Journal: eLife

Article Title: Drug-induced changes in connectivity to midbrain dopamine cells revealed by rabies monosynaptic tracing

doi: 10.7554/eLife.93664

Figure Lengend Snippet: ( A ) Schematic and timeline of viral injections into mice. ( B ) Representative images of the BNST, GPe, and DCN of DAT-Cre, GAD2-Cre, and vGlut2-Cre mice. Green indicates RABV-labeled cells. Scale, 200 μm. ( C ) Bar plot showing percent of RABV-labeled cells in each input region for DAT-Cre, GAD2-Cre, and vGlut2-Cre mice. ( D ) PCA of input labeling from brains from DAT-Cre, GAD2-Cre, and vGlut2-Cre mice. For this and other figures, ellipsoids were centered at the average coordinate of a condition and stretched one standard deviation along the primary and secondary axes. ( E ) Box plot comparisons of PC1. One-way ANOVA p = 0.010, pairwise t -tests DAT-Cre vs. GAD2-Cre multiple comparisons adjusted p = 0.0087, DAT-Cre vs. vGluT2-Cre p = 0.46, GAD2-Cre vs. vGluT2-Cre p = 0.19. n = 8 for DAT-Cre, n = 13 for GAD2-Cre, and n = 7 for vGluT2-Cre. ( F ) Box plot comparisons of PC2. One-way ANOVA p = 0.016, pairwise t -tests DAT-Cre vs. GAD2-Cre multiple comparisons adjusted p = 0.65, DAT-Cre vs. vGluT2-Cre p = 0.016, GAD2-Cre vs. vGluT2-Cre p = 0.051. ( G ) Heatmap of the contributions of each brain region, or feature, in the data to PCs 1–3. For this and all figures, error bars = ± 1 SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Strain, strain background ( Mus musculus , males and females) , GAD2-Cre , The Jackson Laboratory , Strain #: 028867, RRID: IMSR_JAX:028867 , .

Techniques: Labeling, Standard Deviation

Journal: eLife

Article Title: Drug-induced changes in connectivity to midbrain dopamine cells revealed by rabies monosynaptic tracing

doi: 10.7554/eLife.93664

Figure Lengend Snippet: ( A ) Schematic of how projection portraits are constructed. A representative slice of the VTA is considered for experiments from the Allen Mouse Brain Connectivity Atlas with injections into relevant brain regions that provide input to the VTA. Projection density values in this slice are averaged and visualized to obtain the projection portrait in the VTA for that group of regions. ( B ) Projection portrait of the VTA based on inputs from regions with a high positive contribution to PC1: BNST, EP, GPe, CeA, and ZI. ( C ) Projection portrait of the VTA based on inputs from regions with a large negative contribution to PC1: anterior cortex, NAcMed, VP, PO, septum, LHb, MHb, and LH. ( D ) Projection portrait of the VTA based on inputs from regions with a high positive contribution to PC2: LHb, MHb, EP, ZI, DCN, and anterior cortex. ( E ) Projection portrait of the VTA based on inputs from regions with a large negative contribution to PC2: NAcMed, NAcLat, NAcCore, DStr, and GPe. ( F ) UMAP embedding of input labeling of brains from DAT-Cre, GAD2-Cre, and vGlut2-Cre mice. ( G ) Correlogram generated using the average Euclidean distance of data points from each other from 20 UMAP embeddings.

Article Snippet: Strain, strain background ( Mus musculus , males and females) , GAD2-Cre , The Jackson Laboratory , Strain #: 028867, RRID: IMSR_JAX:028867 , .

Techniques: Construct, Labeling, Generated

Journal: eLife

Article Title: Drug-induced changes in connectivity to midbrain dopamine cells revealed by rabies monosynaptic tracing

doi: 10.7554/eLife.93664

Figure Lengend Snippet: ( A ) PCA plot showing PC1 and PC2 of brains from DAT-Cre and GAD2-Cre mice. DAT-Cre mice were anesthetized with K/X and treated with saline or anesthetized with isoflurane and treated with saline or one of the drugs cocaine, methamphetamine, amphetamine, nicotine, morphine, or fluoxetine. GAD2-Cre mice were anesthetized with isoflurane and treated with saline or cocaine. ( B ) Box plot of PC1, one-way ANOVA p < 0.0001, pairwise t -tests DAT-Cre saline vs. DAT-Cre fluoxetine multiple comparisons adjusted p = 0.99, DAT-Cre saline vs. DAT-Cre K/X p = 1.0, DAT-Cre saline vs. DAT-Cre drugs p = 0.23, DAT-Cre saline vs. GAD2-Cre p < 0.0001, DAT-Cre fluoxetine vs. DAT-Cre K/X p = 1.0, DAT-Cre fluoxetine vs. DAT-Cre drugs p = 0.46, DAT-Cre fluoxetine vs. GAD2-Cre p < 0.0001, DAT-Cre K/X vs. DAT-Cre drugs p = 0.31, DAT-Cre K/X vs. GAD2-Cre p < 0.0001, DAT-Cre drugs vs. GAD2-Cre p < 0.0001. n = 4 for DAT-Cre saline and DAT-Cre K/X, n = 5 for DAT-Cre fluoxetine, n = 24 for DAT-Cre drugs, and n = 17 for GAD2-Cre. ( C ) Heatmap of the contributions of each brain region to PC1–PC3 for data shown in panel A. ( D ) Representative images of brain slices from DAT-Cre mice showing, from left to right, the NAc, BNST, GPe, and PBN. Scale, 500 μm. ( E ) Bar plot showing percentage of RABV-labeled cells in each input region for the DAT-Cre mice shown in panel A. ( F ) PCA plot showing PC1 and PC2 of brains from DAT-Cre mice anesthetized with isoflurane and treated with cocaine, methamphetamine, amphetamine, nicotine, morphine, fluoxetine, or saline, or anesthetized with K/X and treated with saline. ( G ) PC2 and PC3 of the same group of brains as shown in panel F. ( H ) Box plot of PC1, one-way ANOVA p = 0.81. ( I ) Box plot of PC2, one-way ANOVA p = 0.0093, pairwise t -tests DAT-Cre saline vs. DAT-Cre fluoxetine multiple comparisons adjusted p = 0.94, DAT-Cre saline vs. DAT-Cre K/X p = 0.87, DAT-Cre saline vs. DAT-Cre drugs p = 0.034, DAT-Cre fluoxetine vs. DAT-Cre K/X p = 0.99, DAT-Cre fluoxetine vs. DAT-Cre drugs p = 0.10, DAT-Cre K/X vs. DAT-Cre drugs p = 0.27. ( J ) Box plot of PC3, one-way ANOVA p = 0.086. ( K ) Heatmap of the contributions of each brain region to PC1–PC3 for data shown in panels F and G.

Article Snippet: Strain, strain background ( Mus musculus , males and females) , GAD2-Cre , The Jackson Laboratory , Strain #: 028867, RRID: IMSR_JAX:028867 , .

Techniques: Saline, Labeling

Journal: eLife

Article Title: Translational control in the spinal cord regulates gene expression and pain hypersensitivity in the chronic phase of neuropathic pain

doi: 10.7554/eLife.100451

Figure Lengend Snippet: ( A ) A schematic illustrating the TRAP approach to assess gene expression in specific cell types. Confirmation of the specificity of IP fractions for inhibitory neurons in the L10a- eGFP ; Gad2 Cre mouse line ( B ) and for excitatory neurons in the L10a- eGFP ; Tac1 Cre mouse line ( C ). Experiments were performed in female mice. Dual flashlight plots (left) show the strictly standardized mean difference (SSMD) versus log 2 FC for genes in IP samples and panels on the right show the top 15 upregulated and downregulated genes for inhibitory neurons at day 4 ( D ) and 60 ( E ), and Tac1 + excitatory neurons at day 4 ( F ) and 60 ( G ) post-spared nerve injury (SNI). Positive Log 2 FC indicates increased expression in SNI compared to sham mice. Parameters for defining data as upregulated in SNI are indicated at the top. includes complete datasets (worksheets 5–8), with yellow highlighting log 2 FC values in IP samples and orange highlighting log 2 FC values in IN samples. ( H ) The number of altered genes in each condition (GAD2 D60: SNI versus sham day 60 in GAD2 + neurons; GAD2 D4: SNI versus sham day 4 in GAD2 + neurons; Tac1 D60: SNI versus sham day 60 in Tac1 + neurons; and Tac1 D4: SNI versus sham day 4 in Tac1 + neurons).

Article Snippet: Eif4ebp1 fl/fl mice ( ) were crossed with Gad2 Cre (The Jackson Laboratory, stock #010802) to generate Eif4ebp1 fl/fl ;Gad2 Cre animals, Pvalb Cre (Jackson Laboratory, stock #008069) to generate Eif4ebp1 fl/fl ;Pvalb Cre animals, Tac1 Cre (The Jackson Laboratory, stock #021877) to generate Eif4ebp1 fl/fl ;Tac1 Cre animals, and Slc17a6 Cre (The Jackson Laboratory, stock #028863) to generate Eif4ebp1 fl/fl ; Slc17a6 Cre animals (corresponding Cre lines were used as controls).

Techniques: Gene Expression, Expressing

Journal: eLife

Article Title: Translational control in the spinal cord regulates gene expression and pain hypersensitivity in the chronic phase of neuropathic pain

doi: 10.7554/eLife.100451

Figure Lengend Snippet: ( A ) A schematic of mTORC1 pathway. Deletion of 4E-BP1 in GAD2 ( B , 4E-BP1 cKO: Eif4ebp1 fl/fl ;Gad2 Cre , Control: Gad2 Cre , n = 9/10) and PV ( C , 4E-BP1 cKO: Eif4ebp1 fl/fl ;Pvalb Cre , Control: Pvalb Cre , n = 8/11) neurons induces mechanical (50% withdrawal threshold) but not heat hypersensitivity. A comparable number of male and female mice was used in B and C. Recording from PV neurons in spinal cord slices (identified by the expression of L10a-eGFP) shows that the deletion of 4E-BP1 in PV neurons (4E-BP1 cKO: Eif4ebp1 fl/fl : L10a- eGFP : Pvalb Cre , Control: L10a- eGFP : Pvalb Cre , n = 8/8 female mice) induces a decrease in firing frequency ( D ) and an increase in rheobase ( E ). No change in membrane capacitance ( F ), resting membrane potential (RMP, G ), and input resistance (Rin, H ) was found. AAVs (AAV-CAG-DIO-eGFP-eIF4E-shRNAmir or AAV-CAG-DIO-EGFP-scrambled-shRNAmir) were injected into the parenchyma of the dorsal horn of Pvalb Cre female mice (illustration and time course are shown in I , n = 8/group), preventing the spared nerve injury (SNI)-induced decrease in PV neuron firing frequency ( J ) and elevation of rheobase ( K ). No changes were found in capacitance ( L ), RMP ( M ), and Rin ( N ). An unpaired two-tailed t -test was used in B, C, E–H . Two-way ANOVA followed by Tukey’s post hoc comparison was used in J–N. Each data point represents an individual animal. Data are plotted as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns – not significant.

Article Snippet: Eif4ebp1 fl/fl mice ( ) were crossed with Gad2 Cre (The Jackson Laboratory, stock #010802) to generate Eif4ebp1 fl/fl ;Gad2 Cre animals, Pvalb Cre (Jackson Laboratory, stock #008069) to generate Eif4ebp1 fl/fl ;Pvalb Cre animals, Tac1 Cre (The Jackson Laboratory, stock #021877) to generate Eif4ebp1 fl/fl ;Tac1 Cre animals, and Slc17a6 Cre (The Jackson Laboratory, stock #028863) to generate Eif4ebp1 fl/fl ; Slc17a6 Cre animals (corresponding Cre lines were used as controls).

Techniques: Control, Expressing, Membrane, Injection, Two Tailed Test, Comparison

Journal: eLife

Article Title: Translational control in the spinal cord regulates gene expression and pain hypersensitivity in the chronic phase of neuropathic pain

doi: 10.7554/eLife.100451

Figure Lengend Snippet: ( A ) Lumbar spinal cord tissue from 4E-BP1 cKO GAD2 ( Eif4ebp1 fl/fl : L10a- eGFP : Gad2 Cre ) and Control ( L10a- eGFP : Gad2 Cre ) mice was immunostained for 4E-BP1. eGFP expression indicates GAD2 + neurons (marked by white arrows). ( B ) Lumbar spinal cord tissue from 4E-BP1 cKO PV ( Eif4ebp1 fl/fl : L10a- eGFP : Pvalb Cre ) and Control ( L10a- eGFP : Pvalb Cre ) mice was immunostained for 4E-BP1. eGFP expression indicates PV + neurons (marked by white arrows). ( C ) Lumbar spinal cord tissue from 4E-BP1 cKO Vglut2 ( Eif4ebp1 fl/fl : Slc17a6 Cre ) and Control ( Slc17a6 Cre ) mice was immunostained for 4E-BP1. Excitatory neurons were identified as NeuN + /Pax2 − (white arrows mark inhibitory neurons). ( D ) Lumbar spinal cord tissue from 4E-BP1 cKO Tac1 ( Eif4ebp1 fl/fl : L10a- eGFP : Tac1 Cre ) and Control ( L10a- eGFP : Tac1 Cre ) mice was immunostained for 4E-BP1. eGFP expression indicates Tac1 + neurons (marked by white arrows). ( E ) AAVs (AAV9-CAG-DIO-eGFP-eIF4E-shRNAmir and AAV9-CAG-DIO-eGFP-scrambled) were injected into the lumbar spinal cord of Pvalb Cre mice, and immunohistochemistry against eIF4E was performed 14 days later. Scale bar is 20 µm in all images. An unpaired two-tailed t -test was used. Each data point represents an individual animal. Data are plotted as mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: Eif4ebp1 fl/fl mice ( ) were crossed with Gad2 Cre (The Jackson Laboratory, stock #010802) to generate Eif4ebp1 fl/fl ;Gad2 Cre animals, Pvalb Cre (Jackson Laboratory, stock #008069) to generate Eif4ebp1 fl/fl ;Pvalb Cre animals, Tac1 Cre (The Jackson Laboratory, stock #021877) to generate Eif4ebp1 fl/fl ;Tac1 Cre animals, and Slc17a6 Cre (The Jackson Laboratory, stock #028863) to generate Eif4ebp1 fl/fl ; Slc17a6 Cre animals (corresponding Cre lines were used as controls).

Techniques: Control, Expressing, Injection, Immunohistochemistry, Two Tailed Test