(A) Schematic of affinity purification of EGFP-tagged polysomes (originating from the target cell population; green polysomes) using anti-GFP antibody-coated beads. (B) Transmission electron micrographs of anti-GFP coated magnetic beads after incubation with extracts taken from HEK293T cells transfected with an empty vector (left panel) or the EGFP-L10a construct (right panel); images acquired at 50,000x magnification, inserts enlarged by a factor of 2.3x.

(A) Immunohistochemistry to EGFP in adult sagittal sections from the D2 BACarray line CP101. (B) Characterization of D2 BACarray line CP101 striatal MSN cells: direct EGFP fluorescence (left panel with high-magnification image insert); enkephalin immunohistochemical staining (middle panel); merge (right panel, with 20 μm scale bar). (C) Immunohistochemistry to EGFP in adult sagittal sections from the D1 BACarray line CP73. (D) Characterization of D1 BACarray line CP73 striatal MSN cells: direct EGFP fluorescence (left panel); enkephalin immunohistochemical staining (middle panel); merge (right panel).

(A) Representative purification of EGFP-tagged L10a and co-purification of untagged ribosomal protein L7 from D1 BACarray animals but not wild-type littermates (D1, samples from D1 BACarray mice; WT, samples from wild-type littermates; IN, 1% Input; UB, 1% Unbound; IP, 6.5% Immunoaffinity purified sample). EGFP-L10a signal is only present in the D1 IP lane because the IP samples were more concentrated relative to IN and UB. (B) Representative purification of 18S and 28S rRNA from D1 BACarray transgenic animals (green) but not wild type littermates (red) as detected by Bioanalyzer PicoChips (Agilent Technologies). 28S rRNA runs at ~47 sec, 18S rRNA runs at ~43 sec, and the Picochip marker peak runs at ~23 sec. (C) Normalized expression values from Affymetrix Mouse Genome 430 2.0 arrays are plotted for D1 and D2 BACarray samples. Middle diagonal line represents equal expression, and lines to each side represent 1.5-fold enrichment in either cell population. Axes are labelled for expression in powers of 10. The probesets of well-studied genes known to be differentially expressed are represented in blue.

(A) Projection of an EGFP-labeled MSN from a BAC D2 mouse. The cell was patched with a pipette containing Alexa 594 (50 μM) for visualization and Fluo-4 (200 μM) for measuring changes in intracellular Ca²⁺ (right). Cells were voltage clamped at −70 mV. A puffer pipette containing sphingosine 1-phosphate (S1P, 10 μM) was positioned near a dendrite, 60–80 μm from the soma (left/cartoon). (B) High magnification images of a dendritic segment (control, left panel) show an increase in Ca²⁺ associated with S1P application (S1P puff, center panel) that reversed with washing (wash, right panel). The change in Ca²⁺ was determined by calculating the percent change in fluorescence of Fluo-4 relative to that of Alexa 594 ( G/R). The blue circle in the first panel indicates the analyzed region of interest (ROI). (C) Time course showing the S1P induced increase in intracellular Ca²⁺ in the ROI from B (orange trace); similar recordings from BAC D1 MSNs (black trace) or 10 μM thapsigargin loaded BAC D2 MSNs (green trace) did not reveal any significant changes in dendritic Ca²⁺ levels with S1P application. (D) Box plot summarizing the S1P effects. Percent increase in fluorescence (ΔG/R) in BAC D2 MSNs (median=146%, range 44 to 294%, n=6); BAC D1 MSNs (median=17%, range 13 to 22%, n=4); and thapsigargin-loaded BAC D2 MSNs (median=4%, range –9 to 10%, n=4).

(A) Representative spontaneous mIPSCs traces from BAC D1 striatonigral neurons (expressing soluble EGFP under the D1 promoter) taken from mice treated for 15 days with saline or (B) cocaine (20 mg/kg/day). (C) Bar graph summary of mean mIPSC frequency showing a significant increase in BAC D1 striatonigral neuron mIPSCs frequency following cocaine treatment. (D) Bar graph summary showing that the number of small amplitude mIPSCs (<75 pA) in equal length records (7 min) increased in BAC D1 striatonigral neurons following cocaine treatment. (E) Representative variance-mean current plots from saline treated (black symbols) and cocaine treated (red symbols) BAC D1 neurons suggesting that the cocaine-induced small amplitude events arise from synapses that have fewer GABA_A receptors (N) per synapse but receptors with an unchanged unitary receptor conductance (N) (saline N=33, N=31pS; cocaine N=29, N=31pS; see for means). (F) Representative spontaneous mIPSCs traces from BAC D2 striatopallidal neurons following saline treatment for 15 days and (G) following cocaine treatment for 15 days. (H) Bar graph summary of mean mIPSC frequency in saline and cocaine treated D2 neurons, showing no effect of treatment condition. (I) Bar graph summary showing that the number of small amplitude mIPSCs (<75 pA) in equal length records (7 min) was not altered by treatment condition in BAC D2 neurons. (J) Representative variance-mean current plots showing that cocaine treatment did not change in the number of receptors per synapse or the unitary receptor conductance in BAC D2 neurons (saline N=34, N=31pS; cocaine N=33, N=30pS; see for means).

(A) Immunohistochemistry to EGFP in adult sagittal sections from the Chat BACarray line DW167. (B) Indirect immunofluorescent characterization of Chat BACarray line DW167 brain stem facial motor nucelus: EGFP staining (left panel); Chat staining (middle panel) and merge (right panel, with 20 μm scale bar). (C) Immunohistochemistry to EGFP in adult sagittal sections from the Pcp2 BACarray line DR166. (D) Indirect immunofluorescent characterization of Pcp2 BACarray line DR166 Purkinje cell neurons: EGFP staining (left panel); Calbindin-D28K staining (middle panel) and merge (right panel, with 20 μm scale bar).

Scatterplots of D1, D2, Chat, and Pcp2 BACarray data compared to a reference mRNA sample reveal hundreds of genes enriched in each cell type (A–D). Green circles indicate Affymetrix biotinylated spike-in controls; blue circles indicate known cell-specific markers; and red circles indicate probesets for known glial genes (negative controls; ). Lines on either side of the diagonal mark 2-fold enrichment. Axes are labelled for expression in powers of 10. Venn diagrams of the top 1,000 enriched probesets (with expression value cut-off >100) for each cell type reveal that each cell type has a unique pattern of enriched genes (E–H).