Novel Expression of Neuropeptide Y (NPY) mRNA in Hypothalamic ...

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Endocrinology 142(11):4771– 4776 Copyright © 2001 by The Endocrine Society

Novel Expression of Neuropeptide Y (NPY) mRNA in Hypothalamic Regions during Development: Region-Specific Effects of Maternal Deprivation on NPY and Agouti-Related Protein mRNA K. L. GROVE, R. S. BROGAN,

AND

M. S. SMITH

Division of Neuroscience, Oregon Regional Primate Research Center, and Department of Physiology and Pharmacology, Oregon Health & Science University, Beaverton, Oregon 97006 During development there is novel expression of NPY mRNA in the dorsomedial hypothalamic nucleus (DMH) and perifornical region (PFR), in addition to the arcuate nucleus (ARH). Furthermore, NPY mRNA levels peak in all regions on postnatal d 16 (P16) and decrease to adult levels by P30. The purpose of the present study was to determine whether NPY and agouti-related protein (AGRP) mRNA expression in the different hypothalamic regions on P11 and P16 are similarly affected by fasting. An examination of the full rostral to caudal extent of the hypothalamus revealed two additional regions displaying novel NPY mRNA expression, the parvocellular division of the paraventricular nucleus (PVH) and lateral hypothalamus (LH). Maternal deprivation for 36 h, used to

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ECENTLY, OUR GROUP characterized the novel expression of NPY mRNA in cells in the dorsomedial hypothalamic nucleus (DMH) and perifornical region (PFR) during postnatal development in addition to the typical expression in the arcuate nucleus (ARH) (1). More specifically, we demonstrated a peak level of expression of NPY mRNA in all three regions on postnatal d (P) 16. However, by P30, significant levels of NPY gene expression were observed only in the ARH, with low levels in the compact zone of the DMH, similar to the adult rat (1). It is well recognized that ARH-NPY neurons are pivotal to the regulation of food intake and energy balance (see reviews in Refs. 2– 4). First, NPY mRNA expression in the ARH increases in response to fasting, including during the early postnatal period (5). Second, NPY mRNA expression in the ARH is elevated in models of hyperphagia, including specific models of obesity (6 – 8) and lactation (9, 10). Finally, ARHNPY neurons coexpress agouti-related protein (AGRP) (11, 12), another potent orexigenic agent involved in the regulation of food intake and energy balance. The role played by these other hypothalamic populations (DMH and PFR) of NPY neurons during development is unknown. Furthermore, it is unknown whether they are important for regulation of energy balance and food intake. The purpose of the present study was to determine whether NPY and AGRP gene expression in different hypothalamic re-

Abbreviations: AGRP, Agouti-related protein; ARH, arcuate nucleus; DMH, dorsomedial hypothalamic nucleus; DMHd, dorsal zone of the DMH; -ir, immunoreactive; LH, lateral hypothalamus; P16, postnatal d 16; PFR, perifornical region; PVH, paraventricular nucleus.

bring about a fast, similarly increased (23–29%) NPY and AGRP mRNA expression in the ARH on P11 and P16. In contrast, NPY expression in the DMH and PFR were significantly decreased (19 –30% and 48 –53%, respectively), whereas NPY mRNA levels in the PVH and LH were not altered by this treatment. The increase in NPY and AGRP mRNA expression in the ARH in response to maternal deprivation suggests that these neuronal populations respond to signals of energy balance. In contrast, NPY expression in the DMH, PFR, PVH, and LH is differentially regulated by maternal deprivation or other factors associated with maternal separation. (Endocrinology 142: 4771– 4776, 2001)

gions respond similarly to negative energy balance, induced by maternal deprivation, during specific stages of postnatal development. Materials and Methods Animals Pregnant rats (Simonsen Laboratories, Inc., Gilroy, CA) were housed individually and maintained on rat chow and water ad libitum. Lights were on from 0700 –1900 h. All animal procedures were approved by the Oregon Regional Primate Research Center institutional animal care and use committee. Animals were checked daily for the presence of pups; the day of delivery was considered P0. Litter sizes were adjusted to eight pups on P2.

Developmental study The purpose of this experiment was to investigate the pattern of NPY mRNA expression in the paraventricular nucleus of the hypothalamus (PVH) and lateral hypothalamus (LH) at different postnatal ages and determine whether the pattern was similar to the postnatal ontogeny of NPY mRNA expression previously described for the DMH and PFR (1). For this study rat pups (n ⫽ 2) were killed by decapitation on P2, P11, P16, and P30, and the brains were removed, frozen, and stored at ⫺80 C until processed for in situ hybridization. The brains were cryostat sectioned at 20-␮m thickness in a one in three series. NPY mRNA levels were determined in one series by in situ hybridization.

Maternal deprivation For the P11 and P16 maternal deprivation groups, pups on P9 and P14 were removed from their dam at 1700 h and placed in a cage with wood shavings on a heating pad (to maintain a environmental temperature of approximately 34 –36 C) for 36 h. This paradigm results in approximately 10% loss of body weight. A second group of pups was left with their dams and allowed to nurse during this time, forming the P11 and P16

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nondeprived groups. The pups were killed by decapitation, and the brains were removed, frozen, and stored at ⫺80 C until processed for in situ hybridization. The brains were cryostat sectioned at a 20-␮m thickness in a one in three series. One series was used for quantitative analysis of NPY mRNA, and a second series was used for AGRP mRNA. It is recognized that this maternal deprivation paradigm results in more than a simple negative energy balance. In pups removed from the dam, the additional stress of dehydration and lack of maternal interaction may also affect some parameters. However, this model does provide important insights into the affects of maternal deprivation (which includes negative energy balance) on the development of the hypothalamic NPY system.

AGRP clone For generation of the rat AGRP clone, total RNA was collected, and PCR was performed as previously described (13, 14). The following primers were used: AGRP forward, 5⬘-ATGCTGACTGCAATGTTGCTG-3⬘; and AGRP reverse, 5⬘-GGTACCTGCTGTCCCAAGCAG-3⬘. These primers are identical to those previously used for the generation of a mouse AGRP clone (14). The PCR product was cloned into pGEMT and sequenced to confirm identity.

In situ hybridization In situ hybridization was performed as previously described (1). The NPY experiment was split into two assays due to the large number of sections analyzed. For NPY, a cRNA probe was transcribed from a 511-bp cDNA (obtained from Dr. S. L. Sabol, NIH, Bethesda, MD) in which 25% of the UTP was 35S labeled (NEN Life Science Products, Boston, MA). The specific activity of the NPY cRNA probe was 5– 6 ⫻ 108 dpm/␮g. For AGRP, a cRNA probe was transcribed from a 300-bp cDNA in which 50% of the UTP was 35S labeled. The specific activity of the AGRP cRNA probe was 1.0 ⫻ 109 dpm/␮g. The saturating concentration of the probes used in the assay was 0.3 ␮g/ml䡠kb. The [35S]UTPlabeled NPY sense probe displays no labeling, whereas the AGRP cRNA sense probe displays a low level of labeling in the hippocampus (data not shown). Brain sections were fixed in 4% paraformaldehyde (pH 7.4) and treated with 0.25% acetic anhydride in 0.1 m triethanolamine (pH 8.0). Sections were then rinsed in 2 ⫻ SSC, dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and air-dried. The sections were exposed to the labeled probes overnight in a moist chamber at 55 C. After incubation the slides were washed in 4 ⫻ SSC, ribonuclease A at 37 C and in 0.1 ⫻ SSC at 60 C. Slides were then dehydrated through a graded series of alcohols and dried. For visualization the probe-labeled sections were exposed to sheet film (Biomax MR, Kodak, Rochester, NY) for an appropriate period of time (NPY, 2–3 d; AGRP, overnight). For histological analysis of the distribution of NPY and AGRP mRNAs, slides were dipped in Kodak NBT2 emulsion (Kodak) diluted 1:1 in 600 mm am-

F IG . 1. Photographs of autoradiograms of postnatal expression of NPY mRNA in the PVH and LH. NPY mRNA was detectable in the PVH on P2 and appeared to peak about P16, but was completely undetectable on P30. NPY mRNA in the LH was undetectable on P2 and P30. 3V, Third ventricle. The black boxes outline the areas of interest at each age group. Values in the lower left corner of the figures represent the approximate rostro-caudal position from bregma relative to the adult rat brain (37). Scale bar, 1.0 mm.

Grove et al. • Novel Hypothalamic NPY Expression

monium acetate, placed in light-tight boxes containing desiccant, and stored at 4 C for 8 –10 d. The slides were developed and counterstained with cresyl violet, and the distribution of silver grains was analyzed by darkfield microscopy.

Quantification of autoradiograms NPY and AGRP mRNAs were quantitated using a Macintosh-based image analysis system from NIH interfaced with a Scion image capture board. The area of exposed film and the average gray level density of labeling (integrated OD) in each region studied were measured using a sampling box encompassing the entire labeled area. The sampling box used to measure labeling in each region was kept constant for all sections from all of the brains. Background labeling was determined by sampling the thalamus, an area of the brain showing an even distribution of exposed emulsion, with no evidence of silver grain clusters (indicating a lack of specific staining to cells). Background labeling, which was less than 5% of total labeling, was then subtracted from total labeling to obtain specific labeling values. For the PVH and ARH, the sampling box encompassed the entire nucleus (containing both hemispheres), whereas bilateral samples were taken for the DMH, PFR, and LH. To obtain the mean integrated OD for each brain region, samples were obtained from sections that were anatomically matched between animals. The numbers of sections sampled were as follows: PVH, 5 sections; PFR, 3 sections; ARH, 15 sections; DMH, 4 sections; and LH, 7 sections.

Statistics To reduce the effect of interassay variability, the data were normalized to the controls within each assay. No direct comparisons were made between the P11 and P16 age groups. The data for each brain region were compared using a t test, with P ⬍ 0.05 considered significant. Data are expressed as the mean ⫾ sem.

Results Characterization of novel NPY gene expression in PVH and LH

Previously while investigating the postnatal development of NPY neurons in the ARH, we identified novel expression of NPY mRNA in the DMH and PFR at specific postnatal ages (1). In the present study we further investigated the expression of NPY mRNA in the complete rostro-caudal extent of the hypothalamus and identified two additional populations of NPY mRNA-expressing neurons in the PVH and LH (Fig. 1). NPY-expressing cells in the PVH were mainly located in the anterior parvocellular part of the nucleus (AP: ⫺1.0 to


⫺1.5) (15). Furthermore, the intensity of the staining was relatively low compared with that in other regions displaying NPY mRNA labeling. NPY-expressing cells in the LH were located at the caudal extent of the hypothalamus (AP: ⫺3.6 to ⫺4.5) (15). Qualitatively, NPY mRNA expression in the LH displayed a similar postnatal development pattern as that previously described for the DMH and PFR (1), with undetectable levels on P2, numerous NPY expressing cells on P11 and P16, and undetectable levels on P30 (Fig. 1). In contrast, NPY mRNA was readily detectable in the PVH on P2, with higher levels on P11 and P16 and undetectable levels on P30 (Fig. 1). Effects of maternal deprivation on NPY gene expression

Not surprisingly, 36 h of maternal deprivation significantly increased NPY mRNA in the ARH on both P11 (29%; P ⫽ 0.002) and P16 (20%; P ⫽ 0.014) compared with that in the control group. In contrast, NPY mRNA in the DMH was reduced on P11 (30%; P ⬍ 0.001) and P16 (16%; P ⫽ 0.10) by this treatment; however, only the reduction on P11 was significantly different. Furthermore, NPY mRNA was significantly reduced in the PFR of maternal deprivation animals on P11 (42%; P ⬍ 0.001) and P16 (53%; P ⬍ 0.001) compared with that in the control group. Maternal deprivation had no significant effect on NPY mRNA in either the PVH or LH on either P11 or P16, although there was a trend toward an increase in the LH on P11 (P ⫽ 0.064). Effects of maternal deprivation on AGRP gene expression

In contrast to the expression of NPY mRNA in numerous hypothalamic regions during postnatal development, AGRP mRNA was present only in the ARH on P11 (Fig. 3) and P16. Similar to NPY mRNA expression in ARH, maternal deprivation for 36 h significantly increased AGRP mRNA on P11 (23%; P ⫽ 0.047) and P16 (26%; P ⫽ 0.014) compared with that in the control group. Discussion

The present study identified novel NPY mRNA expression in the LH and PVH at specific postnatal ages (Fig. 1) in addition to the expression in the DMH and PFR previously reported (1). This is in contrast to the expression of AGRP mRNA during postnatal development that was present exclusively in the ARH (Fig. 3), as it is in the adult rat, where AGRP is colocalized with NPY (11, 12). Although this is the first known report of NPY gene expression in the LH and PVH, NPY immunoreactive (-ir) cell bodies have been demonstrated in magnocellular neurons in the PVH and in the dorsomedial portion of the LH during late gestation in the rat brain; however, the NPY cells disappeared at birth (16). Furthermore, NPY-ir cells in these areas were evident into the postnatal period and adulthood in rats that had the ARH ablated by monosodium glutamate treatment during the neonatal period (16, 17). However, the NPY mRNA expression in the PVH during postnatal development, reported in the present study, appeared to be limited to the anterior parvocellular division; however, the exact cellular localization remains to be confirmed. Interestingly, although neo-

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natal monosodium glutamate treatment results in a near complete loss of AGRP and NPY neurons within the ARH and a loss of AGRP-ir throughout the brain, the concentration of NPY-ir fibers within the hypothalamus is not dramatically changed (17, 18). This may be partially explained by the presence of NPY neurons in the brainstem that also provide efferent projections to the hypothalamus (19, 20). However, it is also likely that some of the NPY-ir fibers may originate from these other populations of hypothalamic NPY neurons. The physiological significance of NPY production in these regions during the postnatal period is unknown; however, all of these regions are important hypothalamic feeding centers (see reviews in Refs. 2– 4). Therefore, local production of NPY within these feeding centers may be necessary for the proper development of normal feeding circuitry. Alternatively, recent evidence suggests that NPY may function as a neuroproliferative factor for olfactory neurons (21); thus, it is possible that NPY-expressing cells in the PVH, DMH, PFR, or LH may function to promote neurogenesis in these regions. It is well recognized that removal of the pups from their dams for extended periods of time results in more than a simple negative energy balance. In this model, pups will have a stress response (as indicated by elevated corticosterone) in response to dehydration and lack of maternal interaction (22, 23), all of which could potentially effect NPY expression. However, it should also be noted that any fasting paradigm, whether performed in pups or adult rats, will stimulate a stress response; in fact, the increased glucocorticoid levels may be one of the mediators of increased NPY gene expression in the ARH in response to fasting in the adult rat (24 –26). It is clear from the present study that maternal deprivation resulted in a similar increase in ARH-NPY (20 – 29%; Fig. 2) and AGRP (23–26%; Fig. 3) mRNA expression, as reported in response to fasting in the adult rat (6, 7, 11). These results are also similar to those previously reported by Kowalski et al. (5), demonstrating that 24 h of maternal deprivation (fasting) increased NPY mRNA levels in the ARH of rat pups as early as P2. These data suggest that ARH-NPY/AGRP neurons are responsive to peripheral signals of changes in energy balance very early in the postnatal period. This is somewhat surprising in light of recent evidence that treatment with exogenous leptin, one of the major peripheral signals of energy balance, has very little effect on body weight, metabolic rate, or food intake until after postnatal d 17 (27, 28). Furthermore, chronic leptin treatment during the first postnatal week has no effect on NPY or AGRP mRNA levels in the hypothalamus (28). However, this study involved quantitative real-time PCR on extracts from the whole hypothalamus (28) and therefore cannot rule out the possibility of changes in NPY expression in specific hypothalamic regions. Taken together, these data suggest that neonatal rats may have leptin resistance, as supported by the elevated endogenous leptin levels (29) yet high levels of hypothalamic NPY and AGRP (1) (Figs. 2 and 3). Therefore, the increase in NPY and AGRP expression the ARH in response to maternal deprivation may not be due to changes in leptin levels, as has been suggested in the adult rat, but, rather, may be due to some other peripheral signal (i.e. corticosterone, insulin, or glucose).

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FIG. 2. Photographs of autoradiograms showing NPY mRNA expression on P11 in maternal deprived (MD) and nondeprived (CTR) rats. Values in the graphs represent the mean ⫾ SEM. For ARH, DMH, and PFR, n ⫽ 8; for P16 CTR, n ⫽ 7. For LH, n ⫽ 7 for all groups. For PVH, n ⫽ 5 for all groups. f, CTR; o, MD. Asterisks indicate a significant difference from the CTR controls (P ⬍ 0.05, as determined by t test). Diagrams adapted from Swanson (15). Values in the upper right corner of the diagram represent the approximate rostro-caudal position from bregma relative to the adult rat brain (37). In the images of the DMH, the dashed line indicates the border of the compact zone (co). Scale bar, 0.5 mm.

In contrast to the increased expression of NPY mRNA in the ARH in response to maternal deprivation, NPY gene expressions in these novel populations of hypothalamic NPY neurons were differentially affected by this treatment. Maternal deprivation decreased the expression of NPY mRNA in the DMH (16 –30%) and PFR (42–53%) on P11 and P16. It is worth noting that the reduction in NPY mRNA expression in the DMH in response to maternal deprivation appeared to be mainly due to a reduction of expression in the dorsal zone

of the DMH (DMHd); however, further study is needed to confirm this. It is unclear from the present study what components of maternal deprivation may be responsible for the decreased expression in the DMH and PFR. Although NPY mRNA expression in the DMHd has been reported in several adult rodent models, including the lactating rat (9, 10), agouti mouse (6), melanocortin 4 receptor knockout mice (6), and diet-induced obese rats (30), it has not been determined whether NPY mRNA expression in the DMHd in these mod-


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FIG. 3. Photographs of autoradiograms showing AGRP mRNA expression on P16 in maternal deprived (MD) and nondeprived (CTR) rats. Values in the graphs represent the mean ⫾ SEM (n ⫽ 4). f, CTR; o, MD. Asterisks indicate a significant difference from the CTR controls (P ⬍ 0.05, as determined by t test). The value in the upper right corner represents the approximate rostro-caudal position from bregma relative to the adult rat brain (37). Scale bar, 2.0 mm.

els is also decreased in response to fasting. Further study is necessary to investigate that mechanism controlling NPY expression in the DMH and PFR. Unlike the changes in NPY mRNA in the ARH, DMH, and PFR in response to maternal deprivation, NPY gene expression in the LH and PVH was unaffected by this treatment. These data indicate that neurons in these regions may not be responsive to peripheral signals of energy balance at this age. Consistent with this hypothesis, orexin mRNA expression in neurons in the LH has also been shown to be unaffected by fasting during this period (31). One factor that may contribute to the unresponsiveness of the PVH and LH to fasting on P11 and P16 is the lack of sufficient innervation by ARHNPY/AGRP neurons. Although in the adult rat ARH-NPY/ AGRP neurons are known to densely innervate all of these regions (18, 32, 33), preliminary studies in our laboratory indicate that NPY/AGRP-immunoreactive fibers do not appear to fully innervate the DMH/PFR area until P10 –11 and the PVH until around P15–16 (Grove, K. L., and M. S. Smith, unpublished observations). In the absence of this innervation, there would be no neuronal substrate to convey the changes in activity of ARH neurons in response to the negative energy balance. Therefore, the differential effect of fasting on NPY mRNA expression in the PVH, PFR, DMH, and LH on P11 and P16 may be dependent on the timing and/or intensity of the innervation of these regions by ARH neurons. This model demonstrates that maternal deprivation at two different postnatal ages alters the hypothalamic feeding circuits in a site-specific manner (increasing AGRP and NPY in the ARH, decreasing NPY in the DMH and PFR, and not affecting NPY in the PVH and LH). Furthermore, acute fasting (maternal deprivation) or chronic caloric restriction specifically during the postnatal period has been demonstrated to have long-term consequences on hypothalamic function (i.e. hypothalamic-pituitary-adrenal axis) and body weight management, resulting in a lean-hypophagic phenotype in adults even after returning the animals to ad libitum feeding (34, 35). The results from the present study suggest that the changes in NPY expression in the ARH, DMH, and PFR at a postnatal stage when hypothalamic feeding circuits are developing (1, 16, 31, 33, 36) may be at least partially responsible for the persistent effects on body weight management; however, this remains to be directly tested.

Acknowledgments Received May 9, 2001. Accepted July 30, 2001. Address all correspondence and requests for reprints to: Kevin L. Grove, Ph.D., Oregon Regional Primate Research Center, Oregon Health & Science University, Beaverton, Oregon 97006. E-mail: grovek@ ohsu.edu. This work was supported by NIH Grants HD-14643, HD-18185, and RR-00163.

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