J. comp. Physiol. 86, 59--63 (1973) �9 by Springer-Verlag 1973

Circadian Effects of Hydrocortisone on the Blood

of the Newt, Notophthalmus viridescens*

Charlene l~eed and Mir iam F. Benne t t

Department of Biology, Sweet Briar College, Sweet Briar, Virginia

Received June 13, 1973

Summary. Differential counts of the leucocytes of newts, Notophthalmus viridescens, were made at four times of day (2 : 00, 9: 00, 14: 00 and 21 : 00), 72 hours after the injection of hydrocortisone acetate (expcrimentals) or distilled water (controls). At all times, increases in neutrophils and decreases in lymphocytes were observed in experimentals as compared to the controls (Table 1). The in- creases in neutrophils in the experimental newts were most pronounced at 14:00, and the decreases in the lymphocytes were greatest at 21:00. The least degrees of neutrophilia and lymphopenia occurred at 9:00. Consequently, circadian varia- tions in response to the hydrocortisone are indicated. The possible mechanism of mediation of the variations is discussed.

Introduction

Changes in the d i s t r ibu t ion of leucocytes in the peripherala~blood of m a m m a l s have been repor ted as responses to t r e a t m e n t wi th some hor- mones or exposure to some types of stress (Dal ton and Selye, 1938; Selye, 1950). Comparab le changes in the blood p ic ture of A m p h i b i a were no ted in animals t r ea t ed wi th ACTH or hydrocor t i sone or exposed to osmotic stress. In jec t ions of the adrena l steroid, hydrocor t isone, increased the numbers of ueut rophi l s (neutrophil ia) and decreased the numbers of l ymphocy tes ( lymphopenia) in adu l t and t adpo le Rana catesbeiana and in adu l t Rana pipiens (Bennet t and Alspaugh, 1964; Benne t t and Harbo t t l e , 1968). The urodele, Notophthalmus viridescens, also showed neu t roph i l i a and lymphopen ia af ter t r e a t m e n t wi th the same steroid, hydrocor t i sone (Bennett , Gaudio, Johnson and Spisso, 1972); wi th the cor t icotrophin, ACTH; or af ter immers ion in saline hyperosmot ic to the an imal ' s b o d y fluids (Bennet t and Johnson, 1973). Indeed, these f indings for the Amph ib i a para l le l the earl ier ones r epor ted for l abo ra to ry mammals .

* Supported in part by National Science Foundation grant, GY-7661, to Sweet Briar College.

60 Ch. Reed and ~ . F. Bennett

Cyclic changes in the white b lood cell p ic tures of some ma mma l s have also been observed. F o r example , a da i ly cycle of var ia t ions in the numbers of leucocytes was found in man, and the phases of this r h y t h m were reversed b y revers ing some of the env i ronmenta l factors to which the subjec ts were exposed (Sharp, 1960a, b). Average numbers of leuco- cytes also v a r y th rough the c i rcadian per iod in r abb i t s (Reifenstein, Fe rguson and Weisko t t en , 1941). As opposed to these var ia t ions Observed in the blood of mammals , the newt, Notophthalmus viridescens, showed no obvious da i ly differences in the re la t ive numbers of whi te blood cells when the animals were k e p t under cons tan t l a b o r a t o r y condi t ions (Bennet t , Reed and Roden, unpub l i shed data) .

However , o ther indica tors of c i rcadian organiza t ion have been found in this newt (Bennet t and StMey, 1960) and in closely re la ted species (Hims ted t , 1971), all of which showed cycles of locomotory ac t iv i ty . A n add i t iona l ind ica tor of pe r iod ic i ty in IVotophthalmus is the va r i a t ion in the effects of ACTI-I on the d i s t r ibu t ion of whi te blood cells. The degrees of neu t roph i l i a and l ymphopen ia differed a t four t imes of day, 2 : 00, 9 : 00, 14 : 00 and 21 : 00 (Bennet t , Reed and Roden, unpub l i shed da ta) . The quest ion wi th which the presen t r epor t is concerned was th is : Does Notophthalmus also respond in a va ry ing manne r th rough the c i rcadian per iod when i t is in jec ted wi th the adrena l steroid, hydro- cortisone, a t the same four t imes of d a y a t which i t was t r e a t e d wi th the cor t icot rophin , ACTH ?

Mater ia ls and Methods

Newts, Notophthalmus viride~.cens, of approximately uniform weight (2 g • 1 g) were obtained from a dealer in western Massachusetts during the summer and fall of 1972. In the laboratory, they were kept in battery jars of pond water in constant darkness at 11.0:~ 0.5~ One hundred and forty animals received one 0.5 cc injection of a 0.2 % suspension of hydrocortisone acetate (Nutritional Bioche- micals Corp., Cleveland). The same number of control animals received injections of 0.5 cc of distilled water. The injections were made through the ventrolateral surface of the posterior trunk at four times per day, 2 : 00, 9: 00, 14: 00 and 21 : 00. Seventy-two hours after injection, blood was withdrawn from the tails o5 the experimental and control salamanders. Two slides were made from the blood of each of 35 experimental and of 35 control animals at each of the four times of day. The slides were stained with Wright's blood stain, and differential counts, based on 100 leucocytes, were made for each animal.

Results

The different ia l counts of the leucocytes of newts, Notophthalmu8 viridescens, made 72 hours a f te r t h e y had been t r ea t ed wi th hydro- cor t isone a t 2 :00, 9:00, 14:00 or 21:00 all ind ica ted increases in neutro- phils and decreases in l ymphocy te s (Table 1). However , the percen t

Circadian Variations in Newts 6i

Table 1. The average percent differences between the counts of neutrophils and lym- phocytes for the experimentals (hydrocortisone-injected) and controls (water- injected) at the four times of day. (+indicates an increase in counts for the

experimentals over the controls; -- indicates a decrease.)

Time of day Neutrophils Lymphoeytes

2:00 + 62.4 -- 8.2 9:00 + 30.6 -- 3.4

14:00 + 98.7 -- 4.6 21:00 + 93.2 -- 15.0

differences between the counts of the leucocytes in the experimental and control animals showed tha t the degrees of neutrophilia and lympho- penia varied in a circadian manner (Table 1). The lowest percent dif- ferences between the numbers of comparable cells of the two groups of animals, and therefore, the least degrees of neutrophilia and lympho- penia occurred at 9:00. The highest percent difference in neutrophils was recorded at 14:00. Lymphocytes varied to the greatest extent at 21:00. The increases in neutrophils in the hormone-treated newts were statistically significant (p<0.001) at all the times of day at which they were studied. The decreases in lymphocytes were significant (p <0.001) only at 2:00, 9:00 and 21:00. Changes in the distributions of the rarer leucocytes, monocytes, eosinophils and basophils, were only slight; no daily variations were seen. Therefore, those data have been omitted from this report.

Discussion

The results of the present study of Notophthalmus injected with hydroeortisone are consistent with those reported for stressed and corticoid-treated mammals (Dalton and Selye, 1938; Selye, 1950); steroid-injected frogs (Bennett and Alspaugh, 1964; Bennett and Har- bottle, 1968); osmotically stressed, hydrocortisone- and ACTH-treated newts (Bennett et al., 1972; Bennett and Johnson, 1973). There was an obvious increase in neutrophils and a decrease in lymphocytes in the experimental Notophthalmus as compared to the control animals (Table 1). Moreover, differences in the degrees of neutrophilia and lymphopenia in the newts that received the hydrocortisone during various periods of the day suggest circadian variations in the responses to the hormone.

Sharp (1960a) reported tha t the numbers of the types of leucocytes generally vary diurnally in man. During the waking hours, there was an increase in the numbers of neutrophils and a decrease in the numbers of lymphoeytes in the peripheral blood of his subjects. The daily cycle

62 Ch. Reed and M. F. Bennett

of changes in the number of leucocytes in man could be reversed by the reversal of the activity-sleep pattern of the subjects, or by the reversal of the lighting conditions to which the subjects were exposed (Sharp, 1960a, b). Another mammal, the rabbit, showed a decrease in the number of lymphoeytes during the day, while the number of neutrophils increased during the same period (Reifenstein, Ferguson and Weiskotten, 1941). But, as was pointed out earlier, Notophthalmus showed no great circadian differences in the relative numbers of lymphocytes and neutrophils when kept under constant laboratory conditions (Bennett, Reed and Roden, unpublished data).

Other indicators of a circadian organization in urodeles have been described by Bennett and Staley (1960) and Himstedt (1971). These investigators established that the maximal locomotory activity of two species of newts (Notophthalmus viridescens and Triturus vulgaris) occurred during the evening and night hours. Now, we report a cyclic response in the blood of Notophthalmus to the adrenal steroid, hydro- cortisone. However, the pattern of this variation was different from that seen after treatment with the eorticotrophin, ACTH. Neutrophilia and lymphopenia in the ACTH-treated newts were very obvious at 2:00, 9:00 and 14:00, while their lowest levels were seen at 21:00 (Bennett, Reed and Roden, unpublished data). We found for hydro- cortisone-treated animals that the highest level of neutrophilia was at 14:00, that for lymphopenia occurred at 21:00 and the lowest levels of neutrophilia and lymphopenia were at 9:00 (Table 1). What charac- teristics of the pathway(s) of hormonal action could explain the varying temporal responses in the blood of Notophthalmus to the two different hormones, ACTH and hydrocortisone ?

The rise in neutrophils and the fall in lymphocytes during the waking hours of man were postulated to be mediated by varying levels of activity of the adrenal cortex (Sharp, 1960 a) which are probably effected, in turn, by variations in titers of endogenous ACTH. As has been emphasized, neutrophilia and lymphopenia are also known to be re- sponses of vertebrate systems to stress or to treatment with exogenous hormones. Is it not probable that in addition, the pituitary-adrenal axis and its hormones are part of the pathway through which circadian cycles in the blood of vertebrates in addition to man (such as the changes in the blood of newts described in this report) are mediated ? The path- way might be: anterior pituitary--~ACTH-~adrenal tissue-+steroids including hydrocortisone--~blood changes. The adrenal tissues could respond differently to injections of ACTH during various phases of the solar day. Consequently, the adrenal might secrete its steroids in varying amounts causing the changes in the distribution of leucocytes to differ in intensity.

Circadian Variations in Newts 63

However , when hydrocor t i sone is injected, as i t was in the invest iga- t ion repor ted here, the anter ior p i t u i t a r y - , A C T H - + a d r e n a l t issue por t ion of the med ia t ing p a t h w a y is by-passed. I n t h a t case, the exo- genous steroid, g iven in the same concent ra t ion a t four t imes of day , p robab ly effects the blood changes di rect ly . Therefore, the c i rcadian differences seen mus t reflect var ia t ions in the responsiveness of react ions which lead to increases in neut rophi ls and decreases in lymphoey tes in the per iphera l blood. F u r t h e r studies are necessary to es tabl ish the va l i d i t y of these suggestions.

References Bennett, M.F. , Alspaugh, J . K . : Some changes in the blood of frogs following

administration of hydrocortisone. Va. J. Sci. 15, 76-79 (1964) Bennett, M. F., Gaudio, C. A., Johnson, A. O., Spisso, J. H.: Changes in the blood

of newts, Notophthalmus viridescens, following the administration of hydro- cortisone. J. comp. Physiol. 80, 233-237 (1972)

Bennett, M.F. , Harbottle, J .A. : The effects of hydrocortisone on the blood of tadpoles and frogs, Rana catesbeiana. Biol. Bull. 135, 92-95 (1968)

Bennett, M.F. , Johnson, A. O.: Osmotic stress, ACTH and the white blood cell picture in newts, Notophthalmu8 viridescens. J. comp. Physiol. 82,333-338 (1973)

Bennett, M. F., Staley, J. : Cycles of motor activity in the newt, Triturus viridescens. Anat. Bec. 187, 339 (1960)

Dalton, A. J., Selye, H. : The blood picture and the blood sugar changes during the Marm reaction. Anat. Rec. 72 (Suppl.), 48-49 (1938)

Himstedt, W. : Die Tagesperiodik yon Salamandriden. Oecologia (Berl.) 8, 194-208 (1971)

Reifenstein, G. H., Ferguson, J. H., Weiskotten, H. G.: Studies on leukocytosis. I. Hourly leukocyte variations of normal rabbits. Amer. J. Path. 17, 219-231 (1941)

Sharp, G .W.G. : Reversal of diurnal leucocyte variations in man. J. Endocr. 21, 107-114 (1960a)

Sharp, G .W.G. : The effect of light on diurnal leucocyte variations. J. Endocr. ~l, 213-218 (1960b)

Selye, H.: In: The physiology and pathology of exposure to stress, p. 1-792. Montreal: Acta, Inc. 1950

Professor Miriam F. Bennett Department of Biology Colby College Waterville, Maine 04901 U.S.A.

Miss Charlene Reed Department of Biological Sciences The Florida State University Tallahassee, Florida 32306 U.S.A.