Many mouths to feed: The control of food intake during lactation

Frontiers in Neuroendocrinology 33 (2012) 301–314

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Review

Many mouths to feed: The control of food intake during lactation Barbara Woodside a,⇑, Radek Budin a, Martin K. Wellman b, Alfonso Abizaid b a b

Center for Studies in Behavioral Neurobiology/Groupe de recherches en neurobiologie comportementale, Concordia University, Montreal, Canada Department of Neuroscience, Carleton University, Ottawa, Canada

a r t i c l e

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Article history: Available online 18 September 2012 Keywords: Energy balance Mother-young interactions Hypothalamus Prolactin Estrogen Leptin Ghrelin

a b s t r a c t Providing nutrients to their developing young is perhaps the most energetically demanding task facing female mammals. In this paper we focus primarily on studies carried out in rats to describe the changes in the maternal brain that enable the dam to meet the energetic demands of her offspring. In rats, providing milk for their litter is associated with a dramatic increase in caloric intake, a reduction in energy expenditure and changes in the pattern of energy utilization as well as storage. These behavioral and physiological adaptations result, in part, from alterations in the central pathways controlling energy balance. Differences in circulating levels of metabolic hormones such as leptin, ghrelin and insulin as well as in responsiveness to these signals between lactating and nonlactating animals, contribute to the modifications in energy balance pathways seen postpartum. Suckling stimulation from the pups both directly, and through the hormonal state that it induces in the mother, plays a key role in facilitating these adaptations. Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.

1. Introduction In the past three decades we have witnessed a dramatic growth in our understanding of the mechanisms through which the brain regulates energy state. Recent research on the central control of energy balance has focused on groups of neurons secreting peptide neurotransmitters that work in concert to alter feeding behavior, metabolic rate and energy expenditure (Abizaid et al., 2006). In this paper we focus on how these mechanisms are modified to respond to the greatest metabolic challenge that mammalian females must face: lactation. The birth of young presents the mammalian mother with an array of new behavioral and physiological challenges. These challenges are particularly intense amongst species that give birth to altricial young, which have very limited motor and thermoregulatory abilities and are completely dependent on the mother for nutrients, predator protection and warmth (Numan and Insel, 2003). In some cases, as in small rodents, the offspring may also depend on their mother for the stimulation that induces urination and defecation (Numan and Insel, 2003). Given the multiplicity of tasks required to successfully rear progeny, it is perhaps not surprising that many systems are recruited to accomplish them and that they entail numerous neural and neurochemical adaptations in the maternal brain (Numan and Insel, 2003; Numan and Woodside, 2010). Recently, considerable attention has been ⇑ Corresponding author. Address: CSBN/GRNC, Concordia University, 7141 Sherbrooke St., W., Montreal, Quebec, Canada H4B 1R6. E-mail address: [email protected] (B. Woodside).

focused on the role of the perinatal nutritional environment in programming neural circuits controlling energy balance and thus the risk for and resilience to metabolic disorders in the developing young (Sullivan et al., 2011). Because the nutritional environment of offspring depends on the mother’s own food intake and energy balance it seems fitting to explore the mechanisms through which these parameters are controlled during lactation. 2. Changes in energy intake and expenditure in lactation In small rodents, altricial young are entirely dependent on their mothers for food for at least the first 2 weeks postpartum and during this time the litter can grow to a mass that exceeds that of the mother herself (Woodside et al., 2008). To support this rapid pup growth, lactating females typically show a large increase in food intake. In rats for example, at the end of the second week postpartum dams nursing eight pups produce more than 70 ml of milk/day (Brommage, 1989) and their food intake can reach as much as 80 g/ day compared to the 20–22 g eaten by cycling females (Woodside et al., 2008). Food intake increases with litter size but not sufficiently to ensure that pups from large litters grow at the same rate as those reared in small litters (Leon and Woodside, 1983). Interestingly, although pups raised in litters of four grow faster than those reared in litters of eight, a ceiling of food intake is not reached when nursing eight pups because food intake increases even further with larger litter sizes (Leon and Woodside, 1983). Rather, the data suggest that mothers do not defend a maximal pup growth for their offspring and that the amount of milk delivered is a function of multiple factors. For example, producing

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enough milk for a litter of eight pups entails a doubling of the mother’s metabolic rate. It has been proposed that it is the mother’s inability to deal with the heat generated by these processes that sets a limit on food intake and milk production at least in some species (Krol et al., 2011; Paul et al., 2010; Speakman and Krol, 2011). In addition to the number of pups nursed, both the quality of the food available and ambient temperature affect amounts ingested by lactating females. If the diet is diluted with cellulose, lactating females increase their intake whereas high quality diets result in lower gram intake but increased pup growth (Leon and Woodside, 1983). In rats, increasing ambient temperature (Ta) results in a decrease in food intake (Leon and Woodside, 1983) and a similar negative relationship between Ta and food intake is seen in Siberian hamsters (Paul et al., 2010). Interestingly when Siberian hamster dams nurse larger litters at a low temperature their food intake is even greater than when faced with the challenge of either nursing large litters or a low ambient temperature alone (Paul et al., 2010), further illustrating that food intake does not reach maximal levels when nursing larger number of pups. The 350% increase in food intake that is typical of lactating rats is a feat that entails changing the timing and size of meals. In early lactation increased food intake is accomplished by increasing meal size, however, as the demands of the young increase, the number of meals does also so that by the peak of lactation more meals are eaten during the light part of the day/night cycle (Strubbe and Gorissen, 1980). Studies in lactating rats have demonstrated that increases in food consumption are accompanied by changes in diet selection. Richter and his colleagues (1938) used the cafeteria diet paradigm to study nutrient selection and showed that the diet preferences of dams differed from those of cycling rats and adapted to match the nutrient requirements of the developing young. Thus, during lactation rats ate more protein as well as fat and increased their intake of some micronutrients notably salt and calcium. Some of these findings have been replicated using simpler paradigms (Cohen and Woodside, 1989; Millelire and Woodside, 1989; Woodside and Millelire, 1987). Changing the patterns of food intake are not the only means through which mammalian females meet the energetic demands of their young. Female rats increase fat stores during pregnancy and these are subsequently utilized during lactation (Naismith et al., 1982). When food availability is restricted maternal fat stores become an important means of maintaining pup growth (Abizaid et al., 2004). The ability of female rats to utilize body fat to subsidize the energetic costs of lactation and to reduce the foraging demand can seen by the results of a recent study from our laboratory. We contrasted food intake and body fat loss, as reflected in changes in circulating leptin levels, among multiparous and age matched primiparous rats and young (lighter) primiparous females (Cerone and Woodside, unpublished observations). In this study, multiparous and age matched primiparous females were from the same cohort and had similar body weights at conception whereas the younger cohort weighed approximately 100 g less at mating. Rats in all groups gained similar amounts of weight, but those in the multiparous and age-matched primiparous groups had higher circulating leptin levels one day after giving birth and showed a much larger decrease in leptin levels than the rats in the young primiparous group by Day 14 postpartum, indicative of a greater fat loss in the older rats across the first 2 weeks postpartum. Food intake relative to body weight was lower in the multiparous and agematched primiparous groups than in the young primiparous group suggesting that in the heavier females body fat was utilized in lieu of ingested food. Not all small rodents store fat during pregnancy. For example, Siberian and Syrian hamsters tend to lose fat stores in pregnancy

but females of these species increase food hoarding during both pregnancy and lactation (Bartness, 1997; Wade et al., 1986). Indeed absence of a food hoard during lactation can increase infanticide in Syrian hamsters (Miceli and Malsbury, 1982a,b). These data together with the fact that heavier hamsters cannibalize fewer of their offspring than lean mothers again suggests an equivalency between external and internal food stores (Schneider and Wade, 1989). This pattern of using stored nutrients, whether internally in the form of white adipose tissue or externally in the form of a food hoard, to subsidize nutrient costs of lactation is also seen for calcium. The skeleton can be regarded as a calcium store with both rats and women exhibiting an increase in skeletal calcium over the course of pregnancy (Miller et al., 1986). This is facilitated by increases in 1,25-dihyroxycholcalciferol (1,25(OH)2D3) levels in the small intestine resulting in a doubling of the absorption of both calcium and phosphorus across the gut wall (Robinson et al., 1982). Skeletal calcium then decreases across lactation and this loss is accentuated in the absence of appropriate dietary calcium (Brommage, 1989). There do not seem to be easily accessible stores of protein for use during lactation, however. Although there is some evidence of increases in maternal lean body mass early in pregnancy this probably serves to support fetal growth. Thus, there seems to be little to protect the developing young from maternal protein insufficiency in lactation, which frequently results in retarded pup growth (Martin-Gronert et al., 2008). The changes in food intake that accompany lactation are complemented by increases in the absorptive capacity of the gut (Cripps and Williams, 1975). Energy expenditure in processes not associated with maternal care are also decreased in lactating rats. For example, there is a reduction in sympathetic activation of brown fat thermogenesis in both lactating rats and mice that recovers when the young are weaned (Trayhurn, 1985, 1989). In addition to a decrease in uncoupling protein (UCP) UCP1 mRNA expression in brown adipose tissue (BAT) there is also a decrease in UCP3 in both brown fat and skeletal muscle (Xiao et al., 2004a,b). However, the absence of brown fat thermogenesis does not completely offset the thermogenic cost of the increased metabolism associated with milk production and lactating rats. Siberian hamsters and mice all have an elevated body temperature that some have suggested influences mother-litter interactions (Leon et al., 1978; Paul et al., 2010; Scribner and Wynne-Edwards, 1994a,b). Energy availability for the suckling litter is also enhanced by a decrease in maternal physical activity (Grota and Ader, 1974) and by delaying energy investment in competing reproductive episodes (Bronson, 1985). Thus, implantation of blastocysts resulting from mating during the postpartum estrous is delayed with the length of this suppression of implantation depending on litter size and energy availability (Lamming, 1978; Woodside et al., 1987, 1981). In the absence of a postpartum mating, ovulation is suppressed and again the length of anovulation will depend on the energetic demands on the mother (Smith and Grove, 2002; Woodside, 1991). Finally, a selective insulin resistance in maternal tissues such as muscle and fat is observed, whereas the mammary gland remains insulin sensitive ensuring that metabolic fuels are channeled to this site for milk production (Vernon, 2005; Vernon and Pond, 1997). In sum, the need to provide milk to meet the evergrowing demands of the developing young requires the coordinated reorganization of both patterns of energy intake and expenditure that ensures that all available energy is channeled towards milk production. Although the loss of body fat during nursing suggests that lactating dams do enter negative energy balance, their level of food intake clearly differentiates them from the more traditional models

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of negative energy balance such as food restriction or food deprivation suggesting that lactation places a unique set of challenges on the energy balance mechanisms. In the sections below we describe the systems of the brain that are critical for maintaining energy balance, how metabolic hormones influence these systems, and how they are changed during pregnancy and lactation so as to meet the energetic challenges of motherhood.

3. Peripheral and central control of energy balance Traditionally, research on the control of food intake and energy expenditure has focused on the hypothalamus and the discovery that circulating signals of energy balance target neurons within this area has supported this contention. However, it is clear that these signals also influence neural activity and behavior through their effects in a number of brain areas including the neuronal populations in the midbrain typically associated with reinforcement and reward as well as cell groups within the brainstem (Abizaid, 2009). Thus, the control of energy balance rests on the coordinated action of neuronal populations in multiple brain areas (Abizaid and Horvath, 2008).

3.1. Peptidergic circuits in the hypothalamus Within the hypothalamus the arcuate nucleus (ARC) has emerged as a critical site for the regulation of energy balance (Abizaid et al., 2006) (see Fig. 1A). Within the ARC, two cell populations play a particularly critical role. One of these secretes neuropeptide Y (NPY) and agouti related peptide (Agrp), whereas the second group secretes pro-opiomelanocortin (POMC) and cocaine amphetamine related transcript (CART). NPY and Agrp are peptides that potently increase food intake and decrease energy expenditure, whereas a-melanocyte stimulating hormone (a-MSH; a peptide derived from the POMC precursor) and CART increase energy expenditure and reduce food intake. These two cell groups are now known as the hypothalamic melanocortin system because both Agrp and a-MSH target melanocortin 3–4 receptors (MC3/ 4), with a-MSH being a natural agonist, and Agrp being a natural antagonist for this receptor (Abizaid et al., 2006). The importance of this system is highlighted by work showing that animals with natural or targeted mutations of the melanocortin receptors, or to the POMC gene become morbidly obese (Abizaid et al., 2006). Similarly, optogenetic stimulation of Agrp cells or exogenous administration of Agrp produces dramatic feeding responses, whereas selective ablation of Agrp cells produce animals that die of starvation (Aponte et al., 2011; Luquet et al., 2005; Wu et al., 2008). The projections of NPY/Agrp and POMC/CART neurons and their receptors are found throughout the hypothalamus and are particularly dense in the lateral hypothalamus (LH) and in the hypothalamic paraventricular, and dorsomedial nuclei (PVN and DMH respectively) (Abizaid and Horvath, 2008). Within the LH and DMH, peptides secreted from the ARC modulate the release of other hypothalamic peptides such as orexin and melanin concentrating hormone (MCH), both of which are implicated in a number of processes including arousal, food seeking, and food reward (Adamantidis and de Lecea, 2008; Guyon et al., 2009). Within the PVN, peptides secreted from the ARC alter the release of corticotropin releasing hormone (CRH) and thyrotropin releasing hormone (TRH). Both of these neuroendocrine peptides dramatically alter food intake and metabolic rate through their actions on the pituitary gland as well as brain stem sympathetic descending pathways that regulate metabolic rate, temperature and adiposity (Fulton et al., 2002).

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The opposing effects of a-MSH and Agrp represent only one level of regulation of the melanocortin system. Indeed, it is now evident that NPY/Agrp cells and POMC/CART cells have reciprocal connections that can alter each other’s activity in relation to changes in energy state. For instance NPY/Agrp neurons have GABAergic projections that directly inhibit the activity of POMC neurons (Kalra et al., 1999). In contrast, the inhibitory effects of POMC neurons on NPY/Agrp cells is mediated by the release of opioid peptides (Yang et al., 2011). In addition, the ARC retains a high degree of synaptic plasticity, and this property may be required for optimal homeostatic regulation (Horvath and Diano, 2004; Kokoeva et al., 2005). The redundancy and plasticity and complexity in these hypothalamic circuits may reflect their importance for the survival of organisms, and allow for adaptations to environments with fluctuating energy resources and demands. The ARC is considered to be a circumventricular organ thought to be less sheltered by the blood brain barrier, and as such to have direct access to higher titers of circulating hormones and metabolic fuels (Horvath and Diano, 2004). Not surprisingly, ARC neurons contain receptors for practically every single circulating hormone known to affect appetite including leptin, ghrelin, insulin, estrogen, progesterone, prolactin, and cytokines (Abizaid et al., 2006). Hormones that produce anorectic effects like leptin and estrogen act on the ARC to favor the secretion of a-MSH and CART, while inhibiting the release of NPY and Agrp. In contrast, anabolic hormones like ghrelin or corticosterone stimulate the release of NPY and Agrp while decreasing the release of a-MSH and CART (Abizaid et al., 2006). These hormones also have very rapid effects on ARC neurons suggesting that activation of hormone receptors in this area alter the stimulation thresholds of these neurons either via changes in their membrane sensitivity to classical neurotransmitters, or by altering sensitivity to nutritional signals such as glucose or free fatty acids (Cowley et al., 2001; He et al., 2006; Liu et al., 2011). Evidence for the latter comes from work that shows not only that NPY/Agrp and POMC/CART cells change their firing properties in response to glucose, fatty acids and drug compounds that inhibit the oxidation of these energy substrates, but also by studies demonstrating that leptin and ghrelin have downstream signaling pathways that influence glucose or fatty acid utilization in the hypothalamus (Andrews et al., 2008; Lam et al., 2005). Finally, both leptin and ghrelin signaling has been linked to the synaptic plasticity that is required for energy regulation (Pinto et al., 2004). There are multiple intracellular signaling cascades underlying hormonal and nutrient sensing in the hypothalamus including downstream signaling cascades that are elicited by hormones like leptin, insulin and ghrelin upon action on their respective receptors. There are some intracellular mechanisms, however, that allow cells to monitor energy availability from a variety of hormones and nutrients, and as such, may be critical for the optimal regulation of energy balance. Of these, the AMP-activated protein kinase (AMPK) has been identified as critical for energy regulation in eukariotic cells, as its activity is modulated by changes in the AMP/ATP or ADP/ATP ratio (Hardie et al., 2012). Within the hypothalamus, a reduction of AMPK activity is associated with decreased feeding and increased energy expenditure, whereas increased AMPK activity is followed by increased feeding responses. It is therefore not suprising that leptin and insulin decrease, whereas ghrelin increases AMPK activity in the hypothalamus (Hardie et al., 2012; Kohno et al., 2008). Similarly, viral induction of overexpression of the a2 isoform of the AMPK protein also leads to increased food intake and decreased responsiveness to anorectic signals like leptin. It is unclear, however, whether AMPK activity within the ARC is responsible for these effects as mice with selective mutations to the a2AMPK isoform in either AGRP or POMC cells show only modest alterations in food intake following leptin treatment compared to control WT mice (Claret et al., 2007). This has led some to

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(A)

(B)

(C)

Fig. 1. (A) Hypothalamic nuclei implicated in the control of energy balance. Peripheral signals alter the activity of neurons in the arcuate nucleus (ARC) of the hypothalamus. Orexigenic hormones like ghrelin will increase the secretion of anabolic peptides such as neuropeptide Y (NPY) and agouti related peptide (AGRP), whereas leptin and insulin will increase the activity of catabolic peptides such as aMSH. These first order neurons stimulate other hypothalamic regions to ultimately modulate a number of regulatory processes that include changes in arousal and feeding behavior, metabolic rate, and cognitive processes associated with hunger. (B) Like neurons in the hypothalamus, VTA dopaminergic neurons are sensitive and respond to metabolic hormones and nutrients. This results in changes in dopamine release in nucleus accumbens, a physiological mechanism important for cognitive and behavioral processes linked to food motivation. (C) Brain stem nuclei including the lateral parabrachial nucleus (LPBN), the area postrema (AP), and the nucleus of the solitary tract (NTS) are also targets of peripheral hunger and satiety signals. In turn these areas have reciprocal connections with various hypothalamic nuclei forming a complex network of brain regions that are important for the integration of signals from circulating metabolic hormones, nutrients and ascending visceral information conveyed through the vagus nerve. Abbreviations: Paraventricular nucleus (PVN), Dorsomedial nucleus of the hypothalamus (DMH), Ventromedial Nucleus of the hypothalamus (VMH), Lateral hypothalamus (LH), fornix (fx), Median eminence (ME), third ventricle (3v), prolactin (PRL), preopiomelanocortin (POMC), corticotropic releasing hormone (CRH), thyroid releasing hormone (TRH) melanocortin concentrating hormone (MCH), orexin (ORX), GABA (gamma amino butyric acid), oxytocin (OT) and norepinephrine (NE).

suggest that AMPK signaling occurring in presynaptic cells projecting to the ARC determines how the melanocortin system will work under conditions of energy surplus or surfeit (Hardie et al., 2012; Yang et al., 2011). Moreover, AMPK signaling in the presynaptic inputs mediates the plastic changes that seem critical for optimal modulation of energy balance (Yang et al., 2011). Nevertheless, the source of these inputs remains to be determined, as is the contribution of AMPK in other hypothalamic and extrahypothalamic brain regions associated with the regulation of feeding and energy balance. In addition, there are other intracellular protein complexes that are capable of gauging changes in nutrient availability like the mammalian target for rapamycin (mTOR), which alone or in combination with the AMPK pathway alter the activity of NPY/ AGRP neurons to modulate feeding responses (Stefater and Seeley, 2010). Finally, recent data suggest that signaling pathways associated with inflammatory responses are stimulated by excess levels of nutrients, particularly free fatty acids, directly affecting ARC POMC and NPY/AGRP neurons (Zhang et al., 2008). Although the melanocortin system may be important for regulating body weight and glucose metabolism, recent work suggests that the feeding responses elicited by stimulation of ARC Agrp

neurons are independent of the melanocortin receptor stimulation (Wu et al., 2008). Thus, although selective ablation of Agrp neurons leads to starvation in mice, this effect is independent of the effects of Agrp on melanocortin receptors (Wu et al., 2009). Rather, it appears that the release of GABA from ARC NPY/Argp neurons into the brain stem parabrachial nucleus is critical for the feeding response that follows stimulation of NPY/Agrp neurons (Wu et al., 2009). 3.2. Brain stem contribution to feeding and energy expenditure It is well established that information provided by circulating metabolic signals, including that transmitted by glucocorticoids, are integrated in a number of brain stem nuclei (Berthoud et al., 2006; Grill and Kaplan, 2001, 2002) (see Fig. 1C). The brain stem is also intimately associated with autonomic responses that follow the activation of the stress axis by psychogenic, metabolic or immunological challenges (Habib et al., 2001; Smith and Vale, 2006; Stratakis and Chrousos, 1995). Within the brain stem, the three divisions of the dorsal vagal complex (DVC) play a key role in integrating metabolic signals, afferent signals relaying

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interoceptive information from the digestive system via the vagus nerve, and information transmitted from cranial nerves that carry taste, texture, and sensory-motor signals from the mouth (Grill and Kaplan, 2001, 2002). Within the DVC itself, the nucleus of the solitary tract (NTS) is the main target for information ascending from the gut via the vagus nerve and the enteric nervous system. The NTS contains noradrenergic neurons that project to, and modulate the activity of, hypothalamic and limbic structures implicated in feeding, including the PVN and central nucleus of the amygdala (CeA) Grill and Kaplan, 2001, 2002; Ritter et al., 2000; Treece et al., 2000. The area postrema (AP), a second division of the DVC, lies outside the blood brain barrier and because of this is especially sensitive to blood borne nutritional signals including those that are relayed by hormones like leptin, insulin, cholecystokinin (CCK) and ghrelin. In addition the AP is also sensitive to changes in circulating plasma levels of glucose (Contreras et al., 1982; Edwards and Ritter, 1982; Gilg and Lutz, 2006; Hyde and Miselis, 1983; van der Kooy, 1984). The AP transduces these signals and conveys them primarily to the NTS where they are integrated with ascending visceral signals, and also directly into the PVN, where they may elicit feeding and autonomic responses (Cai et al., 1996; Castaneyra-Perdomo et al., 1992; Diaz-Regueira and Anadon, 1992; Ellenberger and Feldman, 1990; Ferguson, 1991; Woulfe et al., 1990). Interestingly, the DVC contains the only group of POMC producing cells outside of the hypothalamus (Cone, 2005). These cells are located in the NTS, and MC3 and MC4 receptors are found in the NTS as well as in the adjacent dorsal motor nucleus of the vagus (DMX), the third division of the DVC. Infusion of melanocortin receptor agonists into the DMX result in marked reductions in food intake at doses that are typically not effective when administered into the cerebroventricular systems, clearly suggesting that there is a melanocortin system in the brain stem that works in parallel to that described in the ARC (Li et al., 2007). The contribution of the brain stem to the regulation of food intake has been evaluated by Grill and his associates using the decerebrate rat model. In this model the brain stem is separated from the hypothalamus by complete transection (Grill and Kaplan, 2001, 2002). This model has been used effectively to demonstrate that the brain stem alone can maintain ingestive behaviors and responses to short term changes in nutritional signals (Grill and Kaplan, 2001, 2002). In addition to the regulation of feeding responses, the brain stem plays a key role in the generation of autonomic responses, including increases in blood pressure, respiration, glucose release from the liver, vasoconstriction, lipolysis, and thermogenesis (Bamshad et al., 1998; Dunn et al., 2004; Krukoff, 1998). Like the ARC, the NTS also projects to the parabrachial nucleus and these projections are important to decrease appetite in response to increases in satiety signals from the gastrointestinal system, or in response to malaise (Wu et al., 2012). Given that NPY/ Agrp neurons release GABA into the parabrachial nucleus to increase appetite, it is clear that the balance between NTS activation of the PB and GABA inhibition from the ARC represent yet another loop regulating appetite and food ingestion (Wu et al., 2012). In fact the parabrachial nucleus is currently emerging as an important second order region integrating information from the hypothalamus, various sensory inputs including visceral, metabolic (i.e. nutrient sensing) and taste inputs to affect behavioral feeding responses (Wu et al., 2012). 3.3. Midbrain and the regulation of feeding The midbrain contains a number of cell groups that are implicated in multiple brain functions that include reward seeking, learning and memory processes, affective states and the generation of locomotor responses (Berridge and Robinson, 1998; Berridge

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et al., 2009). Their role in the modulation of feeding responses is considered within the context of these processes. Dopaminergic cells of the ventral tegmentum (VTA) and substantia nigra have been implicated in the modulation of food intake through their projection to the striatum, prefrontal cortex, hippocampus and amygdala (Fields et al., 2007; Margolis et al., 2006; Robinson et al., 2007) and because the VTA is itself a target of circculating signals of energy balance (see Fig. 1B). The dopaminergic projections from the VTA to cells in the nucleus accumbens, has received a great deal of attention because of their importance in mediating behaviors geared to obtaining both natural rewards such as food and sex, as well as their involvement in the avoidance of aversive stimuli (Baler and Volkow, 2006; Volkow and Wise, 2005; Wang et al., 2004; Wise, 2004, 2005, 2006). Moreover, this system is also stimulated by drugs of abuse and plays a key role in the development of addiction to these substances (Volkow and Wise, 2005; Wise, 2004, 2005, 2006). Destruction of this pathway results in hypophagia similar to that reported in animals with LH lesions (Oltmans et al., 1977; Salamone et al., 1993, 1990; Zigmond and Stricker, 1972), and dopamine deficient mice die of starvation unless they are given exogenous L-DOPA (Palmiter, 2007). Not surprising is the fact that the nucleus accumbens projects to the LH and modulates food intake through this pathway (Kelley, 2004). Fasting increases dopaminergic tone and re-feeding produces an increase in dopamine release into the nucleus accumbens (Wang et al., 2002). Anticipation of food and other rewards also produce dopamine release from the VTA into the accumbens (Blackburn et al., 1992; Richardson and Gratton, 1996). Projections from the VTA to limbic and prefrontal cortical regions are thought to be involved in learning and decision making although much work needs to be conducted to better understand the role of these pathways in the regulation of feeding (Phillips et al., 2003). The dopaminergic projections from cells in the substantia nigra to the dorsal striatum are associated with the generation of movement, and destruction of this pathway leads to severe motor impairments as occurs in Parkinson’s disease (Fuxe et al., 2006). Nevertheless, recent studies have also linked this circuit with the generation of operant responses to obtain food. In these studies, dopamine deficient mice showed deficits in learning and low levels of operant responses for food compared to their wild type littermates (Palmiter, 2007). Restoration of dopamine signaling in the dorsal portion of the striatum, a region selectively targeted by the substantia nigra, also fully restored the levels of operant responses to obtain food in dopamine deficient mice (Palmiter, 2007). These data have prompted many to re-evaluate the role of the substantia nigra in modulating food seeking behaviors. Finally, serotonergic components of the raphe nuclei have been implicated in the regulation of food intake (Halford et al., 2007; Heisler et al., 2003). Indeed, there are serotonergic projections to hypothalamic and limbic centers involved in the regulation of food intake (Brown and Molliver, 2000; Heym and Gladfelter, 1982; Jankowski and Sesack, 2004; Silva et al., 2002; Vertes et al., 1999). Several lines of evidence demonstrate that serotonin has an inhibitory effect on food intake, including clinical and experimental data on the anorectic effects of serotonin reuptake inhibitors (SSRIs) Halford et al., 2007. The effects of serotonin on food intake and metabolic function are mediated at least in part through the stimulation of POMC cells in the hypothalamic ARC, as well as through direct effects of serotonin onto cells of the PVN (Heisler et al., 2003). The role of these midbrain aminergic groups in regulating energy balance has recently been reconsidered in light of evidence that they are sensitive to direct stimulation by metabolic hormones and substrates like glucose (Abizaid et al., 2006; Fulton et al., 2006; Hommel et al., 2006; Levin, 2000; Orosco et al., 1991). Indeed, receptors for glucocorticoids as well as receptors

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for ghrelin, leptin, and insulin are found in these regions, and direct application of these hormones onto these regions can modulate feeding in rodents (Abizaid et al., 2006; Carlini et al., 2004; Fulton et al., 2006; Guan et al., 1997; Harfstrand et al., 1986; Hommel et al., 2006; Naleid et al., 2005; Zigman et al., 2006). 4. The impact of lactation on energy balance pathways Given the energetic demands of providing milk for offspring, one would expect some changes in both the orexigenic and anorexigenic pathways described in the previous section to occur in the brains of lactating rats and that is exactly what is observed. The expression of POMC and CART mRNA within the ARC is reduced in rats in midlactation (Days 10–13 postpartum) (Mann et al., 1997; Smith, 1993) whereas the levels of mRNA for NPY and AGRP are increased (Chen et al., 1999; Smith, 1993). Recent work shows that the increase in Agrp expression in the ARC may be of particular importance for lactational hyperphagia. In particular, selective ablations of Agrp neurons dramatically attenuate feeding in pregnant and lactating mice without affecting pregnancy, parturition or lactation itself (Phillips and Palmiter, 2008). Whether this effect is dependent on melanocortin signaling, or is mediated through GABAergic effects on the parabrachial nucleus is yet to be determined. In addition to alterations in neuropeptide expression within the ARC, lactating rats also show NPY mRNA expression within the non compact zone of the dorsal medial hypothalamus (DMH), a site where NPY production is not observed in food restricted or deprived rats, but is seen in mice with targeted deletions of the melanocortin 4 receptor and those with diet-induced obesity (Li et al., 1998). Similarly, induction of expression of the precursor peptide for melanin concentrating hormone (ppMCH), as well as the protein itself (MCH), within the medial preoptic area (MPOA) has also been reported towards the end of lactation (Day 19 postpartum) Knollema et al., 1992; Rondini et al., 2010. MCH potently increases food intake, but it has been suggested that its role in the MPOA during late lactation is to maintain the suppression of ovulation in the face of declining suckling stimulation from the pups at this time (Rondini et al., 2010).

stimulation induced Fos expression in a number of brain stem areas implicated in the regulation of food intake and sympathetic activation, including the lateral portion of the parabrachial nucleus (Li et al., 1999). Given the emerging role of the lateral parabrachial nucleus in integrating visceral, metabolic and taste information with hypothalamic outflow, these data suggest that sensory input from nursing young may have a direct influence on all of these processes. Considerable attention has been paid to the role of midbrain systems and in particular to the DA projections from the VTA to NAcc in maternal motivation. For example, the seminal work of Numan and colleagues (Numan, 2007) has revealed the importance of this system in triggering the onset of maternal behavior. The work of other researchers notably Morrell and Fleming and their groups have examined how this system is differentially activated in response to exposure to pups, food or cocaine in mothers compared to cycling rats (Afonso et al., 2009; Fleming et al., 1994; Mattson and Morrell, 2005). Results of these studies indicate that basal DA tone is reduced immediately postpartum and that very early in lactation the DA response to food presentation is lower in mothers than in virgin females (Afonso et al., 2009). Moreover, whereas in the early postpartum mothers prefer cues associated with pups to those associated with cocaine, relative preference for these cues is reversed at later stages of lactation (Mattson et al., 2003). Whether these changes in preference for pups over other reinforcers influence the way in which females share their time between nursing and other offspring-associated behaviors like anogenital licking and activities which entail leaving the nest site such as foraging and sleeping, however, is not clear. Overall, the changes in energy balance systems observed in lactating rats are such that orexigenic pathways are potentiated whereas those that reduce food intake and/or increase energy expenditure are suppressed (see Fig. 2). Such modifications are similar to those that have been observed in food restricted or food deprived rats and might be expected in states in which energy expenditure is greater than energy intake. However, there are some key differences, between food restricted and lactating rats. First, there is evidence for changes not only in levels of circulating signals of energy balance but also in the response to those signals. Second, changes in energy balance pathways are not solely

4.1. Potential contributions of changes in midbrain and brainstem systems to food intake during lactation Little is known about the role of the brain stem in modulating appetite during lactation. There are, however, a number of changes within brain stem nuclei that might contribute to lactational hyperphagia. For instance, feeding and neuronal responses in the brain stem to drugs that prevent the utilization of glucose or free fatty acids are attenuated in lactating females compared to cycling females (Abizaid and Woodside, 2002). This suggests that nutrient sensing in lactating rats is altered, or that more circulating nutrients are available for utilization thereby diminishing the effects of these drugs through competition for entry into cells. The effects of satiety signals in the brain stem may also be altered. For instance, the effects of CCK on appetite are mediated in part by the stimulation of prolactin releasing peptide neurons in the NTS (Bechtold and Luckman, 2006). Animals with targeted deletions of this peptide show increased food intake, primarily because their feeding bouts are longer. Lactating rats show decreased expression of prolactin releasing peptide in the brain stem, suggesting that meal duration in lactating rats may be determined, in part, by these changes in this region (Morales and Sawchenko, 2003). In addition to hormones, the suckling stimulus itself may modulate responses of the brain stem to changes in nutrients and satiety signals. Indeed, Smith and her colleagues found that suckling

Fig. 2. A comparison of the relative influence of peripheral hormones on energy balance systems within the ARC between nonlactating (left) and lactating (right) rats. Lactation is associated with an increase in PRL and glucocorticoid secretion while decreasing plasma leptin, insulin, and estrogen. This, as well as an increase in hypothalamic sensitivity to ghrelin, leads to an overall increase in the expression of NPY and AGRP in the ARC favoring an increase in orexigenic signals. Abbreviations as in Fig. 1A.

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dependent on the energetic drain associated with milk production and delivery. Third, lactating rats have a unique hormonal profile that includes alterations in levels of peptide and steroid hormones that themselves can influence energy balance systems. In this section we review the evidence for these differences and discuss how they might be expected to influence the central control of energy balance. 4.2. Changes in circulating metabolic hormones and their effects The last several years have seen an explosion in the identification of peripheral hormones that have been shown, or proposed, to contribute to the control of metabolism. The list of such signals now includes insulin, leptin, ghrelin, adiponectin, resistin, obestatin and many others. Although there is strong evidence for a central effect of many of these signals on energy balance pathways, lactation associated changes in circulating levels of and sensitivity to these signals have only been documented for leptin and ghrelin. Circulating leptin levels decline with the decrease in fat stores (Brogan et al., 1999; Johnstone and Higuchi, 2001; Woodside et al., 2000) that are observed in mid lactation and peripheral insulin levels are also decreased postpartum (Denis et al., 2003). Although the reduction in circulating leptin reported during lactation might be expected to lead to an increase in food intake, there is considerable evidence to suggest that the restoration of circulating levels to those seen in cycling rats is not sufficient to reduce food intake in lactating rats. For example, Xu et al. (2009b) demonstrated that food intake was not affected following peripheral administration of leptin at doses designed to mimic levels observed in cycling rats. This manipulation was sufficient to increase POMC mRNA expression in the arcuate nucleus but had no effect on Agrp and NPY mRNA expression levels (Xu et al., 2009b). Our own studies have shown that chronic subcutaneous infusion of a much higher dose of leptin produced only a transient decrease in food intake when administered on Day 8 of lactation (Woodside et al., 2000). Similarly, intravenous administration of either 0.2 or 0.4 mg of leptin did not suppress food intake in lactating rats although these same doses were effective in cycling rats (Suzuki et al., 2010). Together these data argue for at least a partial insensitivity to peripheral leptin levels during the postpartum period. This may not be a general characteristic of lactation in all small rodents, however. Results of studies in lactating Brandt voles in which various doses of leptin were administered via chronic subcutaneous infusion from Days 10 to 17 of lactation showed that leptin administration was sufficient to reduce hypothalamic NPY and Agrp gene expression as well as to increase POMC expression. The highest dose of leptin administered also significantly decreased food intake (Cui et al., 2011). Whether the differences in the ability of peripherally administered leptin to modulate POMC mRNA levels and food intake that is seen between lactating voles and rats represents a species difference or variation in time, dose and/or duration of leptin administration remains to be determined. Because lactating rats enter the postpartum period with a greater amount of stored white adipose tissue and consequently higher leptin levels than age-matched cycling females a reduction in the ability of high circulating leptin to reduce food intake might in turn facilitate higher caloric consumption during early lactation. Such a modification is likely to be less important later in lactation when circulating levels of leptin are low. A decrease in the behavioral effectiveness of peripheral leptin could result either from failure of leptin to cross the blood–brain barrier or blunted responsiveness of central receptors. In turn, changes in the central response might result either from of a downregulation in receptor density as described in Brogan et al. (2000), inhibition of leptin signaling pathways or some combination of these factors.

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Downregulation of leptin receptors in the VMH (Brogan et al., 2000) suggests at least some attenuation of this peptide’s action in the hypothalamus during lactation. Although these authors did not see a downregulation of leptin receptors within the ARC itself, other researchers have observed a downregulation of leptin signaling in this area. Roy and his colleagues, using a hypothalamic slice preparation, demonstrated that leptin induced pSTAT3 activation in the ARC of cycling rats but did not do so in slices obtained from lactating rats (Roy et al., 2007). The behavioral relevance of these changes, however, are uncertain because Suzuki et al. (2010) showed that intracerebroventricular (icv) administration of 10 lg of leptin reduced food intake in both lactating and cycling rats. In our own preliminary work we have administered lower doses of leptin icv than that used by Suzuki et al. and found that rats in late lactation responded with decreases in food intake similar to those seen in cycling rats to both doses. Interestingly, in early lactation rats were less responsive to the lower dose (Budin and Woodside, 2011). Although these data suggest a decrease in sensitivity to the behavioral effects of leptin in early lactation they contrast markedly with the pronounced leptin resistance that is observed during the latter half of pregnancy (Ladyman and Grattan, 2004, 2005). Given the increased energetic demands of lactation, one would expect that ghrelin levels would be higher in lactating females than in nonlactating females. Surprisingly, most studies show lower or no differences in circulating ghrelin levels in lactating females (Abizaid et al., 2008). This apparent paradox may be simply explained by the fact that the increased consumption of calories during lactation leads to continued presence of food in the stomach leading to lower plasma ghrelin levels. It is also likely that the partitioning of resources so as to favor nutrient diversion to milk seen during lactation requires lowers levels of circulating ghrelin to facilitate the utilization of fat stores for milk production. In the brain, however, sensitivity to ghrelin may actually increase to favor lactational hyperphagia, a hypothesis that is supported by studies showing that hypothalamic ghrelin receptor expression is elevated in lactating rats (Abizaid et al., 2008). Interestingly there is a rapid downregulation of receptor expression after the litter is removed suggesting that the increase may be dependent on suckling stimulation (Abizaid et al., 2008). Both leptin and ghrelin modulate NPY/AGRP neuron activity, thus the inability of leptin replacement in lactating rats to suppress NPY/AGRP mRNA expression might reflect a greater contribution of ghrelin in modulating this pathway during lactation (Xu et al., 2009a). Recent work from our own laboratory suggests that ghrelin may indeed make an important contribution to food intake during the latter part of lactation. In preliminary work we have shown that on Day 10 postpartum dams are more sensitive to the orexigenic actions of ghrelin, administered into the cerebral ventricles, than cycling females (Abizaid et al. 2010) and a low dose of ghrelin antagonist was effective in reducing food intake in late lactation (D14–16 postpartum) but not in cycling or early lactating rats (Budin and Woodside, 2011). The increased food intake of lactating rats is not the only mechanism through which energy is made available for milk production. Because ghrelin acts at the fat pad to increase fat storage (Abizaid and Horvath, 2008), decreases in circulating ghrelin levels might be expected to favor lipolysis and the liberation of free fatty acids from maternal fat stores to be used in the milk. Activation of NPY/AGRP neurons is associated with a suppression of sympathetic output whereas activation of POMC and hence the melanocortin system increases it (Abizaid et al., 2006). Thus, the changes in hypothalamic neuropeptide levels observed in lactation are consistent with decreased sympathetic output and the increase in central ghrelin signaling might contribute not only to a ghrelin mediated increase in food intake but also to suppression of sympathetic activity.

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As described in an earlier section, ghrelin and leptin target both midbrain DA systems as well as brain stem nuclei. How the changes in circulating levels of these hormones that are observed during lactation affect these facets of the energy balance systems remains to be investigated.

5. Influence of lactational hormones on energy balance A combination of decreased sensitivity to leptin and a potential increase in sensitivity to ghrelin would be expected to facilitate the hyperphagia required by lactating rats. One might suppose that these changes are simply driven by the energetic drain of lactation, however, there is considerable evidence to suggest that lactating rats are modestly hyperphagic in the absence of transferring nutrients to the young (Cotes and Cross, 1954; Fleming, 1976; Woodside, 2007). The hormonal state of lactating females depends on suckling stimulation rather than on milk delivery, thus, it is possible that suckling-induced changes in hormonal status contribute to the altered energy balance of lactating dams. Most of the studies evaluating the contribution of suckling independent of milk delivery to food intake during lactation have used one of two models: the acute resuckling model (Smith and Grove, 2002), or the galactophore-cut or ligation model (Cotes and Cross, 1954; Fleming, 1976; Woodside and Popeski, 1999). In the acute resuckling model, lactation is established and then during the second week of lactation the young and mother are separated for 48 h. Dams readily accept and suckle their young when the dyad is reunited and because suckling is reestablished rapidly prior to the restoration of full lactation there is a period of time during which one can assess the acute effects of suckling independent of the energetic demands of lactation. Experiments using this model have shown that 24 h suckling following separation induces an increase in NPYmRNA expression in both the arcuate nucleus and the dorsomedial hypothalamus (Abizaid et al., 2008; Smith, 1993). In the galactophore-cut (GC) model, the tubes connecting the mammary gland to the nipple are transected. After recovery from surgery, females are mated and pregnancy and parturition proceed normally as does the pups’ attachment to the nipple. The offspring continue to suckle at nipples that do not deliver milk and in order to maintain the health of the pups cross fostering between galactophore-cut and intact foster mothers. The result of the maintenance of suckling stimulation is that circulating prolactin (PRL) levels in GC females are similar to those of intact lactating females (Woodside et al., 2000) as is the length of lactational infertility (Woodside and Popeski, 1999). GC females differ from intacts, however, because they do not lose fat during the postpartum period and maintain high circulating leptin levels. In spite of this, GC females continue to show a higher food intake (about 20%) than cycling females (Woodside et al., 2000). Together, data obtained using both models suggest that the hormonal state of lactation is sufficient to increase food intake and to induce at least some of the changes in hypothalamic peptide expression seen in intact nursing rats. The hormonal profile of lactation is quite distinct from that of any other point in the life cycle and its maintenance depends on the presence of nursing young. Suckling stimulation from the young stimulates both PRL release from the anterior pituitary (Mattheij et al., 1979) and bolus oxytocin (OT) release from the posterior pituitary anterior pituitary (Wakerley et al., 1973). In turn, PRL stimulates milk production as well as progesterone synthesis and release from the ovary (Tomogane et al., 1976, 1969). OT, on the other hand, triggers coordinated smooth muscle contraction in the alveoli of the mammary gland resulting in milk delivery ejection the nipple (Wakerley et al., 1973). Suckling induced PRL release increases with number of young suckled

(Mattheij et al., 1984) and an active transport mechanism in the choroids plexus provides a route for PRL to access the brain (Walsh et al., 1987). PRL receptors are expressed in a number of hypothalamic nuclei including the MPOA, ARC, VMN, and PVN (Augustine et al., 2003; Bakowska and Morrell, 1997; Chiu et al., 1992; Kokay et al., 2006; Kokay and Grattan, 2005). Thus, pituitary PRL can gain access to the brain and modulate numerous functions (see review in Grattan and Kokay (2008)). Little OT from the peripheral circulation accesses the brain but there is an increase in extracellular OT within the supraoptic nucleus (SON) concomitant with the suckling-induced (Neumann et al., 1995) release of this hormone from the posterior pituitary. Serum steroid hormone levels are also changed during lactation. Basal levels of glucocortioids are increased throughout the postpartum period in the nursing mother (Walker et al., 1992) although corticotropin releasing hormone mRNA decreases in mid-late lactation (Walker et al., 2001). Glucocorticoids play a critical role in maintaining milk synthesis (Plucinski and Baldwin, 1976) and modulate some aspects of maternal behavior (Rees et al., 2004). Ovulation is suppressed for most of lactation with an associated reduction in estrogen, luteinizing and gonadotropin releasing hormone levels (Ford and Melampy, 1973; Smith and Grove, 2002). Many of these hormones have been shown to influence energy balance independent of their role in milk production and delivery and thus might contribute to changes in energy balance during lactation. The potential contributions of each of these hormones to the metabolic state of lactation animals as well as the mechanisms through which they exert their effects are described below. 5.1. Changes in anorectic hormones in lactation: estrogen, OT and CCK Following the postpartum ovulation, which typically occurs within the first 36 h after parturition, estrogen levels remain low for most of the first 2 weeks postpartum. They rise to levels similar to those of the morning of diestrus by Day 10–11 postpartum in mothers nursing eight pups (Smith and Neill, 1977) and ovulatory levels of estrogen are typically reached around Day 21 postpartum (Hansen et al., 1983). In contrast to estrogen, the levels of the other major ovarian steroid, progesterone are high for most of the first 2 weeks postpartum. However, progesterone has little effect on either food intake or body weight in ovariectomized rats when administered alone at physiological levels (Yu et al., 2011). The anorectic effects of estrogen are well-established. In virgin rats, ovariectomy results in a rapid increase in food intake and body weight and both of these are suppressed with estrogen replacement (Asarian and Geary, 2006; Wade, 1972). In addition to decreasing food intake estrogen also increases energy expenditure both by increasing locomotor activity and sympathetic outflow (Saleh et al., 2003). Thus this hormone has wide ranging effects on energy balance systems. In a series of studies, Clegg and her colleagues have documented that estrogen has its effects on energy balance in part by changing the response of central pathways to peripheral signals (Meyer et al., 2011). Thus, estrogen increases the ability of centrally administered leptin and insulin to decrease food intake in both gonadectomized male and female rats (Clegg et al., 2006, 2007). Recently this group has shown that the inhibitory effect of estrogen on food intake is mediated through ER alpha receptor positive POMC neurons within the hypothalamus. Although the metabolic stimulating effects of estrogen are also mediated through ER alpha, the critical receptors are localized to steroidogenic factor 1 (SF1) positive neurons (Xu et al., 2011). Estrogen also decreases sensitivity to the orexigenic effects of ghrelin. Interestingly ovariectomized rats, like lactating rats, showed increases in ghrelin receptor density within the hypothalamus, suggesting that the reduction of estrogen during lactation might contribute to increased levels of

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this receptor (Abizaid et al., 2008; Clegg et al., 2007). Results of studies by Gao et al. (2007) suggest that another mechanism through which estrogen modulates the pathways subserving energy balance is to stimulate a reorganization of synaptic inputs to POMC neurons such that excitatory inputs are increased. Given these effects of estrogen, the low levels of circulating estrogen postpartum could contribute to the hyperphagia of lactation. It is noteworthy, however, that circulating levels of estrogen increase to those typical of diestrus females prior to the period of the peak of lactational hyperphagia which typically occurs around Days 14– 16 postpartum (Woodside, 2007). Decreases in levels of an anorectic agent such as estrogen are quite consistent with the hyperphagia of lactation. However, lactation is also associated with increases in both peripheral (Wakerley and Lincoln, 1971) and central release of OT (Neumann et al., 1995) which is a potent satiety hormone. It is unlikely that peripheral oxytocin affects food intake although there is evidence that increased circulating levels of this hormone decrease sympathetic activation (Michelini, 2001). Within the brain OT is synthesized within magnocellular neurons in the SON and in both magnocellular and parvocellular neurons of the PVN. Both of these neuronal populations have been implicated in the control of food intake. The magnocellular neurons of both the PVN and SON project to the posterior pituitary and activation of these neurons leads to OT release into the circulatory system. Some parvocellular OT neurons project to the NTS and a subset of these may potentiate the satiating effects of leptin and of cholecystekinin (CCK) (Olson et al., 1991). Circulating levels of CCK are increased in lactating rats although there is no evidence of increased levels in the CSF (Linden, 1989; Linden et al., 1990). In addition, exogenous administration of this hormone induces a greater anorectic response in virgin than in lactating rats (Linden, 1989) suggesting that there may be some alterations in the ability of OT to augment the response to CCK during this reproductive state. In addition, both leptin and estrogen have been shown to influence activation of brain stem areas after administration of CCK (Geary, 2001). The decrease in circulating levels in both these hormones during lactation might be expected to reduce the effectiveness of this satiety factor. During lactation the bolus release of OT from the posterior pituitary is the proximal stimulus for milk ejection (Insel et al., 1997). MCR4 receptors are localized to these magnocellular neurons and these neurons are strongly activated by alpha MSH administration which results in a large increase in dendritic release of OT. Oxytocin released in this way acts both locally on oxytocin receptors (OTRs) in magnocellular neurons, perhaps to modulate synaptic plasticity, and by diffusion throughout the hypothalamus and forebrain to activate OTR at distal sites. The latter mechanism may be one route through which OT exerts its anorectic effects (Douglas et al., 2007). Interestingly, pregnant rats are less responsive than cycling rats both to the anorectic effects central OT administration and the orexigenic effects of treatment with an OTR antagonist (Douglas et al., 2007). Whether there is a similar lack of effect of these agents on food intake in lactating rats remains to be investigated. 5.2. Changes in orexigenic hormones: PRL and glucocorticoids In contrast to estrogen, OT and CCK, evidence suggests that PRL is orexigenic. Several studies have shown that peripheral injections of PRL lead to an increase in food intake in nonlactating rats but this treatment also suppresses estrous cyclicity (Noel and Woodside, 1993). Thus any orexigenic effects of peripherally administered PRL are confounded with the removal of the food intake suppressing effects of estrogen. Bidaily or chronic infusion of PRL into the lateral ventricles, however, also increases food

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intake in female rats without affecting cyclicity (Sauve and Woodside, 2000). Chronic infusions of PRL also reduce the effect of central leptin administration on both food intake and body weight. This behavioral effect of PRL is associated with a suppression of leptin signaling within the ventromedial hypothalamus and the paraventricular nucleus (Naef and Woodside, 2007). There was, however, no diminution of leptin induced phosphorylation of STAT3 in the ARC. These data are strikingly similar to studies showing that rats become leptin resistant in the last trimester of pregnancy and that this effect depended on the high levels of hypothalamic PRL-R activation at this time (Ladyman and Grattan, 2004). Interestingly, bidaily cerebroventricular injections of PRL given to mimic the bidaily surges of prolactin typical of the early part of pregnancy did not induce leptin resistance even in the presence of high progesterone levels (Augustine and Grattan, 2008). Such bidaily injections of PRL when administered into the paraventricular nucleus of the hypothalamus do, however, increase food intake in cycling rats suggesting that PRL might stimulate food intake by multiple pathways one of which is through the induction of leptin resistance (Sauve and Woodside, 2000). Evidence that PRL actually contributes to the hyperphagia of lactation comes from both the galactophore-cut and resuckling paradigms. Suppressing PRL release with bromocryptine suppresses the food intake of GC females to levels seen in cycling females and restoring PRL levels by direct icv infusion restores food intake (Woodside and Popeski, 1999). These data suggest that PRL acts centrally to contribute to the hyperphagia of lactation. Other evidence in support of a role of PRL in modulating energy balance pathways in lactating rats was obtained by Smith and Chen (2004) using the acute resuckling model. These authors showed that restoring the suckling stimulus after 24 h of mother litter separation was sufficient to induce the pattern of NPY mRNA expression in the DMH and ARC typical of lactating rats. However, when suckling stimulation was combined with suppression of PRL by treatment with bromocryptine the induction of NPY in the DMH, but not the arcuate nucleus, was eliminated. Replacement of PRL restored NPY gene expression in the DMH. Thus, PRL can have a site specific effect on NPY gene expression within the hypothalamus. PRL receptor expression is not limited to the brain and mammary gland. PRLR have been localized on a wide variety of tissues including white fat (Freemark et al., 2001), brown adipose tissue (Pearce et al., 2003), the liver and the pancreas (Amaral et al., 2004). How PRL’s action on these tissues influences energy utilization and storage during lactation remains to be determined. Circulating PRL levels increase with the number of young suckled, thus it is possible that central PRLR activation is one means through which the energy requirements of the nursing young are transmitted to the dam and influence her energy intake. In this way, PRL can be seen as serving a dual role in the provision of nutrients to the young: it is a major lactogenic hormone which acts at at the mammary gland to promote milk production, and it also acts at a number of central sites to facilitate food intake. Both the neuroendocrine and behavioral responses to stress are attenuated during lactation in a wide number of species, and PRL may play an important role in this effect (Numan and Woodside, 2010). Nevertheless, in spite of an attenuated stress response, basal circulating glucocorticoid levels are elevated in lactating rats (Walker et al., 1992), which influences both milk production and maternal behavior. Peripherally, glucocorticoids are catabolic and contribute to the availability of metabolic fuels for milk. Centrally, glucocorticoids have orexigenic effects by increasing activity of NPY neurons (Misaki et al., 1992). Moreover, glucocorticoids, like estrogens, have been shown to have effects on the organization of synaptic input to NPY/Agrp and POMC neurons within the arcuate nucleus (Gyengesi et al., 2010). However, the effect of

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Fig. 3. Suckling stimulation alone is responsible for changes in peptide and steroid hormones including PRL, estrogen, and glucocorticoids that target brain stem nuclei as well as the hypothalamus to modulate energy state and food intake. Abbreviations as in Fig. 1A. The dotted line describing the pathway between the ARC and the LPBN suggests increased AGRP/GABA tone in suckled rats that would promote increased feeding, although this needs to be verified experimentally.

glucocorticoids is to increase both the number of inhibitory synapses on POMC neurons and the number of excitatory inputs on NPY neurons (Gyengesi et al., 2010). In sum, evidence suggests that, with the exception of central OT release, the hormonal profile of lactating rats is consistent with a predominance of orexigenic over anorectic signals. Moreover, the changes in hormone levels observed during lactation would be expected to influence multiple brain sites involved in modulating energy balance and these are summarized in Figs. 2 and 3. Suckling stimulation from the pups is critical both for provoking the release of some hormones such as PRL and OT while reducing the levels of others by suppressing reproductive function. Hence, the young themselves contribute to the alterations in metabolic state of the mother not only because they impose a nutrient drain through the delivery of milk but also by creating in the nursing mother a hormonal state that facilitates the increase in food intake and decrease in energy expenditure related to other activities. Although the milk delivery independent increases in food intake are modest, the fact that they occur in the face of increased levels of anorectic signals such as leptin suggests that they are powerful and may reflect the ability of hormones such as PRL and estrogen to alter the organisms response to metabolic signals. Circulating PRL levels increase with the number of young suckled, thus it is possible that central PRLR activation is one means through which the energy requirements of the nursing young are transmitted to the dam and influence her energy intake. In this way, PRL can be seen as serving a dual role in the provision of nutrients to the young: it is a major lactogenic hormone which acts at at the mammary gland to promote milk production, and it also acts at a number of central sites to facilitate food intake. To the extent that PRL has also been implicated in the suppression of the ovarian axis during lactation this hormone might well be seen as the route through which many of the metabolic changes seen during lactation are orchestrated (Woodside et al., 2008).

6. Conclusion Successful rearing of young requires dramatic alterations in the physiology and behavioral repertoire of female mammals. In this

paper we have described the adaptations that occur in the neural pathways controlling food intake and energy balance in lactating rats together with some of the stimuli that induce these changes. Although the hormonal state of lactating rats predisposes them to hyperphagia, the vast majority of their food intake is driven by the energetic costs of milk production and delivery. One issue that deserves much more attention is how signals relating to these costs are conveyed to the neural pathways controlling food intake. Given the relative insensitivity of lactating rats to changes in circulating leptin levels described earlier, it is unlikely that a decrease in peripheral leptin concentrations concomitant to a decrease in body fat plays an important role. The availability of metabolic fuels has been shown to have profound effects on food intake. The induction of acute glucoprivation by administration of 2-deoxy-D-glucose and inhibition of free fatty acid oxidation by methylpalmoxirate or sodium mercaptoacetate induce food intake in both rats and hamsters (Darling and Ritter, 2009; Schneider et al., 1997). Thus the direct sensing of metabolic fuel availability might be expected to make an important contribution to the hyperphagia of lactation. However, lactating rats, are relatively insensitive to 2DG administration both with respect to its effects on ingestive behavior and its ability to induce Fos protein expression in brain areas associated with increased food intake (Abizaid and Woodside, 2002). Whether there is also insensitivity to inhibition of fatty acid oxidation remains to be investigated. In addition, little is known about changes in the AMPK or mTOR pathways during lactation, nor whether increased caloric intake and energy demands during lactation stimulate inflammatory pathways, and as such these remain important avenues of research to explore. It is likely that the relative contribution of milk delivery dependent and independent factors on the stimulation of food intake in lactating rats depends on the number of young suckled as well as their age. A newborn rat, for example, weighs 5–6 g is blind, deaf and moves very little, whereas a 14 day old rat pup weighs about 45 g has developed fur opened its eyes, and ears and is highly mobile. Both, however, are dependent on their mother for food. Thus, the adaptations required to meet the needs of the young must also change with the developing young. This is true of the mother’s ingestive behavior as it is of other aspects of maternal care such as the need to build a more insulating nest for newborns than for

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older pups. That female mammals do adapt both to the changing needs of the young and the environment in which they raise them reflect the complexity of central and peripheral homeostatic processes as well as their flexibility. Using this model to elucidate the multiple factors brought into play to allow females to meet the nutritional needs of their young will not only provide further insight into maternal physiology but also increase our basic understanding of energy balance regulation both in physiological and pathophysiological conditions. Acknowledgments This research was supported by grants from the National Sciences and Engineering Research Council to A.A. and B.W. The authors would like to thank Megan Sheppard and Joanna Pohl for their careful reading of earlier versions of this manuscript. References Abizaid, A., 2009. Ghrelin and dopamine: new insights on the peripheral regulation of appetite. J. Neuroendocrinol. 21, 787–793. Abizaid, A., Horvath, T.L., 2008. Brain circuits regulating energy homeostasis. Regul. Pept. 149, 3–10. Abizaid, A., Woodside, B., 2002. Food intake and neuronal activation after acute 2DG treatment are attenuated during lactation. Physiol. Behav. 75, 483–491. Abizaid, A., Kyriazis, D., Woodside, B., 2004. Effects of leptin administration on lactational infertility in food-restricted rats depend on milk delivery. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R217–225. Abizaid, A., Gao, Q., Horvath, T.L., 2006a. Thoughts for food: brain mechanisms and peripheral energy balance. Neuron 51, 691–702. Abizaid, A., Liu, Z.W., Andrews, Z.B., Shanabrough, M., Borok, E., Elsworth, J.D., Roth, R.H., Sleeman, M.W., Picciotto, M.R., Tschop, M.H., Gao, X.B., Horvath, T.L., 2006b. Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite. J. Clin. Invest. 116, 3229–3239. Abizaid, A., Schiavo, L., Diano, S., 2008. Hypothalamic and pituitary expression of ghrelin receptor message is increased during lactation. Neurosci. Lett. 440, 206– 210. Abizaid, A., Budin, R., Wellman, M., Woodside, B., 2010. Functional role of ghrelin during lactation. Parental Brain Conference, August Edinburgh. Adamantidis, A., de Lecea, L., 2008. Physiological arousal: a role for hypothalamic systems. Cell. Mol. Life Sci. 65, 1475–1488. Afonso, V.M., King, S., Chatterjee, D., Fleming, A.S., 2009. Hormones that increase maternal responsiveness affect accumbal dopaminergic responses to pup- and food-stimuli in the female rat. Horm. Behav. 56, 11–23. Amaral, M.E., Cunha, D.A., Anhe, G.F., Ueno, M., Carneiro, E.M., Velloso, L.A., Bordin, S., Boschero, A.C., 2004. Participation of prolactin receptors and phosphatidylinositol 3-kinase and MAP kinase pathways in the increase in pancreatic islet mass and sensitivity to glucose during pregnancy. J. Endocrinol. 183, 469–476. Andrews, Z.B., Liu, Z.W., Walllingford, N., Erion, D.M., Borok, E., Friedman, J.M., Tschop, M.H., Shanabrough, M., Cline, G., Shulman, G.I., Coppola, A., Gao, X.B., Horvath, T.L., Diano, S., 2008. UCP2 mediates ghrelin’s action on NPY/AgRP neurons by lowering free radicals. Nature 454, 846–851. Aponte, Y., Atasoy, D., Sternson, S.M., 2011. AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training. Nat. Neurosci. 14, 351–355. Asarian, L., Geary, N., 2006. Modulation of appetite by gonadal steroid hormones. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361, 1251–1263. Augustine, R.A., Grattan, D.R., 2008. Induction of central leptin resistance in hyperphagic pseudopregnant rats by chronic prolactin infusion. Endocrinology 149, 1049–1055. Augustine, R.A., Kokay, I.C., Andrews, Z.B., Ladyman, S.R., Grattan, D.R., 2003. Quantitation of prolactin receptor mRNA in the maternal rat brain during pregnancy and lactation. J. Mol. Endocrinol. 31, 221–232. Bakowska, J.C., Morrell, J.I., 1997. Atlas of the neurons that express mRNA for the long form of the prolactin receptor in the forebrain of the female rat. J. Comp. Neurol. 386, 161–177. Baler, R.D., Volkow, N.D., 2006. Drug addiction: the neurobiology of disrupted selfcontrol. Trends Mol. Med. 12, 559–566. Bamshad, M., Aoki, V.T., Adkison, M.G., Warren, W.S., Bartness, T.J., 1998. Central nervous system origins of the sympathetic nervous system outflow to white adipose tissue. Am. J. Physiol. 275, R291–R299. Bartness, T.J., 1997. Food hoarding is increased by pregnancy, lactation, and food deprivation in Siberian hamsters. Am. J. Physiol. 272, R118–125. Bechtold, D.A., Luckman, S.M., 2006. Prolactin-releasing Peptide mediates cholecystokinin-induced satiety in mice. Endocrinology 147, 4723–4729. Berridge, K.C., Robinson, T.E., 1998. What is the role of dopamine in reward: hedonic impact, reward learning, or incentive salience? Brain Res. Brain Res. Rev. 28, 309–369.

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