THE MEDIAL PREFRONTAL CORTEX EXERTS THE NICOTINE-INDUCED FACILITATION OF OBJECT RECOGNITION MEMORY ENCODING AND RETRIEVAL IN MICE
Authors: Esaki *Hirohito , Imai Kanta , Nishikawa Keisuke , Izumi Shoma , Inutsuka Ayumu , Nagayasu Kazuki , Kaneko Shuji , Nishitani Naoya , Deyama Satoshi , Kaneda Katsuyuki 
Published: February 2025

Abstract

Background
We have recently reported that nicotine facilitates object recognition memory (ORM) encoding via stimulation of nAChRs and the subsequent inhibition of voltage-dependent potassium channels in mouse medial prefrontal cortex (mPFC) pyramidal cells (Esaki et al., 2021a, 2021b). Although these findings suggest that nicotine activates mPFC excitatory neurons, it remains unclear whether the activation of mPFC excitatory neurons is involved in the nicotine-induced ORM enhancement.

Aim and Objectives
We examined whether nicotine facilitates ORM encoding and retrieval via activation of mPFC neurons using the novel object recognition test (NOR). We also examined possible involvement of the projection from the mPFC to the perirhinal cortex (PRH), a brain region associated with ORM (Winters et al., 2008), in nicotine-induced ORM enhancement. Finally, we investigated whether ORM engram cells exist in the mPFC.

Methods
Male C57BL/6J mice (7–12 weeks) were allowed to explore the chamber with two objects for 10 min during the NOR training and test sessions. The test session was conducted 24 h after the training session with changing one of the two objects. The activity of mPFC neurons were regulated by AAV vector-mediated DREADD techniques.

Results
Subcutaneous (s.c.) injection of nicotine (0.1 mg/kg) 10 min before the training session significantly facilitated ORM encoding. Chemogenetic silencing of mPFC excitatory neurons before the training session inhibited the nicotine-induced facilitation of ORM encoding. Conversely, chemogenetic activation of mPFC excitatory neurons before the training session facilitated ORM encoding. Additionally, selective silencing of PRH-projecting mPFC neurons by injecting AAV-hSyn-DIO-DREADDs into the mPFC and AAV-retro-hSyn-Cre into the PRH, before the training session attenuated the nicotine-induced facilitation of ORM encoding. Activation of PRH-projecting mPFC neurons before the training session facilitated ORM encoding. On the other hand, s.c. injection, but not intra-mPFC infusion (0.3 μ g/side), of nicotine before the test session facilitated ORM retrieval, suggesting that nicotine facilitates ORM retrieval by acting on brain regions other than the mPFC. However, silencing of mPFC neuronal activity inhibited the systemic nicotine-induced facilitation of ORM retrieval. Moreover, activation of mPFC excitatory neurons significantly facilitated ORM retrieval. Since activation of mPFC excitatory neurons is important for ORM encoding and retrieval, we hypothesized that nicotine reactivates mPFC neurons during the test session, which were activated during the training session. By injecting AAV-cFos-Cre-ERT2 and AAV-hSyn-DIO-DREADDs into the mPFC and intraperitoneal injection of 4-hydroxytamoxifen (40 mg/kg), DREADDs were selectively expressed in mPFC neurons that had been activated during the training session. Reactivation of DREADD-expressed mPFC neurons before the test session facilitated ORM retrieval. Conversely, silencing of DREADD-expressed mPFC neurons before the test session inhibited the systemic nicotine-induced facilitation of ORM retrieval.

Discussion and Conclusion
These results demonstrate that nicotine facilitates ORM encoding via activating the mPFC-PRH pathway and ORM retrieval via indirectly activating mPFC neurons. Moreover, the results suggest for the first time that nicotine facilitates ORM retrieval via the reactivation of engram cells in the mPFC.

References
1.Esaki, H., Izumi, S., Fukao, A., Ito, S., Nishitani, N., Deyama, S., Kaneda, K. (2021a) Nicotine enhances object recognition memory via stimulating α4β2 and α7 nicotinic acetylcholine receptors in the medial prefrontal cortex of mice. Biol Pharmaceutical Bull, 44: 1007-1013.
2.Esaki, H., Izumi, S., Fukao, A., Nishitani, N., Deyama, S., Kaneda, K. (2021b) Nicotine enhances object recognition memory through inhibition of voltage-dependent potassium 7 channels in the medial prefrontal cortex of mice. J Pharmacol Sci, 147: 58-61.
3.Winters, B.D., Saksida, L.M., Bussey, T.J. (2008) Object recognition memory: neurobiological mechanisms of encoding, consolidation and retrieval. Neurosci Biobehav Rev. 32: 1055-1070.