Striatal CB1 and D2 receptors regulate expression of each other, CRIP1A and δ opioid systems

Abstract

Although biochemical and physiological evidence suggests a strong interaction between striatal CB1 cannabinoid (CB1 R) and D2 dopamine (D2 R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB1 R or D2 R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB1 R and D2 R knockdown reduced striatal dopaminergic-stimulated [(35) S]GTPγS binding, and D2 R knockdown reduced pallidal WIN55212-2-stimulated [(35) S]GTPγS binding. Decreased D2 R and CB1 R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB1 Rs or D2 Rs, and over-expression of CRIP1a. Down-regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D-Pen2, D-Pen5]-enkephalin-stimulated [(35) S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB1 R or D2 R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system.

Fig. 1

Effects of adeno-associated viral (AAV)-shRNA-mediated in vivo knockdown of CB₁R and D₂R on mRNA and protein levels. A mixture of three AAV-shRNAs specifically against CB₁R or D₂R was stereotaxically injected unilaterally into the dorsal striatum of adult male Sprague–Dawley rats. (a, b) qPCR analysis of shRNA-mediated knockdown of CB₁R (a) or D₂R (b) over a 56 day post-AAV injection period. Knockdown of CB₁Rs occurred concurrently with decreased D₂R mRNA levels and increased cannabinoid receptor interacting protein 1a (CRIP1a) mRNA levels (a). Knockdown of D₂R occurred concurrently with decreased CB₁R mRNA levels, and increased CRIP1a mRNA levels (b). (c, d) Immunohistochemical analysis of AAV-mediated knockdown of CB₁Rs (c) and D₂Rs (d). At 17 days post-AAV injection, 40 μm brain slices were obtained from the dorsal striatum (checkered bar), globus pallidus (striped bar), and entopeduncular nucleus (solid bar), and antibody staining was performed as described in the text. shRNA-mediated knockdown of CB₁Rs resulted in significantly decreased CB₁R protein levels in all three brain regions and significantly decreased D₂R levels in the dorsal striatum and entopeduncular nucleus (c). shRNA-mediated knockdown of D₂R resulted in significantly decreased D₂R protein levels in all three brain regions and significantly decreased CB₁R protein levels in the dorsal striatum and entopeduncular nucleus (d). Knockdown of CB₁R or D₂R significantly increased protein levels of CRIP1a in both the dorsal striatum and its outflow projections (c, d). Data are shown as mean ± SEM (n = 5; *p < 0.05, ^#p < 0.01 paired Student’s two-tailed t-test).

Fig. 2

Effects of adeno-associated viral (AAV)-mediated over-expression of cannabinoid receptor interacting protein 1a (CRIP1a) in the rat striatum. (a, b) AAV-CRIP1a was unilaterally injected into rat dorsal striatum. (a) Animals were killed 3, 5, 10, and 17 days post-AAV-CRIP1a injection, and 2 × 2 mm brain punches were taken at the site of injection for qPCR analyses. Neuron-specific enolase (eno2) served as the reference standard and data were analyzed using the ΔΔCT method with the contralateral side serving as the control. AAV-CRIP1a treatment effects on CRIP1a, CB₁R, and D₂R transcript levels are shown as means ± SEM (n = 5, *p < 0.05, ^#p < 0.01 paired Student’s two-tailed t-test). (b) Immunohistochemical analysis from 40 μm coronal brain slices was performed at 17 days post-AAV-CRIP1a treatment. Injection of AAV-CRIP1a significantly increased CRIP1a protein levels in the dorsal striatum (checkered bar) and the outflow projections to the globus pallidus (striped bar) and entopeduncular nucleus (solid bar), but did not significantly alter the immunoreactive protein levels of CB₁R, D₂R or D₁R. Data are shown as means ±SEM (n = 5; *p < 0.05, ^#p < 0.01 paired Student’s two-tailed t-test).

Fig. 3

Influence of CB₁R or D₂R knockdown or CRIP1a over-expression on receptor-mediated G protein activation and downstream signaling. [³⁵S]GTPγS binding assays and immunohistochemical analyses for phosphoERK and phosphoCREB were performed on coronal brain slices from rats killed 17 days post-adeno-associated viral injection, as described in the text. (a, b) Striatal (checkered bars) or globus pallidus (striped bars) 20 μm brain slices were subjected to [³⁵S]GTPγS binding stimulated by the D₂R agonist N-propylnorapo-morphine (NPA) [3 μM] (a) or the CB₁R agonist WIN55212 [3 μM] (b). Data are reported as a percent of the contralateral control as 100% for each rat (means ± SEM, n = 4 rats) for [³⁵S]GTPγS binding (each with three technical replicates), and analyzed for differences between ipsilateral versus contralateral side by paired Student’s two-tailed t-test (*p < 0.05, ^#p < 0.01). Significant reductions in D₂R-stimulated G protein activity in the striatum were observed following knockdown of either the CB₁R or D₂R (a). A significant reduction in CB₁R-stimulated G protein activity in the globus pallidus was observed after knock-down of the D₂R receptor (b). (c, d) Immunohistochemical analyses of phosphoERK/total ERK and phosphoCREB/total CREB ratios following knockdown of CB₁R or D₂R, or over-expression of CRIP1a, were performed as described in the text. PhosphoERK and phosphoCREB protein levels were calculated as a ratio of phosphorylated to total ERK and CREB, respectively. No changes due to CB₁R or D₂R, or over-expression of CRIP1a were observed in total ERK or total CREB protein levels in any region (data not shown). Data are reported as a percent of the contralateral control as 100% for each rat (means ± SEM, n = 5 rats), and analyzed as the difference between ipsilateral versus contralateral side by paired Student’s two-tailed t-test (*p < 0.05, ^#p < 0.01, ^$p < 0.001). PhosphoERK/total ERK ratios were significantly reduced in both the striatum (checkered bar) and its outflow projections to the globus pallidus (striped bar) and entopeduncular nucleus (solid bar) following knockdown of CB₁R or over-expression of CRIP1a. PhosphoERK/total ERK ratios following knockdown of D₂R were changed in a bidirectional manner, with a significant decrease in the dorsal striatum and significant increases in the globus pallidus and entopeduncular nucleus (c). The phosphoCREB/total CREB ratio in the globus pallidus was significantly increased following D₂R knockdown (d).

Fig. 4

Knockdown of CB₁R and D₂R decreases striatal opioid peptide precursors and increases DOR1 transcript levels. Pro-enkephalin, pro-dynorphin, and opioid receptor mRNA levels were quantitated by qPCR on striatal brain punches collected 17 days after a single unilateral injection into the dorsal striatum of adeno-associated viral (AAV)-shCB₁R (a), AAV-shD₂R (b), or AAV over-expressing CRIP1a (c). Data were analyzed using the ΔΔCT method with the contralateral side as the control (100%), and are shown as means ± SEM (n = 5 rats). Differences between data from ipsilateral versus contralateral sides were statistically determined using a paired Student’s two-tailed t-test (*p < 0.05, ^#p < 0.01). Significant decreases in pro-enkephalin and pro-dynorphin and increases in DOR1 transcripts were observed as indicated.

Fig. 5

Opioid receptor levels and G protein activation following striatal knockdown of CB₁R or D₂R or over-expression of CRIP1a. Data are shown as immunoreactive MOR1 and DOR1 protein levels as a percent of the contralateral control value (100%) (means ± SEM, n = 4 rats) in the striatum (checkered bar), globus pallidus (striped bar) and entopeduncular nucleus (solid bar), and analyzed by a paired Student’s two-tailed t-test (*p < 0.05, ^#p < 0.01, ^$p < 0.001. (a) Knockdown of CB₁R or D₂R, and over-expression of CRIP1a in the dorsal striatum did not significantly alter MOR1 protein levels in any regions measured. (b) Knockdown of CB₁R or D₂R and over-expression of CRIP1a significantly increased DOR1 protein levels as indicated. (c) Striatal brain slices were subjected to [³⁵S] GTPγS binding stimulated by the MOR1 agonist D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin [10 μM] (checkered bar) or the DOR1 agonist [D-Pen2, D-Pen5]-enkephalin [10 μM] (solid bar). Data are reported as a percent of the contralateral control side (100%) (means ± SEM, n = 4 rats) from [³⁵S]GTPγS binding measured in triplicates), and were analyzed using a paired Student’s two-tailed t-test (*p < 0.05, ^$p < 0.001).