Tottering Mice Research Articles

Mutations in the Cacnl1a4 calcium channel gene are associated with seizures, cerebellar degeneration, and ataxia in tottering and leaner mutant mice.

Tottering and leaner, two mutations of the mouse tottering locus, have been studied extensively as models for human epilepsy. Here we describe the isolation, mapping, and expression analysis of Cacnl1a4, a gene encoding the alpha subunit of a proposed P-type calcium channel, and also report the physical mapping and expression patterns of the orthologous human gene. DNA sequencing and gene expression data demonstrate that Cacnl1a4 mutations are the primary cause of seizures and ataxia in tottering and leaner mutant mice, and suggest that tottering locus mutations and human diseases, episodic ataxia 2 and familial hemiplegic migraine, represent mutations in mouse and human versions of the same channel-encoding gene.

Absence Epilepsy in Tottering Mutant Mice Is Associated with Calcium Channel Defects

Mutations at the mouse tottering ( tg) locus cause a delayed-onset, recessive neurological disorder resulting in ataxia, motor seizures, and behavioral absence seizures resembling petit mal epilepsy in humans. A more severe allele, leaner ( tg la ), also shows a slow, selective degeneration of cerebellar neurons. By positional cloning, we have identified an α 1A voltage-sensitive calcium channel gene that is mutated in tg and tg la mice. The α 1A gene is widely expressed in the central nervous system with prominent, uniform expression in the cerebellum. α 1A expression does not mirror the localized pattern of cerebellar degeneration observed in tg la mice, providing evidence for regional differences in biological function of α 1A channels. These studies define the first mutations in a mammalian central nervous system–specific voltage-sensitive calcium channel and identify the first gene involved in absence epilepsy.

Reduced function of gamma-aminobutyric acidA receptors in tottering mouse brain: role of cAMP-dependent protein kinase.

The single-locus mutant mouse tottering (tg) displays spontaneous seizures that resemble those in human petit-mal epilepsy. In order to examine alterations in GABAA receptor function which could arise as a result of this mutation, the influx of 36Cl- was determined using microsacs (membrane vesicles) isolated from the brain of tg/tg and coisogenic C57BL/6J (+/+) control mice. In microsacs from both tg/tg and +/+ strains, the maximum level of 36Cl- uptake induced by 50 microM GABA was observed during five seconds of incubation at 28 degrees C. Compared to +/+, the GABA-dependent 36Cl- uptake in tg/tg microsacs was significantly lower and faded rapidly during longer incubations. The levels of gated 36Cl- uptake in tg/tg microsacs were 45 +/- 6.3%, 65 +/- 9.9%, and 33 +/- 6.1% of control (+/+) values for 3-, 5-, and 10-s incubations, respectively. GABAA receptor-specific agonists (30 microM), muscimol, isoguvacine and THIP (4,5,6,7-tetrahydroisoazolo-[5,4-c]pyridin-3-ol) induced 36Cl- influx in the order muscimol > GABA > isoguvacine > THIP. This order was similar for both strains, but the agonist-dependent influx was always significantly lower in tg/tg compared to +/+. Treatment of the microsacs with 10 microM H-89, a membrane-permeant inhibitor of the cAMP-dependent protein kinase (protein kinase A, PKA), was without effect on GABA-gated 36Cl- uptake in +/+, but increased the gated uptake in tg/tg microsacs by 44 +/- 16%. PKA was assayed using [gamma-32]ATP and kemptide as the substrate. Triton X-100 (0.1%) increased both the basal and 8-Br-cAMP dependent PKA activity in microsacs by 3-4 four fold, showing that most of the enzyme was intravesicular. In the presence of Triton, the basal activity of PKA in the tg/tg preparations was twice that of +/+, while the strain difference was no longer apparent in assays containing 8-Br-cAMP. The data suggest that an abnormal elevation of protein kinase A activity in tottering mouse brain contributes to an impairment of GABAA receptor function. It is suggested that the resulting loss of inhibition could play a role in induction of the seizures which characterize the mutant phenotype.

Mutant mouse strains as models for in vivo radiotracer evaluations: [ 11C]methoxytetrabenazine ([ 11C]MTBZ) in Tottering mice

The regional brain distribution of [ 11C]methoxytetrabenazine ([ 11C]MTBZ), a high affinity radioligand for the vesicular monoamine transporter, has been examined in normal and tottering mice, which are neurological mutants. The tottering mice show significantly higher radiotracer accumulation (150–195% of controls) in all brain regions examined (striatum, cortex, hippocampus, hypothalamus, cerebellum and thalamus). The increases in [ 11C]MTBZ binding correlate ( r = 0.91) with the reported increases in norepinephrine concentrations in these regions, and are consistent with the noradrenergic hyperinnervation characteristic of the tottering mutant mouse. These studies demonstrate that a mutant mouse model of increased innervation can be used to evaluate the sensitivity of an in vivo radiotracer measure, in this case [ 11C]MTBZ binding to vesicular transporters, to an increase in the numbers of binding sites in specific regions of the brain.

Analysis of voltage-gated and synaptic conductances contributing to network excitability defects in the mutant mouse tottering

1. Intracellular current- and voltage-clamp recordings were carried out in CA3 pyramidal neurons from hippocampal slices of adult tg/tg mice and their coisogenic C57BL/6J (+/+) controls with the use of the single-electrode switch-clamp technique. The principal aim of this study was to investigate the mechanisms responsible for the tg gene-linked prolongation (mean 60%) of a giant synaptic response, the potassium-induced paroxysmal depolarizing shift (PDS) at depolarized membrane potentials (Vm -47 to -54 mV) during synchronous network bursting induced by 10 mM potassium ([K+]o). 2. To examine the role of intrinsic voltage-dependent conductances underlying the mutant PDS prolongation, neurons were voltage clamped by the use of microelectrodes filled with 100 mM QX-314 or QX-222 chloride (voltage-gated sodium channel blockers) and 2 M cesium sulphate (potassium channel blocker). The whole-cell currents active during the PDS showed a significantly prolonged duration (mean 34%) at depolarized Vms in tg/tg compared with +/+ cells, indicating that a defect in voltage-dependent conductances is unlikely to completely account for the mutant phenotype. 3. Bath application of 40 microM (DL)-2-aminophosphonovalerate (DL-APV) produced a 30% reduction in PDS duration in both genotypes but failed to significantly alter the tg gene-linked prolongation compared with the wild type. These data indicate that the mutant PDS abnormality does not result from a selective increase of the N-methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic component. 4. Blockade of gamma-aminobutyric acid-A (GABAA) transmission with picrotoxin (50 microM) or bicuculline (1-5 microM) completely eliminated the difference in PDS duration between the genotypes. Furthermore, although both GABAA receptor antagonists increased the mean PDS duration in +/+ neurons, they did not significantly alter it in tg/tg neurons. These findings are consistent with a reduction in GABAA receptor-mediated synaptic inhibition during bursting in the tg CA3 hippocampal network. 5. To test this hypothesis, bursting CA3 pyramidal neurons were loaded intracellularly with chloride by the use of KCl-filled microelectrodes to examine the effect of reversing the hyperpolarizing chloride-dependent GABAA receptor-mediated inhibitory postsynaptic component of the PDS. Chloride loading prolonged PDS duration in both genotypes, but the increase was greater in +/+ than in tg/tg neurons, indicating that a smaller GABAA inhibitory postsynaptic potential (IPSP) component was reversed in the mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of the effects of increased potassium and of adenosine on hippocampal neurons from normal and genetically epileptic tg/tg mice.

Tottering mice are an experimental model of genetically determined generalized epilepsy of the absence type. We investigated possible mechanisms underlying epileptogenic hyperexcitability in these mice by studying input/output (I/O) curves of the extracellular response of CA1 neurons to stratum radiatum stimulation in hippocampal slices maintained in vitro. Increases in extracellular potassium are considered to contribute to epileptogenesis, whereas adenosine has been proposed to be an endogenous antiepileptic agent. Moderate elevations (+2 mM) of extracellular K+ concentrations induced a significantly smaller increase of this response (leftward shift of the input/output curves) in slices from epileptic mice as compared with controls. Perfusion of slices with adenosine 10 microM decreased excitability in both groups of slices, especially with regard to response threshold. Adenosine more effectively decreased the responses elicited by low-intensity stimulation than those elicited by high intensity. No significant difference between the groups of slices was observed. On the basis of the present data, it is unlikely that the previously observed hyperexcitability of hippocampal neurons of tottering mice results from a genetically altered sensitivity to moderate increases in [K+]o or to adenosine.

A burst-dependent hippocampal excitability defect elicited by potassium at the developmental onset of spike-wave seizures in the Tottering mutant

Hippocampal CA3 pyramidal neurons in the adult epileptic mutant mouse tottering (tg) show normal intrinsic membrane properties, yet fire abnormally prolonged paroxysmal depolarizing shifts (PDS) during in vitro exposure to elevated extracellular potassium solutions. Intracellular recordings in immature mutants reveal that this network burst abnormality is present during the developmental period that coincides with the onset of seizures in the mutant (19–20 postnatal days), and is significantly more pronounced at this age than at adulthood. These data are inconsistent with the hypothesis that the mutant PDS prolongation represents a secondary consequence of a prolonged history of repeated seizures and suggest that it may reflect a cellular epileptogenic phenotype more directly related to the primary neuropathological expression of the tg gene.

Development of the paramedian lobule of the cerebellum in wild-type and tottering mice.

The mutant mouse tottering, (tg/tg), and the compound heterozygote mouse (tg/tg1a) exhibit three neurological disorders: ataxia, petit mal-like absence seizures and myoclonic intermittent movement disorders which are independent of the absence seizures. The tottering mouse carries an autosomal recessive single gene mutation on chromosome 8, and behavioral symptoms are first observed in the 3rd to 4th week of age. Using an additional genetic marker, Oligosyndactyly (Os), it is possible to distinguish tg/tg and tg/tg1a mice from wild-type mice at birth; nonaffected heterozygous littermates carry the Os mutation while tottering and compound heterozygous mice do not carry the Os gene. Similar to neurons found elsewhere in the brain, cerebellar Purkinje cells in both the wild-type and mutant mice were found to decrease in diameter with maturation. Forebrain weight, hindbrain weight, Purkinje cell dimensions and the thickness of the molecular layer in the paramedian lobule of the cerebellum in mutant mice were found to be reduced in mutants after, but not prior to the onset of behavioral symptoms.

Possible mechanisms underlying hyperexcitability in the epileptic mutant mouse tottering.

Tottering mice present a useful experimental model of genetically determined generalized epilepsy of the absence type. In electrophysiological recordings from hippocampal slices in vitro we found that the postsynaptic excitability (firing threshold) of pyramidal neurons in the CA1 area of tg/tg slices was significantly higher than that of normal slices. In spite of this hyperexcitability, in vitro epileptiform discharges were not observed spontaneously, or upon provocation by intracellular depolarizing pulses, or in response to moderate elevations (+2 mM) in extracellular potassium. The latter elevations actually induced significantly smaller increases in the CA1 synaptic responses of tg/tg as compared to normal slices. The hyperexcitability of tottering neurons could not be explained in terms of altered membrane electrical properties or any reduction of synaptic inhibition or increased capacity for long-term potentiation. Responses to noradrenaline, histamine and adenosine, as well as to the release of N-methyl-D-aspartate channels--by eliminating Mg(2+)--were comparable in tg/tg and normal slices. These studies show that hyperexcitability can be co-inherited with epilepsy and in this model its expression can be maintained in vitro. The neuronal mechanism of this expression remains elusive, as it does not appear to include some features known to be shared by experimental models of chemically or electrically induced epilepsy.

A comparison of recurrent inhibition and of paired-pulse facilitation in hippocampal slices from normal and genetically epileptic mice

Tottering mice exhibit inherited generalized epilepsy of the ‘absence’type. In hippocampal slices from these mutant mice studied in vitro, pairing an alvear antidromic stimulus to an orthodromic one revealed a strong recurrent inhibition (RI) of CA1 pyramidal neurons. RI was maximal at 10 ms inter-pulse interval (IPI 70% decrease of population spike, PS) gradually decreasing to 15% at 320 ms IPI. At 10 ms IPI it shifted the input/output curves to the right and decreased maximum PS. In the group of slices from epileptic mice the early part of RI (2.5–60 ms) was indistinguishable from that of normal mice, with respect to both its strength and its lability to activity-dependent decrement induced by a train of antidromic stimuli (8 s, 5 Hz). However, the delayed part (80–320 ms) was slightly stronger in the epileptic group. Also in this group only the train of antidromic pulses caused a significant and lasting decrease in the unconditioned orthodromic PS. Paired-pulse facilitation was equally strong in the 2 groups of slices. It is concluded that mechanisms underlying epileptogenic hyperexcitability in the tottering mutant may not include a failure of inhibition, at least in the CA 1 area of the hippocampus. On the contrary some inhibitory mechanisms may be stronger.

Glutathione levels in specific brain regions of genetically epileptic ( tg/tg) mice

The tottering ( tg/tg) mouse is a genetic model of human generalized epilepsy; these mice exhibit spontaneous absence seizures accompanied by bilaterally synchronous spike-wave discharges (6). The mechanism(s) for seizure activity are unknown in these mice. Several recent studies have suggested that membrane lipid peroxidation may be causally involved in some forms of experimentally induced epilepsies (18). Since reduced glutathione (GSH) is the most important free radical scavenging compound in vivo that can prevent membrane lipid peroxidation, the objective of this study was to investigate GSH concentrations in specific central nervous system regions of genetically epileptic, tg/tg, mice as compared to age-matched controls. Three brain regions, cerebellum, hippocampus, and occipital cortex, were dissected, weighed and the concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) were measured in each of these tissues. GSH content was significantly lower in the occipital cortex of tg/tg mice compared to controls; no differences were observed in the other two brain regions examined. Total GSH content (GSH plus 2 × GSSG) paralleled GSH concentration differences. GSSG content from tg/tg mice was lower in the hippocampus and occipital cortex, compared to controls. This is the first report of an association between decreased central nervous system glutathione concentrations and seizure activity in animals exhibiting generalized seizures.

Neurotransmitter Receptors in Adult Tottering (tg/tg) Mice

Neurotransmitter receptor binding was analyzed in adult tottering (tg/tg) and control wild-type mice. Saturation studies were performed to analyze the density of muscarinic cholinergic receptors in whole brain, cortical, and hippocampal homogenates of 8-9-week-old animals. Scatchard plot analysis was also performed to determine the density and affinity of alpha-adrenergic and beta-adrenergic receptors. No significant difference in Bmax or Kd values was identified between adult tottering and control mice in any of the tissue preparations. The amount of radioligand binding to 5-hydroxytryptamine 1A (5-HT1A), non-5-HT1A, 5-HT2, dopamine D2, and benzodiazepine receptors was also identical in tottering and control mice. These findings suggest that the epilepsy expressed by adult tottering mice does not result from alterations in the density or affinity of the neurotransmitter receptors studied.

Altered hippocampal network excitability in the hypernoradrenergic mutant mouse tottering

A latent, gene-linked alteration of hippocampal network excitability in tg/tg mutant mice was unmasked in vitro by convulsant-activated synchronous neuronal discharges. Exposure to elevated extracellular potassium ions or 4-aminopyridine, but not picrotoxin, revealed an abnormally prolonged network discharge duration in the mutant CA3 pyramidal cell region. In both phenotypes, noradrenaline, and a selective β-noradrenergic receptor agonist, isoproterenol, reversibly accelerated the frequency of the discharges. These findings identify an intrinsic alteration in the excitability of an isolated neuronal network in a model of inherited generalized spike-wave epilepsy, and further implicate noradrenergic mechanisms in the temporal modulation of hippocampal synchronization and epileptogenesis.

Long-term enhancement of postsynaptic excitability after brief exposure to Mg 2+-free medium in normal and epileptic mice

Brief exposure to Mg 2+-free medium (MFM) enhanced the population response of CA1 neurons to stratum radiatum stimulation in hippocampal slices from normal (+/?) and epileptic tottering (tg/tg) mice. The enhancement was maintained in both groups for at least 2 h following reperfusion with normal medium (NM). Excitability curves obtained from the extracellular records suggest that, while both synaptic activation and postsynaptic excitability are enhanced during MFM perfusion, only the latter enhancement is maintained at significant levels after reperfusion with NM. The long-term increase in postsynaptic excitability was comparable in strength to that produced by long-term potentiation (LTP) inducing tetanic stimuli, was accompanied by an increase in the slope of the population spike/field excitatory postsynaptic potential (PS/fEPSP) curve and did not appear to depend on the induction of epileptiform activity by MFM. Both the short- and the long-term effects of MFM on synaptic activation and postsynaptic excitability were qualitatively similar in normal and epileptic mice and any quantitative differences were not statistically significant. Thus, epileptogenesis in the tottering mutant may not involve a change in the NMDA receptor-mediated control of excitability, at least in the CA1 area of hippocampus.

Membrane properties, response to amines and to tetanic stimulation of hippocampal neurons in the genetically epileptic mutant mouse tottering.

The petit-mal seizures of the "tottering" mutant mouse (tg) have been attributed to an exaggerated noradrenergic projection from locus coeruleus to the telencephalon (Noebels 1984). In order to investigate the possible epileptogenic mechanisms involved, we have compared hippocampal slices from epileptic (tg/tg) and phenotypically healthy (tg/+) mice. Resting potentials, action potentials and afterpotentials, membrane impedances and time constants were not significantly different in 11 neurons from each group. Bath application of noradrenaline, isoproterenol and histamine or a transient exposure to Mg++-free medium caused a long lasting increase in extracellularly recorded population spikes induced in CA1 by electrical stimulation of stratum radiatum. Isoproterenol blocked the calcium dependent afterhyperpolarization and accommodation of firing. Tetanization of afferent fibres evoked post-tetanic potentiation and long-term potentiation. All these results are qualitatively similar to those previously described in rats and guinea pigs and have revealed no significant difference between tg/tg and tg/+ mice.

The functional states of the thalamus and the associated neuronal interplay.

The functional states of the thalamus and the associated neuronal interplay.

Caffeine blocks absence seizures in the tottering mutant mouse.

The neurological tottering mutant mouse is characterized by frequent "absence" seizures accompanied by bilateral synchronous spike and wave EEG bursts. Under anesthesia, adult homozygous tottering mice were implanted with permanent epidural electrodes, and at least 7 days elapsed before electrocorticograms in unrestrained mice were scored for seizure incidence and duration. Caffeine (5, 10, 15 mg/kg, n = 8) injected intraperitoneally (i.p.) at the fourth hour of 8-h recording sessions significantly (p less than 0.001 for 10 and 15 mg) decreased seizure incidence as compared with control saline injections. Spike and wave bursts were eliminated during the 30 min after injection and reached 50% preinjection levels between the first and the second hour after injection. Another central nervous system (CNS) stimulating drug, amphetamine (1 mg/kg; n = 5), under identical conditions failed to decrease seizure incidence in this mutant.

Central adrenergic receptor changes in the inherited noradrenergic hyperinnervated mutant mouse tottering

Adrenergic receptor binding characteristics were analyzed in the mutant mouse tottering ( tg/tg), a single gene locus autosomal recessive mutation causing hyperinnervation by locus coeruleus neurons of their target regions, which results in epilepsy, Instead of the expected down-regulation of receptors due to the hyperinnervation, both [ 3H]prazosin ( α 1-receptor and [ 125I]iodopindolol (β-receptor) binding were normal in the tg/tg hippocampus, spinal cord and slightly increased in the cerebellum. This lack of postsynaptic receptor modulation in the target cells, combined with increased levels of norepinephrine due to the aberrant axon growth, may be critical factors in the expression of the abnormal spike-wave absence seizures in the tg/tg mouse.

Absence of modification in GABA and benzodiazepine binding and in choline acetyltransferase activity in brain areas of the epileptic mutant mouse tottering

1. In the tottering mutant mouse, which suffers from epilepsy and cerebellar ataxia, we examined whether possible changes in GABA, benzodiazepine receptors and choline acetyltransferase (ChAT) activity are implicated in the pathophysiology of these animals. 2. No alteration in GABA A and GABA B binding could be detected in cerebellar membranes of epileptic mice as compared to normal mice. 3. Benzodiazepine receptor density and affinity showed no statistical difference in cerebellar membranes of epileptic and normal mice. 4. The activity of ChAT determined in the cortices of epileptic and normal mice did not differ significantly between the two groups.

Congenital defects among the offspring of epileptic fathers: role of the genotype and phenytoin therapy in a mouse model.

The progeny of coisogenic C57BL/6J mice homozygous for either the tottering (tg/tg) or wildtype (+/+) allele were examined to determine the role of parental seizure disorders and paternal anticonvulsant drug therapy on the incidence of congenital malformations. Pregnancy outcomes in which one, both, or neither of the parents had an epileptic genotype were compared for various maternal and fetal reproductive parameters. In addition, the progeny from phenytoin-treated male tottering (tg/tg) mice with a spontaneous seizure disorder and from control untreated (+/+) male mice were similarly examined for evidence of congenital defects. The results of these experiments suggest that neither the parental genotype with respect to seizure disorders nor paternal anticonvulsant treatment with phenytoin is responsible for an increased incidence of congenital malformations.