Investigating the dynamics of seizure propagation and generation in an in vitro model of temporal lobe epilepsy using the intact isolated mouse hippocampus

The hippocampus plays a crucial role in epilepsy, a neurological condition characterized by recurrent seizures, affecting up to 1% of the world's population. However, our understanding of the mechanisms generating seizures and responsible for seizure propagation remain unclear. In this thesis, I study (1) the nature of recurrent seizures in the hippocampus, (2) the propagation of seizures along the septotemporal axis and (3) the intracellular changes in electrophysiological activity in different types of hippocampal neurons during the seizure transition period. Although my hypotheses target specific questions related to the neurophysiology of epilepsy, the findings are generalizable and can relate to neural dynamics and the function of other brain structures. In conclusion, in this thesis I demonstrate that the intact isolated mouse hippocampus can be utilized in vitro to study the neurophysiology of epilepsy. I further show that the spread of seizures in this structure involves multiple neuronal networks along the septotemporal axis and requires the activation of the CA3 region to sustain and generate this activity. Lastly, examining the intracellular activity of pyramidal cells and interneurons during the seizure transition period reveals a dynamic interplay between inhibition leading to dominant rhythmic simultaneous excitation prior to seizure onset. Intracellular correlates and the dynamics of seizure transition . As the neural dynamics and mechanisms responsible for seizure transition remain unknown in epilepsy, the next area of my thesis focused on the intracellular activity in individual neurons in the intact hippocampus. I show that hippocampal interneurons and pyramidal cells exhibit a characteristic inhibitory-excitatory sequence during this transition period. I characterize the rate of change and reversal potentials of these intracellular activities, demonstrating that IPSPs are the driving force during the early transition period, followed by compound EPSPs in the later transition period. Somatic or dendritic intermittent pressure injection of a GABAA agonist demonstrates that IPSPs are maintained throughout the transition, indicating that prior to seizure onset, there is a dynamic change in the interaction between neuronal clusters in the hippocampus, leading to an alteration in pre-synaptic driving forces that orchestrate the hyperexcitability and hypersynchrony observed during seizures. Generation of recurrent seizures in the intact isolated mouse hippocampus. Current in vitro models of epilepsy utilize brain slices to better understand the cellular mechanisms of this disease. However, the slicing procedure damages superficial neurons and local circuitry and constrains information flow to a two-dimensional plane. In this thesis, I present a model of chronic, recurrent, spontaneous seizures using the intact isolated mouse hippocampus, in which the entire three-dimensional circuitry of this structure is preserved. Utilizing low Mg2+ artificial cerebral spinal fluid (ACSF), recurrent, spontaneous seizures are measured electrophysiologically, and pharmacological, morphological and spectral analyses were designed to characterize this epileptiform activity. Spatial analysis of dual site recordings along the septotemporal hippocampus reveals a characteristic seizure propagation pattern, with seizures first emerging in the temporal region of the hippocampus and propagating septally. This section of the thesis concluded that the whole hippocampus with intact local circuitry, treated with low Mg2+, provides a realistic model of recurrent spontaneous seizures, which may be utilized to further study the dynamics of seizure generation and propagation. Propagation of seizures along the septotemporal hippocampal axis . The next area of my thesis explored the spread of seizures in greater detail using the intact isolated hippocampus and was motivated by our poor understanding of the fundamental mechanisms of seizure propagation. Using multisite electrophysiological recordings in two seizure models and voltage sensitive dye imaging, the initiation and propagation of seizures in the intact structure was spatiotemporally characterized. After seizure onset in the temporal hippocampus, seizure activity became bidirectional along the septotemporal axis at different frequencies, behavior compatible with coupled neuronal network oscillators. When the intact hippocampus was separated along the septotemporal axis, independent bidirectional activity was observed in the two halves, and region-specific cuts to the tissue reveal that the CA3 region is critical for seizure generation and propagation. These data provide novel insights into the dynamic, complex multifocality of seizure onset and propagation, revealing that the current concept of a single seizure "focus" is incomplete.