Neuropharmacology. 1971,10,315-323
Pergamon Press.Printed inGt.Britain.
POSTNATAL DEVELOPMENT OF A NOREPINEPHRINE RESPONSE TO LIGHT IN THE RAT PINEAL AND SALIVARY GLANDS R. Y. MOORE and RUTH A. SMITH Department of Pediatrics, Anatomy and Medicine (Neurology), and the Joseph P. Kennedy Jr. Mental Retardation Research Center, University of Chicago, Chicago, Illinois 60637 (Accepted 24 September 1970) Summary-Under the control of environmental lighting, the pineal and salivary glands of the rat showed a 24-hr rhythm in norepinephrine content. In animals maintained in diurnal lighting, thenorepinephrine content of both tissues was highest at the end of a dark period and lowest at the end of a light period. When animals were placed in constant darkness, the norepinephrine levels remained high, whereas in constant light, they remained low. At birth, the norepinephrine content of the salivary and pineal glands was low, but adult levels were attained between 6 and 10 days of age. At 6 days, no norepinephrine response to light was evident in either the pineal or salivary glands. Between the eighth and tenth days, constant light levels became significantly lower than constant dark levels in both tissues, demonstrating the development of a response to Iight. The 24-hr rhythm took 10 days to develop in animals kept in diurnal light. The present experiments indicated that visual control of these sympathetic neurons was functional by the tenth day of life in the rat. In the newborn rat, control of the salivary gland norepinephrine response to light could be mediated by extraretinal photoreceptors, but in the adult animal, the eye was essential.
A NUMBERof diurnal rhythms are demonstrable in mammalian organisms (HALBERG, 1969). Among the tissues exhibiting such phenomena, the pineal gland has been a particularly rich source, with rhythms in its content of serotonin (QUAY, 1963), norepinephrine (WURTMAN and AXELROD, 1966), tyrosine hydroxylase (MCGEER and MCGEER, 1966) and hydroxyindole-0-methyltransferase (HIOMT; AXELROD et al., 1965). The pineal HIOMT and norepinephrine rhythms appear to be completely controlled by environmental light (AXELROD et al., 1965; WURTMAN et al., 1967) and, in this regard, differ from the pineal serotonin rhythm and circadian rhythms in other tissues which may become free-running in the absence of the usual visual cues (SNYDER et al., 1965; HALBERG, 1969). The dependence of such rhythms on environmental lighting has led WURTMA~ (1967) to consider them separately from other 24-hr rhythms, for, in the strict sense, they represent only responses to light. To the present time, no rhythm of this type has been observed in a tissue other than the pineal gland, although WURTMAN and AXELROD(1966) have presented preliminary evidence for a similar norepinephrine rhythm in the rat submaxillary gland. In the present study the characteristics of the norepinephrine rhythm of the salivary gland were found to be identical to those of the pineal norepinephrine rhythm, suggesting that this type of response to light is a more general property of tissues innervated by the superior cervical ganglion. In addition, the postnatal development of the pineal and salivary gland norepinephrine responses to light was studied. There is evidence available on the development of the pineal 315 F
316
R. Y. MOORE and RUTHA. SMITH
serotonin rhythm (ZWEIG et al., 1966), the plasma corticosterone rhythm (ALLEN and KENDALL, 1967) and the pineal HIOMT rhythm (KLEIN and LINES, 1969). The pineal serotonin rhythm is apparent at 10 days of age, whereas the plasma corticosterone and pineal HIOMT rhythms are not developed until more than 30 days of age. This discrepancy, and the observation that the pineal serotonin rhythm may be mediated by extraretinal photoreceptors (ZWEIG et al., 1966), suggested that a developmental analysis of the pineal and salivary gland norepinephrine responses to light would contribute further to our understanding of functional maturation in the developing visual system and its relation to the development of sympathetic control mechanisms.
METHODS Environmental
lighting and the rhythm of salivary gland NE
In the first part of the experiment, 24 adult male rats (Holtzman strain), approximately 85 days of age, were placed in diurnal lighting with lights on from 7 a.m. to 7 p.m. Groups of 4 animals were put in clear plastic cages with wire tops (Carworth, Cat. No. 18750). The cages were kept on a rack of metal shelves, 30 cm apart, with a single row of 15 W fluorescent light fixtures mounted on the underside of each shelf. The timing of the lighting in the room and in the rack was controlled by 24-hr timers. Both the room lights and the rack light were provided by daylight fluorescent bulbs. With the lights on, the rats were exposed to 25-50 ft-candles of light, depending upon their exact position in the cages. The room was shielded from external lighting and the temperature was maintained at approximately 24°C. After 7 days in diurnal lighting, the animals were killed by decapitation at 8 a.m., 1 p.m., 7 p.m. and 11.30 p.m. The submaxillary glands were freed from the surrounding tissue, weighed, homogenized in O-4 N perchloric acid and analyzed for norepinephrine content by a modification of the method of ANTON and SAYRE(1962) in which 2 M tris buffer was used to neutralize the perchloric acid extract (NEFF and COSTA, 1966). In a repetition of the experiment, the animals were fasted for 2 days before death to counter the possibility that diurnal variation in food or water intake was responsible for the rhythm. In a second part of the first experiment, another 24 adult male rats were placed in diurnal lighting with a reversed cycle (lights on from 7 p.m. to 7 a.m.). After 7 days on this schedule of diurnal lighting, the animals were each killed at the same time of day as in the previous experiment and their submaxillary glands analyzed for norepinephrine content. A third part of the experiment examined the norepinephrine content of the salivary glands in animals maintained in constant light or darkness. Another 48 adult rats were placed in plastic cages. Half of these were kept in constant light in racks as described above. Both the room and the rack lights were on at all times. The room was entered only for animal care. Twenty-four animals were kept in constant dark in the second of 2 interconnected photographic darkrooms. These rooms were also entered only for animal care. After 7 days in continuous light or darkness, the animals were each killed at the same time of day as the animals kept in diurnal lighting and their submaxillary glands were analyzed for norepinephrine content. The postnatal
development
of the salivary gland NE rhythm
Pregnant female rats (Holtzman strain), at 7 days gestation, were placed in diurnal lighting (lights on from 7 a.m. to 7 p.m.) throughout the experiment. These animals were
Development
of diurnal rhythms of NE in rat pineai and salivary gland
317
housed individually in plastic cages, and were provided with paper strips for nest building. The dates of birth of the litters were noted and the pups were kihed on the first, third, sixth, eighth and tenth postnatal days. For comparison, go-day-old adult male animals were placed in diurnal iighting 7 days before death. On each postnatal day, 6 samples were analyzed from pups killed at 8 a.m. and 6 samples from those killed at 8 p.m. The samples from days 1 and 3 each contained the glands pooled from two animals but, thereafter, each sample contained glands from only one pup. No attempt was made to separate the pups by sex. Each group of samples contained pups from two or more litters. The norepinephrine content of the glands was analyzed by the method of ANTONand SAYRE(I 962), modified as above. Assay of NE levels in salivary glands from newborn rats maintained in constant lighting conditions
Pregnant female rats, again at approximately 7 days gestation, were placed individually in cages and maintained in constant light or darkness throughout the experiment. The dates of parturition were recorded and the pups were killed at 8 a.m. on postnatal days 6 and 9. The submaxillary glands from one animal were used for each sample, and 6 samples were analyzed from the constant light and constant dark groups for each day. Adult animals (85 days of age) were placed in constant light or darkness at the time the pups were born and kitled 7 days later. The norepinephri~e content of the salivary glands from these animals was measured for comparison with that of the newborn animals. Assay of NE levels in pinealglazdsfrom
newborn rats maintained in constant lighting conditions
The animals were killed at 1, 6, 10 and 14 days of age. As in the previous experiment, the newborn animals were born and raised in continuous lighting conditions. A group of adult animals at approximately 85 days of age was divided into two subgroups which were placed in continuous light and in continuous darkness 7 days before death. Four pineals were pooled from the adult rats to make up each of the 4 samples anaIyzed for the effects of constant light and darkness. For the groups of newborn animals, seven to eleven pineals were pooled to make up each of the 4 samples. Assay of the NE response to light in the salivary glands of blinded newborn rats
As in the previous experiments pregnant female animaIs were obtained and housed individually in constant light or darkness. The dates of parturition were noted and on the fourth postnatal day half of the pups in each of several litters were subjected to bilateral orbital enucleation under hypothermia. Before the surgical procedure, the animals were placed in a freezer until they ceased to move in response to stimulation. At this point the eyelids were slit open and the eyes removed. The animals were then warmed under an incandescent light and returned to their mothers when they were warm to touch and moved about normally. Control animals were anesthetized but not operated upon. At 14 days of age, 6 animals from each group (constant light, control; constant light, blinded; constant dark, control; constant dark, blinded) were killed and their salivary glands removed, weighed and analyzed for norepinephrine content as described above. At 22 days of age, 6 animals from each of the blinded groups were killed and their salivary glands analyzed for norepinephrine content. Recovery values were obtained for norepinephrine from both salivary gland and pineal tissue. Recoveries from the salivary gland tissue of newborn and adult rats averaged 29 % (ranging from 27 to 32%). This was lower than that obtained from any tissue but it was
318
R. Y. M~~RE and
RUTH
A. SMITH
consistent throughout the experiment. Brain tissue, run simultaneously with salivary gland tissue, gave a recovery of 71%. Recoveries of norepinephrine from pineai tissue averaged 69 %. In the results below, the salivary gland norepinephrine values are corrected for recovery whereas the pineal values are left uncorrected.
RESULTS
Environmental lighting and the rhythm ofsalivary gland NE
The norepinephrine content of the rat salivary gland exhibits a 24-hr rhythm with the highest levels occurring at 8 a.m. and the lowest at 7 p.m. when the animals are kept in normal diurnal lighting. When the cycle of lighting is reversed, the rhythm is reversed so that the highest content of norepinephrine continues to occur at the end of a dark period and the lowest at the end of a light period. Maintaining the animals in either continuous light or darkness abolishes the diurnal rhythm and salivary gland norepinephrine levels remain low and high, respectively, for the light and dark conditions. The rhythm was the same in fasted animals and in animals that had food available ad lib. (Table 1). TABLE I. EFFECTSOF LIGHTING ON SALIVARY GLAND NOREPINEPHRINE CONTENT OF ADULT RATS
Lighting condition 8 a.m. Normal cycle (lights on 7 a.m. to 7 p.m.) Reversed cycle (lights on 7 p.m. to 7 a.m.) Constant light Constant dark
Norepinephrine content t~g/glS.E.) 1 p.m. 7 p.m.
11.30p.m.
2.33H.26
1~7410~17
I.51 +0.14
I *87&0.33
1~33+0.08 1.46”0,15 2.17&O+22
I .81 *O-20 I .14&O-22 1,91+0.19
2.10+0.22 1~09+0*18 2~04+0*20
1.4730.21 1+18$10*17 Z~lOrtO~14
The difference between the 8 a.m. and 7 p.m, values is statistically significant (P ~0.05; two-tailed t-test) for both the normal and reversed cycles. Differences between norepinephrine levels at different times of day in the constant light and constant dark groups are not significantly different (P r 0.05), although at each point the constant dark value is significantly higher than the constant light value (P <.0.05).
The postnatal development of the salivary gland NE rhythm
At 1 day of age, the salivary gland norepinephrine content was less than 0.5 PgLglg and no evidence of a rhythm was apparent. The levels remained low through the sixth postnatal day. Between the sixth and eighth days there was a sharp rise in norepinephrine content, but the values of assays made at 8 a.m. and 7 p.m. were not significantly different. Between the eighth and tenth days, however, there was cfear evidence of an effect of light and the 7 p.m level dropped significantly below the 8 a.m. level. This was the beginning of the rhythm which was then maintained into the adult period (Fig. 1). NE levels in salivary glands from newborn rats maintained in constant lighting conditions
At 6 days of age, the salivary gland norepinephrine levels were nearly identical for the constant light and constant dark groups, and they were close to the levels shown at 6 days by animals raised in diurnal lighting. On the ninth day the constant fight animals exhibited norepinephrine levels signi~cantly lower than the constant dark animals. These values were within the range we have observed in a number of analyses from adult animals where constant
Development
of diurnal rhythms of NE in rat pineal and sanvary gland
DAYS
AFTER
319
EllRTH
FIG. 1. Postnatal development of a diurnal rhythm in ~orepinep~ine content of the submaxillary gland of the rat. The animals were maintained in diurnal lighting (lights on from 7 a.m. to 7 p.m.) from birth. Norepinephrine levels are shown as the mean of the 8 a.m. (filled circles) and 7 p.m. (open circles) samples for each day. The vertical bars represent the standard error of the mean.
light values have varied from 0.8 to 1a7pg/g, and constant dark values from 1a9 to 2-6 pg/g. If the animals have been in constant light or darkness for more than 6 days, the difference between the two groups has nearly always been significant (Table 2). TABLE 2. SALNARY ADULT
Age
6 days 9 days 85 days
GLAND NOREP~NEPHRINE LEVELS IN NEWBORN RATS KEPT
IN CONSTANT
LIGHT
Lighting condition Constant dark
Constant light
l.OSSrO.16 1+46&0*14 0‘96*0,11
AND
OR DARK
1.16$,0.09 1.95&0.11 2Mf0.36
P
N.S. < 0.05 -=z 0.05
Animals killed at 6 and 9 days of age were kept in constant light or dark from birth. Adult animals were kept in constant light or dark for 7 days. Norepinephrine levels expressed as pg/g of tissue * S.E. P values were obtained with a two-tailed f-test. N.S. refers to P > 0.05.
NE levels inpinealglandrfrom
newborn rats maintainedin constant lighting conditions
At 1 day of age, pineal norepinephrine levels were very low and even pooling 10-12 glands did not give sufficient norepinephrine to reliably exceed blank values. By 6 days, however, norepinephrine levels could be determined and, as with the salivary gland, no difference was noted between the constant light and constant dark groups (Table 3). A significant difference appeared by 10 days when the constant light values fel markedIy in comparison with the (i-day level. Thus, the development of the pineal norepinephrine response to light appeared at the same time as the salivary gland response to light. The constant dark pineal norepinephrine levels at 10 and 14 days of age also appeared to be significantly lower than the adult values, but this was not a real difference. Because of the large numbers and small size of the pineal glands pooled for each sample in this experiment,
320
R. Y.
MGORE
and RUTH A.
SMITH
TABLE3. PINEALNOREPINEPHRINE LEVELSIN NEWBORN AND KEPTINCONSTANT
ADULT RATS
LIGHTORDARK
Lighting condition Constant light
Age
6 days 10 days 14 days 85 days
61ri:.O*2 1*9&0*9
1.3&O-l 2-210.2
Constant dark
P
5910.7 4510.3 4.3 40.1 13.4-co.3
N.S.
Animals killed at 6, 10 and 14 days were kept in constant lighting conditions from birth. Eighty-five-day-old animals were kept in constant light or dark for 7 days. Norepinephrine levels expressed as ng/pineal 3S.E. Pvalues obtained with a two-tailed t test. N.S. refers to values,P z 0.05.
it was not possibIe to weigh them, hence the norepinephrine levels are expressed as ng~pineal. A separate group of pineal gIaads from IO-day-oId animals kept in constant dark were weighed and found to average 0*56&0*03 mg, as compared to 1*72-&0*14mg for a group pineal glands from adult animals (approximately 90 days of age) kept in constant dark. With these weights taken into account, the lo-day pineal has reached adult levels of norepinephrine content. The NE response to light in the salivary glands of blinded newborn rats
Control and blinded animals raised in constant light and darkness showed the same differences in salivary gland norepinephrine content as were noted in the third experiment (Table 4). This indicates that the salivary gland norepinephrine response to light can be maintained by extraretinal photoreceptors in the young rat. The response to light in the blinded animal was lost by 22 days of age since constant light-blinded and constant darkblinded groups did not differ at this point (I *83~tO*14vs. 2.02*0*11 pg norepinephrine per g salivary gland for the 2 groups). TABLE
4.
SALIVARYGLANDNOREPINEPH~INER~PONSETOLIGHTIN~E
Lighting condition Group
Control Blinded
Constant light
Constant dark
P
1.37rto.09 1.58 &O-O8
2,11+0.11
< 0.05
1.97ztO.08
The blinded animals were operated on at 4 days of age and killed at 14 days of age. Salivary gland norepinephrine is expressed as rg/g & SE. The differences between constant light and constant dark values are significant for both groups (P values obtained with a two-tailed t-test). DISCUSSION
A 24-hr rhythm in the norepinephrine content of the pineal and submaxillary glands of the rat was first demonstrated by WURTMAN and AXELROD (1966). Subsequent studies indicated that the pineal norepinephrine rhythm is closely controfled by diurnal
Developmentof diurnal rhythms of NE in rat pineal and salivarygland
321
variations in environmental lighting. Reversal of the usual pattern of light and dark periods reverses the timing of the rhythm whereas blinding, or placing the animals in constant light or dark, abolishes the rhythm and maintains norepinephrine levels at high or low values depending upon the conditions imposed (WURTMANet al., 1967). In the present study the 24-hr rhythm in salivary gland norepinephrine appeared to be under visual control in the same manner as the pineal norepinephrine rhythm. In this, it is like the rhythm in the activity of the pineal melatonin-forming enzyme, HIOMT (WURTMAN et al., 1963; AXELROD et al., 1965). The photic control of these rhythms appears to be mediated by the eyes, the accessory optic component of the central retinal projection, unknown pathways through the brainstem reticular formation and spinal cord, and preganglionic sympathetic fibers innervating the superior cervical ganglion (WURTMANet al., 1963, 1964, 1967; AXELRODet al., 1965; MOOREet al., 1968). From the information available at present, it seems that the effect of light may be to activate central pathways which, in turn, stimulate the cervical sympathetics and thus cause a fall in the norepinephrine content of the salivary glands and pineal. Support for this view is obtained from observations that exposure to light and stimulation of the preganglionic sympathetics have similar effects. Exposure to light produces a decrease in pineal and salivary gland norepinephrine (WURTMAN et al., 1967; present study) and in pineal HIOMT content (AXELROD et al., 1965). Stimulation of the preganglionic sympathetics also produces a fall in salivary gland norepinephrine (FREDHOLMand SEDVALL,1966; SEDVALLet al., 1968) and in pineal HIOMT (BROWNSTEIN and HELLER,1968). After the cessation ofelectrical stimulation, there is a repletion of salivary gland norepinephrine (FREDHOLM and SEDVALL,1966) as occurs with the exposure of animals to darkness. Thus, the diurnal rhythm in salivary gland and pineal norepinephrine can be viewed as reflecting the state of activity of the postganglionic sympathetic neurons as these are influenced by visual input to the central nervous system. In this instance, the mechanism for maintenance of the rhythm is evident, as is that for its loss under constant lighting conditions. Two factors would appear to influence the development of a pineal and salivary gland norepinephrine rhythm in the newborn animal. The first of these is the development of the postganglionic sympathetic innervation and the second is the development of central pathways mediating visual control of the cervical sympathetics. Histochemical studies have shown the appearance of norepinephrine-containing neurons with the parenchyma of the pineal in the second day of life in the rat (MACHADOet al., 1968), and by the tenth day the gland was said to have an adult appearance. In the submaxillary gland (DE CHAMPLAIN et al., 1970), axons were noted in the hilus at birth and throughout the parenchyma by 4 days. By the third week an almost adult pattern of fluorescence was achieved. These observations agree closely with our findings that the norepinephrine content of both tissues is low at birth but rises rapidly to reach adult levels during the second week. A similar pattern of development of salivary gland norepinephrine content was noted by IVERSEN et al. (1967). Electron-microscopic studies of the growth of sympathetic innervation to these tissues have not been reported, but YAMAUCHIand BURNSTOCK(1969) observed a marked increase in innervation in the vas deferens during the first 10 postnatal days. The histochemical and electron-microscopic observations on fluorescence, taken together with the chemical data, indicate that the sympathetic innervation to the pineal and salivary glands reaches a functional state of maturation during the second week of life in the rat, and, thereafter, is under photic control. This could be taken as evidence that the central visual pathways mediating the peripheral response are also functionally mature, but this
322
R. Y. MOORE and RUTHA. SMITH
conclusion would not be entirely justified. Concomitant with the development of the sympathetic innervation to the salivary glands and pineal, the pineal gland develops the capacity to produce serotonin, and normal, adult levels of this amine are also achieved during the second week of life (ZWEIG et al., 1966; HAKANSONet al., 1967; ZWEIG and SNYDER, 1968). A diurnal rhythm in pineal serotonin content appears by 6 days of age (ZWEIG et al., 1966), slightly in advance of the pineal and salivary gland norepinephrine response to light. Photic control of both the pineal serotonin rhythm (ZWEIG et al., 1966) and the salivary gland norepinephrine content (present study) can be mediated in the early newborn period by an extraretinal photoreceptor. Recent evidence (WETTERBERG et al., 1970) suggests that the receptors mediating light-induced changes in the pineal serotonin rhythm are present in the Harderian glands but it is not clear whether these affect the pineal, and presumably the salivary glands, via the central nervous system or through some other mechanism. It is evident, however, that the duration of useful function of such receptors is brief since the eyes become essential to maintain changes both in the pineal serotonin rhythm and in salivary gland norepinephrine content by approximately 20 days of age (ZWEIGet al., 1966; present study). At this point it would appear that both the central pathways and peripheral sympathetic mechanisms are functionally mature. Despite this, the pineal HIOMT rhythm does not become evident until after 30 days of age (KLEIN and LINES, 1969) when the gland is first capable of producing adult levels of enzyme (ZWEIG and SNYDER,1968). There is a similar delay in the development of the plasma corticosterone rhythm until after 30 days of age (ALLENand KENDALL,1967) but, in this case, the adrenal is capable of responding to ACTH and the pituitary-adrenal axis can respond to stress prior to the development of the rhythm. There is no ready explanation for these differences but they do serve to emphasize the complexity of the factors controlling the development of diurnal rhythms and that further study is required for their elucidation. Acknowledgements-We are grateful to Mrs. HAMIDAQAVI, Mr. M. MCILHANY and Drs. JEAN SIMONOWITZ and R. RAPPORTfor their help in carrying out these experiments. The work was supported by research grants NS-O.5002 and HD-04583 from the National Institutes of Health, USPHS.
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