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