Lack of GPR88 enhances medium spiny neuron activity and alters motor- and cue- dependent behaviors Albert Quintana, Elisenda Sanz, Wengang Wang, Granville P. Storey, Ali D. Güler Matthew J. Wanat, Bryan A. Roller, Anna La Torre, Paul S. Amieux, G. Stanley McKnight, Nigel S. Bamford and Richard D. Palmiter Supplementary Figures and Tables Supplementary Figure 1. Intact striatal cell populations in Gpr88 Cre/Cre mice No differences in the staining for markers of striatal interneurons (Chat, Calretinin, NPY, Parvalbumin) or MSNs (Dynorphin, Enkephalin) were observed in striata of Gpr88 Cre/Cre (Cre/Cre) mice compared to Gpr88 +/+ (+/+; n=3 each). Enkephalin immunoreactivity is primarily observed in the terminals. Hence, globus pallidus is shown. Scale bars: Chat to Parvalbumin: 250 µm. Dynorphin: 125 µm. Enkephalin: 1000 µm. 1
Supplementary Figure 2. Locomotor responses and pain sensitivity of Gpr88 Cre/Cre mice in several behavioral tests (a) Swim speeds of Gpr88 +/+ (n=12) and Gpr88 Cre/Cre (n=9) in each of the probe trials in the Morris water maze. (Student T-test, n.s.). (b) Distance traveled by Gpr88 +/+ (n=14) and Gpr88 Cre/Cre (n=10) mice during the probe trials in the Morris water maze (Student T-test, n.s.) (c-d) Latencies to swim to the decision point (end of stem, c; F (1,19) =7.31, ** p<0.01, genotype, rmanova, turn-phase, Bonferroni post-test) or the platform (d, F (1,19) =5.29, * p<0.05 (turn-phase) and F (1,19) =7.46, *** p<0.001(cue-phase), genotype, Bonferroni post-test) of Gpr88 +/+ (n=12) and Gpr88 Cre/Cre (n=9) mice in the U-maze test (e) Latencies to escape after the tone in a two-way active avoidance test of Gpr88 +/+ (n=14) and Gpr88 Cre/Cre (n=10) mice. Student T-test (n.s.). (f) Latencies to escape a shock in a two-way active avoidance test of Gpr88 +/+ (n=14) and Gpr88 Cre/Cre (n=10) mice. Student T-test (n.s.). 2
Supplementary Figure 3. Electrode placement and control experiments for striatal in vivo multi-unit recording 3
Supplementary Figure 3. Electrode placement and control experiments for striatal in vivo multi-unit recording (a) Representative image of an hematoxylin staining showing electrode placement (left panel, white arrow). Scale bar: 250 µm. Diagrams show correct electrode placement in the dorsal striatum (CPu) of all recorded animals (n=6 Gpr88 +/+, n=6 Gpr88 Cre/Cre ). (b,c) Locomotor activity (b; F(1,12)=9.44, ** p<0.01, *** p<0.001, genotype, rmanova Bonferroni post-test) and number of rearings (c; F(1,12)=7.04, * p<0.05, genotype, rmanova Bonferroni post-test) of Gpr88 +/+ (n=8) and Gpr88 Cre/Cre (n=6) mice after 10- min daily exposure to the recording environment and analyzed using Ethovision software (Noldus). (d,e) Examples of multi-unit clusterings and waveforms for Gpr88 +/+ (d) and Gpr88 Cre/Cre (e). Units 2 and 3 (d) and 2,3,4 (e) presented valley widths greater than 300µs and were considered putative MSNs. For simplicity, waveforms for some clusters is not shown (dark gray). Non clustered areas are shown in light gray. No differences in clustering statistics were observed from recordings containing putative MSNs (for single unit clusters, comparisons are made against unsorted spikes, Gpr88 +/+ vs. Gpr88 Cre/Cre : F=10.88±4.69 vs. 15.38±8.59; J3=1.28±0.31 vs. 1.47±0.44; PsF=17603±5962 vs. 18282±4714; DB 0.32±0.03 vs. 0.27±0.02; Dn=1.56±0.11 vs. 1.85±0.20,; for multi-unit clusters, Gpr88 +/+ vs. Gpr88 Cre/Cre : F=19.53±2.90 vs. 21.15±4.64; J3=8.57±1.92 vs. 7.20±2.09; PsF=69669±26758 vs. 49874±10443; DB 0.60±0.15 vs. 0.37±0.07; Dn=1.56±0.29 vs. 1.94±0.33). All values are given as mean ± SEM. 4
Supplementary Figure 4. K+ Current responses in MSNs and altered D1R- and D2R- mediated responses in Gpr88 Cre/Cre mice 5
Supplementary Figure 4. K+ Current responses in MSNs and altered D1R- and D2R- mediated responses in Gpr88 Cre/Cre mice (a)voltage-current relationships for the mean raw and Cs+-sensitive currents and current densities in Gpr88 +/+ and Gpr88 Cre/Cre MSNs. (b) Voltage-step protocol and representative traces in MSNs from Gpr88 +/+ (n=10) and Gpr88 Cre/Cre (n=7) mice showing evoked currents, effects of 1 mm Cs+ and digitally subtracted Cs+-sensitive currents. Calibration bars refer to all traces. (c) Representative sections of retrobead labeling of direct and indirect pathway MSNs. For direct pathway MSN labeling, red retrobeads were stereotaxically injected in the substantia nigra pars reticulata (SNr). Colocalization with GFP (green) in the striatum (CPu) denotes targeting of SNr (direct) projecting MSNs (white arrows). For indirect pathway MSN labeling, green retrobeads were stereotaxically injected in the Globus pallidus external part (GPe). Colocalization of green beads with GFP (red) in the striatum denotes targeting of GPe (indirect) projecting MSNs (white arrows). cp: cerebral peduncle. LV: Lateral Ventricle. Scale bar: Injection site: 250 µm; Close-ups: 17 µm (d) Mean tonic GABA currents in whole-cell patch clamp recordings of retrobead-labeled direct (D1+; n=5 cells, 3 mice) or indirect (D2+; n=6 cells, 3 mice) MSNs from Gpr88 Cre/Cre mice show equivalent responses following bicuculline. Grey line denotes Gpr88 +/+ values (from Fig. 5c) as reference. (e-f) Evoked EPSCs from retrobead-labeled direct (D1+; n=7 cells, 3 mice) or indirect (D2+; n=9 cells, 3 mice) MSNs from Gpr88 Cre/Cre mice MSNs had similar mean peak evoked currents with increasing stimulation intensity (e) and similar average stimulation currents required to reach threshold (f).grey lines denote Gpr88 +/+ values (from Fig. 5k,l) as reference. (g) Locomotor activity (90 min) of Gpr88 +/+ (n=5) and Gpr88 Cre/Cre (n=5) mice after daily (5-day) amphetamine administration (sal: saline). F(1,8)=9.80, *p<0.05, genotype, rmanova. (h) Locomotor activity (60 min) of Gpr88 +/+ (n=6) and Gpr88 Cre/Cre (n=6) mice in response to increasing doses of D1R agonist SKF-81297. F(1,10)=9.18, *p<0.05, genotype, rmanova; Bonferroni post-test). (i) Locomotor activity (3 h) of Gpr88 +/+ (n=6) and Gpr88 Cre/Cre (n=5) mice in response to increasing doses of D2R agonist quinpirole. F(1,9)=49.91, ** p<0.01, *** p<0.001, genotype, rmanova; Bonferroni post-test). 6
Supplementary Figure 5. Full-length blots from Figure 6b Images of western blot gels with molecular weight markers from Figure 6b. 7
Supplementary Figure 6. Virally-mediated GPR88 transduction 8
Supplementary Figure 6. Virally-mediated GPR88 transduction (a) Schematic representation of the AAV-based viral vector used for in vitro neuronal transduction: AAV-CBA-GPR88-TdTomato. (b) Neuronal primary cultures transduced with AAV-CBA-GPR88 show targeting of the fusion protein to the membrane Colocalization of TdTomato with PSD95 (b, arrows). Bottom row: Higher magnification image from top row (dashed square). (c) TdTomato-expressing boutons are found in close proximity of the pre-synaptic marker bassoon (arrows). Bottom row: Higher magnification image from top row (dashed square). Bar: b (top row): 35 µm; (bottom row): 10 µm. c (top row):10 µm; (bottom row): 5.75 µm. (d) Extensive rostro-caudal TdTomato expression was observed in VR-KO mice, which was restricted to the striatum. LV: Lateral ventricle. 3V: Third ventricle. CPu: Caudate-Putamen. HPC: Hippocampus. Scale bar: 1000 µm. 9
Supplementary Table 1. Characterization of Gpr88-Cre mediated reporter expression. Description of nuclei showing Gpr88-Cre dependent recombination after breeding with floxed-stop TdTomato reporter mice. The percentage of red cells in each positive nucleus was estimated. 10
Supplementary Table 2. Striatal monoamine and metabolite content in Gpr88 Cre/Cre mice Striatal monoamine and metabolites content in Gpr88 +/+ and Gpr88 Cre/Cre mice (n=9 each). Striatal punches (1mm diameter) were snap-frozen in liquid nitrogen and contents determined by HPLC (CMN/KC Neurochemistry Core Lab, Vanderbilt University). *p<0.05, Student t-test, two-tailed. Supplementary Table 3. Striatal gene expression changes in Gpr88 Cre/Cre mice (a) Gene ontology analysis of the selected genes in an Illumina Beadarray analysis in striatal punches of Gpr88 +/+ and Gpr88 Cre/Cre mice (n=2) each. Selection criteria was a fold-difference >1.2 and DiffScore> 13 or p<0.05. The top two over-represented families are shown. 11
Supplementary Table 3 (cont.). Striatal gene expression changes in Gpr88 Cre/Cre mice (b) Genes upregulated in Gpr88 +/+ striata in an Illumina Beadarray analysis in striatal punches of Gpr88 +/+ (WT) and Gpr88 Cre/Cre (KO) mice (n=2) each. Selection criteria was a fold-difference >1.35 and DiffScore>13 or p<0.05. 12
Supplementary Table 3 (cont.). Striatal gene expression changes in Gpr88 Cre/Cre mice (c) Genes upregulated in Gpr88 Cre/Cre striata in an Illumina Beadarray analysis in striatal punches of Gpr88 +/+ (WT) and Gpr88 Cre/Cre (KO) mice (n=2) each. Selection criteria was a fold-difference >1.35 and DiffScore>13 or p<0.05. 13
Supplementary Table 4. qrt-pcr and genotyping primer sequences All qrt-pcr primers, except those for Gpr88h/m, were selected using primerbank database. Sequences are provided 5 to 3. 14