benzodiazepines influence melatonin secretion of the pineal organ of the trout in vitro

J Pined Res 1994:17:69-78 Piinred in the Unrred SIRICS of Amcrk- -u l l rights rexrved

Copvrrghl 0 Munkspard. 1994

Journal of Pineal Research ISSN 0742-3098

Benzodiazepines influence melatonin secretion of the pineal organ of the trout in vitro

Meissl H, YBiiez J , Ekstrom P, Grossman E. Benzodiazepines influence melatonin secretion of the pineal organ of the trout in vitro. J. Pineal Res. 1994;17:69-78. OMunksgaard, 1994

Abstract: The effect of benzodiazepines (BZP) on melatonin release was investigated in the pineal gland of the rainbow trout, Oncorhynchus mykiss, maintained under in vitro perifusion culture conditions. Melatonin and the methoxyindoles 5-methoxytryptophol (5-MTOL), 5-methoxyindoleacetic acid (5-MIAA), and 5-methoxytryptamine (5-MT) were determined directly in samples of the superfusion medium by HPLC with electrochemical detection. Melatonin release was significantly increased by addition of diazepam and clonazepam in a dose-related and reversible manner. The effects of benzodiazepines were more pronounced in light-adapted pineal organs, when melatonin secretion is low, than under scotopic conditions. When the perifusion medium was replaced by a medium containing low calcium, high magnesium concentrations, melatonin release was considerably decreased by 70% in light-adapted and 20% in dark-adapted pineal organs. Addition of diazepam to low Ca2+, high Mg2+-medium reversed the decrease of melatonin release and produced a clear rise in its secretion rate. Addition of the BZP antagonist flumazenil to the perifusion medium slightly decreased melatonin release in the light- and dark-adapted state, whereas the peripheral receptor antagonist PK 1 1 195 did not alter melatonin release. The effect of diazepam is reduced by simultaneous addition of flumazenil to the superfusion medium, suggesting that the effects of diazepam are receptor-mediated. The methoxyindoles 5-MTOL, 5-MIAA, and 5-MT showed no significant changes of their release pattern after diazepam application in light- and dark-adapted pineal organs. These results suggest that BZP can influence melatonin production and release by an intrapineal action possibly on the melatonin synthesizing photoreceptor cell.

Hilmar Meissl,' Julian Yaiiez,' Peter Ekstrom,' and Eberhard Grossmann' 'Max-Planck-Institute for Physiol. and Clin. Res., W.G. Kerckhoff-Institute, Bad Nauheim, Germany: 2Dept. of Zoology, University of Lund, S-22362 Lund, Sweden

Key words benzodiazepinesaiazepammelatonin- 1 GABA-photoreceptor cell-photosensitivity-HPLC-

electrochemical detection-trout ' Address reprint requests to Dr Hilmar Meissl, Max-Planck-lnstitut fur Physiologische und

1 Klinische Forschung, W G Kerckhoff-lnstrtut. Parkstr 1, D-61231 Bad Nauheim, Germany

Received April 1, 1994, accepted August 1, 1994

Introduction The role of the pineal organ in physiological functions that are related to the circadian and circannual control of endocrine activity of certain mammals is well established [Reiter, 19871.

These functions seem to be mediated by the photoperiodic-dependent secretion of the pineal hormone melatonin with its elevated nocturnal and low daytime levels. The marked day-night rhythm of melatonin secretion makes the hormone a likely candidate for the transduction of photoperiodic information. It was suggested that melatonin may play a role in a variety of functions such as

reproduction, metabolism, seasonality, immunity, and thermoregulation (for review see Yu and Reiter [1993]). Melatonin has been also postulated to be associated with pathophysiological processes in cancer, epilepsy, and seasonal affective disorders [Maestroni and Conti, 1993; Blask et al., 19931. Several studies have additionally indicated that melatonin may exert sedative and anticonvulsant effects in humans and mammals [Anton-Tay et al., 197 1 ; Romijn, 19781. These psychopharmacologi- cal effects of melatonin are similar to those produced by tranquilizing drugs like benzodiazepines and barbiturates [Niles, 19891, which act as a result

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Meissl et al.

of an increase in the efficiency of GABAergic transmission [Richards et al., 19861. It was shown by Cardinali and coworkers that pinealectomy dis- rupts circadian rhythmicity of brain GABA and benzodiazepine binding [Acuiia-Castroviejo et a]., 1986a,b] and that low doses of melatonin counteract the modifications of GABA and benzodiazepine binding observed after pinealectomy [Rosenstein and Cardinali, 19901. In the rat brain, melatonin is able to enhance GABAA binding in vitro, whereas diazepam binding was inhibited, indicating that the effects of melatonin are possibly a result of a direct action on benzodiazepine binding sites [Niles, 19891. These results support the view that some melatonin actions are linked to allosteric modulation of the GABAA receptor complex.

On the other hand, drugs acting on the GABA receptor complex may influence melatonin synthesis and secretion of the pineal gland in mammals, including humans [Wakabayashi et al., 1991a,b; Cardinali et al., 19861, where GABA receptors as well as benzodiazepine binding sites are detected [Basile et al., 1986; Cardinali et al., 1987; Ebadi and Chan, 1980; Lowenstein et al., 19841. The effect of GABA on melatonin synthesis of the rat pineal seems to be predominantly inhibitory in vitro [Rosenstein et al., 19891, although earlier studies reported some controversial results [cf. Mata et al., 1976; Balemans et al., 19831. Diazepam also seems to influence melatonin content of the rat pineal in vivo and in vitro. This effect is possibly mediated by the action on both GABA and peripheral benzodiazepine receptors in the rat pineal [Cardinali et al., 1986, Wakabayashi et al., 1991a,b].

In the current study, we investigated the effects of benzodiazepines on melatonin secretion of the pineal organ of the trout in vitro. We chose the trout pineal as experimental model for the following reasons: 1 ) it contains functionally active photoreceptor cells that are comparable to retinal rods and cones [cf. Meissl and Dodt, 1981; Meissl and Ekstrom 1988 a,b]; 2) the direct photoreception seems to control melatonin production without the involvement of an endogenous oscillator [Gem and Greenhouse, 19881; 3) we have no indication for a catecholaminergic innervation of the trout pineal, as in the mammalian pineal, that may influence melatonin production [Ekstrom and Meissl, 19891 and 4) we have evidence for the contribution of a GABAer- gic mechanism modulating dark and light adaptation properties of the pineal organ [Meissl and Ekstrom, 1991).

If a neurotransmitter system can influence light sensitivity of the photoreceptive pineal system, it should effect also melatonin production and release. In the present investigation, therefore, we made an

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attempt to obtain more information about the possible contribution of the intrapineal neuronal circuitry on melatonin formation and release.

Material and methods

Experiments were performed on pineal organs of adult rainbow trout, Oncorhynchus mykiss (former- ly Salmo gairdneri). The animals were obtained from a commercial hatchery and kept in large aquaria (200 1) with oxygenated fresh water (8- 10°C) under a LD cycle of 12: 12 for at least 3 weeks before each experiment that was conducted throughout the year.

Pineal organs were quickly removed from freshly decapitated trout, placed on small pieces of filter paper, washed in cold Hank’s buffer, and then transferred to the experimental perifusion chamber. Each perifusion apparatus contained six chambers, of which four chambers were usually used in one experiment. One chamber of 100 IJ .~ volume held one pineal organ, only in some experiments where we used smaller trout, two pineal organs were incubated in the same chamber. Pineal organs were continuously superfused with modified Hanks’ buffer (0.85 mM CaCl,, 0.547 mM MgSO,, 3.63 mM KCI, 0.398 mM KH,PO,, 2.81 mM NaHCO,, 92.56 mM NaCl, 0.23 mM Na,HPO,, 3.75 mM glucose, 7.5 mM Hepes, 0.95 pM tryptophan, pH 7.4) at a flow rate of 0.5 ml/hr provided by a microprocessor controlled peristaltic pump (Gilson MP3). The temperature was kept constant at 16°C by a cryostate (Huber, Germany). For blocking synaptic transmission in some experiments, CaCl, content was reduced to 0.135 mM and MgSO, enhanced to 6.08 mM.

For all experiments we used a standardized experimental protocol to minimize possible arti- facts. Pineal organs were light adapted for exactly 2 hr after their removal from the brain before the first collection period started in the light adapted (LA) group. These experiments were performed on the first day. Then the organs were dark-adapted over- night for 15 hr in the absence of any light influence. Experiments were then repeated on the second day in the dark-adapted preparation. The experiments were finished 34 hr after the dissection of the pineal organs, although the pineal organs synthesize and secrete melatonin at this time at an almost un- changed rate.

To obtain sufficient amounts of methoxyindoles for HPLC measurements samples were collected in intervals of 60 min. Furthermore, since the sensitivity of our method is lower than with a radioim- munoassay for melatonin, we used an adapting light

Benzodiazepines and melatonin secretion

Effects of benzodiazepines on melatonin release of light- and dark-adapted pineal organs in vitro The release of melatonin into the superfusion medium varied considerably between glands, but the secretion rate of an individual gland showed a relatively constant rate depending only on irradi- ance and temperature. For the present experiments we chose a constant temperature of 16"C, since this temperature seems to be in the optimal range for pineal photoreception [Tabata and Meissl, 19931. Additionally, we selected a dim light of 520 nm and 1 1.81 log photons/cm2/s for light adaptation. Under these light and temperature conditions, the explanted organ released melatonin in an amount between 30 and 70% of that of the dark-adapted state. Brighter light intensities reduced melatonin output considerably, so that it was difficult to detect minor changes after the addition of drugs.

The melatonin output under the present experimental conditions was between 0.5 and 4 ng/pineal organ/hr in dim light (520 nm, 11.8 1 log photons/ cm2/sec) and up to 10 ng/pineal/hr in the dark adapted state. Figure 1A shows the melatonin release of 19 trout pineal organs that were light adapted for 2 hr. The average melatonin release was 2.51 ? 0.45 nglpineal orgadhr under these light conditions. Addition of 100 pM diazepam to the superfusion medium increased melatonin release considerably to 5.93 ? 0.97 nglorgadhr. Subse- quent exchange of the superfusion medium with normal medium reduced the melatonin output to the previous value. The individual pineal organs showed distinct variations in the diazepam-induced change of melatonin release. Eighteen pineal organs of a total of nineteen showed a clear increase of melatonin output of up to 776% of the control value. In only one pineal organ diazepam decreased melatonin secretion, but subsequent washing with normal medium did not restore the original melatonin output of this organ.

In dark-adapted pineal organs, diazepam also caused an increase of melatonin release, but this increase was lower than in light-adapted organs (Fig. 1B). The melatonin output increased from 3.39 ng/orgadhr to 4.00 ng/organ/hr during diazepam application, i.e., on the average by 18%, and fell to the original level after subsequent washing with Hank's medium. Comparing the individual organs, the effect of diazepam was also not as clear as in the light-adapted group. Thirteen pineal organs showed an increase (more than 10% above control value) of melatonin release during diazepam, four organs a slight decrease, and two showed an un- changed melatonin output.

Increasing concentrations of diazepam led to a steady increase of melatonin release, when the

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in the mesopic range. In fully light-adapted pineal organs the release of melatonin was below the detection limit.

Illumination of the experimental chamber was provided by a 150 W Xenon arc. Light energy was adjusted by neutral density filters. Monochromatic light of 520 nm, which is the maximum photopic spectral sensitivity of most pineal photoreceptors [Meissl and Ekstrom, 1988a1, was provided by an interference band filter (Type AL, Schott, Mainz). The intensit for the adapting light was 6.5 X 10" photondcm /sec at 520 nm. Under these adaptation conditions the pineal organ releases melatonin at a rate of approximately 70% of the dark adapted value.

The methoxyindoles melatonin, 5-methoxytryptamine (5-MT), 5-methoxyindoleacetic acid (5- MIAA) and 5-methoxytryptophol (5-MTOL) were separated by high performance liquid chromatogra- phy (HPLC) and measured by electrochemical detection. Aliquots of the superfusion medium were injected directly into the chromatograph, which consisted of a Gynkotek M-480 pump, an autosam- pler (Spark, Holland) with a Rheodyne 7010 injec- tor and a 100 PI loop, a Spherisorb ODS I1 column (125 X 4.6mm, 5 pm particle size) and a Gynkotek M20 electrochemical detector or a Coulochem I1 detector, ESA. The potential of the glassy carbon working electrode of the Gynkotek detector was set at +0.9 V relative to the reference electrode. The solvent system consisted of 18% acetonitril, 50 mM sodium acetate, 0.05 mM EDTA, and 50 mM citric acid.

Values are presented as mean values * S.E.M. For statistical analysis mean values were compared with the Wilcoxon signed rank test.

Y

Results

The isolated pineal organ of the rainbow trout responds similarly as in situ to changes in the light environment with alterations in its endocrine and physiological status. Under in vitro perifusion culture, the trout pineal synthesizes and secretes melatonin with elevated levels in darkness and low levels in light. Because the melatonin output of the pineal seems to respond differentially to reductions or increases in the illumination level, depending on whether the organs were light or dark adapted [Gern et al., 19921 we used in the present study a fixed experimental protocol. The organs were light- adapted for exactly 2 hr before the measurement began, or they were dark adapted for 15 hr. This protocol minimized the rapid changes observed during light onset and offset.

Meissl et al.

LA

6

4

2 n

r'

g o Control Dmepam Wash Wash

I T

Control b p a m Wash Wash Fig. 1. Effect of 100 FM diazepam on melatonin release of explanted, light-(A) and dark (B)-adapted pineal organs of the trout. (A) The organs were light-adapted for 2 hr to 520 nm, 1 1.81 log photons/cm2/sec prior to the first collection period. Mean +- SEM, n = 19, *P < 0.001 vs. control. (B) Average melatonin release of dark-adapted pineal organs. The pineals were dark-adapted for 15 hr before the first collection period started. Mean values -t_ SEM, n = 19. PO = pineal organ.

organs were maintained in light conditions. The threshold concentration for diazepam was at about 10 pM (Fig. 2A).

This concentration was shown to be effective in in vitro experiments in the rat pineal organ [Lowen- stein et al., 19851. After 15 hr dark-adaptation, diazepam did not produce such clear changes in melatonin release (Fig. 2B). The lack of a significant increase of melatonin release may be a result of a deterioration of the conditions of the pineal organs on the second day of organ culture, since the net output of the organs in darkness was lower than under light-adapted conditions. Using clonazepam,

72

''1 A 12

- 4 II LA *

T

G I 0 a 0 \ Control 1pM 10pM 100pM 500pM Wash

c Diazepam Y

DA

0 ' Control 1pM 10pM 100pM 500pM Wash Wash

Diazepam Fig. 2. Effects of increasing concentrations of diazepam on melatonin release of isolated, perifused pineal organs of the trout in the light-adapted (A) and dark-adapted state (Bf. Light conditions: 520 nm; 6.48 X 10" photons/cm2/sec. Mean values of four pineal organs * SEM; *P < 0.01 vs. control. LA = light-adapted; DA = dark-adapted.

a shorter acting benzodiazepine, melatonin release decreased at first slightly at concentrations between 1 and 100 pM, but increased with a dose of 500 pM. Subsequent washing with Hank's medium reduced melatonin output to the previous control value. The slight decrease was only observed in two out of four organs tested, whereas all four organs showed a distinct increase of the secretion rate at 500 pM.

In another series of experiments, we attempted to identify the possible cellular site of benzodiazepine action, whether it is presynaptic at the melatonin synthesizing photoreceptor cells or postsynaptic on second-order neurons. For this purpose we superfused the pineal organs with a medium containing low calcium and high magnesium levels, i.e., a medium that we usually use in electrophysiological experiments to block synaptic transmission (blocking medium). The idea behind these experiments was that a blockade of synaptic transmission should


A

u P 3

LA DA

4 T

3 C .- 5 2 2

= I 1 8

Control low Wash Wash Control low Wash Wash Ca2+

high Mg2+

Control low Ca2+ Wash high+Mg2+ Diazepam

Ca2+ high Mg2+

LA

Control low Ca2+ Wash highAMg2+ Diazepam

Fig . 3 . (A) Effects of Hank's medium containing low Ca2+ (0.13 mM) and high Mg2+ (6.08 mM) concentrations on melatonin release of explanted pineal organs maintained under light- (left) and dark-adapted conditions (right). (B) Addition of 500 pM diazepam to low Ca2+, high Mg2+ Hank's medium increased melatonin release by more than 600%. Light adaptation: 520 nm, 6.48 X 10" photons/cm2/s. n = 4, * P < 0.001 vs. control.

remove a possible influence of second-order neurons on the photoreceptor cells that are believed to produce melatonin.

Reduction of the calcium content and enhancement of magnesium clearly diminished melatonin release of explanted pineal organs (Fig. 3A). After 1 hr perifusion with blocking medium, melatonin output was reduced to one-third of the control value in light-adapted pineal organs. After dark-adaptation, the reduction was lower and reached a mean value of 35% (Fig. 3B). Addition of diazepam (500 pM) to the blocking medium increased melatonin release considerably to levels that are clearly above the initial control level. We observed an average increase of 440% in light-adapted and of 150% in dark-adapted pineal organs.

The addition of the benzodiazepine antagonist flumazenil (Anexate, Roche) to the perifusion medium in concentrations between 10 nM and 100 pM

LA

T T

Control 0.01 0.1 1 10 100 Wash Wash pM Flumazenil

DA

Control 0.01 0.1 1 10 100 Wash Wash pM Flumazenil

Fig. 4. Effects of increasing concentrations of flumazenil on melatonin release in light-(LA) and dark- (DA) adapted pineal organs. Flumazenil decreased melatonin output by about 50% in the light and about 30% in darkness. Light conditions as in Fig. 1-3. Mean 2 SEM, n = 4.

decreased melatonin output slightly (Fig. 4). One hundred pM of flumazenil reduced melatonin output on the average by 20% in light- and dark- adapted pineal organs, but the individual variations between different organs ranged from a slight increase to a complete inhibition. The diazepam- induced increase of melatonin release was considerably diminished by simultaneous addition of 100 pM flumazenil. Table 1 describes an experiment where melatonin release was determined on two consecutive days with a slightly different experimental protocol. On the first day diazepam was first added to the perifusion medium; thereafter, flumazenil was added together with diazepam. Diazepam increased the secretion rate of melatonin from 1 to 7.5 ng/hr. This value dropped to about 4.5 ng after

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Meissl et al.

TABLE 1. The effect of flumazenil (100 pM) and diazepam (100 pM) on melatonin release of light-adapted pineal organs in vitroa

Day 1

Treatment Control Diazepam Diazepam + Diazepam Wash1 Wash2 flumazenil

Melatonin (ng/organ/hr) 0.99 rt 0.15 7.42 rt 1.43 4.49 rt 0.60 9.14 * 2.16 6.11 * 1.81 3.52 * 0.60

Day 2

Treatment Control Flumazenil Wash Diazepam + Diazepam Wash flumazenil

Melatonin (ng/organ/hr) 0.45 rt 0.12 0.36 * 0.05 0.57 k 0.20 1.10 * 0.23 3.97 * 1.48 1.69 2 0.48 -

aMean values -e SEM, n = 4.

addition of the antagonist flumazenil, but was again enhanced after removal of flumazenil from the superfusion medium. Similar effects were observed on day 2 with the exception that the basal rate of melatonin release was at about 50% of day 1.

The peripheral benzodiazepine antagonist PK 1 I 195 did not alter melatonin release when used in a concentration of 10 pM and it did not prevent the diazepam-induced increase of melatonin release of light-adapted pineal organs (Fig. 5). In darkness, all four pineal organs showed a relatively stable rate of melatonin secretion irrespective of the treatment.

Effects of benzodiazepines on the release of 5- rnethoxytryptophol (5-MTOL), 5-methoxyindoleacetic acid (5-MIAA), and 5-methoxytryptamine (5-MT)

In several experiments, we measured, simulta- neously with melatonin, the other methoxyindoles 5-MTOL, 5-MIAA and 5-MT during diazepam application. In contrast to the effect of diazepam on melatonin release, 5-MTOL, 5-MIAA, and 5-MT showed no significant change on their release after addition of 100 pM diazepam in light- or dark- adapted pineal organs (Fig. 6 ) .

Discussion

The results presented here demonstrate that benzodiazepines at concentrations of more than 10 pM increase melatonin synthesis and secretion of photoreceptor cells of the isolated, superfused pineal organ of the rainbow trout. The stimulatory effect on melatonin release is most pronounced under conditions when melatonin synthesis is relatively low, i.e, when the pineal organ is exposed to mesopic light intensities. In completely dark- adapted pineals, the effect is diminished, but still demonstrable. The effects of benzodiazepines seem to be opposite to those of GABA or the GABA,

Control

* * T

+ Pk

DA

Control Pk Wash Diaz. Wash Diaz. Wash + Pk

Fig. 5. Melatonin release of pineal organs of the trout in vitro during application of the peripheral BZP antagonist PK11195 (10 pM) and of diazepam (100 pM). The BZP antagonist PKl1195 did not affect melatonin release at a concentration of 10 p,M and there was no clear antagonism of diazepam action. Mean -t SEM, n = 4, *P < 0.05 vs. control.

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I Control Diazepam Wash Wash

sion by preventing transmitter release from the presynaptic terminals as it was shown in the retina of fish [cf. Dowling and Ripps, 1973; Kaneko and Shimazaki, 19751, which has many similarities with the photoreceptive pineal organ [Meissl, 19861. Such a blockade of neurotransmission should remove a possible neuronal feedback of second-order neurons onto photoreceptor cells. The persistence of the diazepam effect in high Mg2+ low Ca2+ medium speaks in favor of a direct action on photoreceptor cells. However, recent studies have provided evidence that calcium is also involved in a sequence of other events including the phototransduction process [for review see Pugh and Lamb, 19901, the release of the transmitter from the receptor pedicle

w 5-MTOL v 5-MIAA

5-MT

A * W 5-MTOL v 5-MIAA

6 DA 5-MT

2

~~ ~~~

Control Diazepam Wash Wash Fig. 6. Release of the rnethoxyindoles 5-rnethoxytryptophol (SMTOL), 5-methoxyindoleacetic acid (5-MIAA) and 5-methoxytryptamine (5-MT) of isolated pineal organs during application of diazepam (100 pM). Mean values * SEM, n = 19.

agonist muscimol, which show a moderate inhibition of pineal melatonin synthesis [Meissl et al., 19931.

One site of benzodiazepine action is possibly presynaptic, i.e., on pineal photoreceptor cells that are believed to be the source for melatonin synthesis and release [Falcbn et al., 1992; Gem et al., 19921. This assumption is based on experiments using a perifusion medium containing a low Ca2+- and high Mg2+-content. We assumed that this increase of extracellular magnesium associated with lowered calcium should be able to block synaptic transmis-

- - and the binding of the transmitter with postsynaptic receptors [Normann et al., 19881. Because of the numerous structural, biochemical, and electrophysiological analogies between retinal and pineal photoreceptors, we do not believe in the possibility of the existence of a different mechanism in the photoreceptive pineal organ [Falcbn et al., 19921. In view of these analogies we can assume that reduction of extracellular calcium should decrease calcium influx across the outer segment plasma membrane and reduce intracellular calcium and, additionally, should interfere with synaptic transmission in the neuronal pineal circuitry. In retinal rods and cones, lowering extracellular calcium produces a depolarization of the membrane and increases the response amplitude [Bertrand et al., 1978; Owen and Torre, 19831. If cobalt (2.5 mM), which blocks voltage-sensitive calcium channels in rods (Bader et al., 1982), is added to low calcium Ringer's solution (8 pM), rods and cones continue to respond to light [Schwartz, 19861. Furthermore, it appears that chemical neurotransmission to horizontal and bipolar cells partly persists under these conditions, which are commonly assumed to interrupt calcium influx. From these considerations, it seems possible that in the photoreceptive pineal organ lowered extracellular calcium and/or addition of cobalt ions a) do not abolish the light response and b) do not interrupt completely the synaptic transmission from second-order neurons to photoreceptor cells. This is supported by previous experiments in the frog pineal, where we have shown that addition of 5 mM CoCI, to the superfusion medium reduces, but does not completely eliminate, the light response of second-order neurons [Meissl and George, 19841. Cobalt added in a concentration of 2 mM to the perifusion medium reduces melatonin release from light- and dark-adapted pineal organs of the trout by about 20-30% (Meissl, unpublished). A similar Co2+-induced reduction of melatonin output in cultured chick pineal cells was

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Meissl et al.

attributed to blockade of Ca2+ channels [Harrison and Zatz, 19891.

Melatonin release in low Ca2+/high Mg2+-medium was clearly diminished in the present experiments, especially in the light. Exposing the pineal of the trout to decreasing concentrations of calcium without enhancing Mg2+ also decreases melatonin production [Begay et al., 19941. From the present experiments it therefore appears that we can not distinguish whether the effects of our blockade medium is caused by an influence on the calcium- dependent phototransduction process or by an interference with chemical neurotransmission.

If we assume a presynaptic action on photoreceptor cells, the mechanism of benzodiazepine action on melatonin release is obscure. Benzodiaz- epines enhance melatonin secretion under normal conditions and after blocking or reducing synaptic transmission, meaning their effects are similar to the effect of a dark stimulus. Darkness depolarizes pineal photoreceptor cells [Meissl and Ekstrom, 19881 and is associated with a higher melatonin release [Meissl and Brandstatter, 19921. In the chick pineal photoreceptor it was suggested that membrane depolarization, like darkness, might promote melatonin synthesis and release [Zatz et al., 1988; Harrison and Zatz, 19891. The clear increase of melatonin release in the trout pineal by BZP seems to be comparable to a dark stimulus, which should depolarize the photoreceptor cell.

If we assume that BZP act in the pineal via the common GAB A,/benzodiazepine complex on the photoreceptor cell, their action should be a depolarization of the photoreceptor to produce an increase of melatonin formation, provided that there exists such a direct correlation between melatonin formation and membrane potential. However, re- cently we have shown that the action of GABA and the GABA, analog muscimol on second-order neurons in the trout pineal may be bifunctional, i.e., inhibitory or excitatory, depending on the state of light- or dark-adaptation [Meissl and Ekstrom, 19911. We assumed that a GABAergic mechanism plays a role in the generation and transmission of luminosity responses and in the modulation of light sensitivity during the different adaptational processes as it was earlier proposed for the pineal of the frog [Meissl and George, 19861. The bifunctional effects of GABA, analogs on electrophysiological activity do not entirely explain the increase of melatonin secretion observed after BZP application because GABA and muscimol action on melatonin output was relatively small, but often inhibitory [Meissl et al., 19931, i.e., opposite to the BZP action. This may indicate that BZP action in the

pineal is located on a side that is separate from the common GABA,/benzodiazepine receptor complex.

An increase of melatonin content by diazepam, clonazepam, and the peripheral agonist Ro 5-4864 in concentrations greater than 10 FM was observed in vitro in the rat pineal [Lowenstein et al., 19851. This increase in melatonin content disappeared, with the exception of the effect of Ro 5-4864, after sympathetic denervation of the pineal gland, i.e., after removal of the major pinealopetal neural input into the gland. Therefore it was assumed that in the rat pineal BZP may decrease transmitter release presynaptically by acting on peripheral BZP binding sites on pineal sympathetic nerves [Lowenstein et al., 19851. At present, we have no evidence for a sympathetic innervation of the teleost pineal [Moller and Van Veen, 19821, but catecholamines, especially norepinephrine, of unknown origin can be detected in pineal extracts of the trout by HPLC (C. Martin, unpublished). They seem to exert a small modulatory influence on the nocturnal rise in N-acetyltransferase activity and melatonin release in cultures of pike pineals [Falcon et al., 19921, but obviously not in cultured trout pineals [Falcon et al., 19911, where we observed only a slight modulation of the electrical activity of luminance neurons by norepinephrine [Martin and Meissl, 19921. We, therefore, do not believe that an influence of BZP on catecholaminergic receptors can account for the dramatic increase in melatonin release.

The question of the site of BZP action in the trout pineal remains open. Benzodiazepine binding sites as recognized by incubation of the pineal with the fluorescent benzodiazepine receptor ligand BO- DIPY Ro-1986 seem to be present throughout the pineal of the trout [Meissl et al., 19931. Preliminary experiments have shown that this binding can be suppressed by simultaneous incubation of the trout pineal with melatonin [Meissl et al., 19931. Fur- thermore, electrophysiological experiments have shown that diazepam exerts an inhibitory effect on pineal luminance neurons with a time course that resembles the intrapineal action of melatonin [Meissl et a]., 19901. This might be an indication that both substances act via the same intrapineal receptor system.

The influence of diazepam on melatonin secretion shows that there exists a close relationship between melatonin and psychotropic drugs, like diazepam. The question whether the tranquilizing effects of diazepam may be potentiated by a sec- ondary enhancement of melatonin release, at least in our model animal with directly photoreceptive pi-

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neal organs, remains open and should be further investigated.

Acknowledgments

The authors wish to thank Monika Euler for skilled technical assistance.

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