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Articles

Essential Amino Acids Affect Interstitial Dopamine Metabolites in the Anterior Piriform Cortex of Rats^{1 ,2}

Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, and the Food Intake Laboratory, University of California at Davis, Davis CA 95616, and ^* Department of Animal Science, University of Illinois at Urbana-Champaign, IL 61801

	ABSTRACT

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The anterior piriform cortex (APC) is essential for the anorectic reactions to an amino acid-imbalanced diet, and it also responds to repletion of the limiting amino acid. In the present study, we examine the dynamic changes of the interstitial dopamine metabolites in the APC following feeding of either an amino acid-corrected or -imbalanced diet. Microdialysates, collected from the APC, were analyzed using HPLC with electrochemical detection. The concentrations were 19.7 ± 4.8 µg/L for 3,4-dyhydroxyphenylacetic acid and 25.1 ± 4.4 µg/L for homovanillic acid, respectively, in the baseline dialysates. After diet treatments, no significant changes occurred in 3,4-dyhydroxyphenylacetic acid in the corrected (n = 7) or imbalanced (n = 9) groups vs. the basal group (n = 7). However, after feeding the threonine-corrected diet, the concentration of homovanillic acid was significantly less (P < 0.01) than after the basal and imbalanced diets. The homovanillic acid level in the corrected group was already significantly lower than in the basal group by 20 min (P < 0.05), and reached its lowest level at 70 min (P < 0.05). The concentrations of homovanillic acid in the corrected group remained at this low level until the end of the experiment. The present results introduce the idea that the dopaminergic system is involved in the feeding responses to essential amino acid repletion.

KEY WORDS: • aversion • feeding • microdialysis • monoamine • rats

	INTRODUCTION

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Amino acid-imbalanced diets contain a single essential amino acid at a concentration that is both growth-limiting and disproportionate to the concentration of the remaining essential amino acids. A depression of food intake occurs rapidly when animals are fed the imbalanced diet (Gietzen et al. 1986 , Harper et al. 1970 , Leung et al. 1968a , Leung et al. 1968b , Rogers and Leung 1977 ). Recognition of repletion of the growth-limiting amino acid (LAA)⁴ is also rapid (Beverly et al. 1990a , Beverly et al. 1990b , Leung and Rogers 1969 , Rogers and Leung 1973 ). The nutritional model used in the present study has been validated over decades and has been reviewed extensively (e.g., Gietzen 1993 , Harper et al. 1970 , Rogers and Leung 1973 , Rogers and Leung 1977 ). After introduction of the imbalanced diet, animals that were pre-treated with a basal diet, containing ~75% of the requirement for protein, show a rapid decrease of the LAA in the plasma (reviewed in Gietzen 1993 , Harper et al. 1970 ). Within 1 h after introduction of the imbalanced diet, a brief increase in protein synthesis also occurs, which further depletes the LAA. The dramatic decreases in the plasma LAA cause reduced transport of the LAA at the blood brain barrier and account for a decrease in the concentration of the LAA in certain brain areas (reviewed in Gietzen 1993 , Gietzen et al. 1998 ). The decrease of the LAA in sensitive areas of the brain is thought to be responsible for the depression in intake of the imbalanced diet. In contrast, repletion of the LAA in the brain increases intake of the imbalanced diet, as shown by infusion of small quantities of the LAA into the carotid artery, which prevent the decreased intake of the imbalanced diet (Leung and Rogers 1969 ). When the same quantity of the LAA is infused into the jugular vein, there is no alleviation of the depression in the imbalanced diet intake (Leung and Rogers 1969 , Tobin and Boorman 1979 ). Presently, it is also clear that a decrease in the LAA concentration in the brain appears only in a few regions including the anterior piriform cortex (APC) (Gietzen et al. 1998 ). After ingestion of the imbalanced diet, the concentration of the LAA in the APC decreases 50%; this can occur before the decreased intake of the imbalanced diet is observed (Gietzen et al. 1986 ). Repletion of the decreased LAA in the APC by infusion of the LAA reverses the decrease in food intake of the imbalanced diet (Beverly et al. 1990a , Beverly et al. 1990b , Beverly et al. 1993 , Monda et al. 1997 ). This feeding response to the repletion of the dietary LAA in the APC is specific for the LAA, because injection of a nonlimiting amino acid into the APC has no effect on the food intake. Injection of the LAA into areas other than the APC including the amygdala also has no such effect. In addition, electrophysiology shows that spontaneous discharge frequency is increased in the lateral hypothalamus after similar injections of threonine into the APC in rats fed a threonine-devoid diet (Monda et al. 1997 ). Taken together, it appears that repletion of the LAA in the APC results in amelioration of the anorectic responses to an imbalanced diet. However, the mechanisms responsible for the behavioral response to the repletion of the LAA in the APC are not known. In recent years, accumulated evidence indicates that dopamine plays a role in the regulation of food intake. Dopamine acts as an appetite suppressant in the hypothalamus and a positive reinforcer in the nucleus accumbens (Hoebel 1997 ). Dopamine neurons of the ventral tegmental area and substantia nigra also construct and distribute information about rewarding events (Schultz et al. 1997 ). In the present study, we studied dynamic changes in the interstitial metabolites of dopamine in the APC during amino acid depletion with a threonine-imbalanced diet (IMB) and repletion with a threonine-corrected diet (COR).

	MATERIALS AND METHODS

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Male Sprague-Dawley rats (Simonsen Laboratory, Gilroy, CA), with body weights averaging 210 g at the time of surgery, were used. Animal care and general protocol for animal use were described (Beverly et al., 1990a , Beverly et al., 1990b , Beverly et al., 1991 ), and were according to the National Institutes of Health guidelines. Animal protocols were approved by the University of California-Davis Animal Use and Care Committee. The rats were housed in individual cages under a 12/12 h light–dark cycle with ambient temperature at 22 ± 2°C, with deionized water freely available.

Diets and diet treatments.

Powdered diets, containing L-amino acids (Ajinomoto, Teaneck, NJ) as the sole protein source, were described in detail previously (Beverly et al. 1991 ). Briefly, the threonine basal (BAS) diet contained 12.7% (w/w) amino acids as the protein equivalent, providing ~75% of the amino-acid requirements for growth, having threonine as the growth LAA. The IMB diet was made by adding a mixture of all the indispensable amino acids, except threonine, to the BAS. The COR diet consisted of the IMB plus an amount of the LAA, up to 0.185% of the diet, to replete the LAA rapidly (Wang et al. 1996 ). All diets contained the necessary vitamins and minerals with cornstarch and sucrose (2:1, w/w) as the carbohydrate source and 5% corn oil as the fat source. Carbohydrates were reduced proportionately when amino acids were added. There were three diet groups in this study. For the experiment, the control group (n = 7) was given the BAS diet; the corrected group (n = 7) was given the COR diet; and the imbalanced group (n = 9) was given the IMB diet.

Stock diet (Ralston Purina, St. Louis, MO) was freely available before the surgery. After the surgery the rat was given the BAS for 1 wk prior to microdialysis to promote a prompt expression of an anorectic response to the imbalanced diet (Leung et al. 1968a ). This period also allowed the animal to recover from the surgery (Beverly et al. 1990 , Beverly et al. 1993 , Specter et al. 1996 ). The BAS diet was removed just before the microdialysis probe was inserted into the APC via the guide cannula on the day of the experiment. The test diets, BAS, IMB or COR, were administered by intragastric gavage during the experiment as described below.

Surgery.

The general protocol for surgery was described in detail (Beverly et al. 1990a , Beverly et al. 1990b , Beverly et al. 1991 ). In brief, a guide cannula for the microdialysis probe (Bioanalytical System Inc. West Lafayette, IN) was implanted into the right side of the brain just above the APC. The coordinates were 2.2 mm anterior to bregma, 4.0 mm lateral to the midline and 4.0 mm below the dura, with an angle of 87° between the guide cannula and the visualized brain surface, according to the stereotaxic map of Paxinos and Watson (1982) . With these coordinates, the microdialysis probe was positioned in layer III, and the active membrane of the probe was between the two extremes of the `hairpin' loop of layer II in the APC. This region is known to be an essential area for recognition of amino acid deficiency (Gietzen and Beverly 1992 , Leung and Rogers 1971 ). The guide cannula was fixed in place with dental cement (Hygenic, Akron, OH) and stainless steel screws anchored into the skull. The rats were given 1 wk on the BAS diet to recover from surgery prior to the microdialysis experiment.

Microdialysis.

There were both in vitro (controls to determine recovery) and in vivo experiments. Microdialysis probes were purchased from Bioanalytical Systems Inc. Probe membrane length was 2 mm and outer-diameter was 0.32 mm, with a molecular weight cut-off of 5000 Daltons. The perfusion medium for microdialysis was a modified Ringers solution containing (in mmol/L) NaCl: 145, KCl: 2.7, CaCl₂: 1.2, MgCl₂: 1.0, and pH 7.2–7.4. The microdialysis pump was purchased from CMA, Stockholm, Sweden. Perfusion flow rate was held constant at 1 µL/min, and the collection period was 10 min for each sample. To prevent degradation of the monoamine and its metabolites, the dialysate was collected into a tube containing 1 µL of 1.75 mol/L of perchloric acid. Immediately after sample collection, the tube containing the dialysate was frozen in dry ice and then transferred and stored at -80°C until analyzed. Before the in vivo experiments, recovery of the microdialysate was determined in a solution that contained dopamine and its metabolites 3,4-dyhydroxyphenylacetic acid and homovanillic acid, 25 µg/L, respectively. The relative recoveries for dopamine and its metabolites were around 30%.

In vivo dialysates were collected from freely moving animals. Baseline sample collection started 3 h after the probe was inserted into the APC via the guide cannula, and three baseline samples were collected. The mean concentration of these three samples was defined as the baseline value. After the baseline samples had been taken, 2 g of the test diet, suspended in 4 mL of deionized water, was introduced gastrically with a feeding tube. Dialysates were sampled at 10 min intervals for 3 h following this diet treatment.

Determination of dopamine and its metabolites.

The concentrations of dopamine and its metabolites were analyzed using HPLC with electrochemical detection, as described previously (Gietzen et al. 1986 ). A 5-µL aliquot of sample was injected onto a 100 x 2 mm 3 µm C18 reverse-phase column (Bioanalytical System Inc.). The mobile phase (pH 3.15) contained (in mmol/L) sodium hydroxide, 37.5; EDTA, 1.04; citric acid, 110; and 1-octanesulfonic acid, 1.71. Flow rate was 0.4 mL/min. A glassy carbon electrode was used for electrochemical detection at +800 mV potential. Concentrations of the substances in the dialysates were quantitated by comparison with external standards. The concentrations of dopamine in the in vivo dialysates were below minimal detection limits. The values representing the concentrations of each molecule are expressed as a percentage of the baseline value for that substance.

Histology.

At the end of the experiment, each rat was anesthetized with ethyl ether (Fisher Scientific, Fair Lawn, NJ) and killed by decapitation. The brain was removed and immersed into 8% paraformaldehyde for histology. The brain was sectioned sequentially at a thickness of 60 µm, and sections were stained with Hematoxylin & Eosin (Polysciences Inc., Warrington, PA). Location of the microdialysis probe was verified under a light microscope. Only animals with probe locations in the APC (as described above) were included in the data set.

Statistical analysis.

The microdialysis data were analyzed across the 180 min collection period after diet treatment by repeated measures ANOVA, using a statistical package (SAS version 6.04, Cary, NC). When a significant difference was found in the repeated measures ANOVA, this was followed by post-hoc comparison of the LS means using the least significant differences test with the Bonferroni correction for multiple comparisons (Snedecor and Cochran 1967 ). A P value of less than 0.05 was considered significant. Values are means ± SEM.

	RESULTS

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The concentrations of 3,4-dyhydroxyphenylacetic acid and homovanillic acid were 19.7 ± 4.8 and 25.1 ± 4.4 µg/L, respectively, in the baseline dialysates before any diet treatments. After the dietary treatments, small changes of 3,4-dyhydroxyphenylacetic acid concentration appeared in two stages. In the earlier stage, 0–40 min after the treatments, 3,4-dyhydroxyphenylacetic acid appeared to increase in the all three groups. This earliest trend may reflect reactions of generalized stress induced by the gastric injection. In the later stage, 41–180 min after the treatments, 3,4-dyhydroxyphenylacetic acid tended to increase only in the COR and IMB groups. However, repeated measures ANOVA indicated that the differences were not significant among these three groups (P ranged from 0.06 to 0.4).

The effects of the diet treatments on changes in the level of homovanillic acid, over the 180 min after diet treatments, differed significantly as shown in Figure 1 (repeated measures ANOVA: P < 0.007). In the BAS diet group, the level of homovanillic acid remained stable during the post-feeding period. Post-hoc analysis showed that after the animals were fed the COR diet, the homovanillic acid concentration was significantly lower than that in the BAS group (P < 0.01). The homovanillic acid level was already significantly lower by 20 min after the diet treatment (P < 0.05), and reached a minimum at 70 min after the diet was fed (P < 0.05). Thereafter, the concentrations of homovanillic acid in the COR group remained at this low level until 180 min after feeding, which was the last time point in this study. The level of homovanillic acid was also significantly lower in the COR group when compared with the IMB group, starting from 20 min after the diet treatment, until the end of the experiment (P < 0.05). In contrast, the concentration of homovanillic acid was slightly, but not significantly (P = 0.18), higher in the IMB diet group than in the BAS diet group. After IMB diet treatment, the maximal level of homovanillic acid occurred at 30 min; it decreased thereafter and returned to the baseline at the end of the experiment.

View larger version (17K): [in this window] [in a new window]   Figure 1. Effects of diet treatments on the concentrations of homovanillic acid, a dopamine metabolite, in dialysates of the anterior piriform cortex (APC). The levels of homovanillic acid are expressed as a percentage of the baseline value. The test diet, 2 g, was administered immediately after the last baseline sample was taken. The number of rats in each group is given on the figure. Each point represents the mean ± SE. Data were analyzed with repeated measures ANOVA followed by least significant means post hoc testing for differences among groups, using the Bonferroni correction for multiple comparisons. Abbreviations: BAS, threonine basal diet; COR, threonine-corrected diet; IMB, threonine-imbalanced diet; *, significant difference between the BAS group and the COR group at that time point (P < 0.05), and , significant difference between IMB group and COR group at that time point (P < 0.05).

	DISCUSSION

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The concentration of homovanillic acid decreased dramatically, though the sums of the concentrations of the dopamine metabolites, 3,4-dyhydroxyphenylacetic acid and homovanillic acid, did not change after feeding the COR diet (data not shown). Because 3,4-dyhydroxyphenylacetic acid is converted to homovanillic acid by the enzyme, catechol-O-methyl transferase (Walker 1984 ), these results suggest that the activity of this enzyme may have been decreased after ingestion of the COR diet. This enzyme is abundant in the brain, and in some tissues it tends to prolong the duration of the response to stimulation. Further work will be necessary to confirm this hypothesis and then to clarify a functional role for decreased catechol-O-methyl transferase activity in an animal after feeding the COR diets. In this study, there were no significant changes of homovanillic acid level after feeding the IMB diet. One possible explanation is that activity of the dopamine system may be increased with ingestion of the COR, but not the IMB diet. The COR diet is preferred in choice situations (reviewed in Gietzen 1993 , Hrupka et al. 1997 , Leung et al. 1968b ). Thus, dopamine in this system could act to support continued ingestion of the better diet, consistent with the work of Hoebel (1997) .

In the present study, only one amino acid (threonine) was the LAA, because in our previous studies monoamine changes in the APC were similar whether threonine or isoleucine was used as the LAA. Also, we observed that after ingestion of a threonine IMB diet, the concentration of threonine was decreased in the APC, and when isoleucine was the LAA, its concentration, but not that of other amino acids, such as threonine, was decreased in this area (Gietzen et al. 1986 , Gietzen et al. 1989 , Gietzen et al. 1998 ). Also, similar responses were seen in response to repletion of various LAA in the APC (Beverly et al. 1990a , Beverly et al. 1990b , Beverly et al. 1991 , Beverly et al. 1993 ). Moreover, behavioral responses to amino acid imbalanced diets were shown to generalize over all of the dietary indispensable amino acids, as reviewed by Gietzen (1993) , Harper et al. (1970) , and Rogers and Leung (1977) .

The present results support previous findings that the APC responds to repletion of the LAA. In the previous studies, the food intake depression was prevented by infusion of small quantities of the LAA into the carotid artery when the imbalanced diet was fed. Infusion of the LAA into the jugular vein required a much larger quantity, than that given into the carotid artery, to alleviate the food intake depression to the imbalanced diet (Leung and Rogers 1969 ). Injection of the LAA into the APC also ameliorated the depressed food intake of an amino-acid-deficient diet (Beverly et al. 1990a , Beverly et al. 1990b , Beverly et al. 1993 ). Additionally, administration of the LAA in physiologically relevant concentrations into the APC caused an increase in the firing rate in neurons from the lateral hypothalamus in animals fed a threonine-devoid diet (Monda et al. 1997 ). We assume that this exogenous repletion of the LAA in the APC would correlate with feeding of the COR diet in the present study. Further, we conclude that the dopaminergic system in the APC is among the transmitter systems involved in the feeding response to repletion of the limiting amino acid.

	FOOTNOTES

 
¹ Presented in part at 27th annual meeting of the Society for Neuroscience, October 25–30, 1997, New Orleans, LA [Wang, C. X., Erecius, L. F. and Gietzen, D. W. (1997) Effect of amino acids on monoamines in anterior pyriform cortex in rats. Soc. Neurosci. Abstr. 24: 400].

² Supported by USDA 94-37200-0655, 97-35200-4477, NIH NS 33347 and DK 35747.

³ To whom reprint requests should be addressed.

⁴ Abbreviations used: APC, anterior piriform cortex; BAS, threonine basal diet; COR, threonine-corrected diet; IMB, threonine-imbalanced diet; LAA, growth-limiting amino acid.

Manuscript received December 24, 1998. Initial review completed May 13, 1999. Revision accepted May 24, 1999.

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