Elsevier

Brain Research Bulletin

Volume 169, April 2021, Pages 196-204
Brain Research Bulletin

Orexin-A potentiates glycine currents by activating OX1R and IP3/Ca2+/PKC signaling pathways in spinal cord ventral horn neurons

https://doi.org/10.1016/j.brainresbull.2021.01.017Get rights and content

Highlights

  • Orexin-A potentiated the glycine currents in the spinal cord ventral horn neurons.

  • OX1R and Ca2+-dependent PKC were involved in the orexin-A effect.

  • IP3 receptor was involved in orexin-A-induced potentiation of the glycine currents.

Abstract

Orexin-A/B modulates multiple physical functions by activating their receptors (OX1R and OX2R), but its effects in the spinal cord motor control remain unknown. Using acute separation (by digestive enzyme) of cells and patch-clamp recordings, we aimed to investigate the effect and mechanisms of orexin-A on the glycine receptors in the spinal cord ventral horn neurons. Orexin-A potentiated the glycine currents by activating OX1R. In Ca2+-free extracellular solution, orexin-A still increased the glycine currents. While, the orexin-A-induced potentiation was blocked when Ca2+ was chelated by internal infusion of BAPTA, and the orexin-A effect was abolished by the IP3 receptor antagonists heparin and Xe-C. The PKC inhibitor Bis-IV nullified the orexin-A effect. In addition, orexin-A did not cause a further enhancement of the glycine currents after bath application of the PKC activator PMA. In conclusion, after OX1R is activated, a distinct IP3/Ca2+-dependent PKC signaling pathway, is likely responsible for the orexin-A potentiation on glycine currents in the spinal cord ventral horn neurons.

Introduction

Orexins, also known as hypocretins, are excitatory neuropeptides produced by neurons in the lateral hypothalamus, including orexin-A (OXA) and orexin-B (OXB) (de Lecea et al., 1998). Orexins act by activating two G protein-coupled receptors, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) (de Lecea et al., 1998; Kukkonen, 2017).The neurons that secrete orexin project widely to the brain and spinal cord, and are involved in the regulation of a variety of physiological functions including sleep/wakefulness (Feng et al., 2020), emotion regulation (Cengiz et al., 2019), reward (Zarrabian et al., 2020), food intake (Liu et al., 2020), behavior and movement (Gao et al., 2017). It has been proved that orexin nerve fibers distribute in the brain and spinal cord and may directly or indirectly participate in central motor control (Friston, 2011; Gao et al., 2017; Hu et al., 2015). Glycine receptor (GlyR) is an important inhibitory receptor, which is highly expressed in the spinal cord and plays an essential role in motor control. GlyR mediates inhibitory synaptic transmission in the spinal motor reflex circuit and involves the regulation of excitability of motor neurons (Webb and Lynch, 2007). It is suggested that GlyR plays an important role in the spinal cord motor control. However, the study of the interaction between orexin-A and GlyR in motor control of the spinal cord has not been reported. Thus, this study used acute separation (by digestive enzyme) of cells and patch-clamp recordings to discover the modulating effect of orexin-A on the currents mediated by GlyRs and its underlying mechanisms in the spinal cord ventral horn neurons. This study provides us with a better understanding to the mechanisms of the spinal cord motor control, as well as a new potential method for the clinical treatment of spinal cord injury.

Section snippets

Animals

Neonatal Sprague-Dawley rats of 7–12 days old were selected and obtained from Qinglongshan Animal Breeding Farm (Nanjing, China). Animal license number is SCXK (Su): 2017−0001. All processes in animal experiments were in line with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and the rules of Wannan Medical College regarding the ethical use of animals.

Chemicals

Glycine, EGTA, BAPTA, Phorbol 12-myristate 13-acetate (PMA), Rp-cAMP, K-gluconate,

Morphology of the spinal cord ventral horn neurons

Spinal cord ventral horn neurons could be observed after being isolated by enzyme digestion combined with acute mechanical separation. The isolated neurons were in good condition with large diverse somata and intact processes (Fig. 1).

Glycine concentration-response relationship

In 5 isolated ventral horn neurons of the spinal cord, the glycine currents with different sizes could be induced by applying 3 μM, 10 μM, 30 μM, 100 μM, 300 μM and 1000 μM glycine, respectively. Normalization processing was done to all the peak currents with

Discussions

Spinal cord is a primary center of motor control within human body. A complex glycinergic system, can directly or indirectly participate in the motor control of the spinal cord. GlyR also plays an important role in motor control, a potential new target for the treatment of dyskinesia including chronic pain, muscle relaxation and spasm (Gallagher et al., 2020; Nanaura et al., 2019; Rauschenberger et al., 2020). In addition, more and more evidences have proved the necessity of orexin-A in motor

CRediT authorship contribution statement

Na Jin: Conceptualization, Methodology, Validation, Investigation, Formal analysis, Writing - original draft. Su-Yue Zhu: Investigation, Visualization. Xin-Yu Yang: Investigation. Cheng Zhen: Investigation. Yan Li: Investigation. Huan-Huan Zhang: Visualization, Writing - review & editing, Funding acquisition. Ai-Ping Xu: Visualization. Meng-Ya Wang: Visualization, Writing - review & editing, Funding acquisition, Project administration. Chao Zheng: Conceptualization, Visualization, Funding

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

This work was supported by National Natural Science Foundation of China (31200828 and 31271155), the Natural Science Research Project Fund for Colleges and Universities in Anhui Province, China (KJ2019A0411, KJ2018A0266), the Key Projects of Outstanding Young Talent Support Program in Colleges and Universities of Anhui Province, China (gxyqZD2016175, gxyq2017034), and the Natural Science Foundation of Anhui Province, China (1908085QC132).

References (35)

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