Understanding Circadian Rhythms: Case Study

When people wake up from a general anaesthetic they often seem confused about what time of the day it is, especially how much time has passed. This suggests that general anaesthesia somehow affects our sense of time. Guy’s research team are interested in studying this phenomenon in order to potentially better manage the care of patients who undergo a general anaesthetic.

Significant advances in understanding of how biological systems work can be made by studying model organisms. The classic model organism is the fruit fly, Drosophila melanogaster, but it is not the only one. Guy’s research group uses the honey bee, Apis mellifera, to investigate how general anaesthesia may affect our perception of time.

The honey bee is unique in the animal kingdom in that they have a ‘continuously consulted clock’. This means that bees are able to ‘consult’ their clock at any time to very accurately determine the exact time of the day. This clock forms the basis of their time compensated sun-compass. Bees navigate using this sun compass. They know where the sun should be at any time of the day and use this to determine the compass directions of a nectar source. 

On finding a good nectar source the bee will fly back to the hive and perform a waggle dance in the darkness of the hive. This dance informs other forager bees in the hive of the direction and distance of the nectar source. The angle of the ‘waggle’ component of the dancing bee (with respect to the vertical) is exactly equal to the angle between the sun and the food source.

As the sun moves across the sky over the course of the day, the dancing bees change the angle of their dance so that they are still communicating the correct direction of the food source. They do this by consulting their internal clock and adjusting the angle of the dance accordingly. 

Guy and his colleagues Dr James Cheeseman and Dr Craig Millar (from the University of Auckland) together with collaborators from the Free University of Berlin, used the fact that bees have a continuously consulted clock to model how general anaesthesia affects our sense of time.

Aim

To test how general anaesthesia affects sense of time in an insect model.

Method

Dr Cheeseman and Dr Millar anaesthetised honey bees for a six hour period using the human anaesthetic agent isoflurane. The bees were then fitted with a harmonic radar transponder (see picture on left) which enables them to track the direction of the bees’ flight with a modified ship radar. The direction of the flight is used to determine what time of the day the bees think it is when they wake up from their anaesthetic. The bees had already been trained to get nectar from a source at a known direction from the hive. After the bees were anaesthetised, they were released and their path was tracked (see Figure 5).

In Figure 5, Diagram a shows the paths of the bees as tracked by the radar. The grey track shows the path of the bees that haven’t been anaesthetised. When released they set off in a direction that effectively heads straight for the hive.

The blue track shows the path of bees that had been anaesthetised for 30 minutes. They were a little more confused but did head in much the same direction as the unanaethetised bees. 

The bees that were anaesthetised for six hours headed off in completely the wrong direction (red track).

Diagram b shows the same data but this time the direction each group of bees headed off in has been plotted against compass direction. 

These results show that, on recovering from a six hour anaesthetic, bees show an angle of orientation which is consistent with the bees perceiving that it is earlier in the day than it really is, i.e., that time has not passed during their anaesthetic.

The next hypothesis the researchers wanted to test was whether this change in orientation is due to the bees’ biological clocks being stopped by the anaesthetic. A PhD student working in Guy’s group (Ms Eva Winnebeck) used behavioural recording of bee activity and the molecular analysis of the expression of two clock genes (period and cryptochrome) to test this hypothesis.

After a six hour anaesthetic with isoflurane, the circadian rhythms of activity of an entire hive of bees appeared to be shifted to a later time zone (i.e., phase delayed) by 4-5 hours (see Figure 6). 

The molecular basis for the behavioural phase shift was established by using real-time quantitative PCR to measure the expression of the clock genes. Eva found that the anaesthetic resulted in the rhythms of expression of cryptochrome mRNA being damped (i.e., the oscillations have a smaller amplitude) and phase delayed (see Figure 7). This shows that anaesthetic effectively stops the biological clock. 

Over 230 million people undergo anaesthesia annually; more than the number of children born each year. These findings have implications for such patients and their caregivers as they provide a scientific explanation of why, on waking from an anaesthetic, many patients feel that they have only just ‘gone to sleep’.

Conclusion

Chronobiology or the study of biological rhythms and the clocks that control them is a unique area of biology because of the direct link that can be made between what is happening at the sub-cellular molecular level and the behaviour of an organism or even an entire population. This, and that fact that chronobiology research has direct applications to human health and sleep, makes it a fascinating area of research.