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Plasma catecholamines in rats: Daily variations in basal levels and increments in response to stress
Physiology& Behavior, Vol. 26, pp. 27-31. PergamonPress and BrainResearch Publ., 1981. Primed in the U.S.A.
Plasma Catecholamines in Rats: Daffy Variations in Basal Levels and Increments in Response to Stress I RICHARD McCARTY 2
Department o f Psychology, University o f Virginia, Charlottesville, VA 22901 RICHARD KVETNANSKY
Institute o f Experimental Endocrinology, Center for Physiological Sciences, Slovak Academy o f Sciences, Bratislava, Czechoslovakia, AND I R W I N J. K O P I N
Laboratory o f Clinical Science National Institute o f Mental Health Bethesda, MD 20205 R e c e i v e d 21 J u l y 1980 McCARTY, R., R. KVETNANSKY AND I. J. KOPIN. Plasma catecholamines in rats: Daily variations in basal levels and increments in response to stress. PHYSIOL. BEHAV. 26(1) 27-31, 1981.---Chronic taft artery catheters were surgically implanted into male Sprague-Dewley rats to allow for repeated sampling of blood. Animals were housed individually and maintained on a controlled photoperind (lights on 0600-1800 hours). In the first experiment, blood samples were obtal_ned 2 days after surgery from animals at 0800, 1600, and 2400 hours and again at 0800 hours on the next day. Plasma levels of the sympathetic transmitter norepinephrine (NE) were higher at 2400 hours compared to all other sampling times. In contrast, plasma levels of the adrenal medullary hormone, epinephrine (EPI), were similar across sampling times. In a second experiment, individual rats were transferred to a shock chamber and after 3 minutes received 10 footshocks (2.0 mA, 0.5 sec duration, every 6 see). Separate groups of rats were stressed in this manner at 0800, 1600, or 2400 hours. Basal and transfer-induced increments in plasma levels of NE and EPI did not vary with time of day. However, footshockinduced increments in plasma levels of EPI were greater at 0800 hours than at 1600 or 2400 hours. Footshock-induced alterations in plasma NE and behavior of rats did not vary with time of day. These results suggest that basal plasma levels of NE are elevated at 2400 hours due to postural changes associated with increased locomotor activity and not to an endogenous circadian rhythm. Furthermore, this effect is consistent within but not across groups of rats. The responsiveness of the adrenal medulla to the intense stress of footshock appears to be greatest in early morning compared to later afternoon and midnight. Plasma catecholamines Norepinephrine Footshock stress Circadian rhythms
Epinephrine
T H E activities of many neural and hormonal systems vary predictably over a 24 hour period (for reviews, see [4, 20, 28]). Several such circadian rhythms have been described for functioning of the sympathetic nervous system and the adrenal medulla. These include daily variations in the following parameters: the concentration of the sympathetic neuro-
Sympathetic nervous system
Adrenal medulla
transmitter, norepinephrine (NE), in the pineal gland and salivary glands [37], the turnover of N E in the pineal gland [5] and heart [23], adrenal and urinary content of epinephrine (EPI) and N E [1, 11, 13], activity of dopamine-~hydroxylase in the adrenal medulla [2], and the vesicular prof'fles of preganglionic nerve fibers innervating adrenal
1Portions of this research were conducted in the Laboratory of Clinical Science, National Institute of Mental Health while R. McCarty was a Research Associate in the Pharmacology-Toxicology Training Program and R. Kvemansky was a Visiting Scientist. Partial support was provided by a grant from the University of Virginia Research Policy Council and by NSF grant SER 76-18457. We thank Mrs. Nargis Cross for her careful secretarial assistance. :Requests for reprints should be addressed to: Dr. Richard McCarty, Department of Psychology, Gilmer Hall, University of Virginia, Charlottesville, VA 22901.
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28
McCARTY, KVETNANSKY AND KOPIN
medullary chromaffin cells [17]. However, few studies have been concerned with more direct measures of circadian variations in sympathetic-adrenal medullary activity. In one such study, Renton and Weil-Malherbe [32] noted that plasma levels of NE and EPI were lower in human subjects during sleep as compared to periods of arousal. Recently, analytical methods have been developed which permit the routine measurement of concentrations of NE and EPI in small volumes of plasma (50-100 #.!) with sensitivities less than 10 pg. Such measurements provide an accurate assessment of the functional state of the sympathetic nervous system and adrenal medulla [19, 22, 29]. In addition, blood samples must be obtained via an indwelling catheter as plasma levels of NE and EPI increase dramatically in response to venous puncture (in humans) or handling and decapitation of animals [6,22]. In the present study, we were interested in two aspects of sympathetic-adrenal medullary function. First, sequential blood samples were obtained from conscious, undisturbed rats over a 24 hour period to determine possible circadian alterations in basal plasma levels of NE and EPI. Next, we exposed rats to inescapable footshock at one of three different times of day to determine possible circadian variations in stesss-induced increments in plasma catecholamines.
sample (0.5 ml), an equal volume of heparinized saline (100 U/ml) was infused slowly into the arterial catheter. Blood samples were centrifuged at 4000 g at 4°C for 10 minutes and the plasma was removed and stored at -20°C. Within 2 weeks, samples of plasma were assayed for content of NE and EPI by a radioenzymatic-thin layer chromatographic method [9, 31, 36]. The sensitivity of the assay (values equal to twice the blank) was less than 4 pg for both amines. A two-tailed t test for paired data was used to evaluate significance of differences between means. RESULTS In blood samples obtained from rats via a chronic tail artery catheter, there is an apparent circadian rhythm in plasma levels of NE but not EPI (refer to Fig. 1). Plasma NE in samples obtained at 2400 hours (mean=485 pg/ml) was approximatiey 50~ higher than the combined mean values for the 3 other sampling times that occurred during the light portion of the light-dark cycle (o<0.05). In contrast, plasma levels of EPI varied only slightly over the entire sampling period, with values ranging from 116-142 pg/ml. DISCUSSION
EXPERIMENT 1 In this experiment, we obtained 4 blood samples from individual rats over a 24 hour period to determine if there was a circadian rhythm in the concentrations of NE and EPI in the circulation. METHOD Adult male Sprague-Dawley rats (300-350 g) were used, Rats were housed in groups of 4-5 per cage and were allowed at least two weeks to acclimate to our laboratory prior to experimentation. The animal room was kept on a 12-hour light-dark photoperiod (light on 0600-1800 hours) at a temperature of 21-23"C. Each rat was anesthetized with pentobarbital (35-40 mg/kg) and a PE50 catheter was placed into the ventral tail artery according to the method of Chiueh and Kopin [6]. Briefly, an incision was made through the tail sheath on the ventral surface and the artery was exposed. The catheter was inserted approximately 2 cm up into the artery and secured with two sutures (size 00). The tubing was then run under the tail sheath to the dorsal side and led subcutaneously under the skin to exit at the back of the neck. A piece of spring wire was placed over the tubing and secured to the rat with adhesive tape. The tubing was filled with heparinized saline (500 u/ml) and the end of the tubing was occluded with a 1 ml disposable syringe. After surgery, rats were housed individually in clear plastic cages (25×25× 15 cm) that contained a water bottle and Purina laboratory chow. The catheter was allowed to hang freely from the top of the cage. The patency of the catheter was maintained with slow infusions of 0.5 ml of heparinized saline in the early morning and later afternoon. Beginning on the second day after surgery, a series of 4 blood samples was collected from each rat. Specific sampling times (_+ 15 rain) were: 2 days post-surgery--0800, 1600 hours and 3 days post-surgery--2400, 0800 hours. Care was taken not to disturb the animals during the entire postoperative period. Immediately after collecting each blood
Our results have demonstrated a pronounced daily variation in plasma levels of NE but not EPI in individually housed laboratory rats. The peak level of plasma NE occurred at 2400 hours and corresponds to a time of increased locomotor activity in the nocturnal laboratory rat [23,33]. However, this apparent circadian rhythm in plasma NE does not appear to be generated by an endogenous mechanism. Rather, the increase in plasma NE at 2400 hours appears to result from an activation of the sympathetic nervous system due to greater levels of locomotor activity. In support of this suggestion, several reports have described increases in plasma NE in response to postural adjustments and physical exercise [3, 22, 35]. Similarly, the 24-hour rhythm in human serum dopamineofl-hydroxylase activity has been reported to result from greater activity during daylight hours [30]. The specific housing conditions and surgical and blood collecting procedures employed in this experiment do not appear to have biased our f'mdings. For example, we have also determined that rats with tail artery catheters that were housed in isolation had significantly higher levels of plasma corticosterone at 1600 and 2400 hours than at 0800 hours. Thus, the typical circadian rhythm in adrenocortical secretory activity [8,14] was maintained under conditions that were similar to those employed in this experiment (McCarty, Kvetnansky, and Kopin, unpublished observations). In addition, the higher plasma levels of NE at 2400 hours did not result from repeated sampling of blood, volume depletion or a decrease in hematocrit as the plasma level of NE at 0800 hours returned to the same range as was determined at 0800 hours on the preceeding day. There is a general lack of agreement between our findings for plasma catecholamines and reports of diurnal variations in plasma and urinary levels of catecholamines. Several reports have described daily variations in plasma and urinary levels of NE and EPI in human subjects, with highest values occurring during times of greatest activity [1, ll, 13. 34]. Although this discrepancy may reflect a species difference in peripheral catecholamine metabolism, it is possible that the mechanisms for removal of catecholamines from the site of
DAILY VARIATIONS IN PLASMA CATECHOLAMINES
29 TABLE
600
1
BASAL PLASMA LEVELS OF NOREPINEPHRINE (NE) AND EPINEPHRINE (EPI) AND INCREMENTS IN RESPONSE TO HANDLING AND FOOTSHOCK AT THREE DIFFERENT TIMES OF DAY SO0
Time (hrs)
Basal
NE (pg/ml)
0800 1600 2400
416 ± 51 364±29 430 ± 58
EPI (pg/ml)
0800
288 ± 26
171 ± 317 1593 ~ 1867[
1600 2400
247 ± 24 208 ± 20
224 ± 60* 120 ± 197
400
at,
! o .r
300
o
AHandling AFootshock 222 ± 51" 306_68" 199 ± 51"
1706 ± 1637 1169± 1657 1562 ± 222? 972 82? 1185 ± 1577
I-
Values are means ± SE for groups of 6--8 rats. *p<0.01, ?p<0.001. Vertical bar denotes significant difference among group means, p<0.05.
0 200
X
I
~k
I
EP,
100
I
)800
I
I
LIGHTS
1800
OUT
2400
I
!
0800
TIME OF DAY
FIG. 1. Basal plasma levels (pg/ml) of norepinephrine (NE) and epinephrine (EFI) in rats over a 24 hour period. Values are means for 11-15 rats per point and vertical bars denote _+ SEM. *p<0.05 compared to values at the 3 other times of day.
release at the nerve terminals or from plasma may also vary over a 24 hour period.
transfer to the shock chamber and immediately after the final footshock. After each blood sample, an equal volume of heparinized saline (100 U/rrd) was infused slowly into the arterial catheter. Blood samples were centrifuged and the plasma was separated and stored at -20°C. As described in Experiment 1, plasma levels of N E and EPI were determined within a 3 week period. The sensitivity of these assays (values equal to twice blank) was less than 9 pg for N E and less than 5 pg for EPI. To assess the possible effects of time of day on behavioral responses to footshock, naive unoperated rats were exposed to inescapable footshock as described above. The grid floor of the shock chamber was divided by lines into four quadrants of equal size. Rats were tested once at 0800, 1600 or 2400 hours (_+30 min) and measures of activity (number of quadrants entered with both forepaws) and the number of rears (both forepaws lifted off the grid floor) were recorded during the 1 min of shock delivery. RESULTS
EXPERIMENT 2 To assess the effects of time of day on the responsiveness of the sympathetic-adrenal medullary system to stressful stimulation, we measured increments in plasma levels of N E and EPI after handling and immediately following 1 minute of intermittent footshock. In separate groups o f rats, we measured behavioral responses to footshock. Different groups of rats were tested at each of the three times of day. METHOD
Adult male Sprasue-Dawley rats (350--400 g) were used. Rats were housed in our laboratory and were surgically prepared with tail artery catheters as described in Experiment 1. Two days after surgery, rats were tested at one o f 3 different times (_+30 min): (a) 0800, (b) 1600, or (c) 2400 hours. F o r each group, basal blood samples were obtained from undisturbed rats immediately prior to testing. Each rat was then transferred to a shock chamber [24] and after 3 minutes a train of 10 scrambled footshocks (2.0 mA, 0.6 sec duration, every 6 sec) was delivered through the grid floor. Blood samples (0.5 ml) were collected beginning 1 minute after
There were no significant differences in basal plasma levels of N E and EPI at 0800, 1600, and 2400 hours when separate groups of rats were sampled at each time (Table 1). Handling and transfer of rats from the home cage to the shock chamber resulted in significant increments in plasma levels of both catecholamines, ranging from 46 to 91% (all p ' s < 0 . 0 1 ) . The transfer-induced increments in plasma N E and EPI were similar across sampling times (v's>0.1). Exposure o f rats to 1 minute o f intermittent footshock resulted in dramatic increments above basal values in plasma N E and EPI, ranging from 320 to 570% (p's<0.001). In addition, there was a significant effect of time of day on footshock-induced increments in plasma levels of EPI, F(2,18)=3.91, p < 0 . 0 5 , with increments at 0800 higher than those at 1600 or 2400 hours. Footshock-induced increments in plasma N E were similar at each of the three sampling times (Table 1). Table 2 summarizes behavioral responses of rats during one minute o f inescapable footshock. Measures of line crossing, F(2,21)--0.9, p > 0 . 5 0 , and rearing, F(2,21)=1.0, p>0.60, did not differ across groups of rats tested at either 0800, 1600 or 2400 hours (Table 2).
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McCARTY, KVETNANSKY AND KOPIN TABLE 2 BEHAVIORAL RESPONSES OF RATS TO INESCAPABLE FOOTSHOCK AT THREE DIFFERENT TIMES OF DAY
Time (hrs)
Line Crossing
Rearing
0800 1600 2400
17.9 -+ 1.0 17.4 _+ 1.4 17.4 _+ 1.6
8.3 ± 0.9 10.1 ± 0.9 9.5 + 1.0
Values are means ± SE for groups of 8 rats.
DISCUSSION
When different groups of rats were utilized for each of the three sampling times within a 24-hour period, we failed to replicate our findings in Experiment 1 of a significant circadian rhythm in basal plasma levels of NE. Thus, the pronounced variations across groups of rats in basal plasma catecholamine levels appear to be sufficient to obscure the pattern of results obtained from sequential sampling of blood from a single group of animals. As we have reported previously [25, 26, 27], handling and transfer of rats from their home cages to the shock chamber evoked significant increments in plasma levels of NE and EPI which did not vary with time of day. Exposure of rats to a train of 10 footshocks delivered over a I minute period was attended by 3--6 fold increments in plasma levels of both catecholamines. In addition, footshock-induced increments in plasma levels of EPI were greater in rats tested at 0800 compared to those tested at 1600 or 2400 hours. Thus, there was a circadian variation in the release of EPI from the adrenal medulla in response to the intense stress of footshock but not to the mild stress of handling and transfer to a novel environment. In a previous report [25], we noted that a significant correlation existed between activity during footshock and increments in plasma NE and EPI for seven different rat strains. However, such a relationship could not explain our observation of a circadian variation of increments in plasma EPI following footshock as measures of activity during footshock were similar across times of day. It is possible that the close proximity of testing (0800 hours) to light onset (0600) may result in a greater disturbance to internal homeostatic controis and precipitate an enhanced discharge of EPI by the adrenal medulla. Many studies that evaluate the peripheral adrenergic responses of rats to stressful stimulation are conducted during the lighted portion of a controlled photoperiod. Our results suggest that some caution should be exercised in testing animals within several hours after light onset as there may be a transient increase in sympathetic-adrenal medullary responsiveness.
GENERAL DISCUSSION Exposure of animals to a variety of physical and psychological stressors is attended by an increase in the secretory activity of several endocrine glands, including the anterior pituitary, the adrenal cortex and the adrenal medulla [15, 19, 21]. As a result of the daily variations in basal plasma levels of several hormones [20,28], some consideration has been
given to evaluating the responsiveness of endocrine tissues to stress at times of day when basal hormone titers vary over a wide range. For example, in laboratory rats and mice mainrained on a standard light-dark photoperiod, plasma glucocorticoid levels are lower between 0600-1200 hours compared to 1800-2400 hours. In addition, the rat adrenal cortex in approximately 2.5 times more responsive to adrenocorticotropin (ACTH) during lights off than lights on [8]. There is some lack of agreement in studies which have examined the effects of time of testing on increments in plasma corticosterone following stressful stimulation. Zimmerman and Critchlow [39] exposed rats to ether vapors or restraint stress in early morning or late afternoon and reported no significant effects of time of day on increments above baseline in plasma levels of corticosterone. In contrast, Dunn and co-workers [10] reported that stress-induced increments in plasma corticosterone were greater when rats were tested in early morning (low basal levels) compared to early evening (high basal levels). It should be noted, however, that the time X Acorticosterone F ratio was not significant when all test times were evaluated. In a further refinement of the approach, Engeland and co-workers [12] reported that the plasma ACTH response of rats to an IP histamine injection was greater in the morning than evening whereas the plasma corticosterone response was somewhat greater in the evening in the same animals. Thus, one can view the adrenocortical response to stressful stimulation as the end result of several factors, including the response of the anterior pituitary via ACTH release, the adrenocortical responsiveness to ACTH, and the kinetics of corticosterone distribution in plasma. In the present experiments, we have observed that basal levels of plasma NE were higher at 2400 hours compared to 0800 and 1600 hours. Basal plasma levels of EPl did not vary over the course of a day. The lack of a consistent daily variation in plasma levels of NE and EPI was somewhat surprising in view of the reports of circadian rhythms in many physiological functions that are influenced, at least in part, by the sympathetic-adrenal medullary activity with a circadian variation in effector responsiveness to NE and EPI. At least two lines of investigation support this view. First, the wellsympathetic-adrenal meduillary activity with a circadian variation in effector responsiveness to NE and EPI. At least two lines of investigation support this vies. First, the welldescribed rhythm in pineal N-acetyl transferase activity, an enzyme involved in melatonin synthesis, is influenced in part by daily variations in the number of B-adrenergic receptor binding sites, with maximal values attained at the onset of darkness [18,38]. Secondly, results of a recent report [16] indicated that barorefiex reactivity and vascular responses to infused NE in human subjects exhibited a circadian rhythm in the absence of a daily variation in supine plasma NE. Taken together, these reports suggest that the responses of effector cells to adrenergic stimulation may vary over the course of a day due to changes in the sensitivity and/or number of adrenergic receptors~ Further, effector changes may well occur in the absence of a rhythm in sympatheticadrenal medullary activity. In summary, we have observed dally variations in basal plasma levels of NE and stress-induced increments in plasma levels of EPI. These circadian rhythms in sympatheticadrenal medullary activity appear to play a minor role in the daily variations in several physiological parameters, including locomotor activity, blood pressure, heart rate and core temperature.
DAILY VARIATIONS IN PLASMA CATECHOLAMINES
31
REFERENCES 1. Akerstedt, T. and J. E. Froberg. Interindividual differences in circadian patterns of catecholamine excretion, body temperature, performance and subjective arousal. Biol. Psychol. 4: 277-292, 1976. 2. Banerji, T. K. and W. B. Quay. Adrenal dopamine-/3hydroxylase activity: 24-hour rhythmicity and evidence for pineal control. Experientia 32: 253--255, 1976. 3. Banister, E. W. and J. Gffffiths. Blood levels of adrenergic amines during exercise. J. appl. Physiol. 33: 674-676, 1972. 4. Block, G. D. and T. L. Page. Circadian pacemakers in the nervous system. A. Rev. Neurosci. I: 19-34, 1978. 5. Brownstein M. and J. Axelrod. Pineal gland: 24-hour rhythm in norepinephrine turnover. Science 184: 163--165, 1974. 6. Chiueh, C. C. and I. J. Kopin. Hyperresponsivity of spontaneously hypertensive rats to indirect measurement of blood pressure. Am. J. Physiol. 234: H-690-H-695, 1978. 7. Convoy, R. T. W. L. and J. N. Mills. Human Circadian Rhythms. London: Churchill, 1970. 8. Dallman, M. F., W. C. Engeland, J. C. Rose, C. W. Wilkinson, J. Shinsako and F. Siedenburg. Nycthemeral rhythm in adrenal responsiveness to ACTH. Am. J. Physiol. 235: R210-R218, 1978. 9. DaPrada, M. and G. Zurcher. Simultaneous radioenzymatic determinations of plasma and tissue adrenaline, noradrenaline and dopamine within the femtomole range. Life Sci. 19:. 1161-1174, 1976. 10. Dunn, J., L. Scheving and P. Millet. Circadian variation in stress evoked increases in plasma corticosterone. Am. J. Physiol. 222: 402--406, 1972. 11. Elmadjian, F., E. T. Lamson and R. Neff. Excretion of adrenalin and noradrenalin in human subjects. J. Endocr. Metab. 16: 222-234, 1956. 12. Engeland, W. C., J. Shinsako, C. M. Winger, J. VernikosDanellis and M. F. Dallman. Circadian patterns of stressinduced ACTH secretion are modified by corticosterone responses. Endocrinology 100: 138-147, 1977. 13. Euler, U. S. yon, S. Hellner-Bjorkman and I. Orwen. Diurnal variations in the excretion of free and conjugated noradrenafine and adrenaline in urine from healthy subjects. Acta physiol scand. 33: Suppl. 118: 10-16, 1955. 14. Guillemin, R., W. E. Dear and R. A. Liebelt. Nycthemeral variations in plasma free coriicosteroid levels of the rat. Proc. Soc. exp. Biol. Med. 101: 394-395, 1959. 15. Hennessy, J. W. and S. Levine. Stress, arousal, and the pituitary-adrenal system: A psycho, endocrine model. Prog. Psychobiol. Physiol. Psychol. g: 133--178, 1979. 16. Hossmann, V., G. A. Fitzgerald and C. T. Dollery. Circadian rhythm of baroreflex reactivity and adrenerglc vascular response. Cardiovasc. Res. 14: 125-129, 1980. 17. Kachi, T., T. K. Banerji and W. B. Quay. Daily rhythmic changes in synaptic vesicle contents of nerve endings on adrenomedullary adrenaline cells, and their modification by pinealectomy and sham operations. Neuroendocrinology 28: 201-211, 1979. 18. Kebabian, J. W., M. Zatz, J. A. Romero and J. Axelrod. Rapid changes in rat pineal ~adrenergic receptor: Alterations in (all) dihydroalprenolol binding and adenylate cyclase. Proc. natn. Acad. Sci. U.S.A. 72: 3735-3739, 1975. 19. Kopin, I. J. Plasma catecholamines: A brief overview. In: Cawcholamines: Basic and Clinical Frontiers, edited by E. Usdin, I. J. Kopin and J. D. Barchas. New York: Pergamon Press, 1979, pp. 897-902. 20. Krieger, D. T. (Editor). Endocrine Rhythms. New York: Raven Press, 1979.
21. Kvetnansky, R., A. Mitro, M. Palkovits, M. Vigas, 1. Albrecht, T. Torda and L. Mikulaj. Effect of stress on catecholamines in individual hypothalamic nuclei and plasma ACTH in rats. In: Cellular and Molecular Bases of Neuroendocrine Processes, edited by E. Endroczi. Budapest: Hungarian Academy of Sciences, 1975, pp. 495-512. 22. Lake, C. R., M. G. Ziegler and I. J. Kopin. Use of plasma norepinephrine for evaluation of sympathetic neuronal function in man. Life Sci. 19: 1673-1686, 1976. 23. Lemmer, B. and K. Wenda. Diurnal rhythms in noradrenaline turnover and motility after reserpine and 6-hydroxydopamine. Pharmac. Biochem. Behav. 10: 361-368, 1979. 24. McCarty, R., C. C. Chiueh and I. J. Kopin. Behavioral and cardiovascular responses of spontaneously hypertensive and normotensive rats to inescapable footshock. Behav. Biol. 22: 405--410, 1978. "25. McCarty, R. and I. J. Kopin. Sympatho-adrenal medullary activity and behavior during exposure to footshock stress: A comparison of seven rat strains. Physiol. Behav. 21: 567-572, 1978. 26. McCarty, R. and I. J. Kopin. Plasma catecholamines and behavior of rats during stress. In: Catecholamines: Basic and Clinical Frontiers, edited by E. Usdin, I. J. Kopin and J. D. Barchas. New York: Pergamon Press, 1979, pp. 1789-1791. 27. McCarty, R. and I. J. Kopin. Stress-induced alterations in plasma catecholamines and behavior of rats: Effects of chlorisondamine and bretylium. Behav. Neural Biol. 27: 249265, 1979. 28. Meier, A. H. Chronoendocrinoiogy of vertebrates. In: Hormonal Correlates of Behavior, Vol. 2, edited by B. E. Eleftheriou and R. L. Sprott. New York: Plenum Press, 1975, pp. 469-549. 29. Micalizzi, E. R. and D. T. Pals. Evaluation of plasma norepinephrine as an index of sympathetic neuron function in the conscious, unrestrained rat. Life Sci. 24: 2071-2076, 1979. 30. Okada, T., T. Fujita, T. Ohta, T. Kato, K. Ikuta and T. Nagatsu. A 24-hr rhythm in human serum dopamine-~ hydroxylase activity. Experientia 30: 605-607, 1974. 31. Peuler, J. and G. A. Johnson. Simultaneous single isotope radio,enzymatic assay of plasma norepinephrine, epinephrine, and dopamine. Life Sci. 21: 625-636, 1977. 32. Renton, G. H. and H. Weil-Malherbe. Adrenaline and noradrenaline in human plasma during natural sleep. J. Physiol. 131: 170-175, 1956. 33. Richter, C. P. Biological Clocks in Medicine and Psychiatry. Springfield, Illinois: Charles C. Thomas, 1965. 34. Turton, M.. D. and T. Deegan. Circadian variations of plasma catecholamine, cortisol and immunureactive insulin concentrations in supine subjects. Clin. Chim. Acta $5: 389-397, 1974. 35. Watson, R. D. S., C. A. Hamilton, D. H. Jones, J. L. Reid, T. J. Stallard and W. A. Littler. Sequential changes in plasma noradrenaline during bicycle exercise. Clin. Sci. 58: 37--43, 1980. 36. Weise, V. K. and I. J. Kopin. Assay of catecholamines in human plasma: study of a single isotope radioenzymatic procedure. Life Sci. 19: 1673--1686, 1976. 37. Wurtman, R. J. and J. Axelrod. A 24 hour rhythm in the content of norepinephrine in the pineal and salivary glands of the rat. Life Sci. $: 665-669, 1966. 38. Zatz, M., J. W. Kebabian, J. A. Romero, R. J. Lefkowitz and J. Azelrod. Pineal/3-adrenerglc receptor: correlation of binding of ~rI-dihydroalprenolol with stimulation of adenylate cyclase. J. Pharmac. exp. Ther. 196: 714-722, 1976. 39. Zimmerman, E. and V. CritcMow. Effects of diurnal variation in plasma corticosterone levels on adrenocortical response to stress. Proc. Sac. exp. Biol. Med. 125: 658-663, 1967.
Plasma Catecholamines in Rats: Daffy Variations in Basal Levels and Increments in Response to Stress I RICHARD McCARTY 2
Department o f Psychology, University o f Virginia, Charlottesville, VA 22901 RICHARD KVETNANSKY
Institute o f Experimental Endocrinology, Center for Physiological Sciences, Slovak Academy o f Sciences, Bratislava, Czechoslovakia, AND I R W I N J. K O P I N
Laboratory o f Clinical Science National Institute o f Mental Health Bethesda, MD 20205 R e c e i v e d 21 J u l y 1980 McCARTY, R., R. KVETNANSKY AND I. J. KOPIN. Plasma catecholamines in rats: Daily variations in basal levels and increments in response to stress. PHYSIOL. BEHAV. 26(1) 27-31, 1981.---Chronic taft artery catheters were surgically implanted into male Sprague-Dewley rats to allow for repeated sampling of blood. Animals were housed individually and maintained on a controlled photoperind (lights on 0600-1800 hours). In the first experiment, blood samples were obtal_ned 2 days after surgery from animals at 0800, 1600, and 2400 hours and again at 0800 hours on the next day. Plasma levels of the sympathetic transmitter norepinephrine (NE) were higher at 2400 hours compared to all other sampling times. In contrast, plasma levels of the adrenal medullary hormone, epinephrine (EPI), were similar across sampling times. In a second experiment, individual rats were transferred to a shock chamber and after 3 minutes received 10 footshocks (2.0 mA, 0.5 sec duration, every 6 see). Separate groups of rats were stressed in this manner at 0800, 1600, or 2400 hours. Basal and transfer-induced increments in plasma levels of NE and EPI did not vary with time of day. However, footshockinduced increments in plasma levels of EPI were greater at 0800 hours than at 1600 or 2400 hours. Footshock-induced alterations in plasma NE and behavior of rats did not vary with time of day. These results suggest that basal plasma levels of NE are elevated at 2400 hours due to postural changes associated with increased locomotor activity and not to an endogenous circadian rhythm. Furthermore, this effect is consistent within but not across groups of rats. The responsiveness of the adrenal medulla to the intense stress of footshock appears to be greatest in early morning compared to later afternoon and midnight. Plasma catecholamines Norepinephrine Footshock stress Circadian rhythms
Epinephrine
T H E activities of many neural and hormonal systems vary predictably over a 24 hour period (for reviews, see [4, 20, 28]). Several such circadian rhythms have been described for functioning of the sympathetic nervous system and the adrenal medulla. These include daily variations in the following parameters: the concentration of the sympathetic neuro-
Sympathetic nervous system
Adrenal medulla
transmitter, norepinephrine (NE), in the pineal gland and salivary glands [37], the turnover of N E in the pineal gland [5] and heart [23], adrenal and urinary content of epinephrine (EPI) and N E [1, 11, 13], activity of dopamine-~hydroxylase in the adrenal medulla [2], and the vesicular prof'fles of preganglionic nerve fibers innervating adrenal
1Portions of this research were conducted in the Laboratory of Clinical Science, National Institute of Mental Health while R. McCarty was a Research Associate in the Pharmacology-Toxicology Training Program and R. Kvemansky was a Visiting Scientist. Partial support was provided by a grant from the University of Virginia Research Policy Council and by NSF grant SER 76-18457. We thank Mrs. Nargis Cross for her careful secretarial assistance. :Requests for reprints should be addressed to: Dr. Richard McCarty, Department of Psychology, Gilmer Hall, University of Virginia, Charlottesville, VA 22901.
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28
McCARTY, KVETNANSKY AND KOPIN
medullary chromaffin cells [17]. However, few studies have been concerned with more direct measures of circadian variations in sympathetic-adrenal medullary activity. In one such study, Renton and Weil-Malherbe [32] noted that plasma levels of NE and EPI were lower in human subjects during sleep as compared to periods of arousal. Recently, analytical methods have been developed which permit the routine measurement of concentrations of NE and EPI in small volumes of plasma (50-100 #.!) with sensitivities less than 10 pg. Such measurements provide an accurate assessment of the functional state of the sympathetic nervous system and adrenal medulla [19, 22, 29]. In addition, blood samples must be obtained via an indwelling catheter as plasma levels of NE and EPI increase dramatically in response to venous puncture (in humans) or handling and decapitation of animals [6,22]. In the present study, we were interested in two aspects of sympathetic-adrenal medullary function. First, sequential blood samples were obtained from conscious, undisturbed rats over a 24 hour period to determine possible circadian alterations in basal plasma levels of NE and EPI. Next, we exposed rats to inescapable footshock at one of three different times of day to determine possible circadian variations in stesss-induced increments in plasma catecholamines.
sample (0.5 ml), an equal volume of heparinized saline (100 U/ml) was infused slowly into the arterial catheter. Blood samples were centrifuged at 4000 g at 4°C for 10 minutes and the plasma was removed and stored at -20°C. Within 2 weeks, samples of plasma were assayed for content of NE and EPI by a radioenzymatic-thin layer chromatographic method [9, 31, 36]. The sensitivity of the assay (values equal to twice the blank) was less than 4 pg for both amines. A two-tailed t test for paired data was used to evaluate significance of differences between means. RESULTS In blood samples obtained from rats via a chronic tail artery catheter, there is an apparent circadian rhythm in plasma levels of NE but not EPI (refer to Fig. 1). Plasma NE in samples obtained at 2400 hours (mean=485 pg/ml) was approximatiey 50~ higher than the combined mean values for the 3 other sampling times that occurred during the light portion of the light-dark cycle (o<0.05). In contrast, plasma levels of EPI varied only slightly over the entire sampling period, with values ranging from 116-142 pg/ml. DISCUSSION
EXPERIMENT 1 In this experiment, we obtained 4 blood samples from individual rats over a 24 hour period to determine if there was a circadian rhythm in the concentrations of NE and EPI in the circulation. METHOD Adult male Sprague-Dawley rats (300-350 g) were used, Rats were housed in groups of 4-5 per cage and were allowed at least two weeks to acclimate to our laboratory prior to experimentation. The animal room was kept on a 12-hour light-dark photoperiod (light on 0600-1800 hours) at a temperature of 21-23"C. Each rat was anesthetized with pentobarbital (35-40 mg/kg) and a PE50 catheter was placed into the ventral tail artery according to the method of Chiueh and Kopin [6]. Briefly, an incision was made through the tail sheath on the ventral surface and the artery was exposed. The catheter was inserted approximately 2 cm up into the artery and secured with two sutures (size 00). The tubing was then run under the tail sheath to the dorsal side and led subcutaneously under the skin to exit at the back of the neck. A piece of spring wire was placed over the tubing and secured to the rat with adhesive tape. The tubing was filled with heparinized saline (500 u/ml) and the end of the tubing was occluded with a 1 ml disposable syringe. After surgery, rats were housed individually in clear plastic cages (25×25× 15 cm) that contained a water bottle and Purina laboratory chow. The catheter was allowed to hang freely from the top of the cage. The patency of the catheter was maintained with slow infusions of 0.5 ml of heparinized saline in the early morning and later afternoon. Beginning on the second day after surgery, a series of 4 blood samples was collected from each rat. Specific sampling times (_+ 15 rain) were: 2 days post-surgery--0800, 1600 hours and 3 days post-surgery--2400, 0800 hours. Care was taken not to disturb the animals during the entire postoperative period. Immediately after collecting each blood
Our results have demonstrated a pronounced daily variation in plasma levels of NE but not EPI in individually housed laboratory rats. The peak level of plasma NE occurred at 2400 hours and corresponds to a time of increased locomotor activity in the nocturnal laboratory rat [23,33]. However, this apparent circadian rhythm in plasma NE does not appear to be generated by an endogenous mechanism. Rather, the increase in plasma NE at 2400 hours appears to result from an activation of the sympathetic nervous system due to greater levels of locomotor activity. In support of this suggestion, several reports have described increases in plasma NE in response to postural adjustments and physical exercise [3, 22, 35]. Similarly, the 24-hour rhythm in human serum dopamineofl-hydroxylase activity has been reported to result from greater activity during daylight hours [30]. The specific housing conditions and surgical and blood collecting procedures employed in this experiment do not appear to have biased our f'mdings. For example, we have also determined that rats with tail artery catheters that were housed in isolation had significantly higher levels of plasma corticosterone at 1600 and 2400 hours than at 0800 hours. Thus, the typical circadian rhythm in adrenocortical secretory activity [8,14] was maintained under conditions that were similar to those employed in this experiment (McCarty, Kvetnansky, and Kopin, unpublished observations). In addition, the higher plasma levels of NE at 2400 hours did not result from repeated sampling of blood, volume depletion or a decrease in hematocrit as the plasma level of NE at 0800 hours returned to the same range as was determined at 0800 hours on the preceeding day. There is a general lack of agreement between our findings for plasma catecholamines and reports of diurnal variations in plasma and urinary levels of catecholamines. Several reports have described daily variations in plasma and urinary levels of NE and EPI in human subjects, with highest values occurring during times of greatest activity [1, ll, 13. 34]. Although this discrepancy may reflect a species difference in peripheral catecholamine metabolism, it is possible that the mechanisms for removal of catecholamines from the site of
DAILY VARIATIONS IN PLASMA CATECHOLAMINES
29 TABLE
600
1
BASAL PLASMA LEVELS OF NOREPINEPHRINE (NE) AND EPINEPHRINE (EPI) AND INCREMENTS IN RESPONSE TO HANDLING AND FOOTSHOCK AT THREE DIFFERENT TIMES OF DAY SO0
Time (hrs)
Basal
NE (pg/ml)
0800 1600 2400
416 ± 51 364±29 430 ± 58
EPI (pg/ml)
0800
288 ± 26
171 ± 317 1593 ~ 1867[
1600 2400
247 ± 24 208 ± 20
224 ± 60* 120 ± 197
400
at,
! o .r
300
o
AHandling AFootshock 222 ± 51" 306_68" 199 ± 51"
1706 ± 1637 1169± 1657 1562 ± 222? 972 82? 1185 ± 1577
I-
Values are means ± SE for groups of 6--8 rats. *p<0.01, ?p<0.001. Vertical bar denotes significant difference among group means, p<0.05.
0 200
X
I
~k
I
EP,
100
I
)800
I
I
LIGHTS
1800
OUT
2400
I
!
0800
TIME OF DAY
FIG. 1. Basal plasma levels (pg/ml) of norepinephrine (NE) and epinephrine (EFI) in rats over a 24 hour period. Values are means for 11-15 rats per point and vertical bars denote _+ SEM. *p<0.05 compared to values at the 3 other times of day.
release at the nerve terminals or from plasma may also vary over a 24 hour period.
transfer to the shock chamber and immediately after the final footshock. After each blood sample, an equal volume of heparinized saline (100 U/rrd) was infused slowly into the arterial catheter. Blood samples were centrifuged and the plasma was separated and stored at -20°C. As described in Experiment 1, plasma levels of N E and EPI were determined within a 3 week period. The sensitivity of these assays (values equal to twice blank) was less than 9 pg for N E and less than 5 pg for EPI. To assess the possible effects of time of day on behavioral responses to footshock, naive unoperated rats were exposed to inescapable footshock as described above. The grid floor of the shock chamber was divided by lines into four quadrants of equal size. Rats were tested once at 0800, 1600 or 2400 hours (_+30 min) and measures of activity (number of quadrants entered with both forepaws) and the number of rears (both forepaws lifted off the grid floor) were recorded during the 1 min of shock delivery. RESULTS
EXPERIMENT 2 To assess the effects of time of day on the responsiveness of the sympathetic-adrenal medullary system to stressful stimulation, we measured increments in plasma levels of N E and EPI after handling and immediately following 1 minute of intermittent footshock. In separate groups o f rats, we measured behavioral responses to footshock. Different groups of rats were tested at each of the three times of day. METHOD
Adult male Sprasue-Dawley rats (350--400 g) were used. Rats were housed in our laboratory and were surgically prepared with tail artery catheters as described in Experiment 1. Two days after surgery, rats were tested at one o f 3 different times (_+30 min): (a) 0800, (b) 1600, or (c) 2400 hours. F o r each group, basal blood samples were obtained from undisturbed rats immediately prior to testing. Each rat was then transferred to a shock chamber [24] and after 3 minutes a train of 10 scrambled footshocks (2.0 mA, 0.6 sec duration, every 6 sec) was delivered through the grid floor. Blood samples (0.5 ml) were collected beginning 1 minute after
There were no significant differences in basal plasma levels of N E and EPI at 0800, 1600, and 2400 hours when separate groups of rats were sampled at each time (Table 1). Handling and transfer of rats from the home cage to the shock chamber resulted in significant increments in plasma levels of both catecholamines, ranging from 46 to 91% (all p ' s < 0 . 0 1 ) . The transfer-induced increments in plasma N E and EPI were similar across sampling times (v's>0.1). Exposure o f rats to 1 minute o f intermittent footshock resulted in dramatic increments above basal values in plasma N E and EPI, ranging from 320 to 570% (p's<0.001). In addition, there was a significant effect of time of day on footshock-induced increments in plasma levels of EPI, F(2,18)=3.91, p < 0 . 0 5 , with increments at 0800 higher than those at 1600 or 2400 hours. Footshock-induced increments in plasma N E were similar at each of the three sampling times (Table 1). Table 2 summarizes behavioral responses of rats during one minute o f inescapable footshock. Measures of line crossing, F(2,21)--0.9, p > 0 . 5 0 , and rearing, F(2,21)=1.0, p>0.60, did not differ across groups of rats tested at either 0800, 1600 or 2400 hours (Table 2).
30
McCARTY, KVETNANSKY AND KOPIN TABLE 2 BEHAVIORAL RESPONSES OF RATS TO INESCAPABLE FOOTSHOCK AT THREE DIFFERENT TIMES OF DAY
Time (hrs)
Line Crossing
Rearing
0800 1600 2400
17.9 -+ 1.0 17.4 _+ 1.4 17.4 _+ 1.6
8.3 ± 0.9 10.1 ± 0.9 9.5 + 1.0
Values are means ± SE for groups of 8 rats.
DISCUSSION
When different groups of rats were utilized for each of the three sampling times within a 24-hour period, we failed to replicate our findings in Experiment 1 of a significant circadian rhythm in basal plasma levels of NE. Thus, the pronounced variations across groups of rats in basal plasma catecholamine levels appear to be sufficient to obscure the pattern of results obtained from sequential sampling of blood from a single group of animals. As we have reported previously [25, 26, 27], handling and transfer of rats from their home cages to the shock chamber evoked significant increments in plasma levels of NE and EPI which did not vary with time of day. Exposure of rats to a train of 10 footshocks delivered over a I minute period was attended by 3--6 fold increments in plasma levels of both catecholamines. In addition, footshock-induced increments in plasma levels of EPI were greater in rats tested at 0800 compared to those tested at 1600 or 2400 hours. Thus, there was a circadian variation in the release of EPI from the adrenal medulla in response to the intense stress of footshock but not to the mild stress of handling and transfer to a novel environment. In a previous report [25], we noted that a significant correlation existed between activity during footshock and increments in plasma NE and EPI for seven different rat strains. However, such a relationship could not explain our observation of a circadian variation of increments in plasma EPI following footshock as measures of activity during footshock were similar across times of day. It is possible that the close proximity of testing (0800 hours) to light onset (0600) may result in a greater disturbance to internal homeostatic controis and precipitate an enhanced discharge of EPI by the adrenal medulla. Many studies that evaluate the peripheral adrenergic responses of rats to stressful stimulation are conducted during the lighted portion of a controlled photoperiod. Our results suggest that some caution should be exercised in testing animals within several hours after light onset as there may be a transient increase in sympathetic-adrenal medullary responsiveness.
GENERAL DISCUSSION Exposure of animals to a variety of physical and psychological stressors is attended by an increase in the secretory activity of several endocrine glands, including the anterior pituitary, the adrenal cortex and the adrenal medulla [15, 19, 21]. As a result of the daily variations in basal plasma levels of several hormones [20,28], some consideration has been
given to evaluating the responsiveness of endocrine tissues to stress at times of day when basal hormone titers vary over a wide range. For example, in laboratory rats and mice mainrained on a standard light-dark photoperiod, plasma glucocorticoid levels are lower between 0600-1200 hours compared to 1800-2400 hours. In addition, the rat adrenal cortex in approximately 2.5 times more responsive to adrenocorticotropin (ACTH) during lights off than lights on [8]. There is some lack of agreement in studies which have examined the effects of time of testing on increments in plasma corticosterone following stressful stimulation. Zimmerman and Critchlow [39] exposed rats to ether vapors or restraint stress in early morning or late afternoon and reported no significant effects of time of day on increments above baseline in plasma levels of corticosterone. In contrast, Dunn and co-workers [10] reported that stress-induced increments in plasma corticosterone were greater when rats were tested in early morning (low basal levels) compared to early evening (high basal levels). It should be noted, however, that the time X Acorticosterone F ratio was not significant when all test times were evaluated. In a further refinement of the approach, Engeland and co-workers [12] reported that the plasma ACTH response of rats to an IP histamine injection was greater in the morning than evening whereas the plasma corticosterone response was somewhat greater in the evening in the same animals. Thus, one can view the adrenocortical response to stressful stimulation as the end result of several factors, including the response of the anterior pituitary via ACTH release, the adrenocortical responsiveness to ACTH, and the kinetics of corticosterone distribution in plasma. In the present experiments, we have observed that basal levels of plasma NE were higher at 2400 hours compared to 0800 and 1600 hours. Basal plasma levels of EPl did not vary over the course of a day. The lack of a consistent daily variation in plasma levels of NE and EPI was somewhat surprising in view of the reports of circadian rhythms in many physiological functions that are influenced, at least in part, by the sympathetic-adrenal medullary activity with a circadian variation in effector responsiveness to NE and EPI. At least two lines of investigation support this view. First, the wellsympathetic-adrenal meduillary activity with a circadian variation in effector responsiveness to NE and EPI. At least two lines of investigation support this vies. First, the welldescribed rhythm in pineal N-acetyl transferase activity, an enzyme involved in melatonin synthesis, is influenced in part by daily variations in the number of B-adrenergic receptor binding sites, with maximal values attained at the onset of darkness [18,38]. Secondly, results of a recent report [16] indicated that barorefiex reactivity and vascular responses to infused NE in human subjects exhibited a circadian rhythm in the absence of a daily variation in supine plasma NE. Taken together, these reports suggest that the responses of effector cells to adrenergic stimulation may vary over the course of a day due to changes in the sensitivity and/or number of adrenergic receptors~ Further, effector changes may well occur in the absence of a rhythm in sympatheticadrenal medullary activity. In summary, we have observed dally variations in basal plasma levels of NE and stress-induced increments in plasma levels of EPI. These circadian rhythms in sympatheticadrenal medullary activity appear to play a minor role in the daily variations in several physiological parameters, including locomotor activity, blood pressure, heart rate and core temperature.
DAILY VARIATIONS IN PLASMA CATECHOLAMINES
31
REFERENCES 1. Akerstedt, T. and J. E. Froberg. Interindividual differences in circadian patterns of catecholamine excretion, body temperature, performance and subjective arousal. Biol. Psychol. 4: 277-292, 1976. 2. Banerji, T. K. and W. B. Quay. Adrenal dopamine-/3hydroxylase activity: 24-hour rhythmicity and evidence for pineal control. Experientia 32: 253--255, 1976. 3. Banister, E. W. and J. Gffffiths. Blood levels of adrenergic amines during exercise. J. appl. Physiol. 33: 674-676, 1972. 4. Block, G. D. and T. L. Page. Circadian pacemakers in the nervous system. A. Rev. Neurosci. I: 19-34, 1978. 5. Brownstein M. and J. Axelrod. Pineal gland: 24-hour rhythm in norepinephrine turnover. Science 184: 163--165, 1974. 6. Chiueh, C. C. and I. J. Kopin. Hyperresponsivity of spontaneously hypertensive rats to indirect measurement of blood pressure. Am. J. Physiol. 234: H-690-H-695, 1978. 7. Convoy, R. T. W. L. and J. N. Mills. Human Circadian Rhythms. London: Churchill, 1970. 8. Dallman, M. F., W. C. Engeland, J. C. Rose, C. W. Wilkinson, J. Shinsako and F. Siedenburg. Nycthemeral rhythm in adrenal responsiveness to ACTH. Am. J. Physiol. 235: R210-R218, 1978. 9. DaPrada, M. and G. Zurcher. Simultaneous radioenzymatic determinations of plasma and tissue adrenaline, noradrenaline and dopamine within the femtomole range. Life Sci. 19:. 1161-1174, 1976. 10. Dunn, J., L. Scheving and P. Millet. Circadian variation in stress evoked increases in plasma corticosterone. Am. J. Physiol. 222: 402--406, 1972. 11. Elmadjian, F., E. T. Lamson and R. Neff. Excretion of adrenalin and noradrenalin in human subjects. J. Endocr. Metab. 16: 222-234, 1956. 12. Engeland, W. C., J. Shinsako, C. M. Winger, J. VernikosDanellis and M. F. Dallman. Circadian patterns of stressinduced ACTH secretion are modified by corticosterone responses. Endocrinology 100: 138-147, 1977. 13. Euler, U. S. yon, S. Hellner-Bjorkman and I. Orwen. Diurnal variations in the excretion of free and conjugated noradrenafine and adrenaline in urine from healthy subjects. Acta physiol scand. 33: Suppl. 118: 10-16, 1955. 14. Guillemin, R., W. E. Dear and R. A. Liebelt. Nycthemeral variations in plasma free coriicosteroid levels of the rat. Proc. Soc. exp. Biol. Med. 101: 394-395, 1959. 15. Hennessy, J. W. and S. Levine. Stress, arousal, and the pituitary-adrenal system: A psycho, endocrine model. Prog. Psychobiol. Physiol. Psychol. g: 133--178, 1979. 16. Hossmann, V., G. A. Fitzgerald and C. T. Dollery. Circadian rhythm of baroreflex reactivity and adrenerglc vascular response. Cardiovasc. Res. 14: 125-129, 1980. 17. Kachi, T., T. K. Banerji and W. B. Quay. Daily rhythmic changes in synaptic vesicle contents of nerve endings on adrenomedullary adrenaline cells, and their modification by pinealectomy and sham operations. Neuroendocrinology 28: 201-211, 1979. 18. Kebabian, J. W., M. Zatz, J. A. Romero and J. Axelrod. Rapid changes in rat pineal ~adrenergic receptor: Alterations in (all) dihydroalprenolol binding and adenylate cyclase. Proc. natn. Acad. Sci. U.S.A. 72: 3735-3739, 1975. 19. Kopin, I. J. Plasma catecholamines: A brief overview. In: Cawcholamines: Basic and Clinical Frontiers, edited by E. Usdin, I. J. Kopin and J. D. Barchas. New York: Pergamon Press, 1979, pp. 897-902. 20. Krieger, D. T. (Editor). Endocrine Rhythms. New York: Raven Press, 1979.
21. Kvetnansky, R., A. Mitro, M. Palkovits, M. Vigas, 1. Albrecht, T. Torda and L. Mikulaj. Effect of stress on catecholamines in individual hypothalamic nuclei and plasma ACTH in rats. In: Cellular and Molecular Bases of Neuroendocrine Processes, edited by E. Endroczi. Budapest: Hungarian Academy of Sciences, 1975, pp. 495-512. 22. Lake, C. R., M. G. Ziegler and I. J. Kopin. Use of plasma norepinephrine for evaluation of sympathetic neuronal function in man. Life Sci. 19: 1673-1686, 1976. 23. Lemmer, B. and K. Wenda. Diurnal rhythms in noradrenaline turnover and motility after reserpine and 6-hydroxydopamine. Pharmac. Biochem. Behav. 10: 361-368, 1979. 24. McCarty, R., C. C. Chiueh and I. J. Kopin. Behavioral and cardiovascular responses of spontaneously hypertensive and normotensive rats to inescapable footshock. Behav. Biol. 22: 405--410, 1978. "25. McCarty, R. and I. J. Kopin. Sympatho-adrenal medullary activity and behavior during exposure to footshock stress: A comparison of seven rat strains. Physiol. Behav. 21: 567-572, 1978. 26. McCarty, R. and I. J. Kopin. Plasma catecholamines and behavior of rats during stress. In: Catecholamines: Basic and Clinical Frontiers, edited by E. Usdin, I. J. Kopin and J. D. Barchas. New York: Pergamon Press, 1979, pp. 1789-1791. 27. McCarty, R. and I. J. Kopin. Stress-induced alterations in plasma catecholamines and behavior of rats: Effects of chlorisondamine and bretylium. Behav. Neural Biol. 27: 249265, 1979. 28. Meier, A. H. Chronoendocrinoiogy of vertebrates. In: Hormonal Correlates of Behavior, Vol. 2, edited by B. E. Eleftheriou and R. L. Sprott. New York: Plenum Press, 1975, pp. 469-549. 29. Micalizzi, E. R. and D. T. Pals. Evaluation of plasma norepinephrine as an index of sympathetic neuron function in the conscious, unrestrained rat. Life Sci. 24: 2071-2076, 1979. 30. Okada, T., T. Fujita, T. Ohta, T. Kato, K. Ikuta and T. Nagatsu. A 24-hr rhythm in human serum dopamine-~ hydroxylase activity. Experientia 30: 605-607, 1974. 31. Peuler, J. and G. A. Johnson. Simultaneous single isotope radio,enzymatic assay of plasma norepinephrine, epinephrine, and dopamine. Life Sci. 21: 625-636, 1977. 32. Renton, G. H. and H. Weil-Malherbe. Adrenaline and noradrenaline in human plasma during natural sleep. J. Physiol. 131: 170-175, 1956. 33. Richter, C. P. Biological Clocks in Medicine and Psychiatry. Springfield, Illinois: Charles C. Thomas, 1965. 34. Turton, M.. D. and T. Deegan. Circadian variations of plasma catecholamine, cortisol and immunureactive insulin concentrations in supine subjects. Clin. Chim. Acta $5: 389-397, 1974. 35. Watson, R. D. S., C. A. Hamilton, D. H. Jones, J. L. Reid, T. J. Stallard and W. A. Littler. Sequential changes in plasma noradrenaline during bicycle exercise. Clin. Sci. 58: 37--43, 1980. 36. Weise, V. K. and I. J. Kopin. Assay of catecholamines in human plasma: study of a single isotope radioenzymatic procedure. Life Sci. 19: 1673--1686, 1976. 37. Wurtman, R. J. and J. Axelrod. A 24 hour rhythm in the content of norepinephrine in the pineal and salivary glands of the rat. Life Sci. $: 665-669, 1966. 38. Zatz, M., J. W. Kebabian, J. A. Romero, R. J. Lefkowitz and J. Azelrod. Pineal/3-adrenerglc receptor: correlation of binding of ~rI-dihydroalprenolol with stimulation of adenylate cyclase. J. Pharmac. exp. Ther. 196: 714-722, 1976. 39. Zimmerman, E. and V. CritcMow. Effects of diurnal variation in plasma corticosterone levels on adrenocortical response to stress. Proc. Sac. exp. Biol. Med. 125: 658-663, 1967.