Hypothermia-dependent and -independent effects of forced swim on the phosphorylation states of signaling molecules in mouse hippocampus

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Abstract

Forced swim (FS) stress induces diverse biochemical responses in the brain of rodents. Here, we examined the effect of hypothermia induced by FS in cold water on the phosphorylation of FS-sensitive signaling molecules in the mouse brain. As we have shown previously, FS in cold water induced a significant increase in the level of tyrosine phosphorylation of SIRPα, a neuronal membrane protein, in mouse hippocampus, while such effect of FS was markedly reduced in mice subjected to FS in warm water. FS in cold water also induced phosphorylation of mitogen-activated protein kinase kinase (MEK) as well as of cAMP response element-binding protein (CREB), or dephosphorylation of α isoform of Ca2+/calmodulin-dependent protein kinase II (αCaMKII) in the hippocampus. These effects of FS on the phosphorylation of these molecules were also lost in mice subjected to FS in warm water. Genetic ablation of SIRPα did not change the phosphorylation states of these molecules in the brain. Forced cooling of anesthetized mice, which induced a marked increase in the phosphorylation of SIRPα, induced dephosphorylation of αCaMKII in the brain, while the same treatment did not affect the phosphorylation level of MEK and CREB. Hibernation also induced an increase and a decrease of the phosphorylation of SIRPα and αCaMKII, respectively, in the brain of chipmunk. These results suggest that hypothermia is a major element that determines the levels of phosphorylation of αCaMKII and SIRPα during the FS in cold water, while it is not for the phosphorylation levels of MEK and CREB.

Highlights

► Forced swim (FS) induces changes in the phosphorylation of proteins in the brain. ► Water temperature is a key determinant of the effects of FS. ► Phosphorylation states of CaMKII and SIRPα are primarily affected by hypothermia. ► Hypothermia does not affect the levels of phosphorylation of MEK and CREB. ► Brain responds to the FS stress in a hypothermia-dependent and -independent manner.

Introduction

Investigation of biochemical responses in the brain to stress provides valuable clues to understand how stress leads to depressive and other mental illnesses. Forced swim (FS) test is widely used as an animal model of behavioral despair or depression [1]. In this behavioral test, mice or rats placed in water show consistent behavioral responses to the unpleasant environment, active swimming followed by profound immobility. This behavioral immobility is thought to represent a state of despair or depression because it is markedly reduced by treatment with various classes of antidepressant [2], [3].

FS is an effective stressor to induce biochemical responses in the animal brain. For example, FS stress induces rapid changes in the activation states of several functional molecules, such as mitogen-activated protein kinase kinase (MEK), cAMP response element-binding protein (CREB), and Ca2+/calmodulin-dependent protein kinase II (CaMKII), in the brain [4], [5], [6]. In our previous study, we have shown that FS induced a significant increase in the level of tyrosine phoshorylation of signal regulatory protein α (SIRPα) in mouse brain [7]. SIRPα (also known as SHPS-1, p84, and BIT) is a transmembrane protein that contains putative tyrosine phosphorylation sites in its cytoplasmic region [8], [9]. Mice expressing a mutant form of SIRPα that lacks most of the cytoplasmic region manifest prolonged immobility in the FS test, suggesting that tyrosine phosphorylation of SIRPα participates in regulation of the behavioral immobility of mice in the FS test [7]. Recently, we found that a decrease in body temperature during FS in cold water is a major cause of the FS-induced tyrosine phosphorylation of SIRPα in the brain [10]. These results indicate that some parts of biological responses in the brain to FS stress might be induced by hypothermia rather than by psychiatric stress.

Here we show that hypothermia induced by immersion in cold water is a major determinant of the levels of phosphorylation of α isoform of CaMKII (αCaMKII), as well as that of SIRPα, in the brain of animals subjected to FS. In contrast, hypothermia is not important for induction by FS of the phosphorylation of MEK and CREB.

Section snippets

Animals

Eight- to thirty-week-old male C57BL/6 mice were studied. Mice that express a mutant form of SIRPα [11] were backcrossed to the C57BL/6N for >10 generations. Mice were bred and maintained at the Institute of Experimental Animal Research of Gunma University under specific pathogen–free conditions. They were housed in an air-conditioned room at 23 °C with a 12-h-light, 12-h-dark cycle.

Male chipmunks (Tamias sibiricus) obtained from ARCLAND SAKAMOTO Co. Ltd. (Niigata, Japan) were housed in an

Importance of low water temperature for FS-induced acute changes in the phosphorylation of MEK, CREB, and αCaMKII in the brain

To examine the effect of water temperature on FS stress-induced biological responses in the brain, we analyzed the phosphorylation states of signaling molecules in mouse brain after exposure to FS in two different water temperatures (23 °C and 37 °C). Consistent with our previous observations [7], [10], immunoblot analysis with pAbs specific for tyrosine-phosphorylated SIRPα (anti-pSIRPα) revealed that the level of tyrosine phosphorylation of SIRPα in the hippocampus was markedly increased by

Discussion

Our present data suggest that water temperature is an important determinant of the FS-induced changes in the phosphorylation levels of MEK, CREB, and αCaMKII in the brain. Difference in water temperature also affects the basal phosphorylation levels of MEK, CREB, and αCaMKII, but not that of SIRPα, in control mice that were allowed to stand in shallow water. In these animals, sensation of cold and warm water may have different effects on the phosphorylation of MEK, CREB, and αCaMKII, but not

Acknowledgments

We thank Y. Niwayama-Kusakari for technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (C), a Grant-in-Aid for Young Scientists (B), and a Grant-in-Aid for Scientific Research on Innovative Areas (“Brain Environment”) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and a grant from Takeda Science Foundation and Life Science Foundation of Japan.

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