Orexin-A potentiates glycine currents by activating OX1R and IP3/Ca2+/PKC signaling pathways in 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).
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