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.

No description available.

<p>Intricate neural circuits underlie all brain functions. However, these neural circuits are highly dynamic. The ability to change, or the plasticity, of the brain has long been demonstrated at the level of isolated single synapses under artificial conditions. Circuit organization and brain function has been extensively studied by correlating neuronal activity with information input. The primary visual cortex has become an important model brain region for the study of sensory processing, in large part due to the ease of manipulating visual stimuli. Much has been learned from studies of visual cortex focused on understanding the signal-processing of visual inputs within neural circuits. Many of these findings are generalizable to other sensory systems and other regions of cortex. However, few studies have directly demonstrated the orchestrated neural-circuit plasticity occurring during behavioral experience. </p> <p>It is vital to measure the precise circuit connectivity and to quantitatively characterize experience-dependent circuit plasticity to understand the processes of learning and memory formation. Moreover, it is important to study how circuit connectivity and plasticity in neurological and psychiatric disease states deviates from that in healthy brains. By understanding the impact of disease on circuit plasticity, it may be possible to develop therapeutic interventions to alleviate significant neurological and psychiatric morbidity. In the case of neural trauma or ischemic injury, where neurons and their connections are lost, functional recovery relies on neural-circuit repair. Evaluating whether neurons are reconnected into the local circuitry to re-establish the lost connectivity is crucial for guiding therapeutic development.</p> <p>There are several major technical hurdles for studies aiming to quantify circuit connectivity. First, the lack of high-specificity circuit stimulation methods and second, the low throughput of the gold-standard patch-clamp technique for measuring synaptic events have limited progress in this area. To address these problems, we first engineered the patch-clamp experimental system to automate the patching process, increasing the throughput and consistency of patch-clamp electrophysiology while retaining compatibility of the system for experiments in <i>ex vivo </i>brain slices. We also took advantage of optogenetics, the technology that enables control of neural activity with light through ectopic expression of genetically encoded photo-sensitive channels in targeted neuronal populations. Combining optogenetic stimulation of pre-synaptic axonal terminals and whole-cell patch-clamp recording of post-synaptic currents, we mapped the distribution and strength of synaptic connections from a specific group of neurons onto a single cell. With the improved patch-clamp efficiency using our automated system, we efficiently mapped a significant number of neurons in different experimental conditions/treatments. This approach yielded large datasets, with sufficient power to make meaningful comparisons between groups.</p> <p>Using this method, we first studied visual experience-dependent circuit plasticity in the primary visual cortex. We measured the connectivity of local feedback and recurrent neural projections in a Fragile X syndrome mouse model and their healthy counterparts, with or without a specific visual experience. We found that repeated visual experience led to increased excitatory drive onto inhibitory interneurons and intrinsically bursting neurons in healthy animals. Potentiation at these synapses was absent or abnormal in Fragile X animals. Furthermore, recurrent excitatory input onto regular spiking neurons within the same layer remained stable in healthy animals but was depressed in Fragile X animals following repeated visual experience. These results support the hypothesis that visual experience leads to selective circuit plasticity which may underlie the mechanism of visual learning. This circuit plasticity process is impaired in a mouse model of Fragile X syndrome. </p> <p>In a separate study, in collaboration with the laboratory of Dr. Gong Chen, we applied the circuit-mapping method to measure the effect of a novel brain-repair therapy on functional circuit recovery following ischemic injury, which locally kills neurons and creates a glial scar. By directly reprogramming astrocytes into neurons within the region of the glial scar, this gene-therapy technology aims to restore the local circuit and thereby dramatically improve behavioral function after devastating neurological injury. We found that direct reprogramming converted astrocytes into neurons, and importantly, we found that these newly reprogrammed neurons integrated appropriately into the local circuit. The reprogramming also improved connections between surviving endogenous neurons at the injury site toward normal healthy levels of connectivity. Connections formed onto the newly reprogrammed neurons spontaneously remodeled, the process of which resembled neural development. By directly demonstrating functional connectivity of newly reprogrammed neurons, our results suggest that this direct reprogramming gene-therapy technology holds significant promise for future clinical application to restore circuit connectivity and neurological function following brain injury.</p>

Epilepsy is characterized by the hyperexcitability of individual neurons and hyper synchronization of groups of neurons (networks). The acquired changes that take place at molecular, cellular and network levels are important for the induction and maintenance of epileptic activity in the brain. Epileptic activity is known to alter the intrinsic properties and signaling of neurons. Understanding acquired changes that cause epilepsy may lead to innovative strategies to prevent or cure this neurological disorder. Advances in in vitro electrophysiological techniques together with experimental models of epilepsy are indispensible tools to understand molecular, cellular and network mechanisms that underlie epileptiform activity. The aim of the study was to investigate the epileptiform activity induced alterations in Ca2+ dynamics in apical dendrites of hippocampal subicular pyramidal neurons in slices and changes in network properties of cultured hippocampal neurons. We have also made attempts to develop an in vitro model of epilepsy using organotypic hippocampal slice cultures. In the first part of the present study, investigations on the basic properties of dendritic Ca2+ signaling in subicular pyramidal neurons during epileptiform activity are described. Subiculum, a part of the hippocampal formation is present, adjacent to the CA1 subfield. It acts as a transition zone between the hippocampus and entorhinal cortex. It receives inputs directly from the CA1 region, the entorhinal cortex, subcortical and other cortical areas. Several forms of evidences support the role of subiculum in temporal lobe epilepsy. Pronounced neuronal loss has been reported in various regions of the hippocampal formation (CA1 and CA3) leaving the subiculum generally intact in human epileptic tissue. It has been observed that epileptic activity is generated in subiculum in cases where the CA3 and CA1 regions are damaged or even absent. However, it is not clear how subicular neurons protect themselves from epileptic activity induced neuronal death. It is widely accepted that epileptiform activity induced neuronal damage is a result of an abnormally large influx of Ca2+ into neuronal compartments. In the present study, combined hippocampus / entorhinal cortical brain slices were exposed to zero Mg2+ + 4-amino pyridine artificial cerebrospinal fluid (ACSF) to generate spontaneous epileptiform discharges. Whole cell current-clamp recordings combined with Ca2+ imaging experiments (by incorporating Oregon green BAPTA-1 in the recording pipette) were performed on subicular pyramidal neurons to understand the changes in [Ca2+]i transients elicited in apical dendrites, in response to spontaneous epileptic discharges. To understand the changes occurring with respect to control, experiments were performed (in both control and in vitro epileptic conditions) where [Ca2+]i transients in dendrites were elicited by back propagating action potentials following somatic current injections. The results show clear distance-dependent changes in decay kinetics of [Ca2+]i transients (τdecay), without change in the amplitude of the [Ca2+]i transients, in distal parts (95–110 µm) compared to proximal segments (30–45 µm) of apical dendrites of subicular pyramidal neurons under in vitro epileptic condition, but not in control conditions. Pharmacological agents that block Ca2+ transporters viz. Na+/Ca2+ exchangers (Benzamil), plasma membrane Ca2+-ATPase pumps (Calmidazolium) and smooth endoplasmic reticulum Ca2+-ATPase pumps (Thapsigargin) were applied locally to the proximal and distal part of the apical dendrites in both experimental conditions to understand the molecular aspects of the Ca2+ extrusion mechanisms. The relative contribution of Na+/Ca2+ exchangers in Ca2+ extrusion was higher in the distal apical dendrite in in vitro epileptic condition. Using computer simulations with NEURON, biophysically realistic models were built to understand how faster decay of [Ca2+]i transients in the distal part of apical dendrite associated with [Ca2+]i extrusion mechanisms affect excitability of the neurons. With a linear increase in the density of Na+/Ca2+ exchangers along the apical dendrite, the decrease in τ decay values of [Ca2+]i transients in distal regions seen in experimental epileptic condition was reproduced in simulation. This linear increase in Na+/Ca2+ exchangers lowered the threshold for firing in response to consecutive synaptic inputs to the distal apical dendrite. Our results thus, show the existence of a novel neuroprotective mechanism in distal parts of the apical dendrite of subicular pyramidal neurons under in vitro epileptic condition with the Na+/Ca2+ exchangers being the major contributors to this mechanism. Although the enhanced contribution of Na+/Ca2+ exchangers helps the neuron in removing excess [Ca2+]i loads, it paradoxically makes the neuron hyperexcitable to synaptic inputs in the distal parts of the apical dendrites. Thus, the Na+/Ca2+ exchangers may actually protect subicular pyramidal neurons and at the same time contribute to the maintenance of epileptiform activity. In the second part of the study, neuronal network topologies and connectivity patterns were explored in control and glutamate injury induced epileptogenic hippocampal neuronal networks, cultured on planar multielectrode array (8×8) probes. Hyper synchronization of neuronal networks is the hallmark of epilepsy. To understand hyper synchronization and connectivity patterns of neuronal networks, electrical activity from multiple neurons were monitored simultaneously. The electrical activity recorded from a single electrode mainly consisted of randomly fired single spikes and bursts of spikes. Simultaneous measurement of electrical activity from all the 64 electrodes revealed network bursts. A network burst represents the period (lasting for 0.1–0.2 s) of synchronized activity in the network and, during this transient period, maximum numbers of neurons interact with each other. The network bursts were observed in both control and in vitro epileptic networks, but the frequency of network bursts was more in the latter, compared to former condition. Time stamps of individual spikes (from all 64 electrodes) during such time-aligned network burst were collected and stored in a matrix and used to construct the network topology. Connectivity maps were obtained by analyzing the spike trains using cross-covariance analysis and graph theory methods. Analysis of degree distribution, which is a measure of direct connections between electrodes in a neuronal network, showed exponential and Gaussian distributions in control and in vitro epileptic networks, respectively. Quantification of number of direct connections per electrode revealed that the in vitro epileptic networks showed much higher number of direct connections per electrode compared to control networks. Our results suggest that functional two-dimensional neuronal networks in vitro are not scale-free (not a power law degree distribution). After brief exposure to glutamate, normal hippocampal neuronal networks became hyperexcitable and fired a larger number of network bursts with altered network topology. Quantification of clustering coefficient and path length in these two types of networks revealed that the small-world network property was lost once the networks become epileptic and this was accompanied by a change from an exponential to a Gaussian network. In the last part of the study, we have explored if an excitotoxic glutamate injury (20 µM for 10 min) that produces spontaneous, recurrent, epileptiform discharges in cultured hippocampal neurons can induce epileptogenesis in hippocampal neurons of organotypic brain slice cultures. In vitro models of epilepsy are necessary to understand the mechanisms underlying seizures, the changes in brain structure and function that underlie epilepsy and are the best methods for developing new antiseizure and antiepileptogenic strategies. Glutamate receptor over-activation has been strongly associated with epileptogenesis. Recent studies have shown that brief exposure of dissociated hippocampal neurons in culture to glutamate (20 µM for 10 min) induces epileptogenesis in surviving neurons. Our aim was to extend the in vitro model of glutamate injury induced epilepsy to the slice preparations with intact brain circuits. Patch clamp technique in current-clamp mode was employed to monitor the expression of spontaneous epileptiform discharges from CA1 and CA3 neurons using several combinations of glutamate injury protocols. The results presented here represent preliminary efforts to standardize the glutamate injury protocol for inducing epileptogenesis in organotypic slice preparations. Our results indicate that glutamate injury protocols that induced epileptogenesis in dissociated hippocampal neurons in culture failed to turn CA1 and CA3 neurons of organotypic brain slice cultures epileptic. We also found that the CA1 and CA3 neurons of organotypic brain slice cultures are resilient to induction of epileptogenesis by glutamate injury protocols with 10 times higher concentrations of glutamate (200µM) than that used for neuronal cultures and long exposure periods (upto 30 min). These results clearly show that the factors involved in induction of epileptiform activity after glutamate injury in neuronal cultures and those involved in making the neurons in organotypic slices resilient to such insults are different, and understanding them could give vital clues about epileptogenesis and its control. The resilience of CA1 and CA3 neurons seen could be due to differences in homeostatic plasticity that operate in both these experimental systems. However, further studies are required to corroborate this hypothesis.

Development of a three dimensional (3D) HYBROT model with targeted in vivo like intact cellular circuitry in thick brain slices for multi-site stimulation and recording will provide a useful in vitro model to study neuronal dynamics at network level. In order to make this in vitro model feasible, we need to develop several associated technologies. These technologies include development of a thick organotypic brain slice culturing method, a three dimensional (3D) micro-fluidic multielectrode Neural Interface system (µNIS) and the associated electronic interfaces for stimulation and recording of/from tissue, development of targeted stimulation patterns for closed-loop interaction with a robotic body, and a deep-tissue non-invasive imaging system. To make progress towards this goal, I undertook two projects: (i) to develop a method to culture thick organotypic brain slices, and (ii) construct a multiphoton imaging system that allows long-term and deep-tissue imaging of two dimensional and three dimensional cultures. Organotypic brain slices preserve cytoarchitecture of the brain. Therefore, they make more a realistic reduced model for various network level investigations. However, current culturing methods are not successful for culturing thick brain slices due to limited supply of nutrients and oxygen to inner layers of the culture. We developed a forced-convection based perfusion method to culture viable 700µm thick brain slices. Multiphoton microscopy is ideal for imaging living 2D or 3D cultures at submicron resolution. We successfully fabricated a custom-designed high efficiency multiphoton microscope that has the desired flexibility to perform experiments using multiple technologies simultaneously. This microscope was used successfully for 3D and time-lapse imaging. Together these projects have contributed towards the progress of development of a 3D HYBROT. ----- 3D Hybrot: A hybrid system of a brain slice culture embodied with a robotic body.

Les récepteurs de type Toll (TLR) sont des récepteurs cellulaires jouant un rôle pivot dans le déclenchement de la réponse immunitaire après une infection ou une blessure, c'est-à-dire une inflammation. L'activation de la signalisation TLR a été associée à l’épilepsie. Dans ce projet, j'utilisai trois modèles distincts pour étudier comment le déclenchement des TLR contribue à l'épileptogenèse. Il existe une corrélation entre les malformations corticales développementales telle la dysplasie corticale focale (FCD) et convulsions fébriles dans les enfants de bas âge. Récemment, une réponse neuro-inflammatoire fut identifiée dans les lésions FCD. Nous postulâmes que l'inflammation induite par le FCD peut augmenter la sensibilité aux crises (chapitre 2). Nous modélisâmes FCD en induisant une congélation-lésion corticale chez le rat néonatal. La lésion corticale déclencha des effecteurs en aval de TLR4, spécifiquement le précurseur de la cytokine Caspase-1, dans l'hippocampe ipsilatéral à la lésion. Les rats lésés développèrent des crises fébriles expérimentales nettement plus rapidement que les rats témoins. Le blocage de l'activité de la Caspase-1 prolongea significativement la latence des crises chez les rats lésés. Nos résultats impliquent l'inflammation médiée par la Caspase-1 en tant que déclencheur des crises fébriles chez les enfants avec FCD préexistante. Des études antérieures déterminèrent que l'activation systémique de la cascade TLR4 abaisse le seuil de crise. Nous étudiâmes si la pénétration des cellules immunitaires périphériques dans le cerveau pendant la stimulation TLR4 favorise l'activité ictal en stimulant la voie TLR4 dans les leucocytes prélevés sur la rate de rat (splénocytes). Ensuite, nous co-cultivâmes des splénocytes avec des coupes organotypiques dérivées du cerveau in vitro (chapitre 3). L'ajout de splénocytes stimulés par TLR4 donna lieu à une neuro-inflammation et à une excitation neuronale accrue. L’ajout de splénocytes non-stimulés n’eut aucun effet pro-inflammatoire ou pro-excitateur dans les coupes organotypiques. De plus, l'inhibition de la Caspase-1 dans des coupes organotypiques co-cultivées avec des splénocytes stimulés diminua la neuro-inflammation et l'hyperexcitabilité neuronale. Nos résultats suggèrent que l'infiltration de leucocytes activés par TLR4 dans le cerveau augmente la prédisposition aux crises via les mécanismes médiés par la Caspase-1. Précédemment, des rapports montrèrent que l'activation de la signalisation TLR3 facilite l'évolution des crises. L'introduction d'un agoniste synthétique TLR3 chez la souris in vivo et des coupes organotypiques hippocampiques in vitro produisirent des mécanismes anti-inflammatoires dépendants de la dose et du temps (chapitre 4). La stimulation TLR3 supprimait les crises d'hippocampe in vivo et réduisait l'excitabilité synaptique dans le réseau hippocampique à la fois in vivo et in vitro. Nous avons déterminé que les effets anticonvulsivants médiés par TLR3 étaient principalement provoqués par les cascades en aval IRF3 / IFN-β. Ainsi, nos données suggèrent que l'activation de TLR3 peut protéger le cerveau contre les crises par la production d'IFN-β. Nos résultats donnent un aperçu des nouveaux mécanismes cellulaires sous-jacents à la modulation inflammatoire de l'excitabilité neurale. Notre découverte des rôles de la Caspase-1 et de l'IFN-β dans l'influence du seuil de crise améliorera notre compréhension des fondements moléculaires de la génération de crises ce qui pourraient améliorer le traitement de l'épilepsie. / Toll-like receptors (TLRs) are cellular receptors that play a pivotal role in initiating immune response following infection or injury, i.e. inflammation. Nevertheless, activation of TLR signaling has been associated with seizure manifestation. In this research, I employed three distinct models to study how triggering TLRs contributes to ictogenesis. There is a correlation between developmental cortical malformations, e.g. focal cortical dysplasia (FCD), and fever-provoked, i.e. febrile, seizures in young children. Recently, neuroinflammation was reported in FCD lesions. Therefore, we posited that FCD-induced inflammation may increase seizure susceptibility (Chapter 2). To recapitulate FCD pathology, we induced a cortical freeze-lesion in neonatal rats. Lesioning the cortex triggered TLR4 downstream effectors, specifically the cytokine precursor Caspase-1, in the hippocampus ipsilateral to the lesion. Further, lesioned rats developed experimental febrile seizures markedly faster than sham control rats. Strikingly, blocking Caspase-1 activity prior to seizure induction significantly prolonged seizure latency in lesioned rats. Our results implicate Caspase-1-mediated inflammation as a main driver of febrile seizures in children with pre-existing brain malformations. In addition, previous reports determined that systemic activation of TLR4 cascade lowers seizure threshold. Hence, we developed an in vitro model to investigate whether penetration of peripheral immune cells into the brain during TLR4 stimulation promotes ictogenic activity (Chapter 3). First, we stimulated TLR4 pathway in leukocytes harvested from rat spleen, i.e. splenocytes. Thereafter, we co-cultured splenocytes with brain-derived organotypic slices in vitro. Adding TLR4-stimulated splenocytes gave rise to neuroinflammation and enhanced neuronal excitation, whereas adding unstimulated splenocytes failed to evoke pro-inflammatory or proexcitatory effects in organotypic slices. Moreover, Caspase-1 inhibition in organotypic slices cocultured with stimulated splenocytes diminished neuroinflammation and neuronal hyperexcitability. Our findings suggest that infiltration of TLR4-activated leukocytes into the brain elevate seizure predisposition via Caspase-1-mediated mechanisms. Beside TLR4 pathway, it was previously shown that activation of TLR3 signaling facilitates seizure evolution. In chapter 4, introducing a synthetic TLR3 agonist to mice in vivo and to hippocampal organotypic slices in vitro yielded anti-inflammatory mechanisms in a dose- and time-dependent manner. Also, we found that TLR3 stimulation suppressed hippocampal seizures in vivo and reduced synaptic excitability in the hippocampal network both in vivo and in vitro. Finally, we determined that TLR3-mediated anticonvulsive effects were chiefly driven by IRF3/IFN-β downstream cascades. Thus, our data suggests that TLR3 activation may protect the brain from seizures through production of IFN-β. Altogether, our findings provide insight into novel cellular mechanisms underlying inflammatory modulation of neural excitability. Furthermore, our discovery of the roles of Caspase-1 and IFN-β in influencing seizure threshold will improve our understanding of the molecular underpinnings of seizure generation, which may ultimately have therapeutic benefits for epilepsy treatment.

Aufgabe der elektrophysiologischen Studie zur Charakterisierung der neuronalen hypothalamischen Plastizität beim Haushuhn war es, den Einfluss des Alters sowie GABAerger Substanzen auf die Feuerrate und die Temperatursensitivität (thermischer Koeffizient: TC) von Hypothalamusneuronen mittels extrazellulärer Ableitungen in Hirnschnitten zu untersuchen. Im Vergleich zu adulten Vögeln und Säugetieren wurde bei juvenilen Hühnern eine hohe neuronale Kältesensitivität nachgewiesen, die offensichtlich eine spezifische Eigenschaft juveniler Vögel ist. Die Ontogenese der neuronalen hypothalamischen Thermosensitivität ist deutlich artspezifisch. Einige Neurone wiesen eine inherente Kältesensitivität auf. Eine mögliche zentrale Rolle kältesensitiver Neurone im Rahmen der Thermoregulation juveniler Hühner wurde postuliert. Muscimol und Baclofen hemmen signifikant die Feuerrate der Hypothalamusneurone, unabhängig von der jeweiligen Thermosensitivität. Demgegenüber bewirken Bicucullin und CGP35348 einem Anstieg der Feuerrate. Nur bei kältesensitiven Neuronen wurde der TC signifikant durch GABAB-Rezeptor-Liganden verändert (signifikant erhöht durch Baclofen und durch CGP35348 gehemmt). Der Effekt von Muscimol und Baclofen auf Feuerrate und TC wurde durch Co-Perfusion mit einer 10-fach höheren Konzentration der entsprechenden Antagonisten Bicucullin und CGP35348 aufgehoben. Der wesentliche GABAerge Einfluss auf thermosensitive und –insensitive Hypothalamusneurone ist mit dem bei Säugetieren nachgewiesenen vergleichbar. Der einzige Unterschied betrifft die GABAB-Rezeptor vermittelte Änderung des TC. Beim Hühnerküken betraf dies die kältesensitiven und beim Säugetier die wärmesensitiven Neurone. Der grundlegende Mechanismus der GABAergen Beeinflussung thermosensitiver und –insensitiver Neurone scheint einen älteren evolutionären Ursprung zu haben. Eine funktionelle Rolle GABAerger Substanzen im Rahmen der zentralen Kontrolle der Körpertemperatur beim Vogel ist möglich. / In the present electrophysiological studies, characterization of neuronal hypothalamic plasticity in the chicken aims to investigate the influence of age during development by extracellular recordings. High neuronal cold sensitivity has been found in juvenile chicken in contrast to adult mammals and birds. High hypothalamic cold sensitivity seems to be a specific characteristic feature in juvenile birds. Between species a species specificity of the early development of neuronal hypothalamic thermosensitivity could be clearly demonstrated. Existence of inherent nature to a certain degree suggests a possible thermoregulatory role of cold-sensitive neurons in chicken. The effects of the GABAergic substances on neuronal tonic activity (firing rate) and temperature sensitivity (temperature coefficient) in hypothalamic neurons have been examined. Muscimol and baclofen in equimolar concentrations significantly inhibited tonic activity, regardless of their type of thermosensitivity. In contrast bicuculline and CGP 35348 increased firing rate. Temperature coefficient was significantly changed by ligands of GABAB receptors, restricted to cold-sensitive neurons. The TC was significantly increased by baclofen and significantly decreased by CGP 35348. Effects of muscimol and baclofen on firing rate and TC were prevented by co-perfusion of appropriate antagonists bicuculline and CGP 35348, respectively in tenfold higher concentration. Thus the main effects of GABA in chicken are similar with that described in mammals. The only difference is in respect of the GABAB receptors mediated change restricted to cold-sensitive neurons in chicken but in mammals only seen in warm-sensitive neurons. However, the results indicate that the fundamental mechanism of GABAergic influence in chicken are conserved during evolution. The response of hypothalamic neurons to temperature changes suggest a possible functional role of GABAergic substances in the control of body temperature in birds.