In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphé nucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.

Aims To evaluate the safety and efficacy of intravitreal injection of liposomes encapsulating infliximab in experimental autoimmune uveoretinitis (EAU) rats. Methods Liposomes containing infliximab were prepared and characterised for mean particle size, entrapment efficiency, polydispersity index (PDI) and zeta potential. In vitro release profile and the stability of infliximab-lip were evaluated. EAU rats were intravitreally injected with saline, infliximab, infliximab-lip or unloaded liposomes. Clinical signs and ocular histological sections were graded. Infliximab concentrations were determined with competitive ELISA. Safety of the intravitreal injections was evaluated by electroretinography (ERG) and histopathological examination. Retinal biodistribution and clearance of rhodamine-conjugated liposomes containing infliximab were evaluated with a laser scanning confocal microscope. Results The mean particle size of infliximab liposomes was 351.3±58 nm and entrapment efficiency was 90.65%±2.68%. PDI and zeta potential of infliximab liposomes were 0.386 and −20.8±9.78 mV, respectively. Stability test data showed that the infliximab-lip was stable for 60 days at room temperature. In EAU rats, intravitreal injection of infliximab and infliximab-lip greatly reduced intraocular inflammation determined by clinical scores and histopathological analyses (n=4). The mean concentrations of infliximab decreased quickly in infliximab injection group and were lower than those in infliximab-lip injection group (n=4 eyes, p<0.05 after 3 days post injection). No retinal toxic effects were detected after intravitreal injection of infliximab-lip in ERG (n=4 rats, p>0.05) and histopathological sections compared with normal rats. Confocal microscopy showed that fluorescent liposomes were observed in almost every layer of the retina and remained detectable for >30 days after injection. Conclusions Intravitreal injection of liposomal infliximab can prolong the persistence of the drug in vitreous body and demonstrated a satisfactory safety and significant therapeutic potentials in EAU. The use of biodegradable particles for therapeutic antibody delivery may provide a promising approach for the treatment of ocular diseases.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and cerebellar ataxia. A mutation in Cacna2d2, the gene encoding the alpha2delta-2 voltage-dependent calcium channel accessory subunit, has been found to underlie the ducky phenotype. The alpha2delta-2 mRNA is strongly expressed in cerebellar Purkinje cells. We show that du/du mice have abnormalities in their Purkinje cell dendritic tree. The mutation in alpha2delta-2 results in the introduction of a premature stop codon and predicts the expression of a truncated protein encoded by the first three exons of Cacna2d2, followed by 8 novel amino acids. We show that both mRNA and protein corresponding to this predicted transcript are expressed in du/du cerebellum and present in Purkinje cells. Whereas the alpha2delta-2 subunit increased the peak current density of the Ca(V)2.1/beta(4) channel combination when co-expressed in vitro, co-expression with the truncated mutant alpha2delta-2 protein reduced current density, indicating that it may contribute to the du phenotype.

Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy (IGE) characterised by onset of typical absence seizures in otherwise normal children of school age. A genetic component to aetiology is well established but the mechanism of inheritance and the genes involved are unknown. Available evidence suggests that mutations in genes encoding GABA receptors or brain expressed voltage-dependent calcium channels (VDCCs) may underlie CAE. The aim of this work was to test this hypothesis by linkage analysis using microsatellite loci spanning theses genes in 33 nuclear families each with two or more individuals with CAE. Seventeen VDCC subunit genes, ten GABA(A)R subunit genes, two GABA(B) receptor genes and the ECA1 locus on 8q24 were investigated using 35 microsatellite loci. Assuming locus homogeneity, all loci gave statistically significant negative LOD scores, excluding these genes as major loci in the majority of these families. Positive HLOD scores assuming locus heterogeneity were observed for CACNG3 on chromosome 16p12-p13.1 and the GABRA5, GABRB3, GABRG3 cluster on chromosome 15q11-q13. Association studies are required to determine whether these loci are the site of susceptibility alleles in a subset of patients with CAE.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and ataxia. The ducky gene was mapped previously to distal mouse chromosome 9. High-resolution genetic and physical mapping has resulted in the identification of theCacna2d2 gene encoding the α2δ2 voltage-dependent calcium channel subunit. Mutations in Cacna2d2 were found to underlie the ducky phenotype in the original ducky (du) strain and in a newly identified strain (du2J). Both mutations are predicted to result in loss of the full-length α2δ2 protein. Functional analysis shows that the α2δ2 subunit increases the maximum conductance of the α1A/β4 channel combination when coexpressedin vitro in Xenopus oocytes. The Ca2+ channel current in acutely dissociateddu/du cerebellar Purkinje cells was reduced, with no change in single-channel conductance. In contrast, no effect on Ca2+ channel current was seen in cerebellar granule cells, results consistent with the high level of expression of theCacna2d2 gene in Purkinje, but not granule, neurons. Our observations document the first mammalian α2δ mutation and complete the association of each of the major classes of voltage-dependent Ca2+ channel subunits with a phenotype of ataxia and epilepsy in the mouse.

To understand the cellular and molecular mechanisms that underlie generalized absence seizures sufficiently well to design rational, efficacious new therapies for patients, it is necessary to turn to animal models to gain insights into these mechanisms. The lethargic (lh/lh) mutant mouse expresses spontaneous absence seizures that share behavioral, electrographic, and anticonvulsant profiles with absence seizures in patients. This validates its use to study the mechanisms that underlie absence seizures. This chapter discusses two scientific approaches that involve the use of lh/lh mice. The first part of the chapter discusses neurobiologic approaches used to investigate critical mechanisms that regulate the synchronized burst firing within the thalamocortical network that generates absence seizures. Two of these critical mechanisms have been studied in detail with lh/lh mice. The first critical mechanism involves the required activation of gamma-aminobutyric acid B (GABAB) receptors to generate absence seizures. Because the numbers of GABAB receptors are increased in thalamocortical populations among lh/lh mice compared with littermates without epilepsy, these receptors appear to play a pathophysiologic role in the expression of absence seizures among lh/lh mice. Moreover, there may be a role for GABAB receptors in the generation of absence seizures among humans, because administration of compounds that activate GABAB receptors can produce absence seizures among humans. These findings suggest that GABAB receptor antagonists may represent a new class of antiabsence compounds that will be efficacious against absence seizures among patients. A second critical mechanism that regulates generation of absence seizures involves GABAA receptors in the nucleus reticularis thalami (NRT), a nucleus that sends GABA-ergic afferents to thalamic relay nuclei. Activation of GABAA receptors in the NRT appears to suppress the generation of absence seizures among lh/lh mice and in other models. Moreover, clonazepam may exert its antiabsence actions through this mechanism. Together, these findings suggest that compounds that selectively activate GABAA receptor isoforms expressed in NRT may represent a class of antiabsence drugs that could have fewer side effects than compounds currently used to treat patients. The second part of the chapter discusses a molecular genetic approach to delineation of the mechanisms that underlie absence seizures. Absence seizures among lh/lh mice are caused by a single-gene defect on chromosome 2. If positional cloning and gene isolation techniques are successful, it will be possible to identify the lh disease gene. Subsequent studies of the lh gene product should greatly increase not only our understanding of the pathophysiologic basis for absence seizures among lh/lh mice but also our ability to seek similar mutations in homologous genes in human families that express absence seizures. Accordingly, strategies and progress in cloning and identifying the lh disease gene are presented.

The mouse is a well-established model for human genetic disorders. An increasing number of single-gene human diseases are being elucidated through the use of mouse models. Recently genes for three of the six well-characterised single locus models for human spike-wave epilepsy have been isolated and published. The tottering mouse has been shown to be due to mutations in the gene encoding the high voltage-activated alpha 1(A) calcium channel subunit. The lethargic mouse has been shown to be due to mutations in the gene encoding another calcium channel subunit, beta 4. The slow-wave epilepsy mouse phenotype is the result of loss of function of the ubiquitous sodium hydrogen exchanger NHEI. These genes and the pathways they are involved in are now candidates for human spike-wave epilepsy. The six mouse models and those genes underlying the spike-wave phenotype are discussed in conjunction with how these mutations were discovered and how they may give rise to the seizure phenotypes. Several nonepilepsy human neurologic disorders have been shown to be allelic with the tottering mouse. The question this raises as to the validity of these models for human spike-wave epilepsy is considered. Finally, the effect these discoveries will have on the understanding and treatment of human spike-wave epilepsy are discussed.

INTRODUCTIONWe tested the hypothesis that genetic variants within the GABA(A) alpha5, beta3 and gamma3 subunit gene cluster on chromosome 15q11-q13 confer genetic susceptibility to common subtypes of idiopathic generalized epilepsy (IGE).MATERIAL AND METHODSNinety-four families were selected from IGE patients with either juvenile myoclonic epilepsy (JME), juvenile (JAE) or childhood absence epilepsy (CAE). Cosegregation was tested between dinucleotide polymorphisms associated with the human GABA(A) alpha5, beta3 and gamma3 subunit gene cluster and three different IGE trait models.RESULTSEvidence against linkage to the GABA(A) alpha5, beta3 and gamma3 subunit gene cluster was found in the entire family set and subsets selected from either CAE or JAE. In 61 families of JME patients, a maximum lod score (Zmax=1.40 at Theta(max)=0.00) was obtained for a broad IGE spectrum ("idiopathic" generalized seizure or generalized spike and wave discharges in the electroencephalogram) assuming genetic heterogeneity (alpha=0.37; P=0.06) and an autosomal recessive mode of inheritance.CONCLUSIONThe possible hint of linkage in families of JME patients emphasizes the need for further studies to determine whether a recessively inherited gene variant within the GABA(A) alpha5, beta3 and gamma3 subunit gene cluster contributes to the pathogenesis of "idiopathic" generalized seizures and associated EEG abnormalities in a proportion of families.

Introduction - We tested the hypothesis that genetic variants within the GABA A α 5 , β 3 and γ 3 subunit gene cluster on chromosome 15q11-q13 confer genetic susceptibility to common subtypes of idiopathic generalized epilepsy (IGE). Material and methods - Ninety-four families were selected from IGE patients with either juvenile myoclonic epilepsy (JME), juvenile (JAE) or childhood absence epilepsy (CAE). Cosegregation was tested between dinucleotide polymorphisms associated with the human GABA A α 5 , β 3 and γ 3 subunit gene cluster and three different IGE trait models. Results - Evidence against linkage to the GABA A α 5 , β 3 and γ 3 subunit gene cluster was found in the entire family set and subsets selected from either CAE or JAE. In 61 families of JME patients, a maximum lod score (Z max =1.40 at θ max =0.00) was obtained for a broad IGE spectrum (“idiopathic” generalized seizure or generalized spike and wave discharges in the electroencephalogram) assuming genetic heterogeneity (α=0.37; P =0.06) and an autosomal recessive mode of inheritance. Conclusion - The possible hint of linkage in families of JME patients emphasizes the need for further studies to determine whether a recessively inherited gene variant within the GABA A α 5 , β 3 and γ 3 subunit gene cluster contributes to the pathogenesis of “idiopathic” generalized seizures and associated EEG abnormalities in a proportion of families.