The relationship between circadian rhythms of visual sensitivity and locomotion in the horseshoe crab, Limulus polyphmeus
The studies of Barlow, Chamberlain, Kass, Dodge and their colleagues have clearly demonstrated the presence of a circadian clock in the brain of horseshoe crabs that controls visual sensitivity. This clock communicates with the eyes via efferent nerves. When these nerves are active, at night, they release a substance on the eyes which makes them much more sensitive to light. They estimate this increase in sensitivity is on the order of 100,000 fold or more. Recently, we devised a method for recording the locomotory activity of horseshoe crabs in the laboratory, using modified running wheels (see Figure 1 below). We also developed a technique that allows us to simultaneously record changes in the sensitivity of one of the Limulus lateral eyes AND locomotion (see Figure 2 below). These two novel methods have allowed us to test the hypothesis that both eye sensitivity rhythms and activity rhythms are controlled by the same clock.
Figure 1.Horseshoe crab running wheel. A horseshoe crab is visible inside the wheel on the left. The slit in the wheel makes it possible to place the horseshoe crab’s tail through the back of the wheel and also secure the front of the carapace to the frame in the front (see yellow cable tie). This prevents the animal from becoming stuck inside the wheel. Wheel rotations are detected by a reed switch secured to the frame. Each time a magnet attached to the wheel passes the switch (shown in picture on the right), it produces a voltage change that we can record on a computer.
Figure 2.Recording horseshoe crab electroretinograms (ERGs). The top image shows a horseshoe crab fitted with an eye cup designed to both apply a light stimulus and record the responses of the eye photoreceptors to these pulses. A single green LED flashes every 30 seconds and this causes the responses shown below. Recordings are only obtained for one eye, so the other eye can detect changes in ambient light and thus entrain the circadian clock to the imposed light:dark cycle. The electrodes on the back of the animal are used to record heart rate. The bottom records show typical ERGs during the day and night. The blue trace is a stimulus marker. Note how the eye is more sensitive to light in the night.
Our preliminary results indicate that eye sensitivity rhythms are strickly circadian and readily entrain to imposed light:dark cycles, while activity rhythms are quite flexible, and can be either tidal, diurnal or nocturnal. Finally, we have determined that tidal rhythms of activity can be entrained by small, imposed, tidal cycles. How horseshoe crabs detect these changes in water depth is an ongoing area of investigation.Below are two figures that illustrate our findings. The first figure shows data from an animal who, for a few days, was nocturnal and thus its activity was correlated with its eye sensitivity rhythm. The second figure is more typical, with a tidal rhythm of activity, but a circadian rhythm of visual sensitivity entrained to the imposed L:D cycle.
Figure 3. Changes in visual sensitivity and locomotion. A. Eight days of data during during which there was a strong tendency of this animal to be most active at night, when visual sensitivity was also the greatest (gray bars). B. An expanded view of the same type of data for only two days. However, during the second day the lights were left on and yet the activity and visual sensitivity rhythms continued to coincide.
Figure 4. Simultaneous expression of both circadian and tidal rhythms. In this animal the visual sensitivity rhythm was, as always, entrained to the imposed L:D cycle, while its activity exhibited a strong tidal rhythm, with bouts of walking taking place approximately every 12.4 hours. This was a very common finding.