Differential regulation of morphine antinociceptive effects by endogenous enkephalinergic system in the forebrain of mice

Abstract

Background: Mice lacking the preproenkephalin (ppENK) gene are hyperalgesic and show more anxiety and aggression than wild-type (WT) mice. The marked behavioral changes in ppENK knock-out (KO) mice appeared to occur in supraspinal response to painful stimuli. However the functional role of enkephalins in the supraspinal nociceptive processing and their underlying mechanism is not clear. The aim of present study was to compare supraspinal nociceptive and morphine antinociceptive responses between WT and ppENK KO mice.

Results: The genotypes of bred KO mice were confirmed by PCR. Met-enkephalin immunoreactive neurons were labeled in the caudate-putamen, intermediated part of lateral septum, lateral globus pallidus, intermediated part of lateral septum, hypothalamus, and amygdala of WT mice. Met-enkephalin immunoreactive neurons were not found in the same brain areas in KO mice. Tail withdrawal and von Frey test results did not differ between WT and KO mice. KO mice had shorter latency to start paw licking than WT mice in the hot plate test. The maximal percent effect of morphine treatments (5 mg/kg and 10 mg/kg, i.p.) differed between WT and KO mice in hot plate test. The current source density (CSD) profiles evoked by peripheral noxious stimuli in the primary somatosenstory cortex (S1) and anterior cingulate cortex (ACC) were similar in WT and KO mice. After morphine injection, the amplitude of the laser-evoked sink currents was decreased in S1 while the amplitude of electrical-evoked sink currents was increased in the ACC. These differential morphine effects in S1 and ACC were enhanced in KO mice. Facilitation of synaptic currents in the ACC is mediated by GABA inhibitory interneurons in the local circuitry. Percent increases in opioid receptor binding in S1 and ACC were 5.1% and 5.8%, respectively.

Conclusion: The present results indicate that the endogenous enkephalin system is not involved in acute nociceptive transmission in the spinal cord, S1, and ACC. However, morphine preferentially suppressed supraspinal related nociceptive behavior in KO mice. This effect was reflected in the potentiated differential effects of morphine in the S1 and ACC in KO mice. This potentiation may be due to an up-regulation of opioid receptors. Thus these findings strongly suggest an antagonistic interaction between the endogenous enkephalinergic system and exogenous opioid analgesic actions in the supraspinal brain structures.

Figure 1

Comparison of the distributions and densities of immunoreactive enkephalin neurons and fibers in WT and KO mice. Low magnification photomicrography of coronal brain sections with immunostaining of enkephalinergic neurons and fibers in WT (A) and KO (B) mice. (C) – (N) show higher magnification photomicrography of immunostained brain regions of WT mice (C – H) and KO mice (I – N) enlarged from square areas indicated in A and B respectively. The brain regions magnified are: (C), (I) lateral globus pallidus, (D), (J) lateral septum, (E), (K) intermediated part of caudate putamen, (F), (L) amygdaloid nuclear region, (G), (M) cingulate cortex and (H), (N) sensory motor cortex.

Figure 2

Comparison of nociceptive behavioural responses and the antinociceptive effect of morphine in WT and KO mice. Results from the von Frey test (A) tail withdrawal test (B), EMG evoked by incremental laser pulse duration (C), and hot plate test (D) are shown. The analgesic effects of morphine (5 mg/kg and 10 mg/kg) on the hot plate test as a function of time (E) and 40 min after morphine administration are also shown (F).

Figure 3

Laser evoked CSD profiles across cortical layers of the S1 in WT and KO mice. (A) Schematic diagram of the recording scheme. The position of the multichannel probe is overlaid with histological sections from the S1. Cortical layers are indicated by roman numerals. Arrow indicates the electrolytic lesion mark in layer V. (B) CSD sweeps across the cortical layers in WT (left panel) and KO (right panel) mice. Gray lines indicate the averaged CSD sweeps from individual mice. Black lines indicated the grand averaged CSD sweeps from WT (n = 18) and KO (n = 16) mice. Sink currents are in the downward direction and source currents are in the upward direction. Sink 1a (early component) and sink 2a and sink 2b (late components) were identified. All of the evoked CSD profile was evoked by laser stimuli with intensity of 10 W and duration of 20 ms. (C) Percent of maximal amplitude change of sink 2a evoked by increment of laser duration.

Figure 4

Effect of morphine on the sink current evoked in the S1 by laser pulses (10 W and 20 ms duration). (A) Example of laser evoked CSD profiles in the S1 before and after morphine treatment (10 mg/kg) in WT (upper panel) and KO (lower panel) mice. (B) Statistical analysis of the effect of morphine on sink 1a, sink 2a and sink 2b in WT and KO mice. Reversibility of the effect was evaluated by treatment with naloxone (0.7 mg/kg). * p < 0.05. ** p < 0.01.

Figure 5

Electrical evoked CSD profiles across cortical layers of the ACC in WT and KO mice. (A) Schematic diagram of the recording scheme and the electrical stimulation parameters: 10 mA, 0.5 ms duration and 0.1 Hz. The position of the multichannel probe is overlaid with histological sections of the ACC. Cortical layers are indicated by roman numerals. Arrow indicates the electrolytic lesion mark in layer V. (B) CSD sweeps across the cortical layers in WT (left panel) and KO (right panel) mice. Gray lines indicate averaged CSD sweeps from individual mice. Black lines indicate the grand averaged CSD sweeps from WT (n = 4) and KO (n = 4) mice. Sink 1, sink 2 and sink 3 were identified. (C) Percent of maximal amplitude change of sink 2 evoked by increment of electrical intensity of stimulus.

Figure 6

Effect of morphine on the sink current in the ACC evoked by high electrical intensity stimulation. (A) Example of CSD profiles evoked by electrical stimulation (10 mA, 0.5 ms duration and 0.1 Hz) in the hind paw before and after morphine treatment (10 mg/kg) in WT (upper panel) and KO (lower panel) mice. (B) Statistical analysis of the effect of morphine on sink 1, sink 2 and sink 3 in WT and KO mice. Reversibility of the effect was evaluated by treatment with naloxone (0.7 mg/kg). * p < 0.05. ** p < 0.01.

Figure 7

Effect of morphine on simultaneously recorded evoked cortical responses in the S1 and ACC. (A) Schematic diagram of the recording scheme of the multichannel probes placed in the S1 and ACC regions. (B) Example sweeps of sink currents in the ACC layer V (upper panel) and S1 layer II/III (lower panel) evoked by the high intensity electrical stimulation (10 mA, 0.5 ms duration and 0.1 Hz) in the hind paw. Example sweeps of evoked cortical sink currents after morphine treatment (5 mg/kg, 10 mg/kg and 20 mg/kg) and reversal by naloxone treatment were demonstrated. (C) Statistical analysis of the effect of morphine on simultaneously recorded S1 and ACC evoked sink currents. Data are presented as mean ± SEM. Amplitude of the sink current after morphine injection is expressed as a percentage of the amplitude of the sink current measured before morphine injection. * p < 0.05.

Figure 8

Effect of GABA_B agonist and antagonist on the evoked sink currents in the cortical layer V of the ACC. (A) The electrical stimulation (10 mA, 0.5 ms duration and 0.1 Hz) was applied in the right paw of the mice. Example sweeps of evoked sink currents in layer V of the ACC under before and after treatment with morphine, SKF 97541 and CGP55845. (B) Statistical analysis of the effects of morphine, SKF 97541 and CGP55845 on the layer V sink current in the ACC. * p < 0.05. ** p < 0.01.

Figure 9

Detection and measurement of μ-opioid receptors by immunostaining and Western blotting. (A) Immuoreactive μ-opioid-receptors were stained in the S1 and ACC of WT and KO mice. Micrographs of μ-opioid-receptors (100× magnification) are shown in the upper panel. Micrographs at higher magnification are shown in the lower panel. Locations that were immunopositive for μ-opioid-receptors are indicated by square boxes in the left panel. (B) Western blot data provided a quantitative measurement of μ-opioid receptors in the cortical region of WT and KO mice. The μ-opioid receptor band is located at 43 kDa (arrow heads). Non-specific binding was used as an internal control (56 kDa). (C) The relative quantities of μ-opioid receptor were measured by taking the ratio of specific and non-specific binding in the S1 and ACC of WT (n = 6) and KO (n = 7) mice. Data are presented as the mean ± SEM.