Journal: eNeuro

Article Title: Essential Role of Somatic Kv2 Channels in High-Frequency Firing in Cartwheel Cells of the Dorsal Cochlear Nucleus

doi: 10.1523/ENEURO.0515-20.2021

Figure Lengend Snippet: Antibody information

Article Snippet: Kv2.1 (L80/21) , Synthetic peptide 837–853 aa of rat Kv2.1 , Mouse IgG3 mAb , NeuroMab catalogue 75-315, RRID: AB_2315863 , 1 μg/ml.

Techniques: Concentration Assay, Purification

Journal: eNeuro

Article Title: Essential Role of Somatic Kv2 Channels in High-Frequency Firing in Cartwheel Cells of the Dorsal Cochlear Nucleus

doi: 10.1523/ENEURO.0515-20.2021

Figure Lengend Snippet: Quantitative analysis of Kv2.1 and Kv2.2 immunoreactivities in cartwheel cells

Article Snippet: Kv2.1 (L80/21) , Synthetic peptide 837–853 aa of rat Kv2.1 , Mouse IgG3 mAb , NeuroMab catalogue 75-315, RRID: AB_2315863 , 1 μg/ml.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

doi: 10.1074/jbc.RA120.016009

Figure Lengend Snippet: The GPR motif is critical for RGS14 suppression of LTP. A , RGS14 is a multifunctional signaling protein, which contains an RGS domain that binds active Gαi/o-GTP, an R1 domain that binds H-Ras-GTP, and the GPR motif (G) that binds inactive Gαi1/3-GDP. Amino acid substitutions are indicated for each domain, which confer a loss-of-function phenotype for that specific domain (see Fig. S1 , A – C ). Each eYFP-RGS14 construct, wild-type and mutant, was placed in AAV expression vector and made into live AAV2/9 virus ( Fig. S1 D ). B , AAV2/9-eYFP-RGS14 was added, either as 1 μL or as 10 μL, to cultured primary neurons (35 mm dish), and after 24 h, infected cells were harvested and cell lysates were subjected to SDS-PAGE and western immunoblot. Expressed YFP-RGS14 and actin were detected with mouse monoclonal anti-RGS14 and rabbit polyclonal antiactin sera, respectively. C , eYFP-RGS14 is expressed in area CA1 of cultured hippocampal slices following infection with 5 nL AAV2/9-eYFP-RGS14 (3.14 × 10 13 VG/mL) by glass pipette. D – F , long-term potentiation (LTP) was performed by pairing Schaffer collateral input with CA1 neuron depolarization (time indicated with the arrow) in cultured hippocampal slices expressing AAV-eYFP-RGS14 following infection with virus as in C . Following the LTP-pairing protocol, EPSCs were assessed for potentiation. Control CA1 neurons (n = 12) and those expressing YFP alone (n = 6) showed robust LTP, while eYFP-RGS14 (n = 5) suppressed induction of LTP. G – I , although the eYFP-RGS14 NE/AA (RGS domain knockout; n = 6) and eYFP-RGS14 R/L (R1 domain knockout; n = 5) suppressed LTP similar to WT RGS14, the eYFP-RGS14 GPR motif knockout QR/AA (n = 6) failed to suppress LTP, suggesting that the GPR motif is a critical mediator of RGS14 function. Data is presented as mean ± SD.

Article Snippet: Sections were then incubated for 24 h at 4 °C in this same NGS blocking/permeabilization buffer now including a mouse primary antibody raised against RGS14 (NeuroMab; clone N133/21) at 1:500 dilution.

Techniques: Construct, Mutagenesis, Expressing, Plasmid Preparation, Virus, Cell Culture, Infection, SDS Page, Western Blot, Transferring, Control, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

doi: 10.1074/jbc.RA120.016009

Figure Lengend Snippet: Rare human genetic variants within the GPR and NES motifs of RGS14 disrupt its association with Gαi1-GDP, suggestive of aberrant cellular trafficking. A , RGS14 missense and silent variants were obtained from GnomAD (Broad Institute) and plotted onto the sequence of RGS14 using Lollipops ( https://github.com/pbnjay/lollipops ) as described previously . Missense variants are plotted on top in teal , with silent variants on the bottom in purple . The GPR motif, which contains an embedded NES, has human variants as shown ( red ). B – C , BRET measurement of RGS14-Luc interaction with Gαi1-eYFP-GDP. RGS14-Luc is recruited from the cytoplasm to the plasma membrane by inactive Gαi1-eYFP-GDP . Inactivation of the GPR in RGS14-Luc (Q515A/R516A; GPRm; n = 3) prevents Gαi1-eYFP-GDP binding and recruitment to the plasma membrane. Similarly, inactivation of the NES (L503A/L504A; NESm; n = 3) prevents RGS14 from being accessible for recruitment in the cytoplasm and therefore cannot associate with Gαi1-GDP at the plasma membrane. Variants L504R or R506Q were placed in RGS14-Luc and tested for their capacity to associate with Gαi1-eYFP-GDP by BRET, indicative of proper recruitment to the plasma membrane. L504R (n = 3) completely abolished Gαi1-GDP association, while R506Q (n = 3) exhibited a partial reduction in Gαi1-GDP association. Data is presented as mean ± SEM.

Article Snippet: Sections were then incubated for 24 h at 4 °C in this same NGS blocking/permeabilization buffer now including a mouse primary antibody raised against RGS14 (NeuroMab; clone N133/21) at 1:500 dilution.

Techniques: Sequencing, Clinical Proteomics, Membrane, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

doi: 10.1074/jbc.RA120.016009

Figure Lengend Snippet: RGS14 is a nucleocytoplasmic shuttling protein in hippocampal neurons. A , RGS14 rapidly shuttles into and out of the nucleus in dividing cells directed by a nuclear localization sequence (NLS) and nuclear export sequence (NES). Studies validated that RGS14 followed these same cellular dynamics in primary hippocampal neurons. B , neurons were infected with AAV2/9-eYFP-RGS14 for 18 h and then imaged using live cell confocal microscopy. Leptomycin B (LMB) was added (final concentration 20 nM) and YFP signal was measured. At time zero, YFP-RGS14 is entirely cytosolic. After 20 min, YFP-RGS14 was detectable in the nucleus, at 40 min YFP-RGS14 was mostly nuclear, and by 80 to 120 min YFP-RGS14 was entirely nuclear. C , wild-type YFP-RGS14 does not colocalize with Hoechst in vehicle-treated conditions, but colocalizes with Hoechst entirely under LMB conditions. D , mutation of the NES (L503A/L504A, NESm) in RGS14 sequesters RGS14 entirely in the nucleus under vehicle-treated conditions. Approximately ten images were collected per condition and representative images are shown.

Article Snippet: Sections were then incubated for 24 h at 4 °C in this same NGS blocking/permeabilization buffer now including a mouse primary antibody raised against RGS14 (NeuroMab; clone N133/21) at 1:500 dilution.

Techniques: Sequencing, Infection, Confocal Microscopy, Concentration Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

doi: 10.1074/jbc.RA120.016009

Figure Lengend Snippet: Human variants LR and RQ disrupt RGS14 nucleocytoplasmic equilibrium to favor the nucleus over the cytoplasm. Primary hippocampal neurons were infected with AAV-eYFP-RGS14 or RGS14 carrying variants LR or RQ for 18 h and then imaged using live cell confocal microscopy. A , wild-type RGS14 does not colocalize with Hoechst in neurons under unstimulated conditions. The RQ variant occupies both the nucleus and the cytoplasm, while the LR variant localizes entirely to the nucleus. B , quantification of YFP (RGS14) and Hoechst overlay for WT RGS14 and each of the variants provides a measure of colocalization. Approximately ten images were collected per condition and representative images are shown. C , RGS14–Luc and Gαi1–eYFP–GDP interactions in live HEK cells were measured as static net BRET. Since the RGS14 NES motif overlaps with the GPR binding site of Gαi1–GDP, an NLS mutation (NLSm) was introduced to prevent nuclear localization. Thus, any change in net BRET from wild-type is due to effects of Gαi1–GDP binding, independent of mislocalization to the nucleus and interaction with XPO1. Net BRET for both RGS14 RQ (n = 3) and RGS14 LR (n = 3) is partially (although not fully) restored when an NLS mutation is introduced. D , measure of RGS14-Luc and Gαi1-eYFP-GDP Net BRET comparing L504R and L504R/NLSm and R506Q and R506Q/NLSm (L504R and R506Q data from Fig. 2 ). Adding the NLS mutation enhances Gαi1–GDP association, indicating that XPO1 and Gαi1–GDP interactions are both impacted by the L504R and R506Q mutations.

Article Snippet: Sections were then incubated for 24 h at 4 °C in this same NGS blocking/permeabilization buffer now including a mouse primary antibody raised against RGS14 (NeuroMab; clone N133/21) at 1:500 dilution.

Techniques: Infection, Confocal Microscopy, Variant Assay, Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

doi: 10.1074/jbc.RA120.016009

Figure Lengend Snippet: The GPR motif of RGS14 contains an embedded class 3 nuclear export sequence that binds XPO1. Structural modeling of the RGS14 GPR/NES motif binding to Exportin 1 (XPO1). A , the NES of RGS14 is embedded within the GPR motif and follows a class 3 pattern: ϕ 1 XX ϕ 2 XXX ϕ 3 XX ϕ 4 . B , the crystallized GPR peptide of RGS14 (PDB 1KJY) was structurally aligned with a similar class 3 NES motif (mDia2 PDB 5UWP) crystallized with XPO1. C – D , the RGS14 NES fits a class 3 binding motif, occupying pockets 0 to 3, but not pocket 4, as reported previously . E , RGS14 WT, but not RGS14 NES, coimmunoprecipitates with Flag-XPO1. Flag-XPO1 and RGS14 WT or NES were transfected into HEK cells and Flag was immunoprecipitated (Flag affinity gel). RGS14 (1:1000) and XPO1 (1:300) protein were measured by immunoblot.

Article Snippet: Sections were then incubated for 24 h at 4 °C in this same NGS blocking/permeabilization buffer now including a mouse primary antibody raised against RGS14 (NeuroMab; clone N133/21) at 1:500 dilution.

Techniques: Sequencing, Binding Assay, Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

doi: 10.1074/jbc.RA120.016009

Figure Lengend Snippet: Human variants LR and RQ disrupt RGS14 binding to Exportin 1 (XPO1) through loss of side-chain interactions. A – B , recombinant RGS14 WT and variants LR and RQ were purified from E. coli and mixed with purified GST-XPO1 and constitutively active His-Ran (GV)-GTP. His-Ran (G/V)-GTP was captured by Ni-NTA affinity pull-down and complexes were assessed by Coomassie ( A ) and immunoblot ( B ). WT RGS14 was recovered in complex with XPO1, but RGS14 NES, RGS14 LR, and RGS14 RQ did not bind XPO1. Blots are representative of five independent experiments. C , structural modeling of the R506Q variant binding to XPO1. Left panel is the wild-type amino acid, arginine, which makes a salt bridge with a nearby glutamate near the binding pocket on XPO1. Mutation to a glutamine ( right panel ) removes this salt bridge, presumably destabilizing the protein–protein interaction. D – E , structural modeling of the L504R variant onto XPO1. The leucine interacts directly with the hydrophobic P2 pocket ( left panel ). Mutation of this amino acid to an arginine ( right panel ) introduces a charged side chain into a hydrophobic pocket. These data mechanistically explain the difference in phenotype (R506Q being a subtler phenotype compared with L504R).

Article Snippet: Sections were then incubated for 24 h at 4 °C in this same NGS blocking/permeabilization buffer now including a mouse primary antibody raised against RGS14 (NeuroMab; clone N133/21) at 1:500 dilution.

Techniques: Binding Assay, Recombinant, Purification, Western Blot, Variant Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

doi: 10.1074/jbc.RA120.016009

Figure Lengend Snippet: Human variants LR and RQ that disrupt RGS14 nuclear-cytoplasmic equilibrium also block RGS14 capacity to inhibit long-term potentiation in hippocampal slices. AAV-eYFP-RGS14 was used to make AAV2/9 viruses, including wild-type RGS14 as well as human variants RQ and LR. Furthermore, a stop codon was generated as follows AAV-YFP-STOP-RGS14, to make a truncated YFP virus lacking RGS14. Virus was injected into mouse CA1 slice cultures and incubated for 1 week, at which point electrophysiological recordings were made to assess LTP. A , expression of YFP-RGS14 in CA1 brain slices. To improve visualization of individual neurons, insets are shown derived from cutout white dashed boxes (WT and R506Q) or as 20× images where individual cell resolution in the cutout was not sufficient (YFP and L504R). The horizontal and vertical green lines in images for YFP and L504R represent the interface where composite confocal images were spliced together. WT RGS14 fills the cytoplasm of neurons. RQ fills both the cytoplasm and nucleus, while LR localizes predominantly to the nucleus, entirely consistent with our data in dissociated neurons. Images are representative of 6 to 9 experiments. B–E , long-term potentiation (LTP) was induced by an LTP pairing protocol in CA1. YFP alone (n = 8) had no effect on LTP, but in contrast, WT RGS14 (n = 6) completely inhibits LTP in CA1, a hippocampal region where RGS14 is not natively found. Potentiation lasting only about 10 min was induced in RQ variant-expressing neurons (n = 9), whereas the LR variant (n = 9) failed to suppress LTP. ANOVA with Tukey’s post-hoc analysis demonstrated a statistically significant difference between YFP alone (control) and WT RGS14 at 5 ( p = 0.03) and 10 ( p = 0.006) min poststimulation, while RGS14 LR was not statistically different from YFP alone at either time point. RGS14 RQ was statistically different from YFP alone at 10 min poststimulation ( p = 0.018), but not 5 min. This suggests that the degree of mislocalization by these variants correlates with ablation of RGS14 function in CA1 neurons. Data is presented as mean ± SEM.

Article Snippet: Sections were then incubated for 24 h at 4 °C in this same NGS blocking/permeabilization buffer now including a mouse primary antibody raised against RGS14 (NeuroMab; clone N133/21) at 1:500 dilution.

Techniques: Blocking Assay, Generated, Virus, Injection, Incubation, Expressing, Derivative Assay, Variant Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

doi: 10.1074/jbc.RA120.016009

Figure Lengend Snippet: The RGS14 variant LR localizes to the nuclei of hippocampal neurons but does not alter hippocampal-based spatial learning in CRISPR/Cas9 mice. Mice carrying the LR variant were generated by CRISPR/Cas9 (RGS14 LR mice). A , expression and localization of RGS14 LR in CA2 hippocampal neurons were confirmed by immunohistochemical staining. Consistent with our slice electrophysiology data, RGS14-LR was predominantly nuclear, while RGS14 WT was localized to soma, dendrites, and spines but excluded from the nucleus of CA2 hippocampal neurons. The heterozygous mice exhibited a mixed nuclear-cytoplasmic phenotype. B , RGS14-LR mice (n = 20) and RGS14 WT (n = 20) littermates were subjected to the Morris Water Maze test. RGS14-LR mice do not exhibit enhanced spatial learning, suggesting a unique nuclear role of RGS14 on suppression of spatial learning.

Article Snippet: Sections were then incubated for 24 h at 4 °C in this same NGS blocking/permeabilization buffer now including a mouse primary antibody raised against RGS14 (NeuroMab; clone N133/21) at 1:500 dilution.

Techniques: Variant Assay, CRISPR, Generated, Expressing, Immunohistochemical staining, Staining

Journal: The Journal of Biological Chemistry

Article Title: Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

doi: 10.1074/jbc.RA120.016009

Figure Lengend Snippet: Proposed working model of RGS14 spatial-dependent inhibition of LTP. A , wild-type RGS14 ( green dot ) is disperse throughout the neuron, occupying multiple compartments including the nucleus, the cytoplasm, and the dendrites and spines ( red box ). Within a dendritic spine ( expanded red box ), Gα-GDP interaction with the G protein regulatory motif (G) brings RGS14 to the microcompartment necessary for inhibition of GPCR signaling by the RGS domain, inhibition of H-Ras/ERK signaling by the Ras binding domains (RBD), and inhibition of Ca ++ signaling, all of which support LTP in the absence of RGS14. B , variant forms of RGS14 ( green dots ) are sequestered in the nucleus and cannot reach dendrites ( red box ). Due to their mislocalization, they cannot interact with, and be recruited by, Gα-GDP to dendritic compartments ( expanded red box ). The lack of RGS14 at these microcompartments allows for unrestricted GPCR, H-Ras/ERK, and Ca ++ signaling, removing the block on LTP.

Article Snippet: Sections were then incubated for 24 h at 4 °C in this same NGS blocking/permeabilization buffer now including a mouse primary antibody raised against RGS14 (NeuroMab; clone N133/21) at 1:500 dilution.

Techniques: Inhibition, Binding Assay, Variant Assay, Blocking Assay

Journal: bioRxiv

Article Title: Endogenous Regulator of G protein Signaling 14 (RGS14) suppresses cocaine-induced emotionally motivated behaviors in female mice

doi: 10.1101/2024.09.12.612719

Figure Lengend Snippet: Immunofluorescence detection of RGS14 in (A) NAc, (B) BNST, and (C) amygdala in WT mice. Anatomical annotations (top) from the Allen Mouse Brain Reference Atlas (atlas.brain-map.org) aligned with tile-scan images of RGS14-immunostained tissue (bottom), pseudocolored to show relative fluorescence intensity. Highlighted atlas regions are approximately outlined in tissue sections. Sections ordered rostral to caudal. Scale bar = 250 µM. Anatomical abbreviations:: BLA: basolateral amygdala, BNST(ju,ov): bed nucleus of the stria terminalis-(juxtacapsular, oval), CeA: central amygdala, Ce(C,L,M): central amygdala-(capsular, lateral, medial), NAc: nucleus accumbens.

Article Snippet: Sections were washed in PBS and blocked for 1 hour in 5% normal goat serum/0.1% Triton-X/PBS, then incubated in mouse α-RGS14 (NeuroMab, Davis, CA, clone N133/21; 1:500) primary antibody in 5% normal goat serum/0.1% Triton-X/PBS at 4°C overnight.

Techniques: Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Endogenous Regulator of G protein Signaling 14 (RGS14) suppresses cocaine-induced emotionally motivated behaviors in female mice

doi: 10.1101/2024.09.12.612719

Figure Lengend Snippet: Immunofluorescence detection of RGS14 with D1R and D2R in RGS14-immunostained striatal tissue from Drd1-tdTomato and Drd2-EGFP reporter mice. A. Drd1-tdTomato tissue immunoreactivity; panels:: yellow (left) : D1R-tdTomato, magenta (middle) : RGS14, white (right ): overlap. B. Drd2-EGFP tissue immunoreactivity:: green (left) : D2R-EGFP, magenta (middle) : RGS14, white (right): overlap. Fluorophores were sequentially scanned in separate channels and computationally merged. Approximate borders of NAc core and shell outlined in white dashes. Scale bar = 250 µM. Anatomical abbreviations:: CPu : caudoputamen, NAc(C,S) : nucleus accumbens-(core, shell).

Article Snippet: Sections were washed in PBS and blocked for 1 hour in 5% normal goat serum/0.1% Triton-X/PBS, then incubated in mouse α-RGS14 (NeuroMab, Davis, CA, clone N133/21; 1:500) primary antibody in 5% normal goat serum/0.1% Triton-X/PBS at 4°C overnight.

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Endogenous Regulator of G protein Signaling 14 (RGS14) suppresses cocaine-induced emotionally motivated behaviors in female mice

doi: 10.1101/2024.09.12.612719

Figure Lengend Snippet: Immunofluorescence detection and relative quantification of RGS14 protein in NAc 2 hours after Coc challenge (i.p.) in naïve and Coc-experienced WT mice. A. Representative images of RGS14-immunostained NAc tissue from naive (left) and Coc-experienced (right) mice, pseudocolored to show relative fluorescence intensity. B. Mean fluorescence intensity (arbitrary units) in NAc core and shell of RGS14-immunostained tissue. Analyzed by Welch’s t-test. Error bars represent mean ± SEM. Symbols: †p < .10, * p < .05, ** p < .01, *** p < .001. Scale bar = 100 µm. Anatomical abbreviations:: AC : anterior commissure, NAc(C,S) : nucleus accumbens- (core, shell).

Article Snippet: Sections were washed in PBS and blocked for 1 hour in 5% normal goat serum/0.1% Triton-X/PBS, then incubated in mouse α-RGS14 (NeuroMab, Davis, CA, clone N133/21; 1:500) primary antibody in 5% normal goat serum/0.1% Triton-X/PBS at 4°C overnight.

Techniques: Immunofluorescence, Quantitative Proteomics, Fluorescence

Journal: bioRxiv

Article Title: Endogenous Regulator of G protein Signaling 14 (RGS14) suppresses cocaine-induced emotionally motivated behaviors in female mice

doi: 10.1101/2024.09.12.612719

Figure Lengend Snippet: A. Diagram of cocaine sensitization protocol (n = 48); cocaine (5mg/kg) or saline administered by i.p. injection. (B-G) Total ambulatory activity (beam breaks/30 min) each day of induction and challenge. B. Total ambulatory activity (error bars: ± S.E.M.) each day for all groups. There is a significant genotype × treatment × day interaction effect over induction (LMM) and a significant genotype × treatment interaction effect during cocaine challenge (Two-way ANOVA). C. Within-subjects changes in ambulatory activity from the first to the last day of induction (Ind1 to Ind5). Locomotor activity was decreased in Sal-treated controls as expected from habituation. Locomotor activity was significantly increased only in the RGS14KO+Coc group (paired pairwise t-tests^). D. In WT mice, there is a significant treatment × day interaction effect over induction (LMM), but no significant differences between WT+Coc and WT+Sal group locomotor activity were detected on any individual day (pairwise t-tests^). E. In RGS14-KO mice, there is a highly significant treatment × day interaction effect over induction (LMM). Locomotor activity is significantly elevated in the RGS14KO+Coc group over RGS14KO+Sal controls at Ind2-Ind5 (pairwise t-tests^) and challenge (Tukey HSD test). F. No significant genotype × day interaction in the Sal treatment group (LMM). G. In the cocaine treatment group, there is a significant genotype × day interaction effect over induction (LMM). Locomotor activity is significantly elevated in the RGS14KO+Coc group over WT+Coc controls at Ind5 (pairwise t-tests^) and challenge (Tukey HSD test).

Article Snippet: Sections were washed in PBS and blocked for 1 hour in 5% normal goat serum/0.1% Triton-X/PBS, then incubated in mouse α-RGS14 (NeuroMab, Davis, CA, clone N133/21; 1:500) primary antibody in 5% normal goat serum/0.1% Triton-X/PBS at 4°C overnight.

Techniques: Saline, Injection, Activity Assay

Journal: bioRxiv

Article Title: Endogenous Regulator of G protein Signaling 14 (RGS14) suppresses cocaine-induced emotionally motivated behaviors in female mice

doi: 10.1101/2024.09.12.612719

Figure Lengend Snippet: A. Diagram of cocaine-conditioned place preference (CPP) protocol (n = 32); cocaine (5 mg/kg) or saline administered by i.p. injection. B. Increased magnitude and duration of cocaine-conditioned place preference in RGS14-KO mice. Time spent on the cocaine-paired side (i.e. preference, Δpre-test) was significantly enhanced in WT and RGS14-KO groups at Post1 and Post2, an effect that is significant only in RGS14-KO mice at Post3. Preference was significantly enhanced in RGS14-KO mice over WT controls at Post2 (Tukey HSD test^). C. Increased magnitude and duration of cocaine-conditioned locomotor hyperactivity in RGS14-KO mice. Distance traveled on the cocaine-paired side (Δpre-test) was significantly enhanced exclusively in RGS14-KO mice at Post1 and Post2. ΔDistance was significantly enhanced in RGS14-KO mice over WT controls at Post1 (Tukey HSD test^). D. Correlation plots of cocaine-conditioned place preference (Δtime) and locomotor hyperactivation (Δdistance) at Post1 (shaded area: 95% confidence interval). There is a trend for a positive correlation in WT mice that is absent in RGS14-KO mice (Pearson’s test). E. RGS14-KO mice spend significantly less total time in the aversive white-floored compartment during pre-test (n = 32, Welch Two Sample t-test). F. Association with cocaine overcomes stronger inherent place aversion in RGS14-KO mice. No significant difference between WT and RGS14-KO groups in total time in the white-floored compartment once paired with cocaine (Post1, n = 16, Welch Two Sample t-test). All error bars represent mean ± SEM. Symbols: †p < .10, * p < .05, ** p < .01, *** p < .001. Carat (^) indicates Bonferroni correction.

Article Snippet: Sections were washed in PBS and blocked for 1 hour in 5% normal goat serum/0.1% Triton-X/PBS, then incubated in mouse α-RGS14 (NeuroMab, Davis, CA, clone N133/21; 1:500) primary antibody in 5% normal goat serum/0.1% Triton-X/PBS at 4°C overnight.

Techniques: Conditioned Place Preference, Saline, Injection