Examining the role of the M-type potassium current in epilepsy using mice that carry KCNQ2 and KCNQ3 gene alterations

Benign Familial Neonatal Convulsions (BFNC) is a human pediatric epilepsy characterized by seizure onset in the first few weeks of life and spontaneous resolution after about 2-4 months. It is considered benign because neuronal development and cognitive abilities are not adversely affected; however, BFNC patients have a 10-fold greater risk of developing adult onset epilepsy than the general public. BFNC is caused by mutations in the KCNQ2 and KCNQ3 genes, which encode the KCNQ2 and KCNQ3 subunits. These subunits underlie the M-type K+ channel (M channel), the outward K+ conductance of which generates the M current [Ik(m>] in neurons. Ik(m> is tonically active at resting membrane potential, and activates in response to membrane depolarization to repolarize the cell membrane. It thereby modulates resting membrane potential and regulates neuronal excitability. It has been assumed that mutations in the KCNQ2 and KCNQ3 genes that precipitate BFNC do so by decreasing Ik(m) function; however, no direct link between KCNQ2/KCNQ3 mutation and altered Ik(m) function has been established. The studies conducted in this dissertation were designed to better address this relationship. We hypothesize that mice carrying Kcnq2 and Kcnq3 gene mutations exhibit evidence of increased neuronal excitability. To test this hypothesis, we used three mouse models of Kcnq mutation to study whole-animal seizure thresholds and single-cell biophysical properties in CA1 hippocampal neurons. We have established that mice carrying Kcnq alterations indeed display reduced Ik(m) function, with corresponding hyperexcitability that is detectable at single-cell and whole-animal levels. We have also detailed several differences in M channel pharmacology that closely parallel differences in whole-animal pharmacosensitivity. The results presented in this dissertation are the first to detail reductions in native neuronal Ik(m) function, as well as increased single-cell neuroexcitability, that result from the expression of BFNC-causing mutations. These whole-animal behavioral and single-cell biophysical studies further confirm the link between attenuated Ik(m) function and increase seizure susceptibility that can result from Kcnq2 and Kcnq3 mutation.Kcnq3 mutation.

EXAMINING THE ROLE OF THE M-TYPE POTASSIUM CURRENT IN EPILEPSY USING MICE THAT CARRY KCNQ2 AND KCNQ3 GENE ALTERATIONS by James F. Otto A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacology Department of Pharmacology and Toxicology The University of Utah August 2006 Copyright © James F. Otto 2006 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by James F. Otto This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. H. Steve White Mark F. Leppert I 1 Michae1jt. Sanguinetti THE UNIVERSITY OF UTAH GRADUATE SCHOOL FIN AL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of James F. Otto in its final form and have found that (1) its format, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. Date Approved for the Major Department William Crow'tJ Chair/Dean Approved for the Graduate Council ABSTRACT Benign Familial Neonatal Convulsions (BFNC) is a human pediatric epilepsy characterized by seizure onset in the first few weeks of life and spontaneous resolution after about 2-4 months. It is considered benign because neuronal development and cognitive abilities are not adversely affected; however, BFNC patients have a IO-fold greater risk of developing adult onset epilepsy than the general public. BFNC is caused by mutations in the KCNQ2 and KCNQ3 genes, which encode the KCNQ2 and KCNQ3 subunits. These subunits underlie the M-type K + channel (M channel), the outward K + conductance of which generates the M current [IK(M)] in neurons. IK(M) is tonically active at resting membrane potential, and activates in response to membrane depolarization to repolari~e the cell membrane. It thereby modulates resting membrane potential and regulates neuronal excitability. It has been assumed that mutations in the KCNQ2 and KCNQ3 genes that precipitate BFNC do so by decreasing IK(M) function; however, no direct link between KCNQ21KCNQ3 mutation and altered IK(M) function has been established. The studies conducted in this dissertation were designed to better address this relationship. We hypothesize that mice carrying Kcnq2 and Kcnq3 gene mutations exhibit evidence of increased neuronal excitability. To test this hypothesis, we used three mouse models of Kcnq mutation to study whole-animal seizure thresholds and single-cell biophysical properties in CAl hippocampal neurons. We have established that mice carrying Kcnq alterations indeed display reduced IK(M) function, with corresponding hyperexcitability that is detectable at single-cell and whole-animal levels. We have also detailed several differences in M channel pharmacology that closely parallel differences in whole-animal pharmacosensi tivity. The results presented in this dissertation are the first to detail reductions in native neuronal IK(M) function, as well as increased single-cell neuroexcitability, that result from the expression of BFNC-causing mutations. These whole-animal behavioral and single­cell biophysical studies further confirm the link between attenuated IK(M) function and increase seizure susceptibility that can result from Kcnq2 and Kcnq3 mutation. v TABLE OF CONTENTS ABSTRACT ........................................................... iv LIST OF FIGURES ..................................................... viii LIST OF TABLES ....................................................... x ACKNOWLEDGEMENTS .............................................. xii CHAPTER 1. INTRODUCTION ..................................................... 1 Mutations in KCNQ2 and KCNQ3 Cause Epilepsy ...................... 2 KCNQ2, KCNQ3, and KCNQ5 Encode Subunits of the M Channel .......... 3 The Role of the M Current in Neuron Physiology ........................ 4 Pharmacological Modulation of the M Current .......................... 10 The Need for Genetic Models ofBFNC ............................... 12 References ...................................................... 16 2. MICE CARRYING THE SZTl MUTATION EXHIBIT INCREASED SEIZURE SUSCEPTIBILITY AND ALTERED SENSITIVITY TO COMPOUNDS ACTING AT THE M CHANNEL .......................... 21 Introduction ..................................................... 22 Materials and Methods ............................................. 23 Results ......................................................... 24 Discussion ...................................................... 27 References ...................................................... 28 3. A SPONTANEOUS MUTATION INVOLVING KCNQ2 REDUCES M CURRENT DENSITY AND SPIKE FREQUENCY ADAPTATION IN MOUSE CAl NEURONS ........................................... 30 Introduction ..................................................... 30 Methods ........................................................ 32 Results ......................................................... 35 Discussion ...................................................... 52 References ...................................................... 58 4. A SURVEY OF ELECTROCONVULSIVE THRESHOLDS IN MICE CARRYING KCNQ AMINO ACID EXCHANGE MUTATIONS KNOWN TO CAUSE HUMAN BFNC ................................... 62 Introduction ..................................................... 62 Methods ........................................................ 64 Results ......................................................... 72 Discussion ...................................................... 80 References ...................................................... 85 5. ALTERED BIOPHYSICAL PROPERTIES OF HIPPOCAMPAL CAl NEURONS IN MICE THAT CARRY Kcnq2 AND Kcnq3 KNOCK-IN POINT MUTATIONS IDENTICAL TO THOSE THAT CAUSE HUMAN BFNC .............................................. 87 Introduction ..................................................... 87 Methods ........................................................ 89 Results ......................................................... 92 Discussion ..................................................... 128 References ..................................................... 135 6. DISCUSSION ...................................................... 137 Aim and Significance ............................................ 137 Summary and Conclusions ........................................ 138 Speculations and Future Directions .................................. 140 References ..................................................... 152 Vll LIST OF FIGURES 1.1 Putative KCNQ2, KCNQ3, and KCNQ5 subunit arrangements that form the M-type K+ channel (M channel) ................................ 5 1.2 G protein-coupled receptor activation regulates M channel activity by a diffusible second messenger ....................................... 8 2.1 In all seizure models tested, Szti mice have significantly decreased seizure thresholds relative to B6 mice .................................. 25 2.2 In the minimal clonic seizure paradigm, Szt i mice display increased sensitivity to linopirdine ............................................. 26 2.3 In the partial psychomotor seizure paradigm, Szt i mice display decreased sensitivity to retigabine ..................................... 27 3.1 Szti reduces M current amplitude and current density in CAl pyramidal neurons of the hippocampus ................................. 38 3.2 Szti reduces the ability of CAl neurons to adapt spike frequency ............ 42 3.3 Szt i confers increased sensitivity to the IK(M) blocking properties ofLPD .......................................................... 45 3.4 Szti confers decreased sensitivity to the IK(M) enhancing properties ofRGB .......................................................... 47 3.5 Szti renders CAl neurons largely insensitive to the KCNQ2 subunit-preferring IK(M) blocker TEA ...................... . ........... 50 4.1 Constructs involved in generating mice carrying the Kcnq2 A306T knock-in mutation, and verification of A306T expression .................. 66 4.2 Constructs involved in generating mice carrying the Kcnq3 G31 OV knock-in mutation, and verification of G31 OV expression .................. 69 4.3 Convulsive current (CC) curves generated from electroconvulsive threshold (ECT) testing in female B6-Q2+/ - and littermate control B6+/+ mice ................................................. 73 5.1 IK(M) amplitude and density are decreased, and deactivation is accelerated in C571B16.129 CAl neurons carrying the Kcnq2 A306T mutation ................................................... 94 5.2 Spike Frequency Adaptation (SF A) is inhibited in C571B16.129 CAl neurons carrying the Kcnq2 A306T mutation ........................ 99 5.3 Processes involving repolarization following a single action potential are facilitated in C57/Bl6.l29 CAl neurons carrying the Kcnq2 A306T mutation ............................................. 102 5.4 IK(M) amplitude and density are decreased, and deactivation is accelerated in C571B16.129 CAl neurons carrying the Kcnq3 0310V mutation ................................................. 106 5.5 Spike Frequency Adaptation (SFA) is inhibited in C57/B16.129 CAl neurons carrying the Kcnq3 0310V mutation ....................... 112 5.6 Processes involving repolarization following a single action potential are facilitated in C571B16.129 CAl neurons carrying the Kcnq3 0310V mutation ............................................ 114 5.7 IK(M) amplitude and density are decreased, and deactivation is accelerated in FVBIN.129 CAl neurons carrying the Kcnq3 0310V mutation ................................................. 118 5.8 Spike Frequency Adaptation (SFA) is inhibited in FVBIN.129 CAl neurons carrying the Kcnq3 031 OV mutation ....................... 122 5.9 Processes involving repolarization following a single action potential are encumbered in FVBIN.l29 CAl neurons carrying the Kcnq3 0310V mutation ............................................ 126 6.1 Analysis of the effects of gender on IK(M) density in Kcnq2 A306T CAl neurons .............................................. 145 ix LIST OF TABLES 2.1 Convulsive current 50 (CCso) values and corresponding 95% confidence intervals (CI9s) for B6 and 8zt1 mice in three ECT testing paradigms .......... 25 2.2 Genotype- and seizure phenotype-dependent changes in sensitivity to M current modulators ............................................... 26 3.1 IK(M) amplitude and density values are reduced across a range of voltage steps in Szt 1 CA 1 neurons . . . . . . . . . . . . . . . . . . . . . . . . . . .. . ........ 37 3.2 Intrinsic electrophysiological properties of CA 1 neurons recorded in brain slices prepared from B6 and Szt 1 mice .......................... 40 4.1 Effects of Kcnq2 A306T and Kcnq3 G31 OV mutations on partial psychomotor (6 Hz) seizure threshold .................................. 75 4.2 Effects of Kcnq2 A306T and Kcnq3 G31 OV mutations on minimal clonic seizure threshold ............................................. 77 4.3 Effects of Kcnq2 A306T and Kcnq3 G31 OV mutations on minimal tonic hindlimb extension (THE) seizure threshold ........................ 78 4.4 Effects of Kcnq2 A306T and Kcnq3 G31 OV mutations on seizure thresholds grouped according to genotype .............................. 79 5.1 IK(M) amplitude and density values are reduced across a range of voltage steps in B6.129 CAl neurons homozygous for the Kcnq2 A306T mutation ............................................. 96 5.2 The Kcnq2 A306T mutation does not affect the passive membrane properties of B6.129 CA 1 neurons .................................... 98 5.3 Active membrane properties ofB6.129 CAl neurons carrying the Kcnq2 A306T mutation ............................................ 105 5.4 IK(M) amplitude and density values are reduced across a range of voltage steps in B6.129 CAl neurons homozygous for the Kcnq3 G310V mutation ............................................ 109 5.5 The Kcnq3 G310V mutation does not affect the passive membrane properties ofB6.129 CAl neurons ................................... 110 5.6 Active membrane properties ofB6.129 CAl neurons carrying the Kcnq3 G31 OV mutation ............................................ 117 5.7 IK(M) amplitude and density values are reduced across a range of voltage steps in FN.129 CAl neurons homozygous for the Kcnq3 G310V mutation ............................................ 121 5.8 The Kcnq3 G310V mutation does not affect the passive membrane properties of FN .129 CA 1 neurons ................................... 124 5.9 Active membrane properties ofFN.129 CAl neurons carrying the Kcnq3 G310V mutation ............................................ 129 6.1 Summary of seizure threshold and electrophysiology parameters affected by the Kcnq2 and Kcnq3 mutations studied ...................... 141 xi ACKNOWLEDGEMENTS This dissertation is dedicated to my family: to my parents, who instilled an appreciation for education at a young age; to Joey, Sue and Jeff, Mike, Lynn, and Tim, whose long talks and other diversions provided much-needed relief from the rigors of graduate school. Finally, I dedicate this dissertation to my husband, partner, and best friend - Greg. I could have never completed this without your continuous validation, love, and support through seemingly endless years of school. I am so lucky to have you. CHAPTER! INTRODUCTION Epilepsy affects at least 50 million people worldwide (Reynolds, 2002), with an annual cost of over 12 billion dollars in medical bills and lost earnings in the United States alone (Begley and Beghi, 2002). The disease state of epilepsy can have numerous causes, but the phenotype of all forms of epilepsy is characterized by repeated seizures, which are paroxysmal neuronal discharges of a hyperexcitable and hypersynchronous nature (Delgado-Escueta et aI., 1999). Seizures can be confined to a discrete focus within the brain (focal seizures), or can spread to other brain regions. Epilepsy is treated pharmacologically with anticonvulsant compounds aimed at altering neuronal bursting properties and decreasing neural circuit synchronization. These drugs are designed to tip the scales of excitability toward inhibition, which can be accomplished by way of various mechanisms. Specifically, the plasma membrane of neurons contains several types of ligand- and voltage-gated ion channels that regulate neuronal excitability and neurotransmission. Aberrant conductance through these ion channels can underlie seizure generation and epileptogenesis (Mody, 1998; Jensen, 1999). Moreover, the overwhelming majority of currently used anti epileptic drugs act at one or more voltage­and/ or ligand-gated ion channels to selectively decrease seizure-related neuronal excitability without affecting nonepileptic activity (Levy et aI., 2002; Rogawski and Loscher, 2004). 2 In recent years, the focus of epilepsy research has undergone a substantial shift toward understanding the genetics of the disorder. As a result, many epilepsies that were previously described as "idiopathic"-lacking a known cause-have now been determined to be caused by genetic mutations. Many of the mutations that cause epilepsy have since been identified as affecting ion channel genes, and often result in severe alterations in ion channel function, or channelopathies (Kaneko et aI., 2002). In recent years, many of the epilepsies previously characterized as "idiopathic" have now been re­categorized as "genetic channelopathy diseases" (Mulley et aI., 2003). Mutations in KCNQ2 and KCNQ3 Cause Epilepsy One of the familial epilepsies, Benign Familial Neonatal Convulsions (BFNC), is caused by mutations in the KCNQ2 or KCNQ3 genes (Biervert et aI., 1998; Singh et aI., 1998); to date, 29 mutations in KCNQ2 and 3 mutations in KCNQ3 have been identified. BFNC-causing mutations include amino acid exchange point mutations, usually contained in the P-loop between transmembrane segments S5 and S6, as well as large sequence insertions or deletions in the C-terminus of KCNQ2 (Singh et aI., 2003). Interestingly, all n1utations identified thus far precipitate a similar phenotype that is characteristic of BFNC. Seizures are generalized and tonic in nature, but often include clonus. Episodes start between postnatal day 2 and month 4, and continue for several weeks to months, after which point they spontaneously resolve (Steinlein, 2001). BFNC is considered benign partly because the patients (with only a few case study exceptions) exhibit normal psychomotor skills and learning ability; consequently, the prognosis is favorable. BFNC patients, however, go on to develop adult-onset epilepsy at a rate 10 times greater than that of the general population (Ronen et aI., 1993). Thus, BFNC 3 patients as a group display increased seizure susceptibility, and might therefore be considered an at-risk population. KCN02, KCN03, and KCN05 Encode Subunits of the M Channel The KCNQ2, KCNQ3, and KCNQ5 genes encode the KCNQ2, KCNQ3, and KNCQ5 subunits that comprise the M-type potassium channel (M channel) (Biervert et aI., 1998; Singh et aI., 1998; Wang et aI., 1998). The current that passes through the M channel is termed the M current [IK(M)], and is present in many neurons in the central nervous system (Marrion, 1997). The KCNQ2, KCNQ3, and KCNQ5 subunits have been shown to coassenlble in several tetrameric configurations in many different cell types, but all M channels formed possess characteristics similar to those of the native neuronal IK(M)' Most channel subunit expression studies to date have been conducted in the Xenopus laevis oocyte, Chinese hamster ovary (CHO) cell, and human embryonic kidney (HEK) cell expression systems. Such studies have established that while KCNQ2, KCNQ3, and KCNQ5 subunits can all form functional homomeric channels, the current amplitude through heteromeric channels is usually increased several fold. For example, in Xenopus oocytes, coexpression of KCNQ2 and KCNQ3 increases current amplitude by 10-fold relative to homomeric KCNQ2 and KCNQ3 channels (Wang et aI., 1998; Schwake et aI., 2000). KCNQ5 homomeric channels also form in Xenopus oocytes, but coexpression with KCNQ3 results in a 5-fold increase in current amplitude (Lerche et aI., 2000). There is also evidence that heteromeric channels containing all three subunits do form and are functional, and that the inclusion of KCNQ5 subunits downregulates the total number of KCNQ2/KCNQ3 heteromeric channels assembled within the same cell (Schroeder et aI., 2000). Heteromeric KCNQ2/KCNQ5 channels also form, but their 4 currents are similar in amplitude to those recorded in their respective homomeric channels. In the mammalian brain, Kcnq2, Kcnq3, and Kcnq5 rnRNA are coexpressed in many brain regions including the hippocampus, neocortex, and striatum (Schroeder et aI., 2000; Saganich et aI., 2001); however, the expression patterns of individual subunits do display subtle differences. In the thalamus, for example, KCNQ3 rnRNA expression is qualitatively greater than that of KCNQ2 (Saganich et aI., 2001). In hindbrain structures such as the cerebellum, it appears that only the KCNQ2 subunit rnRNA are appreciably expressed. It is assumed, therefore, that many different M channel subpopulations containing different subunits exist in the brain in various types of neurons, and in different stoichiometric arrangements (Schroeder et aI., 2000). Indeed, work by Edward Cooper's group at the University of Pennsylvania has established that in CAl pyramidal neurons, KCNQ2 subunits can be expressed with or without KCNQ3 or KCNQ5 (Devaux et aI., 2004). This finding is remarkable because it is often assumed that if a homomeric channel is expressed but produces an especially weak current in model systems (i.e., KCNQ2), then it must not play a significant role in neuron physiology. The discrete location of KCNQ2 at the nodes of Ranvier, for instance, observed by Devaux et al. suggests the opposite. Refer to Figure 1.1 for a list of putative M channel subunit configurations. The Role of the M Current in Neuron Physiology IK(M) was first described as an outward potassium current that is tonically active at resting membrane potential (-65 m V), does not inactivate, and is blocked in response to 5 Figure 1.1. Putative KCNQ2, KCNQ3, and KCNQ5 subunit arrangements that form the M-type K+ channel (M channel). A, Two-dimentional structures of the KCNQ subunits. Like most voltage-gated K+ channels, the M channel is presumed to be a heterotetrameric channeL Each subunit contains a voltage-sensor ( +++) within transmembrane region 4. B, Heterotetrameric subunit arrangements that are likely to form and be functional in neurons. Subunit configurations are only representative, as exact arrangements are not currently known. 6 A B KCNQ Subunits Homomeric Channels Q2 Q2 Q3 Q5 Q3 Heteromeric Cha.nnels Q2/Q3 Q3/Q5 Q5 Q2/Q5 Q2/Q3/Q5 7 muscarinic acetylcholine receptor activation (Brown and Adams, 1980; Constanti and Brown, 1981). The "M" current is thus named for its sensitivity to muscarine. IK(M) activates in response to membrane depolarization, repolarizing the membrane, and effectively resisting further depolarization. IK(M) is also the only outward current sustained in the range of action potential initiation (Marrion, 1997); moreover, it plays a major role in setting and maintaining the resting membrane potential of many neurons (Brown et aI., 1990). Kcnq2 and Kcnq3 mRNA are highly expressed in several areas pertinent to seizure generation and propogation, including the hippocampus, the neocortex, and striatum (Saganich et aI., 2001; Shah et aI., 2002). Specifically, in CAl pyramidal neurons of the hippocampus, KCNQ2 and KCNQ3 subunit proteins are largely coexpressed at the axon initial segment (AIS), where action potentials are generated (Devaux et aI., 2004). Indeed, IK(M) provides a mechanism by which cells regulate their own excitability; in response to a prolonged membrane depolarization (representing continuous stimulatory input), IK(M) repolarizes the membrane and attenuates action potential frequency (Goh and Pennefather, 1987; Castaldo et aI., 2002). Chemical block of the M channel slows membrane repolarization, delaying the decay phase of the action potential as well as fast afterdepolarization (Yue and Yaari, 2004). In addition, as previously mentioned, KCNQ2 is discretely and independently expressed at the nodes of Ranvier, presumably to regulate saltatory conduction down the axon (Devaux et aI., 2004). IK(M) is regulated by the activation of various G protein-coupled receptors (GPCR) and involves a diffusible intracellular signal, most likely Ca2 + (Selyanko et al., 1992; Marrion, 1993) (Figure 1.2). The KCNQ2 subunit contains binding residues for 8 Figure 1.2. G protein-coupled receptor activation regulates M channel activity by a diffusible second messenger. Upon binding of the appropriate ligand to the mACh, BK2, or 5HT 2/3 G protein-coupled receptor, Gq/11 protein activation cleaves P1P2 via PLCP activation, producing 1P3 and DAG. IP3 promotes the release of Ca2 +, which binds to CaM and inhibits M channel activity. The specific effects of p-adrenergic and opioid, 5HTl, or SP receptor activation causes changes in cAMP levels in opposing directions, and all produce mixed modulation of IK(M) in different systems (dotted lines). Lines with arrows indicate a stimulatory effect, while lines with bars indicate an inhibitory effect. M channel mACh, BK2 5HT2/3 ~-adrenergic /~ I .... 1 I ,," ,I / / l I I \ , .... .... ... ... ..... ..... , ~ ....... 1-,,', DAG + IP 3 '\ \ '\ ~ '\ PKC \ \ , , Ca2+ release .... .... ..... / ,. ".'" / / / I I I I .......... _----- ..... ' ------".,- --------------------- ,. ,. ,. opioid, 5HT1, SP ,. ,. ". ". ,. / / / J I I \0 10 calmodulin, which act as the sensor for free cytosolic Ca2 + (Gamper and Shapiro, 2003). While the precise mechanism remains a controversial topic, it is generally accepted that this modulation occurs through the Gq/ll family of G proteins. For example, activating the Gqll1-coupled muscarinic acetylcholine (mACh), bradykinin II (BK2), and serotonin II and III (5HT 2/3) receptors also inhibits IK(M) via the conversion of PIP2 to DAG and IP3, which stimulates the activation of PKC and Ca2 +, respectively (Mochidome et al., 2001). The IK(M) inhibition that results from Gq/ll protein activation can be reversed by lowering cytosolic Ca2+ levels (Beech et aI., 1991). IK{M) regulation via activation of the Gas and Gila protein subfamilies through ~ adrenergic and serotonin type I (5HT1), opioid, and substance P (SP) receptors, respectively, is not as well characterized. Activation of each of these receptors has produced conflicting results depending on the systems in which they were analyzed. For example, activating the Gila protein with SP inhibits IK(M) in bullfrog sympathetic neurons (Adams et aI., 1983), but Gi/a activation by somatostatin enhances IK{M) in hippocampal CA3 neurons (Tallent and Siggins, 1997). Pharmacological Modulation of the M Current It has been established that IK(M) plays a major role in neuron physiology, although the molecular identity of the M channel was only discovered relatively recently. To date, only a few pharmacological agents are currently known to directly act at the M channel and thus n10dify IK{M). Of specific interest is the enhancer, retigabine (RGB; D- 23129). Consistent with the idea that increasing IK(M) counteracts neuronal excitability, RGB has been found to effectively reduce or block seizure activity in a wide variety of animal epilepsy models (Dailey et al., 1995; Rostock et aI., 1996; Tober et aI., 1996). Retigabine (RGB) is structurally different from all currently used anticonvulsants, and 11 has a higher protective index than n1any of the commonly prescribed anticonvulsants (Rostock et al., 1996). Consistent with its ability to augment M-channel currents, RGB has been found to effectively hyperpolarize and reduce action potential generation in projection neurons located in layers II and III of the entorhinal cortex (Hetka et al., 1999). Although RGB prevents seizure spread and epileptogenesis, its anticonvulsant efficacy cannot be solely ascribed to IK(M) enhancement. Indeed, RGB was previously determined to enhance GABAergic neurotransmission, but the effects on postsynaptic GABAA receptor activity were not well understood until recently. Experiments in the hippocampal slice preparation suggested that RGB could increase synthesis of the inhibitory neurotransmitter y-aminobutyric acid (GABA) (Kapetanovic et aI., 1995). In cultured rat cortical neurons, RGB potentiated cr currents induced by subsaturating concentrations of exogenously applied GABA (Rundfeldt and Netzer, 2000). Work by our group determined the GABA system-related mechanism of RGB to be postsynaptic at the GABAA receptor (Otto et aI., 2002). In this study, monosynaptically connected cultured n10use cortical neurons were used to determine that RGB enhances the amplitude and decay kinetics of GAB A-mediated inhibitory postsynaptic currents (IPSCs). We analyzed the effects of RGB on spontaneous miniature IPSCs in the presence of TTX, and observed a similar trend. These results indicate that RGB acts as an allosteric modulator of the postsynaptic GABAA receptor. The M channel blocker linopirdine (LPD; DuP-996) was originally identified as a depolarization-dependent neurotransmitter release enhancer (Aiken et aI., 1996). LPD has been found to increase neuronal excitability and induce spontaneous epileptiform activity 12 in the brain slice preparation (Zhu et aI., 2000; Okada et aI., 2002), and is assumed to have proconvulsant activity in the whole-animal. At low doses, LPD subtly and preferentially increased cholinergic neurotransmission, and was therefore initially investigated as a cognitive and memory enhancer (Brioni et aI., 1993), It is apparent that IK(M) serves as a novel and potentially quite useful pharmacological target, not relegated only to epilepsy. IK(M) enhancement could in fact prove a useful strategy for the treatment of many pathologies involving neuronal circuits with slightly elevated excitability. These might include, but are certainly not limited to, neuropathic pain and anxiety. Conversely, slight IK(M) block might be used to help overcome deficits in cholinergic tone associated with diseases involving memory loss and decreased cognition, such as dementia and schizophrenia (Cork et aI., 1987). The Need for Genetic Models of BFNC Although the link between KCNQ2 and KCNQ3 gene mutation and human epilepsy is well-established, the effects of these mutations on native (neuronal) IK(M) function have not been investigated. Given the function of IK(M) in regulating neuroexcitability and the fact that blocking IK(M) is proconvulsant, it has been assumed that mutations in the KCNQ2 and KCNQ3 genes cause BFNC by decreasing IK(M) function (Castaldo et aI., 2002). Several groups are currently investigating the effects that BFNC-causing mutations have on IK(M) function. In Xenopus laevis oocytes, subunits expressing the Kcnq3 G310V and Kcnq2 A306T mutations known to cause BFNC in two families, reduce KCNQ2/KCNQ3 current amplitudes (Schroeder et aI., 1998). It should be noted, however, that results obtained in expression systems often do not agree with those obtained in the native system (DOff, 1993; Lewis et aI., 1997; Sivilotti et aI., 1997). 13 In a targeted knock-out study, heterozygous Kcnq2+1 - mice displayed increased sensitivity to the proconvulsant pentylenetetrazole, and made less KCNQ2 transcript with no change in KCNQ3 (Watanabe et aI., 2000). This study established an increase in seizure susceptibility associated with partial Kcnq2 loss, but did not attempt to quantify IK(M) function specifically. More recently, it was shown that conditional transgenic overexpression of a dominant-negative Kcnq2 G279S mutation reduces IK(M) amplitude and increases neuronal excitability in mice (Peters et a!., 2005). The Tet-Off system used to drive mutant Kcnq2 overexpression in this study, however, significantly interferes with the expression of both wild-type KCNQ2 and KCNQ3 subunits. Due to the pervasive alterations in M channel subunit expression levels, these results are presumably detached from any real disease pathology. For the reasons outlined above, there is clearly a need for better genetic mouse models of BFNC that more closely parallel actual mutations that precipitate the disease. A cleaner genetic model of BFNC would help us better understand the long-term consequences of altered IK(M) function in seizure susceptibility, as apposed to the short­term effects of acutely modifying IK(M) via drug application. In fact, the need for point mutation knock-in, rather than whole gene knock-out, models of epilepsy has been repeatedly cited at the annual meeting of the An1erican Epilepsy Society. Indeed, knock­in mice carrying only the precise mutations that cause BFNC would provide the ideal model system in which to examine IK(M) function and seizure susceptibility. Once such studies are established, these mice could be used to examine the putative changes in other ion channel systems that arise in response to altered IK(M). These results could help to better characterize, for instance, the changes that take place between the 14 phases of spontaneous remission in infants and adult-onset epilepsy observed in some BFNC patients. This relationship has only begun to be examined in detail. It has been shown that blocking IK(M) with linopridine (LPD) enhances depolarization-induced neurotransmission in the hippocampi of postnatal day (P)0-7 rats, but not of P14-28 rats (Okada et aI., 2003). In the same study, blocking GABAA receptors in hippocampi of PO- 7 mice inhibited neurotransmission. This is actually consistent with the well-documented developmental shift in the role of GABA from an excitatory to an inhibitory neurotransmitter in the perinatal and neonatal periods (Owens et aI., 1996). Therefore, it is possible that GABA system maturation underlies the spontaneous seizure resolution observed in BFNC patients. It has not been established whether mutations that cause BFNC, when expressed in normal physiological fashion, do in fact alter the native neuronal IK(M). We hypothesize that mice carrying Kcnq2 and Kcnq3 mutations exhibit evidence of decreased IK(M) function and increased neuroexcitability that can be determined by electroconvulsive threshold (ECT) testing as well as single-cell electrophysiology. To test this hypothesis, the experiments contained herein were conducted in several mouse models of Kcnq mutation. The first mouse model utilized was Szti, which carries a deletion mutation that starts at the genomic DNA encoding the KCNQ2 C-terminus. A related mutation was previously identified as the cause of BFNC in a Czech family (Pereira et aI., 2004). The results of the studies conducted in the Szti mouse describe the consequences of C­terminal deletion on whole-animal seizure threshold and pharmacosensitivity, as well as single-cell IK(M) function, pharmacology, and neuroexcitability. The second set of experiments were conducted in knock-in mice that were genetically engineered to carry 15 the exact amino acid exchange mutations that cause BFNC in two families: Kcnq2 A306T and Kcnq3 G310V (Singh et aI., 2003). The results from these studies describe the consequences of these point mutations on whole-animal seizure threshold, and single­cell IK(M) function and neuroexcitability. Understanding the role of IK(M) in BFNC is imperative for discovering better treatments for the disease, and due to the significant role of IK(M) in neurophysiology, will likely lead to advancements in anticonvulsant drug development. We predict that the mouse models of Kcnq2 mutation and BFNC will prove invaluable model systems in which to examine the effects of M channel subunit mutation on neuroexcitability and seizure threshold. 16 References Adams PR, Brown DA, Jones SW (1983) Substance P inhibits the M-current in bullfrog sympathetic neurones. Br J Pharmacol 79:330-333. Aiken SP, Zaczek R, Brown BS (1996) Pharmacology of the neurotransmitter release enhancer linopirdine (DuP 996), and insights into its mechanism of action. Adv PharmacoI35:349-384. Beech DJ, Bernheim L, Mathie A, Hille B (1991) Intracellular Ca2+ buffers disrupt muscarinic suppression of Ca2+ current and M current in rat sympathetic neurons. Proc Natl Acad Sci USA 88:652-656. Begley CE, Beghi E (2002) The economic cost of epilepsy: a review of the literature. Epilepsia 43 Suppl 4:3-9. Biervert C, Schroeder BC, Kubisch C, Berkovic SF, Propping P, Jentsch TJ, Steinlein OK (1998) A potassium channel mutation in neonatal human epilepsy. Science 279:403-406. Brioni JD, Curzon P, Buckley MJ, Americ SP, Decker MW (1993) Linopirdine (DuP996) facilitates the retention of avoidance training and improves performance of septal­lesioned rats in the water maze. Pharmacol Biochem Behav 44:37-43. Brown DA, Adams PR (1980) Muscarinic suppression of a novel voltage-sensitive K+ current in a vertebrate neurone. Nature 283:673-676. Brown DA, Gahwiler BH, Griffith WH, Halliwell JV (1990) Membrane currents in hippocampal neurons. Prog Brain Res 83:141-160. Castaldo P, del Giudice EM, Coppola G, Pascotto A, Annunziato L, Taglialatela M (2002) Benign familial neonatal convulsions caused by altered gating of KCNQ2/KCNQ3 potassium channels. J Neurosci 22: 1-6. Constanti A, Brown DA (1981) M-Currents in voltage-clamped mammalian sympathetic neurones. Neurosci Lett 24:289-294. Cork LC, Kitt CA, Struble RG, Griffin JW, Price DL (1987) Animal models of degenerative neurological disease. Prog Clin BioI Res 229:241-269. Dailey JW, Cheong JH, Ko KH, Adams-Curtis LE, Jobe PC (1995) Anticonvulsant properties of D-20443 in genetically epilepsy-prone rats: prediction of clinical response. Neurosci Lett 195:77-80. Delgado-Escueta A V, Wilson WA, Olsen RW, Porter RJ (1999) New waves of research in the epilepsies: crossing into the third millennium. Adv Neurol 79:3-58. 17 Devaux JJ, Kleopa KA, Cooper EC, Scherer SS (2004) KCNQ2 is a nodal K+ channel. J Neurosci 24: 1236-1244. Dorr RT (1993) Clinical properties of yeast-derived versus Escherichia coli-derived granulocyte-macrophage colony-stimulating factor. Clin Ther 15:19-29; discussion 18. Gamper N, Shapiro MS (2003) Calmodulin mediates Ca2+-dependent modulation of M­type K+ channels. J Gen PhysioI122:17-31. Goh JW, Pennefather PS (1987) Pharmacological and physiological properties of the after-hyperpolarization current of bullfrog ganglion neurones. J Physiol 394:315- 330. Hetka R, Rundfeldt C, Heinemann U, Schmitz D (1999) Retigabine strongly reduces repetitive firing in rat entorhinal cortex. Eur J PharmacoI386:165-171. Jensen FE (1999) Acute and chronic effects of seizures in the developing brain: experimental models. Epilepsia 40 Suppll:S51-58; discussion S64-56. Kaneko S, Iwasa H, Okada M (2002) Genetic identifiers of epilepsy. Epilepsia 43 Suppl 9:16-20. Kapetanovic 1M, Yonekawa WD, Kupferberg HJ (1995) The effects of D-23129, a new experimental anticonvulsant drug, on neurotransmitter amino acids in the rat hippocampus in vitro. Epilepsy Res 22:167-173. Lerche C, Scherer CR, Seebohm G, Derst C, Wei AD, Busch AE, Steinmeyer K (2000) Molecular cloning and functional expression of KCNQ5, a potassium channel subunit that may contribute to neuronal M-current diversity. J BioI Chern 275:22395-22400. Levy RH, Mattson R, Meldrum B, Perucca E (2002) Antiepileptic Drugs. In: Antiepileptic Drugs, Fifth Edition (Press R, ed). Lewis TM, Harkness PC, Sivilotti LG, Colquhoun D, Millar NS (1997) The ion channel properties of a rat recombinant neuronal nicotinic receptor are dependent on the host cell type. J Physiol 505 ( Pt 2):299-306. Marrion NV (1993) Selective reduction of one mode of M-channel gating by muscarine in sympathetic neurons. Neuron 11 :77 -84. Marrion NV (1997) Control of M-current. Annu Rev Physiol 59:483-504. 18 Mochidome T, Ishibashi H, Takahama K (2001) Bradykinin activates airway parasympathetic ganglion neurons by inhibiting M-currents. Neuroscience 105:785-791. Mody I (1998) Ion channels in epilepsy. Int Rev Neurobiol42: 199-226. Mulley JC, Scheffer IE, Petrou S, Berkovic SF (2003) Channelopathies as a genetic cause of epilepsy. Curr Opin NeuroI16:171-176. Okada M, Wada K, Kamata A, Murakami T, Zhu G, Kaneko S (2002) Impaired M­current and neuronal excitability. Epilepsia 43 Suppl 9:36-38. Okada M, Zhu G, Hirose S, Ito KI, Murakami T, Wakui M, Kaneko S (2003) Age­dependent modulation of hippocampal excitability by KCNQ-channels. Epilepsy Res 53:81-94. Otto JF, Kimball MM, Wilcox KS (2002) Effects of the anticonvulsant retigabine on cultured cortical neurons: changes in electroresponsive properties and synaptic transmission. Mol PharmacoI61:921-927. Owens DF, Boyce LH, Davis MB, Kriegstein AR (1996) Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated­patch recordings and calcium imaging. J Neurosci 16:6414-6423. Pereira S, Roll P, Krizova J, Gentoll P, Brazdil M, Kuba R, Cau P, Rektor I, Szepetowski P (2004) Complete Loss of the Cytoplasmic Carboxyl Terminus of the KCNQ2 Potassium Channel: A Novel Mutation in a Large Czech Pedigree with Benign Neonatal Convulsions or Other Epileptic Phenotypes. Epilepsia 45:384-390. Peters HC, Hu H, Pongs 0, Storm JF, Isbrandt D (2005) Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior. Nat Neurosci 8:51-60. Reynolds EH (2002) Introduction: epilepsy in the world. Epilepsia 43 Suppl6: 1-3. Rogawski MA, Loscher W (2004) The neurobiology of antiepileptic drugs. Nat Rev Neurosci 5:553-564. Ronen GM, Rosales TO, Connolly M, Anderson VE, Leppert M (1993) Seizure characteristics in chromosome 20 benign fan1ilial neonatal convulsions. Neurology 43: 1355-1360. Rostock A, Tober C, Rundfeldt C, Bartsch R, Engel J, Polymeropoulos EE, Kutscher B, Loscher W, Honack D, White HS, Wolf HH (1996) D-23129: a new anticonvulsant with a broad spectrum activity in animal models of epileptic seizures. Epilepsy Res 23:211-223. 19 Rundfeldt C, Netzer R (2000) Investigations into the mechanism of action of the new anticonvulsant retigabine. Interaction with GABAergic and glutamatergic neurotransmission and with voltage gated ion channels. Arzneimittelforschung 50:1063-1070. Saganich MJ, Machado E, Rudy B (2001) Differential expression of genes encoding subthreshold-operating voltage-gated K+ channels in brain. J Neurosci 21:4609- 4624. Schroeder BC, Kubisch C, Stein V, Jentsch TJ (1998) Moderate loss of function of cyclic-AMP-modulated KCNQ2/KCNQ3 K+ channels causes epilepsy. Nature 396:687-690. Schroeder BC, Hechenberger M, Weinreich F, Kubisch C, Jentsch TJ (2000) KCNQ5, a novel potassium channel broadly expressed in brain, mediates M-type currents. J BioI Chern 275:24089-24095. Schwake M, Pusch M, Kharkovets T, Jentsch TJ (2000) Surface expression and single channel properties of KCNQ2/KCNQ3, M-type K+ channels involved in epilepsy. J BioI Chern 275:13343-13348. Selyanko AA, Stansfeld CE, Brown DA (1992) Closure of potassium M-channels by muscarinic acetylcholine-receptor stimulants requires a diffusible messenger. Proc R Soc Lond B BioI Sci 250:119-125. Shah MM, Mistry M, Marsh SJ, Brown DA, Delnlas P (2002) Molecular correlates of the M-current in cultured rat hippocampal neurons. J Physiol 544:29-37. Singh NA, Westenskow P, Charlier C, Pappas C, Leslie J, Dillon J, Anderson VE, Sanguinetti MC, Leppert MF (2003) KCNQ2 and KCNQ3 potassium channel genes in benign familial neonatal convulsions: expansion of the functional and mutation spectrum. Brain 126:2726-2737. Singh NA, Charlier C, Stauffer D, DuPont BR, Leach RJ, Melis R, Ronen GM, Bjerre I, Quattlebaum T, Murphy JV, McHarg ML, Gagnon D, Rosales TO, Peiffer A, Anderson VE, Leppert M (1998) A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat Genet 18:25-29. Sivilotti LG, McNeil DK, Lewis TM, Nassar MA, Schoepfer R, Colquhoun D (1997) Recombinant nicotinic receptors, expressed in Xenopus oocytes, do not resemble native rat sympathetic ganglion receptors in single-channel behaviour. J Physiol 500 ( Pt 1): 123-138. Steinlein OK (2001) Genes and mutations in idiopathic epilepsy. Am J Med Genet 106:139-145. 20 Tallent MK, Siggins GR (1997) Somatostatin depresses excitatory but not inhibitory neurotransmission in rat CAl hippocampus. J Neurophysiol 78:3008-3018. Tober C, Rostock A, Rundfeldt C, Bartsch R (1996) D-23129: a potent anticonvulsant in the amygdala kindling model of complex partial seizures. Eur J Pharmacol 303: 163-169. Wang HS, Pan Z, Shi W, Brown BS, Wymore RS, Cohen IS, Dixon JE, McKinnon D (1998) KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science 282:1890-1893. Watanabe H, Nagata E, Kosakai A, Nakamura M, Yokoyama M, Tanaka K, Sasai H (2000) Disruption of the epilepsy KCNQ2 gene results in neural hyperexcitability. J Neurochem 75:28-33. Yue C, Yaari Y (2004) KCNQ/M channels control spike afterdepolarization and burst generation in hippocampal neurons. J Neurosci 24:4614-4624. Zhu G, Okada M, Murakami T, Kamata A, Kawata Y, Wada K, Kaneko S (2000) Dysfunction of M-channel enhances propagation of neuronal excitability in rat hippocampus monitored by multielectrode dish and microdialysis systems. Neurosci Lett 294:53-57. CHAPTER 2 MICE CARRYING THE SZTl MUTATION EXHIBIT INCREASED SEIZURE SUSCEPTIBILITY AND ALTERED SENSITIVITY TO COMPOUNDS ACTING AT THE M CHANNEL Otto JF, Yang Y, Frankel WN, Wilcox KS, White HS (2004) Mice carrying the Szti mutation exhibit increased seizure susceptibility and altered sensitivity to compounds acting at the M channel. Epilepsia 45: 1009-1016. Reprinted with permission from The International League Against Epilepsy and The American Epilepsy Society E)lilel'xia. 45(9): I()09-IOI6, 2004 Blackwell Publisilin!!, Inc. 2004 International Lcuguc Agninsl Epilepsy Laboratory Research Mice Carrying the Szti Mutation Exhibit Increased Seizure Susceptibility and Altered Sensitivity to Compounds Acting at the M-Channel *James F. Otto, tYan Yang, tWayne N. Frankel, *Karen S. Wilcox, and *H. Steve White * AllticOIw,IIsalll Drug Developmel/t Program. Depw1111ent (~r Phannac%gy and Toxicology, Ullil'ersily of Uwh, Salt Lake City, Utah: and tl11e j(Jck.w)Il Laboratory, Bar HarhOl; Maine, U.S.A. Summary: PUlpose: Mutations in the genes that encode sub­units of the M-lypc K+ channel (KCNQ2/KCNQ3) and nicotinic acetylcholine receptor (CHRNA4) cause epi lepsy in humans. The purpose of lhis study was to examine the effects of the Sr.f] mu­tation, which not only deletes most of the C-terminus of mouse KClIlJ2, but also renders the Clmra4 andAlj'gap-] genes hemizy­gous, on seizure susceptibility anti sensitivity to drugs lhal target thc M-type K+ channel. Methods: The proconvulsant effects of the M-channel blocker linopirdinc (LPn) and anticonvulsant effects of the M-channel enhancer retigabine (RGB) were assessed by electroconvulsive threshold (ECT) testing in C57BLl6J-Szt]/+ (S;::.I]) and liUer­mate control C57BLl6J+/+ (B6) mice. Thc effects of the 3':.11 mutation on minimal clonic, minimal tonic hindlimb extension, and partial psychomotor seizures were evaluated by varying stimulation intensity and frequency. Many causes for epilepsy have been identified, but in the majority of cases, the seizure etiology remains unre­solved. As a result, many forms of epilepsy are described as "idiopathic." Advances in epilepsy research have iden­tified a strong genetic component to many of these id­iopathic epilepsies, including the rare pediatric disorders benign familial neonatal convulsions (BFNC) and autoso­mal dominant nocturnal frontal lobe epilepsy (ADNFLE). BFNC, a generalized epilepsy, js caused by mutations in the KCNQ2 and KCNQ3 genes that underlie the neuronal M clln'enL [IK(M)l (1,2): and ADNFLE, a partial epilepsy, is caused by mutations in the CHRNA4 gene, which en­codes the 0'4 subunit of the nicotinic acetylcholine (nACh) Accepted April 25.2004. Address correspondence und reprint requests to Dr. H.S. White ut Anticonvulsanl Drug Development Progmm. DCpilrlment of Phul'm<lcol­ogy and Toxicology. University of Uwb, 20 S 2030 E, Room 408. Salt Luke CilY, UT 84112. U.S.A. E-mail: [e-mail]1u f009 ReslIlls: 3':.t1 mouse seizure thresholds were signitkanlly re­duced relative to B6 littermates in the minimal clonic, minimal tonic hindlimb extension, and partial psychomotor seizure mod­els. Mice were injected with LPD and RGB and subjected to ECT testing. Tn the minimal clonic seizure model, 8:;'11 mice were significantly more sensitive to LPD than were B6 mice Imedian effective dose (EDso) 3.4 ± I.J mg/kg ancl7.6 ± 1.0 mg/kg, respectively J; in the parlial psychomotor seizure model, Sztl mice were significantly less sensitive to RGB than were 86 mice (EDso = 11.6 1.4 mglkg and 3.4 ± J.3 mg/kg, respec­tively). COl/elusions: These results suggest that the Szt 1 mutation alters baseline seizure susceptibility and pharmacoscllsitiv­ity in a naturally occurring mouse model. Key Words: Seizure susceptibility-M currcnt-KCNQ2-CHRNA4- Electroconvulsive threshold. receptor (3). BFNC is characterized by seizures that start between postnatal day 2 and month 4 and continue for several weeks to months, after which point, they sponta­neously resolve (4). BFNC is considered benign, partly because patients exhibit normal psychomotor skills and learning ability; consequently, the prognosis is favorahle. Patients are, however, at a 15-times greater risk for devel­oping epilepsy as adults (5). ADNFLE is characterized by brief, violent, partial seizures of frontal lobe origin that occur during light sleep. The average age at onset is "" 10 years, and seizures oflen either remit or at least improve after puberty; however, as is the case with BFNC, patients have a higher incidence of epilepsy in adulthood (6). Thus these patients might be described as having long-term in­creased seizure susceptibility. Potassium ion t1ux through the voltage-gated M-type K + channel generates the neuronal M current IIK(fv1) J, which plays a critical role in setting and maintaining the 22 1010 .I. F. OTTO ET AL. resting membrane potential of many neurons, and thus helps establish baseline neuroexcitability (7). In animal models, agents that decrease IK(M) ampliLude are pro­convulsant (8), whereas IKfM) enhancers are anticonvul­sant (9, 10); therefore a putative link exists between de­creased IK(M) function and increased seizure susceptibility. In Xellopus oocytes, it has been shown that TK(M) ampli­tude is diminished by the inclusion of KCNQ2 or KCNQ3 M-channel subunits containing BFNC-causing mutations (11). It is therefore implied that mutations in the KCNQ2 and KCNQ3 genes precipitate IK(M) hypoful1ctionality in BFNC patients, the consequence of which is increased seizure susceptibility. Presynaptic nACh receptors containing the 0!4-subunit SUbtype also playa critical role in modulating neurotrans­mitterrelease (12,13) and neuroexcitability (14,15). In an­imal models, agents that increase conductance through the nACh receptor (e.g., nicotine) are proconvulsant (16), and agents that decrease nACh receptor activity (e.g., scopo­lamine, caramiphen) are anticonvulsant (17). An oocyte expression study of one ADNFLE-causing CHRNA4 mu­tation suggests that slich mutations increase the potency but decrease the efficacy of ACh (18). Thus it is not clear how CHRNA4 mutations directly increase neuroexcitabil­ity in ADNFLE. Previously. in efforts to characterize beller the roles of the KCNQ2 and CHNRA4 genes in epilepsy, sep­arate groups have generated mice with targeted dele­tions of the KCIlQ2 and Chma4 genes. Kcnq2-null mice die shortly after birth, and hemizygous mice exhibit decreased transcript expression and increased suscepti­bility to pentylenetetrazol (PTZ)-induced seizures (19). Chma4-11ull mice are viable and exhibit increased sensi­tivity to PTZ-induced seizures as well as elevated anxiety­like behavior (20). In related work, Yang et al. (2]) identified a 300-kb spontaneous deletion mutation, Szt I, which deletes the region of the Kcnq2 gene that encodes the majority of the KCNQ2 C-terminus, as well as the Chma4 (nACh-receptor 0!4 subunit) and AI/gap I [guano­sine triphosphatase (GTPase)-activating protein that inac­tivates adenosine diphosphate (ADP)-ribosylation factor I J genes. Mice heterozygous for the Szt] mutation were identified in a suhpopulation of stock C57BLl6J (B6) mice as the result of a large-scale electroconvulsive threshold (ECT) screen (21). Mice that are heterozygous for this deletion mutation exhibit a decreased seizure threshold. With the Szt] deletion encompassing this unique combi­nation of epilepsy-related genes, the Szll mOllse presents a model in which to study polygenic epilepsy in a naturally occurring system. ECT testing was chosen as a standard­ized acute seizure-threshold test to examine the effects of this mutation on seizure susceptibility and pharmacosen­sitivity in SZII mice. The aim of this study was to (a) ex­amine further the effects or the 52t] mutalion on seizure thresholds in several electroconvulsive paradigms, and Epill'ps;a, \.'r!J. 45, No. I). 2004 (b) characterize differences in pharmacosensitivity to pro­and anticonvulsant agents that act at the M-channel. MATERIALS AND METHODS Sztl and B6 mice Eight- to 12-week-old coisogenic male and female C57BL/6J-Sl.t 1/ + (Szt 1) mice () 5-25 g) and their C57BL/6J-B6+1+ (B6) Iittermates were obtained from a research colony at the Jackson Laboratory (Bar Harbor, Maine) and used for all ECT testing. Animals were al­lowed free access to food and water and were housed in a temperature- and light-controlled (12 h onl12 h off) envi­ronment. All animal care and experimental manipulations were approved by the Institutional Animal Care and Use Committee (IUCAC) of the University of Utah and are in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Drugs For all behavioral experiments involving drug injection, linopirdine (LPD) and retigabine (RGB) sllspensions were made in 0.5% methyl cellulose (MC) and sonicated for ) 2 min before i.p. injection. Drug solutions were freshly prepared on the day of each experiment. All chemicals were purchased from Sigma (ST. Louis, MO, U.S.A.) un­less otherwise noted. RGB was generously supplied by VIATRIS (Frankfurt, Gennany). Baseline seizure thresholds Baseline convulsive current (CC) curves were con­structed for mice of both genotypes. A drop of tetracaine (0.5%) was administered to each eye just before testing. Three different stimulation protocol s were used in an effort to differentiate the effects of IK(M} modulators on limbic (6 Hz), forebrain (minimal clonic), and hindbrain (mini­mal tonic hindlimb extension) seizure thresholds. Partial psychomotor seizure testing was conducted with a Grass S48 stimulator (6-Hz, 0.2-ms rectangular pulse width, 3-s duration, varying current intensities). The phenotype for these seizures is rhythmic face movements, forelimb clonus, dorsal neck flexion, rearing and falling, and/or transient gait wobblines5/ataxia (22). Minimal clonic and minimal tonic hindlimb extension (THE) seizure testing (60-Hz, 0.2-1115 sinusoidal current pulse, varying current intensities) was conducted with a stimulatorprevious)y de­scribed (23). Minimal clonic seizures are characterized by rhythmic face and forelimh clonus, rearing and falling, and ventral neck flexion. Minimal THE seizures are charac­terized by a tonic-clonic tlexion-extension sequence that stalts with tonic forelimb extension, followed by hindlimb flexion, and terminates in full THE (180 degrees to the torso) (24-26). The previously described staircase eSlimation proce­dure (27) was used to generate population seizure thresh­olds for each mouse genotype. Using these data, full CC 23 24 Sztl MOUSE SEIZURE THRESHOLDS 1011 curves were generated and statistical significance was de­termined using Probit analysis in the statistical program MINITAB (State College, PA, U.S.A.). CCI - 99 values also were calculated for each genotype and seizure type. Sep­arate CC curves were constructed for male and female mice. Behavioral effects of M -current modulation After establishing baseline thresholds for each seizure phenotype in both mouse genotypes, the effects of two compounds that directly modulate the M-channel (LPD, a blocker, and RGB, an enhancer) were tested. LPD and RGB were prepared in 0.5% MC on the day oftesting, such tharO.O I ml/gofbody weight was i.njecled. An drugs were administered by intraperitoneal (Lp.) injection. Mice were injected with either LPD or RGB (10 mg/kg) and tested for partial psychomotor or minimal clonic seizures 15 min after injection (the previously determined time-lo-peak er­fect for both drugs). Control mice received an equivalent volume of 0.5% Me. Mice received either LPD or RGB injections and were stimulated at the previously estab­lished CCIO and CC)o values, respectively. A subsequent study was conducted to test whether the proconvulsant ef­fects of LPD and the anticonvulsant effects of RGB could be mitigated when the two drugs were administered con­currently. For this study, both drugs were co-injected (10 mg/kg each), and B6 and Szf I mice were stimulated at their respective CCso values. Results obtained from this combination study were compared with those obtained from a sludy in which LPD and RG8 were studied sep­arately in hOlh seizure tests at the CCso value for each genotype. Groups of nine to 15 mice were used for this set of experiments. Linopirdine and retigabine dose-response curves Based on results from the initial LPD and RGB studies (see Table 2), full dose-response curves were constructed for RGB in the partial psychomotor model and for LPD in the minimal clonic model. For these experiments, RGB doses of 1.25,2.5,5.0, 10.0, 12.25, 15.0, and 20.0 mg/kg and LPD doses of 1.25, 2.5, 5.0, 7.5, 10.0, 15.0, and 20.0 mg/kg were used. Each successively lower dose was made by serial dilution. Groups of only four to six mice were used for most doses because of the scarcity of age-matched Sui and littermate control B6 mice available from the Jackson laboratory research colony. Toxicity The effect of high doses of LPD and/or RGB (2: 10 mg/kg total drug) on motor function was assessed by the rotarod test (28,29). Rotarod testing was conducted im­mediately before ECT testing by placing the mouse on a I-inch diameter rod, textured with small ridges to aid in gripping, rotating at 6 rpm. Motor function was considered impaired if the mouse fell off the rotarod 3 times during a I-min testing period. Data analysis For baseline seizure threshold estimates, seizure inci­dence was determined at several different stimulus inten­sities according to the staircase estimation procedure (27). Convulsive current (CC) curves were then constructed from these data by Probit analysis, and CCI - 99 values were calculated using Minitab 13 (State College, PA, U.S.A.). CC curves for Sztl mice were compared with those of wild-type B6 mice for each seizure type, and seizure thresholds were considered significantly different at p < 0.05. The effect of drug treatment on seizure incidence at a given CC value was determined for each genotype. Fisher's Exact test was used for within-genotype analy­sis of drug versus Me effects; one-tailed tests were used for the effects of LPD and RGB alone, and two-tailed tests were used for the effects of LPD/RGB coapplica­tion. The two-tailed Mantel-Haenszel X 2 test was used for between-genotype drug-effect comparisons. Significance was determined at p < 0.05. RGB and LPD dose-response curves for B6 and S2tl mice were constructed using Probit analysis and compared for genotype-dependent shifts in dose-response curves. Dose-response curves obtained in B6 and Sz! I mice were considered significanUy different at p < 0.05. RESULTS Sztl mutant mice display decreased seizure thresholds relative to wild-type B6 mice As reported previollsly (21), the Szl} mutation (when expressed on a B6 background) significantly decreases minimal clonic and minimal THE seizure thresholds. In the present study, CC curves were constructed for par­tial psychomotor seizures (Fig. I A), as well as minimal clonic (Fig. I B) and mini mal THE extension seizures (Fig. I C). The data presented here not only confirm, but also extend, the findings of Yang et al. by showing that S2f} mouse seizure thresholds are significantly lower than B6 mouse thresholds for all three seizure phenotypes. Cal­culated CCso values and corresponding 95% confidence intervals (CI9S ) are sUlllmarized in Table 1. Female B6 and Sztl mice disl)lay lowered seizure thresholds relative to male B6 and Sztl mice Previous studies demonstrated that the seizure threshold of female B6 mice is lower than that of their male counter­parts (30,31). Consistent with these findings, the seizure thresholds of both female B6 and S2f 1 mice were signif­icantly decreased relative to male B6 ancl Szt I mice in the minimal clonic and pm1ial psychomotor seizure Illod­els. For example, CC,o values obtained ill the partial psy­chomotor seizure test were as follows: 86 male, 22.4 ± 0.7; B6 female, 19.5 ± 0.7; S2tl male, 19,6 ± 0.5; Sztl fe­male, 17.7 ± 0.5. Furthermore, Sal decreased the partial EpilclJsia, "hI. 45. No. V, 20()4 25 1012 J. F OTTO ET AL. A Partial Psychomotor 1.0 0.8 ~ :N:l 0.6 'iii (/) 0.. 0.4 I or- 0.2 'p < 0.05 0.0 15 20 25 stimulus (mA) B Minimal Clonic 1.0 ...... ... ... ... - 86 0.8 ... " ~ , -- Szt1 '4 ~ 0.6 , 'iii , (/) , 0.. , 0.4 \ I \ or- , A' 0.2 , ... . *p < 0.01 , ... ...... 0.0 ... ------.-- 6 7 8 9 stimulus (mA) c Minimal THE 1.0 .. • ... \ - 86 \ 0.8 ~ ~ ' .... -- Szt1 ::l \ .~ 0.6 \ CD \ (/) \ 0.. 0.4 \ I \ or- \ A' 0.2 \ , 'p < 0.005 " ... ...... 0.0 A - - - - - - - - - - - .. 8 10 12 14 16 stimulus (mA) FIG. 1. In all seizure models tested, Szt1 mice have signifi­cantly decreased seizure thresholds relative to 86 mice. A-C: partial psychomotor, minimal clonic, and minimal tonic hindlimb extension (THE) seizure threshold curves generated in 86 (solid line) and Szt1 (dashed line) mice. Convulsive current (CC) curve data are expressed in terms of one-seizure probability (1-Pseizure)' Circles (e) and triangles (.t.) indicate individual data points used to construct CC curves in 86 and Szt1 mice, respectively. A leftward shift in the CC curve reflects a decrease in seizure threshold. Epilepsio, \;h/. 45. No.9. 2(){)4 TABLE 1. Convlllsive cllrrellt 50 (CC50) values and corresponding 95% confidel1ce intervals (CI95 ) for 86 and Szt I mice ill three ECT testing paradigms Seizure type B6 Sal Partial psychomotor 22.4 19.6<1 (20.l:i-24.0) ( 18.4-20.8) Minimal clonic 7.8 6.7(1 (7.4-8.2) (6.2-7.2) Minimal THE 12.7 9.6" (11.8-13.6) (9.0--10.3) S:,r I mice exhibit significanl1y reduced seizure thresholds relative to B6 mice in all three seizure types tested. "p < 0.05. psychomotor seizure CC50 values of both male and female mice by 2.8 rnA, SztJ mice exhibit altered responsiveness to drugs acting at the M-channel Minimal clonic seizures Consistent with its known anticonvulsant profile, ROB (10 mg/kg) significantly decreased seizure susceptibility to ECT testing in both Szt/ and jittermale control mice (Table 2), When tested at the CCy(j, nine of 10 B6 mice treated with MC displayed a minimal clonic seiZllre versus four of 10 treated with ROB; eight of 10 5zt} mice treated with MC seized versus three of 10 treated with ROB. Conversely, the lK(M) blocker LPD (10 mg/kg) increased seizure susceptibility in both groups. When tesled at the CCI(}, two of 15 B6 mice treated with MC versus six of to treated with LPD displayed a minimal clonic seizure, whereas one of 10 52t} mice treated with MC seized versus 19 of20 treated with LPD. Interestingly, a between-strains statistical comparison of these data re­vealed that LPD was significantly more proconvulsant in 52tl mice than in B6 mice (Mantel-Haenszel X2 test, p < 0.05). Furthermore, not only did a higher propOltion of LPD-treated 5zt} mice display mini mal clonic seizures compared with B6 (19 of 20 vs. six of 10, respectively), but also J 2 of the 19 Szt} mice that displayed a minimal clonic seizure progressed directly to the more severe min­imal THE seizure. This seiZllre spread was not observed in any of the LPD-lrealed B6 mice that exhibited minimal clonic seizures. When mice were co-injected with LPD/ROB (10 mg/kg each) and subjected to ECT testing, another notable genotype-specific difference arose (Table 2): whereas co­injected B6 mice showed no difference in seizure inci­dence compared with vehicle control (12 of 25 vs. 12 of 22, respectively), co-injected Szti mice seized at a signifi­cantly higher rate compared with vehicle control (13 of 15 vs. seven of 15, respectively). The seizure rate observed 26 Szti MOUSE SEIZURE THRESHOLDS 1013 TABLE 2. Cellofype- lind seizure phenotype-dependelll changes ill sel1sifivifJ' to M current modulators Partial Minimal psychomotor Slim. intensity clonic seizure seizure treatment B6 S;,;ll B6 SZli (lCCX): MC 9/10 811 0 9111 7/9 RGB 4110b 3110b 2111/1 7/9f! cCCIO: MC 2/15 1110 III 0 1110 LPD 6/10/) 19/20b<, 6/1(Y' 711l'f "Cso: MC 12/22 7115 6/11 5/9 LPD+RGB !2/25 1.1/15b(' 2111 7/9" .lCC5(): MC 5110 4/10 5111 5/9 LPD 8/10 1011 oJ' 10111" W9b gCCS!): MC 7/12 6112 5111 5/9 RGB 2/12" 2/12 O/Il" 5/9" Data are reported as number or mice seizing/number of mice tested. Note that for groups treated with vehicle contwlmcthyl cellulose (MC), the fraction l1f mice seizing approximates each predetermined CC value. RGB, retigabinc; LPD, linopirdine. "Mice treated with RGB were tested at the previously determined CC')o value for each seizure type "p < 0.05 (within-genotype drug elreel, Fisher's Exal.:t le"I), "Mice treated with LPD were tested at the CC 10 value for each seizure type "Mice treated with LPD/RGB were tested at the CC50 value for I.:ach seizure type ('p 0.05 (between-genotypes drug effect, Muntel-Haenszel X2 lest). r·gThc effects of LPD and RGB were tested independently ut the CC5!1 vulues for both seizure Iypes, Data included in this table were acquired from female mice. Some data from male mice show similar trends in phanllueoresponsivcncss (data not shown). in co-injected Szt/ mice was comparable to that observed when Szl/ mice were treated with LPD only (13 of 15 vs. 19 of 20, respectively). Partial psychomotor seizures With a protocol identical to the one outlined in the pre­vious section, the etfects of LPD, RGB, and LPD/RGB were examined in the partial psychomotor seizure model (Table 2). When tested althe CC9()' RGB (10 mg/kg) sig­nificantly decreased the seizure incidence in B6 mice (nine of I I for MC vs. two of I 1 for RGB) but had no effect in Sr.t/ mice (seven of Ilille for MC vs. seven of nine for RGB). When tested at the CClO, LPD increased the seizure incidence in both B6 (one of 10 for MC vs. six of 10 for LPD) and Sz/ / mice (one of 10 for MC vs. seven of 101'01' LPD). Unlike the results obtained in the minimal clonic seizure test, no notable genotype-dependent difference in LPD sensitivity was noted in the psychomotor seizure test. When mice were co-injected with LPD/RGB and sub­jected to ECT testing, seizure incidence was decreased in B6 mice (six of 11 for MC vs. two of 11 for LPD/ROB), but slightly increased in S':,t/ mice (five of nine in MC VS. seven of nine in LPD/RGB). Although neither change was signiticant (Fisher's Exact test), the Muntel-Haenszel X2 statistic revealed that LPD/RGB co-injection was sig­nificantly more proconvulsant in Szt/ mice than in B6 mice. Taken together, the data obtained in the minimal clonic and partial psychomotor seizure tests reveal both genotype-dependent and seizure type-dependent differ­ences in pharmacosensitivity. Several strains of mice display a propensity for respi­ratory an'est after THE seizures, and death may follow if the mice are not artificially resuscitated (32). Although 86 mice rarely underwent respiratory arrest after mini­mal THE extension seizures, Szll mice usually did (data not shown), and this condition was always lethaJ if mice were not resuscitated. With successive breedings, it be­came virtually impossible to resuscitate Sztl mice after minimal THE seizures. The observed high mortality rate in response to THE seizures, compounded with the general paucity of Szt / and Iittermate control B6 mice, precluded pharmacology experiments in thc minimal THE seizurc model. LPD and RGB EDso values are significantly shifted in Sztl mice The seizurc type-dcpendcnt differences in pharma­coscnsitivity presented in Table 2 indicated the need for complete LPD and RGB dose-response curves. LPD curves were constructed to test the hypothesis that in the minimal clonic seizure model, LPD is a more potent pro­convulsant in Szt/ mice (Fig. 2). Similarly, ROB curves were constructed to test the hypothesis that in the partial psychomotor seizure model, ROB is a less potent anticon­vulsant in Szti mice (Fig. 3). The resulting data support this hypothesis and demonstrate that in the minimal clonic 1.0 -.. ...... • ... " -B6 OJ 0.8 , ..... c .. -- Szt1 'N '\ '03 , (/) 0,6 '".. C \ 0 , :g 0.4 '~ ~ , .. 0.2 .... .. .. 'p < 0.05 .."... ...... . 0.0 Jt:: ...... ____ o 10 20 LPD dose (mg/kg) FIG. 2. In the minimal clonic seizure paradigm, Szt1 mice display increased sensitivity to linopirdine (LPD). LPD dose­response data are expressed in terms of i-fraction of mice seizing (i-fraction seizing). Circles (.) and triangles ( .... ) indicate individual data points used to construct dose-response curves in B6 (solid line) and Szf1 (dashed line) mice, respectively. The leftward shift in the curve induced by the Szf1 mutation reflects increased LPD potency. Epilep.I'ifl. vbl. 45. No.9. 2004 27 1014 J. F. OTTO ET AL. 1.0 OJ 0.8 c: 'N 'm 0.6 (/J c: 'f05 0.4 ~ 0.2 .p < 0.005 0.0 ", " " - 86 '\J. \ -- Szt1 \ \ \ \ \ \ \ \ J. '\ ~\ " '" ..... - o 10 20 RGB dose (mg/kg) FIG. 3. In the partial psychomotor seizure paradigm, Szt1 mice display decreased sensitivity to retigabine (RG8). RG8 dose­response data are expressed in terms of fraction of mice seizing (fraction seizing). Circles (e) and triangles (j,) indicate individual data points used to construct dose-response curves in 86 (solid line) and Szt1 (dashed line) mice, respectively. The rightward shift in the curve induced by the Szt1 mutation reflects decreased RG8 potency. seizure model. Sztl mice are more sensitive to the procon­vulsant effect of LPD than are B6 mice (ED50s: 3.4 ± J.l mg/kg vs. 7.6 ± 1.0 mg/kg, respectively). Likewise, in the partial psychomotor seizure model. Szt 1 mice are less sensitive to the anticonvulsant effects of ROB than are B6 mice (ED50s: 11.6 ± 1.4 mg/kg vs. 3.4 ± 1.3 mg/kg, respectively). Motor impairment, as determined by the ro­tarod test, was observed in only three of nine Szt 1 mice in response to 20 mg/kg ROB injection. None orthe B6 mice tested at the highest ROB dose (i.e., 12.5 mg/kg) exhibited impairment. DISCUSSION Previously it was demonstrated that the Szt 1 mutation lowers the seizure threshold of B6 mice (21). The experi­ments presented here were designed to characterize further the seizure threshold shift induced by the Szt I mutation and assess changes in seizure susceptibility nfter treat­ment with proconvulsant and anticonvulsant drugs. The results obtained from this study support the hypothesis that a deletion mutation affecting a combination of genes implicated in epilepsy alters both seizure susceptibility and pharmacosensitivity. Specifically, mice with a muta­tion in Kcnq2 and hemizygous deletion of Chma4 and Ar./iJap I do display reduced seizure thresholds and altered sensitivity to the M-channel-modifying drugs LPD and ROB. In addition, the changes in drug sensitivity are de­pendent on the seizure type elicited. The results presented here demonstrate that Szt I mice are more sensitive to the proconvulsant properties of LPD in the minimal clonic model, and less sensitive to the anticonvulsant properties of ROB in the partial psychomotor model, than are B6 mice. EJlilepsia. \f){. 45, No.9, 20()4 The seizure type-dependent differences in responsive­ness to M-channel modulators may be explained in part by the fact that the partial psychomotor and minimal clonic seizure types result from activation of different brain ar­eas. For example, clos expression studies have demon­strated that the stimulus required to elicit psychomotor seizures (at threshold levels) strongly activates the neo­cortex (22), whereas the stimulus required to induce min­imal clonic seizures activates both forebrain and midbrain structures. Kcnq2 and Chrna4 transcripts are abundant in several brain regions that are relevant to epiJeptogenesis, including the cerebral cortex, thalamus, and hippocampus (20,33). However, it has not yet been determined if and where KCNQ2 subunits carrying the Szt/ mutation (and thereby Chrna4 hemizygosity) are expressed. Further ex­periments would prove useful in this regard. The results from the LPD/ROB co-injection experi­ments are particularly interesting, as they suggest that Szt 1 mice are more susceptible to seizure spread from fore­brain/ limbic areas to the hindbrain. For example, in Sztl mice, the effects of LPD prevail over those of ROB in the minimal clonic seizure model; although a higher fraction of LPD/ROB co-injected Szt1 mice seized vs. MC-injected SuI mice, none progressed to minimal THE seizures. Re­callihat when Szt I mice were treated with LPD alone and tested for minimal clonic seizures, 12 of the 19lhat seized progressed directly to minimal THE seizures. The results from the LPD/ROB coadministration studies suggest that in B6 mice, the anticollvulsant effects of ROB attenuate the proconvulsant effects of LPD, whereas in Sztl mice, the effects of LPD predominate over those of ROB. The stimuli required toelidt minimal clonic and minimal THE seizures are similar, except that minimal THE seizures re­quire greater current intensity. This finding suggests that although ROB does not protect against minimal clonic seizures in Szt I mice in the presence of LPD, it may pre­vent seizure spread from limbic/forebrain structures to the hindbrain. Considering that KCNQ2 C-terminal deletion muta­tions are among the mutations identified in BFNC pa­tients (34), it is not surprising that mice carrying the Szt I mutation exhibit decreased seizure thresholds. Although CHRNA4 hemizygosity is not implicated in ADNFLE pa­tients, previous mouse studies suggest that Chma4 hem­izygosity most likely contributes to the reduced seizure threshold of the Szt 1 motlse. In contrast to Kcnq2 and Chrna4, the consequences of a decrease in Arfgapl ex­pression have not been characterized. Whereas it has been shown that in the presence of OTP, Arfgap I promotes vesicle formation and cargo sorting in the Oolgi complex (35), the role of Arfgap I in regulating synaptic vesicle formation has not been established. Whereas we can conclude that the S2t I mutation de­creases baseline seizure threshold, we cannol determine whether all of the affected genes contribute to the change 28 Szt I MOUSE SEIZURE THRESHOLDS 1015 in seizure susceptibility. It is noteworthy, however, that the Jackson Laboratory has identitied two new mouse geno­types lhat carry only Kcnq2 point mutations and exhibit decreases in seizure threshold that are at least as severe as those reported in the Szt/ mouse (Yang and Frankel, un­published results). These preliminary results suggest that Kcnq2 mutations alone may be sufficient to reduce elec­troshock seizure thresholds. The results from this study serve to illustrate the SZli mouse's altered sensitivity to IKIM) modifying drugs, but the allercd pharmacosensi­tivity observed in this mouse is by no means limited to M-channel-modifying drugs. Further experiments to es­tablish the etfects of compounds acting at the nACh re­ceptor and compounds that target other epilepsy-reJated receptors and ion channels would prove useful in estab­lishing the Szli mouse as a model of increased seizure susceptibi lity. Although spontaneous seizures have not yet been reported, the S·(',t I mouse does provide a natu­rally occurring hyperexcitable model. system with which to investigate several issues pertinent to epilepsy research, including epileptogenesis, seizure susceptibility, the "sec­ond hit" hypothesis, and disease-modification strategies (36). Finally, electrophysiology experiments, in either ex­pression systems or in vitro preparations, could be used to differentiate the possible roles of the individual gene mutations/deletions in this hyperexcitable mouse. Acknowledgment: We thank Drs. Harold Wolf and Misty Smith-Yockman for encouragement and critical reading, as well as Barbara Beycr for gcnoLyping and Carolync Dunbar for IlHlin­taining the mouse breeding colony at the Jackson Laboratory. This work was supported by NS-40246 (W.N.F. and H.S.W.), NS3134H (W.N.F.), Primal), Children's Medical Center Foun­dation 51001142 (K.S.W.), and a TJL postdoctoral fellowship (Y.Y.). REFERENCES I. Singh NA. Charlicr C, Staufrer D. cl al. A novel potassiulll channel gcne. KCNQ2, is Illutatcd in un inhcrited epilepsy of newborns. Nat Gel1el 1998; 18:25-9. 2. Bil.:rvert C, Schroeder BC, Kubisch C, et al. A potassium chan­nel mutation in lIeonatal hUIll,ll1 epilepsy. Sch'lIee 1998;279:403- 6. 3. Steinlein O. Detection of a Cral polymorphism within exon 5 of the humall neuronal nicotinic acetylcholine receptor alpha 4 suhunit gene (CHRNA4). HI/III Gellet 1995;96: 130. 4. Steinlein OK. Genes and mutations in idiopathic epilepsy. Am.l Med GCllet 200 I; I 06: 139-45. 5. Ronen GM, Rosales TO, Connolly M, et al. Seizure characteristics in chromosome 20 benign familial neonatal convulsions. NeulV/oRY 1993;43: 1355-60. 6. Steinlcin O. Schuster V, Fischer C, et a!. Benign familial neonu­tal convulsions: wnlirll1ation or genetic heterogeneity and fur­ther evidence ror it second tOClIS on chromosome Bt(. HI/m Geller 1995;95:411-5. 7. Brown DA, Gahwiler B}-l. Griffith WH, etal. Membrane currents in hippocnmpal neurons. Prog HI'l/;1I Res 1990;83:141-60. 8. Flagllleycr I, Gchert 1. van del' Staay Fl. General pharmacology of the putative cognition enhalleer linopirdine. Arzlleil1lifte/jiJrsc/IUllg 1995;45:45~9. 9. Dost R, Rundrcldt C. The anticonvulsant retigahinc potenlly suppresses epilept.iform discharges in the low Cu++ and low Mg++ model in the hippocampal slice preparation. Epi/ep.l'Y Res 2000;38:53-66. 10. Armand V. Rundfeldt C, Heinemann U. Effects of retigabine (D-23129) on different patterns of epileptiform activity in­duced by 4-aminopyridinc in !'at entorhinal eortex hippocam­pal slices. Naunyn Schmiedebergs Arch Phanllacoi 1999;359:33- 9. I I. Schroeder BC, Kubiseb C. Stei n V, et aL Moderate loss of function of cyc.lic-AMP-moduluted KCNQ2/KCNQ3 K+ channels causes epilepsy. Nallm: 1998;396:687-90. 12. Gioanni Y, Rougeot C, Clarke PB, ot al. Nicotinic receptors in the rat prefrontal cortex: increase in glutamale releuse and facilita­tion of mediodorsal thalamo-cortical transmission. Eur J Nelll'o:wi 1999; II: 18-30. 13. McGehee DS, Heath MJ, Gelber S, et a!. Nicotine enhancement or fasl excitatory synaptic transmission in CNS by presynaptic recep­tors. Science 1995 ;269: 1692-6. 14. Alkonlion M, Pereira EF, Eisenberg HM, et al. Nicotinic receptor activation in human cerebral cortical interneurons: a mechanism far inhibition and disinhibition of neuronal networks . .I Ncumsci 2000;20:66-75. 15. Porter JT. Cauli B, Tsuzuki K, et al. Selective excitation of sub­types of neocortical inlerneurons by nicotinic receptors. J Nel/rosei 1999; 19:5228-35. 16. Dal11[lj MI, Glassco W, Dukat M, et al. Pharmacological characteri­zation of' nicotine-induced seizures in mice . .I Pharmacal Exp Ther 1999;291: 1284-91. 17. Raveh L, Brandeis R, Gilat E, el al. Anticholinergic llnd tlntiglutamatergic agents protect against soman-induced hrain damuge and cognitive dysfunction. 'foxicol Sci 2(X)3;75: 108- 16. 18. Lenige!' T, Kananura C, Hufnagel A, Cl a1. A new Chr1l(l4 mutation with low pc net ranee in nocturnal fronHtl lobe epilepsy. E.'pilepsia 2003 :44 :981-5. 19. Watanabe H, Naguta E, KoslIkui A. et a!. Disruption orllle epilepsy KCNQ2 gene results in neural hyperexcitability . .I Neuroehem 2000;75:28-33. 20. Ross SA. Wong Jy, Clifford]1, et al. Phenotypic charactcriwtion of an alpha 4 neuronal nicotinic acetylcholine receplor subunit knock­out mOllse. J Neul'Osci 2000;20:6431-41. 21. Yang Y, Beyer BJ, Otto IE et <II. Spontaneous deletion of epilepsy gene arthologs ill a mutant mouse with n low electroconvulsive threshold. Hunt Mol Genet 2003; J 2:975- 84. 22. Barton ME, Klein BD, Wolf HH, et a1. Pharmacological character­ization of the 6 Hz psychomotor seizure model of pm1iaJ epilepsy. EIJilepsy Res 2001;47:217-27. 23. Woodbury L, Davenpoli V. Design and use of a new eleclroshock seizure apparatus, and analysis of factors altering seizure thresh­old and paltern. Arch lilt Plwrtlw('odynlJlll 77,er 1952;92:97- 107. 24. Peterson S. Electroshock. Tn: Peterson TL, ed. Neuropharmacology met/lOlly ill epilep.\·y research. Boca Raton, FL: CRC Press, 1998: 1- 26. 25. Toman J. Neuropharmacologic considerations in psychic seizures. Neurology 1951; 1 :444-60. 26. Wh.ite HS, Woodhead JH, Wilcox KS, et a1. General principles: dis­covery and preclinical development of untiepileptie drugs. In Levy H R et aI., cds.Allfiepile]Jlic drugs. Philadelphia: Li ppincoll Williams & Wilkins, 1998:36-48. 17. Finney D. Pmbit lIIwlysi.l': A statistiml treatmell1 (~t' rite sig­moid response (,11!'l'e. Ctllnhridge: Cambridge University Press, 1971. 28. White HS, Patel S, Meldrum BS. Anticonvulsunt profile of MDL 27,266: an orally active, broad-spectrum antil:onvulsmH agenl. Epilepsy Res 1992: 12:217-26. 29. Dunham M, Miya T. A note on a simple apparatus ror detect­ing neurologicul deficit in rats and mice. J Alii P/tarlll(lcol AssoL' 1957;46:208-9. 30. Frankel WN. Taylor L, Beyer 13, et al. Electroconvubive thresholds of inhred mouse strains. Genomics 200 I ;74:306-12. Epilepsia. \l.-JI. 45, No.9, 2004 29 1016 1. F. OTTO ET AL. 31. Ferraro TN, Golden GT, Snyder R, el al. Genelie inllu­ences Oil electrical seizure thre"hold. Brain Res 1998;813:207- 10. 32. Blumer J, Simpson JM, Lucas SV, et 411. Toxicogcnics of niridazole in inbred mice . .1 Plwrll1£1cul E\p Ther 1980;212:509-13. 33. Sagunich MJ, Machado E. Rudy B. Dirrerential expression of genes encoding subthreshold-operating voltage-gated K+ channels in hrain. J Neumsci 200 I ;21 :4609-24. 34. Pereira S, Roll p. Krizova J, et al. Complete loss of the cyto- El'i/ejJ:iia, "b/. 45. No.9, 200.J plasmic carboxyl terminus of the KCNQ2 potassium channel: a novel mLltation in a large Czech pedigree with benign neonatal convulsions or other epileptic phenotypes. Epilepsill 2004;45:384- 90. 35. Yang IS, Lee SY, Gao M. et al. ARFGAP I promotes the fonmtioll of COPI vesicles. suggesting function as a component of the coat. J Cell Biol2002; 159:69-78. 36. Walker Me, White HS, Sandel' .IW. Disease lTlodilication in partial epilepsy. Bmill 2002; 125: 1937-50. CHAPTER 3 A SPONTANEOUS MUTATION INVOLVING KCNQ2 REDUCES M CURRENT DENSITY AND SPIKE FREQUENCY ADAPTATION IN MOUSE CAl NEURONS Introduction The M current [IK(M)] is a slowly activating voltage-gated K+ channel that is tonically active at resting membrane potential and activates more strongly at depolarizing potentials (Brown and Adams, 1980; Constanti and Brown, 1981). IK(M) activation repolarizes the cell and regulates neuronal excitability by controlling the generation and frequency of action potentials (Marrion, 1997). Accordingly, direct M-channel blockers and enhancers facilitate and inhibit action potential generation, respectively (Aiken et aI., 1995; Otto et al., 2002). In relation to epilepsy, the M-channel blocker linopirdine is proconvulsant while the enhancer retigabine is anticonvulsant (Flagmeyer et aI., 1995; Rostock et aI., 1996; Dost and Rundfeldt, 2000; Otto et aI., 2004), and KCNQ2 and KCNQ3 gene mutations cause Benign Familial Neonatal Convulsions (BFNC), an idiopathic generalized human epilepsy (Biervert et aI., 1998; Singh et aI., 1998). Consistent with the role of IK(M) and the fact that mutations in KCNQ2 and KCNQ3 subunits cause epilepsy, mutant KCNQ2/KCNQ3 channels expressed in Xenopus oocytes display decreased current amplitude (Schroeder et aI., 1998). Studies were recently conducted in mice conditionally overexpressing the Kcnq2 G279S mutation 31 (Peters et aI., 2005). IK(M) amplitude was reduced and neuronal excitability was increased in these mice; however, the acute mutant Kcnq2 overexpression was found to significantly alter the expression of wild-type KCNQ2 and KCNQ3. Thus, the effects of a naturally-occurring Kcnq mutation on the native neuronal IK(M) have still not been characterized. To this end, Yang and colleagues identified a spontaneous deletion mutation in mice, Szt1, which deletes the genomic region encoding most of the KCNQ2 subunit C­terminus, as well as the Chrna4 (nicotinic acetylcholine receptor U4 subunit) and Arfgap1 (GTPase-activating protein that inactivates ADP-ribosylation factor 1) genes (Yang et aI., 2003). In a phenotype similar to that of a previous Kcnq2 knock-out model (Watanabe et aI., 2000), SztllSzt1 mice die of lung atelectasis shortly after birth. Szt11+ mice are viable and display decreased seizure threshold and altered sensitivity to drugs that modify the M-channel (Otto et aI., 2004), but the specific contribution of the Kcnq2 component of this deletion has not been characterized. Since the hippocampus is heavily implicated in epilepsy, and Kcnq2, Kcnq3, and Kcnq5 mRNA are highly expressed in the CAl pyramidal cell layer (Shah et aI., 2002), we sought to characterize IK(M) function in CAl hippocampal neurons of Szt11+ (Szt1) and wild-type mice using the perforated patch electrophysiology technique in the acute brain slice preparation. The experiments presented here were designed to test the hypothesis that Szt1 alters baseline IK(M) function and pharmacology. We conclude that CAl neurons in Szti mice exhibit decreased IK(M) amplitude and current density compared to that of wild-type B6 littermates. Moreover, action potential accommodation is compromised in Szti CAl neurons. These results are the first to show that a naturally-occurring Kcnq2 mutation 32 attenuates native IK(M) amplitude, and consequently increases neuronal excitability. We also detail several differences in IK(M) pharmacology in Sztl CAl neurons, including increased sensitivity to LPD and decreased sensitivity to ROB. In addition, Sztl CAl neurons are largely insensitive to tetraethylammonium (TEA), a blocker of KCNQ2 subunit-containing M-channels. These results shed significant light on the consequences of M-channel mutation as it relates to neuroexcitability and seizure generation, and thus accentuate IK(M) as a therapeutic target for the treatment of epilepsy. Methods Sztl and B6 Mice Eight- to twelve-week-old coisogenic male C57BL/6J-Sztll+ (Sztl) mice (15-25 g) and their C57BL/6J-B6+/+ (B6) littermates, obtained fron1 a research colony at the Jackson Laboratory (Bar Harbor, Maine), were used for all electrophysiology experiments. Animals were allowed free access to food and water and were housed in a temperature- and light-controlled (12 hr onl12 hr off) environment. All animal care and experimental manipulations were approved by the Institutional Animal Care and Use Committee (IUCAC) of the University of Utah, and are in accordance with the National Institutes of Health Ouide for the Care and Use of Laboratory Animals. Drugs For electrophysiology experiments, stock solutions of ROB (0.01-0.10 M) were made in 50% DMSO on the day of the experiment and added to the brain slice perfusion Ringer (see below). The working concentration ofDMSO was kept below 0.01%. Stocks ofLPD (0.01-0.10 M) were made in 10% HCI and frozen. Aliquots ofLPD were thawed 33 and added to perfusion Ringer the day of each experiment. Tetraethylammonium (TEA) was added directly to perfusion Ringer. For drug applications, slices were perfused with drug-containing Ringer for at total of 30-60 min, until full effect was achieved and remained constant for at least 10 min. All chemicals were purchased from Sigma except RGB, which was generously supplied by VIATRIS (Frankfurt, Germany). Acute Brain Slice Preparation Brain slices were prepared from B6 and Szti mice In a manner similar to previously described methods (Barton et aI., 2004). Briefly, mice were anesthetized with sodium pentobarbital (25 mg/kg), decapitated, and their brains quickly removed and placed in oxygenated Ringer solution containing (in mM): 200 sucrose, 26 NaHC03, 10 glucose, 3 KCl, 2 MgS04, 2 CaCh, 1.4 NaH2P04. Brains were trimmed, mounted on a chuck, and 350 Ilm thick coronal slices were cut using a Vibratome slicer (Vibratome). Slices were then transferred to a holding chamber and allowed to incubate for> 1 hr in oxygenated Ringer similar to the above solution, but with 126 mM NaCl in place of sucrose. The NaCl Ringer pH was maintained at 7.37-7.40 with NaOH and continuous bubbling with 95% 0 2/5% CO2, and mOsm was 305-310. Electrophysiological Measurements Whole-cell perforated patch recordings were obtained from CAl pyramidal neurons in the acute brain slice preparation using a MultiClamp 700A amplifier (Axon Instruments). Signals in voltage-clamp and current-clamp modes were acquired at 10 KHz and 20 KHz, and filtered at 2 KHz and 10 KHz, respectively, for offline analysis using Clampfit 8. Glass capillaries (World Precision Instruments, Inc.) were pulled to 34 2.0-3.2 MQ resistances using a micropipette electrode puller (Sutter Instrument Co.). Input and series resistance values of 80-120 MQ and <15 MQ, respectively, were used as selection criteria for accepting recordings. Capacitance compensation and bridge balance functions were used for voltage-clamp and current-clamp experiments, respectively. Amphoterecin B (0.45-0.5 mg/mL) was dissolved in the intracellular solution containing (in mM): 140 potassium gluconate, 10 HEPES, 10 KCl, and 0.2 MgCb (PH adjusted to 7.28 with KOH; mOsm = 290). The external NaCl Ringer solution was supplemented with picrotoxin (50 ~M) and NBQX (10 ~lM) to block GABAA receptor- and non-NMDA receptor-mediated responses, respectively. The flow rate of all perfusing solutions was fairly rapid at 75-125 mLIhr. Data Analysis IK(M) amplitude was measured as the relaxation current in response to a voltage step protocol from -20mV to return potentials of -50mV, -60mV, and -70mV. IK(M) density was calculated as IK(M) amplitude (pA) / whole-cell capacitance (PF) for each cell. Kinetic analysis was performed by fitting the deactivation phase of the IK(M) trace with the standard single-component exponential fit equation: j(t) Ai· e-thi + C. The Y axis was zeroed at the steady state for current deactivation at each hyperpolarizing step, and the fits were extrapolated to zero time. The effects of LPD and RGB were monitored with respect to holding current in response to the -20 m V step, as well as IK(M) amplitude in response to the return step. Half-maximal inhibition (IC50) and enhancement (EC50) values were calculated using the Hill equation: Y/Ymax Yo + [axb/(cb + xb)]. The effects of TEA on IK(M) amplitude were 35 also examined. Strain-dependent differences in drug effects were determined by two-way ANOVA analysis using Prism 4.0. Passive membrane properties and action potential trains were recorded in current­clamp mode using a series of current steps ranging from -80 pA to + 180 pA. Action potential generation was monitored in slices prepared from B6 and Szti mice, in the presence and absence of LPD and RGB. Membrane potential was maintained at -65 m V by direct current injection (in control and drug conditions) as needed. The ability of CAl neurons to accommodate action potential frequency was determined by plotting interspike interval number vs. normalized spike frequency. Each cell served as its own control, and the frequency of each subsequent interspike interval was normalized to that of the first interspike interval. Significant differences in SF A between strains and drug treatments were then determined by nonlinear regression analysis (Prism 4.0). Decreased steepness of the fit is interpreted as decreased SFA. Statistical significance was determined by comparison of the best-fit lines. Results Intrinsic Electrophysiological Properties of B6 and Sztl Neurons Previous characterization of Szti mice established that the Szti mutation reduces seizure threshold and alters sensitivity to M-channel modifying drugs (Otto et aI., 2004); however, these changes in whole-animal behavior could not necessarily be attributed to the Kcnq2 component of Szti. Therefore, we recorded IK(M) in CAl neurons in brain slices prepared from B6 and Szt i mice in an effort to characterize changes in IK(M) specifically. Several fundamental membrane properties were evaluated in CAl pyramidal neurons in brain slices prepared from B6 and Szti mice. The Szti mutation does not 36 significantly alter resting membrane potential, holding current (at -20 m V), input and series resistance (at -70 mY), action potential half-width, action potential threshold, IK(M) deactivation kinetics, or cell capacitance (as mentioned above). These data are summarized in Table 3.1. Effects of Sztl Mutation on IK(M) Amplitude and Density Mice of both genotypes exhibited a functional IK(M) with a characteristic deactivation tail (Fig. 3.1Aa). In slices prepared from Szti mice, CAl neurons exhibited decreased IK(M) amplitudes relative to B6 slices (Fig. 3.1Ab), a trend that was evident throughout a range of hyperpolarizing steps (from a command potential of -20 m V to -50, -60, and -70 m V return steps; Fig. 3.1B). Exponential fits revealed no significant differences in the kinetics of IK(M) deactivation between B6 and Szti CAl neurons (Fig. 3.lAc). Since a decrease in current amplitude could be explained by a decrease in CAl neuron size, whole-cell capacitance was measured, and IK(M) amplitude was corrected to IK(M) density. Whole-cell capacitance measures were similar for CAl neurons in Szti and B6 slices (lIS.7 ± 3.9 pF (n = 40) and 111.3 ± 3.8 pF (n 38), respectively); therefore, in Szti slices, IK(M) density in CAl neurons was significantly decreased relative to B6 slices (Fig 3.lC). IK(M) amplitude and density values for B6 and Szti CAl neurons in response to several return voltage steps are shown in Table 3.2, and tau values obtained from deactivation phase exponential fits are listed in Table 3.1. 37 Table 3.1. IK(M) amplitude and density values are reduced across a range of voltage steps in Szti CA 1 neurons. IK(M) anlplitude (pA) IK(M) density (pA/pF) Step from -20 mV B6 Sztl B6 Sztl -SOmV 53.3 ± 4.6 42.3 ± 2.0* 0.46 ± 0.03 0.38 ± 0.02* (12) (13) -60mV 33.9 ± 2.4 23.2 ± 1.2* 0.29 ± 0.02 0.21 ± 0.01 * (34) (28) -70mV 13.0 ± 3.0 5.8 ± 1.7* 0.12 ± 0.02 0.05 ± 0.02* (12) (12) IK(M) amplitude values were measured at various potentials in response to hyperpolarizing steps from -20 mV to -50, -60, and -70 mY. In response to all three hyperpolarizing return steps, Szti CAl neurons exhibited significantly decreased IK(M) amplitude relative to that of B6 mice. Szti CAl neurons also show a significantly decreased IK(M) density at the same hyperpolarizing return steps (*, P < 0.05; ANOVA). Current densities were calculated by dividing the IK(M) amplitude of each cell by its capacitance. The numbers of cells recorded from (in parentheses) are identical for IK(M) amplitude and density values within the same genotype. 38 Figure 3.1. Sztl reduces M current amplitude and current density in CAl pyramidal neurons of the hippocampus. Aa, Sample IK(M) traces recorded from B6 (black) and Sztl (gray) CAl neurons in response to the -20 to -60 mV step. The dashed line represents the zero-current level for both traces. Ab, Inset traces at an amplified scale illustrate the decreased M current amplitude observed in Sztl slices. The dotted lines show the peak amplitudes of IK(M) observed in each genotype. Note that the IK(M) deactivation kinetics are similar in B6 and Sztl slices (also see Table 3.1). B, Sztl significantly decreases IK(M) amplitude across a range of return voltage steps (*P < 0.01, -70 mV and -50 mV steps; *p < 0.001, -60 mV step). C, IK(M) density is reduced in Sztl CAl neurons relative to B6 littermates (*P < 0.01, -70 mV and -50 mV steps; *p < 0.001, -60 mV step). A a B6 B ~40 :J ::; 30 c.. E to ~10 :::s::: ~ 0 • B6 Szt1 J40PA 400 ms o Szt1 -70 -60 -50 return step (mV) 39 b • •B 6• C G:' 0.5 c.. • B6 < 0.4 DSzt1 -c.. ~0.3 +-I 'V:; c: 0.2 Q) "'0 _0.1 ~ :::s::: ~ 0 -70 -60 -50 return step (mV) 40 Table 3.2. Intrinsic electrophysiological properties of CAl neurons recorded in brain slices prepared from B6 and Sztl mice. Membrane B6 n Sztl n Resting Membrane -60.1 ± 0.4 19 -59.7 ± 0.4 19 Potential (mV) Input Resistance (MQ) 111.3 ± 4.2 38 105.1 ± 3.9 40 Series Resistance (MQ) 13.2 ± 0.5 38 13.4 ± 0.5 40 Spike Half-width (msec) 1.52 ± 0.04 19 1.53 ± 0.04 18 Spike Threshold (m V) -46.2 ± 0.7 19 -45.0 ± 0.7 18 IK(M) Deactivation 157.4 ± 13.0 17 140.1 ± 14.6 17 Kinetics (-r in msec) None of the parameters measured differed between CAl neurons in slices prepared from B6 and Sztl mice (P> 0.05; ANOVA). Input resistance and series resistance were taken from values reported by SealTest and MultiClamp Commander functions. Spike half­width and spike threshold values were calculated from the first action potential that was generated in response to the lowest depolarizing current injection. Tau (.,;) values were calculated by fitting the IK(M) deactivation phase with a single exponential equation. Functional Consequences of Reduced IK(M) in Spike Frequency Adaptation 41 IK(M) activation regulates action potential generation and facilitates spike frequency adaptation (SF A); in response to membrane depolarization, IK(M) repolarizes neurons and decreases input resistance, which attenuates action potential frequency (Goh and Pennefather, 1987; Yue and Yaari, 2004). To test whether the decreased IK(M) density observed in Szti slices causes any observable changes in neuronal excitability, we monitored SF A in CA 1 neurons in response to prolonged depolarization steps. In response to a +120 pA depolarizing current injection of 800 msec duration, action potential trains were elicited in B6 and Szti slices (Fig. 3.2A). Interspike interval number vs. normalized frequency plots were created and the data were fit with a single exponential equation. The nonlinear fits were then compared by regression analysis to examine differences in SFA (Prism 4.0; GraphPad Software, San Diego, CA). Szti CAl neurons (n 10) exhibited significantly less SF A across the course of the response train compared to B6 CAl neurons (n 13; Fig 3.2B). Application ofLPD (10 ~M) to B6 (n = 7) and Szti (n = 9) CAl neurons significantly inhibits SFA (Fig 3.2C, 3.2D). It is particularly noteworthy that SFA in B6 CAl neurons can essentially be converted to that of untreated Szti neurons by applying LPD (Fig 3.2C). These results suggest that a hypofunctional IK(M) compromises the ability of neurons to modulate action potential firing, and further confirm the significant role of IK(M) in regulating neuronal excitability. Pharmacologic Effects of LPD in CAl Neurons Previous experiments demonstrated that Szti mice are hypersensitive to the proconvulsant effects of LPD (Otto et aI., 2004). To better describe LPD pharmacology 42 Figure 3.2. Sztl reduces the ability of CAl neurons to adapt spike frequency. A, Sample traces from CAl neurons in B6 and Sztl slices illustrate SFA from the beginning to the end of the +120 pA, 800 msec long, depolarizing current injection. In the B6 control trace, spike frequency has decreased such that later inters pike intervals are longer than the first. Notice in the Sztl control trace, spike frequency is relatively uniform from the beginning to the end of the trace. Interspike interval number 8 is shown in both traces to illustrate differences in interspike frequency in response to prolonged depolarization. Application of LPD (10 ~M) decreases SFA in both B6 and Sztl slices. B, Spike frequency throughout the depolarizing step was normalized to the frequency of the first interspike interval. Sztl slices exhibit significantly less SFA than B6 slices, as indicated by the decreased steepness of the best-fit line (*P < 0.0005, nonlinear regression analysis). C, In the presence of LPD, B6 SFA is significantly reduced (*P < 0.0001) to a level resembling that of untreated Sztl slices (B6+LPD vs. Sztl control, not significant). D, LPD also significantly decreases SFA in Sztl slices (*P < 0.01). 43 A +120 pA ~r------~----L-__ _ ~r-----------~ __ __ B6 control + 10 LPD JllllllIlIllllll L 1518 Szt 1 control 151 8 ~ + 10 LPD B D C'" 1.0 '~"'" 0 Szt1 control OJ ',1' J:: 0.8 \, \,".~ • B6 control V') 0.6 t.. ' .. ~" "0 + ~"'~ ~ 0.4 +, -~' H-~ E't, ,,"t .. & * '- 0.2 ~·T-·t_+ 0.2 o c: 0.0 +-r--r-or--'I'"_r__r_-r--I 0.0 +-r-.......... ...--.--r--r~--r"""I 0.0 +--~,.....,...,........,,.....,.....- 1 3 579 1 3 5 7 9 11 1 3 5 7 9 11 151 number 44 in these mice, we examined the effects of LPD on IK(M) amplitude and holding current (at a -20mV holding potential) in B6 (n 5-12 per dose) and Szti (n 5-13 per dose) CAl neurons. LPD dose-dependently blocked IK(M) and reduced holding current amplitudes (Fig 3.3A), but more potently inhibited IK(M) amplitude in Szti slices (Szti: ICso = 1.7 0.1 JlM; B6: 1Cso 4.0 ± 0.1 JlM; Fig 3.3B). This Szti-induced shift in LPD 1Cso was significant (P < 0.005). Holding currents in B6 and Szti CAl neurons appear to be equally sensitive to LPD in this paradign1 (Fig 3.3C). These data suggest that the Szti mutation results in increased LPD potency in some, but not all, aspects of IK(M) function. Pharmacologic Effects ofRGB in CAl Neurons Szti mice exhibit a decreased sensitivity to the anticonvulsant effects of RGB (Otto et aI., 2004). We therefore examined the effects of RGB on IK(M) amplitude and holding current in CAl neurons of B6 (n = 5-10 per dose) and Szti (n = 5-12 per dose) CAl neurons. The effects of RGB on slices prepared from each group were quite disparate. Sample traces illustrate differences in the responsiveness of B6 and Szti neurons to RGB (1 0 ~M; gray traces) (Fig. 3.4A). For example, B6 slices exhibited dose­dependent enhancement of IK(M) amplitude in the presence of RGB; remarkably, in Szti slices, RGB was not able to significantly enhance IK(M) amplitude at any concentration tested (Fig. 3.4B). Given this fact, it is particularly surprising that RGB (10 ~lM) still retained its ability to prolong IK(M) deactivation kinetics in Szti slices, just as it did in B6 slices (Fig 3.4A insets, Fig 3.4C). Consistent with a previous study (Passmore et aI., 2003), RGB does not allow complete current inactivation at high concentrations, which precludes accurate measurement of IK(M) amplitude; thus, it was not possible to construct full RGB dose-response curves. 45 Figure 3.3. Szti confers increased sensitivity to the IK(M) blocking properties of LPD. A, Sample traces show the inhibitory effects of LPD on the holding current at a -20m V holding potential and IK(M) amplitude. Traces in control (black) and 10 JlM LPD (gray) conditions illustrate that Szti slices exhibit increased sensitivity to the IK(M) blocking effects of LPD. Inset sample traces have zeroed baselines and magnified Y axes to more clearly illustrate these effects. B, LPD dose-response curves show that Szti CAl neurons exhibit increased sensitivity to LPD with respect to IK(M) amplitude (Szti: ICso = 1.7 ± 0.1 JlM; B6: ICso 4.0 ± 0.1 JlM). A statistical comparison identifies the shift in ICso as significant (*P < 0.005). C, B6 and Szti CAl neurons appear equally sensitive to the LPD-induced decrease in holding current at the -20 m V holding potential. A control 10 LPD --2-0--m,LV- __-_60m_V _ 86 J15 pA 150 ms Szt1 control 10 LPD ",,::::::===--~---J 100 pA 400 ms B 100 :E 80 ~ 1--1 -0 60 ~ "co""" 40 u CR 20 .86 o Szt1 c 120 """" 100 C ....J aJ o t: 80 ~:::J -C: U o on 60 uc CR:.c 40 -0 ..c. 20 .86 6, . .... -0.. 0 Szt1 .",'1.. 'i'''''~'''i 8.1 1 10 100 O~~----------- LPD (J.LM) 0.1 1 1 0 LPD (J.LM) 100 46 47 Figure 3.4. Szti confers decreased sensitivity to the IK(M) enhancing properties of RGB. A, Sample traces in control (black) and 10 f.lM ROB (gray) illustrate that Szti CA 1 neurons exhibit decreased sensitivity to the IK(M) enhancing effects of ROB. B, Szti slices exhibit dramatically decreased sensitivity to the IK(M) amplitude enhancing effects of ROB. Although 3 f.lM and 10 f.lM ROB significantly enhance IK(M) amplitude in B6 slices, IK(M) amplitude is not enhanced by ROB at any concentration in Szti slices (t p < 0.05, Two-way ANOV A). Notice, however, that ROB does prolong IK(M) deactivation kinetics in Szti slices (3.4A inset, 3.4C). C, ROB significantly and similarly prolongs the IK(M) tau of deactivation in CAl neurons of B6 and Szti mice. D, Dose-response curves show that Szti slices are approximately 10-fold less sensitive to the effects of ROB on holding current at -20 m V (Szti: ECso = 31.2 ± 0.04 f.lM; B6: ECso = 3.5 ± 0.04 f.lM). The ROB ECso shift in Szti CAl neurons was significant at P < 0.05. Interestingly, the Hill coefficient was also increased in Szti slices (B6: nR = 2.0; Szti: nR = 5.0). A 10 RGB control B -20mV ---,1-__-60_mV_ _ 86 Szt1 10 RGB 0,. control I - -- - - - --- - - - - - -- - - J140 pA ____ IV~~~=",,,,,_£,=:~ 400 ms c t 3 10 30 B6 Szt1 RGB (J.tM) D ~ 70 ~~60 :; ~50 u Q)40 onu .5~30 32.c 20 Oc .c Q) 10 ~ 0 1 10 100 RGB (J.1M) 48 49 Szti CAl neurons exhibited a 10-fold decrease in RGB potency with respect to holding current enhancement (B6: EDso 3.5 ± 0.04 ~M; Szti: EDso = 31.2 0.04 J.lM; Fig. 3.4D), which was evident throughout a range of depolarized membrane potentials (data not shown). These RGB data suggest that the drug's potency and efficacy are severely reduced in Szti CAl neurons. In addition, the IK(M) pharmacology data as a whole suggest that LPD and RGB can affect some aspects of IK(M) physiology, but not others; thus, these results suggest the existence of multiple populations of M -channels with differing KCNQ stoichiometric arrangements. Effects of TEA on IK(M)in CAl Neurons The differential LPD and RGB pharmacodynamics observed in B6 and Szti CAl neurons suggested that Sztl results in altered M-channel subunit composition. To further test this hypothesis, we examined the effects of TEA (10 mM), a KCNQ2 subunit­preferring IK(M) blocker (Hadley et aI., 2000), on IK(M) amplitude in B6 and Szti CAl neurons. TEA significantly blocked IK(M) in B6 CAl neurons (n = 5) at all voltage steps tested (Fig. 3.5). Szti CAl neurons (n = 5) were largely insensitive to TEA, only displaying significantly reduced IK(M) amplitude at the -50 and -40 m V return steps. Moreover, two-way ANOV A analysis revealed that at every return step, Szti CAl neurons displayed significantly decreased TEA sensitivity relative to B6 CAl neurons (Fig. 3.5B). These data support our hypothesis that M-channel subunit composition differs in Szti and B6 CAl neurons. 50 Figure 3.5. Sztl renders CAl neurons largely insensitive to the KCNQ2 subunit­preferring IK(M) blocker TEA. A, Sample traces in control (black) and 10 mM TEA (gray) illustrate that Sztl CAl neurons exhibit decreased sensitivity to the IK(M) blocking effects of TEA. B, Sztl slices display significantly decreased sensitivity to the IK(M) blocking effects of TEA. 10 mM TEA significantly blocks IK(M) in B6 CAl neurons at every return step tested (*P < 0.01, paired t-test). TEA only significantly reduces IK(M) in Sztl neurons at the -50 and -40 m V steps (*P < 0.05, ANOV A). At all return steps tested, Sztl slices display less sensitivity to TEA than B6 slices (tP < 0.02, two-way ANOV A, drug effect vs. genotype comparison). A -2I0LmV-_ -_60m_V _ 86 control 10 TEA Szt1 control ~. 10 TEA !II ~ __ J140PA 400 ms B Q) 1 • B6 -g D Szt1 ~1 c.. E 80 to ~ 60 ::s::: 1--1 e.., c o u ~ -70 -60 -50 -40 return step (mV) 51 52 Discussion Compounds that block IK(M) are proconvulsant (Flagmeyer et aI., 1995), while those that enhance IK(M) are anticonvulsant (Rostock et al., 1996), and in oocytes, mutations in KCNQ2 and KCNQ3 genes decrease KCNQ2IKCNQ3 channel function. Therefore, it has been assumed that KCNQ mutations decrease native (neuronal) IK(M) function, thereby increasing neuronal excitability, and eventually causing the seizures observed in BFNC patients (Castaldo et aI., 2002). However, until now, it has not been determined whether such mutations actually affect the native neuronal IK(M). We have shed significant light on the consequences of naturally-occurring KCNQ2 mutation using mice heterozygous for the Szti mutation, which deletes the genomic DNA encoding the KCNQ2 C-terminus, and all of CHRNA4 and ARFGAP-1. It was recently shown that conditional transgenic overexpression of a dominant­negative Kcnq2 G279S mutation reduces IK(M) amplitude and increases neuronal excitability in mice (Peters et aI., 2005). The Tet-Off system used to drive conditional Kcnq2 overexpression in this study, however, significantly interfered with the expression of wild-type KCNQ2 and KCNQ3 subunits. Due to the pervasive alterations in M­channel subunit expression levels, these results might be considered to be detached from any real disease pathology. The results presented here are the first direct evidence of decreased native neuronal IK(M) amplitude and current density in a naturally-occurring animal model of KCNQ2 mutation. This may be due to a depolarizing shift in the IK(M) voltage of activation or decreased peak channel conductance, a distinction that cannot be made in the acute brain slice recording paradigm. There were, however, no observed changes in 53 deactivation kinetics as a function of voltage; thus, the data imply that Szt i does not alter the voltage-dependence of IK(M) activation. We have also shown that IK(M) hypofunctionality increases the excitability of CA 1 neurons by compromising their natural ability to accommodate action potential frequency. Finally, the reduction in IK(M) amplitude and SFA, and altered pharmacology in Szti CAl neurons are consistent with the decreased seizure threshold and altered pharmacosensitivity, respectively, that were previously reported in the Szti mouse behavioral study (Otto et aI., 2004). Kcnq2 mRNA is reduced in Sztil+ mice by 30-40%, and is seemingly absent in Szti+l+ mice (Yang et aI., 2003), suggesting that the truncated form of the protein is not likely expressed. Furthermore, the KCNQ2 C-terminus is required for proper KCNQ2/KCNQ3 channel assembly (Schwake et aI., 2003). In CAl pyramidal neurons, KCNQ2 protein is normally concentrated at the Nodes of Ranvier and the axon initial segment (AIS), the anatomical site of action potential initiation (Devaux et aI., 2004). Therefore, one possible explanation for alterations in SF A and lK(M) density in the Szt i mouse is that KCNQ2 protein expression is decreased at the AlS. While it is possible that a trace amount of truncated Kcnq2 mRNA is being generated for the Szti allele, causing a dominant-negative effect, the phenotypic similarities between Szti mice and Kcnq2 null mice (Watanabe et aI., 2000; Yang et aI., 2003), and the inability to detect mRNA encoding the truncated protein suggest a KCNQ2 haploinsufficiency, consistent with the proposed human disease mechanism (Steinlein, 2004). It is especially interesting that the SF A of B6 neurons can essentially be converted to that of untreated Szti slices by partially blocking lK(M) with 10 J1.M LPD. This result is consistent with the hypothesis that proper lK(M) function is an important component of 54 normal SFA (Goh and Pennefather, 1987; Aiken et ai., 1995). Another group, however, has shown that LPD does not affect SF A in cultured mouse and rat superior cervical ganglion neurons (Romero et aI., 2004), although neurons of this population are not implicated in epilepsy. This discrepancy in LPD effect could be explained by the clear differences in the baseline SFA of cultured SCG neurons vs. hippocampal CAl neurons in the acute brain slice preparation. We have also detailed several differences in Szti CAl neuron IK(M) pharmacology, including increased LPD potency with respect to IK(M) amplitude, and decreased RGB potency with respect to both IK(M) amplitude and holding current. These results are noteworthy in that they imply that M-channels with differing KCNQ stoichiometries exist in CAl pyramidal neurons. For instance, Szti CAl neurons are more sensitive to the IK(M) amplitude blocking properties of LPD, but their sensitivity to the effects on holding current is no different from that of B6 slices. Szti CAl neurons were also far less sensitive to the holding current enhancing properties of RGB, and although RGB did not enhance IK(M) amplitude at any concentration (0.3-100 f.lM; data not shown) or in response to any voltage step protocol (-20 mY, -30 mY, and -40 mV depolarizing potentials; only -20 m V data shown), it did prolong IK(M) deactivation kinetics in Szti CAl neurons. At resting membrane potential (-70 m V), where IK(M) is tonically active at low levels, RGB was still able to hyperpolarize Szti CAl neurons to a similar degree as was observed in B6 slices. The most substantial evidence for altered subunit stoichiometry in Szti CAl neurons is the considerable difference in sensitivity to the KCNQ2-preferring IK(M) blocker, TEA. B6 CAl neurons showed significant block at all return steps tested, 55 indicating the presence of KCNQ2 subunits. Sztl CAl neurons, however, were largely insensitive to TEA. Moreover, Sztl CAl neurons exhibited decreased sensitivity to TEA relative to that of B6 CAl neurons. This considerable difference in TEA sensitivity was statistically significant as determined by two-way ANOVA analysis. These data strongly suggest that the Szt 1 mutation decreases KCN Q2 subunit expression, and therefore qualitatively reduces KCNQ2 subunit inclusion in the M-channel. The drastic difference in RGB potency observed in Szti and B6 mice is curious; simply altered M-channel stoichiometry might not fully explain these results. The Sztl mutation most likely renders mice hemizygous for Kcnq 2, and we therefore presume that less KCNQ2 subunit protein is included in Sztl M-channels. RGB ECso values obtained in Szt 1 slices are approximately one order of magnitude above those obtained in B6 slices, but RGB ECso values previously reported in expression systems for KCNQ2/KCNQ3 and KCNQ3/KCNQ5 currents were determined to be quite similar: 1.9 (Tatulian et aI., 2001) and 1.4 J.!M (Wickenden et aI., 2001), respectiVely. It should be noted, however, that these ECso values were obtained in Chinese hamster ovary cells (CHO), which lack the biological compensatory mechanisms of a native CAl neuron that may be triggered by a robust change in subunit expression. Specifically, IK(M) function is tightly regulated by many intracellular factors, including a diffusible second messenger and phosphorylation state (Cruzblanca et aI., 1998; Suh and Hille, 2002). It is possible that in response to the Sztl mutation, the expression of intracellular regulators that are crucial to IK(M) function could be drastically altered. To date, the role of intracellular second messengers in regulating drug activity at the M-channel has not been examined. Finally, it has been documented that results obtained in expression systems often do not 56 closely parallel those obtained in native systems (Dorr, 1993; Lewis et aI., 1993; Sivilotti et aI., 1997). Whatever the molecular mechanisms underlying the pharmacodynamic discrepancies presented here, it is particularly compelling that the M -channel pharmacology is so remarkably consistent with previous in vivo electroconvulsive threshold studies (Otto et aI., 2004), in which Sztl mice displayed decreased LPD sensitivity and quite robustly decreased ROB sensitivity. It has been established that IK(M) is a major contributor to the resting membrane potential of many neurons. Pharmacological block or enhancement of IK(M) depolarizes or hyperpolarizes resting membrane potential, respectively (Aiken et aI., 1995; Otto et aI., 2002; Peretz et aI., 2005). Surprisingly, however, we found no significant change in resting membrane potential between B6 and Szt 1 mice. This suggests that in response to the Sztl mutation, and subsequent reduced expression of the KCNQ2 subunit, compensatory mechanisms may have arisen to prevent a significant change in resting membrane potential. Altered expression of numerous voltage-gated ion channel currents in Sztl mice (i.e., potassium leak currents) might account for this lack of effect. The Sztl mutation does alter another epilepsy-related gene, Chrna4, which is implicated in Autosomal Dominant Frontal Lobe Epilepsy (ADNFLE) (Steinlein et aI., 1995; Hirose et aI., 1999). Chrna4 hemizygosity may appear to contribute to, or even be fully responsible for, the reduction of seizure threshold observed in the Sztl mouse; but it might in fact temper the effects of Kcnq2 hemizygosity, as many of the human ADNFLE­causing point mutations in CHRNA4 observed to date are actually gain-of-function, producing receptors with increased ACh sensitivity (Bertrand et aI., 2002; Scheffer and Berkovic, 2003). Indeed, it has not been determined yet whether nACh receptor 57 hypofunction or hyperfunction underlies ADNFLE. More specific to the Szt 1 mutation, Chrna4/+ mice have no known seizure phenotype and EEG's are similar to WT mice (McColl et aI., 2003), and extensive seizure threshold testing has revealed only modest differences, with mildly increased sensitivity in some chemoconvulsive models and even resistance in others (Ross et aI., 2000; Wong et aI., 2002). On these grounds, we suggest that decreased II«M) amplitude and subsequently inhibited SF A are the primary source of the lowered seizure threshold observed in the Sztl mouse, and may underlie seizure generation in human BFNC patients. Finally, although KCNQ2 and KCNQ3 mutations are associated with human epilepsy, it is easy to imagine that a current so crucial to regulating neuronal excitability could be a useful pharmacological target for the treatment of many pathologies involving aberrant neuronal excitability. 58 References Aiken SP, Lampe BJ, Murphy P A, Brown BS (1995) Reduction of spike frequency adaptation and blockade of M -current in rat CA 1 pyramidal neurones by linopirdine (DuP 996), a neurotransmitter release enhancer. Br J Pharmacol 115:1163-1168. Barton ME, White HS, Wilcox KS (2004) The effect of CGX-I007 and CI-I041, novel NMDA receptor antagonists, on NMDA receptor-mediated EPSCs. Epilepsy Res 59: 13-24. Bertrand D, Picard F, Le Hellard S, Weiland S, Favre I, Phillips H, Bertrand S, Berkovic SF, Malafosse A, Mulley J (2002) How mutations in the nAChRs can cause ADNFLE epilepsy. Epilepsia 43 SuppI5:112-122. Biervert C, Schroeder BC, Kubisch C, Berkovic SF, Propping P, Jentsch TJ, Steinlein OK (1998) A potassium channel mutation in neonatal human epilepsy. Science 279:403-406. Brown DA, Adams PR (1980) Muscarinic suppression of a novel voltage-sensitive K + current in a vertebrate neurone. Nature 283 :673-676. Castaldo P, del Giudice EM, Coppola G, Pascotto A, Annunziato L, Taglialatela M (2002) Benign familial neonatal convulsions caused by altered gating of KCNQ2/KCNQ3 potassium channels. J Neurosci 22: 1-6. Constanti A, Brown DA (1981) M-Currents in voltage-clamped mammalian sympathetic neurones. Neurosci Lett 24:289-294. Cruzblanca H, Koh DS, Hille B (1998) Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons. Proc Natl Acad Sci USA 95:7151-7156. Devaux 11, Kleopa KA, Cooper EC, Scherer SS (2004) KCNQ2 is a nodal K+ channel. 1 Neurosci 24: 1236-1244. Dorr RT (1993) Clinical properties of yeast-derived versus Escherichia coli-derived granulocyte-macrophage colony-stimulating factor. Clin Ther 15:19-29; discussion 18. Dost R, Rundfeldt C (2000) The anticonvulsant retigabine potently suppresses epileptiform discharges in the low Ca ++ and low Mg++ model in the hippocampal slice preparation. Epilepsy Res 38:53-66. Flagmeyer I, Gebert I, van der Staay F 1 (1995) General pharmacology of the putative cognition enhancer linopirdine. Arzneimittelforschung 45:456-459. 59 Goh JW, Pennefather PS (1987) Pharmacological and physiological properties of the after-hyperpolarization current of bullfrog ganglion neurones. J Physiol 394:315- 330. Hadley JK, Noda M, Selyanko AA, Wood IC, Abogadie FC, Brown DA (2000) Differential tetraethylammonium sensitivity of KCNQ 1-4 potassium channels. Br J PharmacoI129:413-415. Hirose S, Iwata H, Akiyoshi H, Kobayashi K, Ito M, Wada K, Kaneko S, Mitsudome A (1999) A novel mutation of CHRNA4 responsible for autosomal dominant nocturnal frontal lobe epilepsy. Neurology 53: 1749-1753. Lewis TB, Leach RJ, Ward K, O'Connell P, Ryan SG (1993) Genetic heterogeneity in benign familial neonatal convulsions: identification of a new locus on chromosome 8q. Am J Hum Genet 53:670-675. Marrion NV (1997) Control of M-current. Annu Rev Physiol 59:483-504. McColl CD, Home MK, Finkelstein DI, Wong JY, Berkovic SF, Drago J (2003) Electroencephalographic characterisation of pentylenetetrazole-induced seizures in mice lacking the alpha 4 subunit of the neuronal nicotinic receptor. Neuropharmacology 44:234-243. Otto JF, Kimball MM, Wilcox KS (2002) Effects of the anticonvulsant retigabine on cultured cortical neurons: changes in electroresponsive properties and synaptic transmission. Mol PharmacoI61:921-927. Otto JF, Yang Y, Frankel WN, Wilcox KS, White HS (2004) Mice carrying the sztl mutation exhibit increased seizure susceptibility and altered sensitivity to compounds acting at the m-channel. Epilepsia 45:1009-1016. Passmore GM, Selyanko AA, Mistry M, AI-Qatari M, Marsh SJ, Matthews EA, Dickenson AH, Brown TA, Burbidge SA, Main M, Brown DA (2003) KCNQIM currents in sensory neurons: significance for pain therapy. J Neurosci 23:7227- 7236. Peretz A, Degani N, Nachman R, Uziyel Y, Gibor G, Shabat D, Attali B (2005) Meclofenamic acid and diclofenac, novel templates of KCNQ2/Q3 potassium channel openers, depress cortical neuron activity and exhibit anticonvulsant properties. Mol PharmacoI67:1053-1066. Peters HC, Hu H, Pongs 0, Storm JF, Isbrandt D (2005) Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior. Nat Neurosci 8:51-60. 60 Romero M, Reboreda A, Sanchez E, Lamas JA (2004) Newly developed blockers of the M-current do not reduce spike frequency adaptation in cultured mouse sympathetic neurons. Eur J Neurosci 19:2693-2702. Ross SA, Wong JY, Clifford JJ, Kinsella A, Massalas JS, Horne MK, Scheffer IE, Kola I, Waddington JL, Berkovic SF, Drago J (2000) Phenotypic characterization of an alpha 4 neuronal nicotinic acetylcholine receptor subunit knock-out mouse. J Neurosci 20:6431-6441. Rostock A, Tober C, Rundfeldt C, Bartsch R, Engel J, Polymeropoulos EE, Kutscher B, Loscher W, Honack D, White HS, Wolf HH (1996) D-23129: a new anticonvulsant with a broad spectrum activity in animal models of epileptic seizures. Epilepsy Res 23:211-223. Scheffer IE, Berkovic SF (2003) The genetics of human epilepsy. Trends Pharmacol Sci 24:428-433. Schroeder BC, Kubisch C, Stein V, Jentsch TJ (1998) Moderate loss of function of cyclic-AMP-modulated KCNQ2/KCNQ3 K + channels causes epilepsy. Nature 396:687-690. Schwake M, Jentsch TJ, Friedrich T (2003) A carboxy-terminal domain determines the subunit specificity of KCNQ K(+) channel assernbly. EMBO Rep 4:76-81. Shah MM, Mistry M, Marsh SJ, Brown DA, Delmas P (2002) Molecular correlates of the M-current in cultured rat hippocampal neurons. J Physio1544:29-37. Singh NA, Charlier C, Stauffer D, DuPont BR, Leach RJ, Melis R, Ronen GM, Bjerre I, Quattlebaum T, Murphy JV, McHarg ML, Gagnon D, Rosales TO, Peiffer A, Anderson VE, Leppert M (1998) A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat Genet 18:25-29. Sivilotti LG, McNeil DK, Lewis TM, Nassar MA, Schoepfer R, Colquhoun D (1997) Recombinant nicotinic receptors, expressed in Xenopus oocytes, do not resemble native rat sympathetic ganglion receptors in single-channel behaviour. J Physiol 500 (Pt 1):123-138. Steinlein OK (2004) Genetic mechanisms that underlie epilepsy. Nat Rev Neurosci 5:400-408. Steinlein OK, Mulley JC, Propping P, Wallace RH, Phillips HA, Sutherland GR, Scheffer IE, Berkovic SF (1995) A missense mutation in the neuronal nicotinic acetylcholine receptor alpha 4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet 11:201-203. 61 Suh BC, Hille B (2002) Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron 35:507-520. Tatulian L, Delmas P, Abogadie FC, Brown DA (2001) Activation of expressed KCNQ potassium currents and native neuronal M-type potassium currents by the anti­convulsant drug retigabine. J Neurosci 21 :5535-5545. Watanabe H, Nagata E, Kosakai A, Nakamura M, Yokoyama M, Tanaka K, Sasai H (2000) Disruption of the epilepsy KCNQ2 gene results in neural hyperexcitability. J Neurochem 75:28-33. Wickenden AD, Zou A, Wagoner PK, Jegla T (2001) Characterization of KCNQ5/Q3 potassium channels expressed in mammalian cells. Br J Pharmacol 132:381-384. Wong JY, Ross SA, McColl C, Massalas JS, Powney E, Finkelstein DI, Clark M, Home MK, Berkovic SF, Drago J (2002) Proconvulsant-induced seizures in alpha(4) nicotinic acetylcholine receptor subunit knockout mice. Neuropharmacology 43:55-64. Yang Y, Beyer BJ, Otto JF, O'Brien TP, Letts VA, White HS, Frankel WN (2003) Spontaneous deletion of epilepsy gene orthologs in a mutant mouse with a low electroconvulsive threshold. Hum Mol Genet 12:975-984. Vue C, Yaari Y (2004) KCNQ/M channels control spike afterdepolarization and burst generation in hippocampal neurons. J Neurosci 24:4614-4624. CHAPTER 4 A SURVEY OF ELECTROCONVULSIVE SEIZURE THRESHOLDS IN MICE CARRYING KCNQ AMINO ACID EXCHANGE MUTATIONS KNOWN TO CAUSE HUMAN BFNC Introduction It has been established that mutations in the KCNQ2 and KCNQ3 genes cause the human seizure disorder Benign Familial Neonatal Convulsions (Biervert et aI., 1998; Pereira et aI., 2004; Tang et aI., 2004). The KCNQ2 and KCNQ3 subunits are molecular correlates of the M-type K + channel, and the current generated by this channel - the M current [IK(M)] is a critical determinant of neuronal excitability. Previous work by o