Norepinephrine and amino acids in prepyriform cortex of rats fed imbalanced amino acid diets

Physiology & Behavior, Vol. 36, pp. 1071-1080.Copyright©PergamonPress Ltd., 1986. Printed in the U.S.A.

0031-9384/86$3.00 + .00

Norepinephrine and Amino Acids in Prepyriform Cortex of Rats Fed Imbalanced Amino Acid Diets I D O R O T H Y W . G I E T Z E N , P H I L I P M. B. L E U N G A N D Q U I N T O N R. R O G E R S 2

Department o f Physiological Sciences, School o f Veterinary Medicine and Food Intake Laboratory, University o f California, Davis, CA 95616 R e c e i v e d 21 A u g u s t 1985 GIETZEN, D. W., P. M. B. LEUNG AND Q. R. ROGERS. Norepinephrine and amino acids in prepyriform cortex of ratsfed imbalanced amino acid diets. PHYSIOL BEHAV 36(6) 1071-1080, 1986.--Monoamines and amino acids were measured in anterior prepyriform cortex (PPC) and anterior cingulate cortex (CC) of male Sprague-Dawley rats after they were offered basal, imbalanced (IMB) or corrected amino acid diets, limited in threonine (THR) or isoleucine (ILE). In the THR study, brains were taken after 2.5 hr of feeding, when intake of THR-IMB was just depressed. In the ILE study the brains were taken after 3.5 hr on ILE-IMB, a less severely imbalanced ration, before the onset of food intake depression. The PPC has been shown to be involved in the acute response of animals to imbalanced amino acid diets. In the PPC from the IMB diet groups, NE was reduced by 30%, but the other monoamines were unchanged. In CC, an area involved in the adaptive, but not the acute feeding response to imbalanced diets, the monoamines were unchanged in the IMB diet groups. In both studies, in both tissues, the limiting amino acids were decreased in the 1MB groups, although the decrease of ILE in the CC failed to reach significance. The remaining indispensable amino acids, added to create the imbalance, were slightly reduced in the THR-IMB group, but not in the ILE-IMB group in both tissues. Thus, the amino acid patterns were altered in the PPC and CC, as they are in whole brains from animals fed imbalanced amino acid diets. These results also suggest that the concentration of NE in the PPC may be associated with the initial food intake response of animals to imbalanced amino acid diets. Amino acid imbalance Cingulate cortex

Rat

Monoamines

Norepinephrine

S E V E R A L lines of evidence point to the importance of neural control in the decreased food intake response of animals given imbalanced amino acid diets. For example, (1) the food intake of rats [13] and cockerels [40] fed amino acid-imbalanced diets was returned to control levels if a small quantity of the most limiting amino acid was infused into the carotid artery. However, infusion of the limiting amino acid into the portal or jugular vein, in 3 times the quantity given in the carotid preparation, did not prevent the food intake depression of rats fed an amino acid imbalanced diet [13]; (2) specific neural lesions have implicated the anterior prepyriform cortex and the anterior cingulate cortex in the control and adaptation, respectively, of food intake in animals fed amino acid imbalanced diets [15,23]. Animals bearing lesions of the prepyriform cortex did not depress their intake of amino acid-imbalanced diets, as did intact animals [15]. Also, in choice studies, rats rejected a diet with an amino acid imbalance and selected a diet with a balanced pattern of amino acids, or, as an "adverse" choice, a

Amino acids

Prepyriform cortex

protein-free diet, which is lethal in the long term [16]. However, animals with lesions of the anterior prepyriform cortex selected the imbalanced diet over the protein-free diet [34], although they were still capable of establishing a learned aversion [22]. Animals bearing lesions of the anterior cingulate cortex did depress their initial food intake of the imbalanced amino acid diet, but adapted more readily to the imbalanced ration [23]; (3) Harper and co-workers [6, 29, 38] found that the concentration of the most limiting amino acid was more severely depressed in the brain than in plasma when rats were fed amino acid-imbalanced diets. The severity of the depression in food intake and growth was associated with the degree of deficiency of the limiting amino acids and the extent of the excess of the amino acid(s) competing for transport into the brain [37,38]. It is established that the catecholamines and serotonin participate in the neural control of feeding [8]. The synthesis and turnover of these neurotransmitters depend in part on the brain content of the amino acid precursors; the plasma

XSupported by NIH grants AM13252, AM07355, and AM07557. 2Requests for reprints should be addressed to Dr. Quinton R. Rogers, Department of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616.

1071

1072

( i l E q Z E N . I~EIjN(I A N I ) R ( ) ( ; E R S TABLE I COMPOSITION OF EXPERIMEN1AL DIETS I":~()F DIET BY WEt(IH'i I Threonine Component

BAS

Dispensable amino acid mixture Indispensable amino acid mixture Imbalanced amino acid mixture L-Threonine L-Isoleucine Vitamin mixture Salt mixture Corn oil Sucrose Starch Choline chloride Total

IMB

lsoleucine COR

BAS

IMB

COR

8.08

8.08

8.08

8.08

8.08

8.08

4.57

4.57

4.57

3.65

3.65

3.65

--

9.86

9.86

--

4.93

4.93

--

--

-. 1.00 5.00 5.00 25.41 50.84 0.10

-1.00 5.00 5.00 22.13 44.26 0.10

0.40 . 1.00 5.00 5.00 21.99 44.00 0.10

100.00

100.00

.

100.00

.

1.00 5.00 5.00 25.72 51.45 0.10

1.00 5.00 5.00 24.08 48.16 0.10

-0, 50 1.00 5.00 5.00 23.92 47.82 I). l0

100.00

100.00

100.00

.

Diets are designated as follows: BAS=basal, IMB=imbalanced, COR=corrected.

TABLE 2 FOOD INTAKE OF RATS OFFERED EITHER A BASAL, IMBALANCED OR CORRECTED DIET Diet

Time Increment (hours)

Cumulative

Threonine Study 0-1

1-2

2-2.5

BAS

1.3 _+0.2

1.6 _+0.2

0.1 _+0.1

3.0 _+0.3

IMB

1.1 _+0.2

0.8* ___0.2

0.3 _+0.1

2.1" _+0.2

fOR

2.1 _+0.6

2.4 _+0.5

0.2 ±0.2

4.7 _+0.8

Isoleucine Study 0-2

2-2.75

2.75-3.5

BAS

3.7 _+0.7

1.7 +0.5

0.9 +0.2

6.3 -+0.8

IMB

3.5 _+0.3

1.5 ___0.5

0.9 -+0.2

5.9 _+0.5

COR

3.2 -+0.6

0.9 +0.4

1.4 +__0.5

5.4 _+0.8

Diets are designated as follows: basal=BAS, imbalanced=IMB, corrected = COR. Values are mean _ standard error for food intake in grams. *=Significantly less than other groups, p<0.05.

amino acid ratios are thought to be the determinants of precursor availability to the brain [26]. It has b e e n p r o p o s e d that shifts in the plasma tryptophan to neutral amino acid ratio, acting via the serotonergic system, may alter the animal's appetite for protein [l] or carbohydrate [43], or the proportions of protein to carbohydrate [2,19], as well as total food intake [2]. On the other hand, tyrosine, and thus the catecholamine system, has been postulated to influence regulation of energy balance and total food intake [l]. H o w e v e r , that plasma amino acid ratios affect self selection or control of protein intake has not been clearly established [4, 30, 31]. N o r is it known how the control of brain m o n o a m i n e synthesis by diet and the resulting plasma amino acid levels may relate to the control of food intake with the ingestion of disproportionate amounts of dietary amino acids. It is, h o w e v e r , clear that the brain is responsive to amino acids. F o r example, microinjections of indispensable amino acids into the zona incerta and lateral hypothalamus inc r e a s e d the discharge frequency of firing of specific neurons [42]. Also, the acidic amino acids and glycine are known to act as neurotransmitters in the central nervous s y s t e m [21], and may well be involved in feeding behavior, although their relationships to the control of protein and amino acid intake have not yet been studied. In particular, the acidic amino acids, glutamic acid and aspartic acid, have been reported to be the major neurotransmitters in the prepyriform cortex, since they were reduced after lesions o f neuronal tracts leading from the olfactory bulb to the prepyriform cortex [7]. In addition, gamma-aminobutyric acid ( G A B A ) and several peptides are currently recognized as being i n v o l v e d in the control of food intake [9, 24, 39]. The purpose of the present study was to measure the m o n o a m i n e and amino acid concentrations in 2 specific brain areas: the prepyriform cortex, already shown to be involved in the initial response of rats to imbalanced a m i n o acid diets [15,25], and the anterior cingulate cortex, a brain a r e a implicated in the adaptive, but not the initial response to these

PREPYRIFORM AMINES AND AMINO ACIDS

1073

CINGULATE CORTEX

i!!iiii!ilTHR BASAL I T H R IMBALANCED 1 THR C O R ~ D 5O0 bJ O~ 4 0 0 F'I-- 3 0 0 W ~, 2 0 0

IOO

NE

FIG. 1. The brain areas, prepyriform cortex and cingulate cortex, that were used for analysis. Adapted from the stereotaxic atlas of Pellegrino and Cushman [27]. Sections were cut I mm thick so that the slices corresponded to 9.6+_1.0 mm anterior to the interaural line.

diets [23]. In the first experiment, the measurements were made on these 2 anterior cortical areas from animals that had just reduced their food intake in response to a threonineimbalanced diet, and in a second experiment, in the same areas from a group of animals that had not yet reduced their intake of a less severely imbalanced diet that was limited in isoleucine. METHOD

Animals Male Sprague-Dawley rats (Bantin and Kingman, Lafayette, CA) weighed 180_+4 g and 187_+2 g at the start of experiments l and 2, respectively. They were housed in hanging wire cages at 22+2°C under a 12:12 lighting schedule, with the lights off between noon (12:00) and midnight (24:00). In addition to the room lighting, two 25 watt red bulbs were kept on at all times to facilitate food intake measurements during the dark phase. The animals were allowed to adapt to the quarters, lighting schedule and a low-protein basal diet for 2 weeks prior to the experiment. During this period, food intake and body weight were measured daily and spillage was carefully recovered. For the 4 days just prior to the experiment, the food cups were removed at the beginning of the light phase to synchronize the first meal of the dark phase. The preweighed food cups were returned at noon. Thus, the animals were food deprived for 12 hours on the 3 days prior to the experiment, as well as on the day of the experiment. Hourly food intake measurements were taken to adapt the animals to the measurement procedure during the dark phase and to obtain baseline food intake data for assignment to experimental groups of 6-7 animals. Group assignments were made such that the 3 groups each had similar feeding patterns prior to the experiment.

Dietary Treatments Purified amino acid diets were used throughout these studies. The diets were the same as those reported previously [13,17]; the composition of the diets is shown in Table 1. There were 2 separate studies, differing in the growth

DA

5HT

FIG. 2. Monoamine concentrations in the prepyriform cortex from animals offered threonine-basal, -imbalanced or -corrected diets. Brains were taken after 2.5 hours of exposure to the diets, when the food intake was determined to be significantly depressed in the imbalanced-diet group. NE=norepinephrine, DA=dopamine, 5HT= serotonin. Bars represent means of 7 animals per group, vertical lines represent the standard error of the mean for the diet treatments indicated in the legend. A star signifies statistical significance at p<0.01.

limiting amino acid upon which the diets were based: in the first study threonine was the most limiting amino acid, and in the second, isoleucine. Briefly, the amino acid-imbalanced diets were prepared by adding a mixture of all the indispensable amino acids except the one to be limiting, to the basal diet. The corrected diets consisted of the appropriate imbalanced diet with the addition of the limiting amino acid (0.4% threonine and 0.5% isoleucine, in experiments 1 and 2, respectively). Thus, the basal and imbalanced diets had the same concentration of the limiting amino acid, while the imbalanced and corrected diets had the same concentrations of all the other indispensable amino acids. All diets contained the necessary vitamins and minerals with starch and sucrose (2:1) as the carbohydrate and 5% corn oil as the fat source.

Threonine-lmbalanced Study The first experiment was designed to determine monoamine and amino acid concentrations in the anterior prepyriform cortex and anterior cingulate cortex of rats fed diets with threonine as the limiting amino acid and killed shortly after the initial depression of food intake by the imbalanced diet group. On the day of the experiment, after 12 hours of food deprivation, one group was given the threonine basal diet, one group the imbalanced diet, and the third group the corrected diet. Food intake was again measured hourly. The animals offered the imbalanced diet had significantly reduced their food intake 2 hours after presentation of the diets (i.e., at 14:00 hours, Table 2). They were decapitated at 14:30. The brains were removed within 45 seconds, frozen in liquid nitrogen and stored at -80°C until analyzed for the concentrations of monoamines and amino acids.

Isoleucine-lmbalanced Study In the second experiment, we used a less severly imbal-

GIETZEN. [,EUNG ANI) i~,()GERS

1074 !III~IHIILI LE BASAL I ILE IMBALANCED ILE CORRECTED

[fh

THR

BASAL IMBALANCED

I

THR

CORRECTED

i~::i~ T H R

500

500

b,I =) ¢/3 4 0 0

b.l u) 0q

400

800 I

7--

I-I-bJ

:500

eool

Go

I-- 300 bJ

c

tOO

o~ 9 0 0 .%

I00

¢:

NE

DA

anced diet, with isoleucine as the limiting amino acid (see Table I). Again, the animals were divided into 3 groups with similar feeding patterns, and offered either an isoleucinebasal, -imbalanced or -corrected diet. The food intake was similar in all 3 diet-treatment groups at the time they were killed, 3.5 hours after diet presentation, although spillage was increased in the imbalanced group. Thus, in the second experiment, the brains were taken before the onset of the food intake depression. Otherwise, the animal treatment, tissue preparation and analysis were essentially the same in both experiments. Determination o f Monoamines and Amino Acids The concentrations of monoamines were measured in the 2 anterior cortical brain areas by high performance liquid chromatography (HPLC) with electrochemical detection according to the method of Wagner et al. [41]. Slices of frozen brain, approximately 1 mm thick, were placed on slides over dry ice. The specific brain sections were dissected from the slices with a scalpel according to the histology of previous lesion studies [15,23]. The coordinates for the slices corresponded to 9.6--9.8___1.0 mm rostral to the interaural line according to the atlas of Pellegrino and Cushman [27], see Fig. 1. Sections were placed into chilled preweighed 1.5 ml microcentrifuge tubes containing 0.6 ml perchloric acid diluent (0.2 M perchloric acid, with 0.5 g disodium EDTA and 100 mg sodium bisulfite per liter). The tubes were then reweighed (Mettler Microbalance, Highstown, N J) for determination of tissue weight. Prepyriform samples weighed 20.2_+1.1 mg; cingulate samples weighed 20.6--+2.5 mg. The protein contents of the tissues, determined by the method of Bradford [3] were: 0.11-+-0.01 mg protein per mg wet tissue weight, and were the same for both prepyriform and cingulate cortices. The samples, kept on ice, were sonicated (Heat Systems sonifier, Plainview, NY) for 10 seconds and centrifuged at 15,000 × g for 10 min in an Eppendorf microcentrifuge housed in a cold room. The supernatants were individually filtered through microfilters, 0.45 /xm pore size (Fischer, Santa Clara, CA); 25/zl of the filtrate was injected

200

I00

5HT

FIG. 3. Monoamine concentrations in the prepyriform cortex from animals offered isoleucine-basal, -imbalanced, or -corrected diets. Brains were taken after 3.5 hours of exposure to the diets; food intake was similar in all 3 groups. The remaining conditions are as described in Fig. 2 for 6 animals per group.

400

NE

DA

~'

]i 5HT

FIG. 4. Monoamine concentrations in the anterior cingulate cortex from animals offered threonine-basal, -imbalanced, or -corrected diets. Conditions are as described in Fig. 2.

illi[klll I LE BASAL I ILE IMBALANCED LE CORRECTED

60C sO0 LLJ C~ 4 0 0

800

I.- 3OC L~

600

20C

40C

IOC

20¢

¢J)

=

0

NE

DA

5HT

FIG. 5. Monoamine concentrations in the anterior cingulate cortex from animals offered isoleucine-basal, -imbalanced, or -corrected diets. Conditions are the same as for Fig. 3.

into the column (C18, reverse phase, dimensions: 100x4.6 mm, Perkin Elmer, Torrence, CA). The mobile phase contained 100 mM formic acid, 0.36 mM octane sulfonic acid, 1.0 mM citric acid, 0.10 mM EDTA, 5.0% (v/v) acetonitrile and 0.25% (v/v) diethylamine at pH 3.1. Flow rate was 1 ml/min. A glassy carbon electrode was used for electrochemical detection at +800 mV potential, with a 2.0 second RC filter (Bioanalytical Systems, West Lafayette, IN). External standards, made up in perchloric acid diluent, contained norepinephrine, epinephrine, dopamine, 5-hydroxyindoleacetic acid, 5-hydroxytryptamine, homovanillic acid and tryptophan (all from Sigma, St. Louis, MO). The external standard was used to identify peaks eluting in the chromatogram according to retention time and conformation. The internal standard was 3,4 dihydroxybenzylamine (Sigma), at a concentration of 1 ng/25 #1, and was included in both the external standard and the homogenizing medium. Recoveries were calculated from the internal standard in each sample.

PREPYRIFORM AMINES AND AMINO ACIDS

1075

TABLE 3 AMINO ACIDS IN THE PREPYRIFORM CORTEX OF ANIMALS OFFERED PURIFIED AMINO ACID DIETS WITH THREONINE AS THE GROWTH-LIMITING AMINO ACID (nmol/mg WET TISSUE WEIGHT) Amino Acid Taurine Aspartic acid Threonine Serine Glutamic acid Glutamine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine GABA IAAs* IAA-threonine~

Basal 9.89 3.84 0.14 2.33 14.30 3.54 1.11 0.83 0.13 0.07 0.04 0.07 0.05 0.06 0.09 0.08 0.26 0.09 3.41 0.92 0.78

-+ 0.92~: _+ 0.41 _+ 0.02~: +- 0.215 _+ 1.36 _+ 0.52 ±_ 0.125 - 0.075 _+ 0.02 _+ 0.01 +-- 0.01 - 0.01 ± 0.01 -+ 0.01 ± 0.01 ± 0.02:~ ± 0.02 _-. 0.01 ± 0.21:~ ± 0.04:~ ± 0.04:~§

Imbalanced 6.75 2.89 0.08 1.64 11.60 2.58 0.75 0.51 0.12 0.08 0.05 0.07 0.08 0.04 0.15 0.03 0.23 0.06 2.45 0.87 0.79

-+ 0.35§ +_ 0.31 +__0.02§ +_ 0.07§ __. 1.16 ±_ 0.27 +_ 0.05§ +_ 0.03§ _ 0.04 _+ 0.01 _ 0.01 +_ 0.00 +_ 0.01 _+ 0.00 --- 0.03 ± 0.00§ _+ 0.01 ___0.00 +_ 0.14§ +_ 0.06~: +_ 0.05:~

Corrected 8.23 3.38 0.25 1.78 12.50 3.05 0.95 0.66 0.12 0.10 0.07 0.10 0.08 0.06 0.17 0.07 0.28 0.08 2.93 1.21 0.96

± 0.62:~§ _+ 0.27 +_ 0.05¶ _+ 0.16§ +__0.97 ___0.42 --- 0.06:~ _+ 0.02§ +_ 0.01 _+ 0.01 _+ 0.01 +_ 0.01 +_ 0.01 _+ 0.01 _+ 0.03 ± 0.025§ ± 0.03 ± 0.01 ± 0.165§ ± 0.07§ +_ 0.05§

*IAAs: Sum of the indispensable amino acids: thr, val, met, ile, leu, phe, trp, lys, his. tlAA-threonine: Sum of the indispensable amino acids not including threonine. Values are mean _ standard error for 7 animals per group. $§¶Differing superscripts indicate significant differences between dietary treatment groups for the indicated amino acid.

The c o n c e n t r a t i o n s o f amino acids were determined in aliquots of the same prepyriform cortex and anterior cingulate cortex samples by c h r o m a t o g r a p h y on an automated amino acid analyzer ( B e c k m a n 121-MB, B e c k m a n Instruments, Palo Alto, CA) as used routinely in this laboratory. C h e m i c a l s for H P L C w e r e H P L C grade, others w e r e reagent grade or the best grade available. The results are expressed per unit wet tissue weight. Statistical significance was determined by analysis o f variance ( A N O V A ) with post hoc evaluation of differences b e t w e e n group m e a n s by the Least Significant Differences test, and correlations were determined by Pearsons Correlation. Statistical significance was set at p < 0 . 0 5 . RESULTS The concentration of norepinephrine was 30% lower in the prepyriform c o r t e x from animals offered the imbalanced diets than from animals given either c o r r e c t e d or basal diets. This o b s e r v a t i o n was consistent in both e x p e r i m e n t s , using 2 different diets, one limited in threonine, F(2,18)=6.69, p < 0 . 0 1 , Fig. 2, and the o t h e r in isoleucine, F(2,14)---11.04, p < 0 . 0 0 5 , Fig. 3. At the s a m e time, there w e r e no changes in d o p a m i n e or serotonin in the prepyriform cortex, nor in the c o n c e n t r a t i o n s o f the m e a s u r e d m o n o a m i n e s in the anterior cingulate c o r t e x e x c e p t for serotonin in the t h r e o n i n e study, which was l o w e r in the c o r r e c t e d group only (Figs. 4 and 5).

The concentrations o f amino acids in the prepyriform cortex from animals offered either basal, imbalanced or corrected diets are listed in Table 3 (threonine-imbalanced study) and Table 4 (isoleucine-imbalanced study). A m i n o acid concentrations in the anterior cingulate c o r t e x are found in Table 5 (threonine-imbalanced study) and Table 6 (isoleucine-imbalanced study). The most limiting amino acids, threonine and isoleucine in e x p e r i m e n t s 1 and 2, respectively, were decreased in both tissues from the imbalanced group, by c o m p a r i s o n with the basal and c o r r e c t e d groups alike (Tables 3-6), although this decrease failed to reach significance in the cingulate c o r t e x in e x p e r i m e n t 2. Specifically, in the threonine-imbalanced study, the concentration of threonine in the prepyriform cortex from the imbalanced group was 57% of that in the basal group; in the anterior cingulate c o r t e x from the imbalanced group, threonine was 50% of that in the basal group. In the isoleucine-imbalanced study, isoleucine in the prepyriform cortex of the imbalanced group was 20% of that in the basal group, but in the cingulate cortex, the concentration o f isoleucine in the imbalanced group was only reduced to 62% o f that in the basal group. In the threonine-imbalanced study, the sums o f all the indispensable amino acids (IAA) in the prepyriform cortex as well as the anterior cingulate c o r t e x differed b e t w e e n the experimental groups. In the prepyriform cortex, the I A A w e r e higher in the c o r r e c t e d group than either of the other 2

1(176

(IIE'I'ZEN. I~EUNG ANI) RI;GERS 'FABLE 4 A M I N O ACIDS IN T H E PREPYRIFORM CORTEX OF A N I M A L S O F F E R E D PURIFIED AMINO ACID DIETS WITH I S O L E U C I N E AS T H E G R O W T H - L I M I T I N G AMINO ACII) (nmol/mg WET TISSUE WEIGHTI

Amino Acid Taurine Aspartic acid Threonine Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Valine Methionine Isolencine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine GABA IAAs* IAAs-isoleucinet

Basal 12.61 2.18 0.17 1.45 0.28 12.98 5.41 0.16 1.06 0.71 0.16 0.06 0.05 0.10 0.01 0.01 0.03 0.18 0.22 0.10 1.58 0.9(I 1/.85

± 0.58 + 0.12 ± 0.025 ± 0.08 ± 0.12 ± 0.99 ± 0.47 ± 0.02 m 0.09 ± 0.06 ± 0.02 ± 0.02 ± 0.012 + 0.01 ± 0.01 ± 0.01 ± 0.01 ± 0.05 ± 0.02 ± 0.01 ± 0.15 ± 0.(17 ± 0.07

lmbalanced 12.50 2.12 0.45 1.32 0.14 13.14 5.56 0.14 0.97 0.64 0.15 0.06 0.01 0.10 0.01 0.01 0.02 0.15 0.26 0.13 1.30 1.20 1.19

_+ 0.43 + 0.16 ± 0.03§ ± 0.06 ± 0.01 ± 1.26 ± 0.32 ± 0.02 _+ 0.07 ± 0.04 ± 0.01 ± 0.01 ± 0.01§ ± 0.01 ± 0.01 ± 0.00 ± 0.01 ± 0.04 ± 0.03 _+ 0.01 ± 0.15 ± 0.115 ± 0.(14

Corrected 13.34 ± 0.94 2.62 ± 0.22 (I.32 ± 0.05§ 1.36 ± 0.12 0.29 ± 0.13 13.18 ± 1.24 6.//7 ± 0.63 1t.14 ±_ 0.41 1.(17 _+ 0.10 (I.68 + 0.05 0.14 + 0.02 (I.05 ± 0.0l 0.06 ± 0.012 0.13 _+ 0.03 0.01 ± 0.01 0.(11 ± 0.01 0.03 ± 0.01 0.14 ± (t.03 0.26 + 0.05 0.11 ± 0.01 1.31 ± 0.18 1.12 +- (1.18 1.06 ± 0.17

*IAAs: Sum of the indispensable amino acids: thr. val. met, ile. leu, phe. trp, lys, his. t IAA-isoleucine: Sum of the indispensable amino acids not including isoleucine. Values are mean -+ standard error for 4-6 animals per group. $§Differing superscripts indicate significant differences between dietary treatment groups for the indicated amino acid.

g r o u p s . In the cingulate c o r t e x , the I A A w e r e h i g h e r in the c o r r e c t e d g r o u p t h a n in the i m b a l a n c e d group. H o w e v e r , the s u m s o f all t h e I A A m i n u s the limiting a m i n o acid, t h r e o n i n e ( I A A - T H R ) , w e r e r e d u c e d in b o t h t i s s u e s f r o m the imbala n c e d g r o u p (82 a n d 49% o f c o n c e n t r a t i o n s in c o r r e c t e d g r o u p in p r e p y r i f o r m a n d cingulate cortices, r e s p e c t i v e l y ) . T h e I A A did n o t differ in e i t h e r tissue in the i s o l e u c i n e study, n o r w e r e t h e r e a n y d i f f e r e n c e s b e t w e e n the g r o u p s w h e n I A A m i n u s i s o l e u c i n e ( I A A - I L E ) was c o n s i d e r e d . W i t h t h e m o r e s e v e r e t h r e o n i n e - i m b a l a n c e d diet, t h e r e w e r e several a l t e r a t i o n s in t h e p a t t e r n o f i n d i v i d u a l a m i n o acids in the p r e p y r i f o r m cortex: the c o n c e n t r a t i o n s o f t a u r i n e , serine, glycine, a l a n i n e , t y r o s i n e , o r n i t h i n e a n d G A B A w e r e all l o w e r in t h e i m b a l a n c e d g r o u p t h a n in the b a s a l g r o u p . In addition, glycine w a s l o w e r in t h e i m b a l a n c e d g r o u p t h a n in t h e c o r r e c t e d g r o u p . H o w e v e r , serine a n d a l a n i n e w e r e also l o w e r in t h e c o r r e c t e d g r o u p t h a n in t h e b a s a l g r o u p , a n d so did n o t differ b e t w e e n b a s a l a n d corr e c t e d g r o u p s (Table 3). B y c o n t r a s t , in t h e i s o l e u c i n e - i m b a l a n c e d s t u d y in w h i c h the i m b a l a n c e w a s less s e v e r e , t h e r e w e r e f e w e r c h a n g e s in t h e p a t t e r n o f a m i n o acids in t h e p r e p y r i f o r m c o r t e x . O t h e r t h a n t h e d e c r e a s e in isoleucine in the i m b a l a n c e d g r o u p a n d t h e i n c r e a s e in this a m i n o acid in t h e c o r r e c t e d g r o u p , t h e

only d i f f e r e n c e in a m i n o acid c o n c e n t r a t i o n s a m o n g the 3 g r o u p s was in t h r e o n i n e , w h i c h was h i g h e r in b o t h imbala n c e d a n d c o r r e c t e d g r o u p s t h a n in the b a s a l g r o u p ( T a b l e 4). In the a n t e r i o r cingulate c o r t e x in the t h r e o n i n e study, isoleucine a n d lysine w e r e i n c r e a s e d in the c o r r e c t e d group, b y c o m p a r i s o n w i t h the i m b a l a n c e d or b a s a l g r o u p , but only t h r e o n i n e differed in all 3 g r o u p s (Table 5). In the isoleucine study, only the c h a n g e s in the c o n c e n t r a t i o n o f isoleucine were similar to t h o s e n o t e d in t h e p r e p y r i f o r m c o r t e x . T h e r e w e r e several significant p o s i t i v e c o r r e l a t i o n s bet w e e n c o n c e n t r a t i o n s o f a m i n o acids in b o t h o f t h e s e tissues u n d e r the p r e s e n t d i e t a r y c o n d i t i o n s . In the p r e p y r i f o r m cortex, the c o n c e n t r a t i o n s o f the n e u r o a c t i v e a m i n o acids. glycine, g l u t a m i c acid a n d a s p a r t i c acid w e r e significantly c o r r e l a t e d in b o t h studies. T h e i n h i b i t o r y a m i n o acid n e u r o t r a n s m i t t e r , GABA, w a s n o t c o r r e l a t e d w i t h t h e o t h e r n e u r o a c t i v e a m i n o acids in the p r e p y r i f o r m c o r t e x in e i t h e r study. H o w e v e r , t h e r e were m a n y m o r e significant c o r r e l a t i o n s b e t w e e n a m i n o acid c o n c e n t r a t i o n s in t h e a n t e r i o r cingulate c o r t e x t h a n in t h e p r e p y r i f o r m c o r t e x in b o t h studies. T h e c o r r e l a t i o n s t h a t w e r e similar in this tissue for b o t h s t u d i e s i n c l u d e d t h o s e n o t e d a b o v e for t h e p r e p y r i f o r m cortex: i.e., b e t w e e n g l u t a m i c acid, a s p a r t i c acid a n d glycine, a l t h o u g h in a d d i t i o n , in this tissue, G A B A was also corre-

P R E P Y R I F O R M A M I N E S A N D A M I N O ACIDS

1077

TABLE 5 AMINO ACIDSIN THE ANTERIORCINGULATECORTEXOF ANIMALSOFFERED PURIFIED AMINO ACID DIETS WITH THREONINE AS THE GROWTH-LIMITING AMINO ACID (nmol/mgWET TISSUE WEIGHT) Amino Acid Taurine Aspartic acid Threonine Serine Glutamic acid Glutamine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine GABA IAAs* IAA-threoninet

Basal

Imbalanced

Corrected

5.59 - 0.62 1.98 -+ 0.30 0.12 +- 0.305 1.41 _+ 0.12 10.33 -+ 1.13 2.41 _+ 0.38 0.80 --- 0.12 0.42 _+ 0.08 0.10 _+ 0.03 0.06 -+ 0.01 0.03 +- 0.015§ 0.06 +- 0.01 0.04 _+ 0.01 0.04 _+ 0.01 0.12 _+ 0.03 0.12 +-_0.025 0.05 -+ 0.01 2.00 +_ 0.18 0.70 -+ 0.125§ 0.58 -+ 0.105§

4.42 _+ 0.67 1.47 +_ 0.38 0.06 _+ 0.02§ 0.95 -+ 0.17 7.32 _+ 1.27 1.59 _+ 0.24 0.48 _ 0.08 0.30 _+ 0.05 0.03 +_ 0.01 0.04 +_ 0.01 0.02 _+ 0.00§ 0.04 _ 0.01 0.05 _+ 0.01 0.02 - 0.01 0.05 -4- 0.01 0.13 -+ 0.025§ 0.04 _+ 0.01 1.54 _+ 0.29 0.44 _+ 0.04§ 0.38 _+ 0.065

5.71 _+ 0.49 2.05 - 0.28 0.30 - 0.085 1.44 _+ 0.17 10.61 _+ 1.22 2.32 _+ 0.25 0.82 _+ 0.09 0.48 +_ 0.05 0.07 _+ 0.03 0.08 -+ 0.02 0.06 _+ 0.025 0.08 _+ 0.01 0.06 -+ 0.01 0.05 -+ 0.01 0.12 _+ 0.04 0.21 _+ 0.03§ 0.10 _+ 0.03 2.48 _+ 0.30 1.07 -+ 0.165 0.77 +_ 0.12§

*IAAs: Sum of the indispensable amino acids: thr, val, met, ile, leu, phe, trp, lys, his. tlAA-threonine: Sum of the indispensable amino acids not including threonine. Values are mean - standard error for 7 animals per group. $§Differing superscripts indicate significant differences between dietary treatment groups for the indicated amino acid.

lated with glycine and the acidic amino acid neurotransmitters. Also, tryptophan was positively correlated with tyrosine in the cingulate cortex. DISCUSSION We report here that the norepinephrine concentration was decreased 30% in the prepyriform cortex of rats offered imbalanced amino acid diets. As noted above, the prepyriform cortex has been shown, by lesion studies, to be important in mediating the control of food intake in rats fed imbalanced amino acid diets [15,25]. The anterior cingulate cortex, which may be considered as a comparison tissue in this acute study, has been shown to be involved in the adaptive, but not the initial phase of food intake depression. No changes in monoamine concentrations were found in the anterior cingulate cortex of the imbalanced diet groups. However, in the prepyriform cortex, similar decreases in norepinephrine concentration were noted in both studies in which diets with different limiting amino acids were used. Studies reporting the effects of norepinephrine on feeding have suggested that increased norepinephrine is facilitatory to feeding [10,35]. Thus, a decrease in norepinephrine associated with or preceding a decrease in imbalaneed diet intake could be considered consistent with these suggestions. However, there are several reports that the role of norepinephrine in feeding depends on the specific brain area in-

volved. For example, reduced norepinephrine in the paraventricular and dorsomedial hypothalamic nuclei led to aphagia, while reductions in norepinephrine in the perifornical hypothalamus led to hyperphagia [20]. This laboratory has previously reported that neither ventral tegmental lesions, designed to deplete norepinephrine stores in the hypothalamus and other areas fed by the ventral tegmental tract [17], nor lesions of the ventral medial hypothalamus [14] altered the animal's response to an imbalanced amino acid diet. However, the source(s) of the fibers feeding into the prepyrfform cortex have not been identified [36], and the effects of decreased norepinephrine in the prepyriform cortex are unknown, at least with regard to feeding responses in the rat. A decrease in the concentration of a neurotransmitter can indicate either increased release or decreased synthesis of the transmitter. If the present result were due to decreased synthesis, it could not be because the tyrosine levels were decreased, since there were no significant changes in tyrosine concentrations in either study. There are several reports of monoamine and amino acid concentrations in whole brains of animals after ingestion of diets differing in protein content [1, 30, 32]; whole brain concentrations of monoamine neurotransmitters were unchanged after feeding several combinations of protein and amino acid diets [31-33]. However, even relatively pronounced changes, if they occur in small brain areas, may

1078

( H E q ' Z E N , I r E U N G A N D R()GI~RS TABLE 6 AMINO ACIDS IN T H E A N T E R I O R C I N G U L A T E CORTEX OF A N I M A L S O F F E R E D PURIFIED A M I N O ACID DIETS WITH I S O L E U C I N E AS T H E G R O W T H - L I M I T I N G A M I N O ACID (nmol/mg W E T TISSUE WEIGHT)

Amino Acid Taurine Aspartic acid Threonine Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Histidine GABA IAAs* IAA-isoleucine?

Basal 13.77 + 1.17 3.07 ± 0.80 0.33 ± 0.19 1.48 ± 0.30 0.16 ± 0.01 14.86 ~ 1.83 5.55 + (I.75 0.30 ± 0.12 1.71 ± 0.59 1.06 + 0.42 0.20 ± 0.05 0.26 ± 0.09 0.08 ± 0.03 0.07 ± 0.02 0.02 + 0.01 0.03 ± 0.02 0.02 ± 0.02 0. I1 ± 0.01 0.15 _+ 0.04 0.16 ± 0.05 1.21 ± 0.17 1.311 + (I.21 1.22 + I).18

Imbalanced 14.39 2.44 0.55 1.27 0.12 12.50 5.11 0.21 1.40 0.87 0.19 0.17 0.05 0.18 0.02 0.03 0.04 0.10 0.21 0.15 1.31 1.57 1.52

± 1.37 ± 0.31 ± 0.10 ± (I.22 ± 0.01 _+ 0.77 ± 0.40 _+ 0.09 ± 0.38 + 0.21 ± 0.04 ± 0.06 ± 0.02 ± 0.06 ± 0.01 ± 0.02 ± 0.02 ± 0.02 ± 0.02 ± 0.03 ± 0.11 + 0.15 + 0.15

Corrected 11.54 2.26 0.25 1.29 0.13 13.09 5.(13 0.09 0.98 0.60 0.14 0.09 0.09 0.10 0.01 (I.02 0.02 0.10 0.31 0.09 1.22 I.II 1.01

+ 0.87 ± 0.23 + 0.02 ± 0.16 + 0.01 ± 1.42 _+ 0.36 ± 0.04 + 0.10 _+ 0.04 +_ 0.01 _+ 0.(14 ± 0.02 ± 0.02 _+ 0.01 ± 0.01 ± 0.01 ± 0.02 ± 0.06 + 0.01 ± 0.15 + 0.08 + 0.08

*IAAs: Sum of the indispensable amino acids: thr, val, met, ile, leu, phe, trp, lys, his. flAA-isoleucine: Sum of the indispensable amino acids not including isoleucine. Values are mean _+ standard error for 4--6 animals per group.

n o t b e n o t e d in studies u s i n g t h e w h o l e brain. T h e b a s a l a m i n o acid diet, w h i c h is relatively b a l a n c e d , a n d the corr e c t e d diet w h i c h is well b a l a n c e d , c o n t a i n a m i n o acids e q u i v a l e n t to 10.5 a n d 17.3% c r u d e p r o t e i n , r e s p e c t i v e l y . T h e s e c o u l d b e c o n s i d e r e d to b e low a n d j u s t a d e q u a t e levels o f d i e t a r y p r o t e i n , b u t e x c e p t for a r e d u c t i o n in o n e o f four c o r r e c t e d g r o u p s , t h e r e w e r e n o c h a n g e s in the s e r o t o n i n c o n t e n t o f t h e s e b r a i n a r e a s , n o r were t h e r e a n y c o n s i s t e n t c h a n g e s in t h e t r y p t o p h a n c o n c e n t r a t i o n s in t h e s e areas. T h u s , o u r r e s u l t s w o u l d n o t s e e m to s u p p o r t the s u g g e s t i o n o f W u r t m a n a n d c o l l e a g u e s [5] a n d A n d e r s o n [1] t h a t the c o n c e n t r a t i o n o f d i e t a r y p r o t e i n affects the w h o l e b r a i n s e r o t o n i n c o n c e n t r a t i o n via c h a n g e s in the b r a i n t r y p t o p h a n concentration. T h e c h a n g e s in a m i n o acid p a t t e r n s r e p o r t e d here are o f i n t e r e s t , since a m i n o acid c o n c e n t r a t i o n s in specific b r a i n a r e a s a f t e r f e e d i n g i m b a l a n c e d a m i n o acid diets h a v e n o t p r e v i o u l s y b e e n i n v e s t i g a t e d . P r e v i o u s r e p o r t s h a v e indicated that whole brain concentrations of the indispensable a m i n o acids a d d e d to c a u s e a n i m b a l a n c e w e r e s o m e w h a t i n c r e a s e d , a n d t h e r e w a s a r e d u c t i o n in t h e c o n c e n t r a t i o n ( s ) o f t h e limiting a m i n o acid(s) in w h o l e b r a i n [29]. W e did o b s e r v e significant d e c r e a s e s in the limiting a m i n o acid in b o t h studies. T h e f o o d i n t a k e o f the a n i m a l s on t h e t h r e o n i n e - i m b a l a n c e d diet w a s d e c r e a s e d , s u c h t h a t t h e n e t i n t a k e o f t h r e o n i n e w a s 70% o f t h a t in t h e b a s a l - d i e t g r o u p , b u t t h e c o n c e n t r a t i o n s o f t h r e o n i n e in t h e s e c o r t e x s a m p l e s

w e r e e v e n f u r t h e r r e d u c e d , to 50-57% o f the basal level. In a d d i t i o n , the c o n c e n t r a t i o n o f isoleucine in the p r e p y r i f o r m c o r t e x w a s d e c r e a s e d , b u t t h e r e w e r e n o d i f f e r e n c e s in f o o d i n t a k e in the isoleucine study. T h e r e f o r e , i n t a k e o f the limiting a m i n o acids did not a c c o u n t fully for t h e i r d e c r e a s e d c o n c e n t r a t i o n s in t h e s e tissues. In parallel e x p e r i m e n t s , the food i n t a k e r e d u c t i o n o f the i s o l e u c i n e - i m b a l a n c e d die! g r o u p r e a c h e d significance by 18:00, 6 h o u r s a f t e r p r e s e n t a t i o n o f the diets ( u n p u b l i s h e d o b s e r v a t i o n s ) . In r e p e a t e d e x p e r i m e n t s w i t h t h e s e diets, the i s o l e u c i n e - i m b a l a n c e d g r o u p did n o t fail to r e d u c e t h e i r food i n t a k e , a l t h o u g h the c o n c e n t r a t i o n o f t h e i m b a l a n c e d m i x t u r e in this diet is less, a n d so t h e i m b a l a n c e is less s e v e r e , a n d t h e rate o f r e d u c t i o n in food intake h a s n e v e r b e e n as s h a r p as t h a t for the t h r e o n i n e - i m b a l a n c e d diet [ 13,14]. T h u s , with r e g a r d to t h e limiting a m i n o acid. the c h a n g e s in t h e a m i n o acid p a t t e r n s in t h e s e 2 b r a i n a r e a s r e s e m b l e d r e s u l t s rep o r t e d for w h o l e brain. In a d d i t i o n , t h e s e v e r a l v a r i a t i o n s in t h e p a t t e r n o f i n d i v i d u a l a m i n o acids in t h e s e b r a i n areas after a more severely imbalanced ration (threoninei m b a l a n c e d study) m a y b e c o m p a r e d w i t h t h e paucity o f alt e r a t i o n s in the less s e v e r e l y i m b a l a n e e d diet (isoleucinei m b a l a n c e d study), in w h i c h food i n t a k e h a d n o t yet dec r e a s e d at the t i m e o f killing. Also, the p r e s e n t results in t h e s e cortical b r a i n a r e a s are in line w i t h t h o s e u s i n g w h o l e b r a i n in t h a t t h e s u m o f t h e i n d i s p e n s a b l e a m i n o acids a d d e d to c r e a t e t h e i m b a l a n c e ( I A A - T H R ) w a s o n l y slightly altered

P R E P Y R I F O R M A M I N E S A N D A M I N O ACIDS

1079

in the imbalanced group of the threonine study [29], rather than greatly increased, as it has been shown to be in the plasma [18]. As noted above, the 2 brain areas used in the present study play different roles in the regulation of food intake in animals given imbalanced amino acid diets. The prepyriform cortex is involved in the immediate reduction of food intake in these animals, while the cingulate cortex is involved in the later adaptation phase of the response. The mechanisms underlying the neural control of the feeding response in this d i e t a ~ paradigm are unknown. The prepyriform cortex is a primary olfactory relay [36], but olfactory bulbectomy did not prevent the reductions in food intake of animals offered the amino acid imbalanced or deficient diets [11]. Thus, olfaction is not considered to be essential in the control of feeding with imbalanced amino acid diets. Neither is taste likely to be involved in the initial response to imbalanced amino acid diets, since food intake was rapidly depressed in rats that were given IV infusions of imbalanced amino acids in order to bypass oral input and the taste pathways [28]. Further, intact rats chose a corrected diet adulterated with quinine over an imbalanced diet without any added taste cues [12]. Reductions in the concentrations of the acidic amino acid neurotransmitters, glutamic acid and aspartic acid have been reported in the prepyriform cortex after olfactory bulbectomy, and so they are postulated to be the primary neurotransmitters in that area [7]. However, these amino acids were altered similarly in both brain areas in both studies, and they were not significantly reduced in the imbalanced group

in either study. Thus, glutamic acid and aspartic acid may not be involved in this feeding response. This interpretation is consistent with the negative results of olfactory bulbectomy, as noted above [I 1]. In sum, in animals offered the imbalanced diet, norepinephrine was reduced in the prepyriform cortex, but not the cingulate cortex, in both studies. The limiting amino acid concentrations were reduced in the prepyriform cortex in both studies, and in the cingulate cortex, the limiting amino acid was reduced only in the threonine-imbalanced study. At the same time, the I A A - T H R were increased in the corrected group both in the prepyriform cortex and cingulate cortex but the I A A - I L E were not significantly altered in either tissue. Since the animals in the present study were intact, and the prepyriform, but not cingulate cortex levels of norepinephrine were reduced after ingesting the imbalanced diet, the concentration of norepinephrine in the prepyriform cortex may be associated with the initial depression of feeding in animals fed an imbalanced amino acid diet. Further studies are in progress to determine what specific effect, if any, the noradrenergic system may play in regulating the feeding response of rats to imbalanced amino acid diets.

ACKNOWLEDGEMENTS We are grateful to Dr. Shri Girl for the use of the HPLC equipment, and to Mrs. Tracy Schuster for excellent secretarial assistance.

REFERENCES 1. Anderson, G. H. Control of protein and energy intake: Role of plasma amino acids and brain neurotransmitters. Can J Physiol Pharmacol 57: 1043-1057, 1979. 2. Blundell, J. E. and P. J. Rogers. Effects of anorexic drugs on food intake, food selection and preferences and hunger motivation and subjective experiences. Appetite 1: 151-165, 1980. 3. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976. 4. Chee, K. M., D. R. Romsos, W. G. Bergen and G. A. Leveille. Protein intake regulation and nitrogen retention in young obese and lean mice. J Nutr 111: 58-67, 1981. 5. Fernstrom, J. D. and R. J. Wurtman. Brain serotonin content: Physiological regulation by plasma neutral amino acids. Science 178: 414--416, 1972. 6. Harper, A. E. Protein and amino acids in the regulation of food intake. In: Hunger: Basic Mechanisms and Clincial Implications, edited by D. Novin, W. Wyrwicka and G. Bray. New York: Raven Press, 1976, pp. 103-113. 7. Harvey, J. A., C. N. Scholfield, L. T. Graham, Jr. and M. H. Aprison. Putative transmitters in denervated olfactory cortex. J Neurochem 24: 445-449, 1975. 8. Hoebel, B. G. Neurotransmitters in the control of feeding and its rewards: Monoamines, opiates and brain-gut peptides. In: Eating and Its Disorders, edited by A. J. Stunkard and E. Stellar. New York: Raven Press, 1984, pp. 15-38. 9. Kelly, J., G. F. Alheid, A. Newberg and S. P. Grossman. G A B A stimulation and blockade in the hypothalamus and midbrain: Effects on feeding and locomotor activity. Pharmacol Biochem Behav 7: 523-541, 1977. 10. Leibowitz, S. F. Brain catecholaminergic mechanisms for control of hunger. In: Hunger: Basic Mechanisms and Clinical Implications, edited by D. Novin, W. Wyrwicka and G. Bray. New York: Raven Press, 1976, pp. 1-31.

11. Leung, P. M. B., D. M. Larson and Q. R. Rogers. Food intake and preference of olfactory bulbectomized rats fed amino acid imbalanced or deficient diets. Physiol Behav 9: 553-557, 1972. 12. Leung, P. M. B., D. M. Larson and Q. R. Rogers. lnfluence of taste on dietary choice of rats fed amino acid imbalanced or deficient diets. Physiol Behav, submitted. 13. Leung, P. M. B. and Q. R. Rogers. Food intake: Regulation by plasma amino acid pattern. Lift, Sci 8: 1-9, 1969. 14. Leung, P. M. B. and Q. R. Rogers. Effect of amino acid imbalance and deficiency on food intake of rats with hypothalamic lesions. Nutr Rep Intern 1: 1-10, 1970. 15. Leung, P. M. B. and Q. R. Rogers, Importance of prepyriform cortex in food-intake response of rats to amino acids. Ant J Physiol 221: 92%935, 1971. 16. Leung, P. M. B. and Q. R. Rogers. Disturbances in amino acid balance. In: Total Parenteral Nutrition, edited by H. Ghadami. New York: John Wiley and Sons, 1975. pp. 25%284. 17. Leung, P. M. B. and Q. R. Rogers. Hyperphagia after ventral tegmental lesions and food intake responses of rats fed disproportionate amounts of dietary amino acids. Physiol Behav 25: 457-464, 1980. 18. Leung, P. M. B., Q. R. Rogers and A. E. Harper. Effect of amino acid imbalance on plasma and tissue free amino acids in the rat. J Nutr 96: 303-318, 1968. 19. Li, E. T. S. and G. H. Anderson. Self-selected meal composition, circadian rhythms and meal responses in plasma and brain tryptophan and 5-hydroxytryptamine in rats. J Nutr 112: 20012010, 1982. 20. Marshall, J. F. Regulation of food intake. In: Adrenergic Activators and lnhibitors, Part 1, edited by L. Szekeres. New York: Springer-Verlag, 1980, pp. 56%578.

1080 21. McGeer, P. L., J. C. Eccles and E. G. McGeer. Specific neuronal participants and their physiological actions. In: Molecular Neurobiology o f the Mammalian Brain. New York: Plenum Press, 1978, pp. 141-363. 22. Meliza, L. L., P. M. B. Leung and Q. R. Rogers. Effect of anterior prepyriform and medial amygdaloid lesions on acquisition of taste-avoidance and response to dietary amino acid imbalance. Physiol Behav 26: 1031-1035, 1981. 23. Meliza, L. L., P. M. B. Leung and Q. R. Rogers, Cingulate lesions and behavioral adaptation to amino aci~l imbalanced diets. Physiol Behav 30: 243-246, 1983. 24. Morley, J. E., A. S. Levine, G. K. Yim and M. T. Lowy. Opioid modulation of appetite. Neurosci Biobehav Rev 7: 281-305, 1983. 25. Noda, K. and K. Chikamori. Effect of ammonia via prepyriform cortex on regulation of food intake in the rat. Am J Phvsiol 231: 1263-1266, 1976. 26. Pardridge, W. M. The role of blood-brain barrier transport of tryptophan and other neutral amino acids in the regulation of substrate-limited pathways of brain amino acid metabolism. J Neural Transm Suppl 15: 43-54, 1979. 27. Pellegrino, L. J. and A. J. Cushman. A Stereotaxic Atlas o f the Rat Brain. New York: Appleton-Century Crofts, 1967. 28. Peng, Y. and A. E. Harper. Amino acid balance and food intake: effect of amino acid infusions on plasma amino acids. Am ,I Physiol 217: 1441-1445, 1969. 29. Peng, Y., J. K. Tews and A. E. Harper. Amino acid imbalance, protein intake, and changes in rat brain and plasma amino acids. Am J Physiol 222: 314-321, 1972. 30. Peters, J. C., D. B. Bellissimo and A. E. Harper. L-Tryptophan injection fails to alter nutrient selection by rats. Physiol Behav 32: 253-259, 1984. 31. Peters, J. C. and A. E. Harper. Protein and energy consumption, plasma amino acid ratios, and brain neurotransmitter concentrations. Physiol Behav 27: 287-298, 1981. 32. Peters, J . C. and A. E. Harper. Influence of dietary protein level on protein self-selection and plasma and brain aminio acid concentrations. Physiol Behav 33: 783-790, 1984.

GIETZEN, LEUNG AN[) ROGERS

33. Peters, J. C. and A, E. Harper. Adaptation ot rats to diets containing different levels of protein: Effects on food intakc, plasma and brain amino acid concentrations and brain neurotransmitter metabolism../Nutr 115: 382-398, 1985 34. Rogers, Q. R, and P, M. B. Leung. The influence of amino acids on the neuroregulation of food intake. Fed Proc 32: 1709-1"719, 1973. 35. Rossi, J., II1, A. J. Zolovick, R. F. Davies and J. Panksepp. The role of norepinephrine in feeding behavior. Neuro~sci Biobehav Rev 6: 195-204, 1982. 36. Shepherd, G. M. Olfactory cortex. In: lhe Synaptic Orgamzation of the Braitl. New York: Oxford University Press, 1979, pp. 289-307. 37. Tews, J. K., Y.-W. L. Kim and A. E. Harper. Induction of threonine imbalance by dispensable amino acids: Relation to competition for amino acid transport into brain, .I ,\;utr 109: 304-315, 1979. 38. Tews, J. K., Y.-W. L. Kim and A. E. Harper. Induction of threonine imbalance by dispensable amino acids: Relationships between tissue amino acids and diet in rats. ,I Nutr 110: 394-408, 1980. 39. Tews, J. K., Q. R. Rogers, J. G. Morris and A. E. Harper. Effect of dietary protein and GABA on food intake, growth and tissue amino acids in cats. Physiol Behav 32: 301-308, 1984. 40. Tobin, G. and K. N. Boorman. Carotid or jugular amino acid infusions and food intake in the cockerel. BrJ Nutr 41: 157--162, 1979. 41. Wagner, J., P. Vitali, M. G. Palfreyman, M. Zraika and S. Huot. Simultaneous determination of 3,4-dihydroxyl~henylalanine, 5-hydroxytryptophan, dopamine, 4-hydroxy-3-methoxyphenylacetic acid, norepinephrine, 3-4-dihydroxyphenylacetic acid, homovanillic acid. serotonin and 5-hydroxyindoleacetic acid in rat cerebrospinal fluid and brain by high performance liquid chromatography with electrochemical detection. ,I Neurochem 38: 1241-1254, 1982. 42. Wayner, M. J., T. Ono, A. DeYoung and F. C. Barone. Effects of essential amino acids on central neurons. Pharmacol Biochem Behav 3: Suppl 1, 85-90, 1975. 43. Wurtman, J. J. and R. J. Wurtman. Drugs that enhance central serotoninergic transmission diminish elective carbohydrate consumption by rats. Lil~" Sci 24: 895-904. 1979.