Analysis of the Anticipatory Behavior Formation Mechanism Induced by Methamphetamine Using a Single Hair.

While the suprachiasmatic nucleus (SCN) coordinates many daily rhythms, some circadian patterns of expression are controlled by SCN-independent systems. These include responses to daily methamphetamine (MAP) injections. Scheduled daily injections of MAP resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. The MAP-induced anticipatory behavior is associated with the induction and a phase advance in the expression rhythm of the clock gene Period1 (Per1). However, this unique formation mechanism of MAP-induced anticipatory behavior is not well understood. We recently developed a micro-photomultiplier tube (micro-PMT) system to detect a small amount of Per1 expression. In the present study, we used this system to measure the formation kinetics of MAP-induced anticipatory activity in a single whisker hair to reveal the underlying mechanism. Our results suggest that whisker hairs respond to daily MAP administration, and that Per1 expression is affected. We also found that elevated Per1 expression in a single whisker hair is associated with the occurrence of anticipatory behavior rhythm. The present results suggest that elevated Per1 expression in hairs might be a marker of anticipatory behavior formation.

A real-time measurement system for gene expression rhythms from deep tissues of freely moving mice under light-dark conditions.

Clock gene expression in most organs of the living body exhibits a diurnal rhythm synchronized with the external 24 h light-dark (LD) cycle via circadian pacemaker suprachiasmatic nucleus (SCN). Disturbances in clock gene expression due to desynchronization of clock gene expression of the external LD cycle are risk factors for developing various diseases. Measuring the in vivo clock genes expression rhythm for a long duration under LD conditions can greatly contribute to understand the pathogenic mechanism of the disease caused by the disturbance of the biological rhythm. However, it is presently difficult to continuously measure gene expression for a long duration under LD conditions. In present study, we succeeded in measuring Period1 (Per1) gene expression under LD conditions using ultraviolet (UV) light with filter cut the visible light range. In addition, we succeeded in measuring the kinetic change of liver Per1 gene expression during the process of desynchronization of behavioral rhythm from the LD cycle by chronic administration of methamphetamine (MAP). In the future, by using this system to measure clock gene expression rhythms of brain tissues such as SCN and peripheral tissues under LD conditions, it could contribute to understand the onset mechanism of diseases induced by the desynchronization mechanism of biological rhythm to the LD cycle.

The analysis of Period1 gene expression in vivo and in vitro using a micro PMT system.

To detect a small amount of Period1 (Per1) expression, we developed a micro-photomultiplier tube (µPMT) system which can be used both in vivo and in vitro. Using this system, we succeeded in detecting Per1 gene expression in the skin of freely moving mice over 240 times higher compared with that of the tissue contact optical sensor (TCS) as previously reported. For in vitro studies, we succeeded in detecting elevated Per1 expression by streptozotocin (STZ) treatment in the scalp hairs at an early stage of diabetes, when glucose content in the blood was still normal. In addition, we could detect elevated Per1 expression in a single whisker hair at the time of diabetes onset. These results show that our µPMT system responds to minute changes in gene expression in freely moving mice in vivo and in mice hair follicles in vitro. Furthermore, Per1 in the hair can be used for a marker of diabetic aggravation.

Period1 gene expression in the olfactory bulb and liver of freely moving streptozotocin-treated diabetic mouse.

Clock genes express circadian rhythms in most organs. These rhythms are organized throughout the whole body, regulated by the suprachiasmatic nucleus (SCN) in the brain. Disturbance of these clock gene expression rhythms is a risk factor for diseases such as obesity. In the present study, to explore the role of clock genes in developing diabetes, we examined the effect of streptozotocin (STZ)-induced high glucose on Period1 (Per1) gene expression rhythm in the liver and the olfactory bub (OB) in the brain. We found a drastic increase of Per1 expression in both tissues after STZ injection while blood glucose content was low. After a rapid expression peak, Per1 expression showed no rhythm. Associated with an increase of glucose content, behavior became arrhythmic. Finally, we succeeded in detecting an increase of Per1 expression in mice hair follicles on day 1 after STZ administration, before the onset of symptoms. These results show that elevated Per1 expression by STZ plays an important role in the aggravation of diabetes.

Stability of d-luciferin for bioluminescence to detect gene expression in freely moving mice for long durations.

Circadian disturbance of clock gene expression is a risk factor for diseases such as obesity, cancer, and sleep disorders. To study these diseases, it is necessary to monitor and analyze the expression rhythm of clock genes in the whole body for a long duration. The bioluminescent reporter enzyme firefly luciferase and its substrate d-luciferin have been used to generate optical signals from tissues in vivo with high sensitivity. However, little information is known about the stability of d-luciferin to detect gene expression in living animals for a long duration. In the present study, we examined the stability of a luciferin solution over 21 days. l-Luciferin, which is synthesized using racemization of d-luciferin, was at high concentrations after 21 days. In addition, we showed that bioluminescence of Period1 (Per1) expression in the liver was significantly decreased compared with the day 1 solution, although locomotor activity rhythm was not affected. These results showed that d-luciferin should be applied to the mouse within, at most, 7 days to detect bioluminescence of Per1 gene expression rhythm in vivo.

Double recording system of Period1 gene expression rhythm in the olfactory bulb and liver in freely moving mouse.

Clock genes express circadian rhythms in most organs. These rhythms are organized throughout the whole body, regulated by the suprachiasmatic nucleus (SCN) in the brain. Disturbance of these clock gene expression rhythms is a risk factor for diseases such as obesity and cancer. To understand the mechanism of regulating clock gene expression rhythms in vivo, multiple real time recording systems are required. In the present study, we developed a double recording system of Period1 expression rhythm in peripheral tissue (liver) and the brain. In peripheral tissue, quantification of gene expression in a steadily moving target was achieved by using a photomultiplier tube (PMT) attached to a tissue contact optical sensor (TCS). Using this technique, we were able to analyze circadian rhythms of clock gene expression over a prolonged period in the liver and olfactory bub (OB) of the brain. The present double recording system has no effect on behavioral activity or rhythm. Our novel system thus successfully quantifies clock gene expression in deep areas of the body in freely moving mice for a period sufficient to analyze circadian dynamics. In addition, our double recording system can be widely applied to many areas of biomedical research, as well as applications beyond medicine.

Mouse period1 gene expression recording from olfactory bulb under free moving conditions with a portable optic fibre device.

Because the disruption of circadian clock gene is a risk factor in many diseases such as obesity and cancer, it is important to monitor and analyzed the expression of the rhythm of the clock gene throughout the body over a long period of time. Although we previously reported on a new gene expression analysis system tracking a target position on the body surface of freely moving mice, the experimental apparatus required a large space. We have therefore developed an in vivo recording system using a portable photomultiplier tube (PMT) system attached to an optical fibre. Directly connecting the target area with the device, we could easily measure the photon counts in a very small space. However, little information is known about the characteristics of optical fibres when exposed to twisting/looping in association with a moving mouse and the effect of the surface of optical fibre. In the present study, we report on the characteristics of optical fibres to detect gene expression rhythm in freely moving mice. Using this portable optical device directly connected with a target area, we were able to measure the circadian rhythm of clock gene expression over a prolonged period in freely moving mice in a small space.