Temporal-spatial pattern of c-fos expression in the rat brain in response to indispensable amino acid deficiency I. The initial recognition phase

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MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 40 (1996) 27-34

Research report

Temporal-spatial pattern of c-fos expression in the rat brain in response to indispensable amino acid deficiency I. The initial recognition phase Yan Wang, Sharon L. Cummings, Dorothy W. Gietzen * Departments of Veterinary Anatomy, Physiology and Cell Biology, and the Food Intake Laboratory, University of California at Davis, Davis CA 95616, USA Accepted 10 January 1996

Abstract Rats reduce their food intake after ingestion of a small amount of an amino acid imbalanced (AA-IMB) diet that induces a pronounced amino acid deficiency. Two hours after ingesting a threonine-IMB diet, just when food intake is depressed significantly, the concentration of threonine is decreased in some but not all brain areas. Neural recognition of this decrease in the limiting amino acid is thought to be the first step in the anorectic responses to AA-IMB diets. To identify the regions of the brain that may be activated upon recognition of an AA-IMB diet, we examined the temporal-spatial distribution of Fos immunoreactive neurons at intervals after introduction of either threonine-IMB or control diets. We found that Fos inununoreactivity in the anterior piriform cortex and immediately surrounding areas, along with the infralimbic cortex, was increased selectively early (by 2 h) after introduction of the AA-IMB diet, and remained elevated through 3 h. The anterior piriform cortex is believed to function in neural recognition of amino acid deficiency. Fos immunoreactivity in the AA-IMB group increased over the control diet groups somewhat later in the dorsomedial nucleus of the hypothalamus. We hypothesize that these areas in the rostral forebrain may serve as neural relays in the early phases of the anorectic responses that occur upon recognition of amino acid deficiency. Keywords: Anterior piriform cortex; Amino acid imbalanced diet; Threonine; Feeding behavior; Dorsomedial nucleus of the hypothalamus; Infralimbic cortex

1. Introduction Animals reduce their food intake rapidly and reliably when fed an amino acid-imbalanced diet (AA-IMB) in which a single growth-limiting amino acid is deficient and other indispensable amino a c i d s are r e p l e t e [10,11,16,35,36]. The anorectic responses to AA-IMB diets are comprised of at least two phases [9]. The first phase is an initial recognition of the amino acid deficiency that results from ingesting AA-IMB. The second phase is an anorectic response [9] in which animals develop an aversion to the diet [25,36]. Central nervous system function is known to be integral to the anorectic responses to AA-IMB [20]. After rats are fed an AA-IMB, the concentrations of

Abbreviations: AA-IMB, amino acid imbalanced diet; APC, anterior piriform cortex; BAS, basal diet; COR, correct diet; DMN, dorsomedial nucleus of the hypothalamus. * Corresponding author. Fax: + 1 (916) 752-7690. 0169-328X/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved

PII SO169-328X(96)O0032-O

the limiting amino acid are altered in areas of the brain which have been implicated in the feeding response [reviewed in [9]]. At 2.5 h after ingestion of a threonine-IMB, the concentrations of threonine in the anterior piriform cortex (APC), anterior cingulate cortex, locus ceruleus and nucleus of the solitary tract are reduced significantly [reviewed in [9]]. Similarly, when isoleucine is the limiting amino acid, it is decreased in the APC [11]. The APC has been considered essential for recognition of amino acid imbalance, based on the following observations. Bilateral lesions of the APC increase intake of an AA-IMB to 8 0 - 9 0 % of controls [20,25]. Injection of the limiting amino acid into the APC also significantly increases intake of AA-IMB [3]. The neural signals generated in the APC upon recognition of essential amino acid deficiency likely are communicated to other brain structures to facilitate the food intake depression and subsequent learned aversion. The present studies were undertaken to identify the regions of the brain that may be activated in (1) the early recogni-

K Wang et al. / Molecular Brain Research 40 (1996) 27 34

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sole protein source, have been described in detail previously [12]. Briefly, the basal diet (BAS) contained 12.7% ( w / w ) amino acids as the protein equivalent, providing about 50% of the amino acid requirements for growth. In the present experiment, the growth-limiting amino acid was threonine. The AA-IMB diet was made by adding a mixture of the indispensable amino acids except threonine (an additional 9.18% of the diet) to BAS. The corrected (COR) diet consisted of the imbalanced diet plus enough of the limiting amino acid (1.6%) to correct the imbalance. This COR diet was nutritionally balanced in amino acid pattern, but was otherwise similar in taste and orosensory properties to the AA-IMB diet. All diets contained the necessary vitamins and minerals with corn starch and sucrose (2: 1, w / w ) as the carbohydrates and 5% corn oil as the fat source. Carbohydrates were reduced proportionately when amino acids were added. Reviews of the nutritional model used in the present studies may be found in refs [9,16]. For food intake measurements, food cups containing the test diets were weighed and placed in the cages. At the intervals specified in the experimental design, cups were removed and reweighed. Food intake was taken as the difference between the two cup weights, corrected for spillage. Beginning 3 days after arrival, rats were fed the lowprotein BAS diet for 10-14 days prior to the experimental day. This prefeeding protocol is necessary for prompt expression of the anorectic response to the AA-IMB diets [23], within a few hours of AA-IMB introduction. For the last 3 prefeeding days, the food cups were removed 3 h prior to the onset of the dark cycle to synchronize the first meal of the dark period. There were 15 groups of rats in a 3 × 5 design with three diet conditions (BAS, IMB, and COR) and five time conditions, from 1-6 h after diet introduction. Mean body weights for the groups ranged from 172.0 _+ 2.1 g (mean _+ S.E.) to 198.4 _+ 12.5 g. There were no significant differences in body weight among the groups. On the experimental day, the animals were fed either threonine-IMB, BAS or COR diet at dark onset, depending on their group assignment. Food intake was increased at 1 h in the COR group and decreased at all

tion of threonine-IMB (the present paper), and (2) the later development of a conditioned taste aversion (the accompanying paper [40]). In both studies, immunohistochemistry was utilized to examine the temporal-spatial pattern of c-fos expression in nerve cell bodies throughout the brain at intervals after introduction of a threonine-IMB diet or one of two control diets, either basal (BAS) or corrected (COR). c-Fos is an immediate-early gene whose transcription can be activated rapidly and transiently within 5 min of stimulation [13,28,38]. c-Fos is not expressed constitutively in the brain; it is low or undetectable in the absence of novel input or strong activation [30,38]. Many physiological stimuli elicit the expression of Fos protein in the nervous system [15], with peak expression having been shown at 30-45 min post-stimulation [15,27]. The half-life of Fos is about 2 h [27]. Elevated c-fos expression has the potential for providing insight into the locations of functional activation in the neuraxis. The time sequence has been utilized successfully to establish the temporal-spatial patterns of c-fos expression in response to specific stimuli in other neural systems [5,29,37]. We hypothesized that the temporal order in which brain regions are activated in response to AA-IMB would provide insight into the neural circuitry of the response to amino acid deficiency. Data from this study suggest that similar early increases in Fos immunoreactivity in response to both threonine-IMB and COR diets reflect neural responses to pre-absorptive sensory signals from novel diets. Later increases, seen selectively in the AA-IMB group, may reveal previously unrecognized relays in the neural responses to the postabsorptive metabolic effects of amino acid deficiency.

2. Materials and methods Male Sprague-Dawley rats were housed individually in hanging wire cages in an artificially illuminated laboratory with light onset at 24:00 h, and a 12-h light/dark cycle, at 22 _+ 2°C and food and water ad libitum. Powdered diets, containing L-amino acids (Ajinomoto, Teaneck, NJ) as the

Table 1 Food intake of experimental animals prior to tissue collection for evaluation of c-Jbs expression in brain areas Diet

BAS (n) IMB (n) COR (n)

Time (h) 1

1.5

2

3

6

2.0 _+ 0.1 ~ 5 1.4 _+ 0.1 '~ 5 3.1 + 0.4 t~ 3

2.9 _+ 0.2 ~ 3 1.5 _+ 0.1 ~ 2 2.7 + 0.2 h 3

2.8 + 0.2 b 3 1.9 + 0. I ~ 3 3.9 + 0.1 ' 3

3.5 _+ 0.3 b 3 2.1 _+ 0.3 '~ 3 3.9 _+ 0.4 b 3

8.3 _+ 1.1 b 3 5.9 ± 0.3 ~' 4 7.6 + 0.2 b 3

Values are mean +_ S.E. for food intake (g). Diets were: basal (BAS), threonine-imbalanced (IMB), or correct (COR). n, N u m b e r s o f animals. Values within a time period not sharing similar superscripts are significantly different according to F i s c h e r ' s Protected Least Significant Differences post-hoc test, done after a significant overall A N O V A with P = 0.0001 for main effects of diet and time.

Y. Wang et al. / Molecular Brain Research 40 (1996) 27-34

time points beginning at 1.5 h in the AA-IMB group, as expected (Table 1). The animals were anesthetized with sodium pentobarbital (65 mg/kg, i.p.) at 1, 1.5, 2, 3, or 6 h after diet introduction at dark onset. Anesthetized animals were perfused transcardially with 0.9% NaC1 followed by a 2% paraformaldehyde-picric acid fixative (pH 7.4). Brains were removed and post-fixed for 1.5 h in the same fixative, then switched to a 10% sucrose solution. Brains were sectioned at 40-80 txm on a sliding freezing microtome and processed for peroxidase-antiperoxidase histochemistry (modified from Sternberger [39]) [4]. Sections were incubated overnight with an affinity purified rabbit antiserum against a peptide corresponding to amino acids 3-16 of human c-fos (1:5000; Santa Cruz, SC-52; lot I013) in phosphate-buffered saline (PBS; pH 7.2) with 0.3% Triton X-100 (PBS-T). The following day, the sections were rinsed 3 X 10 min in PBS, incubated with sheep anti-rabbit IgG (Cappel, West Chester, PA; 1 : 300 PBS-T; 1 h, RT), rinsed 3 X 10 min in PBS, incubated with rabbit peroxidase-antiperoxidase (Cappel, West Chester, PA; 1:500 PBS-T; 1 h, RT), and exposed to PBS containing 0.5% 3,3'-diaminobenzidine tetrahydrochloride and 0.03% H202 for 10-12 rain. After a final rinse in PBS, the sections were mounted on gelatin-coated slides, dried overnight, dehydrated through alcohols, cleared with xylene and coverslips were applied. Sections were observed and photographed using an Olympus BH2 microscope.

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Controls for the specificity of the antiserum were performed by incubating sections with antiserum that had been pretreated overnight at 4°C with an excess of the peptide against which the antiserum had been raised (SC52p, Santa Cruz; 10 Ixg/ml of diluted Fos antiserum). All of the staining observed was found to represent immunohistochemically specific localization, because no staining was found on sections that served as absorption controls. It is understood that a specific antibody will bind to its corresponding antigen, but also may bind to unknown molecules with identical or very similar amino acid sequences. Thus, the present descriptions are of Fos-like immunoreactivity. For the sake of brevity the reaction product will be referred to as Fos immunoreactivity. Differences of c-fos expression among diet groups were evaluated in all areas of the brain. Fos is expressed in a graded fashion with individual cells depending on the level of physiological stimulus. Low levels of expression are not differentiable from the background staining present with peroxidase immunohistochemistry. Thus, cell-counting did not reflect gene expression accurately. Therefore, quantification of c-fos expression and Fos immunoreactivity was achieved by scoring, on a scale of 0-5, the intensity of staining, the number of immunoreactive neurons, or a combination of these factors. Validity of the scoring protocol was evaluated using two independent observers, both of whom were blind to the groups from which the sections were taken. Differences among the raters were not signifi-

Fig. 1. Immunoperoxidase photomicrographs of 75 txm coronal sections of the APC which have been incubated with an antiserum to Fos protein. Few Fos-immunoreactive neurons were present in animals ingesting a basal diet (A). Fos expression increased in the APC and EN within 1 h of ingesting a threonine-imbalanced diet (B). Fos levels returned to normal levels by 6 h subsequent to ingesting a threonine-imbalanced diet (C). APC, anterior piriform cortex; EN, dorsal endopiriform nucleus; lot, lateral olfactory tract.

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et al. / Molecular Brain Rese,rch 40 (1996) 27-34

cant. Data were analyzed by PC-SAS (release 6.04, Cary, NC) using the General Linear Model for analysis of variance with the Fisher's protected LSMEANS for post-hoc testing of differences among the three diet groups for each independent time interval. Statistical significance was assumed at P < 0.05.

5

4

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2

I

3. Results o

In neurons of all brain areas examined from the BAS diet group, Fos immunoreactivity did not change over time. Thus, the level of c-f))s expression in the BAS group was taken as the basal level for comparisons among the experimental groups. Scores for basal levels in the APC ranged from 1.7 +_ 0.3 to 2.0 _+ 0.2. The number and staining intensity of Fos-immunoreactive neurons in the APC increased similarly in the COR (to 4.0 _+ 0.1) and threonine-IMB groups (to 4.5 and 4.4 ± 0.2) at I h and 1.5 h. A N O V A results yielded F2. ~ = 31.0, P = 0 . 0 0 0 7 at 1 h and F2, 6 = 7 5 . 5 5 , P = 0 . 0 0 0 1 at 2 h. However, Fos immunoreactivity remained elevated through 3 h in the threonine-IMB group (4.2 ± 0.3, F~,~ = 21.27, P _< 0.002 at 2 h, with significance due to AA-IMB diet. P = 0.0006; Figs. I and 2A). In contrast, at 2 h, Fos immunoreactivity in the COR group decreased significantly compared to the threonine-IMB group (to 2,8 _+ 0.3), but still was significantly higher than the basal level. At 3 h, the AA-IMB group still was elevated while the COR group was not ( F ~ = 5.38, P_< 0.05, difference from COR --- P _< 0.02). At 6 h, c-fi)s expression in both groups had returned to basal levels (Figs. 1 and 2A). In a pattern much like that in the APC, Fos immunoreactivity in the nearby dorsal endopiriform nucleus and claustrum increased, relative to BAS, in the AA-TMB and COR groups from 1-1.5 h (Table 2). At 2 h and 3 h, c-los expression decreased to basal levels in the COR group, but Fos immunoreactivity did not return to basal levels in threonine-IMB group until 6 h (values and significance are given in Table 2). The dorsal endopiriform nucleus and claustrum were considered together because of their similar c-fos expression and close anatomical proximity, even though their functional roles differ (see below). The olfactory tubercle, another nearby area that receives input from the olfactory bulb [14] showed no differences from basal c-fos expression at any time point (Table 2). In the anterior cingulate cortex, at the level of the APC, Fos immunoreactivity in the AA-IMB and COR groups increased significantly over the basal level at 1 h, showed a not-significant trend to be elevated over both COR and BAS in threonine-IMB animals at 1.5 and 2 h, and returned fully to basal level at 6 h (Table 2). However, the levels of Fos immunoreactivity in the cingulate cortex never reached greater than 1.75, just 1 unit above basal. A different pattern was seen in the infralimbic cortex. Fos immunoreactivity was increased in both AA-IMB and

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3

a:

2

e

t

0

C tt. ~"

3

1

1

1.5

2

TIME

3

8

(h)

Fig. 2. c-Fos immunoreactivity(IR score) in the anterior piriform cortex (A), the infralimbic cortex (B), and the dorsomedial nucleus of the hypothalamus (C). Times (h) indicated on abscissa refer to time after first introduction of threonine-imbalanced diet (black bars), corrected diet (hatched bars) or basal diet (open bars), n = 3-5 rats/group. " Significant differences fi'om BAS diet group within a time period (P ~ F. NS = P > 0.05, not significant. Values are means _ S.E > for expression of c-fi)s in designated brain areas graded on a scale of 0-5. Data determined at the same anterior-posterior level as seen in Fig. 1. * Significantly different from BAS group ( P < 0.05) by Fisher's Protected Least Significant Differences test.

c-fos expression in the APC through 3 h in the threonine-

had returned to basal levels (Fig. 2C). No changes in c-fos expression were seen in the other hypothalamic areas examined. Data for the lateral hypothalamic area and paraventricular nucleus are presented in Table 3. The ventromedial hypothalamic nucleus contained a few scattered cells which did not appear to differ in number or intensity among the diet groups at any time.

IMB group supports the idea that at least part of the recognition signal for amino acid deficiency is sensed at the level of the APC. The early increases (1-1.5 h) in c-fos expression, which were similar in both AA-IMB and COR groups, suggest that the APC did not distinguish any early preabsorptive differences between threonine-IMB and the COR diets, both of which were novel for these rats, that would be due to taste and mouth-feel. Later, in the absorptive period (2-3 h), the effects of the decreased limiting amino acid may have been detected within the APC as the animals recognized the difference between the threonineIMB and the COR diets, i.e., the amino acid deficiency induced after ingesting the AA-IMB diet. The changes in Fos immunoreactivity in the dorsal endopiriform nucleus (sometimes called the deep piriform cortex [33]), claustrum, and infralimbic cortex paralleled those in the APC, suggesting that these areas also may be involved in the

4. Discussion Rats with lesions of the APC do not display the normal rejection of AA-IMB diets [20]. Moreover, replacement of the limiting amino acid (in nanomole amounts) into the APC, but not other brain areas, increases intake of the AA-IMB diet [3]. These observations have led to the suggestion that the APC houses the chemosensor for amino acid deficiency [3,9,20,35,36]. The persistent elevation of

Table 3 Effects of basal, imbalanced and corrected diets on expression of c-fos in hypothalamic areas Brain area

Paraventricular nucleus BAS IMB COR Lateral hypothalamus BAS IMB COR

Time (h)

1

1.5

2

3

6

2.00 5:0.16 2.17 ___0.17 2.08 ± 0.08

2.00 + 0.08 2.00 -4- 0.14 2.00 ± 0.08

1.67 + 0.17 2.00 _+ 0.10 2.08 ± 0.22

1.75 + 0.25 2.08 + 0.08 1.75 ± 0.38

1.83 ± 0.17 1.81 ___0.19 1.75 ± 0.14

1.25 ± 0.08 1.00 ± 0.50 1.83 ± 0.17

1.22 ± 0.02 1.17 ± 0.33 1.50 ± 0.29

1.00 ± 0.29 1.13 ± 0.16 1.67 ± 0.33

1.08 ± 0.08 1.33 ± 0.17 1.00 ± 0.50

0.98 ± 0.13 1.31 __+0.19 1.33 ± 0.44

Values are means __+S.E. for expression of c-fos in designated brain areas graded on a scale of 0-5. There were no significant differences among these values. Diet abbreviations are the same as in Table 1.

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Y. Wang et al. / Molecular Brain Research 40(1996) 27-34

early recognition of the amino acid deficiency. Counting of only the most intensely stained cells in selected areas of the APC and infralimbic cortex also suggested that early discrimination of the IMB diet occurred in both areas (data not shown). The sense of smell, per se, is not thought to be essential for recognition of AA-|MB diets, because anosmic (olfactory bulbectomized) rats are fully capable of recognizing and developing aversions to these diets [19]. Still, the APC, which is a primary olfactory cortex, appears to be associated with recognition of amino acid deficiency. Thus, neural elements related to olfaction likely play some yet to be defined role in the responses to amino acid deficiency, This is consistent with observations that certain lesions in the amygdala are able to disrupt odor but not taste aversion learning [8], and with the involvement of rhinencephaliclimbic, rather than gustatory systems in the model studied here. Nevertheless, the presence of differences in the APC, along with the lack of differences in c-los expression in the olfactory tubercle, which also receives primary input from the olfactory bulb [14] suggests that selective expression of c-fos in the APC and endopiriform cortex after 2 h in the AA-IMB group is not due solely to olfactory input. These factors further support our contention that the differential responses between AA-IMB and COR groups are due to the different metabolic effects of the two diets, rather than their olfactory characteristics. The infralimbic cortex has been called the 'autonomic motor cortex' [17] and may be involved in integrating autonomic responses with olfactory cues in reproductive and feeding behaviors [32]. Fos immunoreactivity in the AA-IMB group increased significantly in this area over both BAS and COR groups at 2 h after introduction of the diets (Fig. 2B). This was about the time of the differential response in the APC. However, it did not remain elevated throughout 3 h, like the pattern of APC responses, but by that point, had decreased to prior levels. Lesions of the anterior cingulate cortex accelerate the usual adaptation of the feeding responses to AA-IMB diets as animals alter their food intake and adapt liver enzyme activity [16] over days (rather than hours). However, lesions of the cingulate cortex do not alter initial feeding responses to AA-IMB [26]. The present study showed small changes in Fos immunoreactivity in the cingulate cortex after ingestion of the AA-IMB diet, suggesting minimal activation of this brain area that was similar to the pattern of changes seen in APC, endopiriform and claustrum, but was only significant for the first hour. This limbic brain area may also play a minor role associated with early recognition of the AA-IMB diet. The claustrum has extensive connections with the motor cortex [24] and receives projections from the immediately overlying piriform cortex [6]. From a neurochemical point of view, the claustrum has been suggested to be a cortical area [7]. It could be hypothesized that the claustrum is involved in the signal transduction from the piriform cot-

tex to the motor cortex. The increased expression of c-los early after introduction of AA-IMB suggests that this area may function in relaying information about recognition of AA-IMB diets to motor components of the feeding responses. The dorsomedial nucleus of the hypothalamus (DMN) receives projections from other regions of the neuraxis involved in food intake [18] and taste [34]; the changes observed with lesions of this area have been termed 'the DMN syndrome', and are characterized by decreased growth and food intake but normal body composition [2]. Fos immunoreactivity increased in the DMN at 2 h after introduction of the threonine-IMB, later than the APC and claustrum and the infralimbic cortex. This time course of c:fi)s expression may indicate that the DMN is not the first step in the initial recognition of the amino acid deficiency, but may be a component of recognition, or of the integrative neural responses to AA-IMB, or both. No changes in Fos immunoreactivity were present in this region with either COR or BAS diets. Thus, the increased expression was induced specifically in response to the threonine-IMB diet. The DMN is the only area of the hypothalamus which showed increased c-fos expression after AA-IMB in this study, and also was the only hypothalamic area to show decreases in measured levels of the limiting amino acid, threonine, after ingestion of a threonine-IMB [{12], Gietzen, unpublished results]. A role for the DMN in the responses to AA-IMB diets also is suggested by a recent study showing increased early intake of an AA-IMB diet by rats bearing DMN lesions [1]. In terms of the neural circuitry of the response to amino acid deficiency, it has been shown that the infralimbic cortex receives direct projections from the molecular layer of the piriform cortex, including the endopiriform nucleus [17]. It also has direct connections with the DMN and the central nucleus of the amygdala [17], which may play a role in subsequent aversive responses to AA-IMB diets {40]. The pifiform cortex projects directly to the claustrum [6], but not to the DMN {33]. It is possible that signals generated in the APC, claustrum and infralimbic cortex upon recognition of indispensable amino acid deficiency are communicated via the infralimbic cortex to the DMN. Usually 0.5 to 1 h is required for naive, ad libitum-fed animals to consume an adequate amount of an AA-IMB diet and to demonstrate the anorectic response to the negative postingestive signals related to amino acid deficiency, while COR diets are eaten readily [10,35,36]. The threonine-IMB and COR diets had similar orosensory properties and ingredients, with the exception of the level of threonine, which differed by just 1.4% of the diet. These differential feeding responses to AA-IMB and COR diets have been well documented [16,21,22] and were seen again in the food intake measurements taken for animals used in the present studies (Table 1). The half-life of Fos is about 2 h [31]. Therefore, it may be argued that the decrease of the Fos immunoreactivity, in the APC for

Y. Wang et al. / Molecular Brain Research 40 (1996) 27-34

example, in the C O R group by 2 h m a y have been due to its natural life span after activation by the n e w diet. If the Fos i m m u n o r e a c t i v i t y in the A A - I M B and C O R groups had decreased at the same time and if this decrease had been consistent in all the brain areas, the decrease of Fos i m m u n o r e a c t i v i t y also might be attributed to the natural time course of c-fos expression. Indeed, the time course of Fos expression could explain the lag b e t w e e n the increased food intake of the C O R group by 1 h, indicating acceptance of the diet at that time (Table 1), and the Fos expression in that group which r e m a i n e d elevated through 2h. In conclusion, in the A P C and surrounding areas, the increase of c-fos expression in the t h r e o n i n e - I M B group over the C O R group at 2-3 h m a y be due to the neural recognition of the different metabolic effects of the A A IMB and C O R diets. The infralimbic cortex m a y also function as an activation site in recognition of the A A - I M B diet. The later onset of c - f o s expression in the D M N indicates that this area m a y be important in subsequent steps in the recognition of the A A - I M B . The effects of a m i n o acid deficiency on c-fos expression in the taste pathway and secondary activation of the conditioned taste aversion system are discussed in detail in the c o m p a n i o n paper [40]. The present study shows that the temporal-spatial pattern of neural activation indicated b y c-fos expression is consistent with the initial recognition of a m i n o acid deficiency in the A P C as previously reported by this and other laboratories. Moreover, additional neural areas i n v o l v e d in this recognition are suggested by the pattern of c-fos expression revealed in this work.

Acknowledgements Supported by U S D A C S R S 94-37200-0655, N I H D K 42274, N S 3 3 3 4 7 and D K 35747 and a Collaborative Research Grant from the U C Davis C o m m i t t e e on Research. The authors also are grateful to K i m b e r l y Dixon, Lesa Erecius and Dave Hinds for expert technical assistance and to Dr. K u n i o Torii for his kind collaboration.

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