Probes for TDTOMATO

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.

Pain thresholds are, in part, set as a function of emotional and internal states by descending modulation of nociceptive transmission in the spinal cord. Neurons of the rostral ventromedial medulla (RVM) are thought to critically contribute to this process; however, the neural circuits and synaptic mechanisms by which distinct populations of RVM neurons facilitate or diminish pain remain elusive. Here we used in vivo opto/chemogenetic manipulations and trans-synaptic tracing of genetically identified dorsal horn and RVM neurons to uncover an RVM-spinal cord-primary afferent circuit controlling pain thresholds. Unexpectedly, we found that RVM GABAergic neurons facilitate mechanical pain by inhibiting dorsal horn enkephalinergic/GABAergic interneurons. We further demonstrate that these interneurons gate sensory inputs and control pain through temporally coordinated enkephalin- and GABA-mediated presynaptic inhibition of somatosensory neurons. Our results uncover a descending disynaptic inhibitory circuit that facilitates mechanical pain, is engaged during stress, and could be targeted to establish higher pain thresholds.

Viral vectors enable foreign proteins to be expressed in brains of non-genetic species, including non-human primates. However, viruses targeting specific neuron classes have proved elusive. Here we describe viral promoters and strategies for accessing GABAergic interneurons and their molecularly defined subsets in the rodent and primate. Using a set intersection approach, which relies on two co-active promoters, we can restrict heterologous protein expression to cortical and hippocampal somatostatin-positive and parvalbumin-positive interneurons. With an orthogonal set difference method, we can enrich for subclasses of neuropeptide-Y-positive GABAergic interneurons by effectively subtracting the expression pattern of one promoter from that of another. These methods harness the complexity of gene expression patterns in the brain and significantly expand the number of genetically tractable neuron classes across mammals.