grafting to further attenuate retrograde neuronal death and to promote functional regeneration.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

Each neuron needs to maintain a careful balance between the changes implicit in experience, and the demands of stability required by its function. This balance tips depending on the neuronal system, but in any role, disease or neural disorders can develop when the regulatory mechanisms involved in neuronal stability fail. The objective of this thesis was to characterize mechanisms underlying neuronal stability and activity maintenance, in the hopes that further understanding of these processes might someday lead to novel interventions for neurological disorders. The pyloric circuit of decapod crustaceans controls the rhythmic contractions of the foregut musculature, and has long been recognized as an excellent model system in which to study neuronal network stability. Recent experimental evidence has shown that each neuronal cell type of this circuit exhibits a unique set of positive linear correlations between ionic membrane conductances, which suggests that coordinated expression of ion channels plays a role in constraining neuronal electrical activity. In Aim 1, we hypothesized a causal relationship between expressed conductance correlations and features of cellular identity, namely electrical activity type. We partitioned an existing database of conductance-based model neurons based on various measures of intrinsic activity to approximate distinctions between biological cell types. We then tested individual conductance pairs for linear dependence to identify correlations. Similar to experimental results, each activity type investigated had a unique combination of correlated conductances. Furthermore, we found that populations of models that conform to a specific conductance correlation have a higher likelihood of exhibiting a particular feature of electrical activity. We conclude that regulating conductance ratios can support proper electrical activity of a wide range of cell types, particularly when the identity of the cell is well-defined by one or two features of its activity. The phenomenon of pyloric network recovery after removal of top-down neuromodulatory input--a process termed decentralization--is seen as a classic model of homeostatic change after injury. After decentralization, the pyloric central pattern generator briefly loses its characteristic rhythmic activity, but the same activity profile is recovered 3-5 days later via poorly understood homeostatic changes. This re-emergence of the pyloric rhythm occurs without the full pre-decentralization set of fixed conductance ratios. If conductance ratios stabilize pyloric activity before decentralization as we showed in Aim 1, then other mechanisms must account for the return of the pyloric rhythm after network recovery. Based on vertebrate studies demonstrating a role for the extracellular matrix (ECM) in activity regulation, we hypothesized in Aim 2 that the ECM was participating in activity maintenance in the stomatogastric nervous system. We used the enzyme chondroitinase ABC (chABC) to degrade extracellular chondroitin sulfate (CS) in the stomatogastric ganglion while in organ culture. Our results are the first to demonstrate the presence of CS in the crustacean nervous system via immunochemistry. Furthermore, we show that while ongoing activity is not disrupted by chABC treatment, recovery of pyloric activity after decentralization was significantly delayed without intact extracellular CS. Our results are the first to show that CS has a role in neuronal activity maintenance in crustaceans, and suggest that CS may be involved in initiating or directing activity maintenance needed in times of neuronal stress.