A missense mutation in the Hspa8 gene encoding heat shock cognate protein 70 causes neuroaxonal dystrophy in rats.

Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by spheroid (swollen axon) formation in the nervous system. In the present study, we focused on a newly established autosomal recessive mutant strain of F344-kk/kk rats with hind limb gait abnormalities and ataxia from a young age. Histopathologically, a number of axonal spheroids were observed throughout the central nervous system, including the spinal cord (mainly in the dorsal cord), brain stem, and cerebellum in F344-kk/kk rats. Transmission electron microscopic observation of the spinal cord revealed accumulation of electron-dense bodies, degenerated abnormal mitochondria, as well as membranous or tubular structures in the axonal spheroids. Based on these neuropathological findings, F344-kk/kk rats were diagnosed with NAD. By a positional cloning approach, we identified a missense mutation (V95E) in the Hspa8 (heat shock protein family A (Hsp70) member 8) gene located on chromosome 8 of the F344-kk/kk rat genome. Furthermore, we developed the Hspa8 knock-in (KI) rats with the V95E mutation using the CRISPR-Cas system. Homozygous Hspa8-KI rats exhibited ataxia and axonal spheroids similar to those of F344-kk/kk rats. The V95E mutant HSC70 protein exhibited the significant but modest decrease in the maximum hydrolysis rate of ATPase when stimulated by co-chaperons DnaJB4 and BAG1 in vitro, which suggests the functional deficit in the V95E HSC70. Together, our findings provide the first evidence that the genetic alteration of the Hspa8 gene caused NAD in mammals.

Imbalance of glutamatergic and GABAergic neurotransmission in audiogenic seizure-susceptible Leucine-rich glioma-inactivated 1 (Lgi1)-mutant rats.

Leucine-rich glioma-inactivated 1 (LGI1) was identified as a causative gene of autosomal dominant lateral temporal lobe epilepsy. We previously reported that Lgi1-mutant rats carrying a missense mutation (L385R) showed audiogenic seizure-susceptibility. To explore the pathophysiological mechanisms underlying Lgi1-related epilepsy, we evaluated changes in glutamate and GABA release in Lgi1-mutant rats. Acoustic priming (AP) for audiogenic seizure-susceptibility was performed by applying intense sound stimulation (130 dB, 10 kHz, 5 min) on postnatal day 16. Extracellular glutamate and GABA levels in the hippocampus CA1 region were evaluated at 8 weeks of age, using in vivo microdialysis techniques. Under naïve conditions without AP, glutamate and GABA release evoked by high-K+ depolarization was more prominent in Lgi1-mutant than in wild-type (WT) rats. The AP treatment on day 16 significantly increased basal glutamate levels and depolarization-induced glutamate release both in Lgi1-mutant and WT rats, yielding greater depolarization-induced glutamate release in Lgi1-mutant rats. On the other hand, the AP treatment enhanced depolarization-induced GABA release only in WT rats, and not in Lgi1-mutant rats, illustrating reduced GABAergic neurotransmission in primed Lgi1-mutant rats. The present results suggest that enhanced glutamatergic and reduced GABAergic neurotransmission are involved in the audiogenic seizure-susceptibility associated with Lgi1-mutation.

Characterization of novel, severely immunodeficient PrkdcΔex57/Δex57 mice.

Severely immunodeficient mice are useful for understanding the pathogenesis of certain tumors and for developing therapeutic agents for such tumors. In addition, engraftment of these mice with human hematopoietic cells can yield information that helps us understand the in vivo molecular mechanisms underlying actual human viral infections. In our present research, we discovered a novel, severely immunodeficient strain of mice having a mutation in exon 57 of the Prkdc gene (PrkdcΔex57/Δex57) in an inbred colony of B10.S/SgSlc mice. Those PrkdcΔex57/Δex57 mice showed thymic hypoplasia and lack of mature T cells and B cells in peripheral lymphoid tissues, resulting in very low levels of production of serum immunoglobulins. In addition, those mice were highly susceptible to influenza viruses due to the lack of acquired immune cells. On the other hand, since they had sufficient numbers of NK cells, they rejected tumor transplants, similarly to Prkdc+/+ mice. Next, we generated Foxn1nu/nuPrkdcΔex57/Δex57Il2rg-/- (NPG) mice on the BALB/cSlc background, which lack all lymphocytes such as T cells, B cells and innate lymphoid cells, including NK cells. As expected, these mice were able to undergo engraftment of human tumor cell lines. These findings suggest that PrkdcΔex57/Δex57 mice will be useful as a novel model of immunodeficiency, while NPG mice will be useful for xenografting of various malignancies.

The rat Downunder (Du) coat color mutation is associated with eye anomalies and embryonic lethality and maps to a 3.9-Mb region on chromosome 3.

Rodent coat color genes have been studied as a bioresource to understand developmental and cellular processes. The Downunder rat is a fancy variety with a marking on its belly that runs from the neck to the breech and appears to mirror the dorsal hooded marking. Here, we established a congenic strain carrying the Downunder (Du) gene in an F344 genetic background. In addition to the ventral marking, Du/+ rats exhibit anophthalmia or microphthalmia with incomplete penetrance. Du/Du embryos die in the early stages of organogenesis. Genetic linkage analysis mapped the Du gene to rat chromosome 3 and haplotype mapping with congenic rats localized the Du locus to a 3.9-Mb region. The Du locus includes two functional genes, glycosyltransferase-like domain-containing 1 (Gtdc1) and zinc finger E-box binding homeobox 2 (Zeb2). Although we found no functional variation within any of Zeb2's exons or intron-exon boundaries, Zeb2 mRNA levels were significantly lower in Du/+ rats compared with wild-type rats. It is known that melanocyte-specific Zeb2 deletion results in the congenital loss of hair pigmentation in mice. Taken together, our results indicate that the Du mutation exerts pleiotropic effects on hair pigmentation, eye morphology, and development. Moreover, the Zeb2 gene is a strong candidate for the Du mutation.

PHF24 is expressed in the inhibitory interneurons in rats.

Noda epileptic rat (NER) is a mutant model for epilepsy that exhibits spontaneous generalized tonic-clonic seizure. Epileptogenesis of NER remains to be elucidated; but it is detected an insertion of an endogenous retrovirus sequence in intron 2 of the PHD finger protein 24 (Phf24) gene, encoding Gαi-interacting protein (GINIP). Phf24 is a strong candidate gene for epileptogenesis in NER. PHF24 modulates GABAB signaling through interacting with Gαi protein. To clarify the epileptogenesis of NER, we investigated a distribution of PHF24-expressing cells in the central nerve system (CNS). While broad expression of PHF24 was observed in the CNS, characteristic expression was noted in the periglomerular layer of the olfactory bulb and the lamina II of the spinal cord in the control rats. These cells showed co-expression with calbindin or calretinin, inhibitory interneuron markers. In the olfactory bulb, 15.6% and 41.2% of PHF24-positive neurons co-expressed calbindin and calretinin, respectively. Immunoelectron microscopy revealed that PHF24 was located in the presynaptic terminals, synaptic membranes and cytoplasmic matrix of neuronal soma. Our data suggested PHF24 is expressed in the inhibitory interneurons and may play important roles in modulation of the GABAB signaling.

Down syndrome cell adhesion molecule like-1 (DSCAML1) links the GABA system and seizure susceptibility.

The Ihara epileptic rat (IER) is a mutant model with limbic-like seizures whose pathology and causative gene remain elusive. In this report, via linkage analysis, we identified Down syndrome cell adhesion molecule-like 1(Dscaml1) as the responsible gene for IER. A single base mutation in Dscaml1 causes abnormal splicing, leading to lack of DSCAML1. IERs have enhanced seizure susceptibility and accelerated kindling establishment. Furthermore, GABAergic neurons are severely reduced in the entorhinal cortex (ECx) of these animals. Voltage-sensitive dye imaging that directly presents the excitation status of brain slices revealed abnormally persistent excitability in IER ECx. This suggests that reduced GABAergic neurons may cause weak sustained entorhinal cortex activations, leading to natural kindling via the perforant path that could cause dentate gyrus hypertrophy and epileptogenesis. Furthermore, we identified a single nucleotide substitution in a human epilepsy that would result in one amino acid change in DSCAML1 (A2105T mutation). The mutant DSCAML1A2105T protein is not presented on the cell surface, losing its homophilic cell adhesion ability. We generated knock-in mice (Dscaml1A2105T) carrying the corresponding mutation and observed reduced GABAergic neurons in the ECx as well as spike-and-wave electrocorticogram. We conclude that DSCAML1 is required for GABAergic neuron placement in the ECx and suppression of seizure susceptibility in rodents. Our findings suggest that mutations in DSCAML1 may affect seizure susceptibility in humans.

Downregulation of aspartoacylase during the progression of myelin breakdown in the dmy mutant rat with mitochondrial magnesium channel MRS2 defect.

OBJECTIVE: The dmy rat is an autosomal recessive mutant that exhibits severe rapid myelin breakdown throughout the central nervous system at 7-8 weeks of age. The dmy rat has a point mutation in Mrs2 gene, which encodes an essential component of the major electrophoretic Mg2+ influx system in the mitochondria. However, it remains unknown how mitochondrial dysfunction leads to the myelin breakdown. METHODS: We focused on the aspartoacylase (ASPA) and mitochondrion-related metabolites to clarify the mechanism of myelin pathology in dmy rats. Aspa mRNA was significantly decreased in both the gray matter and the ventral white matter of spinal cord in the dmy rats from 4 to 8 weeks of age. Very faint immunohistochemical expression for ASPA was noted in the gray and white matter of the affected dmy rats at 8 weeks. Liquid chromatography mass spectrometry revealed no different amount of N-acetylaspartate (NAA), which is synthesized from aspartate and acetyl-coenzyme A (CoA) in neurons, in the brain and spinal cord between the dmy and control rats. CONCLUSION: Our results indicated that the pyruvate dehydrogenase activity might be reduced due to the loss of Mg2+ transport activity in the mitochondria of the dmy rats, suggesting acetyl CoA production might be reduced. The number of oligodendrocytes was well preserved until 7 weeks. It is intriguing that prior to the myelin destruction at 7-8 weeks, disrupted expression of Aspa mRNA and ASPA protein undergoes from early stage of myelinogenesis. These data indicate that ASPA expression would be a useful index to evaluate a function of oligodendrocyte in the dmy rat.

Increased seizure sensitivity, emotional defects and cognitive impairment in PHD finger protein 24 (Phf24)-null rats.

Phf24 is known as Gαi-interacting protein (GINIP) and is associated with the GABAB receptor. To study the function of Phf24 protein in the central nervous system (CNS), we have newly developed Phf24-null rats and investigated their behavioral phenotypes, especially changes in seizure sensitivity, emotional responses and cognitive functions. Phf24-null rats did not exhibit any spontaneous seizures. However, they showed a higher sensitivity to pentylenetetrazol (PTZ)- or pilocarpine-induced convulsive seizures. Phf24-null rats also showed an elevated susceptibility to kindling development with repeated PTZ treatments, suggesting that Phf24 acts as an inhibitory modulator in epileptogenesis. Although young Phf24-null rats showed normal gross behaviors, elevated spontaneous locomotor activity, especially in terms of the circadian dark period, emotional hyper-reactivity, reduced anxiety behaviors in the elevated plus-maze (EPM) test, and cognitive deficits in the Morris water maze test were explicitly observed at older age (20-week-old). The present results suggest that Phf24 is essential for proper functioning of the CNS, especially in preventing epileptogenesis and controlling emotional and cognitive functions.

Down-Regulation of Astrocytic Kir4.1 Channels during the Audiogenic Epileptogenesis in Leucine-Rich Glioma-Inactivated 1 (Lgi1) Mutant Rats.

The dysfunction of astrocytic inwardly rectifying potassium (Kir) 4.1 channels, which mediate the spatial potassium-buffering function of astrocytes, is known to be involved in the development of epilepsy. Here, we analyzed the Kir4.1 expressional changes in Leucine-Rich Glioma-Inactivated 1 (Lgi1) mutant rats, which is a model of autosomal dominant lateral temporal lobe epilepsy in humans, to clarify the role of astrocytic Kir4.1 channels in Lgi1-related epileptogenesis. Priming acoustic stimulation (at postnatal day 16) conferred seizure susceptibility on Lgi1 mutant rats, which evoked audiogenic seizures with test stimulation at eight weeks. In the seizure-susceptible Lgi1 mutant rats (before test stimulation), astrocytic Kir4.1 expression was down-regulated region-specifically in the cerebral cortex, hippocampus, and amygdala. In addition, prophylactic treatments of Lgi1 mutant rats with valproic acid (VPA, 30 mg/kg and 200 mg/kg) for two weeks prevented both the development of seizure susceptibility and the down-regulation of Kir4.1 expression in astrocytes. The present study demonstrated for the first time that the astrocytic Kir4.1 expression was reduced in the Lgi1-related seizure model, suggesting that the down-regulation of Kir4.1 channels in astrocytes is involved in audiogenic epileptogenesis caused by Lgi1 mutation. In addition, VPA seemed to have a prophylactic effect on Lgi1-related seizures.

[Function of synaptic vesicle protein 2A (SV2A) as a novel therapeutic target for epilepsy].

Epilepsy is a chronic neurologic disease characterized by recurrent seizures, affecting nearly 1% of the population. Synaptic vesicle protein 2A (SV2A) is a membrane protein specifically expressed in synaptic vesicles and is now implicated in the pathogenesis of epileptic disorders. This is because 1) Sv2a-knockout mice exhibit severe seizures, 2) SV2A serves as a specific binding site for certain antiepileptics (e.g., levetiracetam and its analogues) and 3) the SV2A expression changes under various epileptic conditions both in animals (e.g., kindling) and humans (e.g., intractable temporal lobe epilepsy and focal cortical dysplasia). Furthermore, it has been shown that a missense mutation in the SV2A gene caused intractable epilepsy, involuntary movements and developmental retardation, indicating a causative role of SV2A dysfunction in epilepsy. In order to explore the mechanism of SV2A in modulating development of epileptogenesis, we recently developed a novel rat model (Sv2aL174Q rat) carrying a missense mutation (Leu174Gln) in the Sv2a gene. These rats were highly susceptible to the kindling development associated with repeated pentylenetetrazole treatments or electrical stimulations of the amygdala. In addition, the Sv2aL174Q mutation specifically impaired depolarization-induced GABA, but not glutamate, release in the hippocampus and amygdala. All this evidence indicates that the SV2A-GABAergic system plays a crucial role in modulating epileptogenesis and encourages discovery research into the novel antiepileptic agents which enhance the function of the SV2A-GABA system.

Rats deficient C-type natriuretic peptide suffer from impaired skeletal growth without early death.

We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.

Tremor dominant Kyoto (Trdk) rats carry a missense mutation in the gene encoding the SK2 subunit of small-conductance Ca2+-activated K+ channel.

Tremor dominant Kyoto (Trdk) is an autosomal dominant mutation that appeared in F344/NSlc rats mutagenized with N-ethyl-N-nitrosourea (ENU). In this study, we characterized and genetically analyzed F344-Trdk/+ heterozygous rats. The rats exhibited a tremor that was especially evident around weaning but persisted throughout life. The tremors of F344-Trdk/+ rats were attenuated by drugs effective against essential tremor (ET) but not drugs used to treat Parkinson's disease-related tremor, indicating that the pharmacological phenotype of F344-Trdk/+ rats was similar to human ET. Using positional candidate approach, we identified the Trdk mutation as a missense substitution (c. 866T>A, p. I289N) in Kcnn2, which encodes the SK2 subunit of the small-conductance Ca2+-activated K+ channel. In vitro electrophysiological studies revealed that the I289N mutation diminished SK2 channel activity. These findings demonstrate that F344-Trdk/+ rats represent a novel model of ET, and strongly suggest that Kcnn2 is the causative gene for the tremor phenotype in F344-Trdk/+ rats.

Identification of Candidate Genes for Generalized Tonic-Clonic Seizures in Noda Epileptic Rat.

The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

Nicotine Elicits Convulsive Seizures by Activating Amygdalar Neurons.

Nicotinic acetylcholine (nACh) receptors are implicated in the pathogenesis of epileptic disorders; however, the mechanisms of nACh receptors in seizure generation remain unknown. Here, we performed behavioral and immunohistochemical studies in mice and rats to clarify the mechanisms underlying nicotine-induced seizures. Treatment of animals with nicotine (1-4 mg/kg, i.p.) produced motor excitement in a dose-dependent manner and elicited convulsive seizures at 3 and 4 mg/kg. The nicotine-induced seizures were abolished by a subtype non-selective nACh antagonist, mecamylamine (MEC). An α7 nACh antagonist, methyllycaconitine, also significantly inhibited nicotine-induced seizures whereas an α4ß2 nACh antagonist, dihydro-ß-erythroidine, affected only weakly. Topographical analysis of Fos protein expression, a biological marker of neural excitation, revealed that a convulsive dose (4 mg/kg) of nicotine region-specifically activated neurons in the piriform cortex, amygdala, medial habenula, paratenial thalamus, anterior hypothalamus and solitary nucleus among 48 brain regions examined, and this was also suppressed by MEC. In addition, electric lesioning of the amygdala, but not the piriform cortex, medial habenula and thalamus, specifically inhibited nicotine-induced seizures. Furthermore, microinjection of nicotine (100 and 300 µg/side) into the amygdala elicited convulsive seizures in a dose-related manner. The present results suggest that nicotine elicits convulsive seizures by activating amygdalar neurons mainly via α7 nACh receptors.

Nicotine evokes kinetic tremor by activating the inferior olive via α7 nicotinic acetylcholine receptors.

Nicotinic acetylcholine (nACh) receptors are implicated in the pathogenesis of movement disorders (e.g., tremor) and epilepsy. Here, we performed behavioral and immunohistochemical studies using mice and rats to elucidate the mechanisms underlying nicotine-induced tremor. Treatments of animals with nicotine (0.5-2mg/kg, i.p.) elicited kinetic tremor, which was completely suppressed by the nACh receptor antagonist mecamylamine (MEC). The specific α7 nACh receptor antagonist methyllycaconitine (MLA) also inhibited nicotine-induced tremor, whereas the α4ß2 nACh antagonist dihydro-ß-erythroidine (DHßE) or the peripheral α3ß4 nACh antagonist hexamethonium showed no effects. Mapping analysis of Fos protein expression, a biological marker of neural excitation, revealed that a tremorgenic dose (1mg/kg) of nicotine region-specifically elevated Fos expression in the piriform cortex (PirC), medial habenula, solitary nucleus and inferior olive (IO) among 44 brain regions examined. In addition, similarly to the tremor responses, nicotine-induced Fos expression in the PirC and IO was selectively antagonized by MLA, but not by DHßE. Furthermore, an electrical lesioning of the IO, but not the PirC, significantly suppressed the induction of nicotine tremor. The present results suggest that nicotine elicits kinetic tremor in rodents by activating the IO neurons via α7 nACh receptors.

Fat Mass Reduction With Adipocyte Hypertrophy and Insulin Resistance in Heterozygous PPARγ Mutant Rats.

Agonist-induced activation of peroxisome proliferator-activated receptor-γ (PPARγ) stimulates adipocyte differentiation and insulin sensitivity. Patients with heterozygous PPARγ dominant-negative mutation develop partial lipodystrophy and insulin resistance. Inconsistent with this evidence in humans, it was reported that heterozygous PPARγ knockout mice have increased insulin sensitivity and that mice with heterozygous PPARγ dominant-negative mutation have normal insulin sensitivity and improved glucose tolerance. In the context of the interspecies intranslatability of PPARγ-related findings, we generated a PPARγ mutant rat with a loss-of-function mutation (Pparg(mkyo)) without dominant-negative activity by using the ENU (N-ethyl-N-nitrosourea) mutagenesis method. Heterozygous Pparg(mkyo/+) rats showed reduced fat mass with adipocyte hypertrophy and insulin resistance, which were highly predictable from known actions of PPARγ agonists and phenotypes of patients with the PPARγ mutation. This report is the first in our knowledge to clearly demonstrate that both alleles of PPARγ are required for normal adipocyte development and insulin sensitivity in vivo. Furthermore, the study indicates that PPARγ regulates mainly adipocyte number rather than adipocyte size in vivo. The choice of appropriate species as experimental models is critical, especially for the study of PPARγ.

A Missense Mutation of the Gene Encoding Synaptic Vesicle Glycoprotein 2A (SV2A) Confers Seizure Susceptibility by Disrupting Amygdalar Synaptic GABA Release.

Synaptic vesicle glycoprotein 2A (SV2A) is specifically expressed in the membranes of synaptic vesicles and modulates action potential-dependent neurotransmitter release. To explore the role of SV2A in the pathogenesis of epileptic disorders, we recently generated a novel rat model (Sv2a(L174Q) rat) carrying a missense mutation of the Sv2a gene and showed that the Sv2a(L174Q) rats were hypersensitive to kindling development (Tokudome et al., 2016). Here, we further conducted behavioral and neurochemical studies to clarify the pathophysiological mechanisms underlying the seizure vulnerability in Sv2a(L174Q) rats. Sv2a(L174Q) rats were highly susceptible to pentylenetetrazole (PTZ)-induced seizures, yielding a significantly higher seizure scores and seizure incidence than the control animals. Brain mapping analysis of Fos expression, a biological marker of neural excitation, revealed that the seizure threshold level of PTZ region-specifically elevated Fos expression in the amygdala in Sv2a(L174Q) rats. In vivo microdialysis study showed that the Sv2a(L174Q) mutation preferentially reduced high K(+) (depolarization)-evoked GABA release, but not glutamate release, in the amygdala. In addition, specific control of GABA release by SV2A was supported by its predominant expression in GABAergic neurons, which were co-stained with antibodies against SV2A and glutamate decarboxylase 1. The present results suggest that dysfunction of SV2A by the missense mutation elevates seizure susceptibility in rats by preferentially disrupting synaptic GABA release in the amygdala, illustrating the crucial role of amygdalar SV2A-GABAergic system in epileptogenesis.

Synaptic vesicle glycoprotein 2A (SV2A) regulates kindling epileptogenesis via GABAergic neurotransmission.

Synaptic vesicle glycoprotein 2A (SV2A) is a prototype synaptic vesicle protein regulating action potential-dependent neurotransmitters release. SV2A also serves as a specific binding site for certain antiepileptics and is implicated in the treatment of epilepsy. Here, to elucidate the role of SV2A in modulating epileptogenesis, we generated a novel rat model (Sv2a(L174Q) rat) carrying a Sv2a-targeted missense mutation (L174Q) and analyzed its susceptibilities to kindling development. Although animals homozygous for the Sv2a(L174Q) mutation exhibited normal appearance and development, they are susceptible to pentylenetetrazole (PTZ) seizures. In addition, development of kindling associated with repeated PTZ treatments or focal stimulation of the amygdala was markedly facilitated by the Sv2a(L174Q) mutation. Neurochemical studies revealed that the Sv2a(L174Q) mutation specifically reduced depolarization-induced GABA, but not glutamate, release in the hippocampus without affecting basal release or the SV2A expression level in GABAergic neurons. In addition, the Sv2a(L174Q) mutation selectively reduced the synaptotagmin1 (Syt1) level among the exocytosis-related proteins examined. The present results demonstrate that dysfunction of SV2A due to the Sv2a(L174Q) mutation impairs the synaptic GABA release by reducing the Syt1 level and facilitates the kindling development, illustrating the crucial role of SV2A-GABA system in modulating kindling epileptogenesis.

Atopic dermatitis-like skin lesions with IgE hyperproduction and pruritus in KFRS4/Kyo rats.

BACKGROUND: Rats showing spontaneous atopic dermatitis (AD)-like skin lesions were observed in the Kyoto Fancy Rat Stock 4 (KFRS4) strain breeding colony. OBJECTIVE: To establish the KFRS4 rat as a model of AD. METHODS: The clinical symptoms of AD-like skin lesions were assessed by scoring the degree of dermatitis and examining scratching behavior. The transepidermal water loss was measured to evaluate skin barrier function. Cells infiltrating the skin lesions were identified using histological and immunohistological analyses. IgE and cytokine levels were measured to examine immune status. An ointment treatment experiment was carried out to characterize dermatitis in the KFRS4 rats. RESULTS: Dermatitis initially appeared around 4 months of age and rapidly worsened from 6 to 8 months of age. The skin lesions accompanied scratching behavior and were predominantly observed in females. The increased transepidermal water loss indicated skin barrier dysfunction. Extensive infiltration of eosinophils, mast cells and lymphocytes was observed in the skin lesions. The plasma IgE level increased in accord with increasing severity of dermatitis. The Th2 and Th17 cytokine mRNA levels were significantly higher in the skin-draining lymph nodes than those in the non-skin-draining lymph nodes. It was demonstrated that betamethasone improved the symptoms of dermatitis. These findings demonstrated that dermatitis in the KFRS4 rats closely resembled that seen in human AD. CONCLUSION: Female KFRS4 rats have the potential to serve as an animal model of human AD.