William A. Gern

Examination of in vitro melatonin secretion from superfused trout (Salmo gairdneri) pineal organs maintained under diel illumination or continuous darkness

Melatonin secretion was measured from rainbow trout (Salmo gairdneri) pineal organs maintained individually under flow-through whole organ culture (superfusion) conditions. Radioimmunoassay of perfusate fractions collected during controlled photic conditions demonstrated that melatonin secretion in vitro remained basal during the photophase and underwent increases in titer during the scotophase. While amounts of melatonin (mel) secreted were characteristic of individual pineal organs, photophase values ranged between 0.25 and 0.75 ng mel/ml and scotophase values ranged from 6 to 10 ng mel/ml of perfusate. Diel melatonin secretion profiles reflected the illumination regimen, with light associated with low melatonin titer in the perfusate and darkness associated with high titer. Light pulses during a normal scotophase resulted in a depression in melatonin secretion regardless of whether it was administered early or late in the dark period. Pulses of darkness given early or late in a normal photophase resulted in increased melatonin secretion. Superfused trout pineal organs did not display endogenous rhythmicity in melatonin secretion when subjected to prolonged exposure to continuous darkness (DD), whether first exposed to entraining light/dark (LD) cycles prior to DD or exposed to DD at the initiation of superfusion. In both studies, elevated melatonin secretion gradually declined over time. But exposure to a 4:4LD cycle after DD resulted in decreased (with light) and increased (with darkness) melatonin secretion. These results demonstrate that the trout pineal organ can be maintained for extended periods of time in superfusion culture, that the trout pineal organ is very responsive to light or dark for regulating melatonin synthesis, and that an endogenous rhythm in melatonin synthesis when organs were maintained in DD was not detectable.

Regulation of Melatonin Production by Pineal Photoreceptor Cells: Role of Cyclic Nucleotides in the Trout (Oncorhynchus mykiss)

Abstract: The light/dark cycle influences the rhythmic production of melatonin by the trout pineal organ through a modulation of the serotonin N‐acetyltransferase (NAT) activity. In static organ culture, cyclic AMP (cAMP) levels (in darkness) and NAT activity (in darkness or light) were stimulated in the presence of forskolin, isobutylmethylxanthine, or theophylline. Analogues of cAMP, but not of cyclic GMP, induced an increase in NAT activity. Light, applied after dark adaptation, inhibited NAT activity. This inhibitory effect was partially prevented in the presence of drugs stimulating cAMP accumulation. In addition, cAMP accumulation and NAT activity increase, induced by forskolin, were temperature dependent. Finally, melatonin release, determined in superfused organs under normal conditions of illumination, was stimulated during the light period of a light/dark cycle by adding an analogue of cAMP or a phosphodiesterase inhibitor. However, no further increase in melatonin release was observed during the dark phase of this cycle in the presence of the drugs. This report shows for the first time that cAMP is a candidate as intracellular second messenger participating in the control of NAT activity and melatonin production by light and temperature.

Plasma melatonin in the neotenic tiger salamander (Ambystoma tigrinum): effects of photoperiod and pinealectomy.

Abstract A nonequilibrium, double-antibody radioimmunoassay was validated for melatonin extracted from tiger salamander plasma. Radioimmunoassay of plasma samples that were collected from salamanders killed at 6-hr intervals, starting at 0600 hr and ending at 2400 hr (12:12 L:D; lights on at 0600 hr), indicated a significant increase in plasma melatonin at 2400 hr and 0600 hr compared to 1200 hr, with the 1800 hr sampling time value being intermediate. Animals which had been pinealectomized, sham pinealectomized, or anesthetized only, and then killed and sampled at either midphotophase or midscotophase, demonstrated that this operation reduced circulating melatonin only during the scotophase. These results indicate the presence of other, non-pineal sources for circulating melatonin and that the pineal body is responsible for the increased titers of melatonin measured during the scotophase.

The Rainbow Trout Pineal Organ: An Endocrine Photometer

Like the pineal organs of other vertebrates, those of teleost fish synthesize and secrete melatonin (N-acetyl-5-methoxytryptamine) (Fenwick, 1970; Gern et al., l978a,b; Falcon et al., 1989a; Kezuka et al., 1989; Zachmann et al., 1991). Unlike mammals, pineal organs in non-mammalian vertebrates are directly photoreceptive and these processes are involved in regulating melatonin synthesis. Pineal melatonin synthesis in chickens (Binkely et al., 1978; Kasal et al., 1979; Takahashi et al., 1980; Takahashi et al., 1989), anoline lizards (Menaker & Wisner, 1983) and probably many other nonmammalian vertebrates (Falcon et al., 1989b; Kezuka et al., 1989) is associated with an endogenous circadian clock. In mammals, experimental evidence does not indicate the presence of a clock within the pineal. Rather in mammals a clock, present in the suprachiasmatic nucleus, regulates pineal melatonin synthesis via neuronal connections from the SCN (Moore & Klein, 1974).

Initial Den Location by Neonatal Prairie Rattlesnakes: Functions, Causes, and Natural History in Chemical Ecology

The concept of adaptation has long been a central idea in the study of the survival and evolution of life on earth (e.g., Williams, 1966). The potential existence of biological adaptation is best thought of as a hypothesis to be tested or an idea to be approached, and not a phenomenon that is automatically inherent in each and every characteristic of a living system1s biology. To be considered an adaptation, sensu stricto, potentially important mechanisms and processes should be rigorously studied to determine if they do in fact figure significantly into an organism’s “fit” with its environment. Once such features of phenotype are determined to be genuinely critical to survival, subsequent analysis of the trait’s mechanisms can lead to much more full explanations of how things actually work, and why they may have evolved into the structural and functional mosaics that we now see.

Effect of Reduced Incubation Temperatures on Survival of Trout Embryos

Abstract The effect on embryo survival of reducing egg incubation temperature from 7°C to 4 or 2°C immediately after 2 h of water hardening was tested for three hatchery species of trout. Embryos incubated at 2°C had significantly lower survival rates to eyeing than embryos incubated at 4 and 7°C. Embryos of fall-spawning brown trout (Salmo trutta) and spring-spawning rainbow trout (Oncorhynchus mykiss) had higher survival rates than cutthroat trout (O. clarki) and domesticated fall-spawning rainbow trout when incubated at 2°C. The rate at which temperatures were reduced from 7 to 4°C or from 7 to 2°C had no effect on embryo survival.

Melatonin in the blood and cerebrospinal fluid of the green sea turtle (Chelonia mydas)

Abstract A melatonin radioimmunoassay was validated for the green sea turtle. A pronounced diurnal rhythm of melatonin content was evident in both serum and cerebrospinal fluid (CSF). In immature turtles, CSF melatonin levels paralleled those in serum but were always significantly lower. Despite the close anatomical juxtaposition of the sea turtles large pineal gland to the brain, our data suggest that the primary route of secretion into the CSF is through the peripheral vasculature via the choroid plexus. Light exposure reduced serum melatonin titers. Adult females that were either mating or nesting exhibited the lowest serum melatonin levels we measured for this species.

Increases in plasma melatonin titers accompanying seawater adaptation of coho salmon (Oncorhynchus kisutch)

Plasma melatonin (MT) concentrations were measured in coho salmon using a radioimmunoassay validated for that species. Plasma MT remained constant in juvenile salmon during the parr to smolt transformation in fresh water. However, upon seawater entry plasma MT concentrations increased significantly, peaked at approximately 24 hr, then returned to levels observed for the fish in fresh water.

The Effect of Light and Temperature on Plasma Melatonin in Neotenic Tiger Salamanders (Ambystoma tigrinum)

Effects of varying illumination and temperature cycles on circulating melatonin (mel) in neotenic tiger salamanders were investigated in three experiments. In Experiment I animals were maintained for seven days on a 12:12LD illumination regimen with either normal high temperature (20?C) during the photophase, low (10?C) temperature during the scotophase or reversed thermal periods. Animals maintained in a normal temperature and photic regimen had 87.7 and 141.7 pg mel/ml plasma (mean values) measured midphotophase and midscotophase, respectively. Animals in a reversed temperature environment had mean plasma melatonin titers of 100.8 and 91.6 pg mel/ ml at midphotophase and midscotophase. In the second experiment, the effect of continuous high or low temperature was examined while animals were under a 12:12LD illumination regimen. Animals held at continuously low (10?C) temperature had a mean plasma melatonin titer of 87.3 and 125.1 pg/ml at midphotophase and midscotophase respectively. Animals maintained in contin- uously high temperatures (20?C) had mean plasma melatonin titers of 87.7 and 82.0 pg/ml. Finally animals maintained under either continuous light or darkness but an alternating thermal regimen did not display significant differences in plasma melatonin. It was observed that high nocturnal temperature can cause a depression in the amount of melatonin measured in the plasma of neotenic tiger salamanders.

Characteristics and non-rhythmicity of retinal hydroxyindole-O-methyltransferase activity in trout (Salmo gairdneri)

The validation and optimization of an hydroxyindole-O-methyltransferase (HIOMT) activity assay system for trout ocular enzyme was undertaken. High-performance liquid chromatographic analysis, cross-validated with a standard thin-layer chromatographic technique verified that HIOMT activity was present in trout ocular tissue by identifying the 3H-melatonin end product. This activity was observed in the neural retina and was restricted to those fractions containing the cytosolic component. The enzyme displayed bimodal pH optima, one at pH 7.6, the other at 8.4. N-Acetylserotonin was O-methylated preferentially and a Km of 4.5 X 10(-7) M was calculated for this substrate. The S-[3H]adenosylmethionine Km was 4.6 X 10(-6) M. Time of incubation and enzyme concentration studies were conducted. The analysis of retinal HIOMT activity in groups of trout killed over a day--night period did not detect a rhythm in enzyme activity. Results of these investigations are discussed in terms of the evolution of melatonin synthesis and functions.