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Department of Physiology, National Institute of Animal Industry, Ministry of Agriculture, Forestry and Fisheries, Inashiki, Ibaraki 305, Japan; and Laboratory of Veterinary Ethology (T.I., Y.M.), Department of Veterinary Medical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
Address all correspondence and requests for reprints to: Hiroaki Okamura, Ph.D., D.V.M., Department of Physiology, National Institute of Animal Industry, Ministry of Agriculture, Forestry and Fisheries, 2 Ikeno-dai, Kukisaki, Inashiki, Ibaraki 305, Japan. E-mail: hokamu{at}niai.affrc.go.jp
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Abstract |
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When estradiol (E2)-treated animals were fasted for 4–5 days, the activity of the GnRH pulse generator was gradually suppressed, and the volley interval at the end of fasting was significantly prolonged, compared with that during the feeding period (67.4 vs. 49.3 min, n = 5, P < 0.01). On the other hand, such a significant effect on the pulse generator was not observed in OVX goats. In the second experiment, the animals received a bolus intracerebroventricular injection of several doses (0, 2, 5, and 20 µg/400 µl) of neuropeptide Y (NPY). Exogenous NPY dose-dependently inhibited the pulse generator activity. At the highest dosage, the 1st posttreatment volley interval was significantly longer than that of the pretreatment (112.4 vs. 32.6 min, n = 5, P < 0.01) in OVX goats. The suppressive effect of NPY was similarly observed in OVX+E2 goats. Further, when NPY was infused (10 µg/200 µl·h for 6 h) into OVX goats, the activity of the GnRH pulse generator was almost completely inhibited during the infusion period.
Hypothalamic sites responding to fasting were immunohistochemically evaluated using an antibody for Fos in castrated goats. Fos-immunoreactive neurons were found in areas adjacent to the third ventricle. Double-labeling immunohistochemistry revealed that a subpopulation of NPY neurons in the arcuate nucleus was activated in response to fasting.
These results demonstrate that: 1) the activity of the GnRH pulse generator is suppressed by fasting in the presence of E2; 2) exogenous NPY inhibits the activity of the GnRH pulse generator regardless of the presence of E2; and 3) several hypothalamic neurons or regions, including those containing NPY in the arcuate nucleus, are activated by fasting. Collectively, these observations suggest that NPY acts as a mediator of undernutrition to the GnRH pulse generator.
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Introduction |
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Neuropeptide Y (NPY), known as a potent orexigenic molecule in the brain (for review, see 13), also participates in the control of LH secretion. It has been shown that intracerebroventricular administration of NPY inhibits pulsatile LH secretion in several ovariectomized (OVX) animal models, such as rat (14), monkey (15), and sheep (16, 17). Morphological studies have demonstrated that food restriction markedly increases hypothalamic NPY gene expression in rats (18) and sheep (9), as well as concentrations of NPY in the cerebrospinal fluid (17) and NPY-immunoreactivity (19) in sheep. Moreover, NPY fibers make close contact with GnRH perikarya in the preoptic area (POA) and with GnRH terminals in the median eminence (ME) (20). These lines of evidence strongly suggest that NPY is one of the pivotal molecules in the brain that transmit undernutritional signals to the regulatory system of GnRH secretion.
The hypothalamic GnRH pulse generator, although its neural component has yet to be identified, governs intermittent GnRH discharge into the pituitary portal circulation and thereby regulates pulsatile LH release (21). A method for monitoring the electrophysiological activities of the pulse generator has been developed in monkeys (22), rats (23), and goats (24, 25, 26, 27) by recording characteristic increase in the frequency of multiple-unit activity (MUA). This method has practical advantages, in that continuous and real-time analysis of pulse generator activity can be done in conscious, unrestricted animals for a long period, which tempted us to investigate central mechanisms for the well-known phenomenon of nutritional control of reproduction. In the present study, OVX goats were fasted or received central administration of NPY in the presence or absence of estradiol (E2), and changes in the activity of the GnRH pulse generator were directly analyzed by the MUA recording. In addition, immunohistochemical observations were conducted using the expression of Fos protein as a marker for neuronal activation (28) to identify hypothalamic substrates involved in the response to undernutrition.
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Materials and Methods |
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Surgery
Six OVX goats were stereotaxically implanted with an array of
bilateral recording electrodes into the arcuate nucleus (ARC)/ME region
under halothane anesthesia, according to the procedure described
previously (24). The electrode consisted of six
Teflon-insulated platinum-iridium wires (75 µm in diameter). For
intracerebroventricular administration of NPY, an 18-gauge stainless
steel guide cannula was also inserted so that the tip was directed to
Monro’s foramen and positioned at 5 mm above the lateral ventricle
(LV). After confirming the position by radio-ventriculographs, the
guide cannula was fixed to the skull and fitted with a 21-gauge stylet
to prevent its occlusion.
MUA recording
The method for recording specific MUA, which reflects the
function of the hypothalamic GnRH pulse generator, has been described
elsewhere (24, 25). Briefly, signals were passed through a
buffer amplifier integrated circuit, which was directly plugged into
the electrode assembly. After further amplification and amplitude
discrimination, MUA signals were stored, as counts per minute, in a
personal computer. A characteristic increase in the MUA (MUA volley)
was considered to be the electrophysiological manifestation of the GnRH
pulse generator. The MUA recording was conducted continuously
throughout the experimental period.
Hormonal treatment
In some experiments, the OVX goats were sc implanted with a
SILASTIC capsule (id, 3 mm; od, 5 mm; length, 20 mm; Dow Corning Corp., Midland MI) filled with crystalline
E2 (Sigma, St. Louis, MO). This
treatment produced luteal phase plasma E2 levels
(4–8 pg/ml) (29). The MUA volley intervals were gradually
prolonged after the E2 treatment and became
stable within 5 days. After confirming that the volley intervals were
maintained at a constant level at least for 3 days, the experiment was
started.
Exp 1: Effect of fasting on the MUA
The effect of acute undernutrition on the activity of the GnRH
pulse generator was examined in the absence (OVX, n = 6) or
presence of E2 (OVX+E2,
n = 5). After a control feeding period, the animals were fasted
for 4–5 days. They were refed at 1200 h of the last day of
fasting, and thereafter treated with the regular feeding regimen. MUA
recording was done throughout the feeding, fasting, and refeeding
periods, and the volley interval values during 12 h (between
0000–1200 h and 1200–2400 h) were averaged for each day. The value at
the end of the fasting period (0000–1200 h) was statistically compared
with that of the feeding period. Plasma LH concentrations were not
determined in this experiment, to avoid the additional stress of
frequent blood sampling.
Exp 2: Effect of NPY administration on the MUA
Sheep NPY (Sigma) was used to make up final
concentrations of 2, 5, and 20 µg NPY in 400 µl of saline
containing 0.4% BSA. After a control period, 400 µl of the NPY
solution or vehicle was injected into the LV, 15 min after the
beginning of one regularly occurring MUA volley, as shown in Fig. 1
. The injection occurred over a period
of 60 sec, through an injection cannula, which was 5 mm longer than the
guide cannula. The injection cannula remained in place for another 60
sec. Doses of NPY and numbers of animals used in this experiment were
as follows; 0 µg (n = 4), 2 µg (n = 4), 5 µg (n =
4), and 20 µg (n = 5) in the absence of
E2; and 0 µg (n = 3), 2 µg (n = 3),
5 µg (n = 3), and 20 µg (n = 5) in the presence of
E2. Each injection was separated from the
previous injection by at least 2 days. Each value of three successive
posttreatment intervals was compared with that of the pretreatment one.
In some animals, blood samples were taken every 5–10 min, and the
synchrony of the MUA volleys with plasma LH pulses was confirmed.
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Exp 3: Immunohistochemical examination of hypothalamic sites
responding to fasting
Male goats, regularly fed (n = 2) or fasted
for 4 days (n = 4), were deeply anesthetized with sodium
pentobarbital (25 mg/kg BW). They were perfused bilaterally through the
carotid arteries with 4 liters PBS, pH 7.4, including 4000 U
heparin/liter, followed by 5 liters 4% paraformaldehyde in 0.1
M phosphate buffer, pH 7.4. The brain block between the
optic chiasma and the anterior edge of the mammillary body was
dissected out and was immersed in the same fixative overnight at 4 C
and then in 20% sucrose in 0.1 M phosphate buffer
at 4 C until it sank. Frontal sections (50 µm) were cut on a freezing
microtome, and they were kept in the cryoprotectant solution
(30) at -20 C.
The free-floating sections were rinsed in PBS containing 0.5% Triton X-100 (PBST) and treated with 3% H2O2 in methanol for 15 min. They were extensively rinsed with PBST and incubated with 10% normal goat serum in PBST including 1% BSA and 0.02% sodium azide (BSA-PBST) for 2 h. Then sections were subsequently incubated with anti-Fos serum (Ab-2, Oncogene Research Products, Cambridge, MA, 1:5000 in PBST) for 72 h at 4 C, biotinylated goat antirabbit IgG (1.8 µl/ml of PBST containing 1% normal goat serum; Vector Laboratories, Inc., Burlingame, CA) for 3 h, and avidin-biotin complex solution (4.5 µl each/ml PBST; Vector Laboratories, Inc., elite kit) for 1 h. Each step was followed by three 15-min washes with PBST. After the last wash, sections were immersed in 0.175 M sodium acetate, pH 5.6, followed by reaction with the chromogen solution consisting of nickel-sulfate (25 mg/ml), 3.3'-diaminobenzidine (Sigma, 0.2 mg/ml), and 0.0025% H2O2 in 0.175 M sodium acetate for 8 min. The reaction was stopped by immersing sections in sodium acetate solution. All reactions were done at room temperature unless otherwise mentioned. Some sections were washed with 0.45% NaCl, dehydrated, mounted on coated slides, and cover-slipped; while the remaining sections were further processed for NPY immunohistochemistry. The Fos-stained sections were extensively washed with PBST and incubated with anti-NPY serum (A609/R2R Ab, UCB-Bioproducts, 1:50,000 in BSA-PBST) for 72 h at 4 C. The following procedure was similar to that for Fos staining except that the sodium acetate solution was substituted with 50 mM Tris-HCl, pH 7.4, and nickel-sulfate was omitted from the chromogen solution. To compare NPY-immunoreactivity between the fed and fasted animals, some sections were stained solely for NPY without the procedure for Fos immunohistochemistry.
The boundaries of the hypothalamic nuclei were delineated on the basis of cresyl-violet-stained sections and the goat brain atlas (31). The sections from the fed and fasted goats that contained similar brain structure were always reacted in parallel under identical conditions. Immunohistochemical controls included substitution of the primary antiserum with preabsorbed antiserum with c-Fos peptide (Oncogen Research Products) or sheep NPY in respective staining procedure, or omission of the second biotinylated antirabbit antibody, all of which gave no reaction product (data not shown).
The extent of colocalization of Fos- and NPY-immunoreactive (ir) materials was estimated at the caudal portion of the ARC in the fasted goats. The numbers of Fos-, NPY-, and Fos/NPY-ir cells were counted under the microscope on two double-labeled sections in each animal. Because only Fos/NPY-ir neurons whose cell bodies were clearly distinguishable from surrounding densely innervated NPY-ir fibers were taken into consideration, the extent of colocalization might be underestimated in this study.
RIA
Plasma concentrations of LH were determined by the
double-antibody RIA as described previously (32). The
minimal detectable concentration was 0.38 ng/ml, and the intraassay
coefficient of variation was 6.8%.
Statistical analysis
All data were analyzed by the General Linear Model procedure of
Statistical Analysis System (SAS/STAT User’s Guide, Release
6.03 Edition, SAS Institute, Inc., Cary, NC). When
significant treatment effects (P < 0.05) were
revealed, the least-squared-means option program was used in post
hoc comparisons of the MUA measures. Differences were considered
to be significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows time course changes
in the MUA volley interval throughout the experimental period, in a
representative goat, with or without the E2
treatment. In the absence of E2, the volley
interval was maintained at a constant level during the feeding period,
and there was no apparent change for 4 days after the onset of fasting.
However, at the end of the fasting period, a subtle increase was
observed in all six animals examined. When the goats were treated with
E2, the volley interval gradually increased after
the E2 implantation but plateaued within a few
days. After the onset of fasting, the volley interval began to increase
again. The influence of fasting on the MUA volley interval became
obvious by the second or third day of treatment, and the prolongation
of the volley interval continued until food was given. Representative
profiles of the MUA during feeding and at the end of fasting periods,
in an OVX+E2 goat, are shown in Fig. 2B. After
refeeding, the volley interval gradually returned to the level that had
existed before fasting, by several days. Although the extent of the
effect of E2 or fasting varied among animals, a
similar pattern was observed in all five goats.
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). When compared at the end
of the feeding period, the MUA volley interval of the
OVX+E2 group was significantly longer than that
of the OVX group (49.3 ± 5.5 vs. 29.8 ± 6.0 min,
P < 0.02). At the end of the fasting period, the
volley interval significantly increased in the presence of
E2 (67.4 ± 7.5 min, P <
0.01). Although there was a tendency toward the increase in the volley
interval at the end of the fasting period, the influence of fasting was
not statistically significant in the absence of
E2 (33.1 ± 3.9 min).
Exp 2: Effect of NPY administration on the MUA
An individual profile of the MUA, in each treatment, and
summarized results are shown in Fig. 3A
and Table 1, respectively. In the absence
of E2, the MUA volley interval was not
significantly altered by 2 or 5 µg NPY, although there was a tendency
toward an increment in the 1st posttreatment volley interval. However,
20 µg NPY strongly suppressed the appearance of the volley
immediately after the injection, which resulted in a significant
increase in the 1st posttreatment interval, being about 3.5 times
larger than the pretreatment interval. In the
OVX+E2 goats, an NPY effect comparable with that
in the OVX animals was observed. In both groups, the suppressive effect
of NPY was only seen for the volley immediately after the injection,
and the 2nd and 3rd posttreatment intervals were not affected by NPY
administration. The vehicle injection had no effect on the MUA in all
goats. Figure 3B demonstrates that the MUA volleys were exclusively
associated with plasma LH pulses regardless of the steroid or NPY
treatment.
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). On the
other hand, an increase in the baseline activity after the NPY
injection was observed in two goats, one without
E2 (Fig. 3A) and the other with and without
E2 (data not shown).
Figure 4 shows representative profiles of
the MUA in an OVX goat that received the vehicle (upper
panel) or NPY (lower panel) infusion, for 6 h,
into the LV. Data obtained from three animals were summarized in Table 2. The control infusion had no effect on
the MUA. On the other hand, the continuous NPY infusion resulted in a
conspicuous suppression of GnRH pulse generator activity. After the
onset of NPY infusion, one or two MUA volleys with a slightly longer
interval were observed, and then the MUA volleys completely disappeared
throughout the infusion period. This strong suppression continued more
than 50 min after the cessation of the infusion. Once the MUA volley
was restored, it occurred more frequently, for several hours, compared
with the preinfusion period. The mean value of the interval between the
MUA volleys observed during the postinfusion period was significantly
smaller than that during the preinfusion period (23.4 ± 0.5
vs. 30.8 ± 1.1 min, P < 0.05).
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). A considerable number of neurons in
the ARC showed Fos-immunoreactivity being predominant at the caudal
portion of the nucleus. Fos-ir neurons were also found scattered in the
medial part of the dorsomedial nucleus of the hypothalamus (DMH) and
periventricular regions. However, there were few Fos-ir cells in the
paraventricular nucleus of the hypothalamus (PVH). Though the number of
Fos-ir cells and their staining density varied among animals, a similar
distribution pattern was observed in all fasted goats. On the other
hand, Fos-ir cells were scarcely seen in fed animals throughout the
hypothalamus. Photomicrographs showing Fos-immunoreactivity in the ARC
of fed and fasted animals are presented in Fig. 6, A and B, respectively.
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). However, not only the
density of NPY-ir fibers, but also the number of NPY-positive cell
bodies, in the ARC were markedly enhanced by fasting (Fig. 6D).
Double-labeling immunohistochemistry revealed that a subpopulation of
NPY-ir neurons in the ARC coexpressed Fos-ir materials (Fig. 6, E and
F). A large portion of those double-labeled cells existed in the region
close to the wall of the third ventricle. It was estimated that they
comprised about 40% of NPY-ir neurons and 2–3% of Fos-ir cells at
the caudal portion of the ARC. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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E2 treatment significantly prolonged the volley
interval (Fig. 2C), possibly reflecting the negative feedback action of
this steroid on the pulse generator, as has been shown in rats
(4), sheep (6), and goats (24).
Fasting induced a significant reduction in the MUA volley frequency in
OVX+E2 goats (Fig. 2C). This result is comparable
with a recent demonstration, in ewe lambs (12), that
chronic food restriction decreases both frequency and amplitude of GnRH
pulse in the pituitary portal circulation. We also observed a tendency
for amplitude of volleys to be smaller at the end of the fasting period
than that observed during the control period (Fig. 2B). Therefore, it
seems that both acute and chronic undernutrition modulate the activity
of the GnRH pulse generator in a similar manner in ruminants. However,
in contrast to previous reports showing that chronic food restriction
inhibits pulsatile GnRH/LH secretion in the absence of gonadal steroids
in sheep (6, 7, 8, 9, 12), we were not able to observe a
significant influence of fasting on the GnRH pulse generator activity
in OVX goats. This discrepancy may be attributable to different
physiological conditions between two undernutrition models. Further
discussion is presented in a latter section.
The interval of the MUA volleys was gradually and continuously
increased as fasting continued, and refeeding immediately began to
restore the prolonged volley interval (Fig. 2A). These results indicate
that the activity of the GnRH pulse generator is highly sensitive to
changes in nutritional state of the body, which are thought to be
informed by several metabolites and hormones, such as glucose (1, 33, 34), fatty acid (1), leptin (4),
insulin (35), and T3 (36). Because the effect
of fasting on MUA becomes apparent within a few days in
E2-treated goats and those changes in MUA are
visible in a real-time manner, this experimental model can be used to
seek such peripheral signals involved in the nutritional control of
GnRH secretion, even though fasting may not accurately reflect the
state of chronic undernutrition occurring in nature.
Because NPY is considered to be an important neurotransmitter that
links nutrition and reproduction (13, 17), the effect of
exogenous administration of NPY on GnRH secretion was investigated in
detail using the MUA technique in the OVX and
OVX+E2 goats. The 1st posttreatment interval of
the MUA volley was prolonged in a dose-dependent manner, after bolus
NPY injections, regardless of the presence or absence of
E2 (Table 1). The inhibitory effect of NPY on the
MUA volley was even more pronounced when the peptide was continuously
infused into the LV (Fig. 4). The present investigation confirms
previous demonstrations that intracerebroventricular administration of
NPY suppresses pulsatile LH secretion in OVX and
OVX+E2 animals (14, 15, 16, 17), and further
provides direct evidence that NPY acts at the hypothalamic level to
suppress the activity of the GnRH pulse generator.
The suppressive effect of NPY was seen only for the volley immediately
after the injection, and the 2nd and 3rd posttreatment intervals were
not affected by 20 µg of NPY; whereas a relatively lower dose (10
µg/h) of NPY infusion almost completely suppressed the occurrence of
the MUA volley (Fig. 4). These results indicate that continuous
activation of receptors for NPY profoundly inhibits a drive to induce
the MUA volley, and thus GnRH release. Five receptor subtypes have been
described for NPY action in rats thus far (37). Recent
studies, using specific pharmacological reagents for respective
receptor subtypes, indicate that the Y5 receptor is involved in the
suppression of LH release by NPY (37); whereas the Y4
receptor (38), together with the Y1 subtype
(39), are responsible for the stimulatory action of NPY on
LH release. Although such functional roles of NPY receptors have not
been well studied, a similar mechanism may exist in ruminants. In the
Shiba goat, the majority of GnRH neurons are located in the medial POA
and project their fibers to the ME (40), as in rats.
Because it has been demonstrated that NPY fibers and terminals make
close contact with GnRH neurons in the POA and their nerve terminals in
the ME in rats (20), NPY may affect GnRH release by acting
at either or both sites. Another possibility is that NPY acts on the
GnRH pulse generator per se to reduce its activities,
although neuronal components of the pulse generator have not yet been
identified. Precise localization of NPY receptors, especially the Y5
subtype, in the hypothalamus might help us to understand this
issue.
In some goats, a decline of the basal level of MUA was observed
immediately after the NPY injection (Fig. 3B), which implies that NPY
inhibits nonspecifically all neural activities in the hypothalamus,
including that of the GnRH pulse generator. However, this is unlikely,
because the NPY injection in other goats induced an elevation of the
basal level while inhibiting the occurrence of the MUA volley (Fig. 3A). These variable responses of the basal level might reflect subtle
differences in the location of the electrodes in the ARC/ME region
between individual animals. The ARC/ME region is a key neural substrate
for the control of endocrine functions, and several of these functions
have been shown to be modulated by central administration of NPY
(41). For example, GH secretion is suppressed
(42), whereas cortisol release is increased
(43), by NPY. Therefore, it seems likely that changes in
the basal level, after the NPY injection, were integrated results of
neural activities responsible for those endocrine functions. Some of
these activities seem to be related to E2 (Fig. 3A), but this was not confirmed in this study.
In this study, the activity of the GnRH pulse generator was significantly suppressed by fasting in the presence, but not in the absence, of E2; whereas both OVX and OVX+E2 animals were similarly inhibited by exogenous NPY. These results may raise a possibility that NPY is not the neural signal mediating the nutritional influence on the GnRH pulse generator. However, when the roles of E2 and the degree of undernutrition achieved by fasting for 4–5 days are taken into account, the steroid-dependent discrepancy between the effects of fasting and exogenous NPY is not surprising. It has been shown that gonadal steroids enhance the responsiveness of the GnRH system to the stimulatory action of NPY (44). However, this is unlikely for the inhibitory action of NPY, because it was not different between the OVX and OVX+E2 animals. It seems plausible that the amount of NPY released endogenously and acting on the GnRH pulse generator is enhanced by E2, because the primary action of gonadal steroids is to facilitate the output of peptide signals that are involved in the control of GnRH secretion (45). It has been demonstrated that estrogen receptors exist in a subpopulation of NPY neurons in the ARC (46, 47) and NPY synthesis and/or release are modulated by gonadal steroids (48). Assuming that those estrogen-sensitive NPY neurons in the ARC innervate the GnRH pulse generator and others do not, as previously suggested (46), then the impact of endogenous NPY on GnRH release in response to fasting could be much greater in the presence of E2. It is also probable that E2 acts presynaptically to enhance NPY transmission to the GnRH regulatory system. Therefore, it is very likely that the activity of endogenous NPY involved in the control of GnRH secretion is less potent in the absence of E2. Indeed, it has been shown, in rats, that the suppression of pulsatile LH release after acute fasting is dependent upon E2 (49).
With regard to the degree of undernutrition, it seemed that 4–5 days of fasting is a milder stress than chronic food restriction in ruminants, because chronic food restriction more severely suppresses pulsatile GnRH/LH secretion, both in the absence (6, 7, 8, 9, 12) and presence of gonadal steroids (6, 11). Moreover, it has been shown that food deprivation for a short period has a lesser influence on NPY mRNA expression than chronic food restriction (18). Consequently, we consider that without such facilitatory actions of E2 in OVX goat, a period of 5 days fasting might not be sufficient to stimulate endogenous NPY release to suppress GnRH pulse generator activity. On the other hand, in the growth-restricted lamb (12), it might be that NPY in the hypothalamus was sustained at high levels and thus NPY receptors were continuously activated during the long period of food restriction (more than 40 weeks); and therefore, undernutrition induced a decrease in GnRH secretion, even without E2. For a clearer conclusion, we are currently preparing further investigations to examine whether the effect of fasting in OVX+E2 goats is reversed by blocking endogenous NPY neuronal activity.
Several neurons in specific hypothalamic nuclei, which are implicated
in the control of ingestion and reproduction (13), were
activated in response to fasting in the male castrated goats (Fig. 5).
Neurons containing Fos were abundant in the ARC, moderate in the DMH,
and of a lesser extent in the PVH. The distribution pattern of Fos
observed in the goat is in agreement with that in mice fasted for
24 h (50), suggesting that similar neural substrates
are involved between monogastric and ruminant species in the neural
processing of undernutrition. The observation that most of these
activated neurons were localized in areas adjacent to the third
ventricle implies that transmission of peripheral signals to the
hypothalamus is mediated, in part, by the ventricular system. Indeed,
glucose is known as an important nutritional signal not only in rats
(34) but also in ruminants (33). Maekawa
et al. (34) recently found glucokinase
immunoreactivity in ependymocytes in the rat brain and suggested their
possible involvement in a glucose-sensing mechanism. However, neural
pathways by which nutritional signals are transmitted to the
hypothalamus are scarcely known in ruminants.
Double-labeling immunohistochemistry revealed that a subpopulation of
NPY neurons in the ARC are activated in response to fasting (Fig. 6F).
This result confirms previous findings that food restriction increases
NPY gene expression and the number of immunoreactive NPY cells in the
ARC of sheep (9, 19) and rats (18), and it
further suggests that NPY might be playing a critical role in
processing the information from undernutrition. However, it should be
mentioned that more than half of the NPY neurons did not express Fos,
which implies that there are discrete subpopulations of NPY neurons in
the ARC as discussed above. Conversely, because there were a number of
Fos-expressing cells that did not contain NPY in the hypothalamus, not
only NPY but also other peptides such as ß-endorphin
(13), galanin (13, 51), and
melanin-concentrating hormone (52) could participate in
the fasting-induced alteration of hypothalamic activity.
In summary, we have demonstrated, in the goat, that: 1) the activity of the GnRH pulse generator is suppressed in proportion to the extent of fasting in the presence of E2; 2) exogenous NPY administered into the LV suppresses the activity of the GnRH pulse generator regardless of the presence of E2; and 3) several hypothalamic neurons/regions, including NPY neurons in the ARC, were activated by fasting. The present electrophysiological and morphological results, together with a large body of previous evidence, suggest that undernutrition stimulates NPY neurons, possibly in the ARC, and that NPY, in turn, acts centrally on the hypothalamic GnRH pulse generator to suppress its activity, and thereby pulsatile release of LH is decreased.
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Acknowledgments |
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Footnotes |
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Received July 18, 2000.
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