Mouse Model Of Absence Epilepsy Research Articles

Synaptic changes in GABAA receptor expression in the thalamus of the stargazer mouse model of absence epilepsy

Absence seizures are known to result from disturbances within the cortico-thalamocortical network, which remains partially synchronous under normal conditions but switches to a state of hypersynchronicity and hyperexcitability during absence seizures. There is evidence to suggest that impaired GABAergic inhibitory function within the thalamus could contribute to the generation of hypersynchronous oscillations in some animal models of absence epilepsy. Recently, we demonstrated region-specific alterations in the tissue expression level of GABAA receptors (GABAARs) α1 and β2 subunits within the thalamus of the stargazer mouse model of absence epilepsy. In the present study we investigated whether changes in these subunits also occur at synapses in the ventral posterior (VP) complex where they are components of phasic GABAAR receptors. Postembedding immunogold cytochemistry and electron microscopy were used to analyze the relative synaptic expression of α1 and β2 subunits in the VP thalamic region in epileptic stargazer mice compared to their non-epileptic littermates. We show that there is a significant increase in expression of α1 and β2 subunits (53.6% and 45.8%, respectively) at synapses in the VP region of stargazers, indicative of an increase in phasic GABAARs at thalamocortical (TC) relay neurons. Furthermore, we investigated whether tissue expression of GABAAR subunits α4 and δ, which constitute part of tonic GABAARs in the VP region, is altered in the stargazer mouse. Semi-quantitative Western blotting showed a significant increase in GABAAR α4 and δ subunits in the VP region of stargazer thalamus, which would indicate an increase in tonic GABAAR expression. Our findings show that there are changes in the levels of both phasic and tonic GABAARs in the VP thalamus; altered GABAergic inhibition within the VP could be one of many mechanisms contributing to the generation of absence seizures in this model.

Complex genetic interactions in a mouse model of absence epilepsy.

Complex genetic interactions in a mouse model of absence epilepsy.

Combining machine learning and simulations of a morphologically realistic model to study modulation of neuronal activity in cerebellar nuclei during absence epilepsy

Epileptic absence seizures are characterized by synchronized oscillatory activity in the cerebral cortex that can be recorded as so-called spike-and-wave discharges (SWDs) by electroencephalogram. Although the cerebral cortex and the directly connected thalamus are paramount to this particular form of epilepsy, several other parts of the mammalian brain are likely to influence this oscillatory activity. We have recently shown that some of the cerebellar nuclei (CN) neurons, which form the main output of the cerebellum, show synchronized oscillatory activity during episodes of cortical SWDs in two independent mouse models of absence epilepsy [1]. The CN neurons that show this significant correlation with the SWDs are deemed to “participate” in the seizure activity and are therefore used in our current study designed to unravel the potential causes of such oscillatory firing patterns. Initially, we set out to study if different types of CN neurons are more prone to show modulated firing patterns during seizure activity. We applied Growing Neural Gas (GNG) [2], an unsupervised clustering algorithm, on the interictal activity, i.e., firing patterns recorded in between seizures, using the measures CV, log-interval entropy, permutation entropy and firing rate. Three main groups of neurons were found by the clustering algorithm, in which the neurons were predominantly participating in the seizures. These can be seen on Figure ​Figure11 as the green, yellow and pale blue crosses (+). Moreover, these three clusters have the highest CV (and therefore more irregular) and higher log-interval entropy (more unpredictable). Further, we used a Gaussian Process Regression model [3] to predict the extent of participation of the neurons in the seizure activity, based on two measures: Z-score of mean power at seizure frequency (6-9Hz) (FFT based Z-score) and modulation frequency. These characterize the extent to which CN neurons phase-lock their spiking activity to the spikes in the EEG during seizures. We achieved a good prediction rate (r = 0.56, p < 0.05 for FFT based Z-score; r = 0.45, p < 0.05 for modulation frequency) using this method. Also, we are using a compartmental model of a CN neuron with realistic morphology [4] to investigate the input conditions that can generate interictal activity found in the participating neurons. Our results indicate that bursting in the Purkinje cell or mossy fiber input can cause behavior that is similar to the interictal activity found in participating neurons. The black dot in Figure ​Figure11 shows the output from the CN neuron model, provided with a bursting Purkinje cell input, when it is subjected to clustering with the experimental data. Desynchronization of the burst occurrence in the input did not alter the position of the data point drastically[0]. Currently, we are in the process of applying an evolutionary algorithm to explore in detail the input conditions that can that lead to the spiking behavior that is associated with seizures. Figure 1 shows 2D projection, using principal component analysis, of the clusters formed as a result of GNG clustering of the CN neuron interictal activity using CV, Log-Interval Entropy, Firing rate and Permutation Entropy. The crosses (+) indicate cells that ...

Regionally specific expression of high-voltage-activated calcium channels in thalamic nuclei of epileptic and non-epileptic rats.

The polygenic origin of generalized absence epilepsy results in dysfunction of ion channels that allows the switch from physiological asynchronous to pathophysiological highly synchronous network activity. Evidence from rat and mouse models of absence epilepsy indicates that altered Ca(2+) channel activity contributes to cellular and network alterations that lead to seizure activity. Under physiological circumstances, high voltage-activated (HVA) Ca(2+) channels are important in determining the thalamic firing profile. Here, we investigated a possible contribution of HVA channels to the epileptic phenotype using a rodent genetic model of absence epilepsy. In this study, HVA Ca(2+) currents were recorded from neurons of three different thalamic nuclei that are involved in both sensory signal transmission and rhythmic-synchronized activity during epileptic spike-and-wave discharges (SWD), namely the dorsal part of the lateral geniculate nucleus (dLGN), the ventrobasal thalamic complex (VB) and the reticular thalamic nucleus (NRT) of epileptic Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) and non-epileptic August Copenhagen Irish (ACI) rats. HVA Ca(2+) current densities in dLGN neurons were significantly increased in epileptic rats compared with non-epileptic controls while other thalamic regions revealed no differences between the strains. Application of specific channel blockers revealed that the increased current was carried by L-type Ca(2+) channels. Electrophysiological evidence of increased L-type current correlated with up-regulated mRNA and protein expression of a particular L-type channel, namely Cav1.3, in dLGN of epileptic rats. No significant changes were found for other HVA Ca(2+) channels. Moreover, pharmacological inactivation of L-type Ca(2+) channels results in altered firing profiles of thalamocortical relay (TC) neurons from non-epileptic rather than from epileptic rats. While HVA Ca(2+) channels influence tonic and burst firing in ACI and WAG/Rij differently, it is discussed that increased Cav1.3 expression may indirectly contribute to increased robustness of burst firing and thereby the epileptic phenotype of absence epilepsy.

Physiological and genetic analysis of multiple sodium channel variants in a model of genetic absence epilepsy

In excitatory neurons, SCN2A (NaV1.2) and SCN8A (NaV1.6) sodium channels are enriched at the axon initial segment. NaV1.6 is implicated in several mouse models of absence epilepsy, including a missense mutation identified in a chemical mutagenesis screen (Scn8aV929F). Here, we confirmed the prior suggestion that Scn8aV929F exhibits a striking genetic background-dependent difference in phenotypic severity, observing that spike-wave discharge (SWD) incidence and severity are significantly diminished when Scn8aV929F is fully placed onto the C57BL/6J strain compared with C3H. Examination of sequence differences in NaV subunits between these two inbred strains suggested NaV1.2V752F as a potential source of this modifier effect. Recognising that the spatial co-localisation of the NaV channels at the axon initial segment (AIS) provides a plausible mechanism for functional interaction, we tested this idea by undertaking biophysical characterisation of the variant NaV channels and by computer modelling. NaV1.2V752F functional analysis revealed an overall gain-of-function and for NaV1.6V929F revealed an overall loss-of-function. A biophysically realistic computer model was used to test the idea that interaction between these variant channels at the AIS contributes to the strain background effect. Surprisingly this modelling showed that neuronal excitability is dominated by the properties of NaV1.2V752F due to “functional silencing” of NaV1.6V929F suggesting that these variants do not directly interact. Consequent genetic mapping of the major strain modifier to Chr 7, and not Chr 2 where Scn2a maps, supported this biophysical prediction. While a NaV1.6V929F loss of function clearly underlies absence seizures in this mouse model, the strain background effect is apparently not due to an otherwise tempting Scn2a variant, highlighting the value of combining physiology and genetics to inform and direct each other when interrogating genetic complex traits such as absence epilepsy.

Altered thalamic GABAA‐receptor subunit expression in the stargazer mouse model of absence epilepsy

Absence seizures, also known as petit mal seizures, arise from disruptions within the cortico-thalamocortical network. Interconnected circuits within the thalamus consisting of inhibitory neurons of the reticular thalamic nucleus (RTN) and excitatory relay neurons of the ventral posterior (VP) complex, generate normal intrathalamic oscillatory activity. The degree of synchrony in this network determines whether normal (spindle) or pathologic (spike wave) oscillations occur; however, the cellular and molecular mechanisms underlying absence seizures are complex and multifactorial and currently are not fully understood. Recent experimental evidence from rodent models suggests that regional alterations in γ-aminobutyric acid (GABA)ergic inhibition may underlie hypersynchronous oscillations featured in absence seizures. The aim of the current study was to investigate whether region-specific differences in GABAA receptor (GABAAR) subunit expression occur in the VP and RTN thalamic regions in the stargazer mouse model of absence epilepsy where the primary deficit is in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression. Immunofluorescence confocal microscopy and semiquantitative Western blot analysis were used to investigate region-specific changes in GABAAR subunits in the thalamus of the stargazer mouse model of absence epilepsy to determine whether changes in GABAergic inhibition could contribute to the mechanisms underlying seizures in this model of absence epilepsy. Immunofluorescence confocal microscopy revealed that GABAAR α1 and β2 subunits are predominantly expressed in the VP, whereas α3 and β3 subunits are localized primarily in the RTN. Semiquantitative Western blot analysis of VP and RTN samples from epileptic stargazers and their nonepileptic littermates showed that GABAAR α1 and β2 subunit expression levels in the VP were significantly increased (α1: 33%, β2: 96%) in epileptic stargazers, whereas α3 and β3 subunits in the RTN were unchanged in the epileptic mice compared to nonepileptic control littermates. These findings suggest that region-specific differences in GABAAR subunits in the thalamus of epileptic mice, specifically up-regulation of GABAARs in the thalamic relay neurons of the VP, may contribute to generation of hypersynchronous thalamocortical activity in absence seizures. Understanding region-specific differences in GABAAR subunit expression could help elucidate some of the cellular and molecular mechanisms underlying absence seizures and thereby identify targets by which drugs can modulate the frequency and severity of epileptic seizures. Ultimately, this information could be crucial for the development of more specific and effective therapeutic drugs for treatment of this form of epilepsy.

Paradoxical proepileptic response to NMDA receptor blockade linked to cortical interneuron defect in stargazer mice

Paradoxical seizure exacerbation by anti-epileptic medication is a well-known clinical phenomenon in epilepsy, but the cellular mechanisms remain unclear. One possibility is enhanced network disinhibition by unintended suppression of inhibitory interneurons. We investigated this hypothesis in the stargazer mouse model of absence epilepsy, which bears a mutation in stargazin, an AMPA receptor trafficking protein. If AMPA signaling onto inhibitory GABAergic neurons is impaired, their activation by glutamate depends critically upon NMDA receptors. Indeed, we find that stargazer seizures are exacerbated by NMDA receptor blockade with CPP (3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid) and MK-801, whereas other genetic absence epilepsy models are sensitive to these antagonists. To determine how an AMPA receptor trafficking defect could lead to paradoxical network activation, we analyzed stargazin and AMPA receptor localization and found that stargazin is detected exclusively in parvalbumin-positive (PV +) fast-spiking interneurons in somatosensory cortex, where it is co-expressed with the AMPA receptor subunit GluA4. PV + cortical interneurons in stargazer show a near twofold decrease in the dendrite:soma GluA4 expression ratio compared to wild-type (WT) littermates. We explored the functional consequence of this trafficking defect on network excitability in neocortical slices. Both NMDA receptor antagonists suppressed 0 Mg 2 +-induced network discharges in WT but augmented bursting in stargazer cortex. Interneurons mediate this paradoxical response, since the difference between genotypes was masked by GABA receptor blockade. Our findings provide a cellular locus for AMPA receptor-dependent signaling defects in stargazer cortex and define an interneuron-dependent mechanism for paradoxical seizure exacerbation in absence epilepsy.

Expanded alternative splice isoform profiling of the mouse Cav3.1/α1G T-type calcium channel

BackgroundAlternative splicing of low-voltage-activated T-type calcium channels contributes to the molecular and functional diversity mediating complex network oscillations in the normal brain. Transcript scanning of the human CACNA1G gene has revealed the presence of 11 regions within the coding sequence subjected to alternative splicing, some of which enhance T-type current. In mouse models of absence epilepsy, elevated T-type calcium currents without clear increases in channel expression are found in thalamic neurons that promote abnormal neuronal synchronization. To test whether enhanced T-type currents in these models reflect pathogenic alterations in channel splice isoforms, we determined the extent of alternative splicing of mouse Cacna1g transcripts and whether evidence of altered transcript splicing could be detected in mouse absence epilepsy models.ResultsTranscript scanning of the murine Cacna1g gene detected 12 regions encoding alternative splice isoforms of Cav3.1/α1G T-type calcium channels. Of the 12 splice sites, six displayed homology to the human CACNA1G splice sites, while six novel mouse-specific splicing events were identified, including one intron retention, three alternative acceptor sites, one alternative donor site, and one exon exclusion. In addition, two brain region-specific alternative splice patterns were observed in the cerebellum. Comparative analyses of brain regions from four monogenic absence epilepsy mouse models with altered thalamic T-type currents and wildtype controls failed to reveal differences in Cacna1g splicing patterns.ConclusionThe determination of six novel alternative splice sites within the coding region of the mouse Cacna1g gene greatly expands the potential biophysical diversity of voltage-gated T-type channels in the mouse central nervous system. Although alternative splicing of Cav3.1/α1G channels does not explain the enhancement of T-type current identified in four mouse models of absence epilepsy, post-transcriptional modification of T-type channels through this mechanism may influence other developmental neurological phenotypes.

Aberrant GABAAReceptor Expression in the Dentate Gyrus of the Epileptic Mutant Mouse Stargazer

Stargazer (stg) mutant mice fail to express stargazin [transmembrane AMPA receptor regulatory protein gamma2 (TARPgamma2)] and consequently experience absence seizure-like thalamocortical spike-wave discharges that pervade the hippocampal formation via the dentate gyrus (DG). As in other seizure models, the dentate granule cells of stg develop elaborate reentrant axon collaterals and transiently overexpress brain-derived neurotrophic factor. We investigated whether GABAergic parameters were affected by the stg mutation in this brain region. GABA(A) receptor (GABAR) alpha4 and beta3 subunits were consistently upregulated, GABAR delta expression appeared to be variably reduced, whereas GABAR alpha1, beta2, and gamma2 subunits and the GABAR synaptic anchoring protein gephyrin were essentially unaffected. We established that the alpha4 betagamma2 subunit-containing, flunitrazepam-insensitive subtype of GABARs, not normally a significant GABAR in DG neurons, was strongly upregulated in stg DG, apparently arising at the expense of extrasynaptic alpha4 betadelta-containing receptors. This change was associated with a reduction in neurosteroid-sensitive GABAR-mediated tonic current. This switch in GABAR subtypes was not reciprocated in the tottering mouse model of absence epilepsy implicating a unique, intrinsic adaptation of GABAergic networks in stg. Contrary to previous reports that suggested that TARPgamma2 is expressed in the dentate, we find that TARPgamma2 was neither detected in stg nor control DG. We report that TARPgamma8 is the principal TARP isoform found in the DG and that its expression is compromised by the stargazer mutation. These effects on GABAergic parameters and TARPgamma8 expression are likely to arise as a consequence of failed expression of TARPgamma2 elsewhere in the brain, resulting in hyperexcitable inputs to the dentate.

Calcium Channel “Gaiting” and Absence Epilepsy

Dysfunction of the Brain Calcium Channel Cav2.1 in Absence Epilepsy and Episodic Ataxia Imbrici P, Jaffe SL, Eunson LH, Davies NP, Herd C, Robertson R, Kullmann DM, Hanna MG Brain 2004;127(Pt 12):2682–2692 The molecular basis of idiopathic generalized epilepsy remains poorly understood. Absence epilepsy with 3-Hz spike–wave EEG is one of the most common human epilepsies and is associated with significant morbidity. Several spontaneously occurring genetic mouse models of absence epilepsy are caused by dysfunction of the P/Q-type voltage-gated calcium channel CaV2.1. Such mice exhibit a primary generalized spike–wave EEG, with frequencies in the range of 5 to 7 Hz, often associated with ataxia, evidence of cerebellar degeneration and abnormal posturing. Previously, we identified a single case of severe primary generalized epilepsy with ataxia associated with CaV2.1 dysfunction, suggesting a possible link between this channel and human absence epilepsy. We now report a family in which absence epilepsy segregates in an autosomal dominant fashion through three generations. Five members exhibit a combination of absence epilepsy (with 3-Hz spike–wave) and cerebellar ataxia. In patients with the absence epilepsy/ataxia phenotype, genetic marker analysis was consistent with linkage to the CACNA1A gene on chromosome 19, which encodes the main pore-forming α1A subunit of CaV2.1 channels (CaV2.1 α1). DNA sequence analysis identified a novel point mutation resulting in a radical amino acid substitution (E147K) in CaV2.1 α1, which segregated with the epilepsy/ataxia phenotype. Functional expression studies using human CACNA1A cDNA demonstrated that the E147K mutation results in impairment of calcium channel function. Impaired function of the brain calcium channel CaV2.1 may have a central role in the pathogenesis of certain cases of primary generalized epilepsy, particularly when associated with ataxia, which may be wrongly ascribed to anticonvulsant medication.

Dysfunction of the brain calcium channel CaV2.1 in absence epilepsy and episodic ataxia.

The molecular basis of idiopathic generalized epilepsy remains poorly understood. Absence epilepsy with 3 Hz spike-wave EEG is one of the most common human epilepsies, and is associated with significant morbidity. Several spontaneously occurring genetic mouse models of absence epilepsy are caused by dysfunction of the P/Q-type voltage-gated calcium channel CaV2.1. Such mice exhibit a primary generalized spike-wave EEG, with frequencies in the range of 5-7 Hz, often associated with ataxia, evidence of cerebellar degeneration and abnormal posturing. Previously, we identified a single case of severe primary generalized epilepsy with ataxia associated with CaV2.1 dysfunction, suggesting a possible link between this channel and human absence epilepsy. We now report a family in which absence epilepsy segregates in an autosomal dominant fashion through three generations. Five members exhibit a combination of absence epilepsy (with 3 Hz spike-wave) and cerebellar ataxia. In patients with the absence epilepsy/ataxia phenotype, genetic marker analysis was consistent with linkage to the CACNA1A gene on chromosome 19, which encodes the main pore-forming alpha1A subunit of CaV2.1 channels (CaV2.1alpha1). DNA sequence analysis identified a novel point mutation resulting in a radical amino acid substitution (E147K) in CaV2.1alpha1, which segregated with the epilepsy/ataxia phenotype. Functional expression studies using human CACNA1A cDNA demonstrated that the E147K mutation results in impairment of calcium channel function. Impaired function of the brain calcium channel CaV2.1 may have a central role in the pathogenesis of certain cases of primary generalized epilepsy, particularly when associated with ataxia, which may be wrongly ascribed to anticonvulsant medication.

Insights from mouse models of absence epilepsy into Ca2+ channel physiology and disease etiology.

1. Changes in intracellular Ca2+ ([Ca2+]i) levels provide signals that allow neurons to respond to a host of external stimuli. A major mechanism for elevating [Ca2+]i is the influx of extracellular Ca2+ through voltage-gated channels (Ca(V)) in the plasma membrane. Malfunction in Ca(V) due to mutations in genes encoding channel proteins are increasingly being implicated in causing disease conditions, termed channelopathies. 2. Seven spontaneous mutations with cerebellar ataxia and generalized absence epilepsy have been identified in mice (tottering, leaner, rolling Nagoya, rocker, lethargic, ducky, and stargazer), and these overlapping phenotypes are directly related to mutations in genes encoding the four separate subunits that together form the multimeric neuronal Ca(V) complex. 3. The discovery and systematic analysis of these animal models is helping to clarify how different mutations affect channel function and how altered channel function produces disease.

Increased number of GABA B receptors in the lethargic ( lh/lh) mouse model of absence epilepsy

This study begins to explore mechanisms underlying the role of GABA B receptors in absence seizures in lethargic ( lh/lh) mice. To test the hypothesis that alterations intrinsic to the GABA B receptor underlie enhanced synaptic activation of these receptors in absence seizures, we measured GABA-displaceable [ 3H]baclofen binding to neocortical plasma membranes prepared from lh/lh and wild (+/+) age-matched congenic mice. The number ( B max) of binding sites was significantly greater (20%) in lh/lh (4.2pmol/mg protein, n = 43 pairs, P < 0.02) than in +/+ mice (3.3 pmol/mg protein) in an age-dependent manner. Interestingly, the subject of lh/lh mice with greater seizure frequency (40–70 seizures/15 min, measured by bipolar electrodes implanted into neocortex; n = 11) had a significantly greater B max (P < 0.003) than the subset with lower seizure frequency (1–20 seizures/15 min; n = 11). The equilibrium dissociation constant ( K d) was unchanged (60 nM in both). The K d of both strains was inhibited to an equal degree by the nonhydrolysable GTP analogue 5′-guanylimido-diphosphate [Gpp(NH)p]. The increased number of GABA B binding sites was selective, because binding to NMDA sites ([ 3H]glutamate binding) and to GABA A sites ([ 3H]muscimol binding) was not significantly different in the two strains. These data suggest that the increased number of GABA B receptors in lh/lh mice underlies enhanced synaptic activation of these receptors. Together with evidence that GABA B receptor activation can produce disinhibition, our data support a role for GABA B receptors in the expression of absence seizures in lh/lh mice.