Endocrinology Vol. 139, No. 5 2322-2328
Copyright © 1998 by The Endocrine Society
The Dipsogenic Effects of Rat Relaxin: The Effect of Photoperiod and the Potential Role of Relaxin on Drinking in Pregnancy1
A. J. S. Summerlee,
D. J. Hornsby2 and
D. G. Ramsey
Department of Biomedical Sciences, Ontario Veterinary College,
University of Guelph, Guelph, Ontario N1G 2W1 Canada
Address all correspondence and requests for reprints to: Dr. Alastair J. S. Summerlee, Office of the Dean of Graduate Studies, University Centre, University of Guelph, Guelph N1G 2W1, Ontario, Canada. E-mail: alastair{at}exec.admin.uoguelph.ca
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Abstract
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Experiments were done to examine whether rat relaxin is dipsogenic and
whether such dipsogenic effects of rat relaxin are related to time of
injection during the light-dark cycle. Female rats were fitted with a
chronic intra-cerebro-ventricular (icv) cannula. Rat relaxin (2.5, 5,
10, 25, 50, or 100 ng/2 µl in 0.9% saline) was injected into the
right lateral ventricle at either morning (0800–1000 h), afternoon
(1400–1600 h), or night (2200–2400 h), and water consumption was
measured. Relaxin caused a dose-dependent dipsogenesis at doses 
5 ng, but the sensitivity and magnitude of the response varied with the
photoperiod. Water consumption was smallest (3.5 ± 0.7 ml at 50
ng) and least sensitive (minimal effective dose at 25 ng) in the
afternoon and maximal (17.7 ± 2.3 ml at 50 ng) and most sensitive
(minimal effective dose 5 ng) at night. The latency from injection to
drinking was 55.8 ± 10.4 sec (mean ± SEM) and
did not vary significantly with either the dose or time of day.
A second set of experiments was done to examine the effects of
neutralizing the central actions of relaxin on drinking behavior in
pregnancy. Pregnant rats were injected daily, through a chronically
implanted icv cannula, with either a specific monoclonal antibody
raised against rat relaxin from day 12 to day 22 of gestation or with
saline as a control. Drinking and eating behavior and weight gain were
monitored every 12 h during pregnancy. There was a significant
decrease in water consumed at night, but no effect on drinking during
the day in relaxin-neutralized rats. These animals also showed a
decrease in weight gain during pregnancy compared with controls and
gave birth to lighter-weight litters.
These data provide evidence that the dipsogenic response to exogenous
rat relaxin in female rats varies with time of injection during the
light-dark cycle and suggest that relaxin in the brain may have a role
in nighttime drinking behavior during the second half of pregnancy.
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Introduction
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CIRCULATING relaxin is essential for
successful birth and lactation in rats (1). Endogenous relaxin promotes
growth and softening of the cervix (2) and growth of the vagina (3) to
allow for delivery of the young. Uterine contractions during the second
half of pregnancy until shortly before birth are reduced by the actions
of relaxin (4), protecting fetuses against premature delivery. Finally,
relaxin stimulates development of mammary nipples (5, 6) so the mother
rat can suckle her young successfully.
Relaxin also has actions on the brain (7). Treatment of rats with
exogenous relaxin suppresses reflex milk ejection (8, 9) and causes a
profound pressor response (10, 11, 12, 13). Relaxin affects the release of a
number of hypothalamic and pituitary peptides including oxytocin and
vasopressin (9, 12, 14, 15), LH (16), and PRL (17, 18). More recently
there have been reports that centrally administered relaxin is
dipsogenic (19, 20), although the physiological significance of these
findings has not been proven.
During the second half of pregnancy in rats there are several changes
in cardiovascular control (21, 22): plasma volume expands, blood
pressure falls, plasma osmolality decreases, and glomerular filtration
rate increases. These changes imply that there is a major shift in the
central thresholds for cardiovascular control in pregnancy. To
accommodate these changes, there is a substantial increase in drinking
(22). Using monoclonal antibodies to neutralize the effects of
endogenous relaxin in the periphery, Zhao and colleagues (23)
demonstrated that endogenous circulating relaxin had marginal effects
on drinking during pregnancy: relaxin-neutralized rats consumed less
water during the day compared with intact controls. Recently Omi and
colleagues (24) expanded these observations by showing that peripheral
injection of exogenous relaxin promotes only moderate increases in
water intake during late pregnancy in rats and does not affect drinking
during the night. These authors suggested that the more likely
explanation was that circulating relaxin might enter the cerebrospinal
fluid and have a central action on drinking. However, they suggested
that it was possible that relaxin produced within the brain could have
a local action on drinking behavior. There is evidence that relaxin is
synthesized within the brain (25), and recent data from our laboratory
suggest that passive neutralization of relaxin in the brain may reveal
roles for relaxin that are different from those observed for relaxin in
the systemic circulation (26).
There were two objective for the current experiments: 1) to
investigate the dipsogenic response to rat relaxin at different
times of the light-dark cycle, and 2) to examine the effects of central
administration of monoclonal antibodies specific to rat relaxin on
drinking behavior of pregnant rats.
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Materials and Methods
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All experiments were carried out in accordance with the
guidelines established by the Canadian Council on Animal Care and were
approved by the Animal Care Committee of the University of Guelph.
Animals
Primiparous pregnant and nonpregnant retired-breeders
Sprague-Dawley rats (230–320 g; Harlan Sprague-Dawley, Indianapolis,
IN) were used in these studies. Pregnant animals were bred at
approximately 90 days. The day on which sperm were found in the vagina
was designated as day 1 of pregnancy, and 0300 h on day 1 was
arbitrarily chosen to represent the estimated time of insemination. The
rats were caged individually in the Central Animal Facility of the
University of Guelph. Food and water were available ad
libitum. All rats were maintained on a 12-h light, 12-h dark
lighting regimen with lights on at 0600 h. The ambient room
temperature was 18 C.
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Surgical preparation of the animals
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Rats were fitted with a chronic indwelling microcannula for
injection into the ventricular system of the brain. The details of the
technique for implantation of the microcannula are described elsewhere
(19). Animals were anesthetized with diazepam (Valium, 10 0.8 mg ip;
Hoffman La Roche Ltd., Etobicoke, Ontario, Canada), xylaxine (Rompun, 4
mg ip; Bayer Inc., Agriculture Division, Animal Health, Etobicoke,
Ontario, Canada), and ketamine hydrochloride (Rogarsetic, 4 mg ip;
Rogar STC Inc., London, Ontario, Canada) and a placed in a stereotaxic
frame (Narishige SR6: Tokyo, Japan). Under strict aseptic conditions a
sterilized microcannula (220A; Kopf Instruments, San Francisco, CA) was
placed with its tip in the right lateral cerebral ventricle:
coordinates = bregma, 2.5 mm lateral to the midline and 3.0 mm ventral
to the cortical surface (27). The cannula was sealed in place using
cold-curing acrylic, and the animals were allowed to recover.
Postoperative analgesic [meperidine HCl (Demerol) 2.5 mg every 4
h im] was given for 24 h. As the operations took 2–3 h to
complete, rats were given 15 ml lactated Ringer’s solution (sc) to
ensure adequate hydration but were not given postoperative antibiotic.
The animals were allowed to recover for at least 2 days before
treatment.
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Rat relaxin and monoclonal antibodies to rat relaxin and
fluorescein
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Rat relaxin was a gift from D. G. Porter. Freeze-dried
hormone was stored at 4 C until use when it was reconstituted in 0.9%
sterile saline. Purified monoclonal antibody to rat relaxin and
antibody to fluorescein were produced according to the methodology
described by Lao Guico-Lamm et al. (28). Details of the
purification and characterization of the monoclonals used in the
current study are described elsewhere (26). Purified antibody was
freeze-dried and stored at 4 C until use when it was reconstituted in
PBS at a concentration of 1 mg protein/ml.
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Exp 1: effect of exogenous rat relaxin on drinking behavior in
rats
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Eight nonpregnant rats were fitted with an icv microcannula as
described above. Five days after implantation, a daily routine was
established for testing the dipsogenic effects of rat relaxin. The
dose-dependent effects of rat relaxin on drinking behavior were
established at three time points in the circadian cycle: morning
(0800–1000), afternoon (1400–1600), and evening (2200–2400). Rats
were treated in a randomized, blinded fashion with one dose per day of
either rat relaxin (2.5, 5, 10, 25, 50, 100 ng relaxin in 2 µl 0.9%
saline), angiotensin II (Sigma Chemical Co., St. Louis, MO; 10 ng in 2
µl saline), monoclonal antibody (MCA-3) to rat relaxin (2 µg in 2
µl saline) plus 50 ng relaxin in 2 µl saline, or 2 µl saline
alone. Four repeats of each dose were tested so each animal received a
total of 36 injections (nine different treatments with four repeats of
each treatment).
With the exception of nighttime injections, water-replete rats were
brought into the laboratory at least 30 min before testing and allowed
free access to water. The animals were lifted from the cage, held
lightly, and injected with the test solution through the neoprine seal
of the microcannula using a sterilized Hamilton 10-µl microsyringe
(701N; Hamilton Co., Reno, NV) and replaced in the observation cage.
(The microsyringe needle was cut down to 
5 mm to make injection
through the seal easier). Water consumption was measured from a
graduated pipette attached to the side of a plexiglass observation
cage. The tip of the pipette was modified in the laboratory to allow
rats to drink without water loss from the tip of the pipette. The
latency to the onset of drinking and the volume of water consumed were
recorded every 5 min for 30 min. For the experiments at night
(2200–2400), the animals were injected in the animal house under red
light illumination.
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Exp 2: effect of central injection of monoclonal antibodies to
relaxin on drinking behavior in pregnancy
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The effect of neutralizing endogenous rat relaxin on drinking
behavior during pregnancy in 12 rats was examined. Rats were fitted
with an icv cannula and left for at least 7 days before mating. The
rats were divided, at random, into three experimental groups: group A
rats were injected each morning (between 0900 h and 1000 h)
from day 12 of pregnancy to day 22 with 1 µl monoclonal antibody to
rat relaxin in PBS icv (concentration 1 mg/ml: n = 6); group B
rats were injected with 1 µl PBS alone intra-cerebro-ventricular
(icv) (n = 6); and group C rats were injected with 1 µl
monoclonal antibody to fluorescein in PBS icv (concentration 1 mg/ml:
n = 4). Rats were weighed daily, and water and food consumption
were monitored twice each day at 0600 h and 2000 h from day
12 of gestation to delivery in rats. Water and food consumption were
calculated by weighing the water bottle and food remaining at each
sample period.
Animals were watched continuously from 0400 h on day 20 of
pregnancy. Observations at night were carried out under red light
illumination. The time to the onset of delivery of the first fetus, the
number of young delivered, and the live birth rate were recorded. The
length of time from putative insemination (0300 h on day 1) to the time
of delivery of the first fetus was calculated.
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Analysis of data
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For Exp 1 (dose dependency), the mean values of each group were
compared by one-way ANOVA and Neuman-Kreuls multiple comparison
procedure to test for statistical difference between pairs of means.
Two-way ANOVA was used for comparing the drinking response at different
times of day. Students’ t test was used for comparison of
means in experiments in which independent groups of means were tested.
Significance was determined at the 5% level.
For Exp 2, daily consumption of water and consumption during either the
light or dark phase of the cycle were analyzed by a linear plot model
for relaxin-neutralized and control-treated rats and the days of
pregnancy. The effect of neutralizing relaxin was tested with an F
statistic of the ratio of the variation between the relaxin-neutralized
(group A), PBS-treated (group B), and monoclonal control group (group
C).
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Confirmation of the sterility of the relaxin and monoclonal
antibody samples
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Evidence indicates that cytokine and endotoxin induce the
release of a variety of hypothalamic peptides (29, 30) and affects body
temperature (31) when injected i.c.v; therefore, the sterility of the
samples tested in the current study was examined for the presence of
bacterial growth and endotoxin. Separate batches of the relaxin or
monoclonal antibody to rat relaxin were made up in sterile solutions
and frozen until use. Each batch was used for a maximum of 7 days. At
the end of the experimental period, the samples were tested for
bacterial or endotoxin contamination (Clinical Pathology Lab., Ontario
Veterinary College, Guelph, Ontario, Canada).
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Confirmation of the site of injection
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At the end of the trials, rats were deeply anesthetized with an
overdose of barbiturate ip and 5 µl Indian Ink injected through the
microcannula before the animal stopped breathing. After death, the
brain was removed, and the presence of ink in the fourth ventricle and
the central canal of the spinal cord was taken as proof that the tip of
the microcannula was in the ventricular system.
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Results
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Exp 1: effect of exogenous rat relaxin on drinking behavior in
rats
Rat relaxin icv caused an almost immediate and significant
drinking response in rats at doses greater than 5 ng compared with
saline-treated controls (Fig. 1
). Most of
the drinking (up to 80% of the volume consumed after treatment)
occurred in the first 15 min after treatment (Fig. 2). There was no dose-dependent effect on
the latency to the observed drinking response in any of the animals
tested. During injections in the morning, the maximal response to
relaxin was similar to drinking observed after 5 ng angiotensin II icv.
Treatment of rats with a mixture of monoclonal antibody to rat relaxin
and 50 ng rat relaxin did not show a drinking response (Fig. 1).
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Figure 1. The dose-dependent dipsogenic effects of rat
relaxin icv Mean (± SEM) volume consumed by female rats in
the 30 min after icv injection of either rat relaxin (dose range
2.5–100ng), saline alone, monoclonal antibody to rat relaxin (MCA-3)
plus 50 ng rat relaxin, or 5 ng angiotensin. Experiments were done
between 0800 and 1000 h. Each datum point represents the average
of four trials at each dose in eight animals. Significant
(P < 0.05) increases above baseline (saline alone)
are shown by the asterisks.
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Figure 2. Mean (± SEM) volume of water consumed
by female rats in 5 min time-bins after icv injection of 50 ng rat
relaxin. Experiments were done between 0800 and 1000 h. Each datum
point represents the average of four trials in eight animals. Data for
this figure were taken from experiments shown in Fig. 1 for treatment
with 50 ng relaxin icv
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There were no significant differences detected between the drinking
responses of individual rats at the same dose; therefore, the data were
pooled for each treatment. Exogenous rat relaxin caused a
dose-dependent dipsogenesis in rats; however, the magnitude and
sensitivity of the response varied with the phase of the light-dark
cycle (Fig. 3). The dipsogenic response
was smallest and least sensitive during the afternoon (1400–1600 h)
and maximal and most sensitive at night (2200–2400 h). The latency
from the time of injection to the onset of drinking did not vary
significantly with either the dose of relaxin or time of
injection (Table 1).
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Table 1. The latency from injection of rat relaxin (icv) on
the latency to an induced drinking response in conscious rats
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Exp 2: effect of central injection of monoclonal antibodies to
relaxin on drinking behavior in pregnancy
Water consumption in control rats (PBS-treated: group B)
significantly increased during the second half of pregnancy: daily
intake increased from about 40 ml on day 12 of pregnancy to
approximately 55 ml on days 18–21 of pregnancy. There was no
statistically significant difference in water consumption of the
PBS-treated rats and rats treated with monoclonal antibody to
fluorescein (group C). The increase in daily consumption in both
control groups (groups B and C) was due to a change in the amount of
water consumed at night (Fig. 4). In
contrast, daytime drinking remained reasonably stable throughout
pregnancy (Fig. 4). Injection of monoclonal antibody to relaxin icv
affected drinking in pregnant rats. There was no significant change in
the amount of water consumed per day throughout pregnancy in the
antibody-treated rats, although there was a slight increase in the
water intake at night. This increase was not statistically significant
at any time between day 12 and day 21 of pregnancy. The amount of water
consumed in the relaxin-neutralized rats (group A) is shown in Fig. 4.
There was no significant difference in the daily food consumption of
the relaxin-intact groups (groups B and C) compared with the rats
treated with monoclonal antibody to rat relaxin (group A). The
exception was day 20 when there was a significant fall in food
consumption in the relaxin-deficient rats. Relaxin-intact rats (groups
B and C) showed significantly greater increases in body weight during
pregnancy compared with the relaxin-neutralized group (group A). A
comparison of food intake of PBS-treated (group B) and
relaxin-neutralized animals (group A) and the effects on body weight
are shown in Fig. 5.
Birth in the relaxin antibody-treated group occurred approximately
24 h in advance of the control groups (Table 2).
There was no significant difference in the live birth rate and the
number of young born between the three treatment groups, but pups in
the relaxin-deficient group were significantly lighter in weight than
pups from the control groups.
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Discussion
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The work in this paper concerns the dipsogenic action of rat
relaxin. There were two objectives to the study: 1) to establish
whether or not exogenous rat relaxin has different effects on drinking
behavior at different times in the light-dark cycle, and 2) to
determine whether or not the action of relaxin in the brain affects
drinking during pregnancy. The results show that the dipsogenic
response exogenous rat relaxin varies during the light-dark cycle, the
most powerful effect being seen at night. They also show that
neutralizing the central actions of relaxin prevents the increase in
drinking that occurs, predominantly at night, in the second half of
pregnancy.
There are several lines of evidence to suggest that the
circumventricular organs mediate the central actions of relaxin on
drinking behavior: lesion of the subfornical organ (SFO) blocks the
action of relaxin on blood pressure (32) and on birth (33); there are
relaxin-binding sites in the SFO and organum vasculosum of the lamina
terminalis (OVLT) (34, 35, 36); icv treatment with relaxin is followed by
c-fos expression within the SFO (37), and finally the SFO
and OVLT contain osmoreceptors that regular thirst and vasopressin
secretion (38, 39, 40). Pathways that emanate from the SFO and OVLT are
involved in the forebrain angiotensin system and use angiotensin II as
a neurotransmitter (41). It has been shown that the action of relaxin
on drinking (19), oxytocin and vasopressin release (14), and on LH
release (16) is negated by specific blockade of the action of
angiotensin II, suggesting that the central actions of relaxin may be
mediated through angiotensin. There are, however, no data suggesting
that relaxin acts in the systemic circulation through the
renin-angiotensin system.
Nocturnal animals tend to drink more at night during the active phase
of the light-dark cycle. This was demonstrated by Zhao and colleagues
(23) in rats. Data from our experiments indicate that rats are
differentially sensitive to exogenous rat relaxin at different times of
the light-dark cycle. The underlying cause of this varying response is
not known. The nucleus medianus (another component of the forebrain
angiotensin system) has been shown to be differentially sensitive to
angiotensin-induced dipsogenesis at different times of the light-dark
cycle (42), and recent evidence from our laboratory (43) shows that the
vasopressinemic but not the oxytocinemic response to exogenous relaxin
depends on the nucleus medianus. It is possible, therefore, that the
nucleus medianus is involved in the effects of the light-dark cycle on
the drinking response to exogenous relaxin. This remains to be
determined.
Two laboratories have reported that exogenous porcine relaxin injected
into brain is dipsogenic (19, 20), which could imply a role for relaxin
in increased drinking during pregnancy. However, recent work indicates
that neutralizing endogenous relaxin in the systemic circulation only
moderately affects water intake during pregnancy in rats (23).
Furthermore, this was supported by experiments in which water intake
was monitored in ovariectomized rats given systemic replacement therapy
with and without relaxin during pregnancy (24). In these experiments
relaxin only moderately increased daytime drinking and did not appear
to affect nighttime drinking. Recently, Summerlee and colleagues (26)
demonstrated that neutralizing the action of relaxin within the brain
had different effects on the physiology of birth compared with the
actions of neutralizing relaxin in the systemic circulation (44). These
data suggest that the actions of relaxin in the brain might be separate
and different from its actions in the periphery. The current work shows
that neutralizing the action of relaxin inside the ventricular system
disrupts the normal increase in drinking that occurs in the second half
of pregnancy in rats and implies that relaxin may have a physiological
role in water balance in late pregnancy. During the second half of
pregnancy in rats, there are several cardiovascular changes (21, 22):
plasma volume expands; blood pressure falls; plasma osmolality
decreases; and glomerular filtration rate increases. These changes
imply a major shift in the central thresholds for cardiovascular
control in pregnancy. It is possible that relaxin might be responsible
for resetting these central thresholds (7). However, previous work
reviewing the possibility of a role for relaxin in the blunted response
to vasoconstrictors during gestation in both normotensive and
hypertensive rats suggests that the changed response may be due to an
action of relaxin on blood vessels in the periphery (45).
Original work on the central actions of relaxin on the brain (46, 47)
was based on the premise that high levels of relaxin in the plasma at
the end of pregnancy might ‘spill over’ and affect the central
nervous system. The circumventricular organs could mediate the actions
of systemic relaxin. However, there is a growing body of evidence to
support the concept that relaxin is synthesized and active within the
brain (7), which could be independent from relaxin in the systemic
circulation. For example, relaxin message is localized in several
discrete regions of the rat brain (25, 34), and relaxin-binding sites
have been demonstrated in a variety of sites (34, 35, 36). As the brain is
separated from the systemic circulation by the blood-brain barrier that
only allows passive access to molecules of less than 2 kDa (48), it is
likely that relaxin in the systemic circulation may not reach the
brain. While there are high concentrations of relaxin binding in some
of the circumventricular organs (34, 36) there are other areas where
the blood-brain barrier is intact that express high concentrations of
receptors. However, to date, there have been no reports of successful
relaxin extraction from neural tissue nor any evidence published that
relaxin is secreted from the brain.
There was a difference in the experimental protocol between the daytime
and nighttime injections. Experiments during the day were carried out
in the laboratory while injections at night were done in the animal
house under re-light illumination to avoid the possibility that moving
the animals at night might disrupt the circadian rhythmicity of the
rats. It is possible that this difference of protocol could explain the
different drinking responses seen at night. This is unlikely, however,
as preliminary experiments, in which rats were brought to the
laboratory for nighttime injections (not reported in this paper),
showed that relaxin caused an increased dipsogenesis at night compared
with the responses during the day.
There is no adequate explanation for the results of water consumption,
food intake, and body weight in relaxin-deficient rats on day 20 of
pregnancy (Figs. 4 and 5). In these animals, food consumption
significantly decreased on day 20 of gestation, yet body weight
increased despite no change in water consumption. One possible
explanation for this anomaly could be the fact that the rats in the
relaxin-deficient groups were observed to spend more time on day 20
(the day before birth occurred) nest building and chewing on bedding
compared with rats in control groups. It is possible that
relaxin-deficient rats ate bedding and feces during day 20, which
resulted in no change in body weight, but this needs further
investigation.
Two sets of experimental data support the contention that there is a
separate central and systemic relaxin ‘system’: Hwang and colleagues
(44) used monoclonal antibodies specific to rat relaxin to establish
the role of relaxin in birth. They showed that neutralizing systemic
relaxin disrupted the process but not the onset of birth in rats. In
contrast, Summerlee et al. (26) injected antibody into the
ventricular system of the brain and showed that the timing, but not the
process, of birth was disrupted. The experiments reported in the
current paper are the second line of evidence: injection of monoclonal
antibodies into the ventricular system affects the drinking response in
pregnant rats by blocking the increase in nighttime drinking. There is
no evidence that relaxin has a physiological action(s) during the
estrous cycle, but the possibility that relaxin may affect drinking
behavior in nonpregnant rats deserves further study.
In conclusion, this paper provides evidence that the dipsogenic
response to injection of exogenous rat relaxin into the ventricular
system of the brain varies with the light-dark cycle. Furthermore,
injection of antibodies specific to rat relaxin into the ventricles
disrupts the increase in nighttime drinking that occurs in the second
half of pregnancy in rats, implying a role for relaxin in the brain on
drinking behavior in pregnancy.
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Acknowledgments
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This paper is dedicated to the memory of David G. Porter. Rat
relaxin used in these experiments was inherited from David. The authors
would like to thank O. David Sherwood for inspiring this work and Drs.
Laura Parry and Brian Wilson for their patience, support, and
encouragement.
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Footnotes
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1 This work was supported by Natural Sciences Engineering Research
Council, Canada. 
2 David Hornsby is a student at St. John’s-Kilmarnock School,
Breslau, Ontario, Canada.
Received October 28, 1997.
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Top
Abstract
Introduction
), or at night between 2200 and 2400h (
). Each
datum point represents four trials at each dose in eight animals.
Asterisks indicate where there were significant
(P < 0.05) differences between the responses at
the three times of day. Note that data for the morning injections are
the same information shown in Fig. 1
) or rats treated with monoclonal antibody to rat
relaxin (MCA-3) icv (
) during the 12-h dark period (upper
panel) or the 12-h light period (lower panel).
Significant (P < 0.05) differences are shown by
the asterisks. Note: there was no statistically
significant difference between the group treated with monoclonal to
fluorescein and rats treated with PBS alone, although these data are
not shown in the figure.