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Endocrinology Vol. 145, No. 2 823-829
Copyright © 2004 by The Endocrine Society

Regulation of Hypothalamic Neuropeptide Y Messenger Ribonucleic Acid Expression during Lactation: Role of Prolactin

Peilin Chen and M. Susan Smith

Division of Neuroscience, Oregon National Primate Research Center, Department of Physiology and Pharmacology, Oregon Health & Science University, Beaverton, Oregon 97006-3499

Address all correspondence and requests for reprints to: Dr. M. Susan Smith, Division of Neuroscience, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006-3499. E-mail: smithsu{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we investigated the role of prolactin (PRL) in the suckling-induced increase in hypothalamic neuropeptide Y (NPY) gene expression in the dorsomedial nucleus of the hypothalamus (DMH) and the caudal portion of the arcuate nucleus of the hypothalamus (ARH-C). Lactating rats were deprived of their eight-pup litters on d 9 postpartum. After 48 h, the animals were randomly divided into two groups: nonsuckled controls and eight pups suckling for 24 h. In addition, some of the suckled animals received two injections of bromocriptine (0.5 mg/rat per injection) to inhibit suckling-induced PRL secretion. Some bromocriptine-treated rats also received ovine PRL (1 mg/rat per injection). In situ hybridization was performed to measure NPY mRNA levels. Suckling for 24 h induced a significant increase in NPY mRNA levels in the DMH and ARH-C. Bromocriptine treatment greatly attenuated the increase of NPY mRNA in the DMH but not in the ARH. Injections of ovine PRL in bromocriptine-treated rats greatly restored DMH NPY mRNA levels but had no additional effects on the ARH NPY expression. Double-label in situ hybridization for NPY and PRL receptor (PRL-R) in the lactating rat brains showed that NPY-positive neurons in the DMH also express PRL-R mRNA. On the contrary, few ARH NPY neurons expressed PRL-R. These data suggest that PRL could act directly on DMH NPY neurons to modulate NPY gene expression during lactation. Thus, the results from the present study demonstrate that NPY neurons in the DMH and ARH are differentially regulated by PRL during lactation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LACTATION IS A NATURAL physiological state occurring after parturition, in which a host of concerted changes in behavior and in the rate of metabolism in various body tissues occur in the dams to ensure that proper adaptations are in place to successfully support the young. It has been shown that the activity of several hypothalamic neuronal systems is altered during lactation; these alterations may mediate some of the physiological adaptations occurring during lactation, such as suppression of ovarian cyclicity, increased food intake, and suckling-induced milk production (1).

During lactation, neuropeptide Y (NPY) neuronal activity has been shown to be greatly increased in the caudal portion of the arcuate nucleus of the hypothalamus (ARH-C) and the dorsomedial nucleus of the hypothalamus (DMH) (2, 3). The functional role of ARH NPY has been extensively studied in various experimental models. It has been suggested that the increased NPY activity in the ARH-C may be important in mediating the sustained hyperphagia and suppression of LH secretion associated with lactation (4, 5, 6, 7). On the other hand, the functional role of the increase in NPY in the DMH is still unknown; however, the DMH has been implicated in the control of food intake and energy balance (8). A similar expression pattern of NPY neurons in the DMH has been reported in mice with high-fat diet-induced obesity (9) and in several lines of mouse genetic obese models (10). Interestingly, all the animal models, including lactation, that exhibit DMH NPY neuronal induction share a commonality, that is, hyperphagia and/or obesity. These data suggest that the activation of DMH NPY may play an important role in the hyperphagia observed during lactation and in some obesity models.

Currently, the mechanisms by which the DMH NPY neurons are activated during lactation are not well understood. It has been shown that the suckling stimulus is essential in triggering this alteration (2, 3). Several factors associated with the suckling stimulus, such as the neural impulses arising from suckling (11) and the elevated levels of prolactin (PRL) (12), have been suggested to be involved in mediating the alterations of NPY activity in the DMH. Our previous studies showed that treatment of lactating rats with bromocriptine, which serves to suppress suckling-induced PRL secretion, caused a significant reduction of DMH NPY mRNA expression. These data suggested that suckling-induced hyperprolactinemia may play an integral part in elevating NPY activity in the DMH. In the present study, PRL replacement was given to bromocriptine-treated dams to examine directly whether PRL is one of the suckling-associated factors that is important in modulating NPY neuronal activity in the DMH during lactation. We also surveyed the anatomical distribution of PRL receptors (PRL-Rs) in the brain, with a special focus on their relationship to the DMH NPY neurons, in an attempt to provide a neuroanatomical basis for the actions of PRL on DMH NPY neurons during lactation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Day 18–19 pregnant Sprague Dawley rats (B & K Universal, Inc., Kent, WA) were housed individually and maintained under a 12-h light, 12-h dark cycle (lights on at 0700 h) and constant temperature (23 ± 2 C). Food and water were provided ad libitum. The pregnant rats were checked for the birth of the pups every morning; the day of delivery was considered d 0 postpartum. All animal procedures were approved by the Oregon National Primate Research Center Institutional Animal Care and Use Committee.

Experimental design
An acute suckling paradigm, as 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 d 2 postpartum, and the pups remained with their mothers until d 9, when the eight-pup litters were removed from the females. On d 11, or 48 h after pup removal, the animals were randomly divided into the following four groups: 1) nonsuckled controls, animals received sc vehicle injections (0 pups + V; n = 7); 2) eight pups suckling for 24 h, animals received vehicle injections (8 pups + V; n = 7); 3) eight pups suckling for 24 h, animals received bromocriptine injections (0.5 mg/rat per injection; 8 pups + B; n = 9); and 4) eight pups suckling for 24 h, animals received injections of bromocriptine (0.5 mg/rat per injection) plus ovine prolactin (oPRL; 1 mg/rat per injection; 8 pups + B + P; n = 10). Resuckling for 24 h was chosen because it consistently induced maximum NPY gene expression in the DMH and ARH after 48 h of pup deprivation (2, 12). This dose of bromocriptine has been shown previously to completely inhibit suckling-induced PRL secretion (12), as was confirmed in the present studies by the absence of milk in the pups’ stomachs at the end of the 24-h resuckling period. The treatment regimen for oPRL, which has been reported to restore PRL-dependent processes (13, 14), resulted in pup stomachs that were full of milk after 24 h of resuckling.

Bromocriptine (Sandoz Pharmaceuticals Corp., East Hanover, NJ) was dissolved in peanut oil containing 25% alcohol (5 mg/ml). oPRL (AFP-10677C, NIDDK-NHPP) was dissolved in 50% polyvinylpyrrolidone (5 mg/ml). Each animal received two injections, with the first treatment at 3 h before returning the litters to the dams on d 11 postpartum, and the second treatment at 12 h after returning the pups.

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 (the full extent of the DMH is dorsal to this area) in a one-in-three series. The slides were stored at -80 C until used for in situ hybridization. Trunk blood was collected to assay for plasma levels of rat PRL by RIA, which was performed by Dr. Marc Freeman at Florida State University, according to methods previously described (15).

In situ hybridization
In the present study, quantitative in situ hybridization was used to measure NPY mRNA levels to serve as an indirect measure of neuronal activity. NPY cRNA probe synthesis, the specificity of the cRNA probe, and procedures for in situ hybridization have been described previously (2, 3). Briefly, the NPY cRNA probe was transcribed from a 511-bp cDNA in which 21% of the uridine 5'-triphosphate (UTP) was 35S-labeled (PerkinElmer, Boston, MA). The specific activity of the probe was 5 to 6 x 108 dpm/µg. The saturating concentration for the probe 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 standard saline citrate (SSC), 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 cRNA probes overnight in moist chambers at 55 C. After incubation, the slides were washed in SSC that increased in stringency, followed by incubation in ribonuclease A, and in 0.1 x SSC at 60 C to remove nonspecific binding. Slides were then dehydrated through a graded series of alcohols and dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed for 5–7 d at 4 C, and developed. After development, the slides were stained with cresyl violet.

Double-label in situ hybridization histochemistry of NPY and PRL-R mRNAs
Fresh-frozen tissue sections from lactating animals (d 12 postpartum; n = 4) were used in this NPY/PRL-R mRNA dual-labeling study. A 365-bp fragment of cDNA coding for the long and short forms of rat PRL-R was generated by PCR and subcloned into the pGEMT vector (Promega, Madison, WI). Antisense and sense rat PRL-R cRNA probes were transcribed from the cDNA in which 25% of the UTP was 33P-labeled (PerkinElmer). The specific activity of the probe was 5–6 x 109 dpm/µg. The saturating concentration for the probe used in the assay was 6 x 107 cpm/ml. The NPY cRNA probe was transcribed from a 511-bp cDNA in which the digoxigenin (dig)-UTP was incorporated. Brain sections were fixed and washed as described above and were exposed to the mixture of 33P-PRL-R (6 x 107 cpm/ml)/dig-NPY (2 µg/ml) cRNA probes in the moist chamber for 15 h at 55 C. After incubation and posthybridization washes, the slides were incubated in alkaline phosphatase-conjugated goat anti-dig antibody (1:2000; Roche Molecular Biochemicals, Basel, Switzerland) at 4 C overnight. The alkaline phosphatase-complexes were visualized with the substrate of nitroblue tetrazolium and 5-bromo-4-chloro-3-inodyl phosphate toluidinum (Roche Molecular Biochemicals). Slides were checked under light microscopy to ensure that the staining intensity of dig-NPY was satisfactory. Slides were then dehydrated, dipped in 3% parlodion followed by NTB-2 emulsion (Eastman Kodak Co.), exposed for 21 d at 4 C, and developed.

Data analysis
Brain sections containing the DMH [corresponding to ARH-C sections in Li et al. (12)] were used for the NPY mRNA analysis (20-µm coronal sections in a 1-in-3 series). The coronal brain sections were anatomically matched across animals from all groups. The hybridization signals were quantified using the OPTIMUS image analysis system, version 6.2 (Media Cybernetics, Silver Spring, MD). Brain section images were captured individually by a charge-coupled device camera (Cohu, Inc., San Diego, CA) and displayed on a computer monitor. The system identified silver grains by the brightness of the image. For the ARH, NPY cells were too close together to analyze individually; an estimate for silver grains over the entire ARH on each tissue section was given as the area occupied by silver grains within the region of interest (ROI). The ROI was kept constant for all the sections analyzed. Similar principles of quantification were used for analyzing NPY mRNA expression levels in the DMH area. A ROI was drawn to encompass all the NPY-positive neurons in the DMH. However, because the NPY neurons were scattering around the compact zone, it was not possible to exclude part of the compact zone, where low levels of NPY mRNA were present in both lactating and nonlactating animals. Therefore, additional steps were taken to ensure that only silver grain clusters associated with NPY neurons were counted. First, the area occupied by silver grains within the entire ROI was read and recorded (value A). Second, on the same image, all the NPY silver grain clusters associated with NPY neurons were retouched with the background color, and a second reading was recorded (value B); this value reflected total background for the ROI. Finally, value B was subtracted from value A to yield the silver grain area occupied by the NPY neurons only. The sum of the silver grain areas was divided by the number of sections quantified for each animal and was expressed as silver grain area per section. To elucidate whether PRL may modulate DMH NPY neuronal activity by increasing the number of NPY neurons or by increasing the intensity of NPY mRNA expression in each neuron, the total number of DMH NPY neurons was counted for each animal. The sum of the silver grain areas was divided by the total number of DMH NPY neurons to yield silver grain area per cell.

Statistical analysis
The data were expressed as grain area per section, NPY-positive cells per section and grain area per cell. The means for each of these measurements were determined for each animal. Data are presented as the mean ± SEM. Differences between groups were evaluated using one-way ANOVA and post hoc Fisher’s tests. Differences were considered significant if P was < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of bromocriptine treatment on PRL levels
PRL levels were significantly elevated by 24 h of suckling (8 pups + V), compared with the low levels in nonsuckled control animals (0 pups + V; Fig. 1Go). Bromocriptine treatment effectively suppressed suckling-induced PRL secretion (8 pups + B; Fig. 1Go). Although the rat PRL RIA did not detect the exogenously administered oPRL (Fig. 1Go), similar treatment has been shown to cause a large increase in oPRL levels (16) and to completely restore PRL-dependent physiological functions inhibited by bromocriptine (13, 14, 17). To support the validity of the PRL treatment, we inspected the stomach of each pup for the existence of milk. All pups from the suckled groups, except those from the 8 pups + B group, had stomachs full of milk after 24 h of suckling.



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  		FIG. 1. Plasma levels of rat PRL as determined by a rat PRL RIA. Suckling induced a significant elevation of plasma levels of rat PRL [8 pups + vehicle (V); solid bar]. Bromocriptine (B) treatment significantly suppressed suckling-induced PRL secretion. Because the PRL antibody used in the assay is specific to rat PRL, the PRL levels remained suppressed in the group treated with bromocriptine and ovine PRL (P) (8 pups + B + P group). *, Significantly different (P < 0.05) from all other groups.

 
NPY gene expression in the DMH
Suckling eight pups for 24 h induced the expression of NPY in a population of neurons located around the compact zone in the DMH (DMHp; 8 pups + V; Fig. 2AGo). This population of NPY neurons was not observed in the nonsuckled control group (0 pups + V; Fig. 2AGo). These data were in agreement with our previous studies (2, 12). Animals that received bromocriptine treatment had a significantly less NPY mRNA signal (silver grain area/section, 6.07 ± 1.28 vs. 27.54 ± 3.68; Figs. 2AGo and 3AGo) than that of the vehicle-treated animals; however, this was still significantly higher than the nonsuckled controls (6.07 ± 1.28 vs. 0.14 ± 0.06; Fig. 3AGo). PRL replacement significantly reversed the effect of bromocriptine treatment on suckling-induced NPY expression, because the NPY mRNA signal of the 8 pups + B + P group was significantly higher than that of the 8 pups + B group (16.46 ± 2.87 vs. 6.07 ± 1.28), although it did not reach the same levels as the 8 pups + V group (Figs. 2AGo and 3AGo). Bromocriptine treatment also resulted in fewer NPY mRNA-positive cells in the DMH induced by suckling stimulus (cells/section, 4.40 ± 0.54 vs. 9.98 ± 0.27; Fig. 3BGo). PRL treatment nearly reversed the effect of bromocriptine on the number of NPY neurons expressed in the DMH (cells/section, 7.32 ± 0.76 vs. 4.40 ± 0.54; Fig. 3BGo), although a significant difference was still apparent between the 8 pups + B + P and 8 pups + V groups (Fig. 3BGo). As the result of this closeness in numbers of NPY cells, when the data were expressed as silver grain area per cell, there was no significant difference between the 8 pups + B + P and 8 pups + V groups (Fig. 3CGo). It should be noted that low levels of NPY mRNA signal were observed in the DMHp in all animals examined, and this expression did not change in response to either suckling or any of the treatments (Fig. 2AGo).



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  		FIG. 2. A, Dark-field photomicrographs of the DMH area from the four treatment groups showing the expression of NPY mRNA. Acute resuckling for 24 h [8 pups + vehicle (V)] induced clusters of silver grains (representative clusters indicated by the arrows) scattered in the DMH, compared with the 0 pups + V group. The low level of signal covering the DMHp was observed in all the animals examined. Bromocriptine (B) treatment significantly blunted NPY mRNA expression (8 pups + B), although PRL replacement partially restored the NPY mRNA expression levels (8 pups + B + P). Scale bar, 200 µm. B, Dark-field photomicrographs of the ARH area from the four treatment groups. The silver grains represent NPY mRNA. Note the marked increase in silver grain expression in all groups that received the suckling stimulus. Bromocriptine and/or ovine PRL (P) treatments did not change the NPY mRNA levels in the ARH. Scale bar, 200 µm.

 


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  		FIG. 3. A, NPY mRNA levels in the DMH area. Resuckling for 24 h induced a significant increase in NPY mRNA levels in this area. Bromocriptine (B) treatment greatly attenuated suckling-induced NPY gene expression. Ovine PRL (P) injections to the suckling rats treated with B partially restored NPY mRNA expression. B, Number of NPY mRNA-positive neurons found in the DMH area in the four treatment groups. C, NPY mRNA levels in the DMH expressed as grain area per cell. It should be noted in panels B and C that there were no NPY cells detected in the 0 pups + vehicle (V) group. D, NPY mRNA levels in the ARH-C region in the four treatment groups. Resuckling for 24 h after a 48-h pup separation induced a significant increase in NPY mRNA levels in this region. Inhibition of PRL secretion by bromocriptine failed to suppress the increase in NPY gene expression induced by the suckling stimulus. Furthermore, exogenous PRL treatment to bromocriptine-treated animals did not alter NPY mRNA expression levels. *, Significantly different (P < 0.05) from 0 pups + V group. {blacktriangleup}, Significantly different (P < 0.05) from 8 pups + V group. {diamondsuit}, Significantly different (P < 0.05) from 8 pups + B group.

 
NPY gene expression in the ARH
In the ARH-C area, 24 h of suckling caused a significant increase in NPY gene expression compared with the nonsuckled group (Figs. 2BGo and 3DGo). In addition, there was no difference in NPY gene expression among the groups of 8 pups + V, 8 pups + B, and 8 pups + B + P (Figs. 2BGo and 3DGo), indicating that inhibition of PRL secretion did not prevent the suckling-induced activation of NPY gene expression in the ARH.

PRL-R expression in the brain
We first performed 33P-PRL-R single-label in situ hybridization on brain sections from lactating and diestrous animals to validate the specificity of the cRNA probe. In agreement with previous studies (18, 19), we detected PRL-R mRNA in discrete regions in the brain from both groups of animals, with the highest hybridizing signal observed in the choroids plexus. Prominent PRL-R mRNA signal was observed mainly in the hypothalamus and limbic structures (data not shown). In the hypothalamus, PRL-R mRNA was observed in the preoptic areas, paraventricular nucleus of the hypothalamus (PVH), ventromedial nucleus, anterior hypothalamic area, ARH, and DMH. In the limbic area, PRL-R mRNA was found in the lateral septum and medial portion of the bed nucleus of stria terminalis. In the midbrain, PRL-R was observed in the periaqueductal gray. The specificity of the PRL-R cRNA probe was confirmed by a lack of hybridization signal on the tissue sections that were hybridized with sense probe.

PRL-R and NPY double-labeling in the DMH
To investigate the anatomical relationship between NPY- and PRL-R-expressing neurons in the DMH and ARH, PRL-R and NPY double-label in situ hybridization was carried out only on the brain sections containing ARH-C. Dig-NPY mRNA-containing neurons were identified under bright-field illumination as dark blue deposits in the cytoplasm. 33P-PRL-R mRNA-containing neurons were identified under bright-field illumination as clusters of black grains clustered over and surrounding the cytoplasm. When both of the signals were examined under bright-field illumination, the majority of the DMH NPY-positive neurons were labeled with PRL-R mRNA signal (Fig. 4Go, A and B). However, in the ARH, most PRL-R-expressing neurons cluster more in the dorsal-medial part of the ARH, whereas NPY-containing neurons congregate in the more ventral-lateral part of the nucleus. Hence, almost no NPY/PRL-R coexpression was observed in the ARH (Fig. 4Go, C and D).



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  		FIG. 4. NPY and PRL-R double labeling in the DMH. A, Lower power bright-field photomicrograph of the DMH showing dig-labeled NPY-positive neurons (blue-black deposit) scattered around the DMHp (outlined by solid white line). B, High magnification of the red boxed area in A showing examples of dig-NPY/ 33P-PRL-R mRNA double-labeled neurons (blue arrows) in the DMH. The inset further illustrates 33P-PRL-R mRNA (as clusters of black grains) scattered over and surrounding the dig-NPY mRNA (as purple-blue staining). C, Representative photomicrograph showing dig-labeled NPY neurons (blue-black deposit) in the ARH. Only occasional NPY/PRL-R double-labeled cells (blue arrow) were observed. D, Higher magnification of the red box in C showing examples of NPY (filled green arrow) and PRL-R (open arrows) single-labeled cells in the ARH. 3V, Third ventricle. Scale bar units, micrometers.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During lactation, a significant elevation of NPY mRNA is observed in neurons located in the ARH-C and DMH. The results for ARH NPY gene expression in the present study are in agreement with those of our earlier studies (2, 3, 12) and others (20), showing a significant increase in NPY gene expression in neurons in the caudal ARH in response to the suckling stimulus. We also showed that bromocriptine does not alter suckling-induced NPY mRNA up-regulation in this area, suggesting that suckling-induced hyperprolactinemia is not essential in modulating NPY gene expression in the ARH during lactation (12). This notion is substantiated in the present study in that oPRL replacement failed to modify NPY gene expression in the ARH. Finally, whereas PRL-R is abundant in the ARH and has been shown to be expressed on tyrosine hydroxylase neurons (21, 22), almost no colocalization of PRL-R and NPY neurons was found in this region. These results are further supported by reports showing that PRL treatment or pituitary graft-induced hyperprolactinemia does not affect ARH NPY gene expression (23, 24), and immunoneutralization of PRL does not reduce NPY expression during lactation (20). It is noteworthy that the bromocriptine-treated dams experience a relatively low energy demand because of the suppression of milk production resulting from the inhibition of PRL secretion, suggesting that 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 suggest that the activation of NPY neurons in the ARH is mediated by incoming neural impulses activated by suckling, and not by elevated levels of PRL or changes in energy balance.

The present study also confirmed our earlier reports that the suckling stimulus activates a second population of NPY neurons located in the DMH (2, 3, 12). Inhibition of elevated PRL by bromocriptine treatment resulted in significantly lower NPY expression levels, and this reduction was reflected by a decrease in the number of NPY neurons activated and in the intensity of mRNA levels of individual neuron. PRL replacement to the bromocriptine-treated animals significantly increased NPY expression levels in the DMH, although the replacement did not completely restore the expression to the levels of control suckling animals. Exogenous PRL reversed the effect of bromocriptine on NPY mRNA expression levels of individual neurons, but only partially restored its effect on the number of NPY neurons activated. The inability of oPRL to completely restore bromocriptine-suppressed DMH NPY mRNA expression could be due to the fact that the oPRL treatment may not completely mimic the physiological actions of endogenous rat PRL. Doherty et al. (16), using a similar regimen, showed that although the serum concentration of oPRL was in the range of several micrograms per milliliter, the treatment was not as effective in suppressing copulatory behavior of male rats as the much lower elevation of serum rat PRL levels produced by ectopic pituitary grafts. In the present study, the physiological potency of oPRL treatment was determined by the fact that all pups from oPRL-treated dams had bellies full of milk, similar to those in the 8 pups + V group, suggesting that the pharmacological oPRL treatment was effective. However, it is possible that some effects of endogenous rat PRL were not fully restored by the oPRL replacement.

The mechanism by which PRL modulates NPY activity in DMH has not been fully explored. Several studies reported the identification of PRL-R in the brain (18, 19, 25, 26, 27), suggesting a possible pathway for PRL to act directly in the brain to modulate NPY neuronal activity. It has been shown previously by immunohistochemistry (25) and receptor autoradiography (27) that PRL-R is found in the DMH, whereas other groups failed to show PRL-R in this area, either by immunocytochemistry or in situ hybridization (18, 19, 26, 28, 29). In the present study, we were able to identify PRL-R mRNA in the DMH area by in situ hybridization. Compared with previous studies, we used a different portion of the PRL-R gene sequence to generate the antisense cRNA probe and a higher energy isotope, 33P instead of 35S, to label the probe. In addition, studies have shown that PRL-R expression levels in several brain areas were altered during lactation (30, 31, 32, 33, 34). Hence, another possible explanation for the discrepancy with earlier studies is that the PRL-R expression in the DMH observed in the present study is a result of lactation-induced up-regulation. However, we observed PRL-R-positive neurons in the DMH in both lactating and nonlactating animals, and the levels between the two groups appeared to be similar.

In the present study, we showed that in the lactating rats, most of the DMH NPY neurons were also PRL-R positive. These results suggest that PRL may act directly on its receptor to activate NPY gene expression in the DMH. In addition to the DMH, PRL-R mRNA is also observed in the medial preoptic area, the lateral septum, and periaqueductal gray areas. Studies from our laboratory have shown that neurons in these areas send direct neural inputs to the DMH and, more importantly, these neurons are activated in response to the suckling stimulus (11). The expression of PRL-R in these areas raises the possibility that PRL may also modulate DMH NPY neuronal activity via indirect pathways by modulating neural populations upstream of the DMH. More studies are needed to resolve this issue.

Another possible signal for activation of DMH NPY neurons is the change in energy balance that is normally associated with milk production. As stated above, the bromocriptine treatment not only blocked PRL secretion but also suppressed milk production, which presumably would alter the status of energy balance compared with that of the suckled, vehicle-treated animals. Conversely, administration of exogenous PRL to replace the suppressed endogenous PRL caused by bromocriptine restored not only PRL levels but also milk production, thus changing energy balance back to the high demand state. However, it has been shown that DMH NPY neurons are activated rapidly after the onset of the suckling [i.e. 90 min (our unpublished observation) to 3 h (Ref. 2), and the expression levels reach maximum levels around 9 h (our unpublished observation) to 12 h (Ref. 2)]. Therefore, the early induction of DMH NPY occurs before any significant milk production and change in energy balance has taken place. It is possible that the altered energy status in lactating animals may be involved in maintaining the expression of NPY in the DMH, even if not in the induction of expression. Future experiments in which both PRL levels and milk production of the dams are independently controlled are needed to elucidate the involvement of energy balance in modulating DMH NPY activity.

Currently, the physiological significance of the suckling-activated DMH NPY neurons during lactation remains unknown. Our previous retrograde tracing study demonstrated that the suckling-activated DMH NPY neurons project to the PVH (19), an area shown to be the key site in the brain in controlling energy homeostasis. These data suggest that one of the possible functions of DMH NPY neurons during lactation may be to modulate activity of the PVH, resulting in the regulation of energy homeostasis. Interestingly, this information suggests that, during lactation, there is an exaggerated NPY input into the PVH, because ARH NPY also provides prominent projections into this nucleus (35, 36, 37). One can argue that this exaggerated NPY input to the PVH is to ensure that high levels of hyperphagia are achieved during lactation to offset the large energy demand due to milk production. On the other hand, previous studies have illustrated that the neural pathways activating the two groups of NPY neurons do not overlap (11, 35, 38). Furthermore, the present study also demonstrated that PRL differentially modulates the activity of the two NPY populations. Finally, anatomical studies show that the projections of neurons from the ARH and DMH only partially overlap (36, 37, 39), suggesting that the function of NPY neurons in the DMH and ARH may not be redundant and each population may serve different physiological functions during lactation.

In conclusion, the present study demonstrated that the suckling stimulus activates two populations of hypothalamic NPY neurons in the DMH and ARH. We also demonstrated that suckling-induced hyperprolactinemia plays a stimulatory role in the activation of NPY neurons in the DMH but not in the ARH. In addition, NPY-positive neurons in the DMH express PRL-R mRNA, whereas most ARH NPY neurons do not express PRL-R mRNA. These data suggest that PRL could act directly on DMH NPY neurons to modulate NPY gene expression during lactation. Therefore, DMH and ARH NPY neuronal populations are differentially regulated by PRL.


   Acknowledgments
 
We thank Drs. Kevin Grove and Chien Li for their comments about the manuscript. We are grateful to Dr. Marc Freeman at Florida State University for performing the rat PRL RIA.


   Footnotes
 
This work was supported by NIH Grants HD14643 and HD18185 and the Oregon National Primate Research Center Grant RR00163.

Abbreviations: ARH-C, Caudal portion of the arcuate nucleus of the hypothalamus; dig, digoxigenin; DMH, dorsomedial nucleus of the hypothalamus; DMHp, compact zone in the DMH; NPY, neuropeptide Y; oPRL, ovine PRL; PRL, prolactin; PRL-R, PRL receptor; PVH, paraventricular nucleus of the hypothalamus; ROI, region of interest; SSC, standard saline citrate; UTP, uridine 5'-triphosphate.

Received September 19, 2003.

Accepted for publication October 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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