Info

Mutant

Chr. Gene Protein

Tottering (tg) 8 Cacnala

Lethargic (lh) 2 Cacnb4

Ducky (du) 9 Cacna2d2

Stargazer (stg) 15 Cacng2

Mocha2j (mh) 10 AP3d

Coloboma (Cm) 2 Cm

P/Q type calcium channel alpha subunit Calcium channel beta 4 subunit Alpha2delta2 subunit Gamma2 subunit Sodium hydrogen exchanger Delta subunit AP3 adaptor protein Microdeletion including SNAP25 and phospholipase C isoform |31

Fletcher et al., 1996 Burgess et al., 1997 Brodbeck et al., 2002 Letts et al., 1998 Cox et al., 1997 Kantheti et al., 2003 Zhang, Hess et al., 1994

Spike wave

Tottering (tg) 8 Cacnala

Lethargic (lh) 2 Cacnb4

Ducky (du) 9 Cacna2d2

Stargazer (stg) 15 Cacng2

Mocha2j (mh) 10 AP3d

Coloboma (Cm) 2 Cm

Convulsions

Dilute lethal (dl) 9 Myo5a

Jittery (ji) 10

Megencephaly (mceph) 6 Kv1.1

Quaking (qk) 17

Staggerer (sg) 9 RORa Torpid (td)

Varitint waddler (Va) 3 mcoln3

Wabbler-lethal (wl) 14

Weaver (wv) 16 GIRK2 Writher (wh)

Convulsions evoked by sensory stimuli

Frings 13 Mass1

Lurcher (Lu) 6 GluR d2

P/Q type calcium channel alpha subunit Calcium channel beta 4 subunit Alpha2delta2 subunit Gamma2 subunit Sodium hydrogen exchanger Delta subunit AP3 adaptor protein Microdeletion including SNAP25 and phospholipase C isoform |31

Myosin Va

Proteolipid protein

Novel protein

Potassium ion channel

Microdeletion, including parkin

Nuclear receptor

Unknown

TRP ion channel

Unknown

G-protein-coupled potassium channel

Novel protein

Glutamate Receptor delta subunit

Fletcher et al., 1996 Burgess et al., 1997 Brodbeck et al., 2002 Letts et al., 1998 Cox et al., 1997 Kantheti et al., 2003 Zhang, Hess et al., 1994

Mercer et al., 1991 Dautigny et al., 1986 Bomar et al., 2003 Peterson, 2003 Lorenzetti, 2004 Matysiak-Scholze, 1997

DiPalma, 2002

Slesinger et al., 1996 unknown

Skratski et al., 2001 Zuo et al., 1997

Periodically new spontaneous mutants are found in both large and smaller breeding colonies in laboratories around the world. When this occurs, the founder animal is back-crossed to determine whether the deviant trait arises from a germline, rather than somatic, mutation and can be propagated for further study. Traditionally a nickname evocative of some behavioral trait is chosen for the new mutant, although the reader should be aware that there is a committee responsible for adherence to the "Standardized Genetic Nomenclature for Mice," which asks that the newly discovered mutation be shown through appropriate genetic testing not to be allelic with other mutants—or if it is, that if differs in phenotype from other allelic mutations at that locus. The final name assigned is that of the mutated gene. For example, the mutant mouse rocker is allelic to tottering. Both are mutations of the calcium channel alpha subunit Cacnala, and both display ataxia and absence epilepsy. However, dendritic arbors of rocker Purkinje cells display a distinct "weeping willow" appearance, whereas those of tottering are unaffected (Fletcher et al., 1996; Zwingman et al., 2001). Strains with mutant and wild-type alleles on the same genetic background (congenic strains) are developed by repeated crossing of the mutant to an inbred strain or by brother-sister mating with forced heterozygosis of the mutant. Proper maintenance of strains requires vigilance and a well-defined breeding scheme. Difficulties with viability and fertility are often encountered with neurologic mutants, and propagation of the mutant may be assisted in various ways (Green, 1975); however, the strain designation must reflect any changes in this background. Because mutants originate from many different colonies, the designation of strain origin can be very informative.

Gene Identification and Induced Mutagenesis Strategies

Early mutant genes were isolated in a lengthy process by mapping the disease locus, followed by laborious strategies to identify genes contained within the region, allowing the final evaluation of each gene for a mutation. With the application of comparative and functional genomic identification strategies, informative marker strains, and publication of the entire sequence of the mouse genome (Waterston et al., 2002), the initial mapping step narrows the disease locus to a precise region containing a small number of genes (positional candidate gene cloning) that can all be sequenced. The process can now lead to rapid identification of the mutation.

Once the fine structure of the single locus mutation has been characterized, mutant alleles are generally found to fall into several different categories. Traits that are transmitted in a Mendelian pattern are often point mutations (e.g., single amino acid substitutions, sequence inversions, or repetitive elements) that lead to gain or loss of function in a single gene. However, there are other possibilities, including duplications or small deletions that encompass several closely linked genes, similar to small contiguous gene deletion syn dromes in man. The pink eye dilution (p) mutation, for example, has seizures and is missing three closely linked subunit genes for GABAa receptors on chromosome 7 (Culiat et al., 1994), and more than 36 variants at this locus have been studied (Johnson et al., 1995).

Although the discovery of rare epileptic mutant pheno-types depends on relatively infrequent spontaneous mutation rates, ancillary strategies have been recently employed to accelerate the pace. Induced (but untargeted) point mutations can be obtained by ethylnitrosourea (ENU) mutagenesis in mice, followed by phenotypic screening for excitability variants (Balling, 2001; Bult et al., 2004; O'Brien and Frankel, 2004). Accelerating the mutation rate by the introduction of random single- base-pair changes is a valuable strategy to generate novel phenotypes. However, the phenotype of either epilepsy or altered seizure threshold must still be ascertained in the mutagenized mice, which then require the same detailed gene isolation procedure as spontaneous mutants. Nevertheless, there are two advantages to this approach: first, the acquisition of novel pheno-types arising from random mutation of as yet unstudied genes; and second, the possibility of generating an expanded series of gain of function alleles that differ from those obtained by targeted deletion strategies. Advanced regional chromosomal saturation methods and "oligogenic chromosomal lesions" are also within the reach of gene targeting technology if required to model a human mutation (van der Weyden et al., 2002).

Validating the Epileptic Phenotype Electroencephalographs Recording

To satisfy the currently accepted definition of an epileptic seizure, the model must display abnormal synchronous electroencephalograph^ (EEG) activity as an electrocortical correlate of any paroxysmal behavior. Not uncommonly a spontaneous epileptiform behavioral disturbance originates as a subcortical event, as evidenced by the lack of change in the EEG. This is the case for the sudden myoclonic jerks in spasmodic mice (mutation of the alphal subunit of the glycine receptor), which is therefore considered a model of the movement disorder, hyperekplexia, or "startle disease," rather than epileptic myoclonus (Buckwalter et al., 1993). Some mice may display both an epileptic seizure as well as nonepileptic paroxysmal dyskinesia, two separable disorders arising in independent brain networks. For example, tottering mice display spike-wave seizures with behavioral arrest involving the thalamocortical circuitry; they also exhibit paroxysmal dyskinesias with dystonic features involving pontocerebellar circuitry that are unaccompanied by EEG discharges (Campbell and Hess, 1998; Noebels and Sidman, 1979). To validate the mutant as a model of epilepsy, repeated spontaneous seizures must be recorded from neocortex or hippocampus in multiple mutants and compared with control recordings in mice of the same strain that are wild-type for the mutant allele.

We have carried out an EEG/photic screen of a large number of mutants in the Jackson Laboratory Mutant Mouse Resource over the years (J.L. Noebels, X. Qiao, and M. Davisson, unpublished). Although the screen validated epilepsy in several mutant strains, more than 100 others, many with neurologic abnormalities, failed to show epilepsy during the EEG monitoring process. Although it is not possible to state categorically that these mice do not have epilepsy at some point in their development, seizures were sufficiently rare to escape detection during the screen (in this case, typically restricted to three affected adult mice for periods from 2 to 10 hours on two or more separate recording days). With that significant caveat, this experience substantially confirms what is well known to clinical neurologists: that seizure disorders, though common, are not an inevitable result of aberrant circuitry or cortical damage and hence represent a specific subset of latent excitability defects in neural networks.

Phenotypic Discovery: Surrogate Markers for Screening

Because episodic seizure disorders require an abnormal EEG to identify correctly the phenotype, many mutant models of epilepsy have gone unrecognized. Although both video monitoring and EEG seizure-detection algorithms are available to facilitate this step, prolonged monitoring may be required to detect rare events, making alternative screening strategies desirable. Unfortunately only a few reliable or specific biochemical indicators of past seizure history are currently available for this purpose. Regional anatomic rearrangements and molecular plasticity of varying longevity are seen in both human and rodent models of limbic epilepsy and can serve as useful (if not specific) indicators of prior seizure events. One such marker is granule cell axonal sprouting, readily visualized by Timm or dynor-phin staining of mossy fibers in the inner molecular layer of the dentate gyrus. Dispersion of granule cell bodies and aberrant axonal reorganization appear to be essentially permanent network changes closely linked to prolonged hip-pocampal seizures (Houser, 1990; Sutula et al., 1989). Molecular markers induced by seizure-induced changes in gene expression and cell death are also abundant in the hippocampus, but they tend to be shorter lasting (Borges et al., 2003) and are drastically modified by genetic background (Morrison et al., 1996; Schauwecker, 2002). Several other molecules that may be useful for indirect detection of recent seizure activity in adult (Frankel et al., 1994; Storey et al., 2002), but not immature (Storey et al., 2002), brains include the reactive transcription factors C-Fos and JunB and the ectopic expression of neuropeptide Y (NPY) in granule cell axons (Tonder et al., 1994). Both mossy fiber sprouting and ectopic axonal NPY expression have been observed in spontaneous epileptic mutants, such as stargazer (Chafetz et al., 1995; Qiao and Noebels, 1993). Identification of seizure-induced neurogenesis among cells located in the infragran-ular layer of the dentate hilus is not a useful screening tool because the cell division must be labeled at the time of the seizure (Parent et al., 1997). C-Fos activation distinguishes between convulsive (Frankel et al., 1994; Morgan et al., 1987) and absence epilepsy models (Morgan et al., 1987). This observation reinforces the genetic evidence for fundamental differences not only in the circuitry but also in the biology of neuronal synchronization among major EEG and clinical seizure types in the brain.

An alternative screening method for detecting epileptic mutants involves acute testing for a lowered seizure threshold induced by auditory activation, convulsant drugs, or maximal electroshock (White et al., 1995). The validity of this approach relies on the assumption that epilepsies arise from circuits that are disinhibited or otherwise more likely to engage in rhythmic bursting; however, this relationship may not always be true. The "seizure threshold" is a separable trait from spontaneous seizures, and mutant mice (like humans) with latent hyperexcitable neural circuits may never display epilepsy; in turn, spontaneously epileptic mice may show only a reduced threshold for triggering seizures if the correct circuit or pattern of activation is applied. For example, P/Q-type calcium ion channel mutants such as tottering and leaner show thalamocortical spike-wave epilepsies; yet cortical excitability is reduced as a result of decreased calcium currents and impaired neurotransmitter release (Ayata et al., 2000; Qian and Noebels, 2000). Mutant mice with a lowered threshold for seizures evoked by one modality may demonstrate an elevated threshold through another pathway. Finally, significant differences in the convulsive threshold exist among inbred strains, which may confound the interpretation of mutations on different backgrounds (Frankel et al., 2001). Nevertheless, the threshold method, though not infallible, has been valuable in identifying certain gene mutations of interest (Yang et al., 2003).

MATCH AND MISMATCH WITH HUMAN SEIZURE DISORDERS: WHAT DOES THE MUTATION MODEL AND HOW WELL?

Inherited lesions in the same gene may show widely disparate phenotypes in mice and humans. The correct explanation, most of the time, is that two different functional alleles are being compared. For example, in the Kv1.1 potassium ion channel gene, dominant human point mutations may alter the kinetics of pore inactivation, thereby prolonging depolarization; such mutations are associated with myokymia, episodic ataxia, or a temporal lobe epilepsy phe-

notype (Browne et al., 1994; Zuberi et al., 1999). In the mouse, a deletion of the same channel leads to a more severe convulsive phenotype with cold-induced neuromyotonia (Smart et al., 1998; Zhou et al., 1998); a truncation of the same channel is linked to a dominant negative syndrome of epilepsy and megencephaly (Petersson et al., 2003). It is noteworthy that insertion of a human ataxia mutation in mouse produces the expected phenotype (Herson et al., 2003). It should also be recalled that many of the human disorders are clinically apparent in heteroygotes, whereas many of the mouse models are studied in their more severe homozygous forms. However, in a variety of murine disorders, an apparently identical functional change may still create dissimilar phenotypes. When this occurs, what conclusions can be drawn about the similarity of the intervening mechanisms? Reconciling species-specific phenotypic differences requires an ability to compare key elements of the disorder in mice with those assembled for human epilepsy syndromes, beginning with the electrographic and behavioral semiology of the seizure phenotype (Blume et al., 2001) and then working backward toward the precise mutation of the gene.

The cortical EEG, which is readily but subjectively described, exhibits important differences in background rhythmicity even between normal rodent and human brains. For example, mice and rats lack spontaneous alpha (712 Hz) rhythms and other complex discharges and sleep stages normally present in the human EEG. Would these background oscillatory differences alter the synchronization patterns of an otherwise identical epileptogenic lesion? The behavioral correlate of the seizure is also often difficult to compare because the semiology of spontaneous seizures in rodents has not been formalized, and many model descriptions are incomplete. Terms such as epileptic seizures, behavioral arrest, convulsions, wild running, and Straub tail phenomenon are typically the only seizure descriptions reported. Ictal durations are rarely recorded, and the changing behavior pattern throughout an ictal episode, well described for the kindling model (Engel et al., 1978), is usually overlooked. Finally, the pharmacological sensitivity and natural history (onset and duration of seizures over the life span), two key descriptors of an epilepsy syndrome, are difficult to determine precisely and rarely mentioned in descriptions of murine epilepsies.

At the neuropathological level, the assumption that an orthologous gene model should show molecular or cellular lesions in the mouse that are identical to those in human may also be premature (at least in some cases). Several basic explanations for potential differences are routinely overlooked. For example, murine genes may be spliced differently than those in human, leading to proteins that differ in functional properties or interaction domains with other molecules. Anatomically, the cellular context and neurobiology of the mouse and human brain vary; for example, some mouse brain regions lack cellular components present in humans. Additional differences may reside in the temporal and regional expression of brain genes, in compensatory gene family members, or directly interacting partners such as channel regulatory subunits, transmitter receptors, or other members of the molecular pathway. Finally, the mouse or human may coexpress an unknown number of mutations in modifying genes that mask the penetrance of the phenotype.

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