A Blog Around The Clock

Persistence In Perfusion

i-710d005c8660d36282911838843a792d-ClockWeb logo2.JPGThis post, from January 25, 2006, describes part of the Doctoral work of my lab-buddy Chris.

Mammals have only one circadian pacemaker – the suprachiasmatic nucleus (SCN). Apparently all the other cells in the body contain circadian clocks, too, but only the SCN drives all the overt rhythms. Without the SCN, there are no rhythms – the peripheral clocks either get out of phase with each other, or their clocks stop ticking altogether.

If you place various tissues in a dish, the SCN cycles indefinitely. All other tissues are capable of only a few oscillations in the absence of a daily signal from the SCN (althogh with the improvement of culturing techniques, the duration has been extended considerably in recent studies). The mammalian pineal secretes melatonin rhythmically, but only in a animal with an intact SCN. The retina also synthesizes melatonin rhythmically, but only if the SCN is present. The melatonin never exits the eye anyway and the clock (and melatonin) in the retina is thought to regulate only local events, e.g., daily rhythms of rod-cone dominance and disk-shedding.

In non-mammalian vertebrates, SCN (although not as precisely located so far) can also be a pacemaker but it is not the only one. In some species, the pineal gland is the pacemaker (e.g., house sparrow, some lizards). In other species it may be the retina. In some, like the pigeon, both the pineal and the eye are pacemakers – only complete removal of both eyes and the pineal renders the whole animal arrhythmic.

Capability to study a pacemaker in a dish is quite important, as it eliminates from the study all the effects of interactions between multiple clocks or feedback from target organs. The SCN of mammals is easy to culture and much progress has been made in understanding the mammalian pacemaker in vitro.

The pineal of a number of species belonging to all vertebrate classes is also relatively easy to culture. The chicken pineal is so easy to keep in a flow-through setup, that there is a whole industry devoted to the study of cell biology of this organ. People who study the chicken pineal even have their own journal – Journal of Pineal Reearch – and their own Gordon Research Conference.

The retina of the eye is a bit more difficult organ to culture. It is very metabolically active and requires high oxygen levels in the culture media. The eyes of lampreys, fish and frogs have been successfully cultured quite a while ago. Eyes of lizards were cultured first about ten years ago or so. Mammalian eye was a little more difficult to do, but nevertheless, Dr.Gianluca Tosini managed to do it in mice and hamsters a few years back, but he is regarded as Grand Master of in vitro chronobiology. While subsequent studies of these tissues were productive in understanding the vertebrate circadian system, the problem with all of these cultured retinae is that none of them is the main pacemaker in its species. In each case, the pacemaker of the animal was either the SCN or the pineal, and what was cultured was a ‘slave oscillator’.
i-3d276b77e12fc776b5674ecfa8cf4356-a1 quail pair.jpg
What was needed was a culture of an ocular clock from an animal in which the eye is the main or sole circadian pacemaker. That meant a bird, preferably the Japanese quail.

Several of the top people in the field tried to culture an avian retina, but to no avail. One could not detect any melatonin secreted by the cultured tissue. Another one measured whopping levels of melatonin but no rhythm at all. Nothing ever got published on those failed attempts, but such information gets exchanged over beer at the conferences.

Perhaps there is really a need for a Journal of Negative, Inconclusive and Unpublishable Results, to deter people from trying, again and again, experiments that have failed before. On the other hand, past failures are not deterrents to everyone. Some people thrive on challenge. One such person is my lab-mate and friend, Christopher Steele, freshly a PhD, happy in his new job up in New England.

His Dissertation was focused on the eye as a pacemaker in the circadian system of Japanese quail. He was particularly interested in the way the two eyes communicate with each other in order to remain synchronized, and in the neural and hormonal signals going from the eyes to the rest of the system, and from the rest of the system to the eyes.

It would have been great for his work if he could isolate the retina and examine how it responds to various hormones and neurotransmitters, or to light pulses and light-cycles. But it was supposed to be impossible to do! But Chris is a veteran of two wars, not to mention endless melatonin hormone assays – nothing is impossible for him. He got the funding, he got the green light from the PI, he got Dr.Tosini to start a collaboration, and he got to work.

Speak of frustration! There is a reason why all those people before gave up on this problem – it’s hard! Over and over and over again, Chris tried and tried, tweaking one little thing at a time, to no avail. A time came when I had a feeling that our advisor was ready to call it quits and was just trying to find a way to suggest that to Chris politely.

But Chris wanted to have another go at it. This time, he decided that all the technical stuff was perfected and doing fine – the method of removing retinae from the eyes, the syringe pumps, the media, the fraction collector, the sterility of the whole setup. Instead, he used his knowledge of biology for a change and decided to supplement the media with precursors of melatonin, either tryptophan or serotonin:
i-cfd83b2708cf91043b260807e70f8f9c-a2 melatonin cascade.jpg

The wells containing retinas perfused with tryptophan-rich media were almost as bad as anything he did before – one cycle, perhaps two, then everything crashes and there is no more melatonin to detect. But with added serotonin, he got this:
i-27a3c3b0fc375127248a415c86e6c42a-a3 retina in vitro.jpg

Ha! It worked. And then it worked again. And again. Of course, this poses all source of new questions. Why did quail retina require supplementation with serotonin while that was not needed for culturing eyes from lampreys, African claw-frogs, green iguanas or hamsters? Would addition of serotonin to flow-through culture also make the quail pineal exhibit melatonin rhythms?

Well, Chris was called up again and spent another year in yet another war. In the meantime, the data just sat there in the corner of the lab, waiting for him to come back. Before he went away, Chris taught me how to use the culture system and I did one run, which did not work, but I knew two days into the experiment why it was not working. Anyway, now I know how to do it and, if anyone ever lets me into a lab again, I intend to use this technique in the future. Once Chris came back, he analyzed the data and the paper is now, finally, out in print.

Now you also understand that the title of this post is a double-entendre. The circadian pacemakers of the quail retina persist in perfusion, but also Chris persisted in trying the perfusion and the persistence paid off.