The Nyctohemeral Rhythm of Plasma Prolactin: Effects of Ganglionectomy, Pinealectomy, Constant Light, Constant Darkness or 6-OH-Dopamine Administration J. S. KIZER, J. A. ZIVIN, D. M. JACOBOWITZ, AND I. J. KOPIN Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20014 ABSTRACT. In male rats maintained on a 12 h light-dark schedule (6 AM-6 PM), there is a nyctohemeral cycle of plasma prolactin which consists of a nadir at 11:30 AM and an apogee at approximately 11:30 PM. In rats exposed to constant darkness, this rhythm persists for 7 days. Seven days of constant light, however, reverses this diurnal variation such that plasma prolactin levels peak at 11:30 AM and reach a nadir at approximately 11:30 PM. In animals maintained on a 12 h light-dark cycle, ganglionectomy and lateral ventricular injections of 6-OH-dopamine (250 /ig) also appear to reverse the diurnal variation of plasma prolactin, whereas a single injection of 6-OH-dopamine (250 fxg) into the third ventricle decreases plasma prolactin values at all time intervals but does not alter the diurnal rhythm. Both sites of 6-OH-dopamine administration markedly deplete hypothalamic dopamine and norepinephrine, but injection of 6-OH-dopamine
into the lateral ventricle destroys the catecholaminergic terminals in the pineal, whereas injection of 6-OH-dopamine into the third ventricle does not. Pinealectomy slightly increases the early morning values of plasma prolactin, but otherwise has no effect on the diurnal variation of prolactin. Five conclusions appear to be justified: 1) there is a nyctohemeral rhythm of plasma prolactin, which is reversed by constant light; 2) the pineal gland probably plays no role in the diurnal regulation of plasma prolactin secretion; 3) the diurnal rhythm of plasma prolactin is controlled by sympathetic input into the brain via the superior cervical ganglion; 4) a rhythm of plasma prolactin develops in constant light which is the exact opposite of the normal diurnal variation; 5) there appears to be a noradrenergic pathway in the hypothalamus or brainstem which stimulates release of prolactin. (Endocrinology 96: 1230, 1975)
are diurnal variations in the X plasma concentrations of corticosterone (1), testosterone (2), growth hormone (3), LH (4) and prolactin (4). In addition, there is evidence that environmental lighting in the form of altered length of a photo periods has a profound influence upon the endocrine system. In rats, constant light induces constant estrus, whereas constant dark induces ovarian atrophy (5,6). Plasma levels of corticosterone are increased in animals exposed to constant light compared to levels in plasma obtained from animals on a diurnal light cycle (7). Plasma prolactin, on the other hand, is decreased in rats exposed to constant light and increased by constant darkness (8). Because one of the major
neurosecretory products of the pineal, melatonin, has been found to influence the hypothalamic-endocrine axis (9), and because the synthesis of this neurohormone by the pineal is light dependent (10,11) much investigative effort has been devoted to the elucidation of the role of the pineal in photo neuroendocrine regulation. Pinealectomy interferes with the regulation of seasonal sexual function in the ferret (12) and golden hamster (13). The pineal also appears to exert long-term inhibitory effects on the gonadal-pituitary axis of the rat (14). In rats, pinealectomy blocks the gonadal atrophy induced by constant darkness (14), and stimulates increases in gonadal weight (14). In addition, pinealectomy has been demonstrated to prevent increases of plasma prolactin in rats maintained in constant darkness (15). On the other hand, there have been relatively few studies of the effects of pinealectomy on the short-term diurnal regulation
Received September 12, 1974. Please direct requests for reprints to: J. S. Kizer, M.D., Laboratory of Clinical Science/NiMH, Building 10, Room 2D-46, Bethesda, Maryland 20014.
1230
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NYCTOHEMERAL RHYTHM OF PLASMA PROLACTIN
of anterior pituitary function. Recently, the nocturnal surge of plasma prolactin in rats was found to be blocked by pinealectomy (16). The authors of this study, however, noted that there are no consistent effects of pinealectomy on plasma levels of other anterior pituitary hormones (16). Furthermore, other workers have demonstrated no effect of pinealectomy on the diurnal variation of plasma testosterone (2). In order to clarify the role of the pineal in the diurnal regulation of plasma prolactin levels, we have examined the diurnal variation of plasma prolactin in constant light, constant darkness, and diurnal light, and after pinealectomy, ganglionectomy and treatment with intraventricular 6OH-dopamine. Materials and Methods Animals Sprague-Dawley, adult male rats weighing 250-300 g were obtained from Zivic-Miller, Allison Park, Pa. Animals were housed 5 per cage (2 sq. ft.) and given free access to water and standard lab chow. Lighting was provided by 16 fluorescent lights (Westinghouse, 40W, cool white) giving approximately 70-80 footcandles of illumination at cage level. Experiments in constant light and constant darkness were performed such that the only environmental cue provided was the changing of food, water and litter every other day. All animals were acclimatized to ambient lighting (12 h light-dark cycles, 6-6) and temperature (23 C) for 7-8 days before surgery, injection of 6-OH-dopamine or exposure to altered light regimens. In all experiments, exposure to altered lighting schedules continued for 8 days. Bilateral superior cervical ganglionectomy was performed under halothane anesthesia. Sham operations consisted of dissecting the carotid sheath free, leaving the ganglia intact. Pinealectomies were performed by Zivic-Miller Laboratories 7 days after exposure to the specified environmental lighting schedule. Sham pinealectomy consisted of opening the skull over the pineal recess but leaving the pineal intact. Sham-sham pinealectomized animals merely received a cut on the head. The pinealectomies were confirmed at autopsy.
1231
After the initial acclimatization period and/or surgery, the animals were exposed to the appropriate lighting regimen for 7 days. On the 8th day, at 5:30 AM (one-half hour before the usual onset of the light period) 11:30 AM, 5:30 PM (one-half hour before the usual onset of the dark period) and 11:30 PM, animals were gently removed from their cages and quickly killed by decapitation. Killing of animals during dark periods was performed with no light other than two dark red 25 watt bulbs. Total elapsed time from initial handling of each (5 animals per cage) until the last animal in each cage was killed was less than 2 min. Previous work (17) and our own observations had demonstrated that rats treated with 6OH-dopamine failed to eat or drink for the first 2-4 days after injection and lost approximately 60-70 g of body weight before resuming an adequate food intake. To control for this nutritional change, animals were starved without food or water for 3 days, and then given free access to food and water for 4 days prior to sacrifice. Injection of 6-OH-dopamine 6-OH-Dopamine (Regis Chemical Co., Chicago, 111.) was dissolved in a solution of ascorbic acid (1 mg/ml) in saline. Final concentration of 6-OH-dopamine was 250 /xg/20 ^tl for injection into the lateral ventricle and 250 ju.g/2.5 fjd for injection into the third ventricle. All injections were performed acutely using stereotactic placement of the injection cannula (David Kopf Instruments, Tujunga, Calif.). After 7 days acclimatization to the normal diurnal lighting schedule (12 h light-dark), animals received either 20 /JL\ (250 /xg 6-OH-dopamine) into the lateral ventricle on 2 successive days or a single 2.5 (A (250 /Ag/6-OH-dopamine) injection into the third ventricle. Control rats received the vehicle solutions. After recovery from anesthesia, animals were transferred to the appropriate lighting regimen for 7 days. Injection of haloperidol Haloperidol was dissolved in 0.02N HC1 containing 2% ethanol to a final concentration of 500 /u,g/ml. Animals received either haloperidol (500 /Ag/kg BW) or the vehicle solution SC 1 h prior to decapitation. Trunk blood was collected in heparinized beakers and the plasma separated by centrifugation.
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1232
KIZER, ZIVIN, JACOBOWITZ AND KOPIN
Assay of prolactin After decapitation, trunk blood was collected in heparinized beakers and the plasma separated by centrifugation. Radioimmunoassay for prolactin was performed using a kit and assay protocol supplied by the NIAMD and results expressed in terms of NIAMD rat prolactin RP-J-1 (Biological Activity 30 IU/mg-crop sac assay). Dissection of hypothalamic nuclei Brains were removed, quickly frozen on dry ice, and serial 300 /xM coronal sections were cut in a cryostat. The median eminence, medial forebrain bundle, the preoptic area, and the periventricular, paraventricular, arcuate and ventromedial nuclei were removed with small needles according to the method of Palkovits (18,19). Assay of dopamine and norepinephrine The hypothalamic nuclear fragments were homogenized in O.IN perchloric acid and'assayed for dopamine and norepinephrine as described by Coyle and Henry (20). Aliquots of the homogenates were assayed for protein by the method of Lowry et al. (21). Statistical analysis Data was analyzed by two-way analysis of variance (22) and comparison of individual means was then carried c it by use of the Newman-Keuls test (23). Tests of the raw data revealed an inhomogeneity of variance which could be attributed to a direct relation between the magnitude of the mean and the magnitude of the standard deviation. Therefore, a natural log transformation of the data was performed, before analysis, to stabilize the variance (24). Chemicals 6-OH-Dopamine hydrobromide was purchased from Regis Chemicals, Chicago, 111. Haloperidol was a gift of McNeill Laboratories, Pittsburgh, Pa. Histofluorescence
techniques
The rats were decapitated and the brains and pineals quickly removed. Tissue blocks were cut and then frozen in isopentane cooled by liquid nitrogen. They were then placed in a
Endo • 1975 Vol 96 • No 5
freeze-drier, and after 5 days were processed for catecholamine fluorescence microscopy (27). The tissue blocks were exposed to dry paraformaldehyde for 1 h and embedded in paraffin. Sections were cut at 14 JAM and examined under the fluorescence microscope. Results Effect of diurnal light, constant light, and constant darkness on plasma prolactin In the presence of a cyclic pattern of environmental lighting consisting of 12 h of light (6:00 AM-6:00 PM) and 12 h of darkness (6:00 PM-6:00 AM) plasma prolactin levels in the rat demonstrate a diurnal rhythm with a nadir at 11:30 AM and an apogee at 11:30 PM. The difference between highest and lowest values is statistically significant (P < 0.01) and persists in constant darkness (P < 0.01). In animals first exposed to a cyclic light pattern and then to constant light this diurnal variation of plasma prolactin was abolished and was replaced by a rhythm with its nadir at 11:30 PM and its peak at 11:30 AM, the inverse of the normal diurnal rhythm (P < 0.01, Fi = 8.89, d f = 6 x 2 4 0 ) 1 . The difference between the highest and lowest values of plasma prolactin under constant light were also statistically significant (P < 0.01). In addition, levels of plasma prolactin in animals exposed to constant light were significantly higher than levels in animals in diurnal lighting at 11:30 AM (P < 0.01) and significantly lower at 11:30 PM (P < 0.01, Fig. 1). Effect of bilateral superior cervical ganglionectomy on the diurnal variation of plasma prolactin In sham-operated rats exposed to a 12 h cycle light pattern, there was a normal diurnal rhythm of plasma prolactin with a nadir at 11:30 AM and a peak from 5:30 PM to 11:30 PM. After bilateral superior cervi1 Fi = F value for interaction; df = degrees of freedom.
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NYCTOHEMERAL RHYTHM OF PLASMA PROLACTIN 25
25
20
20 -
Ganglionectomy N»22
5
" -
11:30 AM
FIG. 1. Effect of constant light, constant darkness, and diurnal light on the nyctohemeral rhythm of plasma prolactin. Each point in this and other figures represents the mean and standard error of plasma prolactin. N in this and all other figures is the number of animals at each point. Statistical comparisons for all figures are given in the test.
T
——I
I
%^0ARK ^ 5:30 AM
y '
15
to
1233
1 ll:3O AM
5:30 PM
11:30 PM
FIG. 2. Effect of ganglionectomy on the nyctohemeral rhythm of plasma prolactin.
(P
into the lateral ventricle destroys the catecholaminergic terminals in the pineal, whereas injection of 6-OH-dopamine into the third ventricle does not. Pinealectomy slightly increases the early morning values of plasma prolactin, but otherwise has no effect on the diurnal variation of prolactin. Five conclusions appear to be justified: 1) there is a nyctohemeral rhythm of plasma prolactin, which is reversed by constant light; 2) the pineal gland probably plays no role in the diurnal regulation of plasma prolactin secretion; 3) the diurnal rhythm of plasma prolactin is controlled by sympathetic input into the brain via the superior cervical ganglion; 4) a rhythm of plasma prolactin develops in constant light which is the exact opposite of the normal diurnal variation; 5) there appears to be a noradrenergic pathway in the hypothalamus or brainstem which stimulates release of prolactin. (Endocrinology 96: 1230, 1975)
are diurnal variations in the X plasma concentrations of corticosterone (1), testosterone (2), growth hormone (3), LH (4) and prolactin (4). In addition, there is evidence that environmental lighting in the form of altered length of a photo periods has a profound influence upon the endocrine system. In rats, constant light induces constant estrus, whereas constant dark induces ovarian atrophy (5,6). Plasma levels of corticosterone are increased in animals exposed to constant light compared to levels in plasma obtained from animals on a diurnal light cycle (7). Plasma prolactin, on the other hand, is decreased in rats exposed to constant light and increased by constant darkness (8). Because one of the major
neurosecretory products of the pineal, melatonin, has been found to influence the hypothalamic-endocrine axis (9), and because the synthesis of this neurohormone by the pineal is light dependent (10,11) much investigative effort has been devoted to the elucidation of the role of the pineal in photo neuroendocrine regulation. Pinealectomy interferes with the regulation of seasonal sexual function in the ferret (12) and golden hamster (13). The pineal also appears to exert long-term inhibitory effects on the gonadal-pituitary axis of the rat (14). In rats, pinealectomy blocks the gonadal atrophy induced by constant darkness (14), and stimulates increases in gonadal weight (14). In addition, pinealectomy has been demonstrated to prevent increases of plasma prolactin in rats maintained in constant darkness (15). On the other hand, there have been relatively few studies of the effects of pinealectomy on the short-term diurnal regulation
Received September 12, 1974. Please direct requests for reprints to: J. S. Kizer, M.D., Laboratory of Clinical Science/NiMH, Building 10, Room 2D-46, Bethesda, Maryland 20014.
1230
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NYCTOHEMERAL RHYTHM OF PLASMA PROLACTIN
of anterior pituitary function. Recently, the nocturnal surge of plasma prolactin in rats was found to be blocked by pinealectomy (16). The authors of this study, however, noted that there are no consistent effects of pinealectomy on plasma levels of other anterior pituitary hormones (16). Furthermore, other workers have demonstrated no effect of pinealectomy on the diurnal variation of plasma testosterone (2). In order to clarify the role of the pineal in the diurnal regulation of plasma prolactin levels, we have examined the diurnal variation of plasma prolactin in constant light, constant darkness, and diurnal light, and after pinealectomy, ganglionectomy and treatment with intraventricular 6OH-dopamine. Materials and Methods Animals Sprague-Dawley, adult male rats weighing 250-300 g were obtained from Zivic-Miller, Allison Park, Pa. Animals were housed 5 per cage (2 sq. ft.) and given free access to water and standard lab chow. Lighting was provided by 16 fluorescent lights (Westinghouse, 40W, cool white) giving approximately 70-80 footcandles of illumination at cage level. Experiments in constant light and constant darkness were performed such that the only environmental cue provided was the changing of food, water and litter every other day. All animals were acclimatized to ambient lighting (12 h light-dark cycles, 6-6) and temperature (23 C) for 7-8 days before surgery, injection of 6-OH-dopamine or exposure to altered light regimens. In all experiments, exposure to altered lighting schedules continued for 8 days. Bilateral superior cervical ganglionectomy was performed under halothane anesthesia. Sham operations consisted of dissecting the carotid sheath free, leaving the ganglia intact. Pinealectomies were performed by Zivic-Miller Laboratories 7 days after exposure to the specified environmental lighting schedule. Sham pinealectomy consisted of opening the skull over the pineal recess but leaving the pineal intact. Sham-sham pinealectomized animals merely received a cut on the head. The pinealectomies were confirmed at autopsy.
1231
After the initial acclimatization period and/or surgery, the animals were exposed to the appropriate lighting regimen for 7 days. On the 8th day, at 5:30 AM (one-half hour before the usual onset of the light period) 11:30 AM, 5:30 PM (one-half hour before the usual onset of the dark period) and 11:30 PM, animals were gently removed from their cages and quickly killed by decapitation. Killing of animals during dark periods was performed with no light other than two dark red 25 watt bulbs. Total elapsed time from initial handling of each (5 animals per cage) until the last animal in each cage was killed was less than 2 min. Previous work (17) and our own observations had demonstrated that rats treated with 6OH-dopamine failed to eat or drink for the first 2-4 days after injection and lost approximately 60-70 g of body weight before resuming an adequate food intake. To control for this nutritional change, animals were starved without food or water for 3 days, and then given free access to food and water for 4 days prior to sacrifice. Injection of 6-OH-dopamine 6-OH-Dopamine (Regis Chemical Co., Chicago, 111.) was dissolved in a solution of ascorbic acid (1 mg/ml) in saline. Final concentration of 6-OH-dopamine was 250 /xg/20 ^tl for injection into the lateral ventricle and 250 ju.g/2.5 fjd for injection into the third ventricle. All injections were performed acutely using stereotactic placement of the injection cannula (David Kopf Instruments, Tujunga, Calif.). After 7 days acclimatization to the normal diurnal lighting schedule (12 h light-dark), animals received either 20 /JL\ (250 /xg 6-OH-dopamine) into the lateral ventricle on 2 successive days or a single 2.5 (A (250 /Ag/6-OH-dopamine) injection into the third ventricle. Control rats received the vehicle solutions. After recovery from anesthesia, animals were transferred to the appropriate lighting regimen for 7 days. Injection of haloperidol Haloperidol was dissolved in 0.02N HC1 containing 2% ethanol to a final concentration of 500 /u,g/ml. Animals received either haloperidol (500 /Ag/kg BW) or the vehicle solution SC 1 h prior to decapitation. Trunk blood was collected in heparinized beakers and the plasma separated by centrifugation.
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1232
KIZER, ZIVIN, JACOBOWITZ AND KOPIN
Assay of prolactin After decapitation, trunk blood was collected in heparinized beakers and the plasma separated by centrifugation. Radioimmunoassay for prolactin was performed using a kit and assay protocol supplied by the NIAMD and results expressed in terms of NIAMD rat prolactin RP-J-1 (Biological Activity 30 IU/mg-crop sac assay). Dissection of hypothalamic nuclei Brains were removed, quickly frozen on dry ice, and serial 300 /xM coronal sections were cut in a cryostat. The median eminence, medial forebrain bundle, the preoptic area, and the periventricular, paraventricular, arcuate and ventromedial nuclei were removed with small needles according to the method of Palkovits (18,19). Assay of dopamine and norepinephrine The hypothalamic nuclear fragments were homogenized in O.IN perchloric acid and'assayed for dopamine and norepinephrine as described by Coyle and Henry (20). Aliquots of the homogenates were assayed for protein by the method of Lowry et al. (21). Statistical analysis Data was analyzed by two-way analysis of variance (22) and comparison of individual means was then carried c it by use of the Newman-Keuls test (23). Tests of the raw data revealed an inhomogeneity of variance which could be attributed to a direct relation between the magnitude of the mean and the magnitude of the standard deviation. Therefore, a natural log transformation of the data was performed, before analysis, to stabilize the variance (24). Chemicals 6-OH-Dopamine hydrobromide was purchased from Regis Chemicals, Chicago, 111. Haloperidol was a gift of McNeill Laboratories, Pittsburgh, Pa. Histofluorescence
techniques
The rats were decapitated and the brains and pineals quickly removed. Tissue blocks were cut and then frozen in isopentane cooled by liquid nitrogen. They were then placed in a
Endo • 1975 Vol 96 • No 5
freeze-drier, and after 5 days were processed for catecholamine fluorescence microscopy (27). The tissue blocks were exposed to dry paraformaldehyde for 1 h and embedded in paraffin. Sections were cut at 14 JAM and examined under the fluorescence microscope. Results Effect of diurnal light, constant light, and constant darkness on plasma prolactin In the presence of a cyclic pattern of environmental lighting consisting of 12 h of light (6:00 AM-6:00 PM) and 12 h of darkness (6:00 PM-6:00 AM) plasma prolactin levels in the rat demonstrate a diurnal rhythm with a nadir at 11:30 AM and an apogee at 11:30 PM. The difference between highest and lowest values is statistically significant (P < 0.01) and persists in constant darkness (P < 0.01). In animals first exposed to a cyclic light pattern and then to constant light this diurnal variation of plasma prolactin was abolished and was replaced by a rhythm with its nadir at 11:30 PM and its peak at 11:30 AM, the inverse of the normal diurnal rhythm (P < 0.01, Fi = 8.89, d f = 6 x 2 4 0 ) 1 . The difference between the highest and lowest values of plasma prolactin under constant light were also statistically significant (P < 0.01). In addition, levels of plasma prolactin in animals exposed to constant light were significantly higher than levels in animals in diurnal lighting at 11:30 AM (P < 0.01) and significantly lower at 11:30 PM (P < 0.01, Fig. 1). Effect of bilateral superior cervical ganglionectomy on the diurnal variation of plasma prolactin In sham-operated rats exposed to a 12 h cycle light pattern, there was a normal diurnal rhythm of plasma prolactin with a nadir at 11:30 AM and a peak from 5:30 PM to 11:30 PM. After bilateral superior cervi1 Fi = F value for interaction; df = degrees of freedom.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 November 2015. at 20:10 For personal use only. No other uses without permission. . All rights reserved.
NYCTOHEMERAL RHYTHM OF PLASMA PROLACTIN 25
25
20
20 -
Ganglionectomy N»22
5
" -
11:30 AM
FIG. 1. Effect of constant light, constant darkness, and diurnal light on the nyctohemeral rhythm of plasma prolactin. Each point in this and other figures represents the mean and standard error of plasma prolactin. N in this and all other figures is the number of animals at each point. Statistical comparisons for all figures are given in the test.
T
——I
I
%^0ARK ^ 5:30 AM
y '
15
to
1233
1 ll:3O AM
5:30 PM
11:30 PM
FIG. 2. Effect of ganglionectomy on the nyctohemeral rhythm of plasma prolactin.
(P
