TRPM4 and PLCß3 contribute to normal behavioral responses to an array of sweeteners and carbohydrates but PLCß3 is not needed for taste-driven licking for glucose.

The peripheral taste system is more complex than previously thought. The novel taste-signaling proteins TRPM4 and PLCß3 appear to function in normal taste responding as part of Type II taste cell signaling or as part of a broadly responsive (BR) taste cell that can respond to some or all classes of tastants. This work begins to disentangle the roles of intracellular components found in Type II taste cells (TRPM5, TRPM4, and IP3R3) or the BR taste cells (PLCß3 and TRPM4) in driving behavioral responses to various saccharides and other sweeteners in brief-access taste tests. We found that TRPM4, TRPM5, TRPM4/5, and IP3R3 knockout (KO) mice show blunted or abolished responding to all stimuli compared with wild-type. IP3R3 KO mice did, however, lick more for glucose than fructose following extensive experience with the 2 sugars. PLCß3 KO mice were largely unresponsive to all stimuli except they showed normal concentration-dependent responding to glucose. The results show that key intracellular signaling proteins associated with Type II and BR taste cells are mutually required for taste-driven responses to a wide range of sweet and carbohydrate stimuli, except glucose. This confirms and extends a previous finding demonstrating that Type II and BR cells are both necessary for taste-driven licking to sucrose. Glucose appears to engage unique intracellular taste-signaling mechanisms, which remain to be fully elucidated.

Ancient AMY1 gene duplications primed the amylase locus for adaptive evolution upon the onset of agriculture.

Starch digestion is a cornerstone of human nutrition. The amylase enzyme, which digests starch, plays a key role in starch metabolism. Indeed, the copy number of the human amylase gene has been associated with metabolic diseases and adaptation to agricultural diets. Previous studies suggested that duplications of the salivary amylase gene are of recent origin. In the course of characterizing 51 distinct amylase haplotypes across 98 individuals employing long-read DNA sequencing and optical mapping methods, we detected four 31mers linked to duplication of the amylase locus. Analyses with these 31mers suggest that the first duplication of the amylase locus occurred more than 700,000 years ago before the split between modern humans and Neanderthals. After the original duplication events, amplification of the AMY1 genes likely occurred via nonallelic homologous recombination in a manner that consistently results in an odd number of copies per chromosome. These findings suggest that amylase haplotypes may have been primed for bursts of natural-selection associated duplications that coincided with the incorporation of starch into human diets.

The role of saliva in taste and food intake.

Saliva is well-described in oral food processing, but its role in taste responsiveness remains understudied. Taste stimuli must dissolve in saliva to reach their receptor targets. This allows the constituents of saliva the opportunity to interact with taste stimuli and their receptors at the most fundamental level. Yet, despite years of correlational data suggesting a role for salivary proteins in food preference, there were few experimental models to test the role of salivary proteins in taste-driven behaviors. Here we review our experimental contributions to the hypothesis that salivary proteins can alter taste function. We have developed a rodent model to test how diet alters salivary protein expression, and how salivary proteins alter diet acceptance and taste. We have found that salivary protein expression is modified by diet, and these diet-induced proteins can, in turn, increase the acceptance of a bitter diet. The change in acceptance is in part mediated by a change in taste signaling. Critically, we have documented increased detection threshold, decreased taste nerve signaling, and decreased oromotor responding to quinine when animals have increases in a subset of salivary proteins compared to control conditions.

A subset of broadly responsive Type III taste cells contribute to the detection of bitter, sweet and umami stimuli.

Taste receptor cells use multiple signaling pathways to detect chemicals in potential food items. These cells are functionally grouped into different types: Type I cells act as support cells and have glial-like properties; Type II cells detect bitter, sweet, and umami taste stimuli; and Type III cells detect sour and salty stimuli. We have identified a new population of taste cells that are broadly tuned to multiple taste stimuli including bitter, sweet, sour, and umami. The goal of this study was to characterize these broadly responsive (BR) taste cells. We used an IP3R3-KO mouse (does not release calcium (Ca2+) from internal stores in Type II cells when stimulated with bitter, sweet, or umami stimuli) to characterize the BR cells without any potentially confounding input from Type II cells. Using live cell Ca2+ imaging in isolated taste cells from the IP3R3-KO mouse, we found that BR cells are a subset of Type III cells that respond to sour stimuli but also use a PLCß signaling pathway to respond to bitter, sweet, and umami stimuli. Unlike Type II cells, individual BR cells are broadly tuned and respond to multiple stimuli across different taste modalities. Live cell imaging in a PLCß3-KO mouse confirmed that BR cells use this signaling pathway to respond to bitter, sweet, and umami stimuli. Short term behavioral assays revealed that BR cells make significant contributions to taste driven behaviors and found that loss of either PLCß3 in BR cells or IP3R3 in Type II cells caused similar behavioral deficits to bitter, sweet, and umami stimuli. Analysis of c-Fos activity in the nucleus of the solitary tract (NTS) also demonstrated that functional Type II and BR cells are required for normal stimulus induced expression.

Temperature Is Sufficient to Condition a Flavor Preference for a Cold-Paired Solution in Rats.

Increasing evidence suggests that stimulus temperature modifies taste signaling. However, understanding how temperature modifies taste-driven behavior is difficult to separate as we must first understand how temperature alone modifies behavior. Previous work has suggested that cold water is more rewarding and "satiating" than warm water, and water above orolingual temperature is avoided in brief-access testing. We explored the strength of cold water preference and warm water avoidance by asking: (1) if cold temperature alone was sufficient to condition a flavor preference and (2) if avoidance of warm stimuli is driven by novelty. We addressed these questions using custom-designed equipment that allows us to monitor and maintain solution temperatures. We conducted two-bottle preference tests, after pairing Kool-Aid flavors with 10 or 40 °C. Rats preferred the flavor paired with cold temperature, both while it was cold and for 1 day while solutions were presented at 22 °C. We then examined the role of novelty in avoidance of 40 °C. Rats were maintained on 10, 22, or 40 °C water in their home cage to increase familiarity with the temperatures. Rats were then subject to a series of brief-access taste tests to water or sucrose at 10 to 40 °C. Rats that had 40 °C experience licked more to 40 °C water, but not sucrose, during brief-access testing. In a series of two-bottle preference tests, rats maintained on 40 °C water had a decreased preference for 10 °C water when paired opposite 40 °C water. Together, these data contribute to our understanding of orosensory-driven behavior with water at different temperatures.

Altering salivary protein profile can decrease aversive oromotor responding to quinine in rats.

Bitter taste is often associated with toxins, but accepting some bitter foods, such as green vegetables, can be an important part of maintaining a healthy diet. It has previously been shown that animals exposed to quinine upregulate a set of salivary proteins (SPs), and those with upregulated SPs have increased rates of feeding on a quinine diet as well as increased brief-access licking to and higher detection thresholds for quinine. These studies suggest that SPs alter orosensory feedback; however, they rely on SPs upregulated by diet exposure and cannot control for the role of learning. Here, we use taste reactivity to determine if SPs can alter bitter taste in animals with no previous bitter diet experience. First, saliva with proteins stimulated by injections of isoproterenol and pilocarpine was collected from anesthetized rats; this "donor saliva" was analyzed for protein concentration and profile. Bitter-naïve rats were implanted with oral catheters and infused with taste stimuli dissolved in saliva that contained all of the SPs from the donors, saliva that was filtered of SPs, water, or artificial saliva. Their orofacial movements were recorded and quantified. We found that presence of quinine increased movements associated with aversive stimuli, but adding SPs to the infusion was sufficient to reduce aversive oromotor responding to quinine. The effect was dependent on the total protein concentration of the saliva, as protein concentration increased aversive responses decreased. Additionally, infusions of whole saliva altered aversive responding to quinine, but not other stimuli (citric acid, NaCl, sucrose). Our work suggests that effect of these SPs is specific and the presence of SPs is sufficient to decrease aversive orosensory feedback to bitter stimuli.

Differential Effects of Diet and Weight on Taste Responses in Diet-Induced Obese Mice.

OBJECTIVE: Previous studies have reported that individuals with obesity have reduced taste perception, but the relationship between obesity and taste is poorly understood. Earlier work has demonstrated that diet-induced obesity directly impairs taste. Currently, it is not clear whether these changes to taste are due to obesity or to the high-fat diet exposure. The goal of the current study was to determine whether diet or excess weight is responsible for the taste deficits induced by diet-induced obesity. METHODS: C57BL/6 mice were placed on either high-fat or standard chow in the presence or absence of captopril. Mice on captopril did not gain weight when exposed to a high-fat diet. Changes in the responses to different taste stimuli were evaluated using live cell imaging, brief-access licking, immunohistochemistry, and real-time polymerase chain reaction. RESULTS: Diet and weight gain each affected taste responses, but their effects varied by stimulus. Two key signaling proteins, α-gustducin and phospholipase Cß2, were significantly reduced in the mice on the high-fat diet with and without weight gain, identifying a potential mechanism for the reduced taste responsiveness to some stimuli. CONCLUSIONS: Our data indicate that, for some stimuli, diet alone can cause taste deficits, even without the onset of obesity.

Rats are unable to discriminate quinine from diverse bitter stimuli.

Compounds described by humans as "bitter" are sensed by a family of type 2 taste receptors (T2Rs). Previous work suggested that diverse bitter stimuli activate distinct receptors, which might allow for perceptually distinct tastes. Alternatively, it has been shown that multiple T2Rs are expressed on the same taste cell, leading to the contrary suggestion that these stimuli produce a unitary perception. Behavioral work done to address this in rodent models is limited to Spector and Kopka (Spector AC, Kopka SL. J Neurosci 22: 1937-1941, 2002), who demonstrated that rats cannot discriminate quinine from denatonium. Supporting this finding, it has been shown that quinine and denatonium activate overlapping T2Rs and neurons in both the mouse and rat nucleus of the solitary tract (NTS). However, cycloheximide and 6-n-propylthiouracil (PROP) do not appear to overlap with quinine in the NTS, suggesting that these stimuli may be discriminable from quinine and the denatonium/quinine comparison is not generalizable. Using the same procedure as Spector and Kopka, we tasked animals with discriminating a range of stimuli (denatonium, cycloheximide, PROP, and sucrose octaacetate) from quinine. We replicated and expanded the findings of Spector and Kopka; rats could not discriminate quinine from denatonium, cycloheximide, or PROP. Rats showed a very weak ability to discriminate between quinine and sucrose octaacetate. All animals succeeded in discriminating quinine from KCl, demonstrating they were capable of the task. These data suggest that rats cannot discriminate this suite of stimuli, although they appear distinct by physiological measures.

Bitter-Induced Salivary Proteins Increase Detection Threshold of Quinine, But Not Sucrose.

Exposures to dietary tannic acid (TA, 3%) and quinine (0.375%) upregulate partially overlapping sets of salivary proteins which are concurrent with changes in taste-driven behaviors, such as rate of feeding and brief access licking to quinine. In addition, the presence of salivary proteins reduces chorda tympani responding to quinine. Together these data suggest that salivary proteins play a role in bitter taste. We hypothesized that salivary proteins altered orosensory feedback to bitter by decreasing sensitivity to the stimulus. To that end, we used diet exposure to alter salivary proteins, then assessed an animal's ability to detect quinine, using a 2-response operant task. Rats were asked to discriminate descending concentrations of quinine from water in a modified forced-choice paradigm, before and after exposure to diets that alter salivary protein expression in a similar way (0.375% quinine or 3% TA), or 1 of 2 control diets. Control animals received either a bitter diet that does not upregulate salivary proteins (4% sucrose octaacetate), or a nonbitter diet. The rats exposed to salivary protein-inducing diets significantly decreased their performance (had higher detection thresholds) after diet exposure, whereas rats in the control conditions did not alter performance after diet exposure. A fifth group of animals were trained to detect sucrose before and after they were maintained on the 3% TA diet. There was no significant difference in performance, suggesting that these shifts in threshold are stimulus specific rather than task specific. Taken together, these results suggest that salivary proteins reduce sensitivity to quinine.

Altering salivary protein profile can increase acceptance of a novel bitter diet.

Bitter taste is often associated with toxins, but accepting some bitter foods, such as green vegetables, can be an important part of maintaining a healthy diet. In rats and humans, repeated exposure to a bitter stimulus increases acceptance. Repeated exposure allows an individual the opportunity to learn about the food's orosensory and postingestive effects. It also alters the salivary protein (SP) profile, which in turn alters taste signaling. We have hypothesized that altering the salivary proteome plays a role in the increased acceptance after repeated exposure. Here we test this and attempt to disentangle the contribution of learning during dietary exposure from the contribution of SPs in increased acceptance of bitter diet. Dietary exposure to quinine or tannic acid and injection of isoproterenol (IPR) result in similar salivary protein profiles. Here we used either the bitter stimulus tannic acid or IPR injection to upregulate a subset of SPs before exposing animals to a novel diet containing quinine (0.375%). Control animals received either a control diet before being exposed to quinine, or a diet containing sucrose octaacetate, a compound that the animals avoid but does not alter SP profiles. The treatments that alter SP expression increased rate of feeding on the quinine diet compared to the control treatments. Additionally, tannic acid exposure altered intake and meal size of the quinine diet. These data suggest that SPs, not just learning about bitter food, increase acceptance of the bitter diet.

TRPM4 and TRPM5 are both required for normal signaling in taste receptor cells.

Peripheral taste receptor cells use multiple signaling pathways to transduce taste stimuli into output signals that are sent to the brain. Transient receptor potential melastatin 5 (TRPM5), a sodium-selective TRP channel, functions as a common downstream component in sweet, bitter, and umami signaling pathways. In the absence of TRPM5, mice have a reduced, but not abolished, ability to detect stimuli, suggesting that a TRPM5-independent pathway also contributes to these signals. Here, we identify a critical role for the sodium-selective TRP channel TRPM4 in taste transduction. Using live cell imaging and behavioral studies in KO mice, we show that TRPM4 and TRPM5 are both involved in taste-evoked signaling. Loss of either channel significantly impairs taste, and loss of both channels completely abolishes the ability to detect bitter, sweet, or umami stimuli. Thus, both TRPM4 and TRPM5 are required for transduction of taste stimuli.

Salivary proteins alter taste-guided behaviors and taste nerve signaling in rat.

Taste stimuli are normally dissolved in saliva prior to interacting with their respective receptor targets. There are hundreds of proteins in saliva, and it has been hypothesized that these proteins could interact with either taste stimuli or taste receptors to alter taste signaling and diet acceptance. However, the impact of these proteins on feeding has been relatively unexplored using rodent models. We have developed a novel technique for saliva collection that allows us to link salivary protein expression with feeding behavior. First, we monitored the microstructure of rats' feeding patterns on a 0.375% quinine diet (Q-diet) while tracking changes in salivary protein expression. We found 5 protein bands were upregulated by diet exposure to Q-diet and upregulation of a subset of these bands were statistically related to increased diet acceptance, including changes in behavioral measures that are thought to represent both orosensory and postingestive signaling. In a second experiment, we measured the licking to a range of quinine solutions (0.01-1.0mM) before and after the animals were exposed to a tannic acid diet that altered salivary protein expression. Rats found the quinine solutions less aversive after salivary protein altering diets. In a third experiment we recorded the response of the chorda tympani (CT) nerve while delivering quinine solutions (0.3-30mM) to the front of the tongue dissolved in either "donor saliva" containing salivary proteins or donor saliva which has had the salivary proteins removed. Donor saliva was collected from a separate group of animals using isoproterenol and pilocarpine. The samples containing salivary proteins resulted in lower nerve responses than those without salivary proteins. Together these data suggest that salivary proteins are capable of altering taste-guided behaviors and taste nerve signaling.

Sex differences in the drinking response to angiotensin II (AngII): Effect of body weight.

Sex differences in fluid intake stimulated by angiotensin II (AngII) have been reported, but the direction of the differences is inconsistent. To resolve these discrepancies, we measured water intake by male and female rats given AngII. Males drank more than females, but when intake was normalized to body weight, the sex difference was reversed. Weight-matched males and females, however, had no difference in intake. Using a linear mixed model analysis, we found that intake was influenced by weight, sex, and AngII dose. We used linear regression to disentangle these effects further. Comparison of regression coefficients revealed sex and weight differences at high doses of AngII. Specifically, after 100ng AngII, weight was a predictor of intake in males, but not in females. Next, we tested for differences in AngII-induced intake in male and females allowed to drink both water and saline. Again, males drank more water than females, but females showed a stronger preference for saline. Drinking microstructure analysis suggested that these differences were mediated by postingestive signals and more bottle switches by the females. Finally, we probed for differences in the expression of components of the renin-angiotensin system in the brains of males and females and found sex differences in several genes in discrete brain regions. These results provide new information to help understand key sex differences in ingestive behaviors, and highlight the need for additional research to understand baseline sex differences, particularly in light of the new NIH initiative to balance sex in biomedical research.

Induction of salivary proteins modifies measures of both orosensory and postingestive feedback during exposure to a tannic acid diet.

There are hundreds of proteins in saliva. Although it has long been hypothesized that these proteins modulate taste by interacting with taste receptors or taste stimuli, the functional impact of these proteins on feeding remains relatively unexplored. We have developed a new technique for saliva collection that does not interfere with daily behavioral testing and allows us to explore the relationship between feeding behavior and salivary protein expression. First, we monitored the alterations in salivary protein expression while simultaneously monitoring the animals' feeding behavior and meal patterns on a custom control diet or on the same diet mixed with 3% tannic acid. We demonstrated that six protein bands increased in density with dietary tannic acid exposure. Several of these bands were significantly correlated with behaviors thought to represent both orosensory and postingestive signaling. In a follow-up experiment, unconditioned licking to 0.01-3% tannic acid solutions was measured during a brief-access taste test before and after exposure to the tannic acid diet. In this experiment, rats with salivary proteins upregulated found the tannin solution less aversive (i.e., licked more) than those in the control condition. These data suggest a role for salivary proteins in mediating changes in both orosensory and postingestive feedback.

Biotransformation enzyme expression in the nasal epithelium of woodrats.

When herbivores come in contact with volatile plant secondary compounds (PSC) that enter the nasal passages the only barrier between the nasal cavity and the brain is the nasal epithelium and the biotransformation enzymes present there. The expression of two biotransformation enzymes Cytochrome P450 2B (CYP2B) and glutathione-S-transferase (GST) was investigated in the nasal epithelia and livers of three populations of woodrats. One population of Neotoma albigula was fed juniper that contains volatile terpenes. Juniper caused upregulation of CYP2B and GST in the nasal epithelium and the expression of CYP2B and GST in the nasal epithelium was correlated to liver expression, showing that the nasal epithelia responds to PSC and the response is similar to the liver. Two populations of Neotoma bryanti were fed creosote that contains less volatile phenolics. The creosote naive animals upregulated CYP2B in their nasal epithelia while the creosote experienced animals upregulated GST. There was no correlation between CYP2B and GST expression in the nasal epithelia and livers of either population. The response of the nasal epithelium to PSC seems to be an evolved response that is PSC and experience dependent.

Preference for sucralose predicts behavioral responses to sweet and bittersweet tastants.

Rats can be classified as either sucralose avoiders (SA) or sucralose preferrers (SP) based on their behavioral responses in 2-bottle preference, 1-bottle intake, and brief-access licking tests. The present study demonstrates that this robust phenotypic variation in the preference for sucralose predicts acceptance of saccharin, an artificial sweetener with a purported concentration-dependent "bitter" side taste and a 0.25 M sucrose solution adulterated with increasing concentrations of quinine hydrochloride (QHCl). Specifically, SA displayed decreased preference for and intakes of saccharin (≥41.5 mM) and sucrose-QHCl (>0.5 mM QHCl) solutions, relative to SP. In a second experiment involving brief-access (30-s) tests, SP and SA did not differ in their unconditioned licking responses across a range of sodium chloride or QHCl solutions (0.03-1 mM). However, the acceptability threshold for sucrose was lower in SA, relative to SP (0.06 and 0.13 M, respectively). Our findings suggest that phenotypic differences in sucralose preference are indicative of a more general difference in the hedonic processing of stimuli containing "bittersweet" or "sweet" taste qualities.

Water restriction and fluid temperature alter preference for water and sucrose solutions.

The role of diet temperature in ingestive behavior is poorly understood. We examined the importance of stimulus temperature and water-restriction state on the preference for and intake of water and sucrose. Using custom-designed equipment that allows us to monitor and maintain solution temperatures during testing (±0.1 °C), we conducted a series of 2-bottle preference tests (10 °C water vs. sucrose 10-40 °C) and brief access tests (10-40 °C water and sucrose). Water-restricted rats preferred cold water over any sucrose concentration (0.0-1.0 M) if the sucrose was 30 or 40 °C, whereas the same rats preferred sucrose at all concentrations and temperatures when unrestricted suggesting that the water-restriction state interacts with temperature preference. In a series of brief-access tests using a Davis Rig (MS-180), rats reduced licking to cold sucrose compared with 20 °C sucrose, suggesting that unlike water, cold temperature reduced the palatability of sucrose.

Rats display a robust bimodal preference profile for sucralose.

Female Sprague-Dawley rats display considerable variability in their preference for the artificial sweetener sucralose over water. While some rats can be classified as sucralose preferrers (SP), as they prefer sucralose across a broad range of concentrations, others can be classified as sucralose avoiders (SA), as they avoid sucralose at concentrations above 0.1 g/L. Here, we expand on a previous report of this phenomenon by demonstrating, in a series of 2-bottle 24-h preference tests involving water and an ascending series of sucralose concentrations, that this variability in sucralose preference is robust across sex, stage of the estrous cycle, and 2 rat strains (Long-Evans and Sprague-Dawley). In a second experiment involving a large sample of rats (n = 50), we established that the ratio of SP to SA is approximately 35-65%. This bimodal behavioral response to sucralose appears to be driven by taste because rats display a similar bimodal licking response to a range of sucralose solutions presented during brief-access tests. Finally, we have shown that sucralose avoidance is extremely robust as 23-h water-deprived SA continue to avoid sucralose in 1-h single-bottle intake tests. Based on their reduced licking responses to sucralose during brief-access (taste driven) tests, and the fact that their distaste for sucralose cannot be overcome by the motivation to rehydrate, we conclude that SA detect a negative taste quality of sucralose that SP are relatively insensitive to.

Maintenance on a high-fat diet impairs the anorexic response to glucagon-like-peptide-1 receptor activation.

Previous data suggests that the adiposity signal leptin reduces food intake in part by enhancing sensitivity to short-term signals that promote meal termination, including glucagon-like peptide 1 (GLP-1). We hypothesized that maintenance on a high-fat (HF) diet, which causes resistance to leptin, would impair GLP-1's ability to reduce food intake. To test this hypothesis, we examined the anorexic responses to intraperitoneal injection of 100 µg/kg GLP-1 and 1 µg/kg exendin-4 (Ex-4), the potent, degradation resistant GLP-1 receptor agonist, in Wistar rats maintained on a low-fat (10%; LF) or HF (60%) diet for 4-6 weeks. Rats maintained on each of these diets were tested twice, once while consuming LF food and once while consuming HF food, to distinguish between effects of acute vs. chronic consumption of HF food. LF-maintained rats tested on LF diet reduced 60-min dark phase intake in response to GLP-1, but HF-maintained rats failed to respond to GLP-1 whether they were tested on HF or LF diet. LF-maintained rats tested on HF diet also showed no response, suggesting that even brief exposure to HF diet can impair sensitivity to GLP-1 receptor activation. Both LF- and HF-maintained rats showed significant anorexic responses to Ex4 at 4h post-treatment, but only LF-maintained rats had significantly reduced intake and body weight 24h after injections. To determine whether the ability of endogenous GLP-1 to promote satiation is impaired by HF maintenance, we examined the response to exendin 3 (9-39) (Ex9), a GLP-1 receptor antagonist. In LF-maintained rats, Ex9 increased intake significantly, but HF-maintained rats reduced food intake in response to Ex9. These data support the suggestion that maintenance on HF diet reduces the anorexic effects of GLP-1 receptor activation, and this phenomenon may contribute to overconsumption of high-fat foods.

Estradiol acts in the medial preoptic area, arcuate nucleus, and dorsal raphe nucleus to reduce food intake in ovariectomized rats.

Estradiol (E2) exerts an inhibitory effect on food intake in a variety of species. While compelling evidence indicates that central, rather than peripheral, estrogen receptors (ERs) mediate this effect, the exact brain regions involved have yet to be conclusively identified. In order to identify brain regions that are sufficient for E2's anorectic effect, food intake was monitored for 48 h following acute, unilateral, microinfusions of vehicle and two doses (0.25 and 2.5 µg) of a water-soluble form of E2 in multiple brain regions within the hypothalamus and midbrain of ovariectomized rats. Dose-related decreases in 24-h food intake were observed following E2 administration in the medial preoptic area (MPOA), arcuate nucleus (ARC), and dorsal raphe nucleus (DRN). Within the former two brain areas, the larger dose of E2 also decreased 4-h food intake. Food intake was not influenced, however, by similar E2 administration in the paraventricular nucleus, lateral hypothalamus, or ventromedial nucleus. These data suggest that E2-responsive neurons within the MPOA, ARC, and DRN participate in the estrogenic control of food intake and provide specific brain areas for future investigations of the cellular mechanism underlying estradiol's anorexigenic effect.