Potential Implications of Acid-Sensing Ion Channels ASIC2 and ASIC4 in Gonadal Differentiation of Dicentrarchus labrax Subjected to Water Temperature Increase during Gonadal Development.

In this study, the expression and implication of acid-sensing ion channels 2 and 4 (ASIC2 and ASIC4) in the gonadal sex differentiation of Dicentrarchus labrax (D. labrax), subjected to increasing water temperatures during gonadal development, were evaluated. Two groups were selected: a control group (CG), in which the average water temperature was maintained at 15 °C and increased to 20 °C in 20 days until weaning; and an experimental group (EG), in which the water temperature was retained at 15 °C for 60 days; thereafter, the temperature was increased daily by 0.5 °C until it reached 20 °C up to the weaning time. Ten fish from the CG and 13 fish from the EG were sampled randomly on the 335th day after hatching (dph). A higher percentage of gonad differentiation in ovaries rather than in testes was observed in the EG compared to the CG (p = 0.01). ASIC2 and ASIC4 were detected for the first time in D. labrax ovaries by indirect immunofluorescence. Both ASIC2 and ASIC4 were expressed in previtellogenic oocytes of ovaries and in scattered cells within some testes, and were most likely intratesticular previtellogenic oocytes in both the CG and EG groups. The CG group showed a higher expression of ASIC4 than the EG cohort (p < 0.05). The results gathered in this study revealed the capacity of water temperature to influence both gonadal differentiation and growth in this gonochoristic fish species, and suggests the possible role of ASIC2 and ASIC4 in gonad differentiation and gamete development in D. labrax.

Follistatin mediates learning and synaptic plasticity via regulation of Asic4 expression in the hippocampus.

The biological mechanisms underpinning learning are unclear. Mounting evidence has suggested that adult hippocampal neurogenesis is involved although a causal relationship has not been well defined. Here, using high-resolution genetic mapping of adult neurogenesis, combined with sequencing information, we identify follistatin (Fst) and demonstrate its involvement in learning and adult neurogenesis. We confirmed that brain-specific Fst knockout (KO) mice exhibited decreased hippocampal neurogenesis and demonstrated that FST is critical for learning. Fst KO mice exhibit deficits in spatial learning, working memory, and long-term potentiation (LTP). In contrast, hippocampal overexpression of Fst in KO mice reversed these impairments. By utilizing RNA sequencing and chromatin immunoprecipitation, we identified Asic4 as a target gene regulated by FST and show that Asic4 plays a critical role in learning deficits caused by Fst deletion. Long-term overexpression of hippocampal Fst in C57BL/6 wild-type mice alleviates age-related decline in cognition, neurogenesis, and LTP. Collectively, our study reveals the functions for FST in adult neurogenesis and learning behaviors.

Walnut oil improves spatial memory in rats and increases the expression of acid-sensing ion channel genes Asic2a and Asic4.

Although Walnut oil (WO) has been reported to enhance cognitive function, the underlying molecular mechanisms are not well understood. This study was designed to assess the effects of WO on spatial memory in rats through modulation of the expression of acid-sensing ion channel genes, Asic2a and Asic4. To investigate the effect of WO on cognitive performance, we supplemented the diet of female rats with WO. The results showed that supplementation with WO at doses of 2.2 and 11 g kg-1 day-1 significantly improved learning and memory. In vitro treatment of rat hippocampal neuronal cells with appropriate doses of WO revealed a significant increase in the expression of Asic2a and Asic4 in a dose-dependent manner at both the mRNA and protein levels. We conclude that WO intake might help to prevent cognitive decline, particularly in the elderly, and that ASIC genes in neurons can be the targets of compounds contained in the oil.

Assessing the role of the acid-sensing ion channel ASIC4b in sodium uptake by larval zebrafish.

Na+ uptake in larval zebrafish (Danio rerio) is coordinated by three mechanisms: Na+/H+-exchanger 3b (NHE3b) expressed in H+-ATPase-rich (HR) cells, an unidentified Na+ channel coupled to electrogenic H+-ATPase expressed in HR cells, and Na+-Cl--cotransporter (NCC) expressed in NCC cells. Recently, acid-sensing ion channels (ASICs) were proposed to be the putative Na+ channel involved in H+-ATPase-mediated Na+ uptake in adult zebrafish and rainbow trout. In the present study, we hypothesized that ASICs also play this role in Na+ uptake in larval zebrafish. In support of this hypothesis, immunohistochemical analyses revealed that ASIC4b was expressed in HR cells on the yolk sac skin at 4 days post-fertilization (dpf). However, neither treatment with the ASIC-specific blocker 4,6-diamidino-2-phenylindole (DAPI) nor morpholino knockdown of ASIC4b reduced Na+ uptake in circumneutral conditions at 4 dpf. However, because ASIC4b knockdown led to significant increases in the mRNA expression of nhe3b and ncc and a significant increase in HR cell density, it is possible that Na+ influx was sustained by increased participation of non-ASIC4b pathways. Moreover, when fish were reared in acidic water (pH = 4), ASIC4b knockdown led to a stimulation of Na+ uptake at 3 and 4 dpf, results which also were inconsistent with an essential role for ASIC-mediated Na+ uptake, even under conditions known to constrain Na+ uptake via NHE3b. Thus, while ASIC4b clearly is expressed in HR cells, the current functional experiments cannot confirm its involvement in Na+ uptake in larval zebrafish.

Distribution of ASIC4 transcripts in the adult wild-type mouse brain.

Acid-sensing ion channel 4 (ASIC4) belongs to the ASIC gene family of neuronal proton-gated cation channels, and is the least understood subtype among the members. Previous studies of ASIC4 expression in the mammalian central nervous system have shown that ASIC4 is abundantly expressed in the spinal cord and in various brain regions, such as the cerebral cortex, the hippocampus, and the cerebellum. However, the detailed distribution of ASIC4 transcripts in mammalian brains still remains to be elucidated. In the present study, radioactive in situ hybridization histochemistry with an ASIC4-specific cRNA probe was performed on wild-type mouse brains, followed by X-gal staining experiments with Asic4-lacZ reporter mice Asic4tm1a(KOMP)Mbp. It was found that ASIC4 mRNAs were widely expressed throughout the wild-type brain, but preferentially concentrated in the olfactory bulb, the piriform cortex, the caudate putamen, the preoptic area, the paraventricular nucleus, the medial habenular nucleus, the pretectal area, the lateral geniculate nucleus, the amygdaloid complex, the superior colliculus, the interpeduncular nucleus, and the granule cell layer of the ventral hippocampus, and these results were in agreement with the X-gal-positive reactions observed in the mutant brain. In addition, X-gal staining combined with immunohistochemistry identified intense signals for ASIC4 transcriptional activity in most of the choline acetyltransferase (ChAT)-positive principal neurons located in the basal forebrain cholinergic nuclei. Our data provide useful information to speculate possible roles of ASIC4 in diverse brain functions.