Nicotine targets a specific subset of acetylcholine receptors (nicotinic acetylcholine receptors or nAChRs) that are normally activated by the excitatory neurotransmitter acetylcholine. Consumption of nicotine by honey bees leads to its accumulation in the hemolymph and tissues throughout the body (du Rand et al., 2017). Thus, it is likely that consumed nicotine can act at multiple sites in the central nervous system. Acetylcholine plays important roles in cognition in both mammals and insects (Gauthier, 2010, Gould and Leach, 2014). Acetylcholine and nicotine increase long term potentiation (LTP), a process believed to underlie learning and memory (He et al., 2000, Matsuyama et al., 2000). Furthermore, memory function in patients with Alzheimer's disease is improved by treatment with nicotine or acetylcholine esterase inhibitors (Haam and Yakel, 2017), which increase activity of acetylcholine receptors. In the honey bee, nAChRs are expressed in regions of the brain involved in olfactory associative conditioning including the subesophageal ganglion, the olfactory sensory neurons, the antennal lobes and the mushroom bodies (Barbara et al., 2008, Dupuis et al., 2012, Goldberg et al., 1999, Kreissl and Bicker, 1989, Wustenberg et al., 2004). The projection neurons carrying olfactory information from the antennal lobes activate the mushroom body cells (Kenyon cells) via nAChRs (Oleskevich, 1999). Furthermore, work in Drosophila has shown that cholinergic mushroom body neurons activate the mushroom body output neurons, which regulate avoidance or approach behaviors, via nAChRs (Barnstedt et al., 2016). Thus, nAChRs play a key role in insect olfactory learning and memory.

Chronic treatment with nicotine has two effects on nAChRs, desensitization and upregulation of receptor expression (Wittenberg et al., 2020). Desensitization occurs after a ligand has bound to the receptor causing its channel to open. The activated receptor is then converted into its desensitized conformation, where the channel is closed and the receptor cannot be re-opened. The desensitized state is distinct from the closed state of the receptor, from which it can be opened once again. In both honey bees and mammals, ongoing activation of nAChRs leads to desensitization thereby decreasing the activity of the receptor (Dupuis et al., 2011, Gould and Leach, 2014, Palmer et al., 2013). In honey bees, neonicotinoid pesticides that act as agonists at nAChRs cause desensitization of nAChRs in the mushroom bodies leading to an inhibition of activation by acetylcholine (Palmer et al., 2013). Thus, although treatment with nicotine leads to an initial increase in neural activity, this is followed by a decrease in response due to the desensitization of AChRs. Normally, nAChRs would likely only have a low level of desensitization as acetylcholine is rapidly broken down by high concentrations of acetylcholinesterase present in the synapse. However, nicotine is not removed via digestion by enzymes or uptake transporters allowing it to linger in the synapse, thus a larger proportion of the nAChRs are in the desensitized conformation in the presence of nicotine. To compensate for the high levels of receptor desensitization in the presence of nicotine, the activity of nAChRs is upregulated (Fenster et al., 1999, Wittenberg et al., 2020). For example, consumption of nicotine by honey bees over three days leads to the upregulation of nAChR subunits (Christen et al., 2016). Thus, while an acute dose of nicotine leads to an increase in nAChR activity, chronic treatment leads to a decrease in activity due to desensitization, followed by an increase in active receptors, suggesting nicotine will have a complex effect on nAChRs depending on the time of exposure.

We examined how prolonged, four day exposure of honey bees fed sublethal doses of nicotine affected learning and memory in a classical conditioning paradigm developed to study olfactory reward learning in bees (Bitterman et al., 1983). Timing between the conditioning trials has been shown to impact memory in humans, rodents and insects, often referred to as the “spacing effect” (Menzel et al., 2001, Philips et al., 2013). Therefore, we used two conditioning protocols, massed and spaced, for studying how nicotine influenced memory production, as previous work revealed that massed conditioned bees were less likely to form a long term memory than spaced conditioned bees (Gerber et al., 1998, Menzel et al., 2001).