Smoking is directly responsible for lung cancer, respiratory disease, and cardiovascular
disease. It follows that smoking is known to be the greatest preventable causes of disease, disability,
and death. In light of the harmful effects of nicotine abuse, the vast majority of smokers claim that
they wish to stop smoking, and yet, few quit attempts result in long-term abstinence. This is in
large part due to the withdrawal syndrome that precipitates upon cessation of nicotine consumption.
Withdrawal from nicotine’s effect on the central nervous system is mediated by the nicotinic
acetylcholine receptors (nAChRs) in the medial habenula (MHb) to interpeduncular nucleus (IPN)
circuit (MHb-IPN). The MHb-IPN is complex – expressing the highest levels and highest diversity
of nAChRs of any system in the brain. Despite the importance of this circuit, the physiological and
pathological role that nAChRs play in the MHb-IPN remains unclear. This is largely due to a lack
of knowledge regarding the expression pattern of nAChRs in the circuit and the effects of chronic
nicotine exposure on the circuit. Therefore, we developed and characterized a photoactivatable
nicotine (PA-Nic) molecule with which to investigate the localization of nAChRs and characterize
the effects of chronic nicotine on the circuit. We found PA-Nic to have wide utility in epiillumination, single-photon, and two-photon laser stimulation paradigms – allowing for broad and
precise spatiotemporal control over nicotine application. We found that MHb neurons exhibited
spontaneous action potential firing and spontaneously oscillated between high and low calcium
states. Acute exposure to nicotine, via uncaging, elicited enhanced action potential firing and
enhanced calcium mobilization in MHb neurons. In order to study the localization of nAChRs
functionally expressed on MHb neurons, we utilized a spatially delimited single-photon laser
stimulation paradigm paired with two-photon laser scanning microscopy to register the PA-Nic
uncaging location with subcellular structural components. By controlling the location of nicotine
uncaging we found that nAChRs were functionally localized to all subcellular locales of MHb neurons, including dendritic arbors and axons, but were most highly expressed at the large
proximal dendrite.
Altered regulation of nAChRs following chronic exposure to nAChR agonists is an
important phenomenon that is thought to sensitize nicotinic signaling in the MHb-IPN and
ultimately underlie nicotine withdrawal. Therefore, we chronically treated mice with nicotine to
test the effects of this exposure on the MHb-IPN circuit. We found that chronic nicotine exposure
enhanced the functional expression of nAChRs at the proximal and distal dendrites as well as on
the axons of MHb neurons. The increase in axonal nAChRs on MHb neurons following chronic
nicotine exposure suggested that terminal presynaptic nAChRs may also be upregulated in
response to nicotine. Activation of presynaptic nAChRs can evoke neurotransmitter release
directly by calcium flux or indirectly by presynaptic depolarization. Therefore, we examined the
effect of chronic nicotine on the MHb synaptic terminals and on IPN neurons themselves. Chronic
nicotine treatment enhanced MHb-IPN excitatory postsynaptic currents in response to subsequent
nicotine exposure. Interestingly, IPN neuron responses to nicotine uncaging were dramatically
prolonged and adapted to multiple exposures of nicotine. This data indicates that the functional
connectivity and sensitivity of the MHb-IPN circuit to nicotine is enhanced by chronic nicotine
exposure. Overall, our studies have yielded a widely generalizable chemical method by which to
create and characterize photoactivatable molecules. Utilization of PA-Nic has improved our
understanding of the role that nAChRs play in the MHb-IPN circuit during nicotine addiction and
withdrawal. Since cholinergic systems are implicated in many disease states, a better
understanding of nicotinic receptor localization and regulation will hopefully help us develop
better models and therapeutic approaches for several diseases.
Funding
Photoactivatable ligands for nicotinic optopharmacology