Suzanne Clark

Mechanism of Positive Allosteric Modulators Acting on AMPA Receptors

Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimers disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1,3-oxazino benzo-1,4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the “hinge” in the ligand-binding core “clamshell” that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.

Efficient unsupervised algorithms for the detection of seizures in continuous EEG recordings from rats after brain injury

Long-term EEG monitoring in chronically epileptic animals produces very large EEG data files which require efficient algorithms to differentiate interictal spikes and seizures from normal brain activity, noise, and, artifact. We compared four methods for seizure detection based on (1) EEG power as computed using amplitude squared (the power method), (2) the sum of the distances between consecutive data points (the coastline method), (3) automated spike frequency and duration detection (the spike frequency method), and (4) data range autocorrelation combined with spike frequency (the autocorrelation method). These methods were used to analyze a randomly selected test set of 13 days of continuous EEG data in which 75 seizures were imbedded. The EEG recordings were from eight different rats representing two different models of chronic epilepsy (five kainate-treated and three hypoxic-ischemic). The EEG power method had a positive predictive value (PPV, or true positives divided by the sum of true positives and false positives) of 18% and a sensitivity (true positives divided by the sum of true positives and false negatives) of 95%, the coastline method had a PPV of 78% and sensitivity of 99.59, the spike frequency method had a PPV of 78% and a sensitivity of 95%, and the autocorrelation method yielded a PPV of 96% and a sensitivity of 100%. It is possible to detect seizures automatically in a prolonged EEG recording using computationally efficient unsupervised algorithms. Both the quality of the EEG and the analysis method employed affect PPV and sensitivity.

EEG spike activity precedes epilepsy after kainate-induced status epilepticus

Purpose:  Chronic epilepsy frequently develops after brain injury, but prediction of which individual patient will develop spontaneous recurrent seizures (i.e., epilepsy) is not currently possible. Here, we use continuous radiotelemetric electroencephalography (EEG) and video monitoring along with automated computer detection of EEG spikes and seizures to test the hypothesis that EEG spikes precede and are correlated with subsequent spontaneous recurrent seizures.

N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ica-27243): A Novel, Selective Kcnq2/q3 Potassium Channel Activator

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are voltage-gated K+ channel subunits that underlie the neuronal M current. In humans, mutations in these genes lead to a rare form of neonatal epilepsy (Biervert et al., 1998; Singh et al., 1998), suggesting that KCNQ2/Q3 channels may be attractive targets for novel antiepileptic drugs. In the present study, we have identified the compound N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243) as a selective activator of the neuronal M current and KCNQ2/Q3 channels. In SH-SY5Y human neuroblastoma cells, ICA-27243 produced membrane potential hyperpolarization that could be prevented by coadministration with the M-current inhibitors 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE-991) and linopirdine. ICA-27243 enhanced both 86Rb+ efflux (EC50 = 0.2 μM) and whole-cell currents in Chinese hamster ovary cells stably expressing heteromultimeric KCNQ2/Q3 channels (EC50 = 0.4 μM). Activation of KCNQ2/Q3 channels was associated with a hyperpolarizing shift of the voltage dependence of channel activation (V½ shift of -19 mV at 10 μM). In contrast, ICA-27243 was less effective at activating KCNQ4 and KCNQ3/Q5 and was selective over a wide range of neurotransmitter receptors and ion channels such as voltage-dependent sodium channels and GABA-gated chloride channels. ICA-27243 (1-10 μM) was found to reversibly suppress seizure-like activity in an ex vivo hippocampal slice model of epilepsy and demonstrated in vivo anticonvulsant activity (ED50 = 8.4 mg/kg) in the mouse maximal electroshock epilepsy model. In conclusion, ICA-27243 represents the first member of a novel chemical class of selective KCNQ2/Q3 activators with anticonvulsant-like activity in experimental models of epilepsy.

Anticonvulsant effects of carbamazepine on spontaneous seizures in rats with kainate-induced epilepsy : Comparison of intraperitoneal injections with drug-in-food protocols

Purpose: The present study evaluated the effectiveness of intraperitoneal (IP) injections and oral administration of carbamazepine (CBZ) in food on the frequency of spontaneous motor seizures in rats with kainate‐induced epilepsy. The purpose was to develop a convenient drug‐in‐food approach for continuous, long‐term administration of potential antiepileptic drugs (AEDs).

Is There a “Critical Period” for Intervention in Posttraumatic Epilepsy?

Penetrating cortical trauma frequently results in delayed development of epilepsy. In the rat undercut model of neocortical posttraumatic hyperexcitability, suppression of neuronal activity by exposing the injured cortex to tetrodotoxin (TTX) in vivo for approximately 2 weeks prevents the expression of abnormal hypersynchronous discharges in neocortical slices. We examined the relation between neuronal activity during the latent period after trauma and subsequent expression of hyperexcitability by varying the timing of TTX treatment. Partially isolated islands of rat sensorimotor cortex were treated with Elvax polymer containing TTX to suppress cortical activity and slices obtained for in vitro experiments 10 to 15 days later. TTX treatment was either started immediately after injury and discontinued after a variable number of days or delayed for a variable time after the lesion was placed. Immediate treatment lasting only 2 to 3 days, and treatment delayed up to 3 days prevented hyperexcitability. Thus a critical period exists for development of hyperexcitability in this model that depends on cortical activity. We propose that the hyperexcitability caused by partial cortical isolation may represent an early stage of posttraumatic epileptogenesis. A hypothetical cascade of events leading to subsequent pathophysiologic activity is likely initiated at the time of injury but remains plastic during this critical period.