Dentate microcircuitry is thought to be involved in filtering, integrating, and relaying extrinsic hippocampal inputs to the hippocampus proper, which contributes to memory formation and retrieval. The axons of granule cells are called mossy fibres (MFs), and contain multiple terminal types that form characteristic synaptic connections with their postsynaptic targets. This diversity of presynaptic release sites that exists on the same MF provides an extremely interesting axonal type to study the organizing principles of presynaptic release regulation. A remarkable set of neurotransmitters and receptors present at the MF synaptic complex allow diverse computational modification of information from the dentate gyrus to the hippocampus. There are several types of glutamate receptors expressed at MF, such as group II/III mGluRs and kainate receptors (KARs). Presynaptic KARs modulate transmission at MF-CA3 pyramidal cell synapses; however, it is not known whether presynaptic KARs affect other synapses made by MFs. The aim of the first part of this thesis was to establish the principles of synapse-specific actions of presynaptic KARs in MFs. Combining electrophysiology and calcium imaging, this study provides compelling evidence that presynaptic KARs and Ca²⁺ stores can be activated by glutamate release from a single action potential in a single MF axon. This contributes to short-term, use-dependent facilitation of presynaptic Ca²⁺ entry and glutamate release exclusively at MF-CA3 pyramidal cell synaps, but not at other MF synapses, on hilar mossy cells or interneurons. Thus, our findings indicate that the presynaptic KARs, coupled with intracellular stores, exist in a synapse-specific autoreceptor mechanism. Activation of KARs strengthened MF-CA3 pyramidal cell synapses by increasing the Ca²⁺ influx at giant boutons, which might also contribute to the KAR-dependent hyper-excitability of the MF circuitry related to the mechanisms of temporal lobe epilepsy (TLE). This makes KARs good potential targets for therapies in CNS disorders such as epilepsy and other neurological and psychiatric disorders. The second part of this thesis was to explore the actions on the hippocampus of purified antibodies from a limbic encephalitis (LE) patient. LE is a CNS disease characterized by subacute onset of memory loss and temporal lobe seizures. The serum of these patients strongly labels MFs apparently co-localizing with the VGKC. The patients improve with immunotherapies that reduce the VGKC antibody levels in the serum, thus, strongly suggesting that these antibodies cause the condition. We found that LE serum IgGs enhance CA3 pyramidal cell excitability by blocking α-DTX sensitive VGKCs, which results in the increased release of glutamate. This, in turn, strengthens and desynchronizes MF and CA3 pyramidal cells synaptic transmission. However, these effects were occluded by α-DTX, a Kv1.1, Kv1.2 and Kv1.6 antagonist which, when applied alone, mimicked the action of the LE IgG, suggesting that they may share similar mechanisms of action. In contrast serum taken from healthy control patients had no significant effect under same recording conditions. Thus, this study provides the first evidence that the LE IgG functionally affects VGKC containing Kv1.1, Kv1.2 and/or Kv1.6 at both presynaptic MF axon terminals as well as the postsynaptic somatodendritic domain of CA3 pyramidal cells. Whatever defines the exact nature of LE IgG action, our results suggest that drugs acting specifically as openers of VGKC might help to protect the hippocampus from immune-mediated damage. In conclusion my data is consistent with the increasingly documented idea that MFs play a critical role in regulating the excitability of the hippocampal circuits and the dysfunction of MF transmission profoundly impairs hippocampal function.

The original Bio-Rad gene gun was unable to transfect acute or organotypic brain slices, as the amount of helium gas used, the distance for the gold-coated microcarriers to travel to target area were not optimised for fragile tissues, such as the brain. Typically, tissues were severely damaged by a helium shock wave and only a few cells were transfected. It was essential to improve gene gun accuracy by restricting the gold particles from being propelled superficially over a wide area. It was also necessary to increase the amount of DNA or dye delivery into intact tissues. Furthermore, for the gene gun to perform successfully on brain slices the helium gas pressure had to be lowered thereby reducing the degree of cell damage incurred during a biolistic delivery. Without knowing it at the time, the modified gene gun had worked particularly well on a variety of other fragile tissues, and not just the brain. However, the modified gun was not optimised for cultured cells as other transfection methods were available. A particularly notable point of this work was the successful labelling of individual Purkinje dendritic spines from live nerve cells in the cerebellum region of the brain. Biolistic images of Purkinje cells show that the distribution of dendritic spines are not random (O’Brien and Unwin, 2006). Spines were shown to grow in elaborate regular linear arrays, that trace short-pitch helical paths around the dendrites. It was apparent that the spines are arranged to maximize the probability that the dendritic arbour would interact with any afferent axon. This was an important discovery as there has been much debate as to how spines develop on a dendritic shaft. There are three general views to this question, each proposing a theory describing a model for spinogenesis. Classification of the three models in relation to our findings is described in chapter six of this thesis. The Investigation of spine morphology by biolistics was further optimized; gold particles were reduced from a micrometre to forty nanometres (O’Brien and Lummis, 2011), demonstrating that it is possible to use gold-coated DNA nanoparticles of this size to transfect tissue revealing exquisite structural detail. It was possible to observe boutons making synaptic contacts with the pyramidal nerve spines in the hippocampal region of the brain. The findings so far have shown spines from the pyramidal shaft are similar to the spines in the cerebellum, forming regular linear arrays. Recent studies had linked defects in the function of presynaptic boutons to the etiology of several neurodevelopment and neurodegenerative diseases, including autism and Alzheimer’s disease. Our discovery could help to understand why there are abnormalities in dendritic spines which are associated with pathological conditions characterized by cognitive decline, such as mental retardation, Alzheimer’s, stroke and schizophrenia (Yuste and Bonhoeffer, 2001). This thesis provides a synthesis of knowledge about biolistic technology. It is presented as a narrative from improving the gene gun transfection efficiency in brain slices to the development of nano-biolistics. The delivery of DNA and fluorescent dyes into living cells by biolistic delivery should enable a detailed map of the anatomical connections between individual cells and groups of cells to be constructed, providing a “wiring diagram” of connections. The implications of this are discussed in Chapter twelve. The original Bio-Rad gene gun was unable to transfect acute or organotypic brain slices, as the amount of helium gas used, the distance for the gold-coated microcarriers to travel to target area were not optimised for fragile tissues, such as the brain. Typically, tissues were severely damaged by a helium shock wave and only a few cells were transfected. It was essential to improve gene gun accuracy by restricting the gold particles from being propelled superficially over a wide area. It was also necessary to increase the amount of DNA or dye delivery into intact tissues. Furthermore, for the gene gun to perform successfully on brain slices the helium gas pressure had to be lowered thereby reducing the degree of cell damage incurred during a biolistic delivery. Without knowing it at the time, the modified gene gun had worked particularly well on a variety of other fragile tissues, and not just the brain. However, the modified gun was not optimised for cultured cells as other transfection methods were available. A particularly notable point of this work was the successful labelling of individual Purkinje dendritic spines from live nerve cells in the cerebellum region of the brain. Biolistic images of Purkinje cells show that the distribution of dendritic spines are not random (O’Brien and Unwin, 2006). Spines were shown to grow in elaborate regular linear arrays, that trace short-pitch helical paths around the dendrites. It was apparent that the spines are arranged to maximize the probability that the dendritic arbour would interact with any afferent axon. This was an important discovery as there has been much debate as to how spines develop on a dendritic shaft. There are three general views to this question, each proposing a theory describing a model for spinogenesis. Classification of the three models in relation to our findings is described in chapter six of this thesis. The Investigation of spine morphology by biolistics was further optimized; gold particles were reduced from a micrometre to forty nanometres (O’Brien and Lummis, 2011), demonstrating that it is possible to use gold-coated DNA nanoparticles of this size to transfect tissue revealing exquisite structural detail. It was possible to observe boutons making synaptic contacts with the pyramidal nerve spines in the hippocampal region of the brain. The findings so far have shown spines from the pyramidal shaft are similar to the spines in the cerebellum, forming regular linear arrays. Recent studies had linked defects in the function of presynaptic boutons to the etiology of several neurodevelopment and neurodegenerative diseases, including autism and Alzheimer’s disease. Our discovery could help to understand why there are abnormalities in dendritic spines which are associated with pathological conditions characterized by cognitive decline, such as mental retardation, Alzheimer’s, stroke and schizophrenia (Yuste and Bonhoeffer, 2001). This thesis provides a synthesis of knowledge about biolistic technology. It is presented as a narrative from improving the gene gun transfection efficiency in brain slices to the development of nano-biolistics. The delivery of DNA and fluorescent dyes into living cells by biolistic delivery should enable a detailed map of the anatomical connections between individual cells and groups of cells to be constructed, providing a “wiring diagram” of connections. The implications of this are discussed in Chapter twelve.

Use-dependent long-term plasticity in synaptic connections represents the cellular substrate for learning and memory. The hippocampus is the most thoroughly investigated brain area for long-term synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD) are both well characterized in glutamatergic excitatory connections between hippocampal principal cells in vitro and in vivo. An increasing number of studies based on acute brain slice preparations report LTP and LTD in excitatory synapses onto postsynaptic hippocampal GABAergic inhibitory interneurons. However, a systematic study of activity-induced long-term plasticity in excitatory synaptic connections to inhibitory GABAergic interneurons in vivo is missing. To determine whether LTP and LTD occur in excitatory synaptic connections to the hippocampal CA1 area GABAergic interneurons types in intact brain, I have used juxtacellular recording to measure synaptically evoked short-delay postsynaptic action potential probability in identified CA1 neurons in the urethane-anaesthetized rats. Plasticity in excitatory synaptic connections to CA1 cell types was measured as a change of afferent pathway stimulation-evoked postsynaptic spike probability and delay. In the study only experiments with monosynaptic-like short-delay (range 3-12 ms) postsynaptic spikes phase-locked to afferent stimulation were used. Afferent fibres were stimulated from the CA1 area of the hippocampus at the contralateral (left) side to avoid simultaneous monosynaptic activation of GABAergic fibres and to exclude antidromic spikes in recorded CA1 cells (in right hemisphere). Plasticity in pathways was tested using theta-burst high-frequency stimulation (TBS, 100 pulses), which is one of the most common synaptic plasticity induction protocols in acute brain slice studies. I discovered that TBS elicited permanent potentiation in single shock-evoked postsynaptic spike probability with shortening or no change in evoked spike latency in various postsynaptic neuron types including three identified pyramidal cells and parvalbumin-expressing (PV+) interneurons. Most fast-spiking PV+ cells showed LTP including an axo-axonic cell and one bistratified cell, whereas two identified basket cells exhibited LTD in similar experimental conditions. In addition, I discovered diverse plasticity in non-fast spiking interneurons, reporting LTP in an ivy cell, and LTD in three incompletely identified regular-spiking CA1 interneurons. I report that the underlying brain state, defined as theta oscillation (3-6 Hz) or non-theta in local field potential, failed to explain whether LTP, LTD or no plasticity was generated in interneurons. The results show that activity-induced potentiation and depression similar to LTP and LTD also occur in excitatory synaptic pathways to various CA1 interneurons types in vivo. I propose that long-term plasticity in excitatory connections to inhibitory interneurons may be take place in learning and memory processes in the hippocampus.

Le cerveau immature est le siège de processus développementaux qui permettent de passer d'une structure primitive à un réseau mature et fonctionnel. L'activité synaptique spontanée générée dans le système nerveux en développement joue un rôle fondamental dans ces processus. Un des principaux moyens par lesquels cette activité peut être traduite en changement phénotypique au niveau neuronal est la sécrétion de neurotrophines. Les neurotrophines sont sécrétées par les neurones et contrôlent toutes les étapes du développement neuronal. Dans l'hippocampe en développement, la neurotrophine principale est le BDNF (brain derived neurotrophic factor). Cette protéine est synthétisée sous forme immature, le proBDNF, dont le rôle est encore méconnu. Durant ma thèse, j'ai montré que le BDNF exerce un contrôle bidirectionnel sur l'efficacité des synapses GABAergiques en développement. La polarité de la plasticité est déterminée par le type d'activité endurée par les neurones et la forme sous laquelle le BDNF est présenté à ces derniers. J'ai ainsi décrit une séquence développementale qui pourrait s'inscrire dans les processus développementaux permettant la maturation du réseau GABAergique dans l'hippocampe de rat. / The immature brain is the place of developmental processes that allow the switch from a primitive structure to a mature and functional network. Spontaneous synaptic activity generated in the developing nervous system plays a fundamental role in these processes. One of the principal ways this activity is translated into phenotypical changes at the neuronal level is the secretion of neurotrophins. Neurotrophins are secreted by neurons and control each step of neuronal development. In the developing hippocampus, the major neurotrophin is BDNF (brain derived neurotrophic factor). This protein is synthetized under an immature form, proBDNF, which role is still poorly known. During my thesis, I showed that BDNF exerts a bidirectional control on the efficacy of developing GABAergic synapses, which polarity is set by the type of activity endured by neurons and the form of BDNF that is presented to them. I described a developmental sequence which could be a part of the developmental processes allowing the maturation of the GABAergic network in the developing rat hippocampus.

Neurotrophe Faktoren haben ein breites Aufgabenfeld und spielen eine wichtige Rolle als Überlebensfaktoren embryonaler Neurone, bei Proliferation und Differenzierung im Nervensystem sowie als Modulatoren synaptischer Plastizität. Im ersten Themenkomplex der vorliegenden Arbeit wurden neurotrophe Faktoren als Modulatoren synaptischer Plastizität und ihr Einfluß auf die BDNF-Regulation im Hippocampus untersucht. Dabei wurde zunächst das selbsthergestellte polyclonale BDNF-Immunserum für die Anwendung in der Immunhistochemie und im Western Blot optimiert, doch es konnten bezüglich BDNF keine Veränderungen in Hippocampi CNTF-defizienter Mäuse gegenüber Wildtyp-Tieren festgestellt werden. Die Ergebnisse der Voruntersuchungen, die im Hippocampus CNTF-defizienter Tiere verminderte BDNF-Level gezeigt hatten, konnten somit nicht verifiziert werden. Im Rahmen dieser Arbeit wurde an CNTF-defizienten Mäusen eine eingeschränkte LTP und LTD nachgewiesen. Zum besseren Verständnis der – laut LTP-Untersuchungen – veränderten Situation an der hippocampalen CA1-Synapse bei CNTF-defizienten Tieren wurden elektronenmikroskopische Bilder dieser Region angefertigt, deren Auswertung keine augenscheinlichen Unterschiede ergab. Im Stratum radiatum der CA1-Region war zudem keine spezifische CNTF-Färbung nachweisbar. Zur Klärung der Frage, ob es IGF-vermittelt nach Training zu hippocampaler BDNF-Hochregulation kommt, wurden Laufradexperimente mit wildtypischen und konditionalen IGF1-Rezeptor-knockout Mäusen durchgeführt und die jeweiligen BDNF-Level untersucht. Dabei wurde BDNF durch Laufradtraining in beiden Genotypen in ähnlichem Maße hochreguliert, was für alternative Wege der BDNF-Hochregulation spricht. Der zweite Themenkomplex befasste sich mit dem Einfluß neurotropher Faktoren auf die Proliferation und Differenzierung in Hippocampus und Cortex. BrdU-Inkorporationsexperimenten zeigten in der Körnerzellschicht des Gyrus dentatus gesteigerte Proliferationsraten bei CNTF-defizienten und CNTF&LIF-defizienten Mäusen, wobei LIF-defiziente Tiere keine veränderten Proliferationsraten zeigten. Untersuchungen an Kulturen cortikaler Vorläuferzellen bestätigten die Hypothese, wonach cortikale Vorläuferzellen zunächst Neurone bilden, die einen Faktor sezernieren, der auf die cortikalen Vorläuferzellen wirkt und sie zur Bildung von Astrozyten veranlasst. Es konnte gezeigt werden, dass CT-1 der Hypothese folgend in vitro und in vivo für die Einleitung der Astrozytogenese im Cortex verantwortlich ist. / Neurotrophic factors are central to many facets of CNS function. They act as survival factors during embryonic development, mediate proliferation, differentiation and survival also in the adult nervous system and play an important role for activity-dependent forms of synaptic plasticity. The first part of this work was addressed to neurotrophic factors as modulators of synaptic plasticity and examined their role for BDNF-regulation within the hippocampal formation. Initially our polyclonal BDNF-immune serum was optimized for the use in immunohistochemistry and Western blot-analysis. No differences concering BDNF-protein in hippocampi of CNTF-deficient mice compared with wildtype were found. Previous data, showing decreased hippocampal BDNF-level in CNTF-deficient mice, could therefore not be verified. Interestingly an impaired LTP and LTD was observed in CNTF-deficient mice.To understand the changed situation at hippocampal CA1-synapse in these mice, leading to an impaired LTP, we used electronmicroscopy, but no apparent differences were seen. In Stratum radiatum of CA1 region no specific CNTF-staining was detectable. To address the question, whether IGF mediates the effect of physical training resulting in BDNF-upregulation within the hippocampus, we performed voluntary running experiments with conditional IGF1-receptor-knockout and with wildtype mice and analysed the BDNF-levels. It was shown that BDNF-upregulation after physical training occurred in both genotypes to a similar extent, suggesting alternative ways of BDNF-upregulation. The second part dealt with the influence of neurotrophic factors on proliferation and differentiation in hippocampus and cortex. Via BrdU-incorporation experiments the different proliferation rates in the subgranular zone of the dentate gyrus were analysed. CNTF-deficient mice and CNTF&LIF-deficient mice showed increased proliferation rates compared with wildtype, whereas LIF-deficient mice had normal proliferation rates. Precursor cells of the embryonic cortex sequentially generate neurons and then glial cells, but the mechanisms regulating this neurogenic-to-gliogenic transition were unclear. Using cortical precursor cultures, which temporally mimic this in vivo differentiation pattern, we demonstrated that cortical neurons synthesize and secrete the neurotrophic cytokine CT-1, which is essential for the timed genesis of astrocytes in vitro. Our data indicate that a similar phenomenon also occurs in vivo.

Die Proliferation von in der Subgranulärzone des Gyrus dentatus ansässigen neuralen Stammzellen ist der erste Schritt der Neuentstehung von Nervenzellen im adulten Organismus, der so genannten adulten Neurogenese, die in bestimmten neurogenen Nischen des ZNS von Säugetieren und des Menschen vorkommt. Die vorliegende Arbeit zeigt, dass das Enzym endotheliale Stickstoffmonoxidsynthase (NOS-III bzw. eNOS) bzw. durch NOS-III gebildetes Stickstoffmonoxid (NO) die Proliferation neuraler Stamm- bzw. Vorläuferzellen im Gyrus dentatus des Hippokampus positiv reguliert, da Mäuse, bei denen das Gen für dieses Enzym deletiert ist, über eine signifikant erniedrigte Stammzellproliferation verfügen. NOS-III-Knockout-Mäuse zeigen außerdem erhöhte Volumina von Substrukturen des Gyrus dentatus. Biometrische Faktoren, wie z. B. Alter, Geschlecht, Körpergewicht, Umgebungsbedingungen, hatten dagegen keinen signifikanten Einfluss auf die adulte Neurogenese. Die Abnahme der adulten Neurogenese bei NOS-III-Knockout-Tieren ist fast vollständig auf die Reduktion der Stammzellproliferation in der Subgranulärzone des Gyrus dentatus zurückzuführen. Ein Netto-Zuwachs an neu gebildeten Neuronen 4 Wochen nach Proliferation kann jedoch durch NOS-III nicht bewirkt werden, was auf eine komplexe Regulation der adulten Neurogenese hinweist. Die Stammzellproliferation im adulten murinen Gyrus dentatus wird jedoch vermutlich unter anderem über im Endothel gebildetes NO (als gasförmiges, parakrines Signalmolekül) vermittelt. / The proliferation of neural stem cells residing in the subgranular layer of the dentate gyrus represents the first step in the formation of new neurons in the adult organism, the so-called adult neurogenesis that occurs in certain neurogenic niches of the mammalian as well as human CNS. The present work shows that endothelial nitric oxide synthase (NOS-III or eNOS) and nitric oxide (NO) formed by NOS-III, respectively, positively regulate the proliferation of neural stem- or precursor cells in the dentate gyrus of the hippocampus since NOS-III knockout mice (NOS III -/-) show significantly reduced stem cell proliferation. Moreover, NOS-III knockout mice exhibit increased volumina of dentate gyrus substructures. In contrast, biometric factors like age, gender, body mass and environmental conditions did not influence adult neurogenesis significantly. Mainly, the decrease of adult neurogenesis in NOS-III knockout animals is attributable to the reduction of stem cell proliferation in the subgranular zone of the dentate gyrus. However, a net increase of new neurons 4 weeks after proliferation cannot be produced by NOS-III pointing to a complex regulation of adult neurogenesis. Yet, stem cell proliferation in the murine dentate gyrus is presumambly mediated inter alia by NO as a gaseous, paracrine molecule produced in the endothelium.

A administração sistêmica da peçonha bruta da cascavel Sul-Americana (Crotalus durissus terrificus, Cdt) induz prejuízos de memória espacial em ratos, sugerindo a ocorrência de dano hipocampal. No presente estudo, foram utilizadas técnicas In vivo e In vitro, a fim de testar os potenciais efeitos centrais do veneno de Cdt e da crotoxina (CTX), principal neurotoxina componente da peçonha bruta da serpente. Foram investigados (1) efeitos comportamentais da administração sistêmica e intrahipocampal do veneno de Cdt e da CTX envolvendo diferentes versões do labirinto aquático de Morris; (2) a morte neuronal, o curso temporal e a ação protetora do soro anticrotálico à exposição de culturas organotípicas de hipocampo a doses crescentes (0,05-1 μg/mL) do veneno bruto de Cdt e de CTX, por meio da coloração fluorescente por Iodeto de propídeo (PI) (que reflete a morte neuronal); e (3) alguns dos possíveis mecanismos fisiopatológicos do envenenamento experimental em culturas de hipocampo utilizando o antagonista de receptores AMPA de glutamato, NBQX, à administração de 0,05 μg/mL do veneno bruto de Cdt, para avaliar a possível participação do glutamato e seus receptores na morte neuronal. A administração sistêmica e intrahipocampal do veneno bruto de cascavel em ratos promoveu marcados prejuízos de memória espacial de referência e operacional. Diferentemente, a CTX promoveu prejuízos comportamentais apenas quando a administração foi intrahipocampal. As culturas expostas ao veneno bruto apresentaram dano sobretudo nas células piramidais de CA1, enquanto as culturas incubadas com CTX não apresentaram danos. Essa seletividade em relação ao CA1 foi mais proeminente nas doses baixas. O soro anticrotálico preveniu a morte neuronal quando administrado até uma hora após a exposição do veneno. O NBQX preveniu parcialmente o dano em CA1 indicando a participação de receptores AMPA de glutamato na cascata de eventos que subjazem os danos a estas células causados pela peçonha bruta de Cdt. Estes achados podem contribuir para a tanto para elucidação dos mecanismos quanto para a conduta utilizada durante o envenenamento crotálico / Systemic administration of the South American rattlesnake (Crotalus durissus terrificus, Cdt) venom in rats permanently disrupts performance in hippocampus-dependent spatial memory tasks, thus indicating the occurrence of damage to this brain area. In vivo and In vitro approaches were employed in the present study to investigate the effects of the venom and its main compound, crotoxin (CTX). We investigated (1) behavioral effects after both systemic and intrahipocampal administration of either venom or CTX in rats on performance in different versions of the Morris\' water maze task; (2) neuronal death, evaluated by the intensity of propidium iodide (PI) fluorescence (neurotoxicity of the venom and of CTX, and the protection by the antivenom, on organotypic hippocampal slice cultures, were evaluated by comparing slices exposed to venom and (1) slices treated only with vehicle - negative control - and (2) slices incubated with 1 mM glutamate - positive control for maximal neuronal death), and the time course of neurotoxic effects of 0.05-1 μg/mL of crude Cdt venom and CTX, and the Brazilian anticrotalid serum on organotypic hippocampal slice cultures; and (3) provide an initial screening of the mechanisms underlying the venom action on cultured hippocampal slices, using the AMPA receptor antagonist NBQX and the crude venom at 0.5 μg/mL dose, to test whether the neuronal damage is mediated by glutamate following the experimental envenomation. Both systemic and intrahippocampal administration of Cdt venom induces permanent disruption of spatial reference memory and spatial working memory in rats. In contrast, CTX only promoted behavioral effects when administered intrahippocampally. Slices exposed to Cdt venom, but not to CTX, exhibited substantial neuronal damage. This effect was particularly prominent in the CA1 sub-field at lower concentrations of the venom. The antivenom prevented damage when applied until 1 hour after the exposure of the venom. The NBQX partially prevented damage to CA1, thus indicating that AMPA receptors play a role in the damage caused by the Cdt venom. These findings may be helpful in the elucidation and management of rattlesnake envenomation

O presente estudo avaliou (1) a memória espacial de ratas Wistar expostas a diferentes formas de experiência maternal [primiparidade (PRIM), sensibilização maternal pela exposição a filhotes adotivos (SENS) e primiparidade com privação de contato com os filhotes por 6 horas diárias (PRIV), em comparação com a nuliparidade (NUL)] em tarefas espaciais de memórias de referência e operacional no labirinto aquático de Morris. Os resultados revelaram ausência de diferenças quando os animais são avaliados após o desmame dos filhotes, enquanto que o período de contato com os mesmos resultou em comportamentos relacionados à ansiedade em ratas SENS. Adicionalmente, ratas PRIV exibiram melhor desempenho no teste de memória operacional, possivelmente devido a um efeito de enriquecimento ambiental pela exposição intermitente e contínua aos filhotes durante o período de lactação. Ainda, comparou- se (2) o desempenho de ratas PRIM e NUL das linhagens Wistar (W) e Sprague-Dawley (SDW) nas mesmas tarefas comportamentais no labirinto aquático. As ratas PRIM-SDW exibiram melhores desempenhos em relação às NUL-SDW e também em relação às PRIM-W no teste de memória de referência, enquanto que no teste de memória operacional, as ratas PRIM-SWD exibiram um desempenho superior em relação às PRIM-W. Adicionalmente, ratas SWD, independentemente da experiência maternal, exibiram estratégias de busca mais eficientes para cumprir a tarefa. Por fim, (3) avaliou-se o desempenho de ratas PRIM e NUL da linhagem SWD expostas a um ambiente enriquecido (AE) em comparação com ratas PRIM e NUL mantidas em gaiolas comuns de laboratório (STD) em tarefas de localização e reconhecimento de objetos, bem como as taxas de neurogênese hipocampal desses animais. Os resultados mostraram que as ratas PRIM-AE foram mais sensíveis à alteração na disposição espacial de um objeto familiar em comparação com as NUL-AE. Adicionalmente, as ratas NUL e STD exibiram mais comportamentos relacionados com ansiedade e estresse. Os resultados anatômicos mostraram que a experiência maternal e a exposição ao AE por 45 dias não geraram alterações na neurogênese hipocampal em ratas SWD. Estes resultados mostram que diferentes formas de experiência maternal exercem alterações distintas sobre o comportamento de ratas, de forma dependente do momento em que os animais são avaliados e da linhagem de ratas utilizadas. Adicionalmente, mostram que o AE gera alterações de memória espacial e produz efeitos ansiolíticos, particularmente em ratas PRIM. / Maternal experience in rats induces changes in brain and behavior. This study compared (1) spatial memory performances of primiparous (PRIM), pup-induced maternal behaviors (SENS), 6h/daily pup-deprived primiparous (DEP) and nuliparous (NUL) Wistar rats in the reference and working memory versions of the Morris water maze task. No differences were found when the animals were tested after pup\'s weaning/exposure. On the other hand, lactation/pup exposure period induced anxiety-like behaviors in SENS rats when tested during this period. In addition, DEP rats showed better performances in the working memory task, which may be an \"environmental enrichment effect\" due the intermittent exposure to the offspring during lactation. We also compared (2) spatial performances of PRIM and NUL rats from 2 different strains, e, Wistar (W) and Sprague-Dawley (SDW) in the same tasks described in the first experiment. The results showed better performances of PRIM-SDW groups relative to both NUL-SWD and PRIM-W in the reference memory task, while PRIM-SWD outperformed PRIM-W rats in the working memory task. Additionally, SWD rats, regardless their reproductive status, showed better performance in relation to their search strategies to find the hidden platform. Lastly, we (3) evaluated performances of PRIM and NUL Sprague-Dawley rats exposed to an enriched environment (EE) compared to NUL and PRIM rats housed in standard laboratory cages (STD) in an object placement and object recognition task. All groups were injected with BrdU in order to assess hippocampal cell proliferation, differentiation/migration, and cell survival in these animals. In the place object task, PRIM-AE rats exhibited better performances compared to NUL-AE rats. In addition, NUL and STD rats showed more anxiety and stress-related behaviors. The anatomical data showed no differences among all groups, indicating that enriched environment in a regimen of 45 days exposure or maternal experience had no influence on the hippocampal neurogenesis in Sprague-Dawley rats. Taken together, these data show that different forms of maternal experience in rats induce different effects on behavior, in a time and strain-dependent manner. Also, the results showed that exposure to an enriched environment induced spatial memory alterations and anxyolitic effects, mainly in PRIM rats.

The hippocampus is a brain structure involved in episodic memory and spatial cognition. Neuronal activity within the hippocampus exhibits intricate temporal patterning, including oscillatory and sequential dynamics, which are believed to underlie these cognitive processes. In individual cells, a temporal activity pattern called phase precession occurs which leads to the organisation of neuronal populations into sequences. These sequences are hypothesised to form a substrate for episodic memory and the representation of spatial trajectories during navigation. In this thesis, I present a novel theory of the phenomenological properties of these neuronal activity sequences. In particular, I propose that the sequential organisation of population activity is governed by the independent phase precession of each cell. By comparison of models in which cells are independent and models in which cells exhibit coordinated activity against experimental data, I provide empirical evidence to support this hypothesis. Further, I show how independent coding affords a vast capacity for the generation of sequential activity patterns across distinct environments, allowing the representation of episodes and spatial experiences across a large number of contexts. This theory is then extended to account for grid cells, whose activity patterns form a hexagonal lattice over external space. By analysing simple forms of phase coding in populations of grid cells, I show how previously undocumented constraints on phase coding in two dimensional environments are imposed by the symmetries of grid cell firing fields. To overcome these constraints, I propose a more complex phenomenological model which can account for phase precession in both place cells and grid cells in two dimensional environments. Using insights from this theory, I then propose a biophysical circuit mechanism for hippocampal sequences. I show that this biophysical circuit model can account for the proposed phenomenological coding properties and provide experimentally testable predictions which can distinguish this model from existing models of phase precession. Finally, I outline a scheme by which this biophysical mechanism can implement supervised learning using spike time dependent plasticity in order to learn associations between events occurring on behavioural timescales. The models presented in this thesis challenge previous theories of hippocampal circuit function and suggest a much higher degree of flexibility and capacity for the generation of sequences than previously believed. This flexibility may underlie our ability to represent spatial experiences and store episodic memories across a seemingly unlimited number of distinct contexts.

The developing brain requires an intricate network of signals to direct proliferation, differentiation and cell fate decisions. Primary cilia are vital organelles with an emerging role regulating several major signalling cascades, in particular the Hedgehog pathway. Talpid3 (Ta3) is 166.7 kD protein found at the distal tip of centrioles. It has been shown to interact with a number of key centriolar proteins and is essential for the formation of primary cilia. A recent mouse model has been designed to conditionally target the highly conserved coiled-coil domain of Ta3 using the Cre/loxP system. This project uncovers the role of Ta3 in the developing brain. It characterises in detail the phenotype of mice with conditional loss of Ta3 in the central nervous system using the Nestin-Cre deleter strain. Morphological and histological analyses demonstrate that significant defects occur postnatally with mice developing severe ataxia and hydrocephaly. Immunohistochemical techniques further characterise the distinct phenotypes of three key brain regions including the cerebellum, cortex and hippocampus. Ta3fl/fl;NesCre mutant mice exhibit defects in the proliferation, organisation, morphology and migration of both neuronal and glial cells. We have shown the mechanistic cause to be the result of widespread loss of primary cilia and a concomitant disruption in the transduction of the Hedgehog signalling pathway. The neural roles of Ta3 are explored further through the optimisation of an in vitro neurosphere system to culture postnatal hippocampal progenitors. The use of a tamoxifen inducible strain allows the timely recombination of Ta3 to study its role in a controlled environment. The cultured cells recapitulate many of the in vivo defects showing loss of primary cilia and reduced migration. Finally, characterisation of the phenotypes seen in the Ta3fl/fl;NesCre mice were shown to resemble neurological traits seen in human conditions with loss of Primary cilia, known as ‘human ciliopathies’. Through clinical collaboration this project demonstrated a human ciliopathy case of Joubert Syndrome with compound heterozygous mutations in TA3. This presents the Ta3fl/fl;NesCre mutant mice as a valuable model system to study a rare but clinically relevant condition.