Probes for INS

Molecular identification and characterization of fear controlling circuitries is a promising path towards developing targeted treatments of fear-related disorders. Three-color in situ hybridization analysis was used to determine whether somatostatin (Sst), neurotensin (Nts), corticotropin releasing factor (Crf), tachykinin 2 (Tac2), protein kinase c delta (Prkcd), and dopamine receptor 2 (Drd2) mRNA co-localize in male mouse amygdala neurons. Expression and co-localization was examined across capsular (CeC), lateral (CeL), and medial (CeM) compartments of the central amygdala. The greatest expression of Prkcd and Drd2 were found in CeC and CeL. Crf was expressed primarily in CeL while Sst, Nts, and Tac2 expressing neurons were distributed between CeL and CeM. High levels of co-localization were identified between Sst, Nts, Crf, and Tac2 within the CeL while little co-localization was detected between any mRNAs within the CeM. These findings provide a more detailed understanding of the molecular mechanisms that regulate the development and maintenance of fear and anxiety behaviors.

Significance Statement Functional and behavioral analysis of central amygdala microcircuits has yielded significant insights into the role of this nucleus in fear and anxiety related behaviors. However, precise molecular and locational description of examined populations is lacking. This publication provides a quantified regionally precise description of the expression and co-expression of six frequently examined central amygdala population markers. Most revealing, within the most commonly examined region, the posterior CeL, four of these markers are extensively co-expressed suggesting the potential for experimental redundancy. This data clarifies circuit interaction and function and will increase relevance and precision of future cell-type specific reports.

Endogenous dynorphin signaling via the kappa-opioid receptor (KOR) in the nucleus accumbens (NAcc) powerfully mediates negative affective states and stress reactivity. Excitatory inputs from the hippocampus and amygdala play a fundamental role in shaping the activity of both NAcc D1 and D2 MSNs, which encode positive and negative motivational valences, respectively. However, a circuit-based mechanism by which KOR modulation of excitation-inhibition balance modifies D1 and D2 MSN activity is lacking. Here, we provide a comprehensive synaptic framework wherein presynaptic KOR inhibition decreases the excitatory drive of D1 MSN activity by the amygdala, but not the hippocampus. Conversely, presynaptic inhibition by KORs of inhibitory synapses on D2 MSNs enhances integration of excitatory drive by the amygdala and hippocampus. In conclusion, we describe a circuit-based mechanism showing differential gating of afferent control of D1 and D2 MSN activity by KORs in a pathway-specific manner.

Behavioral and molecular characterization of cell-type-specific populations governing fear learning and behavior is a promising avenue for the rational identification of potential therapeutics for fear-related disorders. Examining cell-type-specific changes in neuronal translation following fear learning allows for targeted pharmacological intervention during fear extinction learning, mirroring possible treatment strategies in humans. Here we identify the central amygdala (CeA) Drd2-expressing population as a novel fear-supporting neuronal population that is molecularly distinct from other, previously identified, fear-supporting CeA populations. Sequencing of actively translating transcripts of Drd2 neurons using translating ribosome affinity purification (TRAP) technology identifies mRNAs that are differentially regulated following fear learning. Differentially expressed transcripts with potentially targetable gene products include Npy5r, Rxrg, Adora2a, Sst5r, Fgf3, Erbb4, Fkbp14, Dlk1, and Ssh3. Direct pharmacological manipulation of NPY5R, RXR, and ADORA2A confirms the importance of this cellpopulation and these cell-type-specific receptors in fear behavior. Furthermore, these findings validate the use of functionally identified specific cell populations to predict novel pharmacological targets for the modulation of emotional learning.

Basolateral amygdala (BLA) principal cells are capable of driving and antagonizing behaviors of opposing valence. BLA neurons project to the central amygdala (CeA), which also participates in negative and positive behaviors. However, the CeA has primarily been studied as the site for negative behaviors, and the causal role for CeA circuits underlying appetitive behaviors is poorly understood. Here, we identify several genetically distinct populations of CeA neurons that mediate appetitive behaviors and dissect the BLA-to-CeA circuit for appetitive behaviors. Protein phosphatase 1 regulatory subunit 1B+ BLA pyramidal neurons to dopamine receptor 1+ CeA neurons define a pathway for promoting appetitive behaviors, while R-spondin 2+ BLA pyramidal neurons to dopamine receptor 2+ CeA neurons define a pathway for suppressing appetitive behaviors. These data reveal genetically defined neural circuits in the amygdala that promote and suppress appetitive behaviors analogous to the direct and indirect pathways of the basal ganglia.

In the hippocampus, the classical pyramidal cell type of the subiculum acts as a primary output, conveying hippocampal signals to a diverse suite of downstream regions. Accumulating evidence suggests that the subiculum pyramidal cell population may actually be comprised of discrete subclasses. Here, we investigated the extent and organizational principles governing pyramidal cell heterogeneity throughout the mouse subiculum. Using single-cell RNA-seq, we find that the subiculum pyramidal cell population can be deconstructed into eight separable subclasses. These subclasses were mapped onto abutting spatial domains, ultimately producing a complex laminar and columnar organization with heterogeneity across classical dorsal-ventral, proximal-distal, and superficial-deep axes. We further show that these transcriptomically defined subclasses correspond to differential protein products and can be associated with specific projection targets. This work deconstructs the complex landscape of subiculum pyramidal cells into spatially segregated subclasses that may be observed, controlled, and interpreted in future experiments.

Despite decades of research on neurobiological mechanisms of psychostimulant addiction, the only effective treatment for many addicts is contingency management, a behavioral treatment that uses alternative non-drug reward to maintain abstinence. However, when contingency management is discontinued, most addicts relapse to drug use. The brain mechanisms underlying relapse after cessation of contingency management are largely unknown, and, until recently, an animal model of this human condition did not exist. Here we used a novel rat model, in which the availability of a mutually exclusive palatable food maintains prolonged voluntary abstinence from intravenous methamphetamine self-administration, to demonstrate that the activation of monosynaptic glutamatergic projections from anterior insular cortex to central amygdala is critical to relapse after the cessation of contingency management. We identified the anterior insular cortex-to-central amygdala projection as a new addiction- and motivation-related projection and a potential target for relapse prevention.