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Endocrinology Vol. 144, No. 11 4783-4789
Copyright © 2003 by The Endocrine Society

Reduced Hypothalamic Neuropeptide Y Expression in Growth Hormone- and Prolactin-Deficient Ames and Snell Dwarf Mice

David L. Hurley, Derin V. Birch, M. Camille Almond, Irma J. Estrada and Carol J. Phelps

Department of Cell and Molecular Biology (D.L.H., M.C.A.), Neuroscience Program (D.L.H., D.V.B., C.J.P.), and Department of Structural and Cellular Biology, School of Medicine (I.J.E., C.J.P.), Tulane University, New Orleans, Louisiana 70118

Address all correspondence and requests for reprints to: Dr. David L. Hurley, Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana 70118. E-mail: david.hurley{at}tulane.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuropeptide Y (NPY)-producing neurons in the hypothalamic arcuate nucleus (ARC) have been implicated in GH feedback in several studies in rats. Ames (df/df) and Snell (dw/dw) dwarf mice carry mutations in transcription factors Prop-1 and Pit-1, respectively, that abrogate detectable expression of GH, prolactin, and TSH. The present study was undertaken to determine whether and to what extent hypothalamic NPY neurons are affected by the lifelong absence of pituitary hormone feedback in hypopituitary Ames and Snell dwarf mice. Total ARC NPY mRNA levels were quantified using in situ hybridization, and numbers of ARC NPY-producing cells were quantified using immunocytochemistry. For in situ hybridization, dwarf and normal coronal brain sections were hybridized with 35S-labeled riboprobe complementary to rat NPY cDNA, and then analyzed for total signal intensity from the entire ARC for each animal as well as for mRNA per neuron. NPY-containing perikarya in ARC were counted in sections of colchicine-treated (intracerebroventricular) dwarf and normal mice. Total ARC NPY mRNA was reduced in df/df mice to 33.6% (P < 0.01) of that in normal littermates, and reduced in dw/dw mice to 46.3% (P < 0.05) of normals, but there was no difference in expression per neuron as determined by reduced silver-grain counting. The decrement in dwarf mice of total ARC NPY mRNA without reduction in mRNA per cell suggested a reduction in NPY-containing neuron number, which was the case as shown by immunocytochemistry. NPY neuronal number in adult Ames dwarf mice (1048 ± 104) was significantly (P < 0.01) reduced to 68% of that in DF/df littermates (1536 ± 73), and significantly (P < 0.05) reduced in Snell dwarf mice to 63% of normals (1138 ± 137 vs. 1726 ± 205). This study represents the first enumeration of NPY-producing neurons in mouse hypothalamus and the first demonstration of lower NPY neuron number in a hypopituitary model. The reduction in total NPY mRNA was greater than that reported in studies of GH-deficient rats, suggesting that NPY expression may be affected by the lifelong absence of prolactin or TSH or both, as well as GH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH PRODUCTION BY the anterior pituitary is controlled by the antagonistic effects of two hypophysiotrophic factors, GHRH and somatostatin [somatotropin release-inhibiting hormone (SRIH)], from neurons in the respective arcuate (ARC) and periventricular (PeN) nuclei of the hypothalamus (1). Pituitary prolactin (PRL) secretion is inhibited by dopamine (DA) produced by neurons of the ARC (2). The role of hormone feedback upon these hypophysiotropic neurons has been studied using hypopituitary models such as Ames (df/df) and Snell (dw/dw) dwarf mice, which bear spontaneous mutations in transcription factors Prop-1 (3) or Pit-1 (4), respectively. In these mice, GH, PRL, and TSH are all undetectable (5, 6, 7, 8, 9). These mutants show reduced hypophysiotropic SRIH and DA and increased GHRH expression with concomitant changes in the differentiation of the neurons that produce these factors (reviewed in Refs. 10 and 11).

Direct trophic effects of GH and PRL on respective hypophysiotropic neurons would require the presence of hormone receptors in those specific neuron populations. GH-receptors (R) are localized predominantly in PeN and ARC (12, 13, 14, 15). Although GH-R is expressed by SRIH neurons in PeN, the majority of GH-R-expressing neurons in ARC are not GHRH producing (13). Kamegai et al. (16) reported that c-fos expression in response to human GH treatment of hypophysectomized rats was in SRIH neurons in PeN, but in ARC was primarily in the neuropeptide Y (NPY)- rather than GHRH-producing neurons. Subsequently, it was shown that hypothalamic NPY-expressing neurons also expressed GH-R mRNA (17), and that NPY mRNA reduced by hypophysectomy was restored by treatment with rat GH (18). Thus, both these studies supported the role postulated earlier for NPY in GH regulation (19).

Those findings also suggested that NPY expression in ARC neurons would be altered if there were a complete developmental absence of GH. In dw/dw dwarf rats with approximately 5% of normal GH (20), NPY mRNA in the hypothalamus measured by in situ hybridization (ISH) was reduced to 52% of that in normal littermates, and increased after bovine GH replacement to 166% of normal (21). In the Sprague Dawley spontaneous dwarf rat (SDR), with no detectable circulating GH (22), levels of NPY mRNA measured in dissected hypothalami by Northern blot hybridization were 70% of those of normal littermates, which was increased to 130% after replacement with rat GH (23). No analysis of NPY in GH-deficient mice has been reported. Therefore, to examine regulation of NPY in the absence of GH and other pituitary hormones in mice, NPY expression in ARC neurons was studied in hypopituitary Ames and Snell dwarf mice by complementary methods: ISH to localize and quantify NPY mRNA, and immunocytochemistry (ICC) to quantify the number of NPY peptide-producing neurons.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue preparation
Ames and Snell dwarf mice and normal littermates were reared in colonies maintained at Tulane University School of Medicine. Dwarf (df/df and dw/dw) and normal (DF/df and DW/dw) mice were reared from matings of heterozygous (+/-) females with -/- males. Fertility was induced in dwarf males by D/L-thyroxine treatment (2 µg, ip, thrice weekly; Sigma-Aldrich, St. Louis, MO), followed by pituitary renal capsule allografts from normal donors, as a source of homologous PRL, beginning at 9 wk of age. The pituitary transplant surgery was performed in anesthetized (inhalant isoflurane) animals. Normal donors were male or female, 2–6 months old; they were euthanized with carbon dioxide and decapitated for pituitary gland removal. Whole single glands were placed under the kidney capsules of recipient animals. After surgery, recipients were observed continuously, and body temperature was supported, until recovery from anesthesia. Breeding colonies were maintained under conditions of controlled temperature (22 ± 2 C), with lights on from 0600–1800 h, and food and water available ad libitum. Procedures for maintenance, recovery surgery, euthanasia, and perfusion were approved by the Tulane University School of Medicine Animal Care and Use Committee.

Adult male and female mice, aged 3–11 months, were assessed. Some dwarf and normal mice were pretreated with intracerebroventricular (icv) colchicine 2 d before euthanasia. Normal mice received 1.5 µl, and df/df mice received 0.5 µl, of the colchicine solution (Sigma-Aldrich; 30 mg/ml sterile saline) over a 5-min period; the colchicine doses were based upon average body weight (bw) (30.9 ± 1.3 g for normal and 10.2 ± 0.4 g for df/df), so that both normals and dwarfs received 1.5 µg/g bw. Stereotaxic coordinates for the icv injections were as follows: 0.3 mm posterior and 0.8 mm posterior to bregma, for both normal and df/df mice; the needle tip was placed 2.3 mm, for normal mice, and 2.0 mm, for df/df mice, ventral to the surface of the brain, according to a stereotaxic atlas of the mouse brain (24). Deeply anesthetized (80 mg/kg bw pentobarbital) animals were perfused transcardially with normal saline followed by buffered 4% paraformaldehyde/0.5% glutaraldehyde (Faglu) fixative (25). Brains were postfixed at least 24 h in Faglu containing 30% sucrose, sectioned on a sliding microtome in the coronal plane at 30 µm into alternate serial sets of six, and preserved in cryoprotectant (26) until used for ISH or ICC.

ISH
Sections from colchicine-treated mice were not used for ISH. Brain sections were mounted on Vectabond-coated slides, heated at 42 C overnight, and stored desiccated at 4 C. Sections were treated before hybridization with Proteinase K and acetylation as described previously (27, 28). NPY riboprobes were synthesized in vitro from Mlu1-linearized (for antisense strand) and SphI-linearized (for sense strand) plasmid templates (courtesy of Dr. Iain C. A. F. Robinson, National Institute for Medical Research, London, UK) using T7 or SP6 RNA polymerases, respectively, according to manufacturer’s instructions (Ambion, Austin, TX) in the presence of [35S]CTP (Amersham Pharmacia Biotech, Piscataway, NJ) at a specific activity of 4.5 x 108 dpm/µg. Probes were sized by electrophoresis on a 5% polyacrylamide gel. Unincorporated radionucleotides were removed using NucAway spin columns (Ambion), and probes were added to hybridization solution at saturating amounts according to length and specific activity (29). Hybridization was performed overnight at 50 C under coverslips in humid conditions. The slides were then rinsed to remove coverslips, treated with ribonuclease A at 50 µg/ml at 37 C for 30 min, and taken through stringent washes to a final concentration of 0.1x standard saline citrate containing 5 mM dithiothreitol at 60 C.

Slides were then air dried before exposure to x-ray film (Biomax MR, Kodak, Rochester, NY) to allow for macroscopic examination. Multiple exposures, from 6–96 h in duration, were collected to ensure consistent exposure quality for each section, and image analysis was performed on exposures of approximately 48 h. Emulsion (NTB-2, Kodak) autoradiography was then performed for 4 times the optimal x-ray exposure (i.e. 8 d) at 4 C. Emulsion-dipped slides were developed in D19 developer (Kodak) precooled to 14.5 C for 2.5 min, followed by immersion for 30 sec in distilled water and 5 min in general purpose fixer (Kodak), and a final wash in distilled water for approximately 1 h. Slides were dehydrated with increasing ethanol series, then stained with eosin Y (Sigma-Aldrich), and coverslipped using Permount (Fisher, Pittsburgh, PA) for examination.

Image analysis
All image analysis was performed on coded slides so that investigators were blind to animal identity or genotype. Densitometric analysis of x-ray films was performed to obtain relative hybridization density over the ARC. Films were scanned (Microtek Scanmaker X12, Macintosh iMac, and NIH Image 1.62 software available at http://rsb.info.nih.gov/nih-image/) and processed using density-slicing mode. The intensity of hybridization was quantified in all sections containing anatomically identifiable ARC. These sections corresponded to those at distances from 1.22–2.46 mm caudal to bregma according to the atlas of Franklin and Paxinos (24). Intensity in ARC as a whole was calculated as the product of the number of pixels above threshold times the average intensity of these pixels, summed over all labeled sections from each mouse. All settings were held constant over the entire measurement session, and a background value determined in an equivalent area over dorsal hypothalamus was subtracted from each reading. Sections from different hybridization runs were included in each analysis session to control for the effects of multiple assays, and varied by less than 5%. Duplicate readings of the same sections in different analysis sessions varied by less than 5%. Relative integrated intensity was calculated as the product of average intensity and pixel number in the selected area, then relative integrated intensities for all sections (five to seven in normals, and four to six in dwarfs) through ARC were totaled for each animal. Means of total integrated intensity were calculated for each mouse genotype.

Grain counting was performed after emulsion autoradiography of the sections using a Nikon (Melville, NY) Optiphot microscope with dark-field illumination and x20 magnification. Video images were analyzed using a real-time video analysis system [C2400 video camera (Hamamatsu, Hamamatsu City, Japan), Data-Translation Frame Grabber video card (Marlborough, MA), and Macintosh Quadra 950 (Apple Computer, Cupertino, CA)] and grain-counting software developed by Dr. D. K. Clifton (University of Washington Medical Center, Seattle, WA) (30). The software identifies labeled cells with clusters of grains having a signal intensity 3-fold higher than background. Grains were counted in areas containing morphologically identifiable regions of ARC. The labeled cells in ARC were selected by the algorithm of the software program to avoid operator prejudice. Grains per cell were analyzed in 10–40 labeled cells in each section from all sections containing ARC (five to seven in normals, and four to six in dwarfs), and then used to calculate average grains per cell for each animal.

ICC
Sets of brain sections were processed free-floating, with representatives of each type (dw/dw, df/df, DW/dw, and DF/df) included in any single run, using identical solutions simultaneously. Sections were incubated first in 1% aqueous sodium borohydride to remove residual fixative and allow antibody access to glutaraldehyde-fixed linkages. NPY ICC was performed using a primary antiserum produced by Dr. J. K. McDonald (Emory University Medical Center, Atlanta, GA) (19) at a dilution of 1:5000. The tissue sections were processed further using biotinylated goat antirabbit IgG and avidin-biotin complex solutions (Vectastain kit, Vector Laboratories, Burlingame, CA). Visualization of NPY immunoreactivity was achieved by color development using 0.02% diaminobenzidene tetrahydrochloride and 0.003% H2O2 in Tris buffer. Sections were then mounted on acid-cleaned and gelatin-coated microscope slides in rostral-to-caudal order.

Neuronal quantification
Cell counts were performed at 180-µm intervals (on 30-µm sections) for NPY-positive cells from anterior to posterior in ARC. Cell counts were corrected for sampling periodicity (i.e. x6, for every sixth section) to obtain total cell numbers. Because the sampling distance (180 µm) exceeded the average size of ARC neurons (13.3 ± 1.3 µm for DA) (31), cell counts were not corrected for sampling error due to missed or recounted cells (32). The accuracy of this method has been verified previously for GHRH- (33) and tyrosine hydroxylase-positive (34) cell types.

Statistical analysis
One-factor ANOVA, followed by Student-Newman-Keuls post hoc tests, was used to assess statistical differences between mouse types using a computer program (SuperANOVA, Abacus Concepts, Berkeley, CA). For all tests, P < 5% was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Representative x-ray film exposures after ISH to detect NPY mRNA are shown in Fig. 1Go for normal adult DF/df (A) and Ames dwarf (df/df) mice (B), and for normal adult DW/dw (C) and Snell dwarf (dw/dw) mice (D). All ISH analyses were performed on sections from mice not treated with colchicine. There was strong hybridization signal localized to the region of the ARC in sections of normal mice, and a less intense but detectable signal in each type of dwarf hypothalamus. Control hybridizations performed with the NPY sense strand probe showed no detectable hybridization over any brain region (data not shown).



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  		FIG. 1. Representative NPY mRNA ISH visualized by x-ray film autoradiography of coronal sections of adult normal (DF/df; panel A), Ames dwarf (df/df; panel B), normal (DW/dw; panel C), and Snell dwarf (dw/dw; panel D) mice. Signal is most intense in ARC. Original magnification, x2.

 
Summation of ISH hybridization intensity over all ARC-containing sections in each animal was used to make the comparison of total ARC NPY mRNA shown graphically in Fig. 2AGo for adult Ames dwarf (df/df) and normal (DF/df) littermate mice, and in B for Snell dwarf (dw/dw) and normal (DW/dw) mice. The effect of genotype on total NPY mRNA assessed by one-factor ANOVA was significant for df mice (F(1,20) = 29.77; P = 0.0001) and for dw mice (F(1,12) = 22.18; P = 0.0005). Post hoc analysis showed that the total amount of ARC NPY mRNA was reduced significantly (P < 0.01) in df/df mice to 33.6% of that in normal littermates, and reduced significantly (P < 0.01) in dw/dw mice to 46.3% of normal.



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  		FIG. 2. Graph of average total image intensity of NPY mRNA hybridization in normal and dwarf mouse ARC. In panel A, the striped column represents total NPY mRNA signal in adult DF/df mice and the solid column represents signal in df/df mice. In panel B, the striped column represents normal DW/dw mice, and the solid column represents dw/dw dwarfs. Each animal value is the sum of intensity measured in five to seven sections in normal mice and in four to six sections in dwarf mice. The numbers of animals analyzed are shown at the base of each column. Vertical bars denote SEM. **, Significant differences between normal and dwarf values of P < 0.01.

 
After emulsion autoradiography, as shown in the dark-field photomicrographs in Fig. 3Go, reduced silver grains were detectable over single neurons in DF/df (A), df/df (B), DW/dw (C), and dw/dw (D) mice. Grain counts of ARC neurons labeled above background showed that there was not a significant difference in the number of grains per neuron between normal and dwarf mice of either type (Fig. 3CGo).



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  		FIG. 3. Representative cellular localization of NPY mRNA hybridization visualized by emulsion autoradiography of normal DF/df (panel A), df/df (panel B), DW/dw (panel C), and dw/dw (panel D) mice. Original objective magnification, x40. Clusters of reduced silver grains (white dots) are present over single neurons viewed under dark-field illumination. Average numbers of reduced silver grains per neuron are given in panel E for normal (striped columns) and dwarf (solid columns) mice of either Ames (left set) or Snell (right set). Vertical bars denote SEM. Grains per cell were measured in 10–40 cells per section in five to seven sections of each normal mouse and in four to six sections of each dwarf animal. The numbers of animals analyzed are shown in the base of each column.

 
Immunostaining for NPY neurons was first attempted in mice that had not been pretreated with colchicine. Representative sections of ARC are shown in Fig. 4Go for normal DF/df (A), dwarf df/df (B), normal DW/dw (C), and dwarf dw/dw (D) mice. Fiber staining occurs throughout ARC and in the internal zone of the median eminence, but few perikarya are visibly distinguishable. Thus, to intensify staining in NPY-containing neuronal cell bodies, mice were pretreated with icv colchicine to block axonal transport. As shown in Fig. 5Go, neuronal perikarya occur in ventral ARC in all types of animals: DF/df (A), df/df (B), DW/dw (C), and dw/dw (D) mice. Mean numbers of positively stained neurons in normal and dwarf mice are shown in Fig. 6Go. As shown in Fig. 6AGo, adult Ames dwarf mice had 1048 ± 104 NPY-positive neurons vs. 1536 ± 73 in DF/df littermates (F(1,14) = 14.81; P = 0.002), a significant (P < 0.01) decrease to 68.2% of the normal number. Adult Snell dwarf mice (Fig. 6BGo) also showed reduced numbers of NPY-immunoreactive neurons, 1138 ± 137 vs. 1726 ± 205 in normals (F(1,14) = 5.68; P = 0.032), a significant (P < 0.05) decrease to 65.9% of normal.



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  		FIG. 4. Representative NPY immunostaining in midcoronal ARC sections from mice that were not colchicine pretreated. A, DF/df; B, df/df; C, DW/dw; D, dw/dw. Each section is oriented with the third ventricle on the left of the frame. Original objective magnification, x20.

 


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  		FIG. 5. Representative NPY immunostaining in midcoronal ARC sections from icv colchicine-treated adult mice. A, DF/df; B, df/df; C, DW/dw; D, dw/dw. Each section is oriented with the third ventricle on the left of the frame. Original objective magnification, x20.

 


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  		FIG. 6. Graph of average numbers of neurons containing NPY peptide as detected by ICC. In panel A, the striped column represents NPY cells in adult DF/df mice, and the solid column represents cells in df/df mice. In panel B, the striped column represents normal DW/dw mice, and the solid column represents dw/dw dwarfs. For normal mice, seven to nine sections from each animal were counted, whereas in dwarf mice, five to eight sections were analyzed. Numbers of animals analyzed are shown at the base of each column. Vertical bars denote SEM; significant differences between normal and dwarf values are denoted as follows: *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Feedback of target hormones on hypophysiotropic neurons is necessary for homeostatic regulation of GH and PRL production (1, 35). Because Ames and Snell dwarf mice lack GH, PRL, and TSH due to mutations in pituitary-specific transcription factors Prop-1 (3) or Pit-1 (4), respectively, they are models for the study of hypophysiotropic neuron development in the absence of pituitary hormone feedback (10). In the present study, a significant decrease in total ARC NPY mRNA was found, to 33.6% in Ames dwarfs and 46.3% in Snell dwarfs, compared with normal mice of each type. However, there was no change in NPY mRNA expression per cell as measured by grain counting. Decreased total NPY mRNA without a change in expression per neuron suggested that dwarf mice had reduced numbers of NPY-producing neurons. This was supported by the finding that NPY-immunopositive neuron number was reduced, in Ames dwarfs to 68.2%, and in Snell dwarfs to 65.9%, of normal. Thus, the decrement in NPY expression in dwarf mouse hypothalamus is due to reduced numbers of NPY-expressing neurons, indicating a deficit in neuronal differentiation.

Colchicine treatment was necessary for enumeration of hypothalamic NPY neurons, because perikaryal peptide content is normally below a threshold for immunoreactive detectability. Colchicine dose was proportional to weight, and the lower quantity delivered to dwarfs was not responsible for the reduced NPY-immunoreactive population. Previous studies in dwarf mice show that, after treatment with colchicine at this dose, numbers of nonhypophysiotropic neurons in clearly neuroanatomically defined regions are comparable with those in normal mice. After icv colchicine, SRIH-immunoreactive cells in ARC of dwarf mice number the same as in normal siblings (36), and dopaminergic neurons in medial zona incerta (area A13) are the same number in dwarfs and normals (37). This is also true for galanin-immunoreactive neurons in ARC (Romero, M. I., and C. J. Phelps, unpublished data). In addition, GHRH-producing neurons in the dwarf ARC number more than twice those of normals after colchicine at these doses (33, 37). Unfortunately, for NPY-producing neurons, there exists no such clearly defined neuroanatomic region that is nonhypophysiotropic; immunoreactive cells in regions such as cortex are not quantified, because that would measure density per area, and might be affected by difference in brain size between dwarf and normal.

The use of ICC to determine a reduction in NPY neuron number provides new insight into feedback effects of GH, because previous studies measured only NPY mRNA levels (21, 23, 38). The decrease in cell number was less than the decrease in total mRNA, per-cell mRNA levels being constant. It should be noted that counting tabulates cells containing peptide above a threshold of detectability, but does not quantify total peptide, whereas ISH of total mRNA is such a measure. Because the final 36-amino acid NPY peptide is the result of several posttranslational cleavage steps from the initial translation product (39), it is possible that regulation of any of these cleavage enzymes could dissociate mRNA levels from peptide levels. For example, NPY mRNA levels do not change in response to genetic disruption of normal feeding in anorexic (anx/anx) mice, but peptide expression is markedly increased (40).

The significance of these results can be evaluated by comparison with reports of NPY mRNA expression in GH-deficient rats (21, 23) and GH-R knockout mice (38). The reduction in NPY mRNA in Snell and Ames dwarf mice was greater than that measured by Northern blot hybridization in hypothalamic extracts of SDR rats (23), which have complete but isolated GH deficiency. In mice with GH feedback blocked by transgenic inactivation of GH-R, hypothalamic NPY mRNA was 52% of normal as determined by Northern blot hybridization (38). ISH, compared with extraction methods required for blotting or quantitative PCR, has the advantage that the region of interest is delineated microscopically. It is possible that the smaller decrement in NPY mRNA reduction in the SDR rats and GH-R-deficient mice might be due to dissections that included nonhypophysiotropic regions that express NPY, particularly given the high abundance of NPY throughout the brain (41). The decline to approximately 35% of normal in both types of dwarf mice was also greater than the decline to 52% measured using ISH in dw/dw rat (21). This greater decrement could be due either to the complete GH deficiency in dwarf mice (9, 42) vs. the 5–10% GH levels present in dw/dw rats (20), or to the absence of PRL and/or TSH in dwarf mice compared with at least normal PRL in dw/dw rat (20, 43, 44, 45).

In addition to being a response to lower GH, reduced NPY levels in dwarf mice could be due to increased food intake or energy balance (40, 46). However, there is proportionally no difference in food intake between normal and Ames dwarf male mice at either 12 or 24 months of age; food consumption per gram of bw is greater in female dwarfs than in normals at 12 months of age (47). Ames dwarf mice exhibit a comparable percentage of lean mass and a reduced percentage of body fat compared with normal controls at 4.5–6 months of age, but are not significantly different from normal mice for percentage of body fat by 18 months of age (48). SDR rats did not differ from normal littermates in a 2-h feeding period (49); there are no studies of feeding in dw/dw dwarf rats. Thus, dwarf mice, and likely dwarf rats, do not exhibit altered feeding or metabolic states that would contribute to changes in NPY mRNA or peptide.

The greater decrement of NPY mRNA in dwarf mice compared with that in GH-deficient rats may be due to loss of feedback from PRL in Ames and Snell dwarfs (7). The role of NPY in PRL regulation was shown when immunoneutralization of NPY in normal male mice significantly increased PRL levels in the circulation, suggesting that NPY inhibits PRL secretion (50). The effect of NPY on PRL is thought to occur via stimulation of DA neurons in the ARC, resulting in inhibition of PRL secretion (35). Thus, in GH- and PRL- deficient dwarf mice, a proportion of NPY expression related to PRL-positive feedback would be expected to decline, similar to that of DA and tyrosine hydroxylase (10). There are transgenic mouse models with altered PRL (51, 52) available for further evaluation of the feedback effects of PRL on NPY expression.

Because these NPY-producing neurons express GH-R (17, 18), the reduction in number of NPY-producing neurons in the absence of several pituitary hormones is likely to result from decreased receptor activation. Similarly, decreased hypophysiotropic SRIH expression in dwarf mice is due to a reduction in neuron number (27, 36), and these SRIH neurons express GH-R (15). PRL-inhibiting dopaminergic neurons are reduced in number in dwarf mice (34). The dopaminergic neurons express PRL-R (53); studies of PRL-R on NPY-producing neurons have not been reported. Although mechanisms of GH-R and/or PRL-R signaling in hypothalamic neurons are poorly understood, the present findings further implicate both GH and PRL feedback in phenotypic differentiation of hypophysiotropic neurons. Further study of this feedback will be to determine the developmental time at which NPY expression is detectably reduced in dwarf mice. As shown in previous work (54, 55), establishing the onset of altered NPY expression may permit the separation of developmental and dynamic feedback effects.


   Acknowledgments
 
We gratefully acknowledge the gifts of the NPY antiserum from Dr. J. K. McDonald and the NPY cDNA from Dr. I. C. A. F. Robinson, and the helpful comments of Dr. Pamela Bennett Houston at Imperial College (London, UK).


   Footnotes
 
This work was supported by Public Health Service Grant NS25987 (to C.J.P.) and National Science Foundation Grant IBN960805 (to D.L.H.).

Abbreviations: ARC, Arcuate nucleus; bw, body weight; DA, dopamine; ICC, immunocytochemistry; icv, intracerebroventricular; ISH, in situ hybridization; NPY, neuropeptide Y; PeN, periventricular nucleus; PRL, prolactin; R, receptor; SDR, spontaneous dwarf rat; SRIH, somatotropin release-inhibiting hormone.

Received June 13, 2003.

Accepted for publication August 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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