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Thomas L. Kash, Danny G. Winder

Neuropeptide Y and corticotropin-releasing factor bi-directionally modulate inhibitory synaptic transmission in the bed nucleus of the stria terminalis. Thomas L. Kash, Danny G. Winder. Introduction. Neuropeptides (NPY) = potent neuromodulators in the CNS Involved in reward pathway

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Thomas L. Kash, Danny G. Winder

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  1. Neuropeptide Y and corticotropin-releasing factor bi-directionallymodulate inhibitory synaptic transmission in the bed nucleusof the stria terminalis Thomas L. Kash, Danny G. Winder

  2. Introduction • Neuropeptides (NPY) = potent neuromodulators in the CNS • Involved in reward pathway • mediated via G-protein coupled receptors • released in a frequency dependent fashion • longer half-life of activity after release

  3. Introduction • Corticotropin Releasing Factor (CRF) involved in stress response • Mediated through the hypothalamus and the amygdala • Both pathways converge at the BNST

  4. Introduction • Life = Stress • Repeated or severe stressors can produce behaviors such as post-traumatic stress disorder and generalized anxiety disorder • BNST provides a substrate for interaction of CRF and NPY in regulating stress and anxiety

  5. Introduction • NPY Anxiety ↓ Reward pathway • CRF Anxiety ↑ Stress response • BNST acts as a scale to create a balance of CRF and NPY

  6. Neves S, Ram P, Iyengar R. G protein pathways. Science 296, 1636-1639 (2002)

  7. Introduction • BNST expresses both NPY/YRs and CRF/CRFRs • CeA releases CRF and GABA to the BNST • Both CRF and NPY modulate GABAergic transmissions

  8. Introduction • Study GABAergic influence on ventrolateral region of the BNST • vlBNST projects to the VTA (reward) and the PVN (stress)

  9. Introduction • IPSC= inhibitory post-synaptic current m = miniature e = evoked

  10. Methods • Male C57B1/6J mice • Decapitated mice and placed brain in ice-cold sucrose aCSF • Slices 300um thick • Rostral slices contained anterior BNST • Stored in heated, oxygenated container w/ aCSF • Transferred to submerged recording chamber • Heated, O2 aCSF for 1h ā experiments

  11. Methods • Slices in chamber and neurons of vlBNST visualized w/ infrared video microscopy • Analyzed eIPSC & EPSC • Electrodes filled w/ pH 7.2 • Twisted nichrome wire • Placed in vlBNST • Cells held @ -50mV & GABAAR-mediated IPSCs evoked @ 0.2 Hz by fiber stim w/ bipolar electrodes

  12. Methods • GABAA-IPSCs (& EPSC) isolated • 3mM kynurenic acid (& 25uM picrotoxin) = block AMPA & NMDA receptor-dependent postsynaptic currents • 1uM CGP 55845 = block GABABR • Signals acquired via Multiclamp 700B amplifier • Input and series resistance continuously monitored

  13. Methods • eIPSC → measured peak amplitude of synaptic response normalized to baseline • Baseline period = 2 min period immediately preceding drug • Value is 2 min avg 15 min p neuropeptide * B peptide 0 3 5 10 15 20 22

  14. Methods • mIPSC analysis • GABAAR-mediated IPSCs isolated → added 0.5uM TTX • recorded in 120s episodes • Ca2+ influx on NPY → 100uM Cd2+ + aCSF • Amplitude and frequency determined from 120s recording w/ cells held @ -70mV • Multi-clamp

  15. Methods • All drugs applied via bath • All peptides used were dissolved in dH2O to 0.1mM conc • Some stored @ -20°C

  16. Results • NPY and CRF influence on inhibitory synaptic transmission in vlBNST • Whole-cell voltage clamp • Local stimulation produced eIPSC from GABAARs • SR95531= GABAAR antagonist blocked response

  17. Results • TTX elicited spontaneous mIPSCs • GABAzine = GABAAR antagonist • mIPSCs blocked • GABAAR mediated

  18. Results • NPY depresses GABA through Y2R • Baseline recordings revealed decreased peak amplitude of eIPSC • Observed in all cells • NPY-induced depression = concentration dependent

  19. Results • NPY13-36 = Y2R agonist • ↓ peak amplitude • [Pro34]-NPY = Y1R agonist & [D-Trp32]-NPY = Y5R agonist • No change

  20. Results • Antagonist testing was preceded by an exposure to NPY (10-15min) • Agonist and antagonist co-applied (5min) • NONE had significant effects on eIPSC without agonist

  21. Results • Non-peptide Y2R antagonist blocked NPY actions • Peptide Y1R antagonist had no effect • Non-peptide Y5R antagonist had no effect • Y2Rs activated NPY-induced eIPSC depression

  22. Results • Paired Pulse Ratio experiments • Pair of eIPSCs w/ 50ms between • Ratio of amplitudes determined • NPY ↑ PPR of eIPSCs • Suggest ↓ release of GABA • NPY ↓ frequency but not amplitude

  23. Results • Presynaptic inhibition of NT release • Modulate Ca2+ entry • Regulate release machinery • At CNS synapse basal mIPSC freq ↓ by using Cd2+ to block Vg Ca2+ channels • Cd2+ + NPY = restores mIPSCs • NPY inhibits GABA via Y2R regulation of Ca2+ influx

  24. Results • 5 min bath of 1um CRF sig ↑ peak amp of eIPSC • 1um Urocortin (CRFR agonist) → similar results • CRF results were concentration dependent [100nM vs 10nM] • CRF antagonist had no sig effects on eIPSC in absence of agonist

  25. Results • Non-peptide CRFR1 antagonist (NBI 27914) blocked both CRF and Ucn I actions • Peptide CRFR2 antagonist (anti-Sauvagine-30) had no sig effect • Ucn I enhanced eIPSCs in CRFR2 knockout mice • So, CRF/Ucn I induced enhancement of eIPSC is d/t CRFR1 activation

  26. Results • CRF • did not alter PPR or mIPSC kinetics • no effect on freq of mIPSCs • Mean amplitude ↑↑ • Shifted cum. amplitude curve to the right • CRF enhanced GABAergic postsynaptic transmission • Change in IPSCs d/t non-specific enhancement via synaptic excitability

  27. Discussion • NPY suppresses GABAergic transmission in vlBNST via Y2R: • NPY effect mimicked by NPY13-36, not by [Pro34]-NPY (Y1 agonist) or [D-Trp32]-NPY (Y5 agonist) • NPY antagonized by Y2R antagonist (BIEE 0246), not by BVD-10 (Y1R antagonist) or L-152804 (Y5R antagonist)

  28. Discussion • Data consistent w/ NPY actions in thalamus & PVN and Y2R expression in BNST • Y2R ↓ GABA release: • NPY ↑ PPR of eIPSCs → ↓ release probability • NPY ↓ mIPSC frequency not amplitude • Cd+2 effect → NPY via Y2R inhibit GABA via presynaptic Ca+2 influx

  29. Discussion • NPY via Y2R → heterocepter on GABAergic terminals • Which YR is activated determines the behavioral outcome • Y1R and maybe Y5R → NPY anxiolytic response • Y2R → anxiogenic response • But, Y2R activation in LC → NPY anxiolytic effects

  30. Discussion • Suppose region-specific activation of YR subtypes will evoke distinct behavioral phenotypes, OR… • Autoreceptor-like functions are anxiolytic

  31. Discussion • Based on evidence that inhibitory projections from the vlBNST contact PVN neurons: ↓ GABAergic input to vlBNST → ↑ GABAergic output to PVN → ↓ stress response

  32. Discussion • In the vlBNST, CRF and Ucn I enhance GABAergic transmission via CRFR1: • CRF & Ucn I antagonized by NBI 27914 (CRFR1), not AS 30 (CRFR2) • Ucn I effects observed in mice lacking CRFR2 • CRFR1 enhance postsynaptic GABAA-R response

  33. Discussion • CRF ↑ mIPSC amplitude not frequency or PPR • Glutamatergic transmission in vlBNST not affected by Ucn I • Presynaptic CRFR1 mediates GABAergic transmission in CeA • Altering mIPSC frequency modulates GABA release → now amplitude as well

  34. Discussion • CRFR1 mediates CRF anxiogenic effects in the BNST • CRF enhancing GABAergic transmission in BNST could potentially reactivate the PVN → stress response

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