The role of GABAergic interneurons and astrocytes in the mechanism of seizure generation and propagation

Within the brain, inhibitory signals originating from different GABAergic interneurons play crucial roles in establishing proper neural circuit operations. GABAergic interneurons are also directly involved in multiple neurological disorders, including epilepsy. The inhibitory signal from these cells can, indeed, counteract seizures and pharmacological agents that strengthen GABAergic transmission represent powerful anticonvulsant drugs extensively used by epileptic patients. Among the diverse classes of interneurons, Parvalbumin-expressing (Pv) interneurons have been proposed to play a central role in seizure control. In both experimental epilepsy models and patients these interneurons have been observed to generate a feedforward inhibition that effectively opposes seizure spread. When Pv interneurons were artificially activated in vivo with optogenetic techniques, ongoing seizures were interrupted. However, studies in different experimental epilepsy models revealed that Pv interneurons may, under certain conditions, favor seizure generation. It is therefore highly debated whether an intense activity of Pv interneurons can ultimately result in a pro- or in an anti-convulsant action. In my Doctorate thesis, I used a mouse brain slice model of focal epilepsy in which the epileptogenic focus can be identified and the role of Pv interneurons in the generation and in the propagation of seizure-like events accurately analyzed by a combination of optogenetic, electrophysiological and imaging techniques. I observed that a selective rhythmic activation of Pv interneurons at the focus failed to prevent seizure generation, and it rather contributed to prolong seizure duration by synchronizing the afterdischarges of the clonic phase. Moreover, this pattern of Pv interneuron activation induced in pyramidal neurons a post-inhibitory rebound spiking that enhances neuronal synchrony and promotes seizure generation. In contrast, a selective activation of Pv interneurons distant from the focus blocked seizure propagation and shortened seizure duration at the focus. I then revealed that the reduced seizure duration at the focus was a direct consequence of the propagation block which probably prevented newly generated afterdischarges to travel backwards to the original focus of seizure initiation. The functional dichotomy of Pv interneurons described in the thesis opens new perspectives to our understanding of how local inhibitory circuits govern generation and spread of focal epileptiform activities. Given the wealth of evidence pointing at Pv interneurons as key players in epilepsy, their disfunction might also be at the basis of genetic epilepsies such as the severe myoclonic epilepsy of infancy (SMEI or Dravet syndrome), a rare epileptic encephalopathy characterized by an elevated mortality rate, early onset and seizures associated with elevated body temperature. SMEI is associated with a loss of function of the NaV 1.1 isoform of the sodium channel α−subunit. Recently, a mouse model of SMEI was generated by specific deletion of the SCN1A gene that codes for NaV 1.1. Importantly, in this model GABAergic interneurons, and in particular Pv interneurons, have been proposed to be specifically affected by the NaV 1.1 deletion, resulting in a reduced excitability of these cells that might leads to network disinhibition and possibly seizures. Therefore, in the framework of a broad characterization of synaptic and network activities in in vitro and in vivo models from NaV 1.1 heterozygous KO mice, I report here evidence that the propagation of focal epileptiform events in brain slices from NaV 1.1 KO mice is faster than in WT mice, hinting at a weakening of feedforward inhibition that restrains seizure spread, most likely deriving from Pv interneuron impaired excitability. Finally, as accumulating evidence supports a novel view of brain function where neurons intensively cooperate with astrocytes, I decided to investigate if a specific communication between GABAergic interneurons and astrocytes may exist. Indeed, neurotransmitters can elicit in astrocytes Ca2+ elevations that, in turn, trigger the release of neuroactive molecules, commonly known as gliotransmitters. However, whether and how GABA, the main inhibitory neurotransmitter, can evoke similar Ca2+ elevations in astrocytes and what is the functional outcome of these GABA-activated astrocytes are questions poorly addressed. These questions are particularly relevant in epilepsy, where we observe a massive GABAergic activity during seizure generation and propagation. To this aim, I found that in somatosensory and temporal cortex slices loaded with the Ca2+ indicator Fluo-4 AM, a large number of astrocytes in the layer 5 exhibited somatic Ca2+ rises in response to GABA or Baclofen (a specific GABAB receptor agonist). Patch-clamp recordings in the presence of TTX also revealed that GABAB activation triggered N-methyl-D-aspartate receptor- mediated slow inward currents (SICs) in principal neurons, suggesting that astrocytes in local circuits can convert an intense inhibitory input into a delayed excitatory output. These data support the presence of a dynamic, bidirectional signaling between GABAergic interneurons and astrocytes, which opens new perspectives for our understanding of the role of these different cell types in brain physiopathology.