Monitoring and controlling GABAergic interneuron subtypes during epileptiform activity

Inhibitory synaptic transmission is of paramount importance for maintaining the delicate balance between excitation and inhibition in the brain. If this balance is perturbed in favour of excitation, epilepsy is likely to develop. Fast synaptic inhibition is mediated by type A γ-aminobutyric acid ionotropic receptors (GABAARs), which are primarily permeable to Cl−. The strength of synaptic inhibition crucially depends on the release of GABA from different populations of presynaptic interneurons and the transmembrane electrochemical gradient for Cl− in postsynaptic cells. GABAAR-mediated inhibition has been shown to oppose epileptic seizures by establishing an inhibitory restraint against spreading excitation. Of the different subtypes of GABAergic interneurons, parvalbumin-expressing (PV) interneurons that target the somatic compartment of excitatory neurons have been strongly implicated in this process. In the context of an epileptic seizure, it is thought that the inhibitory restraint is overwhelmed by runaway excitation, and the seizure front is able to spread from the pathologic epileptic focus into adjacent healthy areas, referred to as the ‘penumbra’.
In the first part of this thesis I assess the potential of using chemogenetic strategies to suppress epileptiform activity by boosting the synaptic output from three major interneuron populations in the rodent hippocampus: PV, somatostatin (SST) and vasoactive intestinal peptide (VIP) expressing interneurons. Electrophysiological recordings in an in vitro model of epilepsy reveal that the interneuron populations exhibit different effects on epileptiform events. Recruiting VIP interneurons does not change the total duration of epileptiform activity. By contrast, recruiting SST or PV interneurons produces robust suppression of epileptiform synchronisation. PV interneurons exhibit the strongest effect per cell, eliciting at least a five-fold greater reduction in epileptiform activity than the other cell types. Consistent with this, I find that in vivo chemogenetic recruitment of PV interneurons suppresses convulsive behaviours by more than 80%.
In the second part of the thesis I use a genetically-encoded reporter to investigate activity-dependent intracellular pH and Cl− concentration transients in pyramidal neurons and PV, SST and VIP interneurons. I demonstrate that pyramidal neurons and interneurons have different pH and intracellular Cl− concentration steady states, and exhibit distinct dynamics during epileptiform events. Compared to the other cell types, PV interneurons maintain a relatively stable intracellular Cl− concentration, even when challenged with epileptiform activity. This suggests that PV interneurons may be more likely to maintain a balance in their excitatory and inhibitory synaptic inputs during seizures.
In the final part of the thesis I investigate the contribution of PV interneurons to inhibitory restraint in an in vitro model of the epileptic penumbra. Although PV interneurons are recruited in response to spreading excitation, they can be overwhelmed as they enter a state referred to as ‘depolarising block’, which is characterized by a decrease in action potential firing. To investigate the impact of this process, I use a light-activated optogenetic tool to induce brief hyperpolarisations of the PV interneuron membrane potential. This successfully reduces depolarising block in PV interneurons, enhances their action potential firing, and reduces the spread of epileptiform activity.
In conclusion, this thesis demonstrates that selective enhancement of inhibitory synaptic pathways offers potential as an anti-seizure strategy, providing valuable insights into the development of therapeutic interventions.

Keywords: vasoactive intestinal polypeptide
synaptic transmission
inhibition
hippocampus
ClopHensorN
chloride
parvalbumin
seizure
CNO
epilepsy
DREADDs
chemogenetic
hM3Dq
interneuron
GABA
optogenetic
depolarising block
archaerhodopsin
somatostatin
Advisor: Akerman, C