Photoactivatable (‘caged’) pharmacological agents have revolutionized neuroscience but the palette of available compounds is limited. We describe a general method for caging tertiary amines using a stable quaternary ammonium linkage that elicits a red-shift in activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncagable via 1- or 2-photon excitation, that is useful for optopharmacology experiments to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

SUMMARYVentral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2+ neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2+ neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure.

Antagonism of nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb) or interpeduncular nucleus (IPN) triggerswithdrawal-like behaviors in mice chronically exposed to nicotine, implying that nicotine dependence involves the sensitization of nicotinic signaling. Identification of receptor and/or neurophysiological mechanisms underlying this sensitization is important, as it could promote novel therapeutic strategies to reduce tobacco use. Using an approach involving photoactivatable nicotine, we previously demonstrated that chronic nicotine (cNIC) potently enhances nAChR function in dendrites of MHb neurons. However, whether cNIC modulates downstream components of the habenulo-interpeduncular (Hb-IP) circuit is unknown. In this study, cNIC-mediated changes to Hb-IP nAChR function were examined in mouse (male and female) brain slices using molecular, electrophysiological, and optical techniques. cNIC enhanced action potential firing and modified spike waveform characteristics in MHb neurons. Nicotine uncaging revealed nAChR functional enhancement by cNIC on proximal axonal membranes. Similarly, nAChR-driven glutamate release from MHb axons was enhanced by cNIC. In IPN, the target structure of MHb axons, neuronal morphology, and nAChR expression is complex, with stronger nAChR function in the rostral subnucleus [rostral IPN (IPR)]. As in MHb, cNIC induced strong upregulation of nAChR function in IPN neurons. This, coupled with cNIC-enhanced nicotine-stimulated glutamate release, was associated with stronger depolarization responses to brief (1 ms) nicotine uncaging adjacent to IPR neurons. Together, these results indicate that chronic exposure to nicotine dramatically alters nicotinic cholinergic signaling and cell excitability in Hb-IP circuits, a key pathway involved in nicotine dependence.This study uncovers several neuropharmacological alterations following chronic exposure to nicotine in a key brain circuit involved in nicotine dependence. These results suggest that smokers or regular users of electronic nicotine delivery systems (i.e., "e-cigarettes") likely undergo sensitization of cholinergic circuitry in the Hb-IP system. Reducing the activity of Hb-IP nAChRs, either volitionally during smoking cessation or inadvertently via receptor desensitization during nicotine intake, may be a key trigger of withdrawal in nicotine dependence. Escalation of nicotine intake in smokers, or tolerance, may involve stimulation of these sensitized cholinergic pathways. Smoking cessation therapeutics are only marginally effective, and by identifying cellular/ receptor mechanisms of nicotine dependence, our results take a step toward improved therapeutic approaches for this disorder. Arvin et al. • Habenulo-Interpeduncular Circuits Modified by Nicotine

The inhibitory activity of (±)-citalopram on human (h) α3β4, α4β2, and α7 nicotinic acetylcholine receptors (AChRs) was determined by Ca 2+ influx assays, whereas its effect on rat α9α10 and mouse habenular α3β4* AChRs by electrophysiological recordings. The Ca 2+ influx results clearly establish that (±)-citalopram inhibits (IC 50 's in μM) hα3β4 AChRs (5.1 ± 1.3) with higher potency than that for hα7 (18.8 ± 1.1) and hα4β2 (19.1 ± 4.2) AChRs. This is in agreement with the [ 3 H]imipramine competition binding results indicating that (±)-citalopram binds to imipramine sites at desensitized hα3β4 with >2-fold higher affinity than that for hα4β2. The electrophysiological, molecular docking, and in silico mutation results indicate that (±)-citalopram competitively inhibits rα9α10 AChRs (7.5 ± 0.9) in a voltage-independent manner by interacting mainly with orthosteric sites, whereas it inhibits a homogeneous population of α3β4* AChRs at MHb (VI) neurons (7.6 ± 1.0) in a voltage-dependent manner by interacting mainly with a luminal site located in the middle of the ion channel, overlapping the imipramine site, which suggests an ion channel blocking mechanism. In conclusion, (±)-citalopram inhibits α3β4 and α9α10 AChRs with higher potency compared to other AChRs but by different mechanisms. (±)-Citalopram also inhibits habenular α3β4*AChRs, supporting the notion that these receptors are important endogenous targets related to their anti-addictive activities.

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