Control of dopamine ascending pathways by central 5-HT system: implications for treatment of...

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


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



disordersdisorders


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


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


• 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



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 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





DA release (%)

Gobert 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


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


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


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)





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)


Ro 60-0175: 5-HTRo 60-0175: 5-HT2C2C agonist agonist


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


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


DA


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




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:







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


the reduction of 5-HT extracellular levels by 8-OH-

DPAT

The effect of SB 206553 occurs independently of endogenous 5-HT


the lesion of 5-HT neuronsthe lesion of 5-HT neurons


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


Constitutive activity of 5-HTConstitutive activity of 5-HT2C2C receptor and basal DA receptor and basal DA

releaserelease


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


-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


DA

(%

of

bas

elin

e)

-30 0 30 60 90 120

100

125

150

175

200

225

250veh.

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


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 ?

http://www.depressionblog.org/images/clozapine.gif

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

?


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



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 )





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




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


Nucleus Accumbens

5-HT2C receptors

SB 242084SB 242084


Intra-NAcIntra-NAc perfusion of 5-HT perfusion of 5-HT2C2C antagonist antagonist


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


Intra-NAcIntra-NAc perfusion of 5-HT perfusion of 5-HT2C2C antagonists antagonists


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


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



vivovivo


• 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


vivovivo



vivovivo


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


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)


DA release & cocaine-induced behaviorsDA release & cocaine-induced behaviors


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


*p<0.05, **p<0.01 vs veh, Tukey’s test

VTAVTA

NAc NAc

5-HT5-HT2C2C

DADA

5-HT5-HT2C2C


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


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


Ro 60-0175Frontal Cortex

TIME (MIN)

PE

RC

EN

T O

F C

ON

TR

OL

Gobert et al., 2000

* p<0.05 ANOVA


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

Date post:	27-Mar-2015
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Control of dopamine ascending pathways by central 5-HT system: implications for treatment of...

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