elevated hypothalamic neuropeptide y levels in rats with dorsomedial hindbrain lesions

7
Ž . Brain Research 755 1997 84–90 Research report Elevated hypothalamic neuropeptide Y levels in rats with dorsomedial hindbrain lesions Gaylen L. Edwards a, ) , B. Douglas White b , Bin He b , Roger G. Dean c , Roy J. Martin b a Department of Physiology and Pharmacology, UniÕersity of Georgia, Athens, GA 30602, USA b Department of Foods and Nutrition, UniÕersity of Georgia, Athens, GA 30602, USA c Department of Animal Science, UniÕersity of Georgia, Athens, GA 30602, USA Accepted 30 December 1996 Abstract Ž . Ž . Lesions centered on the area postrema AP and adjacent nucleus of the solitary tract APrmNTS-lesions are reported to result in Ž . increased consumption of highly palatable diets. Recent studies suggest that neuropeptide Y NPY may cause a preference for carbohydrate-rich diets. Thus, it is possible that NPY may play a role in the enhanced intake of highly palatable diets by APrmNTS-lesioned rats. In the studies reported here, we found that lesions centered on the AP result in increased levels of NPY-immunoreactivity in the paraventricular nucleus of the hypothalamus. Additionally, steady-state NPY mRNA in the basomedial hypothalamus including the arcuate nucleus was elevated. Enhanced NPY was not found throughout the hypothalamus however, as NPY-immunoreactivity was not elevated in the lateral hypothalamus or the tissue bordering the anteroventral third ventricle. These data suggest the possibility that elevated hypothalamic NPY, particularly in the arcuate and paraventricular nuclei, may contribute to the altered food intake and energy balance observed in rats with lesions centered on the AP. q 1997 Elsevier Science B.V. All rights reserved. Keywords: Area postrema; Nucleus of the solitary tract; Arcuate nucleus; Paraventricular nucleus; Ingestive behavior; Food intake; Diet selection 1. Introduction The region of the dorsomedial medulla including the Ž . area postrema AP has been implicated in the control of body weight. After lesions centered on the AP, animals w x undergo a significant body weight loss 6,7,9,11 . Addi- tionally, the lesioned animals have marked changes in appetite, including an increased intake of highly palatable w x diets 7,15,16 and a marked preference for novel diets w x 12,24 . These data suggest that the AP may be important in diet selection as well as body weight regulation. Recent studies indicate that neuropeptides in the hypo- thalamus appear to be important in diet selection. Prevalent Ž . among these is neuropeptide Y NPY , a very potent w x orexigenic agent 1,3–5,10,13,14,18–23,25–27 . NPY is reported to be elevated at times when animals have an increased drive for food such as after food deprivation w x 18,23 . Moreover, NPY is suggested to specifically en- ) Ž . Corresponding author. Fax: q1 706 542-3015. E-mail: [email protected] w x hance carbohydrate intake 22,26 . For this reason, we were interested in the possibility that NPY is involved in the changes in diet selection observed in rats with lesions centered on the AP. In the studies reported here we have tested the hypothesis that rats with lesions centered on the AP had elevated levels of NPY in hypothalamic nuclei implicated in the control of diet selection. A preliminary wx report of these data is presented in abstract form 8 . 2. Materials and methods Ž . Male Sprague-Dawley rats Harlan, Indianapolis, IN were utilized in all studies. The animals were housed in suspended stainless steel cages in a temperature-controlled room on a 12 hr12 h lightrdark cycle. All animals were Ž allowed ad libitum access to pelleted rat chow Purina . 5012, Purina Mills, St. Louis, MO and water unless otherwise noted in the test protocol. Lesions of the AP and immediately adjacent nucleus of Ž . the solitary tract APrmNTS-lesion, n s 17 were pro- duced by aspiration according to a modification of a 0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0006-8993 97 00107-8

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Ž .Brain Research 755 1997 84–90

Research report

Elevated hypothalamic neuropeptide Y levels in rats with dorsomedialhindbrain lesions

Gaylen L. Edwards a,), B. Douglas White b, Bin He b, Roger G. Dean c, Roy J. Martin b

a Department of Physiology and Pharmacology, UniÕersity of Georgia, Athens, GA 30602, USAb Department of Foods and Nutrition, UniÕersity of Georgia, Athens, GA 30602, USA

c Department of Animal Science, UniÕersity of Georgia, Athens, GA 30602, USA

Accepted 30 December 1996

Abstract

Ž . Ž .Lesions centered on the area postrema AP and adjacent nucleus of the solitary tract APrmNTS-lesions are reported to result inŽ .increased consumption of highly palatable diets. Recent studies suggest that neuropeptide Y NPY may cause a preference for

carbohydrate-rich diets. Thus, it is possible that NPY may play a role in the enhanced intake of highly palatable diets byAPrmNTS-lesioned rats. In the studies reported here, we found that lesions centered on the AP result in increased levels ofNPY-immunoreactivity in the paraventricular nucleus of the hypothalamus. Additionally, steady-state NPY mRNA in the basomedialhypothalamus including the arcuate nucleus was elevated. Enhanced NPY was not found throughout the hypothalamus however, asNPY-immunoreactivity was not elevated in the lateral hypothalamus or the tissue bordering the anteroventral third ventricle. These datasuggest the possibility that elevated hypothalamic NPY, particularly in the arcuate and paraventricular nuclei, may contribute to thealtered food intake and energy balance observed in rats with lesions centered on the AP. q 1997 Elsevier Science B.V. All rightsreserved.

Keywords: Area postrema; Nucleus of the solitary tract; Arcuate nucleus; Paraventricular nucleus; Ingestive behavior; Food intake; Diet selection

1. Introduction

The region of the dorsomedial medulla including theŽ .area postrema AP has been implicated in the control of

body weight. After lesions centered on the AP, animalsw xundergo a significant body weight loss 6,7,9,11 . Addi-

tionally, the lesioned animals have marked changes inappetite, including an increased intake of highly palatable

w xdiets 7,15,16 and a marked preference for novel dietsw x12,24 . These data suggest that the AP may be importantin diet selection as well as body weight regulation.

Recent studies indicate that neuropeptides in the hypo-thalamus appear to be important in diet selection. Prevalent

Ž .among these is neuropeptide Y NPY , a very potentw xorexigenic agent 1,3–5,10,13,14,18–23,25–27 . NPY is

reported to be elevated at times when animals have anincreased drive for food such as after food deprivationw x18,23 . Moreover, NPY is suggested to specifically en-

) Ž .Corresponding author. Fax: q1 706 542-3015. E-mail:[email protected]

w xhance carbohydrate intake 22,26 . For this reason, wewere interested in the possibility that NPY is involved inthe changes in diet selection observed in rats with lesionscentered on the AP. In the studies reported here we havetested the hypothesis that rats with lesions centered on theAP had elevated levels of NPY in hypothalamic nucleiimplicated in the control of diet selection. A preliminary

w xreport of these data is presented in abstract form 8 .

2. Materials and methods

Ž .Male Sprague-Dawley rats Harlan, Indianapolis, INwere utilized in all studies. The animals were housed insuspended stainless steel cages in a temperature-controlledroom on a 12 hr12 h lightrdark cycle. All animals were

Žallowed ad libitum access to pelleted rat chow Purina.5012, Purina Mills, St. Louis, MO and water unless

otherwise noted in the test protocol.Lesions of the AP and immediately adjacent nucleus of

Ž .the solitary tract APrmNTS-lesion, ns17 were pro-duced by aspiration according to a modification of a

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 00107-8

( )G.L. Edwards et al.rBrain Research 755 1997 84–90 85

w xpreviously published procedure 7 . Briefly, this involvedŽ wanesthetizing each rat with methoxyflurane Metafane ,

.Pittman Moore, NJ , clipping and vacuuming the hair fromthe dorsum of the head and neck and placing the animal ina headholder with the neck flexed. The clipped area of skin

Ž wwas then cleaned with chlorhexidine Nolvasan , Fort.Dodge, IA 3 times. An incision was made from just

rostral to the occipital crest to the midcervical region andthe underlying musculature dissected to expose the fora-men magnum. The atlantooccipital ligament and underly-ing meninges were incised to expose the dorsal surface ofthe brainstem. The AP was then visualized through adissecting microscope and aspirated through a bluntedpiece of 30-gauge stainless steel tubing connected to avacuum line. The musculature was sutured with an ab-sorbable material and the skin sutured with silk to closethe surgical wound. The animals were monitored post-surgically until upright and walking, then returned to their

Ž .home cage. Sham-operated, control rats SHAM, ns17underwent a similar procedure during which the brainstemwas exposed, but instead of aspirating the AP, the surfaceof the AP was gently touched with the tip of a cottonswab. No testing was initiated for at least 6 weeks post-surgery to allow APrmNTS-lesioned rats to fully recoverfrom the surgery. The animals were weighed at least 3times per week during the recovery period and maintainedon pelleted rat chow.

Analysis of NPY mRNA and peptide was conducted in2 separate groups of APrmNTS-lesioned and SHAM rats.

ŽIn the first group, the animals APrmNTS-lesion, ns9;.SHAM, ns9 were euthanatized between 09:00 and 11:00

h by decapitation and the brain tissue rapidly collectedusing a macrodissection technique in which the basomedial

Ž .hypothalamus, including the arcuate nucleus ARC , wasŽdissected and placed in RNAzol CinnarBiotecx, Friend-

.wood, TX for extraction of the mRNA. The anteriordorsomedial aspect of the hypothalamus including the

Ž .paraventricular nucleus PVN , was also dissected andplaced in 200 ml 1 M acetic acid in 80% ethanol forextraction of the peptide. The medulla oblongata wasplaced in 10% formalin solution with 10% sucrose forfixation and subsequent histological analysis of theAPrNTS region and reconstruction of the lesions in thedorsomedial medulla.

Ž .The second group of APrmNTS-lesioned ns8 andŽ .SHAM ns8 rats was first tested for intake of a highly

Ž .palatable food lemon cookie before analysis of hypotha-lamic NPY mRNA and peptide. This test consisted of firstweighing the animals and removing the normal chow dietat approximately 09:00 h. Consumption of the lemoncookie was measured by placing 2–3 cookies in each cageand allowing each animal to consume the cookie for 30min at which time the remaining cookie plus crumbscollected below each animal’s cage was weighed. Thecookies were replaced and at 60, 120 and 180 min theremaining cookie plus crumbs was weighed and returned

to the animal’s cage. Behavioral testing was conducted atleast 10 days before the animals were euthanatized. Theanimals were euthanatized between 09:00 and 11:00 h bydecapitation and brain tissue collected for analysis of NPYmRNA and peptide in the hypothalamus. Unlike the firstgroup, the whole forebrain was rapidly frozen on dry ice.The hindbrain including the dorsomedial medulla wasplaced in 10% formalin with 10% sucrose for fixation andsubsequent histological analysis of the APrNTS regionand reconstruction of the lesions. The forebrain was subse-quently placed in a cryostat and 300–500 mm sections cutthrough the hypothalamus. Tissue punches were then madeof the ARC and placed in RNAzol for NPY mRNAanalysis using a section of 18-gauge tubing. Tissue puncheswere also collected from the PVN, ventromedial nucleus of

Ž .the hypothalamus VMH , and the lateral hypothalamicŽ .nucleus LH for analysis of NPY peptide as described

above.NPY content was analyzed by radioimmunoassay. Tis-

sues for peptide analysis were sonicated in 200 ml of 1 MŽ .acetic acid in 80% ethanol and centrifuged 10 000=g

for 10 min. The supernatant was collected and an addi-tional 200 ml of 1 M acetic acid in 80% ethanol was addedto the pellet. The samples were again sonicated and cen-

Ž .trifuged 10 000=g for 10 min. The resulting supernatantwas added to the first supernatant. The supernatants andpellets were lyophilized and stored at y708C until furtherprocessed.

NPY levels were determined from the lyophilized su-Žpernatants by a commercial RIA kit Peninsula, Belmont,

. Ž .CA . Radioimmunoassay buffer 500 ml was added to thesamples. Each sample was assayed in duplicate with anamount equivalent to 10 ml of diluted sample and 5 ml ofdiluted sample assayed.

The lyophilized pellet was resuspended in 0.05 N NaOH.The protein concentration was determined by the Bradford

Ž .protein assay BioRad, Richmond, CA . NPY levels werethen expressed as the NPY-immunoreactivity per mg pro-tein in the pellet formed during initial extraction of NPY.

NPY mRNA was quantitated by extraction of totalRNA from the ARC punches using a previously describedtechnique. This involved homogenizing each sample inRNAzol according to the manufacturer’s instructions toisolate total RNA. Briefly, chloroform was added to thehomogenized tissue and the mixture vortexed and cen-

Ž .trifuged 10 000=g at 48C for 15 min. The aqueous layerwas collected and the RNA precipitated by the addition ofan equal volume of isopropanol. Samples were incubatedat y208C for 45 min, centrifuged and the supernatantaspirated. The RNA pellets were washed and stored under75% ethanol until the samples could be further processed.Just prior to analysis the samples were centrifuged and theethanol aspirated. Pellets were dried under a vacuum for

w10 min and resuspended in 40 ml of Tris-EDTA buffer 10Ž .xmM Tris-HCl, 1 mM EDTA pH 7.5 .

For each sample, 3 serial dilutions were dotted onto a

( )G.L. Edwards et al.rBrain Research 755 1997 84–9086

Žcharged nylon membrane Gene Screen Plus, Dupont,.Wilmington, DE according to the manufacturer’s instruc-

tions. A standard curve that would be used to relate theŽ .optical density darkness of the dots from the autoradio-

graph to a relative amount of NPY mRNA blotted wasproduced by isolating total RNA from the hippocampus of2 rats. The hippocampal RNA was pooled and 12 serial

dilutions were prepared. Aliquots from the 12 serial dilu-tions were blotted on the charged nylon membrane induplicate. Additionally, a Northern analysis of the hip-pocampal RNA was run concurrently with the dot blots to

Ž .verify specificity of the probe binding data not shown .Membranes were prehybridized for 1 h in a phosphate

Ž .buffer 0.25 M NaPO , 0.25 M NaCl, 1 mM EDTA and4

Ž .Fig. 1. Photomicrographs of a coronal section through the caudal medulla at the level of the AP in a rats with an intact AP A and a rat with a lesionŽ .centered on the AP B . Note that the lesion includes the entire AP with minimal damage to the adjacent NTS. See text for additional description of the

lesion. AP s area postrema; NTS s nucleus of the solitary tract; X s dorsal motor nucleus of the vagus; XII s hypoglossal nucleus; 4V s fourthcerebral ventricle. Bar s 500 mm.

( )G.L. Edwards et al.rBrain Research 755 1997 84–90 87

50% formamide with 7% SDS at 62–648C. A 32 P-riboprobecomplementary to rat NPY mRNA was prepared with an

Ž .in vitro transcription kit Promega, Madison, WI andŽadded to the phosphate buffer. The cDNA insert used to

derive the probe was provided by Janet Allen, Harvard.Medical School, Boston, MA. The membranes were hy-

bridized overnight and washed with increasingly stringentphosphate buffers. The membranes were exposed to X-rayfilm for 1 week.

The autoradiographs produced by exposure of the X-rayfilm to the membrane were quantified by laser densitom-

Ž .etry Molecular Dynamics, Sunnyvale, CA . Using thestandard curve described above, the relationship betweenthe optical density of the dots and the relative amount ofNPY mRNA blotted was determined. This relationship wasused to calculate the relative amount of NPY mRNA in thesamples. Membranes were stripped of radioactivity and

32 Žrehybridized with a P-labeled actin probe Oncor,.Gaithersburg, MD . Autoradiographs were made and quan-

tified by densitometry. The relationship between the opti-cal density of the dots and the relative amount of actinmRNA blotted was determined from the standard curve.This relationship was used to calculate the relative amountof actin mRNA in the samples. The amount of NPYmRNA was based on the amount of the actin mRNAblotted, with the final value being the average of the 3serial dilutions.

The formalin-fixed medulla was frozen in a cryostat andsectioned through the region of the obex to evaluate theextent of the lesions centered on the AP. Serial sectionsŽ .30 mm were cut and mounted on gel-coated slides. Thesections were dried, stained with cresyl violet and cover-slipped. The sections were then evaluated microscopicallyfor any remaining AP tissue and for the amount of damageto the adjacent NTS. The damage to the adjacent NTS wasestimated by determining the amount of NTS remainingbetween the solitary tract and the edge of the lesioncomparing this with the amount of NTS present in SHAMrats.

Statistical analysis was performed using analysis ofvariance to compare behavioral data. Where significantdifferences were noted post-hoc Newman-Keuls tests wereperformed to determine significant differences at specifictime points. Data from the mRNA and peptide studieswhere comparisons were made between the 2 groups wereanalyzed using t-tests. Differences were determined to besignificant when a P-0.05 was observed.

3. Results

Histological analysis revealed that all animals withlesions centered on the AP included in the statisticalanalysis had the entire AP removed with minimal damage

Ž .to the adjacent NTS Fig. 1 . Measurement of the remain-ing NTS revealed that the lesion impinged no more than

ŽFig. 2. Three-hour cookie intake by rats with APrmNTS-lesions v,. Ž .ns7 and SHAM rats `, ns8 . Note that rats with an APrmNTS-le-

sion consume a significantly greater quantity of the cookie diet thanŽ) .SHAM rats P-0.0001 and that this response is evident at 30 min.

50 mm from the border of the AP. One animal initiallyincluded in the APrmNTS-lesioned group did not have theentire AP removed. In fact, greater than 50% of the APwas intact in this animal and no damage was noted in theadjacent NTS. Based on the histological evidence thisanimal was removed from the statistical analysis althoughthe behavioral and neurochemical data were similar to ratsin the SHAM group. Additionally, recovery of tissue sam-ples from 2 APrmNTS-lesioned rats in group 1, 2APrmNTS-lesioned rats in group 2, and 2 SHAM rats ingroup 2 were insufficient to detect actin mRNA. Thus,data from these animals were not included in the statisticalanalysis of mRNA data.

All animals with lesions centered on the AP had asignificant weight loss during the first 2–3 weeks aftersurgery. Subsequent to this period of weight loss, theanimals gained weight at a rate that was not different fromcontrol animals although the body weight of theAPrmNTS-lesioned group was significantly lower than

Žthe SHAM group APrmNTS-lesioned 297"10 g vs..SHAM 454"8 g, P-0.0001 . This observation is in

Fig. 3. Steady-state hypothalamic NPY mRNA levels in the basomedialhypothalamus including the arcuate nucleus of rats with APrmNTS-le-

Ž . Ž .sions ns13 and SHAM rats ns15 based on actin mRNA. Note thatNPY mRNA levels in APrmNTS-lesioned rats are significantly elevatedcompared to NPY mRNA levels in SHAM rats. ) P-0.01.

( )G.L. Edwards et al.rBrain Research 755 1997 84–9088

accord with earlier reports indicating weight loss in ratsw xwith lesions centered on the AP 6,9,11 . It is important to

note that weight loss occurred for approximately 15 days,after which the APrmNTS-lesioned rats gained weight

Ž .and by the time of testing 2–3 months post-lesion weregaining weight at a normal rate over the 10 days prior to

Žthe end the experiment APrmNTS-lesioned 1.6"1.3.grd vs. SHAM 1.8"0.7 grd .

Ž .Intake of a highly palatable diet lemon cookie wassignificantly higher than in SHAM rats in the second

Žgroup of APrmNTS-lesioned rats over a 3 h test Fig. 2,.P-0.001 . This effect was apparent at 30 min and per-

sisted throughout the study period.Steady-state levels of hypothalamic NPY mRNA cor-

rected for actin, as determined by dot blot analysis, weresignificantly higher in rats with lesions centered on the AP

Ž .compared to the SHAM group P-0.01 . Also it isimportant to note that the difference was evident in thefirst group of rats with the basomedial hypothalamusmacrodissected and in the second group where the regionof the ARC was punched using an 18-gauge needle. Theamount of tissue removed by the macrodissection tech-nique appears to be similar to the amount of tissue re-moved by the punch technique. Both techniques removetissue including the ARC in the basomedial hypothalamusfor approximately 2 mm rostrocaudally. Since the datawere similar in both groups, we combined the analysis andpresentation of the data in Fig. 3.

Levels of NPY were elevated in the dorsomedial regionof the hypothalamus including the PVN in the first group

Ž .of rats examined Fig. 4, P-0.001 . In the second group,the tissue was collected using a punch technique and thePVN, LH and anterior hypothalamic region surrounding

Ž .the third ventricle AV3V were dissected. Levels of NPYwere only elevated in samples containing the PVN from

Žrats with APrmNTS-lesions in the second study Fig. 5,.P-0.02 . NPY levels in the LH and AV3V were equiva-

Fig. 4. NPY immunoreactivity in the dorsomedial hypothalamus of ratsŽ . Ž .with APrmNTS-lesions ns9 and rats with SHAM-lesions ns9 .

Samples in this study were collected using a macrodissection techniquethat included the PVN and surrounding tissue. Note that NPY levels aresignificantly elevated in the APrmNTS-lesioned rats compared to theSHAM group. ) P-0.001.

Fig. 5. NPY immunoreactivity in the PVN, LH and the AV3V of ratsŽ . Ž .with APrmNTS-lesions ns6 and SHAM-lesioned rats ns6 . Tissue

was microdissected from thick sections prepared on a cryostat for analy-sis of NPY immunoreactivity in this study. Note that NPY was elevatedin only the PVN of APrmNTS-lesioned rats. Levels of NPY in the LHand AV3V were equivalent in APrmNTS-lesioned and SHAM rats.) P-0.02.

lent in the APrmNTS-lesioned group and the SHAMŽ .group Fig. 5, P)0.1 .

4. Discussion

Data from these studies are the first to show that ratswith lesions centered on the AP have significantly elevatedlevels of NPY in the hypothalamus during the light phaseof the lightrdark cycle. Specifically, NPY mRNA is ele-vated in the basomedial hypothalamus including the ARCand NPY peptide is elevated in the PVN. Interestingly,NPY peptide was not found to be elevated in the AV3Vregion or LH. Thus, it is possible that the elevation ofNPY is specific to the PVN in rats with APrmNTS-le-sions. The role of elevated NPY in the ARC and PVN ofrats with APrmNTS-lesions is unclear. Previous workindicating that NPY may be involved in carbohydrateappetite suggests the possibility that elevated NPY in thehypothalamus of APrmNTS-lesioned rats may be relatedto the elevated appetite for highly palatable, sweet foods

w xreported in earlier studies 7,16 . Alternatively, it has alsobeen established that lesions centered on the AP result in amarked weight loss during the first 2–3 weeks after thelesion, primarily due to a decrease in food intake duringthe 2–3 week period immediately following surgeryw x6,9,11 . Since food deprivation is also reported to increaseNPY levels, it is conceivable that the weight loss con-tributes to the elevation in hypothalamic NPY. It is impor-tant to emphasize that in these studies the rats with lesionscentered on the AP were allowed to recover for at least 90days before hypothalamic NPY levels were determined.Several studies point to the fact that rats with lesions of theAP are consuming a normal or near-normal amount offood at this time and despite efforts to force them to gainweight, the lesioned animals will maintain the lower body

w xweight 6,9,11,16 . Thus, it appears as though the

( )G.L. Edwards et al.rBrain Research 755 1997 84–90 89

APrmNTS-lesioned rats have attained a new steady-statebody weight. Since the APrmNTS-lesioned rats do notappear to be food-deprived once fully recovered fromsurgery, it seems unlikely that food deprivation would leadto the elevated hypothalamic NPY levels observed in thesestudies.

It should also be emphasized that the elevation inhypothalamic NPY was only observed at one time pointduring the early light phase of the lightrdark cycle. Thiscorresponds to the time when rats with lesions of the APare reported to consume increased amounts of highly

w xpalatable diet 7 . In fact, the second group of animalsanalyzed for hypothalamic NPY were first found to have

Ž .an elevated intake of a highly palatable diet lemon cookieduring the early light phase. Thus, in the second group ofrats, elevated intake of a highly palatable diet occurred at atime of the lightrdark cycle when levels of hypothalamicNPY were elevated.

In addition to a proposed role in carbohydrate appetite,recent studies suggest that NPY induces fat selection in

w xrats that prefer high-fat diets 14,25 . These observationssuggest that NPY stimulation of food intake may not bespecific to carbohydrate, but instead may simply enhancean animal’s intake of any preferred food. In the case of theAPrmNTS-lesioned rat, increased activity of hypothala-mic NPY systems may simply increase intake of thepreferred palatable diet without necessarily inducing acarbohydrate appetite. It is interesting to note that althoughintake of a preferred palatable diet is elevated, previousstudies indicate that intake of chow is not elevated in ratswith lesions centered on the AP during the light phase of

w xthe lightrdark cycle 6,9 . Thus, it is possible that theintake stimulated by NPY during the light phase requires adiet more palatable the normal pelleted chow diet.

The mechanism by which a lesion centered on the APcan result in elevation of NPY in the hypothalamus is alsonot clear. There is little evidence that neurons in the APproject directly to the hypothalamus and most investigatorssuggest that information from the AP ascends to thehypothalamus after relaying through other hindbrain nucleisuch as the ventrolateral medulla, nucleus of the solitary

w xtract or parabrachial nucleus 15 . Thus, it is likely that oneof these sites is involved in activation of NPY in thebasomedial hypothalamus. Although NPY-immunoreactiveneurons are reported to exist in projections from hindbrain

w xnuclei to the hypothalamus 17 , there is no indication fromour data that NPY neurons in the hindbrain control NPYneurons in the ARC. Studies by Sahu and co-workerssuggest that hindbrain NPY projections contribute to NPYin the PVN but not the ARC, in that they have observedthat midbrain knife-cuts that sever the projections fromhindbrain nuclei to the hypothalamus decrease NPY in the

w xPVN, but not in the ARC 19,20 . It should also be notedthat a NPY-containing pathway is described traveling fromthe ARC to the PVN and this pathway contributes to NPY

w xlevels in the PVN 2 .

It is interesting that our data indicate that hypothalamicNPY is activated in rats with lesions centered on the AP.As noted above, elevated NPY in the hypothalamus isusually associated with increased appetite and increasedweight gain. Rats with lesions of the AP, including the ratsin this study, lose weight shortly after the lesion andremain at a lower body weight than age-matched controlrats, although they do gain weight at a normal rate oncerecovered. We do not know that the elevation in hypotha-lamic NPY in APrmNTS-lesioned rats persists throughoutthe lightrdark cycle. Daily, 24 h food intake is not ele-

w xvated in rats with lesions centered on the AP 6,7,9,11,16 .Thus, it is possible that NPY levels are elevated during thedaylight hours and then return to levels similar to intactcontrol animals during the dark phase. This might accountfor the increased intake of highly palatable diets during thelight phase, while daily caloric intake remains unaltered.

Finally, it is important to recall that NPY is reported tobe involved in numerous functions. In addition to stimulat-ing food intake, NPY is reported to be important inreproduction, regulation of the hypothalamo-pituitary-

Žadrenal axis and autonomic function to list a few see Ref.w x .13 for review . Thus, it is conceivable that the elevationin hypothalamic NPY that we observe in rats with lesionscentered on the AP may function in a system other thanfood intake. Future studies will determine the role thatelevated NPY in rats with lesions centered on the AP playsin body energy balance.

In summary, we found that lesions centered on the APresult in increased NPY mRNA in the basomedial hypo-thalamus and elevated NPY-immunoreactivity in the PVN.Based on earlier reports suggesting that NPY is an impor-tant orexigenic agent, it is possible that this increase inactivity of hypothalamic NPY systems contributes to theincreased consumption of highly palatable or novel foodsby rats with lesions centered on the AP.

Acknowledgements

We wish to thank Ms. Joyce D. Power for assistance inconducting behavioral studies. These studies were sup-ported by a University of Georgia Biotechnology Award.

References

w x1 Beck, B., Stricker-Krongrad, A., Burlet, A., Max, J.-P., Musse, N.,Nicolas, J.-P. and Burlet, C., Macronutrient type independently ofenergy intake modulates hypothalamic neuropeptide Y in Long-Evans

Ž .rats, Brain Res. Bull., 34 1994 85–91.w x2 Bai, F.L., Yamano, M., Shiotani, Y., Emson, P.C., Smith, A.D.,

Powell, J.F. and Tahyama, M. An arcuato-paraventricular and dorso-medial hypothalamic neuropeptide Y containing system which lacks

Ž .noradrenalin in the rat, Brain Res., 331 1985 172–178.w x3 Billington, C.J. and Levine, A.S., Hypothalamic neuropeptide Y

regulation of feeding and energy metabolism, Curr. Opin. Neuro-Ž .biol., 2 1992 847–851.

( )G.L. Edwards et al.rBrain Research 755 1997 84–9090

w x4 Billington, C.J., Briggs, J.E., Harker, S., Grace, M. and Levine,A.S., Neuropeptide Y in hypothalamic paraventricular nucleus: a

Ž .center coordinating energy metabolism, Am. J. Physiol., 266 1994R1765–R1770.

w x5 Clark, J.T., Kalra, P.S. and Kalra, S.P., Neuropeptide Y stimulatesfeeding but inhibits sexual behavior in rats, Endocrinology, 117Ž .1985 2435–2442.

w x6 Contreras, R.J., Kosten, T. and Bird, E., Area postrema: part of theŽ .autonomic circuitry of caloric homeostasis, Fed. Proc., 32 1984

923–927.w x7 Edwards, G.L. and Ritter, R.C., Ablation of the area postrema

causes exaggerated consumption of preferred foods in the rat, BrainŽ .Res., 216 1981 265–276.

w x8 Edwards, G.L., White, B.D., Zhao, W., He, B., Dean, R.G. andŽ .Martin, R.J., Lesions of the area postrema AP radjacent nucleus of

Ž .the solitary tract NTS result in enhanced hypothalamic neuropep-Ž . Ž . Ž .tide Y NPY levels Abstract , Ann. NY Acad. Sci., 739 1994

337–338.w x9 Hyde, T.M. and Miselis, R.R., Effects of area postremarcaudal

medial nucleus of the solitary tract lesions on food intake and bodyŽ .weight, Am. J. Physiol., 244 1983 R577–R587.

w x10 Kalra, S.P., Clark, J.T., Sahu, A., Dube, M.G. and Kalra, P.S.,Control of feeding and sexual behaviors by neuropeptide Y: physio-

Ž .logical implications, Synapse, 2 1988 254–257.w x11 Kenney, N.J., Kott, J.N., Tomoyasu, N., Bhatia, A.J., Ruiz, S. and

McDowell, M.M., Body weight of rats following area postremaŽ .ablation: effect of early force-feeding, Am. J. Physiol., 256 1989

R939–R945.w x12 Kenney, N.J., Tomoyasu, N. and Burkhart, M.K., Food aversion

Ž .induced by area postrema ablation, Appetite, 22 1994 205–220.w x13 Lehmann, J. Neuropeptide Y: An overview, Drug DeÕ. Res., 19

Ž .1990 329–351.w x14 Lynch, W.C., Grace, M., Billington, C.J. and Levine, A.S., Effects

of neuropeptide Y on ingestion of flavored solutions in nondeprivedŽ .rats, Physiol. BehaÕ., 54 1993 877–880.

w x15 Miselis, R.R., Shapiro, R.E. and Hyde, T.M., The area postrema. InŽ .P.M. Gross Ed. , CircumÕentricular Organs and Body Fluids, CRC

Press, Boca Raton, FL, 1987, pp. 185–207.w x16 Ritter, R.C. and Edwards, G.L., Area postrema lesions cause over-

consumption of palatable foods but not calories, Physiol. BehaÕ., 32Ž .1984 923–927.

w x17 Sawchenko, P.E., Swanson, L.W., Grazanna, R., Howe, P.R.C.,Bloom, S.R. and Polak, J.M., Colocalization of neuropeptide Yimmunoreactivity in brain stem catecholaminergic neurons that pro-ject to the paraventricular nucleus of the hypothalamus, J. Comp.

Ž .Neurol., 241 1985 138–153.w x18 Sahu, A., Kalra, P.S. and Kalra, S.P., Food deprivation and ingestion

induce reciprocal changes in neuropeptide Y concentrations in theŽ .paraventricular nucleus, Peptides, 9 1988 83–86.

w x19 Sahu, A., Kalra, S.P., Crowley, W.R. and Kalra, P.S., Evidence thatNPY-containing neurons in the brainstem project into selected hy-pothalamic nuclei: implication in feeding behavior, Brain Res., 457Ž .1988 376–378.

w x20 Sahu, A., Dube, M.G., Kalra, S.P. and Kalra, P.S., Bilateral neuraltransections at the level of mesencephalon increase food intake andreduce latency to onset of feeding in response to neuropeptide Y,

Ž .Peptides, 9 1989 1269–1273.w x21 Stanley, B.G., Anderson, K.C., Grayson, M.H. and Leibowitz, S.F.,

Repeated hypothalamic stimulation with neuropeptide Y increasesdaily carbohydrate and fat intake and body weight gain in female

Ž .rats, Physiol. BehaÕ., 46 1989 173–177.w x22 Stanley, B.G., Daniel, D.R., Chin, A.S. and Leibowitz, S.F., Par-

aventricular nucleus injections of peptide YY and neuropeptide YŽ .preferentially enhance carbohydrate ingestion, Peptides, 6 1985

1205–1211.w x23 Stanley, B.G., Magdalin, W., Seirafi, A., Nguyen, M.M. and Lei-

bowitz, S.F. Evidence for neuropeptide Y mediation of eatingproduced by food deprivation and for a variant of the Y1 receptor

Ž .mediating this peptide’s effect, Peptides, 13 1992 581–587.w x24 Tomoyasu, N. and Kenney, N.J. Response to palatability after area

postrema lesions: a result of learned aversion, Am. J. Physiol., 257Ž .1989 R1075–R1080.

w x25 Welch, C.C., Grace, M.K., Billington, C.J. and Levine, A.S., NPYŽ .induces fat selectin in fat-preferring rats Abstract , FASEB J., 7

Ž .1993 A88.w x26 Welch, C.C., Grace, M.K., Billington, C.J. and Levine, A.S., Prefer-

ence and diet type affect macronutrient selection after morphine,Ž .NPY, norepinephrine, and deprivation, Am. J. Physiol., 266 1994

R426–R433.w x27 White, J.D., Neuropeptide Y: a central regulator of energy home-

Ž .ostasis, Regul. Peptides, 49 1993 93–107.