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Endocrinology Vol. 149, No. 2 544-550
Copyright © 2008 by The Endocrine Society

Role of Agouti-Related Protein-Expressing Neurons in Lactation

Colin T. Phillips and Richard D. Palmiter

Howard Hughes Medical Institute and Department of Biochemistry, University of Washington, Seattle, Washington 98195

Address all correspondence and requests for reprints to: R. D. Palmiter, Howard Hughes Medical Institute and Department of Biochemistry, Box 357370, University of Washington, Seattle, Washington 98195. E-mail: palmiter{at}u.washington.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypothalamic neurons that express agouti-related protein (AgRP) and neuropeptide Y (NPY) are thought to be important for regulation of feeding, especially under conditions of negative energy balance. The expression of NPY and AgRP increases during lactation and may promote the hyperphagia that ensues. We explored the role of AgRP neurons in reproduction and lactation, using a mouse model in which AgRP-expressing neurons were selectively ablated by the action of diphtheria toxin. We show that ablation of AgRP neurons in neonatal mice does not interfere with pregnancy, parturition, or lactation, suggesting that early ablation allows compensatory mechanisms to become established. However, ablation of AgRP neurons after lactation commences results in rapid starvation, indicating that both basal feeding and lactation-induced hyperphagia become dependent on AgRP neurons in adulthood. We also show that constitutive inactivation of Npy and Agrp genes does not prevent pregnancy or lactation, nor does it protect lactating dams from diphtheria toxin-induced starvation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LACTATION IMPOSES tremendous energetic demands on the mother. In rodents the combined weights of the pups at weaning can exceed that of the dam by 3-fold. Not surprisingly, the dam adjusts to this energy demand by increased feeding; nevertheless, she remains in negative energy balance throughout lactation (1, 2, 3). The mechanisms that support enhanced maternal feeding during lactation are not well established, but most investigators assume that hypothalamic melanocortin system is involved and that it may be supplemented by additional adaptations (1, 2, 3, 4, 5).

The melanocortin system includes the proopiomelanocortin (POMC)-producing neurons in the arcuate (ARC) nucleus of the hypothalamus. These neurons process POMC to {alpha}MSH that is released in paraventricular nucleus and in several other brain regions, where it acts on postsynaptic cells bearing G{alpha}s-coupled melanocortin-4 receptors (MC4Rs). The POMC neurons respond to and integrate many hormonal and neurochemical signals that reflect energy balance (6, 7, 8, 9). Under conditions of positive energy balance, activation of POMC neurons (melanocortin signaling) inhibits feeding and promotes metabolism, whereas in negative energy balance, melanocortin signaling has the opposite physiological effects. Intracranial administration of {alpha}MSH (or peptide mimetics) inhibits feeding (10, 11), whereas inactivation the genes that encode POMC, the protease that converts POMC to {alpha}MSH, or MC4R results in obesity (12, 13).

The melanocortin pathway in the ARC is counterbalanced by neighboring neurons that produce agouti-related protein (AgRP), neuropeptide Y (NPY), and {gamma}-aminobutyric acid (GABA) (14, 15). AgRP neurons directly inhibit the POMC neurons through a GABA-dependent mechanism (16). AgRP neurons also project to most of the same postsynaptic cells as POMC neurons (17), where they appear to block the action of {alpha}MSH three ways: 1) AgRP directly antagonizes the binding of {alpha}MSH to MC4R (7, 18); 2) NPY activates G{alpha}i-coupled Y1 and Y5 receptors on the MC4R-bearing target neurons (19, 20); and 3) GABA inhibits postsynaptic cells via GABAA receptors. Thus, activation of AgRP neurons promotes feeding by blocking melanocortin signaling. Although there is strong evidence establishing that intracranial delivery of AgRP or NPY stimulates feeding (21, 22, 23), inactivation of either or both of the genes encoding these peptides has minimal effects on body weight regulation (24, 25, 26). Nevertheless, the rapid ablation of AgRP neurons in adult mice produces starvation, indicating that these neurons are essential for maintenance of feeding behavior (27, 28). Surprisingly, ablation of AgRP neurons in neonatal mice, before the neurons are mature, allows compensation such that adult mice grow normally, despite the absence of these neurons (27).

During lactation, the expression of POMC declines, and the expression of NPY and AgRP in the ARC increases, as might be expected in negative energy balance (29, 30, 31). In addition, a population of NPY-expressing neurons in the dorsal medial hypothalamus (DMH) that is otherwise dormant becomes activated during lactation (32, 33). These NPY-expressing neurons in the DMH may augment or even replace the AgRP/NPY-expressing neurons in the ARC and, thus, support the hyperphagia associated with lactation (34). It is also conceivable that other, as yet unidentified, neuronal circuits become activated to either sustain or enhance feeding during lactation.

Although neonatal ablation of AgRP neurons still permits normal feeding, it remains plausible that AgRP neurons are necessary for lactation-induced hyperphagia. Although the focus of this study was on lactation-induced feeding, we were prepared for the possibility that ablation of AgRP neurons in neonates might interfere with aspects of reproduction, such as establishing or maintaining pregnancy. We also sought to determine whether ablation of AgRP neurons in lactating adults would affect feeding. We imagined three possibilities: 1) feeding is completely independent of AgRP neurons during lactation; 2) lactation-induced hyperphagia (that portion in excess of normal feeding) is independent of AgRP neurons; or 3) all aspects of feeding during lactation depend on AgRP neurons. In the first case, ablation of AgRP neurons during lactation was predicted to have no effect, but subsequent weaning might lead to starvation. In the second case, ablation of AgRP neurons would suppress basal but not lactation-induced feeding, and as a consequence, the dam might be unable to support a normal litter. In the third case, the lactating dam would starve immediately after ablation of AgRP neurons, as observed with nonlactating females.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and procedures
Generation of mice with the human diphtheria toxin receptor (DTR) cDNA targeted to the Agrp locus (AgrpDTR/+) was described (27); they are on a mixed 129/Sv x C57Bl/6 genetic background. For some experiments homozygous AgrpDTR/DTR mice were used; they are equivalent to Agrp-null because the endogenous Agrp gene is disrupted. We also bred NpylacZ/+ mice (24), in which a lacZ gene with nuclear localization signal is integrated into the Npy locus, with AgrpDTR/+ mice to generate AgrpDTR/DTR,NpylacZ/lacZ mice that lack expression of both AgRP and NPY. C57/Bl6 mice from our colony were used for the control experiment. All mice were housed in a temperature and humidity controlled room with a 12-h light, 12-h dark cycle with chow (Purina 5053; Purina Mills, LLC, St. Louis, MO) and water ad libitum, unless otherwise stated. All experiments were performed with approval of the Animal Care Committee at the University of Washington.

For experiments with neonatally ablated mice, AgrpDTR/+ mice were bred with each other, and their pups were injected within the first few days after birth with diphtheria toxin (DT) (List Biological Laboratories, Campbell, CA). DT was dissolved in PBS at 5 µg/ml and injected sc at 10 µl/g (50 µg/kg body weight). After weaning, female AgrpDTR/+ mice were identified by genotyping tail DNA by PCR. As a biological test of AgRP neuron ablation, the ghrelin mimetic compound A (35), which was provided by Merck & Co., Inc. (Whitehouse Station, NJ), was administered to AgrpDTR/+ mice by gavage (3 mg/kg body weight), and consumption of solid chow was measured after 4 h as described (36). Two females (~16 wk old) were then mated with a C57Bl/6 male. Upon visible signs of pregnancy (~5 d before parturition), the female was transferred to an individual cage and provided with liquid diet (PMI Micro-Stabilized Rodent Liquid Diet LD 101; TestDiet, Richmond, IN). Powdered diet (230 g) was added to water (770 ml) and mixed with an electrical handheld blender to prepare an emulsion with 1 Kcal/g. The diet was either prepared fresh each day or stored at 4 C for up to 7 d before use. The diet was provided in test tubes with spouts available through the cage hopper in place of standard chow. When the pups were more than 16 d old, the test tubes were placed as high up in the hopper as possible to minimize the amount of liquid diet consumed by the pups. Body weight and food intake by the dam were monitored daily at about 1200 h each day. Body weights of the pup were measured daily starting at d 7.

For experiments in which AgRP neurons were ablated in lactating adults, two females (three different genotypes were tested; see figure legends 3–5) were housed with a C57/Bl6 male. Upon visible signs of pregnancy (~5 d before parturition), the female was transferred to an individual cage and provided with liquid diet as described previously. DT was injected im (50 µg/kg) on d-7 and -9 lactation. Food intake and body weights were measured as described previously. In one experiment control dams were pair fed such that they lost the same weight as DT-treated dams.

Histochemistry and immunohistochemistry
Immediately after euthanasia by CO2 asphyxiation, mice were transcardially perfused at room temperature with PBS, followed by ice-cold PBS containing 4% paraformaldehyde. Brains were removed, postfixed overnight in 4% paraformaldehyde, and then stored in 70% alcohol. Paraffin-embedded sections (8 µm) were dewaxed in xylenes and a graded series of ethanol, rinsed twice in PBS, and then subjected to microwave antigen retrieval by heating at approximately 99 C in 1 mM EDTA (pH 7.5) for 15 min. After blocking rabbit polyclonal anti-NPY (Peninsula Laboratory Inc., San Carlos, CA) was used at 1:1000 dilution in Tris-buffer saline [137 mM NaCl, 2.7 mM KCl, and 25 mM Tris-HCl (pH 7.5)] containing 3% normal donkey serum, and visualized with Cy3-labeled secondary antibody (1:500; Jackson Immunolaboratory, West Grove, PA).

For β-galactosidase histochemistry, the procedure was the same except that after overnight post fixation, the brains were transferred to a 30% sucrose solution, kept at 4 C overnight, and then frozen in 2-methylbutane. Brains were stored at –80 C, and 30-µm sections were cut on a cryostat. Sections were incubated with X-gal (1 mg/ml; Research Products International Corp., Mt. Prospect, IL) for 24 h, followed by brief washes with PBS. Sections were mounted on slides, counterstained with 0.1% nuclear fast red in 5% aluminum sulfate for 3 min, dehydrated, and coverslips were applied.

Bright-field and immunofluorescent pictures were captured digitally using a CoolSnap camera (Roper Scientific GmbH, Ottobrunn, Germany) attached to a Nikon microscope (Nikon Corp., Tokyo, Japan). All paired photomicrographs were obtained using the same image acquisition settings.

Statistical analysis
Data were analyzed by the Student’s t test; significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ablation of AgRP neurons in neonates does not interfere with pregnancy or lactation
Heterozygous AgrpDTR/+ pups were either injected with DT within the first few days after birth or untreated (naive). At approximately 12 wk of age, the feeding response of females to the ghrelin mimetic was examined as a preliminary test to ensure ablation of AgRP neurons because our previous studies showed that loss of AgRP neurons prevented ghrelin-induced feeding (36). As expected, the DT-treated group of females did not respond to the ghrelin mimetic, compound A, whereas the naive group increased food consumption approximately 5-fold (4-h food consumption by DT-treated mice: vehicle: 0.14 ± 0.07 g, compound A, 0.20 ± 0.09 g, P = 0.56; naive mice: vehicle, 0.17 ± 0.03 g, compound A, 0.91 ± 0.06 g, P < 0.0001).

At approximately 16 wk of age, females of both groups were housed with wild-type (WT) males. When the females were obviously pregnant, they were transferred to individual cages with a liquid diet. All females of both groups became pregnant and had litters. The average time to parturition from the time the females were set up with the males was the same (~29 d) for both groups of females, and the average litter size did not differ between groups (Table 1Go).


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  		TABLE 1. Effects of neonatal AgRP neuron ablation on pregnancy and lactation

 
Consumption of liquid diet by dams that had litters of six or more pups was recorded for 18 d starting at parturition. Although there was a small difference in food consumption during the first few days, over the 18-d period, there was no significant difference in cumulative food consumption by the two groups of mice (Fig. 1Go). The average liquid diet consumed by the females of both groups on d 10 were not different (Table 1Go), and the cumulative liquid diet consumed during the first 12 d of lactation consumed by the DT-treated mice (384 ± 14 g) was the same as that consumed by the naive mice (371 ± 10 g; P = 0.45). By d 16 of lactation (before the pups began to drink from the liquid dispenser), the dams consumed approximately 44 g of liquid diet/d, whereas virgin females consumed approximately 18 g/d (P < 0.001; Fig. 1Go).


Figure 1
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  		FIG. 1. Liquid diet consumed during lactation by neonatally DT-treated AgrpDTR/+ mice (gray squares, n = 5) and untreated (naive) littermate controls (solid circles, n = 7). Liquid diet consumed by virgin females that were treated with DT as neonates (open squares, n = 4) or not treated with DT (open circles, n = 5).

 
A second group of DT-treated and naive females was killed on d 10 g lactation to examine NPY expression in the hypothalamus by immunohistochemistry. NPY staining in the ARC of nonlactating, control females was present in both cell bodies and processes (Fig. 2CGo). During lactation, NPY staining in cell bodies and processes within the ARC of the control mice became more prominent (Fig. 2FGo). By contrast, the DT-treated, lactating females had virtually no NPY staining in the ARC (Fig. 2IGo), in agreement with previous observations with nonlactating females (27, 36). NPY staining in the DMH appeared to be entirely within processes of both nonlactating and lactating control females; staining was more prominent in the lactating females, which displayed abundant NPY-labeled boutons (Fig. 2Go, B and E, inset). DT treatment eliminated most of the NPY staining from the DMH (Fig. 2HGo). As a control for the immunohistochemistry procedure, NPY staining in the cortex of the same sections was the same under all three conditions (Fig 2Go, A, D, and G) as predicted because neither energy balance nor DT treatment would be expected to affect NPY staining in cell bodies or processes of cortical interneurons.


Figure 2
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  		FIG. 2. Photomicrographs of coronal brain sections through the hypothalamus at the level of the ARC stained with antibodies against NPY. A–C, Immunohistochemistry from a naive control AgrpDTR/+ nonlactating female showing NPY staining in cortex (A), DMH (B), and ARC (C). D–F, Immunohistochemistry from control AgrpDTR/+ female on d 20 of lactation with eight pups showing NPY staining in cortex (D), DMH (E), and ARC (F). G–I, Immunohistochemistry from DT-treated AgrpDTR/+ dam on d 21 of lactation with eight pups showing NPY staining in cortex (G) but greatly reduced staining in DMH (H) and ARC (I). The areas indicated by the small squares are enlarged to reveal NPY-stained cell bodies in the ARC (F) and NPY-stained boutons in the DMH (E). Pictures for each set are from the same coronal section (approximating Fig. 46 in Ref. 49 ); all sections were stained at the same time, and pictures were acquired under the same settings.

 
We conclude from these experiments that the loss of AgRP neurons shortly after birth does not compromise the ability of females to become pregnant, give birth to a normal number of pups, increase food intake during lactation, or to nourish the pups throughout lactation. Because virtually all AgRP neurons are gone after DT treatment, and most neonatally ablated mice become resistant to subsequent challenge with DT, we propose that neonatal ablation allows other neuronal circuits involved in the control of feeding to compensate (27).

Ablation of AgRP neurons in lactating females leads to starvation
For the next experiments, either WT females or homozygous AgrpDTR/DTR females (Agrp-null) were bred with WT males and moved to cages with liquid diet available when they were obviously pregnant. Food consumption was monitored beginning at parturition. The females were injected with DT (50 µg/kg) on d 7 and 9 of lactation. Consumption of liquid diet by the WT females continued to increase during the next 5 d (Fig. 3AGo), the females maintained their body weight (Fig. 3BGo), and the pups continued to gain weight (Fig. 3CGo). By contrast, consumption of the liquid diet by the AgrpDTR/DTR females began to decrease within the first day of DT treatment and continued to decline for the next 4 d (Fig. 4AGo); their body weight steadily declined to 80% of their original weight (Fig. 4BGo), although the weight of their pups continued to increase (Fig. 4CGo). The experiment was terminated on d 12 to prevent suffering and to prepare the brains for histology. As expected, there was little NPY staining in the ARC (data not shown).


Figure 3
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  		FIG. 3. Lactation-induced hyperphagia by C57Bl/6 females (n = 6), and effect of DT treatment on dam and litter body weight (BW). A, Daily liquid diet consumption by dams. DT (50 µg/kg) was injected on d 7 and 9 (arrows). B, Body weight of dams as percentage of their weight on d 7. C, Body weight of entire litter starting at d 7.

 

Figure 4
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  		FIG. 4. Lactation-induced hyperphagia by AgrpDTR/DTR females (n = 8), and effect of DT treatment on dam and litter body weight (BW). A, Daily liquid diet consumption by dams. DT (50 µg/kg) was injected on d 7 and 9 (arrows). B, Body weight of dams as percentage of their weight on d 7. C, Body weight of entire litter starting at d 7. Note decline in food consumption, loss of body weight by dams after DT treatment, but pups continue to grow.

 
A third group of AgrpDTR/DTR females that also lacked NPY (NpylacZ/lacZ) was treated with DT on d 7 and 9 of lactation. This group of mice was included to determine whether chronic inactivation of the Npy and Agrp genes might initiate a compensatory mechanism(s) that would make the mice resistant to the sudden loss of AgRP neurons by DT treatment. In addition, these mice provide an alternative method of analyzing NPY expression because the lacZ gene was targeted to the Npy locus, allowing expression of β-galactosidase with a nuclear localization signal instead of NPY (37). These females also gradually stopped consuming the liquid diet (Fig. 5AGo), the females lost weight (Fig. 5BGo), but the pups continued to gain weight during the 5 d after DT treatment (Fig. 5CGo).


Figure 5
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  		FIG. 5. Lactation-induced hyperphagia by AgrpDTR/DTR, NpylacZ/lacZ females (n = 4), and effect of DT treatment on dam and litter body weight (BW). A, Daily liquid diet consumption by dams. DT (50 µg/kg) was injected on d 7 and 9 (arrows). B, Body weight of dams as percentage of their weight on d 7. C, Body weight of entire litter starting at d 7. Note decline in food consumption, loss of body weight by dams after DT treatment, but pups continue to grow.

 
On d 12 of lactation, when the AgrpDTR/DTR, NpylacZ/lacZ females had lost approximately 20% of their body weight, coronal sections through the hypothalamus were examined by histochemistry with X-Gal, as substrate for β-galactosidase that produces a blue precipitate. The DT-treated females had few β-galactosidase-positive cells in the ARC (Fig. 6FGo) compared with AgrpDTR/DTR, NpylacZ/lacZ females that had not been treated with DT (Fig. 6CGo), confirming nearly complete ablation. β-Galactosidase staining in the ARC was also abundant after pair feeding a lactating control female, so she lost the same body weight as a DT-treated female (Fig. 6IGo). There were no β-galactosidase-positive nuclei in the DMH in the control or the DT-treated lactating females (Fig. 6Go, B, E, and H). β-Galactosidase expression in nuclei of cortical interneurons was the same under all of the treatments (Fig. 6Go, A, D, and G).


Figure 6
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  		FIG. 6. Photomicrographs of coronal brain sections through the hypothalamus at the level of the ARC stained after histochemical staining for β-galactosidase, a marker of Npy gene expression. A–C, Histochemistry of a section from a control AgrpDTR/DTR, NpylacZ/lacZ female (not treated with DT) on d 12 of lactation, showing a few NPY-stained cell bodies in cortex (A) and ARC (C), but none in the DMH (B). D–F, Histochemistry of a section from a lactating AgrpDTR/DTR, NpylacZ/lacZ female on d 11 of lactation that had been treated with DT 4 d earlier showing loss of β-galactosidase staining in ARC (F). G–I, Histochemistry of a section from a control AgrpDTR/DTR, NpylacZ/lacZ female not treated with DT, but pair fed to the mouse in D–F on d 12 of lactation showing staining in cortex (G) and ARC (I) is comparable to that of the nonpair-fed lactating female. Pictures for each set are from the same coronal section (approximating Fig. 46 in Ref. 49 ); all sections were stained at the same time, and pictures were acquired under the same settings.

 
We conclude that DT treatment ablated most of the AgRP neurons, which resulted in the progressive loss of feeding by the lactating dam, even when the genes encoding AgRP and NPY were constitutively inactivated.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The NPY neurons in the ARC region of the hypothalamus, which also express AgRP (38), are thought to promote feeding under conditions of negative energy balance, such as fasting (39) or lactation (29), which imposes enormous caloric demands. Thus, it was surprising that inactivation of the genes for either Npy, Agrp, or both, has minimal effects on body weight regulation (24, 25, 26). However, the effect of inactivating both of these genes on lactation-induced feeding had not been investigated. The restricted expression of AgRP to hypothalamic neurons that express NPY provided the opportunity to target selectively these cells for ablation (27, 28). To our astonishment, the selective ablation of NPY/AgRP-expressing neurons in neonates had minimal effects on body weight regulation, whereas their ablation in adult mice led to rapid starvation (27). These results suggest that the loss of these AgRP neurons, before they are fully mature (40), allows compensatory mechanisms to develop that are sufficient to maintain feeding by adult mice (27). However, it was unknown whether these compensatory mechanisms would extend to conditions of extraordinary energetic demand. Although mice with a neonatal loss of AgRP neurons seem to grow normally, they are abnormal in several important ways. First, they lose their responsiveness to ghrelin (35, 36, 40, 41). Second, they have an abnormal response to refeeding after a fast (36), and third, they show extreme weight loss during social isolation (Luquet, S., Colin T. Phillips, and Richard D. Palmiter, unpublished observations). Thus, there were ample reasons to suspect that animals with targeted deletions of AgRP neurons might lack the ability to show an appropriate adaptation to the increased energy demands of lactation.

Although NPY has been implicated in the regulation of reproduction (42, 43, 44), there is little information on the role of AgRP or other transmitters, such as GABA, made by the AgRP neurons on reproduction. The AgRP neurons are situated in the ARC near neurons that express kisspeptin, which regulates GnRH production (45, 46), and tyrosine hydroxylase, which produces dopamine as a negative regulator of prolactin production and release by lactotropes (47, 48); thus, it was conceivable that the loss of AgRP neurons would impair pregnancy and/or lactation.

The first series of experiments established that ablation of AgRP neurons in neonates does not interfere with pregnancy, parturition, or lactation. The loss of feeding response to the ghrelin mimetic and the nearly complete absence of NPY-positive cell bodies in the ARC confirm previous findings that DT treatment ablates most of the neurons in the ARC that express NPY and AgRP (27, 36). We suggested previously that compensatory mechanisms emerge after neonatal AgRP neuron ablation to allow feeding in adulthood (27). The experiments described here suggest that these compensatory mechanisms extend to reproduction and lactation-induced feeding as well. Apparently, there are no hormonal or neuroendocrine signals required for pregnancy or lactation-induced hyperphagia that absolutely depend on AgRP neurons for normal physiological responses. For example, if ghrelin signaling were required, then lactation-induced hyperphagia should be compromised in the neonatally, AgRP-ablated mice.

The second set of experiments demonstrates that lactation-induced hyperphagia becomes dependent upon AgRP neurons. Ablating AgRP neurons on d 7 of lactation inhibits feeding and results in rapid weight loss by the dam. Thus, after mice develop into adults, feeding becomes dependent on AgRP neurons, i.e. the compensatory mechanisms that are effective in neonates either do not occur in the adult, or they are engaged too slowly to prevent starvation. Consequently, the first possibility mentioned in the Introduction of this paper is untenable. The consumption of liquid diet by the dam increased from approximately 12 to approximately 30 g/d by d 7 of lactation. If only the 12 g/d of prelactation consumption were dependent on AgRP neurons, then ablating AgRP neurons on d 7 should cause diet consumption to decrease from 30 to 18 g/d; however, it decreased to approximately 9 g/d (Fig. 4BGo). Furthermore, the rate at which food consumption was declining on d 5 after DT treatment was accelerating rather than approaching a plateau. Therefore, the possibility that lactation-induced hyperphagia is independent of AgRP neurons is also implausible. For animal welfare considerations, we could not let the experiment continue after the dams lost approximately 20% of their body weight. Thus, we cannot be certain that feeding would have declined to zero under these circumstances, but it is clear that the dams’ feeding response was severely impaired. The excess demand of the suckling pups probably caused the body weight of the dams to decrease to the cutoff point before they completely stopped consuming the liquid diet. Note that the litter weight continued to increase even as the dams were losing weight, which suggests that killing AgRP neurons does not impede lactation. Therefore, these experiments suggest that feeding under conditions of extraordinary energy demand, not just basal feeding, depends on AgRP neurons. Consequently, we suggest that neurons are not recruited during lactation that can compensate for the loss of AgRP neurons.

The induction of NPY in the ARC is thought to reflect a response to the reduced energy balance as the pups begin to consume milk and a response to an inductive signal that originates with sucking by the pups (29, 33). Lactation also recruits a population of neurons in the DMH of rats to express NPY, which has been postulated to supplement the feeding requirements during lactation (32, 33). POMC neurons appear to regulate the expression of NPY in the DMH by inhibiting local GABAergic interneurons that express MC4R, and this action is counterbalanced by the activity of neighboring AgRP neurons (34). The expression of NPY in the DMH is also regulated by prolactin, as reflected by the fact that inhibition of prolactin secretion with bromocriptine reduces the expression of NPY in the DMH (5, 34, 42). In the rat, NPY-expressing neurons in the DMH do not express AgRP; thus, we did not expect them to be killed by DT. To our surprise, we were unable to detect NPY-containing cell bodies in the DMH of lactating control (mice), despite the fact that cell bodies staining for NPY were obvious in the ARC, and there was extensive NPY labeling of neuronal processes and boutons in the DMH of lactating females. We were also unable to detect nuclear β-galactosidase labeling in the DMH of lactating NpylacZ/lacZ females; β-galactosidase labeling is a substitute for in situ hybridization because the gene product accumulates in the nuclei of cells that normally express NPY (37). Thus, in the mouse we could not corroborate evidence in the rat that neurons residing within the DMH are recruited to express NPY during lactation. This lack of NPY neuronal recruitment in the DMH of the mouse could reflect a biological difference between species (2). It is notable that NPY can be expressed in the DMH of mice under some circumstances (e.g. obese mice with ectopic expression of agouti or AgRP; for review, see Ref. 2). Our experiments show that virtually all NPY expression in the DMH is lost upon ablation of the AgRP neurons in the ARC, indicating that most of the NPY in the DMH is derived from these cells.

Previous experiments have established that inactivation of Npy and Agrp genes does not interfere with maintenance of energy balance (26). We extend these observations here by showing that mice constitutively lacking these two peptides become pregnant and increase their feeding during lactation similar to WT mice. Furthermore, these mice display the same starvation phenotype as mice expressing NPY and AgRP when AgRP neurons are ablated. It has been suggested that chronic loss of NPY, as in complete Npy gene knockout, elicits a compensatory mechanism that allows mice to feed independently of this orexigenic peptide (25, 39). The same argument could apply to the chronic loss of AgRP after gene inactivation. If the compensatory mechanism elicited by the loss of these neuropeptides is the same as that that occurs after ablation of AgRP neurons in the neonate, then we would have predicted that lactating AgrpDTR/DTR, NpylacZ/lacZ females would have compensated and, therefore, would be resistant to starvation after ablation of AgRP neurons. Clearly, the mice lacking both AgRP and NPY remained sensitive to AgRP neuron ablation (Fig. 5BGo). Thus, the postulated compensatory mechanism elicited by the loss of these neuropeptides is different than those elicited by the loss of AgRP neurons. We conclude that the loss of something other than those two neuropeptides initiates the circuit-based compensatory mechanism when AgRP neurons are ablated in the neonate, and we suggest that it may be GABA.


   Acknowledgments
 
We thank Robert Steiner and Serge Luquet for advice during the planning stages of this project, Glenda Froelick for help with histology, Lisa Beutler for help with β-galactosidase detection, Qi Wu for establishing the double-knockout colony of mice, Don Marsh and Merck & Co., Inc., for supplying the ghrelin mimetic, and Robert Steiner for helpful comments on the manuscript.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online November 1, 2007

Abbreviations: AgRP, Agouti-related peptide; ARC, arcuate nucleus; DMH, dorsal medial hypothalamus; DT, diphtheria toxin; DTR, diphtheria toxin receptor; GABA, {gamma}-aminobutyric acid; MC4R, melanocortin-4 receptor; NPY, neuropeptide Y; POMC, proopiomelanocortin; WT, wild type.

Received August 21, 2007.

Accepted for publication October 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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