Spinal astrocytes contribute to the circadian oscillation of glutamine synthase, cyclooxygenase-1 and clock genes in the lumbar spinal cord of mice

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Abstract

Spinal astrocytes have key roles in the regulation of pain transmission. However, the relationship between astrocytes and the circadian system in the spinal cord remains poorly defined. In the current study, the circadian variations in the expression of several clock genes in the lumbar spinal cord of mice were examined by using real-time PCR. The expression of Period1, Period2 and Cryptochrome1 showed significant circadian oscillations, each gene peaking in the early evening. The expression of Bmal1 mRNA also exhibited a circadian pattern, peaking from around midnight to early morning. The mRNA levels of Cryptochrome2 were slightly, but not significantly altered. Molecules related to pain transmission were also investigated. The mRNA expression of glutamine synthase (GS), and cyclooxygenases (COXs), known to be involved in various spinal sensory functions, showed rhythmicity with a peak in the early evening, although the expression of the neurokinin-1 receptor, subunits of the N-methyl-d-aspartate receptor, and glutamate transporters did not change. In addition, we found that protein levels of GS and COX-1 were also high at midnight compared with midday. Furthermore, we examined the effect of intrathecal fluorocitrate (100 pmol), an inhibitor of astrocytic metabolism, on the expression of oscillating genes in lumbar spinal cord. Fluorocitrate significantly suppressed astrocyte function. Furthermore, the circadian oscillation of clock gene expression and GS and COX-1 expression were suppressed. Together, these results suggest that a significant circadian rhythmicity of the expression of clock genes is present in the spinal cord and that the components of the circadian clock timed by astrocytes might contribute to spinal functions, including nociceptive processes.

Highlights

► Circadian rhythms in clock genes expression were detected in the spinal cord. ► Glutamine synthase and cyclooxygenase-1 mRNA was also under circadian influence. ► Inhibition of spinal astrocyte function disrupted circadian mRNA expression. ► Spinal cord functioning is regulated by circadian oscillation.

Introduction

Most organisms have a biological clock marked by circadian rhythm (Dunlap, 1998). This clock has a crucial role in not only the regulation of various physiological functions but also in the development of some diseases (Hashiramoto et al., 2010, Huang et al., 2011, Lavebratt et al., 2010). Circadian rhythm in mammals is achieved by the transcriptional and posttranscriptional regulation of several clock genes including Period (Per), Bmal1, Clock, and Cryptochrome (Cry) (Sassone-Corsi, 1998). Although the pacemaker of this rhythm resides in the hypothalamic suprachiasmatic nucleus (SCN), other parts of the brain as well peripheral tissues are known to have their own clock systems which are dependent on the activity of the SCN (Balsalobre, 2002, Balsalobre et al., 1998). In fact, the neuronal projection pathways of the SCN are relatively well delineated, which shows that the SCN has extensive access to other brain and peripheral regions.

The spinal cord has important roles in the transmission of information from the external environment to the brain through sensory and motor neuronal pathways. Furthermore, sympathetic preganglionic neurons originate in the intermediolateral nucleus of the thoracic spinal cord, the major component of sympathetic output from the CNS. Previous studies have suggested spinal functions to be under circadian control. Perissin et al. (2004) have demonstrated that, by using receptor autoradiography, the availability of melatonin binding, which is a key hormone in the control of neuroendocrine circadian rhythm, was decreased during the dark-phase compared to the light-phase in the spinal cord of mice. It has recently been reported that spinal cord injury patients with a cervical-level lesion exhibited disturbances in circadian variation of core body temperature, and this phenomenon may be related to the decrease or absence of melatonin release at the cervical level (Thijssen et al., 2011). Furthermore, the expression of tyrosine hydroxylase and nitric oxide synthase displayed circadian patterns in the intermediolateral nucleus, and thus the alteration of those genes with circadian patterns in the thoracic spinal region could lead to changes in autonomic function and its excitability (Clemens et al., 2005). Although these previous observations in total indicate the possibility that the spinal cord is equipped with an independent clock system, circadian variations in the expression of clock genes and other molecules which might be associated with sensory neuronal activity still remain to be identified.

Astrocytes are recognized as the most abundant type of glial cells in the CNS, and have several important roles in maintaining CNS functioning. Several studies have suggested that astrocytes could act as a crucial player in the generation of circadian rhythm. Prosser et al. (1994) have shown that blockade of astrocytes activity in the SCN by the astrocyte metabolic inhibitor fluorocitrate disturbs circadian generation of systemic neuronal and locomotor activities. Furthermore, astrocytes themselves express several clock genes and exhibit circadian oscillation, which is expressed after treatment with explants of adult SCN (Prolo et al., 2005). In addition, the noradrenaline-mediated expression of various clock genes has been previously demonstrated in cultured spinal cord astrocytes (Sugimoto et al., 2011). However, it is unknown whether astrocytes themselves might have a role in the circadian regulation of the clock genes or other genes at the spinal level.

The current study investigated changes in circadian expression of both clock genes and several genes thought to be involved in neuronal transduction and signaling cascades within the lumbar spinal cord, such as the substance P (SP) receptor (neurokinin receptor-1; NK-1R), N-methyl-d-aspartate (NMDA) receptor subunits, glutamate transporters, glutamine synthase (GS), and cyclooxygenases (COXs). In addition, the effect of inhibiting spinal astrocytic metabolism by intrathecal administration of fluorocitrate was examined to determine whether astrocyte activity might affect circadian variations of spinal clock genes expression.

Section snippets

Animals

Male ICR mice, weighing 20–25 g were housed at 22 ± 2 °C with a 12 h light/dark (LD) cycle (lights on at 8:00) and given access to food and water ad libitum. For experiments under constant darkness (DD) conditions, mice were kept under LD conditions then switched to the DD condition, and maintained in the DD condition for 10 days prior to tissue harvesting. All procedures and handling of animals were performed according to the guidelines set by the Animal Care and Use Committee of Hiroshima

Circadian changes in clock genes expression in lumber spinal cord of mice

We first examined the daily expression of typical clock genes in the lumbar spinal cord of mice under light–dark (LD) condition. As shown in Fig. 1, the mRNA levels of Per1 and Per2 showed significant circadian oscillations. Expression of Per1 started to increase in the later part of the light period, and peaked at 20:00 (F5,54 = 8.34, P < 0.001 by one way-ANOVA; P < 0.05, 8:00, 12:00, 4:00 vs. 20:00, by post hoc Tukey–Kramer test; n = 8–14 mice per time point). Expression of Per2 peaked at around

Discussion

The current study is the first to demonstrate circadian variations in the expression of typical clock genes, including Per1, Per2, Cry1, and Bmal1, in the spinal cord of mice. Opposing circadian patterns of expression of Per1 and Per2, compared with Bmal1, were found as in other tissues such as the SCN or liver. In addition, significant circadian expression of GS and COX-1 at both mRNA and protein levels was found. Furthermore, the disruption of astroglial function led to the dramatic

Acknowledgements

This work was supported in part by Grants-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science, and by grants from the Takeda Science Foundation and the Japanese Research Smoking Association. We wish to thank the Analysis Center of Life Science, Hiroshima University for the use of their facilities. We also thank Dr. Aldric T. Hama for his careful editing of the manuscript.

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