Circadian rhythm of ornithine decarboxylase activity in small intestine of fasted rats.

The aim of this study was to determine whether the circadian changes in ornithine decarboxylase (ODC) activity of different segments of the small intestine were governed by factors other than food intake. First, the effects of fasting on mucosal ODC activity were examined. The results indicate that mucosal ODC activity in 24 hr and 48 hr fasted rats decreased significantly compared with ad libitum-fed rats. Second, the circadian rhythm of mucosal ODC activity was characterized by measuring mucosal ODC activity in fasted rats at four time points (09:00, 15:00, 21:00, and 03:00 hr; light period: 06:00-18:00 hr). The results from this study indicate that there is a detectable baseline ODC activity in different segments of fasting intestine. In duodenum, mucosal ODC activity was highest at 15:00 hr (light period), a time at which the rat was normally not eating. In jejunum and ileum, mucosal ODC activity increased between 21:00 and 03:00 hr (dark period). The observation that small intestine exhibits a distinct circadian rhythm of ODC activity in fasted rats suggests that not only food but also intrinsic factors can modulate physiologic oscillations in mucosal ODC activity.     

Time-Dependent Effects of Trichloroethylene on Motor Activity in Rats

Circadian variations in acute and subacute neurobehavioural effects of trichloroethylene (TRI: 1.2 g/kg i.p.) were investigated in the rat under a light: dark = 12:12 hr cycle. An acute effect of TRI evaluated by decreased muscle tone was maximal during the early dark phase (21:00). A subacute effect of TRI was evaluated by a continuous recording of spontaneous locomotor activity in the rat. The circadian rhythm in spontaneous locomotor activity was extensively impaired by the injection of TRI for three consecutive days. Spectral analysis of spontaneous locomotor activity showed that ultradian periods became more dominant than the circadian period, and the 1//fluctuation of the spectrum disappeared after the injection of TRI. The effect of TRI on the circadian rhythm in spontaneous locomotor activity was circadian-phase dependent, and the treatment of TRI at 09:00 provoked greater circadian rhythm impairment than that at 21:00. The mechanisms of the time-dependent effect of TRI on neurobehaviour are the subject of further investigation.     

Time-Dependent Effects of Trichloroethylene on Motor Activity in Rats

Circadian variations in acute and subacute neurobehavioural effects of trichloroethylene (TRI: 1.2 g/kg i.p.) were investigated in the rat under a light: dark = 12:12 hr cycle. An acute effect of TRI evaluated by decreased muscle tone was maximal during the early dark phase (21:00). A subacute effect of TRI was evaluated by a continuous recording of spontaneous locomotor activity in the rat. The circadian rhythm in spontaneous locomotor activity was extensively impaired by the injection of TRI for three consecutive days. Spectral analysis of spontaneous locomotor activity showed that ultradian periods became more dominant than the circadian period, and the 1//fluctuation of the spectrum disappeared after the injection of TRI. The effect of TRI on the circadian rhythm in spontaneous locomotor activity was circadian-phase dependent, and the treatment of TRI at 09:00 provoked greater circadian rhythm impairment than that at 21:00. The mechanisms of the time-dependent effect of TRI on neurobehaviour are the subject of further investigation.     

A blood plasma inhibitor is responsible for circadian changes in rat renal Na,K-ATPase activity

Rhythmic changes in activity following a circadian schedule have been described for several enzymes. The possibility of circadian changes in Na,K-ATPase activity was studied in homogenates of rat kidney cortex cells. Male Sprague-Dawley rats were kept on a schedule of 12h light (06:00-18:00 h) and 12 h darkness (18:00-06:00 h) for 2 weeks. At the end of the conditioning period, one rat was killed every 2 h, until completion of a 24 h cycle. Outermost kidney cortex slices were prepared, homogenized and assayed for Na,K-ATPase activity. The whole procedure was repeated six times. Na,K-ATPase activity shows an important oscillation (2 cycles/24 h). Peak activities were detected at 09:00 and 21:00 h, whereas the lowest activities were detected at 15:00 and 01:00-03:00 h. The highest activity was 40+/-3 nmoles Pi mg protein(-1)min(-1) (09:00 h), and the lowest was 79+/-3 nmoles Pi mg protein(-1)min(-1) (15:00 h). The amount of the Na+-stimulated phosphorylated intermediate is the same for the 09:00 h and 15:00 h homogenates. Preincubation of 09:00 h kidney cortex homogenates with blood plasma drawn from rats at either 03:00 h or 15:00 h, significantly inhibited their Na,K-ATPase activity. This inhibition was not seen when the preincubation was carried out with either 09:00 h or 21:00 h blood plasma. The striking oscillation (2 cycles/24 h) of the Na,K-ATPase activity of rat kidney cortex cells is ascribed to the presence of an endogenous inhibitor in blood plasma.     

Neuropeptide Y normalizes renin secretion in adrenalectomized rats without changing blood pressure

In the periphery, neuropeptide Y is present in plasma, in the adrenal medulla as well as in sympathetic nerve endings and in the juxtaglomerular apparatus. The aim of the present study was to assess the effect of this peptide on renin secretion. Normotensive rats were adrenalectomized or sham-operated and made hypertensive with methylprednisolone acetate (20 mg/kg s.c. once weekly). Deoxycorticosterone pivalate (10 mg/kg s.c. once weekly) was also given to prevent mineralocorticoid deficiency. Two weeks after initial surgery, 12 adrenalectomized and 8 sham-operated conscious rats were infused for 30 min with neuropeptide Y (0.1 micrograms/min) whereas 8 other adrenalectomized and 9 sham-operated conscious rats received under similar conditions the vehicle of neuropeptide Y (10 microliter/min). Neither before nor during the infusions was there a significant difference in blood pressure and heart rate between the 4 groups of animals. Plasma renin activity, measured at the end of the infusion, was 30.5 ng/ml/hr in the adrenalectomized group receiving vehicle and 6.3 ng/ml/hr in that infused with neuropeptide Y (p less than 0.001). This latter value did not differ from that found in sham-operated rats. These results suggest that neuropeptide Y may play an important role in regulating renin secretion.     

Chronopharmacokinetics of imipramine and desipramine in rat forebrain and plasma after single and chronic treatment with imipramine.

Daily variations in the pharmacokinetics of imipramine (IMI) could contribute to circadian phase-dependent effects of the drug. Therefore, the chronopharmacokinetics of IMI and its metabolite, desipramine (DMI), were studied after single and chronic application. Male rats were synchronized to a 12:12 hour light:dark (L:D) regimen with lights on from 07:00 to 19:00 (dark, 19:00-07:00). In single-dose experiments rats were injected with IMI (10 mg/kg) i.p. or i.v. at 07:30 or 19:30 and groups of rats were killed 0-22 hours thereafter. After chronic application of IMI in drinking water (approximately 15 mg/kg/d) groups of rats were killed during the 14th day of treatment at 02:00, 08:00, 14:00, and 20:00, respectively. Brain and plasma concentrations of IMI and DMI were determined by reversed-phase high-performance liquid chromatography with ultraviolet detection. After single i.p. application of IMI, maximal brain concentrations (Cmax) of IMI and DMI were nearly twofold higher in darkness (IMI, 4.8 micrograms/g; DMI, 1.8 micrograms/g) than in light (IMI, 2.85 micrograms/g; DMI, 0.85 microgram/g). Also, the area under the curve (AUC) (0-22 hours) was about 1.6-fold greater in darkness than in light for IMI and DMI; half-lives were not circadian phase dependent. After i.v. injection of IMI, the AUC in brain was also about 30% greater in darkness than in light. After chronic application of IMI in drinking water, brain concentrations of IMI and DMI varied more than threefold within 24 hours. The data demonstrate that the pharmacokinetics of IMI and DMI are circadian phase dependent. It is assumed that circadian variations in drug distribution are more likely to contribute to the drug's chronopharmacokinetics than variations in the drug's metabolism. The 24-hour variations in the drug's concentrations after chronic IMI application in drinking water can be explained by the drinking behavior of the rats, which by itself is altered by IMI.     

Chronopharmacokinetics of Imipramine and Desipramine in Rat Forebrain and Plasma After Single and Chronic Treatment with Imipramine

Daily variations in the pharmacokinetics of imipramine (IMI) could contribute to circadian phase-dependent effects of the drug. Therefore, the chronopharmacokinetics of IMI and its metabolite, desipramine (DMI), were studied after single and chronic application. Male rats were synchronized to a 12:12 hour lightdark (L:D) regimen with lights on from 07:00 to 19:00 (dark, 19:00-07:00). In single-dose experiments rats were injected with IMI (10 mg/kg) i.p. or i.v. at 07:30 or 19:30 and groups of rats were killed 0-22 hours thereafter. After chronic application of IMI in drinking water (≈ 15 mg/kg/d) groups of rats were killed during the 14th day of treatment at 02:00, 08:00, 14:00, and 20:00, respectively. Brain and plasma concentrations of IMI and DMI were determined by reversed-phase high-performance liquid chromatography with ultraviolet detection. After single i.p. application of IMI, maximal brain concentrations (C_max) of IMI and DMI were nearly twofold higher in darkness (IMI, 4.8 μg/g; DMI, 1.8 μg/g) than in light (IMI, 2.85 Mg/g; DMI, 0.85 Mg/g). Also, the area under the curve (AUC) (0-22 hours) was about 1.6-fold greater in darkness than in light for IMI and DMI; half-lives were not circadian phase dependent. After i.v. injection of IMI, the AUC in brain was also about 30% greater in darkness than in light. After chronic application of IMI in drinking water, brain concentrations of IMI and DMI varied more than threefold within 24 hours. The data demonstrate that the pharmacokinetics of IMI and DMI are circadian phase dependent. It is assumed that circadian variations in drug distribution are more likely to contribute to the drug's chronopharmacokinetics than variations in the drug's metabolism. The 24-hour variations in the drug's concentrations after chronic IMI application in drinking water can be explained by the drinking behavior of the rats, which by itself is altered by IMI.     

Circadian and circannual variation of the carrageenin inflammatory effect in rat

The circadian variation of edema produced by carrageenin (carr.) administration into plantar tissue was studied in rats kept under a 12 light - 12 dark regimen. Three doses were used (125, 250 and 500 micrograms per rat) injected at different time (02.00, 08,00, 14.00 and 20.00 h). With the high doses, the level of edema for the four hour period after carr. administration was similar whatever the hour of injection. In contrast, with the lower dose (125 micrograms) a circadian rhythm in the intensity of the edema produced was observed, showing a maximum of susceptibility during the light span. Repetitive experiments performed at different periods of the year validated this finding. Comparing mean mesors, analysis of this data showed two distinct levels of inflammation, with the lower level observed in autumn and winter indicating evidence for a circannual variability.     

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.     

Evaluation of the inhibitory effect of circulating corticosteroids on circadian stimulated and stress induced secretion of ACTH in rats

Male rats were bilaterally adrenalectomized in order to measure the extent of inhibition exerted by endogenous corticosteroids on both basal ACTH secretion along its circadian rhythm and ether-stress induced ACTH secretion. In intact controls, plasma ACTH levels at the circadian maximum exceeded by 4 times the circadian minimum, and ACTH response 15 min after ether-stress surpassed the circadian minimum by 20 times. In adrenalectomized rats, the daily minimum was 8 times that of the controls. Nevertheless the circadian maximum was 3 times above the rhythm's minimum, while the maximal stress response (15 min) surpassed the circadian minimum by 8 times. In adrenalectomized rats supplemented with a solid source of corticosterone inducing a stable plasma corticosterone level equivalent to the controls' circadian minimum (3 micrograms/100 ml), the ACTH rhythm still fluctuated twice as high as in intact controls. The tonic feed-back inhibition exerted by endogenous corticosteroids on ACTH secretion appeared thus significantly stronger than the GABAergic inhibition to the corticotropic system which was previously studied under similar standard conditions.     

Orcadian Rhythm in the Torque Developed by Elbow Flexors During Isometric Contraction Effect of Sampling Schedules

Time-dependent changes in elbow flexion torque have been documented according to two different sampling schedules. Seven physical education students took part in the first series of experiments, and 7 other similar subjects in the second. In both sets of experiments, the subjects performed isometric contractions: maximal and submaximal at 90° in the first experiments and maximal at different angular positions in the second. After a 30-minute rest period, the torque developed was measured at 00:00, 06:00, 09:00, 12:00, 15:00, 18:00, and 21:00h on the day of the experiment. These subjects remained in the laboratory for 24h. In the second series of experiments, the torque developed was measured at 01:00, 05:00, 09:00, 13:00, 17:00, and 21:00h over the subsequent 6 days with only one test session per day. In this case, there was an interval of 20h between two successive test sessions. In the first experiment, a significant time-of-day effect was observed for the torque of the elbow flexors under isometric conditions with an acrophase at 17:58h. The 24h normalized mean score was 92.85% with an amplitude of 7.63% of the daily mean. In the second series of experiments, there was evidence of a circadian rhythm in the torque developed by the elbow flexors at every angle position, especially at 90°, the angle investigated in the first set of experiments. The peak torque was calculated to have occurred at 17:55h. The amplitude of the rhythm was equal to 6.99% of the daily mean. There were no statistically significant differences in the characteristics of the circadian rhythm observed between the two experimental designs. We concluded that an experiment extending over several days could be employed to evaluate circadian rhythms in muscular activity reliably. (Chronobiology International, 14(3), 287–294, 1997)     

Twenty-Four-Hour Variations in the Sensitivity of Rat Aorta to Vasoactive Agents

The presence of time-dependent variations in the in vitro sensitivity of aorta preparations to either vasoconstricting or relaxing agents was investigated in rats maintained in light from 08: 00 to 20: 00 and in darkness from 20: 00 to 08: 00. Rat thoracic aorta rings were obtained from animals sacrificed at four different times of the day. The rat aorta was found to be more sensitive to the constricting effect of phenylephrine at 15: 00, and of 5-hydroxytryptamine at 21: 00. On the other hand, both endothelium-dependent and -independent relaxations were more remarkable at 03: 00 than at other times of the day. These variations represented significant circadian rhythms when analyzed by analysis of variance. Different in vitro responsiveness to these agents might reflect changes in the sensitivity and/or number of related receptors in vascular preparations. In conclusion, the circadian time of animal sacrifice to obtain vascular preparations constitutes an important aspect of the research method and a key determinant of findings. (Chronobiology International, 13(6), 465-475, 1996)     

Time and dose dependent effect of indomethacin on BCG induced PMN migration in mice.

The effect of three doses of indomethacin (Indo) on BCG-induced PMN migration at different times of day was studied in Swiss mice kept on a lighting regimen of LD 12:12, with L from 07:00 to 19:00. Experimental granulomas were induced by subcutaneous implantation of BCG-impregnated cell traps for a time span of 480 min. Doses of 1, 3 and 9 mg/kg of Indo were given orally one hour before trap implantation, at 01:00, 05:00, 09:00, 13:00, 17:00 and 21:00 hr. In sham animals, the maximal PMN count occurred at 17:00 hr. In treated mice, Indo increased or decreased the number of PMN/mm2 as a function of time of administration. Cell migration was inhibited at 17:00 hr by all 3 Indo doses, while the number of PMN in the cell trap increased at 21:00 hr. Various dose-effects were obtained at the other times of day. Several hypotheses are proposed to explain the conflicting data. The results indicate the importance of the time of drug administration in biology.     

Circadian variations of serotonin in plasma and different brain regions of rats

Most of the physiological processes that take place in the organism follow a circadian rhythm. Serotonin is one of the most important neurotransmitters in our nervous system, and has been strongly implicated in the regulation on the mammalian circadian clock, located in the suprachiasmatic nuclei (SCN). The present study analysed the levels of serotonin over a period of 24 h in the plasma and in different brain regions. The model used was of male Wistar rats, 14 +/- 2 weeks of age (n = 120), maintained under conditions of 12 h light and 12 h dark, and food and water ad libitum. The serotonin levels were measured by ELISA every hour at night (20:00-08:00 h) and every 4 h during the daytime (08:00-20:00 h). Ours results show that the maximum levels of serotonin in plasma were obtained at 09:00 and 22:00 and a minor peak at 01:00 h. In hypothalamus there was a significant peak at 22:00 and two minor peaks at 17:00 and 02:00 h; the same occurred in hippocampus with a significant peak at 21:00, and two secondary peaks at 24:00 and 05:00 h; in cerebellum there were two peaks at 21:00 and 02:00 h, while in striatum and pineal there were peaks at 21:00 h and 23:00, respectively. In conclusion, the higher levels of serotonin were during the phase of darkness, which varies depending on the region in which it is measured.     

Twenty-Four-Hour Variations in the Sensitivity of Rat Aorta to Vasoactive Agents

The presence of time-dependent variations in the in vitro sensitivity of aorta preparations to either vasoconstricting or relaxing agents was investigated in rats maintained in light from 08: 00 to 20: 00 and in darkness from 20: 00 to 08: 00. Rat thoracic aorta rings were obtained from animals sacrificed at four different times of the day. The rat aorta was found to be more sensitive to the constricting effect of phenylephrine at 15: 00, and of 5-hydroxytryptamine at 21: 00. On the other hand, both endothelium-dependent and -independent relaxations were more remarkable at 03: 00 than at other times of the day. These variations represented significant circadian rhythms when analyzed by analysis of variance. Different in vitro responsiveness to these agents might reflect changes in the sensitivity and/or number of related receptors in vascular preparations. In conclusion, the circadian time of animal sacrifice to obtain vascular preparations constitutes an important aspect of the research method and a key determinant of findings. (Chronobiology International, 13(6), 465–475, 1996)     

Corticohypophysiotropic and corticotropic activity in adrenalectomized male and female rats after psychic stress

We show modifications in the hypothalamic CRF activity and plasma ACTH concentration in adult rats of both sexes, which were five day sham-operated or adrenalectomized and killed either under basal conditions or after a 3 min period or psychological stress. 1. Under basal conditions, the inhibition of the basal release of ACTH is suppressed in 5 day adrenalectomized rats and a sex difference appears: plasma ACTH concentration is twice as great in females than in males. 2. After a 3 min period of psychological stress, the usual increase in hypothalamic CRF activity observed in sham-operated rats, which seems to be sex-related, does not appear in adrenalectomized male or female rats. However, in adrenalectomized rats, the maximal increase in plasma ACTH concentration occurred more rapidly, with a rate 10 times as great in males and 4 times as great in females, than in sham-operated rats. Differences between the sexes in the maximal increase in plasma ACTH concentration remains 1,6 times as great in females than in males. 3. Our results confirm that corticosteroids exert: (1) a tonic feedback inhibition of the basal release of ACTH, (2) a fast feedback inhibition of the stress induced release of ACTH; the promote an increase in the hypothalamic CRF content. Relative intensity of these two inhibitory mechanisms seems to be sex-related.     

Dim light melatonin onset in alcohol-dependent men and women compared with healthy controls

Sleep disturbances in alcohol-dependent (AD) individuals may persist despite abstinence from alcohol and can influence the course of the disorder. Although the mechanisms of sleep disturbances of AD are not well understood and some evidence suggests dysregulation of circadian rhythms, dim light melatonin onset (DLMO) has not previously been assessed in AD versus healthy control (HC) individuals in a sample that varied by sex and race. The authors assessed 52 AD participants (mean?±?SD age: 36.0?±?11.0 yrs of age, 10 women) who were 3-12 wks since their last drink (abstinence: 57.9?±?19.3 d) and 19 age- and sex-matched HCs (34.4?±?10.6 yrs, 5 women). Following a 23:00-06:00?h at-home sleep schedule for at least 5 d and screening/baseline nights in the sleep laboratory, participants underwent a 3-h extension of wakefulness (02:00?h bedtime) during which salivary melatonin samples were collected every 30?min beginning at 19:30?h. The time of DLMO was the primary measure of circadian physiology and was assessed with two commonly used methodologies. There was a slower rate of rise and lower maximal amplitude of the melatonin rhythm in the AD group. DLMO varied by the method used to derive it. Using 3 pg/mL as threshold, no significant differences were found between the AD and HC groups. Using 2 standard deviations above the mean of the first three samples, the DLMO in AD occurred significantly later, 21:02?±?00:41?h, than in HC, 20:44?±?00:21?h (t?=?-2.4, p?=?.02). Although melatonin in the AD group appears to have a slower rate of rise, using well-established criteria to assess the salivary DLMO did not reveal differences between AD and HC participants. Only when capturing melatonin when it is already rising was DLMO found to be significantly delayed by a mean 18?min in AD participants. Future circadian analyses on alcoholics should account for these methodological caveats.     

Circadian variations in the responsiveness of human gallbladder to sulfated mineral water.

It is well known that the intake of sulfate-containing natural mineral waters leads to contraction of the gallbladder, probably induced by the release of cholecystokinin (CCK). As early as 1959, there were some hints in the literature of circadian variations in gallbladder response; to find out whether this applies with sulfate as a stimulus, a pretest for basic information about gallbladder reaction to sulfate-containing mineral water was carried out on 19 healthy volunteers. On this basis, 15 healthy subjects of both sexes were then studied. After 6h of fasting, 500 mL of a sulfate-containing mineral water (2,800 mg SO4(2-)/L) were ingested within 5 min. The size of the gallbladder was registered ultrasonographically before and 15, 30, 60, and 120 min after drinking. The experiments were carried out seven times at different hours of the day for each volunteer. After the intake of the mineral water, the mean gallbladder size decreased significantly, followed by an increase after 60 min (P < .001). Significant circadian spontaneous variation in gallbladder size was detected (acrophase around 09:00; amplitude was 30.0% of daily average, P < .001). The contraction induced by the sulfate-containing water was most marked in the early morning hours and minimal around mid-day; the amplitude of this variation accounting for 29.0% of the daily average (P < .01). In contrast, the postdrinking relaxation was maximal around 18:00 and minimal around 9:00 (amplitude 38.5%. P < .001). These results show that the basal size of the gallbladder and its reaction to stimuli show a marked circadian variation: Whereas contractibility is maximal in the morning, dilatation is stronger in the afternoon.     

Diurnal changes of ERP response to sound stimuli of varying frequency in morning-type and evening-type subjects

In order to study the cognitive function rhythm related to the auditory frequency system for people who prefer to be active in the morning and at night, we conducted an experiment during morning (09:00), evening (17:00) and late-night (01:00) periods. On the basis of a morningness/eveningness questionnaire, six moderately morning-type subjects (M-types) and seven evening-type subjects (E-types) were selected. Diurnal variation of event-related potential (ERP) were assessed under low-frequency (250/500 Hz) and high-frequency (1000/2000 Hz) condition using an oddball task. M-types were tested during the morning (09:00) and evening (17:00) periods, and E-types were tested during the evening (17:00) and midnight (01:00) periods. Subjects were asked to press a button when the target stimulus was detected. We found that the P300 amplitude at 09:00 was significantly greater than that at 17:00 for M-types, was significantly greater at 17:00 than that at 01:00 for E-types. A significant difference of P300 latency and P300 amplitude was observed at 17:00 between M-types and E-types. The P300 amplitude obtained after a low-frequency stimulus was significantly greater than that after a high-frequency stimulus at 09:00 for M-types, and at 01:00 for E-types. These results revealed that stimulus frequency had effects on the diurnal changes of human cognitive function, and circadian typology had a direct effect on the diurnal change of human cognitive function. This study has extended the previous findings of auditory P300 studies on diurnal variations in terms of circadian typology and stimulus parameter.     

Cardiovascular effects of dynorphin A (1–13) in conscious rats and its modulation of morphine bradycardia over time

The short-term cardiovascular effects of dynorphin A (1–13), as well as its effects upon morphine bradycardia were investigated. In unanesthetized, unrestrained rats, intracerebroventricular (ICV) dynorphin A (1–13) injections (10–20 μg) produced a dose-related pressor effect, whereas intravenous (IV) dynorphin A (1–13) (1.0 mg/kg) produced a depressor effect; these responses persisted less than five min. Heart rate was not significantly altered by these doses or routes of administration. Dynorphin A (1–13) also produced behavioral effects in the unanesthetized animals, such as wet dog shakes in response to IV administration and wet dog shakes accompanied by barrel rolling in response to ICV administration. To evaluate the effects of dynorphin A (1–13) pretreatment on the bradycardic response to IV morphine, rats were pretreated with 10 μg dynorphin A (1–13) ICV four, six or eight hours prior to challenge with morphine sulfate (0.1 mg/kg IV). Four hour pretreatment with dynorphin A (1–13) (tested at 14:00 hr) resulted in a potention of morphine bradycardia, with six hours pretreatment (tested at 16:00 hr) no effect was observed, and eight hours following dynorphin A (1–13) pretreatment (tested at 18:00 hr) morphine bradycardia was attenuated. Additionally, the bradycardic response to IV morphine alone became more exaggerated as rats approached their nocturnal activity cycle. These data further establish that dynorphin A (1–13) exerts a potent, long lasting modulatory effect on morphine bradycardia and emphasize the importance of circadian variables in altering the magnitude of cardiovascular responses to opioid agonists.