Introduction
The fact that humans and animals we usually come in contact with do sleep has been known since long time ago. Research has also been carried out in some invertebrate animals, although it has been of behavioral nature and not aimed at identifying the reason for sleep.
Kayser and Steiner-Kayser1,2 showed that, under certain conditions, bees lose muscle tone, drop the antennae and remain still, a behavior they interpreted as a state of sleep. In turn, Tobler investigated numerous invertebrate animals and described that they spent several hours of the day motionless; she explained that passiveness as the sleeping behavior.3-5 Moreover, in numerous investigations on the Drosophila fly, periods of immobility were also interpreted as equivalent to sleep. However, there is no evidence that these animals had the sleep characteristics clearly outlined by Hobson.6
Our working group was not particularly interested in sleep in the crayfish, but in brain’s processing of sensory signals and their effect on behavior. For that investigation, a freshwater crustacean, the crayfish, was used because its size facilitates the implantation of electrodes onto its brain and the performance of electrophysiological records while the animal seems to live a more or less normal life.
Crayfishes were kept in aquariums with tap water from which chlorine was removed by strongly stirring (“old water”), which appeared not to bother the crayfishes, which can remain for several months under these conditions, even if the water gets muddy with their excretions; the water was changed when the latter occurred. Occasionally, the water in the aquariums evaporated and the animals tended to lay on their side on the surface when only 5 to 7 cm of water were left, a behavior frequently observed by those who work with crayfishes (Fig. 1). Having no explanation for that position, we ventured several theories, including that they might be sleeping. However, that seemed so unlikely that we abandoned the idea.
On one occasion, a crayfish with electrodes implanted onto the brain placed itself in that recumbent position; at that moment we thought it would be adequate to record its brain activity. Surprisingly, the oscilloscope records showed slow waves with a frequency of approximately 10 Hz. Shortly after, the animal regained its vertical position at the bottom of the aquarium and we observed the multitude of characteristic spikes of normal brain record. In view of this finding, we decided to wait for the crayfish to put itself on its side to obtain new records. We performed the corresponding tests on an animal while sleeping, to see if the pattern corresponded to that cerebral state (Fig. 2).
Signal analysis
Initially, the records were analyzed using the fast Fourier transform to obtain frequencies, a strategy turned out not being the best for studying the encephalographic records, and thus using the wavelet transform was decided.
It should be remembered that the Fourier transform allows the analysis of a series of periodic signals, but only delivers a space of frequency and intensity. Conversely, the wavelet transform provides a much more complete space, where intensity, frequency and time coincide, and hence it is more useful for identifying where and when frequency changes occur in the brain records.
Sleep records
The first indications of a change in the cerebral state of the crayfish were postural change and immobility, as well as the change in the type of signals, which went from a continuous chain of spikes of between 20 and 50 µV in amplitude to a series of slow waves of between 10 to 20 cps (Fig. 3).
In a subsequent stage, the crayfish brain was mapped to locate the region that produces the slow waves. The crayfish was placed in a tank with water where it was fixed to a stem leaving all its appendages free. We made a hole in the cephalothorax to expose the dorsal and anterior surface of the brain, in order to enable choosing the region to be recorded (Fig. 4).
To compare the sleep records of the crayfish with those of other animals, including humans, it is necessary to take into account various factors. Humans have two types of sleep, one called REM (rapid eye movements) and another non-REM (without rapid eye movements) during which dreams occur. Although it is possible for the crayfish to move some of its appendages, it is not possible to determine if it does, and this model would therefore be only applicable to the non-REM sleep state.
In summary, as it can be clearly appreciated in Figure 5, the origin of the transition from an active crayfish to a sleeping one occurs with the change of a record dominated by spikes to one with slow waves, which occurs in the central brain complex. Meanwhile, other brain regions continue with records dominated by unitary spikes. The central complex of the crayfish is homologous with that of insects.