Effect of p-chlorophenylalanine on the reproduction of the rat (author's transl)]

The effect of p-chlorophenylalanie (p-CPA) --300 mg/kg-- on reproduction has been studied in the female rat. Groups of animals were injected with a dose of 300 mg/kg of p-CPA 48 hours before proper copulation conditions at different moments along the ovarian cycle. Presence of spermatozoa in the vaginal frotis was negative in treated rats; in the control groups however, positivity was found in variable proportions according to the phase of the ovarian cycle: 30 ad 90% in diestrus and proestrus respectively. Treated animals showed continuous diestrus phases and diffuse luteinitation of the ovary. The results may indicate that a decrease of cerebral 5-HT, caused by p-CPA, lessens the reproductive behaviour of the female rat through mechanisms depending probably on the liberation of gonadrotrophins.     

Sex differences in secretion pattern of neonatal rat Harderian gland under various environmental lighting conditions

1. Secretion pattern of Harderian gland of neonatal rats maintained under (a) diurnal lighting conditions; (b) continuous light or (c) continuous darkness was studied at light microscopy level. 2. All animals were placed in especially designed cages at 13:00 hr on day 1 and studied on day 7 at 13:00 and 23:00 hr, respectively. 3. Acini with intraluminal secretion were counted in glands from each animal and the results were separately grouped for male and female animals. 4. A diurnal rhythm in secretion pattern of rat Harderian gland in neonatal period was demonstrated. 5. A statistically significant difference was observed in the gland secretion pattern between males and females at both, 13:00 and 23:00 hr when the animals were kept under diurnal lighting conditions. 6. Under continuous light or continuous darkness, the diurnal rhythm in secretion pattern was lost and no significant differences were seen when data from males were compared to those from female neonates. 7. Results are discussed in terms of the possible function of Harderian gland as element of an extraretinal photoreceptor system involved in the regulation of pineal function in neonatal rats.     

Environmental lighting and muricidal behaviour in the male Wistar rat.

Effects of different conditions of environmental lighting on the appearance of the muricidal behaviour in male Wistar rats have been studied. The animals were kept under different conditions of environmental lighting: 1) natural day light alternated with the dark of the night; 2) sodium, continuous light emitted by a sodium steam lamp; 3) neon, continuous light emitted by fluorescent neon tubes. The continuous sodium steam light increased the percentage of animals becoming muricide when compared to animals bred in a natural environment with a normal succession of day-night lighting. On the contrary, this percentage decreased if the rats of the same group are exposed to continuous light emitted by fluorescent neon tubes. As the exposure of rats to an environment under continuous light causes a reduction of the cerebral content of serotonin, the muricidal behaviour provoked in naturally non-muricide rats by this type of lighting could be related to this depletion.     

Adrenal dehydrogenases in alloxan diabetic rats following continuous exposure to light

The enzymes delta5-3beta-hydroxysteroid dehydrogenase delta5-3beta-HSD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were demonstrated histochemically in the adrenal cortex of female rat. The activities of these enzymes were increased significantly in the alloxan-treated rats kept in LD (light: darkness) cycles of 10:14 h. Continuous light exposure to diabetic animals appeared to decrease delta5-3beta-HSD and g-6-PDH in comparison to the diabetic rats kept in 10 h illumination. The evidence indicates that suppression of adrenal steroidogenesis in diabetic rats after exposure to continuous light is due to the alteration of pentose phosphate pathway.     

Chick pineal serotonin acetyltransferase: a diurnal cycle maintained in vitro and its regulation by light.

We have reproduced in vitro the diurnal cycles in levels of serotonin acetyltransferase activity found in the chick pineal gland in vivo. The more closely the lighting conditions of culture matched those under which the birds were raised, the closer was the similarity between cycles in levels of enzyme activity in vitro and in vivo. Repetitive cycles in levels of acetyltransferase activity persisted in culture for at least 4 days under a diurnal cycle of illumination, and at least 2 days in continuous darkness. When glands were explanted into culture in the light phase of a cycle, short periods of further exposure to light markedly stimulated subsequent increase of acetyltransferase in the dark (after a short lag). Prolonged exposure to light in culture markedly inhibited increase of enzyme activity. Cycles in the levels of enzyme activity in glands cultured under altered light cycles were regulated primarily by changes in illumination. However, the endogenous biological 'clock' remained at least partly entrained to the original light cycle. Increase of acetyltransferase activity in vitro was markedly stimulated by theophylline plus compound Ro. 20.1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone) under all lighting conditions. Kinetics (to the time of attaining maximum levels in situ) of the increase under diurnal lighting and in constant darkness were indistinguishable from those in vivo. A high concentration of dl-propranolol markedly stimulated an increase in acetyltransferase activity in glands cultured in constant darkness but had little effect on glands under diurnal lighting or continuous illumination.     

Serotoninergic mechanisms in ovarian cycle (author's transl)]

Influence of 5-HT on the rat follicle rupture mechanism was studied by the administration of p-clorophenyl-alanine (p-CPA) (300 mg/kg) at different stages of the ovarian cycle. The ovarian cycle of treated rats was subject of investigation, as well as the histology of tubes and ovaries. Administration of p-CPA at 10 hours of metestrus phase induces an inhibition of ovulation. Predominant diestrus phases in the ovarian cycle and luteinitation of ovaries suggested an increase in progesterone secretion probably owed to a correspondingly greater prolactine secretion. Adequate 5-HT levels in central structures proved to necessary for normal ovulation.     

Endogenous patterns of photomechanical movements in teleosts and their relation to activity rhythms

Summary The retinal rods, cones and epithelial pigment of most lower vertebrates display rhythmic photomechanical (retinomotor) migrations in response to changes in ambient lighting conditions. This study examines the extent of these migrations in the absence of the daily changes in illumination (constant darkness and constant light) in three species of teleosts. Salmo trutta, a crepuscularly active fish, showed two peaks of light adaptation occurring around dawn and dusk when kept in constant darkness. Tinca tinca, a nocturnal species, also showed an endogenous rhythm during extended periods of darkness, but, unlike Salmo trutta, it was light-adapted throughout what would normally have been day. At the maximal extent of migration under conditions of continual darkness, the pigment migrated 59% as much as it did during a normal light/dark cycle. Nannacara anomala, a tropical diurnally active species, showed a similar but more pronounced rhythm than Tinea tinea for all 3 days of experimental darkness, behaving essentially identically to fish exposed to a light/dark cycle. Nannacara anomala also displayed a weak rhythm when kept in constant light.It is concluded from these and previous results that the pattern of endogenous photomechanical movement depends both on the activity pattern of a species and on the constancy of the lighting conditions to which it has been exposed during its lifetime.     

Direct photosensitivity of chick pinealocytes as demonstrated by visinin immunoreactivity

Summary Visinin, a calcium-binding protein isolated from the soluble fraction of homogenized chick retinae, has been recognized immunocytochemically in the pinealocytes of various submammals. In the chick pineal organ, continuous environmental light induced an increase in population density of visinin-immunoreactive pinealocytes. In semi-quantitative, dot-immunoblotting analysis, the amount of visinin in the pineal organs of chicks kept under continuous light for 3 days was 4–8 fold more abundant than that under continuous darkness for the same duration. Eye-enucleation and organ culture experiments clarified that this lighting effect was exerted directly on the pineal organ through the skull, and not via the neural pathway including the retinohypothalamic projection. These data suggest the existence of direct photosensitivity in the chick pinealocyte itself and the possible involvement of visinin in photoreception of the pineal organ as well as the retina of chicks.     

Food-Entrained Feeding and Locomotor Circadian Rhythms in Rats Under Different Lighting Conditions

It has been suggested that two endogenous timekeeping systems, a light-entrainable pacemaker (LEP) and a food-entrainable pacemaker (FEP), control circadian rhythms. To understand the function and interaction between these two mechanisms better, we studied two behavioral circadian rhythmicities, feeding and locomotor activity, in rats exposed to two conflicting zeitgebers, food restriction and light-dark cycles. For this, the food approaches and wheel-running activity of rats kept under light-dark (LD) 12:12, constant darkness (DD), or constant light (LL) conditions and subjected to different scheduled feeding patterns were continuously recorded. To facilitate comparison of the results obtained under the different lighting conditions, the period of the feeding cycles was set in all three cases about Ih less than the light-entrained or free-running circadian rhythms. The results showed that, depending on the lighting conditions, some components of the feeding and wheel-running circadian rhythms could be entrained by food pulses, while others retained their free-running or light-entrained state. Under LD, food pulses had little influence on the light-entrained feeding and loco-motor rhythms. Under DD, relative coordination between free-running and food-associated rhythms may appear. In both cases, the feeding activity associated with the food pulses could be divided into a prominent phase-dependent peak of activity within the period of food availability and another afterward. Wheel-running activity mainly followed the food pulses. Under LL conditions, the food-entrained activity consisted mainly of feeding and wheel-running anticipatory activity. The results provide new evidence that lighting conditions influence the establishment and persistence of food-entrained circadian rhythms in rats. The existence of two coupled pacemakers, LEP and FEP, or a multioscillatory LEP may both explain our experimental results.     

Food-Entrained Feeding and Locomotor Circadian Rhythms in Rats Under Different Lighting Conditions

It has been suggested that two endogenous timekeeping systems, a light-entrainable pacemaker (LEP) and a food-entrainable pacemaker (FEP), control circadian rhythms. To understand the function and interaction between these two mechanisms better, we studied two behavioral circadian rhythmicities, feeding and locomotor activity, in rats exposed to two conflicting zeitgebers, food restriction and light-dark cycles. For this, the food approaches and wheel-running activity of rats kept under light-dark (LD) 12:12, constant darkness (DD), or constant light (LL) conditions and subjected to different scheduled feeding patterns were continuously recorded. To facilitate comparison of the results obtained under the different lighting conditions, the period of the feeding cycles was set in all three cases about Ih less than the light-entrained or free-running circadian rhythms. The results showed that, depending on the lighting conditions, some components of the feeding and wheel-running circadian rhythms could be entrained by food pulses, while others retained their free-running or light-entrained state. Under LD, food pulses had little influence on the light-entrained feeding and loco-motor rhythms. Under DD, relative coordination between free-running and food-associated rhythms may appear. In both cases, the feeding activity associated with the food pulses could be divided into a prominent phase-dependent peak of activity within the period of food availability and another afterward. Wheel-running activity mainly followed the food pulses. Under LL conditions, the food-entrained activity consisted mainly of feeding and wheel-running anticipatory activity. The results provide new evidence that lighting conditions influence the establishment and persistence of food-entrained circadian rhythms in rats. The existence of two coupled pacemakers, LEP and FEP, or a multioscillatory LEP may both explain our experimental results.     

Effect of Different Photoperiod Exposure on [3H]Imipramine Binding and Serotonin Uptake in the Rat Brain

Seasonal rhythmicity in the occurrence of acute depressive episodes and the therapeutic efficacy of light exposure suggest the possible involvement of the pineal gland or other biological oscillators in the pathophysiology of depressive illness. We have performed studies to clarify whether different light/dark (LD) cycle schedules may induce changes in the biochemical targets of antidepressants in the rat CNS. In particular, we have investigated the effect of short- (LD 8:16) or long-day (LD 14:10) photoperiods on different biochemical parameters of serotonergic neurons. A significant increase in the density of [3H]imipramine ([3H]IMI) binding and in the Vmax of 5-[3H]hydroxytryptamine (5-[3H]HT) uptake was found in the hypothalamus of LD 8:16-with respect to LD 14:10-exposed rats, whereas no difference was found in the kinetic properties of postsynaptic 5-HT receptors and in 5-HT metabolism in the hypothalami and cerebral cortices of rats exposed to the two different photoperiods. A seasonal rhythm of [3H]IMI binding sites and 5-HT uptake seems to exist only in certain brain areas, such as the hypothalamus, because no differences were found in the cerebral cortex of LD 14:10- and LD 8:16-accustomed rats. [3H]IMI binding and 5-HT uptake were significantly increased in the hypothalamus of rats accustomed to a light/dark-inverted cycle (DL 10:14) and killed 6 h after the stopping of lighting in comparison to rats exposed to normal LD 14:10 cycles and killed 6 h after the beginning of lighting. Therefore, a circadian modification of the serotonergic presynaptic sites seems to be present and related to light/dark exposure. Because the existence of endogenous compounds able to modulate [3H]IMI binding and 5-HT uptake, other than 5-HT, has been postulated in the mammalian brain, the involvement of these substances in the periodic changes observed could be suggested.     

Reflex factors that modify the effect of p-chlorophenylalanine on ovulation and reproduction in rats (author's transl)]

The appearance of reflex ovulation in rats under the influence of the copulation, has been studied. Evaluation of insemination, fecundation, number of embryos, ovarian cycle and ovulation in several groups of rats treated with doses of p-CPA (300 mg/Kg, i.p.), have been made. The application of p-CPA 48 hours before the estrus phase, inhibits the ovulation. This increases significantly through copulation. Reproduction is totally inhibited by p-CPA in animals under copulation conditions during 24 hours of the estrus phase. The inhibition is desappears if placed under copulation conditions during a full cycle. Inseminated rats had a 33% decrease in number of embryos with respect to the control group. The application op-CPA 24 hours before the estrus phase does not produce any inhibition effect. The decrease observed in the number of embryos is non-significant. When p-CPA is applied, instead, 48 hours before the estrus phase, there is total inhibition of the reproductive conduct.     

The manifestation of the motor activity circadian rhythm of blinded rats depends on the lighting conditions during lactation

The purpose of this experiment was to examine the effect of different lighting conditions during lactation on the functioning of the circadian pacemaker in the adult rat in absence of the retinal input. We reared one group of rats under constant light (LL-rats) and the other under constant darkness (DD-rats). After weaning they were placed under light-dark cycles of 24h period for 29 days to eliminate the aftereffects of the previous lighting. All the animals were then binocularly enucleated and motor activity was recorded. Results reveal that, before and after the enucleation, the expression of the circadian rhythm was stronger in DD- than in the LL-rats. Our results indicate that lighting conditions during lactation modify the functioning of the circadian pacemaker.     

The Manifestation of the Motor Activity Circadian Rhythm of Blinded Rats Depends on the Lighting Conditions During Lactation

The purpose of this experiment was to examine the effect of different lighting conditions during lactation on the functioning of the circadian pacemaker in the adult rat in absence of the retinal input. We reared one group of rats under constant light (LL-rats) and the other under constant darkness (DD-rats). After weaning they were placed under light-dark cycles of 24h period for 29 days to eliminate the aftereffects of the previous lighting. All the animals were then binocularly enucleated and motor activity was recorded. Results reveal that, before and after the enucleation, the expression of the circadian rhythm was stronger in DD- than in the LL-rats. Our results indicate that lighting conditions during lactation modify the functioning of the circadian pacemaker.     

Effect of light during lactation on the phasic and tonic responses of the rat pacemaker

The circadian system in mammals generates endogenous circadian rhythms and entrains them to external cycles. Here, we examine whether the lighting conditions under which rats are reared affect the properties of the circadian pacemaker. We maintained three groups of rats under constant darkness (DD-rats), constant bright light (LL-rats) or light-dark cycles of 24 hours (LD-rats) during lactation. We then studied motor activity rhythm under constant light of four intensities, and under seven light-dark cycles with periods ranging between 22 and 27 hours. Results show that neither the tau nor the phase angle to the external cycle differed between groups. Differences were found in the amplitude of the circadian rhythm and in the number of rats that became arrhythmic under LL. We conclude that the light received during lactation affects the strength of the circadian pacemaker and its sensitivity to light.     

The effect of testosterone on serum gonadotropins of castrated rats kept under different lighting conditions.

Testosterone feedback sensitivity was measured as the ability of testosterone propionate to decrease serum LH and FSH of long-term castrated (4 wk) rats under four different lighting conditions: periodic light (12L:12D), constant light (LL), constant darkness (DD), and dim night illumination (1 lx) with a 12L:12D photoperiod. Rats were exposed to the different lighting conditions for 1 wk, during which they received daily testosterone propionate (125 micrograms or 250 micrograms s.c.). At the end of the experiment the rats were decapitated at 1100 h, and serum gonadotropin levels were measured by RIA. Serum LH of the rats kept under LL was reduced to the level of the intact rats with the smaller testosterone dose (125 micrograms/day). Under all other lighting conditions only the large dose (250 micrograms/day) was able to restore the serum LH concentration to the level of the intact rats. Serum FSH was restored only partially, and the effect was the same with both doses and similar under all lighting conditions. We conclude that the increase in testosterone negative feedback sensitivity was not caused by the lack of periodicity of illumination alone, but that sufficient intensity of lighting throughout the 24 h was needed as well.     

Diurnal Variation in the Function of Serotonin Terminals in the Rat Hypothalamus

The high-affinity binding of [3H]imipramine is associated with the serotonin (5-hydroxytryptamine; 5-HT) transporter in the brain and in platelets. In the rat hypothalamus it has been reported that the density of these sites is increased in the dark period of the day, and this could result in an alteration in the release of 5-HT. The electrically evoked release of [3H]5-HT was thus studied in preloaded hypothalamic slices prepared from rats kept under 12:12 h light/dark or dark/light schedules. The fractional release of [3H]5-HT evoked by electrical stimulation, but not by the 5-HT releasing agent fenfluramine, was significantly decreased during the dark period when compared with the light period. The effects of the 5-HT reuptake blocker citalopram, of the two 5-HT autoreceptor agonists 5-methoxytryptamine and RU 24969, and of the 5-HT autoreceptor antagonist methiothepin on the release of [3H]5-HT were the same in both groups of rats. In conclusion, the release of [3H]5-HT from prelabelled rat hypothalamic slices is decreased during the dark period of the day. This modification is not reflected by changes in the effects of citalopram, an inhibitor of 5-HT reuptake, to modify the overflow of [3H]5-HT. The sensitivity and efficacy of agonists of the 5-HT autoreceptor are the same during the light and dark periods of the day.     

Effect of p-chlorophenylalanine on the acquisition of tolerance to barbital

The effect of p-chlorophenylalanine (p-CPA) pretreatment on barbital tolerance in the rat as measured by motor impairment on the moving belt test was examined in two separate studies. The first used a 2 X 2 design with doses of p-CPA (125 mg/kg) or water, and sodium barbital (300 mg/kg) or water. The treatments continued for 28 days with tests every 7 days. The p-CPA dose used was previously shown to produce and maintain greater than 95% depletion of brain serotonin (5-HT). Tolerance developed to the test doses, and even greater tolerance to the chronic treatment doses. In both cases the p-CPA slowed the development of tolerance without altering the acute response to the challenge dose of barbital. The second study involved only a p-CPA-barbital group and a water-barbital group. In this case treatment lasted for up to 8 days, with separate subgroups being tested only once each at 2-day intervals, in order to prevent the tests from affecting the rate of tolerance development. This experiment confirmed that p-CPA slowed the development of barbital tolerance. The present findings provide additional support for the possibility that 5-HT may be involved in the development of tolerance to sedatives (e.g., alcohol, pentobarbital).     

Continuous light increases N-acetyltransferase activity in the optic lobe of the giant freshwater prawn Macrobrachium rosenbergii de Man (Crustacea: Decapoda).

Giant freshwater prawns, Macrobrachium rosenbergii de Man, were reared under three different lighting conditions: continuous darkness (DD), 12 hr of light and 12 hr of darkness (LD 12:12) and continuous light (LL). After one month, the prawns were sacrificed and optic lobes isolated from the eyestalks were determined for N-acetyltransferase (NAT) activities and melatonin concentrations. Gonads were weighed and examined under light microscopy. The optic lobes from LL prawns contained significantly higher activities of NAT than those from LD 12:12 prawns. The melatonin concentrations and size and histological features of the gonads from the three groups of prawns did not differ. The results indicate that continuous light increases NAT activities in the optic lobe of M. rosenbergii but has no drastic effect on gonadal growth.