Physiology & Behavior, Vol. 22, pp. 817-819. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Melatonin Mediation in Sedative Effect of Serotonin in Goldfish NOBUHIRO
SATAKE 1
Sensory Sciences Laboratory, University o f Hawaii, Honolulu, H I 96822 ( R e c e i v e d 21 S e p t e m b e r 1978) SATAKE, N. Melatonin mediation in sedative effect of serotonin in goldfish. PHYSIOL. BEHAV. 22(5) 817-819, 1979.-Intracranial injection of serotonin caused sedation, measured in terms of surfacing responses, in sham operated goldfish but not in pinealectomized fish. In unoperated fish, melatonin induced sedation with faster onset of the effect than serotonin. That the effect of serotonin on sedation was mediated by melatonin was evidenced by the fact that inhibition of melatonin synthesis by scotophobin prevented the sedative effect of serotonin injection. Serotonin
Surfacing response
Goldfish
Pineal gland
IT H A S been known, since the middle of 20th century, that serotonin (5-HT) has a profound effect on the induction of sleep (sedation) in mammals and birds [8]. The sedative effect of 5-HT was found to be shared by one of the products of 5-HT metabolites, melatonin [9]. Recently, the effect of melatonin on sedation was explained as such that melatonin increases 5-HT concentration and that the increase in 5-HT concentration might be the reason for the effect found in the presence of melatonin [3]. This hypothesis appeared reasonable at that time, since melatonin was found to inhibit monoamine oxidase [4], which in turn decreases the breakdown of 5-HT to biologically inactive 5-hydroxyindole acetic acid. It was also reasonable to think that the inhibition of 5-HT breakdown into melatonin would not interfere with the sedative effect of 5-HT and might even increase its effect. Increase in brain 5-HT concentration was found to be regulated by daily food-intake and plasma neutral amino acids [5, 6, 14]. Since the type o f daily food consumption influences 5-HT concentration and 5-HT might induce a basic physiological state (sleep), it was important to gain a clear understanding of how 5-HT actually influences sedation. In order to study this, the effects of 5-HT in goldfish were examined, which resulted in a rather surprising finding.
Biological Effect of Melatonin on Sedation The sedative effect of 5-HT injection in goldfish was manifested by gulping responses from fish as they came up to the surface of the water [10]. Normally, fish restricted in a narrow space, as they were, will stay on the bottom and rarely come up to the surface. Fish, intracranially injected with low dosages of anaesthetic (2-8/xg tricaine), showed a dose dependent effect which was manifested by surfacing responses for 10 to 40 min. Using this phenomena, the effects of 5-HT in goldfish were examined, melatonin and an inhibitor of melatonin synthesis to clarify the role of melatonin in sedation in goldfish.
Melatonin
Scotophobin
METHOD The fish used were 23 common goldfish of 8 cm length
(Carassius auratus), kept in individual 2-gal tanks, aerated, fed daily and illuminated from 8 a.m. to 8 p.m. Experiments were conducted between 10 a.m. to 3 p.m. The apparatus used for the experiments (Fig. 1) was a rectangular transparent aquarium (135 x 90 x 260 mm high) with one wall being black (135 x 260 mm). A removable black wall with holes (holes not shown in the figure) was inserted in the tank, 25mm from the black wall, leaving a small rectangular space (135 x 25 x 260 mm), in which each fish was placed for the experiments. The tank was constantly aerated in the large side of the compartment. Surfacing of a fish was detected by a photodetector system (30 mm below the water surface). The light for the detector system was filtered with nearinfrared filter (Kodak No. 89B). A duration of interruption of the photobeam on the surface could be counted (in sec) and recorded automatically on a printout counter. The whole apparatus was enclosed in a large box to prevent any illumination. Chemicals used for the experiments were serotonin oxalate (5-HT), N-acetylserotonin (NAS) and melatonin (Sigman Chemical Co.). Scotophobin A [13] was supplied by Dr. G. Ungar. Different groups of fish (3 fish per group except for melatonin plus scotophobin group which had 2 fish) received an intracranial injection of 50/zg 5-HT (SE group), 10 /xg melatonin (M), 50/xg 5-HT plust 10/xg N A S ( S E + N ) , 50 /xg 5-HT plust 2.5/xg scotophobin (SE+SC), 10/xg melatonin plus 2.5/xg scotophobin (M+SC) in 15/xl of 0.9% saline or 15 /zl of saline alone (S). Pinealectomized fish were operated (anaesthetized with 1/8000 dilution of tricaine) under a dissecting microscope. Sham operated fish went through the same procedure without removal of the glands. All the operated fish were used for the experiments after a one month recovery period for resealing of skull openings. Following the experiments, these
'The author wishes to thank Dr. M. E. Bitterman for his advise and encouragement.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/050817-03502.00/0
818
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30 M I NUTES-BLOCKS
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FIG. 2. The mean surface time (in sec) per min was measured in pinealectomized (n=3) and sham operated (n=3) fish. An injection of 50/zg 5-HT in 15/zl saline was given to all the fish just prior to the experiment. Responses were monitored (one min duration) every 5 min and printed every 30 min for 3 hr.
FIG. I. The apparatus used for surfacing responses. The apparatus was a transparent Plexiglas aquarium (135 x 90 x 260 mm high) with a black wail on one side. A removable black partition with holes
(holes not shown in the figure) was inserted in the tank, 25 mm from the black wall. Water level was maintained to the height of 140 mm from the bottom. Response detector system (photocell and the light source, covered with Kodak near-infrared filter No. 89B) was installed 30 mm below water level and 12.5 mm from the black wail. The whole apparatus was enclosed in a large wooden box. Time spent on the surface of water was counted in sec and recorded automatically.
fish were examined under a microscope, which did not reveal any apparent sign of regeneration of the glands. Immediately after injection, each fish was placed in the apparatus and the time spent on the surface was monitored for 3 hr, with one min recordings every 5 min, beginning 5 min after an injection. Data were accumulated and recorded every 30 min. RESULTS
Figure 2 shows the effect of 5-HT on the time spent on the surface of water in pinealectomized and sham operated fish. In sham operated (Sham) fish, 5-HT caused a gradual increase in the surfacing response within 1.5 hr and decreased gradually thereafter. 5-HT did not show a similar effect in pinealectomized (Pineal.) fish. An overall analysis of variance showed a significant Groups effect, F(1,4)=105.1, p
Melatonin Mediation in Sedative Effect of Serotonin in Goldfish NOBUHIRO
SATAKE 1
Sensory Sciences Laboratory, University o f Hawaii, Honolulu, H I 96822 ( R e c e i v e d 21 S e p t e m b e r 1978) SATAKE, N. Melatonin mediation in sedative effect of serotonin in goldfish. PHYSIOL. BEHAV. 22(5) 817-819, 1979.-Intracranial injection of serotonin caused sedation, measured in terms of surfacing responses, in sham operated goldfish but not in pinealectomized fish. In unoperated fish, melatonin induced sedation with faster onset of the effect than serotonin. That the effect of serotonin on sedation was mediated by melatonin was evidenced by the fact that inhibition of melatonin synthesis by scotophobin prevented the sedative effect of serotonin injection. Serotonin
Surfacing response
Goldfish
Pineal gland
IT H A S been known, since the middle of 20th century, that serotonin (5-HT) has a profound effect on the induction of sleep (sedation) in mammals and birds [8]. The sedative effect of 5-HT was found to be shared by one of the products of 5-HT metabolites, melatonin [9]. Recently, the effect of melatonin on sedation was explained as such that melatonin increases 5-HT concentration and that the increase in 5-HT concentration might be the reason for the effect found in the presence of melatonin [3]. This hypothesis appeared reasonable at that time, since melatonin was found to inhibit monoamine oxidase [4], which in turn decreases the breakdown of 5-HT to biologically inactive 5-hydroxyindole acetic acid. It was also reasonable to think that the inhibition of 5-HT breakdown into melatonin would not interfere with the sedative effect of 5-HT and might even increase its effect. Increase in brain 5-HT concentration was found to be regulated by daily food-intake and plasma neutral amino acids [5, 6, 14]. Since the type o f daily food consumption influences 5-HT concentration and 5-HT might induce a basic physiological state (sleep), it was important to gain a clear understanding of how 5-HT actually influences sedation. In order to study this, the effects of 5-HT in goldfish were examined, which resulted in a rather surprising finding.
Biological Effect of Melatonin on Sedation The sedative effect of 5-HT injection in goldfish was manifested by gulping responses from fish as they came up to the surface of the water [10]. Normally, fish restricted in a narrow space, as they were, will stay on the bottom and rarely come up to the surface. Fish, intracranially injected with low dosages of anaesthetic (2-8/xg tricaine), showed a dose dependent effect which was manifested by surfacing responses for 10 to 40 min. Using this phenomena, the effects of 5-HT in goldfish were examined, melatonin and an inhibitor of melatonin synthesis to clarify the role of melatonin in sedation in goldfish.
Melatonin
Scotophobin
METHOD The fish used were 23 common goldfish of 8 cm length
(Carassius auratus), kept in individual 2-gal tanks, aerated, fed daily and illuminated from 8 a.m. to 8 p.m. Experiments were conducted between 10 a.m. to 3 p.m. The apparatus used for the experiments (Fig. 1) was a rectangular transparent aquarium (135 x 90 x 260 mm high) with one wall being black (135 x 260 mm). A removable black wall with holes (holes not shown in the figure) was inserted in the tank, 25mm from the black wall, leaving a small rectangular space (135 x 25 x 260 mm), in which each fish was placed for the experiments. The tank was constantly aerated in the large side of the compartment. Surfacing of a fish was detected by a photodetector system (30 mm below the water surface). The light for the detector system was filtered with nearinfrared filter (Kodak No. 89B). A duration of interruption of the photobeam on the surface could be counted (in sec) and recorded automatically on a printout counter. The whole apparatus was enclosed in a large box to prevent any illumination. Chemicals used for the experiments were serotonin oxalate (5-HT), N-acetylserotonin (NAS) and melatonin (Sigman Chemical Co.). Scotophobin A [13] was supplied by Dr. G. Ungar. Different groups of fish (3 fish per group except for melatonin plus scotophobin group which had 2 fish) received an intracranial injection of 50/zg 5-HT (SE group), 10 /xg melatonin (M), 50/xg 5-HT plust 10/xg N A S ( S E + N ) , 50 /xg 5-HT plust 2.5/xg scotophobin (SE+SC), 10/xg melatonin plus 2.5/xg scotophobin (M+SC) in 15/xl of 0.9% saline or 15 /zl of saline alone (S). Pinealectomized fish were operated (anaesthetized with 1/8000 dilution of tricaine) under a dissecting microscope. Sham operated fish went through the same procedure without removal of the glands. All the operated fish were used for the experiments after a one month recovery period for resealing of skull openings. Following the experiments, these
'The author wishes to thank Dr. M. E. Bitterman for his advise and encouragement.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/050817-03502.00/0
818
SATAKE
50Z
~:40-
\
3o-
d
W
SHAM
p 2oI I
iii {D Pc"
I
~
U3
0
/
~
_
,......-----4)
I
I
I
I
I
I
I
2
3
4
5
6
30 M I NUTES-BLOCKS
/
FIG. 2. The mean surface time (in sec) per min was measured in pinealectomized (n=3) and sham operated (n=3) fish. An injection of 50/zg 5-HT in 15/zl saline was given to all the fish just prior to the experiment. Responses were monitored (one min duration) every 5 min and printed every 30 min for 3 hr.
FIG. I. The apparatus used for surfacing responses. The apparatus was a transparent Plexiglas aquarium (135 x 90 x 260 mm high) with a black wail on one side. A removable black partition with holes
(holes not shown in the figure) was inserted in the tank, 25 mm from the black wall. Water level was maintained to the height of 140 mm from the bottom. Response detector system (photocell and the light source, covered with Kodak near-infrared filter No. 89B) was installed 30 mm below water level and 12.5 mm from the black wail. The whole apparatus was enclosed in a large wooden box. Time spent on the surface of water was counted in sec and recorded automatically.
fish were examined under a microscope, which did not reveal any apparent sign of regeneration of the glands. Immediately after injection, each fish was placed in the apparatus and the time spent on the surface was monitored for 3 hr, with one min recordings every 5 min, beginning 5 min after an injection. Data were accumulated and recorded every 30 min. RESULTS
Figure 2 shows the effect of 5-HT on the time spent on the surface of water in pinealectomized and sham operated fish. In sham operated (Sham) fish, 5-HT caused a gradual increase in the surfacing response within 1.5 hr and decreased gradually thereafter. 5-HT did not show a similar effect in pinealectomized (Pineal.) fish. An overall analysis of variance showed a significant Groups effect, F(1,4)=105.1, p
