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Impairment of cocaine-mediated behaviours in mice by clinically relevant Ras-ERK inhibitors

  1. Alessandro Papale
  2. Ilaria Maria Morella
  3. Marzia Tina Indrigo
  4. Rick Eugene Bernardi
  5. Livia Marrone
  6. Francesca Marchisella
  7. Andrea Brancale
  8. Rainer Spanagel
  9. Riccardo Brambilla  Is a corresponding author
  10. Stefania Fasano  Is a corresponding author
  1. Cardiff University, United Kingdom
  2. IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Italy
  3. Heidelberg University, Germany
  4. IRCCS-San Raffaele Scientific Institute, Italy
Research Article
Cite this article as: eLife 2016;5:e17111 doi: 10.7554/eLife.17111
12 figures

Figures

Molecular modeling of RB1 and RB3.

(A) RB1 peptide (in black) is shown bound to ERK (in red). Graphic was made using UCSF Chimera software, PDB id. 2FYS. (B) RB3 peptide (black ribbon) is shown bound to Ras (green). Graphic was made using UCSF Chimera software, aligning the crystal structure of the CDC25 domain of RasGRF1 (PDB id. 2IJE) with the crystal structure of a ternary Ras:SOS:Ras*GDP complex (PDB id.1XD2).

https://doi.org/10.7554/eLife.17111.002
Figure 2 with 1 supplement
RB1 and RB3 inhibit ERK, histone H3 and S6 ribosomal protein activation in an ex-vivo model of acute striatal slices in a dose-dependent manner.

(A–F) Dose response curves of RB1 and RB3 for ERK, Ac-H3 and S6 activation. 200 μm thick striatal slices were freshly prepared from 2-month old mice and transferred into a perfusion chamber for 1 hr at 32°C. Slices were pre-treated with either different doses of peptides (RB1 and RB3) or scrambled controls (scramble RB1 or scramble RB3). After 1 hr, one group of slices for each condition was fixed in PFA 4% for 15 min, while another group was stimulated with glutamate 100 μM for 10 min prior to fixation. 18 μm cryo-sections were processed for immunohistochemistry with anti-phospho p44/p42 MAP kinase (Thr202/Tyr204) or for immunofluorescence co-labelling the slices with anti-phospho (Ser10) - acetyl (Lys14) H3 or anti-phospho S6 (Ser235/236) and the neuronal marker NeuN. Neuronal quantification was performed with ImageJ software by counting the number of phospho-ERK positive cells and the number of phospho-Ac-H3 or phospho-S6 among NeuN positive neurons in each slice. The level of activation is expressed on the Y-axis as arbitrary units (AU). Doses are reported in a logarithmic scale (Log10) on the X-axis. The IC50 was calculated for each specific response using GraphPad Prism software.

https://doi.org/10.7554/eLife.17111.003
Figure 2—figure supplement 1
RB1 and RB3 show a dose-dependent inhibition of ERK activation and its substrates H3 and S6.

The highest doses of RB1 and RB3 prevented glutamate-induced ERK activation (A,B, scale bar 100 µm), H3 activation (C,D scale bar 30 µm) and S6 activation (E,F scale bar 30 µm), whereas the lowest doses of RB1 and RB3 failed to inhibit ERK, H3 and S6.

https://doi.org/10.7554/eLife.17111.004
RB1 and RB3 have an inhibitory effect on cocaine-induced ERK phosphorylation.

RB1 (20 mg/kg), RB3 (20 mg/kg), or the scrambled peptides (Scr) were administered to wild-type mice 1 hr before an acute cocaine (25 mg/kg) or saline injection. After 5 min mice were perfused. Immunohistochemistry was performed with anti-phospho p44/42 MAP kinase (Thr202/Tyr204, scale bar 30 μm). Quantification of phospho-ERK positive cells shows a significant inhibitory effect of RB1 and RB3 on cocaine-induced ERK activation in the ventral striatum. Two-way ANOVA: effect of RB1 F1,14 = 18.73, p<0.001, effect of cocaine F1,14 = 111.87, p<0.0001, effect of interaction F1,14 = 23.95, p<0.001; Bonferroni’s post-hoc, Scr RB1 saline (n=5) vs Scr RB1 cocaine (n=3) p<0.001, Scr RB1 cocaine (n=3) vs RB1 cocaine (n=5) p<0.001. Two-way ANOVA: effect of RB3 F1,14 = 6.26, p<0.05, effect of cocaine F1,14 = 52.11, p<0.0001, effect of interaction F1,14 = 16.06, p<0.01; Bonferroni’s post-hoc, Scr RB3 saline (n=5) vs Scr RB3 cocaine (n=3) p<0.001, Scr RB3 cocaine (n=3) vs RB3 cocaine (n=5) p<0.01. **p<0.01, ***p<0.001. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.005
RB3, but not RB1 has an inhibitory effect on cocaine-induced S6 phosphorylation.

RB1 (20 mg/kg, i.p.), RB3 (20 mg/kg, i.p.) or the scrambled peptides (Scr RB1 and Scr RB3, 20 mg/kg, i.p.) were administered to wild type mice 1 hr before an acute cocaine (25 mg/kg) or saline injection. After 20 min mice were perfused. Co-labelling was performed with anti-phospho S6 ribosomal protein (Ser235/236, in green) and NeuN (in red, scale bars 30 μm). Neuronal quantification shows that RB3 completely prevented cocaine-induced S6 phosphorylation in the ventral striatum. Two-way ANOVA: effect of RB3 F1,14 = 25.29, p<0.001, effect of cocaine F1,14 = 26.30, p<0.001, effect of interaction F1,14 = 25.88, p<0.001; Bonferroni’s post-hoc, Scr RB3 saline (n=4) vs Scr RB3 cocaine (n=5) p<0.001, Scr RB3 cocaine (n=5) vs RB3 cocaine (n=5) p<0.001. RB1 does not exert any significant effect on cocaine-induced S6 activation. Two-way ANOVA: effect of RB1 F1,36 = 0.01, p>0.05, effect of cocaine F1,36 = 46.63, p<0.001, effect of interaction F1,36 = 1.65, p>0.5; Bonferroni’s post-hoc, Scr RB1 saline (n=10) vs Scr RB1 cocaine (n=10) p<0.0001, Scr RB1 cocaine (n=10) vs RB1 cocaine (n=10) p<0.001. ***p<0.001, ****p<0.0001. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.006
RB1 and RB3 have an inhibitory effect on cocaine-induced H3 phosphorylation.

RB1 (20 mg/kg, i.p.), RB3 (20 mg/kg, i.p.) or the scrambled peptides (Scr RB1 and Scr RB3, 20 mg/kg, i.p.) were administered to wild type mice 1 hr before an acute cocaine (25 mg/kg) or saline injection. After 20 min mice were perfused. Co-labelling was performed with anti-phospho (Ser10)-acetyl (Lys14) histone H3 (in green) and NeuN (in red, scale bars 30 μm). Neuronal quantification shows a significant inhibitory effect of RB1 and RB3 on cocaine-induced H3 activation in the ventral striatum. Two-way ANOVA: effect of RB1 F1,32 = 10.29, p<0.01, effect of cocaine F1,32 = 19.91, p<0.001, effect of interaction F1,32 = 13.99, p<0.001; Bonferroni’s post-hoc, Scr RB1 saline (n=9) vs Scr RB1 cocaine (n=9) p<0.0001, Scr RB1 cocaine (n=9) vs RB1 cocaine (n=9) p<0.0001. Two-way ANOVA: effect of RB3 F1,14 = 9.90, p<0.01, effect of cocaine F1,14 = 14.84 p<0.01, effect of interaction F1,14 = 6.09, p<0.05, Bonferroni’s post-hoc, Scr RB3 saline (n=4) vs Scr RB3 cocaine (n=5) p<0.01, Scr RB3 cocaine (n=5) vs RB3 cocaine (n=5) p<0.01. **p<0.01, ****p<0.0001. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.007
Figure 6 with 1 supplement
PD325901 prevents cocaine-induced ERK phosphorylation in vivo.

Mice received an injection of different inhibitors or vehicle followed by cocaine (25 mg/kg, i.p.) or saline injection 1 hr later. 5 min after the stimulation, mice were perfused and ERK phosphorylation in the ventral striatum was determined. (A) PD325901 (25 mg/kg, i.p.) completely blocked ERK phosphorylation. Two-way ANOVA, effect of pre-treatment F1,80 = 125.76 p<0.0001, effect of cocaine F1,80 = 34.66 p<0.0001, effect of interaction F1,80 = 26.25 p<0.0001; Bonferroni’s post-hoc, PD Saline vs PD Cocaine: p>0.05, VEH Saline vs VEH Cocaine: p<0.0001. (B) Mice were pre-treated with Trametinib (GSK1102212) (5 mg/kg, i.p.), vehicle or PD325901 (25 mg/kg, i.p.) as a positive control for the inhibition. Trametinib did not prevent ERK phosphorylation. Two-way ANOVA, effect of pre-treatment F2,69 = 39.06 p<0.0001, effect of cocaine F1,69 = 49.26 p<0.0001, effect of interaction F2,69 =11.51 p<0.001; Bonferroni’s post-hoc, VEH Saline vs VEH Cocaine: p<0.0001; TRA Saline vs TRA Cocaine: p<0.0001; PD Saline vs PD Cocaine: p>0.05. (C) Mice were pre-treated with Selumetinib (AZD6266) (50 mg/kg), vehicle or PD325901 (25 mg/kg, i.p.) as a positive control for the inhibition. Selumetinib failed to prevent ERK phosphorylation. Two-way ANOVA, effect of pre-treatment F2,72 = 20.91 p<0.0001, effect of cocaine F1,72 = 24.49 p<0.0001, effect of interaction F2,72 = 6.62 p<0.01; Bonferroni’s post-hoc, VEH Saline vs VEH Cocaine: p<0.01; SEL Saline vs SEL Cocaine: p<0.0001; PD Saline vs PD Cocaine: p>0.05. (D) Mice were pre-treated with Dabrafenib (GSK2118436) (50 mg/kg), vehicle or PD325901 (25 mg/kg, i.p.) as a positive control for the inhibition. Dabrafenib displayed a partial, but not significant, inhibitory effect. Two-way ANOVA, effect of pre-treatment F2,66 = 10.74 p<0.0001, effect of cocaine F1,66 = 10.25 p<0.05, effect of interaction F2,66 = 3.08 p>0.05; Bonferroni’s post hoc, VEH Saline vs VEH Cocaine: p<0.0001; DAB Saline vs DAB Cocaine: p>0.05; VEH Cocaine vs DAB Cocaine: p>0.05. VEH=Vehicle, PD=PD325901, TRA=trametinib, SEL=Selumetinib, DAB=Dabrafenib, AU=arbitrary unit, ns=not significant, **p<0.01, ****p<0.0001. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.008
Figure 6—figure supplement 1
PD325901 inhibits ERK phosphorylation in an ex-vivo model of acute striatal slices in a dose-dependent manner.

200 μm thick striatal slices obtained from 2-month old mice were transferred into a perfusion chamber for 1 hr at 32°C in the presence of different doses of PD325901 or vehicle. After 1 hr, one group of slices for each condition was fixed in PFA 4% for 15 min, while another group was stimulated with glutamate 100 μM for 10 min prior to fixation. 18 μm cryo-sections were processed for immunohistochemistry with anti-phospho p44/p42 MAP kinase (Thr202/Tyr204). ERK activation is determined by counting the number of phospho-ERK positive neurons in each slice. The level of activation is expressed on the Y-axis as arbitrary units (AU). Doses are reported in logarithmic scale (Log10) on the X-axis. The IC50was calculated for the specific response using GraphPad Prism software. As shown in the graph, PD325901 is effective in reducing glutamate-induced ERK activation with an IC50 of 1.15 nM. Scale bar 100 µm.

https://doi.org/10.7554/eLife.17111.009
Dose response curve of MEK inhibitor PD325901 on ERK phosphorylation in vivo.

Different doses of PD325901 were administered i.p. to wild-type mice 1 hr before the saline or cocaine (25 mg/kg) injection. After 5 min mice were perfused. Immunohistochemistry was performed with anti-phospho p44/42 MAP kinase (Thr202/Tyr204, lower panel, scale bar 30 μm). Quantification of phospho-ERK positive cells in the dorsal striatum (upper panel, mean with SEM) shows that PD325901 inhibited ERK in a dose-dependent manner. Two-way ANOVA, effect of PD325901 F7,78 = 2.43, p<0.05, effect of cocaine F1,78 = 4.81, p<0.05, effect of interaction F7,78 = 3.47, p<0.01; Bonferroni’s post-hoc, vehicle saline (n=9) vs vehicle cocaine (n=15) p<0.0001, vehicle cocaine (n=15) vs PD325901 0.25 mg/kg (n=4) p>0.05, vehicle cocaine (n=15) vs PD325901 0.5 mg/kg (n=5) p<0.05, vehicle cocaine (n=15) vs PD325901 1 mg/kg (n=5) p<0.05, vehicle cocaine (n=15) vs PD325901 2.5 mg/kg (n=15), p<0.0001, vehicle cocaine (n=15) vs PD325901 5 mg/kg (n=5), p<0.01, vehicle cocaine (n=15) vs PD325901 10 mg/kg (n=5), p<0.01, vehicle cocaine (n=15) vs PD325901 25 mg/kg (n=5), p<0.01. *p<0.05, **p<0.01, ****p<0.0001, all the statistical significances are referred to the vehicle cocaine group indicated by the arrow. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.010
Figure 8 with 4 supplements
A single dose of PD325901 at 10 mg/kg blocks the retrieval of cocaine-associated memory in the conditioned place preference paradigm.

(A) Experimental design of experiments reported in B, C and D. (B) The rewarding properties of cocaine (20 mg/kg i.p.) were first measured on day 8 (test 1). Mice treated with cocaine or saline during conditioning were further divided in 2 groups and received an acute injection of the MEK inhibitor, PD325901 (10 mg/kg i.p.), or DMSO vehicle 1 hr before test 1. A single administration of PD325901 fully inhibited CPP. A two-way ANOVA revealed a significant effect of drug F1,35 = 4.305, p<0.05, effect of PD325901 F1,35 = 4.534, p<0.05 and the interaction between these two factors F1,35 = 10.713, p<0.001; Bonferroni’s post-hoc, cocaine PD325901 (n=12) vs cocaine vehicle (n=12), p<0.0001; cocaine vehicle (n=12) vs saline vehicle (n=8), p<0.01. (C) Two weeks later (test 2) mice were re-tested in the place conditioning apparatus in a drug-free state. In mice that were previously administered PD325901, a lack of preference is still observed. A two-way ANOVA revealed a significant effect of drug F1,35 = 4.748, p<0.05, effect of PD325901 pre-treatment F1,35 = 16.877, p<0.0001 and interaction between these two factors F1,35 = 22.248, p<0.0001; Bonferroni’s post-hoc, cocaine PD325901 (n=12) vs cocaine vehicle (n=12), p<0.0001; cocaine vehicle (n=12) vs saline vehicle (n=8), p<0.0001; cocaine vehicle (n=12) vs saline PD (n=7), p<0.001. (D) One day 23, mice underwent a cocaine (20 mg/kg, i.p.) re-exposure conditioning session in the drug-paired compartment. 24 hr later (day 24, test 3), animals were retested for preference in a drug- and PD325901-free state. Re-exposure to cocaine in the drug-paired compartment was not able to rescue the expression of cocaine preference in PD325901-treated mice. A two-way ANOVA revealed a significant effect of drug F1,35 = 5.100, p<0.05, effect of PD325901 pre-treatment F1,35 = 7.069, p<0.01, and the interaction between these two factors F1,35 =19.404, p<0.0001; Bonferroni’s post-hoc, cocaine PD325901 (n=12) vs cocaine vehicle (n=12), p<0.0001; cocaine vehicle (n=12) vs saline vehicle (n=8), p<0.0001. ***p<0.0001. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.011
Figure 8—figure supplement 1
PD325901 does not influence levels of locomotion during the conditioned place preference.

Locomotion (expressed as distance travelled) in the CPP apparatus during the pre-test- on day 1 in the absence of any treatment and 1 hr after PD325901 injection during the probe test on day 8. In the pre-test, no statistically significant difference was present among groups (one-way ANOVA, distance travelled, F3,38 = 0.311, p=0.817), indicating that all groups used for the experiment did not differ in their basal levels of explorativity. On day 8, half of the mice received an injection of PD325901 (10 mg/kg) or vehicle in their home cage and 1 hr later were let free to explore the CCP apparatus. PD325901 did not appear to affect explorativity as distance travelled of the PD325901-treated and the vehicle-treated groups did not statistically differ (one-way ANOVA, F3,38 = 0.224, p=0.879). Two-way ANOVA for repeated measures showed that levels of locomotor activity were comparable among the four groups (F3,35 = 0.513, p=0.676). There was a significant main effect of time on spontaneous locomotion across the two days of testing (F1,35 = 27.287, p<0.0001), but no significant time x group interaction (F3,35 = 0.78, p=0.971), demonstrating that there were no differences in the rate of explorativity of the four groups. ****p<0.0001. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.012
Figure 8—figure supplement 2
Acute administration of PD325901 at 10 mg/kg had no effect on locomotion.

The horizontal activity of freely moving mice was monitored in one single session starting 30 min after PD325901 injection (10 mg/kg) and recorded every 5 min for a total of 6 trials. At the end of this session, mice received a cocaine challenge (20 mg/kg) and the effect of PD325901 pre-treatment on the hyper-locomotion induced by cocaine was evaluated for the subsequent 30 min. An acute administration of PD325901 had no effect on spontaneous locomotion. Two way ANOVA for repeated measures, showed that levels of locomotor activity were comparable among the groups (F3,36 = 1.195, p=0.326). There was a significant main effect of time on spontaneous locomotion across the 30 min of testing (F5,180 = 128.833, p<0.0001), but no significant time x group interaction (F15,180 = 0.549, p=0.909), demonstrating that there were no differences in the rate of explorativity of all groups. Analysis of the percentages of habituation to the environment confirmed that there were no differences among groups (One-way ANOVA, F3,39 = 0.180, p=0.909). A significant enhancement of locomotion was observed upon cocaine challenge, without any difference between PD325901 and vehicle pre-treated groups (Three-way ANOVA, Coc group effect: F1,36 = 236.787, p<0.0001; PD group effect: F1,36 = 1.216, p=0.278; interaction Coc group x PD group: F1,36 = 0.042, p=0.839). Moreover, a significant effect of habituation across the 6 time points was found (Three-way ANOVA, time effect: F5,180 = 22.851, p<0.0001). A significant interaction with time was found for the cocaine group effect but not for the PD group effect (Three-way ANOVA, interaction time x cocaine group: F5,180 = 12.016, p<0.0001; interaction time x PD group: F5,180 = 1.127, p<0.348). Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.013
Figure 8—figure supplement 3
TRAMETINIB does not interfere with the retrieval of cocaine-associated memory in the conditioned place preference paradigm.

(A) Experimental design of the experiment reported in B. (B) The rewarding properties of cocaine (20 mg/kg) were measured on day 8 (test 1). Mice treated with cocaine (n=24) or saline (n=18) during conditioning were further divided in 2 groups and received an acute injection of the MEK inhibitor, Trametinib (10 mg/kg, i.p.), or vehicle (10 mg/kg i.p.) 1 hr before test 1. A single administration of Trametinib did not interfere with the expression of CPP. A two-way ANOVA revealed a significant effect of cocaine F1,38 = 27.321, p<0.0001, no effect of Trametinib F1,38 = 0.164, p=0.688 and no interaction between these two factors F1,38 = 0.006, p=0.938; Bonferroni’s post-hoc, cocaine Trametinib (n=12) vs cocaine-vehicle (n=12), p>0.05; cocaine-vehicle (n=12) vs saline vehicle (n=10), p<0.01; cocaine-Trametinib (n=12) vs saline-Trametinib (n=8), p<0.01. ⏏⏏ p<0.01. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.014
Figure 8—figure supplement 4
Retrieval of cocaine place preference is partially inhibited by RB1 and RB3 peptides.

(A) Experimental design of experiments reported in B, C and D. (B) The rewarding properties of cocaine (20 mg/kg, i.p.) were first measured on day 8 (test 1). Mice treated with cocaine or saline during conditioning were further divided in 2 groups and received an acute injection of the RB1/RB3 mix (20 mg/kg, i.p.) or scrambled mix (20 mg/kg, i.p.) 1 hr prior to testing. RB1/RB3-treated mice showed a reduction in CPP relative to scrambled control subjects. A two-way ANOVA revealed a significant effect of drug F1,36 = 12.518, p<0.01, peptides F1,36 = 9.152, p<0.01 and interaction between these two factors F1,36 = 16.106, p<0.001; Bonferroni’s post-hoc, cocaine scrambled mix (n=12) vs cocaine RB1/RB3 mix (n=12), p<0.0001; cocaine RB1/RB3 mix (n=12) vs saline RB1/RB3 mix (n=8), p>0.05; cocaine scrambled mix (n=12) vs saline scramble mix (n=8), p<0.0001. (C) Two weeks later (test 2, day 22), mice were then re-tested in the place conditioning apparatus in a drug-free state. Mice that previously received the RB1/RB3 combination spent significantly less time than controls in the drug-paired compartment. A two-way ANOVA revealed a significant effect of drug F1,36 = 41.186, p<0.001, peptides F1,36 = 4.327, p<0.05 and interaction between these two factors F1,36 =15.912, p<0.01; Bonferroni’s post-hoc, cocaine RB1/RB3 mix (n=12) vs cocaine scramble mix (n=12), p<0.01; cocaine RB1/RB3 mix (n=12) vs saline RB1/RB3 mix (n=8), p>0.05; cocaine scramble mix (n=12) vs saline scramble mix (n=8), p<0.0001. (D) One day 23, all groups underwent a cocaine (20 mg/kg, i.p.) re-exposure in the drug-paired compartment. Twenty-four hours later (test 3, day 24), animals were retested for preference in a drug- and peptide-free state. CPP was still partially inhibited in RB1/RB3 pre-treated mice. A two-way ANOVA revealed a significant effect of drug F1,36 = 54.408, p<0.001, peptides F1,36 = 10.361, p<0.05 and interaction between these two factors F1,36 =8.857, p<0.01; post-hoc Bonferroni, cocaine RB1/RB3 mix (n=12) vs cocaine scramble mix (n=12), p<0.05; cocaine RB1/RB3 mix (n=12) vs saline RB1/RB3 mix (n=8), p<0.05; cocaine RB1/RB3 mix (n=12) vs saline scramble mix (n=8), p<0.05; cocaine scramble mix peptides (n=12) vs saline scramble mix (n=8), p<0.0001. *p<0.05, **p<0.01, ***p<0.0001. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.015
A single dose of PD325901 at 10 mg/kg attenuates cue responding following cocaine self-administration.

(A) Experimental design of cocaine self-administration experiments reported in B, C, D, and E. (B) Mice first underwent 7d of daily 1 hr cocaine self-administration sessions (days 1–7). There were no differences in cocaine self-administration based on treatment group prior to drug manipulations, as indicated by a similar number of lever presses [a three-way ANOVA (lever x day x treatment) revealed significant main effects of lever F1,29 = 115.048, p<0.0005 and day F3.5,101.1 = 3.460, p<0.05, and a significant lever x day interaction F3.7,106.9 = 4.080, p<0.05, but no other significant effects (p>0.05)] and (C) reinforcers [a two-way ANOVA (day x treatment) revealed a significant effect of day F3.1,89.4 = 6.341, p<0.005, but no other effects (p>0.05)]. (D) DMSO vehicle (10 ml/kg i.p., n = 18) or PD325901 (10 mg/kg i.p., n = 13) was administered 30 min prior to a 10-min retrieval trial on day 8, during which lever pressing resulted in the CS but no cocaine. No difference in the groups was observed. A two-way ANOVA (lever x genotype) revealed a main effect of lever F1,29 = 48.429, p<0.0005, but no other significant effects (p>0.05). (E) During 10 subsequent daily 1 hr CS-only sessions, PD325901-treated mice demonstrated decreased responding for the cocaine-associated CS. A three-way ANOVA (lever x day x treatment) revealed a significant lever x treatment interaction F1,29 = 5.090, p<0.05, with follow-up ANOVAs revealing a significant difference between responding on the active F1,29 = 4.250, p<0.05, but not inactive F1,29 = 0.007, p=0.932, lever. *p<0.05. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.016
A single dose of PD325901 at 10 mg/kg does not affect acquisition of the Novel Object Recognition.

(A) After a 5 min habituation session in the empty arena on day 1, mice were injected with PD325901 (10 or 25 mg/kg) or vehicle one hour before the 10 min training session with two identical objects on day 2 and were then evaluated for their 24 hr long-term memory in a 10 min testing session on day 3. (B) PD325901 10 mg/kg had no effect on basal preferences, as for both groups percentages of exploration of the two objects resulted to be comparable (Paired-samples t-test, PD325901: t9 = 0.220, p=0.831; VEH: t9 = −0226, p=0.827). (C) Total time of object exploration was equivalent between groups, meaning that PD325901 injection had no influence on the motivational state of the animals (Independent-samples t-test: t18 = −0.083, p=0.935). (D) On day 3, animals underwent the test for their 24 hr long-term memory. For both groups, the percentage of time spent exploring the novel object was significantly higher than the percentage of time spent exploring the familiar object (Paired-samples t-test, PD325901: t9 = 14.788, p<0.0001; VEH: t9 = 13.623, p<0.0001), indicating a significant learning. (E) The discrimination index (D.I.) of the PD325901-injected group was similar to the vehicle injected group (PD325901: 0.337 ± 0.023, VEH: 0.346 ± 0.025; Independent-samples t-test: t18 = −0.258, p=0.799) and above chance level (One-sample t-test, PD325901: t9 = 14.819, p<0.0001; VEH: t9 = 13.615, p<0.0001), indicating that 10 mg/kg of PD325901 did not affect the acquisition process in this memory test. (F) The recognition index (R.I.), based on the ability of an animal to recognize a same object at different time points, showed that the memory of the PD325901-treated group was equivalent to the vehicle treated group (PD325901: 0.339 ± 0.023, VEH: 0.347 ± 0.030; Independent-samples t-test: t18 = −2.17, p=0.830) and significantly above chance level (One-sample t-test, PD325901: t9 = 14.697, p<0.0001; VEH: t9 = 11.628, p<0.0001). PD325901 (n = 10), VEH (n =10). (G) PD325901 25 mg/kg had no effect on basal preferences, as for both groups percentages of exploration of the two objects were similar (Paired-samples t-test, PD325901: t9 = −0.250, p=0.808; VEH: t8 = 0.137, p=0.895). (H) Total time of object exploration was equivalent between groups, meaning that a higher dose of PD325901 had no impact on the motivational state of animals (Independent-samples t-test: t17 = 0.326, p=0.749). (I) On day 3, animals underwent the test for their 24 hr long-term memory. For both groups, the percentage of time spent exploring the novel object was higher than the percentage of time spent exploring the familiar object (Paired-samples t-test, PD325901: t9 = 3.551, p<0.01; VEH: t8 = 7.819, p<0.0001). Importantly, the time spent exploring the familiar object was significantly lower in the vehicle treated group than in the PD325901 treated group (Independent-samples t-test: t17 = 3.561, p<0.01) indicating that PD325901 injection partially perturbed the recognition of the familiar object. (J) The D.I. of the PD325901-injected group was lower, by 39.75%, indicating that 25 mg/kg of PD325901 significantly interfered with memory acquisition (PD325901: 0.137 ± 0.039, VEH: 0.345 ± 0.044; Independent-samples t-test: t17 = −3.560, p<0.01) but still above chance level (One-sample t-test, PD325901: t9 = 3.548, p=0.006; VEH: t8 = 7.818, p<0.0001). (K) Analysis of the R.I. showed that for both groups the percentage of time spent exploring the familiar object significantly decreased compared with the one observed during the first exposition to the same object (Paired-samples t-test, PD325901: t9 = 3.339, p=0.009; VEH: t8 = 9.798, p<0.0001), but the level of recognition memory in the PD325901-treated group was strongly diminished, by 39.77% (PD325901: 0.139 ± 0.042, VEH: 0.349 ± 0.037; Independent-samples t-test: t17 = −3.690, p<0.01), although still above chance level (One-sample t-test, PD325901: t9 = 3.287, p<0.01; VEH: t8 = 9.356, p<0.0001). PD325901 (n = 10), VEH (n = 9). ⏏⏏⏏⏏ p<0.0001, **p<0.01. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.017
A single dose of PD325901 at 10 mg/kg does not affect retrieval of the Novel Object Recognition.

(A) After a 5 min habituation session in the empty arena on day 1, mice were allowed to explore two identical objects in a 10 min training session on day 2. On the subsequent day, one hour before the test session mice received an injection of PD325901 (10 or 25 mg/kg) or vehicle and were then evaluated for their 24 hr long-term memory. (B) On day 2, no statistically significant difference was present between the basal preferences, as for both groups percentages of exploration of the two objects resulted to be comparable (Paired-samples t-test, PD325901: t9 = 0.587, p=0.572; VEH: t8 = 0.059, p=0.955). (C) On day 3, PD325901 did not alter the exploratory behaviour as the percentage of time spent exploring the novel object was significantly higher than the percentage of time spent on the familiar object for both groups, (Paired-samples t-test, PD325901: t9 = 10.886, p<0.0001; VEH: t8 = 7.424, p<0.0001), indicating a significant learning. (D) Total time of object exploration was equivalent between groups, meaning that PD325901 injection had no influence on the motivational state of the animals (Independent-samples t-test: t17 = −0.264, p=0.795). (E) The discrimination index (D.I.) of the PD325901-injected group was similar to the Vehicle injected group (PD325901: 0.375 ± 0.034, VEH: 0.347 ± 0.047; Independent-samples t-test: t17 = 0.493, p=0.628) and above chance level for both groups (One-sample t-test, PD325901: t9 = 10.871, p<0.0001; VEH: t8 = 7.426, p<0.0001), indicating that the retrieval of recognition memory was intact. (F) The recognition index (R.I.) showed that the recognition memory of the PD325901-treated group was equivalent to Vehicle treated group (PD325901: 0.380 ± 0.030, VEH: 0.349 ± 0.041; Independent-samples t-test: t17 = 0.617, p=0.545) and significantly above chance level (One-sample t-test, PD325901: t9 = 12.667, p<0.0001; VEH: t8 = 8.466, p<0.0001). PD325901 (n = 10), VEH (n = 9). (G) On day 2, no statistically significant difference was present between the basal preferences, as for both groups percentages of exploration of the two objects resulted to be comparable (Paired-samples t-test, PD325901: t9 = 0.813, p=0.437; VEH: t8 = 2.176, p=0.061). (H) On day 3, animals received an injection of 25 mg/kg of PD325901 and one hour later were evaluated in a 10 min session. For both groups, the percentage of time spent exploring the novel object was significantly higher than the percentage of time spent on the familiar object (Paired-samples t-test, PD325901: t9 = 3.286, p<0.01; VEH: t8 = 15.233, p<0.0001). Nevertheless, the time spent exploring the familiar object was significantly lower in the vehicle treated group than in the PD325901 treated group (Independent-samples t-test: t17 = 7.896, p<0.0001), indicating that PD325901 injection partially perturbed the recognition of the familiar object. (I) Total time of object exploration was equivalent between groups, meaning that PD325901 injection had no influence on the motivational state of the animals (Independent-samples t-test: t17 = 0.857, p=0.403). (J) The D.I. of the PD325901-injected group was lower, by 23.24%, indicating that 25 mg/kg of PD325901 significantly interfered with the retrieval of recognition memory (PD325901: 0.087 ± 0.027, VEH: 0.376 ± 0.025; Independent-samples t-test: t17 = −7.898, p<0.0001), although still above chance level (One-sample t-test, PD325901: t9 = 3.283, p=0.009; VEH: t8 = 15.264, p<0.0001). (K) Analysis of the R.I. showed that the percentage of time spent exploring the familiar object significantly decreased compared with the one observed during the first exposition to the same object for both groups (Paired-samples t-test, PD: t9 = 3.478, p<0.01; VEH: t8 = 10.260, p<0.0001), but the level of recognition memory in the PD325901 treated group is strongly diminished, by 23.83% (PD325901: 0.093 ± 0.027, VEH: 0.390 ± 0.027; Independent-samples t-test: t17 = −7.856, p<0.0001), although still above chance level (One-sample t-test, PD: t9 = 3.575 p<0.01; VEH: t8 = 14.672, p<0.0001). PD325901 (n = 10), VEH (n =9). ⏏⏏⏏⏏ p<0.0001, ****p<0.0001. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.018
A single dose of PD325901 at 10 mg/kg does not affect fear memory in the Inhibitory avoidance test.

(A) Animals were placed in a lighted arena and when they entered the dark compartment, they received an aversive footshock (0.2 mA × 2”). Immediately after training, animals received an injection of PD325901 (10 or 25 mg/kg, i.p.) or vehicle and were then evaluated for their memory on the subsequent day. (B) 10 mg/kg of PD325901 spared the consolidation of the inhibitory-avoidance learning. Before PD325901 treatment all groups showed equivalent latency to enter the dark compartment (One-way ANOVA, F2,43 = 0.557, p=0.577). PD325901 treatment given immediately after training had a significant effect on memory consolidation on day 2 (Two-way ANOVA, treatment effect F2,43 = 37.848, p<0.0001). In particular, only the higher dose of PD325901 disrupted consolidation when compared with VEH (Bonferroni’s post-hoc, PD325901 10 mg vs VEH: p=0.667; PD325901 25 mg vs VEH: p<0.0001) and a dose-dependent effect was found for PD325901 (Bonferroni’s post-hoc, PD325901 25 mg vs PD325901 10 mg: p<0.0001). Mice treated with 10 mg/kg of PD325901 and vehicle treated mice both showed significantly increased latency 24 hr after training (Paired-samples t-test, PD325901 10 mg: t15 = −9.695, p<0.0001; VEH: t15 = −11.129, p<0.0001), whereas mice injected with 25 mg/kg of PD325901 did not (Paired-samples t-test, PD325901 25 mg: t13 = −2.569, p=0.068), showing impaired memory consolidation. VEH (n =16), PD325901 10 mg (n = 16), PD325901 25 mg (n = 14). (C) Animals were placed in a lighted arena with free access to a dark compartment. When they entered the dark compartment, they received an aversive footshock (0.2 mA × 2”). On the subsequent day, one hour before the test session mice received an injection of PD325901 (10 or 25 mg/kg, i.p.) or vehicle and were then evaluated for their inhibitory-avoidance learning. (D) 10 mg/kg of PD325901 spared the retrieval of the inhibitory-avoidance learning. Before PD325901 treatment all groups showed equivalent latency to enter the dark compartment (One-way ANOVA, F2,32 = 0.092, p=0.913). On day 2, PD325901 treatment altered the memory performance (Two-way ANOVA, treatment effect: F2,32 = 9.536, p<0.001). Specifically, only 25 mg/kg of PD325901 significantly compromised memory retrieval when compared with VEH (Bonferroni’s post-hoc, PD325901 10 mg vs VEH: p=1.000; PD325901 25 mg vs VEH: p<0.001) and a dose-dependent effect was found for PD325901 (Bonferroni’s post-hoc, PD325901 25 mg vs PD325901 10 mg: p<0.01). Mice treated with 10 mg/kg of PD325901 and vehicle treated mice both showed significantly increased latency 24 hr after training (Paired-samples t-test, PD325901 10 mg: t10 = −7.493, p<0.0001; VEH: t13 = −4.804, p<0.0001), while mice injected with 25 mg/kg of PD325901 did not (Paired-samples t-test, PD325901 25 mg: t9 = −1.418, p=0.190), demonstrating a significantly impaired memory. VEH (n = 14), PD325901 10 mg (n = 11), PD325901 25 mg (n = 14). **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean with SEM.

https://doi.org/10.7554/eLife.17111.019

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