Probes for SST

The spinal dorsal horn (SDH) is comprised of distinct neuronal populations that process different somatosensory modalities. Somatostatin (SST)-expressing interneurons in the SDH have been implicated specifically in mediating mechanical pain. Identifying the transcriptomic profile of SST neurons could elucidate the unique genetic features of this population and enable selective analgesic targeting. To that end, we combined the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) method and Fluorescence Activated Nuclei Sorting (FANS) to capture tagged SST nuclei in the SDH of adult male mice. Using RNA-sequencing (RNA-seq), we uncovered more than 13,000 genes. Differential gene expression analysis revealed more than 900 genes with at least 2-fold enrichment. In addition to many known dorsal horn genes, we identified and validated several novel transcripts from pharmacologically tractable functional classes: Carbonic Anhydrase 12 (Car12), Phosphodiesterase 11 A (Pde11a), and Protease-Activated Receptor 3 (F2rl2). In situ hybridization of these novel genes showed differential expression patterns in the SDH, demonstrating the presence of transcriptionally distinct subpopulations within the SST population. Overall, our findings provide new insights into the gene repertoire of SST dorsal horn neurons and reveal several novel targets for pharmacological modulation of this pain-mediating population and treatment of pathological pain.

The dorsal horn of the spinal cord is critical to processing distinct modalities of noxious and innocuous sensation, but little is known of the neuronal subtypes involved, hampering efforts to deduce principles governing somatic sensation. Here we used single-cell RNA sequencing to classify sensory neurons in the mouse dorsal horn. We identified 15 inhibitory and 15 excitatory molecular subtypes of neurons, equaling the complexity in cerebral cortex. Validating our classification scheme in vivo and matching cell types to anatomy of the dorsal horn by spatial transcriptomics reveals laminar enrichment for each of the cell types. Neuron types, when combined, define a multilayered organization with like neurons layered together. Employing our scheme, we find that heat and cold stimuli activate discrete sets of both excitatory and inhibitory neuron types. This work provides a systematic and comprehensive molecular classification of spinal cord sensory neurons, enabling functional interrogation of sensory processing.

Although neuropeptide Y (NPY) has potent anxiolytic actions within the basolateral amygdala (BLA), selective activation of BLA NPY Y2receptors (Y2R) acutely increases anxiety by an unknown mechanism. Using ex vivo male rat brain slice electrophysiology, we show that the selective Y2R agonist, [ahx5-24]NPY, reduced the frequency of GABAA-mediated miniature inhibitory post synaptic currents (mIPSC) in BLA principal neurons (PN). [ahx5-24]NPY also reduced tonic activation of GABAB receptors (GABABR), which increased PN excitability through inhibition of a tonic, inwardly-rectifying potassium current (KIR ). Surprisingly, Y2R-sensitive GABABR currents were action potential-independent, persisting after treatment with tetrodotoxin. Additionally, the Ca2+-dependent, slow afterhyperpolarizing K+ current (IsAHP ) was enhanced in roughly half of the Y2R-sensitive PNs, possibly from enhanced Ca2+ influx, permitted by reduced GABABR tone. In male and female mice expressing tdTomato in Y2R-expressing cells (tdT-Y2R mice), immunohistochemistry revealed that BLA somatostatin interneurons (SST IN) express Y2Rs, as do a significant subset of BLA PNs. In tdT-Y2R mice, [ahx5-24]NPY increased excitability and suppressed the KIR in nearly all BLA PNs independent of tdT-Y2R fluorescence, consistent with presynaptic Y2Rs on SST INs mediating the above effects. However, only tdT-Y2R-expressing PNs responded to [ahx5-24]NPY with an enhancement of the IsAHP Ultimately, increased PN excitability via acute Y2R activation likely correlates with enhanced BLA output, consistent with reported Y2R-mediated anxiogenesis. Furthermore, we demonstrate: 1) a novel mechanism whereby activity-independent GABA release can powerfully dampen BLA neuronal excitability via postsynaptic GABABRs, and 2) that this tonic inhibition can be interrupted by neuromodulation, here by NPY via Y2Rs.SIGNIFICANCE STATEMENTWithin the basolateral amygdala (BLA), neuropeptide Y (NPY) is potently anxiolytic. However, selective activation of NPY2-receptors (Y2R) increases anxiety by an unknown mechanism. We show that activation of BLA Y2Rs decreases tonic GABA release onto BLA principal neurons (PN), probably from Y2R-expressing somatostatin interneurons some of which co-express NPY. This increases PN excitability by reducing GABAB receptor (GABABR)-mediated activation of G-protein-coupled, inwardly-rectifying K+(GIRK) currents. Tonic, Y2R- sensitive GABABR currents unexpectedly persisted in the absence of action potential firing, revealing, to our knowledge, the first report of substantial, activity-independent GABABR activation. Ultimately, we provide a plausible explanation for Y2R-mediated anxiogenesis in vivo and describe a novel and modulatable means of damping neuronal excitability.