Fluoride (F–) is an inorganic ion naturally present in the environment. However, rising F– levels due to anthropogenic activities can be toxic to several plants and animal taxa (Camargo, 2003). As F can cross the blood-brain barrier, the central nervous system is particularly sensitive to overexposure to this ion (Harrison and Gerstein, 2002). Previous studies showed that F– not only alters learning and memory formation but can also impair neurotransmission, and induce oxidative stress and apoptosis (Dondossola et al., 2022, Liu et al., 2014, Pereira et al., 2011).
However, the vast majority of the studies investigating the effects of F– in the central nervous system are performed in rodent models and use a concentration of F– that is much higher than that naturally occurring in wild environments (natural F– levels~0.1–0.4 mg/L) (City of Calgary, water treatment report 2021; Jiang et al., 2014; Wiley et al., 2022).
In this study, we investigated the effects of F exposure on the cognitive functions of an aquatic invertebrate, the pond snail Lymnaea stagnalis. This model has been extensively used in Ecotoxicology and Neuroscience since the 1970s (Rivi et al., 2020). As L. stagnalis possesses an open circulatory system, it represents a valid tool in which to investigate the behavioural and molecular effects induced by many compounds that can be easily absorbed and reach the central ring ganglia (Amorim et al., 2019, Benjamin and Kemenes, 2009, Fodor et al., 2020, Rivi et al., 2021b, Rivi et al., 2020).
Our model is capable of configural learning, a higher form of learning, in which snails develop a landscape of fear when experience an appetitive stimulus (i.e., carrot slurry) along with a predatory cue (i.e., the crayfish effluent), resulting in a suppression of the food cue as it now elicits a fear state rather than increased feeding (Swinton et al., 2019). We recently demonstrated that a 45-minutes exposure to a concentration of F– similar to that naturally occurring in the aquatic environment (0.01–0.3 mg / L of F–) (CEPA 1994), before, during or after a configural learning procedure impairs memory formation and induces long-lasting effects, as snails were still unable to form a configural learning memory when trained one week after the F– exposure (Wiley et al., 2022). These one-trial low exposure effects were surprising and thus called for further investigation into the mechanisms responsible for obstructing memory formation.
Learning and memory formation involves the activation of several highly conserved molecular pathways. Among the key molecules involved in long-term memory (LTM) formation in L. stagnalis, attention should be paid to the cyclic AMP response-element binding protein (CREB1) (Batabyal et al., 2021a, Ribeiro et al., 2003, Wan et al., 2010) and the ionotropic glutamate receptor N-methyl D-aspartate (NMDA). The orthologue of the subunit 1 of the NMDA receptor found in L. stagnalis – the LymGRIN1 – has proven to be necessary during the acquisition phase of LTM formation following conditioning (Li and Tsien, 2009).
Apart from the molecules directly involved in the memory formation cascade we also find chaperone proteins such as the Heat shock protein 70 (HSP70) to be upregulated across taxa during several learning tasks that involve synaptic plasticity or stress-induced learning (Rivi et al., 2021a, Sunada et al., 2016). Along with the effect on learning and memory, HSP70 is activated and modulates a variety of homeostatically stressful situations, including exposure to the predator, facilitating the consolidation of memory of fear conditioning (Kagawa and Mugiya, 2002).
Finally, previous studies from mammals indicate that F– induces the depletion of various antioxidant agents (Lu et al., 2017), and the resulting oxidative stress is associated with cognitive impairment (de Lima et al., 2005). A key target involved in oxidative stress is Cytochrome c Oxidase (CCO). Under physiological conditions, this enzyme acts as the rate-limiting step of the respiratory chain, catalysing the reduction of oxygen for energy metabolism. Thus, its activity is an indicator of the oxidative capacity of the cells. CCO is one of the most ancient and highly conserved enzymes known and the orthologous of the subunit 1 of this target in Lymnaea, LymCCO1, has been characterized (Bargues et al., 2012).
In the current study, we first show that a low concentration of F– (0.7 mg/L NaF equivalent to 0.3 mg/L F–) affects configural learning memory formation and then we measured the mRNA levels of LymCREB1, LymGRIN1, LymHSP70, and LymCCO1 in L. stagnalis central ring ganglia to elucidate the putative mechanisms underlying the molecular cascades involved in configural learning memory formation and the impairing effects induced by F–.
We used a lab-bred strain (the W-strain) of Lymnaea stagnalis (Linnaeus 1758) maintained at the University of Calgary since the 1980s. The founding population originated from polders in Utrecht in The Netherlands in the 1950s and was then maintained at the Vrije University in Amsterdam from which our population originated. We housed snails in artificial pond water (0.25 g L?1 of Instant Ocean in deionized water, Spectrum Brands, Madison, WI, USA) supplemented with CaCO3 to ensure calcium
We first tested the effect of F– on configural learning memory. Two-way Repeated Measures ANOVA revealed a significant interaction effect between the two treatment groups (i.e., configural learning training and exposure to F– before training) and the carrot exposures before and after the training session (F1,14 = 56.21, P < 0.0001). Consistent with our previous studies (Batabyal et al., 2021a; Kagan and Lukowiak, 2019; Swinton et al., 2019), we found that the 45-minutes simultaneous exposure to
In the current study, transcriptional effects are found, which may help to elucidate the possible molecular basis of memory impairment caused by a single low dose of F– exposure in an aquatic invertebrate.
In our previous study (Wiley et al., 2022), we demonstrated that both the acute and long-term effects of a single F– exposure block the memory for configural learning. To explore the mechanisms through which F– affects learning and/or memory formation in L. stagnalis, in the current study, we
In summary, this – to our knowledge – is the first study investigating the possible molecular mechanisms of memory impairment in an invertebrate species which is exposed to naturally occurring levels of F–.
The exposure of F–-naïve snails to a concentration of F– similar to that found in local (i.e., Canadian) ponds (i.e., 0.3?mg/L) for 45?min before the configural learning procedure not only blocks memory formation but also induces significant transcriptional effects in the central ring