Physiol. Genomics Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiol. Genomics 16: 38-46, 2003. First published October 14, 2003; doi:10.1152/physiolgenomics.00123.2003
1094-8341/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
16/1/38    most recent
00123.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arens, J.
Right arrow Articles by Korf, H.-W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arens, J.
Right arrow Articles by Korf, H.-W.
Received 25 July 2003; accepted in final form 22 September 2003.
Physiological Genomics 16:38-46 (2003)
1094-8341/03 $5.00 © 2003 American Physiological Society

Age-dependent hypothalamic expression of neuropeptides in wild-type and melanocortin-4 receptor-deficient mice

Janine Arens1,2, Kim M. Moar3, Sandra Eiden2, Karin Weide2, Ingrid Schmidt2, Julian G. Mercer3, Eckhart Simon2 and Horst-W. Korf1

1 Dr Senckenbergische Anatomie, Institut fuer Anatomie II, Fachbereich Medizin, Johann Wolfgang Goethe-Universitaet, D-60590 Frankfurt/Main
2 Max-Planck-Institut fuer Physiologische und Klinische Forschung, W. G. Kerckhoff-Institut, Arbeitsgruppe Energiebilanz und Adipositas, D-61231 Bad Nauheim, Germany
3 Division of Energy Balance and Obesity, Rowett Research Institute, Aberdeen Centre for Energy Balance and Obesity, Aberdeen, Scotland AB21 9SB, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 Perspectives
 REFERENCES
 
In young (35- to 56-day-old) and middle-aged (9-mo-old) wild-type (+/+) and melanocortin-4 receptor (MC4R)-deficient (+/-, -/-) mice, expressions of neuropeptide Y (NPY), agouti-related protein (AGRP), pro-opiomelanocortin (POMC), and cocaine-and-amphetamine-regulated transcript (CART) were analyzed in the arcuate nucleus (ARC) and adjacent regions comprising the dorsomedial (DMN) and ventromedial (VMN) nucleus. In the ARC of young mice, NPY and AGRP expression increased and POMC and CART expression decreased with body fat content. Adjusting for the influence of body fat content by ANCOVA showed that the levels of NPY, POMC, and CART were highest and of AGRP lowest in young -/- mice. In the middle-aged mice, feedback from body fat content was weakened. For -/- mice ANCOVA revealed higher NPY and AGRP, lower POMC, and unchanged CART expression levels relative to young -/- mice. In the DMN and VMN, POMC and AGRP signals were absent at each age. CART was expressed in the DMN independent of age, fat content, and genotype. For NPY expression, an age-dependent induction was found in the DMN and VMN; it was absent in the young but present in the middle-aged mice, showing close positive correlations between body fat content and the numbers of NPY-labeled cells which were further enhanced in -/- mice. Thus MC4R deficiency augments age-induced NPY expression in the DMN and VMN with no feedback from body fat content. Negative feedback control by body fat content on ARC neuropeptide expression is present in young animals but vanishes with age and is modulated by MC4R deficiency.

orexigenic and anorexigenic neuropeptides; arcuate hypothalamic nuclei; ventromedial hypothalamic nuclei; dorsomedial hypothalamic nuclei; central leptin signaling; in situ hybridization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 Perspectives
 REFERENCES
 
FOOD INTAKE IS CONTROLLED by complex neuronal circuits in the hypothalamus. The arcuate nucleus (ARC) is a site where orexigenic [neuropeptide Y (NPY) and agouti-related protein (AGRP)] and anorexigenic [pro-opiomelanocortin (POMC) and cocaine-and-amphetamine-regulated transcript (CART)] neuropeptides are produced. Their expression is modulated by leptin, a hormone synthesized by adipocytes, which suppresses and stimulates, respectively, the orexigenic and anorexigenic peptides. The medial part of the ARC comprises the highest number of NPY-containing cell bodies which synthesize large amounts of this neuropeptide and project mainly to the paraventricular nucleus (PVN) of the hypothalamus. Other sources of hypothalamic NPY are the dorsomedial (DMN) (18, 19) and the ventromedial (VMN) hypothalamic nuclei, but in general these nuclei express lower amounts of NPY (18). The lateral part of the ARC contains neurons which synthesize POMC, the precursor of {alpha}-melanocyte-stimulating hormone ({alpha}-MSH) (35). The DMN, PVN, and the lateral hypothalamus (LH) contain high numbers of the melanocortin-4 receptor (MC4R). These are activated by the anorexigenic peptide {alpha}-MSH. AGRP is coexpressed with NPY in the same population of neurons in the ARC (14). It acts as the natural antagonist of {alpha}-MSH at MC4R via competitive displacement (31) and can be considered as a neuropeptide for precise adjustment of the {alpha}-MSH/MC4R system. CART is coexpressed with POMC in neurons of the ARC (6); other sources of CART are the LH, PVN, and DMN.

Induction of NPY expression in the DMN is reported for lactating rats (25), diet-induced obese mice (13), tubby mice (12), obese Agouti yellow mice and obese homozygous (-/-) MC4R-deficient mice (21), but obesity is not necessarily associated with detectable NPY expression in the DMN, because obese ob/ob mice did not display any expression (21). NPY expression in DMN neurons is under the control of the melanocortinergic system (21); {alpha}-MSH normally binds at MC4R in the DMN and inhibits the NPY expression in this nucleus. The lack of MC4R might induce NPY expression in the DMN (21). In previous studies in the melanocortin-4 receptor gene knockout (MC4r-KO) model, the induced NPY expression in the DMN was only found in obese homozygous (-/-) mice but not in heterozygous (+/-) and wild-type (+/+) mice of unspecified age (21).

Here we present results obtained by investigating the expressions of NPY, POMC, AGRP, and CART in the ARC and the adjacent hypothalamic tissue comprising the DMN and VMN of 9-mo-old mice of each genotype and, for reasons of comparison, corresponding expression data obtained for young (35- to 56-day-old) mice. The results extend and complement previous findings on NPY and POMC expression in the ARC of young mice, which showed significant common regressions with body fat content for animals, irrespective of genotype at MC4r (34). In addition, we report that induced expression of NPY occurs in the DMN and VMN at the age of 9 mo not only in -/- but also in +/- and +/+ mice and that its expression level is distinctly dependent on body fat content and genotype.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 Perspectives
 REFERENCES
 
Animals.
We used male and female 35-day-old, 56-day-old, and 9-mo-old +/+, +/-, and -/- mice raised in our colony in Bad Nauheim founded in 1999 with heterozygous breeding pairs from the original MC4r-KO line (16) kindly provided by D. Huszar (Millennium Pharmaceuticals). Genotyping followed the recently described procedure (34). The set of 35- and 56-day-old animals used for the present study includes animals for AGRP and CART analysis in the ARC in which NPY and POMC expression had previously been analyzed (34). Animals were maintained on standard pelleted food (type 1314; Altromin, Lage, Germany) at 25°C in a 12:12-h light/dark cycle.

Determination of body fat content.
Two hours before the end of their daily light phase, the mice were exposed to CO2 gas for about 30 s and then decapitated. Brains were quickly removed and frozen on dry ice. Blood was collected in heparinized tubes on ice and centrifuged. Brains and plasma aliquots were stored at -80°C. Carcass mass was determined after removing stomach and intestine and emptying the bladder. Body fat content (fat mass as percentage of carcass mass) was evaluated by drying the carcasses at 75°C to constant weight followed by total body chloroform extraction in a Soxhlet apparatus and drying again to constant weight (27).

Plasma measurements.
For leptin measurements we used a mouse RIA kit (Linco, St. Charles, MO). As previously described (8), measurements were independently duplicated, and variability was decreased by correcting the data for interassay variability and buffer dilution using internal correction factors.

In situ hybridization.
In situ hybridization was carried out on coronal hypothalamic slices. The animals investigated covered the entire range of body fat contents found in body composition analysis for each age group. Neuropeptide expression in the ARC was quantified by densitometry in three (AGRP, 79 animals; CART, 81 animals) or four (NPY, 96 animals; POMC, 96 animals) independent experimental sets. In addition, areas adjacent to the ARC comprising DMN and VMN were controlled for neuropeptide expression and, in the case of CART, quantified in a subset of 55 animals. Each set comprised animals of all genotypes. Additionally, nonradioactive in situ hybridization for NPY and POMC was carried out in one experimental set to count neuropeptide-positive cells in the DMN and VMN. Neuropeptide expression was determined in adjacent 20-µm-thick coronal sections, equivalent to bregma -0.34 mm to -2.54 mm in the mouse brain according to Franklin and Paxinos (9). The sections were mounted on poly-L-lysine-coated slides and stored at -80°C. Prior to hybridization, sections were fixed in 4% paraformaldehyde. For histological control of the locations of ARC, DMN, and VMN, parallel sections were stained with cresyl violet or neutral red.

Antisense and sense probes were prepared for each investigated mRNA. For prepro-NPY mRNA, the probes were generated from a 0.5-kb fragment of rat cDNA cloned into BlueScript M13(-) vector. AGRP and POMC cDNA fragments were cloned from Siberian hamster hypothalamic cDNA and ligated into pGEM-T Easy as described in detail elsewhere (28). CART cDNA was amplified from total RNA from GH3 (rat pituitary) cells by random-primed RT-PCR amplification using techniques described in detail elsewhere (1).

For radioactive in situ hybridization, antisense and sense NPY, POMC, AGRP, and CART probes were labeled with 35S-UTP, as described (27). The 35S-labeled riboprobes were used at a concentration of ~2 x 107 cpm/ml. After hybridization, slides were treated with RNase A, desalted with a final high-stringency wash, dehydrated, and apposed to Hyperfilm ß-max (Amersham). Autoradiographic images of the ARC were quantified using the Image-Pro Plus system (Media Cybernetics, Silver Spring, MD). Data were standardized with 14C-autoradiographic scales (Amersham Biosciences, Little Chalfont, UK). Gene expression was measured as the integrated intensity of the autoradiographic signal, i.e., as the background-corrected optical density integrated over all pixels in the hybridization area. Normally three sections of each brain were analyzed and data were averaged.

For nonradioactive in situ hybridization, antisense and sense NPY and POMC probes were labeled with digoxigenin (DIG)-11-UTP, according to the manufacturer’s instructions (Boehringer, Mannheim, Germany). After hybridization, slides were desalted with a final high-stringency wash. Specific labeling was visualized by incubation with antidigoxigenin serum coupled to alkaline phosphatase followed by 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT). Slides were then air dried and embedded with Entellan (Merck). Cells expressing NPY were counted separately in the VMN and DMN of each brain in three sections passing through the maximal extension of the two nuclei by an investigator without knowledge of genotype and fat content. Counts were averaged for each nucleus and pooled for statistical analysis, since the labeling in the VMN and DMN showed no differential changes.

For both assays, specificity was confirmed by control experiments using the sense probes. No hybridization signals were observed under these conditions.

Statistical evaluation.
For a given neuropeptide, the densitometric data obtained in each in situ hybridization run were standardized by z-transformation, using the SigmaStat program (SPSS, Chicago, IL), to permit common evaluation of the different runs. Regression analysis was then applied to the relationship between the standardized data on NPY, POMC, AGRP and CART expression and body fat content. Because the preceding study (34) had shown that males and females might differ in the absolute levels of neuropeptide expression and body fat mass, but not in the correlation of the standardized expression data with body fat content, males and females were commonly evaluated in this study. In addition, the general relationship between neuropeptide expression and body fat content of the mice was analyzed for potential influences of MC4R deficiency and age. Analysis of covariance (ANCOVA) was used to disclose the influence of genotype as categorical variable independently from that of continuous covariates, such as fat content or plasma leptin concentration, thereby "partializing out" (2) the covariate influence. ANCOVA was carried out with the Statistica program (StatSoft, Tulsa, OK).

NPY-expressing neurons marked by nonradioactive in situ hybridization in the DMN and VMN were counted in specimens from six animals of each genotype. Correlations between fat content and the number of NPY-expressing neurons determined in individual animals were calculated for each genotype. Regressions were analyzed for differences in slope or y-intercept. For the entire sample, ANCOVA was applied with genotype as factor and fat content or plasma leptin concentration as covariate to partialize out its influence.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 Perspectives
 REFERENCES
 
Differential neuropeptide expression in the ARC.
Proceeding from a previous study on a smaller sample of young +/+, +/-, and -/- mice of the MC4r-KO strain (34), the present investigation not only reexamined the observed influences of genotype and body fat content on NPY and POMC expression in the ARC but also analyzed corresponding effects on AGRP (coexpressed with NPY) and CART (coexpressed with POMC). The additional analysis of age as a factor of potential influence on neuropeptide expression was based on the comparison of 9-mo-old with 35-day- and 56-day-old mice of each genotype. The standardized neuropeptide expression data and percent body fat content were related to each other. In Fig. 1 these relationships are illustrated for the sample of 35- and 56-day-old animals by regressions with 95% confidence limits, in line with previous observations (34; and I. Schmidt, unpublished data for AGRP and CART). The common regressions document that at this age the dependence of neuropeptide expression from body fat content prevailed over potential genotype differences. The correlation found for the samples of the 35- and 56-day-old mice was negative for NPY expression (Fig. 1A) and for the coexpressed AGRP (Fig. 1C). POMC expression was positively correlated with body fat content (Fig. 1B); the same tendency was also seen for the coexpressed CART (Fig. 1D).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 1. Relationship between body fat content (logarithmic scale) and the expression data standardized for each experimental run commonly for young and middle-aged mice (see METHODS). Data for the 35- or 56-day-old mice are represented by regression lines with 95% confidence limits for neuropeptide Y (NPY; A), agouti-related protein (AGRP; C), pro-opiomelanocortin (POMC; B), and cocaine-and-amphetamine-regulated transcript (CART; D) in the hypothalamic arcuate nucleus (ARC). Validity of common regressions for +/+, +/-, and -/- mice had previously been demonstrated for NPY and POMC expression data which form part of the present evaluation (34) and for AGRP and CART (I. Schmidt, unpublished data). Correlation coefficients: r = -0.57 (A), r = 0.52 (B), r = -0.59 (C), all P < 0.001; and r = 0.22 (D), P < 0.1. The squares represent the expression data from 9-mo-old mice [+/+ (solid black symbols), +/- (gray symbols), and -/- (open symbols)] for which no significant correlations with body fat content were found. Expression is presented in arbitrary units (a.u.).

 
Our study confirmed previous findings (34) that the amount of body fat is closely reflected by plasma leptin levels (r = 0.9, not shown). The correlations between the standardized neuropeptide expression data and the plasma leptin concentration of the 35- to 56-day-old animals (not shown) were similar to the correlations between neuropeptide expression and body fat content shown in Fig. 1. Correlation coefficients were r = -0.53 for NPY expression, 0.49 for POMC expression (both P < 0.001), and -0.39 for AGRP expression (P < 0.01). The correlation between CART expression and plasma leptin concentration was not significant (r = 0.11).

In contrast, in the 9-mo-old animals neither body fat content nor plasma leptin concentration was significantly correlated with the standardized expression data for any of the neuropeptides. Therefore, those data pairs are presented individually in Fig. 1, with different genotypes being indicated by different symbol fillings. Although there is a considerable overlap in the range of body fat contents of the middle-aged +/+ and +/- animals with that of the younger animals (5–35%), the body fat contents of a substantial number of the 9-mo-old MC4R-deficient mice are higher, ranging between 35 and 60%. For NPY, the expression data are distributed over the same range as in the younger animals, but, in relation to fat content, most of these data are higher than those represented by the regression for the younger animals (Fig. 1A). For POMC, the expression data of the 9-mo-old animals of all three genotypes tend to be lower than in the younger animals. Especially in relation to body fat content, these data are clearly lower than those represented by the regression for the younger mice (Fig. 1B). For AGRP (Fig. 1C), expression data and body fat content in the 9-mo-old mice are related to each other within the same range as in the younger animals only for body fat contents <35%. At higher fat contents, however, AGRP expression levels are distinctly higher in these middle-aged animals and thus reveal a tendency similar to that found for the coexpressed NPY. For CART, on the other hand, the expression data of the 9-mo-old +/+ animals tend to be lower than those of the younger animals, whereas those of -/- mice mostly correspond to the range of the younger animals (Fig. 1D).

For statistical evaluation of genotype influences, the expression data were analyzed separately for the samples of the young and of the middle-aged animals by ANCOVA with genotype as factor and percent body fat content as covariate to partialize out its influence. The overall genotype effects were significant in the younger animals for the expression of each neuropeptide (P < 0.05 for CART and P < 0.01 for the others). By contrast, the middle-aged animals showed a significant dependence on genotype at MC4r only for AGRP expression (P < 0.01). Corresponding statistical evaluation with plasma leptin as covariate also confirmed genotype effects for all analyzed neuropeptides in the younger animals but revealed no significant genotype effects on either of the neuropeptides in the middle-aged animals.

Figure 2 demonstrates the direction of the genotype-dependent changes of the expression data in the young and middle-aged mice for each of the investigated neuropeptides. After partializing out the effect of body fat content by ANCOVA, NPY expression is highest in the -/- mice of each age group. The middle-aged animals, moreover, display a distinctly higher level of expression (P < 0.01 for the overall effect of age), with the difference to the younger animals being significant in the post hoc test for the +/- and the -/- genotype. POMC expression increased with the number of the defective alleles in the younger animals. This increase contrasts with a marked decrease in expression in the middle-aged -/- mice (P < 0.05 for the post hoc test and P < 0.01 for interaction between age and genotype). The genotype effects on AGRP expression are also deviant for the two age groups (P < 0.01 for interaction between genotype and age). The tendency for a decrease in expression with increasing gene dose seen in the younger animals is completely reversed in the middle-aged animals for which post hoc testing confirms significantly higher expression levels in the older compared with the younger +/- and -/- mice (P < 0.05). Only the levels of CART expression and their genotype dependence are similar in the two age groups.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 2. Expression data in the ARC from different radioactive in situ hybridization runs for each neuropeptide standardized commonly for young and middle-aged mice to permit evaluation of age influences (see METHODS) for NPY (A), POMC (B), AGRP (C), and CART (D). Results (least square means ± SE) of ANCOVA with genotype and age as factors and fat content as covariate to partialize out its influence. Significance of overall effects is presented in the text. Significant differences between age groups for a given genotype, according to post hoc testing, are indicated. *P < 0.05. **P < 0.01. ***P < 0.001. Diamonds indicate 35- to 56-day-old mice, and squares indicate 9-mo-old mice. Genotypes are also indicated: black (+/+), gray (+/-), and white (-/-).

 
Selective induction of NPY expression in the DMN and VMN.
According to radioactive hybridization analysis, expressions of AGRP and POMC remained confined to the ARC, independent of age and genotype. CART was expressed in each age group adjacent to the ARC especially in the region corresponding to the DMN, in line with previously reported evidence (18). Densitometric data for CART expression were obtained from 35-day-old (n = 23), 56-day-old (n = 16), and 9-mo-old (n = 16) animals. ANCOVA showed that the standardized expression data were related neither to fat content or leptin (as covariates) nor to age and genotype as categorical factors (lowest P = 0.35). Age-related changes were observed only for NPY expression. Although the signal remained strictly confined to the ARC in the 35- and 56-day-old animals, it had clearly expanded toward the DMN and VMN at the age of 9 mo. Visually the in situ hybridization autoradiographies indicated that the strength of the signal in these two nuclei adjacent to the ARC was genotype dependent. However, the high intensity of the radioactive signal in many of these preparations did not allow for a selective quantification of the NPY signal in the DMN and VMN. Therefore, nonradioactive in situ hybridization was applied in order to quantify NPY expression by counting labeled cells in the DMN and VMN.

Nonradioactive in situ hybridization confirmed the absence of NPY-expressing cells in the DMN or VMN of 35- and 56-day-old mice (n = 19), irrespective of genotype at MC4r. At 9 mo of age, however, labeled cells were clearly present in both nuclei and in each genotype (n = 6 each). Figure 3 compares examples of sections hybridized with the DIG-labeled probe from 56-day-old +/+ mice (Fig. 3, A–C) with corresponding sections from 9-mo-old animals (Fig. 3, D–F). In 9-mo-old +/+ animals, NPY-labeled cells are not restricted to the ARC, but occur also in the VMN and DMN. The cells in the VMN and DMN are scattered and thus countable, unlike in the ARC, where the NPY-labeled cells are densely clustered at each age. At variance with the radioactive in situ hybridization, the borders between the ARC and the DMN and VMN could be easily determined by the nonradioactive method and are indicated in the sections. Figure 4 illustrates for the 9-mo-old mice the genotype-dependent increase of the number of NPY-labeled cells in both the DMN and VMN. The NPY-labeled cells in the VMN and DMN were counted separately in coronal sections from six specimens of each genotype, but there was no evidence for differential increases in NPY expression in the two nuclei. Therefore the cell counts in the VMN and DMN were pooled for the statistical analysis assessing the relationship between the number of NPY-expressing cells with genotype and fat content. At variance with NPY, an induced POMC expression also could not be detected by nonradioactive in situ hybridization in the VMN and DMN in any group of animals investigated here (data not shown).



View larger version (91K):
[in this window]
[in a new window]
  		Fig. 3. Coronal sections through the ARC (left), ventromedial nucleus (VMN, middle), and dorsomedial nucleus (DMN, right) in the hypothalamus of +/+ mice. Cells expressing NPY are labeled by nonradioactive in situ hybridization. A–C: slices from 56-day-old animals. D–F: slices from 9-mo-old animals. 3V, third ventricle. The borders of the nuclei are indicated by dotted lines. Arrows point to some of the labeled cells located near the focal plane of the microscope when taking the photograph. For details, see text.

 


View larger version (120K):
[in this window]
[in a new window]
  		Fig. 4. Coronal sections through the VMN (left) and DMN (right) in the hypothalamus of +/+ (A and B), +/- (C and D), and -/- (E and F) 9-mo-old mice. Cells expressing NPY are labeled by nonradioactive in situ hybridization. For details, see text.

 
In Fig. 5A, cell counts and body fat content obtained for the individual animals are plotted against each other, and regressions are calculated separately for each genotype. The regressions are generally close and positive, different from the negative correlation between body fat content and NPY expression in the ARC of younger animals. For the +/+ and +/- mice (both r > 0.9) they are virtually identical in slope and intercept, whereas the regression found for the -/- mice is similarly close but runs significantly steeper (P < 0.05 for difference in slope compared with the common regression for +/+ and +/- mice). The results of regression analysis are supported by ANCOVA applied to the entire set of NPY expression data, with genotype as factor and fat content as covariate. As expected, it confirms the highly significant positive relation to fat content (P < 0.001) and a significant genotype influence (P < 0.05).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 5. A and B: linear regressions (all r > 0.9) between numbers of NPY-expressing cells (neurons) in the DMN and VMN of 9-mo-old animals (n = 18) and percentage of body fat content (A) and plasma leptin concentration (B) calculated separately for each genotype: +/+ is indicated by black squares and solid line; +/- is indicated by gray squares and long-dashed line; and -/- is indicated by white squares and short-dashed line. For each relationship, the regressions obtained for +/+ and +/-mice are virtually identical, permitting calculation of common regressions (see text). For the -/- mice, the regression of cell counts with fat content (A) is significantly steeper, and the regression of cell counts with plasma leptin (B) is shifted to a significantly higher level compared with the corresponding common regressions for the +/+ and +/- mice. For details, see text.

 
The plasma leptin concentrations measured in the same group of animals were found to increase exponentially with body fat content (r = 0.93 for the log-linear correlation). Accordingly, leptin concentration and NPY expression are closely correlated, too (r > 0.9), as shown in Fig. 5B. Regressions for +/+ and +/- mice are again virtually identical. The regression found for the -/- mice runs in parallel at a significantly higher level (P < 0.05 for difference in y-intercept) than the common regression for the +/+ and +/- animals.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
Perspectives
 REFERENCES
 
The results of the present study on the MC4r-KO obesity model convey three main messages concerning neuropeptide expression in neurons of the ARC, the primary neural target of the lipostatic hormone leptin, and the VMN and DMN, two downstream targets. First, they confirm that, in mice of 1–2 mo of age, expressions of the antagonistic peptides NPY and POMC in the ARC are oppositely related in a counter-regulatory manner to body fat content and plasma leptin concentration, irrespective of genotype at MC4r (34). As a novel result, changes of AGRP and CART expression were found to parallel those of NPY and POMC, respectively, with which they are coexpressed (Fig. 1). At the age of 9 mo, however, ARC neuropeptide expression is no longer significantly correlated with body fat content. Second, genotype at MC4r significantly influences the expression of all four neuropeptides in the ARC in young mice, whereas in middle-aged mice genotype dependence is significant only for AGRP and, moreover, opposite in direction to that in younger animals (Fig. 2). Third, the expression of NPY is restricted to the ARC in 2-mo-old mice but has expanded to the neighboring DMN and VMN at the age of 9 mo. POMC and AGRP are not expressed in these nuclei up to this age, and the constitutive expression of CART in the DMN is not affected by age, fat content, and genotype. Most importantly, at 9 mo of age the number of NPY-expressing DMN and VMN neurons correlates positively with body fat content and plasma leptin concentration, i.e., the relationship is opposite to the negative feedback response found for NPY expression in the ARC of the younger mice. Moreover, an excess increase in the number of NPY-expressing DMN and VMN neurons is seen in the -/- mice (Fig. 5). The data suggest that MC4R deficiency initiates a lifelong process of deteriorating central signaling within the multifactorial control system for energy balance.

Age-dependent changes of neuropeptide expression in the ARC.
The present study has disclosed modulatory influences of body fat content and MC4R deficiency during the time span from shortly after weaning to the end of the fertile period. During this period body fat content increases with age preceding its decline toward senescence. This biphasic course of fat content has, so far, not been particularly considered in studies investigating animals from early adulthood to old age. This would explain why the reported age-dependent changes in hypothalamic neuropeptide expression are, in part, contradictory (11, 10, 20, 23, 26, 30). The interpretation of age-related changes of neuropeptide expression patterns is further complicated by the possibility of growing leptin resistance with age, especially as obesity develops (33, 26). Studies at an age when animals were certainly leptin responsive and thus capable of negative feedback control of body fat content have shown that hypothalamic NPY mRNA levels are lower in 9-wk-old, sexually mature mice than in 6-wk-old, sexually immature mice (4). This suggests a suppressive negative feedback effect of increasing fat content on the expression of the orexigenic NPY. Previous studies in genetically obese mice (32) and rats (17) at ages up to 40 wk have not distinguished between primary influences of genotype and secondary effects of changing fat content on changes of hypothalamic NPY expression with age. Moreover, analyzing neuropeptide expression patterns in total hypothalami and not in distinct nuclei, as in the present study, may have precluded the disclosure of evidence for cause-and-effect relationships underlying the complex age-dependent patterns of neuropeptide expression demonstrated in the present study on wild-type and MC4R-deficient animals.

How do age, fat content, and genotype at MC4r interact in changing ARC neuropeptide expression?
The present study was designed to cover the period from young adulthood to the end of the fertile period during which leptin or insulin resistance is known to develop, i.e., a period of life carrying an increasing adiposity risk for humans as well. Here we found that influences of both body fat content and MC4R deficiency on neuropeptide expression in the ARC are distinct in the young mice but have become obscured in the middle-aged animals (Fig. 1). Growing leptin resistance with advancing age and increasing fat deposition, which is particularly prominent in MC4R-deficient mice, would explain why NPY expression in the ARC progressively escapes the negative feedback control exerted by the lipostatic hormone. The influence of MC4R deficiency on neuropeptide expression (disclosed after adjusting for differences in fat content by ANCOVA) also differs distinctly between young and middle-aged animals. Young animals display enhancing effects of MC4R deficiency on the expression of both the anorexigenic POMC and the coexpressed neuropeptide CART but divergent effects on the orexigenic NPY and the coexpressed AGRP, with the former being highest and the latter lowest in -/- mice of this age. In the middle-aged animals only the relation between CART expression levels and genotype remains similar to that found in the young animals. NPY expression is significantly stronger than in the younger +/- and -/- mice, and the same tendency is found for the +/+ mice. POMC expression is massively decreased in the middle-aged -/- mice, in contrast to the enhancing effect of the MC4R deficiency in the younger animals. The genotype effect on AGRP expression is opposite to the suppressive genotype influence in the younger animals, with the levels for the middle-aged +/- and -/- animals being equally enhanced relative to the +/+ mice of the same age. Taken together, the changing neuropeptide expression patterns with increasing age suggest that MC4R deficiency progressively alters the relationships between the central peptidergic pathways originating in the ARC which normally act as mutually inhibitory controllers of energy balance. This even includes altered relationships between peptides that are coexpressed. The resulting regulatory disturbance is characterized by the progressive impairment of body fat content-related feedback control.

Changing relationships among coexpressed neuropeptides in other animal models.
Changes in the levels of coexpressed neuropeptides in connection with disturbances of energy balance are described in various animal models. In Sprague-Dawley and lean heterozygous (+/fa) and obese homozygous (fa/fa) Zucker rats, the relationship between expression of NPY and AGRP was found to be differentially dependent on starvation and genotype, presumably due to differential sensitivities to leptin and other controlling factors, including insulin (22). Coexpression of POMC and CART in a distinct set of ARC neurons is probably less stringent, and quantitative differences in their expression have been reported in response to leptin administration to ob/ob mice (7). Altered relationships between coexpressed neuropeptides are thus not uncommon, although the factors controlling such changes are still obscure. The highly complex interactions between the peptidergic neurons under consideration (3, 5, 15), even to the extent of positive feedback influences (6), may be influential. Differential susceptibilities to MC4R deficiency, age, and other specific physiological parameters, e.g., feeding conditions (22, 29), may underlie the altered relationships between coexpressed neuropeptides seen in the present study.

Induced NPY expression in DMN and VMN reflects age, body fat content, and genotype at MC4r.
The results of radioactive in situ hybridization analysis have identified NPY as the only neuropeptide changing its expression in the DMN and VMN during the investigated age period, whereas POMC and AGRP signals were absent at each age and CART was expressed independent of age in the DMN in line with previous observations (18). Quantitative evaluation of induced NPY expression by nonradioactive in situ hybridization confirmed the absence of the NPY signal in the younger animals but its presence in the middle-aged animals. The tight positive correlation between the number of NPY-labeled cells in the DMN and VMN and body fat content (or plasma leptin concentration) is opposite to what would be expected if induced NPY expression in these nuclei would be subject to negative feedback control by body fat content. Rather, the observed genotype-dependently enhanced NPY expression in the DMN and VMN would suggest that its central hyperphagic action caused enhanced fat deposition and the consecutive rise of plasma leptin concentration. It is important to note that the tight positive correlation between NPY expression in the DMN and VMN and body fat content is established at an age at which NPY expression in the ARC still tends to decrease with increasing body fat content, albeit no longer significantly, suggesting remnants of its counter-regulatory control, probably by leptin as a fat content-dependent lipostatic signal.

Induced expression of NPY in the DMN and VMN has been previously described for several genetic models of obesity, including Agouti yellow mice (21), tubby mice (12), and -/- MC4r-KO mice (21). Although the age of the MC4r-KO mice was not specified in this study, their body weight suggests that they were 5–6 mo old (21, 16). At this age induced NPY expression was present in -/- but not yet in +/- and +/+ animals (21). All three models of obesity have in common a reduced functionality of the melanocortinergic system. Moreover, induced NPY expression has been observed in the DMN and VMN of rats in states of increased energy demand, i.e., during lactation (25), intense exercise, and food restriction (24). Induced NPY expression in the DMN is also found in diet-induced obese mice (13). Apart from confirming the age dependence of induced NPY expression, the present study demonstrates for the first time its tight positive relation to body fat content and plasma leptin concentration in wild-type as well as MC4R-deficient mice. The absence of the NPY signal in the 2-mo-old mice of all genotypes, its earlier occurrence in -/- mice (21), and its upward shift in the 9-mo-old -/- relative to the +/- and +/+ mice, indicate that the age-dependent onset of induced NPY expression in the DMN and VMN is enhanced by the complete lack of MC4R. The demonstration of NPY-expressing cells in the DMN and VMN appearing well after adulthood, although at an age usually exceeded by the life span of laboratory mice, does not exclude the possibility that in later stages NPY expression in +/- mice may also deviate from that in +/+ mice. The absence of POMC and AGRP expression and the stable constitutive expression of CART in the middle-aged animals also does not exclude further changes of the neuropeptide expression pattern in the DMN and VMN with advancing age.


   Perspectives
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 Perspectives
REFERENCES
 
By combining systemic analysis of energy balance with determination of neuropeptide expression, we were able to disclose modulation of age-related changes in the expression of orexigenic and anorexigenic neuropeptides in the ARC by body fat content and MC4R deficiency. It is also evident that the strength of the age-related induction of NPY expression in the DMN and VMN depends on the genotype (see also Ref. 21). The NPY signal in these nuclei, once emerged, obviously is not under negative feedback control by body fat content. Rather, the strong orexigenic action of NPY would explain the tight positive relation between its expression level and fat deposition. Further studies of comparable design are necessary to investigate systemic parameters, e.g., food intake and plasma insulin levels, which might be responsible for this positive correlation. Likewise, intervening variables other than leptin which might contribute to the negative feedback of body fat on ARC neuropeptide expression in young animals, even those lacking MC4R, have to be identified. Equally essential is the elucidation of the previously discussed highly complex, mutual interactions between the orexigenic and anorexigenic central peptidergic neurons in order to understand their involvement in the observed age-related changes. At the cell- and neuro-biological level, this requires quantification of signal peptides, analysis of signal-receptor interactions at the cellular level, and identification of the underlying neuronal circuits. Detection of neuropeptide expression at the level of single hypothalamic neurons, like in the present study, can be an important tool to achieve this aim. However, all these techniques need to be combined with the tools of systemic physiology applied at defined functional states (e.g., different ages, feeding conditions), to provide meaningful information on the functional relevance of changes seen in the neuronal networks that control energy balance and on the critical sites of neuronal interactions, where age, genotype, and systemic factors exert their influences.


   ACKNOWLEDGMENTS
 
We are deeply indebted to Dennis Huszar (Millennium Pharmaceuticals, Cambridge, MA) for providing us with breeding pairs of the original MC4r knockout line. We thank Johannes Hebebrand and Anke Hinney, Marburg, for initiating the study and diligently providing the organizational and financial framework. We acknowledge the help of Christine Pauli in some parts of the study.

This work was supported by a doctoral stipend to J. Arens (Graduiertenkolleg 361: "Neuronale Plastizität: Moleküle, Strukturen, Funktionen," University Frankfurt/Main) and by the Federal Ministry of Education and Research in the framework of the National Genome Research Network Neuronetz Marburg (01GS0118), the German Science Foundation (Schm 680/4), and the Scottish Executive Environment and Rural Affairs Department (to K. M. Moar and J. G. Mercer).


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: I. Schmidt, Max-Planck-Institut, W.G. Kerckhoff-Institut, Parkstrasse 1, D-61231 Bad Nauheim, Germany (E-mail: ingrid.schmidt{at}kerckhoff.mpg.de).

10.1152/physiolgenomics.00123.2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 Perspectives
 REFERENCES
 

  1. Adam CL, Moar KM, Logie TJ, Ross AW, Barrett P, Morgan PJ, and Mercer JG. Photoperiod regulates growth, puberty and hypothalamic neuropeptide and receptor gene expression in female Siberian hamsters. Endocrinology 141: 4349–4356, 2000.[Abstract/Free Full Text]
  2. Briere J and Elliott DM. Sexual abuse, family environment, and psychological symptoms: on the validity of statistical control. J Consult Clin Psychol 61: 289–290, 1993.[CrossRef]
  3. Broberger C, Landry M, Wong H, Walsh JN, and Hökfelt T. Subtypes Y1 and Y2 of the neuropeptide Y receptor are respectively expressed in pro-opiomelanocortin- and neuropeptide-Y-containing neurons of the rat hypothalamic arcuate nucleus. Neuroendocrinology 66: 393–408, 1997.[ISI][Medline]
  4. Chua SC Jr, Leibel RL, and Hirsch J. Food deprivation and age modulate neuropeptide gene expression in the murine hypothalamus and adrenal gland. Mol Brain Res 9: 95–101, 1991.[Medline]
  5. Cowley MA, Smart JL, Rubinstein M, Cerdán MG, Diano S, Horvath TL, Cone RD, and Low MJ. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411: 480–484, 2001.[CrossRef][Medline]
  6. Dhillo WS, Small CJ, Stanley SA, Jethwa PH, Seal LJ, Murphy KG, Ghatei MA, and Bloom SR. Hypothalamic interactions between neuropeptide Y, agouti-related protein, cocaine- and amphetamine-regulated transcript and alpha-melanocyte-stimulating hormone in vitro in male rats. J Neuroendocrinol 14: 725–730, 2002.[CrossRef][ISI][Medline]
  7. Dhillon H, Ge YL, Minter RM, Prima V, Moldawer LL, Muzyczka N, Zolotukhin S, Kalra PS, and Kalra SP. Long-term differential modulation of genes encoding orexigenic and anorexigenic peptides by leptin delivered by rAAV vector in ob/ob mice. Relationship with body weight change. Regul Pept 92: 97–105, 2000.[CrossRef][Medline]
  8. Eiden S, Preibisch G, and Schmidt I. Leptin responsiveness of juvenile rats: proof of leptin function within the physiological range. J Physiol 530: 131–139, 2001.[Abstract/Free Full Text]
  9. Franklin KBJ and Paxinos G. The Mouse Brain in Stereotaxic Coordinates. New York: Academic, 1997.
  10. Gayle D, Ilyin SE, Romanovitch AE, Peloso E, Satinoff E, and Plata-Salamán CR. Basal and IL-1ß-stimulated cytokine and neuropeptide mRNA expression in brain regions of young and old Long-Evans rats. Mol Brain Res 70: 92–100, 1999.[Medline]
  11. Gruenewald DA, Marck BT, and Matsumoto AM. Fasting-induced increases in food intake and neuropeptide Y gene expression are attenuated in aging male brown Norway rats. Endocrinology 137: 4460–4467, 1996.[Abstract]
  12. Guan XM, Yu H, and Van der Ploeg LHT. Evidence of altered hypothalamic pro-opiomelanocortin/ neuropeptide Y mRNA expression in tubby mice. Brain Res Mol Brain Res 59: 273–279, 1998.[Medline]
  13. Guan XM, Yu H, Trumbauer M, Frazier E, Van der Ploeg LHT, and Chen H. Induction of neuropeptide Y expression in dorsomedial hypothalamus of diet-induced obese mice. Neuroreport 9: 3415–3419, 1998.[ISI][Medline]
  14. Hahn TM, Breininger JF, Baskin DG, and Schwartz MW. Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nat Neurosci 1: 271–272, 1998.[CrossRef][ISI][Medline]
  15. Hansen MJ and Morris MJ. Evidence for an interaction between neuropeptide Y and the melanocortin-4 receptor on feeding in the rat. Neuropharmacology 42: 792–797, 2002.[CrossRef][Medline]
  16. Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield LA, Burn P, and Lee F. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88: 131–141, 1997.[CrossRef][ISI][Medline]
  17. Jhanwar-Uniyal M and Chua SC Jr. Critical effects of aging and nutritional state on hypothalamic neuropeptide Y and galanin gene expression in lean and genetically obese Zucker rats. Mol Brain Res 19: 195–202, 1993.[Medline]
  18. Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, and Kalra PS. Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 20: 68–100, 1999.[Abstract/Free Full Text]
  19. Kamegai J, Minami S, Sugihara H, Hasegawa O, Higuchi H, and Wakabayashi I. Growth hormone receptor gene is expressed in neuropeptide Y neurons in hypothalamic arcuate nucleus of rats. Endocrinology 137: 2109–2112, 1996.[Abstract]
  20. Kaneda T, Makino S, Nishiyama M, Asaba K, and Hashimoto K. Differential neuropeptide responses to starvation with ageing. J Neuroendocrinol 13: 1066–1075, 2001.[CrossRef][ISI][Medline]
  21. Kesterson RA, Huszar D, Lynch CA, Simerly RB, and Cone RD. Induction of neuropeptide Y gene expression in the dorso medial hypothalamic nucleus in two models of the Agouti obesity syndrome. Mol Endocrinol 11: 630–637, 1997.[Abstract/Free Full Text]
  22. Korner J, Savontaus E, Chua SC Jr, Leibel RL, and Wardlaw SL. Leptin regulation of Agrp and NPY mRNA in the rat hypothalamus. J Neuroendocrinol 13: 959–966, 2001.[CrossRef][ISI][Medline]
  23. Kowalski C, Micheau J, Corder R, Gaillard R, and Conte-Devolx B. Age-related changes in cortico-releasing factor, somatostatin, neuropeptide Y, methionine enkephalin and ß-endorphin in specific rat brain areas. Brain Res 582: 38–46, 1992.[Medline]
  24. Lewis DE, Shellard L, Koeslag DG, Boer DE, McCarthy HD, McKibbin PE, Russell JC, and Williams G. Intense exercise and food restriction cause similar hypothalamic neuropeptide Y increases in rats. Am J Physiol Endocrinol Metab 264: E279–E284, 1993.[Abstract/Free Full Text]
  25. Li C, Chen P, and Smith MS. The acute suckling stimulus induces expression of neuropeptide Y (NPY) in cells in the dorsomedial hypothalamus and increases NPY expression in the arcuate nucleus. Endocrinology 139: 1645–1652, 1998.[Abstract/Free Full Text]
  26. Li H, Matheny M, Tümer N, and Scarpace PJ. Aging and fasting regulation of leptin and hypothalamic neuropeptide Y gene expression. Am J Physiol Endocrinol Metab 275: E405–E411, 1998.[Abstract/Free Full Text]
  27. Markewicz B, Kuhmichel G, and Schmidt I. Onset of excess fat deposition in Zucker rats with and without decreased thermogenesis. Am J Physiol Endocrinol Metab 265: E478–E486, 1993.[Abstract/Free Full Text]
  28. Mercer JG, Moar KM, Ross AW, Hoggard N, and Morgan PJ. Photoperiod regulates arcuate nucleus POMC, AGRP, and leptin receptor mRNA in Siberian hamster hypothalamus. Am J Physiol Regul Integr Comp Physiol 278: R271–R281, 2000.[Abstract/Free Full Text]
  29. Mizuno TM, Makimura H, Silverstein J, Roberts JL, Lopingco T, and Mobbs CV. Fasting regulates hypothalamic neuropeptide Y, agouti-related peptide, and proopiomelanocortin in diabetic mice independent of changes in leptin or insulin. Endocrinology 140: 4551–4557, 1999.[Abstract/Free Full Text]
  30. Morley JE. Decreased food intake with aging. J Gerontol Series A Bio Sci Med Sci 56 (Spec No 2): 81–88, 2001.
  31. Ollmann MM, Wilson BD, Yang YK, Kerns JA, Chen Y, Gantz I, and Barsh GS. Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein. Science 278: 135–138, 1997.[Abstract/Free Full Text]
  32. Rizk NM, Liu LS, and Eckel J. Hypothalamic expression of neuropeptide-Y in the New Zealand obese mouse. Int J Obes 22: 1172–1177, 1998.[CrossRef][Medline]
  33. Scarpace PJ, Matheny M, Zhang Y, Tümer N, Frase CD, Shek EW, Hong B, Prima V, and Zolotukhin S. Central leptin gene delivery evokes persistent leptin signal transduction in young and aged-obese rats but physiological responses become attenuated over time in aged-obese rats. Neuropharmacology 42: 548–561, 2002.[CrossRef][ISI][Medline]
  34. Weide K, Christ N, Moar KM, Arens J, Hinney A, Mercer JG, Eiden S, and Schmidt I. Hyperphagia, not hypometabolism, causes early onset obesity in melanocortin-4 receptor knockout mice. Physiol Genomics 13: 47–56, 2003. First published January 14, 2003; 10.1152/physiolgenomics. 00129.2002.[Abstract/Free Full Text]
  35. Williams G, Bing C, Cai XJ, Harrold JA, King PJ, and Liu XH. The hypothalamus and the control of energy homeostasis. Different circuits, different purposes. Physiol Behav 74: 683–701, 2001.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 HypertensionHome page
L. S. Tallam, A. A. da Silva, and J. E. Hall
Melanocortin-4 Receptor Mediates Chronic Cardiovascular and Metabolic Actions of Leptin
Hypertension, July 1, 2006; 48(1): 58 - 64.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Kuramochi, T. Onaka, D. Kohno, S. Kato, and T. Yada
Galanin-Like Peptide Stimulates Food Intake via Activation of Neuropeptide Y Neurons in the Hypothalamic Dorsomedial Nucleus of the Rat
Endocrinology, April 1, 2006; 147(4): 1744 - 1752.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
M. Liang and B. Ventura
Physiological genomics in PG and beyond: July to September 2005
Physiol Genomics, October 17, 2005; 23(2): 119 - 124.
[Full Text] [PDF]

Home page
 EndocrinologyHome page
C. Fekete, D. L. Marks, S. Sarkar, C. H. Emerson, W. M. Rand, R. D. Cone, and R. M. Lechan
Effect of Agouti-Related Protein in Regulation of the Hypothalamic-Pituitary-Thyroid Axis in the Melanocortin 4 Receptor Knockout Mouse
Endocrinology, November 1, 2004; 145(11): 4816 - 4821.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
16/1/38    most recent
00123.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arens, J.
Right arrow Articles by Korf, H.-W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arens, J.
Right arrow Articles by Korf, H.-W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.