br Results br Discussion We first tested
Discussion We first tested for any adverse effect of ATR on learning and memory with a MWM test, which is used to assess hippocampal spatial learning and memory abilities in rodents (Vorhees and Williams, 2006). In the place navigation test, the escape latency – or time spent locating the hidden platform – is a critical parameter reflecting the spatial learning abilities of the subject (Duva et al., 1997). Here, we found that the MWM escape latency was not affected by ATR treatment, thus indicating that the spatial learning abilities of rats may not be sensitive to developmental exposure to ATR. Using the spatial probe test, we selected three parameters reflecting spatial memory abilities. When measuring the percentage of time spent in the target quadrant, and the time spent in the target annulus, we found that ATR treatment decreased spatial memory ability in a dose-dependent manner. Interestingly, the average platform crossing time of the low-dose group was lower than the high-dose group. Indeed, although platform crossing time is a parameter commonly used to measure spatial memory abilities, it has several limitations, including being influenced by the size of the platform, water tank properties and even the tracing software used (Vorhees and Williams, 2006). Thus, the percentage of time spent in the target quadrant and the time spent in the target annulus may serve as more reliable measures of spatial memory ability. Our results of the spatial probe test demonstrate that ATR treatment may affect spatial memory abilities, especially in the high-dose group. These results are also in agreement with previous studies, which suggest that exposure to ATR impairs performance in other hippocampus-dependent learning and memory tasks in rodents, such as the NOR and Y-maze (Bardullas et al., 2011, Kale et al., 2018, Lin et al., 2013). In mammals, the hippocampus is the key Deferiprone structure region responsible for encoding and consolidating information, transforming short-term memory into long term memory. Anatomically, the CA1 and DG regions of hippocampus are critical for spatial learning and memory (Kesner et al., 2004, Nakazawa et al., 2004, Bartsch et al., 2010). A previous study demonstrated that exposure to ATR at a dose of 100 µg/kg BW reduced the total number of neurons with perikaryal swelling and astrocytic formations in the DG area of female mice (Giusi et al., 2006). In this study, analysis by transmission electron microscopy suggested that ATR treatment induces the degeneration of mitochondria and nuclei in neurons of both the DG and CA1 areas. In the present study, in addition to mitochondrial and nuclear defects, we also observed an increased number of lysosomes and unclear synaptic clefts in CA1 neurons following ATR treatment. Lysosomes are critical regulators of cellular homeostasis, and perform multiple functions such as the degradation of macromolecules and nutrient recycling (Ferguson, 2018). Mitochondria support diverse cellular functions such as the formation of reactive oxygen species, ATP generation and apoptosis (Erpapazoglou et al., 2017). Notably, mitochondrial and lysosomal dysfunction is a common theme in neurodegenerative disease, including Alzheimer’s and Parkinson’s disease (de la Monte et al., 2000, Cannon et al., 2009, Gowrishankar et al., 2015, Blazquez-Llorca et al., 2017). We therefore hypothesized that the observed ultrastructural defects in different hippocampal sub-regions may be related to the deficits in learning and memory induced by ATR. Other pesticides, such as paraquat and parathion, have also been observed to cause similar hippocampal deficits in rats (Chen et al., 2010, Canales-Aguirre et al., 2012). It is generally accepted that hippocampus-dependent synaptic plasticity is the foundation of learning and memory. The cellular process of long-term memory formation, known as long term potentiation (LTP), is a well described series of cellular events involved in hippocampus-dependent synaptic plasticity (Kullmann and Lamsa, 2007). Previous studies have shown that the ERK1/2 signaling pathway plays a key role in hippocampal LTP. Specifically, phosphorylation of ERK1/2 is a critical event in LTP induction (Selcher et al., 1999). In this study, we found that the mRNA levels of ERK1/2 were both decreased with high-doses of ATR, whereas no significant differences in the protein levels of ERK1/2 were seen. However, the protein levels of p-ERK1/2 were reduced in a dose-dependent manner. ERK1/2 can be phosphorylated directly by the immediate upstream kinases MEK1/2, with the activation of MEK1/2 also being phosphorylation-dependent via mitogen-activated protein kinase kinases, such as the RAF proteins (Raman et al., 2007). Here, we also found that the mRNA levels of MEK1/2 decreased with high-doses of ATR, with MEK1/2 and p-MEK1/2 protein levels also reduced, especially in the high-dose group. Thus, we are able to infer that the MEK/ERK cascade may be involved in ATR-induced hippocampal dysfunction.