2. Overview of the striatum
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.
dorsal
ventral

3. Neurotransmission and neuromodulation:
Adrenaline (ca )
Acetylcholine (ca )
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)

4. Endocannabinoid signaling in the striatum
Corticostriatal LTD
CB1 immunoreactivity
Gerdeman et al., Nat Neurosci (2002)
Egertova and Elphick, J Comp Neurology (2000)

5. Anatol Kreitzer
Sheela Singla
Brad Grueter
Percy Luu

6. Experimental Methods
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 mM) present to block GABAA-mediated currents.
Experiments performed at room temperature (RT) and °C

7. Tetanus induces endocannabinoid-mediated LTD
(eCB-LTD)
Time (min)

8. eCB-LTD requires mGluRs

9. Endocannabinoids are released by activation of postsynaptic mGluRs
DHPG (100 uM)

10. eCB-LTD requires D2 receptors
EPSC (% baseline)
sulpiride=D2 antagonist

11. Model of endocannabinoid release and LTD in the striatum
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)

12. The striatum is not homogeneous
(2 populations of medium spiny neurons)
dorsal
ventral

13. Basal ganglia motor circuit: direct pathway
Motor Cortex
Premotor
Cortex
Glutamate
Caudate/
Putamen
GPe
GPi/SNr
GABA
Dopamine +
Direct pathway MSNs express:
Dopamine D1 receptors, Muscarinic
M4 receptors, Dynorphin, Substance P
Motor
Thalamus
SNc
STN

14. Basal ganglia motor circuit: indirect pathway
Motor Cortex
Premotor
Cortex
Glutamate
Caudate/
Putamen
GPe
GPi/SNr
GABA
Dopamine
Indirect pathway MSNs express:
Dopamine D2 receptors, Enkephalin -
Motor
Thalamus
SNc
STN

15. Basal ganglia motor circuit:
Parkinson disease
Motor Cortex
Premotor
Cortex
+ Glutamate
Striatum
GPe
GPi/SNr
- GABA
Dopamine + -
Motor
Thalamus X
SNc
STN

16. Nucleus accumbens direct and indirect pathways?
NAc VP
VTA/Substantia Nigra
mPFC
BLA
Ventral
Subiculum D1 D2

17. M4- and D2-GFP BAC-transgenic mice label direct
and indirect pathway medium spiny neurons 1 2 3
1. CPu
2. GPe
3. SNr
M4-GFP
D2-GFP
GENSAT Program (N. Heintz, M. Hatten, W. Yang)

18. Direct and indirect pathway synapses display
different paired-pulse ratios

19. Indirect pathway synapses have a higher
release probability

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

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

22. Indirect pathway medium spiny neurons are more excitable

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

24. Endocannabinoid-mediated LTD occurs only at indirect pathway synapses
Time (min)

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

26. Indirect pathway eCB-LTD requires dopamine D2 receptor activation
AM251: cannabinoid CB1 receptor antagonist
sulpiride: dopamine D2 receptor antagonist

27. CB1 receptor agonists inhibit transmitter release at both direct and indirect pathway synapses
WIN55,212: cannabinoid CB1 receptor agonist
AM251: cannabinoid CB1 receptor antagonist

28. Postsynaptic mGluR-driven endocannabinoid release occurs primarily at indirect pathway synapses

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

30. Indirect pathway eCB-LTD is absent in an animal model of Parkinson disease
Time (min)

31. Endocannabinoid biosynthesis and inactivation
Calcium
Phosphatidylethanolamine (PE)
N-arachidonoyl PE
N-Acyltransferase
FAAH
Arachidonic acid
Ethanolamine +
MGL
glycerol
Phosphatidylinositol (PI)
Diacylglycerol (DAG)
Phospholipase Cb
Receptor activation
(mGluR or mAChR)
NAPE-PLD
Anandamide (AEA)
DAG Lipase
2-arachidonoylglycerol (2-AG)
(URB597)
(URB754)

32. Rescue of indirect pathway LTD in a Parkinson’s disease model (reserpine)
EPSC (% baseline)
quinpirole: dopamine D2 receptor agonist
URB754: monoacylglycerol lipase (MGL) inhibitor
URB597: FAAH inhibitor

33. Rescue of indirect pathway LTD in a Parkinson’s disease model (6-OHDA)
quinpirole: dopamine D2 receptor agonist
URB597: FAAH inhibitor

34. An important role for dopamine D2 receptors and cannabinoid CB1 receptors in movementwt
D2 -/-
D2 -/- mouse
catalepsy
Locomotor activity
Baik et al. (1995), Nature wt
D2-/-
CB1 -/- mouse
Zimmer et al. (1999), PNAS

35. Inhibition of eCB degradation enhances D2-receptor-mediated recovery from catalepsy in animal models of Parkinson disease

36. Inhibition of eCB degradation enhances D2-receptor-mediated recovery of locomotor activity in animal models of Parkinson disease

37. Basal ganglia motor circuit: Parkinson’s disease treatments
Motor Cortex
Premotor
Cortex
+ Glutamate
Caudate/
Putamen
GPe
eCB biosynthesis
GPi/SNr
- GABA
Dopamine +
(L-DOPA) -
Motor
Thalamus
Pallidotomy X
SNc
STN
STN Pacemaker

38. 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

39. Nathaniel Heintz (Rockefeller)
Anatol Kreitzer
Sheela Singla
Brad Grueter
Percy Luu
Nathaniel Heintz (Rockefeller)
X. William Yang (UCLA)

40. 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.
LTD
LTD?
cannabinoids

41. Testing the effect of synaptic activity on LTD
pathway 1: no
synaptic stimulation
pathway 2: 0.05 Hz-1 Hz
synaptic stimulation

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

43. WIN-induced LTD is activity-dependent
10 min WIN
20 min WIN

44. eCB-LTD is activity-dependent
path 1, path 2

45. Imbalanced activity in striatal motor circuits in vivo in a Parkinson disease model
(direct)
(indirect)
Mallet et al (2006) J Neurosci

46. Muscarinic M1 receptors do not regulate striatal endocannabinoid release or eCB-LTD
100 Hz tetanus
pirenzepine: muscarinic M1 receptor antagonist
sulpiride: dopamine D2 receptor antagonist

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

48. Indirect pathway synapses have larger NMDA/AMPA current ratios

49. Inhibition of eCB degradation enhances D2-receptor-mediated recovery from catalepsy in animal models of Parkinson disease

50. Inhibition of eCB degradation enhances D2-receptor-mediated recovery of locomotor activity in animal models of Parkinson disease

51. Signaling through CB1 receptors
THC (partial agonist)
CB1R
2-AG (endogenous agonist)
heterotrimeric G-protein
agonist
Anandamide (endogenous agonist)
Adapted from DiMarzo et al., Nature Reviews Drug Discovery (2004)

52. Endocannabinoid biosynthesis and inactivation
Phosphatidylethanolamine (PE)
Phosphatidylinositol (PI)
Receptor activation
(mGluR or mAChR)
N-Acyltransferase
Calcium
Phospholipase Cb
Calcium
N-arachidonoyl PE
Diacylglycerol (DAG)
DAG Lipase
2-arachidonoylglycerol (2-AG)
NAPE-PLD
Calcium
Anandamide (AEA)
FAAH
Arachidonic acid
Ethanolamine +
MGL
Arachidonic acid +
glycerol

53. Inhibition of endocannabinoid degradation enhances D2-receptor-mediated recovery of movement in a Parkinson disease model

54. D2 receptor activation enhances endocannabinoid release in response to brief mGluR activation
DHPG

55. heterotrimeric G-protein
Signaling through CB1 receptors
THC (partial agonist)
heterotrimeric G-protein
2-AG (endogenous agonist)
agonist
Anandamide (endogenous agonist)
Adapted from DiMarzo et al., Nature Reviews Drug Discovery (2004)

56. Endocannabinoid biosynthesis and inactivation
Calcium
Phosphatidylethanolamine (PE)
N-arachidonoyl PE
N-Acyltransferase
FAAH
Arachidonic acid
Ethanolamine +
MGL
glycerol
Phosphatidylinositol (PI)
Diacylglycerol (DAG)
Phospholipase Cb
Receptor activation
(mGluR or mAChR)
NAPE-PLD
Anandamide (AEA)
DAG Lipase
2-arachidonoylglycerol (2-AG)
(URB597)
(URB754)

57. CB1 receptor distribution in rat brain
(from Pettit et al., 1998)

58. Endocannabinoids are not released by medium spiny neuron depolarization
100 50
EPSC (% baseline)
-50
1 sec depol (n=4)
5 sec depol (n=3) A
10 ms
200 pA
( )
control
t=10 s D

59. mGluR-mediated endocannabinoid release requires L-type calcium channels
DHPG (100 uM)
EPSC (% baseline)

60. mGluR-mediated endocannabinoid release is enhanced by subthreshold depolarization
DHPG (100 uM)
EPSC (% baseline)
Time (min)

61. Medium spiny neurons: up and down states
Down state: -60 to –90 mV
calcium 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 Neurosci
Carter and Sabatini (2004), Neuron
Stern et al.(1998), Nature

62. Imbalanced activity in striatal motor circuits in vivo in a Parkinson disease model
(direct)
(indirect)
Mallet et al (2006) J Neurosci