Introduction

Fear learning and cue- and context-evoked fear-responses are essential to assess potentially deadly situations and escape potential predators based on prior experience. This capacity becomes vital when a young animal becomes increasingly independent from its mother and starts to explore the world on its own, which corresponds to the late pre-weaning and early weaning period. To facilitate the development of those crucial skills, the fear circuit undergoes significant changes during brain development, and as a result fear processing in early life and adulthood differ substantially (Meyer and Lee, 2019, Tallot et al., 2016). Therefore, genes known to impact fear learning and/or processing in adulthood might have different effects during earlier stages of development.

Performing Pavlovian fear conditioning at different developmental stages provides a way to probe effects across development. Pavlovian fear conditioning is a well-established model to assess fear memory with age-dependent conditioned responses (Akers et al., 2012, Akers et al., 2014, Pattwell et al., 2011, Taugher-Hebl et al., 2025). Contextual fear learning begins to emerge at P15, though it is not stably evoked 24 h after training until P17 (Akers et al. 2012). This contextual fear memory acquired at P17 is reduced to about 50% 1 week after training (Akers et al. 2014). However, the expression of contextual fear memory undergoes further developmental fluctuations. For example, juvenile mice (P29 - 33) have been reported to show hardly any context-evoked freezing but do exhibit significant cue-evoked freezing at 24–48 h after training (Pattwell et al. 2011). These developmentally regulated behavioral changes are accompanied by a significant structural and functional maturation of the fear circuit and its key structure, the basolateral amygdala (BLA), a region essential for fear memory acquisition and retrieval. In this region two types of neurons essential for Pavlovian fear conditioning are principal neurons and parvalbumin-positive interneurons (Yau et al., 2021, Wolff et al., 2014, Krabbe et al., 2018). While principal neurons are critical for plasticity during fear memory acquisition, parvalbumin-positive interneurons are essential for fear memory acquisition and expression. Within the first postnatal month, both neuron populations undergo significant maturation. On a morphological level, these developmental changes include increasing spine densities in principal neurons (Berdel and Moryś, 2000, Ryan et al., 2016). On a molecular level, these changes include increasing parvalbumin expression in parvalbumin-positive interneurons, alterations in NMDA and glutamate receptor subunit expression, and changes in protein phosphorylation (e.g., of CREB, GluA1). These developmental changes raise questions about how genes that affect fear memory in adulthood impact fear across development. There are several possibilities: 1) a uniform effect across ages, 2) a genetic effect that increases with ongoing development 3) and a genetic effect that is only present after maturation, in adulthood. Moreover, effects on cued and context could track together or could dissociate. To probe these possibilities, it is necessary to study genes relevant to adult fear learning and fear memory at different developmental timepoints.

One molecule that is well-positioned to play a developmental role in fear learning and fear memory is the acid-sensing ion channel−1A (ASIC1A). ASIC1A is a cation channel that is activated by extracellular acidosis (Storozhuk et al., 2021, Zhu et al., 2022, Wemmie et al., 2013, Zha, 2013). It is localized to dendritic spines, where it has been implicated in dendritic spine dynamics, synaptic plasticity and neurotransmission (Zha et al., 2006, Kreple et al., 2014; Du et al., 2014; Liu et al. 2016; Chiang et al. 2015; Wemmie et al. 2002; González-Inchauspe et al. 2017). In adult mice, ASIC1A is expressed in multiple regions of the fear circuit, including the BLA, where it is present in interneurons and principal cells and where it plays a key role in long-term potentiation (Wemmie et al., 2003, Du et al., 2017, Du et al., 2014, Chiang et al., 2015, Taugher et al., 2017, Pidoplichko et al., 2014, Wemmie et al., 2002). Adult mice that lack ASIC1A (Asic1a_–/–_ mice) have deficits in a variety of fear-related behaviors such as cued and context fear conditioning, TMT-evoked freezing and avoidance, CO2-evoked freezing and avoidance, acoustic startle, and center avoidance in the open field (Wemmie et al., 2003, Coryell et al., 2007, Ziemann et al., 2009, Taugher et al., 2014, Price et al., 2014). Though most studies on the role of ASIC1A have used adult mice, it has been reported that ASIC1A is expressed in the brain as early as embryonic day 12 (Alvarez de la Rosa et al. 2003), raising the possibility that it may play important roles throughout development.

Therefore, we hypothesized that the effects of ASIC1A on fear memory acquisition and recall might differ in young vs. adult mice. To test this hypothesis, we conditioned separate groups of mice, ranging from the pre-weaning period (P17) to adulthood (P83). Specifically, we tested fear memory acquisition, cue-evoked fear memory 24 h and 15 days after training and context-evoked fear memory 48 h and 16 days after training.