Overview of the striatum

dorsal

ventral

•Dorsal striatum important for motor planning and adaptive behaviors. Main inputs arise from motor cortex.

•Disorders involving dorsal striatum include Parkinson’s disease and Huntington’s disease.

•Ventral striatum (nucleus accumbens) is important for reward. Main inputs arise from amygdala and prefrontal cortex.

•Drug addiction, depression are disorders involving the striatum.

Neurotransmission and neuromodulation:• Adrenaline (ca. 1900-) • Acetylcholine (ca. 1900-)• Neuropeptides (1930-)• Amino Acids (GABA and Glutamate) (1950-)• Monoamines (5-HT and Dopamine) (1950-)• Endogenous opioids (1975-) • Endogenous cannabinoids (1992-)

-CB1 receptor cloned (1990)-discovery of endogenous endocannabinoids (1992)-modulation of synaptic function (2001)

Egertova and Elphick, J Comp Neurology (2000)

CB1 immunoreactivity

Gerdeman et al., Nat Neurosci (2002)

Corticostriatal LTD

Endocannabinoid signaling in the striatum

Anatol KreitzerSheela Singla

Brad Grueter

Percy Luu

• Acute coronal brain slices – recordings performed from medium spiny neurons (MSNs) in dorsolateral striatum of 3- to 4-week-old rats and mice

• CsMeSO3 based-internal solution, 0.2 mM EGTA, QX-314 to block Na currents

• External saline: 2 mM Ca, 1 mM Mg. Picrotoxin (50 μM) present to block GABAA-mediated currents.

• Experiments performed at room temperature (RT) and 30-32 °C

Experimental Methods

100

50

0

EP

SC

(% b

asel

ine)

403020100

control (n=15) AM251 (n=6)

Tetanus induces endocannabinoid-mediated LTD(eCB-LTD)

Time (min)

100

50

0

EP

SC

(% b

asel

ine)

403020100Time (min)

LY341495 (n=5)

eCB-LTD requires mGluRs

100

50

0

EP

SC

(% b

asel

ine)

403020100Time (min)

hp:-50 (n=11) hp: -50, AM251 (n=5)

DHPG (100 uM)

Endocannabinoids are released by activation of postsynaptic mGluRs

sulpiride=D2 antagonist

eCB-LTD requires D2 receptors

100

50

0403020100

Time (min)

sulpiride (n=5)

EP

SC

(% b

asel

ine)

1. mGluR activation, combined with subthreshold depolarization, leads to endocannabinoid (eCB) synthesis and release

2. Dopamine D2 receptor activation enhances eCB synthesis, while D2 inhibition reduces eCB synthesis

3. Released eCBs bind to presynaptic CB1 receptors and induce presynaptic LTD

4. Neurotransmitter release is reduced for tens of minutes or more

(Kreitzer and Malenka, 2005)

Model of endocannabinoid release and LTD in the striatum

dorsal

ventral

The striatum is not homogeneous (2 populations of medium spiny neurons)

Caudate/Putamen

Motor Cortex

MotorThalamus

PremotorCortex

Glutamate

GABA

GPeGPi/SNr

STN

Dopamine

SNc

+

Direct pathway MSNs express:Dopamine D1 receptors, MuscarinicM4 receptors, Dynorphin, Substance P

Basal ganglia motor circuit: direct pathway

Caudate/Putamen

Motor Cortex

MotorThalamus

PremotorCortex

Glutamate

GABA

GPeGPi/SNr

STN

Dopamine

SNc

-

Indirect pathway MSNs express:Dopamine D2 receptors, Enkephalin

Basal ganglia motor circuit: indirect pathway

Basal ganglia motor circuit: Parkinson disease

Striatum

Motor Cortex

MotorThalamus

PremotorCortex

+ Glutamate

- GABA

GPeGPi/SNr

STN

Dopamine

SNc

+

-

X

NAc

VP

VTA/Substantia Nigra

mPFC

BLAVentral Subiculum

D1 D2

Nucleus accumbens direct and indirect pathways?

12 3

1. CPu2. GPe3. SNr

M4-GFP

D2-GFP

M4- and D2-GFP BAC-transgenic mice label direct and indirect pathway medium spiny neurons

GENSAT Program (N. Heintz, M. Hatten, W. Yang)

2.0

1.5

1.0A2/

A1

5002500ISI (ms)

2.0

1.5

1.0A2/

A1

5002500ISI (ms)

1.5

1.0A2/

A1

5002500ISI (ms)

direct (D2-)indirect (D2+)

1.5

1.0

A2/

A1

5002500ISI (ms)

direct (M4+)indirect (M4-)

D2+

200 pA50 ms

M4+

200 pA50 ms

Aa Ab

Ba Bb

Direct and indirect pathway synapses display different paired-pulse ratios

1.0

0.5

0.0

Cum

. Pro

b.

210Inter-event Interval (s)

1.0

0.5

0.0

Cum

. Pro

b.

500Amplitude (pA)

20

10

0

Mea

n A

mp

(pA

)

4

2

0

Mea

n Fr

eq (H

z)

A B C

20 pA0.2 s

Direct Indirect

D I D I

(4)

(5) (4) (5)

Indirect pathway synapses have a higher release probability

0 100 2000.5

1.0

1.5

PP

R

Interstim interval (msec)

D2(+) D2(-)

Direct and indirect pathway synapses display different paired-pulse ratios in nucleus accumbens

0

1

2

3

4

5

6

7

D2(-)D2(+)

*

Ave

rage

mE

PS

C F

requ

ency

(Hz)

0

5

10

15

20

25

D2(-)D2(+)Ave

rage

mE

PS

C A

mpl

itude

(pA

)

Indirect (D2+) pathway synapses have a higher release probability in nucleus accumbens?

40

20

0

Freq

(Hz)

4002000I (pA)

directindirect

2

1

0ISI (

norm

.)

100Spike #

directindirect

Aa Ab B

C

Direct Indirect

20 mV

100 ms

Indirect pathway medium spiny neurons are more excitable

0 100 200 300 4000

5

10

15

20

25

30

**

Freq

uenc

y (H

z)I (pA)

D2(+) D2(-)

Indirect (D2+) pathway medium spiny neurons are more excitable in nucleus accumbens

Direct Indirect

100

50

0

EPSC

(% b

asel

ine)

403020100

direct (D2 neg) (n=8)indirect (D2 pos) (n=6)

Endocannabinoid-mediated LTD occurs only at indirect pathway synapses

Time (min)

0 10 20 30 400

50

100

150

EP

SC

Am

plitu

de (%

Bas

elin

e)

Time (min)

D2(-)

0 10 20 30 400

50

100

150

EP

SC

Am

plitu

de (%

Bas

elin

e)

Time (min)

D2(+)

Endocannabinoid-mediated(?) LTD occurs only at indirect pathway synapses in nucleus accumbens

Indirect pathway eCB-LTD requires dopamine D2 receptor activation

100

50

0

EPSC

(% b

asel

ine)

40200Time (min)

AM251sulpiride5-HT, NE antagonists

AM251: cannabinoid CB1 receptor antagonistsulpiride: dopamine D2 receptor antagonist

CB1 receptor agonists inhibit transmitter release at both direct and indirect pathway

synapses

100

50

0

EP

SC

(% b

asel

ine)

40200Time (min)

WINAM251

directindirect

WIN55,212: cannabinoid CB1 receptor agonistAM251: cannabinoid CB1 receptor antagonist

Postsynaptic mGluR-driven endocannabinoid release occurs primarily at indirect pathway

synapses

100

50

0

EPSC

(% b

asel

ine)

40200Time (min)

DHPG

directindirect

0 10 20 30 40 50 600

50

100

150

100μM RS-DHPG

EP

SC

Am

plitu

de (%

Bas

elin

e)

Time (min)

D2(-)

0 10 20 30 40 50 600

50

100

150

100μM RS-DHPG (-50mV)

EP

SC

Am

plitu

de (%

Bas

elin

e)Time (min)

D2(+) (-50mV)

Postsynaptic mGluR-driven LTD occurs primarily at indirect pathway synapses in nucleus accumbens

Indirect pathway eCB-LTD is absent in an animal model of Parkinson disease

100

50

0

EP

SC

(% b

asel

ine)

40200

indirect (n=6)indirect, reserpine treated (n=7)

Time (min)

Calcium

Phosphatidylethanolamine (PE)

N-arachidonoyl PE

N-Acyltransferase

FAAH

Arachidonic acid Ethanolamine+

MGL

Arachidonic acid + glycerol

Calcium

Phosphatidylinositol (PI)

Diacylglycerol (DAG)

Phospholipase CβReceptor activation(mGluR or mAChR)

NAPE-PLD

Anandamide (AEA)

DAG Lipase

2-arachidonoylglycerol (2-AG)

Calcium

Endocannabinoid biosynthesis and inactivation

(URB754)(URB597)

quinpirole: dopamine D2 receptor agonistURB754: monoacylglycerol lipase (MGL) inhibitorURB597: FAAH inhibitor

Rescue of indirect pathway LTD in a Parkinson’s disease model (reserpine)

100

50

040200

Time (min)

reserpine-treated, quinpirole (n=5)reserpine-treated, URB754 (n=6)

EPSC

(% b

asel

ine) 100

50

0

EP

SC

(% b

asel

ine)

40200Time (min)

reserpine, URB597

quinpirole: dopamine D2 receptor agonistURB597: FAAH inhibitor

Rescue of indirect pathway LTD in a Parkinson’s disease model (6-OHDA)

150

100

50

0

EP

SC

(% b

asel

ine)

40200Time (min)

6-OHDA6-OHDA, quinpirole

100

50

0

EPS

C (%

bas

elin

e)

40200

Time (min)

6-OHDA, URB597

An important role for dopamine D2 receptors and cannabinoid CB1 receptors in movement

wt D2 -/- wt D2 -/-

D2 -/- mousecatalepsy Locomotor activity

Baik et al. (1995), Nature

CB1 -/- mouse

Zimmer et al. (1999), PNAS

D2-/-wt

Inhibition of eCB degradation enhances D2-receptor- mediated recovery from catalepsy in animal models

of Parkinson disease

Inhibition of eCB degradation enhances D2-receptor- mediated recovery of locomotor activity in animal

models of Parkinson disease

Caudate/Putamen

Motor Cortex

MotorThalamus

PremotorCortex

+ Glutamate

- GABA

GPeGPi/SNr

STN

Dopamine

SNc

+

-

X

(L-DOPA)

STN Pacemaker

Pallidotomy

eCB biosynthesis

Basal ganglia motor circuit: Parkinson’s disease treatments

Conclusions•

eCB-LTD in the striatum is restricted to indirect

pathway cells

•

it is synapse specific because it requires presynaptic activity in addition to CB1 receptor activation

•

eCB-LTD is prevented by dopamine depletion but can be rescued by a D2 agonist and an inhibitor of eCB

degradation

•

inhibition of eCB

degradation improves motor performace

in animals models of Parkinson

disease

•

D2(+) and D2(-) cells in the accumbens

also have different synaptic properties

Anatol KreitzerSheela Singla

Brad Grueter

Percy Luu

Nathaniel Heintz (Rockefeller)

X. William Yang (UCLA)

LTD

LTD?

cannabinoids

Is presynaptic eCB-LTD activity-dependent?

-Spillover onto synapses that did not participate in depolarizing the postsynaptic cell could cause LTD.

-Synapse specificity may be achieved if LTD depended on presynaptic activity and not just receptor activation.

pathway 1: no synaptic stimulation

pathway 2: 0.05 Hz-1 Hzsynaptic stimulation

Testing the effect of synaptic activity on LTD

100

50

0EPS

C (%

bas

elin

e)

40200

Time (min)

100

50

040200

Time (min)

A B

striatum

WINAM251 WIN AM251

cerebellum

A CB1 agonist is sufficient to induce LTD in the striatum, but not the cerebellum

10 min WIN 20 min WIN

WIN-induced LTD is activity-dependent

path 1, path 2

eCB-LTD is activity-dependent

Mallet et al (2006) J Neurosci

(direct) (indirect)

Imbalanced activity in striatal motor circuits in vivo in a Parkinson disease model

100

50

0

EP

SC

(% b

asel

ine)

3020100

Time (min)

pirenzepine

hp: -70 mV hp: -50 mV

100

50

040200

Time (min)

sulpiridesulpiride and pirenzepine

A B 100 Hz tetanus

pirenzepine: muscarinic M1 receptor antagonistsulpiride: dopamine D2 receptor antagonist

Muscarinic M1 receptors do not regulate striatal endocannabinoid release or eCB-LTD

1.5

1.0

PPR

5002500ISI (ms)

indirect (D2+)indirect (D2+), dopamine-depleted 100

50

0

EP

SC

(% b

asel

ine)

40200Time (min)

WINAM251

dopamine-depleted

Dopamine-depleted mice do not have altered paired-pulse plasticity or sensitivity to cannabinoids

0.6

0.4

0.2

0.0N

MD

A/A

MPA

ratio

A

100 pA

20 ms

direct

indirect

direct indirect

A

A1

2

3 *

Indirect pathway synapses have larger NMDA/AMPA current ratios

Inhibition of eCB degradation enhances D2-receptor- mediated recovery from catalepsy in animal models

of Parkinson disease

Inhibition of eCB degradation enhances D2-receptor- mediated recovery of locomotor activity in animal

models of Parkinson disease

Signaling through CB1 receptors

Adapted from DiMarzo et al., Nature Reviews Drug Discovery (2004)

heterotrimeric G-protein

CB1R

agonist

Anandamide (endogenous agonist)

2-AG (endogenous agonist)

THC (partial agonist)

Endocannabinoid biosynthesis and inactivation

Calcium

Phosphatidylethanolamine (PE)

N-arachidonoyl PE

N-Acyltransferase

FAAH

Arachidonic acid Ethanolamine+

MGL

Arachidonic acid + glycerol

Calcium

Phosphatidylinositol (PI)

Diacylglycerol (DAG)

Phospholipase CβReceptor activation(mGluR or mAChR)

NAPE-PLD

Anandamide (AEA)

DAG Lipase

2-arachidonoylglycerol (2-AG)

Calcium

Inhibition of endocannabinoid degradation enhances D2-receptor-mediated recovery of movement in a

Parkinson disease model

40

20

0Des

cent

Lat

ency

(s) 800

400

0Dis

tanc

e Tr

avel

ed (c

m)

A BCatalepsy Locomotor Activity

rese

rpin

e-tre

ated

+ qu

inpi

role

+ qu

inpi

role

/UR

B75

4*

+ U

RB7

54

rese

rpin

e-tre

ated

+ qu

inpi

role

+ qu

inpi

role

/UR

B75

4

+ U

RB7

54

120 *

D2 receptor activation enhances endocannabinoid release in response to brief

mGluR activation

100

50

0

EPS

C (%

bas

elin

e)

3020100Time (min)

controlquinpirole

DHPG

Signaling through CB1 receptors

agonistheterotrimeric G-protein

THC (partial agonist)

2-AG (endogenous agonist)

Anandamide (endogenous agonist)

Adapted from DiMarzo et al., Nature Reviews Drug Discovery (2004)

Calcium

Phosphatidylethanolamine (PE)

N-arachidonoyl PE

N-Acyltransferase

FAAH

Arachidonic acid Ethanolamine+

MGL

Arachidonic acid + glycerol

Calcium

Phosphatidylinositol (PI)

Diacylglycerol (DAG)

Phospholipase CβReceptor activation(mGluR or mAChR)

NAPE-PLD

Anandamide (AEA)

DAG Lipase

2-arachidonoylglycerol (2-AG)

Calcium

Endocannabinoid biosynthesis and inactivation

(URB754)(URB597)

CB1 receptor distribution in rat brain

(from Pettit et al., 1998)

100

50

0

EP

SC

(% b

asel

ine)

100500-50

1 sec depol (n=4)

5 sec depol (n=3)

A

10 ms

200 pA

( ) control

( ) control

t=10 sΔ

t=10 sΔ

Endocannabinoids are not released by medium spiny neuron depolarization

DHPG (100 uM)

mGluR-mediated endocannabinoid release requires L-type calcium channels

100

50

0403020100

Time (min)

hp:-50 (n=11) hp:-50, nitrendipine (n=8)

EPSC

(% b

asel

ine)

DHPG (100 uM)

mGluR-mediated endocannabinoid release is enhanced by subthreshold depolarization

100

50

0403020100

hp: -70 (n=9) hp: -50 (n=11)

Time (min)

EPSC

(% b

asel

ine)

Down state: -60 to –90 mVcalcium influx mediated by T- and R-type VSCCs

Up state: -40 to –70 mV,calcium influx mediated by L- and R-type VSCCs

Wilson and Kawaguchi (1996), J NeurosciCarter and Sabatini (2004), Neuron

Stern et al.(1998), Nature

Medium spiny neurons: up and down states

Mallet et al (2006) J Neurosci

(direct) (indirect)

Imbalanced activity in striatal motor circuits in vivo in a Parkinson disease model