orexin/hypocretin enhances synaptic strength in vta dopamine neurons
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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
5
10
15
20
25
30
35
40
45
[Orexin A] nM
NM
DA
Am
pli
tud
e (%
in
crea
se)
0 10 20 30 40 50 600
50
100
150
200
100 nM orexin A
Time (min)
NM
DA
R A
mp
litu
de
(% b
asel
ine)
*
**
**
OxA/Hcrt1 does not potentiate AMPA EPSCs in VTA neurons
0 10 20 30 40 500
50
100
150
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
50
100
150
200
1 M SB 334867100 nM orexin A
Time (min)
NM
DA
R A
mp
litu
de
(% 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
0.2
0.4
0.6
0.8
1.0
SB 334867
*A
MP
AR
/NM
DA
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
0.4
0.6
0.8
1.0
SB 334867
*A
MP
AR
/NM
DA
R R
ati
o
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
10
15
20
25*
AM
PA
R m
EP
SC
Am
pli
tud
e (p
A)
control 15 min 3-4 hours APV0
1
2
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
10
15
20
25*
AM
PA
R m
EP
SC
Am
pli
tud
e (p
A)
control 15 min 3-4 hours APV0
1
2
3 **
*
AM
PA
R m
EP
SC
Fre
qu
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
0 1 2 3 4 5 6 70
10000
20000
30000
cocaine + vehiclecocaine + SB 334867saline + vehiclesaline + SB 334867
Days
Dis
tan
ce T
rave
led
(cm
)
Behavioral sensitization is blocked with intra-VTA injections of OXR1/Hcrt1R antagonist
0 1 2 3 4 5 6 7 80
10000
20000
30000
40000
Days
Dis
tan
ce T
rave
led
(cm
)
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
150
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
2.5
5.0
7.5
10.0R
ein
forc
ers
naive vehicle vehicle vehicle0
50
100
150
Pre
sses
naive vehicle vehicle vehicle0
10
20
30
Bre
akp
oin
t
OXR1/Hcrt1 antagonist reduces “motivation” in progressive ratio test in cocaine self-administering rats
n=12
Activ
e
Inac
tive
Activ
e
Inac
tive
0
50
100
150
200
250
Vehicle SB334867
*
Pre
sses
Naive Vehicle Vehicle SB3348670.0
2.5
5.0
7.5
10.0
12.5
**
Rei
nfo
rcer
s
Naive Vehicle Vehicle SB3348670
10
20
30
40
50
60
*
Bre
akp
oin
t
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
50
100
150
200
250
300
350
400
450VehicleSB334867
Time (min)
Cu
mu
lati
ve P
resses
n=12Rat=S5
0 50 100 150 200 2500
100
200
300
vehicleSB 334867
Time (min)
Cu
mu
lati
ve
Pre
sses
Food self administration
Naive Vehicle Vehicle Vehicle0
100
200
300
400
500
600
Pre
sses
Naive Vehicle Vehicle Vehicle0123456789
10111213
Rein
forc
ers
Naive Vehicle Vehicle Vehicle0
102030405060708090
100110
Bre
akp
oin
t
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
5
10
15
20R
ein
forc
ers
Naive Vehicle Vehicle SB3348670
25
50
75
100
125
Bre
akp
oin
t
Active Inactive Active Inactive0
100
200
300
400
500
Vehicle SB334867
Pre
sses
Orexin/Hcrt 1 receptor signaling is not involved in motivation for food
Naive Vehicle Vehicle SB 334867 0123456789
101112
Rei
nfo
rcer
s
Active Inactive Active Inactive0
50
100
150
200
250
300
350
400
450
Vehicle SB 334867
Pre
sses
Naive Vehicle Vehicle SB 334867 0
10
20
30
40
50
60
70
80
90
Bre
akp
oin
t
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
100
200
300
400
vehicleSB 334867
Time (min)
Cu
mm
ula
tive
P
ress
es
Cocaine Self-Administration increases OxA/Hcrt1 potentiation of NMDARs
n=8
0 10 20 30 40 50 600
50
100
150
200
100 nM orexin A
Time (min)
NM
DA
R e
EP
SC
(%
Bas
elin
e)
n=8
0 10 20 30 40 50 600
50
100
150
200
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
100
150
200
100 nM orexin A
Time (min)
NM
DA
R e
EP
SC
(%
Bas
elin
e)
n=7
0 10 20 30 40 50 600
50
100
150
200
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
25
50
75
100
125
150
175 *
NM
DA
R e
EP
SC
s (
% B
asel
ine)
0 10 20 30 40 50 600
50
100
150
200
100 nM orexin A
* * * ** * * **
foodcocaineshamnaive
Time (min)
NM
DA
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
25
50
75
100
125
150
175
200
100 nM orexin A
Time (min)
NM
DA
R A
mp
litu
de
(% 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
250
500
750
1000
*
Naive Cocaine Cocaine SB+Cocaine
Pre
sses
Naive Cocaine Cocaine SB + Cocaine0
5
10
15
**
**
Rei
nfo
rcer
s
OXR1 antagonist reduces breakpoint in the presence of cocaine
n=12
Naive Cocaine Cocaine SB + Cocaine0
100
200
**
Bre
akp
oin
t
Dopamine is required for the orexin-mediated reduction in food seeking
n=11
A I A I A I A I0
500
1000
1500
2000
2500
*
Naive GBR GBR SB + GBR
Pre
sses
Naive GBR GBR GBR+SB0
10
20
*
Rei
nfo
rcer
s
n=9
Naive GBR GBR SB + GBR0
100
200
300
400
*
Bre
akp
oin
t
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
50
100
150
200
250
300
350
400
450
Naive Vehicle Vehicle SB+Cocaine
Pre
sses
Naive Vehicle Vehicle SB + Cocaine0123456789
101112
**
Rei
nfo
rcer
s
Naive Vehicle Vehicle SB + Cocaine0
10
20
30
40
50
60
70
80
90
Bre
akp
oin
t
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
500
750
Pre
sses
Naive Vehicle Vehicle SB 3348670
5
10
15R
ein
forc
ers
Naive Vehicle Vehicle SB 3348670
50
100
150
200
Bre
akp
oin
t
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
200
300
400
500
Pre
sses
Naive Vehicle Vehicle SB3348670123456789
10111213
Rei
nfo
rcer
s
Naive Vehicle Vehicle SB3348670
20
40
60
80
100
120
Bre
akp
oin
t
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)
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