Loss of dopamine D1 receptors and diminished D1/5 receptor-mediated ERK phosphorylation in the periaqueductal gray after spinal cord lesion

Abstract

Neuropathic pain resulting from spinal cord injury is often accompanied by maladaptive plasticity of the central nervous system, including the opioid receptor-rich periaqueductal gray (PAG). Evidence suggests that sensory signaling via the PAG is robustly modulated by dopamine D1- and D2-like receptors, but the effect of damage to the spinal cord on D1 and D2 receptor protein expression and function in the PAG has not been examined. Here we show that 21days after a T10 or C6 spinothalamic tract lesion, both mice and rats display a remarkable decline in the expression of D1 receptors in the PAG, revealed by western blot analysis. These changes were associated with a significant reduction in hindpaw withdrawal thresholds in lesioned animals compared to sham-operated controls. We investigated the consequences of diminished D1 receptor levels by quantifying D1-like receptor-mediated phosphorylation of ERK1,2 and CREB, events that have been observed in numerous brain structures. In naïve animals, western blot analysis revealed that ERK1,2, but not CREB phosphorylation was significantly increased in the PAG by the D1-like agonist SKF 81297. Using immunohistochemistry, we found that SKF 81297 increased ERK1,2 phosphorylation in the PAG of sham animals. However, in lesioned animals, basal pERK1,2 levels were elevated and did not significantly increase after exposure to SKF 81297. Our findings provide support for the hypothesis that molecular adaptations resulting in a decrease in D1 receptor expression and signaling in the PAG are a consequence of SCL.

Keywords: SKF 81297; behavior; extracellular signal regulated kinase; hyperalgesia; neuropathic pain; spinal cord injury.

Figure 1

D1 dopamine receptor protein levels are reduced in the PAG of rats following SCL. A, Basal expression and cellular distribution of D1 and D2 receptors, ERK1,2 and actin in the PAG. Cyto = cytoplasm, DSM = detergent soluble membranes, and DRM = detergent resistant membranes. B, Expression of D1 receptors in the PAG of sham and SCL animals 7 and 21 days after surgery, detected with a D1-specific antibody. C, Quantitation of D1 receptor protein indicates that, compared to sham animals, D1 levels are significantly reduced 7 days after SCL, and remain at low levels through 21 days after injury (F(2, 20) = 11.99, p = 0.0004, ANOVA with Tukey-Kramer post hoc test). (*) indicates significant difference relative to sham surgery. The number of experimental animals per group is indicated in the graph.

Figure 2

The SCL-induced decreases in withdrawal threshold and D1 receptor protein are species-independent. A, Mechanical withdrawal thresholds progressively decline over time following SCL in mice. B, Western blot analysis showing D1 receptor protein levels in three Sham and three SCL animals, compared to β-actin levels. C, Quantification of D1 receptor protein levels in Sham and SCL animals as a function of β-actin levels reveals a significant reduction in D1 receptors after SCL (t(4) = |3.02|, p = 0.039, two-tailed t-test). (*) indicates significant difference relative to sham surgery. The number of animals per condition is indicated in the graph.

Figure 3

Elevation of ERK phosphorylation in the PAG following D1/5 receptor stimulation. A, Systemic exposure of mice to SKF 81297 elicits an increase in pERK1,2 levels in the PAG 15 min after i.p. injection. B, Quantitation of pERK1,2 reveals a significant enhancement of ERK activation in response to agonist (t(13) = |2.44|, p = 0.03). C, ERK phosphorylation is transient; D, pERK levels were not significantly different 30 min after SKF 81297 administration (t(12) = |2.29|, p = 0.93). Data were analyzed using the two-tailed t-test with (*) indicating significant difference relative to saline-injected controls. The number of experimental animals per condition is indicated in the graphs.

Figure 4

Coupling of ERK phosphorylation to D1/5 receptor stimulation in the PAG is impaired in animals with SCL. A, In PAG slices derived from sham animals, the number of pERK1,2-positive cells is low compared to slices from sham animals perfused with SKF 81297 (B), where the number of pERK1,2 immunoreactive cells increases significantly 15 min after stimulation. By contrast, the number of pERK1,2-positive cells in unstimulated PAG slices derived from SCL animals (C) is not significantly different from pERK1,2 levels in PAG slices from SCL that were exposed to SKF 81297 for 15 min. Scale bar for A-D, 400 μm; A1-D1, 100 μm; A2-D2, 50 μm. Statistical information is presented in Table 1.