DREADDnoughts Join in the Battle for Seizure Control

Chemical-Genetic Attenuation of Focal Neocortical Seizures.

Kätzel D, Nicholson E, Schorge S, Walker MC, Kullmann DM. Nat Commun 2014;5:3847.

Focal epilepsy is commonly pharmacoresistant, and resective surgery is often contraindicated by proximity to eloquent cortex. Many patients have no effective treatment options. Gene therapy allows cell-type specific inhibition of neuronal excitability, but on-demand seizure suppression has only been achieved with optogenetics, which requires invasive light delivery. Here we test a combined chemical–genetic approach to achieve localized suppression of neuronal excitability in a seizure focus, using viral expression of the modified muscarinic receptor hM4Di. hM4Di has no effect in the absence of its selective, normally inactive and orally bioavailable agonist clozapine-N-oxide (CNO). Systemic administration of CNO suppresses focal seizures evoked by two different chemoconvulsants, pilocarpine and picrotoxin. CNO also has a robust anti-seizure effect in a chronic model of focal neocortical epilepsy. Chemical–genetic seizure attenuation holds promise as a novel approach to treat intractable focal epilepsy while minimizing disruption of normal circuit function in untransduced brain regions or in the absence of the specific ligand.

Commentary

Despite improvements in localizing the epileptogenic zone, many patients are not candidates for resective brain surgery to treat their focal epilepsy. Reasons include overlap of the epileptogenic zone with eloquent cortex, the presence of multiple epileptic foci, or the inability to localize the seizure onset zone. Thus, novel antiseizure therapies are needed for medically refractory patients, ideally strategies that work in an on-demand fashion. The responsive neurostimulator is one such approach that was recently approved for focal epilepsy (1). Optogenetic seizure suppression is another strategy with promising results in animal models (2). However, this technique requires both gene therapy and invasive devices for light delivery. Recently, a “chemical–genetic” method has been developed for pharmacological manipulation of activity in specific neural circuits (3). This method involves a modified receptor responsive to an exogenous ligand, leaving endogenous transmitter systems unaffected. A variation of this approach has been used in an epilepsy model (4), but Kätzel and colleagues are the first to apply this innovative paradigm to suppress seizures “on demand” in vivo.

The authors use the hM4Di receptor, a G protein-coupled receptor (GPCR) variant of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) (3). This is an inhibitory DREADD that suppresses neuronal activity. A mutation in the M₄ muscarinic acetylcholine receptor renders the channel almost completely insensitive to the endogenous ligand, acetylcholine, but highly sensitive to clozapine-N-oxide (CNO). Receptor activation suppresses action potentials by opening inwardly rectifying potassium channels (3, 5). It also acts on presynaptic muscarinic receptors to prevent neurotransmitter release (6 and the present study). The authors obtain spatial and cell type selectivity by focal injection of the DREADD into the M1 forelimb primary motor cortex of rats using an adeno-associated viral (AAV) vector containing a Ca²⁺/calmodulin-dependent protein kinase IIα (CamkIIα) promoter to drive hM4D_i expression in excitatory neurons. Using a hemagglutinin (HA)-tagged hM4D_i construct, they show that deep layer cortical pyramidal cells co-express CamKIIa and hM4D_i. Animals chronically expressing the gene (4–12 weeks) showed no behavioral abnormalities in the presence vs. absence of CNO during a motor coordination task.

The authors next asked whether selective silencing of hM4D_i receptor-expressing neurons inhibits seizures in an intracortical pilocarpine model. Pilocarpine injected into M1 generated clusters of discrete motor seizures with EEG showing up to 90 minutes of large amplitude, 0.5–2 Hz spike-wave discharges, simple (SS) and complex spike (CS) waveforms, and runs of 5–12 Hz small amplitude waves denoted as intermediate frequency (IF) discharges. These metrics and EEG power were used to measure the effectiveness of silencing infected neurons within M1. The IF discharges correlated with severe convulsive behavioral seizures, but behavioral seizures were not quantified. Immediately after the pilocarpine infusion, a single IP injection of CNO or vehicle was given. Compared to vehicle, CNO reduced the electrographic seizure duration, EEG power, and IF discharges. The CNO effect was quite rapid with onset within 10 minutes after chemoconvulsant infusion.

The GPCR DREADDs utilize the same downstream targets as the endogenous muscarinic receptor (i.e., cAMP and arrestin) and may act to specifically antagonize the downstream activity of pilocarpine. To address this potential confound, the authors repeated the experiment with a separate cohort of rats using the GABA_A receptor blocker picrotoxin injected into M1. Picrotoxin elicited both EEG and motor seizures, and CNO treatment reduced EEG power and IF activity, with a more modest effect on seizure duration compared to pilocarpine-induced seizures. The authors next evaluated the effect of CNO on behavioral seizures. CNO significantly reduced (by 39.5%) the number of convulsive seizures induced by picrotoxin, providing direct evidence that inhibitory DREADD receptor activation in neurons within the focus attenuates seizures. They also demonstrated that CNO had no effect on seizure severity in the absence of the hM4D_i receptor (but with a control vector injected), confirming that the observed reductions are a consequence of DREADD activity.

A critical question is whether hM4Di receptor activation suppresses spontaneous seizures in established epilepsy. The authors thus tested the effectiveness of CNO in reducing spontaneous epileptiform activity using the tetanus toxin model of focal neocortical epilepsy (7). Tetanus toxin and the hM4D_i vector were co-injected, presumably into the rat motor cortex, evoking epileptiform activity (<1 second discharges) beginning after 4 days and persisting for 8 weeks. During this period, rats averaging at least 2 epileptiform events/hour were treated with two IP injections of CNO or vehicle over 2 hours and monitored for epileptiform activity for 3.5 hours using frequency of bursts, coastline index, and high frequency power as measures. After 24 hours to allow CNO to clear, animals were switched from CNO to vehicle or vice versa and reassessed in a crossover design. CNO significantly reduced all three measures of epileptiform activity. Although interesting, the analysis involved only interictal epileptiform activity and did not directly address the authors’ question, that is, no evidence of an effect of DREADD activation on electrographic or behavioral seizures was provided.

Because muscarinic receptors can be located presynaptically, in the final experiment, the authors tested whether hM4D_i receptor activation inhibits excitatory synapses (presumably by reducing transmitter release). They injected the vector into the CA3 subfield of 4-week-old rats and made hippocampal slice recordings 10–11 weeks later. The authors found that in vitro CNO treatment of the slices decreased CA1 field excitatory post-synaptic potentials evoked by Schaffer collateral stimulation. Thus, activation of the hM4Di receptor appears to suppress neuronal activity through both synaptic and intrinsic mechanisms, consistent with recent findings by another group (6).

These experiments generate considerable optimism. First, AAV is effective in delivering hM4Di to a sufficient number of neurons to attenuate focal seizures. This occurred in the apparent absence of behavioral adverse effects of the DREADD. Second, the transient and reversible inhibition is temporally and spatially precise, providing an opportunity to advance studies of the specific neural circuits implicated in epileptogenesis. However, much work remains for therapeutic translation of this approach. Seizures were not completely suppressed after acute chemoconvulsant treatment in this study. Although not unexpected for focal status epilepticus, a critical question is whether hM4D_i receptor activation will suppress spontaneous seizures once animals become chronically epileptic. This key issue was not addressed. Another remaining question is the minimum level of inhibition necessary to inhibit seizures. For instance, it is not stated whether there was any variability in the antiseizure efficacy of DREADD activation related to the efficiency or spatial extent of infection. Such variability will undoubtedly differ based upon the involved networks and etiology of a given epilepsy. The question is critical not only for suppressing seizures but also in terms of potential negative consequences of DREADD activation in the setting of focal epilepsies arising from eloquent cortex, as suppressing normal activity may cause undesirable adverse effects. In this vein, more work is needed to determine how DREADD activation influences interictal behavior in animals with chronic epilepsy. Nonetheless, further studies of this novel chemical–genetic approach to prevent or ameliorate seizures offer great hope for eventual on-demand antiepileptic drug therapy.