Circuits underlying hyperexcitability and epilepsy in the piriform cortex

Abstract

The piriform cortex, the largest structure in the olfactory system, is essential for odor processing and plays a key role in generating and propagating epileptic seizures. Balancing excitatory and inhibitory circuits in this area is crucial, as disruptions can lead to epilepsy. A significant contributor to such imbalances is disinhibition - the inhibition of inhibitory interneurons by other inhibitory interneurons - which increases the excitability of principal neurons. This disinhibition enhances overall circuit excitability, potentially giving rise to abnormal, hyper-synchronized electrical activity. Despite its importance, the disinhibitory circuits within the piriform cortex are not well understood. To address this, the first part of this thesis mapped the connectivity of GABAergic interneuron subclasses in the deep piriform cortex of GAD67-GFP mice. Four subclasses were reliably identified: fast-spiking (FS), neurogliaform (NG), regular-spiking (RS), and bitufted (BT) cells. Following this classification, extracellular stimulation was used to determine whether these neurons received inhibitory inputs. It was found that FS and NG cells received significantly larger inhibitory inputs than RS and BT cells. To investigate the sources of these inputs, dual patch-clamp experiments were conducted, revealing that FS and NG cells established more chemical synaptic connections to other interneurons than BT and RS cells. Additionally, electrical synapses were identified, with NG-to-BT connections being particularly prevalent. Given the piriform cortex's epileptogenic nature, the second part of this thesis explored changes in the piriform cortex using a transgenic mouse model with a pathogenic HCN1 (M294L) channel mutation. Immunohistochemistry and pharmacological analyses confirmed HCN1 channel expression in the piriform cortex. Electrophysiological recordings revealed that pyramidal neurons from mutant mice exhibited depolarised resting membrane potentials and increased excitability. This increased excitability was attributed to the loss of voltage dependence in the mutant HCN channels, as evidenced by voltage-clamp experiments. Collectively, these findings suggest alterations in the piriform cortex associated with the HCN1 mutation. Interneuron subclasses in mutant mice were also examined, focusing on NG and FS cells. NG cells mirrored the hyperexcitable patterns of pyramidal neurons, while FS cells showed unchanged resting membrane potentials but reduced excitability. To investigate the mechanisms underlying these contrasting behaviours of FS cells and pyramidal neurons/NG cells, computational modelling was done. The simulations suggested that differences in the location of the axon initial segment (AIS) and the distribution of HCN1 channels could explain the observed firing patterns. Specifically, a proximal AIS with mutant HCN1 channels on the soma replicated the hyperexcitability of pyramidal neurons, whereas a distal AIS with mutant HCN1 channels between the soma and AIS replicated the reduced excitability of FS cells. Overall, this thesis stresses the roles of FS and NG cells as disinhibitory interneurons in the piriform cortex, emphasising their contributions to modulating global inhibition. However, the second part shows that these contributions can be complex, depending on the type of epilepsy. In the HCN1 epilepsy model, NG cells exhibit enhanced intrinsic excitability which may partially counteract the elevated excitability of pyramidal neurons. In contrast, FS cells exhibit decreased excitability, potentially contributing to elevated circuit excitability. However, since both FS and NG cells also strongly inhibit other interneurons, their impact on the circuit is likely to be multi-factorial. These findings highlight the need for further investigation to fully understand the interplay between inhibition and disinhibition and its consequences for generating hyperexcitability and epilepsy in the piriform cortex. Show more