Encoding and predicting aversive events are critical functions of circuits that support survival and emotional well-being. Maladaptive circuit changes in emotional valence processing can underlie the pathophysiology of affective disorders. The lateral habenula (LHb) has been linked to aversion and mood regulation through modulation of the dopamine and serotonin systems. We have defined the identity and function of glutamatergic (Vglut2) control of the LHb, comparing the role of inputs originating in the globus pallidus internal segment (GPi), and lateral hypothalamic area (LHA), respectively. We found that LHb-projecting LHA neurons, and not the proposed GABA/glutamate co-releasing GPi neurons, are responsible for encoding negative value. Monosynaptic rabies tracing of the presynaptic organization revealed a predominantly limbic input onto LHA Vglut2 neurons, while sensorimotor inputs were more prominent onto GABA/glutamate co-releasing GPi neurons. We further recorded the activity of LHA Vglut2 neurons, by imaging calcium dynamics in response to appetitive versus aversive events in conditioning paradigms. LHA Vglut2 neurons formed activity clusters representing distinct reward or aversion signals, including a population that responded to mild foot shocks and predicted aversive events. We found that the LHb-projecting LHA Vglut2 neurons encode negative valence and rapidly develop a prediction signal for negative events. These findings establish the glutamatergic LHA-LHb circuit as a critical node in value processing.

The dorsal striatum plays a central role in the selection, execution, and evaluation of actions. An emerging model attributes action selection to the matrix and evaluation to the striosome compartment. Here, we use large-scale cell-type-specific calcium imaging to determine the activity of striatal projection neurons (SPNs) during motor and decision behaviors in the three major outputs of the dorsomedial striatum: Oprm1 + striosome versus D1 + direct and A2A + indirect pathway SPNs. We find that Oprm1 + SPNs show complex tunings to simple movements and value-guided actions, which are conserved across many sessions in a single task but remap between contexts. During decision making, the SPN tuning profiles form a complete representation in which sequential SPN activity jointly encodes task progress and value. We propose that the three major output pathways in the dorsomedial striatum share a similarly complete representation of the entire action space, including task-and phase-specific signals of action value and choice.

The dorsal striatum plays a central role in motor and decision programs, such as the selection and execution of particular actions and the evaluation of their outcomes. A standard circuit model has emerged based on the striatal organization where projection neurons with specific molecular and longrange connectivity identities encode discrete and possibly opposing action signals. We used large-scale cell-type specific imaging of calcium signals during motor and decision behaviors to map the activity of individual striatal projection neurons (SPNs) that form the three major output pathways of the striatum -SPNs in the D1+ direct, the A2A+ indirect, and the Oprm1+ patch pathway. We found that during exploration or choice behaviors SPNs showed a pathway-independent representation of the discrete phases and action variables. The tuning of individual SPNs was action and context-dependent, together covering the entire task space, and included pathway-independent representation of decisionvariables such as action value in a dynamic choice task. We propose that the three major SPN pathways broadcast in parallel the complete representation of the task space to downstream targets, including task-and phase-specific signals of action value and choice. The selection of specific actions is based on computations that produce prediction and evaluation of the action outcome, and correct action selection is essential for the survival of all species. Action selection computations have been associated with neuron activity in basal ganglia circuits, where the striatum plays a central role in integrating information from cortical and subcortical circuits, which is then propagated to downstream targets for action execution 1-3 . The striatum has been neuroanatomically divided into two major output pathways: the direct pathway targeting the globus pallidus interna (GPi) and the substantia nigra (SN), and the indirect pathway targeting the globus pallidus externa (GPe) 4,5 . A circuit model has emerged based on the dichotomous organization of the striatum, where the direct and indirect pathway differentially control motor programs and explain the pathophysiology of movement disorders 6-8 . In this model, the striatal pathways regulate motor behaviors through antagonistic signals. Neurochemical definitions can further divide the striatum into compartments 9,10 , for example into patch (also known as striosome) and matrix compartments where the striatal patches exhibit high levels of mu opioid receptor (MOR) expression 11,12 and form a distinct pathway that projects to the GPi and SN 13,14 . Distinct gene expression patterns can be used to genetically target and visualize the direct, indirect, and patch pathway 15-17 . Altogether, evidence from neuroanatomy and physiology have supported that the direct and indirect pathways have opposing effects on behavior. In support of this circuit model, optogenetic manipulation of the direct and indirect striatal pathways has shown their differential role in reinforcement as well as action [18][19][20]...

Excitatory projections from the lateral hypothalamic area (LHA) to the lateral habenula (LHb) drive aversive responses. We used patch-sequencing (Patch-seq) guided multimodal classification to define the structural and functional heterogeneity of the LHA–LHb pathway. Our classification identified six glutamatergic neuron types with unique electrophysiological properties, molecular profiles and projection patterns. We found that genetically defined LHA–LHb neurons signal distinct aspects of emotional or naturalistic behaviors, such as estrogen receptor 1-expressing (Esr1+) LHA–LHb neurons induce aversion, whereas neuropeptide Y-expressing (Npy+) LHA–LHb neurons control rearing behavior. Repeated optogenetic drive of Esr1+ LHA–LHb neurons induces a behaviorally persistent aversive state, and large-scale recordings showed a region-specific neural representation of the aversive signals in the prelimbic region of the prefrontal cortex. We further found that exposure to unpredictable mild shocks induced a sex-specific sensitivity to develop a stress state in female mice, which was associated with a specific shift in the intrinsic properties of bursting-type Esr1+ LHA–LHb neurons. In summary, we describe the diversity of LHA–LHb neuron types and provide evidence for the role of Esr1+ neurons in aversion and sexually dimorphic stress sensitivity.

The GPi-LHb pathway is the main output of the basal ganglia suggested to shape motivated behaviors. We show here that Sst+ GPi-LHb neurons send direct feedback to key nodes of the basal ganglia: the GPe, the striatal striosomes, and dopamine neurons in the SNc. Chronic silencing of this pathway did not affect learning or execution of value-guided choices, but severely disrupted the ability to adapt choice-behavior and seek an alternative reward location after task reversal. Calcium imaging revealed that Sst+ GPi neurons did not signal outcome value or value updates during reversal learning. Instead, progressive suppression of the Sst+ GPi activity was linked to increased commitment to one choice, and activity increased during exploration of alternative choices. We propose that GPi Sst+ neurons drive behavioral flexibility through a direct feedback signal to balance the activity of key nodes in the basal ganglia.