University of Groningen Melatonin on-line Drijfhout, Willem Jan IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1996 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Drijfhout, W. J. (1996). Melatonin on-line: Development of trans pineal microdialysis and its application in pharmacological and chronobiological studies. Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 05-04-2019
Summary At certain times, magic remedies become popular that are supposed to cure almost all diseases. Recently melatonin has become known in this respect. An important trigger for this increased interest was a book with the suggestive title The melatonin miracle, written by the researchers Pierpaoli and Regelson. Without any trace of reserve, claims are made on effectiveness in cardiovascular diseases, depressions, insomnia, cancer, immune deficiency etc. Very appealing are the suggested rejuvenation and the increase in life-span by up to 25%. Unfortunately, such popular writings change the image of a new and promising research field to a kind of alternative medicine and invoke a certain contempt by some people. A second book, written by the American researcher Russell Reiter, also has an intriguing title: Melatonin, your body s natural wonder drug. Also in this book, the broad clinical applicability of melatonin is addressed, but a distinct difference is made between facts and hypotheses. The thorough knowledge of the author concerning the research field, contributes to a fine overview of today s knowledge and the many blank spots still present, concerning this promising hormone. Melatonin was discovered by the end of the fifties, but its ubiquitous action only became known during the last five to ten years. Melatonin is synthesized in the pineal gland, a small, pea-sized organ in the brain. Remarkably, its production is limited to the night period. In the presence of light, synthesis will directly be inhibited. The resulting concentrations in plasma therefore show a marked day-night rhythm, with night-time concentrations about 10-15 fold higher than daytime levels. Furthermore it is interesting that this rhythmicity in melatonin production persists even in the complete absence of any other day-night rhythm, such as the light-dark cycle. It appears that the melatonin production is regulated by a clock in the brain. This clock is called the suprachiasmatic nucleus (SCN) and has the intrinsic capability of inducing all kinds of rhythmic processes in the body. This little watch in our brain, together with some other brain areas, including the pineal gland, is called the biological clock. Time information from the suprachiasmatic nucleus is electrically transmitted to other brain areas. The transmission of time information to other parts of the body is performed by a hormonal signal, namely melatonin. The moments of increase and decrease of melatonin concentrations provide information about the time of day. The length of increased melatonin concentrations is informative for the time of year (long and short nights). Because the average night-time concentrations decrease gradually with age, even information about the body s age can be transduced. An important observation was that, although the suprachiasmatic nucleus is controlling melatonin production, once produced, this melatonin can also influence the suprachi - asmatic nucleus. This gives melatonin the potential to adjust the biological clock. The effects of melatonin on sleeping disorders and jet-lag is based on this mechanism. In addition, this is the reason why melatonin-like compounds are presently under development as a new class of drugs. Besides this effect on the clock, apparently, a number of other processes in the body are influenced by melatonin. A stimulation of the immune system is suggested and melatonin is reported to act as a radical scavenger. Radicals are highly reactive specimens that cause DNA-damage and can induce cancer. Many of the research focused on these Summary 201
properties is still in the phase of animal experiments, some have not yet passed the test-tube. Only few conclusions can be drawn concerning the clinical relevance of these actions. In chapter 1, these matters are thoroughly discussed: the mechanisms in the pineal gland, involved in the control of melatonin production, the various physiological actions, the target sites, the present achievements on melatonin-like compounds and especially potential clinical applications in humans are reported. Also the construction of the biological clock and its specific characteristics, a research field known as chronobiology, is described. The research that is presented in this thesis is directed towards the regulation of melatonin production, also in relation to the suprachiasmatic nucleus, and the use of melatonin measurements in the development of melatonin-like compounds to clinically useful drugs. An extremely important part of the research was the development of a new technique, which enabled the measurement of melatonin production in freely moving animals, mainly rats. This technique is based on the principle of dialysis and shows a marked resemblance with an artificial kidney. In the artificial kidney, however, a large bunch of fibres is used, while in microdialysis only one fibre is sufficient. To make the handling of such a small fibre possible (the outside diameter is about a quarter of a millimeter), it is fixed in a construction called cannula. By inserting a cannula in the pineal gland, it was possible to collect small samples from the gland and measure the concentration of melatonin in it. This sampling and melatonin measurement could be carried out on-line and automated, so that the amount of melatonin produced was measured continuously throughout the day. Chapter 2 provides the reader with a detailed overview of the development of this technique, the procedures followed and the materials used. Also in this chapter, the very sensitive analytical assays are characteri zed, essential for the validation of this new research method. The introduction of a new experimental method should be accompanied by a series of experiments that characterize and show the scope of the method. Chapter 3 provides this characterization and the exploration of possible usage. The differences in melatonin production during day and night, as well as the sensitivity for TTX, a compound that reversibly inhibits the neural input of the gland, clearly show that this on-line method of melatonin measurements provide a reliable marker of the actual production in the gland. Furthermore, it appeared that, although a low basal level of melatonin production was still present during the day, only the high night-time concentrations were caused by active neural stimulation (see chapter 4). A major advantage of the technique used, was that samples are taken every 20 minutes. Compared to alternative techniques, where sampling often takes place at intervals of hours, microdialysis allows close monitoring of fast changing processes. This dynamic behaviour is exploited in two experiments, in which a short stimulation and an inhibition of the innervation were applied. It appeared that melatonin production was only reactive towards short-term inhibition, an effect that was explained by the mechanism underlying the melatonin synthesis. Finally, in this chapter the influence of the suprachiasmatic nucleus and a related brain area (dorsomedial hypothalamus, DMH) on melatonin production are investigated. By lesioning the suprachiasmatic nucleus, it appeared that this nucleus was absolutely necessary for the rhythmicity in melatonin production, not for its amplitude. In addition, the dual-probe 202 Summary
method proved that it was possible to study the connections between the pineal gland and the brain in detail. In this dual-probe method, one cannula was positioned in the pineal gland and a second in the DMH. The marked effects of compounds infused into the DMH on melatonin production in the pineal gland, make this an extremely useful method for functional models in the study of mutual interactions between brain areas. The most important intermediary between the suprachiasmatic nucleus and the pineal gland is the sympathetic nervous system. Nerve endings in the pineal gland release the neurotransmitter noradrenaline at night, which initiates and maintains melatonin production in the pineal gland by activation of β-adrenergic receptors. In chapter 4, the attention is mainly focused on this sympathetic innervation. Various adrenergic compounds were used to investigate the relation between β-adrenergic receptor stimulation and melatonin production. Also the regulating role of α-adrenergic receptors on β-adrenergic receptor stimulation was demonstrated. An important next step was the direct measurement of noradrenaline itself. Therefore, it was necessary to introduce a recently developed new assay, which is described in chapter 2 in great detail. The day-night changes in noradrenaline release appeared to be even more pronounced than those of melatonin itself. Noradrenaline levels showed characteristics of an on/off switch with extremely short intermediate periods of increase and decrease. The moments of turning on and off were also symmetrical to on- and offset of the night. These remarkable characteristics can be interpreted as direct measurements of the actual clock. The sympathetic innervation was further characterized by TTX, a reversible inhibitor of neural activity, yohimbine, which acts on presynaptic regulation, and cocaine, which inhibits the re-uptake of noradrenaline in the nerve terminals. Besides the sympathetic innervation, almost all organs in the body are under the control of the parasympathic nervous system as well. The most important neurotransmitter of this system is acetylcholine. Thus far, data on parasympathic input in the pineal gland were rather controversial. In chapter 5, the effect of cholinergic compounds on the melatonin production is described. It appeared that especially during night-time, this influence was pronounced inhibitory, and mediated by muscarinic receptors. Also, the reason for controversy on this subject is becoming clear in chapter 5. It appeared that the parasympathic influence was not directly aimed at the melatonin production itself, but indirectly, through the sympathetic nervous system. Such presynaptic effects are often difficult to measure in in vitro models, where the innervation is generally absent. From the present data, it appears that a pronounced parasympathic inhibition of melatonin production is certainly possible. The extent to which this inhibition is present under normal physiological conditions remains unclear, because attempts to measure the parasympathic innervation directly have failed thus far. As mentioned before, melatonin plays an important role in the biological clock. The release occurs rhythmically, with peak values during the night. Such rhythms, with a period of approximately one day are called circadian rhythms. From chapter 6 on, the focus is mainly put on circadian rhythmicity. Because the experimental protocol includes a surgery (implantation of the cannula in the pineal gland ) the day before the experi - ments, it is interesting to know whether such an operation perturbs circadian rhythmicity. To address this point, a number of experiments are described in chapter 6, in which circadian rhythms of activity and body temperature are measured before and after the Summary 203
surgery. These measurements were made possible by previously implanted transmitters in the rat s abdomen. These transmitters continuously transmit data on activity and temperature, which are picked up by a receiver placed underneath the cage. To get a good impression on specific effects of implantation of a cannula in the pineal gland, this surgery was compared to a standard microdialysis surgery. Also the effects of different kinds of anaesthesia were measured. It appeared that the rhythmicity was indeed drastically perturbed, especially after the surgery. This was mainly due to effects on height of the activity and temperature rhythms. The underlying clock mechanism did not seem to be disturbed. The effects of surgery lasted substantially longer than those of anaesthesia alone. Differences between the two types of surgery, or between the different kinds of anaesthesia were minimal. Especially the fact that the underlying clock mechanism was not affected, was important for further studies. As will become clear in later chapters, the melatonin rhythm is not affected by the surgery. An important property of melatonin, which is the basis for its positive effects on sleep-wake cycles and jet-lag, is the so-called entrainment. When organisms do not have any change between light and dark in their environment, the so-called free-running conditions, their own clock (the suprachiasmatic nucleus) will impose the day-night rhythm. One of the disadvantages of this is that the biological clock will deviate from the normal clock. The period of the biological clock is not exactly 24 hours. In humans, one day of the biological clock lasts for about 25 hours. Normally, this is no problem, because light-dark changes in the environment are capable of adjusting the clock each day, making it run on time. Under free-running conditions, this is not the case and after several days, the biological clock will differ from normal time substantially. In such a situation, melatonin is capable of taking over the function of light, when it is applied dail y at a specific time point. This is called entrainment. In short, a biological clock that does not run correctly, can be corrected by melatonin. This entraining effect is used in chapter 7. A model is described, in which melatoninlike compounds can be tested on their ability to adjust the biological clock, just like melatonin. Such testing is important in the development of clinically useful drugs. Rats were placed in continuous darkness for a period of 2-4 weeks. At the end of this period, a complete 24 h cycle of melatonin production was measured with microdialysis and compared to the rhythmic melatonin production under normal light-dark conditions. When no compounds were administered during that period of 2-4 weeks, the melatonin profile appeared to be completely shifted compared to the control situation. As was expected, this was not the case when melatonin was administered. A number of melatonin-like compounds were tested in this way, one of which acted indeed like melatonin. A remarkable finding in these experiments was that the height of night-time melatonin production was decreased after chronic treatment with melatonin. In contrast, chronic dosing of melatonin-like compounds resulted in an increase of the nightly melatonin production. The mechanism behind these effects is not clear yet. Further research should determine which of the possible explanations is the right one. From a therapeutical point of view, it is a very interesting finding. Until now, no experiments indicated a decrease in endogenous melatonin production after chronic melatonin dosing. Because it is generally agreed that higher levels are preferable to lower levels, melatonin-like com- 204 Summary
pounds may be better to use than melatonin itself. In order to come to a final conclusion on this point, a confirmation of this finding in human studies is necessary. A lot of people will be familiar with the phenomenon jet-lag. A sudden change of the day-night rhythm, caused by passing several time zones, generally results in sleeping problems and a depressed mood. In chapter 8, an artificial jet-lag is applied, by suddenly changing the rhythm in which the lights are turned on and off. The 24 h melatonin profile was measured at several days after this artificial jet-lag. During the first days after the artificial jet-lag, surprisingly there was no melatonin production at all. During subsequent days, the normal pattern was slowly restored, while during this transient state, certain strange characteristics were noticed. The high time-resolution with which the melatonin was measured in the on-line system, enabled the deduction of several specific characteristics of the suprachiasmatic nucleus from this transient state. Based on these characteristics, it was hypothesized that the suprachiasmatic nucleus is not one clock, but consists of at least two clocks, that respond differently to jet-lag. This difference in response is partly responsible for the problems that occur during jet-lag. Additional research will have to confirm this hypothesis. Finally, in chapter 9, some concluding remarks are made. The various results of all experiments described in this thesis are put in a broader perspective. To stimulate discussions, some parts of these final conclusions have a somewhat subjective and speculative character. The many directions for further research in this chapter, can be the starting point for future projects. Summary 205
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