Genetic mapping of ASIC4 and contrasting phenotype to ASIC1a in modulating innate fear and anxiety

Abstract

Although ASIC4 is a member of the acid-sensing ion channel (ASIC) family, we have limited knowledge of its expression and physiological function in vivo. To trace the expression of this ion channel, we generated the ASIC4-knockout/CreERT(2)-knockin (Asic4(Cre) (ERT) (2)) mouse line. After tamoxifen induction in the Asic4(Cre) (ERT)(2)::CAG-STOP(floxed)-Td-tomato double transgenic mice, we mapped the expression of ASIC4 at the cellular level in the central nervous system (CNS). ASIC4 was expressed in many brain regions, including the olfactory bulb, cerebral cortex, striatum, hippocampus, amygdala, thalamus, hypothalamus, brain stem, cerebellum, spinal cord and pituitary gland. Colocalisation studies further revealed that ASIC4 was expressed mainly in three types of cells in the CNS: (i) calretinin (CR)-positive and/or vasoactive intestine peptide (VIP)-positive interneurons; (ii) neural/glial antigen 2 (NG2)-positive glia, also known as oligodendrocyte precursor cells; and (iii) cerebellar granule cells. To probe the possible role of ASIC4, we hypothesised that ASIC4 could modulate the membrane expression of ASIC1a and thus ASIC1a signaling in vivo. We conducted behavioral phenotyping of Asic4(Cre) (ERT)(2) mice by screening many of the known behavioral phenotypes found in Asic1a knockouts and found ASIC4 not involved in shock-evoked fear learning and memory, seizure termination or psychostimulant-induced locomotion/rewarding effects. In contrast, ASIC4 might play an important role in modulating the innate fear response to predator odor and anxious state because ASIC4-mutant mice showed increased freezing response to 2,4,5-trimethylthiazoline and elevated anxiety-like behavior in both the open-field and elevated-plus maze. ASIC4 may modulate fear and anxiety by counteracting ASIC1a activity in the brain.

Expression of acid-sensing ion channels and selection of reference genes in mouse and naked mole rat

“…A comparison of ASIC1a and ASIC1b expression in human DRG also found that ASIC1b was higher in DRG neurons than ASIC1a , but again a single reference gene was used ( GAPDH ) [55]. We also find that ASIC4 is the least abundant ASIC in DRG, which is in accordance with previous data showing that ASIC4 is sparsely expressed in DRG [8, 36, 37]. …”

“…Previous reports have reached little consensus about ASIC3 expression in the brain, but here we demonstrate that ASIC3 is strongly expressed in all tissues investigated, and a RT-PCR study by Meng et al also showed ASIC3 transcripts present in different parts of the brain, in addition to DRGs [35]. Lastly, as others have shown with a variety of techniques [8, 36, 37], ASIC4 was highly expressed in all brain regions analyzed as well as the spinal cord, where it was more abundant than other ASICs.…”

“…3b’ – g’). It has been suggested that ASIC4 plays a modulatory role and can interact with polyubiquitin to downregulate other ASICs [58] and recent evidence suggests that it can counteract the activity of ASIC1a in the brain to modulate fear and anxiety behavior [8]. Thus, it can be speculated that one of these functions, or another as of yet unknown function of the subunit, has been lost or made redundant in naked mole-rat, although the physiological properties of naked mole-rat ASIC4 are currently unknown.…”

“…However, later work using a combination of western blot and RT-PCR showed that it was present in hippocampus, amygdala, caudate putamen, prefrontal cortex and hypothalamus, as well as and DRG [35]. Initial studies examining the expression of ASIC4 using dot blot, northern blot and RT-PCR showed that it was present in the brain, spinal cord and DRG [36, 37], work that has been supported by more recent investigation using ASIC4 transgenic marker mice [8]. By contrast with mice and rats, there is very little known about ASIC expression in naked mole-rats, although one study used RT-PCR to demonstrate that transcripts for ASIC1a , ASIC1b , ASIC2a , ASIC2b and ASIC3 are present in DRG, but expression of ASIC4 was not determined [14].…”

Human ASIC1a mediates stronger acid‐induced responses as compared with mouse ASICIa

“…Other ASIC subunits, e.g., ASIC3 and -4, are also present in mouse brain. However, the expression of ASIC3 and -4 is restricted to specific nuclei and/or cell types (37)(38)(39). In dissociated human neurons, acid-activated currents also suggest the main contribution from ASIC1a and ASIC2a (18).…”