Paradoxical hyperalgesia induced by mu-opioid receptor agonist endomorphin-2, but not endomorphin-1, microinjected into the centromedial amygdala of the rat

Abstract

The effects of endomorphin-2 or endomorphin-1 microinjected into the centromedial amygdala on the thermally-induced tail-flick response were studied in male CD rats. Microinjection of endomorphin-2 (8.7-35.0 nmol) given into the centromedial amygdala time- and dose-dependently decreased the tail-flick latencies. On the other hand, endomorphin-1 (8-32.6 nmol) given into the same site did not cause any change of the tail-flick latency. However, endomorphin-1 (32.6 nmol) or endomorphin-2 (35.0 nmol) given into the basolateral site of amygdala did not affect the tail-flick latency. Pretreatment with the antiserum against dynorphin A(1-17) (200 microg) significantly reversed the decrease of the tail-flick latency induced by endomorphin-2. The decrease of the tail-flick latency induced by endomorphin-2 was also blocked by the endomorphin-2 selective micro-opioid receptor antagonist 3-methoxynaltrexone (6.4 pmol) and by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (30 nmol), but not by the kappa-opioid receptor antagonist nor-binaltorphimine (6.6 nmol). It is concluded that endomorphin-2, but not endomorphin-1, given into the centromedial amygdala stimulates a 3-methoxynaltrexone-sensitive mu-opioid receptor subtype to induce the release of dynorphin A(1-17), which then acts on the NMDA receptor, but not kappa-opioid receptor for producing hyperalgesia. This conclusion is further supported by the additional findings that dynorphin A(1-17) (2.3 nmol) given into the centromedial amygdala also caused the decrease of the tail-flick latency, which was similarly blocked by the NMDA receptor antagonist MK-801 (30 nmol), but not kappa-opioid receptor antagonist nor-binaltorphimine (6.6 nmol).

Fig. 1

Coronal sections of the atlas of Paxinos and Watson (1997) showing the injection sites for drugs and saline vehicle at the centromedial and basolateral amygdala.

Fig. 2

Effect of endomorphin-1 and endomorphin-2 microinjected into the centromedial amygdala on the thermal tail-flick response. Groups of rats were microinjected with different doses of endomorphin-1 (8.0–32.6 nmol; A), endomorphin-2 (8.7–35.0 nmol; B) or vehicle (0.5 µl) into the centromedial amygdala and the tail-flick responses were measured at different times thereafter. Two-way ANOVA followed by Bonferroni’s post-test was used to test the difference between groups. For the group of rats injected with endomorphin-1 versus vehicle (A): interaction F_{(27, 240)} = 0.77, treatment F_{(3, 240)} = 14.89, time F_{(9, 240)} = 7.56. For the group of rats injected with endomorphin-2 versus vehicle (B): interaction F_{(27, 270)} = 2.43, treatment F_{(3, 270)} = 70.30, time F_{(9, 270)} = 9.39; N = 6 to 8 rats in each group; * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 3

Effect of endomorphin-1 and endomorphin-2 microinjected into the basolateral amygdala on the thermal tail-flick response. Groups of rats were microinjected with endomorphin-1 (32.6 nmol), endomorphin-2 (35.0 nmol) or vehicle (0.5 µl) into the basolateral amygdala and the tail-flick responses were measured at different times thereafter. Two-way ANOVA followed by Bonferroni’s post-test was used to test the difference between groups. For the group of rats injected with endomorphin-1 versus vehicle into the basolateral amygdale: interaction F_{(5, 66)} = 0.82, treatment F_{(1, 66)} = 2.05, time F_{(5, 66)} = 2.23; For the group of rats injected with endomorphin-2 versus vehicle: interaction F_{(5, 78)} = 0.48, treatment F_{(1, 78)} = 0.19, time F_{(5, 78)} = 2.58; N = 6 to 8 rats in each group.

Fig. 4

Pretreatment with antiserum against dynorphin A(1–17) (A/S Dyn) blocks the hyperalgesia induced by endomorphin-2 from the centromedial amydala. Groups of rats were pretreated with different doses (20 to 200 µg) of A/S Dyn or normal rabbit serum given into the centromedial amygdala 1 h before microinjection of endomorphin-2 into the centromedial amygdala and the tail-flick responses were measured 30 min after endomorphin-2 injections. Each column represents the mean and the vertical bar represents the S.E.M. with 6 to 8 rats in each group. Two-way ANOVA followed by Bonferroni’s post-test was used to test the difference between groups. For rats pretreated with A/S Dyn versus vehicle followed by endomorphin-2 challenge, interaction F_{(3, 53)} = 15.72, treatment F_{(1, 53)} = 0.004, time F_{(3, 53)} = 7.86; * p < 001.

Fig. 5

Effects of the pretreatment with 3-methoxynaltrexone (3-MNX), norbinaltorphimine (nor-BNI) or MK-801 on the hyperalgesia induced by endomorphin-2 from the centromedial amygdala. Groups of rats were pretreated with 3-MNX (6.4 pmol) 25 min, nor-BNI (6.6 nmol) 24 h, MK-801 (30 nmol) 20 min or saline vehicle given into the centromedial amygdala before microinjection of endomorphin-2 and the tail-flick responses were measured 30 min after endomorphin-2 injection. Each column represents the mean and the vertical bar represents the S.E.M. with 6 to 8 rats in each group. Students’s t test was used to test the difference between groups; For rats pretreated with nor-BNI versus vehicle followed by endomorphin-2 challenge t=1.65; For rats pretreated with MK-801 versus vehicle followed by endomorphin-2 challenge t=8.77; * p < 0.01, ** p < 0.001.

Fig. 6

Effect of dynorphin A(1–17) microinjected into the centromedial amygdala on the thermal tail-flick response. Groups of rats were microinjected with different doses of dynorphin A(1–17) (0.23–2.3 nmol) or vehicle (0.5 µl) into the centromedial amygdala and the tail-flick responses were measured 10 min thereafter. One-way ANOVA followed by Dunnett’s post-test was used to test the difference between groups, N = 6 to 8 rats in each group; * p < 0.001.

Fig. 7

Effects of the pretreatment with nor-binaltorphimine (nor-BNI) or MK-801 on the hyperalgesia induced by dynorphin A(1–17) from the centromedial amygdala. Groups of rats were pretreated with nor-BNI (6.6 nmol) 24 h, MK-801 (30 nmol) 20 min or saline vehicle given into the centromedial amygdala before microinjection of dynorphin A(1–17) and the tail-flick responses were measured 10 min after dynorphin A(1–17) injection. Each column represents the mean and the vertical bar represents the S.E.M. with 6 to 8 rats in each group. Students’s t test was used to test the difference between groups; For rats pretreated with nor-BNI versus vehicle followed by dynorphin A(1–17) challenge t=1.77; For rats pretreated with MK-801 versus vehicle followed by dynorphin A(1–17) challenge t=5.26; * p < 0.001.