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Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, Oregon 97006; and the Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201-3098
Address all correspondence and requests for reprints to: Dr. M. Susan Smith, Division of Neuroscience, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: smithsu{at}ohsu.edu
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Abstract |
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Introduction |
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Neuropeptide Y (NPY) neuronal activity has been shown to be greatly increased in two discrete areas in the hypothalamus during lactation: the caudal portion of the arcuate nucleus (ARH) and the dorsomedial nucleus of the hypothalamus (DMH) (1, 2). It has been suggested that the increased NPY activity in the caudal portion of the ARH may be important in mediating the increased food intake and the suppression of LH secretion associated with lactation (3, 4, 5, 6). Currently, the functional role of the increased NPY in the DMH is still unknown.
In contrast, the activity of the tuberoinfundibular dopaminergic (TIDA) system in the ARH is greatly suppressed during lactation. Dopamine (DA) production as well as the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the DA biosynthetic pathway, are significantly reduced (7, 8). Under normal conditions, DA produced from the TIDA system is believed to be the main PRL-inhibiting factor that is tonically released to inhibit PRL secretion from lactotrophs in the anterior pituitary (for reviews, see Refs. 9, 10). In addition, the activity of the TIDA neurons is regulated by PRL, such that elevated PRL increases the activity of these neurons and the secretion of DA into the median eminence (for reviews, see Ref. 11). During lactation, however, this feedback regulation of PRL is not operative, because elevated PRL levels are not associated with increased TIDA activity. This apparent dissociation between TIDA activity and PRL secretion may be one of the mechanisms by which high levels of PRL are sustained during lactation.
Currently, the mechanisms by which the activities of these neuronal systems are altered during lactation are not completely understood. It has been suggested that the suckling stimulus is important in triggering these changes (2, 12). Several factors associated with the suckling stimulus, such as the elevated levels of PRL and neural impulses, are possible candidates for mediating the alterations in NPY and the TIDA activities. Thus, in the present study, bromocriptine, a DA D2 receptor agonist, was used to inhibit suckling-induced PRL to characterize its role in modulating NPY and TIDA neuronal activities during lactation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental design
An acute suckling paradigm, previously described (2), was used
in the present study to control the onset of the suckling stimulus more
precisely. Briefly, lactating animals had their litters adjusted to
eight pups on day 2 postpartum, and the pups remained with their
mothers until day 9. At that time, the eight-pup litters were removed
from the females. On day 11, the animals were randomly divided into the
following three groups: 1) nonsuckled controls, animals received two sc
vehicle injections (0 pups control; n = 7); 2) eight pups suckling
for 24 h, animals received two vehicle injections (8 pups; n
= 8); and 3) eight pups suckling for 24 h, animals received two
bromocriptine injections (0.5 mg/rat·injection; 8
pups+B; n = 8). Resuckling for 24 h was chosen because
24 h of the suckling stimulus, after 48 h of pup deprivation,
was necessary to consistently observe significant changes in NPY gene
expression in the ARH (2).
Bromocriptine (Sandoz Pharmaceuticals Corp., East Hanover, NJ) was dissolved in peanut oil containing 25% alcohol (5 mg/ml). Bromocriptine or vehicle was administered sc 3 h before returning litters to the dams on day 11 postpartum; a second injection was given 12 h after returning the pups. The dose of bromocriptine used in the present study has been shown previously to have no direct effect in the brain (13, 14, 15).
After 24 h of suckling, the animals were killed by decapitation, and the brains were quickly removed, frozen on dry ice, and stored at -80 C. Coronal brain sections (20 µm) were collected through the ARH in a one in three series. The slides were stored at -80 C until used for in situ hybridization. Trunk blood was also collected and was assayed for rat PRL by RIA. The assay was performed by Dr. Marc Freeman at Florida State University according to methods previously described (16).
In situ hybridization
In the present study, quantitative in situ
hybridization was used to measure the messenger RNA (mRNA) levels for
TH and NPY, respectively, to serve as an indirect measure of neuronal
activity. The activity of central neurons has been shown to be related
to the cellular levels of mRNA encoding their rate-limiting enzyme or
in peptidergic neurons to the levels of prepropeptide mRNA (17, 18).
Gene expression of TH and NPY in TIDA and NPY neurons, respectively,
exhibits a tight parallel relationship with neuronal activity (2, 8, 19, 20).
NPY and TH cRNA probe synthesis, the specificity of the cRNA probe, and procedures for in situ hybridization have been described previously (1, 2, 8). Briefly, the NPY cRNA probe was transcribed from a 511-bp complementary DNA (cDNA) in which 21% of the UTP was 35S labeled (DuPont-New England Nuclear, Boston, MA). The TH cRNA probe was transcribed from a 300-bp cDNA using 50% 35S-labeled UTP. The specific activity for both probes ranged from 1–3 x 108 dpm/µg. The saturating concentration for both probes used in the assay was 0.3 µg/ml·kb.
The brain sections were fixed in 4% paraformaldehyde and treated with a fresh solution containing 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), followed by a rinse in 2 x SSC (standard saline citrate), dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and then air-dried. The slides were exposed to the respective cRNA probes overnight in moist chambers at 55 C. After incubation, the slides were washed in SSC that increased in stringency, in ribonuclease, and then in 0.1 x SSC at 60 C; dehydrated through a graded series of alcohols; and dried. Slides were dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed for 6–7 days at 4 C, and developed. After development, the slides were stained with cresyl violet.
Data analysis
The ARH was divided into four subdivisions (ARH-A, -B, -C, and
-D), as described in previous studies (1, 2), using the rat brain atlas
of Paxinos and Watson (21). We have shown previously (1, 2) that the
suckling stimulus only affects the NPY neurons located in the caudal
portion of the ARH (ARH-C). On the other hand, even though the suckling
stimulus suppresses TH gene expression throughout the entire ARH (8),
TIDA neurons are most numerous in the rostral portion of the ARH (ARH-A
and ARH-B). Thus, the ARH-C-containing sections were used for the NPY
study, and the brain sections containing ARH-A and ARH-B were used for
the TH study. As the suckling-induced NPY expressing neurons in the DMH
occupied the same plane as ARH-C (2), the tissue sections covering the
ARH-C subdivision were also used to analyze NPY gene expression in the
DMH.
The coronal brain sections were anatomically matched across animals from all groups. The hybridization signals were quantitated using the HARMONY image analysis system by VIDEK (Rochester, NY). The system identified silver grains by the brightness of the image. An estimate for silver grains over the entire ARH (for ARH-NPY and TH) or the entire DMH (for DMH-NPY) on each tissue section was given as the area occupied by silver grains within the marked area.
Statistical analysis
The data were expressed as the area occupied by grains per
section. The mean area occupied by grains per section was determined
for each animal. Data are presented as the mean ±
SEM. Differences between groups were evaluated using
one-way ANOVA and post-hoc Scheffe’s tests. Differences
were considered significant if P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Bromocriptine treatment
effectively blocked the PRL elevation induced by the suckling stimulus
(Fig. 1).
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and 3). In addition, there was no difference
in NPY gene expression between the 8 pups and the 8 pups+B groups
(Figs. 2 and 3), indicating that inhibition of PRL secretion did
not prevent the suckling-induced activation of NPY gene expression in
ARH.
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). This population of NPY neurons
was not observed in the nonsuckled control group (Fig. 4). In the
bromocriptine-treated group (Fig. 4), the signal appeared
to be less intensive compared with that in vehicle-treated animals
(Fig. 4). Quantitatively, the levels of NPY mRNA signal in both the 8
pups and the 8 pups+B groups were significantly higher than those in
the 0 pups control group (Fig. 5). In
addition, the NPY mRNA levels in the 8 pups group were significantly
greater than those observed in the 8 pups+B group (Fig. 5). It should
be noted that low levels of NPY mRNA signal were observed covering the
compact zone of the DMH in all animals examined (Fig. 4), and this
expression did not change in response to suckling.
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). The levels of
expression were comparable to those found in normal diestrous females
(8). TH gene expression in both the vehicle- and
bromocriptine-treated suckled groups were significantly
lower than that in the nonsuckled controls (Figs. 2 and 6). In addition, TH mRNA levels in
bromocriptine-treated suckled animals were significantly
higher than those in the 8 pups group (Figs. 2 and 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of examining ARH NPY gene expression in the present study are in agreement with those of our earlier studies (1, 2) and others (22), showing a significant increase in NPY gene expression in neurons in the caudal ARH in response to the suckling stimulus. In addition, the ineffectiveness of bromocriptine treatment in altering suckling- induced increases in ARH NPY gene expression indicates that suckling-induced hyperprolactinemia is not important in modulating ARH NPY activity. These results are in agreement with reports showing that PRL treatment does not affect ARH NPY gene expression (23), and immunoneutralization of PRL does not reduce NPY expression during lactation (22). In addition, because of the inhibition of PRL secretion, milk production in bromocriptine-treated dams would be minimal; therefore, these animals do not experience a significant change in energy balance. Thus, the activation of NPY in the ARH in the bromocriptine-treated animals occurs in the absence of a change in energy balance. These results thus suggest that the activation of NPY neurons in the ARH is mediated by incoming neural impulses activated by suckling, not by changes in energy balance.
In addition to the ARH NPY neurons, the present study also confirmed our earlier report (2) that the suckling stimulus activates a second population of NPY neurons located in the DMH. The blunted NPY expression after the inhibition of elevated PRL by bromocriptine treatment suggests that the activation of the DMH NPY neurons is, to a significant degree, mediated by PRL. The full activation of the NPY neurons is probably achieved by the combination of the suckling-induced neuronal inputs to the DMH and the hyperprolactinemia. The mechanism by which PRL modulates NPY activity in DMH is unknown. Identification of PRL receptors (PRL-R) in the brain (24, 25, 26, 27, 28) suggests that PRL may act directly in the brain to modulate NPY neruonal activity. It has been shown by immunocytochemistry (25) and receptor autoradiography (24) that PRL-R were found in the DMH, whereas in situ hybridization failed to show PRL-R-positive neurons in this area (26, 27, 28). These results suggest that PRL’s action in DMH may be presynatpic. On the other hand, PRL-R have also been found in the central nuclei of the amygdala, the medial preoptic area, the lateral septum, and the periaqueductal gray (24, 28) areas, which have been shown to be activated by the suckling stimulus during lactation (29, 30, 31, 32). The expression of PRL-R in these areas raises the possibility that PRL may modulate DMH NPY neuronal activity indirectly. Currently, there is very little known about the significance of suckling-activated DMH NPY neurons during lactation. Recently, a retrograde tracing study conducted in our laboratory demonstrated that the suckling-activated DMH NPY neurons project to the paraventricular nucleus of the hypothalamus (33), suggesting that these NPY neurons may modulate paraventricular nucleus of the hypothalamus activity during lactation.
The suppression of TIDA neuronal activity during lactation has been previously reported (7, 8, 12), although the mechanism by which TIDA activity is suppressed is still not understood. The involvement of PRL in regulating the TIDA neurons during lactation has largely been dismissed, because the short loop feedback regulation, in which elevated PRL levels (34, 35, 36) are normally associated with increased TIDA activity, clearly does not exist during lactation. In addition, exogenous PRL administration failed to activate TIDA neurons during lactation (7). Recently, it was shown that inhibition of suckling-induced PRL in midlactation caused a suppression of DOPA accumulation in the median eminence (37), suggesting that PRL may play a stimulatory role in regulating TIDA activity during lactation. However, the interpretation of the results is complicated by the following concerns: 1) the suppression of DOPA could not be reversed by coadministration of PRL; and 2) the methods used for measuring TIDA activity. TIDA activity was assessed by treating the animals with a decarboxylase inhibitor, NSD 1015. After treatment, the animals were killed, and DOPA accumulation in the median eminence fragments was measured (11). However, the midbrain dopaminergic cell groups (A8, A9, and A10), other than TIDA neurons, also terminate in the median eminence (38). Therefore, the changes in DOPA may not solely reflect the activity of TIDA neurons.
In the present study, we used an acute suckling model to examine the possible role of PRL in regulating TIDA neurons. In this model, the animals were first deprived of pups for 48 h before receiving 24 h of resuckling. The period of pup separation allows the TIDA activity as well as TH gene expression to recover to basal levels (8, 20). Secondly, in situ hybridization was used so as to be able to specifically study the changes in TIDA neurons. Consistent with previous reports (8, 37), this paradigm confirmed that the acute suckling stimulus greatly inhibited TH gene expression. Surprisingly, TH mRNA levels in these neurons were partially restored when hyperprolactinemia was prevented by bromocriptine treatment. These results suggest that, at least in response to the acute effects of suckling, elevated PRL is involved in the suppression of TH gene expression and, possibly, TIDA neuronal activity. Theoretically, the inhibitory effect of PRL on TIDA neurons observed in the present study should ensure that hyperprolactinemia is maintained during lactation. More importantly, the suckling stimulus appears to be critical in changing the stimulatory effects of PRL on TIDA activity into inhibitory effects. The mechanisms by which elevated PRL negatively modulates TH gene expression are not clear. Recently, it has been shown that TIDA neurons in the ARH express PRL-R (39). This provides anatomical evidence that PRL can affect TIDA neurons directly by binding to its own receptor. On the other hand, it is also possible that PRL can modulate TH expression indirectly by acting on neurons that connect to the TIDA neurons. Anatomical studies have demonstrated the direct contact between POMC-positive neuronal terminals and TIDA cell bodies (40, 41). It has been shown that µ-opioid receptor antagonists can prevent suckling-induced suppression of TIDA activity (42). Furthermore, immunoneutralization of suckling-induced PRL causes a decrease in the number of activated ß-endorphin-positive neurons during lactation (43). Therefore, it is possible that PRL may modulate TIDA activity by acting through the POMC system in the ARH.
Anatomical and pharmacological evidence also suggests that ARH NPY can
directly modulate TH gene expression and TIDA neuronal activity
(44, 45, 46). Thus, upon activation by the suckling stimulus, the ARH NPY
neurons may directly modulate TH expression in the TIDA neurons. The
possibility that PRL may modulate TH expression through the ARH NPY
neurons is ruled out by the present studies, which show that ARH NPY
expression remained elevated regardless of changes in TIDA neurons that
were affected by the suppression of PRL. Taken together, these results
suggest that during lactation the activity of TIDA neurons is probably
modulated by both PRL-dependent and PRL-independent mechanisms. Thus,
we hypothesize that suckling-induced PRL acts either directly on TIDA
neurons or indirectly through other systems, such as the POMC system,
but not the NPY system, to modulate TH gene expression during lactation
(Fig. 7). In addition, PRL-independent
mechanisms, such as suckling-activated ARH NPY neurons, can modulate
TIDA neurons directly (Fig. 7). It is plausible that the negative
effect of PRL on TH expression is the result of interactions between
these two mechanisms. More studies are needed to resolve this
issue.
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Acknowledgments |
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Footnotes |
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Received April 7, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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opiate receptor subtypes. J
Neuroendocrinol 8:771–776[CrossRef][Medline]
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,
Significantly different (P < 0.05) from 8 pups
group.
