Article Figures & Data
Figures
- Figure 1.
PME changes the dorsal striatal proteome and leads to numerous network changes A–D, Volcano plots (left; blue circles, proteins decreased in PME vs PSE; red circles, proteins increased in PME vs PSE, which reach the level of significance) and heatmaps (right) identifying the top 30 differentially expressed proteins in the DLS of males (A) and females (B), and in the DMS of males (C) and females (D; Extended Data Figs. 1-1, 1-2, full dataset in the DLS and DMS, respectively). The top five hub proteins identified from the network of differentially expressed proteins are plotted as triangles on volcano plots. For more information, see Table 1 and Extended Data Figure 1-3. E, F, Pathway analysis of enriched KEGG and Reactome terms (represented as pink and blue dots, respectively) among the significant differentially expressed proteins for the DLS in males (E) and DMS in males (F). Highlighted and numbered terms include retrograde endocannabinoid signaling (1), alcoholism (2), cGMP–PKG signaling pathway (3), vesicle-mediated transport (4), opioid signaling (5), transmission across chemical synapses (6), L1CAM interactions (7), NGF-stimulated transcription (8), axon guidance (9), and nervous system development (10) in the DLS (E); and cGMP–PKG signaling pathway (1), axon guidance (2), sphingolipid signaling pathway (3), neuronal system (4), cell–cell communication (5), vesicle-mediated transport (6), axon guidance (7), nervous system development (8), and neurexins and neuroligins (9) the DMS (F; for full enrichment analysis results, see Extended Data Fig. 1-4; n = 8 PME (4 males, 4 females) and 8 PSE (4 males, 4 females).
- Figure 2.
PME alters the dorsal striatal phosphoproteome and kinase pathways. A–D, Volcano plots (left; blue circles, phosphopeptides decreased in PME vs PSE; red circles, phosphopeptides increased in PME vs PSE, which reach the level of significance) and heatmaps (right) identifying the top 30 differentially expressed phosphopeptides the DLS in males (A) and females (B) and DMS males (C) and females (D; Extended Data Figs. 2-1, 2-2, full dataset in the DLS and DMS, respectively) Phosphorylated protein hubs of the network of differentially expressed phosphorylated proteins are identified as various triangle symbols on each respective volcano plot (Table 1, Extended Data Fig. 1-3 for more information). E–H, The results of a kinase substrate enrichment analysis demonstrating the top 30 kinases dysregulated. E–H, Right, Blue bars represent kinases decreased in PME versus PSE and red bars represents proteins increased in PME versus PSE, which reach the level of significance for DLS in males (E) and females (F) and DMS males (G) and females (H). Extended Data Figure 2-3, KSEA scores; Extended Data Figures 2-4, 2-5, 2-6, 2-7, coral treeplots. n = 8 PME mice (4 males, 4 females) and 8 PSE mice (4 males, 4 females).
- Figure 3.
Overlap in differentially abundant proteins and phosphopeptides. A–D, Venn diagrams demonstrating the overlap in significantly increased global proteins (blue), decreased global proteins (yellow), increased phosphopeptides (green), and decreased phosphopeptides (red) in PME relative to PSE mice for the DLS of males (A) and females (B) and DMS of males (C) and females (D).
- Figure 4.
PME impairs medium spiny neuron basal glutamate transmission. A, Representative voltage-clamp traces of sEPSCs from MSNs of the DLS in PME (red) and PSE (blue) male (left) and female (right) adolescent offspring. Calibration: 500 ms, 15 pA. B, In the DLS, PME significantly reduced the frequency of sEPSC events (ANOVA: exposure, p = 0.0002). C–E, There was no effect present on amplitude (C), rise time (D), or decay time (E) in the DLS. n = 6 PME mice (3 males; 3 females), 37 neurons (18 males; 19 females), and 6 PSE mice (3 males; 3 females), 37 neurons (18 males; 19 females). F, Representative sEPSC traces from MSNs of the DMS in PME (red) and PSE (blue) male (left) and female (right) adolescent offspring. Calibration: 500 ms, 30 pA. G, H, In the DMS, there was not an effect of exposure on either the frequency of events (G) or the amplitude of responses (H). I, However, PME significantly reduced the rise time of sEPSCs (ANOVA: exposure, p = 0.047). J, The decay was not impacted by PME. n = 6 PME mice (3 males; 3 females), 27 neurons (12 males; 15 females); and 6 PSE mice (3 males; 3 females), 27 neurons (15 males; 12 females). *p < 0.05.
- Figure 5.
PME effects on AMPA/NMDA currents. A, Representative voltage-clamp traces of AMPA and NMDA current traces from MSNs of the DLS PME (red) and PSE (blue) male (left) and females (right) adolescent offspring. The cell was first held at −80 mV, and AMPAR-mediated EPSCs were electrically evoked. For the NMDAR current, the cell was then held at +40 mV, and an EPSC was again evoked. As the AMPAR component of EPSCs at −80 mV was not apparent 100 ms following the electrical stimulus (i.e., the measured current returned to baseline), the NMDAR-mediated portion of the EPSC at +40 mV was calculated as the average of the measured current over the following 25 ms (i.e., 100–125 ms poststimulus; see dotted lines in A for the PME-male trace). Calibration: 100 ms, 100 pA. B, In the DLS, a significant sex effect, but no exposure-related effects, was present on AMPA/NMDA current. n = 6 PME mice (3 males; 3 females), 25 neurons (13 males; 12 females); and 6 PSE mice (3 males; 3 females), 22 neurons (11 males; 11 females). C, Representative AMPA and NMDA current traces from MSNs of the DMS in PME (red) and PSE (blue) male (left) and female (right) adolescent offspring. Calibration: 100 ms, 100 pA. D, A significant sex effect was also present in the DMS, but no exposure-related effects were present. n = 6 PME mice (3 males; 3 females), 23 neurons (10 males; 13 females); and 6 PSE mice (3 males; 3 females), 24 neurons (13 males; 11 females). *p < 0.05.
- Figure 6.
Excitability of dorsal striatal medium spiny neurons is minimally impacted by PME. A, Representative current-clamp traces of action potential firing from MSNs of the DLS in PME (red) and PSE (blue) male (left) and female (right) adolescent offspring. Calibration: 100 ms, 25 mV. B, In the DLS, action potential frequency at various current steps were not significantly different between exposures. C, Female PME MSNs did reveal a slight but significant increase in resting membrane potential compared with PSE-females (ANOVA: interaction, p = 0.0062; female PME vs female PSE, p = 0.032). D–G, No other exposure related effects were discovered for input resistance (D), threshold potential (E), peak action potential amplitude (F), or action potential half-width (G); n = 6 PME mice (3 males; 3 females), 36 neurons (18 males; 18 females); and 6 PSE mice (3 males; 3 females), 36 neurons (18 males; 18 females). H, Representative current-clamp traces of action potential firing from MSNs of the DMS in PME (red) and PSE (blue) male (left) and female (right) adolescent offspring. Calibration: 100 ms, 25 mV. I, J, In the DMS, the frequency of action potentials was not affected by sex or exposure (I), nor was the resting membrane potential (J). K, However, the input resistance was significantly decreased in PME-males compared with PSE-males (ANOVA: exposure × sex, p = 0.012; Sidak’s post hoc test, p = 0.014). L, PME did not alter the threshold potential. M, PME significantly increased the peak action potential amplitude, although this was primarily because of PME-males (ANOVA: exposure, p = 0.022; Sidak’s post hoc test, p = 0.017). N, The action potential half-width was not significantly impacted by PME. n = 7 PME mice (4 males; 3 females), 34 neurons (18 males; 16 females); and 6 PSE mice (3 males; 3 females), 36 neurons (20 males; 16 females). *p < 0.05.
- Figure 7.
PME ablates endocannabinoid-mediated long-term depression in the DLS of males. A, Representative electrically eEPSC traces from the DLS before and after HFS (three trains of 1 s, 100 Hz stimulation separated by 10 s) plus postsynaptic depolarization (0 mV) in PME (red) and PSE (blue) male mice. Calibration: 50 ms, 100 pA. B, Time series of eEPSC amplitude averages in the DLS over the 35 min recording session. Blue circles, PSE-males; red circles, PME-males. C, D, HFS plus depolarization was capable of significantly reducing the eEPSC amplitude compared with baseline in PSE-males (paired t test: p < 0.0001; C), but not in PME-males (paired t test: p = 0.42; D). E, The percentage reduction in eEPSC amplitude during the final 10 min of recording was significantly blunted in PME-males compared with PSE-males (Student’s t test, p = 0.0007); n = 5 PME mice (7 neurons) and 5 PSE mice (7 neurons). F, Representative eEPSC traces from the DMS before and after HFS plus depolarization in PME (red) and PSE (blue) male mice. G, Time series of eEPSC amplitude averages in the DMS over the 35 min recording session. Blue circles, PSE-males; red circles, PME-males. H, I, In the DMS, the protocol reduced eEPSC amplitudes in both PSE-males (paired t test: p = 0.037; H) and PME-males, although the p value did not quite reach the level of significance in PME-males (paired t test, p = 0.079; I). J, The percentage reduction in eEPSC amplitude during the final 10 min of recording was not significantly different between exposure groups (Student’s t test, p = 0.53); n = 5 PME mice (7 neurons) and 5 PSE mice (8 neurons). K, Representative eEPSC traces from the DLS before and after HFS plus depolarization in PME (red) and PSE (blue) female mice. L, Time series of eEPSC amplitude averages in the DLS over the 35 min recording session. Blue circles, PSE-females; red circles, PME-females. M, N, HFS plus depolarization did not impact eEPSC amplitudes in either PSE-females (paired t test, p = 0.31; M) or PME-females (paired t test, p = 0.69; N). O, The percentage reduction in eEPSC amplitude did not differ between exposure groups (Student’s t test, p = 0.49); n = 6 PME mice (6 neurons) and 6 PSE mice (6 neurons). P, Representative eEPSC traces from the DMS before and after HFS plus depolarization in PME (red) and PSE (blue) female mice. Q, Time series of eEPSC amplitude averages in the DMS over the 35 min recording session. Blue circles, PSE-females; red circles, PME-females. R, HFS plus depolarization produced a mild potentiation in PSE-females as eEPSC amplitudes increased (paired t test, p = 0.046). S, However, PME-females exhibited a significant reduction in eEPSC from baseline (paired t test, p = 0.032). T, The percentage change in eEPSC amplitude was significantly different in the DMS between exposure groups (Student’s t test, p = 0.0037); n = 6 PME mice (7 neurons) and 6 PSE mice (7 neurons). *p < 0.05.
- Figure 8.
Direct activation CB1 receptors does not rescue endocannabinoid-mediated long-term depression in the DLS of PME-males. A, Representative eEPSC traces from the DLS before and after WIN55,212-2 (1 μm, 10 min) application in PME (red) and PSE (blue) male mice. Calibration: 50 ms, 100 pA. B, Time series of electrically eEPSC amplitude averages in the DLS over the 35 min recording session. Blue circles, PSE-males; red circles, PME-males. C, D, Application of the CB1 agonist WIN55,212-2 (1 μm) significantly reduced the eEPSC amplitude compared with baseline in PSE-males (paired t test, p = 0.0001; C), but not in PME-males (paired t test, p = 0.18; D). E, The percentage reduction in eEPSC amplitude during the final 10 min of recording was significantly different between exposure groups (Student’s t test, p = 0.018); n = 3 PME mice (8 neurons) and 3 PSE mice (8 neurons). F, Representative eEPSC traces from the DMS before and after WIN55,212-2-2 (1 μm, 10 min) application in PME (red) and PSE (blue) mice. G, Time series of eEPSC averages in the DMS over the 35 min recording session. Blue circles, PSE-males; red circles, PME-males. H, I, Application of the CB1 agonist WIN55,212-2 significantly reduced the eEPSC amplitude compared with baseline in both PSE-males (paired t test, p = 0.032; H) and in PME-males (paired t test, p = 0.015; I). J, The percentage reduction in eEPSC amplitude did not differ between PME-males and PSE-males (Student’s t test, p = 0.31); n = 2 PME mice (4 neurons) and 2 PSE mice (4 neurons). *p < 0.05.
Tables
- Table 1
Hub proteins from differential protein and phosphopeptide expression networks in the DMS and DLS of males and females
Gene Protein MCC DLS hub proteins for global proteome network in males Copb1 Coatomer subunit β-1 242 Dynll2 Dynein light chain 2, cytoplasmic 241 Capza2 F-actin-capping protein subunit α-2 240 Copb2 Coatomer subunit β-2 240 Copg1 Coatomer subunit γ-1 240 DMS hub proteins for global proteome network in males Btbd1 BTB/POZ domain-containing protein 1 5760 Asb6 Ankyrin repeat and SOCS box protein 6 5760 Kbtbd7 Kelch repeat and BTB (POZ) domain containing 7 5760 Fbxw10 F-box/WD repeat-containing protein 10 5760 Fbxo7 F-box only protein 7 5760 DLS hub proteins for phosphoproteome network in males Sptbn1 Spectrin beta chain, nonerythrocytic 1 7 Ank2 Ankyrin-2 6 Ank3 Ankyrin-3 6 Ank1 Ankyrin-1 6 Rpl37 60S ribosomal protein L37 6 DLS hub proteins for phosphoproteome network in females Gapvd1 GTPase-activating protein and VPS9 domain-containing protein 1 6 Reps1 RalBP1-associated Eps domain-containing protein 1 6 Arrb1 β-Arrestin-1 6 Dnm3 Dynamin-3 6 Dlg2 Disks large homolog 2 1 DMS hub proteins for phosphoproteome network in males Hspa8 Heat shock cognate 71 kDa protein 4 Cask Peripheral plasma membrane protein CASK 3 Epb4.1l1 Band 4.1-like protein 1 3 Epn2 Epsin-2 2 Camk2b Calcium/calmodulin-dependent protein kinase type II subunit β 2 DMS hub proteins for phosphoproteome network in females Vcl Vinculin 4 Cyfip1 Cytoplasmic FMR1-interacting protein 1 2 Lrrc7 Leucine-rich repeat-containing protein 7 2 Arhgef2 Rho guanine nucleotide exchange factor 2 1 Cask Peripheral plasma membrane protein CASK 1 - Table 2
Estimated changes in kinase activity from differential phosphopeptide expression networks in the DMS and DLS of males and females (FDR, <0.05)
Gene name Kinase name Enrichment z score FDR DLS of males Cdk9 Cyclin-dependent kinase 9 6.68 <0.0001 Ttbk2 Tau tubulin kinase 2 3.87 0.0039 Prkca Protein kinase C α 3.41 0.0154 DLS of females Pak1 P21 activated kinase 1 −4.11 0.0003 Pak2 P21 activated kinase 2 −4.11 0.0003 Prkaca Protein kinase cAMP-activated catalytic subunit α −4.11 0.0003 Clk4 CDC like kinase 4 −2.71 0.0222 Prkcd Protein kinase C δ −2.71 0.0222 Prkci Protein kinase C ι −2.71 0.0222 Rock2 Rho associated coiled-coil containing protein kinase 2 −2.71 0.0222 Prkcz Protein kinase C ζ −2.44 0.0424 DMS of males Cdk9 Cyclin dependent kinase 9 5.83 <0.0001 Prkci Protein kinase C ι −4.15 0.0011 Pak3 P21 activated kinase 3 −3.58 0.0078 Pak1 P21 activated kinase 1 −3.22 0.0217 DMS of females Csnk1E Casein kinase 1 ϵ 3.38 0.0083 Clk2 CDC like kinase 2 3.47 0.0083 Mapk11 Mitogen-activated protein kinase 11 −3.40 0.0083 Mapk12 Mitogen-activated protein kinase 12 −3.40 0.0083 Cdc42Bpa CDC42 binding protein kinase α −3.54 0.0083 Cdc42Bpb CDC42 binding protein kinase β −3.54 0.0083 Sgk3 Serum/glucocorticoid regulated kinase family member 3 3.16 0.0151 Prkaa2 Protein kinase AMP-activated catalytic subunit α 2 3.01 0.0221 Prkci Protein kinase C ι 2.90 0.0279 Cdk9 Cyclin-dependent kinase 9 2.65 0.0427 Prkaa1 Protein kinase AMP-activated catalytic subunit α 1 2.67 0.0427 Clk1 CDC like kinase 1 2.72 0.0427 Mapk13 Mitogen-activated protein kinase 13 −2.65 0.0427
Figure 1-1
Global proteome DLS. This file contains the full proteomics results for males and females in the DLS. Download Figure 1-1, XLS file.
Figure 1-2
Global proteome DMS. This file contains the full proteomics results for males and females in the DMS. Download Figure 1-2, XLS file.
Figure 1-3
Hub protein analysis. This file contains the full hub protein analysis results for males and females in both the DLS and DMS. Download Figure 1-3, XLS file.
Figure 1-4
Pathway analysis. This file contains the full KEGG and Reactome pathway analysis results for males and females in both the DLS and DMS. Download Figure 1-4, XLS file.
Figure 2-1
Phosphoproteome DLS. This file contains the full phosphoproteomics results for males and females in the DLS. Download Figure 2-1, XLS file.
Figure 2-2
Phosphoproteome DMS. This file contains the full phosphoproteomics results for males and females in the DMS. Download Figure 2-2, XLS file.
Figure 2-3
KSEA kinase scores. This file contains the full KSEA analysis results and kinase scores for males and females in both the DLS and DMS. Download Figure 2-3, XLS file.
Figure 2-4
Kinome treeplots representing the full KSEA results for males in the DLS. The results from kinase-substrate enrichment analysis were mapped onto kinome treeplots via Coral in which branch color corresponds to significance level, node color corresponds to the z score of enrichment, and node size corresponds to the magnitude of enrichment for kinase pathways in the DLS of males. Download Figure 2-4, TIF file.
Figure 2-5
Kinome treeplots representing the full KSEA results for females in the DLS. The results from kinase-substrate enrichment analysis were mapped onto kinome treeplots via Coral in which branch color corresponds to significance level, node color corresponds to the z score of enrichment, and node size corresponds to the magnitude of enrichment for kinase pathways in the DLS of females. Download Figure 2-5, TIF file.
Figure 2-6
Kinome treeplots representing the full KSEA results for males in the DMS. The results from kinase-substrate enrichment analysis were mapped onto kinome treeplots via Coral in which branch color corresponds to significance level, node color corresponds to the z score of enrichment, and node size corresponds to the magnitude of enrichment for kinase pathways in the DMS of males. Download Figure 2-6, TIF file.
Figure 2-7
Kinome treeplots representing the full KSEA results for females in the DMS. The results from kinase-substrate enrichment analysis were mapped onto kinome treeplots via Coral in which branch color corresponds to significance level, node color corresponds to the z score of enrichment, and node size corresponds to the magnitude of enrichment for kinase pathways in the DMS of females. Download Figure 2-7, TIF file.
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