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Through the use of chronic experimental animal models, the majority of in vitro investigations of temporal lobe epilepsy have demonstrated enhanced network activity within the subdivisions of the hippocampal formation. However, clinical evidence in combination with in vivo and in vitro studies indicates that structures external to the hippocampus contribute to the genesis of seizure activity. To address the effects of limbic network excitation, I have utilized combined hippocampal---entorhinal cortex brain slices from pilocarpine-treated rats that display chronic seizures. / My investigations have focused upon three structures, the subiculum, entorhinal cortex and the insular cortex. The experiments in the pilocarpine-treated subiculum demonstrated increased network excitability that was attributed to a more positive GABAA receptor mediated inhibitory post-synaptic potential (IPSP) reversal point coupled with a reduced IPSP peak conductance. Utilizing RT-PCR analysis and immunohistochemical staining we observed a decline in K+-Cl- cotransporter mRNA expression and a reduced number of parvalbumin-positive, presumptive inhibitory interneurons. My second project assessed the network hyperexcitability in layer V of the lateral entorhinal cortex. This is the first study to report spontaneous bursting, in the absence of epileptogenic agents, in the epileptic entorhinal cortex. We attributed this level of network excitation to reduced GABAA receptor mediated inhibition and increased synaptic sprouting. In the final project, we extended our slice preparation to include the insular cortex, a structure external to the temporal lobe. Our investigations identified a mechanism of NMDA receptor dependent synaptic bursting that masked GABA A receptor mediated conductances.

Excitotoxicity can lead to increases in intracellular Na+ and Ca2+ concentrations via the glutamatergic NMDA receptors, which can lead to cell death. Detailing the time-dependent degradation of neuronal components in response to excitotoxic challenge may help elucidate the sequence in which these signaling pathways are initiated and further, associate these pathways with topographic cellular demise. Using organotypic hippocampal slice culture technique, tissue from neonatal rat pups was exposed to NMDA, APV, or co-exposed for 24, 72 or 120 hours. Fluorescent microscopy of propidium iodide (PI) was used to evaluate neuronal membrane damage, changes in the density of mature soma (NeuN) and NMDA NR2B subunits were measured using immunohistochemical procedures. After 24 hours of exposure, the CA1 showed an increased PI signal and a decrease in NeuN marker, indicating somatic injury occurs shortly after excitotoxic challenge; these effects were blocked by co-administration of APV. Also in the CA1, loss of NR2B subunits, heavily expressed in dendritic processes, declined following 72 hours of exposure. Because somatic injury precedes loss of distal NR2B subunits, it is possible that these events involve different mechanisms, findings that may be relevant in the development of therapies to target neurodegeneration resulting from excitotoxicity.

Epileptogenesis is the complex process of the brain developing epileptic acitivity. Due to the role of glutamate and the hippocampus in synaptic plasticity a dysregulation in glutamate neurotransmission and hippocampal dysfunction are implicated in the process of epileptogenesis. However, the exact causal factors that promote epileptogenesis are unknown. We study presynaptic proteins that regulate glutamate neurotransmission and their role in epileptogenesis. The presynaptic protein, tomosyn, is believed to be a negative regulator of glutamate neurotransmission; however, no one has studied the effects of this protein on glutamate transmission in vivo. Furthermore, evidence suggests that mice lacking tomosyn have a kindling phenotype. Thus, in vivo glutamate recordings in mice lacking tomosyn have the potential to elucidate the exact role of tomosyn in glutamate neurotransmission and its potential relationship to epileptogenesis. Here we used biosensors to measure glutamate in the dentate gyrus (DG), CA3, and CA1 of the hippocampus in tomosyn wild-type (Tom+/+), heterozygous (Tom+/-), and knock out (Tom-/-) mice. We found that, in the DG, that glutamate release increases as tomosyn expression decreases across genotype. This suggests that tomosyn dysregulation in the DG leads to an increase in glutamate release, which may explain why these mice have an epileptogenic phenotype.

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This dissertation explored the cognitive processes and neural substrates underlying the simulation and construction of novel mental representations, by manipulating factors influencing construction ability. Across four experiments, subjects constructed novel events by relating multiple cue words to a single context word in order to make a coherent representation. Experiments 1 and 2 tested whether memory deficits related to age and amnesia due to medial temporal lobe lesions affect event construction performance. Both older adults and patients with amnesia showed deficits in event construction, with poorer performance at increasing mnemonic loads. Moreover, older adults’ construction ability was associated with memory performance, suggesting that associative encoding processes are crucial for simulation tasks. Experiments 3a and 3b examined whether semantic congruency between items and context influences event construction and subsequent memory. In Experiment 3a, younger adults constructed imagined events with from cue words that were typically or atypically related to the context word. Atypical events were less coherent, and were rated as poorer in quality and more difficult to construct. Experiment 3b also showed an advantage for typical trials on a cued recall test, suggesting the congruency of an imagined event with prior knowledge has a strong influence on its subsequent retrieval. Experiment 4 used fMRI to determine the neural correlates of imagining. Constructing imagined events activated the hippocampus, medial prefrontal regions, and default mode network regions in comparison to a baseline condition. Moreover, clusters of activation in the anterior hippocampus were positively correlated with construction task performance across all task conditions, whereas activity in the medial frontal poles varied with individual differences in the typicality of imagined events. Posterior hippocampus was associated with the novelty of imagined events, but did not correlate strongly with the anterior hippocampus or task performance. Taken together, these studies suggest that these regions are crucial when constructing a novel imagined event, regardless of the nature of the stimuli. In particular, the hippocampus may be necessary to bind items during the construction process, especially as representations become increasingly complex.

Cannabis has been used medicinally for millennia, but the cannabinoid (CB) field exploded with the identification of its endogenous receptors and endocannabinoids (eCBs). In vitro experimentation established that eCBs alter synaptic plasticity at presynaptic nerve terminals; however, the characterization of the eCB system (ECS) in vivo remains incomplete. This study aimed to determine the mechanism of in vivo eCB-mediated hippocampal synaptic plasticity and to analyze the effects this plasticity had on spatial working memory (SWM). With in vivo recordings of field excitatory postsynaptic potentials (fEPSPs) in anesthetized mice and rats as well as pharmacological manipulation of the ECS and glutamate receptor antagonism, it was found that eCBs, both anandamide (AEA) and 2-arachnidonyl glycerol (2-AG), caused LTD at hippocampal CA3-CA1 synapses. Induction of eCB-LTD occurs via a sequential activation of cannabinoid type-1 receptor (CB1R) and NR2B-containing NMDA receptor (NR2BR) and is expressed through the endocytosis of AMPA receptors (AMPARs). Increased eCB tone also caused an impairment of SWM for over 24 hours in the Delayed Non-Match-To-Sample (DNMTS) T-maze. This study provides the first evidence that an acute administration of eCB degradative enzyme inhibitors not only produces an in vivo LTD at hippocampal CA3-CA1 synapses that requires CB1R, NR2BR, and AMPAR, but also impairs SWM, a phenomenon also caused by an acute injection of exogenous CBs.

Current concepts of hippocampal circuitry assume a large population of excitatory principal neurones whose activity is largely governed by a network of local-circuit GABAergic interneurones. The diversity of hippocampal local-circuit neurones and their synaptic control over principal cell activity was investigated in vitro, in order to define their synaptic connections and functional roles. Single and dual intracellular recordings were made from local-circuit neurones and pyramidal cells in area CA1 of the rat hippocampal slice. Interneurones were tentatively distinguished from pyramidal cells based on their firing as well as their membrane properties. Intracellular labelling of recorded cells with the marker biocytin revealed a diversity of cell types based on differential dendritic and axonal morphology and synaptic connections. The physiological data revealed that all types of interneurone tested evoked inhibitory postsynaptic potentials (IPSPs) in simultaneously recorded pyramidal cells. The IPSPs had fast rise and decay kinetics and the ones tested pharmacologically, were mediated by GABA_A receptors. Similarly, individual interneurones were also shown to innervate other local-circuit interneurones in addition to pyramidal cells, the evoked effects being qualitatively similar in both types of postsynaptic targets. The postsynaptic effect and functional role of one type of hippocampal interneurone, the basket cell, was investigated in greater detail. Basket cell-evoked IPSPs were reliable, but showed some frequency-dependent attenuation. Moreover, basket cell IPSPs were found to interact with intrinsic pyramidal cell conductances to elicit rebound depolarisations and facilitate action potential generation. More detailed investigation showed that basket and axo-axonic cells were particularly effective in entraining pyramidal cell firing and sub-threshold membrane potential oscillations. Through these powerfully tuned mechanisms, sub-types of local-circuit interneurone provide a powerful mechanism to synchronise the activity of pyramidal cells. These results demonstrate a remarkable diversity of GABAergic local-circuit neurones in the hippocampal CA1 area and suggest that specific subtypes of cell mediate different functions.