Elevations in extracellular potassium (K+e) accompany seizure-like events (SLEs), but elevated K+ may also participate in seizure cessation. The objective of this thesis was to investigate the possibility that K+ may undergo a pro- to anti-convulsant switch in the epileptic juvenile (postnatal day 17-21) rat hippocampus. Field recordings were performed in the CA1 pyramidal layer. SLEs and primary afterdischarges (PADs) were induced with 0.25 mM Mg/5 mM K+ perfusion or tetanic stimulation of the Schaffer collaterals respectively. In these seizure models, elevating [K+]e beyond 7.5 mM showed anticonvulsant properties. The addition of ZD7288, a blocker of the hyperpolarization activated nonspecific cationic current (Ih) and allowed SLEs to continue even in elevated [K+]e. This suggests that [K+]e switches from being pro- to anti-convulsant, in part due to an elevated [K+]e-induced potentiation of Ih. Ih likely contributes to this anticonvulsant behavior by decreasing membrane resistance and subsequently attenuating summation of incoming EPSPs.

Mevinphos (Mev) is an orgnophosphate insectide used for suicidal purposes in Taiwan; seizure and cardiovascular depression are commom syptoms observed in organophosphate-poisoned patients. The amygdala (AMG) is part of the limbic system and the basolateral nucleus of AMG (BLA) is one of the most seizure-prone brain structures. The central neucleus of AMG (CeA) is thought to play a central role in behavioral, physiological response and cardiovascular regulation. However, detailed mechanisms in Mev-induced seizure and cardiovascular depression by an action on AMG are lacking. Based on electroencephalographic (EEG) activity to indicate neuronal electrical activity and arterial blood pressure (AP) and heart rate (HR) to indicate cardiovascular responses, the present study investigated whether Mev acts on AMG to elicit seizure or cardiovascular depression. Microinjection of Mev into BLA of adult male Sprague-Dawley (SD) rats maintained under propofol anesthesia increased EEG activity in AMG, cortex and CA3 of hippocampus leading to seizure initiation; however AP, HR, respiration rate (RR) and the power density of low-frequency (LF) component of AP was not significantly changed. Microinjection of Mev into BLA also time-dependently increased protein level and mRNA of cytokines interleukin (IL)-12, IL-13, tumor suppressor factor alpha (TNF£\) and interferon gamma (IFN£^) and cyclooxygenase (COX) activity in AMG. Microinjection of Mev into CA3 induced less seizure activity in cortex and CA3 than that induced by microinjection of Mev into BLA. In addition, microinjection Mev into CA3 did not induce seizure in AMG. These results suggest that Mev acted on BLA to initiate limbic seizures. Intraperitoneal injection of muscarinic receptor antagonist atropine (ATR), which can pass the blood-brain barrier (BBB), activator of GABAergic neurotransmission midazolam (MDZ) or antiinflaamatory agent pentoxifylline (PTX) and Lisofylline (LSF), but not muscarinic receptor antagonist atropine methyl nitrate (AMN), which can not pass BBB, inhibited Mev-induced seizure and increase of cytokines in AMG by an action on BLA. Microinjection of ATR, COX-1 inhibitor naproxen (NPX) or COX-2 inhibitor NS-398, antiserum against receptor of IL-12, IL-13, TNF£\ or IFN£^, but not nicotinic receptor antagonist mecamylamine (MEL), into BLA inhibited Mev-induced seizure and increase of cytokines and COX activity in AMG by an action on BLA. However, caspase 3 activity and DNA fragmentation at AMG were not changed by microinjection of Mev into BLA. Microinjection of Mev into CeA induced a decrease in AP and RR leading to cardiovascular depression and an increase of power desity of LF, accompanied with insignificant HR and EEG activity change. Microinjection of Mev into CeA induced the time-dependently increase of caspase 3 activity and DNA fragmentation leading to apoptosis in AMG. Microinjection of ATR or caspase 3-dependent apoptosome inhibitor NS-3694, but not MEL, into CeA inhibited cardiovascular depression and the increase of caspase 3 activity and DNA fragmentation induced by Mev action on CeA. However, the levels of cytokines were not changed by Mev treatment. Intravenous injection of Mev did not induce changes of partial pressure of oxygen, blood flow and the level of superoxide anion in AMG. In addition, microinjection of Mev into BLA or CeA did not affect the level of superoxide anion in AMG. These results suggest that AMG mediates the initiation of seizure and cardiovascular depression induced by Mev. Furthermore, inflammation in BLA and apoptosis in CeA individually play an important role in Mev-induced seizure and cardiovascular depression.

Hypoglycemia is a severe side effect of insulin overdose in the diabetic population and can result in various neurological sequalae including seizures, coma, and brain death. There is still a limited understanding of the generation and propagation of hypoglycemic seizures, which may exacerbate hypoglycemia-induced neuronal damage. Moreover, glucose reperfusion after a period of transient hypoglycemia has been shown to cause neuronal hyperexcitability which can have further damaging effects. Gap junctional communication can be involved in the spread of hypoglycemic injury in two ways: 1) by providing a cytoplasmic continuity in which seizures can easily propagate and 2) by engaging the astrocytic network in metabolic compensation as well as enhancing astrocytic buffering of K+. This study aims to investigate the role that gap junctions play during brain energy deprivation. Results from these experiments show that gap junction blockade can have a neuroprotective role during hypoglycemia and glucose reperfusion.

Research over the last two decades has revealed that the process of neurogenesis continues in some brain regions throughout adulthood in mammals. The dentate gyrus of the hippocampal formation displays adult neurogenesis and is known to be critical for some forms of learning and memory. Adult neurogenesis has been proposed to play an important role in hippocampal function. Seizures have been found to increase the rate of adult neurogenesis as well as alter the development of newborn neurons. Seizure-induced changes in neurogenesis have been proposed to underlie some of the cognitive impairments seen in epileptic patients. This thesis examines changes in neurogenesis in the dentate gyrus in the kindling model of temporal lobe epilepsy in rats, and explores the potential impact of these changes on hippocampal function. Progenitor cell proliferation and net neurogenesis were found to be increased in kindled rats, but increased proliferation was not sustained and returned to baseline by 8 days. This increase was greater in dorsal than ventral dentate gyrus but did not differ among the blades of the granule cell layer. Kindled seizures were found to enhance the survival of newborn neurons when presented during the second week of their development. These survived for at least 1 month after kindling (6 weeks after their birth). Electrical stimulation at current intensities below afterdischarge threshold failed to alter progenitor cell proliferation, while currents above threshold greatly increased it. There was no relationship found between stimulation current intensity and the rate of cell proliferation. Bilateral kindling of the perforant path failed to alter learning or long-term memory in a water maze test in spite of a large increase in neurogenesis in the dentate gyrus. Unkindled rats with more new neurons showed better learning performance, but had poorer long-term memory in the task. Kindled rats showed a disruption of these relationships. This thesis found that neurogenesis was greatly increased in the kindling epilepsy model, but that performance in a commonly used learning/memory task was unaltered. The impact of kindled seizures on water maze performance is questioned as well as the relevance of enhanced neurogenesis to cognitive impairments in epilepsy.

Research over the last two decades has revealed that the process of neurogenesis continues in some brain regions throughout adulthood in mammals. The dentate gyrus of the hippocampal formation displays adult neurogenesis and is known to be critical for some forms of learning and memory. Adult neurogenesis has been proposed to play an important role in hippocampal function. Seizures have been found to increase the rate of adult neurogenesis as well as alter the development of newborn neurons. Seizure-induced changes in neurogenesis have been proposed to underlie some of the cognitive impairments seen in epileptic patients. This thesis examines changes in neurogenesis in the dentate gyrus in the kindling model of temporal lobe epilepsy in rats, and explores the potential impact of these changes on hippocampal function. Progenitor cell proliferation and net neurogenesis were found to be increased in kindled rats, but increased proliferation was not sustained and returned to baseline by 8 days. This increase was greater in dorsal than ventral dentate gyrus but did not differ among the blades of the granule cell layer. Kindled seizures were found to enhance the survival of newborn neurons when presented during the second week of their development. These survived for at least 1 month after kindling (6 weeks after their birth). Electrical stimulation at current intensities below afterdischarge threshold failed to alter progenitor cell proliferation, while currents above threshold greatly increased it. There was no relationship found between stimulation current intensity and the rate of cell proliferation. Bilateral kindling of the perforant path failed to alter learning or long-term memory in a water maze test in spite of a large increase in neurogenesis in the dentate gyrus. Unkindled rats with more new neurons showed better learning performance, but had poorer long-term memory in the task. Kindled rats showed a disruption of these relationships. This thesis found that neurogenesis was greatly increased in the kindling epilepsy model, but that performance in a commonly used learning/memory task was unaltered. The impact of kindled seizures on water maze performance is questioned as well as the relevance of enhanced neurogenesis to cognitive impairments in epilepsy.

Iatrogenic hypoglycemia is a limiting factor for managing diabetes mellitus and can have severe outcomes such as seizures and coma. Although several studies have investigated the central nervous system consequences of hypoglycemia, the effects of seizures, as well as possible treatment strategies, have yet to be elucidated in juvenile animals. The objective of this thesis was to establish an in vivo model of severe hypoglycemia and seizures in juvenile diabetic and non-diabetic rats. In both groups there existed a similar blood glucose threshold for seizures, and mortality only occurred following severe seizures, particularly with repeated seizures that were unresponsive to treatment. While the administration of anticonvulsants temporarily mitigated seizures, glucose administration was required to prevent mortality. Abnormalities in the hippocampal and brainstem electroencephalograms (EEG) were observed in hypoglycemic animals without a clear correlate to convulsive activity.

Iatrogenic hypoglycemia is a limiting factor for managing diabetes mellitus and can have severe outcomes such as seizures and coma. Although several studies have investigated the central nervous system consequences of hypoglycemia, the effects of seizures, as well as possible treatment strategies, have yet to be elucidated in juvenile animals. The objective of this thesis was to establish an in vivo model of severe hypoglycemia and seizures in juvenile diabetic and non-diabetic rats. In both groups there existed a similar blood glucose threshold for seizures, and mortality only occurred following severe seizures, particularly with repeated seizures that were unresponsive to treatment. While the administration of anticonvulsants temporarily mitigated seizures, glucose administration was required to prevent mortality. Abnormalities in the hippocampal and brainstem electroencephalograms (EEG) were observed in hypoglycemic animals without a clear correlate to convulsive activity.

O floroglucinol é um composto fenólico precursor de diversas moléculas com atividades biológicas já descritas na literatura, com destaque para a antidepressiva. O modelo experimental com zebrafish tem sido bastante utilizado em várias linhas de pesquisa biológica, como, por exemplo, para avaliação da atividade anticonvulsivante. A partir de estudos que mostram uma correlação entre compostos antidepressivos e anticonvulsivantes, nosso grupo administrou floroglucinol e dois derivados sintéticos (composto 7 e composto 8) em zebrafish para observação da atividade locomotora e exploratória no open tank e, posteriormente, à avaliação através do modelo convulsivo induzido por pentilenotetrazol (PTZ). Além disso, os níveis de captação de glutamato e a toxicidade dos compostos foram avaliados em cérebro total de zebrafish. O comportamento dos animais não sofreu alteração em relação ao controle para nenhum dos compostos testados. O composto 7 aumentou significativamente o tempo para os animais atingirem a primeira convulsão além de reduzir a intenidade da crise convulsiva. Também se observou aumento na captação de glutamato para esse composo, sem sinais de toxicidade envolvidos. Desta forma, nossos resultados contribuem para a busca de compostos potencialmente ativos frente a crises convulsivas induzidas por PTZ. / Phloroglucinol, a phenolic compound, which is precursor of several molecules with biological activities are described in the literature, mainly for antidepressant activity. The zebrafish experimental model has been widely used in many kinds of biological research, for example, to evaluate the anticonvulsant activity. From studies that shows correlation between antidepressants and anticonvulsant compounds, our group managed phloroglucinol and two synthetic derivatives (compound 7 and compound 8) in zebrafish in order to observe the locomotor and exploratory activity on open tank and subsequently, conduct the evaluation through the seizure model induced by pentylenetetrazol (PTZ). Furthermore, glutamate uptake and toxicity levels of the compounds were evaluated in zebrafish’s whole brain. The animals' behavior did not change compared to control for any of the tested compounds. The compound 7 increased significantly the time for the animals reach the first seizure and reduce the seizure intensity. It was also observed an increase in glutamate uptake for this compound without signs of toxicity involved. Thus, our results contribute to the search for potentially active compounds against seizures induced by PTZ.

O floroglucinol é um composto fenólico precursor de diversas moléculas com atividades biológicas já descritas na literatura, com destaque para a antidepressiva. O modelo experimental com zebrafish tem sido bastante utilizado em várias linhas de pesquisa biológica, como, por exemplo, para avaliação da atividade anticonvulsivante. A partir de estudos que mostram uma correlação entre compostos antidepressivos e anticonvulsivantes, nosso grupo administrou floroglucinol e dois derivados sintéticos (composto 7 e composto 8) em zebrafish para observação da atividade locomotora e exploratória no open tank e, posteriormente, à avaliação através do modelo convulsivo induzido por pentilenotetrazol (PTZ). Além disso, os níveis de captação de glutamato e a toxicidade dos compostos foram avaliados em cérebro total de zebrafish. O comportamento dos animais não sofreu alteração em relação ao controle para nenhum dos compostos testados. O composto 7 aumentou significativamente o tempo para os animais atingirem a primeira convulsão além de reduzir a intenidade da crise convulsiva. Também se observou aumento na captação de glutamato para esse composo, sem sinais de toxicidade envolvidos. Desta forma, nossos resultados contribuem para a busca de compostos potencialmente ativos frente a crises convulsivas induzidas por PTZ. / Phloroglucinol, a phenolic compound, which is precursor of several molecules with biological activities are described in the literature, mainly for antidepressant activity. The zebrafish experimental model has been widely used in many kinds of biological research, for example, to evaluate the anticonvulsant activity. From studies that shows correlation between antidepressants and anticonvulsant compounds, our group managed phloroglucinol and two synthetic derivatives (compound 7 and compound 8) in zebrafish in order to observe the locomotor and exploratory activity on open tank and subsequently, conduct the evaluation through the seizure model induced by pentylenetetrazol (PTZ). Furthermore, glutamate uptake and toxicity levels of the compounds were evaluated in zebrafish’s whole brain. The animals' behavior did not change compared to control for any of the tested compounds. The compound 7 increased significantly the time for the animals reach the first seizure and reduce the seizure intensity. It was also observed an increase in glutamate uptake for this compound without signs of toxicity involved. Thus, our results contribute to the search for potentially active compounds against seizures induced by PTZ.

The pro-inflammatory cytokine interleukin 1 (IL-1) is involved in numerous physiological and pathological processes. It contributes to thermoregulation, sleep, feeding behaviour and notably to the exacerbation of non-communicable disorders such as cancer, heart disease, stroke and epilepsy, which are the greatest cause of mortality worldwide. Given this important role, IL-1 is tightly regulated, with regulation mechanisms present at the level of its synthesis, activation and receptor engagement. However, when studying IL-1 in vitro, little notice is taken of the disease microenvironment in which it acts. Acidosis is a hallmark of disease, often due to poor perfusion resulting in a shift to anaerobic respiration, a build-up of lactic acid and poor clearance of CO2. Additionally, highly active infiltrating immune cells favour anaerobic respiration and can contribute to this local acidosis. This thesis utilised primary cell cultures, cell lines and reporter cells to explore the mechanisms of IL-1 signalling under disease-relevant acidic conditions. Subsequently, a murine seizure model was developed to further explore IL-1 signalling in disease conditions in vivo. This work demonstrated that acidic pH itself did not induce IL-1β release, however, it did promote release of minimally active 20 kDa IL-1β in response to damage associated molecular patterns (DAMPs) such as ATP, monosodium urate crystals or calcium pyrophosphate dihydrate crystals. The cleavage of pro-IL-1β into 20 kDa IL-1β was mediated by cathepsin D and was also induced on addition of lactic acid to the culture media. This 20 kDa IL-1β was not further cleaved to the active mature 17 kDa IL-1β thus its production limits the spread of inflammation. The intranasal administration of kainic acid induces seizures in C57Bl/6J mice, however, IL-1β was not observed acutely in this model thus the presence of 20 kDa IL-1β in vivo was not confirmed. In recent years, the contribution of IL-1 to disease has become well established. However, despite successes in the development of novel therapeutics targeted at blocking IL-1 activity, such as anakinra, canakinumab or rilonacept to treat cryopyrin associated periodic syndromes, a number of studies have demonstrated poor efficacy and only minor improvements in patients when targeting IL-1. Thus further knowledge of the mechanisms of IL-1 signalling in disease is required to understand this system and develop improved novel therapeutics.