Characterization and pharmacological applications of a new technique

8/3/2019 Characterization and pharmacological applications of a new technique

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C h a p t e r 3

Characterizat ion andpharmacological applicat ions

of a new technique

This chapter describes the characterization of a new technique to measuremelatonin contents in the pineal gland of freely moving rats, by means ofon-line microdialysis. Furthermore some exploratory pharmacologicalexperiments are described, indicating the potential of this technique.

Basal output during daytime was low, but well within the limits of theanalytical capabilities. A marked increase was recorded during night-time.TTX was used to investigate the possible neuronal drive behind melatoninproduction. Such a drive appeared to be present during night, because

melatonin levels decreased substantially during TTX perfusion. Duringdaytime however, even high concentrations of TTX were not able to alterthe melatonin production, indicating that no neuronal drive was respon-sible for the low daytime output of melatonin.

To exploit the high time resolution of the technique, the effects of shortterm changes in noradrenaline release (input) on melatonin secretion(output) were studied. A one minute light pulse was applied aroundmidnight and resulted in an immediate decrease of both noradrenaline andmelatonin. While noradrenaline returned to basal levels in 40 min, mela-tonin did not reach the baseline within 2.5 h. This discrepancy in

correlation between noradrenaline and melatonin indicates a rapid inac-

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3. Characterization and pharmacological applications of a new technique 73


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3.1 Int roduct ion

Over the last decades, extensive research has been performed on the pineal gland and itsneuroendocrine hormone melatonin. Now that much is known about the biosynthesisand its regulation,348,396 the important role of melatonin as part of the biological clock

has become evident.46,212,280 Thus far, however, most of these studies have been doneinvitro with isolated glands, or in vivo by decapitation and dissecting the gland.

The microdialysis technique has found a widespread use for in vivo monitoring ofneurotransmitter release in a variety of neuronal tissues.377,406 Dialysates are relativelyclean and can easily be analyzed by HPLC, because the samples can be injected directlywithout clean-up procedure. The high sensitivity for electro-active compounds likecatecholamines136 and the fact that the animals are conscious during the experimentscontributed to its popularity.

Microdialysis of the pineal gland was first described by Azekawa et al.19 They used anI-shaped cannula to collect the samples. While melatonin was determined by HPLC andelectrochemical detection,103,120 one of the problems they encountered was the level ofsensitivity. Although their detection limit for melatonin was as low as 5pg/sample,some of the daytime samples could not be measured. However, for circadian studiesdaytime values are of crucial importance. The use of fluorescence detection applied inour studies dealt with that problem and allowed a variety of different kinds of microdia-lysis experiments in the pineal gland.

This chapter describes some of those experiments, starting with a characterization ofthis relatively new technique. Data on basal output, day/night variation, and the possibleinvolvement of a neuronal drive in melatonin production, assessed by the use of TTX,

provide a basic understanding of the kind of experiments described in this thesis.The role of the sympathetic nervous system in stimulating melatonin production iswell known.348,280 However, the dynamics of the coupling between noradrenaline andmelatonin in terms of its time dependency, is largely unknown. Short manipulations ofthe noradrenergic innervation may reveal thedynamics of this noradrenaline/melatonincoupling.

It is well known that short light pulses during the dark period are very effective insuppressing pinealN-acetyltransferase activity35,133 and melatonin production.150 De-pending on light intensity, pulses of several minutes to even milliseconds397 suffice toinhibit pineal metabolism. Furthermore, a short stimulation of sympathetic neurons can

be achieved by depolarization with potassium chloride.408

Also in the pineal gland thishas been described as being useful to generate a short sympathetic stimulation duringdaytime.58

In chapter 4, the relationship between noradrenergic innervation and melatoninproduction is extensively studied, but in this chapter we focus on the dynamics of changesin sympathetic innervation on pineal melatonin productionin vivo, made possible by thehigh time resolution.

The neural pathways involved in the regulation of the circadian rhythm in pineal activityare only partly known. Information from the SCN reaches the sympathetic system via anot well established pathway in which the dorsal hypothalamus takes a central posi-

tion.30,119,157

Recently, a GABA-containing projection from the SCN to the paraventricu-



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lar and dorsomedial hypothalamus was described (see Fig.1.15, page 37).38 In addition,the GABA-containing innervation of the dorsomedial and posterior hypothalamus hasbeen implicated in the control of sympathetic outflow.208,209,321,400 Accordingly, in thischapter, a study is described which determines whether the GABAergic projection fromthe SCN might be involved in depressing the activity of neurons in the hypothalamuswhich are associated with the control of the circadian melatonin rhythm, using the dualprobe approach. These experiments are part of a large study addressing the role of GABAreceptors in the dorsomedial hypothalamus(DMH) in the regulation of melatonin andcorticosterone148 and was a collaboration with Dr. A. Kalsbeek, Netherlands Institute forBrain Research.

The activity of the SCN is in opposite phase with many other rhythms.310The inhibitoryprojection of the SCN to other areas as suggested,148 may well result in a stimulation ofmelatonin production after lesioning the SCN. Under such conditions, generally mostof the endogenous circadian rhythmicity is lost.217The present study describes the effects

of SCN-lesions as measured with microdialysis and was carried out in collaboration withDr. G. Boer, Netherlands Institute for Brain Research and Prof. Dr. W. Rietveld,University of Leiden.

The fact that both circadian information and data on absolute melatonin productionare obtained simultaneously, is an advantage of this approach and another exploratoryapplication of trans pineal microdialysis.

3.2 Experimental setupAnimals were used as described on page 58, except for the SCN-lesions, where maleWistar albino rats were obtained from the University of Leiden. Animals underwentsurgery as described on page 59, one or two days before the experiments.

Melatonin production was measured as described on page63, under basal conditionsboth in daytime and during light switch off, until several hours in the dark period. In asecond experiment, melatonin was measured during TTX perfusion (10-6 M, 1 h) bothin the light and dark period. In the light period, melatonin was also measured followingperfusion with TTX in a concentration of 10-5 M (1 h).

The dynamics of the coupling between noradrenergic innervation and resulting mela-tonin production was done by measuring both melatonin (page63) and noradrenaline(off-line, page 67) in separate experiments. Their response to a 1 min light pulse (300lux) at circadian time (CT) 18 and perfusion with potassium (60 mM, 30 min) duringthe light period was recorded.

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SCN-lesion experimentsSCN lesions were made under anaesthesia (Nembutal 60 mg/kg i.p.). A stainless steelelectrode (diameter 0.4 mm) was inserted bilateral in the SCN with the aid of a DavidKopf stereotaxic apparatus. The coordinates used for lesions were: 1.6 mm anterior frombregma, 0.5 mm lateral from midline, and 9.3 mm ventral below the dura, accordingto the atlas of Pellegrino. Lesions were made by leading a 1.5 mA anodal DC for 15 sthrough the electrode. To ensure that lesioned rats were completely arrhythmic, feedingand drinking behaviour was measured continuously for 4 weeks as described in detail byRietveld et al.145 Ten arrhythmic rats were then transported by car from the Departmentof Physiology of the University of Leiden where the lesions were applied, to the Depart-ment of Medicinal Chemistry of the University of Groningen. Upon arrival they wereadapted to their new environment for one week before a microdialysis probe wasimplanted as described on page 59. One day after surgery, the melatonin rhythm wasmeasured for 24 h. After the microdialysis experiments, animals were returned to Leiden

for a further recording of drinking behaviour for two weeks. Finally the rats wereanaesthetized with Nembutal (60 mg/kg i.p.) and perfused with 10% buffered formal-dehyde. The brains were removed, kept in buffered formaldehyde for 1 week and thencut in 50m frozen sections through the SCN area. The location and extent of the lesionswas verified and defined on the basis of Kluver-Barrera myelin stained sections.

Figure 3.1 The effect of TTX on melatonin production. TTX was perfused in aconcentration of 10-6 M ( , n= 4) during night-time and in concentrations of 10-6 M ( ,n= 4) and 10-5 M ( , n= 3) during daytime. All perfusions lasted for 1 h and started at t= 0 min. Melatonin is expressed as percentage of mean day- or night-time levels,whichever is applicable and is presented as the mean S.E.M.



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Dual probeThe dual probe experiments were carried out after implanting both a dialysis probe inthe pineal (page 59) and in the dorsomedial hypothalamus (DMH) during one surgicalprocedure. The DMH probe was implanted by Dr. A. Kalsbeek, Netherlands Institutefor Brain Research, as described elsewhere.148 The U-shaped dialysis probe consisted ofa dialysis fiber (molecular weight cut-off 6000 Da, 3 mm total length), glued into theends of two parallel 25G stainless steel tubings. It was implanted just lateral to the DMH,at coordinates with flat skull of 2.8 mm caudal to bregma, 1.6 mm lateral to the midlineand 8.0 mm below the brain surface. The loop of the probe was positioned in therostro-caudal direction along the DMH.

During the experiments, melatonin was measured as described before (page63).Noradrenaline was measured (on-line, page 66) in two animals that showed a markedeffect on melatonin. The DMH probe was continuously perfused with Ringers solutionat a flow rate of 3.0 l/min. Perfusion occurred by replacing Ringers solution withmuscimol in a concentration of 10-5 M. Perfusion lasted for 40 min, although in oneexperiment two perfusions of 20 and 40 min respectively were carried out.

Figure 3.2 Day/night differences in the production of melatonin. Melatonin wassampled from the pineal gland from 1 h before until 5 h after lights off. Data are expressedas percentage of mean daytime level and presented as the mean S.E.M. (n= 6).



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3.3 Results

Basal levels of melatoninThe basal values varied among different animals, which indicated little differences in theexact placement of the cannula. The average output of melatonin during daytime in the

dialysates was 0.8 0.1 fmol/min (mean SEM; n= 28). Generally, this implicated thattypical daytime dialysates contained about 4 pg (16 fmol) in 20 min samples and 5 pg(24 fmol) in 30 min samples.

TTX sensitivity of melatonin productionInfusion of the sodium channel blocker TTX did not affect melatonin production duringthe light period. In Fig. 3.1 the results are shown of a 1 h perfusion with 10-6 M and 10-5

M TTX respectively. No significant change in melatonin output was observed. Duringthe dark period however, perfusion with 10-6M TTX for one hour resulted in a markeddecrease of melatonin levels to 28.5 3.2 % of basal levels (Fig. 3.1).

Day/night differences in melatonin productionIn Fig. 3.2, the pattern of melatonin contents in the dialysates is shown during the lasthour of the light period and the first five hours of the dark period. An increase inmelatonin levels was observed, reaching a plateau value after four hours of about 1500% compared to average daytime levels. In absolute amounts, this reflected an output ofmelatonin of about 12.3 1.9 fmol/min.

Figure 3.3 The effect of SCN lesions on melatonin production. Melatonin was measuredfor 24 h in constant darkness. Animals had their SCN lesioned and were arrhythmic indrinking behaviour. Melatonin is expressed as percentage of mean basal levels during the

first two hours of the experiment and presented as the mean S.E.M. (n= 6).



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Effect of SCN-lesioning on melatonin productionFig. 3.3 shows the effect of lesioning the SCN on pineal melatonin production. Theanimals did not show any circadian variation in their melatonin production. However,the absolute amount of melatonin released was different. In three animals the output wasmuch higher than under normal conditions. Their average output was 204.0 61.5fmol/sample, which was in the range of normal night-time production. In the other threeanimals, basal output was 12.9 7.3 fmol/sample, which was comparable to normaldaytime production. The average basal output of all six animals was 108.4 50.9fmol/sample. It appeared to be difficult to correlate differences in basal output with thesize and location of the lesion. In one animal, in which the absolute output was 27.1fmol/sample, the SCN was not completely lesioned. From the drinking behaviour re-cordings after the microdialysis experiments, it appeared that in this case some rhyth-micity was restored. However, two other animals which had normal basal levels, didhave complete lesions and did not show any rhythmicity in drinking behaviour whatso-

ever. The animals that showed high basal output of melatonin appeared to have alsocorrect lesions and did not show rhythmicity in drinking behaviour after the microdialysisexperiments.

Effect of one minute of light on melatonin and noradrenaline releaseIn Fig. 3.4, the effect of a one minute light pulse (300 lux) at circadian time (CT) 18 onboth noradrenaline and melatonin are shown. Since CT 0 is the beginning of the lightperiod, CT 18 is defined as the middle of the dark period. As is clear from the data, amaximal decrease of noradrenaline levels occurred within one sample (20 min), whichwas 24.5 6.2 % of basal night-time levels. This effect was significant for 40 min. At t= 80 min, the noradrenaline release had returned to baseline values. For melatonin, thiseffect lasted longer and the effect was even more pronounced. The initial decrease wassimilar to the decrease in noradrenaline, but lowest levels were reached at t= 60 min(6.6 2.7 %). Especially the recovery took longer than for noradrenaline. Significantlower levels were measured until t = 140 min, but basal levels of melatonin were notobtained within 2.5 h after the light pulse.

Effect of potassium chloride on melatonin and noradrenaline releaseStimulation of noradrenaline release with potassium in a concentration of 60 mM for aperiod of 30 min around CT 7, increased the low noradrenaline levels. As is shown inFig. 3.5, the increase in noradrenaline release was rapid and significant to 286 60 %

above baseline. At T = 60 min, levels had returned to basal values. Since basal pinealoutput of noradrenaline under daytime conditions was very low, cocaine, a re-uptakeinhibitor of noradrenaline, was added to the perfusion medium in a concentration of10-6 M. This resulted in an average basal output of 7.8 0.5 fmol/sample. As shown inchapter 4, adding cocaine up to a concentration of 10-5 M did not change the pharma-cology of the system qualitatively. Unlike the light pulse experiment, melatonin produc-tion did not follow the pattern of noradrenaline release. Throughout the experiment, nochanges in melatonin production were observed.



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Figure 3.4 The effect of a one minute light pulse on melatonin ( , n= 5) andnoradrenaline ( , n= 4). Lights were turned on for 1 min at t= 0 min (CT18, midnight).Data are expressed as percentage of mean night-time levels and presented as the meanS.E.M. Asterisks (*) indicate statistical significance (p< 0.05).

Figure 3.5 The effect of potassium chlorideon melatonin ( , n= 5) and noradrenaline( , n= 5). Potassium was perfused in a concentration of 60 mM for 0.5 h, starting at t=0 min. Noradrenaline was determined in the presence of cocaine (10-6 M) throughoutthe experiment. Data are expressed as percentage of mean night-time levels and presentedas the mean S.E.M. Asterisks (*) indicate statistical significance (p< 0.05).



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Effect of muscimol in DMH on pineal melatonin and noradrenaline releasePerfusion with muscimol in a concentration of 10-5 M in the DMH caused an immediateand rapid decrease of melatonin levels (Fig. 3.6 and 3.7). A 40 min perfusion causedmelatonin levels to decrease to approximately 40 % of the basal levels within 60 min.Recovery to basal levels commenced 100 min after the start of the muscimol perfusion,but was sometimes obscured by circadian decrease at the end of the night (Fig.3.6).Noradrenaline levels in the pineal showed decreases to about 30 % of basal levels,following muscimol perfusion (Fig 3.7).

Figure 3.6 The effect of muscimol in the DMH on pineal melatonin production.Muscimol was perfused in the DMH in a concentration of 10-5 M for 20 and 40 min,starting at t = 19 h and t = 21 h respectively. At t= 24 h, lights went on. Melatonin isexpressed as percentage of mean nightime level and presented as a single experiment.



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3.4 Discussion

Basic considerationsThe first results indicated that it is well possible to monitor day- and night-time produc-tion of melatonin directly in the pineal gland of freely moving animals. Therefore it may

be a promising method for studying the neuroendocrine regulation of melatonin. Azek-awa et al.19,20 were the first who used this technique to study the melatonin levels in thepineal gland. They used electrochemical detection in their studies, with a detection limitof 5 pg/sample, which made it impossible to see daytime melatonin levels in some animals.The fluorescence detection, used in our experiments, is more sensitive (detection limitof 1 pg/sample), specific for indolic compounds and very stable. Therefore, approxi-mately 90% of the operated animals could be used in the experiments during daytime.The remaining 10% of the animals could not be used, due to incorrect placement of thecannula, or technical problems, such as obstruction of the cannula etc.

The use of a trans pineal cannula prevented us from hitting the sinuses just above thepineal gland and caused only minor damage to brain tissue. The sharpened point of thetungsten wire cutting through the fleeces surrounding the gland, made it more easy toposition the cannula exactly in the pineal. Rats recovered very fast from surgery, andcould be used for experiments the subsequent day.

Figure 3.7 The effect of muscimol in the DMH on melatonin (left panel) andnoradrenaline (right panel). Muscimol was perfused in the DMH in a concentration of10-5 M for 40 min, starting at t= 0 min. Both melatonin and noradrenaline are expressedas percentage of mean night-time level and presented as the mean S.E.M. (n= 5 formelatonin, n= 2 for noradrenaline).



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uridine incorporation into the RNA following isoproterenol stimulation. In addition,they reported a lack of effect of isoproterenol in changing the tissue level of ornithinedecarboxylase.271 Controversially, an increase in melatonin production in Y79 humanretinoblastoma cells is described following RNA synthesis inhibitors.142

In conclusion, it was shown that noradrenaline release and melatonin production inthe rat pineal gland are closely coupled. Decreases in noradrenaline release are immedi-ately followed by decreases in melatonin production, whereas increased noradrenalinerelease is followed by increased melatonin only after a lag time. Short term increases innoradrenaline release will therefore not influence the melatonin production duringdaytime. Microdialysis by its nature is able to detect these time dependent changes withrelatively high time resolution and is therefore suitable to study the relationship betweeninnervation and output of peripheral tissues in conscious rats.

Dual probe method opens even more possibilitiesIn the present study a pronounced GABAergic inhibition of melatonin production wasdemonstrated at the level of the DMH. Infusion of muscimol in the DMH rapidlydecreased melatonin, by reducing the noradrenergic input. Recently a GABA containingprojection from the SCN to PVN and DMH was described (see Fig.1.15, page 37).38

Furthermore, elevated plasma melatonin concentrations in SCN-lesioned animals couldbe inhibited by infusion of muscimol into the DMH.148 Taken these data together withthe present data, apparently there is a GABA-containing projection from the SCN to theDMH that transmits an inhibitory signal on melatonin secretion, mediated via thesympathetic innervation of the pineal gland. This tonic GABAergic inhibition of sympa-thetic innervation at the level of the hypothalamus may not be restricted to melatoninproduction. Previous studies have shown similar effects on other components of sympa-thetic outflow as well, i.e. heart rate, blood pressure and plasma noradrenaline.208,209,321,400

Because SCN-lesions cause increased daytime heart rate and blood pressure meas-ures,145,402 it may be that these components of the sympathetic system are under theinhibitory control of an SCN derived GABAergic input to the hypothalamus, similar tothe pineal gland.

The use of dual probe microdialysis has revealed important information on neuronalconnections between different brain areas. Implanting probes in a combination of twobrain areas such as substantia nigra and striatum,309 septum and hippocampus228are goodexamples. The combination DMH and pineal is yet another example of the additional

value of this method to anatomical studies. Combinations of SCN/PVN and pineal wouldbe a logical next step.


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Characterization and pharmacological applications of a new technique