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Nutritional Neurosciences

Diets Deficient in Indispensable Amino Acids Rapidly Decrease the Concentration of the Limiting Amino Acid in the Anterior Piriform Cortex of Rats¹

Thomas J. Koehnle^*,,2, Matthew C. Russell^*, Andrew S. Morin^*, Lesa F. Erecius^* and Dorothy W. Gietzen^*,

^* School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology and Animal Behavior Graduate Group, University of California-Davis, Davis, CA 95616

²To whom correspondence should be addressed. E-mail: koehnle{at}bns.pitt.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Diets deficient in an indispensable amino acid have long been known to suppress food intake in rats. Detection of dietary deficiency takes place in the anterior piriform cortex (APC). Recent studies showed that the response to amino acid deficiency takes as little as 15 min to develop, but few data exist to correlate the concentration of amino acids in the APC with this rapid response. The purpose of this study was to measure the concentration of amino acids in the APC in a behaviorally relevant time frame. Rats were preconditioned by consumption of a basal diet for 7–10 d, and then given a test diet with either a control or deficient amino acid profile. Both the threonine- and leucine-deficient diets reliably depleted threonine and leucine concentration in the APC within 30 min, respectively. The control diets and a diet lacking the dispensable amino acid glycine did not lead to amino acid depletion. In combination with previous studies, the present results show that the decrease in the concentration of indispensable amino acids in the APC may be the initial sensory signal for recognition of dietary amino acid deficiency.

KEY WORDS: • amino acid imbalance • anterior piriform cortex • threonine • leucine

Omnivores, including humans, must obtain sufficient quantities of indispensable amino acids from their diet to maintain growth and protein synthesis. Indispensable amino acids are typically maintained within a narrow physiologic range in the brain (1). However, after the ingestion of diets with disproportionate amino acid profiles, the limiting indispensable amino acid is acutely decreased in plasma (2) and suffers a competitive disadvantage for transport into the brain (3–5). This is accompanied by an acute decrease of the limiting amino acid in the brain (6,7). In particular, diets limiting in the indispensable amino acid threonine significantly decrease threonine concentration in the anterior cingulate cortex, the locus coeruleus, the nucleus of the solitary tract, and the anterior piriform cortex (APC)³ [reviewed in (8)].

In addition to their effects on amino acid concentration in the brain, diets with disproportionate amino acid profiles dramatically reduce food intake in rats over a period of hours [reviewed in (9–11)]. Lesions of the APC abolish this anorectic response (12,13). In addition, injection of the limiting indispensable amino acid into the APC restores food intake (14–16). These observations led to the conclusion that the APC is the site responsible for detection of amino acid deficiency in mammals.

Despite the richness of the literature on the correspondence between amino acid concentrations and food intake behavior from 1 to 3 h after ingestion of disproportionate diets, recent studies showed that the behavioral response to amino acid deficiency is far more rapid than previously thought, requiring a mean of only 15 min from first exposure to the test diet (17,18). Rats consistently reduce both first meal food intake and first meal duration in response to deficient diets. Though there was some earlier indication that the response to amino acid repletion might take as little as 30 min (19,20), only one study examined food intake responses to amino acid deficiency in the same short time frame (21). Moreover, there are essentially no data relating the rapid behavioral response to the level of the limiting amino acid in the brain.

To date, we have only partially closed the gap between our knowledge of the role of the concentration of the limiting indispensable amino acid in the APC and the rapid behavioral response. Neurons cultured from the APC are rapidly depleted of intracellular threonine upon threonine deprivation in vitro, a change correlated with increases in the intracellular concentrations of serine, glycine, valine, isoleucine, and phenylalanine (22). We demonstrated recently that injection of threonine into the APC of rats fed a threonine-imbalanced diet increased first meal food intake and first meal duration relative to saline-injected rats (14). The studies described below begin to fill in the rest of this gap, and raise new and interesting questions about the mechanism of amino acid detection in the APC.

The purpose of these studies was 4-fold: 1) to determine whether threonine concentration in the APC after ingestion of a threonine-imbalanced diet is decreased at earlier times than previously thought; 2) to extend previous findings to the new dietary paradigms used in the study of the effects of disproportionate diets (18); 3) to generalize the effects on threonine to other indispensable amino acids such as leucine; and 4) to determine whether decreases in threonine in the APC result in increases in the concentration of other amino acids, as was documented for APC neurons in vitro (22).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    General. The experiments described below were approved by the UC Davis Animal Care and Use Committee. All rats were housed and handled following NIH guidelines for animal care and use. Male albino rats were purchased from commercial vendors (Simonsen and Harlan) and housed individually in hanging wire cages in a vivarium with a 12-h light:dark cycle, maintained at 22°C.

All diets used in these experiments were prepared in our laboratory from purified ingredients and free L-amino acids, and were described previously (18). Briefly, after arrival in our laboratory, all rats were given a basal diet for 7–10 d. The basal diet has a full complement of all indispensable amino acids at 50% of the requirement for maximal growth, except for the most limiting amino acid [for exact values see (18)]. The threonine basal diet contained 40% of the requirement for threonine, whereas the leucine basal diet contained 40% of the requirement for leucine. After the basal feeding period, all rats were fed novel test diets. The corrected diet contained all indispensable amino acids increased to 100% of the required level [for exact values see (18)]. The threonine-imbalanced diet contained all amino acids except threonine raised to 100% of the required level; threonine was kept at 40%. The threonine-devoid diet contained all amino acids raised to 100% of requirement, except threonine, which was omitted. The leucine-devoid diet contained all amino acids raised to 100% of requirement, except leucine, which was omitted. The basal-threonine diet was similar to the threonine basal diet, except that it was totally devoid of threonine. The basal-leucine diet was similar to the threonine basal diet, except that it was totally devoid of leucine. Finally, the basal-glycine diet was similar to the threonine basal diet, except that it was totally devoid of the dispensable amino acid glycine.

Over the basal feeding period, the rats had free access to their food 24 h/d, except when cage maintenance was conducted. Cage maintenance was done during the light cycle to avoid interference with the experiments. On the last day of the basal feeding period, food was removed from each rat for a specified period before the experiments began. Rats in all experiments were assigned to their respective groups so that each group had a similar mean body weight.

On the experimental day, each rat was offered its test diet for a specified period of time (or food deprived, see below) on a rotating schedule to permit efficient harvesting of tissue samples at 3-min intervals. At the designated time, each rat was removed from its cage and taken to the surgery room, where it was killed by decapitation. The APC [from 9.60 to 9.80 ± 1.00 mm rostral to the interaural line (7)] was quickly dissected on a plate chilled with crushed dry ice, and the tissue was placed in a clean, dry microcentrifuge tube. Tubes were weighed before and after the addition of the APC sample to determine the wet weight of the sample. The tubes were stored at –80°C for later processing. For amino acid analysis, each tube was first thawed in a water bath at 4°C. The tissue was then homogenized and analyzed for amino acid content as described below.

    Expt. 1. Time course of threonine in the APC. To determine whether the concentration of threonine was decreased at times earlier than the traditionally assayed 2.5 h [reviewed in (8)] 64 rats (weight, 150–180 g) were purchased from Simonsen. They were given a stock diet (Purina #5001) for 3 d after delivery to allow adaptation to the vivarium before being fed the threonine basal diet.

After 10 d of consuming the threonine basal diet, the rats were divided into 8 groups of 8 individuals balanced for mean body weight. All groups were deprived of food for 3 h before the start of the experiment. One group was killed at the start of the dark cycle without having eaten anything (unfed group), whereas the remaining groups were given the threonine-imbalanced diet and killed at 30, 60, 90, 120, 150, 180, and 210 min on a rotating, randomized schedule. For amino acid analysis, each tube was first thawed in a water bath at 4°C. The tissue was then homogenized by sonication in 200 µL aqueous sulfosalicylic acid (3 g/100 g deionized water). Each sample was then centrifuged at 14,000 x g for 30 min at 4°C. The supernatant was analyzed for amino acid content. Amino acid analysis was done using an automated system (Beckman AA analyzer #7300, Beckman) as previously described (7). Food intake was recorded at 30-min intervals on a digital balance, with spillage captured and subtracted at the same point. Rats consuming <0.06 g of the threonine-imbalanced test diet during any interval were excluded from the data set before analysis because ingestion of the diet was necessary to generate metabolic consequences.

    Expt. 2. Rapid depletion of APC amino acids by devoid diets. Rats (n = 52; weight, 100–110 g) were purchased from Harlan and immediately fed a basal diet. Three groups received the threonine basal diet and one received the leucine basal diet. All rats were fed this diet for 7 d. On the last basal day, access to the diet was limited to 3 h. All rats were then deprived of food overnight (21 h) until the start of the experiment, to ensure prompt eating and large meals. This step eliminated the need to remove rats from the analysis on the basis of inadequate food intake.

For this experiment, the rats were divided into 4 groups (n = 13/group). One group received no food (unfed), one group received the threonine-corrected control diet, one group received the threonine-devoid diet, which is similar to the threonine-imbalanced diet except that it was completely devoid of threonine, and the final group, which had been fed the leucine basal diet, received the leucine-devoid diet. On the basis of our recent findings (14,17,18), we assayed APC amino acids at only one time point. By 21 min after the first exposure to a diet with a disproportionate amino acid profile, 85% of the rats eating the deficient diet had ended their first meal, but only 50% of control rats had (Fig. 1). Therefore, all rats except the unfed group were killed after 21 min of exposure to the test diet on a rotating, randomized schedule at 3-min intervals. Food intake was recorded on a digital balance, and spillage was captured and subtracted after the conclusion of the experiment. The unfed group was killed beginning at 21 min after the start of the dark cycle.

View larger version (17K): [in this window] [in a new window]   FIGURE 1 Rats reliably reduce the duration of their first meal when encountering an amino acid–deficient diet. Data shown are adapted from previous studies in our laboratory (14,17,18). Control diet: filled circles, n = 52; deficient diet, open circles, n = 53. In Experiments 2 and 3 of the present study, APC samples were taken 21 min after exposure to various test diets (vertical bar). At that time point, 85% of rats consuming deficient diets had concluded their first meal, and only 50% of rats eating control diets had done so. The 2 curves begin to diverge after only 6 min. Rats consuming control and deficient diets took 24.02 ± 1.53 and 14.35 ± 0.98 min to complete their first meals, respectively.

    Expt. 3. Depletion of APC amino acids using modified basal diets. The traditional method of using a basal diet and then an imbalanced or devoid test diet (9,10) confounds the effects of dietary novelty with amino acid imbalance because of the greatly increased levels of indispensable amino acids added, and decreased carbohydrates, in those diet recipes (18). Although the threonine-corrected diet controls for this novelty, it activates many of the same brain areas as the threonine-imbalanced diet (25–27). Therefore, we began to use modified basal diets as the appropriate test diets in our studies of the mechanism of amino acid detection in the APC (18).

Rats (n = 65; weight, 100–110 g) were purchased from Harlan and immediately fed a basal diet. Three groups of 13 rats were fed the threonine basal diet and 2 groups of 13 the leucine basal diet. All rats were fed these diets for 7 d. On the last basal day, access to the diet was limited to 3 h. All rats were then deprived of food overnight (22 h) until the start of the experiment. Food intake was recorded on a digital balance, and spillage was captured and subtracted after the conclusion of the experiment.

On the experimental day, 1 group received the threonine basal diet, 1 group received a modified threonine basal diet, completely devoid of threonine (basal-threonine group). In addition, 1 group received the leucine basal diet, and 1 received a modified leucine basal diet, completely devoid of leucine (basal-leucine group). Finally, 1 group received a modified threonine basal diet, completely devoid of the dispensable amino acid glycine (basal-glycine group). All rats were killed after 21 min of exposure to the test diet on a rotating, randomized schedule at 3-min intervals. APC samples were prepared for amino acid analysis using the AccQ Tag method outlined above.

    Statistical analyses. Data are means ± SEM except as noted. The amino acid values from the APC are expressed as nmol/mg wet tissue. Because tests of statistical significance do not indicate the direction or magnitude of effects (28,29) and allow the Type 2 error rate to vary freely (29), effect sizes are reported in addition to P-values. Effect sizes were calculated according to the recommendations of Cohen (30) and are expressed in terms of Cohen’s d, where d is the difference between groups as a multiple of pooled SD. By convention, 0.5 < d < 0.8 are considered moderate effects, and d > 0.8 are considered large. In the results presented below, effects in which d < 0.5 are not reported. For the planned comparisons examining effects on leucine and threonine reported below, was set to 0.05.

In Expt. 1, data were analyzed using a one-factor (time) ANOVA. All effects are given relative to the unfed group. Planned comparisons were made for threonine at each time point, with the hypothesis that the threonine concentration would be lower in fed than in unfed rats at each time. Post-hoc tests, where conducted, were made using Fisher’s least significant difference (LSD) test.

In Expt. 2, data were analyzed using a one-factor (diet) ANOVA. All effects are given relative to both the unfed and threonine-corrected group. Planned comparisons were made between the unfed, threonine-devoid, and threonine-corrected groups for threonine concentration, and the leucine-devoid and unfed group for leucine concentration. We expected threonine concentration to be decreased relative to unfed and threonine corrected rats in the threonine devoid group. Post-hoc tests, where conducted, were made using Fisher’s LSD test.

In Expt. 3, data were analyzed using a one-factor (diet) ANOVA. All effects are given relative to the basal-glycine group and the appropriate basal control group. Planned comparisons were made between the threonine concentration in the basal-threonine group and the threonine basal and basal-glycine group, and also between the leucine concentrations in the basal-leucine, leucine basal, and basal-glycine groups. We hypothesized that threonine would be decreased in the basal-threonine group relative to the basal-glycine and threonine basal groups, and that leucine would be decreased in the basal-leucine group relative to the basal-glycine and leucine basal groups. We did not expect any changes in glycine concentration in the basal-glycine group relative to any other group in this experiment. Post-hoc tests, where conducted, were made using Fisher’s LSD test.

For post-hoc testing, results were considered significant only when the omnibus ANOVA for an individual amino acid yielded P < 0.01. This approach minimizes the probability of Type 1 error to an overall rate of 5% within each experiment. In Experiments 2 and 3, the concentrations of the amino acids leucine, isoleucine, methionine, proline, and phenylalanine were very low (below AccQ Tag detection limits) in some APC samples. Hence, these amino acids had a more variable sample size (see Results).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Expt. 1. Over the course of the experiment, 4 rats were excluded for failure to eat the requisite 0.06 g during the relevant 30-min interval. Rats remaining in the experiment ate 1.02 ± 0.10 g during each 30-min interval (range: 0.14–3.74). Additionally, 3 samples were accidentally destroyed during dissection, and a further 10 samples had read errors on the Beckman analyzer. Of the 64 original rats used in this study, 47 were retained for the final analysis, with no fewer than 5 individuals in each group (Table 1). For Experiments 2 and 3, we adopted procedural changes to avoid this attrition.

The concentrations of glycine, valine, isoleucine, and phenylalanine did not differ between the rats that were not fed and fed rats at the 30-min time point (omnibus P-values = 0.540, 1.00, 0.836, and 0.142, respectively).

    Expt. 2. The effects documented in Expt. 1 showed decreased threonine at 30 min in the APC, but this was not significant due to the relatively low concentration of Thr in the APC and the small sample size. Therefore, we extended the period of food deprivation from 3 h during the light cycle to overnight, and switched to the use of diets devoid of amino acids to ensure sufficient food intake and larger effect sizes. Using this approach, no rats were excluded due to insufficient food intake. One rat was excluded from the unfed group because its APC sample was lost during tissue processing. Rats in this experiment ate 1.22 ± 0.08 g during the 21-min feeding period, excluding rats in the unfed group. Rats in the leucine devoid group had lower food intake than rats in the threonine-devoid and threonine-corrected groups (P = 0.003 and P = 0.047, respectively; Fig. 2). The food intake of the threonine-devoid and threonine-corrected groups did not differ (P = 0.112). Amino acid concentrations were somewhat higher in Expts. 2 and 3 than in Expt. 1, likely due to the difference in method of analysis, body size, vendor, and overnight food deprivation.

View larger version (26K): [in this window] [in a new window]   FIGURE 2 Food intake of rats fed the threonine basal diet or the leucine basal diet followed by no food, a threonine-corrected diet, a threonine-devoid diet, or a leucine-devoid diet (Expt. 2; n = 52) or followed by a threonine basal, a basal-threonine, a basal-leucine, or a basal-glycine diet (Expt. 3; n = 65). Values are means ± 95% CI. All rats were food deprived overnight before each experiment was conducted. Only the leucine-devoid group had significantly decreased food intake (P < 0.05). There were no consistent effects of indispensable amino acid deficiency within or among experiments.

    Expt. 3. Two rats were excluded from the analysis of the basal-threonine group; 1 rat was accidentally removed from its cage out of its proper order, and 1 sample was lost during tissue processing. One rat was excluded from the basal-leucine group due to a sample lost in preparation for amino acid analysis. The remaining rats in this experiment ate 1.53 ± 0.05 g over the 21-min feeding period. The groups did not differ in terms of food intake (P = 0.23; Fig. 2).

Rats fed the basal-threonine diet had a large decrease in threonine concentration in the APC relative to rats fed the threonine basal control diet (P = 0.032, Table 3). Rats fed the basal-glycine and basal-threonine diets did not differ in threonine concentration (P = 0.494).

Isoleucine concentrations were not altered by any dietary treatment (omnibus P = 0.126). The basal-threonine diet increased leucine relative to the threonine basal diet (P = 0.006). Serine concentration was decreased in rats fed the basal-threonine diet relative to both the basal-glycine and threonine basal controls (P = 0.003 and 0.006, respectively). Glycine concentration was decreased in rats fed the basal-threonine diet relative to both the basal-glycine and threonine basal controls (P = 0.050 and 0.081, respectively). Consumption of the basal-threonine diet increased phenylalanine concentration relative to both the basal-glycine and threonine basal controls (P = 0.008 and 0.015, respectively).

The basal-leucine diet group had decreased threonine relative to the leucine basal diet group (P = 0.013), and increased threonine relative to the basal-glycine diet group (P < 0.002). Rats fed the basal-leucine diet had decreased serine concentration relative to the basal-glycine and leucine basal controls (P < 0.001 and < 0.002, respectively). Glycine was increased in rats fed the basal-leucine diet relative to those fed the leucine basal diet (P = 0.057), and decreased relative to those fed the basal-glycine diet (P = 0.005). Consumption of the basal-leucine diet greatly decreased serine relative to all controls (P < 0.006), and greatly increased phenylalanine relative to the leucine basal and basal-glycine controls (P = 0.040 and P < 0.001, respectively).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
To the best of our knowledge, this is the first report of such a rapid effect of dietary manipulation on amino acid concentration in the APC. It confirms the utility of the modified basal diets (18) as a standard practice in the effort to understand the neural basis for recognition of indispensable amino acid deficiency. This report also extended earlier findings that the limiting amino acid is decreased in the APC at times beyond 30 min.

The present results showed decreases in the dietary limiting amino acid between 18 and 56% within 30 min of ingestion of a deficient diet. Although the present results did not include studies of plasma amino acid levels, the preponderance of historical evidence favors direct depletion of plasma and then brain amino acid pools after ingestion of amino acid–deficient diets, although brain concentrations fell more rapidly and to a greater extent than plasma concentrations (6). After the discovery that small amounts of the deficient amino acid infused into the carotid artery prevented recognition of amino acid–deficient diets (31), much effort was expended to learn which region of the brain was responsible for recognition of amino acid deficiency. After the APC was isolated as the primary recognition site (12), Gietzen and colleagues (7) reported that ingestion of diets imbalanced with respect to threonine significantly reduced threonine concentration in the APC after 2.5 h. A later report by Feurte and co-workers (32) demonstrated a significant decrease in plasma threonine concentration between 30 and 60 min after ingestion of a diet devoid of threonine. However, careful reexamination of their findings (32) demonstrates that there was an 20% decrease in plasma threonine concentration within 30 min (Cohen’s d = approximately –1.109), a large effect of the threonine devoid diet, and of the same order as the effects seen in the APC in the present study. Retrospective power analysis revealed that the design of Feurte et al. (32) had a power of 53%, highlighting the problems of relying on statistical testing to make inductive inferences [see also (14,28,29)],

It is possible, however, that the decrease in the local concentration of the limiting amino acids in the APC was not the result of depletion of the plasma pool per se, but rather occurred via an increase in local uptake or disposal of amino acids. In addition to the data on rapid alterations in plasma concentrations cited above, 2 lines of evidence argue against this possibility. First, the basal and basal-devoid diets (and the corrected and devoid diets) used in these studies cannot be discriminated or identified on the basis of taste or smell alone [reviewed in (8,11,18,21)], making it unclear how the APC, a portion of primary olfactory cortex, could be differentially activated by deficient and control diets. Second, manipulations of the peripheral nervous system, such as total subdiaphragmatic vagotomy, do not alter food intake until many hours after first encountering the diet (8,11,21,36), ruling out the possibility that peripheral mechanisms could directly modulate amino acid metabolism in the brain. More work will have to be done to further confirm the intimate relation between dietary, plasma, and brain levels of indispensable amino acids.

This report showed that indispensable amino acids are consistently and rapidly decreased in the APC by dietary deficiency. The indispensable amino acids threonine and leucine were consistently reduced in all of the experiments described above within 30 min. Curiously, leucine levels in rats fed the threonine basal diet (Expt. 3, Table 3) were not consistently higher than leucine levels in those fed the leucine basal diet, despite the latter diet having 10% less leucine. Threonine levels did demonstrate the appropriate reciprocal relation across experiments. It is important to note, however, that in all of the basal diets, amino acid contents approached only 50% of the requirement for maximal growth (9,10). Threonine and leucine are handled by separate transport mechanisms (22,35), and leucine plays an important role in both muscle and the metabolism of glutamate in the brain (33). This makes direct comparisons between the effects of different amino acid deficiencies problematic, although we recorded alterations in feeding behavior in response to a lack of the indispensable amino acid isoleucine, but not the dispensable amino acids serine or arginine (18). Consistent with these effects being related to indispensable amino acids, dietary deficiency of the dispensable amino acid glycine did not decrease glycine in the APC. Indeed, rats fed the basal-glycine diet had increased levels of glycine relative to all groups in Expt. 3 except the threonine basal group from which it did not differ (Tables 1, 2, 3). The effect of deficiency on glycine also provides limited support for previous observations of the effects of amino acid deficiency on APC neurons in vitro (22). Clearly, more work will have to be done to determine whether the differential effects of various basal diet treatments can be replicated.

Depriving neurons cultured from adult rat APC of threonine in vitro resulted in decreased threonine concentration within the cells after only 15 min of incubation (22), and parallel increases in the intracellular concentration of serine, glycine, valine, isoleucine, and phenylalanine. The results reported from Expt. 3 gave tentative support to an increase in phenylalanine and glycine concentration in response to indispensable amino acid deficiency. The remainder of the results did not support previous findings from isolated neurons in vitro, although the tissues used in the studies reported here comprised glial and cortical capillary compartments as well as neurons. More work is warranted to determine whether neurons in vivo accumulate other amino acids in response to deficiency of an essential amino acid.

It is possible that the level of the dietary limiting amino acid in the APC directly and linearly modulates food intake, but this is unlikely given the results of these and other studies. We found in the past that rats do not modulate food intake linearly. Rather, the ingestion of amino acid–deficient diets causes a total cessation of eating (17,18). In a system designed to maintain homeostasis, a correlation between the regulated variable and the behavior used to compensate for deviations in that variable is not automatically expected (34). For example, in a room with a thermostat set to 25°C, there should be no correlation between the amount of time the heater runs and room temperature; room temperature will remain constant whether the heater has to run constantly or intermittently. Similarly, the results reported above provided no evidence for a correlation between the amount of food eaten and the level of the limiting amino acid in the APC (Table 4). The APC concentration of the limiting amino acid was also not correlated with the amount of the limiting amino acid ingested. Because the limiting amino acid represents a constant proportion of the diet, the correlation between total food intake and APC concentration, and the intake of a particular amino acid and APC concentration are mathematically identical. One does expect, however, that a change in the value of the regulated variable would produce a change in behavior. As outlined above, the decreased level of the dietary limiting amino acid in the APC corresponded to the time at which most rats fed a deficient diet stop eating (17,18).

In conclusion, the present findings, together with past behavioral and biochemical studies, illustrate that the rapid rejection of amino acid–deficient diets corresponds to decreases in the level of the limiting indispensable amino acid in the APC of rats. Ongoing and future studies in our laboratory will try to understand how this initial signal is transduced to alterations in neural activity.

	FOOTNOTES

 
¹ Funded by National Institutes of Health Grants NS-33347 and NS-43210, and U.S. Department of Agriculture grant 2000–01049.

³ Abbreviations used: APC, anterior piriform cortex; basal-threonine, threonine basal diet minus the indispensable amino acid threonine; basal-leucine, leucine basal diet minus the indispensable amino acid leucine; basal-glycine, threonine basal diet minus the dispensable amino acid glycine.

Manuscript received 14 January 2004. Initial review completed 15 February 2004. Revision accepted 2 June 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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