Highlights d Rostromedial tegmental (RMTg) neurons are activated by punishment-related stimuli d Distinct RMTg afferents communicate distinct punishmentrelated signals d These RMTg afferents drive correspondingly distinct aspects of punishment learning d Negative valence encoding in the ventral tegmental area depends on the RMTg

Lateral habenula (LHb) neurons are activated by negative motivational stimuli and play key roles in the pathophysiology of depression. Prior reports suggested that rostral entopeduncular nucleus (rEPN) neurons drive these responses in the LHb and rostromedial tegmental nucleus (RMTg), but these influences remain untested. Using rabies viral tracers, we demonstrate disynaptic projections from the rEPN to RMTg, but not VTA, via the LHb in rats. Using in vivo electrophysiology, we find that rEPN or LHb subpopulations exhibit activation/inhibition patterns after negative/positive motivational stimuli, similar to the RMTg, while temporary inactivation of a region centered on the rEPN decreases LHb basal and burst firing, and reduces valence-related signals in LHb neurons. Additionally, excitotoxic rEPN lesions partly diminish footshock-induced cFos in the LHb and RMTg. Together, our findings indicate an important role of the rEPN, and possibly immediately adjacent hypothalamus, in driving basal activities and valence processing in LHb and RMTg neurons.

Withdrawal from amphetamine increases anxiety and reduces the ability to cope with stress, factors that are believed to contribute to drug relapse. Stress-induced serotonergic transmission in the central nucleus of the amygdala is associated with anxiety states and fear. Conversely, increases in stress-induced ventral hippocampal serotonin have been linked to coping mechanisms. The goal of this study is to understand neurobiological changes induced by amphetamine that contribute to stress-sensitivity during withdrawal. We tested the hypothesis that limbic serotonergic responses to restraint stress would be altered in male Sprague-Dawley rats chronically pre-treated with amphetamine (2.5 mg/kg, ip.) followed by two weeks withdrawal. Amphetamine withdrawal resulted in increased stress-induced behavioral arousal relative to control treatment, suggesting that drug withdrawal induced a greater sensitivity to the stressor. When microdialysis was used to determine the effects of restraint on extracellular serotonin, stress-induced increases in serotonin were abolished in the ventral hippocampus and augmented in the central amygdala during amphetamine withdrawal. Reverse dialysis of the glucocorticoid receptor antagonist mifepristone into the ventral hippocampus blocked the stress-induced serotonin increase in saline pre-treated rats, suggesting that glucocorticoid receptors mediate stress-induced serotonin increases in the ventral hippocampus. However, mifepristone had no effect on stress-induced serotonin increases in the central amygdala, indicating that stress increases serotonin in this region independent of glucocorticoid receptors. During amphetamine withdrawal, the absence of stress-induced increases in ventral hippocampus serotonin combined with enhanced stress-induced serotonergic responses in the central amygdala may contribute to drug relapse by decreasing stress-coping ability and heightening stress responsiveness.

The rostromedial tegmental nucleus (RMTg), a GABAergic afferent to midbrain dopamine (DA) neurons, has been hypothesized to be broadly activated by aversive stimuli. However, this encoding pattern has only been demonstrated for a limited number of stimuli, and the RMTg influence on ventral tegmental (VTA) responses to aversive stimuli is untested. Here, we found that RMTg neurons are broadly excited by aversive stimuli of different sensory modalities and inhibited by reward-related stimuli. These stimuli include visual, auditory, somatosensory and chemical aversive stimuli, as well as “opponent” motivational states induced by removal of sustained rewarding or aversive stimuli. These patterns are consistent with broad encoding of negative valence in a subset of RMTg neurons. We further found that valence-encoding RMTg neurons preferentially project to the DA-rich VTA versus other targets, and excitotoxic RMTg lesions greatly reduce aversive stimulus-induced inhibitions in VTA neurons, particularly putative DA neurons, while also impairing conditioned place aversion to multiple aversive stimuli. Together, our findings indicate a broad RMTg role in encoding aversion and driving VTA responses and behavior.

The aversive properties associated with drugs of abuse influence both the development of addiction and relapse. Cocaine produces strong aversive effects after rewarding effects wear off, accompanied by increased firing in the lateral habenula (LHb) that contributes to downstream activation of the rostromedial tegmental nucleus (RMTg). However, the sources of this LHb activation are unknown, as the LHb receives many excitatory inputs whose contributions to cocaine aversion remain uncharacterized. Using cFos activation and in vivo electrophysiology in male rats, we demonstrated that the rostral entopeduncular nucleus (rEPN) was the most responsive region to cocaine among LHb afferents examined and that single cocaine infusions induced biphasic responses in rEPN neurons, with inhibition during cocaine's initial rewarding phase transitioning to excitation during cocaine's delayed aversive phase. Furthermore, rEPN lesions reduced cocaine-induced cFos activation by 2-fold in the LHb and by a smaller proportion in the RMTg, while inactivation of the rEPN or the rEPN-LHb pathway attenuated cocaine avoidance behaviors measured by an operant runway task and by conditioned place aversion (CPA). These data show an essential but not exclusive role of rEPN and its projections to the LHb in processing the aversive effects of cocaine, which could serve as a novel target for addiction vulnerability.

Although cocaine is powerfully rewarding, not all individuals are equally prone to abusing this drug. We postulate that these differences arise in part because some individuals exhibit stronger aversive responses to cocaine that protect them from cocaine seeking. Indeed, using conditioned place preference (CPP) and a runway operant cocaine self-administration task, we demonstrate that avoidance responses to cocaine vary greatly between individual high cocaine-avoider and low cocaineavoider rats. These behavioral differences correlated with cocaine-induced activation of the rostromedial tegmental nucleus (RMTg), measured using both in vivo firing and c-fos, whereas slice electrophysiological recordings from ventral tegmental area (VTA)-projecting RMTg neurons showed that relative to low avoiders, high avoiders exhibited greater intrinsic excitability, greater transmission via calcium-permeable AMPA receptors (CP-AMPARs), and higher presynaptic glutamate release. In behaving animals, blocking CP-AMPARs in the RMTg with NASPM reduced cocaine avoidance. Hence, cocaine addiction vulnerability may be linked to multiple coordinated synaptic differences in VTA-projecting RMTg neurons.

In order to respond appropriately to threats in the environment, the brain must rapidly determine whether a stimulus is important and whether it is positive or negative, and then use that information to direct behavioral responses. Neurons in the amygdala have long been implicated in valence encoding and in fear responses to threatening stimuli, but show transient firing responses in response to these stimuli that do not match the timescales of associated behavioral responses. For decades, there has been a logical gap in how behavioral responses could be mediated without an ensemble representation of the internal state of valence that has rapid onset, high signal-to-noise, and is sustained for the duration of the behavioral state. Here, we present the amygdalostriatal transition zone (ASt) as a missing piece of this highly conserved process that is of paramount importance for survival, which does exactly this: represents an internal state (e.g. fear) that can be expressed in multiple motor outputs (e.g. freezing or escape). The ASt is anatomically positioned as a 'shortcut' to connect the corticolimbic system (important for evaluation) with the basal ganglia (important for action selection) with the inputs of the amygdala and the outputs of the striatum - ideally poised for evaluating and responding to environmental threats. From in vivo cellular resolution recordings that include both electrophysiology and calcium imaging, we find that ASt neurons are unique in that they are sparse coding, extremely high signal-to-noise, and also maintain a sustained response for negative valence stimuli for the duration of the defensive behavior - a rare but essential combination. We further show that photostimulation of the ASt is sufficient to drive freezing and avoidance behaviors. Using single-nucleus RNA sequencing and in situ RNA labelling we generate a comprehensive profile of cell types and gene expression in the ASt, and find the ASt is genetically distinct from adjacent striatal and amygdalar structures. We also find that the ASt has a greater proportion of neurons expressing Drd2 than neurons expressing Drd1a, a unique feature compared to other regions of the striatum. Using in vivo calcium imaging, we show that that this Drd2+ population robustly encodes stimuli of negative valence, and in loss-of-function experiments find that optogenetic inhibition of Drd2+ ASt neurons causes a striking reduction in cue-conditioned fear responses. Together, our findings identify the ASt as a previously-unappreciated critical missing link for encoding learned associations and directing ongoing behavior.