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Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7401
Address all correspondence and requests for reprints to: Dr. Lydia A. Arbogast, Department of Physiology, School of Medicine, Southern Illinois University at Carbondale, Carbondale, Illinois 62901-6512. E-mail: larbogast{at}som.siu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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The endogenous opioid peptides have also been implicated in the
suckling-induced PRL rise. The acute suckling-induced PRL rise is
blocked by naloxone (NAL) (11, 12), as well as specific µ and 
opioid receptor antagonists (12). Although it is not known which of the
endogenous opioid peptides contribute to the suckling-induced PRL
release, ß-endorphin (11, 13), as well as specific µ-selective
opioid peptides (14), can acutely increase PRL release in postpartum
rats. NAL (11), as well as µ, , and 
(13) receptor antagonists
can block ß-endorphin-induced PRL release in postpartum rats.
Although it is not clear which of the endogenous opioid systems contribute to the elevated PRL levels in response to the suckling stimulus, there is evidence that opioid gene expression in hypothalamic areas is different during lactation than other endocrine states in female rats. The POMC cellular mRNA signal levels (15) or number of POMC mRNA-containing cells (16) in the arcuate nucleus and periarcuate area were less in lactating rats than in diestrous rats. However, few of the TIDA perikarya receive contacts from ß-endorphin terminals (17, 18), although one cannot rule out interactions at the level of the median eminence. In contrast, proenkephalin mRNA levels in the arcuate nucleus were higher during lactation, compared with diestrous females (19). The induction of enkephalin immunoreactivity in the TIDA neurons during lactation (20) furnishes a way for enkephalin to act as an autocrine regulator of TIDA neurons during lactation. Prodynorphin mRNA levels in the paraventricular nucleus were also higher in lactating than in diestrous rats (21). Indeed, about 70% of the TIDA neurons have contacts with dynorphin terminals (17), and immunoneutralization of dynorphin results in increased TIDA neuronal activity in male rats (22).
It seems that the endogenous opioid peptides exert their action at the
hypothalamic level to alter PRL secretion (23, 24, 25). Although a
nondopaminergic mechanism(s) may also be involved in the opiate
stimulation of PRL release (26, 27), a number of studies indicate that
the endogenous opioid peptides interact with the TIDA neurons. The
endogenous opioid peptides (28, 29, 30, 31, 32), morphine (30, 31, 33, 34) and
specific agonists (35, 36) suppress TIDA neuronal activity. This opioid
peptide-dopamine interaction seems to be physiologically important
during the nocturnal PRL surge in pregnant rats (37). Recently,
Callahan et al. (38) reported that µ-, but not -opioid
receptor antagonists increase DOPA accumulation in the median eminence
of both pup-deprived and pup-suckled dams after a 4–5 h separation
period. The objective of this study was to evaluate whether the opioid
effect on dopaminergic neurons is physiologically important in the
regulation of PRL release during lactation.
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Materials and Methods |
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Exp 1: constant suckling stimulus
The lactating rats with litters were placed in the experimental
cages on day 6 of lactation. An extension tubing was connected to the
cannula between 0800–0900 h on day 7 of lactation to allow for blood
sampling without disturbing the rats. The litters were weighed and
returned to the dams. An infusion of NAL (60 mg/kg·h; iv) or
heparinized saline (20 U/ml heparin; 300 µl/h; iv) was initiated at
0900 h, immediately after an initial (0 min) blood sample. The
concentration of NAL was adjusted to individual body weights so that
the vol of infusion was 300 µl/h. To evaluate the contribution of
behavioral changes, the rats were observed at 2-h intervals and scored
with a plus (lactating dam with pups in nest) or minus (dam away from
pups). In addition, the attachment of pups to the nipples was noted.
After observation, blood samples (350 µl) were collected from the
jugular vein cannula at 2-h intervals for a 12-h period. After each
sample, the extension tubing and cannula were filled with a
predetermined volume of either NAL or heparinized saline to completely
fill all tubing. The extension tubing was immediately reconnected to
the pump so that the infusion would continue uninterrupted. The
interruption of the infusion caused by sampling was less than 2 min
every 2 h. For in situ hybridization studies, the rats
were rapidly decapitated at 2100 h after the 12-h infusion period;
the brains were collected and frozen immediately in Histofreeze 2000
(Fisher Scientific, St Louis, MO) at -70 C. In experiments to evaluate
TH activity, the rats were injected with m-hydroxybenzylhydrazine
dihydrochloride (NSD 1015; 25 mg free base/kg; iv), an
L-aromatic amino acid decarboxylase inhibitor, after the
12-h blood sample and rapidly decapitated after an additional 15 min.
The NAL/vehicle infusion was continued after the NSD 1015 injection.
The stalk-median eminence (SME) was dissected with fine scissors,
frozen immediately on dry ice, and stored at -20 C until analyzed for
catecholamine content. In both groups, the litters were weighed at the
completion of the experiment.
Exp 2: acute sucking stimulus
An extension tubing was connected to the jugular vein cannula at
the start of the experiment and a 0 h blood sample collected. The
pups were then removed from the dams and weighed. NAL (60 mg/kg·h;
iv) or heparinized saline infusion was initiated. Blood samples were
collected at 3, 6, and 9 h during the infusion period. After
11 h of infusion, the litters were weighed again and returned to
the dams immediately after a 0 min blood sample was collected. Blood
was collected at 15, 30, 45, and 60 min after the onset of the suckling
stimulus (4 pups attached to nipples). After the 60-min blood sample,
NSD 1015 (25 mg/kg; iv) was injected. The lactating rats were rapidly
decapitated after 15 min, the SME dissected, and DOPA accumulation
determined. The litters were weighed at the end of the 1-h suckling
episode.
Determination of DOPA accumulation
The SME was homogenized in 120 µl of 0.1 N perchloric acid and
centrifuged at 10,000 x g for 2 min. The content of
DOPA in the SME was determined by HPLC with electrochemical detection,
as described previously (39). The pellet was solubilized in 0.5 N
sodium hydroxide and analyzed for protein content by the method of
Bradford (40).
In situ hybridization for TH mRNA
Coronal sections (15 µM) were cut through the
arcuate nucleus and thaw mounted on Superfrost Plus microscope slides
(Fisher Scientific). Brain sections were fixed in 4% paraformaldehyde
and subjected to an in situ hybridization procedure, as
described previously (39). After prehybridization steps, the brain
sections were hybridized overnight at 45 C with 2 x
106 dpm of a specific antisense 35S-labeled
cRNA probe for TH with a specific activity of 5 x 108
dpm/µg. The probe was synthesized using SP6 RNA polymerase from a
1.1-kb BamHI/EcoRI insert subcloned into a pSP65
vector. After ribonuclease treatment and a series of posthybridization
washes that increased in stringency, the slides were dipped in Ilford
Emulsion (K-5), diluted 0.25 g/ml water. The autoradiographs were
exposed for 4 weeks, developed by standard photographic methods, and
poststained lightly with hematoxylin.
The anatomical locations of the tissue sections were determined using the rat brain atlas of Paxinos and Watson (41). The rostral border was -2.5 mm, and the caudal border was -3.1 mm from the bregma. Approximately 18 cells/tissue section (i.e. 300–350 cells/rat), selected from all areas of the arcuate nucleus, were analyzed. TH mRNA-containing cells were identified, under darkfield optics, as a cluster of reduced silver grains with an identifiable cell nucleus. The number of grains in individual mRNA-containing cells was measured under x 400 darkfield illumination by a computerized image-processing system (Georgia Instruments, Roswell, GA).
PRL determinations
The blood was centrifuged at 10,000 x g for 5
min, and plasma PRL levels were determined using the rat PRL RIA kit
provided by NIDDK. PRL RP-3 was used as the reference preparation and
[125 I]PRL (New England Nuclear, Boston, MA) as the
radiolabeled antigen. The limit of sensitivity for the assay was 1
ng/ml. The intra- and interassay coefficients of variation were 12.1%
and 9.1%, respectively.
Statistical analysis
Results are expressed as the mean ± SE. The n
for all groups refers to the number of experimental animals. When two
groups were compared, the data were analyzed by Student’s t
test (42). For circulating PRL levels, the data were analyzed by ANOVA
for repeated measures, and multiple comparisons were made with
Fisher’s least-significant procedures (42, 43).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows circulating PRL
concentrations at 2-h intervals in control and NAL-treated rats. An
initial fall in circulating PRL levels during the first 2 h was
observed in both groups, likely because of manipulations to attach the
extension tubing and weighing the pups. In control rats, PRL levels
were consistently elevated to approximately 200 ng/ml during 4–12 h of
saline infusion. However, plasma PRL levels in the NAL-treated rats
were markedly lower (P < 0.05) than control
values.
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, left panel). Similarly, TH
mRNA signal levels in the arcuate nucleus were increased
(P < 0.05) 2.7-fold after 12 h of NAL infusion
(Fig. 2, middle panel; Fig. 3). In contrast, TH mRNA signal levels in
the medial zona incerta were similar in control and NAL-treated rats
(Fig. 2, right panel).
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). In contrast, the litters from the
NAL-treated rats did not gain weight during the 12-h period. For
comparison, the litter weight loss associated with 12 h of
separation from normal dams was determined. The separated litters lost
significantly (P < 0.05) more weight (approximately 2
g/8 pups) than litters from NAL-treated dams during the 12-h
period.
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Acute suckling induced PRL rise and TH activity
A separate experiment evaluated the effect of NAL on the acute
suckling-induced PRL rise. The pups were removed at the beginning of
the experiment and returned to the dams after an 11-h separation
period. At the time the pups were removed, PRL levels were high (380
ng/ml) in the two groups of rats before the onset of treatment (Fig. 5). Within 3 h after pup removal,
PRL levels were comparably less than 2 ng/ml in both the control and
NAL-treated rats and remained low and similar for the 11-h separation
period. In the control rats, PRL levels were markedly elevated, to 650
ng/ml, within 15 min after the pups were returned. In sharp contrast,
PRL levels were only 56 ng/ml in the NAL-treated rats after the pups
were returned. The suckling-induced PRL levels remained elevated to
350–400 ng/ml for 60 min in the control rats, whereas the
suckling-induced PRL rise was markedly suppressed, to less than 100
ng/ml at 15–60 min after the onset of suckling in the NAL-treated
rats.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A decrease in TIDA neuronal activity has been clearly implicated in maintaining the high PRL levels associated with a prolonged suckling stimulus (4, 5, 6, 7, 8, 9). This study supports the inverse relationship between TIDA neuronal activity and PRL secretion in the lactating rat. Furthermore, these data endorse the notion that the endogenous opioid peptides are an essential component in maintaining elevated PRL levels by acting on the TIDA neurons. TH activity in the SME was increased after 12 h of NAL infusion. This occurred when the dams were constantly exposed to the pups and after a 1-h suckling episode in dams deprived of their pups for an 11-h period. It can be inferred from these data, that the TH activity was elevated after the 11-h separation period and that the suckling stimulus resulted in a decrease in TH activity within 60–75 min after the onset of suckling. Although the experimental designs were somewhat different, these data are in general agreement with previous reports that the suckling stimulus acutely decreases dopamine turnover and TH activity in the median eminence (5, 7).
The prolonged blockade of opioid receptors in the present study also revealed that opioidergic input affects the expression of the TH gene in the TIDA neurons. TH mRNA signal levels in the arcuate nucleus were markedly increased after the 12-h NAL infusion. In fact, it is notable that the increase in TH gene expression caused by the NAL infusion is similar to that associated with pup separation (8, 9). The 12-h duration for the NAL infusion was selected because TH mRNA content in the arcuate nucleus is not significantly increased until 6 h after separation and continues to increase between 6 and 24 h after pup removal (8). The increases in TH gene expression after pup separation (9) and NAL infusion (this study) seem to be specific to the TIDA neurons and are not observed in dopaminergic neurons in the medial zona incerta.
The data are in general agreement with those by Callahan et
al. (38) that a µ-selective, but not -selective, opioid
receptor antagonist increased DOPA accumulation in the median eminence
of both pup-deprived and pup-suckled dams after a 4- to 5-h separation
period. The differences in timing and mode of antagonist administration
[i.e. 4 h for µ-selective antagonist and 45 min for
-selective antagonist, by Callahan et al. (38), and 12-h
continuous administration of NAL in the present study] make it
difficult to draw conclusions about the specific receptor subtype
involved in regulating TH gene expression. However, given that NAL has
a high affinity for µ-opioid receptors, moderate affinity for
-opioid receptors, and relatively low affinity for -opioid
receptors (44), it is likely that either µ- and/or -type receptors
may be involved in the opioidergic effects on TH activity and mRNA
levels in the TIDA neurons in the constantly suckled lactating
model.
The physiological consequences on the pups during the 12-h NAL infusion were remarkable. The pups of the NAL-treated dams failed to gain weight during the 12-h infusion period, whereas pups of control dams gained a significant amount of weight. However, the pups apparently did receive some milk during the NAL infusion, because complete separation from dams resulted in pup weight loss. This could not be explained by disruption of maternal behavior, because the time control and NAL-treated dams spent with the pups was similar. Observation of the pups indicated that the pups in the NAL-treated group were suckling vigorously when the dams were with the nest. Indeed, opiates disrupt maternal behavior, and NAL can prevent this disruption (45, 46). Although there is some controversy on the effect of endogenous opioid peptides on oxytocin secretion, it is unlikely that milk ejection failure caused the lack of weight gain in the present study. Hartman et al. (47) reported that NAL treatment did not alter the suckling-induced oxytocin release in conscious, normally hydrated rats, as with the dams in the present study. Reports of an opioid effect on oxytocin neurons indicate that the opioid action on oxytocinergic neurons is to restrain oxytocin release and that NAL augments oxytocin secretion (48).
The apparent escape of PRL release from the NAL-induced suppression was also somewhat striking. Although slightly variable among animals, this phenomenon seems to be synchronized at 6 h and 10 h after the initiation of NAL infusion. In a physiological sense, this may reflect the importance of lactation to sustaining mammalian species and, thus, the multiple mechanisms to maintain adequate lactation.
It is unclear whether the NAL effects on PRL secretion solely involve changes in TIDA neuronal activity. There is evidence that decreased dopaminergic activity may not completely account for opiate-induced PRL release and that PRL-releasing factor(s) may also be involved (26, 27). Indeed, one cannot rule out the possibility that the endogenous opioid peptides could alter nondopaminergic neurons, as well, and that effects on other factors could also contribute to the NAL suppression of suckling-induced PRL release. This study also does not identify which of the major opioidergic neuronal systems contribute to the control of PRL secretion during lactation. However, proenkephalin expression in the arcuate nucleus (19) and dynorphin expression in the paraventricular nucleus (21) are higher in lactating rats than in diestrous rats.
In conclusion, the endogenous opioid peptides are an integral component in the suckling-dependent elevation of PRL secretion during lactation. Moreover, the endogenous opioid peptides reduce TIDA neuronal activity in response to suckling and contribute to the marked suppression of TH gene expression during lactation. Without the opioidergic input, normal lactation seems to be severely compromised. These data support the notion that the TIDA neurons are an intermediate in the essential opioidergic enhancement of PRL secretion in response to suckling.
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Acknowledgments |
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Footnotes |
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Received December 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-opioid antagonists.
Brain Res 567:224–230[CrossRef][Medline]
-Opioid receptor-mediated regulation of central
dopaminergic neurons in the rat. Eur J Pharmacol 249:107–112[CrossRef][Medline]
opiate receptor subtypes. J
Neuroendocrinol 8:771–776[CrossRef][Medline]
-opioids
acting on the rat neurohypophysis. J Physiol 469:365–386This article has been cited by other articles:
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