Day/night variations in membrane bound leucyl-2-naphthylamide hydrolysing activity in rat anterior hypothalamus, pituitary and retina

It is well known that the suprachiasmatic nucleus of the anterior hypothalamus acts as pacemaker regulating circadian rhythms in mammals. The daily variations of neuropeptides as well as their receptors depend on this system (Moore 1983). Aminopeptidases play an important role in regulating the activity of brain peptides (Bauer 1982). In this study we investigated membrane bound leucyl-2-naphthylamide hydrolysing activity in the anterior hypothalamus, pituitary and retina of adult male rats at six time points of a 12:12h light:dark schedule (light from 7:00 to 19:00 h), in order to analyse its day/night variation. The fluorometric assay evidenced significant differences between the three regions: in the anterior hypothalamus being higher during the dark period compared with the light period and in the pituitary higher during the light period compared with the dark period. In the retina the levels of this activity showed a higher heterogeneity during the day. Day - night differences in membrane bound leucyl-2-naphthylamide hydrolysing activity may reflect differences in its susceptible endogenous substrates.     

Day–Night Differences in Membrane‐Bound Pyroglutamyl‐2‐Naphthylamide‐Hydrolyzing Activity in Rat Hypothalamus,Pituitary, and Retina

Membrane‐bound pyroglutamyl‐2‐naphthylamide‐hydrolyzing enzyme activity was analyzed fluorometrically in the anterior hypothalamus, pituitary, and retina of adult male rats to investigate day–night differences. Six groups (n=6 per group) were assessed—three during the light span and three during the dark span—under a standard 12 h–12 h light–dark cycle (light on from 07:00 to 19:00 h) and controlled temperature environment, with food and water available ad libitum. In the hypothalamus, enzyme activity levels were higher for time points of the dark than the light period. In contrast, the pituitary and retina exhibited the highest levels at the time points of the light period. The pituitary and retina also exhibited significant differences between the clock‐hour means of the light period. Day–night differences in membrane‐bound pyroglutamyl‐2‐naphthylamide‐hydrolyzing activity may reflect differences in its susceptible endogenous substrates.     

Day-night differences in membrane-bound pyroglutamyl-2-naphthylamide-hydrolyzing activity in rat hypothalamus, pituitary, and retina

Membrane-bound pyroglutamyl-2-naphthylamide-hydrolyzing enzyme activity was analyzed fluorometrically in the anterior hypothalamus, pituitary, and retina of adult male rats to investigate day-night differences. Six groups (n=6 per group) were assessed—three during the light span and three during the dark span—under a standard 12 h-12 h light-dark cycle (light on from 07:00 to 19:00 h) and controlled temperature environment, with food and water available ad libitum. In the hypothalamus, enzyme activity levels were higher for time points of the dark than the light period. In contrast, the pituitary and retina exhibited the highest levels at the time points of the light period. The pituitary and retina also exhibited significant differences between the clock-hour means of the light period. Day-night differences in membrane-bound pyroglutamyl-2-naphthylamide-hydrolyzing activity may reflect differences in its susceptible endogenous substrates.     

Diurnal opposite variation between angiotensinase activities in photo–neuro–endocrine tissues of rats

Central and peripheral renin–angiotensin systems (RASs) act in a coordinated manner for the physiologic functions regulated by neuroendocrine events. However, whereas the diurnal rhythm of peripheral circulatory and tissue RASs is well known, the circadian behaviour of their components in central photo–neuro–endocrine structures, key elements for the control of circadian rhythms, has been barely studied. In the present study, we analysed the aspartyl- (AspAP) and glutamyl-aminopeptidase (GluAP) (aminopeptidase A) activities, the angiotensinases responsible for the metabolism of Ang I to Ang 2–10 and Ang II to Ang III, respectively, in the retina, anterior hypothalamus and pituitary at different light and dark time-points of a 12:12 h light:dark cycle (7–19 h light), using arylamide derivatives as substrates. The results demonstrated that while retina and pituitary exhibited their highest levels of AspAP activity in the light period and the lowest in the dark one, the contrary occurred in the hypothalamus – the lowest levels were observed in light conditions and the highest in darkness. The outcome for GluAP showed the highest levels in the light period and the lowest in the dark one in the three tissues analysed. In conclusion, changes in angiotensinase activities throughout the daytime may cause changes of their respective substrates and derived peptides and, consequently, in their functions. This observation may have implications for the treatment of hypertension.     

Daily Changes of GABA and Taurine Concentrations in Various Hypothalamic Areas Are Affected by Chronic Hyperprolactinemia

This study was designed to characterize, in anterior, mediobasal, and posterior hypothalamic and median eminence, the 24h changes of gamma aminobutyric acid (GABA) and taurine (TAU) contents in adult male rats and to analyze whether chronic hyperprolactinemia may affect these patterns. Rats were turned hyperprolactinemic by a pituitary graft. Plasma prolactin (PRL) levels increased after pituitary grafting at all time points examined. A disruption of the circadian rhythm was observed in pituitary-grafted rats, whereas GABA and TAU content followed daily rhythms in all areas studied in controls. In the mediobasal hypothalamus, two peaks for each amino acid were found at midnight and midday. In the anterior hypothalamus, GABA and TAU showed only one peak of concentration at midnight. In the posterior hypothalamus, the values of both GABA and TAU were higher during the light as compared to the dark phase of the photoperiod. In the median eminence GABA content peaked at 20:00h, the time when TAU exhibited the lowest values. Hyperprolactinemia abolished the 24h changes of GABA in the mediobasal hypothalamus and reduced its content as compared to controls. Hyperprolactinemia advanced the diurnal peak of TAU to 12:00h in the mediobasal hypothalamus and did not modify the 24:00h peak. In the anterior hypothalamus, hyperprolactinemia increased GABA and TAU contents during the light phase while it decreased them during the dark phase of the photoperiod. In the posterior hypothalamus hyperprolactinemia did not modify GABA or TAU patterns as compared to controls. In the median eminence hyperprolactinemia increased the 20:00h peak of GABA and shift advanced the decrease in TAU content at 20:00h and its maximum at 24:00h as compared to controls. These data show that GABA and TAU content exhibit specific daily patterns in each hypothalamic region studied. PRL differentially affects the daily pattern of these amino acids in each hypothalamic region analyzed.     

Daily changes of GABA and taurine concentrations in various hypothalamic areas are affected by chronic hyperprolactinemia

This study was designed to characterize, in anterior, mediobasal, and posterior hypothalamic and median eminence, the 24h changes of gamma aminobutyric acid (GABA) and taurine (TAU) contents in adult male rats and to analyze whether chronic hyperprolactinemia may affect these patterns. Rats were turned hyperprolactinemic by a pituitary graft. Plasma prolactin (PRL) levels increased after pituitary grafting at all time points examined. A disruption of the circadian rhythm was observed in pituitary-grafted rats, whereas GABA and TAU content followed daily rhythms in all areas studied in controls. In the mediobasal hypothalamus, two peaks for each amino acid were found at midnight and midday. In the anterior hypothalamus, GABA and TAU showed only one peak of concentration at midnight. In the posterior hypothalamus, the values of both GABA and TAU were higher during the light as compared to the dark phase of the photoperiod. In the median eminence GABA content peaked at 20:00h, the time when TAU exhibited the lowest values. Hyperprolactinemia abolished the 24h changes of GABA in the mediobasal hypothalamus and reduced its content as compared to controls. Hyperprolactinemia advanced the diurnal peak of TAU to 12:00h in the mediobasal hypothalamus and did not modify the 24:00h peak. In the anterior hypothalamus, hyperprolactinemia increased GABA and TAU contents during the light phase while it decreased them during the dark phase of the photoperiod. In the posterior hypothalamus hyperprolactinemia did not modify GABA or TAU patterns as compared to controls. In the median eminence hyperprolactinemia increased the 20:00h peak of GABA and shift advanced the decrease in TAU content at 20:00h and its maximum at 24:00h as compared to controls. These data show that GABA and TAU content exhibit specific daily patterns in each hypothalamic region studied. PRL differentially affects the daily pattern of these amino acids in each hypothalamic region analyzed.     

Regional concentrations of melatonin in the rat brain in the light and dark period

The levels of melatonin in five brain regions, whole brain, pineal and serum samples were studied in rats adapted under a photoperiod of 12h light and 12h dark. It was found that the melatonin levels for all the tissues obtained in the dark period were significantly higher than those obtained in the light period. Regional study of melatonin levels in the brain in the light and dark period demonstrated a high level in the hypothalamus, intermediate levels in the mid-brain, cerebellum and pons-medulla and low level in the telencephalon. Our findings indicate that melatonin in the brain is unevenly distributed and that there are diurnal rhythms of melatonin in all the five brain regions studied.     

Day-night variation in the in vitro contractility of aorta and mesenteric and renal arteries in transgenic hypertensive rats

TGR(mREN2)27 (TGR) rats develop severe hypertension and an inverted circadian blood pressure profile with peak blood pressure in the daytime rest phase. The present study investigated the in vitro responsiveness of different arteries of TGR rats during day and night. Twelve-week-old TGR rats and normotensive Sprague-Dawley (SPRD) controls, synchronized to 12h light, 12h dark (LD 12:12) (light 07:00 19:00), were killed at 09:00 (during rest) and 21:00 (during activity), and endothelium-dependent relaxation by acetylcholine and vascular contraction by angiotensin II were studied by measuring isometric force in ring segments of abdominal aorta and mesenteric and renal arteries. In SPRD rats, consistent day-night variation was found, with greater responses to angiotensin II during the daytime rest span. In TGR rats, biological time-dependent differences were found in the renal vasculature, but not in the aorta and mesenteric artery. Relaxation of SPRD rat aorta and mesenteric artery by acetylcholine was greater at 09:00, whereas in TGR rats, day-night variation was absent (mesenteric artery) or inverted (aorta). In conclusion, based on the study of two time points, daynight variation in vascular contractility of aorta and mesenteric artery is blunted in TGR rats, whereas renal artery segments showed an unchanged daynight pattern compared to SPRD controls. (Chronobiology International, 18(4), 665 681, 2001)     

Circadian rhythms of blood clotting time and coagulation factors II, VII, IX and X in rats

The 24-hr variations in clotting times and vitamin K-dependent blood coagulation factors were studied in rats kept on a 12-hr light-dark cycle (light on: 0600-1800 hours). Clotting times were determined under a binocular microscope by measuring the time required for the formation of the first fibrin thread. Factors II, VII and X were analyzed by the prothrombin test while the factor IX was quantified using the activated partial thromboplastin time assay. Results indicated that the clotting times were significantly longer during the dark (activity) period with a peak at 1:00 and a trough at 17:00. Similarly, a variation was found in factor activity levels: prothrombin (II), factor VII and factor X had higher activities during the light span (rest period). The highest activities found at 13:00 and 09:00 were statistically different from the minimum activity levels obtained at 21:00. Factor IX did not show a significant circadian variation.     

DAY-NIGHT VARIATION IN THE IN VITRO CONTRACTILITY OF AORTA AND MESENTERIC AND RENAL ARTERIES IN TRANSGENIC HYPERTENSIVE RATS*

TGR(mREN2)27 (TGR) rats develop severe hypertension and an inverted circadian blood pressure profile with peak blood pressure in the daytime rest phase. The present study investigated the in vitro responsiveness of different arteries of TGR rats during day and night. Twelve-week-old TGR rats and normotensive Sprague-Dawley (SPRD) controls, synchronized to 12h light, 12h dark (LD 12:12) (light 07:00 19:00), were killed at 09:00 (during rest) and 21:00 (during activity), and endothelium-dependent relaxation by acetylcholine and vascular contraction by angiotensin II were studied by measuring isometric force in ring segments of abdominal aorta and mesenteric and renal arteries. In SPRD rats, consistent day-night variation was found, with greater responses to angiotensin II during the daytime rest span. In TGR rats, biological time-dependent differences were found in the renal vasculature, but not in the aorta and mesenteric artery. Relaxation of SPRD rat aorta and mesenteric artery by acetylcholine was greater at 09:00, whereas in TGR rats, day-night variation was absent (mesenteric artery) or inverted (aorta). In conclusion, based on the study of two time points, daynight variation in vascular contractility of aorta and mesenteric artery is blunted in TGR rats, whereas renal artery segments showed an unchanged daynight pattern compared to SPRD controls. (Chronobiology International, 18(4), 665 681, 2001)     

Light-dark cycle and mitotic index in Asellus aquaticus (L.) (Crustacea,Isopoda)

The circadian variation of the mitotic index during spermiohistogenesis was studied in Asellus aquaticus (L.). The actual number of metaphases and prometaphases was determined at the end of each hour of light or darkness over a 24 h period in animals bred under LD 12:12. The number of the metaphases and prometaphases decreases during the light period and sharply increases in the last 3 hrs of the dark period. This variation in the proliferative activity suggests that photoperiod can play a role in the synchronization of mitosis.     

Placing ocular mutants into a functional context: a chronobiological approach

The behavior of mammals is characterized by a 24-h cycle of rest and activity which is a fundamental adaption to the solar cycle of light and darkness. The pacemaker of this circadian clock is localized in the ventral part of the hypothalamus, the so-called suprachiasmatic nuclei (SCN), and is entrained by light signals mediated by the eye. The eye is directly connected via the retinohypothalamic tract (RHT) to the SCN. Light that reaches the retina elicits glutamate release at the synaptic terminals of the RHT and influences the neurons in the SCN in a manner that alters the behavioral state of the animal. A light pulse that reaches the retina at the beginning of the night elicits a delay of the clock phase, whereas a light pulse that reaches the retina at the end of the dark period leads to an advance of the clock phase. This advance or delay can be quantified by measuring the change in onset of wheel-running activity. Such measurements have, and continue to provide, a remarkably powerful assay of how light is detected and transduced to regulate circadian rhythms. The methods used for such measurements in mice are described in the following article.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Rapid Phase Reversal and Response to Shorter than 24-Hour Cycles (IV)

N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is responsible for circadian rhythms in melatonin. The NAT activity rhythm has circadian properties such as persistence in constant conditions and precise control by light and dark. Experiments are reported in which chicks (Gallus domesticus), raised for 3 weeks in 12 h of light alternating with 12 h of dark (LD12:12), were exposed to 1-3 days of light-dark treatments during which NAT activity was measured in their pineal glands. (a) In LD12:12, NAT activity rose from less than 4.5 nmol/pineal gland/h during the light-time to 25-50 nmol/pineal gland/h in the dark-time. Constant light (LL) attenuated the amplitude of the NAT activity rhythm to 26-45% of the NAT activity cycle in LD12:12 during the first 24 h. (b) The timing of the increase in NAT activity was reset by the first full LD12:12 cycle following a 12-h phase shift of the LD12:12 cycle (a procedure that reversed the times of light and dark by imposition of either 24 h of light or dark). This result satisfies one of the criteria for NAT to be considered part of a circadian driving oscillator. (c) In less than 24-h cycles [2 h of light in alternation with 2 h of dark (LD2:2), 4 h of light in alternation with 4 h of dark (LD4:4), and 6 h of light in alternation with 6 h of dark (LD6:6)], NAT activity rose in the dark during the chicks' previously scheduled dark-time but not the previously scheduled light-time of LD12:12. In a cycle where 8 h of light alternated with 8 h of dark (LD8:8), NAT activity rose in both 8-h dark periods, even though the second one fell in the light-time of the prior LD12:12 schedule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian variation in the acetylcholinesterase activity of specific rat brain areas

Abstract

Acetylcholinesterase (AChE) activity of the adenohypophysis, cerebellum, cerebral cortex, hypothalamus, amygdala, hippocampus, midbrain, pons, medulla oblongata and caudate nucleus was determined by a spectro‐photometric method in adult, male rats adapted toan LD 12:12cycle. Results of the study show that AChE activity is highest during the light phase and lowest during the dark phase of the cycle in all the brain areas studied except the adenohypophysis, cerebellum, hippocampus and hypothalamus. These findings expand earlier observations on the circadian variation in rat brain AChE activity and suggests a relationship with reported circadian variation in the acetylcholine levels of rat brain.     

Variation in the Behavior of the Amphipod Gammarus pulex

Variation in the locomotion behavior of Gammarus pulex was studied using the Multispecies Freshwater Biomonitor®. Behavior was recorded individually under a 12:12 h light:dark cycle for a period of 7 d during which the amphipods were not fed. Gammarids behaved differently during the first 7 h in the laboratory chambers. Most specimens were active and decreased their activity but some were relatively inactive after the start of the experiment and increased their locomotion. At least 2 h are needed by G. pulex to accustom to the conditions. No significant relationships existed between type of behavior during the acclimation period or acclimation time and gender or length of the gammarids. There was a large variation in time spent on locomotion during the first night. In general, males were significantly more active than females. Within males, a distinction can be made between very active and less active specimens. The 7-d observation period showed that only 70% of the specimens demonstrated a clear day–night rhythm with higher activity during the night. Unexpectedly, approximately 10% of the specimens were more active during daytime. Furthermore, in this study locomotion became relatively stable only after 4 d. Inter-individual variation in behavior must be taken into account when using behavior as an endpoint in ecotoxicological bioassays.     

Circadian rhythm of active amino acid transport in rat liver

Abstract

The circadian change of the encephalic photosensitivity of quail has been demonstrated. In this study the diurnal variation of the retinal photosensitivity was investigated by the electroretinogram (ERG) to explore the phase relation between the retinal and encephalic systems. The b‐wave, a major component of the ERG, was used as a measure of retinal photosensitivity.

1. In the bird maintained in LD12:12 the b‐wave amplitude of the ERG stayed at a high level for the first 10 h of the light period, and decreased abruptly around the time of light‐off. The decreased level continued until midnight. Thenceforth the b‐wave amplitude recovered progressively to the daytime level before the time of next light on.

2. In the bird maintained in LD16:8, the decrease of the retinal sensitivity in the light phase was initiated prior to the time of expected light‐off, and the onset time of decreasing tendency was advanced by 2 h, with dark adaptation for 30 min given immediately before the ERG measurement. Upon continuous light exposure, the b‐wave amplitude always remained at low level.

3. Periodic changes in retinal sensitivity persisted when the environmental dark phase was temporarily extended for 19 to 30 h.

These observations suggest that the diurnal rhythm of the neural retina in quail might be generated endogenously and appears to control the encephalic photosensitive system.     

Treatment-time-dependent difference of ketamine pharmacological response and toxicity in rats

Circadian rhythms impact many physiological functions that may affect drug pharmacological response. Ketamine is a dissociative agent commonly used for surgical anesthesia in rats. The aim of the present study was to analyze the central nervous system (CNS) depression and lethality of ketamine injected intraperitoneally at different times during the 24 h. The study was conducted in October 2001, spring in the Southern hemisphere. Female prepuberal Sprague-Dawley rats synchronized to a 12h light:12h dark cycle (light, 07:00h-19:00h) were studied. Ketamine (40 mg/kg) was administered to one of six different clock-time treatment groups (n=6-7 rats each). Duration of latency period, ataxia, loss of righting reflex (LRR), post-LRR ataxia, and total pharmacological response were determined by visual assessment. To investigate acute toxicity, ketamine lethal dose 50 (148.0 mg/kg) was also administered as a single injection to six different treatment-time groups of rats. Significant temporal differences and circadian rhythms were detected in drug-induced post-LRR ataxia and total pharmacological response duration. The longest pharmacological response occurred in rats injected during the light (rest) phase and the shortest response in the dark (activity) phase. No circadian rhythm was detected in acute toxicity. The study findings indicate that the duration of CNS depression of ketamine in rats exhibits circadian rhythmic variation.     

A corticotropin-releasing hormone antisense oligodeoxynucleotide reduces spontaneous waking in the rat

We have previously hypothesized that corticotropin-releasing hormone (CRH) is involved in the regulation of physiological waking. In this study, we tested the hypothesis that reduction of CRH peptide would reduce spontaneous wakefulness of rats. We administered intracerebroventricularly into rats at several circadian time points antisense or sense DNA oligodeoxynucleotides (ODNs) corresponding to the initiation codon of CRH mRNA and determined subsequent effects on wakefulness and sleep of the rat. Our results indicate that CRH antisense oligodeoxynucleotides reduce spontaneous wakefulness during the dark (active) period, but not during the light (rest) period of the light/dark cycle. The alterations in time spent awake are due to reduced wake bout numbers, rather than a change in wake bout duration. These reductions in wakefulness were mirrored by increases in slow-wave sleep, while rapid eye movement sleep was not affected. Corticosterone, used as an index of CRH in the hypothalamus, was reduced by CRH antisense oligodeoxynucleotides during the same time that spontaneous wakefulness was reduced, suggesting CRH peptide modulation as the mediator of this response. In contrast, CRH sense oligodeoxynucleotides did not alter any parameter of this study during either the dark or light period. These findings provide additional support for the hypothesis that CRH is involved in the regulation/modulation of wakefulness.     

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.