Control of dopamine ascending pathways Control of dopamine ascending pathways
by central 5-HT system: by central 5-HT system:
implications for treatment of Parkinson’s implications for treatment of Parkinson’s
diseasedisease
Control of dopamine ascending pathways Control of dopamine ascending pathways
by central 5-HT system: by central 5-HT system:
implications for treatment of Parkinson’s implications for treatment of Parkinson’s
diseasedisease
Bordeaux 2 University – INSERM U862
Bordeaux - France
July, 7 -13, 2007, Catania, Italy
Umberto Spampinato
Summer School of Neuroscience (5)
5-HT/DA interaction: anatomical basis 5-HT/DA interaction: anatomical basis 5-HT/DA interaction: anatomical basis 5-HT/DA interaction: anatomical basis
DAVTA / SN DR
5-HTDA
Frontal cortex
n. Accumbens
striatumDA
DA
5-HT
5-HT5-HT3
Na+/K+ channel
5-HT1A
5-HT1BAMPc
5-HT4
5-HT6
5-HT7
AMPc
5-HT2A
5-HT2C
IP3/DAG
5-HT
innervates DA ascending pathways
DA neuron activity
highly represented
in DA regions
DAVTA / SN DR
5-HTDA
Frontal cortex
n. Accumbens
striatumDA
DA
5-HT
5-HT5-HT3
Na+/K+ channel
5-HT1A
5-HT1BAMPc
5-HT4
5-HT6
5-HT7
AMPc
5-HT2A
5-HT2C
IP3/DAG
5-HT
Schizophrenia
Depression
Drug Addiction
Parkinson’s disease
target for improved treatment of DA related diseases
5-HT/DA interaction and neuropsychiatric 5-HT/DA interaction and neuropsychiatric
disordersdisorders
5-HT/DA interaction and neuropsychiatric 5-HT/DA interaction and neuropsychiatric
disordersdisorders
5-HT5-HT systemsystem and Parkinson’s diseaseand Parkinson’s disease5-HT5-HT systemsystem and Parkinson’s diseaseand Parkinson’s disease
• Preclinical data (rat):
central 5-HT transmission Catalepsy
central 5-HT transmission (SSRI) Catalepsy
SSRI:
recent preclinical (MPTP monkey) and clinical studies:
pas Δ L-dopa-induced dyskinesia / park symptoms
• Clinical data:
Park symptoms dyskinesiacentral 5-HT transmission (SSRI)
DAVTA / SN DR
5-HTDA
Frontal cortex
n. Accumbens
striatumDA
DA
5-HT
5-HT5-HT3
Na+/K+ channel
5-HT1A
5-HT1BAMPc
5-HT4
5-HT6
5-HT7
AMPc
5-HT2A
5-HT2C
IP3/DAG
5-HT
Schizophrenia
Depression
Drug Addiction
Parkinson’s disease
5-HT/DA interaction and neuropsychiatric 5-HT/DA interaction and neuropsychiatric
disordersdisorders
5-HT/DA interaction and neuropsychiatric 5-HT/DA interaction and neuropsychiatric
disordersdisorders
selective action on 5-HT receptor subtypes
MPTP monkey
5-HT5-HT1 1 receptorsreceptors and Parkinson’s diseaseand Parkinson’s disease5-HT5-HT1 1 receptorsreceptors and Parkinson’s diseaseand Parkinson’s disease
• 5-HT1AR agonists
LID
Park patients
8-OH-DPAT
sarizotan (low doses)
↑ duration of L-dopa action
• 5-HT1BR agonists
MDMA (ecstasy): 5-HT1A/ 1B stim ( )
buspirone
5-HT5-HT2 2 receptors and Parkinson’s disease receptors and Parkinson’s disease 5-HT5-HT2 2 receptors and Parkinson’s disease receptors and Parkinson’s disease
• 5-HT2C antagonism is effective in animal models of Parkinson’s disease
• 5-HT2A/2C antagonism catalepsy
anti-parkinsonian action DA agonists
L-dopa induced dyskinesia (MPTP primates)
• 5-HT2C receptor binding is increased in the SN ret of parkinsonian patients
- atypical antipsychotic (clozapine)
- antidepressant mirtazapine
L-dopa-induced psychosis, LID / tremor
tremor - LID
• 5-HT2A/2C antagonism may participate to the therapeutic benefit of:
mechanisms ?
5-HT5-HT2C2C receptor receptor5-HT5-HT2C2C receptor receptor
DAVTA / SN DR
5-HTDA
Frontal cortex
n. Accumbens
striatumDA
DA
5-HT
5-HT5-HT3
Na+/K+ channel
5-HT1A
5-HT1BAMPc
5-HT4
5-HT6
5-HT7
AMPc
5-HT2A
5-HT2C
IP3/DAG
5-HT2C
key factor for the 5-HT-DA interaction
Schizophrenia
Depression
Drug addiction
Parkinson’s disease
5-HT5-HT2C2C receptor: cellular location receptor: cellular location5-HT5-HT2C2C receptor: cellular location receptor: cellular location
• non-DA neurons (GABA)Eberle-Wang et al., 1997
SN
GAD + 5-HT2CR mRNA positive
GAD mRNA positive
double-labeling in situ hybridization
• VTA-DA neuron subpopulationBubar & Cunningham, 2007
TH TH + 5-HT2CR5-HT2CR
indirect and direct control
of DA neuron activity
VTAVTA
5-HT5-HT2C2C
DADA
5-HT5-HT2C2C
5-HT2C receptor control
of DA pathways:
controlling NAc DA release
DA transmission
composite responses involving different populations
of 5-HT2C receptors in multiple brain nuclei
and/or DA transmission
5-HT5-HT2C2C Rs control DA release and/or transmission Rs control DA release and/or transmission5-HT5-HT2C2C Rs control DA release and/or transmission Rs control DA release and/or transmission
DA releaseDA release
Fr Cortex / NAc Fr Cortex / NAc electrophysiological
biochemical
behavioral
studies
5-HT5-HT2C2C receptor in the basal ganglia receptor in the basal ganglia5-HT5-HT2C2C receptor in the basal ganglia receptor in the basal ganglia
highly represented
Cortex
GPe
NSTGPi
SNr
NPP
VL
Striatum/NAc
SNc/VTA
DA
RD/RM
5-HTAlexander et Crutcher, 1990; Obeso et coll., 2001
key role in controlling basal ganglia output
5-HT2C
5-HT5-HT2C 2C receptor: molecular and functional receptor: molecular and functional propertiesproperties
5-HT5-HT2C 2C receptor: molecular and functional receptor: molecular and functional propertiesproperties
• Molecular property
COOHTM1TM2TM3TM4TM5TM6TM7
NH2
constitutive activity
G-protein coupled receptor family
activation of intracellular signaling in
the absence of agonist stimulation
- log[drug], M
IP3 a
cc
um
ula
tio
n
(% b
asa
l)
150
0
- 50
100
10 -11 10 -9 10 -7 10 -5
5-HT - 5HT2C agonist
5HT2C inverse agonist
inverse agonist
↓ constitutive activity
pre-mRNA editing variety of receptor isoforms
with different G protein coupling
COOHTM1TM2TM3TM4TM5TM6TM7
NH2
Tecott et al., 1995
• Functional property
“ 5-HT2C receptor mediates tonic inhibition
of neuronal network excitability ”
fine-tuning of DA neuron activity
G-protein coupled receptor family
5-HT5-HT2C 2C receptor: molecular and functional receptor: molecular and functional propertiesproperties
5-HT5-HT2C 2C receptor: molecular and functional receptor: molecular and functional propertiesproperties
AimsAimsAimsAims
Role of central 5-HT2C receptors in the control of
the mesoaccumbens and the nigrostriatal DA pathways
• DA effects of antipsychotic drugs Haloperidol – Clozapine
• Basal DA neuron activity
role of the 5-HT2CR constitutive activity
agonists versus inverse agonists effects
• Mesoaccumbens DA pathway & VTA / NAc 5-HT2C Rs
monitoring NAc DA release by in vivo microdialysis
ATV
SNc
StriatumStriatumNAcNAc
microdialysis probes
time (min)
Dialysates (30 µl)
HPLC-ECDPerfusion Pump(flow rate: 2 µl/min-1)
Experimental procedure : intracerebral microdialysisExperimental procedure : intracerebral microdialysisExperimental procedure : intracerebral microdialysisExperimental procedure : intracerebral microdialysis
Striatum
1 mm
NAc
-30 0 30 60 90 120 150 180
100
150
200
Time (min)
Representation of the resultsRepresentation of the results
Experimental procedureExperimental procedureExperimental procedureExperimental procedure
DA
(%
of
bas
eli
ne
± s
.e.m
.)
I.I. 5-HT 5-HT2C2C receptors and basal DA receptors and basal DA releaserelease
I.I. 5-HT 5-HT2C2C receptors and basal DA receptors and basal DA releaserelease
Mesulergine, a non selective 5-HT2C antagonist
Tonic and inhibitory control on DA neuron activity
Cumulative i.v. doses of mesulergine (MES) on basal DA neuron firing rate in the VTA
Prisco et al., 1994 Time (min)
-30 0 30 60 90
DA
(%
of
bas
elin
e)
100
120
140
160
180
200
0.5 mg/kg0.2 mg/kg0.1 mg/kgcontrols
Dose-response of mesulergine (0.1/0.2/0.5 mg/kg) on DA release at terminals (NAC/Striatum)
Spampinato et al., 1997
SNc
Striatum
VTA
NAC
selective
5-HT2C agents
Tonic and inhibitory control
I. 5-HTI. 5-HT2C2C receptors and basal DA release receptors and basal DA release I. 5-HTI. 5-HT2C2C receptors and basal DA release receptors and basal DA release
SB 206553 5-HT2C/2B antagonist
SB 242084 5-HT2C antagonist
SB 243213 5-HT2C antagonist
Ro 60-0175 5-HT2C agonist
SNc
Striatum
VTA
NAC
5-HT2C agents
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
Tonic and inhibitory control
preferential control
of the
mesoaccumbens DA pathway ?
Sensitivity / magnitude
n. accumbensstriatum
Willins & Meltzer, 1998
Di Giovanni et al., 2000
Di Giovanni et al., 2000
Di Matteo et al., 1999
Gobert et al., 2000
-10 -20 -30 -40-30 -20-40 -10 0
5-HT2C agonists
0
n.d.
mCPP
MK-212
Ro60-0175
1
1
5
1
2.5
+60 +40+80 +20 0
5-HT2C antagonists
n.d.
0 +20 +40 +60 +80
SB 206553
SB 242084
n.d.
n.d.
1
2.5
10
5
2.5
10
5
Porras et al., 2002
Di Matteo et al., 1998
Di Matteo et al., 1998
Di Matteo et al., 1999
Di Matteo et al., 1999
DA release (%)
Gobert et al., 2000
Di Giovanni et al., 2000
10 Gobert et al., 2000
mg/kg
mg/kg
Effect of 5-HT2C agents on DA release assessed by in vivo microdialysis
Do 5-HT2C receptors exert a preferential
control of the mesoaccumbens DA pathway ?
SNc
Striatum
VTA
NAC
Dose-response studies
Simultaneous monitoring of DA release in both regions
5-HT2C agents
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
SB 206553 : 5-HTSB 206553 : 5-HT2B/2C2B/2C antagonist antagonist
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
SB 206553 : 5-HTSB 206553 : 5-HT2B/2C2B/2C antagonist antagonist
Tonic inhibitory control on DA release in both the striatum and NAC
**p<0.01, ***p<0.001 Fisher’s test
Striatum NAC
DA
(%
of
bas
elin
e)
Time (min)
-30 0 30 60 90 120 150 180
100
150
200
Veh.
-30 0 30 60 90 120 150 180
100
150
2001 mg/kg **5 mg/kg ***10 mg/kg ***
Veh. 1 mg/kg 5 mg/kg ***10 mg/kg ***
5-HT5-HT2B2B receptors and basal DA release receptors and basal DA release5-HT5-HT2B2B receptors and basal DA release receptors and basal DA release
5-HT5-HT2B2B antagonist antagonist
SB 204741SB 204741
NA
c D
A (
% o
f b
as
eli
ne
)
Vehicle
BW 723C86, 1 mg/kg, ip
Di Matteo et al., 2000
Vehicle
SB 204701, 10 mg/kg, sc
NA
c D
A (
% o
f c
on
tro
l)
Gobert et al., 2000
5-HT5-HT2B2B agonist agonist
BW 723C86BW 723C86
Time (min)
5-HT2B receptors: no influence on basal DA release
effect of SB 206553 → R 5-HT2C
NAC = striatum
-30 0 30 60 90 120 150
100
125
150veh.
1 mg/kg
3 mg/kg
10 mg/kg
Time (min)
-30 0 30 60 90 120 150
DA
(%
of
bas
elin
e)
100
125
150veh.
1 mg/kg
3 mg/kg
10 mg/kg
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
SB 242084 : 5-HTSB 242084 : 5-HT2C2C antagonist antagonist
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
SB 242084 : 5-HTSB 242084 : 5-HT2C2C antagonist antagonist
Striatum NAC
**
**** **
Tonic inhibitory control on DA release in both the striatum and NACTonic inhibitory control on DA release in both the striatum and NAC
*p<0.05, **p<0.01 Fisher’s test
Striatum NAC
*p<0.05, **p<0.01, ***p<0.001 PLSD test
Time (min)
DA
(%
of
ba
se
lin
e)
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
SB 243213 : 5-HTSB 243213 : 5-HT2C2C antagonist antagonist
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
SB 243213 : 5-HTSB 243213 : 5-HT2C2C antagonist antagonist
Tonic inhibitory control on DA release in both the striatum and NACTonic inhibitory control on DA release in both the striatum and NAC
-30 0 30 60 90 120 150
100
120
140
160 veh.
SB 243213 (1)
SB 243213 (3)
SB 243213 (10)
***
***
-30 0 30 60 90 120 150
100
120
140
160 veh.
SB 243213 (1)
SB 243213 (3)
SB 243213 (10)
*********
60
80
100
120
vehicle 0.3 1 3 mg/kgvehicle 0.3 1 3 mg/kg
60
80
100
120
***
DA
(%
of
bas
elin
e)
-30 0 30 60 90 120 150 180
60
80
100
120
140
NAC
-30 0 30 60 90 120 150 180
60
80
100
120
140
striatum
Time (min)
Ro60-0175 (3 mg/kg) vehicle
Time (min)
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
Ro 60-0175: 5-HTRo 60-0175: 5-HT2C2C agonist agonist
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
Ro 60-0175: 5-HTRo 60-0175: 5-HT2C2C agonist agonist
Phasic inhibitory control on DA release in both the striatum and NAC
SNc
Striatum
VTA
NAC
Frontal Cortex
5-HT2C receptors
Similar in both the striatum and the NAC
- inhibitory control
- tonic and phasic
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
ConclusionsConclusions
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
ConclusionsConclusions
DA release
TM
1TM
2TM
3TM
4TM
5TM
6TM
7
DA
tonic inhibitory control
Constitutive activity of 5-HTConstitutive activity of 5-HT2C 2C receptorreceptor
and basal DA releaseand basal DA release
Constitutive activity of 5-HTConstitutive activity of 5-HT2C 2C receptorreceptor
and basal DA releaseand basal DA release
« Activation of 5-HT2C receptors inhibits … DA release in the NAc in a manner
which is independent of the inhibition of 5-HT neurons »Willins and Meltzer, Brain Res, 1998
5-HT2C
5-HT
DR
5-HT
5-HT
5-HT
5-HT
endogenous 5-HT tonic inhibitory control ?
SNc
Striatum
VTA
NAC
5-HT2CR antagonists
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
DA
tonic inhibitory control
magnitude of effect: SB 206553 >> SB 242084, SB 243213
in vitro studies:
SB 206553
SB 242084
SB 243213
different
pharmacological
properties
0
25
50
75
100
125
150
10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5
0
25
50
75
100
125
150
0
25
50
75
100
125
150
10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -510 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5
SB 206553
SB 242084
SB 206553 + SB 242084(100 nM)
IP A
ccu
mu
lati
on
(% b
asal
)
Effect of 5-HTEffect of 5-HT2C2C antagonists on IP antagonists on IP
accumulation in CHO cells expressing the 5-accumulation in CHO cells expressing the 5-
HTHT2C 2C receptor receptor
Effect of 5-HTEffect of 5-HT2C2C antagonists on IP antagonists on IP
accumulation in CHO cells expressing the 5-accumulation in CHO cells expressing the 5-
HTHT2C 2C receptor receptor
SB 206553: 5-HT2C inverse agonist
SB 242084: 5-HT2C weak efficacy agonist
-log [DRUG] M
In vitro:
-100
-80
-60
-40
-20
0
20
SB 206553clozapine30 nM
300 nM1 uM
IP A
ccu
mu
lati
on
% b
asal
act
ivit
y
SB 243213
SB 243213: 5-HT2C antagonist
SB 206553: SB 206553: 5-HT 5-HT2C2C inverse agonist inverse agonistIn vitro:
Effect of 5-HTEffect of 5-HT2C2C antagonists on IP antagonists on IP
accumulation in CHO cells expressing the 5-accumulation in CHO cells expressing the 5-
HTHT2C 2C receptor receptor
Effect of 5-HTEffect of 5-HT2C2C antagonists on IP antagonists on IP
accumulation in CHO cells expressing the 5-accumulation in CHO cells expressing the 5-
HTHT2C 2C receptor receptor
agonist and inverse agonist:
- opposite effects- opposite effects
- both blocked by antagonists- both blocked by antagonists
DA release
SB 206553SB 206553
inverse agonist inverse agonist in vivo ?in vivo ?
SB 206553SB 206553
inverse agonist inverse agonist in vivo ?in vivo ?
SB 242084 SB 242084 vsvs Ro60-0175 Ro60-0175SB 242084 SB 242084 vsvs Ro60-0175 Ro60-0175
SB 242084 (1 mg/kg, ip.) 30 min before Ro 60-0175 (3 mg/kg, ip.)
* p<0.05, ** p<0.01, ***p<0.001 PLSD test
DA
( %
of
bas
elin
e )
Vehicle+
Vehicle
Vehicle+
Ro60-0175
SB242084+
Vehicle
SB242084+
Ro60-0175
Vehicle+
Vehicle
Vehicle+
Ro60-0175
SB242084+
Vehicle
SB242084+
Ro60-0175
70
80
90
100
110
120
130 Striatum NAC
*****
* *
SB 242084 inhibits the effect of the 5-HT2CR agonist Ro 60-0175
70
80
90
100
110
120
130
SB 243213 (1 mg/kg, ip.) 30 min before Ro 60-0175 (3 mg/kg, ip.)
DA
( %
bas
elin
e ) Striatum NAC
Vehicle+
Vehicle
Vehicle+
Ro60-0175
SB243213+
Vehicle
SB243213+
Ro60-0175
Vehicle+
Vehicle
Vehicle+
Ro60-0175
SB243213+
Vehicle
SB2432123+
Ro60-0175
******
SB 243213 SB 243213 vsvs Ro60-0175 Ro60-0175SB 243213 SB 243213 vsvs Ro60-0175 Ro60-0175
* **
* p<0.05, ** p<0.01, ***p<0.001 PLSD test
SB 243213 inhibits the effect of the 5-HT2CR agonist Ro 60-0175
SB 242084 (1 mg/kg, ip.) 30 min before SB 206553 (5 mg/kg, ip.)
******
**
+ +
DA
( %
of
bas
elin
e )
Striatum NAC
100
125
150
Vehicle+
Vehicle
Vehicle+
SB206553
SB242084+
Vehicle
SB242084+
SB206553
Vehicle+
Vehicle
Vehicle+
SB206553
SB242084+
Vehicle
SB242084+
SB206553
•p<0.05, *** p<0.001 vs controls; + p<0.05 vs « SB 206553 »
SB 242084 SB 242084 vs SB 206553vs SB 206553SB 242084 SB 242084 vs SB 206553vs SB 206553
SB 242084 inhibits the excitatory effect of SB 206553
SB 206553 acts as a 5-HT2C inverse agonist in vivo
SB 243213 (1 mg/kg, ip.) 60 min before SB 206553 (5 mg/kg, ip.)
SB 243213 SB 243213 vs SB 206553vs SB 206553SB 243213 SB 243213 vs SB 206553vs SB 206553D
A (
% b
asel
ine
)
Vehicle+
Vehicle
Vehicle+
SB206553
SB243213+
Vehicle
SB243213+
SB206553
Vehicle+
Vehicle
Vehicle+
SB206553
SB243213+
Vehicle
SB243213+
SB206553
***
**
Striatum NAC
100
125
150 ***
**
** p<0.01, ***p<0.001 PLSD test
SB 243213 inhibits the excitatory effect of SB 206553
SB 206553 acts as a 5-HT2C inverse agonist in vivo
-30 0 30 60 90 120 150
100
125
150
175
veh.
8-OH-DPAT
SB 206553
PAT/SB 206553
Time (min)
-30 0 30 60 90 120 150
DA
(%
of
bas
elin
e)
100
125
150
175Striatum NAC
8-OH-DPAT (0.1 mg/kg, sc.) 5 min before SB 206553 (5 mgkg, ip.)
SB 206553-induced DA release is SB 206553-induced DA release is unaffected by
the reduction of 5-HT extracellular levels by 8-OH-
DPAT
SB 206553-induced DA release is SB 206553-induced DA release is unaffected by
the reduction of 5-HT extracellular levels by 8-OH-
DPAT
The effect of SB 206553 occurs independently of endogenous 5-HT
SB 206553-induced DA release is SB 206553-induced DA release is unaffected by
the lesion of 5-HT neuronsthe lesion of 5-HT neurons
SB 206553-induced DA release is SB 206553-induced DA release is unaffected by
the lesion of 5-HT neuronsthe lesion of 5-HT neurons
SB 206553 (5 mg/kg, ip.)
NAC
-30 0 30 60 90 120 150
100
125
150
175Striatum
Time (min)
-30 0 30 60 90 120 150
DA
(%
of
bas
elin
e)
100
125
150
175
Sham-lesioned
5,7-DHT5,7-DHT
The effect of SB 206553 occurs independently of endogenous 5-HT
TM
1TM
2TM
3TM
4TM
5TM
6TM
7
DA
tonic inhibitory control
Constitutive activity of 5-HTConstitutive activity of 5-HT2C2C receptor and basal DA receptor and basal DA
releaserelease
ConclusionsConclusions
Constitutive activity of 5-HTConstitutive activity of 5-HT2C2C receptor and basal DA receptor and basal DA
releaserelease
ConclusionsConclusions5-HT2C
5-HT
DR
5-HT
5-HT
5-HT
5-HT
Constitutive activity of 5-HT2C receptors
participates in the tonic inhibitory control of
nigrostriatal and mesoaccumbens DA neuron activity
in vivo SB 206553: 5-HT2CR inverse agonist in vivo
II. 5-HTII. 5-HT2C 2C receptors and antipsychotic receptors and antipsychotic drugsdrugs
II. 5-HTII. 5-HT2C 2C receptors and antipsychotic receptors and antipsychotic drugsdrugs
basal stimulated
constitutive activity ?
• haloperidol
• clozapine
( - )( - )
5-HT2C receptors
DA release
Haloperidol 0.01 mg/kg Haloperidol 0.01 mg/kg Haloperidol 0.01 mg/kg Haloperidol 0.01 mg/kg
*****
SB 206553 (5 mg/kg, ip.)
-30 0 30 60 90 120
100
125
150
175
200
225
250veh.
SB 242084
haloperidol
SB 242084 haloperidol
****
SB 242084 (1 mg/kg, ip.)
Striatum
+ +
+
Involvement of constitutive activity of 5-HT2C receptors
inverse agonist ≠ antagonist
veh.
SB 206553
haloperidol
SB 206553 haloperidol
-30 0 30 60 90 120
DA
(%
of
bas
elin
e)
100
125
150
175
200
225
250
*p<0.05, **p<0.01, ***p<0.001 vs controls; +++p<0.001 vs haloperidol group PLSD test
Time (min)
SB 206553 (5 mg/kg, ip.) SB 242084 (1 mg/kg, ip.)
• importance of inverse agonism in vivo
N. Accumbens
-30 0 30 60 90 120
100
125
150
175
200
225
250
veh.
SB 206553
haloperidol
SB 206553 haloperidol
DA
(%
of
bas
elin
e)
-30 0 30 60 90 120
100
125
150
175
200
225
250veh.
SB 242084
haloperidol
SB 242084 haloperidol
******
****
+ +
+
Haloperidol 0.01 mg/kg Haloperidol 0.01 mg/kg Haloperidol 0.01 mg/kg Haloperidol 0.01 mg/kg
Involvement of constitutive activity of 5-HT2C receptors
**p<0.01, ***p<0.001 vs controls; +++p<0.001 vs haloperidol group PLSD test
Time (min)
NACStriatum
Haloperidol (0.01 mg/kg, sc.) 30 minutes after SB 243213 (1 mg/kg, ip.)
-60 -30 0 30 60 90 120
100
125
150
175
200
225
250veh.
SB 243213
haloperidol
SB 243213 haloperidol
DA
(%
of
bas
elin
e)
***
-60 -30 0 30 60 90 120
100
125
150
175
200
225
250
***
***p<0.001 PLSD test
Haloperidol and SB 243213 Haloperidol and SB 243213 Haloperidol and SB 243213 Haloperidol and SB 243213
blockade of 5-HT2C receptors : no effect on haloperidol-induced DA release
Δ haloperidol effect : related to the constitutive activity of 5-HT2C receptors
Time (min)
nM range inverse agonist
5-HT2C
5-HT5-HT2C 2C receptors and antipsychotic drugsreceptors and antipsychotic drugs 5-HT5-HT2C 2C receptors and antipsychotic drugsreceptors and antipsychotic drugs
in vitro
in vivo ?
Striatum NAC
-30 0 30 60 90 120 150 180
100
125
150
175
veh.
0.3 mg/kg
1 mg/kg3 mg/kg
Time (min)
-30 0 30 60 90 120 150 180
DA
(%
of
ba
se
line
)
100
125
150
175
***
****
***
****
*p<0.05, ***p<0.001 PLSD test
Clozapine increases DA release in both the striatum and NAC
Clozapine and DA release Clozapine and DA release in vivoin vivoClozapine and DA release Clozapine and DA release in vivoin vivo
75
100
125
150
175
Clozapine and 5-HTClozapine and 5-HT2C 2C receptors receptors Clozapine and 5-HTClozapine and 5-HT2C 2C receptors receptors
Striatum NAC
***
*** ***
Ro 60-0175 (3 mg/kg, ip.) 15 min before clozapine (1 mg/kg, sc.)
**p<0.01, ***p<0.001 PLSD test
**
DA
( %
of
bas
elin
e )
Vehicle+
Vehicle
Ro60-0175+
vehicle
vehicle+
CLOZ
Ro60-0175 +
CLOZ
Vehicle+
Vehicle
Ro60-0175+
vehicle
vehicle+
CLOZ
Ro60-0175 +
CLOZ
Clozapine occupies 5-HT2C receptors in vivo
CLOZAPINE(1 mg/kg)
Clozapine: 5-HTClozapine: 5-HT2C2C inverse agonist inverse agonist in vivoin vivo ? ?Clozapine: 5-HTClozapine: 5-HT2C2C inverse agonist inverse agonist in vivoin vivo ? ?
SIGNIFICANT INTERACTION
5-HT2C antagonist or
inverse agonist properties ?
5-HT2C inverse
agonist?
Clozapine does not act as a 5-HT2C antagonist
Ro 60-0175 (3)
**
***
Striatum
75
100
125
150
175
Ro CloV Ro / Clo
SB CloV SB / Clo
NON SIGNIFICANT IN
TERACTION
Striatum / N
AC
SB 206553 (5)
100
125
150
175
***
**
SB 242084
SIG
NIF
ICA
NT
IN
TE
RA
CT
ION
?
Clozapine: 5-HTClozapine: 5-HT2C2C inverse agonist inverse agonist in vivoin vivo ? ?Clozapine: 5-HTClozapine: 5-HT2C2C inverse agonist inverse agonist in vivoin vivo ? ?
NACStriatum
SB 242084 (0.3 mg/kg, ip.) 15 min before clozapine (1 mg/kg, sc.)
SB 242084 inhibits the effect of clozapine
veh.
SB 242084
clozapine
SB 242084 + clozapine
-30 0 30 60 90 120
100
120
140
160
***
++
-30 0 30 60 90 120
100
120
140
160
+
***
DA
( %
of
bas
elin
e )
Time (min)
***p<0.001 vs controls; +p<0.05, ++p<0.01 vs clozapine group PLSD test
SNc
Striatum
ATV
NAC
DA
DAergic effects
(low doses)
5-HT2C
DA
inverse agonist property in vivo
DADA
NAc NAc
DADA
VTAVTA
5-HT5-HT2C2C
5-HT5-HT2C2C
DA release
Relative contribution of VTA and NAc
5-HT2C receptors
in the control of NAc DA release
III. VTA and NAc 5-HTIII. VTA and NAc 5-HT2C2C receptors receptorsIII. VTA and NAc 5-HTIII. VTA and NAc 5-HT2C2C receptors receptors
Phasic inhibitory control: 5-HT2C R agonist Ro 60-0175Ro 60-0175
Tonic inhibitory control: 5-HT2C R inverse agonist SB 206553SB 206553
DADA
NAc NAc
DADA
VTAVTA
5-HT5-HT2C2C
5-HT5-HT2C2C
microdialysis probe
DA
shell
1 - Ro 60-01751 - Ro 60-0175 : 3 mg/kg, ip.
2 - SB 2065532 - SB 206553 : 5 mg/kg, ip.: 5 mg/kg, ip.
reverse dialysis (0.1- 1 µM)
SB 242084
SB 242084
(0.1- 0.5 µg / 0.2 µl)
SB 243213
• 5-HT5-HT2C2C antagonists antagonists
VTA and NAc 5-HTVTA and NAc 5-HT2C2C receptors receptorsVTA and NAc 5-HTVTA and NAc 5-HT2C2C receptors receptors
experimental procedure
Injection canula
1 - Ro 60-0175-induced DA release 1 - Ro 60-0175-induced DA release
Intra-VTAIntra-VTA injection of 5-HT injection of 5-HT2C2C antagonists antagonists
1 - Ro 60-0175-induced DA release 1 - Ro 60-0175-induced DA release
Intra-VTAIntra-VTA injection of 5-HT injection of 5-HT2C2C antagonists antagonists
SB 242084SB 242084
Time ( min )
SB 242084SB 242084 prevents the inhibitory effect of Ro 60-0175
***p<0.001 vs veh, ++p<0.01, +++p<0.001
vs Ro 60-0175 , PLSD test
0.5µg/0.2µl
-30 0 30 60 90 12060
80
100
120
++
+***
veh / veh
SB 242084 / veh
veh / Ro 60-0175
SB 242084 / Ro 60-0175
NA
c D
A
(% o
f b
asel
ine)
0.1µg/0.2µl
***+
+60
80
100
120
-30 0 30 60 90 120
veh / veh
SB 243213 / veh
veh / Ro 60-0175
SB 243213 / Ro 60-0175
-60 -30 0 30 60 90 120
***+
++
NA
c D
A
(% o
f b
asel
ine)
60
80
100
120
0.1µg/0.2µl
Time ( min )
1 - Ro 60-0175-induced DA release 1 - Ro 60-0175-induced DA release
Intra-VTAIntra-VTA injection of 5-HT injection of 5-HT2C2C antagonists antagonists
1 - Ro 60-0175-induced DA release 1 - Ro 60-0175-induced DA release
Intra-VTAIntra-VTA injection of 5-HT injection of 5-HT2C2C antagonists antagonists
SB 243213
***p<0.001 vs controls; +++p<0.001 vs RO 60-0175, PLSD test
-60 -30 0 30 60 90 120
++
+***
60
80
100
120
0.5µg/0.2µl
VTA 5-HT2C Rs participate in the
phasic inhibitory control of DA release
SB 243213 prevents the inhibitory effect of Ro 60-0175
2 - SB 206553-induced DA release 2 - SB 206553-induced DA release
Intra-VTAIntra-VTA injection of 5-HT injection of 5-HT2C2C antagonists antagonists
2 - SB 206553-induced DA release 2 - SB 206553-induced DA release
Intra-VTAIntra-VTA injection of 5-HT injection of 5-HT2C2C antagonists antagonists
SB 242084SB 242084
NA
c D
A (
% o
f b
asel
ine)
time (min)
-30 0 30 60 90 12080
100
120
140
160
180
0.1 µg/0,2µl
***
v eh / veh
SB 242084 / veh
veh / SB 206553
SB 242084 / SB 206553
***p<0.001 vs veh PLSD test
-30 0 30 60 90 120
0.5 µg/0,2µl
***
80
100
120
140
160
180
SB 242084SB 242084 has no influence on SB 206553-induced DA release
VTA 5-HT2C Rs do not participate in the tonic inhibition
of DA release related to the CA of 5-HT2C Rs
VTAVTA
DADANAc NAc
5-HT5-HT2C2C
(-)DADA
5-HT5-HT2C2C
DA release
VTA and NAc 5-HTVTA and NAc 5-HT2C2C receptors receptorsVTA and NAc 5-HTVTA and NAc 5-HT2C2C receptors receptors
Nucleus Accumbens
5-HT2C receptors
SB 242084SB 242084
1 - Ro 60-0175-induced DA release 1 - Ro 60-0175-induced DA release
Intra-NAcIntra-NAc perfusion of 5-HT perfusion of 5-HT2C2C antagonist antagonist
1 - Ro 60-0175-induced DA release 1 - Ro 60-0175-induced DA release
Intra-NAcIntra-NAc perfusion of 5-HT perfusion of 5-HT2C2C antagonist antagonist
SB 242084SB 242084 prevents the inhibitory effect of Ro 60-0175
NAc 5-HT2C Rs participate in the phasic inhibitory
control of DA release
**p<0.01, ***p<0.001 vs veh; ++p<0.01; +++p<0.001 vs RO 60-0175 group PLSD test
Time (min)
NA
c D
A (
% o
f b
asel
ine)
-30 0 30 60 90 12060
80
100
120
**+
+
0.1 µM
-30 0 30 60 90 12060
80
100
120
***+
++
1 µM
veh / veh
SB 242084 / veh
veh / Ro 60-0175
SB 242084 / Ro 60-0175
2 - SB 206553-induced DA release 2 - SB 206553-induced DA release
Intra-NAcIntra-NAc perfusion of 5-HT perfusion of 5-HT2C2C antagonists antagonists
2 - SB 206553-induced DA release 2 - SB 206553-induced DA release
Intra-NAcIntra-NAc perfusion of 5-HT perfusion of 5-HT2C2C antagonists antagonists
SB 242084SB 242084 v eh / veh
SB 242084 / veh
veh / SB 206553
SB 242084 / SB 206553
time (min)
-30 0 30 60 90 120
100
120
140
160
***+
++
NA
c D
A (
% o
f b
asel
ine)
-30 0 30 60 90 120
100
120
140
160
***+
++
***p<0.001 vs veh; +++p<0.001 vs SB 206553 PLSD test
SB 242084SB 242084 prevents the excitatory effect of SB 206553
1µM0.1µM
NAc 5-HT2C Rs participate in the tonic inhibitory control
exerted by the CA of 5-HT2C Rs
Basal DA releaseBasal DA release
intra-VTA and intra-NAc administrationintra-VTA and intra-NAc administration
Basal DA releaseBasal DA release
intra-VTA and intra-NAc administrationintra-VTA and intra-NAc administration
vehSB 206553
**p<0.01 vs veh test PLSD
NA
c D
A (
% o
f b
asel
ine
)
time (min)
-30 0 30 60 90 120
100
120 **
1µM
110
130
-30 0 30 60 90 120
100
120
time (min)
0.5 µg/0.2 µl
110
130
SB 206553SB 206553
Intra-NAc application of the 5-HT2C inverse agonist SB 206553
increases basal DA release in the NAc
VTAVTA
DADANAc NAc
5-HT5-HT2C2C
DADA
5-HT5-HT2C2C
• VTA and NAc 5-HT2C Rs both participate in the
overall inhibitory control of NAc DA release
• The NAc may represent a primary site of action for
the effects of the CA of 5-HT2C Rs
VTA and NAc 5-HTVTA and NAc 5-HT2C2C receptors receptorsVTA and NAc 5-HTVTA and NAc 5-HT2C2C receptors receptors
DA release
≠ ≠ levels of 5-HTlevels of 5-HT2C 2C R CA in the NAc and VTA related to R CA in the NAc and VTA related to RNA editingRNA editing
lowers the CA and modulates the desensitization state of 5-HTlowers the CA and modulates the desensitization state of 5-HT2C2C Rs Rs
generatesgenerates tissue-specific expression of 5-HT tissue-specific expression of 5-HT2C2C R isoforms with R isoforms with
distinct biological propertiesdistinct biological properties (Niswender et al., 1999; Marion et al., 2004(Niswender et al., 1999; Marion et al., 2004)
phasic & tonic
phasic
DADA
NAC / StriatumNAC / Striatum
DADA
VTAVTASNSN
5-HT5-HT2C2C receptor control of DA ascending pathways in receptor control of DA ascending pathways in
vivovivo
ConclusionsConclusions
5-HT5-HT2C2C receptor control of DA ascending pathways in receptor control of DA ascending pathways in
vivovivo
ConclusionsConclusions
• inhibitory tonic / phasic control
• involvement of constitutive activity in vivo
- clozapine: inverse agonist in vivo
- DAergic effects of antipsychotic drugs
• region-dependent control by constitutive activity
- NAc → primary site of action
- Role of mRNA editing ( ↓ constitutively active isoforms)
in the control of DA neuron excitability
5-HT2C receptor
“fine tuning”
DA pathways
DADA
NAC / StriatumNAC / Striatum
DADA
VTAVTASNSN
need for studies
assessing the functional significance
and the therapeutic potential
of inverse agonism in vivo
5-HT5-HT2C2C receptor control of DA ascending pathways in receptor control of DA ascending pathways in
vivovivo
ConclusionsConclusions
5-HT5-HT2C2C receptor control of DA ascending pathways in receptor control of DA ascending pathways in
vivovivo
ConclusionsConclusions
Long term studies ……
studies in animal models of Parkinson’s disease….
5-HT2C receptor
“fine tuning”
DA pathways
VTAVTA
NAcNAc
5-HT5-HT2C2C
DADA
5-HT5-HT2C2C
DADA
Sylvia Navailles
Delphine Moison
Dimitri Ryczko
« Neuropharmacologie et Neurochimie « Neuropharmacologie et Neurochimie
Fonctionnelle »Fonctionnelle »
Université Bordeaux 2
INSERM U 862
France
Pr William Clarke & Dr Kelly Berg
Department of Pharmacology, University of TexasHealth Science Center, San Antonio, USA
Philippe De
Deurwaerdère
Guillaume Lucas
Grégory Porras
Umberto Spampinato
Striatum
SNc
DADA
n. Accumbens
VTA
5-HT2A
DA synthesis DA synthesis
DA transmission
5-HT tone
DA cell firing DA cell firing5-HT3
state-dependent facilitatory controltonic inhibitory control
5-HT2C DA cell firing
5-HT receptors and in vivo DA release: conditional 5-HT receptors and in vivo DA release: conditional
involvementinvolvement5-HT receptors and in vivo DA release: conditional 5-HT receptors and in vivo DA release: conditional
involvementinvolvement
Selective modulation of one DA pathway depending
on the state of DA neuron activity
Rational basis for therapeutics
DA transmission
DA cell firing5-HT4
Factors ?
• Preclinical data (rat):
5-HT5-HT and Parkinson’s diseaseand Parkinson’s disease5-HT5-HT and Parkinson’s diseaseand Parkinson’s disease
• Clinical data:
central 5-HT transmission5-HT2C antagonism
Catalepsy
central 5-HT transmission (SSRI)5-HT4 agonists
Catalepsy
5-HT2C antagonism motor response (L-dopa, DA agonists)
5-HT4 agonist (cisapride)
central 5-HT transmission (SSRI)
5-HT1A agonism (buspiron)
5-HT2A/2C - 5-HT3 antagonists
atypical antipsychotic (clozapine)
5-HT2/3 + α2 antagonism (mirtazapine)
Park (tremor)
Park symptoms dyskinesia
dyskinesia
(mianserin, ondansetron) Dopa-induced psychosis(no change motor score)
Dopa-induced psychosis, tremor
tremor- dyskinesia
5-HT5-HT2C2C receptors: receptors:
DA release & cocaine-induced effectsDA release & cocaine-induced effects
5-HT5-HT2C2C receptors: receptors:
DA release & cocaine-induced effectsDA release & cocaine-induced effects
control / contributioninvolvementVTA 5-HT2C R NAc 5-HT2C R
Inhibitory effect on DA release
Phasic inhibitory control of DA release
yes
Basal DA release
yes
no
Cocaine-induced DA release
Cocaine-induced behaviors
yes (ago)
yes (ago)
Involvement in the systemic effect of 5-HT2C agents
Behaviors
DA releaseno
no
yes
yes
no
yes (ago/antag)
no (ago/antag)
yes (antag)
yes (antag)
5-HT5-HT2C2C receptors: receptors:
DA release & cocaine-induced behaviorsDA release & cocaine-induced behaviors
5-HT5-HT2C2C receptors: receptors:
DA release & cocaine-induced behaviorsDA release & cocaine-induced behaviors
ip. Intra-VTA Intra-NAc
5-HT2C agents administration
Ro-induced DA antagonist
agonist
antagonistBasal DA release 0
000
0agonist
antagonistbasal behaviors 0
00 0
0
agonist
antagonistCocaine- behaviors
0
agonist
antagonistCocaine- DA
00
5-HT5-HT2C2C receptors and DA neuron firing rate receptors and DA neuron firing rate 5-HT5-HT2C2C receptors and DA neuron firing rate receptors and DA neuron firing rate
Tonic and phasic inhibitory control on DA neuron activity
VTA VTA
SB 242084SB 242084 Ro 60-0175Ro 60-0175
Di Matteo et al., 1999
*p<0.05, **p<0.01 vs veh, Tukey’s test
VTAVTA
NAc NAc
5-HT5-HT2C2C
DADA
5-HT5-HT2C2C
ConclusionsConclusions
5-HT2C receptor control
of the mesoaccumbens DA pathway:
controlling NAc DA release
DADADA
transmission(+/-) Change of Change of
• behaviorbehavior• cellular activitycellular activity
composite responses involving different populations
of 5-HT2C receptors in multiple brain nuclei
and/or DA transmission
(-)
(-)
releaserelease
5-HT5-HT2C2C Rs control DA release and/or transmission Rs control DA release and/or transmission5-HT5-HT2C2C Rs control DA release and/or transmission Rs control DA release and/or transmission
DA releaseDA release
DAVTAVTA
DA transmission
firing
regulation of DA exocytosis
via DA neuron firing
modulation of DA transmission may occur
independently from changes of DA release
biochemical
electrophysiological
behavioral
studies
Fr Cortex / NAc Fr Cortex / NAc
“DARP-32”Svenningsson et al., Pnas, 2001
5-HT2C
5-HT5-HT2C2C
5-HT5-HT2C2C
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
SB 206553 - SB 242084
Frontal Cortex
Gobert et al., 2000
TIME (MIN)
PE
RC
EN
T O
F C
ON
TR
OL
* p<0.05 ANOVA
SB 206553 SB 242084
5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release5-HT5-HT2C2C receptors and basal DA release receptors and basal DA release
Ro 60-0175Frontal Cortex
TIME (MIN)
PE
RC
EN
T O
F C
ON
TR
OL
Gobert et al., 2000
* p<0.05 ANOVA
Clozapine: 5-HTClozapine: 5-HT2C2C inverse agonist inverse agonist in vivoin vivo ? ?Clozapine: 5-HTClozapine: 5-HT2C2C inverse agonist inverse agonist in vivoin vivo ? ?
NACStriatum
SB 243213 (1 mg/kg, ip.) 30 min before clozapine (1 mg/kg, sc.)
SB 243213 inhibits the effect of clozapine
-60 -30 0 30 60 90 120
100
120
140
160
******
veh.
SB 243213
clozapine
SB 243213 + clozapine
-60 -30 0 30 60 90 120
100
120
140
160
***
**
Time (min)
DA
( %
of
bas
elin
e )
**p<0.01, ***p<0.001 vs controls PLSD test
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