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Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, Oregon 97006; and Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201-3098
Address all correspondence and requests for reprints to: Dr. M. Susan Smith, Division of Neuroscience, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: smithsu{at}ohsu.edu
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Abstract |
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Introduction |
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Recently, it has been shown by several laboratories that the levels of neuropeptide Y (NPY) are significantly elevated in a number of hypothalamic areas in chronic lactating animals (4, 5, 6); this is further confirmed by the increase in NPY messenger RNA (mRNA) levels in the hypothalamus (7, 8). Most of the NPY-containing neurons in the hypothalamus are found in the hypothalamic arcuate nucleus (ARH) (9). NPY in the hypothalamus has been shown to participate in regulating several physiological functions, including induction of the feeding response (for review, see Refs. 10 and 11), hypothalamic neuroendocrine regulation (12, 13, 14), and pituitary hormone secretion (15, 16, 17, 18, 19). The diverse functions of NPY make the peptide a candidate for modulating many of the alterations in hypothalamic function during lactation.
Several key issues have yet to be addressed in considering the involvement of NPY in modulating hypothalamic neuronal activity during lactation. First, it has not been determined if the increase in NPY neuronal activity can be induced by the suckling stimulus. Second, if the activation is indeed associated with the suckling stimulus, then the time course of the activation needs to be determined so as to assess whether it correlates with the time course of suckling-induced changes in other events associated with lactation, such as suppression of LH secretion and increase in food intake.
The aims of the present study were to determine 1) if NPY neurons in the hypothalamus can be activated by the suckling stimulus; and 2) the time course of the activation in response to the suckling stimulus.
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Materials and Methods |
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Experimental design
An acute resuckling paradigm was designed so that changes in
hypothalamic neuronal activity could be correlated with changes in
hormone secretion. The paradigm was validated in ovariectomized
lactating rats suckling eight pups so as to more easily assess changes
in LH secretion. As shown in Table 1
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removal of the eight-pup suckling stimulus resulted in a significant
reduction in PRL levels to values approaching the basal levels observed
in nonlactating animals (data not shown). The significant rise in LH
following removal of the suckling stimulus reflects the ovariectomized
state of the animals. Return of the eight-pup litters and resuckling
for 24 h reversed these changes, resulting in increased PRL
secretion and suppression of LH secretion (Table 1).
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From the first experiment, it was found that although 12 h of resuckling did not result in a significant change in NPY mRNA levels in the ARH, it did induce a strong NPY signal in the dorsomedial nucleus of the hypothalamus area (DMH). To more finely determine the time course of activation of NPY-expressing cells in the DMH area, a second experiment was conducted using the same paradigm described above. Three groups of animals were used: 0 pups (nonresuckled controls, n = 3) and eight-pup litters resuckled for 3 (n = 3) or 6 (n = 3) h. The brains were processed for in situ hybridization to identify NPY-positive neurons in the DMH area.
In situ hybridization
Coronal brain sections (20 µm) were collected through the ARH,
and the slides were stored at -80 C until used for in situ
hybridization. NPY complementary RNA (cRNA) probe synthesis and the
specificity of the cRNA probe and procedure for in situ
hybridization have been described previously (7). Briefly, the NPY cRNA
probe was transcribed from a 511-bp complementary DNA in which 21% of
the UTP was 35S-labeled (Dupont-New England Nuclear, Boston
MA). The saturating concentration of the probe used in the assay was
0.3 µg/ml·kb. The specific activity of the probe ranged from
1–3 x 108 dpm/µg. The brain sections were fixed in
4% paraformaldehyde and treated with a fresh solution containing
0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0),
followed by a rinse in 2 x SSC, dehydrated through a graded
series of alcohols, delipidated in chloroform, rehydrated through a
second series of alcohols, and then air dried. The slides were exposed
to the NPY cRNA probe overnight in moist chambers at 55 C. After
incubation, the slides were washed in SSC that increased in stringency,
in RNase, and then in 0.1 x SSC at 60 C and rehydrated through
graded series of alcohols and dried. Slides were dipped in NTB-2
emulsion (Eastman Kodak, Rochester, NY), exposed for 6–7 days at 4 C
and developed. After development, the slides were stained with cresyl
violet.
Data analysis
NPY mRNA in the ARH. The ARH was divided into four
subdivisions as described in a previous study (7), using the rat brain
atlas of Paxinos and Watson (21). Briefly,
1) ARH-A corresponded to the retrochiasmatic area rostrally, to the elongation of the third ventricle caudally (plate 19).
2) ARH-B continued caudally to the beginning of DMH (plate 20).
3) ARH-C contained the compact zone of the DMH (plate 21).
4) ARH-D began with the disappearance of the DMH, to the end of the ARH (plate 22).
The coronal brain sections were anatomically matched across animals from all groups. NPY mRNA was quantitated using the VIDEK HARMONY image analysis system by VIDEK (Rochester, NY). The system identified silver grains by the brightness of the image. An estimate for silver grains over the entire ARH on each tissue section was given as the area occupied by silver grains within the marked area (because of the close proximity of most NPY cells in the ARH, it was not possible to analyze individual cells). The marked area was constant (1.0 mm x 1.6 mm) for each reading and included the entire ARH. The area occupied by silver grains was typically between 5 and 15% of the marked area.
NPY mRNA in the DMH. NPY mRNA was visualized using dark field optics. The induction of NPY mRNA, represented by clusters of silver grains, was assessed qualitatively.
Statistical analysis
The data for NPY mRNA in the ARH were expressed as the area
occupied by grains per section. The mean area occupied by grains per
section for each subdivision of the ARH was determined for each animal.
Data are presented as mean ± SEM. Differences between
groups within a subdivision were evaluated using one-way ANOVA and post
hoc Scheffé’s tests. Differences were considered significant if
P < 0.05.
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Results |
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). In
contrast, the ARH-C area showed a significant increase in NPY mRNA
levels when the animals were resuckled for 24 h compared with the
nonresuckled control (Fig. 1). As shown in Fig. 2, the increase in NPY mRNA in response
to suckling was due in part to the recruitment of new cells expressing
NPY mRNA; these cells were most evident in the dorsolateral and
ventrolateral portions of the ARH-C area. NPY mRNA levels also appeared
to be increased in previously expressing cells in the ventromedial
portion of the ARH-C. Because of the high density of silver grains over
many of the cells in the ventromedial portion, the magnitude of the
suckling-induced increase may even be an underestimate. The 12-h
resuckled group showed a moderate increase in mRNA content, which
failed to reach significance when compared with the control group (Fig. 1).
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). Under the nonsuckled condition (0
pups, Fig. 3), a low level of NPY mRNA signal was observed in the
compact zone of the DMH. When viewed under higher magnification, the
silver grains appeared to be associated with cell bodies in the compact
zone. After 3 h of resuckling (8 pups, 3 h, Fig. 3), clusters
of NPY mRNA were observed in the DMH, although there was variation in
the intensity of the signal level found among the animals. After 6
h of resuckling, the NPY mRNA signals were consistently observed in all
the animals examined (8 pups, 6 h, Fig. 3). By 24 h of
resuckling, the level of activation was further increased (8 pups,
24 h, Fig. 3) and was similar to that observed in chronic
lactating animals (9). As shown in Fig. 3, the low levels of
hybridization signal detected over the compact zone of the DMH were
observed in all the groups; there was no consistent change in silver
grains over the compact zone in response to the suckling stimulus. The
distribution of NPY expressing cells throughout the DMH in animals
resuckled for 24 h is shown in Fig. 4. This group of NPY neurons was
scattered around the compact zone of the DMH and extended laterally to
the perifornical area of the hypothalamus. Rostrally, few cells were
seen when the compact zone emerged from the medial base of the DMH. The
density of the cells increased quickly and peaked when the compact zone
was fully extended. Caudally, the density decreases rapidly and the
cell group disappeared soon after the compact zone disappeared. The
rostral-caudal expression of NPY neurons in the DMH occupied the same
plane as ARH-C, in which the increase in NPY mRNA was observed.
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Discussion |
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Available anatomical evidence suggests that NPY neurons in the ARH send projections to many hypothalamic areas, including the paraventricular nucleus of the hypothalamus (PVH) and the medial preoptic area (22, 23). The ARH-PVH NPY projection has been shown to play an important role in mediating energy metabolism by regulating food intake. During lactation, there is significant energy demand due to milk production; this demand is met primarily by increased food intake (5, 6, 24). The central role of NPY in inducing the feeding response makes it a possible candidate in mediating the hyperphagia during lactation. In fact, elevated NPY concentrations in PVH, DMH, and ARH and NPY mRNA levels in the ARH and DMH have been reported in the chronic lactating rat (5, 6, 7, 8). The present study further demonstrates that NPY neurons are activated by the acute suckling stimulus. Taken together, the sustained activation of NPY neurons by the suckling stimulus throughout the course of lactation is likely to play an important role in mediating the increase in food intake to meet the energy demands resulting from milk production.
In contrast to NPY neurons in the ARH, there is little known about the suckling-induced NPY neurons in the DMH. Preliminary data from tract tracing studies in our laboratory have shown that NPY neurons in the DMH project to PVH (unpublished observations), suggesting that DMH NPY neurons may also participate in modulation of PVH neuronal activity. It is yet to be determined if DMH NPY neurons interact with ARH NPY neurons in the PVH. In addition, the neuronal phenotypes of the PVH neurons controlled by the DMH NPY neurons remain unknown.
The functional significance of suckling-induced activation of NPY neurons in the DMH also remains to be elucidated. In the female rat, this population of NPY neurons does not appear to be activated under basal conditions, nor in response to stimuli that activate NPY neurons in the ARH, such as food deprivation (25) and streptozotocin-induced diabetes (26). In contrast to the female, the data from male rats are less clear. There are some reports of NPY mRNA expression in the DMH of the male under basal conditions, although it is not possible to determine the location of the cells and the level of expression from the data that has been described (27, 28). On the other hand, a study published by Kershaw et al. (29) showed no evidence of NPY mRNA expression in the area of the DMH in the male under basal conditions, except for the low levels of expression found in the compact zone. Preliminary data from our laboratory also indicate that there is no NPY mRNA expression in the DMH in the male (unpublished observation). Furthermore, there are no studies in the male reporting increased expression of NPY in the DMH in response to any stimulus. Thus, in the rat, suckling is the only stimulus that has been shown to activate NPY neurons in the DMH.
Recently, NPY mRNA levels in the hypothalamus were examined in obese, melanocortin receptor type 4 (MC4-R) knock-out mice and agouti mice (30). Both types of animals lack functional MC4 receptors, either due to the knock out of receptor gene expression or to the functional blockade by endogenous agouti protein (agouti mice). It was surprising that ARH NPY mRNA levels were not altered in these animals compared with normal animals, but there was a significant activation of NPY in the DMH area in both MC4-R knock-out and agouti mice. Interestingly, the pattern and distribution of the NPY-expressing neurons in the DMH area in the two mouse models are very similar to those found in the lactating animals. The MC4 receptor, a member of the receptor family for melanocortin peptides including ACTH, MSH, and opioid peptides (31) has been shown to play an important role in the induction of obesity in these two animal models (30, 32). The data from the MC4-R studies raise the possibility that the function of the MC4-R may be compromised in the lactating rat. The reduction of MC4-R activity may be a factor in the expression of NPY in neurons in the DMH area, which, in turn, may participate in the regulation of increase in food intake during lactation. In fact, the hypothalamic POMC neurons, one of the sources of ligands for activating the MC4-R, showed decreased activity during lactation (7). This data indirectly supports the idea that the function of the MC4-R is compromised during lactation. Taken together, it is possible that the expression of NPY neurons in the DMH area found in these two obese mouse models, as well as in the lactating rat, may play an unique and important role in mediating energy metabolism. Lactation may provide an interesting model to study the role of the DMH NPY neurons, as well as the possible interaction between the ARH NPY neurons and the DMH NPY neurons in regulating energy metabolism and food intake.
In addition to a possible role in regulating food intake during
lactation, increased NPY activity may also modulate reproductive
function through changes in GnRH and LH secretion. NPY in the
hypothalamus has been shown to play an important role in modulation of
GnRH neuronal activity and LH secretion (see reviews in Refs. 17 and
33). It is well documented in the rat that NPY can stimulate GnRH and
LH secretion during proestrus and in response to steroid priming
(34, 35, 36). However, chronic elevations of NPY in the brain have been
shown to suppress LH secretion (18, 19). During lactation in the rat,
pulsatile LH secretion as well as ovarian cyclicity are suppressed (37, 38). The suppression of LH could be accounted for by a diminished
release of GnRH from the hypothalamus (38), although this relationship
has not been established. Interestingly, the time course described in
the present study for the increase in NPY activity is inversely
correlated with the time course of the suppression of LH secretion in
response to acute suckling (Table 1
). The relationship between the
increase in NPY neuronal activity and the suppression of LH secretion
by the suckling stimulus raises the possibility that specific
populations of NPY neurons in the hypothalamus may be linked to the
regulation of LH secretion during lactation. However, further
investigation is needed to understand whether this alteration of NPY
neuronal activity by the suckling stimulus is causally involved in the
modulation of LH secretion.
Important questions remain as to the nature of the signals induced by suckling that are responsible for activating hypothalamic NPY neurons. The neural impulses from suckling appear to ascend through the brain stem to the thalamus and enter the hypothalamus by ventral and dorsal routes (39, 40), resulting in the secretion of oxytocin and PRL. Thus, it is possible that neural signals or the elevated level of hormones induced by the suckling stimulus, or both, could be responsible for the activation of specific populations of NPY neurons.
In summary, these studies show that the acute suckling stimulus can activate two populations of hypothalamic NPY neurons, one in the caudal portion of the ARH and one in the DMH. The increase in NPY neuronal activity may play an important role in modulating changes in hypothalamic function during lactation.
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Acknowledgments |
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Footnotes |
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Received October 22, 1997.
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