Orexin/Hypocretin enhances synaptic strength in VTA dopamine

neurons

Stephanie Borgland, Ph.D.Ernest Gallo Clinic and Research Center, UCSF

Orexin/Hypocretin and Reward Orexin/Hypocretin increases VTA neuron firing

(Korotkova et al., 2003) Intra-VTA orexin/hypocretin increases dopamine in

the Nucleus Accumbens (Narita et al., 2006) or PFC (Vittoz and Berridge, 2006)

Orexin/Hypocretin neurons are activated when rats prefer morphine in a CPP paradigm which is blocked by intra VTA hypocretin antagonist (Harris et al., 2005)

CPP for morphine is abolished in orexin/hypocretin peptide knockout mice (Narita et al., 2006).

Orexin/hypocretin i.c.v. reinstates cocaine seeking (Boutrel et al., 2006)

How does orexin/hypocretin mediate the rewarding effects

of drugs?

Can ox/hcrt cause synaptic plasticity in dopamine

neurons?

Why is synaptic plasticity in the VTA important?

Glutamatergic synaptic plasticity plays a key role in neural plasticity relevant to addiction• Induction of behavioral sensitization is

dependent on activation of NMDA receptors in the VTA (Kalivas and Alesdatter, 1993)

Synaptic plasticity of dopamine neurons in the VTA may play a key role in the reinforcement of reward.

Patch Clamp Recording from VTA neurons

AMPA EPSC-70 mV

NMDA EPSC+40 mV

Currents

OxA/Hcrt1 concentration-dependently increases NMDAR EPSCs in VTA neurons

n=12

1 10 1000

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[Orexin A] nM

NM

DA

Am

pli

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in

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100 nM orexin A

Time (min)

NM

DA

R A

mp

litu

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(% b

asel

ine)

*

**

**

OxA/Hcrt1 does not potentiate AMPA EPSCs in VTA neurons

0 10 20 30 40 500

50

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100 nM orexin A

Time (min)

AM

PA

EP

SC

(%

bas

elin

e)

n=8

OxA/Hcrt1 OxB/Hcrt2

Pre-pro ox/hcrt

OXR2/Hcrt2R

Gq Gi/oGq

OXR1/Hcrt1R

Orexin/Hypocretin Pharmacology

OxA/Hcrt1 mediated potentiation of NMDAR EPSCs is inhibited by OX1/hcrt1 receptor antagonist

n=8

0 10 20 30 40 50 600

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1 M SB 334867100 nM orexin A

Time (min)

NM

DA

R A

mp

litu

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(% b

asel

ine)

GLU

NMDARNR1

NR1/NR2A/?NR2A/?

AMPA

PFCPPN

PLC/PKC

OXA

OXA

OXAOXA

OXR1

+

NR1

NR2A/?

1

2

LH-hcrt neuron

VTA Neuron

OxA/Hcrt1 on AMPAR synaptic transmission

Activation of NMDARs is an important component for VTA long term potentiation (LTP)

AMPAR/NMDAR is measure of LTP

Glu

AMPA

NMDA

Ca++

CaMKII

OxA/Hcrt1, CRF, cocaine

NMDARs AMPARs

NMDAR activation precedes AMPAR plasticity

Time (?)NMDARs

AMPARs

Stress, cocaine, other drugs of abuse

Does orexin A potentiation of NMDARs facilitate cocaine-mediated AMPAR plasticity?

NMDAR activation precedes AMPAR plasticity

OXR1/Hcrt1R antagonist blocks cocaine induced potentiation of AMPAR/NMDAR ratio

n=5

n=11

n=4 n=4

Saline Cocaine Saline Cocaine0.0

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1.0

SB 334867

*A

MP

AR

/NM

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R R

ati

o

5 days cocaine or saline +/- OXR1 antagonist

OXR1/Hcrt1R antagonist blocks cocaine induced potentiation of AMPAR/NMDAR ratio

5 days cocaine or saline +/- OXR1 antagonist

n=5 n=11n=4 n=4

Saline Cocaine Saline Cocaine0.0

0.2

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1.0

SB 334867

*A

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/NM

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R R

ati

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OXR1 antagonist

15 minutes 3-4 hours

Slices are incubated with OxA/Hcrt1for 5 minutes

VTA

Recording Recording

Does OxA/Hcrt1 increase AMPA/NMDA ratio?

OxA/Hcrt1 increases AMPAR/NMDAR hours after application

n=8 n=8 n=8

control 15 min 3-4 hours0.0

0.2

0.4

0.6

0.8

1.0**

*

AM

PA

R/N

MD

AR

Rat

io

OxA/Hcrt1 causes a late phase increase in AMPAR mediated synaptic transmission

control 15 min 3-4 hours

control 15 min 3-4 hours APV0

5

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25*

AM

PA

R m

EP

SC

Am

pli

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A)

control 15 min 3-4 hours APV0

1

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3 **

*

AM

PA

R m

EP

SC

Fre

qu

ency

(H

z)

(7) (6) (7)

OxA/Hcrt1 causes a late phase increase in AMPAR mediated synaptic transmission that is NMDAR dependent

control 15 min 3-4 hours

control 15 min 3-4 hours APV0

5

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AM

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control 15 min 3-4 hours APV0

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Fre

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ency

(H

z)

(7) (6) (7) (7)

Does OxA/Hcrt1 mediated plasticity of dopamine neurons have behavioral consequences?

Activation of NMDARs is an important component for VTA LTP (Bonci & Malenka, 1999) and the development of cocaine sensitization (Kalivas & Alesdatter, 1993)

Behavioral sensitization is a progressive increase in locomotor response to the same cocaine dose.

Since cocaine sensitization is dependent on NMDAR activation in the VTA, we hypothesized that OxA/Hcrt1 may have a role in behavioral sensitization to cocaine.

OXR1/Hcrt1R antagonist blocks cocaine sensitization

n=13

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30000

cocaine + vehiclecocaine + SB 334867saline + vehiclesaline + SB 334867

Days

Dis

tan

ce T

rave

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(cm

)

Behavioral sensitization is blocked with intra-VTA injections of OXR1/Hcrt1R antagonist

0 1 2 3 4 5 6 7 80

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Days

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Cocaine (ip)

Saline (ip)

Intra-VTA vehicle (12)

Intra-VTA vehicle (8)

Intra-VTA SB334867 (12)

Intra-VTA SB334867 (10)

Orexin/hypocretin has a profound role in altering synaptic plasticity in a neural circuit important for motivation

Does orexin/hypocretin signaling mediate motivated behavior?

ie. if orexin/hypocretin receptors are blocked, will rats work as much to get cocaine?

Hypothesis

Self-administration Progressive Ratio

Surgery FR1 FR3 FR5

Nai

veV

ehic

le

SB

334

867

Veh

icle

Progressive ratio

0.5 mg/infusion cocainePaired with tone and light

1 2 3 4

0.0 2.5 5.0 7.5 10.0 12.5 15.00

50

100

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200cocainefood

Reinforcers

Lev

er P

ress

es

Vehicle treated rats do not reduce pressing for the duration of the experiment

n=8

Naive Vehicle Vehicle Vehicle0.0

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OXR1/Hcrt1 antagonist reduces “motivation” in progressive ratio test in cocaine self-administering rats

n=12

Activ

e

Inac

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Activ

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Vehicle SB334867

*

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Naive Vehicle Vehicle SB3348670.0

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Naive Vehicle Vehicle SB3348670

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Bre

akp

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SB 334867 10 mg/kg

Cumulative response shows the pattern of presses for cocaine in vehicle and SB334867 treated rats

0 25 50 75 100 125 150 175 2000

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Time (min)

Cu

mu

lati

ve P

resses

n=12Rat=S5

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vehicleSB 334867

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lati

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Food self administration

Naive Vehicle Vehicle Vehicle0

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Naive Vehicle Vehicle Vehicle0123456789

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akp

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n=9

Orexin/Hcrt 1 receptor signaling is not involved in motivation for food

n=10

SB 334867 10 mg/kg

Naive Vehicle Vehicle SB3348670

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ein

forc

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Naive Vehicle Vehicle SB3348670

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akp

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Active Inactive Active Inactive0

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Vehicle SB334867

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Orexin/Hcrt 1 receptor signaling is not involved in motivation for food

Naive Vehicle Vehicle SB 334867 0123456789

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nfo

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Active Inactive Active Inactive0

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Vehicle SB 334867

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Naive Vehicle Vehicle SB 334867 0

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akp

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n=12

SB 334867 20 mg/kg

0 25 50 75 100 1250

50100150200250300350400450500550 Vehicle

SB334867

Time (min)

Cu

mm

ula

tive

P

ress

es

Cumulative response shows the pattern of presses for food in vehicle and SB334867 treated rats

0 25 50 75 100 125 150 175 2000

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vehicleSB 334867

Time (min)

Cu

mm

ula

tive

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Cocaine Self-Administration increases OxA/Hcrt1 potentiation of NMDARs

n=8

0 10 20 30 40 50 600

50

100

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100 nM orexin A

Time (min)

NM

DA

R e

EP

SC

(%

Bas

elin

e)

n=8

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50

100

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100 nM orexin A

Time (min)

NM

DA

R e

EP

SC

s (%

Bas

elin

e)

Food Cocaine

OxA/Hcrt 1 mediated potentiation of NMDAR is not different between food restricted (sham) and naïve rats

0 10 20 30 40 50 600

50

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100 nM orexin A

Time (min)

NM

DA

R e

EP

SC

(%

Bas

elin

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n=7

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100 nM orexin A

Time (min)

NM

DA

R e

EP

SC

(%

Bas

elin

e)

Naive Sham

n=9

Cocaine self-administration potentiates OxA/Hcrt1-mediated synaptic plasticity in the VTA

naive sham food cocaine0

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175 *

NM

DA

R e

EP

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s (

% B

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100 nM orexin A

* * * ** * * **

foodcocaineshamnaive

Time (min)

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R E

PS

C (

% B

asel

ine)

Summary OxA/Hcrt1 potentiates NMDA currents in DA neurons of

the VTA. OxA/Hcrt1 enhance:

• synaptic strength in the mesolimbic system• burst firing of DA neurons, and increase in DA

release.

OxA/Hcrt1 causes a late phase increase in AMPAR mediated synaptic transmission• Facilitating dopamine’s role in reinforcement?

Summary -2

OXR1/Hcrt1R antagonist blocks cocaine sensitization, indicating that activation of orexin/hypocretin 1 receptors in the VTA is required for the development of sensitization.

Orexin/hypocretin signaling is involved in “motivation” for cocaine but not food seeking

Cocaine self-administration potentiates orexin/hypocretin-mediated plasticity in the VTA

Acknowledgements

Sharif Taha

Federica Sarti

Shao-Ju Chang

Billy Chen

Funding: NARSAD

NIDA (A.B.)

State of California (A.B.)

Antonello BonciHoward Fields

Prolonged application causes a long-lasting increase in NMDAR EPSCs

0 10 20 30 40 50 60 70 800

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100 nM orexin A

Time (min)

NM

DA

R A

mp

litu

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(% b

asel

ine)

n=6

Does OxA/Hcrt1 potentiate NMDARs in dopamine neurons?

Cameron et al., 1996 Neurosci 77:

Biocytin-TR TH-FITC Merge

7/8

0/2

OxA/Hcrt1 increases NMDAR EPSCs in VTA dopaminergic neurons

Orexin plays a gatekeeper role in that it enables neuroplasticity in excitatory synapses in the VTA

1. Ox/hcrt potentiation of NMDA promotes burst firing and increases DA release.

2. Ox/hcrt late phase potentiation of AMPARs may prolong burst firing.

3. This plasticity may underlie the intensification of goal-directed behavior.

Jones & Bonci 2005: 5:20-5

OXR1 antagonist reduces food self-administration in the presence of cocaine

n=12

A I A I A I A I0

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Naive Cocaine Cocaine SB+Cocaine

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OXR1 antagonist reduces breakpoint in the presence of cocaine

n=12

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Dopamine is required for the orexin-mediated reduction in food seeking

n=11

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Naive GBR GBR SB + GBR

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n=9

Naive GBR GBR SB + GBR0

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Dopamine is required for the orexin-mediated reduction in food seeking

SB 334867 attenuates potentiation of breakpoint by a single injection of cocaine

A I A I A I A I0

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OXR1 signaling needed for cocaine PR but not food PR

Is increased dopamine required for orexin release? • Can blocking orexin receptors in food treated rats reduce

breakpoint after injection of GBR12909 (5 mg/kg) or cocaine (15 mg/kg)?

Is orexin signaling involved only for highly motivational

substances?• If you increase the reinforcing value of the reward, ie. Sucrose or

high fat, will the orexin antagonist reduce seeking?

Does cocaine potentiate orexin release/signaling?

SB 334867 (10 mg/kg) does not reduce motivation for sucrose

Active Inactive Active Inactive0

250

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sses

Naive Vehicle Vehicle SB 3348670

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15R

ein

forc

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akp

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Vehicle SB334867

n=12

SB 334867 10 mg/kg

SB 334867 (20 mg/kg) does not reduce motivation for sucrose

Vehicle SB334867

n=9

SB 334867 20 mg/kg

Active Inactive Active Inactive0

100

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sses

Naive Vehicle Vehicle SB3348670123456789

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Naive Vehicle Vehicle SB3348670

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Bre

akp

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OXR1 signaling needed for cocaine PR but not food PR

Is increased dopamine required for orexin release? • Can blocking orexin receptors in food treated rats reduce

breakpoint after injection of GBR12909 (5 mg/kg) or cocaine (15 mg/kg)?

Is orexin signaling involved only for highly motivational

substances?• If you increase the reinforcing value of the reward, ie. Sucrose or

high fat, will the orexin antagonist reduce seeking?

Does cocaine/dopamine potentiate orexin release/signaling?• Is there a change in pre-pro orexin in the LH?

• Is there an alteration of orexin-mediated plasticity in the VTA?

Future experiments

Are sucrose pellets not reinforcing enough? Is orexin signaling involved in motivation for high fat pellets?

Does chronic cocaine change levels of pre-pro orexin or orexin A released?

Is the OXR1 antagonist mediating the reduction in cocaine seeking acting in the VTA?

Orexin and Self-Administration

Single injection (icv) of OxA induced persistent elevations of ICSS thresholds in drug naïve rats (Boutrel et al., 2006)

OxA (icv) reinstated cocaine & food self admin (2 wk extinction) (Boutrel et al., 2006)

OxA induced reinstatement was partially blocked by adrenergic and CRF antagonists (Boutrel et al., 2006)

OXR1 antagonist (ip) blocked footshock induced reinstatement (Boutrel et al., 2006)

OxA (icv) for 3 consecutive days did not alter cocaine self administration (Boutrel et al., SFN 2004)

OxA (icv) did not alter progressive ratio for cocaine (0.25 mg/infusion; Boutrel et al., SFN 2004)