1. The study found that activation of α1-adrenergic receptors (α1ARs) or corticotropin releasing factor receptors type 2 (CRF2Rs) in dopamine neurons enhances the induction of long-term potentiation of NMDA receptors (NMDAR-LTP) through increasing intracellular calcium release. 2. In vitro, acute stress hormones like norepinephrine or CRF facilitated NMDAR-LTP by amplifying calcium-induced calcium release via IP3 receptors. 3. In vivo, acute social defeat stress strengthened the acquisition of cocaine-conditioned place preference, an effect prevented by blocking α1ARs and CRF2Rs in the ventral te

1. 0 10 20 30
0.5
1.0
1.5
2.0
Phe
CRF
Phe 0.5 µM+CRF
Phe 1µM+CRF
Time(min)
IK(Ca)(norm.)
0.5
µM
0.5
µM
+C
R
F
1
µM1
µM
+C
R
F
0
10
20
30
40
50
60
IK(Ca)facilitation(%
0 10 20 30 40 50
0.5
1.0
1.5
2.0
Phe 0.5 µM
Phe 0.5 µM + CRF
LTP
Time (min)
NMDAREPSC(norm.)
Phe
0.5
µMPhe
0.5
µM
+
C
R
F
0
10
20
30
40
50
***
IK(Ca)facilitation(%)
-20 20 40 60
-20
20
40
60
r2 = 0.65
IK(Ca) facilitation (%)
NMDAR-LTP(%)
Phe
0.5
µMPhe
0.5
µM
+
C
R
F
0
10
20
30
40
50
*
NMDAR-LTP(%)
0 10 20 30 40 50
0.5
1.0
1.5
2.0
IP3 alone
IP3 + CRF
LTP
Time (min)
NMDAREPSC(norm.)
0 10 20 30 40 50
0
20
40
60
80
100
120
1
2IP3
+
CRF
LTP
Time (min)
NMDAREPSC(pA)
0 nM 100 nM
0
10
20
30
40
[CRF]
IK(Ca)facilitation(%)
0 nM 100 nM
0
10
20
30
40
[CRF]
NMDAR-LTP(%)
-20 20 40
-20
20
40
r2=0.47
IK(Ca) facilitation (%)
NMDAR-LTP(%)
0 10 20 30 40 50
0.5
1.0
1.5
2.0
CRF 100nM
Control
LTP
CRF
Time (min)
NMDAREPSC(norm.)
0 10 20 30 40 50
0
50
100
1
2
LTP
CRF
100 nM
Time (min)
NMDAREPSC(pA)
0 10 20 30
0.5
1.0
1.5
2.0
100 nM
300 nM
CRF 30 nM
Time(min)
IK(Ca)(norm.)
CRF
100 nM
0 10 20 30
0.5
1.0
1.5
2.0
1st EPSC
2nd EPSC
Time(min)
NMDAREPSC(norm.)
0.5 µM 1 µM
0
10
20
30
40
50
60
**
[Phe]
IK(Ca)facilitation(%)
-20 20 40 60 80
20
40
60
80
r2=0.45
IK(Ca) facilitation (%)
NMDAR-LTP(%)
0.5 µM 1 µM
0
10
20
30
40
50
60
**
[Phe]
NMDARLTP(%)
0 10 20 30 40 50
0
50
100
1
2
LTP
Phe
1 µM
Time (min)NMDAREPSC(pA)
0 10 20 30 40 50
0.5
1.0
1.5
2.0
Phe 1 µM
Phe 0.5 µMPhe
LTP
Time (min)
NMDAREPSC(norm.)
0 10 20 30
0.5
1.0
1.5
2.0
Phe 0.5 µM
1 µM
Time (min)
IK(Ca)(norm.)
0.5 µM 1 µM
0
10
20
30
40
50
60
**
[Phe]
IK(Ca)facilitation(%)
0 10 20 30
0.5
1.0
1.5
2.0
1st EPSCPhe
1 µM 2nd EPSC
Time(min)
NMDAREPSC(norm.)
1st EPSC 2nd EPSC
0
10
20
30
40
50
60
[Phe]
NMDAREPSCincrease(%)
References
1. Sinha R. Chronic stress, drug use, and vulnerability to addiction. Ann.N.Y.Acad.Sci., 2008.
2. Kauer JA and Malenka RC. Synaptic plasticity and addiction. Nat Rev Neurosci., 2007.
3. Sombers L. et al. Synaptic overflow of dopamine in the nucleus accumbens arises from neuronal activity in the ventral tegmental area. J Neurosci.,
2009.
4. Harnett MT. et al. Burst-timing-dependent plasticity of NMDA receptor-mediated transmission in midbrain dopamine neurons. Neuron., 2009.
5. Refojo D. et al. Glutamatergic and dopaminergic neurons mediate anxiogenic and anxiolytic effects of CRHR1. Science, 2011.
6. Grenhoff J. et al. Alpha 1-adrenergic effects on dopamine neurons recorded intracellularly in the rat midbrain slice. Eur J Neurosci., 1995.
7. Cui G. et al. Diferential regulation of action potential- and metabotropic glutamate receptor-induced Ca2+ signals by inositol 1,4,5-trisphosphate in
dopaminergic neurons. J Neurosci., 2007.
Background
- Stressful life experiences are well-known risk factors for the development of
drug addiction1.
- Addictive drugs are thought to hijack the synaptic plasticity mechanisms in brain
circuits involved in reward learning, specially the dopaminergic system in the
ventral tegmental area (VTA)2.
- Activation of NMDA receptors (NMDARs) is necessary for dopamine (DA)
neuron burst firing in response to drugs and drug-conditioned cues3. Therefore,
long term potentiation (LTP) of NMDARs in DA neurons may contribute to the
increased motivational valence of drug-associated cues.
- Mechanistically, NMDAR-LTP induction requires burst-evoked Ca2+ induced-
Ca2+ release (CICR) amplified by preceding production of IP3 due to metabotropic
glutamate receptors activation4.
- Norepinephrine (NE) and corticotropin releasing factor (CRF) release in the VTA
following acute stress5,6 can quickly modulate CICR through α1 adrenoreceptors
(α1ARs) or CRF2 receptors (CRF2Rs), respectively.
Hypothesis
- In vitro, acute activation of α1ARs and CRF2Rs would enhance the induction of
NMDAR-LTP in DA neurons through IP3Rs modulation (see scheme below).
- In vivo, an acute stressor applied right before conditioning would enhance the
acquisition of conditioned placed preference (CPP), a widely used model of
reward conditioning.
Jorge Tovar-Diaz, Matthew Pomrenze, Russell J Kan & Hitoshi Morikawa
Stress acutely promotes calcium-dependent glutamatergic synaptic
plasticity in VTA via differential actions of CRF and norepinephrine
Methodology
- In vitro
- Whole-cell voltage clamp recordings were performed in VTA DA neurons (naïve
male Sprague Dawley rats, 4-6 weeks old). Cells were held at -55 mV in all
recordings.
- Ca2+ signals were indirectly measured through IK(Ca) currents. We have
previously demonstrated that IK(Ca) is generated by unclamped action potentials
and is TTX and apamine sensitive7.
- Excitatory postsynaptic currents (EPSCs) where induced with a bipolar
electrode. NMDAR-mediated EPSCs were pharmacologically isolated with a
cocktail of AMPA, GABAA, GABAB, and D2 blockers and recorded in low Mg++
aCSF.
- Different concentrations of phenylephrine (Phe) and CRF were directly perfused
in the bath.
- LTP protocol. We used a “simultaneous pairing” protocol, where presynaptic
stimulation and postsynaptic burst firing were delivered at the same time. Ten
pairings every 20 sec (bipolar electrode stimulation, 30 stim at 30 Hz) paired with
burst firing (5 unclamped action potentials at 20 Hz).
- Caged IP3 was loaded in the recording pipette and uncaged with UV light.
- In vivo
- Social defeat stress was performed by direct exposure (5 min) to a highly
territorial resident male, followed by protected exposure through a perforated
plastic wall (25 min).
- Conditioned place preference (CPP). Ten min after social defeat, rats were
injected with cocaine (5 or 10 mg/kg, i.p.) and placed in the conditioning box.
Results
Conclusions
- In vitro
- Activation of α1ARs increases CICR through IP3 production, enabling
induction of NMDAR-LTP by a simultaneous pairing protocol.
- Activation of CRF2Rs facilitates CICR and thus NMDAR-LTP, but only when
IP3 is provided from another source.
- CRF2Rs and α1ARs work in a cooperative manner through IP3-dependent
CICR to promote NMDAR-LTP.
- In vivo
- Acute social defeat stress enhances the acquisition of cocaine-CPP only at
low doses of the drug.
- Intra-VTA atagonisms of CRF/adrenergic transmission prevents the
effect of stress on CPP acquisiton..
Funded by NIH grants DA015687 and AA015521
1.
α1AR activation facilitates NMDAR-LTP by increasing Ca2+ release from internal stores
Model of acute stress enhancement
of NMDAR-LTP in DA neurons!
1) Sustained presynaptic stimulation induces
IP3 production through mGluRs activation."
2) Burst firing induces Ca2+ influx through
voltage gated Ca2+ channels."
3) Acting as a coincident detector, IP3R-
mediated CICR enables NMDAR-LTP."
4) Acute activation of α1ARs increases IP3
production, while CRF2Rs increases IP3Rs
sensitivity, thus facilitating NMDAR-LTP
induction."
In vivo, an acute stress applied previous to
conditioning, could increase the valence of the
cues to be associated with the reward, thus
strengthening their conditioning."
2. CRF alone does not affect Ca2+ release nor NMDAR-LTP
3. CRF increases IP3-induced Ca2+ release, thus facilitating NMDAR-LTP
4. CRF increases α1AR-induced Ca2+ release, thus
facilitating NMDAR-LTP
5. Acute stress enhances cocaine-CPP, effect prevented
by intra-VTA blockage of CRF2Rs and α1ARs
Time
course
of
CRF
effect
on
a)
IK(Ca),
n=
5
(30
nM),
8
(100
nM)
and
4
cells
(300
nM)
and
b)
NMDAR
EPSC,
n=
5
cells
(100
nM).
c)
RepresentaJve
experiment
and
summary
Jme
course
of
CRF
(100
nM)
on
NMDAR-­‐LTP
(n=
10
cells).
d)
Summary
bar
graphs
of
CRF
100
nM
effect
on
IK(Ca)
and
NMDAR-­‐LTP.
a)
Time
course,
average
bar
graph
and
representaJve
traces
of
an
acJon
potenJal-­‐induced
IK(Ca)
alone
(black),
IP3-­‐facilitated
(blue)
and
IP3+CRF-­‐
facilitated
(red)(n=
8
cells).
**p<0.009
paired
t
test.
b)
IP3-­‐induced
(n=
8
cells)
and
IP3+CRF-­‐induced
(n=
11
cells)
NMDAR-­‐LTP
summary
Jme
course
and
representaJve
experiment.
c)
Summary
bar
graphs
of
IP3-­‐induced
and
IP3+CRF-­‐induced
IK(Ca)
facilitaJon
(***p<0.001
unpaired
t
test)
and
NMDAR-­‐LTP
(***p<0.001
unpaired
t
test)
and
their
correlaJon.
IP3
[10mM]
was
loaded
in
the
recording
pipet
and
acJvated
with
UV
light
(1mW*100ms)
through
the
microscope
objecJve.
Acute
social
defeat
enables
low
dose-­‐cocaine
condiJoning.
a)
Change
in
percentage
of
Jme
spent
in
the
drug-­‐paired
side
a_er
condiJoning
with
low
or
high
doses
of
cocaine.
***
p
<
0.005
two-­‐way
ANOVA.
n=
8rats/group.
b)
A
mix
of
antagonists
K41498/prazosin
(30
µM/30
µM
in
0.3µl)
prevents
the
effect
of
stress
on
CPP
acquisiJon.
Preliminary
result
(n=
4
rats
each
group).
a)
b)
c)
d)
a)
c)
b)
d)
a)
Phenylephrine
(Phe)-­‐induced
increase
in
IK(Ca),
Jme
course
and
summary
bar
graph.
n=
7
(0.5
µM)
and
9
cells
(1
µM).
**
p<0.005,
t
test.
b)
Phe
does
not
affect
basal
NMDAR
EPSC
amplitude.
n=
5
cells.
c)
RepresentaJve
experiment
and
d)
Summary
Jme
graph
of
of
Phe-­‐induced
NMDAR-­‐LTP.
n=
7
(0.5
µM)
and
10
(1
µM)
cells.
e)
Summary
bar
graphs
of
Phe
1
µM
on
IK(Ca)
and
NMDAR-­‐LTP
and
their
correlaJon.
**p<0.002
t
test.
a)
b)
c)
e)
c)
a)
Time
course,
summary
bar
graphs
and
representaJve
traces
of
CRF
effect
on
Phe-­‐
facilitated
IK(Ca).
n=
8
(Phe
0.5µM)
and
9
(1µM)
cells.
b)
LTP
summary
Jme
course,
bar
graphs
and
correlaJon
of
CRF
effect
on
Phe-­‐induced
IK(Ca)
facilitaJon
and
NMDAR-­‐LTP.
*p<0.03
and
***p<0.007
unpaired
t
test.
100 pA
100 ms
Time (s)
10 pA
500 ms
10 pA
50 ms
1
2
10 pA
50 ms
1
2
20 pA
50 ms
50 pA
100 ms
a)
b)
mGluR
G
Ca
2+
Store
IP3
IP3 R
IP3
PLC
NMDR-LTP
mGluR
G
q
PLC
VGCC
Glu
G q
q
Phasic firing
Afferent stimulation
NM DAR
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+Ca2+
IP3
q
IP3
Ca2+
Ca2+
Ca2+
Gs
PKA
CRF2R
1 AR
Acute stress
NE CRF
NE
CRF
2+
P
Glu
Glu
Glu
Glu
1
2
3
4
Stim
Burst
Stim
Burst
100 pA
200 ms
IK(Ca) alone
IK(Ca) + IP3 flash
IK(Ca) + IP3 flash + CRF
20 pA
20 ms
1
2
IP3
CRF
0 10 20 30
0.5
1.0
1.5
2.0
Time (min)
IK(Ca)(norm.)
IP3 IP3 + CRF
0
10
20
30
40
50
60
**
IK(Ca)facilitation(%)
IP3 IP3 + CRF
0
10
20
30
40
50
60
***
IK(Ca)facilitation(%)
0 20 40 60 80 100
0
20
40
60
80
100
r2=0.64
IK(Ca) facilitation (%)
NMDAR-LTP(%)
IP3 IP3 + CRF
0
10
20
30
40
50
60
***
NMDAR-LTP(%)
50 pA
100 ms
IK(Ca) alone
IK(Ca) + Phe
IK(Ca) + Phe + CRF
Pre Post Pre Post
45
50
55
60
65
70 Stress
Control
***
10 mg/kg 5 mg/kg
Preferencefor
cocaine-pairedside(%)
Pre Post
45
50
55
60
65
70
CRF2/!1 antagonists
PBS
5 mg/kg
Preferencefor
cocaine-pairedside(%)