Carol A. Robbins

Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy

Voltage-gated sodium channels (NaV) are critical for initiation of action potentials. Heterozygous loss-of-function mutations in NaV1.1 channels cause severe myoclonic epilepsy in infancy (SMEI). Homozygous null Scn1a−/− mice developed ataxia and died on postnatal day (P) 15 but could be sustained to P17.5 with manual feeding. Heterozygous Scn1a+/− mice had spontaneous seizures and sporadic deaths beginning after P21, with a notable dependence on genetic background. Loss of NaV1.1 did not change voltage-dependent activation or inactivation of sodium channels in hippocampal neurons. The sodium current density was, however, substantially reduced in inhibitory interneurons of Scn1a+/− and Scn1a−/− mice but not in their excitatory pyramidal neurons. An immunocytochemical survey also showed a specific upregulation of NaV1.3 channels in a subset of hippocampal interneurons. Our results indicate that reduced sodium currents in GABAergic inhibitory interneurons in Scn1a+/− heterozygotes may cause the hyperexcitability that leads to epilepsy in patients with SMEI.

Deletion of the KV1.1 Potassium Channel Causes Epilepsy in Mice

Mice lacking the voltage-gated potassium channel alpha subunit, K(V)1.1, display frequent spontaneous seizures throughout adult life. In hippocampal slices from homozygous K(V)1.1 null animals, intrinsic passive properties of CA3 pyramidal cells are normal. However, antidromic action potentials are recruited at lower thresholds in K(V)1.1 null slices. Furthermore, in a subset of slices, mossy fiber stimulation triggers synaptically mediated long-latency epileptiform burst discharges. These data indicate that loss of K(V)1.1 from its normal localization in axons and terminals of the CA3 region results in increased excitability in the CA3 recurrent axon collateral system, perhaps contributing to the limbic and tonic-clonic components of the observed epileptic phenotype. Axonal action potential conduction was altered as well in the sciatic nerve--a deficit potentially related to the pathophysiology of episodic ataxia/myokymia, a disease associated with missense mutations of the human K(V)1.1 gene.

SEIZURES AND NEURONAL DAMAGE IN MICE LACKING VESICULAR ZINC

Synaptically released zinc has neuromodulatory capabilities that could result in either inhibition or enhancement of neuronal excitability. To determine the net effects of vesicular zinc release in the brain in vivo, we examined seizure susceptibility and seizure-related neuronal damage in mice with targeted disruption of the gene encoding the zinc transporter, ZnT3 (ZnT3-/- mice). ZnT3-/- mice, which lack histochemically reactive zinc in synaptic vesicles, had slightly higher thresholds to seizures elicited by the GABA(A) antagonist, bicuculline, and no differences in seizure threshold were seen in response to pentylenetetrazol or flurothyl. However, ZnT3-/- mice were much more susceptible than wild-type mice to limbic seizures elicited by kainic acid, suggesting that the net effect of hippocampal zinc on acute seizures in vivo is inhibitory. The hippocampi of ZnT3-/- mice showed typical seizure-related neuronal damage in response to kainic acid, demonstrating that damage to the targets of zinc-containing neurons can occur independently of synaptically released zinc. Mice lacking the neuronal zinc-binding protein metallothionein III (MT-III) are also more susceptible to kainic acid-induced seizures. Double knockout (ZnT3 and MT3) mice show the same response to kainic acid as ZnT3-/- mice, suggesting that ZnT3 and MT-III function in the same pathway.

Mouse strain differences in kainic acid sensitivity, seizure behavior, mortality, and hippocampal pathology.

Genetic influences contribute to susceptibility to seizures and to excitotoxic injury, but it is unclear if/how these susceptibilities are linked. This study assessed the impact of genetic background on mouse strain seizure susceptibility, seizure phenotype, mortality, and hippocampal histopathology. A subcutaneous (s.c.) kainic acid multiple injection protocol was developed. Five mouse strains were tested: a and b) C57BL/6J and 129/SvJ, strains commonly used in gene targeting experiments; c) C3HeB/FeJ, a strain with reported sensitivity to the convulsant effects of kainic acid (KA); d) 129/SvEms, a strain reportedly susceptible to hippocampal excitotoxic cell death; and e) a mixed genetic background strain (129/SvJXC57BL/6J) from which targeted gene deletion experiments have been carried out. Histopathological features were examined at early (7-10 day), delayed (2-4 month), and late (6-13 month) time points.Mouse background strains can be genetically segregated based on excitotoxin sensitivity, seizure phenotype, mortality, and hippocampal histopathology. When injected with KA, C3HeB/FeJ and C57BL/6J strains were resistant to cell death and synaptic reorganization despite severe behavioral seizures, while 129/SvEms mice developed marked pyramidal cell loss and mossy fiber sprouting despite limited seizure activity. The mixed background 129/SvJXC57BL/6J group exhibited features of both parental strains. In the mouse strains tested, the duration or severity of seizure activity was not predictive of subsequent hippocampal pyramidal cell death and/or synaptic reorganization. Unlike rats, mice exhibiting prolonged high-grade KA-induced seizure activity did not develop subsequent spontaneous behavioral seizures.

Kainic acid-induced mossy fiber sprouting and synapse formation in the dentate gyrus of rats

In the kainic acid (KA) model of temporal lobe epilepsy, mossy fibers (MFs) are thought to establish recurrent excitatory synaptic contacts onto granule cells. This hypothesis was tested by intracellular labeling of granule cells with biocytin and identifying their synaptic contacts in the dentate molecular layer with electron microscopic (EM) techniques. Twenty‐three granule cells from KA‐treated animals and 14 granule cells from control rats were examined 2 to 4 months following KA at the light microscopic (LM) level; four cells showing MF sprouting were further characterized at the EM level. Timm staining revealed a time‐dependent growth of aberrant MFs into the dentate inner molecular layer. The degree of sprouting was generally (but not invariably) correlated with the severity and frequency of seizures. LM examination of individual biocytin‐labeled MF axon collaterals revealed enhanced collateralization and significantly increased numbers of synaptic MF boutons in the hilus compared to controls, as well as aberrant MF growth into the granule cell and molecular layers. EM examination of serially reconstructed, biocytin‐labeled MF collaterals in the molecular layer revealed MF boutons that form asymmetrical synapses with dendritic shafts and spines of granule cells, including likely autaptic contacts on parent dendrites of the biocytin‐labeled granule cell. These results constitute ultrastructural evidence for newly formed excitatory recurrent circuits, which might provide a structural basis for enhanced excitation and epileptogenesis in the hippocampus of KA‐treated rats. Hippocampus 10:244–260, 2000

Age-dependent differences in flurothyl seizure sensitivity in mice treated with a ketogenic diet.

Despite strong clinical data confirming the anticonvulsant efficacy of a ketogenic diet (KGD) in pediatric patients, corroborative experimental data in young animals are limited. In the present study, the effects of a KGD on flurothyl seizure susceptibility were examined in normal juvenile mice after a dietary duration of 3, 7, or 12 days, and in adult mice for 15 days. In all groups of KGD-treated mice, blood beta-hydroxybutyrate levels were significantly elevated over those measured in controls. The present KGD was anticonvulsant (i.e. delayed onset) against the first (clonic) flurothyl-induced seizure for juvenile mice treated for either 7 or 12 days, but not for juvenile mice and adult mice fed the diet for 3 and 15 days, respectively. While this KGD was not anticonvulsant against the second (tonic extension) seizure induced by flurothyl in any of the juvenile groups, it significantly delayed tonic extension in the adult group. In addition, juvenile mice fed a KGD exhibited a lower mortality rate following flurothyl-induced seizures compared to mice fed a standard diet. In our discussion of animal models of the KGD, we highlight the need to understand better the impact of important variables such as dietary composition, genetic background, and mode of seizure induction in the study of the KGD.

Kv1.1 and Kv1.2: Similar channels, different seizure models

Voltage‐gated K+ channels (Kv) represent the largest family of genes in the K+ channel family. The Kv1 subfamily plays an essential role in the initiation and shaping of action potentials, influencing action potential firing patterns and controlling neuronal excitability. Overlapping patterns with differential expression and precise localization of Kv1.1 and Kv1.2 channels targeted to specialized subcellular compartments contribute to distinctive patterns of neuronal excitability. Dynamic regulation of the components in these subcellular domains help to finely tune the cellular and regional networks. Disruption of the expression, distribution, and density of these channels through deletion or mutation of the genes encoding these channels, Kcna1 and Kcna2, is associated with neurologic pathologies including epilepsy and ataxia in humans and in rodent models. Kv1.1 and Kv1.2 knockout mice both have seizures beginning early in development; however, each express a different seizure type (pathway), although the channels are from the same subfamily and are abundantly coexpressed. Voltage‐gated ion channels clustered in specific locations may present a novel therapeutic target for influencing excitability in neurologic disorders associated with some channelopathies.

Morphology of cerebral lesions in the Eker rat model of tuberous sclerosis.

Tuberous sclerosis (TSC) is an autosomal dominant disorder, caused by mutations of either the TSC1 or TSC2 gene. Characteristic brain pathologies (including cortical tubers and subependymal hamartomas/giant astrocytomas) are thought to cause epilepsy, as well as other neurological dysfunction. The Eker rat, which carries a spontaneous germline mutation of the TSC2 gene (TSC2+/−), provides a unique animal model in which to study the relationship between TSC cortical pathologies and epilepsy. In the present study, we have analyzed the seizure propensity and histopathological features of a modified Eker rat preparation, in which early postnatal irradiation was employed as a “second hit” stimulus in an attempt to exacerbate cortical malformations and increase seizure propensity. Irradiated Eker rats had a tendency toward lower seizure thresholds (latencies to flurothyl-induced seizures) than seen in non-irradiated Eker rats (significant difference) or irradiated wild-type rats (non-significant difference). The majority of irradiated Eker rats exhibited dysplastic cytomegalic neurons and giant astrocyte-like cells, similar to cytopathologies observed in TSC lesions of patients. The most prominent features in these brains were hamartoma-like lesions involving large eosinophilic cells, similar to giant tuber cells in human TSC. In some cells from these hamartomas, immunocytochemistry revealed features of both neuronal and glial phenotypes, suggesting an undifferentiated or immature cell population. Both normal-appearing and dysmorphic neurons, as well as cells in the hamartomas, exhibited immunopositivity for tuberin, the protein product of the TSC2 gene.

BOLD-fMRI of PTZ-induced seizures in rats

PURPOSE To develop a non-invasive method for exploring seizure initiation and propagation in the brain of intact experimental animals. METHODS We have developed and applied a model-independent statistical method--Hierarchical Cluster Analysis (HCA)--for analyzing BOLD-fMRI data following administration of pentylenetetrazol (PTZ) to intact rats. HCA clusters voxels into groups that share similar time courses and magnitudes of signal change, without any assumptions about when and/or where the seizure begins. RESULTS Epileptiform spiking activity was monitored by EEG (outside the magnet) following intravenous PTZ (IV-PTZ; n=4) or intraperitoneal PTZ administration (IP-PTZ; n=5). Onset of cortical spiking first occurred at 29+/-16 s (IV-PTZ) and 147+/-29 s (IP-PTZ) following drug delivery. HCA of fMRI data following IV-PTZ (n=4) demonstrated a single dominant cluster, involving the majority of the brain and first activating at 27+/-23s. In contrast, IP-PTZ produced multiple, relatively small, clusters with heterogeneous time courses that varied markedly across animals (n=5); activation of the first cluster (involving cortex) occurred at 130+/-59 s. With both routes of PTZ administration, the timing of the fMRI signal increase correlated with onset of EEG spiking. CONCLUSIONS These experiments demonstrate that fMRI activity associated with seizure activity can be analyzed with a model-independent statistical method. HCA indicated that seizure initiation in the IV- and IP-PTZ models involves multiple regions of sensitivity that vary with route of drug administration and that show significant variability across animal subjects. Even given this heterogeneity, fMRI shows clear differences that are not apparent with typical EEG monitoring procedures, in the activation patterns between IV and IP-PTZ models. These results suggest that fMRI can be used to assess different models and patterns of seizure activation.

Deafness and retinal degeneration in a novel USH1C knock-in mouse model.

Usher syndrome is the leading cause of combined deaf–blindness, but the molecular mechanisms underlying the auditory and visual impairment are poorly understood. Usher I is characterized by profound congenital hearing loss, vestibular dysfunction, and progressive retinitis pigmentosa beginning in early adolescence. Using the c.216G>A cryptic splice site mutation in Exon 3 of the USH1C gene found in Acadian Usher I patients in Louisiana, we constructed the first mouse model that develops both deafness and retinal degeneration. The same truncated mRNA transcript found in Usher 1C patients is found in the cochleae and retinas of these knock‐in mice. Absent auditory‐evoked brainstem responses indicated that the mutant mice are deaf at 1 month of age. Cochlear histology showed disorganized hair cell rows, abnormal bundles, and loss of both inner and outer hair cells in the middle turns and at the base. Retinal dysfunction as evident by an abnormal electroretinogram was seen as early as 1 month of age, with progressive loss of rod photoreceptors between 6 and 12 months of age. This knock‐in mouse reproduces the dual sensory loss of human Usher I, providing a novel resource to study the disease mechanism and the development of therapies.