Audiogenic Seizure Susceptibility Research Articles

MGluR7 allosteric modulator AMN082 corrects protein synthesis and pathological phenotypes in FXS

Fragile X syndrome (FXS) is the leading cause of inherited autism and intellectual disabilities. Aberrant protein synthesis due to the loss of fragile X messenger ribonucleoprotein (FMRP) is the major defect in FXS, leading to a plethora of cellular and behavioral abnormalities. However, no treatments are available to date. In this study, we found that activation of metabotropic glutamate receptor 7 (mGluR7) using a positive allosteric modulator named AMN082 represses protein synthesis through ERK1/2 and eIF4E signaling in an FMRP-independent manner. We further demonstrated that treatment of AMN082 leads to a reduction in neuronal excitability, which in turn ameliorates audiogenic seizure susceptibility in Fmr1 KO mice, the FXS mouse model. When evaluating the animals’ behavior, we showed that treatment of AMN082 reduces repetitive behavior and improves learning and memory in Fmr1 KO mice. This study uncovers novel functions of mGluR7 and AMN082 and suggests the activation of mGluR7 as a potential therapeutic approach for treating FXS.

MGluR5 Negative Modulators for Fragile X: Treatment Resistance and Persistence.

Fragile X syndrome (FXS) is caused by silencing of the human FMR1 gene and is the leading monogenic cause of intellectual disability and autism. Abundant preclinical data indicated that negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5) might be efficacious in treating FXS in humans. Initial attempts to translate these findings in clinical trials have failed, but these failures provide the opportunity for new discoveries that will improve future trials. The emergence of acquired treatment resistance (“tolerance”) after chronic administration of mGluR5 NAMs is a potential factor in the lack of success. Here we confirm that FXS model mice display acquired treatment resistance after chronic treatment with the mGluR5 NAM CTEP in three assays commonly examined in the mouse model of FXS: (1) audiogenic seizure susceptibility, (2) sensory cortex hyperexcitability, and (3) hippocampal protein synthesis. Cross-tolerance experiments suggest that the mechanism of treatment resistance likely occurs at signaling nodes downstream of glycogen synthase kinase 3α (GSK3α), but upstream of protein synthesis. The rapid emergence of tolerance to CTEP begs the question of how previous studies showed an improvement in inhibitory avoidance (IA) cognitive performance after chronic treatment. We show here that this observation was likely explained by timely inhibition of mGluR5 during a critical period, as brief CTEP treatment in juvenile mice is sufficient to provide a persistent improvement of IA behavior measured many weeks later. These data will be important to consider when designing future fragile X clinical trials using compounds that target the mGluR5-to-protein synthesis signaling cascade.

Cannabinoids in Audiogenic Seizures: From Neuronal Networks to Future Perspectives for Epilepsy Treatment.

Cannabinoids and Cannabis-derived compounds have been receiving especial attention in the epilepsy research scenario. Pharmacological modulation of endocannabinoid system's components, like cannabinoid type 1 receptors (CB1R) and their bindings, are associated with seizures in preclinical models. CB1R expression and functionality were altered in humans and preclinical models of seizures. Additionally, Cannabis-derived compounds, like cannabidiol (CBD), present anticonvulsant activity in humans and in a great variety of animal models. Audiogenic seizures (AS) are induced in genetically susceptible animals by high-intensity sound stimulation. Audiogenic strains, like the Genetically Epilepsy Prone Rats, Wistar Audiogenic Rats, and Krushinsky-Molodkina, are useful tools to study epilepsy. In audiogenic susceptible animals, acute acoustic stimulation induces brainstem-dependent wild running and tonic-clonic seizures. However, during the chronic protocol of AS, the audiogenic kindling (AuK), limbic and cortical structures are recruited, and the initially brainstem-dependent seizures give rise to limbic seizures. The present study reviewed the effects of pharmacological modulation of the endocannabinoid system in audiogenic seizure susceptibility and expression. The effects of Cannabis-derived compounds in audiogenic seizures were also reviewed, with especial attention to CBD. CB1R activation, as well Cannabis-derived compounds, induced anticonvulsant effects against audiogenic seizures, but the effects of cannabinoids modulation and Cannabis-derived compounds still need to be verified in chronic audiogenic seizures. The effects of cannabinoids and Cannabis-derived compounds should be further investigated not only in audiogenic seizures, but also in epilepsy related comorbidities present in audiogenic strains, like anxiety, and depression.

Morphological and molecular correlates of altered hearing sensitivity in the genetically audiogenic seizure-prone hamster GASH/Sal

Rodent models of audiogenic seizures, in which seizures are precipitated by an abnormal response of the brain to auditory stimuli, are crucial to investigate the neural bases underlying ictogenesis. Despite significant advances in understanding seizure generation in the inferior colliculus, namely the epileptogenic nucleus, little is known about the contribution of lower auditory stations to the seizure-prone network. Here, we examined the cochlea and cochlear nucleus of the genetic audiogenic seizure hamster from Salamanca (GASH/Sal), a model of reflex epilepsy that exhibits generalized tonic–clonic seizures in response to loud sound. GASH/Sal animals under seizure-free conditions were compared with matched control hamsters in a multi-technical approach that includes auditory brainstem responses (ABR) testing, histology, scanning electron microscopy analysis, immunohistochemistry, quantitative morphometry and gene expression analysis (RT-qPCR). The cochlear histopathology of the GASH/Sal showed preservation of the sensory hair cells, but a significant loss of spiral ganglion neurons and mild atrophy of the stria vascularis. At the electron microscopy level, the reticular lamina exhibited disarray of stereociliary tufts with blebs, loss or elongated stereocilia as well as non-parallel rows of outer hair cells due to protrusions of Deiters’ cells. At the molecular level, the abnormal gene expression patterns of prestin, cadherin 23, protocadherin 15, vesicular glutamate transporters 1 (Vglut1) and -2 (Vglut2) indicated that the hair-cell mechanotransduction and cochlear amplification were markedly altered. These were manifestations of a cochlear neuropathy that correlated to ABR waveform I alterations and elevated auditory thresholds. In the cochlear nucleus, the distribution of VGLUT2-immunolabeled puncta was differently affected in each subdivision, showing significant increases in magnocellular regions of the ventral cochlear nucleus and drastic reductions in the granule cell domain. This modified inputs lead to disruption of Vglut1 and Vglut2 gene expression in the cochlear nucleus. In sum, our study provides insight into the morphological and molecular traits associated with audiogenic seizure susceptibility in the GASH/Sal, suggesting an upward spread of abnormal glutamatergic transmission throughout the primary acoustic pathway to the epileptogenic region.

Analysis of gene variants in the GASH/Sal model of epilepsy

Epilepsy is a complex neurological disorder characterized by sudden and recurrent seizures, which are caused by various factors, including genetic abnormalities. Several animal models of epilepsy mimic the different symptoms of this disorder. In particular, the genetic audiogenic seizure hamster from Salamanca (GASH/Sal) animals exhibit sound-induced seizures similar to the generalized tonic seizures observed in epileptic patients. However, the genetic alterations underlying the audiogenic seizure susceptibility of the GASH/Sal model remain unknown. In addition, gene variations in the GASH/Sal might have a close resemblance with those described in humans with epilepsy, which is a prerequisite for any new preclinical studies that target genetic abnormalities. Here, we performed whole exome sequencing (WES) in GASH/Sal animals and their corresponding controls to identify and characterize the mutational landscape of the GASH/Sal strain. After filtering the results, moderate- and high-impact variants were validated by Sanger sequencing, assessing the possible impact of the mutations by “in silico” reconstruction of the encoded proteins and analyzing their corresponding biological pathways. Lastly, we quantified gene expression levels by RT-qPCR. In the GASH/Sal model, WES showed the presence of 342 variations, in which 21 were classified as high-impact mutations. After a full bioinformatics analysis to highlight the high quality and reliable variants, the presence of 3 high-impact and 15 moderate-impact variants were identified. Gene expression analysis of the high-impact variants of Asb14 (ankyrin repeat and SOCS Box Containing 14), Msh3 (MutS Homolog 3) and Arhgef38 (Rho Guanine Nucleotide Exchange Factor 38) genes showed a higher expression in the GASH/Sal than in control hamsters. In silico analysis of the functional consequences indicated that those mutations in the three encoded proteins would have severe functional alterations. By functional analysis of the variants, we detected 44 significantly enriched pathways, including the glutamatergic synapse pathway. The data show three high-impact mutations with a major impact on the function of the proteins encoded by these genes, although no mutation in these three genes has been associated with some type of epilepsy until now. Furthermore, GASH/Sal animals also showed gene variants associated with different types of epilepsy that has been extensively documented, as well as mutations in other genes that encode proteins with functions related to neuronal excitability, which could be implied in the phenotype of the GASH/Sal. Our findings provide valuable genetic and biological pathway data associated to the genetic burden of the audiogenic seizure susceptibility and reinforce the need to validate the role of each key mutation in the phenotype of the GASH/Sal model.

Alterations in brainstem auditory processing, the acoustic startle response and sensorimotor gating of startle in Wistar audiogenic rats (WAR), an animal model of reflex epilepsies

While acute audiogenic seizures in response to acoustic stimulus appear as an alteration in sensory-motor processing in the brainstem, the repetition of the stimulus leads to the spread of epileptic activity to limbic structures. Here, we investigated whether animals of the Wistar Audiogenic Rat (WAR) strain, genetically selected by inbreeding for seizure susceptibility, would have alterations in their auditory response, assessed by the auditory brainstem responses (ABR) and sensory-motor gating, measured as pre-pulse inhibition (PPI), which could be related to their audiogenic seizures susceptibility or severity. We did not find differences between the amplitudes and latencies of ABR waves in response to clicks for WARs when compared to Wistars. Auditory gain and symmetry between ears were also similar. However, hearing thresholds in response to some tones were lower and amplitudes of wave II were larger in WARs. WARs had smaller acoustic startle reflex amplitudes and the percentages of startle inhibited by an acoustic prepulse were higher for WARs than for Wistars. However, no correlation was found between these alterations and brainstem-dependent seizure severity or limbic seizure frequency during audiogenic kindling. Our data show that while WARs present moderate alterations in primary auditory processing, the sensory motor gating measured in startle/PPI tests appears to be more drastically altered. The observed changes might be correlated with audiogenic seizure susceptibility but not seizures severity.

A behavioral test battery for mouse models of Angelman syndrome: a powerful tool for testing drugs and novel Ube3a mutants

BackgroundAngelman syndrome (AS) is a neurodevelopmental disorder caused by mutations affecting UBE3A function. AS is characterized by intellectual disability, impaired motor coordination, epilepsy, and behavioral abnormalities including autism spectrum disorder features. The development of treatments for AS heavily relies on the ability to test the efficacy of drugs in mouse models that show reliable, and preferably clinically relevant, phenotypes. We previously described a number of behavioral paradigms that assess phenotypes in the domains of motor performance, repetitive behavior, anxiety, and seizure susceptibility. Here, we set out to evaluate the robustness of these phenotypes when tested in a standardized test battery. We then used this behavioral test battery to assess the efficacy of minocycline and levodopa, which were recently tested in clinical trials of AS.MethodsWe combined data of eight independent experiments involving 111 Ube3a mice and 120 wild-type littermate control mice. Using a meta-analysis, we determined the statistical power of the subtests and the effect of putative confounding factors, such as the effect of sex and of animal weight on rotarod performance. We further assessed the robustness of these phenotypes by comparing Ube3a mutants in different genetic backgrounds and by comparing the behavioral phenotypes of independently derived Ube3a-mutant lines. In addition, we investigated if the test battery allowed re-testing the same animals, which would allow a within-subject testing design.ResultsWe find that the test battery is robust across different Ube3a-mutant lines, but confirm and extend earlier studies that several phenotypes are very sensitive to genetic background. We further found that the audiogenic seizure susceptibility phenotype is fully reversible upon pharmacological treatment and highly suitable for dose-finding studies. In agreement with the clinical trial results, we found that minocycline and levodopa treatment of Ube3a mice did not show any sign of improved performance in our test battery.ConclusionsOur study provides a useful tool for preclinical drug testing to identify treatments for Angelman syndrome. Since the phenotypes are observed in several independently derived Ube3a lines, the test battery can also be employed to investigate the effect of specific Ube3a mutations on these phenotypes.

Expressional change of astrocytic Kir4.1 channel during epileptogenesis in audiogenic seizure-susceptible, Lgi1 mutant rats

Background: Leucine-rich glioma-inactivated 1 (LGI1) was identified as a causative gene of autosomal dominant lateral temporal lobe epilepsy (ADLTE). We previously reported Lgi1 mutant rats, carrying a missense mutation (L385R), showed audiogenic seizure susceptibility. However, its mechanisms underlying seizure induction remain unknown. Astrocytes are recognized as an active partner of neurons in regulating neural activity. Specifically, dysfunction of astrocytic inwardly rectifying potassium (Kir) 4.1 channels mediating spatial potassium buffering is reported to be involved in the generation of generalized tonic-clonic seizures (GTCS).

A genetic context for the study of audiogenic seizures.

Here, the genetic context for the study of audiogenic seizures is four single-gene, spontaneous mutations that occurred in the Behavior Genetics Laboratory at the University of Chicago from 1959 to 1969. Three of these increased the incidence of audiogenic seizures, and one of these decreased the incidence of audiogenic seizures. The genetics of one of these mutants is described in detail, and the effect of diet on the same mutant is also described in detail. Research on genetic and environmental effects on the cortical EEG of audiogenic seizures is reviewed; this research included two of these mutants. The cortical EEG associated with audiogenic seizures in this study was consistent with audiogenic seizures being a type of brain stem epilepsy as had been proposed by others. Also, I proposed that brain stem pathophysiology is the same regardless of the genetic or environmental pathway to audiogenic seizure susceptibility. Research is also reviewed using these mutants to determine whether or not a strain association between glutamic acid decarboxylase (GAD) activity in whole brain and susceptibility to audiogenic seizures is pleiotropic and whether or not a strain association between nucleoside triphosphatase (NTPase) activity in the granule cell layer of the dentate fascia of the hippocampus and susceptibility to audiogenic seizures is a lineal or collateral pleiotropy. Lastly, pleiotropy as an explanation for strain comorbidities in aggressive behavior and audiogenic seizures is considered. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

Glycogen Synthase Kinase-3 Inhibitors Reverse Deficits in Long-term Potentiation and Cognition in Fragile X Mice

Identifying feasible therapeutic interventions is crucial for ameliorating the intellectual disability and other afflictions of fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism. Hippocampal glycogen synthase kinase-3 (GSK3) is hyperactive in the mouse model of FXS (FX mice), and hyperactive GSK3 promotes locomotor hyperactivity and audiogenic seizure susceptibility in FX mice, raising the possibility that specific GSK3 inhibitors may improve cognitive processes. We tested if specific GSK3 inhibitors improve deficits in N-methyl-D-aspartate receptor-dependent long-term potentiation at medial perforant path synapses onto dentate granule cells and dentate gyrus-dependent cognitive behavioral tasks. GSK3 inhibitors completely rescued deficits in long-term potentiation at medial perforant path-dentate granule cells synapses in FX mice. Furthermore, synaptosomes from the dentate gyrus of FX mice displayed decreased inhibitory serine-phosphorylation of GSK3β compared with wild-type littermates. The potential therapeutic utility of GSK3 inhibitors was further tested on dentate gyrus-dependent cognitive behaviors. In vivo administration of GSK3 inhibitors completely reversed impairments in several cognitive tasks in FX mice, including novel object detection, coordinate and categorical spatial processing, and temporal ordering for visual objects. These findings establish that synaptic plasticity and cognitive deficits in FX mice can be improved by intervention with inhibitors of GSK3, which may prove therapeutically beneficial in FXS.

Behavioral and EEG effects of GABAergic manipulation of the nigro-tectal pathway in the Wistar audiogenic rat (WAR) strain II: An EEG wavelet analysis and retrograde neuronal tracer approach

The role of the substantia nigra pars reticulata (SNPr) and superior colliculus (SC) network in rat strains susceptible to audiogenic seizures still remain underexplored in epileptology. In a previous study from our laboratory, the GABAergic drugs bicuculline (BIC) and muscimol (MUS) were microinjected into the deep layers of either the anterior SC (aSC) or the posterior SC (pSC) in animals of the Wistar audiogenic rat (WAR) strain submitted to acoustic stimulation, in which simultaneous electroencephalographic (EEG) recording of the aSC, pSC, SNPr and striatum was performed. Only MUS microinjected into the pSC blocked audiogenic seizures. In the present study, we expanded upon these previous results using the retrograde tracer Fluorogold (FG) microinjected into the aSC and pSC in conjunction with quantitative EEG analysis (wavelet transform), in the search for mechanisms associated with the susceptibility of this inbred strain to acoustic stimulation. Our hypothesis was that the WAR strain would have different connectivity between specific subareas of the superior colliculus and the SNPr when compared with resistant Wistar animals and that these connections would lead to altered behavior of this network during audiogenic seizures. Wavelet analysis showed that the only treatment with an anticonvulsant effect was MUS microinjected into the pSC region, and this treatment induced a sustained oscillation in the theta band only in the SNPr and in the pSC. These data suggest that in WAR animals, there are at least two subcortical loops and that the one involved in audiogenic seizure susceptibility appears to be the pSC-SNPr circuit. We also found that WARs presented an increase in the number of FG+ projections from the posterior SNPr to both the aSC and pSC (primarily to the pSC), with both acting as proconvulsant nuclei when compared with Wistar rats. We concluded that these two different subcortical loops within the basal ganglia are probably a consequence of the WAR genetic background.

Glycogen synthase kinase-3: a promising therapeutic target for fragile x syndrome.

Recent advances in understanding the pathophysiological mechanisms contributing to fragile X syndrome (FXS) have increased optimism that drug interventions can provide significant therapeutic benefits. FXS results from inadequate expression of functional fragile X mental retardation protein (FMRP). FMRP may have several functions, but it is most well-established as an RNA binding protein that regulates translation, and it is thought that by this mechanism FMRP is capable of affecting numerous cellular processes by selectively regulating protein levels. The multiple cellular functions regulated by FMRP suggest that multiple interventions may be required for reversing the effects of deficient FMRP. Evidence that inhibitors of glycogen synthase kinase-3 (GSK3) may contribute to the therapeutic treatment of FXS is reviewed here. Lithium, a GSK3 inhibitor, improved function in the Drosophila model of FXS. In mice lacking FMRP expression (FX mice), GSK3 is hyperactive in several brain regions. Significant improvements in several FX-related phenotypes have been obtained in FX mice following the administration of lithium, and in some case other GSK3 inhibitors. These responses include normalization of heightened audiogenic seizure susceptibility and of hyperactive locomotor behavior, enhancement of passive avoidance learning retention and of sociability behaviors, and corrections of macroorchidism, neuronal spine density, and neural plasticity measured electrophysiologically as long term depression. A pilot clinical trial of lithium in patients with FXS also found improvements in several measures of behavior. Taken together, these findings indicate that lithium and other inhibitors of GSK3 are promising candidate therapeutic agents for treating FXS.

Hypersensitivity to mGluR5 and ERK1/2 Leads to Excessive Protein Synthesis in the Hippocampus of a Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS) is caused by loss of the FMR1 gene product FMRP (fragile X mental retardation protein), a repressor of mRNA translation. According to the metabotropic glutamate receptor (mGluR) theory of FXS, excessive protein synthesis downstream of mGluR5 activation causes the synaptic pathophysiology that underlies multiple aspects of FXS. Here, we use an in vitro assay of protein synthesis in the hippocampus of male Fmr1 knock-out (KO) mice to explore the molecular mechanisms involved in this core biochemical phenotype under conditions where aberrant synaptic physiology has been observed. We find that elevated basal protein synthesis in Fmr1 KO mice is selectively reduced to wild-type levels by acute inhibition of mGluR5 or ERK1/2, but not by inhibition of mTOR (mammalian target of rapamycin). The mGluR5-ERK1/2 pathway is not constitutively overactive in the Fmr1 KO, however, suggesting that mRNA translation is hypersensitive to basal ERK1/2 activation in the absence of FMRP. We find that hypersensitivity to ERK1/2 pathway activation also contributes to audiogenic seizure susceptibility in the Fmr1 KO. These results suggest that the ERK1/2 pathway, and other neurotransmitter systems that stimulate protein synthesis via ERK1/2, represent additional therapeutic targets for FXS.

Vlgr1 is required for proper stereocilia maturation of cochlear hair cells

Very large G-protein coupled receptor (Vlgr1b) is the largest known G-protein coupled receptor. Its function is unknown, although mice with deletion of Vlgr1 (Vlgr1b together with other splicing variants, Vlgr1c, Vlgr1d and Vlgr1e) are known to exhibit audiogenic seizure susceptibility and VLGR1 is reported to be the gene responsible for Usher type 2C syndrome. We demonstrated here that Vlgr1-mutated mice suffered from a hearing defect because of inner ear dysfunction, as indicated by auditory brainstem response (ABR) and distortion product oto-acoustic emissions (DPOAE). The expression of Vlgr1 was identified in the developing hair cells perinatally, and the translated products were seen to be localized in the base of stereocilia on hair cells using confocal microscopy. This Vlgr1 localization was limited to the base of stereocilia within approximately 200-400 nm from the apical surface of hair cells, as shown by immunoelectron microscopy. The Vlgr1-mutated mice exhibited malformation of the stereocilia; the cochlear hair bundles were apparently normal at birth but then became disarranged at postnatal day 8. Furthermore, the stereocilia in the mutant mice became slanted and disarranged thereafter. These results indicate that loss of Vlgr1 resulted in abnormal development of stereocilia formation.

Audiogenic seizure susceptibility is reduced in fragile X knockout mice after introduction of FMR1 transgenes

The Fmr1 knockout (KO) mouse is characterized by an increased audiogenic seizure (AGS) susceptibility and is considered a good animal model for epilepsy and seizures in the human fragile-X (FRAX) syndrome. Here, we tested the hypothesis that the reintroduction of the FMR1 gene is able to revert the AGS susceptibility characterizing Fmr1 KO mice. To this aim, two groups of Fmr1 KO transgenic mice, which have additional copies of the human FMR1 gene (YAC) or FMR1 cDNA (G6) were used. AGS susceptibility of these mice was examined and compared to that of Fmr1 KO, wild type, and wild-type animals in whom the FMR1gene was also introduced (over-expressed). Mice were tested at different ages because AGS susceptibility is age dependent. The intensity of response was scored and the results were analyzed by means of 2-way analysis of variance to evaluate the effects of age and genetic condition. We found that AGS susceptibility rescue is complete in the G6 mice and partial in YAC mice. Our data indicate that the introduction of the human FMR1 gene in Fmr1 KO mice is able to revert the Fmr1 KO epileptic phenotype.

Fmrp is required for the establishment of the startle response during the critical period of auditory development

Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the FMR-1 gene product FMRP. In addition to the hallmark cognitive defect, other symptoms are also apparent including hyperactivity, seizures and sensory abnormalities including a characteristic increase in sensitivity to auditory, tactile, visual, and olfactory stimuli. Fragile X is a developmental disorder with the first symptoms apparent in the first year of life but little is known about the role of FMRP in developmental processes. The sensory hyperreactivity of fragile X can be reproduced in fmr-1 knockout (KO) mice evident as abnormal audiogenic startle response and increased audiogenic seizure susceptibility. Here, we studied the onset and emergence of the startle deficit in fmr-1 KO mice during development. The startle response was first detectable at the end of the 2nd postnatal week in wild-type mice. The amplitude of startle response showed a substantial increase until the 4th postnatal week followed by a further but moderate increase up to adulthood. Expression of the fmr1 gene was detectable in the startle circuit before the onset and throughout the development of the startle response. Although the onset and amplitude of the startle response were not altered in fmr1 KO mice until the 3rd–4th postnatal week, beyond this age it failed to develop further resulting in an overall response deficit in adult KO mice. This indicates that although Fmrp is dispensable at the initial steps of startle response development, it is necessary for the full development of the response.

The Mass1frings mutation underlies early onset hearing impairment in BUB/BnJ mice, a model for the auditory pathology of Usher syndrome IIC

The human ortholog of the gene responsible for audiogenic seizure susceptibility in Frings and BUB/BnJ mice (mouse gene symbol Mass1) recently was shown to underlie Usher syndrome type IIC (USH2C). Here we report that the Mass1 frings mutation is responsible for the early onset hearing impairment of BUB/BnJ mice. We found highly significant linkage of Mass1 with ABR threshold variation among mice from two backcrosses involving BUB/BnJ mice with mice of strains CAST/EiJ and MOLD/RkJ. We also show an additive effect of the Cdh23 locus in modulating the progression of hearing loss in backcross mice. Together, these two loci account for more than 70% of the total ABR threshold variation among the backcross mice at all ages. The modifying effect of the strain-specific Cdh23 ahl variant may account for the hearing and audiogenic seizure differences observed between Frings and BUB/BnJ mice, which share the Mass1 frings mutation. During postnatal cochlear development in BUB/BnJ mice, stereocilia bundles develop abnormally and remain immature and splayed into adulthood, corresponding with the early onset hearing impairment associated with Mass1 frings . Progressive base–apex hair cell degeneration occurs at older ages, corresponding with the age-related hearing loss associated with Cdh23 ahl . The molecular basis and pathophysiology of hearing loss suggest BUB/BnJ and Frings mice as models to study cellular and molecular mechanisms underlying USH2C auditory pathology.

C-Fos immunohistochemical mapping of the audiogenic seizure network and tonotopic neuronal hyperexcitability in the inferior colliculus of the Frings mouse

The Frings mouse is a model of audiogenic seizure (AGS) susceptibility. The genetic locus responsible for the AGS phenotype in the Frings mouse has been named monogenic audiogenic seizure-susceptible ( MASS1). MASS1 is unique in that it is one of only two identified seizure loci that are not associated with an ion channel mutation. Furthermore, Frings mice display a robust AGS phenotype demonstrating very high and prolonged susceptibility to sound-induced tonic extension seizures. The purpose of this investigation was to use c-Fos immunohistochemistry to map the brain structures involved in the Frings AGS and to examine neuronal hyperexcitability in the inferior colliculus, the brain structure that is recognized as the site of AGS initiation. AGS mapping revealed that intense seizure-induced neuronal activation was mostly limited to structures involved in a brainstem seizure network, including the external and dorsal nuclei of the inferior colliculus, as observed in other AGS rodents. Acoustically induced c-Fos expression in the central nucleus of the inferior colliculus to sub-AGS threshold tone stimulations displayed a greater level of neuronal activation in AGS-susceptible Frings, DBA/2J and noise-primed C57BL/6J mice compared to AGS-resistant C57BL/6J and CF1 mice. The AGS-susceptible mice also displayed c-Fos immunoreactivity that was more focused within the tonotopic response domain of the inferior colliculus compared to AGS-resistant mice. Furthermore, Frings mice displayed significantly greater tonotopic hyper-responsiveness compared to other AGS-susceptible mice.

A moderate and transient deficiency of maternal thyroid function at the beginning of fetal neocorticogenesis alters neuronal migration.

Epidemiological studies and case reports show that even a relatively minor degree of maternal hypothyroxinemia during the first half of gestation is potentially dangerous for optimal fetal neurodevelopment. Our experimental approach was designed to result in a mild and transient period of maternal hypothyroxinemia at the beginning of corticogenesis. Normal rat dams received the goitrogen 2-mercapto-1-methyl-imidazole for only 3 d, from embryonic d 12 (E12) to E15. Maternal thyroid hormones decreased transiently to 70% of normal serum values, without clinical signs of hypothyroidism. Dams were injected daily with 5-bromo-2'-deoxyuridine (BrdU) during 3 d, from E14-E16 or E17-E19. Their pups were tested for audiogenic seizure susceptibility 39 d after birth (P39) and killed at P40. Cells that had incorporated BrdU were identified by immunocytochemistry, and quantified: numerous heterotopic cells were found, whether labeled at E14-E16 or E17-E19, that were identified as neurons. The cytoarchitecture and the radial distribution of BrdU-labeled neurons was significantly affected in the somatosensory cortex and hippocampus of 83% of the pups. The radial distribution of gamma-aminobutyric acidergic neurons was, however, normal. The infusion of dams with T4 between E13 and E15 avoided these alterations, which were not prevented when the T4 infusion was delayed to E15-E18. In total, 52% of the pups born to the goitrogen-treated dams responded to an acoustic stimulus with wild runs, followed in some by seizures. When extrapolated to man, these results stress the need for prevention of hypothyroxinemia before midpregnancy, however moderate, and whichever the underlying cause.

Changes in peripheral energy metabolism during audiogenic seizures in rats

Plasma glucose and lactate, hepatic glycogen and epididymal adipose tissue lipogenesis and lipolysis were studied in Wistar audiogenic rats (WARs), a genetic model of epilepsy, under three experimental conditions, i.e., before, 3 min after and 10 min after seizures induced by intense sound exposure. Plasma glucose increased 3 min after the seizure onset and rose to a peak after 10 min. Hepatic glycogen decreased significantly in susceptible audiogenic rats compared to nonepileptic controls, even before sound stimulation. A marked (∼10-fold) rise was observed in plasma lactate levels 3 and 10 min after the seizures compared to the response of the control group. Lipogenic activity showed a marked decrease even after stimulation with 25 ng/ml insulin. Based on these results, WARs showed reduced isoproterenol-stimulated lipolysis compared to control animals, whereas basal levels only differed significantly at 10 min after seizure activity. In conclusion, it can be inferred from these results that (a) the increase in plasma glucose after stimulation might result from sequential interaction of autonomic activation at seizure onset; (b) excessive muscular activity was at least partially responsible for the steady rise in plasma lactate concentrations; (c) audiogenic seizures, which increase adrenergic activity, induce desensitization of the β-adrenergic lipolytic pathway in epididymal adipose tissue; (d) genetic selection for audiogenic seizure susceptibility results in pronounced alterations at multiple levels of metabolic regulation.