Potentiating α₂ subunit containing perisomatic GABA_A receptors protects against seizures in a mouse model of Dravet Syndrome

Abstract

Acknowledgements:This work was supported by NIH grants R01 NS084959 (J.A.K.) and R01MH099114 (A.C.) and a seed grant from the American Epilepsy Society (A.C.). We thank Nick Brandon from AstraZeneca for providing AZD7325.Abstract GABA A receptor potentiators are commonly used for the treatment of epilepsy, but it is not clear whether distinct GABA A receptor subtypes contribute to seizure activity, and whether targeting receptor subtypes will have disproportionate benefit over adverse effects. Here we demonstrate that the α 2 / α 3 selective positive allosteric modulator (PAM) AZD7325 preferentially potentiates hippocampal inhibitory responses at synapses proximal to the soma of CA1 neurons. The effect of AZD7325 on synaptic responses was more prominent in mice on the 129S6/SvEvTac background strain that has been demonstrated to be seizure resistant in the model of Dravet syndrome (Scn1a +/-) and in which the α 2 GABA A receptor subunits are higher relative to in the C57BL/6J strain. Consistent with this, treatment of mice with AZD7325 is associated with a higher temperature threshold for hyperthermia-induced seizures in Scn1a +/mice without apparent sedative effects. Our results in a model system indicate that selective targeting α 2 is a potential therapeutic option for Dravet syndrome. Significance StatementThe GABA A receptor is a target of several antiepileptic drugs (AEDs), but whether specific subtypes of GABA A receptors are critical for seizure suppression is not known. We demonstrated that the α 2 / α 3 selective positive allosteric modulator (PAM) AZD7325 potentiates hippocampal inhibitory synaptic responses in mice in a strain-dependent manner. The results are consistent with our previous findings that uncovered strain dependent differential expression of the α 2 subunit in seizure resistant and protective strains for Scn1a heterozygous deletion, a mouse model of Dravet syndrome. Treatment of mice with AZD7325 was associated with a higher temperature threshold for hyperthermia-induced seizures in Scn1a +/mice indicating α 2 is a potential therapeutic target in Dravet syndrome. Materials & Methods AnimalsAll experimental procedures were carried out in accordance with the policies and protocols approved by the Northwestern University IACUC. Three separate mouse strains were used in the series of experiments; C57BL/6J (Strain # 000664, The Jackson Laboratory), 129S6/SvEvTac (Taconic), and [C57BL/6J x 129S6/SvEvTac] F1 strains. Male Scn1a tm1Kea (Scn1a +/-) mice on the 129S6/SvEvTac background (129.Scn1a +/-)(Miller et al., 2014) were crossed with inbred female C57BL/6J or 129S6/SvEvTac mice to generate Scn1a +/mice with F1 (F1.Scn1a +/-) or 129S6/SvEvTac (129.Scn1a +/-) backgrounds, respectively. Both male and female Scn1a +/or wild-type littermates (Scn1a +/+ ) were used for experiments. Slice preparation for electrophysiologyHorizontal hippocampal slices (350 μm) were prepared from postnatal day 14 -16 (P14 -P16) mice as described previously (Fernandes et al., 2015;Nomura et al., 2016). Briefly, brain ...

Gabra2is a genetic modifier of Dravet syndrome in mice

“…Finally, modifier genes and pathways can provide novel targets for therapeutics. Previously, we used AZD7325, a GABAA α2/α3selective PAM, to modulate the neuronal phenotype of Scn1a +/mice and showed it had protective effects against hyperthermia-induced seizures (28). The current study confirmed that AZD7325 can be used to distinguish the GABAA receptor type in perisomatic synapses and directly demonstrated that the Edited allele contributes to an enriched α2 subunit content.…”

“…We previously demonstrated that perisomatic inhibitory synapses of hippocampal CA1 neurons had a greater abundance of α2-containing GABAA receptors in 129 mice compared to B6 mice [28(28). α2 GABAA receptor mediated currents can be distinguished by the use of the selective α2/α3 positive allosteric modulator (PAM) AZD7325, which has a larger effect on slowing the decay kinetics of inhibitory postsynaptic currents (IPSCs) enriched in α2 subunits (28). To determine whether the Edited allele affected synaptic GABAA receptors, we recorded perisomatic IPSCs in CA1 neurons in B6/B6 and Edited/B6 mice.…”

“…Recording electrodes had tip resistances of 3 -5 MΩ when filled with internal recording solution containing the following (in mM): 135 CsCl, 20 HEPES, 2 EGTA, 2 Mg-ATP, 0.5 Na-GTP, and 10 QX-314 (pH 7.25). Asynchronous IPSCs (aIPSCs) were recorded from perisomatic synapses where α2 GABAA receptor subunits are enriched (28,45). Perisomatic aIPSCs were recorded in voltage clamp mode (-70 mV) in the recording strontium based ACSF containing the following (in mM): 125 NaCl, 2.4 KCl, 1.2 NaH2PO4, 25 NaHCO3, 25 glucose, 6 SrCl2, 0.5 CaCl2, and 2 MgCl2 with blockers of excitatory responses, CNQX (10 µM) and D-APV (50 µM), equilibrated with 95% O2 and 5% CO2.…”

“…Perisomatic aIPSCs were recorded in voltage clamp mode (-70 mV) in the recording strontium based ACSF containing the following (in mM): 125 NaCl, 2.4 KCl, 1.2 NaH2PO4, 25 NaHCO3, 25 glucose, 6 SrCl2, 0.5 CaCl2, and 2 MgCl2 with blockers of excitatory responses, CNQX (10 µM) and D-APV (50 µM), equilibrated with 95% O2 and 5% CO2. Perisomatic aIPSCs were evoked by electrical stimuli given through monopolar extracellular stimulating electrodes filled with ACSF and placed in stratum pyramidale and were analyzed within a 50-500 ms window following the stimulus artifact (28,(45)(46)(47). Access resistance (Ra) was continuously monitored and experiments were omitted if the Ra changed > 20 % during the recordings.…”

Developing more effective seizure therapies requires more selective drugs

“…In this edition of The Journal of Physiology , Nomura et al . () explore the importance of α 2 subunit‐containing GABA A receptors in determining seizure severity in a mouse model of Dravet syndrome.…”