Vol. 275, Issue 2, R466-R470, August 1998
Ontogeny of neuropeptide Y expression in response to
deprivation in lean Zucker rat pups
Timothy J.
Kowalski1,
Thomas A.
Houpt1,
Jeongwon
Jahng2,
Nori
Okada3,
Streamson C.
Chua Jr.3, and
Gerard P.
Smith1
1 E. W. Bourne Laboratory, New
York Hospital-Cornell Medical Center;
2 Laboratory of Molecular
Neurobiology, Burke Medical Research Institute, Department of Neurology
and Neuroscience, White Plains 10605; and
3 Laboratory of Human Behavior and
Metabolism, The Rockefeller University, New York, New York 10021
 |
ABSTRACT |
Hypothalamic
neuropeptide Y (NPY) activity is believed to play an important role in
the response to food deprivation in adult rats. Little is known,
however, about the role of the hypothalamic NPY system in the control
of food intake in the preweanling rat. To address this issue, we
examined the effect of deprivation on arcuate nucleus preproNPY
expression in lean Zucker rat pups, using in situ hybridization.
PreproNPY expression within the arcuate nucleus was localized to cells
in the medial portion. Twenty-four hours of food, water, and maternal
deprivation significantly increased the relative abundance of preproNPY
mRNA in pups on postnatal day (P)
2,
P9,
P12, and
P15 by 14-31%. This response,
however, was not observed on P5. The
absence of an effect on P5 and the magnitude of the response at the other ages tested were not correlated with the amount of weight lost during deprivation.
in situ hybridization; arcuate nucleus; neonate; starvation
| |
INTRODUCTION |
NEUROPEPTIDE Y (NPY) is an orexigenic peptide when
administered centrally and is most potent when injected into the
paraventricular nucleus of the hypothalamus (PVN) (25). The arcuate
nucleus is the primary site of NPY synthesis in the hypothalamus (1), and an arcuo-paraventricular NPYergic projection exists (2). This
arcuo-paraventricular NPYergic projection is believed to play a role in
feeding behavior because its activity is correlated with deprivation
state; arcuate nucleus preproNPY mRNA, PVN immunoreactive NPY, and release of NPY into the PVN all increase during periods of
food deprivation and return to basal levels with refeeding (4, 12, 20).
Hypothalamic NPY expression, peptide levels, and release rates are
higher in many animal models of obesity (21, 30), and chronic
intracerebroventricular administration of NPY mimics many of the
physiological and behavioral symptoms present in obesity (33). These
observations suggest that increased hypothalamic NPY activity is
involved in the etiology of obesity and its associated metabolic and
behavioral disturbances, such as hyperphagia, hyperinsulinemia, and
hypercorticosteronemia.
Despite its apparent importance in the ingestive and metabolic response
to food deprivation and in animal models of obesity, little is known
about the ontogeny of the hypothalamic NPY system. Studies in neonatal
rats have demonstrated that administration of NPY into the PVN can
increase both milk and water intake at postnatal
day (P)
2 and selectively increase milk intake
via an intraoral catheter on P15 (5).
Although pups at P15 are behaviorally responsive to exogenous NPY (e.g., they increase food intake), it is
unknown if an increase in NPY expression in the arcuate nucleus due to
nutritional deprivation is present at this young age. Information
regarding the ontogeny of presynaptic responses to deprivation in the
arcuo-PVN NPYergic pathway is required to achieve a better
understanding of the role of NPY in normal feeding behavior and in the
development of abnormal ingestive behaviors, such as hyperphagia and
anorexia. To address this issue, we investigated the effect of food,
water, and maternal deprivation on arcuate nucleus NPY expression in
lean Zucker rat pups on P2-P15.
The relative abundance of preproNPY mRNA in fed and deprived animals was quantified using in situ hybridization to provide anatomic resolution. The results demonstrate that a 24-h period of deprivation results in significantly higher preproNPY mRNA in the arcuate nucleus
as early as P2, with a transient loss
of responsiveness at P5.
| |
METHODS |
Animals.
Lean Zucker rats aged 2, 5, 9, 12, and 15 days were used in the
experiments. All animals were the progeny of Zucker heterozygotes (+/fa) derived from the Vassar
College colony (Poughkeepsie, NY). Mating pairs and pregnant females
were housed in Plexiglas containers with wood shavings as bedding.
Animals received pelleted chow and water ad libitum and were maintained
on a 12:12-h light-dark cycle (0700-1900) at 22 ± 2°C.
Pregnant females were checked daily for pups, and the day pups were
first seen was termed P0. On
P4, animals assigned for study on
P9,
P12, or
P15 were ear clipped for
identification and tissue collection (for genotyping, see Determination
of
genotype
at
Lepr). Pups studied at
P2 or
P5 were marked for identification
using a dorsal subcutaneous injection of ink before the deprivation
period. A total of 128 pups (+/+, n = 39;
+/fa,
n = 89) from 18 litters were used,
with 3-5 litters used at each age. The pups used in this study
were derived from litters of 7-12 animals, with 14 of the 18 litters having 9-12 pups. Not all littermates were used in this
study (only +/+ and +/fa animals were used).
Food deprivation.
Twenty-four hours before tissue preparation (see
Tissue
preparation), the dam was removed
from the home cage. Pups were weighed and randomly assigned to one of
two groups; one-half of the pups were left in the home cage, and the
dam was returned, and the other pups were housed together in a
humidified incubator at a temperature that maintained thermoneutrality
(34 ± 1°C for pups aged 2 or 5 days and 33 ± 1°C for
pups aged 9, 12, or 15 days) under constant dim light. Those housed
away from the dam in the incubator for the 24-h period were deprived of
both food and water but not sibling interaction.
Tissue preparation.
Twenty-four hours after the start of the deprivation period
(0700-0900), animals were weighed and given an overdose of
pentobarbital sodium intraperitoneally. When unresponsive, animals were
transcardially perfused with 0.9% NaCl, 0.5%
NaNO2, and 100 U/ml heparin
followed by ice-cold 4% buffered paraformaldehyde (4%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). Brains were
removed, postfixed for 24 h in 4% buffered paraformaldehyde, and
cryoprotected in 30% sucrose at 4°C for 48 h before sectioning.
In situ hybridization.
Forty-micrometer frontal sections were cut using a sliding microtome
and placed into ice-cold 2× saline sodium citrate (SSC, 0.15 M
NaCl/0.015 M sodium citrate). Six to ten sections through the midregion
of the arcuate nucleus were taken. Hybridizations were carried out in
glass vials, and sections from a starved and fed animal were hybridized
in the same vial. The tissue was prehybridized in 1 ml of 60%
formamide, 0.02 M Tris pH 7.4, 1 mM EDTA, 10% dextran sulfate, 0.8%
Ficoll, 0.8% polyvinyl-pyrrolidone, 0.8% BSA, 2× SSC, 0.1 M dithiothreitol, and 1.6 mg/ml herring sperm DNA for 2 h at
48°C. After 2 h, radiolabeled probe was added (~1.0 × 107
counts · min
1 · vial1)
and incubated for 16-20 h at 48°C. Hybridization was performed using a 511-bp
[
-35S]dATP random
prime-labeled cDNA-encoding preproNPY (10). Sections were then
sequentially rinsed in 2×, 1×, 0.5×, 0.25×, and
0.125× SSC for 15 min at 48°C and placed into 0.1 M phosphate
buffer for mounting. The tissue sections were mounted onto
gelatin-coated slides, air dried, and apposed to X-ray film (
-max,
Amersham, Arlington Heights, IL) for 12-24 h. Each litter was
processed together, and slides were exposed to the same film. Slides
were then dipped into photoemulsion (Kodak, Rochester, NY) and exposed at 4°C for 1 wk for histological verification of probe labeling.
Probe hybridization in the arcuate nucleus was quantified by
densitometry of autoradiograms using the MCID system
(Imaging Research). The relative optical density (ROD) of hybridization signal in the arcuate nucleus, the area of hybridization in the arcuate
nucleus (A), and the product of the ROD and A (ROD × A, a measure
of total hybridization in the arcuate nucleus) were determined from
6-10 sections/animal. The mean ROD, A, and ROD × A values
from each animal were used in the calculations.
Determination of genotype at Lepr.
Genotypes (+/+,
+/fa) were determined according to a
previously described method (6). Briefly, the A to C mutation at nt 880 of Leprfa
introduces an Msp 1 restriction site,
which can be used to detect the number of copies of the
Leprfa allele.
Tissue was digested with proteinase K, and genomic DNA was extracted
using the Quiagen QIAamp Tissue Kit. Primers
(5'-TGAAGCCCGATCCACCGCTGG-3' and
5'-CTCTCTTACGATTGTAGAATTCTC-3') were used to generate a
143-bp PCR fragment. PCR was performed on a Perkin-Elmer DNA thermal cycler using the following conditions: 92°C (2 min), 1 cycle; 92°C (30 s), 55°C (30 s), 72°C (1 min), 35 cycles; and
72°C (5 min), 1 cycle. The Advantage genomic PCR kit (Clontech) was
used for PCR reactions. The PCR fragments were digested with
Msp 1 (80 mU/µl final concentration)
for 1.5 h and electrophoresed on a 2% agarose-2% low-melting-point
agarose gel. Digestion yields an uncut 143-bp product for the wild-type
allele, whereas the mutant allele
(fa) yields both a 106-bp and a
37-bp product.
Statistical analysis.
Results are presented as means ± SE. Initially, the ROD, A, and ROD × A measures within each litter were normalized to the mean value
of fed +/fa littermates because
+/fa pups were the most numerous in
our sample. No discernible differences were seen between
+/+ and
+/fa animals (Fig.
1). The ROD, A, and ROD × A values
were therefore normalized to the mean values of fed littermates, and
the normalized values of all litters tested at each age were pooled for
analysis. Differences in body weights and preproNPY mRNA abundance
between deprived and fed groups at each age were determined using a
two-way ANOVA, with the state (fed vs. deprived) and litter as
independent variables. Student's
t-test was used to reevaluate data at
ages at which no litter effects were observed. Differences in response
to deprivation across ages were determined using ANOVA with age as the
independent variable and the normalized ROD of starved animals as the
dependent variable. A P value <0.05 was considered significant.
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Fig. 1.
Normalized relative optical density (ROD) values for individual
+/+ ( ) and
+/fa ( ) animals in fed (F) and
deprived (D) state at each postnatal (P) age tested. ROD values of each
animal are normalized to mean ROD of fed
+/fa littermates.
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| |
RESULTS |
A significantly higher ROD was seen in deprived pups at
P2,
P9,
P12, and
P15 but not at
P5 (Fig.
2). Litter effects on ROD were observed
only at P15. No difference in A was
observed with deprivation at P5,
P9,
P12, or
P15, but a significantly larger A was
seen at P2 (+16.7 ± 4.6%). The
pattern of the ROD × A values with deprivation in pups aged
2-12 days was similar to that seen with the ROD values, namely,
higher ROD × A values at P2 (+41 ± 7%), P9 (+27 ± 10%), and
P12 (+50 ± 19%) and no effect of
deprivation at P5 (+12 ± 10%).
Unlike the ROD value, however, the ROD × A value was not
significantly higher with deprivation at
P15 (+19 ± 11%). This observation
was likely due to the modest but significant increase in ROD with
deprivation (+18 ± 4.7%) and large variance in A (coefficient of
variation = 20% in deprived animals at this age). Litter effects on A
and ROD × A were not seen at any age.
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Fig. 2.
Normalized ROD of fed and deprived animals at 5 ages tested (mean ± SE). * Significantly different from fed group
(P < 0.05).
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Analysis of the emulsion-coated cresyl violet-stained sections showed
that the preproNPY mRNA in the arcuate nucleus is localized to small
cells within the medial region (Fig. 3), as
previously demonstrated (4). The deprivation-induced increase in
preproNPY mRNA was specific to the arcuate nucleus at
P12. This was evidenced by no
differences in preproNPY mRNA in the cortex or the thalamic reticular
nucleus of fed and deprived animals (cortex, 1.00 ± 0.03 vs. 0.95 ± 0.02; reticular nucleus, 1.00 ± 0.04 vs. 0.99 ± 0.05;
normalized ROD in fed vs. deprived animals, respectively).
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Fig. 3.
Dark-field photomicrographs of prepro-neuropeptide Y (preproNPY) mRNA
hybridized in arcuate nucleus of a fed
(A) and a deprived
(B) pup aged
P12 (×50 magnification).
Bright-field photomicrographs of preproNPY mRNA hybridized in arcuate
nucleus of a fed (C) and a deprived
(D) pup aged
P12 (×200 magnification).
PreproNPY mRNA within arcuate nucleus is localized to small cells in
medial portion (arrows in C). III,
third ventricle. Bar = 100 µm in A
and B and 25 µm in
C and
D.
|
|
A significant effect of age (F = 2.71, P < 0.05) on the response to
deprivation (expressed as normalized ROD of deprived animals) was seen.
Post hoc analysis revealed that the ROD on
P5 was significantly smaller than the
ROD on P2 and
P12, but not on
P9 or
P15, and that the ROD on
P12 was significantly larger than
P9. A significant effect of age on the
relative change in body weight was observed in fed
(F = 2.6, P < 0.05) and starved
(F = 5.9, P < 0.001) animals (Table
1). Animals on
P9 gained significantly more weight
than those on P2,
P5, and
P15. Weight loss in animals aged 2 days was significantly larger than weight loss at other ages; weight
loss in animals aged 9 days was significantly smaller than those aged 15 days.
These data demonstrate that the failure to respond to deprivation on
P5 was not due to inadequate weight
loss of the starved animals or weight gain of those left with the dam.
Furthermore, the pattern of weight loss and gain across the other ages
does not appear to account for the differences in the magnitude of the
preproNPY expression with deprivation.
| |
DISCUSSION |
This study demonstrates that a 24-h period of food, water, and maternal
deprivation increases the relative abundance of preproNPY mRNA in the
arcuate nucleus of lean Zucker rat pups as early as P2. The relative increase in preproNPY
mRNA abundance (expressed as normalized ROD) seen with deprivation at
the ages tested was between 15 and 30%. This range is smaller than
that seen in adult rats, in which 24- to 96-h food deprivation
increases arcuate nucleus preproNPY mRNA 50-100% (4, 11, 21, 29).
The reason for the difference in the magnitude of response between
neonates and adults is not known, but it could be due to the
differences in the nature of the deprivation paradigm used as well as
the immaturity of brain structures or humoral signals involved in mediating this response.
The mechanism by which food deprivation increases arcuate nucleus
preproNPY expression in adult rats is not clear and may involve one or
several hormones and neurotransmitters such as insulin,
glucocorticoids, serotonin, dopamine,
corticotropin-releasing hormone, and leptin (3, 7, 14, 22,
23, 27). Adult arcuate nucleus cells have receptors for insulin (15),
glucocorticoids (9), and leptin (17), and studies in vivo have
demonstrated that administration of insulin or leptin decreases and
glucocorticoids increase preproNPY expression and release (22, 23, 31). Similar evidence exists for serotonin and dopamine, with serotonergic agents decreasing and dopaminergic antagonists increasing NPY expression or peptide levels (7, 14). The ontogeny of these putative
arcuate nucleus NPY regulatory systems and their involvement in
deprivation-induced changes in hypothalamic NPY, however, have not been
investigated.
The higher arcuate nucleus preproNPY mRNA level after deprivation was
not observed at P5. This was not due
to less change in body weight gain or loss in these animals relative to
other ages (Table 1). This is interesting because food- and
water-deprived animals will independently ingest more milk than fed
animals of the same age at P6 (8) and
P5 (T. Kowalski, unpublished
observations). It is not clear if the absence of a significant response
to deprivation was due to the inability to detect a small increase or
if it represents nonresponsiveness of NPY-expressing arcuate nucleus
neurons at this age. One possible explanation for the lack of a
significant increase in arcuate nucleus NPY mRNA at
P5 with deprivation may be the
transient decline in circulating corticosterone after birth and low
abundance of brain type II glucocorticoid receptor at this age (16).
Several studies in adult rats indicate that glucocorticoids are
required for increased arcuate nucleus NPY expression (13, 28) and that
arcuate nucleus NPY-expressing neurons have been shown to contain type
II glucocorticoid-like immunoreactivity (9).
Our results indicate that at ages when NPY injection into the PVN
increases intraoral milk and water intake
(P2) or selectively increases milk
intake (P15) (5), deprivation
increases the expression of NPY (Fig. 2). These data, as well as
studies demonstrating that pups ingesting food away from the dam
increase their intake with deprivation (8), indicate that the NPY
system may be operating from birth. It is not known, however, when the
arcuate nucleus NPYergic projections to the PVN are established,
although NPY-like immunoreactivity is seen in both the arcuate nucleus
and PVN as early as embryonic day
18 (32).
Because our animals were deprived of food and water, osmotic effects of
water deprivation on NPY expression may have modified the response.
Studies in adult rats have demonstrated that water deprivation also
increases arcuate nucleus NPY mRNA, but to a lesser extent than food
deprivation, whereas both food and water deprivation yields results
similar to food deprivation alone (18). The effect of food deprivation
on NPY expression in hydrated, normovolemic pups is required to
determine if dehydration changes the response to food deprivation
alone.
It is possible that maternal deprivation also contributed to our
results. Several studies have revealed that the absence of maternal
interaction modifies the development of the
hypothalamic-pituitary-adrenal axis (19). Evaluation of the role of
glucocorticoids in the regulation of hypothalamic NPY expression at
these ages is needed to address this issue. The influence of
maintaining the deprived animals under constant light is unknown.
Animals at P2 deprived under the same
light cycle as the colony room, however, showed a similar response
[1.00 ± 0.01 vs. 1.20 ± 0.02, normalized ROD of fed
(n = 3) and deprived
(n = 3) pups, respectively]. The
effect of lighting conditions at the other ages tested requires further investigation.
In summary, deprivation of food, water, and maternal interaction
significantly increases the relative amount of NPY mRNA within the
arcuate nucleus of lean Zucker rats aged 2, 9, 12, and 15 days but not
5 days. Although effects of heterozygosity in Zucker rat pups have been
shown for fat mass and fat-free dry mass (24, 26), preproNPY expression
appeared to be no different between the
+/+ and
+/fa genotypes in both the fed and
deprived states. The mechanisms responsible for the deprivation-induced
increase in arcuate nucleus NPY mRNA, as well as the changes in
hypothalamic NPY synthesis, release, and responsiveness in preweanling
animals remain to be determined.
Perspectives
These data demonstrate that arcuate nucleus NPYergic neurons are
responsive to nutritional and maternal deprivation as early as
P2. It is interesting that
hypothalamic NPY expression is responsive to deprivation at ages when
rat pups demonstrate a behavioral response to both deprivation
[ingesting more milk when tested in independent ingestion
paradigms after deprivation (8)] or central administration of NPY
(5). Unfortunately, the causal relationships among these observations
have not been demonstrated. This will require developmental studies
that correlate NPY expression, peptide level, and release with intake
in independent ingestive tests as a function of maternal or nutritional
deprivation. When these results are available, it will be possible to
compare the controls of NPY that operate in preweanling rats with those
that have been described in adult rats.
| |
ACKNOWLEDGEMENTS |
This work was funded by National Institute of Mental Health Grants
MH-00149, T32 MH-18390, and MH-15455 and National Institutes of Health
Grant DC-03198.
| |
FOOTNOTES |
Address for reprint requests: T. J. Kowalski, E. W. Bourne Laboratory,
New York Hospital-Cornell Medical Center, 21 Bloomingdale Road, White
Plains, NY 10605.
Received 3 September 1997; accepted in final form 24 April 1998.
| |
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Am J Physiol Regul Integr Compar Physiol 275(2):R466-R470
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Abstract
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