Study of a Model of Focal Limbic Epilepsy in Rats

Epilepsy is a neurological disorder characterized by the recurrence of spontaneous, unprovoked epileptic seizures. Mesial temporal lobe epilepsy (more briefly, MTLE) is a very common form of epilepsy which is featured by the occurrence of focal limbic seizures, and associated to a specific neuropathological alteration, the so-called Ammon’s horn sclerosis (AHS, from now on abbreviated as AHS), whose main features are a selective loss of the CA1 and CA3/4 section of the Ammon’s horn (CA, from Latin Cornu Ammonis, abbreviated as CA), a selective cell loss of inhibitory interneurons in the hilus of the dentate gyrus (DG), and the abnormal sprouting of granule cells mossy fibers (the so called mossy fiber sprouting, MFS). The onset of spontaneous recurrent seizures (SRS) is the hallmark of a good model of epilepsy. For temporal lobe epilepsy (TLE), the most used models consist in administering systemically chemoconvulsants inducing limbic status epilepticus (i.e. seizures lasting for more than 30’, SE) and evaluating the occurrence of SRS. However, in these models, the widespread involvement of different structures which complicates the interpretation of experimental findings obtained with this experimental approach, since any morphological/functional effect of these models might be due either to the direct action of the chemoconvulsant or to the SE. The morphological features of many structures of the limbic system are highly phylogenically conserved through the evolution from rodents to primates and humans; it has been recently shown that it is possible to evoke limbic seizures and SE from a small structure, the deep extent of the anterior piriform cortex (from now on abbreviated as APC) by focally infusing picomolar concentration of chemoconvulsants; this structure roughly corresponds to the periamygdaloid cortex in humans. It is the brain region most densely innervated by the noradrenergic fibers originating from the nucleus locus coeruleus (LC), and we recently showed that microinfusing bicuculline (a GABA A receptor antagonist) into the APC of rats with a lesion of LC (induced by a selective neurotoxin, DSP-4, i.p.), induces SE, similarly to the SE obtained by microinfusing into the APC of rats with an intact noradrenergic system, cychlotiazide+ bicuculline. LC plays a critical role in modulating several models of seizures, and it lays a critical role in plastic mechanisms and neuroprotection in the brain. Thus, we compared the group DSP-4+bicuculline and cyclotiazide+bicuculline, to evaluate whether the focal SE evoked from the APC is capable of inducing SRS and AHS, and whether LC plays a significant role in this phenomena. We found that: a) despite a similar duration and severity of SE in the two models of SE, in the group DSP-4+bicuculline there was a higher incidence of SRS; b) the cell loss in the hippocampal DG hilus and CA3 was higher in the group DSP-4+bic, while MFS was more intense in the group cyclotiazide+bicuculline; also the loss of parvalbumin-positive neurons was more represented in the DSP-4+bicuculline group, while GFAP expression (an index of reactive gliosis), was similar in the two groups. In conclusion, our study confirms that focal induction of SE from the APC represents a good model of TLE, and that NE released from the fibers originating from the LC plays a significant role both in the hippocampal damage occurring after SE, and in the incidence of SRS. Differently from what observed in other models, our findings challenge a prominent role of MFS in the occurrence of SRS, since this phenomenon was less intense in the group with more frequent SRS (DSP-4+bicuculline) than in the one with an intact LC

Cognitive Impairment and Aberrant Plasticity in the Kindling Model of Epilepsy

Epilepsy is a neurological disorder that affects approximately 1% of the population worldwide. Although motor seizures are the best known feature of epilepsy, many patients also experience severe interictal (between-seizure) behavioral and cognitive comorbidities that have a greater negative influence on quality of life than seizure control or frequency. To study the characteristics of these interictal comorbidities and the neural mechanisms that underlie them, I use the kindling model of epilepsy. Kindling refers to the brief electrical stimulation of a discrete brain site that produces a gradual and permanent increase in the severity of elicited seizure activity. The repeated seizures associated with kindling induce robust structural and functional plasticity that appears to be primarily aberrant. Importantly, the aberrant plasticity evoked by repeated seizures is thought to contribute to the pathophysiology of epilepsy and its associated behavioral and cognitive comorbidities. Unfortunately, the relationship between aberrant plasticity and cognition dysfunction following repeated seizures remains poorly understood. The aim of this dissertation is to gain a better understanding of the effects of repeated convulsions on aberrant neural plasticity and interictal behavior. In Chapter 2, I will examine the effect of short- and long-term amygdala kindling on amygdala- and hippocampal-dependent forms of operant fear conditioning. To evaluate whether kindling alters neural circuits important in memory, I will analyze post-mortem measures of neural activity following the retrieval of fearful memories. In Chapter 3, I will evaluate whether deficits in operant fear learning and memory are a general consequence of convulsions induced by kindling stimulations or whether these deficits occur following kindling of specific brain regions. To evaluate whether aberrant plasticity following kindling of different brain regions contributes to learning and memory deficits, I will make post-mortem examinations of the inhibitory neurotransmitter neuropeptide Y and its Y2 receptor. In Chapter 4, I will investigate the relationship between hippocampal neurogenesis and cognition. Specifically, I will determine whether kindling of different brain regions induces an aberrant form of hippocampal neurogenesis that contributes to cognitive dysfunction. In Chapter 5, I will investigate whether kindling of different brain regions alters different subpopulations of hippocampal GABAergic interneurons, in terms of number and morphological features. Finally, Chapter 6 will provide preliminary evidence that the cognitive impairments associated with kindling can be ameliorated through intrahippocampal infusions of recombinant reelin. The collection of studies in this dissertation improves our understanding of the relationship between aberrant plasticity and cognitive impairments associated with repeated convulsions

Effects of Mammalian Target of Rapamycin Inhibition on Circuitry Changes in the Dentate Gyrus of Mice after Focal Brain Injury

Post-traumatic epilepsy is a common outcome of severe traumatic brain injury (TBI). The development of spontaneous seizures after traumatic brain injury generally follows a latent period of little to no symptoms. The series of events occurring in this latent period are not well understood. Additionally, there is no current treatment to prevent the development of epilepsy after TBI (i.e. antiepileptogenics). One cell signaling pathway activated in models of TBI and in models of epilepsy is the mammalian target of rapamycin (mTOR). mTOR activity is sustained for weeks after the initial insult in models of TBI, and the inhibition of mTOR using rapamycin has shown promising pre-clinical outcomes in rodent models. This makes rapamycin an ideal therapeutic to test various outcomes associated with epileptogenesis after TBI. The results from this study suggest that rapamycin treatment after controlled cortical impact reduces aberrant axonal sprouting of ipsilateral dentate granule cells, prevents increased neurogenesis in the subgranular zone, and differentially alters phasic and tonic inhibition in dentate granule cells. However, rapamycin treatment did not prevent all forms of axon sprouting in the dentate gyrus or cell loss in selected regions of the hippocampus. Collectively these results support a role of mTOR activity in both excitatory and inhibitory plasticity in the mouse dentate gyrus after TBI

Refractoriness within the semantic system: investigations on the access and the content of semantic memory

The starting purpose of this project was to investigate some issues related to the mechanisms underlying the efficient access to concepts within the semantic memory systems. These issues were mainly related to the role of refractoriness in explaining the comprehension deficits underlying semantic access. The insights derived from this first approach were then used to formulate and test hypotheses about the organization of the contents of the semantic system itself. The first part of the thesis presents an investigation of the semantic abilities of an unselected case-series of patients affected by tumours to either the left or right temporal lobes in order to detect possible semantic access difficulties. Semantic access deficits are typically attributed to the semantic system becoming temporarily refractory to repeated activation. Previous investigations on the topic were mainly based on single case reports, mainly on stroke patients. The rare examples of group studies suggested moreover the possibility that the syndrome might not be functionally unitary. The tasks used in the study were two word-to-picture matching tasks aimed to control for the typical variables held to be able to distinguish semantic access from degradation syndromes (consistency of access, semantic relatedness, word frequency, presentation rate and serial position). In the group of tumour patients tested access deficits were consistently found in patients with high grade tumours in the left posterior superior temporal lobe. However, the patients were overall only weakly affected by the typical temporal factors (presentation rate and serial position) characterizing an access syndrome as refractory. The pattern of deficit, together with the localization data, suggested that the deficit described is qualitatively different from typical semantic access syndromes and possibly caused by the disconnection of posterior temporal lexical input areas from the semantic system. In the second study we tried to answer the question whether semantic access deficits are caused by the co-occurrence of two causes (refractoriness and a lexicalsemantic disconnection) or whether the presence of refractoriness in itself is sufficient to induce all the behavioural effects described in access syndromes. A second aim of the study was moreover to investigate the precise locus of refractory behaviour, since refractory effects have also been reported in naming tasks in which the possibility exists that the interference might be located at a post-semantic lexical stage of processing. To address these issues a series of three behavioural experiments on healthy subjects was conducted. The tasks used were speeded versions of the same word-to picture matching tasks used in the previous study. A speeded paradigm was adopted in order to induce a mild refractory state also in healthy participants. The results showed that it was possible to induce, in the group of subjects tested, a performance similar to that of refractory semantic access patients. Since no post-semantic stage of processing is assumed to be necessary to perform these tasks it was argued that refractoriness arises due to interference occurring between representations within the semantic system itself. In the second part of the project, the finding that refractoriness arises due to interference involving semantic representations themselves, was used to investigate issues related to the organization of the content within the semantic memory. In particular, a second series of behavioural experiments was performed to investigate whether the way an object is manipulated is indeed a feature that defines manipulable objects at a semantic level. The tasks used were speeded word-to-picture matching tasks similar to those previously described. A significantly greater interference was found in the recognition of objects sharing similar manipulation than in the recognition of objects sharing only visual similarity. Moreover the repeated presentation of objects with similar manipulation created a \u2018negative\u2019 serial position effect (with error increasing over presentations), while the repeated presentation of objects sharing only visual similarity created an opposite \u2018positive\u2019 serial position effect (learning). The role of manipulability in the semantic representation of manipulable objects was further investigated in the last study of this work. In a second unselected group of brain tumour patients the ability to name living things and artifacts was investigated. Artifacts were manipulable objects, varying in the degree of their manipulability. Results from both behavioural and Voxel-based Lesion Symptom Mapping (VLSM) analyses showed that the only patients showing a selective deficit in naming artifacts (particularly highly manipulable objects) were patients with lesions in the posterior middle and superior portions of the left temporal lobe, an area lying within the basin of those regions involved in processing object-directed actions and previously linked to the processing of manipulable objects in a wide range of studies. The results of these last two studies support \u2018property-based networks\u2019 accounts of semantic knowledge rather than \u2018undifferentiated network\u2019 accounts. Overall this series of studies represents an attempt to better understand the mechanisms that underlie the access to semantic representations and, indirectly, the structure of representations stored within semantic networks. The insights obtained about the mechanisms of access to stored semantic representations were used as a tool to investigate the structures of the same semantic representations. A combination of different approaches was used (from behavioural speeded interference paradigms on healthy subjects, to neuropsychological case series investigations, as well as Voxel-based Lesion Symptom Mapping technique), to \u2018cross-validate\u2019 the results obtained at any level of analysis