Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Feb 17.
Published in final edited form as: Physiol Behav. 2013 Jan 10;0:109–114. doi: 10.1016/j.physbeh.2013.01.003

Overexpression of neuropeptide Y in the dorsomedial hypothalamus increases trial initiation but does not significantly alter concentration-dependent licking to sucrose in a brief-access taste test

Yada Treesukosol 1, Sheng Bi 1, Timothy H Moran 1
PMCID: PMC3615091  NIHMSID: NIHMS435052  PMID: 23313404

Abstract

Evidence in the literature raises the possibility that alterations in neuropeptide Y (NPY) in the dorsomedial hypothalamus (DMH) may contribute to hyperphagia leading to body weight gain. Previously, we have shown that compared to AAVGFP controls, adeno-associated virus (AAV)-mediated overexpression of NPY in the DMH of lean rats resulted in significantly higher body weight gain that was attributed to increased food intake, and this was further exacerbated by a high-fat diet. Here, we tested AAVNPY and AAVGFP control rats in a brief-access taste procedure (10-s trials, 30-min sessions) to an array of sucrose concentrations under ad libitum and partial food and water access conditions. The test allows for some segregation of the behavioral components by providing a measure of trial initiation (appetitive) and unconditioned licks at each concentration (consummatory). Consistent with previous findings suggesting that NPY has a primary effect on appetitive function, overexpression of DMH NPY did not significantly alter concentration-dependent licking response to sucrose but when tested in a non-restricted food and water schedule, AAVNPY rats initiated significantly more sucrose trials compared to AAVGFP controls in a brief-access taste test.

Keywords: NPY, hypothalamus, adeno-associated virus, sucrose, appetitive, taste

Introduction

Following its discovery [38], neuropeptide Y (NPY) has been shown to be widely distributed in the mammalian central [1] and peripheral nervous systems [17, 26]. Central administration of NPY in rats increases food intake [11, 24, 37] and, with long-term administration, induces increases in body weight and body fat [36, 44]. Consistent with evidence pointing to the importance of hypothalamic peptide systems in energy balance mechanisms, NPY has been found throughout the hypothalamus [10, 17] including localization in the arcuate nucleus and dorsomedial hypothalamus (DMH) [9–10, 17, 42]. Running wheel access and food restriction [25], as well as running wheel access alone [21], have been shown to induce elevated NPY protein or mRNA levels in the DMH. Furthermore, elevated expression of NPY in the DMH has been observed in a number of obesity rodent models, including in homozygous melanocortin 4 receptor (MC4-R) knockout mice [22], obese tubby mice [19], diet-induced obese mice [18] and obese brown adipose tissue-deficient (uncoupling protein-promoter-drive diphtheria toxin A) mice [41]. Increased expression of DMH NPY has also been found in young pre-obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats and OLETF rats that have been pair-fed to match food intake of lean LETO controls [8], thus raising the possibility that alterations in NPY in the DMH precedes the hyperphagia that leads to weight gain, in at least some of these obesity models.

More recently, our group has used the adeno-associated virus (AAV) system to either increase or decrease Npy gene expression in the DMH of rats. Compared to AAVGFP controls, lean rats with overexpression of DMH NPY displayed significantly higher body weight gain, which appeared to be attributed by an increase in chow intake and was further exacerbated by the presentation of a high-fat diet [43]. In contrast, knockdown of NPY in the DMH via AAV-mediated RNAi in OLETF rats, significantly reduced daily food intake, body weight gain, lowered hyperglycemia and decreased fat accumulation compared to control OLETF rats, to a degree similar to that of the lean LETO group [43].

Ingestive behavior can be thought of as consisting of an appetitive component which involves behavior that brings the animal to the stimulus and a consummatory component which describes behavior following stimulus contact with the oral cavity [see 13]. Based on previous findings in the literature, it has been suggested that the main feeding effects of NPY are on increases of appetitive but not consummatory behavior. A number of studies have shown that intracerebroventricular (ICV) administration of NPY increases intake of a sucrose solution when presented in a one-bottle test which involves both appetitive and consummatory behavior, but not when the solution is infused intraorally, a measure which focuses more on the consummatory component of ingestion [3–4, 30–31].

In contrast, prior behavioral training and stimulus exposure have been shown to interact with the effects of NPY so that, for example, ICV NPY administration can elicit increases in intraoral sucrose intake [7]. Furthermore, ICV NPY administration has been shown to increase the size of meal, which can be regarded as a measure of consummatory behavior with little or no significant change in meal frequency, which can be thought of as a measure of appetitive behavior [23, 28]. Meal pattern analysis revealed that the decrease in chow intake observed in OLETF rats with knockdown of NPY expression in the DMH, was primarily attributed to a decrease in meal size compared to OLETF controls [43]. Consistent with these results, NPY knockdown in the DMH of intact rats produces a nocturnal and meal size-specific feeding effect [43]. Licking microstructure analysis has revealed an increase in meal frequency for water, saccharin and sucrose in response to ICV NPY in rats. However, for sucrose, NPY also elicited an increase in meal size [5]. Collectively, these findings suggest that in addition to increasing appetitive feeding, under certain test conditions (e.g. prior training, stimulus exposure and caloric content of stimulus), NPY can also increase consummatory components of behavior.

In the current study, unconditioned licking responses to a sucrose concentration array are compared between rats with AAV-mediated overexpression of Npy in the DMH (AAVNPY) and their AAVGFP controls. The brief-access taste procedure allows for some segregation between appetitive (spout approach measured as number of trials initiated) and consummatory (lick responses across the concentration range within each 10-s trial) behaviors. The two groups were tested in non-deprived (ad libitum access to chow and water) and partial food-and water restricted (~10 g chow, ~20 ml water) conditions. If DMH NPY is primarily involved in behaviors that bring the animal to the stimulus, we would expect AAVNPY animals to initiate significantly more trials compared to their AAVGFP controls, with little or no group difference in licking across the sucrose concentration range. Alternatively, if the ingestive effects of NPY are via orosensory alterations, this may also impact hedonic responses to oral stimuli thus resulting in group differences in concentration-dependent lick responses.

Materials and Methods

2.1 Subjects

Sixteen male Sprague-Dawley rats (Charles River Breeders) weighing 351.4 ± 3.4 g on the first day of behavioral training were individually housed in hanging wire mesh cages in a room where humidity, temperature and a 12 h – 12 h light-dark cycle (lights on at 7:00 am) were automatically controlled. Animals were provided ad libitum chow (Prolab RMH 1000) and distilled water, excepted where noted. Behavioral testing sessions were conducted during the light cycle. All procedures were approved by the Institutional Animal Care and Use Committee at The Johns Hopkins University.

Behavioral testing began after at least 7 days acclimation in the lab environment. During behavioral training, the rats were placed on a water-restricted schedule. Water access was removed from the home cages no more than 23 hours before testing and water was available only during the daily training sessions. Ad libitum access to water resumed in the home cages after the last training session. A partial food and water restriction condition to encourage sampling without imposing a 24-h deprivation schedule and to provide a condition to compare responses during different states of deprivation was included. Rats were presented ~10 g of chow and ~20 ml of water in the home cages for approximately 23 hours before testing as adapted from studies in mice [16] and since used to test rats [e.g. 29]. Body weight was measured every day during water or partial food and water restriction conditions and did not fall below 85% of weight during ad libitum feeding.

2.2 Taste stimuli

All solutions were prepared daily with distilled water and presented at room temperature. Six concentrations of sucrose (0.01, 0.03, 0.06, 0.1, 0.3, and 1.0 M; Sigma Aldrich, St. Louis MO) were used.

2.3 Behavioral Procedure

Training and testing were conducted in a lickometer (Davis MS-160, DiLog Instruments, Tallahassee FL) as previous described elsewhere [eg. 16, 32]. The rat was placed in the testing chamber of the apparatus and given access to a single spout positioned approximately 5 mm behind a slot. The spout was connected to a glass container holding a taste stimulus. A small fan was positioned above the chamber wall slot to direct a current of air past the drinking spout and to minimize potential olfactory cues from the stimulus. The rat initiated a trial by licking the spout. At the end of each trial (10 s), the shutter closed. During each 8-s intertrial interval, a motorized block moved to change the tube presentation and the shutter reopened for the next trial. Concentrations were presented in randomized blocks (without replacement). Animals were able to initiate as many trials as possible during the 30-min sessions.

2.4 Behavioral training and testing

Animals were trained and tested at the start of the experiment and ~ 4 weeks after bilateral AAV injection into the DMH (post-surgical testing). For the first 5 days of behavioral training and testing, animals were placed on a ~23 h water restriction schedule in which water was available only during the daily 30-min sessions. On days 1 and 2, rats were presented with a stationary spout of water for 30 minutes. Total number of licks and inter-lick-interval were measured. On days 3 and 4, 7 tubes of water were prepared and presented one at a time in 10-s trials across 30-min sessions. On day 5, the 7 tubes were prepared with varying sucrose concentrations and presented in a similar manner.

After two days of rehydration, animals were presented the same array of sucrose concentrations for four consecutive days alternating testing conditions between non-restricted (ad libitum access to food and water) and partial-food-and-water states, as outlined in Table 1.

Table 1.

Experimental Design

Condition Days Stimulus
Water restricted 2 Stationary water
Water restricted 2 Multiple presentations of water
Water restricted 1 Multiple concentrations of sucrose
Two days hydration, no testing
Ad lib water + food 1 Multiple concentrations of sucrose
Partial food and water restriction 1 Multiple concentrations of sucrose
Ad lib water + food 1 Multiple concentrations of sucrose
Partial food and water restriction 1 Multiple concentrations of sucrose
Open in a new tab

2.5 Data analysis

Total licks and interlick interval (ILI) values to stationary water on day 2 were compared using two-sample t-tests. Only ILIs that were between 50 and 250 ms were included for analysis. Values that were less than 50 ms were considered as double licks and ILI values greater than 250 ms were considered pauses between licking bursts [see 2, 14, 15].

For a given animal during each test condition (non-restricted and partial-food-and-water restricted), the mean number of licks at each concentration was calculated by collapsing all trials across the two sessions. The mean number of licks to water was subtracted from the mean number of licks at each concentration, yielding a Licks Relative to Water value. This measure has been used in previous studies [20, 35, 39–40] to produce concentration-response curves that are adjusted to a water baseline. The Licks Relative to Water value for each sucrose concentration was compared using analyses of variance (ANOVAs). The total number of trials initiated across the two sessions of each test condition was compared. The statistical rejection criterion of 0.05 was used for all analyses.

Curves were fit to mean data for each group by using the following logistic function:

f(x)=a(1+10(x−c)b)

where × = log10 stimulus concentration, a = asymptotic lick response adjusted for water, b = slope and c = log10 concentration at the inflection point.

2.6 AAV-mediated NPY expression vector

Recombinant viral vector AAVNPY and AAVGFP prepared previously using the AAV Helper-Free System (Statagene) [43] was utilized. Briefly, the full length of rat Npy cDNA was first cloned into the pAAV-IRES-hrGFP vector that also contains the marker gene of humanized Renilla green fluorescent protein (hrGFP). Three plasmids of pAAVNPY (or pAAVGFP as a control), pHelper (carrying adenovirus-derived genes), and pAAV-RC (carrying AAV-2 replication and capsid genes) were cotransfected into the AAV-293 cells according to the manufacturer’s protocol (Stratagene). Three days after transfection, cells were harvested, and the recombinant viral vector AAVNPY (or AAVGFP) was purified using an AAV purification kit (Virapur) and concentrated using Centricon YM-100 (Millipore) according to the manufacturers’ protocols. Viral titers were determined using quantitative PCR and 1 × 109 particles/site were used for each viral injection.

2.7 NPY overexpression in the DMH

After pre-surgical training and testing, rats were assigned to one of two groups so that the groups did not significantly differ in Licks Relative to Water values, body weight on the last day of pre-surgical testing, mean inter-lick interval (ILI) to water and number of trials initiated to water and sucrose (data not shown). One group received bilateral DMH injections of AAVNPY. The control group received bilateral DMH injections of AAVGFP. Rats were anesthetized with a mixture of ketamine hydrochloride (100 mg/kg) and xylazine (20 mg/kg) delivered via intraperitoneal injection and placed in a stereotaxic apparatus. Supplemental doses were administered as necessary. Recombinant AAV vectors (0.5 μl/site, ~1 × 109 particles/site) were bilaterally injected into the DMH using the coordinates: 3.1 mm caudal to bregma, 0.4 mm lateral to the midline and 8.3 mm ventral to the skull surface. Injections were administered with an injector (33 gauge; Plastics One, Roanoke, VA) connected via tubing (PE 20) to a Hamilton syringe. A stepper-motorized nanoliter injection pump (Stoelting) allowed for delivery at the rate of 0.1 μl/min for 5 minutes. After injection, 5 minutes elapsed before the injector was slowly removed. In both surgical groups the incision was closed. Saline was injected subcutaneously following surgery. Two animals died during surgery. There were no significant group differences in any of the measures compared after pre-surgical behavioral testing data from these animals were removed from the study. Animals regained body weight within 4–10 days following surgery. Post-surgical testing was conducted ~4 weeks following AAV injection.

2.8 Qualitative in situ hybridization

During the light cycle, animals were decapitated and brains were removed and frozen on dry ice. Coronal brain sections of 14 μm in thickness were mounted on superfrost slides. Plasmids of NPY were linearized by recommended restriction enzymes. As described previously [9, 43], 35S-labeled antisense riboprobes were used to determine levels of NPY mRNA. Sections were pretreated with acetic anhydride and ethanol then incubated in a hybridization buffer at 55°C for at least 16 hours. Slides were rinsed in 2× SSC at 55°C three times, rinsed in a RNAse A bath at 37°C for 30 minutes, rinsed in 2× SSC twice at 55°C for 5 minutes, then rinsed twice in 0.1 × SSC at 55°C for 15 minutes. Slides were dehydrated in ethanol, air-dried and exposed to film BioMax ZAR Film (Eastman Kodak Company) for 3 to 10 days. Sections were assessed to qualitatively verify overexpression of NPY in the DMH of the AAVNPY animals.

Results

3.1 In situ hybridization

Rats in the AAVNPY group showed dense expression of Npy mRNA in the DMH areas compared to AAVGFP rats. These results indicate that AAVNPY animals had NPY overexpression in the DMH (Figure 1).

Figure 1.

Figure 1

Open in a new tab

Representative image of in situ hybridization with 35S-labeled antisense riboprobe of Npy to confirm AAV-mediated NPY overexpression in DMH

3.2 Licks to Water

Two-sample t-tests revealed no significant group differences in post surgery total licks (t(12) = −0.274, p=0.789) or interlick interval values (t(12) = 0.896, p=0.388) to water when tested to a stationary spout of water for 30 min (Figure 2).

Figure 2.

Figure 2

Open in a new tab

Mean ± SE (A) total licks and (B) ILI, to a stationary spout of water were not significantly different between the AAVGFP (white bars) and AAVNPY (black bars) groups.

3.3 Water-Restricted

When tested post surgery in a water-restricted state, the number of trials initiated to 10-s trials to water (t(12) = −1.728, p=0.110) and to a concentration array of sucrose (t(12) = −0.416, p=0.685) did not significantly differ between the two groups (Figure 3).

Figure 3.

Figure 3

Open in a new tab

Mean ± SE number of 10-s trials initiated in a 30-min session to (A) water, and (B) an array of sucrose concentrations, for AAVGFP (white bars) and AAVNPY (black bars) rats when tested in a water-restricted condition

3.4 Sucrose

Licking responses across the sucrose concentration array tested did not significantly differ between the two groups. When tested in a non-restricted state, two-way ANOVAs comparing Licks Relative to Water values of the two groups did not reveal a significant main effect of group (F(1,12)=0.639, p=0.440), showed a main effect of concentration (F(5,60)=46.616, p<0.001) and no significant interaction (F(5,60)=0.345, 0.884, Figure 4A). Similarly, there was no main effect of group (F(1,12)=0.2.460, p=0.143), a main effect of concentration (F(5,60)=118.509, p<0.001) and no significant interaction (F(5,60)=0.467, 0.799) when Licks Relative to Water values to sucrose under a partial-food-and-water restricted schedule were compared between the two groups (Figure 4C). Although unconditioned licking responses across the concentration range did not significantly differ between the two groups, AAVNPY animals initiated significantly more trials than control rats when tested in a non-restricted state (t(12) = 2.761, p=0.017; Figure 4B) although this did not reach significance under the partial food-and-water restricted schedule (t(12) = 1.143, p=0.275; Figure 4D).

Figure 4.

Figure 4

Open in a new tab

(A) Mean ± SE licks relative to water and (B) trials initiated to a sucrose concentration array when animals were tested in a non-restricted state across two test sessions. (C) Mean ± SE licks relative to water and (D) trials initiated to a sucrose concentration array when animals were tested in a partial food and water restricted state across two test sessions

Discussion

Consistent with evidence in the literature pointing to the principal ingestive effects of NPY on the appetitive components of behavior, overexpression of DMH NPY did not significantly alter concentration-dependent licking response to sucrose but when tested under an ad libitum food and water condition, AAVNPY rats initiated significantly more trials to sucrose compared to AAVGFP controls.

Overexpression of DMH NPY did not significantly alter taste-guided consummatory behavior as measured by concentration-dependent licking in this procedure. Although sensory thresholds and affective responses measure different functional aspects of taste and can be experimentally dissociated [see 33, 34], these measures are not necessarily mutually exclusive. Based on these findings, it appears that overexpression of DMH NPY has little effect on the sensory response to sucrose. Consistent with this, it has been previously reported that ICV NPY did not significantly alter taste reactivity to brief intraoral infusions of sucrose or quinine solutions [30]. Thus, the orexigenic effects of NPY are not likely attributed to alterations in taste sensory function.

In striking contrast, when tested under ad libitum access to food and water conditions, AAVNPY animals initiated significantly more trials to sucrose in the brief-access taste test, compared to their AAVGFP controls. These findings are consistent with studies using other behavioral measures [3–4, 30–31] that suggest the effect of NPY on ingestion is primarily through increasing appetitive behaviors. As a comparison, when tested under the partial food and water restricted condition, the group difference in trial initiation did not reach statistical significance. This group difference appears to be less apparent when tested in the ~23 h water-restricted condition. Thus with increasing degree of water (and chow) restriction, the effect of overexpression of NPY in the DMH on trial initiation is less robust. One possible explanation is that although the brief-access test relies on the hedonic component of the stimulus to drive a behavioral response, with increasing degrees of restriction, the animals also become increasingly sustenance-driven to initiate trials to obtain fluid and calories. Under these conditions, the measures are confounded by ceiling effects that are possibly masking the excitatory effects of NPY.

The two groups did not significantly differ in total licks from a stationary spout of water in the 30-min sessions suggesting overexpression of DMH NPY is insufficient to increase water intake induced by water deprivation. These data are consistent with studies in which NPY administered to the lateral or third ventricles failed to significantly increase overall water intake compared to controls [5, 27]. In contrast, small dose-dependent increases in water intake were observed following NPY injections into the fourth ventricle [12] and the paraventricular nucleus (PVN) [37]. At least in AAVNPY animals, the increased ingestive behavior does not appear to effect or be driven by water intake. Additionally, in the current study, interlick interval to water, a measure of local lick rate, did not significantly differ between the two groups, thus the hyperphagia and increased trial initiation observed in AAVNPY animals is not likely driven by changes in oromotor function.

The findings in the current study support the role of NPY in increasing appetitive behavior thus raising an apparent discrepancy with meal pattern analysis data that reveal changes in meal size as opposed to meal frequency [23, 28, 43] as an effect of NPY manipulation. However, it has been shown that under certain test conditions ICV NPY administration can increase intraoral sucrose intake [7] or increase both meal size and frequency to sucrose [6], thus collectively these findings suggest that in addition to increasing appetitive feeding, NPY can influence consummatory components. In the current study, there were no significant group differences in concentration-dependent licking to sucrose thus if these consummatory influences are playing a role, they are not likely driven by enhanced orosensory taste function.

Previously, we reported that overexpression of DMH NPY in lean rats resulted in significantly increased body weight and food intake. Consistent with previous findings suggesting that NPY has a primary effect on appetitive function, here, we show that overexpression of DMH NPY did not have a significant effect on unconditioned lick responses to a sucrose concentration array, but when tested in a non-restricted food and water schedule, AAVNPY rats initiated significantly more sucrose trials compared to their AAVGFP controls. These data thus suggest that the orexigenic effect of overexpression of DMH NPY is primarily driven by an increase in the appetitive component of ingestive behavior with little or no alterations on orosensory or oromotor function.

Highlights.

  • Rats with adeno-associated virus (AAV)-mediated overexpression of NPY (AAVNPY) in the DMH were tested with a sucrose concentration array in a brief-access taste test

  • AAVNPY rats initiated significantly more trials than AAVGFP controls when tested in a non-deprived state

  • Consistent with the hypothesis that the main feeding effects of NPY are on appetitive function

  • No significant group differences in lick response across the sucrose concentrations

  • DMH NPY overexpression does not appear to alter the consummatory component of behavior as tested in this procedure

Acknowledgements

We would like to acknowledge Yonwook J Kim, Ryan Purcell and Dr. Nu-Chu Liang for their technical help in these experiments. Supported by NIH DK019302 (T.H.M) and DK074269 (S.B).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Contributor Information

Yada Treesukosol, Email: yadatree@jhmi.edu.

Sheng Bi, Email: sbi@jhmi.edu.

Timothy H. Moran, Email: tmoran@jhmi.edu.

References

  • 1.Allen YS, Adrian TE, Allen JM, Tatemono K, Crow TJ, Bloom SR. Neuropeptide Y distribution in rat brain. Science. 1983;221:877–879. doi: 10.1126/science.6136091. [DOI] [PubMed] [Google Scholar]
  • 2.Allison J, Castellan NJ., Jr Temporal characteristics of nutritive drinking in rats and humans. J Comp Physiol Psychol. 1970;70:116–125. [Google Scholar]
  • 3.Ammar AA, Negardh R, Fredholm BB, Brodin U, Sodersten P. Intake inhibition by NPY and CCK-8: A challenge of the notion of NPY as an "Orexigen". Behav Brain Res. 2005;161:82–87. doi: 10.1016/j.bbr.2005.01.014. [DOI] [PubMed] [Google Scholar]
  • 4.Ammar AA, Sederholm F, Saito TR, Scheurink AJ, Johnson AE, Sodersten P. NPY-leptin: opposing effects on appetitive and consummatory ingestive behavior and sexual behavior. Am J Physiol Regul Integr Comp Physiol. 2000;278:R1627–R1633. doi: 10.1152/ajpregu.2000.278.6.R1627. [DOI] [PubMed] [Google Scholar]
  • 5.Baird J-P, Gray NE, Fischer SG. Effects of neuropeptide Y on feeding microstructure: dissociation of appetitive and consummatory actions. Behav Neurosci. 2006;120:937–951. doi: 10.1037/0735-7044.120.4.937. [DOI] [PubMed] [Google Scholar]
  • 6.Baird J, Gray NE, Fischer SG. Effects of neuropeptide Y on feeding microstructure: Dissociation of appetitive and consummatory actions. Behav Neurosci. 2006;120:937–951. doi: 10.1037/0735-7044.120.4.937. [DOI] [PubMed] [Google Scholar]
  • 7.Benoit SC, Clegg DJ, Woods SC, Seeley RJ. The role of previous exposure in the appetitive and consummatory effects of orexigenic neuropeptides. Peptides. 2005;26:751–757. doi: 10.1016/j.peptides.2004.12.012. [DOI] [PubMed] [Google Scholar]
  • 8.Bi S, Ladenheim EE, Schwartz GJ, Moran TH. A role for NPY overexpression in the dorsomedial hypothalamus in hyperphagia and obesity of OLETF rats. Am J Physiol Regul Integr Comp Physiol. 2001;281:R254–R260. doi: 10.1152/ajpregu.2001.281.1.R254. [DOI] [PubMed] [Google Scholar]
  • 9.Bi S, Robinson BM, Moran TH. Acute food deprivation and chronic food restriction differentially affect hypothalamic NPY mRNA expression. Am J Physiol Regul Integr Comp Physiol. 2003;285:R1030–R1036. doi: 10.1152/ajpregu.00734.2002. [DOI] [PubMed] [Google Scholar]
  • 10.Chronwall BM, Dimaggio DA, Massar VJ, Pickel VM, Ruggiero DA, O'Donohue TL. The anatomy of neuropeptide-Y-containing neurons in rat brain. Neuroscience. 1985;15:1159–1181. doi: 10.1016/0306-4522(85)90260-x. [DOI] [PubMed] [Google Scholar]
  • 11.Clark JT, Kalra PS, Crowley WR, Kalra SP. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology. 1984;115:427–429. doi: 10.1210/endo-115-1-427. [DOI] [PubMed] [Google Scholar]
  • 12.Corp ES, Melville LD, Greenberg D, Gibbs J, Smith GP. Effect of fourth ventricular neuropeptide Y and peptide YY on ingestive and other behaviors. Am J Physiol. 1990;259:R317–R323. doi: 10.1152/ajpregu.1990.259.2.R317. [DOI] [PubMed] [Google Scholar]
  • 13.Craig W. Appetites and aversions as constituents of instincts. Biol Bull. 1918;34:91–107. doi: 10.1073/pnas.3.12.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Davis JD, Smith GP. Analysis of the microstructure of the rhythmic tongue movements of rats ingesting maltose and sucrose solutions. Behav Neurosci. 1992;106:217–228. [PubMed] [Google Scholar]
  • 15.Davis JD, Smith GP. Learning to sham feed: behavioral adjustments to loss of physiological postingestional stimuli. Am J Physiol Regul Integr Comp Physiol. 1990;259:R1228–R1235. doi: 10.1152/ajpregu.1990.259.6.R1228. [DOI] [PubMed] [Google Scholar]
  • 16.Glendinning JI, Gresack J, Spector AC. A high-throughput screening procedure for identifying mice with abberant taste and oromotor function. Chem Senses. 2002;27:461–474. doi: 10.1093/chemse/27.5.461. [DOI] [PubMed] [Google Scholar]
  • 17.Gray TS, Morley JE. Neuropeptide Y: anatomical distribution and possible function in mammalian nervous system. Life Sci. 1986;38:389–401. doi: 10.1016/0024-3205(86)90061-5. [DOI] [PubMed] [Google Scholar]
  • 18.Guan X-M, Yu H, Trumbauer M, Frazier E, Van der Ploeg LHT, Chen H. Induction of neuropeptide Y expression in dorsomedial hypothalamus of diet-induced obese mice. Neuroreport. 1998;9:3415–3419. doi: 10.1097/00001756-199810260-00015. [DOI] [PubMed] [Google Scholar]
  • 19.Guan X-M, Yu H, Van der Ploeg LHT. Evidence of altered hypothalamic pro-opiomelanocortin/ neuropeptide Y mRNA expression in tubby mice. Brain Res Mole Brain Res. 1998;59:273–279. doi: 10.1016/s0169-328x(98)00150-8. [DOI] [PubMed] [Google Scholar]
  • 20.Jiang E, Blonde G, Garcea M, Spector AC. Greater superficial petrosal nerve transection in rats does not change unconditioned licking responses to putatively sweet taste stimuli. Chem Senses. 2008;33:709–723. doi: 10.1093/chemse/bjn039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Kawaguchi M, Scott KA, Moran TH, Bi S. Dorsomedial hypothalamic corticotropin-releasing factor mediation of exercise-induced anorexia. Am J Physiol Regul Integr Comp Physiol. 2005;288:R1800–R1805. doi: 10.1152/ajpregu.00805.2004. [DOI] [PubMed] [Google Scholar]
  • 22.Kesterson RA, Huszar D, Lynch CA, Simerly RB, Cone RD. Induction of neuropeptide Y gene expression in the dorsal medial hypothalamic nucleus in two models of the agouti obesity syndrome. Mol Endocrinol. 1997;11:630–637. doi: 10.1210/mend.11.5.9921. [DOI] [PubMed] [Google Scholar]
  • 23.Leibowitz SF, Alexander JT. Analysis of neuropeptide Y-induced feeding: dissociation of Y1 and Y2 receptor effects on natural meal patterns. Peptides. 1991;12:1251–1260. doi: 10.1016/0196-9781(91)90203-2. [DOI] [PubMed] [Google Scholar]
  • 24.Levine AS, Morley JE. Neuropeptide Y: a potent inducer of consummatory behavior in rats. Peptides. 1984;5:1025–1029. doi: 10.1016/0196-9781(84)90165-7. [DOI] [PubMed] [Google Scholar]
  • 25.Lewis DE, Shellard L, Koeslag DG, Boer DE, McCarthy HD, McKibbin PE, Russell JC, WIlliams G. Intense exercise and food restriction cause similar hypothalamic neuropeptide Y increases in rats. Am J Physiol. 1993;264:E279–E284. doi: 10.1152/ajpendo.1993.264.2.E279. [DOI] [PubMed] [Google Scholar]
  • 26.Lundberg JM, Terenius L, Hokfelt T, Martling CR, Tatemoto K, Mutt V, Polak J, Bloom S, Goldstein M. Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta Physiol Scand. 1982;116:477–480. doi: 10.1111/j.1748-1716.1982.tb07171.x. [DOI] [PubMed] [Google Scholar]
  • 27.Lynch WC, Grace M, Billington CJ, Levine AS. Effects of neuropeptide Y on ingestion of flavored solutions in nondeprived rats. Physiol Behav. 1993;54:877–880. doi: 10.1016/0031-9384(93)90295-q. [DOI] [PubMed] [Google Scholar]
  • 28.Marin Bivens CL, Thomas WJ, Stanley BG. Similar feeding patterns are induced by perifornical neuropeptide Y injection and by food deprivation. Brain Res. 1998;782:271–280. doi: 10.1016/s0006-8993(97)01289-4. [DOI] [PubMed] [Google Scholar]
  • 29.Mathes CM, Spector AC. The selective serotonin reuptake inhibitor paroxetine does not alter consummatory concentration-dependent licking of prototypical taste stimuli by rats. Chem Senses. 2011;36:515–526. doi: 10.1093/chemse/bjr011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Sederholm F, Ammar AA, Sodersten P. Intake inhibition by NPY: role of appetitive ingestive behavior and aversion. Physiol Behav. 2002;75:567–575. doi: 10.1016/s0031-9384(02)00648-0. [DOI] [PubMed] [Google Scholar]
  • 31.Seeley RJ, Payne CJ, Woods SC. Neuropeptide Y fails to increase intraoral intake in rats. American Journal of Physiology. 1995;268:R423–R427. doi: 10.1152/ajpregu.1995.268.2.R423. [DOI] [PubMed] [Google Scholar]
  • 32.Smith JC. The history of the "Davis Rig". Appetite. 2001;36:93–98. doi: 10.1006/appe.2000.0372. [DOI] [PubMed] [Google Scholar]
  • 33.Spector AC. Linking gustatory neurobiology to behavior in vertebrates. Neurosci Biobehav Rev. 2000;24:391–416. doi: 10.1016/s0149-7634(00)00013-0. [DOI] [PubMed] [Google Scholar]
  • 34.Spector AC, Glendinning JI. Linking peripheral taste processes to behavior. Curr Opin Neurobiol. 2009;19:370–377. doi: 10.1016/j.conb.2009.07.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Spector AC, Redman R, Garcea M. The consequences of gustatory nerve transection on taste-guided licking of sucrose and maltose in the rat. Behav Neurosci. 1996;110:1096–1109. [PubMed] [Google Scholar]
  • 36.Stanley BG, Kyrkouli SE, Lampert S, leibowitz SF. Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity. Peptides. 1986;7:1189–1192. doi: 10.1016/0196-9781(86)90149-x. [DOI] [PubMed] [Google Scholar]
  • 37.Stanley BG, Leibowitz SF. Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus. Life Sci. 1984;35:2635–2642. doi: 10.1016/0024-3205(84)90032-8. [DOI] [PubMed] [Google Scholar]
  • 38.Tatemoto K, Carlquist M, Mutt V. Neuropeptide Y--a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature. 1982;296:659–660. doi: 10.1038/296659a0. [DOI] [PubMed] [Google Scholar]
  • 39.Treesukosol Y, Blonde GD, Spector AC. T1R2 and T1R3 subunits are individually unnecessary for normal affective licking responses to polycose: implications for saccharide taste receptors in mice. Am J Physiol Regul Integr Comp Physiol. 2009;296:R855–R865. doi: 10.1152/ajpregu.90869.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Treesukosol Y, Smith KR, Spector AC. Behavioral evidence for a glucose polymer taste receptor that is independent of the T1R2+3 heterodimer in a mouse model. . J Neurosci. 2011;31:13527–13534. doi: 10.1523/JNEUROSCI.2179-11.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Tritos NA, Elmquist JK, Mastaitis JW, Flier JS, Maratos-Flier E. Characterization of expression of hypothalamic appetite-regulating peptides in obese hyperleptinemic brown adipose tissue-deficient (uncoupling protein-promoter-driven diphtheria toxin A) mice. Endocrinology. 1998;139:4634–4641. doi: 10.1210/endo.139.11.6308. [DOI] [PubMed] [Google Scholar]
  • 42.White JD, Kershaw M. Increased hypothalamic neuropeptide Y expression following food deprivation. Mol Cell Neurosci. 1990;1:41–48. doi: 10.1016/1044-7431(90)90040-b. [DOI] [PubMed] [Google Scholar]
  • 43.Yang L, Scott KA, Hyun J, Tamashiro KLK, Tray N, Moran TH, Bi S. Role of dorsomedial hypothalamic neuropeptide Y in modulating food intake and energy balance. J Neurosci. 2009;29:179–190. doi: 10.1523/JNEUROSCI.4379-08.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Zarjevski N, Cusin I, Vettor R, Rohner-Jeanrenaud F, Jeanrenaud B. Chronic intracerebroventricular neuropeptide-Y administration to normal rats mimics hormonal and metabolic changes of obesity. Endocrinology. 1993;133:1753–1758. doi: 10.1210/endo.133.4.8404618. [DOI] [PubMed] [Google Scholar]

RESOURCES