Critical Review

Neural Mechanisms in the Responses Amino Acid Deficiency1

DOROTHY W. GIETZEN

to

Department of Physiological Sciences, School of Veterinary Medicine and Food Intake Laboratory, University of California-Davis, Davis, CA 95616
acid provides adaptive advantage to animals. Torii et al. (1987) showed that rats eating a lysine-dficient diet can learn to select from 15 water bottles con taining various solutions (including lysine, other amino acids and saccharin) the one that contains lysine. When given a lysine-replte diet these rats quickly select a saccharin solution, which rats nor mally prefer. Many authors have reported choice situ ations in which the animals have demonstrated selection for a necessary amino acid, albeit having fewer alternatives (e.g., Booth and Simson 1971, Gietzen et al. 1992, Halstead and Gallagher 1962, Rogers and Harper 1970). The basic question under lying the studies to be reviewed here is: What mechanisms allow such exquisite sensitivity to di etary amino acids?

ABSTRACT Food intake is rapidly and reliably reduced when animals are offered diets that result in an essential amino acid deficiency, such as those used in the imbalanced amino acid diet (1MB)paradigm. There seem to be at least three phases in the responses of rats to 1MB: 1) In order to respond to a dietary challenge, the animals must first recognize that challenge. The available data suggest that before the behavioral effects occur, a de cline in the concentration of an essential amino acid is sensed in a specific brain area, the prepyriform cortex. This recognition phase is associated with localized decreases in the concentrations of the limiting amino acid, norepinephrine and cyclic AMP and with altered protein synthesis. 2) Subsequent to recognition of the deficiency, a conditioned taste aversion develops, mediated in part by serotonin at the level of the vagus. 3) Finally, in the absence of a choice, the animals adapt to an 1MB(but not a diet devoid of one or more essential amino acids) in ~1 wk. Damage to certain extrahypothalamic brain areas or liver denervation accelerates adap tation to 1MB, suggesting both central and peripheral control in the adaptation phase. The resulting behavioral responses provide adaptive advantage to an animal in the selection of a diet with an appropriate balance of amino acids. J. Nutr. 123: 610-625, 1993. INDEXING KEY WORDS:

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HISTORICAL

PERSPECTIVE

Amino acid imbalances were defined by Harper (1976) as resulting from "additions to a low protein diet of one or more amino acids, other than the one that is growth limiting, in amounts that individually are not toxic. They cause depressions in food intake and growth that are readily prevented by a sup plement of the growth-limiting amino acid." Nitrogenous constituents have been recognized as important in the diet for over 150 y (Magendie 1816), and diets containing inadequate amounts of amino acids have been associated with a marked depression of growth and food intake since the early part of the 20th century (Osborne and Mendel 1914, Rose 1931, Willcock and Hopkins 1906). Perhaps the first use of

feeding behavior neruous system anorexia amino acid imbalance rats serotonin norepinephrine essential amino acids protein synthesis

Animals, particularly omnivores and generalist her bivores, need mechanisms that provide for selection of a balanced diet to meet all of their nutrient require ments. For most nonruminants, this includes the in dispensable (essential) amino acids. Because they are not stored (Berg and Rose 1929), in the event of a deficiency, amino acids are utilized from body protein, resulting in negative nitrogen balance (Munro 1976). Clearly, the capacity to distinguish balance from imbalance among the amino acids in the diet and to select for the growth-limiting essential amino
0022-3166/93 $3.00 1993 American Institute of Nutrition.

'Supported by USDA: CSRS 90 37200-5440 and NIH grants no. DK42274; NIH DK35747 to the Food Intake Laboratory via the Clinical Nutrition Research Unit, University of California-Davis, Sandoz Research Institute, and a gift of dietary amino acids from Nutri-Quest (Chesterfield, MO). 1992. Accepted 30 November 1992.

Received 9 September 610

NEURAL RESPONSES TO AMINO ACID DEFICIENCY

611

60 hr
50

3 hr

5 hr

6 hr

o E o i

40-

O O

30

20-

10

ni i i i i i i i i i i i 11i11 11i i 11i i i i 11

l l l l l l l l l l l l l

tS^egs..2 - ^o?o iiEw- 9stt t.*

FIGURE 1 Effect of feeding imbalanced amino acid or basal control diet on concentrations of amino acids in plasma. Plasma was collected at various times, as indicated, after a single meal of the test diet. Closed circles: basal diet; X: threonineimbalanced diet; values are ^mol/100 mL plasma, n = 5 per group. Amino acids are indicated by their conventional abbreviations along the abscissa. From Leung et al. (1968b), reproduced with permission. For comparison with Figure 4, (values given in ^mol/100 mL)--10 = values as nmol/mL.

an imbalanced amino acid diet (1MB)2'3was in studies using gelatin (which is devoid of tryptophan and defi cient in several essential amino acids) as the source of dietary protein (reviewed by Jackson et al. 1928). Then, with the isolation of threonine, the final amino acid discovered to be required for growth (McCoy et al. 1935), the protein fraction of the diet could be composed entirely of amino acids, and the effects of each indispensable amino acid could be studied with precision. The effects of 1MB on food intake and growth have been observed in several animal species (Harper et al. 1970) and have been the subject of several reviews (Elvehjem and Krehl 1955, Gietzen et al. 1986a and 1988a, Harper and Rogers 1965, Harper et al. 1970, Jackson et al. 1928, Leung and Rogers 1987, Rogers and Leung 1973 and 1977, Salmon 1958). Amino acid-imbalanced diets can be distinguished from defi cient diets, because either reducing the added amino acids or increasing the limiting one will restore balance to an 1MB, whereas only increasing the limiting amino acid will remedy a deficient diet. It should be noted that requirement studies using graded reductions in the limiting amino acid can also produce an imbalanced situation (Millward and Rivers 1988). The observation that a reduction in food intake occurs before the growth retardation led to the reali

zation that the depression in food intake is the primary event (Harper and Rogers 1965); animals fed 1MB grow to the level of control animals if food intake is maintained. This demonstrated that the depression in food intake is the cause rather than the result of the growth failure (Harper et al. 1970). These historical studies show clearly that amino acids play an important role in the control of food intake. In the search for the mechanisms underlying the responses to 1MB, the one biochemical observation that has consistently been made is a marked im balance in the plasma amino acid pattern, which be comes apparent within a few hours after a meal of 1MB (Leung et al. 1968b; Fig. 1) and which resembles that of a severe amino acid deficiency (Harper and Rogers 1965). The anorectic response can be seen as early as 28 min after introduction of a threoninedevoid diet and is well correlated with a decreased

Abbreviations used: AC, anterior cingulate cortex; CNS, central nervous system; 5HIAA, 5-hydroxyindole amino acid; 5HT, serotonin; 1MB, amino acid-imbalanced diet; LH, lateral hypothalamus; NE, norepinephrine; PPC, anterior prepyriform cortex; PVN, paraventricular nucleus of the hypothalamus; SCOP, scopolamine; VMH, ventromedial hypothalamus. 3Diets have been described in detail (Beverly et al. 1991b, Hammer et al. 1990b).

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concentration of threonine in the plasma (Gietzen et al. 1986a). Thus, the use of 1MB provides a paradigm for the study of a rapidly occurring, robust, nutri tionally induced decrease in food intake due to recog nition of an amino acid deficiency. This review will focus on studies of the neural mechanisms underlying the responses of rats to amino acid deficiency. The 1MB feeding paradigm used by the author, as well as by Rogers and Leung (reviewed in 1977) and others, has a distinguished history and was reviewed comprehensively by Harper et al. (1970). The responses will be viewed in the context of 1} the initial metabolic factors occurring with ingestion of 1MB, i.e., the recognition phase; 2) the aversive responses that require a period of learning; and 3) the adaptive phase, which is seen over several days.

PHASE ONE: RECOGNITION OF AMINO ACID DEFICIENCY

Recognition of the amino acid deficiency does not require intact olfactory (Leung et al. 1972), hepatic (Bellinger et al. 1992), vagai (Jiang 1992, Washburn et al. 1992), gastric (Stickney et al. 1976) or adrenal (Hammer et al. 1990b) systems. Several other potential physiological variables have been studied and nearly as many negative results reported (reviewed in Harper and Rogers 1965). By definition (see above), the amino acids individually added to 1MB are not toxic, because in the absence of a choice animals will adapt and grow on 1MB. Although im portant in learned responses to 1MB (Chambers 1990, Mori et al. 1991, Rogers and Leung 1977), taste is unlikely to be essential for the initial recognition of 1MB that occurs in the first hour after diet in troduction (Rogers and Leung 1977); palatability does not contribute to the initial recognition of 1MB, be cause 1MB intake is not reduced during the first meal (Rogers and Leung 1977). Rather, in studies dating back to the late 1960s, several lines of evidence have supported the idea that the brain is the site of recognition in this model. The primary observation was that the food intake depression of rats (Leung and Rogers 1969) and cock erels (Tobin and Boorman 1979) fed 1MB was prevented after a small quantity of the most limiting amino acid was infused into the carotid artery, be ginning a few hours before 1MB was fed. Infusion of the limiting amino acid into the portal or jugular vein required a much greater quantity than that given into the carotid artery to prevent the food intake depression (Leung and Rogers 1969). In addition, Tews and colleagues (Tews et al. 1978, 1979 and 1981) have shown that the plasma amino acid imbalance creates a disadvantage for the limiting amino acid in its competition for transport into the brain, reducing the

concentration of the growth-limiting amino acid in the brain. Reduced concentrations of the limiting amino acid in whole brain were measured within 2-3 h after consumption of 1MB (Peng et al. 1972) and were well correlated with the onset of the feeding depression (reviewed in Gietzen et al. 1986a). These separate findings demonstrate that the brain has an important role in the food intake responses of animals ingesting amino acid-imbalanced or devoid diets. The work of Tews et al. (1978, 1979 and 1981) described the mechanism for establishing amino acid imbalance in the brain (Tackman et al. 1990) but did not provide evidence for localization within the brain. In a series of neuroanatomical experiments, Leung and Rogers (1987) studied the effects of various brain lesions on intake of 1MB, devoid and high protein diets (Table 1). Among the results was the obser vation that limbic-rhinencephalic neural areas that relay olfactory information, such as the anterior prepyriform cortex (PPC) and amygdala, are essential for the initial depression of food intake in rats fed 1MB (Firman and Kuenzel 1988, Leung and Rogers 1971, 1973 and 1987, Noda 1975, Noda and Chikamori 1976, Rogers and Leung 1973) or devoid diets (Leung and Rogers 1971). Indeed, the PPC has been suggested to be the body's amino acid "chemosensor" (Beverly et al. 1990a and 1990b, Leung and Rogers 1973). To determine whether the decreased concentration of the limiting amino acid, which had already been observed in whole brain by Peng et al. (1972), occurs uniformly throughout the brain, 14 microdissected brain areas, including the PPC, were assayed for con centrations of the limiting amino acid after 1MB feeding. The results (Table 2) show that, although decreases in the limiting amino acid in the 1MB groups were not seen in all brain areas, the concentra tions of threonine in the PPC, anterior cingulate cortex (AC), locus ceruleus and nucleus of the solitary tract from the threonine-imbalanced diet group were significantly reduced. Thus, regional differences in the concentrations of amino acids were seen within the brain at 2.5 h, when food intake had just become depressed. It is interesting that after ingestion of 1MB the concentration of the limiting amino acid was not altered in any of the three hypothalamic areas for merly thought to control feeding (Gietzen et al. 1989) or in the amygdala. A reduction of the limiting amino acid did occur in other brain areas (listed above and in Table 2), suggesting that those areas, along with the PPC, have the potential to serve as sensors of an amino acid decrease. However, whereas either PPC or amygdala lesions resulted in animals that did not avoid the 1MB, the limiting amino acid was decreased in the PPC but not in the amygdala. Moreover, the amygdala has been linked to conditioned taste aversion (Meliza et al. 1981), which can develop only after recognition of some negative stimulus

NEURAL RESPONSES TO AMINO ACID DEFICIENCY

613

TABLE 1

Effects of electrolytic lesions in various brain regions, or removal of pituitary or olfactory bulbs of rats, on intake of amino acid-unbalanced, amino acid-devoid or high protein diets1
Initial Response Lesion Anterior amygdala Anterior cingulate cortex Anterior prepyriform cortex Area postrema Dorsolateral hippocampus Lateral septum Medial amygdala Nucleus of solitary tract Posterior amygdala Raphe nuclei Thalamic taste nuclei Ventral tegmental nuclei Ventromedial hypothalamus 1MB DEV HI 1MB Adaptation DEV HI

T T

T T
T

4*

Hypophysectomy 4 4 Olfactory bulbectomy 'Symbols used: 4 or T, decreased or increased intake of indicated diet, relative to that animal's baseline control intake; ", increased intake with mild but not severe imbalance; ?*, food intake greater than control over time but not true adaptation. Diet abbreviations: 1MB, imbalanced amino acid diet; DEV, diet devoid of an essential amino acid; HI, 75% casein diet. Data taken from Leung and Rogers (1987|, Leung et al. (1986a, 1987a and 1987b), Rogers and Leung (1973 and 1977).

(Chambers 1990). Thus, the PPC remains, from these studies, the primary candidate for the brain area that may serve as the essential amino acid chemosensor.

TABLE 2 Concentration of threonine in rat brain areas taken 2.5 h after introduction of threonine-basal, -imbalanced or -corrected diets1 Diet

The limiting amino acid in the prepyriform cortex

The concentration of the limiting amino acid in the PPC, measured after feeding basal diet or 1MB (Gietzen et al. 1986b), was used in determining the concentration of amino acid to use for injections into the PPC. In this series of studies, the effects of replacing the limiting amino acid precisely into the PPC were established (Beverly et al. 1990a, 1990b, 1991a, 199Ib and 1991c). After injections of threonine into the PPC, rats did not decrease their intake of a threonine-imbalanced diet whereas, as expected, threonine had no effect on intake of an isoleucineimbalanced diet. Similarly, isoleucine injected into the PPC reversed the feeding depression with an isoleucine- but not a threonine-imbalanced diet. The optimum dose of threonine, in improving intake of a threonine-imbalanced diet, was ~2 nmol (given in bilateral injections of 0.5 ^L), and intake of the basal diet was not affected using similar injections (Beverly et al. 1990a and 1990b; solid bars in Fig. 2). Similar

Brain area Amygdala Anterior cingulate cortex Area postrema Hippocampus Lateral hypothalamus Locus coerulus Nucleus of the solitary tract Parabrachial nucleus Paraventricular nucleus Prepyriform cortex Raphe nuclei Septum Tegmentum Ventromedial hypothalamus

Basal

Imbalanced

Corrected

\unolfg wet tissue 0.14 0.00 0.15 0.01 0.24 0.01 0.12 0.30 0.06 0.02' 0.30 0.08 0.20 0.03 0.21 0.06 0.36 0.06 0.27 0.05 0.28 0.04 0.31 0.05 0.25 0.03 0.29 0.07 0.30 0.02 0.25 0.04 0.15 0.00* 0.24 0.02 0.21 0.04 0.37 0.14 0.18 0.02 0.14 0.02 0.25 0.07 0.35 0.11 0.29 0.03 0.12 0.05- 0.32 0.28 0.15 0.42 0.18 0.04 0.31 0.02' 0.25 0.08 0.22 0.04 0.33 0.30 0.03 0.48 0.22 0.04 0.44 0.03 0.09 0.04 0.05 0.04 0.07 0.13

0.24 0.02 0.26 0.04

0.36 0.04

'Values are means SE,n = 4-6/group. 'Significantly reduced, P < 0.05 by Fisher's protected least significant difference test after ANOVA. Data taken from Gietzen et al. (1986a, 1986b, 1988b and 1989).

614
100

GIETZEN

75

VEHICLE SO PUnOMYCIN

CD S

25

aCSF

THREONINE

FIGURE 2 Effects of threonine, with and without puromycin, injected into the prepyriform cortex in 0.3-fjL volumes, on intake of a threonine-imbalanced diet. Bars represent means for food intake over 24 h as a per centage of each animal's baseline intake of the basal diet. Error bars: SE. Solid bars: artificial cerebrospinal fluid (aCSF), injected with a control injection of aCSF (vehicle) or L-threonine (6.67 mmol/L), as indicated on the abscissa; n = 5 per group. Hatched bars: puromycin (100 /mol/L)injected with aCSF or threonine. Injections were made 0.5 h prior to introduction of the imbalanced diet. Adapted from data in Beverly et al. (1991a).

injections made 2 mm posterior to the PPC or into the amygdala were ineffective, showing regional specificity as well (Beverly et al. 1990a). Injection of threonine 6 h before onset of the dark cycle also resulted in increased intake of the threonine-imbalanced diet during the first 6 h of the dark cycle. Total intake of the 1MB was higher (85% of baseline) than that seen with injections made closer to the time of diet introduction, although timing of a vehicle injection did not alter intake of the 1MB at any time point. Thus, the effect of in jecting the limiting amino acid into the PPC lasted at least 6 h, and we have noted a delay of at least 3 h before increased intake of the 1MB was observed, if the threonine was injected into the PPC just before feeding. Delivering two half-optimal doses, several hours apart, brought intake to the same level as a single injection of the optimal dose given just before feeding. Conversely, when threonine (2 nmol) was injected twice (6 h and 0.5 h before feeding), the orexigenic effect of the 2-nmol dose was reduced, as it was in dose-response studies using 4 nmol of threonine in a single dose. The results indicate that some integral of the changes in the concentrations of amino acids is recognized in the PPC, influencing intake of 1MB (Beverly et al. 1993). It was hypothesized that diffusion or metabolism could account for the time lag between injection of the limiting amino acid into the PPC and the in creased intake of 1MB. To examine these questions, first, diffusion of the limiting amino acid was deter mined by measuring the amount of L-[14C]threonine

in the PPC and surrounding areas at various times after injection. Diffusion of label was rapid (<15 min) but limited: 85-90% of the recovered label was within 1 mm of the injection site (Beverly et al. 1991c). Biochemical studies have shown that protein syn thesis is controlled by the most limiting amino acid in the diet (Ip and Harper 1973), and decreases in an essential amino acid have been associated with aber rations of protein synthesis in single cell systems as well (Straus and Takemoto 1988). Therefore, as an index of metabolism in the PPC, effects on protein synthesis were evaluated in homogenates of the PPC by injecting L-[3H]leucine with and without the limiting amino acid. The radioactive label in the free amino acid pool was cleared rapidly,- <10% of the recovered isotope remained in the acid-soluble fraction at 2 h and <4% at 6 h after injection into the PPC (Beverly et al. 1991 c), but the effects of injection could still be seen as increased intake of an 1MB that was not offered until 6 h after the injection. Accumu lation of 3H in the acid-soluble precipitate indicated that, rather than the time lag being due to diffusion of the limiting amino acid itself, a change in metabolism in the PPC, such as protein synthesis, may be in volved in affecting the time course of onset of the feeding response after injection of the limiting amino acid into the PPC (Beverly et al. 1991c). Using blockers of protein syntheses, Beverly et al. (1991a) showed that the behavioral effects of replacing the limiting amino acid into the PPC also depend on intact protein synthesis. Co-injection of microliter volumes of puromycin4 (100 /mol/L)or actinomycin D (10 tmol/L) with the amino acid blocked the limiting amino acid-induced increase in 1MB intake but had no effect on intake of the basal diet (Fig. 2, hatched bars). The effect of puromycin was seen whether the drug was injected with the amino acid or 6 h earlier. Performance in the choice paradigm, in

4Drugs used: actinomycin D (Sigma Chemical, St. Louis, MO); clonidine hydrochloride, 2,6-dichloro-Af-2-midazolidinylidene benzenamine hydrochloride (Research Biochemicals, Natick, MA); ICS 205-930, 3(x-tropanyl-lH-indole-3-carboxylic acid ester (a gift from Sandoz Research Institute, East Hanover, NJ|; MDL72,222, laH,3a,5a-H-tropan-3-yl-3,5-dichlorobenzoate (a gift from Merrill Dow, Cincinnati, OH); 8-OH-DPAT, (+)-8-hydroxydipropylamino tetralin (Research Biochemicals); ondansetron, 1,2,3,9-tetrahydro9-methyl-3-[2-methyl-lH-imidazol-l-ylmethyl]-4H-carbazol-4-one hydrochloride, formerly known as GR38032F (a gift from Glaxo Research Group, Middlesex, U.K.); phentolamine mesylate, Regitin 3-[[4,5-dihydro-lH-imidazol-2-yl)methyl)(4-methylphenyl)-amino]phenol mesylate (Research Biochemicals); pimozide, 1-(1-[4,4-b4'(4-fluorophenyl)butyl]-4-piperidinyl]-1,3-dihydro-2Hbenzimidazol-2-one (Research Biochemicals); puromycin, 3'[aamino-p-methoxy-hydrocinnamamido)-3'-deoxyN,N-dimethyladenosine (Sigma); Q-ICS, 3-tropanyl-indole-3-carboxylate methiodide (Research Biochemicals); scopolamine, (-)-methscopolamine bromide (Research Biochemicals).

NEURAL RESPONSES TO AMINO ACID DEFICIENCY

615

ILE BASAL ILE IMBALANCED ILE CORRECTED

FREE AMINO ACIDS IN PREPYRIFORMCORTEX

15.0
5OO Ul
0) 40 H l_ 300 UJ

13.0 11.0 9.0 7.0 5.0 3.0

1 v
- THR BASAL DIET oo THR IMBALANCED DIET

x. e*
c

o, eoo
100

1.0

NE

DA

5HT

o
C

0.20 0.15 0.10 0.05 O

FIGURE 3 Concentrations of the neurotransmitters norepinephrine (NE), dopamine (DA) and serotonin (5HT) in the prepyriform cortex of rats fed isoleucine-limiting diets as indicated. Bars: mean; error bars: SE for 6-7 rats per group. Values are ng/g wet tissue wt. *Significantly lower NE in the group fed the imbalanced diet (P < 0.05). Rats were given access to the test diets for 3.5 h after onset of the dark period prior to collection of the tissues. Reproduced with permission from Gietzen et al. (1986b).

which the animals are given a choice between 1MB and a protein-free diet, was returned to normal (i.e., the animals preferred the protein-free diet, as do uninjected animals) after either puromycin or actinomycin D. Thus, both the increased intake of an 1MB and the reversal of aversion to the 1MB, when the limiting amino acid was injected into the PPC, re quire de novo protein synthesis (Beverly et al. 1991a). These findings, in association with the amino acid profile described above and in Gietzen et al. (1986b), support the suggestion that a reduction in the concen tration of the limiting amino acid, specifically in the PPC, but only with attendant changes in protein syn thesis, is involved in the initial recognition of 1MB. The data are consistent with a rapidly turning-over protein, which is ) not synthesized after ingestion of 1MB, and 2) increased in IMB-fed animals after in jection of the limiting amino acid. Inability to make a crucial protein in the PPC could lead to a neural signal that protein synthesis is at risk. Thus, effects on neurotransmission are clearly indicated.

FIGURE 4 Free amino acids in the prepyriform cortex from animals killed 2.5 h after introduction of threoninebasal, -corrected or -imbalanced diets. Values are means in nmol/mg fresh tissue. As in Figure 1, amino acids are abbreviated along the abscissa, but note differing order. Samples were microdissected samples of prepyriform cortex, homogenized in acid medium, centrifuged and ana lyzed in an automated amino acid analyzer (Beckman, Palo Alto, C A); n = 5-6 animals per group. *P < 0.05 by ANOVA. Reproduced with permission from Gietzen et al. (1986a).

norepinephrine

in the prepyriform cortex

The monoaminergic neurotransmitter systems play important roles in the control of feeding (reviewed in

Hoebel 1984). To estimate the role of monoamine neurotransmitters in the neural responses to 1MB, the concentrations of several monoamines and their metabolites were measured by HPLC with elec trochemical detection in microdissected brain areas that were taken 2.5 h after introduction of basal, 1MB or corrected diet. Amino acid concentrations in aliquots of the same samples were also measured by automated amino acid analyses. The concentrations of both norepinephrine (NE, Fig. 3) and the limiting amino acid (Table 2) were decreased in the PPC of rats fed either of two 1MB (Gietzen et al. 1986b). A decreased concentration of NE could be due to either increased release or decreased synthesis. Although the increased tyrosine in the PPC of IMB-fed animals (Fig. 4) could reflect a lower rate of tyrosine utilization for NE synthesis, the available evidence favors increased

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GIETZEN

NE release (see below). The limiting amino acid was also decreased in the AC, a nearby cortical area (Table 2), but NE levels were unchanged in the AC. As noted in Table 1, the AC is involved in the adaptive phase of the response but is not critical for the initial feeding depression. These findings suggest that either 1) both the limiting amino acid and NE must be altered in concert to serve as the initial signal to the brain,- or 2) the depression in the limiting amino acid (or the disproportion of amino acids) causes the acti vation of the NE system in specific responsive brain areas, such as the PPC. Support for the involvement of NE in the ventral cortex, in protein selection, has also been provided by Gibson et al. (1987). In any event, it seems that reduction of the limiting amino acid in a responsive brain area is a necessary first step in the recognition of amino acid deficiency (Beverly et al. 1990a and 1990b, Gietzen et al. 1986b). Exami nation of the amino acid profile in the PPC (Fig. 4) shows that any amino acid disproportion must be due to reduction, rather than increase, in the concentra tions of amino acids in that area at 2.5 h after 1MB feeding. This is in keeping with the work of Harper and Tews (1988) showing that the brain is protected from large influxes of amino acids (compare Fig. 1, for plasma, and Fig. 4, for PPC, after threonine 1MB). In the ventromedial hypothalamus (VMH), an area earlier thought to contain the "satiety center" (Stellar 1954), the concentration of NE was increased in IMBfed animals, suggesting that release was inhibited in that area. Inhibition of NE release from cells in the VMH would be consistent with a depression in feeding, because Grossman (1960) showed an increase in feeding after extracellular NE injections into the VMH. This is also consistent with an integrative role, such as assimilation of input from other brain areas in the control of feeding, rather than a signal-recognition role for the VMH. Also, the level of the limiting amino acid was not depressed in any of the three hypothalamic areas examined, and NE concentrations were unchanged in the lateral hypothalamus (LH) and paraventricular nucleus (PVN), which are two other hypothalamic areas thought to be associated with feeding (Gietzen et al. 1989). The lack of depression of the limiting amino acid in the hypothalamus is con sistent with the lack of effect of both VMH and LH lesions on 1MB intake (Leung and Rogers 1970, Scharrer et al. 1970) and again supports the proposal that extrahypothalamic structures support the initial recognition of amino acid deficiency. Still, the in creased concentration of NE in the VMH suggests that hypothalamic areas may be involved in other aspects of the response. Evidence for effects in the LH will be discussed later. In studies such as the amino acid injections described above, clonidine was injected bilaterally into the PPC (see Table 3 for dosage) prior to feeding trials with an 1MB. Clonidine is an alpha2 agonist that

TABLE 3

Effect of Clonidine injection into the prepyriform cortex (PPC) on cumulative intake of isoleucine imbalanced diet by rats1
Time2Dose33 h6h12 h% h24

control0.001150569.069.791.8107.212.5a6.2a24.3b11.6b68.070.085.1102.6 of 4.4a 2.8 5.8a 4.6 5.1a 2.9 4.5b73.877.277.887.9 4.1 'Values are means SE for imbalanced diet intake as a per centage of control (baseline basal diet) intake for each animal; n = 6 for the 1.0- and 1.5-pg dose groups, n = 7 for the 0.0- and 0.5-/ig dose groups. Values for a time interval bearing different superscript letters are significantly different, P < 0.05 by least squares means after significant overall ANOVA. Data are adapted from Gietzen and Beverly (1992). Time after introduction of the imbalanced diet, at the onset of darkness. 3Doses in g/L = total dose per rat. Injections were 1/2 of each dose, given in 0.5-^L volumes, bilaterally, simultaneously into the prepyriform cortex.

decreases NE activity by acting at a presynaptic site, providing feedback inhibition onto presynaptic autoreceptors of NE neurons (Scatton 1990). If the in jection was made within the precise stereotaxic coor dinates identified by early lesion studies (Leung and Rogers 1971), intake of the 1MB was increased (Gietzen and Beverly 1992). The results (Table 3) provide additional evidence for activation of the NE system in the PPC in the anorectic response, because inhibition of NE release resulted in increased intake of 1MB. Clonidine can inhibit the production of cAMP via the alpha2 receptor (Starke et al. 1989), and cAMP is essential for the activity of catabolite activator protein, which is necessary to initiate transcription, at least in prokaryotes (Lewin 1990). Therefore, cAMP concentrations were measured in the PPC of rats fed either threonine-imbalanced or basal control diets and killed at differing times after dark onset, using a protein binding assay (Amersham, Arlington Heights, IL). To control for any generalized changes in cAMP such as would be expected from stress or other stimuli, the data for the PPC were compared with those for the AC from each animal. The AC was used as a control area, because it had not shown any changes in NE concentration after ingestion of 1MB (Gietzen et al. 1986b). The concentration of cAMP was decreased in PPC, reaching a nadir at 2.5 h after introduction of 1MB (Fig. 5). It is tempting to hypothesize that this decrease in cAMP in the PPC, after consumption of 1MB, provides a link between the neurochemical changes and protein synthesis in the PPC after ingestion of 1MB.

NEURAL RESPONSES TO AMINO ACID DEFICIENCY

200

617

3
B
100
O. e CL

1.5 2 HOURS

2.5

3.5

FIGURE 5 Concentrations of cyclic AMP (cAMP) in the prepyriform cortex (PPC) as a percentage of the cAMP con centration in the control tissue: anterior cingulate cortex (AC). Solid line: basal diet; broken line: threonine-imbalanced diet. Markers: means; error bars: SE; n = 4-5 rats per group. Brain areas were microdissected from brains of animals killed at times indicated on the abscissa (h) after introduction of basal or imbalanced diets. Tissues were taken into acidified ethanol, homogenized and centrifuged and the supernatant assayed for the concentration of cAMP using an assay kit from Amersham (Arlington Heights, IL). 'Significantly reduced concentration of cAMP at 2.5 h after introduction of the imbalanced diet, P < 0.05.

Our findings are consistent with the view that a reduction in the concentration of the limiting amino acid in the PPC is necessary but not sufficient for providing the initial signal to the animal that it should find an alternative dietary source for its amino acids. Subsequent neurochemical events, including activation of the NE system, inhibition of adenyl cyclase activity and alterations in protein synthesis, also seem to be involved. Within the PPC, roles for NE, cAMP and probably other neuroactive com pounds seem to be indicated. Still, a full under standing of the neurochemical events in the PPC, which will explain its role as the amino acid chemosensor in the recognition of amino acid defi ciency, awaits further research.

THE SECOND PHASE: LEARNED RESPONSES

After recognition of the amino acid-deficient state, the animals develop an aversion to the 1MB, or to cues associated with the diet, and a preference for cues associated with repletion (Booth and Simson 1971, Halstead and Gallagher 1962, Leung et al. 1968a, Rogers and Harper 1970, Torii et al. 1987, Treneer and Bernstein 1981). It is generally assumed that taste cues are used in these conditioning situa tions (Garcia et al. 1955, Mori et al. 1991, Rogers and Leung 1977). However, that taste is not the cue for recognition of the initial metabolic event signaling

amino acid deficiency is indicated by the following observations. Food intake is rapidly depressed in animals that have been given an intravenous infusion of imbalanced amino acids, which bypasses oral input and therefore the taste pathways (Peng and Harper 1969). Also, rats choose a corrected diet adulterated with quinine over an 1MB without any added taste cues (Leung et al. 1986b). Choice studies have shown that the animals cannot distinguish the corrected diet from the 1MB by taste or smell, although they can become confused for a few days if the diets contain added flavorings as cues and the flavor cues are switched (Rogers and Leung 1977). Thus, rats seem to use taste as a marker for identification of acceptable or unacceptable diets, but only after they have made the appropriate metabolic distinctions. Baker and Booth (1989) demonstrated that olfactory condi tioning is also involved, and Deutsch et al. (1989) reported that rats have an unlearned specific appetite for protein that is mediated by olfactory systems (Heinrichs et al. 1990). Whatever the sensory mo dality used, the ability to make appropriate associa tions would support selection of a more adequate diet. Booth and colleagues (Baker et al. 1987, Gibson and Booth 1986) have argued that learning is required to demonstrate protein preference. A preference for flavors associated with threonine repletion clearly re quired a period of learning (Gietzen et al. 1992). We have also observed that the preference for the threonine-associated flavor was not seen in animals eating a threonine-adequate diet, but could be shown later, in the absence of additional training, if the animals were again depleted of threonine. That is, the learned preference for threonine was state dependent, occurring only in deficient animals (unpublished ob servation). Also, birds selected diets adequate in lysine or valine over diets containing only 25% of their requirements. These results did not become sig nificant until after 2-4 d (Murphy and King 1989), again suggesting that learning had taken place. Additional evidence for separation of the initial recognition phase from a subsequent learned com ponent is shown in the exhaustive series of brain lesion studies by Leung and colleagues, in which the brain areas that were shown to be associated with the initial responses were not the same as those involved in adaptation to the 1MB (Leung and Rogers 1987). Accelerated adaptation to an 1MB after lesioning was thought to result from impaired learning. The brain areas in which lesions improved adaptation but had no effect on initial recognition of 1MB include the AC, the thalamic taste nuclei, dorsolateral hip pocampus, septum and ventral tegmentum (Table 1). As reviewed in Leung and Rogers (1987), the AC, hippocampus and septum are brain areas that have been associated with learning, lesions of the ventral tegmentum would damage NE pathways (Ungerstedt 1971), and the implicated thalamic nuclei are, by

618
21***

GIETZEN

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FIGURE 6 Ratio of the serotonin metabolite, 5-hydroxyindole acetic acid (5HIAA), to serotonin (5HT), an index of serotonergic activity, in three brain areas, taken 3.5 h after introduction of either isoleucine -basal, -imbalanced or -corrected diets as indicated. Brain areas (RAPHE, raphe nuclei, both dorsal and medial; HIP, dorsal hippocampus; LC, locus ceruleus) were microdissected from frozen brains and analyzed by HPLC with electrochemical detection. Values are means SE for 4-6 rats per group. **Significantly increased from both basal and corrected control diets. 'Value from imbalanced group significantly greater than that from the basal group, both P < 0.05. Data are adapted from Gietzen et al. (1987).

FIGURE 7 Effects of serotonergic neural activity on food intake (g/24 h). Animals were injected either with saline (1 mL/kg body wt) quipazine (QUIP, 5 mg/kg intraperitoneally), a serotonin agonist that stimulates serotonin receptors, or 8-OH-DPAT (DPAT, 500 /tg/kg subcutaneously), a serotonin autoreceptor agonist that decreases serotonergic transmission. One hour after in jection, the rats were offered either the isoleucine-basal (BAS) or -imbalanced (1MB) diets as indicated. Differing superscript letters: significantly different at P < 0.05. Bars: means,- error bars: SE; n = 6 per group. Drawn from data given in Gietzen et al. (1987).

definition, part of the taste pathway. Thus, these areas may be involved in different aspects of the learned aversive responses that develop secondary to recognition of a deficient state. Although lesions of the LH had no effect on responses to 1MB (Scharrer et al. 1970), microiontophoretic injections of essential amino acids evoke neuronal responses in that area (Wayner et al. 1975). Also, Panksepp and Booth (1971) injected a balanced amino acid solution into a lateral hypothalamic area, the dorsal perifornical area, where stimulation in duces feeding. They observed depressed food intake after injections of amino acid mixtures, but ,not after saline or glucose. More recently, Ono and colleagues (Tabuchi et al. 1991) used an elegant experimental paradigm in which single neurons can be recorded extracellularly in precise brain areas of awake, be having animals. They recorded neuronal activity in several behavioral states, including while the animals were drinking various solutions. These workers have clearly demonstrated lysine-responsive neurons in the LH of lysine-dficient animals that had learned to select a lysine solution. The lysine-sensitive neurons increase their firing rate only when a lysine-dficient animal is drinking from a solution containing lysine. Thus, the LH must be considered among the brain areas that mediate the responses to amino acid defi ciency. However, because a period of preparation is necessary in this model, the role of the LH may be associated with the learning phase of the responses to

amino acid deficiency. Alternatively, the LH could be activated by connections with the taste pathway (Chambers 1990), because the neurons respond when the animal is tasting a solution it has learned to prefer during its deficiency state.

Serotonin

and the learned

aversion

Increases in serotonin (5HT) neurotransmission have been associated with a depression in feeding (Blundell 1984). Therefore, because 1MB provokes a decrease in food intake, we questioned whether the 5HT system would be stimulated in animals fed an isoleucine-imbalanced diet (Gietzen et al. 1987 and 199Ib). As an approximate index of neuronal activity in the 5HT system, the ratio of the metabolite, 5-hydroxyindole acetic acid (5HIAA), to the parent transmitter, 5HT, was measured. The ratio was elevated in three microdissected brain areas, in cluding the hippocampus, raphe nuclei and locus ceruleus of rats fed an 1MB (Fig. 6). Also, pharmaco logical treatments that stimulate 5HT receptors generally, such as the nonselective agonist, quipazine, exacerbated the depression in feeding, whereas the autoreceptor agonist, 8-OH-DPAT, which reduces 5HT transmission, brought 1MB intake to 97% of control basal diet intake (Gietzen et al. 1987 and 1991b; Fig. 7). At the dose used for these studies (5 mg/kg), quipazine did not produce the "serotonin syn drome" in our rats. These results prompted the sug gestion that, because of the known anorectic effect of 5HT, increased neural activity in the 5HT system,

NEURAL RESPONSES TO AMINO ACID DEFICIENCY

619

ui

VEHICLEICS 250-930LUOUIz0)#f1007550250IGU

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BASAL MILD DIET SEVERE

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FIGURE 8 Effects of ICS 205-930 pretreatment on intake of basal, mild isoleucine-imbalanced, or severe isoleucineimbalanced diets. Values are food intake in g/24 h. Injec tions were saline vehicle (1 mL/kg) or ICS 205-930 (9 mg/kg) given intraperitoneally l h prior to introduction of the test diets. Bars: means,- error bars: SE; n = 6-7 animals per group, a: Significantly less than basal, P < 0.05; b: significantly less than basal, P < 0.01. Reproduced from Hammer et al. (1990a) with permission.

FIGURE 9 Preference for saccharin in the isoleucinecorrected diet when tested in a choice between the cor rected diet without a taste cue and the corrected diet with added saccharin on the day after conditioning. On the con ditioning day, animals were given either ICS 205-930 or saline as for Figure 8, and offered the corrected diet (COR), the corrected diet with saccharin (CORSAC), the isoleucineimbalanced diet (1MB) or the imbalanced diet with added saccharin (IMBSAC) as indicated. Percent saccharin preference on the test day = [CORSAC intake (g)/total diet intake (g)] x 100. Bars: means; error bars: SE, n = 8 animals per group. "Significantly less preference for saccharin P < 0.05. Data are adapted from Terry et al. (1991).

after ingestion of 1MB, is part of the neural pathway associated with the reduction of 1MB intake. Based on several trials using antagonists of various neurotransmitter receptors, we have reported that it is the 5HT3 receptor subtype that underlies the role of 5HT in the feeding responses to 1MB (Gietzen et al. 1991b, Hammer et al. 1990a). Blockade of the 5HT3 receptor, with the specific antagonists ICS 205-930, MDL72,222 or ondansetron, restored feeding in this model (Fig. 8). These drugs are selective for the 5HT3 receptor (ICS 205-930 and MDL72,222 can also act at the 5HT4 receptor, but ondansetron does not) and do not act at 5HTi or 5HT2 receptors (Peroutka 1988). Peripheral administration of antagonists of anoradrenergic (phentolamine) or dopaminergic (pimozide) receptors had no effect, nor did any drugs among several 5HT antagonists that block 5HTj or 5HT2 receptors or combinations of those receptors (Hammer et al. 1990a). These results are consistent with the known anorectic effect of 5HT and suggest that the 5HT3 receptor may mediate the 5HT-induced anorectic response to diets, such as 1MB, that produce an amino acid deficiency. Antagonism of the 5HT3 receptor has been shown to block chemotherapy-induced vomiting in the range of doses that we used (Costali et al. 1988), and we have reported similar blockade of lithium chlorideinduced saccharin aversion using ICS 205-930 (Gietzen et al. 1991a), although this has not proved to be a robust effect (unpublished results). Therefore, in order to test whether the 5HT3 antagonists were indeed blocking the aversive response to amino acid

imbalance in the diet itself, we used a classical condi tioning paradigm with diets containing powdered sodium saccharin (2 g/kg diet). On the training day, animals were assigned to groups in which each rat was given an injection of either a 5HT3 antagonist or vehicle 30 min before receiving one of four diets: plain 1MB, saccharin-flavored 1MB, plain control diet or saccharin-flavored control diet. On the next day, we tested for an aversion to saccharin, expected in the group given a vehicle injection and fed the 1MB con taining saccharin. Only in the control group that had been fed the saccharin-flavored 1MB after injection with vehicle on the conditioning day was there a rejection of the saccharin-containing diet (saccharin preference = 8.3-13.5%). In all the other groups, ac ceptability of the saccharin-containing diet was sig nificantly higher (Fig. 9). Saccharin preference ranged from 50 to 79% in the group that had been injected with drug and fed the saccharin-flavored 1MB during training (P < 0.008). Thus, the appropriate control animals did develop an aversion to saccharin, whereas the pretreatment with either of two 5HT3 antagonists prevented the saccharin aversion in their counter parts. These data also demonstrate that the serotonergic system, at the 5HT3 receptor, seems to be involved in this aspect of the aversive effects of 1MB (Terry et al. 1991).

Peripheral studies
Ever since the pioneering studies of Leung and Rogers (1969 and 1971) showing that the brain was

620

GIETZEN

involved in the responses to 1MB, and the work of Tews et al. (1978, 1979 and 1981) demonstrating the effects of transport at the blood brain barrier, the focus of studies on the responses to 1MB has been on the central nervous system (CNS) (Gietzen et al. 1986a, Rogers and Leung 1973 and 1977, Torii et al. 1987). Nonetheless, the 5HT3 antagonists, which re stored intake of 1MB to control levels, had all been given peripherally, so one could not be certain that these drugs were acting in the CNS. That 5HT might not be acting centrally in the anorectic responses to 1MB was also suggested by the lack of effect of raphe lesions on 1MB intake (Leung and Rogers 1987) and the report that no changes attributable to 5HT were seen in whole brain after 1MB feeding (Tackman et al. 1990). Moreover, peripheral effects of amino acids (Russek 1971, Sharma and Nsset 1962, Tews et al. 1988, Yox and Ritter 1988) and of 5HT (Fletcher and Burton 1984) have been reported. Therefore, two ex periments were conducted to determine whether the effects of these antagonists are mediated through central or peripheral 5HT mechanisms. First, ICS 205-930 was injected centrally, into either the lateral ventricle (doses, 0.3 pmol to 10 nmol) or the cisterna magna (62 nmol). The intake of rats fed an isoleucineimbalanced diet was not affected by either of these central injections. Second, rats received an intraperitoneal injection of saline, ICS 205-930 or a quaternary derivative of ICS 205-930 (Q-ICS) that should not cross the blood brain barrier. Both ICS 205-930- and Q-ICS-injected rats ate significantly more 1MB than did saline-injected rats,- intake of the 1MB did not differ between ICS 205-930- and QICS-injected groups. From these results, it seems that ICS 205-930 acts in the periphery, rather than in the CNS, to restore intake of 1MB (Hrupka et al. 1991). In order to determine where, peripherally, the 5HT3 antagonists may be acting, a series of selective denervation experiments was undertaken. Among other things, the effect of vagotomy on the food intake depression associated with 1MB was inves tigated. One month after undergoing either a subdiaphragmatic vagotomy or sham surgery, rats were given an injection of either ICS 205-930 or saline and then fed either the basal diet or 1MB. Intake of the 1MB by vagotomized animals increased by 20% over that by the sham-vehicle group, and 1MB intakes by ICS 205-930-injected and vehicle-injected vagoto mized rats were similar: 66.8 and 61.2% of BAS, both (P < 0.0001) greater than for the sham-vehicle group fed 1MB. There seemed to be two aspects of the responses to 1MB after vagotomy: 1}intake of the 1MB by the vagotomized animals increased, with or without drug treatment; and, 2) the effect of the 5HT antagonist was blunted in vagotomized animals (see the four bars for SHAM and VAGX on the left side of Fig. 10). Because vagotomy can cause reduced numbers of 5HT3 receptors (reviewed in Tyers 1990),

VEH ICS

SHAM

VAGX

SCOP

FIGURE 10 Effect of vagotomy (VAGX) or scopolamine (SCOP) on intake of a severe isoleucine-imbalanced diet. VAGX or SHAM animals were subjected to complete subdiaphragmatic vagotomy, or a sham operation, 1 mo before the experiment. Rats were injected with either saline or ICS 205-930 (ICS), as for Figure 8, as indicated. Animals indi cated by SCOP were also given subcutaneous injections of scopolamine (0.5 mg/kg) at the time of the ICS 205-930 injections. Values for vehicle controls for the SCOP animals did not differ from those for the SHAM group. Values (bars): means; error bars: SE, for imbalanced diet intake over 6 h as g/100 g body wt; n = 8 per group. Differing letters over SE bars indicate significant differences (P < 0.05). Data are adapted from Jiang (1992).

these data are consistent with a role for the vagus in the 5HT3- mediated responses to 1MB. The question remained whether vagai affrents (that carry sensory information to the brain) or efferents (that provide motor input to vagally inner vated structures) were mediating these changes. To block vagai efferent (motor) activity, we used scopolamine methyl bromide (SCOP), which blocks only peripheral muscarinic receptors for 6-8 h after injection (Brown 1990). The animals were injected with either SCOP or vehicle and with either ICS 205-930 or vehicle prior to introduction of the test diets. In the absence of ICS 205-930 (i.e., comparing sham and SCOP groups given just a vehicle injection, dark bars in Fig. 10), SCOP had no effect on intake of 1MB. However, the effects of ICS 205-930 were again blunted in the animals with both 5HT and muscarinic receptors blocked (compare the three ICS 205-930-treated groups, hatched bars in Fig. 10). Be cause the increase in 1MB intake by vagotomized rats was not seen when the efferents were blocked by SCOP (i.e., only affrents,which carry sensory infor mation, were functioning), that increase may be mediated by vagai sensory fibers. However, when given with ICS 205-930, SCOP decreased the 5HT effect, as had vagotomy. That is, the second aspect, the blunting of the orexigenic effect of the 5HT an tagonist by vagotomy, was seen similarly after SCOP. Thus, another aspect of the increased intake of 1MB caused by ICS 205-930 may be due to vagai motor activity (Jiang 1992).

NEURAL RESPONSES TO AMINO ACID DEFICIENCY

621

In summary, the second, or learned, phase of the responses to IMB-induced amino acid deficiency likely has both central and peripheral components. Numerous studies, such as those using brain lesions and recording from neurons in various brain areas, have shown that CNS structures are involved in the development and maintenance of an aversion to 1MB. Undoubtedly, learning takes place in the CNS. Still, a role for the CNS in the 5HT3 mediation of the aversion to 1MB has been elusive in our hands, while vagotomy diminished the responses to the 5HT3 an tagonists. This points directly to a peripheral com ponent as well. Vagally mediated responses to gas trointestinal (Yox and Ritter 1988) and hepato-portal (Tanaka et al. 1990) amino acids have been demon strated, and ample evidence is available for vagai input to CNS structures involved in feeding (e.g., Bray 1985, Oomura 1988). It remains to be determined how, and with which structures, the vagus communi cates in its participation in the responses to 1MB.

120 100
80

60

01234567 DAYS

THE THIRD PHASE: ADAPTATION

If animals are not given an alternative diet, they will eat the 1MB for energy and adapt to near full food intake over ~7 d. This is not extinction of the learned aversion, as can be shown in choice studies,- even after full adaptation to 1MB, rats select a protein-free diet over an 1MB if given the choice (Rogers and Leung 1977). It should also be noted that animals do not adapt to a diet that is devoid of an essential amino acid (Harper et al. 1970). Meal patterns are modified during the adaptation period, with reductions in both meal size and frequency (Rogers and Leung 1973). However, as noted above, several brain areas were implicated in the adaptive phase, because animals bearing lesions in those areas showed facilitated adap tation to 1MB (Leung and Rogers 1987). The liver has been suggested as part of the "recog nition system" that is involved in the response to amino acids (Russek 1971). In collaboration with Bel linger and colleagues, total hepatic-denervated rats were used to determine 1} whether the innervation of the liver is important in the responses to amino acidimbalanced diets; and 2} whether the hepatic nerves carry information involved in the peripheral 5HT3 receptor mediation of the feeding responses (Bellinger et al. 1992). Early on the first day of 1MB con sumption, the intakes of all of the rats were sup pressed, compared with their ingestion of the basal diet, showing that liver innervation is not essential for 1MB recognition. Over the first day, vehicle-in jected animals had similar 1MB intake (sham operated animals: 6.36 0.76 g 1MB eaten/24 h, mean SE for 11 rats, compared with liver-denervated animals: 7.62 0.63 g, n = 8; no significant difference). After 12 h of feeding, ICS 205-930-injected rats in both the liver-

FIGURE 11 Effect of total liver denervation on intake of a severe isoleucine-imbalanced diet (1MB). Markers indicate means for 24 h imbalanced diet intake expressed as a per centage of each animal's baseline intake of the isoleucinebasal diet. "Denervate" animals were subjected to total liver denervation 2 wk prior to introduction of 1MB. Values are means SE for 9-11 rats per group. Solid line: denervated group,- broken line: sham operated group. 'Significantly increased 1MB intake by denervated animals on that day, P < 0.05. Data are adapted from Bellinger et al. (1992).

denervated and the sham-operated groups were eating significantly more than their counterparts that had been injected with vehicle. Thus, liver innervation was not essential for the improved feeding response after ICS 205-930. However, over the next 2 d, regardless of drug treatment, both denervated groups ate more 1MB and lost significantly less body weight than the sham-operated groups. In a longer trial over 7 d, both total liver-denervated and sham-operated animals initially decreased their intake of the 1MB and responded similarly to ICS 205-930, as before. Over the 7-d period, the denervated groups again showed a marked acceleration of their adaptation to the 1MB, reaching full adaptation by d 5, whereas controls did not achieve full adaptation until the 7th d (Bellinger et al. 1992; Fig. 11). These data suggest that the innervation of the liver may play some role in adaptation to the 1MB. Metabolic or hormonal changes after liver dener vation could be invoked to explain these results. Adrenalectomized animals were less able to tolerate the 1MB over time, suggesting that some adrenal factor aids the adaptation phase (Hammer et al. 1990b). Both hepatic nerves and hormonal systems have been shown to be directly involved in regulating liver metabolism (Jungermann et al. 1987). For ex ample, the vagai efferent system influences the ac tivity of tyrosine transaminase (Black and Reis 1971). Also, excess branched-chain amino acids can induce branched-chain keto acid dehydrogenase, the ratelimiting enzyme in branched-chain amino acid catabolism (Block et al. 1985), and serine-threonine dehydratase activity is increased severalfold after

622

GIETZEN

histidine 1MB feeding (Anderson et al. 1969), although not to the extent seen after feeding 50% protein. Still, it is doubtful that the development of an aversion to the 1MB involves the liver, because ICS 205-930 pre vents the development of an aversion to 1MB in intact rats, and the denervated and sham-operated animals responded similarly to ICS 205-930. Nonetheless, neural input from the liver could play some role in the learned responses to 1MB as a factor in the adaptive phase of the response. Certainly, the brain has influences on the liver; hypothalamic stimulation has strong influences on vagai nerve activity (Yoshimatsu et al. 1988) and on tryptophan pyrrolase activity (Shimazu 1964). An interesting hypothesis involving histidine has been advanced by Mercer (Mercer et al. 1990). In his scheme, 1MB or protein-deficient intake contributes to a decrease in the liver enzyme, histidine ammonia lyase, leading to increased systemic and brain concen trations of histidine. Subsequently, the increased histidine (or histamine, the transmitter for which histidine is the precursor) could affect brain areas and induce the anorectic response. However, the in creased histidine concentrations reported (Gifford et al. 1987) did not occur until after over 4 d, and we saw no changes in histidine concentration in PPC, AC (Gietzen et al. 1986b), VMH, LH or PVN (Gietzen et al. 1989) within the first few hours of ingestion of 1MB. Thus, histidine is unlikely to be the mediator of the neural signal that coincides with the initial reduction of 1MB intake within the first 12 h. Nonetheless, after 2 wk, the levels of histidine are remarkably well correlated with food intake in rats fed either low protein or 1MB (Mercer et al. 1989). Thus, the effects of histidine merit further investi gation, especially at later time points after in troduction of amino acid-deficient diets. In summary, several factors could be involved in the increased adaptation to 1MB, such as the fol lowing: 1} impoverished learning mechanisms fol lowing the lesions in areas such as the hippocampus or septum; 2) the beneficial effects of the altered meal patterns, resulting in a reduced influx of imbalanced amino acids in the plasma; 3) increased food intake driven by an increasing energy deficit; or 4} ongoing metabolic changes that may be occurring in the liver and conveyed to brain structures. The relative contri butions of these factors are still to be determined.

are unique in the rapidity with which they can be depleted (Harper 1964). How 1MB results in plasma patterns similar to those seen in amino acid defi ciency is indeed relevant to the natural situation,animals can encounter diets that are limiting in an essential amino acid, such as corn (which is limiting in tryptophan and lysine) or wheat (in which lysine is limiting). For example, wild primates would ex perience an 1MB situation if they were subsisting on high fat seeds (Oftedal 1991). If supplies have been scarce, the animals are likely to have been feeding previously on low protein items in the wild. This would provide a prefeeding situation similar to the paradigm used extensively in laboratory studies of 1MB. Parallels in human malnutrition are unfor tunately not scarce. Therefore, the adaptive advantage of a mechanism for avoiding a deficient or imbalanced food and selecting something containing a better nutritional balance is clear. In the efforts to understand the mechanisms that may regulate feeding responses to the amino acid deficiency caused by 1MB, our working hypotheses are that there are three coordinated phases involved, each mediated separately. First, the PPC may serve as the amino acid chemosensor in the early recognition of amino acid deficiency. Protein synthesis and other neurochemical changes in the PPC seem to be in volved, perhaps mediated by NE and cAMP. Second, there is good evidence for a serotonergic component (probably mediated by the 5HTs receptor) in the learned (aversive) phase of the response, which in volves vagally innervated structures but not the liver. Also clearly implicated in the learned responses to amino acid deficiency are the neurons in the LH that respond when an animal has learned to drink a so lution containing the dietary limiting amino acid. Third, in addition to the several brain areas that have been implicated, the liver seems to play a role in the adaptive phase. The evidence for these three phases has been reviewed, and the hypotheses are available for further testing and elaboration. Thus, there has been considerable progress in un derstanding the neural mechanisms of the responses to amino acid deficiency induced by 1MB since the review by Jackson et al. (1928). Still, considerable work will be necessary before we can be secure in the knowledge that we understand precisely the mechanisms that animals use to recognize and re spond to a deficiency of an indispensable amino acid.

CONCLUSIONS
ACKNOWLEDGMENTS
The use of 1MB provides a well-controlled, robust paradigm for the study of a rapidly induced nutri tional deficiency, which would be difficult to replicate with other nutrients, because amino acids The constructive comments of Q. R. Rogers and R. A. Freedland about an earlier version of the manu script are much appreciated.

NEURAL RESPONSES TO AMINO ACID DEFICIENCY

623

LITERATURE CITED
Anderson, H. L., Benevenga, N. J. & Harper, A. E. (1969) Effect of prior high protein intake on food intake, serine dehydratase activity and plasma amino acids of rats fed amino acidimbalanced diets. J. Nutr. 97: 463-474. Baker, B. J. & Booth, D. A. (1989) Genuinely olfactory preferences conditioned by protein repletion. Appetite 13: 223-227. Baker, B. ]., Booth, D. A., Duggan, J. P. & Gibson, E. L. (1987) Protein appetite demonstrated: learned specificity of protein-cue preference to protein need in adult rats. Nutr. Res. 7: 481-487. Bellinger, L. L., Gietzen, D. W. & Williams, F. E. (1993) Liver denervation, 5HT3 receptor antagonist and intake of imbalanced amino acid diet. Brain Res. Bull, (in press) Berg, C. P. & Rose, W. C. (1929) Tryptophane and growth. J. Biol. Chem. 82: 479-484. Beverly, f. L., Gietzen, D. W. & Rogers, Q. R. (1990a) Effect of dietary limiting amino acid in prepyriform cortex on food intake. Am. I. Physiol. 259: R709-R715. Beverly, J. L., Gietzen, D. W. & Rogers, Q. R. (1990b) Effect of dietary limiting amino acid in prepyriform cortex on meal pat terns. Am. J. Physiol. 259: R716-R723. Beverly, J. L., Gietzen, D. W. & Rogers, Q. R. (1991a| Protein synthesis in the prepyriform cortex: effects on intake of an amino acid-imbalanced diet by Sprague-Dawley rats. J. Nutr. 121: 754-761. Beverly, ]. 1., Gietzen, D. W. & Rogers, Q. R. |1991b) Threonine concentration in the prepyriform cortex has separate effects on dietary selection and intake of a threonine-imbalanced diet by rats. J. Nutr. 121: 1287-1292. Beverly, J. L., Hrupka, B. J., Gietzen, D. W. & Rogers, Q. R. (1991c| Distribution of dietary limiting amino acid injected into the prepyriform cortex. Am. J. Physiol. 260: R525-R532. Beverly, J. L., Hrupka, B. J., Gietzen, D. W. & Rogers, Q. R. (1993) Timing and dose of amino acids injected into prepyriform cortex influence food intake. Physiol. & Behav. (in press) Black, I. B. & Reis, D. J. (1971) Cholinergic regulation of hepatic tyrosine transaminase activity. J. Physiol. (Camb.) 213: 421-433. Block, K. P., Soemitro, S., Heywood, B. W. &. Harper, A. E. (198S) Activation of liver branched-chain a-keto acid dehydrogenase in rats by excesses of dietary amino acids. J. Nutr. 115: 1550-1561. Blundcll, J. E. (1984) Serotonin and appetite. Neuropharmacology 23: 1537-1551. Booth, D. A. & Simson, P. C. (1971) Food preferences acquired by association with variations in amino acid nutrition. Q. J. Exp. Psychol. 23: 135-145. Bray, G. A. (1985) Autonomie and endocrine factors in the regu lation of food intake. Brain Res. Bull. 14: 505-510. Brown, J. H. (1990) Atropine, scopolamine, and related antimuscarinic drugs. In: Goodman and Gilman's The Pharmacological Basis of Therapeutics (Gilman, A. G., Rail, T. W., Nies, A. S. & Taylor, P., eds.), pp. 150-165. Pergamon Press, New York, NY. Chambers, K. C. (1990) A neural model for conditioned taste aver sions. Annu. Rev. Neurosci. 13: 373-385. Costali, B., Naylor, R. J. & Tyers, M. B. (1988) Recent advances in the neuropharmacology of S-HTj agonists and antagonists. Rev. Neurosci. 2: 41-65. Deutsch, f. A., Moore, B. O. & Heinrichs, S. C. (1989) Unlearned specific appetite for protein. Physiol. & Behav. 46: 619-624. Elvehjem, C. A. & Krehl, W. A. (1955) Dietary interrelationships and imbalance in nutrition. In: Borden's Review of Nutrition Research, pp. 69-84. Borden, New York, NY. Firman, ]. D. & Kuenzel, W. J. (1988) Neuroanatomical regions of the chick brain involved in monitoring amino acid-deficient diets. Brain Res. Bull. 21: 637-642. Fletcher, P. J. & Burton, M. J. (1984) Effects of manipulations of peripheral serotonin on feeding and drinking in the rat. Pharmacol. Biochem. Behav. 20: 835-840. Garcia, J., Kimeldorf, D. J. & Koelling, R. A. (1955) Conditioned

aversion to saccharin resulting from exposure to gamma radi ation. Science (Washington, DC) 122: 157-158. Gibson, E. L., Barber, D. f. & Booth, D. A. (1987) 5HT and CA precursor levels during protein selection. Soc. Neurosci. Abstr. 13: 15 (abs.). Gibson, E. L. &. Booth, D. A. (1986) Acquired protein appetite in rats: dependence on a protein-specific need state. Experentia (Basel) 42: 1003-1004. Gietzen, D. W. & Beverly, J. L. (1992) Clonidine in the prepyriform cortex blocked anorectic response to amino acid imbalance. Am. I. Physiol. 263: R885-R890. Gietzen, D. W., Duke, C. M. &. Hammer, V. A. (1991a) Amino acid imbalance, an nutritional model: serotonin^ mediation of aversive responses. Physiol. & Behav. 49: 981-985. Gietzen, D. W., Hammer, V. A., Beverly, ]. L. & Rogers, Q. R. (1991b) The role of serotonin (5-HT) in feeding responses to amino acids. In: Kynurenine and Serotonin Pathways (Schwartz, R., Young, S. N. & Brown, R. R., eds.), pp. 389-404. Plenum Press, New York, NY. Gietzen, D. W., Leung, P.M.B., Castonguay, T. W., Hartman, W. J. & Rogers, Q. R. (1986a| Time course of food intake and plasma and brain amino acid concentrations in rats fed amino acidimbalanced or deficient diets. In: Interaction of the Chemical Senses with Nutrition (Karc, M. R. & Brand, J., eds.), pp. 415-456. Academic Press, New York, NY. Gietzen, D. W., Leung, P.M.B. & Rogers, Q. R. (1986b) Norepinephrine and amino acids in prepyriform cortex of rats fed imbalanced amino acid diets. Physiol. & Behav. 36: 1071-1080. Gietzen, D. W., Leung, P.M.B. & Rogers, Q. R. (1989) Dietary amino acid imbalance and neurochcmical changes in three hypothalamic areas. Physiol. & Behav. 46: 503-511. Gietzen, D. W., McArthur, L. H., Theisen, J. C. & Rogers, Q. R. (1992) Learned preference for the limiting amino acid in rats fed a threonine-deficient diet. Physiol. &. Behav. 51: 909-914. Gietzen, D. W., Rogers, Q. R. & Leung, P.M.B. (1988a) Dispropor tionate amino acid diets and anorexic responses in rats: the role(s) of limbic brain areas and noradrenergic and serotoninergic systems. In: Amino Acid Availability and Brain Function in Health and Disease (Heuther, G., ed.). NATO ASI Series, vol. H20, pp. 147-157. Springer-Verlag, Berlin, Germany. Gietzen, D. W., Leung, P.M.B. &. Rogers, Q. R. (1988b) Regional differences in the concentration of the limiting amino acid, threoninc (THR) after ingesting a THR imbalanced diet. FASEB (. 2: Al 195 (abs). Gietzen, D. W., Rogers, Q. R., Leung, P.M.B., Semon, B. & Piechota, T. (1987) Serotonin and feeding responses of rats to amino acid imbalance: initial phase. Am. J. Physiol. 253: R763-R771. Gifford, C. D., Dodds, S. J., Johnson, L. K., Smith, D. L. .Mercer, L. P. (1987) Metabolic adaptation to protein deficiency in rats: histidine. Nutr. Res. 7: 617-627. Grossman, S. P. (1960) Eating or drinking elicited by direct adrenergic or cholinergic stimulation of hypothalamus. Science (Washington, DC) 132: 301-302. Halstead, W. C. & Gallagher, B. B. (1962) Autoregulation of amino acids intake in the albino rat. J. Comp. Physiol. Psychol. 55: 107-111. Hammer, V. A., Gietzen, D. W., Beverly, J. L. & Rogers, Q. R. (1990a) Serotoninj receptor antagonists block anorectic responses to amino acid imbalance. Am. J. Physiol. 259: R627-R636. Hammer, V. A., Gietzen, D. W., Sworts, V. D., Beverly, J. L. & Rogers, Q. R. (1990b| Adrenal hormones and the anorectic re sponse and adaptation of rats to amino acid imbalance. J. Nutr. 120: 1617-1623. Harper, A. E. (1964) Amino acid toxicities and imbalances. In: Mammalian Protein Metabolism (Munro, H. N. & Allison, J. B., eds.), pp. 81-134. Academic Press, New York, NY. Harper, A. E. (1976) Protein and amino acids in the regulation of food intake. In: Hunger: Basic Mechanisms and Clinical Impli-

624

GIETZEN
amino acid imbalance on dietary choice in the rat. J. Nutr. 95: 483-492. Leung, P.M.B., Rogers, Q. R. & Harper, A. E. (1968b) Effect of amino acid imbalance on plasma and tissue free amino acids in the rat. J. Nutr. 96: 303-318. Lewin, B. (1990) Genes IV. Oxford University Press, New York, NY. Magendie, M. F. (1816) Sur les proprits nutritives des substances qui ne contiennent pas d'azote. Anal. Chim. Physiq. 3: 66-77. McCoy, R. H., Meyer, C. E. & Rose, W. C. (1935) Feeding experi ments with mixtures of highly purified amino acids. VIII. Iso lation and identification of a new essential amino acid. J. Biol. Chem. 112: 283-302. Meliza, L. L., Leung, P.M.B. & Rogers, Q. R. (1981) 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. Mercer, L. P., Dodds, S. f., Schweisthal, M. R. & Dunn, J. D. (1989) Brain histidine and food intake in rats fed diets deficient in single amino acids. J. Nutr. 119: 66-74. Mercer, L. P., Dodds, S. J., Weber, M. D. & Dunn, J. D. (1990) Histidine, histamine, and the neuroregulation of food intake: a review and hypothesis. Nutrition 6: 273-277. Millward, D. J. & Rivers, J.P.W. (1988) The nutritional role of indispensable amino acids and the metabolic basis for their requirements. Eur. J. Clin. Nutr. 42: 367-393. Mori, M., Kawada, T., Ono, T. & Torii, K. (1991) Taste preference and protein nutrition and L-amino acid homeostasis in male Sprague-Dawley rats. Physiol. & Behav. 49: 987-995. Munro, H. N. (1976) Eukaryote protein synthesis and its control. In: Protein Metabolism and Nutrition (Cole, D.J.A., Boorman, K. N., Buttery, P. f., Lewis, D., Neale, R. J. & Swan, H., eds.), pp. 3-18. Butterworths, London, U.K. Murphy, M. E. & King, J. R. (1989) Sparrows discriminate between diets differing in valine or lysine concentrations. Physiol. & Behav. 45: 423-430. Noda, K. (1975) Possible effect of blood ammonia on food intake of rats fed amino acid imbalanced diets. J. Nutr. 105: 508-516. Noda, K. & Chikamori, K. (1976) Effect of ammonia via prepyriform cortex on regulation of food intake in the rat. Am. J. Physiol. 231: 1263-1266. Oftedal, O. T. (1991) The nutritional consequences of foraging in primates: the relationship of nutrient intakes to nutrient re quirements. Philos. Trans. R. Soc. Lond. B. 334: 161-170. Oomura, Y. (1988) Chemical and neuronal control of feeding moti vation. Physiol. & Behav. 44: 555-560. Osborne, T. B. &. Mendel, L. B. (1914) Amino acids in nutrition and growth. J. Biol. Chem. 17: 325-349. Panksepp, J. & Booth, D. A. (1971) Decreased feeding after injec tions of amino-acids into the hypothalamus. Nature (Lond.) 233: 341-342. Peng, Y. & Harper, A. E. (1969) Amino acid balance and food intake: effect of amino acid infusions on plasma amino acids. Am. J. Physiol. 217: 1441-1445. Peng, Y., Tews, J. K. & Harper, A. E. (1972) Amino acid imbalance, protein intake, and changes in rat brain and plasma amino acids. Am. J. Physiol. 222: 314-321. Peroutka, S. J. (1988) 5-Hydroxytryptamine receptor subtypes: molecular, biochemical and physiological characterization. Trends Neurosci. 11: 496-500. Rogers, Q. R. & Harper, A. E. (1970) Selection of a solution con taining histidine by rats fed a histidine-imbalanced diet. J. Comp. Physiol. Psychol. 72: 66-71. Rogers, Q. R. & Leung, P.M.B. (1973) The influence of amino acids on the neuroregulation of food intake. Fed. Proc. 32: 1709-1719. Rogers, Q. R. & Leung, P.M.B. (1977) The control of food intake: when and how are amino acids involved? In: The Chemical Senses and Nutrition (Kare, M. R. & Maller, O., eds.), pp. 213-249. Academic Press, New York, NY. Rose, W. C. (1931) Feeding experiments with mixtures of highly purified amino acids. 1. The inadequacy of diets containing

cations (Novin, D., Wyrwicka, W. & Bray, G., eds.|, pp. 103-113. Raven Press, New York, NY. Harper, A. E., Benevenga, N. J. &. Wohlhueter, R. M. |1970| Effects of ingestion of disproportionate amounts of amino acids. Physiol. Rev. 50: 428-558. Harper, A. E. & Rogers, Q. R. (1965) Amino acid imbalance. Sym posium proceedings, Institute of Biochemistry, Univ. Glasgow 24: 173-190. Harper, A. E. & Tews, J. K. |1988| Nutritional and metabolic control of brain amino acid concentrations. In: Amino Acid Availability and Brain Function in Health and Disease (Heuther, G., d.), pp. 3-12. NATO ASI Series, vol. H20, Springer-Verlag, Berlin, Germany. Heinrichs, S. C., Deutsch, J. A. & Moore, B. O. (1990) Olfactory self-selection of protein-containing foods. Physiol. & Behav. 47: 409-413. Hoebel, B. G. (1984) Neurotransmitters in the control of feeding and its rewards: monoamines, opiates and brain-gut peptides. In: Eating and Its Disorders (Stunkard, A. J. & Stellar, E., eds.), pp. 15-38. Raven Press, New York, NY. Hrupka, B. J., Gietzen, D. W. &. Beverly, J. L. (1991) ICS 205-930 and feeding responses to amino acid imbalance: a peripheral effect? Pharmacol. Biochem. Behav. 40: 83-87. Ip, C.C.Y. & Harper, A. E. 1974)Liver polysome profiles and protein synthesis in rats fed a threonine-imbalanced diet. f. Nutr. 104: 252-263. Jackson, R. W., Sommer, B. E. &. Rose, W. C. (1928) Experiments on the nutritive properties of gelatin. J. Biol. Chem. 80: 167-186. Jiang, J. C. (1992) The Serotoni^ Receptor and Imbalanced Diet Intake: Specificity and Locus of Action. Master's thesis, University of California-Davis, Davis, CA. Jungermann, K., Gardemann, A., Beuers, U., Balle, C., Sannemann, I., Beckh, K. & Hartmann, H. (1987) Regulation of liver metab olism by the hepatic nerves. In: Advances in Enzyme Regu lation, vol. 26 (Weber, G., d.), pp. 63-88. Pergamon Press, New York, NY. Leung, P.M.B., Gietzen, D. W. &.Rogers, Q. R. (1986a).Effect of area postrema lesions on dietary choice of rats fed disproportionate amounts of dietary amino acids. Soc. Neurosci. Abstr. 12: 1556 labs.). Leung, P.M.B., Gietzen, D. W. & Rogers, Q. R. (1987a) Effect of nucleus of solitary tract (SOL) lesions on food intake responses of rats fed amino acid imbalanced or deficient diet. Fed. Proc. 46: 1484 (abs.|. Leung, P.M.B., Gietzen, D. W. & Rogers, Q. R. (1987b) Effect of nucleus of solitary tract lesions on food intake responses and dietary choices of rats fed diets containing protein in excess. Soc. Neurosci. Abstr. 13: 332 (abs.). Leung, P.M.B., Larson, D. M. &. Rogers, Q. R. (1972) Food intake and preference of olfactory bulbectomized rats fed amino acid imbalanced or deficient diets. Phyisol. & Behav. 9: 553-557. Leung, P.M.B., Larson, D. M. & Rogers, Q. R. (1986b) Influence of taste on dietary choice of rats fed amino acid imbalanced or deficient diets. Physiol. & Behav. 38: 255-264. Leung, P.M.B. & Rogers, Q. R. (1969) Food intake: regulation by plasma amino acid pattern. Life Sci. 8: 1-9. Leung, P.M.B. & Rogers, Q. R. (1970) Effect of amino acid im balance and deficiency on food intake in rats with hypothalamic lesions. Nutr. Rep. Int. 1: 1-10. Leung, P.M.B. & Rogers, Q. R. (1971) Importance of prepyriform cortex in food-intake response of rats to amino acids. Am. J. Physiol. 221: 929-935. Leung, P.M.B. & Rogers, Q. R. (1973) Effect of amygdaloid lesions on dietary intake of disproportionate amounts of amino acids. Physiol. & Behav. 11: 221-226. Leung, P.M.B. & Rogers, Q. R. (1987) The effect of amino acids and protein on dietary choice. In: Umami: A Basic Taste (Kawamura, Y. &. Kare, M. R., eds.), pp. 565-610. Marcel Dekker, New York, NY. Leung, P.M.B. & Rogers, Q. R. & Harper, A. E. (1968a) Effect of

NEURAL RESPONSES TO AMINO ACID DEFICIENCY nineteen amino acids. J. Biol. Chem. 94: 155-165.
Russek, M. (1971) Hepatic receptors and the neurophysiological mechanisms controlling feeding behavior. In: Neurosciences Re search (Ehrenpries, S. & Solnitzky, O. C., eds.), pp. 213-282. Academic Press, New York, NY. Salmon, W. D. (1958) The significance of amino acid imbalance in nutrition. Am. J. Clin. Nutr. 6: 487-494. Scatton, B. (1990) Operation of presynaptic -adrenoceptors in vivo in the rat brain. Ann. N. Y. Acad. Sci. 604: 514-527. Scharrer, E., Baile, C. A. & Mayer, J. (1970) Effect of amino acids and protein on food intake of hyperphagic and recovered aphagic rats. Am. J. Physiol. 218: 400-404. Sharrna, K. N. & Nasset, E. S. (1962) Electrical activity in mesenteric nerves after perfusion of gut lumen. Am. J. Physiol. 202: 725-730. Shimazu, T. (1964) Role of the hypothalamus in the induction of tryptophan pyrrolase activity in rabbit liver. J. Biochem. (Tokyo) 55: 163-171. Starke, K., Gthert, M. Kilbinger, H. (1989) Modulation of neurotransmitter release by presynaptic autoreceptors. Physiol. Rev. 69: 864-989. Stellar, E. (1954) The physiology of motivation. Psychol. Rev. 61: 5-22. Stickney, G. G., Leung, P.M.B., Rogers, Q. R., Lepkovsky, S. & Schmidt, P. (1976) The effect of total gastrectomy on free feeding patterns in rats. Fed. Proc. Al73 (abs.). Straus, D. S. & Takemoto, C. D. (1988) Amino acid limitation negatively regulates insulin-like growth factor-II mRNA levels and E-domain peptide secretion at a post-transcriptional step in BRL-3A rat liver cells. J. Biol. Chem. 263: 18404-18410. Tabuchi, E., Ono, T., Nishijo, H. & Torii, K. (1991) Amino acid and NaCl appetite, and LHA neuron responses of lysine-dficient rat. Physiol. & Behav. 49: 951-964. Tackman, J. M., Tews, J. K. & Harper, A. E. (1990) Dietary dis proportions of amino acids in the rat: effects on food intake, plasma and brain amino acids and brain serotonin. J. Nutr. 120: 521-533. Tanaka, K., Inoue, S., Nagase, H., Takamura, Y. & Niijima, A. (1990) Amino acid sensors sensitive to alanine and leucine exist in the hepato-portal system in the rat. J. Auto. Nerv. Syst. 31; 41-46. Terry, V. R., Gietzen, D. W. & Rogers, Q. R. (1991) Serotonin-3 receptor and conditioned taste aversion (CTA) to saccharin paired with an amino acid imbalanced diet. Int. J. Obesity 15 (suppl. 3): 29 (abs.). Tews, I. K., Bradford, A. M. &. Harper, A. E. (1981) Induction of

625

lysine imbalance in rats: relation to competition for lysine transport into the brain in vitro. J. Nutr. Ill: 954967. Tews, J. K., Good, S. S. & Harper, A. E. (1978) Transport of threonine and tryptophan by rat brain slices: relation to other amino acids at concentrations found in plasma. J. Neurochem. 31: 581-589. Tews, J. K., Kim, Y-W.L. & Harper, A. E. (1979) Induction of threonine imbalance by dispensable amino acids: relation to competition for amino acid transport into brain. J. Nutr. 109: 304-315. Tews, J. K., Repa, J. ]. & Harper, A. E. (1988) Induction of condi tioned taste aversion in rats by GABA or other amino acids. Physiol. & Behav. 42: 591-597. Tobin, G. & Boorman, K. N. (1979) Carotid or jugular amino acid infusions and food intake in the cockerel. Br. J. Nutr. 41: 157-162. Torii, K., Mimura, T. & Yugari, Y. (1987) Biochemical mechanism of umami taste perception and effect of dietary protein on the taste preference for amino acids and sodium chloride in rats. In: Umami: A Basic Taste (Kawamura, Y. & Kare, M. R., eds.), pp. 513-564. Marcel Dekker, New York, NY. Treneer, C. M. &. Bernstein, I. L. (1981) Learned aversions in rats fed a tryptophan-free diet. Physiol. & Behav. 27: 757-760. Tyers, M. B. (1990) 5-HT3 receptors. Ann. N.Y. Acad. Sci. 600: 194-205. Ungerstedt, U. (1971) Stereotaxic mapping of the monoamine pathways in the rat brain. Acta Physiol. Scand. Suppl. 367: 1-48. Washburn, B. S., Cummings, S. L., Theisen-Jiang, J. C., Bellinger, L. L., Williams, F. E. & Gietzen, D. W. (1992) The effect of subdiaphragmatic vagotomy on intake of an amino acid imbalanced diet. First Independ. Cong. Soc. Study Ingest. Behav., Princeton, NJ (abs.). Wayner, M. J., Ono, T., De Young, A. & Barone, F. C. (1975) Effects of essential amino acids on central neurons. Pharmacol. Bi ochem. Behav. 3 (suppl. 1): 85-90. Willcock, E. G. & Hopkins, F. G. (1906) The importance of in dividual amino-acids in metabolism; observations on the effect of adding tryptophane to a dietary in which zein is the sole nitrogenous constituent. J. Physiol. (Lond.) 35: 88-102. Yoshimatsu, H. A., Niijima, A., Oomura, Y. & Katafuchi, T. (1988) Lateral and ventromedial hypothalamic influences on hepatic autonomie nerve activity in the rat. Brain Res. Bull. 21: 239-244. Yox, D. P. & Ritter, R. C. (1988) Capsaicin attenuates suppression of sham feeding induced by intestinal nutrients. Am. J. Physiol. 255: R569-R574.