1. Neuroscience
Download icon

Translational control by eIF2α phosphorylation regulates vulnerability to the synaptic and behavioral effects of cocaine

  1. Wei Huang
  2. Andon N Placzek
  3. Gonzalo Viana Di Prisco
  4. Sanjeev Khatiwada
  5. Carmela Sidrauski
  6. Krešimir Krnjević
  7. Peter Walter
  8. John A Dani
  9. Mauro Costa-Mattioli  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. University of California, San Francisco, United States
  3. Howard Hughes Medical Institute, University of California, San Francisco, United States
  4. McGill University, Canada
  5. Perelman School of Medicine, United States
Research Article
Cite this article as: eLife 2016;5:e12052 doi: 10.7554/eLife.12052
6 figures

Figures

Figure 1 with 4 supplements
Enhanced susceptibility of adolescent mice to cocaine-induced synaptic potentiation and behavior.

(ab) Left, Representative traces of AMPAR and NMDAR EPSCs recorded in VTA DA neurons 24 hr after a single i.p. injection of saline or cocaine. A low dose of cocaine (5 mg/kg) induced LTP, as determined by the increase in the AMPAR/NMDAR ratio (a, Right, p<0.001, n=11/10 saline/cocaine, t19=8.09) as well as CPP (c, p<0.0001, n=11, t20=7.487) in adolescent mice (5 weeks old), but not in adult mice (3–5 months old, b, Right, p=0.951, n=8/9/7 saline/5 mg/kg cocaine/10 mg/kg cocaine, F2,22=27.20; c, p=0.3289, n=9, t16=1.007). A higher dose of cocaine (10 mg/kg) induced LTP in VTA DA neurons (b, Right, p<0.01 vs. saline or 5 mg/kg cocaine, n=8/9/7 saline/5 mg/kg cocaine/10 mg/kg cocaine, F2,22=27.20) and CPP in adult mice (d, p<0.0001, n=15, t28=5.750). (e) DHPG (100 μM, 5 min) evoked LTD in VTA DA neurons of adult mice (p<0.001, n=6, t10=19.38), but not in adolescent mice (p=0.10, n=7, t12=1.76).

https://doi.org/10.7554/eLife.12052.003
Figure 1—figure supplement 1
Identification of lateral VTA DA neurons in mouse midbrain slices.

(a) Stable pacemaker firing at 1–5 Hz was recorded from neurons in the lateral VTA in cell-attached mode. (b) At Vh=-55 mV, spike width was measured from the start of the inward deflection to the outward peak. Cells with spike widths >1.0 ms were taken as dopaminergic. (c) Cells only in the ventrolateral VTA with a large (>150 pA) hyperpolarization-activated current (Ih), and a large (>150 pA) leak current were studied.

https://doi.org/10.7554/eLife.12052.004
Figure 1—figure supplement 2
Adolescent mice are more susceptible than adult mice to cocaine-induced LTP in VTA DA neurons.

Adolescent (5 weeks old, n=6-11 per group) or adult mice (3–5 months old, n=6-9 per group) were i.p-injected with saline or cocaine at indicated doses and whole-cell recording were performed in VTA DA neurons. LTP, manifested by an increase in AMPAR/NMDAR ratio, was induced at a lower dose of cocaine (5 mg/kg, F5,77=22.15, p<0.001 vs. saline) in adolescent mice than in adults (10 mg/kg, F5,77=22.15, p<0.01 vs. saline or 5 mg/kg cocaine).

https://doi.org/10.7554/eLife.12052.005
Figure 1—figure supplement 3
VTA slices from adolescent mice more susceptible to cocaine-induced LTP in vitro.

(a) Scheme of experimental procedure (b) Direct application of a low concentration of cocaine (1 μM) increased AMPAR/NMDAR ratio 3–5 hr post-treatment in VTA DA neurons of adolescent mice, as compared to adult mice (n=5-11 per group, F1,32=6.56, p>0.01 Eif2s1S/A vs. wild-type control).

https://doi.org/10.7554/eLife.12052.006
Figure 1—figure supplement 4
Basal p-eIF2α phosphorylation levels are similar in the VTA of adult and adolescent mice.

Western blots are shown on top and quantification of eIF2α levels is shown below (n=4, p>0.05).

https://doi.org/10.7554/eLife.12052.007
Figure 2 with 1 supplement
A low dose of cocaine selectively reduces p-eIF2α in the VTA of adolescent mice.

(ab) A low dose of cocaine (5 mg/kg) reduced p-eIF2α in the VTA of adolescent (p<0.05, n=5 per group, t8=3.029), but not adult mice (p=0.329, n=3 per group, t4=1.110). A higher dose of cocaine (10 mg/kg) was needed to reduce p-eIF2α in VTA of adult mice (p<0.001, n=6 per group, t10=4.640). (c) Schematic of mTORC1- and eIF4E-mediated translation. In abilescnt mice, a low dose of cocaine (5 mg/kg) did not significantly alter phosphorylation of S6K at Thr-389 (d), 4E-BP1 at Thr-37 and Thr-46 (e) and eIF4E at Ser209 (f). Western blots are shown on top and quantification for each phospho-protein/total-protein is shown at the bottom (n=3/3 saline/cocaine; S6K, p=0.3467, t4=01.066a; 4E-BP1, p=0.5031, t4=0.7351; eIF4E, p=0.5669, t4=0.6233). Plots are mean ± s.e.m.

https://doi.org/10.7554/eLife.12052.008
Figure 2—figure supplement 1
Doses of cocaine which lower p-eIF2α in the VTA have no effect in nucleus accumbens (NAc). 

(a) Scheme of the experimental procedure (b) A low dose of cocaine (5mg/kg) or a higher dose of cocaine (10 mg/kg) had no effect on p-eIF2 in the NAc of adolescent (p=0.678, n=3 per group, t4=0.4) or adult mice (p=0.18, n=3 per group, t4=1.6), respectively. Plots are mean ± s.e.m.

https://doi.org/10.7554/eLife.12052.009
Figure 3 with 3 supplements
Decreasing p-eIF2α makes adult mice more susceptible to cocaine-induced LTP and behavior.

(ab) A low dose of cocaine (5 mg/kg) induced both LTP in VTA DA neurons (a, p<0.05, n=5, t8=4.193) and CPP in adult Eif2s1S/A mice (b, p<0.01, n=7, t12=3.411) compared to Eif2s1S/S mice (a, p=0.89, n=5, t8=0.14; b, p=0.2170, n=7, t12=1.303). (cd) A low dose of cocaine (5 mg/kg) elicited LTP (c, p<0.001, n=6, t10=3.43) and CPP (d, p=0.1761, n=8 vehicle+cocaine, t14=1.425; p<0.0001, n=16 ISRIB+cocaine, t30=2.433) in ISRIB-injected adult mice compared to vehicle-injected mice. (ef) DHPG (100 μM, 5 min) induced LTD in WT adult VTA DA neurons (e, p<0.001, n=5, t8=20.3) and vehicle-injected WT adult mice (f, p<0.001, n=5, t8=5.17), but not in Eif2s1S/A mice (e, p=0.26, n=7, t12=1.2) and ISRIB-injected mice (f, p=0.42, n=4, t6=0.86).

https://doi.org/10.7554/eLife.12052.010
Figure 3—figure supplement 1
eIF2α phosphorylation is reduced in VTA from adult Eif2s1S/A mice. 

Western blots (top) show reduction in p-eIF2α in Eif2s1S/A mutant mice compared to wild-type littermates (Eif2s1S/S). Quantification of eIF2α phosphorylation vs. total-eIF2α is shown below (p<0.01, n=3 per group, t4=6.67).

https://doi.org/10.7554/eLife.12052.011
Figure 3—figure supplement 2
Decreasing p-eIF2α makes VTA slices from adult mice more susceptible to cocaine-induced LTP in vitro. 

Direct application of a low concentration of cocaine (1 μM) increased AMPAR/NMDAR ratio 3–5 hr post-treatment in VTA DA neurons of Eif2s1S/A mice, as compared to wild-type controls (n=5-11 per group, F1,32=6.56, p<0.01 Eif2s1S/A vs. wild-type control).

https://doi.org/10.7554/eLife.12052.012
Figure 3—figure supplement 3
In adult mice, systemic administration of ISRIB alone failed to induce LTP in VTA DA neurons and CPP.

ab. i.p. injection of ISRIB (2.5 mg/kg) alone did not induce LTP (a, p=0.79, n=6/3 ISRIB/vehicle, t7=0.28) or CPP (b, p=0.329, n=9, t16=1.008), as indicated by the lack of potentiation of VTA DA neurons and difference between average pre- and post-test preference scores, respectively.

https://doi.org/10.7554/eLife.12052.013
Figure 4 with 1 supplement
Increasing p-eIF2α in young mice blocks cocaine-induced LTP and behavior.

(a) Schematic showing Sal003 mechanism of action. (bc) Infusion of Sal003 into the VTA blocked cocaine-induced potentiation (c, p<0.001, n=5 per group, t8=3.81) and increased p-eIF2α in the VTA (p<0.01, n=7/6 vehicle/Sal003, t11=3.172). (d) Schematic of experimental design. (e) Direct application of cocaine (1 μM) induced LTP 3–5 hr post-treatment (p<0.05, n=11/6 vehicle/cocaine, F2,20=7.48), whereas Sal003 prevented it (p<0.05, n=11/6, vehicle/cocaine+Sal003, F2,20=7.48, cocaine vs. cocaine+Sal003). Representative traces of AMPAR and NMDAR EPSCs (top). (f) Infusion of Sal003 into the VTA blocked CPP (p<0.05, n=7 vehicle+cocaine, t12=2.592; p=0.1147, n=10 Sal003+cocaine, t18=1.892) in adolescent mice. (g) Application of Sal003 (20 μM, 10 min), a selective inhibitor of eIF2α phosphatases, induced LTD in VTA DA neurons from adolescent mice (p<0.001, n=6, t10=9.517). Plots are mean ± s.e.m.

https://doi.org/10.7554/eLife.12052.014
Figure 4—figure supplement 1
Sites of Sal003 infusions into VTA at seven rostrocaudal planes and corresponding increase in p-eIF2α. 

Coordinates are posterior to bregma and cannula tip placements are from mice infused with Sal003 (1 μl; 20 μM) and vehicle (1 μl).

https://doi.org/10.7554/eLife.12052.015
Decreasing OPHN1 levels in VTA DA neurons makes adult mice more susceptible to cocaine-induced LTP.

(a) A low dose of cocaine (5 mg/kg) induced LTP in adult Ophn1-shRNA injected VTA DA neurons (a, Right, p<0.01, n=5, t8=5.464); above representative traces of AMPAR and NMDAR EPSCs (top). (b) Low doses of cocaine (5 mg/kg) induced CPP in mice locally injected with Ophn1-shRNA (p<0.01, n=14, t26=3.600), but not in control mice injected with scrambled shRNA (p=0.7829, n=4, t6=0.2882). (c) Sal003 (20 μM) blocked the cocaine-induced LTP in the VTA of control shRNA-injected mice (p<0.01, n=6/6/7 vehicle/cocaine/cocaine+Sal003, F2,16=13.03), but failed to do so in Ophn1-shRNA VTA DA neurons (p=0.29, n=6/6/11, vehicle/cocaine/cocaine+Sal003, F2,20=4.29, cocaine vs. cocaine+Sal003; p<0.05 vehicle vs. cocaine or cocaine+Sal003). (d) Representative sample traces of AMPAR EPSCs. (ef) I-V plots. (g) Cocaine increased the rectification index in control-shRNA injected VTA neurons while Sal003 blocked it (p<0.001, n=6/6/7 vehicle/cocaine/cocaine+Sal003, F2,16=30.30, cocaine vs. vehicle or cocaine vs. cocaine+Sal003), whereas both cocaine and cocaine+Sal003 increased the rectification index in VTA DA neurons from Ophn1-shRNA-injected mice (p<0.05, n=6/6/11 vehicle/cocaine/cocaine+Sal003, F2,20=3.92, vehicle vs. cocaine or cocaine+Sal003; p=0.80 cocaine vs. cocaine+Sal003). Plots are mean ± s.e.m.

https://doi.org/10.7554/eLife.12052.016
Multiple drugs of abuse reduce p-eIF2α in VTA of adult mice.

(a) i.p. injection of nicotine (1 mg/kg), ethanol (2 g/kg), or methamphetamine (1 mg/kg) reduces p-eIF2α in VTA (n=5 per group; Saline vs. nicotine, p<0.05, t8=2.879; ethanol, p<0.001, t8=6.278 methamphetamine, p<0.001, t8=5.449).

https://doi.org/10.7554/eLife.12052.017

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)