Exploring Embryo-Ototoxic Effects: Insights into Deodorant-Induced Hair Cell Damage in Zebrafish.

Our study investigated the embryo-ototoxic effects of deodorant2 (DA2) on zebrafish embryos, which serve as valuable model organisms due to genetic and physiological similarities to humans. We focused on understanding DA2's impact on zebrafish hair cells, which are vital for sensory perception and balance regulation. DA2, provided by the Ministry of Environment, Republic of Korea, was used at 460 µg/mL in dimethyl sulfoxide (DMSO), with a 0.43% DMSO solvent control group. Three experiments, each using 10 zebrafish specimens from each group, showed an initial 13% hair cell count reduction in the DA2-exposed group. Subsequent experiments demonstrated reductions of 37% and 22%, each with one mortality case. Statistical analysis revealed a significant 24% hair cell count reduction in the DA2-exposed group. We also assessed DA2's impact on zebrafish behavior. Although not statistically significant, differences in distances traveled (0.33-0.39, 95% confidence interval: -0.46-1.1, p = 0.2033) and latencies (-0.016-0.018, 95% confidence interval: -0.052-0.021, p = 0.1917) hinted at negative effects. These results highlight DA2's ototoxic properties affecting zebrafish auditory systems and behavior. Further investigation into DA2's effects on aquatic organisms and potential mitigation strategies are essential. These findings contribute to understanding DA2's safety profile, benefiting aquatic ecosystems and human health assessments.

Serine/threonine kinase 31 promotes PDCD5-mediated apoptosis in p53-dependent human colon cancer cells.

Although programed cell death 5 (PDCD5) is an important protein in p53-mediated proapoptotic signaling, very little is known about PDCD5-related cell death. In this study, we report that serine/threonine kinase 31 (STK31) interacts with PDCD5, which maintains the stability of PDCD5. STK31 overexpression significantly activated PDCD5 stabilization and p53-mediated apoptosis in response to etoposide (ET). However, STK31 knockdown did not enhance apoptosis by ET treatment. Moreover, when STK31 was depleted, PDCD5 inhibited the activation of the p53 signaling pathway with ET, indicating that the PDCD5-STK31 network has an essential role in p53 activation. Importantly, STK31 activated the p53 signaling pathway by genotoxic stress through positive regulation of PDCD5-mediated apoptosis. We thus demonstrated that overexpression of STK31 greatly inhibited tumorigenic growth and increased the chemosensitivity of HCT116 human colorectal carcinoma cells. Taken together, these findings demonstrate that the STK31-PDCD5 complex network regulates apoptosis of cancer cells, and STK31 is a positive apoptosis regulator that inhibits tumorigenesis of colon cancer cells by inducing PDCD5-mediated apoptosis in response to genotoxic stress.

Demographically-Based Evaluation of Genomic Regions under Selection in Domestic Dogs.

Controlling for background demographic effects is important for accurately identifying loci that have recently undergone positive selection. To date, the effects of demography have not yet been explicitly considered when identifying loci under selection during dog domestication. To investigate positive selection on the dog lineage early in the domestication, we examined patterns of polymorphism in six canid genomes that were previously used to infer a demographic model of dog domestication. Using an inferred demographic model, we computed false discovery rates (FDR) and identified 349 outlier regions consistent with positive selection at a low FDR. The signals in the top 100 regions were frequently centered on candidate genes related to brain function and behavior, including LHFPL3, CADM2, GRIK3, SH3GL2, MBP, PDE7B, NTAN1, and GLRA1. These regions contained significant enrichments in behavioral ontology categories. The 3rd top hit, CCRN4L, plays a major role in lipid metabolism, that is supported by additional metabolism related candidates revealed in our scan, including SCP2D1 and PDXC1. Comparing our method to an empirical outlier approach that does not directly account for demography, we found only modest overlaps between the two methods, with 60% of empirical outliers having no overlap with our demography-based outlier detection approach. Demography-aware approaches have lower-rates of false discovery. Our top candidates for selection, in addition to expanding the set of neurobehavioral candidate genes, include genes related to lipid metabolism, suggesting a dietary target of selection that was important during the period when proto-dogs hunted and fed alongside hunter-gatherers.

Hypoxia adaptations in the grey wolf (Canis lupus chanco) from Qinghai-Tibet Plateau.

The Tibetan grey wolf (Canis lupus chanco) occupies habitats on the Qinghai-Tibet Plateau, a high altitude (>3000 m) environment where low oxygen tension exerts unique selection pressure on individuals to adapt to hypoxic conditions. To identify genes involved in hypoxia adaptation, we generated complete genome sequences of nine Chinese wolves from high and low altitude populations at an average coverage of 25× coverage. We found that, beginning about 55,000 years ago, the highland Tibetan grey wolf suffered a more substantial population decline than lowland wolves. Positively selected hypoxia-related genes in highland wolves are enriched in the HIF signaling pathway (P = 1.57E-6), ATP binding (P = 5.62E-5), and response to an oxygen-containing compound (P≤5.30E-4). Of these positively selected hypoxia-related genes, three genes (EPAS1, ANGPT1, and RYR2) had at least one specific fixed non-synonymous SNP in highland wolves based on the nine genome data. Our re-sequencing studies on a large panel of individuals showed a frequency difference greater than 58% between highland and lowland wolves for these specific fixed non-synonymous SNPs and a high degree of LD surrounding the three genes, which imply strong selection. Past studies have shown that EPAS1 and ANGPT1 are important in the response to hypoxic stress, and RYR2 is involved in heart function. These three genes also exhibited significant signals of natural selection in high altitude human populations, which suggest similar evolutionary constraints on natural selection in wolves and humans of the Qinghai-Tibet Plateau.

Genome sequencing highlights the dynamic early history of dogs.

To identify genetic changes underlying dog domestication and reconstruct their early evolutionary history, we generated high-quality genome sequences from three gray wolves, one from each of the three putative centers of dog domestication, two basal dog lineages (Basenji and Dingo) and a golden jackal as an outgroup. Analysis of these sequences supports a demographic model in which dogs and wolves diverged through a dynamic process involving population bottlenecks in both lineages and post-divergence gene flow. In dogs, the domestication bottleneck involved at least a 16-fold reduction in population size, a much more severe bottleneck than estimated previously. A sharp bottleneck in wolves occurred soon after their divergence from dogs, implying that the pool of diversity from which dogs arose was substantially larger than represented by modern wolf populations. We narrow the plausible range for the date of initial dog domestication to an interval spanning 11-16 thousand years ago, predating the rise of agriculture. In light of this finding, we expand upon previous work regarding the increase in copy number of the amylase gene (AMY2B) in dogs, which is believed to have aided digestion of starch in agricultural refuse. We find standing variation for amylase copy number variation in wolves and little or no copy number increase in the Dingo and Husky lineages. In conjunction with the estimated timing of dog origins, these results provide additional support to archaeological finds, suggesting the earliest dogs arose alongside hunter-gathers rather than agriculturists. Regarding the geographic origin of dogs, we find that, surprisingly, none of the extant wolf lineages from putative domestication centers is more closely related to dogs, and, instead, the sampled wolves form a sister monophyletic clade. This result, in combination with dog-wolf admixture during the process of domestication, suggests that a re-evaluation of past hypotheses regarding dog origins is necessary.

Fast and accurate site frequency spectrum estimation from low coverage sequence data.

MOTIVATION: The distribution of allele frequencies across polymorphic sites, also known as the site frequency spectrum (SFS), is of primary interest in population genetics. It is a complete summary of sequence variation at unlinked sites and more generally, its shape reflects underlying population genetic processes. One practical challenge is that inferring the SFS from low coverage sequencing data in a straightforward manner by using genotype calls can lead to significant bias. To reduce bias, previous studies have used a statistical method that directly estimates the SFS from sequencing data by first computing site allele frequency (SAF) likelihood for each site (i.e. the likelihood a site has each possible allele frequency conditional on observed sequence reads) using a dynamic programming (DP) algorithm. Although this method produces an accurate SFS, computing the SAF likelihood is quadratic in the number of samples sequenced. RESULTS: To overcome this computational challenge, we propose an algorithm, 'score-limited DP' algorithm, which is linear in the number of genomes to compute the SAF likelihood. This algorithm works because in a lower triangular matrix that arises in the DP algorithm, all non-negligible values of the SAF likelihood are concentrated on a few cells around the best-guess allele counts. We show that our score-limited DP algorithm has comparable accuracy but is faster than the original DP algorithm. This speed improvement makes SFS estimation practical when using low coverage NGS data from a large number of individuals. AVAILABILITY AND IMPLEMENTATION: The program will be available via a link from the Novembre lab website (http://jnpopgen.org/).

Genome-wide SNP and haplotype analyses reveal a rich history underlying dog domestication.

Advances in genome technology have facilitated a new understanding of the historical and genetic processes crucial to rapid phenotypic evolution under domestication. To understand the process of dog diversification better, we conducted an extensive genome-wide survey of more than 48,000 single nucleotide polymorphisms in dogs and their wild progenitor, the grey wolf. Here we show that dog breeds share a higher proportion of multi-locus haplotypes unique to grey wolves from the Middle East, indicating that they are a dominant source of genetic diversity for dogs rather than wolves from east Asia, as suggested by mitochondrial DNA sequence data. Furthermore, we find a surprising correspondence between genetic and phenotypic/functional breed groupings but there are exceptions that suggest phenotypic diversification depended in part on the repeated crossing of individuals with novel phenotypes. Our results show that Middle Eastern wolves were a critical source of genome diversity, although interbreeding with local wolf populations clearly occurred elsewhere in the early history of specific lineages. More recently, the evolution of modern dog breeds seems to have been an iterative process that drew on a limited genetic toolkit to create remarkable phenotypic diversity.

Characterizing bias in population genetic inferences from low-coverage sequencing data.

The site frequency spectrum (SFS) is of primary interest in population genetic studies, because the SFS compresses variation data into a simple summary from which many population genetic inferences can proceed. However, inferring the SFS from sequencing data is challenging because genotype calls from sequencing data are often inaccurate due to high error rates and if not accounted for, this genotype uncertainty can lead to serious bias in downstream analysis based on the inferred SFS. Here, we compare two approaches to estimate the SFS from sequencing data: one approach infers individual genotypes from aligned sequencing reads and then estimates the SFS based on the inferred genotypes (call-based approach) and the other approach directly estimates the SFS from aligned sequencing reads by maximum likelihood (direct estimation approach). We find that the SFS estimated by the direct estimation approach is unbiased even at low coverage, whereas the SFS by the call-based approach becomes biased as coverage decreases. The direction of the bias in the call-based approach depends on the pipeline to infer genotypes. Estimating genotypes by pooling individuals in a sample (multisample calling) results in underestimation of the number of rare variants, whereas estimating genotypes in each individual and merging them later (single-sample calling) leads to overestimation of rare variants. We characterize the impact of these biases on downstream analyses, such as demographic parameter estimation and genome-wide selection scans. Our work highlights that depending on the pipeline used to infer the SFS, one can reach different conclusions in population genetic inference with the same data set. Thus, careful attention to the analysis pipeline and SFS estimation procedures is vital for population genetic inferences.

Protective effects of Y-27632 against cisplatin-induced ototoxicity: A zebrafish model Y-27632 and cisplatin-induced ototoxicity.

Cisplatin is an effective chemotherapy agent against various solid malignancies; however, it is associated with irreversible bilateral sensorineural hearing loss, emphasizing the need for drug development to prevent this complication, with the current options being very limited. Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine protein kinase involved in various cellular processes, including apoptosis regulation. In this study, we used a transgenic zebrafish model (Brn3C: EGFP) in which hair cells within neuromasts are observed in green under fluorescent microscopy without the need for staining. Zebrafish larvae were exposed to cisplatin alone or in combination with various concentrations of Y-27632, a potent ROCK inhibitor. Hair cell counts, apoptosis assessments using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay, FM1-43FX labeling assay and behavioral analyses (startle response and rheotaxis) were performed to evaluate the protective effects of Y-27632 against cisplatin-induced ototoxicity. Cisplatin treatment reduced the number of hair cells in neuromasts, induced apoptosis, and impaired zebrafish larval behaviors. Y-27632 demonstrated a dose-dependent protective effect against cisplatin-induced hair cell loss and apoptosis. These findings suggest that Y-27632, as a ROCK inhibitor, mitigates cisplatin-induced hair cell loss and associated ototoxicity in zebrafish.

Measuring Optokinetic Reflex and Vestibulo-Ocular Reflex in Unilateral Vestibular Organ Damage Model of Zebrafish.

One-sided vestibular disorders are common in clinical practice; however, their models have not been fully established. We investigated the effect of unilateral or bilateral deficits in the vestibular organs on the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) of zebrafish using in-house equipment. For physical dislodgement of the otoliths in the utricles of zebrafish larvae, one or both utricles were separated from the surrounding tissue using glass capillaries. The video data from VOR and OKR tests with the larvae was collected and processed using digital signal processing techniques such as fast Fourier transform and low-pass filters. The results showed that unilateral and bilateral damage to the vestibular system significantly reduced VOR and OKR. In contrast, no significant difference was observed between unilateral and bilateral damage. This study confirmed that VOR and OKR were significantly reduced in zebrafish with unilateral and bilateral vestibular damage. Follow-up studies on unilateral vestibular disorders can be conducted using this tool.

Protective effects of esomeprazole against cisplatin-induced ototoxicity: an in vitro and in vivo study.

In this study, we aimed to identify novel compounds that could afford protection against cisplatin-induced ototoxicity by employing both cell- and zebrafish (Danio rerio)-based screening platforms. We screened 923 US Food and Drug Administration-approved drugs to identify potential compounds exhibiting protective effects against cisplatin-induced ototoxicity in HEI-OC1 cells (auditory hair cell line). The screening strategy identified esomeprazole and dexlansoprazole as the primary hit compounds. Subsequently, we examined the effects of these compounds on cell viability and apoptosis. Our results revealed that esomeprazole and dexlansoprazole inhibited organic cation transporter 2 (OCT2), thus providing in vitro evidence that these compounds could ameliorate cisplatin-induced ototoxicity by directly inhibiting OCT2-mediated cisplatin transport. In vivo, the protective effects were validated using zebrafish; esomeprazole was found to decrease cisplatin-induced hair cell damage in neuromasts. Furthermore, the esomeprazole-treated group showed a significantly lower number of TUNEL-positive cells than the cisplatin-treated group. Collectively, our findings revealed that esomeprazole exerts a protective effect against cisplatin-induced hair cell damage in both HEI-OC1 cells and a zebrafish model.

Noise-induced hearing loss in zebrafish model: Characterization of tonotopy and sex-based differences.

Hearing loss caused by frequent and persistent exposure to loud noise is one of the most common diseases in modern society. Many studies have demonstrated the characteristics of noise-induced hearing loss in human and non-human vertebrate models, including frequency-specific noise-induced hearing loss and sex-biased differences. Zebrafish (Danio rerio) is a useful hearing research model because its lateral line is easy to access and because of its detailed perception of sound. Despite the increasing popularity of zebrafish as a model for NIHL, a better understanding of this model is needed to determine sex differences in NIHL. To study the features of zebrafish as they relate to an NIHL model, we tested various phenotypes after frequency-specific noise stimulation. The degree of damage to hair cells and hearing loss were investigated after exposing zebrafish to 200 Hz and 1 kHz continuous waves and broadband white noise with a bandwidth from 50 Hz to 1 kHz. After exposure to all frequencies, the larvae showed lateral line hair cell damage, which is superficially located. In adult zebrafish, the threshold of auditory-evoked potential signals is elevated. Moreover, the number of hair cells remarkably decreased in the rostral region of the saccule, after exposure to 1 kHz and white noise, whereas zebrafish exposed to 200 Hz noise showed a decrease in hair cells in the caudal region. Moreover, male zebrafish were found to be more vulnerable to noise than female zebrafish, as is the case in humans and other mammals. Cortisol levels also increased in the noise-exposed male group, as compared to the noise-exposed female and control male groups. However, there was no difference in cortisol levels when the noise-exposed female group was compared to the control female group. Our study demonstrates not only that noise-induced hearing loss is frequency-dependent but also that the degree of hearing loss is affected by sex in zebrafish, emphasizing the need to consider sex in NIHL studies.

Principal component analysis under population genetic models of range expansion and admixture.

In a series of highly influential publications, Cavalli-Sforza and colleagues used principal component (PC) analysis to produce maps depicting how human genetic diversity varies across geographic space. Within Europe, the first axis of variation (PC1) was interpreted as evidence for the demic diffusion model of agriculture, in which farmers expanded from the Near East approximately 10,000 years ago and replaced the resident hunter-gatherer populations with little or no interbreeding. These interpretations of the PC maps have been recently questioned as the original results can be reproduced under models of spatially covarying allele frequencies without any expansion. Here, we study PC maps for data simulated under models of range expansion and admixture. Our simulations include a spatially realistic model of Neolithic farmer expansion and assume various levels of interbreeding between farmer and resident hunter-gatherer populations. An important result is that under a broad range of conditions, the gradients in PC1 maps are oriented along a direction perpendicular to the axis of the expansion, rather than along the same axis as the expansion. We propose that this surprising pattern is an outcome of the "allele surfing" phenomenon, which creates sectors of high allele-frequency differentiation that align perpendicular to the direction of the expansion.

Analysis of behavioral changes in zebrafish (Danio rerio) larvae caused by aminoglycoside-induced damage to the lateral line and muscles.

Zebrafish behavior is influenced by the lateral line hair cells and muscles. Drug-induced behavioral changes can serve as indicators in the evaluation of drug toxicity. The aminoglycoside family of antibiotics comprise a number of agents, including neomycin (NM) and gentamicin (GM). We hypothesized that NM and GM exert different effects on zebrafish larvae through their action on the lateral line and muscle fibers, inducing different swimming behavioral patterns such as locomotor behavior and the startle response. In this study, 125 µM NM and 5, 10, 20 µM GM induced hair cell damage in the anterior and posterior lateral lines of zebrafish larvae. However, unlike GM, 125 µM NM also caused muscle damage. Locomotor behavior was decreased in the 125 µM NM-exposed group compared to the group exposed to GM. Furthermore, 125 µM NM exposure induced significantly different patterns of various indices of startle behavior compared with the GM exposure groups. Additionally, the larvae exhibited different startle responses depending on the concentration of GM. These results suggest that GM may be the drug-of-choice for analyzing behavioral changes in zebrafish caused by damage to the lateral line alone. Our study highlights the importance of confirming muscle damage in behavioral analyses using zebrafish.

Evaluation of Ototoxicity of an Antifog Agent and the Suspected Underlying Mechanisms: An Animal Study.

Use of rigid endoscopes has become widespread in middle ear surgeries, thereby attracting attention to the safety of antifog agents. However, few studies on the ototoxicity of antifog agents have been conducted. The purpose of this study was to evaluate hair cell damage and the underlying mechanisms caused by antifog agents using zebrafish larvae. We exposed zebrafish larvae at 3 days postfertilization to various concentrations of the antifog agent, Ultrastop (0.01, 0.02, 0.04, and 0.08%) for 72 hours. The average number of hair cells within 4 neuromasts of larvae, including supraorbital (SO1 and SO2), otic (O1), and occipital (OC1), in the control group were compared to those in the exposure groups. Significant hair cell loss was observed in the experimental groups compared to that in the control group (P < .01; control: 53.88 ± 4.85, 0.01%: 45.08 ± 11.70, 0.02%: 41.36 ± 12.00, 0.04%: 35.36 ± 16.18, and 0.08%: 15.60 ± 7.53 cells). Concentration-dependent increase in hair cell apoptosis by terminal deoxynucleotidyltransferase (TDT)-mediated dUTP-biotin nick end labeling assay (control: 0.00 ± 0.00, 0.01%: 3.48 ± 2.18, 0.02%: 9.64 ± 5.75, 0.04%: 17.72 ± 6.26, and 0.08%: 14.60 ± 8.18 cells) and decrease in the viability of hair cell mitochondria by 2-(4-[dimethylamino] styryl)-N-ethylpyridinium iodide assay (control: 9.61 ± 1.47, 0.01%: 8.28 ± 2.22, 0.02%: 8.45 ± 2.72, 0.04%: 7.25 ± 2.44, and 0.08%: 6.77 ± 3.26 percentage of total area) were observed. Antifog agent exposure can cause hair cell damage in zebrafish larvae, possibly by induction of mitochondrial damage with subsequent apoptosis of hair cells.

Assessment of hair cell damage and developmental toxicity after fine particulate matter 2.5 µm (PM 2.5) exposure using zebrafish (Danio rerio) models.

OBJECTIVES: Particulate matter (PM) exposure has become one of the most serious problems. The aim of the present study was to evaluate the hair cell damage and possible developmental toxicity caused by PM2.5 exposure using a zebrafish model. METHODS: Zebrafish embryos were exposed to various concentrations of PM2.5. Developmental toxicity was evaluated based on general morphology score (GMS) system and Panzica-Kelly score, and by measurement of body length and heart rate. To evaluate hair cell damage, the average number of total hair cells within four neuromasts exposed to various concentrations of PM2.5 was compared with that of the control group. RESULTS: Morphological abnormalities evaluated by the GMS system and Panzica-Kelly score were rare and body length tended to be shorter in the PM2.5-exposed groups. Heart rate decreased significantly in the PM2.5-exposed group. Additionally, significant hair cell damage was observed after PM2.5 exposure. It was dose-dependent and more severe after a longer period exposure (10 dpf). CONCLUSIONS: In zebrafish embryos, exposure of PM2.5 in the early stages of life decreased heart rate and caused significant hair cell damage in a dose-dependent manner.

Enhancement of Seed Dehiscence by Seed Treatment with Talaromyces flavus GG01 and GG04 in Ginseng (Panax ginseng).

Seed dehiscence of ginseng (Panax ginseng C. A. Mayer) is affected by moisture, temperature, storage conditions and microbes. Several microbes were isolated from completely dehisced seed coat of ginseng cultivars, Chunpoong and Younpoong at Gumsan, Korea. We investigated the potential of five Talaromyces flavus isolates from the dehiscence of ginseng seed in four traditional stratification facilities. The isolates showed antagonistic activities against fungal plant pathogens, such as Cylindrocarpon destructans, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia nivalis, Botrytis cinerea, and Phytophthora capsici. The dehiscence ratios of ginseng seed increased more than 33% by treatment of T. flavus GG01, GG02, GG04, GG12, and GG23 in comparison to control (28%). Among the treatments, the reformulating treatment of T. flavus isolates GG01 and GG04 showed the highest of stratification ratio of ginseng seed. After 16 weeks, the reformulating treatment of T. flavus isolates GG01 and GG04 significantly enhanced dehiscence of ginseng seed by about 81% compared to the untreated control. The candidate's treatment of T. flavus GG01 and GG04 showed the highest decreasing rate of 93% in seed coat hardness for 112 days in dehiscence period. The results suggested that the pre-inoculation of T. flavus GG01 and GG04 found to be very effective applications in improving dehiscence and germination of ginseng seed.

Clustering of 770,000 genomes reveals post-colonial population structure of North America.

Despite strides in characterizing human history from genetic polymorphism data, progress in identifying genetic signatures of recent demography has been limited. Here we identify very recent fine-scale population structure in North America from a network of over 500 million genetic (identity-by-descent, IBD) connections among 770,000 genotyped individuals of US origin. We detect densely connected clusters within the network and annotate these clusters using a database of over 20 million genealogical records. Recent population patterns captured by IBD clustering include immigrants such as Scandinavians and French Canadians; groups with continental admixture such as Puerto Ricans; settlers such as the Amish and Appalachians who experienced geographic or cultural isolation; and broad historical trends, including reduced north-south gene flow. Our results yield a detailed historical portrait of North America after European settlement and support substantial genetic heterogeneity in the United States beyond that uncovered by previous studies.

Roles of adenosine receptors in the regulation of kainic acid-induced neurotoxic responses in mice.

Kainic acid (KA) is a well-known excitatory and neurotoxic substance. In ICR mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the hippocampal CA3 pyramidal neurons. In the present study, the possible role of adenosine receptors in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. It has been shown that 3,7-dimethyl-1-propargylxanthine (DMPX; A2 adenosine receptors antagonist, 20 microg) reduced KA-induced CA3 pyramidal cell death. KA dramatically increased the phosphorylated extracellular signal-regulated kinase (p-ERK) immunoreactivities (IR) in dentate gyrus (DG) and mossy fibers. In addition, c-Jun, c-Fos, Fos-related antigen 1 (Fra-1) and Fos-related antigen 2 (Fra-2) protein levels were increased in hippocampal area in KA-injected mice. DMPX attenuated KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. However, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX; A1 adenosine receptor antagonist, 20 microg) did not affect KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. KA also increased the complement receptor type 3 (OX-42) IR in CA3 region of hippocampus. DMPX, but not PACPX, blocked KA-induced OX-42 IR. Our results suggest that p-ERK and c-Jun may function as important regulators responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Activated microglia, which was detected by OX-42 IR, may be related to phagocytosis of degenerated neuronal elements by KA excitotoxicity. Furthermore, it is implicated that A2, but not A1, adenosine receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

Effects of MK-801 and CNQX on various neurotoxic responses induced by kainic acid in mice.

Effects of MK-801 (a NMDA receptor blocker) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; a non-NMDA receptor blocker) on several neurotoxic responses induced by kainic acid (KA) were examined in ICR mice. In a lethality test, intracerebroventricular (i.c.v.) pretreatment of MK-801 (1 microg), but not CNQX (0.5 microg), attenuated the time to lethality induced by KA (0.5 microg) administered i.c.v. In the memory test (a passive avoidance test), MK-801, but not CNQX, prevented the memory loss induced by KA (0.1 microg). The damage induced by KA (0.1 microg) administered i.c.v. in the hippocampus was markedly concentrated in the CA3 pyramidal neurons. Both MK-801 and CNQX blocked the pyramidal cell death in CA3 hippocampal region induced by KA. In the immunocytochemical study, KA dramatically increased the phosphorylated ERK (p-ERK) and decreased the phosphorylated CREB (p-CREB) in the hippocmapus. Both MK-801 and CNQX attenuated, in part, the increased p-ERK and the decreased p-CREB induced by KA. In addition, both MK-801 and CNQX partially reduced the increased c-Fos and c-Jun protein expression in hippocampus induced by KA. Our results suggest that both NMDA and non-NMDA receptors are involved in supraspinally administered KA-induced pyramidal cell death in CA3 region of hippocampus in the mouse and the p-ERK and the dephosphorylation of CREB protein may play an important role in CA3 region cell death of the hippocampus induced by KA administered supraspinally. Furthermore, c-Fos and c-Jun proteins may serve as third messengers responsible for CA3 pyramidal cell death induced by supraspinally administered KA.