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Is Involved in the Signaling of Indispensable Amino Acid Deficiency in the Anterior Piriform Cortex of the Brain in Rats1,2
Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616
3To whom correspondence should be addressed. E-mail: dwgietzen{at}ucdavis.edu.
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ABSTRACT |
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(p-eIF2[Ser51]) and translation of its downstream product, c-Jun, were increased (47%, P < 0.005, and 55%, P < 0.025, respectively) in APC from rats offered devoid, but not corrected diets, compared with those offered basal diets. This was not seen in other brain areas. In cells intensely labeled for cytoplasmic p-eIF2, there was intense fluorescence for c-Jun in the nucleus. Thus, p-eIF2, which is pivotal in the initiation of global protein translation, and its downstream product, the leucine zipper protein, c-Jun, are increased in the mammalian APC within the time frame necessary for the behavioral response. We suggest that p-eIF2 and c-Jun participate in signaling nutrient deficiency in the IAA-sensitive neurons of the APC.
KEY WORDS: • nutrient sensor • translation initiation • feeding behavior • essential amino acids • c-Jun
Maintenance of the amino acid precursors for protein synthesis is crucial for biological organisms. When amino acids are depleted, various adaptive responses, each appropriate to repletion of the limiting amino acid for that organism, are called into action. In spite of considerable work on the issue, the cellular sensors for amino acids are unknown. When de novo synthesis is available, as in deficiencies affecting plants and microorganisms, the appropriate enzymes are activated or synthesized to provide the needed amino acid. Although the yeast, Saccharomyces cerevisiae, serves as a well-studied model for the effects of amino acid limitation on the translation of amino acid–synthesizing enzymes in eukaryotic cells (1), the responses to amino acid deprivation in animals are more complex.
Half of the amino acids from which proteins are made cannot be synthesized by animals; these are the dietary-essential (indispensable) amino acids (IAA).4 In addition, there is no storage form for free amino acids in animals (2), and metabolic recycling accounts for only about two thirds of the daily IAA requirements. Protein degradation can provide free amino acids, but at the expense of existing body protein (3). Therefore, IAA levels must be maintained by regular ingestion of balanced proteins; this requires appropriate diet selection based on accurate, timely feedback about the IAA status of the organism. The behavioral rejection of diets inducing IAA deficiency has long been the hallmark indicating sensing of an IAA imbalance (4). Generalist feeders, from mollusks (5) to mammals [reviewed in (4)], reject diets leading to IAA deficiency and quickly select other foods, or initiate foraging for a better ration. Later, rats develop a conditioned aversion to such diets; still later, if they have no choice, they adapt to an imbalanced diet, but not to a diet devoid of an IAA (4). Thus the ability to sense and respond to IAA limitation is highly conserved and is considered adaptive for omnivores.
The anterior piriform cortex (APC) of rats (6) and its counterpart in birds (7) are brain areas shown by ablation studies to be essential for the behavioral rejection of diets inducing IAA deficiency. This rapid sensing, which occurs during the first meal, can be prevented by injecting 1–2 nmol of the limiting IAA into the APC (8–10). These injections are specific, both biochemically for the limiting IAA and anatomically for the APC (8). Taken together, these findings support the role of the APC as the IAA chemosensor. Although the mechanisms involved in sensing IAA deprivation are unknown in multicellular organisms (11), it is clear that the neurons of the APC are activated shortly after rats begin to ingest an IAA-deficient diet (12–14). Activation of the APC diminishes by 6 h (12,14), but appears to affect neuronal areas associated with feeding circuits within 30 min (15). The feeding circuitry in rats is complex, but clearly includes the lateral hypothalamus (15,16), which is likely involved in the well-described hypophagia of omnivores given IAA-deficient or imbalanced rations (4).
Signals arising from assorted stressors, including amino acid limitation, are conserved [see Table 2 in (17)]. They converge at the point of phosphorylation of serine 51 in the subunit of eukaryotic initiation factor 2 (eIF2). In amino acid deficiency, the general control nonderepressing- (GCN-) kinase, GCN2, is activated by uncharged tRNA (18), with subsequent phosphorylation of eIF2. The phosphorylated form of eIF2 (p-eIF2) inhibits global protein synthesis by inhibiting eIF2B, the crucial factor for global protein translation (17). However, not all protein translation is stalled. When eIF2 is phosphorylated, a small subset of upstream open reading frames is inhibited, resulting in increases of selected leucine-zipper (B-ZIP) proteins, such as ATF4 (19), c-Jun (20), and the CCAAT/enhancer-binding protein (C/EBP) homologous protein, CHOP (21). Importantly, p-eIF2 is the key regulatory signal at the pivotal point between the network of upstream pathways that signal cellular stress, and the downstream switch from global protein synthesis to translation of adaptive proteins (22).
In yeast, downstream evidence for the persistent phosphorylation of eIF2, after activation of GCN2 by amino acid depletion, includes increased GCN4. Therefore, we also conducted tests to determine whether either of two partial mammalian orthologs of GCN4, c-Jun (20) or ATF4 (19), is increased in the APC of rats. We compared immunoblot (Western) analysis and immunohistochemistry for p-eIF2, c-Jun, and ATF4 in brain areas from rats fed a basal diet (BAS) (23,24) or the same diet with modified threonine content. The experimental diets were either devoid of threonine (TBD) or replete with threonine (corrected, TBC).
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MATERIALS AND METHODS |
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75% of the rats’ requirement for protein and 50% of their requirement for the limiting IAA for maximum growth, meets all other known nutrient requirements of rats. The rapid recognition of an IAA-deficient diet, whether imbalanced or devoid of the limiting IAA, is facilitated if the rats were prepared appropriately by feeding BAS for 7–10 d (25). In this well-established nutritional model (4), the BAS prefeeding regimen causes a modest depletion of labile protein and reduces circulating free threonine, thus allowing a rapid recognition of the acute TBD-induced deficiency (25). As noted above, the TBD diet is the same as the BAS diet, but is devoid of threonine. The corrected (TBC) diet is the same as BAS, but corrected by the addition of threonine (4 g/kg of diet), with a corresponding subtraction of carbohydrate. The rats were randomly divided into two groups, food-deprived for 18 h (during the last 6 h of the dark phase of the light-dark cycle, and the 12 h of the light phase when rats normally eat little). At the onset of the next dark phase, they were allowed access to either a BAS or a TBD for 20 min. In a separate study conducted in exactly the same way, the diets were BAS or TBC. Food intake was measured by difference and corrected for spillage. Rats that consumed at least 1 g of food were included in the studies. Unless otherwise specified, reagents were purchased at the finest grade available from Sigma-Aldrich. Immunoblot (Western) tissue collection. Twenty minutes after introduction of the diets, rats were decapitated. The brains were rapidly removed and placed into liquid nitrogen for 10–15 s, then placed on dry ice and the areas to be analyzed were dissected. Tissues from 5 rats were pooled for each immunoblot. Protein analysis was done according to the protocol for monoclonal antibodies issued by Cell Signaling Technology, with the following modifications. Tissues were homogenized in extraction buffer (0.25mol/L Tris HCl pH 6.8, 25 g/L SDS, 2 mmol/L EDTA, 2 mmol/L EGTA, 10% v:v glycerol and 5% v:v 2-mercaptoethanol). Protease inhibitors and two sets of phosphatase inhibitors (P8340, P2850, and P5726, respectively, Sigma) were added to the extraction buffer each at a 1:100 dilution. The homogenates were heated at 100°C for 10 min then centrifuged at 10,000 x g for 10 min. The supernatant was divided into 100-µL aliquots that were stored at -80°C. Protein levels in the samples were analyzed via the Bio-Rad DC Protein Assay. Colorimetric absorbances were read on a Multiskan Ascent 96-well plate reader (Labsystems Oy).
Brain areas.
The tissues were dissected from transverse sections of frozen brain, at 0.70–3.70 mm rostral to Bregma. The APC was cut as described in (26), and divided into an area medial to the lateral olfactory tract (APClot) and a more ventrorostral segment (APCvr) also known as the Area Tempestas, a highly chemosensitive area described by Gale (27), and more recently by Ekstrand et al. (28). The APClot is the injection site previously used (8,15) for IAA repletion studies. The APCvr, APClot, and a section of neocortex corresponding to the primary motor cortex (29) were examined by Western blots and immunohistochemistry. A segment of cerebellar cortex was taken and analyzed by immunoblot. In addition, a segment of tissue dorsal to the rhinal fissure was taken as the "perirhinal" area, and the dorsal endopiriform cortex, medial to the APCvr; these were examined by immunohistochemistry.
Immuno (Western) blotting.
Samples were diluted 1:2 in an SDS reducing buffer and underwent electrophoresis on precast gels (either NuPAGE Novex 7% Tris Acetate for 70 min at 150 V or NuPAGE Novex 10% BisTris for 50 min at 200 V, both from Invitrogen). The volume of sample added to each well was adjusted so that the same amount of protein was added to each well. Biotinylated protein standards (Bio-Rad) were diluted 1:50 and run in lane 1 of the gel to provide markers for molecular weights between 6.5 and 200 kDa. The proteins were electrotransferred to a polyvinylidene difluoride membrane at 30 V for 1 h. Protein detection was done according to the Cell Signaling protocol, except that the blocking buffer was used rather than the primary antibody dilution buffer for the antibody dilutions, the incubations were done for 1 h at room temperature then overnight at 4°C, and the washes were carried out for 15 rather than 5 min each. For p-eIF2, we incubated the membrane with antibody for 1 h at room temperature and then for 48 h at 4°C. All antibodies were certified specific as purchased from Cell Signaling, or other vendors, where indicated below. The primary antibodies used were phosphorylated eIF2 (rabbit polyclonal IgG for Phospho-eIF2 on ser51, diluted 1:500), eIF2 (rabbit polyclonal IgG for eIF2, diluted 1:500), c-Jun (rabbit polyclonal IgG to c-Jun, diluted 1:1000), or ATF4 (CREB-2 [C20]Santa Cruz Biotechnology, diluted 1:200). Membranes were placed on Kodak X-OMAT AR film (Eastman Kodak) for 15 min to 2 h. Films were scanned and the appropriate bands were analyzed with ImageQuant 5.1 (Molecular Dynamics). Values calculated by the software are reported as volume density (integrated intensity of all the pixels in the spot) compared with total volume of the spot on the blot after subtraction of background and defined here as density units (U).
Immunohistochemistry.
After 20 min of access to either BAS or TBD as above, different rats were anesthetized with a ketamine cocktail as we have reported previously (30). Transverse sections were taken and prepared as previously described (3). Fluorescent immunohistochemistry was used to label the slides for eIF2 and p-eIF2, or double label them with p-eIF2/c-Jun. The primary antibodies used were either anti-phospho-eIF2 or anti-eIF2 as used on the Western blots above. The primary antibody was removed and the sections were washed 3 times for 10 min in PBS. AlexaFluor 488 anti-rabbit secondary antibody (Molecular Probes), diluted 1:200, was incubated with the sections for 2 h at 24°C. Sections were washed 3 times for 10 min with PBS and then either mounted with cover slips or doubled labeled with the appropriate second primary antibody. Those slides (previously labeled for p-eIF2) that were double labeled were then incubated with the primary antibody to c-Jun as above, but diluted 1:200. The primary antibody to c-Jun was removed and the sections were washed and incubated with the anti-rabbit secondary antibody, washed again and mounted as above. The brain sections were imaged using a confocal microscope (Zeiss 510). The commercially available antibodies used here are reported by the suppliers to be specific for the protein indicated, and not to cross-react with other proteins. Values were calculated from at least 6 measurements taken from 8 rats during 3 separate experiments.
Statistical analysis. At least 3 separate experiments were done for the results of immunoblotting (5 rats pooled/blot) and immunohistochemistry (6–8 rats counted/experiment) reported here. Data from >2 groups were subjected to ANOVA with post-hoc testing using Fischer’s Protected Least Significant Differences Test, where appropriate. If only 2 groups were compared, Student’s t test was used. Differences were considered significant at P < 0.05.
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were increased (P < 0.05) in the APC of brains taken after the rats had been eating TBD; they did not differ after TBC consumption compared with those eating the BAS for just 20 min. In the other brain areas studied, neocortex, endopiriform nucleus, perirhinal cortex, and cerebellum, there were no changes in either the immunoblots or the immunohistochemistry due to the diet treatments.
Phosphorylated eIF2 and c-Jun protein after TBD.
After rats had access to TBD for 20 min there were increases in phosphorylated eIF2 (Fig. 1, row 1) and no changes in the nonphosphorylated form of eIF2 (Fig. 1, row 2), compared with BAS. In the Western blots, c-Jun, one of the two putative GCN4 orthologs tested (19,20), was increased after the TBD treatment (Fig. 1, row 3). In contrast, ATF4, the other of the B-ZIP proteins suggested to serve as a mammalian ortholog of GCN4 (19), did not differ in abundance among diet treatments for APC (Fig. 1, band 4) or any brain area tested. Neither p-eIF2 nor c-Jun was increased in either area of the APC in rats fed the corrected (TBC) diet (Fig. 2).
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and the nonphosphorylated form, eIF2, indicated that there were no differences between the APClot and the APCvr for these proteins, the data were combined. Optical density in the APC immunoblot bands for p-eIF2 were 10.1 ± 1.15 U in rats fed BAS vs. 14.9 ± 1.15 U for rats fed TBD (P < 0.01), whereas there were no differences noted in other brain areas, including the neocortex and cerebellum (Fig. 3A). In the corrected (TBC) comparison, again the APClot and APCvr, did not differ; optical densities for the Western bands were as follows: BAS, 18.8 ± 1.6 and TBC, 14.8 ± 2.0 U, clearly not increased as it was in rats fed the TBD diet. The ratio TBC:BAS for combined APC values was 0.79 ± 0.08, which tended to be less than the expected ratio of 1 (P = 0.06), again supporting the lack of an increase after consumption of the TBC diet. The nonphosphorylated form of eIF2 was decreased in the APC of rats fed TBD, i.e., BAS 13.4 ± 0.5 U vs. TBD 11.5 ± 0.7U (P = 0.03). When expressed as a normalized ratio of the phosphorylated to nonphosphorylated band densities (p-eIF2:eIF2), the ratio for the APC was 0.75 ± 0.09 for rats fed the BAS diet vs. 1.28 ± 0.11 for those receiving the TBD treatment (P < 0.001).
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Positive immunohistochemical labeling for p-eIF2 was increased in the APC after rats were allowed access to TBD for 20 min (Fig. 4). Quantification was based on the area of p-eIF2 labeling:total area, determined by computerized densitometry. Both the APClot and the APCvr were responsive to the deficient diet (TBD), as indicated by increased phosphorylation of eIF2 in both areas (each P < 0.01), consistent with the Western blot analysis described above. In the other areas tested, including dorsal neocortex at the same anterior-posterior level as the APC, the perirhinal area at that same level, and the endopiriform nucleus, there were no such increases in p-eIF2. In these measures, the ratio of TBD/BAS for the APClot was 2.01 ± 0.18 vs. 3.17 ± 0.26 for the APCvr, which did not differ. The p-eIF2 positive cells appeared to be in layers II and III of the APC, many of which are the glutamatergic output cells of layer II of the APC (Fig. 5A: the cell body layer lies diagonally upward to the right). The labeling appeared chiefly in the cytoplasm of large cells in this area (Fig. 5B). Finally, immunohistochemical labeling for both p-eIF2 and c-Jun indicated that in cells highly fluorescent for cytoplasmic p-eIF2, c-Jun labeling was found in the nucleus. In these cells, c-Jun labeling was present in the nucleus, surrounded by punctate p-eIF2 fluorescence in the cytoplasm (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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(17). This halts general protein translation and decreases the translation of a few open upstream reading frames on mRNA, resulting in increased translation of GCN4 (31). Thus, our key finding that p-eIF2 was increased in APC after only 20 min of consuming an IAA-devoid diet is important. In rats, as in the yeast model, amino acid deprivation rapidly caused an increase in p-eIF2 in the amino acid–sensitive neurons of the APC. It is also interesting that not all cells responded in this way, suggesting that a small population of IAA-sensitive cells may be present within layer II of the APC. We showed previously that AA repletion via injecting just 2 nmol of the limiting IAA into the APC prevents the hypophagic response within 20 min (9). This shows the early effects of decreasing IAA in this brain area, and it seems intuitive that a decrease in the limiting IAA in the APC should be the initial event in an IAA deficiency situation. The next step in this model is the activation of amino acids for charging of the cognate tRNA. Uncharged tRNA.
The uncharged tRNA-GCN2-peIF2-GCN4 pathway in rats was the subject of the present and previous studies. Because IAA are by definition not synthesized in animals, the adaptations involving amino acid synthesis, useful to single cell organisms and perhaps for the dispensable amino acids in animals, are not available for the IAA. The histidyl tRNA synthetase binding domain appears to be conserved in rodents (32). This domain can bind a variety of uncharged tRNAs (33), suggesting that the yeast model for activating eIF2 via GCN2 may serve higher eukaryotes as well, at least for the dispensable amino acids [reviewed in (38)]. Alternatively, signals activated by uncharged tRNA directly (35) could be part of the rapid signaling in response to IAA deprivation, resulting in behavioral changes leading to acquisition of the necessary IAA.
Earlier, in investigating the role of tRNA charging, we saw decreased food intake 20 min in rats after inhibition of threonyl- tRNA synthetase using L-threoninol, the synthetase inhibitor (39). This is consistent with a link between uncharged tRNA in the APC and food intake. However, the issue of uncharged tRNA in animals in vivo has been controversial. The Km for the amino acyl tRNA synthetases tend to be very low (40), and the in vivo concentrations of the IAAs rarely fall to such an extent that tRNAs are uncharged over the time course usually measured (hours). Shenoy and Rogers (41) reported a decrease in isoleucyl-tRNA charging in rat liver after ingestion of an isoleucine-imbalanced diet that was reversed with feeding a corrected (complete) diet. However, they were unable to link this to a decrease in the concentration of the limiting IAA in the tissue. More recently, we measured tRNA charging in rat APC after 2 h of consuming an IAA-imbalanced diet, and found, using two different assay methods, that charging, compared with that in the BAS and corrected diet groups, was either increased or unchanged, but not decreased, as expected (42). An increase in tRNA charging for a dietary limiting IAA could occur by 2 h if sufficient catabolism of cellular protein had taken place by that time because amino acids can regulate proteolysis (3). It may be that if IAA-deficiency signaling results from uncharged tRNA, such rapid signaling may be transitory, as was seen with the monoaminergic systems in this model (43). To our knowledge, neither tRNA charging nor direct activation of GCN2 in APC has been evaluated after only 20 min of IAA deprivation. As an indirect method for evaluating the activation of GCN2 (also known as EIF2AK3), or its analogous kinases, one can measure the phosphorylation level of eIF2(Ser51) (22).
Phosphorylation of eIF2.
Although not all mammalian cells respond to amino acid limitation by increasing p-eIF2 (44), the present results showed increased p-eIF2 in the APC of rats within 20 min, the time of their initial behavioral response to the IAA deficiency (23). The specific eIF2 kinase that is activated in the rat APC by IAA deficiency is unknown, but it could be GCN2 (38), PERK (45), or other kinase(s), perhaps as yet unidentified (38). Alternatively, a decrease in phosphatase activity, rather than increased activity of the kinase systems, could result in increased levels of p-eIF2 (46,47). Whatever the route to increased phosphorylation, p-eIF2 appears to be the unique focal point among the various pathways affecting protein translation in the signaling of IAA deficiency.
Proteins downstream of p-eIF2.
B-ZIP proteins translated further downstream from eIF2 in the responses to nutrient deprivation include GCN4 in yeast (31), ATF4 [CREB-2, (19)], C/EBPß (48), CHOP (49), c-fos (14), and c-Jun (the present results). The DNA binding components of the GCN4 and c-Jun were shown to be interchangeable (50). The B-ZIP proteins were reviewed recently, and the human variants were compared with GCN4 (51). In this comparison, both ATF4 and c-Jun were near GCN4 in the dendrograms; their relative positions in these figures depended on which parts of the molecule were being compared. It is interesting that c-Jun, a protein known to be translated when there are high levels of p-eIF2 in human tissues, was elevated in the APCvr, whereas ATF4, the putative GCN4 ortholog in mice (19) was unaffected, at least at the 20 min time point corresponding to the behavioral response (Fig. 1). The lack of an effect on ATF4 is not due to its absence in the tissue because rat brain contains large amounts of ATF4, thought to be involved in long-term memory (52).
c-Jun was increased in APCvr also by 20 min after introduction of an IAA-depleting diet. Notably, upregulation of the ortholog, ATF4, seen within 2 h of amino acid deprivation, requires de novo protein synthesis (48). Thus, the early (20 min) time for detection of the increase in c-Jun suggests that this may be due to a reduction in the degradation, as occurs with GCN4 (53), rather than increased translation, as is reported for ATF4 (48). The effects of IAA-deficient preparations on proteolysis are the object of current study in this laboratory.
As another alternative, the increase in c-Jun could be due to parallel signaling events, such as those related to the extracellular signal related kinase family of mitogen-activated protein kinases (MAPK). We reported previously that MAPK is phosphorylated in the APC after introduction of an IAA-devoid diet (13). In addition, inhibition of p70s6 kinase (a member of the mTOR pathway) occurs within 15 min of incubation of cells in an amino acid–deficient medium (54). Therefore, several signaling pathways appear to be activated at least within 20 min of IAA deprivation.
Activation of APC neurons.
Several lines of evidence showed that the output neurons of the APC are activated in IAA deficiency (12–14). How the neurons become activated is not yet understood, but phosphorylation events clearly must be involved. Upregulation of cationic amino acid transport during amino acid starvation is dependent on phosphorylation of eIF2 (22). Recent results using APC neurons in primary culture showed that system A, the amino acid transporter that carries threonine along with sodium into the cell (55), is upregulated within minutes of threonine deprivation, an effect dependent on phosphorylation (56). This is consistent with the rapid upregulation of system A in other cell types (57) and with a rapid increase in intracellular sodium, which would activate the neurons of the APC by direct depolarization (55).
In summary, these findings suggest that kinase activity in the APC, likely related to the increased p-eIF2 seen here, is integral to the signaling of IAA deficiency in rats. It is not yet known whether the phosphorylation of eIF2 in IAA deficiency is via activation of GCN2 by uncharged tRNA, or secondary to other mechanisms, such as another kinase, calcium-mediated events, decreased phosphatase activity (46,47), or increased transporter (56,57) activity. Nonetheless, the present results show that phosphorylation of eIF2, a key well-conserved regulator in the molecular responses to IAA deficiency, is increased within 20 min of introduction of an IAA-deficient diet, along with a potential downstream ortholog of GCN4, c-Jun, in the excitable APCvr. Clearly, phosphorylation events and translation of B-ZIP proteins take place in this highly chemically sensitive area of the brain early enough to be part of the initial recognition of IAA deficiency, shown by the behavior of rats within 20 min (23). It is tempting to suggest that these excitable neurons of the APC, the brain area essential for the rejection of IAA-deficient diets by rats, could fully explain the mechanisms for IAA sensing and lead to discovery of the mammalian IAA sensor.
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in response to dietary threonine deficiency. FASEB J. 17: A681.3 (abs.) online]. 
2 Supported by National Institutes of Health grants NS33347 and NS43210 and U.S. Department of Agriculture grant NRI 2000 01049.
4 Abbreviations used: APC, anterior piriform cortex; APClot, anterior piriform cortex medial to the lateral olfactory tract; APCvr, ventrorostral anterior piriform cortex; BAS, threonine basal diet; B-ZIP, leucine-zipper; C/EPB, CCAAT/enhancer-binding protein (C/EBP); CHOP, C/EPB homologous protein; eIF2, eukaryotic initiation factor 2; GCN2, general control nonderepressing- (GCN-) kinase; IAA, indispensable amino acid; MAPK, mitogen-activated protein kinases; neocortex, cortical area most dorsal from the APC; p-eIF2(Ser51), the phosphorylated form of eIF2 at serine 51; perirhinal, cortex in the plane of the APC and immediately dorsal to the rhinal fissure; TBC, threonine basal corrected diet; TBD, threonine basal devoid diet; tRNA, transfer ribonucleic acid; U, density units.
Manuscript received 20 October 2003. Initial review completed 23 November 2003. Revision accepted 6 January 2004.
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