Autism spectrum disorder (ASD) is one of the most common neurodevelopmental disorders and is thought to be closely associated with genetic factors. It is noteworthy that many ASD-associated genes reported by genome-wide association studies encode proteins related to synaptic formation, transmission, and plasticity. Therefore, it is essential to elucidate the relationship between deficiencies in these genes and the relevant ASD-related phenotypes using synaptic and behavioral phenotypic analysis of mice that are genetically modified for genes related to ASD (e.g., knockout or mutant mice). In this review, we focus on the behavioral-, cellular-, and circuit-level phenotypes, including synaptic formation and function, of several knockout mouse models with genetic mutations related to ASD. Moreover, we introduce our recent findings on the possible association of the dense-core vesicle secretion-related gene CAPS2/CADPS2 with ASD by using knockout mice. Finally, we discuss the usefulness and limitations of various mouse models with single gene mutations for understanding ASD.
A magnitude 9.0 great earthquake, the 2011 off the Pacific coast of Tohoku Earthquake, occurred on March 11, 2011, and subsequent Fukushima Daiichi Nuclear Power Station (Fukushima NPS) accidents stirred up natural radiation around the campus of Fukushima Medical University (FMU). FMU is located in Fukushima City, and is 57 km to the northwest of Fukushima NPS. Due to temporary failure of the steam boilers, the air conditioning system for the animal rooms, all autoclaves, and a cage washer could not be used at the Laboratory Animal Research Center (LARC) of FMU. The outside air temperature dropped to zero overnight, and the temperature inside the animal rooms fell to 10°C for several hours. We placed sterilized nesting materials inside all cages to encourage rodents to create nests. The main water supply was cut off for 8 days in all, while supply of steam and hot water remained unavailable for 12 days. It took 20 days to restore the air conditioning system to normal operation at the facility. We measured radiation levels in the animal rooms to confirm the safety of care staff and researchers. On April 21, May 9, and June 17, the average radiation levels at a central work table in the animal rooms with HEPA filters were 46.5, 44.4, and 43.4 cpm, respectively, which is equal to the background level of the equipment. We sincerely hope our experiences will be a useful reference regarding crisis management for many institutes having laboratory animals.
Frogs possess pain receptors and pathways that support processing and perception of noxious stimuli however the level of organization is less well structured compared to mammals. It was long believed that the experience of pain was limited to ‘higher’ phylums of the animal kingdom. However, it is now commonly accepted that amphibians possess neuro-anatomical pathways conductive of a complete nociceptive experience. Xenopus laevis frogs have been one of the most popular aquatic research models for developmental studies and genetic research. These frogs have been extensively use in research for their eggs, that can be collected following hormonal stimulation either naturally or by surgical intervention. Many anaesthetics have been used in amphibians such as bath solutions of MS-222, benzocaine and eugenol as well as systemic injections of ketamine or tiletamine, barbiturates, propofol and gas administrations of methoxyflurane, halothane and isoflurane. Most of these anaesthetic drugs produce variability in depth and duration of anaesthesia. MS-222 appears to be one of the most reliable anaesthetics. This review will focus on the evidence of pain perception in frogs and will compare the effectiveness and limitations of different anaesthetics used in Xenopus leavis frogs.
The aim of this study was to analyze the response of gene expression caused by etoposide (VP-16) in the fetal mouse brain. Four miligrams/kilogram of VP-16 was intraperitoneally injected into pregnant mice on day 12 of gestation (GD 12). Gene expression profiling of the VP-16-treated fetal mouse brain by DNA microarray was performed. The expression changes of the target genes of p53 were also examined by real-time RT-PCR. VP-16 induced S-phase accumulation, G2/M arrest, and eventually apoptosis of neuroepithelial cells in the fetal brain. DNA microarray analysis revealed that 8 of cell cycle control- and apoptosis-related genes were upregulated and that 5 of DNA damage, repair, replication, and transcription genes were also upregulated in the fetal telencephalons at 4 h after VP-16 treatment (HAT). The results of real-time RT-PCR demonstrated that the expression of topoisomerase IIα was increased at 4 and 8 HAT. The expression of pro-apoptotic factors such as puma, noxa, bax, and cyclin G was also increased from 4 to 12 HAT. These results suggest that VP-16 induces DNA damage, DNA repair, cell cycle alternation, and apoptosis in the fetal mouse brain. In addition, VP-16-induced apoptosis is mediated through the mitochondrial pathway in a p53-related manner. The present study will provide a better understanding of the mechanisms of VP-16-induced fetal brain injury.
In order to provide healthy experimental animals, it is important to find and remove animals that have been accidentally exposed to various stresses during breeding. This study focuses mouse health-care management. Here we used human olfaction and gas chromatography-mass spectrometry to assess odor intensity and determine the concentrations of odor components. The feces were collected from mice that were exposed to 4 different stresses (no bedding chips, shaking, fasting, and movement restriction). These stresses caused a change in odor intensity as assessed by 6 panelists. Seventeen components were identified as dominant components in the odor that was emitted from feces. The concentration of each compound was converted to relative values versus its odor threshold levels in order to select ones effective for the quality of the odor. As a result, 12 selected components were found to be a useful set for the recognition of mice bred under different stress conditions. The present results may provide useful information for the development of standard fecal odor materials that may be used for the training of mouse care personnel.
An unidentified Helicobacter species, strain MIT 01-6451, was frequently detected in mice obtained from domestic commercial and academic institutions in Japan. To partially characterize this strain, its distributions in the gastrointestinal tract and hepatobiliary system of mice were investigated. In gastrointestinal tissues, this strain was detected in all cecum, colon, and feces samples tested, whereas fewer mice were positive in the ileum, jejunum, and duodenum. Interestingly, strain MIT 01-6451 was also detected in most stomach samples and in 33% of gallbladder samples. One mouse was found to be infected with multiple Helicobacter species. Fourteen copies of 16S rRNA genes were cloned from the tissues of this mouse. One had the highest level of sequence homology with H. canadensis, while 13 had the highest level of homology with the H. ganmani type strain or strain MIT 01-6451. Twelve of these 13 16S rRNA genes were mosaic sequences, being partially derived from H. ganmani and strain MIT 01-6451. These results suggest that H. ganmani and Helicobacter sp. MIT 01-6451 are prevalent in specific-pathogen-free mouse colonies in Japan and that lateral gene transfer probably occurs among Helicobacter species during coinfection.
Prediabetes model mice in which more than one gene associated with diabetes is knocked down simultaneously are potentially useful for pharmaceutical and medical studies of diabetes. However, the effective conditions for sufficient knockdown in vivo are dependent on the intrinsic properties of the target genes. It is necessary to investigate which genes are applicable or not to the in vivo knockdown method. In this study, insulin receptor substrate 1 and 2 (Irs-1, Irs-2) were selected as target genes. Effective siRNAs against the respective genes were designed, and their efficacy was confirmed by cell-based experiments. Based on the results of siRNAs, shRNA expression vectors against Irs-1 and Irs-2 were constructed, respectively. Their efficacy was also confirmed by cell-based experiments. A hydrodynamic method was applied to the delivery of the vectors to mice. This method was found to be effective for predominant delivery to the liver by demonstrative delivery of an EGFP expression vector and successive histochemical analysis. Fifty micrograms of the shRNA expression vector was injected into the tail vein. After 24 h, the liver, pancreas, and muscle were isolated, and the expression levels of Irs-1 and Irs-2 were analyzed by quantitative RT-PCR. In the liver, Irs-2 was effectively knocked down to 60% of the control level, but Irs-1 was not influenced even under the same conditions. The protocol developed here is feasible for the selection of genes fit for in vivo knockdown method.
The murine norovirus (MNV), which belongs to the Caliciviridae family, is prevalent in laboratory mice. Since this virus affects macrophages and dendritic cells, infected mice are not suitable for immunological investigations, making it important to detect MNV infections accurately. When we tested RNA extracts derived from mouse feces for MNV detection using nested RT-PCR with a set of MNV-specific primers reported by Goto et al. (Exp. Anim. 58: 135-140, 2009), we found that these primers amplified not only an MNV-specific signal but also amplified a relatively weak signal with a size almost identical to that of the specific signal. Analysis of the nucleotide sequence of this amplified signal revealed that it was at least 98% identical to the exophosphatase gene of a commensal bacterium, Bacteroides vulgatus. Subsequent analysis showed that the signal amplified with a pair of nested primers was from DNA derived from B. vulgatus, which is sometimes present in SPF laboratory mouse feces, and the nested primers used were both partly homologous with the B. vulgatus nucleotide sequence. We thus designed a new set of nested RT-PCR primers that was not cross-reactive with the B. vulgatus genome. PCR products amplified by the newly designed primers were at least 89.3% identical to the MNV RNA polymerase gene in all cases. Our findings demonstrated that the primer set we designed was suitable for detecting an MNV-specific signal without cross-reacting with B. vulgatus DNA in mouse feces.
The tail-flick (TF) test is the most frequently used method to measure pain levels and assess the effects of anesthesia. In this study, we performed the TF test in rats sedated via an indwelling epidural catheter and then examined the effectiveness of this method in evaluating the local anesthetic effects. First, an epidural catheter was inserted into the epidural space, and anesthetic [lidocaine (L) or lidocaine including adrenaline (AL)] or normal saline (NS) was administered. Under sedation, we measured the dose for disappearance of the TF response, time to TF response recovery, onset and regression of local anesthesia, as well as the effect of an added agent on its continuation. The time course of TF latency (% maximum possible effect) in the NS group did not change during the experiment. In the AL group, TF latency increased significantly more than baseline during the 30-min period after injection. This was also significantly higher than the latency in the NS group and the L group. In the L group, the TF latency increased significantly above baseline for 20 min after injection and was significantly higher than that in the NS group. Due to the fact that we were able to detect the effect of local anesthesia onset and regression, as well as the local anesthesia continuation action of an additive agent, in rats sedated via an indwelled epidural catheter, we consider our method to be an improvement over conventional methods.
Heterohybridomas, created to secrete monoclonal antibodies, are generally unstable over long-term culture because of chromosome elimination during culture. To evaluate the karyotype of heterohybridomas, we used simultaneous genomic in situ hybridization (GISH), which can distinguish unambiguously between parental genomes in allopolyploid species. Using GISH, we discriminated gerbil and mouse chromosomes in heterohybridomas with high sensitivity. The GISH technique will allow evaluation of the stability and crossability of heterohybridomas made from the fusion of mouse cells with those of related species.
Ggt1dwg/dwg mice are spontaneous mutant mice with a nucleotide deletion in the Ggt1 gene. They are characterized by dwarfism, cataract, and coat color abnormality. These abnormalities in the external appearance of Ggt1dwg/dwg mice closely resemble those of previously reported GGT1-deficient mice, Ggt1tm1Zuk/tm1Zuk (Ggt1-/-) and Ggt1enu1/enu1, generated by gene targeting or ENU mutagenesis. However, whether the pathological features of Ggt1dwg/dwg mice are also similar to those of the Ggt1-/- and Ggt1enu1/enu1 mice remains unclear. To clarify the pathogenesis of Ggt1dwg/dwg mice, we physiologically and histologically investigated the abnormalities of Ggt1dwg/dwg mice in this study. First, we analyzed the activity of GGT1 and GSH levels in Ggt1dwg/dwg mice. GGT1 activity in the Ggt1dwg/dwg mice was reduced to approximately 4.0% of that in the wild-type mice. Plasma and kidney GSH levels were markedly increased, while eye and liver GSH levels were markedly decreased, in the Ggt1dwg/dwg mice. Notably, no significant difference in survival rate was observed between the Ggt1dwg/dwg and wild-type mice, whereas high mortality was reported in the Ggt1-/- and Ggt1enu1/enu1 mice. Growth retardation, degeneration of lens fibers, and an increased number of osteoclasts in the Ggt1dwg/dwg mice were reversed by administration of N-acetyl-L-cysteine, a precursor of GSH synthesis. Thus, we conclude that the abnormalities of Ggt1dwg/dwg mice are caused by alteration of the GSH levels due to the depression of GGT1 activity and that Ggt1dwg/dwg mice will be a useful model for GGT deficiency with peculiar features.