Karen S. Wilcox

Discovery of antiepileptic drugs

SummarySince 1993, the Anticonvulsant Drug Development Program has contributed to the successful development of nine clinically effective drugs for the symptomatic treatment of epilepsy. These include felbamate (1993), gabapentin (1994), lamotrigine (1994), fosphenytoin (1996), topiramate (1996), tiagabine (1997), levetiracetam (1999), zonisamide (2000), and oxcarbazepine (2000). Despite the apparent success of the current discovery process, a significant need persists for more efficacious and less toxic antiepileptic drugs (AEDs). This is particularly true for patients whose seizures remain refractory to the currently available AEDs. This chapter will review the current process for AED discovery employed by the Anticonvulsant Drug Development Program at the University of Utah and other laboratories working toward the common goal of discovering better therapeutic options for patients living with epilepsy. It will discuss some of the inherent advantages and limitations of the primary animal models employed, while offering insight into potential future directions as we seek to better understand the pathophysiology underlying acquired epilepsy, therapy resistance, and epileptogenesis.

Calcium-Dependent Paired-Pulse Facilitation of Miniature EPSC Frequency Accompanies Depression of EPSCs at Hippocampal Synapses in Culture

Two forms of evoked neurotransmitter release at excitatory synapses between cultured hippocampal neurons have been described. After an action potential, it has been shown that transmitter initially is released synchronously, and this is followed by a period of “slow” asynchronous release. The “fast” synchronous component of release at these synapses has been found routinely to demonstrate paired-pulse and tetanic depression, whereas the short-term plasticity of asynchronous release has not been investigated. In the present experiments, we have used the whole-cell patch-clamp technique to record from pairs of neurons in a low-density hippocampal culture preparation to determine both the properties and underlying mechanisms of short-term plasticity of asynchronous release. It was found that an increase in miniature EPSC (mEPSC) frequency accompanied both single and multiple stimuli, and this mEPSC increase was facilitated during paired stimuli, even when the evoked synchronous release was depressed. In addition, both the activity-dependent depression of evoked EPSCs and facilitation of asynchronous mEPSC release were dependent on Ca accumulation in the nerve terminal. However, the Ca-dependent mechanisms underlying these two processes could be distinguished by the differential effects of two membrane-permeant calcium chelators, BAPTA-AM and EGTA-AM. Frequency-dependent depression of evoked EPSCs involves a rapid rise in intraterminal Ca, which likely triggers a process that proceeds in a Ca-independent manner, whereas the asynchronous release may be linked more directly to a sustained increase in intraterminal Ca.

Mouse models of human KCNQ2 and KCNQ3 mutations for benign familial neonatal convulsions show seizures and neuronal plasticity without synaptic reorganization

The childhood epilepsy syndrome of benign familial neonatal convulsions (BFNC) exhibits the remarkable feature of clinical remission within a few weeks of onset and a favourable prognosis, sparing cognitive abilities despite persistent expression of the mutant KCNQ2 or KCNQ3 potassium channels throughout adulthood. To better understand such dynamic neuroprotective plasticity within the developing brain, we introduced missense mutations that underlie human BFNC into the orthologous murine Kcnq2 (Kv7.2) and Kcnq3 (Kv7.3) genes. Mutant mice were examined for altered thresholds to induced seizures, spontaneous seizure characteristics, hippocampal histology, and M‐current properties of CA1 hippocampal pyramidal neurons. Adult Kcnq2A306T/+ and Kcnq3G311V/+ heterozygous knock‐in mice exhibited reduced thresholds to electrically induced seizures compared to wild‐type littermate mice. Both Kcnq2A306T/A306T and Kcnq3G311V/G311V homozygous mutant mice exhibited early onset spontaneous generalized tonic‐clonic seizures concurrent with a significant reduction in amplitude and increased deactivation kinetics of the neuronal M‐current. Mice had recurrent seizures into adulthood that triggered molecular plasticity including ectopic neuropeptide Y (NPY) expression in granule cells, but without hippocampal mossy fibre sprouting or neuronal loss. These novel knockin mice recapitulate proconvulsant features of the human disorder yet show that inherited M‐current defects spare granule cells from reactive changes in adult hippocampal networks. The absence of seizure‐induced pathology found in these epileptic mouse models parallels the benign neurodevelopmental cognitive profile exhibited by the majority of BFNC patients.

Innate but not adaptive immune responses contribute to behavioral seizures following viral infection.

Purpose:  To examine the role of innate immunity in a novel viral infection–induced seizure model.

Seizures following picornavirus infection

Purpose: We demonstrate the establishment and characterization of a novel virus infection‐induced seizure model in C57BL/6 mice.

A rat brain slice preparation for characterizing both thalamostriatal and corticostriatal afferents.

The striatum, the primary input nucleus of the basal ganglia, is crucially involved in motor and cognitive function and receives significant glutamate input from the cortex and thalamus. Increasing evidence suggests fundamental differences between these afferents, yet direct comparisons have been lacking. We describe a slice preparation that allows for direct comparison of the pharmacology and biophysics of these two pathways. Visualization of slices from animals previously injected with BDA into the parafascicular nucleus revealed the presence of axons of thalamic origin in the slice. These axons were especially well-preserved after traversing the reticular nucleus, the location chosen for stimulation of thalamostriatal afferents. Initial characterization of the two pathways revealed both non-NMDA and NMDA receptor-mediated currents at synapses from both afferents and convergence of the afferents in 51% of striatal efferent neurons. Annihilation of action potentials was not observed in collision experiments, nor was current spread from the site of stimulation to striatum found. Differences in short-term plasticity suggest that the probability of release differs for the two inputs. The present work thus provides a novel rat brain slice preparation in which the effects of selective stimulation of cortical versus thalamic afferents to striatum can be studied in the same preparation.

Interleukin-6, Produced by Resident Cells of the Central Nervous System and Infiltrating Cells, Contributes to the Development of Seizures following Viral Infection

ABSTRACT Cells that can participate in an innate immune response within the central nervous system (CNS) include infiltrating cells (polymorphonuclear leukocytes [PMNs], macrophages, and natural killer [NK] cells) and resident cells (microglia and sometimes astrocytes). The proinflammatory cytokine interleukin-6 (IL-6) is produced by all of these cells and has been implicated in the development of behavioral seizures in the Theilers murine encephalomyelitis virus (TMEV)-induced seizure model. The assessment, via PCR arrays, of the mRNA expression levels of a large number of chemokines (ligands and receptors) in TMEV-infected and mock-infected C57BL/6 mice both with and without seizures did not clearly demonstrate the involvement of PMNs, monocytes/macrophages, or NK cells in the development of seizures, possibly due to overlapping function of the chemokines. Additionally, C57BL/6 mice unable to recruit or depleted of infiltrating PMNs and NK cells had seizure rates comparable to those of controls following TMEV infection, and therefore PMNs and NK cells do not significantly contribute to seizure development. In contrast, C57BL/6 mice treated with minocycline, which affects monocytes/macrophages, microglial cells, and PMNs, had significantly fewer seizures than controls following TMEV infection, indicating monocytes/macrophages and resident microglial cells are important in seizure development. Irradiated bone marrow chimeric mice that were either IL-6-deficient mice reconstituted with wild-type bone marrow cells or wild-type mice reconstituted with IL-6-deficient bone marrow cells developed significantly fewer behavioral seizures following TMEV infection. Therefore, both resident CNS cells and infiltrating cells are necessary for seizure development.

Increased coupling and altered glutamate transport currents in astrocytes following kainic-acid-induced status epilepticus

Profound astrogliosis coincident with neuronal cell loss is universally described in human and animal models of temporal lobe epilepsy (TLE). In the kainic acid-induced status epilepticus (SE) model of TLE, astrocytes in the hippocampus become reactive soon after SE and before the onset of spontaneous seizures. To determine if astrocytes in the hippocampus exhibit changes in function soon after SE, we recorded from SR101-labeled astrocytes using the whole-cell patch technique in hippocampal brain slices prepared from control and kainic-acid-treated rats. Glutamate transporter-dependent currents were found to have significantly faster decay time kinetics and in addition, dye coupling between astrocytes was substantially increased. Consistent with an increase in dye coupling in reactive astrocytes, immunoblot experiments demonstrated a significant increase in both glial fibrillary acidic protein (GFAP) and connexin 43, a major gap junction protein expressed by astrocytes. In contrast to what has been observed in resected tissue from patients with refractory epilepsy, changes in potassium currents were not observed shortly after KA-induced SE. While many changes in neuronal function have been identified during the initial period of low seizure probability in this model of TLE, the present study contributes to the growing body of literature suggesting a role for astrocytes in the process of epileptogenesis.

The challenge and promise of anti-epileptic therapy development in animal models

Translation of successful target and compound validation studies into clinically effective therapies is a major challenge, with potential for costly clinical trial failures. This situation holds true for the epilepsies-complex diseases with different causes and symptoms. Although the availability of predictive animal models has led to the development of effective antiseizure therapies that are routinely used in clinical practice, showing that translation can be successful, several important unmet therapeutic needs still exist. Available treatments do not fully control seizures in a third of patients with epilepsy, and produce substantial side-effects. No treatment can prevent the development of epilepsy in at-risk patients or cure patients with epilepsy. And no specific treatment for epilepsy-associated comorbidities exists. To meet these demands, a redesign of translational approaches is urgently needed.

A Spontaneous Mutation Involving Kcnq2 (Kv7.2) Reduces M-Current Density and Spike Frequency Adaptation in Mouse CA1 Neurons

The M-type K+ current [IK(M)] activates in response to membrane depolarization and regulates neuronal excitability. Mutations in two subunits (KCNQ2 and KCNQ3; Kv7.2 and Kv7.3) that underlie the M-channel cause the human seizure disorder benign familial neonatal convulsions (BFNC), presumably by reducing IK(M) function. In mice, the Szt1 mutation, which deletes the genomic DNA encoding the KCNQ2 C terminus and all of CHRNA4 (nicotinic acetylcholine receptor α4 subunit) and ARFGAP-1 (GTPase-activating protein that inactivates ADP-ribosylation factor 1), reduces seizure threshold, and alters M-channel pharmacosensitivity. Genomic deletions affecting the C terminus of KCNQ2 have been identified in human families with BFNC, and truncation of the C terminus prevents proper KCNQ2/KCNQ3 channel assembly in Xenopus oocytes. We showed previously that Szt1 mice have a reduced baseline seizure threshold and altered sensitivity to drugs that act at the M-channel. Specifically, the proconvulsant M-channel blocker linopirdine and anticonvulsant enhancer retigabine display increased and decreased potency, respectively, in Szt1 mice. To investigate the effects of the Szt1 mutation on IK(M) function explicitly, perforated-patch electrophysiology was performed in CA1 pyramidal neurons of the hippocampus in brain slices prepared from C57BL/6J-Szt1/+ and control C57BL/6J+/+ mice. Our results show that Szt1 reduces both IK(M) amplitude and current density, inhibits spike frequency adaptation, and alters many aspects of M-channel pharmacology. This is the first evidence that a naturally occurring Kcnq2 mutation diminishes the amplitude and function of the native neuronal IK(M), resulting in significantly increased neuronal excitability. Finally, the changes in single-cell biophysical properties likely underlie the altered seizure threshold and pharmacosensitivity reported previously in Szt1 mice.