How eyes talk to each other?

One of the important questions in the study of circadian organization is the way multiple clocks in the body communicate with each other in order to produce unified rhythmic output.

In the case of mammals, the two pacemakers are the left and the right suprachiasmatic nucleus (SCN). The tow nuclei are anatomically close to each other and have direct nerve connections between them, so it is not difficult to imagine how the two clocks manage to remain continuously coupled (syncronized) to each other and, together, produce a single output, thus synchronizing all the rhythms in the body.

In the case of those non-mammalian vertebrates in which the pineal organ is the pacemaker, the answer is even easier - there is only one organ keeping time and, of course, it produces a single output.

However, in animals like quail, in which the pacemakers reside in the eyes, it is by no means clear how the two pacemakers manage to keep in sync with each other. There are no direct nerve connections between the two eyes, after all, so the communication is either indirect or bypasses the nervous system and utilizes hormones instead.

This is the question that my lab buddy Chris tackled in his doctoral Dissertation. Here, I will explain briefly some of his work - only the part that has already been published as I do not feel I am at liberty to divulge yet unpublished data.

In Japanese quail, the main pacemakers reside in the eyes. Without eyes, the rhythms of activity, body temperature and the synthesis of melatonin by the pineal decay into arrhythmicity. But, the operative word here is "decay". The rhythms do not suddenly disappear the moment the eyes are gone. It takes one to two weeks before the rhythm gradually loses its coherence to the point that no statistical software can detect any 24-h periodicity in the data. During that period, which is quite long, the rhythms are generated by the bird's SCN.

Thus, the SCN in quail are also pacemakers. They can drive overt rhythms for a period of time on their own. But they are not robust enough on their own. For them to function more long-term, they require daily input either from the pacemakers in the eyes, or the direct light-dark information from the environment via the extraretinal photoreceptors located deep inside the brain.

When one monitors and measures overt rhythms of locomotor activity or body temperature, one monitors the outputs of the SCN. It is not certain that such rhythms accurately portray the activity of the eye clocks.

So, Chris performed a series of experiments in which he tested if eyes are indeed tightly coupled to each other and if the measurable outputs do fairly represent the behavior of the clocks in the eyes. While the measurable output of the SCN is body temperature, the measurable output from the eyes in melatonin secretion. Eyes contribute about 1/3 to 1/2 of all circulating melatonin in this species, almost all of which is synthesized and released during the night (the rest is provided by the pineal gland, also almost exclusively during the night). So, Chris did pairs of experiments, i.e., two experiments would be very similar in logic and the protocol, but one would measure body temperature and the other one melatonin. The restuls of such paired experiments could then be compared to see if the behavior of the eyes and the behavior of the SCN matches each other.

First, he exposed quail to constant darkness for very long periods of time (months!) and continuously monitored body temperature. The period, amplitude and the general clarity of the rhythms remained constant over the whole period of time, indicating that the circadian system as a whole, or at least the SCN (which are dependent on eyes to remain coherent), were tightly coupled. At the same time, melatonin levels in both (left and right) eyes were strongly correlated to each other at all times - both very low during the subjective day and both very high during the subjective night:

Blood levels of melatonin also remained rhythmic over many months in intact birds, while in the absence of eyes, the pineal was not capable of providing rhythmic output of melatonin indicating that the whole system, SCN included, was arrhythmic:

OK, so these two experiments showed that the circadian system as a whole, eyes included, is tightly coupled over long term in constant conditions. They also strengthened the case for the eyes being the dominant pacemakers in the system. But how do the two eyes communicate with each other in order to remain coupled? Chris started resolving this question by uncoupling the outputs of the eyes and monitoring how long it took for the two eyes to-recouple again.

He did it by placing opaque black patches over the eyes of quail (they looked like little pirates with those patches). In control birds he placed patches over both eyes simultaneously, took them off 12 hours later, on again 12 hours hence and so on. In this way, the pineal and the extraretinal photoreceptors were receiving information that the environment was in constant light (LL), while the eyes were perceving a light-dark cycle (LD12:12).

In the treatment group, he placed the patches in an alternating fashion: 12 hours over the left eye, 12 hours over the right eye and so on. That way, each eye perceived a light-dark cycle, yet the two eyes got entrained 12 hours out of phase with each other.

Again, he did this in a pair of experiments - one measuring ocular melatonin and the other measuring the SCN output of body temperature. In a previous study, melatonin measurements were made during the first day of constant darkness following the alternating eye-patch protocol and the large difference between melatonin levels in left and right eyes was found (as opposed to roughly equal levels if the eyes were patched in synchrony). Chris wanted to know how long it takes for the eyes to get back together again, so he assayed melatonin after two and after five days in DD following the patching protocol and found that the eyes are still quite uncoupled on Day2 but completely coupled again on Day5:

In order to do the same experiment while monitoring body temperature, Chris had to somewhat modify the protocol. Handling the birds induces short spikes in body temperature which make the analysis of temperature rhythms difficult. So, after several days of alternate-phase (or in-phase) eye-patching, he placed the birds into skeleton photoperiods consisiting of one hour of light, 11 hours of dark, 1 hours of light and 11 hours of dark over several days. Each of 1-hour light pulses served as a dawn signal to one eye and as a dusk signal to the other eye (in out-of-phase patching; they were perceived the same in the in-phase patched birds), thus keeping the eyes uncoupled from each other for several days without human handling, thus providing clean body temperature records.

In the in-phase patched controls, once the birds were placed in constant darkness the rhythms started free-running from the phase of the last dawn-simulating light pulse:

In the out-of-phase birds, once the birds were placed in cionstant darkness, it took about 5 days for two separate components of the rhythm to fuse together into a single output, with the extrapolated phase exactly in the middle between the dawn-mimicking and the dusk-mimicking light pulses:

Putting all the data together into some circular statistics shows that quails' eyes consistently recoupled to a phase somewhere in the middle, between the two phases when they were experimentally uncoupled, suggesting that, over the five days, one eye phase-advanced about 6 hours, while the other eye phase-delayed about 6 hours until they recoupled completely:

These data showed how surprisingly strong the coupling between the two eyes is. Also, Chris showed that body temperature rhythms, which are easy to continuously measure, track the behavior of the eyes, thus eliminating the need for further experiments measuring ocular melatonin which are difficult, require more birds, and provide only snapshots in time instead of continuous observation of the phase of the pacemakers. Thus, the alternate-patching protocol can be useful for further studies of mechanisms of coupling - treating the animals in various ways (hormones, neurotransmitters, surgeries) and measuring which treatments affect the rate of recoupling of the two eyes which would indicate if the eyes communicate with each other via the nervous system (e.g, via the SCN) or via hormones (e.g., melatonin).

Reference:
Ocular clocks are tightly coupled and act as pacemakers in the circadian system of Japanese quail, Christopher T. Steele, Bora D. Zivkovic, Thomas Siopes, and Herbert Underwood, Am J Physiol Regul Integr Comp Physiol 284: R208-R218, 2003.