The lateral habenula (LHb) balances reward and aversion by opposing activation of brain reward nuclei and is involved in the inhibition of responding for cocaine in a model of impulsive behavior. Previously, we reported that the suppression of cocaine seeking was prevented by LHb inactivation or nonselective antagonism of LHb mAChRs. Here, we investigate mAChR subtypes mediating the effects of endogenous acetylcholine in this model of impulsive drug seeking and define cellular mechanisms in which mAChRs alter LHb neuron activity. Using in vitro electrophysiology, we find that LHb neurons are depolarized or hyperpolarized by the cholinergic agonists oxotremorine-M (Oxo-M) and carbachol (CCh), and that mAChRs inhibit synaptic GABA and glutamatergic inputs to these cells similarly in male and female rats. Synaptic effects of CCh were blocked by the M 2 -mAChR (M 2 R) antagonist AFDX-116 and not by pirenzepine, an M 1 -mAChR (M 1 R) antagonist. Oxo-Mmediated depolarizing currents were also blocked by AFDX-116. Although M 2 R activation inhibited excitatory and inhibitory inputs to LHb neurons, the effect on excitation was greater, suggesting a shift in excitatory-inhibitory balance toward net inhibition. Activation of VTA inhibitory inputs to LHb neurons, via channelrhodopsin-2 expression, evoked IPSCs that were inhibited by M 2 Rs. Finally, we measured LHb-dependent operant response inhibition for cocaine and found it impaired by antagonism of M 2 Rs, and not M 1 Rs. In summary, we show that a cholinergic signal to LHb and activation of M 2 Rs are critical to enable inhibition of responding for cocaine, and we define cellular mechanisms through which this may occur.

The lateral habenula (LHb) plays a central role in balancing reward and aversion by opposing the contributions of brain reward nuclei. Using a rat cocaine self-administration model, we previously found that LHb inhibition or non-selective blockade of LHb muscarinic acetylcholine receptors (mAChRs) led to persistent cocaine seeking despite its signaled unavailability. As understanding roles for the LHb and cholinergic signaling in behavioral control is important to psychiatric illness and addiction, we examine how mAChRs act on LHb neurons using in vitro electrophysiology. We find that different groups of LHb neurons are depolarized or hyperpolarized by the cholinergic agonist carbachol (CCh), and that CCh could inhibit GABAergic and glutamatergic synaptic inputs to these cells. Presynaptic CCh effects were reversed by the M2 mAChR (M2R) antagonist AFDX-116, but not by pirenzepine, an M1R antagonist. Contemporaneous measurement of CCh effects on synaptic inhibition and excitation in LHb neurons showed a smaller effect on inhibition, suggesting a net shift in synaptic integration toward greater inhibition by mAChRs. Synaptic currents elicited by light-activation of ventral tegmental area (VTA) axons in the LHb, following channelrhodopsin-2 transfection of VTA, were also inhibited by M2Rs, suggesting the VTA as at least one M2R-sensitive LHb afferent. Finally, Go-NoGo cocaine seeking studies showed that blockade of LHb M2Rs, and not M1Rs, triggered continued cocaine seeking. These data identify LHb M2Rs as a potential control point of LHb function that enables withholding responses for cocaine and define cellular mechanisms through which mAChRs modulate LHb activity.