Endomorphin-2 Inhibits the Activity of the Spinoparabrachial Projection Neuron through Presynaptic Mechanisms in the Spinal Dorsal Horn in Rats

Abstract

Background/Aims: Spinal dorsal horn (SDH) is one of the most important regions for analgesia produced by endomorphin-2 (EM2), which has a higher affinity and specificity for the µ-opioid receptor (MOR) than morphine. Many studies have focused on substantia gelatinosa (SG, lamina II) neurons to elucidate the cellular basis for its antinociceptive effects. However, the complicated types and local circuits of interneurons in the SG make it difficult to understand the real effects of EM2. Therefore, in the present study, we examined the effects of EM2 on projection neurons (PNs) in lamina I. Methods: Tracing, immunofluoresence, and immunoelectron methods were used to examine the morphological connections between EM2-immunoreactive (-ir) terminals and PNs. By using in vitro whole cell patch clamp recording technique, we investigated the functional effects of EM2 on PNs. Results: EM2-ir afferent terminals directly contacted PNs projecting to the parabrachial nucleus in lamina I. Their synaptic connections were further confirmed by immunoelectron microscopy, most of which were asymmetric synapses. It was found that EM2 had a strong inhibitory effect on the frequency, but not amplitude, of the spontaneous excitatory postsynaptic current (sEPSC) of the spinoparabrachial PNs in lamina I, which could be reversed by MOR antagonist CTOP. However, their spontaneous inhibitory postsynaptic current (sIPSC) and intrinsic properties were not changed after EM2 application. Conclusion: Applying EM2 to the SDH could produce analgesia through inhibiting the activities of the spinoparabrachial PNs in lamina I by reducing presynaptic neurotransmitters release from the primary afferent terminals.

Morphological investigations of endomorphin-2 and spinoparabrachial projection neurons in the spinal dorsal horn of the rat

“…Stereotaxic microinjections followed the procedures described in our previous studies ( Yin et al, 2014 , 2018 ). All rats were anesthetized with intraperitoneal ( i.p. )…”

“…The procedures in immunofluorescence histochemical multi-staining were the same as those in our previous studies ( Hui et al, 2010 ; Yin et al, 2014 , 2018 ). The second to the seventh sets of the SC sections were adopted to examine the multi-staining of EM2/FG and triple-labeling of EM2/MOR/FG, EM2/MOR/NeuN, EM2/SP/FG, EM2/VGLUT2/FG, and EM2/GAD67/FG, respectively.…”

“…Endomorphin (EM) had remarkable affinity and selectivity for the μ-opioid receptor (MOR), which included EM1 (Tyr-Pro-Trp-Phe-NH2) and EM2 (Tyr-Pro-Phe-Phe-NH2) ( Zadina et al, 1997 ; Gu et al, 2017 ). It was considered that EM1 played an important role in brain nucleus such as the periaqueductal gray ( Chen et al, 2016 ), while EM2 mainly functioned in the spinal dorsal horn (SDH) ( Tseng et al, 2000 ; Yin et al, 2018 ; Lu et al, 2022 ). EM2 plays a pivotal role in inhibiting nociceptive information transmission at the spinal cord level ( Stone et al, 1997 ; Zadina et al, 1997 ).…”

“…EM2 plays a pivotal role in inhibiting nociceptive information transmission at the spinal cord level ( Stone et al, 1997 ; Zadina et al, 1997 ). EM2-immunoreactive (-ir) fibers existed in the superficial lamina of the SDH ( Yin et al, 2018 ; Lu et al, 2022 ), which was released following the dorsal root entry zone receiving electrical stimulation ( Williams et al, 1999 ; Heinke et al, 2011 ). Intrathecal ( i.t. )…”

“…Thus, the morphological features of the PNs in lamina I are important for us to understand how the nociceptive information were transmitted to the brain and how the central sensitization for the chronic pain was induced. Our previous study has shown that EM2-ir axons directly formed synapses with the spinoparabrachial PNs and applying EM2 inhibited the activities of these PNs through reducing presynaptic release of glutamate from the primary afferent terminals ( Yin et al, 2018 ).…”