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Transcript of Neuropeptide Y and corticotropin-releasing factor bi-directionally modulate inhibitory synaptic...
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 pathwaymediated via G-protein coupled
receptorsreleased in a frequency dependent
fashionlonger half-life of activity after release
Introduction
Corticotropin Releasing Factor (CRF) involved in stress response Mediated through the hypothalamus
and the amygdalaBoth pathways converge at the BNST
Introduction
Life = StressRepeated 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
Introduction
NPY
Anxiety ↓
Reward pathway
CRF
Anxiety ↑
Stress response
BNST acts as a scale to create a balance of CRF and NPY
Neves S, Ram P, Iyengar R. G protein pathways. Science 296, 1636-1639 (2002)
Introduction
BNST expresses both NPY/YRs and CRF/CRFRs
CeA releases CRF and GABA to the BNST
Both CRF and NPY modulate GABAergic transmissions
Introduction
Study GABAergic influence on ventrolateral region of the BNST
vlBNST projects to the VTA (reward) and the PVN (stress)
IntroductionIPSC= inhibitory post-synaptic current
m = miniature e = evoked
MethodsMale C57B1/6J miceDecapitated mice and placed brain in ice-
cold sucrose aCSFSlices 300um thickRostral slices contained anterior BNST
Stored in heated, oxygenated container w/
aCSFTransferred to submerged recording chamber
Heated, O2 aCSF for 1h ā experiments
Methods
Slices in chamber and neurons of vlBNST visualized w/ infrared video microscopy
Analyzed eIPSC & EPSC Electrodes filled w/ pH 7.2Twisted nichrome wirePlaced in vlBNST
Cells held @ -50mV & GABAAR-mediated IPSCs evoked @ 0.2 Hz by fiber stim w/ bipolar electrodes
Methods
GABAA-IPSCs (& EPSC) isolated3mM 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
Methods
eIPSC → measured peak amplitude of synaptic response normalized to baseline
Baseline period = 2 min period immediately preceding drugValue is 2 min avg 15 min p neuropeptide
0 3 5 10 15 20 22
B peptide *
Methods
mIPSC analysisGABAAR-mediated IPSCs isolated → added
0.5uM TTXrecorded in 120s episodesCa2+ influx on NPY → 100uM Cd2+ + aCSFAmplitude and frequency determined from
120s recording w/ cells held @ -70mVMulti-clamp
Methods
All drugs applied via bath All peptides used were dissolved in dH2O
to 0.1mM concSome stored @ -20°C
Results
NPY and CRF influence on inhibitory synaptic transmission in vlBNSTWhole-cell voltage clamp
Local stimulation produced eIPSC from GABAARs
SR95531= GABAAR antagonist blocked response
Results
TTX elicited spontaneous mIPSCsGABAzine = GABAAR antagonist
mIPSCs blockedGABAAR mediated
Results
NPY depresses GABA through Y2RBaseline recordings revealed
decreased peak amplitude of eIPSCObserved in all cells
NPY-induced depression = concentration dependent
Results
NPY13-36 = Y2R agonist ↓ peak amplitude
[Pro34]-NPY = Y1R agonist &
[D-Trp32]-NPY = Y5R agonistNo change
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
Results
Non-peptide Y2R antagonist blocked NPY actions
Peptide Y1R antagonist had no effectNon-peptide Y5R antagonist had no
effectY2Rs activated NPY-induced
eIPSC depression
Results
Paired Pulse Ratio experimentsPair of eIPSCs w/ 50ms betweenRatio of amplitudes determined
NPY ↑ PPR of eIPSCsSuggest ↓ release of GABA
NPY ↓ frequency but not amplitude
ResultsPresynaptic inhibition of NT release
Modulate Ca2+ entryRegulate release machinery
At CNS synapse basal mIPSC freq ↓ by using Cd2+ to block Vg Ca2+ channels
Cd2+ + NPY = restores mIPSCsNPY inhibits GABA via Y2R
regulation of Ca2+ influx
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
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
ResultsCRF
did not alter PPR or mIPSC kineticsno effect on freq of mIPSCsMean amplitude ↑↑Shifted cum. amplitude curve to the
rightCRF enhanced GABAergic
postsynaptic transmissionChange in IPSCs d/t non-specific
enhancement via synaptic excitability
Discussion
NPY suppresses GABAergic transmission in vlBNST via Y2R:
1. NPY effect mimicked by NPY13-36, not by [Pro34]-NPY (Y1 agonist) or [D-Trp32]-NPY (Y5 agonist)
2. NPY antagonized by Y2R antagonist (BIEE 0246), not by BVD-10 (Y1R antagonist) or L-152804 (Y5R antagonist)
DiscussionData consistent w/ NPY actions in
thalamus & PVN and Y2R expression in BNST
Y2R ↓ GABA release:1.NPY ↑ PPR of eIPSCs → ↓ release
probability2.NPY ↓ mIPSC frequency not amplitude3.Cd+2 effect → NPY via Y2R inhibit
GABA via presynaptic Ca+2 influx
DiscussionNPY via Y2R → heterocepter on
GABAergic terminalsWhich YR is activated determines
the behavioral outcomeY1R and maybe Y5R → NPY anxiolytic
responseY2R → anxiogenic response
But, Y2R activation in LC → NPY anxiolytic effects
Discussion
Suppose region-specific activation of YR subtypes will evoke distinct behavioral phenotypes, OR…
Autoreceptor-like functions are anxiolytic
Discussion
Based on evidence that inhibitory projections from the vlBNST contact PVN neurons:↓ GABAergic input to vlBNST →
↑ GABAergic output to PVN →
↓ stress response
Discussion
In the vlBNST, CRF and Ucn I enhance GABAergic transmission via CRFR1:1.CRF & Ucn I antagonized by NBI 27914
(CRFR1), not AS 30 (CRFR2)2.Ucn I effects observed in mice lacking
CRFR2
3.CRFR1 enhance postsynaptic GABAA-R response
DiscussionCRF ↑ mIPSC amplitude not frequency
or PPRGlutamatergic transmission in vlBNST
not affected by Ucn IPresynaptic CRFR1 mediates
GABAergic transmission in CeAAltering mIPSC frequency modulates
GABA release → now amplitude as well
Discussion
CRFR1 mediates CRF anxiogenic effects in the BNST
CRF enhancing GABAergic transmission in BNST could potentially reactivate the PVN → stress response