Kim, Yoo Rim
(Department of Physiology, Seoul National University College of Medicine)
,
Shim, Hyun Geun
(Department of Physiology, Seoul National University College of Medicine)
,
Kim, Chang-Eop
(Department of Physiology, Seoul National University College of Medicine)
,
Kim, Sang Jeong
(Department of Physiology, Seoul National University College of Medicine)
The superficial dorsal horn of the spinal cord plays an important role in pain transmission and opioid activity. Several studies have demonstrated that opioids modulate pain transmission, and the activation of ${\mu}$-opioid receptors (MORs) by opioids contributes to analgesic effects in ...
The superficial dorsal horn of the spinal cord plays an important role in pain transmission and opioid activity. Several studies have demonstrated that opioids modulate pain transmission, and the activation of ${\mu}$-opioid receptors (MORs) by opioids contributes to analgesic effects in the spinal cord. However, the effect of the activation of MORs on GABAergic interneurons and the contribution to the analgesic effect are much less clear. In this study, using transgenic mice, which allow the identification of GABAergic interneurons, we investigated how the activation of MORs affects the excitability of GABAergic interneurons and synaptic transmission between primary nociceptive afferent and GABAergic interneurons. We found that a selective ${\mu}$-opioid agonist, [$D-Ala^2$, $NMe-Phe^4$, Gly-ol]-enkephanlin (DAMGO), induced an outward current mediated by $K^+$ channels in GABAergic interneurons. In addition, DAMGO reduced the amplitude of evoked excitatory postsynaptic currents (EPSCs) of GABAergic interneurons which receive monosynaptic inputs from primary nociceptive C fibers. Taken together, we found that DAMGO reduced the excitability of GABAergic interneurons and synaptic transmission between primary nociceptive C fibers and GABAergic interneurons. These results suggest one possibility that suppression of GABAergic interneurons by DMAGO may reduce the inhibition on secondary GABAergic interneurons, which increase the inhibition of the secondary GABAergic interneurons to excitatory neurons in the spinal dorsal horn. In this circumstance, the sum of excitation of the entire spinal network will control the pain transmission.
The superficial dorsal horn of the spinal cord plays an important role in pain transmission and opioid activity. Several studies have demonstrated that opioids modulate pain transmission, and the activation of ${\mu}$-opioid receptors (MORs) by opioids contributes to analgesic effects in the spinal cord. However, the effect of the activation of MORs on GABAergic interneurons and the contribution to the analgesic effect are much less clear. In this study, using transgenic mice, which allow the identification of GABAergic interneurons, we investigated how the activation of MORs affects the excitability of GABAergic interneurons and synaptic transmission between primary nociceptive afferent and GABAergic interneurons. We found that a selective ${\mu}$-opioid agonist, [$D-Ala^2$, $NMe-Phe^4$, Gly-ol]-enkephanlin (DAMGO), induced an outward current mediated by $K^+$ channels in GABAergic interneurons. In addition, DAMGO reduced the amplitude of evoked excitatory postsynaptic currents (EPSCs) of GABAergic interneurons which receive monosynaptic inputs from primary nociceptive C fibers. Taken together, we found that DAMGO reduced the excitability of GABAergic interneurons and synaptic transmission between primary nociceptive C fibers and GABAergic interneurons. These results suggest one possibility that suppression of GABAergic interneurons by DMAGO may reduce the inhibition on secondary GABAergic interneurons, which increase the inhibition of the secondary GABAergic interneurons to excitatory neurons in the spinal dorsal horn. In this circumstance, the sum of excitation of the entire spinal network will control the pain transmission.
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문제 정의
In this study, we investigated how activation of MORs affects spinal GABAergic interneurons which receive monosynaptic inputs from primary nociceptive fibers. The μ-opioid agonist DAMGO induces an outward current in GABAergic interneurons.
가설 설정
(B) In voltage clamp mode, DAMGO produced reproducible outward currents of about 39.55±2.16 pA in GABAergic interneurons.
제안 방법
29 and an osmolarity of 300 mOsm and the resistance of the pipettes was 3-5 MΩ. All experiments in this study were acquired using multiclamp 700B patch-clamp amplifier (Axon Instruments), and the signals were filtered at 2 kHz. To record evoked excitatory postsynaptic currents (EPSCs) of GABAergic interneurons in SG, the attached dorsal root was stimulated through a suction electrode with a constant current stimulator (A360; WPI) at 0.
From the lumbar segment, transverse spinal slices with L4 or L5 dorsal roots were prepared using a microm HM 650V vibratome (Walldorf, Germany), and the slices were incubated at 34°C for 30 min for a recovery.
And GIRK channels coupled to MORs are mainly found in excitatory neurons in the spinal dorsal horn [24]. Here, we identified GABAergic interneurons using transgenic mice expressing green fluorescent protein (GFP) and examined the effect of DAMGO on these interneurons. In our study, outward currents was produced in GABAergic interneurons by DAMGO treatment and these neurons exhibited inwardly rectifying I-V relationship, which imply that GIRK channels are activated by DAMGO in GABAergic interneurons.
Given the role of GABAergic interneurons in pain control, it is important to understand how these interneurons are regulated by the activation of MORs. In this study, using transgenic mice that allow GABAergic interneuron identification, we investigated the effect of MOR activation by DAMGO in GABAergic interneurons which directly interact with primary nociceptive C fibers. Our observation suggests that DAMGO reduce the activity of GABAergic interneurons and the analgesic action of DAMGO is the result of total sum of excitation and inhibition throughout the entire spinal neural network.
In this study, we propose a possible mechanism to explain how DAMGO induces analgesic effects in spinal dorsal horn. Our experiments were limited to GABAergic interneurons expressing eGFP under the control of the glutamate acid decarboxylase (GAD) 65 promoter.
All recorded neurons were obtained from lamina ΙΙ of the spinal dorsal horn. To confirm whether recorded GABAergic neurons directly receive excitatory inputs from nociceptive C fibers, we recorded evoked excitatory postsynaptic currents (EPSCs) of GABAergic interneurons while stimulating the attacheddorsal root, and the latency to produce EPSCs was calculated. Fig.
데이터처리
All data are represented as mean±s.e.m. Two-way RM ANOVA and Two-tailed paired t-test were used to determine the significance in statistical comparisons.
성능/효과
To prepare the spinal lumbar slices, we used 4-weeks-old transgenic mice. The mice were anesthetized with 10% urethane (i.p. injection, 1.5 mg/kg) and a laminectomy was performed after enough anesthesia was confirmed. During the laminectomy, the spinal cord from each mouse was extracted and quickly transferred to the ice-cold cutting solution consisted of (in mM) 95 NaCl, 1.
In addition, DAMGO does not act on specific cell populations to exert analgesic effects, but on the entire spinal neural network, including MORs expressed in primary afferent fibers and excitatory neurons in the spinal dorsal horn. Therefore, a possible explanation for our findings is that, the analgesic action of DAMGO is the result of the total sum of excitation and inhibition throughout the entire spinal neural network.
후속연구
Our experiments were limited to GABAergic interneurons expressing eGFP under the control of the glutamate acid decarboxylase (GAD) 65 promoter. Considering that several subtypes of interneurons coexist in spinal circuits, further studies are required to provide a deeper understanding of how opioids act as analgesics in the spinal cord using dual patch recording or Ca2+ imaging which permit simultaneous recording of multiple interneurons.
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