Signal toxicity caused by nicotinic acetylcholine receptor agonist and development of detection method using two-photon microscope

<p> By acting on receptors in the brain, some chemicals exhibit "signal toxicity" that disrupts intracellular and intercellular signaling, causing unexpected effects. In particular, in brain function, a complex signal network between neurons and glial cells is formed, and disruption of this network can be a critical endpoint of signal toxicity. However, “disruption of brain function” due to signal toxicity is often not accompanied by cell death and accompanying brain tissue degeneration, and cannot be detected by current neurotoxicity test methods. In this study, we used neonicotinoids (NNs) and nicotine, which are agonists of the nicotinic acetylcholine receptor (nAChR), as model compounds, and detected changes in neuronal activity in the somatosensory cortex using a two-photon microscope. The aim was to detect disruption of brain function associated with signal toxicity.</p><p> C57BL/6N were orally administered ACE at 20 mg/kg (with reference to LOAEL in general pharmacology test of the central nervous system) and their blood was collected over time. It was analyzed quantitatively using LC-MS/MS. Then, we exposed mice to ACE at 20 mg/kg or 7.1 mg/kg (with reference to NOAEL to rats), and their behavior was observed using openfield (OF) test 1 hour after the administration at the former concentration, and using elevated plus maze (EPM) test 1 hour after the administration at the latter concentration. In addition, C57BL/6J were administered ACE at 30 mg/kg (with reference to NOAEL in the subacute toxicity test) and Nicotine, which is the typical agonist of nAChR, at 1.6 mg/kg (with reference to 1/2 LD50), and the neuronal activity in somatosensory cortex was observed over time by using two-photon microscope. </p><p> As a result, ACE absorbed quickly and the Tmax of ACE was around 25 minutes after administration. In comparison, dm-ACE, which is one of its major metabolites, absorbed a bit slower and the Tmax was around 150 minutes. An hour after the administration, the activity of mice decreased, and the anxiety-like behavior increased. In the somatosensory cortex, the firing frequency of nerve cells decreased, and the synchronous firing increased, 1 hour and 2.5 hour after ACE administration. Similarly, the firing frequency tended to decrease, and the synchronous firing increased, 5 minutes and 30 minutes after the administration of Nicotine. This suggest that changes in neuronal activity after administration of ACE are the result of ACE acting as a ligand for nAChR because we can see the same trends between ACE and Nicotine. In summary, it was observed that administration of ACE at a concentration that is originally non-toxic, such as NOAEL, affects the behavior and neural activities. This suggests that cranial nerve activity is responsible for changes in behavior.</p>