Zoletil anaesthesia in chronobiological studies

Zoletil anaesthesia does not affect vital functions; however, there is no literature evidence regarding the effect of zoletil anaesthesia on the circadian rhythm(s) of vital functions. The aims of this study, with respect to dependence on the light–dark (LD) cycle under in vivo conditions in spontaneously breathing zoletil-anaesthetized rats, were to assess ECG parameters that may to predict development of ventricular arrhythmias and to assess arterial acid–base balance, which have the direct impact on the heart electrophysiology. The experiment was performed using zoletil-anaesthetized (30 mg/kg, i.p) female Wistar rats after adaptation to LD cycle (12 h:12 h). LD differences were found in heart rate, rectal temperature, electrophysiological myocardial parameters and acid–base balance. Animals were in systemic acidosis, hypoxia and hypercapnia in the both lighted periods of the regimen day. These results suggest that zoletil can be used in chronobiological studies investigating in vivo electrophysiological myocardial properties in rat models.     

Chronobiological perspectives on myocardial electrophysiological parameters under three types of general anaesthesia in a rat model

The specific aim of the present study, with respect to dependence on the light–dark (LD) cycle under in vivo conditions in spontaneously breathing rats was to review initial state in electrophysiological parameters that may predict the development of heart rhythm disorders in pentobarbital (40 mg/kg), ketamine–xylazine (100 + 15 mg/kg) and zoletil (30 mg/kg) anaesthetized animals. The study was performed using female Wistar rats that were adaptated to an LD cycle (12 h:12 h). Heart rate, PQ and QT intervals were evaluated for their dependence on the LD cycle. The longest PQ interval duration is under zoletil anaesthesia in the light period and the longest QT interval duration is under ketamine–xylazine anaesthesia in both light periods. We concluded that the most significant predisposition toward the development of ventricular arrhythmias originating from disorders of impulse production and conduction occurred under zoletil anaesthesia in the light period; those resulting from disorders in the dispersion of refractory periods occurred under ketamine–xylazine anaesthesia in both the light periods.     

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day^1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).Although circadian rhythms in running-wheel activity are typically linked to the SCN clock^6-8, circadian oscillators in many other regions of the brain and body^9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN^15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators^17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.     

Circadian rhythms of heart rate in freely moving and restrained American lobsters,Homarus americanus

While circadian rhythms of locomotion have been reported in the American lobster, Homarus americanus, it is unclear whether heart rate is also modulated on a circadian basis. To address this issue, both heart rate and locomotor activity were continuously monitored in light-dark (LD) cycles and constant darkness (DD). Lobsters in running wheels exhibited significant nocturnal increases in locomotor activity and heart rates during LD, and these measures were significantly correlated. In DD, most lobsters exhibited persistent circadian rhythms of both locomotion and heart rate. When heart rate was monitored in restrained lobsters in LD and DD, most animals also demonstrated clear daily and circadian rhythms in heart rate. Overall, this is the first demonstration of circadian rhythms of heart rate in H. americanus, the expression of which does not appear to be dependent on the expression of locomotor activity.     

Lighting requirements of nocturnal primates in captivity: A chronobiological approach

From the results of chronobiological studies in 11 Aotus lemurinus (trivirgatus) griseimembra, 3 Galago garnettii, 5 Galago senegalensis, and 6 Microcebus murinus, inferences can be made on the most suitable lighting conditions for nocturnal primates kept in captivity. In each species studied light controls the daily periodic course of activity in a dual way. First, the light-dark (LD) cycle acts as the main Zeitgeber, entraining the endogenous circadian timing system (CTS) to the environmental periodicity. Second, the prevailing light intensity has a direct species-specific inhibiting or enhancing effect, masking the level of activity predetermined by the CTS. Marked inhibition of activity is caused especially by low light intensities during dark-time (D-time), which can also lead to drastically reduced food intake (e.g., in Aotus). Therefore, high-amplitude LD cycles should be applied which guarantee a stable external and internal synchronization of the various circadian rhythms of the organism, with a D-illumination intensity high enough to prevent light-induced impairments of the behavior of the animals. Up to now LD cycles of 12:12 h (100–1,000:0.5–0.01 lx; ≥5,000°K) have proved to be most suitable. Only in Microcebus should the D-illumination be reduced to about 10^?4 lx. Moreover, it must be considered that species with a photoperiodically controlled reproduction cycle require specific alterations of the L-time:D-time ratio.     

Forced desynchronization model for a diurnal primate

The circadian system is organized in a hierarchy of multiple oscillators, with the suprachiasmatic nucleus (SCN) as the master oscillator in mammals. The SCN is formed by a group of coupled cell oscillators. Knowledge of this coupling mechanism is essential to understanding entrainment and the expression of circadian rhythms. Some authors suggest that light-dark (LD) cycles with periods near the limit of entrainment may be good models for promoting internal desynchronization, providing knowledge about the coupling mechanism. As such, we evaluated the circadian activity rhythm (CAR) pattern of marmosets in LD cycles at lower limits of entrainment in order to study induced internal dissociation. To that end, two experiments were conducted: (1) 6 adult females were under symmetrical LD cycles T21, T22 and T21.5 for 60, 35 and 48 days, respectively; and (2) 4 male and 4 female adults were under T21 for 24 days followed by 18 days of LL, back to T21 for 24 days, followed by 14 days of LL. The CAR of each animal was continuously recorded. In experiment 1, vocalizations were also recorded. Under Ts shorter than 24 days, a dissociation pattern was observed for CAR and vocalizations. Two simultaneous circadian components emerged, one with the same period as the LD cycle, called the light-entrained component, and the other in free-running, denominated the non-light-entrained component. Both components were displayed in the CAR for all the animals in T21, five animals (83.3%) in T21.5 and two animals (33.3%) in T22. Our results are in accordance with the multioscillatory nature of the circadian system. Dissociation is partial synchronization to the LD cycle, with at least one group of oscillators synchronized by relative coordination and masking, while another group of oscillators free runs, but is also masked by the LD cycle. Since only T21 promoted the emergence of both circadian components in the circadian rhythms of all marmosets, it was considered the promoter period of circadian rhythm dissociation in this species, and is proposed as a good animal model for forced desynchronization in non-human diurnal primates.     

H Fluxes in Excised Samanea Motor Tissue : II. Rhythmic Properties

Homogeneous groups of cells were excised at regular intervals from opposing (extensor and flexor) motor tissue of Samanea saman (Jacq) Merrill maintained in white light for 34 hours. H⁺ fluxes between the tissue and bathing solution were then monitored during 30 minutes of darkness. Flux rates in both cell types vary with circadian rhythms. Flexor cells secrete H⁺ to the medium during two-thirds of the circadian cycle and take up H⁺ during the remainder of the cycle, while extensor cells take up H⁺ from the medium during the entire cycle.     

Endocrine and cardiovascular rhythms differentially adapt to chronic phase-delay shifts in rats

Disturbances in regular circadian oscillations can have negative effects on cardiovascular function, but epidemiological data are inconclusive and new data from animal experiments elucidating critical biological mechanisms are needed. To evaluate the consequences of chronic phase shifts of the light/dark (LD) cycle on hormonal and cardiovascular rhythms, two experiments were performed. In Experiment 1, male rats were exposed to either a regular 12:12 LD cycle (CONT) or rotating 8-h phase-delay shifts of LD every second day (SHIFT) for 10 weeks. During this period, blood pressure (BP) was monitored weekly, and daily rhythms of melatonin, corticosterone, leptin and testosterone were evaluated at the end of the experiment. In Experiment 2, female rats were exposed to the identical shifted LD schedule for 12 weeks, and daily rhythms of BP, heart rate (HR) and locomotor activity were recorded using telemetry. Preserved melatonin rhythms were found in the pineal gland, plasma, heart and kidney of SHIFT rats with damped amplitude in the plasma and heart, suggesting that the central oscillator can adapt to chronic phase-delay shifts. In contrast, daily rhythms of corticosterone, testosterone and leptin were eliminated in SHIFT rats. Exposure to phase shifts did not lead to increased body weight and elevated BP. However, a shifted LD schedule substantially decreased the amplitude and suppressed the circadian power of the daily rhythms of BP and HR, implying weakened circadian control of physiological and behavioural processes. The results demonstrate that endocrine and cardiovascular rhythms can differentially adapt to chronic phase-delay shifts, promoting internal desynchronization between central and peripheral oscillators, which in combination with other negative environmental stimuli may result in negative health effects.     

Changes in phase-angle under light–dark cycles influenced by nonphotic stimulation

ABSTRACT

Most work looking at nonphotic effects on circadian rhythms is conducted when animals are held under freerunning conditions, usually constant darkness. However, for nonphotic effects to be functionally significant, they should be demonstrable under conditions in which most animals live, i.e., a 24-hr light–dark cycle (LD). Syrian hamsters held in LD 6:18 were administered nonphotic stimulation in the form of a 3-hr confinement to a novel wheel starting about 6 hr before the start of their normal nightly activity bout. This resulted in a 2.5-hr advance of their activity rhythm on the next day that gradually receded to about 1.5 hr over the next 10 days. When hamsters held in LD 6:18 were given five novel wheel confinements over 13 days their nightly activity onset advanced 3 hr and remained at that phase for at least 2 weeks. Home cage wheel deprivation experiments indicated that high levels of home cage activity are necessary to maintain the advanced phase. These results show that nonphotic stimulation can have large, long-lasting effects on daily rhythms in LD and suggest a possible mechanism whereby nocturnal rodents might achieve phase flexibility in response to seasonal changes.     

Blunted diurnal firing in lateral habenula projections to dorsal raphe nucleus and delayed photoentrainment in stress-susceptible mice

Daily rhythms are disrupted in patients with mood disorders. The lateral habenula (LHb) and dorsal raphe nucleus (DRN) contribute to circadian timekeeping and regulate mood. Thus, pathophysiology in these nuclei may be responsible for aberrations in daily rhythms during mood disorders. Using the 15-day chronic social defeat stress (CSDS) paradigm and in vitro slice electrophysiology, we measured the effects of stress on diurnal rhythms in firing of LHb cells projecting to the DRN (cells^LHb→DRN) and unlabeled DRN cells. We also performed optogenetic experiments to investigate if increased firing in cells^LHb→DRN during exposure to a weak 7-day social defeat stress (SDS) paradigm induces stress-susceptibility. Last, we investigated whether exposure to CSDS affected the ability of mice to photoentrain to a new light–dark (LD) cycle. The cells^LHb→DRN and unlabeled DRN cells of stress-susceptible mice express greater blunted diurnal firing compared to stress-näive (control) and stress-resilient mice. Daytime optogenetic activation of cells^LHb→DRN during SDS induces stress-susceptibility which shows the direct correlation between increased activity in this circuit and putative mood disorders. Finally, we found that stress-susceptible mice are slower, while stress-resilient mice are faster, at photoentraining to a new LD cycle. Our findings suggest that exposure to strong stressors induces blunted daily rhythms in firing in cells^LHb→DRN, DRN cells and decreases the initial rate of photoentrainment in susceptible-mice. In contrast, resilient-mice may undergo homeostatic adaptations that maintain daily rhythms in firing in cells^LHb→DRN and also show rapid photoentrainment to a new LD cycle.

Daily rhythms are disrupted in patients suffering from mood disorders, and it is known that the lateral habenula and dorsal raphe nucleus contribute to circadian timekeeping and regulate mood. This study shows that stress-susceptible mice have blunted and inverted diurnal firing rhythms in lateral habenula cells that project to the dorsal raphe nucleus, and have a slow rate of photoentrainment to a new light cycle.     

Temporal phasing of locomotor activity, heart rate rhythmicity, and core body temperature is disrupted in VIP receptor 2-deficient mice

Neurons of the brain's biological clock located in the hypothalamic suprachiasmatic nucleus (SCN) generate circadian rhythms of physiology (core body temperature, hormone secretion, locomotor activity, sleep/wake, and heart rate) with distinct temporal phasing when entrained by the light/dark (LD) cycle. The neuropeptide vasoactive intestinal polypetide (VIP) and its receptor (VPAC2) are highly expressed in the SCN. Recent studies indicate that VIPergic signaling plays an essential role in the maintenance of ongoing circadian rhythmicity by synchronizing SCN cells and by maintaining rhythmicity within individual neurons. To further increase the understanding of the role of VPAC2 signaling in circadian regulation, we implanted telemetric devices and simultaneously measured core body temperature, spontaneous activity, and heart rate in a strain of VPAC2-deficient mice and compared these observations with observations made from mice examined by wheel-running activity. The study demonstrates that VPAC2 signaling is necessary for a functional circadian clock driving locomotor activity, core body temperature, and heart rate rhythmicity, since VPAC2-deficient mice lose the rhythms in all three parameters when placed under constant conditions (of either light or darkness). Furthermore, although 24-h rhythms for three parameters are retained in VPAC2-deficient mice during the LD cycle, the temperature rhythm displays markedly altered time course and profile, rising earlier and peaking ～4-6 h prior to that of wild-type mice. The use of telemetric devices to measure circadian locomotor activity, temperature, and heart rate, together with the classical determination of circadian rhythms of wheel-running activity, raises questions about how representative wheel-running activity may be of other behavioral parameters, especially when animals have altered circadian phenotype.     

Electrogenic Properties of the Na+,K+-ATPase Probed by Presteady State and Relaxation Studies

Electrical measurements on planar lipid bilayers, patch/voltage clamp experiments, and spectroscopic investigations involving a potential sensitive dye are reviewed. These experiments were performed to analyze the kinetics of charge translocation of the Na⁺,K⁺-ATPase. High time resolution was achieved by applying caged ATP, voltage-jump, and stopped-flow techniques, respectively. Kinetic parameters and the electrogenicity of the relevant transitions in the Na⁺,K⁺-ATPase reaction cycle are discussed.     

A chronobiological study on the relation between heart rate and QT interval duration in newborn infants

The relation between heart rate and QT interval is the result of the autonomic nervous system control on cardiac function in healthy adults; accordingly, chronobiological studies have shown that adult subjects have circadian rhythms of heart rate (expressed as R-R interval) and QT interval in phase. We have employed chronobiological methods to study heart rate and QT interval relation in 10 newborn infants, who are known to have an immature cardiac control. Findings from this study indicate that not all the newborns show circadian rhythms of heart rate and QT interval and that when both rhythms are present they do not correlate like in the adults. Likely, this lack of relationship between heart rate and QT interval in newborns is due to different maturational stages of the newborns studied. As a practical implication, in newborn infants, mathematical correction of QT interval by heart rate is not a reliable method.     

ACTIVITY RHYTHMS OF WILD AND LABORATORY GOLDEN HAMSTERS (MESOCRICETUS AURATUS) UNDER ENTRAINED AND FREE-RUNNING CONDITIONS

The golden hamster (Mesocricetus auratus) is one of the most frequently used laboratory animals, particularly in chronobiological studies. One reason is its very robust and predictable rhythms, although the question arises whether this is an inbreeding effect or rather is typical for the species. We compared the daily (circadian) activity rhythms of wild and laboratory golden hamsters. The laboratory hamsters were derived from our own outbred stock (Zoh:GOHA). The wild hamsters included animals captured in Syria and their descendants (F1). Experiments were performed under entrained (light: dark [LD] 14h:10h) and under free-running (constant darkness, DD) conditions. Locomotor activity was recorded using passive infrared detectors. Under entrained conditions, the animals had access to a running wheel for a certain time to induce additional activity. After 3 weeks in constant darkness, a light pulse (15 min, 100 lux) was applied at circadian time 14 (CT14). Both laboratory and wild hamsters showed well-pronounced and very similar activity rhythms. Under entrained conditions, all hamsters manifested about 80% of their total 24h activity during the dark portion of the LD cycle. The robustness of the daily rhythms was also similar. However, interindividual variability was higher in wild hamsters for both measures. All animals used the running wheels almost exclusively during the dark portion of the LD cycle, although the wild hamsters were three times more active. The period length, measured in constant darkness, was significantly shorter in wild (23.93h ± 0.10h) than in laboratory hamsters (24.06 ± 0.07h). The light-induced phase changes were not different (about 1.5h). In summary, these results indicate that the laboratory hamster is not much different from the wild type. (Chronobiology International, 18(6), 921–932, 2001)     

Specific Modulation of two Neuronal Digitalis Receptors by Anaesthesia

Abstract

Three isoenzymes of digitalis receptors (α₁, α₂, α₃) in the brain and only one in the kidney (α₁) can be distinguished by their ouabain affinities and their responsiveness to sodium. Since we have reported modulations for these digitalis receptors by their fatty acid membrane environment, anaesthesics could bind on and modulate either directly these receptors or indirectly by disturbing membrane lipids. The aim of this study was to evaluate this anaesthetic action on apparent ouabain affinities and sodium dependence of cerebral and renal Na⁺,K⁺-ATPase isoenzymes activities. Rat brain and kidney membrane fractions with pentobarbital-induced anaesthetized state were compared to an unanaesthetized state for their (1) fatty acid composition of total membrane phospholipids, (2) responsiveness to ouabain and (3) Na⁺ dependence of digitalis receptors. An anaesthesia period of 10 minutes induced (1) a fatty acid modification of brain membranes and (2) a significant sensibilization to ouabain for the α₂ and α₃ isoforms of digitalis receptors (α2, IC₅₀; 8.2 ± 0.5 × 10⁷ mol/l vs 4.5±0.2 × 10⁷ mol/l; α₃, IC₅₀; 6.0±0.3 × 10^-8 mol/l vs 2.5±0.1 × 10^-8 mol/l). In contrast, the ouabain affinity of the α₁ subunit expressed in kidney and brain membranes was unaltered. No anaesthetic effect was observed on the Na⁺ dependence of the α₁ isoenzyme in the brain (4 mmol/l) and the kidney (8 mmol/l). Pentobarbital induced a desensibilization for α₂-receptors (8.3±0.5 vs 16.0±1.4 mmol/l Na⁺) and a sensibilization for α₃-receptors (14.4±0.8 vs 10±1.3 mmol/l Na⁺). These altered properties could be related to a selective modification of the fatty acid composition and/or to the presence of a specific binding site for pentobarbital on these two neuronal digitalis receptors.     

THERMOCYCLIC AND PHOTOCYCLIC ENTRAINMENT OF CIRCADIAN LOCOMOTOR ACTIVITY RHYTHMS IN SLEEPY LIZARDS,TILIQUA RUGOSA

Australian sleepy lizards (Tiliqua rugosa) exhibit marked locomotor activity rhythms in the field and laboratory. Light-dark (LD) and temperature cycles (TCs) are considered important for the entrainment of circadian locomotor activity rhythms and for mediating seasonal adjustments in aspects of these rhythms, such as phase, amplitude, and activity pattern. The relative importance of 24 h LD and TCs in entraining the circadian locomotor activity rhythm in T. rugosa was examined in three experiments. In the first experiment, lizards were held under LD 12:12 and subjected to either a TC of 33:15?°?C in phase with the LD cycle or a reversed TC positioned in antiphase to the LD cycle. Following LD 12:12, lizards were maintained under the same TCs but were subjected to DD. Activity was restricted to the thermophase in LD, irrespective of the lighting regime and during the period of DD that followed, suggesting entrainment by the TC. The amplitude of the TC was lowered by 8?°?C to reduce the intensity and possible masking effect of the TC zeitgeber in subsequent experiments. In the second experiment, lizards were held under LD 12.5:11.5 and subjected to one of three treatments: constant 30?°?C, normal TC (30:20?°?C) in phase with the LD cycle, or reversed TC. Following LD, all lizards were subjected to DD and constant 30?°?C. Post-entrainment free-run records revealed that LD cycles and TCs could both entrain the locomotor rhythms of T. rugosa. In LD, mean activity duration (α) of lizards in the normal TC group was considerably less than that in the constant 30?°?C group. Mean α also increased between LD and DD in lizards in the normal TC group. Although there was large variation in the phasing of the rhythm in relation to the LD cycle in reversed TC lizards, TCs presented in phase with the LD cycle most accurately synchronized the rhythm to the photocycle. In the third experiment, lizards were held in DD at constant 30?°?C before being subjected to a further period of DD and one of four treatments: normal TC (06:00 to 18:00 h thermophase), delayed TC (12:00 to 00:00 h thermophase), advanced TC (00:00 to 12:00 h thermophase), or control (no TC, constant 30?°?C). While control lizards continued to free-run in DD at constant temperature, the locomotor activity rhythms of lizards subjected to TCs rapidly entrained to TCs, whether or not the TC was phase advanced or delayed by 6 h. There was no difference in the phase relationships of lizard activity rhythms to the onset of the thermophase among the normal, delayed, and advanced TC groups, suggesting equally strong entrainment to the TC in each group. The results of this experiment excluded the possibility that masking effects were responsible for the locomotor activity responses of lizards to TCs. The three experiments demonstrated that TCs are important for entraining circadian locomotor activity rhythms of T. rugosa, even when photic cues are conflicting or absent, and that an interaction between LD cycles and TCs most accurately synchronizes this rhythm. (Author correspondence: david.j.ellis@adelaide.edu.au)     

TURNOVER OF CELL-RENEWING POPULATIONS UNDERGOING CIRCADIAN RHYTHMS IN CELL PROLIFERATION

Evidence is presented in support of the concept of partial synchrony of cells as the cause for circadian rhythms in DNA synthesis and mitotic activity. The nonuniform age distribution of cells in cycle indicated that equations based on total asynchrony were not applicable for calculation of cytokinetic parameters in cellrenewing populations undergoing circadian rhythms. The integration of the circadian mitotic curve is introduced as a simple and accurate method for determination of proliferation rate and turnover time. An approximately linear increase in the labeling index following repeated injections of ³H-thymidine demonstrated that nearly 100% basal cells in hamster cheek pouch epithelia were in cycle during a turnover time. These experiments suggest that if there is a G₂ phase in cellrenewing tissues, this is short with respect to turnover time and that it may be a specific compartment where the control of cell proliferation operates.     

Chronopharmacology of pancuronium in rats anesthetized by CT 1341 (Alfatésine)]

A circadian variation in the curarizing ability of pancuronium bromide (Pavulon) has been documented in an homogenous group of 100 Wistar AF-SPF adult male rats anaesthetized by the steroid anaesthetic althesin (Alfatésine, CT 1341). Animals were maintained at 24 +/- 2 degrees C and synchronized with natural light 06.00 to 18.00 and darkness (october 1977). We observed significant circadian rhythms for both of these agents: first the induction of anaesthesia by althesin was markedly pronounced at 15.30 and varied with season. Secondly the maximum effect of pancuronium was recorded at 08.00. When rats were anaesthetized by sodium pentobarbital under similar experimental conditions but during a different season (january to may) we observed a similar circadian rhythm for pancuronium. These data indicate that: a) the type of anaesthesia used in the protocol may not be of importance in demonstration of a curarizing rhythm in the rat but, b) the possibility of a seasonal component being present and effecting this rhythm needs to be investigated.     

Signaling of Pigment-Dispersing Factor (PDF) in the Madeira Cockroach Rhyparobia maderae

The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF''s importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca²⁺ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca²⁺ baseline concentration and frequency of oscillating Ca²⁺ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca²⁺ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1–4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca²⁺ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K⁺ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K⁺ and Na⁺ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance.     

EFFECTS OF L-DOPA ON CIRCADIAN RHYTHMS OF 6-OHDA STRIATAL LESIONED RATS: A RADIOTELEMETRIC STUDY

Temporal variation in the motor function of Parkinson's disease (PD) patients suggests the potential importance of a chronobiological and chronopharmacological approach in its clinical management. We previously documented the effects of striatal injection of 6-OHDA (as an animal model of PD) on the circadian rhythms of temperature (T), heart rate (HR), and locomotor activity (A). The present work assessed the possible influence of L-Dopa on these same rhythms in the 6-OHDA animal model of PD. The study began after a four-week recovery period following surgical implantation of telemetric devices to monitor the study variables and/or anaesthesia. The study was divided into an initial one-week control period (W1) for baseline measurement of T, HR, and A rhythms. Thereafter, stereotaxic 6-OHDA lesioning was done. and a second monitoring for two weeks followed (W2, W3). Rats were then randomly divided into two groups: eight control rats received, via a mini-osmotic pump implanted subcutaneously, the excipient saline; the other eight rats received L-Dopa (100?mg/kg SC/day). After a seven-day period (W4), the pumps were removed and the T, HR, and A rhythms were monitored for two weeks (W5 and W6). To control for 6-OHDA striatal dopamine-induced depletion, 12 other rats were injected by identical methods (eight rats with 6-OHDA and four controls with saline) and sacrificed at W1, W3, and W5 for dopamine striatal content determination. To verify the delivery of levodopa from the osmotic pumps, plasma levels of levodopa and its main metabolites 3-OMD, DOPAC, and HVA were determined on separate group of rats receiving the drug under the same experimental conditions (osmotic pumps delivering continuously 10 µl/h for seven days, 100?mg/kg/subcutaneously). Our results agree with previously reported rhythmic changes induced by 6-OHDA—loss of circadian rhythmicity or changes in the main parameters of the registered rhythms. When circadian rhythmicity was abolished, L-Dopa treatment improved or accelerated recovery of the circadian rhythms, the effect being more pronounced for the HR rhythm. When circadian rhythms were not abolished but perturbed, L-Dopa treatment did not improve the 6-OHDA-induced changes in the T and A mesor (24?h mean level), while a significant effect was observed for HR. It appears that constant-rate L-Dopa infusion is unable to totally balance dopamine depletion; taking into account the circadian pattern of many structures implicated in drug effect, a sinusoidal delivery of L-Dopa must be evaluated in future experiments. (Author correspondence: bernard.bruguerolle@univmed.fr)