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Molecular modulation of dopamine
transmission by TAAR1
Raul R. Gainetdinov
Italian Institute of Technology, Genova, Italy
St. Petersburg State University, St. Petersburg, Russia;
Skolkovo Institute of Science and Technology, Moscow, Russia
ECNP Congress-2014, Berlin, Germany
TAAR1 knockout (TAAR1-KO) mice
• Supersensitivity to behavioral and neurochemical effects of
amphetamine (Wolinsky et al., Genes, Brain and Behavior, 2007)
• Deficit in prepulse inhibition
• Enhaced behavioral and neurochemical
effects of amphetamine
• DA D2 receptors are supersensitive
• Indicated as a model related to schizophrenia
Similarly, supersensitivity to psychostimulants was observed
in other strains of TAAR1-KO mice developed independently
(Linderman et al., 2008, Di Cara et al., 2011)
Enhanced stimulatory effects of amphetamines and
b-PEA in TAAR1-KO and heterozygous mice
d-Amphetamine (3 mg/kg, i.p.)
2000
TA1-WT (n=8)
TA1-HET (n=8)
TA1-KO (n=8)
1000
500
Total distance
(cm/5min)
Total distance
(cm/5min)
1500
L-Amphetamine (10 mg/kg, i.p.)
TA1-WT
TA1-KO
1500
1000
500
0
0
0
30
60
90
120
0
150
30
60
90
120
Time (min)
Time (min)
b-PEA (50 mg/kg, i.p.)
Total distance
(cm/5min)
1500
TA1-WT (n=12)
TA1-HET (n=12)
TA1-KO (n=10 )
1000
500
0
0
30
60
90
Time (min)
Suggests inhibitory action of TAAR1 on DA-dependent hyperactivity
Selective TAAR1 ligands
• First selective TAAR1 antagonist EPPTB,
selective for mouse TAAR1, less active for rat
and human form (Bradaia et al., PNAS, 2009)
•
•
•
Regulates the firing frequency of DA neurons in VTA
Blocks the TAAR1-mediated activation of an inwardly rectifying K(+) current..
Increases the potency of DA at D2 receptors
Unfortunately, poor solubility and BBB permeability complicates the use of EPPTB in studies in vivo
First selective full TAAR1 agonist
RO5166017, with high potency and
efficacy at mouse, rat, monkey and human
TAAR1 (Revel et al., PNAS, 2011)
•
•
•
•
Regulates firing rate of both DA and 5-HT neurons potentially via interference with autoreceptor regulation
Potential anxiolytic effect in stress-induced hyperthermia test in mice
Reduces cocaine and NMDA antagonist induced hyperactivity in mice
Reduces hyperactivity of DAT-KO mice
Dopamine transporter knockout
(DAT-KO) Mice
Normal
Neurotransmission
Lack of
Dopamine Transporter
DAT
DA
DA
TH
Gainetdinov and Caron, Annu. Rev. Pharmacol. 2003
TH
Extracellular DA is elevated 5-fold
DA storage is reduced 20-fold
Hyperactivity of DAT-KO mice
Validation of selectivity of newly developed TAAR1 agonist
(in collaboration with F. Hoffmann-La Roche Ltd )
• TAAR1 agonist inhibits hyperactive DAT-KO mice
• This effect is absent in double KO mice lacking
DAT and TAAR1
• Indicates an inhibitory action of TAAR1 on DAdependent hyperactivity
The full agonist
The partial agonist
Potential antipsychotic-like activity:
•
•
•
Block cocaine and NMDA antagonists (PCP, L-687,414) - induced hyperactivity
Potentiate effects of Olanzapine on psychostimulant-induced hyperacitivity
Brain activation pattern reminiscent of the antipsychotic drug Olanzapine
Potential antidepressant-like activity:
• Porsolt forced-swim test in rats
• The differential reinforcement of low-rate behavior paradigm in monkeys
Potential pro-cognitive activity:
• The object retrieval paradigm in Cynomolgus macaques
• Attentional set-shifting in rats
TAAR1 partial agonist RO5263397 increases wakefulness in rats
Potential mechanisms involved:
1. Role of TAAR1 in presynaptic regulation of DA
transmission
2. Role of TAAR1 in the regulation of postsynaptivc
D2Rs
3. Modulation of glutamate NMDA receptors in the
prefrontal cortex by TAAR1
Role of TAAR1 in presynaptic regulation of DA transmission
• TAAR1-KO mice have altered firing rate of
VTA dopaminergic neurons in slices
• p-tyramine decrease firing rate only in WT
• TAAR1 antagonist EPPTB reverse tyramine effect
• TAAR1 antagonist EPPTB increase firing rate in
WT but not TAAR1-KO mice
• D2 DA receptor binding is increased in TAAR1-KO
mice (Seeman et al, Synapse, 2007)
• Suggest an interaction of TAAR1 and D2 dopamine
receptor signaling or regulation
(Bradaia, et al., PNAS , 2009)
Role of TAAR1 in presynaptic regulation of DA transmission
HPLC Analysis of total tissue content
Leo et al.,
Neuropharmacology,
2014
HVA, but not DA and DOPAC levels are increased in the striatum of TAAR1-KO mice
Increased extracellular levels of DA in the Nucleus Accumbens but not
Striatum of TAAR1-KO mice
(Quantitative Low Perfusion Rate Microdialysis in Awake Mice)
Leo et al., Neuropharmacology, 2014
Fast Scan Cyclic Voltammetry (FSCV)
FSCV is a real-time electrochemical technique that detects
electrochemically active neurotransmitters by their redox properties.
Applied waveform
DA :-400 mV to 1V to -400
mV; scan rate 300V/s
Applied waveform
Background
current
Ammine
Oxidation pick
(mV vs Ag/AgCl)
Reduction pick
(mV vs Ag/AgCl)
Dopamine
+500
-400
Adrenaline
+500
-100 and -400
Noradrenaline
+500
-100
Serotonin
+500
-100 and -800
Voltamogram
An increased evoked DA release in the Nucleus Accumbens but not
Striatum slices from TAAR1-KO mice
*
No alterations in measures of DA uptake (half-life; tau) are found in TAAR1 KO mice
TAAR1 agonist reduces DA evoked overflow in Dorsal STR and NAc
of control animals but does not affect TAAR1 KO mice
TAAR1 agonist
RO5166017
N= 10, one way ANOVA
But no alteration in DA half-life and Tau (indications of DA uptake)
TAAR1 antagonist influences DA release in the NAc but
not in the Dorsal STR
TAAR1 antagonist N-(3-Ethoxy-phenyl)-4-pyrrolidin-1yl-3-trifluoromethyl-benzamide (EPPTB)
N= 10, one way ANOVA
But no alteration in DA half-life and Tau (indications of DA uptake)
TAAR1 agonist potentiates effect of Quinpirole on evoked
DA release in the NAc of WT but not TAAR1-KO mice
WT
Quinpirole
0.1 mM
Wash
*
*
*
Two-pulse assessment of autoreceptor
activity suggests impaired D2R autoreceptor
function in TAAR1-Ko mice
Demonstrate that TAAR1 regulates sensitivity of D2R autoreceptos
Role of TAAR1 in the regulation of postsynaptic D2R receptors
D2R-related AKT3/GSK3 signaling pathway
D2R binding and D2R-related signaling are altered
in the striatum of TAAR1-KO mice
D2R but not D1R number is altered
AKT/GSK3 signaling pathway is activated
No alterations in the
phosphorylation of pERK1/2,
pCREB, pDARPP32
Haloperidol-induced catalepsy is reduced in TAAR1-KO mice
Bar test
TAAR1 and D2R form a heterodimer in HEK-293 cells
Bioluminescence Resonance
Energy Transfer (BRET) assay
TAAR1 and D2R form an heterodimer in cells
that may be disrupted by haloperidol
Role of TAAR1 in prefrontal cortex-related processes
Prefrontal cortex is implicated in cognitive behavior and in general
in executive functions
Dysfunction of this areas are linked to psychiatric disorders such as
schizophrenia, ADHD and bipolar disorder
VTA neurons project in different part of prefrontal cortex
TAAR1 is expressed and modulate DA VTA neurons and is also
expressed in the PFC
phMRI shows anypsychotic
-like pattern of brain activation
induced by TAAR1 agonists
mPFC Pre-Limbic Area
Bregma 1.94
VI
V
Recording
Layer V
Patch-clump recordings in pyramidal neurons
II I
III
Stimulation
Layer III
Evoked post-synaptic currents are altered
Evoked Post-Synaptic Currents (EPSCs)
Alterations in amplitude and and kinetics of EPSCs suggest changes in composition
of postsynaptic glutamate receptors
NMDA-dependent currents are reduced
NMDA/AMPA ratio is
changed
NMDA mEPSCs are
reduced
Indicates NMDA receptor-dependent synaptic deficits in PFC neurons of TAAR1-KO mice
Glutamate NMDA receptor composition and phosphorylation
is altered in the prefrontal cortex of TAAR1-KO mice
Taken together, these data indicate glutamate NMDA receptor
hypofunction n the prefrontal cortex of TAAR1-KO mice
Conclusions
• Stimulation of TAAR1 exerts an inhibitory action
on dopamine-dependent locomotor activity
• TAAR1 ligands can modulate dopamine release
• TAAR1 can form a heterodimer with D2R and this
interaction may be important for downstream
signaling and behavior both at the level of
presynaptic autoreceptors and postsynaptic
receptors
• TAAR1 modulates glutamate NMDA receptor
function in the prefrontal cortex and related
functions
ACKNOWLEDGEMENTS
Ali Salahpour
University of Toronto
Bernard Masri
INSERM, Tolouse
Larry Barak
Duke University, Durham
Marc Caron
Stefano Espinoza
Damiana Leo
Ilya Sukhanov
Ludmilla Mus
Tatyana Sotnikova
Italian Institute of Technology, Genova, Italy
Duke University, Durham
Fabio Fumagalli,
University of Milan, Milan, Italy
Marius Hoener, F. Hoffman La-Roche,
Basel, Switzerland
Supported in part by F. Hoffman La-Roche, Basel, Switzerland and San Paolo Foundation, Torino, Italy