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.

Single-cell RNA sequencing has generated catalogs of transcriptionally defined neuronal subtypes of the brain. However, the cellular processes that contribute to neuronal subtype specification and transcriptional heterogeneity remain unclear. By comparing the gene expression profiles of single layer 6 corticothalamic neurons in somatosensory cortex, we show that transcriptional subtypes primarily reflect axonal projection pattern, laminar position within the cortex, and neuronal activity state. Pseudotemporal ordering of 1,023 cellular responses to sensory manipulation demonstrates that changes in expression of activity-induced genes both reinforced cell-type identity and contributed to increased transcriptional heterogeneity within each cell type. This is due to cell-type biased choices of transcriptional states following manipulation of neuronal activity. These results reveal that axonal projection pattern, laminar position, and activity state define significant axes of variation that contribute both to the transcriptional identity of individual neurons and to the transcriptional heterogeneity within each neuronal subtype.