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Endocrinology, doi:10.1210/en.2005-1331
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Endocrinology Vol. 147, No. 6 3050-3059
Copyright © 2006 by The Endocrine Society

Maternal Perinatal Undernutrition Alters Neuronal and Neuroendocrine Differentiation in the Rat Adrenal Medulla at Weaning

Olivier Molendi-Coste1, Luca Grumolato1, Christine Laborie, Jean Lesage, Eric Maubert, Hafida Ghzili, Hubert Vaudry, Youssef Anouar, Christophe Breton and Didier Vieau

Unité Propre de Recherche et de l’Enseignement Supérieur Equipe Associée 2701 (O.M.-C., C.L., J.L., C.B., D.V.), Laboratoire de Neuroendocrinologie du Développement, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France; Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 413 (L.G., H.G., H.V., Y.A.), Laboratoire de Neuroendocrinologie Cellulaire et Moléculaire, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23, Université de Rouen, 76821 Mont-Saint-Aignan, France; and INSERM-Avenir (E.M.), Groupement d’Intérêt Public Cyceron, 14000 Caen, France

Address all correspondence and requests for reprints to: Prof. Didier Vieau, Unité Propre de Recherche et de l’Enseignement Supérieur Equipe Associée 2701, Laboratoire de Neuroendocrinologie du Développement, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France. E-mail: didier.vieau{at}univ-lille1.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Epidemiological studies suggest that chronic adult diseases, such as type 2 diabetes and hypertension, can be programmed during fetal and early postnatal life. The nervous system regions governing vegetative functions and the hypothalamic-pituitary-adrenal axis are particularly sensitive to the perinatal nutritional status. Despite recent reports demonstrating that the activity of the sympathoadrenal system can be altered by early life events, the effects of maternal nutrient restriction on the adrenal medulla remain unknown. Using a rat model of maternal perinatal 50% food restriction (FR50) from the second week of gestation until weaning, immunohistochemical experiments revealed alterations in chromaffin cell aggregation and in nerve fiber fasciculation in the adrenal medulla of FR50 pups. These morphological changes were associated with enhanced circulating levels of catecholamines after decapitation (epinephrine by 55% and norepinephrine by 41%). Using macroarrays, we identified several genes whose expression was affected by maternal nutrient restriction. Semiquantitative RT-PCR confirmed the overexpression of four genes involved in neuroendocrine differentiation and neuronal plasticity (chromogranin B, growth-associated protein 43, neurofilament 3, and Slit2) in the adrenal glands of FR50 rats. Using in situ hybridization, we showed that these genes are solely expressed in the adrenal medulla. Together, our results suggest that perinatal maternal undernutrition markedly alters the differentiation of the adrenal medulla during postnatal life, resulting in enhanced activity of chromaffin cells at weaning. These alterations may persist in adulthood and participate to the programming of chronic adult diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN HUMANS, INTRAUTERINE growth retardation (IUGR) is frequently associated with the development of several pathologies, including insulin resistance, type 2 diabetes, and hypertension in later life (1, 2, 3). Such an association between IUGR and the appearance of diseases in adult life has led to the fetal programming hypothesis (1), which implies that adverse environmental factors acting in utero program the development of fetal tissues, producing dysfunctions and diseases in adults. This concept is supported by numerous studies in animal models. In rodents, uterine artery ligation or major caloric restriction lead to severe nutrient restriction in the fetus, producing IUGR and subsequent permanent hypertension, dysregulation of glucose metabolism, and insulin resistance in later life (4, 5).

In mammals, it is well established that maturation of the nervous system depends in part on perinatal nutritional factors and postnatal environmental stimulation. Brain regions governing vegetative functions and the hypothalamic-pituitary-adrenal (HPA) axis are particularly sensitive to such cues acting in prenatal and postnatal periods (6, 7). The sympathetic nervous system and the adrenal medulla, i.e. the sympathoadrenal system (SAS), are also affected by nutritional factors during development (6). It has been shown that protein restriction in maternal food during pregnancy reduces norepinephrine (NE) turnover in several tissues in adults (6). Likewise, limitation of food availability during lactation, using animals reared in large litters, abolishes the stimulatory effect of dietary sucrose on the cardiac sympathetic nervous system activity in rat (8). These observations strongly suggest that neonatal nutrition may influence the development and the activity of the SAS.

Sympathoadrenal (SA) progenitor cells originating from pluripotent stem cells in the embryonic trunkal neural crest aggregate at the dorsal aorta and subsequently migrate into a ventrolateral direction to colonize the adrenal gland, in which they lose neuronal traits and finally differentiate into neuroendocrine chromaffin cells (9, 10). The final step of chromaffin cell differentiation is characterized by the appearance of a large number of epinephrine (E)-producing cells resulting from the induction of phenylethanolamine-N-methyl transferase (PNMT) gene expression by glucocorticoids (11). However, after birth, some chromaffin cells that still express neuronal-type markers, such as the major synaptosomal growth-associated protein 43 (GAP43), retain their noradrenergic phenotype (12). The differential expression of adhesion molecules determines the architecture of the adrenal gland by segregating chromaffin cells into homophenotypic groups of either E or NE cells (13). Proliferation of chromaffin cells occurs mainly during fetal and neonatal development. However, in contrast to sympathetic neurons, chromaffin cells retain their ability to proliferate after birth and throughout the whole life span in rat, at least in vitro (14, 15).

We have shown previously that maternal 50% food restriction (FR50), during the last week of gestation and lactation, disturbs the activity of the HPA axis at weaning under both resting and stress conditions (16). In particular, we demonstrated that ether inhalation-induced plasma ACTH increase is reduced in FR50 pups and that the plasma corticosterone values in these animals are lower than baseline after this stressful procedure. In addition, FR50 adult male rats still show impaired adrenocortical responsiveness to stress and present elevated basal plasma corticosterone levels compared with controls, as well as an altered responsiveness of the SA axis to restraint stress (17). However, although numerous studies have deciphered the consequences of perinatal maternal undernutrition on the HPA axis activity in the offspring, the impact of early life nutrient restriction on SA development and adrenal medullary function has never been reported. Because it has been shown clearly that putative noxious signals (i.e. hypoxia, glucocorticoid hormones, and placental restriction) may profoundly affect chromaffin cell development (18, 19, 20), we hypothesized that perinatal maternal undernutrition might alter the neuronal and neuroendocrine differentiation of the adrenal medulla in the male rat offspring at weaning. To test this hypothesis, we compared the adrenomedullary organization, function, and gene expression in 21-d-old male rats malnourished during their perinatal life and in age-matched control animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and regimen
Male (300–350 g) and female (250–300 g) Wistar rats were purchased from Charles Rivers Laboratories (L’Arbresle, France). The animals were housed five per cage with a controlled light cycle (12 h-light, 12-h dark cycle; lights on at 0700 h) and temperature (22 ± 2 C) with free access to food (regular rat chow number 113, containing 22% protein, 5% fat, and 53% carbohydrates; UAR, Villemoisson-sur-Orge, France) and tap water. After 14 d of acclimation, females were mated with a male for one night. Day 0 of pregnancy was defined as the day immediately after the night during which males were present, if spermatozoa were found in the vaginal smears. Pregnant rats were then housed in individual cages. Animal use accreditation by the French Ministry of Agriculture (number 04860) has been granted to our laboratory for experimentation with rats. All animal experiments were conducted in accordance with the European Communication Council Directive of November 24, 1986 (86/609/EEC).

Two groups of pregnant rats were studied. In the control group, dams were fed ad libitum during gestation [from embryonic d 0 (E0) to E21] and the 3 wk of lactation [from E21 to postnatal day 21 (P21)]. In the FR50 group, females received 50% of ad libitum intake determined by the amount of food consumed by control females from E14 until P21 in a pilot study. Briefly, FR50 females received 12 g/d food from E14 to E21. Then, available food was gradually increased from 12 to 22 g/d (E21 to P6), from 27 to 31 g/d (P7–P13), and from 35 to 40 g/d (P14–P21). Dams delivered spontaneously during the night between E21 and P1, and pups were weighed in the morning of P1 and at weaning (P21). To improve survival of very small pups, litter size was reduced to eight pups per litter at birth in both groups. Experiments were conducted only on male pups.

Plasma and tissue collections
At weaning (P21), pups were decapitated between 0800 and 1100 h. Decapitation was properly conducted by skilled staff with well-maintained equipment, ensuring rapid death and plasma collection. Trunk blood samples were collected in tubes prerinsed with 5% EDTA and centrifuged. Plasma samples were stored at –80 C until determination of circulating catecholamines. Usually, the right adrenals were frozen in liquid N2 for catecholamines or gene expression assessment. The left adrenals were frozen on dry ice for in situ hybridization studies or postfixed for 24 h in 4% paraformaldehyde in phosphate buffer and cryoprotected by incubation for 24 h in PBS 0.05 M containing 20% sucrose for immunohistochemistry and histology. The adrenal glands were cut into serial 12-µm sections, mounted on gelatin-coated slides, and directly stored at –80 C for immunohistochemistry and histological analysis or dried at 63 C for 1 min before performing in situ hybridization.

Histology
After hematoxylin and picro-indigo-carmin staining, the relative proportion of medulla and cortex was determined using a scanner coupled with the computer-assisted image analysis software Multi-Analyst (Bio-Rad, Hercules, CA). Five adrenals were analyzed for each experimental group, and five adjacent slices from the median region of the medulla were analyzed for each adrenal gland.

Immunohistochemistry
For chromaffin cell staining, sections of adrenal glands were immunostained with a polyclonal rabbit anti-tyrosine hydroxylase (TH) antibody (Jacques Boy Institut, Reims, France). Immunostaining of differentiated adrenergic chromaffin cells was performed with a polyclonal rabbit anti-PNMT antibody (Chemicon, Temecula, CA). Immunostaining of NE chromaffin cells and nerve fibers was performed with a monoclonal mouse anti-GAP43 antibody (Sigma, St. Louis, MO).

Adrenal sections were pretreated with 1.5% H2O2 in 20% methanol and 0.2% Triton X-100 to inactivate endogenous peroxidases, and unspecific labeling was blocked with 2% donkey serum (Sigma) in 0.1 M PBS for 30 min. Sections were then rinsed with 0.1 M PBS and incubated with anti-GAP43 (1:500 dilution) overnight at room temperature. Sections were subsequently incubated with a biotinylated antimouse secondary antibody (1:300 dilution) (Chemicon) for 2 h at room temperature, and labeling was amplified with biotin and avidin-horseradish peroxidase reagents as described in the ABC kit (Vector Laboratories, Burlingame, CA). Color development of immunoreactivity was performed for 15 min, using diaminobenzidine tetrahydrochloride (Sigma-Aldrich, Saint Quentin Fallavier, France).

For double-immunofluorescence staining, tissue slices were incubated with anti-TH or anti-PNMT antibody, respectively (1:1000 dilution), and anti-GAP43 antibody (1:500 dilution) overnight at room temperature. Then, sections were incubated with a mixture of fluorescein isothiocyanate-conjugated goat antirabbit and tetramethylrhodamine isothiocyanate-conjugated goat antimouse secondary antibodies (1:300 dilution) (Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature. Sections were then rinsed in PBS and mounted with Fluorescent Mounting Medium (DakoCytomation, Carpinteria, CA).

Sections were examined with a Zeiss (Gottingen, Germany) Axioplan Microscope II equipped with x20 and x40 objectives and appropriate fluorescent barrier filters.

The determination of NE clusters and nerve fiber density was performed with Multi-Analyst software (Bio-Rad) on selected representative pictures (see Fig. 2Go). Five adrenals were analyzed from each experimental group, and five adjacent sections from the median region of the medulla were analyzed for each adrenal gland. The area, number, and total area of NE clusters as well as the section area, number, and total section area of nerve fibers were normalized to the area of medulla examined.


Figure 2
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  		FIG. 2. GAP43 and PNMT immunohistochemistry in 21-d-old animals adrenal glands from ad libitum (A, C, E) and restricted (B, D, F) fed dams. In A and B, double immunofluorescence was performed for PNMT (green) and GAP43 (red), showing confined expression of GAP43 to NE chromaffin cells. In C and D, diaminobenzidine tetrahydrochloride staining was performed for GAP43, showing only NE-labeled chromaffin cells clusters (arrows). A and C show the typical organization of adrenal glands from control animals, with large clusters of NE chromaffin cells (*). B and D show morphological alterations of adrenals from FR50 animals, consisting of more numerous NE clusters (*) with reduced size. E and F show GAP43 staining in intramedullary nerve fibers (arrows) in control (E) and FR50 (F) adrenals. Scale bars: A–D, 40 µm; E, F, 20 µm. Co, Adrenal cortex.

 
HPLC
Adrenal glands were sonicated in 1 ml ice-cold 0.2 M HClO4 and centrifuged at 20,000 x g at 4 C for 20 min. The supernatants were used to determine NE and E contents by HPLC coupled with electrochemical detection as described previously (21). Catecholamine assays in plasma samples were also performed by HPLC after alumina extraction as described previously (21).

Macroarray preparation
A microarray containing more than 15,000 mouse embryonic/placental cDNA probes (22) obtained from the National Institute on Aging mouse 15K cDNA library and corresponding to 15,264 Unigene clusters (for details, see http://lgsun.grc.nia.gov/cDNA/15k.html) as well as the suppression substractive hybridization (SSH) method were used to identify genes overexpressed in pheochromocytoma PC12 cells after differentiation by 100 nM pituitary adenylate cyclase-activating polypeptide (PACAP38) (23). Clones identified by microarray and SSH analyses were amplified with universal primers and the DyNAzyme EXT DNA polymerase, following the instructions of the manufacturer (Ozyme, Saint-Quentin en Yvelines, France) in a PCRexpress thermal cycler (Hybaid, Paris, France) and used as probes to make a macroarray. The quality of the amplified DNA was checked by migration on a 1% agarose gel. The PCR products contained in a 384-well plate were directly printed on Hybond NX membrane (Amersham Biosciences, Les Ulis, France) using a ChipWriter system (Virtek, Waterloo, Ontario, Canada). These filters were denatured with a 0.4 M NaOH, 0.1 M NaCl solution for 5 min and neutralized with 40 mM Na2HPO4/NaH2PO4 buffer (pH 7.2) for 5 min. The macroarrays were hybridized with target cDNAs derived from control and FR50 pups as follows: total RNA was isolated using the Tri-Reagent (Sigma-Aldrich) purified on RNeasy Mini Spin Columns (Qiagen, Courtaboeuf, France) and quantified by spectrophotometry. The quality of the RNA was checked by ethidium bromide staining of the 28S and 18S ribosomal RNA on a formaldehyde-agarose gel. cDNAs were labeled during synthesis in the presence of [{alpha}-33P]dCTP (PerkinElmer, Courtaboeuf, France) according to standard National Human Genome Research Institute protocols (http://www.hngri.nih.gov/UACORE/protocols.html). Images of the hybridized macroarrays obtained from a PhosphorImager (Amersham Biosciences) were quantified with the XdotsReader software (Cose, Dugny, France). Hybridization signals were normalized to those of a glyceraldehyde-3-phosphate dehydrogenase probe that was printed at several locations of the macroarray. Three independent experiments were performed for each hybridization, and mean values were calculated.

Semiquantitative RT-PCR
RNA was extracted and purified from adrenals using the TRIzol reagent (Invitrogen, Strasbourg, France). The quality of total RNA was assessed by determining the 260/280 absorbance ratio and by gel electrophoresis in agarose. The semiquantitative RT-PCR method used here has been described and validated previously (24).

Briefly, 3 µg total RNA was reverse transcribed into cDNA using 3 µg random hexamers and 200 U Moloney murine leukemia virus reverse transcriptase (Invitrogen). One thirtieth of the first-strand synthesis reaction was amplified using 1 U Taq DNA polymerase (Qbiogen, Illkirch, France) and 2 µM of each forward and reverse primers. The cycling parameters were as follows: 94 C for 1 min 30 sec, 60 C for 1 min 30 sec, and 72 C for 2 min. Negative control RT-PCRs were performed by omitting RT from the reaction mixture. The position of the primers as well as the predicted size of the amplification products are summarized in Table 1Go. Cyclophilin B (Cyclo B) was used as an internal standard. Moreover, the priming sites were separated by an intron, thus preventing amplification of any contaminating genomic DNA. Pilot experiments were conducted to determine the optimal cycle number for each primer pair for linear semiquantitative amplification. Each experiment was performed in triplicate and gave similar results. After amplification, the samples were separated on a 1% gel agarose, visualized by ethidium bromide, and quantified by Multi-Analyst software (Bio-Rad).


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  		TABLE 1. Primers and probes used for macroarray validation

 
In situ hybridization
Sections (12 -µm-thick) were mounted on gelatin-coated slides, dried, and kept at –80 C. In situ hybridization was performed as described previously (25). cDNAs for chromogranin B (CgB), GAP43, neurofilament 3 (NeuF3), and Slit2 were subcloned into pGEM-T easy. The CgB probe was a 472-bp fragment linearized with SacI (antisense probe). The GAP43 probe was a 454-bp fragment linearized with Apa1 (antisense probe). The NeuF3 probe was a 444-bp fragment linearized with Apa1 (antisense probe). The Slit2 probe was a 486-bp fragment linearized with SacI (antisense probe). Riboprobes were labeled using [35S]UTP (1.300 Ci/mmol; PerkinElmer, Paris, France) and synthesized according to the previously described procedure (25). Controls with the sense probes did not produce any specific hybridization signals. All slides were exposed to x-ray films (Biomax-MR; Eastman Kodak, Le Pontet, France) for 6 h for the CgB probe, 7 d for the GAP43 and NeuF3 probes, and 20 d for the Slit2 probe.

Statistical analysis
All data are presented as mean ± SEM. Statistical analysis was performed using the unpaired Student’s t test and the SigmaStat software (Systat Software, Port Richmond, CA). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adrenal morphology
Anatomical characteristics of adrenal glands from 21-d-old animals from ad libitum fed (control) and food-restricted (FR50) dams are presented in Figs. 1Go and 2Go. Consistent with a previous report (16), the absolute adrenal weight was reduced in 21-d-old FR50 pups [17.1 ± 0.38 mg in control (n = 35) vs. 10.4 ± 0.24 mg in FR50 (n = 35); P < 0.001], whereas the relative adrenal weight was significantly increased (3.70 ± 0.07 mg/10 g body weight in control vs. 4.01 ± 0.08 mg/10 g body weight in FR50; P < 0.01). However, as shown in Fig. 1CGo, the proportion of medulla or cortex to the total adrenal area was not different between the two groups (medulla/adrenal area, 16 ± 0.6% in control vs. 16 ± 0.9% in FR50, P = 0.9691; cortex/adrenal area, 85 ± 0.6% in control vs. 84 ± 0.9% in FR50, P = 0.9691), indicating that the mass loss affected to the same extent the two tissues.


Figure 1
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  		FIG. 1. Pictures of median adrenal gland histological slices from control (A) and FR50 (B) in 21-d-old animals. C represents the area measurements expressed as medulla/adrenal and cortex/adrenal proportions for control (white bars) and FR50 (gray bars) adrenal glands. Data represent means ± SEM (n = 5 animals per group). Scale bars, 1 mm. Co, Adrenal cortex; Me, adrenal medulla.

 
The adrenal gland organization from control and FR50 rats (P21) was studied by immunohistochemistry (n = 8 per group). As shown in Fig. 2Go, double-immunofluorescence labeling for PNMT and GAP43 revealed that GAP43 was only expressed in NE chromaffin cells but was undetectable in PNMT-expressing E chromaffin cells in both control (Fig. 2AGo) and FR50 (Fig. 2BGo) adrenal medulla. The number of TH- and PNMT-immunoreactive cells in the adrenal medulla was similar in FR50 and control rats (data not shown).

Using the GAP43 and PNMT labeling, the organization of chromaffin cells was examined. The shape and dimensions of E and NE chromaffin cells were similar in control and FR50 adrenals. Within the glands from control rats, NE chromaffin cells formed few large clusters preferentially localized at the periphery of the medulla (Fig. 2Go, A and C, 100% of the glands examined), whereas, in the FR50 adrenal glands, NE chromaffin cells were organized in much smaller isolated groups of cells scattered throughout the medulla (Fig. 2Go, B and D, 75% of the glands examined). As summarized in Table 2Go, the relative area of NE clusters was reduced in FR50 medulla (5.94 ± 0.22% in controls vs. 1.44 ± 0.12% in FR50; P < 0.05), whereas the relative number of NE chromaffin cell clusters was increased in FR50 medulla compared with controls (2.2 ± 0.26 clusters in controls vs. 6.89 ± 0.11 clusters in FR50; P < 0.001). However, the total relative area was not different between the two groups (13.18 ± 1.55% in control vs. 9.93 ± 0.89% in FR50; P = 0.18). In contrast, the architecture of E chromaffin cell clusters was not different between control and FR50 medulla.


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  		TABLE 2. NE clusters and nerve fiber density

 
Maternal perinatal food restriction also altered the organization of the intraadrenal innervation. In the adrenal medulla of control rats, fibers labeled with GAP43 formed few large bundles that innervated some chromaffin cell groups (Fig. 2EGo), whereas, in the adrenals of FR50 rats, the fiber network innervating cell clusters was mainly composed of more numerous and smaller processes scattered within the medulla (Fig. 2FGo, 75% of the glands examined). The relative section area and number of nerve fibers are reported in Table 2Go. The relative section area was significantly reduced in FR50 adrenals (0.15 ± 0.02% in controls vs. 0.023 ± 0.002% in FR50; P < 0.001), whereas the relative number increase was not significant in FR50 medulla compared with controls (3.03 ± 0.57 fibers in controls vs. 4.73 ± 1.52 fibers in FR50; P = 0.29). However, the apparent reduction of the relative total density of nerve fibers was not significant between control and FR50 animals (0.44 ± 0.07% in control vs. 0.11 ± 0.03% in FR50; P = 0.11).

Plasma and adrenal catecholamine concentrations
To determine whether the structural alterations observed in the adrenal medulla of FR50 pups have physiological consequences, we evaluated adrenal contents as well as plasma levels of E and NE by HPLC analysis after decapitation procedure. As shown in Fig. 3Go, the relative E and NE adrenal contents were similar in controls and FR50 pups (0.37 ± 0.042 µg E/mg gland in control vs. 0.41 ± 0.053 µg E/mg gland in FR50; 0.11 ± 0.009 µg NE/mg gland in control vs. 0.11 ± 0.014 µg NE/mg gland in FR50). The E/NE ratio was also unchanged in FR50 compared with control pups (3.34 ± 0.18 in control vs. 3.66 ± 0.16 in FR50). In contrast, plasma E and NE levels were increased by 55% (8.91 ± 1.3 ng/ml in control vs. 13.8 ± 0.76 ng/ml in FR50; P < 0.05) and by 41% (2.4 ± 0.2 ng/ml in control vs. 3.4 ± 0.36 ng/ml in FR50; P < 0.05), respectively. However, the E/NE ratio in plasma was not affected in FR50 compared with control pups (3.8 ± 0.55 for control vs. 4.35 ± 0.52 for FR50).


Figure 3
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  		FIG. 3. Adrenal content, plasma levels of E and NE, and E/NE ratio in 21-d-old animals from ad libitum (white bars) and restricted (gray bars) fed dams. Data represent means ± SEM (n = 8 animals per group). **, P < 0.01, FR50 vs. control.

 
Analysis of gene expression in the adrenal gland of food-restricted animals
Using a macroarray, we compared the expression of 384 genes, previously shown to be regulated during PACAP-induced differentiation of PC12 cells (23), in the adrenals of weaning control and FR50 animals. Results are presented in Table 3Go and expressed as FR50/control ratios. Among the 384 genes coated on the macroarray, only one gene was underexpressed (ratio < 0.7), whereas 20 genes were overexpressed (ratio > 1.5) in FR50 compared with control animals. These genes were classified in functional categories, as shown in Table 3Go.


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  		TABLE 3. Genes differentially expressed in 21-d-old animals adrenal glands

 
To validate the variations of gene expression obtained using the macroarray, we performed semiquantitative RT-PCR analysis. CgB, GAP43, NeuF3, and Slit2 genes, known to be involved in neuroendocrine and/or neuronal differentiation or implicated in secretory activity, were chosen as candidates. We also analyzed the expression of TH, PNMT, and Cyclo B mRNAs, three genes that were not included in the macroarray analysis (Fig. 4AGo). Interestingly, the expression of TH (1.07 ± 0.16 in control vs. 0.96 ± 0.15 in FR50), PNMT (1.73 ± 0.28 in control vs. 1.61 ± 0.26 in FR50), and Cyclo B (1.95 ± 0.18 in control vs. 2 ± 0.14 in FR50) was not impaired by maternal perinatal food restriction compared with controls (Fig. 4BGo). In contrast, CgB (1.13 ± 0.11 in control vs. 1.71 ± 0.25 in FR50; P < 0.05), GAP43 (1.13 ± 0.11 in control vs. 1.71 ± 0.25 in FR50; P < 0.05), NeuF3 (0.89 ± 0.08 in control vs. 1.47 ± 0.21 in FR50; P < 0.05), and Slit2 (0.78 ± 0.11 in control vs. 1.68 ± 0.24 in FR50; P < 0.05) mRNA levels were significantly increased in P21 FR50 adrenals (Fig. 4BGo). These results are in agreement with values obtained by the macroarray analysis (Fig. 4CGo and Table 3Go).


Figure 4
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  		FIG. 4. Levels of mRNA of the selected representative genes quantified by semiquantitative RT-PCR in 21-d-old animals adrenal glands from ad libitum [control (C)] and restricted (FR50) fed dams. Each experiment was realized on five animals per group in triplicate, and results were normalized to the expression level of an internal standard (Cyclo B). The results are indicated in B, whereas A shows an example (CgB) of electrophoresis after amplification in semiquantitative conditions. C shows the FR50/control expression ratio. *, P < 0.05; **, P < 0.01, FR50 vs. control.

 
In situ hybridization analysis of gene expression
In situ hybridization experiments showed that CgB, GAP43, NeuF3, and Slit2 mRNAs are mainly expressed within the adrenal medulla in both control (Fig. 5Go) and FR50 (data not shown) adrenal glands. In contrast to the other transcripts, GAP43 labeling was scattered in groups of medullary cells, probably due to its restricted expression in NE chromaffin cell clusters (Fig. 5Go). No hybridization signal was observed using the sense riboprobes (data not shown).


Figure 5
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  		FIG. 5. In situ hybridization of selected gene mRNAs in 21-d-old animal adrenal glands from control dams. Experiments were realized on 10 animals per group, and all gave the same results. Scale bars, 1 mm. Co, Adrenal cortex.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We observed previously that, in rat, maternal perinatal FR50 results in a reduction of the absolute weight of the adrenal gland at weaning and impairs the activity of the HPA axis from postnatal development to adulthood (16, 17). We show here that the mass loss of the adrenal gland affected to the same extent the medulla and the cortex, suggesting that the SAS functionality could also be altered. However, the relative mass of neonatal adrenals compared with the body mass was increased in undernourished rats, suggesting that adrenal growth is maintained within an optimal range through compensatory mechanisms. The maintenance of the adrenal mass appears to be of primary importance for neonates because circulating glucocorticoids and catecholamines play essential roles in the adaptation to extrauterine life (26).

Our results reveal that maternal FR50 alters the structural organization of the adrenal medulla of weaning rats. Clusters of NE chromaffin cells were smaller, more abundant, and more widely distributed in the medulla in FR50 adrenals compared with controls. Development of chromaffin cells begins at approximately E14–E16 and is characterized by the differentiation of SA precursors to neuroendocrine cells and segregation of chromaffin cells into homophenotypic groups. Differential expression of adhesion molecules between E and NE chromaffin cells determines the formation of clusters in the adrenal medulla. Indeed, coexpression of L1 and neural cell adhesion molecule might be responsible for the association of NE chromaffin cells in homophenotypic aggregates by a process of differential adhesion, because E chromaffin cells lack L1 (13). During the differentiation of SA cells to neuroendocrine cells, the expression of some neuronal markers, such as NeuF proteins, L1, and GAP43, is repressed in differentiated E chromaffin cells and maintained in differentiated NE cells (12, 27). Because GAP43 is associated with synaptic plasticity, its presence in chromoblasts and its persistence only in NE chromaffin cells suggests a possible role in the migration and/or aggregation of chromaffin cells into the adrenal medulla. As reported by Grant et al. (12), predictive NE chromaffin cells form few large clusters as early as E16.5. Thereafter, small NE clusters observed in the medulla of FR50 weaning rats could correspond not only to a delayed settling of chromaffin tissue but also to a transitory or permanent remodeling of its structure. At weaning, intense GAP43 staining was detected in nerve fibers that innervate the adrenal medulla of both control and FR50 rats. In FR50 rats, nerve bundles were thinner and more scattered throughout the medulla, suggesting a profound alteration in their fasciculation. However, we do not know to what extent these modifications affect sympathetic activity. During ontogeny, preganglionic sympathetic nerves invade the adrenal gland at E15 and are identified in apposition to chromaffin cells at E17 (9). However, synaptic connections become functional at the end of the first postnatal week and are completed by the third postnatal week (28). The development of sympathetic innervation during postnatal life implicates coordinated growth and expansion of nerve endings within the innervated tissue. Indeed, several growth factors produced by chromaffin cells regulate the development and the maintenance of the preganglionic innervation (9, 29, 30). During development of the nervous system, GAP43 promotes axonal pathfinding, neurite outgrowth, and synaptic plasticity (31, 32). It is also required for selective fasciculation to maintain topographic organization of axons (33). During the second postnatal week, the expression of GAP43 in the adrenal medulla decreases to a level comparable with that observed in the adults (12), a phenomenon that corresponds to the establishment of the definitive organization of the medulla. Our macroarray data confirmed by RT-PCR indicate that GAP43 mRNA expression was increased in the adrenal glands of FR50 pups and was restricted to the medulla, as shown by in situ hybridization experiments. It is thus possible that maternal FR50 somehow alters gene expression in the adrenal medulla, which provokes a remodeling of the structure of chromaffin cells and the distribution of neuronal networks, in weaning pups. The structural changes may in turn trigger a plastic response as a compensatory and/or adaptive reaction to provide an opportunity for aggregation of chromaffin cells and settlement of innervation.

Interestingly, the mRNA expression of Slit2 was also increased in the adrenal medulla of FR50 pups. To our knowledge, this is the first demonstration of Slit2 expression in the rat adrenal medulla. Slit2 belongs to the chemotropic factor superfamily and plays important roles in neuronal, glial, and neural crest cell migration (34, 35, 36, 37), as well as in axon elongation/branching (38), acting via Roundabout receptors. In addition, Slits provide repulsive cues to migrating non-neuronal cells (39). High levels of Slit2 mRNA expression in the adrenal medulla of FR50 rats could be involved in the remodeling of chromaffin cell aggregates and/or nerve fiber fasciculation and could account, at least in part, for the structural alterations reported here. Together, morphological alterations and overexpression of genes implicated in morphogenesis and plasticity may reflect a delayed or adaptive settling of chromaffin tissue and innervation.

The question then arises as to whether such alterations of the adrenal medulla structure have physiological and functional consequences. Several studies reported that maternal and neonatal diet restriction retards the functional maturation of chromaffin medullary cells (8, 40). Similarly, IUGR (41) and prenatal exposure to dexamethasone (42) disturb the storage and synthesis capacities of the adrenal gland. However, the impact of maternal FR50 on the adrenal medulla during the perinatal period differs from the effects reported in previous studies. In FR50 rats, TH and PNMT mRNA levels as well as the adrenal content in catecholamines were unaffected. Conversely, maternal undernutrition led to enhanced circulating levels of both E and NE, although the E/NE ratio remained unchanged. However, plasma levels of catecholamines measured in our experimental conditions do not correspond to stress-free baselines or to fully stress-procedure-induced levels but indicate differential nonspecific effect of decapitation between control and FR50 animals. The source of circulating NE may be the adrenal medulla, extraadrenal chromaffin cells, or sympathetic neurons. The proportional relationship between adrenal and circulating E/NE ratio in the two groups indicates that NE levels in plasma is mainly due to production from the adrenal medulla at this stage of development. Together, these data suggest that the rise in plasma catecholamine levels after decapitation is probably due to increased release from the adrenal gland as a consequence of excessive mobilization of secretory granule stores and/or enhanced granule biogenesis. The increase in CgB mRNA expression in the adrenal medulla of FR50 pups is in agreement with this hypothesis. It has been shown that suppression of CgB expression in neuroendocrine PC12 cells leads to a reduction in the number of (intrinsic) secretory granules, whereas expression of CgB in non-neuroendocrine cells, which normally do not contain any secretory machinery, led to granules biogenesis (43). In addition, it has been shown that CgB interacts with the 1,4,5-triphosphate receptor/Ca channels (44, 45) and with proteins involved in the formation of fusion complex (46), suggesting that CgB could be involved in Ca2+-dependent exocytotic processes. Moreover, the expression of Ca2+-dependent actin-binding proteins such as gelsolin, phafin, or adducin 3, which induce the cytoskeleton remodeling necessary for exocytosis, was also augmented in FR50 adrenals (Table 3Go). These observations suggest that the adrenal medulla of FR50 pups remains at a high level of activity during postnatal life, probably as a mechanism of specific physiological adaptation to neonatal adverse environment elicited by maternal undernutrition. As reported previously, catecholamine release from neonatal adrenal medulla occurs despite the immaturity of the splanchnic innervation and is under the influence of a non-neurogenic mechanism during the first week of extrauterine life (28). The neurogenic control is fully completed by the end of the third postnatal week. Because the organization of adrenal medulla innervation is altered in FR50 rats at weaning, it is tempting to speculate that the sensitivity of chromaffin cells to the non-neurogenic control could persist in those animals and thus be implicated in the enhanced plasma levels of catecholamines. In addition, neuropeptides produced by the adrenal medulla, such as vasoactive intestinal peptide, neuropeptide Y, and PACAP, can induce CA release via autocrine and/or paracrine mechanisms (9). Interestingly, the mRNA expression of PACAP and its receptor PAC1-R are increased in FR50 adrenals at weaning as assessed by real-time RT-PCR (data not shown).

Recent reports suggested that perinatal life adrenomedullary hyperactivity may have adverse outcomes on cardiovascular and metabolic homeostasis in adulthood, such as high blood pressure and insulin resistance (47, 48, 49). Therefore, the alterations observed in FR50 pups could participate to the programming of the metabolic disorders developed by FR50 adult rats (50). It would be important to investigate whether such alterations in the catecholamine status persist in FR50 adult rats and contribute to the pathogenesis of some diseases.

In conclusion, our results based on morphological, functional, and transcriptional analyses demonstrate that maternal perinatal undernutrition markedly alters the processes that take place in the adrenal medulla during postnatal life. Together, these data suggest that maternal nutrient restriction delays and/or irremediably alters the structural and functional maturation of the adrenal gland, which may have pathophysiological consequences.


   Acknowledgments
 
We thank Dr. Y. Guéraldel and Prof. J. Mazurier (Institut Fédératif de Recherches 118, University of Lille 1, Villeneuve d’Ascq Cedex, France) for providing the HPLC analysis equipment. We are grateful to V. Montel and A. Dickès-Coopman for technical assistance.


   Footnotes
 
This study was supported by the Lille-Amiens-Rouen-Caen (LARC-Neuroscience) network, the Institut National de la Santé et de la Recherche Médicale Unite 413, the Conseil Régional de Haute-Normandie, and the Conseil Régional du Nord-Pas-de-Calais.

O.M.-C., L.G., C.L., J.L., E.M., H.G., H.V., Y.A., C.B., and D.V. have nothing to declare.

First Published Online February 23, 2006

1 O.M.-C. and L.G. contributed equally to this work. Back

Abbreviations: CgB, Chromogranin B; Cyclo B, cyclophilin B; E, epinephrine; E21, embryonic d 21; FR50, food restriction 50%; GAP43, growth-associated protein 43; HPA axis, hypothalamo-pituitary-adrenal axis; IUGR, intrauterine growth retardation; NE, norepinephrine; NeuF3, neurofilament 3; P21, postnatal d 21; PACAP, pituitary adenylate cyclase-activating polypeptide; PNMT, phenylethanolamine-N-methyl transferase; SA, sympathoadrenal; SAS, SA system; SSH, suppression substractive hybridization; TH, tyrosine hydroxylase.

Received October 19, 2005.

Accepted for publication February 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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