Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

A large percentage of individuals with intractable epilepsies have an accompanying cortical malformation, the underlying cellular mechanisms of which are poorly understood. It is known however that in an animal model for one such malformation, polymicrogyria, epileptogenesis occurs most easily from an adjacent area termed the paramicrogyral region (PMR). Previous studies implicate SS interneurons as a potential contributor to this pathology, which lead to our hypothesis: in PMR, SS interneurons exert a higher modulatory influence on excitatory pyramidal cells, as compared to the same by SS interneurons within homologous control cortex. Using a freeze-lesion model for polymicrogyria in transgenic mice that selectively express either Channelrhodopsin or Archaerhodopsin optogenetic channels in these cells, we assessed the contribution of SS interneurons as it potentially differs between layer V of PMR and control cortex. These studies provided the following biological examples in support of previous extrapolations that indicate SS over-activation within PMR: (1) SS interneuron mediated evocation of inhibitory events in layer V excitatory neurons is more robust in PMR than in control. Similarly, electrically-evoked inhibitory events in these excitatory neurons trend towards being larger, signifying a larger contribution by interneurons. (2) SS interneuron mediated suppression of electrically-evoked responses trends towards being stronger in PMR; and (3) the selective silencing of SS interneurons might not impart an effect on spontaneous inhibitory postsynaptic events.

Stroke is the most common cause of acquired epilepsy in persons 35 and older. The massive increase in extracellular glutamate during stroke causes a cascade of intracellular events that can lead to cell death or the molecular changes that initiate the development of epilepsy. In addition, many studies point to a modulatory role of the endocannabinoid system in controlling seizures. Animal models of stroke induced acquired epilepsy have been difficult to develop. Therefore, this dissertation was initiated to develop an organotypic hippocampal slice culture model of acquired epilepsy and examine the changes in distribution and function of the endogenous CB1 receptor system. We utilized 4-aminopyridine and glutamate to induce separate excitotoxic injuries to slice cultures. Both injuries produced significant cell death acutely following the injury. After a latency period, we observed a significant increase in the number of slice cultures that displayed electrographic seizures in both injury models. Western blot analysis demonstrated that the cannabinoid CB1 receptor protein was significantly upregulated following injury with glutamate. Immnohistochemical studies demonstrated that this receptor upregulation was likely specific to the glutamatergic terminals. Electrophysiological experiments were performed to study endocannabinoid modulation of inhibitory and excitatory signaling in the CA3 pyramidal cells. We demonstrated that depolarization induced suppression of excitation (DSE) was enhanced in slice cultures that had undergone glutamate injury. This indicated that the upregulation of CB1 receptors following glutamate injury was physiologically functional, as it enhanced cannabinoid control of the excitatory signaling. These studies support the hypothesis that there is a functional alteration of CB1 receptors in the epileptic state that acts to suppress seizures. The development of an organotypic hippocampal slice culture model of stroke acquired epilepsy provides a unique tool to study the neuronal plasticity changes associated with epileptogenesis. It also provides a practical model to study pharmacological agents that may be useful in preventing or treating epilepsy.

Epilepsy, defined by recurrent seizures, is the one of the most prevalent neurological disorders worldwide (World Health Organization 2007). While many forms of epilepsy are well-controlled by anti-epileptogenic medications, a significant portion of patients have intractable, i.e. untreatable, seizures. The etiology of these seizures is varied, but a significant cause, particularly for patients with intractable epilepsy is developmental malformation. In these cases, an error or interruption during the development of the neocortex produces a structural alteration. Such patients may have other neurological problems, but seizures are the most common symptom. The neuronal mechanisms that link malformation and cortical hyperexcitability are not well understood. Here we have sought to examine potential mechanisms that result from microgyria, a malformation characterized by excessive numbers of small gyri.The presence of epileptiform activity indicates that the normal balance of excitation and inhibition has shifted . Two functions of inhibition within neocortex are to prevent spread of excitation, and to modulate the timing of surrounding excitation. Although seemingly contradictory, increasing some forms of inhibition can result in an increase in synchronous excitatory activity. We hypothesize that for certain malformation epilepsies, the inhibitory processes that control timing are increased, creating a hyper-synchronous cortex, while the inhibitory processes that control horizontal spread are decreased, allowing the propagation of such activity. Here we have examined the network effect of selectively modulating the inhibitory cells that control vertical or columnar cortical synchrony. This modulation is performed via activation of metabotropic glutamate receptors found on the vertically-projecting interneurons but not on those inhibitory cells that control horizontal spread of activity. Our results suggest that the network effect of activating these interneurons is altered in malformed, epileptogenic cortex.

Cortical malformations from altered development are common causes of human epilepsy. The cellular mechanisms responsible for the epileptic state of cortex remain unclear and a significant portion of these cases do not respond to treatment. Previous electrophysiological recordings in the Jacobs lab in a rat polymicrogyria model indicated an increased response to group I metabotropic glutamate receptor agonists in the region adjacent to the malformation (PMZ). In addition there was a novel response in low threshold spiking (LTS) interneurons via mGluR5 activation. To determine whether cell specific expression of these receptors was altered in malformed cortex immunohistochemical stains were performed for group I mGluRs along with non-overlapping interneuron subtype specific markers, a neuronal marker and general inhibitory cell marker. There was no altered mGluR5 expression seen in the PMZ. There was an altered expression seen in PMZ mGluR1α labeled cells and cells in other cortical regions.

This study was conducted to characterize cellular changes occurring during the progression of status epilepticus (SE) that could lead to the maintenance of increased membrane excitability. SE was induced by injection of pilocarpine after which rats were monitored both electrographically and behaviorally. After various lengths of time in SE, specific brain regions were isolated for biochemical study. SE resulted in an early maintenance of PKA activity in both cortical homogenate and crude synaptoplasmic membrane (crude SPM) fractions. At subsequent stages of SE there was a significant increase in PKA activity in both homogenate and crude SPM fractions. Wester blot analysis showed that alteration of PKA protein expression was not responsible for the increase in PKA activity. These results show that SE has a significant duration-dependent effect on PKA activity. Combined with other cellular changes these findings, could represent a mechanism for the formation for potentiated seizure states like epilepsy.

Epilepsy has been previously attributed to either increased excitation or decreased inhibition. With this closed frame of mind, modern medicine has been unable to develop a permanent treatment against the mechanisms of epilepsy. In order to treat patients with intractable seizures, especially those caused by developmental malformations, it is essential to understand the entirety of mechanisms that could possibly play a role in the abnormal cortical function. One such developmental malformation is known as polymicrogyria. Epileptogenesis occurs in an area laterally adjacent to this malformation known as the paramicrogyral region (PMR). Past studies have narrowed down the potential cause of this increased network excitation to a certain type of inhibitory interneuron, the somatostatin (SS) interneuron. Additionally, previous studies have shown an increase in the mGlu5 receptor on this interneurons in the PMR region only and not in control tissue, meaning that targeting these receptors as treatment will not affect normal functioning tissue. These results lead to our hypothesis: blockade of the mGluRs will decrease the 2 activity of SS interneurons and thereby prevent the generation of epileptiform activity and increased SS output in malformed cortex. Utilizing the freeze-lesion model for microgyria in transgenic mice expressing Channelrhodopsin optogenetic channels in SS interneurons, we assessed the contribution of these SS interneurons in four different animal groups: control or PMR treated with either Gabapentin, a current AED (antiepileptic drug), or MTEP, an mGlu5 receptor antagonist. We tested the effects of these two drugs on SS interneuron output to determine whether they decrease the over activation in the PMR that has been previously studied. The following study revealed no correlation between Gabapentin-treated animals and a decrease in epileptiform activity. Additionally, no significant difference was seen between the MTEP-treated groups in the protocols that were measured.

Fast postsynaptic inhibition in the brain is mediated by ionotropic GABA_A receptors (GABA_ARs), which are activated by the release of the neurotransmitter GABA from presynaptic interneurons. The GABA_AR is primarily permeable to chloride ions (Cl-) and therefore the transmembrane gradient for Cl- sets the reversal potential of the receptor (E_GABA-A). When intracellular Cl^- concentrations are relatively low, E_GABA-A is more negative than the membrane potential and GABA_AR responses will have a hyperpolarising and inhibitory effect upon the postsynaptic cell. In contrast, when intracellular Cl^- concentrations are relatively high, E_GABA-A will be more positive and GABA_AR activation will have a depolarising effect. How a neuron controls its intracellular Cl^- concentrations is a fundamental question that has direct relevance to hyperexcitability conditions such as epilepsy. Recently, it has become clear that Cl^- homeostasis is altered in epileptic tissue such that postsynaptic inhibition via the GABA_AR is reduced and, under some conditions, GABA_AR signalling may even be excitatory. In my thesis I explore some of the mechanisms and factors that are responsible for regulating postsynaptic GABA_AR signalling in the context of epileptic seizure activity in the rat hippocampus. In the first series of experiments I combined pharmacological approaches with electrophysiological recordings from pyramidal neurons in the CA3 region of the hippocampus to trigger seizure activity. My results show that intense neuronal activity during a seizure leads to a transient accumulation of intracellular Cl^-, which generates a pronounced depolarising shift in E_GABA-A. Under these conditions, GABAergic synapses become excitatory and contribute to ongoing neuronal activity rather than exerting their normal inhibitory role. I found that the same seizure activity also induces the release of a neuromodulator called adenosine, which serves to limit the deleterious effects of excitatory GABA_AR responses. Adenosine exerts these effects by activating downstream potassium channels, which increase the postsynaptic cell’s membrane conductance and, in doing so, ‘shunt’ incoming GABA_AR responses. In the second series of experiments I examined Cl^- homeostasis and E_GABA-A in the context of neonatal seizures. One of the main mechanisms by which neurons maintain their intracellular Cl^- levels is through the activity of ion transporter proteins that reside in the membrane and move Cl^- either into, or out of, the cell. I discovered that the intracellular trafficking of an important Cl^- transporter protein, NKCC1, correlates with changes in Cl^- homeostasis. Using a combination of biochemical and molecular techniques, I then identified a novel molecular association between NKCC1 and a motor protein, Myosin Va, which has been implicated in the intracellular trafficking of membrane proteins. Using electrophysiological recordings I found that Myosin Va is required for NKCC1’s contribution to Cl^- homeostasis, which may be important for E_GABA-A changes in epilepsy. In the final series of experiments I developed methods to study the temporal dynamics in E_GABA-A during a single seizure. These revealed a Cl^- unloading mechanism that emerges at the end of a seizure and which depends upon hyperpolarisation of the postsynaptic membrane potential. This mechanism aids E_GABA-A recovery after the seizure and moves E_GABA-A to more hyperpolarised values. This mechanism could boost postsynaptic inhibition after a seizure and thereby help to protect against further seizure episodes. In conclusion, this work extends our understanding of postsynaptic GABAergic transmission in the context of epileptic seizure activity and suggests new mechanisms that could be relevant for the development of rational anti-epileptic treatments.

<p>Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations. </p><p>We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization. </p><p>Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.</p><p>Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association. </p><p>Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases. </p><p>Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.</p> / Dissertation

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucie Chaloupková Supervisor: Prof. MUDr. Radomír Hrdina, CSc. Title of diploma thesis: Treatment of epilepsy Epilepsy is a serious chronic disease affecting all ages which can be characterized by recurrent epileptic attacks. It affects about 1,3-4 % of the population and endangers the patient's life at every incoming attack. Long-term treatment must be preceded by thorough diagnosis and classification of the disease, which can be very difficult. The aim of the treatment is to prevent recurrent epileptic attacks, or at least mitigate them while minimizing the side effects of the treatment and reducing the negative impact on the quality of life. When choosing a suitable drug for an adult, the doctor usually decides individually depending on the type of epileptic attack. In children, the choice of the treatment often depends on the diagnosed type of syndrome, which appears more in the lower age category than in adult patients. The doctor should follow expert standards of the treatment of epilepsy, and also their own most recent experience and knowledge gained during the course of lifelong learning. The therapy begins with monotherapy, and only when not successful, it is necessary to...