Article
Reference
Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour
PASCOLI, Vincent Jean, TURIAULT, Marc, LUESCHER, Christian
Abstract
Drug-evoked synaptic plasticity is observed at many synapses and may underlie behavioural adaptations in addiction. Mechanistic investigations start with the identification of the molecular drug targets. Cocaine, for example, exerts its reinforcing and early neuroadaptive effects by inhibiting the dopamine transporter, thus causing a strong increase in mesolimbic dopamine. Among the many signalling pathways subsequently engaged, phosphorylation of the extracellular signal-regulated kinase (ERK) in the nucleus accumbens is of particular interest because it has been implicated in NMDA-receptor and type 1 dopamine (D1)-receptor-dependent synaptic potentiation as well as in several behavioural adaptations.
A causal link between drug-evoked plasticity at identified synapses and behavioural adaptations, however, is missing, and the benefits of restoring baseline transmission have yet to be demonstrated. Here we find that cocaine potentiates excitatory transmission in D1-receptor-expressing medium-sized spiny neurons (D1R-MSNs) in mice via ERK signalling with a time course that parallels locomotor sensitization. Depotentiation [...]
PASCOLI, Vincent Jean, TURIAULT, Marc, LUESCHER, Christian. Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour.
Nature, 2012, vol. 481, no. 7379, p. 71-75
PMID : 22158102
DOI : 10.1038/nature10709
Available at:
http://archive-ouverte.unige.ch/unige:26937
Disclaimer: layout of this document may differ from the published version.
i
Supplementary information for
In vivo reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behavior
Vincent Pascoli, Marc Turiault & Christian Lüscher
a b
c d
400
300
200
100
0
40 30 20 10 0 -1 0
Norm. EPSC (%)
D1+ D1 -
400
300
200
100
0
40 30 20 10 0 -1 0
Norm. EPSC (%)
D2+
D2 -
400
300
200
100
0
40 30 20 10 0 -1 0
D1+ D1-
Time (min)
Norm. EPSC (%)
HFS
HFS
HFS
400
300
200
100
0
40 30 20 10 0 -1 0
HFS
D2- D2+
Time (min)
Norm. EPSC (%)
f e
drd1a-EGFP mice+/– drd2-EGFP mice+/–
1 week ip saline
recordings
1 week ip cocaine
recordings
1 week ip saline
recordings
1 week ip cocaine
recordings
400
300
200
100
0
40 30 20 10 0 -1 0
Norm. EPSC (%)
HFS
400
300
200
100
0
40 30 20 10 0 -1 0
Norm. EPSC (%)
D2- D2+
HFS 1 week
recordings ip
SL327 -1 hour
cocaine
D1+ D1- 1 week
recordings ip
SL327 -1 hour
cocaine
drd1a-EGFP mice+/– drd2-EGFP mice+/–
drd1a-EGFP mice+/– drd2-EGFP mice+/–
Pascoli et al., Suppl. Figure 1
Time (min) Time (min)
Time (min) Time (min)
Suppl. Figure 1: Characterization of HFS-LTP in drd1a-EGFP mice and drd2-EGFP mice. Double- controlled experiment showing separated data obtained from EGFP positive or negative neurons of drd1a- EGFP mice or drd2-EGFP mice. Green symbols represent EGFP positive neurons from mice of either genotype (gray for EGFP negative), square symbols are used for EGFP positive neurons from drd1a-EGFP mice and EGFP negative neurons from drd2-EGFP mice (triangles are used for EGFP negative neurons from drd1a-EGFP mice and EGFP positive neurons from drd2-EGFP mice), filled symbols are used for cocaine and empty symbols for saline (if no other treatment indicated). Scale bars: 10 ms and 20 pA. Normalized EPSC (%) as a function of time and overlay of averaged (20 trials) traces of AMPAR EPSCs before (black line) and after (gray line) HFS are represented. Symbols represent average of 6 trials. (a) No difference detected in EGFP positive (n = 7) versus EGFP negative (n = 7) neurons from saline-treated drd1a-EGFP mice (210 ± 24.2 % versus 235 ± 35.5 %, P = 0.571). (b) No difference detected in EGFP positive (n = 8) versus EGFP negative (n = 6) neurons from saline-treated drd2-EGFP mice (223 ± 34.9 % versus 209 ± 25.7
%, P = 0.757). (c) Significant reduction of LTP magnitude in EGFP positive (n = 10) versus EGFP negative (n = 10) neurons from cocaine-treated drd1a-EGFP mice (119 ± 14.6 % versus 246 ± 15.0 %, P ≤ 0.001). (d) Significant reduction of LTP magnitude in EGFP negative (n = 10) versus EGFP positive (n =11) neurons from cocaine-treated drd2-EGFP mice (111 ± 9.3 % versus 232 ± 20.7 %, P ≤ 0.001). (e) No difference detected in EGFP positive (n = 6) versus EGFP negative (n = 4) neurons from SL327+cocaine-treated drd1a- EGFP mice (201 ± 44.6 % versus 197 ± 34.9 %, P = 0.949). (f) No difference detected in EGFP positive (n = 7) versus EGFP negative (n = 7) neurons from SL327+cocaine-treated drd2-EGFP mice (252 ± 37.5 % versus 199 ± 18.8 %, P = 0.229).
iii
cocaine saline 2 ,000
1 ,500 1 ,000 500
Locomotion (1/4 turns) 0 WT
drd1a-EGFP drd2-EGFP
–/– +/– –/– +/–
* *
* *
*
Pascoli et al., Suppl. Figure 2
Suppl. Figure 2: Similar acute locomotor response to cocaine in drd1a-EGFP and drd2-EGFP mice.
Acute locomotor activity in response to cocaine in WT or heterozygous BAC transgenic mice in which enhanced green fluorescent protein (EGFP) expression was driven by either D1R (drd1a-EGFP) or D2R (drd2-EGFP) genes. Bars represent mean ± s.e.m. of quarter-turns in the circular corridor for 60 min after injection. Cocaine induced locomotor hyperactivity when compared to saline injected mice for all genotypes.
No significant differences were detected between genotypes. Differences were analyzed with a two-way analysis of variance for matching data [interaction between genotype and treatment; F(4,161) = 0.235, P = 0.918; effect of genotype F(4,161) = 0.676, P = 0.610 ; effect of treatment F(1,164) = 122.826, P < 0.001].
Bonferroni was as follows: *P < 0.001 WT C57BL/6 cocaine (n = 20) versus saline (n = 13) ; *P < 0.001 WT drd1a-EGFP cocaine (n = 18) versus saline (n = 16) ; *P < 0.001 heterozygous drd1a-EGFP cocaine (n
= 30) versus saline (n = 9) ; *P < 0.001 WT drd2-EGFP cocaine (n = 18) versus saline (n = 10) ; *P = 0.002 heterozygous drd2-EGFP cocaine (n = 25) versus saline (n = 6).
400 300 200 100 0
40 30 20 10 0 -1 0
D1R-MSNs D2R-MSNs
Time (min)
Norm. EPSC (%)
HFS 1 week ip
cocaine
recordings
Pascoli et al., Suppl. Figure 3
Suppl. Figure 3: Cocaine disrupts HFS-induced LTP in D1R expressing MSNs of the NAc core . AMPAR EPSCs evoked by electrical stimulation of glutamatergic inputs were recorded from medium spiny neurons in NAc core under whole-cell voltage-clamp mode at a holding potential of –70 mV before and after high frequency stimulation (HFS, 100 pulses at 100 Hz repeated 4 times at 0.1 Hz paired with depolarization at 0 mV). Normalized EPSC (%) as a function of time and overlay of averaged (20 trials) traces of AMPAR EPSCs before (black line) and after (gray line) HFS are represented. Symbols represent average of 6 trials. In slices from drd1a- or drd2-EGFP mice treated with cocaine a week before recording, LTP is abolished in D1R-MSNs, n = 11, 9 ± 7.1 % when compared to D2R-MSNs, n = 10, 224 ± 27 %, P < 0.001. Scale bars: 10 ms and 20 pA. Error bars : s.e.m.
SL327+cocaine vehicle+cocaine 2000
1500 1000 500
Locomotion (1/4 turns) 0
Pascoli et al., Suppl. Figure 4
drd1a -EGFP
+/–
drd2-EGFP
+/–
Suppl. Figure 4: SL327 does not affect acute locomotor response to cocaine. Effect of administration of SL327 (40 mg/kg, ip, 10 ml/kg) 1 hour prior to cocaine on acute locomotor activity in response to cocaine in heterozygous BAC transgenic mice in which enhanced green fluorescent protein (EGFP) expression was driven by either D1R (drd1a-EGFP) or D2R (drd2-EGFP) genes. Bars represent mean ± s.e.m. of quarter- turns in the circular corridor for 60 min after injection. Cocaine-induced locomotor hyperactivity is not modified by administration of the MEK inhibitor. T-test analysis was as follows: drd1a-EGFP cocaine (n = 30) versus SL327+cocaine (n = 14), P = 0.497 ; drd2-EGFP cocaine (n = 25) versus SL327+cocaine (n = 9), P = 0.881.
5 4 3 2 1 Sensitization index 0
1 week 1 month saline or
cocaine
1 month
cocaine 1 week
*
Pascoli et al., Suppl. Figure 5
Suppl. Figure 5: A two-injection protocol induces locomotor sensitization to cocaine in mice.
Locomotor sensitization induced by cocaine in C57BL/6 mice in the two-injection protocol with 1 week or 1 month interval between the injections. Scatter plots of individual sensitization indices and bars represent mean ± s.e.m. of sensitization index. On day 1, mice received saline (n = 12 or 10) or cocaine (n = 13 or 10), respectively, while for the second injection on day 8 or 30 both groups received cocaine and quarter-turns in the circular corridor during the 60 min after injection were recorded. Sensitization index at 1 month (n = 13) were compared to sensitization at 1 week (1.1 ± 0.26 versus 2.3 ± 0.37, P = 0.010).
v NAc shell
infralimbic ac
NAc
mPFC
dorsal prelimbic
ventral prelimbic
NAc core NAc shell
Suppl. Figure 6: Histological verification of ChR2-GFP expression. Representative coronal slices at the level of the anterior commissure (ac, Bregma 1.5 mm) and in the middle of the medial prefrontal cortex (mPFC, Bregma ~ 1.85 mm). Insets represent high magnification confocal images at the positions indicated by the color-coded rectangles for the same mouse (blue nuclear staining with Hoechst, scale bars 20 µm).
The strongest GFP-expression is in the infralimbic cortex and there is a ventro-dorsal gradient of the partial infection of the prelimbic mPFC. Note that the axons in the NAc core are very sparsely labeled compared to the NAc shell (same image as Fig. 4a). The openings in the coronal slices represent the artifacts of the optical stimulation fiber cannulae (removal and slice fixation lead to tissue alterations, such that the lumen is overestimated). Note that the tips of the optical fiber are positioned well passed the NAc core directly aiming at the NAc shell. The bright spots represent non-specific fluorescence.
a
b c
PPR
3
2
1
0
before after 120
100 80 60 40 20 0
40 30 20 10 0 -10
Norm. EPSC (%)
Time (min) 1 Hz
AP5 vehicle
vehicle
Suppl. Figure 7: Characterization of LFS-LTD of optogenetic evoked AMPAR EPSCs.
(a) Example traces (overlay of averaged (20 trials) traces) of AMPAR EPSCs evoked by light stimulation recorded from MSNs in the NAc shell of mice infected with AAV coding for eGFP-ChR2 under whole-cell voltage-clamp mode at a holding potential of –70 mV before (black line) and after (gray line) low frequency stimulation (LFS, light pulses of 4 ms at 1 Hz for 10 min). Recordings were done in presence of the
NMDAR antagonist (AP5, 100 µM) or vehicle. (b) Normalized EPSC (%) as a function of time before and after LFS are represented. Symbols represent average of 6 trials. In presence of AP5, LFS did not depress AMPAR EPSCs (AP5, n = 6, 96 ± 8.3 % compared to vehicle, n = 11, 49 ± 4.3 %, P < 0.001). (c) Paired- pulse ratio (PPR) measured with a 50 ms inter-stimulus interval is not modified by LFS (Paired t-test : after, n = 6, 0.85 ± 0.22 versus before, 0.82± 0.11, P = 0.73. Scale bars: 20 ms and 20 pA. Error bars : s.e.m.
PPR
3
2
1
0
D1R-MSNs D2R-MSNs 1 week
cocaine ip injection
recordings
AAV-ctrl AAV-ChR2
AAV-ctrl D1R-MSNs
D2R-MSNs
Pascoli et al., Suppl. Figure 8
Laser 1 Hz
AAV-ChR2 AAV-ctrl
AAV-ChR2
Suppl. Figure 8: Laser treatment does not affect paired pulse ratio (PPR). Acute slices of cocaine- and laser-treated drd1a-EGFP mice or drd2-EGFP mice were prepared on day 8 (45 min after laser treatment), and the PPR of electrically evoked EPSC was recorded at an interpulse interval of 50 ms. (a) Overlay of average trace of AMPAR EPSCs recorded either in D1R-MSNs or in D2R-MSNs NAc shell. (b) The PPR was not modified by laser treatment neither in D1R-MSNs nor in D2R-MSNs (AAV-ChR2, n = 7, 1.4 ± 0.07 versus AAV-ctrl, n = 7, 1.6 ± 0.11, P = 0.26; AAV-ChR2, n = 6, 1.7 ± 0.20 versus AAV-ctrl, n = 5, 1.6 ± 0.06, P = 0.53, respectively). These results suggest that the laser treatment did not affect the transmitter release probability and is in line with a postsynaptic expression mechanism of the optogenetic depotentiation. Scale bars: 20 ms and 20 pA. Error bars : s.e.m.
vii
Pascoli et al., Suppl. Figure 9
Laser 1 Hz
1600
1200
800
400
Locomotion (1/4 turns) 0
ns
#
ns ns
challenge 1challenge 2 challenge 1challenge 2 challenge 1challenge 2
AAV-cont AAV-ChR2
salinecocaine b
Suppl. Figure 9: A cocaine challenge injection is not sufficient to re-prime locomotor sensitization. (a) Diagram of the experiment. Mice that received the chronic cocaine treatment as in Fig. 5d (laser treatment 45 min before the first challenge) received a second challenge injection on day 16. (b) Bar graphs of locomotor responses to the two challenge injections (mean ± s.e.m. of quarter-turns in the circular corridor for 30 min after injection). Differences between groups were analyzed with a multiple-way repeated-measures analysis of variance for matching data [interaction between challenge and treatment F(5,41) = 0.36, P = 0.700 ; effect of challenge F(1,41) = 0.035, P = 0.851 ; effect of treatment F(2,41) = 16.94, P < 0.001]. Bonferroni analysis yielded: # P = 0.019 for AAV-ChR2 infected mice versus AAV-control infected mice pretreated with cocaine.
Responses to challenge 2 were not different from response to challenge 1 in the 3 groups.
Locomotion (1/4 turns for 10 min)
Pascoli et al., Suppl. Figure 10
AAV-ctrl AAV-ChR2
800 600 400 200 0
AAV-ctrl
cocaine challenge laser 1 Hz for 10 min
- - +
-2
Locomotor activity measurments
saline
34 5 15
2 1 0 -1
cocaine
ns
Suppl. Figure 10: Laser treatment alone has no effect on locomotor activity. The locomotor activity was recorded during laser treatment in AAV-ChR2 infected mice (n = 6) and in AAV-control infected mice (n = 6) that were treated with five daily injections of cocaine and after a withdrawal of 10 days. Graph represents mean ± s.e.m. of quarter-turns in the circular corridor for the duration (10 min) of the laser protocol (Illumination of the NAc through optic fibers with laser-evoked light pulses of 4 ms pulses at 1 Hz for 10 min). Locomotor activity is not enhanced during laser treatment in sensitized mice (t-test: P = 0.972, AAV- ChR2 versus AAV-Ctrl). Locomotor activity in AAV-control infected mice (n = 6) injected with a cocaine challenge is also reported.
Methods
Mice. C57BL/6 or heterozygous BAC transgenic mice, in which enhanced green fluorescent protein
(EGFP) expression was driven by either D1R (drd1a-EGFP) or D2R (drd2-EGFP) gene regulatory elements were back-crossed
31in C57Bl/6 mice for 3 to 4 generations, were used. Mice were housed in groups of 3-4 except for those implanted with guide cannulae, in which case animals were housed separately. All animals were kept in a temperature- and hygrometry-controlled environment with a 12h light/12h dark cycle. Mice were injected i.p. with 20 mg/kg cocaine, 40 mg/kg SL327 (dissolved in 25 % DMSO) or 0.9% saline (injection volume 10 ml/kg). Immediately after injection, mice were placed in the locomotor recording apparatus for 1 hour. The chronic treatment consisted of 5 daily injections of cocaine (15 mg/kg i.p.) followed by 10 d of withdrawal.
All procedures were approved by the Institutional Animal Care and Use Committee of the University and Canton of Geneva.
Locomotor sensitization. Locomotor activity was measured as the number of quarter-turns (1/4
turns) entirely crossed by a mouse in a circular corridor. Locomotor chamber apparatus was placed under a video tracking system (Any-maze, Stoelting, US) and measurements were made
automatically by the software. After 3 days of habituation to the test apparatus, mice underwent the experimental procedure, which consisted of two sessions of 60 min separated by one week (or one month), called day 1 and day 8 (or day 30). During the day 1 session, mice received saline or cocaine and were placed immediately in the corridor for 60 min. One week or one month later (day 8 or day 30), a second session was performed during which all mice were injected with cocaine before being placed in the circular corridor for 60 min. Light stimulation protocol (600 pulses of 4 ms at 1 Hz) was done 45 min before the second cocaine injection. To compare the effects of various times after the first injections or various virus infections, locomotor activity (LA) in response to the second cocaine injection was normalized to the mean LA of saline-pretreated mice and the
sensitization index was calculated by dividing the normalized locomotor response to the second injection by the normalized response to the first injection. Locomotor sensitization was also
evaluated during challenge sessions that followed a chronic treatment (5 days of cocaine 15 mg/kg, 10 days withdrawal). Light stimulation protocol was done 45 min or 5 days before the challenge injection of cocaine.
Virus stereotaxic injection of ChR2-eGFP-AAV or control-eGFP-AAV. AAV1 viruses1
produced
at the University of North Carolina (Vector Core Facility) were injected into the infralimbic cortex
of 15-20 g WT or BAC transgenic mice. Anesthesia was induced and maintained with isoflurane
ix
µl/min. In all experiments the viruses were allowed a minimum of 3 weeks to incubate before any
other procedures were carried out. As a control, some mice were injected with an AAV containing only GFP.
Cannula implantation. Following anesthesia and craniotomy over the NAc, two holes were drilled
around the craniotomy and screws were placed in the holes. Two weeks after viral injections, guide cannulae (Plastics One, Roanoke, VA) were lowered slowly into position using stereotaxic
coordinates (bilaterally AP +1.5, ML ±1.6, DV 4.1, 15° angle) and cemented in place using dental cement (Lang Dental MFG Company, Wheeling, IL) to encase the base of the guide cannulae and the screws. Once the cement had dried, a dummy cannula (Plastics One) was placed inside each guide cannula to prevent infection.
Slice electrophysiology. Coronal 200-250 µm slices of mouse forebrain were prepared in cooled
artificial cerebrospinal fluid (ACSF) containing (in mM) NaCl 119, KCl 2.5, MgCl 1.3, CaCl2 2.5,
Na2HPO4 1.0, NaHCO3 26.2 and glucose 11, bubbled with 95% O2 and 5% CO2. Slices were kept
at 32-34° C in a recording chamber superfused with 2.5 ml/min ACSF. Visualized whole-cell
voltage-clamp recording techniques were used to measure holding and synaptic responses of
medium-sized spiny neurons (MSNs) of the NAc shell, identified in some experiments by the
presence of the green fluorescent protein (GFP) of BAC transgenic mice by using a fluorescent
microscope (Olympus BX50WI, fluorescent light U-RFL-T). The holding potential was –70 mV,
and the access resistance was monitored by a hyperpolarizing step of –14 mV with each sweep,
every 10 s. Experiments were discarded if the access resistance varied by more than 20%. Synaptic
currents were evoked by stimuli (50-100 µs) at 0.1 Hz through bipolar stainless steel electrodes
placed at the cortex-NAc border. The internal solution contained (in mM) 140 K-gluconate, 5 KCl,
130 CsCl, 10 HEPES, 0.2 EGTA, 2 MgCl2, 4 Na2ATP, 0.3 Na3GTP, and 10 sodium creatine-
phosphate. Currents were amplified (Multiclamp 700B, Axon Instruments), filtered at 5 kHz and
digitized at 20 kHz (National Instruments Board PCI-MIO-16E4, Igor, WaveMetrics). The liquid
junction potential was small (–3 mV), and therefore traces were not corrected. All experiments were
carried out in the presence of picrotoxin (100 µM). The magnitude of HFS (100 pulses at 100 Hz
repeated 4 times at 0.1 Hz paired with depolarization at 0 mV)-induced LTP was determined by
comparing average EPSCs that were recorded 20-40 min after induction to EPSCs recorded immediately before induction.
Miniature EPSCs were recorded in the presence of tetrodotoxin (0.5 µM). The frequency, amplitudes, and kinetic properties of these currents were then analyzed using the Mini Analysis software package (v4.3, Synaptosoft, Leonia, NJ). Cocaine or light illumination of ChR2-induced changes in cumulative miniature EPSC amplitude and inter-event interval distribution were
analyzed for statistical significance using the nonparametric two-sample Kolmogorov-Smirnov test (KyPlot) with a conservative critical probability level of P < 0.05.
PPR ratio was calculated by dividing the second evoked EPSC by the first with a 50 ms interval in between. AMPAR-EPSCs evoked with ChR2 stimulation by 4 ms light pulses (LED, Thorlabs) were recorded in the same conditions as electrically-evoked synaptic currents. LFS (1 Hz for 10 min) was applied with light pulses and the magnitude of LTD was determined by comparing average EPSCs that were recorded 20-30 min after induction to EPSCs recorded immediately before induction.
In vivo stimulation of infralimbic cortex projections in the NAc shell. Virus injected and
