A promising dreadded strategy: exploring the rol of astrocyte signalling on synaptic plasticity and refractory epilepsy

Temporal lobe epilepsy(TLE) is the most common, but also the most difficult-to-treat, type of epilepsy in adults. TLEi s characterized by the occurrence of focal seizures, originating in medial temporal structures, and is often associated with severe comorbidities, such as cognitive dysfunction and mood disorders. Additionally, TLE is frequently drug-resistant and in spite of continued endeavours to discover new treatments, the fraction of pharmacoresistant patients has stagnated over the last 30 years. In addition, the treatment of co-occurring psychiatric disorders is challenging and there is currently no therapy available for ameliorating cognitive dysfunction. Clearly, there is a strong urge for new therapies. Therefore, the focus in epilepsy research nowadays lies on the exploration of innovative strategies, such as targeting nonneuronal cells, and on finding a therapy for epilepsy-related comorbidities. In consonance with this angle, one of the main aims of this doctoral dissertation was to investigate the role of astrocytes on hippocampal synaptic plasticity, a molecular mechanism related to memory functioning. Furthermore, we evaluated if modulation of astrocytes could be a promising strategy in the treatment of intractable, focal seizures as well as for mood and memory comorbidities in a clinically relevant experimental model for TLE. For astrocyte modulation, we employed the state-of-the-art chemogenetic strategy or the so-called DREADDs(Designer Receptors Exclusively Activated by Designer Drugs). DREADDs are G protein-coupled receptors that are genetically engineered in such a way that they are only activated by a designer drug, i.e., clozapine-N-oxide (CNO) and not by their endogenous ligands. Astrocyte-specific expression of DREADDs was obtained via gene therapy, namely through adeno-associated viral (AAV) vector delivery of the DREADD construct under an astrocyte-specific promotor. In a first published manuscript (chapter III),we compared the effects of activation of the two most common DREADDs, Gq-and Gi-DREADD, expressed in astrocytes on intracellularCa2+transients as well as on synaptic plasticity in CA1 in ex vivo hippocampal slices. We found that both Gq-and Gi-DREADD agonism induced de novosynaptic potentiation, a mechanism known to be involved in memory consolidation, which was to date only reported for the activation of the Gq-DREADD. Interestingly, stimulation of Gq-DREADDs, but not Gi-DREADDs, induced long-lasting increases in intracellular Ca2+events. Thus, our study indicated that selective modulation of astrocytes leads to long-lasting alterations in the hippocampal network activity, which can be achieved via two distinct underlying pathways. Furthermore, with this study, we confirmed functionality of the DREADD approach in an ex vivo setting, paving the way for its use in vivo. In a second published manuscript(chapter V), we showed for the first time that the Barnes maze is a valid alternative to test spatial learning and memory and that particularly spatial learning is affected in the intrahippocampalkainic acid(IHKA) mouse model for pharmacoresistant TLE. To date, the impact of chemogenetic modulation of astrocytes has not yet been studied in chronic epilepsy. In chapter VI, we explored the efficacy of either the Gq-or Gi-DREADDs exclusively expressed in astrocytes of the seizure focus, to reduce spontaneous, focal seizures in addition to improving cognitive dysfunction and anhedonia in the IHKA model. We showed that approximately 24-hours after activation of either Gq-or Gi-DREADDs in astrocytes the seizure coverage was significantly reduced, which lasted for a 10-hour period. Unfortunately, continued, chronic CNO treatment did not result in chronic seizure suppression and after the10-hour-lasting Gq- or Gi-DREADD-mediated anticonvulsive effects, the seizure coverage returned to baseline. Moreover, daily CNO administrations in Gq-DREADD expressing epileptic mice for10 consecutive days eventually resulted in an aggravation of the seizure burden, i.e., prolonged seizures. Furthermore, investigation of spatial learning and memory demonstrated that Gq-DREADD activation in non-epileptic mice exhibited improved spatial learning, while their Gq-DREADD activated, epileptic counterparts did not. In contrast, spatial learning and memory was unaffected upon Gi-DREADD activation in non-epileptic mice, but long-term spatial memory deteriorated upon Gi-DREADD activation in epileptic mice. In addition, no significant DREADD-mediated alterations in anhedonia-like behaviour after chronic CNO treatment were observed in either epileptic or non-epileptic mice. In conclusion, selective modulation of astrocytes has shown to be a promising strategy for the treatment of intractable seizures in TLE. Via patch clamping we attempted to get a first insight into the astrocytic DREADD-mediated alterations in the epileptic and non-epileptic hippocampal network on dentate gyrus neurons, more than20 hours after activation, i.e., at the time of the anticonvulsive effect of the DREADDs. Nevertheless, the results obtained within the context of this dissertation call for further investigations in order to clarify the underlying mechanism(s)of the DREADD-mediated anticonvulsive actions. Indeed, too little is known about the impact of DREADD-mediated modulation of reactive astrocytes on the molecular signature, intracellular Ca2+signallingandsynaptic plasticity in the epileptic hippocampal network. Future experiments of astrocytic DREADD-mediated effects in epileptic conditions involving molecular profiling of astrocytes, ex vivo and in vivoCa2+imaging,slice electrophysiology as well as the exploration of different CNO dosing regimen, are required to reveal the true power of DREADD-mediated astrocyte modulation as a therapeutic strategy for treating pharmacoresistant TLE.