Abnormal nociception and opiate sensitivity of STOP null mice exhibiting elevated levels of the endogenous alkaloid morphine

Abstract

Background: Mice deficient for the stable tubule only peptide (STOP) display altered dopaminergic neurotransmission associated with severe behavioural defects including disorganized locomotor activity. Endogenous morphine, which is present in nervous tissues and synthesized from dopamine, may contribute to these behavioral alterations since it is thought to play a role in normal and pathological neurotransmission.

Results: In this study, we showed that STOP null brain structures, including cortex, hippocampus, cerebellum and spinal cord, contain high endogenous morphine amounts. The presence of elevated levels of morphine was associated with the presence of a higher density of mu opioid receptor with a higher affinity for morphine in STOP null brains. Interestingly, STOP null mice exhibited significantly lower nociceptive thresholds to thermal and mechanical stimulations. They also had abnormal behavioural responses to the administration of exogenous morphine and naloxone. Low dose of morphine (1 mg/kg, i.p.) produced a significant mechanical antinociception in STOP null mice whereas it has no effect on wild-type mice. High concentration of naloxone (1 mg/kg) was pronociceptive for both mice strain, a lower concentration (0.1 mg/kg) was found to increase the mean mechanical nociceptive threshold only in the case of STOP null mice.

Conclusions: Together, our data show that STOP null mice displayed elevated levels of endogenous morphine, as well as an increase of morphine receptor affinity and density in brain. This was correlated with hypernociception and impaired pharmacological sensitivity to mu opioid receptor ligands.

Figure 1

Immunodetection of morphine-like compounds in the brain of WT and STOP animals. A. Control experiment using anti-morphine immunoadsorbed mouse monoclonal antibody (same incubation time as in B and C). B. Immunodetection of morphine-like molecules in WT mouse brain. Sagittal slices were incubated with a mouse monoclonal anti-morphine antibody and visualized with an HRP-conjugated donkey anti-mouse IgG. C. Immunodetection of morphine-like compounds in STOP null brain. Boxes indicated the areas of interest. Ca-c panels correspond to the areas defined by the boxes. Arrows indicate eM immunoreactive neurons. S1HL, hind limb primary somatosensory (S1HL) cortex.

Figure 2

Quantification of the morphine contents in different mouse brain areas of WT and STOP null mice using a morphine-specific ELISA. Amounts of endogenous morphine (ng) present in the cerebellum, brainstem and the remaining brain tissue, per gram of wet tissue. The values correspond to the morphine amount determined for STOP null (n = 10 animals; white bars) and WT (n = 13 animals; grey bars) mice in different cerebral areas as indicated. For spinal cord, the values correspond to the morphine amount determined for STOP null (n = 9 animals; white bars) and WT (n = 9 animals; grey bars) mice. Amounts of eM in the STOP null and WT groups were found statistically different (mean +/- SD, Mann-Whitney test using Bonferroni correction; **: p < 0.01; ***: p < 0,001).

Figure 3

Characterization of morphine in mouse brain extracts. Morphine qualification was performed in selected reaction monitoring (SRM) mode with the following transitions: m/z 286.2 → 165.1 (see also additional file 1). SRM trace of the specific transition for morphine in A: standard samples of morphine (50, 100, 500 and 1000 fmol); B: WT brain animals and C: STOP null brain animals.

Figure 4

[³H]-Morphine binding in WT and STOP null mice. A : Saturation analysis of [³H]-morphine binding to the MORs in wild-type and STOP null mice. Data are presented as means ± S.E.M. n = 3. B : Transformation of the data by linear regression_Scatchard plots. Data are presented as means ± S.E.M. n = 3. C : Comparison of the B_max values of MORs in wild-type and STOP null mice. D : Comparison of the K_d (C) values of MORs in wild-type and STOP null mice. Data are presented as means ± S.E.M. n = 3, Statistical significance between STOP null versus WT mice is indicated as follow: * p < 0.05, ** p < 0.01, *** p < 0.001 by Student t-test. E : Western blot analysis showing MOR immunolabel present in 50 μg of brain proteins of wild-type and STOP null mice (n = 3). No cross reactivity was found for the secondary antibody used.

Figure 5

Thermal Nociceptive thresholds and behaviors of WT and STOP null mice. A. Mechanical von Frey thresholds of WT (n = 17) and STOP null (n = 16) mice. B. Thermal hot nociceptive thresholds (B1) and behaviors (B2: total number of jumps between 30 and 43°C) for WT (n = 16) and STOP null mice (n = 16). C. Thermal cold nociceptive thresholds (C1) and behaviors (C2: total number of jumps between 20 and 0°C) for WT (n = 10) and STOP null mice (n = 18). Statistical significance between STOP null versus WT mice is indicated as follow: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Effects of morphine and naloxone on mechanical nociceptive threshold. A. Effects of a single subcutaneous morphine injection, at 10 mg/kg (A1) and 1 mg/kg (A2), on mechanical von Frey threshold for WT (n = 6) and STOP null mice (n = 6). B. Effects of a single subcutaneous naloxone injection, at 1 mg/kg (B1) and 0.1 mg/kg (B2), on mechanical von Frey threshold of WT (n = 6) and STOP null (n = 6) mice. Statistical significance is indicated as follow: * p < 0.05, ** p < 0.01.